【作者】 高琳；

【导师】 李庆业；

【作者基本信息】 北京中医药大学 ， 中医方剂学， 2009， 博士

【摘要】 理中丸是张仲景治疗脾阳虚证的传统名方,由干姜、人参、白术、炙甘草四味药组成,功能温中散寒,补气健脾。干姜作为理中丸的君药,在全方的配伍结构中处于核心地位,在对脾胃虚寒证的治疗中发挥着关键性作用。为了从实验的角度对君药在方剂中核心配伍地位的内涵进行揭示,本实验以理中丸中的君药干姜作为研究切入点,借助中医脾阳虚证候模型,对干姜及其不同有效部位对理中丸调节消化吸收及能量代谢功能的影响进行了比较研究,从药味和化学成分群两个层次的药效物质基础的角度,对君药干姜决定理中丸主要功效的核心地位进行了揭示。全文共分为脾阳虚模型的制备和干姜及其成分对理中丸药效学影响的比较两大部分。1“石膏-知母”复合法脾阳虚模型的制备1.1目的以清热泻火药石膏-知母代替常用的大黄、番泻叶类苦寒泻下药,并结合饥饱失常、劳倦过度的方法进行造模,以期建立更符合中医“寒凉伤阳、饥饱伤气和劳倦伤脾”致病思路的中医脾阳虚证候模型。1.2方法模型动物隔日足量喂食,每日在25℃水中游泳,直至大鼠连续三次吐泡下沉,或沉入水底,3秒内不能自行浮出水面,并以三种不同给药频率灌胃给予石膏一知母煎液,比较石膏-知母不同给药方法对制备脾阳虚模型的影响。方法1:造模1-6日隔日灌胃4℃的石膏-知母煎液（浓度为4:1,下同）,剂量为10ml/kg（下同）;造模7-12日,每同灌胃石膏-知母煎液,自然恢复15天。方法2:隔日灌胃给予4℃的石膏-知母煎液,剂量为10ml/kg,连续20天,自然恢复12天。方法3:每R灌胃给予4℃的石膏一知母煎液,2ml/只,连续12天,自然恢复9天。观察造模动物的一般生物学特征改变,检测体温、体重、食量的变化,以及血中D-木糖、6AS、MLT含量和肠组织中N0水平等与消化吸收功能及能量代谢相关指标。1.3结果三种造模方法均能引起造模动物一般生物学特征的改变,出现腹泻、倦怠、体温下降、体重减轻等表现。其中方法2的影响最小,模型动物恢复较快;方法1和方法3都能造成模型动物一般生物学特征的明显改变,且以方法3引起的模型症状变化更加明显且稳定。在消化吸收及能量代谢的相关指标方面,造模方法3能使血中GAS、MLT、D-木糖含量降至正常水平以下,使肠运动抑制性神经递质NO的水平降低,体温减低明显、食量减低、体重增长减慢。造模方法1对消化吸收相关药理指标的改变与造模方法3的影响相似,但对模型动物的体重和食量的影响不明显;造模方法2除能使模型动物的体重减轻外,对其他各指标均无显著影响。1.4结论对比实验采用的三种石膏-知母的给药方法,方法3的造模效果最为显著和稳定,即以每日灌胃给药,连续12天,恢复9天,是本实验证实的最有效的脾阳虚证候模型制备方法。2干姜及其不同有效部位对理中丸消化吸收功能及代谢的影响2.1目的通过对理中丸中君药干姜,以及干姜中挥发性成分和水溶性成分对理中丸主要功效影响的研究,从药味和化学成分群两个层次,证实君药干姜及干姜中的主要活性成分群对理中丸整体功效的决定性影响,以初步揭示理中丸中君药核心地位的药理学和化学内涵。2.2方法采用石膏-知母复合造模法制备大鼠脾阳虚证候模型,造模期为14天1;治疗期间各给药组分别给予治疗药物,连续给药8天。供试药物分别为理中丸、四君子汤供试液,干姜水溶性成分和不同剂量挥发性成分（大、中、小剂量比为2:1:0.5）分别代替干姜原药材配伍入理中丸,组成的干姜水溶性成分理中配方和大、中、小剂量干姜挥发性成分理中配方供试液,其中挥发性成分的中剂量相当于干姜的常规剂量。造模及治疗过程中,对各组大鼠的体温、体重、食量进行测定;治疗结束时,测定下列两组指标。与消化吸收有关的指标有:胃的排空率和肠推进率;血中D-木糖（D-xylose）、胃泌素（GAS）、胃动素（MLT）含量,胆碱酯酶（CHE）活性,胃组织中H⁺-K⁺-ATP酶活性,空肠组织中NO含量;肠中血管活性肠肽（VIP）和肠间质细胞（ICC）含量（免疫组化法）;测定肠肌间神经丛中胆碱能神经（AchE阳性神经）和氮能神经（NOS阳性神经）的分布（组织化学法）及累计光密度;观察胃、肠的病理改变,以及干姜挥发性成分理中配方中、大剂量组大鼠肝的病理改变。与能量代谢相关的指标有:空腹血糖值,血中肌酸激酶（CK）活性、乳酸（LD）水平;胃、空肠、骨骼肌线粒体ATP酶活性;肌糖元的含量;下丘脑中5-羟吲哚乙酸（5-HIAA）含量。2.3结果为了对不同层次的研究问题分别进行说明,研究结果分为三个层次进行表述。2.3.1理中丸与四君子汤对脾阳虚证消化吸收功能和能量代谢的影响差异在胃的运动和消化功能方面,四君子汤可提高血中MLT、GAS水平,增加胃的排空率,但作用明显弱于理中丸。四君子汤对正常的胃H⁺-K⁺-ATP酶活性有明显抑制作用,但理中丸作用不明显。在肠的消化吸收功能方面,四君子汤和理中丸均可降低肠推进率,且无明显差异。四君子汤主要是通过降低血中CHE活性水平引起的,对NO无明显影响。虽然四君子汤对提高模型大鼠D-木糖吸收率和增加食量方面的作用优于理中丸,但两方在增加模型动物体重方面的作用无显著差异。并且对胃肠病理形态的观察显示,四君子汤对胃、肠粘膜的保护作用较理中丸弱。在对能量代谢的影响方面,理中丸能改善造模引起的CK活性降低、LD和肌糖元含量升高;降低下丘脑中5-HIAA含量,升高体温。理中丸对造模引起的代偿性血糖升高、骨骼肌线粒体中Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性增强均有明显的抑制作用。理中丸除了对胃组织线粒体中的Na⁺-K⁺-ATP酶的活性有所降低外,对胃肠线粒体中的其他ATP酶活性均无明显影响。而四君子汤不能纠正造模引起的大鼠体温降低,对下丘脑中5-HIAA的含量没有明显影响;除了对血中CK的活性降低和对胃线粒体中ATP酶的抑制作用强于理中丸外,对其他指标的影响均弱于理中丸。2.3.2干姜挥发性成分和水溶性成分对理中丸调节脾阳虚模型消化吸收功能和能量代谢作用的影响在对消化吸收功能的影响方面,干姜挥发性成分理中配方和干姜水溶性成分理中配方均能提高脾阳虚模型血中GAS和MLT水平,干姜挥发性成分理中配方的作用优于干姜水溶性成分理中配方,也优于理中丸原方;但两者在增加胃排空率方面的作用无明显差异。干姜水溶性成分理中配方对血中CHE的抑制作用较强,同时能升高模型大鼠肠组织中降低的NO水平,降低模型大鼠肠中ICC数量,但对肠中VIP的含量无明显影响。干姜挥发性成分理中配方对血中CHE活性的抑制作用弱于干姜水溶性成分理中配方,也不能提高空肠中抑制性神经递质NO的释放,且对肠组织VIP的含量也无明显影响,仅在抑制肠中ICC的数量方面作用显著。然而比较干姜挥发性成分理中配方、干姜水溶性成分理中配方和理中丸原方对大鼠小肠推进率的抑制作用,三者并未显示出明显差异。此外,干姜挥发性成分理中配方对模型大鼠D-木糖的吸收率的增加和促进体重增长方面的作用,优于干姜水溶性成分理中配方和理中丸。在对胃肠组织形态的保护方面,干姜挥发性成分理中配方的作用更优。在对能量代谢的影响方面,干姜挥发性成分理中配方能有效降低下丘脑5-HIAA水平,体温明显升高;同时能有效降低血中CK的活性和LD的含量,并能抑制造模引起的骨骼肌ATP酶活性及血糖的代偿性增高,使之恢复正常水平。