In-vitro antioxidant, antimutagenic and cancer cell growth inhibition activities of Rhododendron arboreum leaves and flowers.

In the current investigation, the active principles of the methanol extracts of Rhododendron arboreum leaves (MEL) and flowers (MEF) were investigated with the help of ultra-high performance liquid chromatography (UHPLC), amino acid analyzer and gas chromatography mass spectrometry (GC-MS). UHPLC revealed different polyphenols present in the extracts. GC-MS identified 20 phytochemicals in leaves and 17 in the flowers, whereas, amino acid analyzer confirmed 11 amino acids in leaves and 10 in the flowers. The extracts were subjected to the investigation of biological activity through analysis of antioxidant activity in different in vitro assays, antimutagenic activity in Ames assay and cancer cell growth inhibition activity by MTT (3-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) assay. MEL showed higher antioxidant activity in lipid peroxidation inhibition assay (95.32 ± 0.37%) than MEF (77.09 ± 4.17%) with IC50103.6 µg/ml for MEL and 271.17 µg/ml for MEF. In nitric oxide scavenging assay, an activity of 94.46 ± 0.32% (IC50 150.13) was observed in MEF followed by 83.71 ± 0.74% (IC50 179.52) in MEL. The antimutagenic activity of both the extracts was evaluated against sodium azide, 4-nitro-O-phenylenediamine and 2-aminofluorene mutagens in TA-98 and TA-100 strains of Salmonella typhimurium. The analysis was carried out using pre- and co-incubation modes. However, both extracts were observed to possess considerable antimutagenic activity against different known mutagens, flowers came out to be more effective than the leaves in terms of % inhibition. The extracts also exhibited significant cancer cell growth inhibition activity, when tested against 3 cancer cell lines namely, Human cervical cancer cell line (HeLa), Breast cancer cell line (MCF7) and Lung cancer cell line (A549). In case of HeLa and A549, MEL showed higher activity of 64.62 ± 2.65 and 75.08 ± 1.68% as compared to 53.11 ± 2.84 and 45.92 ± 2.43% in MEL, respectively. The EC50 values for MEL in HeLa and A549 were noted to be 232.76 and 155.38 µg/ml, respectively, whereas, MEF had EC50 of 395.50 µg/ml in HeLa and 660.26 µg/ml in A549. Further, MEF showed higher cytotoxicity in MCF7 cell line (84.93 ± 1.17%) followed by the MEL (73.57 ± 1.27%) with EC50 value of 95.16 µg/ml for MEF followed by 172.19 µg/ml for MEL. The biological activities of the extracts can be attributed to the phyto-constituents identified by sophisticated instruments. The biological activities of the extracts can be attributed to the active principles identified by sophisticated instruments.

Effect of Rhododendron arboreum Leaf Extract on the Antioxidant Defense System against Chromium (VI) Stress in Vigna radiata Plants.

In the current investigation, we studied role of Rhododendron leaf extract in Vigna radiata grown under chromium metal stress. We observed that seed treatment with Rhododendron leaf extract resulted in the recuperation of seedling growth under chromium toxicity. Seed treatment with Rhododendron leaf extract significantly improved the contents of anthocyanin and xanthophyll pigments under stress. The antioxidative defense system triggered after Rhododendron extract treatment, resulting in the increased actions of antioxidant enzymes. Oxidative stress induced by the assembly of reactive oxygen species was reduced after Rhododendron extract treatment under chromium toxicity as indicated by the enhanced contents of non-enzymatic antioxidants, namely ascorbic acid, tocopherol, and glutathione. Furthermore, Rhododendron leaf extract treatment under chromium metal stress also encouraged the biosynthesis of organic acids, polyphenols, as well as amino acids in Vigna radiata. Statistical analysis of the data with multiple linear regression also supported that Rhododendron leaf extract can effectively ease chromium metal-induced phytotoxicity in Vigna radiata.

Choice of contraceptive methods in public and private facilities in rural India.

