Study On Material Basis And Mechanism Of Coked Atractylodes Rhizome Non-Volatile Invigorating Spleen And Stopping Diarrhea

1 ObjectiveAtractylodes Rhizome is a commonly used medicine for invigorating spleen and removing dampness.The raw Atractylodes Rhizome has strong ability of removing dryness,dispelling wind and dispersing cold,and it is mainly used for dampness in Middle-Jiao,rheumatic pain,numbness of skin,pain of feet and knees,and cold.But the coked Atractylodes Rhizome can strengthen the spleen function and mainly used to treat diarrhea caused by spleen deficiency and dampness.In this study,the main incremental components for the effects of tonifying spleen and stopping diarrhea from the coked Atractylodes Rhizome will be found and researching the effects of tonifying spleen and stopping diarrhea of various non-volatile extracts before and after the coked from Atractylodes Rhizome.Then the processing principle of Atractylodes Rhizome,its main material basis and mechanism of action were explained preliminary.2 methods and results2.1 Study on the pharmacodynamics of each extract for strengthening spleen and stopping diarrheaThe essential oil in the raw and coked Atractylodes Rhizome was extracted by methods of steam distillation.The water extract was extracted with dichloromethane and n-butanol saturated by water respectively,then the parts of dichloromethane,n-butanol and rafied-water were obtained.The model of splenasthenic diarrhea was selected,and Shen Ling Bai Zhu pill was the positive drug.The indexes of diarrhea index,intestinal propulsion rate,gastric residue rate,plasma MTL,serum GAS,VIP,TNF-?,IL-10 and colonic AQP₃ were used to study the pharmacodynamics of the extracts from the raw and coked Atractylodes Rhizome.The results showed that in terms of the macro diarrhea index,the coked group was significantly lower than the corresponding parts and the same dose of the raw material group?P<0.05?,and the diarrhea index of the medium and high dose of the coked group was reduced to no statistical difference from that of the positive drug group?P>0.05?.In the regulation of gastrointestinal motility?intestinal propulsion rate,gastric residue rate,plasma MTL,serum GAS,VIP?,the part of n-butanol>the part of dichloromethyl>the part of water,and the part of coked n-butanol significantly better than the part of raw product n-butanol in the regulation of gastrointestinal motility?P<0.05?.In terms of the regulation of inflammatory factors,the effect of n-butanol was significantly better than that of dichloromethane and water,and the effect of n-butanol on inflammation of coked was significantly better than that of n-butanol at the corresponding dose?P<0.05?.In addition,in terms of the expression of colon AQP₃,the high,medium and low dose groups of coked n-butanol were significantly better than those of other parts,but it was no significant difference from the normal group and the positive group?P>0.05?.2.2 Influence of each extracted part for damp evilThe model for syndrome of accumulation of dampness in middle-Jia was selected and Xiang Sha Wei Ling pill was the positive drug.Using kidney AQP₂,plasma ALD and electrolyte levels as indicators,the effects of extraction parts of raw and coked Atractylodes Rhizome on metabolisms of water and electrolyte in model animals were investigated.Results:AQP₂ and ALD were significantly increased in the model group?P<0.01?,indicating that water wetting stopped in model body.At the same time,the plasma K⁺concentration of the model rats was significantly lower than that of the normal group?P<0.05?,while the concentrations of Na⁺and Cl^-were increased,but there was no significant difference?P>0.05?.After administration,the kidney AQP₂ and plasma ALD of rats in the positive drug group,the medium and high doses proups of n-butanol from raw Atractylodes Rhizome were significantly decreased,and the plasma K⁺level in the high dose group was increased to no significant difference from the normal group?P>0.05?.But the effects of other groups were not significant.2.3 Determinations of main incremental components from Atractylodes Rhizome before and after cokedBy comparing HPLC fingerprints of n-butanol extracted parts from raw and coked Atractylodes Rhizome,it was found that the fingerprints of n-butanol extracted parts of the coked Atractylodes Rhizome had five corresponding chromatographic peaks with significantly increased area at 3.4,4.5,9.0,15.4 and 23.6 min respectively,and two new chromatographic peaks with smaller area were generated at 5.4 and 8.6 min respectively.Combined with the literatures of Atractylodes Rhizome and the accumulation of the research group's