Samples of E. drupifera (Thonn) Stapf. were collected from a local farm in Calabar municipality and were identified at the herbarium of the Department of Botany, University of Calabar.

Fresh leaf samples (3.5kg) were oven dried (40 ⁰ C) and ground, yielding 1.86kg.of powder. The powder was extracted with a mixture of ethanol: water (ratio 70:30) by a conventional reflux method for 1hr. The total volume of the mixture was 21.5L. The extraction was repeated 3 times and the filtered hydro-ethanolic extract was mixed and evaporated under reduced pressure. The concentrated extract was frozen and finally freeze-dried to yield 162 of powder. Thus, 1mg of lyophilized extract was obtained from 11.5g of dry leaf powder.

Male white albino mice (20-25g) were assigned to 12 groups of 20 animals per group. Each group was injected (i.p) with one of the following: 10, 20, 40, 80, 120, 200, 250, 300, 350, 400, 450, and 500µg/kg of the crude extract. The maximum volume injected was 0.5ml. The animals were returned to their home cages and given free access to food and water. The mortality in each cage was assessed 24 hours after administration of the extract. The percentage mortalities were converted to probits (a probability unit) and plotted against the log₁₀ of the dose of the extract. Regression lines were fitted by the method of least squares and confidence limits for the LD₅₀ value was calculated by the method of Litchfield and Wilcoxon (1949).

Normal white Wistar rats of either sex (200-280g) were anaesthetized with pentobarbital (35mg/kg.i.p). Surgery and cannulations were performed as described by Eno, et al (2000). The blood pressure from the carotid artery was recorded using Washington, F. T. 400 transducer connected to a recorder (Washington Model 400 MD/2C). For simultaneous heart rate counting, the transducer signal was fed into a biotachometer in one of the polygraph channels. By means of a table heater, the rectal temperature of the animals was maintained at 37 ± 1 °C.

In another group, hypertension was induced using silicone rubber moulds containing deoxycorticosterone acetate (DOCA, 15mg/100g). These were implanted S.C to induce DOCA- salt hypertension. They were given normal saline (0.9% NaCl₂ w/v) in place of drinking water, and fed with rat cubes ad libitum. The animals were considered hypertensive after a period of eight weeks. However, animals with the mean arterial pressure below 160mmHg were not considered hypertensive and were therefore not included in the study. The crude extract (5-80µg/kg.iv) were administered to these hypertensive rats and their BP and heart rate monitored as earlier described.

In another group of normotensive rats, attempts were made to elucidate the possible mechanism of action of the crude extract on BP. Some standard pharmacological agents were employed to influence the action of the extract on blood pressure.

Forty normotensive white Wistar rats (180-250g) were obtained. The crude extract was injected (i.v) as a 0.2ml bolus at eight different increasing doses to construct a dose-response curve. The doses (2, 4, 8, 16, 32, 64, 130 and 260µg/kg. were injected at an interval of 5 min, giving cumulative doses of, 2, 6, 14, 30, 62, 126, 250 and 510µg/kg. extract. The effect of a dose was calculated by averaging the last minute of blood pressure and heat rate recording preceding the following dose. In preliminary experiments, a single dose of the extract produced an effect lasting more than 80min. In another group of rats, the dose-response experiment was repeated but the animals received the extract 2min. after atropine (0.2mg/kg.i.v) pretreatment. The mean arterial BP was calculated from the formula, MAP=DP + 1/3 (SP-DP), where DP and SP are the diastolic and systolic pressures respectively.

Regression lines with confidence limits were calculated for the linear portions of log concentration-response curves. The significance of differences in slopes was used as a measure of parallelism of the two lines. Log concentration limits at 50% of the maximum response were used in the analysis of the significance of the concentration differences as described by (Birmingham et al 1970). Maximum responses were compared by paired Student's t-test.

Noradrenline, mepyramine and propranolol were obtained from Sigma (USA) while Atropine sulphate was obtained from the British Drug House (BDH).

Phytochemical studies (Table 1) revealed that E. drupifera leaf extract contained large amount of Saponins and polyphenols. Tannins, flavonoids, phelobatinns and anthraguinones were probably absent. Sodium and particularly potassium were the cations in very high concentration in the extract. Chloride and possibly phosphorus were also in very high levels in the extract as the anions. With a pH of 5.8, the extract must have been very acidic.

2. Acute Toxicity Test

3. Effects of Leaf Extract on Blood Pressure and Heart Rate

(i) Dose-Effect Relationship

Before the administration of E. drupifera leaf extract, the control mean arterial pressure (MAP) in pentobarbital anaesthetized rats was about 85±3.8mmHg (SEM, n=6). Slow intravenous injection of the extract produced a fall in the BP of rats in a dose-dependent manner. Fig 1 shows the extract-induced decrease in BP. (ED₅₀ = 134.90µg/kg). The maximum decrease in BP (36.2mmHg) was achieved with the extract at a cumulative dose of 560µg/kg cumulative dose represents about 42.35 % (%control) depression of BP by the leaf extract. In general, the effect of the extract on the rat BP was quick in onset and the recovery time increased with increasing concentration of the extract.

