| BACKGROUNDGlycyrrhiza flavonoids have significantly pharmacological functions,such as liver protection,anti-oxidation,anti-tumor,enzyme inhibition and so on.Glycyrrhiza flavonoids are effective raw materials which have been applied for whitening cosmetics.However,it show whitening effect poor thanks to poor solubility and low local bioavailability in the skin.Microsponge is a kind of drug carrier system that can improve the retention of the drug,prolong the release time of drug and improve the bioavailability of drug in the skin.Moreover,it also improves the stability of drug in the preparation.It has a great significance in the treatment of topical disease especially in whitening.OBJECTIVEBased on the study of microsponge,we would like to prepare glycyrrhiza flavonoids into the glycyrrhiza flavonoids microsponge which improve the local bioavailability of the drug.It can raise the retention of the drug and make drug form a reservoir in the skin so that strengthen the efficacy of glycyrrhiza flavonoids.METHODS1.Pre-formulation studies of glycyrrhiza flavonoids microspongeA new method developed for determining content of six components of glycyrrhiza flavonoids simultaneously by HPLC.And then,the oil-water partition coefficients of six components of glycyrrhiza flavonoids were investigated by an n-octanol-water system.In order to screen out an appropriate receptor fluid in in-vitro release studies,the equilibrium solubility of six components of glycyrrhiza flavonoids in various solvents were investigated.2.The preparation of glycyrrhiza flavonoids microspongeThe central composite rotatable design-response surface methodology(CCD-RSM)was applied to design the preparation technology of glycyrrhiza flavonoids microsponge,and the parameters such as content of polyvinyl alcohol,the ratio of drug/ethyl-cellulose and stirring rate were chosen as variables.Afterwards,the optimal preparation conditions of the glycyrrhiza flavonoids microsponge were analyzed with high production yield,high loading efficiency and low particle size distribution value by Design-Expert 8.0 software.At last,the morphology of the microsponge was observed by scanning electron microscopy(SEM),and the particle size of the microsponge was determined by the Mastersize 2000 laser diffraction particle size analyzer.3.In vitro release experiment of glycyrrhiza flavonoids microspongeFirstly,glycyrrhiza flavonoids microsponge ointment and glycyrrhiza flavonoids ointment were prepared by emulsification method.Secondly,the saline solution containing 30%PEG-400 was used as receptor fluid and the skin of nude mice was used as permeation membrane,to investigate the differences of permeation behavior in skin between two kinds of ointments by Franz diffusion cells.The cumulative permeation amount of the drug was plotted against the time to obtain the permeation curve of the six components of glycyrrhiza flavonoids from two kinds of ointments.The release kinetics of the six components of glycyrrhiza flavonoids were calculated,and their release patterns were analyzed using different model fitting equation.Lastly,we determined the amount of drug deposited at 4 hours,8 hours,12 hours and 24 hours in the skin,and investigated the difference of in vitro drug-deposition between two kinds of ointment.4.The evaluation of whitening effect about glycyrrhiza flavonoidsIn our research,the rat model of hyperpigmentation was built to evaluate the whitening effect of glycyrrhiza flavonoids and the B16 mouse melanoma cells was used to elucidate the its preliminary mechanism.The rat model of hyperpigmentation was established by UVB irradiation and treated with glycyrrhiza flavonoids.The melanin contents in skin of different groups were measured by L-DOPA staining method,the expressions of tyrosinase were carried out by Western Blot analysis.In order to study the effects of glycyrrhiza flavonoids on cytotoxicity of the B16 mouse melanoma cells,the cells were treated with drugs of different concentrations,and then,the cell proliferations were measured by MTT assay.The effects of drugs on melanogenesis and activity of tyrosinase in B16 mouse melanoma cells were carried out by NaOH-assay and L-DOPA-oxidation-assay,respectively.Eventually,the expression level of tyrosinase in B16 mouse melanoma cells were determined by western blot analysis,and discussed the mechanism of whitening effects in glycyrrhiza flavonoids.RESULTS1.Pre-formulation studies of glycyrrhiza flavonoids microspongeThe chromatographic conditions of HPLC were as follows:Agilent 5 HC-C18 column(250mm×4.6mm,5?m),the mobile phase were mixed up by acetonitrile,methanol and 0.2%phosphoric acid water(15:10:75),the flow rate was 1.Oml/min,and the column temperature was 30 ?,the injection volume was 10?l,the detection wavelength was 300 nm.The standard curves equation in methanol and 30%PEG-400 physiological saline were established respectively,and they have a good linear relationship between the peak areas for various concentrations.In addition,the equilibrium solubility of six components of glycyrrhiza flavonoids in normal saline solution which contained 30%PEG-400 could meet the demand of in vitro release studies.For glycyrrhiza flavonoids,the oil-water partition coefficient of liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,licochalcone A and glabridin were 0.1958,0,3983,0.7809,0.6574,1.672 and 0.4602,respectively.2.The preparation of glycyrrhiza flavonoids