Abstract
This study evaluated the effects of cysteine-stabilised peptide fraction (CSPF) of aqueous extract of Morinda lucida leaf on selected cardiovascular disease indices in mice. Sixty adult Swiss Albino mice were randomly divided into 6 groups (n = 10). Group A served as control and received 5% DMSO. Half of the mice in groups B, C, D, E and F received 31.25, 62.5, 125, 250, and 500 mg/kg body weight of CSPF respectively for 7 days while the other half received the various doses for 28 days. After the experimental period, selected cardiovascular disease indices were determined in the mice. The results revealed that CSPF significantly reduced (p < 0.05) atherogenic index, plasma concentrations of total cholesterol and LDL-cholesterol but significantly increased (p < 0.05) plasma HDL-cholesterol concentration at higher doses after 28 days of administration. Plasma lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase activities were not significantly altered (p > 0.05) at all doses of the CSPF after 7 and 28 days of administration compared to controls. After 7 days of CSPF administration, the activities of heart Ca2+, Mg2+-ATPase and Na+–K+-ATPase were not significantly altered (p > 0.05) but heart Mg2+-ATPase activity was significantly increased (p < 0.05) at 250 mg/kg body weight compared to controls. Also, 28 days of CSPF administration at all doses had no significant effect (p > 0.05) on the activities of heart Mg2+-ATPase and Na+–K+-ATPase of mice compared to controls but heart Ca2+–Mg2+-ATPase activity was significantly increased (p < 0.05) at the highest dose with no significant alteration (p > 0.05) at other doses compared to controls. Generally, CSPF administration had no significant effect (p > 0.05) on haematological parameters after 7 and 28 days. These results suggest that CSPF may not predispose subjects to cardiovascular diseases.
Keywords: Morinda lucida, Cysteine-stabilised Peptides, Leaf, Cardiovascular diseases
Introduction
Plants have always been a major source of therapeutic and bioactive compounds over the centuries [1]. The structural diversity of plant-derived therapeutic bioactive compounds has made them very interesting class of compounds in drug discovery [2]. An interesting class of such bioactive compounds are the cysteine-stabilised peptides (CSP), which have recently been attracting research interest because of their stability and bioactivities which can be harnessed for rational drug development [3–5]. CSP have been reported to have haemolytic, insecticidal, anticancer, immunomodulatory, uterotonic and anti-HIV activities [6]. Our earlier study showed that the leaf of Morinda lucida expresses CSP and the cysteine-stabilised peptide fraction (CSPF) of the leaf displayed antimalarial and antioxidant properties [5]. Morinda lucida Benth (Rubiaceae) is a tropical West Africa rainforest tree popularly called Brimstone tree whose leaf extract is used in folk medicine for the management of hypertension, malaria, ulcers, and gonorrhoea [7]. However, it has not been evaluated whether CSP from Morinda lucida leaf predisposes subjects to cardiovascular diseases. Therefore, this study was carried out to evaluate the effect of CSPF of aqueous extract of Morinda lucida leaf on selected cardiovascular disease indices in mice.
Materials and Methods
Chemicals
Trifluoroacetic acid and acetonitrile were obtained from Sigma Chemical Company, St. Louis, Mo, USA. C18 is a product of Phenomenex, Aschaffenburg, Germany. Dichloromethane, methanol, sodium dihydrogen tetraoxophosphate (V) dihydrate, ammonium molybdate, ascorbic acid, tetraoxosulphate (VI) acid, magnesium chloride hexahydrate, Tris KCl, ethylene–glycol tetraacetic acid, adenosine triphosphate, sodium dodecyl phosphate, and sodium chloride were obtained from BDH Chemicals Limited, Poole England.
Plant Material
Morinda lucida leaf was obtained from Ibadan, Oyo State, Nigeria. It was identified and authenticated at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria, where a voucher specimen (DPHUI:1626) was deposited.
Animals
Sixty adult Swiss mice were obtained from the animal holding unit of the Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Ilorin, Nigeria.
Preparation of CSPF
CSPF was prepared from Morinda lucida leaf as described by Hellinger et al. [6]. Morinda lucida leaves were dried under shade and pulverised into powder. The powder obtained (500 g) was percolated in 6 L of dichloromethane-methanol mixture (1:1, v/v) and left for 18 h at room temperature. The mixture was filtered using muslin cloth to remove plant debris. The filtrate was transferred into a separating funnel where equal volume of distilled water was added; thereafter the aqueous layer was collected, concentrated on rotary evaporator and freeze dried to obtain the crude peptide extract (CPE).
