以慈航丹方药探讨含挥发性成分复方用“半仿生提取法”研究的模式
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摘要
目的：以慈航丹方药为例,通过半仿生提取法(SBE法)制备分子量≤1000Da提取物为最终产物,探讨含挥发性成分的中药复方药效物质用“SBE法”研究的基本思路与模式。
方法：①首先对慈航丹方药进行基源、性状鉴定与质量鉴定研究；②将方中含挥发性药物(当归、川芎、香附)用超临界流体萃取,以均匀设计U7(75)表发布点试验,在饮片、设备、技术参数相同的基础上作SFE-CO2萃取,萃取物另器保存；以藁苯内酯、盐酸川芎嗪、α-香附酮、总萃取物为指标,综合评判,优选出方药超临界萃取(SFE-CO2)较佳工艺条件；③将超临界萃取后的药渣与方中益母草混合,用半仿生法提取,采用均匀设计U9(91×33)表发布点试验,以盐酸水苏碱、盐酸川芎嗪、阿魏酸、总糖及分子量≤1000Da提取物为指标,综合评判,优选出SBE法制备分子量≤1000Da提取物的条件；④在乙醇浓度30%-80%范围内,采用比例分割法,以上述相同的指标和评判方法,优选出方药醇提制备分子量≤1000Da提取物的较佳醇浓度；⑤以上述相同的指标和评判方法,作方药3种方法(SBE法、WE法、AE法)分子量≤1000Da提取物的多指标成分比较；⑥因化学等值不一定生物等效,故作方药3种方法分子量≤1000Da提取物的活血调经、止痛、抗炎等主要药效及毒性的研究比较；⑦对方药3种方法分子量≤1000Da提取物药液及其兔含药血清作HPLC指纹图谱的研究比较；⑧对方药3种方法分子量≤1000Da提取液的兔含药血清作大鼠离体子宫平滑肌作用的研究比较；⑨最后综合评判分析,优选出慈航丹方药的最佳提取方法和工艺条件。
结果：①方中所用的中药皆符合《中国药典》2005版一部各项下规定,均为合格的饮片；②优选出慈航丹方中含挥发性成分的中药超临界流体萃取的工艺条件为：萃取压力30MPa、萃取温度48℃、解析温度Ⅰ60℃、解析温度Ⅱ30℃、萃取时间4.0h。③优选出慈航丹方药的SBE法工艺条件为：3煎用水的pH依次为：6.0、7.5、8.8；提取时间依次为2.0h、1.5h、1.5h。④优选出醇提的最佳浓度为80%；⑤3种方法提取液的综合评判值的顺序为：YSBE>YAE>YWE;⑥以SBE液的含药血清HPLC指纹图谱,优选出兔的最佳采血时间为1.5h；给药剂量为7.0g·Kg-1(生药·体重-1)；3种方法提取液及其含药血清的HPLC指纹图谱比较研究结果表明,以SBE组色谱中特征峰峰数最多,特征峰总面积最大,共有峰重叠率最高；⑦3种方法提取液的主要药效试验研究结果表明,SBE液的活血调经、止痛、抗炎作用的效果最强；该试验结果与用多指标综合评判的提取工艺优选结果及指纹图谱的比较研究结果相吻合；最大耐受量试验动物无一死亡,3种方法提取液的毒理学研究结果无显著性差异；⑧3种方法提取液含药血清对大鼠离体子宫平滑肌的收缩频率、收缩张力和收缩幅度均有抑制作用；与对照组比较,均有显著性差异(P<0.05)；与SBE组相比,AE组、WE组亦有显著性差异(P<0.05),其中SBE液的抑制作用最强；⑨综合评判结果表明,慈航丹方药的3种提取方法中,以SBE法为最佳。
结论：慈航丹方药的提取,以先将含挥发性成分的中药用SFE-CO2萃取,其药渣与方中益母草混合,再用SBE法提取,经离心-膜滤法制得分子量≤1000Da提取物,其多指标综合评判效果最佳；SBE法分子量≤1000Da提取液及其含药血清与其他提取方法(AE法、WE法)分子量≤1000Da提取液作相应比较,以SBE法提取液的药效物质含量高,药理作用最强。因此,慈航丹方药以SBE法提取为佳。
本研究将化学成分(盐酸水苏碱、盐酸川芎嗪、阿魏酸、总糖)及分子量≤1000Da提取物、主要药效、分子量≤1000Da提取液的指纹图谱及其含药血清指纹图谱有机结合起来,对慈航丹方药进行了SBE法系统的研究,进一步证明了该法研究设计的科学性、合理性,丰富和完善了“用灰思维方式建立中药半仿生提取法”的基本模式。以分子量≤1000Da提取物量为最终产物,能体现中药整体、综合、客观、模糊的特点,为选用现代药物剂型提供了技术支撑,拓展了中药现代化研究的思路与方法。
Objective:Taking Cihang dan prescription as an example to probe into the basic mode on extraction of available substances through the final product of molecular weight≤1000 Da contraction of traditional Chinese medicines compound containing volatile ingredients based on the composite evaluate indexes by comprehensive evaluation extracted by semi-bionice extraction (SBE) method.
