Multiple migrations from East Asia led to linguistic transformation in NorthEast India and mainland Southeast Asia.

NorthEast India, with its unique geographic location in the midst of the Himalayas and Bay of Bengal, has served as a passage for the movement of modern humans across the Indian subcontinent and East/Southeast Asia. In this study we look into the population genetics of a unique population called the Khasi, speaking a language (also known as the Khasi language) belonging to the Austroasiatic language family and residing amidst the Tibeto-Burman speakers as an isolated population. The Khasi language belongs to one of the three major broad classifications or phyla of the Austroasiatic language and the speakers of the three sub-groups are separated from each other by large geographical distances. The Khasi speakers are separated from their nearest Austroasiatic language-speaking sub-groups: the "Mundari" sub-family from East and peninsular India and the "Mon-Khmers" in Mainland Southeast Asia. We found the Khasi population to be genetically distinct from other Austroasiatic speakers, i.e. Mundaris and Mon-Khmers, but relatively similar to the geographically proximal Tibeto Burmans. The possible reasons for this genetic-linguistic discordance lie in the admixture history of different migration events that originated from East Asia and proceeded possibly towards Southeast Asia. We found at least two distinct migration events from East Asia. While the ancestors of today's Tibeto-Burman speakers were affected by both, the ancestors of Khasis were insulated from the second migration event. Correlating the linguistic similarity of Tibeto-Burman and Sino-Tibetan languages of today's East Asians, we infer that the second wave of migration resulted in a linguistic transition while the Khasis could preserve their linguistic identity.

Genome-wide analysis of DNaseI hypersensitivity unveils open chromatin associated with histone H3 modifications after areca nut with lime exposure.

Research over the years revealed that precocious anaphase, securin overexpression, and genome instability in both target and nontarget cells are significantly associated with the increased risk of areca nut (AN) and lime-induced oral, esophageal, and gastric cancers. Further, hyperphosphorylation of Rb and histone H3 epigenetic modifications both globally and in the promoter region of the securin gene were demonstrated after AN + lime exposure. This study aims whether the extract of raw AN + lime relaxes chromatin structure which further facilitates the histone H3 epigenetic modifications during the initial phase of carcinogenesis. Three groups of mice (10 in each group) were used. The treated group consumed 1 mg/day/mice of AN extract with lime ad libitum in the drinking water for 60 days. The dose was increased by 1 mg every 60 days. Isolated nuclei were digested with DNaseI and 2 kb and below DNA was eluted from the agarose gel, purified and PCR amplified by using securin and GAPDH primers. Securin and E2F1 expression, pRb phosphorylation, and histone epigenetic modifications were analyzed by immunohistochemistry. The number of DNA fragments within 2 kb in size after DNaseI treatment was higher significantly in AN + lime exposed tissue samples than in the untreated one. The PCR result showed that the number of fragments bearing securin gene promoter and GAPDH gene was significantly higher in AN + lime exposed DNaseI-treated samples. Immunohistochemistry data revealed increased Rb hyperphosphorylation, upregulation of E2F1, and securin in the AN + lime-treated samples. Increased trimethylation of histone H3 lysine 4 and acetylation of H3 lysine 9 and 18 were observed globally in the treated samples. Therefore, the results of this study have led to the hypothesis that AN + lime exposure relaxes the chromatin, changes the epigenetic landscape, and deregulates the Rb-E2F1 circuit which might be involved in the upregulation of securin and some other proto-oncogenes that might play an important role in the initial phases of AN + lime mediated carcinogenesis.

Securin overexpression correlates with the activated Rb/E2F1 pathway and histone H3 epigenetic modifications in raw areca nut-induced carcinogenesis in mice.

