Cardioprotective Potentials of Anacardium occidentale Nuts Methanolic Extract in Diabetes-Induced Cardiac Dysfunction in Rats

Background: The unwanted adverse toxicity displayed by synthetic antidiabetic medicine leads to the search for effective natural medicine to combat diabetes complications. This study investigated the cardioprotective of Anacardium occidentale nuts methanolic in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Forty male adult Wistar were used and fed with HFD for 6 weeks before diabetes induction. The rats were grouped into 5 groups, 8 rats/group. Group I: normal control; Group II: diabetic control; Group III & IV: diabetic rats + 100 mg/kgb.wt & 200 mg/kgb.wt Anacardium occidentale nuts methanolic extract; Group V: diabetic rats + 200 mg/kgb.wt metformin. The rats were sacrificed on the experiment’s last day, blood samples were collected and the hearts were isolated for biochemical parameters estimation.

Results: Food intake, water intake, plasmas insulin, Fasting Blood Glucose (FBG), glycosylated hemoglobin (HbA1c), cardiac enzymes, lipid profile, inflammatory cytokines, malondialdehyde, fibrotic marker, caspase-3 in cardiac of diabetic rats were elevated (p < 0.05) significantly. Body weight, cardiac antioxidant, and anti-apoptotic marker levels diminished (p < 0.05) significantly in diabetic rats. 100 mg/kgb.wt & 200 mg/kgb.wt of Anacardium occidentale nuts methanolic extract administration significantly suppressed the plasma insulin, FBG, HbA1c, cardiac lipid profile, cardiac enzymes biomarker, cardiac inflammatory cytokines, cardiac malondialdehyde, cardiac fibrotic marker, cardiac caspase-3, food intake & water intake and increased the body weight, cardiac antioxidant & cardiac anti-apoptotic marker in the diabetic rats.

Conclusion: Anacardium occidentale nuts attenuate cardiac injury in diabetes. It could be a natural medicine to manage diabetes-cardiovascular complications.

The prevalence of diabetes mellitus escalates significantly [1]. According to the International Diabetes Federation, it is projected that diabetes will affect approximately 783.2 million individuals by 2045 [2].

Diabetes mellitus is described as a chronic metabolic disorder due to impairments in pancreatic β-cells insulin secretion, insulin action, or both which disrupt the metabolism of carbohydrates, lipids, and proteins, leading to chronic hyperglycemia [3-5]. Noticeably, untreated sustained hyperglycemia is implicated in the pathogenesis of diabetes-related macro-vascular and micro-vascular complications, including nephropathy, neuropathy, retinopathy, and cardiovascular diseases [6].

Cardiovascular complications are recognized as the foremost cause of mortality and morbidity in type 1 and type 2 diabetic patients [7]. Cardiovascular complications cause 80% of mortality in diabetes conditions [8].

Diabetes treatment strategies in recent decades have advanced. Nevertheless, anti-diabetic medications have severe side effects [9]. The World Health Organization (WHO) has shifted attention to the use of medicinal plants to manage diabetes mellitus [10,11]. Medicinal plants possess notable anti-diabetic compounds including flavonoids, alkaloids, phenolics, and tannins with few or no adverse effects [12].

Anacardium occidentale Linn (A. occidentale) globally known as the cashew tree is a member of the Anacardiaceae family and is grown widely in tropical countries. A. occidentale has been used as a folk remedy for treating a range of diseases including diabetes mellitus [13]. The species of this plant possess abundant phenolic compounds and flavonoids in their leaves, bark, fruits, and nuts. These compounds exhibit potent anti-inflammatory and antioxidant properties, providing cellular protection [14]. Anti-inflammatory, anti-oxidative, and analgesic activities of the nuts have been previously reported [15]. Also, in mild hyperhomocysteinemia rats, oral administration of A. occidentale nuts was reported to counteract biochemical changes, oxidative stress, pro-inflammatory cytokine release, histological tissue injuries, fibrosis, and apoptosis in the kidney, colon, and liver [16]. However, research on A. occidentale nuts to protect and

manage cardiac complications in diabetes has never been elucidated. This recent study scientifically investigated the potential of A. occidentale nuts methanolic extract to attenuate cardiac injury in high-fat diet/streptozotocin-induced diabetic rats.

