中药地锦草抗HBV活性成分研究
|
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。
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.
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。
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.
引文
[1]王晓军,张荣珍,胡苑笙等.我国病毒性肝炎流行现状研究[J].疾病监测,2004, 19(8):290-292.
[2]苏成豪,许龙善.乙型肝炎流行病学研究进展[J].旅行医学科学,2008,14(1):5-8.
[3] Dienstag JL. Hepatitis B Virus Infection[J]. N Engl J Med,2008,359:1486-500.
[4] Lok ASF, McMahon BJ. Chronic Hepatitis B[J]. Hepatology,2007,45(2):507-539.
[5]钟敬波.抗乙肝病毒核苷类药物的研究进展[J].中国热带医学,2007, 7(11):4112-4113.
[6]王彩娟.抗乙肝病毒药物市场前景分析[J].世界临床药物,2007,28(7):444-448.
[7]余凌虹,刘耕陶.五味子联苯环辛烯类木脂素成分的结构与药理活性关系和药物创新[J].化学进展,2009,21(1):66-76.
[8]阎莉,郑作文.双环醇的研究进展[J].科学技术与工程,2006,6(9):1232-1238.
[9]林敬楠,罗光汉.苦参素治疗慢性乙型肝炎的临床研究进展[J].国际流行病学传染病学杂志,2007,34(6):409-411.
[10]朱铉,郑哲洙,崔京浩.水飞蓟素及其制剂的研究进展[J].中国野生植物资源, 2001,20(3):47-49.
[11]蒋道荣.甘草甜素对慢性乙型肝炎患者血清病毒标志的影响[J].南通医学院学报,1994,14(2):231-231.
[12] Shin MS, Kang EH, Lee YI. A flavonoid from medicinal plants blocks hepatitis B virus-e antigen secretion in HBV-infected hepatocytes[J]. Antiviral Research, 2005,67:163-168.
[13] Kang EH, Kown TY, Oh GT et al. The flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis B virus-e antigen[J]. Antiviral Research,2006,72:100-106.
[14] Wang GF, Shi LP, Ren YD et al. Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro[J]. Antiviral research,2009, available online.
[15] Xu J, Wang J, Deng F et al. Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro[J]. Antiviral Research,2008, 78:242-249.
[16] Lee SJ, Lee HK, Jung Mk et al. In vitro antiviral activity of 1,2,3,4,6-Penta- O-galloyl-β-D-glucose against Hepatitis B virus[J]. Biol Pharm Bull,2006,29(10): 2131-2134.
[17] Li JY, Huang H, Zhou W et al. Anti-hepatitis B virus activities of geranium carolinianum L. extracts and identification of active compounds[J]. Biol Pharm Bull, 2008,31(4):743-747.
[18] Zhou Z, Zhang Y , Ding XR et al. Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo[J]. Antiviral Research,2007,74:59-64.
[19] Zhao YL, Cai GM, Hong X et al. Anti-hepatitis B virus activities of triterpenoid saponin compound from Potentilla anserine L.[J]. Phytomedicine,2008,15:253-258.
[20] Huang RL, Huang YL, Ou JC et al. Screening of 25 compounds isolated from Phyllanthus species for anti-Human Hepatitis B Virus in vitro[J]. Phytother Res,2003, 17:449-453.
[21] Ma WH, Lu Y, Huang H et al. Schisanwilsonins A-G and related anti-HBV lignans from the fruits of Schisandra wilsoniana[J]. Bioorg Med Chem Lett, 2009,19:4958-4962.
[22]孙燕,李晶,李治.蒽醌对HBV稳定产毒细胞株HepAD38 HBV DNA复制的抑制作用[J].中国组织化学与细胞化学杂志,2008,17(1):9-13.
[23] Huang RL, Chen CC, Huang HL et al. Anti-Hepatitis B virus effects of wogonin isolated from Scutellaria baicalensis[J]. Planta Med,2000,66:694-698.
[24]辛秀,袁琳,王兴等.槲皮素对肝脏的药理作用研究进展[J].中国中医药信息杂志,2008,15(suppl.):102-104.
