Cordycepin（3'-去氧腺苷）是冬虫夏草（Cordyceps militaris）的主要活性成分，是冬虫夏草品质检测的化学标志物，具有重要的医药开发价值。目前获取cordycepin的方法复杂昂贵。本研究通过物理结晶探索了一种经济简便的从冬虫夏草发酵液中分离纯化cordycepin的方法。首先将冻干的粉状发酵液（LPFL）和纯甲醇（1g /100mL，w/v）混合，然后反复溶解和结晶直到沉淀成白色。通过冷冻干燥沉淀物得到纯化产品。经高效液相色谱、质谱和红外光谱鉴定，物质为cordycepin，纯度为94.26％。与现有方法相比，这种方法简单且成本低。此外，通过体外测试确定了cordycepin的功能活性。结果表明，cordycepin导致人类结肠癌Caco-2细胞死亡和形态变化，并显著抑制Caco-2细胞的增殖，半数抑制浓度（IC50）为107.2μg/mL。Cordycepin可以导致Caco-2早期凋亡，并导致细胞周期停留在G2期。Caco-2细胞凋亡和细胞周期阻滞在一定范围内呈剂量依赖性。这些结果改进了cordycepin纯化方法，在癌症抑制机制方面提供了洞见，并为cordycepin的进一步开发和临床应用提供重要参考。© 2023 The Authors.

Cordycepin (3 '-deoxyadenosine) is the main active component of Cordyceps militaris, which is a chemical marker for quality detection of Cordyceps militaris and has important medicinal development value. Existing methods for obtaining cordycepin are complex and costly. In this study, an economical and simple method for separation and purification of cordycepin from Cordyceps militaris fermentation liquid through physical crystallization was explored. First, lyophilized powdered fermentation liquid (LPFL) and pure methanol (1 g/100 mL, w/v) were mixed, and then repeatedly dissolved and crystallized until the precipitation was white. Purified product was obtained by freeze-drying the precipitate. The substance was determined to be cordycepin by high performance liquid chromatography, mass spectrometry and infrared spectroscopy, and the purity was 94.26%. Compared with the existing methods, this method is simple and low cost. In addition, the functional activity of cordycepin was determined by in vitro test. The results exhibited that cordycepin caused death and morphological changes in human colon cancer Caco-2 cells, and significantly inhibited the proliferation of Caco-2 cells, with a half-maximal inhibitory concentration (IC50) of 107.2 μg/mL. Cordycepin could induce early apoptosis of Caco-2 and caused cell cycle arrest in the G2 phase. Caco-2 cell apoptosis and cell cycle arrest showed dose dependence to cordycepin over a certain range. These results improved cordycepin purification method, provided insights into the mechanism of cordycepin in cancer inhibition, and would provide important reference for further development and clinical application of cordycepin.© 2023 The Authors.