BACKGROUND: Raw areca nut (RAN) consumption induces oral, esophageal and gastric cancers, which are significantly associated with the overexpression of pituitary tumor transforming gene 1/securin and chromosomal instability (CIN). An association of Securin/PTTG1 upregulation and gastric cancer in human was also demonstrated earlier. Since the molecular mechanism underlying securin upregulation remains unclear, this study intended to investigate the association of securin upregulation with the Rb-E2F1 circuit and epigenetic histone (H3) modification patterns both globally and in the promoter region of the securin gene. METHODS: Six groups of mice were used, and in the treated group, each mouse consumed 1 mg of RAN extract with lime per day ad libitum in the drinking water for 60 days, after which the dose was increased by 1 mg every 60 days. Histopathological evaluation of stomach tissues was performed and securin expression was analysed by immunoblotting as well as by immunohistochemistry. ChIP-qPCR assays were performed to evaluate the recruitment of different histone modifications in the core promoter region of securin gene as well as its upstream and downstream regions. RESULTS: All mice developed gastric cancer with securin overexpression after 300 days of feeding. Immunohistochemistry data revealed hyperphosphorylation of Rb and upregulation of E2F1 in the RAN-treated samples. Increased trimethylation of H3 lysine 4 and acetylation of H3 lysine 9 and 18 both globally and in the promoter region of the securin gene were observed by increasing the levels of lysine-N-methyltransferase 2A, lysine-acetyltransferase, EP-300 and PCAF after RAN treatment. ChIP-qPCR data revealed that the quantity of DNA fragments retrieved from the immunoprecipitated samples was maximum in the -83 to -192 region than further upstream and the downstream of the promoter for H3K4Me3, H3K9ac, H3K18ac and H3K9me3. CONCLUSIONS: RAN-mediated pRb-inactivation induced securin upregulation, a putative E2F1 target, by inducing misregulation in chromatin remodeling in its promoter region, which led to transcriptional activation and subsequent development of chromosomal instability. Therefore, present results have led to the hypothesis that RAN-induced changes in the epigenetic landscape, securin overexpression and subsequent elevation of chromosomal instability is probably byproducts of inactivation of the pRb pathway.

Mitigation of X-ray induced DNA damages and expression of DNA-repair genes by antioxidative Potentilla fulgens root extract and its ethyl-acetate fraction in mammalian cells.

Potentilla fulgens is a medicinal plant in North-East India whose root is reported to have anti-diabetic, anticarcinogenic and antioxidant properties. The potential of hydro-alcoholic extract of P. fulgens root (PRE) for providing protection to mammalian cells exposed to ionising radiation was investigated in this study. The methanolic extract of PRE shows an enhanced radical scavenging ability in a concentration dependent manner. PRE-pre-treatment to stimulated human blood lymphocytes (HBLs) reduced the frequency of deletion and exchange aberrations induced by X-irradiation. Similar protection of chromosome aberrations was also observed in mouse bone marrow cells (BMCs) where mice were given PRE extract (1 mg extract/day/mice) ad libitum in the drinking water for 45 days before whole-body X-irradiation. Of the various extracts prepared by partitioning of the methanol extract, the ethyl-acetate (EA) fraction was found to possess better antioxidant, radical scavenging and DNA-damage reduction activities. PRE-pre-treatment also reduced the radiation-induced cell-cycle delay effectively in HBL. In HEK-293 cells, PRE reduced radiation-induced G2-block in cell kinetics. Interestingly, PRE-treatment alone increased the concentration of endogenous glutathione (GSH) in mouse BMC and in stimulated HBL along with the elevated expression of γ-glutamyl-cysteine synthetase heavy/catalytic subunit, a key determinant of GSH synthesis. Studies on expression of two DNA-repair genes revealed that there was a marked increase in the expression of GADD45 and H2AX genes after X-irradiation in stimulated HBL, and such expression was reduced significantly if PRE-treatment was given prior to radiation. The present findings show the ability of PRE to reduce radiation-induced DNA damages probably by free radical scavenging whereas modulation of expression of DNA-repair genes' and endogenous GSH-increment emerge as effective strategies. The present study is the first report on the selected medicinal plant species that suggests it to be a potential natural radioprotector when used as root extract or its EA fraction for mitigating radiation toxicity.

Glutathione S-transferasesP1 AA (105Ile) allele increases oral cancer risk, interacts strongly with c-Jun Kinase and weakly detoxifies areca-nut metabolites.