干姜水溶性成分理中配方对下丘脑中5-HIAA不但没有降低作用,反而使其含量升高,对造模引起的体温降低无明显影响。除了对骨骼肌中ATP酶的抑制作用较为强烈外,干姜水溶性成分理中配方对其他各指标的改善作用均弱于干姜挥发性成分理中配方。2.3.3不同剂量干姜挥发性成分对理中丸调节脾阳虚模型消化吸收功能和能量代谢的影响干姜挥发性成分中剂量配伍能显著提高血中GAS和MLT水平,使之达到正常值以上,并能显著提高胃排空率。干姜挥发性成分大、小剂量理中配方虽也具有相似的作用,但作用较干姜挥发性成分中剂量配方弱。各干姜挥发性成分理中配方主要通过影响胆碱能神经功能和空肠ICC数量来抑制肠的运动功能。干姜挥发性成分中剂量配方对ICC数量的减少作用最强,大剂量配方对抑制血中CHE活性的作用最突出。对肠中NO含量的影响中,除干姜挥发性成分小剂量能使其含量上升外,姜醚中、大剂量表现为降低的作用,但中剂量配方能使肌间神经丛中氮能神经的累积光密度值（IOD）增高。从对改善肠推进率的作用来看,大、中、小剂量的干姜挥发性成分理中配方间没有明显差异。但对胃肠组织形态的保护方面,中剂量配方的作用更为显著。对能量代谢功能的影响方面,中剂量和大剂量干姜挥发性成分理中配方可有效降低下丘脑5-HIAA水平,并使模型大鼠体温上升至正常水平;小剂量干姜挥发性成分理中配方虽然对下丘脑5-HIAA水平也有降低作用,但对造模后大鼠的体温降低未显示出明显改善作用。三剂量理中配方均能有效降低血中CK活性和LD水平,以及肌糖元含量,其中中剂量和大剂量干姜挥发性成分理中配方的作用更加明显。对于造模引起的血糖及骨骼肌线粒体中Ca²⁺-Mg²⁺-ATP酶活性代偿性增高,三剂量理中配方均有降低作用,中剂量配方对血糖的降低作用最突出,而小剂量配方对ATP酶的影响较明显。实验中发现,大剂量干姜挥发性成分理中配方能导致血中GOT活性有所升高,使肝小叶中央静脉周围肝组织出现少量的细胞坏死,提示干姜挥发性成分配伍剂量过大时,有可能引起肝脏的损害。而小剂量和中剂量干姜挥发性成分配伍对肝脏的功能未见损害作用。2.4结论在对脾阳虚证模型的治疗中,理中丸所代表的温阳健脾法是针对脾阳虚证进行治疗的基本立法,干姜是理中丸实现温阳散寒作用的核心药物;而在干姜所含的挥发性成分和水溶性成分中,干姜的挥发性成分成分对理中丸功效的影响最为突出,对理中丸调节脾阳虚证模型消化吸收功能和能量代谢具有决定性作用。随着干姜挥发性成分在理中配方中配伍剂量的增加,理中配方的功效的作用强度发生改变,以挥发性成分中、大剂量配伍时,其疗效显著提高,但大剂量干姜挥发性成分理中配方时,存在对模型动物的肝脏产生损害倾向,因此从本实验的结果判断,干姜挥发性成分以中剂量配伍时,其疗效最佳。通过上述的研究可以证明,君药干姜在理中丸配伍结构中的核心地位和功效上的决定性作用并不是方剂学理论中纯粹理论层面上的假设,而是具有重要的事实基础的。对方剂药效物质基础的揭示,又会对方剂配伍理论的认识产生至关重要的影响。更多还原

【Abstract】 Lizhong Pills invented by Zhongzhongjing in Treatise on Cold-Attack is a famous formula used to treat deficiency of spleen YANG,being consist of dried ginger,ginseng,rhizoma atractylodis macrocephalae and radix glycyrrhizae preparata,possessing the function of warming the middle to dispel cold and reinforcing the spleen-qi.As the main herb of Lizhong Pills, dried ginger is the core structure of the compatibility,and plays a key role in treating deficiency of spleen Yang.An experiment was designed to explore a key position of the main herb in a formula.Being selected as a sample, Lizhong Pills and dried ginger was observed in the experiment concerning the regulating efficacy on the models of animals suffering from deficiency of spleen YANG.The different impacts on digestion,assimilates and energy metabolizability of the models were observed in the research,when every kinds of test formulations of Lizhong Pills which contains dried ginger or different components of dried ginger were given to the models respectively.It is indicated that the effects of Lizhong Pills mainly rely on dried ginger, the main herb in the formula,which is the same as the traditional consideration.Just the single change of the main herb concerning about dried ginger or its’ different components could transform the efficacy of the whole formula.This paper is mainly comprised of two components:preparation of models of rats suffering from deficiency of spleen Yang;comparison of the pharmacodynamics impacts of different Lizhong Pills formulations made up by dried ginger or its different components in Lizhong Pills to the different groups of the models.1 Establish the model of deficiency of spleen YANG induced by " cypsum fibrosum and anemarrhenae " with complex factors1.1 