TanninsTannins and tannin-like substances are widespread in nature and are probably present in all plant materials. Those are polyphenolic compounds divided into two main groups - hydrolysable and condensed.Hydrolysable tannins contain a polyhydric alcohol usually, if not always, glucose esterified with gallic acid or with hexahydroxydiphenic acid. Condensed tannins are mostly flavonols and are probably polymers of flavan 3-ol (catechin) and these cannot be hydrolyzed to simple components. Among the cereals, sorghum has been found to contain higher amounts of polyphenols. Even though high polyphenol seeds are immune to attack by birds and diseases, they display impaired nutritional quality, lower digestibility and reduction of food consumption. The tannins are estimated by the following two methods: (i) Folin-Denis Method: This is based on the non-stoichiometric oxidation of the molecules containing a phenolic hydroxyl group. (ii) Vanillin Hydrochloride Method: Vanillin method is specific for dihydroxy phenols and is particularly sensitive for meta-substituted, di and tri hydroxybenzene containing molecules. (i) Folin-Denis Method Principle Tannin-like compounds reduce phosphotungstomolybdic acid in alkaline solution to produce a highly colored blue solution, the intensity of which is proportional to the amount of tannins. The intensity is measured in a spectrophotometer at 700nm. Materials» Folin-Denis ReagentDissolve 100g sodium tungstate and 20g phosphomolybdic acid in 750mL distilled water in a suitable flask and add 50mL phosphoric acid. Reflux the mixture for 2h and make up to one liter with water. Protect the reagent from exposure to light. » Sodium Carbonate Solution Dissolve 350g sodium carbonate in one liter of water at 70-80°C. Filter through glasswool after allowing it to stand overnight. » Standard Tannic Acid Solution Dissolve 100mg tannic acid in 100mL of distilled water. » Working Standard Solution Dilute 5mL of the stock solution to 100mL with distilled water. One mL contains 50mg tannic acid. Procedure
CalculationCalculate the tannin content of the samples as tannic acid equivalents from the standard graph.References 1. Schanderl, S H (1970) In: Method in Food Analysis Academic Press New York p 709 (ii) Vanillin Hydrochloride Method Principle The vanillin reagent will react with any phenol that has an unsubstituted resorcinol or phloroglucinol nucleus and forms a colored substituted product which is measured at 500nm. Materials» Vanillin Hydrochloride ReagentMix equal volumes of 8% hydrochloric acid in methanol and 4% vanillin in methanol. The solutions must be mixed just before use, and avoid using even if it is slightly colored. » Catechin-Stock Standard Solution Prepare a standard solution containing 1mg catechin/mL methanol. » Working Standard: Dilute the above stock solution ten times. 10mL to 100mL (100mg/mL). » Preparation of the Extract Extract 1g of ground seed in 50mL methanol. Mix occasionally by swirling. After 20-28h, centrifuge and collect the supernatant. Procedure
CalculationFrom the standard graph, calculate the amount of catechin, i.e., tannin in the sample as per the absorbance values and express the results as catechin equivalents.References 1. Robert, EB (1971) Agro J 63 511. |
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