四逆散对大鼠胃酸分泌、胃蛋白酶活性及预防应激性溃疡的基础研究
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摘要
前言
消化性溃疡是一种常见的消化道疾病，在我国传统医学中，许多中药通过调理脾胃等作用，在治疗消化性溃疡方面取得了很好的疗效。四逆散出自《伤寒论》，原为肝气郁结、气机不利、阴郁于里、不能外达、四肢厥冷而设，方中有四味药：柴胡、白芍、枳实、甘草。其中柴胡透邪疏郁，枳实破气消积，白芍养阴柔肝，甘草益脾和胃。近些年来，通过动物实验及临床研究，证明四逆散对消化性溃疡，特别是胃溃疡有明显的疗效。在作用机制方面：佐藤等人的研究表明，其对基础胃酸分泌有抑制作用；可增加胃粘膜血流量。从单味药的角度，甘草可抑制胃酸分泌、抑制胃蛋白酶的活性；柴胡及白芍有预防应激性溃疡的作用；枳实在单独使用时对消化性溃疡的作用还未见报道。在该方剂中，各药对药效的影响有主有次，有单独作用，也有综合效应。为了了解四逆散中的各味药对消化性溃疡的作用的大小和交互作用，我们以胃酸、胃蛋白酶及溃疡的大小作为实验指标，用正交设计法分析了四味药对胃酸分泌、胃蛋白酶的活性及预防应激性溃疡的单独效应和四味药的不同组合的效应。
目的
本实验通过建立大鼠应激性溃疡、乙酰水杨酸所致溃疡模型，用正交设计法，观察四逆散及各组成药味对胃酸分泌、胃蛋白酶活性、预防应激性溃疡的作用，及其各药味间的交互作用。
材料和方法
实验材料：动物：雄性Wistar大鼠；研究药物：柴胡（河北X白
芍（河南入积实（四川X甘草（内蒙古厂其中柴胡：5克、白芍：4
克、积实：二克、甘草：1．5克，按正交设计表L；／2‘勺的各实验组安
排。每组加蒸馏水350毫升，浸泡30分钟以后，以文火煎制30分
钟，滤出液以干燥系统处理至浸膏粉剂，置4t条件备用。仪器：
分光光度仪（UV－260人手术器械、灌胃针、注射器、试管、水浴箱、
滴定管、秤、天平机；试剂：牛血红蛋白人－酪氨酸、Foin试剂刀．3
N及0．ZN氯化氢刀．3N三氯乙酸刀．olN及0．SN氢氧化钠。实
验方法：将大鼠随机分5组，每组16只，随机编号。分取各组浸膏
粉剂，以上各为成人（按50公斤标准）一日量的药量，分别溶于20
毫升蒸馏水中，在制备应激性溃疡模型前10分钟分别经口灌注相
应药物p 体重人7 ／J’时后把胃取出，认定渍疡、测量溃疡
的长径。在乙酚水杨酸所致胃溃疡模型的建立中，截扎幽门后立
即灌药，模型建立后收集胃液，测定胃液总酸度及胃蛋白酶活性。
数据处理：取5组数据的均值，采用直观分析法和方差分析法。
实验结果
对胃酸分泌的影响：用直观分析法将这四味药按SR值大小
排列，顺序如下：甘草（D，142人柴胡（A，79．6X四逆散去白芍
（ACD，68．4）、四逆散（ABCD，29．2）、白芍与甘草（BD，24）、柴胡
与白芍（AB，22．8X柴胡与甘草（AD，22X四逆散去柴胡（BCD，
21．幻、四逆散去积实（ABD，－2．4X积实（C，－9．2X柴胡与积实
（AC，－17．6）积实与甘草（CD，－37．8）、四逆散去甘草（ABC，－
40．4X白芍闷，－73．6人从方差分析上看甘草的作用非常显著
·2·
（P＜o＊门，柴胡、四逆散去白芍、四逆散、白芍、四逆散去甘草、积
实与甘草的影响显著（P＜o．05厂但白芍、四逆散去甘草、积实与
甘草SR为负值，表明在该方剂中上述三种组合非但不能抑酸，而
且会促进胃酸分泌。积实的影响不显著u＞O．05人
另外，虽然甘草及柴胡都有抑酸作用，但二者合用后作用不明
显瞩＞o．05入说明二者之间的交互作用为负效应，但二者与积实
合用后，P＜o．05，说明积实于二者之间为正效应。
对胃蛋白酶活性的影响：用直观分析法将这四味药按SR值
大小排列＊ 序如下：积实（C，7205）、甘草（D，71 19）、积实与甘草
（D，5274）、四逆散去积实（ABD，299 n、柴胡与甘草（AD，1547）。
柴胡与积实（AC，821入白芍（B，－133入柴胡（A，一451X四逆散
去柴胡（BCD，－4m5）、四逆散（ABCD，－403）、四逆散去白芍
（ACD，－5929人方差分析表明，积实、甘草、四逆散去帜实、积实
与甘草、四逆散去柴胡、四逆散＼四逆散去白芍的作用显著（P＜
0．05厂但后三者的 8 R为负值，说明其交互作用为负效应。柴胡
及白芍的影响不显著（P＞0．05人
对预防应激性溃疡的作用：用直观分析法将这四味药按SR
值大d排列，D序女下：甘草p，13．54）、柴胡（A，10．98）、柴胡与
甘草（AD，5．46X四逆散去甘草（ABC，3．3X白芍（B，1．86入积实
与甘草（CD，1．62）、四逆散（ABCD，1．46）、白芍与甘草（BD，
0．82X积实＊，－0．22厂余均为负值，从方差分析上看甘草及柴
胡的作用非常显著P＜O．01X白芍的影响显著汀＜0刀5人积实
无显著性差异（P＞o．05人方剂中四逆散、柴胡与甘草、四逆散去
甘草、积实与甘草的作用显著（P＜o．05人四逆散去白芍、柴胡与
积实合用的作用显著u＜o．05入但其SR值为负，说明其交互作
用为负效应。
·3·
讨
