Background: Myocardial infarction (MI) is the leading cause of cardiovascular deaths worldwide. Musa paradisiaca has been reported to contain secondary metabolites with antioxidant properties. This study investigated the possible cardioprotective effects of aqueous ripped Musa paradisiaca peel extract (MPPE) in an isoproterenol (ISO)-induced MI rat model relative to aspirin as a standard drug. Methods: The MPP was extracted in distilled water using cold extraction; thereafter, MPPE was screened for secondary metabolites using standard biochemical methods. Investigation on the acute toxicity of MPPE was done in compliance with ARRIVE guidelines. Cardioprotective effects of the extract were established using biochemical assays (ELISA technique), an electrocardiogram, and a histological examination. We analyzed the data using a graphical prism version 5.03. Results: The screening of MPPE revealed the presence of secondary metabolites, including flavonoids and phenols. The LD₅₀ was above 5000 mg/kg. Rats administered ISO developed MI evidenced by increased cardiac troponin-I (cTn-I), pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), malondialdehyde, and ST segment elevation on the ECG. Further, there was a reduction in antioxidant enzymes and membrane-bound Na⁺/K⁺ ATPase activities. Pre-treatment with MPP promoted restoration of cardiomyocytes with no side effect compared to aspirin. Significantly, it increased CAT, SOD, and Na+/K+ ATPase activities and decreased pro-inflammatory cytokines, MDA, and cTn-I, thereby reducing the elevation of ST-segment on the ECG to near normal. Results from the histopathological study support the cardioprotective effects of MPP. Conclusion: The MPP confers protection to the myocardium through its antioxidant and anti-peroxidation properties that act as possible mechanisms in ISO-induced MI in rat models.

Anti-inflammatory Antioxidant Isoproterenol Musa paradisiaca Myocardial infarction

Myocardial infarction (MI), characterized by the hypoxic state of myocardial tissue sequel to uneven perfusion of coronary blood supply compared to demand, is a leading cause of cardiovascular morbidity and mortality in men1. MI causes myocardial ischemic injury and damage to the cardiomyocytes. The repair of damaged hearts in patients is challenging due to the limited ability of the cardiomyocytes to regenerate post-mitotically. Prolonged evident ischemia in chronic MI eventually leads to permanent myocardial cell injury or death2.

Citizens from low and middle-income countries, such as sub-Sahara Africa, including Nigeria, are more vulnerable to MI, with an increasing number of cases recorded in adults owing to habits and lifestyle such as excessive consumption of junk food, a lack of physical exercise, an over-labored working condition, and poverty3.

Previous reports suggest that the accumulation of reactive oxygen species (ROS) causing oxidative changes to the macro-molecules during ischemic damage played a pivotal role in the pathogenesis of MI with the deleterious consequence, evidenced by the elevation of cardiac troponin-I (cTn-I), the upregulation of the pro-inflammatory cytokines interleukine-1 (IL-1β) and interleukine-6 (IL-6), and tissue necrotic factor (TNF-α)2. In addition, MI manifests by impaired sodium-potassium ATPase activity and electrocardiogram alterations together with histopathology changes4, 5.

Therapy with agents possessing antioxidant properties is of great interest due to the overwhelming evidence implicating oxidative stress sequel to increased production of free radicals during the pathogenesis of MI6. However, the limitations (such as a high level of toxicity and low aqueous solubility) observed following the use of synthetic antioxidants in preventing cardiovascular diseases have shifted the attention of researchers to the use of naturally derived antioxidants, owing to their high safety margin, cultural acceptability, and promised efficacy7.

Banana is the common term used to refer to several hybrids in the genus Musa. Plantain, Musa paradisiaca, is considered a good food source of natural antioxidants against cancer and heart disease8. It is widely consumed without apparent toxic effects, and its antioxidant properties have been traced to its presence of vitamin C, vitamin E, β-carotene, and dopamine. The report showed that peels of Musa paradisiaca constitute about 40% of the total weight of the fresh fruit and displayed reasonable antimicrobial and antioxidant activities in both ethyl acetate and water-soluble extracts9, 10. In addition, several secondary metabolites (flavonoids, phenols, tannins, alkaloids, and glycosides) have also been isolated from plantain peels suggesting its antioxidant and anti-inflammatory effects8.

