Screening of immunomodulatory components in Yu-ping-feng-san using splenocyte binding and HPLC

Yu-ping-feng-san (YPFS) is a widely used immunomodulatory herbal medication used in traditional Chinese medicine, but the active molecules remain obscure. To screen for bioactive components we combined splenocyte binding with high performance liquid chromatography (SB-HPLC). After enrichment by splenocyte binding, two YPFS components (C1 and C2) were analyzed by HPLC. Compound C2 was identified as linoleic acid (LA) based on UV absorption and mass spectrometry. Silica gel chromatography was used to purify compound C1 from Radix Saposhnikoviae, a major constituent of YPFS. This allowed identification of the molecule as panaxynol (PAN) based on EI-MS and NMR spectrometry. Bioassay in vitro demonstrated that PAN significantly inhibited splenocyte proliferation induced by concanavalin A (ConA) in a concentration-dependent manner, whereas LA had no significant effect on splenocyte proliferation. In vivo, PAN was found to attenuate allergic contact dermatitis in a mouse model of delayed-type hypersensitivity (DTH), a pharmacological activity not previously reported for this molecule. It is suggested that PAN contributes to the anti-DTH effects of YPFS. SB-HPLC provides a rapid and efficient method for the identification of potential immunomodulatory components in traditional Chinese medicines.