Chromatographic Analysis and Anti-Oxidative Property of Naoxinqing Tablet, a Proprietary Preparation of Diospyros Kaki Leaves

Abstract

The Naoxinqing (NXQ) tablet is a standardised proprietary herbal product containing an extract of persimmon leaves (Diospyros kaki) for the management of cardio- and cerebrovascular diseases. Although previous reports suggested that the efficacy of NXQ is at least partly mediated by its anti-oxidative property, the anti-oxidative effect of the major components of NXQ has not been studied systematically. For quality control purposes, only analytical methods limited to 3 marker analytes have been reported, the extent to which the other components affect efficacy has not been explored. In this study, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC MS/MS) method for the identification of seven analytes (kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hypericin), quercetin-3-O-glucoside (isoquercitin), kaempferol, 3,4-dihydroxybenzoic acid (protocatechuic acid), and furan-2-carboxylic acid (pyromucic acid) and quercetin) in the NXQ. This is the first method reported and validated for the quantification of the seven major secondary metabolites in NXQ. The results for the quantified analytes were then compared in 15 different batches of NXQ. The variation observed in the seven components highlights the need to quantify key bioactive components to ensure product consistency. Radical scavenging activity and abundance was used to rank the analytes. The anti-oxidative effects of NXQ were examined using cultured human vascular endothelial cells (EA.hy926). Corrected 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) activity results revealed that quercetin and kaempferol have the strongest anti-oxidant capacity in the extract. Both quercetin and kaempferol significantly inhibited the hydrogen peroxide (H₂O₂)-induced EA.hy926 cell injury and intracellular reactive oxygen species (ROS) generation. In conclusion, we established and validated an UPLC-MS/MC method for the analysis of major bioactive components in the NXQ and demonstrated that its anti-oxidative property may play a critical role in cerebrovascular protection.

Keywords: Naoxinqing; UPLC MS/MC analysis; anti-oxidative; endothelial cells; reactive oxygen species.

Figure 1

Ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) chromatogram of NXQ extract.1 = Succinic acid, 2 = 3,4-dihydroxybenzoic acid, 3 = furan-2-carboxylic acid, 4 = quercetin-3-O-galactoside, 5 = quercetin-3-O-glucoside, 6 = quercetin-3-O-rutinoside, 7 = myricetin, 8 = kaempferol-3-O-glucoside, 9 = quercetin, 10 = kaempferol.

Figure 2

(A) Effects of NXQ (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± SD **p <0.001 vs control (CLT) group; ^###p <0.001 vs. H₂O₂ group. (B) Effects of quercetin (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± S.D. *** p <0.0001 vs. control (CLT) group; ^###p <0.001 vs. H₂O₂ group. (C) Effects of kaempferol (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± SD **p <0.001 vs control (CLT) group; ^##p <0.001 vs. H₂O₂ group.

Figure 2

(A) Effects of NXQ (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± SD **p <0.001 vs control (CLT) group; ^###p <0.001 vs. H₂O₂ group. (B) Effects of quercetin (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± S.D. *** p <0.0001 vs. control (CLT) group; ^###p <0.001 vs. H₂O₂ group. (C) Effects of kaempferol (10–500 µg mL⁻¹) on H₂O₂-induced intracellular ROS generation in EA.hy926 cells. (n = 3). Data are presented as means ± SD **p <0.001 vs control (CLT) group; ^##p <0.001 vs. H₂O₂ group.