This study was performed on adult Wistar rats of both sexes with an average age of 4 - 6 weeks, weighing between 100 and 120 g. Animals were purchased and acclimatized in the animal house of the Faculty of Science (University of Buea, Cameroon) for 7 days. The rats were housed in wire-bottomed cages under natural environmental conditions. The animals were given food and water ad-libitum. All animals were strictly handled according to guidelines for the care and use of laboratory animals. An authorization (UB-IACUC N˚ 25/2025) was obtained from the University of Buea Institutional Animal Care and Use Committee. The animals were given food and water ad-libitum.

Fresh Perseaamericana (P.americana) leaves were collected from Buea, washed properly with tap water and shed dried. The shed-dried leaves were ground using an electric blender; the powder obtained was then kept at 4˚C in a sealed plastic packet until experiment, to avoid microbial contamination. Briefly, plant powder (1000 g) was macerated at room temperature in 5 L of water for 48 h (with occasional stirring), followed by filtration using Whatman filter paper N˚ 1. The residues were macerated again in the correspondent solvent for 48 h followed by filtration. The filtrates obtained were pooled, concentrated in an oven at 45˚C to yield the aqueous extract.

Thirty (30) adult Wistar rats were randomly distributed into six (6) groups of five (5), and then treated for twenty-eight (28) days. Groups 1-3 were the three controls: neutral (receiving water 10 mL/kg, peros daily), negative (receiving ISO 150 mg/kg, sc, 2 days to sacrifice) and positive (receiving ISO 150mg/kg, sc, 2 days to sacrifice + propranolol 10 mg/kg, peros, 10 days to sacrifice) respectively. Test groups (4 - 6) received perosPerseaamericana leaf aqueous extract at 50, 100 or 200 mg/kg respectively, daily during the experimental period and ISO (150 mg/kg, sc) 2 days to sacrifice (Table 1) Myocardial infarction was induced in the rats using subcutaneous injection of 150 mg/kg/day Isoprenaline (ISO) for two consecutive days (27th and 28th) at 24 hours interval [30].

Table 1. Animal distribution and treatment.

Note: ISO: Isoprenaline.

Food and water consumption, and body weight were recorded during the period of treatment. Specific quantities of feed and water were given to each group daily and their consumptions were determined by subtracting the left-overs from the originally given quantities. Average food and water consumptions were calculated weekly. Furthermore, the relative weight of the heart was calculated following the formula:

where:

HW = Heart Weight;

BWAS = Body Weight at Sacrifice;

RHW = Relative Heart Weight.

Rats were sacrificed on day 29. Blood (4 mL) was collected by cardiac puncture with a syringe, put in dry tube and allowed to rest for 5 mins. Thereafter, blood was centrifuged at 3000 revolutions per min (−5˚C) for 10 minutes, then the serum collected and stored at −28˚C in the freezer, for further analyses. Serum levels of creatine-kinase (CK-MB) and troponin I were assayed using ELISA kits following the instructions of the manufacturer.

The heart was also collected, cleaned, and the weight recorded. It was then preserved in 4% formalin for two weeks and then carried to the laboratory of Animal Physiology of the University of Yaounde I for histopathological examinations. The tissues were removed from the fixatives, dehydrated, included in paraffin and sections of 2 µm thick were made using a microtome. Tissue sections were further mounted on glass slides, rehydrated, stained with Haematoxylin and Eosin stain (HE) [31] and finally examined under a light microscope (×200). Micrographs were snapped with the aid of a digital camera attached to the eyepiece of the microscope.

Statistical evaluation: Data were recorded in MS Excel and analyzed using GraphPad Prism (version 8.3). Results are presented as tables and graphs, with values expressed as the mean, ±SEM. Data were evaluated using a one-way ANOVA followed by a Tukey’s multiple comparisons test, with statistical significance set at P < 0.05.

3.1.1. Effects of Perseaamericana Leaf Aqueous Extract on Food Intake

During 28 days administration of rats with P.americana (50 - 200 mg/kg), ISO (150 mg/kg), and propranolol (10 mg/kg), there was a haphazard change in the quantity of food consumed by the rats in all groups from day 0 to 28. However, the mean food consumption did not vary among the neutral control, and P.americana groups treated. From day 21 - 28, food consumption was 632.8 ± 44.79 g in the negative control group against 754.6 ±58.42, 666.92 ± 72.36, or 812.6 ± 61.23 g in animals treated with P.americana leaf aqueous extract respectively at 50, 100, or 200 mg/kg (Figure 1).

Note: Each point on the line represents the mean, ±SEM, n = 5.

Figure 1. Effects of Persea americana leaf aqueous extract on food consumption of treated rats.

3.1.2. Effects of Perseaamericana Leaf Aqueous Extract on Water Consumption

Note: Each point on the line represents the mean, ±SEM, n = 5.

Figure 2. Effects of Persea americana leaf aqueous extract on water consumption of treated rats.

During 28 days of treatment of rats with P.americana (50 - 200 mg/kg), 2 days of ISO (150 mg/kg), and 10 days of propranolol (10 mg/kg), there was a variation in water consumption of the rats in all groups from day 0 to 28. However, the mean water consumption did not vary significantly from the neutral control, negative control, positive control and P.americana treated groups. During the two days of ISO administration, there was a marked reduction in water consumption in all the groups (Figure 2). During the last week, water consumption was 493.4 ± 23.48 mL in the negative control group vs. 506.8 ± 48.18 or 590.2 ± 56.37 mL in animals treated with P.americana leaf aqueous extract respectively at 50 or 100 mg/kg (Figure 2).

