Zingiber officinale extract protects against diabetic hepatopathy via gut-liver axis modulation: Enhanced efficacy with metformin in type 2 diabetic rats

2.1 Experimental animals and study design

Sixty adult male Wistar rats (age: 6 months; body weight: 180–200 g) were procured from the Laboratory Animal Center at King Abdulaziz University, Jeddah, Saudi Arabia. Animals were maintained under standardized laboratory conditions (temperature: 22 ± 2°C; relative humidity: 50–60%; 12:12 h light-dark cycle) with ad libitum access to standard rodent chow and filtered water. Rats were randomly allocated into six experimental groups (n = 10 per group) using computer-generated randomization: (i) Control (C): Non-diabetic rats fed a standard diet, (ii) Type 2 Diabetic (T2D): Diabetic control rats induced by HFD and STZ, (iii) Ginger monotherapy (G): Non-diabetic rats receiving ginger extract (200 mg/kg/day) by oral gavage, (iv) T2D + Ginger (T2D+G): Diabetic rats treated with ginger extract (200 mg/kg/day) by oral gavage, (v) T2D + Metformin (T2D+M): Diabetic rats administered metformin (200 mg/kg/day) by oral gavage, (vi) T2D + Combination Therapy (T2D+G+M): Diabetic rats receiving both ginger extract (200 mg/kg/day) and metformin (200 mg/kg/day) by oral gavage.

Dose Rationale: The ginger extract dose (200 mg/kg/day) was selected based on recent preclinical evidence demonstrating optimal therapeutic efficacy in metabolic syndrome models without adverse effects (Soleimani et al., 2023, Ahmed et al., 2024). This dose corresponds to approximately 2.2 g/day in humans using allometric scaling (Reagan-Shaw et al., 2008), which falls within the clinically recommended range of 1-3 g/day for glycemic control (Daily et al., 2023). Recent pharmacokinetic studies confirm this dose achieves therapeutic plasma concentrations of key bioactive compounds (6-gingerol: 0.8-1.2 μg/mL) associated with AMPK activation and anti-inflammatory effects (Zhang et al., 2024). Similarly, the metformin dose (200 mg/kg/day) represents a standard preclinical dose validated in multiple T2DM rodent models and translates to approximately 1500 mg/day in humans (Foretz et al., 2014; Kumar et al., 2023), consistent with first-line clinical practice guidelines (American Diabetes Association, 2024).

2.2 Induction of T2DM

A validated T2DM model was employed, consisting of an 8-week HFD (45% kcal from fat) to induce insulin resistance, followed by a single intraperitoneal injection of low-dose STZ (30 mg/kg; Sigma-Aldrich, USA) after overnight fasting (Wei et al., 2003). Diabetes induction was confirmed by measuring fasting blood glucose concentrations (≥200 mg/dL) at 72 hours post-STZ administration, with verification through two consecutive measurements.

2.3 Preparation and standardization of Zingiber officinale extract

Fresh ginger rhizomes (Zingiber officinale Roscoe) were procured from local markets in Makkah, Saudi Arabia, and authenticated by a certified botanist at the Herbarium of King Abdulaziz University (voucher specimen: ZO-2024-05). The rhizomes were thoroughly washed, peeled, and thinly sliced (2-3 mm thickness), and shade-dried at room temperature for 14 days. Dried rhizomes were ground into fine powder using a mechanical grinder and stored at –20°C until extraction. Aqueous extraction was performed by soaking 100 g of ginger powder in 1 L of distilled water at 60°C for 4 h with continuous magnetic stirring. The mixture was filtered through whatman no. 1 filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator (Buchi R-210, Switzerland) at 50°C. The concentrated extract was subsequently lyophilized using a freeze dryer (Alpha 1–2 LD Plus, Martin Christ, Germany) at –50°C and 0.05 mbar for 48 h, yielding a dried powder (yield: 8.2% w/w). The lyophilized extract was stored in airtight amber containers at –20°C until use.