[25] Cao Y, Tan NH, Chen JJ et al. Bioactive flavones and biflavones from Selaginella moellendorffii Hieron.[J]. Fitoterapia,2009,available online.
[26]张娟,陈建宗,张金平等.黄芪甲甙体外抗乙型肝炎病毒的作用[J].第四军医大学学报,2007,28(4):2291-2293.
[27] Chen HC, Chou CK, Lee SD et al. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells[J]. Antiviral Research,1995,27:99-109.
[28] Jiang ZY, Zhang XM, Zhou J et al. Two New Sesquiterpenes from Alisma orientalis[J]. Chem Pharm Bull,2007,55(6):905-907.
[29] Zhao GQ, Yin ZF, Dong JX. Antiviral efficacy against hepatitis B virus replication of oleuropein isolated from Jasminum officinale L. var. grandiflorum[J]. Journal of Ethnopharmacology,2009,125:265-268.
[30] Yan MH, Cheng P, Jiang ZY et al. Periglaucines A-D, anti-HBV and–HIV-1 alkaloids from Pericampylus glaucus[J]. J Nat Prod,2008,71:760-763.
[31] Wu YR, Ma YB, Zhao YX et al. Two new quaternary alkaloids andanti-Hepatitis B virus active constituents from corydalis saxicola[J]. Planta Med, 2007,73:787-791.
[32]中国科学院中国植物志编辑委员会.中国植物志[M].北京:科学出版社, 1997,44(第三分册):49-50.
[33]韩广轩,谷莉,王立新等.中药地锦草的研究概况[J].药学实践杂志,2001,19(5): 308-311.
[34] Yoshida T, Amakura Y, Liu YZ, et al. Tannins and related polyphenols of Euphorbiaceous Plants.XI. Three new hydrolysable tannins and a polyphenol glucoside from Euphorbia humifusa[J]. Chem Pharm Bull,1994,42(9):1803-1807.
[35]裴英鸽.地锦草化学成分及生物活性研究.兰州大学研究生学位论文. 2007:40-64.
[36] Liu ZQ, Chen GT, Zhang JQ, et al. Four new lanostane triterpenoids from Euphorbia humifusa[J]. Helvetica Chemica Acta,2007,90:2245-2250.
[37] Deng F, Tang N, Xu J, et al. Newα-pyrrolidinonoids and glycosides from Euphorbia humifusa[J]. Journal of Asian Natural Products Research,2008,10(6): 531-539.
[38]柳润辉,王汉波,孔令义.地锦草化学成分的研究[J].中草药,2001,32(2):107-108.
[39]柳润辉,孔令义.地锦草脂溶性成分研究[J].天然产物研究与发,2005,17(4): 437-439.
[40]饶光宇,陈秀芬.地锦草保肝作用研究[J].中药药理与临床,1996,2:24-25.
[41]曹瑞珍,魏永春,强欣等.地锦草总黄酮预防四氯化碳所致急性肝损伤的实验研究[J].时珍国医国药,2007,18(1):85-86.
[42]李治建,古力娜·达吾提,斯拉甫·艾白.地锦草提取物体外抗真菌作用研究[J].时珍国医国药,2008,19(12):2958-2960.
[43]董鹏,唐万斌,郭连芳.地锦草的止血作用研究[J].武警医学院学报,1996, 5(1):22-24.
[44]陈福星,陈文英,褚景生等.地锦草水煎液对小鼠IFN-γ的影响[J].中国兽医杂志,2007,43(4):46-47.
[45]曹瑞珍,魏永春,张国文等.地锦草总黄酮对D-半乳糖衰老模型小鼠抗氧化作用的研究[J].卫生研究,2007,36(3):387.
[46] Nawwar MAM, Hussein SAM, Merfor I. NMR spectral analysis of polyphenols from punica granatum[J]. Phytochemistry,1994,36(3):793-798.
[47] Latte KP, Kolodziej H. Pelargoniins, new ellagitannins from Pelargonium reniforme[J]. Phytochemistry,2000,54:701-708.
[48] Nonaka G, Tanaka T, Nishioka I. Tannins and related compounds. Part 3. A newphenolic acid, sanguisorbic acid dilactone, and three new ellagitannins, sanguiins H-1, H-2, and H-3, from sanguisorba officinalis[J]. J C S Perkin Trans I,1982,1067-1073.
