Studies On Quality Standard For Compound Loutuopengzi Ointments And Hydrophilic Gels

Objective: To improve the quality standard for compound Loutuopengzi ointments(CLO). To set up quality control methods for determining active ingredients in Colchicibulbous and Hyoscyami semen. Meanwhile, based on the researches of quality control forColchici bulbous and Hyoscyami semen and preparation of alkaloids effective parts ofPeganum harmala semen, Colchici bulbous and Hyoscyami semen, to develop a newdosage form of compound Loutuopengzi hydrophilic gel (CLHG), a multi-fractions TCM,and to explore its permeation effects and drug release behavior of CLHG in vitro.Methods: CLO was identified with ethyl acetate-methanol-ammonia water (17:2:1) asdeveloping agent in silica gel HSGF254plate with bismuth potassium iodide test solutionand0.8mol/L Sodium sodium nitrite solution as the visualization reagent. Thesimultaneously determination of vasicine, harmaline and hamine was achived on a BostonLuna C18column (150mm×4.6mm,5μm) with acetonitrile-ammonium acetate buffersolution as mobile phase (gradient elution), and the detective wavelength was set at265nm. The determination of colchicine in Colchici bulbous was performed on SymmetrixODS-H column and mobile phase consisted of acetonitrile-0.1%tri-fluoroacetic acid(28∶72). The flow rate was1.0mL/min and the UV detective wavelength was set at350nm.The determination of scopolamine and hyoscyamine in hyoscyami semen wasperformed on Symmetrix ODS-H column and mobile phase consisted of acetonitrile-0.1%trifluoroacetic acid (14∶86). The flow rate was1.0mL/min and the UV detectivewavelength was set at210nm.The enrichment of total alkaloids was separated and purifiedon five types of macroporous resins with special structures. Colchicine was determined byHPLC. The performance of colchicine on macroporous resins including LSA5B, LSA21,LSD001, HPD600, and D101were compared according to their adsorption and desorptionproperties, when the conditions of the processing volume, the pH value of loading solution,adsorption flow rate, and desorption flow rate being investigated. To optimize theformulation and technology of CLHG, central composite design-response surface methodology was employed, with percutaneous cumulative permeation amount ofharmaline and harmine as index, and also the skin permeability effect of the dosages ofGel matrix, azone and triethanolamine were investigated. A second-order polynomialequation was fitted to the data, and the resulting eqution was used to predict the responsesin the optimal region. The response variable was found to be highly dependent on theformulation variables. Results: Peganum harmala semen, colchici bulbous, andhyoscyami semen in CLO can be identified by TLC using vasicine, harmaline, hamine,scopolamine, atropine, and colchicine as indexes. The simultaneous determination ofvasicine, harmaline and hamine in CLO could be used as quality control method. Thevaluation results shown that the linear ranges of vasicine, harmaline and hamine were1.0056~201.1200μg/mL (r=0.9998),1.0016~200.3200μg/mL (r=0.9998),0.8599~171.9887μg/mL (r=0.9996), respectively. The recoveries were98.12%,97.13%, and98.29%, with RSD all being less than2.0%. The mass fraction of vasicine, harmaline andhamine in three batches of CLO were0.17%~0.34%,0.27%~1.23%, and0.77%~1.17%, respectively. But, the determination results for CLO fluctuated with significantvariance, it indicated that the homogeneity of CLO was a potential problem and needed tobe solved further. Colchicine could be used as index to establish a HPLC method toeffectively control the quality of colchici bulbous.The calibration curve of was linear inthe ranges of2.5~160.0μg/mL (r=0.9999) for colchicine. The average recoverie was96.55%, with RSD0.95%.The mass fractionof colchicine in7batches of holchici bulbuswas0.02%~0.13%. Atropine and scopolamine, as index ingredient, could be used toestablish a HPLC determination method to quickly control the quality of hyoscyamisemen. The linear ranges of scopolamine and hyoscyamine were4.02~513.64μg/mL(r=0.9999) and4.00～512.00μg/mL (r=0.9998), and the average recoveries were98.41%and98.79%. LSA5B was demonstrated with a remarkable capability forenrichment of alkaloids from colchici bulbous and hyoscyami semen. The optimalconditions for total alkaloids from colchici bulbous were composed of the pH value ofloading solution of2.5and flow rate of1.5BV/h. The gradient elution program of waterfor8BV then70%ethanol for20BV was used to wash impurities and enrich totalalkaloids with saturated absorption of26.49mg/g colchicine and flow rate of1.5BV/h.The desorptive efficiency could exceed98%. The optimum conditions for the enrichmentprocess of hyoscyami semen alkaloids were composed of the flow rate of1.5BV/h, thesaturated absorption of scopolamine and hyoscyamine were2.835and4.947mg/g. Thegradient elution program of water for4BV then50%ethanol for10BV was used to wash impurities and enrich for total alkaloids from hyoscyami semen with saturated absorptionof2.835mg/g,4.947mg/g of scopolamine and hyoscyamine with loading flow rate of1.5BV/h. The desorptive efficiency could exceed74%and86%. The optimal formulation ofCLHG could be obtained at1.00%of carbomer-934,1.50%of azone, and2.00%oftriethanolamine. The deviations between measured values and predicted values werewithin5%. The central composite design-response surface methodology can well predictthe relationship between the factors and evaluation indexes. Conclusions: Established themethod of qualitative and quantitative is rapid, simple, sensitive, precise and stable, whichcould be used for the quality control of colchici bulbous, Hyoscyami semen and CLO andlaid the foundation for improving quality standards of CLO. LSA5B was demonstratedwith a remarkable capability for enrichment of Colchici bulbous and Hyoscyami semen.The colchicine content of Holchici bulbus was enhanced25folds higher thanpre-treatment solution by using LSA5B resin under the optimum technological conditions.The hyoscyamine and scopolamine content of Hyoscyami semen were enhanced18foldshigher than pre-treatment solution by using LSA5B resin under the optimum technologicalconditions. The central composite design and response surface methodology is useful forthe formula optimization of CLHG. The prediction accuracy of the established model isgood. The gels matrix prepared by carbopol, which was transparent, delicate, smooth,uniform, sticky and easy to clean and obtain the perfect transdermal effect. The researchprovided a firm foundation for redeveloping on the new dosage form in quality controllingwith CLHG and studying on pharmacodynamics,transdermal absorption and vivometabolism.