The peptic ulcer is one kind of common alimentary disease. In the traditional Chinese medicines, a lot of medicines are more effective for the peptic ulcer through recuperating the spleen and stomach. The powder for treating cold limbs (PTCL) which treated the stagnation of liver - qi , disturbance of qi, stagnation of yin and so on before comes from the 《Treatise on Febrile Disease
》. There are four kinds of medicines-. Thorowax root, White peony root, Immature bitter orange and Licorice root. The thorowax root could relieve the depressed liver. The white peony root could enrich blood and astingting yin fluid and nourish the liver. The immature bitter orange could relieve stagnation of to remove food. The licorice root could invigorate the spleen and stomach. Recently some results of animal experience and clinical research explain PTCL is effective for the peptic ulcer, especially for the gastric ulcer. It was reported PTCL inhibit the output of gastric acid and increase the GMBF. The licorice root could inh
ibit the output of gastric acid and pepsin. The thorowax root and white peony root could prevent the stress - induced gastric ulcer. The immature bitter oranges effect for the peptic ulcer hasnt been reported. In this recipe, there are major medicines and minor medicines and there are sole effects and synthetic effects. In order to understand the effects and interaction of every medicine of PTCL for the gastric uleer , we took the gastric acid , gastric pepsin and ulcer length as the target and analyzed the effects of every medicines and different combinations of four kinds of medicines with the orthogonal design. The orthogonal design is one
kind of the scientific statistical method, which could arrange the more factors and compare with every factor and analyze the interactions and help us find the best combination of the recipe.
Objectives
The present study was to investigate the effects of PTCL and the different combinations of PTCL s four kinds of medicines and interactions of four kinds of medicines for the output of gastric acid and pepsin and the length of gastric ulcer with the orthogonal design through producing the animal models.
