【作者】 李芳；

【导师】 孙秀梅；

【作者基本信息】 山东中医药大学 ， 中药学， 2009， 博士

【摘要】 目的:以桂枝茯苓丸方药为研究对象,采用多指标成分综合评价的方法,进一步探讨含挥发性成分的中药复方药效物质用“半仿生提取法（SBE法）”研究的基本思路与模式。方法:首先对桂枝茯苓丸方药进行文献综述和饮片质量鉴定研究。提取工艺研究时,先将方中含挥发性成分的中药（桂枝、牡丹皮、赤芍、桃仁）用超临界流体萃取,以均匀设计U₇（7⁴）表布点实验,桂皮醛、肉桂酸、丹皮酚、总萃取物为指标,综合评判,优选出方药超临界CO₂萃取（SFE-CO₂）的较佳工艺条件;再将超临界萃取后的药渣与方中剩余的茯苓混合,用SBE法提取,以U₉（9¹×3³）表布点实验,芍药苷、肉桂酸、苦杏仁苷、茯苓酸、总多糖、干浸膏为指标,综合评判,优选出SBE法较佳工艺条件。在优选出该方药SFE-CO₂法和SBE法较佳提取工艺的基础上,用比例分割法优选SBE液和WE液（水提液）较佳醇沉浓度,以及该方药醇（AE）提的较佳浓度;在此基础上,对SBE法、WE法、SBAE法（半仿生提取醇沉法）、WAE法（水提醇沉法）、AE法（醇提法）的5种提取液进行了多指标成分、HPLC指纹图谱比较。因为化学等值不一定生物等效。又对5种提取液作活血化瘀、抗实验性痛经、抗炎、增强免疫和改善大鼠乳腺增生作用的主要药效学及急性毒性比较。同时对5种含药血清进行了HPLC指纹图谱和对缩宫素所致大鼠离体子宫平滑肌活动影响作用的比较研究。最后综合评判,优选出桂枝茯苓丸方药的较佳提取工艺条件。在提取工艺研究的基础上,进行了桂枝茯苓丸的剂型改革研究,确定了桂枝茯苓软胶囊的制备工艺,制定了质量标准（草案）。结果:（1）优选出的桂枝茯苓丸方药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36℃、分离温度32℃、萃取时间3.5h。（2）优选出桂枝茯苓丸方药的SBE法工艺条件为:3煎用水pH依次为5.0、7.5、8.0;提取时间依次为2.0h、1.5h、1.5h。（3）SBE液醇沉较佳浓度为60%。（4）WE液醇沉较佳浓度为70%。（5）醇提较佳浓度为70%。（6）5种方法提取液（SBE、SBAE、WE、WAE、AE）指标成分的综合评判值顺序为:Y_SBE＞Y_AE＞Y_WE＞Y_SBAE＞Y_WAE。（7）5种方法提取液指纹图谱相对应的特征峰总面积以SBE液最大。（8）5种方法提取液血清指纹图谱以SBE液含药血清色谱峰特征峰总面积最大,共有峰重叠率最高。（9）5种方法提取液主要药效学综合评价结果以SBE液最佳,最大耐受量试验动物无一死亡。（10）5种方法提取液及含药血清对大鼠离体子宫平滑肌活动试验表明,SBE液及其含药血清综合评价值最大。（11）确定了桂枝茯苓软胶囊的最佳成型工艺,制订了桂枝茯苓软胶囊的质量标准,以方便有效地控制其内在质量。结论:桂枝茯苓丸方药提取,以先将含挥发性成分的中药（桂枝、牡丹皮、赤芍、桃仁）用SFE-CO₂萃取,其药渣与方中剩余的茯苓混合,再用SBE法提取为佳。SBE法提取液及其含药血清与其他提取方法（WE法、SBAE法、WAE法、AE法）作相应比较,药效物质含量高,药理作用强。本研究再一次验证了灰思维方式指导下的SBE法研究设计是科学、合理和先进的,丰富和完善了“用灰思维方式建立中药半仿生提取法”的基本研究模式,拓展了中药研究开发的思路,并为其他含挥发性成分中药复方药效物质的提取及二次开发提供了有益的借鉴,也为快速提高中药现代化水平探索了一条新途径。更多还原