A preloaded C18 solid phase extraction column was preconditioned with methanol, activated with solvent B [acetonitrile-distilled water mixture (9:1, v/v)] and equilibrated with buffer A [distilled water-trifluoroacetic acid mixture (100:0.05, v/v)]. A portion of the concentrated aqueous extract was loaded onto the C18 column and eluted with increasing concentrations (20, 80, and 100%) of solvent B to obtain the peptides and remove other hydrophilic compounds. The 80% solvent B fraction was concentrated on a rotary evaporator and freeze-dried (Gallenkhamp) to obtain the cysteine-stabilised peptide fraction (CSPF), with a percentage yield of 0.5%. This was kept at − 20 °C for further use.
Animal Handling and Grouping
The animals were treated in compliance with the ethical standards as approved by the University of Ilorin Ethical Committee (UERC/ASN/2015/067) in agreement with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health [8]. The mice were randomly divided into 6 groups of 10 mice each. Animals in group A served as the control and were orally administered 5% DMSO while animals in groups B, C, D, E and F were administered 31.25, 62.5, 125, 250 and 500 mg/kg body weight of CSPF (dissolved in 5% DMSO) respectively. The administration lasted for 7 days for half of the mice in each group and for 28 days for the other half.
Sample Collection and Preparation
Five (5) mice from each group were sacrificed after 7 days of CSPF administration while the remaining 5 were sacrificed after 28 days of CSPF administration using slight diethyl ether anaesthesia. Small portion of the blood was collected into ethylene-diaminetetraacetic acid (EDTA) bottles for haematological studies while the remaining portion was collected in heparin bottles for biochemical analyses. Blood collected into heparin bottles was centrifuged at 3000 rpm for 5 min; the plasma was separated into plain sample bottles and kept frozen for biochemical analyses. The mice were dissected and the hearts were excised, freed of fat, cleansed of blood and weighed. The heart was homogenised in ice-cold 0.25 M sucrose solution (1:5, w/v); the homogenate was centrifuged at 5000 rpm for 5 min in a refrigerated centrifuge (Eppendorf 5804 R, Harmburg, Germany) and the supernatant was collected and used for biochemical analyses.
Haematological Parameters
Red blood cell count (RBC), white blood cell count (WBC), haemoglobin concentration (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), percentage neutrophil, percentage lymphocyte and platelet count (PLT) were estimated using Sysmex Automated Haematological Analyzer KX21 (SYSMEX Corporation Kobe, Japan).
Biochemical Assays
The concentration of total protein was estimated using the method of Gornall et al. [9]. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were determined by the methods of Reitman and Frankel [10], Wright et al. [11] and Wroblewski and La Due [12] respectively. Mg2+-ATPase activity was evaluated according to the method of Ronner et al. [13] as modified by Fleschner and Kraus-Friedmann [14]. Ca2+, Mg2+-ATPase and Na+, K+-ATPase activities were determined according the methods of Ronner et al. [13] and Bewaji et al. [15]. The plasma concentrations of total cholesterol (TC) and triglyceride (TAG) were determined according to the method of Tietz [16]. The concentrations of plasma high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were determined according to the method of Friedewald [17]. Atherogenic index (AI) was determined according to the method of Lamarche [18] using the formula: Total Cholesterol concentration/HDL-cholesterol concentration.
Statistical Analysis
The data are presented as mean ± standard error of mean. Data were analysed using one-way ANOVA and Duncan Multiple Range test using GraphPad 6 (GraphPad Software California, USA). Differences at p < 0.05 were considered significant.
Results
Heart-Body Weight Ratio of Mice Treated with Cysteine-Stabilised Peptide Fraction of Aqueous Extract of Morinda lucida Leaf
The results showed that there was no significant change (p > 0.05) in heart-body weight ratio after 7 and 28 days of administration of the CSPF at all doses compared to controls (Fig. 1).
Fig. 1.
