Phytochemical screening

Qualitative & Quantitative phytochemical screening of herbs.

1. Phytochemical Screening of plants Dr. SINDHU K. M . V. Sc . SCHOLAR, DEPT . OF VPT, COVAS, Pookode.

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

3. Qualitative Quantitative S teroids, R educing sugars, T riterpenoids, S ugars , A lkaloids, P henolic compounds, F lavonoids, S aponins, T annins, Anthroquinones, A mino acids . Determination of total a lkaloids, T otal f lavonoids , Total phenolics, Total saponins, T otal tannins, Total glycosides.

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

5. Qualitative analysis methods

6. 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.

7. Detection of carbohydrates

8. Detection of reducing sugars

9. Detection of saponins

10. Detection of phytosterols

11. Detection of phenolic compounds:

12. Detection of tannins:

13. Detection of flavonoids:

14. Detection of proteins and amino acids:

15. Detection of terpenoids:

16. Detection of cardiac glycosides

17. Test for fixed oils and fats:

18. Test for gums and mucilages

19. 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) .

20. 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.

21. Determination of total alkaloids (Harborne J, 1973) Weigh 5 g of the sample & add 5g sample into a 250 ml beaker. A dd 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. A dd 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

22. 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.

23. Determination of total tannins (Van-Burden T, Robinson W . 1981). W eigh 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.

24. 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 t he samples over a hot water bath for 4 hour with continuous stirring @ about 55°C. Filter t he 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.

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

26. Qualitative Quantitative steroids, reducing sugars, triterpenoids, sugars , alkaloids, phenolic compounds, flavonoids, saponins, tannins, Anthroquinones, amino acids . Determination of total a lkaloids, T otal f lavonoids , Total phenolics, Total saponins, T otal tannins, Total glycosides.

27. Steps Involved in the Extraction of Medicinal Plants 1. Size reduction 2. Extraction 3. Filtration 4. Concentration 5. Drying 6. Packing 7. Storage

28. Size reduction

29. Different extraction methods maceration, infusion , percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, phytonic extraction (with hydrofluorocarbon solvents).

30. Different methods of drying Natural drying  Sun drying  Shade drying II. Artificial drying  Tray drying  Vacuum dryers  Spray dryers

31. GARBLING / DRESSING Process of removal of sand & foreign organic part from same plant. Eg : removal of excessive stem. removal of excessive stalk in case of cloves. careful removal of rhizomes from roots & rootlets. removal of iron piece using magnet in caster beans. bark removal from gum acacia.

32. Packing Pressed & balded  leaf drugs like senna , vinca . Amber color bottle  cod liver oil ( protect from direct sunlight) Closed container  asfoetida (prevent loss of volatile oil) Kerosine tins  colophony, balsam of tolu . Goat skin  aloe preparations. Packing in big masses  colophony (control auto oxidation). Packing one side the other  cinnamon bark /quill (prevent volatilization of oils). Simple packing  crude drugs from root & seeds.

33. Storage & preservation of crude drugs Sterile, Closed containers, Moisture free, air tight. Physical & chemical damages Insect & mould attacks

34. References HERIN 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 . Google images.