Materials and Methods
Materials; Male Wistar rats. Drugs: Thorowax root(5g) ,White peony root(4g) , Immature bitter orange (2g) and Licorice root (1.5 g). It was grouped based on the table of orthogonal design { L16 (215) }. Every group of drugs was boiled for 30 min by gentle fire after soaked in the distilled water 350 ml and the medicine solution was took into the dry fine powder by dryness system . The dry fine powder was placed in the surroundings of 4t. Instruments: the spectropho-tometer ( UV - 260 ) , the appliances for operation, the syringe for stomach, syringes, the tubes, the water bath box , the burets, the scale. Reagent: Hb of cattle, L-tyrosine, Folin reagent,0. 3N,0.2N HC1,0.01N,0.5N NaOH,0.3N trichloracetic acid. Methods: The rats were grouped randomly. Every group had 16 rats. Every group of dry fine powder was dissolved in the 20ml of distilled water. Drugs (20ml/kg) were given 10 min before water immersion. The stomach
was removed 7 h after water immersion. At last, the ulcer was confirmed and measured. During producing the acetylsalicylic acid (ASA) -induced gastric ulcer, drugs(20ml/kg) were given immediately after pylorus ligature and the gastric juice was collected and analyzed for its acidity and pepsin output 7 h later. Data was analyzed by the direct analysis and variance analysis.
Results
Output of gastric acid; The licorice root markedly inhibited the gastric acidity output( p <0. 01) . The thorowax root, PTCL and PTCL without white peony root inhibited the gastric acidity output ( p < 0.05 ). White peony root enhanced the gastric acidity output. The immature bitter orange hadnt marked difference (p >0.05) .
Although the licorice root and thorowax root inhibited the gastric acidity output, the combination of both hadnt marked difference (p > 0.05). It explained the interaction of both was negative effect. If t
消化性溃疡是一种常见的消化道疾病，在我国传统医学中，许多中药通过调理脾胃等作用，在治疗消化性溃疡方面取得了很好的疗效。四逆散出自《伤寒论》，原为肝气郁结、气机不利、阴郁于里、不能外达、四肢厥冷而设，方中有四味药：柴胡、白芍、枳实、甘草。其中柴胡透邪疏郁，枳实破气消积，白芍养阴柔肝，甘草益脾和胃。近些年来，通过动物实验及临床研究，证明四逆散对消化性溃疡，特别是胃溃疡有明显的疗效。在作用机制方面：佐藤等人的研究表明，其对基础胃酸分泌有抑制作用；可增加胃粘膜血流量。从单味药的角度，甘草可抑制胃酸分泌、抑制胃蛋白酶的活性；柴胡及白芍有预防应激性溃疡的作用；枳实在单独使用时对消化性溃疡的作用还未见报道。在该方剂中，各药对药效的影响有主有次，有单独作用，也有综合效应。为了了解四逆散中的各味药对消化性溃疡的作用的大小和交互作用，我们以胃酸、胃蛋白酶及溃疡的大小作为实验指标，用正交设计法分析了四味药对胃酸分泌、胃蛋白酶的活性及预防应激性溃疡的单独效应和四味药的不同组合的效应。
目的
本实验通过建立大鼠应激性溃疡、乙酰水杨酸所致溃疡模型，用正交设计法，观察四逆散及各组成药味对胃酸分泌、胃蛋白酶活性、预防应激性溃疡的作用，及其各药味间的交互作用。
材料和方法
实验材料：动物：雄性Wistar大鼠；研究药物：柴胡（河北X白
芍（河南入积实（四川X甘草（内蒙古厂其中柴胡：5克、白芍：4
克、积实：二克、甘草：1．5克，按正交设计表L；／2‘勺的各实验组安
排。每组加蒸馏水350毫升，浸泡30分钟以后，以文火煎制30分
钟，滤出液以干燥系统处理至浸膏粉剂，置4t条件备用。仪器：
分光光度仪（UV－260人手术器械、灌胃针、注射器、试管、水浴箱、
滴定管、秤、天平机；试剂：牛血红蛋白人－酪氨酸、Foin试剂刀．3
N及0．ZN氯化氢刀．3N三氯乙酸刀．olN及0．SN氢氧化钠。实
验方法：将大鼠随机分5组，每组16只，随机编号。分取各组浸膏
粉剂，以上各为成人（按50公斤标准）一日量的药量，分别溶于20
毫升蒸馏水中，在制备应激性溃疡模型前10分钟分别经口灌注相
应药物p 体重人7 ／J’时后把胃取出，认定渍疡、测量溃疡
的长径。在乙酚水杨酸所致胃溃疡模型的建立中，截扎幽门后立
即灌药，模型建立后收集胃液，测定胃液总酸度及胃蛋白酶活性。
数据处理：取5组数据的均值，采用直观分析法和方差分析法。
实验结果
对胃酸分泌的影响：用直观分析法将这四味药按SR值大小
