Preparation And In Vitro And In Vivo Evaluation Of Temperature-Sensitive In-Situ Gel Of Phellodendron Amurense And Angelica Dahurica

Objective:In view of the problem that the snake bite is mainly powder,which leads to mechanical stimulation and poor patient compliance,this paper uses Phellodendron amurense and Angelica dahurica as the prescription of traditional Chinese medicine to develop an environment-responsive gel-Phellodendron amurense and Angelica dahurica（Yellow-white）thermosensitive in situ gel.The purpose of this study is to develop a new external preparation of traditional Chinese medicine,which is easy for patients to accept and can quickly eliminate the pain and swelling of the affected area caused by snake bite,and provide reference for the development of compound external preparation of traditional Chinese medicine.Methods:（1）HPLC was used to determine berberine hydrochloride in Phellodendron amurense and imperatorin in Angelica dahurica.Taking berberine hydrochloride transfer rate and imperatorin transfer rate as indexes,and ethanol concentration,ethanol dosage,extraction time and extraction times as investigation factors,L₉（3⁴）orthogonal design was used to optimize the optimum extraction process of Cortex Phellodendri and Radix Angelicae Dahuricae.（2）The yellow-white temperature-sensitive gel was prepared by“cold solution method”.The gelling temperature was the evaluation index.The single factor combined with Box-Behnken design was used to optimize the optimal prescription of yellow-white thermosensitive in-situ gel,and the appearance properties,gelation temperature,gelation time,p H value,micromorphology,osmotic pressure,rheological properties,stability and percutaneous permeability of the prescription were evaluated.（3）To establish an LC-MS/MS method for the determination of berberine hydrochloride and imperatorin,and investigate the in vivo transdermal pharmacokinetics of Yellow-white temperature-sensitive gel by in vivo microdialysis coupled with LC-MS/MS.（4）Rats were randomly divided into groups and the right leg snake bite model was replicated by intramuscular injection of Agkistrodon halys venom.The drug efficacy was investigated by comparing the swelling degree,pain degree,serum and tissue inflammation factor levels,histopathological examination and other indicators before and after the administration of yellow-white temperature-sensitive gel.（5）To establish the quality standard of Yellow-white thermosensitive in-situ gel,Phellodendron amurense and Angelica dahurica in Yellow-white thermosensitive in-situ gel were qualitatively identified by TLC,and Berberine hydrochloride in Phellodendron amurense and imperatorin in Angelica dahurica were determined by HPLC.Results:（1）A method for the determination of berberine hydrochloride in Phellodendron amurense and imperatorin in Angelica dahurica was established.The extraction technology of Phellodendron amurense and Angelica dahurica was defined:the best extraction technology of Phellodendron amurense was 8 times of 60%ethanol as solvent,extracting for 3 times,each time for 1 hour;The best extraction process of Angelica dahurica is 10 times of 75%ethanol as solvent,extracting for 3 times,each time for 1 hour.The extraction process is stable and has good reproducibility.（2）The optimal prescription of yellow-white thermosensitive gel is:20.10%of poloxamer 407,2.54%of poloxamer 188,2.00%of polyethylene glycol 6000,and the gelation temperature is（34.2±0.2）℃.After optimization,the temperature-sensitive gel is yellow-brown evenly dispersed solution below 34.2℃,and yellow-brown semi-solid gel above 34.2℃;Gelling time（53.6±0.8）s;p H（5.78±0.02）;Scanning electron microscopy showed that the microstructure of the gel was a network structure,arranged neatly,with loose pores and interconnected;Osmotic pressure is 1302m Osmol/kg;Good rheological properties;The cumulative transdermal permeation of berberine hydrochloride and imperatorin in vitro for 24 h was（395.72±35.04）and（14.07±1.34）μg·cm^-2,respectively,which accorded with Higuchi kinetic equation.（3）An LC-MS/MS method was established for the simultaneous determination of berberine hydrochloride and imperatorin,and the methodological investigation met the determination requirements.In the microdialysis sampling process,30%ethanol normal saline was used as the perfusion fluid at the perfusion rate of 1μl·min^-1,and sampling was conducted every 0.5 h as the sampling interval.The in vitro recovery rates of the probes determined by the increment method and the decrement method were almost identical.The in vivo recovery rate determined by the decrement method was used to correct the measured data.The Cmax of berberine hydrochloride and imperatorin in Huangbai temperature-sensitive gel was（7.12±1.65）ng·ml^-1and（39.14±6.26）ng·ml^-1,and the Tmax was（4.50±0.00）h and（1.17±0.29）h.T_1/2was（2.09±1.56）and（9.20±5.67）h,AUC_（0→t）was（17.36±4.15）ng·h ml^-1and（165.13±47.70）ng·h·ml^-1,AUC_{（0→∞）}was（21.92±10.39）ng·h·ml^-1and（439.62±246.59）ng·h·ml^-1,MRT_{（0→∞）}was（4.67±2.41）h and（14.70±8.24）h.（4）10-30 min after injection of snake venom,compared with the blank group,the swelling of the right hind leg of the rats in the model group,the temperature sensitive in situ gel group and the positive drug group was obvious（P<0.01）.Compared with the model group,the swelling degree of the right hind leg in the thermosensitive in-situ gel group decreased after 48 h（P<0.05）,and the gait of the right hind leg improved obviously after 12 h,and the contents of IL-1β,IL-6,TNF-αin serum and IL-1β,IL-6,TNF-α,PGE in swollen muscle decreased significantly（P<0.05）.（5）After qualitative identification,the TLC identification method of Phellodendron amurense was as follows:saturated in concentrated ammonia water for 15～20 min,then used ethyl acetate-isopropyl alcohol-formic acid（3∶3∶1）as developing agent,placed in ultraviolet lamp（365 nm）and observed in sunlight,and TLC identification method of Angelica dahurica was as follows:petroleum ether（30～60℃）-diethyl ether（1∶1）as developing agent The content of berberine hydrochloride and imperatorin in yellow-white thermosensitive in-situ HPLC should not be less than0.16%and 0.005%,respectively.Conclusion:The yellow-white thermosensitive in situ gel was developed in this paper,and it has obvious inhibitory effect on swelling,pain and inflammatory factors caused by snake bite.The quality standard of the preparation was established,which laid a foundation for further study of the preparation for clinical treatment of snake bite.