Synergistic degradation of catechin and minocycline hydrochloride in alkaline solution

2.1. Chemicals

(+)-Catechin (C₁₅H₁₄O₆) was purchased from Toronto Research Chemicals Inc. (Toronto, ON). MCH (C₂₃H₂₈ClN₃O₇) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Monopotassium phosphate (KH₂PO₄), dipotassium phosphate (K₂HPO₄), and FCR were purchased from Sigma-Aldrich (St. Louis, MO). A Milli-Q system was used to prepare ultra-pure water for the aqueous solutions.

2.2. Effect of pH on (+)-catechin and MCH solutions

The effect of pH on catechin and MCH was determined using an enzyme-linked immunosorbent assay (ELISA) reader provided by Thermo Fisher Scientific (Multiskan, Waltham, MA). Solutions were prepared as follows: (A) catechin (1 mM) was dissolved in 0.1 M phosphate-buffered saline (PBS) (pH 6, 8, and 10); (B) MCH (0.1 mM) was dissolved in PBS (pH 6, 8, and 10); and (C) a mixture of 0.1 mM MCH and 1 mM catechin was dissolved in PBS (pH 6, 8, and 10). Portions of the MCH and catechin solutions were incubated in the dark for 2 or 24 h. The absorbance of catechin solutions was recorded in the wavelength range of 300-800 nm using an ELISA reader.

2.3. Effect of pH on a mixture of catechin and MCH solution using a high-performance liquid chromatography-mass spectrometry (HPLC-MS)

The effect of pH on catechin and MCH solutions in PBS was evaluated using HPLC-MS, as described in previous studies [23-32]. Solutions were prepared as described in Section 2.2. The solutions were analysed using an Agilent 1200 Series HPLC system coupled with a 6410B triple quadrupole tandem MS equipped with electrospray ionization (ESI) as the ionizing source (Agilent Technologies, Palo Alto, CA). The MS analysis was conducted in negative ion mode. The operating parameters were as follows: drying gas (N₂) at a temperature and flow rate of 350°C and 11 mL/min, respectively; a nebulizer gas pressure of 50 psi; a capillary voltage of 3700 V; and a temperature of 280°C. Data were collected over a full-scan range of 100-1,000 atomic mass units (amu). The data were processed using Agilent Mass Hunter Workstation software (version B.06.00).

The solutions were eluted at 40°C using a Poroshell 120 EC-C18 column (length = 150 mm, inner diameter = 4.6 mm, and particle size = 2.7 µm) provided by Agilent Technologies (Palo Alto, CA). The solutions were prepared as described in Section 2.2 and filtered through a Millipore 0.45 μm filter (Brighton, MA) before each analysis.

MCH and catechin, along with their degradation products, were separated using HPLC, with a mobile phase comprising methanol as solvent A and 0.1% formic acid as solvent B. The elution profile was established using a linear gradient of solvent A as follows: 0-3 min, 1%-10%; 3-10 min, 10%-20%; 10-16.5 min, 20%-75%; 16.5-20 min, 75%-50%; and 20-23 min, 50%-1%. For each analysis, a 5 μL reaction solution was injected and eluted at a flow rate of 400 μL/min.

2.4. Total phenol content

The total phenol content of catechin was evaluated using the FC method with slight modifications [31]. A phenolate anion produced by the dissociation of a phenolic proton can reduce FCR [24]. To perform the assay, we mixed 250 μL of 1 mM catechin solution with 250 μL of 1 N FCR and 4.5 mL of PBS (pH 6, 8, and 10). The mixture was incubated at room temperature for 25 mins. The phenolic content was determined by measuring the extent of the reduction of Mo⁶⁺ to Mo⁵⁺, with absorbance recorded at 730 nm [24] using an ELISA reader.