Methods:Fistly, distinguishing the source、the character and definiting the contents identification of the prepared slices of Chinese crude drugs respectively according to Chinese pharmacopeiachp of 2005; only if they are fit for the pharmacopoeia quality standard, they can be used in the experiment. Secondly, extracting the volatile ingredients of Rhizoma Chuanxiong、Rhizoma Cyperi、Radix Angelicae Sinensis by supercritical carbon dioxide extraction (SFE-CO2) through U7(75) homogeneous design based on the same conditions of medicine、equipment and technical parameter and taking ligustilid、chuanxionggzine hydrochlorid、α-Cyperone and toal yield as evaluating indexes to optimize four operational variables of SFE-CO2 (extraction pressure、extraction temperature、desorb temperature and extraction time); the total productivity of SFE-CO2 extraction was stored in other container. Thirdly, getting the dregs of three medicinal material extracted by SFE-CO2 and Herba Leonuri together and extracted by SBE through U9(91×33) homogeneous design based on adding all the indexes of the productivity of Chuanxionggzine hydrochlorid、Ferulic acid、Stachydrine Hydrochoride、D-Glucose anhydrous and molecular weight≤1000 Da dry extraction processed by standardized, comparing the composite evaluate indexes of each method by weighted summ and finally choosing the best method of the preparation of molecular weight≤1000 Da contraction. Fourthly, Optimizing the best consistency of ethanol(30%-80%) for extracting the prescription by proportional subsection method through the same method as mentioned above to ge the final product of molecular weight≤1000 Da contraction.Fifthly, extracting the Cihang dan prescription by SBE、by water and eighty percent of ethanol respectively, got the there contractions of molecular weight≤1000 Da and optimized the best method by comparing the composite evaluate indexes through the same deal methods.Sixth, as the matter of the same component not mean the same effect, comparing the results of studying on the main pharmacodynamics and the acute toxicity test including the effects of dysmenorrhea、regulate menstruation、neuroleptanalgesia and the toxicology.Seventh, comparing the results of High Performance Liquid Chromatography (HPLC)fingerprint of Cihang dan prescription extracted by three methd through the contractions of molecular weight≤1000 Da and the serun containing drugs of rabbits.Eighth, comparing the effects of serun containing drugs of molecular weight≤1000 Da of rabbits on the smooth muscle of isotated uterine in the rats of Cihang dan prescription extracted by three methods.Finally, Optimizing the best conditions of Cihang dan prescription extracted by three methods by composite evaluate indexes of each method by weighted sum.
Results:①All the prepared slices of Chinese crude drugs of Cihang dan prescription were fit for the pharmacopoeia quality standard of 2005 respectively and can be used in the experiment.(2)Combinating manufacturing fact, the optimal extracting procedure was as follows:the extraction pressure is 30Mpa; the extraction temperature is 48℃, the desorb temperature I is 60℃, the desorb temperature II is 30℃and the extracting time is 4.0h.③The best conditions of Cihang dan prescription is that the pH value of three extractions is 6.0、7.5、8.8 respectively and the extraction time is2.0h、1.5h、1.5h in turn.④The best consistency of ethanol for extracting the prescription is 80 percent.⑤The composite evaluate indexes of Y of each method by weighted sum of CihangDan prescription extracted by three methods is as follows:YSBE>YAE>YWE.⑥Haven had optimized the best sampling blood time of the rabbits was 1.5h by the HPLC fingerprints of the serun containing drugs of rabbits of Cihang dan prescription extracted by SBE and the dosage of the rabbits was 7.0g·Kg-1 (crude drug-body weight-1).Facts proved that the characteristic peak numbers and the total areas of SBE group both were the most by comparing the results of the HPLC fingerprints of the serun containing drugs of rabbits and three contractions of Cihang dan.⑦The results showed that the resultant effects of promoting blood circulation to restore menstrual flow、acesodyne and anti-inflammatory of SBE extraction is the best through the experiments of elementary pharmacodynamics and the results were in agreement with that of comprehensive evaluating by multi-indexed process and the research result of HPLC fingerprints of Cihang dan prescription extracted by three methods, but the research result of toxicology of the three extractions were not statistical significance differentially, none of the rats was dead in the test.⑧Comparing to the control group, all the serun containing drugs of the extractions of the Cihang dan prescription extracted by three methods can significantly reduce the contraction of the amplitude、the frequency and the tension of the rats'in vitro uterine smooth muscle (P<0.05); Comparing to the SBE groups, the AE group and the WE group both were statistical significantly differentially(P<0.05), and of which the SBE groups produced the greatest effect (P<0.05).⑨It proved that the method of SBE is the best one of three methods through composite judgement.
