Studies On The Anti-diarrheal Effect And Its Mechanism Of The Aerial Part Of Rubia Cordifolia

Objective:The aim of this study was to investigate the safety and the anti-diarrheal efficacy of the aqueous extract and the different polarity parts of Rubia cordifolia’saerial part.Using classic pharmacological models to explore the anti-diarrheal mechanism of Rubia cordifolia’s aerial part.To provide the pharmacological data for its clinical application and the experimental basis for the development of ethno anti-diarrheal drug.Methods:1.To investigate the antibacterial effect of the aqueous extract,petroleum ether extract,ethyl acetate extract of Rubia cordifolia’s aerial part in vitro on Staphylococcus aureus,Escherichia coli and Shigella using 2-fold broth dilution method.The MIC and the MBC of each extract on three different bacteria were determined.2.To investigate the anti-rotavirus effect of petroleum ether extract,ethyl acetate extract,and butyl alcohol extract of Rubia cordifolia s aerial part in vitro using MA-104 cell model.3.To observe the safety of aqueous extract and ethyl acetate extract of Rubia cordifolia’s aerial part using the acute toxicity tests.The experimental mice were administered with different does of aqueous extract and ethyl acetate extract to determine the 50%lethal dose(LD50)and the maximum tolerated dose(MTD).4.In the first part of the study,Senna leaf decoction was used to induce diarrhea in mice.Totally 50 mice were randomly allocated into five groups.The model control group was treated with distilled water.The positive control group was treated with loperamide 4mg/kg.The test groups were administered 500,1000,or 2000mg/kg aqueous extract of Rubia cordifolia’s aerial part.The time to initial semi-solid feces was recorded.Meanwhile,the number of solid,semi-solid and liquid feces of each mouse were recorded to calculated the EI.5.In the first part of the study,the intestinal transit experiments were used to evaluate the anti-diarrheal activity of aqueous extract of Rubia cordifolia’s aerial part.In normal intestinal transit test,50 mice were randomly divided into five groups.The negative group was given distilled water,the positive group was given loperamide 4mg/kg.The test groups were given 500,1000,or 2000mg/kg aqueous extract of Rubia cordifolia’s aerial part.Then the red Chinese ink was administered through gastric gavage.The intestinal transit percentage of the red Chinese ink was determined.In castor oil induced intestinal transit test,castor oil was given before the red Chinese ink,and the intestinal transit percentage of the red Chinese ink was determined either.In the second part of the study,the intestinal transit experiments were used to evaluate the anti-diarrheal activity of ethyl acetate extract of Rubia cordifolia’s aerial part.50 mice were randomly divided into five groups.The model control group was given distilled water,the positive group was given loperamide 4mg/kg.The test groups were given 415,830,1660mg/kg ethyl acetate extract of Rubia cordifolia’s aerial part.Then the castor oil and red Chinese ink was administered in succession through gastric gavage.The intestinal transit percentage of the red Chinese ink and the weight of intestinal content were determined.6.In the second part of the study,the ulcerative colitis(UC)model on rat was established by enema administration of Trinitrobenzenesulfonate(TNBS).72 rats were divided into 9 groups.The health control group and model control group were given distilled water.The positive control group was administered dexamethasone 0.3mg/kg.The test groups were given aqueous extract of Rubia cordifolia’s aerial part 250,500,1000mg/kg,and ethyl acetate extract of Rubia cordifolia’s aerial part 208,415,830mg/kg respectively.The macroscopic and the microscopic damage were measured.The indicators of oxidative stress MDA,MPO and GSH-PX were assessed by biochemical colorimetric method.ELISA was used to detect the level of IL-1β and TNF-α.Results:1.The results of MIC and MBC showed the aqueous extract of Rubia cordifolia’s aerial part inhibited the growth of Shigella at the dose of 0.25g/ml(2.09g/ml crude drug).The aqueous extract of Rubia cordifolia’s aerial part didn’t inhibit the growth of Staphylococcus aureus and Escherichia coli at highest dose.The MIC of the petroleum ether extract of Rubia cordifolia’s aerial part on all three bacteria were 262.1g/ml(crude drug).The MBC of its on Shi was 524.1g/ml(crude drug),on E coli and Sau were both 262.1g/ml.The MIC of ethyl acetate extract of Rubia cordifolia’s aerial part on Shi,E coli and Sau were 4.5g/ml,4.5g/ml,9.0g/ml(crude drug)respectively,the MBC were 4.5g/ml,9.0g/ml and 35.8g/ml(crude drug).2.The petroleum ether extract,ethyl acetate extract and butyl alcohol extract of Rubia cordifolia’s aerial part could inhibit rotavirus multiplication significantly at the dose of 0.51g/ml,0.28g/ml and 0.18g/ml(crude drug).3.In acute toxicity test,the aqueous extract of Rubia cordifolia’s aerial part didn’t produce any mortality