毛冬青相关有效成分筛选及其抑制磷酸二酯酶活性的研究

发布时间：2018-06-23 07:39

本文选题：毛冬青 + 磷酸二酯酶　；参考：《广州中医药大学》2017年博士论文

【摘要】：目的:磷酸二酯酶(PDEs)具有水解细胞内第二信使环磷酸腺苷(cAMP)或环磷酸鸟苷(cGMP)的功能,从而终结这些第二信使所传导的生化作用。11种PDE酶家族中的7种PDE酶,已被发现在心脏组织中表达,提示PDE酶在心血管系统病生理过程中的重要作用。近年来,PDE酶作为新的心血管疾病治疗靶点,成为一个新的研究热点。毛冬青作为一个常用中药,具有活血通脉、消肿止痛、清热解毒等功效,在冠心病、心绞痛和脉管炎等疾病治疗方面应用广泛。课题组在前期的研究中,发现毛冬青对心血管疾病特别是心衰方面有着良好的药理作用,也有文献表明毛冬青甲素具有PDE酶的抑制活性,然而毛冬青药材的作用基础和可能的靶点等尚未有较系统的揭露。本文旨在建立一种联合应用超滤技术和液质联用技术的快速筛选方法,对毛冬青中潜在的PDE酶抑制成分进行研究,探讨毛冬青对心血管疾病药理作用的可能机制。方法:1.毛冬青根中主要成分的LC-MS分离与鉴定毛冬青根粉碎至80目,以80%甲醇超声提取,提取物溶液在高效液相色谱仪中,以乙腈-0.1%甲酸水的流动相体系进行梯度洗脱分离,二极管阵列检测器检测后,在线联用电喷雾离子-离子阱-飞行时间质谱仪进行分析,将所得的色谱和质谱行为数据与文献对照,并对相应的多级质谱碎片进行质谱裂解分析,得到初步的化合物结构解析结果,部分化合物进一步与标准品的色谱保留时间和质谱行为数据进行对照,验证结构鉴定结果。2.毛冬青根中4种绿原酸类和4中皂苷类成分的含量测定毛冬青根粉碎至80目,以80%甲醇超声提取,提取物定容为0.05 g · mL~(-1)(以生药计)的供试品溶液。精密称取各对照品,分别加入甲醇配制成含如下浓度的对照品溶液,绿原酸12.5μg·mL~(-1),异绿原酸B 29.65μg·mL~(-1),异绿原酸A30.05μg·mL~(-1),异绿原酸C34.5μg·mL~(-1),毛冬青皂苷B1 169.5μg·mL/1,毛冬青皂苷B2 130.5μg·mL~(-1),毛冬青皂苷A1324.05μg·mL~(-1),冬青素A728.05μg·mL~(-1),按比例稀释,绘制各化合物的标准曲线。对照品和供试品在相同的色谱条件下进行测定,以混合对照品溶液进行方法学考察,包括精密度、定量限、检测限等,并对样品的稳定性进行考察,最后测定得到各化合物成分在毛冬青根中的含量。3.毛冬青根中与PDE Ⅰ酶结合成分的LC-MS分析制备浓度为0.1 g· mL~(-1)(以生药计)的毛冬青根提取物溶液,与20 U·mL~(-1)的PDE Ⅰ酶溶液进行孵育,置于容量4 mL截留分子量为10 kDa的超滤管中进行分离,分离后超滤内管的药物-蛋白复合物以50%甲醇洗脱,收集并以氮气吹干,定容后进行高效液相-二极管阵列检测器-电喷雾离子-离子阱-飞行时间质谱仪联用进行分析,将所得的的色谱和质谱行为数据与毛冬青根提取物的数据进行对照,并对相应的多级质谱碎片进行质谱裂解分析,得到初步的化合物结构解析结果,部分化合物进一步与标准品的色谱保留时间和质谱行为数据进行对照,验证结构鉴定结果。4.毛冬青根中主要成分与PDE Ⅰ酶结合的活性研究制备浓度为0.1 g·L~(-1)(以生药计)的毛冬青根提取物溶液,分别与浓度为0,10U·mL~(-1),20U·mL~(-1)的PDEⅠ酶溶液进行孵育,后置于容量4mL截留分子量为10 kDa的超滤管中进行分离,分离后超滤内管的药物-蛋白复合物以50%甲醇洗脱,收集并以氮气吹干,定容到200μL进行分析。同时,精密称取各对照品,分别加入甲醇配制成含如下浓度的对照品溶液,异绿原酸B 10.24μg·mL~(-1),异绿原酸A 8.9624μg·mL~(-1),异绿原酸C7.8424μg·mL~(-1),毛冬青皂苷B168.824μg·mL~(-1),毛冬青皂苷B2 53.6μg·mL~(-1),毛冬青皂苷A167.2μg·mL~(-1),冬青素A 475.2μg·mL~(-1),按比例稀释,绘制各化合物的标准曲线,并计算得到各化合物在供试品中的浓度。按照色谱峰增强因子的计算公式,计算各有效成分在不同浓度PDEⅠ酶溶液中的富集程度,研究相应的结合活性。5.毛冬青根中活性成分对PDEⅠ酶的半数抑制浓度(IC50)的测定毛冬青根中的主要活性成分在不同浓度下与PDE Ⅰ酶混合孵育,用环核苷磷酸二酯酶活性试剂盒对PDE Ⅰ酶的活性进行检测,使用多功能酶标仪于620 nm处测定OD值,然后根据标准曲线求出对应的5'-AMP释出量,与空白对照组相比,求得相应浓度下的酶活性抑制率。以抑制率平均值对lg[药物浓度]作图,用GraphPad Prism 5软件进行非线性拟合,求得相应药物的IC50。6.毛冬青根中活性成分对PDE5A、PDE9A酶的半数抑制浓度(IC50)的测定毛冬青根中的主要活性成分在不同浓度下分别与PDE5A、PDE9A酶混合孵育,用环核苷磷酸二酯酶活性试剂盒对PDE5A、PDE9A酶的活性进行检测,使用多功能酶标仪于620 nm处测定OD值,然后根据相应的标准曲线求出对应的5'-GMP释出量,与空白对照组相比,求得相应浓度下的酶活性抑制率。以抑制率平均值对lg[药物浓度]作图,用GraphPad Prism 5软件进行非线性拟合,求得相应药物的IC50。结果:1.毛冬青根中主要成分的LC—MS分离与鉴定在对毛冬青根提取的条件进行优化的基础上,建立了分析毛冬青根中主要成分的HPLC色谱条件;在文献总结、质谱条件优化的基础上,建立了液质联用分析毛冬青根中主要成分的质谱条件。通过对毛冬青根的甲醇提取物进行的分析,分离鉴定出11个化合物,分别为绿原酸、tortosideA、异绿原酸B、异绿原酸A、异绿原酸C、3,4,5-三咖啡酰奎宁酸、毛冬青皂苷B3、毛冬青皂苷B2、毛冬青皂苷A1、毛冬青皂苷B1、冬青素A。2.毛冬青药材中4种绿原酸类及4种皂苷的含量测定建立了 HPLC法同时测定毛冬青根中8个成分含量。绿原酸,异绿原酸B、异绿原酸A、异绿原酸C,毛冬青皂苷B2、毛冬青皂苷Ai、毛冬青皂苷B1,冬青素A的质量浓度分别在 1.25～12.5、2.96～29.6、3～30、3.45～34.5、13.05～130.5、32.4～324、16.95～169.5、72.8～728 μg· mL~(-1)范围内与峰面积呈良好的线性关系,平均加样回收率(n=9)均高于95%,RSD值均小于3.0%。3批样品测定结果分别为:0.13～0.15、0.23～0.27、0.31～0.37、0.27～0.28、1.24～1.65、2.42～2.86、2.18～2.81、8.29～10.44 mg·g~(-1)。方法学验证及3批样品的测定结果表明,该方法简便、准确,适用于同时测定毛冬青根中8个成分的含量测定。3.毛冬青根中与PDE Ⅰ酶结合成分的LC-MS分析应用超滤技术,对毛冬青根提取物中具有与PDE Ⅰ酶结合活性的主要成分进行富集、分离,并应用HPLC-DAD-IT-TOF-MS联用技术对相关成分进行分析,将所得的化合物的色谱行为和质谱数据与毛冬青根提取物中的数据进行比较分析,鉴定出11个具有PDEⅠ酶结合活性的化合物成分,分别为绿原酸、tortoside A、异绿原酸B、异绿原酸A、异绿原酸C、3,4,5-三咖啡酰奎宁酸、毛冬青皂苷B3、毛冬青皂苷B2、毛冬青皂苷A1、毛冬青皂苷B1、冬青素A。