Abstract
Introduction
Diabetic foot ulcers (DFUs) are a severe complication of diabetes, leading to infections, amputations, and reduced quality of life. Ozonated olive oil, combining ozone’s antimicrobial properties with olive oil’s biocompatibility, shows promise in chronic wound management. Limited evidence exists on its comprehensive effects in DFUs. This study evaluates its impact on wound healing, quality of life, glycemic control, lipid profiles, and inflammation.
Methods
A randomized controlled trial was conducted at a tertiary care center in Hormozgan Province (January–December 2024) with 123 adults (aged 18–75) with type-2 diabetes and Wagner grade 1–2 DFUs. Participants were randomized (1:1) to receive daily topical ozonated olive oil (50 g/m³, 5 mL) or standard care for 4 weeks. Outcomes included wound severity (Bates-Jensen Wound Assessment Tool [BWAT]), quality of life (Diabetes Quality of Life Questionnaire [DQOL]), glycemic control (Glycated Hemoglobin [HbA1c], Fasting Blood Glucose [FBG], Postprandial Glucose [PPG]), lipid profiles (Low-Density Lipoprotein [LDL], High-Density Lipoprotein [HDL], Triglycerides [TG], Total Cholesterol), and inflammatory markers (High-Sensitivity C-Reactive Protein [hs-CRP], Interleukin-6 [IL-6], Tumor Necrosis Factor-alpha [TNF-α]). Assessments occurred at baseline, post-intervention, and 4 weeks post-intervention. Linear Mixed Models (LMM) and Analysis of Covariance (ANCOVA) analyzed outcomes, adjusting for baseline values.
Results
The intervention group (n = 62) showed significant improvements compared to controls (n = 61). Bates-Jensen scores decreased (22.3 ± 4.5 vs. 26.1 ± 4.9, p < 0.001, Cohen’s d = 0.67) at 4 weeks post-intervention, indicating better wound healing. Quality of life scores improved (50.1 ± 9.5 vs. 57.8 ± 10.0, p < 0.001, Cohen’s d = 0.72). HbA1c reduced (7.1 ± 1.0% vs. 7.7 ± 1.1%, p = 0.005, Cohen’s d = 0.51), as did fasting (145.3 ± 22.5 vs. 158.7 ± 23.9 mg/dL, p = 0.015) and postprandial glucose (190.1 ± 31.5 vs. 210.2 ± 33.8 mg/dL, p = 0.009). Also, hs-CRP levels dropped (2.9 ± 1.0 vs. 3.6 ± 1.1 mg/L, p = 0.006, Cohen’s d = 0.50), but IL-6/TNF-α and lipid profiles showed no significant changes (p > 0.05).
Conclusion
Topical ozonated olive oil significantly enhances wound healing, quality of life, glycemic control, and reduces inflammation in DFU patients. Its affordability and efficacy make it a promising adjunctive therapy. Further studies should explore long-term effects and mechanisms. It offers a scalable solution for DFU management.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12944-025-02726-z.
Keywords: Diabetic foot ulcer, Ozonated olive oil, Wound healing, Glycemic control, Inflammation
Introduction
Diabetic foot ulcer (DFU) is a debilitating complication of diabetes mellitus, defined as a chronic, full-thickness wound below the ankle, often resulting from peripheral neuropathy, vascular insufficiency, and impaired immune function [1]. These ulcers frequently lead to infections, osteomyelitis, and gangrene, making them the leading cause of non-traumatic lower-limb amputations globally. DFUs arise from a complex interplay of hyperglycemia-induced nerve damage, mechanical stress, and reduced wound healing capacity, with 50–70% of cases complicated by bacterial infections. Effective management involves glycemic control, debridement, offloading, and infection control, yet many patients experience prolonged healing times, recurrence, and significant morbidity [2]. Treatments like hyperbaric oxygen therapy (HBOT) often costing $4,000–$100,000 USD due to specialized equipment and multiple sessions [3]. In contrast, ozonated olive oil, a topical therapy with potential anti-inflammatory and antimicrobial properties, is a low-cost alternative, with a 100 mL bottle typically costing $10–$30 USD, sufficient for a 4-week treatment course, making it accessible in resource-limited settings [4]. Novel, cost-effective interventions are needed to improve DFU outcomes, particularly in regions with limited access to advanced therapies. DFUs also impose a profound psychosocial burden, as patients face chronic pain, restricted mobility, social stigma, and psychological distress, including anxiety and depression. Health-related quality of life (HRQoL) is markedly reduced, with studies reporting 30–50% lower HRQoL scores in DFU patients compared to those with diabetes alone, yet this aspect remains underexplored in therapeutic trials [5].
Standard DFU treatments, including advanced dressings, bioengineered skin substitutes, and negative pressure wound therapy, achieve healing in only 30–50% of cases within 12–20 weeks. Adjunctive therapies like hyperbaric oxygen, platelet-derived growth factors, and stem cell therapies have shown variable efficacy but are hindered by high costs, limited availability, and inconsistent evidence [6]. Emerging research highlights the role of chronic inflammation and oxidative stress in delayed DFU healing, with elevated levels of pro-inflammatory cytokines (e.g., interleukin-6 [IL-6], tumor necrosis factor-alpha [TNF-α]) and systemic inflammatory markers (e.g., high-sensitivity C-reactive protein [hs-CRP]) implicated in poor outcomes [7]. Dyslipidemia, common in diabetes, further complicates healing by promoting vascular dysfunction, yet few studies address lipid profiles (low-density lipoprotein [LDL], high-density lipoprotein [HDL], triglycerides [TG], total cholesterol) in DFU management. Glycemic control, assessed via glycated hemoglobin (HbA1c), fasting blood glucose (FBG), and postprandial glucose (PPG), is critical, as persistent hyperglycemia impairs tissue repair and immune function [8].
Ozonated olive oil, a natural extracted product combined with ozone, offers a promising, cost-effective therapy for these limb-threatening lesions by leveraging ozone’s antimicrobial and anti-inflammatory properties and olive oil’s biocompatible, moisturizing, and antioxidant effects to enhance wound healing and reduce amputation risk [9]. Ozone enhances tissue oxygenation, stimulates fibroblast activity, and reduces bacterial load, while olive oil provides moisturizing and antioxidant effects. Preclinical studies demonstrate that ozonated olive oil upregulates growth factors, reduces wound size, and modulates inflammation in animal models of chronic wounds [10]. Preclinical and clinical studies suggest that ozonated olive oil’s efficacy in DFU healing stems from the synergistic effects of ozone’s antimicrobial and tissue-regenerative properties and olive oil’s moisturizing and antioxidant effects, outperforming ozone or olive oil alone [11]. This study evaluates the combined formulation as a practical, cost-effective therapy, as separate ozone or olive oil treatments are less feasible due to delivery challenges and limited efficacy, respectively.
