Extraction And Activity Analysis Of Total Triterpenoids From Mycelium Of Sanghuangporus Sanghuang Liquid Fermentation

Sanghuangporus sanghuang is a traditional and precious medicinal fungus in China,which has the function of antibacterial,anti-tumor and scavenging free radicals.Due to the traditional controversy over the species and the scientific name of S.sanghuang,the research and development of the application have been greatly affected,and it was not clarified until recently(2016).The active substance represented by triterpenoids is an important pharmaceutical ingredient,but there is still little concern about the true triterpenoids of the S.sanghuang.In this study,S.sanghuang liquid fermentation mycelium was used as the experimental material to explore the extraction method of total triterpenoids and the corresponding biological activity analysis.The main contents and results of the study are as follows:1.The ITS-rDNA sequence of obtained S.sanghuang strain was compared by GenBank comparison analysis for the authoritative verification.The result of liquid fermentation of S.sanghuang showed that the optimum fermentation time for the total triterpenoids production was 11 days.The ultrasonic extraction process of the total triterpenes of 5.sanghuang mycelium was optimized by response surface method,and the optimal extraction method was that the ultrasonic extraction was under the condition of 100 W for 20 min with the ethanol concentration of 80%and the temperature of 60℃.According to this method,the total triterpenoids extraction was extracted with a yield up to 13.30 mg/g.2.The triterpenoids and organic acids in the total triterpenes of S.sanghuang was analyzed by qualitative assay of chemical constituents,and no flavonoids and polysaccharides were detected.By further infrared spectroscopy analysis,the corresponding functional groups of the absorption peak were compared to determine the presence of lanostane triterpenoids.Combined with GC-MS analysis,other small molecules were found in the total extracts of S.sanghuang total triterpenoids,in which a total of 34 compounds were isolated and the structures of 31 compounds were obtained.The content of its components accounted for 96.04%of the total chromatographic effluents,of which the acids accounted for 48.78%.3.Bacteriostatic activity determination showed that the extract with 2 times MIC of S.sanghuang total triterpenoids had obvious inhibitory effect on 4 kinds of test bacteria such as Escherichia coli,and the absorbance at OD260nm was significantly increased.It was speculated that triterpene destroyed the cell membranes of the bacteria and caused massive leakage of intracellular substances.Scanning electron microscopy confirmed that the cell morphology was significantly mutated,and the cells appeared shrunken and the cells were depressed.Among the four tested bacteria,the MIC was 20 mg/mL.Staphylococcus aureus was the most sensitive to the total extract of S.sanghuang total triterpenoids,and the diameter of the inhibition zone after the action was 10.22 mm.The diameters of the inhibition zones against Bacillus subtilis and Bacillus thuringeinsis were 9.28 mm and 9.32 mm,respectively.The bacteriostatic effect of Escherichia coli was relatively insignificant,and the diameter of the inhibition zone after the total extract of S.sanghuang total triterpenoids was 8.75 mm.4.To test the effect of the extract of S.sanghuang total triterpenoids on tumor cells,we found that it inhibited the proliferation of colon cancer cells Caco-2,and destroyed the morphology of cells which could hardly grow on the culture plate wall.In combination with flow cytometry,it was found that the cell cycle of colon cancer cell Caco-2 was blocked,and programmed cell apoptosis was induced in the cancer cells,and early and late apoptosis were increased accordingly.With the increase of S.sanghuang total triterpenoids extract concentration in a certain range,the programmed apoptosis of cells also increased.5.The results of determination of the antioxidant capacity of the total triterpenoids extract of S.sanghuang showed that it had a strong scavenging ability for free radicals such as 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).and when the concentration of S.sanghuang total triterpenoids reached 300 μg/mL,the scavenging abilities of four kinds of free radicals reached more than 90%.Totally,this kind of extract of S.sanghuang has good development and application potential.