| Ipomoea stolonifera (IS) is a medicinal herb in Shantou, Guangdong Province, China.The ancient literature of Chaoshan medicine documents its anti-rheumatoid-arthritis effect. IS has been widely used as a folkloric medicine to treat inflammatory disorders, especially rheumatic arthritis. The use of IS has gained wide attention in the last few decades because of its remarkable curative effects, especially for rheumatic arthritis. However, the anti-inflammatory effect of IS on arthritis has not yet been reported. We aimed to investigate the anti-inflammatory activity of the extract of IS using several experiment animal models of inflammation.Objective:To study the analgesic and anti-inflammatory effects and mechanism of Ipomoea stolonifera (IS)Methods1 Groups:All rats were randomly assigned into five groups:(control), postive,100,200, and 400mg/kg.All other groups were established with various in vivo models of both acute and chronic inflammations.2 A writhing response was induced by injection of 0.7% acetic acid in saline (0.1 mL/10 g) intraperitoneally in mice and arctigenin (150,300, and 600mg/kg) dissolved in 0.5% CMC was administrated orally 1 h before the injection of acetic acid. The number of writhes occurring between 10 and 20 min after the acetic acid injection was counted.3 Acute inflammation model:Croton oil-induced ear edema in mice. 100um mixed liquor Croton oil:ethanol:pyridine:ether (1:10:20:69) was applied to right ear of mice. And the same volume of solvent was applied to the right ear as a control. Four hours later, the animals were sacrificed, and each ear was perforated with a metal punch to provide an 8-mm diameter disc. Edema was assessed by subtracting the weight of the disc from the right control ear from the weight of the disc from the left treated.Carrrageenan-induced paw oedema in rats.IS (100,200, and 400 mg/kg) dissolved in 0.5% carboxymethyl cellulose (CMC) was administrated orally 1 h prior to carrageenan application, and 1% carrageenan in saline (0.1 mL) was injected subcutaneously into the right hind paw of the rats. The perimeter of ankle joint was measured initially and then 1,2, 3.4,5and 6 h after the carrageenan injection.4.Chronic inflammation model:Cotton pellet-induced granuloma in rats.Under ether anesthesia, sterile cotton pellets weighting 50±1mg were implanted subcutaneously in both the pars inguinalis region of each rat through a single needle incision, one on each side. The second group was served as drug control and received dexamethasone daily at a dose of 2.5 mg/kg orally for 7 days. The same volume of CMC was given orally to the first group of animals as vehicle control. IS at doses of 100,200 and 400 mg/kg were administered orally to the other three groups of rats, respectively, for 7 days from the day of cotton pellet implantation. On the eighth day, the granuloma tissue was dissected out carefully and dried at 60℃to constant weight. The increase in dry weight of the pellets was taken as the measure of granuloma formation.5. Tissue samples of each ear from the Croton oil-induced ear edema model were assessed biochemically with the neutrophil and eosinophil marker enzymes, MPO. Tissue samples of paw from Carrrageenan-induced paw oedema in rats were assessed production of PGE2.6. Adjuvant-induced chronic arthritisRats were divided into five groups each comprising six animals. Arthritis was induced by intradermal injection of Complete Freund's Adjuvant (0.1 ml) into the right hind paw. The adjuvant contained heat-killed Mycobacterium tuberculosis (10 mg) in paraffin oil (1 ml).7. Carrageenan-induced pleurisy in rats The rats were divided into five groups. The experimental and positive drug groups of animals were treated with IS at a dose of 100,200 and 400 mg/kg and dexamethasone at a dose of 2.5 mg/kg, respectively. The same volume of normal CMC was respectively administered to the vehicle and model groups of rats. The drugs were given orally once per day for 7 days. One hour after the last administration of drugs, rats were lightly anaesthetized under ether and then 0.2 ml of normal saline alone or containing 1% carrageenan were injected into the pleural cavity of each rat. Four hours after the injection of carrageenan, rats were slightly anaesthetized and blood samples were taken from the eyepit. The serum was separated and stored at-20℃for the measurement of MDA and SOD. The animals were then sacrificed under an overdose of ether and the pleural cavities were exposed. The exudate volumewas