Biochemical and toxicological investigation of karanjin, a bio-pesticide isolated from Pongamia seed oil.

Karanjin, a furanoflavonol from Pongamia pinnata L is used in agricultural practices for its pesticidal, insecticidal and acaricidal activities. It is commercially available as a bio-pesticide targeting a wide variety of pests. The present study was intended to evaluate the biochemical interactions of karanjin with bovine serum albumin (BSA) and study its toxicological effects on mammalian and bacterial cell lines. Karanjin bound to BSA at a single site with a dissociation constant of 19.7 µM. Evaluation of BSA-karanjin interactions at three different temperatures indicated the involvement of static mode of quenching. Binding experiments in the presence of warfarin and computational docking analysis indicated that karanjin bound closer to the warfarin binding site located in the Subdomain IIA of BSA. Using Förster resonance energy transfer analysis the distance between TRP 213 of BSA and karanjin was found to be 20 Å. Collective results from synchronous fluorescence spectra analysis, differential scanning calorimetry, and circular dichroism analysis indicated that binding of karanjin induced conformational changes in the secondary structure of BSA. Karanjin exhibited low toxicity against human cervical cancer cells and normal mouse fibroblast L929 cells and modestly inhibited the growth of B. subtilis and E. coli cells. The data presented in this study provides insights for understanding the binding interactions of karanjin with BSA and its possible toxicological effects on mammalian cell lines and bacteria.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 µM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ±â€¯3 µM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-ß tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.