Chemicals and Drugs: Streptozotocin, methanol, glucose, phosphate buffer, ketamine, and xylazine.

Experimental animals

Forty adult Wistar rats weighing (250 g – 300 g) were purchased from the Animal Research House of the Physiology Department, Ladoke Akintola University of Technology (LAUTECH), Ogbomoso, Oyo State, Nigeria. The animals were kept in a cleaned propylene cage and fed with standard pelletized feed with water ad libitum for 7 days to acclimatize under the pathogen-free environmental conditions of temperature (25 ± 2 °C), relative humidity (45% ± 5%) and 12:12 hour’s light/dark cycles. All experimental procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals protocol and approved by the Faculty of Basic Medical Science Ethics Research Committee Ladoke Akintola University of Technology (Ethical Approval Number: ERCFBMSLAUTECH:021/01/2024).

A. occidentale nuts collection and extraction

A. occidentale plant was identified at LAUTECH Agriculture Research Farm, authenticated, and assigned a voucher number LH0533 by Dr. A. T. J. Ogunkunle of the Biology Department, LAUTECH. The freshly plunged A. occidentale nuts were washed thoroughly with distilled water, air-dried, and removed from the outer coat before extraction. The nuts were grinded to powder form, kept in an air-tight container, and 500 g of the nut’s powdered form was extracted with 95% methanol in a Soxhlet apparatus to form a semi-solid. The semi-solid was then evaporated in a rotary evaporated pressure and the solid formed was collected and stored at -4 °C until needed.

Diabetes induction

The animals were fed with a High-Fat Diet (HFD) for 6 weeks before diabetes induction. Then, subjected to overnight fasting (12 hours) before diabetes. The animals were injected intraperitoneally with a repeated single dose of freshly prepared streptozotocin (35 mg/kgb.wt) to induce diabetes and given a 2% glucose solution to prevent drug-induced hypoglycemic death. Diabetes induction was authenticated after 72 hours of streptozotocin injection via the animals’ tail prick venous blood using a glucometer (Accu-check) and test stripes. Animals with fasting blood glucose ≥ 200 mg/dL were confirmed diabetic and picked for the experimental study.

Experimental animal grouping

Forty rats were grouped into five groups, 8rats/group. Group I served as control rats, and Group II-V were HFD/STZ-induced diabetic rats treated with different doses of A. occidentale nuts methanolic extract as follows:

Group I: Normal control

Group II: Diabetic control

Group III: Diabetic + 100 mg/kgb.wt AONM Extract (Low dose)

Group IV: Diabetic + 200 mg/kgb.wt AONM Extract (high dose)

Group V: Diabetic + 200 mg/kgb.wt Metformin (reference drug)

A. occidentale nuts methanolic extract was administered for consecutive 6 weeks. Food intake, water intake, and body weight were recorded daily with weighing balance. Weekly fasting blood glucose levels were measured using the glucose-oxidase/peroxidase (GOD-POD) method via the pricked tail vein blood with a digital Accu-Chek glucometer and test strips and recorded throughout the treatment period of the experiment.

Blood Pressure and Electrocardiographic (ECG) parameters recording

The mean Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DSP) were measured using a non-invasive tail-cuffed method.

