OBJECTIVE To establish an efficient method for the separation and purification of polyphenols from Smilax glabra Roxb (SGR) and to elucidate their anti-inflammatory and analgesic mechanisms based on network pharmacology.

METHODS Using the capacity factor k as the theoretical framework, a pH/ethanol binary-gradient macroporous resin (MR) separation system was established and combined with recycling preparative high-performance liquid chromatography (prep-HPLC) to separate and purify seven polyphenols from SGR. Potential targets related to the anti-inflammatory and analgesic effects of the seven polyphenols were predicted by network pharmacology, and molecular docking was performed to validate their binding affinities with the core targets. ADMET prediction was further conducted to evaluate the drug-like properties of the seven polyphenols. The analytical results of network pharmacology were verified by Western blot experiments using RAW 264.7 cells.

RESULTS The ethanol extract of SGR was preliminarily divided into three fractions by MR, with recovery rates of all fractions exceeding 70%. Subsequently, seven high-purity polyphenol monomers (>95%) were further obtained by recycling prep-HPLC, namely 5-O-caffeoylshikimic acid, neoastilbin, astilbin, neoisoastilbin, isoastilbin, engeletin, and isoengeletin. Network pharmacology and molecular docking analysis indicated that the core targets for the anti-inflammatory and analgesic effects of polyphenols from SGR were SRC, MAPK14, MMP2, CASP3, and CASP8. ADMET prediction showed that 5-O-caffeoylshikimic acid possessed good pharmacokinetic properties and drug-like characteristics. Cell experiments verified the regulatory effect of 5-O-caffeoylshikimic acid on the core anti-inflammatory and analgesic targets p-SRC, p-P38, MMP2, and CASP8 in an inflammatory cell model.

CONCLUSION This study successfully establishes an efficient separation method for polyphenols from SGR, and predictes and verifies its anti-inflammatory and analgesic mechanisms at the molecular level, providing a theoretical basis for the development and application of SGR.