The effects of Mixodin supplementation on migraine headache characteristics, oxidative stress and inflammatory biomarkers in patients with migraine: a double-blind, placebo-controlled, randomized trial

Abstract

Background

Migraine is a prevalent neurological disorder closely linked to oxidative stress and neurogenic inflammation. Curcumin, gingerol, and piperine are natural compounds with well-established anti-inflammatory and antioxidant properties; however, clinical evidence on their combined effects in migraine remains limited.

Aim

This study aimed to evaluate the effects of eight weeks of Mixodin supplementation on inflammatory and oxidative stress biomarkers, and clinical migraine characteristics, in patients with migraine.

Methods

This randomized, double-blind, placebo-controlled trial enrolled 60 patients with migraine, who were randomly assigned to receive two Mixodin capsules daily (each containing 300 mg curcumin, 7.5 mg gingerol, and 3.75 mg piperine) or placebo for eight weeks. Serum hs-CRP, NO, MDA, TOS, TAC, and SOD were measured before and after the intervention. Headache severity, frequency, and duration were recorded using VAS and a headache diary.

Results

Mixodin supplementation significantly reduced serum Hs-CRP (− 0.87 ± 0.19 vs. − 0.16 ± 0.09 mg/L; P = 0.001) and NO levels (− 5.53 ± 1.53 vs. + 3.51 ± 1.79 µmol/L; P = 0.042) compared with placebo. Additionally, eight weeks of Mixodin supplementation resulted in a trend toward increased SOD activity and TAC, and a trend toward decreased TOS; however, these changes did not reach statistical significance when compared with the control group (all P > 0.05). No significant change was observed in MDA levels. Mixodin supplementation significantly reduced headache severity (-1.93 ± 0.30vs. -0.36 ± 0.21; P = 0.001) compared with placebo, whereas frequency and duration did not differ significantly between groups (P = 0.737 and P = 0.873, respectively).

Conclusion

Eight weeks of Mixodin supplementation significantly improved hs-CRP, NO levels, and headache severity among migraine patients, suggesting a promising role for this combination as an adjunctive therapeutic approach in migraine management. Further large-scale trials with longer follow-up are needed.

Trial registration

Iranian Registry of Clinical Trials (www.irct.ir) (ID: IRCT20241009063312N1).

Introduction

Migraine is a common, severe, and disabling neurological disorder with an estimated incidence of 14 per 100 individuals worldwide [45]. The disorder causes recurring severe headaches that frequently lead to nausea, vomiting, and light and sound sensitivity in patients, resulting in significant social and psychological impacts [3]. The exact pathophysiology of the disorder remains unclear, but research indicates that oxidative stress and neurogenic inflammation are key contributors to its development and progression [9, 17]. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defenses. Increased levels of oxidative stress markers, including malondialdehyde (MDA) and total oxidative status (TOS), have been observed alongside decreased antioxidant capacity in individuals with migraine [28, 46]. This disruption of oxidative balance contributes to various migraine-related issues, including neuronal damage, vascular instability, and enhanced pain signaling that can trigger migraine attacks [15]. Furthermore, inflammatory mediators such as C-reactive protein (CRP) are significantly elevated during migraine attacks, indicating that neurogenic inflammation is a critical component of the disorder's pathophysiology [30].

Although several therapeutic options are available for migraine management, current treatments have significant limitations, including potential side effects, medication-overuse headaches, and incomplete effectiveness [2]. These limitations strongly motivate further research into natural therapeutic agents with improved safety profiles. Curcumin, the primary bioactive compound in turmeric (Curcuma longa), has demonstrated anti-inflammatory and antioxidant effects in numerous clinical and preclinical studies by enhancing endogenous antioxidant enzymes and reducing pro-inflammatory cytokines [32]. Similarly, gingerol, the active ingredient in ginger (Zingiber officinale), exhibits notable anti-inflammatory, analgesic, and neuroprotective properties by modulating cyclooxygenase and lipoxygenase pathways and through serotonin receptor antagonism [16]. However, both curcumin and gingerol have low oral bioavailability due to extensive hepatic metabolism and poor intestinal absorption [41, 44]. Piperine, an alkaloid from black pepper (Piper nigrum), helps overcome these pharmacokinetic challenges by inhibiting hepatic and intestinal glucuronidation. This inhibition significantly raises plasma levels and prolongs the half-life of co-administered compounds [31].

