Potentiality of Actinomycetia Prevalent in Selected Forest Ecosystems in Assam, India to Combat Multi-Drug-Resistant Microbial Pathogens.

Actinomycetia are known for their ability to produce a wide range of bioactive secondary metabolites having significant therapeutic importance. This study aimed to explore the potential of actinomycetia as a source of bioactive compounds with antimicrobial properties against multi-drug-resistant (MDR) clinical pathogens. A total of 65 actinomycetia were isolated from two unexplored forest ecosystems, namely the Pobitora Wildlife Sanctuary (PWS) and the Deepor Beel Wildlife Sanctuary (DBWS), located in the Indo-Burma mega-biodiversity hotspots of northeast India, out of which 19 isolates exhibited significant antimicrobial activity. 16S rRNA gene sequencing was used for the identification and phylogenetic analysis of the 19 potent actinomycetia isolates. The results reveal that the most dominant genus among the isolates was Streptomyces (84.21%), followed by rare actinomycetia genera such as Nocardia, Actinomadura, and Nonomuraea. Furthermore, seventeen of the isolates tested positive for at least one antibiotic biosynthetic gene, specifically type II polyketide synthase (PKS-II) and nonribosomal peptide synthetases (NRPSs). These genes are associated with the production of bioactive compounds with antimicrobial properties. Among the isolated strains, three actinomycetia strains, namely Streptomyces sp. PBR1, Streptomyces sp. PBR36, and Streptomyces sp. DBR11, demonstrated the most potent antimicrobial activity against seven test pathogens. This was determined through in vitro antimicrobial bioassays and the minimum inhibitory concentration (MIC) values of ethyl acetate extracts. Gas chromatography-mass spectrometry (GS-MS) and whole-genome sequencing (WGS) of the three strains revealed a diverse group of bioactive compounds and secondary metabolite biosynthetic gene clusters (smBGCs), respectively, indicating their high therapeutic potential. These findings highlight the potential of these microorganisms to serve as a valuable resource for the discovery and development of novel antibiotics and other therapeutics with high therapeutic potential.

Snake venom nerve growth factor-inspired designing of novel peptide therapeutics for the prevention of paraquat-induced apoptosis, neurodegeneration, and alteration of metabolic pathway genes in the rat pheochromocytoma PC-12 cell.

Neurodegenerative disorders (ND), associated with the progressive loss of neurons, oxidative stress-mediated production of reactive oxygen species (ROS), and mitochondrial dysfunction, can be treated with synthetic peptides possessing innate neurotrophic effects and neuroprotective activity. Computational analysis of two small synthetic peptides (trideca-neuropeptide, TNP; heptadeca-neuropeptide, HNP) developed from the nerve growth factors from snake venoms predicted their significant interaction with the human TrkA receptor (TrkA). In silico results were validated by an in vitro binding study of the FITC-conjugated custom peptides to rat pheochromocytoma PC-12 cell TrkA receptors. Pre-treatment of PC-12 cells with TNP and HNP induced neuritogenesis and significantly reduced the paraquat (PT)-induced cellular toxicity, the release of lactate dehydrogenase from the cell cytoplasm, production of intracellular ROS, restored the level of antioxidants, prevented alteration of mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) production, and inhibited cellular apoptosis. These peptides lack in vitro cytotoxicity, haemolytic activity, and platelet-modulating properties and do not interfere with the blood coagulation system. Functional proteomic analyses demonstrated the reversal of PT-induced upregulated and downregulated metabolic pathway genes in PC-12 cells that were pre-treated with HNP and revealed the metabolic pathways regulated by HNP to induce neuritogenesis and confer protection against PT-induced neuronal damage in PC-12. The quantitative RT-PCR analysis confirmed that the PT-induced increased and decreased expression of critical pro-apoptotic and anti-apoptotic genes had been restored in the PC-12 cells pre-treated with the custom peptides. A network gene expression profile was proposed to elucidate the molecular interactions among the regulatory proteins for HNP to salvage the PT-induced damage. Taken together, our results show how the peptides can rescue PT-induced oxidative stress, mitochondrial dysfunction, and cellular death and suggest new opportunities for developing neuroprotective drugs.

