Mechanistic studies on polyphenol rich fractions of Kangra tea by HPTLC and NMR for their antioxidant activities

Abstract

Abstract: The antioxidant activity in tea is largely driven by its polyphenolic content, however, the antioxidant reaction mechanism and the compounds involved are not well characterized. Therefore, in this study, we performed in-depth profiling of the antioxidant reaction mechanism of Green Tea (GT), Black Tea (BT), and their polyphenolic fractions with free radical using state-of-the-art analytical techniques. The polyphenol enriched fractions from GT and BT were isolated using column chromatography. Catechins were isolated and characterized by diverse spectroscopic techniques. Samples were screened for their antioxidant activity by HPTLC and further evaluated using a spectrophotometer. The free radical reactions with GT, BT, enriched fractions viz, GT Polyphenols (GTP), BT Polyphenols (BTP), and isolated catechins were studied using the ¹³C NMR technique. The highest polyphenol content was found in GTP (795.4 ± 0.012 mg/g) whereas GT (321.0 ± 0.028 mg/g) showed maximum flavonoids content. Individual catechins isolated from GTP were EGCG, ECG, EGC, EC and C. Antioxidant activity followed the order EGCG > ECG > EGC > EC > GTP > C > BTP > GT > BT. In GT, the antioxidant reaction mechanism showed single electron and H-transfer in all catechins, which involved the transformation of the hydroxyl group to the carbonyl group. Whereas in BT theaflavins, conversion of the benzotropolone ring to the six-membered ring was observed.

Supplementary information: The online version contains supplementary material available at 10.1007/s13197-021-05297-w.

Keywords: Antioxidant; Camellia sinensis; DPPH; Flavonoids; Tea polyphenols.

Fig. 1

Representative HPLC chromatogram of standard mixture of catechins, GTP, GT, BT and BTP viz gallic acid (1), GC (2), EGC (3), C (4), caffeine (5), EGCG (6), EC (7), ECG (8)

Fig. 2

A TLC fingerprints with DPPH of C, EC, EGC, ECG, EGCG, GC, GTP and BTP before incubation at white light; B TLC fingerprints of DPPH with C, EC, EGC, ECG, EGCG, GC, GTP and BTP after incubation at white light; Mobile phases, toluene: acetone: formic acid (18:18:4.2), respectively

Fig. 3

A and B DPPH radicals scavenging effect C and D ABTS radicals scavenging effect, n = 3, mean ± S.D. E Correlation between DPPH and ABTS (EGCG, ECG, AA, EC, EGC, C); F Correlation between DPPH and ABTS (GTP, TX, BTP, GT, BT). G Unutilized DPPH in the rate of reaction GT, BT, GTP, BTP, TR. H EGCG, AA, EGC, EC, ECG with DPPH in respect to time and unutilized ABTS in the rate of reaction of (I) GT, BT, GTP, BTP, TR. J EGCG, AA, EGC, EC, ECG with ABTS in respect to time