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Studies on the Camptothecin Production and Bio-activity of Endophytic Fungi Isolated from Camptotheca Acuminate

Author: LuRong
Tutor: FanMeiZhen;HuFengLin
School: Anhui Agricultural University
Course: Microbiology
Keywords: endophytic fungi camptothecin molecular identification anti-tumor activity anti-fungi activity
CLC: S182
Type: Master's thesis
Year: 2008
Downloads: 114
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With the study object of endophytic fungi of camptotheca acuminate in Anhui Province based on separation,screening,fermentation conditions,extraction process of camptothecin,the production of camptothecin and new anti-cancer drugs by microbial fermentation were researched in this paper.There were 197 strains of endophytic fungi were isolated from the roots,branches, leaves,fruits,and bark of C.acuminata in Hefei,Xuancheng,Huangshan of Anhui province.And 13 strains could produce camptothecin or their analogues were screened preliminarily by thin-layer chromatography(TLC) methods.But only 1 strain named LR051 has fermented metabolites and camptothecin have the similary absorption peaks analyzed by the high-performance liquid chromatography(HPLC).Based on the results of an orthogonal experiment for LR051 fermentation conditions with the content of camptothecin as an estimate,effects of nutritional and non-nutritional factors on the camptothecin production of the strain LR051 were studied.The result showed that the optimal composition of medium was:glucose 2%,potato 20%,peptone 0.5%, MgSO4·7H2O 0.2%,CaCl2 1%and MnSO4·H2O 0.006%.According to extraction effect of different solvents and the physical and chemical character tests of active components,an optimal extraction procedure was determined as follows:the liquid culture was condensed at 47℃to 1/5 volume of the broth,3 times volume of 95%ethanol was added to remove large molecule impurities such as polysaccharide,and then,supernatant was condensed and extracted with 2 times volume of chloroform twice.Finally,the extract was condensed to 1/10 volume of the ethylacetate phase.The concentrated substance derived was crude camptothecin sample.Then got the pure samples by Silica column thechnology and detect the purity by TLC and HPLC-MS technique.Its molecular formular was C20H16N2O4 and molecular weight was 348,and this compound was known as 4-Ethyl-4-hydroxy-1H-pyrano [3’,4’:6,7]in-dolizino[1,2-6]quinoline-3,14(4H,12H)-dione.In 197 for measuring strains,46 strains could inhibite the growth of CHO cells,have 23.4%in the total strains.The strains isolated from the bark had the highest anti-tumor activity,up to 45.0%;from the sticks followed by 40.6%;the leaves was lowest,only to 8.7%.The strain isolated from different geographical origin,there was a significant difference in anti-tumor activity.The strains isolated from Huangshan strains had the highest proportion,reaching 42.9%,followed by Xuancheng for 35.6%,Hefei had the lowest percentage of 25.9.A strain named LR126 got from Anhui Huangshan isolated from the fruits of C.acuminata could inhibite CHO cells more than 50%,and inhibite Hela cells over 90%.Because this strain has no obvious sporulation structure,the type was confirmed by ITS-rDNA sequence analysis method.The sequence was submitted to National Center for Biotechnology Information(NCBI),compared with DNA sequence in GeneBank by Blaster,and was confirmed that it was Rhizopycnis sp..According to the results of Anti-tumor experiment,in order to found some strains that had good functions in the two areas of anti-fungi and anti-tumor,even to explored whether there was any relevance between anti-tumor and anti-fungi activity,20 high toxicity of anti-tumor strains were selected to carry out further experiments.The results showed that 35.0 percent of the strains have a respectful activity against the four pathogenic microorganisms at the same time.So,the endophytic fungi of C.acuminata could be a good source of get bioactive substance.The fermentation product of strain LR004 had a high anti-fungi activity,showed the best activity against Rhizoctonia cerealis,Glomerella cingulata,Phytophthora capsici and Scleratinia sckerotiorum.Morover,LR004 had good thermal stability,sunshine stability and storing stability,but a little worse in pH stability.

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