Taxifolin-3-O-glucoside from Osbeckia nepalensis Hook. mediates antihyperglycemic activity in CC1 hepatocytes and in diabetic Wistar rats via regulating AMPK/G6Pase/PEPCK signaling axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Osbeckia nepalensis Hook. f. is an ICMR documented plant well known for its antidiabetic uses among the folk people of Northeast Region of India. In-depth study with scientific substantiation of the plant may uphold the therapeutic potential against the treatment of type 2 diabetes mellitus (T2DM). AIM OF THE STUDY: The present study evaluates the traditionally claimed prophylactic potential of O. nepalensis and its extracts along with the isolated compound taxifolin-3-O-glucoside (TG) against the downregulation of T2DM related hepatic gluconeogenesis through in vitro, in vivo and in silico conditions as a means of ameliorating hyperglycemia. MATERIALS AND METHODS: Antidiabetic potential of O. nepalensis was carried out in both CC1 hepatocytes (in vitro) and STZ-induced diabetic male Wistar rats (in vivo). Enriched bioactive fraction and bioactive molecules were isolated through bioactivity-guided fractionation, yielding two major molecules, taxifolin-3-O-glucoside and quercitin-3-O-rhamnoside. The bioactivity of taxifolin-3-O-glucoside was validated through immunoblotting techniques aided by in silico molecular docking and simulations. RESULTS: Methanolic extract of O. nepalensis and taxifolin-3-O-glucoside (TG) isolated thereof enhanced the uptake of glucose in CC1 hepatocytes and downregulates the gluconeogenic enzymes (G6Pase and PEPCK) and its related transcription factors (FOXO1, HNF4α and PGC1α) through the stimulation of AMPK phosphorylation in in vitro condition. Moreover, in in vivo experiments, the in vitro most active fraction BuSFr1 (consisting of the two active major compounds taxifolin-3-O-glucoside and quercitin-3-O-rhamnoside) exhibited a substantial decrease in elevated blood glucose level and increase the glucose tolerance as well as plasma insulin level. In silico molecular docking and simulations for TG with the protein G6Pase inferred the docking sites and stability and showed taxifolin-3-O-glucoside as more potent and non-toxic as compared to quercitin-3-O-rhamnoside. CONCLUSION: The traditionally claimed antidiabetic effect of O. nepalensis has been proved to be effective in lowering the blood glucose level through in vitro, in vivo and in silico analysis which will pave a way for the development of antidiabetic phytopharmaceutical drugs which can be validated through further clinical studies.

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.

Chemical and biochemical characterization of Ipomoea aquatica: genoprotective potential and inhibitory mechanism of its phytochemicals against α-amylase and α-glucosidase.

Ipomea aquatica, also known as water spinach, is an aquatic non-conventional leafy vegetable and is considered a healthy and seasonal delicacy in ethnic food culture. The study revealed the presence of rich chemical and biochemical composition in I. aquatica and antioxidant activities. Moreover, the plant extracts demonstrated significant DNA damage prevention activity against UV/H2O2-induced oxidative damage. High-resolution mass spectrometric analysis by UPLC-qTOF-MS/MS resulted in the identification of over 65 different compounds and 36 important secondary metabolites. Most of the compounds identified represented polyphenolic compounds, viz. polyphenol glycosides and phenolic acids, followed by alkaloids and terpenoids. A UPLC-DAD method was developed and quantified for 10 different polyphenolic compounds. Out of all the metabolites examined, a significant number of compounds were reported to have various bioactive properties, including antibacterial, antiviral, antitumor, hepatoprotection, and anti-depressant effects. The plant extracts were found to contain various compounds, including euphornin, lucidenic acid, and myricitin glycosides, which possess significant medicinal value. Metabolite analysis utilizing GC-MS revealed the presence of various fatty acids, amino acids, sugars, and organic acids. The analysis revealed the presence of essential unsaturated fatty acids such as α-linolenic acid as well as beneficial substances such as squalene., The evaluation of glycemic control activity was carried out by comprehending the inhibitory potential of α-amylase and α-glucosidase, outlining the kinetics of the inhibition process. The inhibitory activities were compared to those of acarbose and revealed stronger inhibition of α-glucosidase as compared to α-amylase. Furthermore, the mechanism of inhibition was determined using in silico analysis, which involved molecular docking and molecular dynamic simulation of the identified IA phytochemicals complexed with the hydrolase enzymes. The study generates convincing evidence that dietary intake of I. aquatica provides a positive influence on glycemic control along with various health-protective and health-promoting benefits.

