Study On Antipruritic Activity Of α-caryophyllene In Mikania Micrantha And β-caryophyllene Nano-preparation

ObjectiveIn China,Mikania micrantha is a highly harmful invasive species,and there is still no good way to curb its spread.In order to solve this problem,the research group screened the anti-itching active ingredients of Mikania micrantha in the early stage and selectedβ-caryophyllene.Because of the low-dose β-caryophyllene ethanol solution sample,it was found that the antipruritic activity of the low-dose β-caryophyllene could not be fully represented the antipruritic activity of the Mikania micrantha plant extract,so the new antipruritic active ingredient in Mikania micrantha was screened again.At the same time,in order to better develop Mikania micrantha and improve the antipruritic activity ofβ-caryophyllene,this study studied the dose-effect relationship,physicochemical properties,content of Mikania micrantha under different conditions and also explore the possibility of the development of β-caryophyllene preparations.In the screening process of the formulation,the prescription was screened by in vitro transdermal test,and the relationship between the formulation prescription and the antipruritic activity was investigated according to the results of the in vitro transdermal test,and the formulation prescription was screened to provide a new idea for the prescription screening of antipruritic drugs in the future.The safety of β-caryophyllene and preparations was investigated to develop a new antipruritic topical drug,which provided a theoretical basis for the transformation of Mikania micrantha with β-caryophyllen.Methods1.Intensive research on anti-itch active ingredientsThrough the steroid test of KM mice,the new antipruritic active ingredients in Mikania micrantha were screened again,and the dose-effect relationship and the synergistic effect with β-caryophyllene were investigated.2.Dynamic monitoring of the content of β-caryophyllene in Mikania micranthaThe content of β-caryophyllene in Mikania micrantha was measured by HPLC.The changes of the content of Mikania micrantha in different months,locations,growth environments and parts were investigated to determine the optimal harvesting conditions.3.Screening of preparation prescriptionsThe stability of β-caryophyllene under different conditions was investigated.The solubility of β-caryophyllene in organic solvents such as n-hexane was determined by static equilibrium method.The solubility of β-caryophyllene in the physiological saline solution containing polyol was investigated by uniform test design.On the basis of effective ointment,the transdermal absorption enhancer was screened by the permeation amount ofβ-caryophyllene as an indicator in vitro,and the anti-itching activity of the ointment was enhanced,and the ointment and nanoemulsion were screened.The cumulative permeation amount of β-caryophyllene at different time points in oil solutions,ointments and nanoemulsions was determined by HPLC.According to the results of the in vitro transdermal test,the antipruritic activity of the preparation was predicted,and the antipruritic activity was verified at the same time,and the formulation prescription was screened out.Finally,the acute toxicity test of β-caryophyllene and formulation was investigated.Result1.Intensive research on antipruritic active ingredientsThe new antipruritic active ingredient α-caryophyllene was screened.The antipruritic activity was not statistically different from the positive drug at the concentration of 80 mg·g-1.The synergistic effect of α-caryophyllene and positive drug was not obvious.The concentration of α-caryophyllene ointment showed a positive correlation with the antipruritic activity.2.Dynamic monitoring of the content of β-caryophyllene in Mikania micranthaThe content of β-caryophyllene in Mikania micrantha is dynamically changed,and leaves,stems and flowers all contain β-caryophyllene.The content of leaves is significantly higher than that of stems,and the inflorescence content is high,but the yield is low.The content in December is higher,and the growth environment is superior to the river.3.Screening of preparation prescriptionsPhysical and chemical properties of β-caryophyllene preparation before formulation:β-caryophyllene has good stability under normal temperature,70℃ condition,strong light irradiation,and β-caryophyllene is easily soluble in organic solvents such as n-hexane.The solubility of β-caryophyllene in the physiological saline solution containing the polyol is 557.9800 μg·mL-1.On the basis of effective ointment,four kinds of transdermal absorption enhancers such as oleic acid were screened by uniform test design,and it was found that oleic acid promoted better.Based on the oleic acid structural formula,five kinds of transdermal absorption enhancers such as oleic acid and linoleic acid were screened again,and it was found that 3%linoleie acid can increase the cumulative permeation ofβ-caryophyllen by more than 11%.The formulation of the preparation was screened by in vitro transdermal test,and the three concentrations of the same concentration ofβ-caryophyllen oil solution,ointment and nanoemulsion were compared and predicted.After the antipruritic activity was verified,the results were in agreement with the prediction,and the prescription was confirmed to be 3%.ConclusionIn the Mikania micrantha,the new antipruritic active ingredients α-caryophyllene,α-caryophyllene and β-caryophyllene were found to have no statistically significant anti-itch activity,but the synergistic effect was not obvious.The concentration ofα-caryophyllene and β-caryophyllene ointment showed a positive correlation with the antipruritic activity.β-caryophyllene is easily soluble in medium and small polar organic reagents and is recommended to be stored in a low temperature,dark environment.The harvesting period of Mikania micrantha was in the riverside growth environment in December,and the content in leaves and inflorescences was higher,and no obvious difference was found in the geographical position.Through the in vitro transdermal experiment,a new transdermal absorption enhancer,linoleic acid,was found to predict the effectiveness of 2%-3%β-caryophyllen nanoemulsion.It was confirmed by animal experiments that linoleic acid has a promoting effect,and 3%β-caryophyllen nanoemulsion is effective.Therefore,through the in vitro transdermal experiment,the experimental results of the antipruritic model animal can be predicted to some extent,and the linear regression equation is fitted as follows:Y=6.7649X+39.888,r=0.9694,which can provide new ideas for the prescription screening of the itch drug.Finally,it was confirmed that the preparation was nanoemulsion,and the prescription was composed of 3%β-caryophyllene,20%absolute ethanol,9%poloxamer-124,4.5%Tween-80,0.5%oleic acid,and 63%water.