Litsea glutinosa extract promotes fracture healing and prevents bone loss via BMP2/SMAD1 signaling.

Estrogen deficiency is one of the main causes for postmenopausal osteoporosis. Current osteoporotic therapies are of high cost and associated with serious side effects. So there is an urgent need for cost-effective anti-osteoporotic agents. Anti-osteoporotic activity of Litsea glutinosa extract (LGE) is less explored. Moreover, its role in fracture healing and mechanism of action is still unknown. In the present study we explore the osteoprotective potential of LGE in osteoblast cells and fractured and ovariectomized (Ovx) mice models. Alkaline phosphatase (ALP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and mineralization assays revealed that LGE treatment increased osteoblast cell differentiation, viability and mineralization. LGE treatment at 0.01 µg increased the expression of BMP2, PSMAD, RUNX2 and type 1 col. LGE also mitigated RANKL-induced osteoclastogenesis. Next, drill hole injury Balb/C mice model was treated with LGE for 12 days. Micro-CT analysis and Calcein labeling at the fracture site showed that LGE (20 mg/kg) enhanced new bone formation and bone regeneration, also increased expression of BMP2/SMAD1 signaling genes at fracture site. Ovx mice were treated with LGE for 1 month. µCT analysis indicated that the treatment of LGE at 20 mg/kg dose prevented the alteration in bone microarchitecture and maintained bone mineral density and bone mineral content. Treatment also increased bone strength and restored the bone turnover markers. Furthermore, in bone samples, LGE increased osteogenesis by enhancing the expression of BMP2/SMAD1 signaling components and decreased osteoclast number and surface. We conclude that LGE promotes osteogenesis via modulating the BMP2/SMAD1 signaling pathway. The study advocates the therapeutic potential of LGE in osteoporosis treatment.

Structure analysis of depsides, dibenzofuran and sugar derivatives from Cladia aggregata (SW) Nyl using ultra-performance liquid chromatography-tandem mass spectrometry.

Cladia aggregate (SW) Nyl is a lichenized fungi in the family Cladoniaceae producing characteristic secondary metabolites of interest. There are only limited chemical studies relating to the genus Cladia. A chemical study of the lichen C. aggregata was conducted and their chemical constituents were elucidated by ultra-performance liquid chromatography-electrospray ionization/triple-quadrupole tandem-mass spectrometry analysis. It is the first time report of structure analysis of its metabolite by liquid chromatography-mass spectrometry/mass spectrometry. The molecular masses for 20 compounds were detected from different fractions. Seven compounds were elucidated with mass spectrometry/mass spectrometry fragmentation pattern analysis. Barbatic acid (12) was identified as the major compound being common to all fractions. The identified compounds belong to depsides (2, 11, 12 and 20), dibenzofurans (13, 18) and sugar derivatives (1) which are usually distributed in lichens.

Quantification of Arbortristoside-A isolated from Nyctanthes arbor-tristis using HPLC: Method development and pharmaceutical applications.

Arbortristoside-A (Arbor-A) is a naturally occurring iridoid glycoside and herbal-based lead molecule with proven medicinal potential. Aiming at the development of an efficient analytical tool for the quantification of Arbor-A in pharmaceutical dosage forms, in the presented work, we developed an economical, fast, and sensitive RP-HPLC-UV method and validated the procedure as per the ICH guidelines, Q2(R1). The chromatographic separation was accomplished under the optimised experimental conditions using an HPLC system with an LC-2010 autosampler, a PDA detector, and a Phenomenex C18 column with the mobile phase composed of a 70:30 (v/v) water-acetonitrile mixture eluting isocratically at a flow rate of 1 mL/min at ambient temperature, and UV detection at 310 nm. Arbor-A showed a sharp peak at the retention time of 5.60 min and exhibited linearity (R2 = 0.9988) with LOD and LOQ of 0.50 µg/mL and 1.50 µg/mL, respectively. The accuracy of the method was 98.33-101.36 % with acceptable intra-day and inter-day precisions as well as robustness (<2% RSD). To ratify the applicability of the presented approach in emerging pharmaceuticals, a nanoformulation loaded with Arbor-A was designed and analysed utilising the provided methodology. The method has also enabled to determine the degradation kinetics of Arbor-A under stress conditions, etcetera, employing forced degradation and short term stability studies.