The Glutathione S-transferases (GSTs) protects cellular DNA against oxidative damage. The role of GSTP1 polymorphism (A313G; Ile105Val) as a susceptibility factor in oral cancer was evaluated in a hospital-based case-control study in North-East India, because the habit of chewing raw areca-nut (RAN) with/without tobacco is common in this region. Genetic polymorphism was investigated by genotyping 445 cases and 444 controls. Individuals with the GSTP1 AA-genotype showed association with the oral cancer (OR = 3.1, 95% CI = 2.4-4.2, p = 0.0002). Even after adjusting for age, sex and habit the AA-genotype is found to be significantly associated with oral cancer (OR = 2.4, 95% CI = 1.7-3.2, p = 0.0001). A protein-protein docking analysis demonstrated that in the GG-genotype the binding geometry between c-Jun Kinase and GSTP1 was disrupted. It was validated by immunohistochemistry in human samples, showing lower c-Jun-phosphorylation and down-regulation of pro-apoptotic genes in normal oral epithelial cells with the AA-genotype. In silico docking revealed that AA-genotype weakly detoxifies the RAN/tobacco metabolites. In addition, experiments revealed a higher level of 8-Oxo-2'-deoxyguanosine induction in tumor samples with the AA-genotype. Thus, habit of using RAN/tobacco and GSTP1 AA-genotype together play a significant role in predisposition to oral cancer risk by showing higher DNA-lesions and lower c-Jun phosphorylation that may inhibit apoptosis.

Methanolic extract of Potentilla fulgens root and its ethyl-acetate fraction delays the process of carcinogenesis in mice.

People of north-eastern states of India consume raw areca-nut (RAN) and lime which could lead to oral, esophageal and gastric cancers. However, the incidence of these cancers are significantly lesser in those who consume pieces of Potentilla fulgens root along with RAN. Since evaluation of anticancer role, if any, of P. fulgens on RAN-mediated genetic alterations in human is difficult because of other compounding factors, this study was undertaken in mice to focus on gastric carcinogenesis since ad libitum administration of RAN extract with lime in drinking water induced stomach cancer due to greater exposure of its lining. A total of 160 mice were used at different time points and either methanol extract of P. fulgens roots (PRE) or mixture of four compounds of ethyl-acetate fraction (EA-mixture) was mixed with mice feed. Histological studies revealed that RAN + lime induced cancer in all the mice and interestingly only 20% developed cancer when PRE/EA-mixture was provided along with RAN + lime. Higher frequency of precocious anaphase and over expression of p53 and Securin genes were significantly reduced by PRE/EA-mixture. Thus PRE/EA-mixture mitigates the RAN-induced tumor-initiating process in stomach by maintaining expression of tumor suppressor and check-point genes under control.

Functional interplay between YY1 and CARM1 promotes oral carcinogenesis.

Coactivator associated arginine methyltransferase 1 (CARM1) has been functionally implicated in maintenance of pluripotency, cellular differentiation and tumorigenesis; where it plays regulatory roles by virtue of its ability to coactivate transcription as well as to modulate protein function as an arginine methyltransferase. Previous studies establish an oncogenic function of CARM1 in the context of colorectal and breast cancer, which correlate to its overexpressed condition. However, the mechanism behind its deregulated expression in the context of cancer has not been addressed before. In the present study we uncover an oncogenic function of CARM1 in the context of oral cancer, where it was found to be overexpressed. We also identify YY1 to be a positive regulator of CARM1 gene promoter, where silencing of YY1 in oral cancer cell line could lead to reduction in expression of CARM1. In this context, YY1 showed concomitant overexpression in oral cancer patient samples compared to adjacent normal tissue. Cell line based experiments as well as xenograft study revealed pro-neoplastic functions of YY1 in oral cancer. Transcriptomics analysis as well as qRT-PCR validation clearly indicated pro-proliferative, pro-angiogenic and pro-metastatic role of YY1 in oral cancer. We also show that YY1 is a substrate of CARM1 mediated arginine methylation, where the latter could coactivate YY1 mediated reporter gene activation in vivo. Taken together, CARM1 and YY1 were found to regulate each other in a positive feedback loop to facilitate oral cancer progression.

Bioprospection of anti-inflammatory phytochemicals suggests rutaecarpine and quinine as promising 15-lipoxygenase inhibitors.

