Total Phenol

0.5ml of (1mg/ml) aqueous ethanolic extract was mixed with 1ml of folin colcateau reagent (v/v) and 2.5ml of 20 per cent Na₂CO₃ was added and the reaction mixture was shaken thoroughly. The reaction mixture was incubated at 40ºC for 20mins and the absorbance was read at 760nm. Tannic acid was used as standard phenolic compound (Mc Donald et al., 2001).

Ferric Reducing Antioxidant Property

2.5ml of 1mg/ml of the aqueous methanolic extract was mixed with 2.5ml of 200mm sodium phosphate (pH 6.6) and 2.5ml of 1 per cent potassium ferricyanide. The reaction mixture was incubated at 50ºC for 20mins 2.5ml of Trichloroacetic acid was added and centrifuged at 650 rpm for 10mins, 5ml of the supernantant was mixed with 5ml of water and 0.1 per cent FeCl₃ was added. The absorbance was read at 700nm (Pullido et al., 2000).

Total Flavonoid

Aluminium chloride colorimetric method was used for flavonoids determination (Cheng et al., 2002). Each plant extract (0.5ml of 1:10g/ml) in methanol were separately mixed with 1.5ml 955methanol, 0.1ml of 10 per cent aluminium 2.8ml of distilled water. It was allowed to stand for 30min; the absorbance of the reaction mixture was measured at 415nm with double Perkin Elmer UV/Visible Spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solution at concentrations at 12.5 to 100µg/ml in methanol.

H₂O₂ Radical Scavenging Activity

The ability of extract to scavenge H₂O₂ was determined according to the method of Ruch et al. (1989) with slight modifications of solutions of H₂O₂was prepared in phosphate buffer (PH7.4) extracts (5-40mg/ml) of the aqueous ethanolic extract were added to 0.6ml of 40mm H₂O₂ solution. Absorbance of H₂O₂was measured at 230nm after 10mins against a blank solution containing phosphate buffer without H₂O₂ solution. per cent of H₂O₂ scavenging was calculated as follows:

Hypotensive Activities of Crude Methanol Extract

Wister albino rats (150–200 g), bred at the animal house of the Department of Physiology, Lagos University Teaching Hospital (LUTH), Lagos Nigeria were used. They were all clinically healthy and maintained in standard environmental conditions of temperature (27.0±0.5ºC), relative humidity (73.0±15 per cent) and 12 hrs darkness and12 hrs light cycle. They were fed a standard diet and water ad libitum. The hypotensive activity was investigated according to the method of Adeboye et al. (1999). The Wistar albino rats were divided into 7 groups of 5 rats each. Groups 2-7 received varying doses (0.05-200mg/kg) of Parinari curatellifolia extract while group 1 (control) received an equivalent volume of distilled water. One hour after the administration of extract, they were anaesthetized with urethane (1.75 mg/kg). As soon as the animals reached the state of surgical anaesthesia, each of them was placed on its back and pinned on a rat dissecting board. Splitting the muscle in front using a forceps and scissors exposed the trachea of the rats. The trachea cannula for rat was dissected pointing to the thorax and tied firmly in place using a soft thread. The femoral artery was located and exposed carefully and a bulldog clip was placed on the artery and it was then cannulated. The bulldog clip was removed and the cannula was pushed in further and then tied securely using a disposable syringe and the cannula was filled with heparinized saline to prevent blood clotting. The femoral vein was also located and exposed carefully and a bull dog clip was put on the vein and then cannulated at one end. Heparinized saline was filled into a three-way tap and the cannula. The other end of the tap was connected to the transducer for blood pressure recording. After the stabilization period of 30 min, the initial blood pressure was taken followed by the administration of extract in volume not exceeding 1 ml/kg body weight. The extract was injected intravenously into the femoral artery and the blood pressure was recorded using a Glass polygraph Model 7D, calibration 1cm = 50 mm Hg. The mean arterial blood pressure (MABP) was calculated using

where,

SP: Systolic pressure.

DP: Diastolic pressure.

Effect of Crude Extract on Isolated Rabbit Hearts Preparations

The animals were anaesthetized with urethane (1.75 mg/kg). The chest was cut open and the heart exposed and the aorta was freed from its attachment to the pulmonary artery. It was then quickly transferred into a basin containing Ringer Locke solution and squeezed gently to remove as much blood as possible from the aorta (Burande et al., 1983). The heart was then tied to the Langendorff’s set up with the aorta tied to the cannula connected to the tube leading from the reservoir of the Ringer Locke solution (temperature 37 º C, flow rate of 7-8 ml/min, pH 7.4). The Ringer Locke solution was continuously bubbled with oxygen. After the stabilization period of 5 minutes, extract was administered at concentration 10 mg/ml (stock) through the side arm of the cannula at various volumes of 0.1, 0.2 and 0.3 ml. The contractility of the heart was recorded on the Glass polygraph model 7D by means of a planar clip placed at the apex of the heart connected through a lever to the transducer for recording (calibration, 2.0 cm = 1.0 g).