microspongeThe optimal preparation conditions of the glycyrrhiza flavonoids microsponge were analyzed with high production yield,high loading efficiency and low particle size distribution value by Design-Expert 8.0 software.The optimal conditions were as follows:the PVA content was 2.69%,the ratio of drug/ethyl-cellulose was 6:1,and the stirring rate was 1100 rpm.Under the optimal conditions,the production yield was 60.76±0.45%,the loading efficiency was 52.84±3.43%and the dispersion coefficient was 1.06±0.12.The mean specific surface area and the volume diameter were 11.01±0.98?m and 24.28±1.09?m,respectively.3.In vitro release experiment of glycyrrhiza flavonoids microspongeThe result of release kinetics showed that release profile of drugs could be best expressed by zero order kinetics.For glycyrrhiza flavonoids ointment,the permeation rate of liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,licochalcone A and glabridin were 0.55±0.16?g·h-1·cm-2,0.72±0.04?g·h·1·cm-2,1.38±0.02?g·h-1·cm-2,0.67±0.05?g·h-1·cm-2,2.73±0.14?g·h?1·cm-2 and 1.00±0.23?g·h-1·cm-2,respectively.For glycyrrhiza flavonoids microsponge ointment,the permeation rate of liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,licochalcone A and glabridin were 1.75±0.02?g-h-1·cm-2,1.02±0.01 ?g·h-1·cm-2,1.75±0.14?g·h-1·cm'2,1.4±0.13?g,g·h-1·cm-2,3.12±0.27?g·h-1·cm-2 and 1.8±1.04?g·h-1·cm-2,respectively.The permeation rate of glycyrrhiza flavonoids microsponge ointment was higher than glycyrrhiza flavonoids ointment.As shown in the in vitro drug-deposition studies at different time,the amount of six components of glycyrrhiza flavonoids which deposited in the skin were much higher for the glycyrrhiza flavonoids microsponge ointment than for the glycyrrhiza flavonoids ointment.4.The evaluation of whitening effect about glycyrrhiza flavonoidsCompared with blank control group,the result of L-DOPA staining method and Western Blot analysis showed that melanin content of model group was significantly increased and expression of tyrosinase was also increased,and it means the rat model of hyperpigmentation was built up successfully.In contrast to the model group,the melanin contents and expression of tyrosinase in rat skin were both decreased after the use of arbutin or glycyrrhiza flavonoids.It means that arbutin and glycyrrhiza flavonoids could inhibit expression of tyrosinase in rat skin of hyperpigmentation which induced by UVB irradiation,and further caused melanin content reduced.After treated with glycyrrhiza flavonoids microsponge ointment,the melanin content in rat skin was lower than glycyrrhiza flavonoids ointment,and the inhibition about expression of tyrosinase was stronger than that of glycyrrhiza flavonoids ointment.It means that glycyrrhiza flavonoids microsponge could make glycyrrhiza flavonoids deposit in the skin,and increased the drug concentration of glycyrrhiza flavonoids in the skin,futher caused whitening effect improved.The cells were respectively treated with different concentrations of glycyrrhiza flavonoids,the result of cytotoxicity showed that glycyrrhiza flavonoids have cytotoxicity when its concentration was above 400?g/ml.The results of melanin contents and tyrosinase activity showed that with the increase of concentration of glycyrrhiza flavonoids,the tyrosinase activity and the relative content of melanin decreased.Furthermore,the result of Western Blot discovered that with the increase of intervention time and concentration of drug,the expression of tyrosinase declined,the best intervention time was 24h and the best concentration of drug was 200?g/ml.In addition,the result of extracellular regulated protein kinases and its phosphorylation showed that phosphorylation of ERK was activated after 60 min intervention of glycyrrhiza flavonoids.CONCLUSION1.Pre-formulation studies showed that the developed method had strong stability and good precision.A good linear relationship was found between the peak areas for various concentrations.The oil-water partition coefficient of six components of glycyrrhiza flavonoids showed they had good diffusion ability into skin.The equilibrium solubility of six components of glycyrrhiza flavonoids in normal saline solution which contained 30%PEG-400 can met the demand of in vitro release studies.2.The studies of preparation technology showed that it is effective and reliable to analyze and optimize the preparation of glycyrrhiza flavonoids microsponge by central composite rotatable design-Response surface methodology(RSM).The obtained three-dimensional charts can be intuitive display the effects of PVA content,drug/ethyl-cellulose ratio and stirring rate on the production yield,loading efficiency and particle size distribution.3.In vitro release studies showed that glycyrrhiza flavonoids microsponge could increase the permeation rate of glycyrrhiza flavonoids,improve the drug retention in skin,and prolong the time of drug sustained release.4.The studies of whitening effect showed that glycyrrhiza flavonoids microsponge could improve the whitening effect of glycyrrhiza flavonoids and then decrease the melanin content.And the research of cell pharmacodynamics showed that glycyrrhiza flavonoids could inhibit the expression of MITF by inducing phosphorylation of ERK,and then inbihit the expression of tyrosinase,further caused melanin content in rat skin reduced,to achieve the whitening effect. |
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