排列，顺序如下：甘草（D，142人柴胡（A，79．6X四逆散去白芍
（ACD，68．4）、四逆散（ABCD，29．2）、白芍与甘草（BD，24）、柴胡
与白芍（AB，22．8X柴胡与甘草（AD，22X四逆散去柴胡（BCD，
21．幻、四逆散去积实（ABD，－2．4X积实（C，－9．2X柴胡与积实
（AC，－17．6）积实与甘草（CD，－37．8）、四逆散去甘草（ABC，－
40．4X白芍闷，－73．6人从方差分析上看甘草的作用非常显著
·2·
（P＜o＊门，柴胡、四逆散去白芍、四逆散、白芍、四逆散去甘草、积
实与甘草的影响显著（P＜o．05厂但白芍、四逆散去甘草、积实与
甘草SR为负值，表明在该方剂中上述三种组合非但不能抑酸，而
且会促进胃酸分泌。积实的影响不显著u＞O．05人
另外，虽然甘草及柴胡都有抑酸作用，但二者合用后作用不明
显瞩＞o．05入说明二者之间的交互作用为负效应，但二者与积实
合用后，P＜o．05，说明积实于二者之间为正效应。
对胃蛋白酶活性的影响：用直观分析法将这四味药按SR值
大小排列＊ 序如下：积实（C，7205）、甘草（D，71 19）、积实与甘草
（D，5274）、四逆散去积实（ABD，299 n、柴胡与甘草（AD，1547）。
柴胡与积实（AC，821入白芍（B，－133入柴胡（A，一451X四逆散
去柴胡（BCD，－4m5）、四逆散（ABCD，－403）、四逆散去白芍
（ACD，－5929人方差分析表明，积实、甘草、四逆散去帜实、积实
与甘草、四逆散去柴胡、四逆散＼四逆散去白芍的作用显著（P＜
0．05厂但后三者的 8 R为负值，说明其交互作用为负效应。柴胡
及白芍的影响不显著（P＞0．05人
对预防应激性溃疡的作用：用直观分析法将这四味药按SR
值大d排列，D序女下：甘草p，13．54）、柴胡（A，10．98）、柴胡与
甘草（AD，5．46X四逆散去甘草（ABC，3．3X白芍（B，1．86入积实
与甘草（CD，1．62）、四逆散（ABCD，1．46）、白芍与甘草（BD，
0．82X积实＊，－0．22厂余均为负值，从方差分析上看甘草及柴
胡的作用非常显著P＜O．01X白芍的影响显著汀＜0刀5人积实
无显著性差异（P＞o．05人方剂中四逆散、柴胡与甘草、四逆散去
甘草、积实与甘草的作用显著（P＜o．05人四逆散去白芍、柴胡与
积实合用的作用显著u＜o．05入但其SR值为负，说明其交互作
用为负效应。
·3·
讨
The peptic ulcer is one kind of common alimentary disease. In the traditional Chinese medicines, a lot of medicines are more effective for the peptic ulcer through recuperating the spleen and stomach. The powder for treating cold limbs (PTCL) which treated the stagnation of liver - qi , disturbance of qi, stagnation of yin and so on before comes from the 《Treatise on Febrile Disease
》. There are four kinds of medicines-. Thorowax root, White peony root, Immature bitter orange and Licorice root. The thorowax root could relieve the depressed liver. The white peony root could enrich blood and astingting yin fluid and nourish the liver. The immature bitter orange could relieve stagnation of to remove food. The licorice root could invigorate the spleen and stomach. Recently some results of animal experience and clinical research explain PTCL is effective for the peptic ulcer, especially for the gastric ulcer. It was reported PTCL inhibit the output of gastric acid and increase the GMBF. The licorice root could inh
ibit the output of gastric acid and pepsin. The thorowax root and white peony root could prevent the stress - induced gastric ulcer. The immature bitter oranges effect for the peptic ulcer hasnt been reported. In this recipe, there are major medicines and minor medicines and there are sole effects and synthetic effects. In order to understand the effects and interaction of every medicine of PTCL for the gastric uleer , we took the gastric acid , gastric pepsin and ulcer length as the target and analyzed the effects of every medicines and different combinations of four kinds of medicines with the orthogonal design. The orthogonal design is one
kind of the scientific statistical method, which could arrange the more factors and compare with every factor and analyze the interactions and help us find the best combination of the recipe.
