IN-VITRO AND IN-VIVO STUDY OF TURMERIC-GINGER SYNERGY IN DIABETIC OXIDATIVE STRESS MANAGEMENT
Prince Ogochukwu Alaebo
, Anthony Cemaluk Chinedum Egbuonu
, Precious Uchechi Okpara
, Juliet Chinonso Njoku
, Chimaobi Anthony Nwede
, Chidera Nnamdi Abalihe
, Thankgod Chidumije Ugboaja
, Livinus Chinonso Chukwu
, Enyinna Wisdom Chukwuka
, John Chiemelie Uchegbusi
, Kenneth Ifechukwu Ataka
Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, P.M.B 7267 Umuahia, Abia State, Nigeria.
Abstract
Background and aim: Oxidative stress is a major factor in both the progression and the adverse effects of diabetes mellitus. Natural Bioactive substances like curcumin (derived from turmeric) and gingerol (from ginger) possess antioxidant properties that may offer therapeutic benefits by enhancing the body's defense mechanisms against oxidative damage.
This study investigates the synergistic antioxidant effects of co-administered curcumin and gingerol in managing oxidative stress associated with diabetes mellitus.
Method: Oxidative stress biomarkers including superoxide dismutase (SOD), malondialdehyde (MDA), catalase, and glutathione were evaluated in experimental rat groups treated with varying doses of curcumin and gingerol. Additionally, in vitro antioxidant activities of turmeric and ginger extracts were assessed using DPPH and FRAP assays.
Results: The combination of 400 mg/kg curcumin and gingerol significantly increased SOD (24.49±6.85 IU/g protein) and catalase (19.02±1.76 kU/g protein) activities (p<0.05) compared to other groups. Glutathione levels also rose notably (5.31±1.26 µg/L), indicating enhanced endogenous antioxidant capacity. Although MDA levels (75.05±24.76 nmol/g protein) were higher than in lower dose groups, they remained lower than in the 200 mg/kg group (135.35±8.51 nmol/g protein), suggesting a dose dependent response. In vitro assays confirmed strong radical scavenging and reducing activities, with turmeric slightly outperforming in FRAP, while ginger matched in DPPH scavenging.
Conclusion: Co-administration of curcumin and gingerol enhances antioxidant defenses more effectively than individual treatments or metformin. This synergy presents a potential adjunct therapy for mitigating oxidative damage in diabetes and merits further clinical investigation.
Keywords: Antioxidant, curcumin, diabetes mellitus, gingerol, oxidative stress, synergistic effect.
INTRODUCTION
Diabetes mellitus is a long-term, progressive metabolic condition marked by high blood glucose levels, insulin resistance, and increased oxidative stress, all of which contribute to complications such as vascular damage, neuropathy, and organ dysfunction. Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the body's intrinsic antioxidant defense mechanisms, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)1. In individuals with type 2 diabetes mellitus (T2DM), there is often an increase in lipid peroxidation indicators like malondialdehyde (MDA), accompanied by a reduction in the activity of key antioxidant enzymes such as SOD and CAT2.
Against this backdrop, there is growing interest in plant-derived nutraceuticals as safer adjunct options to conventional therapy. Curcumin the principal curcuminoid in turmeric (Curcuma longa) has been clinically demonstrated to enhance serum total antioxidant capacity and SOD activity, while significantly reducing MDA levels in individuals with T2DM2.
Ginger (Zingiber officinale) contains bioactive phytochemicals particularly gingerols and shogaols that exhibit Ginger exhibits strong antioxidant and anti-inflammatory properties. In studies involving diabetic animal models, supplementation with ginger at doses between 200 and 400 mg/kg per day has shown beneficial effects elevated SOD, CAT, and glutathione levels, while reducing MDA in liver, kidney, and neural tissues. The mechanistic underpinnings involve activation of Nrf2-mediated antioxidant pathways, suppression of NF‑κB signaling, and regulation of lipid metabolism and glucose utilization.
While research has traditionally focused on single herb interventions, emerging data support synergistic anti‑inflammatory and antioxidant interactions when turmeric and ginger are combined. In vitro studies using RAW 264.7 macrophages showed that combined turmeric‑ginger (GT) extracts inhibited NF‑κB translocation and miR‑155‑5p expression more effectively than either extract alone (e.g., reduced LPS‑induced pro‑inflammatory cytokines)3.
