Pyrazole Scaffolds Having Promising Pharmacological Potential: A Review.

Heterocyclic compounds have diverse structure, which makes them broad and economical therapeutic agents. Pyrazole is a five-membered aromatic ring that contains two nitrogen atom and is responsible for the diverse functions of heterocyclic compounds. This implies the need of reviewing the work that already has been done on the potential of pyrazole scaffolds in the field of medicinal chemistry for the synthesis of new compounds with more effective antimicrobial, antioxidant, anticancer, anti-tuberculosis, and anti-inflammatory activities.

Molecular insights into enhanced resistance of Papaver somniferum against downy mildew by application of endophyte bacteria Microbacterium sp. SMR1.

Downy mildew is one of the most serious diseases of Papaver somniferum. Endophytes isolated from different parts of P. somniferum were screened for their ability to enhance resistance against downy mildew caused by the obligate biotrophic oomycete Peronospora meconopsidis. Two endophytes (SMR1 and SMR2) reduced the downy mildew on three P. somniferum genotypes (Sampada, J-16, and I-14). SMR1 (Microbacterium sp.) also enhanced the resistance of P. somniferum against downy mildew under field conditions. The biochemical markers of plant susceptibility under biotic stresses (proline and malondialdehyde) were found to be reduced in P. somniferum upon SMR1 treatment. To understand the mechanisms underlying the enhanced resistance to downy mildew in SMR1 endophyte-treated P. somniferum genotype J-16, we compared the expression profiles using the next-generation RNA sequencing approach between P. somniferum pretreated with SMR1 and untreated endophyte-free control plants following exposure to downy mildew pathogen. Comparative transcriptome analysis revealed differential expression of transcripts belonging to broad classes of signal transduction, protein modification, disease/defense proteins, transcription factors, and phytohormones in SMR1-primed P. somniferum after infection with downy mildew pathogen. Furthermore, enhanced salicylic acid content was observed in SMR1-primed P. somniferum after exposure to downy mildew pathogen. This study sheds light on molecular mechanisms underlying enhanced resistance to downy mildew in SMR1-primed P. somniferum. Finally, we propose that the SA-dependent defense pathway, the hallmark of systemic acquired resistance, is activated in SMR1-primed P. somniferum, triggering the endophyte-induced resistance.

The Bioactive Potential of Culturable Fungal Endophytes Isolated From the Leaf of Catharanthus roseus (L.) G. Don.

INTRODUCTION: Endophyte is considered a source of natural bioactive secondary metabolites that provides an array of bioactive lead compounds. The present study was aimed to determine the antimicrobial and anti-inflammatory potential of fungal endophytes isolated from Catharanthus roseus. METHODS: A total of seven fungal endophytes crude extract were screened against bacterial pathogens. Of these, Curvularia geniculata CATDLF7 crude extract exhibited the most potent inhibitory activity against bacterial pathogens. Hence, CATDLF7 crude extract was subjected to chromatographic separation. This purification leads to the isolation of six pure compounds (1PS - 6PS). Of these, 3PS was found to be a major constituent and most effective against clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentration (MIC) values ranging from 100 to 200 µg/ml. Based on the spectroscopic data, 3PS was characterized as α,ß- dehydrocurvularin. This compound also showed synergistic interaction with norfloxacin and reduced its MIC up to 32-folds with a fractional inhibitory concentration index (FICI) of 0.09. RESULTS: To understand the possible antibacterial mechanism of action, α,ß-dehydrocurvularin alone (100 µg/ml) exhibited efflux pump inhibitory potential by 0.84 fold decreasing in ethidium bromide (EtBr) fluorescence. In addition, α,ß-dehydrocurvularin inhibited inflammatory cytokines TNF-α and IL-6 production, which is further validated by molecular docking scores -4.921 and -5.641, respectively, for understanding orientation and binding affinity. CONCLUSION: Overall, the results highlighted identifying bioactive compound α,ß-dehydrocurvularin, which could be used as an antimicrobial and anti-inflammatory agent.

Innate endophytic fungus, Aspergillus terreus as biotic elicitor of withanolide A in root cell suspension cultures of Withania somnifera.

