Camptotheca Acuminata Separation Of Endophytic Fungi And Its Metabolites 10 - Hydroxycamptothecin

In this paper,Camptotheca acuminate Decne,a Chinese endemic medicinal plant,was selected as material.The tissues distribution of endophytic fungi in C.acuminata and isolation method were preliminary studied.The result indicated that,the superficial disinfection for endophytic fungi isolation is better by 75%ethanol combined with 0.1%HgCl₂ method.And the HgCl₂ disinfection time is 4～5min to C.acuminat's root and stem,for C.acuminata leafs HgCl₂ disinfection time should be controlled about 2min.Under this superficial disinfection condition,totally 38 endophytic fungi has been isolated.Mainly distributes in the leaf(39.3%) and the bark (37.1%),the number of endophytic fungi from root(15.9%) and seed(7.7%) to be few,has not been isolated from the xylem.TLC conditions and HPLC methods were discussed in this paper.We identified that Start agent system was chloroform/methanol(volume ratio 9:1);we also got a perfect HPLC method:The sample was analyzed on a phenomenex Gemini C18 column(250mm?.60mm,5祄),with mobile phase of H₂O and methanol(80:20) at flow rate 0.8mLç©–in^-1 and detection at wavelength of 266nm.Thin layer chromatography(TLC) method and the highly effective liquid chromatography(HPLC) were employed to detect metabolites of A5 and A9.The results indicated the mycelium of this 2 strains may include 10-hydroxycamptothecine or similar compounds.By using traditional taxonomic methods,ITS sequencing and phylogenetic analysis,the endophytic fungus A5 was identified as Botryosphaeria berengeriana and the endophytic fungus A9 was identified as Diaporthe phaseolorum.Test on culture media and cultural conditions for A9,showed that carbon source and nitrogen source presented the most significant effect on the production of bioactivity.The optimized medium was composed of glucose 2%,soya-bean cake 2.6%,potassium dihydrogen phosphate 0.3%,epsom salt 0.3%.The suitable shaking culture conditions are as follows:initial pH of medium 6.0,shaking frequency 220r/min,incubation temperature 28℃,incubation time 6 days.Under this condition, Its content is 80礸/L.compared with the original strain,Its production increased about 23%.