For over 25 years, aspirin (acetylsalicylic acid) has been known for its role in preventing MI and ischemic stroke through its anti-thrombotic effect11. Aspirin displays an antiplatelet effect at low doses, thus prolonging bleeding time by selectively inhibiting cyclooxygenase (COX)-I, an enzyme that produces the platelet antagonist thromboxane A2, and acting as an effective vasoconstrictor12. Based on the chemical constituent and reported activities of plantain peel, the present study aims to evaluate the cardioprotective effects of the aqueous extract of riped Musa paradisiaca peels following isoproterenol-induced myocardial infarction.

A commercial grade standard of H906 (25 g) marketed as Isoprenaline HCl (98% HPLC) was obtained from AK Scientific, Inc, USA. Aspirin, the standard drug used, was procured from Bioraj pharmaceuticals, Nigeria. Pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and cTn-I test kits were obtained from Junjiang Inter. Bldg Shanghai, China, and Calbiotech, California, USA, respectively. Other reagents and chemicals used were of analytical grades.

Freshly ripped peels of Musa paradisiaca were collected from a major food canteen at Malete, Kwara State, Nigeria. The plant was identified at the herbarium of the Department of Plant Biology, University of Ilorin, Nigeria, and the voucher specimen (UIL/001/2019/1381) was deposited at the herbarium for reference purposes. After rinsing in distilled water, the peels were air-dried to a constant weight in the laboratory at room temperature (25 ± 2.0 °C) and pulverized to a powdered form using a kitchen blender (Nakai Japan, China). The powdered samples were kept in an airtight container until ready for use.

One kilogram of powdered Musa paradisiaca peels was extracted in 5 Litres (L) of distilled water using the cold maceration method13. Thereafter, the solution was filtered with Whatman No 1 filter (Sigma Aldrich, St. Louis, MO, USA). The filtrate was concentrated in-vacuo, using a hot air oven (Surgifield Medical, Germany) set at 30 °C and thereafter preserved at 4 °C in a refrigerator until required for use. The extract was subjected to GC-MS. The Percentage yield (22.4 %) was determined, after which the extract was reconstituted with distilled water to a final concentration of 200 mg/mL.

The Musa paradisiaca peel extract was evaluated for the presence of secondary metabolites by using standard methods14.

Bioactive compounds contained in the extract were analyzed in a GC-MS (Shimadzu, Kyoto) QP2010S that was fitted with a 1.4 µm column Rxi-5sil MS 30-meter length, 0.25 µm film thickness, and 0.25 mm inner diameter. The carrier gas was helium with a flow rate of 0.98 ml/min, column temperature 80 ºC, initial temperature 70 ºC, injector temperature 260 ºC, and detector temperature 300 ºC. The programmed linear temperature varied from 70 to 280 ºC at a rate of 10 ºC/min, operating in electron impact mode. The samples were injected in splitless mode. The interface temperature was kept at 280 ºC. Oven temperature programming was varied from 80 °C to 260 ºC at 10 ºC/min. The pressure of the carrier gas was kept at 63.6 kPa. The individual bioactive compounds were identified using the NIST 11 and WILEY 8 library based on the RT. Consequently, the name, molecular weight (MW), and structure of the components of the test materials were ascertained15.

A total of 48 apparently healthy male Wistar rats (250 - 330 g) were acquired from an experimental animal unit of the Department of Veterinary Pharmacology and Toxicology, University of Ilorin, Nigeria. The rats were distributed at random into 6 polypropylene cages (8 animals each) lined with wood shavings, renewed every 24 h. The animals were given access to pelletized vital feed (UAC, Nigeria) while water was provided ad libitum. Optimum temperature and humidity were maintained at 28 ± 2 ℃ and 65 ± 10%, respectively.

