A novel UPLC-MS/MS method for simultaneous quantification of trigonelline, 4-hydroxyisoleucine, and diosgenin from Trigonella foenum-graecum extract: Application to pharmacokinetic study in healthy and type 2 diabetic rats.

Trigonelline (TR), 4-hydroxyisoleucine (4-HI), and diosgenin (DG) are the main bioactives of the purified standardized extract of the popular plant Trigonella foenum-graecum L. (TFG), and it has been proven effective for the treatment of various diseases. However, to the best of our knowledge, no study has investigated the pharmacokinetic parameters of purified standardized T. foenum-graecum extract in normal and diabetic Wistar rats. The present study has developed and validated a rapid, reliable, and sensitive simultaneous ultra-performance liquid chromatography MS method to estimate these bioactives. The chromatographic separation was achieved using methanol, acetonitrile, and 0.1% formic acid with the ideal gradient flow system on a BEH Shield RP 18 column. A positive electrospray ionization mode was selected to estimate m/z values of TR (138.14 > 94.63), 4-HI (148.19 > 74.08), and DG (415.54 > 271.33). The method was robust and reproducible over the linearity range of 60-5000, 6-5000, and 15-5000 ng/mL for TR, 4-HI, and DG, respectively. Using this novel validated method, we investigated the pharmacokinetic parameters of bioactives using Phoenix WinNonlin version 8.0 (Certera) in normal and diabetic rats. The assay was successfully applied for the estimation of pharmacokinetic parameters using noncompartmental analysis. This investigation shows that the absorption rate increased, whereas distribution and elimination processes slowed down in diabetic rats compared with normal rats.

Pre-clinical pharmacokinetic and pharmacodynamic modelling study of 4-hydroxyisoleucine using validated ultra-performance liquid chromatography-tandem mass spectrometry.

A reliable and sensitive ultra-performance liquid chromatography-tandem mass spectrometry-based method has been developed for the estimation of 4-hydroxyisoleucine (4-HI), a potent insulinotropic and hypolipidemic agent. The extraction of 4-HI from plasma was accomplished by the protein precipitation technique using l-isoleucine as an internal standard. The separation of analytes was achieved with a mobile phase consisting of acetonitrile and 0.1% formic acid in an isocratic flow system on a BEH Shield RP-18 column (150 mm × 2.1 mm, 1.7 µm). 4-HI and l-isoleucine were detected using an electrospray ionization (ESI) ion source, using multiple reaction monitoring (MRM) in positive ion mode. The precursor to product ion transitions of 4-HI and l-isoleucine were found at m/z values of 148.19 > 74.02 and 132.17 > 69.04, respectively. As per the guidelines for bioanalytical methods, all validation parameter results were within the acceptable range. The method exhibited a robust and reproducible linearity range of 1-5000 ng mL-1 with a coefficient of regression of 0.9999. The method was successfully applied for the estimation of pharmacokinetic parameters after oral administration of 4-HI (10 mg kg-1) in Wistar rats, by using Thoth Pro (version: 4.3) software. Herein, the two-compartment model was statistically fitted based on AIC and SBC values for evaluation of the pharmacokinetic parameters of 4-HI. Pharmacodynamic studies were also performed by measuring the levels of triglyceride and total cholesterol, and showed that the pharmacokinetic and pharmacodynamic data of 4-HI correlated with each other.

Drug discovery from plant sources: An integrated approach.

New drug discovery is facing serious challenges due to reduction in number of new drug approvals coupled with exorbitant rising cost. Advent of combinatorial chemistry provided new hope of higher success rates of new chemical entities (NCEs); however, even this scientific development has failed to improve the success rate in new drug discovery. This scenario has prompted us to come out with a novel approach of integrated drug discovery, where Ayurvedic wisdom can synergize with drug discovery from plant sources. Initial steps in new drug discovery involve identification of NCEs, which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation. The sources of many of the new drugs and active ingredients of medicines are derived from natural products. The starting point for plant-based new drug discovery should be identification of the right candidate plants by applying Ayurvedic wisdom, traditional documented use, tribal non-documented use, and exhaustive literature search. Frequency analysis of the ingredients of the ancient documented formulations and analysis of their Ayurvedic attributes may provide an in-depth idea of the predominance of particular Ayurvedic characteristics based on which appropriate candidate plants may be selected for bioactivity-based fractionation. The integration of Ayurvedic wisdom with drug discovery also brings the need for a paradigm shift in the extraction process from sequential to parallel extraction. Bioassay-guided fractionation of the identified plant may lead to standardized extract or isolated bioactive druggable compound as the new drug. This integrated approach would lead to saving of cost and time, coupled with enhanced success rate in drug discovery.

Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?

AIM: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study. MATERIALS AND METHODS: The effects of extract on glucose uptake, insulin receptor-ß (IR-ß), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days. RESULTS: The effect of extract promoted glucose uptake, IR-ß and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC(50) 17.4 µg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment). CONCLUSION: The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B.

Ayurvedic processed seeds of nux-vomica: neuropharmacological and chemical evaluation.

The effect of detoxification on Strychnos nux-vomica seeds by traditional processing with aloe and ginger juices (B), by frying in cow ghee (C), and by boiling in cow milk (D) was investigated. The ethanolic extracts of these samples were subjected to spontaneous motor activity (SMA), pentobarbitone-induced hypnosis, PTZ induced convulsions, diazepam-assisted protection, and morphine-induced catalepsy. All samples reduced SMA and inhibited catalepsy. The seeds processed in milk (D) showed the lowest strychnine content in the cotyledons, exhibited marked inhibition of PTZ induced convulsions and maximal potentiation of hypnosis, and were the safest (LD(50)).