【作者】 李绍林；

【导师】 涂瑶生；

【作者基本信息】 广州中医药大学 ， 中医学， 2012， 博士

【摘要】 湿疹为临床常见病,多发病,是由机体内外激发因子引起的一种迟发型变态反应,并伴随激烈瘙痒或皮肤炎症现象。西药多采用激素、抗生素或抗组胺类药物治疗该病,长期使用,毒副作用大,临床使用受限。湿疹方是治疗湿疹的临床验方,由黄连、苦参、牡丹皮及冰片等4味中药组成,具有清热解毒、燥湿止痒、调节机体免疫的功效,该方临床以洗剂外用,疗效确切,但存在药效成分不明确,提取制备方法原始、粗简,缺乏标准,内在质量难以控制,载药量小,作用时间短,生物利用率低等缺点,同时洗剂使用和携带均不方便,影响了其临床推广应用。为了提升该方质量稳定性,增强药效成分的透皮性能,提高疗效及患者的使用依从性,在前期洗剂的研究基础上,本课题对湿症方进行了系统研究,取得了较满意的结果。本文首先通过湿疹方提取、纯化工艺路线的研究,确定了湿疹方的最佳提取纯化工艺路线,优化了处方中各中药的提取、纯化工艺,通过处方前研究掌握了各纯化物的理化性质,综合应用微乳与凝胶制备技术,将湿疹方纯化物药物,制备成了复方湿疹微乳凝胶剂,并对其进行质量评价。全文主要研究内容如下：1湿疹方提取纯化工艺路线的确定针对本制剂制备的需要,分别采用药效学方法,以小鼠接触性湿疹模型中耳肿胀抑制率为评价指标,探讨本品处方中药材分煎、合煎以及不同提取组分物对药效的影响。结果表明,与模型对照组比较,单煎组与合煎组对小鼠接触性湿疹均有明显的抑制作用(P<0.01),且两组之间无明显差别(P>0.05),提示,处方中药材可采用单独提取,所得提取纯化物加入处方量冰片制备成制剂；采用正交试验法,以小鼠接触性湿疹中耳肿胀抑制率为考察指标,考擦本品处方量黄连、苦参、牡丹皮不同提取纯化物与冰片混合后的药效,结果表明,黄连总生物碱纯化物、苦参总生物碱纯化物、丹皮酚、冰片混合物作用最好,初步确定处方各中药提取纯化工艺路线,为湿疹方各中药的提取方法和后续的处方工艺研究奠定了基础。2提取纯化工艺的优化(1)指标成分的含量测定：采用HPLC法分别对黄连总生物碱纯化物指标成分“HLJ”(盐酸小檗碱、巴马汀、黄连碱及表小檗碱总和)、苦参总生物碱纯化物指标成分“K”(苦参碱与氧化苦参碱总和)及丹皮酚进行了含量测定,建立的方法灵敏准确,简单实用,为本课题的研究提供了可靠的检测手段。(2)黄连提取纯化工艺优化：以HLJ的转移率为考察指标,优选黄连提取纯化工艺。采用单因素与正交试验结合法,确定黄连提取工艺为：取黄连药材饮片,加8倍量1.5%(m/m)硫酸溶液回流提取3次,每次40分钟,HLJ提取率高达93%；提取液以酸碱置换及盐析相结合的方法,制得黄连总生物碱纯化物,总碱含量(以HLJ计)大于80%,得率7.70%,方法工艺稳定,可行。(3)苦参提取纯化工艺优化：以K的转移率为考察指标,优选苦参提取纯化工艺。采用单因素试验结合星点设计.效应面法,确定苦参提取工艺为：取苦参药材饮片,加11倍量65%的乙醇提取2次,每次65分钟,K提取率大于90%,总固物得率大于23%；提取液过732型阳离子树脂柱,制得苦参总生物碱纯化物,总碱含量(以K计)大于41%,得率4.67%,方法工艺稳定,可行。(4)牡丹皮提取纯化工艺优化：采用水蒸气蒸馏法,以丹皮酚收率为考察指标,确定丹皮酚制备工艺为：取牡丹皮药材饮片,加8倍量水,浸泡2小时,共水蒸馏,收集药材5倍量馏出液；馏出液冷藏,取沉淀,制得丹皮酚,丹皮酚含量大于96%,得率1.11%,方法工艺稳定,可行。3处方前研究对黄连总生物碱纯化物、苦参总生物碱纯化物、丹皮酚制剂处方设计前的主要理化性质进行了测试。结果表明,HLJ微溶于水,K易溶于水,其油水分配系数均小于1,其中,K的油水分配系数大于HLJ,丹皮酚易溶于有机溶剂,油水分配系数较大；稳定性试验结果表明,黄连总生物碱纯化物、苦参总生物碱纯化物对光、湿、热稳定,丹皮酚易升华挥发；以黄连总生物碱指标成分HLJ以及丹皮酚为考察指标,对湿疹方的体外透皮性能进行了考察,结果表明,黄连总生物碱中HLJ透皮速率为6.36ug.cm-2.h-1,丹皮酚透皮速率为6.83ug.cm-2.h-1,吸收机制均符合为零级过程。以上工作为剂型的选择和制备工艺的确定提供了依据。4复方湿疹微乳凝胶制备工艺研究为提高药物的透皮性能,采用微乳作为药物载体,结合凝胶剂的特点,制备复方湿疹微乳凝胶。在复方湿疹微乳制备研究中,通过单因素及正交试验筛选出微乳的油相、表面活性剂、助表面活性剂、水相分别为：肉豆蔻酸异丙酯(IPM)、Cremephor EL(EL)、丙三醇、去离子水；通过体外透皮试验,以药物透皮性能为考察指标,确定微乳各相的百分含量,筛选出最佳微乳处方为：IPM6.5%,EL-丙三醇(1：1)58.5%,水35%,所制得的复方湿疹微乳药物透皮效果最佳。对空白微乳理化性质进行了考察,结果显示微乳粒径分布范围10-100nm, Zeta电位为负。微乳对提取纯化物具有增溶性,对试验内的药物、温度、pH值均具有较好的稳定性。以复方湿疹微乳凝胶样品的光泽性、粘稠度、均匀度、涂展性、稳定性为考察指标,筛选出凝胶基质为卡波姆940(CP-940),用量为0.5%,结果表明微乳加入凝胶基质CP-940制成微乳凝胶后,体系的电势能提高,稳定性增强。透射电镜观察呈圆球形,粒径在100nm内。验证试验显示,复方湿疹微乳凝胶制备工艺稳定、可行。5复方湿疹微乳凝胶质量评价生物学评价：通过量效关系的考察确定复方湿疹微乳凝胶的载药量为0.9%,其中黄连总生物碱纯化物含量为0.47%,其余各提取纯化物折算为原处方生药比例加入。小鼠实验性湿疹试验中,相同生药载药量的原湿疹方洗剂与复方湿疹微乳凝胶剂比较,后者较前者药效得到显著提高(P<0.01)。安全性考察试验,结果显示复方湿疹微乳凝胶制剂无不良刺激、无过敏性、无急性毒副作用,复方湿疹微乳凝胶药效强、安全性好。理化评价：对复方湿疹微乳凝胶体理化常数进行评价,分别建立了TLC定性鉴别及HPLC定量分析的方法,方法准确、稳定、简便。通过透射电镜观察微乳呈圆球形,粒径小于100nm；体外透皮试验显示,复方湿疹微乳凝胶较原湿疹方洗剂药物的透皮性能显著提升(P<0.01),其中黄连总生物碱指标成分HLJ透皮速率提高了约16倍,丹皮酚透皮速率提高了约10倍；初步稳定性试验显示,复方湿疹微乳凝胶稳定性良好。本文研制的复方湿疹微乳凝胶制剂,较原湿疹方洗剂,药物透皮性能得到显著提升,药效增强,产品药效成分明确,质量均一、稳定、可控,提高了用药的方便性、舒适性、安全性,具有良好的市场应用前景。更多还原

【Abstract】 Eczema is a common, frequently-occurring disease, it is a kind of delayed allergic reaction, caused by evocators both from outside and inside, accompanying with intense itching or skin inflammation. Western medicine such as hormones, antibiotics, or anti-histamine, is used for curing this disease. But its clinical application is limited in this area, because of side effects for long-term use. Shi-zhen Prescription is used for curing eczema, composed of coptidis rhizoma, sophora flavescens, paeonia suffruticosa and borneol, with