Heart-body weight ratio of mice after 7 and 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf. Values are means of 5 replicates ± SEM. Values for each day with the same superscripts are not significantly different (p > 0.05)
Lipid Profile of Mice Treated with Cysteine-Stabilised Peptide Fraction of Aqueous Extract of Morinda lucida Leaf
Administration of the CSPF for 7 days caused no significant change (p > 0.05) in plasma TC and HDL-C at all doses compared to controls (Table 1). It was however observed that plasma TAG was significantly increased (p < 0.05) by CSPF at doses above 31.25 mg/kg body weight compared to control (Table 1). Administration of CSPF for 7 days led to significant reduction (p < 0.05) in plasma LDL-C at all doses investigated compared to control (Table 1). AI of the CSPF-treated groups was significantly reduced (p < 0.05) at the highest dose with no significant alteration (p > 0.05) at lower doses compared to control after 7 days of administration (Table 1). Administration of CSPF for 28 days caused a significant reduction (p < 0.05) in plasma TC at the dose of 500 mg/kg body weight with no significant alteration (p > 0.05) at lower doses compared to control (Table 2). Administration of CSPF for 28 days did not cause any significant alteration (p > 0.05) in plasma TAG at all doses but significantly increased (p < 0.05) plasma HDL-C at doses higher than 62.5 mg/kg body weight with a significant reduction (p < 0.05) in plasma HDL-C at 62.5 mg/kg body weight compared to controls (Table 2). Administration of CSPF for 28 days led to a significant increase (p < 0.05) in plasma LDL-C at the dose of 31.25 mg/kg body weight but caused a significant decrease (p < 0.05) in plasma LDL-C at doses higher than 62.5 mg/kg body weight compared to control (Table 2). Also, administration of the CSPF for 28 days resulted in significantly lower (p < 0.05) AI at doses above 62.5 mg/kg body weight compared to control (Table 2).
Table 1.
Plasma lipid profile of mice after 7 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf
| Treatment | Total cholesterol concentration (mg/dl) | Triglyceride concentration (mg/dl) | High density lipoprotein cholesterol concentration (mg/dl) | Low density lipoprotein cholesterol concentration (mg/dl) | Atherogenic index |
|---|---|---|---|---|---|
| Control | 130.19 ± 16.89a | 74.78 ± 4.23a | 65.35 ± 7.78a | 49.88 ± 13.44a | 2.00 ± 0.20a |
| 31.25 mg/Kg b wt | 126.07 ± 8.08a | 68.72 ± 7.66a | 76.73 ± 15.40a | 35.61 ± 14.36b | 1.70 ± 0.33a |
| 62.5 mg/Kg b wt | 124.42 ± 7.37a | 92.97 ± 8.46b | 69.61 ± 7.78a | 36.22 ± 10.58b | 1.81 ± 0.23a |
| 125 mg/Kg b wt | 117.83 ± 9.92a | 96.01 ± 9.45b | 72.46 ± 9.26a | 26.18 ± 12.58b | 1.65 ± 0.22a |
| 250 mg/Kg b wt | 120.30 ± 12.50a | 88.93 ± 7.66b | 80.98 ± 22.80a | 21.54 ± 12.88b | 1.59 ± 0.47a |
| 500 mg/Kg b wt | 123.74 ± 2.91a | 94.99 ± 6.59b | 79.11 ± 5.94a | 25.04 ± 8.14b | 1.56 ± 0.15b |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with different superscripts are significantly different (p < 0.05)
Table 2.
Plasma lipid profile of mice after 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf
| Treatment | Total cholesterol concentration (mg/dl) | Triglyceride concentration (mg/dl) | High density lipoprotein concentration (mg/dl) | Low density lipoprotein concentration (mg/dl) | Atherogenic index |
|---|---|---|---|---|---|
| Control | 106.45 ± 3.29a | 80.62 ± 2.57a | 33.59 ± 15. 84a | 56.74 ± 16.55a | 4.86 ± 1.23a |
| 31.25 mg/Kg b wt | 111.43 ± 5.91a | 79.11 ± 1.68a | 34.38 ± 8.18a | 61.23 ± 12.39b | 4.07 ± 1.23a |
| 62.5 mg/Kg b wt | 99.54 ± 4.97a | 78.42 ± 0.40a | 26.29 ± 4.88b | 57.58 ± 1.97a | 4.22 ± 0.70a |
| 125 mg/Kg b wt | 104.10 ± 1.72a | 77.61 ± 2.25a | 52.41 ± 6.68c | 36.17 ± 7.56c | 2.12 ± 0.35b |
| 250 mg/Kg b wt | 101.89 ± 1.59a | 79.00 ± 0.00a | 60.38 ± 7.26c | 25.70 ± 6.99c | 1.78 ± 0.26b |
| 500 mg/Kg b wt | 98.16 ± 2.82b | 75.18 ± 1.33a | 59.60 ± 11.76c | 23.49 ± 5.02c | 2.01 ± 0.63b |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with different superscripts are significantly different (p < 0.05)
Activities of Selected Heart Enzymes of Mice Treated with Cysteine-Stabilised Peptide Fraction of Aqueous Extract of Morinda lucida Leaf
Administration of CSPF of aqueous extract of Morinda lucida leaf for 7 and 28 days caused no significant alteration (p > 0.05) in the activities of AST and LDH in the heart and plasma of mice at all doses investigated compared to controls (Figs. 2 and 3). There was no significant change (p > 0.05) in ALP activity in the heart at all doses after 7 and 28 days of CSPF administration compared to control (Fig. 4).
