【作者】 李林福；

【导师】 刘振丽；

【作者基本信息】 中国中医科学院 ， 中药学， 2008， 硕士

【摘要】 和其他国家的草药、天然药物相比,中药的一个显著特色是炮制后才能用于临床。古人云:“制药如练兵,率未练之兵不能克敌制胜,用未制之药难得药到病除”,由此可知,炮制对中药的重要性可见一斑。然而,炮制之所以对药物的药性、功效影响如此之大,究其根本是炮制对其内在物质基础的改变。研究中药炮制前后化学成分组成的变化,对阐明中药炮制的原理和制订质量标准具有重要意义。何首乌为蓼科植物何首乌Polygonum multiflorum Thunb.的干燥根茎。生何首乌具有解毒、消痈、润肠通便之功效,用于瘰疬疮痈、风疹瘙痒、肠燥便秘、高血脂。制何首乌为何首乌的炮制加工品,具有补肝肾、益精血、乌须发、强筋骨的功效,用于血虚萎黄、眩晕耳鸣、须发早白、腰膝酸软、肢体麻木、崩漏带下、久疟体虚、高血脂。为阐明中药何首乌炮制前后功效的改变,制订科学合理的制何首乌的质量评价方法,本文对制何首乌的化学成分进行了提取分离,在此基础上建立了区别何首乌与制何首乌的薄层层析鉴别方法,研究了制何首乌的HPLC特征图谱分析方法及与主要有效成分含量的关系,就不同炮制工艺和方法对何首乌中主要有效成分含量的影响进行了研究。1文献综述综合评价分析了何首乌在本草学、炮制学、质量控制、化学成分、药理学、临床应用等相关领域的研究文献,为制定切实可行的研究方案,确定研究考察指标及研究工作起点奠定了基础。2实验研究2.1制何首乌化学成分研究采用50%乙醇对制何首乌进行提取,提取液减压浓缩,加水混匀,依次用乙酸乙酯、正丁醇进行提取。乙酸乙酯提取物经过柱层层析分离,得到10个化合物。通过颜色反应、熔点测定以及光谱数据(UV、MS、¹H-NMR、¹³C-NMR)的测定确定了其结构。10个化合物分别为大黄素（emodin）、大黄素甲醚（physcion）、丁二酸（succinic acid）、5-羟甲基糠醛（5-hydroxymethylfurfural）、对羟基苯甲醛（4-Hydroxybenzaldehyde）、没食酸（garllicacid）、对苯二酚（hydroquinone）、2,3,5,4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside）、大黄素-8-O-β-D-葡萄糖苷（emodin-8-O-β-D-glucoside）、大黄素甲醚-8-O-β-D-葡萄糖苷（physcion-8-O-β-D-glucoside）。其中丁二酸（succinicacid）和5-羟甲基糠醛（5-hydroxymethylfurfural）为首次在制何首乌中发现。2.2制何首乌质量标准研究2.2.1建立了区别何首乌生品与炮制品的薄层层析鉴别方法目前对何首乌生品与炮制品采用相同的薄层层析鉴别方法。本文在制何首乌化学成分研究基础上,选择分离得到的5-羟甲基糠醛为对照品,采用硅胶GF₂₅₄薄层板,以石油醚（60～90℃）—乙酸乙酯（1∶1）为展开剂,展开,取出,晾干,紫外光灯（254 nm）下检视,制何首乌色谱中,在与对照品色谱相应的位置上显相同颜色的斑点,何首乌色谱在相应的位置上没有显相同颜色的斑点。2.2.2制何首乌HPLC特征图谱研究在对15个批次制何首乌饮片研究的基础上,建立了制何首乌高效液相特征图谱分析方法,最多检出峰18个。结合主要有效成分2,3,5,4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（简称:二苯乙烯苷）的含量测定结果,显示特征峰的检出率与含量之间呈现正相关,二苯乙烯苷含量高,则特征峰检出率高。符合药典标准的制何首乌样品特征峰检出率在88%以上,共有的色谱峰16个。2.2.3制何首乌中二苯乙烯苷含量测定对不同地区采购的15个批次的制何首乌中二苯乙烯苷的含量进行了测定,研究结果表明制何首乌中二苯乙烯苷含量为0.127～4.15%,表明不同地区样品之间存在很大差异,有些不符合《中华人民共和国药典》2005年版一部制何首乌中二苯乙烯苷含量不得低于0.70%的规定。2.3炮制对何首乌中二苯乙烯苷含量的影响针对全国不同省市何首乌炮制工艺不统一,缺乏规范的炮制工艺参数的现状,研究了不同炮制工艺即豆汁蒸、豆汁炖、清蒸、豆汁高压蒸和豆汁屉上蒸以及不同炮制时间对何首乌中主要有效成分二苯乙烯苷含量的影响。结果显示二苯乙烯苷含量:生片＞豆汁高压蒸片＞豆汁炖片＞豆汁蒸片＞清蒸片＞豆汁屉上蒸片。何首乌采用黑豆汁炖,随炮制时间的延长,二苯乙烯苷含量逐渐下降。更多还原