The development of Mixodin, which contains curcumin, gingerol, and piperine [24], represents a promising polypharmacological strategy that may benefit migraine treatment. This multi-target therapeutic approach can address the complex mechanisms underlying migraine attacks. While each ingredient has shown potential therapeutic properties relevant to migraine pathophysiology [2], combining these compounds may yield synergistic effects with more favorable outcomes. Although these compounds show promise, clinical evidence regarding their combined effects on oxidative stress and inflammation in migraine is limited. Therefore, this randomized, double-blind, placebo-controlled trial investigated the effects of Mixodin supplementation on oxidative stress and inflammatory markers, as well as clinical migraine characteristics, in patients with migraine.

Materials and methods

Study design

This randomized, double-blind, placebo-controlled, parallel-group trial assessed the effects of Mixodin supplementation on migraine headache characteristics, oxidative stress and inflammatory biomarkers in 60 patients with migraine headaches. The study included two research groups and an 8-week follow-up period. The primary outcome was the between-group difference in migraine headache characteristics, including the frequency, severity, and duration of migraine attacks at 8 weeks. Secondary outcomes included serum levels of hs-CRP, NO, MDA, TOS, TAC, SOD, and OSI. The study protocol was approved by the Ethics Committee of Isfahan University of Medical Sciences (IUMS) (Approval Code: IR.MUI.MED.REC.1403.231) and registered with the Iranian Registry of Clinical Trials (IRCT20241009063312N1), available at https://www.irct.ir [5]. The study adhered to the ethical principles of the Declaration of Helsinki and was conducted and reported in accordance with the CONSORT guidelines [8].

Study population

All volunteer participants were recruited from the neurology clinic affiliated with IUMS in Isfahan, Iran, between December 2024 and April 2025. Eligible patients were 20 to 60 years old and had been diagnosed with migraine by a neurologist (FK) based on the criteria outlined in the International Classification of Headache Disorders, 3rd edition (ICHD-3) [35], and at least one year had passed since their initial migraine diagnosis. Participants were excluded if they had any other type of headache, including tension-type, cluster, trigeminal autonomic cephalalgias, medication-overuse, or menstrual-related headaches. Other exclusion criteria included pregnancy or breastfeeding, failure to attend scheduled study visits, known hypersensitivity or adverse reactions to the prescribed supplement, and any change in medication type or dosage within the past month. Individuals with a history of cardiovascular disease, hypertension, diabetes, kidney, liver, or thyroid disorders, other neurological conditions, or smoking were also excluded. Furthermore, participants were ineligible if they followed specific diets (e.g., vegetarian, Atkins, or ketogenic) or consumed supplements containing active ingredients found in Mixodin (curcumin, gingerol, or piperine). The study’s aims were thoroughly explained to all patients, and written informed consent was obtained before enrollment.

Random assignment and masking

Participants were randomly assigned to the Mixodin supplementation group or the placebo group using a web-based randomization system from Sealed Envelope (https://www.sealedenvelope.com). The system used a permuted-block design with a block size of four to ensure balanced 1:1 allocation. Stratification was based on sex (male or female) and age (20–39 and 40–60 years) to control for potential confounding factors. A trained individual, not involved in randomization or patient recruitment, coded the Mixodin supplements and placebos. All investigators, research coordinators, participants, and data analysts remained blinded to treatment assignments throughout the study to maintain objectivity and reduce bias.

Intervention

This study evaluated the effects of Mixodin supplementation versus placebo in patients with migraines. Participants were randomly assigned in a 1:1 ratio to the intervention or control group for eight weeks. The intervention group (n = 30) took two Mixodin capsules daily, each containing 300 mg of curcumin, 7.5 mg of gingerol, and 3.75 mg of piperine. The control group (n = 30) received two placebo capsules daily, formulated with the same inactive fillers as the Mixodin capsules (calcium phosphate, Avicel, and gelatin). Participants received 60 capsules of either the intervention or placebo twice during the study: once at baseline and again at the follow-up visit at the end of the fourth week. To monitor compliance, patients were asked to return any empty or unused supplement packets at each visit. Adherence was tracked through weekly phone calls, capsule counts, and inquiries about potential adverse effects. Patients were advised to maintain their usual exercise, medication, and dietary routines during the intervention period and to avoid taking any supplements containing active ingredients found in Mixodin. Compliance was calculated using the formula: Compliance rate = (Capsules taken/Capsules prescribed) × 100.