ECG was recorded using a three-lead non-invasive with electrodes positioned in lead II and sampled at 1 kHz. RR interval, PR interval, P-wave, QRS complex, QT interval, and heart rate were measured. QT was corrected from the QR interval using the Bazett formula [17]:

$\text{QTc }=\frac{\text{QT}}{\left(\text{RR}/\text{f}\right)\text{ 1}/\text{2}}$ where f = 150 ms

Biochemical assay

At the end of the treatment period, the rats were fasted overnight after the last A. occidentale nuts methanolic extract low and high doses (100 mg/kgb.wt & 200 mg/kgb.wt) were administered. The animals were anesthetized with ketamine (40 mg/kgb.wt) and xylazine (20 mg/kgb.wt) and sacrificed by cervical dislocation. Fasting blood samples were collected from the apex beat of the rats’ hearts and the hearts were isolated immediately after blood collection, rinsed in normal saline, and homogenized with freshly prepared cold phosphate-buffered. The blood samples were centrifuged at 3,500 rpm for 15 minutes at 4 °C and the heart tissues homogenates were centrifuged at 10000 rpm for 10 minutes at 4 °C. After centrifugation, the clear supernatant plasma obtained was used for biochemical parameters determination.

Glycated hemoglobin (HbA1c) was estimated using a rat hemoglobin HbA1c assay kit following the manufacturer’s instructions.

Enzyme-Linked Immunosorbent Assay (ELISA) was utilized to measure the levels of insulin, total protein, Creatine Kinase-Myocardial Band (CK-MB), cardiac troponin I, Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), Brain Natriuretic Peptide (BNP), Transforming Growth Factor-β1 (TGF-β1), B-cell lymphoma-2 (Bcl-2) and caspase-3 in rats. Each assay employed a specific ELISA kit designed for rats, following the manufacturer’s instructions.

Lactate Dehydrogenase (LDH) and aspartate aminotransferase (AST) were determined using a spectrophotometer and assay method with an available commercial kit.

Cardiac lipid profile including Total Cholesterol (TC), Triglycerides (TG), and High-Density Lipoprotein cholesterol (HDL-C), was determined using enzymatic colorimetric methods with commercially available assay kits, following the manufacturer’s protocol. Low-Density Lipoprotein Cholesterol (LDL-C) was calculated using the Friedewald equation: LDL-C = TC - (HDL-C + TG/5) [18]. Cardiovascular Risk Indices (CRI) were calculated as the ratio of TG to HDL-C.

The Atherogenic Coefficient (AC) and Castelli’s Risk Index-1 (CRI-1) were computed using the following formulas:

Atherogenic Coefficient (AC) = (TC - HDL-C)/HDL-C.

Castelli’s Risk Index-1 (CRI-1) = TC/HDL-C.

To determine the cardiac oxidative stress Malondialdehyde (MDA) and antioxidants’ Catalase (CAT) and Superoxide Dismutase (SOD) activities, ELISA assay kits were employed as per the manufacturer’s guidelines.

Statistical analysis

Data were presented as the standard error of means (Mean ± SEM) and were analyzed with a statistical package for social science (SPSS version 21.0 software) using one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test to determine the statistically significant difference between groups. Data at p < 0.05 was considered statistically significant.

Effect of A. occidentale nuts methanolic extract on body weight and relative heart weight in HFD/STZ-induced diabetic rats

Diabetic-induced rats displayed a significant (p < 0.05) reduction in body and relative organ weights compared with normal control rats. The administration of 100 mg/kgb.wt (low dose) and 200 mg/kgb.wt (high dose) A. occidentale nuts methanolic extract improved the body and relative organ weights compared with diabetic control rats (Table 1).

Effect of A. occidentale nuts methanolic extract on cardiac oxidative stress markers and antioxidants in HFD/STZ-induced diabetic rats

Antioxidants’ Superoxide Dismutase (SOD) and Catalase (CAT) levels in cardiac of diabetic-induced rats reduced (p < 0.05) significantly and oxidative stress markers’ Malondialdehyde (MDA) increased (p < 0.05) significantly in comparison with the normal control. The low dose (100 mg/kgb.wt) and high dose (100 mg/kgb.wt) A. occidentale nuts methanolic extract administration increased the cardiac SOD and CAT and reduced MDA when compared with diabetic control rats (Table 2).