15-Lipoxygenase (15-LOX) belongs to the family of nonheme iron containing enzymes that catalyzes the peroxidation of polyunsaturated fatty acids (PUFAs) to generate eicosanoids that play an important role in signaling pathways. The role of 15-LOX has been demonstrated in atherosclerosis as well as other inflammatory diseases. In the present study, drug-like compounds were first screened from a set of anti-inflammatory phytochemicals based on Lipinski's rule of five (ROF) and in silico toxicity filters. Two lead compounds-quinine (QUIN) and rutaecarpine (RUT) were shortlisted by analyzing molecular interactions and binding energies of the filtered compounds with the target using molecular docking. Molecular dynamics simulation studies indicate stable trajectories of apo_15-LOX and docked complexes (15-LOX_QUIN and 15-LOX_RUT). In vitro 15-LOX inhibition studies shows that both QUIN and RUT have lower inhibitory concentration (IC50 ) value than the control (quercetin). Both QUIN and RUT exhibit moderate antioxidant activities. The cell viability study of these compounds suggests no significant toxicity in HEK-293 cell lines. Further, QUIN and RUT both did not show any inhibition against selected Gram-positive and Gram-negative bacterial species. Thus, based on our present findings, rutaecarpine and quinine may be suggested as promising 15-LOX inhibitor for the prevention of the atherosclerosis development.

Secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro inhibitory activities of Nardostachys jatamansi on key enzymes linked to hyperglycemia, hypertension and cognitive disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D. Don) DC., 'Spikenard' or 'Jatamansi', a highly valued, aromatic herb from alpine Himalayas has a long history of use as ethnomedicine and dietary supplements in Ayurveda, Unani and Chinese system of medicine since Vedic ages (1000-800 BC). In Ayurveda and traditional system of medicine, the species is used as stimulant, sedative, brain tonic or mind rejuvenator, antidiabetic, cardio tonic, and in the treatment of various neurological disorders such as insomnia, epilepsy, hysteria, anxiety and depression. It is considered as Sattvic herb in Ayurveda and is now commercially marketed either as single or poly-herbal formulations by many companies in national and international markets. AIM OF THE STUDY: The species has become threatened in its natural habitats due to over exploitation and illegal trade of its rhizomes for drug preparation in herbal and pharmaceutical industries. Considering the increasing demand and tremendous medicinal importance of this threatened plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory activities on key enzymes linked to hyperglycemia, hypertension and cognitive disorders in different plant parts of wild and in vitro-raised plants with respect to different solvent systems for its sustainable utilization. MATERIALS AND METHODS: Anti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. In vitro anti-hyperglycemic (α-amylase and PTP1B), anti-hypertensive (angiotensin-converting enzyme), anti-tyrosinase and anti-inflammatory potential (5-lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro-raised plants with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of rootstock extracts of wild and in vitro-derived plants were against cancer (HCT-116, MCF-7 and OE33) and two normal (HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and in vitro-derived plants was carried out by quantitative gas chromatography-mass spectrometry (GC-MS). RESULTS: In vitro-raised plantlets showed comparative higher yield of various secondary metabolites with a significantly high antioxidant activity as compared to the wild plants. Methanolic rootstock extracts of both wild and in vitro-derived plants of N. jatamansi exhibited significant AChE (IC50 36.46 ±â€¯2.1 and 31.18 ±â€¯2.6 µg/ml, respectively) and BuChE (IC50 64.6 ±â€¯3.5 and 60.12 ±â€¯3.6 µg/ml, respectively) inhibitory potential as compared to standard inhibitor galanthamine (IC50 0.94 ±â€¯0.03 and 4.45 ±â€¯0.5 µg/ml). Methanolic rootstock extract of in vitro-derived plants showed significant α-amylase (IC50 90.69 ±â€¯2.1 µg/ml), PTP1B (IC50 24.56 ±â€¯0.8 µg/ml), angiotensin-converting enzyme (IC50 42.5 ±â€¯3.6 µg/ml) and tyrosinase (IC50 168.12 ±â€¯3.6 µg/ml) inhibitory potential as compared to standard acarbose (IC50 52.36 ±â€¯3.1 µg/ml), ursolic acid (IC50 5.24 ±â€¯0.8 µg/ml), captopril (IC50 32.36 ±â€¯2.5 µg/ml) and kojic acid (IC50 = 54.44 ±â€¯2.3 µg/ml). Both the methanolic rootstock and leaf extracts of tissue culture-derived plants exhibited promising anti-5-LOX and anti-hyaluronidase activities against the known inhibitor of 5-LOX and hyaluronidase. Furthermore, methanolic rootstock extracts of both wild and in vitro-derived plants exhibited promising cytotoxic effects to HCT-116, MCF-7 and OE33 cell lines as compared to the normal HEK and MEF after 12 h of treatment. Secondary metabolite profiling of wild and in vitro-derived plants by quantitative GC-MS analysis revealed the presence of different classes of terpenoids and phenolic acids might be responsible for its effective biological activities. CONCLUSION: In vitro-derived plants revealed a substantial anti-cholinesterases, anti-hyperglycemic anti-inflammatory, anti-hypertensive and anti-tyrosinase potential with higher yield of various bioactive metabolites and significantly higher antioxidant activity which substantially explain medicinal importance of N. jatamansi in traditional medicine, used for centuries in different Ayurvedic formulations. The present findings suggest that cultured plants could be a promising alternative for the production of bioactive metabolites with comparative biological activities to the wild plants.

