Bokka Ramesh

Effects of an integrated yoga program in modulating psychological stress and radiation-induced genotoxic stress in breast cancer patients undergoing radiotherapy.

Effects of an integrated yoga program in modulating perceived stress levels, anxiety, as well as depression levels and radiation-induced DNA damage were studied in 68 breast cancer patients undergoing radiotherapy. Two psychological questionnaires—Hospital Anxiety and Depression Scale (HADS) and Perceived Stress Scale (PSS)—and DNA damage assay were used in the study. There was a significant decrease in the HADS scores in the yoga intervention group, whereas the control group displayed an increase in these scores. Mean PSS was decreased in the yoga group, whereas the control group did not show any change pre- and postradiotherapy. Radiation-induced DNA damage was significantly elevated in both the yoga and control groups after radiotherapy, but the postradiotherapy DNA damage in the yoga group was slightly less when compared to the control group. An integrated approach of yoga intervention modulates the stress and DNA damage levels in breast cancer patients during radiotherapy.

Apoptogenic activity of ethyl acetate extract of leaves of Memecylon edule on human gastric carcinoma cells via mitochondrial dependent pathway

OBJECTIVE To evaluate the anti-proliferative and apoptogenic activity of ethyl acetate extract from the leaves of Memecylon edule (EtAc-LME) in MKN-74, NUGC gastric cancer cells and non cancerous gastric mucous cells (GES-1), and to explore the mechanism of EtAc-LME induced apoptosis. METHODS The mechanism of EtAc-LME induced apoptosis was explored by analysing the activation of pro-caspases, PARP cleavage, expression of cytochrome-c (Cyt-c) was determined by western blotting, mRNA expression of Bcl-2, Bax by RT-PCR, loss of mitochondrial potential using DiOC6 dye, annexin binding assay and its influence on cell cycle arrest by flow cytometry. RESULTS The results indicated that EtAc-LME inhibited the gastric cancer cell growth in dose-dependent manner and cytotoxicity was more towards the gastric cancer cells (NUGC and MKN-74) compared to normal gastric cells (GES-1), suggesting more specific cytotoxicity to the malignant cells. Over expression of Cyt-c and subsequent activation of caspases-3 and down regulation of Bcl-2 and loss in mitochondrial potential in EtAc-LME treated MKN-74 and NUGC cells suggested that EtAc-LME induced apoptosis by mitochondrial dependent pathway. CONCLUSIONS The present findings suggest that ethyl acetate extract of Memecylon edule induces apoptosis selectively in gastric cancer cells emphasizing the importance of this traditional medicine for its potential in the treatment of gastric cancer.

Comparison of conventional and supported liquid extraction methods for the determination of sitagliptin and simvastatin in rat plasma by LC–ESI–MS/MS

Three extraction methods were compared for their efficiency to analyze sitagliptin and simvastatin in rat plasma by LC–MS/MS, including (1) liquid–liquid extraction (LLE), (2) solid phase extraction (SPE) and (3) supported liquid extraction (SLE). Comparison of recoveries of analytes with different extraction methods revealed that SLE was the best extraction method. The detection was facilitated with ion trap-mass spectrometer by multiple reactions monitoring (MRM) in a positive ion mode with ESI. The transitions monitored were m/z 441.1→325.2 for simvastatin, 408.2→235.1 for sitagliptin and 278.1→260.1 for the IS. The lower limit of quantification (LLOQ) was 0.2 ng/mL for sitagliptin and 0.1 ng/mL for simvastatin. The effective SLE offers enhanced chromatographic selectivity, thus facilitating the potential utility of the method for routine analysis of biological samples along with pharmacokinetic studies.

