發(fā)布時間：2018-06-23 07:39

  本文選題：毛冬青 + 磷酸二酯酶��； 參考：《廣州中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：目的:磷酸二酯酶(PDEs)具有水解細(xì)胞內(nèi)第二信使環(huán)磷酸腺苷(cAMP)或環(huán)磷酸鳥苷(cGMP)的功能,從而終結(jié)這些第二信使所傳導(dǎo)的生化作用。11種PDE酶家族中的7種PDE酶,已被發(fā)現(xiàn)在心臟組織中表達(dá),提示PDE酶在心血管系統(tǒng)病生理過程中的重要作用。近年來,PDE酶作為新的心血管疾病治療靶點,成為一個新的研究熱點。毛冬青作為一個常用中藥,具有活血通脈、消腫止痛、清熱解毒等功效,在冠心病、心絞痛和脈管炎等疾病治療方面應(yīng)用廣泛。課題組在前期的研究中,發(fā)現(xiàn)毛冬青對心血管疾病特別是心衰方面有著良好的藥理作用,也有文獻(xiàn)表明毛冬青甲素具有PDE酶的抑制活性,然而毛冬青藥材的作用基礎(chǔ)和可能的靶點等尚未有較系統(tǒng)的揭露。本文旨在建立一種聯(lián)合應(yīng)用超濾技術(shù)和液質(zhì)聯(lián)用技術(shù)的快速篩選方法,對毛冬青中潛在的PDE酶抑制成分進行研究,探討毛冬青對心血管疾病藥理作用的可能機制。方法:1.毛冬青根中主要成分的LC-MS分離與鑒定毛冬青根粉碎至80目,以80%甲醇超聲提取,提取物溶液在高效液相色譜儀中,以乙腈-0.1%甲酸水的流動相體系進行梯度洗脫分離,二極管陣列檢測器檢測后,在線聯(lián)用電噴霧離子-離子阱-飛行時間質(zhì)譜儀進行分析,將所得的色譜和質(zhì)譜行為數(shù)據(jù)與文獻(xiàn)對照,并對相應(yīng)的多級質(zhì)譜碎片進行質(zhì)譜裂解分析,得到初步的化合物結(jié)構(gòu)解析結(jié)果,部分化合物進一步與標(biāo)準(zhǔn)品的色譜保留時間和質(zhì)譜行為數(shù)據(jù)進行對照,驗證結(jié)構(gòu)鑒定結(jié)果。2.毛冬青根中4種綠原酸類和4中皂苷類成分的含量測定毛冬青根粉碎至80目,以80%甲醇超聲提取,提取物定容為0.05 g · mL~(-1)(以生藥計)的供試品溶液。精密稱取各對照品,分別加入甲醇配制成含如下濃度的對照品溶液,綠原酸12.5μg·mL~(-1),異綠原酸B 29.65μg·mL~(-1),異綠原酸A30.05μg·mL~(-1),異綠原酸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),按比例稀釋,繪制各化合物的標(biāo)準(zhǔn)曲線。對照品和供試品在相同的色譜條件下進行測定,以混合對照品溶液進行方法學(xué)考察,包括精密度、定量限、檢測限等,并對樣品的穩(wěn)定性進行考察,最后測定得到各化合物成分在毛冬青根中的含量。3.毛冬青根中與PDE Ⅰ酶結(jié)合成分的LC-MS分析制備濃度為0.1 g· mL~(-1)(以生藥計)的毛冬青根提取物溶液,與20 U·mL~(-1)的PDE Ⅰ酶溶液進行孵育,置于容量4 mL截留分子量為10 kDa的超濾管中進行分離,分離后超濾內(nèi)管的藥物-蛋白復(fù)合物以50%甲醇洗脫,收集并以氮氣吹干,定容后進行高效液相-二極管陣列檢測器-電噴霧離子-離子阱-飛行時間質(zhì)譜儀聯(lián)用進行分析,將所得的的色譜和質(zhì)譜行為數(shù)據(jù)與毛冬青根提取物的數(shù)據(jù)進行對照,并對相應(yīng)的多級質(zhì)譜碎片進行質(zhì)譜裂解分析,得到初步的化合物結(jié)構(gòu)解析結(jié)果,部分化合物進一步與標(biāo)準(zhǔn)品的色譜保留時間和質(zhì)譜行為數(shù)據(jù)進行對照,驗證結(jié)構(gòu)鑒定結(jié)果。