effects of removing pathological heat and removing toxicity, relieving itching by eliminating dampness and regulating the body’s immune systerm. The prescription in form of lotion is commonly applied in clinic with its exact accurative effect. Meanwhile, its shortcomings such as uncertain pharmacodynamic materials, crude preparation with low standards, quality control difficulties, small drug loading, short action time, low bioavailability, inconvenient application and taking along, and so on, limited its clinical generalization. In order to promote the prescription quality stability, enhance the ability of drug’s skin penetration, enhance the curative effect and patient compliance, systematic research was made on Shi-zhen prescription, on the basis of former studies.In this paper, firstly, the optimal extraction and purification processes were determined by the research on extraction and purification of Shi-zhen prescription. Secondly, the physicochemical properties of the purified materials were mastered by the preformulation studies, thirdly, with integrated application of microemulsion and gel’s advantage, the purified materials were made into "Shi-Zhen compound microemulsion-based gels", finally, physicochemical and biology evaluation of the new preparation was made.The main content of the research in this paper was as follows.1Determination of the extraction and purification processes of Shi-zhen prescriptionFor the preparation of microemulsion-based gels, with the pharmacodynamics methodology, taking the inhibition rate of mice ears-swelling as the investigation factor, studies on the different influence on prescription’s efficacy by decocting separately and together.The result showed as follows:Both single-decoction and mixed-decoction group had a significant effect on inhibiting mice ears-swell ing (P<0.01), compared with the model group, with no significant difference between single-decoction and mixed-decoction group (P>0.05). The result showed that the Shi-zhen prescription could be decocted Separately.the preparation could be made of the purified material and borneol.Taking the inhibition ratio of mice ears-swelling during the mice contact eczema test as the investigation index,studies on the coptidis rhizomas’、sophora flavescens’and paeonia suffruticosa’s different extracted and purified materials’s effection on the prescription’s efficacy by orthogonal experimental design.The result showed that the combination of the purified matetials of total alkaloids from coptidis rhizome, total alkaloids from sophora flavescens, paeonol and borneol had the strongest efficacy. The extraction and purification processes were determined preliminarily. The above findings laid the foundation for the subsequent studies on formulation and processes.2Optimization of the extraction and purification processes(1) Determination of ingredient index’s content:Studies on content’ determination of HLJ(berberine hydrochloride, palmatine, coptisine and epiberberine), K(matrine and oxymatrine)and paeonol, which respectively stand for ingredient index of the purified materials:total alkaloids from coptidis rhizome, total alkaloids from sophora flavescens and crystals from moutan cortex. The established methods for content’s determination were sensitive, accurate, simple and practical.