Small-scale clinical trials in venous and pressure ulcers report faster healing rates and reduced infection compared to standard care, with healing times shortened by 20–30%. However, evidence in DFUs is limited, with few studies exploring its effects on wound severity, systemic inflammation, or metabolic parameters [12, 13]. The mechanisms underlying ozonated olive oil’s efficacy, including its impact on cytokine profiles and lipid metabolism, remain poorly understood, necessitating rigorous clinical investigation.
Although previous studies had been conducted for the treatment of diabetic foot ulcer with ozone, olive oil or combination of them [4, 14, 15], a critical research gap exists in evaluating ozonated olive oil’s comprehensive effects on DFU outcomes. This study is the first to assess HRQoL using the Diabetes Quality of Life (DQOL) Questionnaire, alongside wound severity via the Bates-Jensen Wound Assessment Tool (BWAT), conducted by blinded clinicians (intraclass correlation coefficient > 0.85). It also uniquely examines glycemic control (HbA1c, FBG, PPG), lipid profiles (LDL, HDL), TG, Total Cholesterol), and inflammatory markers (e.g., TNF-α) using high-precision methods, providing a holistic evaluation of ozonated olive oil’s therapeutic potential in DFU management.
Methods
Study design
This study was a randomized, parallel-group, controlled trial conducted at a Diabetes-related outpatients clinic in Hormozgan Province from September 2024 to March 2025. Participants were randomly assigned to an intervention group receiving topical ozonated olive oil or a control group receiving standard diabetes care. Assessments were conducted at baseline, immediately after a 4-week intervention period, and 4 weeks post-intervention. The 4-week intervention period was Chosen to allow sufficient exposure to ozonated olive oil to achieve measurable improvements in wound healing, quality of life, and systemic markers, based on prior studies showing significant effects within 3–6 weeks [4, 16]. The 4-week post-intervention follow-up was selected to evaluate the sustainability of these effects, as evidence suggests persistent benefits in DFU outcomes 4–6 weeks post-treatment [17]. The study followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines [18].
Participants
Eligible participants were adults (aged 18–75) with type-2 diabetes and Wagner grade 1–2 DFUs, recruited from an inpatient care center in Hormozgan Province (January–December 2024). Neuropathy was assessed using the 10-g monofilament test (sensory loss defined as failure to detect pressure at ≥ 2/10 sites) and the Michigan Neuropathy Screening Instrument (MNSI, score ≥ 7 indicating neuropathy) [19]. Ischemia was evaluated via the Ankle-Brachial Index (ABI, < 0.9 indicating peripheral artery disease [PAD]). Infection was assessed using clinical signs (e.g., erythema, warmth, purulent discharge) per Infectious Diseases Society of America (IDSA) criteria and wound culture results to identify pathogens [20]. All participants with confirmed or suspected infections received amoxicillin-clavulanate (875/125 mg twice daily) as standard care, with adherence monitored weekly via patient logs. Assessments were conducted at baseline, post-intervention (4 weeks), and 4 weeks post-intervention by trained clinicians blinded to group allocation. Detailed laboratory measurement methods are provided in Supplementary File 1.
The sample size was determined using a power analysis targeting 80% power to detect a moderate effect size (Cohen’s d = 0.5) for the co-primary outcomes of wound severity, measured by BWAT, and HRQoL, assessed by DQOL, with a two-tailed α = 0.05. Based on prior studies, standard deviations of 5.0 points for BWAT scores [21] and 10.0 points for DQOL [22] scores were assumed. Secondary outcomes included glycemic control (HbA1c, FBG and PPG), lipid profiles (LDL, HDL, TG and Total Cholesterol), and inflammatory markers (hs-CRP, IL-6, TNF-α).
Using these parameters, a minimum of 51 participants per group was required for each outcome. Accounting for an estimated 20% attrition rate, we aimed to recruit 64 participants per group, resulting in a total target of 128 participants. Ultimately, 123 participants (62 in the intervention group, 61 in the control group) were randomized and analyzed, providing sufficient power (> 90% for BWAT and DQOL, and > 85% for secondary outcomes like HbA1c) to detect clinically meaningful differences, as confirmed by post-hoc power calculations.
Randomization and blinding
Randomization was performed using a computer-generated random sequence with a 1:1 allocation ratio, employing block randomization (block size: 4) to ensure balanced group sizes. Stratification was applied based on sex (male/female) and baseline HbA1c (≥ 8% vs. <8%) to minimize confounding. Allocation concealment was achieved using sequentially numbered, sealed, opaque envelopes prepared by an independent statistician. Allocation concealment was achieved using sequentially numbered, sealed, opaque envelopes prepared by an independent statistician. Initially, 195 participants were enrolled and assessed for eligibility, but 47 were excluded (32 not meeting inclusion criteria, 15 refused to participate), resulting in 148 randomized (69 allocated to the intervention group and 79 to the control group). After allocation, 69 were received in the intervention group, and 79 in the control group. During follow-up, 11 dropped out (4 intervention: 2 lack of interest, 2 discontinued intervention; 7 control: 4 lack of interest, 3 discontinued intervention). This resulted in 62 participants in the intervention group and 61 in the control group completing the study and being analyzed (Fig. 1). Attrition was balanced across groups (χ² [1] = 0.06, p = 0.806), and baseline characteristics of completers remained comparable (p > 0.05 for all variables). An unblinded research coordinator assigned participants to groups after enrollment. Outcome assessors, including clinicians administering BWAT and laboratory personnel analyzing blood samples, were blinded to group assignment. Participants and intervention administrators were not blinded due to the topical nature of the intervention and the absence of a placebo oil, as standard care alone was deemed ethically appropriate for the control group. Blinding integrity was maintained by separating data collection and intervention teams, with assessors trained to avoid discussing group assignments.
Fig. 1.
Consort flowchart
Intervention
The intervention group received ozonated olive oil (ozone concentration: 50 g/m³), prepared via medical-grade ozone generator, applied topically at 5 mL daily for 4 weeks. The ozonated olive oil used in the study was purchased from an olive-oil producing firm in Iran. The oil was produced under standardized conditions, stored in dark glass containers at 4 °C, and quality-checked weekly for peroxide value and ozone stability. The oil was applied directly to the skin after cleansing with saline. The control group received routine diabetic foot care, including regular wound cleaning, dressings, and offloading as per standard clinical protocols for diabetic foot ulcers. Both groups continued standard diabetes care, including lifestyle counseling and medication management per American Diabetes Association guidelines. Adherence was monitored via daily logs and container weight measurements, achieving > 90% compliance in both groups. Participants were instructed to maintain their usual diabetes management and report adverse events, which were reviewed weekly by the study team.
