连翘叶乙醇提取物抗小鼠蓖麻油性腹泻活性及其对家兔离体空肠平滑肌自发性收缩的影响研究

Anti-diarrhea Activity of Ethanol Extract from Forsythia Suspensa Leaves and Its Effect on Spontaneous Contraction of the Isolated Rabbit Intestinal Smooth Muscle

背景中医药治疗腹泻具有疗效迅速、不良反应少、费用低廉等优势。连翘具有清热解毒、消肿散结的功效,目前就其治疗腹泻方面的研究鲜有报道。目的观察连翘叶乙醇提取物(FS ext)对小鼠蓖麻油性腹泻的影响,并研究其对家兔离体空肠平滑肌自发性收缩的影响及其作用机制。方法2018年6—12月,于四川省南充市采集连翘叶并经提取得到FS ext,采用高效液相色谱法分析FS ext的化学成分;取60只禁食4 h(可自由饮水)的小鼠随机分为0.5 g/kg FS ext试验组、1.0 g/kg FS ext试验组、2.0 g/kg FS ext试验组、4.0 g/kg FS ext试验组、8.0 g/kg FS ext试验组、16.0 g/kg FS ext试验组,每组10只,分别给予0.5 g/kg、1.0 g/kg、2.0 g/kg、4.0 g/kg、8.0 g/kg、16.0 g/kg的FS ext进行灌胃,记录14 d内小鼠的死亡情况和毒性迹象,建立相应的剂量-效应曲线,从而确定FS ext的半数致死剂量(LD50),以确定其安全性。另取50只小鼠随机分成阴性组、阳性组、0.25 g/kg FS ext试验组、0.50 g/kg FS ext试验组、1.00 g/kg FS ext试验组,每组10只,阴性组给予0.9%氯化钠溶液(20 ml/kg)灌胃,阳性组给予洛哌丁胺(4 mg/kg)灌胃,0.25 g/kg FS ext试验组、0.50 g/kg FS ext试验组、1.00 g/kg FS ext试验组分别给予0.25、0.50、1.00 g/kg FS ext。给药30 min后,小鼠均给予蓖麻油0.4 ml灌胃,观察4 h内以下指标:初始半固体粪便时间,固体、半固体、液体粪便数量。采用腹泻指数(EI)评估腹泻的严重程度。取42只健康家兔,制备离体空肠平滑肌,观察FS ext对离体空肠平滑肌自发性收缩的影响:分别用乙酰胆碱(ACh,10μmol/L)和氯化钾(KCl,60 mmol/L)预处理离体空肠平滑肌,来探究FS ext对其诱导的离体空肠平滑肌自发性收缩的影响;用含FS ext或维拉帕米(1μmol/L)的无Ca2+高K+台式液预处理离体空肠平滑肌,然后累计CaCl2进一步研究其机制。结果FS ext中主要含连翘苷和连翘酯苷A等化学成分;小鼠按不同剂量的FS ext进行灌胃后,在观察期内未出现任何死亡或毒性迹象。阳性组初始半固体粪便时间长于阴性组、0.25 g/kg FS ext试验组、0.50 g/kg FS ext试验组(P<0.05);1.00 g/kg FS ext试验组初始半固体粪便时间长于阴性组、0.25 g/kg FS ext试验组(P<0.05)。阴性组EI高于阳性组、1.00 g/kg FS ext试验组(P<0.05);0.25 g/kg FS ext试验组、0.50 g/kg FS ext试验组EI高于阳性组(P<0.05)。FS ext(0.01~10.00 mg/ml)以剂量依赖性的方式抑制家兔离体空肠平滑肌的自发性收缩,并抑制由ACh和KCl诱导的空肠收缩,同时,FS ext也能抑制CaCl2诱导的空肠平滑肌自发性收缩,并使CaCl2量-效关系曲线右下移,与维拉帕米引起的CaCl2量-效关系曲线变化类似。结论FS ext有明显的抗小鼠蓖麻油性腹泻作用,并能抑制家兔离体空肠平滑肌的自发性收缩及ACh和KCl诱导的空肠收缩,其机制可能与阻断M受体和抑制L-型钙离子通道有关。

