Antifilarial activity of diterpenoids from Taxodium distichum

Vikas Kushwaha, Kirti Saxena, Richa Verma, Shiv K. Verma, Deepali Katoch, Neeraj Kumar, Brij Lal, P. Kalpana Murthy, Bikram Singh

Lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi and B. timori, is a debilitating disease with an adverse social and economic impact. The infection remains unabated in spite of treatment with existing antifilarial drugs diethylcarbamazine (DEC) and ivermectin which are chiefly microfilaricides. There is therefore, need for macrofilaricides, embryostatic agents and better microfilaricides. In the present study we explored the antifilarial potential of crude extract and its molecular fractions of the plant Taxodium distichum using in vitro assay systems and rodent models of B. malayi infection. Ethanolic extract (A001) of aerial parts of T. distichum was solvent fractionated and sub-fractionated. Four molecules, 3-Acetoxylabda-8(20), 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated from the fractions and their structure determined by spectroscopic analysis. The extract, subfractions and molecules were evaluated for antifilarial activity against B. malayi by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction and motility assays in vitro and in two animal models, Meriones unguiculatus and Mastomys coucha, harbouring B. malayi infection. A001 was effective in killing microfilariae (mf) and adult worms in vitro. The diterpenoid K003 produced 100 % reduction in motility of both mf and adult worms and > 80 % inhibition in MTT reduction potential of adult female worms. In B. malayi-M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals. K003 was embryostatic (> 95 %) in this model. In the B. malayi-M. coucha model, K003 killed ~54 % of adult worms (macrofilaricidal activity) and rendered > 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 post-initiation of treatment. Ethanolic extract of aerial parts of the plant T. distichum possesses potent antifilarial activity and the active principle was localised to K003 which showed significant macrofilaricidal activity and late suppression of peripheral microfilaraemia and some embryostatic activity. These findings indicate that labdane diterpenoid molecule(s) may provide valuable leads for design and development of new macrofilaricidal agent(s). To the best of our knowledge, this is the first report on antifilarial efficacy of products from the plant T. distichum.