HPLC-DAD analysis: Phytochemical characterization was performed using high-performance liquid chromatography coupled with diode-array detection (HPLC-DAD, Agilent 1260 Infinity II, USA). The chromatographic separation was achieved on a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm particle size; Agilent Eclipse XDB-C18) maintained at 30°C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) with the following gradient elution: 0–5 min (10% B), 5–20 min (10–40% B), 20–35 min (40–80% B), and 35–40 min (80% B), followed by re-equilibration. The flow rate was set at 1.0 mL/min, the injection volume was 20 μL, and the detection wavelength was 280 nm. Quantification of major bioactive compounds was performed using authentic standards (Sigma-Aldrich, USA): 6-gingerol (purity ≥98%), 8-gingerol (purity ≥95%), 10-gingerol (purity ≥95%), and 6-shogaol (purity ≥98%). Standard calibration curves were constructed using five-point serial dilutions (10–500 μg/mL, R² ≥ 0.998). The ginger extract contained: 6-gingerol (18.7 ± 1.2 mg/g), 8-gingerol (6.3 ± 0.8 mg/g), 10-gingerol (3.1 ± 0.5 mg/g), and 6-shogaol (4.8 ± 0.6 mg/g). Total phenolic content was determined using the Folin-Ciocalteu method and expressed as gallic acid equivalents (42.3 ± 2.1 mg GAE/g extract).

2.4 Biochemical analysis

Weekly body weights, fasting glucose, OGTT, IST, insulin (ELISA), and HOMA indices were measured (Ayala et al., 2010, Matthews et al., 1985). Hepatic antioxidant enzymes (CAT, SOD, GPx, and GST) and lipid profile assays were performed (Weydert and Cullen, 2010, Rifai et al., 2018).

2.5 Histopathological and immunohistochemical analysis

Liver tissues were stained with H&E (Fischer et al., 2008), glycogen assessment by PAS staining (McManus, 1948), and immunohistochemistry for BCL2 expression (Kim et al., 2016).

2.6 Gut microbiome analysis

Fecal DNA extraction (Godon et al., 1997), sequencing quality (Caporaso et al., 2010), OTU assignment (Bolyen et al., 2019), and functional predictions (Douglas et al., 2020) were conducted.

2.7 Statistical analysis

Sample size calculated (Mishra et al., 2019), data analyzed using ANOVA, Tukey’s post-hoc, eta-squared effect size (Cohen, 1988), and FDR-corrected multiple comparisons (Benjamini and Hochberg, 1995). Statistical significance set at p <0.05.

2.8 Ethical approval

Approved by Umm Al-Qura University Ethical Committee (approval: HAPO-02-K-012-2025-06-2799) following ARRIVE guidelines (Percie du Sert et al., 2020).

2.9 Study limitations

Limitations include accelerated disease progression compared to human T2DM (King, 2012; Furman, 2015), single-time microbiome analyses potentially missing dynamics (Heydemann, 2016), exclusive male subjects, and predictive rather than direct metabolomic analyses. Future studies should incorporate multi-omics, longer durations, both sexes, and direct metabolomics for deeper mechanistic insights.

3.1 Metabolic parameters and anthropometric measurements

STZ-induced diabetes mellitus resulted in substantial weight loss, with diabetic animals (T2D) exhibiting significant body weight reduction compared to healthy controls (Table 1). Zingiber officinale extract administration (T2D+G) significantly attenuated this weight loss, achieving modest weight recovery to 275.1 ± 7.8 g (p <0.05 vs. diabetic controls). Metformin monotherapy (T2D+M) demonstrated similar moderate weight recovery to 273.4 ± 7.1 g (p <0.05), while dual therapy (T2D+G+M) produced optimal weight restoration to 287.5 ± 8.9 g, reaching levels comparable to healthy controls (p < 0.01 vs. diabetic controls).