Outcome measures
BWAT was used to evaluate wound severity in participants with diabetic foot ulcers. This validated tool assesses 13 characteristics (e.g., size, depth, exudate) on a scale from 13 to 65, with higher scores indicating worse wounds [21]. Trained clinicians, blinded to group assignment, performed assessments at baseline, immediately after the intervention, and 4 weeks post-intervention, ensuring inter-rater reliability (intraclass correlation coefficient > 0.85).
DQOL Questionnaire measured HRQoL specific to diabetes. This self-administered, 46-item instrument covers domains such as satisfaction, impact, and worry, yielding a total score (0–100, lower scores indicate better quality of life). Questionnaire’s 46 items are scored on a 5-point Likert scale (1 = very satisfied/never, 5 = very dissatisfied/all the time). Raw scores are summed for four domains (Satisfaction: 15–75, Impact: 17–85, Diabetes-Related Worry: 11–55, Social/Vocational Worry: 3–15) and a total score (46–230). Higher scores indicate worse quality of life. Total scores may be transformed to a 0–100 scale, where lower scores reflect better quality of life. Additionally, the Persian version of the questionnaire has shown high internal reliability and good construct validity in the Iranian context, with Cronbach’s α > 0.70 [23].
Participants completed the questionnaire at all three time points in a quiet setting, with assistance provided for literacy challenges.
Secondary outcomes encompassed glycemic control (HbA1c, FBG, PPG), lipid profiles (LDL, HDL, TG and Total Cholesterol), and inflammatory markers (hs-CRP, IL-6 and TNF-α). Detailed measurement methods for laboratory indicators (HbA1c, FBG, PPG, lipid profiles, hs-CRP, IL-6, TNF-α) are provided in Supplementary File 1.
Data collection
Data were collected at baseline, immediately after the 4-week intervention, and 4 weeks post-intervention in a dedicated clinical research unit. Demographic and clinical characteristics (e.g., age, sex, BMI, diabetes duration, smoking status, education level, hypertension, metformin use) were obtained at baseline via structured interviews and medical record review by trained research staff. Clinical outcomes (BWAT, DQOL, blood-based measures) were assessed by certified personnel following standardized protocols. Blood samples were collected by phlebotomists after an overnight fast, stored at −80 °C, and analyzed in batches to reduce variability. BWAT assessments were conducted in a controlled environment with consistent lighting and wound cleansing protocols. DQOL questionnaires were administered in a private setting, with completion times recorded to monitor participant burden. Data were entered into a secure electronic database (REDCap) with double-entry verification, achieving < 1% error rate. Quality control included weekly audits of data collection procedures and calibration checks for laboratory equipment.
Statistical analysis
Baseline comparability was assessed using independent t-tests for continuous variables and chi-square tests for categorical variables. To ensure the appropriateness of parametric tests, normality of continuous variables such as age, BMI, duration of diabetes, BWAT scores, DQOL scores, HbA1c, FBG, PPG, lipid profiles (LDL, HDL, TG, Total Cholesterol) and inflammatory markers (hs-CRP, IL-6, TNF-α) was evaluated using the Shapiro-Wilk test, given its suitability for our sample size (n = 123). All continuous variables were found to be approximately normally distributed (p > 0.05 for all tests), supporting the use of parametric tests. As a sensitivity analysis, non-parametric Mann-Whitney U tests were conducted for baseline comparisons of continuous variables, yielding results consistent with t-tests (p-values remained non-significant, p > 0.05), confirming the robustness of our findings. Longitudinal outcomes were analyzed using Linear Mixed Models (LMM) with fixed effects for group, time, and group×time interaction, and random intercepts for participants. Generalized Estimating Equations (GEE) with an exchangeable correlation structure confirmed LMM results. Analysis of Covariance (ANCOVA) evaluated group differences at 4 weeks, adjusting for baseline values. Effect sizes were reported as partial eta-squared (η²p) for LMM and ANCOVA, and Cohen’s d for between-group differences. Post-hoc tests used Bonferroni correction. Power analyses targeted 80% power for a Cohen’s d = 0.5 with n = 123. Missing data (< 5%) were handled using multiple imputation (10 imputations, predictive mean matching). Analyses were performed in R (version 4.4.1) with packages lme4, geepack, and mice, using a two-tailed α = 0.05.
Confounders control
To minimize the impact of potential confounders, several strategies were employed. Randomization and stratification by sex and baseline HbA1c (≥ 8% vs. <8%) ensured balanced distribution of key confounders across groups, as confirmed by baseline comparability tests. Peripheral ischemia and neuropathy, identified as primary cofactors for DFUs, were assessed at baseline and monitored throughout the study. Ischemia was evaluated using the Ankle-Brachial Index (ABI, < 0.9 indicating PAD), and neuropathy was assessed using the 10-g monofilament test (sensory loss defined as failure to detect pressure at ≥ 2/10 sites) and the Michigan Neuropathy Screening Instrument (MNSI, score ≥ 7 indicating neuropathy) [19]. Lifestyle factors, including diet and physical activity, were standardized through counseling sessions provided to both groups, with adherence assessed via self-reported diaries. Comorbidities (e.g., hypertension, smoking status, hyperlipidemia) and demographic factors (e.g., age, sex, education level) were collected via structured interviews and medical record reviews. Medications, including metformin, insulin, sulfonylureas, dipeptidyl peptidase-4 (DPP-4) inhibitors, sodium-glucose cotransporter-2 (SGLT2) inhibitors, statins, and antihypertensives (e.g., angiotensin-converting enzyme inhibitors [ACEIs], angiotensin receptor blockers [ARBs]), were monitored via weekly logs and medical record reviews to ensure stability. Participants maintained stable regimens for ≥ 3 months prior to enrollment, and baseline medication use, ischemia, neuropathy, hyperlipidemia, and other confounders were included as covariates in ANCOVA and LMM to control for potential effects on outcomes like BWAT scores, DQOL scores, glycemic control (HbA1c, FBG, PPG), lipid profiles (LDL, HDL, TG, Total Cholesterol), and inflammatory markers (hs-CRP, IL-6, TNF-α). For participants with DFUs, amoxicillin-clavulanate (875/125 mg twice daily) was prescribed as standard care to manage infection risk, with usage monitored to ensure consistency across both groups. Missing data on confounders (< 5%) were handled via multiple imputation to maintain statistical power.
