Preparation Of DHAvD Flexible Liposome Gel And Preliminary Study On Its Anti-allergic Effect

Purposes:Dihydroavenanthramide D(Dihydroavenanthramide D,DHAvD)has excellent anti-allergic and anti-inflammatory pharmacological activity and it is widely used in personal care and cosmetic fields as an anti-sensitivity ingredient,but its low solubility in water limits its application in the field of medicine.Therefore,this study intends to encapsulate DHAvD in flexible liposomes to solve the problem of its low solubility,enhance its transdermal absorption and overcome the problem that traditional preparation methods are difficult to penetrate effectively through the skin barrier.In order to facilitate its application,this study further made the DHAvD flexible liposome(DHAvD-FLip)into gel(DHAvD-FLip-gel),which could be easily retained in the skin and at the same time improve the stability of the DHAvD-FLip.Methods:1.The High Performance Liquid Chromatography(HPLC)method was established for the determination of contentnt of DHAvD-FLip and DHAvD-FLip-gel.HPLC was used to determine the equilibrium solubility and the oil-water partition coefficient of DHAvD.2.The DHAvD-FLip were prepared by ethanol injection-membrane extrusion method;The encapsulation efficiency of DHAvD-FLip was determined by ultrafiltration centrifugation;The encapsulation efficiency and particle size were used as evaluation indicators,the effect of prescription process on the preparation of DHAvD-FLip was investigated by single factor experiments;Box-behnken response surface methodology was used to optimize the Formulation;The DHAvD-FLip was characterized by appearance,micromorphology,particle size distribution and potential,encapsulation rate and drug loading.3.The gel matrix and matrix content were selected with appearance, formability,glossiness,uniformity,malleability,fluidity and viscosity as evaluation indexes;The moisture loss rate was used as evaluation indexes to screen the content of moisturizing agent;The type and content of osmotic promoters were selected with unit cumulative permeability of DHAvD as evaluation index.The quality of DHAvD-FLip-gel was evaluated with appearance,p H,viscosity and content as indicators.The stability of DHAVD-Flip-gel was investigated by influence factor test,centrifugal stability test and acceleration test.4.Franz vertical diffusion cell was used to investigate the transdermal effect of DHAvD-FLip and DHAvD-FLip-gel in isolated rat skin.The transdermal mechanism was investigated by HE staining,Differential Scanning Calorimetry(DSC)and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy(ATR-FTIR).5.The Passive Cutaneous Anaphylaxis model of KM mice was established by using ovalbumin as sensitizer and the pharmacodynamic effect of DHAvD-FLip-gel on Type I Hypersensitivity Reaction was investigated.Result:1.The established HPLC method can be used for the determination of contentnt of DHAvD-FLip and DHAvD-FLip-gel.The solubility of DHAvD in water is 0.1127 mg/m L.The HLB values of DHAvD in n-octanol-water and n-octanol-pbs buffer systems are respectively 2.073 and1.009.2.The optimized Formulation and process of DHAvD-FLip were as follows:The drug concentration is 5 mg/m L;the drug-lipid ratio is 1/21(w/w);the ratio of tween 20 to phospholipid is 1/3.65(w/w);the ratio of cholesterol to phospholipid is 1/15(w/w);the ratio of organic phase to water phase is1/2.5(v/v);the preparation temperature is 50℃;the rotation speed is 300 rpm;the dropping rate is 0.5 m L/min;The extrusion temperature through the membrane is 55°C;the membrane extrusion process is to extrude liposomes through 400 nm,200 nm and 100 nm polycarbonate membranes 7 times each.DHAvD-FLip is a pale yellow translucent liquid with blue opalescence and the particles are quasi-circular or oval when observed under TEM,with an average particle size of 66.7±1.6 nm,an average PDI of 0.075±0.015,an average potential of-19.78±0.71 m V,the average encapsulation efficiency was 72.89±0.81%and the average drug loading was 2.53±0.03%.3.The DHAvD-FLip-gel Formulation obtained by the experimental screening were as follows:DHAvD-FLip is 70%,glycerol is 10%,propylene glycol is 5%,PE9010 is 0.7%,carbopol 940 is 0.4%,vitamin E is 0.2%,EDTA-2Na is 0.02%and water residue.The DHAvD-FLip-gel is a pale yellow,uniform and fine semi-solid preparation with p H of about 6.49.The average content of DHAvD-FLip-gel was 3.112±0.079 mg/g.The DHAvD-FLip-gel is unstable under high temperature(60℃)and strong light irradiation conditions,the DHAvD-FLip-gel is placed at a relative humidity of75%±5%and a temperature of 30℃±2℃for two months,its drug content,uniformity and appearance have not changed significantly.4.The cumulative permeation per unit area of DHAvD-FLip and DHAvD-FLip-gel for 24 hours was 103.70±34.16μg/cm~2and 42.98±9.84μg/cm~2,respectively,and the intradermal retention was 7.03±2.28μg/cm~2and 2.82±0.12μg/cm~2,respectively.The two preparations fit well in the first-order release model equations,The transdermal mechanism of DHAvD-FLip and DHAvD-FLip-gel is mainly to promote drug penetration by changing the lipid structure,keratin structure and hydration of the stratum corneum and increasing the fluidity of intercellular lipids.5.The low-dose(0.0259 mg/cm~2),medium-dose(0.0519 mg/cm~2) and high-dose(0.0778 mg/cm~2)groups of DHAvD-FLip-gel were significantly reduced the degree of dye exudation in the model group mice and reduced the vascular permeability of the sensitized mice.The inhibition rates of DHAvD-FLip-gel low,medium,high dose groups and positive drug groups(hydrocortisone butyrate cream)on the vascular permeability of sensitized mice were 19.04±3.20%,32.59±3.21%,36.32±4.97%and37.76±6.56%,respectively.Conclusion:DHAvD-FLip prepared in this study has small particle size and uniform distribution;DHAvD-FLip-gel is uniform,delicate and has good stability.The DHAvD-FLip-gel has good therapeutic effect on PCA mouse model,providing a new strategy of allergic skin diseases caused by TypeⅠHypersensitivity Reaction.