摘要
“热毒宁注射液”是由青蒿、金银花和栀子三味常用中药组成的复方中药注射液,具有清热、疏风、解毒之功效,临床上常用于治疗外感风热所致感冒、咳嗽,症见高热、微恶风寒、头痛、身痛、咳嗽痰黄；上呼吸道感染、急性支气管炎见上述证候。
本文采用流感病毒致应激负荷小鼠模型,以对甲型H1N1流感病毒FM1株感染应激负荷小鼠的开始发病时间、生存质量和生存时间为考察指标,对“热毒宁注射液”成品及其经HP-20大孔吸附树脂柱分离得到的子馏分(RDN-1/RDN-2/RDN-3)进行活性评价,确定馏分RDN-3(95%乙醇洗脱部位)为“热毒宁注射液”的抗病毒活性部位。
综合运用硅胶柱色谱、ODS柱色谱、Sephadex LH-20柱色谱、Toyopearl HW-40柱色谱以及反相MPLC、HPLC等多种色谱学分离手段,从活性部位(RDN-3)中共分离得到了63个化合物,通过理化性质和现代波谱学手段鉴定了其中56个化合物的结构,包括12个环烯醚萜类化合物,12个木脂素类化合物,3个香豆素类化合物,5个倍半萜类化合物,7个黄酮类化合物,4个咖啡酰奎宁酸类化合物,11个酚酸类化合物以及2个其他类型成分。其中japonicaside A(化合物6),jasmigeniposide B(化合物7),7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one(化合物13),7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one (化合物14),2-carboxy-4-methyl-α-methylene-3-(3-oxobutyl)-cyclohexaneacetic acid(化合物15),1,3,4,5-tetrahydro-7-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-8-methoxy-1,4-Methano-2-benzoxepin-10-methanol (化合物27)为新化合物。
本论文还建立了“热毒宁注射液”成品的HPLC特征图谱,并对图谱中的主要色谱峰进行了化学分离和指认,完成了27个化合物的分离和结构鉴定,从中发现了3个新的环烯醚萜苷类成分,它们分别是6"-O-trans-feru loylgenipin gentiobioside(化合物B18),2'-O-trans-caffeoylgardoside(化合物B23)和jasmigeniposide A(化合物B20)；化学指认工作完成了色谱峰总峰面积78.8%色谱峰的指认。
采用体外抗RSV(呼吸道合胞病毒)、EV-71(肠道病毒71型)、H3N2(甲型流感病毒H3N2)及H1N1(甲型流感病毒H1N1)对分离得到的36个化合物进行了体外抗病毒活性评价,活性结果显示：异绿原酸C及其甲酯、异绿原酸B、木犀草素、槲皮素和京尼平,对流感病毒(H1N1)表现出一定的抑制效果,其中木犀草素和异绿原酸C甲酯的ECso值小于50μM,SI值大于10,可以关注,有待进一步研究；此外,槲皮素、木犀草素、异绿原酸B甲酯、异绿原酸C及其甲酯、异绿原酸A及其甲酯还表现出一定的体外抑制流感病毒(H3N2)的活性,木犀草素的ECso值小于50gM,SI值大于10,且在显微镜下可观察到明显的细胞保护作用,值得关注,有待进行深入研究；而体外抗EV-71病毒的活性结果显示,异鼠李素和木犀草素有一定的抑制活性,但其细胞毒性较大,选择指数(SI)较小,其体外抑制EV-71病毒的活性可能是其细胞毒性造成的；咖啡酰奎宁酸类化合物(新绿原酸及其甲酯、隐绿原酸及其甲酯、异绿原酸A、B、C及它们的甲酯化产物)均对RSV表现出高效低毒的抗病毒效果,其中4个单取代的咖啡酰奎宁酸及其甲酯化产物的ECso在10uM到30uM之间,SI(选择指数)大于30；6个二咖啡酰奎宁酸类化合物的EC50小于10uM,SI(选择指数)大于100,提示该类成分具有较好的体外抗RSV病毒活性。
本研究的创新之处还在于对“热毒宁注射液”进行活性成分追踪分离时,发现复方中产生新的化学成分(即人工产物),我们选取其中含量较大,结构较有代表性的化合物6为例,对其结构、来源及稳定性进行了研究。本文采用高效液相-高分辨质谱及多级质谱碎裂和丰度等信息对标准品(分离得到的化合物6)和十二个不同批次的“热毒宁注射液”成品进行质谱分析,通过对比保留时间及二级碎裂行为的一致性,我们确定每个批次的热毒宁注射液中均含有该化合物。并通过UV、IR、NMR以及高分辨质谱分析最后确定了该化合物的结构和来源,直接证明了中药复方“热毒宁注射液”在制剂生产过程中产生了新的化学成分。这从一个方面说明了中药复方并不是单味药的简单加和,为研究中药复方配伍应用的合理性、科学性提供了一个实例,另一方面也说明用现代科学方法研究中药复方药效物质基础的重要性,为揭示中药复方配伍应用的科学内涵和制定科学的质量检测标准提供科学依据。这也是首次在“热毒宁注射液”制剂生产过程中发现的,并在成品中鉴定的稳定存在的新人工产物,该发现对“热毒宁注射液”质量标准的提升具有潜在的研究价值和意义。
上述工作部分揭示了“热毒宁注射液”抗病毒的药效物质基础,为制定科学合理的质量控制方法提供了依据,同时也为复方药效物质基础的研究提供了一种可能的思路与方法。
TCM prescription "Re-Du-Ning Injection" consisted of three common used Chinese material medica, including Lonicera japonica Thunb (Jinyinhua), Gardenia jasminoides Ellis (Zhizi), Artemisia annual (Qinghao). It has been proved to be widely used for the treatment of wind-heat cold, cough, fever, upper respiratory tract infection and acute bronchitis in clinical practice.