previous studies on the pharmacodynamic components of Atractylodes Rhizome,the content of the main pharmacodynamic components in Atractylodes Rhizome was determined.Results:the contents of 5-HMF,atractyloside A and tannin in the coked Atractylodes Rhizome were significantly higher than those in the raw Atractylodes Rhizome,and the increment was 18 times,50%and 125%respectively.Meanwhile,the adsorption capacity of the coked Atractylodes Rhizome increased by 15%.2.4 pharmacological and pharmacodynamic study of the main incremental component“invigorating spleen and stopping diarrhea”2.4.1 Influence on diarrhea indexAfter 1 h of Rhubarb gavage,the mice in the model group presented obvious diarrhea symptoms successively,and the diarrhea index reached above 0.9,while the normal group had no loose stools.Compared with the model group,the diarrhea index of mice in each dose group decreased with the delay of the first time of defecation.Except for the low dose group of 5-HMF,the diarrhea index of each group was significantly different from that of the model group?P<0.05?.Among them,the high dose group of 5-HMF,the high dose group of atractyloside A,the high?medium?low dose groups of tannin,and the combined high?medium?low dose groups had extremely significant differences?P<0.01?.2.4.2 Influence on gastrointestinal motilityMethod by Rhubarb causing diarrhea combined with hunger was used to establish the model.Results:in the model group,the intestinal propulsion rate,plasma MTL and serum GAS were significantly decreased?P<0.01?,while gastric residue rate and serum VIP were significantly increased?P<0.01?.After the treatment,5-HMF,atractyloside A,tannins and combined dosage groups were all showed on enhancing gastrointestinal motility.Particularly,the high dose group of 5-HMF,atractyloside A,the medium dose group of tannins and the combined medium?low dose groups had obvious effect on the regulation of gastrointestinal dynamics,and the three factors shows certain synergy in promoting gastrointestinal dynamics.Meanwhile,the effect of tannins high dose and combination high dose was weakened on gastrointestinal motility.2.4.3 Influence on digestive tract inflammationELISA results showed that serum TNF-?was significantly increased?P<0.01?and IL-10 was significantly decreased?P<0.01?in the model group,accompanied by obvious diarrhea symptoms and significant digestive tract inflammation.TNF-?level in each dose group was significantly lower than that in the model group?P<0.01?,and IL-10 level was significantly higher than that in the model group?P<0.05?.There were no significant differences in TNF-?levels between the positive drug group,the high dose 5-hmf group,the combined high dose group and the normal group?P>0.05?,and no significant differences in IL-10 levels between the positive drug group,the high dose tannin group,the combined medium dose group and the normal group?P>0.05?.At the same time,the results of colon section HE staining showed that the positive medicine group,the high-dose of 5-HMF group,the high dose of ractyloside A group,the medium dose of tannins and combination high dose group was obviously returns on inflammation.The distribution of columnar and goblet cells was more regular,and the morphology of villi was clear with less breakage.2.4.4 Influence on water and salt metabolismELISA and biochemical tests showed that AQP₃ in the colon of the model rats was significantly decreased?P<0.01?,AQP₂ in the kidney was significantly increased?P<0.05?,and plasma ALD was significantly increased?P<0.01?,but there was no statistical difference between the model group and the normal group on plasma electrolyte level.In terms of the regulation of aquaporin,except for the low dose of5-HMF,the expression level for AQP₃ in the colon of other groups was significantly higher than that of the model group?P<0.05?.The effect of single administration on renal AQP₂ was not significant,only the expression level of AQP₂ in the combined high-dose group was significantly reduced,and there was no difference between it and normal group?P>0.05?.In terms of plasma ALD,only the combined high-dose group showed significant efficacy.These results suggest that 5-HMF,atractyloside A and tannins may have a synergistic effect on the regulation of body water and liquid metabolism,and there is a dose-dependent relationship.2.5 Preliminary study on the mechanism of"invigorating spleen and stopping diarrhea"of the coked Atractylodes Rhizome2.5.1 Influence on the structure of intestinal flora structureThrough sequencing of intestinal flora 16S rDNA Amplicon,the changes of intestinal flora structure in the model group and each group were