Identification and Quantification of Free Radical Scavengers in Pu-erh Tea by HPLC-DAD-MS Coupled Online with 2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) Diammonium Salt Assay

Pu-erh tea, a well-known traditional beverage in China, has attracted more attention because of its beneficial health effects and special flavor and taste. Generally, it is believed that Pu-erh tea with a longer preservation period has better quality and taste. Antioxidant activity is one of the major beneficial activities of tea. In this study, a HPLC-DAD-MS coupled with 2,2'-azinobis (3-ethylbenzthiazoline-sulfonic acid) diammonium salt (ABTS) assay was employed for identification and quantification of free radical scavengers in different samples of Pu-erh tea. Among 12 main peaks detected in Pu-erh raw tea, 11 compounds were identified as gallic acid, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin, caffeine, (-)-epicatechin, (-)-epigallocatechingallate, rutin, (-)-epicatechingallate, quercetin-3-glucoside, and kaempferol-3-glucoside by comparison of their UV and MS data with standard compounds or literature data, respectively. The contents of 12 investigated compounds were also determined or estimated using caffeine, (-)-epicatechin, or rutin as standard. ABTS assay showed that 10 out of 12 compounds were free radical scavengers. Their total amount was used as the marker for evaluation of free radical scavenging activities of different Pu-erh teas, which indicated that the activity of different Pu-erh teas varied; Pu-erh raw tea was stronger than the ripe one, and the activity decreased with the increase of preservation period.