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新疆一枝蒿抗病毒有效组分及其药理作用研究
阿吉姑·阿布都热西提
学位类型博士
导师阿吉艾克拜尔·艾萨
2016-06-04
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词新疆一枝蒿 抗病毒有效部位 制备工艺 质量标准研究 生物活性
摘要

一枝蒿(Artemisia rupestris L.),菊科 (Compositae) 蒿属植物岩蒿的全草,广泛分布于中国新疆,中亚,欧洲等地。一枝蒿是维吾尔医常用药材,具有解毒、健胃消食、抗过敏、抗菌、抗病毒及保肝等功效,主要用于治疗感冒、咽炎、荨麻诊及肝炎等症。为了更深入的了解一枝蒿药效作用的物质基础,以及更好的开发这一新疆传统药材,本论文对新疆一枝蒿抗病毒有效部位的制备工艺、化学成分分析、质量标准以及抗流感病毒和抑菌活性进行了系统研究。具体研究内容如下:1.新疆一枝蒿抗病毒有效部位提取纯化富集工艺的研究以一枝蒿酮酸、总黄酮及出膏率为指标,对新疆一枝蒿抗病毒有效部位的提取纯化工艺进行研究。通过单因素实验和正交试验,确定了新疆一枝蒿抗病毒有效部位的最佳提取条件,即料液比为14倍、70%乙醇溶液在80℃温度下将新疆一枝蒿药材提取2次,每次2小时,合并滤液。按照优化的提取工艺提取测得新疆一枝蒿有效部位中总黄酮含量为12.58%,一枝蒿酮酸平均含量为1.38%,而原药材中总黄酮含量为1%,一枝蒿酮酸平均含量为0.3%,该方法操作简便、工艺稳定可靠。在提取工艺的基础上,考察了8种不同极性的大孔树脂对一枝蒿酮酸和总黄酮的静态吸附及解析能力,筛选出HPD450型为其最佳大孔吸附树脂。通过考察最佳上样浓度、上样流速、最大上样量、除杂溶液用量和洗脱乙醇浓度及用量等工艺参数,确定了新疆一枝蒿抗病毒有效部位的富集纯化工艺为:上样浓度为0.2g/mL溶液,以2BV/h吸附速率进行吸附,上样量6BV时接近饱和,4BV纯化水除杂,再分别以4BV柱体积30%乙醇和3BV柱体积70%乙醇洗脱,合并乙醇洗脱液,减压浓缩,浸膏真空干燥,粉碎过80目筛,即得该有效部位。经过该富集方法纯化后的有效部位中,总黄酮的含量为22.82%,一枝蒿酮酸的含量为7.19%,与原药材中总黄酮和一枝蒿酮酸的含量相比,均得到了很大的提高。2.新疆一枝蒿有效部位的化学成分分析和质量标准研究采用HPLC-QTOF-MS/MS技术,结合制备型高效液相色谱等分离技术和NMR等结构鉴定方法,从新疆一枝蒿有效部位中鉴定出60个化合物。质谱研究发现,一枝蒿有效部位的大孔树脂30%乙醇洗脱部位的主要成分为黄酮苷类和绿原酸类化合物;而70%乙醇洗脱部位中主要含有倍半萜类、黄酮苷元及色原酮类化合。通过大孔树脂、制备液相色谱等分离纯化手段,从一枝蒿有效部位中分离纯化了6个化合物,通过核磁波谱解析和质谱,确定其结构为金腰素乙 (54)、一枝蒿酮酸 (34)、芹菜素-6,8-C-二-β-D-葡萄糖苷 (1)、3,5-二咖啡酰基奎宁酸 (19)、5,4′-二羟基-3′,5′-二甲氧基黄酮-7- o -葡萄糖醛酸苷 (25) 和5,4¢-二羟基-3′-甲氧基-7-o-葡萄糖醛酸苷(26)。利用高效液相色谱分析技术,通过与标准品在HPLC-DAD中保留时间与紫外光谱的比对,指认了该有效部位中的包含主要成分在内的15种化学成分,并同时对其中9个成分进行了含量测定。结果表明, 经纯化之后9个成分含量从原药材中的0.57%提高到20%,主要成分在有效部位中得以有效富集,含量有明显的提高。同时,建立了新疆一枝蒿抗病毒有效部位的质量标准,建立了原料药中一枝蒿酮酸、蒙花苷的薄层色谱鉴别方法和高效液相色谱(HPLC)含量测定的方法;通过紫外分光光度法建立了总黄酮的含量测定方法。本研究建立的方法简便、稳定、专属性强,为新疆一枝蒿抗病毒有效部位的质量控制提供了科学依据。3.新疆一枝蒿抗病毒有效部位的药理作用研究对新疆一枝蒿有效部位的体内、外抗流感病毒和抑菌生物活性进行了研究。一枝蒿有效部位的抑制甲型流感病毒的IC50为2.62μg·mL-1,小于利巴韦林组的IC50(8.15μg·mL-1),说明其具有显著的体外抑制甲型流感病毒作用。体内实验研究表明,与模型组比较,一枝蒿有效部位对PR8感染小鼠死亡具有显著的保护作用,并且可以显著延长感染小鼠的存活天数,存活时间随着给药剂量的增大而延长,说明一枝蒿有效部位在小鼠体内也具有较好的抗流感病毒作用。抑菌实验发现,新疆一枝蒿有效部位对金黄色葡萄球菌及大肠杆菌的MIC分别为3.9 mg/mL和31.25 mg/mL,其中对金黄色葡萄球菌的抑菌能力较强;此外,新疆一枝蒿有效部位中、大剂量组对大肠杆菌和金黄色葡萄球菌感染小鼠有一定的保护作用。

