【作者】 杨江萍；

【导师】 谢鸣；

【作者基本信息】 北京中医药大学 ， 方剂学， 2012， 博士

【摘要】 方剂是中医临床用药的主要形式,复方的效用不是所组成的各单味药及其化学成分的简单相加,而是其多种组分共存及共同作用的结果。研究方剂效用物质基础是中药现代化基础研究的重要领域,对于在物质层面揭示方剂的科学原理和促进我国中药质量水平的提高都有重大意义。本课题是以中医方剂学学理为基础,以小柴胡汤中核心功能药对的水煎液为研究对象,选择与柴胡—黄芩药对功效密切相关的退热作用为效应指标,考查柴-芩药对中的化学部位及其配伍与退热效用之间的关系；在获得优化部位配伍的基础上,进一步探查有效组分配伍的退热作用机制。研究一发热小鼠模型的研究方法：NIH雄性小鼠,25-30g,30只,小鼠于23℃左右室温下适应性饲养3-5d,第4、5d每日测体温(肛温)2次。取肛温的平均值作为基础体温,选用体温波动范围不超过38.0℃,且2次测体温波动均不超过0.4℃的小鼠。将符合实验条件的小鼠随机分为正常组、模型组1组、模型2组,每组10只。实验前6h禁食不禁水。小鼠模型1和模型2组分别给予110μg/kg,200μg/kg的LPS腹腔注射,诱发动物发热；正常组注射等体积的生理盐水。分别测定各组小鼠腹腔注射后0,30,60,120,180,240,330min时的肛温和各时间点的体温差值△T(实测肛温-初始肛温),绘制各组平均体温的变化曲线。结果：与正常组比较,模型1和模型2组小鼠30min后的各时间点的肛温均呈显著性升高(P<0.01,P<0.05)；与模型2组比较,模型1组60min后各时间点的肛温均见显著性降低(P<0.05)。结论：腹腔注射200μg/kg的LPS可明显引起NIH小鼠的体温升高,且其发热持续时间较长。研究二不同分离方法对柴-芩药对退热效用部位的探讨(一)水提醇沉法分离柴胡—黄芩水提液的研究实验1.柴芩水煎液与合并液对LPS诱导的发热小鼠△T的影响方法：用水提醇沉法将柴胡—黄芩水提液分离为A、B、C、D四个化学部位。观察各部位合并液与水提液对脂多糖诱导的小鼠发热模型的降温作用。结果：与正常组比较,模型组小鼠30min后各时间点的肛温均显著性升高(P<0.05)；与模型组相比,柴芩水煎液组与柴芩各部位合并液组小鼠30min后各时间点的肛温均呈显著性降低(P<0.05)；与柴芩水煎液组相比,柴芩分离各部位合并液组小鼠各时间点的肛温变化无明显差异(P>0.05)。结论：柴胡-黄芩药对水提液经醇沉所获各部位合并液的降温作用与柴黄-黄芩水提液作用相当,表明分离过程未引起其退热有效成分的损失实验2、柴胡—黄芩各部位配伍与退热作用关系的研究方法：将柴胡-黄芩水提液中分离的各部位按正交L.(2)7表分组配伍,依次配制成配方DA、CA、CD、BA、BD、BC、ABCD组(各部位剂量相当于原药材1g/mL的用量折算),在造模前半小时,按相当于原药10g/kg的浓度给NIH小鼠灌胃各部位配伍药液；测定各组小鼠腹腔注射LPS (200μg/kg)后60min、180min的肛温。结果：各部位组及CD交互组均能明显降低发热小鼠的肛温(p<0.01)。结论：各部位均有不同程度的解热作用,各部位之间存在着复杂的交互关系。(二)大孔树脂分离法探查柴胡—黄芩水提液的退热效用部位实验一大孔树脂初步分离柴胡—黄芩水提液方法：柴胡-黄芩水提液经AB-8型大孔树脂柱净化,依次用水,20%,40%,60%和90%乙醇梯度洗脱,通过化学反应检识,合并组成相近的流份得到a、b、c、d4个部位；观察各部位对脂多糖诱导的小鼠发热模型的退热作用。结果：柴胡-黄芩水提液经分离得到的4个部位分别为糖类(a)、皂苷类(b)、皂苷及黄酮苷类(c)、黄酮苷元及皂苷元(d)；4个部位均具有不同程度的退热作用(p<0.05),其中以d部位的退热作用最好(p<0.01)。结论：柴胡-黄芩水提液由大孔树脂分离出的各部位均有一定的退热作用。实验二、柴胡黄芩各有效部位配伍与退热作用的关系方法：将经大孔吸附树脂柱分离的柴胡-黄芩水提液中的4个部位a、b、c、d,按正交L8(2)7表分组配伍,方法同研究二中实验二。结果：柴芩各部位配伍组中以柴芩cd组、柴芩abcd组、柴芩abd组退热作用较好,其中柴芩cd组作用最优。柴芩最佳部位配伍为cd组。结论：柴胡-黄芩水提液由大孔吸附树脂柱分离所获部位及其配伍均有一定的退热作用,cd部分为其主要效用基础。研究三、柴胡黄芩有效部位配伍的退热作用机制的探查方法：选取柴胡-黄芩水提液经大孔吸附树脂柱分离所得部位,进行ab、abd、cd、abcd不同配伍,观察各部位配伍对发热小鼠模型相关指标的影响。结果：(1)与正常组比较,模型组小鼠外周血清中TNF-α、β-EP含量,下丘脑TNF-α、PGE2、cAMP、AVP含量均显著性升高(P<0.05、P<0.01)；(2)与模型组比较,柴-芩cd组、柴-芩abcd组小鼠外周血清中TNF-α、β-EP的含量均显著性降低(P<0.05),下丘脑中TNF-α、PGE2、cAMP的含量均显著性降低(P<0.05)；柴-芩abcd组小鼠下丘脑PGE2含量显著性降低(P<0.01),柴芩abd组小鼠下丘脑的cAMP含量显著性降低(P<0.01)；柴-芩各配伍组小鼠下丘脑AVP值均有降低作用趋势(P>0.05)。结论：LPS诱导的发热小鼠模型的发热机制涉及中枢介质(PGE2、cAMP、AVP)内生致热原TNF-α、脑肠肽(p-EP)多个环节的变化。柴芩相关部位配伍的退热作用机制主要涉及对体温中枢正调质PGE2、cAMP的调节及抑制内生致热原TNF-α、β-EP合成或释放,但不同部位配伍组的退热作用环节有所差异；退热效应大小与退热作用的环节多少有关。更多还原

【Abstract】 The research of the effective fraction about antifebrile action from Bupleurum Chinese-Scutellaria and its compatibility mechanismTCM formula is the main form of clinical medication. The utility of the compound is not composed of the single herb medicine and the chemical composition of the simple addition is the kinds of components and coexistence the result of joint action.Prescriptions research foundation is the modernization of TCM utility of the important research areas, for the material level in formula of scientific principle and reveal to promote the quality of traditional Chinese medicine to raise the level of all have important significance.This subject is based on