Preparation Of Anti-inflammatory Cream Of Polygala Japonica Houtt.extract

Objective:The Polygala japonica Hout.is a commonly used traditional Chinese medicinal with anti-inflammatory,analgesic,anti-tumor and anti-nervous pharmacological activities.It is often used for the treatment of pain and swelling,bruises and snake and insect bites.It has been included in the 2015 and 2020 editions of the Chinese Pharmacopoeia.In view of the current situation that the curative effect of Polygala japonica Hout.is definite but the research and development work is weak,In this paper,Polygala japonica Hout.was extracted with ethanol solution,and the extraction was purified with macroporous resin to obtain the total saponins from Polygala japonica Hout..The vitro antibacterial and antibacterial activity of total saponins of Polygala japonica Hout.and the anti-inflammatory test of Polygalaxanthone III were studied.Based on the efficacy of the above substances,the star point design-response surface and other test optimization methods were used to screen out the optimal prescription and preparation technology of the Polygala japonica Hout.total saponins cream,and the preliminary quality of the Polygala japonica Hout.cream was carried out.The research lays the foundation for the resource utilization and product development of Polygala japonica Hout..Methods:（1）Extraction and purification of Polygala japonica Hout.total saponins.The vanillin-glacial acetic acid-perchloric acid colorimetric method and the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method were used to determine the total saponins and total flavonoids in the Polygala japonica Hout..The single factor was used to optimize the best extraction method of the Polygala japonica Hout.extract.The types of macroporous resins were screened by static adsorption,on the basis,parameters such as the loading concentration,loading volume,washing volume,elution solvent and elution volume of the Polygala japonica Hout.extract were optimized.（2）Examination of extract from Polygala japonica Hout..TLC and HPLC chromatographic methods were used for the determination of the components of the Polygala japonica Hout.extract refer to the Pharmacopoeia.The R_fand the t _Rwere used for qualitative identification.A Evaporative Light Scattering Detector detector was used to measure the content of Polygalasaponin F before and after purification.（3）In vitro antibacterial test and vitro Oxidation test of Polygala japonica Hout.total saponins.The bacteriostatic zone and minimum inhibitory concentration MIC test of total saponins of Polygala japonica Hout.were carried out by the filter paper method and double dilution method respectively;and take Vc as a positive control,the scavenging ability of total saponins on DPPH,ABTS and TPTZ three free radicals were investigated.Anti-inflammatory test of Polygalaxanthone III.The anti-inflammatory test of Polygalaxanthone III was carried out using the zebrafish inflammation model induced by cutting tail,the anti-inflammatory ability was evaluated according to the number of macrophages and neutrophils at the inflammation site,and the anti-inflammatory mechanism of Polygalaxanthone III was studied by the model of zebrafish inflammation induced by lipopolysaccharide.（4）Anti-inflammatory test of Polygalaxanthone III.The anti-inflammatory test of Polygalaxanthone III was carried out using the zebrafish inflammation model induced by cutting tail,the anti-inflammatory ability was evaluated according to the number of macrophages and neutrophils at the inflammation site,and the anti-inflammatory mechanism of Polygalaxanthone III was studied by the model of zebrafish inflammation induced by lipopolysaccharide.（5）Preparation and quality research of Polygala japonica Hout.extract.Cream.Single-factor and star-point design-response surface method were used to optimize the formulation and process of preparing Polygala japonica Hout.saponin cream and verify it,The study on the quality of Polygala japonica Hout.extract cream was carried out and the preliminary quality standard was established.A method for the determination of total saponins in cream was established,and the appearance,physical properties,rheological properties,as well as preliminary stability tests such as influencing factor tests,have been carried out.Results:（1）The best extraction method of Polygala japonica Hout.was ultrasonic extraction with 70%ethanol.The contents of total saponins and total flavonoids in the extract before purification were 7.207%and0.7295%,respectively.The best purification process of total saponins is:using HPD-826 resin,the concentration of the sample solution is 0.2g/m L,the sample volume is 3 BV,the amount of washing and impurity removal is 8 BV,the elution solvent is 80%ethanol,the elution amount is 4 BV,the extraction rate after purification was（8.918±0.27）%,The purity is（74.24±0.08）%.