4.毛冬青根中主要成分与PDE Ⅰ酶结合的活性研究应用色谱峰增强因子,对其中7个主要活性成分与PDE Ⅰ酶结合活性进行分析,初步筛选出可能具有PDEⅠ酶抑制活性的化合物,按照结合活性大小,依次为异绿原酸A毛冬青皂苷B2异绿原酸C冬青素A异绿原酸B毛冬青皂苷B1毛冬青皂苷A1。5.毛冬青根中活性成分对PDEⅠ酶的半数抑制浓度(IC50)的测定异绿原酸B、异绿原酸A、异绿原酸C、西地那非、毛冬青皂苷B1、毛冬青皂苷B2、毛冬青皂苷A1及冬青素A对PDEⅠ酶抑制活性(IC50)分别为779.5 μM,1516μM,106μM,44.79 μM,459.5 μM,313.1 μM,158 μM,250 μM。6.毛冬青根中活性成分对PDE5A、PDE9A酶的半数抑制浓度(IC50)的测定异绿原酸B、异绿原酸A、异绿原酸C、西地那非、毛冬青皂苷B1、毛冬青皂苷B2、毛冬青皂苷A1及冬青素A对PDE5A酶抑制活性(IC50)分别为193.5 μ M,436.8μM,104μM,0.43μM,1801.7μM,48.8μM,22.4μM,176.6μM;对PDE9A酶抑制活性(IC50)分别为104 μM,213.7μM,1452 μM,2568 μM,3944 μM,8810 μM,105 μM,4546 μM。结论:首次建立了一种联合应用超滤技术和液质联用技术对潜在的PDE酶抑制剂进行快速筛选的方法。从毛冬青根甲醇提取物中,分离鉴定出11个具有PDEⅠ酶结合活性的有效成分,并进一步对其中7个化合物其PDE酶的抑制活性进行了验证。结果显示毛冬青皂苷A1和毛冬青皂苷B2对PDE5A的抑制活性最强,对于了解毛冬青对心血管疾病的良好药理作用提供了一定的依据。同时,这一方法也为从毛冬青及其他中药材中快速筛选潜在的PDE酶抑制剂提供了有效的应用参照。
[Abstract]:Objective: phosphodiesterase (PDEs) has the function of hydrolyzing second messenger cyclic adenosine (cAMP) or cyclophosphate (cGMP) in the cell, thus terminates the biochemical action of these second messengers and the 7 PDE enzymes in the PDE enzyme family of.11, which has been found in the cardiac tissue, suggesting that the PDE enzyme is important in the physiological process of cardiovascular system disease. In recent years, as a new target for the treatment of cardiovascular disease, PDE enzyme has become a new research focus. As a common Chinese medicine, Mao Dongqing has the efficacy of activating blood circulation, eliminating swelling and relieving pain, clearing heat and detoxification and so on. It should be widely used in the treatment of diseases such as coronary heart disease, angina pectoris and vasculitis. In the earlier study, we found Mao Dongqing Cardiovascular diseases, especially heart failure, have good pharmacological effects, and there are also references to the inhibitory activity of PDE enzyme. However, the basis of the action and possible targets of the medicinal herbs have not been systematically revealed. The aim of this paper is to establish a combination of ultrafiltration and liquid chromatography for rapid screening. The potential mechanism of PDE enzyme inhibition in Holly hairy holly was studied, and the possible mechanism of Mao Dongqing's pharmacological action on cardiovascular disease was explored. Methods: the LC-MS separation of main components in 1. hairy holly root was separated and identified by LC-MS and identification of Radix Ilex root to 80 orders, 80% methanol ultrasonic extraction, extract solution in high performance liquid chromatograph, and acetonitrile -0.1% formic acid water The flow phase system was separated by gradient elution. After the detector was detected by diode array detector, an on-line electrospray ion trap time of flight mass spectrometer was used for analysis. The obtained chromatographic and mass spectrometric data were compared with the literature, and the corresponding multistage mass spectrometric fragmentation was analyzed. The preliminary structure analysis of the compound was obtained. As a result, some compounds were further compared with the chromatographic retention time and mass spectrometric data of the standard products. The results