A

PROJECT REPORT ON
IN-VITRO Evaluation of Antioxidant Potential of Coleus
Scutellariodes Nanoparticle using FRAP Assay
Submitted In Partial Fulfillment of the Requirements
For The Degree Of

Bachelor of Pharmacy

Under the guidance of: Submitted by:

Prof. (Dr.) Shailendra K.Saraf Divya Singh
Asst. Prof. Shristy Srivastava B.PHARM, IVTH YEAR
1900560500035
Faculty of Pharmacy
B.B.D.N.I.I.T.,Lucknow

To the
Faculty of Pharmacy
Dr. Abdul Kalam Technical University, Lucknow
(Formerly Uttar Pradesh Technical University, Lucknow)
2022-2023
 ACKNOWLEDGEMENT

This project report could not have been completed without the tremendous help provided
to me by my teachers. Firstly, I would like to thank my Director Dr. Shailendra K. Saraf
for his valuable instructions and constructive comments. It was for his organizational skill
that whole project was so efficiently carried out. I am also grateful to my project guide
Asst. Prof. Shristy Srivastava for her help and support. I am thankful to all those known
and unknown sources that helped me in completing such task. Express a word of thanks to
my friends for their constant support, suggestion and encouragement during preparation of
this project. Finally, I thank God for giving me the loving siblings and affectionate
parents, who blessed me with everything all throughout my life. My gratitude to them
cannot be expressed in words. To them I owe my wonderful today and a dream filled
tomorrow.

DIVYA SINGH
BBDNIIT, Lucknow.
 CERTIFICATE

This is to certify that Ms. Divya Singh has satisfactory completed the project work entitled
IN - VITRO Evaluation of Antioxidant Potential of Coleus Scutellariodes Nanoparticle
using FRAP assay’’ Of the elective subject practice school, as per ABDUL KALAM
TECHNICAL UNIVERSITY, LUCKNOW, for B.Pharm. 7th Sem. In the academic year
2022-23.

Prof.(Dr.) Shailendra K. Saraf

Director of pharmacy
BBDNIIT,LUCKNOW
 DECLARATION

I here by declare that the studies described in this report entitled were carried out by me in the
Faculty of Pharmacy, BBDNIIT, Lucknow under the guidance of Prof. (Dr.) Shailendra
K. Saraf and Asst. Prof. Shristy Srivastava. Further, these works has not been submitted in
the part or in fulfillment to obtain any other degree.

DIVYA SINGH
B.PHARMA, IVTH YEAR
BBDNIIT LUCKNOW
 CONTENT

S.NO TOPIC PAGENO.

Acknowledgement 2
1

2 Certificate 3

Declaration 4
3

Introduction 6-7
4

Antioxidants 8-13
5

Nanoparticle 14- 17
6

7 Procedure 18- 20

Observation 20-23
8

Conclusion 24
9

References 25
10
 INTRODUCTION

Free radicals are toxic by-products for naturally as a result of aerobic metabolism in our body.
They are any species capable of independent existence and contain one or more unpaired
electrons which react with other molecules by oxidation or reduction reaction [1]. Free radicals
include reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Generation of oxidants due to endogenous factors(metabolism, infections, exercise, ageing) or

exogenous factors (exposure to radiations, metal catalyzed reaction sand oxygen free radicals as
pollutants in the atmosphere) that are beyond the antioxidant capacity of a biological system
gives rise to oxidative stress.

The generation of oxidative stress is harmful to the body and may cause per oxidation of
membrane lipids leading to loss of membrane integrity and cell death, denaturation of proteins
including enzymes, ion channels and strand breakage in DNA. Therefore, they can be related to
certain path physiological conditions such as arthritis, hemorrhagic shock, coronary artery
diseases, cataract, cancer, AIDS as well as related degenerative brain disorders.

Antioxidants are compounds that can decrease oxidative stress and minimize the incidence of
pathological conditions caused by the oxidants. The mechanism of action of these antioxidant
compounds includes inhibition of the enzymes or chelating of trace elements involved in free
radical production, scavenging of reactive species and up-regulation or protection of antioxidant
defense.