Bone Fracture-healing Properties and UPLC-MS Analysis of an Enriched Flavonoid Fraction from Oxystelma esculentum.

Oxystelma esculentum has been used as a folk medicine to treat jaundice, throat infections, and skin problems. In the current study, the bone fracture-healing properties of a flavonoid-enriched fraction (Oxy50-60F) of O. esculentum were investigated in Swiss mice using a drill-hole injury model. Oxy50-60F (1 mg/kg/day, 5 mg/kg/day, and 10 mg/kg/day) was administered orally (from the next day) after a 0.6 mm drill-hole injury in mice femur mid-diaphysis for 7 days and 14 days. Parathyroid hormone (40 µg/kg; 5 times/week) was given subcutaneously as the positive control. Confocal imaging for bone regeneration, micro-architecture of femur bones, ex vivo mineralization, hematoxyline and eosin staining, measurement of reactive oxygen species, and gene expression of osteogenic and anti-inflammatory genes were studied. Quercetin, kaempferol, and isorhamnetin glycosides were identified in the active fraction using mass spectrometry techniques. Our results confirm that Oxy50-60F treatment promotes fracture healing and callus formation at drill-hole sites and stimulates osteogenic and anti-inflammatory genes. Oxy50-60F administration to fractured mice exhibited significantly better micro-CT parameters in a dose-dependent manner and promoted nodule mineralization at days 7 and 14 post-injury. Oxy50-60F also prevents ROS generation by increasing expression of the SOD2 enzyme. Overall, this study reveals that Oxy50-60F has bone regeneration potential in a cortical bone defect model, which supports its use in delayed-union and non-union fracture cases.

Hesperidin: Enrichment, forced degradation, and structural elucidation of potential degradation products using spectral techniques.

RATIONALE: Hesperidin (HES) is a well-known citrus bioflavonoid phyto-nutraceutical agent with polypharmacological properties. After 2019, HES was widely used for prophylaxis and COVID-19 treatment. Moreover, it is commonly prescribed for treating varicose veins and other diseases in routine clinical practice. Pharmaceutical impurities and degradation products (DP) impact the drug's quality and safety and thus its effectiveness. Therefore, forced degradation studies help study drug stability, degradation mechanisms, and their DPs. This study was performed because stress stability studies using detailed structural characterization of hesperidin are currently unavailable in the literature. METHODS: In the HES enrichment method crude HES was converted to its pure form (98% purity) using column chromatography and then subjected to forced degradation under acid, base, and neutral hydrolyses followed by oxidative, reductive, photolytic, and thermal stress testing (International Conference on Harmonization guidelines). The stability-indicating analytical method (SIAM) was developed to determine DPs using reversed-phase high-performance liquid chromatography (C18 column with methanol and 0.1% v/v acetic acid in deionized water [70:30, v/v] at 284 nm). Further, structural characterization of DPs was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In addition, in silico toxicity predictions were performed using pKCSM and DataWarior freeware. RESULTS: HES was found to be susceptible to acidic and basic hydrolytic conditions and yielded three DPs in each, which were detected using designed SIAM. Of six DPs, three were pseudo-DPs (short lived), and the remaining were characterized using LC-MS/MS and NMR spectroscopy. The tentative mechanism of the formation of proposed DPs was explained. The proposed DPs were found inactive from in silico toxicity predictions. CONCLUSIONS: Hesperidin was labile under acidic and basic stress conditions. The potential DPs were characterized using LC-ESI-MS/MS and NMR spectral techniques. The proposed mechanism of formation was hypothesized. In addition, to identify and characterize the DPs, a SIAM, which has broad biomedical applications, was successfully developed.

