| ObjectiveThe active ingredients of Hemsleya macrosperma were extracted to prepare Hemsleya macrosperma enteric-coated pellets(HMECP)using extrusion spheronization technology after optimization of prescription factors and process parameters.The pharmacodynamic effect of HMECP was studied on rats with ulcerativecolitis indced by the dextran sulfate sodium(DSS).The present study provides a newmethod for the treatment of colitis with traditional Chinese medicine and provides a theoretical basis for the development of new preparations.Methods(1)Identification and determination of the quality of Hemsleya macrosperma: The molecular identification method was used to identify the species of Xuedan.Hemsleya macrosperma was identified by Thin layer chromatography(TLC)and its water content was determined.A HPLC method was established determine the content of cucurbitacin IIa.(2)Extraction process of extract of Hemsleya macrosperma:The extraction process of extract of Hemsleya macrosperma was optimized by single factor experiment and orthogonal test.(3)Preparation process of HMECP: The pellets core was prepared by extrusion-spheronization technology.With yield and roundness as indexes,a single factor test was used to investigate the formulation factors and process parameters in the preparation of the pellet core.The orthogonal test was used to optimize the process parameters of the pellet core.Fluidized bed coating technology was employed to coat pellet core and the release rate was measured to determine the best prescription and process conditions for coating.Releasing behavior of HMECP in vitro was investigated by simulating the conditions of the human gastrointestinal tract.(4)Pharmacodynamic study of HMECP: Total of 60 SD rats were randomly divided into six groups: normal control group,DSS model group,sulfasalazine(SASP)positive control group and HMECP at low,middle and high dose of treatment groups(0.95 mg/kg,1.89 mg/kg,3.78 mg/kg).The model of ulcerative colitis was established by 4% DSS induction.Body weight,fecal traits,blood in the stool,colon length,colon weight,histopathological damage and the degree of inflammatory cell infiltration were observed after administration.And the levels of Interleukin-1β(IL-1β),Interleukin-10(IL-10),Interleukin-6(IL-6)and Tumor Necrosis Factor(TNF-α)in serum and the content of myeloperoxidase(MPO)and Nitric oxide(NO)in colon tissue were detected by ELISA method.The contents of short-chain fatty acids(SCFAs)in the feces of different groups of rats were simultaneously determined by GC-MS.Results(1)Xuedan belongs to the cucurbitaceae,Hemsleya,Hemsleya macrosperma.The results of TLC identification and water content of Hemsleya macrosperma are consistent with the standard.The content of cucurbitacin IIa in Hemsleya macrosperma is(1.25 ± 0.09)%.(2)The optimal extraction process of extract of Hemsleya macrosperma were add 10 times of 95% ethanol and extract 3 times,each time for 2 h at 80℃.The transfer rate and content of cucurbitacin IIa were(81.73±1.56)% and(20.63 ± 0.37)%,respectively.(3)The core of HMECP was prepared by extrusion spheronization,and the determination of cucurbitacin IIa as an index component in the pellet core by HPLC was established.The optimal prescription for the pellet core was to add starch 63 g,microcrystalline cellulose(MCC)27 g,drug 5 g,wetting adhesive80 ml of 1% Hydroxypropyl Methyl Cellulose(HPMC)water solution,Polyvinylpolypyrrolidone cross-linked(PVPP)5 g.The best process conditions for preparation were as follows: extrusion speed was set at 30 Hz,and spheronization speed was set at 20 Hz for 120 s.The pellet core obtained by this method has high yield and good roundness.Study in vitro release test found that the cumulative release rate of the pellet core within 2 h reached(83.27 ± 1.09)%.The release rate can meet the requirements for the pellet.The contents of cucurbitacin IIa in HMECP were detected in different dissolution media by HPLC.No.Ⅲ polyacrylic resin was used as the coating material,which gained 20% of weight.The prepared HMECP was showed a good release rate by fluidized bed coating technology.The cumulative release dose within 2 hours not more than 10% in simulated gastric fluid,and reached about(75.54±0.50)% at 9 h in simulated intestinal fluid.