ObjectivesWith using heat-clearing and fire-purging drug cypsum fibrosum and anemarrhenae,insteading of the common usage of rhubarb and folium sennae,accompanied with irregular diet and overstrained,the model of deficiency of spleen-YANG is to be established,which is much close to the pathopoiesis theory of " frigidity and chilly damaging yang,irregular diet injuring qi,overstrain impairing the spleen" in TCM.1.2 MethodsModel rats is fully fed every other day,given the molding drug of cypsum fibrosum -anemarrhenae（concentration 4:1） with 3 different frequency,and arranged to swim everyday in 25℃water until exhausted（performing as spitting bubble 3 times continuously or sinking unable to float by itself）.The influence of each modeling method could be compared through the courses.Method 1:given the modeling drug（dosage 10ml/kg） every other day from 1d to 6d,and everyday from 7d to 12d,accompanied with 15 days’ spontaneous recovery.Method 2:given the modeling drug（dosage 10ml/kg） every other day, 20 days continuously,accompanied with 12 days’ spontaneous recover.Method 3:given the modeling drug（2ml/each rat） everyday,12 days continuously,accompanied with 9 days’ spontaneous recover.Observe the variation of model rats’ general preference,detect the change of correlate indexes of digestive and absorptive function and energy metabolism such as body temperature,body weight,food intake and serum D-xylose,GAS and MLT levels and the content of NO in the tissue of jejunum etc. 1.3 ResultsAll methods could induced the changes of general preferences of model rats,appearring diarrhea,lassitude,temperature drop,loss of weight etc.Among the Methods,Method 2 shows the weakest function,with the model rats recovering fast.The modeling effedt of Method 1 and 3 appears stronger than method 2,among which method 3 cause the most distinct and steadiest pathological change.In the aspect of influenced indexes in digestive and absorptive function and energy metabolism,method 3 could decrease the serum levels of D-xylose,GAS,MLT,and lower the content of NO which is the mainly inhibitory neurotransmitter in jejunum,bring down body temperature, degrade food intake and slower body weight grower.There is no significouldt variation between the function of Method 1 and 3 in the indexes of digestive and absorptive function,except the effect on body weight and food intake;as well,model 2 shows no significouldt influence on the indexes besides body weight lose.1.4 ConclusionsContrast the three modeling methods,model 3 is the most obvious and steady method for the establishment of deficiency of spleen YANG.2 comparison on the efficacy of Lizhong Pills influenced by dried ginger and its different components on digestive and absorptive functions and energy metabolism2.1 ObjectivesThis research was designed to observed the different influences of Lizhong Pills formula on the models,which contains dried ginger or its main group of components（volatile oil and water solubles）.It would be explored from two levels of material base,single herb dried