Cardiovascular disease (CVD) is one of the most common causes of morbidity and mortality in patients with type 2 diabetes 1. Individuals with type 2 diabetes are more likely to develop cardiovascular disease 2. Diabetes mellitus is a multifactorial disorder characterized by hyperglycaemia, polydipsia, polyuria, and glycosuria 3. CVD is affected by conditions such as elevated cholesterol, fibrinogen, clotting factors, platelet function, glucose metabolic disturbances, and smoking 4. Therapeutic interventions are necessary to reverse diabetes-induced cardiovascular damage, and cardioprotective drugs are particularly important during hypoglycaemic management. Ischaemia-reperfusion (I-R) injury results in functional and structural heart defects, primarily due to uncontrolled excess cytosolic calcium (Ca²⁺) and oxidative stress 5, 6, 7. Reperfusion augments reactive oxygen and nitrogen species,4, 8 and aging hearts can exhibit increased sensitivity to IR injury 9. Herbal consumption has increased worldwide. Although herbs are traditionally safe, they can interact with drugs. Approximately 15–20% of patients with prescriptions take herbal supplements, with less than 40% reporting to physicians, often out of fear of being judged 10. Most physicians are not aware of herb-drug interaction hazards,11 making it imperative to conduct reliable research 12.

Garlic (Allium sativum) is an ancient culinary spice and folk medicine with antidiabetic and cardioprotective properties 13. It prevents cardiovascular and metabolic diseases owing to its high bioactive compound content 14. Repaglinide (REP) enhances cardiovascular function and glucose metabolism by inducing insulin secretion, although its rapid absorption and metabolism are different from sulfonylureas 15. Therefore, complementary herbs must be used to support their efficacy. Short-acting hypoglycemics assist in correcting the initial insulin secretion deficiencies in type 2 diabetes 16. Calcium channel blockers, such as amlodipine (AML), block calcium entrance, which is advantageous for heart and vessel function 17. AML is used to treat hypertension and angina with few cardiac effects and assists in healing after ischaemia 18, 19. The current study was conducted to evaluate whether garlic could enhance the cardioprotective effects of AML and REP during ischaemia-reperfusion-induced myocardial injury in diabetic rats.

Instruments and Chemicals: The study used an analytical balance (Shimadzu, Japan), autoanalyser (Qualigens, Mumbai), centrifuge, colorimeter, perfusion pump, Polyrite (RMS, Chandigarh), Student's Physiograph (INCO, India), spectrophotometer, and reagents including calcium chloride, CK-MB kits, LDH kits, sucrose, D-glucose, EDTA, ethanol, heparin, ketamine, magnesium sulfate, NBT, propranolol, and others.

Experimental Animals: Male or female rats with body weights of 175–250 g were maintained in a well-ventilated animal house at a temperature of 25 ± 5°C under a light-dark cycle of 12:12 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (KCP/IAEC-27/2009-10), and animals were handled according to the regulations of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

Preparation of Garlic Homogenate: Locally brought garlic bulbs were peeled, chopped, and ground into paste. A garlic homogenate (GH) concentration of 0.1 gm/ml, equivalent to 250 mg/kg body weight, was prepared in suspension in distilled water and administered to the rats within 30 min of preparation.

Induction of Diabetes: Rats were fasted for 24 h prior to the induction of diabetes. Diabetes was induced by a single intraperitoneal injection of alloxan (150 mg/kg body weight). Three days after alloxan administration, blood glucose levels were measured, and rats with serum glucose levels ≥300 mg/dl were selected for further experimentation.

Experimental protocol: Sprague-Dawley rats of both sexes were separated into two groups (normal and diabetic), and each group was further separated into eight groups, with six animals in each group. The groups were distributed as follows: Group I (Control), Group II (GH, 30 days oral treatment), Group III (REP, 10 days oral), Group IV (AML, 10 days oral), Group V (GH+REP), Group VI (GH+AML), Group VII (REP+AML), and Group VIII (GH+REP+AML).

