Study On Preparation,Quality Evaluation And Preliminary Pharmacodynamics Of Compound Schizonepeta Granule

Objective:In this paper,the compound Schizonepeta granule were prepared by traditional prescription that has been clinically practiced for many years and has definite curative effect.The formulation and preparation process were optimized,and the quality evaluation,efficacy,security of the granule were studied.Methods:（1）In extraction technology study,based on the inhibitory rate of ear swelling and hydroxyl radical scavenging rate,the anti-inflammatory and anti-oxidation effects of different components of the extract were investigated to determine the effective parts.Moreover,based on the contents of puleone,total flavonoids and total polysaccharide as indexes,all factors,such as extraction solvent,soaking time,extraction time,solid-liquid ratio and extraction times,were optimized by single factor experiment and central composite design-response surface methodology（CCD-RSM）.（2）In preparation technology experiments,based on the contents of puleone,total flavonoids and total polysaccharide as indexes,the concentration process were optimized by single factor experiment.The optimal formulation of compound Schizonepeta granule were selected with sensory evaluation,molding rate and hygroscopicity percentage as indexes.Based on the water content,and contents of puleone,total flavonoids as indexes,the drying process were optimized by single factor experiment combined with CCD-RSM.（3）Quality evaluation studies of compound Schizonepeta granule were executed.Firstly,6 Chinese medicine in granule were qualitatively identified by TLC.The physical and chemical properties of granule,such as appearance,solubility water content,dry weight loss,pile density,angle of repose and hygroscopicity,were determined.Secondly,the pulegone and total flavonoids contents were measured in granule by HF-LPME-HPLC method and Na NO₂-Al（NO₃）₃colorimetric method,respectively.Thirdly,based on the reaction of granule with Fenton reagent,the hydroxyl radical scavenging rate was determined.Finally,according to the 2020 version of Chinese Pharmacopoeia,the stability of the granule was investigated to preliminarily determine its storage conditions.（4）Preliminary studies on safety,efficacy and pharmacokinetics of the compound Schizonepeta granule were carried out.Firstly,the safety of granule was evaluated by acute toxicity test in mice.Secondly,the mice experiments of acetic acid-induced writhing test,tracheal phenol red excretion test and xylene-induced ear edema were performed to detect the number of writhes,amount of phenol red excretion and ear swelling degree.Thirdly,the pharmacokinetics of pulegone in compound Schizonepeta granule were preliminarily studied using rats as test animals.Results:（1）The essential oil,alcohol-soluble components and water-soluble components in The extract of the prescription could significantly inhibit the ear swelling of mice,and had antioxidant activity with the scavenging rates of hydroxyl radical of 47.5%,70.2%and13.4%,respectively.Under the optimum conditions,the pulegone of extracted solution achieved effective enrichment and obtained good linearity（r=0.9990）.The rutin and glucose obtained good linearity with correlation coefficients higher than 0.9998.The precision and accuracy of the three methods can meet the requirements,indicating that the methods can be used for the determination of the contents of analytes in the extracted solution.On the basis of this determination,the optimal extraction process was as follows:the volatile oil was extracted by steam distillation,and the residue and the other 14medicines were soaked in 70%ethanol solution of 12 times volume for 4 h and extracted by reflux twice for 2.5 h each time.It is proved that the extraction method is stable and feasible.（2）The optimum concentration process,formulation ratio and drying process were obtained as follows:the extract was concentrated to 1 m L/g（measured by crude drug amount）under low pressure at-0.085MPa at 50℃,and then further concentrated into an extractum with a concentration of 0.26 g/g（measured by crude drug amount）under normal pressure at 60℃.An appropriate amount of extractum and the essential oil of Schizonepeta were mixed,and the mixtures and triples of extraneous materials（maltodextrine:powdered sugar=0.5:3.5）were used for preparation of granule by wet method.Then,the wet granule were tiled with a thickness of 1 cm and dried at 68℃for2.5h.It has been proved that the preparation process is stable and feasible.（3）The results of quality evaluation:Firstly,the compound Schizonepeta granule are brown,fragrant and bitter,and dissolved in hot water with slight turbidity.Moreover,the water content,weight loss,bulk density,angle of repose,and the moisture absorption percentage were 2.1%,1.1%,0.3 g/m L,26.6^o,and 13.7%,respectively.In addition,the TLC identification results of granule showed that the separation effect of each sample spot was good with clear colors,and the negative granule had no interference.Secondly,the pulegone and rutin of granule obtained good linearity with correlation coefficient higer than 0.9990.The precision and accuracy of the two methods can meet the requirements,indicating that the methods can be used for the determination of the contents of analytes in the granule.Thirdly,the average hydroxyl radical scavenging rate was 71.5±1.7%.Finally,the results of stability test showed that the percentage of each index decreased less than9.6%when placed under high temperature and strong light for 10 days.However,when placed in 75%humidity environment for 10 days,the percentage of each index decreased as high as 91.8%.The results of acceleration test for 3 months at 40℃and 70%humidity showed that all the indexes had no change basically when the granule were sealed well.（4）The results of safety,efficacy and pharmacokinetic tests:Firstly,During the observation period,all mice in the dose group survived healthily,and there was no significant difference in body weight growth rate and organ index between the does group and the blank control group（P>0.05）.Secondly,compared with the blank control group,the ear swelling degree decreased significantly（P<0.05）,the numbers of writhes decreased significantly（P<0.01）in mice of granule high and medium dose groups,and the tracheal phenol red excretion of mice was significantly increased in high,medium and low dose groups（P<0.01）.Finally,the developed HF-LPME-HPLC method obtained good linearity（r=0.9984）,which could be used for the determination of pulegone in plasma and pharmacokinetic tests of rats.Its main pharmacokinetic parameters are as follows:T_max0.15 h,C_max0.83 mg/L,t_1/2zof 6.15 h,MRT of 6 h,CL_Z/Fof 126.6 L/h/kg,which provided the basis for further study of volatile oil.Conclusion:In this paper,the compound Schizonepeta granule was prepared through prescription screening,preparation process optimization and quality control,and the granule were safe,non-toxic and had good anti-inflammatory,analgesic,expectorant effects and antioxidant activity,which had been proved by safety test,effectiveness test,hydroxyl radical scavenging rate test and pharmacokinetic test.It had laid a good foundation for the further development and clinical application of compound Schizonepeta granule and had practical value for the prevention and treatment of chronic pharyngitis.