上市品种“康复新液”的药学再评价

【作者】 吴红梅；

【导师】 杨明；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 消化性溃疡(peptic ulcer, PU)是常见病、多发病,积极防治尤为重要。康复新液为美洲大蠊Periplaneta americaana (L.)提取制成的单方制剂,临床上通过口服主要用于消化性溃疡的治疗。本文基于古代本草知识,从临床有效性出发,围绕康复新液生产工艺内外物质的抗消化性溃疡药效差异、工艺内药效物质基础及相关机理、质量评价,多方面、多学科结合、多层次开展上市品种“康复新液”药学再评价的系统研究,以期为康复新液临床使用的安全性、有效性及其“二次开发”提供理论依据和技术支撑。1、工艺再评价以实际生产工艺为核心,采用药效(大鼠幽门结扎型胃溃疡模型与水浸应激型胃溃疡模型)和化学成分分析相结合的方法,对康复新液现有工艺的科学合理性进行再评价。研究结果表明：现有工艺除去特殊腥臭气味的油脂不仅无防治消化性溃疡作用,反而加重溃疡的程度；工艺外未被提取出的粗多糖及蛋白质也未显示出抗消化性溃疡的活性；抗消化性溃疡活性成分仍集中于康复新液。从而初步阐明康复新液原料美洲大蠊发挥抗消化性溃疡的活性物质与油脂、多糖、大分子量的蛋白质无关,现有康复新液生产工艺具有一定的科学合理性。2、康复新液中间体各洗脱物的成分分析与药效评价根据康复新液中间体成分疏水性强弱的不同,采用大孔吸附树脂对其进行梯度洗脱,分离制得水洗脱物、25%乙醇洗脱物、50%乙醇洗脱物及75%乙醇洗脱物四个部位,结合化学与药效(水应激型胃溃疡模型)实验对康复新液中间体各洗脱物进行活性追踪及成分分析。结果：康复新液中间体经分离后,水洗脱物不仅未发挥防治消化性溃疡作用,反而加重溃疡的程度,提示水洗脱物中的氨基酸类、糖类、复合核苷碱基类成分及表皮生长因子与抗消化性溃疡作用关系不大；25%乙醇洗脱物有防治消化性溃疡趋势；50%乙醇洗脱物有较好的防治溃疡作用；75%乙醇洗脱物防治溃疡的药效最显著,且药效较纯化之前更佳,表明康复新液抗消化性溃疡的活性物质主要集中在75%乙醇洗脱物中,且多肽类成分纯度高达90%,从而提示康复新液抗消化性溃疡作用主要与多肽类成分有关。3、药效机制采用酶联免疫法(Elisa法)检测血清中5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)的含量;采用免疫组织化学技术检测胃组织中热休克蛋白(HSPs). H+/K+-ATP酶、前列腺素(PGE2)、表皮生长因子(EGF)的数量,探索康复新液及各洗脱物抗溃疡作用的机制。结果：在应激性溃疡中,HSPs及EGF未体现保护胃组织作用。水洗脱物不仅不能抗溃疡作用,反而增加溃疡面的形成并有炎症发生,与未能抑制H+/K+-ATP酶活性并刺激PGE2炎症介质的释放有关。康复新液抗溃疡作用是通过抑制5-HT、NE的释放及H+/K+-ATP酶活性并通过抑制PGE2的合成有关；25%乙醇洗脱物有抗溃疡作用趋势,与其能够抑制5-HT、NE等有关；50%乙醇洗脱物抗溃疡效果较稳定,与其能够抑制5-HT、NE分泌及对PGE2的抑制作用相对较强有关；75%乙醇洗脱物抗溃疡作用最为显著并有明显的抗炎作用,除了与该部位能够抑制5-HT、NE的分泌有关外,更与其能够显著抑制H+/K+-ATP酶活性而抑制胃酸分泌,及抑制PGE2的合成减少炎症发生和促进DA含量升高等综合因素发挥明显的抗溃疡作用。同时各洗脱物抗溃疡的药效研究结果具有可重复性。4、康复新液中间体75%乙醇洗脱物的成分结构鉴定研究利用气质联用仪对75%乙醇洗脱物进行成分分析,研究结果提示除油脂是康复新液生产过程及制备75%乙醇洗脱物的关键环节,除油脂不完全,将会残留相对百分含量较高的具有细胞毒作用的棕榈酸;同时大孔树脂中的有毒物质未残留于75%乙醇洗脱物中，可安全使用。采用基质辅助激光解析／离子化-飞行时间质谱(MALDI-TOF-MS)测定75%乙醇洗脱物肽类成分的分子量,结果分子量分布在500-3300之间,m/z520.34响应最高为主要物质,另外还存在m／z1348.7432,m/Z1943.0382等多种含量较少的多肽成分。5、康复新液及其原料药材的质量再评价模式采用HPLC方法,建立了前处理简单、信息量丰富的康复新液及原料药美洲大蠊药材的指纹图谱,并对约材、制剂进行整体性评价,结果：来源于科伦药业及荷花池市场的美洲大蠊约材与云南各基地生产美洲大蠊药材指纹图谱有一定差异;美洲大蠊约材与黑胸大蠊的特征峰有差异,可作为鉴别依据；A药业生产的10批康复新液除个别批号外,其他均较为均一、稳定。在建立药材及制剂指纹图谱的基础上结合药效研究结果,将具有加重溃疡的水洗脱物的指纹图谱与10批美洲大蠊药材及康复新液指纹图谱的共有模式进行比较分析,得出水洗脱物与药材及制剂共有峰,将这些共有峰剔除后再次对药材及制剂HPLC指纹图谱进行相似度评价及共有峰的聚类分析,并以本文研究所用具有抗溃疡作用的科伦药业美洲大蠊药材及批号为M1204122康复新液为参照。结果显示,荷花池美洲大蠊单独归为一类,科伦药业美洲大蠊与云南各基地的药材归为一类,提示除来源于荷花池美洲大蠊药材外,其他药材在抗溃疡方面具有较好的一致性；10批A药业生产的康复新液虽归为2类,但批号为M1204122康复新液与其他批次间的相似度及相关系数非常高,表明A药业生产的康复新液抗溃疡作用较为稳定。采用电导率、表面张力、渗透压、粘度等参数对康复新液进行测定,从物理性质角度对康复新液进行再评价。研究结果表明,康复新液的质量也较为稳定。综上所述,康复新液抗消化性溃疡的物质基础及作用机理已较为明确,探讨了基于剔除无效部位成分峰的指纹图谱评价模式及理化参数表征的质量再评价模式,为质量评价提供了新思路。康复新液现有生产工艺具一定合理性且具有上升空间,对消化性溃疡的防治作用也可显著提升。为此,我们建议,在康复新液生产或二次开发过程中,尽可能除去油脂并监测棕榈酸的含量、并富集多肽类成分以充分保证临床的有效性及质量可控性。更多还原

【Abstract】 Peptic ulcer (peptic ulcer, PU) is a frequent disease and common disease. Kangfuxin liquid is the single preparation, made by Periplaneta americaana (L.) extract, oral administration for the treatment of peptic ulcer in clinical. In this thesis, based on ancient herbal medicine, departure from the clinical efficacy, around the Kangfuxin liquid’s substance inside and outside anti-peptic ulcer pharmacodynamic differences of the Production, combined material basis for efficacy in the process and related mechanisms, quality assessment, we carry out the listed species Kangfuxin liquid pharmaceutically re-evaluation system, for the clinical use of safety, efficacy, and its secondary development to provide a theoretical basis and technical support.1.The process reviewThe actual production process as the core, the efficacy (The rat pylorus ligated gastric ulcer model and Water immersion stress gastric ulcer model) combined with chemical analysis to scientific rationality re-evaluation of the existing process of Kangfuxin liquid. The results showed that:the existing process which removed special fishy smell of grease didn’t have the role of prevention and treatment of peptic ulcer, actually worsened the extent of