Fresh plants were purchased from the New Plant Nursery, George, South Africa in August and September, 2008; identified by the taxonomist at the Department of Biodiversity and Conservation Biology of the University of the Western Cape (UWC), Bellville; and deposited at the herbarium with voucher numbers 6955 and 6956.
Fresh leaves weighing 2.4 kg were washed, and dried in an oven at 30°C until a constant mass was obtained. Dried leaves were ground into powder. Methanol leaf extract (MLE) was prepared by Soxhlet extraction and the excess solvent removed at 40°C using a rotavapor. The dried black paste obtained was placed in a −20°C freezer before being dried further using a freeze-drier. The final dried extract (76.6 g or 3.2% yield) was stored in a brown bottle in a refrigerator at −4°C. Fresh MLE was dissolved with drops of dimethylsulfoxide (DMSO) and the required concentration made up with 0.9% normal saline and filtered before infusion into the rat to prevent the formation of emboli.
Healthy male SHR weighing 300–350 g, aged less than 5 months old were obtained from the Animal Unit at the University of Cape Town, South Africa; and were kept under laboratory conditions in the animal room, School of Pharmacy, UWC; and given water and feed ad libitum. Room temperature was kept at 24°C, with a 12:12-h light–dark cycle.
Atropine (atropine sulfate salt monohydrate), muscarine ((+)-muscarine chloride) and captopril were purchased from Sigma-Aldrich, South Africa. Solvents were 0.9% saline (Adcock Ingram, South Africa) and dimethylsulfoxide (DMSO, Merck Chemicals, South Africa). Atropine and muscarine were dissolved in 0.9% saline, while T. violacea and captopril were initially dissolved with drops of DMSO, and made up to the required dilution (concentration) using 0.9% saline. Fresh drug solutions were prepared at the beginning of each experiment and kept on ice during the course of the experiment.
In- vivo experiments
Rats were anaesthetized with 6% sodium pentobarbitone (Kyron Laboratories, South Africa) at a dose of 40 mg/kg intraperitoneally, and fastened in a supine position on a heated rat-operating table (BioScience), whose temperature was maintained at 37.3 ± 0.5°C by a thermostat. A temperature probe (AD Instruments) was inserted into the rectum to monitor the body temperature. The trachea was cannulated to maintain airflow during the experiment and an oxygen mask placed close to the opening of the tracheal cannula to maintain adequate supply of oxygen (Afrox, South Africa) to the rat. The right external jugular vein was cannulated with a small polyethylene cannula to allow intravenous infusion of drugs via a syringe placed on the two-way injection Ascor AP 22 syringe pump (Poland). The left femoral artery was cannulated with a small polyethylene cannula filled with heparin (Intramed, South Africa) 100 IU/ml normal saline . The femoral cannula was connected to a BP transducer attached to a BP amplifier (ADInstruments, Australia) and Power Lab 4/20 T (ADInstruments, Australia) for recordings of the BP and HR onto a Chart 5.0 for Windows software (ADInstruments, Australia). Rats were allowed a 15-min stabilization period to ensure that BP and HR parameters were stable before any further experiment, and each group consisted of 8 rats. Drugs were infused, flushed with 0.1 ml of normal saline and results recorded within 3 min of infusion. Pressures and HR were allowed to return to baseline values (10–15 min) before further doses were infused.
In conscious SHR, systolic BP was measured using non-invasive tail-cuff plethysmography (ADInstruments, Australia), between 09:00 and 16:00 h. The SHRs were warmed at 37°C for 10 min and allowed to rest in a glass restrainer, and a black conical plastic piece with a nose opening, placed over the head region of the rat, to cover the eyes of the rat, and consequently allow the rat to rest better; before the tail-cuff plethysmography. An average of 5 consecutive readings was recorded. Rats were trained every 3 days for BP measurement for two weeks prior to the start of the actual experiment [26–29].
a) Muscarinic receptors
Dose–response experiments (DRE) for muscarine (0.16 -10 μg/kg) and atropine (0.02 - 20.48 mg/kg) were performed in the SHR. The dose at which 80% of the maximum effect obtained was noted.
Previously , 80% of the maximum effect of T. violacea, was achieved at 60 mg/kg. Therefore, this dose (60 mg/kg), half of it (30 mg/kg), and twice this dose (120 mg/kg) were used in this study. T. violacea (30, 60 and 120 mg/kg) was infused into eight SHRs with the BP and HR values allowed to return to baseline, prior to the administration of the subsequent dose. Another set of eight SHRs were pre-treated with atropine (5.12 mg/kg). In this study, T. violacea or muscarine was infused 20 min [30–33] after atropine was infused into rats. T. violacea (30, 60 and 120 mg/kg) was then infused, with the BP and HR values allowed to return to baseline, prior to the subsequent dose of the MLE being administered.
b) T. violacea effect compared with muscarine chloride after atropine pre-treatment.
Eight SHRs were pre-treated with atropine (5.12 mg/kg), before T. violacea (60 mg/kg) was infused into these rats. The BP and HR returned to baseline, before muscarine (2.5 μg/kg) was infused into the same rat.
The effects of the MLE and the standard drugs on SBP, DBP, MAP and HR were evaluated.
c) Body weight, BP, HR and plasma aldosterone levels
The SHRs used in this protocol were allowed 14 days of acclimatization to the restrainers [26–29]. The body weight, SBP and HR of the rats were measured on the first day of study, and the rats were randomly divided into three groups of eight each. The intraperitoneal injection given to the rats during the next 21 days were T. violacea (60 mg/kg), captopril (10 mg/kg) or 0.2 mls of the vehicle (DMSO + NS). The BP and HR of the rats were measured using the non-invasive tail cuff plethysmography during the intervention period. On day 22, invasive BP measurement was done, and blood was collected from the femoral artery of the SHR after the BP and HR values had stabilized and recorded. Blood was stored in ethylenediaminetetraacetic acid (EDTA) tubes and rapidly spun at 10 000 rpm, in a centrifuge to separate the plasma from the blood cells. The plasma obtained was then stored in another set of EDTA tubes and placed in a – 40°C freezer. Plasma samples were sent to the Veterinary Hormone Laboratory, Faculty of Veterinary Science, University of Pretoria (South Africa), for analysis of plasma aldosterone levels.
The paired and/or unpaired Student’s T test was used to calculate statistical significance (p < 0.05), using the Microsoft Excel software.
The methodology and ethics adhered to in this study were approved by the Ethics Committee of the University of the Western Cape, and the registration number obtained was 09/7/35. All experimental procedures used in this study were conducted in accordance with the guidelines provided by the European Community guidelines (EEC Directive of 1986; 86/609/EEC).