Ascorbic Acid PDF

BIOCHEMISTRY RESEARCH TRENDS
ASCORBIC ACID
PROPERTIES, SYNTHESIS AND
APPLICATIONS
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ASCORBIC ACID
PROPERTIES, SYNTHESIS AND
APPLICATIONS
EMMA PARSONS
EDITOR
New York
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Library of Congress Cataloging-in-Publication Data

Names: Parsons, Emma, editor.
Title: Ascorbic acid : properties, synthesis and applications / editor, Emma Parsons.
Description: Hauppauge, New York : Nova Science Publisher's, Inc., [2016] |
Series: Biochemistry research trends | Includes bibliographical references and index.
Identifiers: LCCN 2016031698 (print) | LCCN 2016039823 (ebook) | ISBN 9781634858861
(hardcover)
Subjects: LCSH: Vitamin C--Physiological effect. | Vitamin C--Therapeutic use.
Classification: LCC QP772.A8 A83 2016 (print) | LCC QP772.A8 (ebook) | DDC 612.3/99--dc23
LC record available at https://lccn.loc.gov/2016031698
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Preface vii
Chapter 1 Overview of the Physiological Reactions of the
Monodehydroascorbate Radical 1
Kazuo Kobayashi
Chapter 2 Production of L-Ascorbic Acid at the Industrial
Scale 29
Sui Kiat Chang
Chapter 3 Recent Progress on the Electrochemical Detection of
Ascorbic Acid 47
Li Fu and Aimin Yu
Chapter 4 Suppressive Effect of Acyl Ascorbate Coexistent with
Tocopherol on Oxidation of Linoleic Acid 91
Yoshiyuki Watanabe, Takahiro Ishigaki, Sachiyo
Fujii and Shuji Adachi
Chapter 5 Stabilization of L-(+)-Ascorbic Acid in an Iron
Fortified Vegetable Product (Cucurbita Moschata
Duchesne Ex Poiret) Using an Alginate Coating 105
María Dolores De’Nobili, Carolina Genevois, Ana
M. Rojas, Silvia Flores and Marina de Escalada Pla
Chapter 6 Stabilization of Ascorbic Acid by
Microencapsulation for Cosmetic Preparations 123
Yhors Ciro and John Rojas
vi Contents
Chapter 7 Alkyl Esters of L-Ascorbic Acid: From Synthesis to

Applications 141
Maria Laura Fanani, Raquel Viviana Vico and
Luciano Benedini
Chapter 8 Design, Preparation and Characterization of Modified
Release Pellets of Ascorbic Acid for Pharmaceutical
Applications 175
John Rojas and David Correa
Chapter 9 Role of Ascorbic Acid in Neuropsychiatric and
Neurodegenerative Disorders 193
Daiane de Bittencourt Fraga, Morgana Moretti and
Ana Lúcia S. Rodrigues
Chapter 10 The Application of Intravenous Megadose Ascorbic
Acid in the Treatment of Animal Cancers 221
Nabhat Thongsoi and Anudep Rungsipipat
Chapter 11 Ascorbic Acid as a Multifunctional Nutrient in
Mammals: Our Understanding Based on Studies
Using Genetically Modified Mice 239
Junichi Fujii, Takujiro Homma and Sho Kobayashi
Index 267
PREFACE
Ascorbic acid (AsH-, vitamin C), an essential nutrient for both human
health and animal health, participates in a wide range of important biochemical
reactions, including the synthesis of collagen, carnitine, and the
neurotransmitter norepinephrine. This book provides current research on
several properties of ascorbic acid. It also reviews the synthesis and
applications of this water-soluble vitamin.
Chapter 1 - Ascorbate (AsH-) plays a vital role in protection against
oxidative stress as a physiological reductant. In most cases, the
monodehydroascorbate radical (As-･) is formed as the primary product of such
reactions. The As- ･ disproportionates rapidly to form AsH- and
dehydroascorbate (As). Within cells, the regeneration of AsH- from As-･ is
important to maintain the antioxidant activity of AsH-. Some of the
mechanisms for the reversible reduction of As-･ to AsH- via specific enzymes
have been identified. One such enzyme is FAD-containing
monodehydroascorbate reductase, which is responsible for the NADH-
dependent recycling of As- ･ to AsH-. This activity participates in AsH-
regeneration in chloroplasts for scavenging hydrogen peroxide by ascorbate
peroxidase. Another example is the cytochrome b561 that acts as AsH--
dependent oxidoreductase transmembranes. Many cytochrome b561 family
members have been identified in various organisms. These proteins are known
to employ an organic radical As-･ as an enzyme substrate. This review focuses
on the reactions of As-･ and their physiological functions.
Chapter 2 - L-Ascorbic acid, also known as vitamin C, is a six-carbon
organic compound with reducing properties. L-Ascorbic acid (L-AA) is
produced using the industrial Reichstein-Grussner process. Industrially
viii Emma Parsons
synthesized L-AA is used in the feed, food, nutrition and pharmaceutical

sector as nutritional supplement and preservative, making use of its
antioxidant properties. Sugars are the main substrate used for the synthesis of
L-AA. D-sorbitol is converted to L-AA via 2-keto-L-gulonic acid as key
intermediate, using a bio-oxidation with Gluconobacter oxydans and several
chemical steps. Recently, industrial production processes use extra bio-
oxidation steps with Ketogluconicigenium vulgare as biocatalyst to convert D-
sorbitol to the intermediate 2-keto-L-gulonic acid without chemical steps. The
enzymes involved are characterized by a broad substrate range with
regiospecificity. Recently, novel enzymes were identified that generate L-AA
directly via oxidation of L-sorbosone, an intermediate of the bio-oxidation of
D-sorbitol to 2-keto-L-gulonic acid. This step opens the possibility for a direct
route from D-sorbitol to L-AA, without the need for chemical rearrangement
of 2-keto-L-gulonic acid. All these steps utilize the concept of biotechnology
by using renewable raw materials to reduce solvent and energy needs.
Chapter 3 - Ascorbic acid (vitamin C) is a water-soluble vitamin, widely
present in many biological systems. The detection of ascorbic acid in various
foods, drugs, physiological fluids is very important for agricultural,
pharmaceutical and biomedical industries. The present review summarises the
recent progress on the detection of ascorbic acid using electrochemical
approaches. The electrochemical behavior of ascorbic acid at common
electrodes is first introduced, followed by a comprehensive review on the
different types of materials used for electrode surface modification to enhance
the electrochemical detection of ascorbic acid. Strategies to minimize the
interference problem during detection and the practical application for real
sample analysis are then discussed. To conclude, further research trends on the
development of electrochemical sensors of ascorbic acid are proposed.
Chapter 4 - Lipids are susceptible to oxidation, which results in
undesirable flavor and the formation of toxic products such as hydroperoxides.
Therefore, lipid oxidation has received much attention because of its
involvement in food deterioration. L-Ascorbic acid and tocopherols are used
as natural and safe antioxidants in foods. Acyl ascorbate has an amphiphilic
structure and the advantages of increased solubility and stability in fat-
containing foods relative to ascorbic acid. Further, the combination of ascorbic
acid and tocopherol is known to allow for synergistic inhibition of oxidation.
In this chapter, the antioxidative properties of acyl ascorbate coexistent with
-tocopherol for the oxidation of linoleic acid are examined. Lauroyl or
palmitoyl ascorbate and -tocopherol were added to linoleic acid at the total
molar ratio of 0.005; the mixture was stored at 65°C and 12% relative
Preface ix
humidity, and then, the oxidation process was measured by gas

chromatography. Linoleic acid without any antioxidant oxidized rapidly. In
contrast, -tocopherol markedly suppressed the oxidation of linoleic acid,
while the addition of lauroyl ascorbate further improved the oxidative stability.
Additionally, the antioxidative effect of palmitoyl ascorbate was as significant
as that of lauroyl ascorbate. On the other hand, the synergistic antioxidative effect
of ascorbic acid and -tocopherol was not remarkable, as acyl ascorbate
showed more effective antioxidative activity than did ascorbic acid in bulky
linoleic acid containing -tocopherol.
Chapter 5 - In previous studies it was observed that in fortified pumpkins
(Cucurbita moschata Duchesne ex Poiret) with iron (Fe) and L-(+)-ascorbic
acid (AA), the latter had a minimum retention because AA stabilization was
adversely affected by the presence of Fe. From this, it was proposed
compartmentalize these two nutrients, being Fe applied to the impregnation of
mesocarp pumpkin, while the AA was dissolved in a coating formulation
consisting of alginate. Pumpkins, cut and blanched, were subjected to a dry
impregnation. Two batches were prepared, one in which the Fe and AA were
incorporated into pumpkin tissue, while in the second batch the vegetable
matrix was only fortified with Fe. The studied systems were: a) Control
fortified pumpkins with Fe and AA, covered with alginate coating (C); b)
fortified with Fe, covered with alginate coating containing AA (F). In addition,
two water activity (aW) conditions 0.920 (F1 and C1) and 0.760 (F2 and C2)
were tested. All systems were contained in plastic trays, packaged in plastic
bags and stored at a constant temperature of 25ºC for 23 days. During storage,
AA concentration as well as color changes were registered. The AA loss and
also b* and Chroma (Chr) parameter reductions fitted to a first order kinetic.
For aW 0.760, the values obtained for the AA initial content were significantly
(p < 0.05) higher, while the rate constants of AA loss were lower. The kinetic
constants for color parameters were one order lower than AA loss. In general,
F systems showed a faster reduction of b* and Chr than controls (C), but the
aw assayed did not affect the rate of color changes. When fortified pumpkins
were tested at aw 0.92 and storage at 18 and 25ºC, it was observed reduction of
AA loss rate only in F1 pumpkin at the lower temperature. The reduction of
color parameters showed an important influence of both compartmentalization
of AA and storage temperature. It could be concluded that AA supporting on
an alginate edible coatings enhances this vitamin retention and then it could be
represent an effective strategy when developing food products fortified with
iron.
x Emma Parsons
Chapter 6 - Ascorbic acid has a variety of biological and dermatological

functions. These functions are closely related to the antioxidant properties of
this compound contributing to maintenance of the sensory properties of
cosmetics products. However, environmental factors such as air, oxygen,
metal ions, temperature, pH, UV and X-ray degrade this compound affecting
the quality of cosmetic products. Currently, the microencapsulation technology
is being used to stabilize this compound. Particularly, in this chapter the
technique of ionic gelation of chitosan with sodium lauryl sulfate is discussed.
Chitosan is biodegradable, biocompatible and soluble at the body temperature
and hence, it is an ideal material for cosmetic applications. Further, it is
positively charged and forms a polyelectrolyte complex with negatively
charged compounds such as sodium lauryl sulfate. The internal gelation was
carried out with solutions of chitosan and sodium lauryl sulfate at
concentrations of 0.5, 1.0 and 1.5% (w/v), containing ~20 mg of ascorbic acid
in different experimental runs. The encapsulation process of ascorbic acid was
performed by processes such as homogenization and sonication-
homogenization, respectively. In both processes, spherical microcapsules of
AA were obtained with a narrow distribution of particle size (0.7-2.1 µm), but
only by using sonication-homogenization at 7000 rpm less cohesive
microcapsules were obtained.
The encapsulation efficiency depended on the processing conditions and
level of parent materials and ranged from ~14 to 90% and ~14 to 71% for
sonication-homogenization and homogenization, respectively. In both
processes, runs having the lowest levels of chitosan (0.5% w/v) were desirable
resulting in particles with spherical morphology, high encapsulation efficiency
and low cohesive behavior. The ionic gelation technique was successful to
stabilize ascorbic acid and hence, it enhanced the shelf-life of these cosmetic
products (21 days) with respect to the free AA (3.1 days).
Chapter 7 - Antioxidants are important additives in food, cosmetics and
pharmaceutics, and vitamin C is one of the best-positioned for industrial
applications. The usage of native L-ascorbic acid is limited to water-
containing media because it is hydrophilic. To overcome this, an extensive
effort has been made to develop new amphiphilic derivatives capable of being
applied in lipophilic environments. The alkyl derivatives obtained through
esterification of the primary hydroxyl group of L-ascorbic acid with carboxylic
acids are the most widely studied compounds from the synthetic,
physicochemical and pharmaceutical point of view, as well as being those
most used in commercial formulations.
Preface xi
This chapter offers a comprehensive review of the bibliography regarding

the main pathways to synthesize these alkyl derivatives, involving enzyme
catalysis and esterification in acidic or alkaline media to obtain the alkanoyl-6-
O-ascorbic acid esters (ASCn). This information is presented together with
their stability properties.
Besides its antioxidant properties, the ASCn family has been used in
different pharmacological preparations for its amphiphilic nature. The
presence of the acyl chain provides the capacity to self-organize into micelles
and coagels when suspended in water. Knowledge of the phase behavior of
surfactants in aqueous media is important for understanding the properties of
these systems and is vital for numerous industrial applications. These, along
with a review of surface properties of the ASCn family, are dealt with in this
chapter.
ASCn amphiphiles, when self-organized at the air-water interface, form
Gibbs and Langmuir monolayers with a large variety of stability, phase states
and surface textures, depending on the length of the acyl chain moiety and
ionization conditions. Furthermore, different members of the ASCn family
have differences in their effectiveness for particular pharmaceutical purposes.
Thus, the physicochemical properties of each member of this drug family may
be determinant for their efficacy in pharmaceutical processes, in particular
those related to surface phenomena and interaction with biomembranes.
Cell membranes are the first contact of lipophilic drugs with cells and
often form a reservoir. Drug/membrane interaction is thus a fundamental
condition for their pharmacological function and a potential regulatory point.
The authors review here some attempts to experimentally explore the
interaction and the general physicochemical effect of ASCn family members
when they are incorporated into model lipid membranes under controlled
conditions.
Finally, the authors review some approaches to the use of ASCn in
biomedicine. Those are based mainly on I) their antioxidant capacity, because
the presence of ASCn derivatives in formulations may prevent the degradation
of drugs sensitive to light exposure, oxidant or reactive oxygen compounds,
and/or II) the capacity of ASCn to form micelles, coagels and liquid crystals,
which can modify some properties of the medium and act as drug delivery
nanostructures. The new strategies in the use of ASCn comprise examples
from liposome-based encapsulated nanoparticles to novel adjuvant activity of
ASCn liquid crystals with high immunological efficiency.
Chapter 8 - Ascorbic acid is an essential vitamin, which cannot be
synthesized and stored in the body. It scavenges free radicals, enhances
xii Emma Parsons
immunity, promotes collagen biosynthesis, provides photoprotection, and

causes melanin reduction. Further, its antioxidant activity could reduce the risk
of cancer. As a result, a daily supplementation of this vitamin is crucial to
prevent and ameliorate these diseases. However, most pharmaceutical products
are intended for immediate release of this vitamin. Conversely, a controlled
release formulation is preferable due to better patient compliance and
application of a single dose in extended time intervals. This chapter explores
the pelletization technology to control the particles size, enhance the process
reliability, reproducibility, and the ascorbic acid release. The goal of this
chapter is to study the influence of polymer type (i.e., microcrystalline
cellulose, kappa carrageenan and hydroxypropyl methylcellulose (HPMC) and
ascorbic acid concentration on shape descriptors, particle characterization and
drug release kinetics. Processing time varied according to the progression of
spheronization process and the rate was kept constant at 12 Hz using a 30 cm
diameter spheronizer equipped with a cross-hatched plate.
On the other hand, the polymer chemistry influenced the morphological
characteristics of the pellets. Further, other hydrophilic polymers such as
HPMC, k-carrageenan, pectin and sodium carboxymethylcellulose were
unsuccessful to produce spherical pellets. All polymers exhibited a biphasic
drug release profile characterized by an initial large drug released (60-80%)
within 1h followed by a slow released within 9h. The increase of drug loading
was in detriment of shape descriptors, mass and breaking strength, especially
at 75% drug load, but increased the compressibility, densification, and
friability of the pellets. Preliminary studies of drug loading in mixtures with
microcrystalline cellulose having loads from 50 to 60% were chosen as ideal
for the formation of spherical pellets and exhibited an immediate drug release.
Conversely, MCC pellets coated with shellac, carnauba wax and the
copolymer of ethyl acrylate, methyl methacrylate and methacrylic acid with a
quaternary ammonium group (Eudragit® RL) showed a slower drug released.
The best controlled release pellet formulation had a coating layer of Eudragit®
RL followed by 10 layers of shellac. These coated pellets were able to release
the drug by diffusion from the core to the coating layer which in turn, swelled
and controlled the drug release.
Chapter 9 - Over the past decade, new insights and advances on
neurobiology have extensively characterized critical roles for ascorbic acid in
central nervous system. Ascorbic acid is proposed as a neuromodulator of
glutamatergic, cholinergic, dopaminergic and GABAergic neurotransmission.
Moreover, it has also shown an important role in support and structure of the
neurons, differentiation process, maturation and neuronal survival.
Preface xiii
Additionally, ascorbic acid is a powerful antioxidant highly concentrated in

the brain and neuroendocrine tissues such as adrenal, therefore its involvement
in neurological disorders should not be dismissed. On the basis of the
extensive research on ascorbic acid biological mechanisms in health and
disease and its involvement in homeostasis of central nervous system, this
chapter presents evidence for associations of this vitamin with schizophrenia,
major depressive disorder, bipolar disorder and neurodegenerative diseases.
Considering the drawbacks of the conventional pharmacotherapy for the
treatment of these neuropathologies, particular attention is attributed to the
biochemistry and neuronal metabolism interface, animal models that are useful
for this area of research, and the human studies that implicate ascorbic acid as
potential therapeutic strategy.
Chapter 10 - Intravenous megadose ascorbic acid (vitamin C, ascorbate)
has been using as a popular chemotherapeutic agent in complementary,
alternative, and integrative medicine over the past 50 years. Because of its
outstanding property in cancer treatment, ascorbic acid has gained increasing
interest in human oncology. As a result, accumulative updated scientific basis
for the use of intravenous megadose ascorbic acid as an adjuvant treatment for
human cancer patients is increasing with new knowledge in the
pharmacokinetics and pharmacodynamics of intravenous and parenteral use. In
addition, new clinical data in humans has given a more complete
understanding of the fundamental aspects of intravenous megadose ascorbic
acid and its therapeutic effect on various types of cancer. These included in
vitro and in vivo studies, as well as, the clinical use in many types of terminal
cancer patients that had underwent standard conventional treatment with
limited improvement. Recently, the results obtained from human clinical trials
in patients with advanced untreatable malignancies revealed the outstanding
anticancer effects of intravenous ascorbic acid without any adverse effects.
This study also demonstrated prolonged survival time, improvement in global
health and quality of life. Studies related with intravenous ascorbic acid on
cancer treatment have been conducting up to the present. According to the fact
that the use of intravenous megadose ascorbic acid has never been reported in
canine cancer treatment, and most preclinical and clinical data presented so far
has only been derived from human studies. It is interesting whether the same
response could be obtained in canine cancer patients, so, preliminary case
studies were conducted to explore the clinical response and the
histopathological features of neoplastic cells after treatment in two aggressive
tumors, mammary tumor and mast cell tumor, commonly found in dogs. The
results were discussed.
xiv Emma Parsons
Chapter 11 - Ascorbic acid (AsA) functions as a cofactor in a variety of

physiological reactions including collagen synthesis and monoamine
production and also functions as an essential antioxidant by scavenging
reactive oxygen species. Hence a deficiency of AsA results in an imbalance of
cellular homeostasis and can be fatal, as is typically observed in scurvy. While
most organisms, including vertebrates, are able to synthesize AsA, primates
and some other animals such as the guinea pig cannot due to a loss of L-
gulono--lactone oxidase (GULO) function, which catalyzes the final step of
the AsA synthesis in the endoplasmic reticulum. Although the mouse is the
most popular animal used in biomedical research, it is not an adequate animal
for studying the physiological and pathological roles of AsA for this reason.
Genetically modified mice that have a defect in processes of the AsA synthesis
have been developed to conquer this disadvantage and are now used in studies
to reveal currently unrecognized functions of AsA in vivo.
At present genetically modified mice that have defects in the last three
steps of the AsA synthetic pathway have been established. The GULO−/−
mouse was first established in 2000 and has since been used as an AsA-
deficient mouse model. Gluconolactonase (GNL) is known as a senescence-
associated marker protein 30 (SMP30) with uncertain functions. The identity
of SMP30 as GNL was demonstrated in 2006 by using SMP30−/− mice that
were established in 2002. In 2010, studies using mice lacking AKR1A and
AKR1B, which are members of the aldo-keto reductase superfamily, revealed
that they catalyze the conversion of D-glucuronate to L-gulonate in the AsA
synthesis pathway. Because the liver is the major organ for producing AsA
and AKR1A is predominantly expressed in the liver, AKR1A contributes
about 85-90% of the AsA that is synthesized in the body of the mouse.
In this review article, the authors provide an overview of the literature
regarding studies in which these genetically modified mice have been used and
attempt to gain insights into the physiological and pathological roles of AsA in
vivo. The authors also note that some of the studies may have limitations
because the gene products may have additional functions in addition to the
synthesis of AsA. Thus the authors will pay attention to this issue in this
review and attempt to obtain a clear vision of the roles of AsA in the mouse.
In: Ascorbic Acid ISBN: 978-1-63485-886-1
Editor: Emma Parsons © 2017 Nova Science Publishers, Inc.
Chapter 1
OVERVIEW OF THE PHYSIOLOGICAL

REACTIONS OF THE
MONODEHYDROASCORBATE RADICAL
Kazuo Kobayashi*
The Institute of Scientific and Industrial Research, Osaka University,
Mihogaoka, Ibaraki, Osaka, Japan
ABSTRACT
Ascorbate (AsH-) plays a vital role in protection against oxidative
stress as a physiological reductant. In most cases, the monodehy-
droascorbate radical (As- ･) is formed as the primary product of such
reactions. The As- ･ disproportionates rapidly to form AsH- and
dehydroascorbate (As). Within cells, the regeneration of AsH- from As-･
is important to maintain the antioxidant activity of AsH-. Some of the
mechanisms for the reversible reduction of As- ･ to AsH- via specific
enzymes have been identified. One such enzyme is FAD-containing
monodehydroascorbate reductase, which is responsible for the NADH-
dependent recycling of As-･ to AsH-. This activity participates in AsH-
regeneration in chloroplasts for scavenging hydrogen peroxide by
ascorbate peroxidase. Another example is the cytochrome b561 that acts as
*
Corresponding author: Kazuo Kobayashi. The Institute of Scientific and Industrial Research,
Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047 Japan. Email:
kobayasi@sanken.osaka-u.ac.jp.
2 Kazuo Kobayashi
AsH--dependent oxidoreductase transmembranes. Many cytochrome b561

family members have been identified in various organisms. These
proteins are known to employ an organic radical As- ･ as an enzyme
substrate. This review focuses on the reactions of As - ･ and their
physiological functions.
ABBREVIATIONS
AsH- ascorbic acid
As-･ monodehydroascorbate radical
As dehydroascorbate
MDA reductase monodehydroascorbate reductase
DCIP 2,6-dichlorophenolindophenol
TEMPO 2, 2’, 6, 6’-terametthypiperidin-1-oxyl radical
CG chromaffin granules
Dcytb duodental cytochrome b
TM transmembrane
EDTA ethylenediaminetetraacetic acid
SOD superoxide dismutase
Keywords: ascorbate, monodehydroascorbate radical, pulse radiolysis,

monoascorbate reductase, cytochrome b561, proton coupled electron
transfer
INTRODUCTION
Ascorbic acid (AsH-, vitamin C), an essential nutrient for human health
[1], participates in a wide range of important biochemical reactions, including
the synthesis of collagen [2], carnitine [3], and the neurotransmitter
norepinephrine [4]. Futhermore, AsH- is critical antioxidant in extracellular
fluids [5] that has been shown to efficiently scavenge reactive oxygen and
radical species in cells [6-8]. AsH- also contributes significantly to the
recycling of plasma membrane-tocopherol (vitamin E) via the reduction of
the -tocopheroxyl radical [9]. In this way, AsH- acts to prevent lipid
peroxidation in membranes [10].
Overview of the Physiological Reactions of the … 3
Under physiological conditions, AsH- typically undergoes a reversible

one-electron oxidation to As-･. The formation of As-･ was first empirically
observed in 1961 during the enzymatic oxidation of AsH- by ESR techniques
[11], and has been observed in many enzymatic and nonenzymatic reactions
[11-17]. In the absence of a reactant for As-･, the radical is spontaneously
disproportionated to AsH- and dehydroascorbate (As) [18]. As is unstable at
physiological pH and undergoes irreversible hydrolysis to 2, 3-diketo-L-
gulonic acid [19, 20], which results in decreased levels of vitamin C.
Therefore, in addition to dietary intake, the regeneration of AsH- from As-･ is
vital to maintain the antioxidant activity of AsH-. Several species, including
humans, higher primates and guinea pigs, have lost the ability to synthesize
AsH- because they lack L-gulonolactone oxidase, the final enzyme in the AsH-
biosynthetic pathways in mammals [21]. Thus, efficient mechanisms for the
absorption, transport, and recycling of AsH- are critical in these species. Some
of the mechanisms for the reversible reduction of As-･ and As to AsH-, that
have been identified so far, are located within cells.
Due to the instability of As- ･, its concentration is generally measured
indirectly in the presence of AsH- and ascorbate oxidase [22, 23]. However, it
is possible to directly investigate the reaction of As-･ using a pulse radiolysis
technique [24-26]. The pulse radiolysis approach allows for the production of
definite amounts of As-･ over a very short time-scale and it is used estimate
the oxidative and catalytic mechanisms of the enzymatic reactions.
Numerous valuable reviews describing ascorbate, its properties and
associated mechanisms of action, have been published over the years [1, 27-
30], but few have covered As-･. Thus, this review aims to fill this void by
collating and discussing the information known so far about As-･, including its
systematic pathways.
1. CHEMICAL PROPERTIES OF ASCORBATE

Ascorbate exists in three states: the fully oxidized form, dehydroascorbate
(As), semiquinone form (As-･) and fully reduced ascorbic acid form (AsH-), as
shown Figure 1. AsH- is an excellent reducing agent and undergoes two
consecutive one-electron oxidations to form As-･ and As. At physiological pH,
ascorbate exists predominantly as the ascorbate monoanion, AsH- [18, 31].
The ESR spectrum of As-･ shows that the radical exists as the anion with the
4 Kazuo Kobayashi
unpaired electron spread over highly conjugated tricarbonyl systems. Thus, its
radical form is stabilized, and has an absorption maximum at 360 nm with an
extinction coefficient of 3300 M-1cm-1 [32].
Figure 1. (A) Titration of ionizable group of ascorbate. The ascorbate monoanion

(AsH-) is the predominant species of ascorbate under physiological conditions. The
three redox states of ascorbate (AsH-, fully reduced form; As-･,
monodehydroascorbate; As, dehydroascorbate).
Figure 2. pH-dependent decay of the second-order rate constants of As-･.
The decay of As- ･ obeys second-order kinetics, and the decay rate
constant increases rapidly as the solution acidity increases from pH 8 to 5
(Figure 2). Bielski et al. [18] firstly hypothesized that the pH-dependent
change in the decay could be attributed to reactions between As-･ and AsH･
with an associated pKa value of 4.25, similar to that observed for the pH-
dependent decay of O2- [33]. However, the ESR of As-･ exhibits the anionic
form over the pH range 1-12 and protonates only at lower pH levels [34, 35].
Bielski et al. [32] then formulated a mechanism for the decay of As- ･ in
solvents of varying ionic strengths and suggested that As- ･ decays via an
initial dimerization to As22-, which may then react with H+ to form AsH- and
As, as seen below in reactions (1), (2), and (3).
(1)
(2)
(3)
The equilibrium constant ([As22-]/[As-･]2 = k1/k-1) was calculated to be 103

M-1. From this value, the radical would not be dimerized more than 2%.
However, the definitive identification of As22- has not yet been achieved.
6 Kazuo Kobayashi
The redox potentials of As- ･/AsH- (390 mV) and As/As- ･ (-210 mV)
suggest that As-･ serves not only as a one-electron donor, but also as a one-
electron oxidant. As- ･ acts as an effective one-electron reductant for low
molecular weight compounds such as Fe3+-ethylenediaminetetraacetic acid
(EDTA) [24], ferricyanide, and 2,6-dichlorophenolindophenol (DCIP) [36]. In
contrast, the reaction of As-･ with ferric hemoproteins such as metmyoglobin
(+53 mV), methemoglobin (+175 mV), and cytochrome b5 (0 mV) could not
be detected, though the redox couple of As/As-･ (-210 mV) is expected to be
favorable for the reduction of these hemoproteins [24]. It may be that As-･
decays prior to accessing the heme that is buried in a hydrophobic pocket.
The univalent oxidation of AsH- to As-･ proceeds via a concerted transfer
of H+ and e-, rather than by a stepwise mechanism with initial proton or
electron transfer. An interesting example was reported in the reaction of AsH-
with the (2, 2’, 6, 6’-tetrametthypiperidin-1-oxyl) (TEMPO) radical in
acetonitrile (reaction 4) [37, 38].
(4)
The reaction rate and equilibrium constants changed upon the addition of
water or other polar solutes in acetonitrile. This implies that changes in the
hydrogen-bond donating environment influences the reactivity of AsH-.
Notably, AsH- has the negative charge with the enolate oxygen, whereas As-･
has a more delocalized triketone-like structure. This suggests that hydrogen
bond donors bind stronger to AsH- than As- ･ . The tuning of ascorbate
properties by the degree of hydrogen-bonding may be very important in
various biological actions of ascorbate.
2. MONODEHYDROASCORBATE REDUCTASE
2.1. Monodehydroascorbate Reduction System
Figure 3 shows recycling of ascorbate in the cell. Ascorbate is transported

into the cell by Na+-dependent active transporters [57, 58] and facilitative
glucose transporters [59]. Both As- ･ and As are reduced back to AsH- by
several enzymatic systems. The reduction of As- ･ in the cytosol has been
assigned using NADH (NADH-cytochrome b5 reductase) or NADPH
(thioredoxin reductase). As- ･ can also be reduced in the lumen of
neuroendocrine secretary vesicles. Iyanagi and Yamazaki showed that
oxidation of NADH by liver microsomes was stimulated in the presence of As-
･, and microsomal NADH-cytochrome b5 reductase was proposed to reduce
As- ･ in the presence of As- ･ [42]. In rat liver, the NADH-dependent As- ･
reductase activity is localized in the outer mitochondrial membrane [43].
Purified rat liver thioredoxin reductase was also shown to catalyze the
NADPH-dependent reduction of As-･ with a Km value of ~3 M [44]. The
NADPH-dependent As-･ reduction markedly decreased by ~75%, in the liver
cytosolic fraction derived from selenium-deficient rats. These results suggest
that thioredoxin reductase can function as a cytosolic As-･ reductase and may
complement cellular AsH- recycling by membrane-bound NADH-dependent
reductase.
Figure 3. Recycling ascorbate in the cell.

8 Kazuo Kobayashi
The reduction of As is probably less important than reaction of As-･. As

can be nonenzymatically reduced to AsH- by GSH, but this reaction is not
efficient [45]. As is also reduced by glutaredoxin [46], protein disulfide
isomerase [46], glutathione transferase [47], 3-hydroxysteroid
dehydrogenase [48], and thioredoxin reductase [49] in reactions using GSH or
NADPH.
2.2. Reaction Mechanism in Monodehydroascorbate Reductase
In plants, As-･ is reduced to regenerate AsH- by an NAD(P)H-dependent

in many plants [50-54]. This activity participates in AsH- regeneration in
chloroplasts for scavenging hydrogen peroxide by ascorbate peroxidase [50,
55]. Hossain and Asada first purified a peculiar enzyme, named
monodehydroascorbate (MDA) reductase, from cucumber. MDA reductase is
soluble monomeric enzyme, with a molecular mass of 47 kDa, that contains 1
molecule of FAD per enzyme molecule. It is distinguished from menadione
reductase [56] and cytochrome b5 reductase by its specificity for electron
acceptors and donors. Both NADH and NADPH serve as electron donors [56].
The As- ･ acts as the specific electron acceptor. In addition to As- ･ ,
ferricyanide and DCIP, p-benzoquinone, and 1,2-naphthoquinone-4-sulfonate
can serve as the electron acceptor, but little or no oxidation of NADH was
observed by cytochrome b5, cytochrome c or methylene blue. The kinetic data
suggests a ping-pong mechanism following reactions (5), (6) and (7):
(5)
(6)
(7)
where E-FAD, E-FADH--NAD(P) and E-FAD ･ --NAD(P) represent the
+ +
oxidized form, the reduced charge-transfer complex, and the semiquinone

forms, respectively. The enzyme FAD is reduced by NAD(P)H to form a
charge-transfer complex, E-FAD(P)H--NAD+ (reaction 5). Then the reduced
enzyme donates the electron to As- ･ through two successive one-electron
transfers, forming the FAD semiquinone, E-FAD ･ --NAD(P)+, as an
intermediate (reactions 6 and 7). This sequence of reactions was observed
using the pulse radiolysis method [25]. When 30 M As-･ was generated in 28
M of the fully reduced form of MDA reductase, the kinetic difference spectra
after pulse radiolysis at 1, 100, and 800 s after pulse radiolysis were obtained
(Figure 4-A). The spectrum observed 1 s after the pulse with an absorption
maximum at 360 nm corresponds to the As- ･. From the kinetic difference
spectra taken at 100 and 800 s (Figure 4-B), As-･ reacts first with the fully
reduced enzyme to form the semiquinone enzyme (reaction 6). Subsequently,
the semiquinone reacts with As-･ to yield the oxidized form of the enzyme
(reaction 7).
Figure 4. (A) Kinetic difference spectra after pulse radiolysis of MDA reductase (A)
and the difference spectra of the semiquinone form of MDA reductase (straight line)
and the oxidized enzyme (dashed line) minus the fully reduced form of the enzyme in
complex with NAD+, respectively (B). (From Ref. 25).
10 Kazuo Kobayashi
Figure 5. Proposed topological model of adrenal cytochrome b561 in the CG membrane.
The second-order rate constants between As-･ and the fully reduced and
semiquinone forms of MDA reductase were measured to be the same at 2.6 ×
108 M-1 s-1. The values of the highest rate constant were obtained for the
reaction of As-･ with biological molecules. The high rate constants of MDA
reductase and the high specificity of As-･ can be attributed to the localization
of the amino acid cationic group residues near the active site, which provides
electrostatic guidance to the anionic substrate, As-･. This proposal is supported
by the fact that the rate constant of the reaction of MDA reductase with As-･
decreases with increasing NaCl concentration [25]. A similar ionic strength
effect was obtained in the reaction of O2- with superoxide dismutase (SOD)
[58]. In order to achieve rapid reaction with O2-, the Fe, Mn, and Cu/Zn
centers of SOD enzymes have evolved so that O2- is guided into the active site
channel using the distribution of electrostatic charges on the surface of the
enzymes [59]. In addition, it seems likely that MDA reductase has an active
site structure that accepts the physiological substrate of As-･.
A similar reaction sequence (5, 6, 7) has been proposed for NADH-
cytochrome b5 reductase. However, the rate constants for MDA reductase are
about 50 times larger than those in b5 reductase (4.3 x 106 M-1 s-1 3.7 x 105 M-1
s-1) [24]. This reflects the difference of the molecular activity between MDA
reductase (200 mol/enzyme s-1) and cytochrome b5 reductase (6 mol/enzyme s-
1) at a concentration of 2 M As-･ [24]. The rate of NADH oxidation by b
5
reductase did not follow saturation kinetics with respect to the steady-state
concentration of As-･ in the range of 0-6 M, whereas the Km value of As-･ in
MDA reductase was 1.4 M. In contrast, the electron transfer from b5
reductase may occur at a flavin edge exposed to the solvent through
bimolecular collision [60, 61]. The difference in the activity is consistent with
the fact that MDA reductase exhibits low sequence homology to bovine
cytochrome b5 reductase (homology score, -67) [62]. MDA reductase exhibits
only a low degree of sequence homology to eukaryote flavoenzymes.
Putidaredoxin reductase from Pseudomonas putida gives the highest
homology score (-390), and 26.8% of residues are conserved between
putidaredoxin reductase and MDA reductase in their sequences. The protein
fold of putidaredoxin reductase [63] is similar to disulfide reductase [64], and
consists of the FAD-binding, NAD-binding, and C-terminal domains. The
major differences are present in their C-terminal domains, and it seems likely
that As-･ binding site is located to this domain.
3. CYTOCHROME B561
3.1. Properties
Cytochrome b561, initially identified in the chromaffin granule (CG) of

bovine adrenal medullae [65, 66], is a family of highly conserved AsH--
dependent oxidoreductase transmembranes. This protein functions as a
regenerator of AsH- inside the neuroendocrine of neural cells. AsH- entrapped
in the neuroendocrine vesicles are consumed by dopamine -hydroxylase and
peptidylglycine -amidating monooxygenase resulting formation of As-･ [4,
67-69]. Intravesicular As- ･ is reduced back to AsH- by membrane-bound
12 Kazuo Kobayashi
cytochrome b561, and subsequently cytochrome b561 is reduced by

extravesicular AsH-, as shown in Figure 5 [4, 70-73]. With recent progress in
genome sequencing, there are many genes encoding proteins homologous to
animal cytochrome b561 [74], insects [69], planarian [74] and a very wide
range of higher plants [75-78]. Humans have six cytochrome b561 family
members; CG cytb; duodenal cytochrome b (Dcytb), lysosomal cytochrome b
(Lcytb); tumor suppressor cytochrome b (101F6); stromal cell-derived
receptor 2 (SDR-)2, and CYB561D1 [73]. These members display typical
characteristic properties of the cytochrome b561 family, such as the presence of
two b-type hemes, similarities in absorption spectra, and reducibility with
AsH-. These members family have not been studied in detail for their
physiological functions except CG cytochrome b561. It was proposed that
Dcytb has a transmembrane ferric reductase activity and catalyzes the
reduction of extravesicular ferric ion using electrons from cytosolic AsH- [79,
80].
3.2. Structure
Lu et al. [81] reported the crystal structure of the ferric form of

cytochrome b561 from Arabidopsis thaliana, which shares 34% identity and
54% similarity with the human form found in chromaffin granules [75].
Cytochrome b561 forms a homodimer, with each protomer consisting of six
transmembrane (TM) helices and two heme groups that are partially exposed
to solvent (Figure 6). The heme group on the cytoplasmic side is coordinated
by His84 from TM3 and His157 from TM5, whereas the heme on the
noncytoplasmic side is coordinated by His51 from TM2 and His118 from
TM4. The closest edge to edge distance between the two heme groups is about
15.3 Å, which exceeds the range, 14 Å or less, for through-space electron
transfer [81]. Notably, a water molecule is located between the heme centers,
4.5 Å from the cytoplasmic heme and 12 Å from the noncytoplasmic heme
(Figure 7). This water molecule and the conserved Phe129, located between
the two heme centers, have potential roles in bridging a tunneling electron
transfer. In this model, the electron transfer route between the two heme
groups occurs via through-space electron tunneling. An alternative interesting
model in electron transfer has been proposed through bonding, via a number of
covalent and hydrogen bonds [82]. This bond based model is illustrated in
Figure 7.
Figure 6. Crystallographic structure of cytochrome b561 from Arabidopsis thaliana. The

structure was produced with PyMol using structure from the Protein Data Bank (PDB
ID codes 4O6Y, 4O79, and 4O7G). (From Ref. 81).
Figure 7. Electron transfer route between the two heme groups of cytochrome b561 from
Arabidopsis thaliana. (From Ref. 81).
14 Kazuo Kobayashi
A B
Figure 8. Recognition of ascorbate by conserved amino acids. (A) Recognition of

ascorbate on the cytoplasmic side. (B) Recognition of As -･ on the noncytoplasmic side
of cytochrome b561 from Arabidopsis thaliana. (From Ref 81).
The negatively charged substrate AsH- or As- ･ binds to the conserved

positively charged residues on either side of the membrane. However, the
binding mode of AsH- and As-･ are quite different. The negatively charged
AsH- is enclosed in a hydrophobic pocket on the cytoplasmic side. The O atom
of the ketone group of AsH- is hydrogen-bonded to the NH2 groups of Lys81
and Arg150, and the hydroxyl group at position 2 of AsH- form two hydrogen
bonds with NH2 of Lys77 and Lys81 (Figure 8-(A)). The hydroxyl group at
position 3 interacts with the residue of Lys77 and the heme 6-propionate. The
five-membered ring of AsH- interacts with the heme group and the TM4
Tyr140 via van der Waals contacts. These residues are highly conserved in
cytochrome b561 from plants to human. In contrast, As-･ is positioned above
the heme group on the noncytoplasmic side, surrounded by two polar residues
and three aromatic amino acids (Figure 8-(B)). As-･ is hydrogen-bonded with
the residues of His106, Tyr115, and Asn186, and is stacked by the benzene
rings of Phe105 and Phe182 (Figure 8-(B)).
3.3. Reaction Mechanisms
The reaction of As-･ with purified cytochrome b561 from bovine adrenal
CG was investigated by pulse radiolysis [26]. Radiolytically generated As-･
rapidly oxidized the reduced form of heme b in cytochrome b561 to form the
oxidized form (8) with a second-order rate constant of 2.6 × 107 M-1 s-1 at pH
7.0. A decrease at 430 nm and an increase at 405 nm in absorbance reflect this
reaction (Figure 9(A)).
Subsequently, the initial changes in absorbance reversed in the time range
of seconds, indicating that re-reduction of cytochrome b561 occurred (Figure
9(B)). The rate constant of this process is independent of the concentration of
AsH- with a first order rate constant of 4 s-1 at pH 7.0. Figure 10 shows the
schematic presentation of reaction scheme after pulse radiolysis of reduced
form of cytochrome b561. In the first step (i), one of the heme b centers in
cytochrome b561 specifically reacts with As- ･, whereas the other does not.
Subsequently, an intramolecular electron transfer to the oxidized heme from
the other heme take place. However, the process cannot be followed directly
by the present method, because the two heme centers cannot discriminated by
the absorption spectra [72, 82]. Under the experimental condition, only half of
the heme in cytochrome b561 is oxidized under As-･ concentration. Therefore,
the rate-limiting step of the whole transmembrane reaction (Figure 10) is the
intramolecular electron transfer of cytochrome b561 from the extravesicular to
the intravesicular region.
16 Kazuo Kobayashi
A B
Figure 9. (A) (B) Absorbance changes after pulse radiolysis of the reduced form of CG
cytochrome b561 measured at 430 and 405 nm in the presence of AsH- and N2O at pH
7.0. (From Ref. 26).
Figure 10. Schematic presentation of the reactions after pulse radiolysis of the reduced
form of CG cytochrome b561.
Table 1. Rate Constants of the Oxidation of heme b by As-･ and

Re-reduction Rates and Their Redox Potentials of bH and bL in
Cytochrome b561 Family
b561 Protein Second-order rate First-order rate E01 (mV) E02 (mV)
constants of constants of re-
oxidation (M-1s-1) reduction (s-1)
bovine CG
cytochrome 2.6 × 107 4.0 170 60
b561
Zea may
Cytochrome
b561
WT 1.0 × 107 4.8 118 11.3
K83A 1.0 × 106 1.2 93.0 44.8
Tumor 5.0 × 107 4.8 109 26
Suppressor
106F6
Cytochrome b561 contains two b-type heme centers with redox potentials
of ~170 mV (high potential, bH) and ~60 mV (low potential bL) [83, 84]. The
difference is essential to facilitate the individual specificities for AsH- and As-
･. We propose that heme centers with a higher redox potential is responsible
for the electron donation to As-･. The physiological transmembrane electron
transport from the cytoplasma to the intravesicular region facilitates a ~100
mV gradient redox potentials between the two heme centers from bL to bH
(Figure 6). This proposal is supported by the treatment of oxidized cytochrome
b561 with diethyl pyrocarbonate. This modification led to the selective loss of
the electron accepting ability of AsH- without affecting electron donating to
As-･ [84, 85]. However, an alternative proposal on the location of hemes of the
bL to bH centers with respect to the cytoplasmic and luminal sides of the CG
membrane has been proposed [86-88]. In this case, the electron transfer from
bH to bL occurs through thermodynamically “uphill” electron transfer. The
electron transfer from cytosolic AsH- to intragranular As-･ is driven by the
membrane potential ( and the pH gradient [89].
Similar rapid oxidation by As-･ and subsequent slower re-reduction were
observed in other members of cytochrome b561 family such as in Zea mays
cytochrome b561 [90] and candidate tumor suppressor 101F6 protein [91].
18 Kazuo Kobayashi
Table I compares the rate constants of the oxidation and re-reduction of heme
b and their redox potentials at pH 7.0. It is noteworthy that the rate constant
for the initial oxidation of 101F6 protein is much faster than that of CG
cytochrome b561 (2-fold) and Zea mays cytochrome b561 (5-fold). The
difference cannot be explained by the redox potential of heme b. This suggests
that the molecular mechanism of the electron donation to As- ･ might be
different from other members of the cytochrome b561 family. The As-･ binding
residues observed in the crystal structures are not conserved in the sequence of
101F6 proteins. On the other hand, it is clear that the intramolecular electron
transfer in the cytochrome b561 family is very similar, where the rate constants
are not significantly different from each other (Table I). The differences in the
redox potential between the two heme groups are almost same (approximately
100 mV). This suggests that the distances between the two heme groups are
equivalent.
Notably, the mutation of Lys83 (corresponding to Lys85 of bovine CG
cytochrome b561 and Lys81 of A. thaliana cytochrome b561) on the cytosolic
side of Zea may cytochrome b561 indicates that Lys83 plays an essential role
for the binding of AsH- and the intramolecular electron transfer [90]. During
the electron transfer process, Lys83 on the cytoplasmic side is involved not
only in AsH- recognition but in catalysis through cycles of protonation and
deprotonation.
CONCLUSION
Free radicals usually decay rapidly through a dimerization or dismutation
pathway. In contrast, O2- and As-･ are the only known free radicals used as the
substrate of enzymes. The mechanisms in the recognition of free radicals of
the substrate and their efficient redox reaction should be verified. Ascorbate
binding pockets in cytochrome b561 have hydrogen-bond-donating or hydrogen
bond accepting character, and the changes in hydrogen-bond environment can
control the reactivity of ascorbate.
ACKNOWLEDGMENTS
I thank the many colleagues and collaborators who contributed to this
research. I thank Emeritus Prof. K. Asada (Kyoto University) and Dr. S. Sano
(Kyoto Prefectural University) for the reaction mechanism of MDA reductase;

and Prof. M. Tsubaki (Kobe University) and Dr. F. Takeuchi (Kobe
University) for the reaction mechanism of cytochrome b561. I am indebted to
Emeritus Prof. T. Iyanagi (Himeji Institute Technology) for valuable advice
and helpful discussions. I also thank the members of the Radiation Laboratory
at the Institute of Scientific and Industrial Research of Osaka University for
assistance in operating the accelerator.
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3668.
BIOGRAPHICAL SKETCH
Name: Kazuo Kobayashi
Affiliation: The Institute of Scientific and Industrial Research, Osaka
University
Education: Bachelor of Engineering, Himeji Institute Technology
Master of Engineering, Osaka University
Doctor of Engineering, Osaka University
Postdoctoral research Karolinska Institute, Department of Toxicology
Research and Professional Experience: Pulse radiolysis, Radiation
Biology, Nitric Oxide
Professional Appointments: Assistant Professor
Honors:
Publications Last 3 Years:
[1] Oxidative Stress Sensing by the Iron-Sulfur Cluster in the Transcription

Factor, SoxR Kazuo Kobayashi, Mayu Fujikawa, and Takahiro Kozawa.
J. Inorganic Biochem. 133, 87-91 (2014).
[2] Mechanistic Studies on Formation of the Dinitrosyl Iron Complex of the
[2Fe-2S] Cluster of SoxR Protein Mayu Fujikawa, Kazuo Kobayashi,
and Takahiro Kozawa. J. Biochem. 156, 163-172 (2014).
[3] Pulse Radiolysis Study of the Dynamics of Ascorbic Acid Free Radicals
within a Liposomal Environment Kazuo Kobayashi, Yumiko Seike,
Akinori Saeki, Takahiro Kozawa, Fusako Takeuchi, and Motonari
Tsubaki. PhysChemPhys 15, 2994-2997 (2014).
[4] Acid generation mechanism in anion-bound chemically amplified resists
used for extreme ultraviolet lithography Yoshitaka Komuro, Hiroki
Yamamoto, Kazuo Kobayashi, Yoshiyuki Utsumi2, Katsumi Ohomori,
and Takahiro Kozawa. Jpn. J. Appl. Phys. 53 (2014) 116503-1-116503-8.
[5] Redox-Dependent DNA Distortion in a SoxR Protein-Promoter
Complex Studied using Fluorescent Probes Mayu Fujikawa, Kazuo
Kobayashi, and Takahiro Kozawa. J. Biochem. 157, 389-397 (2015).
28 Kazuo Kobayashi
[6] Binding of Promoter DNA to SoxR Protein Decreases the Reduction

Potential of the [2Fe-2S] Cluster Kazuo Kobayashi, Mayu Fujikawa, and
Takahiro Kozawa. Biochemistry 54, 334-339 (2015).
[7] The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes
Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon
Thioether Bonds. Tadashi Nakai, Hiroto Ito, Kazuo Kobayashi, Yasuhiro
Takahashi, Hiroshi Hori, Motonari Tsubaki, Katsuyuki Tanizawa, and
Toshihide Okajima. J. Biol. Chem. 292, 11144-11166 (2015).
Chapter 2
PRODUCTION OF L-ASCORBIC ACID

AT THE INDUSTRIAL SCALE
Sui Kiat Chang

Department of Nutrition and Dietetics, School of Health Sciences,
International Medical University Malaysia. Kuala Lumpur, Malaysia
ABSTRACT
L-Ascorbic acid, also known as vitamin C, is a six-carbon organic
compound with reducing properties. L-Ascorbic acid (L-AA) is produced
using the industrial Reichstein-Grussner process. Industrially synthesized
L-AA is used in the feed, food, nutrition and pharmaceutical sector as
nutritional supplement and preservative, making use of its antioxidant
properties. Sugars are the main substrate used for the synthesis of L-AA.
D-sorbitol is converted to L-AA via 2-keto-L-gulonic acid as key
intermediate, using a bio-oxidation with Gluconobacter oxydans and
several chemical steps. Recently, industrial production processes use
extra bio-oxidation steps with Ketogluconicigenium vulgare as
biocatalyst to convert D-sorbitol to the intermediate 2-keto-L-gulonic
acid without chemical steps. The enzymes involved are characterized by a
broad substrate range with regiospecificity. Recently, novel enzymes
were identified that generate L-AA directly via oxidation of L-sorbosone,
an intermediate of the bio-oxidation of D-sorbitol to 2-keto-L-gulonic
acid. This step opens the possibility for a direct route from D-sorbitol to
L-AA, without the need for chemical rearrangement of 2-keto-L-gulonic
30 Sui Kiat Chang
acid. All these steps utilize the concept of biotechnology by using

renewable raw materials to reduce solvent and energy needs.
Keywords: L-Ascorbic acid (L-AA), 2-keto-L-gulonic acid, fermentation, L-

Sorbosone, microbial oxidation
INTRODUCTION
Vitamin C is widely distributed in plants (80-90% in fruits and vegetables)
and animals [1]. Some cereals and other foods and beverages have been
fortified with Vitamin C. Vitamin C is also available in caplets, tablets,
capsules, drink mix packets, in multi-vitamin formulations, in multiple
antioxidant formulations [1]. L-AA is a widely used food additive with many
functional roles, which is mainly based on its oxidation-reduction properties.
Its functional roles include its uses as a nutritional food additive, antioxidant,
browning inhibitor, reducing agent, flavor stabilizer, modifier and enhancer,
color stabilizer, dough modifier and in many other capacities. Ascorbyl
palmitate was developed to provide an ascorbic acid form with greater lipid
solubility for use in cooking purposes and antioxidant preparations [2]. The
annual production of synthetic L-AA is about 110 000 tonnes with the
fluctuating market price of about 10 US dollar per kg. About 50% of L-AA
production is used in the pharmaceutical industry, followed by the use as
antioxidant in food (25%) and beverages industry (15%). The remaining 10%
of L-AA production is used for animal feed applications. However, the feed
sector is the major application area for other vitamins compared to L-AA [3].
Right from the start, the key step in the industrial production of the
vitamin was a microbial oxidation converting D-sorbitol to L-sorbose. This is
followed by two further oxidation reactions, chemical or biotechnological.
Until today, the immediate product of these oxidations is 2-keto-L-gulonic
acid, which is isolated, purified, and chemically rearranged to L-AA [3]. Many
pathways have been shown to be able to convert a carbohydrate source to L-
AA, but only those using L-sorbose as intermediate achieved commercial
application. This demonstrated their superior technical and economic
efficiency [3]. Another way to 2-keto-L-gulonic acid, utilizing D-glucose with
2,5-diketo-D-gluconate as an intermediate, attracted much attention in the
1980s. However, these processes were not used industrially. Microbial routes
directly affording L-AA, based on the natural biosynthetic pathways, have
Production of L-Ascorbic Acid at the Industrial Scale 31
been reported in the past. These pathways build on the conversion of a

common carbohydrate source, e.g., D-glucose, to L-gulose or L-galactose,
which then serve as substrates for suitable oxidases for the oxidation to L-AA
[3].
This review chapter will present the main industrial processes utilized for
the synthesis of L-AA from the past until present. Besides, this chapter will
also highlight the increasing importance of biotechnology and bioengineering
during the industrial production of L-AA. Moreover, the prospect of
converting D-sorbitol directly to L-AA purely by biotechnological means,
avoiding 2-keto-L-gulonic acid as an intermediate to achieve efficient
processes will also be discussed.
CHEMISTRY (GENERAL AND PHYSICAL PROPERTIES) OF

