王不留行中刺桐碱的分离鉴定及抗炎活性研究

Isolation,Identification and Its Anti-inflammatory Activity of Hypaphorine from Vaccaria segetalis

利用有机溶剂萃取、制备型薄层层析、半制备高效液相色谱三步分离法从王不留行中分离出生物碱物质,鉴定并探讨其抗炎活性。王不留行种子经石油醚脱脂、乙醇回流提取,二氯甲烷、乙酸乙酯、水饱和正丁醇依次萃取,获得水饱和正丁醇相(组分A3)。组分A3经制备型薄层层析分离,展开剂为正丁醇-乙酸乙酯-水(4∶1∶5,上层),于紫外灯254 nm、365 nm下检视,分为7个组分(组分B1~B7)。含量高且峰形简单的组分B2经半制备高效液相色谱分离,甲醇-水作为流动相,获得1个单体化合物(样品I)。经HPLC分析,样品I纯度高于98%。通过化学反应、UV、IR、LC-MS、1H NMR、13C NMR多种波谱分析方法对该化合物进行结构解析,确定其分子式为C14H18N2O2,化学名称为N,N,N-三甲基色氨酸,中文名为刺桐碱(hypaphorine),属于吲哚类生物碱。再采用二甲苯致小鼠耳廓肿胀的炎症模型,观察刺桐碱的抗炎效果。连续给药6 d后,刺桐碱剂量为25 mg·kg-1时,可显著抑制小鼠耳廓肿胀,肿胀抑制率为52.02%。与阿司匹林(200 mg·kg-1)的肿胀抑制率(67.48%)相比,无显著性差异。本研究首次采用一条新工艺路线获得王不留行刺桐碱,并发现其在整体水平上具有抗炎活性,有望成为化学一类新药的候选分子。

By solvent extraction,preparative thin-layer chromatography and semi-prepared high performance liquid chromatography successively,a kind of alkaloid was isolated from vaccaria segetalis and its anti-inflammatory activity was explored.Vaccaria segetalis seeds were defatted with petroleum ether following the reflux extraction with ethanol.Then the residual was further extracted with methylene chloride,ethyl acetate,n-butanol saturated with water.The n-butanol fraction(constituent A3)was isolated by preparative thin layer chromatography with following procedure∶n-butanol∶ethyl acetate∶water=4∶1∶5 as developer,reviewing under the UV light at 254 and 365 nm,and divided to seven fractions(constituent B1~B7).Constituent B2 of high content and simple composition was subjected to semi-preparative high performance liquid chromatography using an ethanol-water gradient system to get a compound(sample I).Sample I occupied the content of 0.215‰in the raw seeds,and its purity was higher than 98%.On the basis of chemical reaction and spectroscopic methods,the formula of sample I was dentified as C 14 H 18 N 2O 2.The chemical name was N,N,N-trimethyl-tryptophan,and the Chinese name was hypaphorine belonging to indole alkaloids.The anti-inflammatory effect of hypaphorine was evaluated by the model of xylene-induced mouse ear edema after continuous administration for 6 days.The study showed that hypaphorine could significantly inhibit mouse ear swelling with an inhibition rate of 52.02%in the dose of 25 mg·kg-1.Compared with aspirin,hypaphorine had a higher biological activity.In this research,a new process route was studied to isolate the hypaphorine from Vaccaria segetalis.It was also found that hypaphorine in Vaccaria segetalis had the activity of anti-inflammatory at the overall level,thus hypaphorine was expected to be a candidate molecule to become a new drug.