The Study Of Extraction Process And Anti-tumor Active Component Of Compound Fomes Fomentarius

Objective To screen the anti-tumor extract group of compound Fomes fomentarius, evaluate its anti-tumor activity in vitro and in vivo and reveal the possible anti-tumor mechanism. Detect its chemical composition preliminarily, optimize its extraction process and provide preclinical research data for the research of the anti-tumor capsule of compound F.fomentarius.Methods①Distilled water or ethanol was used for extraction of compound F. fomentarius. The MTT assay was used to measure the inhibition effect in vitro to A549, Hela, QGY of distilled water extract of compound F. fomentarius and ethanol extract of compound F.fomentarius. Two methods were used for further separation of distilled water extract of compound F. fomentarius. One was precipitated by ethanol and divided into two parts, the supernatant (S3) and the precipition (S2). The MTT assay was used to measure the inhibition effect in vitro to A549, Hela, QGY of the two parts above. The organic solvent extraction technology was also used for further separation of distilled water extract of compound F.fomentarius. The MTT assay was used to measure the inhibition effect in vitro to Eca109,A549,SGC-7901,DU145of the four parts, the chloroform extraction component (S-1), the ethyl acetate extraction component (S-2), the n-butanol extraction component (S-3) and the apueous phase component (S-4).②The system pre-test method was used to detect the chemical composition preliminarily. The content of total glycosides, total triterpene, total flavonoids and total phenolic were determined by colorimetry. Orthogonal test was used to optimize its extraction process of S3.③The MTT assay was used to measure the inhibition effect in vitro to A549, Eca109, Saos-2, U2os, DU145, HCT116. Apoptosis of A549was tested by flow cytometry. The maximum tolerance testing assay was used to messure the acute toxicity of S3. S180-bearing and Lewis tumor-bearing mice models were estabilished. Five groups, model group, cyclophosphamide (CTX) group, high-(0.15g·kg-1), middle-(0.1g·kg-1) and low-dose (0.05g·kg-1) of S3, were studied for the two models. Then the effects of S3on tumor growth, thymus and spleen indexs, tumor necrosis factor (TNF-a) and macrophage colony-stimulating factor (M-CSF) levels were determined.④Sephadex G-25was used for atmospheric separation of S3, eluent was distilled water, merge and collect the same component. The MTT assay was used to measure the inhibition effect in vitro to SMMC-7721of the collected components. S180-bearing mice model was estabilished. Five groups, model group, cyclophosphamide (CTX) group, high-(0.04g·kg-1), middle-(0.015g·kg-1) and low-dose (0.005g·kg-1) of S3, was studied for the model. Then the effects of S3on tumor growth, thymus and spleen indexs were determined.Results①The IC50of distilled water extract of compound F. fomentarius to A549, Hela and QGY were(0.42±0.02),(0.46±0.12) and (0.44±0.06) g·L-1respectively, and to ethanol extract of compound F. fomentarius, the IC50were (0.57±0.09),(0.53±0.13) and (0.56±0.10) g·L-1respectively. The IC50of S2to A549, Hela and QGY were (0.87±0.02),(0.82±0.20) and (1.01±0.28) g·L-1respectively, and to S3, the IC50were (0.32±0.14),(0.29±0.06) and (0.38±0.14) g·L-1respectively. S-lshowed a stronger inhibitory effect compared with the other three components. The IC50of S-2to Ecal09, A549, SGC-7901, DU145were0.17,0.16,0.13and0.11g·L-1respectinely.②There were organic acid, sugar, polysaccharide and nucleoside, saponins, lactone, coumarone and nucleoside, plant sterol and terpene composition, volatile oil and grease in S3. The total sugar content of S3was more than33.00%, the total triterpenoid content was more than5.50%, the total flavonoids content was more than7.80%, the total phenolic content was more than10.00%. The optimum extraction process was extraction temperature100℃, extraction time1.5h, extraction times2and solid to liquid ratio of1:15. Extract three batches of herbs according to the optimum extraction, the yield of S3were4.14%,4.32%and4.36respectinely, and the total glucoside content were35.20%,35.83and35.59%respectinely.③S3has strong inhibition to A549, Eca109, DU145, HCT116, Saos-2and U2os and there was a relationship of time and dose-dependent.14.8%of totle cells apoptosis after A549treating with S3at1g·L-1for24h. The maximum tolerance of the KM mice to S3was more than10g·kg-1. Compared with dodel group, high-dose group of S3could inhibit tumor growth and reduce M-CSF levels of S180-bearing mice (P<0.05), middle-dose group could inhibit tumor growth and reduce M-CSF levels of Lewis tumor-bearing mice (P<0.05), high-dose group could increase thymus indexs, high-, middle-and low-dose groups of S3could elevate TNF-a levels (P<0.05, P<0.01)④Four components were collected, they were S31, S32, S33and S34. S31and S32showed a promoting role to the growth of the tumor cell SMMC-7721, while S33and S34showed an inhibition to tumor cells growth, and the IC50of S33and S34to SMMC-7721were (0.03±0.00) and (0.04±0.00) g·L-1respectively. S33and S34showed a weak inhibition to tumor growth of S180-bearing mice, and showed no significant difference in thymus and spleen indexs (P>0.05).Conclusion S3showed higher anti-tumor activity in vitro compared with S2and has significant inhibitory effect to a variety of tumor cells, further investigation revealed that S3exerted an antitumor activity maybe via apoptosis-induced cell death. S3has no overt toxicity to mice, could exhibitory effect on the growth of transplanted S180and Lewis tumor, probably via increasing index of immune organ and the regulation of TNF-a and M-CSF expression. Further investigation maybe developed for a new anti-tumor medicine.