VITAMIN C
L-Ascorbic acid (C6H8O6) is the trivial name for vitamin C, which is the
accepted biochemical nomenclature name by International Union of Pure and
Applied Chemistry-International Union of the Biochemistry (IUPAC-IUB).
The systematic chemical designation is 2,3-endiol-L-gulonic acid-γ-lactone,
which was formerly known as hexuronic acid (Figure 1) [4]. Vitamin C is the
generic name for all compounds exhibiting qualitatively the biological activity
of ascorbic acid [5, 6]. These include esters of ascorbic acid (AA), such as
ascorbyl palmitate and synthetic forms with 100% relative activity, such as 6-
deoxy-L-ascorbic acid with 33% relative activity and the primary oxidized
form of L-ascorbic acid (L-AA) [7]. L-AA is used widely as a food additive
with many functional roles, based mainly on its oxidation-reduction properties
[6].
L-AA has chiral centers at carbon 4 and 5 and can exist in four
stereoisomeric forms. Enantiomeric pairs are L- and D-ascorbic acid and L-
and D-araboascorbic acid. L-AA and D-araboascorbic acid (more commonly
known as D-isoascorbic acid) or erythorbic acid, are epimers differing in
orientation of hydrogen and hydroxyl on carbon 5 [8]. Structures of various
AA forms are shown in Figure 1. Isoascorbic acid is not present in foods but is
synthesized commercially for its antioxidant capacities and commonly used in
cured meat products to prevent oxidation and protect color. The stereoisomers
have no biological activity other than a small amount from isoascorbic acid
(2.5-5% relative to L-AA) [9].
32 Sui Kiat Chang
Figure 1. Structures of L-Ascorbic acid and related compounds. Adapted for use with
permission from the publisher [2].
Figure 2. Outline of the chemical L-AA synthesis according to Reichstein and

Grussner (1934). L-AA is derived from D-glucose by inversion of the carbon skeleton.
Next, D-sorbitol is oxidized using Gluconobacter oxydans, followed by further
oxidation of the primary alcohol to 2-keto-L-gulonic acid (2KGA). Finally, 2-keto-L-
gulonic acid is rearranged to produce ascorbic acid (Asc). Adapted with permission
from the publisher [3].
L-AA and L-dehydroascorbic acid (L-DHA) are the primary dietary

sources of vitamin C. L-DHA is considered to have as high as 80%
bioequivalency to L-AA [5]. However, recent study reported that it can have
as low as 10% of the activity of L-AA [10]. If this finding relates to the
biological activity of L-DHA in humans, many current available food
composition values that combine L-AA and L-DHA to present a value for total
ascorbic acid would need to be revised to give a clear picture of the active
amount of ascorbic acid in specific foods [6]. L-AA is a white to slightly
yellow crystalline powder with high water solubility (30 g 100 mL-1) at room
temperature. It forms salts, the most important of which are the sodium and
34 Sui Kiat Chang
calcium salts, the aqueous solutions of which are strongly acidic. Its salt has
higher water solubility [6].
THE REICHSTEIN PROCESS: THE MAJOR INDUSTRIAL

PROCESS IN THE PRODUCTION OF L-AA
Industrial L-AA production has been traditionally based on the Reichstein
process which is an efficient four-steps process to convert D-glucose to L-AA,
one biochemical and three chemical. In the first step, D-glucose is
hydrogenated to D-sorbitol followed by the oxidation of D-sorbitol to L-
sorbose that is commonly carried out using a bacterial fermentation [11].
Other proposed and reported biotechnological steps have focused on the
microbial conversion of D-sorbitol, L-sorbose or D-glucose to 2-keto-L-
gulonic acid that is the last intermediate in the Reichstein process [12].
Currently, the main industrial process to produce L-AA is a two-step
fermentation process [3].
First, D-sorbitol is oxidized to L-sorbose using Gluconobacter oxydans.
Figure 2 demonstrated the schematic flow of the synthesis of L-AA proposed
by Reichstein and Grussner. The chemical structures of the key intermediates
demonstrated in Figure 2 are in the form of Fischer projections. The L-AA is
derived from D-glucose by inversion of the carbon skeleton. Subsequently, a
mixed fermentation with Ketogulonicigenium vulgare and Bacillus
megaterium, to convert L-sorbose to 2-keto-L-gulonic acid which will be
subsequently converted to L-AA.
a) Oxidation of L-Sorbose to 2-keto-L-Gulonic Acid and

Rearrangement to L-AA
Oxidation of L-sorbose to 2-keto-L-gulonic acid was achieved by treating

with potassium permanganate, which is an oxidizing agent. After this
oxidation, the resulting diacetone-2-keto-L-gulonic acid was hydrolyzed to the
free acid, which was isolated and crystallized as the 2,6-hemiketal isomer.
Subsequently, the compound was rearranged into the 1,4-lactone, upon heating
under acidic conditions, which was then enolized to L-AA. The rearrangement
gives higher yields, if 2-keto-L-gulonic acid is esterified with methanol to
yield methyl-2-keto-L-gulonic acid. L-AA was produced after treating with
sodium methoxide in methanol, followed by acidification. During the

subsequent years, the synthesis was optimized so that each individual step
could be carried out in greater yield (>90%) producing L-AA with higher
overall yield (>50%) from glucose. As a result of the improvements made, the
Reichstein/Grüssner synthesis became the industrial standard for the
production of L-AA until the 1990s [13]. Palladium-catalyzed air oxidation
had replaced permanganate or bleach (NaOCl) oxidation. This should result in
a more environmental friendly process [14, 15]. Moreover, the palladium-
catalyzed air oxidation had demonstrated preference for the C1 hydroxy group
of L-sorbose [16]. However, the selectivity was not a main factor to allow the
oxidation to proceed without the need to protect the other hydroxy groups in
the molecule.
The D-sugars found naturally must be backward-integrated in order to
ensure a cost-efficient synthesis of L-AA at the industrial scale. The
Reichstein/Grüssner synthesis accomplishes this task by the “inversion of the
carbon skeleton” of D-glucose such that its C2 carbon becomes C5 of L-
sorbose by an oxidation step exclusively at C5 of D-sorbitol. Acetic acid
bacteria are well known as vinegar producers (ethanol oxidation) and also for
their ability to only partially oxidize various sugars and sugar alcohols [17]. Of
the various genera of acetic acid bacteria, Gluconobacter sp. is particularly
effective in partially oxidizing sugars and sugar alcohols. Following the
empirical Bertrand-Hudson rule “polyols with a cis-arrangement of two
secondary hydroxy groups in D-configurations to the adjacent primary alcohol
group are oxidized to the corresponding ketones” [18] the secondary alcohol at
C5, but not at C2, of D-sorbitol is oxidized to the carbonyl group, providing L-
sorbose with a yield of 100%.
BACTERIAL FERMENTATION PROCESSES FOR

THE PRODUCTION OF L-AA
Bacteria have been identified to be able to transform glucose to 2,5-keto-

D-gluconic acid and this product to 2-keto-L-gulonic acid, the precursor of L-
AA. When the corresponding strains are used together, it is possible to get 2-
keto-L-gulonic acid directly from glucose [19]. Further, new strains have been
constructed by introducing a gene from a strain responsible for the second step
into a strain responsible for the first step. By using one of the few strains, the
transformation can be done in a single step with only one single strain.
However, the traditional process still remains the best [19].
36 Sui Kiat Chang
The Two-Step 2-Keto-L-Gulonic Acid Fermentation Process
The conversions occurring during the fermentation of D-sorbitol with

Ketogulonicigenium vulgare were based on the individual substrate
specificities of Ssda and Ssdb enzymes [3]. The ssdb and ssda 1 genes encode
the key enzymes of Ketogulonicigenium vulgare for the conversions of D-
sorbitol to L-sorbose and L-sorbose to 2-keto-L-gulonic acid oxidation,
respectively. First, D-sorbitol is converted to L-sorbose by Ssdb, followed by
further oxidation to L-sorbosone. Second, L-sorbosone is oxidized to 2-keto-
L-gulonic acid by the Ssda dehydrogenases, mainly Ssda1 [3]. L-sorbosone
oxidation is also catalyzed by Ssdb via the cyclic 1,5 pyranoside isomer and
the 2-keto-L-gulono-1,5-lactone as immediate oxidation products. Moreover,
the Ssda dehydrogenases oxidize L-sorbitol to D-glucose and L-gulose. After
cyclization to the corresponding pyranosides, the aldoses are further oxidized
by Ssdb and other enzymes [3].
The current industrial microbial 2-keto-L-gulonic acid processes comprise
of a two-step regime to prevent D-sorbitol being oxidized back to the aldoses
by the Ssda dehydrogenases [3] (Figure 4). As in the Reichstein process, D-
sorbitol is first oxidized to L-sorbose by Gluconobacter oxydans. The
complete utilization of the substrate took less than 24 h, which results in
product with a concentration of more than 200 g/L [20]. Subsequently, the
water-diluted broth is inoculated with a Ketogulonicigenium vulgare culture
together with the helper strain, usually Bacillus megaterium, to initiate the
second step. In this step, L-sorbose is converted to 2-keto-L-gulonic acid,
reaching final 2-keto-L-gulonic acid titers of 100 g/L in less than 40 h [20].
Sodium hydroxide is used to neutralize the acid produced during the
fermentation run. The overall yield of 2-keto-L-gulonic acid production from
D-sorbitol is around 90% [20]. The first step yielded higher volumetric
productivity compared to the second step with higher product concentration
reached in half the time, the fermenter volume for the first step is much
smaller than that required during the second step [20]. The oxygen transfer
rate required during both fermentation runs is below 100 mmol/L × h,
facilitating the usage of air-lift fermenters with great savings on investment
and operational costs [20].
The biomass in the fermentation broth is removed using cation exchange,
one of the main nanofiltration techniques, to obtain the product in the
protonated form (acidic 2-keto-L-gulonic acid), and hence, crystallization of 2-
keto-L-gulonic acid [20]. The chemical conversion from 2-keto-L-gulonic acid
to L-AA is achieved as in the Reichstein process, mainly through the 2-keto-L-
gulonic acid methyl ester [20]. Recently, Takagi et al. [21] demonstrated that
the second conversion step can also be carried out in continuous mode. A high
content of complex components (3% corn steep liquor, 7% baker’s yeast) in a
single culture broth of Ketogulonicigenium vulgare DSM4025 produces 2-
keto-L-gulonic acid at a steady-state concentration of 112.2 g/L 2-keto-L-
gulonic acid for 140 h [21]. Subsequently, the productivity reduced. The
dilution rate was kept between 0.035 and 0.043 per hour resulting in a
volumetric 2-keto-L-gulonic acid productivity of 3.90 to 4.80 g/L/h. The
average molar conversion yield of 2-keto-L-gulonic acid from L-sorbose was
91.3% [21].
In another scenario, Takagi et al. [22] demonstrated that the continuous
mixed culture fermentations with Xanthomonas maltophilia IFO 12692 as a
helper strain, 2-keto-L-gulonic acid production from L-sorbose with
DSM4025 could be kept in a stable, continuous mode for more than 1,300 h.
Instead of a larger stirred-tank reactor, two smaller, similar sized fermenters
were connected in series. At a dilution rate of 0.0380 per hour and a steady
concentration of 113 g/L 2-keto-L-gulonic acid, the volumetric productivity
(volumes of both fermenter) was 2.15 g/L/h. The molar conversion yield was
90.1%. About 70% of the conversion was reached in the first of the two
serially connected fermenters [22].
The One-Step 2-Keto-L-Gulonic Acid Fermentation Process
The specific activity of Gluconobacter to convert D-sorbitol to L-sorbose

is much higher compared to the specific activity of Ketogulonicigenium to
convert the sugar alcohol to unwanted aldoses and the subsequent oxidation
products [3]. Hence, the simultaneous inoculation of a D-sorbitol containing
culture broth with both strains is possible, with the condition that the right
ratio between the two strains in the inoculum is met [3]. However, it must be
ensured that the substrate is already oxidized to L-sorbose by Gluconobacter
before Ketogulonicigenium functions to convert D-sorbitol to D-glucose and
L-gulose in considerable amounts. There are two advantages of this step. First,
fewer fermenters are needed and thus, fewer unit operations are required
compared to two-step processes, saving the operational costs [3]. Besides,
there is no need of an additional helper strain to stimulate the oxidizing
capabilities of Ketogulonicigenium, since Gluconobacter can take over this
function [3].
38 Sui Kiat Chang
Figure 3. Biochemistry of the oxidation of L-sorbose to 2-keto-L-gulonic acid in

Gluconobacter oxydans. FAD-linked sorbose dehydrogenase (Sdh) is responsible for
L-sorbose to L-sorbosone oxidation. NAD(P)-linked sorbosone dehydrogenase (Sndh)
is the key enzyme for L-sorbosone for the oxidation of 2-keto-L-gulonic acid. Adapted
with permission from the publisher [3].
In a patent filed by Hoshino et al. [23], the one-step 2-keto-L-gulonic acid

process was suggested for the first time. For instance, the simultaneous
inoculation of Ketogulonicigenium vulgare DSM4025 with Gluconobacter
oxydans IFO3291 in a broth containing D-sorbitol 140 g/L 2-keto-L-gulonic
acid was achieved at a molar conversion yield of 89% within 48 h. According
to a patent application filed by Stoddard et al. [24], the one-step 2-keto-L-
gulonic acid process can also be conducted with a combination of
Ketogulonicigenium robustum B-21630 and Gluconobacter oxydans IFO3293
or ATTC621. As a result, 110 g/L 2-keto-L-gulonic acid from D-sorbitol was
produced at a conversion yield of approximately 100% within 70 h. The
cultivation medium was very rich in complex components by using a 31.5 g/L
corn steep liquor as dry solids [24].
Alternatively, another approach demonstrated by Asakura et al. [25]
achieves the rapid conversion of D-sorbitol to L-sorbose with a
Ketogulonicigenium vulgare DSM4025 derivative strain that has the ssdb gene
amplified on a vector. The overexpression of the Ssdb enzyme helps to reduce
the loss of D-sorbitol as D-glucose/L-gulose due to the undesired activity of
the Ssda enzymes on D-sorbitol [25]. An efficient conversion of D-sorbitol to
2-keto-L-gulonic acid could be achieved with Ketogulonicigenium vulgare
strain using a complex growth medium containing autoclaved yeast, without
the need to co-cultivate with another helper strain. 99 g/L 2-keto-L-gulonic
acid were retrieved from 150 g/L of L-sorbitol in the fermentation of 51 h
[25]. Besides, further improvements of the conversion yield by using stronger
promoters for Ssdb expression have also been reported [3].
Figure 4. Outline of the industrial L-AA production based on the fermentation of 2-

keto-L-gulonic acid with Ketogulonicigenium vulgare. L-Sorbose is produced
separately using Gluconobacter oxydans as in the original Reichstein process. The
final conversion of 2-keto-L-gulonic acid to L-AA follows the original Reichstein
process. Adapted with permission from the publisher [3].
40 Sui Kiat Chang
The Importance of the Helper Strain for 2-Keto-L-Gulonic Acid

Production by Ketogulonicigenium Vulgare
The 2-keto-L-gulonic acid productivity of Ketogulonicigenium processes

at high intensity, industrial-scale level has only been achieved upon cultivation
of Ketogulonicigenium vulgare with complex media components, preferably
corn steep liquor for cost efficiency [26, 27] and after co-cultivation with a
helper strain, usually Bacillus megaterium [3]. Ketogulonicigenium vulgare is
particularly dependent on the supply of growth media externally in large-scale
industrial processes [3]. Complex media components, such as, yeast extract,
peptone and beef extracts could contribute to high-performing processes. The
function of the helper strain can be exerted by various microorganisms, such
as, various Bacillus strains and Gluconobacter oxydans, could help to bridge
the nutritional gap corn steep liquor cannot provide, rather than to supply a
specific growth factor [3].
Direct Microbial Production of L-AA
The 2-keto-L-gulonic acid fermentation by Ketogulonicigenium vulgare

has reached a cost-efficient level with all the efforts in strain and process
optimization [3]. 2-keto-L-gulonic acid pathway is different from the natural
biosynthetic routes, where L-AA is formed directly from the oxidation of
precursor molecules (Figure 5) with appropriately preformed 1,4-lactone
linkage (L-gulono-1,4-lactone in animals, L-galactono-1,4-lactone in plants).
Enzymes converting L-gulono-1,4-lactone to L-AA are from bacteria, such as,
Ketogulonicigenium [28]. These enzymes are different compared to the
mammalian gulono-1,4-lactone dehydrogenase, besides sharing the same
cofactor, which is FAD. The use of these natural L-AA-forming enzymatic
steps in biotechnological production processes has been limited by the nature
of these L-sugar-derived lactone precursor molecules and the lack of efficient
production methods for these compounds [3, 19]. It was, therefore, a great
discovery when L-AA formation directly from L-sorbosone, the intermediate
of the efficient 2-keto-L-gulonic acid formation route, was identified in those
two species already in the focus for 2-keto-L-gulonic acid production for
decades: Ketogulonicigenium vulgare [29] and Gluconobacter oxydans [30].
Figure 5. L-AA biosynthesis pathways in animals (top) and plants (bottom). Both
biosynthetic routes are significantly different. In animals, the L-configuration of L-AA
is the result of the inversion of the carbon skeleton while in plants it results from
epimerization at C5. Only the final step of oxidation of the 1,4 lactone precursor to L-
AA is similar in both plant and animal pathways. Adapted for use with permission
from the publisher [3].
The original Reichstein process had already made use of renewable raw
materials and a biotechnological step to achieve the regioselectivity required to
convert D-sorbitol to L-sorbose [3]. The implementation of 2-keto-L-gulonic
acid fermentation further reduced the need for solvents and energy. Recently, a
review chapter [3] highlighted the way for direct oxidation of L-sorbose to L-
AA, without the need for a chemical conversion of 2-keto-L-gulonic acid to L-
AA. This procedure will help to reduce solvent and energy requirements.
Additional opportunities may be found in ongoing developments for a more
sustainable provision of the starting materials D-glucose [31] or D-sorbitol
[32]. The scientific community is expecting more exciting discoveries
regarding the optimization of the production of L-AA with lower costs in the
future.
CONCLUSION
The production of L-AA applying the fermentation pathway still follows
the original concept of Reichstein. It uses D-glucose as the starting material,
followed by the inversion of the carbon skeleton to L-sorbose, oxidation to 2-
42 Sui Kiat Chang
keto-L-gulonic acid, and finally rearrangement to L-AA. The process of

Reichstein’s synthesis is simple and common, with limited reaction steps,
utilizing renewable starting material D-glucose. It is the ultimate choice to
meet future challenges, to meet production cost, product quality and
environmental sustainability.
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[12] Bremus, C., Herrmann, U., Bringer-Meyer, S. and Sahm, H. (2006). The
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[13] Crawford, T. C. and Crawford, S. A. (1980). Synthesis of L-ascorbic
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[22] Takagi, Y., Sugisawa, T. and Hoshino, T. (2010). Continuous 2-keto-L-
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44 Sui Kiat Chang
[23] Hoshino, T., Ojima, S. and Sugisawa, T. (1991). Fermentation process

for producing 2-keto-L-gulonic acid. EP0518136B1.
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strains and use thereof in fermentation processes for 2-keto-L-gulonic
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Development of chemically defined media supporting high cell density
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59, 190-196.
[29] Sugisawa, T., Miyazaki, T. and Hoshino, T. (2005). Microbial
production of L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and
L-sorbosone by Ketogulonicigenium vulgare DSM 4025. Microbiol.
Ferment. Technol. Commun., 69, 659-662.
[30] Berry, A., Lee, C., Mayer, A. and Shinjoh, M. (2003). Microbial
production of L-ascorbic acid. EP2348113.
[31] Chundawat, S., Beckham, G., Himmel, M. and Dale, B. (2011).
Deconstruction of lignocellulosic biomass to fuels and chemicals. Annu.
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[32] Silveira, M. M. and Jonas, R. (2002). The biotechnological production of
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Reviewed by:
Michal Janiak, PhD.

Department of Chemical and Physical Properties of Food, Institute of
Animal Reproduction and Food Research of Polish Academy of Sciences,
Olsztyn, Poland.
Email address: m.janiak@pan.olsztyn.pl.
Krishnamurthy Nagendra Prasad, Ph.D.

Chemical Engineering Discipline,

School of Engineering,
Monash University, Malaysia
Email: knag76@gmail.com.
Chapter 3
RECENT PROGRESS ON THE

ELECTROCHEMICAL DETECTION
OF ASCORBIC ACID
Li Fu and Aimin Yu*

Faculty of Science, Engineering and Technology,
Swinburne University of Technology, Australia
ABSTRACT
Ascorbic acid (vitamin C) is a water-soluble vitamin, widely present
in many biological systems. The detection of ascorbic acid in various
foods, drugs, physiological fluids is very important for agricultural,
pharmaceutical and biomedical industries. The present review
summarises the recent progress on the detection of ascorbic acid using
electrochemical approaches. The electrochemical behavior of ascorbic
acid at common electrodes is first introduced, followed by a
comprehensive review on the different types of materials used for
electrode surface modification to enhance the electrochemical detection
of ascorbic acid. Strategies to minimize the interference problem during
detection and the practical application for real sample analysis are then
discussed. To conclude, further research trends on the development of
electrochemical sensors of ascorbic acid are proposed.
Keywords: Ascorbic acid, electrochemical detection, electrode modification

48 Li Fu and Aimin Yu
INTRODUCTION
Ascorbic acid or vitamin C is an essential dietary nutrient for a variety of
biological functions. Ascorbic acid is a hexanoic sugar acid with two
dissociable protons (pKa 4.04 and 11.34). Therefore, under physiological
conditions, it exists as an ascorbate anion. It is a fundamental signal for the
biosynthesis of collagen. Besides, ascorbic acid is widely used in
biomedical chemistry, diagnostics and the identification of food ingredients.
Ascorbic acid can be found in many biological systems such as fresh
vegetables and fruits. Recommended dietary allowances for ascorbic acid have
been established for adult women of 75 mg/day and 90 mg/day for men. The
excess of ascorbic acid could lead to gastric irritation, and one of its
metabolites, oxalic acid, causes renal problems. Ascorbic acid is also an
excellent reducing agent, well known for its high antioxidant activity with the
ability to neutralize free radicals in biological systems. However, its
antioxidant property becomes active only in aqueous media and in the absence
of heavy metal cations. The reason is heavy metal cations are prooxidants,
which could react with acerbic acid and convert it to dehydroascorbate. Due to
the crucial role of ascorbic acid in biochemistry and industrial applications, it
is very important to develp simple and accurate methods for the routine
analysis of acerbic acid.
A wide variety of analytical techniques, such as titrimetric method,
fluorimetry [1], spectrometry [2], chemiluminiscence [3], liquid
chromatography [4] and enzymatic methods [5] have been reported for the
determination of ascorbic acid. Titration is the simplest technique among these
methods. Dichlorophenol indophenol, potassium iodate and bromate are
common agents used for titrimetric determination of ascorbic acid [6].
However, the main drawback of this method is the lack of specificity. HPLC is
a more selective method but requires long time sample preparation and
complex operation process. The other analytical methods such as
spectrophotometric, enzymatic and chemiluminiscence approaches are not
very popular due to their complicated analytical process.
Recently there is considerable interest in the development of
electrochemical sensors for the detection of ascorbic acid. Ascorbic acid is
electro-active and can be electrochemically oxidized at many conventional
electrodes [7]. The reaction mechanism involves the formation of an ascorbate
anion intermediate, which is electrochemically oxidized to diketolactone.
Diketolactone then dehydrates to form dehydroascorbic acid, which
subsequently rearranges to another enediol and is further oxidized at higher
Recent Progress on the Electrochemical Detection of Ascorbic Acid 49
potentials [8, 9]. The oxidation reaction is irreversible, thus only the anodic
oxidation peak is observed during the cyclic voltammetric (CV) scan. pH was
found to strongly affect the oxidation process due to the involvement of proton
in the oxidation process. There are two protons interchanging in the pH range
of 2-4.5, while only one proton in the pH range of 4.5-8. When pH is over 8,
two protons are involved in the oxidation. However, it was impossible to
identify the oxidation products when pH is higher than 8 due to the instability
of ascorbic acid and dehydro-ascorbic acid in basic condition [9].
Though it is possible to directly measure the oxidation current of ascorbic
acid at common electrodes, this method often suffers from high overpotential,
electrode fouling from oxidation products, as well as interference from some
other biological molecules such as dopamine and uric acid which undergo
oxidation within the similar potential window as ascorbic acid. Therefore,
electrode surface modification was widely applied for enhancing the analytical
performance towards the electrochemical detection of ascorbic acid. The
following section gives a comprehensive review on various materials recently
used for electrode modification.
MATERIALS USED FOR ELECTRODE MODIFICATION

Carbon Based Nanomaterials
Carbon might be the most widely used material in electroanalysis and

electrocatalysis due to their high electric conductivity and electrocatalytic
activity, ability to accumulate analyte and alleviate surface fouling, as well as
the capabilityfor surface functionalization. They are chemically stable in most
solutions and able to perform well in a wide range of temperatures. Carbon
electrodes such as carbon paste and glassy carbon electrodes (GCE) are
commercially available and have the advantages of low background current,
low cost and easy surface regeneration. In this section, we mainly focus on the
application of carbon based nanomaterials as electrode surface modifiers for
the construction of ascorbic acid sensors.
Since the first report on the electrochemical behavior of C60 in 1990,
fullerenes have been widely studied in the field of electrochemistry. Fullerenes
have been found to be able to lower the overpotential and enhance the
electrochemical reaction rate. Therefore, fullerenes have been used as both
redox catalysts and mediators in the fabrication of electrochemical sensors.
Some fullerene based electrochemical sensors have been demonstrated for

ascorbic acid measurement with excellent performance [10].
Recent studies have indicated that carbon nanotubes (CNT) can enhance
the electrochemical reactivity of some electroactive biomolecules and promote
their electron-transfer reactions. CNT-modified electrodes have also been
shown to be able to accumulate biomolecules on electrode resulting in the
sensitivity enhancement. Single-walled carbon-nanotubes (SWCNT) and
multi-walled carbon-nanotubes (MWCNT) were both used for constructing
ascorbic acid sensors.
Beitollahi et al. [11] reported the construction of a SWCNT paste
electrode by mixing SWCNT and 2,2 ′ - [1,2-ethanediylbis
(nitriloethylidyne)]-bis-hydroquinone. The prepared electrode showed a
promising electrochemical performance towards the detection of ascorbic acid.
Fernandes et al. [12] reported an ascorbic acid electrochemical sensor based on
N-doped SWCNT and Fe3O4 nanoparticles (NPs). Fe3O4 NPs were wrapped
by SWCNT which exhibited an excellent performance towards the detection of
ascorbic acid. Sing and co-workers also fabricated an ascorbic acid sensor
based on the SWCNT-tungsten oxide NPs [13].
For MWCNT studies, Zare and co-workers compared the performance of
MWCNT modified GCE with bare and activated GCE for the detection of
ascorbic acid [14]. A similar work was also done by Noroozifar et al. [15]. For
used as electrode modifier, surface modification was commonly adopted to
overcome the hydrophobicity of MWCNT. For example, poly(tyrosine) was
used for modify the MWCNT surface and consequently applied for ascorbic
acid sensor fabrication [16]. Besides MWCNT, MWCNT based composite
materials have been extensively explored for the electrode surface
modification. For example, composites of MWCNT-cobalt (II) phthalocyanine
[7a], MWCNT-chitosan [17], MWNT/Au NPs/HS(CH2)6Fc [18], MWCNT-
polyglycine-ZnO NP [19], MWCNT-tetradecyltrimethylammonium bromide
[20], MWCNT-cobalt(II) tetra-neopentyloxy phthalocyanine [21], MWCNT-
poly(2,5-dimethoxy aniline) [22], MWCNT-MgB2 [23], MWCNT-Li+-carbon
paste [24], MWCNT-tetra-β-isoheptyloxyphthalocyanine cobalt (II) [25],
MWCNT-poly(acrylic acid) [26], MWCNT-Pt NPs-polypyrrole [27], B-doped
MWCNT [28], MWCNT-TiO2-Au NPs [29], MWCNT/poly(N-
isopropylacrylamide) [30], N-doped MWCNT-MnFe2O4 [31] and MWCNT-
copper-phthalocyanine [32] have been reported for the enhancement of
ascorbic acid measurement.
Helical CNT is a kind of CNT that has a particular 3D helical structure,
which exhibits catalytic activity and promotes electron transfer. Zhang and co-
workers developed an ascorbic acid sensor based on poly(diallyl

dimethylammonium chloride) surface functionalzed helical CNT [33].
Carbon nanofibers (CNFs) with diameters varying from 10 to 500 nm,
have the similar mechanical strength and electric properties as CNTs. Recent
electrochemical studies have shown that CNFs were capable of promoting the
kinetics of electron transfer reaction. Huang et al. [34] demonstrated the
preparation of a palladium NP-loaded CNFs by electrospinning and
subsequent thermal treatment. The oxidation overpotential of ascorbic acid
was significantly reduced compared with those obtained at the bare carbon
paste electrode. The calibration curve for ascorbic acid was obtained in the
range of 2–200 μM with a detection limit of 0.7 μM.
Carbon nanohorn (CNH) is synthesized by laser ablation of pure graphite
with a high production rate, high yield and without using metal catalyst. It is
essentially metal-free and can be directly used for electrochemical biosensing
study. Zhu and co-workers prepared a single-walled carbon nanohorn
(SWCNH) modified GCE for the electrochemical determination of ascorbic
acid [35]. Zhang and co-workers fabricated a novel carbon
nanohorns/poly(glycine) based sensor for ascorbic acid detection [36].
Hollow carbon microspheres (HCMS) with a unique structure of hollow
core and carbon shell have high specific surface area, low density, high surface
permeability and good electronic properties. Nitrogen-doped HCMS was
prepared and its catalytic activity towards the electro-oxidation of ascorbic
acid was investigated. [37].
Graphene has attracted strong scientific interest in recent years due to its
unique physicochemical properties: high surface area, excellent thermal
conductivity, electric conductivity and strong mechanical strength. Graphene
is the basic building block for graphitic materials of all other dimensionalities.
In electrochemical sensor fabrication, reduced graphene oxide (RGO) is the
most used graphene material due to its conductivity and reasonable water
soulubility. In some cases, graphene has been directly used as electrode
surface modifier. For example, Keeley et al. [38] proposed an electrochemical
ascorbic acid sensor based on DMF-exfoliated graphene, which achieved a
linear detect range of 0.4 to 6.0 mM, with a 0.12 mM detection limit. Similar
work was also carried out by another research group [39]. Alternatively,
graphene was added into carbon paste electrodes to enhance the
electrochemical performance of electrodes [40]. Compared with conventional
carbon paste electrode, the addition of graphene showed an improved
electrochemical response towards the electrocatalytic oxidation of ascorbic
acid with a lowered overvoltage, pronounced current response and good

sensitivity.
Other graphene composites that have been prepared include polymer,
noble metals, metal oxide or hydroxide, and hemin functionalized graphene.
Polymer functionalized graphene composites, such as polyaniline-GO fibrous
nanocomposite was successfully prepared and applied for the determination of
ascorbic acid [41]. The electrochemical behavior of ascorbic acid using a
poly(L-arginine)-GO modified GCE has been studied by cyclic voltammetry
and chronoamperometry [42]. Noble metals incorporated graphene composites
have been showed excellent property towards the detection of ascorbic acid
due to their excellent electrochemical and electrocatalytic properties of both
components. For example, Sun et al. [43] prepared an ascorbic acid sensor
based on graphene-Pt NPs modified GCE. In comparison to the bare GCE and
graphene modified GCE, a large electrochemical potential difference was
achieved by using graphene-Pt nanocomposites modified electrode. A similar
work was reported by Wang and co-workers. They prepared an ascorbic acid
sensor based on Pd NPs-graphene/chitosan/GCE [44]. Moreover, Au
nanoplates/graphene composites were demonstrated to be effective for the
determination of ascorbic acid [45]. Similar works were reported by other
groups as well [46]. Li and co-workers demonstrated the synthesis of Ag
nanowire/RGO nanocomposites for the ascorbic acid sensing [47]. Bi-metallic
system has also been applied for incorporating graphene. For example, Jiang
and co-workers reported the preparation of Au@Pd-RGO nanocomposites by
one-step synthesis method and then used for ascorbic acid detection [48].
Some metal oxides and hydroxides are good candidates for enhancing the
electrochemical properties of electrodes. Therefore, several papers were
focused on the development of graphene-metal oxides or hydroxides
nanocomposites. Nickel hydroxide has the advantages of low cost, high
electrocatalytic effect, biocompatibility and high chemical stability. A
synergistic effect of the catalytic activity of nickel hydroxide together with the
high conductivity and surface area of graphene sheets was observed. Nancy
and co-workers synthesized a graphene/nickel hydroxide composite for the
electrochemical determination of ascorbic acid [49]. See and co-workers
reported the fabrication of an ascorbic acid electrochemical sensor using
graphene-iron oxide, which was synthesized by in-situ one-step chemical
method at room temperature [50]. Xie et al. [51] proposed a facile ultrasonic
synthesis of graphene-SnO2 nanocomposite and applied for ascorbic acid
determination. Rajamani et al. fabricated an electrochemical ascorbic acid
sensor based on the reduced graphene oxide-TiO2 nanocomposite [52].

Graphene-ZnO composite was also prepared for ascorbic acid detection [53].
Hemin is an iron porphyrin derivative, and is well known as the active
center of heme-proteins. Hemin has abilities to mimic various enzymes.
Development of novel materials as biomimetic catalysts with enzyme-like
activity is highly desired. There has been some attempt to use graphene as a
platform for loading hemin molecules. Zou and co-workers demonstrated the
successful functionalization of GO using hemin and applied as a bio-catalyst
for the electrochemical determining ascorbic acid [54].
In addition, element (or material) doped graphene has also been
demonstrated to be able to enhance the electrochemical determination of
ascorbic acid. Sheng et al. [55] reported the preparation of a nitrogen doped
graphene (NG) by thermally annealing the mixture of graphite oxide and
melamine. Due to its unique structure and properties originating from nitrogen
doping, NG showed highly electrocatalytic activity towards the oxidation of
ascorbic acid. Similar work was reported by another group as well [56]. Zhang
and co-workers recently reported the synthesis of graphitic carbon nitride
nanosheets doped GO and applied this material for the detection of ascorbic
acid [57].
Composites made of two different types of carbon nanomaterials have also
been reported for the fabrication of electrochemical sensors. Sun et al. [58]
prepared a MWCNT/GO nanoribbon core–shell composite modified GCE and
used for electrochemically detecting ascorbic acid. Li et al. synthesized a
MWCNT bridged mesocellular graphene foam nanocomposite and used for
ascorbic acid detection [59]. Zhang et al. [60] reported the hollow nitrogen-
doped carbon spheres-RGO for electrochemically detecting ascorbic acid.
Other carbon based materials including nitrogen doped porous carbon
nanopolyhedra [61], pyrolytic carbon [62], nitrogen doped porous graphitic
carbon [63], nitrogen doped carbon [64], exfoliated graphite paper [65],
graphene sheet-graphene nanoribbon hybrid [66], MWCNT-porous
graphitized carbon [67], nitrogen doped carbon sphere [68], were also
synthesized and successfully applied for the construction of ascorbic acid
electrochemical sensors.
Silica Based Materials
Mesoporous silica is one type of silica material with distinctive properties

such as large surface area, tunable pore sizes and biocompatibility.
Mesoporous silica has obtained much attention and broad applications in

catalysis, drug delivery, sorption and separation. For example, silica modified
electrode could accumulate electroactive analytes onto electrode to enhance
the current signal. In addition, silica can be grafted with a variety of functional
groups/materials to tune their surface properties [69]. For example, Au NPs on
a thiol-functionalized silica network was prepared for ascorbic acid
electrochemical detection [70].
Metal Hexacyanoferrates
Metal hexacyanoferrates are a class of polynuclear mixed-valence

compounds which possess special properties such as ion-exchange selectivity,
electrochromism, magnetism and electrocatalysis. A sol–gel method was used
for constructing an ascorbic acid sensor based on the terbium hexacyanoferrate
modified carbon ceramic electrode [71]. Li et al. [72] proposed an
electrochemical ascorbic acid sensor based on the incorporation of a
ferricyanide mediator with a polyelectrolyte–calcium carbonate microsphere.
Propargyl-functionalized ferrocene has been modified on the electrode through
reacting with azide terminal modified Au electrode via copper(I) catalyzed
azides and alkynes cycloaddition reaction [73]. Study showed that the surface
coverage value from electrochemical impedance spectroscopy has a linear
response to the logarithm of ascorbic acid concentration in the range of 5.0 pM
to 1.0 nM with a detection limit of 2.6 pM. Farhadi et al. [74] reported an
ascorbic acid sensor based on Th(IV)-hexacyanoferrate/ carbon composite.
Isaac et al. [75] reported the synthesis of a DNA-Prussian blue-carbon paste
biosensor for the detection of ascorbic acid. Sun et al. [76] reported the
synthesis of Na2H4[C3H5N2]4[Sb2W20FeII2(H2O)6O70]·12H2O and tested its
electrocatalytic activity towards the oxidation of ascorbic acid.
Conducting Electroactive Polymers
Conductive polymers are materials discovered just two decades ago.

Originally heralded for their high conductivity/weight ratio, it is the unique
chemical and electronic properties they possess that now arouse much
attention. Most polymers have strong adherence to the electrode surface and
remain stable during measurement, which makes them good candidates for
electrode surface modification.
Some reports showed electroactive polymer-coated electrode displayed

excellent selectivity and sensitivity for the determination of ascorbic acid. For
example, Yao et al. [77] reported the in-situ electro-polymerization of
eriochrome black T on the surface of a GCE in alkaline solution by CV. The
modified GCE exhibited excellent electrocatalytic activity towards the
oxidations of ascorbic acid. Zhang and co-workers demonstrated the electro-
polymerization of acid chrome blue K and then successfully used for ascorbic
acid detection [78]. Kumar and co-workers demonstrated an anodic
polymerization of the azo dye DB71 on GCE and then used for ascorbic acid
determination [79]. Other conductive polymers including poly(p-toluene
sulfonic acid) [80], poly (l-serine) [81], poly(acid chrome blue K) [82],
poly(malachite green) [83], polymethylene blue [84], poly(fuchsine acid) [85],
p-phenylenediamine [86], poly-niacinamide [87], poly (N, N-dimethylaniline)
[88], dopamine polymer-3,4,9,10-perylenetetracarboxylic acid [89], poly
(xanthurenic acid) [90], poly(indoleacetic acid) [91], poly(diphenylamine)-
phosphotungstic acid [92], poly (o-aminophenol) [93], polyortho-
phenylenediamine [94], osmium(II) copolymer [95], poly(alizarin red S) [96],
poly (acriflavine) [97], poly(hydroxymethylated-3,4-ethylenedioxythiophene)
[98], polyoxometalate silicotungstic acid-polyelectrolyte [99], poly(N-
acetylaniline)/poly-(4-styrenesulfonic acid-co-maleic acid) composite film
[100], polypyrrole nanofibers [101] and polycysteine 102 were investigated for
ascorbic acid sensing.
Among common conductive polymers, polyaniline has been widely
studied and used in rechargeable batteries and electro-catalysis due to its high
conductivity, good stability and redox reversibility [103]. Several studies
demonstrated the direct use of polyaniline for ascorbic acid analysis [104].
However, polyaniline shows nearly no electroactivity when pH > 4. Chemical
doping was used to overcome this problem and make it usable for
biomolecules detection in neutral condition. For example, a composite film of
polyaniline-p-aminobenzene sulfonic acid modified GCE was fabricated via an
electrochemical oxidation procedure and applied for the electro-catalytic
oxidation of ascorbic acid [105]. A similar work was carried out by Zhang and
co-workers [106]. They reported the electrochemical copolymerization of
aniline and o-aminophenol on GCE and then used for electro-oxidation of
ascorbic acid. The incorporation of metallic NPs into polyaniline is of great
interest because of their strong electronic interactions between the NPs and the
polymer matrixes. It has been reported that the electrocatalytic properties of
NPs could be enhanced by the conductive polymeric matrixes. Additionally,
the conductivity of the hybrid systems is largely improved. Yang et al. [107]
reported the synthesis of Au NPs-polyaniline core–shell nanocomposites by

one-step chemical oxidative polymerization of aniline using chloroaurate acid
as the oxidant and Au NPs as the seeds. The synthesized Au NPs-polyaniline
core–shell nanocomposite exhibited an outstanding performance towards the
detection of ascorbic acid. In addition, polyaniline-polysulfone composite
[108], Naphthol green B doped polypyrrole film [109], polyaniline films
containing Au NPs [110], polyaniline-poly(acrylic acid) [111], graphene-
copper phthalocyanine-polyaniline [112], polyaniline–thiol composite [113],
poly(3, 4-ethylene dioxythiophene)-polyaniline [114], CuGeO3-polyaniline
nanowire [115], polyaniline-tetracyanoquinodimethane-ormosil [116], SiO2-
Au NPs-polyaniline [117], polyaniline–keggin iron–clay composite [118] and
graphene-CuPc-polyaniline nanocomposites [119] have also been studied for
ascorbic acid sensing.
On the other hand, conducting polymers with entrapped metal or metal
oxide NPs have received great interest due to their probable electronic
interactions between NPs and functional groups of the polymer. The porous
structure of conducting polymer allows metal particles dispersed into polymer
matrix and generates additional electrocatalytic sites. For example, Au NPs
were distributed into poly(4-aminothiophenol) modified electrode for
electrochemical determination of ascorbic acid [120]. Characterization showed
the presence of uniformly distributed Au NPs with sizes of 8–10 nm. Tian et
al. [121] reported the modification of GCE with a novel vanadium oxide
polypropylene carbonate and then used for ascorbic acid determination. The
electrode was prepared by casting a mixture of vanadium tri(isopropoxide)
oxide and poly(propylenecarbonate) onto the GCE surface. This modified
electrode exhibited a superior electrocatalytic response to the oxidation of
ascorbic acid and consequently used for analyzing the scorbic acid content in
fruit samples. Cu NPs were also been incorporated within the conducting
polymer for the advanced electrochemical sensors desgin. For example, Cu
NPs immobilized polyaniline modified GCE was reported for the oxidation of
ascorbic acid [122]. Besides, poly(3, 4-ethylene dioxythiophene)-sulphated β-
cyclodextrin was applied for ascorbic acid detection as well [123].
Molecular imprinting techniques are becoming more commonly accepted
as useful method for the recognition and isolation of biological target
molecules. Molecularly imprinted polymers are potential candidates to replace
natural receptors and enzymes due to their advantages such as superior
stability, low cost and easy preparation. Polypyrrole-based film was fabricated
as molecularly imprinting electrochemical sensor for the determination of
ascorbic acid [124]. The polypyrrole-based film was prepared by incorporation
of ascorbic acid during the electropolymerization of pyrrole onto a pencil

graphite electrode in aqueous solution using a CV method. The polypyrrole-
based film exhibited a high selectivity and sensitivity toward ascorbic acid
with a linear calibration curve in the range of 0.25 to 7.0 mM. Zhao and co-
workers proposed disposable electrochemical ascorbic acid sensor based on
molecularly imprinted poly(o-phenylenediamine)-modified dual channel
screen-printed electrode and can be successful applied for detecting ascorbic
acid in organic juice [125].
Metals
Noble metals (such as Au, Ag, Pt, Pd, Ru and their alloys) are excellent
electrode materials due to their electro-conductivity and electrocatalytic
properties [126]. For Au nanostructures, Ahn et al. [127] investigated the
biosensing ability of the dendritic Au rod towards ascorbic acid. It is well
known that conducting porous layers on the electrode surface is favorable for
electrochemical reactions to take place. Three dimensional nanoporous gold
thin film was formed on the electrode using an electrochemical deposition
method [128]. Other reports using Au nanostructures for ascorbic acid
determination were also carried out [129]. Ag electrode possesses high
conductivity and good stability in aqueous solution. In biosensor application,
Ag ceramic graphite composite sensors have shown high performance for
rapid, sensitive and selective determination of many biomolecules. Li et al.
[130] reported the construction of an ascorbic acid biosensor based on Ag-
Ag2S modified electrode. Sun et al. [131] reported the preparation of silver
doped poly(glutamic acid) modified GCE for ascorbic acid detection. For Pd
nanostructures, Ramakrishnan et al. [132] demonstrated an ascorbic acid
electrochemical sensor based on Pd NPs-decorated graphene and carbon
nanotube. For Pt, Rageh and co-workers measured the detection limit of
ascorbic acid using a Pt electrode and was found to be 9.24 μM [133].
Many electrodes of pure metals exhibit unsatisfactory sensitivity,
selectivity and are easy poisoned by adsorbed intermediates, which are critical
issues for practical applications. Bimetallic systems were introduced for
overcoming these disadvantages. For example, Zhang and co-workers
demonstrated the synthesis of carbon-supported Pd-Ni NPs and subsequently
used for electrode surface modification and detection of ascorbic acid [134].
Yan et al. [135] fabricated an ascorbic acid electrochemical sensor based on
Pd-Pt NPs anchored graphene. Manjo et al. [136] used Pd NPs supported Po-
Pd hollow spheres for ascorbic acid sensing. Zhao and co-workers

demonstrated the synthesis of hierarchical nanoporous Pt-Cu alloy for the
electrochemical determination of ascorbic acid [137].
Other transition metals and their compounds were also used for
constructing electrochemical sensors. For example, He and co-workers used
Cu NPs as building component for the fabrication of ascorbic acid
electrochemical sensor [138]. Cu4(OH)6SO4, a transition metal compound-
based cheap catalyst, exhibited an electrocatalytic property towards the
detection of ascorbic acid [139]. Other transition metals and their compounds
including cobalt (II) tetrakisphenylporphyrin [140], niobium oxide [141],
copper germanate nanowire [142], ruthenium oxideNPs [143], bismuth oxide
[144], NiCo2O4/Nano-ZSM-5 nanocomposite [145], iron porphyrin
immobilized Nb2O5-SiO2 [146], carbon-supported NiCoO2 NPs [147],
manganese (III) complex [148], NiO NPs-(9,10-dihydro-9,10-
ethanoanthracene-11,12-dicarboximido)-4-ethylbenzene-1,2-diol[149], iridium
oxide [150], MoS2-reduced graphene oxide [151], were investigated for
ascorbic acid detection as well.
In addition, Mn-SnO2 [152], Cu2O [153], one-dimensional magnesium
oxide [154], copper (II)-4,4’-dicyanamidobiphenyl ligand [155], titanium
dioxide [156], zirconium NPs decorated reduced graphene oxide [157],
hexaaza macrocyclic copper (II) complex [158], were also investigated for
their ability of electrochemical detection of ascorbic acid.
Ionic Liquids
Room temperature ionic liquids are generally regarded as compounds

entirely composed of organic cations and various inorganic anions that exist in
the liquid state around room temperature. Ionic liquids have many unique
physical and chemical properties such as wide electrochemical window, high
ionic conductivity and good solubility. Therefore, the modification of
electrode using ionic liquids can effectively enhance the electrochemical
properties compared with that of bare electrodes. Sun et al. [159] reported the
use of room temperature ionic liquid N-butylpyridinium hexafluorophosphate
as a binder to make an ionic liquid modified carbon paste electrode. The
electrochemical oxidation of ascorbic acid on the ionic liquid modified carbon
paste electrode was studied. Pandurangachar et al. [160] reported the
electrochemical deposition of 1-butyl-4-methyl-pyridinium tetrafluroborate
ionic liquid on carbon paste electrode using CV method in 0.1 M nitric acid.
An enhanced electrochemical performance towards ascorbic acid oxidation

was observed. Other ionic liquids such as 1-n-butyl-3-methylimidazolium
tetrafluoroborate [161], was also used for the fabrication of electrochemical
sensors of ascorbic acid.
Other Materials
Surfactants, a kind of amphiphilic molecules with a hydrophilic head on

one side and a hydrophobic tail on the other side, have been widely applied in
electrochemistry to improve the property of the electrode/solution interface.
Ascorbic acid is an anion which can be attracted to the positively charged
ionic micelles. Cao and co-workers reported the successful determination of
ascorbic acid at cetylpyridine bromide/chitosan composite film-modified GCE
[162]. Avendañoa et al. [163] reported the surface modification of GCE using
cationic surfactant cetyltrimethyl ammonium bromide and used for ascorbic
acid detection.
Self-assembled monolayer provides a means for controlling the chemical
nature of the electrode interface. The use of self-assembled monolayer
modified electrodes to improve the selectivity and sensitivity of Au electrode
has been reported [164]. Sun et al. [165] reported an ascorbic acid
electrochemical sensor based on the triazole self-assembled monolayer
modified Au electrode.
Besides, other organic materials including epinephrine [166],
metalloporphyrin intercalated layered niobate [167], ytterbium fluoride NPs
[168], methionine [169], 2,2 ′ -[3,6-Dioxa-1,8-
octanediylbis(nitriloethylidyne)]-bis-hydroquinone [170], DL-norvaline [171],
dihydroxyalkanedithiol [172], cysteine [173], phenanthroline 174, clenbuterol
[175] and metalloporphyrin–graphene [176] were also employed for electrode
surface modification due to their specific properties for ascorbic acid
electrochemical determination. For inorganic materials, the application of
zeolite [138], lignin [177], iodide ions [178], sodium do-decyl benzene
sulphate [179] and methylene blue intercalated into calcium phosphate [180]
were also used for the fabrication of ascorbic acid sensors.
The analytical performances of some most illustrative modified electrodes
in electrochemical determination of ascorbic acid are summarized in Table 1.
Table 1. Analytical performances of various modified electrodes for the

measurement ofascorbic acid.
Detection Electrode modifier Linear detection Detection limr Reference

methods range
DPV DMF-exfoliated graphene 0.4 to 6 mM 0.12 mM [38]
Amperometry Pd NPs supported on 0.02 to 2.28 mM ― [159, 181]
graphene oxide
DPV Tetrabromo-p- 4.0 to 10.0 μM 0.62 μM [182]
benzoquinone modified
carbon paste electrode
DPV Nitrogen doped graphene 5.0 μM to 1.3 mM 2.2 μM [55]
SWV Fullerene-C60-modified 1 nM to 5.0 μM 0.26 nM [183]
gold electrode
LSV Single-walled carbon 30 to 400 μM 5 μM [35]
nanohorn
Amperometry Graphene doped carbon 0.1 to 106 μM 0.07μM [40]
paste electrode
DPV Au NPs-poly(4- 0.01 to 1 μM ― [120]
aminothiophenol)
DPV Cetylpyridine 0.01 to 2 mM ― [162]
bromide/chitosan
composite film
CV Poly(caffeic acid) film 0.02 to 1.2 mM 9 μM [184]
CV Cu-zeolite-graphene 0.02 to 0.2 mM 0.011 mM [138]
CV Nano TiO2-PEDOT film ― 0.02 μM [185]
Amperometry Carboxylated MWCNT 2 to 206 μM 0.9 μM [186]
and Polyaniline
DPV Cathodically pretreated 19 to 210 μM 19 μM [187]
boron-doped diamond
electrode
Amperometry Clark oxygen electrode 0.1 to 0.55 mM 0.023 mM [188]
SWV Triazole SAM modified 0.1 to 10 μM 0.01 μM [165]
gold electrode
DPV Poly(vinyl alcohol) 2 to 50 μM ― [189]
DPV Carbon ionic liquid 0.5 to 7 mM 0.2 mM [190]
electrode
DPV Cresol red modified 50 to 500 µM 10 μM [191]
glassy carbon electrode
Amperometry Poly(p-aminobenzene ― ― [192]
sulfonic acid)
DPV Electrochemically 0.5 to 60 μM 0.5 μM [193]
reduced graphene oxide
DPV Electrospun carbon 2 to 64 μM 2 μM [194]
nanofibers
SWV Carbon ceramic electrode 10 to 200 μM 0.36 μM [195]
Amperometry Sol–gel composite 16 to 5000 μM 8.6 μM [196]

containing copper and
MPS
DPV Choline and acetylcholine 7 to 19 μM 0.9 μM [197]
modified glassy carbon
DPV 2,2-Bis(3-amino-4- 0.5 to 100 μM 0.1 μM [198]
hydroxyphenyl)hexafluor
opropane
LSV Highly oxidized electrode 0.1 to 70 mM 0.1 mM [199]
SWV Gold modified electrode 0.3 to 100 μM 0.3 μM [200]
cationic self-assembled
monolayer
DPV Modified carbon paste 100 to 600 μM 0.62 μM [201]
electrode by tetrabromo-
p-benzoquinone
Amperometry Bppg electrode modified 1 to 50 μM 0.4 μM [202]
with MWCNTs
DPV Poly(bromocre sol 0.02 to 0.7 mM 6.5 μM [203]
purple)
MINIMIZATION OF INTERFERENCES IN
ELECTROCHEMICAL DETERMINATION OF ASCOBIC ACID
Dopamine, uric acid, glucose and hydrogen peroxide are the main
interfereneces when measuring ascorbic acid in biological samples. This is
because their electro-oxidation potentials overlap with that of ascorbic acid.
For example, the oxidation peak of uric acid is very close to ascorbic acid
which strongly affects the accurate measurement of current from ascorbic acid.
There have been lots of efforts to minimize this influence. It was reported that
the addition of copper ions could separate the current signal of interference
from ascorbic acid at single crystal Au (111) electrode [204]. Alternatively,
forming a monolayer of dimercaptothiadiazole on Au electrode could also
separate the signals of uric acid and ascorbic acid [205]. However, the
formation of the dimercaptothiadiazole film has to be in a non-aqueous
condition. Otherwise the formed dimercaptothiadiazole film would be too
thick for this purpose. Polypyrrole–tetradecyl sulfate ultrathin film was
another example to be used on Au electrode for the separation of ascorbic acid
with dopamine and uric acid [206].
Another way to minimize the interference is to reduce the oxidation
overpotential of ascorbic acid by surface modification of electrodes. Sripriya
et al. [207] demonstrated a modification process at a GCE using a chitosan
film incorporating cetylpyridine bromide. Results showed that the oxidation

potential of the ascorbic acid decrease 200 mV compared with that of bare
GCE. In addition, some functional groups on pretreated carbon electrodes
were also found effective in lowering the overpotential. For example, surface
functional groups formed on GCE after being electro-oxidized in a mild acidic
media (0.1 M H2SO4), was able to decrease the over-potential of ascorbic acid
comparing with bare GCE. This pretreated electrode was successfully applied
for simultaneous determination of ascorbic acid, dopamine and uric acid [208].
This electrode had the advantages of very easy to fabricate and 100% reusable
for multi-analysis.
As discussed above, carbon materials were extensively studied for
ascorbic acid determination due to their excellent electrocatalytic property.
Many carbon material modified electrodes have been demonstrated to be
effective on separating the signal of ascorbic acid from those of interferences.
For example, fullerene-C60 modified Au electrode was successfully used for
separating oxidation peaks of dopamine and ascorbic acid [183]. Carbon
nanotubes exhibited a better performance. A MWCNT-polyaniline modified
Au electrode was able to detect ascorbic acid without interference from co-
existing molecules including oxalic acid, glucose, fructose, citric acid, lactose,
starch, sucrose, tartaric acid and sodium chloride [186]. Similar results were
reported by other studies as well [209]. On the other hand, graphene exhibits
unusual electronic conductivity, high specific surface area, high mechanical,
thermal and chemical stabilities. Therefore, it has been extensively studied for
electrochemical sensor fabrication in the past 10 years. Many reports found
that the selectivity of graphene was even better than carbon nanotube. For
example, chitosan modified graphene could be effectively used for separating
oxidation peaks of ascorbic acid, dopamine and uric acid during the
electrochemical sensing [210]. The main reasons for the excellent selectivity
of the carbon based materials can be ascribed to the following three aspects:
the excellent electro-conductivity of carbon nanaomaterials could facilitate the
electron transfer performance of electrodes. Secondarily, carbon based
materials could selectively catalyze the electrochemical reaction of different
molecules resulting in the separation of oxidation potentials.
Despite carbon based nonmaterials, metal based nanostructures also
exhibit excellent selectivity towards the detection of ascorbic acid by
separating the electro-oxidation potentials of interferences. As an example,
Mahshid et al. [211] demonstrated the modification of TiO2 nanotubes
electrode by electrodeposition of Pd, Pt and Au NPs. The Au/Pt/Pd modified
TiO2 nanotube electrode represented a high sensitivity towards the individual
detection of dopamine, uric acid and ascorbic acid as well as simultaneous

detection of three components in a mixed solution of different acidity. In this
work, the maximum peak separation of uric acid and ascorbic acid was 0.15 V,
which allows the simultaneous determination of uric acid and ascorbic acid in
a mixed solution.
APPLICATIONS FOR REAL SAMPLE ANALYSIS

