肠激安胶囊制剂学及干预腹泻型肠易激综合征机理研究
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摘要
肠易激综合征(irritable bowel syndrome，IBS)是一种常见的慢性非器质性肠功能紊乱性疾病，以反复发作的腹痛、腹泻、便秘或腹泻与便秘交替出现为主要症状，缺乏病理形态学改变及生化学指标异常改变依据，因此将该病归入功能性肠病(functional bowel disease，FBD)的范畴，以往IBS被认为是由于肠功能紊乱和痛感异常所引起，但近年来许多研究显示IBS与内脏的高敏感性有关，一般认为该病是一种具个体差异性的多病因、异质性疾病。
IBS是消化道常见的功能性疾病，约占胃肠道疾病的30％～50％，临床以腹泻型IBS为常见，患者常因此就诊。近年来，国内外大量文献报道心理因素与该病的发病密切相关，患者常伴有焦虑、忧郁等精神和心理异常，这可能涉及到中枢神经系统对肠神经系统的调整失常，胃肠激素的变化以及局部神经、肌肉各种受体的改变，脑—肠轴的存在等多种因素。目前西医治疗IBS主要以止痛剂、止泻剂、止痉剂、5-HT_3受体拮抗剂等药物(如易蒙停，阿托品等)对症治疗，再配以调整肠道菌群和植物神经功能紊乱或肠道黏膜保护剂的药物(如谷维素、丽珠肠乐、培菲康、思密达等)，这些药物对症状的缓解有效，但远期疗效尚不理想，且毒副作用难以接受。
中医在治疗胃肠疾病方面积累了几千年的丰富经验和大量有效方剂，因此，中医药治疗IBS方面有其优势，疗效肯定，副作用小，越来越受到国内外市场重视。祖国医学认为，肝主疏泄，调畅气机，而机体情绪的变化与气血的运行密切相关，研究表明，肝主疏泄的功能在机体心理应激中起着决定性的作用，肝是机体调节心理应激反应的核心。动物实验表明，调肝方药对束缚应激所引起的神经内分泌免疫功能紊乱具有一定的调节作用。腹泻的发生在中医学中则主要是脾失健运，水湿不化的结果，因此，肝脾两脏功能失调在腹泻型肠易激综合征发病过程中的重要作用是肯定的，从肝脾两脏对腹泻型肠易激综合征加以治疗，疗效也是肯定的。临床常用的治疗方剂及治疗方法有三白散、补脾益肠丸、益母草煎剂、健脾调气汤、针刺疗法、艾灸疗法、保留灌肠、穴位注射、耳穴贴压、按摩等，中医中药已经在IBS方面进行了有效的尝试，在治疗方面积累了丰富的经验。
医院制剂由于其可靠的临床背景，历来是中药新药开发的摇篮，许多著名的新药如复方丹参滴丸、通络生骨胶囊等都出自医院制剂。肠炎灵片作为医院制剂在广州中医药大学第一附属医院临床使用十多年，主要用于治疗腹泻型IBS、慢性结肠炎、腹痛腹泻等症，临床疗效满意。但由于基础研究工作缺乏，制剂工艺落后、质量标准不完善，未能进行有效开发。
肠激安胶囊由我院传统制剂肠炎灵片经改良研制而成，具有健脾疏肝，清热祛湿、涩肠止泻的功效，组方以四君子汤合痛泻要方加味组成，以党参、白术、茯苓健脾渗湿为君药，防风、延胡索、木香、陈皮、白芍疏肝理气醒脾为臣药，佐以黄连、火炭母、救必应、石榴皮清利湿热，邪去则土安，甘草和中佐白芍以柔肝缓急，全方具健脾疏肝、清利湿热、涩肠止泻之效。预实验提示，其挥发油可能通过抑制组织胺的兴奋性而发挥作用；对新斯的明引起的肠推进亢进具有拮抗作用，以及拮抗小鼠的痉挛疼痛反应，较好地解释了该制剂在临床上明显缓解患者腹泻、腹痛的功效，临床及动物实验结果更显示了新药开发的可行性。
要研制安全有效、稳定可控的新制剂，成熟先进的提取分离技术不可或缺。超临界二氧化碳萃取技术(SFE-CO_2)是目前最先进的物理萃取技术，可以广泛应用于中草药领域，并具有萃取效率高、生产周期短、有效成分不破坏、不残留有机溶剂、工艺简便、节约劳动力、减少三废污染等优越性，特别适用于含挥发性成分中药材的提取。
腹泻型IBS目前尚无公认的动物模型。直肠注射醋酸造模的方法属于外周致敏IBS动物模型，是基于重复的伤害性刺激或者持续的伤害性刺激都可以导致疼痛的高敏感性。临床研究也发现IBS患者在其患病前往往有急性胃肠道感染的病史，炎症性肠病患者在缓解期产生肠易激的症状。选择大鼠新生期的刺激是由于新生期的大鼠神经系统处于发育期，比较脆弱和敏感，易受造型变化的影响。表现在：①缺乏有效的背角神经元下行抑制系统；②新生脊髓比成年后更易兴奋。如果在这一时期，动物经受持续有害刺激可以引起神经痛觉传导通路的长时期的过敏，在成年后，尽管当初的有害刺激早已消除，肠道始终处于过敏状态。从这一点看乳鼠的新生期肠道内的慢性炎症刺激，可以在成年后引起慢性内脏敏感性增高，是一个较符合IBS的基本特征的模型，值得进一步尝试。
目前国内外对IBS的研究多集中在临床阶段，以患者为研究对象，个体差异大，缺乏对照和可比性，基于动物实验的基础研究较少。本课题从大鼠肠道敏感性、5-HT、肥大细胞、肥大细胞的脱颗粒状态等角度系统探讨肠激安胶囊的作用机理，较详细地阐述肠激安胶囊的作用途径和机理。
我国治疗肠道疾病的中药制剂有丸、散、片、胶囊、口服液等，大多依据传统工艺水提、醇提、水蒸气蒸馏提取挥发油，加热蒸发浓缩、热风循环干燥等，热敏性有效成分损失较多，批间差异大，质量和疗效不稳定，且绝大多数有效的方剂依然停留在医院制剂或个体给药的汤剂阶段，并未进行有效开发或形成产业化。采用现代制药技术，以新剂型、新工艺和稳定可控的质量标准对传统中药进行开发和改良，是中药现代化的必然趋势。
本项目采用SFE-CO_2技术提取分离复方中药的有效成分，以药理药效实验为评价指标，将疗效确切的医院制剂按照中药新药的技术要求进行研究和评价，并采用气相色谱—质谱(GC-MS)指纹图谱、高效液相色谱(HPLC)，薄层色谱(TLC)等多种检测方法跟踪和控制工艺参数，以安全有效、稳定可控的研究原则对肠激安胶囊进行临床前开发，对提高药物的稳定性，减少服用量和降低毒副作用，探讨肠激安胶囊的作用机理具有重要意义。
随着现代生活节奏的加快和生活方式及饮食结构的改变，IBS发病率有逐年上升的趋势，而至今为止未见同类中成药上市使用。肠激安胶囊根据腹泻型肠易激综合征的不同临床表现，结合中医病因病机，从肝脾两脏对腹泻型肠易激综合征加以综合治疗，组方合理，临床基础扎实，国内外市场前景广阔，具有良好的应用价值和开发前景。
○、肠激安胶囊生产工艺研究
1．肠激安胶囊药材来源及检验
目的：对肠激安胶囊原料药材进行来源和质量检验。方法：采用性状鉴别，粉末显微鉴别、TLC鉴别，浸出物、总灰分、水分、酸不溶性灰分、农药残留测定，HPLC测定目标成分含量。结果：各药材性状，鉴别，检查、含量测定等均符合要求。结论：按照《中国药典》2005年版有关规定检查，本批所有原料药材质量符合规定，可以进行后续研究。
2．超临界萃取工艺技术条件的优化
目的：优化白术、防风、木香、陈皮超临界萃取工艺的技术条件。方法：根据SFE-CO_2提取的影响因素，选定萃取压力、萃取温度、萃取时间3个因素，每个因素取3个水平，采用L_9(3~4)正交表优化提取工艺条件，并将优化条件进行2次重复性实验，即：取处方量白术、防风、木香、陈皮粗粉共308g，置1000ml的超临界二氧化碳萃取装置中，调节萃取温度45℃，萃取压力25MPa，解析压力6MPa，解析温度45℃，CO_2流量为21.7L／h，萃取3h。得萃取物1和萃取物2，分别称定萃取物的量和测定木香烃内酯、去氢木香内酯总量，计算木香烃内酯、去氢木香内酯的提取率。结果：从极差分析及方差分析结果认为：影响萃取物得量及木香烃内酯、去氢木香内酯总量的因素按影响大小排列为A(萃取压力)、C(萃取时间)、B(萃取温度)，其中A和C对萃取物的量及木香烃内酯、去氢木香内酯总量的影响有统计学意义，为主要因素。处方中的木香、白术、防风、陈皮的超临界二氧化碳萃取物平均得率为3.72％，木香烃内酯、去氢木香内酯总量为995mg，与投料药材相比，该工艺木香烃内酯、去氢木香内酯的提取率为95.5％。结论：依据正交试验结果，SFE-CO_2技术条件最佳组合为A3B1C3，即：萃取压力26MPa，萃取时间3h，萃取温度45℃，两次重复实验结果基本相同，说明临界二氧化碳萃取工艺稳定、重复可行。
3．水提工艺技术条件的优化
目的：选取最优化的水提工艺技术条件。方法：根据设计的工艺路线，白术、防风、木香、陈皮超临界二氧化碳萃取后的残渣连同党参、火炭母、白芍、石榴皮、救必应、甘草、茯苓采用水提方式。选取加水量，提取时间，提取次数3个因素，每个因素取3个水平，选用L_9(3~4)正交表进行试验，以总固物重量、芍药苷的含量为指标，考察工艺的最佳参数，并将优化条件进行2次重复性实验。结果：极差分析及方差分析结果认为：影响总固物得量和芍药苷含量的因素按影响大小排列均为C(提取次数)、A(水用量)、B(提取时间)，其中C与A对总固物得量及芍药苷总量的影响均有统计学意义，为主要因素。处方中白芍等药材的水提物总固形物量平均为138g，芍药苷总量为2121mg，与投料药材相比，该工艺芍药苷的提取率为88％。结论：依据正交试验结果，水提工艺的最佳组合为A3B1C3，由于因素B(提取时间)对总固物得量和芍药苷含量影响均较小，考虑到实验室小试和大批量生产的差异及提取完全，综合能源和时间因素，故在B因素的三个水平中选择B2：即加8倍水，提取3次，每次1小时。两次重复实验结果基本相同，说明工艺稳定、重复可行。
4．干燥工艺选择及优化
目的：以芍药苷为指标成分，对白芍等水提清膏进行喷雾干燥、真空干燥和常压干燥工艺的筛选及干燥条件的优化。方法：对三种干燥工艺下得到的干燥物进行重量计算和芍药苷含量测定，计算芍药苷总转移率，选择此条件进行重复实验，确定干燥方式。以干燥条件对药液可喷性、粘壁现象、干燥时间为影响因素，计算芍药苷的干燥工艺保留率和总转移率，确定干燥参数。结果：实验结果显示，对于常压干燥及真空干燥，长时间的加热使指标成分芍药苷损失严重；喷雾干燥方法工艺可行，干燥时间短，产物干爽，指标成分芍药苷损失较少，与其它干燥工艺比较，是生产上可行的干燥方式。重复实验结果显示，喷干粉量为367g，与喷雾干燥前清膏相比，该工艺芍药苷的保留率为79.5％，与投料药材相比，该工艺芍药苷的总转移率为70.0％。两次重复实验结果基本相同，说明工艺稳定、重复可行。因此，本工艺干燥方式选择喷雾干燥。选择干燥参数结果为：入口风温度180℃、出口风温度100℃，抽气参数为95％，泵参数为25％。重复性实验结果表明：水提清膏的喷干粉得量为366g，芍药苷量为1609mg，与喷雾前清膏相比，该工艺芍药苷的转移率为75.82％，与投料药材相比，该工艺芍药苷的总转移率为66.08％。两次重复实验结果基本相同，说明工艺稳定、重复性良好。结论：干燥工艺选择喷雾干燥，筛选出的干燥参数稳定可行。
5．制备颗粒工艺参数优化
目的：优化制备颗粒的工艺参数。方法：取处方量的干膏粉，加入延胡索、黄连细粉及超临界萃取物，混匀，选择不同参数在干压制粒机上进行干压制粒，制成20～40目的颗粒，并对颗粒性状进行观察比较。结果：颗粒的硬度、细粉的比例与干压制粒机的进料速度、压辊转速和制粒速度均密切相关。结论：最优干压制粒参数为：进料速度48rpm，压辊转速30rpm，制粒速度30rpm。
二、肠激安胶囊质量标准制定
1、TLC鉴别