【Abstract】 In comparison with the herbs and natural medicines of other countries,an outstanding characteristic of Chinese materia medica was that Chinese materia medica must be processed before used for clinic.The ancients said,"Processing is as much as training,leading the untrained soldiers cannot overcome the enermy,using the unprocessed drugs is hard to cure the diseases",therefore,the processing is very important to Chinese materia medica.But the basic reason that the processing has great affect to the drug property and efficacy was the change of those internal substances after processed.Study the chemical constituents’ changes of before and after processing have important significance to clarify the processing principle of Chinese materia medica and to formulate the quality standard.Heshouwu,the dried root of Polygonum multiflorum Thunb.The crude have the efficacy of detoxication,eliminating carbuncle and loosening bowel to relieve constipation,and used to treat scrofula,sores,carbuncle,rubella,pruritus,constipation and hyperlipidemia.Radix Polygoni Multiflori Preparata,the processed products of Radix Polygoni Multiflori,have the efficacy of reinforcing liver and kidney,tonifying blood and essence,blacken beard and hair, strengthen the tendons and bones,and used to cure hemopenia,etiolate,vertigo,tinnitus, beard and hair graying early,waist and knees lassitude,limb numbness,metrorrhagia and leucorrhea,chronic malaria and body deficiency,hyperlipidemia.This study want to clarify the change of Polygonum multiflorum Thunb.before and after processed and formulate the scientific reasonable qualify evaluation method of Radix Polygoni Multiflori Preparata by extracting and separating the chemical constituents of Radix Polygoni Multiflori Preparata. On this basis,the thin-layer chromatography（TLC） identification method for distinguishing the Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata was established,the HPLC fingerprint spectrum analysis method of Radix Polygoni Multiflori Preparata have been studied,and the main effective constituents contents in Polygonum multiflorum Thunb. which affected by different processed technology and method have been studied.1 Literature reviewThe research literatures of Polygonum multiflorum Thunb.in the fields of bencaology, preparation,quality control,chemical constituent,pharmacology and clinical application were summed up and analyzed.Based on this the feasible research scheme was formulated,the research investigation index and the researches originating point were determined.2 Experimental research2.1 Study on chemical constituents of Radix Polygoni Multiflori Preparata.Accordingto the modern research,the stilbene glucoside was the main active constitute of Polygonum multiflorum Thunb.,but not the only active constitute,the research shows that its content will decrease after processing.In this experiment,the roots of P.multiflorum were extracted with EtOH,and the EtOH solution obtained evaporated to dryness.The EtOH extract was suspended in water,partitioned successively with,with the resulting EtOAc and n-BuOH solutions evaporated to dryness in vacuo.Ten compounds were isolated by chromatographic fractionation in the EtOAc and n-BuOH extract,and identified by UV,MS,¹H-NMR, ¹³C-NMR.The ten compounds were emodin,physcion,succinie acid,5-hydroxymethyl furfural,4-Hydroxybenzaldehyde,garlic acid,hydroquinone,2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside, emodin-8-O-β-D-glucoside,physcion-8-O-β-D-glucoside.The succinic acid and 5-hydroxymethylfurfural were the first found in Radix Polygoni Multiflori Preparata.2.2 Study on quality standard of Radix Polygoni Multiflori Preparata.2.2.1 the thin-layer chromatography（TLC） identification method for distinguishing the Radix Polygoni Multiflori and Radix Polygoni Multiflori Preparata was established.Now the crude and processed product of Polygonum multiflorum Thunb.were identified by the same TLC method.Based on the study of chemical constitutes in Radix Polygoni Multiflori Preparata,this study choose 5-HMF which was the new product after processing as the reference substance,adopt the silica GF254 thin layer plate with the developing solvent of petroleum benzene（60℃-90℃）-EtOAc（1:1）,and detected by UV detection at 254 nm,the same color stains were detected on the spectrum of both Radix Polygoni Multiflori Preparata and reference substance,the same color stain was not found in Radix Polygoni Multiflori.2.2.2 Study on the HPLC fingerprint spectrum of Radix Polygoni Multiflori Preparata. Based on the study of 15 Radix Polygoni Multiflori Preparata,the HPLC fingerprint spectrum analysis method of Radix Polygoni Multiflori Preparata was established.2.2.3 Assay of the 2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside,emodin-8-O-β-D-glucoside, physcion-8-O-β-D-glucoside in Radix Polygoni Multiflori Preparata.The contents of stilbene glucoside in ten groups Radix Polygoni Multiflori Preparata buying from different places were determined,the results show that the content of stilbene glucoside in was 0.127～4.150%,and there have a great difference between the different place sample,even some not meet the pharmacopoeia of people’s republic of chinesestandard that the content of stilbene glucoside in Radix Polygoni Multiflori Preparata can’t below 0.70%.2.3 Effect of processing on the main chemical constituents in Polygonum multiflorum Thunb..Aiming at current situation that the disunity of processing technology in different city and the lack of processing technology parameter standard,we study the effect of different processing technology such as black bean sauce steaming,black bean sauce stewing,water stewing,black bean sauce steaming at high pressure,black bean sauce stewing up the lattice and different processing time on the main chemical constituent stilbene glucoside in Polygonum multiflorum Thunb..The results show the contents of stilbene glucoside as: crude＞black bean sauce steaming at high pressure＞black bean sauce stewing＞black bean sauce steaming＞water stewing＞black bean sauce stewing up the lattice.In the processing of black bean sauce stewing,the content of stilbene glucoside in Polygonurn multiflorum Thunb. decreased with processing time.更多还原