Study On Anti Hepatitis B Virus Activities And Its Mechanisms Of Suosuo Grape

Chronic virus hepatitis is a serious infectious disease. According to a statistics, there are about 2 billions people who infected hepatitis B virus in the world. Among them, about 3.5 to 4 hundreds of millions people are chronic hepatitis B patients. They are 6 percent in the whole world people. Chronic hepatitis B patient or carrier is the risk factor of liver cirrhosis and hepatic cellular cancer. About 15 percent to 25 percent chronic hepatitis B patients or carriers will succumb to diseases which correlate to hepatitis B virus infection at the last. With the entrance of World Health Organization, there are 1 million people succumbed to diseases correlate to hepatitis B virus infection in the whole world. It is the ninth disease in spectrum of death. About 75 percent of patients distribute in Asia and Pacific. Our country is the high epidemic region. People who carry hepatitis B virus are 1.3 hundreds of millions in China. The ratio of infection is 10 percent in average and several regions are beyond 15 percent such as Guangdong province and Hubei province. Among them, more than 30 millions carriers have developed into chronic hepatitis B patients and about 2 millions carriers will change into patients. According to an investigation of economics of disease, our country has to cost one hundred of billions RMB to treat chronic hepatitis, liver cirrhosis and hepatic cellular cancer. It is obvious that chronic hepatitis B not only has seriously endangered people's health, but also brought about heavy economic and social burden.The pathogenesis of chronic hepatitis B is comparative complex. It involves several links, such as virus replication, release of inflammation medium, dysfunction of immune system, injury from free radicals, et al. Among them, continuous virus replication and immune clearance of body are two basic pathogenic factors of chronic hepatitis B and continuous replication of hepatitis B virus is the main cause why chronic hepatitis B is very difficult recovery. At present, comprehensive measures in clued anti virus, immune regulation, anti inflammatory, improving liver function and anti hepatic fibrosis are used to therapy chronic hepatitis B in the world. The measure of anti virus is the key. Now, interferon or lamivudine is the generally acknowledged effective medicine applied to the clinical. These drugs can improve the prognosis, prevent liver cirrhosis and delay hepatic failure or hepatic cancer by drug's function of anti virus or immune regulation. But these drugs also have some limitations, such as expensive price, rebound phenomenon after drug suspension and point mutation after long term use. Searching new, cheap and effective drugs for therapy chronic hepatitis B is always the common goal of the whole world because hepatitis B virus infection is a great problem puzzled all people.In china, it has a longterm history that people adapted the Chinese herbs medicine to therapy chronic hepatitis B and its clinical application is gradually extensive. At present, many studies about its chemical components, function mechanism, application range and therapeutic effect are being developed continuously and deeply. SuoSuo grape is a kind of Woodiness Liana and belongs to Vitaceae branch, vitis catalog. It mainly distributes in East Asia is a kind of little red seedless grape. It mainly produced in Turpan, Hetian and Shanshan of Xinjiang and was recorded as a kind of drug in Shen Nong Materia Medica, Compendium of Materia Medica and Urghur Materia Medica. Its fruit tastes sweet and week acid, nontoxic. It can be oral administration by the method of juice or alcohol. It has obvious functions of supplement of qi-blood, strong the muscle and joint, inducing urination and is used to treat weakness of qi-blood, cough, palpitation, night sweat, rheumatism and arthralgia, et al. in Chinese medicine and Urghur medicine. Urghur use it to therapy children's measles and hepatitis in folk. But there was no systematic reports relating to phytochemistry, pharmacology and mechanism of SuoSuo grape presently.This subject developed systematic research around chemical constituents, immune regulation function, anti virus function of SuoSuo grape's and its possible mechanisms in order to reveal the substance basis of SuoSuo grape anti virus and its possible mechanisms, provide the scientific evidence for Urghur medicine using it to therapy virus disease included chronic hepatitis B and promote the theoretical development of Urghur medicine. Meanwhile, it also aims to bring into a new method for exploitation and utilization of abundant grape resource in Xinjiang to produce some new effective and cheap drugs and promote the development of region's economy and society.1 The study on chemical constituents of SuoSuo grapeObjective: To analyze the chemical constituents of SuoSuo grape, determine theirs contents and study the reducing ability and free radical scavenging effect of polysaccharides from SuoSuo grape.Methods: The lipid-soluble position of SuoSuo grape was extracted with ethanol and by silica gel column chromatography and its water-soluble position was extracted by polyamide column chromatography. Chemical structures elucidated by physicochemical properties and spectroscopic analysis.Polysaccharides