Ethical approval
This research was approved by the ethical committee of our institution. The rats were handled according to the guidelines of the National Institute of Health (NIH) on the care and use of laboratory animals.
Drug and chemicals
All the chemicals, reagents, and drug used for the research were of analytical grade.
Collection of sample
The leaves of Cola hispida plant were freshly obtained from Ajuona Nsukka Local Government Area of Enugu State, without causing any damage to the plant. The leaves were identified by Ozioko Alfred, a taxonomist at Bioresources Development and Conservation Programme, Nsukka, Enugu State, Nigeria. The plant leaves were deposited in the herbarium of Bioresources Development and Conservation Programme, Nsukka, Enugu State, Nigeria, with a voucher number: InterCEDD/16304.
Plant material preparation
Cola hispida leaves were thoroughly washed using distilled water, kept at room temperature to dry, then grounded into a fine powder for extraction. A known quantity of the sample (750 g) was softened in 3.75 L methanol and kept for 48 h at room temperature. The mixture was filtered using Whatman No. 1 filter paper and then concentrated using the rotary evaporator to obtain chocolate-like semi-solid extract.
Solvent-solvent partitioning (fractionation) of Cola hispida methanol leaf extract
A quantity (20 g) of the crude extract was further re-extracted by the process of partitioning using 20% methanol, ethyl-acetate, and n-hexane in order of increasing polarity. This quantity (20 g) of the crude methanol extract was re-dissolved into 250 ml of 20% of the solvent followed by addition of n-hexane in the same proportion, stirred to mix and then allowed to settle on standing for 20 min for complete separation into two partitions; then, the n-hexane partition was collected in a flat bottom flask. The above process was repeated three times with the remaining 250 ml of 20% methanol until the n-hexane partition is colorless. Then, 250 ml of ethyl-acetate was added to the remaining partition of the 250 ml of 20% methanol; the ethyl-acetate partition was collected into flask. The process was repeated three times until the ethyl-acetate partition became colorless. The different solvent partitions were collected and concentrated, and the solvent (ethyl-acetate) which showed a better result in the pilot study was selected for further studies.
Screening of secondary metabolites
Phytochemical screening of the Cola hispida leaf constituents was in accordance with the method of Harborne [17] and Trease and Evans [18]. Quantitative analyses were done as described by Harborne [19] and Soni and Sosa [20].
Determination of cardioprotective and antioxidant properties of Cola hispida leaf fraction
A total of thirty (30) experimental albino rats (187–224 g) were purchased from Department of Zoology and Environmental Biology and kept in aluminum cages in an animal house with temperatures steadied at 25 °C and 55% relative humidity. All the experimental animals were allowed for 7 days of acclimatization with daily access to their feed and water all through the experiment. The advisory notes of the National Institute of Health on the management of research animals were maintained.
Treatment procedures
The thirty experimental albino rats were separated into six experimental groups (A, B, C, D, E, and F) in a random manner, each consisting of five experimental albino rats. A 100 mg kg−1 b.wt of vitamin E and varied oral administration of the fraction was given to groups C and D–F respectively while groups A and B received only distilled water and were also fed for 21 days after acclimatization.
Induction of myocardial injury
On the 21st day, after 2 h of fraction and vitamin E administration, group B–F were intraperitoneally treated with equal doses of 2.5 mg kg−1 b.wt of Dox while group A was administered only distilled water.
Group A: distilled water only
Group B: 2.5 mg kg−1 b.wt Dox only
Group C: 100 mg kg−1 b.wt vitamin E + 2.5 mg kg−1 b.wt Dox
Group D: 100 mg kg−1 b.wt, fraction + 2.5 mg kg−1 b.wt Dox
Group E: 200 mg kg−1 b.wt, fraction + 2.5 mg kg−1 b.wt Dox
Group F: 400 mg kg−1 b.wt, fraction + 2.5 mg kg−1 b.wt Dox
Collection of blood sample
This followed the procedure described by Agbafor et al. [21] which after an overnight fast, blood was collected through cardiac puncture after the rats were euthanized using chloroform. The blood samples were stored in a plain specimen container with no anticoagulant. The rats were humanely sacrificed, and the hearts were immediately surgically cut out, perfused with cold normal saline and homogenized using 0.25 M sucrose in 0.2 M phosphate buffer at pH 7.4.
Determination of cardioprotective property of Cola hispida
The Randox kits were used to determine the serum level of creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) following manufacturer’s guidelines.
Heart homogenate preparation
The heart tissue was homogenized in 1.5% KCl with 10 mM phosphate buffer, pH 7.4 in ethylenediaminetetraacetic acid (EDTA), and centrifuged at 12,000 rpm for 20 min. An aliquot of the supernatants’ prepared tissue homogenate was drawn for each biochemical analysis.
Determination of antioxidant property
The concentration of malondialdehyde (MDA) of the tissue homogenate was measured following the method described by Wallin et al. [22]. Myocardial activities of the antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were assayed respectively by methods described by Fridovich [23], Aebi [24], and Paglia and Valentine [25].
Estimation of nutrient constituents of Cola hispida fraction
The method described by AOAC [26] and Pearson [27] was used to determine the vitamin and mineral presence respectively.
Preparation of tissue
The tissue samples were trimmed, washed, and dehydrated using ethanol in increasing percentage (60, 70, 80, and 90% proportions). The tissue samples were cleaned in xylene, immersed in paraffin, and broken up into sections at about 5-μ thickness. They were then treated with hematoxylin and eosin (H and E stain) and viewed at ×400 magnifications [28].
Data analysis
Analysis of data was done using SPSS version 21.0 on the test statistic, one-way analysis of variance (ANOVA), and Duncan Multiple Range Test to ascertain the statistical difference among the mean and the interaction between the variables. The statistical differences obtained were adjudged statistically significant at p<0.05.