Potential pharmacological quality control markers in the traditional Japanese medicine Hangeshashinto: identifying anti-inflammatory ingredients through a cell-based bioassay and multicomponent analysis

Context: The traditional Japanese medicine Hangeshashinto (HST) is a multicompound drug used for stomatitis. The identification of HST's active ingredients is essential for understanding its mechanism of action and establishing pharmacological quality control markers, but remains unclear due to its multicomponent drug.

Objective: To systematically explore anti-inflammatory ingredients in HST using a combined approach involving a cell-based bioassay and multicomponent analysis, and to identify potential quality marker compounds.

Materials and methods: A cell-based bioassay modeling stomatitis was established using human oral keratinocytes (HOK), with interleukin (IL)-1β-induced prostaglandin E2 (PGE2) as an inflammatory indicator. The PGE2 inhibitory effects of quality-controlled 101 HST manufacturing lots were compared. Multicomponent analysis was performed on the 101 HST samples, correlating the intensity of 121 component peaks with the pharmacological activities. Ingredients with the highest correlation coefficients were validated for their PGE2 inhibitory effects.

Results: A total of 101 HST samples (30 µg/mL: approximately IC₅₀) showed consistent inhibition of IL-1β-induced PGE2 production in HOK (41.33%-67.38% with 100% as IL-1β). Correlation analysis between PGE2 inhibitory activities and peak intensity of 121 components revealed the contribution of each ingredient to the pharmacological effect. Eight ingredients (glycycoumarin, neoglycyrol, [6]-shogaol, [8]-shogaol, [10]-shogaol, [6]-gingerol, [8]-gingerol, and [10]-gingerol) with the highest correlation coefficients (below -0.30) exhibited PGE2-inhibitory potential.

Conclusion: The standardization of current quality control markers contributes to the stability of HST's anti-inflammatory effects. The eight components discovered by this integrated analysis method may become novel quality control marker ingredients for further improving the pharmacological quality of HST.

Hangeshashinto; Kampo; LC-MS; correlation analysis; inflammation; multicomponent drug; phenotypic assay; prostaglandin E2; quality maker; stomatitis.