Study On The Preparation Method Of Eupolyphaga Sinensis Ethanol Extract And Its Hydrogel Cataplasm Based On Fracture Healing Activit

Objective:The present study was undertaken to investigate the preparation methods of the alcohol extracts of Eupolyphaga sinensis,and the samples was made into hydrogel cataplasm.Methods:The Eupolyphaga sinensis extracts were separately prepared by the soak or homogenate methods with 50%ethanol.The effects of different treatments:temperature(4℃～60℃),time(4 h～4 d),solid-liquid ratio(4～12 times)and agitation on the total yield and the total protein yield were measured.The extracting conditions were optimized through orthogonal test based on single factor.Effects of ethanol extracts of Eupolyphaga sinensis on proliferation of MC3T3-E1 osteoblasts were assayed by MTT methods.Bone glaprotein,bone alkaline phosphatase and β-crosslaps were detected with the method of Elisa kit in vivo.The methods of sensory assessment,primary adhesive and gel strength were used to select hydrogel cataplasm matrix.Results:Whether stirring did not affect the amount of substance extracted by the two methods.With increasing the treating temperature,the total yield and the total protein yield increased which displayed in two methods.The ratio of solid to liquid has great influence on yield of the homogenate method.The optimized extraction process was decided by orthogonal experiment which was at 60℃,10 times of solid-liquid ratio,4 d,non-agitation for the soak method and at 50℃,8 times of solid-liquid ratio,12 h and non-agitation for the homogenate method.The maximum total yield by the soak or the homogenate method were 11%and 10.2%,respectively,which was not statistically different.MTT assay showed that most of the samples promoted the MC3T3-E1 osteoblasts cells proliferation when the concentration was lower than 0.5mg/mL.The promotion ability of the samples prepared by homogenate methods was stronger than by the soak methods.When the concentration of the sample was 0.2 mg/mL,the cell proliferation rate of the homogenate and soaking method reached to 171.59%and 48.45%,respectively.Compared with negative control,the value of bone glaprotein,bone alkaline phosphatase and β-crosslaps were the highest by the way of the samples extracted by the homogenate method in vivo.The mean of sensory assessment of polyacrylic acid(NP700)was higher than polyacrylic acid(NP800),so does gel strength.The primary adhesive of polyacrylic acid(NP700)and polyacrylic acid(NP800)was obedient to Chinese Pharmacopoeia 2015,vol.4.Conclusion:The amount of the total yields didn’t show different between the two methods but the ability to promote fracture healing of the samples extracted by the homogenate method was the best in vitro and in vivo.The ability to promote fracture healing of the samples extracted by the homogenate method medium dose was the best.The matrix of hydrogel cataplasm was polyacrylic acid(NP700).