抗禽流感天然产物筛选及八仙草化学成分研究
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摘要
高致病性禽流感(Highly Pathogenic Avian Influenza, HPAI)自1878年在意大利首次暴发以来,已在世界范围内发生了20多次,每次暴发都造成了严重的经济损失和社会恐慌。目前有效治疗高致病性禽流感病毒(Highly Pathogenic Avian Influenza Virus)的药物比较少。
本课题选择A/Tiger/Harbin/01/2002株简称(HAB/01)H5N1病毒感染MDCK细胞为活性筛选模型,通过细胞病变法(Cytopathic Effect,CPE)作为初步筛选的活性评价方法,对40种中草药进行体外活性筛选。首次发现了几种抗H5N1病毒作用显著的中草药。首先发现八仙草(Galium aparine L)有较强的抗H5N1病毒作用。通过活性追踪发现,八仙草的抗H5N1病毒活性成分集中在正丁醇25%~50%乙醇洗脱部位,抗病毒作用呈明显的效量依赖关系。
八仙草为茜草科拉拉藤属一年生草本,该植物资源丰富,目前国内还没有开展相关的化学成分研究和开发。本课题通过生物活性跟踪并结合化学导向的研究路线,对八仙草抗H5N1病毒活性部位进行系统分离,综合运用多种分离手段对有效部位进行系统的分离纯化。分离得到33个单体化合物。通过理化测定和采用ESI-MS、1H-NMR、13C-NMR、HMQC、HMBC、NOESY等波谱手段,鉴定了26个化合物。分别是:去乙酰车叶草酸(1),1-羟基-3-异戊烯基-2-萘甲酸甲酯-4-O-β-D-木糖苷(2),3-异戊烯基-2-萘酸甲酯-1,4-二-O-β-D-葡萄糖苷(3)1,3,6-三羟基-2-甲基蒽醌-3-O-α-鼠李糖(1→2)-O-β-D-葡萄糖苷(4),1, 3-二羟基蒽醌(5),7S,8R,8'R-(-)-落叶松脂醇-4,4'–二-O-β-D-葡萄糖苷(6)3′,4′,3,5,7-五羟基黄酮(7),3′,4′,5,7-四羟基黄酮(8),3′,5,7-三羟基黄酮(9),4′-甲氧基-5-羟基黄酮-7-O-α-鼠李糖(1→6)-β-D-葡萄糖苷(10),4′,5,7-三羟基黄酮-3-O-α-鼠李糖(1→2)-β-D-葡萄糖苷(11),4′-甲氧基-3′5,7-三羟基黄酮-3-O-α-鼠李糖(1→6)-β-D-葡萄糖苷(12),尿嘧啶(13),胞嘧啶(14),尿嘧啶核苷(15),胸腺嘧啶脱氧核苷(17),腺嘌呤核苷(18),对羟基苯甲酸(19),原儿茶酸(20),香草酸(21),咖啡酸(22),β-谷甾醇(23),β-胡萝卜苷(24),正十六烷酸(25),杜鹃花酸(26),其中(2)为新化合物。
通过细胞病变法(Cytopathic Effect,CPE)对分离得到的部分化合物进行抗H5N1病毒活性评价,结果发现去乙酰车叶草酸(1)有明显的抗H5N1病毒活性,药物浓度为37.5μg/mL时,CPE结果为一个“+”,且有较好的剂量依赖关系。
Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype capable to infect and kill humans highlights the urgent need for new and efficient countermeasures against this viral disease. In the dissertation, we demonstrate that the extract from the Galium aparine exerts a potent anti-influenza virus activity in MDCK cell cultures infected with H5N1 subtype.
Galium aparine is a plant of the Rubiaceae family, distributed in the southern provinces of China. It is used in traditional Chinese folk medicine for the treatment of influenza. However, the investigations on the chemical constituents of plant have been few.
