Study On The Material Basis And Mechanism Of Dashanzha Pill Based On Crataegi Fructus Before And After Charring Process

Object:Dashanzha Pill is a representative formula for promoting appetite and eliminating food stagnation in clinical practice.It is used for treating loss of appetite and dyspepsia caused by food stagnation,etc.However,the formula’s material basis and mechanism of effect are still unclear.The main ingredient of this formula,Crataegi Fructus,has strong irritation in raw form,but when charred,it can moderate the irritation and enhance the therapeutic effect of eliminating food.To study the similarities and differences in the composition and efficacy between Dashanzha Pill composed of raw and charred Crataegi Fructu,qualitative identification of chemical components in vitro and in vivo,identification of main components,and establishment of fingerprint profiles were carried out.Additionally,we comparatively analyzed the gastrointestinal motility indexes,endoplasmic reticulum stress PERK/e IF2αsignaling pathway-related factors and intestinal flora in rats with high-fat food induced dyspepsia after different Dashanzha Pill treatment.This study will provide a reference for studying Dashanzha Pill and processing of traditional Chinese medicine.Method:1.LC/MS study of the chemical composition of Dashanzha Pill composed of raw and charred Crataegi Fructu in vivo and in vivoThe Dashanzha Pill composed of raw and charred Crataegi Fructu were prepared according to the Chinese Pharmacopoeia.The data of different Dashanzha Pill extracts and rat serum containing drugs were collected by ultra-performance liquid chromatography-high resolution mass spectrometry in positive and negative ion modes.And the chemical composition and blood migration components were identified by combining with reference substances and proprietary database.2.Study on the quality difference of the Dashanzha Pill composed of raw and charred Crataegi Fructu based on HPLCThe HPLC method was used to establish ursolic acid determination and fingerprint.The common peaks of different Dashanzha Pill were determined by using the " Chinese Medicine Fingerprint Similarity Evaluation System(2012 Version)".The 10 batches of raw and charred Dashanzha pill were distinguished and compared by cluster analysis,principal component analysis and orthogonal partial least squares-discriminant analysis.3.The effects of Shengshanzha Pill and Jiaoshanzha Pill on PERK/e IF2α pathway in gastric sinus tissues in rats with dyspepsiaA high-fat diet was used to induce a dyspepsia model in SPF-grade SD rats,and saline and corresponding drug interventions were given to the Control group,Model group,Domperidone group,Shengshanzha Pill group and Jiaoshanzha Pill group respectively.The pharmacodynamic indicators,including general status,body weight,food intake,gastric residual rate,intestinal propulsion rate,gastric acidity and HE staining,as well as gastrointestinal motility indexes MTL,GAS,VIP in serum were measured to compare the similarities and differences between the efficacy of raw and charred Dashanzha Pill.The gene and protein expressions of related factors GRP78,PERK,e IF2α in rat gastric sinus tissues were also measured to investigate the relationship between the appetizing effects of different Dashanzha Pill and the endoplasmic reticulum stress PERK/e IF2α signaling pathway.4.The effect of Dashanzha Pill composed of raw and charred Crataegi Fructu on the intestinal flora in rats with dyspepsiaFecal samples were collected and the intestinal flora was sequenced by 16 S r RNA sequencing technology according to the bacterial V4～V5 variable region.And the Alpha diversity,Beta diversity,flora structure and differential species of bacterial community were analyzed to investigate the effect of different Dashanzha Pill on the intestinal flora of rats with dyspepsia.Result:1.LC/MS study of the chemical composition of Dashanzha Pill composed of raw and charred Crataegi Fructu in vivo and in vivoA total of 115 compounds were identified in the extracts of Dashanzha Pill composed of raw and charred Crataegi Fructu,of which 8 components,including D-Saccharic acid,catechin,D-fructose,Astragalin and Stearamide,were unique to Shengshanzha Pill.And 8components,including Gluconic acid,Salicylic acid,Naringin,Hexadecanamide and Oleamide,were unique to Jiaoshanzha Pill.There were not detected in the blank serum but 50 components in the serum of rats after drugs administration.There were 30 components in the serum given Shengshanzha Pill and 34 components in the serum given Jiaoshanzha Pill.There were 14 samel blood components in Shengshanzha Pill and and Jiaoshanzha Pill.16 blood components in Shengshanzha Pill were different from Jiaoshanzha Pill,mainly flavonoids.And 20 blood components of Jiaoshanzha Pill were different from those of Shengshanzha Pill,mainly flavonoids,organic acids and their