Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790.

Contents Introduction Materials and methods Results and discussion Conclusion

Introduction Tannins gallotannins hydrolyzable tannins condensed tannins high molecular weight polyphenolics found in higher plants gallotannins ellagitannins

Structural formulas of representative hydrolyzable tannins from plants Structural formulas of representative hydrolyzable tannins from plants. Compound 1 is pentagalloyl glucose, comprised of the gallate (G,2) esters of glucose. Compound 3 is the simple ellagitannin eugeniin, comprised of both the gallate esters and a single HHDP (G−G,4) ester. Compound 5 is EGCG, the gallate ester of the flavanoid epigallocatechin. Compound 6 is oenothein B, a macrocyclic hydrolyzable tannin. All four of thehydrolyzable tannins react in acidic methanol to yield methyl gallate (7)

Introduction Individual hydrolyzable tannins can usually be determined by HPLC, but methods for conveniently screening large numbers of samples with variable hydrolyzable tannin compositions have not been developed. The KIO3 method has been employed in some studies of oaks and maples. The purpose of this work was to optimize the KIO3 method for analysis of hydrolyzable tannins.

Materials and methods HPLC Analysis of Methanolysis Products Reagents tannin acid，gallic acid, methyl gallate, epigallocatechin gallate (EGCG) Methanolysis and pH Adjustment anhydrous methanol, 85 ℃, 20h, pH: 5.5±0.1 Analysis 30 ℃ water bath， 50 min after KIO3 was added, recorded absorbance at 525 nm HPLC Analysis of Methanolysis Products C18 (100×4.6 mm),1.0 mL/min , ƛmax 525 nm, gradient elution

Results and Discussion 1 2 + KIO3 A B ƛmax 525 nm yellow Why KIO3 ？ Hypochlorite ? the lifetime of A was shorter Potassium periodate (KIO4) ? past the red intermediate to the final product

Improve Disadvantages of KIO3 method different color yields in rates of reaction for structurally different gallotannins Improve Why not gallic acid ？ Methyl gallate 1 2 + KIO3 A B ƛmax 525 nm yellow

Methanolysis Why 85℃? avoid the disappearance of some methyl gallate formation of unidentified phenolic products observed at higher temperatures Why anhydrous methanol? traces of water caused production of a mixture of gallic acid and methyl gallate

How to control pH? KIO3 Reaction too acidic, the chromophore A did not accumulate too basic, oxidation was accelerated and only the yellow end product Reaction of KIO3 with methyl gallate as a function of pH. Methyl gallate solutions dissolved in methanol were diluted with a series of solutions comprised of ethanolamine and ammonium acetate buffer prepared at various pH values between 4.5 and 7.0.

KIO3 Reaction product A was temperature-dependent and time-dependent How to control temperature? at room temperature, there were significant day-to-day variations 30 ℃ water bath How to control time? 50 min after KIO3 was added, recorded absorbance at 525 nm

a linear relationship between methyl gallate and absorbance at 525 nm: Abs = 0.0132 μg + 0.0701 where R2 = 0.996, standard error of Y = 0.0303, and standard error of slope = 0.000 19.

A Methanolysis samples (s) of tannic acid B . EGCG C. Acer leaf powder Conversion of hydrolyzable tannins to methyl gallate by 20 h of methanolysis at 85°C.

Theoretical Production of Methyl Gallate from Hydrolyzable Tannins Yield of methyl gallate is calculated from molecular weights of tannin and methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

Example:red maple leaves Analysis was done with the original KIO3 method (A) Analysis was done with the new method described (B)

Theoretical Production of Methyl Gallate from Hydrolyzable Tannins Yield of methyl gallate is calculated from molecular weights of tannin and methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

Conclusion The new KIO3 method will be useful for examining hydrolyzable tannin in ecological, physiological, and nutritional studies of plants.