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基于不同来源黄芩物质基础拮抗流感病毒的实验研究
【作者】 宋琳莉；
【作者基本信息】 北京中医药大学 ， 中医基础理论， 2009， 博士
【摘要】 目前从黄芩中提取出60多种化学成分,以黄酮类化合物为主,如黄芩苷,黄芩素,汉黄芩苷,汉黄芩素,黄芩新素,其中以黄芩苷的含量较高被药典作为检测黄芩的质量标准。但不同来源黄芩中不仅总黄酮的含量差异较大,其主要活性成分的含量亦存在差异。物质基础的不同有可能是黄芩发挥不同药理作用的原因。黄芩具有广谱的抗病毒作用,其药效学研究,虽已深入到细胞、分子水平,但以水煎液、总提物及单一成分为研究对象,或仅仅将几种成分的药理作用作简单的对比,对于各有效成分之间的整合作用未见报道,而各有效成分的共存及合理配比有可能是其药效作用的物质基础。因此研究以黄芩主要活性成分的合理配比作为体现黄芩的药效质量,可为其它中药材的药效学研究提供借鉴。我国是流感多发区,几乎每年都有局部地区的爆发流行,其发病率居传染病首位。由于流感病毒容易变异形成新型株,给疫苗预防带来很大困难,因而目前流感的治疗尚无特效药。中医学在防治流感方面积累了丰富的理论和实践经验。对中医药抗流感进一步深入研究,可促进中医药更好地应用于流感防治,对于促进人类健康具有重大现实意义。本论文理论探讨部分主要从中药化学组成成分的复杂性、有效组分交互作用的复杂性及中药作用对象——人的开放性及复杂性三个角度出发,阐述中药药效物质基础系统具有复杂性特征,提出只有在复杂性科学理论、方法和技术指导下,借鉴现代中药药理学、药化学及数学计算机等多学科的研究方法,进行中药药效物质基础的研究,才能反映出其固有的本质特征。本论文实验研究部分主要以中医药学理论为基础,中药药理学为主要研究方法,以流行性感冒为切入点,开展黄芩抗病毒药效学实验,比较不同来源黄芩在抗流感病毒方面的药效学差异。同时在药物主要有效成分提取,谱效关系分析的基础上,进行有效成分的配比实验,从而找出其抗流感病毒的最佳物质基础。并进一步作黄芩拮抗流感病毒机制研究,以期为黄芩的质量检测及创新药物的深入研究提供实验依据。实验方法:1将ICR小鼠随机分为空白组、模型组、利巴韦林组、河北醇提组、河北水提组、山西醇提组、山西水提组、大连醇提组、大连水提组。小鼠用乙醚轻度麻醉后,空白组以生理盐水滴鼻,其它小鼠以流感病毒亚洲甲型鼠肺适应株(FM1)稀释液滴鼻感染。感染一小时后给药,连续至感染后的第六天。观察不同来源黄芩提取物对造模小鼠死亡的保护作用,肺指数、肺组织中病毒血凝滴度的影响。2采用灰色关联度分析方法,结合高效液相色谱法,进行不同来源黄芩谱效关系分析,以确定黄芩成分配比物组最佳成分,继而进行药效学验证。将ICR小鼠随机分为空白组、模型组、利巴韦林组、河北水提组、黄芩成分配比物组。小鼠用乙醚轻度麻醉后,空白组以生理盐水滴鼻,其它小鼠以病毒稀释液滴鼻感染。感染一小时后给药,连续至感染后的第六天。观察河北水提组与黄芩成分配比物组对造模小鼠死亡的保护作用,肺指数、肺组织中病毒血凝滴度的影响。3将ICR小鼠随机分为空白组、模型组、黄芩成分配比物组。小鼠用乙醚轻度麻醉后,空白组以生理盐水滴鼻,其它小鼠以病毒稀释液滴鼻感染。感染一小时后给药,连续至感染后的第三天。光镜、电镜观察小鼠肺部组织形态结构改变,TUNEL法检测肺组织细胞凋亡情况,免疫组织化学染色法检测肺组织Bcl-2、Bax、Fas、FasL蛋白表达含量,ELISA法测定血清TNF-α的表达改变,Western Blotting法测定Caspase-3、NF-κB p65蛋白的表达变化。实验结果:1利巴韦林组、河北水提组死亡率与模型组比较有非常显著性差异(P＜0.001),河北醇提组、山西水提组及大连醇提组死亡率与模型组比较有非常显著性差异(P＜0.01),山西醇提组、大连水提组死亡率与模型组比较有显著性差异(P＜0.05)。利巴韦林组、河北水提组平均存活天数与空白组比较无差异(P＞0.05),河北醇提组平均存活天数与空白组比较有显著性差异(P＜0.05),山西水提组、大连醇提组平均存活天数与空白组比较有非常显著性差异(P＜0.01),大连水提组平均存活天数与空白组比较有非常显著性差异(P＜0.001)。利巴韦林组、河北水提组、山西醇提组、大连醇提组、大连水提组肺指数与模型组比较有非常显著性差异(P＜0.001),河北醇提组、山西水提组肺指数与模型组比较有非常显著性差异(P＜0.01)。河北水提组、山西醇提组、大连水提组肺指数与利巴韦林组比较无差异(P＞0.05),大连醇提组肺指数与利巴韦林组比较有显著性差异(P＜0.05),河北醇提组、山西水提组肺指数与利巴韦林组比较有显著性差异(P＜0.01)。利巴韦林组及黄芩各提取物组病毒血凝滴度与模型组比较有非常显著性差异(P＜0.001),河北水提组、大连水提组病毒血凝滴度与利巴韦林组比较无差异(P＞0.05),大连醇提组病毒血凝滴度与利巴韦林组比较有显著性差异(P＜0.05),河北醇提组、山西醇提组、山西水提组病毒血凝滴度与利巴韦林组比较有非常显著性差异(P＜0.001)。2利巴韦林组、河北水提组死亡率与模型组比较有非常显著性差异(P＜0.001),黄芩成分配比物组死亡率与模型组比较有非常显著性差异(P＜0.01),利巴韦林组、黄芩成分配比物组死亡率与河北水提组相比无差异(P＞0.05)。利巴韦林组、河北水提组、黄芩成分配比物组平均存活天数与模型组比较有非常显著性差异(P＜0.001)。利巴韦林组、黄芩成分配比物组平均存活天数与河北水提组相比较无差异(P＞0.05)。利巴韦林组、河北水提组、黄芩成分配比物组肺指数与模型组比较有非常显著性差异(P＜0.01),利巴韦林组、黄芩成分配比物组肺指数与河北水提组比较无差异(P＞0.05)。利巴韦林组、河北水提组及黄芩成分配比物组病毒血凝滴度与模型组比较有非常显著性差异(P＜0.01),利巴韦林组、黄芩成分配比物组病毒血凝滴度与河北水提组比较无差异(P＞0.05)。3大体观察显示,空白组小鼠肺脏呈淡粉红色,含气,无实变区。模型组可见感染小鼠肺脏大多数均有两个肺叶以上成片的实变区,呈现暗褐色外观。黄芩成分配比物组病变程度较模型组明显减轻,褐色实变区较少。