目的：建立肠激安胶囊中延胡索、黄连、救必应、石榴皮、火炭母、白术的薄层鉴别方法。方法：取肠激安胶囊内容物，经有机溶剂提取，过滤，浓缩，定容，制成供试品溶液。另分别精密取对照品，加有机溶剂溶解，定容，制成对照品溶液。以不含目标物质的处方，按制剂工艺及供试品溶液制备方法，制成阴性样品溶液。三者分别点于同一硅胶G薄层板上，展开剂展开，在紫外或可见光下检视对应的薄层斑点。结果：供试品色谱中，在与对照药材色谱相应的位置上，显相同颜色的薄层斑点，阴性对照无干扰。结论：延胡索、黄连、救必应、石榴皮、火炭母、白术的薄层鉴别方法稳定可行，可以作为肠激安胶囊定性鉴别方法。
2、砷盐、重金属、水分、崩解时限、微生物限度检查
目的：检查三批肠激安胶囊中砷盐、重金属、水分、崩解时限、微生物限度是否符合规定。方法：依据《中国药典》2005版一部检测方法。结果：砷盐含量低于2ppm，重金属含量低于10ppm，水分6.73％，崩解时限4.5min，未检出大肠埃希菌，细菌数、霉菌、酵母菌数、大肠菌群均＜10个／g。结论：砷盐、重金属、水分、崩解时限均符合《中国药典》2005版一部规定，微生物限度检查结果符合口服给药制剂微生物限度标准。
3、特征成分含量测定
目的：测定白芍所含特征性成分芍药苷，木香所含特征成分木香烃内酯、去氢木香内酯的含量，制定合理的含量限度。方法：高效液相色谱法(HPLC)。色谱柱：Phenomenex luna C18柱(250mm×4.60mm，5μm)；乙腈—水为流动相梯度洗脱；流速：0.8mL·min~(-1)；柱温：30℃；检测波长为230nm；进样量：10μL。理论板数按芍药苷峰计算不低于2000，按木香烃内酯、去氢木香内酯峰计算均不低于3000。结果：芍药苷在0.1516～3.032ug，木香烃内酯在0.1116～2.232ug，去氢木香内酯0.1014～2.028ug范围内线性关系良好，相关系数r=0.9999；精密度试验、稳定性试验、重复性、回收率试验结果：三种特征成分峰面积积分值RSD均小于2％；芍药苷回收率100.90％，RSD=1.75％，木香烃内酯回收率101.12％，RSD=1.80％；去氢木香内酯回收率101.37％，RSD=1.22％。样品测定结果：芍药苷((?)±s)∶3.38±0.05mg，RSD(％)∶1.43，木香烃内酯((?)±s)∶1.66±0.03mg，RSD(％)∶1.82，去氢木香内酯((?)±s)∶1.87±0.02mg，RSD(％)∶1.05。结论：经考察，HPLC法同时测定三种特征成分的精密度，重复性，稳定性良好，可以作为制剂含量测定的方法。根据测定结果，并按药典最低含量限度，白芍含芍药苷(C23H28011)不得少于1.6％，按处方投料每粒含0.105g白芍药材，以40％的提取转移率计算，本品含量限度确定为：每粒含白芍以芍药苷(C23H28011)计，不得少于1.25mg；根据测定结果，按药典最低含量限度，木香按干燥品计算，含木香烃内酯、去氢木香内酯的总量不得少于1.8％，按处方投料每粒含0.042g木香药材，以40％的提取转移率计算，结果本品含量限度确定为：每粒含含木香烃内酯(C_(15)H_(20)O_2)和去氢木香内酯(C_(15)H_(18)O_2)的总量不得少于1.05mg。
4．肠激安胶囊超临界CO_2萃取物GC-MS指纹图谱
目的：建立肠激安胶囊超临界CO_2萃取物GC-MS指纹图谱，进一步控制制剂的内在质量，减少批间差异。方法：超临界CO_2萃取物，超声提取，用GC-MS法进行指纹图谱分析，结合计算机检索技术对分离的化合物进行结构鉴定，应用色谱峰面积归一化法计算各成分的相对百分含量。结果：肠激安胶囊超临界CO_2萃取物各组分均得到较好分离，共分离出13个成分，鉴定了10个化合物。结论：用GC-MS法建立肠激安胶囊超临界CO_2萃取物的指纹图谱方法可行。
三、肠激安胶囊主要药效学试验
目的：根据肠激安胶囊的功能主治，研究该药健脾疏肝、清热祛湿、涩肠止泻的作用。方法：止泻试验、小肠推进运动实验、抗炎试验、镇痛试验。结果：本制剂能延长小鼠排黑粪潜伏期，减少排黑粪数量，减慢小肠蠕动速度，具有一定的止泻作用；能降低毛细血管的通透性，具有一定的抗炎作用；能延长小鼠扭体发生潜伏期，减少扭体发生的次数，对化学因素引起的疼痛有较好的镇痛作用。结论：肠激安胶囊具有一定的止泻、抗炎及镇痛作用，为临床治疗腹泻型肠易激综合征(IBS)，慢性结肠炎、腹痛腹泻等提供了药效学依据。
四、腹泻型IBS大鼠模型的建立及其内脏敏感性评估
目的：建立腹泻型IBS大鼠模型，观察模型大鼠排便次数、直结肠扩张阈值的变化以及致痉剂对模型大鼠离体肠管舒缩运动的影响。方法：取出生后第8d的大鼠，每天给予直肠内醋酸刺激，持续2周，复制内脏高敏感大鼠模型。新生大鼠分别于造模结束后第14天，15天，16天，观察4h内大鼠排便的粪点数。并在出生后第6周及第8周进行直肠扩张，评估其腹部收缩反射(AWR)阈值；测定致痉剂作用于模型大鼠离体肠管舒缩运动的升高幅度。结果：与正常组比较，模型组大鼠排便次数显著增多，粪点不成型。直肠扩张时，模型组大鼠腹部抬高和背部拱起的压力阈值较正常组显著降低；离体肠管在加入致痉剂后模型组升高值均明显大于正常组。结论：新生期肠道内的慢性炎症刺激，可以在成年后引起慢性腹泻及内脏敏感性增高，模型大鼠的离体小肠对致痉剂的反应性增高，符合IBS的基本特征，造模方法可行。
五、肠激安胶囊干预腹泻型IBS模型大鼠的机理研究
目的：研究肠激安胶囊对IBS模型大鼠的干预机理。方法：将新生SD大鼠随机分为正常组、模型组、肠激安高剂量组、中剂量组、低剂量组、补脾益肠丸组。正常组乳鼠每天直肠给予0.9％生理盐水，其余各组从出生后第8—21天，每天给予直肠内醋酸刺激，复制内脏高敏感大鼠模型，分别于造模结束后第14天，15天，16天，观察4h内大鼠排便的粪点数，并在出生后第6周及第8周进行直肠扩张，评估其腹部收缩反射(AWR)阈值；第8周后，分别灌胃给予生理盐水(正常组及模型组)、肠激安药液3.76g／kg体重、1.88g／kg体重、0.94g／kg体重、补脾益肠丸6.76g／kg体重，每日一次，共两周，给药后再次观察大鼠排便情况及评估其腹部收缩反射(AWR)阈值。用流式细胞仪测定大鼠血中T细胞亚群分布的百分率；光镜观察大鼠肠组织的5-HT免疫组化染色情况。光镜观察大鼠结肠组织中肥大细胞脱颗粒情况及肥大细胞计数；电镜观察MC细胞及其毗邻结构。结果：肠激安胶囊能显著减少腹泻大鼠排便次数，具有止泻作用；能升高IBS大鼠的肠道敏感压力阈值；腹泻型IBS模型动物的细胞免疫功能下降，中药干预两周后，能使大鼠血中CD_4~+的值逐渐升高，CD_8~+和CD_4~+／CD_8~+的比值逐渐恢复正常，改善机体的免疫功能；能明显降低IBS大鼠5-HT分泌明显增多的情况，通过改善因5-HT的异常升高而导致肠道动力异常和内脏感觉过敏从而改善腹泻症状。光镜观察结果显示：各组结肠粘膜表面未见溃疡或糜烂，腺体大小及形态正常；腺体粘液上皮细胞及杯状细胞形态未见异常；间质内少量炎细胞浸润，各组结肠组织无器质性病变，符合IBS临床病理表现，中药的干预对IBS引起的慢性腹泻肠粘膜轻度水肿及炎症反应有一定作用，模型组结肠粘膜固有层肥大细胞计数及脱颗粒现象较正常组明显增多，高剂量组粘膜固有层肥大细胞计数及脱颗粒现象较模型组明显减少，且与正常组无显著差异，提示高剂量的肠激安药液能减少肥大细胞数量，改善模型大鼠结肠组织中肥大细胞的脱颗粒情况，并使之恢复正常。中剂量组也能明显改善肥大细胞脱颗粒现象，显示了肥大细胞稳定剂的效果，在降低肠道敏感性方面起着重要作用，这一作用可能是肠激安治疗肠易激综合征的主要作用机理之一。电镜下观察到肥大细胞位于肠粘膜固有层中，胞浆中含有许多电子密度较高的颗粒；模型组肥大细胞部分有脱颗粒现象，空泡形成；各组均可见肥大细胞与神经纤维毗邻。因此，肠道激活状态的MC可能在IBS中所表现的以动力改变、感觉异常为特征的内脏高敏感性的病理生理过程中起到关键性作用。结论：肠激安胶囊干预腹泻型IBS的途径和作用机理为：①减少稀便次数；②升高肠道敏感性压力阈值；③改善大鼠机体的细胞免疫功能；④改善因5-HT的异常升高而导致肠道动力异常和内脏感觉过敏；⑤改善由IBS引起的慢性腹泻肠粘膜轻度水肿及炎症反应；⑥减少肥大细胞数量，改善结肠组织中肥大细胞的脱颗粒情况，具有肥大细胞稳定剂的作用。研究结果为进一步揭示IBS的发病机制和干预措施创立了必要的研究基础，显示了中药干预腹泻型IBS的多环节、多靶点的综合作用。
六、本课题的特点和创新性：
1．IBS发病率高、病程长，恢复慢，本研究将临床疗效确切的医院制剂进行开发，针对疾病特点，使传统方剂与现代制药技术相结合，采用先进的SFE-CO_2技术、喷雾干燥技术等，优化制备工艺，更好发挥药效，减少刺激性，并利用GC-MS、HPLC等现代分析技术严格控制其质量，建立挥发性成分指纹图谱，制剂工艺具有先进性及新颖性。
2．目前已建立的与IBS相关的动物模型多是根据体外单因素建立的符合IBS某一方面的病理生理机制的动物模型，而病证结合模型未见有报道。情志因素与IBS症状的发作和加重有关，且情志所伤多引起肝郁。本实验选用的模型虽然是由化学炎症刺激引起，但由于刺激处在乳鼠期，属于早期生活事件引起的动物模型，因此其对乳鼠也是一种情志因素刺激。新生期肠道内的慢性炎症刺激，可以在成年后引起慢性内脏敏感性增高，符合IBS的基本特征。由此建立的腹泻型IBS大鼠模型具有稳定性及重复性好，可操作性强，可量化评估的特点，对治疗药物的筛选和评价具有重要现实意义。
3．目前国内外对IBS的研究多集中在临床阶段，以患者为研究对象，个体差异大，缺乏对照和可比性，基于动物实验的基础研究较少。本课题从大鼠肠道敏感性、5-HT、肥大细胞、肥大细胞的脱颗粒状态等角度系统探讨肠激安胶囊的作用机理，丰富了IBS的基础研究内容，较详细地阐述了中药多靶点的综合作用。
Irritable bowel syndrome (IBS) is a common chronic and non-organic intestinal disorder disease. Its main symptoms are recurrent abdominal pains, diarrhea, constipation or the alternating of diarrhea and constipation. Because of lack of evidence of pathologic and morphological change and abnormal biochemical index, it is included in IBS as FBD (Functional bowel disease, FBD). In the past, IBS was considered to be caused by disorders of bowel function and abnormal pains. However, in recent few years, many researches have demonstrated that IBS is related to the high sensitivity of the bowel. Now it is usually considered to be a heterogeneity disease with multiple causes caused by individuality.