Longitudinal fasting blood glucose monitoring revealed progressive hyperglycemia development in diabetic animals (Table 1). At study endpoint (day 56), diabetic controls exhibited severe hyperglycemia (280.2 ± 13.7 mg/dL) compared to healthy controls (95.1 ± 4.5 mg/dL). Ginger supplementation yielded substantial glycemic improvement throughout the study period, with final glucose concentrations of 215.8 ± 10.9 mg/dL at day 56. The combined intervention (T2D+G+M) demonstrated superior and consistent efficacy with glucose levels maintained at 165.7 ± 8.2 mg/dL by day 56 (p < 0.001 vs. diabetic controls). Significant glycemic improvements were observed as early as day 14 post-treatment initiation, with statistical significance maintained throughout the monitoring period.

3.2 Insulin sensitivity and glucose homeostasis

Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) analysis revealed severe insulin resistance in diabetic animals (2.9-fold elevation: 8.2 ± 1.1 vs. 2.8 ± 0.4, p < 0.001), as demonstrated in Table 2. Ginger extract treatment produced a remarkable HOMA-IR reduction (52.3%, p <0.0001), surpassing metformin’s efficacy (45.7%, p < 0.01). Combination therapy achieved optimal restoration of insulin sensitivity with 68.5% HOMA-IR reduction (p < 0.001).

HOMA-β assessment revealed marked β-cell dysfunction in diabetic animals (44.7 ± 4.3% of control values, p < 0.01), as shown in Table 2. Therapeutic interventions progressively restored pancreatic function: metformin improved β-cell capacity to 72.1 ± 4.5% of control values, ginger extract restored function to 88.3 ± 5.1% of control, and combined treatment achieved optimal restoration to 97.2 ± 5.3% of control values. OGTT analysis demonstrated significantly impaired glucose clearance in diabetic animals, with baseline glucose AUC approximately 2.5-fold higher than in healthy controls (Table 2). Combination therapy produced optimal glucose tolerance with approximately 47% AUC reduction compared to diabetic controls.

3.3 Hepatic antioxidant defense systems

Diabetes significantly compromised hepatic antioxidant defense systems, and combined intervention normalizing enzyme activity to near-control levels with 78-85% enzyme activity recovery across all measured parameters (Table 3).

CAT activity was reduced to approximately 38% of control levels (from 27.0 ± 2.3 U/mL to 10.2 ± 1.4 U/mL, p < 0.05), as illustrated in Table 3. Therapeutic interventions demonstrated progressive enzyme restoration: metformin treatment achieved partial recovery to 16.5 ± 1.8 U/mL, while ginger supplementation showed superior restoration to 19.2 ± 2.0 U/mL. SOD activity exhibited similar diabetes-induced suppression patterns, declining from control levels of 142.1 ± 9.5 U/mL to 68.3 ± 6.2 U/mL in diabetic animals (Table 3). GPx activity was substantially compromised in diabetic liver tissue, declining from control levels of 398.4 ± 20.1 U/mL to 263.2 ± 16.8 U/mL, as presented in Table 3. GST activity was pathologically elevated in diabetic animals, increasing from control levels of 52.1 ± 3.8 U/mL to 98.3 ± 7.2 U/mL, reflecting compensatory responses to oxidative stress (Table 3).

3.4 Lipid metabolism modulation

T2D induction precipitated severe dyslipidemia characterized by marked atherogenic profile alterations, as demonstrated in Table 4. Combination therapy normalized lipid profiles across all measured parameters with 80-90% improvement.

Diabetic animals exhibited significant LDL elevation (from 1.9 ± 0.3 to 3.7 ± 0.4 mmol/L), elevated FFAs (from 1.4 ± 0.2 to 2.2 ± 0.3 mmol/L), substantial triglyceride accumulation (from 1.1 ± 0.2 to 2.5 ± 0.3 mmol/L), and total cholesterol elevation (from 12.1 ± 1.8 to 22.3 ± 2.9 mmol/L), as shown in Table 4. Concurrently, protective HDL cholesterol was significantly depleted (from 3.6 ± 0.4 to 2.0 ± 0.3 mmol/L). Therapeutic interventions demonstrated progressive LDL reduction efficacy: metformin monotherapy achieved modest improvement (reduction to 2.9 ± 0.3 mmol/L), while ginger extract showed superior LDL-lowering effects (reduction to 2.5 ± 0.3 mmol/L). Combination therapy produced optimal LDL normalization (reduction to 2.0 ± 0.3 mmol/L, approaching control levels) and HDL restoration (3.4 ± 0.4 mmol/L, approaching control levels).

