Phytochemistry

Phytochemistry

1. T Phytochemical Screening of plants And their Biological Activity Dr . Gurumeet C Wadhawa Karmaveer Bhaurao Patil College,Vashi

2. Medicinal plants Me d ici n al pla n ts c on s t itute an effective so u r ce o f both traditional and modern medicines Herbal medicine has been shown to have genuine utility A b o u t 80% of ru r al p o p ulation d e p e n d s o n it as p ri m a r y health care. [WHO, (2005)]

3. Medicinal plants are the richest bio-resource drugs of traditional systems of medicine modern medicines nutraceuticals food supplements folk medicines pharmaceutical intermediates chemical entities for synthetic drugs

4. Natural bioactive compounds found in different parts of plant (fruit, flower, stem, leaf, root) Provide definite physiological action on the human body Bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids W i del y u s e d i n t h e hu m a n t h e r ap y , vet er i n ar y , agricult ure, scientific research and countless other areas Have inhibitory effects on all types of microorganisms in vitro Phytochemicals

5. Ext r action ……… is the separation of medicinally active portions of plant tissues using selective solvents through standard procedures The basic parameters influencing the quality of an extract Plant part used as starting material Solvent used for extraction Extraction procedure

6. Choice of solvents Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure Property of a good solvent in plant extraction Low toxicity Ease of evaporation at low heat Promotion of rapid physiologic absorption of the extract Preservative action

7. The factors affecting the choice of solvent Quantity of phytochemicals to be extracted Rate of extraction Diversity of different inhibitory compounds extracted Ease of subsequent handling of the extracts Toxicity of the solvent in the bioassay process Potential health hazard of the extractants

8. Solvents used for active component extraction Water A n t hoc y ani n s Starches Tannins Saponins Terpenoids Polypeptides Lectins Ethanol Tannins Polyphenols P o l y a c et y l e nes Flavonols Terpenoids Sterols Alkaloids Methanol Anthocyanins Terpenoids Saponins Tannins Xan t hox y l l in e s Totarol Quassinoids Lactones Flavones Phenones Polyphenols Chloroform Terpenoids Flavonoids Ether Alkaloids T e rpeno i d s Coumarins Fatty acids Acet o n e Phenol Flavonols

9. General techniques of medicinal plant extraction Plant tissue homogenization Maceration Infusion Percolation Digestion Decoction Soxhlet extraction (Hot continuous extraction) Sonication (Ultrasound extraction)

10. Plant tissue homogenization

11. M a ce r ati o n Who le //coarsely powdered crude drug is plalaced in a stoppered containiner with the solv ent Allow to stand @ room Temperature for a perioiod off at least 3 days wit h aggititatioion unt i l the solu ble mattter gets disisssoolvlved The mix ture then is strainined, ,the marc (the damp solilid m aterial )is presssseed The companied liquid are clarified by filtration or decantation after standining

12. I n f u s i o n

13. D i g e sti o n A f o r m of m ac e r a ti o n in w h i c h gentle he a t is u sed during the process of extraction Us e d w hen m ode r a t e ly e l eva t e d t e m p e r a t u r e is not objectionable T h e s o lvent e f f i c i enc y o f the m ens t r u u m is the re b y increased Microwave digestion system

14. Decoction Suitable for extracting  water-soluble, heat-stable constituents Typically used in preparation of Ayurvedic extracts

15. Percolation • • Used most frequently to extract active ingredients in the preparation of fluid extracts The solid ingredients are moistened with an appropriate amount of the specified menstruum Allowed to stand for approximately 4 hours in a well closed container, After stand time, the mass is packed & the top of the percolator is closed The mixture is allowed to macerate in the closed percolator for 24 h

16. • Additional menstruum is added as required, until the percolate measures about three-quarters of the required volume of the finished product • The m a r c is then p r e s s e d and the exp r e ss e d l i quid i s added to the percolate • Sufficient menstruum is added to produce the required volume • The mixed liquid is clarified by filtration or by standing followed by decanting

17. Soxhlet Extraction (Hot Continuous Extraction)

18. Sonication (Ultrasound Extraction) • Involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz Increases the permeability of cell walls & produces cavitation Disadvantage D eleter i o u s e ff ect of ultrasound energy m edicinal plants thr o u gh f o r m a t ion o f ( > 20 k H z) on the a c t ive c o nst i tue n t s of free r a d ic a ls and c o n s equently undesirable changes in the drug molecules

19. Effect of extracted plant phytochemicals depends on The nature & origin of the plant material Degree of processing Moisture content Particle size

20. Variation in extraction methods Length of the extraction period Solvent used pH of the solvent Temperature Particle size of the plant tissues Solvent-to-sample ratio

21. Phytochemicals have two categories: Primary & Secondary constituents. The phytochemical analysis  Commercially value. Great interest in pharmaceutical companies for the production of the new drugs for curing of various diseases.

