Tannin Assay

TANNINS ESTIMATION Keshab Barman, Deepak K. Dubey, Mayank Tandon, Thirumeignanam, D. and Dr.S.N. Rai. TANNINS ESTIMATION. . Dairy Cattle Nutrition Division, N.D.R.I., Karnal, India. Sample Preparation and Tannins Extraction The sample was dried at 55±10 C and ground to pass through a sieve of 1mm diameter. Tannins extraction was done using 400 mg ground sample in conical flask with 40 ml diethyl ether containing 1 per cent acetic acid (v/v) and mixed to remove the pigment material. Carefully discarded the supernatant after 5 minute and 20 ml of 70 per cent aqueous acetone was added and sealed the flask with cotton plug covering with aluminum foil and kept in electrical shaker for 2 h for extraction. Then it was filtered through Whatman filter paper No. 1 and sample was kept in refrigerator at 4 0C until analysis. (Barman, 2004) Estimation of Total Phenol and Tannins It was estimated according to the procedure of Makkar et al., (1993). Reagents a) Folin Ciocalteu reagent (1 N): commercially available Folin Ciocalteu reagent (2N) was diluted with an equal volume of distilled water. The resultant solution was kept in a brown color bottle and stored in refrigerator at 4 0C. b) Sodium carbonate (20%): fifty gram of sodium carbonate (NaCO310H2O) was dissolved in distilled water and made the volume to 250 ml with distilled water. c) Insoluble polyvinyl polypyrrolidone (PVPP, P 6755): it was obtained from the Sigma Company, U.S.A. d) Standard tannic acid solution (0.5mg/ml): twenty-five mg tannic acid (T-0125) obtained from the Sigma Company, U.S.A. was dissolved in 50 ml distilled water. It was prepared freshly just before use. Analytical procedure Fifty micro litre (µl) of tannins extract for each sample was taken in test tube and volume was made to 1.0 ml with distilled water. Then, 0.5 ml Folin Ciocalteu reagent was added and mixed properly. Then 2.5 ml 20 per cent sodium carbonate solution was added and mixed it and kept for 40 minutes at room temperature. Optical density was taken at 725 nm in spectrophotometer and concentration was estimated

from the standard curve. Total phenol was estimated as tannic acid equivalent and expressed on dry matter basis. Non-tannins phenol was estimated by precipitating tannins with polyvinyl polypyrrolidone (PVPP), which binds tannins. 200 mg PVPP was taken in test tube and then 2.0 ml distilled water and 2.0 ml tannins extract was added. Vortex it and kept in refrigerator for 15 minutes at 4 0C. Then the mixture was again vortex and filtered through Whatman filter paper No. 1. Filtrate was taken for estimation of non tannin phenol. 150 µl of filtrate was taken in test tube and volume was made to 1.0 ml with distilled water and then processed like that of total phenol estimation. Concentration of non tannin phenol was calculated from the standard curve and expressed on DM basis. Total tannins were calculated by subtracting non tannin phenol from total phenol. The standard was prepared (Fig 3.1) from the stock solution of tannic acid (0.5 mg/ml) using 0,10,20,30,40 and 50 µl in test tubes and volume was made to 1.0 ml. It gives a tannic acid concentration of 0, 5,10,15,20 and 25 µg respectively. Then 0.5 ml Folin reagent and 2.5 ml 20 per cent sodium carbonate were added. Whole content was mixed properly and after 40 minutes reading was taken at 725nm in spectrophotometer. Estimation of Condensed Tannin (Proanthocyanidin) Condensed tannin was estimated according to the method of Porter et al., (1986). Reagents a) Butanol-HCℓ reagents (butanol- HCℓ, 95:5 v/v): 950 ml n-butanol was mixed with 50 ml of concentrated HCℓ. b)

Ferric reagent (2% ferric ammonium sulphate in 2N HCℓ): 16.6 ml concentrated HCℓ was diluted to 100 ml with distilled water to make 2N HCℓ. To this 2.0 g ferric ammonium sulphate was dissolved. The reagent was stored in an amber colour bottle.

Analytical procedure 0.5 ml of tannins extract was taken in test tube in triplicate and 3.0 ml butanol HCℓ and 0.1 ml of ferric reagent was added. Tube was vortex to ensure proper mixing. The mouth of the tube was covered with glass marble and then boiled it for 60 minutes. Similarly blank was prepared for each sample but without heating the reagent. The tube was cooled to room temperature and reading was taken at 550 nm using

spectrophotometer. Condensed tannins as leucocyanidin equivalent was calculated as below % Condensed tannins= (A550nm X 78.26 X dilution factor)/(% dry matter) 0.7

0.6

OD (at 725 nm)

0.5

0.4

0.3

0.2

0.1

0 0

5

10

15

20

25

30

35

Tannin acid (µg) Fig 3.1: Standard curve of tannic acid Determination of Hydrolysable Tannin Hydrolysable tannin was estimated by subtracting condensed tannins from total tannin phenol. References: Barman, K. 2004. Biodegradation of tanniniferous feeds and their influence on nutrient utilization and productivity of the dairy animals. Ph.D thesis. Submitted to NDRI, Karnal. Makkar, H.P.S., Blummel, M., Borowy, N.K. and Becker, K. 1993. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J.Sci. Food Agric., 61:161-165. Porter, L.J., Hrstich, L.N., Chan, B.J., (1986). The conversion of proanthocyandins prodelphinidins to cyanidin and delphinidin. Phytochemistry. 25, 223-230.