The multifaceted role of glutathione S-transferases in cancer.

Glutathione S-transferases (GSTs) are phase II detoxifying enzymes involved in the maintenance of cell integrity, oxidative stress and protection against DNA damage by catalyzing the conjugation of glutathione to a wide variety of electrophilic substrates. Though enzymes of the glutathione synthesis and salvage pathways have been well characterized in the past, there is still a lack of comprehensive understanding of their independent and coordinate regulatory mechanisms in carcinogenesis. The present review discusses implication of GST in cancer development and progression, gene polymorphism, drug resistance, signaling and epigenetic regulation involving their role in cancer. It is anticipated that GST especially the GSTP1 class can be developed as a biomarker either used alone or in combination with other biomarkers for early cancer detection and/or diagnosis as well as for future targeted preventive and therapeutic interventions with dietary agents.

Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach.

Radiation induced DNA double-strand breaks (DSB) are the major initial lesions whose misrejoining may lead to exchange aberrations. However, the role of glutathione (GSH), a major cellular thiol, in regulating cell's sensitivity to DNA damaging agents is not well understood. Influence of endogenous GSH on the efficiency of X-rays and bleomycin (Blem) induced DNA DSBs end-joining has been tested here cytogenetically, in human lymphocytes and Hct116 cells. In another approach, oligomeric DNA (75bp) containing 5'-compatible and non-compatible overhangs mimicking the endogenous DSB were for rejoining in presence of cell-free extracts from cells having different endogenous GSH levels. Frequency of aberrations, particularly exchange aberrations, was significantly increased when Blem was combined with radiation. The exchange aberration frequency was further enhanced when combined treatment was given at 4°C since DNA lesions are poorly repaired at 4°C so that a higher number of DNA breaks persist and interact when shifted from 4°C to 37°C. The exchange aberrations increased further when the combined treatment was given to Glutathione-ester (GE) pre-treated cells, indicating more frequent rejoining of DNA lesions in presence of higher cellular GSH. This is further supported by the drastic reduction in frequency of exchange aberrations but significant increase in incidences of deletions when combined treatment was given to GSH-depleted cells. End-joining efficiency of DNA DSBs with compatible ends was better than for non-compatible ends. End-joining efficiency of testicular and MCF7 cell extracts was better than that of lungs and Hct116 cells. Cell extract made from GE-treated MCF-7 cells provided more efficient end-joining than from untreated and GSH-depleted cells. However, direct addition of GSH to the cell-free extracts showed considerable reduction in end-joining efficiency. Present data indicate that higher endogenous GSH favours rejoining of DNA DSBs (both restitution and illegitimate reunion) which in turn produce more exchange aberrations.

Mechanism of apoptosis induction in human breast cancer MCF-7 cell by Ruviprase, a small peptide from Daboia russelii russelii venom.

