菊花降血压有效部位的筛选及相关实验研究
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摘要
高血压是一种发病率、致残率、死亡率较高的疾病。目前的降血压的化学合成药物大都存在作用时间短、毒副作用大、依赖性较强,治疗效果存在局限性等问题。我国医学典籍中关于中约降血压的记载颇多,中药资源丰富,价格低廉,且其毒副作用小。
现在药理学研究表明,菊花提取物(Chrysanthemum morifolium extract,CME)具有治疗心血管系统疾病的作用。对菊花进行初步的研究,筛选降血压的有效部位,开发降血压新药。有非常重要的现实意义。
本研究的主要内容有:(1)采用血管紧张素转化酶-底物-血管紧张素转化酶抑制剂(ACE-HHL-ACEI)反应体系,以高效液相色谱法测定反应体系中马尿酸含量,衡量ACE抑制剂的活性,追踪菊花降血压有效部位;(2)比较菊花的水、50%乙醇和95%乙醇提取物对ACE抑制作用的活性;(3)采用石油醚、乙酸乙酯和丙酮有机溶剂初步萃取分离菊花95%乙醇提取物,并研究丙酮萃取的丙酮用量、不同时间和不同温度所得的萃取物对ACE抑制作用的影响;(4)采用硅胶柱层析和半制备高效液相色谱进行进一步分离提纯菊花中丙酮萃取物;(5)将有效部位的成分的定性检测和降高血压的动物实验研究。
通过以上实验,得到以下研究结果:
(1)高效液相色谱法测定ACE-HHL-ACEI反应体系中马尿酸含量,马尿酸的峰面积与浓度的线性关系的相关系数r=0.9996,马尿酸的加样回收率为97.889%～105.061%,相对标准偏差(RSD)为2.53%,精密度的相对偏差(RSD)为1.97%。
(2)菊花95%乙醇提取物的IC_(50)为70.52+2.04mg/mL,比50%乙醇提取和水提物对ACE抑制作用的活力强(P＜0.05)。
(3)通过石油醚、乙酸乙酯和丙酮有机溶剂对菊花95%乙醇提取物初步萃取分离,得到丙酮萃取物的IC_(50)为14.47±1.26 mg/mL。比95%乙醇提取物对ACE抑制作用的活性明显增强(P＜0.01)。
(4)通过硅胶柱层析的方法对丙酮萃取物进行再次分离,得到对ACE抑制作用最强的甲醇洗脱部位(组分5),该部位的IC_(50)为0.71±0.05mg/mL,对ACE抑制作用的活性进一步增强(与丙酮萃取物比较,P＜0.01),然而半制备高效液相分离该部位,获得的18个化合物对ACE抑制作用的活性没有继续增强。说明菊花降血压的有效部位是甲醇洗脱部位(组分5)。
(5)通过对甲醇洗脱部位化学成分的定性检测,可知有效部位为黄酮类化合物。体内实验结果表明低剂量(1 mg/Kg·BW)和高剂量(5mg/Kg·BW)的甲醇洗脱部位对自发性高血压大鼠血压具有降低作用(与阴性对照组比较,p＜0.01),血清ACE活力显著降低(与阴性对照组比较,P＜0.01)。
Hypertension is a disease of high morbidity, deformity and mortality rate. Previously, most of the drugs for curing hypertension on the market have a lot of problems: the effect can't last long, drugs have many side effects and high drug dependency, limited in curing effects and so on. There are many records about antihypertensive Chinese traditional medicines (CTM) in medical books in our country. CTMs are rich in resources, very cheap and low in side effects. It makes very good sense to find effective CTMs to take the place of synthetic medicine in curing hypertension or decrease side effects of synthetic medicine.
Modern pharmacological researches indicate that Chrysanthemum extract can help to cure cardiovascular diseases. So it is important to do researches on Chrysanthemum extract to exploit new effective medicine to cure hypertensive.