Another in vitro investigation determined that mixtures of turmeric and ginger extract in optimized ratios (e.g., 5:2 w/w) significantly enhanced Nrf2 and HO‑1 protein expression, leading to greater suppression Such as nitric oxide, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) production than single extracts3.
However, preclinical evidence evaluating turmeric–ginger synergy in diabetic oxidative stress models remains limited. Although curcumin or ginger alone have shown improvements in SOD, CAT, GSH, and reductions in MDA in diabetic rodents2, little in vivo work has tested combination dosing to define potential synergistic antioxidant effects. One animal study combining aqueous ginger extract and curcumin reported superior reductions in hyperglycemia, HbA1c, dyslipidemia, and oxidative stress markers compared to either component alone but detailed dose–response data and multiple oxidative biomarkers were not systematically evaluated4.
In this context, a comprehensive experimental design that integrates both in vitro antioxidant assays (e.g., DPPH, FRAP) and in vivo diabetic models evaluating biomarkers such as SOD, CAT, GSH, and MDA across varying combination doses are crucial. In vitro methods specifically highlight the direct free radical-scavenging and reducing capacities of turmeric and ginger extracts, while in vivo models (e.g., alloxan‑induced diabetic rats) assess functional outcomes on oxidative biomarkers and tissue integrity (Table 1). Integrating both approaches fills a critical gap by demonstrating whether higher combination doses effectively enhance endogenous enzyme defenses and suppress lipid peroxidation, potentially outperforming standard treatment such as metformin5.
The current study seeks to assess the dose-dependent effects of curcumin and gingerol on oxidative stress and related biochemical markers in diabetic models gingerol combinations on key oxidative stress biomarkers: SOD, CAT, GSH, and MDA in Alloxan induced diabetic rat models, alongside in vitro assays (DPPH, FRAP) assessing radical scavenging and reducing capacity. We hypothesize that higher-dose combinations will synergistically enhance antioxidant enzyme activities and mitigate lipid peroxidation more effectively than either agent alone or metformin, and that in vitro assays will support these findings by demonstrating superior radical‑scavenging and reducing power.
This integrated investigation spanning in vitro and in vivo modalities is poised to deliver deeper insight into turmeric–ginger synergy, informing future clinical translation as a complementary antioxidant strategy in diabetes management.
METHODS
Collection of plant material
The fresh rhizomes of turmeric (Curcumalonga) and ginger (Zingiber officinale) was harvested from National Root Crops Research Institute, Umudike, Abia State, Nigeria. The plant materials, the rhizomes were carefully washed under running tap water to eliminate soil and surface contaminants, then rinsed with distilled water. They were cut into smaller segments to enhance the drying process and subsequently air-dried at ambient temperature (25–28 °C) for 10–14 days until a constant weight was achieved. Once fully dried, the samples were finely ground using a mechanical grinder, weighed and stored in airtight containers under at room temperature until they were extracted.
Plant Extraction
The authenticated plant materials were brought to the Biochemistry Department laboratory and air-dried for two weeks. After drying, the samples were ground into a fine powder using a Warring blender (Quilink QBL-20 L40 model). Methanolic extraction was performed following the method described by Achuba⁵. Specifically, 500 g of each powdered sample (turmeric and ginger) was soaked in 80% methanol and allowed to undergo fermentation for 72 hours. After the fermentation period, the mixtures were filtered sequentially using cotton wool and Whatman filter paper. The filtrates were then concentrated using a rotary evaporator and further dried in a water bath to yield a crude extract in powdered form.
Study Protocol
The study protocol adhered to the guidelines and declarations of Animal Research Ethics6 and the World Medical Association7 concerning the use of animals in biomedical research. It also complied with the animal rights regulations of Michael Okpara University of Agriculture, Umudike, Abia State.
Preparation of Extract for Animal Administration
A stock solution of the rhizome extract was prepared by dissolving 2 g of the crude methanol extract in 20 ml of water, resulting in a concentration of 100 mg/ml. Doses were administered individually based on the animals' body weight (mg/kg).
