左金丸抗幽门螺杆菌药效物质筛选及其现代制剂研究
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摘要
中药药效物质基础一直是当今中医药研究的热点和难点。中医药理论认为,炮制和配伍可不同程度地改变中药的药性,即活性。为了寻求中药药效物质基础研究的新突破,本文在中医药理论的指导下,以黄连不同炮制品以及主含黄连的左金丸及其经典类方为研究对象,主要采用生物热动力学方法,同时结合常规药理试验验证和化学组分关联分析,从复方组成、炮制规格、有效部位三个层面,较系统地考察左金丸抗幽门螺杆菌等药效物质基础;开展基于复方有效部位的左金漂浮片制备工艺研究;在此基础上,探索构建一套主要基于生物热动力学表达的清热类中药药效物质筛选模式和方法。主要研究结果如下:
1.生物热动力学分析的方法学考察:用安瓿法测定的细菌代谢热谱图与生物学培养过程中的各个阶段,如停滞期、指数生长期、稳定期及衰亡期能很好吻合。细菌代谢的热谱图完整地描述了细菌的代谢过程。细菌生长代谢的热谱图的指数生长段符合InP_t=InP_0+kt的线性方程。
2.生物热动力学研究:采用生物热动力学方法,以大肠杆菌、金黄色葡萄球菌、痢疾杆菌及幽门螺杆菌为模型生物体,测定了黄连不同炮制品、黄连与吴茱萸不同剂量配比类方水提物的生物热谱图以及系列生物热动力学参数。结果表明细菌生长速率常数k、代谢产热量Q等生物热动力学参数可以量化地表征黄连不同炮制品及类方的生物活性差异。k与Q值的排列顺序为:吴茱萸制黄连＞酒制黄连＞姜制黄连＞生黄连＞胆汁制黄连＞盐制黄连＞醋制黄连;反左金丸＞茱萸丸＞甘露散＞左金丸。Q值变化规律与其传统的寒热药性认知相吻合,说明生物热动力学可作为评价中药药性/活性的较好的研究方法。
3.常规药理实验研究:采用常规的体外抑菌试验方法进行了黄连不同炮制品及左金丸类方抗幽门螺杆菌试验研究,结果表明:左金丸类方及黄连不同炮制品对幽门螺杆菌的体外抑菌效果呈现差异,以醋制黄连及左金丸活性最强。本实验结果验证和支持了生物热力学试验结果。
4.化学组分研究:建立了黄连不同炮制品水提液、左金丸类方水提液、左金丸生物碱部位及非生物碱部位的HPLC指纹图谱,并分析了其化学属性,将化学组分进行合成资料的主成分分析。统计结果表明黄连不同炮制品指纹图谱的差异主要体现在化学组分Y3、药根碱含量的变化;左金丸类方指纹图谱的差异主要体现在吴茱萸碱、吴茱萸次碱、化学组分Y7、Y8的含量变化。聚类分析结果表明黄连不同炮制品的聚类与中药药性呈现出一定关联;左金丸类方中以反左金丸与其它三类方的差异最为明显。
5.左金丸抗幽门螺杆菌物质的确定:左金丸类方的药性/活性差异研究表明四类方中以左金丸抗幽门螺杆菌作用最强;黄连不同炮制品的药性/活性差异研究表明醋制黄连抗幽门螺杆菌作用最强;左金丸不同化学部位的药性/活性差异研究表明生物碱部位为左金丸抗幽门螺杆菌的主要药效物质。
6.基于生物热动力学表达的清热类中药及复方药效物质筛选模式和方法的构建:通过对清热药中的代表性药物—黄连及黄连与吴茱萸不同配伍类方—左金丸、茱萸丸、甘露散、反左金丸的研究,初步构建了基于生物热动力学表达的清热类中药及复方药效物质筛选模式,即通过生物热动力学研究,结合化学组分分析及常规药理学试验研究,运用主成分分析,典型相关分析等数理统计分析手段,进行生物热动力学-常规药理-化学组分的关联分析,探求与功能主治相关的药效物质基础。
7.左金漂浮片的药学研究:在了解左金丸抗幽门螺杆菌有效部位的基础上,初步探讨了左金漂浮片制备工艺的相关内容。结果认为D101大孔吸附树脂可达到富集、纯化左金生物碱部位的目的;通过先优化漂浮性能工艺,再优化满足药物释放度工艺的方法可制备出既具较佳的漂浮性能,又具备合格药物释放度的漂浮片,其释药机制为药物扩散、骨架溶蚀协同作用;释放条件对左金漂浮片的漂浮性能及药物释放度无明显影响。
At present, the study of active essence of Chinese Materia Medica(CMM) is the concerned point. The theory of Traditional Chinese Medicine(TCM) revealed that the preparation and prescription compatibility can change active essence of CMM at different degree and then change the property of CMM. In order to explore the new breakthrough of study of active essence of CMM, in this paper, we investigated systematically the anti-Helicobacter pylor(Hp) active essence of Zuojingwan from three aspects of prescription compatibility, preparation and active fraction and carried out the preparing technic study of Zuojing Sustained Floating Tablets(ZJSFT), taking preparations of Coptidis Rhizoma (CR) and Zuojing similar formula as objects, adopting biothermodynamic as main method, and combining with chemical basis analysis and pharmacology experiment. Based on this, we tried to establish a new mode and method of active essence screening of heat cleaning CMM . The main results as following:
1. Investigation on biothermodynamic method: The result showed that the biothermodynamic spectrogram of bacterial metabolism was well coincided with phases of biologic cultivation such as lag phase, exponential growth phase, stable phase and decline phase. The biothermodynamic spectrogram can integritly describe the bacterial metablic phases. The profile of exponential growth phase fit with linear eqution: InPt=InP0+kt. It indicated that microcalorimetry had such superiorities as actual time, on-line, repeat well, easy to manipulate.