The median lethal dose (LD₅₀) was determined in accordance with the Organization of Economic Co-operation Development guidelines by a standard two-phase approach described by Lorke16. In the first phase, nine apparently healthy male Wistar rats (270 – 310 g) were distributed at random into 3 groups of 3 animals. The rats had fasted for 12 hours. Animals in each group (groups 1, 2, and 3) were given an aqueous extract of Musa paradisiaca peels (MPP) orally, at 10, 100 and 1000 mg/kg body wt. respectively. Next, the rats were closely observed for 48 hours for signs of mortality or toxicity. Three rats were individually separated into 3 groups in the second phase of the approach. A rat in each group (groups 4, 5, and 6) was orally administered with MP extract at 1600, 2900, and 5000 mg/kg body wt. respectively. They were later monitored from the first 24 hours until 14 days for possible toxicity signs (sedation, salivation, lethargy, and muscular weakness) and mortality. The estimated value of LD₅₀ was thereafter determined by the previously described method13.

Myocardial infarction was induced in this study by a twice subcutaneous injection of isoproterenol hydrochloride (85 mg/kg body wt.) of 20 mg/ml concentration at a 24-hour intervals as previously described4 on days 15 and 16 of the study. The resultant elevation of cardiac troponin-I (cTn-I) in rats confirmed the occurrence of myocardial infarction.

A total of 48 rats were used in this 16-day study. The animals were assigned at random into 6 groups (n = 8) and treated as follows:

Group 1 (normal group): Rats were administered 1 ml of distilled water orally for 16 days.

Group 2 (model group): Myocardial infarction was induced by subcutaneous injection of isoproterenol (85 mg/kg body wt.) at 24-hour intervals on days 15 and 16 of the study.

Groups 3, 4, 5, and 6 (preventive groups): For 14 days, rats in groups 3, 4, and 5 were pre-treated with Musa paradisiaca peel extract (100, 200, and 400 mg/kg body wt. respectively). Also, rats in group 6 were pre-treated with aspirin (30 mg/kg body wt.) for 14 days. Myocardial infarction was then induced with a twice subcutaneous injection of isoproterenol (85 mg/kg body wt.) at 24-hour intervals on days 15 and 16 of the study.

Next, the rats were anesthetized using ketamine (100 mg/kg, intramuscular, i.m.) and xylazine (10 mg/kg, i.m.) on day 17 after fasting for 24 hours. Electrocardiogram readings of all rats were then taken, after which blood was withdrawn from all the rats by cardiac puncture into disposable blood collecting tubes devoid of anticoagulant (Shandong Medical Instrument Co., Ltd, Zibo, China). The blood samples were allowed to clot within 2 hours at 37 °C, followed by centrifugation for 15 minutes at 1000 × g to obtained serum. The sera obtained as supernatants were collected using a plastic pipette into another sterile sample bottle. The rats were later sacrificed by cervical dislocation. The heart tissues were immediately harvested, rinsed in phosphate buffer solution, PBS (0.01 M and pH 7.4) to remove excess blood, and then weighed. The tissues were homogenized in PBS (pH 7.4) using a mortar and pestle and then thawed at 8 °C, after which they were centrifuged at 2000× g for 20 minutes to give tissue homogenates. The serum and tissue homogenates were used to carry out biochemical assays as previously described17.

On day 17 of the study, ketamine (100 mg/kg, i.m.) and xylazine (10 mg/kg, i.m.) were used to anesthetize the rats. The electrodes from EDAN (the ECG machine connected to the laptop) (Heal force biotech, Shanghai, China) were then fixed to the rat's forelimbs (skin above the elbow), hind limbs (just above stifle joint), and the heart18. On paper, the ECG recordings were produced using speed and sensitivity of 50 mm/s and 10 mm/mv respectively from the experimental animals, and ECG wave alterations were noted.

Serum levels of cardiac troponin-I (cTn-I) and the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were estimated by the Enzyme-Linked Immunosorbent Assay (ELISA) technique using the standard kit (Calbiotech, California, USA, and Junjiang Inter. Bldg Shanghai, China) in accordance with manufacturer's instructions. The sodium-potassium ATPase activities in the tissue homogenate were measured by estimating the level of phosphorus liberated from the test samples19. The serum activities of superoxide dismutase (SOD) and Catalase (CAT) were determined according to the previously described method20. Serum lipid peroxide activities were determined by the previously described method21. Also, serum electrolytes (Na⁺, K⁺, Ca^2+, and Cl^-) levels were determined following previously described protocols22.

