Study On The Effect Of Mao Jian Green Tea On The Gastrointestinal Motility Of Rats And Its Mechanism Of Action

BackgroundMao Jian Tea（MJT）is a representative tea series of the Shanxi herbal teas six major producing areas,which is made from the tender leaves of Dracocephalum rupestre Hance,a perennial herb of the Lamiaceae family（also known as Mao Jian Cao）.According to“Chinese Materia Medica”,“National Compilation of Chinese Herbal Medicine”,and the“Highland herbal medicine treatment manual”,the whole herb of Mao Jian Cao can be used as medicine,which is pungent and bitter,cool in nature,and belongs to the lung and spleen meridians,with the effect of draining wind and clearing heat,cooling the liver and stopping bleeding.It is used for treating wind-heat headache,external sensation of food,distention and fullness in the chest and epigastrium,headache due to wind-damp,sore throat and cough.In the northern counties of Ningwu,Wuzhai,Shenchixian,and Kelan,it has been a custom to use D.rupestre for tea-making and believed to have the function of promoting digestion and tonifying the stomach.Depending on the tea-making and fermentation processes,MJT is mainly divided into Mao Jian Black Tea（MJBT）and Mao Jian Green Tea（MJGT）.As one of the recommended tourism products in Ningwu County,the sales volume of MJT in2020 alone has reached over 20,000 catties,and it has been exported to Southeast Asia.However,due to the lack of modern pharmacological research on the digestive and stomach-tonifying effects of MJT,there is still insufficient scientific evidence for its functional promotion.To promote the high-quality development of MJT industry in our province,the research team chose MJGT,which has a refreshing taste and is more widely accepted by the public,as the research object,and used modern pharmacological research techniques to conduct the first study on the effects of MJGT hydro extract（MJGT＿HE）on normal rat gastrointestinal motility and explored the feasibility of using MJGT ethanol extract（MJGT＿EE）for irritable bowel syndrome with constipation（IBS-C）animal model.The study aims to clarify the mechanism of MJGT on"abdominal distention"and provide new practical and theoretical basis to promote the development of the characteristic medicinal tea industry in our province.Objectives1.To clarify the regulatory effect of MJGT＿HE on the gastrointestinal tract,and preliminarily elucidate its mechanism of action and active ingredients.2.To explore the improvement effect of MJGT＿EE on IBS-C related symptoms and analyze the mechanisms.Methods1.According to the conversion between human and rat drug dosages,three concentration groups of MJGT＿HE,high（MJGT＿HE＿H）,medium（MJGT＿HE＿M）,and low（MJGT＿HE＿L）were set.After continuous 30 days of gavage administration to rats,changes in indicators such as gastric emptying rate,small intestinal propulsion rate,and serum gastrointestinal hormone secretion levels were used to preliminarily judge the effect of MJGT＿HE on gastrointestinal motility.The effect of MJGT＿HE on the spontaneous activity and frequency of gastrointestinal tissues was studied by in vitro experiments on the stomach,duodenum,jejunum,and ileum,to further clarify its action site.By using ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-ESI-MS）and high performance liquid chromatography（HPLC）,the main active ingredients and their contents were preliminarily determined.Then,the standard samples of the target active ingredients were set up with five concentration gradients to study the tension and frequency of the four gastrointestinal segments,and to identify the rules and screen the key active ingredients.Finally,the inter-group microbial differences were analyzed by 16S r DNA sequencing.2.An IBS-C rat model was constructed using infant separation combined with ice water gavage method.The effect of MJGT＿EE on gastric emptying rate and small intestinal propulsion rate in the rat model was studied.The changes in colonic tissue morphology and5-HT secretion were observed by hematoxylin-eosin staining and