Effect of Crude Extract on Salt-Induced Hypertension

Wister albino rats (150–200 g), bred at the animal house of the Department of Physiology, Lagos University Teaching Hospital (LUTH), Lagos Nigeria, were used. They were all clinically healthy and maintained in standard environmental conditions of temperature (27.0±0.5ºC), relative humidity (73.0±15 per cent) and 12 hrs darkness and12 hrs light cycle for two weeks before inducing hypertension. The Wistar albino rats were divided into 6 groups of 4 rats each. Groups 3-6 were fed with 8 per cent salt diet for six weeks after which varying doses (200-800mg/kg) of Parinari curatellifolia extract were administered for two weeks. Group 2 was given 8 per cent salt diet alone for six weeks while group 1(control) received an equivalent volume of distilled water throughout the experimentation period. They were all anaesthetized with 5ml/kg urethane-chloralose (25 per cent urethane and 1 per cent chloralose). As soon as the animals reached the state of surgical anaesthesia, each of them was placed on its back and pinned on a rat dissecting board. Splitting the muscle in front using a forceps and scissors exposed the trachea of the rats. The trachea was cannulated. The carotid artery was located and exposed carefully and a bulldog clip was placed on the two ends of the artery and it was then cannulated towards the heart (1 per cent heparin in 9 per cent NaCl). The bulldog clip was removed and the cannula was pushed in further and then tied securely using a disposable syringe and the cannula was filled with heparinized saline to prevent blood clotting. Heparinized saline was filled into a three-way tap and the cannula. The other end of the tap was connected to the transducer for blood pressure recording. After the stabilization period of 30 min, the blood pressure was taken and recorded using a Glass polygraph Model 7D, calibration 1cm = 40 mm Hg. The mean arterial blood pressure (MABP) was calculated using:

where, PP = Pulse.

Effect of Crude Extract on Serum Biochemical Indices

Creatinine was measured by Heinegard and Tiderstrom (1973) using jafe reaction (Slot, 1965).Urea was measured by an enzymatic method of Jansen et al., 1970.

Cholesterol was estimated according to Katayama et al. (1999).

Triglycerides was estimated using the Hantzchcondensation reaction method outlined in tietz

Lipid Peroxidation Assay

Thiobarbituric acid reactive species (TBARS) production was determined as described by Ohkawa et al.(1979).

Superoxide Dismutase Activity (SOD)

The liver homogenate was adequately diluted by NaCl 0.9 per cent to a factor of 20 for the estimation of superoxide dismutase (SOD) assay according to the method described by Misra and Firdovich, 1972. Briefly, epinephrine undergoes autooxidation rapidly at pH 10.0 to produce adrenochrome, a pink colored product that will be detected at 480 nm in kinetic mode using UV/VIS spectrophotometer. The amount of enzyme required to produce 50 per cent inhibition is defined as one unit of enzyme activity. The SOD is expressed as per cent control.

Catalase Activity (CAT)

Catalase (CAT) activity was measured by the method of Aebi (1974). An aliquot of liver and kidney supernatants (10 µL) was added to a quartz cuvette and the reaction was started by the addition of freshly prepared H₂O₂ (30 mM) in phosphate buffer (50 mM, pH 7.0). The rate of H₂O₂ decomposition was measured spectrophotometrically at 240nm during 120 seconds.

Glutathione Peroxidase Activity (GPx)

GPx activity estimation is based on the following principle: GPx catalyses the oxidation of glutathione by cumen hydroperoxide (Chance, 1951). In the presence of hydrogen peroxide, glutathione is immediately converted to the reduced form. The decrease in absorbance at 340 nm is measured.

Results

Phytochemical Constituents

Phytochemical screening revealed the presence of alkaloids, terpenes, glycosides and flavonoids (Table 20.1).

In vitro Antioxidants Activities

The results revealed that the percentage of DPPH radical scavenge varied from (17.05 per cent) to (68.18 per cent) (600mg/ml–1000mg/ml), total phenolic content (12.73mg/g tannic acid equivalent), total flavonoids (22.38mg/g Quercertin), ferric reducing power (13.92mg/g ascorbic acid) and the percentage of hydrogen peroxide inhibition 76.38-96.85 per cent (5-40mg/ml) (Table 20.2).

Effect on Blood Pressure of Normotensive Albino Rats and Isolated Rabbit Heart Preparation

Parinari curatellifolia seed extract produced a dose dependent (0.05–200mg/kg) statistically significant (P<0.05) reduction in systolic blood pressure (120.84–45.30), diastolic blood pressure (98.4.19-19.63), mean arterial blood pressure (111.74–15.10) and an increase in the percentage change in mean arterial blood pressure (0-39 per cent) as compared with the control (Table 20.3). Equally the extract produced dose dependent (4-12 mg/kg) statistically significant (P<0.05) decreases in the force of contraction of isolated rabbit heart preparation (8-3g) and heart rate (708-350 beats/min) (Table 20.4).

Table 20.1:The Phytochemical Constituents of Parinari curatellifolia

Table 20.2:Antioxidant Properties of Parinari curatellifolia

Table 20.3: Effect of Parinari curatellifolia on Blood Pressure of Bormotensive Albino Rats

^*

Stay updated, free articles. Join our Telegram channel

Join