Optimization Of Extraction Process Of Blumea Balsamina And Evaluation Of Its Anti-inflammatory Activit

Blumeatin is a flavonoid.Previous studies have found that blumeatin shows good anti-inflammatory activity in vitro.At present,there are few reports on the extraction process of blumeatin from Blumea balsamifera（L.）DC,and it’s purification process is complicated.In order to obtain a suitable technological route for industrial production of enaxanthin,improve the evaluation of its anti-inflammatory activity,and investigate its pharmacokinetic characteristics in vivo.Based on the establishment of the method for the determination of blumeatin content,the extraction solvent,solvent concentration,extraction time,and liquid-solid ratio were selected by single factor,and the reflux extraction process of blumeatin was optimized by response surface method.The obtained blumeatin was enriched and purified by water sedimentation method,macroporous resin column chromatography and crystallization,and its structure was identified.On the basis of acute toxicity limit test,the anti-inflammatory activity was evaluated by ear swelling induced by xylene and capillary permeability induced by acetic acid in mice.Finally,the determination method of blumeatin in plasma was established,the drug time curve was drawn,and the pharmacokinetic parameters were calculated.The test results show that the optimal extraction conditions were as follows:ethanol concentration 89.80%,liquid-to-solid ratio 15.17:1,extraction at 80℃for2.02 h.The actual yield was 1.97 mg/g,which was basically consistent with the predicted value（1.98 mg/g）.The extract was subjected to water sedimentation process:the extract（concentration of blumeatin 2.40 mg/m L）was added to 4 times the amount of water,and centrifuged to precipitate,AB-8 macroporous resin was used for enrichment and purification:the loading solution concentration was 0.40 mg/m L,the loading amount was 6.38 mg/g,the diameter/height ratio of the resin was 1:6,the loading flow rate was 1 BV/h,and the elution was eluted with 50%ethanol.After collecting the 2-6 BV effluent,it was crystallization several times with 80%methanol at a solid-liquid ratio of 1:100,and the purity of blumeatin was≥95%.However,it was identified as 5,3’,4’-trihydroxy-7-methoxyflavanone by NMR,and the structure of the originally named blumeatin（5,3’,5’-trihydroxy-7-methoxyflavanone）had some errors in the analysis,mainly due to the positioning of the hydroxyl group on the B-ring.In the physiological activity study,no poisoning or death occurred in mice after oral administration of more than 5 g/kg of blumeatin for 14 days.The time-effect study showed that the inhibition rate of ear swelling was the highest in mice injected with 20 mg/kg and 5 h,reaching 35.58%.The dose-effect study showed that the inhibition rate of ear swelling was the highest in mice injected with 7.50 mg/kg and 5h,reaching 75.03%,which was not significant（P（29）.0.05）to that of 10 mg/kg dexamethasone.blumeatin（7.5,15 and 30 mg/kg groups）could significantly inhibit acetic acid-induced capillary permeability in mice（P（27）0.05）,and 7.50 mg/kg of blumeatin had the best effect,but the effect decreased with the increase of concentration.HPLC method for the determination of plasma concentration of blumeatin linear range was 0.02-20μg/m L（R²=0.9959）,and the lower limit of quantification was 0.02μg/m L.The highest plasma concentration of blumeatin was observed 2 h after intraperitoneal injection.The peak concentration(C_max),half-life(T_1/2),area under the curve(AUC_0-∞),plasma clearance（Cl）and apparent volume（V）of distribution of blumeatin were:1.55±0.26μg/m L,2.20±0.37 h,5.70±0.62 L/h·mg,35.39±3.45 h·kg/L and 111.43±17.79 L/kg.Blumeatin could still be detected in plasma 12 hours after administration of drugs.The results showed that the optimized extraction and purification process was stable and feasible,and the operation was simple,and a large amount of blumeatin could be obtained.In vivo experiments showed that blumeatin had low toxicity,and a certain amount of blumeatin had good anti-inflammatory activity in vivo,and the anti-inflammatory effect was similar to that of dexamethasone.The drug-time curve of intraperitoneal injection conformed to the two-compartment open model.The pharmacokinetic parameters suggested that blumeatin may be distributed in the metabolic organs of the body in large quantities,resulting in its low blood concentration.This study provides a material basis and theoretical reference for the in-depth study of blumeatin,provides a theoretical basis for the pharmacological evaluation of blumeatin,and provides some new ideas for the utilization of medicinal resources of Blumea balsamifera（L.）DC.