Figure 1

Figure 1: Dose-effect relationship. The curves show blood pressure changes (mmHg) following cumulative injection of leaf extract (2 - 516µg/kg.iv.) to a group of normotensive rat ( ? ) and to another group ( ? ) that received atropine (0.2mg/kg.iv.) pretreatment. Data are the mean values ± SEM (n = 6, p<0.05).

A similar dose-effect experiment was repeated in another group of normal rats (control MAP = 83. 85 ± 9.4 mmHg, n=6). In this group, the animals were pretreated with atropine sulphate (0.2mg/kg.i.v) to block the muscarinic cholinoceptors, before injecting the extracts (2-516µg/kg.i.v cumulative dose range) as described in the methods section. In atropine-treated animals, the extract-induced decrease in BP was reversed. The concentration response curve (ED₅₀ =83.18µg/kg) was shifted to the left and the atropine-antagonism appeared to be surmounted with higher doses of the extract Fig.1. The maximum decrease in BP (86.24 mmHg) of rats pretreated with atropine before the injection of extract, was not significantly different from the matching controls.

The control mean systolic and diastolic arterial pressures were 166.3 ±12.8 and 110. 4 ± 14.2 mmHg respectively, for the hypertensive group of rats. Following the administration of the crude extract (5-80µg/kg.i.v), both systolic and diastolic pressures were significantly decreased (p<0.05) in a dose-dependent fashion (Fig. 2).

Figure 2

Figure 2: Histograms showing the effects of graded doses (5 - 80µg/kg.i.v) of leaf extract on the systolic ( ? ) and diastolic ( ? ) blood pressures of DOCA-induced hypertensive rats. Each bar represents the mean value ± SEM, p<0.05; n = 6.

However, the decreases appeared to be more marked in the diastolic than the systolic pressures. The maximum dose of extract tested (80µg/kg.i.v) produced about 24. 7% and 63.6% (% controls) depression of the systolic and diastolic pressures respectively. In general, the extract appeared to have very mild reducing effect on the systolic pressures when compared to its effect on diastolic pressures.

The mean control heart rate (HR) of the hypertensive rats, measured simultaneously with the BP was about 460 beats/ min before administering the extract (Fig. 3). Following the injection of the crude extract (5-80µg/kg. body wt), the HR showed a dose-dependent decrease. The highest dose of extract injected (80µg/kg) also produced the maximum decrease in HR (120±14 beats/min) which represents about 73.91 % (% control) decrease produced by the crude extract.

Figure 3

Figure 3: Histograms showing the effects of graded doses (5 - 80µg/kg.i.v) of leaf extract on Heart rate measured simultaneously with the blood pressure of DOCA-induced hypertensive rats. Each bar represents the mean valve ± SEM. P<0.05, n = 6.

Attempts were made in order to elucidate the possible mechanism of action employed by the crude extract (Figs. 4 and 5) using normotensive rats propranolol (0.5µg/kg) caused a reduction in BP and this condition was aggravated by the extract (10.µg/kg) administered about 10 min after the injection of the drug (Fig. 4a). Noradrenaline-induced increase in BP was as well depressed by the extract (10.µg/kg.i.v) (Fig. 4b). This reduction was by about 23.5% (p<0.05). However, in atropine-treated (1.0µg/kg) animals, the extract (10.µg/kg.iv) failed to depress the BP and even tended to raise it (Fig. 4c). Figure 5 shows the effects of administering high doses (above the ED₅₀ value) of the extract on blood pressure. In all animals tested with this dose of extract (0.2mg/kg.iv), results showed decreased pulse rate and increased pulse pressure (Fig. 5). This was the lowest dose of extract that could produce this effect. Propranolol (0.5µg/kg.iv) and mepyramine (2mg/kg.iv) failed to ameliorate this effect (Fig. 5a and b) while ACh in combination with the extract, aggravated it (Fig. 5c), resulting to the death of these animals. However, these responses (decreased pulse rate and increased pulse pressure) were readily reversed by the administration of atropine sulphate (1.5µg/kg.iv) (Fig. 5b).

Figure 4

Figure 4: Representative tracing on arterial blood pressure (BP) of normotensive rats. Record shows the influence of propranolol (Prop), noradrenaline (NA) and atropine sulphate (Atro) on extract (Ext.)-induced decrease in BP.

Figure 5

Figure 5: Representative tracing on arterial BP. of normotensive rats. Record shows the effects of a high dose (0.2mg/kg.i.v) of the extract (above LD value) on the blood pressure and the influence of propranolol (Prop.), mepyramine (mepy.), atropine (Atro.) and acetylcholine (Ach) on the BP.

Figure 6

Table 1: Chemical characteristics of leaf extract.

Phytochemical and ionic composition of crude aqueous extract from E. drupifera leaves. Values are determined in the laboratories of the National Fertilizer Company of Nigeria “NAFCON”. NIGERIA. ( + = present; ++ = greater presence, - = not present)

We are grateful to Mr. D. D Dakat of the University of Jos, Jos, Nigeria for his technical assistance. Our thanks also go to Prof. Jones Akpan (a pharmacologist) for a very stimulating discussion and to Miss Idorenyin D. Udo for typing the manuscript.