In order to verify the reliability of the electrochemical method for the
analysis of ascorbic acid in field applications, various real samples were used
for electrochemical sensor tests.
Vitamin C tablet is the common pharmaceutical product sample chosen
for sensor tests [17, 112, 132, 134, 141, 145, 159]. Zhang and co-workers
measured the content of vitamin C tablet using their electropolymerized acid
chrome blue K ascorbic acid sensor [78]. The typical procedure for sample
preparation could be described as follow: the tablet was accurately weighed
and ground to fine powder. An amount of the powder equivalent to
approximately 200 mg of vitamin C was weighed and added into 50 mL of
water. The mixture was shaken and filtered into a 100 mL volumetric flask.
The residue was washed several times with water and the solution was diluted
to the mark. A certain volume of the solution was diluted with electrolyte and
then transferred to an electrolysis cell for the determination of ascorbic acid.
Besides the pharmaceutical vitamin C tablet, some vitamin C supplements
were also used for testing such as Rubex [38], vitamin C syrup [124], ampoule
[11], effervescent tablet [109] and chewable tablet [81, 109].
Due to the clinic needs, the determination of ascorbic acid in human urine
was investigated [10, 49, 54, 56, 61-62, 65, 73, 132]. Commonly, human urine
sample was diluted using electrolyte without any pretreatment. The
concentration of ascorbic acid was then detected in the diluted human urine
directly.
Human serum was also employed as the media for ascorbic acid
determination [49, 54, 56, 107, 128, 132]. Commonly, ascorbic acid was
dissolved in human serum (1%) in comparison with the same mixtures in
buffer solution to make simulated sample. Besides human serum, rat serum
was also used as real sample for electrode testing [212]. Moreover, the calf
plasma was also tested in a report [10].
Detecting ascorbic acid content in fruit sample is important for agriculture
and food industrial. In order to evaluate the validity of the modified electrode
for the determination of ascorbic acid, recovery studies were carried out on
samples to which known amount of ascorbic acid standards were added.
Orange juice [121, 125, 169], pear juice [121], peach juice [121], pineapple
juice [121], watermelon [121], grape [121], green chilli [169], papaya [169],
lemon [169], strawberry [121] and tomato [121, 213] were used for real
sample analysis.
Electrochemical monitoring extracellular ascorbic acid was achieved using
a thin-layer electrochemical flow cell [214]. The thin-layer electrochemical
flow cell consists of a thin-layer radial flow block equipped with a GCE as
working electrode, an Ag/AgCl electrode (3 M NaCl) as reference electrode
and a stainless steel as counter electrode. To achieve the specificity for
ascorbic acid measurements, the GCE was modified with the heat-treated
SWCNT. A similar work was carried out by Zhang et al. [215]. An in vitro
real-time direction of the ascorbic acid level in rat liver tissues was achieved
using RuO2 nanorwires grown on electrospun TiO2 nanofibers [216].
CONCLUSION
This review summarized the recent development of chemically modified
electrodes for electrochemical determination of ascorbic acid. A wide range of
newly introduced nanomaterials and electrocatalytic compounds were used to
improve the sensitivy of detection by enhancing the electro-oxidation of
ascorbic acid at electrodes and effectively reducing the interferences from
common electroactive compunds. Based on our summary, carbon based
materials and conductive polymers were intensively studied as low-cost and
high performance electrode surface modifiers for the fabrication of ascorbic
acid electrochemical sensors. Most proposed sensors were successfully applied
for the detection of ascorbic acid content in many real samples including fruits
and vegetables as well as pharmaceutical products. Several reports also
conducted the ascorbic acid content detection in living cells.
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BIOGRAPHICAL SKETCH
Name: Dr Aimin Yu
Affiliation: Swinburne University of Technology
Education: PhD
Research and Professional Experience: Dr Yu has 20 years’ experience

in the research filed of synthesis, functionalization and application of
nanostructured materials.
Professional Appointments: Associate Professor
Publications Last 3 Years: Dr Yu has published 40 journal articles and

three book chapters in the last three years.
Chapter 4
SUPPRESSIVE EFFECT OF ACYL ASCORBATE

COEXISTENT WITH TOCOPHEROL ON
OXIDATION OF LINOLEIC ACID
Yoshiyuki Watanabe1,*, Takahiro Ishigaki1,

Sachiyo Fujii1 and Shuji Adachi2
1
Department of Biotechnology and Chemistry, Faculty of Engineering,
Kindai University, Hiroshima, Japan
2
Division of Food Science and Biotechnology, Graduate School of
Agriculture,
Kyoto University, Sakyo-ku, Kyoto, Japan
ABSTRACT
Lipids are susceptible to oxidation, which results in undesirable
flavor and the formation of toxic products such as hydroperoxides.
Therefore, lipid oxidation has received much attention because of its
involvement in food deterioration. L-Ascorbic acid and tocopherols are
used as natural and safe antioxidants in foods. Acyl ascorbate has an
amphiphilic structure and the advantages of increased solubility and
stability in fat-containing foods relative to ascorbic acid. Further, the
*
Corresponding author: Yoshiyuki Watanabe. Department of Biotechnology and Chemistry,
Faculty of Engineering, Kindai University, 1 Umenobe, Takaya, Higashi-Hiroshima,
Hiroshima 739-2116, Japan. E-mail address: wysyk@hiro.kindai.ac.jp.
92 Yoshiyuki Watanabe, Takahiro Ishigaki, Sachiyo Fujii et al.
combination of ascorbic acid and tocopherol is known to allow for

synergistic inhibition of oxidation. In this chapter, the antioxidative
properties of acyl ascorbate coexistent with -tocopherol for the
oxidation of linoleic acid are examined. Lauroyl or palmitoyl ascorbate
and -tocopherol were added to linoleic acid at the total molar ratio of
0.005; the mixture was stored at 65°C and 12% relative humidity, and
then, the oxidation process was measured by gas chromatography.
Linoleic acid without any antioxidant oxidized rapidly. In contrast, -
tocopherol markedly suppressed the oxidation of linoleic acid, while the
addition of lauroyl ascorbate further improved the oxidative stability.
Additionally, the antioxidative effect of palmitoyl ascorbate was as
significant as that of lauroyl ascorbate. On the other hand, the synergistic
antioxidative effect of ascorbic acid and -tocopherol was not
remarkable, as acyl ascorbate showed more effective antioxidative
activity than did ascorbic acid in bulky linoleic acid containing -
tocopherol.
Keywords: acyl ascorbate, linoleic acid, oxidation, synergistic effect,

tocopherol
1. INTRODUCTION
In many food systems, lipid oxidation is one of the main concerns to be
addressed because it causes rancidity and nutritional deterioration. Lipid
oxidation proceeds because of a free radical chain reaction between
unsaturated acyl groups in the lipid and active oxygen species and is a
complicated process including three stages defined as initiation, propagation,
and termination [1]. The initiation stage involves the formation of free radicals
through abstraction of hydrogen atoms from an unsaturated acyl group in the
presence of an initiator such as lipid peroxyl radicals. In the propagation stage,
lipid peroxyl radicals are formed, which can then produce lipid
hydroperoxides and new alkyl radicals by abstraction of the hydrogen from
unsaturated acyl group in other molecule. Eventually, the termination stage is
reached when two free radicals react with each other and produce non-radical
species [2].
-Tocopherol is popular as a natural and lipophilic antioxidant for foods,
since it quickly scavenges the radicals and breaks the chain. Thus,
antioxidative activities of tocopherols and their related compounds in various
reaction systems have been studied [3-5]. L-Ascorbic acid is also well known
Suppressive Effect of Acyl Ascorbate … 93
as a hydrophilic vitamin with high reductivity because of its enediol-lactone

resonant structure and is extensively used as an additive in foods and
cosmetics [6]. The combination of -tocopherol and ascorbic acid results in a
synergistic inhibition of lipid oxidation [7-10]. It has been found that the -
tocopherol radical reacts with ascorbic acid and that -tocopherol remains
almost unchanged until ascorbic acid is exhausted. 6-O-Palmitoyl and 6-O-
stearoyl L-ascorbates are lipophilic derivatives of ascorbic acid, which are
synthesized and used as a fat-soluble antioxidants and surfactants in foods, in
particular in bread-making for strengthening dough and for softening bread
crumbs [11]. Acyl ascorbate, which is the lipophilic derivative of ascorbic acid
with acyl chains, is amphiphilic and has increased solubility relative to
ascorbic acid in lipid rich foods [12]. Previous studies have reported the
lipase-catalyzed syntheses and the antioxidative abilities of various acyl
ascorbates [13-15]. Enzymatic synthesis of acyl ascorbate would be more
advantageous than a chemical procedure due to the simplicity of its reaction
process and its high regioselectivity. In addition, use of an immobilized lipase
make the continuous production of acyl ascorbate possible [16, 17]. The
antioxidative properties of acyl ascorbate on lipid oxidation in bulk as well as
in disperse and microcapsule systems have been examined, determining that
the antioxidative abilities depend on the acyl chain length in the ascorbate
molecule and the molar ratio to lipid in each system [18-22]. Although there
have been some reports on the antioxidative ability of saturated acyl ascorbate
with long acyl chains and the synergistically suppressive effect with -
tocopherol on bulky lipid oxidation [23-28], the application of acyl ascorbate,
such as lauroyl ascorbate, with medium chain length has hardly been
investigated.
In this chapter, the antioxidative property of acyl ascorbate in combination
with -tocopherol for the oxidation of linoleic acid is examined. Lauroyl and
palmitoyl ascorbates were synthesized through lipase-catalyzed condensation
of ascorbic acid with the corresponding fatty acids and were used to
investigate the dependence of the antioxidative ability on acyl chain length.
The kinetics of the oxidation in a bulk lipid have been widely studied and the
overall oxidation process of lipid has been described by a kinetic expression of
the autocatalytic type in terms of the fraction of unoxidized substrate [29-31].
Therefore, the expression for the entire oxidation process of linoleic acid in the
presence of acyl ascorbate with -tocopherol was applied and the kinetic
parameters were estimated to evaluate the synergistically suppressive effect of
acyl ascorbate with -tocopherol on the oxidation.
2. MATERIALS AND METHODS

2.1. Materials
L(+)-Ascorbic acid (purity >99.5%) was purchased from Nacalai Tesque,

Inc., Kyoto, Japan. Linoleic acid (purity >99%) and trimethylsilyl
diazomethane solution were obtained from Sigma-Aldrich Fine Chemicals, St.
Louis, MO, US. Lauric and palmitic acids were purchased from Wako Pure
Chemical Industries, Osaka, Japan, and Yoneyama Yakuhin Kogyo, Osaka,
respectively. Immobilized lipase from Candida antarctica, Chirazyme® L-2 c.-
f. C2 was obtained from Roche Molecular Biochemicals, Mannheim,
Germany. All other chemicals were of analytical grade and were purchased
from either Wako Pure Chemical Industries or Yoneyama Yakuhin Kogyo.
2.2. Enzymatic Synthesis and Purification of Acyl Ascorbate
Saturated acyl ascorbate was synthesized through the condensation of

ascorbic acid and a fatty acid [19]. 15 mmol of ascorbic acid and 45 mmol of
lauric or palmitic acid were weighed in an amber glass bottle equipped with a
screw cap. One gram of immobilized lipase from C. antarctica, Chirazyme®
L-2 C2, and 300 mL of acetone were added to the bottle. The headspace of the
bottle was filled with nitrogen gas to suppress the oxidative degradation of
ascorbic acid during the reaction and then tightly sealed. The bottle was
immersed in a water-bath (Personal-11, Taitec Co., Ltd., Saitama, Japan) at
50°C with vigorous shaking to commence the condensation reaction. After 24
h, 6-O-lauroyl or palmitoyl ascorbate was isolated from the reaction mixture
according to previously reported procedures [14] with minor modifications.
The immobilized lipase was removed by filtration (Filter paper 1 (185 mm ),
Advantec Toyo Kaisha, Ltd., Tokyo, Japan) from the reaction mixture, and
then the solvent was removed by a rotary evaporator (RE200, Yamato
Scientific Co., Ltd., Tokyo). The concentrate was washed at least three times
with 50 mL of n-hexane to remove the unreacted lauric or palmitic acid. The
mixture was dissolved in 150 mL of 2-butanone, and washed with 50 mL of
water at least three to remove the unreacted ascorbic acid. The organic phase
was separated and excess organic solvent was removed by rotary evaporation.
Each isolated product was dried for one day in a desiccator under dark
conditions using a Petri dish containing phosphorus pentoxide. The purity of
acyl ascorbate was determined via high-performance liquid chromatography

(HPLC) analysis. The HPLC equipment constituted of a pump (LC-10AT,
Shimadzu Co., Ltd., Kyoto), UV detector (245 nm, SPD-10A, Shimadzu Co.,
Ltd.), and COSMOSIL 5C18-AR-II column (4.6 mm  × 250 mm, Nakalai
Tesque, Inc.). Following adequate dilution, the product solution (20 L) was
applied to the column and eluted using a methanol/water/phosphoric acid
mixture (90:10:0.1 by vol.) as the eluent at a flow rate of 1.0 mL/min. In the
case of insufficient purification, the desired product was obtained by
recrystallization in n-hexane or acetone.
2.3. Measurement of Linoleic Acid Oxidation
The oxidation of linoleic acid in the presence of -tocopherol and acyl

ascorbate was measured as follows: First, linoleic acid and -tocopherol were
separately dissolved in n-hexane at concentrations of 0.071 mol/L and 0.036
mol/L, respectively. Ascorbic acid, lauroyl, and palmitoyl ascorbate solutions
were prepared using methanol as a solvent at a concentration of 0.036 mol/L.
The linoleic acid solution was mixed with the -tocopherol, ascorbic acid,
lauroyl and palmitoyl ascorbate solutions to obtain the intended molar ratios of
the antioxidants to linoleic acid. The mixture (25 L each) was placed in flat-
bottomed glass cups (1.5 cm i.d. and 3.0 cm height). The n-hexane and
methanol was then evaporated under reduced pressure. The cups were put in a
plastic container (21 cm × 15 cm × 7 cm height) in which a Petri dish filled
with a saturated lithium chloride solution was placed to maintain the relative
humidity (RH) at 12%. The container was stored in the dark at 65°C. The cups
were removed intermittently and 300 L of a methyl myristate solution,
methanol/benzene/methyl myristate = 20/80/0.05 by vol., was added to each
cup. Methyl myristate was used as the internal standard for gas
chromatography (GC) analysis of unoxidized linoleic acid. 20 µL of 2.0 mol/L
trimethylsilyldiazomethane solution with n-hexane as solvent were then
poured into the cup to convert unoxidized linoleic acid to methyl linoleate [32].
The mixture was put into the amber vial and was left for 30 min at room
temperature after filling the headspace with nitrogen gas. After evaporation of
methanol, benzene, and hexane in a desiccator under reduced pressure, the
remainder was then dissolved with 500 L of n-hexane. The amount of
unoxidized linoleic acid was measured by GC (G-3500, Hitachi, Ltd., Tokyo)
with a flame ionization detector. One µL of the n-hexane solution was injected
into a capillary column, the dimensions of which were 0.25 mm in diameter

and 30 m in length, with polyethylene glycol (ZB-WAX, Shimadzu Co., Ltd.).
The column temperature was 180°C and the injection or the detection
temperature was 200°C.
3. RESULTS AND DISCUSSION
3.1. Effect of -Tocopherol Content on Oxidation of Linoleic

Acid
Figure 1 shows the oxidation process at 65°C and 12% RH of linoleic acid
in the absence and the presence of -tocopherol. Linoleic acid without any
antioxidative additive oxidized rapidly and the fraction of unoxidized linoleic
acid was 0.24 after 9 h. The addition of -tocopherol to linoleic acid
significantly improved its oxidative stability. The fraction of unoxidized
linoleic acid with -tocopherol at 0.005 molar ratio to linoleic acid was 0.71
after 11 h. The oxidative stability of linoleic acid was enhanced by increasing
the -tocopherol content. The fraction of unoxidized linoleic acid with -
tocopherol at the molar ratio of 0.01 was 0.85 for at least 30 h.
1.2
Fraction of unoxidized linoleic acid
1.0
0.8
2
ln [(1-Y)/Y]
0.6
0
-2
0.4
-4
0.2 0 10 20 30 40
t [h]
0
0 10 20 30 40
Time [h]
Fig. 1. Oxidation at 65 oC and 12% RH for linoleic acid in the
Figure 1. Oxidation at 65°C
absence and
of an 12% RH for
antioxidative linoleic
additive acidtheinpresence
(⭕ ) and the absence
of of an
-tocopherol at 0.005 (□) and 0.01 (△) molar ratio to linoleic
antioxidative additive (⭕) and the presence of -tocopherol at 0.005 (□) and 0.01 (△)
acid. The solid curves were obtained from the estimated
molar ratio to linoleic acid. The
parameters, k andsolid
Y0, curves
for the were obtained
oxidation. The from the estimated
inset shows
parameters, k and determination
Y0, for the oxidation. The insetk, shows
of the rate constant, determination
in the rate expression of of the rate
constant, k, in the the
rateautocatalytic
expressiontype.
of the autocatalytic type.
Table 1. Kinetic parameters of the autocatalytic type equation for the

oxidation of linoleic acid in the presence or absence of -tocopherol and
Table 1. Kinetic parameters of the autocatalytic type equation
ascorbate.
for The ascorbate
the oxidation is either
of linoleic acidascorbic acid, or
in the presence lauroyl,
absenceor
of palmitoyl
-tocopherol and ascorbate. ascorbate
The ascorbate is either ascorbic
acid, lauroyl, or palmitoyl ascorbate.
Molar ratio of -tocopherol to

linoleic acid
Parameters 0 0.005 0.01
k [h-1] 0.400 0.178 0.062
Y0 0.942 0.944 0.967
Molar fraction of lauroyl ascorbate

coexistent with -tocopherol a)
Parameters 0.25 0.50 0.75
k [h-1] 0.163 0.149 0.147

Y0 0.988 0.986 0.988
Type of ascorbate coexistent

with -tocopherol a, b)
Parameters Ascorbic acid Palmitoyl ascorbate
k [h-1] 0.277 0.143

Y0 0.989 0.985
a
The molar ofa)the
Theadditive to linoleic
molar ratio acid is 0.005.
of the additive to linoleic acid is 0.005.
b
Molar fraction of the fraction
b) Molar ascorbateofcoexistent with
the ascorbate -tocopherol
coexistent with -tocopherol
is 0.50.
is 0.50.
As previously reported, the entire oxidation process of unsaturated fatty
acid and its glyceride could be expressed by the following kinetic equation of
the autocatalytic type [33, 34]:
dY
 kY (1  Y ) (1)
dt
where Y is the fraction of the unoxidized substrate, t is time, and k is the rate
constant for the oxidation process. Under the condition of Y = Y0 at t = 0, the
integration of the above equation gives:
1Y 1  Y0
ln  kt  ln (2)
Y Y0
where Y0 is the initial fraction of unoxidized substrate and determines the
induction period based on the mathematical feature format of the equation.
Equation 2 was applied to the oxidation of linoleic acid mixed with -
tocopherol at various concentrations. Linear plots of ln [(1 - Y)/Y] versus t for
the oxidation are shown in the inset of Figure 1. The k and Y0 values were
calculated from the slope and the intercept, respectively, of a linear regression
analysis. The estimated parameters of the kinetic equation are shown in Table
1. The rate constant for the oxidation of linoleic acid, k, decreases with the
addition of -tocopherol. Furthermore, k decreases with increases in -
tocopherol. On the other hand, Y0 for the oxidation of linoleic acid in the
presence of -tocopherol at the molar ratio of 0.005 is equivalent to Y0 for the
oxidation in the absence of -tocopherol while Y0 for the oxidation in the
presence of -tocopherol at the molar ratio of 0.01 is a little higher than those
of the formers. In summary, -tocopherol contributes to both lowering the
oxidation rate and prolonging the induction period of the oxidation.
3.2. Synergistic Antioxidative Effect of Lauroyl Ascorbate and

-Tocopherol
The oxidative stabilities of linoleic acid in the presence of lauroyl

ascorbate and -tocopherol at 65°C and 12% RH were examined. The total
amount of lauroyl ascorbate and -tocopherol was 0.005 of the molar ratio of
linoleic acid and the molar fractions of lauroyl ascorbate in the antioxidative
additives were 0.25, 0.50, and 0.75. Figure 2 shows the oxidation of linoleic
acid with lauroyl ascorbate and -tocopherol at the various molar fractions, as
well as without any additives and with only -tocopherol as the additive. The
coexistence of lauroyl ascorbate with -tocopherol increased the oxidative
stability of linoleic acid and the suppressive effect on the oxidation by the
coexistence was greater than that of only -tocopherol. The k and Y0 values for
the oxidation of linoleic acid in the presence of lauroyl ascorbate and -
tocopherol were also determined using the kinetic equation of the autocatalytic
type. The estimated parameters are shown in Table 1. The k value for the
oxidation of linoleic acid with both antioxidants is lower than that with only -
tocopherol. This indicates the synergistic effect of lauroyl ascorbate and -
tocopherol on suppressing the oxidation. The k value slightly decreases as the

lauroyl ascorbate content increases. At the same time, the Y0 values for the
three molar fractions are very high and nearly equivalent. These results
suggest that the induction period of the oxidation is prolonged by the
combination of lauroyl ascorbate with -tocopherol and the synergistic
inhibition reaches the maximum level when the molar amount of lauroyl
ascorbate is at least equal to the molar amount of -tocopherol.
1.2
1.0
0.8
0.6
0.4
0.2
0
0 10 20 30 40
Time [h]
Figure 2. Oxidation
absenceatof65°C and 12% RH additive
an antioxidative for linoleic
(⭕ )acid
and in thepresence
the absenceof of an
antioxidative -tocopherol
additive (⭕) andandthe
lauroyl
presence of -tocopherol
ascorbate. The molar andratio of the
lauroyl ascorbate. The
molar ratio ofantioxidative additive
the antioxidative to linoleic
additive acid
to linoleic is is0.005.
acid 0.005.The
The molar
molar fractions of
fractions of lauroyl ascorbate coexistent with -tocopherol are
lauroyl ascorbate coexistent with -tocopherol are 0 (□), 0.25 (△), 0.50 (◇), and
0 (□), 0.25 (△), 0.50 (◇), and 0.75 (▽). The solid curves
0.75 (▽). Thewere
solidobtained
curves were
from obtained fromparameters,
the estimated the estimated parameters,
k and Y0, for thek and Y0, for
the oxidation.oxidation.
3.3. Relationship between Acyl Chain Length of the Ascorbate

and the Suppressive Effect on the Oxidation
The effect of acyl chain length of acyl ascorbate coexistent with -

tocopherol on the oxidation of linoleic acid was investigated. Figure 3 shows
the oxidation of linoleic acid with -tocopherol and ascorbic acid, lauroyl
ascorbate, or palmitoyl ascorbate at 65°C and 12% RH. The molar ratio of the
antioxidative additives, which are -tocopherol and one of the three ascorbates,
to linoleic acid is 0.005, and the molar fractions for -tocopherol and the
ascorbate molecules are equivalent. The kinetic parameters in Equation 2 were
calculated based on these results and are shown in Table 1.
1.2
1.0
0.8
0.6
0.4
0.2
0
0 10 20 30 40
Time [h]
Figure 3. Oxidation
absence of an at 65°C and 12%
antioxidative RH for
additive (⭕ linoleic
) and theacidpresence
in the absence
of of an
antioxidative additive
ascorbic acid (□),(⭕) and the
lauroyl presence
ascorbate (△), ofand
ascorbic acidascorbate
palmitoyl (□), lauroyl ascorbate
(△),(◇)and palmitoyl with -tocopherol.
coexistentascorbate (◇) coexistent Thewith -tocopherol.
molar ratio of Thethe molar ratio of the
antioxidative additive to linoleic acid is 0.005 and the molar
antioxidative additive to linoleic acid is 0.005 and the molar fraction of the ascorbate
in thefraction of the ascorbate in the antioxidative additive is 0.50. The
antioxidative additive is 0.50. The solid curves were obtained from the estimated
solid curves were obtained from the estimated parameters, k and Y0,
parameters, k and Y0, for the oxidation.
for the oxidation.
In the coexistent system of ascorbic acid with -tocopherol, both k and Y0

values are greater than those of the single antioxidant system with -
tocopherol. Therefore, relative to the presence of only -tocopherol, the
coexistence of ascorbic acid elongates the induction period of the oxidation
but increases the oxidation rate. The reason for this phenomenon is not clear
but the high hydrophilicity of ascorbic acid might weaken the synergistic
effect with -tocopherol in bulky linoleic acid. The k and Y0 values for the
coexistence of palmitoyl ascorbate and -tocopherol are almost equal to those
in the coexistent system with lauroyl ascorbate, indicating that the
hydrophobicity among saturated acyl ascorbates with acyl chain lengths from
12 to 16 does not affect the synergistic antioxidative effect of acyl ascorbate
with -tocopherol.
CONCLUSION
Kinetic analysis using the oxidation rate equation of the autocatalytic type
reveals that the coexistence of ascorbic acid or acyl ascorbate with -
tocopherol synergistically retards the oxidation of linoleic acid. The
coexistence of lauroyl or palmitoyl ascorbate with -tocopherol prolongs the
induction period of the oxidation and lowers the oxidation rate, while the
synergistically suppressed antioxidative effect of ascorbic acid on the radical
chain reaction during the propagation stage of the oxidation is weak. The
synergistic effects of the ascorbates are mainly ascribed to the elongation of
the induction period.
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Chapter 5
STABILIZATION OF L-(+)-ASCORBIC ACID IN

AN IRON FORTIFIED VEGETABLE PRODUCT
(CUCURBITA MOSCHATA DUCHESNE EX
POIRET) USING AN ALGINATE COATING
María Dolores De’Nobili, Carolina Genevois,

Ana M. Rojas, Silvia Flores and
Marina de Escalada Pla
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
Argentina
ABSTRACT
In previous studies it was observed that in fortified pumpkins
(Cucurbita moschata Duchesne ex Poiret) with iron (Fe) and L-(+)-
ascorbic acid (AA), the latter had a minimum retention because AA
stabilization was adversely affected by the presence of Fe. From this, it
was proposed compartmentalize these two nutrients, being Fe applied to
the impregnation of mesocarp pumpkin, while the AA was dissolved in a
coating formulation consisting of alginate. Pumpkins, cut and blanched,

Corresponding author: Silvia Flores, e-mail: skflores14@gmail.com.
106 María Dolores De’Nobili, Carolina Genevois, Ana M. Rojas et al.
were subjected to a dry impregnation. Two batches were prepared, one in

which the Fe and AA were incorporated into pumpkin tissue, while in the
second batch the vegetable matrix was only fortified with Fe. The studied
systems were: a) Control fortified pumpkins with Fe and AA, covered
with alginate coating (C); b) fortified with Fe, covered with alginate
coating containing AA (F). In addition, two water activity (aW) conditions
0.920 (F1 and C1) and 0.760 (F2 and C2) were tested. All systems were
contained in plastic trays, packaged in plastic bags and stored at a
constant temperature of 25ºC for 23 days. During storage, AA
concentration as well as color changes were registered. The AA loss and
also b* and Chroma (Chr) parameter reductions fitted to a first order
kinetic. For aW 0.760, the values obtained for the AA initial content were
significantly (p < 0.05) higher, while the rate constants of AA loss were
lower. The kinetic constants for color parameters were one order lower
than AA loss. In general, F systems showed a faster reduction of b* and
Chr than controls (C), but the aw assayed did not affect the rate of color
changes. When fortified pumpkins were tested at aw 0.92 and storage at
18 and 25ºC, it was observed reduction of AA loss rate only in F1
pumpkin at the lower temperature. The reduction of color parameters
showed an important influence of both compartmentalization of AA and
storage temperature. It could be concluded that AA supporting on an
alginate edible coatings enhances this vitamin retention and then it could
be represent an effective strategy when developing food products fortified
with iron.
Keywords: edible coating, alginate, L-(+)-ascorbic, fortified foods, iron, color
INTRODUCTION
Iron deficiency remains one of the world’s greatest public health
problems. Globally it is the main contributor to anaemia, affecting 47% of pre-
school age children and 25% of school age children worldwide. Inadequate
intake qualitatively or quantitatively is the commonest cause of deficiency in
children in the industrialized world. As iron is poorly absorbed, a typical
western diet will be barely sufficient in meeting daily requirements.
Worldwide management strategies again focus on dietary improvements,
as well as the control of hook worm and malaria infections to reduce levels of
iron deficiency (Petit et al., 2011).
From a nutritional standpoint, it is important to know the amount of
dietary mineral available for absorption and utilization, i.e., its bioavailability.
The first stage towards bioavailability comprises mineral solubility in the
Stabilization of L-(+)-Ascorbic Acid in an Iron Fortified Vegetable … 107
intestinal tract, i.e., the so-called “bioaccessibility” (Salovaara et al., 2002;

Cilla et al., 2009; 2011; Briones-Labarca et al., 2011; Parada and Aguilera
2007). In relation to bioaccessibility, it is important to consider the efficacy of
mineral fortificants present in the food tested, as influenced by the interplay of
promoter factors, such as organic acids and inhibitory factors, such as calcium,
casein and polyphenols (Cilla et al., 2011; Perales et al., 2006; 2007). Ascorbic
acid (AA) is the most potent enhancer of iron (Fe) absorption, both in its
natural form in fruits and vegetables and when added as the free compounds
(Nayak and Nair, 2003). -Carotene has also been demonstrated to improve Fe
absorption in vitro and in human absorption studies, by a mechanism that is
yet to be elucidated but could be associated with its provitamin-A activity
(García-Casal et al., 2003). It was also reported that lycopene, lutein, and
zeaxanthin significantly increased Fe absorption in human beings from corn
and wheat meals (García-Casal et al., 2000, 1998).
On the other hand, the processes applied involve food matrix changes and
then the subsequent effect on minerals and micronutrients bioavailability. For
instance, Frontela et al. (2011) concluded that breadmaking process has
important effects on iron, calcium, and zinc availability. The raw material used
(wheat flour and whole-wheat flour) as well as the stage of the processing,
play key roles in mineral availability values. Fermentation and baking stages
reduced the phytate content of samples, increasing dialysis of iron and calcium
after processing. Cilla et al. (2011) reported that high-pressure processing
improves Ca and P bioaccesibility in different milk-based fruit beverages
when comparing with thermal treatments.
Commonly available fortified foods include non-structured and
formulated foods. In contrast, impregnation or vacuum impregnation allows
the introduction of physiologically active compounds into vegetal tissues
without disrupting their cellular structure, but inducing changes in their
behaviour during further processing. In this sense, some authors (Barrera et al.,
2004; Fito et al. 2001) analyzed the feasibility of vacuum impregnation
treatment in the development of products fortified with Ca and Fe from fresh
fruits and vegetables. De Escalada Pla et al. (2009) employed osmotic
dehydration in order to obtain a functional food fortified with Fe and AA
based on pumpkin (Cucurbita moschata, Duchesne ex Poiret). More recently,
Genevois et al., (2014) could improve the efficiency of the fortifying process
applying dry infusion technique. Nevertheless, color changes in vegetal tissue
due to iron and AA presences and also the process applied, were reported.
Edible coatings are fine layers of digestible material added to a food
product. Many polysaccharide-based coatings have demonstrated to provide
protection against external microbial contamination, to extend the shelf life of

food, to act as a barrier to water vapor or gas, and also can be used to
incorporate additives, such as antimicrobials, antioxidants, dyes, etc. (Han,
2000; Cuppett, 1994; Flores, 2007; Campos et al., 2011). In previous studies,
colleagues have reported that compartmentalizing AA in a network of edible
film helps achieve stabilization as it may prevent AA interactions with oxygen
and other preservatives, nutrients or food components. Besides the films may
provide antioxidant activity localized at the interfaces (León and Rojas, 2007;
Pérez et al., 2009, 2013, De’Nobili et al., 2013). Alginic acid is a natural
polysaccharide harvested from brown algae (Jothisaraswathi et al., 2006).
Alginate hydrogels have been particularly attractive in tissue engineering
applications, as these gels retain structural similarity to the extracellular
matrices in tissues and can be manipulated to play several critical roles.
Alginates are also very useful because of their utility in preparing hydrogels at
mild pH and temperature conditions, suitable for sensitive biomolecules
(Pawar and Edgar, 2012).
The aim of this work was to study the possibility of stabilization of L-(+)-
ascorbic acid in an iron fortified product based on Cucurbita moschata
Duchesne ex Poiret using edibles coatings of alginate and to analyze different
strategies. The food color stabilization was also studied at different process
and storage conditions.
MATERIAL AND METHODS

Chemicals
Food grade sucrose and glucose (Anedra, Argentina) were employed.

Cargill (Mechelen, Belgium) alginate was also used in this study. The
additives: FeSO4.7H2O (Merck, Argentina), potassium sorbate (Sigma, US),
L-(+)-ascorbic acid (Merck, Argentina), citric acid and glycerol (Sintorgan,
Argentina) and other chemicals used were of analytical grade.
Preparation of the Pumpkin Fortified with Fe and AA
Dry Infusion Process of Pumpkin Cylinders

Pumpkin (Cucurbita moschata Duchesne ex Poiret) obtained in a local
supermarket was carefully washed and rinsed with distilled water. Then,
cylinders of 29 mm diameter and 10 mm thickness were cut from the

mesocarp using a stainless steel cork borer. The cylinders were blanched with
water vapor for 8 minutes, rapidly cooled for 1 minute by immersion in water
at 0°C and then submitted to a dry infusion (Alzamora, Guerrero, Nieto and
Vidales, 2003) with some modifications according to Genevois et al. (2014).
Briefly, pumpkin cylinders were placed in a plastic bowl and sprinkled with
powdered glucose (33 g/100 g pumpkin) and sucrose (23 g/100 g pumpkin).
Water from vegetal tissue began to flow from the pumpkin cylinder to the
surrounding. In that moment, citric acid (0.15 g/100 g pumpkin), potassium
sorbate (0.19 g/100 g pumpkin) and Fe by adding FeSO4.7H2O (0.17 g/100 g
pumpkin, equivalent to 34 mg Fe/100 g pumpkin), were added to the liquid
solution and the orbital shaking started up. Citric acid was added in order to
decrease pH values below 5, since sorbate is more effective, as an
antimicrobial, in this range of pH (Damodaran, Parkin and Fennema, 2008).
The dry infusion was carried out at 20°C up to equilibrium on an orbital shaker
(Vicking S.A., Argentina) at 35 revolutions per minute (rpm).The described
conditions of infusion were selected taking into account previous assays.
Equilibrium was reached at 72 hours when pumpkin cylinders and the
surrounding solution achieved the same aw and pH values. Once the dry
infusion was concluded, the cylinders were drained through a stainless steel
strainer.
Two batches were carried out: one as described previously (F) and the
other, control system (C) was performed in similar conditions used for F but
fortified also with AA by mean of the addition of 0.9 g AA/100 g pumpkin to
the dry infusion media.
Coating Procedure, Packaging and Final Storage

Two different formulations were employed for edible coatings
preparations, one containing 2% w/w sodium alginate, 1% w/w glycerol,
0,05% w/w potassium sorbate and AA (4% w/w); was used to coat pumpkins
pieces coming from F. The other one was prepared similarly but without AA
addition. This coating was employed to cover pumpkin cylinders coming from
control system (C). In both cases, preparations were homogenized as stated by
Del Nobili et al. (2013). Briefly, alginate; glycerol, as a plasticizer, and
potassium sorbate, as antimicrobial agent, were dissolved in distilled water at
T 90°C. For F coating, the AA was previously dissolved in cold water and then
added to the final cooled solution.
Then pumpkin pieces were immersed for one minute in the respective
solutions. Immediately after, they were immersed for one more minute in a
solution of calcium chloride (2% w/w) to constitute the coating on the pieces
of plant tissue.
A first batch, remained with aw achieved after coating constitution, aw 0.92
(F1 and C1). Pumpkin systems were introduced into low density polyethylene
bags of 80 µm thickness and stored at two different room temperatures: 18 and
25ºC, in order to studied temperature effect on product stabilization.
On the other hand, in order to study the effect of water activity on product
stabilization, another batch was carried out. One part of this remained at aw
0.92, meanwhile the other part, was submitted to air drying in a chamber at
30°C in order to reduced aw to 0.76 (F2 and C2). Finally, each pumpkin
system (F2, C2) were introduced into low density polyethylene bags of 80 µm
thickness, provided with an easy-to-close Ziploc® closing. The bags were
filled with the corresponding 5 pieces (40 g) and stored at 25°C.
Then, the effect of compartmentalize AA into the alginate coating in the
Fe fortified food was analyzed in the maximum aw (0.92) at two different
storage temperature; and at the maximum room temperature (25°C) with two
different water activity.
The dry infusion process and the coating were performed at least twice.
Product Characterization
The following properties were measured:
♦ a w:
Pumpkin cylinders were reduced to a puree with the aid of a homogenizer

Ultra Turrax (IKA, US) at 6500 rpm for 20 seconds. The water activity (aw)
was measured with a hygrometer (Aqualab, US) at 20°C. Determinations were
performed at least in duplicate on all the systems during storage.
♦ Color
Color parameters were evaluated using a photocolorimeter (Minolta,

Japan) in the CIE L*a*b* space [L*: lightness, a*: greenness - redness, b*:
blueness - yellowness] under illuminant D65 and with the observer at an angle
of two degrees.
The value of Chroma parameter was also calculated. This parameter
describes color intensity (Olivera et al., 2008) and was calculated as Chroma =
(a2 + b2)1/2. The determinations were performed on the samples after applying
infusion and coating and throughout 23 days. The averages of four

measurements are reported.
Nutritional Characterization: Determination of L-(+)-Ascorbic Acid

AA was determined according to De’Nobili et al. (2013). Briefly, twenty
grams of samples were weighed and then broken in the presence of 20 g of
oxalic acid solution of 1% (w/v) with the aid of a homogenizer (Ultra Turrax,
Germany). The homogenate was diluted to 100 mL with oxalic acid solution
1% (w/v). The suspension was homogenized by stirring and then centrifuged
at 5000 rpm for 10 min at 6°C (Eppendorf, Germany). Aliquots of the
supernatant were taken for spectrophotometric determination of AA, upon
reaction with 2,6-dichlorophenol–indophenol. The determinations were
performed on the samples in triplicate throughout 23 days of storage.
The AA content was measured during storage and AA concentration
(mg/g) was fitted to a pseudo first order kinetic (De’Nobili et al., 2013).
Statistical Analysis
The results are reported as the average and their standard deviation (SD).
The statistical analyses of results were performed by applying ANOVA (:
0.05), followed by Bonferroni Test. The Statgraphics Centurion XV (version
15.2.06 StatPoint, Inc. 2007, US) was used for non linear regressions and for
statistical treatment of data.
RESULTS AND DISCUSION

After dry infusion and coating processes, it was possible to obtain a ready
to eat pumpkin based snack fortified with Fe and AA. The resulting aw of the
products was 0.92 ± 0.03 for both systems assays (F1 and C1) without
significant differences between them. In order to depress the aw of the fortified
pumpkin, a fraction of each systems was submitted to air drying till aw of 0.76
± 0.02 was reached (C2 and F2 systems). The samples were stored at 25ºC and
AA retention as well as color changes were studied during storage.
Influence of aw and Compartmentalization on AA Loss
The AA contents at the beginning of the storage (C0) are resuming in

Table 1. It was observed that control systems, C1 and C2, suffered an
important AA reduction since C0 values, for aw 0.76 and 0.92, were significant
lower (p < 0.05) than the corresponding initial AA content for F1 and F2. Two
reasons could explain this result. The dry infusion process applied could not
incorporate AA in the vegetable tissue as expected. On the other hand, 72 hs
were needed to reduced aw from ≈ 0.993 till equilibrium (aw 0.92), during this
time, AA solution remained at 20°C along with solvated Fe2+ ions. These
conditions resulted worst for AA retention when comparing with AA support
in a coating (F1 and F2). The AA loss involved a first step of the non-
enzymatic browning reactions chain (León and Rojas, 2007). Such irreversible
degradation of AA can occur through hydrolysis simultaneously or
competitively to AA oxidation when oxygen is present, producing 2-keto-L-
gulonic acid which is a reactive molecule that suffers successive
transformations that involve dehydrations and decarboxylations producing
different browning active compounds (Genevois et al., 2016; De’Nobili et al.,
2013; Pérez, Flores, Marangoni, Gerschenson, and Rojas, 2009).
Table 1. AA loss parameters values fitted to first-order model, from

pumpkin coated with alginate stored at 25ºC and different water activity
aw Systems C0 (mg/g) k x 104 (min-1) R2 DW

c a
0.92 F1 20 ± 1 1.3 ± 0.2 98.2 2.1
C1 6.5 ± 0.2a 1.2 ± 0.1a 99.0 2.5
0.76 F2 14.1 ± 0.3b 0.37 ± 0.03c 99.0 3.0
C2 6 ± 0.2a 0.64 ± 0.08b 95.3 1.2
C0: initial ascorbic acid content in pumpkin and the corresponding standard deviations
(SD). Values expressed as mg/g pumpkin.
k: kinetic rate constant. Values expressed as min-1
R2: determination coefficients.
DW: Durbin-Watson statistic.
Similar letters denote no significant (p ≥ 0.05) differences between the mean values.
The incorporation of AA in F1 and F2 was conducted once dry infusion

finished and in addition, the support of AA in alginate edible coating could
have exerted a protective action on the vitamin, avoiding the negative
interactions with Fe or other pumpkin tissue components. This protective
action could be corroborated through the rate constant k, at certain conditions

(Table 1 and Table 3) as will be discussed later. In addition, oxygen transfer
from surrounding to the product was promoted during air drying because the
convection system of the chamber triggering a significant reduction (p < 0.05)
of AA initial content in the alginate coating for F2 system in comparison with
F1 (Table 1).
Table 1 shows the results of the fit of experimental data to a first-order
kinetic model from pumpkin coated with alginate at different aw and stored at
25°C. All studied systems could be properly fitted to the proposed model since
the R2 ranged values from 0.95 to 0.99 and the DW parameter was higher than
1.2. Therefore, the AA concentration [CAA(t)] in each identified coated
pumpkin, changed with the storage time (t) according to the pseudo-first order
kinetic law (eq. (1)). Hence, the pseudo first order kinetic rate constant of the
AA hydrolysis (k) was the slope calculated through the linear regression of the
experimental data according to a first order reaction whose differential
equation is as follows:
1 𝑑𝐶𝐴𝐴
𝑟𝐴𝐴 = − = 𝑘𝐴𝐴 × 𝐶𝐴𝐴 (𝑡) (1)
𝜐𝐴𝐴 𝑑𝑡
where νAA is the stoichiometric coefficient for AA hydrolytic reaction (νAA =

1), rAA is the AA-reaction rate/unit volume at a constant temperature (25.0°C),
CAA(t) is the AA proportion remaining at any time (t). The rate constants of
AA hydrolysis (k) are reported in Table 1 for all the systems. It must be
remarked that all the values obtained are in the order of those reported for a
similar product (de Escalada Pla, 2009). In that opportunity, the authors did
not use coatings, but osmotic solution coverage. The disadvantage was that a
lot of nutrients were lost if the product was not consumed together with the
osmotic solution. From the results it can be seen that the kinetic rate constant
(k) was higher for systems with aw 0.92 (C1 and F1) in comparison with
pumpkin cylinders of aw 0.76 (C2 and F2). As expectable, the hydrolytic
destruction of AA was mainly affected by the aw of the systems (Table 1),
which is directly related to the water mobility in the film network and in the
pumpkin tissue (Leon et al., 2009). The k of control system with aw 0.76 (C2)
is nearly the half rate of AA degradation than that at aw 0.92 while the
pumpkin with alginate based edible coating containing AA, showed a k
reduction around 70%. Consequently, the average life of the C2 and F2
products are increased. Half-life times of AA in the coated pumpkins were
also calculated according to the following equation, derived from equation (1):
0.693
𝑡1⁄ = 𝑘𝐴𝐴
(2)
2
It could be observed that half-life time for systems of aw 0.92 were 4.0 and
3.7 days for C1 and F1 systems respectively. Similarly, the half-life of 7.5
days of fortified tissue (C2) could be extended to 13 days when AA was in the
coating (F2) for systems of aw 0.76.
As regards to AA compartmentalization, significant differences (p < 0.05)
in k values were observed for the lower analyzed aw (0.76) where k for F2 was
approximately 50% reduced in comparison with C2. This would indicate that
the AA impregnated into the plant tissue fortified with Fe, is suffering more
daily degradation if compared with the AA retained in the alginate coating.
This shows the protective effect of the alginate coating on the AA stability
even under adverse conditions, such as being applied on food fortified with Fe
(De’Nobili et al., 2013). However, for aw 0.92, such differences were
minimized probably due to the important influence of aw on the increase of
reactions rate.
Influence of aw and Compartmentalization on Color Changes
During the storage time at 25ºC, the pumpkin cylinders showed a

darkening in all systems. From all the color parameters tested, the b* and Chr
were selected as the better parameters to represent the changes observed. The
changes in b* and Chr parameters could be well fitted to a pseudo-first order
kinetic model (eq. (1)), showing R2 and DW values higher than 0.808 and 1.1
respectively (Table 2).
It was determined at time zero b* values trended to be lower for F2 and
C2 (aw 0.76) than F1 and C1, respectively, equilibrated at aw 0.92 (Table 2).
These tendencies could be explained by an additional loss of the yellow color
during the drying step at 30ºC in C2 and F2 samples. Genevois et al. (2016)
also observed higher Chr and b* values in a pumpkin based snack fortified
with AA and covered with a k-carrageenan edible coating supporting Fe, in
comparison with a similar snack but coated with a starch. The authors
explained these results in terms of a higher quantity of retained water in the k-
carrageenan coating structure (aw 0.954). Conversely, pregelatinized tapioca
starch coating was subjected to air drying treatment which reduced the aw
(0.879) and it could have influenced the browning of the pumpkin cylinders
through three phenomena: the pigment concentration by reducing the amount
of water available in the product, the AA deteriorative reactions (de Escalada

Pla et al., 2009) and the carotenoids degradation by exposure to heat and
oxygen, with a consequent increase in cis-isomers (Lago-Vanzela, do
Nascimento, Fontes, Mauro, Kimura, 2013).
Table 2. Parameters of color fitted to first-order model for pumpkin

coated with alginate stored at 25ºC and different water activity
Color aw Systems b0 or Chr0 k x 105 (min-1) R2 DW

b* 0.92 F1 20.4 ± 0.9a 4.6 ± 0.6b 96.3 1.2
b
C1 23 ± 1 2.8 ± 0.6a 88.1 1.13
a
0.76 F2 18 ± 2 3 ± 1a 85.9 1.8
C2 20 ± 1a 2.3 ± 0.6a 80.8 1.1
ab
Chr 0.92 F1 27 ± 1 5.5 ± 0.6b 97.7 1.2
a
C1 30 ± 2 3.3 ± 0.8a 91.3 1.8
b
0.76 F2 24 ± 2 5 ± 1b 83.0 1.3
C2 30 ± 2a 3.2 ± 0.8a 87.6 1.7
k: kinetic rate constant.
Different letters in the same column denote no significant (p ≥ 0.05).
The highest (p < 0.05) color loss was observed through Chr for F1 and F2
systems. The rate constant, k, for Chr reduction corresponding to pumpkin
covered with alginate coating supporting AA, resulted significant higher than
controls for both aw tested. Taking into account that the Chr value describes
the intensity of color, it could be deduced that the C systems, was able to keep
better color intensity of the fortified pumpkin. Possibly, the AA supported into
alginate coating would not be available to protect the pigments of the pumpkin
tissue from reaction with Fe and the consequent formation of compounds that
degrade the yellow color. Consequently, faster reduction of Chr during storage
was observed for F1 and F2 systems. This Chr changes were in part explained
trough b* parameter (Table 2 and Table 4). On the other hand, the aw effect on
color changes could be only observed in k constant of b* for F1 and F2
systems, being that of F1 the highest (p < 0.05) value, (4.6 ± 0.6) x 10-5 min-1.
In the case of aw 0.76, F2 systems followed the same trend but without
significant differences (p > 0.05) in comparison with C2 (Table 2).
Table 3. Parameters values of AA loss fitted to first-order model, from

pumpkin coated with alginate with water activity of 0.92 and stored at
two different temperatures
T (°C) Systems C0 (mg/g) k x 104 (min-1) R2 DW

b b
18 F1 17 ± 2 0.6 ± 0.2 88.0 2.4
C1 5.7 ± 0.3a 2.2 ± 0.3a 97.0 2.2
b
25 F1 19 ± 2 2.0 ± 0.5a 91.6 3.5
C1 6.6 ± 0.6a 2.0 ± 0.4a 93.2 2.1
C0: initial ascorbic acid content in pumpkin and the corresponding standard deviations
(SD). Values expressed as mg/g pumpkin.
k: kinetic rate constant Values expressed as min-1
Similar letters in the column, denote no significant (p ≥ 0.05) differences between the
mean values.
Table 4. Parameters of color fitted to first-order kinetics for pumpkin

coated with alginate with water activity of 0.92 and stored at two different
temperatures
Color T (°C) Systems b0 or Chr0 k x 105 (min-1) R2 DW

b* 18 F1 23.1 ± 0.6a 2.5 ± 0.2b 96.2 2.9
a a
C1 23.4 ± 0.3 1.2 ± 0.1 96 2.9
25 F1 23 ± 1a 4.3 ± 0.9c 86.8 1
C1 23 ± 1a 2.0 ± 0.4b 82.6 1.8
b b
Chr 18 F1 26.6 ± 0.9 2.8 ± 0.4 93.9 3
C1 25.9 ± 0.5b 1.3 ± 0.1a 93.5 3
25 F1 28 ± 1b 6.5 ± 0.7d 96.9 1.1
b b
C1 26 ± 1 2 ± 0.3 93 2.6
k: kinetic rate constant.
Different letters denote no significant (p ≥ 0.05) differences between rows.
It is important to remark that such color evolution at aw 0.92 occurred with

a rate approximately one order lower than AA loss while at aw 0.76 color and
AA degradation rates trended to be similar (Table 1). The former tendency
was also observed with natural as well as AA fortified orange juice (Remini et
al., 2015). Accordingly, at the end of the storage, it is expected a retention of
95% for any system regarding color changes in contrast with AA retention of
78.5, 39, 91 and 65% for F1, C1, F2 and C2 respectively.
Influence of Temperature and Compartmentalization on AA

