lactuca virosa leaves extract antioxidant activity.pptx

lactuca virosa leaves extract antioxidant activity

1. WELCOME A.K.R.G College of Pharmacy 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. A.K.R.G College of Pharmacy 4

5. Taxonomical Classification Kingdom : Plantae Botanical name : Lactuca virosa Kingdom : Plantae Order : Asterales Family : Asteraceae Genus : Lactuca Species : L. Virosa A.K.R.G College of Pharmacy 5 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. A.K.R.G College of Pharmacy 8

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. A.K.R.G College of Pharmacy 9

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. A.K.R.G College of Pharmacy 10

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: T hree extracts were dissolved individually in 5ml distilled water & filtered. Then the filtrates were separately treated with Molish’s, Benedict’s & Fehling’s reagent. A.K.R.G College of Pharmacy 11

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. A.K.R.G College of Pharmacy 12

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. A.K.R.G College of Pharmacy 13

14. In Vitro Evaluation of Anti- Oxidant Activity by Hydrogen Peroxide scavenging assay (H 2 O 2 ) 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. A.K.R.G College of Pharmacy 14

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. A.K.R.G College of Pharmacy 15

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. A.K.R.G College of Pharmacy 16

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). A.K.R.G College of Pharmacy 17

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: X 100   A.K.R.G College of Pharmacy 18

19. Percentage yield of Lactuca virosa extracts A.K.R.G College of Pharmacy 19 S. No Extract Percentage yield 1 Water 11.60% 2 Methanol 18.2% 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 Anti Hydrogen peroxide scavenging Test Percentage Inhibition of H 2 O 2 free radical by extract/ascorbic acid at 230 nm. S.No Concentrations (µg/ml) % inhibition of H 2 O 2 free radical by extract/ascorbic acid at 230nm Methanolic extract (Mean ± S.D) Ascorbic acid (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 Percentage Inhibition of H 2 O 2 free radical by extract/ascorbic acid at 230 nm.

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. A.K.R.G College of Pharmacy 24

25. From the graph IC 50 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. A.K.R.G College of Pharmacy 25

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. A.K.R.G College of Pharmacy 26

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30. A.K.R.G College of Pharmacy 30 Anna Stojakowska , Janusz Malarz , Agnieszka Szewczyk , Wanda Kisiel . Caffeic acid derivatives from a hairy root culture of Lactuca virosa. Acta Physiol Plant; 2012, 34: 291–298 Sima Besharat   Mahsa Besharat  and Ali Jabbari. Wild lettuce (Lactuca virosa) toxicity. BMJ Case Reports; 2009: bcr06.2008.0134. A. Wesołowska , A. Nikiforuk, K. Michalska , W. Kisiel , E. Chojnacka -Wojcik. Analgesic and sedative activities of lactucin and some lactucin -like guaianolides in mice. Journal of Ethnopharmacology; 2006, 107: 254–258 Rekha Singhal. Medicinal Plants and Primary Health Care: The Role of Gender Journal of Health Management 2005; 7: 277-278 .

31. Knowing is not enough we must APPLY ! Willing is not enough We must DO ! A.K.R.G College of Pharmacy 31