Vitamin C-Assisted Fenton Oxidation Improves Oxidative Killing of Helicobacter pylori In Vitro

Summary

This study examines how vitamin C modulates the kinetic behavior of a Fenton reaction under in vitro conditions. Chemical-dye probe and bacterial assays show that controlled vitamin C dosing alters the reaction's oxidative behavior and is associated with increased antibacterial activity against Helicobacter pylori in a simplified model system.

Abstract

Helicobacter pylori infection remains a major global health burden, and the increasing prevalence of antibiotic resistance underscores the need for alternative antibacterial strategies. This study examined whether vitamin C dosing could modulate Fenton oxidation and enhance oxidative antibacterial activity against H. pylori under defined acidic in vitro conditions. Oxidative activity was assessed using Acid Orange 7 decolorization as an indirect chemical readout and a terephthalic acid fluorescence assay as a relative indicator of hydroxyl radical-associated product formation in iron–peroxide reactions supplemented with vitamin C. Antibacterial efficacy was evaluated by exposing standardized H. pylori suspensions in urea-containing saline at pH 3 to hydrogen peroxide and ferrous iron with or without stepwise vitamin C supplementation, followed by colony-forming unit enumeration. Vitamin C supplementation altered oxidative readouts and was associated with greater reductions in viable bacteria compared with conventional Fenton chemistry. Rescue experiments using deferoxamine and thiourea attenuated antibacterial activity, supporting the hypothesis that antibacterial activity is associated with iron-mediated and radical-related oxidative processes. Live/dead fluorescence staining and scanning electron microscopy provided qualitative visual observations consistent with the colony-forming unit (CFU) enumeration results. Collectively, these findings indicate that controlled vitamin C dosing can modulate Fenton-based oxidative reactions and enhance antibacterial activity against H. pylori in a simplified, acidic in vitro model.

Introduction

H. pylori, a Gram-negative bacterium with a spiral shape that belongs to the genus Helicobacter, has been shown through population health studies to have a high prevalence of infection across geographical areas, sex, and age, with a global prevalence rate of approximately 50%¹. The International Agency for Research on Cancer (IARC) has classified H. pylori as a Group 1 carcinogen, underscoring its strong connection with gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. At present, the clinical management of H. pylori infection primarily relies on pharmacological regimens. The most extensively studied agents include proton pump inhibitors (PPIs), which suppress gastric acid secretion; bismuth compounds, which enhance antibiotic efficacy; and antibiotics such as amoxicillin, clarithromycin, and metronidazole. However, monotherapy has been shown to be insufficient for complete eradication, leading to the widespread use of combination regimens, commonly referred to as triple therapy. This approach typically consists of a PPI, clarithromycin, and either amoxicillin or metronidazole, administered for approximately two weeks³. With the increasing prevalence of antibiotic-resistant H. pylori strains, the efficacy of triple therapy has declined markedly, prompting the adoption of quadruple therapy combining a PPI, bismuth, and two antibiotics³. Nevertheless, these regimens still fundamentally depend on antibiotics, and antibiotic resistance remains a major challenge in clinical management^4,5. Consequently, the exploration of alternative, antibiotic-independent antibacterial strategies has attracted growing interest.

Beyond its direct pathological effects, H. pylori infection has also been associated with alterations in gastric micronutrient homeostasis. Clinical and epidemiological studies have reported reduced concentrations of vitamin C in the gastric juice and serum of infected individuals⁶, which may be related to mucosal inflammation, impaired secretion, or increased oxidative consumption^7,8,9. In parallel, H. pylori infection has been linked to iron deficiency through multiple mechanisms, including reduced gastric acidity, impaired dietary iron absorption, and bacterial competition for available iron¹⁰. These observations highlight the complex biochemical environment accompanying H. pylori infection and underscore the relevance of redox- and iron-related processes in acidic gastric conditions.

Reactive oxygen species (ROS) have gained considerable interest in antimicrobial research because of the oxidative properties of these species. ROS refer to a class of chemically reactive molecules formed during the partial reduction of oxygen, such as superoxide anion (O₂•⁻), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), and hydroxyl radicals (•OH), among others. In biological systems, ROS are extensively involved in normal physiological metabolism and are essential for diverse cellular regulatory activities¹¹. Notably, numerous studies have demonstrated that excessive ROS can disrupt bacterial cell membranes through lipid peroxidation, leading to alterations in membrane structure and function, ultimately resulting in bacterial inactivation¹². Accordingly, ROS-based antimicrobial approaches have attracted interest as a potential avenue for investigating non-antibiotic antibacterial effects against H. pylori^13,14,15,16. Among various ROS-generation methods, the Fenton reaction has garnered significant attention for its highly efficient radical production. The Fenton reaction is an advanced oxidation process¹⁷ in which ferrous ions (Fe²⁺) react with H₂O₂ under acidic conditions to produce hydroxyl radicals (•OH), which possess strong oxidative potential and can oxidize intracellular biomolecules.

