Isolating, Identifying The Endophytes And Researching On Their Metabolite From Paris Polyphylla Var. Chinensis Franch

Paris polyphylla Var. chinensis Franch is belong to Trilliaceae, Paris polyphylla genus, which is a kind of valuable Chinese herbal medicine of perennial herbs. Paris polyphylla is used as Chinese folk medicine for a few hundred years, because it can help to cure carbuncle swollen, sore throats, snake bitten, epilepsy disease et al. And it recorded in Pharmacopoeia of the People’s Repulic of China (2010). According to the pharmacological studies, Paris polyphylla have the ability of detoxication, analgesia, antibiosis, anticancer. So it was the vital composition in’Gong xue ning’,’RDQ’, ’Yunnan Baiyao’. But the production of Paris polyphylla is scarce every year, by the reason of its solw growth, low propagation coefficient, the lack of plant technology. Moreover, due to the huge market benefits, a large number of wild resources were excavated by farmers, beacese of that the wild Paris polyphylla resources reducing year by year. So exploreing alternatives of Paris polyphylla and developing artificial planting technology is the solution. Reference to some report, its endophytes can synthetize the same active ingredient, which can be extracted for the scale production. This study used endophytes of Paris polyphylla collected in Qionglai Sichuan try to find the endophytes which can produce saponins or saponins analogue, which can afford the research foundation to solve the lack of Paris polyphylla resources.Samples were collected from Dujiangyan, Baoxing, Yucheng, Mingshan and Tianquan in Sichuan. Based on the Tissue section method, we make an initial study on the Isolation and identification of the endophytic bacteria and the endophytic fungi. It was found 82 endophytic fungi, with 52 purified. Using ITS sequence, Identifying 26 endophytic fungi of them belonging to 7 genus.105 endophytic bacteria were isolated from the samples with 48 purified. Using 16S rDNA sequence, Identifying 35 endophytic bacteria belonging to 16 genus. After ultrasonic crushing of the fermentation liquor of endophytes, extracting, rotary evaporation. And then we use HPLC method detected it, by the contrast of Paris polyphylla saponin Ⅰ, Ⅱ, Ⅵ, Ⅶ and diosgenin. It is found that 4 endophytic fungi (DJY—10、DJY—7、MS—9、BX—15) and 3 endophytic bacteria (BX—9、YC—3、MS—6) of which had at least one with the same highly effective liquid phase on the peak time compared with the standard panel. The retention time of a peak in the HPLC of DJY—7, MS—9, BX—15, YC—3 corresponded to that of saponin Ⅰ. The retention time of a peak in the HPLC of YC—3 corresponded to that of saponin Ⅱ. By the way, BX—15、YC—3、MS—6 corresponded to saponin Ⅵ. BX—15 corresponded to saponin Ⅶ. And all the 7 endophytes corresponded to diosgenin. BX—15 and YC—3 had more retention time of a peak with standard panel,which had the potential to become the alternatives of Paris polyphylla.According to sequence data, DJY—10 and MS—9 were identified as Fusarium oxysporum. DJY—7,BX—9,YC—3 and MS—6 were identified as Trichoderma hamatum Bain, Ochrobactrum Holmes, Bacillus Cohn, pseudomonas aeruginosa, respectively. BX—15 was not identified because of sequence problem. Within the Trichoderma and Ochrobactrum, we found saponin produced endophytes at first time. That also indicate we should do the further research on Paris polyphylla endophytes.