The document summarizes the phytochemical screening and antioxidant activity of a poly herbal formulation. It begins with an introduction to antioxidants and their importance. It then describes the objective to investigate the pharmacological screening of ethanol and aqueous extracts of the polyherbal drug to justify its use as an antioxidant. The document outlines the various materials, extraction methods, and assays used to evaluate the antioxidant activity including hydrogen peroxide scavenging, reducing power, nitric oxide scavenging, and DPPH free radical scavenging activities. The results of these assays on the ethanolic extract showed significant free radical scavenging and antioxidant activity. The conclusion states that the polyherbal formulation is a potential source of natural antioxidants that could

1. PHYTOCHEMICAL SCREENINGPHYTOCHEMICAL SCREENING
AND ANTIOXIDANT ACTIVITY OFAND ANTIOXIDANT ACTIVITY OF
POLY HERBAL FORMULATIONPOLY HERBAL FORMULATION
1
Vishnu Institute of Pharmaceutical
Education & Research
C. Anusha Reddy
Ch. Divya
N. Sravanthi Reddy
G. Sujana
N. SudhakarGuide:
Mr. K. Ramanajaneyulu
M.Pharm, (PhD)
Dept: Pharmaceutical Chemistry

2. 2
CONTENTS
• Introduction
a. Need for investigation
b. Objectives of the work
c. Antioxidant systems
• Phytochemical screening
•Extraction
• Materials and methods
• Methods of screening
• Results and discussion
• Conclusion

3. 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…
3

4. 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.
4

5. 5
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.

6. OBJECTIVE OF WORK
The 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.
6

7. PLAN OF WORK
7
PRODUCT
EXTRACT
PHYTOCHEMICAL SCREENING
ANTIOXIDANT
ACTIVITY

8. 8

9. ANTIOXIDANT ACTIVITY
9

10. MECHANISM OF ANTIOXIDANT
10

11. MODES OF ACTION OF ANTIOXIDANT
11
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

12. 12
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
Water soluble vitamin

13. SELECTED FORMULATION
13
S.No NAME OF THE DRUG LATIN NAME
1. Red oxide Shodita gairika
2. Shunti Zingeber
3. Mercury

14. LITERATURE REVIEW
SHUNTI:
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
•
14

15. LITERATURE REVIEW
MERCURY:
• 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.
15

16. LITERATURE REVIEW
SHODITHA 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.
16

17. 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.
17

18. 18
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

19. 19
MATERIALS & METHODS
EXTRACTION:
150gm of powder was percolated with 500ml ethanol as
solvent
Filtered, extract is concentrated by evaporation
Dried
Resulting material was weighed

20. METHODS
1. Hydrogen peroxide scavenging activity
2. Reducing power assay
3. Nitric oxide scavenging activity
4. DPPH free radical scavenging activity
20

21. MATERIALS
21
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

22. 22
HYDROGEN PEROXIDE RADICAL
SCAVENGING ASSAY (8)
1ml Extract / Standard ( 25- 800 ug/ml) + 0.6ml 40mM H2O2
incubate for 10 minutes
Absorbance – 230 nm

23. 23
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

24. 24
Graph for hydrogen peroxide scavenging activity
Series1: standard , series2: ethanolic extract
Graph was plotted between concentration Vs percent scavenging.

25. 25
REDUCING POWER ASSAY(9)
1 ml extract+ 2.5 ml phosphate buffer(6.6)
+2.5 ml of potassium ferricyanide
50°C 20 min incb
Add 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

26. 26
Reducing Power Activity
CONCENTRA
TIONS
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

27. 27
Graph for reducing power activity:
Series1: standard , series2: ethanolic extract
Graph was plotted between concentration and percent scavinging.

28. DPPH ASSAY (7)
28
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

29. 29
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

30. 30
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

31. 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
31

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Vishnu Institute of Pharmaceutical
Education & Research

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