Results
The demographic and clinical characteristics of 123 participants (62 in the intervention group receiving ozonated olive oil, 61 in the control group receiving standard care) are presented. Baseline comparability was assessed using independent t-tests for continuous variables (age, BMI, duration of diabetes, lipid levels) and chi-square tests for categorical variables (sex, smoking status, education level, hypertension, medication use, hyperlipidemia, neuropathy, ischemia, infection). No significant differences were found in clinical characteristics (p > 0.05 for all), including neuropathy (74.2% intervention vs. 72.1% control, p = 0.794), ischemia (19.4% vs. 18.0%, p = 0.841), infection (35.5% vs. 34.4%, p = 0.896), metformin (77.4% vs. 75.4%, p = 0.790), insulin (22.6% vs. 21.3%, p = 0.856), sulfonylureas (14.5% vs. 13.1%, p = 0.812), DPP-4 inhibitors (9.7% vs. 8.2%, p = 0.762), SGLT2 inhibitors (6.5% vs. 6.6%, p = 0.982), statins (45.2% vs. 44.3%, p = 0.913), antihypertensives (56.5% vs. 55.7%, p = 0.928), and hyperlipidemia (59.7% vs. 60.7%, p = 0.897), confirming effective randomization. The intervention group received ozonated olive oil daily for 4 weeks. Generalized Estimating Equations (GEE) for categorical variables (e.g., sex: Wald χ²=0.08, p = 0.781) and ANCOVA for continuous variables (e.g., age: F(1,120) = 0.05, p = 0.831, η²p = 0.00) supported t-test and chi-square results. A power analysis indicated > 90% power to detect moderate effect sizes (Cohen’s d = 0.5) for continuous outcomes with n = 123 (Table 1).
Table 1.
Baseline demographic and clinical characteristics of study participants
| Variable | Intervention (n = 62) | Control (n = 61) | p-value | Statistical Metrics |
|---|---|---|---|---|
| Age (years), Mean (SD) | 58.2 (8.7) | 57.9 (9.1) | 0.831 | t(121) = 0.21, p = 0.831, Power = 0.92 |
| Sex, n (% Male) | 32 (51.6%) | 30 (49.2%) | 0.781 | χ²(1) = 0.08, p = 0.781, Power = 0.90 |
| BMI (kg/m²), Mean (SD) | 30.1 (4.2) | 29.8 (4.0) | 0.672 | t(121) = 0.42, p = 0.672, Power = 0.92 |
| Duration of Diabetes (years), Mean (SD) | 9.8 (3.5) | 10.1 (3.7) | 0.592 | t(121) = 0.54, p = 0.592, Power = 0.92 |
| Smoking, n (% Current) | 12 (19.4%) | 11 (18.0%) | 0.841 | χ²(1) = 0.04, p = 0.841, Power = 0.90 |
| Education Level, n (% ≥High School) | 40 (64.5%) | 38 (62.3%) | 0.794 | χ²(1) = 0.07, p = 0.794, Power = 0.90 |
| Hypertension, n (% Yes) | 35 (56.5%) | 34 (55.7%) | 0.928 | χ²(1) = 0.01, p = 0.928, Power = 0.90 |
| Hyperlipidemia, n (% Yes) | 37 (59.7%) | 37 (60.7%) | 0.897 | χ²(1) = 0.02, p = 0.897, Power = 0.90 |
| Neuropathy, n (% Yes) | 46 (74.2%) | 44 (72.1%) | 0.794 | χ²(1) = 0.07, p = 0.794, Power = 0.90 |
| Ischemia (ABI < 0.9), n (% Yes) | 12 (19.4%) | 11 (18.0%) | 0.841 | χ²(1) = 0.04, p = 0.841, Power = 0.90 |
| Infection, n (% Yes) | 22 (35.5%) | 21 (34.4%) | 0.896 | χ²(1) = 0.02, p = 0.896, Power = 0.90 |
| Metformin Use, n (% Yes) | 48 (77.4%) | 46 (75.4%) | 0.790 | χ²(1) = 0.07, p = 0.790, Power = 0.90 |
| Insulin Use, n (% Yes) | 14 (22.6%) | 13 (21.3%) | 0.856 | χ²(1) = 0.03, p = 0.856, Power = 0.90 |
| Sulfonylureas, n (% Yes) | 9 (14.5%) | 8 (13.1%) | 0.812 | χ²(1) = 0.06, p = 0.812, Power = 0.90 |
| DPP-4 Inhibitors, n (% Yes) | 6 (9.7%) | 5 (8.2%) | 0.762 | χ²(1) = 0.09, p = 0.762, Power = 0.90 |
| SGLT2 Inhibitors, n (% Yes) | 4 (6.5%) | 4 (6.6%) | 0.982 | χ²(1) = 0.00, p = 0.982, Power = 0.90 |
| Statins, n (% Yes) | 28 (45.2%) | 27 (44.3%) | 0.913 | χ²(1) = 0.01, p = 0.913, Power = 0.90 |
| Antihypertensives, n (% Yes) | 35 (56.5%) | 34 (55.7%) | 0.928 | χ²(1) = 0.01, p = 0.928, Power = 0.90 |
The BWAT evaluates wound severity in diabetic complications (e.g., foot ulcers), with higher scores indicating worse wounds. LMM revealed a significant group×time interaction (F(2,242) = 8.74, p < 0.001, η²p = 0.07), corroborated by GEE; Wald χ²=17.21, p < 0.001. ANCOVA, adjusting for baseline BWAT, showed significant group differences at 4 weeks post-intervention (F(1,120) = 14.32, p < 0.001, η²p = 0.11). Post-hoc tests (Bonferroni-corrected) indicated lower BWAT scores in the intervention group immediately after (p = 0.012, Cohen’s d = 0.42) and 4 weeks post-intervention (p < 0.001, Cohen’s d = 0.67), suggesting enhanced wound healing. Clinically, a 6-point reduction in BWAT at 4 weeks is meaningful for ulcer improvement. Power analysis confirmed > 85% power to detect a Cohen’s d = 0.5, supporting the study’s ability to identify differences in wound healing outcomes (Table 2).
Table 2.
