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. 2026 Jan 5;11(2):3264–3272. doi: 10.1021/acsomega.5c10089

Creation and Functional Evaluation of a Novel Antioxidant Compound via UV-Induced Structural Transformation of Epicatechin from White Wine Pomace

Mana Tsukada †,‡,*, Yuki Odanaka §, Yuka Koike ∥, Masaya Fujishiro ⊥, Takehiko Sambe †, Kiyoshi Fukuhara #
PMCID: PMC12824729  PMID: 41585642

Abstract

Plant-derived polyphenols, particularly flavonoids, have attracted considerable attention for their antioxidant activity and their potential to prevent aging and lifestyle-related diseases. White wine pomace, a winemaking byproduct, contains abundant nonpigmented polyphenols; however, most remain unutilized and are typically discarded. Herein, epicatechin (EC), a major flavonoid in white wine pomace, was irradiated with ultraviolet (UV) light at 302 nm, producing a novel compound, designated as compound X. Compound X was isolated and purified by preparative high-performance liquid chromatography (HPLC). Structural characterization using nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and mass spectrometry (MS) revealed a bicyclo[2.2.2]­octane-type skeleton featuring an intramolecular C–C bond between the A and B rings and a ketone group at the C-5 position. Electron spin resonance (ESR) analysis showed that compound X had substantially stronger hydroxyl radical (·OH)–scavenging activity than epigallocatechin gallate (EGCg, IC50 = 395.9 μM), vitamin C (IC50 = 1327 μM), Trolox (IC50 = 1436 μM), and EC (IC50 = 2755 μM), with an IC50 of 71.42 μM. In all five pomace samples tested, a peak corresponding to compound X (m/z 289.0712) was observed following UV irradiation, confirming conversion of flavan-3-ols such as EC into compound X. These results highlight the potential to generate high-value bioactive compounds from white wine pomace via photoinduced structural modification, offering a sustainable approach for valorizing underutilized agro-industrial resources.

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1. Introduction

Plant-derived polyphenols, particularly flavonoids, have gained considerable attention in the food, pharmaceutical, and cosmetic industries because of their antioxidant activity and their potential to prevent aging and lifestyle-related diseases. Among these, grape-derived polyphenols are noted for their strong antioxidant capacity and their potential as functional materials. ,

Approximately 70 million tons of grapes are produced annually worldwide, generating substantial pomaceprimarily seeds, skins, and stemsduring winemaking. Approximately 70% of grape-derived polyphenols remain in the pomace rather than being transferred to the wine. Grape seeds are rich in flavan-3-ols, proanthocyanidins, and phenolic acids, which display diverse bioactivities, including antioxidant, anti-inflammatory, anticancer, antidiabetic, and cardioprotective effects. White grape seeds contain high levels of catechins and proanthocyanidins, contributing to their high total phenolic content.

The polyphenolic composition of pomace varies with grape cultivar and winemaking methods. Red wine pomace contains high levels of anthocyanins, with malvidin-3-O-glucoside often predominant. Conversely, white wine pomace is enriched in nonpigmented polyphenols, including flavonols (e.g., quercetin, myricetin), flavan-3-ols (e.g., catechin, procyanidins), and phenolic acids (e.g., hydroxycinnamic acids). − Numerous studies have validated that these compounds are retained in white pomace after vinification, supporting its strong antioxidant potential. , Comparative studies on Chilean cultivars also indicate that Sauvignon Blanc and Chardonnay (white pomace) exhibit higher antioxidant activity, total phenolic content, and proanthocyanidin levels than Cabernet Sauvignon and Carménère (red pomace), likely reflecting their abundance of nonpigmented polyphenols.

Despite its high polyphenol content, white wine pomace remains underutilized. Disposal requires considerable energy and releases substantial CO2 during incineration. Effective use of this resource could reduce food waste, promote resource recycling, and advance sustainability.

Recent studies have increasingly examined physical or chemical modification of natural compounds to enhance or introduce new functions. In pharmaceutical development, unmodified natural compounds are seldom used directly; semisynthetic or fully synthetic derivatives are typically employed to improve bioactivity, selectivity, and pharmacokinetic properties. Yao et al. reported that structural modifications can preserve the original active scaffold while enhancing solubility, stability, and bioactivity, including antitumor effects, through approaches such as semisynthesis and prodrug design. Similarly, Atanasov et al. emphasized that structural transformation and synthetic strategies are central to optimizing pharmacokinetic profiles and selectivity of natural product derivatives.