Ischaemia reperfusion-induced myocardial dysfunction: The rats were anaesthetised with sodium pentobarbital (35 mg/kg, i.p.) at the end of the treatment schedule. The heart and pancreas were collected. Hearts were perfused using a modified Langendorff apparatus with Krebs-Henseleit solution, carbogen-gassed, at 37°C, and a constant flow rate of 5 ml/min. The contractile force was recorded using a grass electromechanical recorder with a displacement transducer. Baseline readings were recorded for 15 minutes after a 15-minute equilibration time, followed by 15 min of global ischaemia and reperfusion. The recovery of inotropic and chronotropic functions was measured, and cardioprotection was checked by assessing the developed tension. Perfusates were harvested during both the pre- and post-ischaemic phases and used for biochemical estimation of LDH and CK-MB. Three excised hearts per group were homogenized in 0.25 M sucrose to prepare heart tissue homogenate (HTH) for subsequent biochemical analyses. Histopathological sections of pancreatic tissue were stained with haematoxylin and eosin (H&E) and observed under a microscope to assess myocardial changes in diabetic rats following different treatments.

Measurement of Antioxidant Status: Total protein was estimated on the basis of coloured complex formation with Folin-Ciocalteu reagent, monitored at 610 nm, using bovine albumin as a standard. Superoxide dismutase (SOD) activity was determined by measuring the inhibition of NBT reduction at 560 nm after hydroxylamine hydrochloride oxidation. Catalase activity was measured by measuring hydrogen peroxide decomposition at 240 nm. Thiobarbituric acid-reactive substances (TBARS) were measured as an indicator of lipid peroxidation, with malondialdehyde (MDA) as a marker, measured at 532 nm.

Cardiac Markers Estimation: CK-MB activity was determined by using enzyme reagent and starter reagent, incubated and read by an autoanalyzer at 340 nm. LDH activity was determined by the oxidation of NADH to NAD⁺ in the presence of pyruvate, and the decrease in absorbance was measured, which was equivalent to the LDH activity in the sample.

Ischemia-reperfusion (ISC) injury in normal rats reduced superoxide dismutase (SOD) and catalase activities (0.97±0.06 and 3.49±0.35 units/mg protein, respectively) and increased TBARS levels (50.01±1.70). GH treatment significantly improved SOD (3.98±0.08), catalase (10.4±0.8), and reduced TBARS (26.66±1.26). REP showed moderate improvement, whereas AML treatment provided greater protection (SOD: 4.7±0.2; catalase: 11.5±0.7; TBARS: 28.89±1.7). Co-treatment with REP+GH and AML+GH further enhanced antioxidant levels, and the combined GH+REP+AML treatment resulted in the highest SOD (12.89±1.2), catalase (43±2.67), and lowest TBARS (13.39±1.84) (Table 1).

In diabetic rats, reperfusion-ischemia injury caused oxidative stress, shown by decreased SOD (0.56±0.09), catalase (6.8±0.98), and increased TBARS (56.98±1.9). GH treatment improved SOD (5.58±0.3) and catalase (16.33±2.88) levels and reduced TBARS (34.98±2.9) levels. REP raised SOD to 4.77±0.8, catalase to 17.18±1.34,

and lowered TBARS to 42.78±3.1. AML treatment resulted in SOD (8.95±0.50), catalase (23.84±2.88), and TBARS (29.88±2.3). Combined treatments (REP+GH, AML+GH, REP+AML) further improved results, with the GH+REP+AML combination showing the highest protection: SOD (13.8±0.61), catalase (48.4±3.75), and TBARS (17.06±0.99) (Table 1). ISC significantly increased CK-MB and LDH activities in both the perfusate and HTH of control rats, with CK-MB (82±4.8 unit/lit, 33±0.7 unit/gm) and LDH (perfusate: 141±10.8 unit/lit, HTH: 32±3.1 unit/gm). GH treatment reduced CK-MB (perfusate: 39±5.4, HTH: 67±3.3) and LDH (perfusate: 65±4.1, HTH: 114±5.2) levels. REP and AML alone partially restored levels, while the GH+REP+AML combination provided maximum protection, with CK-MB (perfusate: 16±1.56, HTH: 124±3.78) and LDH (perfusate: 13.99±1.09, HTH: 187±4.77) at their lowest levels (Table 2).