22. Qualitative Quantitative Steroids, Reducing sugars, Triterpenoids, Sugars, Alkaloids, Phenolic compounds, Flavonoids, Saponins, Tannins, Anthroquinones, Amino acids. Determination of total alkaloids, Total flavonoids, Total phenolics, Total saponins, Total tannins, Total glycosides.

23. Standard procedures Sofowara (1993). Trease and Evans (1989). Harborne (1973).

24. Qualitative analysis methods

25. Detection of alkaloids The individual extract is dissolved in dilute hydrochloric acid and filter. The filtrate was further tested with following reagents for the presence of alkaloids.

26. Filtrate was treated with potassium bismuth iodide solution (Dragendroff’s reagent). Formation of orange red precipitate indicated the presence of alkaloids. Dragendroff’s Test: Hager’s Test: Filtrate was treated with saturated aqueous solution of picric acid (Hager’s reagent). Presence of alkaloids were confirmed by the formation of yellow coloured precipitate. Mayer’s Test: Filtrate was treated with potassium mercuric iodide solution (Mayer’s reagent). Formation of a whitish yellow or cream coloured precipitate indicated the presence of alkaloids.

27. Detection of carbohydrates Dissolve 2g extract in 5 ml distilled water & filter it. The filtrates were used to test for the presence of c a r b o h y d rate s . Molisch’s Test: Filtrate was treated with 2 drops of alcoholic α- naphthol solution in a test tube, shaken Add conc. sulphuric acid from the side of the test tube. Development of a violet ring @ the junction of two liquid confirmed the presence of carbohydrates

28. Detection of reducing sugars Benedict’s test: Filtrate was treated with Benedict’s reagent & boil in a thermostatic water bath for 5 minutes. Formation of an orange red precipitate indicated the presence of reducing sugars. Fehling’s Test Filtrate was acidified with dil. Hydrochloric acid, neutralized with alkali & heated with Fehling’s A & B solutions. Formation of red precipitate indicated the presence of reducing sugars.

29. Detection of saponins Froth Test: Extract was diluted with distilled water to 20 ml & shaken in a graduated test tube for 15 minutes. Formation of 1 cm layer of foam indicated the presence of saponins. Foam Test: Small quantity of the extract was shaken with 2 ml of water. Persistence of foam produced for ten minutes indicated the presence of saponins.

30. Detection of phytosterols Small quantity of extract dissolved in 5 ml of chloroform Salkowski’s Test: On adding a few drops of conc. Sulphuric acid. Allow the solution to stand Formation of brown ring indicated the presence of phytosterols LibermannBurchard’s test: The chloroform extracted solution was treated with few drops of acetic anhydride. Boil & cool. Add conc. sulphuric acid. Formation of a bluish green colour solution confirmed the presence of phytosterols.

31. Detection of phenolic compounds: Ferric Chloride Test: Treat the extract with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicated the presence of phenols. Lead Acetate Test: Treat the extract with 3ml of 10% lead acetate solution. A bulky white precipitate indicated the presence of phenolic compounds.

32. Detection of tannins: Take 0.5 g of the dried powdered plant Boil 0.5g sample in 20 ml of water in a test tube. Filter the above mixture Add few drops of 0.1% ferric chloride. Development of a brownish green or a blue-black colouration indicated the presence of tannins

33. Detection of flavonoids: Treat the extract with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on further addition of dilute acid, indicated the presence of flavonoids. Alkaline Reagent Test Treat the extract with few drops of lead acetate solution. Formation of yellow precipitate indicated the presence of flavonoids. Lead acetate Test: Add a few drops of ferric chloride solution to the extract solution. Development of intense green colour indicates the presence of flavonoids. Ferric chloride Test:

34. Detection of proteins and amino acids: Millon’s Test: Treat the test solution with few drops of Millon’s reagents. when warmed , a white precipitate is formed which changes to a brick red or disappears: indicates the presence of proteins & A.A. Biuret Test: Treat the test solution with few drops of 2% of copper sulphate solution Add 1ml of ethanol followed by excess of potassium hydroxide pellets formation of pink colour in the extract layer indicates the presence of Pr. Ninhydrin Test: Add Ninhydrin reagent to the test solution & boiled for few minutes. Formation of blue colour indicated the presence of amino acids.

35. Detection of terpenoids: Salkowski test: Mix 2 ml of chloroform to extract solution carefully added conc. Sulphuric acid (3 ml) to form a layer. A reddish brown colouration of the interface indicated the presence of terpenoids.

