Isolation And Identification Of Secondary Metabolites From The Liquid Culture Of Suillus Luteus(L.:Fr) Gray

Secondary metabolites play an important role in human life because many secondary metabolites show a variety of biological activity such as antibacterial antitumor,anti-inflammatory and so on. Edible medicinal fungus is big natural source of the secondary metabolites. The secondary metabolites in them may have unique structures and biological activity that may be lead compounds in the researching and development of new drugs. This may have important reference value to deal with various complex diseases of people.Submerged fermentation is a kind of efficient culture method for enrichment of secondary metabolites of fungi. A large number of secondary metabolites of fungi and mycelium can be obtained. Solid-state fermentation is also an effective method to produce many secondary metabolites which different from submerged fermentation.In order to study the secondary metabolites of Suillus luteus(L.:Fr) Gray which was collected in Tibetan areas. Fermentation products of Suillus luteus(L.:Fr) Gray by submerged fermentation and solid state fermentation were extracted. Ethyl acetate extract has been separated and purified. The cytotoxicities of part compounds were tested by MTT assay.The optimum formula of the liquid fermentation is potato200g/L, glucose 20g/L, yeast extract powder 5g/L, KH2PO4 3g/L, MgSO41.5g/L, VB110mg/L. The best time of the fermentation is 8.5d according to the OD600 that has the minimum value and the dry weight of mycelium is 0.9186g/ml. The 50 L fermentation tank is used for the liquid culture. The culture condition is liquid volume(60%), inoculum quantity(5%), temperature(26 ℃), pH(natural), tank pressure(0.05Mpa), ventilation(0.6v/v.m), rotation rate(160r/min). The content of reducing sugar first increase and then decrease, finally reach the minimum value; the content of protein always rise when the fermentation continue and the content of extracellular polysaccharide reach the highest value over time.Petroleum ether extract 6.2g, ethyl acetate extract 16.5g, n-butanol layer extract 123.7g was obtained. Crude extracellular polysaccharides 127.2g, dry weight of mycelium 421.3g of the liquid fermentation of S.luteus was obtained.The optimum formula of the solid-state fermentation is rice+0.5g/L ammonium tartrate.Ethyl acetate extract 20 g.The secondary metabolites of liquid fermentation and solid fermentation was studied. Modern separation methods such as thin layer chromatograpHy, sil ica gel- column chromatograpHy, HPLC etc, and spectral techniques including 13C-NMR, 1H-NMR, DEPT were employed for isolation and identification of th e compounds. Structures of 11 compounds were elucidated by detailed spectros copic analysis and compared with literature data. They are cycl-o(Tyr-Pro), cycl o-(Val-Pro), glycerol, cyclo-(pHe-Pro), xyloside, erthy ritol, P-hydroxytoluene, c ompound 8(Is to be identified), dibutyl phthalate, oleic acid, neo-inositol.This is the first time that these compound were isolated from fermentation of S. lu teus.Vitro antitumor experiment(determined by MTT method) was carried on for the part of the compounds andfermentation extract. The results of the antitumor vitro experiment showed that compound 2 had the best activity to the S180 cellline, the IC50 value is 0.073mg/ml. Extract of liquid fermentation had the best activity to the B16 cellline, the IC50 value is 0.0735mg/ml, and it also had the best activity to the MCF-7 cell-line with the IC50 value of 0.0828mg/ml.