3.5 Hepatic histopathological assessment

C and G exhibited preserved hepatic architecture with normal hepatocyte morphology, intact sinusoidal structure, and well-defined central veins, as demonstrated in Fig. 1(a-b). These groups showed typical hepatic lobular organization with healthy hepatocytes displaying clear cytoplasm and prominent nuclei.

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Fig. 1.

Comprehensive hepatic histopathological analysis using hematoxylin and eosin (H&E) staining. (a,b) Control and ginger-only groups displayed standard hepatic architecture with intact hepatocytes, clear sinusoidal structure, and well-organized lobular arrangement. (c) Diabetic liver demonstrating severe pathological alterations, including hepatocellular degeneration, inflammatory infiltration, sinusoidal congestion, and architectural disruption. (d) Metformin treatment showed moderate histological improvement with reduced inflammation and partial architectural recovery. (e) Ginger extract treatment demonstrates superior hepatoprotective effects with markedly improved cellular morphology and reduced pathological changes (red circle). (f) Combination therapy achieves optimal hepatic restoration with near-complete recovery of typical architecture (red circle). (g-l) Higher-magnification views showing detailed morphological improvements in the treatment groups, including restored sinusoidal architecture, improved portal tract organization, and enhanced hepatocellular integrity (red circle).

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D demonstrated severe hepatopathology, including extensive cellular degeneration, inflammatory cell infiltration (particularly around portal areas and central veins), sinusoidal congestion, hepatocellular ballooning, and early fibrotic changes (Fig. 1c). The hepatic architecture was significantly disrupted with loss of normal lobular organization and evidence of hepatocellular injury.

Metformin monotherapy (D+M) produced moderate histological improvements with reduced inflammatory infiltration and decreased hepatocellular degeneration compared with diabetic controls (Fig. 1d). However, some degree of architectural disruption remained evident. Ginger extract treatment (D+G) achieved superior hepatoprotective effects with markedly improved cellular morphology, reduced inflammation, and preservation of hepatic architecture (Fig. 1e). Hepatocytes showed improved cytoplasmic organization and reduced signs of degeneration. Combination therapy (D+G+M) demonstrated optimal hepatic restoration with near-complete recovery of standard hepatic architecture, minimal inflammatory infiltration, and well-preserved hepatocellular morphology approaching control group characteristics (Fig. 1f-l).

Quantitative histopathological analysis demonstrated significant improvements across all measured parameters (Table 5). Inflammatory response assessment revealed hepatic inflammation scores that were significantly elevated in diabetic animals (1.9 ± 0.4 vs. 1.2 ± 0.3 in controls, p <0.001). Therapeutic interventions produced progressive anti-inflammatory effects: metformin treatment reduced inflammation scores to 1.5 ± 0.3 (p < 0.05 vs. diabetic), and ginger extract achieved superior reduction to 1.4 ± 0.3. In contrast, combination therapy produced optimal anti-inflammatory effects with scores approaching control levels (1.3 ± 0.3). Vascular congestion analysis revealed a significant elevation in diabetic liver tissue (increase from control levels of 48.3 ± 5.7% to 83.2 ± 8.9%, p < 0.05). Treatment interventions demonstrated dose-dependent improvements: metformin reduced congestion to 68.1 ± 7.6%, ginger extract achieved reduction to 63.4 ± 7.1%, and combination therapy produced optimal vascular restoration to 53.7 ± 6.2%, approaching control levels.

3.6 Hepatic glycogen storage assessment

Periodic Acid-Schiff (PAS) staining revealed significant alterations in hepatic glycogen storage patterns across experimental groups, as demonstrated in Fig. 2. Control animals (Panel a) exhibited abundant hepatic glycogen deposits with intense, uniform PAS-positive staining (deep magenta coloration) throughout the hepatocyte cytoplasm, indicating normal carbohydrate storage capacity. The ginger-only treatment group (Panel b) maintained glycogen storage patterns comparable to controls, with robust PAS positivity and preserved hepatocellular glycogen content.