the ulcer; the crude polysaccharide and protein had not been extracted in the process also did not show anti-ulcer activity; anti-peptic ulcer active ingredient was still concentrated in Kangfuxin liquid. Thus initially clarify the Kangfuxin liquid raw materials Periplaneta americaana (L.) active substance of anti-peptic ulcer had no relate to the fats, polysaccharides, and high molecular weight protein. So the existing Kangfuxin liquid production process has a certain scientific rationality.2.Element analysis and pharmacodynamic evaluation of each eluate of Kangfuxin liquid intermediateAccroding to Hydrophobic strength of different Kangfuxin liquid intermediate composition, the use of macroporous resin, gradient elution, Obtained water eluate,25%ethanol material,50%ethanol elution and75%ethanol eluate four parts, Combined with chemical and efficacy (Gastric ulcer model of water stress) experiment carried out by activity tracking and component analysis of each eluate of Kangfuxin liquid intermediates. Results:Kangfuxin liquid intermediates were separated and the water eluate did not play against peptic ulcer, actually worsened the extent of the ulcer, suggesting that the amino acids, carbohydrates, composite nucleotide bases constituents and epidermal growth factor in the water of eluate had little to do with anti-peptic ulcer effect;25%ethanol elution had trend to prevention and treatment of peptic ulcer; better prevention and treatment of ulcers in50%ethanol elution; efficacy of75%ethanol ulcer was the most significant, and better effective than before purification, showed that the active substance of the Kangfuxin liquid to anti-ulcer was mainly concentrated in75%ethanol elution, and the purity of the peptide component up to90%, suggesting that Kangfuxin solution’s anti-peptic ulcer effect is related to peptide component.3.Efficacy mechanismBy enzyme linked immunoassay (Elisa method) detected the content of5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) in serum; Immunohistochemical techniques detection the number of heat shock proteins (HSPs), H+/K+-ATPase enzyme, prostaglandin (PGE2), epidermal growth factor (EGF) in gastric tissue, we explored the antiulcer mechanism of Kangfuxin solution and its elution. Results:In the stress ulcer, HSPs and EGF did not reflect the protection role of gastric tissue. The water eluate didn’t have anti-ulcer effect and even increased the formation of ulcers and inflammation, caused by failing to inhibit H+/K+-ATPase activity and stimulate PGE2inflammatory mediators release. Kangfuxin liquid antiulcer’s action is based of the inhibition of5-HT, NE release and H+/K+-ATPase enzyme activity, and inhibition of PGE2synthesis;25%ethanol elution had a trend to have antiulcer effect, it is possible to suppress the5-HT, NE, and et al;50%ethanol elution was more stable in anti-ulcer effect, and it had a strong relate to the inhibition of5-HT, NE secretion and of PGE2synthesis;75%ethanol elution antiulcer action was most significant in anti-inflammatory effects, not only can inhibit the secretion of5-HT and NE, but also are able to significantly inhibit the H+/K+-ATPase enzyme activity and inhibition of gastric acid secretion, and inhibition of PGE2synthesis to reduce the occurrence of inflammation and promote DA elevated levels et al. These factors played a significant role in anti-ulcer. At the same time, each eluted material’s anti-ulcer efficacy had a good repeatability.4.Composition and structure identification of the Kangfuxin liquid intermediate75%ethanol elutionComponent of75%ethanol elution were analysised by GC-MS, the results suggested that remove of the grease in Kangfuxin liquid production process and the preparation of75%ethanol elution is the key link, and removing of grease incomplete