M.phil Thesis Saif

In vitro antioxidant and inhibitory effects of Myristica fragrans,
Illicuim verum, Curculigo orchioeides, Glycyrrhiza glabra and

Embelia ribes against lipid peroxidation in mice brain and liver
This thesis is submitted in partial fulfilment of the
Requirement for the degree of Master of Philosophy
By
Saif Ur Rehman
Thesis Submitted in Partial Fulfilment of Requirements for the Degree of

Master OF PHILOSOPHY
In Bio Chemistry
DEPARTMENT OF Biological Sciences, GOMAL UNIVERSITY
DERA ISMAIL KHAN (KPK) PAKISTAN
2014
In the name of Allah, the Most Merciful, the Most
Kind
Dedication
To
MY MOTHER
For
Leading me into intellectual pursuits and unconditional support in my studies
To
MY FATHER
For
The uncompromising principles that guided me throughout my student life
To
MY SISTERS
For
Their kindness and to create a friendly environment
To
MY TEACHERS
For
Their guidance and kind patronage throughout my research pursuits
DECLARATION
I, Saif ur Rehman, M.Phil scholar of the Department of Biological
sciences, ,GomalUniversityD.I.Khan, hereby declare that this research work entitled “In vitro
antioxidant and inhibitory effects of Myristica fragrans, Illicuim verum, Curculigo
orchioeides, Glycyrrhiza glabra and Embelia ribes against lipid peroxidation in mice brain
and liver” is done by me under the kind supervision of Dr.Asma Saeed and that nothing has
been incorporated in this dissertation without acknowledgment and that to the best of my
knowledge and belief it does not contain any material previously published or any material
previously submitted for a degree in any University; where due reference is not made in the
text.
SAIF UR REHMAN
CERTIFICATE OF APPROVAL
“In vitro antioxidant and inhibitory effects of Myristica fragrans,
Illicuim verum, Curculigo orchioeides, Glycyrrhiza glabra and
Embelia ribes against lipid peroxidation in mice brain and liver”
The thesis prepared by Mr. Safi.Ur.Rehman under my guidance in partial
fulfillment of the requirements for the degree MASTER OF PHILOSOPHY IN BIO
CHEMISTRY is hereby approved for the submission to the Department of
Biological Sciences, Gomal University D. I. Khan, KPK Pakistan.
Dr.ASMA Saeed
Supervisor
ROLL NO 13
SESSION
RESEARCH SUPERVISOR AND DR. ASMA SAEED
INTERNAL EXAMINER
DEAN
EXTERNAL EXAMINER
ORAL EXAMINATION HELD ON:
PhD SCHOLAR
ACKNOWLEDGMENTS
All praise is for the Supreme Being, “ALLAH” who guides me in darkness, and bring
out my abilities and best efforts in a very commendable manner to complete my research
work, and all the respect is for the Last Prophet “MUHAMMAD” (Peace be upon Him) who
enabled us to recognize our Creator. Almighty Allah never spoils the efforts. I am thankful to
Almighty Allah the most Gracious, the Merciful who blessed me with good health, talented
teachers, affectionate family, brothers, sisters, and friends who provided an opportunity to
complete this M.PHIL research work.
I owe a profound debt of gratitude and thanks to my research supervisor Dr.ASMA for his
encouragement and advice to carry out this research work. Dr. ASMA door is always open
for discussions. I am greatly appreciative of his expertise and guidance without which this
project would not have been possible. He has a sound, critical and healthy research attitude
towards the ANTI OXIDANTS studies. I deem it a great honor and no word is too eloquent
to express my sense of gratitude to him. .
I express my most sincere gratitude to my Research Co-Supervisor Dr. Muhammad Akram,
Faculty of Medical & Health Sciences, University of Poonch, Rawalakot, for his invaluable
guidance, constant supervision, monitoring, encouragement and instruction to carry out the
research work embodied in this thesis.
. I am very thankful to Prof. Dr. Abdul Haleem, Chairman Biological sciences for providing
the necessary research facilities to conduct my research work. I highly appreciate and
acknowledge the help extended by Asmatullah Khan, during the in-vitro studies of my
project. Also, many thanks to Dr.Jabbar Khan, Dr.Hafeezullah and Dr.Abid Hussain for their
help and guidance. Special thanks to all my teachers at Department of Biological sciences,
Gomal University, D.I.Khan. Thanks to my friends at Department of Biological sciences,
especially Malik Muhammad Irfan, Muhammad Sohaib, Hafiz Muhammad Rafie, Asghar Ali
Asghar, Khuramur Rehman. I am also grateful to the office and laboratory staff for their
cooperation and friendly attitude especially Malik Shahjahan, Muhammad Naeem,
Muhammad Ayaz and at last my best friends Abdul Hamid,Iftikhar Ahmad,Hamza
Altaf,Nazir Suleman,Imran Quyyum and Ehitasham Ahmad and in last but not the least
Dr.Muhammad Asif.
This dissertation is dedicated to my family, for without their blessings nothing would have
been possible. My sisters and brothers have been a driving force behind me for my
accomplishments and achievements. Thanks a lot for everything that you have been doing for
me.
SAIF. UR. REHMAN.

CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBRIVIATION
ABSTRACT
INTRODUCTION
1 Definition of Antioxidant
1.1.1 Classification of Antioxidants
1.1.2 Primary Antioxidants
1.1.2.1 Secondary Antioxidants
1.1.2.2 Miscellaneous antioxidants
1.1.2.3 Sources of antioxidants
1.1.3 Synthetic antioxidants
1.1.3. 1 Natural antioxidants
1.1.3. 2 Lipid Oxidation
1.1.4 Natural Antioxidants in Plant Sources

1.1.5 Tocopherols
1.1.6 Flavonoids
1.1.8. Carotenoids
1.1.9 Other Natural Antioxidants
1.1.10 Phytosterols
2 LITERATURE REVIEW
Therapeutic plants: source of impressive antioxidants
2.1.
2.2. Free radical generation and oxidative stress

2.3. Effect of antioxidants from different plant fractions on oxidative stress
2.4. In vitro assays estimating the antioxidant capabilities of medical plants
2.5 Overview of plants.
2.5.1 Myristica fragrans
2.5.2 Plant description
2.5.3 Chemical constituents.
2.5.4
Pharmacological actions
2.5.5
Medicinal uses
2.6 Illicium verum

2.6.1
Plant description
2.6.2 Chemical constituents

2.6.3 Pharmacological actions
2.6.4 Medicinal uses.
2.7.
Curculigo orchioides
2.7.1
Plant description
2.7.2
Chemical constituents.
2.7.3
Pharmacological actions.

2.8
Glycyrrhiza glabra
2.8.1
Plant description
2.8.2
Chemical constituents.

2.8.4 Medicinal uses
2.9 Embelia ribes Brum
2.9.1
Plant description

2.9.4
Medicinal uses
3 MATERIALS AND METHODS

3.1. Materials
3.2
Preparation of plant extract
3.3
Test animals
.
3.4 In vitro assays.
3.4.1.
DPPH radical-scavenging
3.4.2. Production of TBARS from animal tissues

3.4.3. Total Antioxidant assay
3.4.4. Phenolics content
3.5 Statistical analysis
3.6.
Aims and objectives
4 RESULTS AND DICUSSION

4.1. Results
Lipid peroxidation in mice brain induced with sodium nitroprusside
4.1.1.
and iron
Lipid peroxidation in mice liver induced with sodium nitroprusside and
4.1.2.
iron
4.1.3. DPPH radical scavenging activity
4.1.4. Total antioxidant activity by phosphomolybdenum assay
4.1.5. Total phenolics content
4.2. Discussion
CONCLUSIONS
REFERENCES
List of Publications
Chapter 1: Introduction
LIST OF FIGURES
Figure Title Page No
No
1.1 The interaction among vitamin C, vitamin E and thiol redox cycles is shown by antioxidant
5
network
1.2 Chemical structures of the phytosterols and cholesterol 10
2.1 Seed of Myristica fragrans 14
2.3 Roots of Curculigo orchioides

2.4
Roots of Glycyrrhiza glabra
2.5 Fruit of Emblica ribes

2.6 Leaf of Emblica ribes
4.1 The effect of aqueous extracts of medicinal plants against lipid peroxidation in
mice brain induced by sodium nitroprusside.
(a).The effect of Myristicafragrans, Illicuim verum, Curculigo orchioeides

against lipid peroxidation in mice brain.
(b) the effect of Glycyrrhiza glabra, Embelia ribes against lipid peroxidation
in mice brain
mice brain induced by iron.
(a).The effect of Myristicafragrans, Illicuim verum, Curculigo orchioeides

against lipidperoxidation in mice brain
(b) the effect of Glycyrrhiza glabra, Embelia ribes against lipidperoxidation in

mice brain
mice liver induced by sodium nitroprusside
(a).The effect of Myristica fragrans, Illicuim verum, Curculigo orchioeides

against lipidperoxidation in mice liver.
(b) The effect of Glycyrrhiza glabra, Embelia ribes in mice liver

mice liver induced by iron.
Page 6
(a).The effect of Myristica fragrans, Illicuim verum and Curculigo orchiodes

against lipidperoxidation in mice liver.
(b) the effect of Glycyrrhiza glabra, Embelia ribes in mice liver

4.5 DPPH radical scavenging activity of different plants.
4.6 Total antioxidant activity measured by phosphomolybdenum reduction
method.
LIST OF TABLES
Table No Title Page No
4.1 Total phenolic content among aqueous extract of medicinal plants 3
Page 7
LIST OF ABBREVIATIONS
M.F Myristica fragrans
I.V Illicuim verum
C.O Curculigo orchioeides
G.G Glycyrrhiza glabra
E.R Embelia ribes
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Abstract
The present study compares the protective properties of Myristica fragrans, Illicuim verum,
Curculigo orchioeides, Glycyrrhiza glabra and Embelia ribes on lipid peroxidation induced by
different pro-oxidants. The extracts showed inhibition against thiobarbituric acid reactive species
(TBARS) induced by different pro-oxidants (10 µM FeSO4 and 5 µM sodium nitroprusside) in
mice brain and liver. Moreover, the free radical scavenging activities of the extracts was
evaluated by the scavenging of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50, 151.50±2.3
µg/ml (Myristica fragrans), 42.41±1.2 µg/ml (Illicuim verum), 71.14±0.6 µg/ml (Curculigo
orchioeides), 26.60±0.45 µg/ml (Glycyrrhiza glabra), and 106.30±1.1 µg/ml (Embelia ribes).
The higher inhibitory effect of Glycyrrhiza glabra could be attributed to its significantly higher
phenolic contents, reducing ability and free radical scavenging activities. Therefore, the
oxidative stress in brain and liver could be potentially managed/prevented by the dietary intake
of Glycyrrhiza glabra, Illicuim verum, Myristica fragrans, Curculigo orchioeides and Embelia
ribes. The higher inhibitory effect of Glycyrrhiza glabra, Illicuim verum plants which justifies
the use of these plants in various degenerative diseases. Myristica fragrans,Curculigo
orchioeides and Embelia ribes relatively showed weak antioxidant activities.
INTRODUCTION
Page 9
For more than five million years, medicinal plants are being used as primary source of relief from
sickness (Newman et al., 2000). Herbal plants were used to be the foundation of several traditional
medicines around the world including countries like India and China (Balandrin et al., 1985). In
modern practice, they occupy an important place in the raw materials used for the manufacturing of
therapeutic products (Taylor et al., 1995). Such plants play their role as neutraceuticals, cosmetics,
food supplements as well as pharmaceuticals. According to the World Health Organization (WHO),
about 75- 80% of population still depend on conventional medication i.e. herbal drugs to meet their
prime health concerns (Aibinu et al., 2007).
In past, plants have been remained major source of human medicines . Through observed
innovation, humans of many cultures have constantly recognized plants having favorable health
effects. The 20th century brought additional understanding of human health and the development
of synthetic or semisynthetic analogs of plant compounds that led to drugs with higher levels of
effectiveness.
From the past few years, researchers are trying to discover the natural antioxidants from easily
accessible mistreated fruits, vegetables and wild plants (Umamaheswari and Chatterjee, 2008;
Kandasamy et al., 2010) as well as their role in detoxifying free radicals which stimulate majorly
lung and liver injuries in the experimental animal model (Kil et al., 2009). A wide variety of such
plant species (250,000 to 500,000) are available which can be employed to isolate pharmacologically
important substances, further used in the manufacturing of various drugs. Stem, leaf, Fruit, flower,
root and twigs of the plant are utilized for the extraction and characterization of medicinally active
compounds (Uniyal et al., 2006). The utilization and demand of therapeutic plants is increasing day
by day because the isolated natural products are nontoxic, available at economical prices and have no
side effects (Britto et al., 2011).
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1.1. ANTIOXIDANTS
1.1.1. Definition of Antioxidant
Antioxidants are group of chemicals that defend biological systems against the possible injurious
effects of reactions or processes that cause oxidation. The U.S FDA explains antioxidants as
“preservatives that specifically retard deterioration, rancidity, or discoloration due to oxidation”
(Specchio, 1992). In most raw materials, the antioxidants exist as natural component. But, during
manufacturing of food and storage the natural antioxidants are normally destroyed or exhausted.
So, to retain the food quality and higher shelf-life the addition of antioxidants to food products is
essential. Antioxidants to be used in food products should be economical, harmless, higher
therapeutic index, stable, and have no or nominal effect on physical properties (color, flavor and
odor) of food products (Reische, 1998; Rajalakshmi and Narasimhan, 1996). The use of
antioxidants in food products is regulated by laws and international standards (Rajalakshmi and
Narasimhan, 1996).
1.1.2. Classification of Antioxidants
Antioxidants have different mechanism of actions such as quenching of secondary lipid
oxidation products, chelating of metals, inactivation of reactive oxygen species and peroxides
and free radical scavenging. Antioxidants are classified as primary and secondary antioxidants
due to their mechanism of action (Rajalakshmi and Narasimhan, 1996).
1.1.2.1. Primary Antioxidants
1. Primary antioxidants donate hydrogen or electrons to lipid free-radicals and disturb
radical chain reactions by converting them into more stable non radical products (a)
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(Rajalakshmi and Narasimhan, 1996). They also react with lipid peroxy and alkoxy
radicals and nonlipid free radicals (b, c) by same mechanism. In fact, the primary
antioxidants are most effective during induction period, where the antioxidants are
utilized and free-radicals are Produced (Reische, 1998). Along H donation, the primary
antioxidants also form lipid-antioxidant complexes when interact with lipid free radicals
(Rajalakshmi and Narasimhan 1996). They can reduce hydro peroxides to hydroxy
compounds (Reische 1998).
AH + R Aͦ + RH
ͦ→
(a) H donation of antioxidant to lipid free radical
AH + ROO. _ A. + ROOH (b) H donation of antioxidant to lipid peroxy radical
AH + RO. _ A. + ROH (c) H donation of antioxidant to lipid alcoxy radical
Following donation of H from antioxidants, the antioxidant radicals formed further interfere with
the chain-propagation reactions by inhibiting the peroxy or alcoxy lipid radicals (d,e). The
antioxidant radicals also react with each other and contribute to termination reactions (f).
ROO. + A. _ ROOA (d) Reaction of peroxy radical with antioxidant radical