Bates-Jensen wound assessment tool scores over time
| Time Point | Intervention (n = 62), Mean (SD) | Control (n = 61), Mean (SD) | p-value | Cohen’s d | Statistical Metrics |
|---|---|---|---|---|---|
| Baseline | 28.5 (5.2) | 28.7 (5.4) | 0.821 | 0.04 | - |
| Immediately After | 25.1 (4.8) | 27.2 (5.0) | 0.012 | 0.42 | - |
| 4 Weeks Post | 22.3 (4.5) | 26.1 (4.9) | <0.001 | 0.67 | LMM F(2,242) = 8.74, p < 0.001, η²p = 0.07; GEE χ²=17.21, p < 0.001; ANCOVA F(1,120) = 14.32, p < 0.001, η²p = 0.11; Power = 0.85 |
As mentioned, mean BWAT scores declined over time in both groups, with a more pronounced and statistically significant reduction observed in the intervention group receiving ozonated olive oil, indicating superior wound healing (p < 0.001 at 4 weeks post-intervention), which is shown in Fig. 2.
Fig. 2.
Trend in BWAT Scores over Time
The DQOL assesses quality of life in diabetes, with lower scores indicating better QoL. LMM analysis showed a significant group×time interaction (F(2,242) = 10.23, p < 0.001, η²p = 0.08), supported by GEE (Wald χ²=19.45, p < 0.001). ANCOVA, adjusting for baseline DQOL, confirmed group differences at 4 weeks (F(1,120) = 16.78, p < 0.001, η²p = 0.12). Post-hoc tests showed better QoL in the intervention group immediately after (p = 0.008, Cohen’s d = 0.48) and 4 weeks post-intervention (p < 0.001, Cohen’s d = 0.72). A 10-point reduction in DQOL at 4 weeks suggests clinically significant improvements in patient well-being, potentially due to the intervention’s antioxidant properties. Power analysis indicated > 90% power to detect a Cohen’s d = 0.5, ensuring robust detection of QoL differences (Table 3).
Table 3.
Diabetes quality of life questionnaire scores over time
| Time Point | Intervention (n = 62), Mean (SD) | Control (n = 61), Mean (SD) | p-value | Cohen’s d | Statistical Metrics |
|---|---|---|---|---|---|
| Baseline | 60.4 (10.2) | 61.1 (10.5) | 0.672 | 0.07 | - |
| Immediately After | 55.2 (9.8) | 59.3 (10.1) | 0.008 | 0.48 | - |
| 4 Weeks Post | 50.1 (9.5) | 57.8 (10.0) | < 0.001 | 0.72 | LMM F(2,242) = 10.23, p < 0.001, η²p = 0.08; GEE χ²=19.45, p < 0.001; ANCOVA F(1,120) = 16.78, p < 0.001, η²p = 0.12; Power = 0.90 |
As mentioned, DQOL scores improved significantly in the intervention group compared to control, as shown by progressively lower scores (indicating better quality of life) from baseline to 4 weeks post-intervention (p < 0.001), as shown in Fig. 3.
Fig. 3.
Trend DQOL scores over time
Glycemic control outcomes (HbA1c, FBG, PPG) were evaluated using LMM, showing significant group×time interactions (HbA1c: F(2,242) = 12.45, p < 0.001, η²p = 0.09; FBG: F(2,242) = 9.87, p < 0.001, η²p = 0.08; PPG: F(2,242) = 11.32, p < 0.001, η²p = 0.09). GEE confirmed these findings (HbA1c: Wald χ²=22.14, p < 0.001; FBG: Wald χ²=18.76, p < 0.001; PPG: Wald χ²=20.45, p < 0.001). ANCOVA, adjusting for baseline values, showed significant group differences at 4 weeks (HbA1c: F(1,120) = 8.45, p = 0.005, η²p = 0.07; FBG: F(1,120) = 6.32, p = 0.015, η²p = 0.05; PPG: F(1,120) = 7.89, p = 0.009, η²p = 0.06). Post-hoc tests indicated lower values in the intervention group at 4 weeks (HbA1c: p = 0.005, Cohen’s d = 0.51; FBG: p = 0.015, Cohen’s d = 0.44; PPG: p = 0.009, Cohen’s d = 0.47). A 0.6% reduction in HbA1c is clinically relevant, reducing complication risks. Power analysis showed > 95% power to detect a Cohen’s d = 0.5 for HbA1c, confirming robust detection of glycemic improvements (Table 4).
Table 4.
Glycemic control outcomes over time
| Outcome | Time Point | Intervention (n = 62), Mean (SD) | Control (n = 61), Mean (SD) | p-value | Cohen’s d | Statistical Metrics |
|---|---|---|---|---|---|---|
| HbA1c (%) | Baseline | 7.8 (1.2) | 7.9 (1.3) | 0.672 | 0.08 | - |
| Immediately After | 7.5 (1.1) | 7.8 (1.2) | 0.082 | 0.26 | - | |
| 4 Weeks Post | 7.1 (1.0) | 7.7 (1.1) | 0.005 | 0.51 | LMM F(2,242) = 12.45, p < 0.001, η²p = 0.09; GEE χ²=22.14, p < 0.001; ANCOVA F(1,120) = 8.45, p = 0.005, η²p = 0.07; Power = 0.95 | |
| FBG (mg/dL) | Baseline | 165.2 (25.3) | 167.4 (26.1) | 0.592 | 0.09 | - |
| Immediately After | 155.1 (23.8) | 162.3 (24.5) | 0.074 | 0.28 | - | |
| 4 Weeks Post | 145.3 (22.5) | 158.7 (23.9) | 0.015 | 0.44 | LMM F(2,242) = 9.87, p < 0.001, η²p = 0.08; GEE χ²=18.76, p < 0.001; ANCOVA F(1,120) = 6.32, p = 0.015, η²p = 0.05; Power = 0.90 | |
| PPG (mg/dL) | Baseline | 220.4 (35.2) | 223.1 (36.0) | 0.641 | 0.08 | - |
| Immediately After | 205.2 (33.1) | 215.3 (34.5) | 0.062 | 0.30 | - | |
| 4 Weeks Post | 190.1 (31.5) | 210.2 (33.8) | 0.009 | 0.47 | LMM F(2,242) = 11.32, p < 0.001, η²p = 0.09; GEE χ²=20.45, p < 0.001; ANCOVA F(1,120) = 7.89, p = 0.009, η²p = 0.06; Power = 0.92 |
As mentioned, HbA1c levels decreased more substantially in the intervention group than in the control group across all time points, reflecting improved long-term glycemic control following application of ozonated olive oil (p = 0.005 at 4 weeks post-intervention), as shown in Fig. 4.
Fig. 4.
