| Acute diarrhea(AD)is a kind of diarrhea disease with rapid onset and short cycle.The main lesion is in the small intestine.The clinical manifestations are abdominal pain,vomiting,and increased frequency of bowel movements.Especially for infants and young children,AD causes dehydration,shortage of trace elements and slow growth and development easily.Studies have shown that patients with Zn in the presence of AD can effectively shorten the duration of diarrhea and the duration of diarrhea.In this paper,a new kind of composite rehydration salt is prepared,which increases the absorption of Zn while supplementing the electrolyte.In this paper,the preparation process is optimized by the concept of quality by design(QbD),and the energy is developed.The Zn content is controlled by the energy dispersive X-ray fluorescence spectroscopy(EDXRF)method.Finally,the mouse model of AD is induced by senna,and the HE staining,cytokine changes,immunohistochemistry,protein imprinting are performed.Experiments are conducted to investigate the mechanism of action of complex rehydration salts.The main research contents are as follows:(1)Preparation of compound rehydration salts are based on QbD concept:The key quality attributes of the evaluation screening are mixing uniformity and loss on drying.The key material properties are the fluidity and particle size of the raw materials.Confirm the prescription:each bag contains 0.375 g of potassium chloride,0.650g sodium chloride,0.725g sodium citrate,3.375g anhydrous glucose,0.140g zinc gluconate,after the risk assessment,the key process parameters are initial mixing and total mixing time.Finally,the design expert software is used to carry out two factors.The horizontal full factor experiment obtains the simulation equation.When the predicted RSD is 0.217%,the mixing uniformity is the highest.The verification experiment shows that the model fit well,and the control space is established for initial mixing is 30 minute and total mixing is 40~45 minute.(2)Quality control of Zn content in composite rehydration salt by EDXRF method:The standard curve of Zn element R~2 is 0.9942,which indicates that the Zn content in the sample has a good linear relationship with X-ray fluorescence intensity.The detection limit of Zn element in this method is 2.32mg/ml,the limit of quantification is 7.04mg/ml.The RSD of the Zn element test results of 6 parallel samples is 1.85%,the accuracy of the three levels in the accuracy test is 98%-102%.The preparation of the two batches of samples and the detection results of the intermediate products also meet the requirements,indicats that it is feasible to use the EDXRF method to detect the Zn content in the composite rehydration salt and it can also be used for process control.(3)Study on the mechanism of action of complex rehydration salts on AD:The senna induces mouse AD model,the low,medium and high doses of compound rehydration salts can significantly reduce the leanness,loose stool and diarrhea index of mice.HE staining observation of small intestine tissue find that the low,medium and high doses of complex rehydration salts can also effectively alleviate the pathological changes of small intestinal mucosa in mice.Enzyme-linked immunosorbent assay is used to observe the changes of inflammatory factors in serum and small intestine.It is found that the complex rehydration salts in low,medium and high dose groups can significantly reduce the levels of IL-8 and TNF-?in serum and small intestine.However,there is no significant change in the anti-inflammatory factors IL-4 and IL-10.Then,through immunohistochemistry experiments,it is found that the positive rate of the compound rehydration salt high-dose group increases slightly.Finally,Western blot is used to detect the expression levels of Claudin,Occludin,ZO-1 and NF-?B,which further confirm that the compound rehydration salt has a significant therapeutic effect on AD. |
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