Polyphenols, which contain multiple hydroxyl groups, are prone to structural changes under external energy sources such as heat or light, altering their stability and biological activity. , Cao et al. demonstrated that hydroxyl group number strongly influences polyphenol stability, with external stress facilitating structural transformations. Dall’Acqua et al. reported that polyphenols with more hydroxyl groups undergo structural modifications more readily during thermal or photonic exposure.

To date, no study has reported the ultraviolet (UV) irradiation–induced formation of a bicyclo[2.2.2]­octane-type structure from epicatechin (EC) or the generation of a structurally rigid polyphenolic compound with enhanced antioxidant activity originating from EC in white wine pomace. Based on these insights, the present study investigated the effects of UV irradiation on the antioxidant activity of white wine pomace. EC, a representative flavonoid abundant in white wine pomace, was selected as a model compound because of its multiple phenolic hydroxyl groups and strong propensity to absorb UV light and undergo photoinduced reactions. The study assessed changes in antioxidant activity following UV irradiation, isolated and structurally characterized UV-induced products using spectroscopic and analytical techniques, and focused on the formation of a novel bicyclo[2.2.2]­octane-type compound. The antioxidant activity of the newly generated compound was compared with that of known phenolic antioxidants, and its formation in UV-irradiated white wine pomace samples was confirmed.

This work addresses two main objectives: valorization of underutilized white wine pomace and generation of structurally novel functional materials through photoinduced modification of natural products, thereby contributing to the sustainable development of high-value bioactive resources.

2. Results

2.1. Effect of UV Irradiation on the Antioxidant Activity of White Wine Pomace Extracts

Aqueous extracts from five white wine pomace samples were irradiated with UV light, and·OH–scavenging activity was measured (Figure ). UV treatment produced a marked increase in antioxidant activity in all samples.

1.

1

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·OH–scavenging activity of white wine pomace extracts prior (black bars) and post (gray bars) UV irradiation. Data are presented as mean ± SD. Statistical significance was assessed using two-way ANOVA; ns ****p < 0.0001.

Two-way analysis of variance (ANOVA) showed that both sample type (row factor) and treatment condition (column factor) significantly influenced antioxidant activity (p = 0.02 and p < 0.0001, respectively), accounting for 4.17% and 85.34% of the total variance. The predicted least-squares means (LS means) were 23.65% for the control group and 41.95% for the UV-irradiated group, with a statistically significant difference of −18.30% ± 1.10 (95% CI, −20.54 to −16.06).

These findings show that UV irradiation significantly enhances the·OH–scavenging activity of white wine pomace extracts, indicating a strong photochemical effect on their antioxidant properties.

UV: ultraviolet; ANOVA: analysis of variance; ns: not significant.

2.2. Isolation, Purification, and Structural Analysis of UV-Induced Products from EC

To identify photochemically derived transformation products, six flavan-3-ols commonly present in white wine pomace were subjected to UV irradiation. Among these, catechin (Cat) and (−)-epicatechin (EC) generated a distinct new signal in the extracted-ion chromatogram at m/z 289.0712 (t_R = 3.52 min; Figure ), which was not observed in the unirradiated controls. Because the EC-derived peak was more pronounced and chemically well-resolved, EC was selected as the substrate for preparative-scale irradiation and isolation of the corresponding product (compound X).

2.

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Mass chromatograms (m/z 289.0712) of flavan-3-ol standards (Cat, EC, EGC, EGCg, procyanidin B2, and procyanidin C1) before (left) and after (right) UV irradiation. Newly formed peaks corresponding to compound X are indicated by arrows. Cat: (+)-Catechin; EC: (−)-Epicatechin: EGC: (−)-Epigallocatechin; EGCg: (−)-Epigallocatechin gallate; Dimer: Procyanidin B2; Trimer: Procyanidin C1.

For isolation, EC (580 mg) was dissolved in H2O (2.0 L) and irradiated for 24 h, after which the reaction mixture was lyophilized, dissolved in acetonitrile containing 0.1% formic acid, and purified by preparative HPLC to afford compound X (tR = 19.8 min). A typical run furnished 2.6 mg (0.44%) of X, and across independent preparations the isolated yield averaged 0.58% ± 0.14%. This protocol was repeated to prepare sufficient material for antioxidant assays and spectroscopic characterization.