In a search for the active constituents from the plant, the 60% ethanol extract of the plant was subjected to the systematical chemical investigation, leading to the isolation of thirty-three compounds by chromatography methods. And twenty-six structures were elucidated on the basis of physical and chemical evidences, and modern spectral analysis (including IR,UV,1H-NMR,13C-NMR,HMQC,HMBC,DEPT,EI-MS,et al.). Their structures were elucidated as deacetyl asperulosidic acid (1),2-carbometho-xy-3-prenyl-1,4-naphthohydroquinone-1-O-β-D-xylopyranoside (2), 2-carbomethoxy-3-prenyl-1,4-naphthohydroquinone-di-β-D-glucoside(3), 2-methyl- 1,3,6-trihydroxy-9,10-anthraquinone-O-α-rhamnosyl(1→2)-β-D-glucoside(4), xantho- purpurin (5), cleamastanin B (6), quercetin (7),luteolin (8) ,apigenin (9), linarin (10), kaempferol-3-O-3-L-rhamnopyranosyl(1→2)-O-β-D-glucopyranoside (11),tamarixeti- n-3-rutinoside (12), uracil (13), cytosine (14), uridin (15), cytidine (16), thymidine (17), adenine-riboside (18), p-hydroxybenzcic acid (19), protocatechuic acid (20), vanillic acid (21), caffeic acid (22) ,β-sitosterol (23),β-daucosterol (24), n-hexadecane (25). azelaic acid (26). Compound 2 was a new compound. The result of the research was meaningful to a widespread usage of the plant, it is also helpful for the development of a new medicine.
At the effective dose of 37.5μg/mL, the compound 1 did not exhibit apparent harming effects on cell viability, metabolism or proliferation, which exerted a potent anti-influenza virus activity in MDCK cell cultures infected with H5N1 subtype.
本课题选择A/Tiger/Harbin/01/2002株简称(HAB/01)H5N1病毒感染MDCK细胞为活性筛选模型,通过细胞病变法(Cytopathic Effect,CPE)作为初步筛选的活性评价方法,对40种中草药进行体外活性筛选。首次发现了几种抗H5N1病毒作用显著的中草药。首先发现八仙草(Galium aparine L)有较强的抗H5N1病毒作用。通过活性追踪发现,八仙草的抗H5N1病毒活性成分集中在正丁醇25%~50%乙醇洗脱部位,抗病毒作用呈明显的效量依赖关系。
八仙草为茜草科拉拉藤属一年生草本,该植物资源丰富,目前国内还没有开展相关的化学成分研究和开发。本课题通过生物活性跟踪并结合化学导向的研究路线,对八仙草抗H5N1病毒活性部位进行系统分离,综合运用多种分离手段对有效部位进行系统的分离纯化。分离得到33个单体化合物。通过理化测定和采用ESI-MS、1H-NMR、13C-NMR、HMQC、HMBC、NOESY等波谱手段,鉴定了26个化合物。分别是:去乙酰车叶草酸(1),1-羟基-3-异戊烯基-2-萘甲酸甲酯-4-O-β-D-木糖苷(2),3-异戊烯基-2-萘酸甲酯-1,4-二-O-β-D-葡萄糖苷(3)1,3,6-三羟基-2-甲基蒽醌-3-O-α-鼠李糖(1→2)-O-β-D-葡萄糖苷(4),1, 3-二羟基蒽醌(5),7S,8R,8'R-(-)-落叶松脂醇-4,4'–二-O-β-D-葡萄糖苷(6)3′,4′,3,5,7-五羟基黄酮(7),3′,4′,5,7-四羟基黄酮(8),3′,5,7-三羟基黄酮(9),4′-甲氧基-5-羟基黄酮-7-O-α-鼠李糖(1→6)-β-D-葡萄糖苷(10),4′,5,7-三羟基黄酮-3-O-α-鼠李糖(1→2)-β-D-葡萄糖苷(11),4′-甲氧基-3′5,7-三羟基黄酮-3-O-α-鼠李糖(1→6)-β-D-葡萄糖苷(12),尿嘧啶(13),胞嘧啶(14),尿嘧啶核苷(15),胸腺嘧啶脱氧核苷(17),腺嘌呤核苷(18),对羟基苯甲酸(19),原儿茶酸(20),香草酸(21),咖啡酸(22),β-谷甾醇(23),β-胡萝卜苷(24),正十六烷酸(25),杜鹃花酸(26),其中(2)为新化合物。
通过细胞病变法(Cytopathic Effect,CPE)对分离得到的部分化合物进行抗H5N1病毒活性评价,结果发现去乙酰车叶草酸(1)有明显的抗H5N1病毒活性,药物浓度为37.5μg/mL时,CPE结果为一个“+”,且有较好的剂量依赖关系。
Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype capable to infect and kill humans highlights the urgent need for new and efficient countermeasures against this viral disease. In the dissertation, we demonstrate that the extract from the Galium aparine exerts a potent anti-influenza virus activity in MDCK cell cultures infected with H5N1 subtype.
