Halogen bonding assisted site-selective C-3 triaryl methylation of indoles and N-triaryl methylation of imidazoles.

Halogen bonding triggered by the Lewis basic nature of acetonitrile catalyzes the site-selective C-3 triaryl methylation of indoles and N-triaryl methylation of imidazoles with trityl chlorides under catalyst-, metal-, and additive-free conditions at room temperature. This method generates a quaternary carbon centre appended to a heterocyclic moiety. UV-Vis and FT-IR analyses indicate the existence of halogen bonding which is the driving force of the reaction. This approach is suitable for a wide range of substrates, furnishing moderate to excellent yields (up to 100%) of triaryl methylated products under ambient reaction conditions. Equimolar amounts of reactants are sufficient to obtain the optimum yield and in some cases pure products can be obtained without column chromatography.

Beneficial effect of the methanolic leaf extract of Allium hookeri on stimulating glutathione biosynthesis and preventing impaired glucose metabolism in type 2 diabetes.

Oxidative stress resulting from the depletion of glutathione (GSH) level plays a vital role in generating various degenerative diseases, including type 2 diabetes (T2D). We tested the hypothesis that depleted glutathione levels can be enhanced and the impaired glucose metabolism can be prevented by supplementing Allium hookeri, a herb rich in organosulfur compounds, in a High Fat (HF) diet-induced T2D Male Sprague Dawley rat model. The experimental rats were divided into three groups (n = 6), namely normal diet, high-fat diet, and high-fat diet treated with A.hookeri methanolic leaf extract (250 mg/kg). Consumption of HF diet along with the plant extract resulted in significant reduction of the body weight (7.08%-14.89%) and blood glucose level (6.5%-16.4%) from the 13th week onward. There was a significant decrease in reactive oxygen species, oxidized glutathione (GSSG) levels, and an increase in GSH level in skeletal muscle tissues supplemented with the plant extract. The protein expressions of the signaling molecules such as GCLC and GR involved in GSH synthesis and of GLUT4 in glucose transport were also upregulated in the skeletal muscle tissues of the plant extract-treated group. Results of in vitro studies with muscle cell line (L6) further demonstrated the beneficial effect of the plant extract in increasing glucose uptake and maintaining the GSH/GSSH equilibrium via regulation of protein expression of GCLC/GR/GLUT4 signaling molecules in sodium palmitate (0.75 mM) treated cells. Overall this study suggests that dietary supplementation with Allium hookeri, can restore the glutathione level and regulate the blood glucose level in T2D.

Cephalotaxus griffithii Hook.f. needle extract induces cell cycle arrest, apoptosis and suppression of hTERT and hTR expression on human breast cancer cells.

BACKGROUND: Cephalotaxus spp. are known to possess anticancer potential. In this present work, for the first time the effects of C. griffithii needle (CGN) extracts on human cancer cells were examined. METHODS: The CGN was successively extracted with petroleum ether (PE), acetone and methanol. The extracts were tested for its effect on proliferation of cancer cells (MTT assay on HeLa, ZR751 and HepG2). Extract that showed the maximum growth inhibitory effect was subjected for mechanism of action study. These included apoptosis (morphological and DNA fragmentation assay), cell cycle (flow cytometry), caspase expression (Western blot) and activity (assay kit), p53 (western blot and TP53 siRNA interference) and telomerase expression (reverse transcriptase PCR) analysis. RESULTS: Among the extracts, PE extract induced maximum cytotoxicity, with highest death occurred in ZR751 cells. Since, PE extract induced cell death was highest among the CGN extracts, with maximum cancer cell death occurred in ZR751 cells; we carried out mechanism study of PE extract induced ZR751 cell death. It was observed that PE extract induced ZR751 cell death was associated with cell cycle arrest and apoptosis by activating both intrinsic and extrinsic apoptotic pathways. Knock down study revealed that p53 is essential for loss of ZR751 cell viability induced by PE extract. Further, PE extract down-regulated hTERT, hTR, and c-Myc expression. Thin layer chromatography analysis indicated the presence of unique phytochemicals in PE extract. CONCLUSION: Based on the observations, we concluded that PE extract of C. griffithii needle contains important phyto-components with multiple cellular targets for control of breast cancer and is worthy of future studies.