RO. + A. _ ROA (e) Reaction of alcoxy radical with antioxidant radical
A. + A. _ AA (f) Reaction of antioxidant radicals
Primary antioxidants even show their activity at very low concentrations.
However, at very high concentrations they may act as prooxidants. The synthetic phenolic
antioxidants are the major primary antioxidants (Rajalakshmi and Narasimhan, 1996; Reische
1998). However, although these antioxidants are highly effective to prevent autooxidation, only
a few of them is approved for food applications. The major considerations of acceptability of
synthetic phenolic antioxidants are potential toxicity and/or carcinogenicity of these compounds.
The examples of these synthetic antioxidants are tertiary butylhydroquinone. Propyl gallate,
Page 12
butylated hydroxytoluene and butylated hydroxyanisole. The natural phenolic antioxidants and
tocopherols can also act as primary antioxidants.
1.1.2.2. Secondary Antioxidants
There are different mechanisms by which secondary antioxidants may act to slow down the rate
of oxidation. These antioxidants are also called synergists, since they enhance the antioxidant
activity of primary antioxidants. However, they do not convert free radicals to more stable
products (Reische, 1998). The major type of these antioxidants contains chelators, singlet
oxygen quenchers, reducing agents and oxygen scavengers (Rajalakshmi and Narasimhan 1996).
Oxygen scavengers and reducing agents act by scavenging oxygen and donating H atoms to
peroxy radicals and primary antioxidants. The H donation to primary antioxidant radicals
regenerates primary antioxidants and this enables using primary
Antioxidants more effectively (Rajalakshmi and Narasimhan, 1996). Example antioxidants in
this group include Vitamin C and its derivatives and sulfides. On the other hand chelators
include ethylenediaminetetraacetic acid (EDTA), polyphosphates, citric acid, citrate esters,
lecithin, phytic acid and tartaric acid (Rajalakshmi and Narasimhan, 1996; Reische, 1998). These
substances form complexes with prooxidant metals such as iron and copper and increase the
effect of oxygen scavengers and primary antioxidants significantly. The metals speed up the
oxidation reactions by acting as catalysts in free radical formation reactions. In initiation step
they can also lower the activation energy. Metal atoms can either interact directly with lipids (g)
or with hydroperoxides (h,i) to form active radical species. These reactions can be periodic with
regeneration of the lower oxidation state of the metals (Reische 1998).
RH + M(n-1) _ Mn+ + H+ + ROO (g) Metal atom -lipid interaction

ROOH + M(n+1)+ _ Mn+ + H+ + ROO
(h) Metal atom (lower oxidation state)- hydroperoxide interaction
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ROOH + Mn+ _ M (n+1)+ + OH- + RO (i) Metal atom (higher oxidation

state)-hydroperoxide interaction
Hydroperoxide degradation is speeded up by the metals in their lower oxidation states as
compared to metals in their higher oxidation states (Reische, 1998). Thus, in presence of metals,
reducing agents such as ascorbic acid act as pro oxidants by converting metals such as Fe+3 and
Cu+2 to their lower oxidation states (Fe+2 and Cu+). Singlet oxygen quenchers, on the other
hand, are secondary antioxidants that deplete high energy of singlet oxygen and dissipate the
energy in the form of heat (Reische, 1998). Photooxidation of unsaturated fats occurs due to high
energy molecule known as singlet oxygen and subsequently hydroperoxides are generated
(Reische, 1998).
1.1.2.3. Miscellaneous antioxidants
Miscellaneous antioxidants are compounds that may act as primary or secondary antioxidants.
Miscellaneous antioxidants comprises of natural phenolic compounds like flavonoids and
related compounds, zinc , nitrites, Maillard reaction products, proteins and nitrates, carotenoids,
amino acids, glucose oxides, superoxide dismutase , catalase and glutathione peroxidase
enzymes. (Rajalakshmi and Narasimhan, 1996). Natural phenolic compounds can act as primary
antioxidants due to their chain breaking properties. Proteins and Maillard reaction products act
as chelator and radical scavenger. Enzyme glucose oxidase is an oxygen scavenger (Labuza and
Breene, 1989), superoxide dismutasecatalase enzyme mechanism degrades reactive oxygen
species to oxygen and water, peroxides are reduced to alcohol by glutathione peroxidase
(Nordberg and Arner, 2001). Zinc prevents iron binding and subsequently lipid peroxidation is
inhibited by Zinc at the membrane level (Rajalakshmi and Narasimhan, 1996).
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1.1.3. Sources of antioxidants
1.1.3. 1. Synthetic antioxidants
The antioxidants utilized in foods are TBHQ, PG, BHA and BHT that are synthetic antioxidants
(Rajalakshmi and Narasimhan, 1996). From these antioxidants, the synthetic phenolic
antioxidants, BHT and BHA, have a particular importance, since they are the most preferred
food antioxidants. These antioxidants are strongly lipophilic. They are also fairly heat stable
antioxidants which are suitable for thermally processed food (Reische, 1998). Moreover, BHA
and BHT are steam volatile. Thus, they easily diffuse into food lipid layers and inhibit oxidation
when incorporated into food packaging materials. On the other hand, TBHQ is a very heat stable
antioxidant which is proper choice for frying applications (Reische, 1998). The other synthetic
antioxidant is PG that is less thermostable in nature due to which it is not appropriate for frying
applications. Since PG combines with copper and iron and undesirable dark color complexes are
formed, its preparations should be combined with chelators (Reische, 1998).
1.1.3. 2. Natural antioxidants
Recently, significant concerns have been raised related to the use of synthetic antioxidants in
foods. Mainly, the suspicious carcinogenic effects of BHA and BHT on laboratory animals
increased the demand of natural antioxidants enormously (Yang et al., 2000; Hwang et al.,
2001). Natural antioxidants are gladly accepted by the consumers because they are not
considered as chemicals. Also, the natural antioxidants have a GRASS (Generally Recognized
As Safe) status and do not require toxicological testing. However, natural antioxidants need
generally a purification before used in food applications because of their lesser effectiveness
than synthetic antioxidants, (Rajalakshmi and Narasimhan, 1996). This makes natural
antioxidants more costly than the synthetic ones. Also, most natural antioxidants effect food
Page 15
color and flavor badly. Their strong flavor is the main limitation for the use of herb and spice
extracts rich in phenolic acids and flavonoids and tea extracts rich in catechins (Reische et al.,
1998). On the other hand, the rosemary extract containing diterpene, phenolics, carnisol and
carnosic acid, is one of the few commercially available odorless and tasteless phenolic extracts
(Medhavi et al., 1996b; Reische et al., 1998).
Ascorbic acid (vitamin C) and its derivatives such as erythorbic acid and ascorbyl palmitate are
other important natural antioxidants that have GRASS status. Due to hydrophilic nature of
ascorbic acid, except the more lipid soluble ascorbyl palmitate, they are not suitable to be
utilized in fat-containing food (Reische et al., 1998). However, after prohibition and restrictions
of using sulfites in processed fresh fruits and vegetables, vitamin C (Ascorbic acid) and its
derivatives became the major substitutes to prevent enzymatic and non-enzymatic browning in
these products (Sapers and Ziolkovski, 1987; Yemenicioglu, 2002). Carotenoids such as lutein,
lycopen, licopen, isozeaxanthin and carotene are lipophilic antioxidants. However, these natural
antioxidants can be only used when their colors like yellow, red etc. are compatible with the
food. Another lipophilic in nature antioxidant group is tocopherols that have vitamin E activity
in the diet (Reische et al.,1998). These natural antioxidants present in high amounts in most oil
seeds and pass into crude oil during pressing and extraction. However, the tocopherols show
high antioxidant activity in animal fats than in edible oils. Proteins, protein hydrolysates,
peptides, amines and amino acids are also an important group of natural antioxidants. The
antioxidant groups in proteins may show Radical scavenging activity (Rajalakshmi and
Narasimhan, 1996). Also, some iron binding proteins can act as chelating agents (Reische et al.,
1998).
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1.1.4. Lipid Oxidation
Lipid oxidation affects the processing, shipment and storage of food containing lipids. Sterols,
triglycerides and phospholipids are lipids that are affected by oxidation process because of
presence of oxygen in the environment. They are prone to be oxidized due to unsaturated
double bonds of lipid molecules. Polyunsaturated fatty acids are found in food stuffs that are
highly susceptible to lipid oxidation. Oxidation of lipids is usually seen in food stuffs that are
mediated by reactive oxygen species and oxygen free radicals (Barry et al., 1995).
Two unpaired valence electrons are found in triplet molecule. Energy is absorbed by the
triplet oxygen molecule through a chemical or physical reaction and excitation of electron
occurs. This excited electron reacts with unsaturated fatty acids and excess energy is released.
Reaction of singlet oxygen with linoleic acid is 1450 times more than triplet oxygen. Rancidity
of cooking oil occurs due to singlet oxygen during dispensation, transport and oil storage.
Following three reactions occur that are involved in lipid peroxidation mediated by free
radicals and reactive oxygen species (Angelo et al., 1996):
Initiation: RH + initiator → R· (1)
ROOH + initiator → ROO·
Propagation: R· + O2 → ROO· (2)
ROO· + RH → ROOH + R·
Termination: R· + R· → R-R (3)
ROO· + R· → ROOR
Page 17
Various chemical and physical factors (initiators) initiate the above mechanisms. The factors
involved are radiations, heating, transition metal ions, reactive oxygen species and temperature.
Preliminary radical residues are produced. Free radical chain reactions are triggered by initiators
and oxidation of the substrate molecule is mediated and hydrogen is released from substrate
molecule as a result fee radical R• is formed. Lipid hydroperoxide (ROOD) or an allytic group of
an unsaturated fatty acid (RH) is site where initiation occurs as shown in equation 1. Rree
radical R• reacts with oxygen and Peroxy radial (ROO•) is produced. Peroxy radial (ROO•)
reacts with another lipid molecule as a result another lipid free radical (R•) and hydroperoxide
(ROOH) is produced causing a flow model of chain reactions until other free radicals neutralize
the free radical, the reaction of which is shown in equation 2. In the last phase, non free radical
products merge into two radicals and flow model of chain reaction stops (Phase 3). In addition,
some free radical scavengers or antioxidants also terminate the chain reaction. Copper and iron
are the transition metal ions that act as catalyst and initiate the chain reactions. Furthermore,
lipoxygenase can initiate the oxidation process acting as initiator and peroxides are generated in
food stuffs containing lipids.
Hydroperoxide is produced initially as a primary product and is converted to several
secondary products in oxidation process. The compounds that are usually produced are
aldehydes, ketones, alcohols, acids and hydrocarbons. Edibility, quality and appearance of food
products is badly affected by these compounds as a result texture, wholesomeness, color, flavor
and nutritional value of food products is impaired (Angelo., 1996).
1.1.5. Natural Antioxidants in Plant Sources
Process of lipid peroxidation can be inhibited or delayed with natural and synthetic antioxidants.
Antioxidants refer to those substances that have ability to prevent oxidation mediated by
prooxidants. Prooxidants are those substances that can cause oxidative reaction. Antioxidant acts
as singlet oxygen scavenger, chelator, reducing agent or free radical scavenger. Many synthetic
anti-oxidants are available but their use is minimum due to adverse effects and toxicity. Tertiary
butylatehydroquinone (TBHQ), butylated hydroxyl toluene pueraria glycoside and butylated
hydroxyl anisole are classical antioxidants allowed as the food additives.
Scientists are now in search of natural antioxidant from the different sources because
synthetic food additives have adverse. Natural antioxidants are found in all parts of plant and
Page 18
are mainly in plant phenolic compounds. Cinnamic acid, tocopherol and coumarins,
polyfunctional organic acids and falvonoid compounds are common phenolic antioxidant
derived from plants (Shahidi et al., 1992).
1.1.6. Tocopherols
Tocopherols prevent oxidation of polyunsaturated fatty acids found in food containing oils or
fats and are widely used in the food industry (Khan et al., 2001). Tocopherols are added in
edible oils in shipment, processing and storage. Tocopherols transfer phenolic hydrogen to the
radical molecule and singlet oxygen is scavenged. Tocopherols act as radical scavengers and
lipid radicals (R•) by tocopherols produced in free radical reactions. Tocopherols (AH 2)
transfers hydrogen atom to the radical R• and RH molecule is produced as a result tocopheryl
semiquinone radical (AH·) is formed. Tocopheryl quinine (A) and tocopherol molecule is
formed by combination of two tocopheryl semiquinone radicals (AH•) with one another or it is
reduced by donation of hydrogen atoms from ascorbic acid. NADH acts as reducing agent and
cause reduction of oxidized ascorbic acid. This mechanism has been shown in figure 1(Packer,
et al., 2001). It is important to mention that tocopherols do not entirely prevent the reaction
but it slow down or interrupt a free radical reaction.
Soybean oil contains more tocotrienols and tocopherols than other plants. Furthermore, 30 to
40% of tocotrienols and tocopherls are varnished during process of oil manufacturing (Erickson.,
1980).
Page 19
Figure 1—The interaction among vitamin C, vitamin E and thiol redox cycles is shown
by antioxidant network (Packer, et al., 2001)
1.1.6 Flavonoids
Flavonoids are in all parts of plants and known as polyphenolic compounds (Nijveldt, et al.,
2001). Flavonoids are categorized chalconcatechins, isoflavones, anthocyanines, flavones and
flavonols. Round about 4,000 flavonoids have been reported, most of them have anti-oxidant
activity. Most important anti-oxidant is Quercetin and catechin and they are widely used in food
industry. (Heim et al., 2002).
Molecular structures of flavonoids are basis for their antioxidant. Antioxidant activity
will be more on hydroxylation of the B ring. Quercetin plays an important role in antioxidation
Page 20
by scavenging free radicals (Namiki, 1990). An additional hydroxyl group is present at 5’
position of the B ring of robinetin and myricetin, their antioxidant activities are significant as
compared to other flavones with no such 5’ hydroxyl group. Antioxidant activity of
isoflavone, i.e., genistein is significant because at the 4’ and 5’ positions of isoflavone are
found both hydroxyl groups (Heim, et al., 2002).
1.1.7. Ascorbic Acids
Ascorbic acid(vitamin C) is an important antioxidant acting as free radical scavenger and
reducing agent. Free radical chain reactions are reduced by hydrogen atoms are donated by
ascorbic acid and its etherified derivatives and double bond of substrate molecule is protected.
Ascorbic acid (vitamin C) converts oxidized tocopherols to its reduced form thus working as a
synergist for tocopherol. glutathione or NADH also donates hydrogen to ascorbic acid (vitamin
C) for regeneration of dehydro ascorbic acid (Johnson, 1979).
1.1.8. Carotenoids
Carotenoids are found in all segments of plants and are considered as a category of natural, fat-
soluble compounds. Carotenoids are involved in photosynthesis and give red, yellow and
orange color to different parts of plants. Molds, yeasts, non-photosynthetic bacteria,
photosynthetic bacteria and algae also contain carotenoids, where they act as protective agent
against oxygen and light induced membrane damage. Due to their antioxidant activities, food
scientists are interested in xanthophylls and carotene. Oxygenated derivatives of carotenes are
known as xanthophylls (lutein) (Delgado et al., 2000)
1.1.9. Other Natural Antioxidants
Antioxidant activities have been reported in many other compounds such as phytosterols
(Boskou, 1998). Antioxidant activity of consol has been reported that is found in rosemary
Page 21
extract. Tea leaves also possess antioxidant activities (Wiseman et al., 1997).
1.1.10. Phytosterols
Phytosterols are triterpenes containing 28 or 30 carbon atoms. Steroid nucleus is found in
phytosterols and is derived from cycartenol. It is found in membranes just like cholesterol
(Moreau, et al, 2002). Phytosterols stabilize the membrane and decrease fluidity of biological
membrane (Guillen and Manzanos, 1998) Figure 2 represents the structures of 4 sterols,
cholesterol and three plant sterols that are usually found in plants.
Phytosterols are present in plant oils, seeds, legumes and cereals. Phytosterols are found
in vegetable oils in concentration of 0.5 to 1.5% (Itoh et al., 1973). Stigmasterol, campesterol
and β-sitosterol are the major sterol found in vegetable oils. β-sitosterol is found in high
concentration as compared to other sterols (Ling and Jones, 1995)..
Page 22
Figure 2—Chemical structures of the phytosterols and cholesterol
Various types of phytosterols are found in plants (Moreau et al., 2002) and among them forty
four phytosterols have been documented in plants, some examples are avenasterol in olives,
brassicasterol in cruciferous plants , peanut coconut, linseed, corn, Linseed oils, castor oils
and cotton seeds contain ergosterol. (Ling et al., 1995).
Over the last 10 years, phytosterols are considered to be cardioprotective (Plat et al.,
2000; Weststrate et al., 1998;). Phytosterols reduce the level of LDL in blood and decreases
cardiovascular disorders (Quilez et al., 2003, Jones, 1999). Daily diet contains naturally
occurring phytosterols, it is believed greater than before reduces risk of cardiovascular
disorders (Ostlund, 2004). The use of phytosterol exerted no adverse effects in human and
animal models except in few cases occasional diarrhea reported (Ling and Jones, 1995;
Becker et al., 1993, Mayr, 1999, Guillen and Manzanos, 1998). β-sitosterol, stigmasterol
and campesterol have antioxidant activity. Retardation of polymerization has been observed
by sterols having ethyldiene group on the side chain at temperature comparable to the ones
used in deep-fat frying. Antioxidation of phytesterols in frying food has been reported
(Boskou, 1998). Briefly, phytosterols are membranes stabilizers, radical scavengers and
antioxidant.
2. LITERATURE REVIEW
Sawarkar et al., 2011 reported that various plants synthesize substances that are beneficial for
human body. In the classical medicine systems including Chinese, Ayurvedic, Unani, and
others, plants and herbs have made the foundation. These systems provided a number of
important drugs which are still in use these days. At the present time, exploration of new
compounds and molecules has taken vaguely different direction where the knowledge of
ethnobotany with ethnopharmacognosy is being utilized for guidance so that the chemists can
look for different novel classes and resource of compounds. In this perspective, tropical floras
because of its diversity play significant role and offer new leads to the scientists (Gurib-
Fakim, 2006).
The dispute of disease and decease, initiated by infectious diseases is clearly evident not only
in third world countries but is also apparent in developed states around the world.
Due to the progress of communicable diseases, pathogens also develop resistance against
existing medicines. So, there is a continuous need to explore the novel leads, most likely with
different Pharmacological actions action especially, against viral, fungal, and bacterial
diseases (Ndhlala et al., 2013). In addition to medical and cultural usage, medicinal plants
also have financial benefits. National as well as global markets are continuously increasing
their demand, and remarkable economic and financial growth is being apprehended through
the trade of therapeutic herbal products.
2.1. Therapeutic plants: source of impressive antioxidants
Primary mechanism of various human pathological circumstances such as digestive tract
disorders, viral infections, diabetes, inflammation, neurodegenerative conditions, and
autoimmune diseases have been examined in several studies (Nafiu et al., 2013). Most of the
human pathologies are in the result of oxidative cellular injury caused by reactive oxygen
species (ROS). It is proved by scientific trials that reactive oxygen species (ROS) induced
cellular damages can be decreased and neutralized by means of therapeutic herbs and foods.
On the basis of past achievements about natural products, a variety of medical vegetation has
been appraised in favor of their antioxidative potential.
Soobratteea et al. (2005) gathered proofs about phenolics as strong pharmacological
antioxidant agents. These scientific, biochemical, and epidemiological evidences supported
the hypothesis that phenolic antioxidants have high chemo protective potential against
infections produced by oxidative stress. The therapeutic behavior of phenolic chemicals was
supported by their impact on gene expression and biological signaling pathways in addition
to their metal chelating and free radical hunting activities. Phenolic compounds, present in
plant based meals, were tested for antioxidant capacities by theircopper phenanthroline
mediated DNA oxidation ability, hypochlorite foraging capacity, Trolox equivalent
antioxidant capacity(TEAC) and ferric reducing antioxidant power( FRAP). Results obtained
from FRAP, TEAC, and hypochlorite foraging assay computed activity order as phenolic
simple acids < hydroxycinnamic acids < flavonol < flavanol aglycones < procyanidin dimer.
In the category of hydroxycinnamic acids and simple phenolics, the potent antioxidant
activity was found for rosmarinic acid and gallic acid respectively while among flavonol
aglycones, antioxidative strength increased from kaempferol through myricetin to quercetin.
In deoxyribose degradation assay, maximum inhibitory activity was displayed by ferulic acid.
Phenolic compounds differ in their effectiveness which depends on antioxidant action
mechanism in each assay. Plants that possessed phenolic compounds are categorized as good
natural antioxidant sources. It is also necessary by further investigating of their mode of
action plus its molecular mechanism,

