Plant collection and extraction
Fresh barks of C. zeylanicum were harvested in Njombe, Moungo Division in the Littoral Region of Cameroon in January 2007. The plant material was identified at the National Herbarium in Yaounde, in comparison to the voucher specimen number SRFC/22309. The barks were air-dried and ground to a fine powder. 800 g of the powder obtained were soaked at room temperature in 3 l of methanol for 48 h, with occasional shaking. After filtration, the filtrate was concentrated by evaporation at 70°C under reduced pressure on a rotary evaporator to afford 45 g of the methanol extract, corresponding to an extraction yield of 5.6%. This extract was dissolved in DMSO (4%) for daily used.
The major phytochemical groups were determined using the Lieberman Buchard, ferric chloride, copo of magnesium and Vanillin-sulphuric acid tests. Sterols, polyphenolic compounds, flavonoids, alkaloids and saponins were identified in the extract.
Animals and experimental design
Male Wistar rats, aged 12–16 weeks and weighing 150 to 250 g were randomly selected from our colony. They were raised in the animal house of the Faculty of Sciences, University of Dschang, Cameroon. Animals were exposed to daily 12 h light–dark cycle with free access to a standard animal diet and tap water. The effects of Cinnamomum zeylanicum methanol extract (MECZ) were examined acutely and chronically in vivo on mean arterial blood pressure (MABP) of rats previously treated with L-NAME.
Experimental protocols used in this study were approved by the Laboratory committee (Laboratory of Animal Physiology and Phytopharmacology, Department of Animal Biology, University of Dschang – Cameroon) according to the standard ethical guidelines for laboratory animal use and care as described in the European Community guidelines; EEC Directive 86/609/EEC, of the 24th November 1986
Blood pressure and heart rate measurement
Blood pressure and cardiac frequency were determined by the invasive method. Brietly, Animals were anaesthetized by intraperitoneal administration of sodium thiopental at the dose of 50 mg/kg and a catheter was implanted in the femoral vein for drug administration. Another catheter was inserted in the left carotid artery for direct blood pressure measurement. Both catheters were filled with glucose-saline heparinized solution. The catheter inserted in the carotid artery was connected to a blood pressure transducer model Ugo Basile PRC 21k-10 coupled to an Ugo Basile Unirecord model 7050 for blood pressure recording. A stabilisation period of 30 minutes was observed before any recording. Heart frequency was determined by the use of pulse intervals.
For acute antihypertensive study, a solution of L-NAME, an inhibitor of nitric oxide synthase was intravenously injection to normotensive Wistar rats (20 mg/kg) after the stabilization period at corresponding volume of 100 μl/100 g bw. MECZ was administered intravenously at the doses of 5, 10 or 20 mg/kg, twenty minutes after L-NAME administration, when the rise in blood pressure induced by L-NAME had reached the maximum
In the chronic study, male Wistar rats were randomly divided into four groups of eight rats each. The first group (control) received a solution of DMSO 4% daily, while the second one (L-NAME group) received L-NAME (40 mg/kg/day) plus the vehicle. The third group was treated every day with a combined solution of L-NAME (40 mg/kg/day) and captopril (LN-Capto; 20 mg/kg/day) while the forth group, received a combination of L-NAME (40 mg/kg/day) and MECZ (LN-MECZ; 300 mg/kg/day). All the treatments were administered daily by gastric intubation on non-anesthetized animals for 4 weeks at the corresponding volume of 1 ml/100 g bw. The intubation was done daily, on non-anesthetized animals. The dose of the extract was determined based on previous results. At the end of the treatment, animals were anaesthetized by intraperitoneal administration of sodium thiopental (50 mg/kg) for blood pressure measurement. Immediately after blood pressure measurement, blood samples were collected from the abdominal artery, and centrifuged at 3000 rpm for 15 minutes. The plasma obtained was kept at −20°C for lipid assay. Thereafter, the heart and the thoracic aorta were collected, washed in saline and weighed. The heart was dissected out for the evaluation of the left ventricular mass. Three organs’ samples were fixed with 10% formalin in saline while the five others were used for NO evaluation.
The concentrations of total cholesterol (TC), high density lipoprotein (HDL) and triglycerides (TG) in plasma were determined spectrophotometrically using a commercially available kit Dialab and Helios Epsilon spectrophotometer. Low density lipoprotein (LDL) content was calculated from the other lipid parameters according to the
 equation: LDL = TC–(TG/5)–HDL. The atherogenic index was calculated using the formula
: Atherogenic Index.
The left ventricle and aorta were homogenized in a carbonate buffer solution, and centrifuged at 4000 rpm for 15 minutes. The supernatant obtained was used to measure spectrophotometrically tissue concentration of nitric oxide (NO) as previously described by
. Briefly, 300 μl of sample were allowed to react with Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid) at room temperature for 10 min, and then the absorbance was read at 530 nm. The NO concentration was determined by using NaNO2 standard curve.
Aortic and left ventricular histological analysis
Fixed left ventricle and aorta were dehydrated and embedded in paraffin. 5 μm thick sections were mounted on glass slides. After deparaffinization and rehydration, they were stained with either Hematoxylin-Eosin or Van Gieson trichrome solutions in order to assess histological injuries and collagen accumulation in tissues. Histological analysis was performed with light microscope. The media thickness of aorta defined as the distance between the internal and external elastic lamina was determined using the following argument. Each photo have a bar corresponding to a given distance (aμm). Knowing that the magnification used was 400, the length (L) of each bar was then calculated using the formula: L = aμm/400.
L-NAME was obtained from Fluka (Germany), sodium thiopental from Rotex Media (Germany), Heparin from Sanofi (France) and captopril was obtained from the Sahib Singh Agencies (India). All test solutions, including Krebs’ solution were freshly prepared in distilled water.
Statistical analysis was performed using GraphPad Prism version 5.0. All results are expressed as means ± standard error of the mean. Data were analysed using one-way analysis of variance (ANOVA) followed by Tukeys’post test. Differences between means were considered to be significant when p < 0.05.