In this thesis, the influenza virus induced stress mouse model was adopted, using disease time, survival quality and survival time as evalulation index. Moreover, among the fractions of "Re-Du-Ning Injection" over Dainon HP-20column, RDN-3(95%ethanol elute) was proved to be the anti-virus active fraction of "Re-Du-Ning Injection" in vivo.
The futher investigation on the chemical constituents of RDN-3led to the isolation of63compounds through various chromatographic techniques. The structures of56compounds were established on the basis of their physic-chemical properties and spectroscopic analysis, including twelve iridoids, twelve lignans, seven flavones, eleven phenolic components, three coumarins, four caffeoylquinic acids and other two compounds. Among them, six compounds were new compounds, namely japonicaside A (compound6), jasmigeniposide B (compound7),7,8,11-trihydroxy-l-hydroperoxy-4-guaien-3-one (compound13),7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one (compound14),2-carboxy-4-methyl-a-methylene-3-(3-oxobutyl)-cyclohexaneacetic acid (compound15),1,3,4,5-tetrahydro-7-hydroxy-5-(4-hydroxy-3,5-dimethoxyphenyl)-8-methoxy-1,4-Methano-2-benzoxepin-10-methanol (compound27)。
Furthermore, this paper has also established "Re-Du-Ning Injection" HPLC characteristic chromatogram, and we also separated and identified the main peaks. The structures of27compounds were established on the basis of their physic-chemical properties and spectroscopic analysis. Among them, three compounds were new iridoid glycosides, namely2'-O-trans-caffeoylgardoside (compound B23), jasmigeniposide B (compound B20),6"-O-trans-feruloylgenipin gentiobioside (compound B18).
The anti-virus (RSV/EV-71/H1N1/H3N2) activities of thirty-six isolated compounds were evaluated in vitro. The results showed that4,5-di-O-caffeoylquinic acid,4,5-di-O-caffeoylquinic methyl ester,3,5-di-O-caffeoylquinic acid, luteolin, quercetin and genipin could inhibit H1N1influenza virus. Among them, we can focus on4,5-di-O-caffeoylquinic methyl ester and luteolin with EC50lower than50μM, and selective index (SI) value greater than10, they are value for further research. Additionally, quercetin, luteolin,3,4-di-O-caffeoylquinic methyl ester,3,5-di-O-caffeoylquinic acid,3,5-di-O-caffeoylquinic methyl ester,4,5-di-O-caffeoylquinic methyl ester,3,5-di-O-caffeoylquinic acid showed anti-influenza virus activity (H3N2). Among them, we can also focus on luteolin with EC50lower than50μM, and selective index (SI) value greater than10, and cell protection can be observed clearly under a microscope. Isorhamnetin and luteolin showed anti-virus activity (EV-71), but its anti-virus activity may be caused by cytotoxicity because of a lower SI value. In addition,5-O-caffeoylquinic acid, methyl5-O-caffeoylquinate,4-O-caffeoylquinic acid, methyl4-O-caffeoylquinate,3,5-di-O-caffeoylquinic acid,3,5-di-O-caffeoylquinic methyl ester,4,5-di-O-caffeoylquinic acid,4,5-di-O-caffeoylquinic methyl ester,3,4-di-O-caffeoylquinic acid,3,4-di-O-caffeoylquinic methyl ester can efficiently inhibit RSV. It is highly value for further research.
The innovation of this research topic is to characterize the new constituent produced in traditional Chinese medicine prescription. The strategy and methodology of this study can be used to other chemical research of compound prescription of traditional Chinese herb drugs.
This work partly revealed the anti-virus material basis of "Re-Du-Ning Injection",and provided evidence for developing the scientific and rational methods for quality control. This investigation also provided a possible research method for the study of bioactive and effective material basis of traditional Chinese medicine prescription.
引文
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