Dereplication of Aporphine Alkaloids by UHPLC-HR-ESI-MS/MS and NMR from Duguetia lanceolata St. -Hil (Annonaceae) and Antiparasitic Activity Evaluation

Although the genus Duguetia is well known for producing alkaloids as chemical constituents, there are no reports of alkaloids identified in the species D. lanceolata. Thus, aiming to identify the chemical composition of this species, the dereplication of alkaloidic phase was performed by use of ultra-high performance liquid chromatography high resolution electrospray ionization tandem mass spectrometry (UHPLC-HR-ESI-MS/MS) and 1H nuclear magnetic resonance (NMR). The chromatographic fractionation of the alkaloid extract from Duguetia lanceolata (Annonaceae) leaves afforded four fractions (I-IV) that were shown to be composed of aporphine alkaloids. 1H NMR analysis and UHPLC-HR-ESI-MS/MS based dereplication allowed the identification of eight alkaloids: glaucine (1), norglaucine (2), isocorydine (3), N-methyllaurotetanine (4), oxoglaucine (5), liriodenine (6), lanuginosine (7), dehydroglaucine (8). Compounds 2,3, 4,6 and 7 were described for the first time in this species, while alkaloids 1,5 and 8 are newly discovered in the genus Duguetia. Additionally, the antiparasitic activity of the four fractions was evaluated in vitro against Leishmania infantum and Trypanosoma cruzi. Fraction I, composed exclusively by 1, displayed activity against Leishmania infantum and Trypanosoma cruzi intracellular amastigotes, with half maximal inhibitory concentration (IC50) values of 7.5 and 28.6 µg mL-1, respectively. Fraction IV (constituted by 2,3 and 4) showed activity against promastigotes of Leishmania infantum with IC50 value of 50.0 µg mL-1, while fraction II (constituted by 5 and 6) showed activity against trypomastigotes of Trypanosoma cruzi, with IC50 values of 83.0 µg mL-1. This work showed that fragmentation in UHPLC-HR-ESI-MS/MS combined with 1H NMR analysis of fractions is useful for identifying alkaloids in mixtures. Additionally, it was also demonstrated the potential of aporphine alkaloids from Duguetia lanceolata St. -Hil (Annonaceae) in the search for new drug candidates for neglected diseases.

尽管番荔枝科（Annonaceae）杜克木属（Duguetia）以产出生物碱类化学成分而广为人知，但目前尚无披针叶杜克木（D. lanceolata）中鉴定出生物碱的相关报道。为此，本研究旨在明确该物种的化学成分组成，采用超高效液相色谱-高分辨电喷雾电离串联质谱（ultra-high performance liquid chromatography high resolution electrospray ionization tandem mass spectrometry, UHPLC-HR-ESI-MS/MS）与1H核磁共振（1H nuclear magnetic resonance, 1H NMR）技术对生物碱相进行了去冗余鉴定。研究人员对披针叶杜克木叶片的生物碱提取物进行色谱分级分离，得到四个组分（I~IV），经分析证实其均含有阿朴菲类生物碱（aporphine alkaloids）。通过1H NMR分析与基于UHPLC-HR-ESI-MS/MS的去冗余鉴定策略，共鉴定出8种生物碱：海罂粟碱（glaucine，1）、去甲海罂粟碱（norglaucine，2）、异紫堇定（isocorydine，3）、N-甲基劳罗坦宁（N-methyllaurotetanine，4）、氧代海罂粟碱（oxoglaucine，5）、鹅掌楸碱（liriodenine，6）、毛叶含笑碱（lanuginosine，7）与脱氢海罂粟碱（dehydroglaucine，8）。其中，化合物2、3、4、6与7为该物种中首次报道的成分，而生物碱1、5与8则为杜克木属内的新发现成分。此外，本研究还对四个组分体外抗婴儿利什曼原虫（Leishmania infantum）与克氏锥虫（Trypanosoma cruzi）的活性进行了评价。仅含有化合物1的组分I对婴儿利什曼原虫与克氏锥虫的胞内无鞭毛体均显示出活性，其半数抑制浓度（half maximal inhibitory concentration，IC50）分别为7.5 µg·mL⁻¹与28.6 µg·mL⁻¹。由化合物2、3与4组成的组分IV对婴儿利什曼原虫的前鞭毛体显示出活性，IC50值为50.0 µg·mL⁻¹；而由化合物5与6组成的组分II对克氏锥虫的锥鞭毛体具有活性，IC50值为83.0 µg·mL⁻¹。本研究表明，将UHPLC-HR-ESI-MS/MS碎片分析与组分的1H NMR分析相结合，可有效用于混合物中生物碱的鉴定。此外，本研究还证实了披针叶杜克木（Duguetia lanceolata St. -Hil，番荔枝科Annonaceae）来源的阿朴菲类生物碱在研发针对被忽视疾病的新型候选药物方面具有潜力。