| Objective: Gastrointestinal motility disorder is a chronic and recurrent digestive tract disease,whose pathogenesis is complex and has not been well clarified.Among them,gastrointestinal motility disorder is one of the main causes of this disease.Lomatogonium rotatum(LR)belongs to Gentianaceae species,and traditionally used to treat liver and gall bladder diseases with the effects of calming "Xieri",clearing heat,detoxification and so on.In addition,LR has the effect of enhancing gut functions.Therefore,according to the taste theory of Mongolian medicine and the functional role of bitter receptors,the current study aimed to clarify the regulatory effect of bitter Mongolian medicine LR on gastrointestinal motility function in a mice model,and reveal its bitter receptors associated mechanism of action.In addition,we also used neural hormones such as dopamine and serotonin to induced gastrointestinal disorder model,and to demonstrate the curative effect of LR on neurotransmitter induced gastrointestinal disorders.The overall findings will further interpret the scientific connotation of Mongolian medicinal taste theory,and provide the new research ideas for the prevention and treatment of gastrointestinal disorders and uncover potential therapeutic targets.Methods: 1.Chemical composition analysis and bitterness detection: the dried LR whole plant was extracted with dichloromethane for 3 times,each time for 3 hours,and then extracted with 95% ethanol for 2 times,each time for 2 hours.Two extracts were combined and vacuum concentrated and then freeze-dried to obtain the extract powder of LR.The chemical constituents of LR extract were determined by ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS).Meanwhile,the electronic tongue technique was used to analyze the taste properties of the extract.2.The effects of LR and swertiamarin on gastric emptying and small intestinal propulsion in normal mice and DA,5-HT induced gastrointestinal motility dysfunction model mice were observed 3.Organ bath was used to evaluate the regulatory effects of LR and ts main bitter ingredients(ST,SO,GA,HP and HC etc.)on ex vivo intestinal smooth muscle contractile actions.In addition,Ach(0.1 u M),Ca CI2(1 mm),bitter receptor inhibitor gallein were used to verify the mechanism of LR on regulating gastrointestinal motility functions.Results:1.UPLC-MS results showed that 30 compounds were identified with the inclusion rate was above 1%,mainly flavonoids,organic nitrogen compounds,coumarin and its derivatives,cycloalkene ether terpenoids.Its main bitter components include swertiamarin,swertiroside,diazobenzene,gentianxanthol,amygdalin,hesperidin,coumarin,etc.,which accounting for about 66% of the total.The results of electronic tongue analysis showed that the LR extract had the highest sensitivity to the acid-bitter taste sensor,and the bitter value increased significantly with the increase of the concentration.2.In vivo experimental results showed that 1g/kg of LR extract significantly promoted gastric emptying in mice(P < 0.01),while 4g/kg of LR extract significantly delayed gastric emptying and intestinal motility in mice(P < 0.001),which indicate that LR extract may have a bidirectional regulatory effect on gastric emptying and intestinal motility in mice.In addition,swertiamarin,the main bitter component of LR could significantly inhibit gastric emptying in mice at the dose of0.2g/kg(P < 0.01).In addition,the extract of LR had a significant inhibitory effect on5-HT induced enhanced gastric emptying(P < 0.05),and had significant synergistic effect on inhibitory effect of DA induced delayed gastric emptying(P < 0.05).3.The ex vivo results showed that(1)different segments of mouse intestinal tissues showed well spontaneous contractile actions,among which ileum had the largest systolic amplitude.(2)The results of ex vivo contractile force of mice ileum showed that the LR extract stimulated the contraction of smooth muscle(P < 0.01)at the concentration of 0.1-0.3mg/m L,and the contraction rate reached 45.10% at 0.3mg/m L.However,LR extract could inhibit the contractility of smooth muscle at the concentration of 0.4mg/m L or above(P < 0.01),and the inhibition rate reached83.21% at the concentration of 6mg/m L,suggesting that the LR extract may have a bidirectional regulatory effect on the contractility of smooth muscle in the ileum of mice.In addition,LR extract had no significant bidirectional regulation on the contractile frequency of ileum smooth muscle,but the inhibitory effect was significantly enhanced with the increase of drug concentration(P < 0.05),and the inhibitory rate reached 94.1% at 6mg/m L.(3)The five bitter chemical components were tested to observe the regulatory effects on contractile tensions and frequency of the isolated ileal smooth muscle.The results showed that in comparison with DMSO,there were no significant differences in the contractile tensions and frequency of the isolated ileal smooth muscle of mice between the three chemical components of ST,SO and GA(P > 0.05).HC and HP could significantly inhibit the tensions and frequency of spontaneous contraction of mouse ileum(P < 0.05),and could achieve complete relaxation at high concentration.However,after rewashing,the smooth muscle of the mouse ileum returned to normal contractile status.(4)The pretreatment with ACh and Ca Cl2,the extract of LR,HC and HP could significantly inhibit the smooth muscle contraction induced by ACh and Ca Cl2,and the inhibitory effect increased with the elevation of concentration(P < 0.05).(5)The results of Gallein pretreatment showed that the contraction of smooth muscle at low concentration was significantly inhibited(P < 0.05),but there was no significant effect on the relaxation effect at high concentration.Conclusion : The current study demonstrated that the extract of the Mongolian medicine LR mainly contained swertiamarin,swertiroside,diazobenzene,gentianxanthol,amygdalin,hesperidin,coumarin,etc.,accounting for about 66% of the total.The bitterness was mainly perceived by acidic bitterness and with the highest intensity.In vivo and ex vivo studies have shown that the extract of the Mongolian medicine LR has a two-way regulation effect on gastric emptying and intestinal smooth muscle contraction,which is low concentration of LR induced contraction while high concentration of LR induced relaxation,and revealed the potential mechanism may associated with the gastrointestinal bitter receptor signaling pathway.Further studies confirmed the potential therapeutic effect of the LR extract on gastrointestinal motility disorders induced by neurotransmitter disorders.Overall,according to the functional role of taste receptors on regulating gastrointestinal motility and Mongolian medicine bitter flavor theory on LR enhancing gut functions,the present study demonstrated the two-way regulatory effects of LR on gastrointestinal motility and identified the main active bitter components and clarified the potential mechanisms related to bitter taste receptors functionalities.These findings may provide new research ideas and potential therapeutic targets for the prevention and treatment of gastrointestinal disorders. |
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