Animal Acclimatization
Twenty healthy male albino rats, weighing between 140 and 190 g, were obtained from the animal house of Dr. Daniel. The animals were housed in the Biochemistry Department’s animal facility at Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. They were allowed to acclimate to the new environment and kept in well-constructed stainless steel cages (30 x 40 x 80 cm) with wire mesh tops to ensure adequate ventilation and airflow. The cages were designed to be free of sharp edges and protrusions to prevent injuries or accidental entrapment of the animals’ limbs.
Experimental Design
After acclimatization 36 healthy mature rats were selected for further experimentation. The animals were divided into six groups, each consisting of six rats. The groups were organized as follows: Six groups (n=6 each):
Treatments were administered orally daily for 21 days. At study end, rats were euthanized; blood and tissues were collected for biochemical assays.
Biochemical Assays
Serum and tissue homogenates were analyzed for activities of antioxidant enzymes such as superoxide dismutase (SOD)⁸, catalase (CAT)⁹, and glutathione peroxidase (GPx)¹⁰ and for malondialdehyde (MDA) concentration as a marker of lipid peroxidation11, using standard colorimetric methods and spectrophotometry.
In vitro DPPH radical scavenging assay:
The antioxidant activity of the individual and combined extracts was assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, following the procedure outlined by reference 12. In brief, a 0.1 mM methanolic DPPH solution was combined with different concentrations of turmeric extract, ginger extract, and their mixture. The samples were incubated in the dark at room temperature for 30 minutes, after which absorbance was recorded at 517 nm using a UV-Vis spectrophotometer. The percentage of DPPH radical scavenging was calculated, and IC₅₀ values were determined to evaluate the antioxidant strength of each extract.
Statistical Analysis
Results are expressed as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA followed by Duncan’s post hoc test, with p-values less than 0.05 considered significant.
RESULTS
This figure illustrates the antioxidant activity of turmeric, measured based on its capacity to neutralize DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radicals. Greater scavenging activity indicates stronger antioxidant potential.
The results likely show a concentration-dependent increase in radical scavenging. This figure shows the Free radical scavenging capacity of ginger extracts assessed by DPPH assay. Similar to turmeric, higher scavenging percentages reflect stronger antioxidant properties. The data can be used to compare the antioxidant strength of ginger with that of turmeric. This figure presents the FRAP assay results for turmeric. FRAP measures the extract's ability to reduce Fe³⁺ to Fe²⁺, indicating reducing power. A higher FRAP value indicates a stronger antioxidant capacity.
This figure displays the FRAP values for ginger, assessing its antioxidant capacity through ferric ion reduction. Comparing this with turmeric helps evaluate which has stronger reducing (antioxidant) properties. FRAP of Ginger.
DISCUSSION
The MDA reduction, although not reaching levels in normal control, indicates partial mitigation of lipid peroxidation but at high dose, the MDA offset still remained above baseline, implying potential oxidative challenges or dose-linked pro-oxidant effects that warrant closer dose optimization. This finding concurs with the research of He et al.13, and Hamdy et al.14, who reported similar dose-dependent antioxidant responses and cautioned about the biphasic effects of curcumin and related phytochemicals on oxidative stress markers. The marked increase in glutathione, often depleted in diabetic conditions, suggests restored redox buffering capacity, consistent with Nrf2-mediated upregulation of antioxidant genes observed in curcumin and gingerol treatment studies13-15.
The combination’s ability to outperform metformin in boosting endogenous defenses (SOD, CAT, GSH) highlights its promise as an adjunct or complementary strategy, although clinical relevance remains to be established. This is supported by research demonstrating curcumin’s potent Stimulation of the Nrf2–ARE signaling cascade, leading to the induction of HO-1, NQO-1, and enzymes such as catalase and SOD16,17. Similarly, gingerol has been reported to stimulate Nrf2 signaling and antioxidant enzyme expression in diabetic models18,19.
The high dose (400 mg/kg) produced the strongest antioxidant response, but also a relatively elevated MDA level compared to normal controls, indicating the need to identify the optimal therapeutic window. Such biphasic dose responses have been documented in phytochemical research13,14, highlighting the importance of balancing efficacy with potential pro-oxidant effects at supraphysiologic doses. Future studies should incorporate histopathological assessments and explore markers of tissue oxidative damage or repair. Additionally, evaluating gene expression (e.g., Nrf2, HO-1, NF-κB) would elucidate mechanistic pathways of synergy beyond enzyme activation20.
