【作者】 田瑛；

【导师】 董俊兴；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物化学， 2010， 博士

【摘要】 乙型肝炎病毒(HBV)感染严重危害人类的生命与健康,现有的抗HBV药物主要为核苷类药物。该类药物在临床上对于抑制病毒复制,缓解病人症状起到了积极的作用,但仍存在不能彻底地清除病毒、停药易反弹、耐药性等主要问题。因此,从中草药中发现并研发具有自主知识产权的新型抗HBV药物是重要途径。前期实验首次发现中药地锦草体外具有抗HBV作用,其活性部位为EHE-2和EHE-3。本研究采用HepG2.2.15细胞模型指导地锦草分离纯化的方法,深入、系统地开展了地锦草抗HBV活性部位的化学成分研究,评价了黄酮类化合物的体外抗HBV活性,初步分析了黄酮类化合物的构效关系,进一步探讨了活性化合物的抗病毒作用机理。地锦草(Euphorbia humifusa Willd.)为大戟科大戟属植物,全草入药,主治菌痢、肠炎、咳血、吐血、便血、崩漏、外伤出血、湿热黄疸、乳汁不通、痈肿疔疮和跌打肿痛等。本研究通过各种色谱分离技术,从地锦草抗HBV活性部位分离得到40个单体化合物,运用MS,1D-NMR,2D-NMR,NOESY,CD和UV等波谱技术,鉴定了其中38个化合物的结构,分别为phyllanthusiin E methyl este(rEHE-2-3-1),短叶苏木酚酸甲酯(EHE-2-3-2),短叶苏木酚酸(EHE-2-3-3),1-O-乙基-3,6-O-(R)-六羟基联苯二甲酰基-(1C4)-β-D-葡萄糖苷(EHE-2-3-4),地锦草酮(EHE-2-3-5),芙芦草素(EHE-2-3-6),橡椀酸双内酯（EHE-2-3-7),1-(2’,3’,4’,5’-四羟基戊基)-6,7-二甲基-喹噁啉-2,3(1H,4H)-二酮(EHE-2-3-8),没食子酸(EHE-2-3-10),没食子酸甲酯(EHE-2-3-11),芹菜素-6,8-二-C-β-D-葡萄糖苷(EHE-2-3-12),7’’-乙基-地榆酸双内酯(EHE-2-3-13),(1R,9S,11R)-5(Z),7(Z)-二烯-6-羰基-12-羟基-丁香烷( EHE-2-3-14 ), 1-O-甲基-6-O-p-二没食子酰基-α-D-葡萄糖苷(EHE-2-3-15),1-O-乙基-6-O-p-二没食子酰基-α-D-葡萄糖苷(EHE-2-3-16),1-O-甲基-6-O-p-二没食子酰基-β-D-葡萄糖苷(EHE-2-3-17),(1R,8S,9S,11R)-5(Z)-烯-6-羰基-8,12-二羟基-丁香烷(EHE-2-3-18),地榆酸双内酯(EHE-2-4-1),短叶苏木酚(EHE-3-4-1),木犀草素-7-O-β-D-葡萄糖苷(EHE-3-4-2),木犀草素-7-O-(6’’-O-阿魏酰)-β-D-葡萄糖苷(EHE-3-4-3),芹菜素-7-O-β-D-葡萄糖苷(EHE-3-4-4),芹菜素-7-O-(6’’-O-没食子酰)-β-D-葡萄糖苷(EHE-3-4-5),木犀草素-7-O-(6’’-O-香豆酰)-β-D-葡萄糖苷(EHE-3-4-6),芹菜素-7-O-β-D-芦丁糖苷(EHE-3-4-7),芹菜素-7-O-β-D-芹糖(1→2)-β-D-葡萄糖苷(EHE-3-4-8),芹菜素(EHE-3-4-9),槲皮素-3-O-α-L-鼠李糖(1→6)-β-D-半乳糖苷(EHE-3-4-10),槲皮素-3-O-β-D-葡萄糖苷和槲皮素-3-O-β-D-半乳糖苷(EHE-3-4-11),鞣花酸(EHE-3-4-12),橙皮苷(EHE-3-4-13),短叶苏木酚酸乙酯(EHE-3-4-15),3,3’-二甲氧基鞣花酸-4-O-β-D-葡萄糖苷(EHE-3-4-16),没食子酸乙酯(EHE-3-4-17),3,3’-二甲氧基鞣花酸(EHE-3-5-1),木犀草素(EHE-3-5-2)和槲皮素(EHE-3-5-3)。所得化合物中,7个新化合物:EHE-2-3-4,EHE-2-3-5,EHE-2-3-13,EHE-2-3-14,EHE-2-3-16,EHE-2-3-18和EHE-3-4-5;24个化合物为首次从该植物分得:EHE-2-3-1 , EHE-2-3-2 , EHE-2-3-3 , EHE-2-3-6 , EHE-2-3-7 , EHE-2-3-8 ,EHE-2-3-11,EHE-2-3-12,EHE-2-3-15,EHE-2-3-17,EHE-2-4-1,EHE-3-4-3,EHE-3-4-6,EHE-3-4-7,EHE-3-4-8,EHE-3-4-9,EHE-3-4-10,EHE-3-4-11,EHE-3-4-13,EHE-3-4-15,EHE-3-4-16,EHE-3-4-17和EHE-3-5-1。采用HepG2.2.15细胞模型,评价了黄酮类化合物的体外抗HBV活性,首次发现了4个抗HBV活性化合物(EHE-3-4-2,EHE-3-4-3,EHE-3-4-4和EHE-3-4-5)。化合物EHE-3-4-2体外主要对HepG2.2.15细胞中HBsAg分泌具抑制作用,IC50为20μg·mL-1(44.64μM);化合物EHE-3-4-3体外对HepG2.2.15细胞中HBeAg分泌的抑制作用明显,IC50为75.81μg·mL-1(121.49μM),对HBsAg分泌抑制率为18.6%(80μg·mL-1);化合物EHE-3-4-4和EHE-3-4-5体外对HepG2.2.15细胞中HBeAg、HBsAg分泌均具剂量依赖性的抑制作用,IC50分别为34.71μg·mL-(180.35μM)和16.27μg·mL-(137.66μM),75.75μg·mL-(1129.71μM)和36.90μg·mL-1(63.18μM)。初步的构效关系分析结果表明:黄酮类化合物结构中糖基数量对其抗HBV活性具有决定作用,表现为黄酮单糖苷>黄酮二糖苷,同时也决定了该类化合物的体外细胞毒性,表现为黄酮苷元>黄酮单糖苷>黄酮二糖苷;黄酮母核结构与化合物的抗HBV活性关系密切,呈现出芹菜素单糖苷>木犀草素单糖苷>槲皮素单糖苷;酰基取代的黄酮糖苷仍具有较好的体外抗HBV活性,表现为没食子酰基>阿魏酰基>香豆酰基。地锦草抗HBV活性成分研究的结果表明,通过对活性部位EHE-2和EHE-3的化学成分研究,分离鉴定了38个单体化合物(15个黄酮类、20个酚类、2个倍半萜类和1个生物碱类化合物),其中7个为新化合物,24个化合物为首次从该植物分得;通过黄酮类化合物抗HBV活性评价,首次发现了4个活性化合物,并初步分析了其构效关系。本研究发现了体外具有较好抗HBV活性的黄酮类化合物,针对活性化合物开展体内药效学研究,有希望成为抗HBV候选药物;该植物的活性部位富含黄酮类化合物,成分集中且体外具有抗HBV活性,开展活性部位的成药性研究,具有研制成中药五类新药的潜力。更多还原

