| Strain Aspergillus tubingensis identified by morphology observation and molecular biology analyses showed the most efficient activity of transforming total saponins from leaves and stems of Panax ginseng.The total metabolite exhibited potential anticancer activity in vitro and in vivo.In order to confirm the substantial mechanism for the high activity of the total metabolite,which was subjected to the open ODS column chromatography,preparative HPLC and crystallization to afford 6 ginsenosides and 1 metabolite of Aspergillus tubingensis.They were identified respectively as gisnenosides Rh4 aglycone(1),Rh4(2),Rg6(3),20(S)-PPT(4),20(R)-Rg2(5),dammara-(20E)22,25-diene-3β,6α,12β,24S-tetrol(6),cyclic pentapeptide(7)identified by ESI-MS,13C-NMR and 1H-NMR.In vitro cytotoxicity activities against human gastric cancer SGC-7901cells,human fibrosarcoma HT-1080 cells and human oral epithelium carcinoma KB-A-1cells were evaluated by MTT method.The Rh4 aglycone and the cyclic pentapeptide had the most strong cytotoxicity activities.The IC50 values of Rh4 aglycone against SGC-7901,HT-1080 and KB-A-1cells were 31.40,12.10,30.56μmol/L respectively,and those of the cyclic pentapeptide were 1.20,1.72,1.64μmol/L respectively.The content of Rh4 aglycone was increased by about 4000%.Hence,the production of ginsenoside Rh4 aglycone in a large amount and the production of the cyclic pentapeptide in the fermentation process were the substantial mechanism for the high activity of the total metabolite.Previous work confirmed that the transformed products by Fusarium sacchari had strong antitumor activities.And it was confirmed that the total content of ginsenosides-Me,-Mx and C-K was up to above 60%.In order to discuss the related substantial constituents and to explore more kinds of active ginsenosids.12 compounds were isolated from the transformed products of the total saponins from Panax notoginseng leaves and stems by Fusarium sacchari,they were 20(R)-PPD(2-1),20(S)-PPD(2-2),20(S)-PPT(2-3),Me(2-4),C-K(2-5),Mx(2-6),20(R)-Rg3(2-7),20(S)-Rh2(2-8),20(R)-Rh1(2-9),20(R)-Rb1(2-10),20(R)-Rb3(2-11),20(R)-Rd(2-12)identified by 13C-NMR.Moreover,the growth inhibitory rates of product C-K against human gastric cancer BGC-823 cells,human colorectal cancer HCT-116 cells and human breast cancer MCF-7 cells were also studied,it was found that C-K showed a certain activities in low and middle dose,and showed strong activities in high dose,the growth inhibitory rates were 85.25%,74.04%,89.43%respectively.The asiaticoside(3-1)was firstly transformed into 3 compounds(derhamno-asiaticoside)(3-2),(derhamno-degluco-asiaticoside)(3-3)and asiatic acid(3-4)by fungus,Fusarium sacchari.The structures of compounds 3-1,3-2,3-3 and 3-4 were identified by 1H-and 13C-NMR analysis.The in vitro anticancer activities of these compounds against BGC-823,HCT-116 and MCF-7 cells were also evaluated.The results showed that compounds 3-1,3-2 and compound 3-3 nearly had no anticancer activity,but compound 3-4 exhibited strong anticancer activity.This is the first time to transform asiaticoside to asiatic acid.F.Sacchari was not only capacity of producing β-D-glycosidase activity,but also rhamnosidase activity.The glycosidase activity from crude enzyme in different temperature(T)and pH was detected.The data showed that the glycosidase produced by Fusarium sacchari active in the conditions(30-70℃,pH 3-7),and exhibited the strongest activity in 40-50℃,pH 5,and the enzyme was acid protease.Previous work discovered that Ginkgo biloba L.endophytic fungi,SY0056 may produce ginkgolide B by HPLC/MS.The fermentation liquid of SY0056 was extracted,purified and identified to afford ginkgolide B.Furthermore,the strain SY0056 was identified as Fusarium oxysporum by morphology observation and molecular taxonomy.The research offers a new idea to produce ginkgolide B by fungal fermentation,which can overcome the natural resource limitation of ginkgolide B isolated from Ginkgo leaves and barks forwards. |
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