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Antihypertensives

Blood Pressure Lowering Effect of an


Olive Leaf Extract (Olea europaea) in
L-NAME Induced Hypertension in Rats
Mohamed T. Khayyala, Mona A. El-Ghazaly b, Dalal M. Abdallaha, Noha N. Nassar a,
Samuel N. Okpanyic, and Matthias-Heinrich Kreuter d

Department of Pharmacology, Faculty of Pharmacy, Cairo University a, Cairo (Egypt), Department of Drug
Radiation Research, National Center for Radiation Research and Technology b, Cairo (Egypt), Steigerwald
Arzneimittelwerk GmbHc, Darmstadt (Germany), and Flachsmann AG d, Zurich (Switzerland)

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Summary
A specially prepared olive leaf extract effects of olive leaf. The special extract,
(EFLA 943) has been tested for its blood EFLA 943, was shown to give consistent
pressure lowering activity in rats ren- results with little individual variability.
dered hypertensive by daily oral doses of The antihypertensive effect of the extract
L-NAME (NG-nitro-L-arginine methyl may be related to a variety of factors
ester, 50 mg/kg) for at least 4 weeks. Oral involving reversal of vascular changes
administration of the extract at different involved in the L-NAME induced hyper-
dose levels at the same time as L-NAME tension.
for a period of 8 weeks showed a dose
dependent prophylactic effect against the
rise in blood pressure induced by L-
NAME, best effects being induced by a
dose of 100 mg/kg of the extract. In rats
previously rendered hypertensive by L-
NAME for 6 weeks and then treated with
that dose of the extract for a further 6
weeks without discontinuation of L-
NAME, normalisation of the blood pres-
sure was observed. The findings confirm
previous reports on the hypotensive

Zusammenfassung Key words


Antihypertensives
Blutdrucksenkende Wirkung eines Oli- methylester) fr mindestens 4 Wochen
EFLA 943, antihypertensive
venbltter-Extraktes (Olea europaea) bei induziert wurde. Eine gleichzeitige Ver-
L-NAME- induzierter Hypertonie der abreichung verschiedener Dosierungen effects, rat
Ratte des Extraktes mit L-NAME ber 8 Wo- Hypertension, experimental
chen ergab eine dosisabhngige prophy- Olea europaea
Die blutdrucksenkende Wirkung eines laktische Wirkung gegen den L-NAME-in- Olive leaf extract
Olivenbltter-Extraktes (EFLA 943) wurde duzierten Blutdruckanstieg. Die beste
an Ratten geprft, bei denen Bluthoch- Wirkung wurde bei der Dosis von 100 Arzneim.-Forsch./Drug Res.
druck durch tgliche orale Gabe (50 mg/ mg/kg KG des Extraktes erzielt. Bei Rat- 52, No. 11, 797802 (2002)
kg KG) von L-NAME (NG-Nitro-L-arginin- ten, bei denen zuvor durch eine 6-w-

Arzneim.-Forsch./Drug Res. 52, No. 11, 797802 (2002)


Khayyal et al. Olive leaf extract EFLA 943 797
Antihypertensiva

chige Gabe von L-NAME eine Hyperten- Berichte ber die hypotensive Wirkung sammenhngen, welche zur Reversibili-
sion induziert worden war, fhrte von Olivenblttern. Es zeigte sich dar- tt vaskulrer Vernderungen fhren, die
anschlieende Behandlung mit dem ber hinaus, da dieser Spezialextrakt mit L-NAME-induzierter Hypertonie
Extrakt (100 mg/kg KG) ber 6 Wochen durchwegs gleichmige Ergebnisse mit assoziiert sind.
bei ununterbrochener L-NAME-Gabe zu geringer individuellen Variabilitt liefert.
einer Normalisierung des Blutdruckes. Die antihypertensive Wirkung des Extrak-
Diese Ergebnisse besttigen frhere tes knnte mit mehreren Faktoren zu-