36. Detection of cardiac glycosides Keller-Killani test Add 1ml of conc. sulphuric acid, Appearance of brown ring @ the interface indicate the deoxysugar characteristic of cardenolides Appearance of a violet ring below the brown ring & a greenish ring in the acetic acid layer confirmed the results. Treat the extract with 2 ml of glacial acetic acid containing one drop of ferric chloride solution.

37. Test for fixed oils and fats: Spot Test: Place small quantity of the extract in between two filter papers. Oil stain produced with any extract showed the presence of fixed oils and fats in the extracts. Saponification test: Add few drops of 0.5N alcoholic potassium hydroxide extract with few drops of phenolphthalein solution. Heat on a water bath for 1-2 hours. Formation of soap indicated the presence of fixed oils and fats in the extracts.

38. Test for gums and mucilages Dilute small quantity of the ethanolic extract with water Add ruthenium red solution. A pink colour production showed the presence of gums and mucilages.

39. Quantitative determination of phytochemicals Total phenols determination: Hagerman A., Muller I., Makkar H. (2000). Total alkaloid determination: Harborne.J. (1973). Total flavonoids determination: Kumaran A, Karunakaran R. (2006). Total tannins determination: Van-Burden T, Robinson W. (1981). Total saponins determination: Obdoni B, Ochuko P. (2001) .

40. Determination of total phenolic compounds(Hagerman A, Muller I, Makkar H, 2000) Weigh accurately 100 mg of the extract of the sample & dissolved in 100 ml of triple distilled water (TDW). Transfer 1 ml of this solution to a test tube & add 0.5 ml 2N of the Folin-Ciocalteu reagent. Add 1.5 ml 20% of Na2CO3 solution & make volume up to 8 ml with TDW followed by vigorous shaking. Finally allow to stand for 2 hours. Take the absorbance at 765 nm. (Spectroscopic determination). Data use : To estimate the total phenolic content using a standard calibration curve obtained from various diluted concentrations of gallic acid.

41. Determination of total alkaloids (Harborne J, 1973) Weigh 5 g of the sample & add 5g sample into a 250 ml beaker. Add 200 ml of 10% acetic acid in ethanol & cover the beaker with aluminum foil Allow to stand for 4 hour. Filter the extract & concentrated on a water bath to one-quarter of the original volume. Add concentrated ammonium hydroxide drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle Collect the precipitate & wash with dilute ammonium hydroxide and then filter. The residue is the alkaloid, which was dried and weighed

42. Determination of total flavonoids (Kumaran A, Karunakaran R. 2006) The method is based on the formation of the flavonoids - aluminium complex which has an absorptivity maximum at 415nm. Mix 100μl of the plant extracts in methanol (10 mg/ml) with 100 μl of 20 % aluminum trichloride in methanol Add a drop of acetic acid, and then diluted with methanol to 5ml. After 40 minutes read the absorption @ 415 nm. Blank samples were prepared from 100 ml of plant extracts and a drop of acetic acid, and then diluted to 5ml with methanol. The absorption of standard rutin solution (0.5 mg/ml) in methanol was measured under the same conditions. All determinations were carried out in triplicates.

43. Determination of total tannins (Van-Burden T, Robinson W. 1981). Weigh 500 mg of the sample & transfer to a 50 ml plastic bottle. Add 50 ml of distilled water & shaken for 1 hour in a mechanical shaker. Filter the above mixture into a 50 ml volumetric flask & make up to the mark. Pipette out 5 ml of the filtrate into a test tube & mix with 2 ml of 0.1 M FeCl3 in 0.I N HCl & 0.008 M potassium ferrocyanide. Measure the absorbance @ 120 nm within 10 min.

44. Determination of total saponins ( Obdoni B, Ochuko P. 2001 ) Ground the samples & 20 g of each were put into a conical flask Add 100 cm3 of 20% aqueous ethanol. Heat the samples over a hot water bath for 4 hour with continuous stirring @ about 55 ° C. Filter the mixture. Re-extract the residue with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over water bath @ about 90 ° C. The concentrate was transferred into a 250 ml separatory funnel & add 20 ml of diethyl ether; shake vigorously.

45. The aqueous layer was recovered while the ether layer was discarded. Repeat the purification process. Add 60 ml of n-butanol & wash twice with 10 ml of 5% aqueous sodium chloride. Heat the remaining solution in a water bath. After evaporation the samples were dried in the oven to a constant weight. The saponins content was calculated using standard formulae .

