琐琐葡萄抗乙肝病毒作用及其机制研究
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摘要
慢性病毒性肝炎是常见的严重危害人类健康的传染性疾病,全球60亿人口中,约20亿人曾感染乙型肝炎病毒(HBV),其中3.5~4亿人为慢性HBV感染者,约占全球人口的6%。慢性乙型肝炎患者或病毒携带者是发展为肝硬化和肝细胞癌的危险因素,15%~25%的感染者或携带者最终将死于与乙肝病毒感染相关的疾病。据WHO报告,全球每年约有100万人死于与乙肝病毒感染相关的肝病,占疾病死因的第九位,其中75%分布在亚太地区。我国是HBV感染的高流行区,病毒携带者约达1.3亿,HBV平均携带率为10%,部分地区如广东、湖北达到15%以上,其中慢性乙型肝炎现症患者约有3000万之众,且每年新增约200万。据一项疾病经济学调查表明,我国因慢性乙型肝炎及其相关疾病(肝硬化、肝癌等)每年造成的直接经济损失高达1122.8亿元人民币。病毒性肝炎、肝硬化不仅严重危害人民的健康,而且给国家带来了严重的社会经济负担。
慢性乙型肝炎的发病机制较为复杂,涉及到病毒复制、炎症介质释放、免疫功能紊乱及自由基损伤等多个环节,其中病毒持续复制和机体免疫清除是慢性乙型肝炎发病的两个基本要素,而HBV在体内持续复制是其迁延难愈的主要原因。因此,国内外对慢性乙型肝炎的治疗也采取了抗病毒、免疫调节、抗炎、改善肝功能和抗肝纤维化等综合措施,其中抗病毒治疗是关键。目前,使用干扰素、拉米夫定等药物进行临床治疗,通过抗病毒和调节机体免疫功能等作用,可诱导疾病缓解,阻止肝硬化进程,防止出现肝衰竭和/或肝细胞癌等严重后果,取得了良好的临床效果,但也存在价格昂贵,停药后反跳和长期服用易出现点突变,产生耐药性等局限性,使乙肝病毒感染始终是困扰人们的巨大难题。因而,寻找新型、价廉、有效的治疗乙型肝炎的药物一直是人们不懈追求的目标。
国内在应用中草药辩证施治或单方治疗慢性乙型肝炎方面已有长期的历史,其临床应用日趋广泛,而对其发挥药理作用的物质基础、作用机制、应用范围和疗效等方面的研究工作也在不断深入。琐琐葡萄(Vitis vinifera L.)为葡萄科(Vitaceae)葡萄属植物山葡萄,属无核红葡萄品系,木质藤本,主要分布在东亚,我国主产于新疆吐鲁番、和田、鄯善等地,是医药文献如《神农本草经》、《本草纲目拾遗》、《维吾尔药志》等所记载的药用葡萄品种之一。其果实,味甜微酸、气微、无毒,可内服、煎汤、捣汁或浸酒,有补气血,强筋骨,利小便之功效,主治气血虚弱,肺虚咳嗽,心悸盗汗,风湿痹痛等,传统医学常用作补气药,有解表透疹、补五脏、退黄等功效;维吾尔医用于治疗脾胃不和、头晕腰酸、神志不安等,民间用于小儿麻疹、肝炎等症,具有良好的临床效果。但是,目前有关对琐琐葡萄系统的植物化学、药理作用及作用机制的研究报道较少。
为了揭示维药琐琐葡萄抗病毒作用的物质基础及其可能的作用机制,为维吾尔医药用琐琐葡萄治疗肝炎等病毒性疾病提供科学依据,促进维吾尔医药的理论发展;同时,也为了充分利用新疆丰富的葡萄资源,变资源优势为经济优势,研制开发安全、有效、价廉的免疫调节制剂或抗病毒民族新药提供新思路,促进地区经济和社会的发展,本课题围绕维药琐琐葡萄的化学成分、抗病毒作用、免疫调节作用及其作用机制开展了系列研究。
1、琐琐葡萄化学成分的研究