from SuoSuo grape was extracted with water, subsided with 95% ethanol, cleared protein with Sevage assay and then dialyzed and purified. Contents of oleanolic acid (VOA) in triterpene (VTT) and polysaccharides (VTP) in SuoSuo grape were assayed with high performance liquid chromatography and phenol-vitriolic colorimetry respectively. Determination and compare of deoxidization activities of VTP (200, 400, 600, 800, 1000μg/ml) and vitamin C (50, 100, 200, 400, 600, 800, 1000μg/ml) and clearing DPPH free radical activities of VTP and vitamin C (40, 80, 160, 320,600,800μg/ml).Results: VTT and VOA were extracted from the lipid-soluble position of SuoSuo grape. The average content of VOA in VTT was 65.52%. Flavones (VTF) and VTP were extracted from the water-soluble position of SuoSuo grape. Five compounds were isolated and elucidated from VTF as isoquercitrin(2),quercetin-3-O-β-D-glucuronatesodium(3),quercetin-3-O-β-D-glucuronopyranoside ethyl ester (4), gallic acid (5), and Caftaric acid (6). The extract and purified method of VTP was established and the content of polysaccharides (VTP) in SuoSuo grape was 8.19%. With increase of concentration, the deoxidization and scavenging DPPH free radical capacity of VVP was increased continuously. VVP (800μg/ml) could clear 90% DPPH free radical. That was close to Vc. Conclusions: SuoSuo grape included three kinds of compounds at least, for example triterpene, flavones and polysaccharides. Among flavones, compound 2, 3, 4, 5 and 6 are obtained from this plant for the first time, and compound 2, 3 and 4 were isolated from Vitis genus for the first time.VTP showed a good market prospect of development because of its high content and strong antioxidant activity.2 The experimental study of SuoSuo grape against HBV in vitroObjective: To investigate the inhibition effects and joint action of VTT, VTP and VTF isolated form SuoSuo grape on HBV in vitro. Methods: Using the HBV genetic transfection hepatocarcinoma line HepG 2.2.15 as cellular model to screen anti-HBV drugs. To observe and compare the effect of VTT, VTP or VTF on the 2.2.15 cell secreting HBsAg, HBeAg and replicating HBV DNA and test theirs toxicity on hepatic cell with MTT. The joint action of VTT, VTP and VTF on the 2.2.15 cell secreting HBeAg were observed by orthogonal experimental design. Results: The result of cytotoxicity test displayed that TC50 of VTT, VTP, VTF and mixture of them were 160.61, 1412.85, 284.91 and 244.77μg/ml respectively. The median effective dose (IC50) of inhibition HBsAg were 62.54, 112.69 and 327.56μg/ml, the therapeutic index of HBsAg were 2.57, 12.54 and 0.87 individually. The median effective dose (IC50) of inhibition HBeAg were 89.28, 204.46 and 215.34μg/ml, the therapeutic index of HBeAg were 1.80, 6.91 and 1.32 individually. The inhibitory effect on HBV DNA was enhanced with the concentration increasing of extracts. The result of orthogonal experimental design showed that there had joint actions among VTT, VTP and VTF on inhibition HBeAg. The strength of inhibition were A×B×C>B×C>B>C in turn. The best combination of extracts was A2B1C1 (using VTT 25μg/ml, VTP 50μg/ml and VTF 50μg/ml together on HepG 2.2.15 showed the best effect of inhabitation HBeAg). Conclusions: VTT, VTP and VTF isolated form SuoSuo grape had antiviral effect in vitro. These activity positions could play the antiviral effect by alone or by joint use.3 The experimental study of SuoSuo grape against duck hepatitis B virusObjective: To observe the anti-duck hepatitis B virus (DHBV) action of extracts from SuoSuo grape. Methods: 30 Ducklings with congenital infection of DHBV screened with fluorescence quantitative PCR were used as the animal model. They were randomly and equally divided into 5 groups: virus control group, LAM group and low, middle, high dose groups of SuoSuo grape extracts. Except control group fed with physiological saline, others were respectively fed with LAM or SuoSuo grape extracts according to setting dose via i.g during 10 days. Blood was collected through the tibial vain. The expression of DHBV DNA was detected by spot blot hybridization before and after SuoSuo grape extracts was given. Results: In middle dose group of SuoSuo grape extracts, level of DHBV DNA in ducks'serum decreased distinctly on the 10th days after treatment. But the low and high dose groups were not showed the same action.4 The study on protective effects of SuoSuo grape on immunological liver injury in vitroObjective: To study the protective effects of VOA, VTT, VTP and VTF isolated form SuoSuo grape on immunological liver injury model in vitro for screening trial objects of animal experiment. Methods: Randomly distribute 6 male SD rats to control group and model group. The immunological hepatotoxicity model was induced by Bacillus Calmette Guerin (BCG) plus lipopolysaccharides (LPS) in vitro. The experiment on the cytotoxicity of VOA, VTT, VTP, VTF and Grz, DDB was done with MTT method. In experiment of the cell activity, the primary rats hepatocytes were separated and cultured with DMSO(200μg/ml), DDB or Grz (5,10,20μg/ml), VOA, VTT, VTP or VTF (5,20,80μg/ml) respectively. The supernatant of cells after 3, 6, 12 and 24h incubations were taken for the estimation of activities of ALT, AST and contents of NO respectively. Results: The result of cytotoxicity test displayed that