immunohistochemistry.Western blot was used for quantitative analysis to detect changes in upstream protein tryptophan hydroxylase（TPH）and downstream protein serotonin transporter（SERT）in the5-HT pathway and visceral sensitivity-related receptors 5-hydroxytryptamine receptor 3（5-HT₃R）and 5-hydroxytryptamine receptor 4（5-HT₄R）,further revealing the mechanism of action of MJGT＿EE.Results1.From the gastric emptying rate and small intestinal propulsion rate experiments,it was found that the low（MJGT＿HE＿L）and medium（MJGT＿HE＿M）concentration groups could promote gastrointestinal motility（p<0.05）,while the high concentration（MJGT＿HE＿H）had an inhibitory effect（p<0.01）.MJGT＿HE＿L and MJGT＿HE＿M could promote the secretion of motilin（MTL）and gastrin（GAS）,and inhibit the generation of vasoactive intestinal peptide（VIP）（p<0.05）,while in MJGT＿HE＿H,except for MTL,there was no significant difference（p>0.01）and the secretion levels of GAS and VIP were opposite to those in MJGT＿HE＿L and MJGT＿HE＿M.Four major active components were identified by HPLC and UPLC-ESI-MS,including eriodictyol,luteolin,eriodictyol-7-O-glucoside and luteolin-7-O-glucoside.These compounds can all regulate the contraction of gastrointestinal segments in vitro,but two glycoside components are predominant.In addition,different concentrations of MJGT＿HEalso affect the composition and abundance of gut microbiota.Specifically,MJGT＿HE＿L increased the abundance of beneficial bacteria,such as Muribaculaceae,Prevotellaceae,and Lactobacillaceae,and inhibited the growth of pathogenic microbes,especially Staphylococcaceae.However,the abundance of these two types of bacteria in MJGT＿HE＿H showed the opposite trend compared to MJGT＿HE＿L.2.An IBS-C（Irritable Bowel Syndrome with Constipation）animal model was established using maternal separation combined with ice water gavage,and the model was evaluated by measuring two indicators,fecal water content（FWC）and the minimum volume threshold of colorectal distention（CRD）.Then,the overall regulatory effect of MJGT＿EE on the gastrointestinal tract was preliminarily evaluated by gastric emptying rate and small intestinal propulsion rate experiments.The results showed that MJGT＿EE significantly increased FWC（p<0.01）and the minimum volume threshold of CRD（p<0.05）,and promoted gastric emptying and small intestinal propulsion（p<0.01）.Its mechanism involved the regulation of protein expression in the 5-hydroxytryptamine（5-HT）pathway by MJGT＿EE.Specifically,it decreased TPH（p<0.05）and increased SERT expression（p<0.05）,inhibited 5-HT secretion（p<0.01）;activated downstream signaling pathways of calmodulin（Ca M）/myosin light chain kinase（MLCK）;increased 5-HT₄R expression（p<0.05）.In addition,MJGT＿EE increased the diversity of intestinal microbiota and the proportion of beneficial bacteria,while affecting the number of intestinal bacteria associated with 5-HT generation.The four active ingredients in MJGT＿EE were the same as those in MJGT＿HE.Conclusion1.MJGT＿HE exhibits a concentration-dependent regulatory effect on gastrointestinal motility,with low（MJGT＿HE＿L）and medium（MJGT＿HE＿M）concentrations promoting gastrointestinal motility,while a high concentration（MJGT＿HE＿H）group exhibited inhibitory effects.The stomach and duodenum are more sensitive to MJGT＿HE.Different concentration treatments also resulted in significant differences in intestinal microbiota.Overall,MJGT＿HE＿L promoted beneficial bacteria and inhibited pathogenic bacteria,while MJGT＿HE＿H had the opposite effect.The main active substances of MJGT＿HE was identified as luteolin,eriodictyol and their corresponding glycosides.2.MJGT＿EE improved the main symptoms of the IBS-C model rats by affecting the5-HT pathway,reducing the accumulation of 5-HT and response receptors.The mechanism of this action also involved the regulation of the intestinal microbiota.In summary,this study clarifies the regulatory effect of MJGT on gastrointestinal motility and reveals its mechanism of action and related active components,laying an important foundation for the promotion and application of MJT.