【作者】 冀宾；

【导师】 石刚刚；

【作者基本信息】 汕头大学 ， 药理学， 2009， 硕士

【摘要】 潮汕地区利用当地中药预防,治疗风湿病有悠久的历史。海滩牵牛(IS)为潮汕地区生长一种中草药,长期以来,该单味中草药在临床治疗风湿病取得了良好的效果。人们用它去风祛湿,活血通络,在治疗风湿病,特别对于类风湿关节炎经常使用,有极好的疗效,表现为：消肿、镇痛、解痉。特别是长期服用后,类风湿关节炎发作期的时间缩短,症状减轻,间隔时间大大延长,未见不良反应。因此,我们对它进行系统研发。为了进一步研究有效部位的化学成分,揭示其抗炎作用和研究相关机理,采用分离手段,从有效部位中得到了其中单体成分。本试验对IS有效部位镇痛和抗炎作用进行究,为将来将其开发成为治疗风湿病的新药提供实验基础。研究目的：将此植物(IS)为本试验的乙醇提物进行化学成分预试验,筛选出有效部位,对有效部位成分做了进一步鉴定。观察IS有效部位对疼痛和炎症动物模型的镇痛和抗炎作用及其初步探讨其作用机制。研究方法：1.实验分组：大鼠随机分成五组：即对照(control)组或模型组(model)、阳性(ibu、DEX)组、EFIS高剂量(400mg/kg)组、中剂量(200mg/kg)组、和低剂量(100mg/kg)组。小鼠随机分成五组：即对照(control)组、布洛芬(ibu)组、EFIS高剂量(600mg/kg)组、中剂量(300mg/kg)组、和低剂量(150mg/kg)组。耳肿胀与角叉菜试验后取致炎耳组织与致炎右足待测。2.小鼠疼痛模型建立：小鼠腹腔注射0.7%冰醋酸0.1ml/10g,制成疼痛模型。测定各组小鼠20 min内扭体反应次数。3.急性炎症模型建立：将100ul(巴豆油：乙醇：吡啶：乙醚=1：10：20：69)均匀涂在小鼠右耳正反两面致炎造成耳肿胀模型。测定各组小鼠耳肿胀度。大鼠右后足跖皮下注射1%角叉菜胶(0.1ml／只)致炎造成足肿胀模型。测定各组大鼠足跖肿胀率。4.慢性炎症模型建立：经腹部切口将两个无菌棉球(50±lmg／个,高压灭菌,各加入氨苄青霉素lmg/0.1ml/个,50℃烘箱烤干)分别植入两侧腹股沟皮下,造成慢性炎症模型。测定各组大鼠肉芽肿净重。5.佐剂关节炎模型建立：大鼠右足皮下注射Freund’s完全佐剂(Freund’s Adjuvant complete)0.1ml/只,造成佐剂关节炎模型。6.耳组织MPO的测定与足组织PGE2测定：比色法测定巴豆油诱导的耳肿胀试验中的小鼠耳组织髓过氧化物酶(MPO)的活性以及对角叉菜胶致大鼠肿胀足组织中炎症介质(PGE2)含量的影响。测定各组大鼠炎症足PGE2渗出量。7.大鼠角叉菜诱导的胸膜炎实验：连续给药7d,末次给药后1h,于大鼠右侧胸腔(尽量靠近胸腔右后角以免伤肺)注入灭菌1%角叉菜胶生理盐水溶液0.2 mL/100 g,4h后脱颈椎处死大鼠,从眼眶静脉取血,硫代巴比妥酸法测定渗出液丙二醛的含量；黄嘌呤氧化酶法测定渗出液超氧化物歧化酶的活性。在横隔周边部剖开胸腔(避免血液渗入胸腔),然后以磷酸盐缓冲盐水液(含肝素5 U/ml-1)2ml灌洗胸膜腔,收集灌洗液准确计量,用1%冰醋酸水溶液作为稀释液,稀释至2ml,按白细胞计数法在倒置显微镜下进行观察计数白细胞。测定白细胞数、收集上清液用于测定蛋白质含量。8.小鼠急性毒性实验测定：取正常健康昆明小鼠,雌雄各半,一次性ig给药。0.75 mg/kg1.5g/kg,3g/kg,与6g/kg四个浓度(药液仅能通过灌胃针头)正丁醇提物的浓缩液,给药后自由饮水进食,雌雄各半,分别给小鼠一次性ig给药。