[49] Nawwar MAM, Hussein SAM. Gall polyphenolics of Tamarix aphylla[J]. Phytochemistry,1994,36(4):1035-1037.
[50] Latte KP. Ferreira D. Venkatraman MS. et al. O-Galloyl-C-glycosylflavones from Pelargonium reniforme[J]. Phytochemistry,2002,59:419-424.
[51]张兰珍,郭亚健,涂光忠等.叶下珠多酚化合物的分离与鉴定[J].中国中药杂志,2000,25(12):724-725.
[52]许文东,林厚文,邱峰等.蛇莓的化学成分[J].沈阳药科大学学报,2007, 24(7):402-405.
[53] Yazaki K, Hatano T, Okuda T. Constituents of Geranium thunbergii Sieb. et Zucc. Part 14. Structures of didehydrogeraniin, furosinin, and furosin[J]. J C S Perkin Trans I,1989,2289-2296.
[54] Hirano Y, Kondo R, Sakai K. 5α-Reductase inhibitory tannin-related compounds isolated from Shorea laeviforia[J]. J Wood Sci,2003,49:339-343.
[55]吴军,屠鹏飞,赵玉英.补阳还五汤中生物碱类化合物的分离与结构鉴定[J].中草药,2005,36(7):965-968.
[56]雷海民,李强,柏冬等.粗根老鹳草化学成分的研究[J].中国中药杂志,2007,32 (4):348-349.
[57]胡浩斌,樊君.短柄五加的化学成分研究[J].中国药学杂志,2009,44(15): 1137-1140.
[58] Lu YR, Foo LY. Flavonoid and phenolic glycosides from Salvia officinalis[J]. Phytochemistry,2000,55(3):263-267.
[59] Tanaka T, Nonaka G, Nishioka I. Tannins and related compounds. XVI. Isolation and characterization of six methyl glucoside gallates and a gallic acid glucoside gallate from Sanguisorba officinalis L.[J]. Chem pharm bull,1984,32(1):117-121.
[60] Nishizawa M, Yamagishi T. Tannins and related compounds. Part 5. Isolation and characterization of polygalloylglucoses from Chinese gallotannin[J]. J C S Perkin Trans I,1982,2963-2968.
[61]林佳,李琰,徐丽珍.石榴叶的化学成分研究[J].中南药学,2005,3(2):70-72.
[62]吴霞,刘净,于志斌等.薰衣草中黄酮类化学成分的研究[J].中国中药杂志,2007, 32(9):821-823.
[63] Iclal S, Mehtap V, Sebnem H et al. Acylated .flavonoids and phenol glycosides from Veronica thymoides subsp. pseudocinerea[J]. Phytochemistry,2004,65:2379-2385.
[64]赵东保,张卫,李明静等.顶羽菊化学成分研究[J].中国中药杂志,2006,31(22): 1869-1872.
[65] Miauno M, Kato M, Iinuma M et al. Acylated luteolin glucosides from Salzx gilglana[J]. Phytochemistry,1987,26(8):2418-2420.
[66]任玉琳,杨俊山.中药泥胡菜化学成分的研究[J].药学学报,2001,36(10):746-749.
[67] Yoshikawa M, Uemura T, Shimoda H et al. Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum Mill. (Parsley) and Structures of 6’’-acetylapiin and a new monoterpene glycoside, petroside[J]. Chem Pharm bull,2000,48(7):1039–1044.
[68]邹忠杰,杨峻山,鞠建华.泥胡菜化学成分的研究[J].中草药,2006,37(9): 1303-1304.
[69] Markham KR, Ternai B, Stanley R et al. Carbon-13 NMR studies of flavonoids- III naturally occurring flavonoid glycosides and their acylated derivatives[J]. Tetrahedron,1978,34:1389-1397.
[70] Brasseur T, Angenot L. Flavonol glycosides from leaves of Strychnos variabilis [J]. Phytochemistry,1986,25(2):563-564.
[71]尹锋,胡立宏,楼凤昌.罗勒化学成分的研究[J].中国天然药物,2004,2(1):20-23.