其他摘要

Artemisia rupestris L. (Compositae) is widely distributed in the Xinjiang Uighur autonomous region of China, central Asia, Europe, etc. It is a well-known traditional Chinese medicinal plant in Xinjiang and pharmacological studies showed its activities of clearing away toxins, strengthening the stomach to promote digestion, anti-hypersusceptibility, antibacteria, antivirus and protecting liver. Clinically, it is frequently used to treat cold, pharyngitis, urticaria, hepatitis and etc. In order to further develop the traditional Uyghur medicinal material, the preparation technology, chemical analysis, quality standards and biological activities of antiviral effective components from Artemisia rupestris L. were studied. Specific contents are as follows:1. Study on the extraction and purification technology of antiviral effective components from Artemisia rupestris L..In the present study,the rupestonic acid,total flavonoid and extraction yield were taken as indexes of extraction process of active fraction from Artemisia rupestris L. and purification process by macroporous resin. The extraction of active fraction from Artemisia rupestris L. was optimized using single factors and then the results of orthogonal test. The optimum extraction conditions were liquid ratio of 14 times, 70% ethanol solution at a temperature of 80 ℃, extract 2 times, 2 hours each time and the combined filtrate. The content of total flavonoids and rupestonic acid in the antiviral effective components were 12.58% and 1.38%, respectively, whereas their contents were 1% and 0.3% in the Artemisia rupestris L., respectively.Eight kinds of different of macroporous resins for the separation and enrichment of rupetonic acid and total flavonoids were screed. The HPD-450 type of resin offered the best adsorption and desorption capacity for the rupestonic acid and total flavonoids than others,and obtained the optimum parameters for adsorption and desorption of HPD-450. The sample concentration was 0.2g / mL , adsorption rate was 2BV/h , the sample volume was 6BV, purified water was 4BV, eluting with 4BV of 30% ethanol and 3BV of 70% ethanol,respectively. Fractions were combined and evaporated to dryness under reduced pressure. The content of total flavonoids and rupestonic acid of the antiviral effective components were 22.82% and 1.38%, respectively.2. Study on the chemical analysis and quality standards of antiviral effective components from Artemisia rupestris L..In this study, HPLC/QTOF-MS/MS was used to analyze components of the extract from Artemisia rupestris L., which showed an antivirus activity. 58 compounds were identified or tentatively characterized on the basis of Mass analysis, chlorogenic acids, flavonoid aglycones, flavonoid glycosides were the main components in the 30% ethanol part, and sesquiterpenoids, sesquiterpenoid derivatives and 2-phenoxy-chromones were the main components in the 70% ethanol part. Six compounds were isolated and purified from the antiviral effective components of Artemisia rupestris L. by macroporous resin and preparative high performance liquid chromatography and their structures were identified as chrysosptertin B (54), rupestonic acid (34), apigenin-6,8-c-di-β-d-glucopyranoside(1),3,5-dicaffeoylquinic acid (19), tricin-7-O-glucu-ronide (25), 5,4′-trihydroxy-3′-methoxy--7-O-glucuronide flavone (26). 15 components were identified from the antiviral effective fraction by HPLC-DAD analysis through comparison with retention time and UV spectrum of their corresponding the reference material. Among them, the contents of 9 components were determined. The quality standards of antiviral effective components from Artemisia rupestris L. were established. The method of TLC and HPLC for identify rupestonic acid and linarin were established. These methods are simple, stable, specific and provide a scientific basis for quality control of the antiviral effective components.3.Study on the biological activities of antiviral effective components from Artemisia rupestris L..In this study, the antiviral and antimicrobial activities of the Artemisia rupestris L. active fraction against H1N1 virus, Staphylococcus aureus and Escherichia coli were evaluated both in vitro and in vivo. Cytopathic effect reduction method was employed to determine anti-influenza activity of active fraction, and result showed that the active fraction possessed anti-virus activity. The IC50 value of the active fraction and ribavirin were 2.62 ± 1.22 μg ? mL-1 and 8.15 ± 0.35 μg ? mL-1, respectively. Among tested microorganisms, the active fraction showed more strong inhibitory effect on Staphylococcus aureus (MIC = 3.9 mg·mL-1) than Escherichia coli. Moreover,results showed that, mortality rate was more significantly attenuated in the treatment of the active fraction (60 and 120 mg/kg) than those in the infected control group. Our results have suggested that Artemisia rupestrisL. active fraction has great potential as a natural medicine for influenza and microbial infections.

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4563
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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阿吉姑·阿布都热西提. 新疆一枝蒿抗病毒有效组分及其药理作用研究[D]. 北京. 中国科学院大学,2016.
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