SWATH-MS based quantitative proteomics analysis to evaluate the antileishmanial effect of Commiphora wightii- Guggul and amphotericin B on a clinical isolate of Leishmania donovani.

Drug resistance and relapse after treatment of visceral leishmaniasis (VL) with the chemotherapeutic drugs has impeded the VL elimination programme especially, in the endemic region of Bihar, India. Currently, Antimonials (Sbv) have been rendered obsolete (Bihar) as frequent treatment failure and relapse in Sbv treated patient's warrants greater vigilance and attention to the limited drugs. A clinical isolate of L.donovani obtained from an Amphotericin B (AmB) relapse patient was evaluated for its susceptibility to AmB and a hyperlipidemic drug Guggul. The evaluation of susceptibility or resistance to any drug still relies on in vitro assay on promastigote and amastigote stages of Leishmania spp. as there are no validated markers which can ascertain drug resistance in Leishmania. The anti-promastigote effect of AmB and Guggul were demonstrated by significant cellular and morphological changes exhibiting apoptosis-mediated cell death. To further illustrate the molecular mechanism of the parasite's response upon exposure to either AmB and Guggul, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) for quantitative proteomics analysis was performed along with computational data analysis; revealing considerable differences in the proteome profiles which could be regarded as putative markers for resistance or drug targets for development of therapeutic antileishmanials.

SWATH-MS based quantitative proteomics analysis to evaluate the antileishmanial effect of Commiphora wightii- Guggul and Amphotericin B on a clinical isolate of Leishmania donovani.

The present study provides comprehensive proteomics analyses of the response of L. donovani parasite to pharamacological stress in vitro. Identification of differentially expressed proteins with associated molecular functions and metabolic pathways, clearly provides an insight into the potential mechanism of the antileishmanial effects as well as a comparative response of the parasite to Guggul and AmB. Treatment of parasite with AmB results in an enhanced modulatory mechanism to counteract the drug induced stress which may have contributed to relapse. In the case of Guggul treatment, an effective antipromastigote activity was observed, which is being reported for the first time. Thus, a deeper understanding of the molecular pathways in the Leishmania parasite in response to pharmacological stress would help in designing novel and effective strategies in targeting the key molecules essential for parasite survival. It will also help in screening of new lead molecules targeting these vital pathways which could be used as an adjunct therapy along with the limited repertoire of antileishmanial drugs.

High-Performance Thin-Layer Chromatography Method for Simultaneous Determination of Antipsychotic and Medicinally Important Five ß-Carboline Alkaloids.

This is the first report on the development and validation of high-performance thin-layer chromatography (HPTLC) method for simultaneous analysis of five antipsychotic and medicinally important ß-carboline alkaloids (ßCAs), namely, harmalol, harmaline, harmine, harmane and norharmane. These ßCAs occurs in both plant and animal system including human being. In the present investigation, their best separation was achieved using an optimized mobile phase, chloroform: methanol: glacial acetic acid (7.8:2.2:0.2, v/v/v) on aluminum TLC plates precoated with silica gel 60 F254. The quantification was performed by densitometric scanning in fluorescence mode at 366 nm. The calibration curves were drawn using linear regression, plotted over the range 25-250 ng band-1 of standard ßCAs with correlation coefficient (R2) between 0.97 and 0.992. Accuracy in terms of recovery (83.95-112.40%), repeatability of application (0.61-2.42%), repeatability of measurement (1.94-3.05%) and intermediate precision (0.62-11.16%) of developed method were simultaneously determined. The limit of detection and limit of quantification were between 4.95-6.59 and 16.50-21.93 ng band-1, respectively. The method was validated according to ICH guidelines and was simple, cost-effective, precise, sensitive and specific for the determination of ßCAs in herbs, Fagonia schweinfurthii, Peganum harmala and Tribulus terrestris. The developed HPTLC method would have importance in forensic and industrial chromatographic analysis and fingerprinting of various herbs and drug formulations containing ßCAs.