[72]沙东旭,刘英华,王龙顺等.叶下珠化学成分的研究[J].沈阳药科大学报,2000, 17(3):176-178.
[73]王亚君,杨秀伟,郭巧生.黄菊花化学成分研究[J].中国中药杂志,2008,33(5): 526-530.
[74]邓安,秦海林.土蜜树果实化学成分的研究[J].中国中药杂志,2008,33(2): 158-160.
[75]李帅,陈若芸,于德泉.三春水柏枝化学成分的研究(II)[J].中草药,2008,39(10): 1459-1461.
[76]段文娟,姜艳,靳鑫等.赤芍的化学成分研究[J].中国药物化学杂志,2009, 19(1):55-58.
[77]漆淑华,吴大刚,罗晓东.桂林乌桕中的香豆素和鞣花酸类化合物[J].天然产物研究与开发,2004,16(4):297-299.
[78]任玉琳,杨俊山.西藏雪莲花化学成分的研究II[J].中国药学杂志,2001,36(9): 590-593.
[79]吉腾飞,杨建波,宋卫霞等.新疆一枝蒿化学成分研究II[J].中国中药杂志,2007, 32(12):1187-1189.
[80] Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci USA,1987,84:1005-1009.
[81]史鸣树,闵建荣主编.乙型病毒性肝炎[M].人民军医出版社,2009:32-33.
[82] Chen B, Lian M, Xu SL et al. A chemical lipid modification of recombinant preS antigen to study the mechanism of HBV attachment to the host cell[J]. Journal of Biotechnology,2008,137:8-13.
[83]史鸣树,闵建荣主编.乙型病毒性肝炎[M].人民军医出版社,2009:40.
[84] Chen M, Sallberg M, Hughes J et al. Immune tolerance split between hepatitis B virus precore and core proteins[J]. J virol,2005,79(5):3016-3027.
[85] Havsteen BH. The biochemistry and medical significance of the flavonoids[J]. Pharmacology & Therapeutics,2002,96:67-202.
[86] Ahn MJ, Kim CY, Lee JS, et al. Inhibition of HIV-1 integrase by galloyl glucoses from Terinalia chebula and flavonol glycoside gallates from Euphorbia pekinensis[J]. Planta Med,2002,68:457-459.
[87] Wu JH, Wang XH, Yi YH et al. Anti-AIDS Agents 54. A Potent anti-HIV chalcone and flavonoids from genus Desmos[J]. Bioorg Med Chem Lett,2003, 13:1813-1815.
[88] Miki K, Nagai T, Suzuki K et al. Anti-influenza virus activity of biflavonoids[J]. Bioorg Med Chem Letters,2007,17:772-775.
[89] Jr BR, Fink RC, McMichael MD et al. Elderberry flavonoids bind to and prevent H1N1 infection in vitro[J]. Phytochemistry,2009,70(10):1255-1261.
[90] Jeong HJ, Ryu YB, Park SJ et al. Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities[J]. Bioorg Med Chem,2009,17:6816-6823.
[91] Zembower DE, Lin YM, Flavin MT et al. Robustaflavone, a potential non-nucleoside anti-hepatitis B agent[J]. Antiviral Research,1998,39:81-88.
[92] Orhan DD, Ozcelik B, Ozgen S, Ergun F. Antibacterial, antifungal, and antiviral activities of some flavonoids[J]. Microbiol Res,2009,available online.
[93] Du J, He ZD, Jiang RW et al. Antiviral flavonoids from the root bark of Morus alba L.[J]. Phytochemistry,2003,62:1235-1238.
[94] Meyer JJM, Afolayan AJ, Taylor MB, Erasmus D. Antiviral activity of galangin isolated from the aerial parts of Helichrysum aureonitens[J]. Journal of Ethnopharmacology,1997,56:165-169.
[95] Semple SJ, Nobbs SF, Pyke SM et al. Antiviral flavonoid from Pterocaulon sphacelatum, an Australian Aboriginal medicine[J]. Journal of Ethnopharmacology, 1999,68:283-288.