to assess the capability of such phytochemicals as prophylactic mediums. Cai et al. (2004)
characterized various classical medicinal plants, including 112 species, for their anti-cancer
and antioxidant abilities. For the systematic evaluation of total antioxidant aptitude i.e. TEAC
(Trolox equivalent antioxidant capacity), advanced ABTS•+ method was adopted. The
observed TEAC values for methanol plant extracts varied from 46.7- 17, 323 Amol Trolox
equivalent per 100 g dry weight while 0.22- 50.3 g of gallic acid equivalent per 100 g dry
weight was the range of total phenolics. Statistically significant linear correlation between
antioxidant activities of herbal extracts and their phenolic contents proved that in tested
traditional herbs, total phenolics were the major antioxidants. Most important types of
phenolics include phenolic acids, tannins, lignans, quinones, curcuminoids, coumarins,
flavonoids, and stilbenes. Levels of phenolic compounds in these herbs were higher than
every day fruits and green vegetables and it was believed that these traditional Chinese plants
might be a good source not only for natural antioxidants but also for valuable chemo
preventive agents.
Valkoa et al. (2007) recognized the reactive oxygen species (e.g. superoxide radical) along
with reactive nitrogen species (RNS) (e.g. nitric oxide) for duality in their function i.e. as
advantageous and harmful species. These reactive species are produced by strongly
synchronized enzymes like NOS (NO synthase) and NAD (P) H oxidase respectively. It was
observed that NAD(P)H exorbitant stimulation or disturbance in mitochondrial electron
transport chain cause ROS overproduction which leads to deleterious damages to the cell
structures (proteins, lipids, membranes, DNA), simply called oxidative stress. On the other
hand, positive achievements of ROS/RNS are affiliated with their moderate levels and
include physiological functions in cellular response to noxia (in function of various signaling
pathways, in resistance against infectious entities). Normally, the ROS- established activities
occur in animal tissues guard the cells against ROS- provoked oxidative stress by maintaining
a redox balance specifically called “Redox homeostasis”. The researchers described the
source and biochemistry of these free radicals, the damage they cause to biological structures,
ability of antioxidants like glutathione to maintain the cellular redox homeostasis, signaling
pathways induced by ROS along with their role in pathophysiological inferences of varied
redox regulation (ageing and human diseases). The main focus was on ROS/RNS-related
pathogenesis of cardiovascular disease, hypertension, neurodegenerative diseases, arthritis,
ageing and cancer (Roy et al., 2009).
Reddy (2011) studied and developed an encapsulation method which retains the antioxidant
effects for a longer duration and polyphenolic contents from plants remain stable at room
temperature and have no toxic effects. Myristica fragrans and Cordyline terminalis contain
polyphenolic contents. Methanolic extract of these plants was prepared under optimum
conditions. He observed that antioxidant activity and polyphenol content remain stable in the
capsulated beads as compared to unencapsulated extracts. Sodium-caseinate beads are
considered to be capable procedure for food supplementation with natural antioxidants.
Traditionally, antioxidants were used to dispose the toxic and harmful derivatives of aerobic
metabolism which are mostly reactive oxygen intermediates simply called ROIs (Mittler,
2002). In current years, it has been reported that plants produce ROIs as signaling substances
in order to control different processes including pathogen defense, apoptosis, abiotic stress
reactions, and systemic signaling. Mahesh and Satish (2008) investigated antimicrobial
activity of various essential medicinal plants against pathogens (Human and plants). The
extracts of these medicinal herbs ( Methanol leaf, root, and bark ) were examined for
fungicidal and bactericidal properties. Considerably high bactericidal activities of leaf
methanol extracts of Ziziphus mauritiana, Tinospora cordifolia, Acacia nilotica, Withania
somnifera, and Sida cordifolia were detected against Escherichia coli(E coli), Pseudomonas
fluorescens, Staphylococcus aureus, and Bacillus subtilis. Fungicidal activity of leaf
methanol extract was founded against Aspergillus flavus Fusarium verticillioides, and
Dreschlera turcic,.
Pacheco and Gonsebatt (2009) studied the protective responses of antioxidants along with
certain antioxidant-associated enzymes towards environmentally stimulated oxidative stress
(OS). Oxygen is a necessary need for proficient energy production in aerobic organisms but
in contradiction it is also involved in generation of chronic noxious stress in cells. In order to
deal with these oxidative environments, diverse protective schemes with adaptation ability
must exist. Oxidative stress results in exceeding the ROS production from capability of
cellular antioxidative defense system to remove these lethal species. Different scientific trials
have shown a relation between environmental factors like (dietary habits and lifestyle) and
various diseases like (cancer, diabetes, atherosclerosis, and neurodegenerative diseases).
There are present large numbers of environmental pollutants having the capacity to get
engage with signaling pathways which are activated as a result of ROS. The similar
sequential events are considered to be cause of various chronic disorders. Research has been
conducted for different in vivo models to determine their oxidative responses and it showed
that mammals ( complex organisms) have typical antioxidant systems in their tissues and
organs. As a result, different organism’s susceptibility against toxic environmental factors is
different. So, by understanding the pathways involved in induction of antioxidant reactions
will help in developing strategies for oxidative damage protection. Kahkonen et al. (1999)
carried out auto-oxidation of the methyl linoleate for this purpose he studied antioxidant
ability of ninety two (92) different phenolic extracts collected from herbs, tree materials,
berries, fruits, cereals, seeds, vegetables , and plant sprouts (comprising both edible and
non-edible plant stuffs). Protocol of Folin-Ciocalteu reagent was used for determination of
total phenolic amount spectrophotometrically and values were determined as gallic acid
equivalents(GAE). Data revealed that from category of edible plants, berries (particularly
crowberry and aronia) contained incredibly high phenolic contents (GAE > 20 mg/ g) and
antioxidant activity while apple extracts presented tenacious antioxidant property despite the
fact that total phenolic amounts were diminutive (GAE < 12.1 mg/ g). Among inedible plant
material like, pine bark, willow, birch phloem, and spruce needles displayed high activities.
Furthermore, beetroot peel and potato peel extracts had extremely high antioxidant abilities.
In order to utilize these plants as source for natural antioxidants, additional characterization
of phenolic constitutions is needed.
Gawlik-Dziki et al. (2013) analyzed the phenolic contents of Chenopodium quinoa leaves
(ChL) to evaluate its nutraceutical ability. They elucidated the in vitro bioavailability,
bioaccessibility, and antioxidative activity of these compounds along with their effect on
properties of cancer cells. In Chenopodium quinoa leaves (ChL) extract, isorhamnetin, rutin,
kaempferol, ferulic, sinapinic, , and gallic acids were spotted and coupled with inhibitory
effects of leaves extract on prostate cancer cell expansion, mobility and cellular proficiency
for gap junctional intercommunication. The extract obtained from imitated digestion along
with chemical extract employed inhibiting effect on the activity of lipoxygenase enzyme,
comparable to their substantial chelating, antiradical, antioxidative, plus reducing power.
These findings indicate the anti-cancer properties of phenolic ChL extract against oxidative
stress as well as ROS- reliant intracellular signaling by means of synergic effects. These
findings proved the suitability of ChL as dietary supplement, because its bioavailability and
distribution is relatively high. Maisarah et al. (2013) conducted the study to analyze different
elements of Carica papaya for their antioxidant potential. For this, methanol extracts (80%)
of plant parts (ripe/ unripe) including seeds, fruits, and small leaves were utilized to carry out
assays for total phenolic/ total flavonoids content (TPC and TFC), in addition to total
antioxidative activity (TAA). The later was discovered by using scavenging methods for
DPPH radical and β-carotene bleaching. For TPC determination, folin-ciocalteu’s model was
used whereas aluminium trichloride method was followed for TFC computation. Results
reported that for β carotene bleaching assay, maximum antioxidant activity was observed in
case of unripe fruit i.e. 90.67 ± 0.29 %, chased by leaves (young), ripe fruit and then seed. On
other side, small leaves exhibited significantly high scavenging activity against DPPH and
calculated EC 50 value was 1.0 ± 0.08 mg/ml whereas EC 50 shown by other fractions was
6.5 ± 0.01 mg/ml for ripe fruit, 7.8 ± 0.06 mg/ml for seeds and 4.3 ± 0.01 mg/ml for unripe
fruit. Surprisingly, leaves extract showed maximal antioxidative content and reported values
were 424.89 ± 0.22 mg GAE/ 100 g d.w for TPC with 333.14 ± 1.03 mg rutin equivalent/ 100
g d.w for TFC. Statistically, positive Pearson correlation of scavenging activity against DPPH
was found with phenolics (r=0.846) and flavonoids (r=0.873). Conversely, no correlation of
beta-carotene decolorizing activity was found with TPC and TFC. Briefly, by considering all
measured parameters, antioxidants were highly incredible in order of seed < fruit (ripe) <
fruit (unripe) < small leaves.
Murugan and Mohan (2012) studied Dioscorea esculenta (Lour). Burkill methanol extract in
vitro for its antioxidant activity and evaluated the flavonoid contents along with total
phenolic compounds. Folin Ciocalteu method was used for the estimation of total phenolic
substances while flavonoids were determined with the help of AlCl3 (aluminum chloride).
Standard methods were used for the verification of reducing power capability and in vitro
antioxidant potential. The value of phenolic contents from D. esculenta methanol extract was
calculated to be 0.79 g/ 100 g while 0.26 g/ 100 g was the computed amount for flavonoids
content. Antioxidant potential of plant extract was assessed by performing assays including
ABTS cation foraging activity, hydroxyl radical foraging activity, superoxide radical foraging
activity, and DPPH radical foraging activity. Ebrahimzadeh et al. (2010) examined the
antioxidant and free radical scavenging activity of certain medicinal plants such as C.
speciosum, V. odorata, B. hyrcana and H. fficinalis L. Var. Angustifolius. Extracts of these
aromatic medicinal foliages were studied for potent antioxidant capability by establishing
various in vitro systems which include flowers of C. speciosum, leaves of B. Hyrcana and V.
Odorata, aerial parts of H. Officinalis L. Var. Angustifolius. The observed IC50 values for
experimental herbs, in case of DPPH (radical) scavenging assay, were increased in order of
BH (113.1 μg/ ml) < VO (245.1 μg/ ml) < HO (311 μg/ ml) < CS (585.6 μg/ ml) while they
showed high effectiveness (25-800 μg/ ml) with order of VO < BH< CS< HO , in case of
reducing power assay. When analysis performed for iron (Fe2+ ) chelating activity, IC50
value found were VO (188 μg/ ml), CS (750 μg/ ml), and HO (980 μg/ ml) while for BH
extract, at concentration of 800 μg/ ml, merely 38% inhibition has been reported. Results for
nitric oxide-foraging activity were not so good and in case of FTC, extracts displayed
extremely low/ moderate antioxidant activity (concentration dependent). IC50 values in
support of H2O2 scavenger hunt were calculated to be 169 μg/ ml, 175 μg/ ml, 640 μg/ ml,
663 μg/ ml for BH, CS, VO and HO respectively. Moreover, the plant extracts were also
examined for concentrations of flavonoids as well as phenolic total compounds. It became
obvious from statistics achieved for in vitro models that all extracts enclosed antioxidant
potency.
Acqua et al. (2008) investigated antioxidant activity of few classical plants from Sardinia in
vitro and specially investigated Rubus ulmifolius for its antioxidant behavior. Several in vitro
techniques like DPPH, TEAC, FC, and BR were applied to assess Rubus ulmifolius species.
Results from all assays recognized that Rubus ulmifolius had highest antioxidant activity
among the range of species. Extracts were phytochemically investigated, numerous phenolic
molecules were isolated namely chlorogenic acid, 5-caffeoylquinic acid, ferulic acid,
kaempferol-3-O-glucuronide , 4-caffeoylquinic acid, kaempferol-3-O-(600-caffeoyl)-b-D-
glucopyranoside, quercetin-3-O-glucuronide, kaempferol-3-O-(600-p-coumaroyl)-b-D-
glucopyranoside, and caffeic acid. Antioxidative aspects of isolated compounds were also
calculated. Lot of studies is reported for many medicinal plants regarding the phenolic
contents and antioxidant activity. Antioxidant of many medicinal plants has remarkable
antioxidant activity. For example; methanolic extract of Mucuna pruriens (seeds) (Rajeshwar
et al., 2005), Thapsia garganica, Artemisia compestris, Anthemis arvensis, Artemisa herba,
Teucrium polium, Ruta montana
(Djeridane et al., 2007), Rheum Ribes (Ozturk et al., 2007), Cyperus rotundus (Nagulendran
et al., 2007), Equisetum arvense L. (Dukic et al., 2008), Morinda Lucida (Oluseyi et al.,
2008), aerial parts of Teucrium polium (Sharififar et al., 2009), bark of Terminalia arjuna
(Shridhar and Gopal 2009). Katalinic et al. (2006) determined polyphenolic contents and total
antioxidant activity of seventy medicinal plants, among them melissae folium, serpylii herba,
spirae herba showed strong antioxidant activity. Amic et al., 2003, studied the relationship
between structural characteristic of flavonoids with their antiradical activity. Results of this
study revealed that free radical quenching activity of polyphenolic compounds strongly
depends on the specific substitution pattern of free hydroxyl moieties on flavonoids structure.
B ring with 3', 4' dihydroxy groups and 3-OH group at ring C are essential features for strong
antioxidant activity.
2. LITERATURE REVIEW
Sawarkar et al., 2011 reported that various plants synthesize substances that are beneficial for
human body. In the classical medicine systems including Chinese, Ayurvedic, Unani, and
others, plants and herbs have made the foundation. These systems provided a number of
important drugs which are still in use these days. At the present time, exploration of new
compounds and molecules has taken vaguely different direction where the knowledge of
ethnobotany with ethnopharmacognosy is being utilized for guidance so that the chemists can
look for different novel classes and resource of compounds. In this perspective, tropical floras
because of its diversity play significant role and offer new leads to the scientists (Gurib-
Fakim, 2006).
The dispute of disease and decease, initiated by infectious diseases is clearly evident not only
in third world countries but is also apparent in developed states around the world.
Due to the progress of communicable diseases, pathogens also develop resistance against
existing medicines. So, there is a continuous need to explore the novel leads, most likely with
different Pharmacological actions action especially, against viral, fungal, and bacterial
diseases (Ndhlala et al., 2013). In addition to medical and cultural usage, medicinal plants
also have financial benefits. National as well as global markets are continuously increasing
their demand, and remarkable economic and financial growth is being apprehended through
the trade of therapeutic herbal products.
2.1. Therapeutic plants: source of impressive antioxidants
Primary mechanism of various human pathological circumstances such as digestive tract
disorders, viral infections, diabetes, inflammation, neurodegenerative conditions, and
autoimmune diseases have been examined in several studies (Nafiu et al., 2013). Most of the
human pathologies are in the result of oxidative cellular injury caused by reactive oxygen
species (ROS). It is proved by scientific trials that reactive oxygen species (ROS) induced
cellular damages can be decreased and neutralized by means of therapeutic herbs and foods.
On the basis of past achievements about natural products, a variety of medical vegetation has
been appraised in favor of their antioxidative potential.
Soobratteea et al. (2005) gathered proofs about phenolics as strong pharmacological
antioxidant agents. These scientific, biochemical, and epidemiological evidences supported
the hypothesis that phenolic antioxidants have high chemo protective potential against
infections produced by oxidative stress. The therapeutic behavior of phenolic chemicals was
supported by their impact on gene expression and biological signaling pathways in addition
to their metal chelating and free radical hunting activities. Phenolic compounds, present in
plant based meals, were tested for antioxidant capacities by theircopper phenanthroline
mediated DNA oxidation ability, hypochlorite foraging capacity, Trolox equivalent
antioxidant capacity(TEAC) and ferric reducing antioxidant power( FRAP). Results obtained
from FRAP, TEAC, and hypochlorite foraging assay computed activity order as phenolic
simple acids < hydroxycinnamic acids < flavonol < flavanol aglycones < procyanidin dimer.
In the category of hydroxycinnamic acids and simple phenolics, the potent antioxidant
activity was found for rosmarinic acid and gallic acid respectively while among flavonol
aglycones, antioxidative strength increased from kaempferol through myricetin to quercetin.
In deoxyribose degradation assay, maximum inhibitory activity was displayed by ferulic acid.
Phenolic compounds differ in their effectiveness which depends on antioxidant action
mechanism in each assay. Plants that possessed phenolic compounds are categorized as good
natural antioxidant sources. It is also necessary by further investigating of their mode of
action plus its molecular mechanism,

