Development of suitable standards for quantitative determination of persimmon phenol contents in Folin-Ciocalteu and vanillin assays

Use of gallic acid (GA) as a standard in the Folin-Ciocalteu assay leads to an underestimation of phenol content in persimmon extracts so does the widely used catechin (C) in vanillin assay for determining condensed tannins in persimmon extracts. To develop suitable standards for addressing the issue of accuracy, we prepared highly purified persimmon tannin (PT) and its three fractions (PT20, PT40 and PT60), as well as three characteristic structural subunits of PT including epicatechin-3-gallate-(4β → 8, 2β → O→7)-epicatechin-3-gallate (A-type ECG dimer), epigallocatechin-3-gallate-(4β → 8, 2β → O→7)-epigallocatechin-3-gallate (A-type EGCG dimer) and epicatechin-(4β → 8)-epicatechin (B-type EC dimer). We compared the color yield of commercially available standards including the dimers GA, EGCG, ECG, C and PT and its three fractions in the Folin-Ciocalteu assay and vanillin assay, respectively. Our results suggested that for estimating phenol content in persimmon extracts, EGCG, ECG, C and GA were poor standards in Folin-Ciocalteu assay. We recommended PT40 as the most suitable standard in determining the phenol content of persimmon extracts. In the vanillin assay, A-type EGCG dimer or A-type ECG dimer were superior than the commonly used C as standards, and ECG was also an alternative when A-type EGCG dimer or A-type ECG dimer were not available.