School of Life Sciences, BK 21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 41566, Republic of Korea.
College of Pharmacy, Chosun University, 375 Seosukdong, Donggu, Gwangju 61452, Republic of Korea.
J Ethnopharmacol. 2019 Mar 1;231:19-28. doi: 10.1016/j.jep.2018.11.005. Epub 2018 Nov 5.
Aster yomena, a perennial herb that grows mainly in South Korea, has been employed in the traditional temple food for antibiotic efficacy. Recently, it was reported that apigenin isolated from A. yomena has a physical antifungal mechanism targeting membrane against Candida albicans.
Our study aimed to investigate the biochemical responses underlying the antifungal activity of apigenin isolated from A. yomena due to lack studies reporting the investigation of intracellular responses of apigenin in C. albicans.
Apigenin was isolated from the aerial parts of A. yomena. To evaluate apigenin-induced inhibitory effects and membrane damages, the measurement of the cell viability assay and the flux of cytosolic components were performed with at various concentrations. Intracellular external potassium and calcium levels were assayed by an ion-selective electrode meter, Fura2-AM and Rhod2-AM, respectively. Mitochondrial dysfunctions were analyzed by using JC-1, Mitotracker Green FM, and MitoSOX Red dye. HDCFDA, glutathione, and MDA assay were used to detect oxidative damage. Also, flow cytometry was carried out to detect apoptotic hallmarks using Annexin V-PI, TUNEL, and FITC-VAD-FMK staining. Tetraethylammoniumchloride (TEA), Ruthenium red (RR), and N-acetylcysteine (NAC) were used as a potassium channel blocker, mitochondrial calcium uptake inhibitor, and reactive oxygen species (ROS) scavenger, respectively.
We confirmed that there was no decrease of cell survival percentages in crude extracts of A. yomena treatment, however, only isolated apigenin has the antifungal effect in C. albicans. Apigenin triggered a dose-dependent mitochondrial calcium uptake followed by mitochondrial dysfunction, loss of the membrane potential and an increase in the mitochondrial mass and ROS. Apigenin also induced intracellular redox imbalance as indicated by the ROS accumulation, glutathione oxidation, and lipid peroxidation. Interestingly, NAC failed the restore the mitochondrial calcium levels and thus alleviate the mitochondrial damages, however, RR reduced the apigenin-induced redox imbalance. Furthermore, apigenin induced apoptosis activation marked by the phosphatidylserine exposure, DNA fragmentation, and caspase activation. The pro-apoptotic effect of apigenin was counteracted by RR and NAC pretreatment. In particular, RR significantly reduced the pro-apoptotic responses.
Apigenin isolated from A. yomena induced mitochondrial-mediated apoptotic pathway, and mitochondrial calcium signaling is main factor in its pathway in C. albicans.
Aster yomena 是一种多年生草本植物，主要生长在韩国，它被用于传统的寺庙食品中，具有抗生素功效。最近，据报道，从 Aster yomena 中分离出的芹菜素具有针对白色念珠菌的物理抗真菌机制，针对细胞膜。
由于缺乏报告芹菜素在白色念珠菌细胞内反应的研究，本研究旨在调查从 Aster yomena 中分离出的芹菜素的抗真菌活性的生化反应。
从 Aster yomena 的地上部分分离出芹菜素。为了评估芹菜素诱导的抑制作用和膜损伤，用不同浓度的细胞活力测定法和胞质成分通量法进行测定。通过离子选择性电极计、Fura2-AM 和 Rhod2-AM 分别测定细胞内外钾和钙水平。线粒体功能障碍通过使用 JC-1、Mitotracker Green FM 和 MitoSOX Red 染料进行分析。使用 HDCFDA、谷胱甘肽和 MDA 测定法检测氧化损伤。此外，通过 Annexin V-PI、TUNEL 和 FITC-VAD-FMK 染色，使用流式细胞术检测凋亡特征。四乙铵氯化物 (TEA)、钌红 (RR) 和 N-乙酰半胱氨酸 (NAC) 分别用作钾通道阻断剂、线粒体钙摄取抑制剂和活性氧 (ROS) 清除剂。
我们证实，Aster yomena 处理的粗提取物中没有降低细胞存活率百分比，但是只有分离出的芹菜素有抗真菌作用。芹菜素触发了剂量依赖性的线粒体钙摄取，随后线粒体功能障碍、膜电位丧失、线粒体质量增加和 ROS 增加。芹菜素还诱导了细胞内氧化还原失衡，表现为 ROS 积累、谷胱甘肽氧化和脂质过氧化。有趣的是，NAC 未能恢复线粒体钙水平，从而减轻线粒体损伤，然而，RR 减轻了芹菜素诱导的氧化还原失衡。此外，芹菜素诱导了凋亡激活，表现为磷脂酰丝氨酸暴露、DNA 片段化和 caspase 激活。RR 和 NAC 预处理可拮抗芹菜素的促凋亡作用。特别是，RR 显著降低了促凋亡反应。
从 Aster yomena 中分离出的芹菜素诱导了线粒体介导的凋亡途径，线粒体钙信号是其在白色念珠菌中的途径中的主要因素。