46. DPPH scavenging test Antioxidant activity measured in terms of free radicle scavenging Quantitative measurement of radical scavenging properties was carried out in a universal bottle. The reaction mixture contained sample under study 50 μL taken or 80% MeOH as blank and 5 mL of a 0.004% (w/v) solution of DPPH in methanol during the antioxidant activity. Different known antioxidants, vitamin E, and butylated hydroxytoluene (BHT, Sigma) were used for comparison or as a positive control. Discoloration was measured at 517 nm after incubation for 30 min. Measurements was taken at least in triplicate. DPPH or antioxidant activity concentration was measured using the following formula : DPPH scavenging effect (%) or antioxidant activity radical’s = Ao – A1 / Ao X100 Where Ao was the absorbance of the control and A1 was Absorbance in presence of sample

47. Alpha -Amylase inhibition Alpha -Amylase inhibition . The Alpha -amylase inhibitory activity was determined using a different methods but one of the newmodified assayof that described in the Worthington Enzyme Manual is good method reported. A total of 500 µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl ) containing 0.5 mg/ mL of Alpha - amylase were pre-incubated at 25 o C for 10 min. After the preincubation , 500 μL of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl ) was added to each tube at timed intervals with constant shaking. The reaction was stopped using 1.0 mL of dinitrosalicylic (DNS) acid color reagent. The test tubes were incubated in a boiling water bath for 5 min and then cooled to room temperature. The Phoboo et al. was reported In Vitro Assays of Anti-diabetic and reaction mixture was diluted by adding 5 to15 mL of distilled water, and the absorbance was measured at 540 nm using the previously described UV-Visible light spectrophotometer. The absorbance readings were compared with the controls that contained buffer instead of sample extract .

48. Alpha -Glycosidase Inhibitory activity In-vitro method Alpha -Glycosidase Inhibitory activity A better method to check activity of compounds for diabetes is Alpha - Glucosidase . Inhibitory activity of Alpha - glycosidase was measured following the protocol described various by McCue et al. for the development of new protocol during recent years The Alpha - glycosidase was usually assayed using 50 µL of sample extracts and 100 µL of 0.1 M phosphate buffer (pH 6.9) containing ,Alpha -glycosidase solution (1 mL ), from various source which was then incubated in 96-well plates at 25°C for 10 min shaking After the pre-incubation period, 50 µL of 5 mM p -nitro phenyl-µ- d - glucopyranoside solution in0.1 M phosphate buffer (pH 6.9) was added to each well at timed intervals. The reaction mixtures were incubated at 25°C for 5 min. Before and after incubation, absorbance readings of the samples were recorded at 405 nm using a micro plate reader and compared with a control that had 50 µ Lof buffer solutions in place of the extract.

49. Assessment of invitro anti-inflammatory activity Inhibition of albumin denaturation The anti-inflammatory activity of Enicostemma axillare was studied by using inhibition of albumin denaturation technique which was studied according to Mizushima et al and Sakat et al followed with minor modifications.The reaction mixture was consists of test extracts and 1% aqueous solution of bovine albumin fraction, pH of the reaction mixture was adjusted using small amount of 1N HCl. The sample extracts were incubated at 37 ºC for 20 min and then heated to 51 º C for 20 min, after cooling the samples the turbidity was measured at 660nm.( UVVisible Spectrophotometer Model 371, Elico India Ltd) . The experiment was performed in triplicate. The Percentage inhibition of protein denaturation was calculated as follows: Percentage inhibition = (Abs Control –Abs Sample) X 100/ Abs control

50. Assay for in vitro cytotoxicity study In vitro cytotoxicity assay of plant extract was performed by using Vero (African green Monkey Kidney), A-549 (Human lung) and Dalton’s Lymphoma Ascites ( Tumour cells) cell line. Briefly, 1×106 cells were suspended in 0.1 ml of phosphate buffered saline (PBS, 0.2 M, pH 7.4) and mixed with 100μl of various concentration (25,50,100,150,200 and 300μg/ml) of plant and standard drug 5-flurouracil. The final volume was adjusted 1 ml with PBS and was incubated at 37°C for 3 h after the incubation was over, the viability of the cells was determined using trypan blue (0.4% in normal saline) method and the percentage of cytotoxicity was determined by calculating percentage inhibition and IC50 value

51. References III. H ERIN SHEEBA D. GRACELIN A. JOHN DE BRITTO & P. BENJAMIN JEYA RATHNA KUMAR.2013. QUALITATIVE AND QUANTITATIVE ANALYSIS OF PHYTOCHEMICALS IN FIVE PTERIS SPECIES. Int J Pharm Pharm Sci, Vol 5(1): 105-107 . Adarsh Krishna T.P., Ajeesh Krishna T.P., Sanyo Raj V.N., Juliet S., Nair S.N., Ravindran R. and Sujith S. 2013.Evaluation of phytochemical constituents and proximate contents of the ethanolic leaf extract of Tetrastigma leucostaphylum (Dennst.) Alstone (Vitaceae) found in Western Ghats of Kerala, India Res. J. Pharmaceutical Sci. 2(10): 1-6. Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur. 2011. Phytochemical screening and Extraction: A Review Internationale Pharmaceutica Sciencia 1(1): 98-106. IV. Google images.