光镜结果显示,空白组肺泡大小正常,肺间质未见炎症反应。模型组肺泡间隔增宽,肺泡壁和细支气管壁大量单个核细胞浸润,形成实变区,显示重度间质性肺炎的病变。黄芩成分配比物组与模型相比病变明显减轻,肺泡间隔轻度增宽,仅在细支气管壁和支气管周围少量肺泡壁有少量单个核细胞浸润,肺组织接近正常。电镜结果显示,空白组Ⅰ型和Ⅱ型上皮细胞正常,微绒毛丰富,细胞核规整,线粒体清晰,肺泡腔洁净,未见炎性细胞。模型组Ⅰ型和Ⅱ型上皮细胞严重退变,微绒毛明显减少,细胞核边集、固缩,可见凋亡小体,线粒体肿胀空泡变性,嗜锇型板层小体数量减少,几乎全部呈排空状,肺泡腔中可见大量坏死脱落细胞和少量红细胞,毛细血管内皮细胞肿胀,炎性细胞浸润。黄芩成分配比物组肺泡Ⅰ型和Ⅱ型上皮细胞接近正常,微绒毛较丰富,个别可见细胞核边集,线粒体肿胀不明显,嗜锇型板层小体排空现象减少,肺泡腔较洁净,毛细血管内皮细胞形态大致正常无肿胀。TUNEL法、免疫组织化学染色法、ELISA法及Western Blotting法结果显示,黄芩成分配比物组凋亡指数、Bcl-2、Bax、Fas、FasL、Caspase-3、NF-κB p65含量表达与模型组比较有非常显著性差异(P＜0.01)。实验结论:1不同来源黄芩提取物对流感病毒FM1感染小鼠均有较好的死亡保护作用,能降低感染小鼠的肺指数及病毒血凝滴度。2黄芩成分配比物对流感病毒FM1感染小鼠有较好的死亡保护作用,能降低感染小鼠的肺指数及病毒血凝滴度。3黄芩中抗流感病毒的药效物质基础不能单一由黄芩苷来表示,以汉黄芩素、黄芩素为代表的一类黄酮物质也是黄芩中抗流感病毒作用的重要物质基础。4黄芩成分配比物能显著改善流感病毒FM1感染引起的小鼠肺部病理改变,同时也能降低感染小鼠的凋亡指数,上调Bcl-2蛋白表达含量,降低Bax、Fas、FasL、TNF-α、Caspase-3、NF-κB p65蛋白表达含量,抑制流感病毒引起的细胞凋亡,防止病毒增殖,从而起到拮抗流感的作用。
【Abstract】 At present researchers extract sixty various chemical constituents from scutellaria,and the main are flavonoids,such as baicalin,baicalein,wogonoside,wogonin,neobaicalein.Especially baicalin is high content and is a quality standard of testing scutellaria in pharmacopoeia.But there are difference content of total flavones and main active component in different sources scutellaria. Various substance basis is a possible reason of scutellaria having different pharmacological action.Scutellaria have wide anti-virus effect.It’s pharmacodynamic study achieve a level of cellular and molecular,and research objects include water decoction、total extraction、single-component、contrast of several ingredients pharmacodynamic study.But there is a blank about integration effect of effective components,coexistence and reasonable ratio of effective components may be substance basis of pharmacodynamic actions.Therefore studying scutellaria reasonable ratio embodying effect quality can provide a reference to other chinese medicinal materials study.Almost every year there is influenza epidemic outbreak of local area,and influenza incidence is the highest in China..Influenza vitus is easy to be a variation,which brings great difficulty for vaccine preventing.So there isn’t specific remedy now.Traditional chinese medicine(TCM) accumulated abundant theories and practical experience.Further study on TCM anti-influenza virus could promote human health.This research topic compared pharmacodynamics difference of different sources scutellaria on anti-influenza virus by using TCM theory and chinese drug pharmacology. Meanwhile on the basis of extracting main active component and analyzing spectrum-effect relationship,making experiments of active component ratio,which found out the