Laboratory of Foodomics, Institute of Food Science Research, CIAL, CSIC, Madrid, Spain.
Methods Mol Biol. 2023;2571:45-55. doi: 10.1007/978-1-0716-2699-3_5.
This methodological work demonstrates the potential of metabolomic approaches based on liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI(+/-)-HRMS) to investigate the antiproliferative capacity of underexplored biomasses (e.g., Passiflora mollissima seeds and Physalys peruviana calyx), by evaluating the molecular changes induced at the metabolite expression levels on HT-29 human colon cancer cells. This protocol describes in detail the optimal conditions to obtain bioactive extracts by pressurized liquid extraction (PLE), the experimental procedure to grow and treat HT-29 human colon cancer cells and CCD-18Co normal human colon fibroblasts with the target extracts, the metabolites extraction from the cytosolic fraction, and subsequent metabolomic fingerprinting. After treatment for 48 and 72 h, the viability of HT-29 colon cancer cells is markedly affected, and metabolites can be extracted for investigation. Following the proposed metabolomic data analysis and interpretation workflow, altered cellular redox homeostasis, as well as inactivation or dysfunction on other metabolic pathways, constitutes valuable biological information to understand the mechanisms underlying the antiproliferative effect.
本方法学工作展示了基于液相色谱与高分辨率质谱联用（LC-ESI(+/-)-HRMS）的代谢组学方法在研究探索较少的生物质（如 Passiflora mollissima 种子和 Physalys peruviana 花萼）的抗增殖能力方面的潜力，通过评估在 HT-29 人结肠癌细胞中代谢物表达水平引起的分子变化。本方案详细描述了通过加压液体提取（PLE）获得生物活性提取物的最佳条件、用目标提取物培养和处理 HT-29 人结肠癌细胞和 CCD-18Co 正常人类结肠成纤维细胞的实验程序、从细胞质部分提取代谢物，以及随后的代谢组学指纹图谱分析。经过 48 和 72 h 的处理后，HT-29 结肠癌细胞的活力明显受到影响，可以提取代谢物进行研究。按照提出的代谢组学数据分析和解释工作流程，改变的细胞氧化还原稳态以及其他代谢途径的失活或功能障碍，为理解抗增殖作用的机制提供了有价值的生物学信息。