目的:分析琐琐葡萄的化学成分,鉴定其结构,测定其含量,初步探讨琐琐葡萄多糖的还原能力及清除自由基能力。方法:采用乙醇提取,硅胶柱层析等方法分离琐琐葡萄脂溶性部位;采用聚酰胺等柱层析方法分离琐琐葡萄水溶性部位,采用1H-NMR、12C-NMR和MS等现代光谱技术进行结构鉴定;采用热水提取、乙醇沉淀、Sevage法除蛋白等方法提取琐琐葡萄多糖(VTP),并采用苯酚-硫酸法测定其含量,比较不同浓度VTP(200、400、600、800、1000μg/ml)和抗坏血酸VC(50、100、200、400、600、800、1000μg/ml)的还原能力以及不同浓度VTP和VC(均为40、80、160、320、600、800μg/ml)清除DPPH·的能力。结果:从琐琐葡萄脂溶性部位中提取分离得到琐琐葡萄总三萜(VTT),并纯化得到齐墩果酸(OA,Ⅰ),采用HPLC测定OA在VTT中的平均含量为65.52%;从琐琐葡萄水溶性部位中提取分离得到琐琐葡萄总黄酮(VTF),并从中分离得到异槲皮苷(Ⅱ)、槲皮素-3-O-β-D-葡萄糖醛酸钠盐(Ⅲ)、槲皮素-3-O-β-D-葡萄糖醛酸乙酯苷(Ⅳ)、没食子酸(Ⅴ)、单咖啡酰酒石酸(Ⅵ)等五个化合物,化合物Ⅱ~Ⅵ为首次从该植物中分离得到,其中化合物Ⅱ、Ⅲ和Ⅳ为首次从该属植物中分离得到;建立了VTP的提取及纯化方法,采用苯酚-硫酸法测定琐琐葡萄中多糖的含量为8.19%,经体外试验研究结果显示VTP的还原能力及清除DPPH·的能力随其浓度的增加而增大,在800μg/mL时,DPPH·清除率达到90%以上,接近VC的清除率,提示VTP具有较好的抗氧化能力。结论:通过琐琐葡萄系统的植物化学研究及结构鉴定,结合本课题组前期研究结果证实,该植物在民间药用治疗疾病的物质基础至少包括三萜、黄酮和多糖三大类化合物,其中,琐琐葡萄多糖含量较高,并具有较好的抗氧化能力,显示出良好的市场开发前景。
2、琐琐葡萄体外抑制HBV的实验研究
目的:研究琐琐葡萄提取物VTT、VTP和VTF的体外抗HBV作用及其联合作用。方法:通过VTT、VTP和VTF作用于体外培养的Hep G 2.2.15细胞,观察和比较VTT、VTP和VTF对HepG 2.2.15细胞分泌HBsAg、HBeAg和HBV DNA的影响及药物的细胞毒性;采用正交试验设计,观察VTT、VTP和VTF对体外培养的Hep G 2.2.15细胞分泌HBeAg的影响。结果:VTT、VTP、VTF及三者共同作用于Hep G 2.2.15细胞8天后,对细胞的半数毒性浓度TC50分别为160.61、1412.85、284.91和244.77μg/ml;VTT、VTP、VTF对HepG 2.2.15细胞分泌HBsAg的半数有效浓度(IC50)依次为62.54、112.69、327.56μg/ml,其治疗指数(TI)依次为2.57、12.54和0.87;对HepG 2.2.15细胞分泌HBeAg的IC50依次为89.28、204.46、215.34μg/ml,其TI依次为1.80、6.91和1.32;而对HepG 2.2.15细胞分泌HBV DNA的抑制作用,则表现为随着VTT、VTP和VTF浓度的增大,抑制作用逐渐增强;正交实验设计结果显示,VTT、VTP和VTF抑制Hep G 2.2.15细胞分泌HBeAg具有显著的交互作用,抑制强度依次为A×B×C>B×C>B>C,药物最佳组合为A2B1C1,即VTT 25μg/ml、VTP 50μg/ml和VTF 50μg/ml共同干预时,对Hep G 2.2.15细胞分泌HBeAg的抑制效果最佳。结论:通过体外抗病毒筛选研究初步证实,琐琐葡萄提取物VTT、VTP、VTF具有一定的体外抗HBV作用,其发挥抗病毒作用的物质至少包含VTT、VTP和VTF,这些活性部位既可单独作用也可联合作用发挥抗病毒效果。
3、琐琐葡萄体内抗鸭乙肝病毒作用的研究