to assess the capability of such phytochemicals as prophylactic mediums. Cai et al. (2004)
characterized various classical medicinal plants, including 112 species, for their anti-cancer
and antioxidant abilities. For the systematic evaluation of total antioxidant aptitude i.e. TEAC
(Trolox equivalent antioxidant capacity), advanced ABTS•+ method was adopted. The
observed TEAC values for methanol plant extracts varied from 46.7- 17, 323 Amol Trolox
equivalent per 100 g dry weight while 0.22- 50.3 g of gallic acid equivalent per 100 g dry
weight was the range of total phenolics. Statistically significant linear correlation between
antioxidant activities of herbal extracts and their phenolic contents proved that in tested
traditional herbs, total phenolics were the major antioxidants. Most important types of
phenolics include phenolic acids, tannins, lignans, quinones, curcuminoids, coumarins,
flavonoids, and stilbenes. Levels of phenolic compounds in these herbs were higher than
every day fruits and green vegetables and it was believed that these traditional Chinese plants
might be a good source not only for natural antioxidants but also for valuable chemo
preventive agents.
Valkoa et al. (2007) recognized the reactive oxygen species (e.g. superoxide radical) along
with reactive nitrogen species (RNS) (e.g. nitric oxide) for duality in their function i.e. as
advantageous and harmful species. These reactive species are produced by strongly
synchronized enzymes like NOS (NO synthase) and NAD (P) H oxidase respectively. It was
observed that NAD(P)H exorbitant stimulation or disturbance in mitochondrial electron
transport chain cause ROS overproduction which leads to deleterious damages to the cell
structures (proteins, lipids, membranes, DNA), simply called oxidative stress. On the other
hand, positive achievements of ROS/RNS are affiliated with their moderate levels and
include physiological functions in cellular response to noxia (in function of various signaling
pathways, in resistance against infectious entities). Normally, the ROS- established activities
occur in animal tissues guard the cells against ROS- provoked oxidative stress by maintaining
a redox balance specifically called “Redox homeostasis”. The researchers described the
source and biochemistry of these free radicals, the damage they cause to biological structures,
ability of antioxidants like glutathione to maintain the cellular redox homeostasis, signaling
pathways induced by ROS along with their role in pathophysiological inferences of varied
redox regulation (ageing and human diseases). The main focus was on ROS/RNS-related
pathogenesis of cardiovascular disease, hypertension, neurodegenerative diseases, arthritis,
ageing and cancer (Roy et al., 2009).
Reddy (2011) studied and developed an encapsulation method which retains the antioxidant
effects for a longer duration and polyphenolic contents from plants remain stable at room
temperature and have no toxic effects. Myristica fragrans and Cordyline terminalis contain
polyphenolic contents. Methanolic extract of these plants was prepared under optimum
conditions. He observed that antioxidant activity and polyphenol content remain stable in the
capsulated beads as compared to unencapsulated extracts. Sodium-caseinate beads are
considered to be capable procedure for food supplementation with natural antioxidants.
Traditionally, antioxidants were used to dispose the toxic and harmful derivatives of aerobic
metabolism which are mostly reactive oxygen intermediates simply called ROIs (Mittler,
2002). In current years, it has been reported that plants produce ROIs as signaling substances
in order to control different processes including pathogen defense, apoptosis, abiotic stress
reactions, and systemic signaling. Mahesh and Satish (2008) investigated antimicrobial
activity of various essential medicinal plants against pathogens (Human and plants). The
extracts of these medicinal herbs ( Methanol leaf, root, and bark ) were examined for
fungicidal and bactericidal properties. Considerably high bactericidal activities of leaf
methanol extracts of Ziziphus mauritiana, Tinospora cordifolia, Acacia nilotica, Withania
somnifera, and Sida cordifolia were detected against Escherichia coli(E coli), Pseudomonas
fluorescens, Staphylococcus aureus, and Bacillus subtilis. Fungicidal activity of leaf
methanol extract was founded against Aspergillus flavus Fusarium verticillioides, and
Dreschlera turcic,.
Pacheco and Gonsebatt (2009) studied the protective responses of antioxidants along with
certain antioxidant-associated enzymes towards environmentally stimulated oxidative stress
(OS). Oxygen is a necessary need for proficient energy production in aerobic organisms but
in contradiction it is also involved in generation of chronic noxious stress in cells. In order to
deal with these oxidative environments, diverse protective schemes with adaptation ability
must exist. Oxidative stress results in exceeding the ROS production from capability of
cellular antioxidative defense system to remove these lethal species. Different scientific trials
have shown a relation between environmental factors like (dietary habits and lifestyle) and
various diseases like (cancer, diabetes, atherosclerosis, and neurodegenerative diseases).
There are present large numbers of environmental pollutants having the capacity to get
engage with signaling pathways which are activated as a result of ROS. The similar
sequential events are considered to be cause of various chronic disorders. Research has been
conducted for different in vivo models to determine their oxidative responses and it showed
that mammals ( complex organisms) have typical antioxidant systems in their tissues and
organs. As a result, different organism’s susceptibility against toxic environmental factors is
different. So, by understanding the pathways involved in induction of antioxidant reactions
will help in developing strategies for oxidative damage protection. Kahkonen et al. (1999)
carried out auto-oxidation of the methyl linoleate for this purpose he studied antioxidant
ability of ninety two (92) different phenolic extracts collected from herbs, tree materials,
berries, fruits, cereals, seeds, vegetables , and plant sprouts (comprising both edible and
non-edible plant stuffs). Protocol of Folin-Ciocalteu reagent was used for determination of
total phenolic amount spectrophotometrically and values were determined as gallic acid
equivalents(GAE). Data revealed that from category of edible plants, berries (particularly
crowberry and aronia) contained incredibly high phenolic contents (GAE > 20 mg/ g) and
antioxidant activity while apple extracts presented tenacious antioxidant property despite the
fact that total phenolic amounts were diminutive (GAE < 12.1 mg/ g). Among inedible plant
material like, pine bark, willow, birch phloem, and spruce needles displayed high activities.
Furthermore, beetroot peel and potato peel extracts had extremely high antioxidant abilities.
In order to utilize these plants as source for natural antioxidants, additional characterization
of phenolic constitutions is needed.
Gawlik-Dziki et al. (2013) analyzed the phenolic contents of Chenopodium quinoa leaves
(ChL) to evaluate its nutraceutical ability. They elucidated the in vitro bioavailability,
bioaccessibility, and antioxidative activity of these compounds along with their effect on
properties of cancer cells. In Chenopodium quinoa leaves (ChL) extract, isorhamnetin, rutin,
kaempferol, ferulic, sinapinic, , and gallic acids were spotted and coupled with inhibitory
effects of leaves extract on prostate cancer cell expansion, mobility and cellular proficiency
for gap junctional intercommunication. The extract obtained from imitated digestion along
with chemical extract employed inhibiting effect on the activity of lipoxygenase enzyme,
comparable to their substantial chelating, antiradical, antioxidative, plus reducing power.
These findings indicate the anti-cancer properties of phenolic ChL extract against oxidative
stress as well as ROS- reliant intracellular signaling by means of synergic effects. These
findings proved the suitability of ChL as dietary supplement, because its bioavailability and
distribution is relatively high. Maisarah et al. (2013) conducted the study to analyze different
elements of Carica papaya for their antioxidant potential. For this, methanol extracts (80%)
of plant parts (ripe/ unripe) including seeds, fruits, and small leaves were utilized to carry out
assays for total phenolic/ total flavonoids content (TPC and TFC), in addition to total
antioxidative activity (TAA). The later was discovered by using scavenging methods for
DPPH radical and β-carotene bleaching. For TPC determination, folin-ciocalteu’s model was
used whereas aluminium trichloride method was followed for TFC computation. Results
reported that for β carotene bleaching assay, maximum antioxidant activity was observed in
case of unripe fruit i.e. 90.67 ± 0.29 %, chased by leaves (young), ripe fruit and then seed. On
other side, small leaves exhibited significantly high scavenging activity against DPPH and
calculated EC 50 value was 1.0 ± 0.08 mg/ml whereas EC 50 shown by other fractions was
6.5 ± 0.01 mg/ml for ripe fruit, 7.8 ± 0.06 mg/ml for seeds and 4.3 ± 0.01 mg/ml for unripe
fruit. Surprisingly, leaves extract showed maximal antioxidative content and reported values
were 424.89 ± 0.22 mg GAE/ 100 g d.w for TPC with 333.14 ± 1.03 mg rutin equivalent/ 100
g d.w for TFC. Statistically, positive Pearson correlation of scavenging activity against DPPH
was found with phenolics (r=0.846) and flavonoids (r=0.873). Conversely, no correlation of
beta-carotene decolorizing activity was found with TPC and TFC. Briefly, by considering all
measured parameters, antioxidants were highly incredible in order of seed < fruit (ripe) <
fruit (unripe) < small leaves.
Murugan and Mohan (2012) studied Dioscorea esculenta (Lour). Burkill methanol extract in
vitro for its antioxidant activity and evaluated the flavonoid contents along with total
phenolic compounds. Folin Ciocalteu method was used for the estimation of total phenolic
substances while flavonoids were determined with the help of AlCl3 (aluminum chloride).
Standard methods were used for the verification of reducing power capability and in vitro
antioxidant potential. The value of phenolic contents from D. esculenta methanol extract was
calculated to be 0.79 g/ 100 g while 0.26 g/ 100 g was the computed amount for flavonoids
content. Antioxidant potential of plant extract was assessed by performing assays including
ABTS cation foraging activity, hydroxyl radical foraging activity, superoxide radical foraging
activity, and DPPH radical foraging activity. Ebrahimzadeh et al. (2010) examined the
antioxidant and free radical scavenging activity of certain medicinal plants such as C.
speciosum, V. odorata, B. hyrcana and H. fficinalis L. Var. Angustifolius. Extracts of these
aromatic medicinal foliages were studied for potent antioxidant capability by establishing
various in vitro systems which include flowers of C. speciosum, leaves of B. Hyrcana and V.
Odorata, aerial parts of H. Officinalis L. Var. Angustifolius. The observed IC50 values for
experimental herbs, in case of DPPH (radical) scavenging assay, were increased in order of
BH (113.1 μg/ ml) < VO (245.1 μg/ ml) < HO (311 μg/ ml) < CS (585.6 μg/ ml) while they
showed high effectiveness (25-800 μg/ ml) with order of VO < BH< CS< HO , in case of
reducing power assay. When analysis performed for iron (Fe2+ ) chelating activity, IC50
value found were VO (188 μg/ ml), CS (750 μg/ ml), and HO (980 μg/ ml) while for BH
extract, at concentration of 800 μg/ ml, merely 38% inhibition has been reported. Results for
nitric oxide-foraging activity were not so good and in case of FTC, extracts displayed
extremely low/ moderate antioxidant activity (concentration dependent). IC50 values in
support of H2O2 scavenger hunt were calculated to be 169 μg/ ml, 175 μg/ ml, 640 μg/ ml,
663 μg/ ml for BH, CS, VO and HO respectively. Moreover, the plant extracts were also
examined for concentrations of flavonoids as well as phenolic total compounds. It became
obvious from statistics achieved for in vitro models that all extracts enclosed antioxidant
potency.
Acqua et al. (2008) investigated antioxidant activity of few classical plants from Sardinia in
vitro and specially investigated Rubus ulmifolius for its antioxidant behavior. Several in vitro
techniques like DPPH, TEAC, FC, and BR were applied to assess Rubus ulmifolius species.
Results from all assays recognized that Rubus ulmifolius had highest antioxidant activity
among the range of species. Extracts were phytochemically investigated, numerous phenolic
molecules were isolated namely chlorogenic acid, 5-caffeoylquinic acid, ferulic acid,
kaempferol-3-O-glucuronide , 4-caffeoylquinic acid, kaempferol-3-O-(600-caffeoyl)-b-D-
glucopyranoside, quercetin-3-O-glucuronide, kaempferol-3-O-(600-p-coumaroyl)-b-D-
glucopyranoside, and caffeic acid. Antioxidative aspects of isolated compounds were also
calculated. Lot of studies is reported for many medicinal plants regarding the phenolic
contents and antioxidant activity. Antioxidant of many medicinal plants has remarkable
antioxidant activity. For example; methanolic extract of Mucuna pruriens (seeds) (Rajeshwar
et al., 2005), Thapsia garganica, Artemisia compestris, Anthemis arvensis, Artemisa herba,
Teucrium polium, Ruta montana
(Djeridane et al., 2007), Rheum Ribes (Ozturk et al., 2007), Cyperus rotundus (Nagulendran
et al., 2007), Equisetum arvense L. (Dukic et al., 2008), Morinda Lucida (Oluseyi et al.,
2008), aerial parts of Teucrium polium (Sharififar et al., 2009), bark of Terminalia arjuna
(Shridhar and Gopal 2009). Katalinic et al. (2006) determined polyphenolic contents and total
antioxidant activity of seventy medicinal plants, among them melissae folium, serpylii herba,
spirae herba showed strong antioxidant activity. Amic et al., 2003, studied the relationship
between structural characteristic of flavonoids with their antiradical activity. Results of this
study revealed that free radical quenching activity of polyphenolic compounds strongly
depends on the specific substitution pattern of free hydroxyl moieties on flavonoids structure.
B ring with 3', 4' dihydroxy groups and 3-OH group at ring C are essential features for strong
antioxidant activity.
2.5. Overview of plants.
2.5.1 Myristica fragrans
Family name. Myristicaceae.
English name. Nutmeg, Mace, Fragrant Nut tree
Urdu name. Jaiphal/Bisbasa/Jawatri
Part used. Seeds
A diocecious or occasionally monoecious ever green, aromatic tree, usually 9-12 m high, but
sometimes reaching a height of 20 m or more. Bark grayish-black, longitudinally fissured in
old trees; leaves elliptic or oblong-lanceolate, coriceous; flowers in umbellate cymes, creamy
yellow, fragrant; fruit yellow, broadly pyriform or globose, 6-9 cm long, glabrous, often
drooping; pericarb fleshy, c. 1.25 cm thick splitting into 2 halves at maturity; seeds broadly
ovoid, arillate, albuminous, with a shell like purplish brown testa; aril red, fleshy laciniate
(Van et al., 1994).
Fig 1.3 Seed of Myristica fragrans