The main contents of this paper are: (1) Mensurate the activity of ACE in ACE-HHL-ACEI system by measuring the content of hippuric acid to tracing the effective substances in Chrysanthemum extract; (2)Compare the ACE inhibiting activity of Chrysanthemum 95%-ethanolic extract (IC_(50)=70.52±2.04mg/mL) is higher than the 50%-ethanolic extract and aqueous extract with each other; (3) Use petroleum ether, ethyl acetate and acetone to isolate and purify ACE inhibitor extracted from Chrysanthemum 95%-ethanolic extract, and study the ACE inhibiting activity of different extracts prepared under different acetone-dosages, time and temperature. (4) Use half-preparative HPLC and silica column chromatography to do further isolation and purification on the active components; (4) Do qualitative detection on effective components and antihypertensive experiments on animals.
The results of these experiments were shown as follows:
(1) Mensurate content of hippuric acid in ACE-HHL-ACEI system: r=0.9996, RSD=2.53%, Precision RSD=1.97%, added Sample Recycling Rate=97.889%-105.061%.
(2) The ACE inhibiting activity of Chrysanthemum 95 % -ethanolic extract (IC_(50)=70.52±2.04mg/mL) is higher than the 50%-ethanolic extract and aqueous extract..
(3) Use organic solvent (petroleum ether, ethyl acetate and acetone) to extract the active component of Chrysanthemum. Results indicate that the ACE inhibiting activity of Chrysanthemum acetonic extract (IC_(50)=14.47±1.26mg/mL) is higher than Chrysanthemum 95%-ethanolic extract (P <0.01).
(4) Chrysanthemum acetonic extract was further isolated by using silica column chromatography. Result indicates that the activity of component (Component 5) washed by methanol is the highest. The IC_(50) of this new isolated extract is 0.71±0.05mg/mL, which is significantly higher than the IC_(50) of Chrysanthemum acetonic extract. Use half-preparative HPLC to do further isolation, and 18 components were observed. The ACE inhibiting activity of these 18 components doesn't get further increase. So the effective antihypertensive component is ethanol-washing part.
(5) By qualitative detection it indicates that the effective components contribute to the ACE inhibiting activity in ethanol-washing part are flavone compounds. Low dosage (1 mg/Kg·BW) and high dosage (5mg/Kg·BW) of ethanol-washing part are given to mice to test the effect in vivo. The results indicate that the components can decrease the blood pressure of hypertension mice (compared with the control group, P < 0. 05) and serum ACE activity was significantly decreased (compared with the control group, P < 0.01).
现在药理学研究表明,菊花提取物(Chrysanthemum morifolium extract,CME)具有治疗心血管系统疾病的作用。对菊花进行初步的研究,筛选降血压的有效部位,开发降血压新药。有非常重要的现实意义。
本研究的主要内容有:(1)采用血管紧张素转化酶-底物-血管紧张素转化酶抑制剂(ACE-HHL-ACEI)反应体系,以高效液相色谱法测定反应体系中马尿酸含量,衡量ACE抑制剂的活性,追踪菊花降血压有效部位;(2)比较菊花的水、50%乙醇和95%乙醇提取物对ACE抑制作用的活性;(3)采用石油醚、乙酸乙酯和丙酮有机溶剂初步萃取分离菊花95%乙醇提取物,并研究丙酮萃取的丙酮用量、不同时间和不同温度所得的萃取物对ACE抑制作用的影响;(4)采用硅胶柱层析和半制备高效液相色谱进行进一步分离提纯菊花中丙酮萃取物;(5)将有效部位的成分的定性检测和降高血压的动物实验研究。
通过以上实验,得到以下研究结果:
(1)高效液相色谱法测定ACE-HHL-ACEI反应体系中马尿酸含量,马尿酸的峰面积与浓度的线性关系的相关系数r=0.9996,马尿酸的加样回收率为97.889%～105.061%,相对标准偏差(RSD)为2.53%,精密度的相对偏差(RSD)为1.97%。
(2)菊花95%乙醇提取物的IC_(50)为70.52+2.04mg/mL,比50%乙醇提取和水提物对ACE抑制作用的活力强(P＜0.05)。
(3)通过石油醚、乙酸乙酯和丙酮有机溶剂对菊花95%乙醇提取物初步萃取分离,得到丙酮萃取物的IC_(50)为14.47±1.26 mg/mL。比95%乙醇提取物对ACE抑制作用的活性明显增强(P＜0.01)。
(4)通过硅胶柱层析的方法对丙酮萃取物进行再次分离,得到对ACE抑制作用最强的甲醇洗脱部位(组分5),该部位的IC_(50)为0.71±0.05mg/mL,对ACE抑制作用的活性进一步增强(与丙酮萃取物比较,P＜0.01),然而半制备高效液相分离该部位,获得的18个化合物对ACE抑制作用的活性没有继续增强。说明菊花降血压的有效部位是甲醇洗脱部位(组分5)。
(5)通过对甲醇洗脱部位化学成分的定性检测,可知有效部位为黄酮类化合物。体内实验结果表明低剂量(1 mg/Kg·BW)和高剂量(5mg/Kg·BW)的甲醇洗脱部位对自发性高血压大鼠血压具有降低作用(与阴性对照组比较,p＜0.01),血清ACE活力显著降低(与阴性对照组比较,P＜0.01)。