[96] Evers DL, Chao CF, Wang X et al. Human cytomegalovirus-inhibitory flavonoids: Studies on antiviral activity and mechanism of action[J]. Antiviral Research,2005,68:124-134.
[2]苏成豪,许龙善.乙型肝炎流行病学研究进展[J].旅行医学科学,2008,14(1):5-8.
[3] Dienstag JL. Hepatitis B Virus Infection[J]. N Engl J Med,2008,359:1486-500.
[4] Lok ASF, McMahon BJ. Chronic Hepatitis B[J]. Hepatology,2007,45(2):507-539.
[5]钟敬波.抗乙肝病毒核苷类药物的研究进展[J].中国热带医学,2007, 7(11):4112-4113.
[6]王彩娟.抗乙肝病毒药物市场前景分析[J].世界临床药物,2007,28(7):444-448.
[7]余凌虹,刘耕陶.五味子联苯环辛烯类木脂素成分的结构与药理活性关系和药物创新[J].化学进展,2009,21(1):66-76.
[8]阎莉,郑作文.双环醇的研究进展[J].科学技术与工程,2006,6(9):1232-1238.
[9]林敬楠,罗光汉.苦参素治疗慢性乙型肝炎的临床研究进展[J].国际流行病学传染病学杂志,2007,34(6):409-411.
[10]朱铉,郑哲洙,崔京浩.水飞蓟素及其制剂的研究进展[J].中国野生植物资源, 2001,20(3):47-49.
[11]蒋道荣.甘草甜素对慢性乙型肝炎患者血清病毒标志的影响[J].南通医学院学报,1994,14(2):231-231.
[12] Shin MS, Kang EH, Lee YI. A flavonoid from medicinal plants blocks hepatitis B virus-e antigen secretion in HBV-infected hepatocytes[J]. Antiviral Research, 2005,67:163-168.
[13] Kang EH, Kown TY, Oh GT et al. The flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis B virus-e antigen[J]. Antiviral Research,2006,72:100-106.
[14] Wang GF, Shi LP, Ren YD et al. Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro[J]. Antiviral research,2009, available online.
[15] Xu J, Wang J, Deng F et al. Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro[J]. Antiviral Research,2008, 78:242-249.
[16] Lee SJ, Lee HK, Jung Mk et al. In vitro antiviral activity of 1,2,3,4,6-Penta- O-galloyl-β-D-glucose against Hepatitis B virus[J]. Biol Pharm Bull,2006,29(10): 2131-2134.
[17] Li JY, Huang H, Zhou W et al. Anti-hepatitis B virus activities of geranium carolinianum L. extracts and identification of active compounds[J]. Biol Pharm Bull, 2008,31(4):743-747.
[18] Zhou Z, Zhang Y , Ding XR et al. Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo[J]. Antiviral Research,2007,74:59-64.
[19] Zhao YL, Cai GM, Hong X et al. Anti-hepatitis B virus activities of triterpenoid saponin compound from Potentilla anserine L.[J]. Phytomedicine,2008,15:253-258.
[20] Huang RL, Huang YL, Ou JC et al. Screening of 25 compounds isolated from Phyllanthus species for anti-Human Hepatitis B Virus in vitro[J]. Phytother Res,2003, 17:449-453.
[21] Ma WH, Lu Y, Huang H et al. Schisanwilsonins A-G and related anti-HBV lignans from the fruits of Schisandra wilsoniana[J]. Bioorg Med Chem Lett, 2009,19:4958-4962.
[22]孙燕,李晶,李治.蒽醌对HBV稳定产毒细胞株HepAD38 HBV DNA复制的抑制作用[J].中国组织化学与细胞化学杂志,2008,17(1):9-13.
[23] Huang RL, Chen CC, Huang HL et al. Anti-Hepatitis B virus effects of wogonin isolated from Scutellaria baicalensis[J]. Planta Med,2000,66:694-698.
[24]辛秀,袁琳,王兴等.槲皮素对肝脏的药理作用研究进展[J].中国中医药信息杂志,2008,15(suppl.):102-104.
[25] Cao Y, Tan NH, Chen JJ et al. Bioactive flavones and biflavones from Selaginella moellendorffii Hieron.[J]. Fitoterapia,2009,available online.