Streptomyces sp. Strain PBR11, a Forest-Derived Soil Actinomycetia with Antimicrobial Potential.

The Actinomycetia isolate PBR11 was isolated from the forest rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The isolate was identified as Streptomyces sp. with 92.91% sequence similarity to their closest type strain, Streptomyces atrovirens NRRL B-16357 DQ026672. The strain demonstrated significant antimicrobial activity against 19 test pathogens, including multidrug-resistant (MDR) clinical isolates and dermatophytes. Phenol, 2,5-bis(1,1-dimethylethyl), is the major chemical compound detected by gas chromatography-mass spectrometry in the ethyl acetate extract of PBR11 (EtAc-PBR11). The presence of the PKS type II gene (type II polyketide synthases) and chitinase gene suggested that it has been involved in the production of antimicrobial compounds. Metabolic profiling of the EtAc-PBR11 was performed by thin-layer chromatography and flash chromatography resulted in the extraction of two bioactive fractions, namely, PBR11Fr-1 and PBR11Fr-2. Liquid chromatography-tandem mass spectrometry analysis of both the fractions demonstrated the presence of significant antimicrobial compounds, including ethambutol. This is the first report on the detection of antituberculosis drug in the bioactive fractions of Streptomyces sp. PBR11. EtAc-PBR11 and PBR11Fr-1 showed the lowest MIC values (>0.097 and >0.048 µg/mL, respectively) against Candida albicans MTCC 227, whereas they showed the highest MIC values (>0.390 and >0.195 µg/mL, respectively) against Escherichia coli ATCC BAA-2469. The effects of PBR11Fr-1 were investigated on the pathogens by using a scanning electron microscope. The results indicated major morphological alterations in the cytoplasmic membrane. PBR11Fr-1 exhibited low cytotoxicity on normal hepatocyte cell line (CC-1) and the percent cell viability started to decline as the concentration increased from 50 µg/mL (87.07% ± 3.22%) to 100 µg/mL (81.26% ± 2.99%). IMPORTANCE Novel antibiotic breakthroughs are urgently required to combat antimicrobial resistance. Actinomycetia are the principal producers of antibiotics. The present study demonstrated the broad-spectrum antimicrobial potential of an Actinomycetia strain Streptomyces sp. strain PBR11 isolated from the PWS of Assam, India, which represents diverse, poorly screened habitats for novel microorganisms. The strain displayed 92.4% sequence similarity with genes of the closest type strain, indicating that the strain may represent a novel taxon within the phylum Actinomycetota. The metabolomics studies of EtAc-PBR11 revealed structurally diverse antimicrobial agents, including the detection of the antituberculosis drug ethambutol, in the bioactive fraction of Streptomyces sp. PBR11 for the first time. The PBR11 strain also yielded positive results for the antibiotic synthesis gene and the chitinase gene, both of which are responsible for broad-spectrum antimicrobial activity. This suggests that the untouched forest ecosystems have a tremendous potential to harbor potent actinomycetia for future drug discovery.

Streptomyces sp. MNP32, a forest-dwelling Actinomycetia endowed with potent antibacterial metabolites.