Figure presents the chromatographic profiles of Cat, EC, and other flavan-3-ols following 24 h of UV irradiation. The relative peak area of compound X increased with irradiation time and reached a maximum at 24 h, concomitant with depletion of the EC peak. Prolonged exposure beyond 24 h did not increase the formation of X and instead resulted in a gradual decline, suggesting that X undergoes secondary photodegradation or overoxidation under excessive UV exposure. Thus, 24 h was identified as the optimal irradiation time for maximizing the accumulation of X under the present conditions.

It should be noted that the values reported above represent isolated yields after multistep purification rather than the true conversion efficiency of the reaction mixture. Given the high redox activity and apparent chemical lability of X, partial degradation during purification is likely, which may have contributed to the modest isolated yield.

2.3. Structural Elucidation of Compound X

The structure of the UV-irradiated EC product was elucidated using nuclear magnetic resonance (NMR) and infrared spectroscopy (IR) spectroscopy (Figure ). The 1H NMR spectrum showed two oxygenated sp3 methine protons, four aromatic (sp2) methine protons, and one methylene group. The 13C NMR spectrum displayed 15 distinct carbon resonances, assigned with the aid of heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple–bond correlation (HMBC) spectra as follows: ten sp2 (aromatic or olefinic) carbons, including four oxygenated quaternary carbons; two oxygenated sp3 methine carbons; one sp3 quaternary carbon; one sp3 methylene carbon; and one carbonyl carbon. The 1H–1H COSY spectrum indicated a partial fragment comprising C-2, C-3, and C-4. Each ring unit was further characterized based on key HMBC correlations. For the A-ring, correlations from H-6 to C-8 and C-10 and from H-8 to C-6 and C-10 were observed. For the B-ring, correlations from H-2′ to C-2, C-1′, and C-3′, and from H-5′ to C-10, C-1′, C-4′, and C-6′ supported the proposed substitution pattern. For the C-ring, correlations from H-2 to C-3, C-4, C-1′, C-2′, and C-6′; from H-3 to C-2 and C-4; and from H-4 to C-2, C-3, C-10, and C-1′ were consistent with intramolecular connectivity. The resonance at 51.2 ppm in the 13C NMR spectrum was assigned to C-10 based on HMBC correlations from H-6 and H-8. Additional correlations from H-5′ to C-6′ and C-10 indicated the formation of a new intramolecular C–C bond between C-10 of the A-ring and C-6′ of the B-ring, generating a bicyclo[2.2.2]­octane-type fused framework.

3.

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Proposed structure of compound X generated from UV-irradiated EC. Both structural representations are equivalent (≡). The right-hand structure shows the standard representation of compound X, while the left-hand structure is based on the catechin skeleton to illustrate the formation of the intramolecular C–C bond between C-10 of the A-ring and C-6′ of the B-ring during the photoinduced transformation. UV: ultraviolet; EC: epicatechin.

The IR spectrum showed absorption bands corresponding to hydroxyl and carbonyl stretching vibrations. The absence of resonances downfield of 200 ppm in the 13C NMR spectrum excluded nonconjugated carbonyls (e.g., aldehydes or simple ketones). Instead, the observed carbonyl was consistent with a conjugated phenolic oxidation product. Carbon shifts for C-3′ and C-4′ (∼145 ppm) indicated intact phenolic groups, excluding oxidation at these positions. Consequently, the carbonyl was attributed to oxidation of a hydroxyl group at either C-5 or C-7 of the A-ring.

Upfield shifts of H-4 and H-5′ relative to EC were consistent with a shielding effect consistent with carbonyl introduction at C-5. Taken together, the spectroscopic data support the presence of a carbonyl at C-5.

Overall, the UV photoproduct of EC was shown to possess a bicyclo[2.2.2]­octane-type framework formed via an intramolecular C–C bond between C-10 of the A-ring and C-6′ of the B-ring, with concurrent oxidation at C-5 yielding a ketone. The UV–Vis spectrum of compound X, reflecting electronic changes associated with the rigidified core, is provided in the Supporting Information (Figure S1).

2.4. Comparative Antioxidant Activity of Compound X and Known Phenolic Antioxidants

·OH–scavenging activity was evaluated by electron spin resonance (ESR) spectroscopy in a concentration-dependent, logarithmic manner. Compound X exhibited more than 50% radical–scavenging activity at concentrations approximately 1 order of magnitude lower than those required for vitamin E (VE) and EC, indicating significantly higher antioxidant potential (Table ). Remarkably, at a low concentration of 1 μM, compound X retained more than 20% scavenging activity, with a steep concentration–response curve.