Diatomaceous earth supported liquid extraction and LC-MS/MS determination of elvitegravir and ritonavir in rat plasma: application to a pharmacokinetic study

A rapid, sensitive and selective bio-analytical method was developed for determination of elvitegravir and ritonavir in rat plasma by high-performance liquid chromatography–tandem mass spectrometry. Sample preparation was based on diatomaceous earth supported liquid extraction using dichloromethane to extract elvitegravir and ritonavir from rat plasma. Chromatographic separation was performed on a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) using 20 mM ammonium formate containing 0.05% trifluoroacetic acid and acetonitrile (35 : 65 v/v) for isocratic elution and mass detection. Calibration curves were linear with regression coefficients greater than 0.997 over the concentration range of 2–5000 ng mL−1 for elvitegravir and ritonavir. Absolute mean recoveries were in the range of 87.31 to 92.18% and no significant interferences were observed at the retention times of the elvitegravir, ritonavir and internal standard. The method exhibited good intra- and inter-day performance in terms of 1.55–7.47% precision and 0–5% accuracy. The developed method has been successfully applied to the pharmacokinetic study of elvitegravir in healthy rats following a single oral administration of a 15 mg kg−1 dose boosted with 5 mg kg−1 ritonavir.

HPTLC METHOD FOR DETERMINATION OF DARUNAVIR IN RAT PLASMA AND ITS APPLICATION IN PHARMACOKINETIC STUDIES

A new, rapid, and sensitive high-performance thin-layer chromatography coupled with electrospray ionization mass spectrometry (HPTLC-ESI/MS) has been developed and validated for the quantification of darunavir in rat plasma and its application in pharmacokinetic studies. Detection and quantification were performed without using an internal standard. Protein precipitation method was followed for extracting darunavir from plasma. A saturated mixture of toluene: acetone: methanol (6:2:2 v/v) was used as mobile phase. Densitometric quantification was performed at λ = 262 nm by reflectance scanning. The method was linear over a concentration range of 5–150 ng/µL. The intra-day and inter-day precisions, expressed as RSD, were in the range of 2.15–2.77 (n = 3) and 2.13–2.47 (n = 3) respectively. The LOD was 1.24 ng/µL and LOQ was 3.85 ng/µL. The method proved to be accurate with a recovery between 94.77–98.44 and it was selective for the active principle tested. Additionally, selectivity of the method was confirmed by mass spectrometry. The mass spectra showed darunavir ion at m/z 569.80 [M +Na]+ being acquired directly from the rat plasma sample bands spiked with darunavir by an elution-based interface. The method was applied for the determination of plasma levels as well as pharmacokinetic study of darunavir administered orally to rats. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Quantification of bergenin from Mallotus philippinensis by HPTLC-MS and study on different extraction methods

A simple and accurate high-performance thin-layer chromatographic (HPTLC) method has been established for the determination of bergenin in the roots and stem-bark powder of Mallotus phillippensis. It has been reported to have a wide array of biological activities like antioxidant, anti-HIV, antiarrhythmic, hepatoprotective, anti-inflammatory and anti-microbial. Methanolic extract of the stem-bark and root powder was used for the experimental work. Separation was performed on 20 × 10 silica gel 60 F254 using ethyl acetate-methanol-acetic acid-formic acid (8:1:0.5:0.5, v/v) as mobile phase and scanned using densitometry at 284 nm. Extracts were prepared by conventional soaking, and accelerated solvent extractor (ASE) was used for the analysis. The results indicate that bergenin was found to be maximum in root ASE extract (6.0% w/w), while the minimum concentration of bergenin was found in stembark cold extract (2.9% w/w).The determination was carried out using the densitometric absorbance mode at 284 nm. The method was validated in terms of selectivity, linearity, precision, accuracy, and robustness. Additionally, peak identity was confirmed by mass spectrometry. The electron spray ionization (ESI) mass spectra showed the [M + Na]+ ions for bergenin detected at m/z 351 being acquired directly from the sample bands by an elution-based interface. By considering the validation results, the method was found to be very simple, accurate, precise, fast, and economical and can be used for routine quality control.