【Abstract】 Objective:Take Guizhi Fuling Pills Decoction as an example to probe into the basic mode on extraction of available substances in Chinese compound medicine that contain volatile ingredients by semi-bionic extraction（SBE）.Methods:Firstly,Guizhi Fuling Pills Decoction was studied on literature,the source and contents identification of cut crude drugs.Of extraction study,First,Using U₇（7⁴）homogeneous design and taking cinnamaldehyde,cinnamic acid,paeonol and total CO₂ extract yield as evaluating indexes to optimize SFE-CO₂ technical conditions for extracting volatile ingredients of Guizhi Fuling Pills Decoction,Then,mixed dregs of medicinal material extracted by SFE-CO₂ with other medicinal material,using U₉（9¹×3³） homogeneous design form and taking Paeoniflorin,Cinnamic acid,Laetrile,Pachymic acid, total polysaccharose and dry extract as indexes,the result was comprehensive evaluated to optimize SBE technical conditions.Next,basede on the optimized condtions,using proportional method to optimize the alcohol precipitation concentration of SBE extracting solution and WE extracting solution and alcohol extraction concentration.Last,the 5 kinds of extracting solution（SBE、WE、SBAE、WAE、AE） were comprehensive compared through the multi-indexes ingredients and HPLC fingerprints chromatograms.Because the same components content not mean the same biologic effects,so we contrasted 5 kinds of extracting solution on effect of hemorrheology of the acute blood stasis rats model,effect of dysmenorrheal rats induced by drug,anti-inflammatory action, immuneomodulation effect and Cyclomastopathy rats model.And acute toxicity was compared.Simultaneously,compare the 5 kinds of solution and medicated serum on contractility of isolated rat uterus in the presense of oxytocine.Finally judged them through comprehensively evaluation and determined the best technique method and technique parameters of Guizhi Fulingpills Decoction.According to the former extraction study,optimizing the shaping technology of Guizhi Fuling soft capsules and establishing quality standard.Results:（1）The optimized SFE-CO₂ extraction conditions are the following:the extraction pressure was 30 MPa;the extraction temperature was 36℃,the separation temperature was 32℃and the extraction time was 3.5h.（2）the optimized SBE extraction conditions are the following:pH values of the water for three extraction is 5.0、7.5、8.0,and extraction time is 2.0h、1.5h、1.5h in turn.（3）the optimized alcohol precipitation concentration for SBE solution is 60%.（4）the optimized alcohol precipitation concentration for WE solution is 70%.（5）the optimized alcohol concentration is 70%. （6）the order of comprehensive evaluated values of the 5 kinds of extracting solution is Y_SBE＞Y_AE＞Y_WE＞Y_SBAE＞Y_WAE.（7）SBE solution has the most total area of corresponding characteristic peaks in the fingerprint chromatograms of 5 kinds of solution.（8）in the serum fingerprint chromatograms of 5 kinds of solution,the SBE solution has the most peak number and total area.（9）Main pharmacodynamic activities test results indicate that the comprehensive value of SBE extract is the largest among extracts by 5 methods and no animals died in maximum tolerated dose experiment.（10）The 5 kinds of solution and their medicated serum could certain degree inhibit the contractility of the isolated uteri,and SBE solution and it’s medicated serum has the max comprehensive value.（11）Optimizing the shaping technology of Guizhi Fuling soft capsules and establishing a quality standard for Guizhi Fuling soft capsules so as to control its quality efficiently and effectively.Conclusion:The best way to prepare Guizhi Fuling Pills Decoction is as follows,first, extracting Herbal pieces With volatile components（Ramulus Cinnamomi,Cortex Moutan, Radix Paeoniae Rubra and Semen Persicae）by SFE-CO₂,then mix dregs with other medicinal materials（Poria Cocos）,and extracted by SBE.Compared with WE,SBAE, WAE and AE method,Guizhi Fuling Pills Decoction extracted by SBE possesses more contents of chemical compositions and better pharmacodynamic functions.This study verified again the scientificity,rationality and advancement of the semi-bionic extraction patterns for compound which based on the gray thinking mode.Provides reference to other researches of valuable substances containing volatile components in traditional Chinese medicine and twice exploitation of chinese herbs.In the meantime,it may open a new avenue for the rapid growth of Modernization of Traditional Chinese Medicine.更多还原