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

Blutdrucksenkende Wirkung eines Oli-
venbltter-Extraktes (Olea europaea) bei
L-NAME- induzierter Hypertonie der
Ratte
Die blutdrucksenkende Wirkung eines
Olivenbltter-Extraktes (EFLA 943) wurde
an Ratten geprft, bei denen Bluthoch-
druck durch tgliche orale Gabe (50 mg/
kg KG) von L-NAME (NG-Nitro-L-arginin-
Antihypertensives
methylester) fr mindestens 4 Wochen
EFLA 943, antihypertensive
induziert wurde. Eine gleichzeitige Ver-
abreichung verschiedener Dosierungen effects, rat
des Extraktes mit L-NAME ber 8 Wo- Hypertension, experimental
chen ergab eine dosisabhngige prophy- Olea europaea
laktische Wirkung gegen den L-NAME-in- Olive leaf extract
duzierten Blutdruckanstieg. Die beste
Wirkung wurde bei der Dosis von 100 Arzneim.-Forsch./Drug Res.
mg/kg KG des Extraktes erzielt. Bei Rat- 52, No. 11, 797802 (2002)
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-
sion induziert worden war, fhrte
anschlieende Behandlung mit dem
Extrakt (100 mg/kg KG) ber 6 Wochen
bei ununterbrochener L-NAME-Gabe zu
einer Normalisierung des Blutdruckes.
Diese Ergebnisse besttigen frhere
Berichte ber die hypotensive Wirkung sammenhngen, welche zur Reversibili-
von Olivenblttern. Es zeigte sich dar- tt vaskulrer Vernderungen fhren, die
ber hinaus, da dieser Spezialextrakt mit L-NAME-induzierter Hypertonie
durchwegs gleichmige Ergebnisse mit assoziiert sind.
geringer individuellen Variabilitt liefert.
Die antihypertensive Wirkung des Extrak-
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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accredited [2, 3]. Early experimental and clinical studies
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]
further investigated the hypotensive effect of a lyophi-
lised decoction of olive leaf and found it to be hypoten-
sive and vasodilator on isolated aortae, the vasodilator
effect being endothelium independent. Other authors,
however, have shown that oleuropein increases nitric
oxide synthase activity, and the effect is blocked by L-
NAME (NG-nitro-L-arginine methyl ester) [12]. Petkov
and Manolov [13] demonstrated the hypotensive as well
as coronary vasodilator and anti-arrhythmic properties
of oleuropein. Oleuropein is metabolised in the body to
calcium elenolate, which is apparently responsible for
many of the pharmacological actions of the olive leaf.
Most of the reported literature dealt with the hypoten-
sive effects of olive leaf in normal animals, and not in
hypertensive ones. Few studies have been conducted
on hypertensive patients. In one study, the extract has
been tested orally on 30 hypertensive patients and
found to have statistically significant effects [14]. The
reported findings on the hypotensive effects of olive leaf
extract were not always consistent, possibly due to the
instability of the extract and to lack of proper standard-
isation of its constituents. The iridoid glycosides of Olea
europaea leaves in particular easily undergo rapid de-
gradation if proper harvesting and extraction technolo-
gies are not implemented. Pure oleuropein, the main
iridoid glycoside in Olea europaea leaf extract, was
shown to be degraded rapidly in aqueous solutions, but
it remains stable in the solubilised extract, EFLA 943,
which has been developed and validated by Flachs-
mann AG (Zurich, Switzerland) to give reproducible and
consistent contents of active ingredients in aqueous
media. This made it necessary to test the activity of the
stabilised extract on one of the acknowledged models
of experimental hypertension, namely that of L-NAME.
the use of rats rendered hypertensive by the administra-
tion of L-NAME.
2. Materials and methods
2.1. Animals
All animals used in this work were male Wistar rats, purchased
from the Agricultural Research Centre in Cairo. The animals
were fed on a standard pellet diet supplied by Alexandria Com-
pany for Oil Industries. They were housed for 12 weeks prior
to starting experimentation at the animal facility of the Faculty
of Pharmacy, Cairo University, at a temperature of 25 1 C
and relative humidity of 60 5 %. The study was carried out
according to international guidelines after approval by the Eth-
ical Committee for Animal Experimentation at the Faculty of
Pharmacy, Cairo University.
2.2. Chemicals
Olive leaf extract (EFLA 943, Batch No. 42128-01) was supplied
as a finely dispersable powder by Steigerwald Arzneimittelwerk
(Darmstadt, Germany) as provided by Flachsmann (Zurich,
Switzerland). Olive leaf was primarily extracted with 80 % m/
m ethanol, and the product purified by a patented procedure
(US Patent 6024998) to remove undesired contaminants and
residues. The solvent was subsequently removed resulting in a
free flowing powder, which was then assayed by HPLC to con-
tain 1826 % m/m oleuropein. The extract was used in a finely
dispersed form (nearly dissolved) in water shortly before use.
L-NAME was obtained from Sigma Chemical Co. (St. Louis,
MO, USA), and was dissolved in water before administration.
The drug was given in a dose of 50 mg/kg orally.
2.3. Experimental design
2.3.1. Induction of hypertension
Animals were rendered hypertensive by treatment with L-
NAME orally in a daily dose of 50 mg/kg for 4 weeks. By the
end of that period, the animals showed a significant rise in
systolic blood pressure. The blood pressure and heart rate were
measured non-invasively at weekly intervals through a tail cuff
using a computer assisted monitoring device (TSE Systems,
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