Objectives
The present study was to investigate the effects of PTCL and the different combinations of PTCL s four kinds of medicines and interactions of four kinds of medicines for the output of gastric acid and pepsin and the length of gastric ulcer with the orthogonal design through producing the animal models.
Materials and Methods
Materials; Male Wistar rats. Drugs: Thorowax root(5g) ,White peony root(4g) , Immature bitter orange (2g) and Licorice root (1.5 g). It was grouped based on the table of orthogonal design { L16 (215) }. Every group of drugs was boiled for 30 min by gentle fire after soaked in the distilled water 350 ml and the medicine solution was took into the dry fine powder by dryness system . The dry fine powder was placed in the surroundings of 4t. Instruments: the spectropho-tometer ( UV - 260 ) , the appliances for operation, the syringe for stomach, syringes, the tubes, the water bath box , the burets, the scale. Reagent: Hb of cattle, L-tyrosine, Folin reagent,0. 3N,0.2N HC1,0.01N,0.5N NaOH,0.3N trichloracetic acid. Methods: The rats were grouped randomly. Every group had 16 rats. Every group of dry fine powder was dissolved in the 20ml of distilled water. Drugs (20ml/kg) were given 10 min before water immersion. The stomach
was removed 7 h after water immersion. At last, the ulcer was confirmed and measured. During producing the acetylsalicylic acid (ASA) -induced gastric ulcer, drugs(20ml/kg) were given immediately after pylorus ligature and the gastric juice was collected and analyzed for its acidity and pepsin output 7 h later. Data was analyzed by the direct analysis and variance analysis.
Results
Output of gastric acid; The licorice root markedly inhibited the gastric acidity output( p <0. 01) . The thorowax root, PTCL and PTCL without white peony root inhibited the gastric acidity output ( p < 0.05 ). White peony root enhanced the gastric acidity output. The immature bitter orange hadnt marked difference (p >0.05) .
Although the licorice root and thorowax root inhibited the gastric acidity output, the combination of both hadnt marked difference (p > 0.05). It explained the interaction of both was negative effect. If t
引文
1．黄盘冰．中西医结合治疗十二指肠球部溃疡近期疗效观察．实用中西医结合杂志，1997；5(10)：413．
2．叶幼咏．止溃汤与雷尼替丁治疗十二指肠溃疡疗效对比观察．中国中西医结合脾胃杂志，1995；(3)：34-35．
3．张守峰，郝莉萍，龚传美等．甘草泻心汤对小鼠的免疫机能和常压缺氧耐受力的影响．中药药理与临床，1997；33(2)：89-93．
4．水野修一．四逆散胃溃疡症治療．日本東洋醫学会，1983．
5．水野修一．四逆散胃溃疡症治療．和汉醫药学会志，1985；2(1)：176．
6．水野修一，原敬二郎，武田省吾．四逆散颗粒胃溃疡治療．和汉医学会志，1986；3(3)：344．
7．锑内悌辅。消化性溃疡汉方制剂效果役割．诊断上治療，1996；2(83)：245-249．
8．佐藤弘．汉方制剂胃分泌及影医．Pharma-Mediea，1988；2(3)：87．
9．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版；199-200．
10．雷载权，张廷模，陈先难等．中华临床中药学．北京：人民卫生出版社，第一版：1439．
11．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版：540．
12．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版：1247．
13．黄泰康，王育良，王荣娣等．中成药学．北京：中国医药科技出版社，第三版：221-222．
14．李岩，陈苏宁，李宇权等．芍药甘草汤、四逆散对胃排空及小肠推进功能影响的拆方研究．中华消化杂志，1996：1(16)：18-21．
15. Okaba S, Takata Y, Takeuchi K, et al. Effects of carbenoxolone Na on acute and chronic gastric ucler models in experimental animals. DigDis, 1976;21(8):618-625.