Fig. 2.
Activities of aspartate aminotransferase in the heart of mice after 7 and 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf. Values are means ± SEM of 5 replicates. Bars for each day with the same alphabet are not significantly different (p > 0.05)
Fig. 3.
Activities of lactate dehydrogenase in the heart of mice after 7 and 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf. Values are means ± SEM of 5 replicates. Bars for each extract with the same alphabet are not significantly different (p > 0.05)
Fig. 4.
Activities of alkaline phosphatase in the heart of mice after 7 and 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf. Values are means ± SEM of 5 replicates. Bars for each day with the same alphabet are not significantly different (p > 0.05)
The results revealed that after 7 days of the CSPF administration, Mg2+-ATPase activity was significantly increased (p < 0.05) in the heart of mice at 250 mg/kg body weight with no significant alteration (p > 0.05) at other doses compared to control (Fig. 5). After 28 days of the CSPF administration, there was no significant alteration (p > 0.05) in heart Mg2+-ATPase activity at all doses compared to control (Fig. 5). Administration of CSPF for 7 days caused no significant (p > 0.05) change in Ca2+, Mg2+-ATPase activities in the heart of mice compared to control (Fig. 5). However, after 28 days of administration, CSPF caused a significant increase (p < 0.05) in Ca2+, Mg2+-ATPase activity in the heart at the dose of 500 mg/kg body weight with no significant alteration (p > 0.05) at lower doses compared to control (Fig. 5). Administration of the fraction for 7 and 28 days caused no significant change (p > 0.05) in Na+, K+-ATPases activity in the heart at all doses administered compared to controls (Fig. 5).
Fig. 5.
Activities of selected ATPases in the heart of mice after 7 and 28 days of administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf. Values are means ± SEM of 5 replicates. Bars for each day with different alphabets are significantly different (p < 0.05)
Hematological Indices of Mice Treated with Cysteine-Satbilized Peptide Fraction of Aqueous Extract of Morinda lucida Leaf
There was no significant change (p > 0.05) in all the red blood cell indices of mice treated with CSPF at all doses after 7 days of administration compared to controls (Table 3). After 28 days of CSPF administration, there was no significant alteration (p > 0.05) in RBC, PCV, Hb and MCHC at all doses administered compared to controls (Table 4). However, MCV and MCH were significantly increased (p < 0.05) at all doses of CSPF administered compared to control after 28 days of administration (Table 4). After 7 days of the CSPF administration, the percentage lymphocyte was significantly reduced (p < 0.05) with a corresponding significant increase (p < 0.05) in percentage neutrophil at the dose of 31.25 mg/kg body weight compared to the controls. All other parameters were not significantly altered (p > 0.05) at all doses compared to controls. After 28 days of CSPF administration, all the white blood cell indices and platelet count were not significantly altered (p > 0.05) compared to controls (Tables 5 and 6).
Table 3.
Red blood cell indices of mice administered cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf for 7 days
| Treatment | RBC (× 1012/L) | PCV (%) | Hb (g/L) | MCV (%) | MCH (ρg) | MCHC (g/L) |
|---|---|---|---|---|---|---|
| Control | 9.20 ± 0.59a | 52.72 ± 4.55a | 12.96 ± 0.92a | 57.34 ± 3.85a | 14.08 ± 0.59a | 24.62 ± 0.64a |
| 31.25 mg/Kg b wt | 9.21 ± 0.86a | 52.32 ± 5.94a | 12.96 ± 1.38a | 56.90 ± 5.07a | 14.10 ± 1.15a | 24.80 ± 1.48a |
| 62.5 mg/Kg b wt | 8.73 ± 0.37a | 47.92 ± 2.66a | 12.14 ± 0.89a | 54.94 ± 2.32a | 13.92 ± 0.91a | 25.32 ± 1.00a |
| 125 mg/Kg b wt | 8.97 ± 1.06a | 49.38 ± 3.19a | 12.42 ± 0.57a | 55.50 ± 5.51a | 13.98 ± 1.31a | 25.22 ± 1.85a |
| 250 mg/Kg b wt | 8.40 ± 2.58a | 46.96 ± 15.23a | 13.08 ± 0.44a | 55.48 ± 2.88a | 17.66 ± 8.59a | 32.34 ± 1.75a |
| 500 mg/Kg b wt | 8.97 ± 0.44a | 49.24 ± 3.40a | 12.28 ± 0.64a | 54.96 ± 3.84a | 13.72 ± 0.99a | 24.98 ± 0.86a |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with the same superscript are not significantly different (p > 0.05)
Table 4.