The hearts were washed with normal saline immediately after harvest and then fixed in 10% buffered neutral formalin, after which the heart tissues and coronary arteries were embedded in paraffin. After this, they were sectioned, stained (hematoxylin and eosin stain), and examined under a light microscope (x100)23. The slides were observed for evidence of inflammatory cells infiltration, necrosis, and edema.

The data were expressed as mean ± standard deviation and subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test using Graphical prism version 5.03 (Graphical Software, San Diego, California, USA). The value of p < 0.05 was considered a significant difference between and within groups and was denoted by letters.

The experimental protocols were conducted in compliance with principles and guidelines of the ARRIVE guideline Faculty of Veterinary Medicine, the University of Ilorin Committees on Animal Use and Care (FVER/007/2019) and following the principles of laboratory animal care (NIH, 2011) guidelines.

Table 1

Phase	Number of rats	Weight of rats (g)	Dose (mg/mg)	Signs of toxicity (OTS/HR)*	Mortality (DR/SR)*
1	3	260	10	0/3	0/3
		271
		310
	3	283	100	0/3	0/3
		286
		256
	3	315	1000	0/3	0/3
		270
		293
2	1	325	1600	0/1	0/1
	1	265	2900	0/1	0/1
	1	275	5000	0/1	0/1

OTS: observed toxicity signs, HR: healthy rats, DR: deceased rats, SR: survived rats

Table 2

Chemical constituents	Indications
Saponin	10.78 ± 0.04
Tannins	5.77 ± 0.25
Phenolics	13.12 ± 0.77
Phlebatannin	Not detected
Steroids	99.40 ± 0.75
Flavonoids	209.20 ± 0.39
Coumarins	Not detected
Anthocyanins	0.17 ± 0.01
Terpenoids	Not detected
Glycosides	4.40 ± 0.44
Triterpenes	120.90 ± 9.33
Alkaloids	6.47 ± 0.27

Data represent mean ± SD of three determinations

Figure 1

Table 3

Peak	Retention Time	Area %	Molecular formula	Molecular weight (g/mol)	Name of the compound
1	10.767	1.73	C2H4O	44.05	Oxirane
2	12.343	0.90	C10H14	134.22	1-methyl-3-(1-methylethyl) benzene
3	13.644	1.29	C10H14	134.22	1,2,3,4-tetramethylbenzene
4	15.790	3.24	C10H8	128.17	Naphthalene
5	16.002	2.58	C5H10N2O	114.15	1-Piperazinecarboxaldehyde
6	17.935	0.72	C8H8O	120.15	2,3-dihydrobenzofuran
7	20.731	6.00	C9H10O2	150.18	1-(2-hydroxy-5-methylphenyl) ethanone
8	21.626	1.33	C11H29O6PSi3	372.57	Acetic acid, [bis[(trimethylsilyl)oxy]-
9	22.977	1.06	C9H17NO	155.24	2-(tetrahydro-2-furanyl)Piperidine
10	24.028	2.97	C10H13NO2	179.22	l-Phenylalanine, methyl ester
11	26.993	7.51	C14H42O7Si7	519.07	Tetradecamethyl cycloheptasiloxane
12	28.870	1.13	C11H11N	157.21	3-Methyl-4-phenyl-1H-pyrrole
13	31.753	2.95	C16H48O8Si8	593.20	Hexadecamethyl cyclooctasiloxane
14	33.893	1.98	C9H10N2O2	178.19	5,6-Dimethoxybenzimidazole
15	35.907	2.79	C18H54O9Si9	667.40	Octadecamethyl cyclononasiloxane
16	36.939	6.29	C17H34O2	270.50	Methyl palmitate
17	37.295	11.05	C16H32O2	256.42	n-Hexadecanoic acid
18	37.489	2.27	C18H23N3	281.40	3,6-Bis(N-dimethylamino)-9-ethylcarbazole
19	38.203	2.19	C19H34O2	294.5	methyl ester 9,12-Octadecadienoic acid
20	38.240	8.92	C19H32O2	292.5	Methyl ester 9,12,15-Octadecatrienoic acid
21	38.321	9.18	C20H40O	296.5	Phytol
22	38.497	20.37	C18H32O	264.4	(Z,Z,Z) 9,12,15-Octadecatrien-1-ol
23	39.247	1.55	C16H17N3O2	283.32	Amonafide