2. The study of biothermodynamic: Adopting the method of biothermodynamic, taking Escherichia coli, Shigella dysenteroae, Staphylococcus aureus, Helicobacter pylori(Hp) as pattern organism, the biothermodynamic spectrograms and parameters of aqueous extracts of the preparations of CR(Shenghuanglian, Jiangzhihuanglian, Wuyuhuanglian, Jiuzhihuanglian, Cuzhihuanglian, Yanhuanglian and Danzhihuanglian) and Zuojing similar formula(Fanzuojinwan, Zhuyuwan, Ganlusan and Zuojinwan) were determined. The result showed the biothermodynamic parameters such as growth rate constant k and heat production Q can exosyndrome the differences of biologic activity of preparations of CR and Zuojing similar formula. The sequence of k and Q : Wuyuhuanglian > Jiuzhihuanglian > Jiangzhihuanglian > Shenghuanglian > Danzhihuanglian > Yanhuanglian > Cuzhihuanglian, Fanzuojinwan> Zhuyuwan > Ganlusan > Zuojinwan. The rule of change coincided with the property of CMM, it also indicated a better method for evaluating the property or activity of CMM was put forward.
3. The study of routine pharmacological experiment:Through routine vitro bacteriostasis experiment, the anti-Hp action of preparations of CR and Zuojing similar formula was studied.The result showed that there were differences between different preparations of CR and Zuojing similar formula, it also indicated that Cuzhihuanglian and Zuojingwan had the best anti-Hp activity. It verified and supported the result of biothermodynamic experiment and showed that biothermodynamic method can be served as an available method to determine the property and activity of CMM.
4. The study of chemical basis: In this paper,chemical HPLC fingerprint chromatograph of aqueous extracts of preparations of CR, Zuojing similar formula, alkaloid fraction and non-alkaloid fraction were established, and the chemical attribute was analyzed. Using principal component analysis as main statistics, taking the internal integral area as the samples, statistical result indicated that the differences of HPLC fingerprint of different samples of preparations of CR mainly reflected on the change of content of chemical basis Y3 and Jateorhicine, and Zuojing similar formular mainly reflected on Evodiamine, rutacarpine,chemical basis Y7 and Y8. The result of hierarchical clustering showed that clustering of different preparations of CR correlated with the property of CMM. Fanzuojingwan significantly differed with the other similar formula.
5. The confirmation of anti-Hp active essences of different chemical parts of Zuojingwan: The study on differences of property or activity of Zuojing similar formula and preparations of CR indicated that Zuojingwan and Cuzhihanglian had the best anti-Hp activities. The study also showed that the alkloid fraction of Zuojingwan was the main anti-Hp active substance.
6. Through study on preparations of CR and Zuojing similar formula, a new mode and method of active essence screening of heat cleaning CMM and prescription compatibility based on biothermodynamics were preliminarily established.The active essence which correlated with function and indication of CMM or prescription compatibility can be explored through the study of biothermodynamics, chemical basis and pharmacological activity combined with statistical method of principal component analysis and canonical correlation analysis.