Effect of A. occidentale nuts methanolic extract on cardiac inflammatory markers, apoptotic and anti-apoptotic markers in HFD/STZ-induced diabetic rats

There was a significant (p < 0.05) increase in cardiac interleukine-6 (IL-6), interleukine-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta1 (TGF-β1) in diabetic-induced rats compared with normal control rats. The administration of 100 mg/kgb.wt (low dose) and 200 mg/kgb.wt (high dose) A. occidentale nuts methanolic extract decreased the level of cardiac IL-6, IL-1β, TNF-α, and TGF-β1 in comparison with diabetic control rats (Table 3).

Cardiac apoptotic marker caspase-3 levels increased (p < 0.05) significantly and anti-apoptotic marker B-cell lymphoma-2 (Bcl-2) lowered (p < 0.05) significantly in cardiac diabetic rats when compared with the control. The low dose (100 mg/kgb.wt) and high dose (100 mg/kgb.wt) A. occidentale nuts methanolic extract administration increased the cardiac level anti-apoptotic Bcl-2 and reduced the apoptotic marker caspase-3 in comparison with the diabetic rats (Table 3).

Diabetes mellitus, an incurable chronic metabolic disease needs ample care for its management. Prolonged hyperglycemia causes many complications related to diabetic mellitus [19]. Cardiovascular ailment is one of the common diabetic complications. Oxidative stress, inflammation, and cell apoptotic are considerably implicated in cardiovascular disease manifestation in diabetes [20]. However, the World Health Organization data revealed that about twenty thousand medicinal plants are available worldwide [21,22]. Natural compounds in medicinal plants display strong pharmacological efficacy for managing many diseases. This study investigates the potential of A. occidentale nuts methanolic extract to attenuate cardiac damage in diabetes.

Diabetes animal models typically displayed clinical symptoms and features noticeable in human diabetes including, elevated blood glucose, decreased plasma insulin, dyslipidemia, frequent urination, polyphagia polydipsia, and loss of body weight [23]. Consistent with Raish, et al. [24] findings, the current experimental streptozotocin-induced diabetic rats developed hyperglycemia, hyperinsulinemia, polyphagia, polydipsia, and a reduction in body weight. Body weight loss in diabetes has been hypothesized to result from metabolic alterations that lead to excessive structural protein catabolism and reduce protein synthesis [25]. Excessive hepatic glycogenolysis and gluconeogenesis and decreased utilization of insulin by peripheral organs lead to hyperglycemia and hyperinsulinemia in diabetes [26]. Obviously, low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract supplement ameliorates the aforementioned clinical features and symptoms in diabetic rats. Inhibition of hepatic gluconeogenesis, increase in hepatic glycogen storage and stimulation of peripheral tissues sensitivity to insulin action for glucose uptake could be likely mechanisms for the hypoglycemic and insulin-normalizing effects of the A. occidentale nuts.

As reported in various findings, hyperglycemia causes glycosylation of proteins which progresses to the formation of chronic elevated glycated hemoglobin HbA1c, a maker of glycemic control in diabetes [27]. This finding observed elevated HbA1c levels in diabetic rats, supports the other report. However, administration of low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract suppressed the HbA1c levels and this might stem from the reduction in blood glucose level upon administration of the A. occidentale nuts extract, parallel the findings of Gothandam, et al. [28].

Diabetes patients frequently encounter alterations in the normal electrocardiography (ECG) pattern mostly the Q-T interval and T-wave. Diabetes specifically leads to prolongation of the QRS and Q-T intervals [29]. QTc stands for corrected Q-T interval and a remarkable prolongation of the Q-T interval serves as a crucial indicator for screening diabetic patients susceptible to sudden cardiac death [30,31]. This study observed bradycardia, prolonged P-wave, QT interval, and QTc intervals, and decreased QRS complex in the ECG pattern of diabetic rats. Moreover, supplements of A. occidentale nuts methanolic extract low (100 mg/kgb.wt) and high (200 mg/kgb.wt) doses to the diabetic rats restored the normal ECG pattern. Many studies have reported the significance of bioactive compounds of medicinal plants in restoring and enhancing cardiac function in diabetic rats via normalizing cardiac electrical activities [32]. This improvement in cardiac ECG patterns noticed in diabetic rats could be attributed to the antioxidants from bioactive compounds of the A. occidentale nuts.