best substance basis on anti-influenza virus.Besides we had study on mechanism and expected to provide experimental basis for scutellaria quality detection and innovative drugs study.Method:1 ICR mice had been divided into blank、model、ribavirin、He Bei ethanol extract、He Bei aqueous extract、Shah Xi ethanol extract、Shan Xi aqueous extract、Da Lian ethanol extract and Da Lian aqueous extract at random.After being anesthetized by ether,blank was dropped saline in nose and other group was dropped A/FM/1/34(FM1) in nose.After infection an hour the mice were treated,administration would last six days.Observed the effect of different sources extracted scutellaria on the death protection、lung index and hemagglutination titer of FM1 infected mice.2 Analyzed spectrum-effect relationship through grey correlation analysis method and high performance liquid chromatography,then we determined the best component of ratio and verified it was right or wrong.ICR mice had been divided into blank、model、ribavirin、He Bei aqueous extract and scutellaria element ratio at random.After being anesthetized by ether,blank was dropped saline in nose and other group was dropped FM1 in nose.After infection an hour the mice were treated,administration would last six days.Observed the effect of different sources extracted scutellaria on the death protection、lung index and hemagglutination titer of FM1 infected mice.3 ICR mice had been divided into blank、model and scutellaria element ratio at random.After being anesthetized by ether,blank was dropped saline in nose and other group was dropped FM1 in nose.After infection an hour the mice were treated,administration would last three days. Observed tissue morphology structure change of mice lung by light and electron microscopic、lung tissue apoptosis by TUNEL、expression content of Bcl-2、Bax、Fas、FasL by immunohistochemistry、serum expression change TNF-αby ELISA and Caspase-3、NF-κB p65 by Western Blotting.Result:1 There were very significant difference from ribavirin、He Bei aqueous extract to model on mortality.Significant difference existsed in He Bei ethanol extract、Shan Xi aqueous extract、Da Lian ethanol extract and model on mortality.There were difference from Shan Xi ethanol extract、Da Lian aqueous extract to model on mortality.There were no difference from ribavirin、He Bei aqueous extract to blank on average survival days,however difference existsed in other extracted scuteltaria and blank.There were very significant difference from ribavirin、He Bei aqueous extract、Shan Xi ethanol extract、Da Lian ethanol extract and Da Lian aqueous extract to model on lung index,significant