ginger or its different components,to confirm that the whole effects of Lizhong Pills do mainly depend on its main herb dried ginger in the formula just as the traditional acknowledgement in TCM.Further more,the connotative meaning of dried ginger used as the main herb in Lizhong Pills egthe aspect of pharmacology and chemistry would be preliminary revealed.2.2 MethodsEach group of model rats of deficiency of spleen-YANG were to be duplicated by method 3 for 14 days,and be treated by corresponding drug samples for 8 days.The drug samples,respectively,are the liquid of Lizhong Pills（contains original dried ginger,namely LZL）,the liquid extract of Sijunzi Decoction（namely SJL）,and the liquid extract of Lizhong Pills formulae with the original dried ginger replaced by its water solubles, （namely LZW） or volatile oil from it（namely,LZV）.Futhermore,the chemical compositions formula with high dosage volatile oil is named LZVH;with medium dosage volatile oil,the LZVM;and with low dosage volatile oil,the LZVL.And the dosage ratio among high dose,medium dose and low dose groups is 2:1:0.5,and the medium dose formula equals to LZL in component.During the course of the experiment,the body temperature,body weight and food intaking ara to detected everyday.After being treated the following measurement indicators would be detected.The measurement Indicators related to digestive and absorptive function:the gastric evacuation rate and small intestinal propulsion rate; serum contents of D-xylose,GAS and MLT;activity of CHE;H⁺-K⁺-ATPase in stomach tissues;contens of ICC,VIP and NO in the tissue of jejunum; IOD（mean） of AchE and NOS in Myenteric plexus（histochemical method）; histopathologic observations on tissue of stomach and jejunum,besides the observation on liver in LZVM and LZVL.The measurement indicators concerning with energy metabolism: fasting blood-glucose;activity of CK and content of LD in serum,activity of ATPase in the mitochondria of gastric,jejunal and skeletal muscle cells; contents of muscle glycogen and 5-HIAA in hypothalamus.2.3 ResultsAll data is organized for three targets. 2.3.1 comparison on the efficacy of LZL and SJL in regulating the digestive and absorptive functions and energy metabolism on model rats of deficiency of spleen YANGOn the aspect of digestive and absorptive functions,SJL could elevate the level of MLT、GAS in serum,and improve the gastric evacuation rate, meanwhile,the effect of LZL is much stronger.SJL could inhibit H⁺-K⁺-ATPase activity of normal stomach,while LZL doesn’t work.Both SJL and LZL could reduce the small intestinal propulsion rate, which is closely related to the activity of CHE,rather than NO in the tissue of jejunum being reduced by SJL.and there is no obvious significance among the groups.Although it seems that the SJL could elevate the absorptivity of D-xylose more effeciently than LZL,and improve the appetite of the modeling rats,it fails to increase the body weight of the models as obviously as the above.Evenmore,according to the pathomorphism of the stomach and the enteric