The plant, Ferdinandusa hirsuta Standl., belongs to the coffee family, Rubiaceae, and sits within the subfamily Ixoroideae. Prior work on related Ferdinandusa species had reported antifungal, antimycobacterial, antioxidant, and toxicity effects against the brine shrimp Artemia salina, but the compounds responsible for those activities had never been identified. The new study, led by Gabriel Santana Crispim of the National Institute for Amazonian Research (INPA) and colleagues, set out to close that gap by combining chemical annotation with biological testing.

The researchers collected leaves and branches on April 18, 2023, dried and ground them, and extracted each part successively with hexane and methanol using ultrasound-assisted maceration. The yields were modest: 3.36 grams of hexane extract from 100.04 grams of leaves and 6.46 grams from 1,152.4 grams of branches, along with 20.25 grams and 19.74 grams of the corresponding methanolic extracts. Because fatty acids and other nonpolar constituents can swamp mass spectrometry signals, the methanolic extracts were first cleaned up on C18 solid-phase extraction cartridges before analysis.

The chemical analysis relied on liquid chromatography coupled to high-resolution mass spectrometry with electrospray ionization in both positive and negative modes. Fragmentation spectra were converted to mzML format and submitted to the GNPS2 platform, which builds molecular networks by clustering spectra with similar fragmentation patterns and matching them against spectral libraries. Crucially, the team did not stop at library matches. They manually verified the fragmentation patterns of each annotation, checking for diagnostic neutral losses such as 162 daltons from a glucosyl moiety, 18 daltons from water, and 44 or 28 daltons from carboxyl or carbonyl groups, as well as retro-Diels-Alder rearrangements characteristic of the secoiridoid skeleton.

Five secoiridoids were putatively identified: secologanoside, secologanin, sweroside, epivogeloside, and swertiamarin, along with one phenolic compound, caffeic acid. Secoiridoids were predominantly annotated in the methanolic extract from leaves, although sweroside and caffeic acid were also detected in the branch extract. The annotations remain tentative, based on fragmentation patterns and library matches rather than isolated, purified compounds, and the authors note that isolation and nuclear magnetic resonance analyses would be needed to confirm the structures definitively. Still, the chemotaxonomic picture is consistent: iridoids are considered important chemical markers of the Ixoroideae, and secoiridoids have been reported in other members of the tribe Condamineeae, to which Ferdinandusa belongs.

One particularly intriguing finding came from the molecular network itself. The epivogeloside annotation at m/z 389.148 had an analog in the network at m/z 375.133, differing by 14 daltons, which could correspond to a hydroxyl group instead of a methoxy group at position 7. Searches of CAS SciFinder and PubChem turned up no secoiridoid with that exact structure, suggesting that an unknown compound may be present in the extract and that isolation work could characterize it. The network also grouped secologanin, sweroside, and swertiamarin into a single molecular family based on their shared neutral losses, and sweroside and swertiamarin have previously been reported in Swertia species of the Gentianaceae, a family with taxonomic affinities to Rubiaceae.

On the biological side, the antibacterial results were modest. Tested at 1,000 micrograms per milliliter against twelve clinically significant bacterial strains, including Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli, the methanolic leaf extract showed inhibition of only about 21 to 30 percent against Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli, while the hexane extracts showed activity only against Staphylococcus aureus, at roughly 25 to 31 percent. Activity below 20 percent was considered inactive. The positive control, oxytetracycline at 125 micrograms per milliliter, inhibited the same strains by 68 to 100 percent. The authors suggest that the low but detectable activity of the methanolic leaf extract could be related to sweroside and swertiamarin, which have been reported elsewhere to inhibit C. freundii, E. coli, and other pathogenic bacteria, though the compounds themselves were not isolated and tested in this study.

The cytotoxicity results were more striking. Using an MTT assay, the team tested the methanolic extracts against four human tumor cell lines: MDA-MB-231 triple-negative breast cancer cells, HL60 acute promyelocytic leukemia cells, K562 chronic myeloid leukemia cells, and CCRF-CEM acute lymphoblastic leukemia cells, with concentrations ranging from 3 to 100 micrograms per milliliter over 24, 48, and 72 hours. The branch methanolic extract showed an IC50 of 30.06 micrograms per milliliter against CCRF-CEM at 48 hours, while the leaf extract required 58.14 micrograms per milliliter at 72 hours for the same line. Against MDA-MB-231 cells, the branch extract reached an IC50 of 48.76 micrograms per milliliter at 72 hours and the leaf extract 41.01 micrograms per milliliter. Both extracts were less potent against HL60 and K562 cells, with significant inhibition occurring mainly at higher concentrations.

Perhaps most importantly, the same statistical models confirmed no significant toxicity toward human peripheral blood mononuclear cells or Vero monkey kidney epithelial cells, even at the highest concentration tested, 100 micrograms per milliliter. Non-linear regression fits for the active models showed R-squared values above 0.95 with steep HillSlopes, indicating a sharp drop in viability once threshold concentrations were reached. The authors point to sweroside, the only secoiridoid annotated in the branch extract, as a possible major contributor to the branch extract’s potency, noting that sweroside has previously been reported to suppress nuclear factor-kappa B signaling in ways relevant to cell proliferation and apoptosis. By contrast, the leaf extract contained secologanoside, secologanin, epivogeloside, and swertiamarin, compounds the literature describes as having more restricted or moderate cytotoxic activity, which the authors suggest may explain the leaf extract’s lower potency against the leukemia models.

The study has clear limitations. The compound annotations are putative, resting on mass spectrometry fragmentation and molecular networking rather than on isolated, structurally confirmed molecules, and the biological activities were measured in cultured cells rather than in animal models or patients. The cytotoxicity findings are in vitro observations and do not establish any therapeutic benefit. Nevertheless, the work establishes a chemical baseline for a genus that had been almost entirely unexplored phytochemically, provides the first report of secoiridoids in Ferdinandusa, and identifies specific extracts and candidate compounds for future isolation, structural characterization, and biological evaluation. The authors frame the results as a foundation for further chemical investigation of F. hirsuta and its relatives, whose bioactive constituents have remained unknown despite decades of reported biological activity.

Subject of Research: First chemical annotation of secoiridoids in Ferdinandusa hirsuta and evaluation of antibacterial and cytotoxic activity of its extracts

Article Title: Annotation of secoiridoids in bioactive extracts of Ferdinandusa hirsuta Standl. (Rubiaceae) using untargeted LC-MS/MS based molecular network

Article References: Crispim, G. S., Moreira, L. D. C., Azevedo, R. G. D., Sousa, F. J. R. D., Pontes, G. S., & Nunez, C. V. (2026). Annotation of secoiridoids in bioactive extracts of Ferdinandusa hirsuta Standl. (Rubiaceae) using untargeted LC-MS/MS based molecular network. Frontiers in Natural Products, 5, Article 1906976. https://doi.org/10.3389/fntpr.2026.1906976

Image Credits: AI Generated

DOI: 10.3389/fntpr.2026.1906976

Keywords: Ferdinandusa hirsuta, secoiridoids, Rubiaceae, LC-MS/MS, molecular networking, GNPS2, cytotoxicity, antibacterial activity, Amazonian plants, natural products, sweroside, triple-negative breast cancer