(2) Optimization of coptidis rhizome-extraction and purification processes.Taking transfer rate of HLJ as evaluation index,the optimized extraction processes of coptidis rhizome were screened by single factor and orthogonal experimental design. The optimum condition of extraction processes were that the tablets ready for decoction were decocted with8-fold1.5%(m/m) sulfuric acid solution for3times,40min each time. The transfer rate of HLJ was above to93%;Total alkaloids was made from extract by methords of acid-base replacement and salting out method. The yield of total alkaloids was about7.70%, and the content of HLJ was more than80%. The processes of preparation were stability and feasible.(3) Optimization of sophora flavescens-extraction and purification processes:Taking transfer rate of K as evaluation index, the optimized extraction processes of sophora flavescens were screened by single factor and central composite design-response surface experimental design. The optimum condition of extraction processes were that the tablets ready for decoction were decocted with11-fold65%alcohol for2times,65min each time. The transfer rate of K was more than90%. The yield of dried extractum was more than23%. The total alkaloids was purified by732cationic resin processes,the content of which was over41%(based on the calculation of K),and the yield of which was about4.67%. The processes of preparation were stability and feasible.(4) Optimization of paeonia suffruticosa-extraction and purification processes: Taking yield of paeonol as evaluation index, The paeonol was extracted from paeonia suffruticosa by steam distillation technology, and the optimal extraction processes were as follows:the tablets ready for decoction were socked for2h, then decocted with8-fold water.5times crude drug-amuout of distillate was collected. the distillate was placed in refrigerator (4℃) for12h, Filtered, and paeonol was gotten, The yield of paeonol was aboutl.11%, and the content of paeonol was about96%。 The processes of preparation were stability and feasible.3Preformulation studiesThe studies on physicochemical properties of the purified matetials of total alkaloids from coptidis rhizome, total alkaloids from sophora flavescens and paeonol showed that HLJ was slightly soluble in water, k was hydrophilic, their oil and water distribution coefficient were both less than1, and k had larger distribution coefficient. In addition, the paenol was soluble in organic solvents, its oil and water distribution coefficient was larger than the former. Stability test results showed that the total alkaloids from coptidis rhizome and the total alkaloids from sophora flavescens were kept unchanged in the condition of light, wet, and heat, but the paenol was easily volatile. We took HLJ and paenol as the index to explore the purified materials’ skin permeation. The result showed that the speed of HLJ’s skin permeation was6.36ug·cm-2·h-1and the speed of paenol’s skin permeation was6.83ug·cm-2·h-1. Their absorption mechanisms belonged to the zero-order kinetics. All of the above provided the basis for the forms of the choice and the determination of the preparation technology.4Preparation of Shi-zhen compound microemulsion-based gelsIn order to improve drug’ ability of skin permeation, microemulsion