of models,the protective action of SJL to the gastric and intestinal mucosa is weaker than LZL’s.On the effect of energy metabolism,LZL could lower the serum CK activity and LD level,depress the content of muscle glycogen,reduce 5-HIAA level in hypothalamus and upgrade body temperature inhibited by modeling.LZL could significently inhibited the increasing of blood glycogen and Ca²⁺-Mg²⁺-ATPase activity in the mitochondria of skeletal muscle cells in models.LZL failed to influence the activity of the ATPase detected in mitochondria of gastric and intestinal tissues,while LZL could reduce the the activity of the Na⁺-K⁺-ATPase.The body temperature of models could not be improved effectively by SJL as well as the 5-HIAA level do in hypothalamus.Besides the effect on serum CK activity,the inhibiting effect of SJL on ATPase activity in mitochondria of gastric tissues is stronger than LZL’s, while the other influences of SJL is weaker than that of LZL. 2.3.2 comparison on the efficacy of LZV and LZW in regulating the digestive and absorptive functions and energy metabolism on model rats of deficiency of spleen YANGOn the aspect of digestive absorption,both LZW and LZV could elevate the MLT,GAS in serum of models.The effect of LZV is more stronger than that of LZW,as well as that of LZL.But there is no differenece in effects observed between LZW and LZV in gastric evacuation rate.LZW could strongly depress the serum CHE activity,while elevate the NO level and reduce the quantity of ICC in the jejunum.But there is no change to the VIP level in the jejunum.The inhibition of LZV to serum CHE activity is weaker than that of LZW, but LZV could not increase the release of NO in the jejunum.Moreover,it could not change the quantity of the VIP in the jejunum,while the number of ICC in the jejunum could be reduced obviously.But there is no obvious difference among the groups of LZV,LZW and LZL.In addition,the effect of LZV in increasing the D-xylose absorption rate and promoting the body weight is better than LZW’s and LZL’s.As to protecting morphology of stomach intestine tissue,LZV has the advantage over the others.On the impact of energy metabolism,LZV could reduce 5-HIAA level in hypothalamus and upgrade the body temperature.At the same time,the serum CK activity and LD level could be depressed effectively by LZV.The skeletal muscle ATPase activity and the fasting blood-glucose increase in blood induced by modeling could be decreased to normal level by LZV.On the contrary,the 5-HIAA level in hypothalamus could be increased by LZW, while there is no obvious influence on the decreased body temperature. Except the stronger inhibition of skeletal muscle ATPase,the effect of LZW on the other Indicators is weaker than LZV’s.2.3.3 Influence on digestive and absorptive functions and energy metabolism of the model of deficiency of spleen-YANG in different dosage of volatile oil in dried gingerLZVM could significantly raise the serum GAS and MLT level exceeding the normal value,and then could elevate the gastric evacuation rate.LZVH