Substance basis and fermentation mechanism study of the analgesic and anti-inflammatory effects of Tibetan medicine Wuwei Ganlu based on JiuQu fermentation.

Background: Wuwei Ganlu, as an integral part of the Tibetan medical system known as "Sowa Rigpa" originates from the formulations documented in the "Four Tantras". It is recognized by UNESCO as an intangible cultural heritage, representing a body of traditional knowledge and practice. Fermentation, a crucial step in the preparation of Wuwei Ganlu, has not been fully understood in terms of its mechanisms and its impact on the material basis and pharmacological efficacy of the medicine.

Objective: By investigating the effects of fermentation on the chemical components and pharmacological activities of Tibetan medicine Wuwei Ganlu, this study aims to reveal the scientific basis for the enhanced efficacy induced by fermentation.

Methods: An analgesic and anti-inflammatory animal model was employed to investigate the effects of Tibetan medicine Wuwei Ganlu at different fermentation stages (from 0 to 5 days) on the number of twisting and serum IL-6 levels in mice, aiming to explore the influence of fermentation on the potentiation of its analgesic and anti-inflammatory effects. Based on this, a systematic analysis of the substance basis of Wuwei Ganlu before and after fermentation was conducted using UPLC-Q-Exactive Orbitrap MS and HPLC-MS/MS methods, identifying key differences in the substance composition and determining the indicative components. Additionally, fungal community changes before and after fermentation were studied using ITS rRNA sequencing, which revealed the differences in the fungal community. The integrity of the plant cell walls in Wuwei Ganlu before and after fermentation was examined under a microscope, and the effects of fermentation on the plant cell walls and cellulase activity were assessed by measuring the enzyme activity during the fermentation process. Finally, the fermentation mechanism of Wuwei Ganlu was further corroborated by studying the changes in content and transformation patterns of specific components, including Rutin, Quercitrin, Hyperoside, Quercetin, and Ephedrine, under the same fermentation conditions.

Results: The analgesic and anti-inflammatory experiments indicated that fermentation significantly enhanced the analgesic and anti-inflammatory effects of Tibetan medicine Wuwei Ganlu, as evidenced by a marked reduction in the number of twisting and IL-6 levels (P < 0.05). Fermentation caused significant changes in the Chemical Compotents of Wuwei Ganlu, increasing the levels of Ephedrine (19.69%), Rutin (16.71%), Quercitrin (21.54%), Quercetin (132.54%), and Hyperoside (110.16%). Fungal community analysis revealed that Saccharomycetaceae was the dominant fungal genus during fermentation, with its abundance significantly increasing after fermentation, while Aspergillus showed relatively low abundance on day 3 of fermentation. The cellulase produced by Saccharomycetaceae promoted the hydrolysis of the plant cell walls. Microscopic observation demonstrated that fermentation led to plant cell walls rupture and fiber structure transparency in the raw materials, which enhanced cellulase activity between days 1 and 3, promoting the release of Ephedrine, Rutin, Quercitrin, Quercetin, and Hyperoside. Under the catalytic action of JiuQu fermentation starter, Rutin, Quercitrin, and Hyperoside were hydrolyzed into Quercetin, while Ephedrine was transformed into Methylephedrone and Cathinone, confirming the fermentation-induced transformation pathways of these compotents. The heatmap of the correlation between chemical components, microbial community, and pharmacological indicators showed that the abundance of Saccharomycetaceae in the fermented samples was significantly correlated with the Twisting inhibition rate and IL-6 levels in mice, as well as Ephedrine and Quercetin, suggesting that Ephedrine and Quercetin may have potential analgesic and anti-inflammatory effects.

Conclusions: The fermentation process significantly enhances the analgesic and anti-inflammatory effects, primarily due to the chemical components changes in Tibetan medicine Wuwei Ganlu induced by fermentation. Fermentation regulates the abundance of dominant microbial communities, particularly Saccharomycetaceae species, and enhances cellulase activity. This enhances the breakdown of plant cell walls, promoting the release of chemical components. Additionally, fermentation facilitates the conversion of glycosides (water-soluble) into aglycones (lipid-soluble), such as the hydrolysis of Rutin, Hyperoside, and hypericin into Quercetin. In summary, the fermentation mechanism of Wuwei Ganlu involves an increase in Saccharomycetaceae abundance and enhanced cellulase activity under the influence of JiuQu fermentation, leading to plant cell walls breakdown and the subsequent release of chemical components. Moreover, the conversion of glycosides to lipid-soluble aglycones during fermentation enhances transdermal absorption, which may also contribute to the potentiation of its effects.