16．刘闺男，曾金伊，王恩华等．D-A-ANGI对大鼠心肌梗死后心脏重塑和瘢痕形成的影响研究．中国医科大学学报，2001；30(4)：266-269．
17．杨锦南，朱明，张玉林等．Effects of aluminum 3-oxo-acety-11 deoxoglycyrrhetinate on gastric ulcer in rats．中国药理学与毒理学杂志，1999；13(2)：106-109．
18．赵宝民，黄裕新，王庆莉等．电针足三里穴对胃酸分泌的影响及与促胃液素、表皮生长因子的关系．世界华人消化杂志，2000；8(3)：276-278．
19．周有元，陈世凤．胃蛋白酶活力测试条件探讨．药物分析杂志，1984；4(3)：154-158．
20．倪宗瓒，于石成，王洁贞等．医学统计学。北京：人民卫生出版社，第二版：97-103．
2．叶幼咏．止溃汤与雷尼替丁治疗十二指肠溃疡疗效对比观察．中国中西医结合脾胃杂志，1995；(3)：34-35．
3．张守峰，郝莉萍，龚传美等．甘草泻心汤对小鼠的免疫机能和常压缺氧耐受力的影响．中药药理与临床，1997；33(2)：89-93．
4．水野修一．四逆散胃溃疡症治療．日本東洋醫学会，1983．
5．水野修一．四逆散胃溃疡症治療．和汉醫药学会志，1985；2(1)：176．
6．水野修一，原敬二郎，武田省吾．四逆散颗粒胃溃疡治療．和汉医学会志，1986；3(3)：344．
7．锑内悌辅。消化性溃疡汉方制剂效果役割．诊断上治療，1996；2(83)：245-249．
8．佐藤弘．汉方制剂胃分泌及影医．Pharma-Mediea，1988；2(3)：87．
9．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版；199-200．
10．雷载权，张廷模，陈先难等．中华临床中药学．北京：人民卫生出版社，第一版：1439．
11．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版：540．
12．阴健，郭力弓，陈德昌等．中药现代研究与临床应用(1)．广东：学苑出版社，第一版：1247．
13．黄泰康，王育良，王荣娣等．中成药学．北京：中国医药科技出版社，第三版：221-222．
14．李岩，陈苏宁，李宇权等．芍药甘草汤、四逆散对胃排空及小肠推进功能影响的拆方研究．中华消化杂志，1996：1(16)：18-21．
15. Okaba S, Takata Y, Takeuchi K, et al. Effects of carbenoxolone Na on acute and chronic gastric ucler models in experimental animals. DigDis, 1976;21(8):618-625.
16．刘闺男，曾金伊，王恩华等．D-A-ANGI对大鼠心肌梗死后心脏重塑和瘢痕形成的影响研究．中国医科大学学报，2001；30(4)：266-269．
17．杨锦南，朱明，张玉林等．Effects of aluminum 3-oxo-acety-11 deoxoglycyrrhetinate on gastric ulcer in rats．中国药理学与毒理学杂志，1999；13(2)：106-109．
18．赵宝民，黄裕新，王庆莉等．电针足三里穴对胃酸分泌的影响及与促胃液素、表皮生长因子的关系．世界华人消化杂志，2000；8(3)：276-278．
19．周有元，陈世凤．胃蛋白酶活力测试条件探讨．药物分析杂志，1984；4(3)：154-158．
20．倪宗瓒，于石成，王洁贞等．医学统计学。北京：人民卫生出版社，第二版：97-103．