A Eno, O Ofem, E Itam
blood pressure, cholinergic, depression, heart rate, leaves
A Eno, O Ofem, E Itam. Blood pressure lowering agent(s) in the leaves of a deciduous shrub-Elaeophorbia drupifera.. The Internet Journal of Nutrition and Wellness. 2006 Volume 4 Number 1.
The crude ethanolic extract was prepared from the leaves of
In the normotensive group, the extract reduced the BP dose-dependently. A dose-response curve constructed gave a sigmoidal shape with an ED50 of 134.9µg/kg.i.v. The maximum decrease produced by the extract (560µg/kg.iv) was about 36.2mmHg, representing about 42.35% decrease (%control). A repeat of the dose-effect experiment with a group of atropinized rats, showed that the dose-response curve shifted to the left. This atropine-antagonism appeared to be surmounted by raising the concentration of the extract. In the hypertensive group, the extract (80µg/kg.i.v) produced a maximum decrease in BP of about 24.7% and 63.6% for the systolic and diastolic pressures respectively. Both decreases were significant (p<0.05 in both cases). Also, the extract (80µg/kg.i.v) markedly depressed the heart rate of hypertensive rats. The depression was up to about 73.91% (P<0.05).
Propranolol (0.5µg/kg.iv) failed to prevent the extract-induced reduction of BP. The extract (10µg.kg.iv) reduced noradrenaline-induced increase in BP, and when it (extract) was given in combination with ACh, the action of the extract was enhanced by ACh.
We conclude that the crude ethanolic extract from the leaves of
Hypertension is a significant health problem because of the percentage of the population affected and the serious consequences of uncontrolled high blood pressure. In general terms, hypertension continues to be a major risk factor for stroke, congestive heart failure and coronary artery disease (Akinkigbe, 2001).
The worldwide increasing demand for medicine from natural sources (Lapa, 1992) has motivated us to search for plants with potential hypotensive activity.
Plants of the family Euphobiaceae are frequently used in indigenous practice of medicine. Their pharmacological properties include anti-tumor, antibacterial and antihypertensive activities (Schiff, 1970). However, little literature is available on the medicinal uses of the species
This local herb is used by traditional herbalists for the treatment of hypertension, diabetes and many other ailments. Ground leaves (paste) are dissolved in either water or soft drink and administered orally in doses determined by age.
In cases of over dosage producing any adverse effect, fresh coconut water is administered to the patient as an antidote (personal comm.).
The present study is aimed at investigating the scientific basis of employing this leaf extract as an antihypertensive agent by our traditional herbalists. This study became necessary following earlier report that the roots of this plant has hypotensive activity (Eno and Owo, 1999).
Materials And Methods
(a) Preparation of Extract
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.
(b) Acute Toxicity Test
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).
(c) Intact Preparation of Normo-and Hypertensive Rats
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,
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
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.
(d) Dose-Effect Relationship
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.
(e) Statistical Analysis
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
(f) Reference Drugs
Noradrenline, mepyramine and propranolol were obtained from Sigma (USA) while Atropine sulphate was obtained from the British Drug House (BDH).
(a) Phytochemical Studies
Phytochemical studies (Table 1) revealed that
2. Acute Toxicity Test
In lethality studies, a dose-mortality relationship (not show) was apparently sigmoidal. A plot of probit (a probability unit) values (% mortality against the log-dose of extract) produced a straight line. From the straight line graph, the LD50 was extrapolated. This value was about 112.6mg dry wt. of extract per kg, mice (mg/kg mice, i.p). No animal died earlier than 8h. post-injection.
3. Effects of Leaf Extract on Blood Pressure and Heart Rate
(i) Dose-Effect Relationship
Before the administration of
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.
(b) Effect of Crude Extract on DOCA-Induced Hypertensive Rats
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).
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.
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).
Phytochemical and ionic composition of crude aqueous extract from
The crude ethanolic extract from
The present studies show clearly that
Blood pressure measurements reflect the status of the cardiovascular system (Milnor, 1980), and the maintenance of an adequate BP in the aorta depends on the product of two factors, the cardiac output and the total peripheral resistance of the vessels (Guyton, 2005). Therefore, the present studies were focused on the effect of the crude extract from
Although, we are unable to explain why
In conclusion, it appears, therefore, that the crude ethanolic extract from the leaves of
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.
Dr. A. E. Eno Physiology Department College of Medical Sciences, University of Calabar, P. M. B.1115, Calabar Cross River State. Nigeria. E-mail: nkineno2000@ yahoo.co.uk Tel: 08032866269. 08055664612.