blood pressure and heart rate were taken for each animal and
the mean values were calculated. On continued administration
of L-NAME the blood pressure continued to rise without any
significant change in heart rate.
2.3.2. Prophylactic antihypertensive effect
of the olive leaf extract
In order to investigate the potential usefulness of the extract in
preventing the development of hypertension induced by L-
NAME, five groups of rats, 10 animals each, were randomly
divided into the following groups:
1. Control group: Animals received no extract medication, but
were given an oral gavage of physiological saline (0.2 ml) daily
for 8 weeks.
2. L-NAME treated group: Animals were treated with L-NAME
orally in a daily dose of 50 mg/kg for 8 weeks.
3. L-NAME + olive leaf extract treated groups: Animals were
treated with L-NAME for 8 weeks as above, but with the conco-
mitant administration of the leaf extract in doses of either 25,
50 or 100 mg/kg for the same length of time. The extract was
given orally as an aqueous solution of the powdered form pre-
pared shortly before use. Although not completely soluble, the
solution contained uniformly finely dispersed particles and did
not necessitate the use of a suspending agent.
The body weights, blood pressure and heart rate of all an-
imals were recorded on a weekly basis as described above.
2.3.3. Effect of olive leaf extract administration
in hypertensive rats
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
L-NAME for 6 weeks, then treatment with the leaf extract was
started for a further 6 weeks whilst continuing the L-NAME ad-
ministration.
Animals were allocated to the following groups:
1. Control group: Fifteen animals were used in this group. They
received no medication but were given an oral dose of saline
(0.2 ml) daily for the duration of the experimental period (12
weeks).
2. Hypertensive group: Forty animals were included in this
group. The rats were rendered hypertensive by treatment with
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-
groups, 20 animals each:
Subgroup A: The rats were given L-NAME alone for 6 more
weeks.
Subgroup B: The animals were given olive leaf extract in a daily
oral dose of 100 mg/kg along with L-NAME for the next 6
weeks.
The body weights, blood pressure and heart rate of all an-
imals were recorded on a weekly basis as described above.
2.4. Statistical evaluation
The standard errors of mean values at the commencement and
at the end of all the experiments were calculated, taking in
consideration not to include animals that may have succum-
bed during the experimental period. The Students t-test for
unpaired data was applied to assess the level of significance
between the means at the start and end of the experimental
period. Probabilities of 0.05 or less at the respective degrees of
freedom were regarded as statistically significant.
Arzneim.-Forsch./Drug Res. 52, No. 11, 797802 (2002)
Antihypertensives
3. Results
3.1. Prophylactic effect of the olive leaf extract
The daily oral treatment with L-NAME in a dose of 50
mg/kg resulted in the development of hypertension
after 4 weeks of administration, the effect gradually in-
creasing over an 8-week period to reach a rise of about
85 % over initial values. The concomitant use of the ex-
tract in a dose of 25 mg/kg was not effective in sup-
pressing the rise in blood pressure induced by L-NAME,
whereas in a dose of 50 mg/kg, it was capable of redu-
cing the rise in blood pressure induced by L-NAME
from 85 % to only 51 %. The changes in blood pressure
of the rats in each group showed little individual variab-
ility, such that the results were very consistent and the
standard errors of the means were always very small.
By the end of 8 weeks, it was evident that the use of
the 100 mg/kg dose of the leaf extract had completely
prevented the rise in blood pressure induced by L-
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NAME (Fig. 1) as shown by the lack of statistical signifi-
cance at p < 0.05 between that group and the normal
control one.
Treatment with L-NAME, with or without the conco-
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-
week experimental period, the increase in weight ran-
ging from 3750 %.
3.2. Effect of the olive leaf extract administration
in hypertensive rats
In this experiment, 20 rats were rendered hypertensive
by daily treatment with L-NAME for a 12-week period.
The average blood pressure of the animals at the begin-
ning of the experiment was 109 mmHg but reached 204
by the end of 12 weeks, constituting a rise of 87 %.
Six weeks after initiating treatment, the average BP had
reached 160 mmHg constituting a rise of about 46 %. In
a separate group, another 20 rats were started on the
olive leaf extract in a daily oral administration of 100
mg/kg after 6 weeks of L-NAME treatment, whilst con-
tinuing treatment with L-NAME. The blood pressure of
the animals which had risen by 46 % after 6 weeks of
treatment with L-NAME alone, started to drop progres-
sively over the next 6 weeks to reach an average of 125
mmHg at the end of the experimental period in spite of
the continued administration of L-NAME, constituting
only a 12 % rise from the initial values, thereby signal-
ling normalisation of the blood pressure.
It was noticed that treatment with the olive leaf ex-
tract produced a dramatic effect within the first 2 weeks
of administration to the hypertensive animals, i.e. by
week 8, but the effect was not maintained because of
the continued administration of L-NAME. The effects of
giving L-NAME alone for 12 weeks compared to those
where treatment with the leaf extract was instituted at
the end of the sixth week are shown graphically in Fig. 2.
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].