Sample size calculation

The sample size was calculated based on previous studies reporting a 25% reduction in pain following ginger treatment in migraine patients [10, 12, 38]. Using a two-sided alpha of 0.05, 80% statistical power (β = 0.20), and accounting for a 2% dropout rate, the required sample size was determined as 30 participants per group (total N = 60). This calculation is consistent with standard methods for randomized controlled trials (RCTs) and similar studies in the field [13, 39].

Assessment of variables

Assessment of anthropometric parameters, dietary intakes, and physical activity

Socio-demographic data, including age, marital status, education level, disease duration, family history, employment status, medication use, and medical history, were collected via validated questionnaires during face-to-face interviews conducted by a trained dietitian under the supervision of the project manager. Anthropometric measurements were obtained with participants wearing light clothing and no shoes. Body weight (BW) was measured to the nearest 0.5 kg using a Seca scale (Seca, Hamburg, Germany), and height to the nearest 0.1 cm. BMI was calculated as weight (kg) divided by height squared (m²). Dietary intake was assessed using a 3-day food record (two weekdays and one weekend day), and food quantities were converted to grams per day using standard Iranian household measures [19]. Nutrient analysis was performed using Nutritionist IV software, adapted for Iranian foods. Physical activity levels were assessed using the International Physical Activity Questionnaire (IPAQ), validated for Iranian adults, and reported as metabolic-equivalent hours per day (MET/h/day) [34]. Blood pressure was measured twice with a mercury sphygmomanometer after a 10-min seated rest, with a 5-min interval between readings. The average of the two readings was recorded. All assessments were performed by the principal investigator (M.A).

Migraine assessment

At the initial visit, patients received instructions on how to complete a headache diary to record details of their migraine attacks. An experienced neurologist (FK) evaluated migraine symptoms, including headache severity (on a 10-cm visual analog scale (VAS)), duration (in hours), and monthly attack frequency, based on diary entries collected at the beginning and end of the trial. Headache severity was rated on a scale from 1 to 10, with patients selecting the number that best represented their pain level.

Biochemical outcome assessment

Fasting blood samples (10 mL) were collected at the beginning and end of the trial and immediately centrifuged at 2,500 rpm for 10 min to separate the serum. The isolated serum was transferred into sterile microtubes and stored at − 80 °C until analysis. Serum Hs-CRP levels were measured using turbidimetric assay kits (BIOREX, Iran), and CGRP levels were evaluated using ELISA kits (Crystal Day BioTec, China). Additional oxidative stress markers, including TAC (CUPRAC method), TOS (modified ferrous oxidation-xylenol orange method), MDA (TBARS assay), NO (Griess method), and SOD (resazurin inhibition method), were measured using standardized kits from Kiazist Life Sciences (Iran), following manufacturer protocols and appropriate quality controls. The oxidative stress index (OSI) was calculated as: OSI = [(TOS, μmol/L)/(TAC, μmol/L)] × 100 [18].

Statistical methods

All statistical analyses were performed using SPSS (version 21.0; SPSS Inc., Chicago, IL, USA), and P-values below 0.05 were considered statistically significant. Analyses followed the intention-to-treat (ITT) principle with the last observation carried forward (LOCF) method. Normality was assessed using Q-Q plots and the Kolmogorov–Smirnov test, confirming a normal distribution. Continuous data are presented as mean ± standard error (SE), and categorical data as frequency and percentage. Baseline characteristics and nutrient intakes were compared between groups using independent samples t-tests and Pearson’s chi-square tests. Within-group changes during the 8-week intervention were analyzed using paired-samples t-tests. The effect of Mixodin on biochemical and migraine parameters was evaluated using analysis of covariance (ANCOVA), with baseline values and NSAIDs use included as covariates in all adjusted between-group comparisons.

