| This paper studied on pharmacognosy, chemical composition and anti-tumor activity of fruiting body of Phellinus yamanoi.On pharmacognosy, microstructure of tube and basidiospore of fruiting body of Phellinus yamanoi were observed by tissue dissociation method. The result indicated that hyphal system are two hyphae system; the diaphragm of hyphae is simple. Generative hyphae are light color, with diaphragm, its diameter is 3.1～4.5μm. In context, most are skeletal hyphae. Skeletal hyphae are brown, non-branching, bending, spread ing interlaced and its paries is thick and has a lumen, its diameter is 3.4～5.1μm. Setas are conical, acuminate thick, its size is 40～60×6～10μm. Basidiospores is spherical,brown, smooth surface,thick-walled, containing a medium-sized droplets, and its size is 5～6×4～5μm.On chemistry, the dried fruit body was crushed and extracted by petroleum ether, chloroform, acetone, methanol and water in turn. Then petroleum ether extract, chloroform extract, acetone extract, methanol extract and water extract were obtained. Petroleum ether extract is divided into the supernatant and crystal two parts. Crystal recrystallized by ether, compound 1 was obtained; Dehydrated the supernate part of petroleum ether extract, and was separated by silica gel column with a solvent of petroleum ether:ethyl acetate (12:1 and 20:1), Components 1 and components 3 were obtained; Components 1 was separated by silica gel column with a solvent of methylene chloride:ethyl acetate (35:1), Components 2 were obtained; Components 2 was separated by silica gel column with a solvent of hexane:acetone (8:1), compound 3 was obtained; Components 3 was separated by silica gel column with a solvent of petroleum ether:acetone (30:1), Components 4 was obtained; Components 4 was separated by silica gel column with a solvent of hexane: acetone (30:1), compound 2 was obtained; Acetone extract was separated by silica gel column with a solvent of petroleum ether:acetone (50:1), Components 5 were obtained; Components 5 was separated by silica gel column with a solvent of methylene chloride:methanol (25:1), Components 6 were obtained; Components 6 was separated by silica gel column with a solvent of petroleum ether:ethyl acetate (4:1), compound 4 was obtained;The three compounds were analyzed by physicochemical examination, NMR and MS and identified as ergosterol peroxide, 1-methyl-18-ethyl-9-en-1,18-diester, 4,6,8 (14),22 (23)-tetraen-3-one ergostane,and 3,7-dihydroxy1-phene-7-butenone, is separated from fruiting body of Phellimis yamanoi for the first time.On anti-tumor activity, The anti-tumor activities of the petroleum ether extract, methanol extract and water extract and the monomer compound were studied in vivo on mice of Hepatoma H22. Inhibition rates of tumor, immune organ indexes and the contents of immune factors were evaluated as indicators. The results showed that the inhibition rates of petroleum ether extract of high dosage group (100mg/Kg) and the monomer compound of middle dosage group (7.5mg/Kg) were 62.21% and 57.67%.Their spleen indexes and thymus indexes are higher than the control group and CTX group, meanwhile IL-2 level is obviously higher than the control group and CTX group (P<0.01). Their TNF-a level is significantly lower than the control group (P<0.01). Hence, the above petroleum ether extract and the monomer compound inhabits Hepatoma H22 cell of mice, and also can improve mice's immune function. And comparing the chemical structure of compound 3, ergosterin and ergosterol peroxide and their inhibitory effects on anti-tumor, we confirm that cyclopentane with more hydrogen philippines of steroid nucleus and its shared substitute group are of the effective structures harbouring anti-tumor effects. The high-dose petroleum ether extraction of Phellinus yamanoi is the active part for anti-tumor effects and 4,6,8(14),22(23)-tetraen-3-one-ergostane is one of the active components. Besides, to determine whether the petroleum ether extraction has the alleviating effect on immunosuppression induced by CTX or not, we analyse the combined effect of the low-dose petroleum ether extraction and CTX.The results indicate that the spleen index, thymus index and the expression of IL-2 in CTX group were significantly declined compared to control group (P<0.01). Moreover, the spleen index, thymus index the expression of IL-2 in the low-dose petroleum ether extraction and CTX combined group were significantly increased compared to CTX group (P<0.01). It is suggested that the petroleum ether extraction of Phellinus yamanoi has the alleviating effect on immunosuppression induced by CTX to a certain degree, and at the same time its anti-tumor effects will not be influenced.We also analyse the influences of petroleum ether extraction, metheol extraction and water extraction on the histomorphology of cancer tissues respectively. The results indicate that the cancer cells in the group treated with petroleum ether extraction show disfussed distribution, distrinct size and more necrotic ratio than control group. Moreover, the cancer cells treated with the high-dose petroleum ether extract group (100mg/kg) show obviously deformation, restrained growth and more cone-shaped cells than the low-dose petroleum ether extract group (50mg/kg). Compared with the control group, more cells with deformation and different volume are found in group treated with low-dose petroleum ether extract combined with the CTX. Besides, the low-dose of methanol extract group (500mg/kg), the other dosages of methanol (1000mg/kg) or water extracts (500mg/kg and 1000mg/kg) have the same effects on cancer cells as the petroleum ether extract treated group. The morphology of cancer cells is almost spherical and do not change significantly, in spite of the volume which is different under treatment of low-dose of compound 3 group (3.5mg/kg). However, the cancer cells treated with the compounds 3 on the dosage of 7.5mg/kg show disfused distribution, restrained growth and more cone-shaped and necrotic cells.In addition, we also measure the effects of compound 3 on SMMC-7721 and MCF-7 cells using the MTT assay. Compared to control group, the OD value of the administrated group was significantly lower, and the compound 3 has different inhibiting effect on the proliferation of SMMC-7721 and MCF-7. when the dosage of compound 3 is up to 31.3ug/mL,the inhibitions ratios were 42.24% and 37.97% on SMMC-7721 and MCF-7, respectively, but the inhibiton do not show a concentration-dependent manner. The half maximal inhibitory concentration of compound 2 for human human liver cancer cells SMMC-7721 and human breast cancer cells MCF-7 in vitro are respectively43.08μg/mL,39.86μg/mL. |
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