Conclusion:The best way to extract the medicinal composition of Cihang dan prescription as follows:firstly extracting herbal pieces with volatile components(Rhizoma、Chuanxiong、Rhizoma Cyperi、Radix Angelicae Sinensis)by SFE-CO2, then blending dregs of decoction Herba Leonuri and extracted by SBE method and got the molecular weight≤1000 Da contraction through "centrifugate-membrane filtration method".It proved that the comprehensive components value of 3 kinds of extraction solution(SBE、AE、WE)consistent with comprehensive pharmacodynamics value.Of the three extractions of the Cihang dan prescription extracted by there methods, the content of pharmacodynamic substances of SBE extraction was the highest and the effect of the pharmacological action of SBE extractions was the strongest, among the three extracting methods, the SBE method was the best.
In the study, all the indexes of the chemical composition、the productivity of the (molecular weight≤1000Da) dry extraction、the elementary pharmacodynamics、the HPLC fingerprints of the extractions of the Cihang dan prescription and the results of the HPLC fingerprints of the serun containing drugs of rabbits of the three extractions of Cihang dan prescription extracted by there methods were organically combined together systematically.The study further richened and embodied the rationality and the scientificalness of the basic mode of establishing semi-bionic extraction method of Chinese medicine by gray thinking method.The study embodied the globality、synthesis objectivity and fuzzyness of Chinese medicine through the results of molecular weight≤1000 Da contractions.The study provides reference to other researches in choosing modern pharmaceutical dosage form and expanded the train of thought and method for the study of modernization of Chinese medicine.
方法：①首先对慈航丹方药进行基源、性状鉴定与质量鉴定研究；②将方中含挥发性药物(当归、川芎、香附)用超临界流体萃取,以均匀设计U7(75)表发布点试验,在饮片、设备、技术参数相同的基础上作SFE-CO2萃取,萃取物另器保存；以藁苯内酯、盐酸川芎嗪、α-香附酮、总萃取物为指标,综合评判,优选出方药超临界萃取(SFE-CO2)较佳工艺条件；③将超临界萃取后的药渣与方中益母草混合,用半仿生法提取,采用均匀设计U9(91×33)表发布点试验,以盐酸水苏碱、盐酸川芎嗪、阿魏酸、总糖及分子量≤1000Da提取物为指标,综合评判,优选出SBE法制备分子量≤1000Da提取物的条件；④在乙醇浓度30%-80%范围内,采用比例分割法,以上述相同的指标和评判方法,优选出方药醇提制备分子量≤1000Da提取物的较佳醇浓度；⑤以上述相同的指标和评判方法,作方药3种方法(SBE法、WE法、AE法)分子量≤1000Da提取物的多指标成分比较；⑥因化学等值不一定生物等效,故作方药3种方法分子量≤1000Da提取物的活血调经、止痛、抗炎等主要药效及毒性的研究比较；⑦对方药3种方法分子量≤1000Da提取物药液及其兔含药血清作HPLC指纹图谱的研究比较；⑧对方药3种方法分子量≤1000Da提取液的兔含药血清作大鼠离体子宫平滑肌作用的研究比较；⑨最后综合评判分析,优选出慈航丹方药的最佳提取方法和工艺条件。
结果：①方中所用的中药皆符合《中国药典》2005版一部各项下规定,均为合格的饮片；②优选出慈航丹方中含挥发性成分的中药超临界流体萃取的工艺条件为：萃取压力30MPa、萃取温度48℃、解析温度Ⅰ60℃、解析温度Ⅱ30℃、萃取时间4.0h。③优选出慈航丹方药的SBE法工艺条件为：3煎用水的pH依次为：6.0、7.5、8.8；提取时间依次为2.0h、1.5h、1.5h。④优选出醇提的最佳浓度为80%；⑤3种方法提取液的综合评判值的顺序为：YSBE>YAE>YWE;⑥以SBE液的含药血清HPLC指纹图谱,优选出兔的最佳采血时间为1.5h；给药剂量为7.0g·Kg-1(生药·体重-1)；3种方法提取液及其含药血清的HPLC指纹图谱比较研究结果表明,以SBE组色谱中特征峰峰数最多,特征峰总面积最大,共有峰重叠率最高；⑦3种方法提取液的主要药效试验研究结果表明,SBE液的活血调经、止痛、抗炎作用的效果最强；该试验结果与用多指标综合评判的提取工艺优选结果及指纹图谱的比较研究结果相吻合；最大耐受量试验动物无一死亡,3种方法提取液的毒理学研究结果无显著性差异；⑧3种方法提取液含药血清对大鼠离体子宫平滑肌的收缩频率、收缩张力和收缩幅度均有抑制作用；与对照组比较,均有显著性差异(P<0.05)；与SBE组相比,AE组、WE组亦有显著性差异(P<0.05),其中SBE液的抑制作用最强；⑨综合评判结果表明,慈航丹方药的3种提取方法中,以SBE法为最佳。
结论：慈航丹方药的提取,以先将含挥发性成分的中药用SFE-CO2萃取,其药渣与方中益母草混合,再用SBE法提取,经离心-膜滤法制得分子量≤1000Da提取物,其多指标综合评判效果最佳；SBE法分子量≤1000Da提取液及其含药血清与其他提取方法(AE法、WE法)分子量≤1000Da提取液作相应比较,以SBE法提取液的药效物质含量高,药理作用最强。因此,慈航丹方药以SBE法提取为佳。
