| Background:Ulcerative Colitis (UC) is a kind of common gastrointestinal disease that easily develops to cancer. The treatment of UC is one of the major clinical tasks at present for the reasons that not only UC’s high recurrence rate, cancer relativeness but also the untreatability by current medicine.Uyghur herb medicine Turkish Gall has been used effectively for the treatment of UC for centuries.However, there is little information about the KuiJieAn enema (KJA) preparation’s mechanisms,stability and toxicity. Therefore,the studies of KJA’s mechanism on UC,stability and toxicity will provide useful experimental evidences for the reasonable use in further clinical research.Objective:(1)To make a clear understanding on KJA’s efficiency and mechanism for UC treatment.(2)To estimate the stability and predict the shelf-life of KJA enema after variety of stability test by determining physical characteristics and chemical composition of enema’s ingredients.(3)To investigate the acute toxicity, chronic toxicity and local stimulation of KJA enema to Colon and Anus.Methods:(1)Mechanism study:UC was induced by enemating2、4-dinitrochlorobenzene(DNCB)and acetic acid in rats.Rats were then randomly divided into following groups:normal control group(which haven’t received any intervene),negative control group,positive control group with5-ASA gavages,KJA gavage group,KJA enema group,KJA enema plus5-ASA gavages group respectively. Each rat’s general state of health was observed before and after treatment. Treatment experiments consist of three different procedures.Rats in each group were selected according to principle of randomization and were sacrificed in the next day after each period of treatment. Clinical signs,body weights,food and water consumption, organ weight, tissues,normal characteristics were observed as general items.For mechanical items blood spacemen were collected to analyze the differentially expressed proteins in UC model group and the KJA treated group compare to control group by using the technologies of2D-DIGE, MALDI-TOF-TOF and Biological information analyse; Colon tissue spaceman to measure integrity of colon for histological analysis;the colonic sample was stained with HE and evaluated by pathologist in double blinded manner. Apoptosis differences of colonic samples in different groups was determined by using Annexin V-FITC/PI and EPICS ALTRA machine; The quality determination of gallic acid after24hour of administration in serum was checked by LC-MS.(2) Stability estimation:Total phenolic,tannins and precipitation et al were determined by the methods according to ChP and related references.The batches dosage form was carried of (i) long-term test:the enema was kept for24months under the condition of (25±2)℃plus RH (60±10)%and for36months under the4℃; sample was taken at the time according to the ChP to confirm its shelf-life,(ii) Classical constant temperature test:the enema was kept at different temperatures of60℃70℃,80℃,90℃, and sample ware taken at different time; the change of different index contents at different time was determined to estimate the shelf-life of the enema,(iii) Stress test:under the condition in the ChP stress test, the enema was explored to high temperature (differently at40℃,60℃)and tungsten lamp (4500±500Lx) for10days with package and without package;the affect of these conditions was determined to measure the enema stability, and accessed by the results of different indexes.(iv) Accelerated test:sample was kept with package under condition of (40±2)℃and RH (75±5)%for6months,and determined whether it qualified to apply for the clinical test and producing.(3)Toxicity evaluation:The acute toxic responses of KJA enema were observed by enemated administration to mice.The rectal mucosa irritation was observed by rectum administration to rabbits.The chronic toxic responses of KJA enema were observed to Wistar rats by enemated administration. Statistical analysis was performed using SPSS16.0for windows,P values less than0.05were considered statistically significant.Date was expressed as the mean±SD.Differences among groups were tested using one-way ANOVA followed by multiple comparisons by LSD test and Dunnett’s T3test.Spearman’s correlation coefficients were calculated to study the relationship between indexes in KJA mechanism research experiment. The enumeration date and sample matching were tested with chi-square test.Result:(1)Mechanism sturdy:Although the expression level of indexes in this experiment was changed in each experimental groups, there was no statistical significance between the UC negative model group and the different treatment groups (P>0.05)at the first period of treatment;At the second, third period of treatment there was statistical significance between the UC negative model group and different treatment groups;Proteomics differences show the candidate biomarkers which have relationship with UC are C4bpa up regulated IgG-2a and Igh-6protein(Fragment)which down regulated in UC mordel compare to normal, and with KJA mechanism are Serpina3k up regulated while Igh-6protein down regulated in KJA treatment UC compare to UC model group.(2) Stability estimation:(ⅰ) Long-term test:Indexes have not happened different change in each observed months.(ⅱ) Classical constant temperature test:The active energies of different indexes were calculated based on Arrhinius formula, the shelf-life was predicted at25℃:1.4year for total phenolic,0.57year for tannins, at4℃:14year for total phenolic,32year for tannins,(ⅲ)Stress test:It confirms that this enema stability has been affected by photo and temperature.This test also indicates that the package bag can improve the enema’s stability.(iv)Accelerated test:KJA qualified to apply for the clinical test and producing.(3) Toxicity evaluation of KJA:When the quite KJA enema dosage was10ml/kg, tested mice showed no toxic responses,no animal death case;there was no significant difference in colon and anus mucous membrane of tested rabbits compared with controlled.When the dosage was differently equal to KJA enema50mL/kg,21mL/kg,4mL/kg, there was no significant adverse effect on the growth rate, hematological and clinical biochemical parameters including liver function. Histological studies showed that the intervention did not induce any pathological changes in the liver, kidney, heart, colon and lung of tested rats compared with control.Conclusion:(1)It is proved that KJA is effective in treating UC, which may be related to inhibition of inflammation and apoptosis rate, regulating the expression of many proteins in plasma, such as C4bpa, Serpina3k, Igh-6and IgG-2a so on;(2) Varieties of stability test results show KJA enema stability correspond with required level and can be used for research in clinical trail;(3)No significant acute toxicity, local mucosal irritation and long-term toxicity was observed which provide a scientific basis for further clinical trials of KJA. |
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