| Diarrhea is a common intestinal disease which brings great harm to the animal industry,especially the pig breeding industry.Although antibacterial drugs play an important role in the prevention and treatment of diarrhea,due to the problems of bacterial resistance and residues,finding anti-diarrheal drugs from traditional Chinese medicine has become a research hotspot.Plantago asiatica L.is the largest genus in the plantago family,with the advantages of wide distribution and low price.In addition,the previous research in this laboratory showed that its water extract,ethyl acetate extraction part and the remaining part had significant anti-diarrheal effects,but its anti-diarrheal pharmacodynamical material basis was unknown and the preparation was single,thus computer simulation technology was used to screen the pharmacodynamical material basis of Plantago asiatica L.in this study.Therefore,Plantago asiatica L.Superfine Powder?PSP?was developed and its anti-diarrheal effect was evaluated providing technical support for its clinical application.The specific results are as follows:?1?The chemical components in ethyl acetate and remaining parts of Plantago asiatica L.was identified by ultra performance liquid chromatography coupled with time-of-flight mass spectrometry,then the homology modeling technology was used to build the opioid receptor and AQP4 protein,these two proteins and the existing Na+/K+-ATPase were molecularly docked with the chemical compounds of the effective parts of Plantago asiatica L..The results showed that there were 14 chemical compounds in the effective parts of Plantago asiatica L.,and the three compounds of Plantago asiatica L.were screened by molecular docking including scutellarein,luteolin and caffeic acid.?2?Establishment of the mouse diarrhea model,observe pathological sections,record of the first diarrheal time,the total number of bowel movements,the number of loose stools and calculation of the total histopathological score were to further verify the anti-diarrheal compounds of Plantago asiatica L..The results show that scutellarein and luteolin can significantly reduce the total number of bowel movements and the number of loose stools,but the effect of caffeic acid is not significant.Therefore,the anti-diarrheal medicinal substance base of Plantago asiatica L.is presumably mainly luteolin and scutellarein.?3?The intestinal fluid accumulation model was established and the volume and weight of intestinal effusion were measured,the charcoal meal model of intestinal motility was established and the toner propulsion rate was recorded,hereafter,the activity of Na+/K+-ATPase,Creatine Kinase and the concentrations of Na+and K+and the changes of gene levels of these two enzymes were determined to preliminarily explore the anti-diarrheal mechanism of luteolin and scutellarein.The results showed that scutellarein and luteolin can increase the concentration of Na+,K+by up-regulating the activity and ckb level of Na+/K+-ATPase affected by up-regulating the activity and Atp1b3 level of Creatine Kinase.Moreover,they also decrease the volume and weight of small intestinal contents to exert anti-diarrheal activity,but have no effects on intestinal motility.?4?Superfine grinding of Plantago asiatica L.was carried out by changing different injection volume and particle sizes to obtain 5 kinds of PSPs.The color,touch and fluidity of PSPs were evaluated by recording the appearance state,and measuring the tap density and angle of repose.Moreover,the microscopic state was observed with optical microscope and scanning electron microscope.The results showed that as the particle size decreased,the angle of repose of PSP gradually decreased,the color changed from light yellow to grayish white,and the microscopic state gradually changed from large particles to small particles as well as showed fragmentation.?5?The chemical structures and active compounds content of PSPs with different particle sizes were characterized by Fourier transform infrared spectroscopy and high-performance liquid chromatography.The results showed that with the decrease of particle size,the main structure of PSP was not changed.The intramolecular hydrogen bond was broken by the mechanical force of superfine grinding,resulting in the changes of the peak value at 1624 cm-1,1264 cm-1,and 1066cm-1.In addition,the contents of its active compounds including luteolin and scutellarein gradually increased,and the content of active compound of PSP with a particle size of 3.55?m was the highest.?6?The establishment of mouse diarrhea model,the observation of the pathological section and the record of the first diarrheal time,the total number of bowel movements,the number of loose stools,and the calculation of the histopathological score were used to evaluate the anti-diarrheal activity of PSP.The results showed that PSP can significantly prolong the first diarrheal time,reduce the total number of bowel movements and the number of loose stools,and decrease the histopathological score,indicating that PSP had a significant anti-diarrheal effect with dose-dependent.To sum up,the anti-diarrheal medicinal substance base of PSP is luteolin and scutellarein.Luteolin and scutellarein can increase the activity of Na+/K+-ATPase and its expression level of gene Atp1b3 by increasing the activity of Creatine kinase and its gene ckb expression level,thereby increasing the concentration of Na+and K+,inhibiting the volume and weight of intestinal effusion and exerting anti-diarrheal activity.In addition,with the decrease in particle size,the appearance,fluidity,and the content of active compounds of PSP change significantly.T he optimal size of PSP is determined from 6.66?m to 3.55?m.Furthermore,PSP has a significant anti-diarrheal activity with dose-dependent,laying a foundation for its clinical application. |
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