Fe²⁺ +H→Fe³⁺+•OH+OH^-

Fe³⁺+ H→Fe²⁺+•OOH+ H⁺

H₂O→OH+•OOH+H₂O

Specifically, at a pH of approximately 2.8–4.5, H₂O₂ undergoes catalytic decomposition by Fe²⁺, generating •OH while Fe²⁺ is oxidized to Fe³⁺. Fe³⁺ then reacts with H₂O₂ to regenerate Fe²⁺, establishing a Fe²⁺/Fe³⁺ redox cycle that sustains continuous ROS production¹⁸. However, the reduction of Fe³⁺ to Fe²⁺ is relatively slow, constituting the rate-limiting step and consequently lowering the overall efficiency of the Fenton reaction. Studies indicate that introducing a suitable reducing agent into the Fenton reaction system can accelerate Fe²⁺ reduction to Fe²⁺, thereby enhancing ROS generation. Vitamin C has a context-dependent redox role. In iron-containing systems, it can promote Fe²⁺ reduction to Fe²⁺ and thereby support Fenton cycling, whereas at higher concentrations or under different reaction conditions, it may also function as an antioxidant or radical scavenger¹⁹.

Based on this background, the present study investigates a vitamin C–assisted Fenton reaction system as a chemically tunable approach for modulating oxidative activity under defined acidic in vitro conditions^20,21. Vitamin C was used as a reducing agent to facilitate Fe²⁺ regeneration and thereby alter the effective oxidative behavior of the Fenton reaction. The experimental system used here was a simplified acidic saline-urea model and was not intended to fully reproduce the physiological gastric microenvironment, which contains mucus, buffering components, host cells, immune factors, and diverse organic substrates. Within this controlled model, oxidative activity was characterized using chemical readouts and evaluated in parallel with bacterial phenotypic responses, providing an experimental basis for probing ROS-associated antibacterial effects against H. pylori in vitro.

Protocol

Ethics statement
This study was an in vitro bacterial study and did not involve human participants, animal subjects, or patient-derived materials. Therefore, institutional review board approval and animal ethics approval were not required.

Evaluation of AO7 degradation in the vitamin C–assisted Fenton system
To assess the effect of vitamin C on the observable oxidative behavior of the Fenton reaction, Acid Orange 7 (AO7) degradation was used as an indirect chemical readout of oxidative activity. This assay does not specifically quantify individual reactive oxygen species. Prepared 100 mL of AO7 solution at 5 mg/L and adjusted the solution to pH 3. Added H₂O₂ and FeSO₄ to final concentrations of 2.2 mM and 0.055 mM, respectively. Added vitamin C either as a single bolus at reaction initiation or in a three-step dosing scheme. For the dose-response experiment, Vc: Fe²⁺ molar ratios of 0, 0.12, 0.24, 0.48, 2, 4, 6, and 8 were used, corresponding to final vitamin C concentrations of 0, 0.0066, 0.0132, 0.0264, 0.110, 0.220, 0.330, and 0.440 mM, respectively, when FeSO₄ is used at 0.055 mM. For the three-step addition condition, divided the same total vitamin C amount into three equal aliquots and added one aliquot at 3, 5, and 7 min after reaction initiation. For example, at a Vc:Fe²⁺ ratio of 0.48, added vitamin C at 0.0088 mM per aliquot, giving a final total concentration of 0.0264 mM.

Stirred the reaction solution continuously in the dark for 10 min while maintaining the temperature below 30 °C. Collected 2 mL samples before and after the reaction. Measured absorbance at 483 nm using a UV-Vis spectrophotometer, and calculated the AO7 degradation rate using Equation 1:

Degradation rate (%) = [(A₀ − A₁) / A₀] × 100% (1)

Here, A₀ represents the absorbance of the sample before the reaction, while A₁ represents the absorbance of the sample after the reaction.