本研究将化学成分(盐酸水苏碱、盐酸川芎嗪、阿魏酸、总糖)及分子量≤1000Da提取物、主要药效、分子量≤1000Da提取液的指纹图谱及其含药血清指纹图谱有机结合起来,对慈航丹方药进行了SBE法系统的研究,进一步证明了该法研究设计的科学性、合理性,丰富和完善了“用灰思维方式建立中药半仿生提取法”的基本模式。以分子量≤1000Da提取物量为最终产物,能体现中药整体、综合、客观、模糊的特点,为选用现代药物剂型提供了技术支撑,拓展了中药现代化研究的思路与方法。
Objective:Taking Cihang dan prescription as an example to probe into the basic mode on extraction of available substances through the final product of molecular weight≤1000 Da contraction of traditional Chinese medicines compound containing volatile ingredients based on the composite evaluate indexes by comprehensive evaluation extracted by semi-bionice extraction (SBE) method.
Methods:Fistly, distinguishing the source、the character and definiting the contents identification of the prepared slices of Chinese crude drugs respectively according to Chinese pharmacopeiachp of 2005; only if they are fit for the pharmacopoeia quality standard, they can be used in the experiment. Secondly, extracting the volatile ingredients of Rhizoma Chuanxiong、Rhizoma Cyperi、Radix Angelicae Sinensis by supercritical carbon dioxide extraction (SFE-CO2) through U7(75) homogeneous design based on the same conditions of medicine、equipment and technical parameter and taking ligustilid、chuanxionggzine hydrochlorid、α-Cyperone and toal yield as evaluating indexes to optimize four operational variables of SFE-CO2 (extraction pressure、extraction temperature、desorb temperature and extraction time); the total productivity of SFE-CO2 extraction was stored in other container. Thirdly, getting the dregs of three medicinal material extracted by SFE-CO2 and Herba Leonuri together and extracted by SBE through U9(91×33) homogeneous design based on adding all the indexes of the productivity of Chuanxionggzine hydrochlorid、Ferulic acid、Stachydrine Hydrochoride、D-Glucose anhydrous and molecular weight≤1000 Da dry extraction processed by standardized, comparing the composite evaluate indexes of each method by weighted summ and finally choosing the best method of the preparation of molecular weight≤1000 Da contraction. Fourthly, Optimizing the best consistency of ethanol(30%-80%) for extracting the prescription by proportional subsection method through the same method as mentioned above to ge the final product of molecular weight≤1000 Da contraction.Fifthly, extracting the Cihang dan prescription by SBE、by water and eighty percent of ethanol respectively, got the there contractions of molecular weight≤1000 Da and optimized the best method by comparing the composite evaluate indexes through the same deal methods.Sixth, as the matter of the same component not mean the same effect, comparing the results of studying on the main pharmacodynamics and the acute toxicity test including the effects of dysmenorrhea、regulate menstruation、neuroleptanalgesia and the toxicology.Seventh, comparing the results of High Performance Liquid Chromatography (HPLC)fingerprint of Cihang dan prescription extracted by three methd through the contractions of molecular weight≤1000 Da and the serun containing drugs of rabbits.Eighth, comparing the effects of serun containing drugs of molecular weight≤1000 Da of rabbits on the smooth muscle of isotated uterine in the rats of Cihang dan prescription extracted by three methods.Finally, Optimizing the best conditions of Cihang dan prescription extracted by three methods by composite evaluate indexes of each method by weighted sum.