BACKGROUND: Client-centric quality of care (QoC) in family planning (FP) services are imperative for contraceptive method adoption and continuation. Less is known about the choice of contraceptive method in India beyond responses to the three common questions regarding method information, asked in demographic and health surveys. This study argues for appropriate measurement of method choice and assesses its levels and correlates in rural India. METHODS: A cross-sectional study was conducted with new acceptors of family planning method (N = 454) recruited from public and private health facilities in rural Bihar and Uttar Pradesh, the two most populous states in India. The key quality of care indicator 'method choice' was assessed using four key questions from client-provider interactions that help in making a choice about a particular method: (1) whether the provider asked the client about their preferred method, (2) whether the provider told the client about at least one additional method, (3) whether the client received information without any single method being promoted by the provider, and (4) client's perception about receipt of method choice. The definition of method choice in this study included women who responded "yes" to all four questions in the survey. The relationship between contraceptive communication and receipt of method choice was assessed using logistic regression analyses, after adjusting for socio-demographic characteristics of the respondents. RESULTS: Although 62% of clients responded to a global question and reported that they received the method of their choice, only 28% received it based on responses about client-provider interactions. Receipt of the information on side-effects of the selected method (Adjusted Odds Ratio [AOR]: 7.4, 95% Confidence Interval [CI]: 3.96-13.86) and facility readiness to provide a range of contraceptive choice (AOR: 2.67, 95% CI: 1.48-4.83) were significantly associated with receipt of method choice. CONCLUSIONS: Findings demonstrated that women's choice of contraceptive could be improved in rural India if providers give full information prior to and during the acceptance of a method and if facilities are equipped to provide a range of choice of contraceptive methods.

Infection prevention preparedness and practices for female sterilization services within primary care facilities in Northern India.

BACKGROUND: In 2014, 16 women died following female sterilization operations in Bilaspur, a district in central India. In addition to those 16 deaths, 70 women were hospitalized for critical conditions (Sharma, Lancet 384,2014). Although the government of India's guidelines for female sterilization mandate infection prevention practices, little is known about the extent of infection prevention preparedness and practice during sterilization procedures that are part of the country's primary health care services. This study assesses facility readiness for infection prevention and adherence to infection prevention practices during female sterilization procedures in rural northern India. METHOD: The data for this study were collected in 2016-2017 as part of a family planning quality of care survey in selected public health facilities in Bihar (n = 100), and public (n = 120) and private health facilities (n = 97) in Uttar Pradesh. Descriptive analysis examined the extent of facility readiness for infection prevention (availability of handwashing facilities, new or sterilized gloves, antiseptic lotion, and equipment for sterilization). Correlation and multivariate statistical methods were used to examine the role of facility readiness and provider behaviors on infection prevention practices during female sterilization. RESULT: Across the three health sectors, 62% of facilities featured all four infection prevention components. Sterilized equipment was lacking in all three health sectors. In facilities with all four components, provider adherence to infection prevention practices occurred in only 68% of female sterilization procedures. In Bihar, 76% of public health facilities evinced all four components of infection prevention, and in those facilities provider's adherence to infection prevention practices was almost universal. In Uttar Pradesh, where only 55% of public health facilities had all four components, provider adherence to infection prevention practices occurred in only 43% of female sterilization procedures. CONCLUSION: The findings suggest that facility preparedness for infection prevention does play an important role in provider adherence to infection prevention practices. This phenomenon is not universal, however. Not all doctors from facilities prepared for infection prevention adhere to the practices, highlighting the need to change provider attitudes. Unprepared facilities need to procure required equipment and supplies to ensure the universal practice of infection prevention.

Antioxidant and Antimutagenic Activities of Different Fractions from the Leaves of Rhododendron arboreum Sm. and Their GC-MS Profiling.

In this era of urbanization and environmental pollution, antioxidants and antimutagens derived from plants are promising safeguards for human health. In the current investigation, we analyzed the antioxidant and antimutagenic effects of the hexane, chloroform, and ethyl acetate fractions of Rhododendron arboreum Sm. leaves and determined their chemical composition. The different fractions inhibited lipid peroxidation, repressed the production of nitric oxide radicals, and prevented deoxyribose degradation. The antimutagenic activity of the leaf fractions was analyzed against 4-nitro-O-phenylenediamine, sodium azide and 2-aminofluorene mutagens in two test strains (TA-98 and TA-100) of Salmonella typhimurium. The experiment was conducted using pre- and co-incubation modes. The best results were obtained in the pre-incubation mode, and against indirect acting mutagen. The presence of a number of bioactive constituents was confirmed in the different fractions by GC-MS analysis. The study reveals the strong antioxidant and antimutagenic activity of R. arboreum leaves. We propose that those activities of R. arboreum might correspond to the combined effect of the phytochemicals identified by GC-MS analysis. To the best of our knowledge, this is the first report on the antimutagenic activity of R. arboreum leaves.

Interaction of 24-epibrassinolide and salicylic acid regulates pigment contents, antioxidative defense responses, and gene expression in Brassica juncea L. seedlings under Pb stress.