The characterization and quantification of structures of Cajanus scarabaeoides phytochemicals and their seasonal variation analysis using ultra-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Cajanus scarabaeoides, belonging to the Fabaceae family, is an underutilized herb and traditionally used to treat several ailments. However, it is not well explored phytochemically. Therefore, mass spectrometry (MS)-based phytochemical analysis was carried out to investigate the bioactive ingredients of the herb. METHODS: A ultra-performance liquid chromatography (UPLC) coupled to photodiode array detection (PDA) and electrospray ionization (ESI) tandem mass spectrometry (UPLC-PDA-ESI-MS/MS) system was used for the qualitative and quantitative analysis of phytochemicals. The chromatographic separation was achieved on the Acquity BEH C18 column (150 × 2.1 mm, 1.7 µm) using a gradient system consisting of three solvents, acetonitrile, methanol, and 0.1% formic acid, used at a flow rate of 0.300 ml/min. RESULTS: Sixteen bioactive ingredients (gallic acid, gallocatechin, epigallocatechin, catechin, procyanidin dimer, epicatechin, procyanidin trimer, isoorientin, orientin, vitexin, isovitexin, quercetin-mono-O-glycoside, isoquercitrin, luteolin-7-O-glucoside, quercetin, and luteolin) were identified and structurally characterized. Consequently, 12 compounds were reported for the first time from C. scarabaeoides, and 13 were quantitatively determined in different seasons. Isoorientin (10.2-7.1% w/w) and orientin (5.78-5.17% w/w) were the most abundant constituents in the dry weight of plant material, followed by vitexin and isovitexin in the rainy season. CONCLUSIONS: The phytochemical investigation has revealed that C. scarabaeoides could be a potential alternate source of bioactive ingredients, namely, isoorientin, orientin, vitexin, and isovitexin, contributing to further exploration of its biological activity. In addition, analytical methods can be used for the rapid identification and quantification of bioactive ingredients in C. scarabaeoides.

Aurintricarboxylic acid mitigates cigarette smoke extract induced oxidative stress and pulmonary inflammation via inhibition of NF-Ò¡B/p65 signaling.

Cigarette smoke (CS) induced emphysema and chronic pulmonary inflammation are major comorbidities of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. CS exposure exacerbates pulmonary inflammation and compromises immunity to various infections. Aurintricarboxylic acid (ATA) is a polyanionic aromatic compound especially recognized for its anti-inflammatory, nucleic acid, and protein interaction inhibition properties. The study was designed to investigate the anti-inflammatory role of ATA against cigarette smoke extract (CSE) induced pulmonary inflammation. Nicotine concentration was quantified in CSE by UPLC/MS technique. In vitro, fluorescence microscopy, and flow cytometry was performed in CSE stimulated alveolar epithelial cells to determine the effect of ATA on oxidative stress-mediated cellular apoptosis. In vivo, pulmonary inflammation was induced in male Wistar rats via a modified non-invasive intratracheal instillation of cigarette smoke extract (100 µl/animal) twice a week for 8 weeks and post-treated with ATA (10 mg/kg) intraperitoneally for 15 days. Lung homogenates were assessed for MDA and GSH. Lung tissues were subjected to western blotting and histopathological analysis. As result, ATA reduced CSE-induced chromatin condensation, fragmentation, cellular apoptosis in alveolar epithelial cells, and apoptotic biomarkers expression including BAX and Caspase-3 in the lungs. ATA reduced inflammation by normalizing redox balance reflected by MDA/GSH levels. ATA obviated airspace enlargement, fiber deposition, and immune cell infiltration. Reduced inflammation was accompanied by inhibition of inflammatory biomarkers TNF-α, TNFR1, TWEAK, and NF-Ò¡B/p65 activation and nuclear translocation. ATA efficaciously diminished the oxidative stress and pulmonary inflammation associated with lung pathogenesis through TNF-α/TNFR1/NF-Ò¡B/p65 signaling pathway. HIGHLIGHTSATA treatment attenuates CSE-stimulated chromatin condensation, fragmentation, and cellular apoptosis in alveolar epithelial cells.ATA treatment inhibits CSE stimulated activation and nuclear translocation of NF-Ò¡B/p65.ATA treatment diminishes CSE-induced oxidant injury, apoptosis, and emphysema-like phenotypic changes in the lungs.ATA inhibits lung inflammation via suppression of the NF-Ò¡B/p65 signaling pathway.