The combination of curcumin and gingerol at high doses (curcumin 400 mg/kg + gingerol 400 mg/kg) produced substantial improvements in oxidative stress biomarkers: SOD activity increased to 24.49 ± 6.85 IU/g protein, catalase to 19.02 ± 1.76 kU/g protein, and glutathione reached 5.31 ± 1.26 µg/L, significantly higher than both negative controls and metformin groups (p < 0.05). MDA, an index of lipid peroxidation, was also reduced compared to the 200 mg/kg group (75.05 vs. 135.35 nmol/g protein), albeit not to baseline levels. These findings suggest a powerful induction of endogenous antioxidant defenses in a dose-dependent manner (Table 1), consistent with previous reports on curcumin’s dose-dependent antioxidant enzyme induction13,16.
In particular, the high-dose combination elevated SOD and catalase far above metformin-treated and normal control levels, highlighting its antioxidant enzyme induction potential. This aligns with prior evidence Curcumin stimulates the Keap1–Nrf2–ARE pathway, enhancing the expression of antioxidant genes like HO-1, SOD, CAT, and glutathione peroxidase in diabetic models16. Similarly, gingerol-based interventions in diabetic rat studies have demonstrated elevated SOD, CAT, and glutathione levels, alongside reduced MDA in brain and renal tissues, reflecting systemic antioxidant protection18,19.
Both in vitro assays (Figure 1 - Figure 4) supporting concentration-dependent radical scavenging activity (DPPH) and ferric reducing antioxidant power (FRAP) for turmeric and ginger extracts reinforce the plausibility of their in vivo synergy. Such antioxidant capacity has been documented for both turmeric and ginger extracts individually, with several studies noting enhanced scavenging activities correlating with improved oxidative stress biomarkers in animal models21,22.
This study is novel in demonstrating dose-dependent antioxidant synergy of combined curcumin and gingerol, with 400 mg/kg outperforming lower doses and metformin in antioxidant enzyme activity. Biomarkers evaluate multiple Oxidative stress indicators such as SOD, CAT, GSH, and MDAsystematically in a combination-treated diabetic model, extending beyond single-agent studies that typically measure only one or two biomarkers13,14. Including metformin as a standard comparator allows direct assessment of how herbal synergy measures against established pharmaceutical therapy16. Correlating in vitro radical-scavenging potency with in vivo enzymatic and molecular outcomes provides mechanistic insight into systemic antioxidant modulation21,23.
Limitation of the study
Although antioxidant enzyme activities and oxidative stress markers were evaluated, the study did not extensively investigate the molecular signaling pathways involved in the turmeric-ginger synergy.The antioxidant activities observed in vitro (e.g., DPPH and FRAP assays) may not fully represent the complex interactions and metabolism of these compounds in living organisms.
CONCLUSION
This study contributes significantly by demonstrating, for the first time in a systematic manner, the dose-responsive synergistic antioxidant potential of curcumin and gingerol combination in diabetic oxidative stress management. The findings offer mechanistic insights grounded in robust in vitro/in vivo correlation and establish groundwork for future translational research.
ACKNOWLEDGMENTS
The author gratefully acknowledges Dr. Samuel O. Onoja for his support in providing the laboratory analysis and results that were vital to this research. Appreciation is also extended to the Department of Biochemistry, Michael Okpara University of Agriculture, Umudike (MOUAU) for providing access to a conducive animal house and well-equipped undergraduate laboratory facilities. Sincere thanks go to my project students for their dedication and valuable contributions, which significantly aided the successful execution of this work.
AUTHORS' CONTRIBUTIONS
Alaebo PO: conceptualization, supervision, writing the original draft. Egbuonu ACC: supervision, methodology, writing, review. Okpara PU: investigation, editing. Njoku JC: investigation, formal analysis, data collection. Nwede CA: Investigation, Resources, Animal Handling. Abalihe CN: laboratory analysis, validation. Ugboaja TC: data collection, investigation. Chukwu LC: writing, review, editing. Chukwuka EW: literature review. Uchegbusi JC: software, data entry. Ataka KI: literature review. Final manuscript was checked and approved by all authors.
DATA AVAILABILITY
Data will be made available on request.
CONFLICT OF INTEREST
None to declare.
REFERENCES