TC50 of VOA, VTT, VTP, VTF, Grz and DDB were 408.53,348.57,676.04,334.33,513.66 and 458.44μg/ml. In the cell activity test, ALT, AST and NO in the supernatant of the normal rat hepatocytes had no significant changes in 24h. Whereas levels of ALT, AST and NO in BCG+LPS group were distinctly increased within 6, 12 and 24h compared with control group (P<0.01). VOA, VTT, VTP and VTF (5,20,80μg/ml) could decreased levels of ALT, AST and NO in different degree and different time point(P<0.01). Among them, the reducing enzymes effects of VTP and VTF showed a certain dose dependence relationship and the mentioned above effects of VTT and VOA were similar with those of DDB and Grz. Conclusions: The results indicated that VOA, VTT, VTP and VTF form SuoSuo grape had significantly protective effects against immunological liver damage induced by BCG plus LPS in vitro.5 The study on protective effects of SuoSuo grape on immunological liver injury in mice and its mechanismsObjective: To study the protective effects of VTT, VTP and VTF isolated form SuoSuo grape on immunological liver injury model in mice and its mechanisms for offering scientific basis of using SuoSuo grape to therapy hepatitis, also for providing experimental foundation of exploitation and utilization of abundant grape resource in Xinjiang to produce some new effective and cheap immune adjusting and liver protection reagents. Methods: 144 healthy Kunming mice were randomly divided into 12 groups: control group, immunological liver injury model group, DDB group and low, middle, high dose groups of VTT, VTP and VTF respectively. Except control group, others were injected BCG by tail vein. Next day, every group was respectively fed with DDB (200mg/kg bw) or VTT, VTP, VTF(50, 150, 300 mg/kg bw) via i.g during 12 days excluded control and model group. On 12th day, mice were injected with LPS or physiological saline (control group) and were killed 12h later. Blood, serum, liver and spleen were sampled. Determination and compare of liver and spleen index between groups. determine and compare activities of ALT, AST, SOD and contents of MDA in liver tissue were determined and compared by using sequential mensuration or colorimetry. The ratio of CD4+/CD8+ was measured with flow cytometer. Immune cytokines included IFN-γ, IL-10, IL-2 and IL-4 in serum were determined with ELISA. Expressions of apoptosis genes Bax and Bcl-2 in liver tissue were observed with immunohistochemical method and histopathological change of liver was observed. Results: (1) Treatment with different dose of VTT, VTP and VTF could significantly reduce liver and spleen index (P<0.01), increase activities of ALT, AST, SOD (P<0.01) and decrease contents of MDA and NO (P<0.01) in liver tissue of immunological liver injury mice. (2) The ratio of CD4+/CD8+ in blood had no significant change, but the high dose group of VTT, VTP or VTF could decrease the secretion of IFN-γand increase the secretion of IL-10. Meanwhile they showed a trend of IL-2 decrease and IL-4 increase. VTT, VTP and VTF could modulate the secretion of Th1 and Th2 cellular factors in a certain extent and resume the balance of Th1 and Th2. (3) Expressions of apoptosis genes Bax and Bcl-2 in liver tissue showed been inhibited and promoted respectively after intervention of VTT, VTP or VTF. And this result had a certain dose dependence relationship among VTP groups. (4) Histopathological observations also showed that VTT, VTP and VTF could ameliorate the liver injury in different degree and those results were also in correlation with the biochemical parameters. Conclusions: Results indicated that VTT, VTP and VTF from SuoSuo grape had significantly protective effects against immunological liver damage induced by BCG plus LPS in mice. Its mechanism might be relative to strong the activity of antioxidation, screen the free radical effectively, decrease the lipid peroxidation products, protect the hepatocytes membrance, improve the hepatocytes function, modulate the secretion of immune cytokines and expressions of apoptosis genes and so on.Results of chemical constituents study, immune modulation and antiviral action showed that the material base of SuoSuo grape developing the effects of immune modulation and antivirus included triterpene, flavones and polysaccharides at least. And these pharmacological mechanisms might be comprehensive effects including to strong the activity of antioxidation, screen the free radical effectively, decrease the lipid peroxidation products, protect the hepatocytes membrance, improve the hepatocytes function, modulate the secretion of immune cytokines and expressions of apoptosis genes, inhibit the secretion of HBsAg, HBeAg and the replication of HBV DNA. Researches above provided basis theoretical foundation for clarifying what was the relation between extracts (VTT, VTP and VTF) and the SuoSuo grape's function of immune modulation or antivirus. Studies above provided the scientific evidence for Urghur medicine using it to therapy virus disease included chronic hepatitis B and promoted the theoretical development of Urghur medicine. Meanwhile, those also brought into a new method and direction for exploitation and utilization of abundant grape resource in Xinjiang to produce some new effective and cheap drugs and promote the development of region's economy and society.