A supramolecular deep eutectic solvent combining beta-cyclodextrin and glycolic acid substantially enhances the extraction yield, structural stability, and biological potency of propolis flavonoids compared with conventional ethanol extraction, according to research published in the Journal of Agriculture and Food Research. Propolis is a complex resinous substance gathered and manufactured by honeybees through mixing plant exudates with beeswax, functioning within hives to seal structural gaps and defend the colony against pathogens and environmental stressors. Because propolis chemical composition varies with botanical origin, geographic location, and seasonal climate, the resin has attracted considerable scientific interest for its antimicrobial, anti-inflammatory, and immunoregulatory properties. To conduct the investigation, researchers collected raw propolis samples from Henan Province in China, freeze-dried the material, sieved the resulting matter through a forty-mesh apparatus, and stored the powder at minus eighty degrees Celsius prior to extraction trials.
Among the bioactive constituents of propolis, flavonoids represent key contributors to its therapeutic potential. Characterized by a two-phenylchromone core, these polyphenolic compounds comprise subclasses including flavones, flavonols, flavanones, flavanonols, isoflavones, isoflavanones, and chalcones, with over one hundred distinct flavonoids identified in propolis. In biological systems, these molecules exhibit antioxidant, anti-inflammatory, antimicrobial, anticancer, and pancreatic lipase inhibitory activities, the latter suggesting utility in regulating dietary lipid digestion. However, industrial and laboratory isolation has traditionally relied on organic solvents like aqueous ethanol, which present low selectivity, environmental concerns, and potential toxicity. Furthermore, propolis flavonoids remain vulnerable to rapid degradation when exposed to heat, ambient light, and oxidative conditions during processing and storage. Developing sustainable extraction systems that simultaneously enhance flavonoid solubility and protect their structural integrity is therefore critical for functional food and pharmaceutical formulations.
To address these extraction limitations, deep eutectic solvents and natural deep eutectic solvents have emerged as versatile, eco-friendly alternatives. Synthesized through the complexation of hydrogen bond acceptors and hydrogen bond donors, these eutectic liquids feature low volatility, adjustable polarity, high solubilization capacity, and biodegradability. Extending this concept, supramolecular deep eutectic solvents, or SUPRADES, incorporate cyclodextrins into the solvent matrix. Cyclodextrins are cyclic oligosaccharides possessing hydrophobic central cavities capable of establishing non-covalent host-guest inclusion complexes with target polyphenols, shielding them from environmental degradation while increasing aqueous solubility. In a SUPRADES system where cyclodextrins act as hydrogen bond acceptors and organic acids serve as hydrogen bond donors, extensive hydrogen bonding occurs between the oligosaccharide hydroxyl groups and acid carboxyl groups. This architecture forms a stable, tunable liquid medium that unites eutectic solubilization with supramolecular encapsulation, permitting simultaneous extraction, molecular recognition, and stabilization of fragile propolis flavonoids.
The authors formulated four candidate SUPRADES systems by pairing alpha-cyclodextrin or beta-cyclodextrin with glycolic acid or gluconic acid at a fixed molar ratio of one to fifty. Initial extraction screening at sixty degrees Celsius, sixty minutes, thirty percent water content, and a thirty milliliter-per-gram solvent-to-solid ratio revealed marked performance differences. The beta-cyclodextrin and glycolic acid system achieved a total flavonoid yield of 115.5 ± 3.7 milligrams of rutin equivalents per gram of propolis, significantly outperforming alpha-cyclodextrin with glycolic acid at 79.7 ± 0.6 milligrams per gram, alpha-cyclodextrin with gluconic acid at 39.8 ± 0.2 milligrams per gram, and beta-cyclodextrin with gluconic acid at 39.9 ± 0.8 milligrams per gram. The researchers explained that beta-cyclodextrin possesses a hydrophobic cavity diameter of 6.0 to 6.5 angstroms, wider than the 4.7 to 5.3 angstrom cavity of alpha-cyclodextrin, thereby accommodating aromatic flavonoid rings more effectively. Furthermore, Kamlet-Taft solvatochromic profiling showed that glycolic acid systems exhibited higher polarity and hydrogen-bond acidity. Pearson correlation analysis confirmed that flavonoid yield correlated positively with solvent density (r = 0.92), polarity (r = 0.91), and hydrogen-bond acidity (r = 0.91), while correlating negatively with electrical conductivity (r = -0.91).