The Actinomycetia isolate, MNP32 was isolated from the Manas National Park of Assam, India, located in the Indo-Burma biodiversity hotspot region of Northeast India. Morphological observations and molecular characterization revealed its identity to be Streptomyces sp. with a 99.86% similar to Streptomyces camponoticapitis strain I4-30 through 16S rRNA gene sequencing. The strain exhibited broad-spectrum antimicrobial activity against a wide range of bacterial human pathogens including WHO-listed critical priority pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii. The ethyl acetate extract was found to disrupt the membrane of the test pathogens which was evidenced through scanning electron microscopy, membrane disruption assay and confocal microscopy. Cytotoxicity studies against CC1 hepatocytes demonstrated that EA-MNP32 had a negligible effect on cell viability. Chemical analysis of the bioactive fraction using gas chromatography-mass spectrometry (GC-MS) showed the presence of 2 major chemical compounds that include Phenol, 3,5-bis(1,1-dimethylethyl)- and [1,1'-Biphenyl]-2,3'-diol, 3,4',5,6'-tetrakis(1,1-dimethylethyl)- which have been reported to possess antimicrobial activity. The phenolic hydroxyl groups of these compounds were proposed to interact with the carbonyl group of the cytoplasmic proteins and lipids leading to destabilization and rupture of the cell membrane. These findings highlight the potential of exploring culturable actinobacteria from the microbiologically under-explored forest ecosystem of Northeast India and bioactive compounds from MNP32 which can be beneficial for future antibacterial drug development.

Phytochemical mediated modulation of COX-3 and NFκB for the management and treatment of arthritis.

In this study, we investigated whether zerumbone (ZBN), ellagic acid (ELA) and quercetin (QCT), the plant-derived components, can modulate the role of COX-3 or cytokines liable in arthritic disorder. Initially, the effect of ZBN, ELA, and QCT on inflammatory process was investigated using in-vitro models. In-silico docking and molecular dynamics study of these molecules with respective targets also corroborate with in-vitro studies. Further, the in-vivo anti-arthritic potential of these molecules in Complete Freund's adjuvant (CFA)-induced arthritic rats was confirmed. CFA increases in TNF-α and IL-1ß levels in the arthritic control animals were significantly (***p < 0.001) attenuated in the ZBN- and ELA-treated animals. CFA-induced attenuation in IL-10 levels recovered under treatment. Moreover, ELA attenuated CFA-induced upregulation of COX-3 and ZBN downregulated CFA-triggered NFκB expression in arthritic animals. The bonding patterns of zerumbone in the catalytic sites of targets provide a useful hint in designing and developing suitable derivatives that can be used as a potential drug. To our best knowledge, the first time we are reporting the role of COX-3 in the treatment of arthritic disorders which could provide a novel therapeutic approach for the treatment of inflammatory disorders.

Kaempferol 3-O-rutinoside from Antidesma acidum Retz. Stimulates glucose uptake through SIRT1 induction followed by GLUT4 translocation in skeletal muscle L6 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Antidesma acidum Retz, a perennial herb is known for its anti-diabetic potential among the traditional health care providers of the tribal communities of Manipur, India. Scientific validation of the ancient knowledge on traditional use of this plant with the help of modern tools and techniques can promote further research and its use in health care. AIM OF THE STUDY: Type 2 Diabetes (T2D) is a complex metabolic disorder and linked with hyperglycemia occurring from insufficiency in insulin secretion, action, or both. The aim of this study was to scientifically validate the traditional myth behind the uses of this plant material against diabetes. More specifically, it was aimed to determine the effect of methanolic extract of A. acidum leaves and/or any of its bioactive phytochemical(s), in enhancing insulin sensitization and subsequently stimulating the insulin signaling cascade of glucose metabolism. MATERIALS AND METHODS: Methanol was used for extraction from the leaf powder of A. acidum followed by bioactivity guided fractionation and isolation of most active component. Biological evaluation was performed to determine the glucose uptake ability against insulin resistance in skeletal muscle (L6) cells. To understand the detailed mechanism of actions of the purified compound, several molecular biology and structural biology experiments such as Western blot, siRNA transfection assay and molecular docking study were performed. RESULTS AND DISCUSSION: Bioactivity guided isolation of pure compound and spectral data analysis led us to identify the active component as Kaempferol 3-O-rutinoside (KOR) for the first time from the leaf of A. acidum. Over expression of NAD-dependent histone deacetylase, Sirtuin 1 (SIRT1) was observed following KOR treatment. SIRT1 plays an important role in the metabolic pathway and over expression of SIRT implies that it involves in insulin signaling directly or indirectly. Molecular docking and simulation study showed the strong involvement between KOR and SIRT1.Treatment with KOR resulted in significant over expression of SIRT1followed by upregulation of insulin-dependent p-IRS, AKT and AMPK signaling molecules, and stimulation of the GLUT4 translocation, which ultimately enhanced the glucose uptake in sodium palmitate-treated insulin resistant L6 myotubes. Further, the effect of KOR on IRS1, AKT and AMPK phosphorylation, GLUT4 translocation, and glucose uptake was attenuated in SIRT1-knockdown myotubes. CONCLUSION: Overall, the results of this study suggest that Kaempferol 3-O-rutinoside is the active component presents in the leaf of A. acidum which increases glucose consumption by inducing SIRT1 activation and consequently improves insulin sensitization. These results may find future applications in drug discovery research against T2DM.