1. IC50 Values and·OH–Scavenging Activities of Compound X and Reference Antioxidants .

compound name IC50 (μM)
compound X 72.41
epigallocatechin gallate (EGCg) 395.9*
epicatechin (EC) 2755*
ascorbic acid (vitamin C, VC) 1327*
Trolox (water-soluble vitamin E analogue) 1436*
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a

Data were derived from nonlinear regression analysis. *p < 0.05 versus compound X (extra sum-of-squares F test).

In comparison, EGCg demonstrated a similar low-concentration scavenging profile (Figure ).

4.

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Concentration–dependent·OH–scavenging activity of compound X compared with reference antioxidants: EC, EGCg, ascorbic acid (vitamin C, VC), and Trolox (water-soluble vitamin E analogue). Data were analyzed by nonlinear regression with a four-parameter logistic model to determine IC50 values. The red dashed line indicates 50% scavenging activity. EC: epicatechin; EGCg: epigallocatechin gallate.

Nonlinear regression analysis of the concentration-dependent inhibition curves showed that compound X had the lowest half-maximal inhibitory concentration (IC50) among all tested compounds (72.41 μM), approximately 5-fold lower than that of EGCg (395.9 μM), indicating the highest radical–scavenging potency (Figure ). Extra sum-of-squares F-tests performed in GraphPad Prism demonstrated that the inhibition curve of compound X was significantly different from those of EGCg, Trolox, VC, and EC (p < 0.05), confirming its superior activity. The 95% confidence interval (CI) for the IC50 of compound X was the narrowest among all samples, reflecting high precision in the estimation.

These results indicate that compound X exhibits stronger antioxidant activity than representative wine-derived polyphenols (EC and EGCg) as well as antioxidant vitamins. Its potent activity at low concentrations underscores its potential as a functional bioactive compound.

2.5. Detection of Compound X in UV-Irradiated White Wine Pomace Samples

Liquid chromatography–time-of-flight mass spectrometry (LC–TOF–MS) analysis of all UV-irradiated white wine pomace samples revealed a new peak at m/z 289.0712, with a retention time and mass consistent with those of the UV-irradiated EC product (compound X) (Figure ). These findings confirm that flavan-3-ols, predominantly EC, present in white wine pomace are converted into compound X upon UV irradiation.

5.

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LC–TOF–MS chromatograms (extracted ion, m/z 289.0712) of white wine pomace extracts from five grape cultivars (Kerner, Trebbiano, Koshu, Niagara, and Merlot) before (left) and after (right) UV irradiation. Peaks corresponding to compound X are indicated by red arrows.

UV irradiation of white wine pomace significantly enhanced its antioxidant activity, with the formation of a novel, highly active EC-derived compound likely serving as a major contributor.

3. Discussion

This study showed that UV irradiation of underutilized white wine pomace significantly enhanced·OH–scavenging activity across all tested samples (Figure ). Two-way ANOVA indicated that the treatment condition (UV irradiation) accounted for more than 85% of the total variation, identifying photoirradiation as the primary factor driving the observed increase in activity. These findings suggest that nonpigmented polyphenols abundant in white wine pomace, particularly flavan-3-ols, are photoresponsive and that light-induced structural modifications can enhance their bioactivity. Detection of compound X in all irradiated pomace extracts confirms that UV-induced structural modification occurs consistently across cultivars, reinforcing the broad applicability of this photochemical effect (Figure ).

The novel compound X, isolated and purified from UV-irradiated EC, was structurally characterized by NMR, IR, and MS analyses. These findings confirmed a bicyclo[2.2.2]­octane-type skeleton featuring an intramolecular C–C bond between C-10 of the A-ring and C-6′ of the B-ring, along with ketonization at C-5 (Figure ). This structural transformation was driven by spatial proximity of the aromatic rings and altered electron-donating properties, which promoted intramolecular reactions under UV irradiation. The pathway is consistent with previous reports of catechin isomerization and condensation reactions induced by UV light. ,, Although UVB-induced transformations of catechins have been previously documented, these reports primarily focused on photolysis and photoisomerization and did not describe the formation of a rigid bicyclo[2.2.2]­octane-type structure. In the present study, extended irradiation at 302 nm in an aqueous environment enabled intramolecular condensation between C-10 and C-6′, likely facilitated by the spatial proximity of the aromatic rings in cis-configured epicatechin. As a result, a stable bicyclic product was obtained. These findings suggest that irradiation wavelength, solvent system, reaction duration, and stereochemical configuration play critical roles in determining whether transient photolysis fragments or isolable rearranged products are favored.

