High-resolution hyaluronidase inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of anti-necrosis constituents in Clausena excavata

On average 100,000 persons are bitten by venomous snakes in China each year, with a mortality rate of 5 – 10% [1]. Gloydius blomhoffi brevicaudus, Deinagkistrodon acutus, Naja naja atra and Trimeresurus stejnegeri bites are most common. Necrotizing enzymes phospholipase A₂, proteases, and hyaluronidase in the snake venom cause local tissue damage. Clausena excavata is a common plant used against snakebite in China. The aim of our study was to explore the anti-necrosis potential of plant C. excavata and identify non-tannin anti-necrosis constituents.

Extracts of C. excavata were tested at 1 mg/mL in hyaluronidase, phospholipase A₂, and protease inhibition assays [2] against G. blomhoffi, D. acutus, N. naja and T. stejnegeri venom. Extracts with over 90% inhibition were fractionated into microplates and biochromatograms were constructed (high-resolution profiling). Bioactive constituents from the biochromatograms were analyzed by HPLC-HRMS-SPE-NMR.

Analysis of HRMS and NMR data obtained via HPLC-HRMS-SPE-NMR led to identification of the metabolites as 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1), lansiumamide I (2), indicolactone (3), N-methyl-3-phenyl-N-[(Z)-styryl]oxirane-2-carboxamide (4), ξ-clausenamide (5), and lansiumamide B (6). The 2,3-trans double bond and 1',2'-cis double bond of 6 might be essential for the inhibition of hyaluronidase by comparing the structure with compound 1, 2, 4 and 5. Compounds 1 – 6 were purified by preparative scale HPLC and subjected to the activity test in the hyaluronidase assay against D. acutus venom. Compounds 1 – 5 showed no activity in the test. The IC ₅₀ value of lansiumamide B (6) was very close to the value of the standard hyaluronidase inhibitor aristolochic acid, which indicate lansiumamide B might be a promising inhibitor against snakebite of D. acutus.

References:

[1] Nie HJ. Modern Clin Med 2007; 33: 234 – 236.[2] Molander, M et al. J. Ethnopharmacol 2014; 157: 171 – 180.