In vitro and in vivo anti-diabetic and hepatoprotective effects of edible pods of Parkia roxburghii and quantification of the active constituent by HPLC-PDA.

ETHNOPHARMACOLOGICAL RELEVANCE: Parkia roxburghii G. Don. is a traditional medicinal plant and its pods are extensively used as food and medicine. It is believed by the traditional healers to have medicinal properties to treat diabetes, hypertension and urinary tract infections (Jamaluddin et al., 1994). MATERIALS AND METHODS: The methanolic extract of pods of P roxburghii and fractions were screened for their α-glucosidase and α-amylase inhibitory activity. Anti-hyperglycemic effects were studied on streptozotocin (45mg/kg b.w.) induced diabetes in albino rats (seven groups, n=7 n=6), using different doses for 14 days. Plasma glucose concentration (HbA1c) was analysed using whole blood, while SGOT, SGPT, TG, TC and uric acid were analysed using serum, employing commercial kits. Quantitative analysis of the major active constituent was carried out by HPLC-PDA. RESULTS: Bioactivity guided chemical investigation of the edible pods of P roxburghii identified sub-fraction EA-Fr 5 which significantly inhibited α-glucosidase (IC50 0.39±0.06 µgmL(-1)), reduced the blood glucose level to normal, and lowered the elevated levels of liver function enzymes SGOT and SGPT in STZ-induced diabetic rats. EA-Fr 5 was found to contain epigallocatechin gallate (1) and hyperin (2) which exhibited significantly higher α-glucosidase inhibitory potency with IC50 0.51±0.09 and 0.71±0.03µM respectively. EA-Fr 5 contained 379.82±2.90mg/g of EGCG, the major active constituent which manifests a broad spectrum of biological activities. CONCLUSION: The present investigation for the first time reports the occurrence of EGCG and hyperin in P roxburghii and substantiates the traditional use of pods of P roxburghii as dietary supplement for management of diabetes with significantly promising α-glucosidase inhibitory potency and anti-hyperglycemic as well as hepatoprotective effects.

Essential oil of Cephalotaxus griffithii needle inhibits proliferation and migration of human cervical cancer cells: involvement of mitochondria-initiated and death receptor-mediated apoptosis pathways.

This study was conducted to determine the effect of Cephalotaxus griffithii needle essential oil (CGNO) on proliferation and migration of human cervical cancer (HCC) cells. CGNO treatment decreased the viability of all the tested HCC (HeLa, ME-180 and SiHa) cells. Morphological and DNA fragmentation analysis of CGNO-treated HeLa cells indicated the involvement of apoptosis in inducing HCC cell death. CGNO increased mitochondrial membrane depolarisation and upregulated the expression of caspase-9, caspase-8, caspase-3 and cleaved-PARP. The activity of caspase-8 and caspase-9 was also significantly increased. Wound healing and transwell migration assay demonstrated that CGNO significantly inhibited the migration of HeLa cells to close a scratched wound and also inhibited their migration through filter towards a chemotactic stimulus. Taken together, these results indicated that CGNO inhibited the proliferation and migration of HCC cells. Of note, CGNO induced HeLa cell death through mitochondria-initiated and death receptor-mediated apoptosis pathway.