Figure 2

Rats in phases 1 and 2 administered with aqueous Musa paradisiaca peel extract of varying doses showed no toxicity signs and mortality during the monitoring period of 14 days (Table 1). In addition, at postmortem, no gross lesion was noticed in the tested rats.

From our study, the presence of some secondary metabolites was evidenced following the phytochemical screening. These include flavonoids, phenolics, saponins, tannins, steroids, anthocyanins, glycosides, triterpenes, and alkaloids. However, coumarins, terpenoids, and phlorotannins were not detected (Table 2).

Table 4

Treatment	Troponil-I (ng/ml)	Na+/K+ ATPase ( μmol/Pi/mg Prot./hr)
Normal control	9.06 ± 0.59	3.06 × 10-4 ± 3.31 × 10-5
ISO	20.75 ± 1.25 a	1.40 × 10-4 ± 3.21 × 10-5 a
C(100) + ISO	17.11 ± 1.60 a	1.80 × 10-4 ± 3.62 × 10-5 a
C(200) + ISO	14.84 ± 1.09 a	2.05 × 10-4 ± 3.09 × 10-5
C(400) + ISO	10.77 ± 2.14 b	2.84 × 10-4 ± 2.93 × 10-5 b
ASP	11.08 ± 1.82 b	2.75 × 10-4 ± 2.60 × 10-5 b

The data are expressed as mean ± SD and analyzed using one-way analysis of variance (ANOVA) and later subjected to Dunnett’s post hoc test for multiple comparisons. The expressed significant differences were considered at p < 0.05, ^ap < 0.05, when compared to normal control; ^bp < 0.05, when compared to ISO control (n = 8).

Table 5

Treatment	SOD ( μ/ml)	CAT ( μmol/ml/mins)	MDA (nmol/mL)
Normal control	1.15 ± 0.22	36.77 ± 1.22	11.24 ± 2.54
ISO	0.24 ± 0.08 a	20.89 ± 2.17 a	27.44 ± 5.01 a
C(100) + ISO	0.35 ± 0.11	21.10 ± 2.24	25.22 ± 5.54
C(200) + ISO	0.58 ± 0.09	26.82 ± 1.50	22.21 ± 3.94 b
C(400) + ISO	0.92 ± 0.17 b	32.24 ± 1.94 b	12.85 ± 1.38 b
ASP	0.84 ± 0.14 b	31.72 ± 1.56 b	13.34 ± 1.84 b

The data are expressed as mean ± SD and analyzed using one-way analysis of variance (ANOVA) and later subjected to Dunnett’s post hoc test for multiple comparisons. The expressed significant differences were considered at p < 0.05. ^ap< 0.05, when compared to normal control; ^bp < 0.05, when compared to ISO control (n = 8)

Figure 3

A total of 23 major peaks were revealed by the GC-MS chromatogram of aqueous Musa paradisiaca peels (Figure 1). The identified compounds are presented with their peak, retention time (RT), area (%), molecular formula, and molecular weight (Table 3). The (Z,Z,Z) 9,12,15-Octadecatrien-1-ol and n-Hexadecanoic acid showed maximum percentage. The compounds that showed moderate percentage include Phytol, Methyl ester 9,12,15-Octadecatrienoic acid, Tetradecamethyl Cyclopentasiloxane, and Methyl palmitate. The compounds 1-methyl-3-(1-methyl ethyl) benzene and 2,3-dihydrobenzofuran showed a minimum percentage.