In STZ-induced diabetic rats, the DC group of rats exhibited highly significant elevation in CK-MB (112.3±3.7) and LDH (296.6±7.6) in perfusate and lowered HTH CK-MB (18.43) and LDH (16.36). GH increased these values to CK-MB perfusate 52±3.4, HTH 70.77, LDH perfusate 195±5.0, and HTH 49.15. REP and AML treatments improved, with the AML+GH and REP+AML combinations having more recovery. The GH+REP+AML group had maximum improvement with CK-MB perfusate at 12.43±0.6, HTH at 134.78, LDH perfusate at 128±3.9, and HTH at 81.39 (Table 2).

Recovery of heart function, as measured by the developed tension and heart rate, was weak in ischaemia-reperfusion rats. GH treatment significantly improved recovery, whereas REP and AML individually improved recovery moderately. Co-treatment with GH and either REP, AML, or a combination of the two drugs resulted in improved recovery, and the GH+REP+AML group showed the best improvement. In diabetic rats, the diabetic control group showed very poor recovery. GH alone significantly enhanced heart function, while REP and AML treatments improved recovery compared with the control. The GH+REP+AML group showed the highest recovery (Table 3).

Oral glucose test: Fasting blood glucose level (FBS) was 60 ± 3.45 mg/dl in the normal control (NC) group and significantly increased to 130 ± 4.78 mg/dl in the diabetic control (DC) group. The GH-, REP-, and AML-treated groups showed FBS levels of 124 ± 2.92, 106 ± 4.11, and 112 ± 5.67 mg/dl, respectively. The combination groups (GH+REP, GH+AML, REP+AML, GH+AML+REP) showed better control, with FBS values between 79 ± 3.74 and 103 ± 3.77 mg/dl. At 30 min post-glucose load, blood glucose peaked in the DC group (330 ± 7.10 mg/dl) compared to that in the NC group (160 ± 3.45 mg/dl), while the GH, REP, and AML groups recorded 310 ± 6.83, 429 ± 8.55, and 230 ± 2.45 mg/dl respectively. Combination therapies, especially GH+AML+REP (214 ± 5.24 mg/dl), showed better glucose control. At 120 min, glucose in the NC group decreased to 110 ± 1.33 mg/dl, while the DC remained high at 375 ± 2.56 mg/dl. REP+AML (292 ± 7.59 mg/dl) and GH+AML+REP (139 ± 2.64 mg/dl) groups showed significant improvement compared to monotherapies. After 240 min, the NC returned to 90 ± 2.65 mg/dl, while GH+AML+REP achieved near-normal levels (89 ± 3.88 mg/dl), indicating the strongest glucose-lowering effect among the treated groups.

Histopathological evaluation: Histopathological analysis revealed that ISC induced severe myocardial damage in both diabetic and non-diabetic rats. GH maintained the myocardial structure, whereas AML and REP alone induced mild-to-moderate damage. GH, in combination with AML or REP, further enhances the architecture. The GH+REP+AML group had an almost normal myocardial structure, with minimal infiltration. In diabetic rats, treatment with GH ensured heart integrity, and triple therapy with GH, REP, and AML showed the maximum cardioprotective effect. (Figure 1 )

Histopathological evaluation revealed that the normal group exhibited no damage to pancreatic β cells. However, the diabetic control group showed clear signs of pancreatic necrosis, along with a marked reduction in β and α cells. REP treatment increased the β cell population within the islets of Langerhans, and when combined with GH, this effect was further enhanced compared to REP alone. Notably, GH alone did not exhibit any significant protective effects. (Figure 2)

This study investigated the therapeutic potential of GH, REP, and AML in reducing ischaemia-reperfusion-induced myocardial damage in diabetic rats. ISC damage in normal rats caused a significant reduction in antioxidant enzyme activities, namely SOD and catalase, and a significant increase in TBARS, an indicator of oxidative stress. These results align with previous observations by Banerjee et al., who found a reduction in antioxidant activity and an increase in oxidative markers following ischaemia-reperfusion injury in rat hearts 20. GH treatment dramatically enhanced antioxidant defense by increasing SOD and catalase activity and decreasing TBARS levels. These findings are similar to those of Bhatti et al., who established the cardioprotective properties of garlic through modulation of oxidative stress in ischaemia-reperfusion injury 21 .