Red blood cell indices of mice administered cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf for 28 days
| Treatment | RBC (× 1012/L) | PCV (%) | Hb (g/L) | MCV (%) | MCH (ρg) | MCHC (g/L) |
|---|---|---|---|---|---|---|
| Control | 9.94 ± 0.34a | 54.10 ± 1.84a | 14.25 ± 0.43a | 54.43 ± 0.29a | 14.35 ± 0.13a | 26.35 ± 0.12a |
| 31.25 mg/Kg b wt | 9.55 ± 0.09a | 52.80 ± 1.16a | 14.28 ± 0.18a | 55.25 ± 1.03b | 14.95 ± 0.27b | 27.05 ± 0.44 |
| 62.5 mg/Kg b wt | 9.04 ± 0.95a | 53.15 ± 1.12 | 14.15 ± 0.32a | 58.78 ± 0.90c | 15.65 ± 0.25b | 26.63 ± 0.31a |
| 125 mg/Kg b wt | 9.43 ± 0.36a | 53.45 ± 1.74a | 14.20 ± 0.30a | 56.75 ± 0.43b | 15.10 ± 0.32b | 26.60 ± 0.37a |
| 250 mg/Kg b wt | 9.25 ± 0.23a | 52.40 ± 0.91a | 14.18 ± 0.17a | 56.70 ± 0.52b | 15.35 ± 0.29b | 27.05 ± 0.25a |
| 500 mg/Kg b wt | 9.27 ± 0.38a | 51.53 ± 1.97a | 14.03 ± 0.62a | 56.60 ± 0.39b | 15.13 ± 0.26b | 27.25 ± 0.63a |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with different superscripts are significantly different (p < 0.05)
Table 5.
White blood cell indices and platelet count of mice administered cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf for 7 days
| Treatment | WBC (× 109/L) | Platelet count (× 109/L) | Lymphocytes (%) | Neutrophils (%) |
|---|---|---|---|---|
| Control | 9.46 ± 3.02a | 1217.40 ± 354.40a | 76.77 ± 3.40a | 23.23 ± 3.40a |
| 31.25 mg/Kg b wt | 8.48 ± 1.85a | 1241.00 ± 339.53a | 58.55 ± 4.66b | 41.45 ± 4.66b |
| 62.5 mg/Kg b wt | 12.94 ± 6.29a | 1454.40 ± 221.85a | 71.38 ± 5.05a | 28.63 ± 5.05a |
| 125 mg/Kg b wt | 13.05 ± 3.27a | 1160.20 ± 364.13a | 83.23 ± 2.58a | 16.78 ± 2.58a |
| 250 mg/Kg b wt | 9.40 ± 4.41a | 1010.20 ± 338.38a | 72.78 ± 6.33a | 27.23 ± 6.34a |
| 500 mg/Kg b wt | 9.98 ± 3.05a | 1354.20 ± 162.87a | 76.25 ± 7.88a | 23.75 ± 7.08a |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with different superscripts are significantly different (p < 0.05)
Table 6.