Assessment of Tissue Specific Distribution and Seasonal Variation of Alkaloids in Alstonia scholaris.

Alstonia scholaris is a well-known source of alkaloids and widely recognized for therapeutic purposes to treat the ailments in human and livestock. However, the composition and production of alkaloids vary due to tissue specific metabolism and seasonal variation. This study investigated alkaloids in leaves, stems, trunk barks, fruits, and flowers of A. scholaris. The impact of seasonal changes on the production of alkaloids in the leaves of A. scholaris was also investigated. One and two-dimensional Nuclear Magnetic Resonance (NMR) experiments were utilized for the characterization of alkaloids and total eight alkaloids (picrinine, picralinal, akuammidine, 19 S scholaricine, 19,20 E vallesamine, Nb-demethylalstogustine N-Oxide, Nb-demethylalstogustine, and echitamine) were characterized and quantified. Quantitative and multivariate analysis suggested that the alkaloids content is tissue specific, illustrating the effect of plant tissue organization on alkaloidal production in A. scholaris. The results suggest that the best part to obtain alkaloids is trunk barks, since it contains 7 alkaloids. However, the best part for isolating picrinine, picralinal, akuammidine, 19 S scholaricine, and 19,20 E vallesamine is fruit, since it shows highest amount of these alkaloids. Undoubtedly, NMR and statistical methods are very helpful to differentiate the profile of alkaloids in A. scholaris.

Furostanol saponins from Asparagus racemosus as potential hypoglycemic agents.

Bioactivity guided phytochemical investigation led to isolation of six undescribed furostanol saponins, furoasparoside A-F along with five known compounds, gallic acid, methyl gallate, quercetin-3-O-ß-glucopyranoside, liquiritigenin 4׳-O-ß-apiofuranosyl-(1 â†’ 2)-ß-glucopyranoside and ß-glucogallin for the first time from the roots of Asparagus racemosus. Isolated saponins were screened for their antidiabetic potential in L6-GLUT4myc myotubes in vitro followed by an in vivo evaluation in streptozocin-induced diabetic rats and db/db mice. Furoasparoside E produced a notable decrease in the postprandial blood glucose profile, in leptin receptor-deficient db/db mice, type 2 diabetes model. The effect of furoasparoside E on GLUT4 translocation was found to be mediated by the AMPK-dependent signaling pathway in L6-GLUT4myc myotubes. Moreover, it emerged as a stable plant metabolite with higher bioavailability and efficacy in in vivo pharmacokinetic studies. Therefore, these studies indicated that furoasparoside E may serve as a propitious lead for the management of type 2 diabetes and its secondary complications from natural source.

Quantitative evaluation of cardiac glycosides and their seasonal variation analysis in Nerium oleander using UHPLC-ESI-MS/MS.

INTRODUCTION: Nerium oleander is an eminent source of structurally diverse cardiac glycosides (CGs), plays a prominent role in the treatment of heart failure, and inhibits the proliferation of cancer cell lines. CGs exert their cardiotonic action by binding to the extracellularly exposed recognition sites on Na+ /K+ -ATPase, an integral membrane protein that establishes the electrochemical gradient of Na+ and K+ ions across the plasma membrane. OBJECTIVE: We aimed to quantitatively determine CGs and their seasonal variation in leaf and stem samples of N. oleander utilizing UHPLC-ESI-MS/MS techniques. METHODS: The UHPLC-ESI-MS/MS analytical method was developed utilizing multiple reaction monitoring (MRM) mode. The Waters BEH C18 (150 mm × 2.1 mm, 1.7 µm) column was used with a 22-min linear gradient consisting of acetonitrile and 5 mM ammonium acetate buffer. RESULTS: In total 21 CGs were quantitatively determined in the seasonal leaf and stem samples of N. oleander along with the absolute quantitation of the three chemical markers odoroside H (244.8 µg/g), odoroside A (231.4 µg/g), and oleandrin (703.9 µg/g). The season-specific accumulation of chemical markers was observed in the order of predominance odoroside A (summer season, stem), odoroside H (winter season, stem), and oleandrin (rainy season, leaf). Besides this, the remaining 18 CGs were relatively quantified in the same samples. CONCLUSION: The developed method is simple and reliable and can be used for the identification and quantification of multiple CGs in N. oleander.

