TANNINS AND OTHER POLYPHENOLS IN SORGHUM: SEPARATION, IDENTIFICATION AND POLYMERIZATION

LESLEY JANE PUTMAN, Purdue University

Abstract

Tannins have been associated with sorghum's ability to defend itself against predators and disease. With an increasing awareness of the diversity of phenolic compounds present in sorghum there is an interest in determining the role of tannins and other polyphenols in plant defense. To study tannins, counter-current chromatography (CCC) and HPLC were used to obtain more homogeneous fractions. HPLC was also used to study some of the properties of tannins. In the biosynthesis of tannins, polymerization was studied to determine whether it is an enzymic process. CCC was used to further fractionate purified tannin. This technique was also found to be useful on a preparative scale for purification of crude tannin extracts. CCC fractionation was on the basis of relative solubility in an aqueous phase compared to 1-butanol. Longer tannin chains were more water soluble and eluted first. A method was developed to separate tannins by HPLC using a short reversed phase column (5 cm), a fast flow rate (3.8 ml/min) and a combination of linear and step gradients. In contrast to CCC, the longer chains eluted later indicating that although they are very water soluble, they are capable of extensive interaction with the column support. This separation method was used to study the effect of various conditions on the stability of tannins. To verify enzymic polymerization of tannin, sorghum seed was exposed to conditions intended to denature any enzymes. Seed was then dried to induce polymerization. Treatment with SDS, TCA, urea and microwaves inhibited polymerization induced by drying. Attempts to isolate an enzyme responsible for tannin polymerization resulted in the identification of a polyphenol oxidase in immature sorghum. An 8-C-glycosyl flavone was identified in immature sorghum seed and was found to be a substrate for the polyphenol oxidase.

Degree

Ph.D.

Subject Area

Biochemistry

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