White blood cell indices and platelet count of mice administered cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf for 28 days
| Treatment | WBC (× 109/L) | Platelet count (× 109/L) | Lymphocytes (%) | Neutrophils (%) |
|---|---|---|---|---|
| Control | 14.03 ± 3.24a | 1711.80 ± 48.10a | 87.30 ± 1.69a | 12.70 ± 1.69a |
| 31.25 mg/Kg b wt | 18.00 ± 2.78a | 1598.80 ± 142.11a | 86.10 ± 2.03a | 13.90 ± 2.03a |
| 62.5 mg/Kg b wt | 11.52 ± 1.76a | 1588.80 ± 132.11a | 87.33 ± 2.67a | 12.68 ± 2.67a |
| 125 mg/Kg b wt | 13.05 ± 3.27a | 1725.50 ± 98.03a | 86.78 ± 1.46a | 13.23 ± 1.46a |
| 250 mg/Kg b wt | 10.82 ± 1.53a | 1716.80 ± 79.96a | 91.63 ± 2.29a | 8.38 ± 2.30a |
| 500 mg/Kg b wt | 13.95 ± 1.44a | 1373.20 ± 165.06a | 85.40 ± 4.86a | 14.6 ± 4.86a |
b wt Body weight
Values are means of 5 replicates ± SEM. Values in the same column with the same superscripts are not significantly different (p > 0.05)
Discussion
Organ-body weight ratios are normally investigated to determine whether the size of the organ has changed in relation to the weight of the whole animal [19]. Increase in heart-body weight ratio may be due to cardiac hypertrophy and increased systemic circulatory resistance which can result in hypertension and other cardiovascular diseases [20]. In this study, administration of the CSPF for 7 and 28 days caused no change in heart-body weight ratio of mice, suggesting that the fraction exerted no adverse effect on the size of the heart.
The levels of major lipids such as TC, HDL-C, LDL-C and TAG are used to determine predisposition to atherosclerosis and associated coronary heart disease (CHD) [21]. The major identifying risk factors are increased serum TC, LDL-C, reduced serum HDL-C and high blood pressure [21, 22]. Increased HDL-C is protective against CHD [21]. AI is a very useful and more reliable indicator of cardiovascular diseases [23], and it has been established that myocardial infarction increases when the AI higher than 5 [24, 25]. The reduction in plasma LDL-C at all doses administered and significant reduction in AI at 500 mg/kg body weight obtained in this study after 7 days of CSPF administration suggest that consumption of the CSPF may be able to lower the risk of cardiovascular diseases. After 28 days of CSPF administration, the reduction in AI at the doses of 125, 250 and 500 mg/kg body weight obtained in this study also support the earlier suggestion that administration of the CSPF may not predispose subjects to cardiovascular diseases.
Increased plasma AST and LDH activities are indicators of heart diseases such as myocardial infarction [16]. This study revealed that the activities of both enzymes in the plasma were not altered after 7 and 28 days of CSPF administration, suggesting that the fraction may not cause myocardial infarction. The observation that there was no leakage of these enzymes into the plasma is also supported by the fact that ALP activity, the marker enzyme for plasma membrane integrity [26], in the heart was not altered after 7 and 28 days of administration.
Mg2+ ATPase helps in maintaining membrane permeability while Na+–K+ ATPase helps in maintaining intracellular Na+ and K+ concentrations, which play a vital role in maintaining membrane potential. In the same vein, Ca2+, Mg2+-ATPase helps in maintaining intracellular Ca2+ concentration [27]. Ca2+ plays a vital role in cellular signalling and muscle contraction [28]. Generally, these enzymes were not affected by CSPF, suggesting that their roles in heart cells were not compromised. The increase in the activity of Ca2+, Mg2+-ATPase in the heart at the dose of 500 mg/kg body weight after 28 days of the CSPF administration suggests that CSPF may activate the enzyme in situ or induce its synthesis after prolonged administration at a high dose. However, cellular signalling, muscle contraction and other roles played by calcium ion in cardiac cells may not be adversely affected by the increase in Ca2+, Mg2+-ATPase activity at this dose [28].
The absence of any alteration in all haematological parameters after 7 days of CSPF administration suggests that the fraction may not adversely affect the production of red blood cell at all doses administered. Thus, the CSPF may not exert any adverse effect on erythropoietin production [29]. The increase in MCV and MCH after 28 days of administration of the fraction at all doses suggests that the fraction may be able to boost the oxygen carrying capacity of the red blood cells and the size of RBCs. The fact that CSPF generally had no effect on WBC and platelet count after 7 and 28 days of administration suggests that the fraction may not adversely affect the immune system. However, the reduced percentage lymphocyte and increased percentage neutrophil at the dose of 31.5 mg/kg body weight after 7 days of administration of CSPF suggests a shift from the adaptive immune system to the innate immune system. However, this was reverted to the range of control after 28 days of administration. The fact that the platelet count was not adversely affected suggests that CSPF may not adversely affect thrombopoietin production [30].
The results of this study suggest that administration of cysteine-stabilised peptide fraction of aqueous extract of Morinda lucida leaf may not predispose subjects to cardiovascular diseases.
Compliance with Ethical Standards
Conflict of interest
The authors declare that there is no conflict of interest.
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