目的:研究琐琐葡萄提取物抗鸭乙型肝炎病毒(DHBV)的作用。方法:采用荧光定量PCR法筛选先天感染DHBV的雏鸭为动物模型,共30只,根据DHBV DNA含量进行分层,随机分为病毒对照组、阳性对照药物拉米夫定组、琐琐葡萄提取物低、中、高剂量组,共5组,每组6只。除病毒对照组给予生理盐水外,其余各组均按设定剂量灌胃给药,给药体积为0.8 ml/只,一天1次,连续10天,斑点杂交法观察用药前(T0)、用药后(T5、T10)及停药后(P5)血清中DHBV DNA表达的变化。结果:琐琐葡萄提取物中剂量组在用药第10天(T10),血清中DHBV DNA均较给药前(T0)明显下降,停药5 d后(P5)有反跳的趋势,但反跳强度不大;低、高剂量组在给药期间未表现出明显抑制DHBV DNA的作用。结论:琐琐葡萄提取物具有一定的抑制DHBV DNA作用,与体外抗病毒试验结果基本一致,显示出较好的临床应用前景。
4、琐琐葡萄对大鼠原代肝细胞免疫损伤保护作用的研究
目的:观察琐琐葡萄提取物VOA、VTT、VTP和VTF在体外对大鼠原代肝细胞免疫性损伤的保护作用,为整体动物实验研究筛选受试物。方法:SD雄性大鼠6只,随机分为正常组与模型组,模型组采用卡介苗(BCG)整体致敏,脂多糖(LPS)离体攻击法建立大鼠原代肝细胞免疫损伤模型;采用MTT法分别测定VOA、VTT、VTP、VTF及阳性对照药物肝立欣(Grz)和联苯双酯(DDB)的细胞毒型;活性筛选试验中,模型组肝细胞分别加入DMEM培养液配制的二甲基亚砜溶液(DMSO)200μg/ml,DDB溶液5、10、20μg/ml, Grz溶液5、10、20μg/ml及VOA、VTT、VTP或VTF溶液5、20、80μg/ml,测定并比较不同培养时间(3、6、12、24 h)各组肝细胞培养上清液中谷丙转氨酶(ALT)、谷草转氨酶(AST)的活性和一氧化氮(NO)的含量。结果:细胞毒性试验结果显示,VOA、VTT、VTP、VTF、Grz和DDB的半数毒性浓度TC50分别为408.53、348.57、676.04、334.33、513.66和458.44μg/ml;细胞活性试验结果显示,正常大鼠肝细胞上清ALT、AST和NO基本未见改变,BCG+LPS损伤后大鼠肝细胞培养上清液中ALT、AST和NO在6、12和24 h较正常大鼠肝细胞明显升高(P<0.01),而从琐琐葡萄中提取的VOA、VTT、VTP或VTF(5,20,80μg/ml),在6、12和24 h均可不同程度地使升高的ALT、AST和NO明显下降(P<0.01),尤其是VTP和VTF的降酶效果呈现一定的剂量依赖关系,与阳性对照药物DDB和Grz作用相似。结论:琐琐葡萄提取物VOA、VTT、VTP和VTF在体外对大鼠免疫性肝损伤具有明显的保护作用。
5、琐琐葡萄对小鼠免疫性肝损伤的保护作用及作用机制研究
目的:研究琐琐葡萄提取物VTT、VTP和VTF对小鼠免疫性肝损伤的保护作用,并对其作用机制进行初步探讨,为阐明维医药用琐琐葡萄治疗肝炎等病提供理论依据,也为充分利用新疆丰富的葡萄资源优势,开发出安全、价廉、有效的抗病毒免疫调节制剂或保肝民族新药提供实验依据。方法:昆明种雄性小鼠144只,随机分为正常对照组、免疫性肝损伤模型组(BCG+LPS组)、DDB阳性对照组(200 mg/kg)以及VTT、VTP和VTF低、中、高剂量组(50、150、300 mg/kg)共12组,测定并比较各组小鼠肝脏、脾脏系数,采用连续监测法或比色法测定并比较各组小鼠肝组织匀浆中ALT、AST和超氧化物歧化酶(SOD)的活性以及丙二醛(MDA)和NO的含量,采用流式细胞仪测定各组小鼠外周血中CD4+/CD8+细胞亚群的比例,采用酶链免疫吸附法(ELISA)测定小鼠血清中干扰素-γ(IFN-γ)、白介素-10(IL-10)、白介素-2(IL-2)和白介素-4(IL-4)细胞因子的变化,采用免疫组化法观察细胞凋亡基因Bax和Bcl-2在小鼠肝组织中表达的变化,并采用HE染色,观察各组小鼠肝脏病理组织学变化。