The individual effects of REP and AML were mild, with partially improved oxidative stress parameters in the present study. The combination treatments, especially the GH+REP+AML group, displayed the strongest protection, with considerably increased SOD and catalase activities, and the lowest TBARS values. This synergy between garlic and REP and AML is consistent with the findings of Asdaq and Inamdar, who also reported a pharmacodynamic interaction between garlic and captopril, which also showed increased myocardial protection in ischaemia-reperfusion injury 22.

In the present study, oxidative stress caused by ischaemia-reperfusion was enhanced in diabetic rats, as indicated by decreased SOD and catalase levels and increased TBARS content compared with normal controls. GH treatment in diabetic rats indicated a significant enhancement in oxidative stress parameters, which aligns with the findings of Sener et al., who showed the antioxidant properties of garlic to prevent ischaemia-reperfusion injury 23. In addition, REP and AML treatments enhanced these parameters, with the combination of GH+REP+AML having the most complete cardioprotection. The synergistic effects of this combination treatment suggest a multi-target strategy to reduce oxidative damage, as previously reported by Iwalokun et al., who showed that Allium sativum (garlic) extracts regulated oxidative stress and diminished tissue damage in animal models 24 .

With regard to myocardial injury biomarkers, CK-MB and LDH activities were greatly increased in the perfusate and heart tissue homogenates (HTH) of control and diabetic rats with ischaemia-reperfusion injury in the present study. GH treatment effectively decreased the levels of these markers, indicating a protective effect against myocardial injury. REP and AML treatments caused partial recovery, but the combination of GH+REP+AML resulted in the greatest reduction in CK-MB and LDH levels. These findings agree with those of Shackebaei et al., who similarly found decreased levels of myocardial injury markers when rats were treated with garlic in an ischaemia-reperfusion model25.

In the present study, restoration of heart function, reflected in the developed tension and heart rate, was impaired in ischaemia-reperfusion rats. Recovery was markedly enhanced by GH, but not by REP or AML, to a modest extent. The GH+REP+AML combination showed optimal functional recovery, corroborating the findings of Patumraj and Jetapai, where the cardiovascular complications in diabetic rats were improved with the use of garlic extracts 26 . The diabetic control group of diabetic rats showed inferior recovery, which was significantly enhanced by GH alone and further augmented by combination therapies, especially GH+REP+AML.

In the present study, oral glucose tolerance tests showed that the combination treatments, particularly GH+REP+AML, maintained blood glucose levels. The glucose-lowering activity of the combination treatment was greater than that of the single treatments, supporting the synergistic potency of GH, REP, and AML, providing a combined metabolic advantage to diabetic rats. These findings complement those of Bhatti et al., who demonstrated improved glucose metabolism after garlic administration in diabetic animals21.

In the present study, histopathological examination indicated extensive myocardial damage in diabetic and non-diabetic ischaemia-reperfusion injury-induced rats. GH therapy maintained myocardial architecture, and the GH+REP+AML combination provided maximum protection, with an almost normal myocardial structure. These findings agree with those of Banerjee et al., who showed that garlic treatment prevents ischaemia-induced myocardial damage and preserves myocardial integrity 20.

The current study demonstrated the cardioprotective effects of garlic combined with repaglinide and amlodipine against ischaemia-reperfusion injury in diabetic rats. These results suggest that combination treatment can be an effective way to counteract oxidative stress, enhance myocardial injury recovery, and offer better cardiovascular protection against diabetes. Further studies are needed to investigate the molecular basis of these synergistic actions and determine their potential therapeutic applications.