[26]张娟,陈建宗,张金平等.黄芪甲甙体外抗乙型肝炎病毒的作用[J].第四军医大学学报,2007,28(4):2291-2293.
[27] Chen HC, Chou CK, Lee SD et al. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells[J]. Antiviral Research,1995,27:99-109.
[28] Jiang ZY, Zhang XM, Zhou J et al. Two New Sesquiterpenes from Alisma orientalis[J]. Chem Pharm Bull,2007,55(6):905-907.
[29] Zhao GQ, Yin ZF, Dong JX. Antiviral efficacy against hepatitis B virus replication of oleuropein isolated from Jasminum officinale L. var. grandiflorum[J]. Journal of Ethnopharmacology,2009,125:265-268.
[30] Yan MH, Cheng P, Jiang ZY et al. Periglaucines A-D, anti-HBV and–HIV-1 alkaloids from Pericampylus glaucus[J]. J Nat Prod,2008,71:760-763.
[31] Wu YR, Ma YB, Zhao YX et al. Two new quaternary alkaloids andanti-Hepatitis B virus active constituents from corydalis saxicola[J]. Planta Med, 2007,73:787-791.
[32]中国科学院中国植物志编辑委员会.中国植物志[M].北京:科学出版社, 1997,44(第三分册):49-50.
[33]韩广轩,谷莉,王立新等.中药地锦草的研究概况[J].药学实践杂志,2001,19(5): 308-311.
[34] Yoshida T, Amakura Y, Liu YZ, et al. Tannins and related polyphenols of Euphorbiaceous Plants.XI. Three new hydrolysable tannins and a polyphenol glucoside from Euphorbia humifusa[J]. Chem Pharm Bull,1994,42(9):1803-1807.
[35]裴英鸽.地锦草化学成分及生物活性研究.兰州大学研究生学位论文. 2007:40-64.
[36] Liu ZQ, Chen GT, Zhang JQ, et al. Four new lanostane triterpenoids from Euphorbia humifusa[J]. Helvetica Chemica Acta,2007,90:2245-2250.
[37] Deng F, Tang N, Xu J, et al. Newα-pyrrolidinonoids and glycosides from Euphorbia humifusa[J]. Journal of Asian Natural Products Research,2008,10(6): 531-539.
[38]柳润辉,王汉波,孔令义.地锦草化学成分的研究[J].中草药,2001,32(2):107-108.
[39]柳润辉,孔令义.地锦草脂溶性成分研究[J].天然产物研究与发,2005,17(4): 437-439.
[40]饶光宇,陈秀芬.地锦草保肝作用研究[J].中药药理与临床,1996,2:24-25.
[41]曹瑞珍,魏永春,强欣等.地锦草总黄酮预防四氯化碳所致急性肝损伤的实验研究[J].时珍国医国药,2007,18(1):85-86.
[42]李治建,古力娜·达吾提,斯拉甫·艾白.地锦草提取物体外抗真菌作用研究[J].时珍国医国药,2008,19(12):2958-2960.
[43]董鹏,唐万斌,郭连芳.地锦草的止血作用研究[J].武警医学院学报,1996, 5(1):22-24.
[44]陈福星,陈文英,褚景生等.地锦草水煎液对小鼠IFN-γ的影响[J].中国兽医杂志,2007,43(4):46-47.
[45]曹瑞珍,魏永春,张国文等.地锦草总黄酮对D-半乳糖衰老模型小鼠抗氧化作用的研究[J].卫生研究,2007,36(3):387.
[46] Nawwar MAM, Hussein SAM, Merfor I. NMR spectral analysis of polyphenols from punica granatum[J]. Phytochemistry,1994,36(3):793-798.
[47] Latte KP, Kolodziej H. Pelargoniins, new ellagitannins from Pelargonium reniforme[J]. Phytochemistry,2000,54:701-708.
[48] Nonaka G, Tanaka T, Nishioka I. Tannins and related compounds. Part 3. A newphenolic acid, sanguisorbic acid dilactone, and three new ellagitannins, sanguiins H-1, H-2, and H-3, from sanguisorba officinalis[J]. J C S Perkin Trans I,1982,1067-1073.