Also, abnormal Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP) in diabetic rats have been established [33]. Dysfunction in these parameters was obvious in this study as the diabetic rats exhibited high SBP and reduced DBP, contrary to the findings of Bulani, et al. [34]. A. occidentale nuts methanolic extract low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose administered to the diabetic rats ameliorates the altered blood pressure and this showed the nuts inhibit the progression of blood pressure impairment in diabetic rats.

Cardiac enzymes are key sensitive biomarkers for cardiac injury in a diabetic heart. These enzymes are abundant in cardiac muscle tissue and damage to this muscle evokes the release of the enzymes into the circulation [35]. Chronic hyperglycemia-induced oxidative stress causes injury to the cardiac muscle cells leading to the release of these enzymes into the blood [36]. Also, studies have reported the release of high Brain Natriuretic Peptide (BNP), a ventricular dysfunction biomarker in diabetic heart failure [37]. Elevated levels of these biomarkers have been previously reported in the hearts of diabetic rats [38], the finding of this study is in line with the report, as the diabetic rats exhibited elevated cardiac enzymes Creatine Kinase-Myocardial Band (CK-MB), troponin- I (cTnP-I), Lactate Dehydrogenase (LDH) and aspartate aminotransferase (AST) and similar with Saklan et al findings [39], overexpression of ventricular dysfunction biomarker BNP was observed. The low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract administration lessened the cardiac enzymes biomarker, indicating cardiomyocyte cells protection of the nuts which might stem from the antioxidant potentials of A. occidentale nuts which harmonized the findings of Adoga, et al. [40].

Dyslipidemia in chronic hyperglycemia is considered to contribute to the pathogenesis of ischemic heart disease and the progression of heart failure by enhancing lipid toxicity and reactive oxygen species production in diabetic individuals [41]. Our results revealed dyslipidemia in the diabetic rats noticed by a high level of cardiac triglycerides (TG), Total Cholesterol (TC) low-density lipoprotein-cholesterol (LDL-C), atherogenic coefficient (AI), and Castelli’s risk index-1 (CRI-1) with low cardiac high-density lipoprotein-cholesterol (HDL-C), consistent with findings of Tangvarasittichai [42]. The levels of cardiac TG, TC, LDL, AI, and CRI-1 were reduced and HDL-C improved on treatment with low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract, suggesting anti-hyperlipidemic and cardioprotective of the nuts and could be due to the suppression and inhibition of intestinal and hepatic acyl-coenzyme A (CoA) activity and this parallel with the reports of Yang, et al. [43].

Oxidative stress is well-known to trigger the mechanism that leads to myocardial contractility loss, cardiac fibrosis, cardiac inflammation, apoptosis of cardiac cells, and DNA damage via excessive generation of reactive oxygen species in cardiac tissues [44-47]. The heart is vulnerable to oxidative stress injury due to the low antioxidant enzymes capacity to scavenge free radicals [48]. Excessive cardiac oxidative stress and low antioxidant enzymes have been reported in the diabetic heart [49]. According to the previous report, elevated cardiac oxidative stress marker malondialdehyde (MDA) and low levels of antioxidants superoxide dismutase (SOD) and catalase (CAT) were observed in the diabetic rats of the current study. Administration of low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract raised the cardiac endogenous antioxidants SOD and CAT and depressed the oxidative stress marker, this indicates cardiac oxidative stress attenuation with potent antioxidants properties of the nuts via the numerous bioactive compounds present in the nuts, which is in line with the findings of Li, et al. [50].

