Optimization Of Processing Technology In The Origin Of Platycodon Grandiflorum Root And Pharmacodynamic Study On Platycodin D In Early Complications Of T2DM

Platycodon grandiflorum（PG）is the dried root of platycodon grandiflorum（Jacq.）A.DC.,a plant of the family platycodon,was first recorded in the"Shennong Bencao Jing".As a plant used for both food and medicine,PG contains abundant active ingredients.Modern research has shown that PG has pharmacological activities such as antitussive,expectorant,anti-obesity,anti-inflammatory,hepatoprotective,and anti-diabetic effects.In order to further ensure the safe use of PG and develop its medicinal value,the production and processing of PG slices and their pharmacological activities were studied.In this article,the optimal production and processing technology for Platycodon grandiflorum slices was established.Traditional methods were used to isolate the monomers of PG,providing a material basis for the screening of active ingredients in the future.Through in vitro cell experiments,it was confirmed that Platycodin D（PD）is the main active ingredient.Based on this,the therapeutic effects and molecular mechanisms of PD on type 2 diabetes mellitus（T2DM）and early complications were analyzed.The main contents of the research are as follows:1.Optimization of initial processing technology for PGThe initial processing of PG from its production region is a crucial step in ensuring the quality of the medicinal material.In this study,we evaluated and analyzed PG slices prepared using different processing methods,and recorded the time and appearance of each method.The main evaluation indexes of PG,including platycodin D（PD）,total saponins,and alcohol-soluble extract,were detected and analyzed.In addition,the contents of other active ingredients of PG,such as total polysaccharides,total flavonoids,and 17 amino acids,were determined.The results showed that there were significant differences in appearance,total saponins,PD,polysaccharides,and alcohol-soluble extract among PG slices prepared using different processing methods.Among them,the PG slices produced by semi-dry cutting and 70°C drying had excellent appearance,and the processing time was 16 hours,which greatly shortened the processing time of PG slices.Under this processing condition,the total saponin content in PG slices was 2.92%,PD content was 0.16%,total polysaccharide content was 21.12%,and alcohol-soluble extract content was 20.1%,which were among the highest compared to other processing methods.Therefore,semi-dry cutting and 70°C drying can be considered as the best processing method for PG slices,laying a foundation for subsequent experiments.Based on the screening of processing methods,we conducted single-factor experiments to investigate the effects of drying temperature and slice thickness on PD,total saponins,total polysaccharides,and alcohol-soluble extract.Using PD content,total saponin content,alcohol-soluble extract content,and the total evaluation value of appearance as evaluation indexes,we used the Box-Behnken response surface method to optimize the best processing parameters.The optimal processing conditions for the initial processing of PG were determined as follows:the water content before slicing was 55%,the drying temperature was 60°C,and the slicing thickness was 4 mm,which provides a scientific basis for standardizing the production of PG slices.It provided the best raw material basis for extraction and separation of platycodon saponins.2.Extraction and separation of platycodon saponins and their structural identificationThe main effective components of PG were isolated and obtained from the processed Platycodon root using traditional chromatographic separation techniques and high-performance liquid chromatography.Modern spectroscopic techniques（MS,1H-NMR,13C-NMR,etc.）were applied to identify 16 saponin compounds,including platycodin D（1）,deapi-platycodin D（2）,polygalacin D（3）,polygalacin D（4）,deapi-Platycoside E（5）,3-O-β-D-glucopyranosyl polygalacic acid（6）,3-O-β-laminaribiosylpolygalacic acid（7）,3-O-β-glucopyranosyl platycodigenin（8）,3-O-β-gentiobiosylplatycodigenin（9）,3-O-β-D-glucopyranosy I-（1→6）-β-D-glucopyranosy I-（1→6）-β-D-glucopyranosyl-2β,3β,16α,23,24-pentahydroxyolean-l2-en-28-oic acid（10）,polygalacin D₂（11）,platycoside E（12）,platycodin D₂（13）,platycodin D₃（14）,platycoside J（15）,platycoside F（16）.The saponins isolated from PG provide material basis for the study of platycodon pharmacological activity.3.Protective effect of PD on D-glucose-induced INS-1 cell damageIn this study,an in vitro cell model of T2DM was established and the isolated saponin monomers were screened for their in vitro activity.It was found that PD and deapi-platycodin D and platycoside E significantly increased cellular activity.Among them,PD had the best activity,so its pharmacological activity was further studied.In INS-1 cell experiments,pretreatment with PD significantly reduced ROS levels and inhibited the increase in mitochondrial membrane potential in INS-1 cells.Hoechst 33258 staining results showed that PD effectively inhibited high glucose-induced cell apoptosis.Molecular docking results also showed that PD has good binding ability with target proteins related to