[49] Nawwar MAM, Hussein SAM. Gall polyphenolics of Tamarix aphylla[J]. Phytochemistry,1994,36(4):1035-1037.
[50] Latte KP. Ferreira D. Venkatraman MS. et al. O-Galloyl-C-glycosylflavones from Pelargonium reniforme[J]. Phytochemistry,2002,59:419-424.
[51]张兰珍,郭亚健,涂光忠等.叶下珠多酚化合物的分离与鉴定[J].中国中药杂志,2000,25(12):724-725.
[52]许文东,林厚文,邱峰等.蛇莓的化学成分[J].沈阳药科大学学报,2007, 24(7):402-405.
[53] Yazaki K, Hatano T, Okuda T. Constituents of Geranium thunbergii Sieb. et Zucc. Part 14. Structures of didehydrogeraniin, furosinin, and furosin[J]. J C S Perkin Trans I,1989,2289-2296.
[54] Hirano Y, Kondo R, Sakai K. 5α-Reductase inhibitory tannin-related compounds isolated from Shorea laeviforia[J]. J Wood Sci,2003,49:339-343.
[55]吴军,屠鹏飞,赵玉英.补阳还五汤中生物碱类化合物的分离与结构鉴定[J].中草药,2005,36(7):965-968.
[56]雷海民,李强,柏冬等.粗根老鹳草化学成分的研究[J].中国中药杂志,2007,32 (4):348-349.
[57]胡浩斌,樊君.短柄五加的化学成分研究[J].中国药学杂志,2009,44(15): 1137-1140.
[58] Lu YR, Foo LY. Flavonoid and phenolic glycosides from Salvia officinalis[J]. Phytochemistry,2000,55(3):263-267.
[59] Tanaka T, Nonaka G, Nishioka I. Tannins and related compounds. XVI. Isolation and characterization of six methyl glucoside gallates and a gallic acid glucoside gallate from Sanguisorba officinalis L.[J]. Chem pharm bull,1984,32(1):117-121.
[60] Nishizawa M, Yamagishi T. Tannins and related compounds. Part 5. Isolation and characterization of polygalloylglucoses from Chinese gallotannin[J]. J C S Perkin Trans I,1982,2963-2968.
[61]林佳,李琰,徐丽珍.石榴叶的化学成分研究[J].中南药学,2005,3(2):70-72.
[62]吴霞,刘净,于志斌等.薰衣草中黄酮类化学成分的研究[J].中国中药杂志,2007, 32(9):821-823.
[63] Iclal S, Mehtap V, Sebnem H et al. Acylated .flavonoids and phenol glycosides from Veronica thymoides subsp. pseudocinerea[J]. Phytochemistry,2004,65:2379-2385.
[64]赵东保,张卫,李明静等.顶羽菊化学成分研究[J].中国中药杂志,2006,31(22): 1869-1872.
[65] Miauno M, Kato M, Iinuma M et al. Acylated luteolin glucosides from Salzx gilglana[J]. Phytochemistry,1987,26(8):2418-2420.
[66]任玉琳,杨俊山.中药泥胡菜化学成分的研究[J].药学学报,2001,36(10):746-749.
[67] Yoshikawa M, Uemura T, Shimoda H et al. Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum Mill. (Parsley) and Structures of 6’’-acetylapiin and a new monoterpene glycoside, petroside[J]. Chem Pharm bull,2000,48(7):1039–1044.
[68]邹忠杰,杨峻山,鞠建华.泥胡菜化学成分的研究[J].中草药,2006,37(9): 1303-1304.
[69] Markham KR, Ternai B, Stanley R et al. Carbon-13 NMR studies of flavonoids- III naturally occurring flavonoid glycosides and their acylated derivatives[J]. Tetrahedron,1978,34:1389-1397.
[70] Brasseur T, Angenot L. Flavonol glycosides from leaves of Strychnos variabilis [J]. Phytochemistry,1986,25(2):563-564.
[71]尹锋,胡立宏,楼凤昌.罗勒化学成分的研究[J].中国天然药物,2004,2(1):20-23.
[72]沙东旭,刘英华,王龙顺等.叶下珠化学成分的研究[J].沈阳药科大学报,2000, 17(3):176-178.
