Phytochemical screening methods

Patil A.S. and Paikrao H.M.
Department of Biotechnology, Sant Gadge Baba, Amravati University, India.


Correspondence: harish.paikrao13@gmail.com

Introduction-
On a recent survey conducted by the World Health Organization (WHO) globally, around 20,000 Medicinal plants are being used profusely either in pharmaceutical industry or in folk medicines. Interestingly, about 1.4% do possess well-established, widely—proven and broadly accepted unequivocally active constituents (Kar, 2007).

Plants known to contain food and mixture of extraordinary chemicals, some of which act beneficially to human and others may have detrimental effects. From many wild plant species, nothing is known as regard their chemical makeup beyond the fact that most contain carbohydrates, fats, protein and chlorophyll (Ngbede et al., 2008). However, the majority of herbal substances used in the manufacturing of medicinals is not or only cursory described in a monograph of pharmacopoeia, especially, if it is only used regionally and/or in traditional medicine. Thus, a researcher has to develop specific methods of phytochemical analysis, with priority on a qualitative and quantitative assay method, to monitor the quality of the product (Harnischfeger, 2005)

Plants synthesize a vast range of organic compounds that are traditionally classified as primary and secondary metabolites although the precise boundaries between the two groups can in some instances be somewhat blurred. Primary metabolites are compounds that have essential roles associated with photosynthesis, respiration, and growth and development. These include phytosterols, acyl lipids, nucleotides, amino acids and organic acids. Other phytochemicals, many of which accumulate in surprisingly high concentrations in some species, are referred to as secondary metabolites. These are structurally diverse and many are distributed among a very limited number of species within the plant kingdom and so can be diagnostic in chemotaxonomic studies. Although ignored for long, their function in plants is now attracting attention as some appear to have a key role in protecting plants from herbivores and microbial infection, as attractants for pollinators and seed-dispersing animals, as allelopathic agents, UV protectants and signal molecules in the formation of nitrogen-fixing root nodules in legumes. (Croteau et al. 2000; Dewick 2002).

Scientist working in the lab

Methods of Phytochemical Screening:

1. Preliminary phytochemical screening
The extract of medicinal plants is analyzed for the presence of Alkaloids, Saponins, Tannins, Cardiac Glycoside, Anthraquinones, Steroid, coumarins, carbohydrates and flavonoids, according to standard methods (Odebiyi and sofowora, 1978; Sofowora, 1982; Williamson et. al., 1996; Banso and Ngbede, 2006; Ngbede et. al., 2008).

For the study of phytochemical analysis, the ethanol extract of the plant leaves, prepared according to standard methods (Sofowora, 1982). Air dry the plant leaves and grind to powder. Transfer the powdered material in to solvent extractor and extract it with 95% ethanol for 72 hours. The extract obtained as a brown gummy solid, stored and used for phytochemical screening.

2. Screening for alkaloids-
Stir 3 grams of extract with ethanol containing 3% tartaric acid. The filtrate shared into 3 beakers and tested for alkaloids as follows-
In to the first beaker, add Hagar’s reagent and in the second beaker, add Mayer’s reagent and in last beaker add Marquins reagent. Precipitations in any of 3 tests indicate the presence of alkaloid (Odebiyi and sofowora, 1978; Banso and Ngbede, 2006)

3. Screening for Saponins – About 0.5 g of the plant extract, shake with water in test tube, frothing which persists on warming considered as preliminary evidence for the presence of Saponins.
Add Few drops of olive oil to 0.5 g of extract and vigorously shake, formation of soluble emulsion in the extract indicates the presence of Saponins (Odebiyi and sofowora, 1978).

4. Screening for tannins- In to 10ml of freshly prepared 10% potassium hydroxide (KOH) in a beaker, add 0.5 g of extract and shake to dissolve. A dirty precipitate is observed which indicates the presence of tannin (Odebiyi and sofowora, 1978; Willimson et al., 1996).

5. Screening for steroids – Dissolve total 100mg of extract in 2 ml of chloroform. Add sulphuric acid carefully to form a lower layer. A reddish brown colour at the interface is indicative of the presence of steroidal ring (Sofowora, 1982)

6. Screening for flavonoids- About 2 g of the powdered leaves, completely detanned with acetone. The residue extracted in warm water after evaporating the acetone in water bath. Filter the mixture while still hot. Cool the filtrate and use.

6.1. Sodium hydroxide test- Add Five ml of 20% sodium hydroxide to equal volume of the detanned water extract. A yellow solution indicates the presence of flavonoids.

7. Screening for Anthraquinones – Take About 0.5 g of the extract in to dry test tube and add 5 ml chloroform and shake for 5 min. filter the extract and the filtrate shake with an equal volume of 100% ammonia solution. A pink violet or red colour in the ammoniacal layer (lower layer) indicates the presence of free anthraquinones. (Bontrager’s test)

8. Screening for cardiac glycoside-
Dissolve Total 100 mg of extract in 1 ml of glacial acetic acid containing one drop of ferric chloride solution, then underlayered with 1 ml of concentrated sulphuric acid. a brown ring obtained at the interface indicates the presence of de-oxysugar characteristics of cardenolides. (Keller Killiani test).

References-
1. Banso A., Ngbde J.E.. Phytochemical screening and invitro antifungal properties of Fagara zanthoxyloides. JFA and E, 2006 ;( 3 and 4): 8-9.

2. Croteau, R., Kutchan, T.M. and Lewis, N.G. (2000) Natural products (secondary metabolites. In B.B. Buchannan, W. Gruissem and R.L. Jones (eds), Biochemistry and Molecular Biology of Plant. American Society of Plant Physiologists, Rockville, MD, pp. 1250–1318.

3. Dewick, P.M. (2002)Medicinal Natural Products: A Biosynthetic Approach, 2nd edn., JohnWiley and Sons, Chichester

4. Harnischfeger G. UNIDO-ICS Training Course on: Development, quality assurance and regulatory aspects of herbal medicinal products, Panama City, Panama, 21 – 25 November 2005.

5. Ngbede J., Yakubu R. A., Nyam D.A. Phytochemical screening for active compounds in Canarium schweinfurthii (Atile) leaves from Jos North, plateau state, Nigeria, Research journal of Biological sciences.2008; 3(9): 1076-1078.

6. Obdeyi A., sofowora E.A. Phytochemical screening of Nigeria medicnal plants. Part II Liyodia. 1978; 403:234-246.

7. Sofowora A. Medicnal plants and traditional medicine in Africa. John Wiley and Sons Ltd. Chichester.England (1982)142-146.

8. Williamson EM., Okpako DG. and Evan F.J. Pharmacological methods in phytotherapy research vol.1. John Wiley and Sons Ltd. Chichester.England, (1996) 9-13.

9. Kar A. Pharmacognosy and PharmacoBiotechnology, 2nd edn., New Age International Publishers. 2007.

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