基于不同来源的黄芩解热药效差异比较及作用机制研究

【作者】 徐珊；

【导师】 孟庆刚；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2008， 硕士

【摘要】 黄芩(Radix Scutellariae)为唇形科植物黄芩(Scutellaria baicalensis Georg)的干燥根,始载于《神农本草经》。本品是临床常用的清热中药,应用历史悠久,药用需求量很大,为我国中药材大宗品种之一,拥有良好的开发前景。综合近几年国内外文献报道发现,目前对于黄芩解热作用机制的研究,仅局限于单一活性成分——黄芩苷及野黄芩苷(黄芩茎叶提取物)解热机制的研究上,对于黄芩提取物中多种活性成分共同作用机制的研究比较少,难以体现出黄芩作为一个整体入药时的解热作用机制。不仅如此,对其解热机制的研究也多集中于体温中枢的正调节介质,但对于致热性细胞因子及体温中枢的负调节介质的作用却未见报道。而且,不同来源黄芩中主要活性成分及其微量元素的含量存在差异。作为道地药材的河北热河黄芩其黄芩苷的含量不及山西、山东地区的黄芩,但其解热等方面的效果却优于其他地区的黄芩;且栽培黄芩的黄芩苷含量较高,但临床还是承认野生黄芩的疗效。因此,黄芩苷含量不能完全代表黄芩的药效质量,在某些药效作用方面(比如解热),黄芩的其它活性成分可能起着关键性作用。由此说明,以黄芩苷的含量作为黄芩的质量检测标准是值得怀疑的。故本课题以大连市科技重大项目《大连市黄芩生产基地建设》(任务书编号:2005e11sf215)和国家自然科学基金《基于不同来源的黄芩物质基础与其药效学相关性研究》(项目编号:30672642)为依托,以中医药学理论为基础,以中药药理学为主要研究方法,针对黄芩的基础功效——清热泻火解毒,开展解热药效学试验,从该药材最常见的入药形式——黄芩水提物入手,研究黄芩作为一个整体入药时的解热作用机制;并比较不同来源黄芩(河北、大连、山西)在解热作用方面的药效学差异,结合中药指纹图谱,找出解热作用方面的谱效关系。实验共分两部分:实验一黄芩提取物解热作用机制研究目的从该药材最常见的入药形式——黄芩水提物入手,分别从致热性细胞因子、体温中枢正调节介质及体温中枢负调节介质的角度,力求全面地考察黄芩作为一个整体入药时表现出的解热作用机制。方法1干酵母致大鼠发热模型的建立以雄性Wistar大鼠作为实验动物,以阿司匹林、地塞米松作为阳性对照药物。将合格大鼠随机分为7组,每组8只,分别为大连黄芩水提物大、中、小剂量组(3组),正常对照组、模型组、阿司匹林阳性对照组和地塞米松阳性对照组。第一次灌胃1小时后,各组背部皮下注射20%的酵母混悬液(正常对照组注射无菌生理盐水)。于造模后发热高峰期第8小时测量大鼠体温。2观察项目与检测方法最大体温上升高度(ΔT/℃):计算体温上升的最大值,即体温最大值与基础体温之差。血清IL-1β:实验8小时后,立即断头处死动物,取躯干血,离心取上清,按放免试剂盒说明书进行加样测定。下丘脑CAMP和IL-1β:实验8小时后,立即断头处死动物,迅速取出全脑,冰浴下于视交叉与灰结节间取下丘脑,投入液氮中固定20分钟后,转入-86℃冰箱保存待测。按放免试剂盒说明书进行加样测定。脑腹中隔区(Ventral septal area, VSA)AVP:实验8小时后,立即断头处死动物,迅速取出全脑,于冰浴下取VSA脑组织,投入液氮中固定20分钟后,转入-86℃冰箱保存待测。测定方法按照放免试剂盒说明书进行。结果在造模后8小时各发热组均进入发热高峰期,由最大体温上升高度可知,黄芩水提物大剂量组较中小剂量组有明显的解热优势(P<0.05),且与以临床剂量折算实验剂量的阿司匹林解热作用相当(P>0.05);黄芩水提物能够显著降低干酵母致热大鼠下丘脑IL-1β及CAMP的含量(与模型组相比P<0.05),也能够降低腹中隔区脑组织AVP的含量(与模型组相比P<0.05),各组大鼠的最大体温上升高度与下丘脑IL-1β、CAMP含量以及腹中隔区脑组织AVP的含量成正相关关系(相关系数r分别为0.911,0.967,0.853;P<0.05);地塞米松组对发热大鼠血清IL-1β含量有明显的降低作用(与模型组相比P<0.05),与正常组相比差异不显著(P>0.05);但其它用药组对发热大鼠血清IL-1β含量的抑制作用并不明显(与模型组相比P>0.05)。结论1本实验成功复制了干酵母致大鼠发热的动物模型。该模型具备以下特点:发热稳定,持续时间长,重现性较好;热度较高容易产生差异;局部有明显炎症,与临床伴内脏或皮肤有明显急性炎症的里热证类似。适合作为研究清热中药黄芩解热药效及其作用机制的动物模型。2黄芩水提物的解热作用表现出明显的剂量相关性,大剂量组解热作用最优,与临床常用剂量的阿司匹林解热作用相当;黄芩水提物的整体解热机制之一可能如下:减少致热性细胞因子IL-1β在体温中枢的含量,减少中枢发热介质CAMP的含量,促进中枢解热介质AVP的释放。实验二不同来源的黄芩提取物解热药效差异比较目的比较不同来源黄芩在解热作用方面的药效学差异。结合中药指纹图谱,找出解热作用方面的谱效关系,寻找黄芩解热作用的物质基础。并为在今后药物主要有效成分提取、组合的基础上,用不同的配比作药效学实验,找出最佳的配比奠定基础,为黄芩的质量检测及创新药物的深入研究提供新的实验依据。方法1干酵母致大鼠发热模型的建立造模及给药方法同前。以阿司匹林作为阳性对照药物。将体温合格的大鼠随机分为9组,每组10只,分别为各地黄芩水提物组(3组)、各地黄芩醇提物组(3组),正常对照组、模型组、阿司匹林阳性对照组。于造模后0.5、1、2、4、6、8、10及12小时各测大鼠体温1次,观察体温变化情况(测量体温与基础体温的差值)。2观察项目与检测方法平均体温变化曲线:以体温上升高度(Δt)为纵坐标,测量时间为横坐标,根据各测量点的均数绘制平均体温变化曲线。最大体温上升高度(ΔT/℃):计算体温上升的最大值,即体温最大值与基础体温之差。体温反应指数(thermal response index, TRI12):计算体温变化曲线与基线之间的面积。结果由平均体温变化曲线、ΔT及TRI12可以得出:以干酵母作为外源性致热原的大鼠发热模型发热比较稳定,造模成功;不同产地的黄芩提取物(水提、醇提)均表现出明显的解热作用(P<0.05);在整个实验过程中,各个产地的黄芩醇提物均较同产地的水提物表现出较优的解热作用(P<0.05);各个产地的黄芩水提物的解热作用均与临床常用剂量的阿司匹林作用相当(P>0.05);不同产地的黄芩提取物相比较,来自于河北的黄芩醇提物较其它两个产地的醇提物解热作用更有优势(P<0.05)。总之,河北黄芩醇提物较其它各组表现出明显的解热作用优势。结论比较不同产地(河北、大连、山西)的黄芩水提物及醇提物的解热药效差异。结果显示,产地为河北的黄芩醇提物较其它各组表现出明显的解热作用优势。在此基础上,将本实验的结果与中药指纹图谱相结合,找出不同产地黄芩提取物解热作用方面的谱效关系,最终找出黄芩在解热药效方面的物质基础。更多还原

【Abstract】 Radix Scutellariae is dry root of Scutellaria baicalensis Georg. The beginning carries is in "Shen Nong Book on Chinese medicine". This is the clinical commonly used refrigeration traditional Chinese medicine. The application history is glorious and demand for medicinal purposes is very big. It is one of large amount varieties of traditional Chinese medicine in our country, which has the good development prospect. From the synthesis of domestic and foreign literature report in recent years, we found that the main active constituent and the trace element content existence difference of Radix Scutellariae from different Originates. Radix Scutellariae from Hebei has better function of relieving fever and less content of baicalin, compare with Radix Scutellariae from Shanxi and Shandong; The grow wild Radix Scutellariae has less content