3.1.3. Effects of Perseaamericana Leaf Aqueous Extract on Body Weight

During 28 days of treatment of rats with aqueous P.americana, ISO (150 mg/kg), and propranolol (10 mg/kg), there was an increase in body weight of the rats in all groups from day 0 to 28. There was a drop in the 50 mg/kg P.americana group in the last week. However, the mean body weight gain did not vary significantly among the neutral control and P.americana treated groups at P < 0.05 (Figure 3). The average percentage increase in body weight throughout the treatment period was 13.58% in the negative control group vs. 23.34, 26.43, or 15.00% in animals treated with P.americana leaf aqueous extract respectively at 50, 100 or 200 mg/kg (Figure 3) during the last week of treatment.

Note: Each point on the line represents the mean, ±SEM, n = 5.

Figure 3. Effects of Persea americana leaf aqueous extract on body weight of treated rats.

During 28 days treatment of rats with P.americana (50 - 200 mg/kg), ISO (150 mg/kg), and propranolol (10 mg/kg), significant differences were not recorded in the mean of heart weights in all the groups treated. The positive control group recorded animals with the highest heart weight to body ratio and Persea americana (200 mg/kg) + ISO recorded the lowest (Table 2).

Table 2. Effects of Perseaamericana leaf aqueous extract on Relative Heart Weight of Isoprenaline induce myocardial infarction in rat.

Note: Each value in the table represents the mean ± SEM, n = 5.

RHW = Relative heart weight

RHW (%) = HW ÷ BWAS × 100,

where:

HW= Heart weight and BWAS= Body weight at sacrifice.

3.2.1. Effects of Perseaamericana Leaf Aqueous Extract on Cardiac Biomarkers (Troponin I/T and Creatine-Kinase-MB) Levels in Isoprenaline-Induced Myocardial Infarct Rats

Effects of Persea americana leaf aqueous extract on Troponin I/T level in serum.

Note: Each bar represents the mean, ±SEM, n = 5, at #P < 0.05, ##P < 0.01, ###P < 0.001, **P < 0.01, ***P < 0.001 ANOVA, followed by Tukey multiple comparison test, #P < 0.05 significantly different from the negative control and *P < 0.05 significantly different from the positive control. The neutral control received only tap water, negative control received Isoprenaline, positive control received propranolol + Isoprenaline, and the test groups received Persea americana (50 mg/kg) + Isoprenaline, Persea americana (100 mg/kg) + Isoprenaline and Persea americana (200 mg/kg) + Isoprenaline.

Figure 4. Effects of Persea americana leaf aqueous extract on troponin level in the various treated groups.

Induction of myocardial infarction provoked an increase in troponin level; 54.96 ± 3.36 µg/L in the negative control vs. neutral control (14.49 ± 2.58 µg/L). Treatment with P.americana aqueous extract at 50, 100, or 200 mg/kg dose-dependently and significantly (P < 0.05, P < 0.01, P < 0.001) decreased the troponin level respectively to 39.97 ± 1.27 µg/L, 31.69 ± 1.75 µg/L, or 29.41 ± 0.01 µg/L (Figure 4). There was a decrease in the positive control animals to 21.23 ± 1.39 µg/L.

3.2.2. Effects of Perseaamericana Leaf Aqueous Extract on Creatine-Kinase-MB (CK-MB) Level in Serum

There was an increase in the level of Creatine-kinase-MB in the other groups compared to the neutral control from 17.88 ± 4.74 µg/L to 61.28 ± 5.07 µg/L (negative control), to 54.39 ± 3.10 µg/L, 42.18 ± 2.62 µg/L and 35.50 ± 3.73 µg/L (in ISO + P.americana 50 mg/kg, 100 mg/kg and 200 mg/kg treated groups) respectively. In the positive control, the level of CK-MB was elevated to 21.03 ± 1.0 µg/L vs. neutral control (Figure 5).

Note: Each bar represents the mean, ±SEM, n = 5, at #P < 0.05, ##P < 0.01, ###P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001 ANOVA, followed by Tukey multiple comparison test, #P < 0.05 significantly different from the negative control and *P < 0.05 significantly different from the positive control. The neutral control received only tap water, negative control received Isoprenaline, positive control received propranolol + Isoprenaline, and the test groups received Persea americana (50 mg/kg) + Isoprenaline, Persea americana (100 mg/kg) + Isoprenaline and Persea americana (200 mg/kg) + Isoprenaline.

Figure 5. Effects of Persea americana extract on CK-MB of the different rat groups.

3.2.3. Effects of Perseaamericana Leaf Aqueous Extract on the Heart Tissue Architecture of Rats with Isoprenaline-Induced Myocardial Infarction

The 6 groups receiving different treatments exhibited many differences in the level of destruction and pathological changes in the cardiac tissue. Generally, treatment with ISO caused the cells to lose the normal appearance, possessed inhomogeneous content, and some fibres and cells lysed. Myocytes showed variable degree of damage induced by ISO. Heart tissues as observed after the histological study indicated a pattern of damage as several long haphazard cuts (infarctus).

Infarction was highest in the negative control (rats administered with ISO only). In the positive control (rats administered with both ISO and its standard antagonist, propranolol), infarcts were less observed. In Groups 4 (aqueous Perseaamericana (50 mg/kg) + ISO), 5 (aqueous Perseaamericana (100 mg/kg) + ISO) and 6 (aqueous Perseaamericana (200 mg/kg) + ISO) the heart was not damaged, the architecture of the heart was similar to those in the neutral and positive controls (Figure 6).

Note: A = Neutral control; B= Negative control; C = E50; D = E100; E = E200; F = Positive control; Ne= Necrotic cell; No = Nucleus of cardiomyocyte; Cm = Cardiomyocyte.

Figure 6. Microhistology of the heart tissue in rats induced Isoprenaline myocardial infarction treated with Perseaamericana leaf aqueous extract (×200).