Effect Of Volatile Oil From A. Longiligulare T.L. Wu On Experimental Ulcerative Colitis And Its Safety Assessment

Objective: To study the effect and mechanism of volatile oil from A.longiligulare T.L.Wu (VOA) on experimental ulcerative colitis (UC) and its safety assessment.Methods: (1) Water vapor distillation method was used to extract VOA; Gas chromatography was applied to analyse the content of bornyl acetate for studying the stability and controllability of VOA. (2) The medium lethal doses (LD50) of mice in acute toxicity test were analyzed by Bliss method. (3) The mice model of UC was established by using 4% dextran sulfate sodium (DSS) solution for 7 days, the sulfasalazine (SSZ) group was considered as positive control, the clinical symptoms and the lesion of colonic mucosa of the mice were observed.(4) The rat model of UC was established by an enema of 2,4-dinitrobenzene-1-chlorobenzene (DNCB) and acetic acid, the SSZ group was considered as positive control, the macroscopical and histological changes of the colon were evaluated. (5) The auris-swell model induced by dimethylbenzene and foot edema and swelling model induced by carrgeen were made to study the anti-inflammation effect by different dosages of VOA. Tests of hot plate and body wrest were used to study the analgesic effect. The anti-diarrhea function on mice with diarrhea induced by castor oil and folia sennae was observed. (6) The activity or content of maleic dialdehyde (MDA), nitric oxide (NO) and super-oxide dismutase (SOD) in the colonic tissue of mice with UC induced by DSS were determined to explore the mechanisms of therapeutic effect by biochemical method. (7) The activity or content of glutathione peroxidase (GSH-Px), nitric oxide synthase (NOS) and super-oxide dismutase (SOD) in the colonic tissue of rats with UC induced by an enema of DNCB and acetic acid were determined to explore the mechanisms of therapeutic effect by biochemical method. (8) Effect of VOA on the expressions of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule 1(ICAM-1) in the colonic tissue of mice with UC induced by DSS were detected by immunohistochemistry. (9) Effect of VOA on the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor-κBp65 (NF-κBp65) in the colonic tissue of rats with UC induced by DNCB and acetic acid were detected by immunohistochemistry. (10)The SD rats were orally given VOA preparation with different doses for 90 days. And rats were observed for 15 days after drug withdrawl. The rat's weights, organ coefficients, the indexes of hematology and biochemisty were determined. Pathological examinations were carried out.Results: (1)The chemical compositions of VOA were stable and controllable. (2)The LD50 of VOA of mice by gavage was 7.3009g/kg. (3) Compared with the model control induced by DSS, the clinical symptoms and the lesion of colonic mucosa in mice were remarkably improved after administration of VOA. (4) Compared with the UC group induced by DNCB and acetic acid, treatment with VOA reduced colon macroscopic and histological damage significantly. (5) VOA could obviously inhibit the auris-swell in mice and the foot edema and swelling in rats, the effect of high dosage can equal to that of indometacin. In hot plate test, each dosage of VOA increased the threshold value of pain significantly, especially the high dosage group. The number of mice's writhe caused by glacial acetic acid was reduced by VOA. There was no significant difference between different dosage group of VOA. It could also decrease the times of wet manure induced by folia sennae, while it was of no effect on diarrhea induced by castor oil.(6) Compared with the model control induced by DSS, the activity of SOD were increased and the contents of MDA and NO were decreased after administration of VOA. (7) Compared with UC group induced by DNCB and acetic acid, treatment with VOA elevated the activities of SOD and GSH-Px and reduced the contents of NOS.(8)Administration with VOA could inhibited the protein expressions of iNOS and ICAM-1 in the colon tissues of mice with UC induce by DSS.(9) Treatment with VOA could inhibited the protein expressions of TNT-αand NF-κBp65 in the colon tissues of rats with UC induce by DNCB and acetic acid.(10) The outer appearance and behavior,the indexes of hematology and biochemistry in administration with VOA groups had not shown significant difference by comparing with control group.The body weight of the animals in the groups with treatment of VOA had shown definite differenence with that of control group.The pathological examination and organ coefficient revealed significant pathological changes in lung and spleen related to VOA.Conclusion: (1)VOA had significant therapeutic effect on UC and anti-inflammation, analgesic and anti-diarrhea effects, whereas the acute toxicity was very low and with stable quality,thereby VOA might be taken as a potent candidated in prevention and therapeutic drug of UC. (2)The anti-UC mechanisms of VOA were possibly related to the following factors:①Inhibiting generation of free radicals and depression of lipid peroxidation reaction;②Modulating the redundant production of NO in the colonic tissue;③Alleviating the adhesive action of cells to colonic tissue;④Curbing the inflammatory cascade effects;⑤Anti-inflammation, analgesia and anti-diarrhea.(3) VOA was found to be long term toxicity to SD rats in lung and spleen with administration of VOA at the dosage of 3800 mg/kg.It was safe to administrate VOA continuously at the dosage of 1900 mg/kg.