Construction And Intestinal Absorption Of Component-based Nanocrystals Submicroemulsion Of Medicinal Formula Guanxinning

Guanxinning is a commonly used oral Chinese patent medicine for the treatment of coronary heart disease in clinic,which is composed of salvia miltiorrhiza and ligusticum Chuanxiong at a ratio of 1:1.However,the existing Guanxinning preparations lack water-insoluble components of tanshinones and ligusticum Chuanxiong oil,which have been proven to have good therapeutic value for coronary heart disease.Tanshinones and ligusticum Chuanxiong oil are difficult to be absorbed in the form of conventional oral dosage forms.Supported by the key research project of Chongqing Science and Technology Joint Traditional Chinese Medicine（2023ZDXM034）,the study aimed to develop a new component-based oral preparation for Guanxinning,which contained not only salvianolic acids and ferulic acids existing in Guanxinning preparations on market,but also tanshinones,volatile oil of ligusticum missing in current preparations,and had good oral biaoavailability.The development of the novel oral preparation could provide a new study mode for oal delivery of other traditional Chinese medicine compound preparations containing water-insoluble active components.（1）Methods for the content determination of each pharmacodynamic component in GuanxinningMethods:HPLC was established for the simultaneous determination of ferulic acid,salvianolic acid B,ligusticolactone A,ligustilide A,cryptotanshinone and tanshinoneⅡA in Guanxinning nanocrystals submicroemulsion.The methodology verification was carried out according to the‘Guidelines for Validation of 9101 Pharmacopoeia Quality Standard Analysis Methods’in the Ch.p.The contents of four kinds of raw materials were determined.Results:The determination was performed on Waters Symmetry C₁₈ column（4.6×250 mm,5μm）with a mobile phase consisted of 0.02%phosphoric acid–acetonitrile with a gradient elution at a flow rate of 1 m L·min^-1.The detection wavelength was 280nm and column temperature was 30℃.The specificity,linearity and range,repeatability,stability and accuracy met the requirements of pharmacopoeia,confirming that the HPLC method could be used for the submicroemulsion determination of the 6 components in Guanxinning.The content of cryptotanshinone and tanshinoneⅡA in tatal tanshinones were 46.81%and 42.55%,respectively.Salvianolic acid B content in total salvianolic acid was70.80%.The content of ligusticide A and ligusticide in ligusticide oil was 6.45%and16.27%,ferulic acids was 98.03%,respectively.（2）Study on the preparation of component-based nanocrystals submicroemulsion of medicinal formula Guanxinning by one-step methodMethods:According to the ratio of salvia miltiorrhiza and ligusticum in Guanxinning and the extraction ratio of each component,the ratio of total ketone of salvia miltiorrhiza,total phenolic acid of salvia miltiorrhiza,ferulic acid and ligusticum oil.One-step process for the preparation of guanxinning nanocrystals submicroemulsion was designed.Single factor experiment was used to investigate the mixing conditions of water and oil,the volume ratio of oil and water,the p H value of water phase,the concentration of drugs and the preparation conditions of final emulsion.Results:Each component was determined to be 2:28:1:40.One-step process for the preparation of guanxinning nanocrystals submicroemulsion was designed.Single factor experiment was used to investigate the mixing conditions of water and oil,the volume ratio of oil and water,the p H value of water phase,the concentration of drugs and the preparation conditions of final emulsion.The final preferred one-step process is as follows:71.5 mg salvia total phenolic acid dissolved in 5 m L pure water,adjust the p H value to 6.5 to obtain water phase.Then 5 mg total Danshen keto and 2.5 mg ferulic acid were dispersed them in 0.1 m L Ligusticum Chuanxiong oil to obtain oil phase,then add the oil phase to the water phase drop by drop,stir at room temperature for 15 min,and then ultrasonic probe under the condition of ice bath with cell crushing instrument,ultrasonic power 500 W treatment,to obtain the final emulsion.（3）Study on the preparation of component-based nanocrystals submicroemulsion of medicinal formula Guanxinning by two-step methodMethods:The two-step method is to prepare the nanocrystalline suspension of total ketone and total salvianolic acid of salvianolic acid by reverse-solvent precipitation method,and then mix with oil containing ferulic acid to prepare emulsion.Using the single factor experiment method,the emulsion appearance,particle size and component content were used as indicators to investigate the organic solvent type,acetone removal method,organic phase volume,water phase volume,salvianolic acid B addition method,water and oil phase mixing conditions and other technical factors in the reverse-solvent method.Results:The final preferred two-step process is as follows:10 mg total ketone of salvia miltiorrhiza were dissolved in 1.5 m L acetone,inject it into 50 m L pure water under ultrasonic stirring,continue ultrasonic stirring for 10 min,and then heat it to 60℃for decompress ultrasonic degassing.The total ketone nanosuspension was prepared by ultrasonic treatment with 1000-1200 W.After 50 nm polycarbonate membrane filtration,the filter layer was dispersed in 10 mlsalvianolic acid B aqueous solution with p H 6.5 to obtain water phase.The oil phase was obtained by dispersing 5 mg ferulic acid in 0.2 m L Ligusticum Chuanxiong oil.The oil phase was added to the water phase drop by drop,stirred at room temperature for 30 min,and ultrasonic power 500 W treatment,to obtain the final emulsion.