Retention
In order to study the effect of different temperature on the AA loss rate,

fortified pumpkin cylinders of aw 0.92 (F1 and C1) were storage at 18 and
25ºC. The results are shown in Table 3. The experimental data of AA
degradation were properly fitted to a pseudo-first order kinetic model. The
calculated R2 ranged from 0.88 to 0.97 while DW parameters for all systems
assayed were higher than 2.1.
As was previously discussed, the initial amount of AA suffered an
important reduction in control systems (C1) at 25ºC as well as 18ºC when
comparing with the corresponding fortified pumpkin coated with an alginate
edible coating supporting AA (F1). Therefore, the compartmentalization of
AA into a coating exerted a positive action from AA retention point of view at
both temperatures analyzed. As it was expected, there were not significant
differences (p > 0.05) in C0 values for these systems at 25ºC or 18ºC.
It is well known that the rates of reactions decrease as temperature is
diminished. The effect of temperature reduction on AA retention was only
observed in F systems (Table 3), when AA was stabilized in alginate coating.
It can be seen in Table 3 that the AA loss rate (k) of the system stored at 18ºC
(F1) was around 73% lower than that of system stored at 25ºC. This rate
reduction was in agreed with reported by other authors. Remini et al. (2015)
studied the kinetic of the AA degradation and color intensity in pasteurized
orange juice during storage. Taking into account an average activation energy
reported by these authors (93 kJ mol-1), a rate constant for F1 system could be
predicted, resulting a value similar than that showed in Table 3. With regard to
control systems (C1 and C2), the decrease in storage temperature performed in
our study was not enough to reduce de AA loss rate in C1 system. Possibly,
the combined addition of Fe and AA into vegetal tissue promoted the AA loss
regardless the seven grades reduction in temperature. It was also observed that
at 25ºC, the AA degradation was similar (p > 0.05) for F1 and C1, suggesting
that at room temperature the AA compartmentalization strategy should be
combined with other preservation factor, for example aw reduction such as was
stated before.
Influence of Temperature and Compartmentalization on Color

Changes
The color characteristics of fortified pumpkins were analyzed by b* and

Chr parameters determination at 25ºC and 18ºC. It was observed in Table 4
that b* and Chr data were satisfactorily fitted to a pseudo-first order kinetic
model since R2 were higher than 0.826 and DW statistics were higher or equal
to 1.
As expected, b* and Chr initial values were similar for F and C systems at
both storage temperature assayed. In addition, there were not significant
differences (p > 0.05) in first values of color parameters for control systems
and coated with alginate coating supporting AA at 18ºC or 25ºC. The Table 4
also shows that the kinetic rate constants were lower at 18ºC than at 25ºC for
b* and Chr. In the case of Chr, k showed reductions of 35% and 57% for C1
and F1 respectively while k for b* was reduced around 41% (F1 or C1). It was
also observed an important influence of compartmentalization strategy on k
values since F1 systems showed a significant (p < 0.05) increase of the color
change rate (b* and Chr) for both temperatures analyzed. Such acceleration in
color loss was related to the AA addition into edible coating, which could
allow the development of browning reactions in the fortified pumpkin matrix.
According to the results, changes in color were, in general, slower than AA
loss since k values for b* and Chr reductions were inferior to the
corresponding k for AA degradation. The estimated retentions for color
parameters after 23 days of storage ranged from 94 to 98%. In the case of AA,
F1 at 18 or 25ºC could retain around 75 or 65% respectively while C1 may
loss its total vitamin content. Similar tendencies were also reported by other
authors. Remini et al. (2015) registered color changes in orange juices as
absorbance decay at 515 nm, they reported lower k reference rate for color
changes as well as activation energy (20 x 10-3 day-1 and 75 kJ mol-1
respectively) than that of AA loss (100 x 10-3 day-1 and 93 kJ mol-1,
respectively). They stated that the effect of storage temperature and deaeration
are the most influent factors on kinetics degradation, while the AA
fortification revealed no significant effect on ascorbic acid content and color
intensity of the orange juices (Remini et al., 2015). In the present work the
best AA retention resulted when this vitamin was supported on the alginate
coating and the lower aw. De’Nobili et al. (2013) stated that enriched alginate
networks in general preserved AA from oxidation and the water
immobilization is a key factor to be controlled.
CONCLUSION
In the present research, a ready to eat food fortified with Fe and AA was
successfully prepared using pumpkin (Cucurbita moschata Duchesne ex
Poiret) as raw material, applying a dry infusion and coating processes. The AA
and color stabilization were tested by the use of an alginate based edible
coating containing AA. The evolution of AA content and Chr and b* color
parameters were properly fitted to a pseudo-first kinetic model. It was
demonstrated that AA initial content was improved when it was incorporated
in the coating, mainly due to the avoiding of AA reactions with matrix
components. In addition, an aw reduction of the product at a level of 0.76 or
the storage temperature reduction at 18ºC helped to slow down the AA loss
rate.
The study of the color changes reveled that all samples suffered slight
darkening during storage. Faster b* and Chr reduction rates were observed for
systems covered with a coating supporting AA. It was attributed to the
development of browning reactions into pumpkin matrix, without the
assistance of AA to prevent Fe interactions with vegetal components and
carotenes degradation. However, in general, the mentioned color changes
occurred at a slower rate than AA loss.
According with these results, it was possible to stabilize AA in an alginate
edible coating loaded with glycerol, potassium sorbate and AA while is
coating a pumpkin fortified with Fe.
This research constitutes a contribution to the innovation of processes and
formulation of a functional food fortified with Fe.
ACKNOWLEDGMENT
The authors acknowledge the financial support from the University of
Buenos Aires (UBACyT GEF 2012–2014 20020110200192; 2014–2017
20020130200237) and National Scientific and Technical Research Council of
Argentina (CONICET; PIP 11220090100531).
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Chapter 6
STABILIZATION OF ASCORBIC ACID

BY MICROENCAPSULATION
FOR COSMETIC PREPARATIONS
Yhors Ciro and John Rojas

Department of Pharmacy, University of Antioquia, Medellín, Columbia
ABSTRACT
Ascorbic acid has a variety of biological and dermatological
functions. These functions are closely related to the antioxidant properties
of this compound contributing to maintenance of the sensory properties of
cosmetics products. However, environmental factors such as air, oxygen,
metal ions, temperature, pH, UV and X-ray degrade this compound
affecting the quality of cosmetic products. Currently, the
microencapsulation technology is being used to stabilize this compound.
Particularly, in this chapter the technique of ionic gelation of chitosan
with sodium lauryl sulfate is discussed. Chitosan is biodegradable,
biocompatible and soluble at the body temperature and hence, it is an
ideal material for cosmetic applications. Further, it is positively charged
and forms a polyelectrolyte complex with negatively charged compounds
such as sodium lauryl sulfate. The internal gelation was carried out with
solutions of chitosan and sodium lauryl sulfate at concentrations of 0.5,

Corresponding author: John Rojas. Cll 67 # 53-108, office 1-157. Medellín, Tel.: 574 219 5472.
Email address: jrojasca@gmail.com.
124 Yhors Ciro and John Rojas
1.0 and 1.5% (w/v), containing ~20 mg of ascorbic acid in different

experimental runs. The encapsulation process of ascorbic acid was
performed by processes such as homogenization and sonication-
homogenization, respectively. In both processes, spherical microcapsules
of AA were obtained with a narrow distribution of particle size (0.7-2.1
µm), but only by using sonication-homogenization at 7000 rpm less
cohesive microcapsules were obtained.
The encapsulation efficiency depended on the processing conditions
and level of parent materials and ranged from ~14 to 90% and ~14 to
71% for sonication-homogenization and homogenization, respectively. In
both processes, runs having the lowest levels of chitosan (0.5% w/v) were
desirable resulting in particles with spherical morphology, high
encapsulation efficiency and low cohesive behavior. The ionic gelation
technique was successful to stabilize ascorbic acid and hence, it enhanced
the shelf-life of these cosmetic products (21 days) with respect to the free
AA (3.1 days).
INTRODUCTION
L-Ascorbic acid (AA) is a water soluble vitamin found naturally in fruits
and vegetables and has several biological and dermatological functions. This
vitamin promotes collagen biosynthesis, provides photoprotection, causes
melanin reduction, scavenges free radicals, and enhances the immune system.
These functions are mainly attributed to its antioxidant properties.
Antioxidants are compounds that can either inhibit or reduce the lipid
oxidation of fats, oils and fatty acids [1]. For this reason, AA is widely
employed as antioxidant to protect the sensory, nutritional and aesthetic
qualities of cosmetic products, foodstuffs and pharmaceutical preparations [2].
However, its high aqueous reactivity and environmental factors such as
moisture, heat, UV light, pH (i.e., neutral and basic pH), oxygen, transition
metal ions (i.e., iron and copper) and X-rays affect the stability of ascorbic
acid [3]. AA degradation can also occur by presence of enzymes such as
ascorbate oxidase and ascorbate peroxidase [4]. As a result, it decomposes into
biologically inactive compounds such as 2,3-diketo-L-gulonic acid, oxalic
acid, L-threonic acid, and L-lyxonic acid. The degradation of AA is also a
major cause of quality issues during processing and storage of cosmetic
products leading to heavy losses and color changes [5].
Currently, microencapsulation is the main strategy used to extent the
shelf-life of AA. It is defined as a technology of packaging solids, liquids, or
gaseous materials from micron to several millimeters in size [6]. In this case, a
Stabilization of Ascorbic Acid by Microencapsulation … 125
membrane surrounds small particles in order to protect and stabilize them.

However, the chemical nature of the wall predominantly affects the degree of
protection of the active core, the microparticle stability and the encapsulation
efficiency. Microcapsules could also reduce the hygroscopicity of hydrophilic
compounds when the capsule is composed by a hydrophobic carrier. Further,
microencapsulation protects core materials during manufacture, storage and
handling from heat, enzymatic and chemical degradation. It also facilitates the
transport of charged molecules across absorptive epithelial cells [7]. Further,
the wall material should eventually release the entrapped compound during use
and consumption [8]. These capsules have different shapes, depending on the
composition and methods used to prepare them. Further, microencapsulation
can also reduce off-flavors produced by certain vitamins such as B1, B2, B3,
Vitamin E and D and reduces reactivity among different active compounds [9].
It also allows for the manufacture of products which are more sensorial
complete and enables the enrichment of various cosmetic products with natural
antioxidants [10]. The utilization of more stable forms of AA is then a crucial
requirement for the development of a robust and stable cosmetic product
having adequate functional properties [11].
There are several microencapsulation methods that can be used to stabilize
sensitive functional compounds. For instance, coacervation which is a
colloidal phenomenon based on the partial immiscibility of two or more
isotropic liquids is one of the most widely used. Other techniques frequently
employed for AA microencapsulation are complex coacervation [12], spray-
drying [13], microfluidic technique [14], liposomes [15], and melt extrusion
[16]. Further, a preparation of a W/O/W emulsion followed by spray-drying
with acrylic compounds has been reported. The latter showed a controlled drug
delivery at different pHs and exhibited a very low permeability [17]. In
another study, AA was encapsulated by spray-drying using gum arabic and
rice starch as wall materials [18]. AA has also been microencapsulated with
polyacylglycerol monostearate by spray-drying and used for milk fortification
[19]. Further, concentrated pea protein has been used as a wall material to
encapsulate AA by spray-drying [20]. Another study employed melt
dispersion, solvent dispersion and spray-drying as techniques to encapsulate
AA [21]. Liposomes have been used as AA carriers produced and stored at
4°C at pH of 3 and 7 [22]. Further, a W/O microemulsion have also been used
to microencapsulate AA [23].
On the other hand, several microencapsulation polymeric materials have
been used to achieve a functional wall material. For instance, ß-cyclodextrin
has been used for the encapsulation of olive leaf extract [24]. Further, kafirin
has been employed for the encapsulation of catechin and sorghum condensed
tannins [25]. Currently, chitosan is one of the most widely used entrapping
materials. It exhibits an excellent biocompatibility, biodegradability,
mucoadhesivity, edibility, water solubility, nontoxicity, and absorption-
enhancing effects [26]. Chitosan is similar in structure to cellulose since it is
made of linear (1-4)-linked monosaccharides. However, unlike cellulose,
chitosan is composed of 2-amino-2-deoxy--D-glucan units combined with
glycosidic linkages. Thus, it is considered as a copolymer of glucosamine and
N-acetyl glucosamine. The primary amine groups render special properties
that make it an ideal material for cosmetic, pharmaceutical, and food
applications. The free amine groups provide a positive charge and react with
many negatively charged surfaces such as the cell membrane and mucus lining
(due to negatively charged sialic acid residues), and other anionic polymers.
Therefore, the adhesive properties of chitosan are due to molecular attractive
forces (i.e., hydrogen bonding) formed by electrostatic interactions between
positively charged amine groups of chitosan and negatively charged surfaces.
Chitosan has the ability to increase membrane permeability, and can be
degraded by lysozyme in serum. Thus, it is expected that chitosan form
positive charges at acidic pH making chitosan water soluble allowing the
polymer to interact with negatively charged materials forming a
polyelectrolyte complex with anions. Therefore, the chitosan solubility
depends on the distribution of free amino and N-acetyl groups. However,
chitosan can be considered as a weak base, insoluble in water and organic
solvents, but soluble in dilute aqueous acidic solutions being able to form gels.
Particle size, density, viscosity, degree of deacetylation, and molecular weight
are important parameters that affect the gelling properties of chitosan. [27].
Chitosan has been widely used in the pharmaceutical field as a tablet
disintegrant and for controlled drug delivery [28] due to its ability to control
the diffusion rate of the encapsulated compounds [29].
The cosmetic industry is currently emphasizing the use of natural rather
than synthetic materials. Therefore, cosmetic products should be developed
based on polysaccharides derived from natural sources. For this reason,
chitosan microcapsules represent a very efficient mean for topical application
of bioactive compounds. In this chapter, the production of chitosan
microcapsules by ionic gelation as a strategy to enhance the shelf-life of AA is
described and discussed.
PRINCIPLES OF THE IONIC GELATION TECHNIQUE

Ionic gelation, also known as “ion-induced gelation,” is a polyelectrolyte
complexation phenomenon which results in the formation of microparticles or
microcapsules. It is commonly performed in aqueous media and dilute
solutions of charged macromolecules such as gelatin, chitosan, gellan, pectin,
gums, alginates, carrageenan, polylysine, etc. In this context, the
macromolecule is dissolved under constant stirring in water with a
concentration below the gel point and then it is set to react with small ions of
opposite charge to form clusters of a polyelectrolyte complex. These clusters
can subsequently be stabilized using other oppositely charged polyelectrolytes
to achieve a gel phase [30].
Figure 1. Structure of (a) intermolecular and intramolecular hydrogen bonding pattern

of chitosan with a 75% with a 75% deacetylation degree (b) ionic interactions between
the chains with sodium lauryl sulfate.
This technique offers several advantages since it requires mild conditions,

renders a more regular surface morphology, narrow particle size distribution
and awards rigidity to the particulate system. Further, it does not require
harmful organic solvents and high shear forces. As a result, capsules produced
by this technique have the general capability to protect the encapsulated
bioactive molecules from external threats and thus their functionality is
prolonged. Further, reversible physical crosslinking by electrostatic interaction
instead of chemical crosslinking prevents the accumulation of undesirable
toxic reagents or organic solvents in the final product [31].
There are several reports of polyelectrolyte complexes formed by ionic
gelation that are used to incorporate several bioactive compounds. For
instance, polymethacrylic acid and the negatively charged cyclodextrin were
able to formed nanocapsules that entrapped insulin [32]. Gelatin crosslinked
with genipin can also form a complex with alginate and were successfully used
to encapsulate some Bifidus spp making them resist to degradation induced by
acid pH [33]. Further, β-lactamases have been encapsulated in a complex
formed by non-amidated or amidated pectin and calcium chloride [34].
Moreover, deacetylated gellan and calcium ions are also able to form capsules
to entrap azathioprine [35]. On the other hand, thermosensitive polymers such
as carrageenan have been used at temperature gradients from 50°C to 25°C
followed by stabilization with ions to induce gelation [36].
PRODUCTION OF CHITOSAN-SODIUM LAURYL

SULFATE MICROCAPSULES
The amine groups of chitosan are positively charged in acetic acid
solution and in consequence, are responsive to complexation with anionic ions
like sodium lauryl sulfate (LSS) inducing a pre-gel phase in diluted solutions.
In this kind of complexation, two solutions are generally prepared, the first one
containing the dissolved biopolymer and the other one containing LSS. The
two dispersions are mixed under strong agitation to induce droplets collision
and hence, the required gelation to form the microparticles. Furthermore, the
size and morphology of the microparticles obtained greatly depend on the
concentration, molecular weight, and high surface energy properties of
chitosan and they are also influenced by the size of the opposite charged LSS.
In order to avoid further variations, the deacetylation degree and molecular
weight of chitosan were maintained at 75% and ~550 kDa, respectively.
Since chitosan is a cationic polymer, the gelling properties are affected by

pH or ionic strength changes. As a result, a pH increase from acidic to basic
values causes deprotonation of the amine groups and as a consequence, a gel
shrinking due to the reduction of the intramolecular electric repulsions inside
the particles. In addition, variation of the ionic strength by increasing the
concentration of salt in the medium reduces the chitosan-anion interaction
which favors particles swelling and the formation of microgels [37].
It is assumed that chitosan oligomers assume a straight chain
conformation when suspended in acetic acid (Figure 1a), due to charge-to-
charge repulsions in the protonated amino groups (NH3+). On reaction with
LSS, chitosan oligomers lose their straight chain conformation and appear
looped probably with entanglements. Since LSS in aqueous media form an
alkaline solution, it contains free sulfate ions (SO4)=. These ions randomly
bind to the (NH3+) sites of chitosan upon immediate addition of LSS resulting
in a complex formation and loop conformation of the chains. This process of
chain conformation is random, but maintains a uniform surface morphology.
In the case of a complete interaction one participating NH3+ group is linked
directly with LSS and this could be denoted as OLSS···NCHI (Figure 1b). The
length of LSS is very suitable for a perpendicular configuration with respect to
the chitosan chains. The OLSS···NCHI distance could be closed to that of LSS
alone of ~23 Å. Even though the chitosan oligomers were initially oriented
parallel to each other, their final configuration is somehow disrupted. This
tendency induces a bias in the orientation of chitosan oligomers that is
expected to affect the CHI/LSS microparticle formation mechanism, especially
at high LSS concentrations. This effect became prominent since the length was
sufficient to permit a weak direct oligomer−oligomer interactions leading to
interruption of interchange hydrogen bonding [38]. Typically, chitosan forms
an intermolecular hydrogen bonding between the hydroxyl group of the C-3
and cyclic oxygen of the adjacent monomer. Further, the nitrogen atom of the
amino group could form a hydrogen bond with the hydroxyl group of C-6. In
addition, the hydrogen atom of C-6 could also form a bond with the carbonyl
group of the acetamide moiety. On the other hand, two possible intermolecular
interactions are possible for chitosan. The first one occurs between the
carbonyl of acetamide moiety and the hydrogen of the amino group; the
second bonding occurs between the oxygen of the acetamide moiety and the
hydrogen of hydroxyl moiety of C-6 (Figure 1a) [39, 40].
The initial aqueous media maintains the amino groups of chitosan as non-
ionized (NH2) and the chains assume a randomly coiled arrangement. These
non-ionized groups act as buffer sites and upon acid addition are transformed
into protonated amines (NH3+) awarding partial expansion of the polymer

chains and particle swelling. Further, in the process of formation of
microcapsules at acid pH several points of chain scission could be formed due
to mechanical stirring or gentle sonic shaking allowing for the formation of
different particle sizes. In addition, during the chitosan-LSS complex
formation a non-bridging oxygen atom binds to the central sulfur atom of LSS
interacting with the amino group of chitosan [39]. As a result, a complete
disruption of the initial intermolecular hydrogen bonding structure of chitosan
occurs.
Thus, particle formation is largely influenced by the amount of ionic
bonds formed with LSS which in turn, depends on the level of LSS employed.
In mildly acidic solutions, the amino groups of chitosan are protonated, thus
attributing a positive charge to these groups. At these mild acidic conditions
LSS anions will be partially neutralized, but in contact with chitosan in the
acid media it is more likely to provide electrons to the positively charged
amino groups [40].
The electrostatic interaction is particularly suited for the incorporation of
AA for two reasons. First, the microcapsule formation process is based on the
electrostatic interaction of oppositely charged compounds. Second, the
incorporation can be achieved in aqueous conditions and this type of ionic
interaction can form a strong aggregation and precipitation phenomena,
especially when high levels of chitosan are used (i.e., 1.5%), limiting the
applicability of these formulations due to the formation of cohesive films upon
drying [41].
The chitosan-anionic complex induced by ionic gelation can be carried out
by several processes such as sonication, homogenization or their combination
(Figure 2). For instance, in the sonication method an acidic dispersion of
chitosan is mixed with LSS at different rates and then submitted to sonication
for ~1h. On the other hand, AA can be added to a chitosan solution followed
by the addition of a LSS solution and homogenized at 7500 rpm for 5 minutes.
The experimental runs for the production of microparticles and the resulting
particle size are listed on Table 1.
Runs that included a high level of chitosan along with a high level of LSS
with no ascorbic acid load favored the formation of spherical microcapsules
(Figure 3). On the contrary, using either sonication or sonication-
homogenization at 7000 rpm for 5 minutes had virtually no effect on the
particle size except for runs obtained with large levels of chitosan and LSS
(Table 1).
Figure 2. Schematics showing the formation of microcapsules by ionic gelation using

sonication and homogenization.
Table 1. Experimental runs for the placebo microparticle production
Run Chitosan level Surfactant level Mean particle size (µm)±SD

(%) (%) Sonication Sonication-
Homogenization
1 0.5 0.5 0.99 ± 0.07 1.38 ± 0.09
2 0.5 1.0 1.55 ± 0.05 1.48 ± 0.05
3 0.5 1.5 1.17 ± 0.03 1.04 ± 0.03
4 1.0 0.5 0.78 ± 0.02 1.63 ± 0.07
5 1.0 1.0 1.28 ± 0.14 1.18 ± 0.07
6 1.0 1.5 1.60 ± 0.03 1.08 ± 0.04
7 1.5 0.5 2.44 ± 0.82 1.04 ± 0.08
8 1.5 1.0 1.66 ± 0.18 1.11 ± 0.04
9 1.5 1.5 1.29 ± 0.04 1.66 ± 0.11
Data correspond to the mean ± standard deviation of 650 particles.
Figure 3. Microphotographs of microparticles and ascorbic acid (AA) microcapsules

produced by ionic gelation using two stirring processes.
Figure 4. Ascorbic acid microcapsules produced by homogenization.
Table 2. Effect of processing on the encapsulation efficiency and particle

size of AA microcapsules
CHI: Ascorbi Encapsulation efficiency (%) Mean particle size (µm)

LSS c acid Homogenization Sonication- Homogeniza- Sonication-
ratio (mg) homogenization tion homogenization
(0.5:0.5) 20 71.3 76.9 1.8 0.9
(0.5:1.0) 20 65.4 63.5 2.1 1.2
(0.5:1.5) 20 14.2 46.9 1.3 0.9
(1.0:0.5) 20 39.9 63.2 1.1 0.7
(1.0:1.0) 20 15.6 74.8 1.0 1.2
(1.0:1.5) 20 55.9 88.7 0.8 1.5
(1.5:0.5) 20 32.5 52.2 0.8 0.8
(1.5:1.0) 20 28.7 59.2 1.1 2
(1.5:1.5) 20 15.2 14.5 1.2 1.4
The CHI:LSS molar ratio and the evolved interactions were crucial for the
formation of particles with a narrow particle size. It is assumed that all ionic
groups of LSS can interact with chitosan amino groups although not all amino
groups of chitosan can be counterbalanced by SO4= even at levels larger than
1%. It is clear that there is an optimal CHI level of less than 1% (w/v) where
microparticles with smaller sizes are produced. At these conditions, the
sonication-homogenization process produced particles with a slightly lower
size than sonication alone.
On the other hand, if only homogenization is employed, particles with a
slightly larger size are obtained. It appears that the optimal CHI:LSS w/w ratio
among these samples is 1:1.5, which rendered microcapsules with sizes of ~1.5
µm, and ~89% encapsulation efficiency, whereas for other CHI:LSS ratios, the
size of the microcapsules varied and the encapsulation efficiency was very
low. The increased efficiency of the microcapsule formation processes

occurred at a CHI levels lower than 1% (Table 2).
Further, the optimum ratio of 1:1.5 is rather fortuitous, since this is a w/w
ratio and not a monomer/LSS ratio. This ratio does not provide a direct insight
into the proportions between individual molecular units on an atomistic level.
In this study, the chitosan oligomer chain was composed of 75% deacetylated
(D-glucosamine) and 25% acetylated (N acetyl-D-glucosamine) monomers
which along with the polymer molecular weights are expected to affect the
interaction of chitosan monomers with LSS anions. In this scenario, it is
probable that the best microparticles formation required high levels of LSS.
Further, the presence of AA had a synergistic effect with LSS affecting the
resulting properties leading to the formation of more spherical microcapsules.
In general, the presence of AA at a high chitosan levels led to the formation of
continuous film due to a coalescence of large microcapsules. Conversely, low
chitosan levels rendered microcapsules with the largest size and large numbers
(Figure 4).
THERMAL STRESS STUDIES

Aqueous solutions and semisolid formulations containing free and
encapsulated AA were placed in a tray oven at 45ºC, at 1:1 and 1:2
AA:excipient ratios. The aqueous solution and the semisolid product
containing free AA had a self-life of 0.9 and 3.1 days as compared to 3.3 and
21.1 days for the encapsulated aqueous and semisolid products, respectively
(Figure 5). It is evident that all degradation patterns followed an order 1
scheme having an r2 larger than 0.9900. In addition, degradation rates were
larger for all formulations containing the free form of AA. Once the shelf-life
of AA was reached cosmetic products acquired a brownish coloration due to
the formation of degradation compounds such as 2,3-diketo-L-gulonic acid,
oxalic acid, L-threonic acid, and L-lyxonic acid.
Figure 5. Thermal stress curves of AA samples stored at 45ºC for two months. AQ_F:
Aqueous dispersion with free AA; AQ_E: Aqueous dispersion with encapsulated AA;
CR_F: Semisolid product with free AA; CR_E: Semisolid product with encapsulated
AA, AD aqueous dispersion, SP: semisolid product, Enc: Encapsulated, k: degradation
constant, SL: shelf-life.
CONCLUSION
The ionic gelation process between chitosan as a polycation in acidic pH
and sodium lauryl sulfate as the anionic partner led to the formation of
encapsulated AA systems which ranged from 0.7 to 2.1 µm in size. Depending
on the processing conditions, particles with different properties were obtained
including differences in size, encapsulation efficiency, and stability. Therefore,
the spherical shaping, encapsulation efficiency and particle size were greatly
influenced by the chitosan:LSS ratio, and processing conditions employed.
Further, low chitosan levels rendered the most spherical, uniform and
abundant particles.
Microencapsulation by ionic gelation increased the AA stability on
aqueous solution and cosmetic formulations and thus, it represents a promising
alternative to overcome problems related to its instability. In the semisolid
formulation, microencapulation protected AA from adverse environmental
conditions, such as light, moisture, oxygen, and interactions with other
compounds in a formulation, and maintained its functionality on extended

period of time. These micro-sized complex systems are of great interest as
drug stabilizing systems.
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Chapter 7
ALKYL ESTERS OF L-ASCORBIC ACID:

FROM SYNTHESIS TO APPLICATIONS
Maria Laura Fanani1,, Raquel Viviana Vico2

and Luciano Benedini3
1
Centro de Investigaciones en Química Biológica de Córdoba, (CIQUIBIC-
CONICET), Departamento de Química Biológica,
Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba, Córdoba, Argentina
2
Instituto de Investigaciones en Fisicoquímica de Córdoba (INFIQC-
CONICET), Departamento de Química Orgánica,
Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba, Córdoba, Argentina
3
Instituto de Química del Sur (INQUISUR-CONICET),
Departamento de Química, Universidad Nacional del Sur,
Bahía Blanca, Argentina
ABSTRACT
Antioxidants are important additives in food, cosmetics and
pharmaceutics, and vitamin C is one of the best-positioned for industrial
applications. The usage of native L-ascorbic acid is limited to water-
containing media because it is hydrophilic. To overcome this, an

E-mail: lfanani@fcq.unc.edu.ar.
142 Maria Laura Fanani, Raquel Viviana Vico and Luciano Benedini
extensive effort has been made to develop new amphiphilic derivatives

capable of being applied in lipophilic environments. The alkyl derivatives
obtained through esterification of the primary hydroxyl group of L-
ascorbic acid with carboxylic acids are the most widely studied
compounds from the synthetic, physicochemical and pharmaceutical
point of view, as well as being those most used in commercial
formulations.
This chapter offers a comprehensive review of the bibliography
regarding the main pathways to synthesize these alkyl derivatives,
involving enzyme catalysis and esterification in acidic or alkaline media
to obtain the alkanoyl-6-O-ascorbic acid esters (ASCn). This information
is presented together with their stability properties.
Besides its antioxidant properties, the ASCn family has been used in
different pharmacological preparations for its amphiphilic nature. The
presence of the acyl chain provides the capacity to self-organize into
micelles and coagels when suspended in water. Knowledge of the phase
behavior of surfactants in aqueous media is important for understanding
the properties of these systems and is vital for numerous industrial
applications. These, along with a review of surface properties of the
ASCn family, are dealt with in this chapter.
ASCn amphiphiles, when self-organized at the air-water interface,
form Gibbs and Langmuir monolayers with a large variety of stability,
phase states and surface textures, depending on the length of the acyl
chain moiety and ionization conditions. Furthermore, different members
of the ASCn family have differences in their effectiveness for particular
pharmaceutical purposes. Thus, the physicochemical properties of each
member of this drug family may be determinant for their efficacy in
pharmaceutical processes, in particular those related to surface
phenomena and interaction with biomembranes.
Cell membranes are the first contact of lipophilic drugs with cells
and often form a reservoir. Drug/membrane interaction is thus a
fundamental condition for their pharmacological function and a potential
regulatory point. We review here some attempts to experimentally
explore the interaction and the general physicochemical effect of ASCn
family members when they are incorporated into model lipid membranes
under controlled conditions.
Finally, we review some approaches to the use of ASCn in
biomedicine. Those are based mainly on I) their antioxidant capacity,
because the presence of ASCn derivatives in formulations may prevent
the degradation of drugs sensitive to light exposure, oxidant or reactive
oxygen compounds, and/or II) the capacity of ASCn to form micelles,
coagels and liquid crystals, which can modify some properties of the
medium and act as drug delivery nanostructures. The new strategies in the
use of ASCn comprise examples from liposome-based encapsulated
Alkyl Esters of L-Ascorbic Acid 143
nanoparticles to novel adjuvant activity of ASCn liquid crystals with high

immunological efficiency.
1. INTRODUCTION
Vitamin C (ascorbic acid) is one of the most commonly used antioxidants
as an additive in food, cosmetics and pharmaceutics (Bauernfeind, 1982;
Rowe, Sheskey, and Quinn 2009). This is mainly due to vitamin C’s natural
origin, which avoids undesired effects and the generation of toxic products
caused by degradation (Bauernfeind, 1982; Linster and Van Schaftingen
2007). In contrast, toxicological research shows that other very effective
antioxidants derived from petrochemicals (such as butylated hydroxytoluene
(BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), tertiary butyl
hydroquinone (TBHQ) and ethoxyquin) induce tumor growth and are
carcinogenic (Karmee, 2011; Lü et al., 2010; Pokorny, Yanishlieva, and
Gordon, 2001).
In this scenario, the employment of vitamin C and its derivatives is
gaining attention. Native L-ascorbic acid is limited to water-containing media
because it is hydrophilic. Extensive efforts have therefore been made in recent
years to develop new amphiphilic derivatives capable of being applied in
lipophilic media (Austria, Semenzato and Bettero, 1997; Rowe et al., 2009;
Spiclin, Gasperlin, and Kmetec, 2001).
A)
B)
Figure 1. Scheme of the synthesis of alkanoyl-6-O-ascorbic acid derivatives. A)

Details of the reaction performed in concentrated sulfuric acid; B) Hydroxyallyl
structure involved in the formation of monoesters of Vitamin C in concentrated
sulfuric acid (Cousins et al., 1977).
A comprehensive review of the bibliography shows that 6-O-alkyl

ascorbic acid esters (ASCn, see Figure 1A), obtained through esterification of
the primary hydroxyl group of L-ascorbic acid with a carboxylic acid, are
those most widely studied from the synthetic, physicochemical and
pharmaceutical point of view, as well as being the most used in commercial
formulations (Karmee, 2011; Pokorny et al., 2001). They are widely applied
because they are highly active surfactants (Benedini, Fanani, et al., 2011;
Mottola et al., 2015; Zulueta Diaz et al., 2016) and to a great extent conserve
their antioxidant properties (Lo Nostro et al., 2000; Palma et al., 2003b).
Among the ASCn family, ascorbyl palmitate (ASC16) also called L-ascorbic
acid 6-palmitate, E304, 3-oxo-L-gulofuranolactone 6-palmitate, or vitamin C
palmitate, is the most widely used (Rowe et al., 2009).
Other amphiphilic forms of vitamin C have also been synthesized through
esterification of L-ascorbic acid with triacylglycerols, L-methyl lactate,
phenylbutyric acid, Bixin or Norbixin (Karmee, 2011), but these compounds
have not yet found the extensive industrial applications reached by the ASCn
esters.
Before discussing the synthetic methods available for obtaining ASCn
esters, it is worth mentioning some issues regarding nomenclature. The
IUPAC name of L-ascorbic acid is (R)-5-((S)-1,2-dihydroxyethyl)-3,4-
dihydroxyfuran-2(5H)-one. However, for simplicity, the common names
employed for ASCn derivatives do not follow the numbering suggested by
IUPAC. In the common nomenclature, position 1 is assigned to the carbonyl
group present in ascorbic acid moiety and the primary hydroxyl group is
assigned position 6, as shown in Figure 1A. We will use here the numbering
adopted by common nomenclature.
To preserve its highly antioxidant activity, any vitamin C derivative needs
to conserve the cyclic moiety, especially at the positions indicated as 2 and 3
in Figure 1A (Palma et al., 2003b). A number of attractive derivatives of L-
ascorbic acid have been reported in the literature (Kote et al., 2013; Thopate,
Dengale, and Kulkarni, 2013; Wittine et al., 2004) but, as many of them lack
the active moiety, they have probably lost or diminished their antioxidant
potential. However, Thopate et al., reported novel 2,3-di-O-alkyl derivatives of
L-ascorbic acid which present pro-oxidant or oxidant activity, and some of
them were found to have enhanced antiproliferative activity against tumor cell
lines compared to L-ascorbic acid (Kote et al., 2013).
2. SYNTHESIS OF ALKYL ESTERS OF L-ASCORBIC ACID

(ASCN)
There are two main pathways to obtain the valuable ASCn derivatives by
esterification of the primary hydroxyl group of L-ascorbic acid with a
carboxylic acid. These involve synthesis by traditional chemical esterification
in acidic media or by enzyme catalysis. Both pathways have advantages and
disadvantages related to the reaction yield, the handling of harsh chemicals
and the overall cost associated with the process. Traditional chemical methods
that employ harsh reaction conditions, such as strong acidic media, in general
give high product yield (over 80% according to the literature) with some
decomposition products or uncreated starting materials. Probably the most
difficult point in the synthesis by traditional esterification, as well as during
purification, is the handling of harsh reactives such as concentrated acids. On
the other hand, enzymatic synthesis is performed in mild conditions but the
overall reaction yields are lower compared to the traditional chemical
methods, and there are some differences in yield depending on the enzyme
source and the carboxylic acid bonded to the L-ascorbic acid moiety.
2.1. Synthesis of ASCn by Traditional Chemical Methods
One of the first reports on ASCn synthesis was performed by Wells et al.,
in 1943 (Swern et al., 1943). In this pioneering work, they tried several media
for the synthesis of fatty acid monoesters of L-ascorbic acids and D-
isoascorbic acids. The media tested involved acids such as sulfuric or
hydrochloric acid and basic media containing pyridine or pyridine/chloroform
mixture. Of all of them, concentrated sulfuric acid (95 per cent) was the media
that gave the best yield. After this pioneering work, many other authors
continued using this method, with some variations mainly in the purification
process.
The reaction that takes place is an esterification between the primary
hydroxyl group present in position 6 of L-ascorbic acid and the carboxylic acid
group present in the fatty acid, as shown in Figure 1A. This is a reversible
reaction and concentrated sulfuric acid acts as solvent and catalyst. The overall
yield of the reaction can be improved by displacing the equilibrium to the
products side by using an excess of fatty acid (Cousins et al., 1977), by
removing H2O through distillation (Capuzzi et al., 1996) or by using molecular

sieves (Mottola et al., 2015).
Lillard et al., presented a detailed study of the reaction mechanism for this
selective monoesterification. They attributed the selective esterification in
position 6 of L-ascorbic acid to the formation of a hydroxyallyl cation (Figure
1B) and to the favored sulfonation of the primary hydroxyl group, which is
more reactive than the secondary group. The selective acylation of the
hydroxyl in position 6 can also be explained by considering that hydroxyls in
position 2 and 3 are involved in, or are in the proximity of, the hydroxyallyl
structure.
2.2. Enzymatic Synthesis
The use of enzymes for industrial catalytic processes is a promising

alternative to conventional industrial chemistry, making processes for
producing large amounts of chemicals sustainable. Enzymes are specific,
selective and, in general, display good activity under mild experimental
conditions. Among the most used enzymes, lipases have gained a clear
predominance. In contrast to most enzymes, lipases exhibit a wide specificity,
recognizing very different substrates (Fernandez-Lafuente, 2010). Lipases are
the main enzymes used in the synthesis of ASCn.
The lipase-mediated reaction is regioselective and, during enzyme
acylation, the primary hydroxyl group of L-ascorbic acid is esterified to an
acyl donor. The nature of the acyl donor accepted by lipases varies, including
fatty acids, fatty methyl esters, fatty acid vynil esters, and triacylglycerols
(Karmee, 2011), depending on the lipase source. The most frequently used
lipases for ASCn synthesis are from Candida antartica (Domínguez de María
et al., 2005; Secundo et al., 2001) and Thermomyces lanuginosus (Fernandez-
Lafuente, 2010), either as soluble lipase preparation or in an immobilized
form. The lipases from the microorganisms mentioned above are commercially
available. Additionally, ASCn was reported to be synthesized with a good
yield, using lipase from Staphylococcus xylosus immobilized onto silica
aerogel (Kharrat et al., 2014).
The effectiveness of the enzyme-catalyzed reaction is highly dependent on
the acyl donor and is considerably modified by the reaction media (type and
percentage of cosolvent, reagent molar ratios, water content, etc.). These
features have been systematically studied by several authors (Adamczak,
Bornscheuer, and Bednarski, 2005; Adamczak and Bornscheuer, 2009; Reyes-

Duarte et al., 2010; Zhao et al., 2011).
The major disadvantage of enzyme-catalyzed ASCn synthesis is the low
yield observed for some acyl donors and the large excess needed of one of the
reagents to be used. Nevertheless, these problems can be overcome if the
enzyme and reagents can be recycled or if the reaction condition can be
properly adjusted for a particular ASCn derivative.
3. STABILITY OF ASCN IN SOLID STATE AND SOLUTION

It is well known that L-ascorbic acid is stable in solid state but is
susceptible to oxidation in water solution or water-containing media, making
its use to some extent impractical because it undergoes oxidation under
aerobic conditions and under light exposure (Austria et al., 1997). A stability
study of solutions of L-ascorbic acid in DMSO-d6, performed with 1H NMR in
function of time and temperature, also showed that in this solution, numerous
olefinic and aromatic signals (furan) appear over time, which must have
resulted either from decomposition or tautomerization (Dabbagh and Azami,
2014).
Some studies have been performed of the stability of lipophilic ASCn
derivatives, especially for the widely used ascorbyl palmitate (ASC16). The
stability of ASC16 was evaluated in topical microemulsions (Austria et al.,
1997; Spiclin et al., 2001) and in solutions of methanol (MeOH),
dimethylsulfoxide (DMSO) and chloroform (CCl3H) (Austria et al., 1997;
Mottola et al., 2015). Also, the stability of different ASCn derivatives, in solid
state and in solution (DMSO and CCl3H), have been studied using FT-IR and
NMR spectroscopy. The derivatives studied included palmitoyl ascorbate
(ASC16), miristoyl ascorbate (ASC14) and lauryl ascorbate (ASC12) (Mottola
et al., 2015).
Austria et al., reported that ASC16 was more stable to oxidation than
native L-ascorbic acid. There were important differences between the ascorbyl
ester and the parent compound, L-ascorbic acid, but esterification with
palmitic acid in position 6 did not totally avoid the hydrolysis of the molecule,
either in MeOH solution or in microemulsions (Austria et al., 1997). They also
reported that only media with high viscoelastic properties were able to reduce
the typical hydrolysis of this compound. Solutions of ASC16 in MeOH (1%
w/v), stored both at room temperature and at 42°C in the dark for 60 days (a
test that simulates accelerated aging during about 12 months’ storage at room
temperature), proved to be more stable than those of L-ascorbic acid, but still
ASC16 had a significant concentration loss after 60 days of storage (recovery
77% at room temperature, 47% at 42°C, measured by HPLC). In contrast,
ascorbic acid underwent high concentration losses (37% recovery at room
temperature and none after 2 months at 42°C), confirming its low stability
(Austria et al., 1997).
The same trend was observed when ASC16 was employed in suitable gel-
like emulsions with high viscoelastic properties (Austria et al., 1997). Kmetec
et al., reported that, after a period of 28 days, the remaining ASC16 in o/w
media and w/o microemulsions, varied from 0% to 40%, depending on the
initial concentration of ASC16. The amount of ASC16 significantly influences
the degradation of the compound, with higher concentrations of ascorbyl
palmitate, as a rule, reducing the extent of its degradation (Spiclin et al., 2001).
30 days
% Transmittance
0 day
3800 3400 3000 2600 2200 1800 1400 1000 600
Wavelength (cm-1)
Figure 2. FT-IR analysis of solid of ASC14 (KBr) at 0 day (solid) and 30 days
(dashed).
We also studied the stability of several members of the ASCn family

including ASC16, ASC14 and ASC12, both in solid state and in solution
(Mottola et al., 2015). In solid state, the stability analysis was performed by
FT-IR spectroscopy. The spectra acquired during a period of 30 days under
ambient conditions showed the characteristic absorption bands of the
compounds which were unmodified with time (Mottola et al., 2015). Similar
behavior regarding solid state stability was previously reported for L-ascorbic
acid (Dabbagh and Azami, 2014). Some representative FT-IR spectra of ASC
14 are shown in Figure 2.
The stability of ASC16, ASC14 and ASC12 in solution was studied
through NMR spectroscopy (Mottola et al., 2015). This study was performed
either with DMSO-d6, a polar aprotic solvent, or with CCl3D, and the results
were compared to those reported by Azami et al., for native L-ascorbic acid
(Dabbagh and Azami, 2014).
The NMR study performed in CCl3D was carried out for ASC16 and
evaluated through 1H, 13C, and HSQC-DEPT over a 30-day period. The
solution (40 mM) was kept at 4ºC and at ambient atmosphere. From the NMR
spectra, it is clearly evident that no change occurs in ASC16 structure for this
period of time, as can be seen in Figures 3 and 4.
Time=30 days
(D2 O added)
Time=4 days
(D2 O added)
D G L
Time=4 days K
J
E F C
Time=0
CCl3H TMS
DMSO
Figure 3. 1H NMR stability study of ascorbyl palmitate (ASC16). The spectra were
measured after sample preparation (time = 0) and at several different times. At 4 days,
deuterium oxide (D2O) was added to identify hydroxyl groups. The integral values of
the signals (not shown) are as expected for the proposed structure. Solvent: CCl3D,
400.13 MHz (some drops of DMSO-d6 were added to enhance solubility), ASC16 =
40 mM. Extracted from Mottola et al., 2015.
The stability study performed in DMSO-d6, a solvent that can be used for
biomedical purposes (Chen and Allen, 2009), was carried out for ASC16,
ASC14, and ASC12. The solutions were kept under ambient conditions and
stored at 4°C. All ASCn behave similarly within the period of time studied (7
days), without the appearance of olefinic or aromatic signals, as occurs with
unmodified vitamin C (Dabbagh and Azami, 2014).
Interestingly, the NMR study in DMSO-d6 allowed us to show the
presence of small amounts of ASCn tautomer/s, the quantity of which varied
with time and depended on the alkyl chain length. The shorter the alkyl chain,
the larger the proportion of tautomer observed. Although no attempts were
made to identify if one or more tautomers were present, it seems plausible that
a greater contribution was given by structure ii shown in Figure 5, but we
cannot discard the presence of compounds iii, iv or their mixture, as proposed
by Berger for vitamin C (Berger et al., 1977).
8 19
17
2 20 15
13 11
3 7 16
56 18 9
4 14 22
12 10
1 21
Time=30 days CCl3H

(D2 O added)
DMSO
Time=4 days
Time= 0 10-19
5 6 7 8 9 20-21
4
12 3 22
Figure 4. 13C NMR stability study of ascorbyl palmitate (ASC16). The spectra were
measured after sample preparation (time = 0) and at several different times. At 4 days,
deuterium oxide (D2O) was added to identify hydroxyl groups. The integral values of
the signals (not shown) are as expected for the proposed structure. Solvent: CCl3D,
400.13 MHz (some drops of DMSO-d6 were added to enhance solubility), [ASC16] =
40 mM. Extracted from Mottola et al., 2015.
Figure 5. ASCn (i) and possible tautomeric forms of ASCn derivatives (ii, iii, iv).
Extracted from Mottola et al., 2015.
NMR spectra of ASCn (5 mM, DMSO-d6) were acquired immediately

after solution preparation and after 7 days. The spectra displayed in Figure 6
for ASC16 and Figure 7 for ASC14, acquired at time zero, show the hydroxyl
groups of the ascorbic acid ring (named A–C) and the proton named D. After a
week, new signals were observed (named a, b, d in Figures 6 and 7), which
can be attributed to a tautomeric form. Upon the addition of deuterium dioxide
(D2O), the signal corresponding to -OH groups disappeared, as expected. The
same feature was present for ASC12 (Mottola et al., 2015).
In order to confirm the presence of the tautomer/s HSQC–DEPT,
experiments with a 50 mM solution of ASC16 were performed at time zero
and after 8 days. These experiments allowed us to confirm the presence of
these tautomeric species, as is shown in Figure 8.
The amount of tautomer/s in ASCn was roughly estimated by integrating
1H NMR signals corresponding to protons D and d (see structures in Figures 6
or 7). The NMR results show that the quantity of tautomer/s increased with
time, and depended on the alkyl chain length, as summarized in Table 1.
8 19
I)- 2
17
15
20 13
3 7 16 11
56 18 9
4 14 22
12 10
1 21
10-19
Time=0
21
5 9 22
12 3 4 6 7 8 20
II)-
H2 O K
DMSO
L
D G
E
J
A B C F
Time=0
III)-
Time=7 days
(D2 O added)
ab
d
Time=7 days D
D d
A B C
Time=0
Figure 6. (I) 13C NMR and (II) 1H NMR spectra of ascorbyl palmitate (ASC16)
acquired after sample preparation (time=0). (III) 1H NMR spectra of ASC16 acquired
at time=0 and at 7 days. At 7 days, the solution was doped with deuterium oxide to
identify hydroxyl groups. Solvent: DMSO-d6, 400.13 MHz, [ASC16] = 5 mM. The
inset shows signals D and d as doublets with their corresponding integrals.
The fact that the tautomeric equilibrium in ASCn derivatives was reached
slowly may be attributed to the formation of aggregates in the organic solvent,
as was reported before for other amphiphiles (Fracaroli, Granados, and Rossi,
2009; Silva et al., 2014). The presence of this tautomeric equilibrium, and
probably the establishment of a hydrogen bonding network between the

ascorbic acid moieties in the self-organized systems, may account for the
decrease in antioxidant activity generally observed for ASCn (Lo Nostro et al.,
2000; Palma et al., 2003b). It has been shown that ASC10 micelles retain
about half of the antioxidant performance for oxygen uptake (Palma et al.,
2003a), while ASC12 and ASC14 retain only 40 and 34% of the antioxidant
activity (Kharrat et al., 2014; Lo Nostro et al., 2000) compared to unmodified
L-ascorbic acid. These reversible molecular features may also explain changes
in phase states, phase transitions and topography observed in the monolayer
experiments (Mottola et al., 2015).
8
17
15
2 13
18 11
3 7 16 9
56 14 20
4 12
10 19
1
10-17
Time= 0
9
19
5 18
1 3 4 67 8 20
2
G K
II)- B III)-
A
E J L
a, b F
C D
Time= 7 days
d
1
ab d ~ 0.10
Time= 2 days
DMSO
1
~ 0.06
Time= 0 G K
D E
A B J
C F H2 O 1
L > 0.01
Figure 7. (I) 13C NMR spectrum of ascorbyl myristate (ASC14) acquired after sample
preparation (time=0). (III 1H NMR spectra of ASC16 acquired at time = 0, 2 and 7
days. (III) The inset shows signals D and d as doublets with their corresponding
integrals. Solvent: DMSO-d6, 400.13 MHz, [ASC14] = 5 mM.
Table 1. Amount of tautomer/s present in DMSO-d6 solutions of ASCn
% Tautomer/sa
0 day 7 days
ASC16 0 2
ASC14 0 10
ASC12 5 10
aThe amount of tautomer/s was estimated by integrating the signal corresponding to proton
d relative to proton D, the signal of which was assigned a value of 1 (see Figures 6 -
7).
I) K L II)
D G
E D
J
F
C
d
22
21
10-19 20
9
8
7
6
5
Figure 8. HSQC-DEPT of ASC16 acquired after solution preparation and at 8 days.

