| Objective:Ginseng(Panax ginseng C.A.Meyer)is a commonly used essential medicine for invigorating vital energy.Fresh ginseng juice has a mellow taste,a cool and moist nature,and has the characteristics of replenishing qi without causing fire.It has a stronger ability to nourish yin and promote fluid production,and has a broad market application value.Fresh ginseng can completely retain its original active ingredients while maintaining its original form without processing.Fresh ginseng that has undergone traditional processing has differences in active ingredient composition and pharmacological activity.At present,the research on the material basis of fresh ginseng is still weak,which limits its product development.Further efforts are needed to clarify the advantageous active ingredients of fresh ginseng.Shengmai Powder is a classic and famous recipe for tonifying the lung and Qi,nourishing yin and promoting fluid production.Fresh ginseng as a medicine helps to better play the prominent advantages of Shengmai Powder in nourishing yin and promoting fluid production.Freeze-dried forming technology is a preparation technology that freeze-dries the precise formulation of active substances and excipients to shape.It can effectively retain the active ingredients in fresh ginseng,providing a dosage form platform for the development of fresh ginseng compound preparations.This project is based on the above background and revolves around the following content for research:to analyze the composition of Saponins in fresh ginseng,screen extraction processes that can fully retain the active components of fresh ginseng,and preliminarily evaluate the pharmacological activity of the dominant saponin components in fresh ginseng combined with network pharmacology,laying the foundation for the development and application of fresh ginseng.Based on the optimized extraction process of fresh ginseng,the freeze-dried formula and process curve are investigated to prepare a Shengmai freeze-dried orally disintegrating tablet using fresh ginseng as medicine,and preliminary establishment of its quality evaluation method.The research on the preparation process of Shengmai freeze-dried orally disintegrating tablets will further broaden the application types of Shengmai preparations.Methods:(1)Using fresh ginseng as the research object,ethanol extracts,homogenates,and decocted extracts were prepared.The saponins were analyzed and identified using UPLC-Q-Exactive MS technology,and the differences in the types and content of saponins in different extraction processes were compared.(2)Taking dominant saponin components as the research object,combined with network pharmacology research methods,PharmMapper was used to predict potential targets of target saponin components,String and DAVID were used to conduct protein interaction,GO process,and KEGG pathway enrichment analysis on potential targets,Cytascape software was used to construct a component-target-pathway network,and AutoDock software was used to conduct molecular docking verification.(3)Taking the prescription Shengmai Powder as the research object,the preparation method of homogenate alcohol precipitation was used,with the content of Rg1,Re,Schisandrin A and the extraction ratio as evaluation indicators.The concentration level and alcohol precipitation concentration conditions during the preparation process were optimized,and the preparation method of Shengmai extract was improved.(4)Taking the Shengmai extract as the research object,selecting the type and proportion of excipients,investigating the drug concentration of the Shengmai extract,and determining the freeze-dried formula of the Shengmai extract based on its appearance characteristics,molding,and adhesion.Taking the heating rate,temperature,and time of sublimation drying and analytical drying as the research objects,the optimal freeze-drying process curve was determined based on the appearance characteristics,molding,adhesion,moisture content,and disintegration time limit.