Guangdong Hanchao Traditional Chinese Medicine Technology Co., Ltd., Guangzhou 510163, China.
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Molecules. 2022 Aug 25;27(17):5435. doi: 10.3390/molecules27175435.
Citri Reticulatae Pericarpium (CRP) is one of the most commonly used food supplements and folk medicines worldwide, and possesses cardiovascular, digestive, and respiratory protective effects partially through its antioxidant and anti-inflammatory functions. The unique aromatic flavor and mild side effects make CRP a promising candidate for the development of anti-inflammatory functional food. However, recent studies show that the crude alcoholic extract and some isolated compounds of CRP show compromised anti-inflammatory activity, which became the main factor hindering its further development. To identify the bioactive compounds with anti-inflammatory potential, and improve the anti-inflammatory effects of the extract, a bioinformatics-guided extraction protocol was employed in this study. The potential bioactive candidates were identified by combing network pharmacology analysis, molecular docking, principal components analysis, k-means clustering, and in vitro testing of reference compounds. Our results demonstrated that 66 compounds in CRP could be grouped into four clusters according to their docking score profile against 24 receptors, while the cluster containing flavonoids and phenols might possess a more promising anti-inflammatory function. In addition, in vitro anti-inflammatory tests of the seven reference compounds demonstrated that hesperitin, naringenin, and gardenin B, which were grouped into a cluster containing flavonoids and phenols, significantly decreased LPS-induced NO, TNF-α, and IL-6 production of macrophages. While the compounds outside of that cluster, such as neohesperidin, naringin, hesperidin, and sinensetin showed little effect on alleviating LPS-induced NO and proinflammatory cytokine production. Based on the chemical properties of selected compounds, ethyl acetate (EtOAc) was selected as the solvent for extraction, because of its promising solubility of flavonoids and phenols. Furthermore, the ethanol alcoholic extract was used as a reference. The chemical profiling of EtOAc and crude alcoholic extract by HPLC/MS/MS also demonstrated the decreased abundance of flavonoid glycosides in EtOAc extract but increased abundance of phenols, phenolic acid, and aglycones. In accordance with the prediction, the EtOAc extract of CRP, but not the crude alcoholic extract, significantly decreased the NO, IL-6, and TNF-α production. Taken together, the results suggested selective extraction of phenols and flavonoids rich extract was able to increase the anti-inflammatory potential of CRP partially because of the synergistic effects between flavonoids, phenols, and enriched polymethoxyflavones. Our study might pave the road for the development of ethyl acetate extract of CRP as a novel functional food with anti-inflammatory function.
橘红（CRP）是世界上最常用的食品补充剂和民间药物之一，具有心血管、消化和呼吸系统保护作用，部分通过其抗氧化和抗炎功能。独特的芳香风味和温和的副作用使 CRP 成为开发抗炎功能性食品的有前途的候选者。然而，最近的研究表明，CRP 的粗醇提取物和一些分离化合物的抗炎活性降低，这成为阻碍其进一步发展的主要因素。为了确定具有抗炎潜力的生物活性化合物，并提高提取物的抗炎效果，本研究采用了一种基于生物信息学的提取方案。通过结合网络药理学分析、分子对接、主成分分析、k-均值聚类和参考化合物的体外测试，确定了潜在的生物活性候选物。我们的结果表明，CRP 中的 66 种化合物可以根据它们与 24 种受体的对接评分图谱分为四个簇，而含有类黄酮和酚类的簇可能具有更有希望的抗炎功能。此外，对七种参考化合物的体外抗炎测试表明，属于含有类黄酮和酚类的簇的橙皮苷、柚皮苷和橙皮苷 B 显著降低了 LPS 诱导的巨噬细胞中 NO、TNF-α 和 IL-6 的产生。而属于含有类黄酮和酚类的簇以外的化合物，如新橙皮苷、柚皮苷、橙皮苷和橙皮苷，对减轻 LPS 诱导的 NO 和促炎细胞因子的产生几乎没有影响。基于所选化合物的化学性质，选择乙酸乙酯（EtOAc）作为溶剂进行提取，因为它对类黄酮和酚类具有良好的溶解性。此外，还使用乙醇醇提取物作为参考。HPLC/MS/MS 对 EtOAc 和粗醇提取物的化学分析也表明，EtOAc 提取物中类黄酮糖苷的丰度降低，但酚类、酚酸和苷元的丰度增加。与预测一致，CRP 的 EtOAc 提取物，而不是粗醇提取物，显著降低了 NO、IL-6 和 TNF-α 的产生。综上所述，结果表明，选择性提取富含酚类和类黄酮的提取物能够部分提高 CRP 的抗炎潜力，这可能是由于类黄酮、酚类和富马酸多甲氧基黄酮之间的协同作用。我们的研究可能为开发具有抗炎功能的 CRP 乙酸乙酯提取物作为新型功能性食品铺平道路。