Chrysin rich Scutellaria discolor Colebr. induces cervical cancer cell death via the induction of cell cycle arrest and caspase-dependent apoptosis.

AIMS: Scutellaria discolor Colebr. has been extensively used in traditional medicine against several diseases. The purpose of this study was to investigate the anticancer potential of S. discolor and to isolate the bioactive principle responsible for the anticancer activity. METHODS: Cytotoxicity experiments were performed on cancer and normal cells using MTT assay. The mechanism of cell death was evaluated using real time PCR array, fluorescence microscopy, flow cytometry and Western blotting. MTT assay guided isolation (partition and column chromatography) was performed to identify the antiproliferative principle. Quantification of the active principle was done using HPLC. KEY FINDINGS: Acetone extract of S. discolor (SDE) inhibited the growth and survival of cancer cells to varying degree, but the inhibition was found to be maximum in cervical cancer cell lines. There was no significant toxicity induced to normal cells. The cell death was mediated through apoptosis. There was increased mitochondrial membrane depolarization, expression of Bax, caspase-9, caspase-3 and cleaved-PARP indicating that SDE-induced caspase dependent apoptosis in HeLa cells. Moreover, SDE caused cell cycle arrest in G2 phase in HeLa cells. Cytotoxicity guided fractionation of SDE led to the isolation of chrysin as the active principle responsible for the antiproliferative activity for cervical cancer cells. Interestingly, chrysin was the major phytochemical constituent present in S. discolor. SIGNIFICANCE: S. discolor is an important anticancer plant and a new source of chrysin.

Anti-diabetic potential of selected ethno-medicinal plants of north east India.

ETHNOPHARMACOLOGICAL RELEVANCE: Through one-to-one interaction with the traditional healers, the present study has identified 15 medicinal plant species traditionally used as remedies to control diabetes. MATERIALS AND METHODS: The methanolic extracts were screened for their α-glucosidase inhibitory activity. Hypoglycemic activity was assessed following glucose, sucrose and starch tolerance test on normal and STZ induced diabetic rats. RESULTS: Ficus cunia extract had the highest α-glucosidase inhibitory potency with IC50 1.39±0.74 µg mL(-1) followed by Schima wallichi (IC50 1.43±0.20 µg mL(-1)) and Wendlandia glabrata (IC50 1.67±0.33 µg mL(-1)). In STZ induced diabetic rat model, F. cunia and W glabrata extracts reduced blood glucose concentration to near normal up to 14 days when administered 48 h after STZ. CONCLUSION: The present study supports the traditional use of some of these medicinal plants in anti-diabetic remedies. The present study contributes to evidence for use of traditional medicine.

HPLC analysis of harringtonine and homoharringtonine in the needles of Cephalotaxus griffithii alkaloid fraction and cytotoxic activity on chronic myelogenous leukaemia K562 cell.

Harringtonine (HT) and homoharringtonine (HHT) are Cephalotaxus alkaloids with considerable antileukaemic activity. The objectives of this research were to (1) determine the content of HT and HHT present in Cephalotaxus griffithii needles alkaloid fraction (CGAF) and (2) compare the antiproliferative activity of CGAF, with that of HT and HHT on chronic myelogenous leukaemia K562 cell. The concentration of HT and HHT was found to be 122.14 and 16.79 mg/g of CGAF, respectively. Treatment of K562 cells with CGAF, HT and HHT decreased the viable cells in a dose- and time-dependent manner. Interestingly, the maximum cell death was found in CGAF, with IC50 value which was 3- to 4.6-fold lower than those of HT and HHT. Our results indicate that HT content in the needles of C. griffithii is higher than HHT, and alkaloids other than HT and HHT in CGAF are predominantly responsible for K562 cell death.