Plant source and extraction
Annona muricata, Carica papaya, Moringa oleifera and Aloe barbadensis were collected in Ogbomoso town (8º08’N4º15ºE) and were authenticated by a taxonomist from the Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria. The voucher numbers of the plants were deposited at the University Herbarium.
Preparation of Poly-herbal extracts (PHE)
Five hundred each of the fresh leaves of the four plants were pulverized using a kitchen blender and subsequently macerated and boiled in water at 100 degrees for 2 hours. It was left alone to cool under room temperature for 3 hours and was then filtered using a clean muslin cloth. The supernatant was freeze-dried and the lyophilized crude extract was stored in an airtight dark bottle and refrigerated until further use.
Acute toxicity and Determination of LD50
Acute toxicity testing was carried out in two phases according to the method described by [25]. Firstly, nine animals were divided into three groups of three animals each. Each group of animals was administered 10, 100, and 1000 mg/kg of the poly-herbal extract, and no mortality was observed. In the second phase, 6 animals were distributed into 3 groups of two animals each and were administered higher 1600, 2900, and 5000 mg/kg of the PHE respectively. No mortality was observed after 24 to 48 hours among animals in all groups.
Animal grouping and experimental design
All animal procedures in this study were performed following the guidelines of the research and ethics committee, Ladoke Akintola University of Technology (LAUTECH) for the use of laboratory animals. Thirty-five (35) healthy male Wistar rats weighing 140-150 g were obtained from the animal house of the Department of Biochemistry, College of Basic of Medical Sciences, LAUTECH. They were housed in ventilated cages on a 12:12 hour light-dark cycle and acclimatized for 2 weeks and were separated into 5 groups of 7 animals each as depicted in Table 1
Table 1: Animal grouping and experimental design
Groups
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Treatment
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1
|
Fed with normal rat chow only (positive control)
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2
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Fed with 16 % salt diet only (negative control)
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3
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Fed with 16% salt diet and 800 mg/kg of the poly-herbal extract once daily
|
4
|
Fed with 16% salt diet and 400 mg/kg of the poly-herbal extract once daily
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5
|
Fed with 16% salt diet and 200 mg/kg of the poly-herbal extract once daily
|
Collection of blood serum and tissue preparation from treated rats
The animals were sacrificed through cervical dislocation on the 29th day after overnight fasting. Blood was collected through cardiac puncture using a 5 ml syringe and transferred into plain sample bottles. The blood samples were centrifuged at 4000 rpm for 10 minutes to obtain the serum. The kidney, liver, and aorta were excised, washed in cold washing buffer, and homogenized in phosphate buffer (10%w/v). The homogenates were centrifuged at 10,000 x gravitational force for 15 minutes at 4°C. The supernatants were collected and stored in the freezer at -18°C.
Antioxidant assays
Antioxidant enzyme activity and oxidative stress markers were estimated in the liver, aorta, and kidneys homogenates.
Determination of superoxide dismutase activity
Superoxide dismutase (SOD) activity was evaluated according to the method of [26]. One and a half mL each of 75 mM of Tris-HCl buffer (pH 8.2), 30 mM EDTA, and 2 mM of pyrogallol were added to 70 µL of tissue homogenate. Change in absorbance was recorded at 420 nm for 3 min in a spectrophotometer.
Determination of glutathione concentration
Glutathione (GSH) activity was estimated according to the procedure of Ellman [27]. One hundred (100 μL) of the tissue homogenate was diluted in 20 mL of phosphate buffer (0.1 M, pH 8). Fourty mL of 0.01 M 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was added to 6mL of the mixture, and absorbance was read at 412 nm.
Determination of glutathione peroxidase activity
Glutathione peroxidase activity was determined according to the method of Reddy et al. [28]. To 3.0 ml of glutathione peroxidase substrate solution, 0.1 ml of the homogenate was added. To the test cuvette, 0.5ml of hydrogen peroxide was added and mixed. The change in absorbance was recorded every 30 seconds for 3 minutes in a spectrophotometer at 430nm.
Determination of catalase activity
Catalase activity was determined according to the method of Clairborrne [29]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.4), 19 mM H2O2 and 20 uL tissue homogenate. The degradation of H2O2 was read spectrophotometrically at 240 nm for 1 min. and the catalase activity was calculated according to the formula: K = 2.303/T x log (A1/A2)
Where: K: Rate of reaction; T: Time interval (minutes); A1: Absorbance at time zero; A2: Absorbance at 60 seconds interval.
Determination of malondialdehydeconcentration
Estimation of malondialdehyde (MDA) concentration as an index of lipid peroxidation was assayed according to the method described by [30] . One mL of 20% trichloroacetic acid was added to 1 ml of the tissue homogenate thereafter 2 mL of 0.67% thiobarbituric acid was added. The mixture was incubated at 100oC for 15 min in a water bath and cooled. Six 8 ml of n-butanol was added and centrifuged at 3000 rpm for 15min. The absorbance of the clear pink supernatant was then read against a blank at 532 nm spectrophotometrically. The concentration of MDA is expressed in nmol / g of the tissue.
Determination of Nitric oxide concentration
The level of nitric oxide (NO) in the tissues was determined according to the method described by [31]. Succinctly, 200 μL of the samples was incubated with 200 μL of Griess reagent at 25 °C in the dark for 30 min. Absorbance was subsequently read at 548 nm.
Serum Lipid Assay
Collected serum samples were analyzed for lipid profile. High-density lipoprotein-cholesterol (HDL-C) was assayed using an assay kit (Elabscience, USA). Triglyceride (TG) content was evaluated by enzymatic method using an assay kit (Randox, England). Total cholesterol (TC) was determined according to the method of Parakh and Jank (1982). Low-density lipoprotein (LDL-C) and Very low-density lipoprotein – cholesterol (VLDL-C) was calculated according to Friedwald et al. (1972).
Statistical analysis
Data obtained in this study were expressed as mean ± SEM and subjected to one-way analysis of variance (ANOVA) using statistical package for social sciences 21.0. Duncan's multiple test was used to identify significance between means at P<0.05