Aehtesham Hussain

Discovery of anti-microbial and anti-tubercular molecules from Fusarium solani: an endophyte of Glycyrrhiza glabra

Glycyrrhiza glabra is a high‐value medicinal plant thriving in biodiversity rich Kashmir Himalaya. The present study was designed to explore the fungal endophytes from G. glabra as a source of bioactive molecules.

Isolation, characterization and HPLC quantification of compounds from Aquilegia fragrans Benth: Their in vitro antibacterial activities against bovine mastitis pathogens

ETHNO-PHARMACOLOGICAL RELEVANCE The underground parts of Aquilegia fragrans are traditionally used for the treatment of wounds and various inflammatory diseases like bovine mastitis. However, there are no reports on the phytochemical characterization and antibacterial studies of A. fragrans. AIM OF THE STUDY To isolate compounds from the methanol extract of the underground parts of A. fragrans and determine their antibacterial activity against the pathogens of bovine mastitis. The study was undertaken in order to scientifically validate the traditional use of A. fragrans. MATERIALS AND METHODS Five compounds were isolated from the methanol extract of the underground parts of A. fragrans using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of isolated compounds in the crude methanol extract. The methanol extract and isolated compounds were evaluated for antibacterial activities against mastitis pathogens using broth micro-dilution technique. RESULTS The five isolated compounds were identified as (1) 2, 4-dihydroxyphenylacetic acid methyl ester (2) β-sitosterol (3) Aquilegiolide (4) Glochidionolactone-A and (5) Magnoflorine. A quick and sensitive HPLC method was developed for the first time for qualitative and quantitative determination of four isolated marker compounds from A. fragrans. The crude methanol extract and compound 5 exhibited weak antibacterial activities that varied between the bacterial species (MIC=500-3000 µg/ml). CONCLUSIONS The above results show that the crude methanol extract and isolated compounds from A. fragrans exhibit weak antibacterial activities. Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species in the treatment of various inflammatory diseases like bovine mastitis.

Novel bioactive molecules from Lentzea violacea strain AS 08 using one strain-many compounds (OSMAC) approach

A new eudesmane sesquiterpenoid (1), and a new homologue of virginiae butanolide E (2) along with butyl isobutyl phthalate (3) were isolated from, actinomycete-Lentzea violacea strain AS08 isolated from north western Himalayas by stressing on modified one strain-many compounds (OSMAC) method. The structures of the new compounds were elucidated by extensive spectroscopic analyses including 1D, 2D NMR along with HR-ESI-MS and FT-IR data. Herein, a distinctive method was added for inspecting secretory profile of the strain by quantification of extract value of cell free supernatant in different types of culture media fallowed by HPLC profiling of respective extracts, which revealed a highly altered metabolic profile of the strain and formed the base for the selection of media. The compounds 1 and 2 showed moderate activity against Gram negative (MIC ∼32-64µgml-1) in comparison to Gram positive bacterial pathogens. Compound 1 exhibited significant activity in human cancerous cell lines (IC50 ∼19.2µM).

Streptomyces puniceus strain AS13., Production, characterization and evaluation of bioactive metabolites: A new face of dinactin as an antitumor antibiotic

A highly active actinobacterial strain isolated from untapped areas of Northwestern Himalayas and characterised as Streptomyces puniceus strain AS13 by 16S rRNA gene sequencing was selected for production of bioactive metabolites. The bioassay-guided fractionation of microbial cultured ethyl acetate extract of the strain, led to isolation of macrotetrolide compound 1 (Dinactin) and compound 2 (1-(2,4-dihydroxy-6-methylphenyl)-ethanone). Structures of the isolated compounds were elucidated by [corrected] interpretation of NMR and other spectroscopic data including HR-ESI-MS, FT-IR. These compounds are reported for first time from Streptomyces Puniceus. Compound 1 exhibited strong anti-microbial activity against all tested bacterial pathogens including Mycobacterium tuberculosis. The MIC values of compound 1 against Gram negative and Gram positive bacterial pathogens ranged between 0.019 - 0.156μgml-1 and 1μgml-1 against Mycobacterium tuberculosis H37Rv. Dinactin exhibited marked anti-tumor potential with IC50 of 1.1- 9.7μM in various human cancerous cell lines and showed least cytotoxicity (IC50∼80μM) in normal cells (HEK-293). Dinactin inhabited cellular proliferation in cancer cells, reduced their clonogenic survival as validated by clonogenic assay and also inhabited cell migration and invasion characteristics in colon cancer (HCT-116) cells. Our results expressed the antimicrobial potential of dinactin and also spotted its prospective as an antitumor antibiotic.

