2.6.1. Determination of condensed tannins content

The condensed tannins content was determined according to the method described by Maxson and Rooney (¹⁹⁷²). About 1.000g of the sample was weighed and mixed with 10 ml of 1% HCl solution in methanol in a screw cap test tube. Then, the tube was shaken for 24 hr at room temperature on a mechanical shaker (Hy‐2(C), Shanghai, China). The solution was centrifuged (sigma 2‐16KC, UK) at 1,000 rpm for 5 min. One ml of supernatant was transferred to another test tube and mixed with 5 ml of vanillin–HCl reagent (prepared by combining equal volume of 8% concentrated HCl in methanol and 4% vanillin in methanol). The D (+)‐catechin was used as a standard for condensed tannins content determination. A 40 mg of D (+)‐catechin was weighed and dissolved in 1,000 ml of 1% HCl solution in methanol, which was used as stock solution from which a series of standard solutions (0, 12, 24, 36, 48, and 60 μg /ml) were prepared by mixing with 5 ml 1% HCl in methanol. The absorbance of samples and the standard solutions were measured at 500nm using UV‐VIS Spectrophotometer (JASCO V‐630, Shimadzu Corporation, Tokyo, Japan) after 20 min. The condensed tannins content was determined from standard curve of catechin, and result was expressed as mg/100g.