Galium aparine is a plant of the Rubiaceae family, distributed in the southern provinces of China. It is used in traditional Chinese folk medicine for the treatment of influenza. However, the investigations on the chemical constituents of plant have been few.
In a search for the active constituents from the plant, the 60% ethanol extract of the plant was subjected to the systematical chemical investigation, leading to the isolation of thirty-three compounds by chromatography methods. And twenty-six structures were elucidated on the basis of physical and chemical evidences, and modern spectral analysis (including IR,UV,1H-NMR,13C-NMR,HMQC,HMBC,DEPT,EI-MS,et al.). Their structures were elucidated as deacetyl asperulosidic acid (1),2-carbometho-xy-3-prenyl-1,4-naphthohydroquinone-1-O-β-D-xylopyranoside (2), 2-carbomethoxy-3-prenyl-1,4-naphthohydroquinone-di-β-D-glucoside(3), 2-methyl- 1,3,6-trihydroxy-9,10-anthraquinone-O-α-rhamnosyl(1→2)-β-D-glucoside(4), xantho- purpurin (5), cleamastanin B (6), quercetin (7),luteolin (8) ,apigenin (9), linarin (10), kaempferol-3-O-3-L-rhamnopyranosyl(1→2)-O-β-D-glucopyranoside (11),tamarixeti- n-3-rutinoside (12), uracil (13), cytosine (14), uridin (15), cytidine (16), thymidine (17), adenine-riboside (18), p-hydroxybenzcic acid (19), protocatechuic acid (20), vanillic acid (21), caffeic acid (22) ,β-sitosterol (23),β-daucosterol (24), n-hexadecane (25). azelaic acid (26). Compound 2 was a new compound. The result of the research was meaningful to a widespread usage of the plant, it is also helpful for the development of a new medicine.
At the effective dose of 37.5μg/mL, the compound 1 did not exhibit apparent harming effects on cell viability, metabolism or proliferation, which exerted a potent anti-influenza virus activity in MDCK cell cultures infected with H5N1 subtype.
引文
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[43]张晓玲,杨桥,代俊,等.黑色素及谷胱甘肽通过抑制NF-κB途径的激活可有效抑制H5N1禽流感病毒感染[J] .中国生物化学与分子生物学报2009, 25 (4): 364-372.
[44] Furuta Y, Takahashi K, Kuno-Maekawa M, et al. Mechanism of action of T-705 against influenza virus [J]. Antimicrob Agents Chemother, 2005, 49(3): 81?986.
[45]赵晓虹,梁剑平,祝艳华,等.金丝桃素蛋白络合物体外抗H5N1亚型禽流感病毒的活性研究[J].中兽医医药杂志, 2006, 25(3): 13-15.
[46]祝艳华,梁剑平,赵晓虹,等.金丝桃素蛋白络合物在鸡体内对H9N2亚型禽流感病毒杀灭效果的研究[J].中兽医医药杂志, 2006, 25(3): 5-8.
[47]王曙阳,梁剑平,陈积红,等.金丝桃素体外抗口蹄疫病毒与宿主细胞吸附作用研究[J].中兽医医药杂志, 2009, 25 (1): 5-8.
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