Reasearch On Preparation And Quality Evaluation Of Microemulsion-based Gels From Shi-zhen Prescription

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.