Result

Study population

A flowchart showing the sample composition and participant dropouts during the 8-week follow-up period is shown in Fig. 1. Initially, 260 patients with migraine were screened according to the study’s eligibility criteria. Of these, 60 participants met the criteria and were randomly assigned to either the Mixodin supplementation group or the placebo group for an 8-week intervention. Two participants (one from each group) dropped out for personal reasons. All 60 participants were included in the final statistical analysis using the ITT approach. No serious adverse events were reported during the study.

Fig. 1.

Number of naloxone cartons ordered and naloxone administered by fiscal month, 2022–2024

The average age of participants was 38.90 ± 4.42 years, and the average weight was 72.79 ± 5.68 kg. Detailed demographic characteristics for each group are presented in Table 1. There were no statistically significant differences between the groups for BMI, weight, age, disease duration, migraine type, physical activity level, family history of migraines, systolic blood pressure (SBP), or diastolic blood pressure (DBP) (all P > 0.05), except for NSAIDs use, which differed significantly (P = 0.035). Similarly, employment status, marital status, and education level did not differ significantly between the groups (P > 0.05). Baseline dietary intake data, including macronutrients (total energy, protein, carbohydrates, and fats) and micronutrients (calcium, potassium, sodium, zinc, magnesium, folate, selenium, and antioxidants such as vitamins C and E), are presented in Table 2. There were no statistically significant differences between the groups at baseline (P > 0.05), as shown in Table 2.

Table 1.

Baseline characteristic of study subjects

Variable		Mixodin group (n = 30)		Placebo group (n = 30)	Pa
Age (year)		39.63 ± 4.65		38.16 ± 4.11	0.201
Weight (kg)		73.58 ± 6.52		71.99 ± 4.66	0.280
Height (m)		1.64 ± 0.05		1.63 ± 0.07	0.498
BMI (kg/m2)		27.30 ± 2.38		27.17 ± 2.45	0.824
SBP (mmHg)		119.80 ± 3.19		118.83 ± 2.57	0.202
DBP (mmHg)		79.30 ± 3.75		78.66 ± 3.55	0.505
Disease duration (year)		3.96 ± 1.44		3.56 ± 1.50	0.298
Physical activity (MET/h/day)		31.16 ± 4.95		30.60 ± 7.82	0.744
Gender (Female)		27 (90%)		26 (86.7%)	0.988
Migraine type	Episodic	19 (63.3%)		18 (60%)	0.791
	Chronic	11 (36.7%)		12 (40%)
Job	Housewife	13 (43.3%)		7 (23.3%)	0.812
	Freelance	5 (16.7%)		11(36.7%)
	Employee	12 (43.3%)		13 (43.3%)
Education status	University graduated	13 (40%)		12 (40%)	0.843
	Diploma	7 (23.3%)		9 (30%)
	Under diploma	10 (33.3%)		9 (30%)
Marital status	Married	14 (46.7%)		13 (43.3%)	0.795
	Single	16 (53.3%)		17 (56.7%)
Familial history	Yes	13 (43.3%)		10 (33.3%)	0.426
	No	17 (56.7%)		20 (66.7%)
Medication	NSAIDs	Yes	16 (53.3%)	8 (26.7%)	0.035
		No	14 (46.7%)	22 (73.3%)
	TCAs	No	21 (70%)	23 (76.7%)	0.559
		Yes	9 (30%)	7 (23.3%)
	Benzodiazepine	No	29 (96.7%)	27 (90%)	0.301
		Yes	1 (3.3%)	3 (10%)
	Sodium valproate	No	23 (76.7%)	25 (83.3%)	0.519
		Yes	7 (23.3%)	5 (16.7%)
	Topiramate	No	26 (86.7%)	24 (80%)	0.488
		Yes	4 (13.3%)	6 (20%)
	Beta blockers	No	22 (73.3%)	23 (76.7%)	0.382
		Yes	8 (26.7%)	7 (23.3%)
	Triptans	No	22 (73.3%)	23 (76.7%)	0.766
		Yes	8 (26.7%)	7 (23.3%)
	SSRIs	Yes	4 (13.3%)	3 (10%)	0.688
		No	26 (86.7%)	27 (90%)
	Gabapentin	No	23 (76.7%)	24 (80%)	0.754
		Yes	7 (23.3%)	6 (20%)
	TeCAs	No	25 (83.3%)	24 (80%)	0.739
		Yes	5 (16.7%)	6 (20%)

