| Endophyte is a special and important group of microorganisms with taxonomic diversity, which can produce various bioactive substances, including insecticidal, antimicrobial and antitumor compounds etc. In this research work, a large amount of endophytes had been screened based on several models and dozens of strains showed activities. Three of them were taxonomically identified and their secondary metabolites were studied.The fermentation samples were prepared from 1280 strains of endophytes. After primary and secondary screening on two models, 28 samples showed high and stable antitumor activities while 8 samples showed strong antibiotic activities. Among them, the strains HCCB1614, HCCB1778 and HCCB2310 were selected for further study.According to their morphology, cultural characteristics, 18S rDNA and ITS sequences, the strains HCCB1614, HCCB1778 and HCCB2310 were indentified as Phomopsis sp., Alternaria sp. and Ascospermum sp., respectively.Five compounds from the metabolites, named as HCCB1778-1～5 respectively, were purified by means of solvent extraction, silica gel chromatography, gelatum LH-20, and HPLC preparation. MS and NMR analyses showed that the molecular weights of these compounds are 242, 254, 270, 352, 352, respectively, and their molecular structures were identified as HCCB1778-1: 6, 8, 10- trimethylbenzopteridine- 2, 4 (3H, 10H)- dione; HCCB1778-2: 4', 7- dihydroxyisoflavone; HCCB1778-3: 4', 5, 7- trihydroxy- 7 methoyisoflavone; HCCB1778-4: Altertoxin-I; HCCB1778-5: 1, 4, 9, 12- tetrahydroxy- 1, 2, 12, 12a- tetrahydroperylene- 3, 10 (11H, 12bH)- dione; respectively. Compounds HCCB1778-3 and 5 showed strong cytotoxyties against A549 and LoVo cell lines. HCCB1778-4, which is a kind of cytotoxins, hosts strong mutagenicity and inhibition of platelet aggregation. HCBB1778-5 is a new compound. Meanwhile, two compounds isolated from the fermentation broth of strain HCCB2310 were identified as 3, 22, 24-trihydroxy-olean-12-ene and succinic acid.High speed counter-current chromatography (HSCCC) was applied to the separation of anti-tumor constituents from an endophyitic fungi HCCB1614 isolated from Trachelospermum jasminosides. A biphasic solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:3:2.5, v/v) was successfully performed. The bioactive compound purified and collected was analyzed by HPLC. A white powder was obtained in purity above 99% with the method. The chemical structure of the bioactive compound was identified by MS, 1H NMR and 13C NMR.Altertoxin I (ATX I), isolated from strain HCCB1778, is one of the common mycotoxins produced by genus Alternaria which is a common food pathogen of fruits and grains, diminishing the value of human foodstuffs. Recent research found that it can be used for the prophylaxis and therapy of deseases in which high blood platelet aggregations occur. To prepare enough quantity of pure ATX I for further research of mutagenicity, toxicology tests and anti-platelet aggregation, a novel method using preparative high-speed counter-current chromatography (HSCCC) was developed for the first time. The ethyl acetate crude extracts of the acetone washes obtained from fermentation liquor of Alternaria sp. was separated by a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (2:5:5:6, v/v). Collected fractions were analyzed by HPLC and identified by EI–MS and NMR analysis. The technique can isolate target compound at milligram amount in one run, which produced 45mg ATX I with purity better than 95% from 18L fermentation culture. The recovery field of ATX I from the crude extracts was 86.8%. This one-step purification method provided a simple and effective tool for obtaining a relatively large amount of ATX I for the purpose of various studies.
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