2.5. Effects of MCH and its degradation on bacterial growth curves

The E. coli DH5α strain (NCBI Taxonomy ID: 668369) was obtained from the Bioresource Collection and Research Centre and cultured overnight in Lysogeny broth at 37°C. Following incubation, 1 mL of the culture was transferred into an Eppendorf micro-centrifuge tube and diluted to obtain an optical density of 0.2 at 600 nm (OD₆₀₀) (approximately 3.0 × 10⁶ colony-forming units/mL). A 2.8 mL bacterial solution was then prepared, to which 0.2 mL of catechin (1 mM) and MCH (0.1 mM) or degraded MCH (D-MCH) was added. To prepare D-MCH, we dissolved 0.1 mM MCH and 1 mM catechin solutions in PBS at pH 8, followed by incubation for 2 h. The bacterial growth curves were monitored by measuring absorbance (OD₆₀₀) using an ELISA reader. For each sample, measurements were obtained every 20 min for a 3 h incubation period at 37°C, with agitation at 150 revolutions per minute (rpm).

2.6. Statistics

All tests were conducted in triplicate. A one-way analysis of variance was used to determine the differences between the two samples. For comparisons between two groups with statistically significant differences, the unpaired Student’s t-test was used. The results are presented as mean ± standard deviation for each experiment, with statistical significance defined as p < 0.05.

3.1. Effect of pH on the color of catechin solution

The effect of pH on the colour of the catechin solution was assessed. As shown in Figure 2(a), the catechin in PBS (pH 6 and 8) was transparent; however, it turned yellow at pH 10. The absorption spectra of 0.17 mM catechin varied with pH. As shown in Figure 2(b), 0.17 mM catechin at pH 10 exhibited a peak at 426 nm.

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Figure 2.

Changes in the (a) colour of 1 mM catechin and (b) absorption spectra of 0.17 mM catechin incubated at different pH levels: (A) 6, (B) 8, and (C) 10 for 2 h, and (D) 10 for 24 h.

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3.2. HPLC-MS analysis of catechin at different pH values

Catechin and its oxidized products were analysed using HPLC-MS. The total ion chromatograms of catechin at different pH (6, 8, and 10) have been shown in Figure 3. Figure 3(a) shows the total ion chromatogram for catechin in PBS at pH 6, with a peak at 17.884 min corresponding to a mass-to-charge ratio (m/z) of 289 (Figure 3e), indicating the presence of quasi-molecular anions [M-H]⁻.

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Figure 3.

Total ion chromatograms of 1 mM catechin incubated at pH (a) 6, (b) 8, and (c) 10 for 2 h and (d) 10 for 24 h in the dark, as determined using HPLC-MS. The inset shows the mass spectrum of (e) catechin alone. (f) Relative content of 1 mM catechin incubated at pH 6, 8, and 10 for 2 h and at pH 10 for 24 h in the dark, as determined by HPLC-MS. Statistically significant differences (p < 0.05) between the two groups are indicated by different letters above each bar.

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Following incubation at pH 10 for 2 h, the total ion chromatogram revealed the presence of several basification products eluted at 8.655, 11.549, 14.422, 15.631, 17.929, 18.458, and 19.190 mins, with ion fragments detected at m/z 575, 575, 591, 577, 289, 303, and 289, respectively, as shown in Figure 3(c). After 24 h of incubation at pH 10, catechin decomposition was observed (Figure 3d). The basification products were eluted at 8.49, 11.4, 14.213, 14.758, 15.355, 16.213, 17.824, 18.399, and 19.190 mins, with ion fragments at m/z 575, 575, 591, 577, 577, 287, 289, 303, and 289, respectively. The relative amount of 1 mM catechin after incubation was 100% at pH 6, 101.3% at pH 8, and 62.0% at pH 10 after 2 h, and 34.6% at pH 10 after 24 h (Figure 3f).

3.3. Effect of pH on MCH

In Figure 4, the effect of pH on the colour of the MCH solution was also investigated. The MCH in PBS (pH 6, 8, and 10) was transparent, as shown in Figure 4(a). The absorption spectra of 0.1 mM MCH at different pH values are shown in Figure 4(b). At pH 10, a peak at 382 nm was observed for MCH.

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Figure 4.

(a) Change in the colour of a 0.1 mM MCH solution incubated at pH (A) 6, (B) 8, and (C) 10 for 2 h and (D) 10 for 24. (b) Absorption spectra of a 0.1 mM MCH solution incubated at pH (A) 6, (B) 8, and (C) 10 for 2 h and (D) 10 for 24 h.