IBS is a common functional disease, nearly accounting for about 30%-50% of the gastrointestinal disease, of which the IBS with the main symptom of diarrhea is the most common among the patients. In recent years, a close relationship with psychological factors is reported both in domestic and international researches in this disease. Most patients have anxiety disorder,depressive illness or other mental and psychological abnormality, which may imply the failing of central nervous system' s adjustment to the intestinal neural system, the change involving hormone and receptors of partial muscle or nerve,the existence of the brain—bowel stalk and other factors. The western medicine nowadays prefers to use analgesics, antidiarrheal agents, antispastics and 5-HT3 receptor antagonist, drugs (such as loperamide and atropine) that relieve clinical symptoms,and drugs (such as oryzanolum, bifidobiogen-livzon, bifid Triple ciable capsule and dioctahedral smectite) that help regulate intestinal flora and vegetative nerve functional disturbance, or protect the bowel films. However, the long-term curative effect of these drugs is far from satisfactory, not to mention their side effects.
The Traditional Chinese Medicine, however, has accumulated lots of experience in treating disease of intestines and stomach. There are also many useful formulas,which have little side effects,but better results. The value of herbs and formulas has gradually realized both at home and broad. In TCM, liver is thought to in charge of dipersing and regulation as well as qi activity. The emotional changes of the physical body are closely related to the flowing and circulation of qi and blood. Many researches have demonstrated that the function of the liver plays a critical role in our psychological reaction. The live is the core of the body in managing its psychological reaction. Animal experiments have showed that the herbs and formulas used for regulating the liver have some kind of effect in immune functions concerning the neurology and endocrine. In TCM, diarrhea is due to the spleen' s failing to transporting,which causes the stagnation of body fluid. Thus, functional disorders of the liver and spleen play a critical role during the process of IBS with diarrhea as the major symptom. The treatment from this point of view is also proved to be very effective. Formulas and treatments often used in clinic include the use of sanbai san, bupi yichang pill, yimucao decoction, jianpi tiaoqi tang, acupuncture, moxibustion, retention enema, injectio ad acumen, ear acupoint pressure therapy, acupuncture, massein and so on. Valid attempts have been made in the treatment of IBS, and also lots of experience has been accumulated. Besides, the new herb praeparatsion exploition is certain to have a broad prospect.
The herb praeparatsion coming from the hospital is always considered to be the cradle of new drug, due to its credible clinical background. Many new drugs, such as fufang danshen drop pill, tongluo shenggu capsule, are all coming from in this way. Changyan ling tablet has been used as hospital clinical praeparatsion for 18 years in our hospital. The drug is mainly used in the treatment of diarrhea IBS, chronic colonitis and so on. However, because of lacking basic research, there are still many problems: our production craft falls behind, the standard for quality is not yet complete. That is why the drug is not effectively developed.
Chang ji' an capsule is made by improving a traditional drug called changyan ling plan, which helps regulate spleen and liver, eliminate heat and wetness evil and antidarrhea with astringent. The capsule is constituted on the basis of sijunzi tang and tongxie yaofang: Dangshen, baizhu, fuling are used as principal drugs to invigorate spleen to excrete dampness; fangfeng, yanhusuo, muxiang, chenpi, baishao are the ministerial drugs for activating QI and invigoratsing spleen, together with huanglian, huotanmu, jiu biying, shiliu pi that can help eliminate damp and heat; gancao and baishao ease the affected liver to relieve pain. The whole formula invigorates the spleen and relives the liver,eliminate heat and damp and alleviates. Pre-experiment showed that the essential oil abstracted from the preparation produced effect probably by restraining excitability of histamine and rivalry bowels accentuation caused by neostigmine. Besides, it also has a counteract to Jimpy mice cramp pain reaction. This perfectly explains why it can alleviate patients' diarrhea and bellyache obviously. In conclusion , as a result of the animal experiment and the clinical research, the exploiture of the preparatsion is much promising.
A mature and advanced technique for the speratsion and abstraction is necessary in the manufacture of a new valid preparatsion. SPE-CO~ is the most advanced physical technique currently. So we can make good use of it in the herbal manufacture. Its advantages such as superiority in extraction efficiency, shorter producing periods, complete valid constituent, less organic solvent residue, simple craft, smaller labor and less pollution. Therefore this technique is applicable to the craft for herbal abstraction.
For the lack of animal models that are generally accepted now, the research of the disease is merely carried out in clinic. The models made by rectal injection with acetic acid is one of the methods to make animal model from outer stimulation. Theoretically speaking, the high sensitivity to ache is caused by continuate or repeated incitement of injury. During our clinical research, the IBS patients are usually found to have a gastrointestinal infection history, and often have irritable bowel syndrome during recovering periods. Generally speaking, thebaby rats' nerve structure is growing, and it is more sensitive and weaker. So it is much easier to have an effect on it. It represents: first, lack of available descending inhibitory system, second, the new-born spinal cord that gets excited much more easily. Therefore, if the animals are stimulated by some harmful injuries during these periods, their nervous tissue that is in charge of pains' transmission will get an allergy, even when they have grown up, and the former destructive stimulus has been eliminated for a long time, their intestine can remains to be highly sensitive. Viewing from this point, the chronic inflammation stimulus to baby rats, can cause a high bowel sensitivity in the adulthood. This tallies with the basic characters of IBS, and this method is worthy of further tries.
The herbal products used for the treatment of intestine disease include pills, dispersers, pieces, capsules, oral liquid and so on, most of which are based on traditional crafts, including water distilling, wine distilling, vapor distilling of naphtha, heating, evaporatsing, concentratsing, cyclic sirocco drying and so on. But much valid composition gets lost during the producing procedure, especially the part that is sensitive to heat. Besides, the quality and effect are not stable at all. That is why most valid herbal preparatsions are not completely empoldered and grow into a industry, their application limits in doctors' prescription. With the help of modern technique, it is an inevitable trend to empolder and develop the traditional herb, and new preparation, and new craft and stable standard are necessary anyway.
In the program, We adopt the SFE-CO2 technique to extract active ingredient from the compound prescription herbs, with the experiment result of pharmacy and curative efficacy as the evaluation index. According to the requirement of the new herbal preparatsion, the research and evaluation are merely carried out on the medicine whose efficacy has already been testified in clinic. We also adopt many new techniques, such as GC-MS fingerprint,HPLC, TLC etc, in order to survey and control the craft parameter. So the pre-clinical research of the new medicine must be safe, valid, stable and also kept in control. Only in this way, could we make sure of the stability, reduce the taken dose, lower the side effect, and explore the mechanism finally. A strict principle in the program is quite significant for the research of the mechanism about chang ji' an capsule.
Along with the quicker modern living rhythm, the change of the life style and the food structure, it seems a trend that the incidence of IBS rises year by year, but not for the use of a patent medicine of the same kind at the same time. Chang ji' an capsule treats IBS with the diarrhea as the main symptom, according to different clinical performance, combining with the etiological factors and pathogenesis, to treat IBS from the liver and spleen, and the formula is quite reasonable, Although it only takes a very short time, the clinical background is quite credible, and the foreground of the new preparatsion is vast both in the domestic and international markets, and it is quite worthy of being made good use of. Section I A Study of the Craft of Chang ji' an capsule production . Part I Chang ji' an capsule medicine material source and examination. Ob]eet~e: To check the source and quality of the raw material medicine. Methods:Char appreciation, powder micro-appreciation, thin-layer chromatography (TCL) appreciation, extract, total ash, moisture content, acid-indissoluble ash, pesticide residue determination and high performance of liquid chromatogram(HPLC) are used to determine the content of the aim ingredient.
Results:each medical material char, appreciation、check、content determination, etc are all consistent with the requirement.
Conclusion: According to The Pharmacopoeia of China 2005 relevant specifications, the medicine material's quality corresponds to the regulation, so it can be follow-up studied.
PartⅡThe grading-up of supercritical fluid extraction's condition.
Objective: To grade up supercritical fluid extraction specification's condition, of baizhu, fangfeng, muxiang, chenpi.
Methods: According to the influencing factor of SFE-CO_2 abstraction, select the three factors: pressure、temperature、time, and every factor has three levels, use L9(3~4) orthogonal table to improve the craft condition of extraction, and take it by two reproducibility experiments, i.e.: Take about 308g of the coarse powder of baizhu, fangfeng, muxiang, chenpi, group it in the HA221-50-06 SFE-CO_2 (1000ml) equipment, accommodate the temperature to 45°C, and extract pressure at 25Mpa. Resolution pressure is at 6Mpa, and resolution temperature is 45°C, the flow rates of CO_2 is 21.7L/h, then carry out 3 hours. Then gains the abstractive product land 2, weigh the product, determine costunolide and dehydro-α-curcumene. Calculate the extraction percentage of costunolide and dehydro-α-curcumene.
Results:The analysis of range and analysis of variance show that: the factors influencing extraction and costunolide dehydro-α-curcumene volume dose are:(A) extractive pressure >(B) extractive time >(C) extractive temperature. Among of them (A) and (C) are main factors, which possess the statistics meaning. So the best combination is A3B1C3: pressure is 26Mpa、time 3h, temperature is 45°C.
Conclusion: SFE-CO_2 average yield of the prescription is 3.72%. The volume dose of costunolide and dehydro-α-curcumene is 995mg. Extraction percentage is 95.5%. The result of the two experiments is the same. It shows that the craft of SFE-CO_2 is stable and the repeated experiment can be made.
PartⅢThe grading-up of water extraction condition.
Objective: To select the best water extraction craft technique condition.
Methods: According to the design of craft route, the remnant of baizhu, fangfeng, muxiang, chenpi left after the extraction of the SFE-CO_2, together with dangsheng, baishao, shiliupi, jiubiying, gancao, fulin, and the three factors water extraction、extraction time and frequency. Every factor has three levels. The experiment is run in L9 (3~4) orthogonal layout. The index is total weight of solid substance, content of peoniflorin. To investigate the best parameter, the best condition experiment will be made twice.
Results: From analysis of range and analysis of variance, it can be seen that the influencing factor of total solid substance and peoniflorin content can be presumed as C (extract frequency) >A (water duty) >B (extract time), effect of C and A are the principal having statistical significance. The best combination is A3B1C3. Due to the small effect of B (extract time), in order to save time and energy source, we choose B2. So the best combination is decided to be A3B2C3, I.e.: 800% water, extract 3 times, each time lasted 1h.
Conclusion: The total solid substance average dose of baishao and other medicine by water extraction is 138g, peoniflorin total amount is 2121mg, extraction percentage of peoniflorin via water extract is 88%. The results of tow repeat experiments are the same. It explains that the craft is stable and repeated experiment can be made.
PartⅣThe choice and optimization of drying craft.
Objective: With peoniflorin as the index constituent, to screen the craft of drying spray、vacuum dehydration and normal pressure drying, and to grade up drying condition.
Methods: Calculate drying product weight from the three drying craft and determinate the content of peoniflorin, then calculate peoniflorin total changing rate, repeating these experiments, and then decide drying method. Using drying condition as effect factor to physic liquor blowing character、adhesive wall phenomenon、drying time, calculate the remaining rates and total changing rates, and decide the drying parameter.
Results: The result of the experiments proves that if heating too long, peoniflorin would be lost seriously when normal pressure drying and vacuum dehydration is used; drying spray has feasibility, time is short, production is clean, loss of peoniflorin is light, so it is better than other methods. The repeated experiment result shows that, weight of dry powder is 367g, compare with the paste before drying spray, remaining rate is 79.5%. Compared with medical material, the changing rates of peoniflorin is 70.0%. The results of two experiments are the same. It explains that the craft is stable and the repeated experiment can be made. So we will choose drying spray for this craft. The parameter results: the temperature of inlet is 180°C, the temperature of outlet is 100°C, air exhausting parameter is 95%, pump parameter is 25%. The result of the repeated experiment indicates: dry Dower of water extract paste is 366g, peoniflorin weight is 1609g, compared with paste before drying spray, changing rates is 75.82% compare with medical material, total changing rates is 66.08%. The two results of the repeated experiments are the same.
Conclusion: The drying spray craft and drying parameter stabilization and the repeat experiment can be made.
PartⅤGrading-up the craft parameter of preparing granules.
Objective:Grading-up the craft parameter of preparing granule.
Methods: Take dry power of prescription dose add yanhusuo、huanglian fine powder and supercritical fluid extraction, misce bene, and choose different parameters when granulate, granulation is 20-40 mesh. Observe the granules characters.
Results: The result indicates that corpuscular firmness、fine grits proportion are close related to feed material speed of granulator、roller rotation speed and granulate speed.
