Triterpenoid saponins from the aerial parts of Phytolacca americana and their anti-influenza A virus activity

Abstract

Phytolacca americana L. (Phytolaccaceae), a perennial species native to North America, is rich in oleanane-type triterpenoid saponins. In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC–QTOF–MS) profiling of the aerial-part extract revealed characteristic fragment ions of phytolaccagenin- and phytolaccagenic acid-type saponins with diverse glycosylation. MS-guided annotation enabled targeted isolation and structural elucidation of two previously undescribed compounds, one oleanane-type triterpenoid saponin (1) and one neolignan (3). Additionally, compound 2, which had been reported only based on mass spectrometric data, was isolated, and its structure was fully elucidated for the first time, together with those of 10 known oleanane-type triterpenoid saponins (4–13). Their structures were elucidated by comprehensive one-dimensional and two-dimensional nuclear magnetic resonance spectroscopic analyses combined with UPLC–QTOF–MS data. Although P. americana extracts have been reported to inhibit influenza A virus infection in previous studies, the bioactive constituents responsible for this antiviral activity have not been identified. The crude extract and isolated saponins were evaluated using complementary in vitro assays. In H1N1-infected A549 cells, compound 11 (3-O-trisaccharide phytolaccagenic acid) reduced viral NS1 RNA levels at 6 and 24 hpi as determined by RT–qPCR. Antiviral activity was further confirmed by plaque reduction and immunoblot analyses, with a plaque-assay IC50 of 57.98 ± 0.26 µM.