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Fig. 2.

(a-l) Hepatic glycogen storage assessment using Periodic Acid-Schiff (PAS) staining. (a) The control group demonstrated abundant hepatic glycogen deposits with intense, uniform PAS-positive staining (deep magenta) throughout hepatocyte cytoplasm. (b) The ginger-only treatment group maintained normal glycogen storage patterns comparable to controls. (c, d) Diabetic liver tissue showed severe glycogen depletion with markedly reduced PAS staining intensity and heterogeneous distribution, indicating compromised glucose storage capacity. (e, f) Ginger extract treatment demonstrated significant glycogen storage restoration with enhanced PAS staining intensity and improved distribution patterns (red circle). (g, h) Metformin monotherapy showed moderate glycogen recovery with improved but incomplete restoration compared to controls (red circle). (i, j) Combination therapy achieved superior glycogen normalization with staining patterns closely resembling healthy controls, indicating optimal glucose storage restoration (red circle).

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Quantitative analysis of glycogen storage parameters confirmed these qualitative observations (Table 6). PAS staining intensity was significantly reduced in diabetic animals (1.2 ± 0.3 vs. 3.8 ± 0.5 in controls, p < 0.001). Therapeutic interventions demonstrated progressive restoration: ginger extract treatment improved intensity to 2.9 ± 0.4, metformin achieved 2.5 ± 0.4, while combination therapy produced optimal normalization at 3.4 ± 0.5 (p <0.001 vs. diabetic). Glycogen content analysis revealed severe depletion in diabetic liver (15.3 ± 2.9 mg/g tissue vs. 42.6 ± 4.8 mg/g in controls, p < 0.001), with combination therapy achieving 91.3% recovery (38.9 ± 4.4 mg/g tissue).

3.7 Apoptotic marker expression assessment

BCL2 immunohistochemical staining revealed significant alterations in anti-apoptotic protein expression across experimental groups, as demonstrated in Fig. 3. Control animals (Panel a) exhibited minimal baseline BCL2 immunoreactivity with weak, scattered cytoplasmic staining in hepatocytes, indicating low physiological anti-apoptotic protein expression under normal conditions. Ginger-only treated animals (Panel b) maintained BCL2 expression patterns similar to controls with minimal immunoreactivity.

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Fig. 3.

BCL2 immunohistochemical expression analysis in hepatic tissue. (a) The control group demonstrated minimal baseline BCL2 immunoreactivity with weak, scattered cytoplasmic staining in hepatocytes under physiological conditions. (b) The ginger-only treatment group maintained expression patterns similar to controls, confirming no alteration of baseline anti-apoptotic mechanisms in healthy tissue. (c, d) Diabetic liver tissue showed markedly elevated BCL2 immunoreactivity with intense, widespread cytoplasmic staining (brown DAB-positive) concentrated in clusters around portal areas (red circles), reflecting compensatory upregulation in response to cellular stress. (e, f) The ginger extract treatment demonstrated modulated BCL2 expression with reduced intensity and more organized distribution (red arrows indicate areas of decreased staining intensity). (g, h) Metformin monotherapy showed similar modulatory effects with decreased immunoreactivity and more physiological staining patterns. (i, j) Combination therapy achieved optimal BCL2 regulation with mild, well-distributed cytoplasmic staining approaching control patterns. (k, l) Additional combination-therapy views confirmed consistent expression normalization throughout hepatic parenchyma. Scale bar = 50 μm, magnification 400X. Data presented as mean ± S.E.M (n = 10 per group), p <0.05 vs. control or diabetic groups as indicated.

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Diabetic liver tissue demonstrated markedly elevated BCL2 immunoreactivity with intense, widespread cytoplasmic staining (Panels c, d). The diabetic hepatocytes showed strong brown DAB-positive staining concentrated in clusters and around portal areas, reflecting compensatory upregulation of anti-apoptotic proteins in response to diabetes-induced cellular stress and oxidative damage. Ginger extract treatment (Panels e, f) modulated BCL2 expression patterns, with a structured and moderate immunoreactivity distribution compared to diabetic controls. Metformin monotherapy (Panels g, h) produced similar modulatory effects on BCL2 expression, with decreased immunoreactivity intensity and more physiological staining patterns. Combination therapy (Panels i-l) achieved optimal BCL2 expression regulation, with immunoreactivity patterns approaching those observed in control animals.

