Optimization Of Realgar Composition And Preparation Of The Kit For Down-regulating Gain-of-function Ras Pathway

Realgar, one of the most common mineral drugs, has been confirmed to be clinically effective for the treatment of refractory or relapsed acute promyelocytic leukemia (APL) and chronic myelogenous leukemia (CML), recently, but its poor water solubility, high toxicity and low bioavailability hampered its further clinical application. CN1839880A provided a bioleaching method for realgar. In contrast with the traditional realgar, realgar bioleaching solution increased in bioavailability, decreased in the toxicity, and improved in anti-cancer activity. However, there is still possibility to promote its activity of realgar bioleaching solution, to reduce its toxicity. The objective is to optimize realgar composition which can down-regulate gain-of-function Ras pathway in Caenorhabditis elegans. Meanwhile, the reagar composition should be less toxic than reagar bioleaching solution. Finally, a kind of kit is developed for screening drug candidates which can down-regulate gain-of-function Ras pathway in Caenorhabditis elegans.Realgar compotion is composed of realgar bioleaching solution, matrine, ginseng extract and astragalus extract. Each component was tested to be qualified before use. Arsenic was measured by ICP-AES, Rg1, Re and Rb1 in ginseng extract were analyzed by HPLC and astragalosides in astragalus extract was detected by HPLC with ELSD detector. Realgar composition was optimized by orthogonal experiments, it indicated that the ingredient with 12.8μg/mL(total arsenic) of realgar bioleaching solution,2μg/mL matrine,1.35 mg/mL (crude drug) of ginseng extrat and 1.0 mg/mL (crude drugs) of astragalus extract was a promising realgar composition.To test the toxicity of realgar composition, several indicators were examined in C. elegans. Realgar could significantly delay the growth and development of the nematodes, but realgar composition could not; Compared to the control group, the brood size was not affected by realgar composition, but it decreased significantly after treatment with realgar bioleaching solution; Egg-laying summit of C. elegans was delayed by realgar bioleaching solution, while realgar composition rescued the delay of egg-laying summit; There are few differences between realgar bioleaching solution and realgar composition on pharyngeal pump rate in C. elegans; Realgar composition could induce the translocation of the DAF-16 from the cytoplasm into the nucleus more obviously than realgar bioleaching solution.To detect drug candidates which could down-regulating gain-of-function Ras pathway rapidly, low costly, effortless and coveniently, a kind of kit was developed. Conditions for large scale cultivation and storage of C. elegans were explored.