of baicalin than cultivates Radix Scutellariae, but clinical acknowledges the curative effect of former. Therefore, the baicalin’s content cannot completely on behalf of Radix Scutellariae ’s drug efficacy quality. In certain drug efficacy function aspect (for instance: relieving fever) , Radix Scutellariae ’s other active constituent is possibly playing the key role. From this explanation,it is dubious that taking the baicalin’s content as Radix Scutellariae ’s quality detection standard. At present , the study of the relieving fever function mechanism research regarding Radix Scutellariae limits in the sole active constituent—baicalin and scutellarin(stem leaf extraction of Radix Scutellariae) ,and research of combined action mechanism of many kinds of active constituent is quite few., which make it difficult to manifest the whole function mechanism of relieving fever. Furthermore, the mechanism research of relieving fever also concentrates in positive control mediums in temperature center, but less in thermal property cell factors and negative control mediums in temperature center. This experiment takes the Chinese medicine pharmacy theory as foundation, and takes traditional Chinese medicine Pharmacology as main research technique, to carry out experiment in view of Radix Scutellariae ’s foundation effect(alleviating heat-producing factors and disintoxicating) ,in order to compare the pharmacodynamics difference of Radix Scutellariae from different Originates(Hebei, Dalian, Shanxi), and combine with traditional Chinese medicine fingerprint atlas to find out the spectrum-effect relationship in relieving fever. Then, research the whole function mechanism of relieving fever.The experiment is divided into two parts:Part 1: The mechanism research of withdraws of Radix Scutellariae in relieving feverObjectiveFrom the angle of thermal property cell factors, positive control mediums and negative control mediums in temperature center to research the whole function mechanism of Radix Scutellariae in relieving fever. Method1 The model’s establishment of dry yeast making rat feverTake male Wistar rat as experimental animal, and take aspirin and dexamethasone as positive control medicine. We divide temperature qualified rats into 7 groups stochastically, each group of 8. They are aqueous extraction of Radix Scutellariae from Dalian-which content large, medium and small dosage groups (3 groups) , normal control group, model group, aspirin and dexamethasone positive control group. One hour after intragastric administration for the first time, back hypodermic injection of 20% yeast suspl is performed in each group(SPSS for normal control group ). Take the temperature at 8 hours -fever plateau.2 Observation item and methodMaximum height of temperature(ΔT/℃):The difference between maximum of temperature and BBT.Blood serum IL-1β:At the time of fever plateau, behead rats immediately, take the trunk blood and centrifugate it for supermatant, and then carry out application of sample according to instruction booklet of radio-immunity exempts box.Hypothalamus CAMP and IL-1β: At the time of fever plateau, behead rats immediately, take out of brain, get the hypothalamus in the middle of chiasm opticum and the tuber cinereum in the ice-bath, fix them in liquid nitrogen for 20min, and preserve them in -86℃icebox. Carry out application of sample according to instruction booklet of radio-immunity exempts box.Ventral septal area AVP: At the time of fever plateau, behead rats immediately, take out of brain, get the ventral septal area in the ice-bath, fix them in liquid nitrogen for 20min, and preserve them in -86℃icebox. Carry out application of sample according to instruction booklet of radio-immunity exempts box.ResultsEach group reach the fever plateau after 8 hours. Make know by the maximum