Functional evaluation revealed that compound X exhibited an IC50 of 71.42 μM for·OH–scavenging activity, substantially lower than that of EGCg (IC50 = 395.9 μM) and antioxidant vitamins C and E (Table ). At 1 μM, compound X retained more than 20% radical–scavenging activity, with a steep concentration–response curve (Figure ). Because EGCg has long served as a benchmark in antioxidant research, the superior activity of compound X highlights the impact of this photoinduced structural modification. The inhibition curve of compound X differed significantly from those of EGCg, VC, Trolox, and EC (p < 0.05, extra sum-of-squares F test), and its IC50 CI was the narrowest among all tested compounds, indicating both higher potency and precise estimation (Table , Figure ).

The high antioxidant activity of compound X was attributed to structural rigidification via intramolecular bridging and enhanced electron-donating capacity, which stabilized phenoxyl radical resonance and promoted hydrogen atom transfer or single-electron transfer mechanisms. This interpretation was supported by the structural characteristics of the precursor EC. EC contains multiple phenolic hydroxyl groups, particularly the 3′,4′-dihydroxy substitution on the B-ring, which act as strong electron donors in photo-oxidation and photoisomerization reactions. Its aromatic rings efficiently absorb UV light (200–400 nm) through π → π* transitions, generating electronically excited states that trigger radical formation, ring opening, and condensation reactions. , In its cis-form, the B-ring is positioned near the A/C rings, facilitating intramolecular hydrogen bonding and electron delocalization, thereby enhancing the electron-donating ability of the 3′,4′-dihydroxy group under photo-oxidative conditions. − These intrinsic structural features explain why EC is particularly suitable as a model compound for UV-induced modification and why compound X is generated with enhanced activity.

From a bioavailability perspective, compound X shows high application potential. Grape pomace–derived polyphenols have demonstrated considerable stability in the gastric environment and favorable intestinal absorption. Human intervention studies indicate that although these compounds are metabolized into various derivatives after ingestion, they retain significant biological activity. Therefore, it is reasonable to expect that a highly active compound such as compound X may exert potent antioxidant effects in vivo.

The environmental implications of this approach are significant. White wine pomace, a byproduct of winemaking, is typically discarded or incinerated, processes that generate CO2 emissions and consume large amounts of energy. The UV irradiation method described here requires only light energy within a narrow wavelength range, offering, in principle, a simple, low-cost, and environmentally friendly processing strategy. The potential use of natural sunlight further enhances sustainability. Additionally, utilizing nonmarketable or surplus fruit as raw material could contribute to resource recycling and reduction of food waste.

Several limitations of this study should be acknowledged. Antioxidant activity was evaluated only in vitro using ESR, and although the structure of compound X is strongly supported by NMR, IR, and MS data, its absolute configuration remains unconfirmed. Moreover, the isolation yield of compound X was low; a representative single run yielded ∼0.44%, whereas the average yield across multiple experiments was 0.58% ± 0.14% (Figure ), underscoring the need to optimize reaction conditions to improve productivity. Stability, safety, and bioavailability assessments are also lacking, emphasizing the necessity for in vivo validation to support practical applications. Complementary antioxidant assays (e.g., DPPH, ABTS, ORAC), theoretical calculations such as density functional theory, and preliminary cellular evaluations are planned for future studies.

Despite these constraints, the unique structural features and potent radical–scavenging properties of compound X provide promising avenues for applications. In the food and nutraceutical sectors, its high antioxidant efficiency at low concentrations suggests potential as an ingredient in functional foods, dietary supplements, or formulations targeting oxidative stress. This potential is supported by previous reports demonstrating the gastrointestinal stability and bioavailability of grape-derived polyphenols. In cosmetic applications, the nonpigmented bicyclic structure and photostable antioxidant properties of compound X indicate suitability for skin-care formulations aimed at mitigating photo-oxidative and aging-related damage. In pharmaceutical and preventive medicine contexts, its superior potency compared with commonly used antioxidants, including EGCg and vitamins C and E, positions compound X as a promising candidate for further exploration in oxidative stress–related disorders. Furthermore, the photoirradiation approach described here provides a sustainable, low-energy strategy for upcycling white wine pomacean underutilized agricultural byproductinto a structurally novel and bioactive compound. These findings highlight the broader significance of photoinduced structural modification as an efficient and environmentally friendly method for generating functional materials from agricultural waste.

