The study evaluates the phytochemical screening and antioxidant activity of Lactuca virosa leaf extract, focusing on its hydrogen peroxide scavenging efficiency. The results reveal that methanolic extracts exhibit a higher percentage yield and contain various beneficial compounds, including alkaloids and flavonoids, while demonstrating significant antioxidant activity. Overall, the findings support the traditional use of Lactuca virosa in herbal medicine due to its marked in vitro antioxidant effects.

1. 1

2. In Vitro Evaluation and Phytochemical screening of Lactuca virosa
Leaf Extract by H2O2 Free Radical Scavenging Activity.
A.K.R.G. College of Pharmacy Nallajerla, W. G. Dist., AP.
Approved By A.I.C.T.E., New Delhi and Affiliated to JNTU, Kakinada
2
Submitted by
CH.SRI CHAKRADHAR REG. NO188N1R0001
B.BABY SHIVANI REG. NO188N1R0011
G.DIVYA REG. NO188N1R0032
J.DEEKSHITHA REG. NO188N1R0036
K.DURGA BHAVANI REG. NO188N1R0051
L.HARITHA DEVI REG. NO188N1R0060
Under the guidance of
Dr. SRINIVAS NANDYALA,M. Pharm., Ph.D

3. INTRODUCTION
 Traditional medicine is “the knowledge, skills and practices based on the
theories, beliefs and experiences indigenous to different cultures, used in
the maintenance of health and in the prevention, diagnosis, improvement
or treatment of physical and mental illness”
 Over the past 100 years, the development and mass production of
chemically synthesized drugs have revolutionized health care in most
parts of the word.
A.K.R.G College of Pharmacy 3

4.  However, large sections of the population in developing countries still rely on
traditional practitioners and herbal medicines for their primary care.
 According to a survey by the National Center for Complementary and
Alternative Medicine herbal therapy or the usage of natural products other than
vitamins and minerals was the most commonly used alternative medicine.
 In India, herbal medicine is a common practice, and about 960 plant species
are used by the Indian herbal industry, of which 178 are of a high volume,
exceeding 100 metric tons per year.
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5. Taxonomical Classification
 Kingdom : Plantae
 Botanical name : Lactuca virosa
 Kingdom : Plantae
 Order : Asterales
 Family : Asteraceae
 Genus : Lactuca
 Species : L. Virosa
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Plant Name: Lactuca virosa (Wild lettuce)

6. AIM& OBJECTIVES
AIM:-
The aim of the present study is to perform the phytochemical
screening and to analyze antioxidant property of Lactua virosa.
A.K.R.G College of Pharmacy 6
OBJECTIVES:-
 To prepare & authenticate Lactuca virosa.
 To collect and authenticate Lactua virosa plant.
 To prepare and preserve the herbarium of leaf of Lactua virosa.
 To extract active constituents from the dried leaves of Lactua virosa different solvent system.
 To carryout phytochemical screening of Lactua virosa respective extracts.
 To perform the anti oxidant study of methanolic leaf extract of Lactua virosa.

7. METHODOLOGY
AUTHENTICATION : Lactuca virosa collected from Avapadu village, East
Godavari District, Andhra Pradesh. The plant was authentified by Dr. D. V.
Swami, Associate professor of Horticulture, Dr. Y. S. R. Horticulture University
Horticulture College & Research Institute, Venkataramannagudem.
A.K.R.G College of Pharmacy 7
HERBARIUM PREPARATION :
The collected plant specimen was pressed on
news paper & dried. The dried specimen was mounted
on the herbarium sheet.