（4）The study of spray drying on solidification of component-based nanocrystals submicroemulsion of medicinal formula GuanxinningMethods:The emulsion preparation by the two routes was compared in appearance,centrifugal stability,content of each pharmacodynamic component,particle size and PDI,and microscopic structure of emulsion.The emulsion with well properties was optimized.It was solidified by spray drying.The types and concentrations of spray-drying excipients were screened based on the appearance,powder yield,redispersability,centrifugal stability and pharmacodynamic component content of spray-drying emulsion.The properties of the selected dry emulsion were compared with the original emulsion.Results:The one-step and two-step emulsions all showed orange-red emulsions with particle sizes of 176.53±1.89 and 169.81±1.08 nm.There was no significant difference in the morphology and surface structure of the two kinds of emulsion droplets observed by fluorescence microscope and scanning electron microscope.However,the contents of ligusticolactone A,ligustilide,cryptotanshinone and tanshinoneⅡA in the emulsion prepared by two-step method were significantly higher than emulsion prepared by one-step method.Therefore,nanocrystals submicroemulsion by two-step was selected for spray drying.With 15%hydroxypropyl-β-cyclodextrin as the carrier,dry emulsion with good powder of orange and less of each pharmacodynamic component can be prepared.Dry emulsion can quickly recover,then dispersed emulsion particle size（373.10±11.00 nm）with 1800×g remained stable after centrifugation was ne doubled to original emulsion（169.81±1.08 nm）.The structure and morphology of the emulsion droplets observed by FIM and SEM are similar to the original emulsion,indicating that the redispersed emulsion also maintains the morphology and structure of the original emulsion.（5）Intestinal absorption effect of component-based nanocrystals dry emulsion of medicinal formula GuanxinningMethods:The absorption effects of six pharmacodynamic components（ferulic acid,salvianolic acid B,ligusticolactone A,ligustilide a,saphenoneⅡA,tanshinoneⅱA）in Guanxinning nanocrystals dry emulsion in duodenum,jejunum,ileum and colon were evaluated by rats’everted intestinal sac model.Results:The results showed that nanocrystals submicroemulsion could effectively improve the absorption of ferulic acid,ligusticolactone A,ligustilide,cryptotanshinone and tanshinoneⅡA in small intestine.Cryptotanshinone and tanshinoneⅡA were not detected in the four intestinal segments in the physical mixture group,indicating that these two components were almost not absorbed by the intestinal tract.In the submicroemulsion group,the cumulative absorption amounts Q（μg）of these two components in duodenum,jejunum,ileum and colon were 14.60,13.98,10.96,10.47 and2.83,2.84,3.40,6.44.The apparent permeability coefficients P_app(×10^-6 cm·s^-1)were1.00,0.98,0.80,0.74 and 0.25,0.27,0.36,0.44.Compared with the bulk drug,the cumulative absorption of ferulic acid in each intestinal segment of nanocrystals dry emulsion was increased by 49.63%,62.29%,39.41%and 50.50%,and P_app was increased by 76.33%,48.60%,62.38%and 66.43%.The cumulative absorption of ligustrin A was 4.12,3.38,4.07 and 3.72 times of that of the bulk drug,respectively,and P_app was increased by 2.11,1.64,1.87 and 1.25 times,respectively.The cumulative absorption of ligustilin was 12.35,8.47,11.31 and 10.09 times of the bulk drug,respectively,and P_app was increased by 9.30,4.91,9.87 and 6.92 times,respectively.The cumulative absorption of salvianolic acid B in nanocrystals submicroemulsion only increased by 40.74%in duodenum,while the cumulative absorption and P_app of other intestinal segments had no significant change.Conclusion:In this study,the main pharmacodynamic components of Guanxinning,total salvia ketone,total salvia phenolic acid,ferulic acid and ligusticum Chuanxiong oil were used as preparation materials to successfully prepare the nanocrystals submicroemulsion preparation of Guanxinning component.Compared with the bulk drug,the oral absorption of water insoluble components（ferulic acid,cryptotanshinone,tanshinoneⅡA）and volatile oil components（ligusticolactone A,ligusticolactone）was significantly improved.