Conclusion: The best granulate parameters are: 48rpm of feed material speed, 30rpm of roller rotation speed and 30rpm of granulate speed.
SectionⅡEstablishment of the quality standards of Changji' an Capsule
PartⅠIdentify by TLC
Objective: To build up a TLC method to identify yanhusuo, huanglian jiubiying, shiliupi, huotanmu, baizhu in Changji' an Capsule.
Methods:Extract the contents in the capsule by organic solvent, filter, condense and determine the volume, to manufacture solution for the article aqua. Also the differentiate nicety to check against the control article, and add organic solvent to dissolve, metered volume, as control article solution. With the prescription not containing target material, press the praeparatsum craft and use the trial article aqua preparative method, as negative sample solution. Three samples are respectively in the thin layer plate of the same gel silica G, with agent spread out, examine under the ultraviolet light, the visible light responding to lamellar spots may be inspected.
Results:The reference substance and the contents in the capsule display the same lamellar spot in the corresponding position in the colour spectrum, while the negative control does not have any interference..
Conclusion: The methods to identify the yanhusuo, huanglian, jiubuying, shiliupi, huotanmu, baizhu in the capsule are stable can be used to identify Changji' an Capsule.
PartⅡThe limit check on arsenic salt, heavy metal, moisture content, disintegration time limit, microbial limit.
Objective: To check the arsenic salt, heavy metal, humidity, disintegratsion time, microbial limit of three batches of Changji' an Capsule.
Methods: Follow the methods containing in Provisions for New Drugs Approval and Chinese Pharmacopeia I edition, 2005.
Results:The content of arsenic salt is less than 2 ppm, the content of heavy metal is less than 10 PPM, the moisture content is 6.73%, disintegration time limit is 4.5 min. No large angstrom sievert bacterium is checked; bacterial number、mold、yeast count、coli group are all<10/g.
Conclusion: The arsenic salt, heavy metal, humidity, disintegratsion time are all consistent with the regulation of Chinese pharmacopeia I 2005 edition. The microbial limit is consistent with the microbial limit standards of praeparatsum for oral use.
PartⅢDetermining of the characteristic compositions
objective: To determine the content of peoniflorin in baishao, costunolide and dehydro-α-curcumene in aucklandiae, and make up a reasonable content limit.
Methods:Column: Phenomenex luna C18(250mm×4.60mm, 5μm);mobile phase: acetonitrile-water, gradient elution; flow rates:0.8mL·min-1; column temperatsure:30℃; detecting wavelength 230 nm; sample size:10μl. The number of theoretical plate can not be less than 2000 according to peoniflorin, and can not be less than 3000 according to costunolide and dehydro-α-curcumene.
Results:The linear relationship is fine if the content of peoniflorin is between 0.1516-3.032 ug, costunolide is between 0.1116-2.232 ug, dehydro-α-curcumene is between 0.1014-2.028 ug, coefficient correlation r=0.9999; the results of density test、stability test、repeatability test: each peak area score's RSD are less than 2%,the recovery rate of peoniflorin is 100.90%, RSD=1.75%;the recovery rate of costunolide is 101.12%, RSD=1.80%; the recovery rate of dehydro-α-curcumene is 101.37%, RSD=1.22%。the result of sample detection: peoniflorin((?)±s): 3.38±0.05mg, RSD(%):1.43, costunolide ((?)±s): 1.66±0.03mg, RSD(%):1.82, dehydro-α-curcumene ((?)±s): 1.87±0.02mg, RSD(%): 1.05.
Conclusion:Three kinds of characteristic compositions are detected by the same method of HPLC with good precision, repeatability and stability. According to the results and the minimum content limit of Chinese pharmacopeiaⅠ, the content of peoniflorin(C23H28011) in baishao could not be less than 1.6%,each capsule contain 0.105g, according to the prescribed drugs, if the transfer rate is 40%, this article content is determined as: Each capsule contains peoniflorin no less than 1.25 mg, accounted with the peoniflorin (C23H28011);According to the measurement result, each capsule contains costunolide and dehydro-α-curcumene of volume cannot be less than 1.8% at the level with the lowest limit content and muxiang. According to the prescription(each capsule contains muxiang 0.042g), then the limit content is: each capsule contains costunolide (C15H2002) and dehydro-α-curcumene (C15H1802) can not be less than 1.05 mg, in 40% transfer-rate. PartⅣThe GC- MS fingerprint of SFE-CO_2 extraction in the Changji' an Capsule
Objective: To build up the GC- MS finger print of SFE-CO_2 extraction in Changji' an Capsule for controlling the praeparatsum's internal quality.
Methods: The SFE-CO_2 extraction, supersound extracting, analysis the finger print by GC- MS, combining the computer index technique to identify the structure of the separated chemical compound, and the relative contents in percentage were calculated with Area Normalization Method.
Results: Each composition in Changji' an Caps SFE-CO_2 extraction are all well separated. There are 13 compositions be separated, 10 compounds structure are identified.
Conclusion: The GC- MS method can be used to establish finger print of the volatil ingredients in Changji' an Caps.
SectionⅢThe experiment of the main pharmacodynamics of Changji' an capsule.
Objective:According to the main pharmacodynamics of changji' an capsule, to study its function of regulate spleen and liver, eliminate heat and wetness evil and antidarrhea with astringent
Methods: Checking diarrhea experiment, small intestines' propulsion experiment, anti-inflammatory experiment, and relieving pain experiment.
Results: This Chinese medicine (which is called changji' an capsule) can prolong incubation period of defecating dark stool and reduce egesta of small rats, and have a certain function of checking diarrhea, reduce the small intestine peristalsis speed, lower the permeability of blood capillary, and achieve an anti-inflammatory effect. It can also prolong the incubation period of the experiment small rats' body stretching; reduce the frequency of body stretching, and have analgesic effect especially on the ache caused by chemistry substance.
Conclusion: Changji' an capsule has certain functions of checking diarrhea, anti-inflammatory and analgesic effect, and provides the pharmacodynamic basis for treating Diarrhea-IBS, chronic colitis, chronic indigestion, stomachache, diarrhea, etc.
SectionⅣEstablishment of rats experiment model with Diarrhea-IBS and the valuation of its internal organs sensitivity
Objective:To build up the rats experiment model with Diarrhea-IBS, observe the times of the rats defecation and the value threshold change of ectocolon and the influence of bowel movement by adding ACH.
Methods:Give the acetic acid to stimulate the rectum of the rats new-born only for 8 days everyday, keep on for 2 weeks, duplicate the model of high sensitivity of internal organs of rats and count the number of egesta of model rats on 14th days, 15th days, 16th days within 4 hours. Evaluate threshold value of rats' belly shrink reflect (AWR) and 6 weeks and 8 weeks after they were born., they have their rectum extended. Measure the rising range of the bowl taken from the model rat movement on which ACH is affected.
Results: Compared with the normal groups, the model groups show defecation times increasingly, and its egesta are not confectioning molding. When the rectum be extended, the press use threshold value and belly raising value of the model group are cut down more obviously than those of the normal groups. After ACH is joined into the isolated bowel, the value of the model group goes up, all obviously higher than those of the normal groups.
Conclusion: Stimulating the bowel of model rats in new-born period by the chronic inflammation can cause chronic bacillary diarrhea and increase the internal organs sensitivity in adult, and increase the reaction of the spasm agent which stimulate the isolated bowel, all those essentially match Diarrhea-IBS basic characteristics.
SectionⅤResearch on the Mechanism of Changji' an Capsule Intervening Diarrhea-IBS Model Rats.
Objective: To study the intervention mechanism which Changji' an capsule has on Diarrhea-IBS model rat.
Methods: Randomly divided rats of new-born into normal group, model group, Chinese herbal medicine in high quantity group, Chinese herbal medicine medium quantity group, Chinese herbal medicine low quantity group and the pill of Bupi yichang pill group. New -born rats in normal group are given 0.9% physiology in rectum, the other groups from 8th day to 21st day are given the acetic acid to stimulate the rectum everyday, keeping on 2 weeks, duplicating the model of high sensitivity of internal organs of rats and counting the number of egesta of model rats on 14th days, 15th days, 16th days within 4 hours. After 8 weeks, infuse the stomach to give respectively the NS, Changji' an capsule liquid 3.76g/ kg weight, 1.88g/kg weight, 0.94g/kg weight and Bupi yichang pill 6.76g/kg weight, once daily, for two weeks, observe the rats again after medicine is taken and evaluate its belly constringency reflect threshold (AWR) value. Measure the rat blood with the cell instrument in a percentage for distribution of T lymph-cell, observes the rat bowel organization of immunity dyeing of 5-HT. Observe those cell degranulation of mast cell and count mast cells number with light microscope, and observe MC cells and their adjacent structures.
Results:Changji' an capsule can raise the press threshold value of sensitivity of the model rats, and has diarrhea checking function to Diarrhea-IBS. It can increase CD_4~+ value in blood gradually, CD_8~+ and the specific value of the CD_4~+ CD_8~+ which recover normally, improve the immunity function. Changji' an can lower increasing secretion 5-HT of the model rats of IBS, improve allergy of internal organ, diarrhea symptom and motive abnormality caused by 5-HT going up unusually, in each group of colon glue surface and no ulcer or debauched was seen, the gland body size and appearances are normal, the glandular organ mucus and cup cell form were seen abnormal, a little amount cell of phlegmasia inside the quality is gradually changed, each colon organization has no change, matching the clinical pathology of IBS performance. The experiments show that the Chinese herbal medical has certain function on the IBS chronic bacillary diarrhea and phlegmasia diseases. The number of mast cells of proper layer and degranulation in model group is higher than those of normal group obviously, and the number of mast cell of proper layer and degranulation in high quantity group is smaller than those of model group, but is not difference with the normal group. It shows that, the medicine liquid can reduce the mast cell quantity, improve the model rats colon organization in the mast cell degranulation, and the instauratsion that make it normal. The medium quantity group also can improve degranulation of mast cells obviously, showing it has the function of stabilizer, this function may be one of main mechanism that changji' an capsule treating IBS, gives an electric mirror, to observe mast cells to locate the bowel to proper layer, implying many grains with higher electronics density in the afterbirth syrup; The mast cells of model group has part of degranulation, empty bubble formation; Each group can be seen to have mast cells and the nerve fibers are adjacent. As a result, it expresses that a MC possibility that activates the appearance is in the IBS expression with the motive change, the felling abnormality for characteristic of internal organs raise the decisive function in the pathologic physiology process of the high sensitivity.
Conclusion: The function mechanisms of Changji' an capsule intervening Diarrhea-IBS are: (1) Improve sensitivity of bowel of the model rats of IBS; (2) Reduce times of loose stools;(3)Improve the cell immunity function of the model rats; (4) Improve bowel abnormality caused by the unusual increase of 5-HT and allergic feeling of internal organs; (5) Relieve intestinal mucosa edema and inflammatoryreaction caused by IBS. (6) Reduce the mast cell quantity, improve the colon organization degranulation, have the function that stabilizes mast cells. The result for further demonstrating the disease occurence mechanism of IBS and taking intervention measures helps establish necessary research foundation. This shows that the overall function of multiple targets of Chinese herbal medicine in its intervention of IBS. Section VI Peculiarities and New ideas in this Study.
Firstly, The IBS is a common chronic disease with characteristic of high incidence rate, long course and slow recovery. This research which exploited hospital praeparatum with exact curative effect, aims at the disease characteristics, makes traditional prescription and modern medicine techniques combine together, It adopts advanced techniques of SFE- CO_2, spraying dry technique of fog etc., excellent craft, and better exertive efficacy of medicine, It reduces stimulus and makes use of the modern and analytical technique of GC- MS, HPLC etc. to control its quality strictly, establishes the volatility composition fingerprint diagram, and makes the craft to have the advanced novelty. Secondly, currently there are many animal related models of IBS, but built up according to the single factor outside the body that mostly matches the IBS animal model on some pathologic physiology mechanism, but the combination of disease and symptoms in models is not reported. Feeling mood factor has something to do with symptom of IBS when it occurs and aggravates, and feeling mood can cause liver gloomy. This experiment chooses to use the model stimulated by the chemistry chronic inflammation, and the incitement placed in the new born period of new born rats, belonging to the animal model which caused by earlier period event, so it is also a kind of feeling mood factor to the rats. Chronic inflammatory stimulation in bowel in new born rats may cause the chronic internal organs sensitivity to increase after they become adult, matching IBS basic characteristics. Thus, building up of the model rats with Diarrhea-IBS have stability and repetitions advantages, the maneuverability is strong, it has characteristic of being evaluated, thus having important and realistic meaning to the further sieving of the treatment medicine. Finally, most researchers of the world concentrate upon clinic study of IBS. there are few studies based on animal experiment. This article studies in detail the mechanism of Changji' an capsule from sensitivity of bowel, 5-HT, mast cell, mast.cell degranulation etc, thus enriching the contents of IBS basic research, and explaining complex action of multitargets.
