| ObjectivesPsoriasis is a chronic skin disease with high recurrence rate and difficult to cure.Yinxieling is an optimized formula created by Professor Xuan guowei initialiy,the famous dermatologist of Guangdong province hospital of TCM,and the PSORI-CM04 is the active ingredients of Yinxieling.The aim of this paper is to prepare the PSORI-CM04 emulsion and evaluate its quality,efficacy and safety.Methods1 Determination of dosage forms and to study the mechanism of absorption preliminarilyHealthy adult male albino guinea pigs were randomly divided into blank control group and model group,and the 5%propranolol cream was applied uniformly on the the ears skin of guinea pig,0.2 mL/ear at a time,3 times a day.After 8 days,psoriasis-like model would be successful when the ears of guinea pigs were redness and even skin lesions.According to the severity of disease and weight of the pigs,the model group was randomly divided into PSORI-CM04 cream group,PSORI-CM04 emulsion group,PSORI-CM04 microemulsion group,model group,MTX microemulsion group and Calcipotriol Betamethasone Ointment group,the medicine dose was 0.2 g/ear.About a week later,the guinea pigs would be sacrificed by celiac anesthesia with 10%chloral hydrate(0.34 mL/100 g),abdominal aortic blood,then cut out the ears,the right ears were saved-80 ℃ refrigerator for content determination,while the left were fixed in 10%formalin solution for pathological slice.The pathological changes were observed and photographed by the optical microscope,and the Baker scores were calculated through randomly selected five horizons of the pathological slice.The dosage form was chosen by the Baker scores from Pathological for animals.The content of paeoniflorin,astilbin,glycyrrhetinic acid in guinea pig ear tissue were extracted and detected by HPLC to discuss the mechanism of absorption preliminarily.2 Preparation of microemulsion2.1 Choice of excipientThe solubility of efficient composition in kinds of different oil phases,surfactants and cosurfactants and the area of the formation of microemulsion in the pseudo ternary phase diagram were preliminarily stuied and to determine the component of the formation of microemulsion.2.2 Prescription optimizationEach of the excipient of microemulsion rates were optimized by the central composite design-response surface method.Equal amounts of the preparation of microemulsion medicated 2 copies,added same volume of red dye Sudan red oil soluble and blue dye methylene blue water soluble respectively,then observed the two dye diffusion velocity in the microemulsion to identify the type.The viscosity was measured by NDJ-1 type rotational viscometer under constant temperature.The size was measured by laser particle size analyzer after diluted 10 times at room temperature,the microemulsion form was observed by transmission electron microscopy(TEM).Combined the efficacy to get the final prescription.3 Pharmacodynamics studyThe method reference to item 1,according to the severity of disease and weight,they were randomly and averagely divided into 6 groups,which were including MTX microemulsion group,blank microemulsion group,Calcipotriol Betamethasone Ointment group,PSORI-CM04 microemulsion group,model group,and blank control group.The changes of patholofical were shown under the optical microscope,and then evaluated the efficacy through the Baker scores.4 Safety evaluation of microemulsionThe safety evaluation included skin stimulant experiment and acute toxicity test.The stimulant experiment include a single skin irritation and multiple dosing skin irritation tests.Healthy New Zealand rabbits were randomly divided into normal saline group,blank microemulsion group and PSORI-CM04 microemulsion group,each group of 6(the intact of 3 and damaged skin of 3).A single stimulus after the treatment for 24h,was observed and recorded 1,24,48 and 72 h after remove drug daub area whether presence of erythema and edema reaction or not.The stimulation was scored and calculated on the basis of skin irritation reaction score standard.Multiple dosing skin irritation daubed 1 times a day timing for 7 d.1 h after removal of drugs,observed and recorded if daub parts had erythema and edema,scored and calculated each animal daily average integral to determine the intensity of skin irritation.Acute toxicity test groups liked the stimulant experiment.24 h after the treatment,observed daily rabbit skin,eyes,mucous membrane and respiratory,activities,etc.,for 