7. The pharmaceutic study on Zuojing sustained floating tablets(ZJSFT): The paper tried to preliminarily explore the preparational technic of ZJSFT based on understanding the anti-Hp active fraction of Zuojingwan. The result indicated that D101 macroporous resin can concentrate and purify the Zuojing alkaloid fraction; The ZJSFT which with best floating performance and qualified cumulative drug release as well can be prepared by optimizing the technic of floating performance first and then adjusting the cumulative drug release. The drug release mechanism was joint actions of drug diffusion, bulk erosion and chalasia. It showed that release condition had no obvious effect on floating performance and cumulative drug release.
1.生物热动力学分析的方法学考察:用安瓿法测定的细菌代谢热谱图与生物学培养过程中的各个阶段,如停滞期、指数生长期、稳定期及衰亡期能很好吻合。细菌代谢的热谱图完整地描述了细菌的代谢过程。细菌生长代谢的热谱图的指数生长段符合InP_t=InP_0+kt的线性方程。
2.生物热动力学研究:采用生物热动力学方法,以大肠杆菌、金黄色葡萄球菌、痢疾杆菌及幽门螺杆菌为模型生物体,测定了黄连不同炮制品、黄连与吴茱萸不同剂量配比类方水提物的生物热谱图以及系列生物热动力学参数。结果表明细菌生长速率常数k、代谢产热量Q等生物热动力学参数可以量化地表征黄连不同炮制品及类方的生物活性差异。k与Q值的排列顺序为:吴茱萸制黄连＞酒制黄连＞姜制黄连＞生黄连＞胆汁制黄连＞盐制黄连＞醋制黄连;反左金丸＞茱萸丸＞甘露散＞左金丸。Q值变化规律与其传统的寒热药性认知相吻合,说明生物热动力学可作为评价中药药性/活性的较好的研究方法。
3.常规药理实验研究:采用常规的体外抑菌试验方法进行了黄连不同炮制品及左金丸类方抗幽门螺杆菌试验研究,结果表明:左金丸类方及黄连不同炮制品对幽门螺杆菌的体外抑菌效果呈现差异,以醋制黄连及左金丸活性最强。本实验结果验证和支持了生物热力学试验结果。
4.化学组分研究:建立了黄连不同炮制品水提液、左金丸类方水提液、左金丸生物碱部位及非生物碱部位的HPLC指纹图谱,并分析了其化学属性,将化学组分进行合成资料的主成分分析。统计结果表明黄连不同炮制品指纹图谱的差异主要体现在化学组分Y3、药根碱含量的变化;左金丸类方指纹图谱的差异主要体现在吴茱萸碱、吴茱萸次碱、化学组分Y7、Y8的含量变化。聚类分析结果表明黄连不同炮制品的聚类与中药药性呈现出一定关联;左金丸类方中以反左金丸与其它三类方的差异最为明显。
5.左金丸抗幽门螺杆菌物质的确定:左金丸类方的药性/活性差异研究表明四类方中以左金丸抗幽门螺杆菌作用最强;黄连不同炮制品的药性/活性差异研究表明醋制黄连抗幽门螺杆菌作用最强;左金丸不同化学部位的药性/活性差异研究表明生物碱部位为左金丸抗幽门螺杆菌的主要药效物质。
6.基于生物热动力学表达的清热类中药及复方药效物质筛选模式和方法的构建:通过对清热药中的代表性药物—黄连及黄连与吴茱萸不同配伍类方—左金丸、茱萸丸、甘露散、反左金丸的研究,初步构建了基于生物热动力学表达的清热类中药及复方药效物质筛选模式,即通过生物热动力学研究,结合化学组分分析及常规药理学试验研究,运用主成分分析,典型相关分析等数理统计分析手段,进行生物热动力学-常规药理-化学组分的关联分析,探求与功能主治相关的药效物质基础。
7.左金漂浮片的药学研究:在了解左金丸抗幽门螺杆菌有效部位的基础上,初步探讨了左金漂浮片制备工艺的相关内容。结果认为D101大孔吸附树脂可达到富集、纯化左金生物碱部位的目的;通过先优化漂浮性能工艺,再优化满足药物释放度工艺的方法可制备出既具较佳的漂浮性能,又具备合格药物释放度的漂浮片,其释药机制为药物扩散、骨架溶蚀协同作用;释放条件对左金漂浮片的漂浮性能及药物释放度无明显影响。
At present, the study of active essence of Chinese Materia Medica(CMM) is the concerned point. The theory of Traditional Chinese Medicine(TCM) revealed that the preparation and prescription compatibility can change active essence of CMM at different degree and then change the property of CMM. In order to explore the new breakthrough of study of active essence of CMM, in this paper, we investigated systematically the anti-Helicobacter pylor(Hp) active essence of Zuojingwan from three aspects of prescription compatibility, preparation and active fraction and carried out the preparing technic study of Zuojing Sustained Floating Tablets(ZJSFT), taking preparations of Coptidis Rhizoma (CR) and Zuojing similar formula as objects, adopting biothermodynamic as main method, and combining with chemical basis analysis and pharmacology experiment. Based on this, we tried to establish a new mode and method of active essence screening of heat cleaning CMM . The main results as following:
1. Investigation on biothermodynamic method: The result showed that the biothermodynamic spectrogram of bacterial metabolism was well coincided with phases of biologic cultivation such as lag phase, exponential growth phase, stable phase and decline phase. The biothermodynamic spectrogram can integritly describe the bacterial metablic phases. The profile of exponential growth phase fit with linear eqution: InPt=InP0+kt. It indicated that microcalorimetry had such superiorities as actual time, on-line, repeat well, easy to manipulate.