Antioxidants are produced naturally in the human body and there is symmetry between the
oxidants generated and the antioxidants present. However, due to oxidant over production,
inadequate supply of nutritional supplement sand/order antioxidant argument this equilibrium is
hindered resulting in oxidative hassle .Therefore, a dietary source rich in antioxidant is
recommended. Antioxidants taken as supplements are particularly important in reducing the
cumulative oxidative damages. Among currently available drugs, synthetic antioxidants do have
potential side effects and carcinogenicity of which can be minimized to a great extent through the
administration of natural compounds. Therefore, interest in medicinal plants and their therapeutic
potential as antioxidants is fast increasing. Still there remain a large number of natural drugs
which are yet to be explored scientifically.

Coleus Scutellariodes, commonly known as coleus, is a species of flowering plant in the family
lamiaceae (the mint or deadnettle family), native to South East Asia through to Australia.
Typically growing to 60–75cm (24–30in) tall and wide, it is a bushy, woody-based evergreen
perennial, widely grown for the highly decorative variegated leaves found in cultivated varieties.
Another common name is painted nettle, reflecting its relationship to deadnettle’s (Lamium
species), which are in the same family. (True nettles and their close kin are in the distant family
Urticaceae.) The synonyms Coleus blumei, Plectranthus Scutellariodes and Solenostemon
Scutellariodes are also widely used for this species.

Coleus is a plant that has been used since ancient times to treat heart disorders such as high blood
pressure and chest pain(angina),as well as respiratory disorders such as asthma. Forskolin is a
chemical found in the roots of the coleus plant. When taken by mouth, coleus is used to treat
allergies, dry eye, skin conditions such as eczema and psoriasis, obesity, painful menstrual periods
,irritable bowel syndrome(IBS), urinary tract infections (UTI), bladder infections, advanced
cancer, blood clots, sexual problems in men, trouble sleeping(insomnia),and convulsions.
Healthcare providers sometimes give coleus intravenously (by IV) for heart failure. Some people
breathe in (inhale) coleus powder for asthma. Coleus drops are used in the eyes to treat glaucoma.
Herbal product manufacturers are now producing Coleus extracts that contain high levels of
forskolin. These preparations are being promoted for the same conditions for which forskolin have
been traditionally used. However, currently there is no reliable scientific information that shows
Coleus extracts taken by mouth are effective.
Antioxidants: Its medicinal and pharmacological applications.

Antioxidants are substances that may protect cells from the damage caused by unstable
molecules known as free radicals. Antioxidants interact with and stabilize free radicals and may
prevent some of the damage free radicals might other wise cause. Free radical damage may lead to
cancer. Examples of antioxidants include beta-carotene, lycopene, vitamins C, E, and A and other
substances.

Sources and origin of antioxidants

Antioxidants are abundant in fruits and vegetables, as well as in other foods including nuts, grains
and some meats, poultry and fish. For example Beta-carotene is found in many foods that are
orange in color, including sweet potatoes, carrots, apricots, pumpkin and mangoes. Some green,
leafy vegetables, including collard greens, spinach and kale. Lycopene is a potent antioxidant
found in tomatoes, watermelon, guava, papaya, apricots. Selenium is a mineral, not an antioxidant
nutrient. However, it is a component of antioxidant enzymes. Plant foods like rice and wheat are
the major dietary sources of selenium in most countries.

Classification of antioxidants

Antioxidants are grouped into two namely;

1. Primary or natural antioxidants

(a) Antioxidants minerals - These are co factor of antioxidants enzymes. Their absence
will definitely affect metabolism of many macromolecules such as carbohydrates.
Examples include selenium, copper, iron, zinc and manganese.
(b) Antioxidants vitamins – It is needed for most body metabolic functions. They
include-vitamin C (Figure 1),vitamin E, vitamin B.

2. Secondary or synthetic antioxidants

These are phenols compounds that perform the function of capturing free radicals and
stopping the chain reactions, the compound include
i. Butyrate hydroxyl anisole(BHA).

ii. Butyrate hydroxyl toluene(BHT).

iii. Propyl gallate (PG) and metal chelating agent(EDTA).

