Studies On Antitumor Active Agents Of Endophytic Fungi Isolated From Pharmaceutical Plants,Taxus Mairei, Torreya Grandis And Cephalataxus Fortunei

Endophytic fungi are the microbes associated with living tissue of plants without causing any noticeable symptoms of disease. They constitute a large, special fungal group, and they are to be found in all or almost all plants. It's reported that some endophytic fungi could produce the same metabolite as that produced by their hosts. For example, some endopytic fungi isolated from Taxus sp. could produce taxol, one of most important antitumor drugs. Moreover, the secondary metabolites of endophytic fungi are abundant, including insecticidal, antimicrobic and antitumor agents. Therefore, endophytic fungi have become a new promising source for screening antitumor drugs.China has very rich pharmaceutical plants resources. Studies on endophytic fungi will promote the development of microbial medicine and help the protection of the valuable and rare plants. We have studied the antitumor activities of the endophytic fungi of several pharmaceutical plants in Fujian province in order to find new antitumor drugs.Altogether there were 172 strains of endophytic fungi isolated from the bark of three plants from Wuyi Mountain and Sanming County in Fujian province: Taxus mairei, Torreya grandis and Cephalataxus fortunei. The antitumor activities of the fermentation broths of these endophytic fungi were tested by MTT method. The result shows that 25 fermentation broths (about 14.5% of the total strains isolated) show noticeable growth inhibition against KB and HL6O tumor cells. The strains with antitumor activity in three plants, T. makei, T. grandis, C. fortunei account for 19.6%, 8.6%, 3.3% respectively among the total strains isolated. The strains with antitumor activity can be grouped into 8 genera by conventional identification. Paecilomyces (28%) and Cephalosporium (24%) were the main genera among the strains identified with antitumor activity. Even in the same genus, the antitumor activities show obvious difference. The diversity of genera and antitumor activities provides us with the possibility of searching antitumor metabolites.We further studied the antitumor metabolites of two strains withhigh antitumor activity, W-TF5 and H-036, which respectively isolated from T mairei and T grandis. Using TLC, UV, HPLC, MS, MIT, Tubulin Assembly methods, we verified that W-TF5 could produce taxol. From organic extract of W-TF5 fermentation broth, we also isolated another antitumor compound by column chromatography and recrystal methods, which was identified as Cytochalsin D (CytD) by X-Ray single crystal diffraction, IR, MS and NMR methods. Moreover, the X-Ray crystal structure of CytD was determined for the first time in the world.The fermentation broth of H-036 has a strong cytotoxicity on HL6O. KB. Hela~ SPC-A-1 and MCF-7 tumor cells. After the broth was extracted with ethyl acetate and condensed, and recrystalized in methanol and acetone repeatedly, a kind of colorless, pure columner shaped crystal was obtained, which identified as Brefeldin A (BFA) by X-Ray single crystal diffraction, lR and NMR methods.Our studies show that endophytic fungi are abundant in T mairei, T grandis and C. fortunei with great diversity. Strain W-1F5, identified as a Tubercularia sp., can produce both Taxol and CytD. This is the first time that finds Tubercularia sp. isolated from Taxus sp., and strain W-TF5 has been found as a new taxol and CytD-producing fungus. Strain H-036, identified as a Paediomyces sp., can produce BFA with high production (~10Omg/L), which is a new BFA-producing fungus. These results indicate that studies on endophytic fungi of pharmaceutical plants are beneficial to develop new microbial medicine.We also primarily studied the effects of CytD and BFA on tumor cell. CytD shows cytotoxicity on all tested tumor cell lines (HL60~ KB~ Hela. MCF-7 and Spc-A-1) with 1C50 ranging from 100 ng/ml to 500 ng/ml. BFA has stronger cytotoxicity than CytD with IC50 ranging from 2.0 ng/ml to 15 ng/ml. CytD can induce G2IM inhibition and apoptosis of HL6O and KB cells. After treated with CytD, KB cells appeared cell b...