IBS是消化道常见的功能性疾病，约占胃肠道疾病的30％～50％，临床以腹泻型IBS为常见，患者常因此就诊。近年来，国内外大量文献报道心理因素与该病的发病密切相关，患者常伴有焦虑、忧郁等精神和心理异常，这可能涉及到中枢神经系统对肠神经系统的调整失常，胃肠激素的变化以及局部神经、肌肉各种受体的改变，脑—肠轴的存在等多种因素。目前西医治疗IBS主要以止痛剂、止泻剂、止痉剂、5-HT_3受体拮抗剂等药物(如易蒙停，阿托品等)对症治疗，再配以调整肠道菌群和植物神经功能紊乱或肠道黏膜保护剂的药物(如谷维素、丽珠肠乐、培菲康、思密达等)，这些药物对症状的缓解有效，但远期疗效尚不理想，且毒副作用难以接受。
中医在治疗胃肠疾病方面积累了几千年的丰富经验和大量有效方剂，因此，中医药治疗IBS方面有其优势，疗效肯定，副作用小，越来越受到国内外市场重视。祖国医学认为，肝主疏泄，调畅气机，而机体情绪的变化与气血的运行密切相关，研究表明，肝主疏泄的功能在机体心理应激中起着决定性的作用，肝是机体调节心理应激反应的核心。动物实验表明，调肝方药对束缚应激所引起的神经内分泌免疫功能紊乱具有一定的调节作用。腹泻的发生在中医学中则主要是脾失健运，水湿不化的结果，因此，肝脾两脏功能失调在腹泻型肠易激综合征发病过程中的重要作用是肯定的，从肝脾两脏对腹泻型肠易激综合征加以治疗，疗效也是肯定的。临床常用的治疗方剂及治疗方法有三白散、补脾益肠丸、益母草煎剂、健脾调气汤、针刺疗法、艾灸疗法、保留灌肠、穴位注射、耳穴贴压、按摩等，中医中药已经在IBS方面进行了有效的尝试，在治疗方面积累了丰富的经验。
医院制剂由于其可靠的临床背景，历来是中药新药开发的摇篮，许多著名的新药如复方丹参滴丸、通络生骨胶囊等都出自医院制剂。肠炎灵片作为医院制剂在广州中医药大学第一附属医院临床使用十多年，主要用于治疗腹泻型IBS、慢性结肠炎、腹痛腹泻等症，临床疗效满意。但由于基础研究工作缺乏，制剂工艺落后、质量标准不完善，未能进行有效开发。
肠激安胶囊由我院传统制剂肠炎灵片经改良研制而成，具有健脾疏肝，清热祛湿、涩肠止泻的功效，组方以四君子汤合痛泻要方加味组成，以党参、白术、茯苓健脾渗湿为君药，防风、延胡索、木香、陈皮、白芍疏肝理气醒脾为臣药，佐以黄连、火炭母、救必应、石榴皮清利湿热，邪去则土安，甘草和中佐白芍以柔肝缓急，全方具健脾疏肝、清利湿热、涩肠止泻之效。预实验提示，其挥发油可能通过抑制组织胺的兴奋性而发挥作用；对新斯的明引起的肠推进亢进具有拮抗作用，以及拮抗小鼠的痉挛疼痛反应，较好地解释了该制剂在临床上明显缓解患者腹泻、腹痛的功效，临床及动物实验结果更显示了新药开发的可行性。
要研制安全有效、稳定可控的新制剂，成熟先进的提取分离技术不可或缺。超临界二氧化碳萃取技术(SFE-CO_2)是目前最先进的物理萃取技术，可以广泛应用于中草药领域，并具有萃取效率高、生产周期短、有效成分不破坏、不残留有机溶剂、工艺简便、节约劳动力、减少三废污染等优越性，特别适用于含挥发性成分中药材的提取。
腹泻型IBS目前尚无公认的动物模型。直肠注射醋酸造模的方法属于外周致敏IBS动物模型，是基于重复的伤害性刺激或者持续的伤害性刺激都可以导致疼痛的高敏感性。临床研究也发现IBS患者在其患病前往往有急性胃肠道感染的病史，炎症性肠病患者在缓解期产生肠易激的症状。选择大鼠新生期的刺激是由于新生期的大鼠神经系统处于发育期，比较脆弱和敏感，易受造型变化的影响。表现在：①缺乏有效的背角神经元下行抑制系统；②新生脊髓比成年后更易兴奋。如果在这一时期，动物经受持续有害刺激可以引起神经痛觉传导通路的长时期的过敏，在成年后，尽管当初的有害刺激早已消除，肠道始终处于过敏状态。从这一点看乳鼠的新生期肠道内的慢性炎症刺激，可以在成年后引起慢性内脏敏感性增高，是一个较符合IBS的基本特征的模型，值得进一步尝试。
目前国内外对IBS的研究多集中在临床阶段，以患者为研究对象，个体差异大，缺乏对照和可比性，基于动物实验的基础研究较少。本课题从大鼠肠道敏感性、5-HT、肥大细胞、肥大细胞的脱颗粒状态等角度系统探讨肠激安胶囊的作用机理，较详细地阐述肠激安胶囊的作用途径和机理。
我国治疗肠道疾病的中药制剂有丸、散、片、胶囊、口服液等，大多依据传统工艺水提、醇提、水蒸气蒸馏提取挥发油，加热蒸发浓缩、热风循环干燥等，热敏性有效成分损失较多，批间差异大，质量和疗效不稳定，且绝大多数有效的方剂依然停留在医院制剂或个体给药的汤剂阶段，并未进行有效开发或形成产业化。采用现代制药技术，以新剂型、新工艺和稳定可控的质量标准对传统中药进行开发和改良，是中药现代化的必然趋势。
本项目采用SFE-CO_2技术提取分离复方中药的有效成分，以药理药效实验为评价指标，将疗效确切的医院制剂按照中药新药的技术要求进行研究和评价，并采用气相色谱—质谱(GC-MS)指纹图谱、高效液相色谱(HPLC)，薄层色谱(TLC)等多种检测方法跟踪和控制工艺参数，以安全有效、稳定可控的研究原则对肠激安胶囊进行临床前开发，对提高药物的稳定性，减少服用量和降低毒副作用，探讨肠激安胶囊的作用机理具有重要意义。
随着现代生活节奏的加快和生活方式及饮食结构的改变，IBS发病率有逐年上升的趋势，而至今为止未见同类中成药上市使用。肠激安胶囊根据腹泻型肠易激综合征的不同临床表现，结合中医病因病机，从肝脾两脏对腹泻型肠易激综合征加以综合治疗，组方合理，临床基础扎实，国内外市场前景广阔，具有良好的应用价值和开发前景。
○、肠激安胶囊生产工艺研究
1．肠激安胶囊药材来源及检验
目的：对肠激安胶囊原料药材进行来源和质量检验。方法：采用性状鉴别，粉末显微鉴别、TLC鉴别，浸出物、总灰分、水分、酸不溶性灰分、农药残留测定，HPLC测定目标成分含量。结果：各药材性状，鉴别，检查、含量测定等均符合要求。结论：按照《中国药典》2005年版有关规定检查，本批所有原料药材质量符合规定，可以进行后续研究。
2．超临界萃取工艺技术条件的优化
目的：优化白术、防风、木香、陈皮超临界萃取工艺的技术条件。方法：根据SFE-CO_2提取的影响因素，选定萃取压力、萃取温度、萃取时间3个因素，每个因素取3个水平，采用L_9(3~4)正交表优化提取工艺条件，并将优化条件进行2次重复性实验，即：取处方量白术、防风、木香、陈皮粗粉共308g，置1000ml的超临界二氧化碳萃取装置中，调节萃取温度45℃，萃取压力25MPa，解析压力6MPa，解析温度45℃，CO_2流量为21.7L／h，萃取3h。得萃取物1和萃取物2，分别称定萃取物的量和测定木香烃内酯、去氢木香内酯总量，计算木香烃内酯、去氢木香内酯的提取率。结果：从极差分析及方差分析结果认为：影响萃取物得量及木香烃内酯、去氢木香内酯总量的因素按影响大小排列为A(萃取压力)、C(萃取时间)、B(萃取温度)，其中A和C对萃取物的量及木香烃内酯、去氢木香内酯总量的影响有统计学意义，为主要因素。处方中的木香、白术、防风、陈皮的超临界二氧化碳萃取物平均得率为3.72％，木香烃内酯、去氢木香内酯总量为995mg，与投料药材相比，该工艺木香烃内酯、去氢木香内酯的提取率为95.5％。结论：依据正交试验结果，SFE-CO_2技术条件最佳组合为A3B1C3，即：萃取压力26MPa，萃取时间3h，萃取温度45℃，两次重复实验结果基本相同，说明临界二氧化碳萃取工艺稳定、重复可行。
3．水提工艺技术条件的优化
目的：选取最优化的水提工艺技术条件。方法：根据设计的工艺路线，白术、防风、木香、陈皮超临界二氧化碳萃取后的残渣连同党参、火炭母、白芍、石榴皮、救必应、甘草、茯苓采用水提方式。选取加水量，提取时间，提取次数3个因素，每个因素取3个水平，选用L_9(3~4)正交表进行试验，以总固物重量、芍药苷的含量为指标，考察工艺的最佳参数，并将优化条件进行2次重复性实验。结果：极差分析及方差分析结果认为：影响总固物得量和芍药苷含量的因素按影响大小排列均为C(提取次数)、A(水用量)、B(提取时间)，其中C与A对总固物得量及芍药苷总量的影响均有统计学意义，为主要因素。处方中白芍等药材的水提物总固形物量平均为138g，芍药苷总量为2121mg，与投料药材相比，该工艺芍药苷的提取率为88％。结论：依据正交试验结果，水提工艺的最佳组合为A3B1C3，由于因素B(提取时间)对总固物得量和芍药苷含量影响均较小，考虑到实验室小试和大批量生产的差异及提取完全，综合能源和时间因素，故在B因素的三个水平中选择B2：即加8倍水，提取3次，每次1小时。两次重复实验结果基本相同，说明工艺稳定、重复可行。
4．干燥工艺选择及优化
目的：以芍药苷为指标成分，对白芍等水提清膏进行喷雾干燥、真空干燥和常压干燥工艺的筛选及干燥条件的优化。方法：对三种干燥工艺下得到的干燥物进行重量计算和芍药苷含量测定，计算芍药苷总转移率，选择此条件进行重复实验，确定干燥方式。以干燥条件对药液可喷性、粘壁现象、干燥时间为影响因素，计算芍药苷的干燥工艺保留率和总转移率，确定干燥参数。结果：实验结果显示，对于常压干燥及真空干燥，长时间的加热使指标成分芍药苷损失严重；喷雾干燥方法工艺可行，干燥时间短，产物干爽，指标成分芍药苷损失较少，与其它干燥工艺比较，是生产上可行的干燥方式。重复实验结果显示，喷干粉量为367g，与喷雾干燥前清膏相比，该工艺芍药苷的保留率为79.5％，与投料药材相比，该工艺芍药苷的总转移率为70.0％。两次重复实验结果基本相同，说明工艺稳定、重复可行。因此，本工艺干燥方式选择喷雾干燥。选择干燥参数结果为：入口风温度180℃、出口风温度100℃，抽气参数为95％，泵参数为25％。重复性实验结果表明：水提清膏的喷干粉得量为366g，芍药苷量为1609mg，与喷雾前清膏相比，该工艺芍药苷的转移率为75.82％，与投料药材相比，该工艺芍药苷的总转移率为66.08％。两次重复实验结果基本相同，说明工艺稳定、重复性良好。结论：干燥工艺选择喷雾干燥，筛选出的干燥参数稳定可行。
5．制备颗粒工艺参数优化
目的：优化制备颗粒的工艺参数。方法：取处方量的干膏粉，加入延胡索、黄连细粉及超临界萃取物，混匀，选择不同参数在干压制粒机上进行干压制粒，制成20～40目的颗粒，并对颗粒性状进行观察比较。结果：颗粒的硬度、细粉的比例与干压制粒机的进料速度、压辊转速和制粒速度均密切相关。结论：最优干压制粒参数为：进料速度48rpm，压辊转速30rpm，制粒速度30rpm。
二、肠激安胶囊质量标准制定
1、TLC鉴别
目的：建立肠激安胶囊中延胡索、黄连、救必应、石榴皮、火炭母、白术的薄层鉴别方法。方法：取肠激安胶囊内容物，经有机溶剂提取，过滤，浓缩，定容，制成供试品溶液。另分别精密取对照品，加有机溶剂溶解，定容，制成对照品溶液。以不含目标物质的处方，按制剂工艺及供试品溶液制备方法，制成阴性样品溶液。三者分别点于同一硅胶G薄层板上，展开剂展开，在紫外或可见光下检视对应的薄层斑点。结果：供试品色谱中，在与对照药材色谱相应的位置上，显相同颜色的薄层斑点，阴性对照无干扰。结论：延胡索、黄连、救必应、石榴皮、火炭母、白术的薄层鉴别方法稳定可行，可以作为肠激安胶囊定性鉴别方法。