In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic fungus Aspergillus terreus 2aWF in shake flask. Culture filtrate of A. terreus 2aWF significantly elicits withanolide A at 6H (12.20 ± 0.52 µg/g FCB). However, with A. terreus 2aWF mycelial extract, withanolide A content was higher at 24H (10.29 µg/g FCB). Withanolide A content was maximum with salicylic acid (0.1 mM) treatment at 24H (8.3 ± 0.20 µg/g FCB). Further, expression analysis of withanolide pathway genes, hydrogen peroxide production, and lipid peroxidation was carried out after 48H of elicitation with 2aWF mycelial extract and culture filtrate. The expression levels of withanolides biosynthetic pathway genes, viz. HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were quantified by real time PCR at 48H of elicitation. In all the treatments, the expression levels of key genes were significantly upregulated as compared to untreated suspension cells. Hydrogen peroxide was noticeably enhanced in SA, mycelia extract and culture filtrate, at 20% (115 ± 4.40 nM/g FCB), 42% (137.5 ± 3.62 nM/g FCB), and 27% (122.8 ± 1.25 nM/g FCB) respectively; however, lipid peroxidation was 0.288 ± 0.014, 0.305 ± 0.041 and 0.253 ± 0.007 (µM/gm FCB) respectively, higher than the control (0.201 ± 0.007 µM/gm FCB).

Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots.

Endophytes have been reported from all plant species from different parts of tissue including root, stem and leaves. Here we report, three fungal endophytes, Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) from Withania somnifera, which could enhance withanolides content in leaf and root. Upon treatment with the above endophytes to 4 weeks old plants in field conditions, W. somnifera elicited withanolide A content (97 to 100%) in leaves without considerable changes in withaferin A content. Furthermore, withanolide A content in roots of 5aWF and 10aWF endophyte treated W. somnifera plants increased up to 52% and 65% respectively. Incidentally, expression profile of withanolide and sterol biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were significantly upregulated in 2aWF, 5aWF and 10aWF fungal endophyte treated plants. Besides, modulation of withanolide biosynthetic pathway genes, fungal endophytes also induce a host resistant related gene, NPR1 resulting in 2, 4 and 16 fold expression levels in 2aWF, 10aWF and 5aWF endophyte treatments respectively, compared to control plants. Overall, our results illustrate that application of native-fungal endophytes 2aWF (96.60%), 5aWF (95%) and 10aWF (147%) enhances plant biomass in addition to withanolide content.

Endophytes of Withania somnifera modulate in planta content and the site of withanolide biosynthesis.

Tissue specific biosynthesis of secondary metabolites is a distinguished feature of medicinal plants. Withania somnifera, source of pharmaceutically important withanolides biosynthesizes withaferin-A in leaves and withanolide-A in roots. To increase the in planta withanolides production, a sustainable approach needs to be explored. Here, we isolated endophytes from different parts of W. somnifera plants and their promising role in in planta withanolide biosynthesis was established in both in-vivo grown as well in in-vitro raised composite W. somnifera plants. Overall, the fungal endophytes improved photosynthesis, plant growth and biomass, and the root-associated bacterial endophytes enhanced the withanolide content in both in-vivo and in-vitro grown plants by modulating the expression of withanolide biosynthesis genes in leaves and roots. Surprisingly, a few indole-3-acetic acid (IAA)-producing and nitrogen-fixing root-associated endophytes could induce the biosynthesis of withaferin-A in roots by inducing in planta IAA-production and upregulating the expression of withanolide biosynthesis genes especially MEP-pathway genes (DXS and DXR) in roots as well. Results indicate the role of endophytes in modulating the synthesis and site of withanolides production and the selected endophytes can be used for enhancing the in planta withanolide production and enriching roots with pharmaceutically important withaferin-A which is generally absent in roots.

Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant.

Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids.

MAIN CONCLUSION: Endophytes reside in different parts of the poppy plant and perform the tissue-specific functions. Most leaf endophytes modulate photosynthetic efficiency, plant growth, and productivity while capsule endophytes modulate alkaloid biosynthesis. Endophytes promote plant growth, provide protection from environmental stresses and are the source of important secondary metabolites. Here, we established that the endophytes of opium poppy Papaver somniferum L. may play a role in the modulation of plant productivity and benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 22 endophytes isolated from leaves, roots, capsules and seeds of the poppy plants were identified. Isolated endophytes were used to inoculate the endophytes free poppy seeds and screened for their ability to improve plant productivity and BIA production. It was evident that the endophytes from leaf were involved in improving photosynthetic efficiency, and thus crop growth and yield and the endophytes from capsule were involved in enhancing BIA biosynthesis. Capsule endophytes of alkaloid-rich P. somniferum cv. Sampada enhanced BIA production even in alkaloid-less cv. Sujata. Expression study of the genes involved in BIA biosynthesis conferred the differential regulation of their expression in the presence of capsule endophytes. The capsule endophyte SM1B (Acinetobacter) upregulated the expression of the key genes for the BIA biosynthesis except thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). On the other hand, another capsule endophyte SM3B (Marmoricola sp.) could upregulate both T6ODM and CODM. Colonization of poppy plant by endophytes isolated from leaves, roots and capsules found to be higher in their respective plant parts confirmed their tissue-specific role. Overall, the results demonstrate the specific role of endophytes in the modulation of host plant productivity and BIA production.