Comprehensive phytochemical study of Galianthe thalictroides: isolation of three new compounds, structural revision of β-carbolines, and evaluation of cytotoxic and anti-wnt activities

Introduction The medicinal plant G. thalictroides (K. Schum.) E. L. Cabral has been traditionally used in Midwestern Brazil for cancer treatment and prevention. Previous studies demonstrated that the chloroform fraction of its roots exhibits significant cytotoxic activity against cancer cell lines. Phytochemical investigation of this fraction led to the isolation of four cytotoxic compounds, including two β-carboline alkaloids and two cyclopeptides, supporting the traditional use of this species. However, several minor metabolites detected in this active fraction were not obtained in sufficient quantities for complete structural elucidation or biological evaluation. In this context, the present study aimed to expand the chemical characterization of Galianthe thalictroides , with a particular focus on previously unresolved metabolites. Additionally, the study sought to explore other extracts and preparations, including the traditional decoction, to provide a more comprehensive understanding of the phytochemical profile and its potential biological relevance. Methods The roots of G. thalictroides were extracted with ethanol and a traditional water decoction was prepared in parallel. The ethanolic extract was subjected to liquid/liquid partitioning with hexane, chloroform, and ethyl acetate. The extracts, fractions, and traditional decoction were analyzed by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS) to establish a comprehensive metabolite profile and generate a molecular networking (MN). The chloroform fraction was purified using medium-pressure liquid chromatography (MPLC-UV) followed by semi-preparative HPLC. Structural characterization of the isolated compounds was achieved through UV, Electronic Circular Dichroism, Nuclear Magnetic Resonance, and High-Resolution Mass Spectrometry data. The chemical shifts and coupling constants of β-carboline derivatives were calculated using the CT web application, which performs a complete Quantum Mechanical Spectral Analysis. Finally, the isolated compounds were evaluated for activity in a dose-dependent manner using BT-20 triple-negative breast cancer cells. This was conducted to determine their cytotoxic potencies and specific Wnt-inhibitory activities via a TopFlash reporter assay. Results and Discussion Using this approach, 26 compounds were isolated from the chloroform fraction and fully characterized. Notably, this study led to the discovery of two new cyclopeptides and one new β-carboline alkaloid, which represent the most significant and original contributions of this work. In addition, the structures of two previously reported β-carboline alkaloids from this species were revised. Molecular networking–guided MS profiling of the extracts, fractions, and traditional decoction enabled the annotation of 13 additional metabolites, thereby expanding the known chemical diversity of G. thalictroides . Biological evaluation confirmed the cytotoxic activity of known compounds and revealed new active analogues. Bouvardins exhibited strong cytotoxicity (IC 50 0.25–14.00 µM), while anthraquinones and β-carboline alkaloids showed moderate activity (IC 50 23–110 μM and 65–193 μM, respectively). Mechanistic studies revealed that β-carboline derivative 24 selectively inhibited the Wnt signaling pathway (IC 50 ≈ 40 µM), whereas its analogue ( 25 ) was inactive. Conclusion Overall, this study significantly advances the chemical and biological understanding of G. thalictroides by integrating compound isolation, structural characterization, and biological evaluation. The discovery of novel bioactive compounds, together with evidence of pathway-specific effects, highlights the plant’s potential as a source of structurally diverse and pharmacologically relevant metabolites for anticancer research.