结果:(1)VTT、VTP和VTF均可不同程度地抵抗免疫性损伤所致小鼠肝脏和脾脏的肿大,有效恢复肝组织ALT和AST活性,升高免疫性肝损伤小鼠肝组织中SOD活性、减少MDA和NO的生成,尤其是VTF升高小鼠肝组织SOD活性具有显著的剂量依赖关系,而降低NO生成作用在各受试物中都显示一定的浓度依赖关系;(2)VTT、VTP和VTF未引起小鼠外周血CD4+、CD8+细胞数量的变化,但各受试物的高剂量组均可不同程度的显著降低IFN-γ的分泌,促进IL-10的分泌,同时也呈现出降低IL-2分泌,促进IL-4分泌的趋势,且VTT和VTF各剂量组间促进IL-10的分泌呈现一定的剂量反应关系,VTT、VTP和VTF可在一定程度上调节Th1和Th2型细胞因子分泌,恢复Th1/Th2细胞的平衡;(3)VTT、VTP和VTF均能不同程度的抑制肝损伤小鼠肝组织中Bax的表达,并促进Bcl-2的表达,恢复凋亡基因Bax和凋亡抑制基因Bcl-2的表达平衡,且VTP各剂量组间对Bax和Bcl-2表达的调节均具有一定的剂量反应关系,而VTF各剂量组间对促进Bcl-2的表达具有一定的剂量反应关系;(4)小鼠肝脏病理组织学检查结果显示,VTT、VTP和VTF均可在一定程度上改善肝组织病理损伤,恢复肝脏功能,与生化检查结果相一致。结论:VTT、VTP和VTF对小鼠免疫性肝损伤具有一定的保护作用,其作用机制可能与提高机体抗氧化物酶活性,有效清除自由基,降低脂质过氧化产物生成,保护肝细胞膜的完整性,改善肝细胞功能,调节Th1和Th2型细胞因子分泌,恢复Th1/Th2细胞的平衡及调节细胞凋亡基因的表达等有关。
综合琐琐葡萄植物化学分析、抗病毒作用、免疫调节作用及机制研究结果初步证实,琐琐葡萄发挥抗病毒作用和免疫调节作用的物质基础至少包含三萜、黄酮和多糖三大类化合物;而抑制HBV HBsAg、HBeAg分泌和HBV DNA合成及提高机体抗氧化物酶活性,有效清除自由基,降低脂质过氧化产物生成,保护肝细胞膜的完整性,改善肝细胞功能,调节机体免疫功能紊乱、抑制肝细胞过度凋亡的综合作用,可能是琐琐葡萄提取物VTT、VTP和VTF发挥抗病毒和免疫调节的药理学作用机制。这为阐明VTT、VTP和VTF与琐琐葡萄抗病毒、免疫调节作用的关系及其可能的作用机制提供了实验依据,也为维吾尔医药用琐琐葡萄治疗乙型肝炎等病毒性疾病提供了科学依据,将促进维吾尔医药的理论发展,为充分利用新疆丰富的葡萄资源,变资源优势为经济优势,进一步研制开发安全、有效、价廉的免疫调节制剂或抗病毒民族新药提供了新的思路,促进地区经济和社会的发展。
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 grape
Objective: 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 vitro
Objective: 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 virus
Objective: 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 vitro
Objective: 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 mechanisms
Objective: 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.
