连翘叶总黄酮的提取及其抗氧化与抗炎活性研究

Extraction of Total Flavonoids from Forsythia suspensa Leaves and Evaluation of Their Antioxidant and Anti-inflammatory Activities

摘要:

目的 探究连翘叶总黄酮（Forsythia suspensa leaf total flavonoids，FLTF）的抗氧化活性及其对RAW264.7巨噬细胞炎症反应的调控机制。方法 采用响应面法优化乙醇-硫酸铵双水相体系提取FLTF的工艺参数，使用UHPLC-Q-TOF-MS/MS技术鉴定FLTF的活性成分；进一步检测FLTF的抗氧化活性及其对RAW264.7巨噬细胞炎症反应的调控作用。结果 FLTF的最优提取工艺为：提取时间108 min、料液比1 g∶88 mL、乙醇体积分数29.50%、硫酸铵质量浓度0.25 g/mL，在此条件下FLTF质量分数达99.52 mg/g。从FLTF中共鉴定出35种化合物，其中黄酮类7种，芦丁为其最主要的单体特征成分。体外抗氧化活性结果表明，FLTF质量浓度为9.6 mg/mL时，Cu²⁺螯合能力最高，为85.25%；FLTF质量浓度为1.2 mg/mL时，DPPH自由基清除率高达97.70%。细胞试验结果表明，FLTF呈剂量依赖性抑制NO的释放，IC₅₀值为14.08 μg/mL；同时可显著下调iNOS与COX-2基因的mRNA相对表达水平及iNOS与COX-2的蛋白质表达水平。结论 FLTF富含芦丁等黄酮类物质，具有较强的体外抗氧化活性，可通过抑制iNOS/COX-2信号通路降低巨噬细胞炎症反应。该研究为实现连翘采收废弃物的资源化利用、构建连翘全株利用的循环经济模式提供了数据支撑。

Abstract:

Objective This study aimed to investigate the antioxidant activity of total flavonoids from Forsythia suspensa leaves (FLTF) and their regulatory effects on the inflammatory response in RAW264.7 macrophages.Method The response surface method was adopted to optimize the extraction parameters of FLTF via an ethanol-ammonium sulfate aqueous biphasic system. The active components in FLTF were identified by UHPLC-Q-TOF-MS/MS. Furthermore, the antioxidant activity of FLTF and the regulatory effects of FLTF on the inflammation of RAW264.7 macrophages were measured.Result The mass fraction of FLTF reached 99.52 mg/g under the optimal conditions: extraction time of 108 min, a solid-to-liquid ratio of 1 g∶88 mL, an ethanol volume fraction of 29.50%, and ammonium sulfate mass concentration of 0.25 g/mL. A total of 35 compounds were identified from FLTF, including 7 flavonoids. Rutin was the most abundant monomeric marker. The in vitro antioxidant assay results showed that FLTF achieved the maximum Cu²⁺-chelating capacity and DPPH radical scavenging rate of 85.25% at the mass concentration of 9.6 mg/mL and 97.70% at the mass concentration of 1.2 mg/mL, respectively. The results of cell experiments showed that FLTF dose-dependently inhibited nitric oxide (NO) release, with an IC₅₀ value of 14.08 μg/mL. Moreover, FLTF downregulated the relative mRNA levels of iNOS and COX-2 genes and the protein levels of iNOS and COX-2.Conclusion FLTF is rich in flavonoids, primarily rutin. FLTF possesses potent antioxidant activity in vitro and reduces the inflammation of RAW264.7 macrophages via suppression of the iNOS/COX-2 pathway. This study provides data support for the utilization of the harvesting waste and establishment of a circular economy pattern characterized by whole-plant utilization of Forsythia suspensa.