Extract and fraction of Musa paradisiaca flower have osteogenic effect and prevent ovariectomy induced osteopenia.

BACKGROUND: Osteoporosis is an asymptomatic bone disorder leading to altered bone microarchitecture, mineralization and strength. Musa paradisiaca has been reported to have antioxidant and anti-inflammatory effects in various diseases. Its impact on postmenopausal osteoporosis has not been investigated yet. PURPOSE: The intention of the current study was to evaluate the bone regeneration and osteoprotective potential of extract and fraction of M. paradisiaca flower in ovariectomized (Ovx) Sprague Dawley (SD) rats, a model of post-menopausal bone loss. The study also aims to identify osteogenic compounds from active fraction. METHODS: Ethanolic extract (MFE) and butanolic fraction (MFE-Bu) from flower of M. paradisiaca were prepared and their efficacy was tested in rat femur osteotomy model at different doses. Effective dose from both extract (250 mg/kg) and fraction (50 mg/kg) were taken for study in osteopenic bone loss model. PTH was taken as reference standard (20 µg/kg/twice a week). Bones were harvested at autopsy for dynamic and static histomorphometry. Serum was collected for ELISA. Pure compounds were isolated from butanolic fraction (MFE-Bu), and were assessed for their osteogenic effect. RESULTS: MFE and MFE-Bu were observed for their potential in bone healing and prevention of bone loss. Both MFE and MFE-Bu promoted new bone regeneration at injury site as assessed by microCT and calcein dye labeling studies. These also led to restoration of bone microarchitecture deteriorated as a result of osteopenia and improved bone biomechanical properties. Extract as well as the fraction exhibited dual bone anabolic and anti-resorptive properties where they elevated serum procollagen type I N-terminal propeptide (P1NP), a bone formation marker and suppressed serum C-telopeptide of type I collagen (CTX-1), a bone resorption marker. As many as four osteogenic compounds were isolated from MFE-Bu. Oleracein-E was found to be the most potent osteogenic agent based on osteoblast differentiation, mineralization assays, qPCR and protein expression studies. CONCLUSION: Our studies demonstrates that ethanolic extract from the flower of M. paradisiaca and its butanolic fraction exhibit dual osteogenic and anti-resorptive potential, and have an advantage over PTH which though promotes bone formation but is also bone catabolic in nature.

Quantitative Analysis of Bioactive Carbazole Alkaloids in Murraya koenigii (L.) from Six Different Climatic Zones of India Using UPLC/MS/MS and Their Principal Component Analysis.

Murraya koenigii (L.) Spreng (Curry leaf) is a commercially important medicinal plant in South Asia, containing therapeutically valuable carbazole alkaloids (CAs). Thus, the quantitative evaluation of these compounds from different climatic zones of India are an important aspect for quality assessment and economic isolation of targeted compounds from the plant. In this study, quantitative estimation of CAs among 34 Indian natural populations of M. koenigii was assessed using UPLC/MS/MS. The collected populations represent the humid subtropical, tropical wet & dry, tropical wet, semi-arid, arid, and montane climatic zones of India. A total of 11 CAs viz. koenine-I, murrayamine A, koenigine, koenimbidine, koenimbine, O-methylmurrayamine A, girinimbine, mahanine, 8,8''-biskoenigine, isomahanimbine, and mahanimbine were quantified using multiple reaction monitoring (MRM) experiments within 5.0 min. The respective range for natural abundance of CAs were observed as 0.097-1.222, 0.092-5.014, 0.034-0.661, 0.010-1.673, 0.013-7.336, 0.010-0.310, 0.010-0.114, 0.049-5.288, 0.031-1.731, 0.491-3.791, and 0.492-5.399 mg/g in leaves of M. koenigii. The developed method shown linearity regression coefficient (r2 >0.9995), LOD (0.003-0.248 ng/mL), LOQ (0.009-0.754 ng/mL), and the recovery was between 88.803-103.729 %. The bulk of these CAs were recorded in their highest concentrations in the humid subtropical zone, followed by the tropical wet & dry zones of India. Further, principal component analysis (PCA) was performed which differentiated the climatic zones according to the dominant and significant CAs contents within the populations. The study concludes that the method established is simple, rapid, with high sample throughput, and can be used as a tool for commercial purposes and quality control of M. koenigii.