or signs of toxicity in LD50 test.In the MTD test,orally administered the aqueous extract up to 13.2g/kg body weight(8.37g crude drug/g)did not result in any morality and toxicity.The LD50 of ethyl acetate extract of Rubia cordifolia’s aerial part was 16.6g/kg.4.In senna leaf-induced diarrhea test,the aqueous extract of Rubia cordifolia’s aerial part 500,1000mg/kg groups significantly inhibited diarrhea by increasing the onset time of semi-solid feces and decreasing the El score compared with the negative control group(p<0.05-0.01).Such inhibitory effects were not noted in the 2000mg/kg group.5.Graded doses of the aqueous extract of Rubia cordifolia’s aerial part didn’t inhibit normal intestinal transit compared with the negative control group.However,in the castor oil-induced intestinal transit test,the aqueous extract show inhibitory effect in every does group,but only the 1000,2000mg/kg groups showed significant differences(p<0.05).Compared to the model control group,the ethyl acetate extract of Rubia cordifolia’s aerial part decreased the intestinal transit significantly(37.2 ± 2.2%,33.0 ± 2.4%,24.0 ±2.5%vs 49.6 ± 2.4%,p<0.01).Meanwhile,the weight of the enterpooling decreased accordingly.6.In the TNBS induced UC model,the macroscopic and microscopic observation tests showed that the rats in the model control group had most severe damage in the colon tissue.Compared to the model control group,the general condition and the tissue morphology were significantly improved in every test group.In the tissue sample detection,the MDA content,MPO activity,IL-1β and TNF-α level were significantly higher,and the GSH-PX activity was significantly lower in model control group compared with the health control group(p<0.01).Compared with the model control group,the MDA content and the TNF-a level decreased significantly(p<0.01)and the GSH-PX activity increased significantly(p<0.01)in test groups.Only ethyl acetate extract of Rubia cordifolia’s aerial part 830mg/kg group decreased IL-1β level significantly compared with the model control group.The MPO activity decreased in every test group,but the difference were not significantly except aqueous extract 1000mg/kg group.In the serum sample detection,the MDA content,MPO activity,IL-1β and TNF-α level were significantly higher,and the GSH-PX activity was significantly lower in model control group compared with the health control group(p<0.05～0.01).Compared to the model group,the MDA content of some test groups decreased significantly(p<0.05).Only the aqueous extract 500mg/kg group increased GSH-PX activity significantly.In the MPO activity detection,every test group except the aqueous extract 1000mg/kg decreased significantly compared with model control group(p<0.01).Similarly,IL-1β and TNF-α level in each test group decreased to some extent compared to model control group,but only positive control group and the aqueous extract 500,1000mg/kg groups in IL-1β detection and the ethyl acetate extract of Rubia cordifolia’s aerial part 415mg/kg group in TNF-α level detection show significant difference(p<0.05～0.01).Conclusion:The aqueous extract of Rubia cordifolia’s aerial part showed anti-bacteria activities in vitro.The acute toxicity test demonstrate the safety profile of the aqueous extract.The senna leaf-induced diarrhea and the castor oil induced intestinal transit test provided evidence regarding the anti-diarrheal effect of the aqueous extract.In addition,based on the results of this study,it can hypothesize that the anti-diarrheal mechanism of the aqueous extract might be partly mediated by anti-secretion activity,followed by decreased fluid accumulation in the intestinal lumen,and by inhibiting the production of prostaglandin.The ethyl acetate extract and the petroleum ether extract of Rubia cordifolia’s aerial part showed antibiosis activities in vitro but not stronger than the aqueous extract.All three different polarity parts of Rubia cordifolia’s aerial part showed anti-rotavirus activity in vitro,but the effect of each was weaker than the aqueous extract.The compounds of different polarity parts of Rubia cordifolia’s aerial part produced synergistic anti-bacteria and anti-rotavirus effect.The ethyl acetate extract of Rubia cordifolia’s aerial part is safe when administered orally.The ethyl acetate extract of Rubia cordifolia’s aerial part has anti-diarrheal effect and the effects might be mediated by anti-secretion activity followed by reduced fluid accumulation in the intestinal lumen.The aqueous extract and the ethyl acetate extract of Rubia cordifolia’s aerial part protected the rats against the TNBS induced colitic inflammation by reducing the oxidative stress productions and decreasing pro-inflammatory cytokine levels.Rubia cordifolia’s aerial part can be developed to a novel alternative therapeutic drug for the treatment of diarrheal in UC patients.The anti-oxidative and anti-inflammatory effect of Rubia cordifolia’s aerial part might also be the underlying mechanism of its anti-diarrheal activity.