慢性乙型肝炎的发病机制较为复杂,涉及到病毒复制、炎症介质释放、免疫功能紊乱及自由基损伤等多个环节,其中病毒持续复制和机体免疫清除是慢性乙型肝炎发病的两个基本要素,而HBV在体内持续复制是其迁延难愈的主要原因。因此,国内外对慢性乙型肝炎的治疗也采取了抗病毒、免疫调节、抗炎、改善肝功能和抗肝纤维化等综合措施,其中抗病毒治疗是关键。目前,使用干扰素、拉米夫定等药物进行临床治疗,通过抗病毒和调节机体免疫功能等作用,可诱导疾病缓解,阻止肝硬化进程,防止出现肝衰竭和/或肝细胞癌等严重后果,取得了良好的临床效果,但也存在价格昂贵,停药后反跳和长期服用易出现点突变,产生耐药性等局限性,使乙肝病毒感染始终是困扰人们的巨大难题。因而,寻找新型、价廉、有效的治疗乙型肝炎的药物一直是人们不懈追求的目标。
国内在应用中草药辩证施治或单方治疗慢性乙型肝炎方面已有长期的历史,其临床应用日趋广泛,而对其发挥药理作用的物质基础、作用机制、应用范围和疗效等方面的研究工作也在不断深入。琐琐葡萄(Vitis vinifera L.)为葡萄科(Vitaceae)葡萄属植物山葡萄,属无核红葡萄品系,木质藤本,主要分布在东亚,我国主产于新疆吐鲁番、和田、鄯善等地,是医药文献如《神农本草经》、《本草纲目拾遗》、《维吾尔药志》等所记载的药用葡萄品种之一。其果实,味甜微酸、气微、无毒,可内服、煎汤、捣汁或浸酒,有补气血,强筋骨,利小便之功效,主治气血虚弱,肺虚咳嗽,心悸盗汗,风湿痹痛等,传统医学常用作补气药,有解表透疹、补五脏、退黄等功效;维吾尔医用于治疗脾胃不和、头晕腰酸、神志不安等,民间用于小儿麻疹、肝炎等症,具有良好的临床效果。但是,目前有关对琐琐葡萄系统的植物化学、药理作用及作用机制的研究报道较少。
为了揭示维药琐琐葡萄抗病毒作用的物质基础及其可能的作用机制,为维吾尔医药用琐琐葡萄治疗肝炎等病毒性疾病提供科学依据,促进维吾尔医药的理论发展;同时,也为了充分利用新疆丰富的葡萄资源,变资源优势为经济优势,研制开发安全、有效、价廉的免疫调节制剂或抗病毒民族新药提供新思路,促进地区经济和社会的发展,本课题围绕维药琐琐葡萄的化学成分、抗病毒作用、免疫调节作用及其作用机制开展了系列研究。
1、琐琐葡萄化学成分的研究
目的:分析琐琐葡萄的化学成分,鉴定其结构,测定其含量,初步探讨琐琐葡萄多糖的还原能力及清除自由基能力。方法:采用乙醇提取,硅胶柱层析等方法分离琐琐葡萄脂溶性部位;采用聚酰胺等柱层析方法分离琐琐葡萄水溶性部位,采用1H-NMR、12C-NMR和MS等现代光谱技术进行结构鉴定;采用热水提取、乙醇沉淀、Sevage法除蛋白等方法提取琐琐葡萄多糖(VTP),并采用苯酚-硫酸法测定其含量,比较不同浓度VTP(200、400、600、800、1000μg/ml)和抗坏血酸VC(50、100、200、400、600、800、1000μg/ml)的还原能力以及不同浓度VTP和VC(均为40、80、160、320、600、800μg/ml)清除DPPH·的能力。结果:从琐琐葡萄脂溶性部位中提取分离得到琐琐葡萄总三萜(VTT),并纯化得到齐墩果酸(OA,Ⅰ),采用HPLC测定OA在VTT中的平均含量为65.52%;从琐琐葡萄水溶性部位中提取分离得到琐琐葡萄总黄酮(VTF),并从中分离得到异槲皮苷(Ⅱ)、槲皮素-3-O-β-D-葡萄糖醛酸钠盐(Ⅲ)、槲皮素-3-O-β-D-葡萄糖醛酸乙酯苷(Ⅳ)、没食子酸(Ⅴ)、单咖啡酰酒石酸(Ⅵ)等五个化合物,化合物Ⅱ~Ⅵ为首次从该植物中分离得到,其中化合物Ⅱ、Ⅲ和Ⅳ为首次从该属植物中分离得到;建立了VTP的提取及纯化方法,采用苯酚-硫酸法测定琐琐葡萄中多糖的含量为8.19%,经体外试验研究结果显示VTP的还原能力及清除DPPH·的能力随其浓度的增加而增大,在800μg/mL时,DPPH·清除率达到90%以上,接近VC的清除率,提示VTP具有较好的抗氧化能力。