| Malignant tumor is one of the very irremediable and high mortality of major diseases.Liver cancer is the third most common types of mortality after gastric cancer and esophaguscancer. Although adjuvant chemotherapy has made some progress, the existing main classchemotherapy drugs are still the traditional cytotoxic chemotherapy drugs, while tumor cellswere in the treatment, normal cells of the human body were caused many adverse side effectswithout ideal therapeutic effects at the same time. Therefore, it is necessary to develop goodtumor suppression effect and side effects of small antitumor drugs, especially natural activeingredients isolated from natural materials have become a current research hotspot.Chroogomphus rutilus is a wild large ectotrophic mycorrhiza fungi which has richnutritions and extensive pharmacological activities. The purpose of this experiment is to screennatural monomer compounds with antitumor activity from Chroogomphus rutilus through H22tumor-burdened mice in vivo, and were combined with antitumor effect in vitro to elucidate itsrole in the treatment of liver cancer process. The anti-tumor mechanism of apoptosis andpossible pathways of apoptosis were further lucubrated. Experimental basis of liver cancertherapy was provided from natural active monomers as antitumor drug.This paper is that chemical components of Chroogomphus rutilus were separated andidentified using gradient extraction. By gas chromatography-mass spectrometry (GC-MS)technique, the chemical compounds and their contents of the petroleum ether extract weremeasured. And according to the national standards, heavy metals and trace elements of its driedfruiting body were evaluated systematically. While the chemical compositions of thechloroform layer, ethyl acetate layer and acetone layer extracts were isolated and identified,ethanol extracts achieved by ethanol extraction method were preliminary separated bymacroporous resin D101, receiving red and yellow components. The red, yellow componentsand ethanol extracts of were tentatively screened through antitumor activity in vivo, selectionof liver cancer H22ascitic fluid tumor mice model. The chemical compound was isolated fromthe active component---xerocomic acid, and further verified its anti-activity of liver cancer invivo. At the same time, tumor tissue pathology was researched by application of HE staining,Bax and Bcl-2immunohistochemical observation. Sulphorhodamine B (SRB) assay was usedto examine xerocomic acid’s cytotoxicity against cancer cells in vitro. Tumor cellmorphological changes were observed under inverted microscope after xerocomic acid treatment. Effects of different concentrations of xerocomic acid on HepG2cell apoptosis weremeasured by flow cytometry Annexin V-FITC/PI double staining. Finally the expressionlevels of Bcl-2, Bax, Cyt-c and PARP induced apoptosis pathway in HepG2cells weremonitored by western blot test.The extraction and separation of chemical components in fruiting body of Chroogomphusrutilus and the identification of the results: five monomer compounds were isolated, and threecompounds were identified as palmitic monoglycerol ester, adenosine and xerocomic acid.These three compounds were obtained from Chroogomphus rutilus for the first time. Thepreliminary anti-activity of H22liver cancer in vivo screening results: the yellow group isdivided into active component of anti-liver cancer, and the monomer compound was furtherseparated and identified----xerocomic acid. The experiments of H22tumor suppression in vivoverified that xerocomic acid was the natural active ingredient of anti-liver cancer. Its tumorinhibition rate highly amounted to57.71%closed to the positive group (57.89%). The SRBassay results showed that xerocomic acid significantly inhibited the proliferation of HepG2、Bel-7402、MCF-7and Hela cancer cells. It has a broad spectrum of tumor suppression effect ina dose-dependent manner and IC50as follows:41.37±0.36μg/ml、37.09±0.69μg/ml、80.84±1.06μg/ml、68.25±0.58μg/ml. After treatment with xerocomic acid at the concentrations of0,25,50and100μg/ml for24h, the apoptosis rates of HepG2cells were0.80±0.15%、5.68±0.27%、19.28±1.21%and21.38±1.98%, respectively, indicating xerocomic acid inducedapoptosis of HepG2cells in a dose-dependent manner. Western blot test results showed that itcan significantly increase the expression of Bax protein, cut down the Bcl-2protein expression,thereby enabling Cyt-c expression in great quantities, eventually reduce the protein expressionof PARP and increase the protein expression of cleaved PARP after treatment with differentconcentrations xerocomic acid.By gas chromatography-mass spectrometry (GC-MS), eight compounds were identifiedfrom petroleum ether extract of Chroogomphus rutilus fruiting bodies, which mainly containsunsaturated fatty acids of Linoleic acid and Vaccenic acid. After testing, four kinds of heavymetal elements as As, Pb, Hg, Cd and essential trace elements as Cu, Zn, Fe, Ca, Se, Cr wereincluded in the dried fruiting bodies of Chroogomphus rutilus, and the contents of heavy metalelements did not exceed the national standard. From the fruit body of Chroogomphus rutilus,palmitic monoglycerol ester, adenosine and xerocomic acid, these three kinds of monomercompounds were isolated and identified for the first time. Xerocomic acid was screenedrepeatedly through the experiment of anti-activity of H22tumor in vivo. In addition, it can notonly inhibit the proliferation of HepG2and Bel-7402, but also have very good effects on MCF-7and Hela tumor cell lines suppression in vitro. It shows that xerocomic acid has abroad-spectrum antitumor effect in vitro. And further research proved that the inhibitionmechanism of HepG2liver cancer cells was apoptosis. With the detection of Bcl-2, Bax, Cyt-cand PARP protein expression levels in the HepG2cell, preliminary judgment was made thatxerocomic acid induced HepG2liver cancer cell apoptosis through mitochondrial endogenousapproaches. |
PDF Full Text Request