Study On The Preparation Of Compound Cream Of "Keqingjia" And Its Quality Standard, Safety, And Pharmacodynamics

Oidiomycosis unguium is a special type of a class of fungal diseases, which predominantly caused by candida albicans. Chemotherapy drugs were widely used for clinical treatment of oidiomycosis unguium. But they are expensive and have serious side effects. Initial study shows that, Radix Sophorae Flavescentis, Rhizoma Smilacis Glabrae and Cortex Dictamni, which usually used in empirical formula of traditional Chinese medicinal, exhibit a strong inhibition effect on candida albicans. Miconazole nitrate, an imidazole ntifungal drug, has a favourable inhibition effect on cutaneous fungus and deep fungus eg.candida albicans. In order to develop a new type antifungal agent, this research devoted on the selection of best extraction technology, combined effect between extracts from magistral formula extracts with Miconazole nitrate in vitro, and the selection of cream prescription. At last, on the base of those studies, investigate the quality standard, safety and pharmacodynamics in vitro of compound "Keqingjia" cream (CKC). The main results as follows:1. Taking minimal inhibitory concentration (MIC) of extracts to Candida albicans as an index, discussing the effect of concentration of ethanol (A), each time of extraction (B), the proportion of three components (C) and solid-liquid ratio (D), to chose the best extraction technology by orthogonal design. The component pretesting of magistral formula extracts was carried out by chemistry method. The result of the experiment shows that the optimum technology which is A3B2C3D1 obtained from the test. That means extract with alcohol concentration of 70%, the proportion of 2:2:1, solid-liquid ratio of 1:10 and extraction time of 2 hours. The yield is about 23%, and MIC is 0.195 mg·mL-1. The component test results revealed that polysaccharides, flavonoids and alkaloids are the major components of magistral formula extracts.2. The checkerboard microdilution method was applied in vitro to study interaction of candida albicans to a combination of magistral formula extracts and miconazole. Through the research of antifungal activities of magistral formula extracts and 2% miconazole nitrate with different proportions, to decide the best compatibility proportion.The results shows that compared with the single agents, the minimal inhibitory concentration (MIC) of the combination of magistral formula extracts reduced from 0.195 mg·mL-1 to 0.049 mg-mL"1, miconazole nitrat reduced from 7.8μg·mL-1 to 3.9μg·mL-1. The value of fractional inhibitory concentration index (FICI) was 0.751. It shows that, there is a synergetic action against candida albicans in a combination of magistral formula extracts and miconazole nitrat. Both of the proportion of 3:land 4:1, have the best antifungal activities and the MIC was 0.097mg-mL-1.3. The formula for oil in water (O/W) cream of "Keqingjia" includes magistral formula extracts, miconazole nitrat, Carbamide, white Vaseline, octadecanol, Tween 80, glyceryl monostearate, and so on. The prescription of compound "Keqingjia" cream was optimized. Through optimize the value of hydrophile-lipophile balance (HLB), and the dosage of mixed emulsifiers and oil phase, to preparing compound "Keqingjia" cream which could come up to the requirements in character, thermostability, cold-resistant and centrifugal test. The results shows that the optimized prescription was shown as follows: the value of HLB was 10.5, the dosage of Tween 80 was 6%, the dosage of mono-stearin was 4%, the dosage of white Vaseline was 8%, and the dosage of octadecyl alcohol was 7%.4. Through characteristic evaluation, identify carbamide and matrine in official method and detection the content of matrine by TLC Scanning, to establish quality standard of compound "Keqingjia" cream (CKC). Result shows that it can identify carbamide and matrine by official method. In TLC scanning, the linear ranges of matrine was 1-9μg (r=0.9951), and the average content of matrine in 4% cream was 0.28%.5. To investigate the safety of compound "Keqingjia" cream (CKC), the test about acute toxic skin reactions, skin irritation and eye irritation were experimented on health rabbits, and the skin allergy test was experimented on health guinea pigs. And all the reactions were kept under observation. The result shows that, neither acute toxic effects nor eye irritant in experimental animals was induced by applications of CKC. No irritant dermal reaction was observed on intact skin. Slight irritation reaction was observed on injured skin after the repeated applications of CKC, but faded away while removing medicine afterwards. No allergic reactions on intact and injured skin of guinea pigs. That means CKC is a safe topical preparation.6. In order to study the effect of CKC, we established oidiomycosis unguium model in vitro. Normal fingernails were divided into 2 groups (control group and model group). The model group's nails cultured 2 weeks in liquid medium which contains candida albicans, to establish the patho-model. Then made pathological section and examined by PAS staining. Groups of model nails were divided into untreated group, base cream group and drug treatment group. And the drug treatment group was divided into three concentration group (2%,4% and 8%). After 3 weeks, observe them by PAS staining method at last. The results show that, in control group, hard keratins were dense and orderly arrangement, structural integrity and uniform and pale colored. In 2 weeks model group, there is an obviously dividing line between diseased region and normal structure. In untreated group, the structure of nails were destroyed completely, and it have a large number of hole-like structure, in which full of fungus, gathered into a mass. In base cream group, the middle layer of nails remains some normal structure, but the structure of outer layer which were destroyed has assumed rarefaction, and easily to shed. In concentration groups, the normal structure has assumed in middle layer of nails, which has not been invaded and destruction by fungus. The higher concentration, the lighter extent of stratum corneum and the outer horny is more easily shed. It's successfully established oidiomycosis unguium model. CKC has a certain effect on oidiomycosis unguium.