Quantitative variables are expressed as mean ± Standard deviation (SD) and qualitative variables expressed as n (%)

BMI Body mass index, DBP Diastolic blood pressure, SBP Systolic blood pressure, NSAIDs Nonsteroidal anti-inflammatory drugs, SSRIs Selective serotonin reuptake inhibitor, TCA Tricyclic Antidepressants, TeCAs Tetracyclic antidepressants

^aP values resulted from independent t-tests for quantitative and Chi-square for qualitative variables between the two groups

Table 2.

Baseline dietary intake of the participants

Variable	Mixodin group (N = 30)	Placebo group (N = 30)	Pa
Energy (kcal/day)	2128.36 ± 89.92	2037.00 ± 79.41	0.449
Carbohydrate (g/day)	262.33 ± 3.59	255.70 ± 4.37	0.246
Protein (g/day)	71.86 ± 1.04	69.56 ± 1.34	0.184
Fat (g/day)	72.80 ± 1.59	70.66 ± 1.64	0.355
Fiber (g/d)	14.15 ± 0.41	13.12 ± 0.39	0.080
Zinc (mg/day)	9.37 ± 0.21	9.04 ± 0.15	0.219
Vitamin E (mg/day)	4.61 ± 0.13	4.49 ± 0.15	0.551
Vitamin C (mg/day)	122.06 ± 4.99	118.76 ± 5.05	0.644
Selenium (mg/day)	0.51 ± 0.05	0.48 ± 0.03	0.563
Folate (μg/d)	390.76 ± 9.92	381.78 ± 12.84	0.501
Sodium (mg/d)	2217.16 ± 41.42	2214.23 ± 47.30	0.963
Potassium (mg/d)	2334.00 ± 27.01	2325.33 ± 43.88	0.867
Calcium (mg/d)	844.46 ± 12.65	820.26 ± 13.53	0.197
Magnesium (mg/day)	229.40 ± 4.76	224.03 ± 4.21	0.402

Variables are expressed as mean ± standard error

^aObtained from independent t test

The mean changes in biochemical markers from baseline to the end of the intervention are presented in Table 3. At the end of the study, Mixodin supplementation resulted in significant reductions in serum Hs-CRP (−0.87 ± 0.19 vs. −0.16 ± 0.09; P = 0.001), and NO (−5.53 ± 1.53 vs. + 3.51 ± 1.79; P = 0.042) compared with the placebo group. Although the OSI significantly decreased within the Mixodin group (− 0.18 ± 0.07; P = 0.017), the between-group comparison did not reach statistical significance (P = 0.146). Additionally, eight weeks of Mixodin supplementation resulted in a trend toward increased SOD activity (105.20 ± 59.96; P = 0.090) and TAC (147.13 ± 74.72; P = 0.059), and a trend toward decreased TOS (− 1.96 ± 1.05; P = 0.073); however, these changes did not reach statistical significance when compared with the control group (all P > 0.05). No significant change was observed in MDA levels (−1.60 ± 0.95 vs. −0.96 ± 0.77; P = 0.798). The effects of Mixodin supplementation on migraine headache characteristics are presented in Table 3. At the end of the study, Mixodin supplementation resulted in significant reductions in headache severity (−1.93 ± 0.30 vs. −0.36 ± 0.21; P = 0.001) compared with the placebo group. Although headache frequency declined significantly in the Mixodin group (−1.03 ± 0.30; P = 0.002), this change was not significantly different from placebo after adjustment (P = 0.737). Moreover, headache duration did not change significantly within either group or between groups (P = 0.873).

Table 3.