Solvent: DMSO-d6, 400.13 MHz. [ASC16] = 50 mM.
4. PHYSICOCHEMICAL PROPERTIES OF BULK

SUSPENSION OF ASCN
Besides its widely proclaimed antioxidant properties, the ASCn family has
been used in different pharmacological preparations for its amphiphilic nature.
The presence of the acyl chain, besides improving its solubility in alcohol and
non-polar solvents, provides the capacity to self-organize into micelles and
coagels when suspended in water (Palma et al., 2007).
Lyotropic liquid crystalline systems have complex phase equilibrium due
to the rich diversity of mesophases formed. Thus, being acutely aware of the
properties, structure and dynamics of water in the polar layer of aqueous
surfactant microstructures is a key issue for understanding both the physical

chemistry of surfactant aggregation and the interactions occurring therein.
Palma et al., (Palma, Lo Nostro, et al., 2002; Palma, Manzo, et al., 2002)
detected a rich variety of supramolecular assemblies formed by 6-O-alkyl
ascorbic acid derivatives in water: ASCn produce stable coagels at low
temperature in 10% water dispersions. Those lamellar liquid crystalline
structures are formed when the ASCn molecules arrange themselves in layers,
yielding a lamellar phase with alternating polar and non-polar layers, and form
micrometric size aggregates at high water content (Mottola et al., 2013).
Coagels are characterized by closely packed lamellar structures, with strongly
bound water between their layers. Gel phases possess a lower degree of
crystallinity and higher water content than coagels. Upon heating, these
coagels form either micellar phases or gels, depending on the length of the
hydrocarbon chain. The shorter chained ASCn (ASC8 and ASC10) exhibit a
coagel-to-micelle phase transition. On the other hand, the long-chained
surfactants (ASC11, ASC12, ASC14, and ASC16) form either coagels or gel
phases, depending on temperature (Palma, Manzo, et al., 2002).
The phase behavior of aqueous surfactants allows access to the
physicochemical properties of the system, which is important for the
understanding and handling of the numerous industrial applications of
surfactants. The phase diagram of the ASC16-water system was determined by
Benedini et al., (Benedini, Schulz, et al., 2011) and a new representation of
this system is shown in Figure 9. Two different lamellar mesophases are
displayed, one in which there is bulk water (Figure 9B) and the other only
hydration water (Figure 9E) (in the first and second hydration layer). When
there is no bulk water, the formation of the high concentration lamellar liquid
crystal is preceded by a transition from hydrated crystals to cubic liquid
crystal. At very high concentration, waxy crystals form with melted
hydrocarbon bilayers, which retain their crystalline structure (because the
polar bilayers are still rigid). This results in a cubic mesophase (Figure 9F),
leading to lamellar liquid crystals on heating. The water in the system is in
three different states: bulk, surface-related and hydration, instead of the two
reported in literature. The different kinds of water in the system affect the
nature of the mesophases formed when the system is heated.
ASCn possess three free–OH groups in positions 2 (pKa = 11.6), 3 (pKa =
4.2), and 5 (secondary hydroxyl groups) and it has been proposed that they
behave as anionic surfactants, but experimental data indicates that they behave
as non-ionic surfactants (Benedini, Schulz et al., 2011; Palma, Lo Nostro et al.,
2002). This effect may be due to a double-layer induced neutralization of the
ASCn molecules when self-organized at low ionic strength (Benedini, Fanani

et al., 2011).
Figure 9. Phase Diagram of ASC16-water system. Micropictures showing the formed

phases. A) Texture of the gel phase after heating and cooling (0.45 w/w fraction), B)
Low concentration lamellar liquid crystal showing low birefringent smooth oily streaks
(0.36 w/w fraction at 65ºC), C) Negative units (bottom right) of lamellar liquid crystal
and also cubic liquid crystal (dark zones) phases and growing gel domains showing
large raw textures similar to positive units (0.75 w/w fraction at 70ºC), D) Lamellar
liquid crystal (mosaic) and gel (right) growing from the mesophase (heating), E) High
concentration lamellar liquid crystal showing birefringent smooth oily streaks and
spherulites (0.95 w/w fraction at 102ºC), F) Cubic liquid crystals appear between TPR
(two phase region) (0.80 w/w fraction at 93ºC).
Either the coagel formed by long-chain ASCn at low water content or the
micrometer-size lamellar (smectic) structure occurring when diluted to
millimolar concentration (Mottola et al., 2013) involves the formation of
amphiphile-water interface planes with a negative net charge, given by the
acidic properties of ASCn. The latter effect leads to the establishment of a
double-layer of counterions (including H+), which are attracted to the surface
(Myers, 1999). In this way, the surface pH becomes lower than the bulk pH,
which may drop up to two pH units depending on ionic strength, thus lowering
the ionization of ASCn (Benedini, Fanani, et al., 2011; Mottola et al., 2013).
An increase in pH overcomes this effect and the surface charge is maintained,
thus inducing desegregation of the large lamellar structures ( 1 µm diameter)
into micelles (20-50 nm size) (Mottola et al., 2013).
5. SURFACE PROPERTIES OF ASCN

When a concentrated solution of ASCn is dissolved in the bulk of an
aqueous solution, the adsorption of these amphiphilic molecules to the
air/water interface resulted in a Gibbs monolayer, evidenced by a decrease in
the surface tension (or an increase of surface pressure) (Davies and Rideal,
1963). The surface pressure reached at equilibrium increases with the
subphase amphiphilic concentration when it falls below a critical monomer
concentration (called equilibrium monomer concentration, EMC). Above this
EMC, usually in the µM range (Mottola et al., 2015), the ASCn molecules
form supramolecular structures such as micelles or lamellas, as commented in
the previous section, in equilibrium with the monomeric form and the
monolayer film. In this concentration range, the system shows a concentration-
independent equilibrium surface pressure. Thus, Gibbs monolayers reflect the
equilibrium between the bulk form of the amphiphile and the organized film at
the interface (Davies and Rideal, 1963; Myers, 1999).
ASCn containing an acyl chain of 12, 14 and 16 C atoms (but not shorter)
induce a significant increase in surface pressure to the 34–40 mN/m range
when assembled as Gibbs monolayers, meaning that they can lower the surface
tension of water from 72 to 32–37 mN/m (Mottola et al., 2015). This finding
confirms that long-chain ASCn are amphiphiles with potent surface activity.
On the other hand, the mean area occupied by ASCn molecules in the Gibbs
monolayer is in the 20–22 Å2.molecule-1 range (Mottola et al., 2015; Palma,
Manzo, et al., 2002). Considering that the cross section of a single acyl chain
occupies 18 Å2.molecule-1 (Gaines, 1968), these values suggest a close
packing of ASCn molecules in Gibbs monolayers.
When a chloroform solution of long-chain ASCn (  12 C) is spread onto
the air/aqueous solution interface, a Langmuir monolayer is established
(Benedini, Fanani et al., 2011; Capuzzi et al., 1996; Mottola et al., 2015). Even
when the monolayer, as spread, does not represent a state of absolutely stable
equilibrium, meaningful experiments can be performed on it (Gaines, 1968).
ASCn Langmuir monolayers can be classified according to their elastic

response upon dilatational stress, in the same way as lipid films.
For liquid-expanded (LE) monolayers, a decrease in the area available for
the amphiphile molecules induces a relatively low increase in surface pressure,
resulting in values of compressibility modulus typically  100 mN/m (Davies
and Rideal, 1963). For liquid-condensed (LC) and solid (S) monolayers, a
larger increase in surface pressure is induced by a small reduction of the
monolayer area, leading to compressibility modulus values several folds
higher than for LE monolayers.
ASC14 forms stable films that behave as LE at neutral pH and show a S
phase at acidic pH and high surface pressures (Mottola et al., 2015). On the
other hand, ASC16 monolayers show LE, LC or S films depending on surface
pressure and subphase pH (Benedini, Fanani et al., 2011; Mottola et al., 2015).
Additionally, ASC18 monolayers were also studied showing condensed (LC or
S) phases both at neutral and acidic subphase pH (Capuzzi et al., 1996). Phase
transitions between those phase states are usually visualized as near-plateau
regions in the isothermal compression curve of the films, as can be seen in the
isothermal compression curve of ASC16 and ASC14 monolayers (Figure
10A).
Both LC and S phases show low compressibility (high compressibility
modulus values) (Davies and Rideal, 1963) and a restricted lateral diffusion
(Zulueta Diaz et al., 2016). S phases are characterized by a regular internal
structure and high shear modulus that restrict the relaxation of the domain
borders and are observed in the micron-scale as sharp-edged structures, as has
been reported by Brewster angle microscopy (BAM). This is a powerful
technique that allows visualization of nanometer thick films placed at the
air/water interface. It requires no probe for such visualization as it can
differentiate monolayer phases by their thickness and refractive index
(Lheveder and Meunier, 2000). It is also sensitive to in-plane anisotropy, such
as that observed for ASC14 and ASC16 films at acidic pH (Figure 10). At this
subphase pH, the ASCn molecules remain in a near-neutral condition and the
S phase appears as growing with preferential orientations relative to the crystal
seed by BAM (Figure 10C, D).
On the other hand, LC phases are liquid phases with viscoelastic character
upon shear (Espinosa et al., 2011). Thus, when there is coexistence with a
more fluid phase (such as LE phases). the LC phases form rounded or flower-
like domains in order to balance intradomain repulsion / line tension forces
(McConnell and Vrljic, 2003). At neutral pH, ASC16 monolayers have a
significant amount of acidic dissociation (30–40% of the molecules are
negatively charged) which prevents the formation of very compact crystalline

phases. Thus, a LC phase is formed, which shows rounded border domains in
the LE/LC coexistence region as visualized by BAM (Benedini, Fanani, et al.,
2011), and is shown below in Figure 12F.
Image computer
60
A B analysis
Surface Pressure (mN/m)
Laser beam
Lasser beam
at at
the Brewster
Brewster angle
angle 2020x x
50 Objective
Objetive
Micro-
40 balance
Compression
30
20
10
0
0 10 20 30 40 50 60 70
2
Mean molecular
Mean molecuar area
area (Å /molecule)
1 mN/m 2 mN/m 15 mN/m 23 mN/m 40 mN/m
15 mN/m 21 mN/m 26 mN/m 31 mN/m 37 mN/m
Figure 10. Langmuir monolayers of ASCn. A) Isothermal compression of Langmuir

monolayers of ASC16 (black, full line) and ASC14 (gray, dashed line) at acidic
subphase pH. The arrows indicate the beginning of the LE  S phase transition upon
compression. B) Schematic representation of the Brewster angle microscopy (BAM)
set-up: the incident light is reflected by the monolayer film and collected by the
objective during the film compression. BAM images of ASC16 (C) and ASC14 (D)
films at acidic pH along the isothermal compression curve. The images were captured
at the surface pressures indicated. Image size: 200 x 250µm. Extracted from (Mottola
et al., 2015).
6. ASCN INTERACTION WITH LIPID MEMBRANES

Cell membranes are the first contact of lipophilic drugs with cells and
often form a reservoir. The drug/membrane interaction is therefore a
fundamental condition for its pharmacological function and a potential
regulatory point.
Some attempts have been made to explore the interaction and the general
physicochemical effect of ASCn family members when they are incorporated
into model lipid membranes under controlled conditions. From the universe of
naturally occurring lipids that populate cell membranes, phosphatidylcholines
(PC) are especially abundant and are the most studied lipid family (Heimburg,
2007). They comprise zwitterionic amphiphilic lipids that, when they contain
at least one double bond in their chains, form lipid membranes, which
represent the simplest model of biomembranes. PC are of widespread use in
both bilayer and monolayer systems, since they usually present Lα (or Liquid-
disordered) phases in bilayers and, their equivalent, an LE phase state in
monolayers throughout the surface pressure range and at room temperature.
These liquid phases (Lα and LE) are thought to be the major phase state found
in cell membranes.
Early attempts to study this were reported by Casal’s group in an
investigation of ASC16 interaction with phospholipid bilayers (Köhler,
Mantsch, and Casal, 1988). They reported that ASC16 stabilizes the gel phase
of PC, shifting the gel stage to a liquid-crystalline stage at higher temperatures.
More recently, several studies have been made of the interaction of ASCn
with biomembranes, using lipid monolayers as a model of biological
membrane. When a concentrated solution of ASCn is injected into the
subphase of a pre-formed lipid monolayer, the further increase in surface
pressure of the films shows the insertion of the amphiphile into the lipid
membrane (Figure 11A). ASC16, ASC14 and ASC12 (but not ASC10)
penetrate phospholipid monolayers in the LE phase state, initially at 30 mN/m
until reaching equilibrium surface pressure, which is 8–16 mN/m higher than
that reached when they are adsorbed into a bare air/water surface (Mottola et
al., 2013, 2015).
The resultant increase in surface pressure after incorporation of the
amphiphilic drug into a lipid monolayer has been traditionally taken as an
indirect measure of the affinity of the amphiphiles to the lipid film. However,
the amount by which the surface pressure increases depends on at least two
factors, (i) the amount of drug incorporated into the lipid film and (ii) the
sensitivity of the lipid film to being laterally compressed by the drug
molecules incorporated, that is, the mechanical properties of the host film
(Zulueta Diaz et al., 2016).
For ASC14 or ASC16, the surface pressure increase after drug penetration
into lipid monolayers and the drug uptake capacity of those lipid films were
found to depend on the phase state of the host lipid film (Figure 11B). Thus,
both the mechanical response and the drug incorporation capacity of the host
membrane regulate the final response in surface pressure of the drug-lipid
system. Hence, monolayers that show an S phase state (such as DSPC)
responded with a greater surface pressure increase to the incorporation of a
lower amount of ASCn than did POPC films, which are in LE state (Zulueta
Diaz et al., 2016). The length of the acyl chain of the ASCn compounds also
induces differential changes in the rheological properties of the host membrane
and subtly regulates the kinetics and extent of the penetration process (Mottola
et al., 2015; Zulueta Diaz et al., 2016).
Because the ASCn family has been mainly used in topical
pharmacological preparations, it is important to inquire how these amphiphilic
drugs are incorporated into the stratum corneum, where the biological function
is highly controlled by the lipid composition (Bouwstra et al., 2003). The
incorporation of ASC14 and ASC16 into model lipid monolayers, which
closely mimic the stratum corneum structure and its barrier function (Školová
et al., 2013), was studied recently (Zulueta Diaz et al., 2016). Those complex
monolayers are formed by a quaternary mixture of cholesterol, ceramide, long-
chain fatty acids and cholesterol sulfate, the four major lipid components of
stratum corneum. Of those key components, long-chain fatty acids and
ceramides typically form S films with high shear modulus (Espinosa et al.,
2011), while cholesterol typically provides a liquid-ordered character,
inducing high lateral mobility and compressibility modulus (Fanani and
Maggio, 2011).
ASCn are scarcely incorporated into S or cholesterol-containing films,
including the mixture that mimics stratum corneum, but are easily
incorporated into PC films in the LE state (Figure 11B). This phenomenon
appears to be independent of membrane diffusional properties, but directly
related to the compressibility properties of the film, given mainly by a high
cholesterol content (Zulueta Diaz et al., 2016). This suggests that cholesterol
makes a significant contribution to the maintenance of the barrier function of
stratum corneum.
Since the penetration process involves enrichment of the lipid membrane
with ASCn molecules, mixed Langmuir films constitute a valuable model for
studying in-plane ASCn/lipid interactions. The enrichment of PC membranes
with ASC16 or ASC14, but not with ASC12, results in the occurrence of
ASCn-enriched domains surrounded by a PC-enriched LE phase (Mottola et
al., 2013, 2015). At neutral subphase pH, those ASCn-enriched domains show
rounded borders, typical of LC phases, which grow in size and relative area
with the ASCn content (Figure 12), following the lever rule for two-phase
equilibrium (Heimburg, 2007). At acidic subphase pH, ASC16 can also induce
the formation of crystalline-like domains (Mottola et al., 2015), which
resemble the structures formed in pure ASC16 films (see Figure 10). Similar
ASC16-enriched domains were recently observed in giant unilamellar vesicles
composed initially of PC and incubated with ASC16 aggregates (unpublished
results), indicating that the phenomenon observed in Langmuir films can be
extrapolated in some way to lipid bilayers.
60 40
ASC16 neccesary to reach 90%

60
A B
Surface Pressure (mN/m)
of the saturation  (mol %)

50 55
equilibrium (mN/m)
30
40 50
30  45 20
20 40
10
10 35
0 30 0
0 10 20 30 40 POPC SM16 DSPC CHO SCM Lipid film
Time (min) LE LC S LO LC Phase state
Figure 11. Penetration of ASC16 into monolayers with different rheological character
A) Representative penetration curve showing the insertion of ASC16 into a pure
distearoyl-PC (DSPC) monolayer, initially at 30 mN/m. The inset shows a schematic
representation of the experimental set-up. B) Equilibrium surface pressures ()
achieved after insertion of ASC16 (gray bars) and mol% of ASC16 necessary to reach
90% saturation in the acceptor monolayers by surface titration (white bars). The lipid
monolayers were composed of palmitoyl-oleoyl-PC (POPC), palmitoyl-sphingomyelin
(SM16), DSPC, cholesterol (CHO) or stratum corneum mimicking membrane (SCM).
The phase state of the corresponding lipid monolayers is denoted by: LE (liquid-
expanded), LC (liquid-condensed), S (solid) and LO (liquid-ordered). Extracted from
Zulueta Diaz et al., 2016.
A B C
44mN/m 39mN/m 26mN/m
D E F
25mN/m 19mN/m 16mN/m
Figure.12. BAM visualization of binary Langmuir monolayers. Representative images

of ASC16-dimiristoyl PC Langmuir monolayers are shown at the onset of the LE-LC
phase transition (2–4 mN/m above the corresponding transition pressure) at the surface
pressure indicated. ASC16 content is 10 (A), 20 (B), 40 (C), 60 (D), 80 (E), or 100 (F)
mol%. Extracted from Mottola et al., 2013.
In recent years, a relationship between the presence of lipid domains and

cellular function has been widely discussed (Edidin, 2003). In this context, it is
not difficult to relate the different pharmacological potential of each ASCn
compound to its capacity of forming condensed domains in biomembranes.
The effective use of ASCn in biomedicine might thus be strongly related to
their surface properties and their ability to interact with lipid membranes.
7. ASCN IN BIOMEDICINE
To generate any therapeutic effect, the target site must be reached by the
drug with no modifications. Sensitive drugs can be degraded by light
exposure, oxidants or reactive oxygen compounds. The presence of ASCn
derivatives in formulations could prevent this in two ways: the first is related
with their antioxidant capacity, which has been discussed earlier. The second
is that aggregates can be formed under certain conditions of temperature,
concentration of the derivative and the presence or not of co-solvents.
Micelles, coagels and liquid crystals, of colloidal size, can limit the access of
oxidizing compounds to the drugs, modifying some properties of the medium,
such as viscosity.
ASCn aggregates have been proposed as drug delivery nanostructures

capable of providing antioxidant protection to sensitive and hydrophobic
drugs. Various aspects of lamellar liquid crystal systems (coagels) formed by
ASCn were explored for dermal delivery system usage and were addressed in
the literature concerning stability (Vicentini et al., 2008) and improved drug
activity (Chorilli et al., 2011; Nesseem, 2001; Palma et al., 2007). The increase
of the chain length of the derivatives enables higher amounts of drug to be
solubilized due to the increase of hydrophobic zones formed in the aggregates.
The ASCn derivative most studied is ASC16. This compound has been
included in formulations applied by various administration routes. In 2001,
Spiclin et al., (Spiclin et al., 2001) used ASC16 in microemulsions, and
compared the stability of these formulations with the presence of ASC16 or
sodium ascorbyl phosphate. The hydrophilic compound was more stable than
the lipophilic one. The authors concluded that the stability of ASC16 is highly
dependent on its initial concentration (such as in other formulations) and its
location in the microemulsion, which established the possibility that the
oxidizing factors could reach the derivative. Furthermore, the amount of
oxygen dissolved in the system and the storage conditions were important for
keeping the ASC16 stable within the formulation.
Recently, liquid crystals containing Tween 80/ lecithin/ isopropyl
myristate/ water were treated with ASC16 in order to develop a skin-compliant
delivery system (Gosenca, Bešter-Rogač, and Gašperlin, 2013). Rheological
studies were carried out showing that the formulation behaves like a non-
Newtonian system, with characteristics of shear-thinning material. Structural
changes in the system were observed after incorporation of ASC16, and were
attributed to micelle formation due to its amphiphilic character. Additionally,
in vitro cytotoxicity of the ASC16-loaded lamellar liquid crystals was
evaluated on a keratinocyte cell line, proving that this dermal delivery system
was physiologically acceptable.
The uses of natural polymers to improve formulations based on ASC16
are increasing. The encapsulation of ASC16 in chitosan particles by droplet
formation via an oil-in-water emulsion was reported, using ionic gelation with
sodium triphosphate pentabasic (TPP) as a cross-linking agent (Yoksan,
Jirawutthiwongchai and Arpo, 2010). The success of ASC16 encapsulation
was confirmed by different techniques such as FT-IR. The ASC16-loaded
chitosan particles obtained were spherical in shape, as observed by scanning
electron microscope (SEM) and by transmission electron microscope (TEM).
Loading capacity and encapsulation efficiency was also evaluated. Increasing
the initial ASC16 concentration led to a modification in the liquid crystalline
state. Also, the amount of ASC16 released from the nanoparticles increased
with the loading capacity and decreased with the degree of cross-linking.
Several strategies were used to enhance ASC16 chemical stability, for it to
be used in industry and pharmacy as an oxygen-scavenging system. Inclusion
complexes of ASC16 and cyclodextrins showing micrometric size-rod shape
were produced for this purpose, and a good oxygen-scavenging capacity was
found after thermal processing (Byun and Whiteside, 2012). Lipid-based
nanostructured systems containing ASC16 also enhanced its chemical
stability. A detailed study was carried out by Teeranachaideekul et al.,
(Teeranachaideekul, Müller, and Junyaprasert, 2007), in which lipid
nanoparticles composed of non-polar lipids in solid state (solid lipid
nanoparticles or SLN) or mixed with more fluid lipids (forming nanostructured
lipid carriers or NLC) were stabilized by different types of surfactants. NLC
containing ASC16 were developed. Their chemical stability was reached by
selecting suitable types of lipid, surfactant, and proper storage conditions, such
as cold temperature and flushing with nitrogen gas or inert gas.
Other nanostructured systems, such as lipid vesicles containing ASC16
were also described (Gopinath et al., 2004; Kristl et al., 2003). Lipid vesicles
or liposomes differ from the above lipid nanostructures in that they contain an
entrapped aqueous compartment. They have the advantage of being able both
to entrap hydrophilic drug solutions and to incorporate hydrophobic drugs in
their membrane phase. Gopinath et al., reported the characterization of lipid
vesicles containing ASC16 called “aspasomes,” with potential application in
disorders implicated with reactive oxygen species when the anti-retroviral
drug AZT was used. These structures, prepared by the film hydration method,
were formed in combination with cholesterol and also a negatively charged
lipid (dicetyl phosphate) and have the capacity to encapsulate AZT aqueous
solution and also enhance its transdermal permeation.
PC liposomes (DMPC) were also loaded with ASC16 to avoiding the
oxidizing effect of amiodarone (AMI), a hydrophobic drug, which is very
useful in the treatment of severe cardiac disease (Benedini et al., 2014). The
employment of liposomes would avoid the use of cosolvents in AMI
formulations, and the incorporation of ASC16 into the liposome structure
would minimize the adverse effects of AMI administration. To evaluate the
partition and integration of AMI and ASC16 in lipid membranes, penetration
studies onto PC monolayers were carried out. The disturbance of the liposome
membranes was studied by generalized polarization (GP), whilst the stability
of liposomes was evaluated experimentally and by means of the Derjaguin-
Landau-Verwey-Overbeek (DLVO) theory. In experimental conditions, all
vesicles showed stability at time 0, but only PC + ASC16 10% + AMI 10%
liposomes kept their size and zeta potential stable during 28 days. These
results are encouraging and suggest that such a system could be suitable for
intravenous AMI delivery formulations. A sketch of this system is shown in
Figure 13.
Figure 13. Effect of AMI on the thermotropic behavior of phospholipid/ASC16

liposomes. A) Schematic representation of liposomes containing DMPC and ASC16
with (left) and without (right) AMI; B) Generalized polarization function (GP) of the
fluorescent probe Laurdan sensing the phospholipid phase transition through the
membrane/water surface properties. Extracted from Benedini et al., 2014.
Other members of the ASCn family have been used in different

pharmacological preparations for its amphiphilic nature. Supramolecular
assemblies of ASC10 have been used to dissolve hydrophobic drugs such as
anthralin, phenacetin, danthron, and griseofulvin (Palma et al., 2007). This
derivative forms micelles above the critical aggregation concentration at which
drugs can be solubilized into their hydrophobic core, and in this way improves
their water solubility. The addition of ASC12 to topical ocular preparations
improves the ocular bioavailability of the hydrophobic drug acetazolamide,

with mild to moderate in vivo irritation effects (Tártara et al., 2012).
For optimization of permeation through mouse skin and distribution of
ibuprofen, two different ASCn coagel systems were compared, based on
ASC12 and ASC16 in water or water/polyethylene glycol (PEG) mixtures
(Saino et al., 2010). The study showed the enhancement activity of coagels
and the synergic effect of their combination with PEG, with ASC12 being
more active than ASC16. Furthermore, medium-long chains (ASC10 and
ASC12) but not ASC16 showed enhanced antileishmanial effect against
visceral and cutaneous parasites (Kharrat et al., 2014).
As commented above, the different pharmacological potential of each
ASCn compound may be strongly related to their surface properties and their
ability to interact with lipid membranes. Drug penetration enhancers can
increase drug diffusion through the cell membrane or skin by modifying the
lipid packing, or can increase the partition coefficient of the active agent.
Medium-long chain ASCn, such as ASC10 or ASC12, form micelles and may
also form lamellar particles in aqueous solution. The presence of those small
lax structures may be of importance in the loading of other hydrophobic drugs
and also responsible for rapid molecular exchange with biomembranes.
New approaches have been reported involving ASC16 liquid crystals with
immunogenic systems. Coagels of ASC16 containing synthetic
oligodeoxynucleotides with unmethylated CpG motifs (CpG-ODN) were used
as a novel vaccine adjuvant (Sánchez Vallecillo et al., 2014). Only the
formulation with ASC16 coagels (OVA/CpG-ODN/Asc16 coagels) resulted in
long-lasting cell-mediated immunization. Furthermore, Coa-ASC16 alone
allows a controlled release of CpG-ODN in vitro and induces local
inflammatory response. This study shows the potential of this formulation,
opening a new avenue for the development of better vaccines.
The success of ASC16 coagel as a fundamental part of vaccines can be
analyzed from its physicochemical properties. ASC16 shows good membrane
restructuring properties with the additional feature of being able to form drug-
enriched condensed domains once incorporated into the lipid membrane
(Mottola et al., 2015; Zulueta Diaz et al., 2016). This, together with an
organization in aqueous solution as highly compacted large lamellar structures
(Mottola et al., 2013; Palma et al., 2007), may account for its good adjuvant
capacity. The effect of these coagels, when used as adjuvants, shows sustained
release of the antigen and a local inflammatory response induced by ASC16 as
a main feature responsible for the immunization obtained (Sánchez Vallecillo
et al., 2015). Evidence of local necrosis in the injection area supported this
hypothesis. In this picture, ASC16 can be expected to be slowly released from

coagel structures and integrated into cell membranes, where it can also
accumulate and laterally separate into rigid domains acting as reservoirs of the
drug and threatening cell survival.
CONCLUSION
In summary, the ASCn family show different physicochemical properties
and biomembrane perturbation activity of different extent depending on the
length of their acyl chains. Some members of this family have been
extensively explored for pharmacological and industrial use but the potential
of some of them remains unknown. This encourages future work to understand
and explore new and novel uses of these compounds. The differential surface
activity and interaction with lipid structures, such as cell membranes and
stratum corneum, of each ASCn member may account for their different
pharmacological potential. Studies of those aspects may thus contribute to the
design of new applications for these types of drugs.
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Chapter 8
DESIGN, PREPARATION AND

CHARACTERIZATION OF MODIFIED
RELEASE PELLETS OF ASCORBIC ACID FOR
PHARMACEUTICAL APPLICATIONS
John Rojas and David Correa

Department of Pharmacy, University of Antioquia, Medellín, Columbia
ABSTRACT
Ascorbic acid is an essential vitamin, which cannot be synthesized
and stored in the body. It scavenges free radicals, enhances immunity,
promotes collagen biosynthesis, provides photoprotection, and causes
melanin reduction. Further, its antioxidant activity could reduce the risk
of cancer. As a result, a daily supplementation of this vitamin is crucial to
prevent and ameliorate these diseases. However, most pharmaceutical
products are intended for immediate release of this vitamin. Conversely, a
controlled release formulation is preferable due to better patient
compliance and application of a single dose in extended time intervals.
This chapter explores the pelletization technology to control the particles
size, enhance the process reliability, reproducibility, and the ascorbic acid
release. The goal of this chapter is to study the influence of polymer type

Corresponding author: John Rojas. Cll 67 # 53-108, office 1-157. Medellín, Tel.: 574 219 5472.
Email address: jrojasca@gmail.com.
176 John Rojas and David Correa
(i.e., microcrystalline cellulose, kappa carrageenan and hydroxypropyl

methylcellulose (HPMC) and ascorbic acid concentration on shape
descriptors, particle characterization and drug release kinetics. Processing
time varied according to the progression of spheronization process and
the rate was kept constant at 12 Hz using a 30 cm diameter spheronizer
equipped with a cross-hatched plate.
On the other hand, the polymer chemistry influenced the
morphological characteristics of the pellets. Further, other hydrophilic
polymers such as HPMC, k-carrageenan, pectin and sodium
carboxymethylcellulose were unsuccessful to produce spherical pellets.
All polymers exhibited a biphasic drug release profile characterized by an
initial large drug released (60-80%) within 1h followed by a slow
released within 9h. The increase of drug loading was in detriment of
shape descriptors, mass and breaking strength, especially at 75% drug
load, but increased the compressibility, densification, and friability of the
pellets. Preliminary studies of drug loading in mixtures with
microcrystalline cellulose having loads from 50 to 60% were chosen as
ideal for the formation of spherical pellets and exhibited an immediate
drug release. Conversely, MCC pellets coated with shellac, carnauba wax
and the copolymer of ethyl acrylate, methyl methacrylate and methacrylic
acid with a quaternary ammonium group (Eudragit® RL) showed a slower
drug released. The best controlled release pellet formulation had a coating
layer of Eudragit® RL followed by 10 layers of shellac. These coated
pellets were able to release the drug by diffusion from the core to the
coating layer which in turn, swelled and controlled the drug release.
INTRODUCTION
Ascorbic acid (AA) also known as Vitamin C, ascorbate, or ascorbate
monoanion is a water-soluble vitamin, which cannot be synthesized and stored
in the body due to the absence of L-gulonolactone oxidase which is the
enzyme responsible for its synthesis [1]. Therefore, the dietary intakes of this
compound as a vitamin supplement awards nutritional and health benefits.
The physiological function of AA is due to its high reducing power. It acts
as singlet oxygen quencher since it neutralizes and stabilizes reactive oxygen
species or other free radicals that can damage DNA [2]. It also promotes
collagen biosynthesis, provides photoprotection, causes melanin reduction, and
enhances immunity. It also reduces the risk of acquiring degenerative diseases
[3, 4] such as cancer, cardiovascular diseases, and cataracts [5, 6].
AA also prevents other diseases through different mechanisms such as
increasing lymphocyte production [7], stimulation of collagen formation
Design, Preparation and Characterization of Modified Release Pellets …177
necessary for ‘walling off’ tumors [8], inhibition of hyaluronidase and

oncogenic microorganisms [9], enhancement of the effect of certain
chemotherapy drugs (i.e., tamoxifen or cisplatin) reduction of the toxicity of
other chemotherapeutic agents (i.e., doxorubicin) and neutralization of
carcinogenic substances in mice and guinea pigs by increasing the life span of
the drug [10, 11]. Further, it has been demonstrated that AA inhibits colorectal
cancer in rodents [12].
PELLETIZATION: THE FUNDAMENTALS

Pellets are defined as spherical or semispherical, free-flowing
agglomerates having a smooth surface, low friability and narrow size
distribution, typically varying between 0.5 and 2.5 mm [13]. This ensures a
reproducible die or gelatin capsule filling leading to a good content uniformity,
ease of coating and packing [14]. Usually, several hundred pellets filled into a
capsule give one dose [15]. The capsule shell dissolves rapidly (i.e., within 5
min.) after application [16].
Pellets offer some important technological advantages over conventional
single-unit solid dosage forms. The process is more reliable, reproducible and
it is possible to control the particles size. In addition, it is easy to scale-up and
it is little dependent on the solubility of drug and polymer. Moreover, they
have a high density and pack more uniformly [17]. Therefore, pellets have a
low production cost, high production rate, reduced lubricity, low risk of
tampering and reduced dust problems [18]. Further, pellets have a high drug
loading ability, especially for drugs having a very low aqueous solubility [19].
This is explained by their high density [20].
They facilitate administration, individual dosing, reproducibility and
improve patient compliance. The defect of an individual unit has no serious
effect on drug efficacy. They have a low surface area-to-volume ratio,
providing an ideal shape for the application of film coating to mask
undesirable tastes, odors and colors, and control drug release. Further, coating
provides stabilization during storage and transportation since it protects the
drug from the action of air, moisture and light. However, film coating of
pellets might cause aggregation or formation of clusters [21].
PELLETS AS A MATRIX DEVICE FOR DRUG RELEASE

Pellets exhibit pharmaceutical advantages over monolithic systems such as
less irritation, a lower risk of side effects due to dose dumping at a focus site
of mucosa since high local concentrations are avoided [22]. They have a lower
tendency of adhering to oesophagus during swallowing [23]. Further, due to
their small particle size (<3 mm) pellets provide a good distribution, and
smoother absorption profile from the GI tract because the beads pass gradually
from the stomach into the duodenum at a steady rate. Pellets spread over a
longer period of time and render a more homogenous distribution in the
gastro-intestinal tract allowing for less variable transit times and drug
absorption [27]. As a result, they render reproducible drug blood levels and
behave like liquids, leaving the stomach rapidly regardless the gastric
emptying rate, feeding or nutritional state of the patient [24].
The pelletization technique has found application for the development of
hydrophilic compounds such as vitamins, drugs, enzymes, acidifiers,
probiotics, peptides, antioxidants and flavors. For instance, ethyl cellulose
microparticles have been developed to protect and control the release of folic
acid in foods and dietary supplements [25, 26].
Pellets are adaptable since a broad range of doses for different age groups
can be combine into one dose [28]. Further, it is possible to combine several
incompatible drugs with different release profiles in the same dosage unit.
Thus, several pellets with varying release profiles can be combined within one
capsule dose allowing the adjustment of the desired release profile [29].
PELLETIZATION METHODS
Several manufacturing techniques can be applied to produce
pharmaceutical pellets, such as solution/suspension layering, direct
pelletization, melt suspension, spray-drying, coacervation, extrusion-
spheronization among others. These methods affect the resulting physical
characteristics of the pellets. However, only the extrusion-spheronization is the
most widely used method due to the ease of operation, throughput with low
waste, high efficiency, robustness, reproducibility, and low costs, rendering
pellets with low friability, high drug loading, high density, narrow size
distribution and short processing times [30, 31]. In this method, the drug and
excipient mixture is first wet-massed and forced through a die plate leading to
an intermediate spaghetti-like product, which is then broken down into shorter

rods, and rounded into spheroids using a fast rotating friction plate. During the
spheronization process particles always tumble, collide, and rotate and are
shaped by centrifugal, frictional, and elastic forces. The most widely accepted
spheronization mechanism establishes that extrudates are broken up into short
cylinders of more or less equal length, and transformed into dumb-bells,
elliptical spheroids, and spherical pellets [32]. Therefore, the quality of the
pellets is affected by complex interactions between the type of processing
equipment, and formulation variables occurring during the different steps. At
the end of the process, drying is used to finalize the textural characteristics of
the product by densification through mechanical shrinkage induced by the
extraction of water [33].
DEVELOPMENT OF ASCORBIC ACID PELLETS

The success of the pelletization process depends on a large number of
factors such as the composition and physico-chemical properties of the raw
materials, and the manufacturing process conditions [34]. Therefore, an ideal
pelletization aid should be water insoluble, have large water absorption and
retention capacity, good binding properties, sufficient large surface area for
interaction with water and other ingredients in the powder mixture, and ability
to enhance drug release [35]. Further, pelletization aids should have an
inherent fluidity permitting flow during the process and self-lubricating
properties as it passes through the die/mesh. These materials must be rigid
enough to get a coherent aggregate as well as brittle enough to be sufficiently
plastic to enable the cylindrical rods to be rolled into spheres by the action of
the friction plate in the spheronizer. Further, the cylinders formed must not
adhere to each other so that the particles do not aggregate and break down into
tiny dumb-bells during spheronisation [36]. To date, only few pelletization
aids have been identified [37]. However, MCC is still the most commonly
used pelletization aid in pharmaceutical applications [32].
In this work, the pelletization technique was used to produce microspheres
containing a 50% ascorbic acid, using hydrophilic polymers such as
microcrystalline cellulose (MCC), k-carrageenan, hydroxypropylmethyl
cellulose (HPMC), pectin, carboxymethyl cellulose (NaCMC) and sodium
alginate. Runs were conducted on a cross-hatched spheronizer having a 30 cm
diameter plate operated at 12 Hz and several processing times. Results indicate
that the amount of water needed for wetting the powder mass varied depending
of the polymer type used. Further, the spheronization time varied according to
the tackiness of the material. However, the initial experimental screening of
AA:polymer (50:50) mixtures determined only MCC, k-carrageenan and
HPMC as the most suitable materials for the pelletization process (Figure 1).
All other materials rendered only sticky granules instead of spherical particles.
This behavior is attributed to the tackiness and swelling ability of these
polymers [38]. Other studies report less conventional swelling materials such
as chitosan, hydroxypropyl cellulose, and -cyclodextrin, in mixtures with
MCC resulting in less spherical pellets [39, 40]. Further, lipophilic materials
such as cetyl alcohol, paraffin, glyceryl trimyristate, glyceryl distearate, stearic
acid, glyceril palmitostearate, gelucire 50/02, glyceryl monostearate, and
cetostearyl alcohol have been used efficiently only in hot-melt extrusion
applications [41].
Drug Loading and Wet-Massing Moisture
Among all polymers used, MCC was the best pelletization material for
AA. The presence of a large surface area and high internal porosity in the fine
capillaries increased its ability to absorb and retain a large amount of water
[42], due to randomly aggregated filamentous microcrystals, improving the
wetted mass plasticity and enhancing the spheronization process. Moreover,
by controlling the movement of water through the plastic mass, MCC prevents
phase separation during extrusion and spheronization [43].
k-carrageenan (50 mL, water 30s) Sodium carboxymethyl cellulose

(80 mL water, 90s)
Hydroxypropylmethyl cellulose Sodium alginate (60 mL water, 90s)

(60 mL water, 60s)
Pectin (60 mL water, 120s) Microcrystalline cellulose

(35 mL water, 60s)
Figure 1. Microphotographs of pellets produced from excipient:ascorbic acid mixtures

(50:50).
Table 1. Pellet properties having different drug loads
Properties Units Ascorbic acid load (%)

0 10 25 50 60 75
Spheronizat (s) 0 30 30 60 60 90
ion time
Wetting mL 64 60 56 35 33 19
Areaa mm2 3.1 ± 1.4 4.4 ± 2.6 6.0 ± 3 5.1 ± 8.0 5.8 ±5.0 1.2 ± 0.9
Aspect N.A. 0.6 ± 0.79 ± 0.75 ± 0.71 ± 0.70 ± 0.68 ±
ratioa 0.0 0.13 0.13 0.20 0.19 0.16
Circularitya N.A. 0.7 ± 0.82 ± 0.78 ± 0.80 ± 0.72 ± 0.72 ±
0.2 0.13 0.11 0.21 0.20 0.18
Feret mm 2.5 ± 1.8 2.8 ± 0.9 3.3 ± 2.0 ± 2.1 2.8 ± 1.7 1.6 ± 0.6
diameter 0.9
Table 1. (Continued)
Perimetera mm 7.8 ± 3.4 8.2 ± 3.4 9.7 ± 6.1 ± 7.0 8.4 ± 5.1 4.7 ± 2.7
3.0
Roundnessa N.A. 0.7 ± 0.78 ± 0.75 ± 0.71 ± 0.70 ± 0.68 ±
0.2 0.13 0.13 0.20 0.19 0.16
Soliditya N.A. 0.8 ± 0.1 0.94 ± 0.92 ± 0.87 ± 0.86 ± 0.86 ±
0.07 0.05 0.13 0.14 0.07
Compressib (%) 8.5 8.6 11.8 12.3 12.2 15.5
ility
Bulk g/cm 0.52 ± 0.54 ± 0.51 ± 0.54 ± 0.52 ± 0.60 ±
densityb 3
0.01 0.01 0.01 0.00 0.01 0.01
Tap g/cm 0.61 ± 0.59 ± 0.58 ± 0.63 ± 0.59 ± 0.71 ±
densityb 3
0.01 0.01 0.00 0.03 0.01 0.00
True g/cm 1.304± 1.594 ± 1.604 ± 1.635 ± 1.642 ± 1.675 ±
densityb 3
0.000 0.001 0.000 0.004 0.000 0.000
c
Flow rate g/s 189.9 ± 107.8 ± 102.0 ± 100.5 ± 39.0 ± 48.4 ±
23.9 33.0 46.7 46.7 17.9 21.7
Friability (%) 2.8 0.7 0.1 0.6 1.0 0.9
Breaking (N) 3.5 ± 52.2 ± 47.7 ± 28.9 ± 25.9 ± 3.5 ±
strengthc 0.1 13.7 8.6 12.1 10.3 0.1
c
Mass (mg) 7.0 ± 12.7 ± 2.9 19.5 ± 82.3 ± 20.7 ± 2.3 ±
1.6 6.4 25.1 10.2 0.6
Porosity (%) 73.2 66.2 67.8 66.8 68.1 64.9
a
Sample size of 650 particles; b Data correspond to the mean ± standard deviation of 3
replicate; c Data correspond to the mean ± standard deviation of 10 replicates.
The effect of MCC was also studied at different AA loadings (i.e., 10, 25,
50, 60, and 75%) and the physical properties of the resulting pellets were
studied (Table 1). As the level of the drug increased, the process became more
difficult to carry out to completion. Thus, drug loads below 60% are
suggested, but a drug load of 50% resulted in the most spherical, robust and
reproducible pellets (Figure 2). On the contrary, a 75% drug load rendered
more granular products and thus it was not suitable to conduct an efficient
pelletization process (Figure 2). It is accepted that MCC functions as a
“molecular sponge” retaining a high percentage of water conferring a degree
of plasticity. During extrusion, the wet mass is compressed until water is
squeezed out and lubricates the particles. The volume of the extrudate will
expand becoming less plastic, and thus, it is easily chopped and rounded into
short lengths due to friction and collision forces in the spheronizer plate [44].
10% 25%
50% 60%
75%
Figure 2. Microphotographs of pellets produced with different loads of ascorbic acid.
Since AA is a water soluble drug, less water for pelletization is needed

than pellets having lower solubility drugs. This is explained by the high
capillarity inside the pellets. These capillary forces could lead to an excess of
water at the surface of the pellets during spheronization. Due to the moisture at
the surface these pellets tend to stick together and densify into large
agglomerates. Thus, AA dissolves in water increasing the volume of the liquid

phase with respect to the solid mass leading to an overwetting of the system.
For this reason, less water is required to form a plastic mass as the AA load
increased [37].
A good wettability or ability to bind moisture is essential for the
petellization process [30]. Moisture provides interparticle cohesion, lubricates
particle contacts, promotes wall slip and can also bear some of the stress
generated during the agglomeration and pelletization processes. However, the
pressure exerted on the liquid phase during extrusion makes moisture to move
around the mass particulate network. This movement might cause dewatering
or “liquid phase migration” [45]. The amount of water needed depends on the
die/screen geometry and the polymer packing and structure and thus, the
internal porosity and friability of pellets decreased with increasing water
concentration [46]. For this reason, the amount of water required for the
pelletization process was inversely related to the AA content. Therefore,
pellets having a high drug content required less moisture and formed a sticky
wet mass rendering less spherical particles having a small size. AA pellets
dried at room temperature prevented their degradation and led to the formation
of hard and less porous pellets. In the drying proces pellets shrank by capillary
pressure due to the high surface tension of water [47].
Table 1 lists the shape descriptors and other particle properties of pellets at
different drug loads. Shape descriptors such as circularity, perimeter and
projected area of the pellets decreased as the drug load increased. This is
explained by the need of a lower amount of water to form a wet mass with
sufficient plasticity to form pellets. If this critical water mass is exceeded a
non-processable sticky mass will be formed. On the contrary, as the drug load
is decreased, a larger amount of water is required since the influence of MCC
is decreased. For this reason, pellets having a drug load lower than 60% were
highly spherical, more porous, larger in size, mass, and flow, and had the best
mechanical properties.
The pellet properties of AA and selected excipients (50:50 ratio) is shown
in Table 2. Shape descriptors indicated that MCC rendered the most regularly-
shaped pellets. However, there were no significant differences in sizes as seen
by the Ferret diameter, projected area and perimeter. Further, the polymer
chemistry played a crucial role in pellet properties. For instance, pellets made
with HPMC were less dense, have a lower flow rate, but exhibit the largest
plasticity as seen by their largest breaking strength.
Table 2. Pellet properties of ascorbic acid and selected excipients at a

50:50 ratios
Properties Units HPMC MCC CARRAGEENAN

Wetting (mL) 36 36.7 33
Areaa mm2 9.2 ± 5.1 7.5 ± 2.6 2.7 ± 1.7
a
Aspect ratio N.A. 0.6 ± 0.0 0.8 ± 0.0 0.7 ± 0.0
Circularitya N.A. 0.6 ± 0.1 0.8 ± 0.1 0.8 ± 0.1
Feret diametera mm 4.3 ± 1.5 3.5 ± 0.7 2.2 ± 0.6
Perimetera mm 12.9 ± 4.6 10.6 ± 2.3 6.6 ± 2
a
Roundness N.A. 0.7 ± 0.1 0.8 ± 0.1 0.8 ± 0.1
Soliditya N.A. 0.9 ± 0.1 1.0 ± 0.0 0.9 ± 0.0
Compressibility (%) 12.1 12.6 12.9
b 3
Bulk density g/cm 0.44 ± 0.01 0.47 ± 0.00 0.56 ± 0.01
Tap densityb g/cm3 0.50 ± 0.02 0.54 ± 0.00 0.65 ± 0.01
True densityb g/cm3 1.43 ± 0.00 1.66 ± 0.00 1.71 ± 0.00
Breaking N 105.1 ± 0.0 14.0 ± 0.1 7.1 ± 1.2
c
strength
Flow ratec g/s 34.4 ± 2.1 80.5 ± 5.0 83.4 ± 4.4
Porosity (%) 69.3 71.5 67.0
a
Sample size of 650 particles; b Data correspond to the mean ± standard deviation of 3
replicate; c Data correspond to the mean ± standard deviation of 10 replicates.
Figure 3. Dissolution profiles of ascorbic acid pellets manufactured with different

excipients.
Drug Release
Drug release kinetics from pellets is typically determined by in-vitro

experimental approaches to predict in-vivo behavior. As clearly appreciable,
the high aqueous solubility of AA favored the rapid release behavior from the
pellets and this profile depended slightly on the solubility of the polymers. For
instance, in MCC, and k-carrageenan pellets AA was washed out from the
pellets leaving the pores in the matrix by diffusion. In this case, MCC and k-
carrageenan act as insoluble matrixes in which the eluting aqueous medium
penetrates the matrix and AA diffuses out to the surrounding environment for
ultimate absorption. On the contrary, HPMC pellets swelled and eroded whilst
releasing the drug. MCC pellets loaded with 50% AA showed the best
morphological, particle and mechanical properties. However, all these pellets
exhibited an immediate drug release due to the high porosity of MCC and
aqueous solubility of AA (Figure 3).
For this reason, MCC pellets were coated with different hydrophobic
polymers such as carnauba wax, shellac and the copolymer of ethyl acrylate,
methyl methacrylate and metacryilic acid with a quaternary ammonium group
(Eudragit®). The erosion, swelling and diffusion characteristics of the pellets
played a key role on the drug release behavior showing the following ranking:
k-carrageenan>MCC>HPMC. Therefore, AA release rates varied according to
the prevalence for erosion, diffusion and swelling for k-carrageenan, MCC and
HPMC, respectively.
Pellet Coating
As seen previously, the non-coated pellet showed an immediate release

pattern within 1h due to damping of the entire AA located at the surface and
outer pores of the pellets followed by a slower release of the drug located in
the inner core. The immersion pellet coating process was used at 25°C
followed for a mild drying between the immersions. Figure 4 shows the
resulting drug release profiles of MCC pellets coated with different layers of
®
film-forming materials. Eudragit RL 30D is an aqueous dispersion of a
copolymer based on ethyl acrylate and methyl methacrylate. Further, carnauba
wax and shellac are biopolymers obtained from natural sources such as the
palm tree named Copernicia prunifera and the insect called Laccifer lacca,
respectively. All these polymers are suitable for controlled-release film coating
applications [48]. A 5% ethanolic dispersion of shellac was used, whereas the
carnauba wax was melted at 60°C to coat pellets followed by a rapid cooling
in a water bath maintained at 25°C. Coating with the carbauba wax dispersion
did not cause a major effect of the release rate of AA because ~70% drug
release was obtained within 4 h. Further, pellets coated with carnauba wax still
maintained a biphasic pattern with a moderate release rate within 4h of the
drug located at the surface followed by a slower release of AA located in the
®
inner core. On the contrary, pellets coated with Eudragit RL 30D and 10
layers of shellac showed the most controlled pattern releasing about 85% of
AA within 12h. In this case, drug release was actively controlled via diffusion
and swelling of the polymer.
Several drug release models were used to fit the experimental release data
obtained from these pellets. However, only the Hixon-Crowell model showed
the best fit. This model explains the release behavior in terms of the particle
regular area as being proportional to the cubic root of its volume. Therefore,
this model evaluates drug release with changes in surface area and diameter of
particles. In all cases, pellets retained their initial spherical shape, and the
coating film expanded maintaining the original shape. Thus, AA dissolution
occurred in planes that were parallel to the sphere surface and expanded
proportionally in such a manner that the initial geometrical shape was kept
constant all the time. Therefore, coated pellets achieved a controlled rate of
drug release.
Figure 4. Dissolution profiles of ascorbic acid pellets coated with several polymers.
CONCLUSION
The polymeric drug delivery system produced by pelletization coated with
®
Eudragit and shellac had a potential to provide a controllable drug release
profile. These coated pellets are advantageous compared to conventional
granules since exhibit a controlled release and masking taste. This allows the
use of less film forming polymer, simplifies the coating process and maintains
a constant drug release rate. Further, the release rate of AA was heavily
affected by the nature of the film-forming material.
ACKNOWLEDGMENTS
The authors thank Mr. Cesar Londoño, Julián Herrera, and Yhors Ciro for
their valuable technical assistance.
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Chapter 9
ROLE OF ASCORBIC ACID IN

NEUROPSYCHIATRIC AND
NEURODEGENERATIVE DISORDERS
Daiane de Bittencourt Fraga, MD1,