Assessment of •OH-associated fluorescence signal using a TA probe
To assess hydroxyl radical-associated fluorescence signals under the same acidic in vitro conditions used in the antibacterial experiments, a cell-free chemical system was established using disodium terephthalate (TA, 1 mM) as a fluorescence probe. This assay was used as a relative indicator of •OH-associated product formation and was not interpreted as an absolute quantification of hydroxyl radical generation. Seven groups were included: (1) blank (1 mM TA in reaction buffer only), (2) H₂O₂ alone, (3) Fe²⁺ alone, (4) Vc alone (stepwise addition), (5) conventional Fenton (Fe²⁺ + H₂O₂), (6) vitamin C–assisted Fenton with bolus Vc addition (Fe²⁺ + H₂O₂ + Vc, bolus), and (7) Vc–assisted Fenton with stepwise Vc addition (Fe²⁺ + H₂O₂ + Vc, stepwise). In the stepwise-addition condition, Vc was introduced at 3, 5, and 7 min after reaction initiation in equal aliquots, whereas the bolus condition received the same total amount of Vc as a single addition.

Aliquots were collected over a 10-min reaction period, immediately alkalinized with NaOH to quench further radical reactions and stabilize fluorescence, and stored at 4 °C prior to measurement. Fluorescence intensity of the TA-derived product was measured using a microplate reader (Ex/Em: 315/425 nm). Radical generation was expressed as fluorescence intensity after blank subtraction and used as a relative indicator of radical generation. At least three independent experiments were performed, and data were presented as time-course profiles and endpoint comparisons at 10 min.

Evaluation of antibacterial performance by colony counting
H. pylori strains were stored at −80 °C in brain heart infusion (BHI) broth containing 25% glycerol. The primary strain used for the main colony-counting assay, rescue assay, matrix-interference assay, live/dead fluorescence staining, and scanning electron microscopy analysis was H. pylori G27. Before each experiment, bacteria were streaked onto Columbia blood agar plates and incubated for 3 days at 37 °C under microaerophilic conditions consisting of 5% O₂, 10% CO₂, and 85% N₂²².

Colony counting assay in the primary strain
Used colony-forming unit (CFU) enumeration to evaluate antibacterial activity under different treatment conditions. Included the following groups: untreated control, H₂O₂ alone, vitamin C alone, conventional Fenton reaction, and vitamin C–assisted Fenton reaction. If an Fe²⁺-alone group was not included in the original experiment, avoid interpreting the primary assay as fully separating iron-only effects from Fenton reaction-mediated oxidative effects. Harvested cultured H. pylori and resuspend the bacteria in physiological saline. Adjusted the optical density at 600 nm (OD₆₀₀) to 0.5, and transferred the suspension into physiological saline containing 3 mM urea adjusted to pH 3. Added H₂O₂, FeSO₄, and vitamin C according to the assigned treatment group. Used final concentrations of 2.2 mM H₂O₂ and 0.055 mM FeSO₄. For vitamin C–assisted Fenton treatment, added vitamin C in three equal aliquots at 3, 5, and 7 min, with a final total concentration of 0.0264 mM.

After 10 min of treatment, each sample was immediately diluted 100-fold in phosphate-buffered saline (PBS, pH 7.4) to neutralize the acidic suspension and reduce carryover of residual oxidants and iron into the plating step. Plated the diluted bacterial suspension onto Columbia blood agar plates and incubated at 37 °C for 3 days under microaerophilic conditions of 5% O₂, 10% CO₂, and 85% N₂. Counted CFUs and calculated antibacterial activity as log₁₀ CFU reduction relative to the untreated control using Equation 2:

log₁₀ CFU reduction = log₁₀(CFU_control) − log10(CFU_treated) (2)

Rescue assay
To examine whether the antibacterial activity of the vitamin C–assisted Fenton system was associated with iron availability and radical-related processes, rescue experiments were performed using deferoxamine (DFO) as an iron chelator and thiourea as a hydroxyl radical scavenger. DFO or thiourea was added to the bacterial suspension 5 min before the addition of FeSO₄ and H₂O₂. DFO and thiourea were used at final concentrations of 100 µM and 5 mM, respectively.

Six experimental groups were included: untreated control, vitamin C–assisted Fenton, vitamin C–assisted Fenton with DFO, vitamin C–assisted Fenton with thiourea, DFO alone, and thiourea alone. All other experimental parameters, including pH, saline/urea background, H₂O₂ concentration, FeSO₄ concentration, vitamin C dosing schedule, treatment duration, dilution/neutralization before plating, and incubation conditions, were identical to those used in the primary colony-counting assay. Antibacterial activity was quantified by CFU enumeration and expressed as log₁₀ CFU reduction relative to the untreated control.