Terpenes, geraniol, terpineol, volatile oil and myristicin
Emollient, tonic, desiccative aphrodisiac, hepatoprotective, stomachic, exhilarant, astringent,
antidiarrheal, carminative, digestive, anti-inflammatory, antiarthritis, nerve stimulant,
sedative, resolvent, antiseptic, uterine tonic, deobstruent, deodorant, antiscrofulous, aperients,
rubefacient, narcotic, stimulant and aromatic.
It is prescribed for the treatment of impotency, cardiac weakness, premature ejaculation; in
different prescription is useful for the treatment of flatulence and diarrhea. Its oil is applied
externally for the treatment of headache, arthritis and paralysis. It is chewed to mask the bad
odour of mouth. It is useful in nausea and vomiting. Infusion of nutmeg is prescribed for
cholera patient to combat thirst. Mace (Javatri) is prescribed in asthma and chronic bowel
complaints. The nutmeg and mace are prescribed to secrete more milk from the breast, and
generally for liver and spleen diseases. Nutmeg had been reported to have aphrodisiac
(Tajuddin, 2005), stomachic, tonic (Burkill, 1935), nervous stimulant, anti-inflammatory,
antidysentric, antifungal, antithrombotic, antihyperlipidemic, astringent and aromatic
properties (Tajuddin, 2005).
The nutmeg is reported to be useful in paralysis and increases blood circulation. It is
demonstrated to have antioxidant property (Murcia et al., 2004; Olaleye et al., 2006). M.
fragrans fruits has antidiarrheal effect that is reported by using its petroleum ether extracts
and memory enhancing activity of n-hexane extract of M. fragrans has been reported in mice
(Parle, 2004)
2.6 Illicium verum
Family name. Illiciaceae
English name. True star anise
Arabic name. Raziyanje khatai
Persian name. Badian – i- khatai
Part used. Seeds