(4)The rats in the normal group had a shiny coat,normal diet,and increased body weight.Rats in the DSS group developed loose stools and bloody stools.So their diets were significantly reduced and their body weight decreased.Rats in each administration group had healthy coat color,spirit and activity.Compared with the DSS group,the weight of the rat treated with HMECP in low dose group and in middle dose group were very significantly increased(P ＜ 0.01).Fecal scores of rats treated with HMECP in low-dose group and middle-dose group were very significantly decreased(P ＜ 0.01),while the high-dose group treatment was significantly decreased(P ＜ 0.05).The colon of the normal group appeared normal.The colon length of the DSS group was significantly shortened;the wet weight increased;the shape became thicker,and edema appeared.The colonic state of rats in each administration group was improved to some extent.Compared with the DSS group,each dose of HMECP could increase the length of the colon and reduce the wet weight of the colon in the rats(P ＜ 0.05),and the ratio of the length and weight of the colon in the middle-dose group was very significantly reduced(P ＜ 0.01).Compared with the DSS group,the ratio of the length and weight of the colon in the SASP group was significantly reduced,but there was no significant difference(P > 0.05).There was no significant difference in the ratio of the length and weight of colon between SASP group and HMECP groups(P > 0.05).In the normal group,the structure of colonic mucosa was normal,and no obvious histopathological damage and inflammatory infiltration were observed.In the DSS group,focal epithelial cells were necrotic and shed,the lamina propria was damaged,and inflammatory cells infiltrate the entire intestinal wall.Compared with the normal group,the difference was very significant(P＜ 0.01).Colon inflammation in SASP group and HMECP groups were significantly improved.Compared with the DSS group,the histopathology scores of the SASP group,HMECP in low-dose group and in middle-dose group were very significantly reduced(P ＜ 0.01),while the HMECP in high-dose group was significantly reduced(P ＜ 0.05).Compared with the DSS group,the spleen index of rats in each administration group decreased to a certain extent.The spleen index of rats in DSS group was(2.77±0.83)mg/g,and the spleen index of SASP group was(2.06±0.23)mg/g,which was very significantly lower than that of DSS group(P ＜ 0.01).The spleen indexes of HMECP in middle-dose group and in high-dose group were(2.31±0.42)mg/g and(2.10±0.59)mg/g,which were significantly lower than that of DSS group(P ＜ 0.05).Compared with the DSS group,the levels of IL-6 and TNF-α in the serum of rats in each dose group of HMECP decreased and the level of IL-10 increased.Compared with the DSS group,the level of IL-6 in the serum of rats in HMECP of low-dose group was significantly reduced(P ＜ 0.05),and the TNF-α level was very significantly reduced(P＜ 0.01),and the IL-10 level was very significantly increased(P ＜ 0.01).The levels of IL-6 and TNF-α in the serum of rats in the HMECP of middle-dose group were very significantly reduced(P ＜ 0.01),and the level of IL-10 was very significantly increased(P ＜ 0.01).The levels of IL-6 and TNF-α in the serum of rats in the HMECP of high-dose group were extremely significantly reduced(P ＜ 0.01),and the level of IL-10 was significantly increased(P ＜ 0.05).Compared with the DSS group,the levels of MPO and NO in the colon tissue of HMECP groups were reduced.The levels of MPO and NO in the colon in the low-dose and middle-dose groups of HMECP were significantly reduced(P ＜ 0.05),and the level of NO in the colon of high-dose group was significantly reduced(P ＜ 0.05).GC-MS method was employed to determine the types of short-chain fatty acids in feces.The results showed that acetic acid,propionic acid and butyric acid were main acids of the short-chain fatty acids in feces,and the relative content of acetic acid was the highest.The contents of acetic acid and butyric acid in middle-dose group of HMECP were significantly higher than that in the DSS group(P ＜ 0.05).ConclusionThe extract of Hemsleya macrosperma were successfully extracted and HMECP were prepared.The preparation process is proven stable and feasible,and the shape and release of the pellets are good.Pharmacodynamic studies have shown that HMECP can improve the growth performance,increase the spleen index of rats,increase the level of anti-inflammatory factor in rat serum and reduce the level of pro-inflammatory factor.HMECP also can decrease the contents of MPO and NO in colon tissue of ulcerative colitis rats.At the same time,HMECP can increase the contents of short-chain fatty acids in rat feces.Present studies show that HMECP can delay and inhibit the occurrence and development of ulcerative colitis in rats. |
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