In summary, this study highlights new opportunities for the valorization of white wine pomace and the sustainable development of functional materials. Compound X exhibits potent antioxidant activity even at low concentrations and may serve as a promising bioactive candidate for applications in food, pharmaceuticals, and cosmetics. Future work should evaluate its stability, safety, and pharmacokinetics, as well as develop scalable production methods to facilitate clinical and industrial applications.

4. Experimental Section

4.1. Grape Pomace Samples

White wine pomace was obtained from Camel Farm Co., Ltd. (Yoichi, Hokkaido, Japan) and Iwasaki Jozo Co., Ltd. (Katsunuma, Koshu, Yamanashi, Japan). Five grape cultivars were used: Kerner, Trebbiano, Koshu, Niagara, and Merlot. Each pomace sample was dried, powdered, and extracted with water (10 mg/mL) overnight. The extracts were centrifuged at 1500g for 10 min. The resulting supernatants were collected for analysis. For antioxidant assays, the aqueous extracts were diluted 10-fold with water to a final concentration of 1 mg/mL before use.

4.2. Chemicals and Reagents

(−)-EC, (−)-EGC, (−)-EGCg, and VE were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan). Procyanidin B2 (dimer) and Procyanidin C1 (trimer) were obtained from Extrasynthese (Lyon, France). (+)-Catechin (Cat) and ascorbic acid (vitamin C, VC) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Acetonitrile containing 0.1% formic acid and formic acid (HPLC grade) were obtained from FUJIFILM Wako Pure Chemical Corporation. Trifluoroacetic acid (first grade) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

The UV irradiation experiments were conducted using a hand-held UV lamp (EL series, 8 W; Funakoshi Co., Ltd., Tokyo, Japan). Disposable methacrylate cuvettes (4.5 mL) were obtained from Fisher Scientific (Waltham, MA, USA).

4.3. UV Irradiation of Samples

For UV irradiation, 2 mL of each sample was placed in disposable methacrylate cuvettes and irradiated from the top surface at a distance of 10 mm with UV light at 302 nm (∼2000 μW/cm2) for 24 h at room temperature.

For EC, 580 mg was dissolved in 2 L of water, and 2 mL aliquots were UV-irradiated under the same conditions. The UV-irradiated EC solutions were freeze-dried, and the resulting powders were dissolved in acetonitrile containing 0.1% formic acid for purification by HPLC.

For white wine pomace aqueous extracts, the original extracts were diluted 10-fold to a final concentration of 1 mg/mL before use. Standard compounds, including EC and other reference reagents, were prepared at 1 mM. These solutions were used for antioxidant activity measurements by ESR and for peak identification by LC–MS.

Additionally, EC solutions (1 mM) before and after UV irradiation were freeze-dried, and the resulting solids were analyzed by HPLC for structural characterization.

4.4. Antioxidant Activity Measurement by ESR

The·OH–scavenging activity of samples was evaluated using ESR spectroscopy with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping reagent.·OH radicals were generated via the Fenton reaction and trapped by DMPO for ESR detection. Briefly, 120 μL of ultrapure water (Milli-Q system, Merck Millipore, Burlington, MA, USA), 20 μL of sample solution, 20 μL of 10 mM FeSO4, 20 μL of 89 mM DMPO, and 20 μL of 10 mM H2O2 were mixed and vortexed for 50 s. The reaction mixture was then aspirated into a 50 μL-capillary tube, and ESR spectra were recorded for 60 s using an EMX-micro ESR spectrometer (Bruker BioSpin, Ettlingen, Germany). VC, Trolox, and EGCg served as reference antioxidants.

ESR measurement parameters were as follows: magnetic field sweep, 3450–3500 G; field modulation frequency, 9.84 GHz; modulation amplitude, 0.1 mT; signal amplitude, 200; sweep time, 10 s; time constant, 0.01 s; microwave frequency, 9.420 GHz; microwave power, 10 mW. Chromium (Cr3+) held in the ESR cavity was used as an internal standard.

