| Functional dyspepsia(FD)is a common gastrointestinal disease with a global incidence of 5-20%.It is defined as disorder of brain-gut interaction by the Roman Ⅳ,which is a group of diseases combined with dynamic disorders,visceral hypersensitivity,mucosal and immune function changes,intestinal flora changes,central nervous processing changes,and gastrointestinal symptoms.Gastrointestinal motility drugs,acid inhibitors and reducing visceral hypersensitivity drugs are clinicly prescribed for treating FD.Houpo,the stem bark of Magnolia officinalis Rehd.et Wils and M.officinalis Rehd.et Wils.var.biloba Rehd.et Wils,was one of traditional Chinese medicines.It was described as being warm in property and bitter and acrid in flavor and could affect the spleen,stomach,lung and large intestine meridians,which had properties of lowering Qi and eliminating fullness and was used for the treatment of phlegm,fullness,constipation and etc..More than 200 kinds of prescriptions including Houpo,have been used for the treatment of various gastrointestinal tract diseases.In this study,we aimed to investigate the effect of Houpo on FD rats and its mechanism based on the’microbiota-gut-brain axis’.Meanwhile,LC-MS/MS technology was applied to investigate the pharmacokinetics and metabolites of Houpo in vivo,which could provide data for excavating the pharmacodynamic material basis of Houpo.1 The effects of Houpo on brain-gut peptide in FD ratsNeonatal gastric irritation and mature alternate-day fasting were used for FD model preparation.Enzyme-linked immunosorbent assay(ELISA),real time quantitative reverse transcription polymerase chain reaction(RT-q-PCR)and immunohistochemistry were used to determine the level of brain-gut peptide in serum and 5-hydroxytryptamine(5-HT)related receptors in colon,respectively.Compared with normal rats,Houpo could promote gastric empty rate,increase the level of calcitonin gene-related peptide(CGRP)and the gene expression of 5-HT receptors such as 5-HT3R,5-HT4R and serotonin reuptake transporter(SERT),transient receptor potential A1(TRPA1)and transient receptor potential C4(TRPA1)in FD rats.2 The effect of Houpo on neurotransmitters and their metabolites in FD ratsThe UPLC-QqQ-MS/MS method was well established to determine fifteen neurotransmitters and their related metabolites like L-tryptophan(Trp),L-Tyrosine(Tyr),Glutamic acid(GLU),L-Kynurenine(KYN),y-aminobutyric acid(GABA),5HT and etc.,which showed good specificity,linearity,precision,accuracy,extraction recovery,matrix effect and stability.It was applied to determine the concentration of above neurotransmitters in hippocampus,cortex,serum,urine and feces of FD rats.The types and levels of the 15 neurotransmitters were different in hippocampus,cortex,serum,urine and feces,among which the level of Trp,Tyr,GLU and GABA were higher in the five biological samples.Compared with control group,the levels of Tyr,KYN and KYN/Trp in hippocampus,cortex and serum were increased and the level of GLU were decreased,the level of 5-HT/Trp level in serum was increased and 5-HT/Trp level in urine was decreased in FD rats.Houpo could improve FD by increasing the level of GLU in cortex,5-HT/Trp in cortex and hippocampus,5-HT/Trp in urine,and decreasing the level of Tyr in cortex,hippocampus and serum,KYN and KYN/Trp in hippocampus and serum,and 5-HT/Trp in serum.3 The effect of Houpo on gut microbiota16S rRNA sequencing was applied to investigate the effect of Houpo on gut microbiota in FD rats.The results of a-diversity and β-diversity analysis showed that species abundance in FD rats was increased compared with the control group,and Houpo significantly decreased the increased species abundance of FD rats.A total of 29 different microbiota were screened by the method of LEfSe and metastat.The level of eight kinds of short-chain fatty acid in rat feces were determined by UPLC-QqQ-MS/MS method.Compared with the control group,the level of isobutyric acid in FD rats was significantly increased(p<0.05),and the level of butyric acid,valeric acid,isovaleric acid and heptanic acid showed an increasing trend.Houpo could downregulate the level of above five short-chain fatty acids in FD rats.In addition,there were the significant correlations between 18 microbiota and 8 kinds of SCFAs by Spearman two-tail method.4 The study on metabolism of Houpo in vivoUPLC-QqQ-MS/MS method was established to determine syringin,magnoloside B,magnoloside A,honokiol and magnolol in Houpo for investigating their pharmacokinetics in vivo.At the dose of 2 g/kg,the concentration-time curves of the glycosides(syringin,magnoloside B and magnoloside A)showed a single peak with the characteristics of fast absorption,short half-life,low AUC(0-t)and high clearance rate.For example,the AUC(0-t),MRT(0-t),t1/2z,CLz/F of magnoloside B were respectively 233.79±113.72 μg·h/L,3.29±1.1 h,4.3±6.86 h,and 226.69±157.56 L/h/kg in normal rats.The concentration-time curves of the lignans(honokiol and magnolol)showed double absorption peaks and had the characteristics of fast absorption,longer half-life,higher AUC(0-t),and lower clearance rate,compared with the glycosides.For example,the AUC(0-t),MRT(0-t),T1/2z and CLz/F of honokiol were respectively 54466.67±23060.79 μg·h/L,9.19±4.44 h,6.68±6.65 h and 0.86±0.57 L/h/kg in normal rats.As for glycosides the MRT was shortened and the CLz/F was increased in FD rats,.As for lignans,the MRT was significantly shortened,the AUC and Cmax showed a decreasing trend.The CLz/F of honokiol showed an increasing trend,while the CLz/F of magnolol showed a decreasing trend in FD rats.Our results indicated that the pharmacokinetic parameters of the above five components in Houpo changed under pathological conditions.In addition,it was found that coexisting ingredients of Houpo could decrease the absorption and increase the elimination of honokiol,while decreased the elimination of magnolol in vivo,compared with those of the mixture of honokiol and magnolol.The level of magnoloside B,magnoloside A,honokiol,magnolol and hydroxytyrosol were determined in urine and feces by using UPLC-QqQ-MS/MS method.It was found that phenylethanoid glycosides were excreted in urine and feces mainly as the form of hydroxytyrosol and magnoloside A,respectively.The excretion rate of honokiol in feces and urine was higher than those of magnolol.The UPLC-Q-TOF-MS method were applied to analyze the metabolites of honokiol,magnolol,magnoloside A in vivo.A total of 33 metabolites of honokiol(7 and 26 metabolites in phase Ⅰ and phase Ⅱ,respectively),13 magnolol metabolites(5 and 8 metabolites in phase Ⅰ and phase Ⅱ,respectively)and 8 metabolites of magnoloside A were identified from the sample of serum,liver,stomach,intestine and feces in normal rats,among which,18 metabolites were firstly identified in this study.In conclusion,the effect of Houpo on FD rats were investigated in terms of gastrointestinal function regulation,brain-gut peptide secretion,neurotransmitter level and gut microbiota.The results showed the Houpo could improve FD symptoms by promoting gastric empty,regulating brain-gut peptide secretion,influencing the level of neurotransmitter and changing gut microbiota structure and short chain fatty acid.Furthermore,the glycosides and lignans of Houpo have the characteristics of fast absorption and elimination in FD rats,and the coexisting ingredients is one of the factors affecting the metabolism of lignans in Houpo. |
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