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3.4. HPLC-MS analysis of MCH at different pH values

The total ion chromatograms for MCH incubated at pH 6, 8, and 10 for 2 h and 10 for 24 h were acquired via HPLC-MS analysis and are shown in Figure 5. As shown in Figure 5(a), the total ion chromatogram of an aqueous MCH solution at pH 6 shows a peak at 18.8 min, corresponding to its elution, as confirmed by its molecular ion at m/z 456 (Figure 5e). The intensity of the MS peak was obtained in [M-H]⁻ mode.

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Figure 5.

(a) Total ion chromatograms of a 0.1 mM MCH solution incubated at pH (a) 6, (b) 8, and (c) 10 for 2 h and (d) 10 for 24 h in the dark, as determined via HPLC-MS analysis. The inset shows the mass spectrum of (e) MCH alone. (f) Relative content of a 0.1 mM MCH solution after incubation at pH 6, 8, and 10 for 2 h and 10 for 24 h in the dark, as determined by HPLC-MS analysis. Statistical differences (p < 0.05) between the two groups are indicated by distinct letters above each bar.

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The MCH content after incubation at pH 6, 8, and 10 for 2 h and 10 for 24 h was determined using HPLC-MS. No statistically significant difference was observed between the relative percentages of MCH incubated at pH 8 and 10 for 2 h or at pH 10 for 24 h (Figure 5f).

As shown in Figure 4(b), the spectra of 0.1 mM MCH exhibited a red shift in the stimulated emission as the pH increased, indicating the behaviour of MCH under alkaline conditions. Notably, an isosbestic point was observed (Figure 4b), indicating the deprotonation of MCH. Proton and charge transfer studies have shown that MCH exists as MCH₂^± (neutral zwitterionic species), MCH⁻, and MC²⁻ at pH 6.4, 9.0, and 11.3, respectively, with deprotonation occurring under alkaline conditions [35]. The relative percentage of MCH was consistent at pH 6, 8, and 10 after 2 h of incubation (Figure 5f). MS ionizes molecules to form molecular ions, providing information about their molecular weight, elemental composition, and fragmentation patterns. As shown in Figure 5, the mass spectra of MCH at different pH values were consistent with that of deprotonated MCH, with a molecular weight of 456 Da, as determined by ESI.

3.5. Changes in catechin solution after the addition of MCH at pH 10

The effect of pH on catechin, as indicated by the observed colour change after the addition of MCH, was assessed. When 1 mM catechin was mixed with 0.1 mM MCH in PBS at pH 10 and incubated for 2 or 24 h, the solution turned yellow, as shown in Figure 6(a).

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Figure 6.

(a) Colour change of (A) 1 mM catechin, (B) 0.1 mM MCH, and (C) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 2 h at pH 10, and (D) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 24 h at pH 10. (b) Absorption spectra of (A) 1 mM catechin, (B) 0.1 mM MCH, and (C) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 2 h at pH 10, and (D) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 24 h at pH 10.

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The spectra of a 1 mM aqueous catechin solution before and after the addition of a 0.1 mM MCH at pH 10 are shown in Figure 6(b). Upon the addition of MCH and subsequent incubation for 2 and 24 h at pH 10, peaks characteristic of catechin appeared at 412 and 431 nm, respectively (Figure 6b).

3.6. HPLC-MS analysis of catechin solution following the addition of an aqueous alkaline MCH solution

The MCH concentrations at pH 8 and 10 after incubation for 2 and 24 h were determined using HPLC-MS. The MCH concentrations after incubation at pH 6, 8, and 10 for 2 h and at pH 10 for 24 h showed no statistically significant differences, as shown in Figure 5(f). At pH 8, the concentrations of MCH (initial concentration = 0.1 mM) were 0.047 and 0.043 mM, corresponding to 47.3% and 42.9%, after 2 and 24 h of incubation with catechin, respectively, as shown in Figure 7(a). No statistically significant difference (p = 0.12) was observed between the relative percentages of MCH at pH 8 for 2 and 24 h incubation with catechin (Figure 7a). At pH 10, the concentrations of MCH were 0.038 and 0.013 mM, corresponding to 37.7% and 12.6%, after 2 and 24 h of incubation with catechin, respectively, as shown in Figure 7(a). These results indicate that the degradation of MCH is enhanced in the presence of catechin under alkaline conditions.

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Figure 7.