Direct injection HILIC–MS/MS analysis of darunavir in rat plasma applying supported liquid extraction

A novel bioanalytical method was developed and validated for the quantitative determination of darunavir (DRV) in rat plasma by employing hydrophilic interaction chromatography and tandem mass spectrometry (HILIC–MS/MS) with supported liquid extraction (SLE). Irbesartan (IRB) was used as an internal standard (IS). The analyte in rat plasma (200 µL) was isolated through SLE using ethyl acetate as the eluting solvent. The chromatographic separation was achieved on Luna-HILIC (250 mm×4.6 mm, 5 μm) column with a mobile phase of 0.1% of formic acid in water:acetonitrile (5: 95, v/v), at a constant flow rate of 1.0 mL/min. The MS/MS ion transitions for DRV (548.1→392.0) and IS (429.2→207.1) were monitored on an ion trap mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) was 0.2 ng/mL and quantitation range was 0.2–5000 ng/mL. The method was validated for its selectivity, sensitivity, carryover, linearity, precision, accuracy, recovery, matrix effect and stability. The method was successfully applied to pharmacokinetic study in rats.

Stability-indicating HPTLC method for analysis of venlafaxine hydrochloride, and use of the method to study degradation kinetics

A new quantitative densitometric HPTLC method for stability indicating analysis of venlafaxine hydrochloride, both in bulk and formulations, has been established and validated. Venlafaxine hydrochloride from formulations was separated and identified on silica gel 60F254 HPTLC plates with butanol-acetic acid-water 6:2:2 (v/v), as mobile phase. Densitometry was performed at 254 nm by reflectance scanning; a well-resolved band was obtained at RF 0.58 ± 0.02. Response to venlafaxine hydrochloride was a linear function of concentration in the range 100–600 ng, with correlation coefficient, slope, and intercept of 0.9984 ± 0.0004, 14.54 ± 0.04, and 141.75 ± 1.76 respectively. The minimum amounts of venlafaxine hydrochloride that could be authentically detected and quantified were 39.32 and 130.89 ng per band, respectively. The drug does not undergo degradation under oxidative or photolytic conditions but acidic and alkaline conditions resulted in degradation, as was evident from additional peaks observed. When the method was used to investigate the alkaline degradation kinetics it was observed the degradation process followed pseudo-first-order kinetics. The Arrhenius plot was constructed and the activation energy was calculated.

Validated High-Performance Thin-Layer Chromatographic Method for the Determination of Dibenzyl Cyclooctadiene Lignans from Schisandra grandiflora

A simple, accurate, and rapid high-performance thin-layer chromatographic (HPTLC) method has been established and validated for the simultaneous quantification of the four biologically active dibenzyl cyclooctadiene lignans, i.e., schisandrin (1), gomisin B (2), deoxyschisandrin (3), and gomisin N (4) from the hexane extract of Schisandra grandiflora (Wall.) Hook. f. & Thoms. Separation was performed on silica gel 60 F254 plates using a saturated mixture of toluene—ethyl acetate—methanol (6:1:1 v/v) as the mobile phase. Quantitation was performed using densitometric absorption—reflection mode at 225 nm. The method was validated for its selectivity, linearity, precision, and accuracy. HPTLC was hyphenated with a mass spectrometer as an additional tool for the confirmation of the markers using an interface. The developed method is simple and convenient which can be applied for the regular quality analysis of the raw plant material used in Chinese traditional herbal formulations.

Liquid chromatographic separation, determination and ESI-MS/MS, FT-IR and NMR characterization of the forced degradation products of cinacalcet

A validated stability indicating RP-HPLC assay of cinacalcet was developed by separating its related substances and degradants on a Phenomenex C8 (250 × 4.6 mm, 5 µm) column using 10 mM aqueous ammonium acetate–acetonitrile as the mobile phase in a gradient mode of elution at a flow rate of 1.0 mL min−1 at 25 °C. The column effluents were monitored by a photo diode array detector set at 270 nm. The method was validated for accuracy, precision and linearity according to ICH guidelines. The limits of quantification of the impurities were in the range of 0.23–0.30 µg mL−1. The forced degradation of cinacalcet was carried out under acidic, basic, thermal, photo, and peroxide conditions, and the degradation products were isolated and characterized by ESI-MS/MS, FT-IR, 1H and 13C NMR spectroscopy. The method was successfully applied not only to quantify the degradation products but also to quantify process related substances of cinacalcet in bulk drugs.