【作者】 俞进；

【导师】 楼宜嘉；

【作者基本信息】 浙江大学 ， 药理学， 2003， 硕士

【摘要】 β-甘草酸（18β-glycyrrhizic acid,β-GA）是甘草的主要活性成分，具有抗炎、抗病毒和抗过敏反应等作用，已知其能够抑制肾脏11β-羟基类固醇脱氢酶（11β-OHSD₂）。α-甘草酸（18α-glycyrrhizic acid,α-GA）是由β-GA经异构化反应而得到的，比β-GA具有更强的保肝抗炎作用，在临床应用时，可见高血压等不良反应，其机制迄今未明。为论证α-GA是否也能降低11β-OHSD₂的活性，揭示其临床应用时出现高血压等不良反应的机制，本研究采用豚鼠离体和整体实验，研究α-GA对11β-OHSD₂的抑制作用，旨在为临床合理用药提供依据。 1．α-GA对离体11β-OHSD₂的抑制作用。 取花豚鼠肾脏皮质，制成匀浆液并提取微粒体酶11β-OHSD₂，加入底物皮质醇进行反应，以底物皮质醇转化为可的松的百分率为指标，研究了α-GA对11β-OHSD₂的抑制作用。结果表明：（1）肾皮质匀浆物阴性对照组皮质醇的转化率为99％；α、β-GA浓度＜0.8mmol／L时，皮质醇的转化率均＞96％；浓度为1.6mol／L时，α、β-GA组的皮质醇转化率分别为98.55％和90.51％；浓度上升至3.2mmol／L时，皮质醇的转化率分别降为62.59％和38.64％。α、β-GA对11β-OHSD₂的抑制作用呈浓度相关性。（2）GA浓度为0.8、0.4、0.2mmol／L时，11β-OHSD₂对皮质醇转化率（％）α、β-GA组分别为38.99±0.85，21.08±1.5（n=3）：53.37±0.63，33.09±0.19（n=3）；60.41±0.86，44.67±0.33（n=3）。和阴性对照组的转化率（％）70.56±3.03（n=3）比较，皮质醇转化率的减小均有显著性意义（P＜0.05）；计算出α、β-GA的IC₅₀值分别为1140.1 p mol/L，346.4pmol/L，说明a一GA抑制豚鼠肾11p代川SDZ的强度小于p{A。（3）制备三批花豚鼠肾皮质11日一OHSDZ悬液，每批各进行三次实验，其皮质醉转化率平均值阴性对照组、Q一GA组、p一GA组分别为70.56%士3.03%、38.9喘士0.85%、21.0既士1.5俄;70.9器士4.6既、38.器士2.2既、21.55%士1.38%;71.80%士2.33%、38.80%士1.9既、21.5既士1.61%。三批酶之间相应平均值经两两脸验，均无显著性差异（乃0.05）:同一批酶各组之间比较，均有显著性差异（只0.05）。提示Q一GA能够抑制离体Hp一OHS山的活性，但强度比p一GA弱。2.11p一OHs加动力学参数测定 取花豚鼠肾皮质微粒体酶H日一OHSDZ，在4个底物浓度时，均加入5个不同浓度a一GA，测定可的松的生成速度，根据MichaeliS一Menten的双倒数方程，求出1 lp一OHSDZ动力学参数。结果:在不同的底物浓度时，a一GA对微粒体酶的抑制作用由双倒数法作图表示，各条直线在Y轴上交于一点，显示Q esGA的竞争性抑制作用;求出吃户15.25 nmol.mg一‘.h一，，龙==l .31p伽1/L。根据Dixon方程，求出尤== 209.2 p mol/L士1 7.1协mol/L。3.花白两种豚鼠np一OHSDZ体外活性的差异 取花、白两种豚鼠，体重相近，同时提取肾皮质微粒体酶np一OHSDZ，加入底物皮质醉进行平行实验，抑制剂选用Q、p一GA，a一甘草次酸，大黄素。结果表明，皮质醉转化率（%）花鼠/白鼠，阴性对照组:70.56士3.03/70.37士1 .42（乃0.05），a一GA组:38.99士0.85/30.01土1.53（只0.05），p一GA组:21.08士1.50/16.50士1.15（只0.05，n=3）。说明a、p弓A对白豚鼠11p一OHSDZ的抑制作用强于花豚鼠。a一甘草次酸，大黄素为抑制剂的实验结果与此一致。提示白豚鼠肾脏微粒体酶1 lp一OHSDZ对抑制剂的敏感性明显强于花豚鼠。4.a一以对整体豚鼠1 lp一OHsDZ活性的抑制作用取雄性白色豚鼠，分组，分别腹腔注射生理盐水（SN），a弓A（75、巧O、300mg/Kg三个剂量组）和p‘A，连续15日;收集第15天的豚鼠尿液，用高效液相测定其中的可的松（E）含量及可的松/皮质醇（E/F）的比值;第16日取豚鼠血，侧定血清钾离子浓度:取肾皮质，提取微粒体酶1 lp刃HSDZ，测定其活性。结果表明，（l）第巧天尿中，E值:SN/。一GA（75.9/Kg），一以（75，g/Kg）/a弓A（3（X）。g/Kg），a一GA（300 mg/Kg）/p一GA（30Om酬Kg），均有显著性差异（只0.05）;E/F值:。一GA（300 mg/Kg）/p‘A（300 mg/Kg），。一GA（150 mg/Kg）/a一GA（300 mg/Kg），均有显著性差异（只0.05）:（2）肾皮质微粒体酶11日一OHSDZ的活性测定以皮质醇的转化率为指标:和SN组比较，除Q一GA（75mg/Kg）组外，其余组均有显著性差异（只0.05）;和p一GA（300 mg/Kg）组比较，各组均有显著性差异（只0.05）。（3）豚鼠血中钾离子浓度，除SN/a弓A（75 mg/Kg）外，其余各组之间均差异显著（代0.05）。提示a一GA能够抑制整体豚鼠肾皮质np一OHSDZ的活性，引起低血钾，尿中可的松含量和可的松/皮质醇比值降低。 综上所述，a一GA能够抑制豚鼠在体和离体的肾皮质1 1 p se0HSDZ活性，降低11p一OHSDZ对皮质醇的转化率，引起低血钾，降低豚鼠尿中可的松含量和可的松/皮质醇比值。Q seGA对np一OHSDZ活性的抑制作用比p弓A弱。白豚鼠肾皮质11p刃HS玩对抑制剂的敏感性明显高于花豚鼠。更多还原