Ruviprase, a 4.4 kDa peptide isolated from Daboia russelii russelii venom demonstrated antiproliferative activity against EMT6/AR1, U-87MG, HeLa and MCF-7 cancer cells with an IC50 value of 23.0, 8.8, 5.8 and 4.0 µg ml(-1), respectively. However, it was nontoxic to non-cancerous human embryonic kidney cell and human peripheral blood lymphocytes. Flow-cytometric analysis confirmed the apoptosis induction in MCF-7 cells by Ruviprase where it induced DNA condensation but did not cause mitotic blockage or chromosomal aberration in treated-cells. Immunofluorescence microscopic analysis indicated Ruviprase induced apoptosis in MCF-7 cells through p53 and p21-mediated pathways. Ruviprase generated reactive oxygen species (ROS), altered the mitochondrial transmembrane potential, and significantly decreased the cellular glutathione (GSH) content of MCF-7 cells. Immunoblotting and quantitative real-time PCR (qRT-PCR) analyses suggested that Ruviprase down-regulated the expression of anti-apoptotic protein Bcl-2, increased cleavage of poly (ADP-ribose) polymerase (PARP) protein, and up-regulated the expression of pro-apoptotic protein Bax, as well as executer protein caspase-7 to induced apoptosis in MCF-7 cells via intrinsic pathway. This is the first report on the characterization of the anticancer potential of a small, non-toxic and anticoagulant peptide purified from Russell's viper venom.

Induction of Apoptosis and Reduction of Endogenous Glutathione Level by the Ethyl-Acetate Soluble Fraction of the Methanol Extract of the Roots of Potentilla fulgens in Cancer Cells.

Potentilla fulgens root traditionally used as a folk remedy in Meghalaya, India. However, systematic evaluation of its anticancer efficacy was limited. We investigated the anticancer potentials of the various extracts prepared by partitioning of the methanol extract of the root with the aim to discover major contributing factors from the most effective fractions. Methanol extract of P. fulgens roots (PRE) was prepared by maceration which was subsequently fractionated into hexane, ethyl-acetate (EA) and n-butanol soluble fractions. Various assays (clonogenic assay, Flow cytometry analysis, western blot, semiquantitative RT-PCR and the level of endogenous glutathione) were used to evaluate different parameters, such as Cell survivability, PARP-1 proteolysis, expression pattern of anti-apoptotic and γ-glutamyl-cysteine synthetase heavy subunit (GCSC) genes in both MCF-7 and U87 cancer cell lines. Since the EA-fraction showed most efficient growth inhibitory effect, it was further purified and a total of nine compounds and some monomeric and dimeric flavan-3-ols were identified and characterized. Three compounds viz., epicatechin (EC), gallic acid (GA) and ursolic acid (UA) were taken on the basis of their higher yield and 10 µg/ml of each was mixed together. The concentration used in this study for PRE, EA- and Hex-fraction was 100 µg/ml, which was higher than the IC50 value. Apoptotic cell death in the PRE, EA-fraction and EC+GA+UA treated cancer cell cultures was significantly greater than in normal cells due to suppression of anti-apoptotic protein Bcl2 following treatment. Depletion of glutathione by downregulating GCSC was also observed. Induction of apoptosis and lowering the level of glutathione are considered to be positive activity for an anticancer agent. Therefore, modulation of GSH concentration in tumor cells by PRE and its EA-fraction opened up the possibility of a new therapeutic approach because these plant products are not harmful to normal cells and may regulate the tumor cellular response to different anticancer treatments. Thus, it would be interesting to examine efficacy of these plant products or EA-fraction in human cancer treatment.

Bio-synthesis of silver nanoparticles using Potentilla fulgens Wall. ex Hook. and its therapeutic evaluation as anticancer and antimicrobial agent.

The present study aims to develop an easy and eco-friendly method for the synthesis of silver nanoparticles using extracts from the medicinal plant, Potentilla fulgens and evaluation of its anticancer and antimicrobial properties. The various parts of P. fulgens were screened and the root extract was found to have the highest potential for the synthesis of nanoparticles. The root extracts were able to quickly reduce Ag(+) to Ag(0) and stabilized the nanoparticles. The synthesis of nanoparticles was confirmed by UV-Visible spectrophotometry and further characterized using Zeta sizer, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray diffraction (XRD). Electron microscopic study showed that the size of the nanoparticle was in the range of 10 to 15 nm and spherical in shape. The studies of phytochemical analysis of nanoparticles indicated that the adsorbed components on the surface of nanoparticles were mainly flavonoid in nature. Furthermore, nanoparticles were evaluated as cytotoxic against various cancer cell lines and 0.2 to 12 µg/mL nanoparticles showed good toxicity. The IC50 value of nanoparticles was found to be 4.91 and 8.23 µg/mL against MCF-7 and U-87 cell lines, respectively. Additionally, the apoptotic effect of synthesized nanoparticles on normal and cancer cells was studied using trypan blue assay and flow-cytometric analysis. The results indicate the synthesized nanoparticle ability to kill cancer cells compared to normal cells. The nanoparticles also exhibited comparable antimicrobial activity against both Gram-positive and Gram-negative bacteria.