The Illicum verum is a medium sized tree is about 8-15 (-20m) tall and 30 cm depth with a
straight rounded trunks and green, glabrous branch lets. The bark is white to bright grey.
Leaves
6-12 cm long, alternate, simple, leathery, entire, shining, glabrous, usually crowded in
bundles at
the end of the branches. Flower large, bisexual, 1-1.5 cm in diameter, white pink to red or
greenish yellow, axillary and solitary. Fruit is capsule like, aggregate is star shaped, radiating
five to ten pointed boat shaped sections about eight on average. Each arm is seed pod. Tough
skinned and rust colored they measure up to 3 cm (1-1/4”) long. Fruits are picked before it
ripens
and dried. Seed are shiny brown or reddish with high oil content .
The fruit contain higher bitter principle, tannins and essential oil (9-10%), consisting of
anethole
(85-90%), α-pinene, limone, β-phellandrene, α-terpineol, farnesol and safrol. They are 14
hydrocarbons components and 22 oxygenated hydrocarbon derivatives and small amount of
nitrogenous compounds ρ-ally anisole, ρ-cumicaldehyde, ρ-allylpen, anisylacetone,
anisaldehyde linoleic acid (1-4 methoxyphenyl)-prop-2-one, foeniculin and palmitic acid.The
new phenylpropanoid glucosides, known phenylpropanoid, alkylglucosides and Seco-
Cycloartane;3,4seco(242)cycloartane4(28),24(diene)3,26-dioic acid.26, methyl ester of
nigranoic
acid from the dichloromethane extract from leaves of illicium verum was identified.
2.6.3 Pharmacological actions.
Insecticidal, antifungal, antibacterial, antioxidant, carminative, stomachic, stimulant,

expectorant, diuretic, digestive, expectorant, deodorant

The Illicium verum herb is reported to be antioxidant, antibacterial and antifungal (Chouksey
et al., 2010: Shan et al., 2007). It can increase production of milk new mother. Some folk
remedies recommended the use to facilitate birth and to increase the libido, as well as to
relieve menopausal discomforts; oil is used in rheumatism. The attributed medicinal
properties are diuretic, expectorant, stimulant, stomachic and carminative. It is used in
abdominal pain, rheumatism and cough. Illicium verum fruit is used in traditional system of
medicines having both culinary and medicinal uses. Its seed oil is used worldwide as
medicine. The fruits are sweet, aromatic, carminative, digestive, stomachic, stimulant,
diuretic, expectorant, deodorant, constipation and insomnia. It relieves colic and is a common
ingredient of cough lozenges and cattle sprays. It is used in asthma, facial paralysis,
abdominal pain, flatulence and dyspepsia(Shan et al., 2007).
2.7. Curculigo orchioides
Family name. Amaryllidaceae
English name. Black Musale
Urdu name. Musli Siah
Arabic name. Musli Aswad
Part used. Roots
Curculigo orchioides belongs to the family Amaryllidaceae. (Figure 1) This is herbaceous
plant and has lateral roots and elongated tuberous rootstock. It has elongated root about -25
cm in length. Leaves are (5-20 x 0.8-1.5 cm). Petioles are short and about of 3 cm. Flowers
are regular, seesile, bisexual and light yellow throughout the year. It has six lobed perianth,
yellow lobes ((0.6-1 x 0.2-0.3 cm), 3 celled ovary, 2mm anther and stamens 6.
Fig 3. Roots of Curculigo orchioides
Glucose, mannose, xylose, glucuronic acid, glycoside, calcium oxalate, fat, mucilage, tannin,
resin, protein, starch, inorganic compounds (Na, P, K and Ca), fibers, sterols, sapogenin,
flavone glycoside, dimethoxy glucopyranoside, curculigoside, phenolic glycoside, behenic
acid, arachidic acid and linolenic acid.

General tonic, aphrodisiac, diuretic, demulcent, aphrodisiac, restorative, .
It is used as general tonic, aphrodisiac and for skin diseases in some ayurvedic formulations
like Marmagulika (Joy et al., 2004). It is aphrodisiac, demulcent, diuretic and antimicrobial
(Kalaigandhi and Poovendran, 2011). It is used as aphrodisiac, rejuvenating, antinociceptive
(Bhandage et al., 2009), and restorative drug. It is also prescribed in urinary infections,
jaundice, impotence, skin disorders, gonorrhea, diarrhea, piles, asthma, cough, deafness and
stress (Sowmya and Kumar, 2010). Further it is also used in skin diseases, as a demulcent,
diuretics, tonic, diarrhea, jaundice, and asthma in combination with aromatics and bitters. It is
prescribed for various ailments in different dosage form designs. It is used in peptic ulcer,
wound infection, helminthiasis, spermatorrhea, ring worm, rheumatism, paralysis, anorexia,
vertigo, headache, hernia, epilepsy, diabetes mellitus and blood cancer (Wu et al., 2005).
Isocrassifoside G, crassifoside G and phenolic glycosides have been isolated from C.
orchioides (Wang and Li, 2007). Syringic acid, phenolic glycosides and antioxidant phenols
have been isolated from roots of C. orchioides (Wu et al., 2005). Phenolic compounds
isolated have various pharmacological effects such as anticancer, antimicrobial,
hypoglycemic properties, wound healer and antioxidant (Nagesh et al., 2009).
2.8 Glycyrrhiza glabra
Family name. Glycyrrhiza glabra
English Name: Liquorice,
Urdu Name: Mulethi, Asal-as-sus.
Parts used: Root

It is undershrub or a hardy herb with multifoliate leaves. It has axillar spikes flowers with
lavender to violet color. It has compressed pod with reniform seeds. It has stout root stock
which has various number of perennial roots. Underground par called licorice is used as drug
Fig 7. Roots of Glycyrrhiza glabra
Main constituents found in liquorice glycyrrhizin that has characteristic sweet taste. Other
constituents found in liquorice are coloring matter, volatile oil, resins, bitter principles,
asparagines, starch, mannite, sucrose and glucose. Anthoxathin glycoside gives yellow color
to liquorice.

The root is tonic, laxative, expectorant, emollient, diuretic, demulcent and antispasmodic.
Liquorice is anti-inflammatory, hepatoprotective, expectorant and anti-anxiety (Kumar, 2012,
Mirmala and Selyaraj, 2011), antioxidant (Latif et al., 2012), anticancer (Taro et al., 2002),
antimalarial (Sianne and Fanie, 2002), antiviral (Taro et al., 2002), antimicrobial (Nitalikar et
al., 2010), anticonvulsant (Yazdi, 2011), anticancer (Lee et al., 2007). It is used in mouth
ulcers and arthritis (Cooper et al., 2007). Hormonal effects of liquorice are similar to
hormones of ovary. The root has also been shown to have a hormonal effect similar to the
ovarian hormone. Liquorice root is prescribed in urinary tract infections and lung infections
(Chakravarthi and Avadhani, 2012). It is used in allergic complaints, arthritis, peptic ulcer,
cough, bronchitis, asthma and Addison's disease (Rathi et al., 2009). It should be used with
care and is not prescribed for patients with high blood pressure and pregnant women (Sahu
and Vaghela, 2011; Kalaiarasi, 2009).
2.9 Embelia ribes Brum.
Family name. Myrsinaceae.
English name. Embelia.
Urdu name. Baberang
Part used. Fruit, leaves, root bark
A large scandent shrub, branches long. Leaves coriaceous 5-9 by 2-3.8 cm, shining above,
and base rounded or acute. Flowers pentamerous, numerous. Calyx about 1.25 mm long.
Petals 5, greenish yellow, free, 4 mm long. Fruit globose, 3-4 mm in diameter, smooth, black
when ripe.
Fig 1. Fruit of Emblica ribes. Fig 2. Leaf of Emblica ribes.
Vilagin, tannins, quercitol, embelin, christembine, embolic acid (
Alterative, analgesic, laxative, appetizer, stomachic, stimulant, diuretic, carminative,
astringent, anthelmintic.
Embelia ribes is used as anthelmintic drug in Ayurvedic system of medicine. It is useful in
flatulence, anorexia and abdominal pain. It is hypolipidemic and is prescribed to treat obesity.
In one study, Tripathi reported its hypoglycemic activity in albino rabbits (Tripathi, 1979).
This plant is also used for fever, skin diseases, diarrhea, cough, chest ailments, abdominal
and cystic tumors. It is also utilized in ascites, dyspnea, dysuria, hemicrania, jaundice, urinary
disorders and constipation. Embelia is also regarded as an effective remedy for tapeworm.
3. MATERIALS AND METHODS
3.1. Materials
Malonaldehyde-bis-dimethyl acetal (MDA), Thiobarbituric acid (TBA), 2, 2-diphenyl-1-
picryl-hydrazyl (DPPH), gallic acid and Folin-Ciocalteus reagents were purchased from
Sigma (St. Louis,MO,USA). Sodium nitroprusside(SNP) was obtained from Merck
(Darmstadt,Germany) and iron(II)sulphate (Biochemical, Lahore).
3.2. Preparation of plant extract

The plant parts were purchased from the local market and authenticated by different Hakims
teaching in Department of Eastern Medicine and Surgery at University of Poonch Rawalakot,
Azad Kashmir. The parts of plant used in our experiments were soaked in boiling water. The
0.1 gram dried powder of plant material was soaked in 100ml boiling water for 15 minutes,
kept to cool at room temperature and then filtered using Wattman filter paper. The residues
obtained were extracted further, twice, and then concentrated in a rotary evaporator. Filtrates
were then dried in an oven to obtain a powder form at 40–50 0C, giving of a 21–23 percent
yield. To obtain the desired concentration of plant for the experiment, serial dilutions of these
were made.
3.3. Test animals
All procedures of using animals were in strict accordance with the NIH Guide for the use and
Care of Laboratory Animals. Two to two and half months old males and females Swiss-
Albino mice (200–250 g), from the animals breeding colony of National Institute of Health
Islamabad, were used for these In vitro studies. The animals were kept in clean cages with a
frequent access to food, water and libitum, in a room with uniformly controlled temperature
(22 _C ± 3) and in a 12 hours light and dark cycle with lights on from 7:00 a.m to 7.00 p.m.
3.4. In vitro assays.
3.4.1. Production of thio-barbituric acid reactive species (TBARS) from animal tissues.
The production thio-barbituric acid reactive species (TBARS) was determined applying a
modified method of Ohkawa et al., 1979. The mice were decapitated by anaesthetizing them
mildly in some quantity of ether and the tissues (brain and liver) were quickly collected in a
Petri-dish placed on ice. One gram quantities of tissues both from brain and liver separately
were homogenized in cold strokes in a Teflon glass homogenizer. These homogenates were
centrifuged at 1400 rev/min for 10 minutes to form a precipitate which was discarded and a
low-speed supernatant tissues were used for performing this assay. The homogenates (100 µl)
were then incubated with or without 50 µl of different oxidants (nitroprusside and iron)
which were freshly prepared and various concentrations of plant aqueous extracts, together
using a proper volume of deionized water, to gain a 300 µl of total volume at 37 0C for 1 h.
The color reaction was performed by adding 200, 500 and 500 µl each of the 8.1% sodium
dodecyl sulphate (SDS), acetic acid buffer (pH 3.4) and 0.6% TBA, respectively. The
reaction mixtures were incubated at 97oC for 1 hour with those of serial dilutions of 0.03 mM
standard MDA. The readings of absorbance were taken after cooling the tubes using a
wavelength of 532 nm in a spectrophotometer.
3.4.2. DPPH radical scavenging.
The DPPH radical-scavenging of the stable radicals, was performed in vitro according to the
method mentioned by Hatano et al., 1988. The prepared aqueous extract(s) (25-200 µg/ml)
were added to an already taken 0.5 ml solution of DPPH (0.25 mM in 95% ethanol). The
added reagents were then properly shaken and allowed to gain room temperature for 30
minutes. The absorbance in a spectrophotometer was determined at 517 nm. Percentage
inhibition was calculated from the control of each solution.
3.4.3. Phenolics content
For determination of total phenolics content, we mixed 2.5 ml 10% Folin–Ciocalteau’s
reagent (v/v), 0.5 ml of the aqueous extract and 2.0 ml of 7.5% sodium carbonate. The
mixture was incubated for 40 minutes at a maintained temperature of 45 0C, and the
absorbance in the spectrophotometer was measured at 765 nm. The Gallic acid was used as a
standard phenol. The three readings were used for taking their mean and the total phenolics
content was expressed as milligrams of gallic acid equivalents/ g of extract.