【Abstract】 As the major effective component of licorice root, & -Glycyrrhizin (18 β - Glycyrrhizic acid, β -GA) has antiinflammatory, antivirusand anti-allergic effects . Through isomerization, it can turn into a -Glycyrrhizin( a -GA), which exhibites a more potential anti-inflammatory effect in hepatitis disease. Due to the inhibitory effects on 11β-hydroxysteroid dehydrogenase (11β-OHSD2), β-GA has many adverse reactions in clinical therapy, such as hypertension, hypokalemia etc. In case of a -GA , similar mild adverse reactions are also reported, however, the related mechanism remaines unknown. The aim of present study is to determinate whether a -GA inhibits the activity of 11β-OHSD2 and reveal the coleration between inhibition of IIβ -OHSD2 and the adverse reactions observed in a -GA treatment. Through experiments both in vivo and in vitro,ve examined either the effects of α-GA on of 11β-OHSD2 or the adverse reactions caused by α-GA treatment, so as to provide evidence for optimized administration in clinical therapy.1. Inhibitory effects of α-GA on 11β-OHSD2 in vitro.Microsomal 11β -OHSD2 was prepared from the honogenate of multicoloured Hartley guinea pig kidney cortex and its activity was determinated from the conversion percentage of cortisol to cortisone . Inhibitory effects of α-GA on 11β-OHSD2 activity in vitro were studied. Results: (1) the conversion percentage of cortisol tocortisone in control with homogenate preparation was 99%. When the concentrations of a. p-GA were below 0.8 mmol/L, the conversion of cortisol percentage was more than 96%. When the concentrations were 1. 6 mmol/L, the conversion percentages of cortisol in a -GA group and P -GA group were 98.55% and 90.51% respectively. When the concentrations were up to 3.2 mmol/L, the percentages of a-GA group and 3-GA group were 62.59% and 38.64% respectively. The inhibitory effects of a-GA on 11P-OHSD2 activity showed dose- dependent. (2) When the concentrations were 0.8,0.4,0.2 mmol/L, the conversion percent rates of cortisol by microsomal IIP -OHSD2 in a -GA group and P-GA group were 38. 99?. 85, 21. 08?. 5(n=3) ; 53. 37?. 63, 33. 09?0. 19(n=3); 60. 41+0. 86, 44. 67 ?. 33 (n=3) respectively. Compared with the conversion percentages of cortisol in control(70.56 + 3.03, n=3), the reduction of the percentage was significant (/<0. 05). The ICsoS were 1140. 1 and 346.4 nmol/L for a-GA and P-GA respectively. This suggested that the inhibitory effects of a-GA on 11P-OHSD2 from Hartley guinea pig kidney were less potent than 3 -GA. (3) Using three batches kidney microsomal 11P-OHSD2, of which every batch was experimented thrice, the average conversion percentage of cortisol in control, a-GA group and 3 -GA group were 70. 56%?3.03%, 38. 99%?. 85%, 21.08%+1. 50%: 70. 93%+4.62%, 38.2%+2.28%> 21. 55% + 1. 38% ; 71. 80% + 2. 33% , 38. 80% + 1. 98% , 21. 50% ?.61% respectively. Using t test, the reciprocal average conversion rates of cortisol in three batches microsomes were compared each other and showed no significant difference(/^0. 05). In one batch microsomes, the conversion rates of cortisol in varying group were compared and showed significant difference(/<0.05). This suggested that a -GA could inhibit the activity of 11P-OHSD2 in vitro, but was less potent than P-GA.2. Determination of kinetic constants of microsomal 110-OHSD2Using 4 concentrations of cortisol,5 different concentrations of a -GA were added into kidney cortex microsomal suspension and yielding velocity of cortisone was determinated. Kinetic constants of microsomal IIP -OHSD2 were calculated from the double reciprocal plots according to Michaelis-Menten equation and Dixon equation. Results: The effects of inhibition by a -GA at varying concentrations of cortisol on the 113 -OHSD2 were shown as a double reciprocal plot. Most of the lines converged on the ordinate, which indicated that the inhibition by a -GA was competitive. H,=15. 25 nmol. mg"1. h"land 10=1. 31 y mol/L were calculated. According to Dixon plot, ?209.2 u mol/L ?7. 1 u mol/L was calculated.3更多还原