compared according to relative abundance values of each bacterial family and genera.Model group rats intestinal Bacteroidetes abundance in significantly lower proportion of intestinal flora,bacteria and thick wall door and deformation abundanceratioincreasedsignificantly,withBacteroidesand Escherchia/Shigella represented by conditional pathogenic bacteria abundance significantly increased,and on behalf of probiotic lactic acid bacteria abundance significantly reduced.After administration,the high dose group of5-hmf,the high dose group of atractyloside A,the high dose group of tannin,and the combined high dose group all showed significant inhibitory effects on bacteroides and escherichia coli.At the same time,the lactobacillus abundance of each group was lower than that of the model group.2.5.2 Influence on the expression levels of 5-HT and 5-HTR in the colonThe results of ELISA and PCR-mRNA for 5-HT4R and 5-HT7R detection showed that 5-HT,5-HT4R and 5-HT7R were highly expressed in colon tissues of the model rats?P<0.01?.Compared with the model group,the overexpression of 5-HT,5-HT4R and 5-HT7R in the colon was significantly down-regulated in each administration group?P<0.05?.Among them,the contents of 5-HT in the colon of the high-dose group of 5-HMF,atractyloside A,tannins and combined administration were restored to the same level as those of the normal group?P>0.05?.The expression levels of5-HT4R in the high-dose group,the medium-dose and high-dose groups of atractyloside A,the low-dose and medium-dose groups of tannin and the combined low-dose,medium-dose and high-dose groups all decreased to no significant difference from the normal group?P>0.05?.The 5-HMF medium and high dose groups,atractyloside A medium and high dose groups,and combined low,medium and high dose groups all recovered to normal level?P>0.05?.3 Conclusion3.1 Coking can increase the dissolution rate of the chemical components of Atractylodes RhizomeThe yield of extract from different parts of coked Atractylodes Rhizome was significantly higher than that of raw Atractylodes Rhizome.It indicats that the processing of coking can improve the dissolution rate of chemical components in coked Atractylodes Rhizome.3.2 The n-butanol extraction site of coked Atractylodes Rhizome is the main pharmacodynamic site of its“invigorating spleen and stopping diarrhea”Based on the pharmacodynamics study of the extracts of raw and coked Atractylodes Rhizome on the model of spleen deficiency and diarrhea,the n-butanol extract of coked Atractylodes Rhizome was significantly stronger than the other extracts in enhancing gastrointestinal motility,promoting digestion,inhibiting digestive tract inflammation and promoting colon's absorption of fecal water.The results showed that the extract of n-butanol from coked Atractylodes Rhizome was the main pharmacodynamic part of“invigorating spleen and stopping diarrhea”.3.3 5-HMF,atractyloside A and tannins were the main incremental components of coked Atractylodes Rhizome that cause its change of efficacyThe HPLC fingerprints of the n-butanol parts of raw and coked Atractylodes Rhizome were compared and analyzed.There are significant incremental chromatographic peaks indeed.The contents of 5-HMF,atractyloside A and tannins were significantly increased in coked Atractylodes Rhizome with strong pharmacological activity.3.4 Synergistic regulation of gastrointestinal dysfunction by 5-HMF,atractyloside A and tannins5-HMF,atractyloside A and tannins can Promote gastrointestinal motility,inhibite intestinal pathogenic bacteria and the conditions of excessive proliferation against digestive tract inflammation,restrain the expressions of5-HT and its receptor 5-HT4R,5-HT7R in colon,restore its normal movement function,promote colon and restrain kidney moisture reabsorption,in to restore the metabolism of the body fluid balance.Achieve the goal of treatment of deficiency of spleen and diarrhea.In conclusion,5-HMF,atractyloside A and tannins contents increased significantly in Atractylodes Rhizome after coked.These lead to it good at"spleen antidiarrheal",and they are the main material basis.The coked Atractylodes Rhizome play efficacy mainly through promoting MTL,GAS and other gastrointestinal hormones to promote gastrointestinal movement,inhibiting the intestinal pathogens,against the digestive tract inflammation,and inhibiting the brain neurotransmitters 5-HT and its receptor 5-HT4R?5-HT7R high expression,adjusting the colon movement,promoting the expression of AQP₃ in colon and inhibit the expression of AQP₂ in kidneys,restore the body's normal metabolic pathways of the water and liquid,playing spleen anti-diarrhea effect.