基于光谱-效应关系分析和NF-κB通路的 Angelica dahurica 根抗炎活性成分筛选

Angelica dahurica 根（ADR）是一种在中国及其他亚洲国家广泛应用的草药，具有对多种炎症性疾病的抗炎作用。然而，ADR的具有活性的成分及其抗炎作用的潜在机制仍不清楚。本研究试图基于光谱-效应关系分析和NF-κB信号通路，发现ADR中的抗炎生物活性化合物并探索其作用机制。采用高效液相色谱-二极管阵列检测（HPLC-DAD）建立ADR样品的色谱指纹，选择了11个共同峰。随后，利用高效液相色谱-四极杆飞行时间质谱（HPLC-Q/TOF-MS）对ADR中的这11个共同峰进行鉴定。同时，通过抑制LPS诱导的RAW264.7细胞中NO、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）生成，评估ADR样品的抗炎活性。利用灰色关联分析（GRA）和偏最小二乘回归（PLSR）分析HPLC指纹中11个共同峰与抗炎效果的关系，筛选潜在的抗炎生物活性成分。结果显示，6种香豆素类化合物，包括 bergapten、xanthotoxin、phellopterin、isoimperatorin、xanthotoxol 和imperatorin，可能是ADR中的潜在抗炎活性成分。进一步验证实验亦表明，这六种香豆素在抑制LPS诱导的RAW264.7细胞中NO、IL-1β、IL-6和TNF-α的生成方面具有显著效果。此外，采用Western杂交分析探讨了两种潜在抗炎生物活性成分（phellopterin 和 isoimperatorin）调控机制，通过检测NF-κB信号通路中的蛋白水平，结果显示phellopterin和isoimperatorin可以显著下调磷酸化的NF-κB p65（p-p65）、磷酸化的IκBα（p-IκBα）及iNOS，并降低p-p65/p65和p-IκBα/IκBα的比例，提示这两种香豆素通过抑制NF-κB通路发挥抗炎作用。

Angelica dahurica root (ADR), a commonly utilized herbal medicine in China and other Asian nations, which has anti-inflammatory effects on diverse inflammatory ailments. However, the bioactive components and underlying mechanism responsible for the anti-inflammatory effect of ADR are still unclear. This work attempted to discover the anti-inflammatory bioactive compounds and explore their underlying mechanism in ADR based on spectrum-effect relationship analysis and NF-κB signaling pathway. Chromatographic fingerprints of ADR samples were established by high performance liquid chromatography with diode array detection (HPLC-DAD), and a total of eleven common peaks were selected. Then, high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q/TOF-MS) was employed for identification of eleven common peaks in ADR Meanwhile, the anti-inflammatory activities of ADR samples were assessed by inhibition of NO, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production in LPS-induced RAW264.7 cells. The spectrum-effect relationships between the eleven common peaks in HPLC fingerprints and anti-inflammatory effects of ADR samples were investigated to identify the potential anti-inflammatory bioactive compounds by grey relational analysis (GRA) and partial least squares regression (PLSR). The spectrum-effect relationship analysis results indicated that six coumarin compounds, including bergapten, xanthotoxin, phellopterin, isoimperatorin, xanthotoxol and imperatorin could be potential anti-inflammatory bioactive compounds in ADR. The further validation experiments also showed that these six coumarins demonstrated significant inhibition of NO, IL-1β, IL-6, and TNF-α production in LPS-induced RAW264.7 cells. In addition, western blot analysis was conducted to explore the mechanisms of two potential anti-inflammatory bioactive compounds (phellopterin and isoimperatorin) by assessing the protein levels in the NF-κB signaling pathway. The western blot results illustrated that phellopterin and isoimperatorin could significantly down-regulate the phosphorylated NF-κB p65 (p-p65), phosphorylated IκBα (p-IκBα) and iNOS, and depress the pro-portion of p-p65/p65 and p-IκBα/IκBα, which indicated that these two coumarins in ADR could potentially exert anti-inflammatory effects by suppressing of NF-κB pathway.