showed that the content of 4 kinds of chlorogenic acids and 4 saponins in.2. hairy holly roots was crushed to 80 orders, and 80% methanol was extracted with 80% methanol, and the extract was determined to be 0.05 g. ML~ (-1). The test products were accurately called the control products, and the control products were prepared by adding methanol into the control solution containing the following concentration, 12.5 g / mL~ (-1) of chlorogenic acid, B 29.65 mu g. ML~ (-1), isochlorogenic acid A30.05, G mL~ (-1), isochlorogenic acid C34.5, 169.5 Mu The saponins A1324.05 mu g / mL~ (-1), Ilex A728.05 mu g. ML~ (-1) were diluted in proportion to draw the standard curves of the compounds. The control products and the tested products were measured under the same chromatographic conditions, and the mixed control solution was investigated, including the precision, the quantitative limit, the detection limit and so on. The stability of the samples was investigated and the final measurement was carried out. The content of each compound in the root of hairy holly was determined by LC-MS analysis of the binding component of PDE I enzyme in the root of.3. hairy holly. The preparation of the extract solution of 0.1 g. ML~ (-1) (-1) was incubated with the 20 U. ML~ (-1) PDE I enzyme solution and was placed in the ultrafiltration tube with the capacity of 4 mL interception molecular weight 10 kDa. After separation, the drug protein complex of the ultrafiltration inner tube was eluted with 50% methanol, collected and dried with nitrogen, and then analyzed by high performance liquid phase diode array detector, electrospray ion trap and time of flight mass spectrometer. According to the analysis of mass spectrometric fragmentation analysis of the corresponding multistage mass spectrometry fragments, the analytical results of the preliminary compound structure were obtained. Some compounds were further compared with the chromatographic retention time and mass spectrometric data of the standard products. The results of the structural identification results were verified by the study of the activity of the main component of.4. hairy holly root and the activity of PDE I enzyme. 0.1 g. L~ (-1) (-1) was incubated with 0,10U / mL~ (-1) and 20U. ML~ (-1) PDE I enzyme solution respectively, then separated from the ultrafiltration tube with a capacity 4mL interception molecular weight of 10 kDa. After separation, the drug protein complex of the ultrafiltration inner tube was eluted with 50% methanol, collected and dried by nitrogen gas. At the same time, it can be analyzed by 200 mu L. At the same time, the control products are niced accurately, and methanol is added into the control solution containing the following concentration. Isochlorogenic acid B 10.24 G. ML~ (-1), isochlorogenic acid A 8.9624, G. ML~ (-1), isochlorogenic acid C7.8424, G mL~, Mao Dongqing saponins. Glucoside A167.2 mu g. ML~ (-1), Ilex A 475.2 mu g. ML~ (-1), draw the standard curve of each compound in proportion, and calculate the concentration of each compound in the sample. According to the calculation formula of the chromatographic peak enhancement factor, the