The effects of eight weeks’ Mixodin supplementation on migraine headache characteristics, inflammatory, and oxidative stress parameters in patients with migraine

Biochemical variable		Mixodin (N = 30)				Placebo (N = 30)				Pb	Pc
		Before	After	Change	Pa	Before	After	Change	Pa
Inflammatory and oxidative stress parameters	Hs-CRP (mg/l)	3.95 ± 0.14	3.07 ± 0.12	−0.87 ± 0.19	< 0.001	3.75 ± 0.06	3.58 ± 0.08	−0.16 ± 0.09	0.076	0.002	0.001
	TAC (nmol equivalent of trolox/ml)	2119.00 ± 52.80	2266.13 ± 60.74	147.13 ± 74.72	0.059	2078.70 ± 56.96	2149.33 ± 79.92	70.63 ± 95.96	0.468	0.249	0.347
	TOS (nmol/ml)	30.09 ± 1.17	28.13 ± 1.26	−1.96 ± 1.05	0.073	30.53 ± 1.40	29.50 ± 1.12	−1.03 ± 1.30	0.436	0.422	0.303
	OSI	1.45 ± 0.07	1.26 ± 0.06	−0.18 ± 0.07	0.017	1.51 ± 0.09	1.43 ± 0.08	−0.07 ± 0.11	0.527	0.126	0.146
	MDA (nmol/mL)	23.17 ± 1.49	21.57 ± 1.13	−1.60 ± 0.95	0.105	22.77 ± 1.20	21.80 ± 1.13	−0.96 ± 0.77	0.220	0.885	0.798
	NO (nmol/ml)	32.76 ± 1.09	27.23 ± 0.91	−5.53 ± 1.53	0.001	28.00 ± 1.24	31.51 ± 1.18	3.51 ± 1.79	0.060	0.006	0.042
	SOD (U/mL)	1741.30 ± 80.73	1846.50 ± 60.63	105.20 ± 59.96	0.090	1698.97 ± 77.67	1711.37 ± 65.10	12.40 ± 39.02	0.753	0.134	0.053
Migraine headache characteristics	Frequency	8.46 ± 0.20	7.43 ± 0.29	−1.03 ± 0.30	0.002	7.91 ± 0.26	7.33 ± 0.29	−0.58 ± 0.34	0.106	0.809	0.737
	Severity	8.68 ± 0.18	6.75 ± 0.29	−1.93 ± 0.30	< 0.001	8.16 ± 0.15	7.80 ± 0.19	−0.36 ± 0.21	0.104	0.004	0.001
	Duration	13.76 ± 0.57	12.90 ± 0.56	−0.86 ± 0.45	0.065	12.93 ± 0.62	12.30 ± 0.41	−0.63 ± 0.49	0.211	0.393	0.873

Variables expressed as mean ± standard error

NO Nitric Oxide, TAC Total Antioxidant Capacity, TOS Total Oxidative Status, OSI Oxidative Stress Index, Hs-CRP High-sensitivity C-reactive protein, MDA Malondialdehyde, SOD Superoxide dismutase

^aObtained from paired t-test

^bObtained from independent t-test

^cObtained from analysis of covariance in the adjusted models (adjusted for baseline value, and taking NSAIDs)

Discussion

The current randomized, double-blind, placebo-controlled trial provides supportive evidence that Mixodin supplementation improves inflammatory and oxidative stress markers in patients with migraine. Our results showed significant reductions in Hs-CRP and NO levels, as well as in migraine severity, after Mixodin supplementation. This suggests that the combined use of curcumin, gingerol, and piperine may offer therapeutic benefits in migraine management by affecting underlying pathophysiological mechanisms. Although positive trends were observed for SOD, TAC, and TOS, these changes did not achieve statistical significance. This may reflect the relatively short intervention period, the sample size being adequate for primary but potentially insufficient for secondary outcomes, or individual variability in response. These findings should be interpreted as preliminary and hypothesis-generating, requiring confirmation in larger trials with longer follow-up.