(a). Relative percentage of 0.1 mM MCH in the presence of 1 mM catechin after incubation for 2 and 24 h at pH 8 and 10. (b) Relative percentage of 1 mM catechin in the presence of 0.1 mM MCH after incubation for 2 and 24 h at pH 8 and 10, as determined by HPLC-MS analysis. Statistical differences (p < 0.05) between the two groups are indicated by distinct letters above each bar.

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The catechin concentrations were determined at pH 8 and 10 after incubation for 2 and 24 h. No statistically significant difference (p = 0.28) was observed between the relative percentages of catechin at pH 8 for 2 and 24 h incubation with MCH (Figure 7b). At pH 10, the concentrations of 1 mM catechin in the presence of 0.1 mM MCH were 0.438 and 0.017 mM after 2 and 24 h of incubation, respectively. The relative percentages of catechins were 62.0% and 34.6% after incubating catechin alone for 2 and 24 h at pH 10, respectively, as shown in Figure 3(f). As shown in Figure 7(b), the relative percentages of catechins in the presence of 0.1 mM MCH were 43.8% and 1.7% after 2 and 24 h of incubation at pH 10, respectively. These results indicate that catechin degradation is enhanced to some degree in the presence of MCH under alkaline conditions.

The total ion chromatograms obtained from the HPLC-MS analysis of catechins in the presence of MCH at pH 10 after incubation for 2 and 24 h have been shown in Figure 8. As shown in Figure 8(a), the total ion chromatogram of the MCH solution at pH 10 shows a peak at 18.8 min with fragmented ion species at m/z 456. As shown in Figure 8(c), the total ion chromatogram of MCH was significantly reduced after 2 h of incubation with catechin at pH 10, with the basification products eluting at 14.743, 15.295, 17.802, 18.384, 18.884, and 19.107 min and characteristic ion fragments detected at m/z 577, 577, 289, 303, 456, and 289, respectively. Figure 8(d) shows the further degradation of the total ion chromatogram of MCH after 24 h of incubation with catechin at pH 10, with the basification products (after adding MCH) eluting at 8.468, 11.341, 14.191, 14.601, 16.108, 17.794, 18.175, 18.369, 18.869, and 19.234 min and characteristic ion fragments detected at m/z 575, 575, 591, 472, 287, 289, 287, 303, 456, and 303, respectively.

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Figure 8.

Total ion chromatograms obtained from HPLC-MS analysis at pH 10 for (a) 0.1 mM MCH incubated for 2 h, (b) 1 mM catechin incubated for 2 h, (c) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 2 h, and (d) 0.1 mM MCH in the presence of 1 mM catechin and incubated for 24 h. The inset shows the mass spectrum of (e) degraded MCH (D-MCH) after basification in the MCH–catechin system.

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3.7. Total phenol content at different pH values

The effect of pH on the phenol content of catechin was evaluated using a modified FC method. As shown in Figure 9, the phenol content of catechin at pH 10 was approximately 1.64-fold higher than that at pH 6. According to the FC method for polyphenol analysis in aqueous alkaline solutions, phenolic compounds tend to dissociate into an anionic phenolate, which reduces Mo⁶⁺ in the FCR to Mo⁵⁺ via electron transfer [31]. However, catechin is unstable at pH 10 and undergoes significant decomposition via electron transfer.

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Figure 9.

Effects of pH on 1 mM catechin in the Folin–Ciocalteu (FC) assay at pH 6, 8, and 10. Statistical differences (p < 0.05) between the two groups are indicated by distinct letters above each bar

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3.8. Effects of incubating MCH in the presence of catechin on bacterial growth curves

MCH is a TC antibiotic that poses a significant environmental threat when TC-containing waste is released [6]. The effects of MCH and D-MCH on bacterial growth curves were examined. The bacterial growth curves for MCH and D-MCH over 0-3 h are shown in Figure 10. There was a significant difference (p < 0.05) between the growth rate of D-MCH and MCH (Figure 10). The growth rate constants for D-MCH and MCH were 0.057 and 0.037 h⁻¹, respectively, indicating that the MCH-catechin system reduces the antibiotic activity of MCH. Furthermore, D-MCH obtained via the MCH-catechin interaction exhibited lower antibacterial activity against E. coli, as shown in Figure 10.

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Figure 10.

