The present study reports production, partial purification, and media optimization for alkaline protease using Bacillus cereus PW3A. A profiling study for protease production indicates maximum enzyme activity (17.22 U/ml) was observed after 48 h of incubation. The studies also showed that the enzyme activity increased with the decrease in carbon content indicating the growth associated with nature protease production. Partial purification of protease was done using ammonium sulfate precipitation and dialysis. Further studies were conducted to assess significant media ingredients influencing protease production using the one-factor-at-a-time approach and Plackett-Burman design. Fructose and yeast extract were identified as the most significant variables. Response surface methodology was applied to optimize the factors for maximizing protease production. The results showed that the production increased from 17.22 U/ml to 47.43 U/ml indicating a three-fold augment in enzyme activity. Characterization of protease showed that the highest enzyme activity was shown at pH 8.0 and temperature 50°C; however, significant enzyme activity was retained till pH 10 and temperature 60°C. Using casein as substrate, the enzyme showed maximum activity V max 39 U/ml and K m 18 μM. The activity was enhanced by MgCl 2 and CuSO 4 and inhibited by HgCl 2 . Since the enzyme has both pH and temperature stability with greater substrate affinity, this protease finds many useful industrial applications.

Background: Non-alcoholic fatty liver disease (NAFLD) is a complex, multifactorial and multi-system disorder. It is one of the major contributors of liver disease worldwide. Among the many factors involved in the pathogenesis of NAFLD, free fatty acids (FFAs) such as palmitic acid induced lipotoxicity promotes steatosis, oxidative stress and insulin resistance that activate apoptotic cascades leading to tissue damage and inflammation. Since NAFLD is a multifactorial metabolic disorder, conventional target-based drug therapies have limited success. Therefore, the use of multicomponent ayurveda herbal formulations could be a promising alternative due to their multitargeted mechanisms of action. The present study investigates the effects and underlying mechanism of actions of an ayurveda formulation, Lodhrasavam (TDU-LS-1), in the in-vitro model of NAFLD. Methods: Lipotoxicity was induced in HepG2 cells by treating the cells with 1mM palmitic acid for 24 hrs followed by drug (TDU-LS-1) treatment for another 24 hrs. The effect of TDU-LS-1 on lipotoxicity was evaluated by MTT assay. The effect of TDU-LS-1 on steatosis was studied by estimating intracellular triglycerides, lipid droplets formation and expression of genes involved in lipid metabolism. Further, to examine the antioxidant activity, DPPH scavenging assay was performed. Results/discussion: TDU-LS-1 was found to increase the antioxidant activity in a concentration dependent manner with an IC50 of 16.45 μg GAE/ml. Palmitic acid induced lipotoxicity in HepG2 cells was reduced by lower concentrations of TDU-LS-1. Also, the results from triglyceride (TAG) assay, Oil-Red-O staining and BODIPY 493/503 confocal imaging suggest that TDU-LS-1 reduces the palmitate induced triglyceride deposition and lipid droplet accumulation in HepG2 cells. Further, the qRT-PCR analysis of TDU-LS-1 at a concentration of 32 μg/GAE revealed that it modulates the expression of SREBP, FASN, SCD1, ACOX, and PPARγ ; that are relevant in hepatic lipid metabolism. Our results suggest that TDU-LS-1 can reduce de novo lipogenesis, peroxisomal lipid peroxidation as well as lipotoxicity in the in vitro palmitate-induced NAFLD model in HePG2 cells.

No abstract

Frontiers of disease biology started recognizing the importance of systems and network medicine approach for managing chronic disease like diabetes. Polyherbal preparations like Ayurveda formulations are known to exert a multicomponent-multitargeted mode of action that makes them an ideal tool for delineating new biological insights into this systemic mechanism of disease manifestation and management. Additionally, these formulations are rich repository for identifying novel ligands that interact with drug targets having systemic effects. The current study aims at identifying DPP4 inhibitory potential and modulation of diabetes associated gene network by a clinically established Ayurvedic anti-diabetic formulation Nisakathakadi Kashaya (NK) using in vitro and in silico methods. DPP4 inhibitory potential of NK was evaluated by standard enzyme inhibition assay. Bioinformatics and computational biology tools were used to identify the potential bioactives responsible for the DPP4 inhibitory activity of NK. Molecular docking and dynamics studies of the identified compounds provided insights about the molecular interactions involved in DPP4 inhibition. Target mapping of phytochemicals using network pharmacology tools viz. STITCH, CHEMBL and BindingDB databases was used to depict the multi-targeted interaction of the formulation and a sub network for diabetes related genes and their relationship with other diabetes associated comorbidities were depicted with the help of EnrichR. NK demonstrated a dose dependent DPP4 inhibition with an IC50 of 2.06 μg GAE/mL. Further, the in silico studies identified three compounds namely Terchebin, Locaracemoside B and 1,2,4,6 Tetra o Galloyl Beta D Glucose showing stable interactions with DPP4 when compared to the standard drug Vildagliptin. Network pharmacology studies with the phytochemicals identified from NK revealed a number of genes like TNF, TGFβ, SOD1, SOD2, AKT1, DPP4 and GLP1R in its protein-protein interaction network which are vital to diabetic progression and complications. This suggests that a polyherbal formulation like NK can exert its action through various phytomolecules present. The present work demonstrated that the polyherbal formulation NK has DPP4 inhibition potential and modulates a large number of diabetes related genes and pathways. The approach adopted in the current study by combining in vitro and in silico methods allowed us to understand the mechanism of DPP4 inhibition by the formulation and also the possible pharmacological networking through which the formulation modulate diverse disease related targets.

No abstract