Types of antioxidants

Ascorbic acid or" vitamin C" is a monosaccharide antioxidant found in both animals and plants.
As one of the enzymes needed to make ascorbic acid has been lost by mutation during human
evolution, it must be obtained from the diet and is a vitamin. Most other animals are able to
produce this compound in their bodies and do not require it in their diets. In cells, it is
maintained in its reduced form by reaction with glutathione, which can be catalyzed by protein
disulfide isomers and glutaredoxins.

Glutathione The free radical mechanism of lipid per oxidation: Glutathione is a cytokine-
containing peptide found in most forms of aerobic life. It is not required in the diet and is instead
synthesized in cells from its constituent amino acids. Glutathione has antioxidant properties since
the thiols group in its cytosine moiety is a reducing agent and can be reversibly oxidized and
reduced.

Melatonin is a powerful antioxidant that can easily cross cell membranes and the blood-brain
barrier. Unlike other antioxidants, melatonin does not undergo redox cycling, which is the ability
of a molecule to undergo repeated reduction and oxidation.

Tocopherols and tocotrienols (vitamin E) is the collective name for a set of eight related
Tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties. Of
these, Tocopherols has been most studied as it has the
Highest bioavailability, with the body preferentially absorbing and metabolizing this form. It has
been claimed that the Tocopherols form is the most important lipid-soluble antioxidant and that it
protects membranes from oxidation by reacting with lipid radicals produced in the lipid per
oxidation chain reaction.

MEDICINAL APPLICATIONS OF ANTIOXIDANTS

Anti-cancer agent in medicinal chemistry

Lanthanides as anti-cancer agents The application of inorganic chemistry to medicine is a

rapidly developing field, Novel therapeutics and diagnostic metal complexes are now having an
impact on medical practice, . A lot of metal –based drugs are widely used in the treatment of
cancer. The clinical success of cisplatin and other platinum complexes is limited by significant
side effects acquired or intrinsic resistance Strategies for developing new anticancer agents
include the incorporation of carrier groups that can target tumor cells with high specificity. Also
of interest is to develop complexes that bind to DNA in a fundamentally different manner than
cisplatin, in an attempt to overcome the resistance pathway that has evolved to eliminate the drug

Lycopene as a potential anti-cancer agent Dietary chemo prevention has emerged as a cost
effective approach to control most prevalent chronic diseases including cancer. In particular,
tomato and products are recognized to confer a wide range of health benefits. Epidemiology
studies have provided evidence that high consumption of tomatoes effectively lowers the risk of
reactive oxygen species (ROS)-mediated diseases such as cardiovascular diseases and cancer by
improving the antioxidant carotenoid reported to be more stable and potent singlet oxygen
quenching agent compared to other carotenoid. In addition to its antioxidants properties,
lycopene shows an array of biological effects including cardio protective, anti-inflammatory,
anti-mutagenic and anti-carcinogenic activities.

Antioxidants therapy in acute central nervous system injury

Free radicals are highly reactive molecules generated predominantly during cellular respiration
and normal metabolism balance between cellular productions of free
Radicals and ability of cells to defend against the oxidative stress(OS). OS has been implicated
as a potential contributor to acute central nervous system (CNS) injury by ischemic or
hemorrhagic stroke or trauma. The production of reactive oxygen species (ROS) may increase,
sometimes drastically leading to tissues damage via several different cellular molecular
pathways. Radicals can cause damage to cardinal cellular components such as lipids, proteins
and nucleic acid e.g. DNA leading to subsequent cell death by modes of necrosis or apoptosis.
The damage can become more widespread due to weakened cellular antioxidant defense systems.
Therefore, treatment with antioxidants may theoretically act as tissue damage and improve both
the survival and neurological outcome, several such agents of widely varying chemical structures
have been investigated as therapeutic agents for acute CNS injury, although, a few of the
antioxidants showed some efficacy in animal models or in small clinical studies.