【Abstract】 Hepatitis B virus (HBV) infection causes major public health problems worldwide. Nucleoside analog are the major drugs for HBV infections. However, significant side effects of these drugs and inevitable drug resistance have been noted. Therefore, it is important for our country to research and develop new anti-HBV drugs from the Traditional Chinese Medicine.Anti-HBV activity of the 70% ethanol extract from Euphorbia humifusa Willd. (EHE) has been found for the first time and then two active fractions including EHE-2 and EHE-3 were enriched from EHE under the guidance of the anti-HBV assay in vitro. In this dissertation, the phytochemistry of the active fractions from Euphorbia humifusa was studied deeply and systematically, anti-HBV activities of the flavones from the active fraction were evaluated, structure-activity relationships of these flavones were explained and further antiviral mechanism of the active compounds were discussed.Euphorbia humifusa Willd. (Euphorbiacea) is used for the treatment of bacillary dysentery, enteritis, cough up blood, blood-spitting, bloody stool, uterine bleeding, bleeding wound, jaundice with damp-heat pathogen and so on.The chemical investigation of the active fractions of this plant resulted in the isolation of 40 compounds by a variety of chromatography methods, and 38 compounds were identified on the basis of spectral data including MS, 1D-NMR, 2D-NMR, NOESY, CD and UV, as follows: phyllanthusiin E methyl ester (EHE-2-3-1), methyl brevifolincarboxylate (EHE-2-3-2), brevifolin carboxylic acid (EHE-2-3-3), 1-O-ethyl-3,6-O-(R)-hexahydroxydiphenoyl-(1C4)-β-D-glucopyranoside (EHE-2-3-4), humifusaone (EHE-2-3-5), furosin (EHE-2-3-6), valoneaic acid dilactone (EHE-2-3-7), 1-(2’,3’,4’,5’-tetrahydroxypentyl)-6,7-dimethyl-quinoxaline- 2,3(1H,4H)-dione (EHE-2-3-8), gallic acid (EHE-2-3-10), methyl gallate (EHE-2-3-11), 6,8-di-C-β-D-glucopyranosylapigenin (EHE-2-3-12), 7’’-ethyl- sanguisorbic acid dilactone (EHE-2-3-13), (1R,9S,11R)-5(Z),7(Z)-diene-6-carbonyl- 12-hydroxyl-caryophyllane (EHE-2-3-14), 1-O-methyl-6-O-p-digalloyl-α-D- glucopyranoside (EHE-2-3-15), 1-O-ethyl-6-O-p-digalloyl-α-D-glucopyranoside (EHE-2-3-16), 1-O-methyl-6-O-p-digalloyl-β-D-glucopyranoside (EHE-2-3-17), (1R,8S,9S,11R)-5(Z)-ene-6-carbonyl-8,12-dihydroxyl-caryophyllane (EHE-2-3-18), sanguisorbic acid dilactone (EHE-2-4-1), brevifolin (EHE-3-4-1), luteolin-7-O-β-D- glucopyranoside (EHE-3-4-2), luteolin-7-O-(6’’-O-trans-feruloyl)-β-D- glucopyranoside (EHE-3-4-3), apigenin-7-O-β-D-glucopyranoside (EHE-3-4-4), apigenin-7-O-(6’’-O-galloyl)-β-D-glucopyranoside (EHE-3-4-5), luteolin-7-O-(6’’-O -coumaroyl)-β-D-glucopyranoside (EHE-3-4-6), apigenin-7-O-β-D-lutinoside (EHE-3-4-7), apigenin-7-O-β-D-apiofuranosyl(1→2)-β-D-glucopyranoside (EHE-3-4-8), apigenin (EHE-3-4-9), quercetin-3-O-α-L-rhamnosyl(1→6)-β-D- galactoside (EHE-3-4-10), quercetin-3-O-β-D-glucopyranoside and quercetin-3-O-β-D-galactoside (EHE-3-4-11), ellagic acid (EHE-3-4-12), hesperidin (EHE-3-4-13), ethyl brevifolincarboxylate (EHE-3-4-15), 3,3’-di-O-methyl ellagic acid-4-O-β-D- glucopyranoside (EHE-3-4-16), ethyl gallate (EHE-3-4-17), 3,3’-di-O-methyl ellagic acid (EHE-3-5-1), luteolin (EHE-3-5-2) and quercetin (EHE-3-5-3). Among these compounds, EHE-2-3-4, EHE-2-3-5, EHE-2-3-13, EHE-2-3-14, EHE-2-3-16, EHE-2-3-18 and EHE-3-4-5 are new compounds. EHE-2-3-1, EHE-2-3-2, EHE-2-3-3, EHE-2-3-6, EHE-2-3-7, EHE-2-3-8, EHE-2-3-11, EHE-2-3-12, EHE-2-3-15, EHE-2-3-17, EHE-2-4-1, EHE-3-4-3, EHE-3-4-6, EHE-3-4-7, EHE-3-4-8, EHE-3-4-9, EHE-3-4-10, EHE-3-4-11, EHE-3-4-13, EHE-3-4-15, EHE-3-4-16, EHE-3-4-17 and EHE-3-5-1 were isolated from the plant for the first time.Through the anti-HBV assay of flavones from the active fraction, 4 active compounds (EHE-3-4-2, EHE-3-4-3, EHE-3-4-4 and EHE-3-4-5) were reported for the first time. EHE-3-4-2 blocked effectively the secretion of HBsAg with IC50 value of 20μg·mL-1 (44.64μΜ). EHE-3-4-3 appeared to significantly downregulate the secretion of HBeAg with IC50 value of 75.81μg·mL-1 (121.49μΜ) and the inhibition ratio of the secretion of HBsAg by this compound was 18.6% (at 80μg·mL-1). EHE-3-4-4 and EHE-3-4-5 blocked the production of HBeAg and HBsAg from HepG2.2.15 in a dose-dependent manner with IC50 values of 34.71μg·mL-1 (80.34μΜ) and 16.27μg·mL-1 (37.66μΜ), 75.75μg·mL-1 (129.71μΜ) and 36.90μg·mL-1 (63.18μΜ), respectively.The structure-activity relationship of these flavones showed that parent structure was closely relevant to their activities (agigenin > luteolin > quercetin). It was found that the number of glucoside determined their activities (flavone monoglucosides > flavone diglucosides) and cytotoxicities (flavones > flavone monoglucosides > flavone diglucosides). In addition, the substitution of acyl group on glucoside may be important to keep their anti-HBV activities (galloyl > feruloyl > coumaroyl).According to the study on the anti-HBV compounds of Euphorbia humifusa, 38 compounds were isolated and elucidated including 15 flavones, 20 phenols, 2 sesquiterpenoids and 1 alkaloid. Among these compounds, 7 compounds are new compounds and 24 compounds were isolated from this plant for the first time. Anti-HBV activities of 4 flavones were reported for the first time and further the structure-activity relationships were revealed.In the future, further active evaluation in vivo of the active compounds would help to determine anti-HBV candidates. And further officinal drug research on the active fractions would develop the fifth new drugs of the Traditional Chinese Medicine.更多还原