Microbial community structure at different fermentation stages of kutajarista, a herbal formulation.

Kutajarista is an Ayurvedic fermented herbal formulation prescribed for gastrointestinal disorders. This herbal formulation undergoes a gradual fermentative process and takes around 2 months for production. In this study, microbial composition at initial stages of fermentation of Kutajarista was assessed by culture independent 16S rRNA gene clone library approach. Physicochemical changes were also compared at these stages of fermentation. High performance liquid chromatography-mass spectrometry analysis showed that Gallic acid, Ellagic acid, and its derivatives were the major chemical constituents recovered in this process. At 0 day of fermentation, Lactobacillus sp., Acinetobacter sp., Alcaligenes sp., and Methylobacterium sp. were recovered, but were not detected at 8 day of fermentation. Initially, microbial diversity increased after 8 days of fermentation with 11 operational taxonomic units (OTUs), which further decreased to 3 OTUs at 30 day of fermentation. Aeromonas sp., Pseudomonas sp., and Klebsiella sp. dominated till 30 day of fermentation. Predominance of γ- Proteobacteria and presence of gallolyl derivatives at the saturation stage of fermentation implies tannin degrading potential of these microbes. This is the first study to highlight the microbial role in an Ayurvedic herbal product fermentation.

Involvement of the PI3K/AKT pathway in the hypoglycemic effects of saponins from Helicteres isora.

AIM OF THE STUDY: Saponins from Helicteres isora have previously been shown to exert antidiabetic effects. The present study explored the underlying mechanisms in C2C12 skeletal muscle cells. MATERIALS AND METHODS: C2C12 cells were incubated with saponins and sapogenin followed by Western blotting and immunofluorescence analysis. RESULTS: Western blotting revealed that incubation with saponins (100 microg/ml) and sapogenin (100 microg/ml) induced the phosphorylation of the phosphatidylinositol-3-kinase (PI3K) as well as of the downstream targets protein kinase B/Akt (at Ser473) and glycogen synthase kinase GSK-3 alpha/beta (at Ser21/9) in a time-dependent manner. In contrast, no phosphorylation of the AMP-sensitive kinase AMPK (at Thr172) was observed. Within 48 h saponins/sapogenin treatment further increased the protein abundance of the insulin-sensitive glucose transporter Glut4. Confocal microscopy confirmed that saponins/sapogenin treatment stimulated Akt phosphorylation and revealed that the treatment was followed by translocation of Glut4 into the cell membrane of C2C12 muscle cells. CONCLUSIONS: Saponins and sapogenin activate the PI3K/Akt pathway thus leading to phosphorylation and inactivation of GSK-3 alpha/beta with subsequent stimulation of glycogen synthesis as well as increase of Glut4-dependent glucose transport across the cell membrane.

Liquid chromatography-mass spectrometry-based quantification of steroidal glycoalkaloids from Solanum xanthocarpum and effect of different extraction methods on their content.

A new liquid chromatography-mass spectrometry (LC-MS)-based method coupled with pressurized liquid extraction (PLE) as an efficient sample preparation technique has been developed for the quantification and fingerprint analysis of Solanum xanthocarpum. Optimum separations of the samples were achieved on a Waters MSC-18 XTerra column, using 0.5% (v/v) formic acid in water (A) and acetonitrile (ACN):2-propanol:formic acid (94.5:5:0.5, v/v/v) (B) as mobile phase. The separation was carried out using linear gradient elution with a flow rate of 1.0mL/min. The gradient was: 0min, 20% B; 14min, 30% B; 20min, 30% B; 27min, 60% B and the column was re-equilibrated to the initial condition (20% B) for 10min prior to next injection. The steroidal glycoalkaloids (SGAs) which are the major active constituents were isolated as pure compounds from the crude methanolic extract of S. xanthocarpum by preparative LC-MS and after characterization were used as external standards for the development and validation of the method. Extracts prepared by conventional Soxhlet extraction, PLE and ultrasonication were used for analysis. The method was validated for repeatability, precision (intra- and inter-day variation), accuracy (recovery) and sensitivity (limit of detection and limit of quantitation). The purpose of the work was to develop a validated method, which can be used for the quantification of SGAs in commercialized S. xanthocarpum products and the fingerprint analysis for their routine quality control.

LC and LC-MS study of stress decomposition behaviour of isoniazid and establishment of validated stability-indicating assay method.

Isoniazid was subjected to different ICH prescribed stress conditions of thermal stress, hydrolysis, oxidation and photolysis. The drug was stable to dry heat (50 and 60 degrees C). It showed extensive decomposition under hydrolytic conditions, while it was only moderately sensitive to oxidation stress. The solid drug turned intense yellow on exposure to light under accelerated conditions of temperature (40 degrees C) and humidity (75% RH). In total, three major degradation products were detected by LC. For establishment of stability-indicating assay, the reaction solutions in which different degradation products were formed were mixed, and the separation was optimized by varying the LC conditions. An acceptable separation was achieved using a C-18 column and a mobile phase comprising of water:acetonitrile (96:4, v/v), with flow rate and detection wavelength being 0.5 ml min(-1) and 254 nm, respectively. The degradation products appeared at relative retention times (RR(T)) of 0.71, 1.34 and 4.22. The validation studies established a linear response of the drug at concentrations between 50 and 1000 microg ml(-1). The mean values (+/-R.S.D.) of slope, intercept and correlation coefficient were 35,199 (+/-0.88), 114,310 (+/-4.70) and 0.9998 (+/-0.01), respectively. The mean R.S.D. values for intra- and inter-day precision were 0.24 and 0.90, respectively. The recovery of the drug ranged between 99.42 and 100.58%, when it was spiked to a mixture of solutions in which sufficient degradation was observed. The specificity was established through peak purity testing using a photodiode array detector. The method worked well on application to marketed formulation of isoniazid, and a fixed-dose combination containing isoniazid and ethambutol HCl. It was even extendable to LC-MS studies, which were carried out to identify the three degradation products. The m/z values of the peaks at RR(T) 0.71 and RR(T) 1.34 matched with isonicotinic acid and isonicotinamide, respectively. The product appearing at RR(T) 4.22 was isolated using preparative LC-MS, and turned out to be a yellow compound that was identified as isonicotinic acid N'-(pyridyl-4-carbonyl)-hydrazide based on mass, FTIR and (1)H/(13)C NMR spectral data. The same was indicated to be responsible for discolouration of isoniazid bulk drug substance and formulations, which is a familiar problem. The mechanism of formation of the said compound is outlined.