enrichment degree of each effective component in the solution of different concentration PDE I enzyme is calculated, and the corresponding binding activity.5. hair is studied. The determination of the active components of active components in holly root to PDE I enzyme concentration (IC50), the main active components in the root of PDE I were incubated with PDE I enzyme at different concentrations, and the activity of PDE I enzyme was detected by the RNA phosphodiesterase activity kit, and the OD value was measured at 620 nm using the multifunctional enzyme labeling instrument, and then according to the standard curve. The corresponding 5'-AMP release quantity was obtained. Compared with the blank control group, the inhibitory rate of the enzyme activity under the corresponding concentration was obtained. With the average inhibition rate of the lg[drug concentration, the GraphPad Prism 5 software was used for nonlinear fitting to obtain the half inhibitory concentration (IC50) of the active components of the corresponding drug IC50.6. hairy holly root to PDE5A and PDE9A enzyme. The main active components in the root of holly Holly were incubated with PDE5A and PDE9A enzyme at different concentrations. The activity of PDE5A and PDE9A was detected by the RNA phosphodiesterase active kit. The OD value was measured at 620 nm using the multifunctional enzyme labeling instrument, and then the corresponding 5'-GMP release amount was calculated according to the corresponding standard curve, and the blank was obtained. Compared with the control group, the inhibitory rate of the enzyme activity under the corresponding concentration was obtained. With the average inhibitory rate of the lg[drug concentration, the GraphPad Prism 5 software was used for nonlinear fitting, and the IC50. results of the corresponding drugs were obtained: 1. the LC MS separation and identification of the main components of the root of holly hairy root were based on the optimization of the conditions for the extraction of Mao Dongqing root. The HPLC chromatographic conditions for the analysis of the main components of the root of holly hairy root were established. On the basis of the literature summary and the optimization of mass spectrometry, the mass spectra of the main components in the root of holly hairy root were established. 11 compounds were separated and identified by the analysis of the methanol extracts of the root of holly. They were chlorogenic acid, tortosideA, and different. Chlorogenic acid B, ISO chlorogenic acid A, ISO chlorogenic acid C, 3,4,5- three caffeoyl quinic acid, Mao Dongqing saponins B3, Mao Dongqing saponin B2, Mao Dongqing saponin A1, Mao Dongqing saponins B1, and Ilex Ilex holqing, 4 kinds of chlorogenic acids and 4 kinds of saponins were determined by HPLC method and the content of 8 components in the root of hairy holly was determined. Chlorogenic acid, isochlorogenic acid B, ISO chlorogenic acid A, ISO chlorogenic acid C, Mao Dongqing saponins B2, Mao Dongqing saponin Ai, Ilex saponins B1, and Ilex A mass concentration in the range of 1.25 ~ 12.5,2.96 to 29.6,3 to 30,3.45 ~ 34.5,13.05 to 130.5,32.4 ~ 324,16.95 ~ 728 micron. Higher than 95%, RSD values were less than 3.0%.3 batch samples: 0.13 ~ 0.15,0.23 ~ 0.27,0.31 ~ 0.37,0.27 ~ 0.28,1.24 ~ 1.65,2.42 ~ 2.86,2.18 ~ 2.81,8.29 ~ 10.44 mg. G~ (-1). Methodological verification and the determination of 3 batch of samples showed that the method was simple and accurate and suitable for the simultaneous determination of the content of 8 components in the root of holly hairy holly The LC-MS analysis of the binding component of PDE I enzyme in.3. hairy holly root was used to enrich and separate the main components of the extract from the extract of Ilex hairy root with the activity of PDE I enzyme, and the related components were analyzed by HPLC-DAD-IT-TOF-MS technology, and the chromatographic behavior and mass data of the obtained compounds were obtained. The data in the extract of holly root were compared and analyzed. 11 compounds with PDE I enzyme binding activity were identified: chlorogenic acid, tortoside A, ISO chlorogenic acid B, ISO chlorogenic acid A, isochlorogenic acid C, 3,4,5- three caffeoyl quinic acid, Mao Dongqing saponins B3, Mao Dongqing saponin B2, Mao Dongqing saponin A1, Mao Dongqing saponin B1, Ilex A.4. hair The activity of the combination of the main components of holly root and PDE I enzyme used the chromatographic peak enhancement factor to analyze the binding activity of 7 main active components and PDE I enzyme, and preliminarily screened the compounds which may have the inhibitory activity of PDE I enzyme. According to the binding activity, the isochlorogenic acid A Ilex B2 ISO chlorogenic acid C Ilex ISO chlorogenic acid was in turn. A ISO chlorogenic acid B hairy holly saponins B1 the active constituents of Ilex A1.5. hairy holly root in the root of PDE I enzyme (IC50) determination of isochlorogenic acid B, ISO chlorogenic acid A, isochlorogenic acid C, sildenafil, Ilex saponins B1, Mao Dongqing saponins B2, Mao Dongqing saponins A1 and Ilex enzyme inhibition activity of 779.5 Mu respectively, 1516 mu M, 106 M, 44.79 mu M, 459.5 mu M, 313.1 mu M, 158 M, and 250 mu M.6., the half inhibitory concentration of PDE5A and PDE9A enzyme (IC50) was used for the determination of isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid, Ilex saponins, Mao Dongqing saponins and Ilex 193.5 mu M, 436.8 mu, 104 mu M, 0.43 mu M, 1801.7 mu M, 48.8 mu M, 22.4 mu M, 176.6 mu M, and PDE9A enzyme inhibition activity (IC50) are 104 mu M, 213.7 mu M, 1452 micron, 3944, 2568 mu. Methods. 11 effective components with PDE I enzyme binding activity were isolated from the Mao Dongqing root methanol extract, and the inhibitory activity of 7 of them was further verified. The results showed that the inhibitory activity of Mao Dongqing saponins A1 and Mao Dongqing saponins B2 on PDE5A was the strongest, and to understand the cardiovascular disease of Mao Dongqing. At the same time, this method also provides effective reference for the rapid screening of potential PDE enzyme inhibitors from Mao Dongqing and other Chinese medicinal materials.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284;R285

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