8. DRYING:
After collecting the leaves, they were left for shade drying on the
floor above the newspapers for 25 days. After that the leaves were dried,
started the extraction process to remove the equilibrium moisture
content.
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9. Preparation of simple methanolic extract
 Extraction for the phytochemical screening was performed by Magnetic stirrer. Dried
leaves were grinded and later sieved through mesh size 120 to get the powder of uniform
size.
 Around weighed into a flask. The extraction was done by using 5g of the powder in 50
ml methanol as a solvent. The temperature was maintained at 45 °C and extraction was
continued for 3 hours.
 Then the methanolic extract was collected and powder was discarded.
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10.  Similarly the extraction procedure was repeated with distilled water and
percentage yield was calculated.
 Then the dried extracts were used for the phytochemical screening and
evaluation studies.
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11. PHYTOCHEMICAL SCREENING
All the extracts were subjected for phytochemical screening to test the presence
of major chemical constituents.
 Alkaloids test: Each of the extracts were dissolved individually in dil. HCl and
filtered. Then the filtrates were separately treated with Mayer’s, Dragondrafts
Reagent & Wagner’s reagent.
 Carbohydrates test: Three extracts were dissolved individually in 5ml distilled
water & filtered. Then the filtrates were separately treated with Molish’s,
Benedict’s & Fehling’s reagent.
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12.  Saponins test: The three extracts were shaken vigorously with 5ml of water
in a test tube for few min following a process known as foam test.
 Glycosides test: Each extract hydrolyzed with dil. HCl then treated with
modified Borntrager's & Legal’s reagent.
 Diterpenes test: It was done by copper acetate test in which 2 drops of copper
acetate solution was added to the aqueous solution of extracts and observed.
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13.  Fats& fixed oils test: It was done by filter paper press test.
 Resins test: It was done by acetone water test in which extracts were treated with 5ml
acetone. Then equal volume of water was added& results were observed.
 Phenols test: It was done by treating with extracts 5ml of FeCl3 solution.
 Tannins test : It was done by gelatin test method in which extracts were treated with
5ml of 1% gelatin solution containing NaCl & results observed.
 Test for Coumarins: It was done by 5ml mixing extract with 3 ml of 2N sodium
hydroxide solution. Observation of no change in colour into dark yellow.
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14. In Vitro Evaluation of Anti-Oxidant Activity by Hydrogen Peroxide
scavenging assay (H2O2) method.
Preparation of pH 7.4 phosphate buffer
2.32 grams of disodium hydrogen phosphate (Na2HPO4), 0.19 grams of
potassium dihydrogen phosphate (KH2PO4) and 8.00 grams of Sodium Chloride
(NaCl) was weighed accurately. These were dissolved in 1000 ml of distilled water
sufficiently.
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15. Preparation of Ascorbic acid stock solution
100mg standard ascorbic acid was weighed accurately and was dissolved
in 100 ml of distilled water. The resulting solution is of concentration
1000µg/ml. From this stock solution the concentrations of 10, 20, 30, 40, 50,
100 and 200µg/ml of ascorbic acid were prepared.
Preparation of hydrogen peroxide solution
1ml of 30% 𝑤/𝑣 hydrogen peroxide is diluted with 100ml of water. Then
the working solution is prepared by 2.266ml of the solution is diluted with
100ml of water to final 20 mM concentration of H2O2.
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16. Preparation of Lactuca virosa stock solution
100mg of Lactuca virosa dried extract powder was weighed
accurately. This was then dissolved in minimum quantity of methanol. The
resulting solution was made up to 100ml with distilled water. The resulting
solution is of concentration 1mg/1ml. From this stock solution the
concentrations of 10, 20, 30, 40, 50, 100 and 200 µg/ml of Lactuca virosa
were prepared.
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17. Estimation of percentage inhibition of hydrogen peroxide.
The test was performed according to the previously described method.
Aliquot of 1 mL of extracts (10, 20, 30, 40, 50, 100 and 200 μg/mL) was
transferred into the test tubes and to that 3mL of phosphate buffer (pH 7.4)
was added and followed by the addition of 6 mL of H2O2 solution (20mM).
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18. The reaction mixture was vortexed and after 10 min of reaction time, its
absorbance was measured at 230 nm.
Ascorbic acid was used as the positive control. The ability of the extracts
to scavenge the H2O2 was calculated using the following equation:
Percentage Inhibition % =
(Abs sample – Abs control)
Abs control
X 100
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19. Percentage yield of Lactuca virosa extracts
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S.No Extract Percentageyield
1 Water 11.60%
2 Methanol 18.2%
11.60%
18.20%
Percentage yield
Water Methanol
RESULTS