1. Introduction With regard to the tolerability of olive leaf constituents,


Elliott et al. [15] found that calcium elenolate was well
The utilization of the medicinal properties of olive leaf
tolerated by rats in daily oral doses of up to 300 mg/kg
(Olea europaea, L.) dates back to the early 1800s when
for one month.
extracts were used for the treatment of malaria. In the
Because of the paucity of literature concerning the
early 1900s, a bitter principle of the leaves was identi-
effect of olive leaf on hypertensive animals, it was de-
fied as oleuropeoside (later designated oleuropein), a
cided to carry out the present investigation with the
polyphenolic iridoid glycoside [1] to which earlier re-
stabilised olive leaf extract. The model used involved
ports of the hypotensive activity of the leaf extracts was

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the use of rats rendered hypertensive by the administra-
accredited [2, 3]. Early experimental and clinical studies
tion of L-NAME.
reported on the hypotensive effect of olive leaf ex-
tracted by different solvents and methods, ranging from
hot water extracts [48], glycerine/ethanol extracts [9],
ethanol/chloroform [10]. In 1991, Zarzuelo et al. [11] 2. Materials and methods
further investigated the hypotensive effect of a lyophi- 2.1. Animals
lised decoction of olive leaf and found it to be hypoten- All animals used in this work were male Wistar rats, purchased
sive and vasodilator on isolated aortae, the vasodilator from the Agricultural Research Centre in Cairo. The animals
effect being endothelium independent. Other authors, were fed on a standard pellet diet supplied by Alexandria Com-
however, have shown that oleuropein increases nitric pany for Oil Industries. They were housed for 12 weeks prior
to starting experimentation at the animal facility of the Faculty
oxide synthase activity, and the effect is blocked by L-
of Pharmacy, Cairo University, at a temperature of 25 1 C
NAME (NG-nitro-L-arginine methyl ester) [12]. Petkov
and relative humidity of 60 5 %. The study was carried out
and Manolov [13] demonstrated the hypotensive as well
according to international guidelines after approval by the Eth-
as coronary vasodilator and anti-arrhythmic properties ical Committee for Animal Experimentation at the Faculty of
of oleuropein. Oleuropein is metabolised in the body to Pharmacy, Cairo University.
calcium elenolate, which is apparently responsible for
many of the pharmacological actions of the olive leaf. 2.2. Chemicals
Most of the reported literature dealt with the hypoten- Olive leaf extract (EFLA 943, Batch No. 42128-01) was supplied
sive effects of olive leaf in normal animals, and not in as a finely dispersable powder by Steigerwald Arzneimittelwerk
hypertensive ones. Few studies have been conducted (Darmstadt, Germany) as provided by Flachsmann (Zurich,
on hypertensive patients. In one study, the extract has Switzerland). Olive leaf was primarily extracted with 80 % m/
been tested orally on 30 hypertensive patients and m ethanol, and the product purified by a patented procedure
found to have statistically significant effects [14]. The (US Patent 6024998) to remove undesired contaminants and
reported findings on the hypotensive effects of olive leaf residues. The solvent was subsequently removed resulting in a
free flowing powder, which was then assayed by HPLC to con-
extract were not always consistent, possibly due to the
tain 1826 % m/m oleuropein. The extract was used in a finely
instability of the extract and to lack of proper standard-
dispersed form (nearly dissolved) in water shortly before use.
isation of its constituents. The iridoid glycosides of Olea
L-NAME was obtained from Sigma Chemical Co. (St. Louis,
europaea leaves in particular easily undergo rapid de- MO, USA), and was dissolved in water before administration.
gradation if proper harvesting and extraction technolo- The drug was given in a dose of 50 mg/kg orally.
gies are not implemented. Pure oleuropein, the main
iridoid glycoside in Olea europaea leaf extract, was 2.3. Experimental design
shown to be degraded rapidly in aqueous solutions, but 2.3.1. Induction of hypertension
it remains stable in the solubilised extract, EFLA 943,
Animals were rendered hypertensive by treatment with L-
which has been developed and validated by Flachs-
NAME orally in a daily dose of 50 mg/kg for 4 weeks. By the
mann AG (Zurich, Switzerland) to give reproducible and end of that period, the animals showed a significant rise in
consistent contents of active ingredients in aqueous systolic blood pressure. The blood pressure and heart rate were
media. This made it necessary to test the activity of the measured non-invasively at weekly intervals through a tail cuff
stabilised extract on one of the acknowledged models using a computer assisted monitoring device (TSE Systems,
of experimental hypertension, namely that of L-NAME. Bad Homburg, Germany; model V2.1). At least 3 readings of