was used as the drug delivery system.Shi-zhen compound microemulsion-based gels was prepared, combined with the characteristics of microemulsion and gels.In the research of Shi-zhen compound microemulsion-based gels’ spreparation, types of oil phase, surfactant, cosurfactant were choosed by single factor and orthogonal experimental design. According to the result, isopropyl myristate (IPM, oil phase), Cremephor EL(EL, surfactant) and glycerol (cosurfactant), and double-distilled water were determined as the composition of the Shi-zhen microemulsion. Percentage composition of microemulsion’s each phase was determined by in vitro transdermal permeation experiment. According to the result of the experiment,6.5%isopropyl myristate(IPM),35%double-distilled water and58.5%Cremephor EL/glycerol(1:1) as the optimal prescription of microemulsion was ascertained. The result showed the optimal microemulsion had the best ability of skin permeation.Studies on physicochemical properties, solubilization and stability of blank micromulsion were investigated primitively. It showed that the microemulsion preparation can increase the purified materials’ solubility and the stability to the purified materials, pH, and temperature in the test. Taking the glossiness, viscosity, degree of homogeneity, stretchability of the preparation as the investigation index, carbomer940was chosed as the optimal gels’ base material, and the content of it was0.5%. microemulsion-based gels’ zetea potential got higher than blank microemulsion, and The new preparation microemulsion-based gels got more stable than blank microemulsion,with diameter less than100nm, showing round shape in the horizon of transmission electron microscope(TEM). Verifying test showed that the processes of preparation for Shi-zhen compound microemulsion-based gels were stability, reasonable and feasible.5Quality evaluation of the Shi-zhen compound microemulsion-based gels Biological evaluation:Drug loading of Shi-zhen compound microemulsion-based gels was determered by the dose-effect relationship experiment, which was0.90%(calculated by the amount of the purified materials, the content of HLJ was0.47%). With the same drug loading, Shi-zhen compound microemulsion-based gels’drug efficacy was improved significantly, compared with the original lotion preparation. The Shi-zhen compound microemulsion-based gels had no side effects of irritation, irritability or acute toxicity to the guinea pigs and New Zealand rabbits.Physicochemical evaluation:Methods of qualitative identification (TLC),and quantitative analysis(HPLC) for microemulsion-based gels were established, which were accurate, stable and reliable. According to the in vitro transdermal permeation experiment, it showed that the component HLJ’s skin penetration was improved about16times, and paeonol’s was improved about10times than the original lotion preparation. Preliminary stability test showed that Shi-zhen compound microemulsion-based gels had good stability.Drugs in the Shi-zhen compound microemulsion-based gels had a higher ability of skin permeation and stronger drug efficacy.compared with the orginal lotion, the Shi-zhen compound microemulsion-based gels performed controllable, homogeneous and stable quality, convenient application and good market prospect.更多还原