الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 217 |  |  |
| | | from | 217 | | |
Abstract
Summary and Conclusion
Acetylcholinesterase inhibitors (AChE-Is) play an important pharmacological role in neurodegenerative diseases specially Alzheimer’s disease. Recent reports showed the ability of some microorganisms that resides plants named, endophytes to produce these inhibitors similarly as were produced by the host plant. In this study, some endophytic fungi and actinomycetes were isolated from different plant species as Cassia fistula, Ziziphus spina-christi, Psidium guajava, Ficus religiosa, Lantana camara, and Huperzia serrate grown in cultivated areas in Egypt and China. Endophytic isolates underwent fermentation, alkaloid extraction, and screening for AChE inhibition. Among these, a molecularly identified Alernaria brassicae AGF041 recovered from H. serrate, was the most potent with the maximum acetylcholinesterase inhibitory activity of 75.5%.
It is the first time to report that, A. brassicae AGF041 produce huperzine A (HupA) as an effective acetyl cholinesterase inhibitor and a well-known promising anti-Alzheimer’s drug. HupA was approved and and marketed as a new anti-AD drug in China while, in the USA, HupA is currently marketed as a food supplement for decreasing memory impairment. The HupA produced from the studied A. brassicae fermentation was found to be identical to the standard HupA sample through analysis by UV-visible spectrophotometer, thin layer chromatography, and a high-performance liquid chromatography.
The liquid fermentation conditions that were represented in thirteen process parameters including physical factors and medium components, were optimized for maximum HupA production based on three sequential factorial designs, Plackett-Burman, full factorial and central composite resulting in a final HupA yield of 42.8 µg/g of dry mycelia.
In order to enhance the production of HupA, the endophytic strain underwent mutagenesis through irradiation by UV and gamma rays. Radiation induces chemical, metabolic or physiological changes to living cells and disturb their organization. Exposure of microorganisms to sublethal doses of radiation produces mutations which may be desired in production of important products such as antibiotics, organic acids, amino acids, vitamins, alcohols and pigments. In the current study, the tested endophytic strain was subjected to UV and gamma radiation at different radiation doses. The effect of each irradiation type on HupA production by A. brassicae, was studied separately through one factor at a time (OFAT) method then, their interaction effect was also tested through response surface methodology. The irradiation mutagenesis resulted in an enhancing yield of HupA (51.9% increase) by the endophytic A. brassicae strain
Additionally, the cytotoxic effect of the obtained HupA extract was evaluated through viability assay using two normal Mammalian cell lines: WI-38 cells (human lung fibroblast normal cells), and HFB 4 (Human normal melanocytes cells). The Cytotoxic concentration (CC50), the concentration required to cause toxic effects in 50% of intact cells was 68.3 ± 0.8 µg/ml and 96.3 ± 1.2 µg/ml for human lung fibroblast and human normal melanocytes, respectively.
As a starting point for larger scale production of HupA, a 6.6 L bioreactor containing 4 L of a total medium volume, was used for a batch cultivation technique using the irradiated fungal culture of A. brassicae. The optimum medium components and other physical parameters obtained from the optimization step, were employed in the bioreactor besides shaking and aeration facilities. The production of HupA was found to reach 261.6 µg/l suggesting a promising alternative way for fungal HupA production in a large scale.