| ObjectiveIt is to study production process of EGCG pellet,establish its quality standards and be preparing for preclinical study;EGCG improves TNBS(C3H3N3O9S)/50.0%ethanol(CH30H)-induced colitis in rats.Methods(1)Preparation of EGCG pellet was influenced by the proportion of EGCG and matrix,nozzle temperature and temperature of preparation for single factor;the tailed degree and the spherical degree of EGCG pellet were obsevered;orthogonal design was used to optimize the production process of EGCG pellet.The TLC method was applied to ensure EGCG of pellet for qualitative research.The HPLC method was applied to set up quality standard of pellet for quantitative analysis.(2)48 SD rats were randomly divided to 6 groups,including SASA groupss(100.Omg/kg/day,n=8).EGCG groups(25.0 mg/kg/day,n=8)、EGCG groups(50.0 mg/kg/day,n=8)、EGCG groups(75.Omg/kg/day,n=8)、normal groups(2.OmLnormal saline,n=8)and model groups(2.OmLnormal saline,n=8).After 1 day by TNBS(C3H3N3O9S)/50.0%ethanol(CH3OH)-induced colitis in rats,EGCG and SASA were irrigated one time a day for 1 week.HPS、CMDI and DAI were estimated.ELISA determined the serum levels of tumor necrosis factor(TNF)-α、interleukin(IL)-10、interleukin(IL)-4 and myeloperoxidase activity(MPO)in colonic tissue.Immunohistochemistry(S-P)method determined the expression of NF-κBp65 in colonic tissue.Resulis(1)Better production process of EGCG pellet was the combination of PEG6000(94g)and stearic acid(6g)melted by water bath heating(80℃)for 50.0 min,EGCG filled and melted with 6 drop head of 2 mm(diameter),nozzle temperature for 55℃,mixed solution for 80℃,constant speed for 350～360/min dropping into dimethyl silicone oil.The TLC spots of EGCGpellet and standard EGCG were clear and same level.The consequence of HPLC analysis for standard EGCG illustrated a good line between 0.06880 and 0.50112 μg.Its average recovery of pellet was 101%and RSD was 3.91%.(2)Rats in normal groups disposed of ethanol(CH3OH)put on weight and defecated regularly,but rats in model group were opposite.While EGCG(25.0,50.0 and 75.Omg/kg/day)or SASA(100.Omg/kg/day)in rats were irrigated on 1 week,bloody stools were not apparent,stool were gradully formed,and weights were slowly incresead in rats.Colonic tissues of the model groups in rats severely adhered to liver、spleen and small intestines and found inflammation,marked hyperemia,necrosis and ulcer,but the colons of normal groups in rats were not or a small inflammation.Treatment with EGCG(50.0、75.0 mg/kg/day)or SASA(100 mg/kg/day)obviously did not increase inflammation and hyperemia in colonic tissues.The DAI and CMDI after 1 week by irrigation of EGCG(25.0、50.0、75.Omg/kg/day)in instill at i on-TNBS rats,were more decreased than model groups;colonic tissues of the model groups were found with the colon little increasing,penetration of many transmural inflammatory cells,ulcerations,congestion of goblet cell and largely fibrosis.The HPS significantly was decreased by administration of EGCG(25.0、50.0、75.0 mg/kg/day);NF-κBp65 protein expression were significantly enhanced in instillation-TNBS colon in rats of model groups.NF-κBp65 protein expression in colonic tissues in rats were decreased by administration of EGCG(50.0.75.Omg/kg/day)significantly,in contrast to the normal groups;MPO activity in the normal groups group was very low.Compared with the model groups,MPO activity in EGCG(25.0、50.0、75.Omg/kg/day)groups increased;Administration of EGCG(25.0、50.0、75.Omg/kg/day)decreased of levels of serum TNF-α and IL-4 and at the same time increased levels of serum IL-10.Conclusions(1)The production process and quality standards on the EGCG pellets has been investigated;(2)TNBS(C3H3N3O9S)/50.0%ethanol(CH30H)-induced colitis in rats are attenuated by 90%EGCG.The potential mechanism might be connected to reduce NF-κB protein expression in colon,reducing serum level of TNF-αand IL-4,and adding serum level of IL-10. |
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