Arzneim.-Forsch./Drug Res. 52, No. 11, 797802 (2002)


798 Khayyal et al. Olive leaf extract EFLA 943
Antihypertensives

blood pressure and heart rate were taken for each animal and 3. Results
the mean values were calculated. On continued administration
3.1. Prophylactic effect of the olive leaf extract
of L-NAME the blood pressure continued to rise without any
significant change in heart rate. The daily oral treatment with L-NAME in a dose of 50
mg/kg resulted in the development of hypertension
2.3.2. Prophylactic antihypertensive effect after 4 weeks of administration, the effect gradually in-
of the olive leaf extract creasing over an 8-week period to reach a rise of about
In order to investigate the potential usefulness of the extract in 85 % over initial values. The concomitant use of the ex-
preventing the development of hypertension induced by L- tract in a dose of 25 mg/kg was not effective in sup-
NAME, five groups of rats, 10 animals each, were randomly pressing the rise in blood pressure induced by L-NAME,
divided into the following groups: whereas in a dose of 50 mg/kg, it was capable of redu-
1. Control group: Animals received no extract medication, but cing the rise in blood pressure induced by L-NAME
were given an oral gavage of physiological saline (0.2 ml) daily from 85 % to only 51 %. The changes in blood pressure
for 8 weeks.
of the rats in each group showed little individual variab-
2. L-NAME treated group: Animals were treated with L-NAME
ility, such that the results were very consistent and the
orally in a daily dose of 50 mg/kg for 8 weeks.
3. L-NAME + olive leaf extract treated groups: Animals were standard errors of the means were always very small.
treated with L-NAME for 8 weeks as above, but with the conco- By the end of 8 weeks, it was evident that the use of
mitant administration of the leaf extract in doses of either 25, the 100 mg/kg dose of the leaf extract had completely
50 or 100 mg/kg for the same length of time. The extract was prevented the rise in blood pressure induced by L-

