Jiangxi Baishen Pharmaceutical Co., Ltd.

The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive.

The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel.

The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca²⁺ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches.

The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca²⁺ influx through the channel opening and ultimately relieving pain.

Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

The content determination method of VD₃ through pos. phase HPLC was established.The exptl. condition was listed below: Ultimate SiO₂ chromatog. column(4.6*250 mm 5 um) as stationary phase, hexane:n-pentanol(99:1) as mobile phase, detection wavelength 265 nm, flow velocity 1 mL/min, column temperature 30°C.It was proved that the content of vitamin D₃ of the standard solution in the range of 0.7497∼1.9992 μg/mL was well proportional to the peak area of the standard solution(r=0.9999, n=6).The average recovery of the experiment was 100.50%, and the precision was 0.24%.This method was simple, reliable, reproducible and suitable for quality control of pharmaceutical production process.

The purpose of the article is to optimize the process of synthesis of celecoxib, a COX-2 selective inhibitor, i.e. 4-[5-(4-methylphenyl)-3-(trifluromethyl)-1H-pyrazol-1-yl]benzsulfamide.The reaction procedure include 2 steps: (1) Claisen condensation of p-methylacetophenone and Et trifluoroacetate to afford a key intermediate; and (2) cyclization of the above mentioned intermediate with sulfonamide phenylhydrazine hydrochloride to afford the final product.In the 1st step, hexane was used as reaction solutionIn the 2nd step, ethanol was used as reaction solutionThe molar yields for the 2 steps were about 0.95 and 0.85 resp.The crude product was refined in ethanol-water system.And the overall yield was about 80.7%.The optimized process has the advantages of simple operation, short reaction time, high yield, etc., and is suitable for industrial production