Research On The Hupa-Producing Endophyte Colletotrichum Gloeosporioides ES026 Isolated From Huperia Serrata

Huperzine A as an ideal drug for the treatment of Alzheimer’s disease, is facing a increasing market demand with the increasing of dementia patients. However, Huperzine A is basically derived from the fern of Huperziaceae plants, and such plants have a weakly reproductive capacity, long growth cycle and a small amount of yield, which seriously restricts the market sources of huperzine A, therefore, to solve its raw material contradictions become a current research focus. In this study, we have conducted a research on H. serrata and its endophyte Colletotrichum gloeosporioides ES026 obtained by our laboratory staff previously. In the current study, HupA produced by C. gloeosporioides ES026 was investigated by optimizing its extracting, detection and activity assay methods, and a shake-flask fermentation approach was used to investigate the influences of conditions and media components on C. gloeosporioides ES026 to optimize HupA production. Intend to lay the foundation for expanding HupA production by C. gloeosporioides ES026 and to provide a reference for the peer research. The main work as follows:Firstly, a HupA extracting method from C. gloeosporioides ES026 was established. In this part, the univariate analytical approach and orthogonal experiments were used to investigate HupA extracting method from H. serrata. With the optimal method, HupA extraction can be completed within 5 h. The method, pretreated with ethanol for 30 mins, next soaked with 0.5% hydrochloric acid binding short time sonicated, and then extracted two times with CHCI3 led to the best extraction result. The average extracted rate reached 156.83 μg·g-1 dw, and was 1.90 times of conventional extraction method. The experimental results showed that the method was also suit for HupA extracting from C. gloeosporioides ES026. Being a time-saving and low-cost method, while improving the extraction rate and reducing environmental pollution, it is more suitable for HupA extraction research at laboratory level.Secondly, HupA HPLC detection method from the total alkaloids of C. gloeosporioides ES026 was established. The univariate analytical approach was used to investigate HupA detecting method by HPLC from the total alkaloid of H. serrata, and then HupA from C. gloeosporioides ES026 was detected by the optimized method. In the method, the temperature of the column compartment was kept at 40℃, the injection volume was set to 20 μL, the flow rate was 1.0 ml/min using 80 mM ammonium acetate (pH6.0)-methanol (6.5:3.5, v/v) as mobile phase, detection time was set 30 min, and the effluent was monitored at 310 nm. With this method, HupA sensitive detection from the total alkaloids of C. gloeosporioides ES026 was achieved, and to some extent, the damage caused by other ingredients of total alkaloids due to not out flowing in time to the column was avoided.Thirdly, the test conditions of HupA inhibition activity on acetylcholinesterase were established. According to the literature combined with univariate analysis method, the effects of reaction temperature, enzyme concentration and substrate usage on the inhibition of HupA standard on acetylcholinesterase activity were investigated. In 200 μL reaction system, the test conditions are:0.1 M pH7.2 PBS,60 μL; 0.1 M ATCh,20 μL; 1 U/mL AChE,10 μL; samples,10 μL; 10 mM DTNB,100 μL; reaction temperature,25℃; reaction time,10-30 min.The forth, the fermentation research of HupA produced by C. gloeosporioides ES026. With the HupA-producing endophyte C. gloeosporioides ES026 isolated by our laboratory staff as the research object, the impacts of different fermentation conditions and medium components on the HupA production of C. gloeosporioides ES026 were investigated. The optimized fermentation conditions are as follows:fermentation temperature,25 ℃; fermentation duration,6 days; shake-flask speed,150 revolutions per minute; volume of liquid,1:5; inoculum volume,2%; length of light per 24 h,9. Optimization of these parameters resulted in a 28.58% increase in HupA yield. The optimized fermentation medium components are as follows:potato broth,200 g/L; sucrose or glucose,10 g/L. A final concentration of 0.5-2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of mycelium pellets for HupA extraction showed a slightly difference in that ethanol treated mycelia gradually became as dark as the non-treated control, while methanol treated mycelia remained grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.Fifth, the preservation and rejuvenation research of C. gloeosporioides ES026. The endophytic fungi and their hosts have established a unique metabolic mechanism during the long-term synergistic growth, and some characteristics of the fungi will deteriorate when being separated from its host. In this study, a variety of programs were tried to rejuvenate C. gloeosporioides ES026 and effectively slowed the fungi recession speed. As follows:No.1, to optimize the culture conditions; No.2, the fungi was grown in slants made of alcohol or water extract from host plants, combined with basic media (0.5% carbon sources plus 100 g/L potato soup extract) and rich media (1.5% carbon sources plus 200 g/L potato soup extract) in turn for short time storage at 4 degree; No.3, as much as possible to reduce the passage number of the fungi; No.4, to rejuvenate the fungi by being reinjected to its host.Sixth, the fermentation physiological characteristics of C. gloeosporioides ES026 were researched. In this part, a random sampling method was adopted to investigate the variation tendency of soluble sugar, soluble total-protein, SOD, PPO and POD during the fermentation. In the hope of accumulating data for further research of C. gloeosporioides ES026 fermentation, and to offer reference for the fermentation physiological characteristics research of other endophytic fungi.