| Protopanaxatriol（PPT）is a ginseng dammarane-type protopanaxatriol saponin,which is obtained by hydrolyzing the Sugar moiety part of protopanaxatriol saponins by structural modification technology using ginseng stems and leaves panaxatriol saponins as raw materials.Its molecular formula is C30H52O4,and its molecular weight is 476.73.After years of research,our research group found that PPT has anti-myocardial infarction biological activity.On this basis,it is planned to be developed into a new drug for the treatment of cerebral thrombosis.According to the data requirements of the"Administrative Measures for Drug Registration"（2020 Edition）for the approval of Class 1 new chemical drugs,combined with the"Chinese Pharmacopoeia"（2020 edition）and the"Technical Guidelines for the Standardization Process of the Establishment of Chemical Drug Quality Standards"（[H]GPH1-1）and other related guidelines,we carry out pre-clinical pharmaceutical research on it,mainly including the optimization of the production process of PPT API and its guttate pills,quality research,establishment of quality standards（draft）and stability research,and a preliminary pharmacodynamic study of PPT against myocardial infarction was carried out at the same time.On the basis of reviewing the research progress of the biological activity and the mechanism of PPT and anti-myocardial infarction drugs,we conducted preclinical pharmaceutical research on PPT,and obtained the following innovative scientific research results:1.the Preparation Process of PPT and its Guttate Pills was Determined.First,the total saponins of ginseng stems and leaves were used to prepare the raw materials of protopanaxatriol saponins by alcohol-alkali precipitation method,and then the PPT was obtained by hydrolyzing of protopanaxatriol saponins and sodium hydroxide.Based on this basic principle,this chapter used ginseng stems and leaves protopanaxatriol saponins as raw materials,sodium hydroxide as hydrolysis power,and1,2-propanediol as solvent for high-temperature degradation reaction to prepare PPT.PPT guttate pills were prepared by using PPT as API and PEG4000 as excipients.（1）The optimization of the production process for preparing PPT.Taking the conversion rate of PPT as evaluation indicator,single factor experiments was used to investigate the effects of four factors（alkalinity,time,temperature and material-liquid ratio）on the preparation of PPT by protopanaxatriol saponins.Combined with response surface experiments,the best semi-synthetic process was determined.Small and pilot tests have verified the stability of the production process.The pure PPT（purity≥98%）was isolated by column chromatography and recrystallization,and the structure of PPT was determined by comprehensive analysis of ~1H-NMR,13C-NMR,MS,IR,UV and crystal coefficient.（2）The determination on the preparation process of PPT guttate pills.According to the research experience of our research group on ginsenoside preparations for many years,the optimal prescription of PPT guttate pills is determined as PPT:PEG4000=1:4.2.the Quality Research on PPT and its Guttate Pills was Conducted,and the Quality Standards（draft）for both has been Established.The research was carried out in accordance with the guiding principles of the"Chinese Pharmacopoeia"（2020 edition）on the establishment of quality standards for API and guttate pills.（1）The quality study of PPT API.The quality research of PPT API includes the study of the properties of PPT,chromatographic identification of PPT,inspection of PPT（mainly including the color of the solution,loss on drying,residue on ignition,chloride inspection,heavy metal inspection and arsenic salt inspection）,content determination,impurities and the crystal form of PPT.The results showed that all the data were in line with the requirements of the"Chinese Pharmacopoeia"（2020 edition）for the establishment of API quality standards.On this basis,the quality standard（draft）of PPT API was established.（2）The quality research of PPT guttate pills.The quality research of PPT guttate pills mainly studies its properties,chromatographic identification,weight difference,dissolution time limit,impurities and content determination.The results showed that all the data were in line with the requirements of the"Chinese Pharmacopoeia"（2020version）on the establishment of quality standards for guttate pills.On this basis,the PPT guttate pills quality standard（draft）was formulated.3.The Stability Study of PPT API and its Guttate Pills has been Completed.Based on the"Chinese Pharmacopoeia"（2020 Edition）9001 Guidelines for Stability Testing of API and Preparations and"Technical Guidelines for Stability Research of Chemical Drugs"（2015 Edition）,the stability research content of API and guttate pills is mainly conducted through influencing factor tests（high temperature,high humidity,strong light）,accelerated test and long-term test,and the analysis method needed to be verified.（1）The stability study of PPT API.The stability study of PPT API mainly studies the properties,melting point,solution color,loss on drying,moisture gain,impurities and content of PPT.The results show that PPT has a certain degree of hygroscopicity in a high-humidity environment.The high temperature,strong light influencing factor experiments,accelerated experiments and long-term experiments all show that there is no obvious change in the investigation items of PPT.（2）The stability study of PPT guttate pills.The research on the stability of PPT guttate pills mainly investigates the properties,disperse during dissolution,impurities and content of the guttate pills.The results show that there is no obvious change in the experimental data of each inspection project.It is speculated that PPT and PPT guttate pills are suitable for storage in dry,sealed,normal temperature packaging bottles,and the validity period of PPT and PPT guttate pills is at least 24 months.4.A Preliminary Study on the Biological Activity of PPT against Myocardial Infarction was Carried Out.Through the In vitro cell experiments,it was found that PPT can reduce the apoptosis of cardiomyocytes,reduce the apoptosis rate of cells,and have certain protective effects on H/R injury of cardiomyocytes.Western blot analysis and molecular docking indicated that PPT may function through the PI3K/AKT signaling pathway.In summary,the preclinical pharmacology research and preliminary pharmacodynamics of PPT were deeply explored in this paper.The preparation process of PPT and its guttate pills was determined,the quality standards（draft）for both has been established,and the stability study and preliminary anti-myocardial infarction pharmacodynamics have been completed.It has laid a solid foundation for PPT API and its guttate pills to enter into clinical research. |
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