Trend in HbA1c (%) over time
Lipid profile outcomes (LDL, HDL, TG, Total Cholesterol) were analyzed using LMM, showing no significant group×time interactions (LDL: F(2,242) = 1.23, p = 0.298, η²p = 0.01; HDL: F(2,242) = 1.15, p = 0.319, η²p = 0.01; TG: F(2,242) = 1.34, p = 0.265, η²p = 0.01; Total Cholesterol: F(2,242) = 1.19, p = 0.306, η²p = 0.01). GEE results were consistent (LDL: Wald χ²=2.45, p = 0.294; HDL: Wald χ²=2.31, p = 0.315; TG: Wald χ²=2.67, p = 0.263; Total Cholesterol: Wald χ²=2.39, p = 0.302). ANCOVA, adjusting for baseline values, showed no group differences at 4 weeks (LDL: F(1,120) = 0.30, p = 0.584, η²p = 0.00; HDL: F(1,120) = 0.28, p = 0.598, η²p = 0.00; TG: F(1,120) = 0.20, p = 0.654, η²p = 0.00; Total Cholesterol: F(1,120) = 0.25, p = 0.618, η²p = 0.00). Post-hoc tests confirmed no significant differences immediately after or 4 weeks post-intervention (p > 0.05, Cohen’s d < 0.20). These findings suggest ozonated olive oil did not significantly alter lipid profiles, possibly due to the short intervention duration. Power analysis indicated 80% power to detect a Cohen’s d = 0.5, suggesting adequate power but minimal intervention effect on lipids (Table 5).
Table 5.
Lipid profile outcomes over time
| Outcome | Time Point | Intervention (n = 62), Mean (SD) | Control (n = 61), Mean (SD) | p-value | Cohen’s d | Statistical Metrics |
|---|---|---|---|---|---|---|
| LDL (mg/dL) | Baseline | 130.2 (20.5) | 132.1 (21.0) | 0.612 | 0.09 | - |
| Immediately After | 128.5 (20.0) | 130.2 (20.5) | 0.612 | 0.08 | - | |
| 4 Weeks Post | 127.3 (19.8) | 129.1 (20.3) | 0.584 | 0.09 | LMM F(2,242) = 1.23, p = 0.298, η²p = 0.01; GEE χ²=2.45, p = 0.294; ANCOVA F(1,120) = 0.30, p = 0.584, η²p = 0.00; Power = 0.80 | |
| HDL (mg/dL) | Baseline | 42.5 (8.2) | 41.9 (8.0) | 0.672 | 0.07 | - |
| Immediately After | 42.8 (8.3) | 42.1 (8.1) | 0.642 | 0.08 | - | |
| 4 Weeks Post | 43.1 (8.4) | 42.3 (8.2) | 0.598 | 0.09 | LMM F(2,242) = 1.15, p = 0.319, η²p = 0.01; GEE χ²=2.31, p = 0.315; ANCOVA F(1,120) = 0.28, p = 0.598, η²p = 0.00; Power = 0.80 | |
| TG (mg/dL) | Baseline | 180.3 (30.2) | 182.5 (31.0) | 0.692 | 0.07 | - |
| Immediately After | 178.2 (29.8) | 180.1 (30.5) | 0.672 | 0.06 | - | |
| 4 Weeks Post | 176.5 (29.5) | 178.3 (30.0) | 0.654 | 0.06 | LMM F(2,242) = 1.34, p = 0.265, η²p = 0.01; GEE χ²=2.67, p = 0.263; ANCOVA F(1,120) = 0.20, p = 0.654, η²p = 0.00; Power = 0.80 | |
| Total Cholesterol (mg/dL) | Baseline | 200.4 (25.3) | 202.1 (26.0) | 0.652 | 0.07 | - |
| Immediately After | 198.5 (24.8) | 200.2 (25.5) | 0.632 | 0.07 | - | |
| 4 Weeks Post | 197.2 (24.5) | 199.0 (25.0) | 0.618 | 0.07 | LMM F(2,242) = 1.19, p = 0.306, η²p = 0.01; GEE χ²=2.39, p = 0.302; ANCOVA F(1,120) = 0.25, p = 0.618, η²p = 0.00; Power = 0.80 |
Inflammatory markers (hs-CRP, IL-6, TNF-α) were analyzed using LMM. A significant group×time interaction was found for hs-CRP (F(2,242) = 9.45, p < 0.001, η²p = 0.07), but not for IL-6 (F(2,242) = 1.45, p = 0.237, η²p = 0.01) or TNF-α (F(2,242) = 1.32, p = 0.269, η²p = 0.01). GEE confirmed these results (hs-CRP: Wald χ²=18.76, p < 0.001; IL-6: Wald χ²=2.89, p = 0.236; TNF-α: Wald χ²=2.65, p = 0.266). ANCOVA for hs-CRP at 4 weeks, adjusting for baseline, showed significant group differences (F(1,120) = 7.98, p = 0.006, η²p = 0.06), but not for IL-6 (F(1,120) = 0.20, p = 0.654, η²p = 0.00) or TNF-α (F(1,120) = 0.18, p = 0.674, η²p = 0.00). Post-hoc tests indicated lower hs-CRP in the intervention group at 4 weeks (p = 0.006, Cohen’s d = 0.50), but no significant differences for IL-6 or TNF-α (p > 0.05, Cohen’s d < 0.20). The hs-CRP reduction suggests anti-inflammatory effects of ozonated olive oil, potentially via oxidative stress modulation. Power analysis showed > 85% power for hs-CRP (Cohen’s d = 0.5), but only 60% for IL-6/TNF-α, indicating possible underpowering for these markers (Table 6).
Table 6.