结论:通过琐琐葡萄系统的植物化学研究及结构鉴定,结合本课题组前期研究结果证实,该植物在民间药用治疗疾病的物质基础至少包括三萜、黄酮和多糖三大类化合物,其中,琐琐葡萄多糖含量较高,并具有较好的抗氧化能力,显示出良好的市场开发前景。
2、琐琐葡萄体外抑制HBV的实验研究
目的:研究琐琐葡萄提取物VTT、VTP和VTF的体外抗HBV作用及其联合作用。方法:通过VTT、VTP和VTF作用于体外培养的Hep G 2.2.15细胞,观察和比较VTT、VTP和VTF对HepG 2.2.15细胞分泌HBsAg、HBeAg和HBV DNA的影响及药物的细胞毒性;采用正交试验设计,观察VTT、VTP和VTF对体外培养的Hep G 2.2.15细胞分泌HBeAg的影响。结果:VTT、VTP、VTF及三者共同作用于Hep G 2.2.15细胞8天后,对细胞的半数毒性浓度TC50分别为160.61、1412.85、284.91和244.77μg/ml;VTT、VTP、VTF对HepG 2.2.15细胞分泌HBsAg的半数有效浓度(IC50)依次为62.54、112.69、327.56μg/ml,其治疗指数(TI)依次为2.57、12.54和0.87;对HepG 2.2.15细胞分泌HBeAg的IC50依次为89.28、204.46、215.34μg/ml,其TI依次为1.80、6.91和1.32;而对HepG 2.2.15细胞分泌HBV DNA的抑制作用,则表现为随着VTT、VTP和VTF浓度的增大,抑制作用逐渐增强;正交实验设计结果显示,VTT、VTP和VTF抑制Hep G 2.2.15细胞分泌HBeAg具有显著的交互作用,抑制强度依次为A×B×C>B×C>B>C,药物最佳组合为A2B1C1,即VTT 25μg/ml、VTP 50μg/ml和VTF 50μg/ml共同干预时,对Hep G 2.2.15细胞分泌HBeAg的抑制效果最佳。结论:通过体外抗病毒筛选研究初步证实,琐琐葡萄提取物VTT、VTP、VTF具有一定的体外抗HBV作用,其发挥抗病毒作用的物质至少包含VTT、VTP和VTF,这些活性部位既可单独作用也可联合作用发挥抗病毒效果。
3、琐琐葡萄体内抗鸭乙肝病毒作用的研究
目的:研究琐琐葡萄提取物抗鸭乙型肝炎病毒(DHBV)的作用。方法:采用荧光定量PCR法筛选先天感染DHBV的雏鸭为动物模型,共30只,根据DHBV DNA含量进行分层,随机分为病毒对照组、阳性对照药物拉米夫定组、琐琐葡萄提取物低、中、高剂量组,共5组,每组6只。除病毒对照组给予生理盐水外,其余各组均按设定剂量灌胃给药,给药体积为0.8 ml/只,一天1次,连续10天,斑点杂交法观察用药前(T0)、用药后(T5、T10)及停药后(P5)血清中DHBV DNA表达的变化。结果:琐琐葡萄提取物中剂量组在用药第10天(T10),血清中DHBV DNA均较给药前(T0)明显下降,停药5 d后(P5)有反跳的趋势,但反跳强度不大;低、高剂量组在给药期间未表现出明显抑制DHBV DNA的作用。结论:琐琐葡萄提取物具有一定的抑制DHBV DNA作用,与体外抗病毒试验结果基本一致,显示出较好的临床应用前景。