height of temperature, large dosage group has better effect than medium and small dosage groups(P<0.05), and has same effect with clinical commonly used dosage aspirin(P>0.05). Aqueous extraction of Radix Scutellariae can cut down the IL-1βand CAMP content of hypothalamus, and AVP content of ventral septal area significantly (compared with model group P<0.05 ) .There are positive correlativity between maximum height of temperature and IL-1β, CAMP content of hypothalamus, and AVP content of ventral septal area (r=0.911,0.967,0.853; P<0.05) . Dexamethasone positive control group can cut down the blood serum IL-1βcontent apparently(compared with model group P<0.05 ), while the other groups cannot(compared with model group P>0.05 ).ConclusionThe effect of relieving fever of aqueous extraction of Radix Scutellariae is dose dependent. One of the whole function mechanism of Radix Scutellariae in relieving fever may be as follows: decrease the content of IL-1βin temperature center, decrease the content of CAMP and advance the releasing of AVP.Part 2: Comparison of relieving fever function about withdraws of Radix Scutellariae from different OriginatesObjectiveComparing the pharmacodynamics difference of relieving fever function about withdraws of Radix Scutellariae from different Originates, and combined with traditional Chinese medicine fingerprint atlas to find out the spectrum-effect relationship in relieving fever.Method1 The model’s establishment of dry yeast making rat feverThe methods of making model and giving medicine are mentioned above. Take aspirin as comparison medicine. We divide temperature qualified rats into 9 groups stochastically, each group of 10. They are aqueous extractions (3 groups) and alcohol extractions (3 groups) of regional Radix Scutellariae groups, normal control group, model group, aspirin positive control group. Take the temperature at 0.5, 1, 2, 4, 6, 8, 10 and 12 hours respectively, and observe the change situation of temperature.2 Observation item and methodThe mean temperature curve: Take ascending height of temperature (Δt) as Y-axis, time as X-axis. Plot average temperature change curve ,according to various measuring points mean value.Maximum height of temperature(ΔT/℃):The difference between maximum of temperature and BBT.Thermal response index(TRI12): The area between average temperature change curve and baseline.ResultsThe fever rat model by dry yeast is quite stable. We had a successful model building. All withdraws of Radix Scutellariae from different Originates appear conspicuous relieving fever effect(P<0.05). Alcohol extractions have better relieving fever effect than aqueous extractions for same originate(P<0.05). Aqueous extractions from all originates have same effect with clinical commonly used dosage aspirin(P>0.05). The aqueous extraction from Hebei has better effect than aqueous extractions from the other two(P<0.05). In a word, the aqueous extraction of Radix Scutellariae from Hebei has the best relieving fever effect.Conclusion1 This experiment make a copy of fever rat model by dry yeast successfully. This model has the following characteristic: fevering stably and long-enduring make good reproducibility; higher temperature makes apparent different; the obvious inflammation is similar to endopyretic syndrome. All of above prove that this model is fit for researching the pharmacodynamics effect and the mechanism of relieving fever.2 Comparing the pharmacodynamics difference of relieving fever function about withdraws of Radix Scutellariae from different Originates.The result is that the aqueous extraction of Radix Scutellariae from Hebei has the best relieving fever effect. And then, combine the result with traditional Chinese medicine fingerprint atlas to find out the spectrum-effect relationship in relieving fever.更多还原