Results:①All the prepared slices of Chinese crude drugs of Cihang dan prescription were fit for the pharmacopoeia quality standard of 2005 respectively and can be used in the experiment.(2)Combinating manufacturing fact, the optimal extracting procedure was as follows:the extraction pressure is 30Mpa; the extraction temperature is 48℃, the desorb temperature I is 60℃, the desorb temperature II is 30℃and the extracting time is 4.0h.③The best conditions of Cihang dan prescription is that the pH value of three extractions is 6.0、7.5、8.8 respectively and the extraction time is2.0h、1.5h、1.5h in turn.④The best consistency of ethanol for extracting the prescription is 80 percent.⑤The composite evaluate indexes of Y of each method by weighted sum of CihangDan prescription extracted by three methods is as follows:YSBE>YAE>YWE.⑥Haven had optimized the best sampling blood time of the rabbits was 1.5h by the HPLC fingerprints of the serun containing drugs of rabbits of Cihang dan prescription extracted by SBE and the dosage of the rabbits was 7.0g·Kg-1 (crude drug-body weight-1).Facts proved that the characteristic peak numbers and the total areas of SBE group both were the most by comparing the results of the HPLC fingerprints of the serun containing drugs of rabbits and three contractions of Cihang dan.⑦The results showed that the resultant effects of promoting blood circulation to restore menstrual flow、acesodyne and anti-inflammatory of SBE extraction is the best through the experiments of elementary pharmacodynamics and the results were in agreement with that of comprehensive evaluating by multi-indexed process and the research result of HPLC fingerprints of Cihang dan prescription extracted by three methods, but the research result of toxicology of the three extractions were not statistical significance differentially, none of the rats was dead in the test.⑧Comparing to the control group, all the serun containing drugs of the extractions of the Cihang dan prescription extracted by three methods can significantly reduce the contraction of the amplitude、the frequency and the tension of the rats'in vitro uterine smooth muscle (P<0.05); Comparing to the SBE groups, the AE group and the WE group both were statistical significantly differentially(P<0.05), and of which the SBE groups produced the greatest effect (P<0.05).⑨It proved that the method of SBE is the best one of three methods through composite judgement.
Conclusion:The best way to extract the medicinal composition of Cihang dan prescription as follows:firstly extracting herbal pieces with volatile components(Rhizoma、Chuanxiong、Rhizoma Cyperi、Radix Angelicae Sinensis)by SFE-CO2, then blending dregs of decoction Herba Leonuri and extracted by SBE method and got the molecular weight≤1000 Da contraction through "centrifugate-membrane filtration method".It proved that the comprehensive components value of 3 kinds of extraction solution(SBE、AE、WE)consistent with comprehensive pharmacodynamics value.Of the three extractions of the Cihang dan prescription extracted by there methods, the content of pharmacodynamic substances of SBE extraction was the highest and the effect of the pharmacological action of SBE extractions was the strongest, among the three extracting methods, the SBE method was the best.
In the study, all the indexes of the chemical composition、the productivity of the (molecular weight≤1000Da) dry extraction、the elementary pharmacodynamics、the HPLC fingerprints of the extractions of the Cihang dan prescription and the results of the HPLC fingerprints of the serun containing drugs of rabbits of the three extractions of Cihang dan prescription extracted by there methods were organically combined together systematically.The study further richened and embodied the rationality and the scientificalness of the basic mode of establishing semi-bionic extraction method of Chinese medicine by gray thinking method.The study embodied the globality、synthesis objectivity and fuzzyness of Chinese medicine through the results of molecular weight≤1000 Da contractions.The study provides reference to other researches in choosing modern pharmaceutical dosage form and expanded the train of thought and method for the study of modernization of Chinese medicine.
引文
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