3.4.4. Total Antioxidant assay
The reduction of molybdenum, from Mo (VI) to Mo (V) was the base for this assay. The
aqueous extracts of plants were applied to form subsequently a green phosphate/Mo(V)
complex at the acidic pH. The aqueous extracts of plants (each of 0.1 mg/ml) ware
mixed with 3ml of the reagent solution (28 mM sodium phosphate, 0.6 M H 2SO4 and 4
mM amonium molybdate). Then the tubes were kept in incubator for 90 minutes at a
temperature maintained at 95 0C. The mixture when withdrawn from incubator was
cooled to room temperature and then the absorbance of the mixture was taken at 695
nm.
3.5. Statistical analysis
The obtained results were mentioned as means ± standard deviation. All the collected data
was statistically analyzed by one way ANOVA and the groups of different means were
compared with each other by Duncan’s multiple range test; The significant considered in all
cases was P < 0.05. The Statistica, a software package, was used for data analysis.
4. RESULTS AND DISCUSSION
4.1 Results
Mice liver and brain homogenates were induced with iron and sodium nitroprusside to cause
lipid peroxidation and the effect of Myristica fragrans, Illicuim verum, Curculigo
orchioeides, Glycyrrhiza glabra and Embelia ribes were determined. These plants were
chosen as they are frequently used as antihypercholestermic agents in a number of herbal
formulations.
4.1.1 Lipid peroxidation in mice brain induced with sodium nitroprusside and iron
Figure4.1 (a) shows the antioxidant effect of Myristica fragrans, Illicum verum and
Curciligo orchiodes in mice brain. The results revealed that treatment with 5 µM sodium
nitroprusside caused a significant (P <0.05) increase in thiobarbituric acid reactive substances
(TBARS) compared to the basal. Three separate controls were used for Myristica fragrans,
Illicuim verum and Curculigo orchioeides. However, treatment with different concentrations
of extracts (50-250 µg/ml) caused a significant decrease in lipid peroxidation. Illicum verum
showed a higher decrease in lipid peroxidation compared to the other plant species. Myristica
fragrans showed a the pro-oxidant effect at 50 µg/ml by causing increase in lipid
peroxidation but from concentration 100 ug/ml and upto 250 µg/ml it shows a regular
decrease in lipid peroxidation. Illicuim verum showed a gradual decrease in lipid
peroxidation at all concentrations (50-250 µg/ml). Curculigo orchioeides also showed
antioxidant effect by a gradual decrease in lipid peroxidation from 50-250 µg/ml extract
concentrations. . The order of decreasing antioxidant activity was Illicuim verum > Curculigo
orchioeides > Myristicafragrans.
Graph4.1 (b) shows the antioxidant effect of Glycyrrhiza glabra, Embelia ribes in mice
brain. The results revealed that treatment with 5 µM sodium nitroprusside caused a
significant (P <0.05) increase in thiobarbituric acid reactive substances (TBARS) compared
to the basal. Two separate controls were used for Glycyrrhiza glabra, Embelia ribes.
Treatment with Glycyrrhiza glabra and Embelia ribes caused a significant decrease in lipid
peroxidation. Glycyrrhiza glabra showed tremendous antioxidant activity at all
concentrations where as Embelia ribes shows pro-oxidant effect at 50 µg/ml extract
concentration but lipid per oxidation is significantly reduced from concentration 100 µg/ml
upto 250 µg/ml. The order of decreasing antioxidant activity was Glycyrrhiza glabra >
Embelia ribes.
M. F I. V C.O
500
400
TBARS(n mol/g.tissue)
300
200
100
0
Bas al Cont r ol 50 100 150 200 250
Extract concentration(microgram/ml)
G.G E.R
500
400
TBARS (nmol/g.tissue)
300
200
100
0
Basal Cont r ol 50 100 150 200 250
Extract concentrations (microgram/ml)
Figure.4.1. The effect of aqueous extracts of medicinal plants against lipid peroxidation in
mice brain induced by sodium nitroprusside.Graph.4.1 (a).The effect of Myristica
fragrans, Illicuim verum, Curculigo orchioeides against lipid peroxidation in mice
brain.Graph.4.1 (b) the effect of Glycyrrhiza glabra, Embelia ribes against lipid
peroxidation in mice brain.
Graph4.2 (a) shows the antioxidant effect of Myristica fragrans, Illicuim verum and
Curculigo orchioeides in mice brain. Here, the TBARS was induced with 10 µM iron. The
results revealed that treatment with Fe (II) caused a significant (P <0.05) increase in
thiobarbituric acid reactive substances (TBARS) compared to the basal. Three separate
controls were used for Myristica fragrans, Illicuim verum and Curculigo orchioeides.
Treatment with different concentrations of Myristica fragrans, Illicuim verum and Curculigo
orchioeides caused a marked decrease in lipid peroxidation. Illicuim verum showed a higher
percentage decrease in lipid peroxidation compared to Myristica fragrans and Curculigo
orchioeides.As at lower concentrations of Myristicafragrans and Curculigo orchioeides had
pro-oxidant effect. The order of decreasing antioxidant activity was Illicuim verum >
Myristicafragrans > Curculigo orchioeides.
Graph4.2 (b) shows the antioxidant effect of Glycyrrhiza glabra, Embelia ribes in mice brain
induced with iron. Treatment with Fe (II) stimulated the TBARS production. However, the
lower concentrations of Glycyrrhiza glabra and Embelia ribes were found to be pro-oxidant
but at high concentrations they showed decrease in lipid per oxidation. The order of
decreasing antioxidant activity was Glycyrrhiza glabra > Embelia ribes.

M.F I.V C.O
600
500
400
300
200
100
0
Basal Cont rol 50 100 150 200 250

Figure. 4.2. The effect of aqueous extracts of medicinal plants against lipid peroxidation in
mice brain induced by iron.Graph.4.2 (a).The effect of Myristicafragrans, Illicuim
verum, Curculigo orchioeides against lipidperoxidation in mice brain Graph.4.2 (b)
the effect of Glycyrrhiza glabra, Embelia ribes against lipidperoxidation in mice
brain.
4.1.2 Lipid peroxidation in mice liver induced with sodium nitroprusside and iron
Figure4.3 (a) shows the antioxidant effect of Myristica fragrans, Illicuim verum and
Curculigo orchioeides in mice liver induced with sodium nitroprusside. Treatment with
sodium nitroprusside stimulated the TBARS production. However, treatment with Myristica
fragrans, Illicuim verum and Curculigo orchioeides caused a significant (P<0.05) decrease in
lipid peroxidation and were found to decrease the TBARS level almost comparable to the
basal. The order of decreasing antioxidant activity was Illicuim verum > Curculigo
orchioeides >Myristica fragrans.
Figure4.3 (b) shows the antioxidant effect of Embelia ribes,Glycyrrhiza glabra in mice liver
induced with sodium nitroprusside. Treatment with sodium nitroprusside stimulated the
TBARS production. Glycyrrhiza glabra showed good antioxidant effect by decrease in lipid
peroxidation at all concentrations where as Embelia ribes showed pro-oxidant effect on
50µg/ml and 100 µg/ml however from concentration 150 µg/ml upto 250 µg/ml it showed
gradual decrease in lipid per oxidation induced by sodium nitroprusside.
The order of decreasing antioxidant activity was Glycyrrhiza glabra > Embelia ribes.
M.F I.V C.O
500
400
a
TBARS(nmol/g.tissue)
300
200
100
0
Basal control 50 100 150 200 250
Extract concentarion(microgram/ ml)

G.G E.R
500
400
TBARS (nmo l /g.ti ssue )
300
200
100
0
Basal Cont rol 50 100 150 200 250
mice liver induced by sodium nitroprusside Figure4.3(a).The effect of Myristica
fragrans, Illicuim verum, Curculigo orchioeides against lipidperoxidation in mice
liver.Figure4.3(b) The effect of Glycyrrhiza glabra, Embelia ribes in mice liver.
Figure 4.4 (a) shows the anti-lipidperoxidative effect of Myristica fragrans, Illicuim verum
and Curculigo orchioeides in mice liver induced with iron. Treatment with iron stimulated
TBARS production. However, treatment with Myristica fragrans, Illicuim verum and
Curculigo orchioeides caused a significant (P<0.05) decrease in lipid peroxidation.These

plants showed gradual decreased in lipid per oxidation However , Illicuim verum showed
abrupt decreased in TBARS even at low concentration.
The order of decreasing antioxidant activity was Illicuim verum > Curculigo orchioeides
>Myristicafragrans.
Figure4.4 (b) shows the antioxidant effect of Glycyrrhiza glabra, Embelia ribes in mice liver
induced with iron. Treatment with iron stimulated the TBARS production. Glycyrrhiza
glabra showed abrupt anti-oxidant affect at low concentration but from concentration 100
µg/ml up to 250µg/ml it showed a gradual decrease in lipid peroxidation. However, treatment
with Embelia ribes showed pro-oxidant effect at 50µg/ml but it showed decrease in lipid per
oxidation as the concentration was increased from 1o0 µg/ml upto 250 µg/ml.
The order of decreasing antioxidant activity was Glycyrrhiza glabra > Embelia ribes.
M.F I.V C.O
500
400
300
200
100
0
Basal Cont rol 50 100 150 200 250

G.G E.R
600
500
TBARS (n mo l /g .ti ssu e )
400
300
200
100
0
Basal Cont r ol 50 100 150 200 250
mice liver induced by iron.Figure.4.4 (a).The effect of Myristicafragrans, Illicuim verum and
Curculigo orchiodes against lipidperoxidation in mice liver.Figure.4.4 (b) the effect of
Glycyrrhiza glabra, Embelia ribes in mice liver.
4.1.3 DPPH radical scavenging activity
Graph (5) shows the DPPH radical scavenging activity of studied plants. All the extracts have
shown high antioxidant activity which is evident by their ability to scavenge DPPH radical to
more than 50%. However, the order of their antioxidant activity is Glycyrrhiza glabra >
Illicuim verum > Curculigo orchioeides > Embelia ribes > Myristicafragrans.
Glycyrrhiza glabra , Illicuim verum and Curculigo orchioeides showed higher % scavenging
of DPPH radical even at lower tested concentrations.
M. F I. V C.O GG E.R
100
% scavenging of DPPH radical
80
60
40
20
0
50 100 150 200 250
Figure.4.5.Antioxidant activity of aqueous extract of medicinal plant. DPPH radical
scavenging activity of different plants. Values are means ± SD (n=3).
4.1.4 Total antioxidant activity by phosphomolybdenum assay

Graph 6 shows the total antioxidant activity of different plants extract expressed as
ascorbic acid equivalent. All the extracts showed their reducing activity. However, the order
of their reactivity was Glycyrrhiza glabra > Illicuim verum > Curculigo orchioeides >
Myristicafragrans > Embelia ribes.
M. F I. V C.O GG E.R
250
Ascorbic Acid equivalent(microgram/ml)
200
150
100
50
0
50 100 150 200 250
Figure.4. 6. Total antioxidant activity measured by phosphomolybdenum reduction method.

Values are mean ± SD (n=3).
4.1.5 Total phenolics content
Mean values for total phenolic content are shown in Table 1. The results revealed that the
highest phenolic content was found in Glycyrrhiza glabra (339±1.25mg/g), Illicuim verum
(319.5±0.2), Curculigo orchioeides (287±4.1 mg/g) followed by Embelia ribes
(263±0.65mg/g) and Myristica fragrans (259± 2.33 mg/g) content low amount of phenolic
content.
Extracts Phenolics mg/g
Glycyrrhiza glabra 339±1.25
Illicuim verum 319.5±0.2
Curculigo orchioeides 287±4.1
Embelia ribes 263±0.65
Myristica fragrans 259± 2.33
Table4.1.Total phenolic content among aqueous extract of medicinal plants. The results are
expressed as means ± SD
Discussion
Oxidative stress is now known to have an association with more than hundred pathologies, as
well as with the normal aging process (Ghasanfari et al., 2012). There is a strong correlation
between the products that cause oxidative damage to DNA and thiobarbituric acid-reactive
substances (TBARS) as a marker of lipid peroxidation (Chen et al., 2005).
It is well known that the production of reactive oxygen species (ROS), catalyzed by metals,do
attack not only on proteins and DNA, but also on other components of cell involving poly-
unsaturated fatty acid residues of phospholipids, which are highly sensitive to oxidation
(Shacter, 2000). Inhibition of lipid peroxidation in rabbit brain homogenate is analogous to
neuro-protection (Muralikrishnan et al., 2008). The brain cells and tissues are prominently
vulnerable to oxidative damage because of its high demand of oxygen utilization, have a high
amount of oxidisable poly-unsaturated fatty acids and the presence of redox-active trace
metals (Fe,Cu). The natural antioxidants in plants have been reported to have a high level of
pharmacological,biologicaland therapeutic actions against oxidative stress caused by free
radicals (Shahidi, 2000). As compared to the normal, Increase in the formation of
thiobarbituric acid reactive substances (TBARS) in iron(II) sulphate (10 µM)-induced
oxidation, suggest the possible damage of cells and tissues with an iron-overload. Free iron in
the mitochondria and in cytosol of cytoplasm can cause considerable oxidative cellular injury
by increasing the production of superoxides, which can react with Fe(III) to regenerate Fe(II)
that participates in the Fenton reaction (Fraga and Oteiza, 2002). Iron overload results in the
formation of products of lipid peroxidation, which have been demonstrated in a number of
tissues, including the liver and kidneys (Houglum et al., 1990). Liver cirrhosis is a serious
disease caused by the storage of iron in the liver. Ratswhenoverloaded with iron showed toxic
effects at different organs, such as cardio myopathy,hepatocellularh ypertrophy, splenic white
pulp atrophy, pancreatic atrophy and hemosiderosis in the heart, liver, spleen, pancreas and
endocrine glands, respectively (Whittaker et al, 1997). The iron toxicity is caused by the
mechanisms which include free radical-mediated peroxidative reactions, which are readily
catalyzed by iron. The protections against the free radicals offered by the aqueous extracts of
Myristica fragrarans, Illicuim verum, Curculigo orchioeides, Glycyrrhiza glabra and
Embelia ribes suggest that they will be beneficial to treat brain and liver toxicities.
Sodium nitroprusside is a drug use for hypertension, whose action is to relax the smooth
muscles of vessels; it dilates the arteries and veins present in peripheries. However, SNP
has also been reported to cause cytotoxicity by the production of nitric oxide and/or
cyanide (Bates et al., 1991). The nitric oxide acts freely, it may also cause damage to
neurons along with other reactive oxygen species (ROS). In mice liver, methionine
synthase has also been deactivated by sodium nitropruside. This deactivation of enzyme
has been due to nitric oxide produced by sodium nitropruside. This induction of
deactivation of enzyme methionine synthase by nitric-oxide could rationally explain the
cytotoxic and cellular effects of this highly reactive molecule (Nicolaou et al., 1997). The
protection offered by aqueous extracts of Myristica fragrarans, Illicuim verum, Curculigo
orchioeides,Glycyrrhiza glabra and Embelia ribes on tissues shows the in vitro
antioxidant activity of aqueous extracts of the above medicinal plants and suggests that
they may be play a significant role in preventing diseases occuring from the overload of
sodium nitropruside. These results also indicate that the extracts of these plants show
protection against various hepatotoxins and neurotoxins (Iron and SNP) at very low
concentrations (less than 250 µg/ml) and, in most of the cases, has the capability to
minimize the production of TBARS to less than the basal level.