Morgana Moretti, PhD2
and Ana Lúcia S. Rodrigues, PhD1
1
Department of Biochemistry,
Universidade Federal de Santa Catarina,
Florianópolis, Brazil.
2
Nutrition Post-Graduate Program,
Universidade Federal de Santa Catarina,
Florianópolis, Brazil.
ABSTRACT
Over the past decade, new insights and advances on neurobiology
have extensively characterized critical roles for ascorbic acid in central
nervous system. Ascorbic acid is proposed as a neuromodulator of
glutamatergic, cholinergic, dopaminergic and GABAergic
neurotransmission. Moreover, it has also shown an important role in
support and structure of the neurons, differentiation process, maturation
and neuronal survival. Additionally, ascorbic acid is a powerful
antioxidant highly concentrated in the brain and neuroendocrine tissues
194 D. de Bittencourt Fraga, M. Moretti and A. L. S. Rodrigues
such as adrenal, therefore its involvement in neurological disorders

should not be dismissed. On the basis of the extensive research on
ascorbic acid biological mechanisms in health and disease and its
involvement in homeostasis of central nervous system, this chapter
presents evidence for associations of this vitamin with schizophrenia,
major depressive disorder, bipolar disorder and neurodegenerative
diseases. Considering the drawbacks of the conventional
pharmacotherapy for the treatment of these neuropathologies, particular
attention is attributed to the biochemistry and neuronal metabolism
interface, animal models that are useful for this area of research, and the
human studies that implicate ascorbic acid as potential therapeutic
strategy.
Keywords: ascorbic acid, central nervous system, neuromodulation,

psychopathologies
1. INTRODUCTION
1.1. Chemistry of Ascorbic Acid
L-ascorbic acid (C6H8O6 - Figure 1), usually known as vitamin C, is a

water-soluble antioxidant vitamin that participates in several vital functions.
The name “ascorbic acid” reflects both chemical and biological properties of
this substance. Its structure contains an enolic hydroxyl group, tautomer of α-
hydroxyketone, which provides the reducing capability and the acid behavior
of this compound. On the other hand, the word “ascorbic” refers to
antiscorbutic property of this vitamin [1].
The outstanding chemical characteristic of ascorbic acid is that it is a
reducing agent; numerous physiological function of this compound depends on
the oxido-reduction properties of this vitamin [2]. Ascorbic acid is stable in its
dry form, however, it quickly oxidizes in solution and when exposed to the air
or heat. In mammals, at physiological pH, deprotonation occurs at the C3
hydroxyl group, forming the ascorbate anion, the endogenous form of this
molecule [3]. The two-step, reversible process of ascorbate oxidation leading
to dehydroascorbate is shown in Figure 1.
Role of Ascorbic Acid in Neuropsychiatric and … 195
Figure 1. Ascorbate oxidation process. In mammals, at physiological pH,

deprotonation of ascorbic acid occurs at the C3 hydroxyl group, forming the ascorbate
anion. The oxidation of ascorbate is a two-step, reversible process. The first step
involves formation of the ascorbyl radical, through either the loss of a hydrogen atom
or electron transfer, followed by rapid deprotonation. Although it may react with other
radicals, it generally decays to dehydroascorbate via deprotonation.
1.2. Biosynthesis, Uptake and Metabolism
Some species synthesize ascorbic acid from D-glucose and D-galactose,

process illustrated in Figure 2. In mammals capable of producing ascorbic
acid, synthesis occurs in the liver; reptiles and birds synthetize this compound
in the kidneys. However, some animals such as fruit bats, monkeys, guinea
pigs, fishes, invertebrates and humans are unable to synthesize this vitamin
because the genetically determined absence of L-gulono-gamma-lactone
oxidase enzyme, required in the final stage of L-ascorbic acid biosynthesis
(Figure 2). Outstandingly, this mutation does not cause problems for
organisms unable to synthesize ascorbic acid, since vitamin C is naturally

present in some foods, added to others, and available as a dietary supplement.
Moreover, this vitamin is easily absorbed, specifically in the gut of the
animals; intestinal uptake is performed by a family of sodium-dependent
transporters, named SVCT1 [4]. In humans, most of ascorbic acid (80-90%) is
absorbed when consumption is up to 100 mg/day, however, at high levels, the
efficiency of absorption rapidly decreases [2]. Once absorbed, ascorbate is
distributed throughout the body via the blood. It is worth mentioning that the
highest ascorbate concentrations are found in the adrenal glands, brain and
spinal cord [3, 4].
Figure 2. L-ascorbic acid biosyntesis in animmals. L-ascorbic acid is mainly

synthesized from glucose through a series of enzymatic reactions. Humans do not
synthesize ascorbic acid due to the presence of the nonfunctional gene for L-gulono-
gamma-lactone oxidase.
The major route by which ascorbate enters the central nervous system
(CNS) involves slow transport from plasma to the cerebrospinal fluid (CSF)
across the epithelium of the choroid plexus, via type 2 sodium-dependent
transporters (SVCT2). If dehydroascorbate is present in blood in substantial
quantities, it can rapidly enter the CNS via glucose transporters (GLUT1) in
the blood–brain barrier endothelium. Once in the CSF, ascorbate or
dehydroascorbate enter the neurons through SVCT2 or GLUT1, respectively;
dehydroascorbate is subsequently reduced to ascorbate and stored. Glial cells
obtain ascorbate by reduction of dehydroascorbate, which undergoes uptake
through GLUT1 [5].
As mentioned before, ascorbate can be oxidized by a reversible, two-step
process, to form dehydroascorbate, which can be chemically converted back to
ascorbate through various enzymatic reactions as well as by the action of
reducing agents present in biological systems [3, 4]. If dehydroascorbate is not
reduced back to ascorbate, it is hydrolyzed irreversibly to 2,3-diketogulonic
acid, which can be metabolized to oxalate, xylose, xilonate, and other products
[6]. The formation of oxalate has clinical significance since hyperoxaluria
(overexcretion of oxalate) can result in oxalate kidney stones in some people.
The main route of elimination of ascorbate and its metabolites is via urine [2].
1.3. Biological Functions
Ascorbate is involved in many physiological functions in living organism,

many of them based on its redox properties [2, 3]. This vitamin has a well-
documented role as a cofactor in enzymatic hydroxylation reactions, such as
hydroxylation catalyzed by prolyl and lysyl hydroxylase enzymes. In these
reactions, hydroxyl groups are added to proline and lysine residues in the
collagen molecule, contributing to increase the stability of the collagen triple
helix [7]. In the absence of ascorbate, collagen is unable to be properly
secreted from fibroblasts or is structurally unstable [8]. Physiological
consequences range from impaired wound healing to capillary fragility, the
hallmark of scurvy.
Ascorbic acid is also essential for synthesis of carnitine, a substance that
plays a critical role in the transport of long-chain fatty acids into the
mitochondria for oxidation and cellular ATP generation. The two
hydroxylation reactions for biosynthesis of carnitine are catalyzed by α-
ketoglutarate-dependent dioxygenases. These enzymes require reduced iron
(Fe2+) and a reducing agent (ascorbic acid) for activity [9]. Another enzyme
that uses ascorbate as co-factor is dopamine β-hydroxylase, the enzyme that

catalyzes the conversion of dopamine to norepinephrine, an important mood
regulator in mammals [10]. Additionally, the steps leading to formation of
primary bile acids include hydroxylation of cholesterol, catalyzed by the
cytochrome P450 enzyme cholesterol 7α-hydroxylase, that also uses ascorbate
as a cofactor [11]. It was reported that hypoascorbemia in guinea pigs results
in atherosclerosis due to accumulation of cholesterol caused by inefficient
cholesterol 7α-hydroxylase [12].
Besides acting as enzyme cofactor, ascorbate can terminate chain radical
reactions; it is therefore a reducing agent and scavenger of radicals [2, 13].
Ascorbate can interact with reactive oxygen species (ROS) and stabilize free
radicals produced during cellular metabolism [6]. ROS are highly reactive and
may oxidize biomolecules such as lipids, proteins and nucleic acids, triggering
different kinds of biological damage, including cell death [14]. Metabolites of
ROS are associated with degenerative processes, such as Parkinson's disease.
In addition, ROS are associated with neurotoxic effects produced by excessive
glutamate release [15], a process implicated in a wide variety of
neuropathological conditions such as Huntington's disease, Alzheimer’s
disease, Parkinson’s disease, epilepsy, schizophrenia and major depressive
disorder [16-20].
Besides the above mentioned functions, studies have also demonstrated
that ascorbate can perform other biological functions, including enhancement
of iron absorption [21], immunomodulation, control of glucocorticoids
synthesis by the adrenal gland [2, 22], putative anti-tumoral action [2, 23, 24],
and antinociceptive effects [25].
1.4. CNS Functions
Notably, vitamin C is more than merely a “micronutrient” in the CNS

because it is present in millimolar concentrations in neuron-rich areas and
exerts important neuromodulatory functions, as it will be discussed below.
The distribution of ascorbate in the CNS is dynamically regulated [26],
and it can vary in concentration between regions of the brain. Structures
belonging to the anterior region of the brain, including hippocampus,
neocortex, amygdala, neostriatum, nucleus accumbens, hypothalamus and
septum, have high concentrations of ascorbate, whereas medial and posterior
structures have reduced concentrations of this molecule [27]. An exception to
this gradient is the cerebellum, that is situated in the posterior region of the
brain and has high concentrations of ascorbate [28].
In addition to the aforementioned biological functions, ascorbate is able to
play important roles in the CNS. It is involved in the formation of the myelin
sheath by Schwann cells; ascorbate absence decreases the synthesis of
collagen type IV, which is essential for basal lamina formation, a crucial
structure in the myelination of axons [29, 30]. Ascorbate is also capable of
regulating the action of sodium-potassium ATPase [31, 32], besides being
essential for catecholamine synthesis (as a cofactor of dopamine β-
hydroxylase, as previously described). It was also demonstrated that ascorbate
modulates acetylcholine release of synaptic vesicles from rat brain
synaptosomes [33] and cultured adrenal chromaffin cells [34].
Ascorbate has also an important neuromodulatory action on both
glutamatergic and dopaminergic neurotransmission [3, 4, 26]. It has been
described that administration of dopamine receptor agonists, such as
methamphetamine, increases the release of ascorbate in the rat striatum and
nucleus accumbens [35, 36]. Moreover, amphetamine, GBR-12909,
apomorphine, and the combined administration of dopamine D1 and D2
agonists facilitates ascorbate release from glutamatergic terminals in the
neostriatum, an effect that is abolished by dopamine receptor antagonists [3].
Regarding glutamatergic neurotransmission, it is known that ascorbate
anion is released from glutamatergic neurons as part of glutamate reuptake
process, in which high-affinity glutamate transporters exchange ascorbate for
glutamate. Although the transporters and specific cell types involved in this
mechanism are not fully known, the glutamate transporters EAAT2 and
EAAT3 seem to be more involved in this process, which can occur in neurons
and glial cells, ensuring a high level of extracellular ascorbate in many regions
of the brain [3, 4]. It was also shown that ascorbate inhibits NMDA receptors
via a redox phenomenon [37]. Additionally, ascorbate has an important role in
neuronal development and maturation, exhibiting antioxidant properties
against excitotoxicity mediated by NMDA receptors [38]. Considering the
studies involving NMDA receptors in the pathophysiology of
psychopathologies [39] and the inhibitory effect that ascorbate has on the
function of these receptors [37], one may suggest that this vitamin can have
valuable effects dependent on this property.
On the basis of ascorbic acid biological mechanisms and its involvement
in homeostasis of CNS, we will present experimental and clinical evidence for
associations of this vitamin with psychopathologies, including schizophrenia,
major depressive disorder, bipolar disorder and neurodegenerative diseases.
Considering the drawbacks of the conventional pharmacotherapy for the

treatment these neuropathologies, special attention will be given to the
biochemistry and neuronal metabolism interface, animal models that are useful
for this area of research, and the human studies that implicate ascorbic acid as
a potential therapeutic strategy.
2. ROLE OF ASCORBIC ACID IN NEUROPSYCHIATRIC AND

NEURODEGENERATIVE DISORDERS: PRECLINICAL
EVIDENCE
2.1. Schizophrenia
Schizophrenia is a chronic disorder of brain function defined by three

categories of symptoms: positive symptoms (delusions, hallucinations and
paranoia), negative symptoms (social withdrawal, anhedonia and apathy) and
cognitive symptoms (deficits in attention and executive functions) [40].
Historically, a dopaminergic hypothesis of schizophrenia has prevailed,
supported by observations that dopamine receptor agonists had psychotic
effects, whereas dopamine receptor antagonists decrease psychotic effects,
although they exhibit no effect on negative symptoms or cognitive
impairments [41, 42]. The glutamate hypothesis of schizophrenia posits that
the function of the NDMA receptor is compromised in the disease. This
hypothesis is based on the observations that NMDA receptor antagonists, such
as ketamine and phencyclidine, are able to mimic in animals several aspects of
negative and cognitive symptoms seem in schizophrenic patients [43, 44].
Amphetamine (AMPH), which sensitizes the mesolimbic dopaminergic
pathway, induces sensitization and sensoriomotor gating in rats, two
preclinical behavioral procedures relevant to schizophrenia-related
psychopathology [45, 46]. As mentioned above, an increase in extracellular
dopamine concentrations induced by AMPH may reflect in elevated release of
ascorbic acid by nerve terminals. Accordingly, it is suggested that ascorbic
acid may regulate the action of dopamine in schizophrenia. In addition, White
and colleagues [47] demonstrated that neoestrial infusion of ascorbic acid
potentiates the anti-amphetamine effects of haloperidol, suggesting that, like
haloperidol, ascorbate exerts its effects at the level of the dopamine receptor.
Interestingly, haloperidol and ascorbic acid could share a common mechanism
of action, since both of them significantly attenuated AMPH-induced
sensitization, in terms of psychotomimetic behaviors (hyperlocomotion and

sensorimotor gating deficits). Furthermore, it is possible that ascorbate
indirectly influences dopamine transmission by modulating the glutamatergic
system. It has been suggested that L-glutamate presynaptically controls the
striatal dopaminergic function by acting on NMDA-receptors located on
dopaminergic terminals. Like dopamine, ascorbate is a neuromodulator closely
associated with glutamatergic transmission [48].
Current proponents of the glutamatergic hypothesis of schizophrenia
postulate that hypo-functional NMDA receptors located on gamma-
aminobutyric acid (GABA)-ergic inhibitory interneurons disinhibit pyramidal
neurons, leading to a paradoxical increase in glutamatergic activity in striatum
and prefrontal cortex, promoting the development of positive and negative
symptoms in this neuropathology [49]. Although there is limited preclinical
evidence regarding the glutamatergic mechanism of ascorbic acid in animal
models of schizophrenia, an important neuromodulatory role of this compound
involves its participation in presynaptic reuptake of glutamate, as well as on
glutamate transport by the ascorbate-glutamate heteroexchange mechanism
[50].
As previously mentioned, ascorbate is a one-electron donor that readily
reacts with a range of ROS to neutralize or decrease their reactivity. There is
an increasing body of evidence demonstrating the occurrence of oxidative
stress in schizophrenia as well as a dysregulation of cellular processes
dependent on enzymatic and non-enzymatic antioxidant system [51]. In
addition, preclinical evidence suggests that antioxidant system deficit and
oxidative stress are sufficient to induce schizophrenia-like behavior in rodents.
Cabungcal and colleagues [52] demonstrated that glutathione (GSH) deficit
during brain development induced schizophrenia-like behavior in adult rats. In
the same study, it was observed that synthesis of ascorbic acid increased to
compensate the GSH deficit. In agreement with these findings, treatment of
GSH-deficient newborn rats with high doses of ascorbic acid decreased
mortality, indicating that this compound can itself serve as an essential
antioxidant [53]. Moreover, a study suggests that GSH deficiency may be
stronger during development that in adulthood; the authors demonstrated that
inhibition of GSH synthesis combined with dopamine reuptake inhibitor
administration in a strain of rodents unable to synthesize ascorbic acid induced
psychomotor and spatial memory deficit [54].
Although the exact mechanisms involved in the beneficial effects of
ascorbic acid in schizophrenia are not completely known, the aforementioned
results clearly indicate that this vitamin not only modulates the glutamatergic
and dopaminergic systems, but also decreases oxidative stress in animal

models of schizophrenia. Heiser et al. [55] demonstrated that antipsychotics
induce the formation of ROS in rats and ascorbic acid reduced the ROS
production induced by olanzapine, haloperidol and clozapine. In sum, the
ability of ascorbic acid to counteract oxidative damage caused by
antipsychotics and the dysfunctional glutamatergic and dopaminergic systems
in schizophrenia makes it a potential therapeutic strategy for the management
of this psychiatric disorder.
For decades, schizophrenia has been treated using antipsychotic drugs
targeting dopamine receptors. However, there are significant side effects
associated with currently available antipsychotics and no mechanistically
novel treatment has emerged to replace them. Considering preclinical evidence
supporting the antipsychotic effect of ascorbic acid and taking into account its
attractive risk-benefit profile, it could be a promising strategy to manage
schizophrenic symptoms, especially as adjunctive therapy along with standard
antipsychotics.
2.2. Major Depressive Disorder
Major depressive disorder is a chronic and debilitating mental illness that

represents a significant cause of mobidity and mortality worldwide. It has been
almost 50 years since the monoamine hypothesis of depression was
articulated. Although several monoamine-based pharmacological drug classes
have been developed for the treatment of major depressive disorder, the
remission rates are low (often less than 60%) [56]. A number of preclinical
studies have been conducted in order to investigate the antidepressant
properties of ascorbic acid. This compound is known to exhibit antidepressant-
like effect in a predictive test for antidepressant activity [57], and was able to
abolish brain oxidative damage induced by chronic unpredictable stress in
mice, with efficacy similar to fluoxetine [58].
The antidepressant effect of ascorbic acid may be due its involvement in
neurotransmitter synthesis. Ascorbate directly donates an electron to dopamine
β-hydroxylase for the conversion of dopamine into norepinephrine.
Furthermore, this compound exerts an indirect role in the recycling of
tetrahydrobiopterin, which is required for the activation of the rate-limiting
enzyme for catecholamine synthesis, tyrosine hydroxylase, as well as
tryptophan hydroxylase in the synthesis of serotonin. Although these functions
of ascorbate are well established, its physiologic relevance to major depressive
disorder is unclear. A preclinical study demonstrated that the dopaminergic

system is involved in the antidepressant-like action of ascorbic acid in the tail
suspension test, through an interaction with dopamine D2 receptors, since the
pre-treatment of the animals with haloperidol or sulpiride, prevented the
antidepressant-like effect evoked by this vitamin [57].
The importance of monoaminergic system in developmental period and
the association between adult anxiety and depressive behavior has been
recently discussed [59]. Popa and colleagues [60] demonstrated that the
treatment with selective serotonin reuptake inhibitors during the first 3 weeks
of postnatal life induced a depressive phenotype and anhedonia into adulthood
in mice. Ascorbate along with other important factors, has been shown to aid
in differentiation of dopaminergic and serotonergic cells during the
development period. A recent study suggested that ascorbate could regulate
embryonic brain cortex monoamine synthesis. The authors found that low
levels of ascorbate led to a decrease in tyrosine hydroxylase, but not
tryptophan hydroxylase levels [61]. These results emphasize the importance of
receiving adequate ascorbate amount during development for the prevention of
depressive symptoms in adult age.
There is growing evidence that the glutamatergic system plays an
important role in the neurobiology and treatment of major depressive disorder.
A number of glutamatergic drugs, including positive modulators of AMPA
receptors, antagonists of NMDA receptor channels and mGluR agonist-
antagonist, exhibit antidepressant-like effects in animal models of depression.
Investigations on the involvement of glutamatergic system in the
antidepressant-like effect of ascorbic acid have also been reported. Moretti and
colleagues [62] demonstrated a synergistic antidepressant-like effect of
ascorbic acid and MK-801 in the mouse tail suspension test, reinforcing the
hypothesis that the anti-immobility effect of ascorbic acid in this test is
dependent on the NMDA receptor inhibition. Later, it was observed that the
antidepressant-like effect of ascorbic acid in an animal model of depression
induced by central administration of TNF-α involves, at least partially, a
reduced p38MAPK phosphorylation, activation of the monoaminergic system, as
well as inhibition of NMDA receptors and nitric oxide synthesis [63].
The prominent role of the NMDA receptors is hypothesized to be one of
the major axes to rapid, robust, and sustained antidepressant effect in
experimental and clinical studies. Particularly, the rapid antidepressant effect
of NMDA receptor antagonists involves ERK1/2 and Akt-mediated mTOR
signaling in the brain. This signaling pathway is activated by ketamine, an
NMDA receptor antagonist which activates several mTOR target proteins
associated with cellular survival and synaptogenesis; these proteins include

4E-BP (eukaryotic translation initiation factor 4E binding protein) and
p70S6K (p70 ribosomal S6 kinase) [64]. In support to the relevant role of
ascorbic acid in depression, it was demonstrated that the antidepressant-like
effect of this vitamin is dependent on activation of mTOR, as well as the
activation of PI3K/Akt, inhibition of GSK-3β and induction of heme
oxygenase-1, reinforcing the notion that these are important targets for
antidepressant activity and contributing to elucidate the antidepressant
mechanisms of ascorbic acid [65].
2.3. Bipolar Disorder
Bipolar disorder is characterized by recurring cycles of depressive and

manic symptoms (Bipolar I) or hypomanic symptoms (Bipolar II). In an initial
phase, this disorder involves an imbalanced neurotransmission;
characteristically an excessive dopaminergic∕glutamatergic transmission and
reduced cholinergic muscarinic transmission. Usually, the chronic phase of
bipolar disorder is characterized by cognitive decline, brain atrophy,
schizoaffective disorder and depression [66]. Classical mood stabilizer drugs
(lithium, valproic acid and carbamazepine) affect the regulation of glutamate
homeostasis and glutamatergic transmission, through K+ buffering, regulation
of calcium-dependent phospholipase A2 or through Ca2+-mediated signaling
pathways. Several lines of evidence suggest the urgent need for more effective
treatments, mainly for depressive phases, that are closely associated with
substance abuse and suicide [67].
Ascorbate modulates intracellular sodium levels, has anti-inflammatory
and antioxidant properties, and increases neurotransmitter synthesis, therefore
it has therapeutic potential for treating bipolar disorder [68].
Intracerebroventricular injection of ouabain induces hyperactivity in rats by
inhibiting the Na+/K+-ATPase enzyme, subsequently increasing intracellular
calcium levels. Ouabain administration in rats is considered a relevant animal
model of mania and induces biochemical alterations that are also seen in brain
from bipolar disorder patients. The brain alterations induced by ouabain are
also associated with lipid and protein oxidation in the prefrontal cortex and
hippocampus [69]. A possible mechanism underlying the effective therapeutic
action of ascorbic acid in bipolar disorder may be associated with its ability to
influence in the Na+/K+-ATPase enzyme activity, as well as to reduce the
oxidative damage [70]. Indeed, ascorbate normally acts as an antioxidant, that
effectively scavenges free radicals and protects the brain against oxidative
stress, particularly in psychiatric disorders [71].
Interestingly, manipulation of the GSK-3β pathway produces both
antimanic and antidepressant effects. Many agents with mood-stabilizing
properties, such as lithium, valproate, and atypical antipsychotics, directly and
indirectly modulate the PI3K, GSK-3β, and Wnt signaling pathways, all of
them implicated in genetic studies of bipolar disorder [72]. Some studies have
reported the influence of ascorbic acid on GSK-3β. Huang and colleagues [73]
showed that ascorbic acid prevented the inactivation of AKT-GSK-3β pathway
mainly by altering ROS levels. In contrast, another study showed that ascorbic
acid had no effect on the GSK-3β phosphorylation in an animal model of
depression, although it reduced the oxidative damage [74]. These results
reinforce the need for more studies on the influence of ascorbic acid on GSK-
3β pathway modulation and its possible effect in bipolar disorder.
Despite the numerous studies of drugs with established antimanic and
antidepressant efficacy and the identification of additional putative treatments
for bipolar disorder, some patients still respond poorly to available medication.
Furthermore, there is a lack of preclinical evidence dealing with the
mechanisms underlying the putative mood-stabilizing effect of vitamin C in
bipolar disorder. Therefore, more preclinical evidence is necessary to better
elucidate the possible beneficial effects this vitamin in this mood disorder.
2.4. Neurodegenerative Diseases
Neurodegenerative disease is an encompassing term for a set of over 600

diseases; the most common are Alzheimer’s disease, Parkinson’s disease and
Huntington’s disease. It is well established in preclinical studies that excessive
activation of glutamate receptors leads to impairment on calcium buffering,
generation of free radicals, and increase in mitochondrial permeability,
suggesting that neurodegenerative diseases with distinct genetic etiologies may
share excitotoxicity and its consequences as a common pathogenic pathway
[75]. Importantly, preclinical studies strongly suggest that ascorbic acid is a
potent neuroprotective agent.
A number of studies have reported the relationship between protein
aggregation and supplementation with vitamin C. The accumulation of toxic
protein aggregates is a hallmark of neurodegenerative diseases; commonly
implicated proteins include tau and amyloid-β (Aβ) in Alzheimer’s disease, α-
sinuclein in Parkinson’s disease and huntingtin in Huntington’s disease.
Murakami and colleagues [76] demonstrated that treatment with vitamin C for
6 months mitigated Aβ oligomers formation and behavioral decline in an
Alzheimer’s disease mouse model. This attenuation of Aβ oligomerization was
accompanied by decreased brain oxidative damage and ratio of soluble Aβ42
to Aβ40, a typical indicator of the progression of this disease. Furthermore, the
authors showed that the intake of vitamin C restored the declined
synaptophysin and the phosphorylation of tau at Ser39. It has been reported
that neurotoxic forms of Aβ peptides stimulate cooper-mediated oxidation of
ascorbate and generation of hydroxyl radicals, a mechanism detailed by
Jamova et al. [77].
Another study suggested that ascorbic acid prevents both the increase of
intracellular calcium and cell death induced by Aβ [78]. Additionally, Dixit et
al. [79] hypothesized that low brain vitamin C could induce oxidative stress
from an early age and accelerate the development of pathological changes
such as Aβ deposition and the associated cognitive deficits in mice. The same
study revealed that mice having a lifelong decrease in vitamin C presented
changes from a much earlier stage of amyloid accumulation. Moreover,
vitamin C suppressed reactivity of the Aβ A11, an antibody that recognizes a
particular conformation of toxic, prefibrilar Aβ oligomers.
In 1977, the discovery that point mutation in the α-sinuclein gene was
responsible for some inherited forms of Parkinson’s disease has dramatically
changed the viewpoint in the pathogenesis of this disease. Studies have
described an important effect of ascorbate on α-sinuclein. For example,
ascorbate can directly reduce α-syn-Cu2+ to α-syn-Cu+, setting up a redox
cycle in which O2 is reduced to H2O2 and several cellular redox species are
continuously exhausted [80]. In addition, Fernandes et al. [81] demonstrated
that ascorbate decreases the formation of α-sinuclein by preventing the
formation of excessive ROS. Furthermore, overexpressed α-sinuclein is
thought to enhance the sensitivity of dopaminergic neurons to oxidative
damage and induce mitochondrial dysfunction. These alterations are important
to understand the mechanisms by which ascorbic acid may be beneficial for
the management of Parkinson’s disease.
Huntington’s disease is a progressive neurodegenerative disorder
characterized by motor and cognitive deficits. There is strongly evidence of
ascorbic acid involvement in this disease. Preclinical studies support that
Huntington’s disease is not simple associated with ascorbic acid deficiency,
but with a deficit on its release into striatal fluid that either directly or
indirectly affects glutamate release by cortical afferents [82]. Rebec and
colleagues [83] showed that ascorbate significantly attenuates the neurological
motor signs in Huntington’s disease animal model, without altering overall

motor activity. In addition, striatal ascorbate deficit was reported in
behaviorally active mice expressing the Huntington’s disease gene, suggesting
that a deficiency in the regulation of striatal ascorbate may be implicated in the
pathogenesis of this disease. [84]. Acuña et al. [85] suggested that ascorbate
homeostasis was altered before the onset of behavioral symptoms in a
Huntington’s disease mouse model. The authors showed that in the pre-
symptomatic stages of the mouse disease model, astrocytes do not release
ascorbate efficiently. In fact, over-supplementation with exogenous ascorbic
acid is enough to produce an adequate ascorbate uptake. The authors also
demonstrated that during the symptomatic stages of Huntington’s disease in
mice, exogenous ascorbic acid is not able to induce SVCT2 translocation to
the plasma membrane, a process that is huntingtin-dependent. Considering the
important effects of ascorbic acid in Huntington’s disease-like behavioral
symptoms, it could be a promising strategy as adjunctive therapy for the
management of this disease.
Finally, the NMDA receptors may be revealed as a common target in
neurodegenerative diseases. Over-stimulation of NMDA receptors has been
found in Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. It
is likely that an increase in extracellular ascorbate is required to prevent
oxidative damage and protofibril formation in these neurodegenerative
diseases [75].
Considering the important role of ascorbic acid in these neuropathologies,
the effects of this vitamin should not be underappreciated. However, more
studies are necessary to elucidate the neurochemical basis of the effects
elicited by ascorbic acid and to better characterize it as a potential therapeutic
strategy in clinical studies.
3. ROLE OF ASCORBIC ACID IN NEUROPSYCHIATRIC AND

NEURODEGENERATIVE DISORDERS: CLINICAL EVIDENCE
3.1. Schizophrenia
Some studies have suggested a possible relationship between ascorbic acid

levels and schizophrenic symptoms. Regarding this issue, in patients with
schizophrenia reduced levels of ascorbic acid in serum [86, 87] and leucocytes
[87], a better index of ascorbic acid status, have been reported.
In a double-blind, placebo-controlled, 8-week study with forty

schizophrenic patients, treatment with vitamin C (500 mg/d) combined with
atypical antipsychotics reversed the increased serum malondialdehyde
(indicative of oxidative stress) and the decreased plasma ascorbic acid levels
and also improved psychiatric scores indicative of suspiciousness, hostility,
excitement, and somatic concern compared to placebo plus atypical
antipsychotics [86]. In line with this study, there is a clinical report showing
beneficial effects of the addition of ascorbic acid to conventional treatment
with neuroleptic for a 37-year-old chronic schizophrenic patient [88]. These
studies suggest that ascorbic acid may be an attractive therapeutic adjuvant in
the treatment of schizophrenia, but further studies regarding this issue are
necessary.
Although ascorbic acid has elicited beneficial effects against
schizophrenic symptoms in few studies, these data are not unequivocal. In a
study conducted in patients with schizophrenia or related psychosis, the
administration of ascorbic acid (1000 mg/day, 16 weeks) as well as vitamin E
did not cause benefit to patients during an acute episode. Curiously, these
vitamins induced psychotic symptoms, especially persecutory delusions in
those patients presenting low blood levels of polyunsaturated fatty acids [89].
3.2. Major Depressive Disorder
Some contradictory results are reported regarding the possible beneficial

effects of ascorbic acid on depressive symptoms in humans, although
preclinical studies consistently indicate that ascorbic acid administration
causes antidepressant-like effects.
It has been shown that poor vitamin C status (indicated mainly by
decreased serum or plasma ascorbate levels) is associated with the occurrence
of depressive symptoms [90, 91, 92]. Evidence that ascorbic acid may play a
role in mood regulation was also given by a clinical report showing that
ascorbic acid relieved depressive symptoms associated with
adrenocorticotropic hormone in a 5-year-old girl treated for chronic hepatitis
[93]. Moreover, the relationship between scurvy (ascorbic acid deficiency) and
depression has been indicated by studies that show the occurrence of
depressive symptoms in individuals with scurvy [94] and by the development
of scurvy reported in a depressive patient [95].
There are some clinical studies that report a positive effect of ascorbic
acid on mood of healthy individuals. In this line, a randomized double-blind,
placebo-controlled 14-day trial of sustained-release ascorbic acid (3000

mg/day) including 42 healthy young adults, showed that ascorbic acid
administration caused an increase in intercourse frequency and improved
mood evaluated by Beck Depression Inventory [96]. In addition, ascorbic acid
intake was shown to be associated with decreased depressive symptoms
among elderly persons dwelling in communities in Japan [97].
Regarding the possible antidepressant effect of ascorbic acid in depressive
patients, there is a positive result in a study in which ascorbic acid was
administered in combination with fluoxetine for pediatric patients in a double-
blind, placebo-controlled pilot trial. A significant decrease in depressive
symptoms was observed in patients treated with ascorbic acid plus fluoxetine
as compared to those that received fluoxetine plus placebo [98]. In line with
these results, an eight-week, double-blind clinical study showed that the
administration of ascorbic acid (500 mg) with antidepressants decreased the
scores of depression evaluated by Hamilton depression rating scale in a small
sample of 22 depressive patients [99].
Conversely, some clinical studies did not find any beneficial effect of
ascorbic acid on depression. In a randomized single-blind study with 45
diabetic patients, the administration of ascorbic acid (1000 mg/d) for six weeks
was able to decrease anxiety level in DASS-21 (Depression Anxiety Stress
Scales 21-item) questionnaire, but did not affect stress and depression scores
[100]. More recently, a study by Sahraian et al. [101] showed no beneficial
effect of the association of citalopram plus ascorbic acid in adult patients with
major depressive disorder as compared with citalopram plus placebo. Also, in
this study ascorbic acid plus citalopram was as effective as placebo plus
citalopram for the treatment of adult patients with suicidal behavior.
3.3. Bipolar Disorder
In a controlled trial involving 40 male psychiatric patients that were

deficient in vitamin C, the supplementation with this vitamin was reported to
reduce both manic and depressive symptoms [102]. The effects of vitamin C in
bipolar disorder and depression were assessed by a double-blind, placebo
controlled trial that indicated that the severity of symptoms of both manic and
depressed patients were lower following a single 3 g dose of vitamin C as
compared to placebo, an effect observed 3-6 h after its administration, but the
beneficial effects of ascorbic acid were more evident in depressed patients
[103]. Another double-blind study that investigated the combined effects of
ascorbic acid with EDTA in bipolar disorder showed that this combined
administration was as effective as the antidepressant amitriptyline for
depressed patients. Although not statistically significant, during week 1
improvement in global scores of the EDTA/ascorbic acid treatment group was
greater than for the amitriptyline group. Also, the ascorbic acid/EDTA group
appeared to respond faster on the Beck and global ratings than the
amitriptyline group, suggesting a possible rapid therapeutic onset of ascorbic
acid. However, in this study manic patients responded better to lithium than to
EDTA/ascorbic acid [104].
3.4. Neurodegenerative Diseases
The possible relationship between ascorbic acid and neurodegenerative

diseases has been investigated, but it is still not well established. It is
remarkable that lymphocyte vitamin C levels were reported to be lower in
patients with severe Parkinson’s disease as compared with those at less severe
stages [105]. However, serum levels of ascorbic acid in Parkinson’s disease
patients were reported to be similar to control group and serum levels of this
vitamin did not correlate with age, age at onset and duration of the disease
[106]. In addition, ascorbic acid levels in leucocytes were higher in
Parkinson’s disease patients as compared to control individuals [107].
Regarding clinical trials using ascorbic acid for treating Parkinson’s
disease patients, a study by Fahn [108] showed that the combined
administration of high dosages of tocopherol and ascorbic acid to patients with
early Parkinson’s disease extended by 2.5 years the time when levodopa
became necessary, suggesting that the combined administration of these
antioxidants may slow the progression of this disease, but in this study it is not
possible to identify the individual contribution of ascorbic acid.
Ascorbic acid was also given to six Parkinson’s disease patients who
experience on-off effects, causing a modest improvement in functional
performance, but no relevant alteration was found in the pattern of on-off
effects, severity of parkinsonism/dyskinesia, or self-assessment ratings [109].
In two large cohorts of men and women who completed detailed and
validated semiquantitative food frequency questionnaires, the intake of
vitamin C was not significantly associated with risk of Parkinson’s disease
[110].
Several studies have examined the relationship between Alzheimer’s
disease and intake of vitamin C, but equivocal results have been provided. A
promising prospective study carried out by Morris et al. [111] with a random
sample of 633 persons 65 years and older showed that none of these
individuals supplemented with ascorbic acid developed Alzheimer’s disease
over the follow up period (mean 4 years). However, a further prospective
study carried out in a stratified random sample of community-dwelling
residents (815 residents 65 years and older free of Alzheimer’s disease at
baseline and followed up for a mean of 3.9 years) found that intake of vitamin
C was not significantly associated with risk of this disease [112]. In addition,
the use of vitamin E and vitamin C supplements in combination in a cross-
sectional and prospective study with elderly (65 years or older) was found to
be associated with reduced prevalence and incidence of Alzheimer’s disease,
but ascorbic acid alone did not decrease risk for this disease [113]. In a study
with over 5000 participants, high intake of vitamin C was associated with
lower risk of Alzheimer’s disease after a mean follow-up period of 6 years
[114].
A study that evaluated plasma ascorbate levels in Alzheimer’s disease
individuals showed lower levels of this vitamin either in home-living
Alzheimer subjects or hospitalized Alzheimer subjects, despite adequate
vitamin C intake [115].
4. CONCLUSION
In conclusion, a number of studies have shown that altered physiological
mechanisms associated with neuropathologies can be ameliorated by
nutritional interventions and this chapter is focused on the possible beneficial
effects of ascorbic acid for the prevention and/or treatment of neuropsychiatric
disorders and neurodegenerative diseases. Although several preclinical and
clinical studies presented herein point out to the role of ascorbic acid as a
possible complementary nutritional strategy for the management of these
neuropathologies, negative results are also present in literature, highlighting
the importance of further studies on this issue.
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Chapter 10
THE APPLICATION OF INTRAVENOUS

MEGADOSE ASCORBIC ACID IN THE
TREATMENT OF ANIMAL CANCERS
Nabhat Thongsoi1 and Anudep Rungsipipat2,

1
Department of Surgery and Theriogenology, Faculty of Veterinary
Medicine,
Khon Kaen University, Khon Kaen, Thailand
2
Companion Animal Cancer Research Unit, Department of Pathology,
Faculty of Veterinary Science, Chulalongkorn University, Bangkok,
Thailand
ABSTRACT
Intravenous megadose ascorbic acid (vitamin C, ascorbate) has been
using as a popular chemotherapeutic agent in complementary, alternative,
and integrative medicine over the past 50 years. Because of its
outstanding property in cancer treatment, ascorbic acid has gained
increasing interest in human oncology. As a result, accumulative updated
scientific basis for the use of intravenous megadose ascorbic acid as an
adjuvant treatment for human cancer patients is increasing with new
knowledge in the pharmacokinetics and pharmacodynamics of

Corresponding author: Anudep Rungsipipat. Companion Animal Cancer Research Unit,
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University,
Bangkok, Thailand 10330. Email: Anudep.R@ chula.ac.th.
222 Nabhat Thongsoi and Anudep Rungsipipat
intravenous and parenteral use. In addition, new clinical data in humans

has given a more complete understanding of the fundamental aspects of
intravenous megadose ascorbic acid and its therapeutic effect on various
types of cancer. These included in vitro and in vivo studies, as well as, the
clinical use in many types of terminal cancer patients that had underwent
standard conventional treatment with limited improvement. Recently, the
results obtained from human clinical trials in patients with advanced
untreatable malignancies revealed the outstanding anticancer effects of
intravenous ascorbic acid without any adverse effects. This study also
demonstrated prolonged survival time, improvement in global health and
quality of life. Studies related with intravenous ascorbic acid on cancer
treatment have been conducting up to the present. According to the fact
that the use of intravenous megadose ascorbic acid has never been
reported in canine cancer treatment, and most preclinical and clinical data
presented so far has only been derived from human studies. It is
interesting whether the same response could be obtained in canine cancer
patients, so, preliminary case studies were conducted to explore the
clinical response and the histopathological features of neoplastic cells
after treatment in two aggressive tumors, mammary tumor and mast cell
tumor, commonly found in dogs. The results were discussed.
Keywords: canine, ascorbic acid, megadose, cancer, intravenous
INTRODUCTION
Nowadays the incidence of cancer increase both in human and pet
population especially in dogs (Baek et al., 2009). This is regarded as a major
public health problem worldwide. Although new therapeutic protocols and
new anti-cancer drugs have been developed, their efficacies of the treatment
are still insufficient. One of the main reasons is that these drugs do not only
destroy cancer cells, but also normal healthy cells resulting in impaired
immune function, gastrointestinal function and wound healing. Over the past
50 years, there have been increased in research investigating the impact of
megadoses of nutritional substances on many degenerative diseases and on
various organ functions. Renewed interest developed in the theory by Linus
Pauling, two-time Nobel Prize winner, and molecular biologist, who stated
that some certain illness was due to abnormal concentrations of essential
nutrients. These abnormal concentrations are determined by genetic
constitution and diets. Pauling advocated the treatment of such illness by the
provision of optimal concentrations of these vital substances called
The Application of Intravenous Megadose Ascorbic Acid … 223
Orthomolecular Medicine (Cook and Botting, 1997). Orthomolecular therapy

is defined by its proponents as the treatment of disease by varying the
concentrations of substances normally present in the human body. Its
proponents claim that many diseases are caused by molecular imbalances that
are correctable by administration of the “right” nutrient molecules at the right
time. Pauling stated that daily supplementation of vitamins in optimum
amounts, together with following a healthy diet, was the most important step
that anyone could take to live a long and healthy life. This approach has been
used to treat various kinds of degenerative diseases including cancer in
humans.
The important megadose vitamin used for treatment of cancer in
Orthomolecular approach is ascorbic acid (vitamin C, ascorbate). An
important review by Pauling, Cameron, and Leibovitz presented the scientific
basis to support the use of ascorbic acid as a therapeutic agent in the treatment
of cancer (Gonzalez et al., 2005). Later, new knowledge on the
pharmacokinetics and pharmacodynamics of ascorbic acid and new clinical
data in humans have given a better complete understanding of the fundamental
aspects of ascorbic acid’s therapeutic effect on cancer. Up to the present,
accumulative updated scientific basis for the use of intravenous megadose
ascorbic acid as adjuvant treatment for cancer patients is increasing. These
included in vitro and in vivo studies, as well as, the clinical use in terminal
cancer patients that had underwent standard conventional treatment with
limited improvement. The results obtained from these studies revealed the
outstanding anticancer effects of ascorbic acid without any adverse effects.
Recently, phase I clinical trial conducted by Hoffer et al. (2008) had confirmed
that intravenous ascorbic acid appears to be safe and free of important
toxicities in patients with advanced untreatable malignancies. In addition,
Yoem et al. (2007) focused the study of intravenous ascorbic acid on the
quality of life in terminal cancer patients. This study also demonstrated
improvement in global health and quality of life along with improvement in all
functions (physical, role, emotional, cognitive and social) and some symptoms
including fatique, nausea, vomiting, sleep disturbance and appetite loss. Since
most preclinical and clinical data presented so far has been derived from
human studies. It is interesting whether the same response could be obtained in
canine cancer patients. Since the use of intravenous megadose ascorbic acid
has never been reported in canine cancer treatment. For this reason,
preliminary case studies were conducted to explore the response on the growth
of neoplastic cells, also with clinical responses in dogs with aggressive tumors.
It might be of great benefit to study the application of this approach in the
purpose of searching novel effective regimen with least adverse effects, low
cost of treatment and enhancement of better quality of life.
Intravenous Megadose Ascorbic Acid and Cancer
Ascorbic acid (vitamin C, ascorbate) is a ketolactone with a molecular

weight of 176.13 g/ml. A basic identified biochemical role for ascorbic acid is
to accelerate hydroxylation reactions in a number of biosynthetic pathways. In
many of these reactions, ascorbic acid directly or indirectly provides electrons
to enzymes that require prosthetic metal ions in a reduced form to achieve full
enzymatic activity. The best-known biochemical role of ascorbic acid is that of
cofactor for prolyl and lysyl hydroxylase enzymes in the biosynthesis of
collagen. The molecular structures of ascorbic acid and its oxidized form
dehydroascorbic acid are similar to that of glucose. The similarity is because
of several hydroxyl groups (OH) that are next to each other. The hypothesis on
ascorbic acid and cancer started as early as in 1949, ascorbic acid use was
proposed for cancer therapy. With the continuity of study, in 1952, ascorbic
acid has been proposed as a chemotherapeutic agent with concurrent
controversies against its anticancer properties among the conventional
physicians and scientists during that time (González et al., 2005). This leaded
to a great number of articles including an array of in vitro, in vivo, animal
model, and human studies, have been publishing to advocate on ascorbic acid
and cancer so far. These demonstrated the outstanding anticancer effects of
megadose ascorbic acid when giving parenterally.
The important actions of ascorbic acid include both antioxidant and pro-
oxidant activities. The former action reduces reactive oxygen species, which is
an important promoter of carcinogenesis. The latter action is selectively
cytotoxic to tumor cells when: (1) the concentration of antioxidant enzymes
(e.g., catalase) are low; (2) more intracellular transition metals (i.e., iron and
copper) are available; and (3) glucose transport of cells are over-expressed
(Zhang, 2004) These lead to an increase in hydrogen peroxide and hydroxyl
radical, causing cancer cells to die.
For more details of its mechanisms of action as a chemotherapeutic agent,
it is well known that ascorbic acid is an effective biologic antioxidant and does
not act as a pro-oxidant under normal conditions. Indeed, it was nicely shown
in humans that blood concentrations of ascorbic acid are tightly controlled as a
function of oral dose (Levine et al., 1996; Levine et al., 2001). As a
consequence, complete plasma saturation occurs at oral doses of ≥400 mg

daily, achieving physiological blood concentrations of 60-100 μM. In contrast,
intravenous infusions of ascorbic acid have been reported to achieve plasma
concentrations up to 20 mM, which is 200 times more than what it is possible
to reach orally (Padayatty et al., 2004). Interestingly, at this range of
pharmacologic concentrations (0.3-20 mM), ascorbic acid exhibits a strong
cytolytic activity in vitro against a wide variety of cancer cells (Prasad, 1980;
De Laurenzi et al., 1995; Kang et al., 2003) which seem to be strikingly more
sensitive than normal cells (Chen et al., 2005). A continued works of Chen and
co-workers showed that millimolar concentrations of extracellular vitamin kill
cancer cells but not normal cells by producing hydrogen peroxide (H2O2),
which is a strong tumoricidal substance. This killing effect is a H2O2
dependent manner (Chen et al., 2005; Chen et al., 2007; Chen et al., 2008). In
local tumor environment, ascorbic acid is postulated to exert local pro-oxidant
effects by generating H2O2 in the interstitial fluid surrounding tumor cells,
killing them or inhibiting their growth, while leaving normal cells intact (Chen
et al., 2005; Chen et al., 2007; Chen et al., 2008). The H2O2 produced seems to
be a key to ascorbic acid’s anti-tumor effect because H2O2 causes cancer cells
to undergo apoptosis, pyknosis and necrosis (Chen et al., 2005). In contrast,
normal cells are considerably less vulnerable to H2O2. The reason for the
increased sensitivity of tumor cells to H2O2 is not clear but may be due to
lower antioxidant defenses, in fact, a lower capacity to destroy H2O2 e.g., by
catalase, peroxiredoxins, and glutathione peroxidase may cause tumor cells to
grow and proliferate more rapidly than normal cells in response to low
concentrations of H2O2. It is well known that H2O2 exerts dose-dependent
effects on cell function, from growth stimulation at very low concentrations to
growth arrest, apoptosis, and eventually necrosis as H2O2 concentrations
increase (Davies, 1999). The work of Chen and co-workers in 2008 confirmed
that intraperitoneal injection of pharmacologic doses of ascorbic acid
decreased the growth and weight of human, rat, and murine tumor xenografts
in athymic, nude mice. These millimolar concentrations of ascorbic acid can
be achieved in humans by parenteral infusion such as intravenous or
intraperitoneal infusion but not by diet or supplements (Padayatty et al., 2004;
Verrax and Calderon, 2009). In summary, the accumulative works of Chen and
co-workers and other scientists strongly suggested that H2O2 is responsible for
the anticancer activity of megadose ascorbic acid giving parenterally.
Besides the mechanism mentioned above, another mechanism of
anticancer effect of megadose ascorbic acid was discovered in 1982 by
Poydock and colleagues. They found that dehydroascorbic acid, the oxidized
form of ascorbic acid has the remarkable ability to eliminate the aggressive
mouse tumors. In 1993, Jakubowski found that cancer cells (but not normal
cells) contain measurable quantities of homocysteine thiolactone. Recently,
Toohey (2008) found that dehydroascorbic acid reacts with homocysteine
thiolactone, converting it to the toxic compound, 3-mercaptopropionaldehyde.
Taken together, these findings suggested that rapidly dividing tumor cells
make unusually large amounts of homocysteine thiolactone and that
administered dehydroascorbic acid enters the cells and converts the thiolactone
to mercaptopropionaldehyde which kills the cancer cells. Another supporting
effect for cancer treatment of intravenous megadose ascorbic acid is the
involving in angiostatic activity that may help host resistance to the growth or
invasiveness of solid tumors (Ashino et al., 2003). This property was
confirmed by in vivo study of Yoem et al. (2009), who stated that carcinostatic
effect of high dose ascorbic acid occurred through inhibition of angiogenesis.
Up to the present, the studies of intravenous megadose ascorbic acid have
reached to preclinical studies in phase I clinical trial in terminal human cancer
patients. The results published demonstrated that the dose up to 1.5 g/kg have
been injected intravenously to cancer patients with minimal adverse effects
(Hoffer et al., 2008). This protocol achieved plasma ascorbic acids
concentration of 10 mM for more than 4 hours, which is largely sufficient to
induce cancer cell death in vitro. Additionally, series of case reports indicated
that intravenous megadose ascorbic acid was associated with long-term tumor
regression in three human patients with advanced renal cell carcinoma, bladder
carcinoma, and B-cell lymphoma (Padayatty et al., 2006). Latest in vitro and
in vivo works including case studies in recent years focus on the studies of
pharmacological dose of ascorbic acid alone or the combination use with
standard chemotherapies used in common human malignancies such as
pancreatic cancer, ovarian cancer, melanoma and hepatocellular (Du et al.,
2010; Espey, et al., 2011; Ma et al., 2014; Rouleau et al., 2016).
THE USE OF INTRAVENOUS MEGADOSE ASCORBIC ACID

IN A TERMINAL STAGE MAMMARY TUMOR DOG
Canine mammary tumors (CMTs) are the most common neoplasms in

intact female dogs (Sleeckx et al., 2011). Approximately 48% of dogs with
mammary carcinoma die or are euthanized within one year after surgical
removal of the primary tumor or recognition of metastases (Graham et al.,

1999). Surgery remains the treatment of choice in CMTs. At present, therapies
used to treat human breast cancer have been adopted, such as several
chemotherapeutic agents and their combinations, hormonal therapy, anti-
cyclooxygenase-2, desmopressin and radiotherapy (Hunley et al., 1993; Morris
et al., 1993; Lombardi et al., 1999; Sorenmo, 2003). However, the efficacy of
these methods is still limited and are also associated with significant toxicities
and certain complications. Like other types of canine cancer, an effective
treatment strategy continues to be warranted.
In human alternative medicine, megadose of ascorbic acid has been
applied to treat and prevent various types of cancer including breast cancer
(Yeom et al., 2007). As the use of megadose ascorbic acid for treatment of
canine mammary tumors has not ever been reported. Therefore, we applied
intravenous megadose ascorbic acid as an alternative treatment in the terminal
stage of canine mammary tumor, which already had metastasized to the lungs,
and had not responded to prior conventional chemotherapy.
Case history: An 11-year-old female, golden retriever, weighing 25 kg,
had mammary mass at cranial and caudal mammary glands of both sides. The
mass had been removed 2 times. Seven months after the second surgical
removal, the dog had right axillary (size: 7 x 3 x 3 cm), right prescapular (size:
12 x 4 x 3 cm) and left prescapular lymph node (size: 6 x 3 x 2 cm)
enlargement. The histopathological diagnosis was mammary adenocarcinoma
with metastases to the lymph nodes. Radiography showed multiple tumor
masses, varying in sizes, invading all lung lobes and mild splenomegaly
(Thongsoi et al., 2012).
Conventional chemotherapy with a single intravenous infusion of
doxorubicin (A.D. mycin; Boryung Pharma, Korea) 30 mg/m2 body surface
area every three weeks for 2 cycles was the initial treatment. Two weeks
following the infusion, the dog developed cancer cachexia and mild anemia
caused by bone marrow toxicity. The dog’s palpable lymph nodes also
remained unreduced in size. As a result, conventional treatment with
doxorubicin was discontinued and replaced by intravenous megadose ascorbic
acid in combination with an organic homemade diet, on the basis of
intravenous doses used in humans (Padayatty et al., 2010). We speculated that
the dog would require lower intravenous doses of ascorbic acid. Regarding the
life-threatening effect of tumor hemorrhage and necrosis that can result from
intravenous AA (Cameron and Pauling, 1993), we used an intravenous dose
that we believed was safe, followed by daily oral doses of ascorbic acid. The
dog was administered 250 mg/kg/day of ascorbic acid (Vitamin C Injection;
T.P. Drug Laboratories, Thailand) over 60 minutes in a 100 ml sterile normal

saline bag intravenously for 14 days. This was followed by 4,000 mg/day
orally of ascorbic acid (Nat-C; MEGA Life Sciences, Thailand) for 30 days.
The organic homemade diet was given to the dog twice daily and included
brown rice, marine fish, organic vegetables and sometimes eggs, cheese and a
brewer’s yeast supplement. The dog was kenneled in the private hospital for
the duration of its treatment.
One week after beginning the megadose of ascorbic acid and organic
homemade diet, the dog developed symptoms of depression, an intermittent
fever, and a productive cough. Blood work done during this period showed
moderate anemia, thrombocytopenia, and mild neutrophilic leukocytosis. Liver
and kidney function tests were within normal limits. By day 16 (2 days after
finishing the course of intravenous ascorbic acid), another CBC was done.
Hematologically, the anemia was improving, even though the neutrophilic
leukocytosis persisted. By day 35, the dog looked better and displayed the
“typical” features of a normal dog. The dog’s weight had increased by 3 kg
(i.e., 25 kg before treatment to 28 kg). All the mentioned lymph nodes gained
partial remission (defined as a greater than 50% reduction in tumor volume).
Remarkably, the right axillary lymph node decreased to 2 x 1 x 1 cm in size
compared to its enlarged size before treatment (7 x 3 x 3 cm). The owner took
the dog home from the private hospital during long holidays and fed the dog
with ordinary food and dessert, then the clinical signs got worse and the dog
died 3 weeks later and necropsy was performed. This dog had survived for 17
months following the first surgery, which is longer than what has been
previously reported.
Gross evaluation demonstrated right axillary, and right and left pre-
scapular lymph node enlargement. The thoracic cavity was filled with
approximately 1,000 ml sero-sanguineous transudate. Multiple white firm
nodules, varying in size, appeared in all lung lobes. Histopathology of the
mass sections showed extensive necrosis; pyknosis of tumor cells and
suppurative areas with intralesional hemorrhage. The tumor cells were loosely
arranged, having an island and glandular structure, with few inflammatory cell
infiltrations. The lymph nodes and lungs showed massive necrosis of
metastasized tumor areas. The cause of death was respiratory failure due to
hydrothorax. Hydrothorax developed from anemia and hypoproteinemia, as a
result of anorexia and cancer cachexia.
Generally, in the advanced terminal stages of tumors in dogs, the disease
will rapidly progress and result in death. Before megadose ascorbic acid
treatment, this dog showed the same pattern of disease as other dogs having
cancer. However, dramatic results were observed during five weeks of

megadose ascorbic acid treatment, starting with large intravenous doses for 14
days followed by large oral doses for 30 days. The lymph node mass
diminished in size and the dog’s quality of life and behavior normalized,
which obviously showed a therapeutic effect from megadose ascorbic acid.
We speculate that parenteral ascorbic acid caused tumor hemorrhage,
which might have resulted in the decrease we observed in the RBCs and
platelets during therapy. The neutrophilic leukocytosis may have been the
result of tumor necrosis and/or the enhancement of the immune system by
ascorbic acid. Nevertheless, these effects were not deemed clinically
detrimental while the dog was treated in the hospital since there was regression
of lymph node size and the dog’s quality of life and behavior normalized.
We believe that the pro-oxidant activities of AA likely caused the massive
destruction of neoplastic cells and the accompanying necrosis and hemorrhage.
Since some of the necrotic areas showed evidence of repair (i.e., they were
being replaced by fibrosis), it is also possible that ascorbic acid was preventing
further tumor dissemination as a result of its function in collagen synthesis
(Zhang, 2004; Li and Schellhorn, 2007).
THE USE OF INTRAVENOUS MEGADOSE ASCORBIC ACID