Regulatory safety pharmacology and toxicity assessments of a standardized stem extract of Cassia occidentalis Linn. in rodents.

Cassia occidentalis Linn (CO) is an annual/perennial plant having traditional uses in the treatments of ringworm, gastrointestinal ailments and piles, bone fracture, and wound healing. Previously, we confirmed the medicinal use of the stem extract (ethanolic) of CO (henceforth CSE) in fracture healing at 250 mg/kg dose in rats and described an osteogenic mode of action of four phytochemicals present in CSE. Here we studied CSE's preclinical safety and toxicity. CSE prepared as per regulations of Current Good Manufacturing Practice for human pharmaceuticals/phytopharmaceuticals and all studies were performed in rodents in a GLP-accredited facility. In acute dose toxicity as per New Drug and Clinical Trial Rules, 2019 (prior name schedule Y), in rats and mice and ten-day dose range-finding study in rats, CSE showed no mortality and no gross abnormality at 2500 mg/kg dose. Safety Pharmacology showed no adverse effect on central nervous system, cardiovascular system, and respiratory system at 2500 mg/kg dose. CSE was not mutagenic in the Ames test and did not cause clastogenicity assessed by in vivo bone marrow genotoxicity assay. By a sub chronic (90 days) repeated dose (as per OECD, 408 guideline) study in rats, the no-observed-adverse-effect-level was found to be 2500 mg/kg assessed by clinico-biochemistry and all organs histopathology. We conclude that CSE is safe up to 10X the dose required for its osteogenic effect.

Structural Analysis of Diastereomeric Cardiac Glycosides and Their Genins Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is an economical and indispensable tool in natural product research to investigate novel metabolites, biomarker discovery, chemical diversity exploration, and structure elucidation. In this study, the structural analysis of 38 naturally occurring cardiac glycosides (CGs) in various tissues of Nerium oleander was achieved by the extensive use of mass spectrometry. The chemical diversity of CGs was described on the basis of characteristic MS/MS fragmentation patterns, accurate mass measurement, and published scientific information on CGs from Nerium oleander. It was observed that only six genins, viz., Δ16anhydrogitoxigenin, Δ16adynerigenin, gitoxigenin, oleandrigenin, digitoxigenin, and adynerigenine, produce 38 diverse chemical structures of CGs. Among them, 20 were identified as diastereomers having a difference in a sugar (l-oleandrose, ß-d-diginose, and ß-d-sarmentose) unit. However, the differentiation of diastereomeric CGs was not possible by only MS/MS fragments. Thus, the diastereomer's chromatographic elution order was assigned on the basis of the relative retention time (RRt) of two reference standards (odoroside A and oleandrin) among their diastereomers. Besides this, the in-source fragmentation of CGs and the MS/MS of m/z 325 and 323 disaccharide daughter ions also exposed the intrinsic structure information on the sugar units. The daughter ions m/z 162, 145, 113, 95, and 85 in MS/MS spectra indicated the abundance of l-oleandrose, ß-d-diginose, and ß-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a ß-d-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Rapid and Simultaneous Analysis of Multiple Classes of Antimicrobial Drugs by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Routine Biomedical, Food, and Soil Analyses.

