野鸢尾苷的制备及其体外抗炎活性机制的初探

Preparation of iridin and study on its anti-inflammatory mechanism in vitro

目的 从中药射干中分离制备野鸢尾苷单体,并初步研其体外抗炎活性及作用机制。方法 采用多溶剂萃取法制备野鸢尾苷;在野鸢尾苷溶液对小鼠单核巨噬细胞RAW 264.7无细胞毒性的浓度范围内,采用脂多糖诱导并建立体外细胞炎症模型,给予不同浓度野鸢尾苷溶液干预,采用Griess法检测细胞上清液中一氧化氮(NO)的含量、ELISA法检测肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)的含量;并结合野鸢尾苷对乙酰胆碱酯酶活性的影响,初步揭示野鸢尾苷的抗炎活性及作用机制。结果 多溶剂萃取法所得的野鸢尾苷单体,经HPLC峰面积归一化法计算其纯度大于98%;给药12.5~100μg·mL-1野鸢尾苷对RAW 264.7无细胞毒性;与模型组比较,在给药12.5~100μg·mL-1野鸢尾苷时,对抑制巨噬细胞中TNF-α、NO、IL-1β、IL-6的释放具有极显著性差异,且呈剂量关系;野鸢尾苷对乙酰胆碱酯酶的活性具有抑制作用,其数半抑制浓度(IC50)为4.09 mg·mL-1,且呈剂量关系。结论 多溶剂萃取法是一种简单、高效分离制备射干中野鸢尾苷的方法;野鸢尾苷的抗炎机制可能是通过胆碱能抗炎通路,调节巨噬细胞释放TNF-α、IL-1β、IL-6炎性介质,并结合调节NO抗炎途径,从而实现多途径的抗炎作用。

OBJECTIVE To isolate and prepare the iridin monomer from the traditional Chinese medicine Belamcandae Rhizoma,and to preliminaily explore the in vitro anti-inflammatory activity of iridin and its mechanism.METHODS Multi-solvent extraction method was applied to separate iridin.In the range of non-cytotoxic concentration of iridin on mouse mononuclear macrophages RAW 264.7,lipopolysaccharide(LPS)was used to induce and establish an in vitro cellular model of inflammation,and different concentrations of iridin were administered.The concentration of NO in cell supernatants was determined by Griess method,and TNF-α,IL-1βand IL-6 were analyzed by ELISA.Combined with the effect of iridin on acetylcholinesterase activity,it preliminarily revealed the anti-inflammatory activity and mechanism of action of iridin.RESULTS The purity of iridin monomer obtained by the multi-solvent extraction method is more than 98%calculated by the HPLC peak area normalization method.The iridin concentration showed no cytotoxicity to RAW 264.7 in the range of 12.5-100μg·mL-1.At the concentration of 12.5-100μg·mL-1,compared with the LPS modeling group,the inhibition of the release of TNF-α,NO,IL-1βand IL-6 in macrophages was significantly different,and showed a dose relationship.Iridin had an inhibitory effect on acetylcholinesterase activity,with a semi inhibitory concentration(IC50)of 4.09 mg·mL-1,in a dose-dependent manner.CONCLUSION Multi-solvent extraction method is a simple and efficient method for the isolation and preparation of iridin in Belamcandae Rhizoma.The anti-inflammatory action of iridin possibly involves the cholinergic anti-inflammatory pathway,which regulates the release of inflammatory mediators such as TNF-α,IL-1βand IL-6 from macrophages.This effect is likely auqmented by the modulation of the NO anti-inflammatory pathway,resulting in a comprehensive and multi-faceted approach to inflammation control.