Protective effect of Clerodendrum colebrookianum leaves against iron-induced oxidative stress and hepatotoxicity in Swiss albino mice.

Liver toxicity due to iron overload leads to oxidative damage of proteins, lipids and nucleic acids which in turn manifests several human diseases. Here, we evaluated the improving effect of Clerodendrum colebrookianum leaf on iron overload induced liver injury along with in vitro iron chelation and the protection of Fenton reaction induced DNA damage was conducted. Iron overload was induced by intraperitoneal administration of iron-dextran into mice. Post oral administration of different doses of the extract (50, 100 and 200 mg/kg body weight) showed significant decrease in different biochemical markers such as liver iron, serum ferritin and serum enzyme levels, along with decreased lipid peroxidation, protein oxidation and collagen content. In addition, the extract effectively enhanced the antioxidant enzyme levels and also exhibited the potential activity of the reductive release of ferritin iron. The protective effect of C. colebrookianum extract on injured liver was furthermore supported by the histopathological studies that showed improvement histologically. In conclusion, the present results demonstrated the hepatoprotective efficiency of C. colebrookianum leaf in iron overloaded mice, and hence, a potential iron chelating drug for iron overload diseases.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines.

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 µM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Qualitative and quantitative analysis of Potentilla fulgens roots by NMR, matrix-assisted laser desorption/ionisation with time-of-flight MS, electrospray ionisation MS/MS and HPLC/UV.

INTRODUCTION: Potentilla fulgens is a commonly used folk medicine by natives of northeast India, Nepal and Bhutan and is rich in polyphenolic and triterpene constituents. OBJECTIVE: To identify chemomarkers in the roots of P. fulgens by an interplay of (13)C-NMR, matrix-assisted laser desorption/ionisation with time-of-flight (MALDI/TOF) MS, electrospray ionisation (ESI) MS/MS and HPLC/UV. MATERIAL AND METHODS: The (13)C-NMR spectrum of crude methanolic extract was recorded in deuterated dimethyl sulphoxide. For MALDI/TOF/MS analysis, 2,5-dihydroxybenzoic acid was used as the matrix. For determination of chemical constituents, two independent simple isocratic HPLC/UV methods for monomeric/oligomeric flavanols and triterpene acids were developed and validated. RESULTS: The (13)C-NMR spectrum of the methanolic extract indicated the presence of B-type oligomeric polyphenolics containing mainly epicatechin/catechin (epicat/cat) and epiafzelechin/afzelechin (epiafz/afz) as the monomeric units. Several isobaric monomeric and oligomeric flavanols and triterpenoids were tentatively identified by MALDI/TOF/MS and ESI/MS/MS. Fourteen compounds (four monomeric and five dimeric flavanols and five triterpene acids) were isolated using repeated column chromatography and semi-preparative HPLC, and were quantitated using HPLC/UV. CONCLUSION: It is evident from these analyses that roots of P. fulgens contain flavans, including oligomeric flavanols, as major constituents followed by triterpene acids. The methods described can be applied to other Potentilla species to identify their constituents.

Precocious anaphase and expression of Securin and p53 genes as candidate biomarkers for the early detection in areca nut-induced carcinogenesis.