Structural and thermal characterization confirmed the formation of a stable supramolecular liquid held together by non-covalent interactions. Proton nuclear magnetic resonance spectra of the beta-cyclodextrin and glycolic acid SUPRADES retained the characteristic proton resonances of both pure precursors without new signals or chemical shifts, confirming that no covalent bonding alterations occurred during solvent synthesis. Fourier transform infrared spectroscopy provided evidence of intermolecular hydrogen bonding. In the SUPRADES spectrum, the hydroxyl stretching band broadened around 3400 reciprocal centimeters, while the carbonyl stretching vibration shifted from 1700 to 1723 reciprocal centimeters, indicating restructuring of the local hydrogen-bonding environment around carboxyl and hydroxyl moieties. Thermogravimetric analysis highlighted differences in thermal decomposition among the components. Pure glycolic acid underwent mass loss starting near 100 degrees Celsius and reached 98.70 percent degradation by 370 degrees Celsius, whereas beta-cyclodextrin exhibited principal decomposition between 300 and 350 degrees Celsius. In contrast, the SUPRADES demonstrated a continuous mass-loss profile totaling 94.22 percent, illustrating modified thermal behavior resulting from supramolecular assembly.
To maximize flavonoid recovery, the research team conducted systematic single-factor evaluations before deploying response surface methodology. In single-factor assays, raising extraction temperature from forty to seventy degrees Celsius enhanced flavonoid yield by accelerating molecular motion and mass transfer, while further heating to eighty degrees induced thermal degradation. Extraction time peaked at sixty minutes before protracted thermal exposure decreased total yields. Modulating water content demonstrated that dilution up to thirty weight percent significantly reduced solvent viscosity and promoted solute diffusion, though higher water fractions weakened intermolecular hydrogen bonds. Similarly, expanding the solvent-to-solid ratio up to forty milliliters per gram enhanced concentration gradients before stabilizing. Building upon these ranges, the team executed a four-variable, three-level Box-Behnken design comprising twenty-nine experimental runs. Second-order polynomial regression demonstrated strong statistical validity, yielding an F-value of 19.34 (p < 0.0001), an R-squared of 0.9508, an adjusted R-squared of 0.9017, and a non-significant lack-of-fit value (p = 0.6170). Ranking factors by F-values showed that solvent-to-solid ratio exerted the greatest impact on extraction yield, followed by extraction duration, water content, and temperature.
The multivariate optimization model predicted an optimal extraction state at 70.6 degrees Celsius, 51.8 minutes, 40.0 percent water content, and a solvent-to-solid ratio of 32.8 milliliters per gram, forecasting a total flavonoid yield of 156.50 milligrams of rutin equivalents per gram. For practical laboratory execution, parameters were adjusted to 70 degrees Celsius, 52 minutes, 40 percent water content, and 33 milliliters per gram. Empirical validation under these conditions produced an observed flavonoid yield of 158.5 ± 3.1 milligrams per gram, closely matching the predicted figure and confirming model reliability. Under identical operating parameters, conventional extraction using ethanol yielded 140.6 ± 1.7 milligrams per gram, demonstrating that the beta-cyclodextrin and glycolic acid SUPRADES delivered a 12.7 percent increase in extraction efficiency. The authors attributed this superior yield to decreased solvent viscosity at 40 percent water content, which enhanced mass transfer, combined with favorable polarity, extensive hydrogen-bonding interactions, and potential host-guest inclusion involving beta-cyclodextrin cavities that promoted flavonoid solubilization from the propolis matrix.