2. The study of biothermodynamic: Adopting the method of biothermodynamic, taking Escherichia coli, Shigella dysenteroae, Staphylococcus aureus, Helicobacter pylori(Hp) as pattern organism, the biothermodynamic spectrograms and parameters of aqueous extracts of the preparations of CR(Shenghuanglian, Jiangzhihuanglian, Wuyuhuanglian, Jiuzhihuanglian, Cuzhihuanglian, Yanhuanglian and Danzhihuanglian) and Zuojing similar formula(Fanzuojinwan, Zhuyuwan, Ganlusan and Zuojinwan) were determined. The result showed the biothermodynamic parameters such as growth rate constant k and heat production Q can exosyndrome the differences of biologic activity of preparations of CR and Zuojing similar formula. The sequence of k and Q : Wuyuhuanglian > Jiuzhihuanglian > Jiangzhihuanglian > Shenghuanglian > Danzhihuanglian > Yanhuanglian > Cuzhihuanglian, Fanzuojinwan> Zhuyuwan > Ganlusan > Zuojinwan. The rule of change coincided with the property of CMM, it also indicated a better method for evaluating the property or activity of CMM was put forward.
3. The study of routine pharmacological experiment:Through routine vitro bacteriostasis experiment, the anti-Hp action of preparations of CR and Zuojing similar formula was studied.The result showed that there were differences between different preparations of CR and Zuojing similar formula, it also indicated that Cuzhihuanglian and Zuojingwan had the best anti-Hp activity. It verified and supported the result of biothermodynamic experiment and showed that biothermodynamic method can be served as an available method to determine the property and activity of CMM.
4. The study of chemical basis: In this paper,chemical HPLC fingerprint chromatograph of aqueous extracts of preparations of CR, Zuojing similar formula, alkaloid fraction and non-alkaloid fraction were established, and the chemical attribute was analyzed. Using principal component analysis as main statistics, taking the internal integral area as the samples, statistical result indicated that the differences of HPLC fingerprint of different samples of preparations of CR mainly reflected on the change of content of chemical basis Y3 and Jateorhicine, and Zuojing similar formular mainly reflected on Evodiamine, rutacarpine,chemical basis Y7 and Y8. The result of hierarchical clustering showed that clustering of different preparations of CR correlated with the property of CMM. Fanzuojingwan significantly differed with the other similar formula.
5. The confirmation of anti-Hp active essences of different chemical parts of Zuojingwan: The study on differences of property or activity of Zuojing similar formula and preparations of CR indicated that Zuojingwan and Cuzhihanglian had the best anti-Hp activities. The study also showed that the alkloid fraction of Zuojingwan was the main anti-Hp active substance.
6. Through study on preparations of CR and Zuojing similar formula, a new mode and method of active essence screening of heat cleaning CMM and prescription compatibility based on biothermodynamics were preliminarily established.The active essence which correlated with function and indication of CMM or prescription compatibility can be explored through the study of biothermodynamics, chemical basis and pharmacological activity combined with statistical method of principal component analysis and canonical correlation analysis.
7. The pharmaceutic study on Zuojing sustained floating tablets(ZJSFT): The paper tried to preliminarily explore the preparational technic of ZJSFT based on understanding the anti-Hp active fraction of Zuojingwan. The result indicated that D101 macroporous resin can concentrate and purify the Zuojing alkaloid fraction; The ZJSFT which with best floating performance and qualified cumulative drug release as well can be prepared by optimizing the technic of floating performance first and then adjusting the cumulative drug release. The drug release mechanism was joint actions of drug diffusion, bulk erosion and chalasia. It showed that release condition had no obvious effect on floating performance and cumulative drug release.
引文
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