Study On The Major Secondary Metabolites Of Endophytic Fungus CH1307 From Cephalotaxus Hainanensis LI

Medicinal plants are the main resources for screening natural drug, but since it was reported that some endophytic fungi can produce the same or similar active components to those of the host, the study on endophytic fungi of medicinal plant has become the hotspot to obtain bioactive compounds. At present, Cephalotaxus hainanensis Li. is the resource for producing anti-cancer drugs homoharringtonine (HHT). Since the plant is extremely limited, it is difficult to meet the growing clinical need. Therefore, it is very important to screen endophytic fungi of Cephalotaxus hainanensis Li. in order to obtain compounds similar to HHT or other active ingredients.In this study, endophytic fungus strain CH1307 (Alternaria tenuissima) isolated from a phloem of Cephalotaxus's stem in the Hainan Danzhou Tropical Botanical Garden was our research object. The preliminary results showed that the strain had the ability to produce HHT, but it was found that its ability to synthesize HHT with dynamic fermentation in shake flask is unstable and the strain morphology is variable after long-term preservation and several-generation subculture. Therefore, in this study the strain was first confirmed that it still has the capability of synthesizing HHT; secondly, the strain was further separated, purified and identified to explore whether strain has changed or not after the several-generation subculture; finally, the strain was cultured with the rice solid fermentation and separation, purification, structure identification of the crude extraction and the physiological activities of the extraction were discussed. The main results are as follows:(1) Detection of HHT in the secondary metabolites of the endophytic fungus CHI 307 from the dynamic shake flask fermentationWith dynamic shake flask fermentation of endophytic fungus CH1307,40 L of fermentation broth and the crude extraction of alkaloids was obtained. The precipitation reaction was used for validating alkaloids. And it was confirmed that the endophytic fungus CH1307 still has a HHT production ability on the basis of ultra performance liquid chromatography mass spectrometry (UPLC-MS/MS) and magnetic resonance spectroscopy (NMR). And the yield was 81.2?g/L.(2) Further isolation, purification and molecular identification of endophytic fungus CH1307The strain CH1307 was further isolated, variability of colony morphology was observed, so CH1307 was further subdivided into five morphological types on the basis of the colony diameter and shape, hypha shape and height, cultivating pigment or not, and the plate color of positive and negative aspects. They were named CH1307a, CH1307b, CH1307c, CH1307d and CH1307e. Through microscopic observation, it was found that hyphae form of these five morphotypes were similar, but only CH1307c had spores. In addition, rDNA-ITS sections of the five morphological type strains were compared and it was found that they were the same. The five morphological type strains CH1307 can be classified as Alternaria sp. on the basis of the rDNA-ITS gene fragment and morphological characters.(3) The physiological activities the crude extraction of the secondary metabolites of strain CH1307c by rice solid fermentationThe strain CH1307c was cultured by solid state fermentation. The secondary metabolites were detected by the test tube qualitative reaction, and they were rich in phenols, alkaloids, flavonoids, saponins, steroids, glycosides and volatile oils and other substances. The crude extraction with ethyl acetate had no inhibitory effects to the seven indicators strains (Escherichia coli, Staphylococcu aureus, Bacillus subtilis, Streptococcus agalactiae, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum f.sp. Cubense). In addition, the crude extraction did not show inhibitory activity to cell lines K562, but showed strong cytotoxic activity to the two kinds of acute leukemia cell lines (NB4 IC50=28?g/mL, HL60 IC50=35?g/mL).(4) Structure identification and activity evaluation of the secondary metabolites of strain CH1307c by rice solid fermentationWith large scale fermentation of strain CH1307c in rice,200.4g of the crude extraction with the ethyl acetate was obtained. The components of the extraction were isolated and purified by normal phase silica gel column chromatography, thin layer chromatography (TLC), semi preparative HPLC and recrystallization. And the 8 single compounds were identified on the basis of NMR. They are Pestalotiopin A(1), ?-Sitosterol(2), Alternariol 5-O-sulfate(3), Alternariol monomethyl ether(4), Scopoletin(5), Cerevisterol(6), Oblongolide V(7), and 3-Methoxyphenoxy(8). Moreover, the eight compounds did not show inhibitory activities to leukemia cell lines K562 and NB4.