PHYTOCHEMICAL SCREENING AND PHYTOCHEMICAL SCREENING AND ANTIOXIDANT ACTIVITY OF POLY ANTIOXIDANT ACTIVITY OF POLY

HERBAL FORMULATIONHERBAL FORMULATION

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Vishnu Institute of Pharmaceutical Education & Research

C. Anusha ReddyCh. DivyaN. Sravanthi ReddyG. SujanaN. SudhakarGuide:

Mr. K. RamanajaneyuluM.Pharm, (PhD)Dept: Pharmaceutical Chemistry

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CONTENTS

• Introductiona. Need for investigationb. Objectives of the workc. Antioxidant systems• Phytochemical screening•Extraction • Materials and methods• Methods of screening• Results and discussion• Conclusion

INTRODUCTION

• Antioxidant is a molecule that inhibits the oxidation of other molecules. • Oxidation is a chemical reaction that transfers the electrons from the

substance to an oxidizing agent. • These oxidation reactions can produce free radicals which starts the chain

reactions and causes damage and death to the cell.• Antioxidants can terminate the chain reactions by removing the free

radicals and inhibit the oxidation reactions.• They do this by oxidizing themselves, so these are often called as

reducing agents such as thiol, ascorbic acid, polyphenols etc…

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INTRODUCTION

• Plantsourced food antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytates and phytoestrogenes have been recognized as having the potential to reduce disease risk.

• The intake of food rich in a-tocopherols, ß-carotene and ascorbic acid has been associated with reduced oxidative-stress related diseases.

• Phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl, thus inhibiting the oxidative mechanism that lead to degenerative diseases [5,6].

• These compounds have antioxidant, antimutagenic and anticarcinogenic activities and also free radical scavenging properties.It was, therefore aimed to investigate its antioxidant activity by various in vitro models.

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NEED FOR INVESTIGATION

Approximately 80% of the world population depends exclusively on plants for their health and healing. Whereas in the developed world, reliance on surgery and pharmaceutical medicine is more unusual but in the recent years, more and more people are complementing their treatment with natural supplements. Furthermore, motivation of people towards herbs are increasing due to their concern about the side effects of drugs, those are prepared from synthetic materials. The people want to concern their own health rather than merely submitting themselves to impersonal health care system. Many botanical and some common dietary supplements are good sources of antioxidants and anti-inflamatory compounds.

OBJECTIVE OF WORKThe study was aimed at investigating the pharmacological

screening of ethanol and aqueous extracts of polyherbal drug with a view to justify the use of the formulation as antioxidant.

• Extraction of polyherbal drug using ethanol extracts using hot percolation technique.

• Invitro screening of the ethanolic extract of polyherbal drug by hydrogen peroxide scavenging activity.

• To perform the reducing power assay on ethanolic extract of polyherbal drug.

• To perform the DPPH and nitric oxide scavenging method on ethanolic extract of polyherbal drug.

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PLAN OF WORK

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PRODUCT

EXTRACT

PHYTOCHEMICAL SCREENING

ANTIOXIDANT ACTIVITY

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ANTIOXIDANT ACTIVITY

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MECHANISM OF ANTIOXIDANT

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MODES OF ACTION OF ANTIOXIDANT

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There are four routes:

•Chain breaking reactions, e.g. alpha-tocopherol which acts in lipid

phase to trap the radical.

•Reducing the concentration of reactive oxygen species e.g. glutathione.

•Scavenging initiating radicals e.g. superoxide dismutase which acts in

aqueous phase to trap superoxide free radicals.

