To investigate and screen the active antibacterial constituents of Niuhuang Shangqing Pill (NSP), the current study developed a two-dimensional liquid chromatography (2DLC) method combining microcalorimetry technique. 60% ethanol extracts from 10 batches of different commercial NSP samples were analyzed and their chemical fingerprint were developed by the comprehensive 2DLC system of Shimadzu Nexera X2. Anti-streptococcus pneumoniae (SP) constituents were determined by microcalorimetry. Thermal kinetic parameters of the SP thermogram affected by 60% ethanol extracts from 10 NSP samples were analyzed by principal component analysis. Spectrum-effect correlation between comprehensive 2DLC fingerprint and the antibacterial activity were analyzed by orthogonal partial least squares (OPLS) and orthogonal partial least squares discriminant analysis (OPLS-DA). Findings showed that peak X1 (unknown), X9 (aloe-emodin), X10 (baicalein), X11 (unknown), X14 (wogonin), X15 (glycyrrhizic acid) and X17 (unknown) are the relevant components that are in positive correlation with inhibitory rate. Regarding inhibitory rate, X17 is the most powerful one, followed by X14, X15, X10, X11, X1 and X9, suggesting that compound X17, wogonin, glycyrrhizic acid and baicalein are the major active antibacterial components of NSP. The current method employing 2DLC with microcalorimetry technique proposes a new insight for screening and identifying antibacterial components in complex herbal formula.
Keywords: Active components; Microcalorimetry; Niuhuang Shangqing Pill; Spectrum-effect relationship; Three-dimensional fingerprint; Two-dimensional liquid chromatography.
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