目的 旨在建立一種基于生物傳感器聯(lián)合超高效液相色譜/串聯(lián)質(zhì)譜（ultra-performance liquid chromatography with electrospray tandem mass spectrometry，UPLC-MS/MS）的中藥苦味關(guān)鍵質(zhì)量屬性辨識方法。以白術(shù)內(nèi)酯類成分為研究對象，通過分子對接與生物傳感技術(shù)揭示其與苦味受體的相互作用機制，實現(xiàn)苦味屬性從傳統(tǒng)經(jīng)驗判定到分子水平表征的跨越，為完善白術(shù)質(zhì)量控制體系提供支撐。方法 采用D101大孔吸附樹脂結(jié)合紫外分光光度法，制備并篩選富含內(nèi)酯類成分的白術(shù)組分；通過UPLC-MS/MS與分子對接技術(shù)，在分子水平解析白術(shù)組分與味覺受體的相互作用特征；采用生物傳感器與UPLC-MS/MS聯(lián)用技術(shù)，實現(xiàn)白術(shù)組分苦味關(guān)鍵質(zhì)量屬性的精準(zhǔn)辨識。結(jié)果 成功制得白術(shù)不同極性組分11個，其中白術(shù)醇提物70%組分的總內(nèi)酯含量最高，為后續(xù)性味研究的理想載體；分子對接實驗顯示白術(shù)組分與II型味覺受體家族14號成員（taste receptor type 2 member 14，TAS2R14）的結(jié)合展現(xiàn)出明顯優(yōu)勢，在分子層面揭示了白術(shù)組分具有強烈趨于苦味屬性的作用特質(zhì)；進一步構(gòu)建苦味生物傳感器，對白術(shù)組分的苦味屬性進行表征，結(jié)果顯示白術(shù)組分與TAS2R14的相互結(jié)合強度為49.0 ng/L，屬于強相互作用，表明以內(nèi)酯類成分為主的白術(shù)組分具有顯著的苦味屬性，該結(jié)果與分子對接結(jié)果相互印證，增強了實驗結(jié)論的可靠性；最后，聯(lián)合UPLC-MS/MS技術(shù)，共辨識得到包括白術(shù)內(nèi)酯II在內(nèi)的苦味關(guān)鍵質(zhì)量屬性13個，在物質(zhì)基礎(chǔ)層面表明白術(shù)組分與苦味受體TAS2R14的結(jié)合受關(guān)鍵質(zhì)量屬性驅(qū)動，為理解白術(shù)內(nèi)酯類成分苦味特性提供直接證據(jù)。結(jié)論 通過多技術(shù)融合的系統(tǒng)性研究，從分子相互作用到物質(zhì)基礎(chǔ)層面揭示了白術(shù)內(nèi)酯類成分苦味屬性的本質(zhì)，所發(fā)現(xiàn)的苦味關(guān)鍵質(zhì)量屬性為建立基于苦味特征的白術(shù)質(zhì)量控制方法奠定了理論基礎(chǔ)，此外，提出的技術(shù)路線為中藥苦味物質(zhì)基礎(chǔ)研究提供了新的方法學(xué)參考。

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

國家重點研發(fā)計劃課題（2023YFC3504505）；魯南集團橫向課題（BUCM-2021-JS-FW-173）；國家自然科學(xué)基金項目（882274110）；北京科技新星交叉項目（20230484458）