•Chelating the transition metal catalysts

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Antioxidant Role Remarks

ENZYMES Superoxide dismutase (SOD) Mitochondrial Cytoplasmic Extracellular

Dismutates O2⁻ to H2O2

ContainsManganese (Mn.SOD)

Contains Copper & Zinc (CuZnSOD)

Contains Copper(CuSOD) Catalase Dismutates H2O2 to H2O Tetrameric hemoprotein

present in peroxisomes

Glutathione peroxidase (GSH.Px)

Removes H2O2 and lipid peroxides

Selenoproteins (contains Se2+) Primarily in the cytosol also mitochondria uses GSH

VITAMINS Alpha tocopherol Breaks lipid peroxidation Lipid peroxide and O2

⁻ and

OH scavenger

Fat soluble vitamin

Beta carotene Scavenges OH, O2⁻ and

peroxy radicals Prevents oxidation of vitamin A binds to transition metals

Fat soluble vitamin

Ascorbic acid Directly scavenges O2⁻,

OH, and H2O2. Neutralizes oxidants from stimulated neutrophils. Contribute to regenerate vitamin E

Water soluble vitamin

SELECTED FORMULATION

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S.No NAME OF THE DRUG LATIN NAME

1. Red oxide Shodita gairika

2. Shunti Zingeber

3. Mercury

LITERATURE REVIEWSHUNTI:It is a dried rhizome of Zingiber officinale belonging to the family Zingiberaceae • It is consumed as delicacy, medicine or spice.• According to preliminary research 9 compounds found in shunti may bind to

serotonin receptors which may influence gastrointestinal function• Reduces muscle pain associated with exercises• Reduces colon inflamation, used in chemotherapy, treat morning sickness• Treat arrithritis, blood thinning and rduces cholesterol but these effects

remain unconfirmed.• Zingerone may have activity against enterotoxigenic Escherichia

coli in enterotoxin-induced diarrohea•

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LITERATURE REVIEWMERCURY:• Mercury is a chemical element with the symbol Hg and atomic number 80.

It is commonly known as quicksilver and was formerly named hydrargyrum (from Greek "hydr-" water and "argyros" silver).

• It remains in use in scientific research applications and in amalgam material for dental restoration.

• Merbromin (Mercurochrome) is a topical antiseptic used for minor cuts and scrapes.

• Thiomersal - preservative in vaccines• Mercury in the form of one of its common ores, cinnabar, is used in

various traditional medicines, especially in traditional Chinese medicine• Mercury compounds are found in some over-the-counter drugs including

topical antiseptics, stimulant laxatives,diaper-rash ointment, eye drops, and nasal sprays.

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LITERATURE REVIEWSHODITHA GAIRIKA:• Latin name: Red Ochre

Vernacular names: Eng: Ochre, Hindi: Geru, Kan: Kemmanu Botanical description: Gairika is an oxide of Iron (Fe2O3). It is a natural mineral pigment found with other iron-

• titanium oxide minerals in igneous and metamorphic rocks as accessory minerals• Actions and uses:

In Anemia: It enhances the red blood cells count and enriches the hemoglobin level. It

• induces blood circulation and is very useful in patients suffering from anemia.

In Hair Loss: It prevents the premature graying of hair and hair damage like brittle hair,

• split ends and rough, dry hair.

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PREPARATION OF LAGHU SUTHASHEKARA RAS

• REFERENCE: Ayurvediya Aushadi Gunadharma Shastra by Vaidya Panchanana Ghandhara Shastri gune.

• PREPARATION OF DRUG:• MATERIALS: Shodhita Gairika 2 parts, Shunti (ginger) 1 part powder• APPARATUS: Kalvayantra, Laddle, Tray• PROCEDURE: gairika and Shunti powder are mixed together and triturated

with fresh juice of Nagavalli (betel leaves) for 1-2 days. Pills weighing about 150mg are prepared out of the paste and dried in shade and collected in glass container and sealed.

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PHYTOCHEMICAL SCREENING

S.NO TEST FOR RESULT

1. Alkaloids +ve

2. Amino Acids -ve

3. Carbohydrates +ve

4. Flavanoids +ve

5. Glycosides +ve

6. Acidic compounds -ve

7. Cellulose -ve

8. Mucilage -ve

9. Tannins -ve

10. Starch +ve

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MATERIALS & METHODS

EXTRACTION:

150gm of powder was percolated with 500ml ethanol as solvent

Filtered, extract is concentrated by evaporation

Dried

Resulting material was weighed

METHODS

1. Hydrogen peroxide scavenging activity2. Reducing power assay3. Nitric oxide scavenging activity4. DPPH free radical scavenging activity

