Madhulika Dixit

Volumetric productivity of camptothecin from the suspension culture of the endophyte Fusarium solani was enhanced up to ~152 fold (from 0.19 μg l-1 d-1 to 28.9 μg l-1 d-1) under optimized fermentation conditions including initial pH (6.0), temperature (32 °C) and agitation speed (80 rpm) with (5% (v/v)) ethanol as medium component. Among various elicitors and precursors studied, tryptamine (0.5 mM) as precursor and bovine serum albumin (BSA) (0.075 mM) as an elicitor added on day 6 of the cultivation period resulted in maximum enhancement of camptothecin concentration (up to 4.5 and 3.4-fold, respectively). These leads provide immense scope for further enhancement in camptothecin productivity at bioreactor level. The cytotoxicity analysis of the crude camptothecin extract from the fungal biomass revealed its high effectiveness against colon and mammary gland cancer cell lines.

Introduction: AMP-activated protein kinase (AMPK)is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined. Methods: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α2β1γ1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG)to α2 subunit was confirmed through isothermal titration calorimetry (ITC)and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. Results: AG interacts with the α2 subunit of AMPK and activates the trimeric kinase with an EC50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC)and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. Conclusion: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. General significance: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.

Volumetric productivity of camptothecin from the suspension culture of the endophyte Fusarium solani was enhanced up to ∼152 fold (from 0.19μgl-1 d-1 to 28.9μgl-1 d-1) under optimized fermentation conditions including initial pH (6.0), temperature (32°C) and agitation speed (80rpm) with (5% (v/v)) ethanol as medium component. Among various elicitors and precursors studied, tryptamine (0.5mM) as precursor and bovine serum albumin (BSA) (0.075mM) as an elicitor added on day 6 of the cultivation period resulted in maximum enhancement of camptothecin concentration (up to 4.5 and 3.4-fold, respectively). These leads provide immense scope for further enhancement in camptothecin productivity at bioreactor level. The cytotoxicity analysis of the crude camptothecin extract from the fungal biomass revealed its high effectiveness against colon and mammary gland cancer cell lines.

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis. © 2013 Kesavan et al.