2、砷盐、重金属、水分、崩解时限、微生物限度检查
目的：检查三批肠激安胶囊中砷盐、重金属、水分、崩解时限、微生物限度是否符合规定。方法：依据《中国药典》2005版一部检测方法。结果：砷盐含量低于2ppm，重金属含量低于10ppm，水分6.73％，崩解时限4.5min，未检出大肠埃希菌，细菌数、霉菌、酵母菌数、大肠菌群均＜10个／g。结论：砷盐、重金属、水分、崩解时限均符合《中国药典》2005版一部规定，微生物限度检查结果符合口服给药制剂微生物限度标准。
3、特征成分含量测定
目的：测定白芍所含特征性成分芍药苷，木香所含特征成分木香烃内酯、去氢木香内酯的含量，制定合理的含量限度。方法：高效液相色谱法(HPLC)。色谱柱：Phenomenex luna C18柱(250mm×4.60mm，5μm)；乙腈—水为流动相梯度洗脱；流速：0.8mL·min~(-1)；柱温：30℃；检测波长为230nm；进样量：10μL。理论板数按芍药苷峰计算不低于2000，按木香烃内酯、去氢木香内酯峰计算均不低于3000。结果：芍药苷在0.1516～3.032ug，木香烃内酯在0.1116～2.232ug，去氢木香内酯0.1014～2.028ug范围内线性关系良好，相关系数r=0.9999；精密度试验、稳定性试验、重复性、回收率试验结果：三种特征成分峰面积积分值RSD均小于2％；芍药苷回收率100.90％，RSD=1.75％，木香烃内酯回收率101.12％，RSD=1.80％；去氢木香内酯回收率101.37％，RSD=1.22％。样品测定结果：芍药苷((?)±s)∶3.38±0.05mg，RSD(％)∶1.43，木香烃内酯((?)±s)∶1.66±0.03mg，RSD(％)∶1.82，去氢木香内酯((?)±s)∶1.87±0.02mg，RSD(％)∶1.05。结论：经考察，HPLC法同时测定三种特征成分的精密度，重复性，稳定性良好，可以作为制剂含量测定的方法。根据测定结果，并按药典最低含量限度，白芍含芍药苷(C23H28011)不得少于1.6％，按处方投料每粒含0.105g白芍药材，以40％的提取转移率计算，本品含量限度确定为：每粒含白芍以芍药苷(C23H28011)计，不得少于1.25mg；根据测定结果，按药典最低含量限度，木香按干燥品计算，含木香烃内酯、去氢木香内酯的总量不得少于1.8％，按处方投料每粒含0.042g木香药材，以40％的提取转移率计算，结果本品含量限度确定为：每粒含含木香烃内酯(C_(15)H_(20)O_2)和去氢木香内酯(C_(15)H_(18)O_2)的总量不得少于1.05mg。
4．肠激安胶囊超临界CO_2萃取物GC-MS指纹图谱
目的：建立肠激安胶囊超临界CO_2萃取物GC-MS指纹图谱，进一步控制制剂的内在质量，减少批间差异。方法：超临界CO_2萃取物，超声提取，用GC-MS法进行指纹图谱分析，结合计算机检索技术对分离的化合物进行结构鉴定，应用色谱峰面积归一化法计算各成分的相对百分含量。结果：肠激安胶囊超临界CO_2萃取物各组分均得到较好分离，共分离出13个成分，鉴定了10个化合物。结论：用GC-MS法建立肠激安胶囊超临界CO_2萃取物的指纹图谱方法可行。
三、肠激安胶囊主要药效学试验
目的：根据肠激安胶囊的功能主治，研究该药健脾疏肝、清热祛湿、涩肠止泻的作用。方法：止泻试验、小肠推进运动实验、抗炎试验、镇痛试验。结果：本制剂能延长小鼠排黑粪潜伏期，减少排黑粪数量，减慢小肠蠕动速度，具有一定的止泻作用；能降低毛细血管的通透性，具有一定的抗炎作用；能延长小鼠扭体发生潜伏期，减少扭体发生的次数，对化学因素引起的疼痛有较好的镇痛作用。结论：肠激安胶囊具有一定的止泻、抗炎及镇痛作用，为临床治疗腹泻型肠易激综合征(IBS)，慢性结肠炎、腹痛腹泻等提供了药效学依据。
四、腹泻型IBS大鼠模型的建立及其内脏敏感性评估
目的：建立腹泻型IBS大鼠模型，观察模型大鼠排便次数、直结肠扩张阈值的变化以及致痉剂对模型大鼠离体肠管舒缩运动的影响。方法：取出生后第8d的大鼠，每天给予直肠内醋酸刺激，持续2周，复制内脏高敏感大鼠模型。新生大鼠分别于造模结束后第14天，15天，16天，观察4h内大鼠排便的粪点数。并在出生后第6周及第8周进行直肠扩张，评估其腹部收缩反射(AWR)阈值；测定致痉剂作用于模型大鼠离体肠管舒缩运动的升高幅度。结果：与正常组比较，模型组大鼠排便次数显著增多，粪点不成型。直肠扩张时，模型组大鼠腹部抬高和背部拱起的压力阈值较正常组显著降低；离体肠管在加入致痉剂后模型组升高值均明显大于正常组。结论：新生期肠道内的慢性炎症刺激，可以在成年后引起慢性腹泻及内脏敏感性增高，模型大鼠的离体小肠对致痉剂的反应性增高，符合IBS的基本特征，造模方法可行。
五、肠激安胶囊干预腹泻型IBS模型大鼠的机理研究
目的：研究肠激安胶囊对IBS模型大鼠的干预机理。方法：将新生SD大鼠随机分为正常组、模型组、肠激安高剂量组、中剂量组、低剂量组、补脾益肠丸组。正常组乳鼠每天直肠给予0.9％生理盐水，其余各组从出生后第8—21天，每天给予直肠内醋酸刺激，复制内脏高敏感大鼠模型，分别于造模结束后第14天，15天，16天，观察4h内大鼠排便的粪点数，并在出生后第6周及第8周进行直肠扩张，评估其腹部收缩反射(AWR)阈值；第8周后，分别灌胃给予生理盐水(正常组及模型组)、肠激安药液3.76g／kg体重、1.88g／kg体重、0.94g／kg体重、补脾益肠丸6.76g／kg体重，每日一次，共两周，给药后再次观察大鼠排便情况及评估其腹部收缩反射(AWR)阈值。用流式细胞仪测定大鼠血中T细胞亚群分布的百分率；光镜观察大鼠肠组织的5-HT免疫组化染色情况。光镜观察大鼠结肠组织中肥大细胞脱颗粒情况及肥大细胞计数；电镜观察MC细胞及其毗邻结构。结果：肠激安胶囊能显著减少腹泻大鼠排便次数，具有止泻作用；能升高IBS大鼠的肠道敏感压力阈值；腹泻型IBS模型动物的细胞免疫功能下降，中药干预两周后，能使大鼠血中CD_4~+的值逐渐升高，CD_8~+和CD_4~+／CD_8~+的比值逐渐恢复正常，改善机体的免疫功能；能明显降低IBS大鼠5-HT分泌明显增多的情况，通过改善因5-HT的异常升高而导致肠道动力异常和内脏感觉过敏从而改善腹泻症状。光镜观察结果显示：各组结肠粘膜表面未见溃疡或糜烂，腺体大小及形态正常；腺体粘液上皮细胞及杯状细胞形态未见异常；间质内少量炎细胞浸润，各组结肠组织无器质性病变，符合IBS临床病理表现，中药的干预对IBS引起的慢性腹泻肠粘膜轻度水肿及炎症反应有一定作用，模型组结肠粘膜固有层肥大细胞计数及脱颗粒现象较正常组明显增多，高剂量组粘膜固有层肥大细胞计数及脱颗粒现象较模型组明显减少，且与正常组无显著差异，提示高剂量的肠激安药液能减少肥大细胞数量，改善模型大鼠结肠组织中肥大细胞的脱颗粒情况，并使之恢复正常。中剂量组也能明显改善肥大细胞脱颗粒现象，显示了肥大细胞稳定剂的效果，在降低肠道敏感性方面起着重要作用，这一作用可能是肠激安治疗肠易激综合征的主要作用机理之一。电镜下观察到肥大细胞位于肠粘膜固有层中，胞浆中含有许多电子密度较高的颗粒；模型组肥大细胞部分有脱颗粒现象，空泡形成；各组均可见肥大细胞与神经纤维毗邻。因此，肠道激活状态的MC可能在IBS中所表现的以动力改变、感觉异常为特征的内脏高敏感性的病理生理过程中起到关键性作用。结论：肠激安胶囊干预腹泻型IBS的途径和作用机理为：①减少稀便次数；②升高肠道敏感性压力阈值；③改善大鼠机体的细胞免疫功能；④改善因5-HT的异常升高而导致肠道动力异常和内脏感觉过敏；⑤改善由IBS引起的慢性腹泻肠粘膜轻度水肿及炎症反应；⑥减少肥大细胞数量，改善结肠组织中肥大细胞的脱颗粒情况，具有肥大细胞稳定剂的作用。研究结果为进一步揭示IBS的发病机制和干预措施创立了必要的研究基础，显示了中药干预腹泻型IBS的多环节、多靶点的综合作用。
六、本课题的特点和创新性：
1．IBS发病率高、病程长，恢复慢，本研究将临床疗效确切的医院制剂进行开发，针对疾病特点，使传统方剂与现代制药技术相结合，采用先进的SFE-CO_2技术、喷雾干燥技术等，优化制备工艺，更好发挥药效，减少刺激性，并利用GC-MS、HPLC等现代分析技术严格控制其质量，建立挥发性成分指纹图谱，制剂工艺具有先进性及新颖性。
2．目前已建立的与IBS相关的动物模型多是根据体外单因素建立的符合IBS某一方面的病理生理机制的动物模型，而病证结合模型未见有报道。情志因素与IBS症状的发作和加重有关，且情志所伤多引起肝郁。本实验选用的模型虽然是由化学炎症刺激引起，但由于刺激处在乳鼠期，属于早期生活事件引起的动物模型，因此其对乳鼠也是一种情志因素刺激。新生期肠道内的慢性炎症刺激，可以在成年后引起慢性内脏敏感性增高，符合IBS的基本特征。由此建立的腹泻型IBS大鼠模型具有稳定性及重复性好，可操作性强，可量化评估的特点，对治疗药物的筛选和评价具有重要现实意义。
3．目前国内外对IBS的研究多集中在临床阶段，以患者为研究对象，个体差异大，缺乏对照和可比性，基于动物实验的基础研究较少。本课题从大鼠肠道敏感性、5-HT、肥大细胞、肥大细胞的脱颗粒状态等角度系统探讨肠激安胶囊的作用机理，丰富了IBS的基础研究内容，较详细地阐述了中药多靶点的综合作用。
Irritable bowel syndrome (IBS) is a common chronic and non-organic intestinal disorder disease. Its main symptoms are recurrent abdominal pains, diarrhea, constipation or the alternating of diarrhea and constipation. Because of lack of evidence of pathologic and morphological change and abnormal biochemical index, it is included in IBS as FBD (Functional bowel disease, FBD). In the past, IBS was considered to be caused by disorders of bowel function and abnormal pains. However, in recent few years, many researches have demonstrated that IBS is related to the high sensitivity of the bowel. Now it is usually considered to be a heterogeneity disease with multiple causes caused by individuality.