THERAPEUTIC PROPERTIES OF ANTIOXIDANTS

Antioxidants are very important in the treatment of fried Reich ataxia, a rare progressive
condition that causes damage to the nervous system. It is inherited in an auto soma recessive
pattern, meaning that, an affected gene must be inherited from each parent for the disease to
develop in their child it is most common recessively inherited worldwide. The progression of the
disease cannot be easily assessed by clinical examination test. Evaluation of diseases is done by
standard neurological scales. Abnormal high levels, oxidative damage to the cells occurs leading
to several pathological conditions, rheumatoid arthritis, hemorrhagic shock, cardiovascular
system disorder, cystic fibrosis, metabolic disorder, gastrointestinal, ulcer genesis and acquired
immunodeficiency. Pharmacological applications of luteinize are as agent in radio immune
therapy and photodynamic.

ANIOXITANT

MECHANISM OF ACTION :
The redox properties of antioxidants play an important role in absorbing and neutralizing free
radicals, quenching singlet and triplet oxygen, or decomposing peroxides. In doing so, the
antioxidants themselves become oxidized. This urges the constant need of antioxidants to
replenish them. The mechanism of antioxidants work has two functions; the first function is that
they act as the give of the hydrogen atom, which is a main function. Antioxidants, which have
such main functions, are referred to as primary antioxidants. They can provide hydrogen atoms at
a faster rate to the lipid radical (R*, Roo*) or change it to a more stable form. It is a chain
breaking step. These function is a secondary one, which is a preventive step. It reduces the rate of
auto-oxidation with a variety of mechanisms beyond the auto-oxidation mechanism of chain
termination by radical conversion of lipids to form more stable i.e. ,by scavenging initiating
radicals, such antioxidants can thwart an oxidation chain from ever setting in motion. The
effectiveness of an antioxidant in the body depends on which free radical is involved, how and
where it is generated, and where the target of damage is present

TYPES OF ANTIOXITANT ASSAY

DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) radical scavenging assay: is a stable free radical

with red Color, based on electron-transfer that produces a violet solution in ethanol. This free
radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving
rise to colorless ethanol solution.

The samples were reacted with the stable DPPH radical in an ethanol solution. The reaction
mixture consisted of adding 0.5 mL of sample, 3 mL of absolute ethanol and 0.3mL of DPPH
radical solution 0.5mM in ethanol. When DPPH reacts with an antioxidant compound, which can
donate hydrogen, it is reduced. The changes in color (from deep violet to light yellow) were read
[Absorbance (Abs)] at 517 nm using a UV-VIS spectrophotometer. The mixture of ethanol
(3.3mL) and sample(0.5mL) serve as blank. The scavenging activity percentage(AA%)was
determined by

AA% Inhibition= [(A0– A1)/A0]×100

A0- absorbance of control

A1-absorbance of the tested sample

Ferric reducing antioxidant power assay: FRAP assay is based on the rapid reduction in ferric
by antioxidants present in the samples forming ferrous, a blue-colored product. A standard
curve was created by adding the FRAP reagent to arrange of Fe2+ solutions of known
concentrations which allows the Fe2+ concentration of the samples to be calculated thereby
determining “antioxidant capacity.”

The FRAP assay is the only assay that directly measures antioxidants in a sample compared to
other assays measuring inhibition of free radicals. The values expressed from the FRAP assay
represent the corresponding concentration of electron-donating antioxidants with the reduction in
the ferric on (Fe3+)to the ferrous ion (Fe2+). FRAP is deemed a suitable assessment for total
antioxidants in plants which are consumed by humans because the only compounds with which
FRAP does not react with are the thiols.

Superoxide anion scavenging activity assay: The superoxide anion radicals are produced in
2ml of phosphate buffer (100mM ,pH7.4) with 78Mn namide adenine dinucleotide (NADH),
50M nitro blue tetrazolium chloride (NBT) and test samples at different concentrations. The
reaction mixture is kept for incubation at room temperature for 5 min. It is then added with 5-
methylphenaziniummethosulphate (PMS) (10 M) to initiate the reaction and incubated for 5 min
at room temperature. The Color reaction between superoxide anion radical and NBT is read at
560 nm. Gallic acid is used as a positive control agent for comparative analysis. The reaction
mixture without test sample is used as control and without PMS is used as blank. The scavenging
activity is calculated as follows, % Scavenging activity=[(Absc – Abss)/ Absc]×100.
Nanoparticle: pharmacological significance

Introduction

Nanotechnology is the fascinating branch of a science which encompasses study of system

having scale size. Targeted drug delivery implies for selective and effective Localization of
pharmacologically active moiety at pre identified targets in therapeutic concentration, while
restricting its access to non target normal cellular linings, thus minimizing toxic effects and
maximizing therapeutic index.
Thecolloidalcarriersbasedonbiodegradableandbiocompatiblepolymeric system like
liposome’s, nanoparticle and micro emulsion and have largely influenced the controlled and
targeted drug delivery concepts.