endoplasmic reticulum stress（ERS）and apoptosis.The combination of Western blotting and molecular dynamics（MD）validation fully demonstrated that PD can reduce the impact of high glucose-induced INS-1 cell damage by regulating the ERS and apoptosis signaling pathways.Therefore,PD may be a promising saponin monomer for improving T2DM and deserves further study.4.Effect of PD on T2DM mice induced by HFD/STZTo further investigate the ameliorative effect of PD on T2DM,a mouse model of type 2diabetes（T2DM）was established by using a high-fat diet（HFD）combined with streptozotocin（STZ）injection,and conduct PD oral administration experiments.Fasting blood glucose（FBG）levels were measured weekly for 4 weeks after STZ injection,and FBG levels greater than 7.8m M were defined as T2DM.After 8 weeks of oral gavage with PD（2.5 mg/kg）,relevant indicators were measured.The results showed that PD treatment（2.5 and 5.0 mg/kg）improved HFD-induced weight gain in mice and reduced fasting blood glucose（FBG）levels,improved glucose tolerance（OGTT）and insulin tolerance（ITT）levels.In the four blood lipid tests,PD administration reduced serum total cholesterol（TC）,triglycerides（TG）,and low-density lipoprotein（LDL）in T2DM mice,controlling the increase in blood lipid levels.In pancreatic and adipose tissue histological staining,PD significantly improved pancreatic damage and fat degeneration caused by T2DM.5.The beneficial effects of PD on early complications of liver and kidney damage induced by T2DM.On the basis of a T2DM mouse model,the study investigated the therapeutic effect and molecular mechanism of PD on early-stage liver and kidney complications of T2DM.The results showed that PD treatment reduced liver histopathological damage and improved liver function as evidenced by decreased levels of aspartate aminotransferase（AST）and alanine aminotransferase（ALT）,thereby restoring abnormal liver function.Moreover,PD reduced liver glycogen breakdown.Molecular docking results showed that PD was closely related to key glucose and lipid metabolism proteins such as AMPK.Western blotting results verified that PD upregulated AMPK phosphorylation expression,downregulated PCK1 and G6Pase expression,and regulated hepatic gluconeogenesis pathways.In terms of lipid metabolism,PD reduced T2DM-induced liver fat accumulation through the AMPK/ACC/CPT-1 fatty acid synthesis and metabolism pathway.In the study of early-stage kidney complications of T2DM,molecular docking and molecular dynamics（MD）methods were first used to study the binding ability of PD with NF-κB and apoptosis-related proteins.The expression of NF-κB and apoptosis-related proteins was detected by immunofluorescence and Western blotting.In vitro experiments were performed using high-glucose cultured RAW 264.7 cells（mouse monocyte macrophages）and HK2 cells（human renal proximal tubular epithelial cells）to verify the relevant mechanism.In vivo experiments showed that PD（2.5 and 5.0 mg/kg）reduced creatinine（CRE）and blood urea nitrogen（BUN）levels in DN mice.PD regulated the NF-κB and apoptosis signaling pathways,reduced serum levels of inflammatory cytokines TNF-αand IL-1βin DN mice,repaired renal apoptosis,and significantly inhibited the occurrence and development of DN.In vitro experiments confirmed that PD regulated the inflammation induced by high glucose in RAW264.7 cells through the NF-κB signaling pathway and inhibited the release of inflammatory cytokines.In HK2 cell experiments,PD was found to inhibit the generation of reactive oxygen species（ROS）and reduce the decrease in mitochondrial membrane potential（MMP）by regulating the NF-κB and apoptosis pathways,thereby inhibiting HK2 cell damage.These data indicate that PD has the potential to prevent early-stage kidney complications of T2DM and is a promising natural hepatorenal protectant.6.The effect and mechanism of PD on T2DM-induced intestinal injury and microbiota changes.First,histopathological sections of the duodenum were stained,and serum levels of LPS and DAO were analyzed in T2DM mice.DNA was extracted from the feces of the mice and high-throughput sequencing was performed,and microbiota analysis was conducted using software such as Mothur and QIIME.The results showed that PD had a certain restorative effect on the duodenal injury caused by T2DM in mice,and its related mechanism may be related to the increased expression of ZO-1 and Occludin proteins,which improved the barrier function of the intestinal mucosa,thereby preventing the influence of external pathogenic factors.In addition,through the detection of the composition of the intestinal microbiota,it was found that PD can significantly improve the richness and composition of the microbiota.These results indicate that PD has a beneficial effect on T2DM-induced duodenal injury and microbiota.In summary,the screening and optimization of the original processing process of PG provide a theoretical basis for the implementation of origin processing.PD regulates liver function through gluconeogenesis and lipid metabolism pathways,and protects the kidney from damage through NF-κB and apoptosis pathways,providing a theoretical basis for PD treatment of T2DM and related complications,as well as the development of new therapeutic drugs for T2DM.