Laccase Enzyme Transforms Quercetin and Caffeic Acid into More Soluble Derivatives

Both compounds are extensively studied for their antioxidant and other biological activities, yet both share a frustrating flaw: they dissolve poorly in water and are chemically unstable under certain conditions, including exposure to light, heat, or changes in pH. That combination of poor solubility and instability has long limited their use in pharmaceutical and food formulations. A new study published in Discover Chemistry reports that a common fungal enzyme may offer a way around this problem, converting the two compounds into a mixture of derivatives that dissolves in water hundreds of times more readily than quercetin itself while retaining measurable biological function.

The research team, led by Karla Verónica Teymennet-Ramírez and Iliana Barrera-Martínez, used a laccase from the white-rot fungus Trametes versicolor to oxidize quercetin and caffeic acid simultaneously in an aqueous buffer. Laccases are multicopper oxidases, enzymes that use molecular oxygen to strip single electrons from phenolic compounds, generating radical intermediates that can couple with one another. The reactions were run under deliberately mild conditions, at pH 4.5 and 30 degrees Celsius, with the enzyme added at 0.05 units per milliliter and the mixture incubated for 24 hours.

Analysis by ultraviolet-visible spectroscopy and ultra-high-performance liquid chromatography coupled to mass spectrometry revealed five major products in the reaction mixture. Three of these were oxidized quercetin derivatives, described by the authors as supramolecular pairs, including structures stabilized by incorporated methanol molecules and one methoxylated variant. The remaining two products arose from caffeic acid chemistry: one was tentatively identified as vanillic acid, based on absorption maxima at 258 and 293 nanometers and a molecular ion at mass-to-charge ratio 167 with a characteristic carbon dioxide loss, and the other appeared to be a genuine coupling product linking quercetin and caffeic acid fragments, showing a molecular ion at m/z 479 with fragment ions attributable to each parent compound.

The reaction showed a clear preference for one substrate over the other. Approximately 97 percent of the quercetin was oxidized over the course of the reaction, while only about 57 percent of the caffeic acid was consumed, leaving 42.6 percent unreacted as determined against calibration curves of authentic standards. Quercetin was therefore the limiting substrate. Time-course monitoring showed that oxidation proceeded rapidly during the first six hours, with a marked drop in absorbance at 375 nanometers, and changed little between 6 and 24 hours, indicating the reaction had essentially reached completion. Products 1 and 5 peaked in abundance after three hours, whereas products 2, 3, and 4 accumulated progressively up to the 24-hour endpoint.

An intriguing detail emerged from experiments in which the order of substrate addition was varied. When quercetin was pre-incubated with the laccase for an hour before caffeic acid was introduced, the resulting spectrum closely matched that of the simultaneous-addition reaction. But when caffeic acid went in first, the product profile shifted, with a weaker band near 300 nanometers, likely because caffeic acid rapidly oxidized and homopolymerized on its own, limiting its opportunity to interact with quercetin. The team therefore standardized on simultaneous addition for all subsequent experiments. Notably, small amounts of products 1 through 4 also appeared in enzyme-free controls, pointing to some non-enzymatic oxidation, but their abundance rose substantially with laccase present, and product 5 was detected only after enzyme addition, supporting its assignment to the enzymatic pathway.

The most striking result concerned water solubility, measured by a gravimetric method in which samples were equilibrated in distilled water for 48 hours and the undissolved residue weighed after drying. Untreated quercetin dissolved at only about 0.14 milligrams per milliliter, while caffeic acid reached 2.07 milligrams per milliliter. The reaction product mixture, designated RPQ+CA, achieved 94.7 milligrams per milliliter, roughly a 677-fold increase over quercetin and a 45.8-fold increase over caffeic acid. The authors attribute this dramatic gain to structural modifications introduced during oxidation, which alter the polarity and intermolecular interactions of the derivatives, including extensive hydrogen-bonding networks in the supramolecular structures.

Stability under ultraviolet light also improved. When samples were exposed to 254-nanometer UV-C radiation, a relatively high-energy condition, untreated quercetin retained 87.3 percent of its spectral area after 24 hours and fell to 65.3 percent after 48 hours, while caffeic acid dropped to 67.4 percent. The RPQ+CA mixture fared considerably better, retaining 97.6 percent after 24 hours and 86.5 percent after 48 hours, a spectral loss approximately 2.6-fold lower than quercetin and 2.4-fold lower than caffeic acid. The authors suggest this photostability may stem from modification of quercetin’s photolabile C2=C3–C4=O system and free hydroxyl groups, extended electron delocalization, and supramolecular stabilization, though they caution that further photochemical studies would be needed to confirm the mechanism.

Functionality came with a trade-off. In the ABTS radical-scavenging assay, expressed as Trolox equivalent antioxidant capacity, the unmodified compound mixture scored 208.1 milligrams per liter Trolox, while the enzymatic products scored 165.6, a reduction of about 20 percent. The authors attribute this to oxidative coupling consuming phenolic hydroxyl groups, the hydrogen- and electron-donating sites responsible for radical scavenging. They frame the outcome as a balance: a partial loss of radical-scavenging capacity exchanged for substantially better solubility and UV stability, with considerable antioxidant activity nonetheless retained.

Biological testing added a further layer of nuance. In cell viability assays using flow cytometry with propidium iodide and thiazole orange staining, both the untreated mixture and the enzymatic products showed low toxicity toward VERO cells, a non-malignant reference line, maintaining viability above 70 percent at all tested concentrations. Against HeLa cervical cancer cells, however, both treatments reduced viability in a dose-dependent manner, with complete inhibition at the highest concentrations. Probit regression analysis yielded a half-maximal inhibitory concentration of 5.03 micrograms per milliliter for the untreated mixture and 12.02 micrograms per milliliter for the enzymatic products, meaning the oxidized mixture was about 2.4-fold less potent on a total sample-mass basis but still retained substantial cytotoxic activity. The authors note that the mixture contains residual parent compounds as well as derivatives, so contributions from each component cannot yet be separated, and they emphasize that VERO cells are not a substitute for normal human cell lines in future work.

The study demonstrates that laccase-catalyzed co-oxidation of mixed phenolic substrates can generate hybrid and supramolecular products with materially altered physicochemical properties, extending prior work that focused mostly on single-substrate oxidation into homodimers and homopolymers. The authors also acknowledge practical hurdles before any application: enzyme cost and recycling, product recovery and purification, and optimization at larger scale. Although the reaction was scaled to 100 milliliters to verify consistency and produce material for the biological assays, a full techno-economic analysis of feasibility remains to be done. For now, the work stands as a proof of concept that a single mild enzymatic step can reshape the solubility, stability, and functional profile of two well-known dietary polyphenols at once.

Subject of Research: Laccase-catalyzed co-transformation of quercetin and caffeic acid into soluble phenolic derivatives

Article Title: Characterization and functional evaluation of laccase-catalyzed synthesis of quercetin and caffeic acid derivatives

Article References: Teymennet-Ramírez, K. V., Casas-Godoy, L., Pacheco-López, N. A., Herrera-Pool, I. E., Flores-Hernández, F. Y., Cerón-Camacho, R., & Barrera-Martínez, I. (2026). Characterization and functional evaluation of laccase-catalyzed synthesis of quercetin and caffeic acid derivatives. Discover Chemistry, 3(1), Article 594. https://doi.org/10.1007/s44371-026-01016-6

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01016-6

Keywords: laccase, quercetin, caffeic acid, Trametes versicolor, enzymatic oxidation, polyphenols, aqueous solubility, UV stability, antioxidant activity, HeLa cells, biocatalysis, phenolic derivatives