（2）Using TLC and HPLC methods,it was detected that the total saponins of Polygala japonica Hout.contained Polygalaxanthone III,Polygalasaponin F,Polygalasaponin V,and Polygalasaponin XXXI.The content of Polygalasaponin F in the purified Polygala japonica Hout.total saponins increased from（0.52±0.01）%Before purification to（6.10±0.34）%.（3）The purified melon seed gold total saponins have varying degrees of inhibitory effects on eight bacterial species when the mass concentration is 200 mg/m L,including Klebsiella pneumoniae,methicillin-resistant Staphylococcus aureus,Candida albicans,Bacillus subtilis and Salmonella Sensitive to drugs,with MICs of 1.5625,3.125,6.25,6.25 and 12.5 mg/m L,respectively.Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus have relatively weak sensitivity to drugs,with the smallest inhibitory concentrations were all greater than 25mg/m L.In addition,total saponins of Polygala japonica Hout.have strong scavenging ability of DPPH and ABTS free radicals,but weak scavenging ability of TPTZ free radicals.Among them,the IC₅₀for DPPH free radicals is 0.1062 mg/m L,and the maximum clearance rate is90.95%;the IC₅₀for ABTS free radicals is 0.2947 mg/m L,and the maximum clearance rate is 93.82%;when the concentration reaches 1mg/m L,the FRAP value is 1.27 mmol/L.The above results indicate that Polygala japonica Hout.total saponins have certain antibacterial activity and antioxidant capacity.（4）The zebrafish toxicity test results show that Polygalaxanthone III is safe to zebrafish at a concentration of≤800μM and an action time of 96 h.Polygalaxanthone III at a concentration of 200～800μM has an inhibitory effect on the inflammation of zebrafish induced by tail cutting.It can inhibit the accumulation of neutrophils and macrophages at the tail cut and promote the reverse migration of neutrophils and macrophages out of the tail cut to play an anti-inflammatory effect.Polygalaxanthone III inhibits the production of ROS and NO in zebrafish induced by LPS.It indicates that its anti-inflammatory mechanism may be through inhibiting the production of ROS and NO.（5）The centrifugal stability,heat-resistant stability and viscosity were used as indicators to screen the Polygala japonica Hout.extract cream prescription.the AHP method,CRITIC method and AHP-CRITIC mixed weighting method determine the weight of each index to be 0.4285,0.4156 and 0.1559,respectively.The star point design-response surface method was used to screen the dosage of stearyl alcohol,cetyl alcohol,and mixed emulsifier in the prescription of Polygala japonica Hout.cream,and the optimal formula was 1.96 g cetyl alcohol,5.17 g stearyl alcohol,and 2.48 g mixed emulsifier,polysorbate 60 1.83 g,glyceryl monostearate 0.65 g,benzyl alcohol 1 g,propylene glycol 5 g,isopropyl myristate 6 g,Polygala japonica Hout.extract 1.25 g,and then add water to 100 g.The quality research results show that the prepared cream is light yellow,easy to spread,p H value is 5.73,particle size is 0.5～2.5μm,the total saponins in the cream are determined by colorimetric method and methodological investigation,the results show this method has strong specificity,good precision and accuracy,centrifugal,heat-resistant and cold-resistant stability test results show that the cream has no oil-water separation and thickening of the paste.The results of rheological investigation show that the cream is a pseudoplastic fluid with shear thinning characteristics,which is conducive to the production and daily use of the cream.The influencing factor experiment shows that the cream is susceptible to humidity and light,and should be stored in a dry and dark place.Conclusion:（1）The total saponins of Polygala japonica Hout.were extracted and purified,which proved the antibacterial and fungal functions of Polygala japonica Hout.total saponins.（2）Experiments have confirmed that Polygala japonica Hout.total saponins have a certain effect of scavenging free radicals.（3）It was found that Polygalaxanthone III,a kind of Xanthones,has a certain anti-inflammatory effect.（4）The development of Polygala japonica Hout.cream can be used for the treatment of bacterial and fungal skin diseases and local inflammation,replacing or reducing the use of antibiotics,and has a good application prospect.