Antimicrobial agents (AMAs) are widely exploited nowadays to meet the high demand for animal-derived food. It has a significant impact on the food chain whose end consumers are human beings. The burden of AMAs on humans comes from either meat or crops cultivated on soil containing high residual antibiotics, which are responsible for the global crisis of antibiotic resistance. Thus, the objective of this study was to design a selective and sensitive liquid chromatography-mass spectrometry (LC-MS)/MS-based simultaneous bioanalytical method for estimation of twenty AMAs in human plasma, raw meat, and soil samples. The selective extraction of all analytes from the above matrices was performed by the solid-phase extraction clean-up method to overcome the interferences. Analytes were separated on a Waters Symmetry Shield C18 (150 × 4.6 mm2, 5 µm) column, using an isocratic solvent system of methanol-0.5% formic acid (80:20, v/v) with 0.75 mL/min flow rate. The average extraction recoveries for all analytes in plasma were ranged from 42.0 to 94.0% with relative standard deviations (RSDs) below ±15%. All of the validation parameters are in accordance with the United State Food and Drug Administration (USFDA) guidelines. Moreover, the method was also valid for a broad plasma concentration range and can be proposed as an excellent method for routine pharmacokinetic studies, therapeutic drug monitoring, clinical analysis, and detection and quantitation of AMA remnants in raw meat as a standard quality control test for human consumption.

Extra-ribosomal functions of Mtb RpsB in imparting stress resilience and drug tolerance to mycobacteria.

Emerging observations suggest that ribosomal proteins (RPs) play important extra-ribosomal roles in maintenance of cellular homeostasis. However, the mechanistic insights into these processes have not been extensively explored, especially in pathogenic bacteria. Here, we present our findings on potential extra-ribosomal functions of Mycobacterium tuberculosis (Mtb) RPs. We observed that Mtb RpsB and RpsQ are differentially localized to cell wall fraction in M. tuberculosis (H37Rv), while their M. smegmatis (Msm) homologs are primarily cytosolic. Cellular fractionation of ectopically expressed Mtb RPs in surrogate host (M. smegmatis) also shows their association with cell membrane/cell wall without any gross changes in cell morphology. M. smegmatis expressing Mtb RpsB exhibited altered redox homeostasis, decreased drug-induced ROS, reduced cell wall permeability and increased tolerance to various proteotoxic stress (oxidative stress, SDS and starvation). Mtb RpsB expression was also associated with increased resistance specifically towards Isoniazid, Ethionamide and Streptomycin. The enhanced drug tolerance was specific to Mtb RpsB and not observed upon ectopic expression of M. smegmatis homolog (Msm RpsB). Interestingly, C-terminus deletion in Mtb RpsB affected its localization and reversed the stress-resilient phenotypes. We also observed that M. tuberculosis (H37Rv) with upregulated RpsB levels had higher intracellular survival in macrophage. All these observations hint towards existence of moonlighting roles of Mtb RpsB in imparting stress resilience to mycobacteria. This work open avenues for further exploration of alternative pathways associated with fitness and drug tolerance in mycobacteria.

Molecular mechanism of apoptosis induction in Jurkat E6-1 cells by Tribulus terrestris alkaloids extract.

The present study demonstrates apoptosis-inducing potential and mechanism of action of Tribulus terristris alkaloid extract in Jurkat E6-1 cancer cell line. Liquid Chromatography-Mass Spectrometry and High Resolution-Mass Spectrometry analysis identified the presence of four N-feruloyltyramine derivatives, namely trans-N-feruloyl-3-hydroxytyramine (1), trans-N-coumaroyltyramine (2), trans-N-feruloyltyramine (3) and trans-N-feruloyl-3-ethoxytyramine (4) in the alkaloid extract. Compounds 2 and 3 have not been yet reported in the alkaloid extract of T. terristris. In silico analysis revealed therapeutic potential of N-feruloyltyramine derivatives and strong binding efficiency to both chains of Tumor Necrosis Factor Receptor 1. Treatment of alkaloids extract to Jurkat E6-1 clone induced dose-dependent cytotoxicity (LC50 140.4 µg mL-1). Jurkat cells treated with alkaloids extract at sub-lethal concentration showed DNA fragmentation, enhancement in caspase-3 activity and phosphatidylserine translocation (apoptosis indicator) compared to control cells. Gene expression analysis using Human Apoptosis RT2 Profiler PCR Array analysis upon alkaloid treatment was found to significantly alter expression of critical genes such as TNFR1, FADD, AIFM, CASP8, TP53, DFFA and NFKB1. These genes are predicted to mediate apoptotic cell death via both intrinsic and extrinsic apoptosis pathway. In summary, we report the identification of new N-feruloyltyramine derivatives from alkaloid extract of T. terristris fruit with probable anti-leukemic and pharmacological potential.

