Identification Of Endophytic Fungus CH1307from Cephalotaxus Hainanensis Li And Its Antitumor Activities

Cephalotaxus mannii Hook.f. was paid much attention because its roots, stems and leaves all have harringtonine compounds with anticancer activity. However it is extremely rare due to slow growth, reproductive problems, over-exploitation and growth condition dete-rioration in the past decades. Under the inspiration that endophytes from Taxaceae could compose taxol, the endophytes isolated from Cephalotaxus manni were under the spotlight.The endophytic fungus, isolated from the phloem of Cephalotaxus manni is capable of synthetizing homoharringtonine. Then, it was identified in molecular biology and morpholo-gy. The homology comparison indicated that the ITSrDNA sequence of the strain had a homology of more than99.8%with some species belonging to Alternaria sp.. The colony in czapek yeast extract agar medium was round with diameter7.0cm, breen on surface and rufous on the back. The surface colony was villiform, slightly thick and unsmooth on the edge. At the same time, light rufous and soluble pigment could be observed. Under the microscope, Alternaria tenuissima CH1307had hazel conidiophores which were monothetic, upright, septate and sometimes branched. The spores were also hazel, and their cell wall was slippy. Generally, several spores together formed a chain.The last one of a chain had verrucif-orm tubers which were backward claviform or ovate. Mature spores had three to seven tabul-as and some of them had one to three mediastinums. These results indicated that the strain belonged to Alternaria tenuissima. And it was named Alternaria tenuissima CH1307.By single factor experiment, the media and culture conditions of Alternaria tenuissima CH1307were optimized for homoharringtonine. The factors included carbon sources, nitro-gen sources, inorganic salts, trace elements, growth factors, pH, medium volume, inoculum size, rotary speed, temperature, incubation time, precursor substances and so on. The results showed that only a narrow scope of rotary speed and temperature was adapted to cell growth and homoharringtonine yield of Alternaria tenuissima CHI307. Besides, Mg2+, Mn2+, growth factors and precursor substances could observably enhance the yield of homoharringtonine.From single factor experiment, four prominent factors were screened out. They were glucose concentration, magnesium sulfate concentration, vitamin B compound concentration and rotary speed. Response surface analysis (RSA) was applied to optimize the fermentation of Alternaria tenuissima CHI307for homoharringtonine. The results manifested that glucose concentration and rotary speed had a remarkable effect on increasing the yield of homoharrin-gtonine. The optimal conditions were as follows:glucose32g/L, peptone2.5g/L, dipotassi-um phosphate1.5g/L, magnesium sulfate0.1g/L, potassium chloride1.0g/L, manganese sulf-ate0.01g/L, vitamin B compound0.125g/L, initial pH nature, medium volume100mL/250 mL, inoculum size2%, cultivate temperature25℃, rotary speed145r/min. Under the first-rank conditions, the tendency chart was drawn by surveying cell growth of Alternaria tenuissima CHI307, change of glucose and transformation of homoharringtonine.The result of antitumor activity research indicated that extracts of exocellular had strong activity to human liver cells SMMC-7721in vitro.