20. S.No Phytochemical test Result
1 Alkaloids test
+ve
2 Carbohydrates test
+ve
3 Saponins test
+ve
4 Glycoside test +ve
5 Cardiac glycosides test +ve
6 Oil test +ve
Phytochemical screening methanolic extract of Lactuca virosa extracts
A.K.R.G College of Pharmacy 20
S.No Phytochemical test Result
7 Resin test
-ve
8 Phenol test
+ve
9 Tannins
+ve
10 Flavonoids test +ve
11 Proteins and Amino acid
test
+ve
12 Coumarins test -ve

21. A.K.R.G College of Pharmacy 21
Phytochemical screening aqueous extract of Lactuca virosa extracts
S No Phytochemical test Result
1 Alkaloids test
+ve
2 Carbohydrates test
+ve
3 Saponins test
+ve
4 Glycoside test +ve
5 Cardiac glycosides test +ve
6 Oil test -ve
S No Phytochemical test Result
7 Resin test
-ve
8 Phenol test
+ve
9 Tannins
+ve
10 Flavonoids test +ve
11 Proteins and Amino acid
test
-ve
12 Coumarins test -ve

22. A.K.R.G College of Pharmacy 22
AntiHydrogen peroxide scavenging Test
Percentage Inhibition of H2O2 free radical by extract/ascorbic acid at 230 nm.
S.No Concentrations(µg/ml)
% inhibitionof H2O2 freeradicalbyextract/ascorbicacidat230nm
Methanolicextract
(Mean± S.D)
Ascorbicacid
(Mean± S.D)
1 10 22.16± 0.05 30.76± 0.67
2 20 32.32± 0.24 53.84± 1.01
3 30 37.16± 1.05 69.23± 0.85
4 40 42.05± 0.13 96.23± 0.71
5 50 48.89± 0.95 96.56± 0.23
6 100 81.63± 0.98 97.45± 0.67
7 200 88.27± 1.02 97.58± 1.13

23. A.K.R.G College of Pharmacy 23
22.16
32.32
37.16
42.05
48.89
81.63
88.27
30.76
53.84
69.23
96.23 96.56 97.45 97.58
0
20
40
60
80
100
120
10 (µg/ml) 20 (µg/ml) 30 (µg/ml) 40 (µg/ml) 50 (µg/ml) 100 (µg/ml) 200 (µg/ml)
%Inhibition
Concentrations (µg/ml)
Methanolic extract Ascorbic acid
Percentage Inhibition of H2O2 free radical by extract/ascorbic acid at 230 nm.
22.16
32.32
37.16
42.05
48.89
81.63
88.27
30.76
53.84
69.23
96.23 96.56 97.45 97.58
0
20
40
60
80
100
120
10 (µg/ml) 20 (µg/ml) 30 (µg/ml) 40 (µg/ml) 50 (µg/ml) 100 (µg/ml) 200 (µg/ml)
%Inhibition
Concentrations (µg/ml)
Methanolic extract Ascorbic acid

24. DISCUSSION
 This study sought to evaluate phytochemical screening and anti oxidant
activity of Lactuca virosa through in vitro method. Inhibition of H2O2 free
radical scavenging method was selected to evaluate the anti oxidant
activity.
 The results of phytochemical screening studies showed that the presence
of alkaloids, carbohydrates, saponins, glycosides, oils, phenols, tannins,
flavonoids, proteins and amino acids.
 In this study we performed the phytochemical screening of successive
water and methanol extracts obtained from extraction.
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25. From the graph IC50 values were determined by using software Graph Pad
Prism version 6.01. The concentration of Ascorbic acid and methanolic
extracts of Lactuca virosa leaves needed for 50% inhibition was found to be
18.86 µg/ml and 52.08 µg/ml respectively.
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26. SUMMARY AND CONCLUSION
 The following conclusions were drawn from the experimental results.
 The methanolic extract showed high percentage yield when compared with aqueous extract.
 The extract of Lactuca virosa leaves contained alkaloids, carbohydrates, saponins, glycosides,
oils, phenols, tannins, flavonoids, proteins and amino acids.
 From the results of H2O2 inhibition activity it has been concluded that methanolic extract of
Lactuca virosa leaves showed inhibitory activity.
 From this study it can be concluded that Lactuca virosa leaves possessed marked in vitro
anti-oxidant effect.
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31. Knowing is not enough
we must APPLY !
Willing is not enough
We must DO!
A.K.R.G College of Pharmacy 31