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given orally as an aqueous solution of the powdered form pre- NAME (Fig. 1) as shown by the lack of statistical signifi-
pared shortly before use. Although not completely soluble, the cance at p < 0.05 between that group and the normal
solution contained uniformly finely dispersed particles and did
control one.
not necessitate the use of a suspending agent.
Treatment with L-NAME, with or without the conco-
The body weights, blood pressure and heart rate of all an-
imals were recorded on a weekly basis as described above.
mitant use of the extract, had no significant effect on
the heart rate of the rats. Both treated and control an-
imals gained in body weight progressively over the 8-
2.3.3. Effect of olive leaf extract administration
week experimental period, the increase in weight ran-
in hypertensive rats
ging from 3750 %.
In order to study whether or not the extract is capable of redu-
cing the blood pressure of rats with established hypertension,
rats were rendered hypertensive by daily oral administration of 3.2. Effect of the olive leaf extract administration
L-NAME for 6 weeks, then treatment with the leaf extract was in hypertensive rats
started for a further 6 weeks whilst continuing the L-NAME ad-
ministration. In this experiment, 20 rats were rendered hypertensive
Animals were allocated to the following groups: by daily treatment with L-NAME for a 12-week period.
1. Control group: Fifteen animals were used in this group. They The average blood pressure of the animals at the begin-
received no medication but were given an oral dose of saline ning of the experiment was 109 mmHg but reached 204
(0.2 ml) daily for the duration of the experimental period (12 by the end of 12 weeks, constituting a rise of 87 %.
weeks). Six weeks after initiating treatment, the average BP had
2. Hypertensive group: Forty animals were included in this
reached 160 mmHg constituting a rise of about 46 %. In
group. The rats were rendered hypertensive by treatment with
a separate group, another 20 rats were started on the
L-NAME orally in a daily dose of 50 mg/kg for 6 weeks. At the
end of this period, they were randomly divided into 2 sub-
olive leaf extract in a daily oral administration of 100
groups, 20 animals each: mg/kg after 6 weeks of L-NAME treatment, whilst con-
Subgroup A: The rats were given L-NAME alone for 6 more tinuing treatment with L-NAME. The blood pressure of
weeks. the animals which had risen by 46 % after 6 weeks of
Subgroup B: The animals were given olive leaf extract in a daily treatment with L-NAME alone, started to drop progres-
oral dose of 100 mg/kg along with L-NAME for the next 6 sively over the next 6 weeks to reach an average of 125
weeks. mmHg at the end of the experimental period in spite of
The body weights, blood pressure and heart rate of all an-
the continued administration of L-NAME, constituting
imals were recorded on a weekly basis as described above.
only a 12 % rise from the initial values, thereby signal-
ling normalisation of the blood pressure.
2.4. Statistical evaluation
It was noticed that treatment with the olive leaf ex-
The standard errors of mean values at the commencement and tract produced a dramatic effect within the first 2 weeks
at the end of all the experiments were calculated, taking in
of administration to the hypertensive animals, i.e. by
consideration not to include animals that may have succum-
week 8, but the effect was not maintained because of
bed during the experimental period. The Students t-test for
unpaired data was applied to assess the level of significance the continued administration of L-NAME. The effects of
between the means at the start and end of the experimental giving L-NAME alone for 12 weeks compared to those
period. Probabilities of 0.05 or less at the respective degrees of where treatment with the leaf extract was instituted at
freedom were regarded as statistically significant. the end of the sixth week are shown graphically in Fig. 2.

Arzneim.-Forsch./Drug Res. 52, No. 11, 797802 (2002)


Khayyal et al. Olive leaf extract EFLA 943 799
Antihypertensiva

90 **
**

change in BP as % of control
70 **
** **
** **
50 **

** **
30

* **
10 *

0 2 4 6 8
-10 Weeks after treatment

L-NAME L-NAME + EFLA943 (25 mg/kg) L-NAME + EFLA943 (50 mg/kg) L-NAME + EFLA943 (100 mg/kg)

Fig. 1: Changes in systolic blood pressure of rats treated with L-NAME (50 mg/kg) given alone or concomitantly with different doses of
olive leaf extract (EFLA 943) over an 8-week period. Each point represents the average change in blood pressure of 10 animals as a

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percentage of normal controls S.E.M. Asterisks indicate significant difference from the normal control rats at p < 0.05 (*) and p < 0.01
(**), respectively.

210 **
**
180 **
** **
150
** ** **
**
BP in mm Hg

*
120 *

90

60

30

0
0 2 4 6 8 10 12
Time in weeks
Control L-NAME L-NAME + EFLA943 (100 mg/kg)

Fig. 2: Changes in systolic blood pressure of animals treated with L-NAME (50 mg/kg orally per day) for 12 weeks (n = 20) and in
animals treated with L-NAME for 6 weeks, then initiated on the olive leaf extract, EFLA 943 (100 mg/kg orally per day) for a further 6
weeks while continuing L-NAME treatment (n = 20) as compared to normal controls (n=15). Each point represents mean values of
blood pressure + S.E.M. Asterisks denote significant difference from control at p < 0.05 (*) and at p < 0.01 (**), respectively.