The most striking finding was a significant reduction in Hs-CRP levels after eight weeks of Mixodin supplementation. This aligns with previous research showing elevated inflammatory markers in migraine patients and supports the anti-inflammatory effects of our intervention. Hs-CRP is a sensitive marker of systemic inflammation and has been consistently elevated in migraine patients compared with healthy controls [23, 30]. The anti-inflammatory mechanisms of Mixodin components are well established in the literature. Curcumin has been shown to inhibit NF-κB activation, a key transcription factor that regulates inflammatory gene expression, and to suppress pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) [25]. A meta-analysis by Gorabi et al. found that curcumin supplementation significantly reduces circulating CRP and Hs-CRP levels across multiple clinical conditions [20], which supports our findings. Gingerol contributes to anti-inflammatory effects by inhibiting cyclooxygenase-2 (COX-2) and 5-lipoxygenase pathways, thereby reducing the synthesis of pro-inflammatory prostaglandins and leukotrienes [22]. The inclusion of piperine in our formulation likely enhanced these effects by increasing the bioavailability of curcumin and gingerol [14]. The observed increase in SOD activity is another significant finding, indicating enhanced endogenous antioxidant capacity after Mixodin supplementation. SOD is a crucial antioxidant enzyme that plays an essential role in protecting cells from oxidative damage [26]. Previous research has shown decreased SOD activity in migraine patients, indicating compromised antioxidant defenses [11, 37, 46]. Our results suggest that Mixodin supplementation may help restore antioxidant enzyme function, potentially reducing migraine frequency and severity.

The antioxidant mechanisms of Mixodin components are complex. Curcumin acts both as a direct free-radical scavenger and as an inducer of antioxidant enzyme expression by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [4]. This transcription factor regulates the expression of many antioxidant genes, including those encoding SOD, catalase, and glutathione peroxidase [27]. Similarly, gingerol exhibits strong antioxidant activity through multiple mechanisms, including direct ROS scavenging, metal ion chelation, and the enhancement of endogenous antioxidant enzyme activity [6]. Although we observed positive trends in TAC (+ 147.13 ± 74.72 nmol/ml; P = 0.059) and TOS (−1.96 ± 1.05 nmol/ml; P = 0.073) in the Mixodin group, these changes did not reach statistical significance compared with the control group, possibly due to the relatively short intervention period or the need for higher doses. The significant decrease in NO levels after Mixodin supplementation is a notable finding with direct implications for migraine pathophysiology. NO plays a crucial role in migraine development by modulating cerebrovascular tone and nociceptive signaling [33]. Elevated NO levels are consistently observed during migraine attacks and are believed to contribute to the characteristic throbbing pain and associated symptoms [21, 36, 42]. The reduction in NO levels following Mixodin supplementation may occur through multiple mechanisms, including curcumin's ability to modulate nitric oxide synthase (NOS) expression and activity, particularly inducible NOS (iNOS), which is upregulated during inflammatory conditions [7].

The significant reduction in headache severity observed in the Mixodin group compared with placebo aligns with findings from previous clinical trials of curcumin-based interventions in patients with migraine. Rezaie et al. reported that eight weeks of curcumin supplementation significantly reduced headache severity compared with placebo, closely paralleling our findings [40]. Furthermore, a meta-analysis of RCTs confirmed that curcumin supplementation significantly reduces migraine severity, while effects on frequency and duration were less consistent across studies [48]. The analgesic and anti-inflammatory properties of curcumin, including inhibition of the COX-2 and iNOS pathways, likely contribute to the reduction in pain intensity observed in our study [1].

Regarding headache frequency and duration, our results showed significant within-group reductions in the Mixodin group; however, these did not reach statistical significance compared with placebo after adjustment. This pattern is similarly reported in the literature, where curcumin's effects on frequency and duration tend to be less pronounced than its effects on severity [40, 48]. Shojaei et al. demonstrated that phytosomal curcumin significantly reduced the frequency, duration, and severity of migraine attacks compared with placebo, suggesting that formulations with enhanced bioavailability may yield broader clinical benefits [43]. The relatively modest effects on frequency and duration in our study may be attributed to the eight-week intervention period or to individual variability in response, warranting further investigation with longer follow-up.

The observed improvements in inflammatory and oxidative stress biomarkers after Mixodin supplementation have important clinical implications for migraine management. This combination's multi-target approach addresses several key disease mechanisms simultaneously, potentially offering benefits over single-compound treatments [29, 47]. The synergistic effects of curcumin, gingerol, and piperine may offer comprehensive protection against the oxidative and inflammatory processes involved in migraine development [28]. Our findings align with several previous investigations examining the effects of individual components on migraine and related conditions. However, given the limited research on the Mixodin combination, our study provides preliminary but promising evidence of the therapeutic potential of this formulation. The robust study design, including randomization, double-blinding, and placebo control, strengthens the reliability of our findings and provides high-quality evidence for the potential therapeutic benefits of this combination.

