本研究将特征分子网络（FBMN）、SIRIUS机器学习辅助结构注释与高速逆流色谱相结合,探索了FBMN与SIRIUS联合分析在虎皮楠生物碱（DAs）发现中的应用。利用FBMN技术对三种虎皮楠属植物进行化学分析,并结合SIRIUS平台对MS/MS特征进行系统注释,筛选出脉叶虎皮楠（Daphniphyllum paxianum）作为研究对象,其蕴含新颖且丰富DAs的潜力最大。进一步采用低损耗分离策略（高速逆流色谱、凝胶色谱和高效液相色谱）,结合多种波谱解析方法以及基于量子力学的计算方法,从中分离并鉴定了13个DAs,包括3个新化合物（1-3）和10个已知化合物（4-13）。其中,化合物1和2分别属于calyciphylline A型和daphnilactone B型DAs,均含有氮杂双环[3.3.1]壬烷结构单元,并且化合物2在C-2位发生了罕见的氧化修饰。化合物3则属于罕见的daphnezomine F型DA,其核心为氮杂双环[5.2.2]十一烷环系,丰富了该类骨架的结构多样性。抗疟疾活性结果显示化合物4可抑制氯喹耐药株Dd2（Plasmodium falciparum Dd2）生长,IC50为27.7 ± 3.7 μM。该研究表明,以FBMN与SIRIUS联合导向为基础,辅以低损耗分离手段,为从复杂天然产物中高效、精准发现结构新颖且具有生物活性的DAs提供了有效途径。
To accelerate the discovery of novel daphniphyllum alkaloids (DAs), an integrated workflow was developed that synergistically integrates feature-based molecular networking (FBMN), machine learning annotation powered by SIRIUS, and high-speed countercurrent chromatography (HSCCC) directed low-sample-loss separation. FBMN analysis was first performed on three Daphniphyllum species to profile their chemical space, leading to the selection of D. paxianum as the most promising source. Subsequent systematic annotation of MS/MS features using the SIRIUS platform predicted multiple known and potentially novel DAs, providing valuable guidance for targeted isolation. Guided by the FBMN and SIRIUS annotation results, a low-sample-loss separation protocol was subsequently implemented, which combined HSCCC, Sephadex LH-20 gel chromatography, and preparative HPLC. The targeted separation approach, in conjunction with comprehensive spectroscopic analysis (IR, HRESIMS, 1D and 2D NMR) and quantum-mechanical computational methods, enabled the efficient isolation and structural elucidation of 13 DAs from D. paxianum, of which three were identified as new compounds (1-3) with various skeletons, along with ten known analogues (4-13). Compounds 1 and 2 belong to the calyciphylline A-type and daphnilactone B-type DAs, respectively, both featuring a 2-azabicyclo[3.3.1]nonane core, with compound 2 characterized by rare C-2 oxidation. Compound 3 is a rare daphnezomine F-type DA with a 2-azabicyclo[5.2.2]undecane core, enriching the structural diversity of DAs. Evaluation of the antiplasmodial activity using SYBR Green Ⅰ method revealed that compound 4 inhibited the growth of the chloroquine-resistant P. falciparum Dd2 strain with a moderate IC50 of 27.7 ± 3.7 μM. Concurrent biocompatibility assessment demonstrated that compound 4 was devoid of detectable cytotoxicity against A549 and HeLa cell lines (cell viability consistently exceeding 75% at 10 μM) and exhibited negligible hemolytic activity (hemolysis rate < 5% at 25 μM). The unity of moderate antiplasmodial efficacy and excellent safety profile highlights compound 4 as a promising candidate for further structural optimization and mechanistic investigation as an antiplasmodial candidate compound. The research demonstrated that the FBMN and SIRIUS dual-guidance strategy, in combination with low-sample-loss separation, constitutes a powerful and efficient approach for the precise discovery of novel and bioactive DAs from complex natural materials.
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