IBS is a common functional disease, nearly accounting for about 30%-50% of the gastrointestinal disease, of which the IBS with the main symptom of diarrhea is the most common among the patients. In recent years, a close relationship with psychological factors is reported both in domestic and international researches in this disease. Most patients have anxiety disorder,depressive illness or other mental and psychological abnormality, which may imply the failing of central nervous system' s adjustment to the intestinal neural system, the change involving hormone and receptors of partial muscle or nerve,the existence of the brain—bowel stalk and other factors. The western medicine nowadays prefers to use analgesics, antidiarrheal agents, antispastics and 5-HT3 receptor antagonist, drugs (such as loperamide and atropine) that relieve clinical symptoms,and drugs (such as oryzanolum, bifidobiogen-livzon, bifid Triple ciable capsule and dioctahedral smectite) that help regulate intestinal flora and vegetative nerve functional disturbance, or protect the bowel films. However, the long-term curative effect of these drugs is far from satisfactory, not to mention their side effects.
The Traditional Chinese Medicine, however, has accumulated lots of experience in treating disease of intestines and stomach. There are also many useful formulas,which have little side effects,but better results. The value of herbs and formulas has gradually realized both at home and broad. In TCM, liver is thought to in charge of dipersing and regulation as well as qi activity. The emotional changes of the physical body are closely related to the flowing and circulation of qi and blood. Many researches have demonstrated that the function of the liver plays a critical role in our psychological reaction. The live is the core of the body in managing its psychological reaction. Animal experiments have showed that the herbs and formulas used for regulating the liver have some kind of effect in immune functions concerning the neurology and endocrine. In TCM, diarrhea is due to the spleen' s failing to transporting,which causes the stagnation of body fluid. Thus, functional disorders of the liver and spleen play a critical role during the process of IBS with diarrhea as the major symptom. The treatment from this point of view is also proved to be very effective. Formulas and treatments often used in clinic include the use of sanbai san, bupi yichang pill, yimucao decoction, jianpi tiaoqi tang, acupuncture, moxibustion, retention enema, injectio ad acumen, ear acupoint pressure therapy, acupuncture, massein and so on. Valid attempts have been made in the treatment of IBS, and also lots of experience has been accumulated. Besides, the new herb praeparatsion exploition is certain to have a broad prospect.
The herb praeparatsion coming from the hospital is always considered to be the cradle of new drug, due to its credible clinical background. Many new drugs, such as fufang danshen drop pill, tongluo shenggu capsule, are all coming from in this way. Changyan ling tablet has been used as hospital clinical praeparatsion for 18 years in our hospital. The drug is mainly used in the treatment of diarrhea IBS, chronic colonitis and so on. However, because of lacking basic research, there are still many problems: our production craft falls behind, the standard for quality is not yet complete. That is why the drug is not effectively developed.
Chang ji' an capsule is made by improving a traditional drug called changyan ling plan, which helps regulate spleen and liver, eliminate heat and wetness evil and antidarrhea with astringent. The capsule is constituted on the basis of sijunzi tang and tongxie yaofang: Dangshen, baizhu, fuling are used as principal drugs to invigorate spleen to excrete dampness; fangfeng, yanhusuo, muxiang, chenpi, baishao are the ministerial drugs for activating QI and invigoratsing spleen, together with huanglian, huotanmu, jiu biying, shiliu pi that can help eliminate damp and heat; gancao and baishao ease the affected liver to relieve pain. The whole formula invigorates the spleen and relives the liver,eliminate heat and damp and alleviates. Pre-experiment showed that the essential oil abstracted from the preparation produced effect probably by restraining excitability of histamine and rivalry bowels accentuation caused by neostigmine. Besides, it also has a counteract to Jimpy mice cramp pain reaction. This perfectly explains why it can alleviate patients' diarrhea and bellyache obviously. In conclusion , as a result of the animal experiment and the clinical research, the exploiture of the preparatsion is much promising.
A mature and advanced technique for the speratsion and abstraction is necessary in the manufacture of a new valid preparatsion. SPE-CO~ is the most advanced physical technique currently. So we can make good use of it in the herbal manufacture. Its advantages such as superiority in extraction efficiency, shorter producing periods, complete valid constituent, less organic solvent residue, simple craft, smaller labor and less pollution. Therefore this technique is applicable to the craft for herbal abstraction.
For the lack of animal models that are generally accepted now, the research of the disease is merely carried out in clinic. The models made by rectal injection with acetic acid is one of the methods to make animal model from outer stimulation. Theoretically speaking, the high sensitivity to ache is caused by continuate or repeated incitement of injury. During our clinical research, the IBS patients are usually found to have a gastrointestinal infection history, and often have irritable bowel syndrome during recovering periods. Generally speaking, thebaby rats' nerve structure is growing, and it is more sensitive and weaker. So it is much easier to have an effect on it. It represents: first, lack of available descending inhibitory system, second, the new-born spinal cord that gets excited much more easily. Therefore, if the animals are stimulated by some harmful injuries during these periods, their nervous tissue that is in charge of pains' transmission will get an allergy, even when they have grown up, and the former destructive stimulus has been eliminated for a long time, their intestine can remains to be highly sensitive. Viewing from this point, the chronic inflammation stimulus to baby rats, can cause a high bowel sensitivity in the adulthood. This tallies with the basic characters of IBS, and this method is worthy of further tries.
The herbal products used for the treatment of intestine disease include pills, dispersers, pieces, capsules, oral liquid and so on, most of which are based on traditional crafts, including water distilling, wine distilling, vapor distilling of naphtha, heating, evaporatsing, concentratsing, cyclic sirocco drying and so on. But much valid composition gets lost during the producing procedure, especially the part that is sensitive to heat. Besides, the quality and effect are not stable at all. That is why most valid herbal preparatsions are not completely empoldered and grow into a industry, their application limits in doctors' prescription. With the help of modern technique, it is an inevitable trend to empolder and develop the traditional herb, and new preparation, and new craft and stable standard are necessary anyway.
In the program, We adopt the SFE-CO2 technique to extract active ingredient from the compound prescription herbs, with the experiment result of pharmacy and curative efficacy as the evaluation index. According to the requirement of the new herbal preparatsion, the research and evaluation are merely carried out on the medicine whose efficacy has already been testified in clinic. We also adopt many new techniques, such as GC-MS fingerprint,HPLC, TLC etc, in order to survey and control the craft parameter. So the pre-clinical research of the new medicine must be safe, valid, stable and also kept in control. Only in this way, could we make sure of the stability, reduce the taken dose, lower the side effect, and explore the mechanism finally. A strict principle in the program is quite significant for the research of the mechanism about chang ji' an capsule.
Along with the quicker modern living rhythm, the change of the life style and the food structure, it seems a trend that the incidence of IBS rises year by year, but not for the use of a patent medicine of the same kind at the same time. Chang ji' an capsule treats IBS with the diarrhea as the main symptom, according to different clinical performance, combining with the etiological factors and pathogenesis, to treat IBS from the liver and spleen, and the formula is quite reasonable, Although it only takes a very short time, the clinical background is quite credible, and the foreground of the new preparatsion is vast both in the domestic and international markets, and it is quite worthy of being made good use of. Section I A Study of the Craft of Chang ji' an capsule production . Part I Chang ji' an capsule medicine material source and examination. Ob]eet~e: To check the source and quality of the raw material medicine. Methods:Char appreciation, powder micro-appreciation, thin-layer chromatography (TCL) appreciation, extract, total ash, moisture content, acid-indissoluble ash, pesticide residue determination and high performance of liquid chromatogram(HPLC) are used to determine the content of the aim ingredient.
Results:each medical material char, appreciation、check、content determination, etc are all consistent with the requirement.
Conclusion: According to The Pharmacopoeia of China 2005 relevant specifications, the medicine material's quality corresponds to the regulation, so it can be follow-up studied.
PartⅡThe grading-up of supercritical fluid extraction's condition.
Objective: To grade up supercritical fluid extraction specification's condition, of baizhu, fangfeng, muxiang, chenpi.
Methods: According to the influencing factor of SFE-CO_2 abstraction, select the three factors: pressure、temperature、time, and every factor has three levels, use L9(3~4) orthogonal table to improve the craft condition of extraction, and take it by two reproducibility experiments, i.e.: Take about 308g of the coarse powder of baizhu, fangfeng, muxiang, chenpi, group it in the HA221-50-06 SFE-CO_2 (1000ml) equipment, accommodate the temperature to 45°C, and extract pressure at 25Mpa. Resolution pressure is at 6Mpa, and resolution temperature is 45°C, the flow rates of CO_2 is 21.7L/h, then carry out 3 hours. Then gains the abstractive product land 2, weigh the product, determine costunolide and dehydro-α-curcumene. Calculate the extraction percentage of costunolide and dehydro-α-curcumene.
Results:The analysis of range and analysis of variance show that: the factors influencing extraction and costunolide dehydro-α-curcumene volume dose are:(A) extractive pressure >(B) extractive time >(C) extractive temperature. Among of them (A) and (C) are main factors, which possess the statistics meaning. So the best combination is A3B1C3: pressure is 26Mpa、time 3h, temperature is 45°C.
Conclusion: SFE-CO_2 average yield of the prescription is 3.72%. The volume dose of costunolide and dehydro-α-curcumene is 995mg. Extraction percentage is 95.5%. The result of the two experiments is the same. It shows that the craft of SFE-CO_2 is stable and the repeated experiment can be made.
PartⅢThe grading-up of water extraction condition.
Objective: To select the best water extraction craft technique condition.
Methods: According to the design of craft route, the remnant of baizhu, fangfeng, muxiang, chenpi left after the extraction of the SFE-CO_2, together with dangsheng, baishao, shiliupi, jiubiying, gancao, fulin, and the three factors water extraction、extraction time and frequency. Every factor has three levels. The experiment is run in L9 (3~4) orthogonal layout. The index is total weight of solid substance, content of peoniflorin. To investigate the best parameter, the best condition experiment will be made twice.
Results: From analysis of range and analysis of variance, it can be seen that the influencing factor of total solid substance and peoniflorin content can be presumed as C (extract frequency) >A (water duty) >B (extract time), effect of C and A are the principal having statistical significance. The best combination is A3B1C3. Due to the small effect of B (extract time), in order to save time and energy source, we choose B2. So the best combination is decided to be A3B2C3, I.e.: 800% water, extract 3 times, each time lasted 1h.
Conclusion: The total solid substance average dose of baishao and other medicine by water extraction is 138g, peoniflorin total amount is 2121mg, extraction percentage of peoniflorin via water extract is 88%. The results of tow repeat experiments are the same. It explains that the craft is stable and repeated experiment can be made.
PartⅣThe choice and optimization of drying craft.
Objective: With peoniflorin as the index constituent, to screen the craft of drying spray、vacuum dehydration and normal pressure drying, and to grade up drying condition.
Methods: Calculate drying product weight from the three drying craft and determinate the content of peoniflorin, then calculate peoniflorin total changing rate, repeating these experiments, and then decide drying method. Using drying condition as effect factor to physic liquor blowing character、adhesive wall phenomenon、drying time, calculate the remaining rates and total changing rates, and decide the drying parameter.
Results: The result of the experiments proves that if heating too long, peoniflorin would be lost seriously when normal pressure drying and vacuum dehydration is used; drying spray has feasibility, time is short, production is clean, loss of peoniflorin is light, so it is better than other methods. The repeated experiment result shows that, weight of dry powder is 367g, compare with the paste before drying spray, remaining rate is 79.5%. Compared with medical material, the changing rates of peoniflorin is 70.0%. The results of two experiments are the same. It explains that the craft is stable and the repeated experiment can be made. So we will choose drying spray for this craft. The parameter results: the temperature of inlet is 180°C, the temperature of outlet is 100°C, air exhausting parameter is 95%, pump parameter is 25%. The result of the repeated experiment indicates: dry Dower of water extract paste is 366g, peoniflorin weight is 1609g, compared with paste before drying spray, changing rates is 75.82% compare with medical material, total changing rates is 66.08%. The two results of the repeated experiments are the same.
Conclusion: The drying spray craft and drying parameter stabilization and the repeat experiment can be made.
PartⅤGrading-up the craft parameter of preparing granules.
Objective:Grading-up the craft parameter of preparing granule.
Methods: Take dry power of prescription dose add yanhusuo、huanglian fine powder and supercritical fluid extraction, misce bene, and choose different parameters when granulate, granulation is 20-40 mesh. Observe the granules characters.
Results: The result indicates that corpuscular firmness、fine grits proportion are close related to feed material speed of granulator、roller rotation speed and granulate speed.
Conclusion: The best granulate parameters are: 48rpm of feed material speed, 30rpm of roller rotation speed and 30rpm of granulate speed.
SectionⅡEstablishment of the quality standards of Changji' an Capsule
PartⅠIdentify by TLC
Objective: To build up a TLC method to identify yanhusuo, huanglian jiubiying, shiliupi, huotanmu, baizhu in Changji' an Capsule.
