Simultaneous quantitative analysis of 11 flavonoid derivatives with a single marker in persimmon leaf extraction and evaluation of their myocardium protection activity

An improved, reliable and comprehensive method for assessing the quality of the ethyl acetate extract from persimmon leaves (EAPL) and its commercial preparation, Naoxinqing (Brain and Heart Clear capsules), has been developed and validated. Based on HPLC-DAD-ESI-Q-TOF-MS analysis, myricetin-3-O--d-galactoside (1), myricetin-3-O-glucoside (2), quercetin-3-O--d-galactoside (3), quercetin-3-O--d-glucoside (4), quercetin-3-O-(2-O-galloyl--d-galactoside) (5), quercetin-3-O-(2-O-galloyl--d-glucoside) (6), kaempferol-3-O--d-galactoside (7), kaempferol-3-O--d-glucoside (8), kaempferol-3-O-(2-O-galloyl--d-galactoside) (9), kaempferol-3-O-(2-O-galloyl--d-glucoside) (10), quercetin (11) and kaempferol (12) were identified from 15 batch samples. A HPLC fingerprint analytical method was established. All compounds, with the exception of compound 2, were simultaneously quantified by the single standard to determine multi-components (SSDMC) method, using kaempferol-3-O--d-glucoside as the internal standard. The rate of analysis was found to be faster with the SSDMC method than with current acid hydrolysis method (Pharmacopoeia of the People's Republic of China 2015 edition) and the results were more intuitive and reliable. Three-dimensional principal component analysis revealed that there were similar characteristics in persimmon leaf from same district. Analysis of the myocardial cell protection activity of 11 monomeric compounds showed that compounds 12, 11 and 10 were the main active ingredients that produce pharmacologic functions in EAPL. Among these compounds, the bioactive constituent of myricetin-3-O--d-galactoside was determined for the first time in Diospyros khaki. Thus, we have established an effective assessment method that can be applied to the comprehensive quality evaluation of EAPL extract and Naoxinqing capsule.