Chemical profiling of the extracts following AB-8 macroporous resin purification via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry identified 103 flavonoids in the SUPRADES extract and 95 in the ethanol extract. In both media, the ten most abundant flavonoids represented approximately three-quarters of total content (76.5 percent in SUPRADES and 74.6 percent in ethanol), with pinocembrin emerging as the predominant compound (13.7 percent in SUPRADES and 15.6 percent in ethanol). However, the two solvent platforms displayed distinct selectivity profiles. The SUPRADES extract contained higher relative proportions of chrysin (13.6 percent), daidzein (7.2 percent), and naringenin (6.9 percent), whereas ethanol favored biochanin A (12.4 percent) and galangin (10.8 percent). Other shared major constituents included quercetin 3,7-dimethyl ether, 7-O-methylchrysin, 3-O-methylquercetin, and 3-O-acetylpinobanksin. These compositional variations reflect distinct solvent polarities, specific hydrogen-bonding affinities, and selective host-guest interactions between cyclodextrin cavities and specific flavonoid subclasses, enabling the supramolecular solvent to target a broader diversity of therapeutic polyphenols.
Beyond extraction efficiency, the study examined flavonoid stability during storage under diverse temperature and illumination conditions over twelve-hour test periods. At an ambient temperature of 25 degrees Celsius, both SUPRADES and ethanol extracts maintained flavonoid retention above 90 percent, though SUPRADES exhibited a markedly slower degradation rate. When subjected to thermal stress at 60 degrees Celsius, degradation accelerated substantially in ethanol, whereas the SUPRADES formulation maintained flavonoid retention above 90 percent throughout the testing duration. In photostability assays, storage in complete darkness yielded retention rates exceeding 88 percent in both systems. However, under direct sunlight and indoor ambient light, the ethanol extract suffered noticeable flavonoid loss, while the SUPRADES extract demonstrated significantly superior photostability. The researchers concluded that this protective stabilization stems primarily from intermolecular hydrogen bonds shielding flavonoid polyhydroxyl groups, potential pi-pi stacking interactions among aromatic rings, and non-covalent host-guest inclusion within beta-cyclodextrin cavities that physically isolates vulnerable moieties from environmental stressors.
In functional evaluations, the SUPRADES propolis extract exhibited notable antioxidant capacity and digestive enzyme inhibition in vitro. While DPPH and ABTS radical scavenging activities were comparable to ethanol extracts, the SUPRADES extract demonstrated significantly higher ferric reducing antioxidant power and hydroxyl radical scavenging, even surpassing ascorbic acid at concentrations below 0.6 milligrams per milliliter. The pure SUPRADES blank showed negligible baseline antioxidant activity, confirming that functional performance derived from extracted flavonoids. Furthermore, the SUPRADES extract strongly inhibited pancreatic lipase, reaching an inhibition rate of 80.0 ± 2.0 percent at 1.0 milligram per milliliter and matching the commercial drug orlistat below 0.6 milligrams per milliliter. Cytocompatibility testing on L929 mouse fibroblasts revealed 94.40 ± 3.45 percent cell viability in CCK-8 assays, with Calcein-AM and propidium iodide staining confirming healthy cellular and nuclear morphology. Although these findings highlight the promise of cyclodextrin-based SUPRADES for developing flavonoid-enriched functional food ingredients, the authors emphasized that current evidence is restricted to in vitro models, noting that future research must elucidate exact molecular binding mechanisms and assess pilot-scale processing economics.
Subject of Research: Supramolecular deep eutectic solvent extraction and stabilization of propolis flavonoids
Article Title: Supramolecular deep eutectic solvents enable high-yield, stable, and bioactive flavonoid extraction from propolis
Article References: Du, T., Zheng, Q.-P., Wang, Y.-T., Sun, L.-K., Zhang, Y.-C., & Zheng, Y.-Z. (2026). Supramolecular deep eutectic solvents enable high-yield, stable, and bioactive flavonoid extraction from propolis. Journal of Agriculture and Food Research, 32, Article 103434. https://doi.org/10.1016/j.jafr.2026.103434
Image Credits: AI Generated
DOI: 10.1016/j.jafr.2026.103434
Keywords: propolis, flavonoids, supramolecular deep eutectic solvents, beta-cyclodextrin, glycolic acid, green extraction, antioxidant activity, pancreatic lipase inhibition, pinocembrin, chrysin, response surface methodology, photostability, thermal stability, solvatochromic parameters, cytocompatibility, polyphenols, food chemistry