Oxidative stress-induced cardiac inflammation has been implicated in the development and progression of cardiac injury in diabetic patients [51]. Excessive production of myocardial pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukine-1β (IL-1β), and interleukin-6 (IL-6) have been reported in the etiology of cardiac function anomalies and fibrosis [52]. In support of the findings of Liang, et al. [53], up-regulation of cardiac cytokines TNF-α, IL-1β, and IL-6 are observed in the heart of diabetic rats of the current study. However, these up-regulated cytokines in the diabetic heart were suppressed with low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract treatment, which evident cardiac anti-inflammatory properties of A. occidentale nuts and this may be attributed to the antioxidant efficacy displayed by suppressing the hyperglycemia induced-oxidative stress in the didactic heart, similar with the report of Arjumand, et al. [54].

Cardiac inflammatory cytokines and reactive oxygen species, the consequences of oxidative stress are crucial mechanisms in the pathogenesis of cardiac fibrosis through superfluous cardiac transforming growth factor-beta 1(TGF-β1) [55]. Noticeably in the current study, a high level of TGF-β1 was expressed in the cardiac of diabetic rats, corroborating the findings of Taye, et al. [56]. Treated with low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose A. occidentale nuts methanolic extract diminished the overexpression of cardiac fibrosis marker TGF-β1. These proved the strong anti-inflammatory and antioxidant potentials of the nuts in protecting the cardiac muscles from being damaged in diabetic conditions which supports the findings of Qu, et al. [57], on amelioration of diabetic cardiomyopathy by Pyrroloquinoline quinone.

In diabetic hearts, three types of cell death have been identified as contributing to the development of cardiac dysfunction: apoptosis, necrosis, and autophagy. Apoptosis, a programmed cell death process, manifests through intrinsic and extrinsic pathways [58,59]. The extrinsic pathway is triggered by factors like TNF-α, which binds to death receptors, initiating apoptosis [60]. Conversely, intrinsic cell death arises from various factors including reactive oxygen species (ROS), inflammatory cytokines, disturbances in calcium levels, and DNA damage [61-63]. Intrinsic apoptosis is characterized by diminished expression of anti-apoptotic genes (e.g., Bcl2), heightened expression of pro-apoptotic genes like Bax, mitochondrial dysfunction, increased release of mitochondrial cytochrome-c, and subsequent activation of caspase-3 and caspase-9, leading to DNA fragmentation [64]. Both extrinsic and intrinsic pathways contribute to cell death in diabetic hearts, observed in both animal models treated with STZ and left ventricular biopsies from patients with type 1 diabetes mellitus, with oxidative damage playing a significant role [65]. Current study findings revealed the establishment of an intrinsic pathway, as there was reduced expression of anti-apoptotic genes B-cell lymphoma-2 (Bcl2) and high expression of pro-apoptotic genes caspase-3 in cardiac diabetic rats. A. occidentale nuts methanolic extract low (100 mg/kgb.wt) and high (200 mg/kgb.wt) dose administration caused an upsurge in the expression of Bcl-2 and a decline in the caspase-3 expression in cardiac of diabetic rats. Inhibition of cardiac inflammatory response, scavenging of cardiac free radicals, and boosting of cardiac anti-oxidant enzymes might be responsible for the anti-apoptotic effect of A. occidentale nuts, corroborating the findings of Tamimi, et al. [66] on apoptotic attenuation properties of Esculeoside A. in diabetic heart.

Findings from this study indicate that Anacardium occidentale nuts attenuated cardiac injury and ameliorated electrocardiographic changes in diabetes. It could be used as a safe and effective natural medicine to manage diabetes-associated complications.

Limitation

The bioactive compound of A. occidentale nuts responsible for the cardio-protective efficacy was not identified in this research. The novel bioactive compound in A. occidentale nuts with this therapeutic effect should be investigated.

Declarations

Authors’ contributions: FO and NO conceived the original idea, and designed and supervised the research. NO, MO SA, AG, PO, ZA, OJ, and ES performed the experiments with the support of FO. FO, NO, and MO performed the data collection. FO, MO, and FE analyzed the data and prepared the manuscript. FO reviewed the manuscript. All authors have read and approved the final manuscript.

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