Research On The Material Basis And Mechanism Of Jiaobaizhu's Anti-gastric Ulcer Based On Nanotechnology

BackgroundThroughout the historical records of the processing of Atractylodes macrocephala Koidz.(AM),the "fre" method has a long history of application.The processing of AM is very important to enhance its spleen and stomach function,which has been proved in clinical practice and modern pharmacology,but its processing mechanism has not been fully revealed.Especially for the study of charred Atractylodes macrocephala Koidz.(CAM),along with the traditional Chinese medicine material basis research ideas have failed to clearly explain its key material basis and pharmacodynamic effect.Through preliminary research and with the help of the research methods and characterization methods in the field of nanoscience,our team found that after the high-temperature carbonization of traditional Chinese medicine,a class of new ingredients with pharmacological activity will be produced,whose structural characteristics belong to nanoparticles,so it was named as nanometer components.This provides a new way of thinking and inspiration for the study of CAM.In recent years,although the treatment of gastric ulcer has made some progress,it still faces many difficulties that are difficult to break through,such as high recurrence rate of gastric ulcer,difficulties to control bleeding complications,drug side effects,bacterial resistance and so on.It is of great significance to improve the curative effect of gastric ulcer by making good use of the advantages of traditional Chinese medicine.The preparation of AM has its unique advantages in the treatment of spleen and stomach diseases.Objective(1)To confirm the anti-ulcer effect of processed AM through the experimental study on the influence of gastric ulcer model,and further explore the material basis.The anti-ulcer bioactive parts were preliminarily located,and the structure and properties of the components were identified and analyzed.(2)To reproduce and optimize the processing process of "fire" method of CAM in the laboratory,and then extract enough anti-ulcer active parts from them and characterize them,so as to further understand the structure,morphology,active groups and other characteristics of the components,and then evaluate its safety.(3)A variety of animal models were used to evaluate the antiulcer activities of the bioactive parts,and to explore its antiulcer mechanism and effect on intestinal microecology.Methods(1)The effects of commercially available AM,Fried AM and CAM on gastric ulcer were compared using alcoholic gastric ulcer model of mice.In order to study the pharmacodynamic substance basis of "CAM",the hydrolytic decoction of pyroatractylodis was dialysed and separated into two parts:the inside and outside solution of the dialysis bag.The effects of the two parts on the gastric ulcer model were compared,and the key antiulcer bioactive parts were screened out.The active parts of antiulcer were identified and analyzed with the help of the characterization technology of nanometer materials.(2)In this paper,we used muffle furnace to heat AM,and established a relatively standard and repeatable processing technology of AM.Then through full purification and dialysis,the preparation method of antiulcer active parts was established.We also compare the efficacy of antiulcer active parts prepared under different conditions,and select the best preparation conditions.The samples prepared under various conditions were characterized in detail of morphological characteristics,optical characteristics,structural characteristics and so on.In the best preparing condition,enough antiulcer fractions were prepared from CAM for the following study.(3)We also evaluated the safety of the bioactive anti-ulcer parts of CAM through cytotoxicity and acute toxicity experiments to obtain its safety parameters.(4)In this study,the gastric ulcer animal model induced by ethanol,water restraint stress,and indomethacin were used to futher study the bioactivities of the antiulcer parts of CAM.Through the evaluation of gastric ulcer in animal model and the observation of gastric histopathology to evaluate the efficacy of anti-ulcer,the its active mechanisms,the bioactive parts of CAM was analyzed by the content detection of gastric tissue related indexes and serum related indexes in animal model.(5)Through the 16s rDNA detection of intestinal flora in rats,combined with the extensive targeted screening and analysis technology of serum metabolomics,the effect of antiulcer parts in CAM on intestinal microecology and the effect on the overall metabolism of rats was preliminarily discussed.Results(1)The three kinds of decoction pieces of commercially sold AM all have anti-gastric ulcer effects on mice.The anti-ulcer effects of fried AM and charred AM are superior to those of raw AM,among which CAM has the highest inhibition rate of ulcer,while there is no significant difference between fried AM and charred AM.(2)The internal and external component solutions of the dialysis bag were obtained by separating and the components inside and outside the dialysis bag.By comparing the anti-ulcer efficacy of the two,it was found that the components in the dialysis bag were the main active parts of anti-ulcer.The characterization and identification results of the active part showed that there were no chromatographic peaks of small-molecule compounds in the HPLC.The particle size of the component was distributed between 1-25 nm,the maximum excitation wavelength was 300 nm,and the maximum emission wavelength was 445 nm.The surface mainly contained active groups such as hydroxyl,carboxyl and amino groups.Based on the above characteristics,it was confirmed that this part belongs to nano components,and was named as "Charred Atractylodis Macrocephalae Rhizoma.nano-components"(CAM-NCs)in this paper.