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

800 Khayyal et al. Olive leaf extract EFLA 943

Rats that are chronically treated with the orally active
nitric oxide synthase inhibitor, L-NAME, have been
shown to develop a marked hypertension [17, 18] as a
result of chronic blockade of nitric oxide synthesis [19].
The model of using L-NAME to induce experimental
hypertension has become a widely accepted method for
testing antihypertensive agents. There is evidence to
suggest that 7 days after treatment with L-NAME activa-
tion of the sympathetic nervous system contributes to
the development of the high blood pressure [20]. More
recent observations have shown that moderate inhibi-
tion of nitric oxide synthase by L-NAME does not seem
to influence the cardiac sympathetic tone. In our study,
the heart rates of animals treated up to 12 weeks with
L-NAME did not show significant changes from con-
trol values.
The present findings indicate that the extract of olive
leaf had a definite dose-dependent prophylactic effect
in preventing the rise in pressure induced by L-NAME,
when both agents were given concomitantly. The best
dose which almost completely prevented the rise in
blood pressure was 100 mg/kg of the extract. Accord-
ingly, that dose of the extract was used in an effort to
see whether giving the extract to animals with estab-
lished hypertension would still be capable of reducing
the high pressure in the presence of L-NAME. The rats
were allowed to develop a conspicuous hypertension
after treatment with L-NAME for 6 weeks, then initiated
treatment with the extract orally for a further 6 weeks,
without stopping the administration of L-NAME. The
extract normalised the blood pressure by the end of the
experimental period, proving once more its efficacy as
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
with L-NAME.
It has been shown that L-NAME induced hyperten-
sion may not only be associated with inactivation of
nitric oxide, but also with generation of free oxygen spe-
cies [21]. The same applies to other models of hyperten-
sion [22]. Evidence shows that oxidative stress induced
by glutathione depletion can cause severe hypertension
in normal rats associated with disturbance in the NO
system [23], and that this can be overcome by the ad-
ministration of free radical scavengers. Since olive leaf
has been demonstrated to possess good anti-oxidant
properties [24, 25], it may well be that the observed ef-
fects may be related to such an action.
It was further demonstrated that aortic vascular re-
activity changes are induced by hypertension caused by
chronic L-NAME administration [26, 27] and that such
reactivity changes can be prevented by some angioten-
sin converting enzyme inhibitors. It has been reported
that the antihypertensive effect of olive leaf might be
associated with inhibiting angiotensin converting en-
zyme [28], a fact which could help to explain the reduc-
Arzneim.-Forsch./Drug Res. 52, No. 11, 797802 (2002)
Antihypertensives
tion in morbidity and mortality in our present experi-
ments. The angiotensin converting enzyme (ACE) in-
hibitor effect of olive leaf, however, seems not to be as-
sociated with oleuropein per se, but with another active
constituent, oleacein [29]. Furthermore, oleuropein
could be cleaved by -glucosidase to a derivative with
high ACE inhibitor activity. High blood pressure is
known to induce remodelling in cardiac and vascular
tissue, which are deleterious to health, possibly through
the involvement of angiotensin and aldosterone. Such
changes are known to be reversed at an early stage of
development by drugs that interfere with the action of
angiotensin, such as the converting enzyme inhibitors.
Olea europaea has also been found to contain an
active constituent, -(3,4-dihydroxy-phenyl)ethanol,
which was found to have calcium channel blocking ac-
tivity [30]. ACE inhibitors combined with calcium chan-
nel blockers exert a synergistic antihypertensive effect
[31]. Combination therapy using agents with different
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mechanisms of action usually results in reducing overall
toxicity and side effects.
It would therefore seem from the present findings
that the beneficial antihypertensive effect of the olive
leaf extract, EFLA 943, could be due to a variety of fac-
tors acting simultaneously. The activity of the stabilised
extract lies probably in its content of oleuropein acting
synergistically with other active principles to exert anti-
oxidant, ACE inhibitory and possibly calcium channel
blocking activities, all of which may contribute to the
efficacy and good tolerability of the extract. Further
studies are under way to further elucidate its mechan-
ism of action.
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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