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  Section: Plant Lab Protocols
 
 
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Methodology for Phenolics

 
     
 
Tannins
 
Tannins 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 Reagent
Dissolve 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
1.
Extraction of Tannin: Weigh 0.5g of the powdered material and transfer to a 250mL conical flask. Add 75mL water. Heat the flask gently and boil for 30 min. Centrifuge at 2,000rpm for 20 min and collect the supernatant in 100mL volumetric flask and make up the volume.
2.
Transfer 1mL of the sample extract to a 100mL volumetric flask containing 75mL water.
3.
Add 5mL of Folin-Denis reagent, 10mL of sodium carbonate solution and dilute to 100mL with water.
4.
Shake well. Read the absorbance at 700nm after 30 min.
5.
If absorbance is greater than 0.7, make a 1 + 4 dilution of the sample.
6.
Prepare a blank with water instead of the sample.
7.
Prepare a standard graph by using 0-100mg tannic acid.
 
 
Calculation
Calculate 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 Reagent
Mix 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
1.
Pipette out 1mL of the supernatant.
2.
Quickly add 5mL of vanillin hydrochloride reagent.
3.
Read in a spectrophotometer at 500nm after 20 min.
4.
Prepare a blank with vanillin hydrochloride reagent alone.
5.
Prepare a standard graph with 20-100mg catechin using the diluted stock solution.
 
 
Calculation
From 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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