4.毛冬青根中主要成分與PDE Ⅰ酶結(jié)合的活性研究制備濃度為0.1 g·L~(-1)(以生藥計)的毛冬青根提取物溶液,分別與濃度為0,10U·mL~(-1),20U·mL~(-1)的PDEⅠ酶溶液進行孵育,后置于容量4mL截留分子量為10 kDa的超濾管中進行分離,分離后超濾內(nèi)管的藥物-蛋白復(fù)合物以50%甲醇洗脫,收集并以氮氣吹干,定容到200μL進行分析。同時,精密稱取各對照品,分別加入甲醇配制成含如下濃度的對照品溶液,異綠原酸B 10.24μg·mL~(-1),異綠原酸A 8.9624μg·mL~(-1),異綠原酸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),按比例稀釋,繪制各化合物的標(biāo)準(zhǔn)曲線,并計算得到各化合物在供試品中的濃度。按照色譜峰增強因子的計算公式,計算各有效成分在不同濃度PDEⅠ酶溶液中的富集程度,研究相應(yīng)的結(jié)合活性。5.毛冬青根中活性成分對PDEⅠ酶的半數(shù)抑制濃度(IC50)的測定毛冬青根中的主要活性成分在不同濃度下與PDE Ⅰ酶混合孵育,用環(huán)核苷磷酸二酯酶活性試劑盒對PDE Ⅰ酶的活性進行檢測,使用多功能酶標(biāo)儀于620 nm處測定OD值,然后根據(jù)標(biāo)準(zhǔn)曲線求出對應(yīng)的5'-AMP釋出量,與空白對照組相比,求得相應(yīng)濃度下的酶活性抑制率。以抑制率平均值對lg[藥物濃度]作圖,用GraphPad Prism 5軟件進行非線性擬合,求得相應(yīng)藥物的IC50。6.毛冬青根中活性成分對PDE5A、PDE9A酶的半數(shù)抑制濃度(IC50)的測定毛冬青根中的主要活性成分在不同濃度下分別與PDE5A、PDE9A酶混合孵育,用環(huán)核苷磷酸二酯酶活性試劑盒對PDE5A、PDE9A酶的活性進行檢測,使用多功能酶標(biāo)儀于620 nm處測定OD值,然后根據(jù)相應(yīng)的標(biāo)準(zhǔn)曲線求出對應(yīng)的5'-GMP釋出量,與空白對照組相比,求得相應(yīng)濃度下的酶活性抑制率。以抑制率平均值對lg[藥物濃度]作圖,用GraphPad Prism 5軟件進行非線性擬合,求得相應(yīng)藥物的IC50。結(jié)果:1.毛冬青根中主要成分的LC—MS分離與鑒定在對毛冬青根提取的條件進行優(yōu)化的基礎(chǔ)上,建立了分析毛冬青根中主要成分的HPLC色譜條件;在文獻(xiàn)總結(jié)、質(zhì)譜條件優(yōu)化的基礎(chǔ)上,建立了液質(zhì)聯(lián)用分析毛冬青根中主要成分的質(zhì)譜條件。通過對毛冬青根的甲醇提取物進行的分析,分離鑒定出11個化合物,分別為綠原酸、tortosideA、異綠原酸B、異綠原酸A、異綠原酸C、3,4,5-三咖啡酰奎寧酸、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.毛冬青藥材中4種綠原酸類及4種皂苷的含量測定建立了 HPLC法同時測定毛冬青根中8個成分含量。綠原酸,異綠原酸B、異綠原酸A、異綠原酸C,毛冬青皂苷B2、毛冬青皂苷Ai、毛冬青皂苷B1,冬青素A的質(zhì)量濃度分別在 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)范圍內(nèi)與峰面積呈良好的線性關(guān)系,平均加樣回收率(n=9)均高于95%,RSD值均小于3.0%。3批樣品測定結(jié)果分別為: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)。