IN TERMINAL STAGE MAST CELL TUMOR DOGS
Mast cell tumors (MCTs) are the most common skin tumors in dogs and
the second most common malignancy found in this species (Goldschmidt and
Hendrick 2002). It accounts for 7-21% of all canine skin tumors. In canines
and humans, malignant mast cell disease is known to link to dysregulation of
the tyrosine kinase receptor, KIT (Newman et al., 2007). Mast cell tumor
(MCT) causes various paraneoplastic syndromes due to their cytoplasmic
granules. Destruction of tumor cells can cause harmful effects including death
from the release of histamine, heparin, and other vasoactive amines (Govier,
2003). The treatment modalities commonly used for canine MCTs include
surgery, radiotherapy, chemotherapy and supportive care. Current
chemotherapeutic treatment for these MCTs generally include one or multiple-
agent regimens with vinblastine, corticosteroids and alkylating agents. The
regimens with either a combination of vinblastine and prednisolone or a
lomustine (CCNU) single-agent protocol are considered standard treatment
(Henry et al., 2007). Unfortunately, these protocols obtained a variable degree

of response, mostly were the partial response.
We instituted megadose ascorbic acid as an alternative new treatment
modality for three terminal stage MCT dogs as in human (Padayatty et al.,
2004) that had already undergone standard therapy with little response. Our
purpose is to investigate the results of treatment and to find out the optimal
dose and route of administration that is practical and has the most effective
outcome in treatment. We found the most serious unexpected systemic
hypersensitivity resulting in death from massive degranulation effect in these
dogs after receiving megadose ascorbic acid.
Case 1 An 11-year-old, 18 kg wt., male, mixed breed dog had been
diagnosed as clinical stage 3a and histopathological grade II MCT. A thoracic
radiograph showed no evidence of lung metastasis and abdominal radiograph
revealed hepatomegaly. The mass was very large, size 33 x 30 x 10 cm at right
inguinal area which was unresectable. The dog was treated with
chemotherapy, prednisolone and vinblastine sulfate with partial response at the
beginning and later with stable disease. Finally, the treatment with megadose
ascorbic acid and natural diet including brown rice, sea fish, organic
vegetables and sometime eggs, supplemented with multivitamin and minerals
tablets were introduced. The dose of ascorbic acid given at the beginning was
130 mg/kg/day intravenously once a week for 3 weeks, after that, the dog
developed anemia and anorexia with 2 sites ulcerated wound at the mass. The
first blood transfusion was done with an improvement of general status. The
treatment continued with ascorbic acid 4,000 mg/day orally for 10 days. The
dog gained weight and looked better in general appearance with good appetite
and healing of ulcerated wound. Later, we changed to 3,500 mg/day of vitamin
C added in 100 ml sterile normal saline given subcutaneously, once a day, for
four days with the same response. At last, the double dose of 3,500 mg/day of
vitamin C added in 100 ml sterile normal saline given subcutaneously twice a
day for four days was introduced. The obvious responses were that the dog
started depressed and had signs of anorexia with purple and edematous of skin
at tumor on the fifth day. The treatment diminished and the dog was sent to
emergency unit due to the progressive deleterious shock signs. The dog died
the next day after receiving the second blood transfusion and necropsy was
performed.
Gross lesions showed massive intratumoral necrosis and hemorrhage with
bloody exudate. Histopathologic findings of mass showed variable features of
dynamic process of destroying tumor cells including infiltration of many
inflammatory cells, mostly neutrophils, and extensive congestion and
hemorrhage. Foamy, swollen, pyknosis of tumor cells in multifocal necrosis

areas was evident. Anaphylaxis and hypovolemic shock caused by massive
degranulation of MCT were diagnosed as a cause of death.
Case 2 A 9-year-old, 30 kg wt, castrated male, mixed breed dog had
clinical stage 3b and MCT grade II at left forelimb, thoracic and inguinal area.
Prednisolone 1 mg/kg/day and ascorbic acid 2,000 mg/day orally were
prescribed without natural food. Two weeks later, the mass was markedly
increased in size with swelling and erythema. Ascorbic acid 7,000 mg added in
100 ml sterile normal saline was given intravenously. The dog developed signs
of, depression, anorexia and ulcerated wound on the next day. The dog died
four days later and necropsy was performed.
Gross lesions showed inguinal mass size 13 x 19 x 4.5 cm. with multifocal
superficial ulceration and intratumoral necrosis. The lung showed severe
congestion with multilobulated mass at mediastinal area size 5 x 4 x 1 cm.
Abdominal cavity was filled with 500 ml of blood exudates. Histopathology
revealed similar lesions to case 1, but in a lesser extent and the same diagnosis
was achieved.
Case 3 A 15-year-old, 11.8 kg wt, male, mixed breed dog, presented with
MCT grade II and mastocytemia. The tumor mass was at the right axillary area
and was treated with surgical excision. Prednisolone at the dose of 1
mg/kg/day was prescribed after surgery. Nine months later, the mass recurred
with continuous swelling at the surgical site, though, prednisolone was in use.
Ascorbic acid 1,000 mg/day PO for 2 wks and 2,000 mg/day PO for 4 wks
were additional prescribed without natural food. The result was that the mass
still increased in size to be 15 x 12 x 12 cm. Blood collection was done with
the result of 86 IU, 1,354 IU, 24 mg% and 0.9 mg% for ALT, ALP, BUN and
creatinine, respectively. Intravenous megadose ascorbic acid added in 100 ml
sterile normal saline at 3,000 mg/day was given and prednisolone was
diminished, 3 days later, the dog developed the signs of anorexia, marked
depression, swelling of left forelimb, edema of abdominal skin, panting and
fever. The dog died on the next day without necropsy.
This is another one of our studies that outstanding cytotoxic effect of
megadose ascorbic acid shown by histopathological pictures was revealed. The
extensive necrosis, hemorrhage areas and loosely arrangement of tumor cells
indicated massive destruction of the mast cells. Nevertheless, rapid massive
death of MCT resulted in fatal degranulation effects from vasoactive amines in
mast cells granules. In addition with the fact that the lack of data in ascorbic
acid dosage for cancer in dogs, adjustment of optimal dosage in MCT and in
other types of canine cancer should be carefully done to yield a suitable
cytotoxic effect with least negative effects such as rapid tumor hemorrhage.
Therefore, a cancer therapy with megadose ascorbic acid should be started at a
low range to ensure that tumor hemorrhage will not occur (Riordan et al.,
1995). From this study, we can presumably conclude from case 1 that ascorbic
acid at the dose of 195 mg/kg/day subcutaneously in combination with 250
mg/kg/day orally seem to be safe and effective. The adverse effects occurred
at the dose of 390 mg/kg/day given subcutaneously. This study is a useful
example for treatment of terminal stage MCT, because it showed that ascorbic
acid treatment effected and improved quality of life in case 1 when used with
appropriate dosage and again, we also observed that the kind of food has great
influence on cancer conditions.
In conclusion, our works are the first preliminary clinical studies of using
megadose ascorbic acid in terminal stage canine cancer patients. It became
evident that megadose ascorbic acid has very interesting anticancer properties
by giving parenterally to yield pharmacological action in canine cancer
patients. Starting with this study, we hope to pave the way for further studies
on the use of intravenous megadose ascorbic acid in the field of veterinary
oncology. We emphasized that the use of megadose ascorbic acid is needed to
be further investigated for their chemotherapeutic properties in dogs and cats
in a larger scale.
The clinical trials in this aspect are warranted to justify its effective value
in animal cancer treatment.
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BIOGRAPHICAL SKETCH
Name: Nabhat Thongsoi, DVM
Affiliation: Department of Surgery and Theriogenology, Faculty of
Veterinary Medicine, Khon Kaen University, Khon Kaen THAILAND 40002
Dr. Nabhat Thongsoi is an officially veterinary surgeon and has been
working at the Department of Surgery and Theriogenology, Faculty of
Veterinary Medicine, Khon Kean University, Thailand. After graduating from
Chulalongkorn University, Bangkok, in 1992. In relation to teaching, Dr.
Nabhat is involved in the teaching of undergraduate and internship veterinary
students. Main research interest is veterinary complementary medicine
particularly application of orthomolecular medicine for treatment of cancer
dogs.
Name: Anudep Rungsipipat, DVM, PhD, DTBVP

Affiliation: Companion Animal Cancer Research Unit,
Department of Pathology, Faculty of Veterinary Science
Chulalongkorn University, Bangkok, THAILAND 10330
Dr. Anudep Rungsipipat is an officially veterinary pathologist and has

been working at the Department of Pathology, Faculty of Veterinary Science,
Chulalongkorn University, Thailand. After graduating from Chulalongkorn
University, Bangkok, in 1992 with second Class Honors in DVM. He got PhD
degree in veterinary pathology and preventive medicine from The United
Graduated School of Veterinary Science, Yamaguchi University (University of
Miyazaki), Japan in 1999. In relation to teaching, Dr. Anudep is involved in
the teaching of undergraduate, research postgraduate and internship veterinary
students. His teaching has earned his the Teacher of the Year 2013 award from
Chulalongkorn University. Dr Anudep’s main research interests include
diagnostic approaches, molecular diagnostic strategies and innovative
applications of companion animal oncology. Key examples of his recent
research achievements include classification, molecular diagnosis and
detection of minimal residual disease in canine and feline lymphomas,
potential effects of herbs for prevention of experimental colon carcinoma
model and application of orthomolecular medicine for treatment of cancer
dogs. In term of professional service, Dr. Anudep is an editor-in-chief The
Thai Journal of Veterinary Medicine, Faculty of Veterinary Science,
Chulalongkorn University and Chair of The Graduated Program of Veterinary
Pathobiology, Department of Pathology and Department of Microbiology,
Faculty of Veterinary Science, Chulalongkorn University.
Publications Last Three Years:
[1] Promtes K, Kupradinun P, Rungsipipat A, Tuntipopipat S, Butryee C

2016. Chemopreventive effects of Eryngium foetidum L leaves on COX-
2 reduction in mice induced colorectal carcinogenesis. Nutrition and
Cancer: 68(1):144-153, DOI: 10.1080/ 01635581.2016.
[2] Rungsipipat A, Sitthicharoenchai P, Marlow P, Prutthithaworn P,
Tangkawattana S 2016. Expression of Metallothionein Protein Relating
to Proliferative Cell Index in Feline Malignant Mammary Tumors Using
High Throughput Tissue Microarray Technique. Comp. Clin. Pathol. 25:
449-457 DOI 10.1007/s00580-015-2208-7.
[3] S. Techangamsuwan, W. Banlunara, A. Radtanakatikanon, A.
Sommanustweechai, B. Siriaroonrat, E. D. Lombardini and A.
Rungsipipat, 2014. Pathologic and Molecular Virologic Characterization
of a Canine Distemper Outbreak in Farmed Civets Vet Pathol published
online 24 September 2014 DOI: 10.1177/0300985814551580.
[4] Chaiyabutr N, Vesaruchapong T, Chanhome L, Rungsipipat A, Sitprija
V 2014. Acute Effect of Russell’s Viper (Daboia siamensis) Venom on
Renal Tubular Handling of Sodium in Isolated Rabbit Kidney. Asian
Biomedicine 8(2): 195-202.
[5] Sailasuta A, Ketpun D, Piyaviriyakul P, Theerawatanasirikul S,
Theewasutrakul P, Rungsipipat A 2014. The Relevance of CD117-
Immunocytochemistry Staining Patterns to Mutational Exon-11 in c-kit
Detected by PCR from Fine-Needle Aspirated Canine Mast Cell Tumor
Cells. Vet. Med. Int. Vol. 2014, Article ID 787498, 8 pages http://dx.
doi.org/10.1155/2014/787498.
[6] Roberts BM, Chumpolkulwong K, Tayamun S, Inamnuay L,

Rungsipipat A, Lombardini ED 2014. Mammary carcinoma in a male
rhesus macaque (Macaca mulatta): histopathology and
immunohistochemistry of ductal carcinoma in situ. J. Med. Primatol.
doi:10.1111/jmp.12110.
[7] Jiranantasak T, Rungsipipat A, Surachetpong S 2014. Histopathological
changes and apoptosis detection in canine myxomatous mitral valve
disease using tissue microarray technique. Comp. Clin. Pathol. 23: 1173-
1178 DOI 10.1007/s00580-013-1759-8.
[8] Rungsipipat A, Chayapong J, Jongchalermchai J, Thongruk T, Manachai
N, Wangnaitham S, Techangamsuwan S 2014. Histopathological and
immunophenotyping classification of spontaneous canine lymphoma in
Bangkok metropolitan. Comp. Clin. Pathol. 23: 213-222.
[9] Manachai N, Lacharoje S, Techangamsuwan S, Rungsipipat A. 2014
Detection of minimal residual disease (MRD) in canine lymphoma
Comp. Clin. Pathol. 23: 199-204.
Chapter 11
ASCORBIC ACID AS A MULTIFUNCTIONAL

NUTRIENT IN MAMMALS: OUR
UNDERSTANDING BASED ON STUDIES USING
GENETICALLY MODIFIED MICE
Junichi Fujii , Takujiro Homma and Sho Kobayashi

*
Department of Biochemistry and Molecular Biology, Graduate School of

Medical Science, Yamagata University, Yamagata, Japan
ABSTRACT
Ascorbic acid (AsA) functions as a cofactor in a variety of
physiological reactions including collagen synthesis and monoamine
production and also functions as an essential antioxidant by scavenging
reactive oxygen species. Hence a deficiency of AsA results in an
imbalance of cellular homeostasis and can be fatal, as is typically
observed in scurvy. While most organisms, including vertebrates, are able
to synthesize AsA, primates and some other animals such as the guinea
pig cannot due to a loss of L-gulono--lactone oxidase (GULO) function,
which catalyzes the final step of the AsA synthesis in the endoplasmic
reticulum. Although the mouse is the most popular animal used in
*
Corresponding author: Department of Biochemistry and Molecular Biology, Graduate School of
Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. Tel. +81-
23-628-5229 Fax +81-23-628-5230. E-mail: jfujii@med.id.yamagata-u.ac.jp.
240 Junichi Fujii, Takujiro Homma and Sho Kobayashi
biomedical research, it is not an adequate animal for studying the

physiological and pathological roles of AsA for this reason. Genetically
modified mice that have a defect in processes of the AsA synthesis have
been developed to conquer this disadvantage and are now used in studies
to reveal currently unrecognized functions of AsA in vivo.
At present genetically modified mice that have defects in the last
three steps of the AsA synthetic pathway have been established. The
GULO−/− mouse was first established in 2000 and has since been used as
an AsA-deficient mouse model. Gluconolactonase (GNL) is known as a
senescence-associated marker protein 30 (SMP30) with uncertain
functions. The identity of SMP30 as GNL was demonstrated in 2006 by
using SMP30−/− mice that were established in 2002. In 2010, studies
using mice lacking AKR1A and AKR1B, which are members of the aldo-
keto reductase superfamily, revealed that they catalyze the conversion of
D-glucuronate to L-gulonate in the AsA synthesis pathway. Because the
liver is the major organ for producing AsA and AKR1A is predominantly
expressed in the liver, AKR1A contributes about 85-90% of the AsA that
is synthesized in the body of the mouse.
In this review article, we provide an overview of the literature
regarding studies in which these genetically modified mice have been
used and attempt to gain insights into the physiological and pathological
roles of AsA in vivo. We also note that some of the studies may have
limitations because the gene products may have additional functions in
addition to the synthesis of AsA. Thus we will pay attention to this issue
in this review and attempt to obtain a clear vision of the roles of AsA in
the mouse.
INTRODUCTION
Ascorbic acid (AsA) functions as a redox cofactor for a variety of
physiological reactions that include collagen synthesis, monoamine
production, and steroidogenesis as well as serving as an antioxidation (Mandl
et al., 2009). AsA synthesis starts from glucose-1-phosphate, a primary
product of glycogenolysis (Figure 1) (Linster and Van Schaftingen, 2007).
Thus, AsA synthesis is tightly associated with glycogen metabolism. The last
step of AsA synthesis is catalyzed by L-gulono-γ-lactone oxidase (GULO)
[EC 1.1.3.8], which is localized at the endoplasmic reticulum (ER) membrane,
with its catalytic domain facing the ER lumen. A mutation in the GULO gene
occurred about 63,000,000 years ago, leading primates being incapable of
synthesizing AsA (Nishikimi et al., 1994), while most animals can synthesize
AsA. The recommended intake of AsA is 100 mg/day for the adult male. An
Ascorbic Acid as a Multifunctional Nutrient in Mammals 241
insufficient intake of AsA (vitamin C) from nutrients is associated with

unhealthy conditions. 2 to 3 months of a diet without AsA results in the
development of scurvy that typically involves aberrant collagen synthesis and
bleeding.
Glycogen
Glucose 1-P (G1P) UDP-Glucose
Glucose 6-P (G6P) L-gulono-

-lactone
G6Pase G6PT GT
Pi
L-gulono-
Glucose Glucose 6-P
-lactone
Gulo
AsA
ER
Blood glucose AsA
Figure 1. Glycogen in the liver is the common source of blood glucose and AsA.
In the liver, the phosphorolysis of glycogen produces glucose-1-phosphate (G1P)
which is then either isomerized to glucose-6-phosphate (G6P) or converted to UDP-
glucose. G6P is transported into the endoplasmic reticulum (ER) via the G6P
transporter (G6PT) and is then dephosphorylated to glucose by G6P-phosphatase
(G6Pase). UDP-glucose is converted by following consecutive reactions to L-gulono-
-lactone, which is then transported into the ER lumen via the gulonolactone
transporter (GT). L-gulono--lactone is consequently oxidized to AsA by GULO.
The penultimate reaction in the AsA synthesis pathway is catalyzed by

gluconolactonase (GNL; EC 3.1.1.17), which catalyzes the dehydration of L-
gulonate to L-gulono-γ-lactone (Figure 2). Kondo et al. (2006) showed that the
senescence marker protein 30 (SMP30) is identical to GNL using the
SMP30−/− mouse. Aldehyde reductase (AKR1A; EC 1.1.1.2) and aldose
reductase (AKR1B; EC 1.1.1.21) have recently been reported to catalyze the
reduction of D-glucuronic acid to L-gulonate using NADPH (Gabbay et al.,
2010; Takahashi et al., 2012). The contributions of AKR1A and AKR1B to
AsA synthesis are 85-90% and 10-15%, respectively. Because the pathway for
AsA synthesis diverges in the early steps, the redundancy in the reaction
pathway may compensate for the gene deficiency and only minimally affects
AsA synthesis.
Glycogen
Glucose-1-P
UDP-Glucose
UDP-Glucuronate
D-Glucuronate -1-P -D-Glucuronide
Glucuronate conjugation D-Glucuronate

Akr1a/Akr1b
(9 : 1)
3-Keto-L-gulonate L-Gulonate
Gnl
L-Gulono--lactone
Gulo
ER
AsA
Figure 2. The pathway for the AsA synthesis depicting enzymes involved in the last
three steps.
Aldehyde reductase (AKR1A) and aldose reductase (AKR1B) differentially catalyze
the NADPH-dependent reduction of D-glucuronate to L-gulonate, which is then
dehydrated to L-gulono-lactone by gluconolactonase (GNL). The resulting L-
gulono--lactone is finally oxidized to L-ascorbic acid by the action of L-gulono-γ-
lactone oxidase (GULO).
Pivotal roles of three classes of genes that catalyze three steps of AsA
synthesis have been unveiled using genetically modified mice lacking the
corresponding genes. In this chapter, we briefly overview the phenotypes of
the genetically modified mice that are defective in GULO, GNL, AKR1A, and
AKR1B. The substrate specificity of some of these enzymes is so general that

a genetic deficit may result in different outcomes in the mice.
GULO−/− MICE AS A PATHOLOGICAL MODEL

GULO catalyzes the oxidative conversion of L-gulono-γ-lactone to L-
ascorbate, the final step of AsA biosynthesis. Cloning of the nonfunctional
GULO gene in humans shows a probable deletion of the regions
corresponding to exons VIII and XI of the rat orthologue (Nishikimi et al.,
1994). The GULO−/− mouse has now been established and shows a total defect
in AsA synthesis (Maeda et al., 2000). Feeding regular chow that contains
about 110 mg of AsA/kg still causes weight loss in the GULO−/− mouse, and
supplementation of AsA (330 mg/liter) is required to support the normal
growth of the mutant mouse. Studies using the GULO−/− mouse has revealed
multiple roles of AsA that are not recognized when conventional mice are
being used (Yu and Schellhorn, 2013). Metabolomic analysis using proton
NMR spectroscopy has been applied to the GULO−/− mouse with or without
AsA administration and changes in metabolites of pathways associated with
other antioxidants, primarily glutathione, and possibly uric acid have been
shown (Duggan et al., 2011).
A defect in AsA synthesis causes aberrant collagen synthesis, resulting in
scurvy. AsA also functions as a non-enzymatic reducing agent for various
natural compounds and metal ions (Foyer and Noctor, 2011). Although AsA is
essential for the survival of the GULO−/− mouse, its absence does not lead to
measurable changes in proline hydroxylation, collagen production,
angiogenesis associated with tumors, or mammary gland growth (Parsons et
al., 2005). In an AsA deficiency, HIF-1 is up-regulated and blocks neutrophil
apoptosis under normoxic conditions, indicating that AsA is required for
resolving inflammation (Vissers and Wilkie, 2007). The efficiency of AsA
uptake depends on the route and composition of the nutrient. For example, the
kiwifruit has been found to be a more effective source of AsA supplementation
than when it is administered via the drinking water (Vissers et al., 2011).
GULO−/− male mice born from GULO+/− mothers show apoptosis of
spermatocytes frequently at 20 days of age. The expression of the heat-shock
protein (Hsp) 70 is impaired, which appears to cause failed meiosis in the
testes of GULO−/− mice (Yazama et al., 2006). An AsA deficiency as well as a
human chorionic gonadotropin (hCG) treatment affects the microRNA
expression in follicles (Kim et al., 2010), implying that AsA may exert more
divergent effects through the expression of the microRNA.
GULO−/− mice show decreased voluntary locomotor activity, diminished
physical strength, and an increased preference for a highly palatable sucrose
reward during scorbutic periods under low AsA supplementation (Ward et al.,
2013). AsA supplementation returns these behaviors to the levels of the
controls. An AsA deficiency is associated with decreased blood glucose levels,
elevated oxidative damage in the cortex, and a decrease in the levels of
metabolites derived from dopamine and serotonin. When GULO−/− mice are
supplemented with low levels of AsA (220 ppm), they are less active in
moving, consistent with a mild motor deficit, but show exaggerated
hyperactivity to a dopaminergic agonist (Chen et al., 2012). Motor
performance decreases in GULO−/− mice without AsA supplementation, but is
not affected by a vitamin E deficiency alone (Pierce et al., 2013). Analyses of
pups derived from heterogenous mice parents show that fetal GULO−/− mice
possess about an equivalent amount of AsA as wild-type mice (Harrison et al.,
2010a). AsA contents in the liver and cerebellum of these mice are markedly
low on postnatal day 10, and malondialdehyde levels, an oxidized lipid
product, concomitantly increase, suggesting that AsA has a significant role in
antioxidation. GULO−/− mice supplemented with low levels of AsA show
elevated oxidative stress in the cortex and cerebellum and a decreased strength
and agility deficit (Harrison et al., 2008). When the AsA contents in the
GULO−/− mice are compared with those in the AsA-supplemented mice, the
cerebellum, olfactory bulbs, and frontal cortex were found to contain the
highest levels of AsA, while the pons and spinal cord contained the lowest
(Harrison et al., 2010b). When low levels of AsA are supplemented, the level
of malondialdehyde in the cortex continues to increase compared to the
cerebellum or pons, suggesting importance of AsA in protection against
oxidative stress in the cortex.
Plasma levels of AsA are decreased in Alzheimer’s disease patients
compared to healthy individuals (Charlton et al., 2004). Transgenic mice that
overexpresses both the amyloid precursor protein (APP) and presenilin 1
(PSEN1), referred to as APP/PSEN1 mice, is a mouse model for amyloid-
plaque formation, a hallmark of and a primary pathogenic agent of
Alzheimer’s disease. An acute AsA deficiency does not affect impaired spatial
learning in GULO−/− mice that possess APP/PSEN1 transgenes. A long-term
AsA deficiency, however, leads to hyperactivity and elevated oxidative stress.
The interaction between AsA and the cholinergic system appears to be
important in the cholinergic degradation associated with Alzheimer’s disease
(Harrison et al., 2010c). Another study, using 5XFAD mice under a GULO−/−
background was reported (Kook et al., 2014). 5XFAD is an early-onset
transgenic mouse model of Alzheimer's disease that shows Aβ pathology with
two APP mutations and two PS1 mutations under regulation by the Thy1
promoter. A reduction of amyloid plaque, amelioration of blood-brain barrier
integrity, and mitochondrial morphology are observed when a higher dose of
AsA is given to the model mice.
Although plolyl-4-hydroxylation is necessary for the proper structural
assembly of oxygen-dependent protein stability of hypoxia-inducible
transcription factors (HIFs), an AsA deficiency showed normal HIF-dependent
gene expression in GULO−/− mice (Nytko et al., 2011). This may be due to
complementation by glutathione for the AsA requirement of HIF plolyl-4-
hydroxylase activity. Nonetheless an AsA insufficiency results in the increased
death of cardiomyocytes, the expression of matrix metalloprotease (MM)-2
and -9, and lipid peroxidation products (Kim et al., 2013). Water restrain stress
accelerates the death of mice as a consequence of cardiac damage. The
development of atherosclerosis is enhanced in GULO and ApoE double
deficient mice (Nakata and Maeda, 2002). Advanced atherosclerotic plaques in
low AsA mice show a reduced amount of collagen, large necrotic cores within
plaques, and reduced fibroproliferation and neovascularization in the aortic
adventitia, which is potentially vulnerable to rupture. Aldehyde
dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin and is
involved in nitroglycerin-dependent vascular relaxation. An AsA deficiency
accelerates the proteasomal degradation of ALDH2, which results in the
vascular tolerance to nitroglycerin (Wölkart et al., 2013). In the lung, an AsA
deficiency inhibits Cl− secretion in the airway epithelium, which appears to be
caused by a decreased expression of the cystic fibrosis conductance regulator
(CFTR) (Kim et al., 2011).
When liver injury is induced in GULO−/− mice by the administration of
concanavalin A, hepatocyte apoptosis and liver damage are induced by an AsA
insufficiency (Bae et al., 2013). Proinflammatory cytokines, TNF- and IFN-
, are elevated in the AsA-deficient GULO−/− mouse model. Although the
levels of interleukin-22, a hepatoprotective cytokine, are high, a defect occurs
in the expression of the receptor IL-22R and activation of downstream
STAT3 signaling. When porphyria cutanea tarda is induced in GULO−/− mice
by treatment with 3,3’,4,4’,5-pentachlorpheny, AsA effectively suppresses the
accumulation of uroporphyrin (Gorman et al., 2007). However, the
suppressive effects of AsA are not observed in the mice that were administered
excessive levels of iron. Thus, AsA was found to suppress the accumulation of
hepatic uroporphyria under conditions of low hepatic iron but was not
effective for the case of high iron. When thioacetamide, a fibrogenic agent, is
administered to GULO−/− mice, an AsA insufficiency causes a decreased
survival rate, aggravation of liver damage, and enhanced fibrosis (Kim et al.,
2014). Because oxidative stress markers are elevated in the case of an AsA
insufficiency, the underlying mechanism appears to be a low ROS-scavenging
ability.
Neutrophil extracellular trap (NET) is a novel mechanism for killing
pathogens, but an excessive formation of NET may injure tissues under
pathogenic conditions such as sepsis. NET produced under sepsis is high in
AsA-deficient GULO−/− mice and becomes attenuated in the AsA-
supplemented mouse (Mohammed et al, 2013). Thus, AsA appears to be
regulating NET formation and, hence, results in protecting tissues under sepsis
conditions. In sepsis, patients die as the result of the multiple organ
dysfunction syndrome. AsA-deficient GULO−/− mice are more susceptible to
the multiple organ dysfunction syndrome, which is effectively attenuated by
an infusion of AsA (Fisher et al., 2014). Because an AsA deficiency in
GULO−/− mice increases the lung pathology of the influenza virus, AsA is
required for an adequate immune response in the lung after an influenza virus
infection (Li et al., 2006). The cytotoxic activity of NK cells against ovarian
cancer cells is reduced in the AsA-deficient GULO−/− mouse (Kim et al.,
2012). In these NK cells, IFN- secretion, the expression of perforin and
granzyme B are also reduced, suggesting that AsA is also involved in natural
immunity. Ascorbate reacts rapidly with oxidants produced by activated
neutrophils in vitro, and neutrophils markedly increase their oxidant
production when mice are infected intraperitoneally with the gram-negative
bacterium Klebsiella pneumoniae (Gaut et al., 2006). When the GULO−/−
mouse is infected with K. pneumoniae, AsA deficient mice are vulnerable to
infection and die. Although oxidized amino acids and F2-isoprostan, a marker
for lipid peroxidation, are elevated by the infection, AsA supplementation does
not affect their levels. Thus, AsA may not protect amino acids or lipids against
oxidative damage during an acute inflammation.
High doses of AsA appear to function as a prodrug for anti-cancer agents
and be applied as a form of therapy for cancer treatment (Du et al., 2012). The
GULO−/− mouse has the potential to permit the efficacy of AsA to be
examined and has been used in some experimental trials as follows. GULO−/−
mice that were implanted with B16FO murine melanoma cells carry smaller
tumors by AsA supplementation (Cha et al., 2011). The serum inflammatory
cytokines IL-6 and IL-1 are concomitantly decreased in these mice as the
result of AsA administration. AsA supplementation also suppresses tumor

metastasis (Cha et al., 2013). Experiments on tumor cell transplantation using
4T1 breast cancer cells showed a reduction in tumor weight and serum
inflammatory cytokine IL-6 levels in AsA-supplemented GULO−/− mice. In
the meantime, AsA-deficient GULO−/− mice are more susceptible to the
carcinogenic effects of nickel subsulfide (Ni3S2). Supplementation with AsA
tends to attenuate the acute toxicity of Ni3S2 and to extend the latency of
transplanted tumors (Kasprzak et al., 2011).
A mouse strain that develops spontaneous fractures (sfx) at an early age
was developed (Beamer et al., 2000) and was found to have the GULO gene
deleted (Jiao et al, 2005). Thus, the sfx mouse is another mouse model for a
GULO deficiency. Because the mouse exhibits an AsA deficiency similar to
the GULO−/− mouse, the phenotypic characteristics of the sfx mouse are
similar to the GULO−/− mouse (Mohan et al., 2005) and has been used in
analyses of gene expression from the viewpoints of antioxidation and bone
formation (Yan et al., 2007; Jiao et al., 2013; Wei et al., 2014; Huang et al.,
2014).
APPLICATION OF GNL−/− MICE AS PATHOLOGICAL

MODELS
Gluconolactonase (GNL), which is identical to the senescence marker
protein 30 (SMP30), catalyzes the penultimate step in AsA synthesis (Kondo
et al., 2006; Aizawa et al., 2013). The GNL−/− mouse has a lower body weight
and a shorter in life span than WT mice but is viable and fertile (Ishigami et
al., 2002). The expression of GNL decreases with the aging process in a
variety of tissues including the heart, lung, liver, and kidney (Mori et al., 2004;
Ishigami et al., 2015). Accelerated senescence is observed in the tubular
epithelia of GNL−/− mice kidneys (Yumura et al., 2006). However, SMP30
was identified as the GNL several years after establishing GNL−/− mice.
GNL−/− mice show an AsA deficiency and their phenotypes are similar to
GULO−/− mice (Kondo et al, 2006; Maruyama et al., 2010). AsA-deficient
GNL−/− mice suffer age-related hearing loss, as judged from an increase in
auditory brainstem response thresholds and a decrease in the number of spiral
ganglion cells (Kashio et al., 2009). Using GNL−/− mice, the absorption and
distribution of AsA in organs after the oral administration of AsA in drinking
water (Iwama et al., 2011) or the intake of potato chips (Kondo et al., 2014a)
have been reported. AsA becomes distributed to the liver and organs in 3 h but
to some organs such as the central nervous systems, testes, and the thyroid
gland, 12 h is required.
The requirement of AsA for a variety of physiological reactions have been
demonstrated using GNL−/− mice. The levels of adrenalin and noradrenalin are
decreased in adrenal glands of AsA-deficient GNL mice (Amano et al., 2014),
which is consistent with the general understanding that AsA functions in their
production. To the contrary, hydroxyproline contents in the skin are not
affected, even though the AsA-deficient GNL−/− mice show, morphologically,
an abnormal epidermis (Arai et al., 2009). Similar phenomena regarding
collagen production have also been reported on GULO−/− mice (Parsons, et al.,
2005) and AKR1A−/− mice (Nishida et al., 2014). Despite the fact that the
contents of skin collagen are not affected by an AsA deficiency in the GNL−/−
mouse (Arai et al., 2009), the collagen content in the lung decreases,
suggesting that the contribution of AsA to collagen synthesis differs with the
tissue. AsA is believed to be required for carnitine biosynthesis, but, again,
results from GNL−/− mice suggest that AsA is not essential for its synthesis
(Furusawa et al., 2008). While the requirement of AsA for the synthesis of
these bioactive substances was shown mostly under in vitro or ex vivo
conditions, there appears to be a compensatory mechanism for AsA in their
syntheses in vivo. Because glutathione coordinately functions with AsA in
certain types of redox metabolism, glutathione is a candidate for compensating
for an AsA deficiency.
In a kainite-induced neurodegenerative disease model, GNL expression is
upregulated, which suggests that GNL has a protective role after brain damage
(Son et al., 2009). In the lung, the incidence of emphysema increases in AsA-
deficient GNL−/− mice at 1–3 months of age (Koike et al., 2010). AsA prevents
smoke-induced emphysema and restores emphysematous lungs in GNL−/−
mice, suggesting a potential therapeutic role of AsA (Koike et al., 2014). It is,
however, noteworthy, that air space size and lipid peroxidation products
increase when excess AsA is present, suggesting a pre-oxidant function of
AsA in lung development under these experimental conditions (Koike et al.,
2010). The phagocytotic removal of secondary necrotic cells is attenuated in
AsA-deficient GNL−/− mice, as observed in aged WT mice (Takahashi et al.,
2016a). GNL−/− mice show a similarity in the enhanced inflammatory
responses induced by secondary necrotic neutrophils in aged WT mice
(Takahashi et al., 2016b).
An increase in the production of reactive oxygen species (ROS) and the
resulting oxidative stress have been reported in AsA-deficient GNL−/− mice.
ROS generation is elevated in the brains of GNL−/− mice, although the major
antioxidative enzymes remain unaffected (Son et al., 2006). While AsA is
present at quite high levels in the lens, UV-induced cataract formation is more
severe in AsA-deficient GNL−/− mice, suggesting a protective role of AsA
against UV irradiation via its antioxidative function (Ishikawa et al., 2012).
Oxidative stress is high in the lungs of GNL−/− mice and is further elevated by
cigarette smoke (Sato et al., 2006). Under oxidative stress conditions, NF-B
appears to be regulated by a GNL-mediated balance between protein kinase
and protein tyrosine phosphatase (Jung et al., 2015) although the precise
molecular mechanism responsible for this is ambiguous. In GNL−/− mice,
oxidative stress markers, such as protein carbonyl groups, are originally high
in the liver. Protein carbonyls, but not lipid peroixidation products, are
suppressed in the liver of AsA-supplemented GNL−/− mice (Amano et al.,
2013; Sato et al., 2014), which is consistent with hydrophilic nature of AsA.
GNL−/− mice also have been used to reveal the potential role of GNL in
glucose metabolism and non-alcoholic fatty liver diseases (Kondo and
Ishigami, 2016). AsA-deficient GNL−/− mice show impaired glucose tolerance
and lower blood insulin levels (Senmaru et al., 2012). Among the transporters
for ascorbic acid or dehydroascorbic acid; SVCT1, SVCT2, and glucose
transporters (GLUT)1, GLUT3, GLUT4, SVCT1 and SVCT2 are upregulated
in the livers of GNL−/− mice (Amano et al., 2010). AsA uptake is actually
elevated in primary cultured hepatocytes from GNL−/− mice. In GNL−/− mice,
triglycerides, cholesterol, and phospholipids accumulate in the liver when fed
a conventional diet (Ishigamai et al., 2004) and show modest impairment in
glucose tolerance with an impaired insulin secretion in the early phase
(Hasegawa et al., 2010). The arazyme is a protease that is produced by a
Gram-negative aerobic bacterium, the Aranicola proteolyticus HY-3 strain,
and appears to protect the livers of GNL−/− mice that had been injured by the
administration of carbon tetrachloride (CCl4) (Park et al., 2008). Because an
AsA deficiency induces CYP2E1 expression and an elevation of ROS, an
oxidative injury in hepatocytes appears to be the underlying mechanism for
binucleation (Park et al., 2010a). Upon the administration of carbon
tetrachloride, GNL−/− mice up-regulate peroxisome proliferator-activated
receptor-gamma (PPAR-) that attenuates the liver fibrosis caused by CCl4
(Park et al., 2010b). A microarray analysis of AsA-deficient GNL−/− livers
shows an increased expression of some genes that are involved in redox
reactions under the regulation of Nrf2 and lipid metabolism, such as Cyp7a1
(Takahashi et al., 2014). Thus AsA is largely involved in maintaining a
healthy liver. However, there is a contradictory report showing that, in GNL−/−
mice, CCl4-induced hepatic injury is aggravated by supplementation with AsA

(Ki et al., 2011). Thus, hepatocytes of GNL−/− mice are more susceptible to
apoptosis induced by TNF- and Fas.
In the Smad3−/− mouse, the overexpression of GNL suppresses apoptosis
induced by radiation (Jeong et al., 2008). Radiation with -rays causes the
upregulation of pro-inflammatory genes in the kidney via the activation of NF-
B in AsA-deficient GNL−/− mice (Chung et al., 2010). A deficiency of AsA
in the fetus and neonates cause tissues damage, including abnormal cardiac
dilation and cognition of the liver and lungs (Kishimoto et al., 2013). Thiol
oxidation associated with endothelial dysfunction is caused by oxidative stress
in GNL−/− mice and triggers coronary artery spasms (Hoshino et al., 2013;
Yamada et al., 2013). The anticancer agent doxorubicin induces cardiac
dysfunction in which ROS appear to be involved. The cardiotoxicity of
doxorubicin is aggravated in AsA-deficient GNL−/− mice (Miyata et al., 2013),
indicating that AsA has a protective role in this model. Exacerbation of
ischemia-reperfusion injury of the heart was observed in GNL−/− mice, even
under AsA-sufficient conditions (Kadowaki et al., 2016), and collateral growth
decreases due to impaired angiogenesis (Yamauchi et al., 2016).
Cardiomyocytes isolated from GNL−/− mice show an increased release of
angiotensin and hydrogen peroxide upon oxidative stress (Mizukami et al.,
2013). These reports suggest that GNL has a protective role under ischemic
conditions. GNL−/− mice with supplementation of AsA (1.5 g/L) still show an
exacerbation of angiotensin II-induced cardiac hypertrophy and remodeling. It
thus appears that the cardioprotective effects of GNL can be attributed to
antioxidation, which is independent of AsA function (Misaka et al., 2013a
2013b), although the underlying antioxidative mechanism is not clear.
There appears to be other GNL functions that are independent from the
AsA supply. Superoxide production is elevated in brain slices of GNL−/− mice
but is effectively suppressed by the administration of hydrogen-rich pure water
(H2) (Sato et al., 2008; Kondo et al., 2008). The overexpression of GNL in
HepG2 cells results in a decrease in the levels of ROS and lipid peroxidation
products, although it is not clear how GNL suppresses oxidative stress (Handa
et al., 2009). When diabetes is induced in GNL−/− mice by injecting them with
streptozotocin, injury in the proximal tubule is exacerbated, even by
supplementing AsA (Okada et al., 2015). Thus, GNL may have other function
than simply AsA synthesis. The double knockout of GNL and SOD1 in animal
models show premature death which is caused by a malfunction of plasma
lipid metabolism and the accumulation of hepatic lipids due to elevated
oxidative stress, even under AsA-supplemented conditions (Kondo et al.,

2014b).
Although diisopropyl phosphorofluoridate (DFP) is hydrolyzed by GNL
(Kondo et al., 2004), the chemical is artificial. Because the administration of
the-adrenergic stimulant, isoproterenol, causes little change in secretory
granules in granular duct cells in GNL−/− mice compared to WT mice, a
modulatory function of GNL in the -adrenergic signal has been proposed
(Ishii et al., 2005). Apoptosis in the crypts of the small intestine of KO mice
increases by -radiation even under AsA-supplemented conditions (Goo et al.,
2013). Mice with a defect in both the leptin receptor (db mutation) and GNL
exhibit increases in small dense-low density lipoprotein (LDL), develop a
severe fatty liver, and show increased endoplasmic reticulum stress that are not
seen in single GNL−/− mice with AsA supplementation (Kondo et al., 2013).
Thus, the alternate function of GNL continues to remain unclear and will
clearly require further investigation.
THE USE OF AKR1A−/− AND AKR1B−/− MICE

AS PATHOLOGICAL MODELS
Aldehyde reductase (AKR1A) and aldose reductase (AKR1B), members

of aldo-keto reductase superfamily, catalyze the reduction of various
aldehydes to their corresponding alcohols in an NADPH-dependent manner.
While AKR1B−/− mice were established by several groups, no phenotypic
changes regarding AsA metabolism have been reported (Ho et al., 2000; Aida
et al., 2000). Gabbay et al. (2010) recently established AKR1A−/− mice and
demonstrated that both AKR1A and AKR1B catalyze the reduction of D-
glucuronic acid in the AsA biosynthetic pathway by using these genetically
modified mice. Takahashi et al. (2012) independently established AKR1A−/−
mice, as well as human AKR1A-transgenic (AKR1A tg/+) mice and reported
detailed analytical data for these mice from the of AsA synthesis. Because
primates do not synthesize AsA, the main role of AKR1A in the liver appears
to be the detoxification of carbonyl compounds. Nevertheless, AKR1A−/− mice
are useful because they can be used as a bona fide model mice for studies on
AsA deficiencies, similar to GULO−/− mice and GNL−/− mice.
Glycogen D-Glucuronate
NADPH
Akr1
NADP+
L-Gulonate
Gnl
H2O
L-Gulono--lactone
ER
O2
Gulo
H2O2
Oxidative
stress
AsA
ER
stress
Figure 3. The GULO-catalyzed oxidation reaction is a potential cause for ER stress.

GULO produces hydrogen peroxide as a byproduct of the oxidation of L-gulono--
lactone to AsA. The resulting hydrogen peroxide is a potential inducer of the ER stress
that, in turn, is associated with cellular damage.
Some phenotypic abnormalities found in the AKR1A−/ − mouse are similar

to GULO−/− and GNL−/− mice and, hence, could be attributed to insufficient
AsA synthesis. However, it is essential to distinguish which function of
AKR1A is responsible for the aberrant phenotypes that have not been reported
in the GULO−/− and GNL−/− mice. When AKR1A−/− mice were anesthetized
for the purpose of a surgical operation, we noticed that pentobarbital showed a
stronger anesthetic effect, characterized by a prolonged sleep time (Ito et al.,
2014). Because AKR1A likely has two potential roles in rodents, i.e., the
detoxification of carbonyl compounds and AsA synthesis, we attempted to
determine the cause for the altered response to pentobarbital. We subsequently
found that AsA played a key role in determining the efficiency of
pentobarbital most likely by altering the responses of the neuronal system to
anesthetics.
AKR1B, the closest aldo-keto reductase superfamily member to AKR1A,

and, together with sorbitol dehydrogenase (SDH), constitute the polyol
pathway. The pathological role of AKR1B and the polyol pathway in the
development of diabetic complications has been well established (Yabe-
Nishimura, 1998). While AKR1A is present in most tissues, its levels are
higher in the liver and kidney, AKR1B is distributed rather ubiquitously.
AKR1A is involved in the detoxification of aldehyde compounds such as
methylglyoxal and 3-deoxyglucosone. the levels of which are elevated under
hyperglycemic conditions (Takahashi et al., 1993; Okado et al., 1996).
AKR1A is also involved in some metabolic pathways that require the
reduction of the aldehyde moieties of intermediary compounds; e.g., the
conversion of prostaglandin H2 to prostaglandin F2 (Hayashi et al., 1989).
Acrolein, a highly reactive α,β-unsaturated aldehyde, is abundantly present in
tobacco smoke and is also produced in the body by lipid peroxidation reactions
as well as being involved in various diseases such as chronic obstructive
pulmonary disease (COPD) (Yadav et al., 2013). We recently reported on the
reductive detoxification of acrolein using mouse embryonic fibroblasts
isolated from AKR1A−/− mice (Kurahashi et al., 2014). For this reason, special
attention should be payed in evaluating results obtained using the AKR1A−/−
mouse based on the AsA requirement.
PERSPECTIVE
The in vivo functions of AsA have been unveiled recently based on
experiments using genetically modified mice. The argument for why primates
have ceased producing AsA is an interesting issue from an evolutional view
point. One possibility is as follows: primates can climb tree to harvest fruits
that are rich in AsA, so they no need to synthesize AsA themselves. Can this
explanation satisfy all researchers? According to this logic, we would not need
to synthesize all amino acids because they can be obtained in the form of meat
and fish, but we are still able to synthesize a half of them from metabolites
other than amino acids. We need essential amino acids that we are unable to
synthesize, such as methionine and lysine, similar to AsA. In addition, some
animals other than primates, e.g., guinea pig, also cannot synthesize AsA and
are defective in the same GULO gene although the origins of their mutations
are completely different (Nishikimi et al., 1992; 1994). Instead of this
hypothesis, the enzymatic reaction of GULO is attractive. During GULO-
catalyzed oxidative conversion of L-gulono-γ-lactone to ascorbic acid,
molecular oxygen is reduced to hydrogen peroxide (Figure 3), which is a well-

known oxidant. The catalytic domain of GULO is exposed to the ER lumen
and hydrogen peroxide is released inside the ER. It is well recognized that the
ER stress, which is induced by also oxidative insult, causes a variety of
diseases such as non-alcoholic liver diseases (Serviddio et al., 2013).
Hydrogen peroxide produced as a byproduct of the GULO reaction would
increase the probability of inducing ER stress, leading to unfavorable liver
conditions. Thus, we propose an alternate hypothesis; animals with a GULO
deficiency have an advantage in maintaining healthy livers by ceasing the
synthesis of AsA synthesis and therefore would flourish during the course of
evolution.
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INDEX
acrylate, xii, 176, 186
# acrylic acid, 50, 56, 68, 78
activation energy, 117, 118
25% acetylated, 134
active compound, 107, 112, 125
2-keto-L-gulonic acid, viii, 29, 30, 31,
active oxygen, 92
33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
active site, 10, 70
43, 44, 112
active transport, 6
acylation, 146
A additives, x, 98, 99, 108, 120, 141, 213
adenine, 23
absorption spectra, 12, 15 adenocarcinoma, 227, 235
accelerator, 19 adenosine, 214
access, 155, 163 adhesive properties, 126
acetaminophen, 69, 81 adjunctive therapy, 202, 207
acetic acid, 35, 43, 128, 129 adrenal gland, 196, 198, 248, 254
acetone, 94 adrenocorticotropic hormone, 208
acetonitrile, 6 adverse conditions, 114
acetylcholine, 61, 87, 199, 214 adverse effects, xiii, 165, 222, 223, 226,
acid, vii, viii, ix, x, xi, xii, xiii, xiv, 2, 3, 232, 235
6, 10, 19, 20, 21, 22, 23, 24, 25, 29, aesthetic, 124
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, aggregation, 130, 155, 166, 177, 205
40, 41, 42, 43, 44, 45, 47, 48, 49, 50, aging process, 247
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, agonist, 203, 244
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, airway epithelial cells, 265
71, 72, 73,74, 75, 76, 77, 78, 79, 80, alcoholic liver disease, 254
81, 82, 83, 84, 85, 86, 87, 88, 89, 91, alcohols, 35, 44, 251
92, 93, 94, 95, 96, 97, 98, 99, 100, Aldehyde reductase (AKR1A), xiv, 240,
101, 105, 107, 108, 109, 111, 112, 241, 242, 248, 251, 252, 253, 261
116, 118, 120, 121, 122, 123, 124, aldehydes, 251
acidic, xi, 34, 35, 37, 62, 76, 126, 129, aldose reductase (AKR1B), xiv, 240,
130, 135, 142, 145, 156, 158, 159, 241, 242, 243, 251, 253, 254
162 algae, 23, 108
acidity, 5, 63
268 Index
alginate, ix, 105, 106, 108, 109, 110, Argentina, 105, 108, 109, 119, 141
112, 113, 114, 115, 116, 117, 118, arginine, 52, 70
119, 120, 121, 128, 138, 179, 181 arterioles, 261
alkaline media, xi, 142 artery, 250, 257, 265
ALT, 231 ascorbic acid, vii, viii, ix, x, xi, xii, xiii,
alternative medicine, 227, 235 2, 3, 19, 20, 22, 23, 24, 25, 30, 31, 33,
American Psychiatric Association, 214 34, 42, 43, 44, 45, 47, 48, 49, 50, 51,
amine, 126, 128, 129, 130, 231 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
amine group, 126, 128, 129, 130, 133 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
amino, 10, 14, 15, 19, 24, 61, 87, 126, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
129, 130, 133, 246, 253, 256, 264 82, 83, 84, 85, 86,87, 88, 89, 91, 93,
amino acid, 10, 14, 15, 19, 24, 246, 253, 94, 95, 97, 99, 100, 101, 105, 108,
256, 264 112, 116, 118, 120, 121, 122, 124,
ammonium, xii, 59, 80, 176, 186 130, 132, 136, 137, 141, 142, 143,
amygdala, 198 144, 145, 146, 147, 149, 151, 153,
anemia, 227, 228, 230 ASEAN, 136
anesthetics, 252 astrocytes, 207, 216
angiogenesis, 189, 226, 236, 243, 250, atherogenesis, 212
265 atherosclerosis, 198, 245
angiotensin II, 250, 261 atherosclerotic plaque, 245, 262
aniline, 50, 55, 67, 77, 78 Au nanoparticles, 70, 89
anisotropy, 158 Austria, 143, 147, 148, 168
annealing, 53
anorexia, 228, 230, 231
ANOVA, 111 B
anti-cancer, 222, 235, 246
anticancer activity, 225 bacillus Calmette-Guerin, 234
antidepressant, 202, 203, 205, 208, 209, Bacillus megaterium, 34, 36, 40, 44
210, 211, 212, 214, 216 bacteria, 35, 41, 43, 86
antioxidant, vii, viii, ix, x, xi, xii, xiii, bacterial fermentation, 34
xiv, 1, 2, 3, 29, 30, 33, 48, 92, 100, bacterium, 246, 249
102, 108, 120, 122, 123, 124, 136, barium sulphate, 192
137, 142, 144, 153, 154, 163, 164, basal lamina, 199, 214
175, 188, 193, 194, 199, 201, 204, base, 43, 107, 126
212, 218, 220, 224, 239, 258, 265 Beck Depression Inventory, 209
antioxidative activity, ix, 92 beneficial effect, 201, 205, 208, 209, 211
antipsychotic, 202, 215 benzene, 15, 59, 85, 95
antipsychotic drugs, 202 beta-carotene, 121
antipsychotic effect, 202 bioavailability, 106, 107, 120, 121, 122,
anxiety, 203, 209, 219 167, 264
apoptosis, 217, 225, 234, 238, 243, 245, biochemistry, xiii, 19, 48, 194, 200
250, 256, 257, 258, 264 biocompatibility, 52, 53, 126
appetite, 223, 230 biodegradability, 126
aqueous solutions, 34 biological activity, 31, 33, 34
Arabidopsis thaliana, 12, 13, 14, 25 biological fluids, 65
biological samples, 61, 65
Index 269
biological systems, viii, 19, 47, 48, 197 cancer, xii, xiii, 21, 175, 176, 177, 188,
biomass, 37, 45 189, 221, 222, 223, 224, 225, 226,
biomaterials, 172 227, 228, 229, 231, 232, 233, 234,
biomolecules, 50, 55, 57, 108, 198 235, 236, 237, 246, 255, 259
biopolymer, 128 cancer cells, 222, 224, 225, 226, 232,
biopolymers, 186, 191 236, 246, 247, 255
biosynthesis, xii, 19, 21, 41, 44, 48, 124, cancer therapy, 224, 232
175, 176, 195, 197, 212, 224, 243, candidates, 52, 54, 56
248, 254, 256, 260, 262 Canine mammary tumors, 226
biosynthetic pathways, 3, 31, 224 capillary, 89, 96, 183, 184, 197
biotechnology, viii, 30, 31 capsule, 125, 177, 178
bipolar disorder, xiii, 194, 199, 204, 205, carbamazepine, 204
209, 216 carbohydrate, 30
blood, 122, 178, 188, 196, 197, 208, carbon, vii, 28, 29, 33, 34, 35, 41, 42,
224, 230, 231, 241, 244, 245, 249 49, 50, 51, 53, 54, 57, 58, 60, 61, 62,
blood pressure, 188 63, 64, 66, 67, 68, 69, 70, 71, 72, 73,
blood transfusion, 230 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
blood-brain barrier, 245 84, 85, 86, 87, 88, 249, 259
body weight, 247 carbon film, 72
bonding, 6, 12, 126, 127, 129, 130, 153 carbon materials, 62
bonds, 12, 14, 130 Carbon nanofibers, 51
bone, 227, 247, 255, 256, 257 Carbon nanohorn, 51, 69
bone form, 247, 255, 257 carbon nanotube nanocomposites, 68
bone marrow, 227 carbon nanotubes, 50, 62, 66, 67, 68, 69,
brain, xiii, 22, 89, 193, 196, 197, 198, 73, 76, 80, 84, 86, 88
199, 200, 201, 202, 203, 204, 206, carbon tetrachloride, 249, 259
212, 213, 215, 216, 217, 248, 250, carbonyl groups, 249
256, 260, 263, 264 carboxylic acid, x, 142, 144, 145
brain damage, 248 carboxylic acids, x, 142
brainstem, 247 carboxymethyl cellulose, 179, 180
Brazil, 193 carcinogenesis, 189, 224, 237
breast cancer, 227, 236, 247, 255 carcinogenicity, 258
breast carcinoma, 189 carcinoma, 189, 226, 235, 237, 238, 256
Brewster angle microscopy, 158, 159 cardiovascular disease, 176
carotene, 121
carotenoids, 115, 121, 220
C case studies, xiii, 222, 223, 226
casein, 107
Ca2+, 120, 204 catabolism, 215
cachexia, 227, 228 catalysis, xi, 18, 24, 54, 55, 142, 145
caffeine, 69, 86 catalyst, 51, 53, 58, 145
calcium, 34, 54, 59, 74, 85, 107, 110, catalytic activity, 50, 51, 52
120, 121, 128, 204, 205, 206, 217 cataract, 249, 258
calcium carbonate, 54, 74 catecholamines, 79
calibration, 51, 57, 82 cation, 37, 146, 254
270 Index
CBC, 228 CNS, 197, 198, 199