NANOPARTICLE: Nano derives from the Greek word ‘’nano’’ which means dwarf or
extremely small. Nanoparticle are solid colloidal particles ranging from1nm and1000nm in
size, they consist of macromolecular material in which the active ingredients is dissolved,
entrapped, or encapsulated, or adsorbed.
Classification of Nanoparticle:

On the basis of dimensions-

1. Zero dimensional 4.Third dimensional e.g. Gold NPs

2. One dimensional e.g. Graphene

3. Two dimensional e.g. Carbon nano tubes

 On the basis of morphology, size, physical, & chemical properties-

1. Organic Nanoparticle e.g. Liposome’s, micelles, ferritin.

2. Inorganic Nanoparticle e.g. Al, Au, Ag, Cd.

3. Carbon Based Nanoparticle e.g. Carbon nano fibers.

Synthesis of Nanoparticle

There are two main approaches for the synthesis of NPs:

A. Top Down Approach

Involve the breaking down of the bulk material into nano sized particle. It is a destructive
method depend either on removal or division of bulk material or miniaturization of bulk
fabrication processes to produce desired structure with appropriate, properties.

B. Bottom-Up Approach
Constructive method is an alternative approach which employs build upapproach
where nanoparticle are build up from clusters which in turn are obtained from atoms.
This approach generally involves sedimentation and reduction technique.
Application of Nanoparticles:

Application of Nanoparticle in Medical Treatments

An increase in the quantity of nanoparticle raises the scattering intensity. Taking advantage
of this feature, the application to specific molecule recognition in a living body tissue is
expected For example, by covering the cancer cell surface it becomes possible to distinguish
a healthy cell from a cancer cell by the presence of antibodies joined to the Au nanoparticle.
Although the Au nanoparticle junction with the antibody is nicely distributed in the healthy
cell, when a cancer cell exists the antibodies are concentrated mostly at the Au nanoparticle.
The imaging at various wavelengths is performed by a change in the shape of the
nanoparticle
Discovery of biomarkers

Nanotechnology is being applied to biomarker-based proteomics and genomics

technologies. Nanoparticle can be used for qualitative or quantitative in vivo or ex vivo
diagnosis by concentrating, amplifying and protecting a biomarker from degradation, in
order to provide more sensitive analysis

Nanoparticle drug delivery systems

The use of pharmacological agents developed using classical strategies of pharmacological

development is frequently limited by pharmacodynamics and pharmacokinetics problems
such as low efficacy or lack of selectivity. Moreover, drug resistance at the target level
owing to physiological barriers or cellular mechanisms is also encountered. In addition, many
drugs have a poor solubility, low bioavailability and they can be quickly cleared in the body
by the reticulo endothelial system. Furthermore, the efficacy of different drugs such as
chemotherapeutical agents is often limited by dose-dependent side effects.

Advantages of Nanoparticle

1. Nanoparticle drug carriers have higher stabilities and capacity

2. Feasibility of incorporation of both hydrophilic and hydrophobic substances

and variable routes of administration.

3. Nanoparticle are biodegradable, nontoxic and capable of being stored for

longer periods.

Disadvantages of Nanoparticle

Polymeric nanoparticle with limited drug loading capacity, slowly biodegradable, on

repeated administration toxic metabolites maybe formed.
Materials and methods

Plants and chemicals

Fresh coleus Scutellariodes leaves were collected from BBD University, Lucknow, India.
Distilled water, silver nitrate was used for preparation of extract (Decoction) and silver
nanoparticle respectively.