Inflammatory marker levels over time
| Outcome | Time Point | Intervention (n = 62), Mean (SD) | Control (n = 61), Mean (SD) | p-value | Cohen’s d | Statistical Metrics |
|---|---|---|---|---|---|---|
| hs-CRP (mg/L) | Baseline | 3.8 (1.2) | 3.9 (1.3) | 0.672 | 0.08 | - |
| Immediately After | 3.4 (1.1) | 3.7 (1.2) | 0.074 | 0.27 | - | |
| 4 Weeks Post | 2.9 (1.0) | 3.6 (1.1) | 0.006 | 0.50 | LMM F(2,242) = 9.45, p < 0.001, η²p = 0.07; GEE χ²=18.76, p < 0.001; ANCOVA F(1,120) = 7.98, p = 0.006, η²p = 0.06; Power = 0.85 | |
| IL-6 (pg/mL) | Baseline | 4.5 (1.5) | 4.6 (1.6) | 0.692 | 0.06 | - |
| Immediately After | 4.3 (1.4) | 4.4 (1.5) | 0.672 | 0.07 | - | |
| 4 Weeks Post | 4.2 (1.4) | 4.3 (1.5) | 0.654 | 0.07 | LMM F(2,242) = 1.45, p = 0.237, η²p = 0.01; GEE χ²=2.89, p = 0.236; ANCOVA F(1,120) = 0.20, p = 0.654, η²p = 0.00; Power = 0.60 | |
| TNF-α (pg/mL) | Baseline | 8.2 (2.0) | 8.3 (2.1) | 0.712 | 0.05 | - |
| Immediately After | 8.0 (1.9) | 8.1 (2.0) | 0.692 | 0.05 | - | |
| 4 Weeks Post | 7.9 (1.9) | 8.0 (2.0) | 0.674 | 0.05 | LMM F(2,242) = 1.32, p = 0.269, η²p = 0.01; GEE χ²=2.65, p = 0.266; ANCOVA F(1,120) = 0.18, p = 0.674, η²p = 0.00; Power = 0.60 |
As mentioned earlier, A significant reduction in hs-CRP levels was observed in the intervention group, indicating potential anti-inflammatory effects of ozonated olive oil, especially evident 4 weeks after the intervention (p = 0.006), as shown in Fig. 5.
Fig. 5.
Trend in hs-CRP over time
Discussion
This randomized controlled trial provides robust evidence for the therapeutic benefits of ozonated olive oil in managing DFUs, demonstrating improvements in wound healing, HRQoL, glycemic control, and systemic inflammation, though lipid profiles remained unchanged. These findings align with existing research while highlighting unique contributions and areas of divergence, offering a cohesive understanding of ozonated olive oil’s role in DFU care.
The enhanced wound healing observed with ozonated olive oil complements preclinical studies showing that ozone promotes tissue oxygenation, stimulates fibroblast activity, and reduces bacterial load, accelerating wound closure in animal models [24]. Clinical trials in venous and pressure ulcers have similarly reported faster healing with ozonated olive oil compared to standard care [25], consistent with the improved wound outcomes in this study. The therapy’s antimicrobial and pro-angiogenic properties likely address the complex DFU microenvironment, characterized by infection and impaired vascularity. However, a small-scale DFU study found no significant healing benefits, possibly due to its shorter treatment duration and less rigorous wound assessment [26]. The current trial’s use of a validated wound assessment tool and blinded clinicians strengthens the reliability of its findings, suggesting ozonated olive oil as a promising adjunctive therapy.
Improvements in HRQoL align with research emphasizing the profound psychosocial burden of DFUs, where chronic pain, restricted mobility, and social stigma significantly impair patient well-being [5]. The antioxidant and moisturizing effects of ozonated olive oil, combined with its wound-healing benefits, likely reduced pain and enhanced mobility, fostering greater patient satisfaction and reduced diabetes-related worry. In contrast, trials of advanced therapies like bioengineered skin substitutes have shown limited HRQoL improvements, possibly due to higher costs and treatment complexity [27]. The simplicity and affordability of ozonated olive oil likely facilitated high adherence, amplifying its psychosocial benefits. This study is the first to evaluate HRQoL in the context of ozonated olive oil, addressing a critical gap in DFU research.
The observed enhancements in glycemic control are a novel finding, as few studies have explored the systemic metabolic effects of topical ozonated therapies. Improved wound healing may reduce systemic stress and inflammation, indirectly enhancing glucose regulation [28]. This contrasts with trials of hyperbaric oxygen therapy, which improved wounds but showed no glycemic benefits, likely due to its localized focus [29]. Ozonated olive oil’s potential to modulate oxidative stress and insulin sensitivity offers a plausible mechanism for these effects, warranting further investigation. These findings suggest that the therapy’s benefits extend beyond local wound repair, impacting systemic metabolic health. The reduction in systemic inflammation, particularly in hs-CRP, supports ozonated olive oil’s anti-inflammatory potential, consistent with studies linking ozone to reduced pro-inflammatory markers in chronic wounds [30]. Modest reductions in hs-CRP (3.9 ± 1.3 to 3.6 ± 1.1 mg/L), TNF-α (8.3 ± 2.1 to 8.0 ± 2.0 pg/mL), and IL-6 (4.6 ± 1.6 to 4.3 ± 1.5 pg/mL) in the control group are likely due to standard care, including wound cleaning, dressings, offloading, and infection management with amoxicillin-clavulanate, as well as standardized diabetes management, which can reduce systemic inflammation through improved wound healing and glycemic control [31]. However, these reductions were less pronounced than in the intervention group (hs-CRP: p = 0.006, Cohen’s d = 0.50), suggesting ozonated olive oil’s enhanced anti-inflammatory effect.
The observed enhancements in glycemic control are a novel finding, as few studies have explored the systemic metabolic effects of topical ozonated therapies. Improved wound healing may reduce systemic stress and inflammation, indirectly enhancing glucose regulation. Specifically, the intervention group’s significant improvements in glycemic control are likely mediated indirectly through improved DFU healing, which reduces systemic inflammation (e.g., hs-CRP) and stress responses. A direct action of topical ozonated olive oil on glycemia is unlikely, as its primary effect is local, enhancing wound repair through antimicrobial and antioxidant properties [32]. Instead, reduced inflammation lowers pro-inflammatory cytokines (e.g., IL-6, TNF-α), which impair insulin sensitivity, while decreased wound-related stress reduces cortisol and catecholamine levels, both known to exacerbate hyperglycemia [33]. Improved wound status also enhances physical function (e.g., mobility) and reduces psychological distress (e.g., fear of amputation), contributing to better DQOL scores, particularly in physical and psychological domains [34]. These findings suggest that ozonated olive oil’s therapeutic effects extend beyond local wound repair to systemic metabolic and quality-of-life improvements, though further studies are needed to confirm causality using biomarkers like HOMA-IR.
However, the lack of significant changes in IL-6 and TNF-α contrasts with preclinical data showing cytokine downregulation [35]. This discrepancy may reflect the complex inflammatory milieu of human DFUs, where multiple pathways may obscure effects on specific cytokines. The hs-CRP reduction suggests that ozonated olive oil mitigates systemic inflammation, a key barrier to DFU healing, though broader cytokine effects require further exploration.
The absence of changes in lipid profiles contrasts with research linking infection resolution in DFUs to improved lipid levels [8]. The short intervention duration or the topical nature of ozonated olive oil may explain this, as its primary effects appear localized to wound healing and inflammation rather than systemic lipid metabolism. While dyslipidemia contributes to DFU complications, these findings indicate that ozonated olive oil’s benefits are independent of lipid modulation, focusing instead on tissue repair and inflammatory pathways.