连续观察七天,记录小鼠活动、饮食、二便、死亡等情况。结果1. EFIS对小鼠镇痛实验的影响IS连续ig给药5d后,各剂量组均减少醋酸所致的小鼠扭体反应次数,具有其镇痛作用,与对照组相比差异非常显著,(P<0.01)且高、中剂量镇痛率高于50%,表明IS对化学性刺激引起的疼痛有明显的抑制作用。2. EFIS对急性炎症模型的影响与对照组比较,阳性对照组、IS高、中、低三个剂量组动物的耳廓肿胀度存在显著性差异(P<0.01);对于巴豆油所致小鼠耳廓肿胀的抑制率,高剂量IS为45.8%,高于阳性对照药布洛芬(32%),中剂量IS的抑制率与阳性药相当,低剂量IS的抑制率(10.7%)低于阳性对照组,说明IS对对巴豆油所致小鼠耳廓肿胀有明显的抑制作用。与对照组比较,对角叉菜胶致大鼠足肿胀的影响IS高剂量组在致炎后1-5 h,中剂量组在致炎后2-4 h足肿胀度与模型对照组的大鼠足肿胀度相比较有明显差异,(P<0.01)可见IS可明显对抗角叉菜胶所致大鼠足肿胀程度。布洛芬也有相似作用。3. EFIS对慢性炎症模型的影响实验结果显示,阳性对照组、IS高剂量组的肉芽组织重量明显轻于空白对照组(p<0.01),且IS大剂量组作用与地塞米松组相当,IS中剂量组与空白对照组肉芽组织重量差异显著,有显著的统计学意义(p<0.05)。说明IS对实验性肉芽组织的增生有较强抑制作用。4. EFIS对佐剂关节炎症模型的影响高剂量组能降低3,6,9,21d的足肿胀程度,中剂量也对3,6d有明显作用,布洛芬作用与高剂量相似。5. EFIS对耳组织MPO、足组织PGE2含量的影响结果表明,与空白对照组比较,阳性对照组、IS高、中、两个剂量组,动物的MPO含量存在显著性差异(P<0.001);对于巴豆油所致小鼠耳廓MPO的抑制率,高剂量IS为63.3%与阳性药相当,阳性对照药布洛芬(66.9%)。中剂量IS的抑制率56.1%,低剂量IS的抑制率(20.1%)低于阳性对照组,说明IS对巴豆油所致小鼠耳廓肿胀中MPO的含量有明显的抑制作用。据此定量测定PGE,布洛芬组和IS高、中剂量组与空白对照组比较,PGE含量(OD值)均降低,差异显著(p<0.01),且IS高剂量组的作用与布洛芬组的作用相当;IS低剂量组的PGE含量也低于空白对照组,差别具有统计学意义(p<0.05)。6. EFIS对角叉菜诱导胸膜炎中渗出量、白细胞、蛋白含量、MDA、及SOD影响结果显示,阳性对照组、IS高剂量组的白细胞数量明显低于空白对照组,且差异极其显著(p<0.01),可见IS能显著减少角叉菜胶所致急性炎症大鼠胸腔白细胞的数量,具有较好的抑制白细胞趋化和游走的作用。IS各剂量组均能降低角叉菜胶所致急性炎症渗出液中蛋白质蛋白含量,减少大鼠胸膜炎渗出液体积产生以及血浆中SOD与MDA生成。7. EFIS的急性毒性实验的结果结果表明,小鼠按最大容量(0.8ml/10g／次)于当日内给四个浓度的IS药液,累计剂连续观察1周,未见动物死亡,所有小鼠皮毛光泽整洁,活动、饮食正常,无稀便及尿血等异常现象。提示IS药液口服给药安全。结论IS有效部位各实验组均有不同程度的抗炎、镇痛作用,其作用机制可能是通减少PGE2、抑制MPO,减少炎症生出以及提高血浆中SOD与降低MDA生成有关。更多还原

【Abstract】 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.。更多还原