will remain on the relative percentage higher levels of palmitic acid with cytotoxicity; toxic substances of macroporous resin do not comprise in the75%ethanol elution, so it can be used safely. Using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), results of the the determination of the molecular weight of the peptide component of the75%ethanol elution showed that the molecular weight distribution between500-3300substances. m/z520.34maximum response was the mainly substance. In addition, there existed some polypeptide component, such as m/z1348.7432, m/z1943.0382et al.5.The quality review mode of Kangfuxin liquid and its raw materialsHPLC methods was used to establish fingerprint of Kangfuxin liquid and its bulk drugs Periplaneta americana herbs, and the fingerprint pre-treatment is simple, informative, as well as omprehensive evaluate of the herbs and preparations. Results: there were differences among the Fingerprint of Periplaneta americaana (L.) from Columbine medicine, Lotus Pond Chinese herbal medicine market and production bases in Yunnan; the Periplaneta americaana (L.) herbs was different from Periplaneta fuliginosa at characteristic peaks, which can be used as a basis for identification;10batches of Kangfuxin solution of A pharmaceutical production were more uniform and stable except several batches.On the basis of establishment medicinal herbs and preparations fingerprint combined with efficacy findings, used the fingerprints of severe ulcers water elution from a comparative analysis of10batches of Periplaneta americaana (L.) herbs fingerprints and Kangfuxin liquid fingerprints Total mode, got total peaks of water eluate, herbs and preparations. After striking out the total peaks, the herbs and preparations’HPLC fingerprint similarity was evaluated and the total peak cluster was analysised, and used the Columbine medicine Periplaneta americaana (L.) herbs, having anti-ulcer effect, lot for M1204122, Kangfuxin liquid as a reference. The results showed that the Periplaneta americaana (L.) of Lotus Pond Chinese herbal medicine market grouped separately as a class, the Columbine medicine and production bases in Yunnan Periplaneta americaana (L.) herbs classified as a category, suggesting that besides the Periplaneta americaana (L.) comes from the Lotus Pond Chinese herbal medicine market, other herbs in the anti-ulcer had a good agreement;10batches of A Pharmaceutical production Kangfuxin liquid although divided into two categories, but the similarity and correlation coefficient of the batch M1204122Kangfixin liquid was very high with other batches, indicating that the A Pharmaceutical production Kangfuxin liquid anti-ulcer effect is more stable.We re-evaluate the Kangfuxin liquid from the perspective of the physical properties by measuring Kangfuxin liquid’s conductivity, surface tension, osmotic pressure, viscosity and other parameters,. The results showed that the quality of Kangfuxin liquid was also stable.In summary, the material basis and mechanism of the Kangfuxin liquid anti-peptic ulcer was more specific. Based on the evaluation model of exclude the invalid parts of the component peak of the fingerprint and the physical and the quality re-evaluation mode of chemical parameters characterize, We provided a new approach for quality evaluation. The existing production processes of Kangfuxin liquid had certain reasonable and room for growth, the role of prevention and treatment of peptic ulcer can also be significantly improved. For this reason, we suggested that Kangfuxin liquid production or secondary development process should remove grease as much as possible and monitor the content of palmitic acid, and enrich peptide ingredients to fully guarantee the effectiveness and quality of clinical controllability.更多还原