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MATERIALS

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Borosil soxhlet extractor,Solvent evaporator,Digital balance. All chemicals and solvents were of analytical grade. O-Phenanthroline, Napthylethylene diamine dihydrochloride(NEDD), Hydrogen peroxide, 2, 2- diphenyl-1-picrylhydrazyl radical (DPPH) were purchased from Prince Trading Academy. Sulfanilamide was purchased form Symed Labs Jeedimetla and Trichloroaceticacid was purchased from Chemicals and Chemicals Hyderabad. The other chemicals used were sodium nitroprusside, ferric chloride, potassium ferricyanide, methanol,ethanol,monosodium dihydrogen phosphate, di-sodium hydrogen phosphate, potassium dihydrogen phosphate . Ascorbic acid was used as standard for whole study

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HYDROGEN PEROXIDE RADICAL SCAVENGING ASSAY (8)

1ml Extract / Standard ( 25- 800 ug/ml) + 0.6ml 40mM H2O2

incubate for 10 minutes

Absorbance – 230 nm

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HYDROGEN PEROXIDE RADICAL SCAVENGING ASSAY

CONC(µg/ml) STANDARD PERCENT

SCAVENGING

ETHANOLIC

PERCENT

SCAVENGING

25 0.155 19.322 0.307 25.11

75 0.181 31.497 0.980 9.77

100 0.185 18.92 1.153 19.95

200 1.205 12.08 1.798 48.52

400 2.155 5.68 2.535 93.2

600 3.047 2.90 2.595 75.82

800 3.180 2.81 2.663 0.38

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Graph for hydrogen peroxide scavenging activity Series1: standard , series2: ethanolic extract Graph was plotted between concentration Vs percent scavenging.

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REDUCING POWER ASSAY (9)

1 ml extract+ 2.5 ml phosphate buffer(6.6)

+2.5 ml of potassium ferricyanide

50°C 20 min incbAdd 2.5 ml of trichloroacetic acid

10 mins 3000rpm

Upper layer+2.5 ml of distiiled water+0.5 ml of FeCl3

Absorbance 700 nm

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Reducing Power Activity

CONCENTRATIONS

STANDARD % SCAVENGED

ETHANOLIC EXTRACT

% SCAVENGED

25 0.886 46.62 0.358 78.433

75 1.667 0 0.290 82.53

100 1.943 17.04 0.262 84.21

200 3.233 94.75 0.292 82.40

400 4.609 177.65 0.397 76.08

600 4.954 198.433 0.677 59.21

800 5.278 217.95 0.886 46.62

Control value:1.66

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Graph for reducing power activity:Series1: standard , series2: ethanolic extractGraph was plotted between concentration and percent scavinging.

DPPH ASSAY (7)

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0.5 ml of sample + 3 ml of ethanol + 0.3 ml of DPPH radical

sol in ethanol

Sample gets reduced

Colour change from deep violet to light yellow

100 mins

Absorbance-517nm

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DPPH Scavenging Activity

CONC(µg/ml) STANDARD PERCENT

SCAVENGIN

G

ETHANOLIC PERCENT

SCAVENGIN

G

25 0.210 0.47 2.525 1096.68

75 0.153 27.48 2.444 1056.39

100 0.124 41.23 2.462 1066.82

200 0.120 43.12 2.227 955.45

400 0.117 44.54 2.304 991.94

600 0.18 14.69 1.947 822.74

800 0.128 39.33 1.613 664.45

CONTROL VALUE: 0.211

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Series1: standard , series2: ethanolic extract Graph was plotted between concentration and percent scavenging

0

200

400

600

800

1000

1200

25 75 100 200 400 600 800

Series1

Series2

CONCLUSION

The results obtained in the present study indicate that poly

herbal ethanolic extracts exhibit significant free radical

scavenging and antioxidant activity.

The overall antioxidant activity might be attributed to its

phytochemical constituents.

Potential source of natural antioxidant that could have great

importance as therapeutic agents

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Vishnu Institute of Pharmaceutical Education & Research

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