Methods:Extract the contents in the capsule by organic solvent, filter, condense and determine the volume, to manufacture solution for the article aqua. Also the differentiate nicety to check against the control article, and add organic solvent to dissolve, metered volume, as control article solution. With the prescription not containing target material, press the praeparatsum craft and use the trial article aqua preparative method, as negative sample solution. Three samples are respectively in the thin layer plate of the same gel silica G, with agent spread out, examine under the ultraviolet light, the visible light responding to lamellar spots may be inspected.
Results:The reference substance and the contents in the capsule display the same lamellar spot in the corresponding position in the colour spectrum, while the negative control does not have any interference..
Conclusion: The methods to identify the yanhusuo, huanglian, jiubuying, shiliupi, huotanmu, baizhu in the capsule are stable can be used to identify Changji' an Capsule.
PartⅡThe limit check on arsenic salt, heavy metal, moisture content, disintegration time limit, microbial limit.
Objective: To check the arsenic salt, heavy metal, humidity, disintegratsion time, microbial limit of three batches of Changji' an Capsule.
Methods: Follow the methods containing in Provisions for New Drugs Approval and Chinese Pharmacopeia I edition, 2005.
Results:The content of arsenic salt is less than 2 ppm, the content of heavy metal is less than 10 PPM, the moisture content is 6.73%, disintegration time limit is 4.5 min. No large angstrom sievert bacterium is checked; bacterial number、mold、yeast count、coli group are all<10/g.
Conclusion: The arsenic salt, heavy metal, humidity, disintegratsion time are all consistent with the regulation of Chinese pharmacopeia I 2005 edition. The microbial limit is consistent with the microbial limit standards of praeparatsum for oral use.
PartⅢDetermining of the characteristic compositions
objective: To determine the content of peoniflorin in baishao, costunolide and dehydro-α-curcumene in aucklandiae, and make up a reasonable content limit.
Methods:Column: Phenomenex luna C18(250mm×4.60mm, 5μm);mobile phase: acetonitrile-water, gradient elution; flow rates:0.8mL·min-1; column temperatsure:30℃; detecting wavelength 230 nm; sample size:10μl. The number of theoretical plate can not be less than 2000 according to peoniflorin, and can not be less than 3000 according to costunolide and dehydro-α-curcumene.
Results:The linear relationship is fine if the content of peoniflorin is between 0.1516-3.032 ug, costunolide is between 0.1116-2.232 ug, dehydro-α-curcumene is between 0.1014-2.028 ug, coefficient correlation r=0.9999; the results of density test、stability test、repeatability test: each peak area score's RSD are less than 2%,the recovery rate of peoniflorin is 100.90%, RSD=1.75%;the recovery rate of costunolide is 101.12%, RSD=1.80%; the recovery rate of dehydro-α-curcumene is 101.37%, RSD=1.22%。the result of sample detection: peoniflorin((?)±s): 3.38±0.05mg, RSD(%):1.43, costunolide ((?)±s): 1.66±0.03mg, RSD(%):1.82, dehydro-α-curcumene ((?)±s): 1.87±0.02mg, RSD(%): 1.05.
Conclusion:Three kinds of characteristic compositions are detected by the same method of HPLC with good precision, repeatability and stability. According to the results and the minimum content limit of Chinese pharmacopeiaⅠ, the content of peoniflorin(C23H28011) in baishao could not be less than 1.6%,each capsule contain 0.105g, according to the prescribed drugs, if the transfer rate is 40%, this article content is determined as: Each capsule contains peoniflorin no less than 1.25 mg, accounted with the peoniflorin (C23H28011);According to the measurement result, each capsule contains costunolide and dehydro-α-curcumene of volume cannot be less than 1.8% at the level with the lowest limit content and muxiang. According to the prescription(each capsule contains muxiang 0.042g), then the limit content is: each capsule contains costunolide (C15H2002) and dehydro-α-curcumene (C15H1802) can not be less than 1.05 mg, in 40% transfer-rate. PartⅣThe GC- MS fingerprint of SFE-CO_2 extraction in the Changji' an Capsule
Objective: To build up the GC- MS finger print of SFE-CO_2 extraction in Changji' an Capsule for controlling the praeparatsum's internal quality.
Methods: The SFE-CO_2 extraction, supersound extracting, analysis the finger print by GC- MS, combining the computer index technique to identify the structure of the separated chemical compound, and the relative contents in percentage were calculated with Area Normalization Method.
Results: Each composition in Changji' an Caps SFE-CO_2 extraction are all well separated. There are 13 compositions be separated, 10 compounds structure are identified.
Conclusion: The GC- MS method can be used to establish finger print of the volatil ingredients in Changji' an Caps.
SectionⅢThe experiment of the main pharmacodynamics of Changji' an capsule.
Objective:According to the main pharmacodynamics of changji' an capsule, to study its function of regulate spleen and liver, eliminate heat and wetness evil and antidarrhea with astringent
Methods: Checking diarrhea experiment, small intestines' propulsion experiment, anti-inflammatory experiment, and relieving pain experiment.
Results: This Chinese medicine (which is called changji' an capsule) can prolong incubation period of defecating dark stool and reduce egesta of small rats, and have a certain function of checking diarrhea, reduce the small intestine peristalsis speed, lower the permeability of blood capillary, and achieve an anti-inflammatory effect. It can also prolong the incubation period of the experiment small rats' body stretching; reduce the frequency of body stretching, and have analgesic effect especially on the ache caused by chemistry substance.
Conclusion: Changji' an capsule has certain functions of checking diarrhea, anti-inflammatory and analgesic effect, and provides the pharmacodynamic basis for treating Diarrhea-IBS, chronic colitis, chronic indigestion, stomachache, diarrhea, etc.
SectionⅣEstablishment of rats experiment model with Diarrhea-IBS and the valuation of its internal organs sensitivity
Objective:To build up the rats experiment model with Diarrhea-IBS, observe the times of the rats defecation and the value threshold change of ectocolon and the influence of bowel movement by adding ACH.
Methods:Give the acetic acid to stimulate the rectum of the rats new-born only for 8 days everyday, keep on for 2 weeks, duplicate the model of high sensitivity of internal organs of rats and count the number of egesta of model rats on 14th days, 15th days, 16th days within 4 hours. Evaluate threshold value of rats' belly shrink reflect (AWR) and 6 weeks and 8 weeks after they were born., they have their rectum extended. Measure the rising range of the bowl taken from the model rat movement on which ACH is affected.
Results: Compared with the normal groups, the model groups show defecation times increasingly, and its egesta are not confectioning molding. When the rectum be extended, the press use threshold value and belly raising value of the model group are cut down more obviously than those of the normal groups. After ACH is joined into the isolated bowel, the value of the model group goes up, all obviously higher than those of the normal groups.
Conclusion: Stimulating the bowel of model rats in new-born period by the chronic inflammation can cause chronic bacillary diarrhea and increase the internal organs sensitivity in adult, and increase the reaction of the spasm agent which stimulate the isolated bowel, all those essentially match Diarrhea-IBS basic characteristics.
SectionⅤResearch on the Mechanism of Changji' an Capsule Intervening Diarrhea-IBS Model Rats.
Objective: To study the intervention mechanism which Changji' an capsule has on Diarrhea-IBS model rat.
Methods: Randomly divided rats of new-born into normal group, model group, Chinese herbal medicine in high quantity group, Chinese herbal medicine medium quantity group, Chinese herbal medicine low quantity group and the pill of Bupi yichang pill group. New -born rats in normal group are given 0.9% physiology in rectum, the other groups from 8th day to 21st day are given the acetic acid to stimulate the rectum everyday, keeping on 2 weeks, duplicating the model of high sensitivity of internal organs of rats and counting the number of egesta of model rats on 14th days, 15th days, 16th days within 4 hours. After 8 weeks, infuse the stomach to give respectively the NS, Changji' an capsule liquid 3.76g/ kg weight, 1.88g/kg weight, 0.94g/kg weight and Bupi yichang pill 6.76g/kg weight, once daily, for two weeks, observe the rats again after medicine is taken and evaluate its belly constringency reflect threshold (AWR) value. Measure the rat blood with the cell instrument in a percentage for distribution of T lymph-cell, observes the rat bowel organization of immunity dyeing of 5-HT. Observe those cell degranulation of mast cell and count mast cells number with light microscope, and observe MC cells and their adjacent structures.
Results:Changji' an capsule can raise the press threshold value of sensitivity of the model rats, and has diarrhea checking function to Diarrhea-IBS. It can increase CD_4~+ value in blood gradually, CD_8~+ and the specific value of the CD_4~+ CD_8~+ which recover normally, improve the immunity function. Changji' an can lower increasing secretion 5-HT of the model rats of IBS, improve allergy of internal organ, diarrhea symptom and motive abnormality caused by 5-HT going up unusually, in each group of colon glue surface and no ulcer or debauched was seen, the gland body size and appearances are normal, the glandular organ mucus and cup cell form were seen abnormal, a little amount cell of phlegmasia inside the quality is gradually changed, each colon organization has no change, matching the clinical pathology of IBS performance. The experiments show that the Chinese herbal medical has certain function on the IBS chronic bacillary diarrhea and phlegmasia diseases. The number of mast cells of proper layer and degranulation in model group is higher than those of normal group obviously, and the number of mast cell of proper layer and degranulation in high quantity group is smaller than those of model group, but is not difference with the normal group. It shows that, the medicine liquid can reduce the mast cell quantity, improve the model rats colon organization in the mast cell degranulation, and the instauratsion that make it normal. The medium quantity group also can improve degranulation of mast cells obviously, showing it has the function of stabilizer, this function may be one of main mechanism that changji' an capsule treating IBS, gives an electric mirror, to observe mast cells to locate the bowel to proper layer, implying many grains with higher electronics density in the afterbirth syrup; The mast cells of model group has part of degranulation, empty bubble formation; Each group can be seen to have mast cells and the nerve fibers are adjacent. As a result, it expresses that a MC possibility that activates the appearance is in the IBS expression with the motive change, the felling abnormality for characteristic of internal organs raise the decisive function in the pathologic physiology process of the high sensitivity.
Conclusion: The function mechanisms of Changji' an capsule intervening Diarrhea-IBS are: (1) Improve sensitivity of bowel of the model rats of IBS; (2) Reduce times of loose stools;(3)Improve the cell immunity function of the model rats; (4) Improve bowel abnormality caused by the unusual increase of 5-HT and allergic feeling of internal organs; (5) Relieve intestinal mucosa edema and inflammatoryreaction caused by IBS. (6) Reduce the mast cell quantity, improve the colon organization degranulation, have the function that stabilizes mast cells. The result for further demonstrating the disease occurence mechanism of IBS and taking intervention measures helps establish necessary research foundation. This shows that the overall function of multiple targets of Chinese herbal medicine in its intervention of IBS. Section VI Peculiarities and New ideas in this Study.
Firstly, The IBS is a common chronic disease with characteristic of high incidence rate, long course and slow recovery. This research which exploited hospital praeparatum with exact curative effect, aims at the disease characteristics, makes traditional prescription and modern medicine techniques combine together, It adopts advanced techniques of SFE- CO_2, spraying dry technique of fog etc., excellent craft, and better exertive efficacy of medicine, It reduces stimulus and makes use of the modern and analytical technique of GC- MS, HPLC etc. to control its quality strictly, establishes the volatility composition fingerprint diagram, and makes the craft to have the advanced novelty. Secondly, currently there are many animal related models of IBS, but built up according to the single factor outside the body that mostly matches the IBS animal model on some pathologic physiology mechanism, but the combination of disease and symptoms in models is not reported. Feeling mood factor has something to do with symptom of IBS when it occurs and aggravates, and feeling mood can cause liver gloomy. This experiment chooses to use the model stimulated by the chemistry chronic inflammation, and the incitement placed in the new born period of new born rats, belonging to the animal model which caused by earlier period event, so it is also a kind of feeling mood factor to the rats. Chronic inflammatory stimulation in bowel in new born rats may cause the chronic internal organs sensitivity to increase after they become adult, matching IBS basic characteristics. Thus, building up of the model rats with Diarrhea-IBS have stability and repetitions advantages, the maneuverability is strong, it has characteristic of being evaluated, thus having important and realistic meaning to the further sieving of the treatment medicine. Finally, most researchers of the world concentrate upon clinic study of IBS. there are few studies based on animal experiment. This article studies in detail the mechanism of Changji' an capsule from sensitivity of bowel, 5-HT, mast cell, mast.cell degranulation etc, thus enriching the contents of IBS basic research, and explaining complex action of multitargets.
引文
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