方法學(xué)驗證及3批樣品的測定結(jié)果表明,該方法簡便、準(zhǔn)確,適用于同時測定毛冬青根中8個成分的含量測定。3.毛冬青根中與PDE Ⅰ酶結(jié)合成分的LC-MS分析應(yīng)用超濾技術(shù),對毛冬青根提取物中具有與PDE Ⅰ酶結(jié)合活性的主要成分進行富集、分離,并應(yīng)用HPLC-DAD-IT-TOF-MS聯(lián)用技術(shù)對相關(guān)成分進行分析,將所得的化合物的色譜行為和質(zhì)譜數(shù)據(jù)與毛冬青根提取物中的數(shù)據(jù)進行比較分析,鑒定出11個具有PDEⅠ酶結(jié)合活性的化合物成分,分別為綠原酸、tortoside A、異綠原酸B、異綠原酸A、異綠原酸C、3,4,5-三咖啡酰奎寧酸、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與PDE Ⅰ酶結(jié)合的活性研究應(yīng)用色譜峰增強因子,對其中7個主要活性成分與PDE Ⅰ酶結(jié)合活性進行分析,初步篩選出可能具有PDEⅠ酶抑制活性的化合物,按照結(jié)合活性大小,依次為異綠原酸A毛冬青皂苷B2異綠原酸C冬青素A異綠原酸B毛冬青皂苷B1毛冬青皂苷A1。5.毛冬青根中活性成分對PDEⅠ酶的半數(shù)抑制濃度(IC50)的測定異綠原酸B、異綠原酸A、異綠原酸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對PDEⅠ酶抑制活性(IC50)分別為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對PDE5A、PDE9A酶的半數(shù)抑制濃度(IC50)的測定異綠原酸B、異綠原酸A、異綠原酸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對PDE5A酶抑制活性(IC50)分別為193.5 μ M,436.8μM,104μM,0.43μM,1801.7μM,48.8μM,22.4μM,176.6μM;對PDE9A酶抑制活性(IC50)分別為104 μM,213.7μM,1452 μM,2568 μM,3944 μM,8810 μM,105 μM,4546 μM。結(jié)論:首次建立了一種聯(lián)合應(yīng)用超濾技術(shù)和液質(zhì)聯(lián)用技術(shù)對潛在的PDE酶抑制劑進行快速篩選的方法。從毛冬青根甲醇提取物中,分離鑒定出11個具有PDEⅠ酶結(jié)合活性的有效成分,并進一步對其中7個化合物其PDE酶的抑制活性進行了驗證。結(jié)果顯示毛冬青皂苷A1和毛冬青皂苷B2對PDE5A的抑制活性最強,對于了解毛冬青對心血管疾病的良好藥理作用提供了一定的依據(jù)。同時,這一方法也為從毛冬青及其他中藥材中快速篩選潛在的PDE酶抑制劑提供了有效的應(yīng)用參照。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R284;R285

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 羅騫;涂星;廖小紅;;毛冬青浸膏對糖尿病潰瘍模型小鼠創(chuàng)面修復(fù)作用及機制研究[J];今日藥學(xué);2016年10期

2 曹利華;鄭雁;辛衛(wèi)云;徐坤;苗明三;;毛冬青總黃酮對小鼠血瘀合并腦缺血耐受模型的影響[J];中國中藥雜志;2016年18期

3 方曉艷;左艇;喬靜怡;馮素香;苗明三;;毛冬青總黃酮提高大鼠腦缺血耐受作用研究[J];南京中醫(yī)藥大學(xué)學(xué)報;2016年05期

4 何鋒云;;毛冬青灌腸治療慢性盆腔疼痛的效果觀察[J];現(xiàn)代診斷與治療;2016年15期

5 林曦;陳娟;吳仕嬌;王碧君;熊天琴;祝晨，

本文編號：2056376