Utilization of industrial waste - Liquid cheese whey for the batch fermentation of lovastatin using Fusarium nectrioides (MH173849) an endophytic fungus: Screening, production and characterization.

In this current research, a novel way of utilizing the plant weed and dairy industrial waste for the cost-effective production of Lovastatin by the novel fungus Fusarium nectrioides (MH173849) under controlled conditions was reported for the first time with scientific evidence. A total of 25 endophytic fungi were isolated from the 90 tissue fragments of Euphorbia hirta (L) and identified based on morphological and microscopical characteristics. All the fungal isolates were screened for Lovastatin production using Neurospora crassa bioassay. Among the 25 fungal isolates, Fusarium sp2, Nigrospora sphaerica, and Fusarium sp 4 showed maximum zone of inhibition and they were further verified by Thin Layer Chromatography. Since the Rf values of Fusarium sp 4 and standard Lovastatin were the same, further characterization was preceded only with Fusarium sp 4. An evolutionary relationship of two positive isolates, Fusarium sp 2 and Fusarium sp 4 was studied with other Lovastatin-producing fungi. Gene sequencing and BLAST revealed that a novel fungus, Fusarium sp 4 was found to be Fusarium nectrioides (MH173849) and it was further used for batch fermentation of Lovastatin in the modified media using liquid cheese whey under controlled conditions, which enhanced the productivity up to 43.40 µg/mL with the minimum purification steps. LC-MS-MS and NMR studies confirmed the production of Lovastatin by F. nectrioides (MH173849) due to the presence of Pyran molecule hydrogen, Hydrogen fusing two molecules as intermediate with triplet signal groups, methylbutanoic acid, and hexahydro naphthalene. Therefore, this fungus may be utilized by industries for the cost-effective production of Lovastatin.

Plant-microbe interactions implicated in the production of camptothecin - An anticancer biometabolite from Phyllosticta elongata MH458897 a novel endophytic strain isolated from medicinal plant of Western Ghats of India.

Endophytic wild fungal strain Phyllosticta elongata MH458897 isolated from medicinal plant Cipadessa baccifera from the Western Ghats region of Sathyamangalam Tiger Reserve Forest. This endophytic fungus has potential of effective anticancer drug Camptothecin (CPT). Endophytic fungi act as key symbionts in-between plants and ecosystem in the biosphere. This recently identified microbial population inside the plants produces many defence metabolites against plant pathogens. Among these defense metabolites, CPT gained much attention because of its effective anticancer activity. The maximum yield of CPT produced by optimizing the various factors like DEKM07 medium, pH 5.6, incubation time using Response Surface Methodology based on Central Composite Design. Extracted CPT is characterized using High Performance Liquid Chromatography and Electrospray ionization-Mass spectrometry. The highest yield of CPT was 0.747 mg/L was produced at optimized factors of dextrose - 50 g L-1, peptone - 5.708 g L-1, magnesium sulphate - 0.593 g L-1, and incubation time - 14 days. In-vitro MTT assay revealed the CPT derivatives were cytotoxic to A-549 cancer cell line (IC50 58.28 µg/ml) as nearly compared to the (IC50 51.08 µg/ml) standard CPT. CPT producing strain P. elongata from C. baccifera has the potential of CPT biosynthesis, and could be an effective anticancer bio metabolite. This compound has been described in the literature to be an effective anticancer metabolite. Our findings support the novel lifesaving anticancer drug from endophytic fungus in forest ecosystem concludes effective utilization of key symbionts will safeguard the humans and forest ecosystem.

Endophytic fungus Diaporthe caatingaensis MT192326 from Buchanania axillaris: An indicator to produce biocontrol agents in plant protection.

The study aims at the Isolation, screening and antibacterial evaluation of Camptothecin (CPT) and its derivatives, an anticancer molecule from endophytic fungus Diaporthe caatingaensis MT192326 of the medicinal plant, Buchanania axillaris. Plant parts were collected from Sathyamangalam Tiger Reserve forest, Tamil Nadu. The fungus was isolated using DEKM07 medium was used as the screening medium for the presence of CPT. The strain with the highest yield of CPT was identified at the molecular level by 18S rDNA sequencing. CPT was isolated and analyzed by UV-Vis spectrophotometry, Thin layer chromatography, High-Performance Liquid Chromatography, Fourier Transform Infrared spectroscopy, and Electron spray ionization-mass spectrometry. The compounds identified by ESI-MS from the fungal extract were studied for their antibacterial assays against procured MTCC bacterial pathogens. The maximum yield of 0.681 mg/L of CPT was produced by the fungus D.caatingaensis. CPT derivatives were identified at m/z of 305, 348 and 389 through ESI-MS analysis. Antibacterial studies revealed that the endophytic fungal extract compounds were studied for antibacterial activities of disc diffusion assay, exhibiting a growth inhibition range of 15-22 mm in nutrient agar plate medium. The Minimum Inhibitory Concentration revealed the antibacterial potential at a lower concentration of 12.5-25 µg/ml with all bacteria studied. The relatively lower antimicrobial efficacy of partially purified bio-metabolites than the positive control streptomycin (3.125) concentration could be due to the presence of derivatives of the compounds that hinder the activity of the biometabolite. This is the first initiative to screen, isolate and analyze the antibacterial assays of CPT and derivatives from endophytic fungus D.caatingaensis of ethnopharmacologically important B.axillaris plant from STRF.

In vitro enzyme inhibitory and cytotoxic studies with Evolvulus alsinoides (Linn.) Linn. Leaf extract: a plant from Ayurveda recognized as Dasapushpam for the management of Alzheimer's disease and diabetes mellitus.

BACKGROUND: Evolvulus alsinoides (Linn.) Linn. (Convolvulaceae) is a therapeutic herb alleviating brain patterns associated with three categories of regulatory principles of the body, mind, and behaviour. In the current research, enzyme inhibition and cytotoxic potentials of E. alsinoides (L.) L. leaf extract has been studied validating its potential application. METHODS: The plant phenolics in the leaf extracts obtained via cold-maceration with solvents viz.: n-hexane, chloroform, ethyl acetate, methanol, and water were quantitatively analyzed. The antioxidant potency was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Ability of Plasma (FRAP) assays at five concentrations (100-500 µg). The enzyme inhibition potential was performed with α-amylase, α-glucosidase, and acetylcholinesterase at seven concentrations (25-500 µg). The experiments were done in triplicates and statistically validated using Minitab-17 and SPSS 22. RESULTS: Water extract contain 45.08 ± 0.02 mg GAE/g, 49.30 ± 0.07 mg GAE/g, 211.21 ± 0.02 mg QE/g tannins, phenolics, flavonoids respectively. Its antioxidant activity was supported by IC50 52.43 ± 0.2 µg/mL (DPPH assay) and 41.58 ± 0.03 (FRAP assay). Methanolic extract inhibits α-amylase with IC50 1.33 ± 0.05 µg/mL. Water extract inhibits α-glucosidase and acetylcholinesterase with IC50 3.58 ± 0.02 µg/mL and 4.46 ± 0.03 µg/mL. Cytotoxicity studies with SH-SY5Y cell-line substantiate the inhibition potential of water extract with IC50 103.0035 µg/mL. DISCUSSION AND CONCLUSIONS: The extracts with potent antioxidant and enzyme-inhibiting activity were determined. The findings of the research are the first report about the inhibition effects of Evolvulus alsinoides (Linn.) Linn extracts against α-amylase, α-glucosidase and acetylcholinesterase. The extracts shall be examined in future studies to evaluate its pharmaceutical potential.