Bovine mastitis: An appraisal of its alternative herbal cure

Bovine mastitis is globally recognized as the most common and costly disease affecting dairy herds. The disease causes huge financial losses to dairy industries by reduced yield and milk quality, deaths and culling of affected cows and also by associated treatment costs. The disease occurs due to invasion of the mammary glands by pathogenic bacteria followed by their multiplication in the milk producing tissues. The most common treatment method available against bovine mastitis is the intra-mammary infusion of antibiotics. However, their use is associated with the problem of antimicrobial resistance. This scenario has made search for alternative treatment approaches necessary. Medicinal plants with their well-established history are an excellent natural product resource used as an alternative therapy. Antibacterial agents from plants can act as important sources of novel antibiotics, efflux pump inhibitors, compounds that target bacterial virulence or can be used in combination with existing drugs. The plants form an essential component of ethno-veterinary medicine used in the treatment of different diseases like bovine mastitis. This review article attempts to provide an overview of the different medicinal plants used in the treatment of bovine mastitis. Antimicrobial studies of these plant species and some of their isolated constituents have been reviewed in detail. It highlights the logic and precedence behind mining this important natural product resource. Our own research findings in this direction and future scope of research are also discussed briefly.

Antituberculotic activity of actinobacteria isolated from the rare habitats

A distinctive screening procedure resulted in the isolation and identification of antituberculotic actinobacteria. In this course, a total of 125 actinobacteria were isolated from various soil samples from untapped areas in Northwestern Himalayas, India. The antibacterial screening showed that 26 isolates inhibited the growth of at least one of the tested bacterial pathogens including Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 11774), Micrococcus luteus (ATCC 10240), Escherichia coli (10536), Pseudomonas aeruginosa (ATCC 10145) and Klebsiella pneumonia (ATCC BAA‐2146). The production media was optimized for the active strains by estimation of their extract value by the quantification of the ethyl acetate extract. The screening of fermentation products from the selected 26 bioactive isolates revealed that 10 strains have metabolites antagonistic against the standard H37Rv strain of Mycobacterium tuberculosis. The characterization by 16S rRNA gene sequencing and phylogenetic analysis demonstrated the diverse nature of these antituberculosis strains. The secondary metabolites of potent, rare strain, Lentzea violacea AS08 exhibited promising antituberculosis activity with minimal inhibitory concentration (MIC) of 3·9 μg ml−1. The metabolites identified by gas chromatography–mass spectrometry (GC–MS) included, Phenol, 2,5‐bis (1, 1‐dimethylethyl), n‐Hexadecanoic acid, Hexadecanoic acid methyl‐ester, Hexadecanoic acid ethyl‐ester and, 9,12‐Octadecadienoyl chloride(Z,Z) are biologically significant molecules.

Abstract 333: Identification of a natural product small molecule inhibitor against Wnt/β-catenin signalling pathway

Wnt/β-catenin signalling pathway plays an important role in many processes like cell proliferation, differentiation, regeneration, carcinogenesis and regulates stem cell pluripotency. β-Catenin, the central component of Wnt/β-catenin signalling pathway has a vast number of binding partners and thus plays different roles in the cells. Mutations of β-catenin are seen in many cancers, including liver cancer, colorectal cancer, lung carcinoma, ovarian cancer and malignant breast tumours. Till date there is not even a single small molecule in clinical use that can block the β-catenin activity or the activities of this signalling pathway. In this study, we established many assays for screening small molecule inhibitors of this signalling pathway. Initially, we created a library of β-catenin deletion and point mutants and carried out a comprehensive comparative analysis of these mutants by luciferase reporter assays, GFP-fluorescence, immunoblotting and protein-protein interactions. We then screened many known and unknown natural and synthetic molecules against the wnt/β-catenin pathway and got 2 hits: C-18 and 055A, bioactive metabolites from actinomycetes. The IC50 (cell toxicity) of these molecules along with salinomycin, a known ionophore and inhibitor of β-catenin, were determined in different human cancer cells by MTT assay. The IC50 of 055A, C-18 and salinomycin in HepG2 cells was 7.5µM, 9.7µM and 25µM respectively. Interestingly, C-18 was ineffective against non-cancerous cell line (HEK-293 cells) even upto 60µM. This shows that C-18 is a very promising hit because it is active against cancer cells and spares non-cancerous cells. The sub lethal doses of these molecules were then tested for β-catenin mediated transcription by luciferase reporter assay in HepG2 cells which harbours a β-catenin deletion responsible for enhanced β-catenin mediated transcriptional activity. Our results show that C-18 and 055A significantly block the β-catenin activity in a dose dependent manner. The inhibition of wnt/β-catenin pathway by C-18 was observed at lower nanomolar concentrations in comparison to 055A and salinomycin which shows activity in micromolar concentrations. The β-catenin target gene expression analysis showed marked decrease in cyclinD1 by immunoblot analysis upon treatment of HepG2 cells with C-18. Interestingly, C-18 treated HepG2 cells failed to show any apoptosis as confirmed by Parp-1 and caspase-3 immunoblotting indicating that C-18 can be targeted to block the activity of wnt/β-catenin pathway, and could act as a lead molecule. Therefore, it warrants further experimentation and validation which are underway to draw the further conclusions. Citation Format: Mohd Saleem Dar, Aehtesham Hussain, Paramjeet Singh, Qazi Parvaiz Hassan, Satdarshan Paul Monga, Mohd Jamal Dar*. Identification of a natural product small molecule inhibitor against Wnt/β-catenin signalling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2017-333