[73]王亚君,杨秀伟,郭巧生.黄菊花化学成分研究[J].中国中药杂志,2008,33(5): 526-530.
[74]邓安,秦海林.土蜜树果实化学成分的研究[J].中国中药杂志,2008,33(2): 158-160.
[75]李帅,陈若芸,于德泉.三春水柏枝化学成分的研究(II)[J].中草药,2008,39(10): 1459-1461.
[76]段文娟,姜艳,靳鑫等.赤芍的化学成分研究[J].中国药物化学杂志,2009, 19(1):55-58.
[77]漆淑华,吴大刚,罗晓东.桂林乌桕中的香豆素和鞣花酸类化合物[J].天然产物研究与开发,2004,16(4):297-299.
[78]任玉琳,杨俊山.西藏雪莲花化学成分的研究II[J].中国药学杂志,2001,36(9): 590-593.
[79]吉腾飞,杨建波,宋卫霞等.新疆一枝蒿化学成分研究II[J].中国中药杂志,2007, 32(12):1187-1189.
[80] Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci USA,1987,84:1005-1009.
[81]史鸣树,闵建荣主编.乙型病毒性肝炎[M].人民军医出版社,2009:32-33.
[82] Chen B, Lian M, Xu SL et al. A chemical lipid modification of recombinant preS antigen to study the mechanism of HBV attachment to the host cell[J]. Journal of Biotechnology,2008,137:8-13.
[83]史鸣树,闵建荣主编.乙型病毒性肝炎[M].人民军医出版社,2009:40.
[84] Chen M, Sallberg M, Hughes J et al. Immune tolerance split between hepatitis B virus precore and core proteins[J]. J virol,2005,79(5):3016-3027.
[85] Havsteen BH. The biochemistry and medical significance of the flavonoids[J]. Pharmacology & Therapeutics,2002,96:67-202.
[86] Ahn MJ, Kim CY, Lee JS, et al. Inhibition of HIV-1 integrase by galloyl glucoses from Terinalia chebula and flavonol glycoside gallates from Euphorbia pekinensis[J]. Planta Med,2002,68:457-459.
[87] Wu JH, Wang XH, Yi YH et al. Anti-AIDS Agents 54. A Potent anti-HIV chalcone and flavonoids from genus Desmos[J]. Bioorg Med Chem Lett,2003, 13:1813-1815.
[88] Miki K, Nagai T, Suzuki K et al. Anti-influenza virus activity of biflavonoids[J]. Bioorg Med Chem Letters,2007,17:772-775.
[89] Jr BR, Fink RC, McMichael MD et al. Elderberry flavonoids bind to and prevent H1N1 infection in vitro[J]. Phytochemistry,2009,70(10):1255-1261.
[90] Jeong HJ, Ryu YB, Park SJ et al. Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities[J]. Bioorg Med Chem,2009,17:6816-6823.
[91] Zembower DE, Lin YM, Flavin MT et al. Robustaflavone, a potential non-nucleoside anti-hepatitis B agent[J]. Antiviral Research,1998,39:81-88.
[92] Orhan DD, Ozcelik B, Ozgen S, Ergun F. Antibacterial, antifungal, and antiviral activities of some flavonoids[J]. Microbiol Res,2009,available online.
[93] Du J, He ZD, Jiang RW et al. Antiviral flavonoids from the root bark of Morus alba L.[J]. Phytochemistry,2003,62:1235-1238.
[94] Meyer JJM, Afolayan AJ, Taylor MB, Erasmus D. Antiviral activity of galangin isolated from the aerial parts of Helichrysum aureonitens[J]. Journal of Ethnopharmacology,1997,56:165-169.
[95] Semple SJ, Nobbs SF, Pyke SM et al. Antiviral flavonoid from Pterocaulon sphacelatum, an Australian Aboriginal medicine[J]. Journal of Ethnopharmacology, 1999,68:283-288.
[96] Evers DL, Chao CF, Wang X et al. Human cytomegalovirus-inhibitory flavonoids: Studies on antiviral activity and mechanism of action[J]. Antiviral Research,2005,68:124-134.