Effects of catechin, D-MCH, and MCH on E. coli growth curves at pH 8

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Catechin is stable under acidic conditions; however, it is significantly unstable under neutral or alkaline conditions. The catechin spectra varied significantly for different pH values. As shown in Figure 2(b), the spectra of the catechin solution at pH 10 showed an absorbance band in the 350-450 nm range. This band can be attributed to the formation of chromogenic catechin derivatives induced by structural rearrangement in aqueous alkaline solutions. This study demonstrated that MCH degradation was significantly increased by the structural rearrangement of catechin incubated at pH 10.

The effect of catechin basification on MCH degradation was examined after incubation at pH 10. When incubated in the presence of 1 mM catechin at pH 10 in the dark for 24 h, 87.4% of MCH was degraded. The structural modification of MCH may significantly alter its properties. To maintain the antibacterial efficacy of TCs, each of the four linearly fused carbon-based rings must be six-membered [1]. Our previous study showed that TC degradation via riboflavin-5′-phosphate (FMN) photolysis markedly reduced the antimicrobial efficacy of TCs, leading to the formation of degraded TCs with the molecular formula C₂₁H₂₂N₂O₈ [3]. The main ion fragment of MCH was detected at m/z 456. However, after 2 h of incubation in the presence of catechin at pH 8 and 10, 52.7% and 62.3% of the MCH degraded, respectively. In addition, although the major ion fragment of MCH was detected at m/z 456, an ion fragment at m/z 472 was observed for the MCH-catechin system at pH 10 (Figure 8). ROS-mediated TC degradation via FMN photolysis significantly decreases antimicrobial activity [3]. E. coli growth curves for MCH and D-MCH over 0-3 h are shown in Figure 10, with a growth rate of 0.057 h⁻¹ for D-MCH and 0.037 h⁻¹ for MCH. It shows that the D-MCH obtained from MCH-catechin treatment is efficient in the preservation of ecosystems due to its lower antibacterial activity. However, the limitations of this study are that (1) the degradation of MCH is reliant on the basification of catechin; i.e., the pH needs to be adjusted in aquatic systems, and (2) the E.coli (a Gram-negative bacillus) growth assay was inhibited in this study, so the effects of MCH–catechin upon basification on the growth of Gram-positive bacteria or protozoan parasites can be further studied by this system.

In solutions, TCs become unstable when irradiated with UV light [8]. However, MCH is more stable under BLIA than DCH. MCH is less photosensitive than DCH [9]. In addition, TC degradation by FMN under BLIA was significantly reduced due to the generation of reactive oxygen species (ROS) [3]. Previous studies have shown that catechin generates ROS during photolytic reactions under BLIA [36], which inhibits the growth of A. baumannii [23]. In this study, MCH was degraded by 69.9% after incubation at pH 10 for 2 h in the presence of 1 mM catechin under BLIA at 6.0 mW/cm² (data not shown), compared with a 62.3% reduction under identical conditions without light treatment (Figure 7), whereas the photolysis of catechin under BLIA at 6.0 mW/cm² for 2 h increased the degradation by 7.6%. It would be of interest to examine whether catechin-treated MCH, after exposure to visible light, could increase the degradation of MCH via ROS generated through a charge transfer process using catechin photolysis for future research.

This study used HPLC-MS to identify the products of catechin and MCH oxidation by atmospheric oxygen and base-catalyzed reactions. The strongly alkaline conditions led to the concurrently increased hydrolysis and oxidation of both catechin and MCH, as shown in Figures 11, respectively. Simultaneously, peroxidation, base-catalyzed cleavage, and atomic rearrangements occurred extensively on the B- and C-rings of flavanol, producing simple aldehydes or acids as intermediates during catechin degradation. Furthermore, many dimeric compounds were generated [37].

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Figure 11.

(a-m) Synergistic degradation mechanism of catechin and MCH in an alkaline solution. The strong alkaline conditions lead to increased hydrolysis and oxidation of both catechin and MCH. The products of catechin and MCH oxidation by atmospheric oxygen and base-catalysed reactions are analyzed by an HPLC-MS assay.