Several limitations of this study should be acknowledged when interpreting the results. First, the relatively short eight-week intervention period may not fully capture the long-term effects of Mixodin supplementation on inflammatory and oxidative stress markers. Second, the study did not assess compliance biomarkers, limiting our ability to objectively verify adherence to the supplementation protocol. Third, detailed monitoring of lifestyle factors, including diet, physical activity, and sleep quality, which may influence inflammatory and oxidative stress outcomes, was not performed. Fourth, the sample size, while adequate for primary outcomes, may have been insufficient to detect statistically significant changes in secondary outcomes such as TAC and TOS. Finally, the optimal dosing regimen for Mixodin components remains to be established, and individual variations in bioavailability and therapeutic response warrant further investigation in larger, longer-duration trials.

Conclusion

In conclusion, our study shows eight weeks of Mixodin supplementation significantly reduces serum hs‑CRP and NO levels and improves headache severity in patients with migraine. These findings suggest that the combination of curcumin, gingerol, and piperine may confer therapeutic benefits by modulating inflammatory and oxidative stress pathways. Although improvements in antioxidant markers did not reach statistical significance, the observed trends merit further investigation. Larger trials with longer follow‑up are needed to confirm these findings and to establish the long‑term efficacy of Mixodin as an adjunctive therapy in migraine management.

Abbreviations

Hs-CRP

High-sensitivity C-reactive protein

NO

Nitric oxide

MDA

Malondialdehyde

TOS

Total oxidant status

TAC

Total antioxidant capacity

SOD

Superoxide dismutase

ROS

Reactive oxygen species

CRP

C-reactive protein

IRCT

Iranian Registry of Clinical Trials

IUMS

Isfahan University of Medical Sciences

ICHD-3

International Classification of Headache Disorders, 3rd edition

CI

Confidence interval

BW

Body weight

BMI

Body mass index

IPAQ

International Physical Activity Questionnaire

MET/h/day

Metabolic-equivalent hours per day

VAS

Visual analog scale

CGRP

Calcitonin gene-related peptide (mentioned in methods)

ELISA

Enzyme-linked immunosorbent assay

TBARS

Thiobarbituric acid reactive substances

OSI

Oxidative stress index

ITT

Intention-to-treat

LOCF

Last observation carried forward

SE

Standard error

NSAID

Non-steroidal anti-inflammatory drug

SBP

Systolic blood pressure

DBP

Diastolic blood pressure

NF-κB

Nuclear factor kappa B

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

TNF-α

Tumor necrosis factor-alpha

COX-2

Cyclooxygenase-2

Nrf2

Nuclear factor erythroid 2-related factor 2

NOS

Nitric oxide synthase

iNOS

Inducible nitric oxide synthase

Authors’ contributions

Mohammad Ali Askari: Methodology, Resources, Data curation, Writing – original draft, Writing – review & editing. Sahar Golpour-Hamedani: Methodology, Formal analysis, Data curation, Writing – original draft. Fariborz Khorvash: Methodology, Writing – review & editing, Project administration. Mahdi Vajdi: Conceptualization, Methodology, Validation, Formal analysis, Resources, Data curation, Writing – original draft, Writing – review & editing, Project administration, Supervision. Afsaneh Yegdaneh: Conceptualization, Validation, Project administration, Supervision.

Funding

The present study was supported by a grant from the Vice-Chancellor for Research, Isfahan University of Medical Sciences (Grant Number: 140389).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study procedure was conducted in accordance with the Declaration of Helsinki and the CONSORT checklist. All participants provided informed written consent. The study protocol was approved by the local Ethics Committee of Isfahan University of Medical Sciences (Approval Code: IR.MUI.MED.REC.1403.231).

Consent for publication

All participants involved in the study provided written informed consent.

Competing interests

The authors declare no competing interests.