cDNA, 264 coagels, xi, 142, 154, 155, 163, 164,
cell death, 198, 206, 217, 226, 262 167, 172
cell line, 122, 144, 164, 189, 233, 262 coatings, ix, 106, 107, 108, 109, 113,
cell membranes, 160, 168 120, 121, 190
cellular homeostasis, xiv, 239 cobalt, 50, 58, 66, 67, 68, 81
cellulose, xii, 122, 126, 176, 178, 179, cognition, 217, 250, 256
180, 181, 190, 191, 192 cognitive deficit, 206, 215
central nervous system (CNS), xii, 193, cognitive impairment, 200, 215
194, 197, 248 collagen, vii, xii, xiv, 2, 48, 124, 175,
ceramic, 54, 57, 61, 73, 81, 87 176, 197, 199, 212, 224, 229, 239,
ceramide, 161 240, 243, 245, 248, 263
cerebellum, 199, 213, 244 color, ix, 30, 33, 106, 107, 108, 110,
cerebrospinal fluid, 197 111, 114, 115, 116, 117, 118, 119,
chain scission, 130 121, 122, 124
chemical, viii, 22, 29, 30, 31, 33, 34, 37, colorectal cancer, 177
41, 45, 52, 54, 56, 58, 59, 62, 65, 93, combined effect, 209
94, 108, 125, 128, 145, 146, 165, commercial, x, 30, 86, 142, 144
179, 194, 251 community, 42, 44, 209, 211, 219, 220
chemical degradation, 125 complex interactions, 179
chemical properties, 58, 179 compliance, xii, 175, 177
chemical stability, 52, 165 complications, 227, 253, 265
chemical structures, 34 composites, 50, 52, 68, 71
chemiluminescence, 65 composition, 34, 121, 125, 161, 179, 243
chemotherapeutic agent, xiii, 177, 221, compounds, x, xi, 6, 31, 32, 41, 54, 58,
224, 227, 235 64, 65, 89, 92, 107, 112, 115, 123,
chemotherapy, 177, 227, 229, 230, 234 124, 125, 126, 128, 130, 134, 136,
chitosan, x, 50, 52, 59, 60, 62, 70, 83, 142, 144, 149, 150, 161, 163, 168,
89, 123, 124, 126, 127, 128, 129, 130, 178, 232, 243, 251, 252, 253
131, 133, 134, 135, 136, 138, 139, compressibility, xii, 158, 161, 176
164, 173, 180 compression, 158, 159
chloroform, 145, 147, 157 condensation, 93, 94
cholesterol, 161, 162, 165, 198, 249 conductance, 245
choline, 87 Conductive polymer, 54
chorionic gonadotropin, 234, 243 conductivity, 49, 51, 52, 54, 55, 57, 58,
choroid, 197 62
chromatography, ix, 20, 48, 65, 92, 95 configuration, 41, 129
chronic obstructive pulmonary disease, construction, 49, 50, 53, 57
253 consumption, 125, 196
cigarette smoke, 249, 259 conversion yield, 37, 38, 40
citalopram, 209 COPD, 253
classification, 25, 237, 238 copolymer, xii, 55, 76, 77, 78, 126, 176,
clinical trials, xiii, 210, 222, 232 186
clozapine, 189, 202 copolymerization, 55
clusters, 127, 177
Index 271
copper, 24, 50, 54, 56, 58, 61, 69, 74, 78, 128, 134, 135, 142, 143, 148, 184,
79, 81, 83, 124, 224 244, 245, 261
coronary artery spasm, 250, 257 degradation rate, 116, 134
cortex, 89, 201, 203, 204, 244 degranulation effect, 230, 231, 235
corticosteroids, 229 degree of crystallinity, 155
cosmetic, x, 93, 123, 124, 125, 126, 134, dehydration, 107, 120, 121, 241
135, 141, 143 delusions, 200, 208
cost, 35, 40, 42, 49, 52, 56, 64, 145, 177, dementia, 255
224 Department of Agriculture, 42
crystal structure, 12, 18, 24 Department of Health and Human
crystalline, 34, 154, 155, 159, 160, 162, Services, 42
164 deposition, 57, 58, 78, 83, 206, 217
crystallinity, 155 depression, 202, 203, 204, 205, 208,
crystallization, 37 209, 213, 216, 217, 218, 219, 228,
crystals, xi, 143, 155, 156, 163, 164, 167 231
cultivation, 39, 40 depressive symptoms, 203, 208, 209,
culture, 36, 37, 38, 44 219
culture broth, 37, 38 derivatives, x, xi, 93, 142, 143, 144, 145,
cycles, 18, 204, 227 147, 151, 152, 155, 163, 164
cyclodextrins, 165 destruction, 113, 229, 231
cyclooxygenase, 227 detection, viii, 47, 48, 49, 50, 51, 52, 53,
cysteine, 59, 77, 81, 85 54, 55, 56, 57, 58, 59, 60, 63, 64, 65,
cystic fibrosis, 245 66, 68, 69, 70, 71, 72, 73, 74, 75, 76,
cytochrome, vii, 1, 2, 6, 7, 8, 10, 11, 12, 77, 78, 79, 80, 82, 84, 87, 88, 89, 96,
13, 14, 15, 16, 17, 18, 19, 22, 24, 25, 237, 238
26, 198 detoxification, 251, 252, 253, 261
cytokines, 245, 246 deviation, 111, 131, 182, 185
cytotoxicity, 164, 235, 265 D-glucose, 30, 33, 34, 35, 36, 38, 40, 42,
195
diabetes, 212, 250, 257, 262
D diabetes insipidus, 257
diabetic nephropathy, 262
damping, 186 diabetic patients, 209, 219
deacetylation, 126, 127, 128 Diagnostic and Statistical Manual of
decay, 5, 18, 20, 118 Mental Disorders, 214
decomposition, 145, 147 diet, 106, 188, 220, 223, 225, 227, 228,
defects, xiv, 240, 263 230, 241, 249, 260
deficiency, xiv, 106, 122, 201, 206, 208, Dietary Guidelines for Americans, 42
212, 215, 217, 239, 242, 243, 244, dietary intake, 3, 176
245, 246, 247, 248, 249, 250, 254, diffusion, xii, 126, 158, 167, 176, 186,
255, 256, 257, 261, 262, 263, 264, 187
265 dilation, 250, 259, 261
deficit, 201, 206, 215, 243, 244 dimerization, 5, 18
degradation, xi, 21, 94, 112, 113, 114, dimethylsulfoxide, 147
115, 116, 117, 118, 119, 124, 125, discrimination, 85
272 Index
disease gene, 207, 218 electrocatalysis, 49, 54, 84, 85

disease model, 207, 248 electrochemical behavior, viii, 47, 49,
diseases, xii, xiii, 175, 176, 194, 199, 52, 85
205, 207, 210, 211, 217, 222, 234, electrochemical deposition, 57, 58
249, 253, 254 Electrochemical detection, 75, 76
disorder, xiii, 194, 198, 199, 200, 202, electrochemical impedance, 54, 76
203, 204, 205, 206, 209, 210, 216, electrochemistry, 49, 59
219, 260 Electrode, 49, 60
dispersion, 125, 130, 135, 186 electrode surface, viii, 47, 49, 50, 51, 54,
distilled water, 108, 109 57, 59, 64
distribution, x, 11, 124, 126, 128, 167, electrodeposition, 63
177, 178, 198, 213, 247, 256, 258 electrodes, viii, 47, 48, 49, 50, 51, 52,
DMF, 51, 60, 69 57, 58, 59, 60, 62, 64, 66, 68, 78, 79,
DNA, 27, 28, 54, 74, 176, 211 84, 87
DOI, 237, 238 electron, 2, 3, 6, 8, 11, 12, 15, 17, 18, 21,
dopamine, 11, 20, 24, 49, 55, 61, 62, 63, 22, 24, 25, 26, 50, 51, 62, 130, 164,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 195, 201, 202, 224
76, 77, 79, 80, 81, 82, 83, 84, 85, 86, electrophoresis, 89
87, 88, 89, 198, 199, 200, 201, 202, electrospinning, 51
214, 215, 244 emphysema, 248, 259
dopaminergic, xii, 193, 199, 200, 202, employment, 143, 165
203, 204, 206, 214, 244 emulsions, 148
dosage, 177, 178, 190, 191, 220, 231 encapsulation, x, 124, 125, 133, 135,
down-regulation, 259, 265 137, 138, 164, 188
drug delivery, xi, 54, 125, 126, 138, 142, endothelial dysfunction, 250
164, 188, 190 endothelium, 197
drug discovery, 216 energy, viii, 30, 41, 117, 118, 128, 218
drug release, xii, 176, 177, 179, 186, engineering, 26, 108, 121
187, 188, 191 enlargement, 227, 228
drugs, viii, xi, 47, 142, 160, 161, 163, environment, 6, 18, 186, 225
164, 165, 166, 167, 168, 177, 178, environmental conditions, 135
183, 189, 190, 202, 203, 204, 205, environmental factors, x, 123, 124
222 environmental sustainability, 42
drying, 103, 110, 111, 113, 114, 121, enzymatic activity, 224
125, 130, 178, 184, 186 enzyme, vii, xi, 1, 3, 8, 9, 11, 18, 22, 23,
D-sorbitol, viii, 29, 30, 31, 33, 34, 35, 24, 36, 38, 40, 41, 44, 53, 56, 124,
36, 38, 40, 41, 44 142, 145, 146, 147, 176, 178, 195,
duodenum, 178 197, 198, 202, 204, 224, 242, 243,
249, 260, 262, 264
epidermis, 248
E epilepsy, 198, 212
epinephrine, 59, 75, 76, 84, 88
economic efficiency, 30 epithelia, 247
edible coating, ix, 106, 109, 112, 113, epithelial cells, 125, 265
114, 117, 118, 119, 121 epithelium, 197, 245, 259
electric conductivity, 49, 51
Index 273
EPR, 25 fluorescence, 65, 77

equilibrium, 5, 6, 109, 112, 145, 152, fluoxetine, 202, 209, 216, 219
154, 157, 160, 162 folic acid, 82, 83, 178, 190
equipment, 95, 179 follicles, 244
erythrocytes, 19 food, viii, ix, x, 29, 30, 31, 34, 48, 64,
ester, 37, 147 65, 82, 91, 92, 106, 107, 108, 110,
estrogen, 234 114, 119, 120, 121, 122, 126, 141,
ethylene, 56, 78 143, 190, 210, 228, 231, 232, 264
Euglena gracilis, 23 food additive, 30, 31, 120
eukaryote, 11 food products, ix, 106
eukaryotic, 23, 26, 204 forebrain, 214
evaporation, 94, 95 formation, viii, xii, 3, 11, 41, 48, 61, 91,
evidence, xiii, 194, 199, 201, 202, 203, 92, 115, 127, 129, 130, 131, 133, 134,
204, 205, 206, 215, 216, 229, 230, 135, 138, 139, 143, 146, 152, 155,
234 156, 159, 162, 164, 176, 177, 184,
evolution, 23, 116, 119, 254 191, 195, 197, 198, 199, 202, 206,
excitotoxicity, 199, 205, 214 207, 213, 214, 215, 217, 244, 246,
executive function, 200 247, 249, 254, 255, 261, 263
experimental condition, 15, 146, 165, fortified foods, 106, 107
248 fractures, 247, 262
exposure, xi, 115, 142, 147, 163 free radicals, xi, 18, 48, 92, 124, 175,
extracellular matrix, 189, 213 176, 198, 205
extrusion, 125, 137, 178, 180, 182, 184, friction, 179, 182
189, 190, 191, 192 frontal cortex, 244
fruits, 30, 48, 64, 76, 79, 107, 119, 124,
253
F fullerene, 50, 62
functional food, 107, 119, 122
fabrication, 49, 50, 51, 52, 53, 58, 59, functionalization, 49, 53, 81, 90
62, 64, 71, 75, 78, 86, 137
FAD, vii, 1, 8, 11, 38, 41
family members, vii, xi, 2, 12, 142, 160 G
fat, viii, 91, 93, 137
fatty acids, 93, 124, 136, 146, 161, 197, GABA, 201
208 GCE, 49, 50, 51, 52, 53, 55, 56, 57, 59,
fermentation, 30, 34, 36, 37, 39, 40, 41, 62, 64
42, 44 GEF, 119
fibroblasts, 197, 253 gel, 54, 61, 73, 74, 87, 127, 128, 129,
fibrosis, 229, 245, 246, 249, 259, 262, 138, 148, 155, 156, 160
263 gelation, x, 123, 124, 126, 127, 128, 130,
films, 56, 77, 78, 108, 120, 121, 122, 131, 132, 135, 138, 139, 164
130, 158, 159, 160, 161 gene expression, 245, 247, 257, 258, 265
fish, 228, 230, 253 genes, 12, 36, 242, 249, 250, 255, 264
flavor, viii, 30, 91 Germany, 94, 101, 111, 170
flour, 107, 121 gestation, 259
fluid, 158, 165, 197, 206, 225, 233 gland, 198, 235, 243, 248, 257
274 Index
glial cells, 199 heme, 6, 12, 13, 14, 15, 17, 18, 24, 25,
glucocorticoids, 198 53, 204
Gluconobacter oxydans, viii, 29, 33, 34, heme oxygenase, 204
36, 38, 39, 40, 41, 43 Hemin, 53
Gluconolactonase (GNL), xiv, 240, 241, hemorrhage, 227, 228, 229, 230, 231
242, 247, 248, 249, 250, 251, 252, heparin, 229
254, 258, 259, 260, 263, 264 hepatic injury, 250, 259, 263
glucose, 6, 30, 33, 34, 35, 36, 38, 40, 42, hepatitis, 208
61, 62, 108, 109, 195, 196, 197, 224, hepatocellular carcinoma, 235
240, 241, 244, 249, 257, 260, 265 hepatocytes, 249, 262
glucose tolerance, 249, 257 hepatomegaly, 230
GLUT, 249 hexane, 94, 95
GLUT4, 249 hippocampus, 198, 204, 211, 214
glutamate, 198, 199, 200, 201, 204, 205, Hollow carbon microspheres, 51
206, 212, 214, 215, 218 homeostasis, xiii, xiv, 194, 199, 204,
glutamate receptor antagonists, 214 207, 239, 254, 256
glutamic acid, 57, 80 homocysteine, 226
glutathione, 8, 22, 24, 201, 215, 218, homovanillic acid, 76
225, 243, 245, 248, 255 hormone, 208, 234
glycerol, 108, 109, 119 human, vii, xiii, 2, 12, 15, 19, 25, 63, 64,
glycine, 24, 51, 69 107, 122, 188, 189, 194, 200, 212,
glycogen, 240, 241 221, 222, 223, 224, 225, 226, 227,
glycol, 96, 167 230, 233, 234, 243, 251, 256, 257,
GnRH, 234 262, 265
gold nanoparticles, 78, 79, 80 human chorionic gonadotropin, 234, 243
granules, 2, 12, 24, 137, 180, 188, 190, human health, vii, 2, 212
191, 192, 229, 231, 251 humidity, ix, 92, 95
Graphene, 51, 53, 60, 76 hybrid, 53, 55, 73, 78
graphene sheet, 52, 53, 70, 73 hydrogels, 108
graphite, 51, 53, 57, 67, 72, 73, 79, 85, hydrogen, vii, 1, 6, 8, 12, 14, 18, 23, 33,
88 61, 76, 92, 126, 127, 129, 130, 153,
growth, 40, 44, 143, 189, 213, 223, 225, 195, 224, 225, 233, 250, 252, 254
226, 233, 236, 243, 250, 255, 258, hydrogen atoms, 92
259 hydrogen bonds, 12, 14
hydrogen peroxide, vii, 1, 8, 23, 61, 76,
224, 225, 233, 250, 252, 254
H hydrolysis, 3, 112, 113, 147
hydroperoxides, viii, 91, 92
half-life, 114 hydrophilicity, 100
hallucinations, 200 hydrophobicity, 50, 100
harmful effects, 229 hydroquinone, 50, 59, 66, 70, 84, 88,
healing, 197, 222, 230 143
health, vii, xiii, 2, 106, 176, 194, 212, hydroxide, 37, 52, 65, 71
222, 223, 236
hearing loss, 247, 258
Helicobacter pylori, 189
Index 275
hydroxyl, x, 14, 33, 129, 142, 144, 145, in vivo, xiii, xiv, 89, 167, 212, 222, 223,
146, 149, 150, 151, 152, 155, 194, 224, 226, 233, 240, 248, 253, 256,
195, 197, 206, 224 262
hydroxyl groups, 149, 150, 151, 152, incidence, 211, 222, 248
155, 197, 224 indirect measure, 160
hydroxypropyl cellulose, 180 individuals, 208, 210, 211, 244
hyperactivity, 204, 244 induction, 98, 99, 100, 101, 189, 204
hyperoxaluria, 197 induction period, 98, 99, 100, 101
hypersensitivity, 230 industrial production, viii, 29, 30, 31
hypertrophy, 250, 261 industry(ies), viii, 30, 47, 126, 165
hypothalamus, 198 infection, 246
hypothesis, 168, 200, 201, 202, 203, inflammation, 243, 246, 256, 259
214, 224, 253 inflammatory cells, 230
hypovolemic shock, 231 inflammatory responses, 248, 264
hypoxia, 245, 264 influenza virus, 246, 261
hypoxia-inducible factor, 264 ingredients, 48, 179
inguinal, 230, 231
inhibition, viii, 92, 93, 99, 177, 201, 203,
I 204, 212, 214, 215, 226
inhibitor, 30, 201, 214, 217
ibuprofen, 167, 191 initiation, 92, 204
ideal, x, xii, 123, 126, 176, 177, 179 injury, 245, 249, 250, 255, 259, 262, 263
identification, 5, 23, 48, 205 inoculation, 38
identity, xiv, 12, 240 inoculum, 38
IFN, 245, 246 insertion, 160, 162
imbalances, 223 insulin, 128, 138, 249, 257, 263
immersion, 109, 120, 186 integration, 97, 165
immobilization, 88, 118 integrity, 245
immune function, 222 intercourse, 209, 219
immune response, 246 interface, xi, xiii, 59, 122, 142, 156, 157,
immune system, 124, 229 158, 194, 200
immunity, xii, 175, 176, 246 interference, viii, 47, 49, 61, 62
immunization, 167 intermolecular interactions, 129
immunofluorescence, 257 interneurons, 201
immunohistochemistry, 238 internship, 236, 237
immunomodulation, 198 intestinal tract, 107, 178
immunomodulator, 234 intracellular calcium, 204, 206, 217
impaired immune function, 222 Intravenous, vi, xiii, 221, 224, 226, 229,
impregnation, ix, 105, 107, 121 231, 235
improvements, 35, 40, 106 inversion, 33, 34, 35, 41, 42
in size, 124, 135, 162, 184, 227, 228, invertebrates, 195
229, 231 iodine, 85
in vitro, xiii, 64, 107, 121, 122, 138, ion-exchange, 54
164, 167, 189, 214, 222, 223, 224, Ionic liquid, 58
225, 226, 246, 248, 259 ionization, xi, 95, 142, 157
276 Index
ions, x, 59, 61, 112, 123, 127, 128, 129, large, xi, xii, 40, 52, 53, 130, 134, 142,
224, 243 146, 147, 156, 157, 167, 176, 179,
IR spectra, 149 180, 183, 210, 226, 229, 230, 245
IR spectroscopy, 149 L-arginine, 52, 70
iridium, 58, 82 laser ablation, 51
iron, ix, 25, 52, 53, 56, 58, 71, 74, 78, latency, 247
82, 105, 106, 107, 108, 120, 121, 122, layering, 178, 190
124, 197, 198, 213, 224, 245, 256 LDL, 251, 260
irradiation, 217, 249 lecithin, 164
ischemia, 89, 250, 265 lesions, 230, 231
ischemia-reperfusion injury, 250 leukocytosis, 228, 229
isoflavone, 136 L-gulono--lactone oxidase (GULO),
isolation, 56, 215 xiv, 239, 240, 241, 242, 243, 244,
issues, 57, 64, 89, 124, 144, 246 245, 246, 247, 248, 251, 252, 253
ligand, 58, 83
light, xi, 25, 43, 124, 135, 142, 147, 159,
J 163, 177
light conditions, 25
Japan, 1, 91, 94, 101, 110, 209, 219,
lignin, 59, 85
237, 239
linoleic acid, viii, 92, 93, 95, 96, 97, 98,
99, 100, 101
K Linus Pauling, 222
lipid metabolism, 249, 250, 264
K+, 204, 214, 217 lipid oxidation, viii, 91, 92, 93, 124, 254
KBr, 148 lipid peroxidation, 2, 245, 246, 248, 250,
keratinocyte, 164 253
Ketogulonicigenium vulgare, 34, 36, 37, lipids, 160, 165, 198, 214, 246, 250, 256,
38, 39, 40, 44 257
ketones, 35 liposomes, 125, 136, 137, 165, 166
kidney, 195, 197, 228, 247, 250, 253 liquid chromatography, 20, 48, 65, 95
kidney stones, 197 liquid crystals, xi, 142, 155, 156, 163,
kill, 225, 232 164, 167
kinetic constants, ix, 106 liquid phase, 158, 160, 184
kinetic model, 113, 114, 117, 118, 119, liquids, 58, 124, 125, 178
122 lithium, 95, 204, 205, 210
kinetic parameters, 93, 99 liver, xiv, 7, 22, 23, 24, 64, 89, 195, 240,
kinetics, xii, 5, 11, 51, 93, 116, 118, 120, 241, 244, 245, 247, 249, 250, 251,
161, 176, 186 253, 254, 255, 257, 258, 259, 260,
KIT, 229 263, 264, 265
liver cells, 257, 265
liver damage, 245
L liver disease, 249, 254, 260, 263
localization, 10, 25, 257
L-(+)-ascorbic, ix, 105, 106, 108 low density polyethylene, 110
lactose, 62
Index 277
L-sorbose, 30, 34, 35, 36, 37, 38, 40, 41, mechanical properties, 120, 161, 184,
42, 43, 44 186
L-sorbosone, viii, 29, 36, 38, 41, 44 media, x, xi, 40, 44, 48, 62, 63, 67, 88,
lumen, 7, 240, 241, 254 109, 127, 129, 130, 141, 142, 143,
Luo, 70, 71, 72, 78, 79, 83, 86, 87, 89 145, 146, 147, 148
lutein, 107 medication, 205
lycopene, 107 medicine, xiii, 221, 227, 235, 236, 263
lymph node, 227, 228, 229 melanin, xii, 124, 175, 176
lymphocytes, 188 melanoma, 226, 234, 246, 255
lymphoma, 226, 238 melt, 125, 178, 180
lysine, 189, 197, 253 membrane permeability, 126
lysozyme, 126 membranes, xi, 2, 23, 26, 142, 160, 161,
163, 165, 167, 168
Mesoporous silica, 53
M metabolism, xiii, 25, 194, 198, 200, 213,
240, 248, 249, 250, 251, 260, 264
macromolecules, 127 metabolites, 48, 197, 243, 244, 253
macrophages, 264 Metal hexacyanoferrate, 54
magnesium, 58, 65 metal ions, x, 123, 124, 224, 243
magnetism, 54 metal oxides, 52
major depression, 213, 218 metals, 52, 57, 58, 224
major depressive disorder, xiii, 194, 198, metastasis, 230, 247, 255
199, 202, 203, 209, 219 methacrylic acid, xii, 176
malaria, 106 methamphetamine, 199
Malaysia, 29, 45 methanol, 35, 95, 147
malignancy, 229, 234 methodology, 82
mammalian cells, 22 methyl methacrylate, xii, 176, 186
mammals, 3, 21, 194, 195, 198, 261 methylene blue, 8, 59
management, 106, 202, 206, 207, 211 mice, xiv, 177, 202, 203, 206, 207, 211,
manganese, 58, 82 213, 217, 218, 225, 233, 236, 237,
manic, 204, 209, 219 240, 242, 243, 244, 245, 246, 247,
manic symptoms, 204 248, 249, 250, 251, 252, 253, 254,
manic-depressive psychosis, 219 255, 256, 257, 258, 259, 260, 261,
manipulation, 205 262, 263, 264, 265
manufacturing, 178, 179 microbial community, 44
mass, xii, 8, 26, 176, 179, 180, 182, 184, microbial oxidation, 30
227, 228, 229, 230, 231 microcapsule, 93, 130, 134
Mast cell tumors, 229 microcrystalline cellulose, xii, 176, 179,
mast cells, 231 190, 191, 192
materials, viii, x, 30, 42, 47, 49, 50, 51, microdialysis, 89
53, 54, 57, 59, 62, 64, 81, 90, 124, microemulsion, 88, 125, 164
125, 126, 145, 179, 180, 186 microgels, 129
matrix, ix, 55, 56, 78, 81, 106, 107, 118, micrometer, 156
119, 120, 121, 137, 186, 189, 213, micronutrients, 107
245 microorganisms, 40, 43, 146, 177
measurement, 50, 54, 60, 61, 64, 86, 111
278 Index
microparticles, 81, 127, 128, 130, 132, nanofibers, 51, 55, 61, 64, 69, 77, 82, 87,
133, 134, 137, 178, 188 89
microRNA, 243 nanohorns, 51, 69
microscope, 164 nanomaterials, 49, 53, 64
microscopy, 158, 159 nanometer, 158
microsomes, 7 nanoparticles, xi, 50, 66, 67, 68, 70, 73,
microspheres, 51, 69, 138, 179 77, 78, 79, 80, 81, 82, 83, 84, 89, 136,
microstructure, 122, 155 138, 139, 143, 165
models, xiii, 19, 89, 187, 194, 200, 201, nanorods, 80, 81
202, 203, 212, 216, 218, 233, 250, nanostructured materials, 90
265 nanostructures, xi, 57, 63, 82, 142, 164,
modifications, 49, 94, 109, 163 165
modulus, 158, 161 Nanostructures, 172, 173
moisture, 124, 135, 177, 183, 184, 192 nanotechnology, 71
molar ratios, 95, 146 nanotube, 57, 62, 63, 66, 67, 68, 88
Molecular imprinting, 56 nanowires, 82, 89
molecular oxygen, 24, 43, 254 National Academy of Sciences, 170
molecular structure, 224 natural compound, 243
molecular weight, 6, 126, 128, 134, 224 natural food, 231
molecules, 10, 40, 49, 53, 56, 59, 62, 87, natural polymers, 164
99, 125, 128, 155, 156, 157, 158, 161, nausea, 223
223 necrosis, 167, 225, 227, 228, 229, 230,
monolayer, 59, 61, 84, 153, 157, 158, 231, 235, 257
159, 160, 162 necrotic core, 245
morphology, x, 124, 128, 129, 245, 262 negative effects, 232
mortality, 188, 201, 202 neocortex, 198
multivariate calibration, 82 neonates, 250
multiwalled carbon nanotubes, 67, 73, neovascularization, 245
86 nephropathy, 262
mutant, 243, 262 nerve, 200
mutation, 18, 195, 206, 240, 245, 251, nervous system, xiii, 25, 194, 197, 248
253 neurobiology, xii, 193, 203
myelin, 199, 214 neurodegeneration, 213
myocardium, 261 neurodegenerative diseases, xiii, 194,
199, 205, 207, 210, 211, 217
neurodegenerative disorders, 212, 217
N neurons, xii, 193, 197, 199, 201, 206,
214, 218
Na+, 6, 204, 214, 217 neuropathologies, xiii, 194, 200, 207,
NaCl, 10, 64 211
NAD, 8, 9, 11, 38 neurotransmission, xii, 193, 199, 204,
NADH, vii, 1, 7, 8, 11, 22, 24 216
nafion, 67, 75, 88 neurotransmitter, vii, 2, 87, 202, 204,
nanocomposites, 52, 56, 68, 70, 71, 77, 212, 216
78, 79 neutral, 55, 76, 78, 124, 158, 162, 257
Index 279
neutral lipids, 257 41, 42, 43, 44, 49, 51, 53, 54, 55, 56,
neutrophils, 230, 246, 248, 264 58, 61, 62, 63, 64, 66, 68, 70, 73, 74,
NH2, 14, 129 75, 76, 77, 78, 81, 84, 85, 87, 91, 92,
niacinamide, 55, 75 93, 95, 96, 97, 98, 99, 100, 101, 112,
nickel, 52, 71, 247, 258 118, 124, 147, 194, 195, 197, 204,
niobium, 58 206, 213, 250, 252, 254, 256, 263,
nitric oxide, 203, 216 264, 265
nitrobenzene, 88 oxidation products, 36, 38, 49
nitrogen, 53, 69, 72, 73, 94, 95, 129, 165 oxidation rate, 98, 100, 101
nitrogen gas, 94, 95, 165 oxidative damage, 202, 204, 205, 206,
NK cells, 246 207, 216, 244, 246
NMDA receptors, 199, 201, 203, 207 oxidative stress, vii, 1, 201, 202, 205,
NMR, 147, 149, 150, 151, 152, 153, 206, 208, 213, 217, 218, 233, 244,
243, 255 246, 248, 250, 256, 260, 263, 264
Nobel Prize, 222 oxide nanoparticles, 66, 81
noble metals, 52, 57 oxygen, x, xi, xiv, 2, 6, 24, 37, 43, 60,
nodes, 227, 228 92, 108, 112, 113, 115, 123, 124, 129,
non-polar, 154, 155, 165 130, 135, 142, 153, 163, 164, 165,
nontoxicity, 126 176, 198, 215, 224, 239, 245, 248,
norepinephrine, vii, 2, 69, 85, 198, 202 254, 256, 260, 261, 262
novel materials, 53
Nrf2, 249
nucleic acid, 198 P
nucleus, 198, 199, 214, 215
nutrients, ix, vii, 2, 48, 105, 108, 113, paclitaxel, 189
122, 220, 222, 223, 241, 243 palladium, 35, 43, 51, 65, 69, 70, 83, 85
nutrition, viii, 29, 265 pancreatic cancer, 226, 233
nutritional state, 178 parallel, 129, 187
paraneoplastic syndrome, 229
parkinsonism, 210
O participants, 211
partition, 165, 167
obstruction, 262 pathogenesis, 206, 207, 215
olanzapine, 202 pathology, 236, 245, 246, 261
oligomerization, 206, 217 pathophysiology, 199, 213
oligomers, 129, 206 pathway, xiv, 18, 40, 42, 200, 203, 204,
omega-3, 218 205, 212, 213, 216, 217, 224, 234,
optimization, 40, 42, 167 240, 241, 242, 243, 251, 253, 256,
organ, xiv, 222, 240, 246, 255 258
organic solvents, 126, 128 PCR, 238
organize, xi, 142, 154 peptides, 24, 178, 206
Orthomolecular Medicine, 223 percolation theory, 189
osmium, 55, 76 perinatal, 22
ovarian cancer, 226, 234, 246, 259 peripheral nervous system, 25
oxidation, viii, 3, 6, 7, 8, 11, 17, 20, 22, permeability, 51, 125, 126, 205
26, 29, 30, 31, 33, 34, 35, 36, 38, 40, permeation, 137, 165, 167
280 Index
permission, iv, 32, 33, 38, 39, 41 PM, 214

permit, 129, 246 point mutation, 206
peroxidation, 2, 214, 245, 246, 248, 250, polar, 6, 15, 149, 154, 155, 165
253 polarization, 165, 166
peroxide, vii, 1, 8, 23, 61, 76, 224, 225, Polyaniline, 60
233, 250, 252, 254 polyelectrolyte complex, x, 123, 126,
persecutory delusion, 208 127, 128
pH, x, 3, 5, 15, 16, 17, 18, 49, 55, 68, 76, polymer, xii, 52, 55, 56, 74, 75, 126,
108, 109, 123, 124, 125, 126, 128, 129, 130, 134, 139, 175, 176, 177,
129, 130, 135, 157, 158, 159, 162, 180, 184, 187, 188
194, 195 polymer chain, 130
phagocytosis, 264 polymer matrix, 55, 56
pharmaceutical, viii, x, xi, xii, 29, 30, polymeric materials, 125
47, 63, 64, 67, 86, 124, 126, 142, 144, polymerization, 55, 56, 78
175, 178, 179, 191 polymers, xii, 54, 55, 56, 64, 126, 128,
pharmaceuticals, 65, 74 164, 176, 179, 180, 186, 187
pharmaceutics, x, 141, 143 polyphenols, 107
pharmacokinetics, xiii, 213, 221, 223, polypropylene, 56, 79
234, 235 Polysaccharides , 107, 126, 138
pharmacology, 261 polyunsaturated fatty acids, 208
pharmacotherapy, xiii, 194, 200 porosity, 180, 184, 186
phase diagram, 155 porphyria, 245, 256
phase transitions, 153 potassium, 35, 48, 65, 108, 109, 119,
phencyclidine, 200, 214 121, 199
phenolic compounds, 89 precipitation, 130
phenotypes, 203, 242, 247, 252, 255 prednisolone, 229, 230, 231
phosphate, 59, 85, 164, 165, 240, 241 prefrontal cortex, 201, 204
phospholipids, 137, 249, 257 premature death, 250
phosphorylation, 203, 205, 206, 217 preparation, 43, 48, 51, 52, 53, 56, 57,
physical characteristics, 178 63, 70, 75, 77, 79, 84, 125, 138, 146,
physical chemistry, 155 149, 150, 151, 152, 153, 154, 190,
physical properties, 182 192
physicians, 224 preservation, 117, 119
physicochemical properties, xi, 51, 142, preservative, viii, 29
155, 167, 168 prevention, 203, 211, 212, 236, 237, 265
physiological mechanisms, 211 primary tumor, 227
physiology, 261 principles, 26
PI3K, 204, 205 probability, 254
placebo, 131, 208, 209, 218, 219 probe, 77, 158, 166
plants, 8, 12, 15, 23, 25, 30, 40, 41 progressive neurodegenerative disorder,
plaque, 217, 244, 260, 262 206
plasma membrane, 2, 21, 207 pro-inflammatory, 250, 255
plasticity, 180, 182, 184 proliferation, 189
platinum, 43, 66, 68, 81, 88 proline, 189, 197, 243
plexus, 197 promoter, 107, 224, 245
Index 281
pro-oxidant, 144, 224, 229, 233 reaction rate, 6, 49, 113

propagation, 92, 101 reactions, vii, xiv, 1, 2, 3, 5, 8, 16, 19,
prostaglandin, 253, 257 22, 30, 50, 57, 112, 114, 115, 117,
protected, 135 118, 119, 136, 196, 197, 198, 218,
protection, vii, 1, 44, 108, 125, 164, 214, 224, 239, 240, 241, 248, 249, 253
244 reactive oxygen, xi, xiv, 2, 142, 163,
protective role, 248, 249, 250, 261 165, 176, 198, 215, 224, 239, 248,
protein family, 25 256, 260, 261
protein oxidation, 204, 263 reactivity, 6, 18, 50, 124, 125, 201, 206
protein sequence, 25 reagents, 65, 128, 147
proteins, vii, 2, 12, 18, 53, 188, 198, receptors, 12, 56, 199, 200, 201, 202,
203, 205 203, 205, 207, 214, 229, 234, 249,
protons, 48, 49, 151 251, 263
psychiatric disorder, 202, 205 recognition, 18, 56, 227
psychiatric patients, 209, 219 recovery, 64, 148
psychopathology, 200 recrystallization, 95
psychosis, 208, 219 recycling, vii, 1, 2, 3, 6, 19, 21, 202, 261
psychotic symptoms, 208, 215 redundancy, 242
public health, 106, 222 refractive index, 158
publishing, 224 regeneration, vii, 1, 3, 8, 20, 23, 49, 233
pure water, 250, 263 regioselectivity, 41, 93
purification, 25, 95, 145 regression, 98, 113, 226, 229
pyrolytic graphite, 88 regression analysis, 98
Reichstein process, 34, 36, 37, 39, 41
relaxation, 158, 245
Q relevance, 202, 215
reliability, xii, 63, 175
quality of life, xiii, 222, 223, 229, 232, remission, 202, 228
236 remodelling, 261
quaternary ammonium, xii, 176, 186 renal cell carcinoma, 226
questionnaire, 209 repair, 19, 229
quinones, 22 repulsion, 158
requirement, 41, 106, 125, 212, 213,
R 245, 248, 253
researchers, 253
radiation, 234, 250, 251, 256, 258 residual disease, 237, 238
Radiation, 19, 27, 250 residues, 10, 11, 14, 18, 26, 63, 126, 197
radiation therapy, 234 respiratory failure, 228
radical reactions, 198 response, xiii, 51, 54, 56, 88, 158, 161,
radicals, xi, 18, 19, 20, 48, 92, 124, 136, 167, 215, 222, 223, 225, 230, 246,
175, 176, 195, 198, 205, 206 247, 252
radiotherapy, 227, 229 restoration, 259
rating scale, 209 restructuring, 167
raw materials, viii, 30, 41, 179 reticulum, xiv, 239, 240, 241, 251
reaction mechanism, 19, 48, 146
282 Index
risk, xii, 175, 176, 177, 178, 189, 202, shear, 128, 158, 161, 164
210, 211, 220, 236 shock, 230, 231, 243
rodents, 177, 201, 252 showing, 114, 131, 156, 158, 162, 164,
rods, 179 165, 186, 208, 249
room temperature, 34, 52, 58, 95, 110, side effects, 178, 202
117, 147, 160, 184 signaling pathway, 203, 204, 205, 212,
Rouleau, 226, 235 217
routes, 31, 40, 41, 164 signals, 61, 147, 149, 150, 151, 152, 153
Royal Society, 21 signs, 207, 228, 230, 231
ruthenium, 58, 81 silica, 53, 73, 146
silver, 57, 71, 77, 79, 80
SiO2, 56, 58, 78, 81, 82
S skeletal muscle, 188
skeleton, 33, 34, 35, 41, 42
saturation, 11, 162, 225 skin, 137, 164, 167, 229, 230, 231, 248,
schizophrenia, xiii, 194, 198, 199, 200, 254
201, 202, 207, 208, 213, 214, 215, sleep disturbance, 223
218 small intestine, 251
schizophrenic patients, 200, 208 social withdrawal, 200
secretion, 212, 214, 245, 246, 249, 257, sodium, x, xii, 34, 35, 59, 62, 65, 67,
259 109, 121, 123, 127, 128, 135, 164,
selective serotonin reuptake inhibitor, 176, 179, 196, 197, 199, 204, 254
203 sodium hydroxide, 65
selectivity, 35, 54, 55, 57, 59, 62, 63 solid state, 147, 148, 165
self-assessment, 210 solid tumors, 226
senescence, xiv, 240, 241, 247, 254, 256, solubility, viii, 30, 34, 58, 91, 93, 106,
257, 259, 260, 261, 263, 265 122, 126, 149, 150, 154, 166, 177,
sensing, 52, 55, 56, 58, 62, 71, 72, 73, 183, 186
74, 77, 80, 82, 83, 89, 166, 262 solution, 5, 55, 57, 59, 63, 64, 78, 85, 94,
sensitivity, 50, 52, 55, 57, 59, 63, 71, 73, 95, 109, 110, 111, 112, 113, 128, 129,
76, 160, 206, 225 130, 134, 135, 147, 148, 149, 151,
sensitization, 200, 215 152, 154, 157, 160, 165, 167, 178,
sensor, viii, 47, 48, 49, 50, 51, 52, 53, 194
54, 56, 57, 58, 59, 62, 63, 64, 65, 66, solvents, 5, 41, 126, 128, 154, 163
69, 70, 71, 72, 73, 74, 75, 76, 78, 79, sorption, 54
81, 82, 83, 84, 85, 86, 87 spatial learning, 244
sensorimotor gating, 201 spatial memory, 201
sepsis, 246, 255 species, xiv, 2, 3, 4, 41, 92, 151, 165,
sequencing, 12, 264 176, 195, 198, 206, 215, 224, 229,
serotonin, 75, 87, 202, 203, 216, 218, 239, 248, 256, 260, 261, 262
244 specific surface, 51, 62
serum, 63, 126, 207, 208, 210, 220, 246 spectrophotometry, 65
sex, 215, 257, 258 spectroscopy, 25, 54, 74, 76, 80, 147,
sex differences, 258 149, 243, 255
shape, xii, 164, 165, 176, 177, 184, 187, spermatogenesis, 265
189, 191
Index 283
spinal cord, 196, 244 Sun, 43, 44, 52, 53, 54, 57, 58, 59, 70,
splenomegaly, 227 72, 73, 74, 80, 84, 102, 213, 233, 235,
stability, viii, xi, 52, 55, 56, 57, 91, 96, 265
98, 114, 124, 125, 135, 137, 142, 147, supplementation, xii, 175, 205, 207, 209,
148, 149, 150, 164, 165, 197, 245 223, 243, 244, 246, 250, 251, 255,
stabilization, ix, 105, 108, 110, 119, 128, 260
177 surface area, 51, 52, 53, 62, 177, 179,
standard deviation, 111, 112, 116, 131, 180, 187, 227
182, 185 surface energy, 128
starch, 62, 114, 120, 121, 125, 137, 191 surface modification, viii, 47, 49, 50, 54,
state, xi, 3, 4, 11, 37, 58, 142, 147, 149, 57, 59, 62
153, 155, 157, 158, 160, 161, 162, surface properties, xi, 54, 142, 163, 166,
165, 178, 218, 264 167, 191
statistics, 118 surface tension, 157, 184
steel, 64, 109 surfactants, xi, 59, 93, 131, 142, 144,
sterile, 228, 230, 231 155, 165, 172
stimulation, 89, 176, 207, 225 surgical removal, 227
stomach, 178, 188 survival, xii, xiii, 22, 168, 193, 204, 222,
storage, ix, 106, 108, 110, 111, 112, 113, 243, 246
114, 115, 116, 117, 118, 119, 122, swelling, 129, 130, 180, 186, 187, 231
124, 125, 147, 164, 165, 177 symptoms, 200, 201, 202, 203, 204, 207,
strategy use, 124 208, 209, 215, 218, 219, 223, 228
stress, vii, 1, 135, 158, 184, 188, 201, synaptic vesicles, 199, 214
202, 205, 206, 208, 209, 211, 212, syndrome, 216, 246
213, 216, 217, 218, 219, 233, 244, synergistic effect, 52, 92, 98, 100, 101,
245, 246, 249, 250, 251, 252, 254, 134
256, 257, 259, 260, 263, 264 synthesis, vii, viii, xiv, 2, 29, 31, 33, 34,
striatum, 199, 201, 214, 218 35, 42, 52, 53, 54, 56, 57, 71, 72, 77,
structure, viii, xii, 6, 11, 12, 13, 20, 24, 78, 79, 80, 81, 83, 89, 90, 93, 139,
25, 50, 51, 53, 56, 72, 91, 93, 107, 143, 145, 146, 147, 176, 195, 197,
114, 126, 130, 143, 146, 149, 150, 198, 199, 201, 202, 203, 204, 212,
154, 155, 156, 158, 161, 165, 170, 215, 229, 239, 240, 241, 242, 243,
171, 184, 193, 194, 199, 228 247, 248, 250, 251, 252, 254, 256,
substance abuse, 204 263
substrate, vii, viii, 2, 10, 14, 18, 24, 29, synthetic methods, 144
31, 36, 38, 93, 97, 98, 146, 243
sucrose, 62, 108, 109, 244
sugar alcohols, 35, 44 T
suicidal behavior, 209, 219
sulfate, x, 61, 85, 123, 127, 128, 129, tamoxifen, 177, 235
135, 161, 230 tannins, 126, 137
sulfur, 130 target, 56, 163, 203, 207, 216, 265
sulfuric acid, 143, 145 technical assistance, 188
techniques, 3, 37, 48, 56, 65, 125, 139,
164, 178
284 Index
technology, x, xii, 119, 123, 124, 175, treatment, xiii, 17, 21, 51, 65, 107, 111,
190 114, 165, 194, 200, 201, 202, 203,
TEM, 164 206, 208, 209, 210, 211, 215, 216,
temperature, ix, x, 34, 52, 58, 96, 106, 217, 218, 221, 222, 223, 226, 227,
108, 110, 113, 117, 118, 119, 123, 228, 229, 230, 232, 233, 234, 236,
128, 147, 148, 155, 160, 163, 165, 237, 243, 245, 246, 255
184 trial, 209, 218, 219, 220, 223, 226, 234
tension, 157, 158, 184 triglycerides, 249
terminals, 199, 200 tryptophan, 72, 80, 82, 202, 203
textural character, 179 tumor, xiii, 12, 17, 143, 144, 213, 222,
Thailand, 221, 228, 236 224, 225, 226, 227, 228, 229, 230,
therapeutic agents, 235 231, 233, 235, 236, 247, 257, 258
therapeutic effect, xiii, 163, 219, 222, tumor growth, 143, 236
223, 229 tumor metastasis, 247
therapeutics, 217 tumor necrosis factor, 257
therapy, 137, 202, 207, 216, 219, 220, tumors, xiii, 177, 222, 223, 226, 227,
223, 224, 227, 229, 230, 232, 233, 228, 229, 233, 234, 235, 243, 246
234, 235, 246 tungsten, 50, 66
thermal treatment, 51, 107 tunneling, 12, 26
thrombocytopenia, 228 type 1 diabetes, 262
thyroid gland, 248 tyrosine, 50, 67, 202, 203, 216, 229, 249,
tissue, ix, 24, 106, 107, 108, 109, 110, 258
112, 113, 114, 115, 117, 120, 121, tyrosine hydroxylase, 202, 203, 216
215, 238, 248, 264 tyrosine kinase receptor, 229
tissue engineering, 108
titanium, 58, 68, 78
TNF, 203, 216, 245, 250, 259 U
TNF-α, 203, 216, 259
tobacco smoke, 253 U.S. Department of Agriculture, 42
tocopherols, viii, 91, 92 uniform, 129, 135
toluene, 55, 74 uric acid, 49, 61, 62, 63, 66, 67, 68, 69,
toxic products, viii, 91, 143 70, 71, 72, 73, 74, 75, 76, 77, 78, 80,
toxicity, 177, 218, 227, 234, 247, 258, 81, 82, 83, 84, 85, 86, 87, 88, 89, 243
265 urine, 63, 197
transcription factors, 245 UV irradiation, 249
transfer performance, 62 UV light, 124
transformations, 36, 112
transfusion, 230 V
transition metal, 58, 124, 224
translation, 204 vaccine, 167
translocation, 207 vacuum, 107
transmission, 164, 201, 204 valuation, 212
transport, 3, 17, 21, 22, 122, 125, 197, vanadium, 56, 79
201, 214, 224 vapor, 108, 109
transportation, 177 variables, 137, 179, 191
Index 285
variations, 128, 145, 218 158, 160, 164, 166, 167, 176, 179,
vasoactive amines, 229, 231 180, 181, 182, 183, 184, 187, 194,
vegetables, 30, 48, 64, 107, 119, 124, 243, 247, 250, 263
228, 230 water absorption, 179
vegetal tissue, 107, 109, 117 water vapor, 108, 109
vertebrates, xiv, 239 wettability, 184
vesicle, 19, 21, 24 Wnt signaling, 205
vinblastine, 229, 230 workers, 50, 51, 52, 53, 55, 57, 58, 59,
viscoelastic properties, 147, 148 63, 225
viscosity, 126, 163 worldwide, 106, 202, 222
visualization, 158, 163 wound healing, 197, 222
vitamin C, vii, viii, x, xiii, 2, 3, 19, 20,
22, 29, 31, 34, 44, 47, 48, 63, 136,
137, 141, 143, 144, 150, 188, 189, X
194, 196, 198, 205, 206, 208, 209,
210, 211, 212, 215, 218, 219, 220, xenografts, 213, 225, 233
221, 223, 224, 230, 234, 235, 236,
241, 254, 255, 256, 258, 259, 260, Y
262, 263, 264
vitamin C deficiency, 212, 254, 255, yeast, 37, 40, 138, 218, 228
262, 264 yield, 9, 35, 37, 38, 40, 51, 145, 146,
vitamin E, 2, 20, 208, 211, 244, 255 147, 231, 232
vitamins, 30, 121, 125, 137, 178, 188, young adults, 209
208, 218, 219, 220, 223, 236 young women, 234
vomiting, 223
Z
W
zinc, 71, 107, 120, 121, 122
Washington, 42, 43, 168, 189 zinc oxide, 71
water, vii, viii, ix, x, xi, 6, 12, 34, 36, 47, zirconium, 58, 83
51, 63, 89, 94, 101, 102, 106, 108, ZnO, 50, 53, 67, 80
109, 110, 112, 113, 114, 115, 116, ZnO nanorods, 80
118, 124, 126, 127, 137, 141, 142,
143, 146, 147, 154, 155, 156, 157,

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BIOCHEMISTRY RESEARCH TRENDS

Additional books in this series can be found on Nova’s website

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Published by Nova Science Publishers, Inc. † New York

Chapter 7 Alkyl Esters of L-Ascorbic Acid: From Synthesis to

synthesized L-AA is used in the feed, food, nutrition and pharmaceutical

humidity, and then, the oxidation process was measured by gas

Chapter 6 - Ascorbic acid has a variety of biological and dermatological

This chapter offers a comprehensive review of the bibliography regarding

immunity, promotes collagen biosynthesis, provides photoprotection, and

Additionally, ascorbic acid is a powerful antioxidant highly concentrated in

Chapter 11 - Ascorbic acid (AsA) functions as a cofactor in a variety of

OVERVIEW OF THE PHYSIOLOGICAL

AsH--dependent oxidoreductase transmembranes. Many cytochrome b561

Keywords: ascorbate, monodehydroascorbate radical, pulse radiolysis,

Under physiological conditions, AsH- typically undergoes a reversible

1. CHEMICAL PROPERTIES OF ASCORBATE

Figure 1. (A) Titration of ionizable group of ascorbate. The ascorbate monoanion

Figure 2. pH-dependent decay of the second-order rate constants of As-･.

The equilibrium constant ([As22-]/[As-･]2 = k1/k-1) was calculated to be 103

Figure 3 shows recycling of ascorbate in the cell. Ascorbate is transported

Figure 3. Recycling ascorbate in the cell.

The reduction of As is probably less important than reaction of As-･. As

2.2. Reaction Mechanism in Monodehydroascorbate Reductase

In plants, As-･ is reduced to regenerate AsH- by an NAD(P)H-dependent

oxidized form, the reduced charge-transfer complex, and the semiquinone

Figure 5. Proposed topological model of adrenal cytochrome b561 in the CG membrane.

Cytochrome b561, initially identified in the chromaffin granule (CG) of

cytochrome b561, and subsequently cytochrome b561 is reduced by

Lu et al. [81] reported the crystal structure of the ferric form of

Figure 6. Crystallographic structure of cytochrome b561 from Arabidopsis thaliana. The

Figure 8. Recognition of ascorbate by conserved amino acids. (A) Recognition of

The negatively charged substrate AsH- or As- ･ binds to the conserved

3.3. Reaction Mechanisms

Table 1. Rate Constants of the Oxidation of heme b by As-･ and

(Kyoto Prefectural University) for the reaction mechanism of MDA reductase;

[12] Skotoland, T. and Ljones, T. (1980). Ascorbate free radical; Dopamine

[23] Kelley, P. M., and Njus, D. (1986). Cytochrome Spectral Changes

[1] Oxidative Stress Sensing by the Iron-Sulfur Cluster in the Transcription

[6] Binding of Promoter DNA to SoxR Protein Decreases the Reduction

PRODUCTION OF L-ASCORBIC ACID

Sui Kiat Chang

acid. All these steps utilize the concept of biotechnology by using

Keywords: L-Ascorbic acid (L-AA), 2-keto-L-gulonic acid, fermentation, L-

been reported in the past. These pathways build on the conversion of a

CHEMISTRY (GENERAL AND PHYSICAL PROPERTIES) OF

Figure 2. Outline of the chemical L-AA synthesis according to Reichstein and

L-AA and L-dehydroascorbic acid (L-DHA) are the primary dietary

THE REICHSTEIN PROCESS: THE MAJOR INDUSTRIAL

a) Oxidation of L-Sorbose to 2-keto-L-Gulonic Acid and

Oxidation of L-sorbose to 2-keto-L-gulonic acid was achieved by treating

sodium methoxide in methanol, followed by acidification. During the

BACTERIAL FERMENTATION PROCESSES FOR

Bacteria have been identified to be able to transform glucose to 2,5-keto-

The Two-Step 2-Keto-L-Gulonic Acid Fermentation Process

The conversions occurring during the fermentation of D-sorbitol with

The One-Step 2-Keto-L-Gulonic Acid Fermentation Process

The specific activity of Gluconobacter to convert D-sorbitol to L-sorbose

Figure 3. Biochemistry of the oxidation of L-sorbose to 2-keto-L-gulonic acid in

In a patent filed by Hoshino et al. [23], the one-step 2-keto-L-gulonic acid

Figure 4. Outline of the industrial L-AA production based on the fermentation of 2-