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Catechin content decreased after alkalization and BLIA at 2.0 mW/cm² for 4 h, with a corresponding increase in B-type proanthocyanidin levels under identical conditions [38]. Under aerobic and alkaline conditions, the photooxidative process for catechin under BLIA generates anionic superoxide radicals and a chromogenic catechin dimer (m/z 577) via electron transfer induced by photosensitized oxidation [23]. As shown in Figure 3(f), 38.0% and 65.4% of 1 mM catechin were degraded after incubation at pH 10 for 2 and 24 h, respectively, with characteristic ion signals at m/z 287, 289, 303, 575, 577, and 591.

The antioxidant activity of flavonoids is significantly influenced by the pH of the medium, and their reducing power is notably higher under alkaline conditions. At higher pH values, catechins release more electrons, facilitating electron flow [39]. Based on solvent effects, including kinetic isotope effects and activation parameters, electron transfer from catechin to dissolved oxygen generates ROS, such as anionic superoxide radicals and peroxyl radical (HOO•), along with the protonation of the incipient hydrogen peroxide (H₂O₂) under specific conditions [40]. The generation of these ROS is significantly enhanced by the deprotonation of phenolic compounds at high pH, forming phenoxy anions that more readily donate electrons compared to their protonated counterparts. The strong oxidizing or reducing ability of flavanols primarily arises from the o-hydroxyl groups on aromatic rings, which are directly related to ROS formation during flavanol autoxidation [37,41,42]. During catechin oxidation, anionic superoxide radicals and semiquinone intermediates play a significant role in the reaction because their stability increases with increasing pH, explaining the pH-dependent nature of the oxidation process [29]. The reaction mechanism of anionic superoxide radicals with phenolic derivatives involves a two-electron transfer process coupled with proton transfer in the transition state, as indicated by negative activation entropy values, which suggests solvent reorganization to facilitate proton transfer [43].

In this study, a mechanism is proposed for the formation of H₂O₂ during the base-catalyzed oxidation of the interaction between MCH and catechin. The process involves reactions Eqs. (1)-(4), including the pH-dependent initiation of superoxide formation, and the redox reaction that involves MCH and catechin. In the final reaction, the redox reaction is driven by the hydroperoxyl radical, resulting in the formation of MCH and catechin redox reaction products and H₂O₂, and promotes the hydroxylation reaction.

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MCH binds in a large, tunnel-shaped active site in close contact with catechin, a cofactor acting as a reductant, and forms pre-orientated adducts for regioselective hydroxylation to form C-11a-hydroxy-minocycline under aerobic conditions [44]. The C11a-C12 double bond of MCH was much more susceptible for free radicals to attack through an electron-withdrawing substituent from adjacent electron-donating groups of catechin, making C11a-C12 the most susceptible position for hydration [45] to hydroxylate by oxygen transfer from H₂O₂ onto the aromatic ring, after which an intermediate epoxide is formed that can spontaneously rearrange into a hydroxyl group on C11a [46]. During this reaction, an intermediate α-hydroxy ketone of MCH is produced. The enol tautomer of the β-diketone is an activated double bond in which the keto-enol motif at C-11a/C-12 undergoes deprotonation, leading to the hydroxylation of C-11a and forming an α-hydroxy ketone at the C-11a-C-12 position [47]. The product of the interaction between MCH and catechin at pH 10 has the molecular formula C₂₃H₂₇N₃O₈ (m/z 472) from C₂₃H₂₇N₃O₇ (m/z 456) of MCH, as shown in Figure 11(d) and Figure 12.

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Figure 12.

Hydroxylation of minocycline (a) minocycline and (b) C-11a-hydroxy-minocycline.

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Flavanol (catechin) degradation occurs spontaneously via autoxidation, and the rate of autoxidation is significantly influenced by factors such as temperature, humidity, and pH. Owing to the stabilizing effects of intramolecular hydrogen bonding and the low-energy transformation of unsaturated conjugated systems, o-/p-polyhydroxy-substituted aromatic groups, such as catechol, pyrogallol, and quinol moieties, donate electrons more efficiently than their m-substituted counterparts, leading to the formation of the o-quinone derivatives found in the original sample [37-48].

The base-catalyzed oxidation of catechin involves three key steps: oligomerization, rearrangement, and hydroxylation [46]. The main oligomerization and rearrangement products formed by the oxidation of polyphenol and hydroxylation are structurally represented by oxygen transfer from H₂O₂ onto the aromatic ring [46]. The roles of electron transfer and oxidative pathways in catechin degradation are as follows: the oxidation of B-rings via the 2e^- pathway (PathⅠ), and the oxidation path of A- and C-rings via the 2e^- pathway (PathⅡ), respectively.