Antioxidants are those substances that tranform the free radicals into non-reactive species
and reduce their negative actions. Their action is at different stages i.e. interception,
prevention, and repair and by different mechanisms: quenching singlet oxygen, reducing
reactive substances by donating hydrogen, acting as chelators of trace metals and trapping
free radicals (Devasagayam et al., 2004). Different research studies show that the
vegetables, fruits, grains, medicinal and other plants are a significant source of bioactive
compounds, polyphenols that have been found to have a very strong free radical
scavenging and antioxidant activity. Free radicals which are involved in the process of
lipid per-oxidation are thought to play a main role in various chronic diseases such as
cardiovascular diseases and cancer (Halliwell, 1992). DPPH• is a role model of a stable
lipophilic radical. The lipophilic radicals initiate their chain reactions by lipid
peroxidation. Antioxidants react with DPPH•, reducing the amount of free radicals
generating from DPPH to the amount of their available hydroxyl groups. Therefore the
absorption at 517 nm is proportional to the amount of residual DPPH •. It is observed
during the discoloration from purple to yellow. The high DPPH radicals scavenging
activity of studied plants suggests their use in those diseases which are arising from free
radicals oxidative stress.
In the total antioxidant assay, which is used to quantitatively evaluate fat-soluble and water-
soluble antioxidant capacity (phosphomolybdenum assay), the plant extracts showed
electron-donating capacity demonstrating its capability to act as chain breakers, changing the
relative free radicals into more stable non-reactive products (Dorman et al., 2003).
Flavonoids are included in that class of polyphenols which are present naturally in nearly all
plant parts (Bravo, 1998). Phenolic compounds donate hydrogen ions, due to this reason they
are good antioxidants (Rice et al., 1995). Flavonoid compounds are of major therapeutic and
scientific value, and above all their antioxidant actions are of more importance. Their great
antioxidant action is of importance because they are capable to scavenge dangerous free
radicals and also reactive oxygen species(ROS) that are produced during various metabolic
processes running inside the cells or tissues lead to oxidation (Bors et al., 1990).
Polyphenolic flavonoids, which are derived from plants, are well known to show their
activity as antioxidant through different mechanisms i.e. preventing lipid peroxidation,
scavenging of ROS and chelation of metal ions (Shahidi, 1997). Hence they have multiple
mechanisms of action; for example, they inhibit those enzymes which are involved in the
generation of reactive oxygen species (ROS), chelation of trace elements such as free copper
and iron, and by donation of hydrogen ions to free radicals, thus protect our bodies from
various diseases such as cancers, heart attacks and strokes. Moreover, the ascorbic acid,
which acts as a chain-breaking antioxidant prevent the formation of free radicals during the
formation of extracellular and intracellular substances throughout the body, includes bone
matrix,collagen and tooth dentine (Beyer, 1994).
Dinesha et al 2014 conducted a study to determine the antioxidant activity of the aqueous
extract of Illicium verum against H2O2 induced DNA damage and human peripheral
lymphocyte cell death. The antioxidant activities were evaluated by lipid peroxidation,
hydroxyl radical scavenging activity, superoxide radical scavenging activity and DPPH (1,1-
diphenyl-2-picryhydrazyl) activity. It was concluded in this study thatIllicium verum
possesses effective prevention ability against H2O2 induced cell death and DNA protection.
Cheng-Hong et al 2012 conducted a study in which results showed that the ethyl
acetate fractions of Illicium verum have free radical scavenging effects and reducing power
effect. The strong correlation between its DPPH and TEAC values with those obtained from
the reducing power assay implied that the antioxidants in the extracts were capable of
scavenging free radicals and reducing oxidants. The antioxidant components of Illicium
verum were also characterized by thin-layer chromatography and GC-MS in this study.
Yadav and Bhatnagar2010 conducted a study to investigate the in vitro inhibition of
lipid peroxidation effect of Illicuim verum. Liver of rats with post mitochondrial supernatant
(PMS) in Tris HCl buffer, pH 7.4 was incubated for 0 and 1 h, with Illicium verum extract in
three different oxidant systems. The results showed that addition of Illicium verum extract to
FeCl (3) medium at 0 h significantly stop the initiation of the lipid peroxidation. It was
concluded that the Illicium verum possesses superoxide radical scavenging activity and strong
reducing power.
Glycyrrhiza glabra is a legume native to India, Southern Europe and other countries
of Asia. Many components have been isolated form Glycyrrhiza glabra including water
soluble, biologically active complex. This complex is composed of flavonoids, triterpene
polysaccharides,saponins, pectins, amino acids, simple sugars, mineral salts and various other
substances (Obolentseva et al., 1999).
Visavadiya and Narasimhacharya, 2006 reported the antioxidant effects and
hypocholesterolaemiceffects of Glycyrrhiza glabra root powder in hypercholesterolaemic
male albino rats in a previous report. A four week administration of Glycyrrhiza glabr root
powder (5 and 10 gm% in diet) to hypercholesterolaemic rat’s led to significant reduction in
plasma, cholesterol, triglycerides, hepatic total lipids, low-density lipoprotein and VLDL-
cholesterol as well as significant increases in HDL-cholesterol levels.
In another study conducted by Visavadiya et al., 2009 to evaluate antioxidant property
of Glycyrrhiza glabra root extracts using in vitro models. The dose-dependent aqueous and
ethanolic extracts demonstrated the scavenging activity against nitric oxide (concentration
that caused 50% inhibition of nitric oxide radicals.
Myristica fragrans also called nutmeg is an aromatic evergreen tree that grows 30–39
ft. with yellow fleshy fruits and high with spreading branches, looking like peach or apricot.
There are many other species of this plant, but M. fragrans is the most common and used as
medicinal plant .
In some previous reports, phenolic compounds belonging to the lignans group have
been reported capable of scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals as
well as chelating with metallic elements to form complexes (Chatterjee et al., 2007; Su et
al., 2007; Gülçin, 2010; Gülçin et al., 2010). Argenteane is a dilignan antioxidant that is
similar in power to vitamin E isolated from Myristica fragrans(Calliste et al., 2010).
Ashish et al, 2013 reported the antioxidant and antimicrobial activities of nutmeg
(Myristica fragrans Houtt) seed extracts in a previous study. Acetone, ethanol, methanol,
butanol and water were used for seeds extraction. All the extracts have shown significant
antioxidant potential. Acetone extract has shown the highest antioxidant potential. This high
antioxidant activity could be due to presence of β-pinene, α-pinene, 1,8-cineole, myrcene,
carvacrol, terpinen-4-ol, isoeugenol and eugenol.
Suchandra et al 2007 reported antioxidant potential of phenolic compounds from fresh
Myristica fragrans and stated that it has the capability to scavenge 1,1′-diphenyl-2-
picrylhydrazyl (DPPH) radical, inhibit lipid peroxidation and protect plasmid DNA damage
upon exposure to gamma radiation suggesting its potential use as a nutraceutical in
preventing oxidative damage to cells.
In another study conducted by Lan et al, 2007 in which Myristica fragrans was
extracted with 80% methanol and 50% acetone.Free radical-scavenging activity against
cation (ABTS+), hydroxyl (HO) radicals, DPPH and peroxyl (ORAC) were evaluated. The
80% methanol extract of Myristica fragrans had greater ORAC, ABTS +and TPC values
compared to the 50% acetone extract. The data indicated that Myristica fragrans may serve
as potential dietary sources of natural antioxidants for improving human nutrition and health.
Venukumar and Latha, 2002 evaluated the antioxidant activity of methanol extract of
rhizomes ofCurculigo orchioides using CCl4-intoxicated rat liver as the experimental model.
The hepatotoxic rats were administered the tested drug for 90 days (daily, orally at the dose
of 70 mg/kg/body weight). Lipid peroxidation in CCl 4-intoxicated rats was exhibited bythe
elevation in the levels of thiobarbituric acid reactive substances (TBARS) and diene
conjugates (CD), and also a distinct diminution in glutathione (GSH) content in the liver.
Devyaniet al, 2010 evaluated the in vitro antioxidant activity of Curculigo orchioides, ans
revealed that Curculigo orchioides possesses potential effects in lipid peroxidation, DPPH
antiradical, nitric oxide scavenging, super oxide scavenging and protection against
superoxide induced damage to erythrocytes supporting the results of our study.
In another study conducted by Krishnamoorthy 2012 who investigated the antioxidant
effect of Curculigo orchioides rhizome extract on antioxidant enzymes in the liver tissue of
male albino rats. The enzyme activities were revealed near normal in Curculigo orchioides
treated rats exhibiting the efficacy of the Curculigo orchioides rhizome in controlling
oxidative stress due to liver toxicities.
Embelia ribes is a large shrub which is found in the hilly parts of India and Pakistan
from the central and lower Himalayas down to Singapore and Sri Lanka. Its dried seeds have
a bitter taste and are prescribed for the treatment of ascites, tumors, mental diseases,
bronchitis, heart diseases, dyspnoea, urinary discharges, jaundice and in snake bites.Major
active compound embelin (embelic acid) 2,5-dihydroxy-3-undecyl-1,4-benzoquinone from
the chloroform extract of Embelia ribes.
Dharmendraet al, 2009 conducted a study to determine the antioxidant potential where
results of biochemical parameters revealed that the administration of CCl 4 to rats caused
significant (P ≤ 0.001) peroxidative damage as evidenced by marker enzymes and antioxidant
defense system through liver and serum contents compare able with that of our results.
Various pharmacological activities of embelin have been reported such as cell lines
like antitumour, anti-inflammatory and analgesic activities (Chitra et al 1994), chemo
preventive (Sreepriya et al, 2005), anticancer activity (Xu et al, 2004), and antioxidant
activity by DPPH method (Joshi et al, 2007).
Conclusion
In the present study following tradional herbal dugs like Myristica fragrans, Illicuim
verum,Curculigo orchioeides, Glycyrrhiza glabra and Embelia ribes were tested with respect
to their anti-oxidant activity against lipid peroxidation in mice brain and liver,totalpheloic
content, DPPH radical-scavenging test and total anti-oxidant assay.
Among all the drugs Glycyrrhiza glabra and Illicuim verum showed highest antioxidant
activity against lipid per oxidation in mice brain and liver induced by Sodiun nitroprusside
and iron. These results were also proved by total phenolic contents because Glycyrrhiza
glabra and Illicuim verum have highest phenolic contents among all five drugs. DPPH
radical scavenging and total antioxidant assay also proved that Glycyrrhiza glabra and
Illicuim verum have higest anti-oxidant activity.
Whereas other 3 drugs Myristica fragrans, Curculigo orchioeides ,Embelia ribes also showed
anti -oxidant activity against lipid per oxidation in mice brain and liver induced by Sodium
nitroprusside and iron but was lower as compared to Glycyrrhiza glabra and Illicuim verum
due to lesser phenolic contents. There lesser anti-oxidant activity was also observed by DPPH
radical scavenging test and total phenolic assay.

Finally, the results in this study indicate that these extracts are capable of reducing lipid
peroxidation and thus can be effectively utilized against diseases arising from oxidative
stress. However, further in vivo studies are required to evaluate their mechanism of action.
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In vitro antioxidant and inhibitory effects of Myristica fragrans,

Illicuim verum, Curculigo orchioeides, Glycyrrhiza glabra and

This thesis is submitted in partial fulfilment of the

Requirement for the degree of Master of Philosophy

Thesis Submitted in Partial Fulfilment of Requirements for the Degree of

Leading me into intellectual pursuits and unconditional support in my studies

The uncompromising principles that guided me throughout my student life

Their kindness and to create a friendly environment

Their guidance and kind patronage throughout my research pursuits

antioxidant and inhibitory effects of Myristica fragrans, Illicuim verum, Curculigo

RESEARCH SUPERVISOR AND DR. ASMA SAEED

ORAL EXAMINATION HELD ON:

complete this M.PHIL research work.

to express my sense of gratitude to him. .

I express my most sincere gratitude to my Research Co-Supervisor Dr. Muhammad Akram,

research work embodied in this thesis.

Gomal University, D.I.Khan. Thanks to my friends at Department of Biological sciences,

cooperation and friendly attitude especially Malik Shahjahan, Muhammad Naeem,

Muhammad Ayaz and at last my best friends Abdul Hamid,Iftikhar Ahmad,Hamza

SAIF. UR. REHMAN.

1.1.4 Natural Antioxidants in Plant Sources

2.2. Free radical generation and oxidative stress

2.6 Illicium verum

2.6.2 Chemical constituents

2.7.4 Medicinal uses.

2.8.3 Pharmacological actions

2.9.2 Chemical constituents.

3 MATERIALS AND METHODS

3.4.2. Production of TBARS from animal tissues

4 RESULTS AND DICUSSION

4.1.4. Total antioxidant activity by phosphomolybdenum assay

4.1.5. Total phenolics content

2.3 Roots of Curculigo orchioides

2.5 Fruit of Emblica ribes

(a).The effect of Myristicafragrans, Illicuim verum, Curculigo orchioeides

(a).The effect of Myristicafragrans, Illicuim verum, Curculigo orchioeides

(b) the effect of Glycyrrhiza glabra, Embelia ribes against lipidperoxidation in

(a).The effect of Myristica fragrans, Illicuim verum, Curculigo orchioeides

(b) The effect of Glycyrrhiza glabra, Embelia ribes in mice liver

(a).The effect of Myristica fragrans, Illicuim verum and Curculigo orchiodes

(b) the effect of Glycyrrhiza glabra, Embelia ribes in mice liver

4.1 Total phenolic content among aqueous extract of medicinal plants 3

I.V Illicuim verum

C.O Curculigo orchioeides

G.G Glycyrrhiza glabra

E.R Embelia ribes

(TBARS) induced by different pro-oxidants (10 µM FeSO4 and 5 µM sodium nitroprusside) in

evaluated by the scavenging of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50, 151.50±2.3

the use of these plants in various degenerative diseases. Myristica fragrans,Curculigo

orchioeides and Embelia ribes relatively showed weak antioxidant activities.

prime health concerns (Aibinu et al., 2007).

side effects (Britto et al., 2011).

1.1.1. Definition of Antioxidant

“preservatives that specifically retard deterioration, rancidity, or discoloration due to oxidation”

essential. Antioxidants to be used in food products should be economical, harmless, higher

1.1.2. Classification of Antioxidants

Antioxidants have different mechanism of actions such as quenching of secondary lipid

due to their mechanism of action (Rajalakshmi and Narasimhan, 1996).

1.1.2.1. Primary Antioxidants

1. Primary antioxidants donate hydrogen or electrons to lipid free-radicals and disturb

compounds (Reische 1998).

ROO. + A. _ ROOA (d) Reaction of peroxy radical with antioxidant radical

Primary antioxidants even show their activity at very low concentrations.