4.5. Statistical Analysis

Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). To evaluate the effects of sample type (row factor) and treatment condition (control vs UV-irradiated, column factor), a two-way ANOVA (repeated-measures design) was performed with a significance threshold of α = 0.05. Type III sums of squares were applied for model fitting, and the significance of main effects and interactions was determined by F-tests. Differences between predicted LS means for treatment effects were also examined.

For inhibition rates (%) of individual compounds, one-way ANOVA followed by Tukey’s multiple-comparison test was used to identify significant group differences (p < 0.05). Concentration–response curves were analyzed by nonlinear regression with a four-parameter logistic model to determine IC50 values and 95% CIs. Differences in IC50 values among compounds were evaluated with the extra sum-of-squares F-test.

4.6. NMR, MS, and IR Analysis

NMR spectra were recorded in DMSO-d 6 using 500 MHz (1H) and 200 MHz (13C) spectrometers. Chemical shifts (δ) are reported in parts per million (ppm) relative to internal standards, and coupling constants (J) are reported in hertz (Hz). Mass spectra were obtained by DART–MS. IR spectra were recorded with a FT–IR spectrometer.

5. Compound X

1H NMR (DMSO-d 6, 500 MHz): δ 1.95 (dd, J = 4.4, 13.2 Hz, 1H), 2.05 (dd, J = 10.7, 13.2 Hz, 1H), 3.97 (ddd, J = 4.4, 7.0, 10.9 Hz, 1H), 4.14 (d, J = 7.9 Hz, 1H), 5.17 (d, J = 1.0 Hz, 1H), 5.21 (d, J = 1.6 Hz, 1H), 6.19 (s, 1H), 6.87 (s, 1H);

13C NMR (DMSO-d 6, 200 MHz): δ 36.4, 50.6, 68.5, 72.0, 98.0, 99.3112.4, 114.2, 124.1, 131.6, 144.3, 144.6, 169.8, 177.6, 179.2; DART–MS m/z: found, 289.0716 [M + H]+, calcd for C15H13O6 289.0707; FT-IR (ATR): 3189 (OH), 1644­(CO), 1574, 1521, 1446 (aromatic ring), 1282 (ether) cm–1.

Supplementary Material

ao5c10089_si_001.pdf (257.8KB, pdf)
ao5c10089_si_002.pdf (669.4KB, pdf)

Acknowledgments

The authors thank Sanae Takemura, a pharmacy student at Showa Medical University School of Pharmacy (Tokyo, Japan), for her dedicated assistance in preparing samples for UV irradiation.

Glossary

Abbreviations

ANOVA

analysis of variance

ATR

attenuated total reflectance

Cat

(+)-catechin

CI

confidence interval

DART–MS

direct analysis in real time–mass spectrometry

DMPO

5,5-dimethyl-1-pyrroline N-oxide

DMSO-d 6

deuterated dimethyl sulfoxide

EC

(−)-epicatechin

EGC

(−)-epigallocatechin

EGCg

(−)-epigallocatechin gallate

ESR

electron spin resonance

FT-IR

Fourier transform infrared spectroscopy

HMBC

heteronuclear multiple–bond correlation

HPLC

high-performance liquid chromatography

HSQC

heteronuclear single-quantum coherence

IC50

half-maximal inhibitory concentration

LC–MS

liquid chromatography–mass spectrometry

LC–TOF–MS

liquid chromatography–time-of-flight mass spectrometry

LS means

least-squares means

m/z

mass-to-charge ratio

MS

mass spectrometry

NMR

nuclear magnetic resonance

·OH

hydroxyl radical

SD

standard deviation

TOF

time-of-flight

UV

ultraviolet

VC

vitamin C

VE

vitamin E

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c10089.

  • Supporting Information 1: UV–Vis spectra of epicatechin and Compound X in methanol (200–900 nm) (PDF)

  • Supporting Information 2: 1D and 2D NMR spectra of Compound X, including ^1H, ^13C, COSY, HMBC, and HSQC, together with the proposed chemical structure and complete signal assignments (PDF)

MT: Conceptualization; ESR methodology and investigation; statistical analysis; writingoriginal draft; writingreview and editing; project administration; funding acquisition. YO: UV irradiation; NMR and IR experiments; data collection. YK: HPLC isolation; NMR data curation. MF: Mass spectrometry methodology and investigation; data curation; visualization. TS: Supervision. KF: Supervision; writingreview and editing. All authors have read and approved the final manuscript.

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 20K15471 and 24K17857 (both to M.T.).

The authors declare no competing financial interest.

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