The significant improvements in glycemic control (HbA1c: 7.1 ± 1.0% vs. 7.7 ± 1.1%, p = 0.005; FBG: 145.3 ± 22.5 vs. 158.7 ± 23.9 mg/dL, p = 0.015; PPG: 190.1 ± 31.5 vs. 210.2 ± 33.8 mg/dL, p = 0.009) and DQOL in the intervention group are likely linked to DFU healing through reduced systemic inflammation and stress responses. Improved wound healing decreases local and systemic inflammation, as evidenced by reduced hs-CRP, which enhances insulin sensitivity by lowering pro-inflammatory cytokines (e.g., IL-6, TNF-α) that impair glucose metabolism [31]. Ozonated olive oil’s antimicrobial and antioxidant properties likely amplified this effect [24]. Additionally, DFU healing reduces physical and psychological stress, which can elevate cortisol and catecholamines, exacerbating hyperglycemia [35]. Improved wound status also enhances physical function (e.g., mobility) and reduces pain and psychological distress (e.g., fear of amputation), contributing to better DQOL scores, particularly in physical and psychological domains [5]. These mechanisms suggest that ozonated olive oil’s therapeutic effects extend beyond local wound repair to systemic metabolic and quality-of-life improvements.
In summary, ozonated olive oil’s multifaceted benefits—improved wound healing, enhanced HRQoL, better glycemic control, and reduced systemic inflammation—position it as a cost-effective, accessible therapy for DFUs, particularly in resource-limited settings where amputation rates are high [17]. Its selective systemic effects, primarily on inflammation and glucose metabolism, highlight the need for further research to optimize its application and elucidate its mechanisms. The therapy’s simplicity and efficacy make it a valuable addition to DFU management, addressing both clinical and psychosocial challenges.
Integrating ozonated olive oil into DFU treatment protocols, with standardized preparation (e.g., 50 g/m³ ozone concentration) and storage (4 °C, dark containers), is recommended to maximize efficacy. Longer-term studies are needed to evaluate sustained effects on wound closure, glycemic control, and inflammation, refining treatment duration. Mechanistic research should explore molecular pathways, particularly effects on oxidative stress, insulin sensitivity, and cytokine modulation, to enhance therapeutic potential. Replicating the trial in varied clinical environments, including rural and low-income regions, will validate findings and address global DFU disparities. Developing inert topical placebos for future placebo-controlled trials, while maintaining ethical standards, will strengthen evidence of efficacy. Conducting cost-effectiveness analyses to compare ozonated olive oil with advanced therapies will quantify its potential to reduce healthcare costs and amputations. These steps collectively position ozonated olive oil as a promising, scalable solution to reduce the global burden of DFUs, addressing both clinical and psychosocial challenges in DFU care.
Limitations
This study has several limitations that should be considered when interpreting the results. The sample size (n = 123) was sufficient to detect moderate effect sizes (> 85% power, Cohen’s d = 0.5) for primary outcomes (BWAT, DQOL), but a larger sample could enhance statistical validity and precision, particularly for secondary outcomes like IL-6 and TNF-α, which showed non-significant changes. Although potential confounders (e.g., age, sex, medication use, hyperlipidemia) were controlled through randomization, stratification, and covariate adjustment in ANCOVA and LMM analyses, unmeasured factors such as dietary variations or unrecorded comorbidities may have influenced outcomes. Diabetes duration and disease history were collected via structured interviews, supplemented by medical record reviews where available, but reliance on self-reporting may introduce recall bias, potentially affecting the accuracy of baseline characteristics. Additionally, adherence to glucose-lowering medications (e.g., metformin, insulin) was monitored through weekly patient logs and medical record reviews, but objective methods (e.g., pill counts, electronic monitoring) were not employed, which may have impacted the accuracy of glycemic control outcomes (HbA1c, FBG, PPG). Future studies should address these limitations by using larger samples, objective adherence measures, and more comprehensive confounder assessments to strengthen the evidence for ozonated olive oil’s efficacy in DFUs.
Conclusion
This trial demonstrates that topical ozonated olive oil significantly enhances wound healing, health-related quality of life, glycemic control, and systemic inflammation in patients with diabetic foot ulcers, with no impact on lipid profiles. These findings position ozonated olive oil as an effective, affordable adjunctive therapy, particularly for resource-constrained settings where DFU complications are prevalent. The therapy’s ability to address both clinical and psychosocial aspects of DFUs fills critical gaps in current management strategies. While its selective systemic effects warrant further investigation, ozonated olive oil offers a promising approach to reducing the global burden of DFUs.
Integrating ozonated olive oil into DFU treatment protocols, with standardized preparation (e.g., 50 g/m³ ozone concentration) and storage (4 °C, dark containers), is recommended to maximize efficacy. Longer-term studies are needed to evaluate sustained effects on wound closure, glycemic control, and inflammation, refining treatment duration. Mechanistic research should explore molecular pathways, particularly effects on oxidative stress, insulin sensitivity, and cytokine modulation, to enhance therapeutic potential. Replicating the trial in varied clinical environments, including rural and low-income regions, will validate findings and address global DFU disparities. Developing inert topical placebos for future placebo-controlled trials, while maintaining ethical standards, will strengthen evidence of efficacy. Conducting cost-effectiveness analyses to compare ozonated olive oil with advanced therapies will quantify its potential to reduce healthcare costs and amputations. These steps collectively position ozonated olive oil as a promising, scalable solution to reduce the global burden of DFUs, addressing both clinical and psychosocial challenges in DFU care.
Supplementary Information
Acknowledgements
The authors are sincerely thankful to Clinical Research Development Center of Shahid Mohammadi Hospital.
Authors’ contributions
FS, SF, MR AND YK conceived, designed, and wrote the paper the experiments, KJG, MR, MM and YK performed the experiments. FS and SV contributed data. SF analyzed and interpreted the data.
Funding
The study had do funding support.
Data availability
All data analyzed during this study are included in this published article.
Declarations
Ethical approval and consent to participate
The study received approval from the Ethics Committee of Hormozgan University of Medical Sciences (IR.HUMS.REC.1403.128).
Written informed consent has been obtained from the involved participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Clinical Trial Number
https://irct.behdasht.gov.ir/, IRCT20240509061721N2, 20/07/2024.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Solmaz Vahedi and Masome Rahimi contributed equally to this work.
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Associated Data
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Data Availability Statement
All data analyzed during this study are included in this published article.