4、琐琐葡萄对大鼠原代肝细胞免疫损伤保护作用的研究
目的:观察琐琐葡萄提取物VOA、VTT、VTP和VTF在体外对大鼠原代肝细胞免疫性损伤的保护作用,为整体动物实验研究筛选受试物。方法:SD雄性大鼠6只,随机分为正常组与模型组,模型组采用卡介苗(BCG)整体致敏,脂多糖(LPS)离体攻击法建立大鼠原代肝细胞免疫损伤模型;采用MTT法分别测定VOA、VTT、VTP、VTF及阳性对照药物肝立欣(Grz)和联苯双酯(DDB)的细胞毒型;活性筛选试验中,模型组肝细胞分别加入DMEM培养液配制的二甲基亚砜溶液(DMSO)200μg/ml,DDB溶液5、10、20μg/ml, Grz溶液5、10、20μg/ml及VOA、VTT、VTP或VTF溶液5、20、80μg/ml,测定并比较不同培养时间(3、6、12、24 h)各组肝细胞培养上清液中谷丙转氨酶(ALT)、谷草转氨酶(AST)的活性和一氧化氮(NO)的含量。结果:细胞毒性试验结果显示,VOA、VTT、VTP、VTF、Grz和DDB的半数毒性浓度TC50分别为408.53、348.57、676.04、334.33、513.66和458.44μg/ml;细胞活性试验结果显示,正常大鼠肝细胞上清ALT、AST和NO基本未见改变,BCG+LPS损伤后大鼠肝细胞培养上清液中ALT、AST和NO在6、12和24 h较正常大鼠肝细胞明显升高(P<0.01),而从琐琐葡萄中提取的VOA、VTT、VTP或VTF(5,20,80μg/ml),在6、12和24 h均可不同程度地使升高的ALT、AST和NO明显下降(P<0.01),尤其是VTP和VTF的降酶效果呈现一定的剂量依赖关系,与阳性对照药物DDB和Grz作用相似。结论:琐琐葡萄提取物VOA、VTT、VTP和VTF在体外对大鼠免疫性肝损伤具有明显的保护作用。
5、琐琐葡萄对小鼠免疫性肝损伤的保护作用及作用机制研究
目的:研究琐琐葡萄提取物VTT、VTP和VTF对小鼠免疫性肝损伤的保护作用,并对其作用机制进行初步探讨,为阐明维医药用琐琐葡萄治疗肝炎等病提供理论依据,也为充分利用新疆丰富的葡萄资源优势,开发出安全、价廉、有效的抗病毒免疫调节制剂或保肝民族新药提供实验依据。方法:昆明种雄性小鼠144只,随机分为正常对照组、免疫性肝损伤模型组(BCG+LPS组)、DDB阳性对照组(200 mg/kg)以及VTT、VTP和VTF低、中、高剂量组(50、150、300 mg/kg)共12组,测定并比较各组小鼠肝脏、脾脏系数,采用连续监测法或比色法测定并比较各组小鼠肝组织匀浆中ALT、AST和超氧化物歧化酶(SOD)的活性以及丙二醛(MDA)和NO的含量,采用流式细胞仪测定各组小鼠外周血中CD4+/CD8+细胞亚群的比例,采用酶链免疫吸附法(ELISA)测定小鼠血清中干扰素-γ(IFN-γ)、白介素-10(IL-10)、白介素-2(IL-2)和白介素-4(IL-4)细胞因子的变化,采用免疫组化法观察细胞凋亡基因Bax和Bcl-2在小鼠肝组织中表达的变化,并采用HE染色,观察各组小鼠肝脏病理组织学变化。