The heart rate of the animals showed no significant 4. Discussion


change over the experimental period, but the animals
gained consistently in body weight reaching 76 to 84 % Inhibition of nitric oxide synthase in mammalian tis-
of their initial values with no statistically significant dif- sues seems to be involved in a multitude of pathophysi-
ferences in the body weight changes between the ex- ological cardiovascular changes including atheroscler-
perimental groups. osis, coronary heart disease, and hypertension [16].

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800 Khayyal et al. Olive leaf extract EFLA 943
Antihypertensives

Rats that are chronically treated with the orally active tion in morbidity and mortality in our present experi-
nitric oxide synthase inhibitor, L-NAME, have been ments. The angiotensin converting enzyme (ACE) in-
shown to develop a marked hypertension [17, 18] as a hibitor effect of olive leaf, however, seems not to be as-
result of chronic blockade of nitric oxide synthesis [19]. sociated with oleuropein per se, but with another active
The model of using L-NAME to induce experimental constituent, oleacein [29]. Furthermore, oleuropein
hypertension has become a widely accepted method for could be cleaved by -glucosidase to a derivative with
testing antihypertensive agents. There is evidence to high ACE inhibitor activity. High blood pressure is
suggest that 7 days after treatment with L-NAME activa- known to induce remodelling in cardiac and vascular
tion of the sympathetic nervous system contributes to tissue, which are deleterious to health, possibly through
the development of the high blood pressure [20]. More the involvement of angiotensin and aldosterone. Such
recent observations have shown that moderate inhibi- changes are known to be reversed at an early stage of
tion of nitric oxide synthase by L-NAME does not seem development by drugs that interfere with the action of
to influence the cardiac sympathetic tone. In our study, angiotensin, such as the converting enzyme inhibitors.
the heart rates of animals treated up to 12 weeks with Olea europaea has also been found to contain an
L-NAME did not show significant changes from con- active constituent, -(3,4-dihydroxy-phenyl)ethanol,
trol values. which was found to have calcium channel blocking ac-
The present findings indicate that the extract of olive tivity [30]. ACE inhibitors combined with calcium chan-
leaf had a definite dose-dependent prophylactic effect nel blockers exert a synergistic antihypertensive effect
in preventing the rise in pressure induced by L-NAME, [31]. Combination therapy using agents with different

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when both agents were given concomitantly. The best mechanisms of action usually results in reducing overall
dose which almost completely prevented the rise in toxicity and side effects.
blood pressure was 100 mg/kg of the extract. Accord- It would therefore seem from the present findings
ingly, that dose of the extract was used in an effort to that the beneficial antihypertensive effect of the olive
see whether giving the extract to animals with estab- leaf extract, EFLA 943, could be due to a variety of fac-
lished hypertension would still be capable of reducing tors acting simultaneously. The activity of the stabilised
the high pressure in the presence of L-NAME. The rats extract lies probably in its content of oleuropein acting
were allowed to develop a conspicuous hypertension synergistically with other active principles to exert anti-
after treatment with L-NAME for 6 weeks, then initiated oxidant, ACE inhibitory and possibly calcium channel
treatment with the extract orally for a further 6 weeks, blocking activities, all of which may contribute to the
without stopping the administration of L-NAME. The efficacy and good tolerability of the extract. Further
extract normalised the blood pressure by the end of the studies are under way to further elucidate its mechan-
experimental period, proving once more its efficacy as ism of action.
an oral therapy for this model of hypertension. Thus,
the olive leaf extract has been shown to reverse the L-
NAME induced dysfunction, when given after the devel-
opment of hypertension, and to prevent the occurrence
of such dysfunction, when given at the outset together 5. References
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Prof. Dr. Mohamed T. Khayyal,
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Department of Pharmacology,
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361 (1996) E-mail: mtkhayyal@hotmail.com

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802 Khayyal et al. Olive leaf extract EFLA 943