目的 旨在建立一种基于生物传感器联合超高效液相色谱/串联质谱（ultra-performance liquid chromatography with electrospray tandem mass spectrometry，UPLC-MS/MS）的中药苦味关键质量属性辨识方法。以白术内酯类成分为研究对象，通过分子对接与生物传感技术揭示其与苦味受体的相互作用机制，实现苦味属性从传统经验判定到分子水平表征的跨越，为完善白术质量控制体系提供支撑。方法 采用D101大孔吸附树脂结合紫外分光光度法，制备并筛选富含内酯类成分的白术组分；通过UPLC-MS/MS与分子对接技术，在分子水平解析白术组分与味觉受体的相互作用特征；采用生物传感器与UPLC-MS/MS联用技术，实现白术组分苦味关键质量属性的精准辨识。结果 成功制得白术不同极性组分11个，其中白术醇提物70%组分的总内酯含量最高，为后续性味研究的理想载体；分子对接实验显示白术组分与II型味觉受体家族14号成员（taste receptor type 2 member 14，TAS2R14）的结合展现出明显优势，在分子层面揭示了白术组分具有强烈趋于苦味属性的作用特质；进一步构建苦味生物传感器，对白术组分的苦味属性进行表征，结果显示白术组分与TAS2R14的相互结合强度为49.0 ng/L，属于强相互作用，表明以内酯类成分为主的白术组分具有显著的苦味属性，该结果与分子对接结果相互印证，增强了实验结论的可靠性；最后，联合UPLC-MS/MS技术，共辨识得到包括白术内酯II在内的苦味关键质量属性13个，在物质基础层面表明白术组分与苦味受体TAS2R14的结合受关键质量属性驱动，为理解白术内酯类成分苦味特性提供直接证据。结论 通过多技术融合的系统性研究，从分子相互作用到物质基础层面揭示了白术内酯类成分苦味属性的本质，所发现的苦味关键质量属性为建立基于苦味特征的白术质量控制方法奠定了理论基础，此外，提出的技术路线为中药苦味物质基础研究提供了新的方法学参考。

Objective This study aims to establish a method for identifying the critical quality attributes (CQAs) of traditional Chinese medicine (TCM) based on biosensors combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Using atractylenolides as the research subject, the interaction mechanism between these compounds and bitter taste receptors will be elucidated through molecular docking and biosensing technology. This approach will achieve a transition from traditional empirical evaluation to molecular-level characterization of bitterness attributes, thereby providing support for improving the quality control system of Baizhu (Atractylodis Macrocephalae Rhizoma, AMR). Methods Preparation and screening of atractylenolides-rich fractions from AMR using D101 macroporous adsorption resin coupled with UV spectrophotometry; Molecular-level characterization of interactions between the obtained fractions and taste receptors via UPLC-MS/MS and molecular docking; Precise identification of CQAs through integrated biosensor technology and UPLC-MS/MS analysis. Results In this study, eleven fractions of AMR with different polarities were successfully prepared. Among them, the 70% ethanol extract fraction of AMR had the highest total lactone content, serving as an ideal carrier for subsequent studies on taste properties. The molecular docking experiment showed that the binding of the components of AMR to taste receptor type 2 member 14 (TAS2R14) exhibited obvious advantages, revealing at the molecular level that the components of AMR have a strong tendency towards the functional characteristics of the bitter taste property. Furthermore, a bitter taste biosensor was constructed to characterize the bitter taste property of the components of AMR. The results showed that the binding strength between the components of AMR and the bitter taste receptor TAS2R14 was 49.0 ng/L, which was a strong interaction, indicating that the components of AMR mainly composed of lactone components have a significant bitter taste property. This result was consistent with the molecular docking results, enhancing the reliability of the experimental conclusion. Finally, by combining with the UPLC-MS/MS technology, a total of 13 CQAs related to bitterness, including atractylenolide II, were identified. At the material basis level, it was demonstrated that the binding of specific chemical components in the components of AMR to the bitter taste receptor TAS2R14 is driven by these CQAs, providing direct evidence for understanding the bitter taste characteristics of atractylenolides components in AMR. Conclusion This study employs a multi-technology integrated approach to systematically investigate the bitterness attributes of atractylenolides in AMR, elucidating their molecular interaction mechanisms and material basis. The identified bitterness CQAs provide a theoretical foundation for establishing a bitterness-characterized quality control method for AMR. Furthermore, the proposed technical strategy offers a novel methodological reference for researching the bitter-tasting substance basis of traditional TCM.

R283.6

国家重点研发计划课题（2023YFC3504505）；鲁南集团横向课题（BUCM-2021-JS-FW-173）；国家自然科学基金项目（882274110）；北京科技新星交叉项目（20230484458）