Hypertension is a disease of high morbidity, deformity and mortality rate. Previously, most of the drugs for curing hypertension on the market have a lot of problems: the effect can't last long, drugs have many side effects and high drug dependency, limited in curing effects and so on. There are many records about antihypertensive Chinese traditional medicines (CTM) in medical books in our country. CTMs are rich in resources, very cheap and low in side effects. It makes very good sense to find effective CTMs to take the place of synthetic medicine in curing hypertension or decrease side effects of synthetic medicine.
Modern pharmacological researches indicate that Chrysanthemum extract can help to cure cardiovascular diseases. So it is important to do researches on Chrysanthemum extract to exploit new effective medicine to cure hypertensive.
The main contents of this paper are: (1) Mensurate the activity of ACE in ACE-HHL-ACEI system by measuring the content of hippuric acid to tracing the effective substances in Chrysanthemum extract; (2)Compare the ACE inhibiting activity of Chrysanthemum 95%-ethanolic extract (IC_(50)=70.52±2.04mg/mL) is higher than the 50%-ethanolic extract and aqueous extract with each other; (3) Use petroleum ether, ethyl acetate and acetone to isolate and purify ACE inhibitor extracted from Chrysanthemum 95%-ethanolic extract, and study the ACE inhibiting activity of different extracts prepared under different acetone-dosages, time and temperature. (4) Use half-preparative HPLC and silica column chromatography to do further isolation and purification on the active components; (4) Do qualitative detection on effective components and antihypertensive experiments on animals.
The results of these experiments were shown as follows:
(1) Mensurate content of hippuric acid in ACE-HHL-ACEI system: r=0.9996, RSD=2.53%, Precision RSD=1.97%, added Sample Recycling Rate=97.889%-105.061%.
(2) The ACE inhibiting activity of Chrysanthemum 95 % -ethanolic extract (IC_(50)=70.52±2.04mg/mL) is higher than the 50%-ethanolic extract and aqueous extract..
(3) Use organic solvent (petroleum ether, ethyl acetate and acetone) to extract the active component of Chrysanthemum. Results indicate that the ACE inhibiting activity of Chrysanthemum acetonic extract (IC_(50)=14.47±1.26mg/mL) is higher than Chrysanthemum 95%-ethanolic extract (P <0.01).
(4) Chrysanthemum acetonic extract was further isolated by using silica column chromatography. Result indicates that the activity of component (Component 5) washed by methanol is the highest. The IC_(50) of this new isolated extract is 0.71±0.05mg/mL, which is significantly higher than the IC_(50) of Chrysanthemum acetonic extract. Use half-preparative HPLC to do further isolation, and 18 components were observed. The ACE inhibiting activity of these 18 components doesn't get further increase. So the effective antihypertensive component is ethanol-washing part.
(5) By qualitative detection it indicates that the effective components contribute to the ACE inhibiting activity in ethanol-washing part are flavone compounds. Low dosage (1 mg/Kg·BW) and high dosage (5mg/Kg·BW) of ethanol-washing part are given to mice to test the effect in vivo. The results indicate that the components can decrease the blood pressure of hypertension mice (compared with the control group, P < 0. 05) and serum ACE activity was significantly decreased (compared with the control group, P < 0.01).
引文
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