The research team, led by scientists at Fundação Ezequiel Dias and partner institutions across Minas Gerais, Brazil, set out to answer a deceptively simple question: what exactly is inside the leaves of this drought-tolerant vine, and does the chemistry change depending on where the plant grows? To find out, they cultivated P. setacea cv. BRS Pérola do Cerrado both in an open-pollinated orchard at the Universidade Federal de Viçosa and in a climate-controlled greenhouse, then harvested young fully expanded leaves, freeze-dried them, and ground them into a fine powder for extraction.

Three extraction strategies were compared: ultrasonic extraction with pure ethanol, a traditional water infusion resembling how local populations prepare medicinal teas, and exhaustive percolation with a 50 percent ethanol-water mixture over 26 days. The yields varied dramatically, from roughly 8 percent for ethanolic sonication to more than 40 percent for hydroalcoholic percolation of greenhouse-grown leaves. But yield alone told only part of the story.

When the researchers measured total phenolic content using the Folin-Ciocalteu assay and total flavonoid content using an aluminum chloride colorimetric method, a striking pattern emerged. Orchard-grown plants consistently produced extracts richer in polyphenols and flavonoids than their greenhouse-grown counterparts, regardless of extraction method. Total polyphenol content ranged from 11.32 to 40.59 milligrams of gallic acid equivalents per gram of extract, while total flavonoids ranged from 3.79 to 33.65 milligrams per gram, calculated as quercetin. The researchers attribute this difference to ultraviolet radiation, which is known to induce flavonoid biosynthesis as a protective sun-screening mechanism in plant epidermal tissues.

All six extracts showed promising antioxidant activity in a β-carotene/linoleic acid lipid peroxidation assay, with IC50 values below 60 micrograms per milliliter. The best performer was an ethanolic extract from greenhouse-grown leaves, with an IC50 of 39.71 micrograms per milliliter. However, when the team selected the hydroalcoholic orchard extract, designated PSHF-01, for deeper analysis and tested it in two additional antioxidant assays, the results were more nuanced. The extract showed no activity in the DPPH free radical scavenging assay or the TBARS liposome peroxidation assay, suggesting that its antioxidant effects are specific to protecting lipid systems from radical-mediated oxidation rather than broadly scavenging stable synthetic radicals.

This assay-dependent behavior is not a contradiction but a reflection of chemistry. The DPPH assay relies on electron transfer to a synthetic nitrogen-centered radical under non-physiological conditions, while the β-carotene bleaching method measures the ability of compounds to interrupt chain oxidation in an emulsified lipid environment. The TBARS assay, meanwhile, detects secondary products of lipid peroxidation such as malondialdehyde. Compounds that partition differently in emulsions or react slowly with DPPH can appear active in one system and silent in another, a well-documented phenomenon in natural products research.

Using thin-layer chromatography, HPLC-UV with co-injection of authentic standards, and ultimately UPLC-PDA-ESI-MS/MS tandem mass spectrometry, the team confirmed the presence of two hallmark passion fruit flavones, isovitexin and isoorientin, and tentatively identified five additional C-glycosylflavones: luteolin-(O-glucopyranosyl)-C-glucopyranoside, (iso)orientin-C-xyloside, vicenin II, (iso)vitexin-2”-O-xyloside, and (iso)orientin-C-rhamnoside. Vicenin II, an apigenin diglucoside, emerged as the likely major constituent based on its dominant mass spectral peak at m/z 593 with characteristic fragments at m/z 473 and 413.

To put a number on the lead compound, the researchers developed and validated an HPLC-DAD analytical method for quantifying isovitexin in PSHF-01. The method showed excellent linearity with a correlation coefficient of 0.9946, a limit of detection of 2.34 micrograms per milliliter, and recoveries between 107 and 116 percent, well within accepted ranges for complex plant matrices. Applying the validated method, they determined that PSHF-01 contains 0.223 milligrams of isovitexin per 100 milligrams of extract, establishing a reproducible marker for future quality control of this emerging botanical material.

Beyond the bench, the team turned to computational prediction using their in-house Active-IT system, a ligand-based virtual screening platform that combines support vector machines and Naïve Bayes classifiers trained on thousands of PubChem bioassay datasets. After rigorous internal and external validation, the models predicted that the P. setacea flavonoids possess hepatic metabolic stability and potential activity against parasitic targets including Leishmania mexicana pyruvate kinase, Trypanosoma brucei RNA editing ligase, and Plasmodium falciparum M17 leucine aminopeptidase. The predictions also flagged anti-inflammatory, antitumor, immunomodulatory, and antiviral profiles consistent with published in vitro data showing that isovitexin and isoorientin inhibit dengue virus replication and that isovitexin suppresses inflammatory signaling through the TLR4/MyD88/NF-κB pathway.

The findings position P. setacea as more than a curiosity of Brazilian biodiversity. Its combination of drought tolerance, resistance to Fusarium and anthracnose, and now a documented C-glycosylflavone-rich leaf chemistry suggests real potential as both a nutraceutical crop and a source of lead compounds for drug discovery. The researchers emphasize that the tentative compound assignments require confirmation by nuclear magnetic resonance spectroscopy and isolation of pure standards, and that future work should explore intracellular reactive oxygen species models, metabolomic profiling under different cultivation conditions, and experimental validation of the predicted molecular targets. For now, the wild vine of the Cerrado has offered chemists a rich molecular blueprint, one that bridges traditional knowledge, agricultural resilience, and modern pharmaceutical exploration in a single leaf.

Subject of Research: Flavonoid profiling and antioxidant activity of Passiflora setacea leaves

Article Title: Flavonoid profiling and antioxidant activity of Passiflora setacea DC. leaves

Article References: Flavonoid profiling and antioxidant activity of Passiflora setacea DC. leaves. (n.d.). https://doi.org/10.3389/fntpr.2026.1901598

Image Credits: AI Generated

DOI: 10.3389/fntpr.2026.1901598

Keywords: Passiflora setacea, flavonoids, isovitexin, isoorientin, vicenin II, antioxidant activity, Cerrado, HPLC-DAD, UPLC-MS/MS, in silico prediction, natural products, Brazilian biodiversity