Background Traditional Chinese medicine has the advantages of rapid acting,less side effects and low cost in the treatment of diarrhea.Forsythia suspensa have the efficacy of clearing heat and detoxicating,reducing swelling and dispersing nodules.However,there are few reports on its effect on diarrhea at present.Objective To observe the effect of ethanol extract from Forsythia suspensa leaves(FS ext)on castor oil-induced diarrhea in mice and to study its effect and mechanism on the contraction of isolated intestinal smooth muscle from rabbits.Methods This study was implemented from June to December,2018.Forsythia suspensa leaves were collected and extracted in Nanchong City,Sichuan Province.The chemical components of FS ext were analyzed by high performance liquid chromatography(HPLC).60 fasted mice(with free access to water)were randomly divided into 0.5 g/kg,1.0 g/kg,2.0 g/kg,4.0 g/kg,8.0 g/kg and 16.0 g/kg FS ext groups,and received gavage administration of 0.5 g/kg,1.0 g/kg,2.0 g/kg,4.0 g/kg,8.0 g/kg and 16.0 g/kg FS ext,respectively,for determining the safety of 6 doses of FS ext.The mortality and toxicity of FS ext within 14 days were recorded,and the corresponding dose-response curve was established to determine the median lethal dose(LD50)of FS ext.Additionally,50 mice were randomly divided into negative and positive groups,and 0.25 g/kg,0.50 g/kg and 1.00 g/kg FS ext test groups,treated by gavage administration of 0.9%sodium chloride solution(20 ml/kg),loperamide(4 mg/kg),0.25,0.50 and 1.00 g/kg FS ext,respectively.30 minutes later,0.4 ml castor oil was administered by gavage to each mouse,and the following indicators were observed within 4 hours:the defecating time of initial semi-solid feces,the number of solid,semi-solid and liquid feces.We used diarrhea index(EI)to evaluate the severity of diarrhea.Then 42 healthy rabbits were used to prepare isolated intestinal smooth muscle in order to carry out the following experiments.Firstly,the effect of FS ext on the spontaneous contraction of isolated intestinal smooth muscle was observed.Secondly,the isolated intestinal smooth muscle was preprocessed with acetylcholine(ACh,10μmol/L)or potassium chloride(KCl,60 mmol/L),the effect of FS ext on the ACh/KCl-induced contraction of isolated intestinal smooth muscle was studied.Finally,the isolated intestinal smooth muscle was preprocessed with Ca2+-free high-K+Tyrode solution containing FS ext or verapamil(1μmol/L),then CaCl2 was accumulated to further study its mechanism.Results The main chemical constituents of FS ext were Forsythin and Forsythoside A.After administration by gavage at different doses of FS ext,there were no signs of death or toxicity in the observation period.The defecating time of initial semi-solid feces of positive group was longer than that of negative group,0.25 g/kg and 0.50 g/kg FS ext groups(P<0.05).The defecating time of initial semi-solid feces of 1.00 g/kg FS ext group was longer than that of negative group and 0.25 g/kg FS ext group(P<0.05).The EI of negative group was higher than that of positive group and 1.00 g/kg FS ext group(P<0.05).The EI of 0.25 g/kg or 0.50 g/kg FS ext group was higher than that of positive group(P<0.05).Besides,FS ext(0.01-10.00 mg/ml)inhibited spontaneous contraction of rabbit isolated intestinal smooth muscle in a dose-dependent manner,and the jejunal contraction induced by Ach or KCl.At the same time,the FS ext also inhibited the contraction of jejunal smooth muscle induced by CaCl2 in a dose-dependent manner,and the CaCl2 dose-response curve was shifted to the lower right,similar to that of verapamil.Conclusion FS ext may have a significant anti-castor oil diarrhea effect in mice,and could inhibit spontaneous contraction of rabbit isolated intestinal smooth muscle,as well as the jejunal contraction induced by ACh or KCl.The mechanism may be related to FS ext-induced blocking of M-receptor and inhibiting of L-type calcium channel.