derivatives.2.Study on the quality difference of the Dashanzha Pill composed of raw and charred Crataegi Fructu based on HPLCThe content of ursolic acid in Shengshanzha Pill was measured to be 2.39 mg/g and in Jiaoshanzha Pill was 1.71 mg/g that was significantly lower.The fingerprint had 13 common peaks in Shengshanzha Pill as well as 13 common peaks in Jiaoshanzha Pill,and 10 common peaks were marked in Shengshanzha Pill and Jiaoshanzha Pill.Gallic acid,5-hydroxymethylfurfural,chlorogenic acid and gibberellin were identified.Cluster analysis,principal component analysis and orthogonal partial least squares-discriminant analysis revealed that all three methods could effectively differentiate the samples into two categories,Shengshanzha Pill and Jiaoshanzha Pill,with consistent results.5-hydroxymethylfurfural,gallic acid and chromatographic peaks 6 and 7 could be used as markers of quality differences between the two drugs.3.The effects of Shengshanzha Pill and Jiaoshanzha Pill on PERK/e IF2α pathway in gastric sinus tissues in rats with dyspepsiaAll the administration groups improved the mental status of rats,increased body weight,intestinal propulsion rate and decreased gastric residual rate,and had a therapeutic effect on high-fat diet-induced dyspepsia.Compared with the Model group,the rats in the Domperidone group,Shengshanzha Pill group and Jiaoshanzha Pill group had significantly increased body weight,increased GAS and MTL content,and decreased VIP content.Meanwhile,there were some differences in the effects between Shengshanzha Pill and Jiaoshanzha Pill,especially the modulating effects of Jiaoshanzha Pill on increasing body weight,promoting intestinal propulsion rate,and promoting gastrointestinal hormone MTL secretion were significantly better than those of Shengshanzha Pill.Compared with the Control group,the m RNA and protein expression of ERS-related factors GRP78,PERK,e IF2α in the gastric sinus tissues of rats in the Model group were significantly increased;compared with the Model group,Shengshanzha Pill could significantly reduce GRP78,PERK,e IF2α m RNA and GRP78 protein expression;while Jiaoshanzha Pill could cause GRP78,PERK,e IF2α m RNA and protein expression were significantly reduced,and the levels of each factor were restored to be no different from those of the Control group.Overall,Jiaoshanzha Pill showed stronger effect than Shengshanzha Pill.4.The effect of Dashanzha Pill composed of raw and charred Crataegi Fructu on the intestinal flora in rats with dyspepsiaThe results showed that the Shannon and Inv Simpson alpha diversity index in rats with dyspepsia significantly decreased,and the regulation of diversity and richness by Dashanzha Pill was not obvious.The beta diversity results showed that there were significant differences between the Control group and the Model group,and between the Model group and each administration group.The relative abundance of Verrucomicrobia,Akkermansiaceae,and Verrucomicrobia in the Model group was significantly higher than that of Control group,and that was suppressed after Shengshanzha Pill and Jiaoshanzha Pill intervening,and Jiaoshanzha Pill was able to restore it to the normal level.In contrast,the trend of Muribaculaceae was opposite,and the relative abundance was significantly reduced,with different degrees of increase after the intervention of Shengshanzha Pill and Jiaoshanzha Pill.They regulated the above species differently,and overall,and Jiaoshanzha Pill was better than Shengshanzha Pill in regulating the disordered flora.Conclusion:The material basis of Dashanzha Pill in vitro and in vivo were dominated by organic acids and flavonoids,along with other components.There were some differences in chemical composition and blood migration components between Shengshanzha Pill and Jiaoshanzha Pill.The content of ursolic acid in Jiaoshanzha Pill decreased significantly.However,the contents of 5-hydroxymethylfurfural and gallic acid were significantly increased.There are obvious differences between them,and they are two different categories.Different Dashanzha Pill had certain ameliorative effects on indigestion symptoms,decreasing gastric residual rate and increasing intestinal propulsion rate,and regulating gastrointestinal hormone secretion.The mechanism of effects might be reducing the expression of endoplasmic reticulum stress PERK/e IF2α pathway-related factors such as GRP78,PERK and e IF2α in gastric sinus tissue to inhibit ERS,and inhibit the overgrowth of Verrucomicrobia and Ackermannia and increase the relative abundance of Muribaculaceae to restore the intestinal flora.In the above aspects Jiaoshanzha Pill was overall better than Shengshanzha Pill.The effect substances and mechanism of Dashanzha Pill treating for appetite and food dissipation were initially revealed,which provided a reference for the application of Crataegi Fructus before and after charring process.