Pyranocarbazoles from Murraya koenigii (L.) Spreng. as antimicrobial agents.

The bioassay guided fractionation of methanolic extract of Murraya koenigii (L.) Spreng. leaves resulted in the isolation of seven pyranocarbazoles. These were evaluated against four bacterial strains and ten Candida sp. including two matched pair of fluconazole sensitive/resistant clinical isolates. Out of seven, three i.e. Koenine (mk279), Koenigine (mk309) and Mahanine (mk347) exhibited significant antibacterial activity MIC90 3.12-12.5 µg/mL against bacterial strains Streptococcus aureus and Klebsiella pneumonia compared with standard drug Kanamycin MIC90 12.5 µg/mL. However, only mk309 was found active against variety of Candida species MIC90 12.5-100 µg/mL. It was observed that hydroxylation at C-6 and C-7 positions in the studied pyranocarbazoles activate the bioactivity. Simultaneously, decrease in Log P value compares with -H and -O-CH3 substituted derivatives. The study is focused on selective antifungal and antibacterial activity of pyranocarbazoles on bacterial strains S. aureus, K. pneumonia and variety of Candida species with structure activity relationship observations.

Ethyl acetate fraction of Eclipta alba: a potential phytopharmaceutical targeting adipocyte differentiation.

Natural products have always fascinated mankind for their miraculous properties. Eclipta alba (E. alba), a medicinal herb has long been used in traditional medicine for curing several pathologies. It has been shown to have anti-diabetic effect as well as hepato-protective activity. Here, in order to address metabolic derangements, the study was designed to evaluate the efficacy of E. alba and its fractions in adipogenesis inhibition and dyslipidemia. Of the crude extract and fractions screened, ethyl acetate fraction of E. alba inhibited adipocyte differentiation in 3T3-L1 pre-adipocytes and hMSC derived adipocytes. It inhibited mitotic clonal expansion and caused cell cycle arrest in G1 and S phase as suggested by western blot analysis and flow cytometry. It was also shown to have lipolytic effects. Oral administration of ethyl acetate fraction of E. alba to hamsters unveiled its anti-adipogenic as well as anti-dyslipidemic activity in-vivo. Mass spectrometry analysis of ethyl acetate fraction confirmed the presence of several bioactive components, projecting it as an effective phytopharmaceutical agent. In conclusion, ethyl acetate fraction of E. alba possesses potent anti-adipogenic as well as anti-dyslipidemic activity and could be projected as an herbal formulation towards obesity.

Chebulinic Acid Isolated From the Fruits of Terminalia chebula Specifically Induces Apoptosis in Acute Myeloid Leukemia Cells.

Chebulinic acid, an ellagitannin found in the fruits of Terminalia chebula, has been extensively used in traditional Indian system of medicine. It has shown to have various biological activities including antitumor activity. The present study aims to investigate the cytotoxic potential of chebulinic acid in human myeloid leukemia cells. Interestingly, chebulinic acid caused apoptosis of acute promyelocytic leukemia HL-60 and NB4 cells but not K562 cells. In vitro antitumor effects of chebulinic acid were investigated by using various acute myeloid leukemia cell lines. Chebulinic acid treatment to HL-60 and NB4 cells induced caspase activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, chromatin condensation, and changes in the mitochondrial membrane permeability. Additionally, inhibition of caspase activation drastically reduced the chebulinic acid-induced apoptosis of acute promyelocytic leukemia cells. Our data also demonstrate that chebulinic acid-induced apoptosis in HL-60 and NB4 cells involves activation of extracellular signal-regulated kinases, which, when inhibited with ERK inhibitor PD98059, mitigates the chebulinic acid-induced apoptosis. Taken together, our findings exhibit the selective potentiation of chebulinic acid-induced apoptosis in acute promyelocytic leukemia cells. Copyright © 2017 John Wiley & Sons, Ltd.