荆芥挥发油体外干预5-L0活性的实验研究及相关方法学评价

【作者】 赵璐；

【导师】 曾南；

【作者基本信息】 成都中医药大学 ， 药理学， 2008， 硕士

【摘要】 荆芥,来源于唇形科植物裂叶荆芥Schizonepeta tenuifolia Briq.的地上干燥部分,是常用解表中药,具有解表散风、透疹之功效。荆芥挥发油含量高,是其主要有效成分之一。前期研究结果表明:荆芥挥发油（STO）抗炎作用确实,且作用机制与影响花生四烯酸的代谢有关。本课题基于前期研究结果,针对5-LO是花生四烯酸代谢过程中的关键酶,将5-LO作为切入点,深入探讨STO抗炎机理。目的:观察STO体外对5-LO活性的影响,进一步探讨其抗炎机制。同时,摸索建立中药有效部位体外干预5-LO活性的筛选方法,并作适当评价。方法:首先采用二甲苯致小鼠耳廓肿胀急性炎症模型对STO抗炎作用进行再次验证;其次建立提取大鼠胸腔白细胞5-LO代谢产物LTB₄和5-HETE及分离测定方法;最后运用反相高效液相色谱（RP-HPLC）紫外检测分离测定LTB₄和5-HETE生物合成水平,观察STO及部分具有抗炎作用的解表药挥发油体外对5-LO活性的影响。结果:1.STO具有良好的抗炎作用:STO连续给药和急性给药两种方式对二甲苯所致小鼠耳廓肿胀均具有明显抑制作用（P＜0.05或＜0.01）。2.提取并分离测定5-LO代谢产物LTB₄和5-HETE的方法建立:以角叉菜胶所致急性胸膜炎模型大鼠的胸腔白细胞为基本反应体系,加入外源性AA与不同浓度的STO,在钙离子载体A₂₃₁₈₇诱导下,提取完整白细胞的LTB₄和5-HE7E,运用RP-HPLC紫外检测分离测定LTB₄和5-HETE。3.STO干预5-LO活性的抗炎机制:STO（二甲基亚砜溶解）和含STO的血清（浆）,体外能明显降低大鼠胸腔白细胞中的LTB₄和5-HETE水平,表现出良好的抑制5-LO活性的作用,且STO的作用呈现剂量依赖性关系;STO中的主要成分薄荷酮（Menthone）、胡薄荷酮（Pulegone）单独使用或二者（M+P）合用,对LTB₄和5-HETE的抑制作用均不及等效量的STO,但M+P的抑制作用优于Menthone、Pulegone的单独使用;以AA所致的足肿胀炎症模型大鼠为供体的含STO血清（浆）对LTB₄和5-HETE的抑制作用优于以正常大鼠为供体的含药血清（浆）。4.方法应用:同等浓度的桂枝、辛夷、香薷、羌活挥发油体外对LTB₄和5-HETE生成的抑制作用较薄荷、菊花挥发油强,初步提示辛温解表药挥发油对5-LO活性抑制作用优于辛凉解表药挥发油。结论:STO抗炎作用确切,其抗炎机制与抑制5-LO活性相关,呈剂量依赖性关系。研究结果补充、完善了荆芥挥发油的抗炎机理,有利于指导其临床合理用药。同时,实验中所建立的体外干预5-LO活性的RP-HPLC测定方法,有利于抗炎、抗过敏中药提取物的体外机制筛选。更多还原

【Abstract】 Herba schizonepetae Briq, the aerial ground part of schizonepeta tenui-folia Briq（family Labiatae）, is one of the commonly used Chinese herb that belongs to the category of relieving Exterior syndrome, which has the effect of expelling wind and treart miseal. The volatile oil is one of the major effective components of Herba schizonepetae Briq., and its contents is high.. The results of previous research showed, Schizonepetae Volatile oil （STO） has obvious anti-inflammation effect, which the anti-inflammatory mechanism be related with the pathway of arachidonic acid （AA）. Based on the previous studies, in this study we concentrated on the 5-lipoxygenase （5-LO）, that is an important enzyme of AA metabolism, to study the effect of STO on the activity of 5-LO in vitro so as to elucidate its mechanisms of anti-inflammatory action.Objective: To investigate the effect of STO on the activity of 5-LO in vitro so as to elucidate its mechanisms of anti-inflammatory action more. Meanwhile, to establish a screening platform for the effective parts of Traditional Chinese Medicine （TCM） that maybe affect the activity of 5-LO in vitro.Methods: First, the ear-swelling induced by xylene were adopted to validate the effect of anti-inflammation of STO in mice. Second, the method of RP-HPLC to extract, separate and detect leukotriene B₄（LTB₄） and 5-hydroxy-6, 8, 11,14-eicosatetraenoic acid （5-HETE） were established and evaluated. At last, the effect of STO and other volatile oil which has anti-inflammatory action on the activity of 5-LO were observed by the established RP-HPLC method in vitro.Results:1. STO exhibited definite anti-inflammation action. STO inhibited the ear-swelling induced by xylene in mice by ig 3 days or ig twice in a day （P< 0.05 or < 0.01） .2. The methods of RP-HPLC to extract, separate and detect LTB4 and 5-HETE were established. Thoracic cavity leukocytes, which from the pleurisy model rat induced by carrageenan, were used as the reaction system in vitro. Exogenous AA, STO in different concentration and calcium ionophore A₂₃₁₈₇ were added into this system to be incubated. The metabolites of 5-LO, LTB4 and 5-HETE, were extracted, separated and detected by means of RP-HPLC.3. Anti-inflammatory mechanism of STO was related to suppress the activity of 5-LO. STO both dissolved in dimethylsulfoxide and contained in serum （blood plasma）, inhibited obviously the activity of 5-LO in rat leukocytes in dose dependent way in vitro, which decreased the production of LTB₄ and 5-HETE. Compared with STO, the synthesis of LTB₄ and 5-HETE were not significantly suppressed by main components of STO, menthone and pulegone used alone or used mixing （M+P）. However, the suppressed effect of M+P on the activity of 5-LO was stronger than menthone and pulegone used alone. Compared with STO drug-serum or plasma from normal rats, the synthesis of LTB4 and 5-HETE were more significantly inhibited by STO drug-serum or plasma from the inflammatory model rats induced by AA.4. The methods of RP-HPLC to extract, separate and detect LTB₄ and 5-HETE were applied to investigate the effect of six kinds volatile oil of drugs for relieving exterior syndrome on the activity of 5-LO in vitro. Compared with Bohe and Juhua in same dose, the synthesis of LTB₄ and 5-HETE were significantly inhibited by Guizhi, Xinyi, Xiangru and Qianghuo. It is suggested that the inhibition effect of volatile oil on the activity of 5-LO, drugs for relieving the exterior syndrome with pungent in flavor and warm in nature were stronger than the drugs of pungent in flavor and cool in nature.Conclusion: The results showed that STO had the significant anti-inflammation action, which the mechanism were related to the inhibition effect on the activity of 5-LO in dose dependence, which be beneficial to guide clinical treatment. Meanwhile, the established research method was sensitive to reflect the action of drug affecting the activity of 5-LO, which could be a screening platform for the anti-inflammatory and anti-allergic drugs in vitro.更多还原