Path Ⅰ: Oxidation of catechin proceeds in two successive steps, with the final product being the o-quinone derivative flavan-3-ol o-quinone, as evidenced by the molecular ion peak at m/z 287 (C₁₅H₁₂O₆), as shown in Figure 11(e).

Path Ⅱ: Li and Deinzer highlighted that, for the accurate identification and characterization of proanthocyanidins, the following aspects need to be considered: 1. Molecular ion peaks (M-H) in the negative ion mode of MS, such as m/z 577 for B-type proanthocyanidin and m/z 575 and 591 for A-type proanthocyanidins derived from catechin monomeric units, indicate oligomer formation. 2. The fragmentation pathway described by Gu et al. provides insights into the hydroxylation of B-rings and bonding between monomeric units. In addition, quinone methide fragmentation, which defines the two monomeric units and the loss of H₂O from the C-ring, indicates the presence of an epicatechin unit [49,50].

Herein, two types of oligomerizations were identified: proanthocyanidin dimers at m/z 577 and prodelphinidin dimers at m/z 591. Mechanistically, proanthocyanidins are formed via the two-electron oxidation of the A-ring of catechin, yielding a p-quinide intermediate, as shown in Figure 11(f). Alternatively, the formation of a carbocation at C-4 of catechin can be hypothesized, which would occur via two-electron oxidation followed by loss of a proton, producing a phenolate carbocation, as shown in Figure 11(g). The phenolate carbocation acts as a strong electrophile and can be attacked by catechin molecules, leading to the formation of catechin dimeric oxidation products, particularly the B-type proanthocyanidin dimer at m/z 577 (C₃₀H₂₆O₁₂) [51,52] (Figure 11h). Furthermore, the B-type proanthocyanidin dimer converts into an A-type dimer through intramolecular oxidation, which shows a molecular ion peak at m/z 575 [52,53], as shown in Figure 11(l). The second type, the prodelphinidin dimer at m/z 591, indicates the presence of an A-type dimer formed from gallocatechin quinide and catechin, as shown in Figure 11(m). During the initial stage of dimer formation, the two-electron oxidation of the A- and C-rings produces a p-quinide intermediate with a rearranged structure.

The ions at m/z 303, identified as p-quinone gallocatechin, were formed via a series of processes involving the hydrolysis, oxidation, and 5′-hydroxylation of the B-ring. Flavan-3-ols are non-planar owing to their saturated C-3 element and represent the most structurally complex subclass of flavonoids, which can form gallocatechin via hydroxylation at the C-5′ position of the B-ring [54,55]. These derivatives are short-lived intermediates and undergo polymerization in aqueous solutions at high pH [37], as shown in Figure 11(j). Subsequently, a p-quinide gallocatechin carbocation can also form after the two-electron oxidation of the B-ring, and a structural rearrangement process promotes prodelphinidin dimer A generation, as evidenced by an ion peak at m/z 591 (Figures 11(k), (l), and (m), respectively).

Under BLIA, catechin undergoes photolysis and polymerization, as indicated by the characteristic m/z ion signal at 577, corresponding to B-type proanthocyanidin [38]. This study demonstrated that, under alkaline conditions (pH 10), both A- and B-type proanthocyanidins were generated and identified by their characteristic ion fragments at m/z 577 and 575, respectively, owing to condensation reactions. The catechin dimer is present in many foods, including apples, blueberries, chocolate, and cranberries [56-58]. In addition to their role as cancer chemopreventive and anti-inflammatory agents, proanthocyanidins can also alleviate the risk of cardiovascular mortality [59]. Therefore, the strengths of this study are as follows: (1) the basification of flavan-3-ols under alkaline conditions effectively produces proanthocyanidin, a naturally occurring antioxidant; (2) catechin is a natural compound with no toxicity in aquatic systems, and as such, the basification of catechin provides a safe means of MCH degradation in water; (3) catechin, when mixed with MCH in an alkaline solution, significantly enhances MCH degradation, indicating potential environmental applications; and (4) the degraded MCH obtained from MCH–catechin treatment is efficient in the preservation of ecosystems due to its lower antibacterial activity.