结果:(1)VTT、VTP和VTF均可不同程度地抵抗免疫性损伤所致小鼠肝脏和脾脏的肿大,有效恢复肝组织ALT和AST活性,升高免疫性肝损伤小鼠肝组织中SOD活性、减少MDA和NO的生成,尤其是VTF升高小鼠肝组织SOD活性具有显著的剂量依赖关系,而降低NO生成作用在各受试物中都显示一定的浓度依赖关系;(2)VTT、VTP和VTF未引起小鼠外周血CD4+、CD8+细胞数量的变化,但各受试物的高剂量组均可不同程度的显著降低IFN-γ的分泌,促进IL-10的分泌,同时也呈现出降低IL-2分泌,促进IL-4分泌的趋势,且VTT和VTF各剂量组间促进IL-10的分泌呈现一定的剂量反应关系,VTT、VTP和VTF可在一定程度上调节Th1和Th2型细胞因子分泌,恢复Th1/Th2细胞的平衡;(3)VTT、VTP和VTF均能不同程度的抑制肝损伤小鼠肝组织中Bax的表达,并促进Bcl-2的表达,恢复凋亡基因Bax和凋亡抑制基因Bcl-2的表达平衡,且VTP各剂量组间对Bax和Bcl-2表达的调节均具有一定的剂量反应关系,而VTF各剂量组间对促进Bcl-2的表达具有一定的剂量反应关系;(4)小鼠肝脏病理组织学检查结果显示,VTT、VTP和VTF均可在一定程度上改善肝组织病理损伤,恢复肝脏功能,与生化检查结果相一致。结论:VTT、VTP和VTF对小鼠免疫性肝损伤具有一定的保护作用,其作用机制可能与提高机体抗氧化物酶活性,有效清除自由基,降低脂质过氧化产物生成,保护肝细胞膜的完整性,改善肝细胞功能,调节Th1和Th2型细胞因子分泌,恢复Th1/Th2细胞的平衡及调节细胞凋亡基因的表达等有关。
综合琐琐葡萄植物化学分析、抗病毒作用、免疫调节作用及机制研究结果初步证实,琐琐葡萄发挥抗病毒作用和免疫调节作用的物质基础至少包含三萜、黄酮和多糖三大类化合物;而抑制HBV HBsAg、HBeAg分泌和HBV DNA合成及提高机体抗氧化物酶活性,有效清除自由基,降低脂质过氧化产物生成,保护肝细胞膜的完整性,改善肝细胞功能,调节机体免疫功能紊乱、抑制肝细胞过度凋亡的综合作用,可能是琐琐葡萄提取物VTT、VTP和VTF发挥抗病毒和免疫调节的药理学作用机制。这为阐明VTT、VTP和VTF与琐琐葡萄抗病毒、免疫调节作用的关系及其可能的作用机制提供了实验依据,也为维吾尔医药用琐琐葡萄治疗乙型肝炎等病毒性疾病提供了科学依据,将促进维吾尔医药的理论发展,为充分利用新疆丰富的葡萄资源,变资源优势为经济优势,进一步研制开发安全、有效、价廉的免疫调节制剂或抗病毒民族新药提供了新的思路,促进地区经济和社会的发展。
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 grape
Objective: 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 vitro
Objective: 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 virus
Objective: 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 vitro
Objective: 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 mechanisms
Objective: 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.
引文
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