and LZVL effect similarly,but weaker than LZVM.Compatibility with the dried ginger volatile oil could restrain the enterokinesia in way of influencing cholinergic neuron（nerve） function and the quantity of ICC.LZVM has the strongest effect in decreasing the number of ICC,at the same time,LZVH plays an important role in lowing serum CHE activity.Comparing the contribution on enhancing NO levels in the jejunum,it is indicated that LZVL increases its level,while LZVM and LZVH decrease it.But LZVM could raise the IOD of nitrergic neuron（nerve） in myenteric nerve plexus.On the improving of total length and propulsion rate of small intestinal, three formulae with the dried ginger volatile oil appear no differences.But LZVM effects significantly in protecting morphology of stomach intestine tissue.On the effect of energy metabolism,LZVM and LZVH both could reduce the 5-HIAA levels in hypothalamus,and upgrade the body temperature to the normal level.Although LZVL also could reduce the 5-HIAA levels in hypothalamus either,but it has no significance in improving the decreased body temperature of modeling rats.All the three formulae could depress the serum CK activity,LD level and muscle glycogen content,in which LZVM and LZVH are more effiency.With the compensated increase of blood glycogen and Ca²⁺-Mg²⁺--ATPase activity in the mitochondria of skeletal muscle cells,all the three groups could influent them,and while LZVM processes the most efficent function in depressing blood glycogen,LZVL does the same in influencing ATPase.In the experiment,it is found that LZVH could increase the serum GOT activity.At the same time,we could find the slight neorobiosis of liver tissue. Contrarily,there is no evidence indicating the damage in liver concerning with LZVL and LZVM.2.4 ConclusionsIn the treatment of deficiency of spleen-YANG,LZL which represented the method of warming YANG and invigorate the spleen,aims directly at the basic method to deficiency of spleen-YANG.As the main herb of LZL,dried ginger plays a key role in warming the middle-jiao to dispelling cold.Comparing the volatile oil and water-soluble contained in the dried ginger,the volatile oil contributes to the most prominent efficacy of LZL,and decisively adjusts the digestive and absorptive functions and energy metabolism on the model.Along with the dosage increasing of dried ginger volatile oil,the potency of LZL enhances correspondingly.But high dosage compatibility of volatile oil tends to lead to slight harm to the liver of model rats.Therefore, according to the results of this research,middle-dosage volatile oil compatibility wins the best potency without no obvious insecurity.It is proved in the research that dried ginger,the major herb of LZL,has the core position and decisive function.This is not only the theory of TCM, but also the modern pharmacological facts.Come to the conclusion that It is vital to reveal material base of effecacy in the formula of Chinese medicine in order to thoroughly understand and master the main principle theory of Fangji.更多还原