Multistrain validation
To provide limited cross-strain support for the antibacterial effect observed in the primary strain, the colony-counting assay was further applied to two additional H. pylori reference strains, ATCC 43504 and ATCC 26695. For each strain, three experimental groups were included: untreated control, conventional Fenton reaction, and vitamin C–assisted Fenton reaction with stepwise vitamin C addition. Experimental conditions and procedures were identical to those used in the primary colony-counting assay. Antibacterial activity was expressed as log₁₀ CFU reduction relative to the untreated control for each strain. Each experiment was performed with n = 3 independent biological replicates, and data were presented as individual data points with mean ± standard deviation.

Matrix interference assay
To evaluate whether organic matrix components could attenuate the antibacterial activity of the vitamin C–assisted Fenton system, a protein interference model was established using bovine serum albumin (BSA). BSA was included at final concentrations of 0, 1, and 3 g·L⁻¹. At each BSA concentration, three experimental groups were included: (1) untreated control, (2) conventional Fenton (Fe²⁺ + H₂O₂), and (3) vitamin C–assisted Fenton with stepwise vitamin C addition. The assay was conducted using the primary strain under the same protocol described in the primary colony counting assay, with identical pH conditions, saline/urea background, reagent concentrations, treatment duration (10 min), and plating procedures. Antibacterial activity was expressed as log₁₀ CFU reduction relative to the untreated control at each BSA concentration. Each experiment was performed at least three times independently. The primary comparison focused on whether the enhanced Fenton condition remained superior to, or at least not weaker than, the conventional Fenton condition under protein-containing conditions.

Bacterial live/dead fluorescence staining
After treatment, the bacterial suspension was diluted 100-fold in PBS as described above to reduce residual acidic and oxidative carryover. Centrifuged the suspension at 1,800 × g for 10 min at room temperature and discarded the supernatant. Resuspended the bacterial pellet in 200 µL of physiological saline. Added SYTO9 and propidium iodide (PI) to final concentrations of 6 µM and 30 µM, respectively. Incubated the suspension in the dark for 20 min at room temperature. Placed 5–10 µL of the stained bacterial suspension onto a clean glass slide and covered it with a coverslip. Acquired fluorescence images using a fluorescence microscope with green and red fluorescence channels under identical exposure settings for all treatment groups. Captured at least three randomly selected fields for each group. Used the images as qualitative visual evidence of live/dead staining patterns rather than as a quantitative measurement of bacterial viability.

Bacterial morphology analysis by scanning electron microscopy
After treatment, the bacterial samples were collected and fixed in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 12 h. After fixation, the samples were washed with phosphate buffer to remove residual fixative. The samples were then sequentially dehydrated in graded ethanol solutions of 50%, 70%, 90%, and 100%. After dehydration, critical point drying was performed. The dried samples were mounted onto SEM stubs and sputter-coated with a thin gold layer of approximately 10 nm. Bacterial morphology was examined using scanning electron microscopy at an accelerating voltage of 5 kV. Representative images were acquired at low and high magnification for each treatment group. SEM images were used as qualitative morphological observations rather than as quantitative evidence of a defined damage mechanism.

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 10.0. All quantitative experiments were performed with n = 3 independent biological replicates, and data are presented as mean ± standard deviation (SD). For TA fluorescence assays, statistical comparisons among multiple treatment groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test. For colony-counting assays in the primary strain involving multiple treatment conditions, statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. For rescue assays, between-group comparisons were performed using one-way ANOVA followed by Dunnett’s multiple-comparisons test, with the vitamin C–assisted Fenton group set as the reference. For limited cross-strain validation experiments, log₁₀ CFU reductions were compared between the conventional Fenton and vitamin C–assisted Fenton groups within each strain using an unpaired two-tailed t-test. For matrix-interference assays, two-way ANOVA was performed with treatment and BSA concentration as factors, including the interaction term. Pairwise comparisons between treatments at each BSA concentration were conducted using Šídák’s multiple-comparisons test. Statistical significance was defined as P < 0.05. Significance levels are indicated as *p < 0.05, ** P < 0.01, ***P < 0.001, and ****p < 0.0001.

Results

Evaluation of AO7 degradation in the vitamin C–assisted Fenton system
AO7 degradation was used as a chemical probe to examine how vitamin C influences the observable oxidative behavior of the Fenton reaction under acidic conditions. In the absence of vitamin C, the conventional Fenton system resulted in partial AO7 degradation over the 10 min reaction period. When vitamin C was introduced, the extent of dye degradation increased, with the effect depending on the molar ratio between vitamin C and Fe2⁺...