difference from He Bei ethanol extract、Shan Xi aqueous extract to model on lung index.There were no difference from He Bei aqueous extract、Shan Xi ethanol extract and Da Lian aqueous extract to ribavirin on lung index and difference existsed in other extracted scutellaria.There were very significant difference from ribavirin and each extracted scutellaria to model on hemagglutination titer,without difference from He Bei aqueous extract and Da Lian aqueouS extract to ribavirin on hemagglutination titer,but difference existsed in other extracted scutellaria and ribavirin.2 Scutellaria element ratio i ncluded Baicalin、wogonin and baicalein through grey correlation analysis method.There were significant difference between scutellaria element ratio and model on mortality、average survival days、lung index and hemagglutination titer.There were no difference between He Bei aqueous extract and scutellaria elemen ratio.3 The lung of blank mice were pink、containing air and without real variable area by general observation.The lung of model infected mice were dark brown and with two lobar real variable areas.Model infected mice had severe interstitial pneumonia lesions including lung epithelial cells degeneration、apoptosis、infiltration of inflammatory cells and so on through light microscope and electron microscope.Lesion degree of scutellaria element ratio were better than model and scutellaria element ratio lung tissue closed to normal.There were significant difference between scutellaria element ratio and model on apoptosis index、Bcl-2、Bax、Fas、FasL、Caspase-3、NF-κ3 p65 by TUNEL、ICH、ELISA and Western Blotting.Conclusion:1 The system on material basis of chinese medicine effect has complexity character including complexity of chemical constituents,interaction and human,We should study it by complex science theory,method and technology,pharmacology,drug chemistry,mathematics computer and so on.2 Different sources extracted scutellaria both had good effect on the death protection、lung index and hemagglutination titer of FM1 infected mice.3 Scutellaria element ratio had good effect on the death protection、lung index and hemagglutination titer of FM1 infected mice.4 Not only Baicalin but also wogonin、baicalein were important sustance basis anti-influenza virus in scutellaria.5 Scutellaria element ratio could significantly improve pathological change of FM1 infected mice lung,decrease apoptosis index、up-regulate Bcl-2 expression content and decrease Bax、Fas、FasL、TNF-α、Caspase-3、NF-κB p65 expression content of FM1 infected mice.It would antagonize FM1 by inhibiting apoptosis and preventing virus proliferation.