Lead (Pb) is considered one the most hazardous pollutant, and its accumulation in soil and plants is of prime concern. To understand the role of plant hormones in combating heavy metal stress, the present study was planned to assess the interactive effects of 24-epibrassinolide (EBL) (10-7 M) and salicylic acid (SA) (1 mM) in regulating growth, pigment contents, antioxidative defense response, and gene expression in Brassica juncea L. seedlings exposed to different concentrations of Pb metal (0.25, 0.50, and 0.75 mM). Reduction in root and shoot lengths, chlorophyll and carotenoid content, and non-enzymatic antioxidants like glutathione, ascorbic acid, and tocopherol in response to Pb toxicity was observed. The enzymatic antioxidants such as guaiacol peroxidase (POD), ascorbate peroxidase (APOX), glutathione peroxidase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate redductase (MDHAR), glutathione-S-transferease (GST), and glutathione peroxidase (GPOX) were lowered in response to Pb treatments. Other antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO) enhanced under metal stress. Co-application of EBL + SA to 0.75 mM Pb-treated seedlings resulted in improvement of root and shoot lengths, chlorophyll, and carotenoid contents. Similarly, glutathione, ascorbic acid, and tocopherol contents were also elevated. Enzymatic antioxidants were also significantly enhanced in response to pre-sowing combined treatment of both hormones. Gene expression analysis suggested elevation in expression of CAT, POD, GR, DHAR, and GST genes by application of EBL. Our results reveal that Pb metal toxicity caused adverse impact on B. juncea L. seedlings, but pre-soaking treatment with EBL and SA individually and in combination help seedlings to counter the ill effects of Pb by improving growth, contents of pigment, and modulation of antioxidative defense system. The combined application of EBL and SA was found to be more effective in ameliorating Pb stress as compared to their individual treatments.

Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in Brassica juncea L. seedlings.

The present study demonstrated the combined effect of 24-epibrassinolide and salicylic acid against lead (Pb, 0.25, 0.50, and 0.75 mM) toxicity in Brassica juncea seedlings. Various parameters including water status, metal uptake, total water- and lipid-soluble antioxidants, metal chelator content (total thiols, protein-bound thiols, and non-protein-bound thiols), phenolic compounds (flavonoids, anthocyanins, and polyphenols), and organic acids were studied in 10-day-old seedlings. Dry matter content and the heavy metal tolerance index were reduced by 42.24 and 52.3%, respectively, in response to Pb treatment. Metal uptake, metal-chelating compounds, phenolic compounds, and organic acids were increased in Pb-treated seedlings as compared to control plants. The treatment of Pb-stressed seedlings with combination of EBL and SA resulted in enhancement of heavy metal tolerance index by 40.07%, water content by 1.84%, and relative water content by 23.45%. The total water- and lipid-soluble antioxidants were enhanced by 21.01 and 2.21%, respectively. In contrast, a significant decline in dry weight, metal uptake, thiol, and polyphenol contents was observed following the application of 24-epibrassinolide and salicylic acid. These observations indicate that Pb treatment has an adverse effect on B. juncea seedlings. However, co-application of 24-epibrassinolide and salicylic acid mitigates the negative effects of Pb, by lowering Pb metal uptake and enhancing the heavy metal tolerance index, water content, relative water content, antioxidative capacities, phenolic content, and organic acid levels.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice.

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

Comparative proteomic analysis of extracellular proteins of Clostridium perfringens type A and type C strains.

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-gamma) and interleukin 2 (IL-2), suggesting a Th1 type immune response.

Overexpression, purification, and immunogenicity of recombinant porin proteins of Salmonella enterica serovar Typhi (S. Typhi).

Porin proteins of Gram-negative bacteria are outer membrane proteins that act as receptors for bacteriophages and are involved in a variety of functions like solute transport, pathogenesis, and immunity. Salmonella enterica serovar Typhi (S. Typhi), a Gram-negative bacterium, is the causative agent of typhoid fever. Porins of S. Typhi have been shown to have a potential role in diagnostics and vaccination. In the present study, the major outer membrane proteins OmpF and OmpC from S. Typhi were cloned in pQE30UA vector and expressed in E. coli. The immunogenic nature of the recombinant porin proteins were evaluated by ELISA by raising hyperimmune sera in Swiss Albino mice with three different adjuvants (i.e., Freund's adjuvant and two human-compatible adjuvants like montanide and aluminium hydroxide gel) and proved to be immunogenic. The recombinant OmpF and OmpC generated in this work may be used for further studies for vaccination and diagnostics.