Quantitative immunohistochemical analysis confirmed the qualitative observations (Table 7). The quantitative data revealed a significant elevation of BCL2 expression in diabetic animals (1.86 ± 0.34-fold increase compared to controls, p <0.001). Therapeutic interventions demonstrated progressive normalization of BCL2 expression: ginger extract treatment reduced expression levels to 1.42 ± 0.28-fold above controls, metformin achieved a similar reduction to 1.51 ± 0.31-fold. At the same time, combination therapy produced optimal normalization with BCL2 levels approaching control values (1.15 ± 0.23-fold).

3.8 Gut microbiome compositional analysis

Comprehensive taxonomic profiling revealed profound diabetes-induced perturbations in gut microbiome architecture (Table 8). Control animals exhibited a characteristic murine gut profile dominated by Firmicutes (78.1 ± 4.2%) and Bacteroidetes (19.5 ± 3.1%). Type 2 diabetes induction precipitated severe dysbiosis characterized by dramatic Firmicutes depletion (11.8 ± 2.7%, p < 0.001), concurrent with pathological expansion of Proteobacteria (35.2 ± 5.8%, p < 0.001). Combination therapy achieved optimal restoration with Firmicutes reaching 61.3 ± 4.6% and Proteobacteria normalized to 8.2 ± 2.1%.

The Firmicutes/Bacteroidetes (F/B ratio) underwent significant modulation across experimental conditions. Control animals maintained an optimal F/B ratio of 4.26 ± 0.45, while diabetes dramatically reduced this ratio to 0.79 ± 0.16 (p < 0.001). Combination therapy produced near-complete normalization at 3.92 ± 0.42. Shannon diversity index was significantly reduced in diabetic animals (2.35 ± 0.21 vs. 4.41 ± 0.23 in controls), with combination therapy achieving maximal recovery to 4.07 ± 0.22.

Critical beneficial genera showed severe depletion in diabetes: Akkermansia virtually disappeared (3.3 ± 0.9% to 0.3 ± 0.2%), while Lactobacillus decreased dramatically (7.1 ± 1.4% to 2.0 ± 0.7%). Combination therapy achieved remarkable restoration: Akkermansia recovered to 2.3 ± 0.6% (70% of control) and Lactobacillus to 5.6 ± 1.2% (79% of control).

Direct SCFA quantification revealed a 57% reduction in diabetic animals compared to controls (26.5 ± 4.8 vs. 61.9 ± 7.1 μmol/g feces). Butyrate decreased by 70% (from 19.4 ± 2.4 to 5.8 ± 1.4 μmol/g), while combination therapy restored levels to 16.1 ± 2.1 μmol/g (84% recovery). SCFA concentrations strongly correlated with Firmicutes abundance (r = 0.78, p < 0.001) and inversely with hepatic inflammation scores (r = -0.72, p < 0.001).

3.9 Comprehensive therapeutic efficacy analysis

The analysis demonstrated that Zingiber officinale extract, particularly in combination with metformin, achieved remarkable restoration across multiple physiological systems with synergistic effects (Table 8). Metabolic recovery reached 83.6% with combination therapy versus 58.9% with ginger alone and 54.3% with metformin alone. Hepatic protection achieved 88.4% recovery with combination therapy compared to 67.2% and 59.1% for individual treatments. Microbiome restoration reached 84.3% with combination therapy compared with 65.2% and 69.8% for monotherapies.

Statistical analysis confirmed robustness across all measured parameters. All primary endpoints achieved statistical significance with large to very large effect sizes (η² = 0.712-0.921). PERMANOVA revealed distinct clustering patterns for microbiome community structure (pseudo-F = 14.6, p = 0.001). Post-hoc power calculations confirmed that achieved power was >90% for all primary outcomes.