University of Agricultural Sciences and Veterinary Medicine, Faculty of Science and Food Technology, Manastur Street 3-5, Cluj-Napoca, 400372, Romania.
Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in Bio-Nano-Sciences, Treboniu Laurean Street 42, Cluj-Napoca, 400271, Romania.
Phytochemistry. 2021 Sep;189:112849. doi: 10.1016/j.phytochem.2021.112849. Epub 2021 Jun 25.
Anthocyanins (AN), natural compounds daily consumed by humans, have outstanding therapeutical potential if administered topically in melanoma pathology. However, the search for efficient therapy development is still in progress, owing to the lack of complete understanding of the AN intracellular path, once they are uptaken by the cells. This target is constrained by the need for an imaging strategy that would enable their intracellular detection and localization in-situ. In this light, diphenylboric acid 2-aminoethyl (DPBA), a non-fluorescent reagent, was here successfully used to form fluorescent complexes with AN. The AN used are the cyanidin aglycon as a free standard molecule (CY), and the glycosylated compounds, extracted and purified from chokeberry fruits (AE). In solution, it was observed that the fluorescence emission increased by 39% (CY@DPBA), and by 34% (AE@DPBA), which concludes that AN form fluorescent complexes with DPBA (CY@DPBA and AE@DPBA). In addition, using NMR (nuclear magnetic resonance) spectroscopy, and HRMS (high-resolution mass spectrometry) analysis, the structure of the CY@DPBA complex was efficiently elucidated. In-vitro experiments showed that the complexes formed after the treatment proved to be non-toxic on B16-F10 cells. The sub-cellular visualization of all AN was monitored by fluorescence microscopy and flow cytometry, demonstrating detectable signals of the non-metabolized CY and glycosylated CY inside melanoma cells. This study reports that the use of DPBA to image AN intracellularly is a sensitive, non-invasive and successful method that can extend its application in broad fields like drug development or metabolism-associated mechanisms.
花色苷（AN）是人类日常食用的天然化合物，如果在黑色素瘤病理学中局部给药，具有突出的治疗潜力。然而，由于对 AN 细胞内途径缺乏全面了解，尽管它们被细胞摄取，高效疗法的研发仍在进行中。这一目标受到需要成像策略的限制，该策略能够使它们在细胞内进行原位检测和定位。有鉴于此，二苯硼酸 2-氨乙基（DPBA），一种非荧光试剂，成功地被用于与 AN 形成荧光配合物。所使用的 AN 是作为游离标准分子的矢车菊苷配基（CY），以及从黑果腺肋花楸果实中提取和纯化的糖苷化合物（AE）。在溶液中，观察到荧光发射增加了 39%（CY@DPBA）和 34%（AE@DPBA），这表明 AN 与 DPBA 形成了荧光配合物（CY@DPBA 和 AE@DPBA）。此外，通过核磁共振（NMR）光谱和高分辨率质谱（HRMS）分析，有效地阐明了 CY@DPBA 配合物的结构。体外实验表明，处理后形成的配合物在 B16-F10 细胞上证明是无毒的。通过荧光显微镜和流式细胞术监测所有 AN 的亚细胞可视化，证明了非代谢的 CY 和糖基化 CY 在黑色素瘤细胞内可检测到信号。本研究报告称，使用 DPBA 对 AN 进行细胞内成像，是一种敏感、非侵入性和成功的方法，可以将其应用扩展到药物开发或代谢相关机制等广泛领域。
