Astragali Radix (AR) is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer (NK) cells. However, owing to the complexity of its composition, the specific active ingredients in AR that act on NK cells are not clear yet. Cell membrane chromatography (CMC) is mainly used to screen the active ingredients in a complex system of herbal medicines. In this study, a new comprehensive two-dimensional (2D) NK-92MI CMC/C₁₈ column/time-of-flight mass spectrometry (TOFMS) system was established to screen for potential NK cell activators. To obtain a higher column efficiency, 3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column. In total, nine components in AR were screened from this system, which could be washed out from the NK-92MI/CMC column after 10 min, and they showed good affinity for NK-92MI/CMC column. Two representative active compounds of AR, isoastragaloside I and astragaloside IV, promoted the killing effect of NK cells on K562 cells in a dose-dependent manner. It can thus suggest that isoastragaloside I and astragaloside IV are the main immunomodulatory components of AR. This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

黄芪（AR）是一种临床使用的草药，具有多种免疫调节活性，可以增强自然杀伤（NK）细胞的活性和细胞毒性。然而，由于其成分的复杂性，AR 中作用于 NK 细胞的具体活性成分尚不清楚。细胞膜层析（CMC）主要用于筛选复杂的草药体系中的活性成分。在本研究中，一种新型综合二维（2D）NK-92MI CMC/C ₁₈建立了柱/飞行时间质谱（TOFMS）系统以筛选潜在的NK细胞活化剂。为了获得更高的柱效，合成了 3-巯基丙基三甲氧基硅烷改性硅胶制备 NK-92MI CMC 色谱柱。该系统共筛选出9种AR成分，10分钟后可从NK-92MI/CMC柱中洗出，对NK-92MI/CMC柱具有良好的亲和力。AR的两种代表性活性化合物异黄芪甲苷I和黄芪甲苷IV以剂量依赖性方式促进NK细胞对K562细胞的杀伤作用。因此可以表明异黄芪甲苷 I 和黄芪甲苷是 AR 的主要免疫调节成分。