Chemical Constituents And Antiproliferative Of Grifola Frondosa On Cancer Cell Line

Grifola frondosa is a kind of precious edible and medical fungi, which belongs to layer of bacteria Basidiomycotina Class of non-Aphyllophorales Polyporaceae tree flowers sp. It can suppress tumor growth, enhance immunity, lower blood glucose, prevent virus, resist radiation, and eliminate free radical. The chemical constituents of Grifola frondosa included polysaccharide, nuclear acid, polyphenol, amino acid and trace elements. It was reported that the compounds of Grifola frondosa with low molecule contained raw polyphenol, however, due to insufficient analysis on Grifola frondosa, it was difficult to know the types, contents of compounds of Grifola frondosa, as well as their pharmacological mechanism.Our study focus on the isolation and purification of compounds with low molecule from Grifola frondosa. And we also investigate their chemical structure and anti-cancer effects. It was found that peroxy bond at 5,8-sites in compounds played an important role in suppressing the proliferation of cancer cells, and it was significant for modification of compounds to suppress tumor growth and accelerating the research on anti-cancer drugs.Isolated with water, ethanol and petroleum ether, which was new and efficient, it was found that water extract of Grifola frondosa contained triterpenoid saponin, ethanol extract contained alkaloid, terpene lactone, coumarin, organic acid, flavonoid, steroid and triterpenoid; petroleum ether extract contained steroid and triterpenoid.Thirteen kinds of constituents were obtained through extraction with 95% ethanol and ethyl acetate, then by silica gel column chromatography. MTT assays were used to determine the activity of various constituents on MCF-7 cell, and the active compounds were obtained finally. Three compounds were obtained by purification with micro recrystallization. And the structures of these compounds were identified by 1HNMR,13CNMR NMR spectroscopy:compound I (5α,8β-epidioxyergosta-6,22diene-3β-ol), compoundsⅡ(ergosterol) and compoundsⅢ(3β, 5α,6β-trihydroxyergosta-7,22-diene).MTT assay and flow cytometry were used to determined the inhibition of these compounds on MCF-7 cells. It was found that these three compounds could suppress the proliferation of cancer cells variously.5α,8β-epidioxyergosta-6,22diene-3β-ol significantly inhibited the proliferation of MCF-7 and led to apoptosis of cells; while ergosterol sterol 3β,5α,6β-trihydroxy ergosterol slightly inhibited proliferation of MCF-7.CompoundsⅠ,Ⅱ,Ⅲhad very similar structure, therefore it was necessary to conduct dynamic structure-activity analysis to find the key groups for anti-cancer. It was showed that peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation of MCF-7.In summary, the main results are as follows:1 For the first time it was report that Grifola frondosa contained coumarin, terpene lactones, flavonoids, organic acids, alkaloids, steroids, triterpenoids and other natural substances. The overall and systemic comprehension over small molecules laid the foundation for the development of complete utilization of Grifola frondosa.2 Three compounds were purified from Grifola frondosa for the first time: 5α,8β-epidioxyergosta-6,22diene-3β-ol; ergosterol and 3β,5α,6β-trihydroxyergosta-7,22-diene. Providing a further undstanding of chemical constituents of Grifola frondosa.3 5α,8β-epidioxyergosta-6,22diene-3β-ol could significant inhibition on MCF-7 and led to apoptosis of cells, providing a profound understanding that the types and contents of compounds in Grifola frondosa, and their relationship with the pharmacological effects of Grifola frondosa.4 Peroxy bond at 5,8-site of the ergot sterols played an important part in inhibiting the proliferation on MCF-7, providing informations for modification of compounds which was the key step during the development of anti-cancer research.5 It was important to improve the traditional method for natural products detction, dramatically reducing the false positive and negetive results. It was also significant to improve a method for recrystallization, enabling the purification of microgram samples.