traditional Chinese medicine pharmacology of scientific theory as the foundation, for the Xiao-Chai-Hu decocting as the research object, select and Bupleurum-scutellaaria baicalensis on efficacy of closely related antipyretic effect as effect indicators, cl on Chai-Qin drugs on chemical fractions and antipyretic effect of compatibility between relationship; in part based on the optimization of compatibility, further exploration of the antipyretic effective component compatibility mechanism.Research 1:The heating methods of mouse modelMethods:the male NIH mouse 25-30g; We raised them for 3-5 days under the room temperature (23℃). On the 4th and the 5th days we will measure every day anus temperature two times, then pick out the continual 2d single body temperature mouse surpasses 38.℃, or two anus temperature fluctuation surpasses 0.4℃which we take the rectal temperature as the average of the basal body temperature.The male NIH mice were assigned into normal group 1, the model group2,10 mice in every group, inject lipopolysaccharide (LPS)/NS solution (110μg/kg,200μg/kg) intraperitoneally to induce fever in mouse. The normal group and the model group were given the same volume saline. The rectal temperature were measured at Omin,30min,60min, 90min,120min,180min,240min,330 min after LPS injection in mouse and each time point temperature differenceΔ(measured rectal temperature-initial rectal temperature),rendering each average temperature change curve.Results:Intraperitoneal injection of200μg/kg dosage’s LPS could cause NIH mice with elevated body temperature obviously,,and the heating duration is longer. The experiment should pay attention to the influence of various factors on rectal temperature. Conclusion:Compared with normal group,200μg model group,110μg model group mice anus temperature raise significantly (P< 0.01, P< 0.05) at different time points after the 30min point; Compared with the 200 u g model group, after 60 min 110μg model group at each time point of anus temperature changes significant differences (P<0.05).Research 2:Exploration of the antipyretic effective fraction from herbal pair of Bupleurum chinese and Scutellaria baicalensis (a) Water extraction and combined alcohol precipitation separation of the decoction Bupleurum chinese and Scutellaria baicalensis herbal pair. Experiment 1:The chai-qin decoction and combined separation on mice induced by LPSΔT heating effect.We used this method to extract the decoction divided into A, B, C, D four chemistry part, determine the separation process of Bupleurum-scutellaaria baicalensis on Antipyretic active ingredients without loss through the observation antipyretic effect on the fever model mice induced by LPS.Experiment 2:The chai-qin parts-Study on the relationship between compatibility and antipyretic action.We combined the separate position according to orthogonal table L8(2)7, The site group and CD interactive group were can significantly reduce the heating mouse rectal temperature (p<0.01). Conclusion:each part and CD interactive group had different degree of antipyretic effect(p<0.01), there