For FRAP Antioxidant assay phosphate buffer saline of pH 6.6 was prepared using
potassium dihydrogen phosphate (13.87gm) and disodium hydrogen phosphate (35.08gm).
other reagents were prepared .i.e.
1% potassium ferrocyanide (1gm in 100 ml){orange},10% trichloroacetic acid
(10gm in 100ml){colorless}, 0.1%ferric chloride(0.1gmin100ml){yellow}

Preparation of the extract

Fresh leaf of plant was washed with distilled water, dried and cut into small pieces, 5 gm of

cut leaf were taken in 150ml of distilled water was boiled at 80-90 o C for 30 min. Further,
the extract was filtered with what man no 1 filter paper, stored and used for further
experiments.

Synthesis of Silver Nanoparticle

The filtered plant extract was transferred to burette to use as reducing agent and capping
agent, 0.2 M silver nitrate solution was prepared to which leaf extract drop wise very slowly
were added until the color of extract gets converted to black- brown color, resulting
information of silver nanoparticle.
FRAP Antioxidant Assay

For the measurement of the reductive ability, we investigated the Fe3+ Fe2+
transformations in the presence of Coleus Scutellariodes NPs following the standard method.
The reducing capacity of a compound may serve as a significant indicator of its potential
antioxidant activity. Like the antioxidant activity, the reducing power of Coleus
Scutellariodes silver nanoparticle increases with increasing concentration.

Preparation of standard solution

3.5 g of ascorbic acid was dissolved in 100 ml of distilled water. Dilutions of this
solution with distilled water were prepared to give various concentrations

Preparation of test sample

Stock solutions of silver nanoparticle were prepared by taking10ml in 100ml distilled water
and further various dilutions were prepared.

Protocol for reducing power

According to this method, the aliquots of various concentrations of the standard and test
sample extracts in 1.0ml of distilled water were mixed with 2.5 ml of (pH 6.6) phosphate
buffer and 3ml of (1%)potassium ferrocyanide.
The mixture was incubated at 50°C in water bath for 20 min after cooling. Aliquots of 3 ml
of (10%) trichloroacetic acid were added to the mixture, which was then centrifuged at
3000rpm for 10min. The upper layer of solution 4ml ml was
mixed with 4 ml distilled water and a freshly prepared 0.5 ml of (0.1%) ferric chloride
solution. Gives bluish color formation. The absorbance was measured from 400- 700 nm in
UV spectrometer. A blank was prepared without adding extract.
Ascorbic acid at various concentrations was used as standard. As illustrated in figures, Fe3+
was transformed to Fe2+ in the presence of silver nanoparticle. This result indicates that
increase in absorbance of the reaction mixture indicates increase in reducing power.

Observation

Plant extract
+
 Concentration(mg/ml) absorbance

1mg/ml 0.145

2 mg/ml 0.237

3 mg/ml 0.335

4 mg/ml 0.4

5 mg/ml 0.532

Absorbance of silver nanoparticle at various concentration in

FRAP assay.

Calibration Curve
0.6
y=0.0937x+0.0487
R²=0.9909
0.5

0.4

0.3

0.2

0.1

0
0 1 2 3 4 5 6

Ferric reducing power determination of silver NPs

 Concentration(mg/ml) Absorbance
1 0.122
2 0.248
3 0.356
4 0.471
5 0.542
Absorbance of Standard ascorbic acid at various concentration in
FRAP assay.

Calibration Curve
0.6
y=0.1063x+0.0289
R²=0.992
0.5

0.4

0.3

0.2

0.1

0
0 1 2 3 4 5 6

Ferric reducing power determination of standard ascorbic acid.

 Conclusion

Reducing power assay method is based on the principle that substances,

which have reduction potential, react with potassium ferrocyanide (Fe3+)

to form potassium ferrocyanide (Fe2+), which then reacts with ferric

chloride to form ferric–ferrous complex that has an absorption maximum
at 700nm. The reducing power of the silver nanoparticle and standard
increases with the increase in amount of sample and standard
concentrations. The reducing power shows good linear relation in both
standard (R2=0.992) and sample extract (R2=0.990). The results of this
study show that silver nano particle of coleus Scutellariodes can be used
easily accessible source of natural antioxidants in pharmaceutical industry.
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nt_Activity_of_some_Medicinal_Species_using_FRAP_Assay
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