Discussion

The present study examined whether vitamin C dosing could modulate the oxidative behavior and antibacterial readouts of a Fenton-based system under defined acidic in vitro conditions. Across the chemical and microbiological assays, stepwise vitamin C supplementation altered the observable reaction behavior and was associated with greater antibacterial activity against H. pylori than the conventional Fenton reaction under the tested conditions. Importantly, this system should be interpreted as a simplifi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acid Orange 7 (AO7)	Aladdin Reagent (Shanghai) Co., Ltd.	O113230	Used as a model dye for the AO7 degradation assay.
Bovine serum albumin (BSA)	Shanghai Macklin Biochemical Technology Co., Ltd.	B885114	Used for the matrix-interference assay.
Brain Heart Infusion (BHI) broth powder	Sichuan Hapyear Bio-engineering Co., Ltd.	N/A	Used for bacterial culture and cryostorage.
Columbia blood agar plates (5% sheep blood)	Changde Bkmam Biotechnology Co., Ltd.	110701028.0	Used for bacterial culture and CFU enumeration.
Critical point dryer (EM CPD300)	Leica Microsystems	EM CPD300	Used for SEM sample preparation after graded ethanol dehydration.
Deferoxamine (DFO)	Shanghai Macklin Biochemical Technology Co., Ltd.	D873692	Used as an iron chelator in the rescue assay.
Disodium terephthalate	Shanghai Macklin Biochemical Technology Co., Ltd.	D835937	Used as the TA fluorescence probe for •OH-associated signal detection.
Ethanol, absolute	Sigma-Aldrich	E7023	Used to prepare the graded ethanol series for SEM sample dehydration.
Fluorescence microscope (DMi8)	Leica Microsystems	DMi8	Used for live/dead fluorescence imaging.
Glutaraldehyde solution	Sigma-Aldrich	G5882	Used for fixation of bacterial samples before SEM.
Glycerol	Sigma-Aldrich	G5516	Used for bacterial cryostorage in BHI broth.
GraphPad Prism 10	GraphPad Software, LLC	Prism 10	Used for statistical analysis and graphing.
Helicobacter pylori strain ATCC 26695	American Type Culture Collection (ATCC)	700392	Additional reference strain used for limited cross-strain validation.
Helicobacter pylori strain ATCC 43504	American Type Culture Collection (ATCC)	43504	Additional reference strain used for limited cross-strain validation.
Helicobacter pylori strain G27	Center of Infectious Diseases, West China Hospital, Sichuan University	N/A	Primary strain used for colony counting, rescue assay, matrix-interference assay, live/dead staining, and SEM.
Hydrogen peroxide solution	Sigma-Aldrich	H1009	Used as the peroxide substrate in Fenton reaction assays.
Iron(II) sulfate monohydrate (FeSO4·H2O)	Shanghai Macklin Biochemical Technology Co., Ltd.	F904376	Used as the ferrous ion source in the Fenton reaction.
Microplate reader (Synergy H1)	Agilent BioTek	Synergy H1	Used to measure TA-derived fluorescence (Ex/Em = 315/425 nm).
Phosphate-buffered saline (PBS), pH 7.4	Sigma-Aldrich	P4417	Used for post-treatment dilution/neutralization before plating and sample washing.
Physiological saline (0.9% sodium chloride solution)	Millipore	567442	Used for bacterial suspension and preparation of the acidic saline-urea model.
Scanning electron microscope (INSPECT F)	FEI Company	INSPECT F	Used for bacterial morphology analysis.
Sodium hydroxide	Sigma-Aldrich	S5881	Used to alkalinize TA assay aliquots and stabilize fluorescence.
Sputter coater (EM ACE200)	Leica Microsystems	EM ACE200	Used to apply the thin gold coating before SEM imaging.
SYTO 9/PI Live/Dead Bacterial Double Stain Kit	Shanghai Maokang Biotechnology Co., Ltd.	MX4234	Used for fluorescence-based live/dead bacterial staining.
Thiourea	Shanghai Macklin Biochemical Technology Co., Ltd.	T81602	Used as a radical scavenger in the rescue assay.
Urea	Shanghai Macklin Biochemical Technology Co., Ltd.	U820349	Used to prepare the acidic saline-urea model.
UV-Vis spectrophotometer (UV-2600i Plus)	Shimadzu Corporation	UV-2600i Plus	Used to measure AO7 absorbance at 483 nm.
Vitamin C (L-ascorbic acid)	Shanghai Macklin Biochemical Technology Co., Ltd.	A800295	Used as the reductant in the vitamin C-assisted Fenton system.