| Paris polyphylla, a valuable medicinal plants mainly distributed in the Sichuan and Yunnan province of China. Paris Polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz and Paris polyphylla Smith var. chinensis (Franch.) Hara are species recorded in Pharmacopoeia of the People's Repulic of China (2010). Paris polyphylla is used as Chinese folk medicine for a few hundred years. For example it is one of the important composition in'Yunnan Baiyao','RDQ','Gongxuening'and so on. As an important medicine of raw materials, the requirements increase every year, but the reserves greatly reduced for its slow growth and low propagation coefficient. The supply has been gradually difficult to meet production requirements. Recent researches have showed that the endophytic fungi usually produced the same secondary metabolites as their hosts. The experimental material P. polyphylla is collected from TianQuan of Sichuan province which is P. polyphylla wild resource-rich regions. The endophytic fungi were isolated at the different growth and developmental stages of P. polyphylla var. chinensis. The endophyte can produce saponins was identified, and the contents of secondary metabolism saponins were determined, the preliminary study on their degeneration were carried out, in order to further exploit and utilize the P. polyphylla resources, and try to use the methods of fermentation to get medicinal composition by endophyte which is forming the foundation for resolve the problem of the wild P. polyphylla resources shortage and provides scientific evidence. The main results were described as following:(1) The isolation of endophytic fungi were carried out with 3 different medium in three different growth stages of P. polyphylla var. chinensis, it was found that 15 different fungi were isolated. Of which,8 were isolated in all the three different growth stages. It is also showed the mean of isolated fungi of the same separation medium in different seasons were quite different. From March to April and December to January the following year, the average number of colonies isolated and the total separated sorts number were higher than that during July to August, and the separation with MPDA medium were relatively better.(2) Microscopic identification was taken in 8 endophyte isolated in all the 3 growth stages of P. polyphylla var. chinensis (WD6H, WD7H, WD9H, CP, A221H, A4H, XIA2X, W2.1H, respectively), which were initially identified as Fusarium, Saprolegniales, Sphaeropsidales, Mucorales, Aspergillus, Aspergillus, Fusarium, Saccardiaceae.(3) Foam test and thin layer chromatography (TLC) were detected for fermentation products of these 8 endophytic fungi, polyphyllins components were identified in secondary metabolites of 4 species (WD6H, WD7H, XIA2X and A221H). Quantitative detection of saponins were detected by ultraviolet spectrophotometry, it turns out that the sapogenin yields in the fermentation broth were 3.61mg/L,2.72mg/L,3.36mg/L and 1.98mg/L.(4) Molecular identification with conserved sequences of fungal ITS sequences was taken for the 4 kinds of saponin produced fungi, it is confirm that WD6H as Fusarium solani, WD7H as Fusarium equiseti, XIA2X as Fusarium oxysporum, A221H as Monascus fumeus.(5) Degradation testing was taken with saponins produced WD6H and A221H fungi. The results showed that the 1st generation mycelium of WD6H can produce 0.68mg/mL saponins, the 5th generation mycelium can only produced 0.012mg/mL saponins; the 1st generation mycelium of A221H can produce 0.49mg/mL saponins, while the 5th generation can only produce 0.076mg/mL saponin. It indicated that after several generations in vitro, the ability of saponins production with endophyte in P. polyphylla var. chinensis has gradually degraded, it will be the focus of future research that how to ensure that endophyte in P. polyphylla var. chinensis can produce saponins stably through inheritance. |
PDF Full Text Request