measured and the pleural cavity was washed with 2ml of ice-cold phosphate-buffered saline (pH 7.2) with heparin (5 U/ml). The exudates and washing were combined as the pleural exudates for the measurement of total protein. Exudates contaminated with blood were discarded. The total leukocyte number in the pleural exudates was counted in a Neubauer Chamber.The levels of MDA and SOD in the pleural rat serum were assayed with MDA, and SOD kits, respectively. The production of total protein in the pleural exudate was measured with total protein kits.8. Acute toxicity testDifferent doses (750mg/kg,1.5 g/kg,3 g/kg,6 g/kg) of IS were made in a 0.5% CMC-Na solution which were homogenized with high speed homogeneous equipment, and were given orally to groups of 5 mice each. During the 7 days after treatment, all the animals were observed and recorded daily and dead animals would be subjected to postmortem examination for determination of the cause of death.Results1. Effect of EFIS on acetic acid-induced writhing response Results show the total number of writhes induced by acetic acid, during 10 min of observation, begun immediately after i.p. injection. The results observed demonstrated the antinociceptive effect of Ipomoea stolonifera when using the dose of 600mg/kg, which is statistically similar to ibuprofen.2. Effect of EFIS on croton oil-induced mice ear oedema and assessment of tissue MPO activityThe oral administration of IS suppressed significantly croton oil-induced ear oedema in mice. The oedema inhibitory rates of IS were 10.3%,30.1%, and 45.8% at doses of 150,300 and 600 mg/kg, respectively. Whereas ibuprofen (300 mg/kg), used as a reference drug, produced 32% inhibitory rate compared to control3. Effect of EFIS on carrageenan-induced paw oedema in ratsThe oral treatment with IS inhibited significantly carrageenan-induced paw oedema in rats. At a dose of 400 mg/kg of IS, oedematization was suppressed and inhibitory rates were 36.6-56.3% at 1-6 h after carrageenan treatment. At a dose of 200 mg/kg, ibuprofen, as a standard drug, produced a greater inhibition of oedema development by 48.9-75.3% at 1-6 h after carrageenan injection.4. Effect of EFIS on cotton pellet-induced granuloma in ratsThe effects of IS and dexamethasone on cotton pelletinduced granuloma in rats.At doses of 100,200 and 400 mg/kg, IS as well as dexamethasone (2.5 mg/kg) inhibited markedly granuloma formation surrounding the pellets compared with control group. Four hundred milligrams per kilogram of IS produced a maximum inhibition of 50.8%, while 100and 200 mg/kg of IS produced 14.6% and 39.4% inhibition in granuloma weight, respectively, when compared to 51.4% for dexamethasone.4. Effect of EFIS on assessment of tissue MPO activity and assessment of tissue PGE2 of paw.MPO activity of croton oil-induced inflamed ear tissues homogenate (1.39±0.329 U/g protein) was significantly increased to compared with the vehicle ear tissues homogenate (0.44±0.01 U/g protein). IS at a dose of 400 (0.51±0.145, P< 0.001) significantly inhibited the MPO activity of croton oil-induced inflamed ear tissues homogenate compared to controls on the dose-dependent manner, respectively.IS inhibited the synthesis of PGE2 in the injected paw of rats caused by carrageenan.6. Effect of EFIS adjuvant-induced chronic arthritis in rats The effects of IS on adjuvant-induced chronic arthritis in rats.As shown in data. IS at a dose of 200,400 mg/kg showed potent inhibitory activity during the first 9 days and the final 21 day.7. Effect of EFIS on carrageenan-induced pleurisy in ratsAt a dose of 400 mg/kg, the oral pre-treatment of IS significantly reduced the pleural exudate volume and the total leukocyte migration, inhibited the production of total protein in the pleural exudates, increased the serum levels of SOD, and also decreased the serum levels of MDA. Dexamethasone used as the standard drug indicated the similar effect.8. Acute toxicityNo animal died during acute toxicity test, neither apparent adverse effect were observed for tested animals with different doses of IS. The result revealed that the extract IS up to an oral dose of 6g/kg body weight was almost nontoxic in mice.ConclusionsIS had a significant analgesic and anti-inflammatory effect which may correlate with the inhibition of PGE2,MPO. inhibited the production of total protein in the pleural exudates, increased the serum levels of SOD, and also decreased the serum levels of MDA.。... |
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