Characterization and selection of endophytic actinobacteria for growth and disease management of Tea (Camellia sinensis L.).

Endophytic microbes are vital for nutrient solubilization and uptake, growth, and survival of plants. Here, 88 endophytic actinobacteria (EnA) associated with five tea clones were isolated, assessed for their diversity, plant growth promoting (PGP), and biocontrol traits, and then used as an inoculant for PGP and disease control in host and non-host plants. Polyphasic methods, including phenotypic and genotypic characteristics led to their identification as Streptomyces, Microbacterium, Curtobacterium, Janibacter, Rhodococcus, Nocardia, Gordonia, Nocardiopsis, and Kribbella. Out of 88 isolates, 35 (39.77%) showed antagonistic activity in vitro against major fungal pathogens, viz. Fusarium oxysporum, Rhizoctonia solani, Exobasidium vexans, Poria hypobrunnea, Phellinus lamaensis, and Nigrospora sphaerica. Regarding PGP activities, the percentage of isolates that produced indole acetic acid, siderophore, and ammonia, as well as P-solubilisation and nitrogen fixation, were 67.05, 75, 80.68, 27.27, 57.95, respectively. A total of 51 and 42 isolates showed chitinase and 1-aminocyclopropane-1-carboxylic acid deaminase activity, respectively. Further, two potent Streptomyces strains KA12 and MA34, selected based on the bonitur scale, were screened for biofilm formation ability and tested in vivo under nursery conditions. Confocal laser scanning microscopy and the crystal violet staining technique revealed that these Streptomyces strains can form biofilms, indicating the potential for plant colonization. In the nursery experiment, they significantly enhanced the shoot and root biomass, shoot and root length, and leaf number in host tea plants. Additionally, treatment of tomato seeds by KA12 suppressed the growth of fungal pathogen Fusarium oxysporum, increased seed germination, and improved root architecture, demonstrating its ability to be used as a seed biopriming agent. Our results confirm the potential of tea endophytic actinobacterial strains with multifarious beneficial traits to enhance plant growth and suppress fungal pathogens, which may be used as bioinoculant for sustainable agriculture.

Prospecting Endophytic Bacteria Endowed With Plant Growth Promoting Potential Isolated From Camellia sinensis.

Endophytes are well-acknowledged inoculants to promote plant growth, and extensive research has been done in different plants. However, there is a lacuna about the endophytes associated with tea clones and their benefit to promote plant growth. The present study focuses on isolating and characterizing the beneficial endophytic bacteria (EnB) prevalent in commercially important tea clones cultivated in North Eastern India as plant growth promoters. Diversity of culturable EnB microbiome, in vitro traits for plant growth promotion (PGP), and applicability of potent isolates as bioinoculant for in vivo PGP abilities have been assessed in the present study. A total of 106 EnB identified as members of phyla Proteobacteria, Firmicutes, and Actinobacteria were related to 22 different genera and six major clusters. Regarding PGP traits, the percentage of isolates positive for the production of indole acetic acid, phosphate solubilization, nitrogen fixation siderophore, ammonia, and 1-aminocyclopropane-1-carboxylic acid deaminase production were 86.8, 28.3, 78.3, 30.2, 95.3, and 87.7, respectively. In total, 34.0, 52.8, and 17.0% of EnB showed notable production of hydrolytic enzymes like cellulase, protease, and amylase, respectively. Additionally, based on the bonitur score, the top two isolates K96 identified as Stenotrophomonas sp. and M45 identified as Pseudomonas sp. were evaluated for biofilm formation, motility, and in vivo plant growth promoting activity. Results suggested strong biofilm formation and motility in K96 and M45 which may attribute to the colonization of the strains in the plants. Further in vivo plant growth promotion experiment suggested sturdy efficacy of the K96 and M45 as plant growth promoters in nursery condition in commercial tea clones Tocklai vegetative (TV) TV22 and TV26. Thus, this study emphasizes the opportunity of commercialization of the selected isolates for sustainable development of tea and other crops.