(5)Taking Shengmai freeze-dried orally disintegrated tablets as the research object,the basic evaluation and process validation of the preparation were carried out based on the appearance characteristics,weight difference,moisture content,disintegration time limit,and taste as indicators;The retention of saponins from fresh ginseng in the preparation was analyzed using UPLC-Q-Empirical MS technology;The fingerprint of Shengmai freeze-dried orally disintegrated tablets was established by HPLC-Q-TOF.Results:(1)Homogenization extraction was beneficial to the retention of active ingredients in fresh ginseng,and malonic acid monoacyl ginsenosides were the dominant saponins in fresh ginseng.Using UPLC-Q-Exactive MS technology 26 saponins were identified from homogenate,ethanol extract,and decoction extract.The relative content of malonic acid monoacyl ginsenosides such as mRg1,mRe,mRf,mRb1,mRb2,and mRd in homogenate extract were higher than that in ethanol extract,while the content in the decoction extract was significantly reduced.(2)Malonic acid monoacyl ginsenosides had the potential to regulate the process of diabetes,cardiovascular disease,neurodegenerative disease,cancer and other diseases.Using mRb2,mRc,mRb1,and mRd as target components,a PPI network was constructed to screen 12 key targets.GO process and pathway enrichment showed that malonic acid monoacyl saponins participate in biological processes such as protein decomposition and interfere with cell enzyme activity,regulating signaling pathways such as AGE-RAGE,IL-17,and TNF to regulate inflammatory,supersession and apoptosis processes in the body.CASP3 and BMP2 were the core targets for the regulation of malonic acid monoacyl saponins,and the relevant results had been verified by molecular docking.(3)The optimized preparation method of Shengmai extract was stable and feasible.Finally,the preparation method of Shengmai extract was determined as follows:approximately weighed 490 g,327 g,and 163 g of fresh ginseng,ophiopogon japonicus,and schisandra chinensis,respectively,added 4900 mL of water according to the ratio of 1:5 material to liquid,soaked for 30 min,homogenized,filtered,centrifuged,concentrated the supernatant by rotary evaporation to a relative density of 1.20,slowly added ethanol until the concentration was 70%,and allowed it to stand overnight at 4℃,took the supernatant and evaporated it to concentrate ethanol,added water to prepare the Shengmai extract.(4)The prepared Shengmai freeze-dried oral disintegrating tablets had a smooth,plump appearance,rapid disintegration,and no mold sticking.The optimal formulation process:added 80 g of 10%mannitol and 0.8 g of 0.1%pullulan,mixed the excipients,extract,and purified water evenly to 800 mL,and prepared a total of 1000 tablets.The optimal freeze drying process curve:pre freezing temperature-50℃,constant for 4 hours,sublimation drying temperature rise rate of 0.2℃·min-1,temperature of-10℃,constant for 9 hours,analytical drying temperature rise rate of 0.3 ℃·min-1,temperature of 15℃,constant for 5 hours,a total of 22.75 hours.(5)The quality evaluation of Shengmai freeze-dried orally disintegrating tablets basically met the requirements.Basic evaluation:The appearance was light red loose cake shaped,with a plump sheet shape,a smooth surface,and no mold sticking.The average sheet weight was about 0.16 g,with a weight difference of-5.45%to 3.53%,moisture content of less than 4%,and disintegration time limit of less than 11 s.The taste was sour and bitter,with a faint fragrance,a cool taste,and it melted immediately upon entry.Chemical evaluation:Shengmai freeze-dried orally disintegrating tablets could effectively retain the malonyl saponins of fresh ginseng,the effective ingredient content in the freeze-dried orally disintegrated tablets for process validation was 0.2934 mg/tablet of Schisandrin A,0.1305 mg/tablet of Rg1,and 0.1297 mg/tablet of Re.Establishment of fingerprint:A total of 15 common peaks were identified,identified mRg1 and mRb2 by HPLC-Q-TOF,comparison of control samples identified Rg1,Re,Rf,Rb1,Rc,Rb2,and Schisandrin.Conclutions:In this study,UPLC-Q-Exactive MS was used to clarify that the homogenization process could effectively retain the malonic acid monoacyl dominant saponins in fresh ginseng,which had potential pharmacological effects in the treatment of diabetes,cardiovascular disease,neurodegenerative disease,cancer and other aspects.The above results provided a theoretical basis for the quality control of fresh ginseng and the basic research of pharmacological substances.Based on the freeze-dried shaping technology platform,a Shengmai freeze-dried orally disintegrating tablet with stable quality and convenient administration was prepared,enriching the market of Shengmai preparations,providing an empirical basis for the development and application of fresh ginseng preparations,and providing the reference for subsequent clinical applications of fresh ginseng. |
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