existed complicated interactive relations between various parts. (b) Study on antifebrile parts for Chai-qin decoction by using macroporous resin column to purify the decoction of Bupleurum Chinese and Scutellaria baicalensis.Experiment 1:Preliminary Separation of the decoction of Bupleurum Chinese and Scutellaria baicalensis by using macroporous resin column.The four fractions, A (polysaccharides), B(saponins), C(saponins and flavonoids), D (flavonoids and aglycones of saponins), purified by AB-8 macroporous resin, all can reduce the temperature of fever mice (p<0.05), and diferent fraction have differen levels. Fraction D showed the best effective on antifebrile action (p<0.01).Conclusion:The decoction of Bupleurum Chinese and Scutellaria baicalensis involved more than one effect of the fever effective fractions.Experiment 2:Study on the effective parts compatibility and antipyretic effect relationship about the fractions of chai-qin.We combined the separate position according to orthogonal table L8(2)7 method of the research in the second experiment of Research 2; Results:in Chai Qin parts compatibility group to Chai Qin group cd, group abcd, Chai Qin group abd antipyretic effect is better, the best part ofChai Qin compatibility were for the CD group. Conclution:The chai-qin water extracted by macroporous adsorption resin column for the separation of the parts and it’s the compatibility were antipyretic effect, part cd was the main utility base.Research 3:The bupleurum chinensis-Huang antifebrile parts compatibility mechanism of action of investigation.We Selected parts of ab, abd, cd, abcd compatibility from Scutellaria baicalensis Radix Bupleuri-water extracted by macroporous adsorption resin column separation, observed Correlation index on the effect of heating model. Results:(1)Compared with the normal group, model group of TNF-α、β-EP content in peripheral serum, TNF-α、PGE2, cAMP, AVP content in hypothalamus, were significantly elevated (P<0.05, P<0.01); (2) Compared with the model group, Chai-Qin cd group and Chai-Qin abed group can obviously reduce the peripheral blood levels of TNF-α、β-EP content (P<0.05), and decreased in hypothalamus of TNF-α、PGE2、cAMP content (P<0.05); Chai-Qin abcd group of hypothalamic PGE2 content were significantly lower (P<0.01), Chai-Qin abd group of hypothalamic cAMP content decreased significantly (P<0.01):on Chai-Qin compatibility group hypothalamic AVP content have decreased trend (P>0.05).Conclusion:The experimental exploration of LPS induction heating mechanism may involve the central medium (PGE2, cAMP, AVP), endogenous pyrogen TNF-α, brain gut peptide (β-EP) of a number of links,Chai-qin antipyretic mechanism of action of the various effective fraction of the compatibility were mainly regulate quenched and tempered PGE2, cAMP of body temperature central,restrain of endogenous pyrogen TNF-α、brain-gut peptideβ-EP synthesis or release to achieve antipyretic effect. But in different parts antipyretic action link is the difference. Antipyretic effect size was related to the antipyretic effect of links number.更多还原