Research over the years has generated enough evidence to implicate areca nut, as a carcinogen in humans. Besides oral, significant rise in the incidence of cancers of the oesophagus, liver and stomach was seen among areca nut chewers. Early diagnosis seems key to understand the initial processes of carcinogenesis which is highly curable. In North-East India, betel quid contains raw areca nut (RAN), lime and small portion of betel leaf without any other constituents. This study was not intended to isolate any active ingredients from the RAN and to look its action. The present objective is to validate the screening of precocious anaphase and analysis of expression of Securin and p53 in non-target cells like human peripheral blood lymphocytes (PBLs) and mouse bone marrow cells (BMCs) as early indicative parameters of RAN + lime-induced cancers. A total of 35 mice were examined at different time points for following ad libitum administration of RAN extract in drinking water with lime. Peripheral blood was collected from 32 human donors of which, 24 were RAN + lime heavy chewers. Expression of genes was assessed by immunoblotting and/or by immunohistochemistry. Histological preparation of stomach tissue of mice revealed that RAN + lime induced stomach cancer. A gradual increase in the frequency of precocious anaphases and aneuploid cells was observed in both RAN + lime-treated mouse BMC and human PBL of RAN heavy chewers. Levels of p53 and Securin were increased in these cells during early days of RAN + lime exposure. The level of Securin was significantly higher in human tumour samples than their adjacent normal counterpart. The expression of Securin was increased significantly in RAN + lime-administered mice as well as in stomach tumour. Present study revealed that precocious anaphase and expression of p53 and Securin in non-target cells are significantly associated with an increased risk of RAN-induced cancer and thus these parameters can be of early diagnostic value.

Two new stereoisomeric antioxidant triterpenes from Potentilla fulgens.

The roots of Potentilla fulgens have been used for a long time as a folk remedy for many ailments without having information on its pharmacological action. Of the various extracts prepared by partitioning of the methanol extract, the ethyl acetate fraction was found to possess better antioxidant and cytotoxic activities. The degree of reduction in cloning efficiencies of MCF-7 cell lines was more with ethyl acetate than with hexane fraction of the root-extract. Hence, this fraction was further purified and nine compounds, including two new ursane type triterpenoids Fulgic acid A (4) and Fulgic acid B (5), were identified and characterized. Other compounds were identified as ursolic acid, euscaphic acid, corosolic acid, epicatechin, catechin, p-hydroxybenzaldehyde and gallic acid. Chemical structures were elucidated by spectroscopic methods, especially ESIHRMS and 2D NMR techniques. The new compounds showed good antioxidant activity and therefore this plant can be a source of natural antioxidants.

Induction of chromosome instability and stomach cancer by altering the expression pattern of mitotic checkpoint genes in mice exposed to areca-nut.

BACKGROUND: There are strong indications for a causal association between areca-nut consumption and cancers. In Meghalaya, India, the variety of areca-nut is used as raw and unprocessed form whose chemical composition and pharmacological actions have been reported. Yet we know little on the initial pathway involved in areca-nut associated carcinogenesis since it is difficult to assess its effects on genetic alterations without interference of other compounding factors. Therefore, present study was undertaken in mice to verify the ability of raw areca-nut (RAN) to induce cancer and to monitor the expression of certain genes involved in carcinogenesis. This study was not intended to isolate any active ingredients from the RAN and to look its action. METHODS: Three groups of mice (n = 25 in each) were taken and used at different time-points for different experimental analysis. The other three groups of mice (n = 15 in each) were considered for tumor induction studies. In each set, two groups were administered RAN-extract ad libitum in drinking water with or without lime. The expression of certain genes was assessed by conventional RT-PCR and immunoblotting. The mice were given the whole RAN-extract with and without lime in order to mimic the human consumption style of RAN. RESULTS: Histological preparation of stomach tissue revealed that RAN induced stomach cancer. A gradual increase in the frequency of precocious anaphase and aneuploid cells was observed in the bone marrow cells with a greater increment following RAN + lime administeration. Levels of p53, Bax, Securin and p65 in esophageal and stomach cells were elevated during early days of RAN exposure while those of different mitotic checkpoint proteins were downregulated. Apoptotic cell death was detected in non-cancerous stomach cells but not in tumor cells which showed overexpression of Bax and absence of PARP. CONCLUSION: Present study suggested (a) RAN induces stomach cancer, however, presence of lime promoted higher cell transformation and thereby developed cancer earlier, (b) perturbations in components of the chromosome segregation machinery could be involved in the initial process of carcinogenicity and (c) the importance of precocious anaphase as a screening marker for identification of mitotic checkpoint defects during early days.