Comprehensive metabolic profiling of three plants of Ardisia based on UPLC-QTOF-MS coupled with bioactivity assays

Ardisia is a large genus of Primulaceae, 734 accepted species worldwide, and most species are used as ethnomedicines for the treatment of bruises, rheumatism, tuberculosis, and various inflammatory diseases. According to our previous ethnobotanical survey, Ardisia gigantifolia Stapf, Ardisia hanceana Mez (Da-luo-san), and Ardisia crenata Sims (Xiao-luo-san) are commonly used in folk medicine for the treatment of rheumatism. Among them, A. hanceana and A. crenata in folklore has "Use Da-luo-san and Xiao-luo-san together for better rheumatism treatment". These three plants can be referred to as the characteristic herbs of Ardisia.

To characterize the metabolites of three plants of Ardisia, clarify the metabolites differences, validate the traditional folk applications, and identify potential biomarkers.

UPLC-QTOF-MS and UPLC-QTRAP-MS metabolomics analysis techniques were utilized to reveal the metabolites of three plants of Ardisia. Combined with multivariate statistical analysis, differentially accumulated metabolites (DAMs) and biomarkers were screened. The inhibition of NO production was measured, and the p_value was determined using one-way ANOVA with Fisher's LSD test, to validate folk traditional application.

A total of 328 metabolites were identified from the three plants' roots using UPLC-QTOF-MS, and 86 DAMs were screened by OPLS-DA. Among them, flavonoids are the main DAMs, especially between Zou-ma-tai group (A. gigantifolia) and the Luo-san group (A. hanceana and A. crenata). UPLC-QTRAP-MS was used to quantify the three DAMs, and it was found that the contents of (+/-)-catechin and gallic acid were higher in the Luo-san group. To verify the traditional application of the three plants, it was found that the activity of the three plants was better at 0.2 mg/mL, and the activity of the Luo-san group was significantly stronger than that of the Zou-ma-tai (p<0.001). Further, 16 potential anti-inflammatory active biomarkers were screened by the two groups, and the relative contents of flavonoids was significantly higher in the Luo-san group, which was inferred to be a possible key active ingredient in the Luo-san group for the treatment of rheumatic diseases. Further network pharmacological methods verified that these flavonoid components may exert their therapeutic effects on rheumatic diseases by modulating multiple signaling pathways such as TNF, 1L-17, NF-κB, and T-cell receptor through key targets such as PTGS2, ESR1, ALOX5, CA2, XDH, and AKR1B1.

This study identified the main active metabolites of the three plants of Ardisia, verified the local folklore that "Use Da-luo-san and Xiao-luo-san together for better rheumatism treatment", and spread the folk traditional knowledge. It is indicated that the Luo-san group have important potential medicinal value and development prospects. It further provides a scientific basis for the conservation and utilization of Ardisia resources as well as sustainable development.