(3)The preparation process of CAM and CAM-NCs was established and optimized by using muff furnace heating method at high temperature.The anti-ulcer effect of CAM-NCs under different conditions was investigated.The optimal preparation condition was selected as 350? heating for 1h based on ulcer inhibition rate and anti-ulcer index.The experiment of tail breakage hemostasis in mice showed that CAM-NCs had hemostasis effect,which verified that it had the common hemostasis effect of carbon nanoparticles.(4)The detail characterization of CAM-NCs was studied,of which morphological structure,optical properties and surface groups were obtained.It was found that the particle size distribution of 350? CAM-NCs was relatively uniform,between 1-10 nm,while the average particle size of 250? and 300? CAM-NCs was larger,with slight agglomeration and overlap,while the average particle size of 400? CAM-NCs was the smallest.The ultraviolet absorption and fluorescence properties of CAM-NCs prepared at different temperatures were similar,mainly containing elements C,O and N,as well as a small amount of elements S and P,with various active functional groups such as hydroxyl group,carboxyl group and amino group on the surface.It indicates that the preparation temperature mainly affects the particle size and dispersion of CAM-NCs s,but has little effect on the fluorescence and ultraviolet characteristics of CAM-NCs and the types of surfactant.(5)Cytotoxicity and acute toxicity experiments showed that the concentration of CAM-NCs in the range of 7.8125 g/ml-2000 g/mL was safe.(6)Effects of CAM-NCs on ethanol-induced gastric ulcer in rats showed that CAM-NCs had an obvious anti-ulcer effect on this model,and the inhibition rate of ulcer could be up to 60%.On the one hand,the mechanism of CAM-NCs may reduce mucosal damage by inhibiting gastric mucosal attack factors,including reducing levels of inflammatory factors such as TNF-a,IL-6 and oxidative stress products such as MDA.On the other hand,the defense ability of gastric mucosa was improved,including the increase of IL-10,PGE2 and the increase of MUC5AC,which played a role in protecting gastric mucosa.At the same time,CAM-NCs can optimize and regulate the alpha diversity and component structure of intestinal flora.(7)Through the study of the influence of CAM-NCs on the rat model of stress gastric ulcer,it was found that the anti-ulcer effect of CAM-NCs on the model was extremely strong,and the inhibition rate could reach over 90%.The mechanism may be related to reducing the levels of inflammatory cytokines TNF-?,IL-1? antioxidant stress(reducing MDA and MPO),increasing the level of PGE2,and increasing the secretion of MUC5AC to protect the gastric mucosa.In addition,for the stress ulcer model,CAM-NCs can reduce the secretion of 5-HT and DA in the brain under stress,reduce the excessive neuroendocrine response caused by stress,regulate the body's energy metabolism and the structure of intestinal flora,improve the damage caused by stress to the body,and relieve the occurrence of gastric ulcer caused by stress.(8)Through the study on the effect of CAM-NCs on indomethacin induced gastric ulcer in mice,it was found that CAM-NCs also had obvious anti-ulcer effect on this model,with the inhibition rate up to 77.6%.The results showed that indomethacin inhibited the synthesis of prostaglandin PGE2,released the inflammatory cytokines TNF-? and IL-1? and activated the cascade reaction of NF-?B signaling pathway in the model mice,while CAM-NCs could reverse the abnormal performance of the model group and make the content of each indicator close to the level of the normal group.ConclusionIn this study,the antiulcer effects of prepared fried AM and charred AM were enhanced compared with that of AM.The nanoparticles produced in the process of processing may be the key material basis for the enhancement of antiulcer effect of CAM.Calcining in muffle furnace at high temperature can reproduce and improve the "fire" process of CAM under laboratory conditions,and the stable and easily controlled CAM can be processed,and then the nanometer components of CAM can be prepared.This ingredient has strong antiulcer activity.In three different gastric ulcer models(ethanol-induced gastric ulcer and stress gastric ulcer),CAM-NCs has a strong anti-ulcer effect,and their common mechanism of action may be inhibiting the inflammatory response,antioxidant stress,and improve the level of PGE2,so as to protect the gastric mucosa.In the ethanol and stress models,CAM-NCs can regulate the diversity and structure of intestinal flora in the two models.Among them,the efficacy of stress ulcer caused by water immersion is the best.The analysis may be related to the reduction of 5-ht and DA in the brain,so as to regulate the neuroendocrine response under stress,relieve the abnormal energy metabolism of the body,and then reduce the stress injury.Therefore,this study confirmed the presence of nanoscale components in the CAM,which had a definite therapeutic effect on three different mouse gastric ulcer models and had a certain regulatory effect on intestinal flora.This study not only provides certain reference for the clinical use of traditional Chinese medicine in treatment of gastric ulcer,but also broadens the way of the modern research of processed drugs.