7 d,and recorded the weight in rabbit on the day 3,5 and 7 days after the treatment.5 Quality control of the microemusionThe content of paeoniflorin,astilbin,glycyrrhetinic acid in microemulsion was detected by HPLC;and the content of β-elemiene and gima ketone in the oil of zedoary turmeric in microemulsion was detected by GC-MS.Oil of zedoary turmeric in microemulsion was detected by TLC.6 Stability studyThe stability of the 3 batches microemulsion were preliminary studied by the content of paeoniflorin,astilbin,glycyrrhetinic acid in microemulsion,pH,appearance observation at the 0,5,10 day in condition of 4 ℃ and room temperature,40 ℃,60 ℃,high humidity and strong light 4500±500 lx,etc.Centrifugation experiments and sample tests were used to further investigate the stability of the microemulsion.Results1 Compared to the blank group,the model group had marked thickening of the stratum spinosum,disordered arrangement,granular layer thinning,neutrophil infiltration and the Baker score increased significantly at the same time(P<0.01).The different dosage forms of Yinxieling effective group can reduce the skin damage.Compared to the model group,the microemulsion and cream were significant,but the emulsion was nonsignificant.The microemulsion had lower scores among these three dosages(microemulsion was 2.81 ±0.66,cream was 3.51 ± 0.62,emulsion was 4.30±0.74),indicated that microemulsion had a better effect,therefore,microemulsion is chosed as the pharmaceutical of Yinxieling.The content of astilbin,paeoniflorin,glycyrrhetinic acid in guinea pig ear tissue respectively was 18.20 μg/mL,3.13 μg/mL,0.52 μg/mL in microemulsion,25.87 μg/mL,4.85 μg/mL,1.14 μg/mL in emulsion,23.48 μg/mL,4.54 μg/mL,and 0.56μg/mL in the cream.Its means that the effective components may be assimilated into the systems.2 Labarifil M 1944 Cs was chosed as the oil phase,RH40 was chosed as the surfactant,and Transcutol HP was chosed as the cosurfactant,and the quality ratio of the surfactant and cosurfactant was 3:1.The central composite design-response surface method combined the efficacy results to which the microemulsion prescription was RH40:Labarifil M 1944 Cs:Transcutol HP:water=6.75:1:2.21:11.37.The content of paeoniflorin,astilbin glycyrrhetinic acid and zedoary turmeric in the microemusion is 4.5%,2.6%,0.3%,and 1.4%respectively.The type of microemulsion was O/W.The average of particle size was 20 nm,polydispersity was 0.191,viscosity value was 0.41 pa · s.Emulsion droplet liked circular,and uniform distribution.3 The pharmacological results showed that compared with the blank group,the model group showed psoriasis-like lesion skin,indicated that the model was successful(P<0.01),and compared with the model group,the skin lesion of animals of other groups showed lower Baker score(P<0.01)except the blank microemulsion group(P>0.05).Compared with MTX group and Calcipotriol Betamethasone Ointment group,the Baker score of the PSORI-CM04 microemulsion group had marginal difference(P>0.05).4 PSORI-CM04 microemulsion and blank microemulsion were applied for complete skin and damaged skin,neither erythema or edema were found during single stimulation experiment,indicated that the microemusion had no stimulatory,and similar results was obtained in the multiple dosing skin irritation study.No death occurred in acute toxicity test,the feeding,hair removal areas of skin,eye conjunctiva,breathing and limbs activity were normal,indicated that PSORI-CM04 microemulsion had no obvious effect on normal growth on New Zealand rabbits,the external use is safe.5 The average contents of each composition in the 3 batches of paeoniflorin,astilbin,glycyrrhetinic acid is 39.19 mg/mL,30.75 mg/mL,3.54 mg/mL respectively.The average contents of β-elemiene and gima ketone in the oil of zedoary turmeric in microemulsion was 56.26μg/mL and 173.17μg/mL respectively.6 Microemulsion had no stratification in the centrifugation experiments and kept clear transparent in the condition of low temperature or package foil.Astilbin in the microemusion and pH value decreased significantly when kept in high temperature,humidity,or illumination,therefore the microemusion should be kept at low temperature,darkness and dry conition.ConclusionPSORI-CM04 microemulsion is a safe external pharmaceuticals with good effect on the psoriasis-like model,and the preparation of it is simple and easy to do. |
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