Bioprocesses optimization and anticancer activity of camptothecin from Aspergillus flavus, an endophyte of in vitro cultured Astragalus fruticosus.

BACKGROUND: Emerging of endophytic fungi as potent camptothecin producers raise the hope for its commercial production, due to their rapid growth and feasibility of metabolic engineering, nevertheless, their loss of productivity with the fungal storage and subculturing is the challenge. Thus, screening for unique fungal isolate with sustainable camptothecin productivity is the objective of this work. RESULTS: The camptothecin productivity of the fungal endophytes of wild and in vitro cultured Astragalus fruticosus was evaluated. Aspergillus flavus ER, endophyte of A. fruticosus explant, was the potent producer (51.7 µg/l), the chemical identity of putative compound was resolved by UV, HPLC and LC-MS/MS analyses. The purified A. flavus camptothecin displayed a significant activity against HEPG-2 (IC50 0.9 mM), MCF7 and HCT29 (IC50 1.2-1.35 mM). The productivity of camptothecin by A. flavus was increased by 1.6 fold with methyljasmonate. Upon Plackett-Burman Design optimization, the yield of camptothecin was enhanced by 3 fold (150 µg/l) comparing to control. The camptothecin biosynthetic machinery of A. flavus was noticed to be attenuated with subculturing, nevertheless, this biosynthetic potency was restored upon addition of A. fruticosus methanolic extract (1%), ensuring the incidence of specific signals from plant tissues that triggers the expression of camptothecin encoding genes. CONCLUSION: This is the first study deciphering the feasibility of A. flavus for sustainable production of camptothecin upon addition of A. fruticosus extracts, that could be a new platform for camptothecin scaling-up.

Purification and Characterization of Ornithine Decarboxylase from Aspergillus terreus; Kinetics of Inhibition by Various Inhibitors.

l-Ornithine decarboxylase (ODC) is the rate-limiting enzyme of de novo polyamine synthesis in humans and fungi. Elevated levels of polyamine by over-induction of ODC activity in response to tumor-promoting factors has been frequently reported. Since ODC from fungi and human have the same molecular properties and regulatory mechanisms, thus, fungal ODC has been used as model enzyme in the preliminary studies. Thus, the aim of this work was to purify ODC from fungi, and assess its kinetics of inhibition towards various compounds. Forty fungal isolates were screened for ODC production, twenty fungal isolates have the higher potency to grow on L-ornithine as sole nitrogen source. Aspergillus terreus was the most potent ODC producer (2.1 µmol/mg/min), followed by Penicillium crustosum and Fusarium fujikuori. These isolates were molecularly identified based on their ITS sequences, which have been deposited in the NCBI database under accession numbers MH156195, MH155304 and MH152411, respectively. ODC was purified and characterized from A. terreus using SDS-PAGE, showing a whole molecule mass of ~110 kDa and a 50 kDa subunit structure revealing its homodimeric identity. The enzyme had a maximum activity at 37 °C, pH 7.4-7.8 and thermal stability for 20 h at 37 °C, and 90 days storage stability at 4 °C. A. terreus ODC had a maximum affinity (Km) for l-ornithine, l-lysine and l-arginine (0.95, 1.34 and 1.4 mM) and catalytic efficiency (kcat/Km) (4.6, 2.83, 2.46 × 10-5 mM-1·s-1). The enzyme activity was strongly inhibited by DFMO (0.02 µg/mL), curcumin (IC50 0.04 µg/mL), propargylglycine (20.9 µg/mL) and hydroxylamine (32.9 µg/mL). These results emphasize the strong inhibitory effect of curcumin on ODC activity and subsequent polyamine synthesis. Further molecular dynamic studies to elucidate the mechanistics of ODC inhibition by curcumin are ongoing.

Molluscicidal Activity of Some Solanum Species Extracts against the Snail Biomphalaria alexandrina.

Background. Snails' species are associated with transmission parasitic disease as intermediate host. Biological control stands to be a better alternative to the chemical controls aimed against snails. The search of herbal preparations that do not produce any adverse effects in the non-target organisms and are easily biodegradable remains a top research issue for scientists associated with alternative molluscicides control. Method. Solvent extracts of fresh mature leaves of S. nigrum, S. villosum, and S. sinaicum were tested against Biomphalaria alexandrina, a common intermediate host of schistosoma mansoni. A phytochemical analysis of chloroform: ethanol extract was performed to search for active toxic ingredient. The lethal concentration was determined. Results. Extracts isolated from mature leaves of Solanum species were found to be having molluscicidal properties. S. nigrum extract was recorded as the highest mortality rate. When the mortality of different solvent extracts was compared, the maximum (P < .05) mortality was recorded at a concentration of 90 ppm of ethanol extract of S. nigrum. Conclusion. Extract of mature leaves of S. nigrum exhibited molluscicidal activity followed by S. sinaicum and the less one was S. villosum. The study provides considerable scope in exploiting local indigenous resources for snails' molluscicidal agents.