Planta Med. 57(199]) 417
Vasodilator Effect of Olive Leaf A. Zarzuelo1, .1 Duarfe1', J. Jiménez', M. Gonzalez', andM. P. Utrilla' 'Department of Pharmacology, School of Pharmacy, University of Granada, E-18071 Granada, Spain 2 Address for correspondence Received: July 13, 1990
We studied the importance of the smooth vascular muscle endothelium in the vasodilator action of the decoction of olive (Olea europaea) leaf The decoction caused relaxation of isolated rat aorta preparations both in the presence (IC50 1.12 0.33 mg/ml) and in the absence (ICse 1.67 0.16 mg/mi) of endothelium. The results indicate that the relaxant activity of the lyophilized decoction is independent of the integrity of the vascular
Materials and Methods Plant material The plant material consisted of leaves of Olea europaea L. (Oleaceae) collected in November1988 in Martos (province of Jadn, Spain). The sample, upon arrival in the laboratory, was powdered and frozen at —10°C until use.
Preparation of extracts A portion of the powdered leaves was used to prepare a decoction (10), which was subsequently lyophilized
endothelium. We also showed that oleuropeoside is a
(yield 39.7%) and refrigerated at 4°C until use.
component responsible for vasodilator activity but, from the results, it seems likely that at least one other principle is to be found in the olive leaf which is either a vaso dilator
crude oleuropeoside (4) (yield 1.19%). We determined the
itself or else potentiates the relaxant effect of oleuropeoside.
Key words Olea europaea, vasodilatation, oleuropeoside, aorta preparations.
Introduction
The other portion of the leaves was used to obtain
oleuropeoside content in the crude residue (yield 48%) by HPLC with a Waters apparatus, using an RP-18 column (25cm x 2.5 cm), a Perkin-Elmer LC-235 diode array detector at 230 nm, and MeOHH20 (7: 3) as the mobile phase at a flow rate of 1 mI/mm, using an
internal standard, pure oleuropeoside.
Hypotensive activity assay Male Wistar rats weighing approximately 200 g were anesthetized with ether. The right jugular vein and the left carotid artery were cannulated for sample administration and blood pressure recording respectively. The latter was recorded continuously by means of a Bell and Howell pressure transducer coupled to a Beckman Dynograph type R41 1 recorder. Twentyfour
The hypotensive action of olive leaf (Olea europaea) has been used for sometime, both empirically as well as in experimental and clinical studies (1, 2, 3). At the
beginning of the 20th century a substance named
oleuropeoside (4) was isolated from olive leaves and credited with the hypotensive action of the leaf decoction (5, 6, 7, 8).
Vasodilator agents are known to act both directly on smooth vascular muscle or to facilitate the release of relaxant factors, upon acting on the endothelium of
hours later, the decrease in diastolic pressure was determined after the i.e. administration of the different doses of the lyophilized decoction. In all cases 0.05 ml fluidIlOO g body weight was injected.
Determination of vasodilator capacity in the rat thoracic aorta with and without endothelium The vasodilator action of the sample was determined in isolated vessels from adult male Wistar rats. Thoracic aortas were excised and fat and connective tissue were removed taking special care to leave the endothelium intact. Spiral (0.3 2cm) strips were cut and suspended in a standard muscle bath
smooth muscle. Among these latter agents are included acetylcholine and its derivatives, which through their ac- chamber in physiologic salt solution of the following composition mM): NaC1 118, KC1 4.7, KH,P04 1.18, Mg504. 7 H,0 1.17, tion on the endothelium favor the release of relaxant fac- (in CaC1,- 2 H,0 1.6, NaHCO, 25.0, and glucose 11.1. In some spiral tors, which in turn stimulate guanylcyclase, and thus in- strips the endothelium was mechanically removed by gently rubcrease the level of cGMP, the agent ultimately responsible for vascular relaxation (9).
bing the intimal surface (11, 12). The strips were maintained at 37°C and aerated with 95% 02, 5% CO2. Vascular strips were attached to a force transducer (Letigraph 2000, Letica) to measure
In this study we set out to confirm the
isometric contraction force. The strips were maintained at a resting tension of 2 g throughout the experiments. After a 1 h equilibration
hypotensive action of olive leaf, and to determine the role of the endothelium in the vascular muscle relaxation caused by this drug. We also aimed to determine whether the relaxant action was related with that of oleuropeoside.
the strips were precontracted with iO M phenylephrine. When the contraction reached a plateau, cumulative concentration-response curves were generated for the sample.
Downloaded by: University of Arizona Library. Copyrighted material.
Abstract
418 Planta Med. 57(7991)
A. Zarzuelo et at
The presence or absence of the endothelium was tested by the ability of acetyicholine to elicit relaxation, as well as by nitroprusside staining at the end of the experiment in selected tissues (13).
of 25—50mg/kg (basal diastolic blood pressure: 89 6mm Hg).
Relaxant effect in isolated aorta
The decrease in tension in response to these
Table 2 presents the IC50 values and
agents was based on the percent of maximal contraction obtained with phenylephrine.
maximum effect obtained with acetylcholine, nitroprusside and lyophilized decoction against phenylephrine-induced contractions (107M) in the presence and in the absence of endothelium. Both nitroprusside and lyophilized olive leaf
The IC55 values were calculated for each strip by
hnear interpolation between the two concentration evoking responses just above and just below 50% of the maximum. The data are expressed as mean S.E.M. obtained from 'n" rats. Statistical
comparisons of data, with and without endothelium were made using Student's t test for paired comparisons. P values less than 0.05 were considered statistically significant.
Drugs The following agents were used: lyophilized de-
coction, acetyicholine chloride (Merck), sodium nitroprusside (Merck), phenylephrine chloride (Landerland), oleuropeoside (Sigma), vasopressin (Sigma). Drugs were dissolved in distilled
were active in aorta with and without endothelium,
whereas acetylcholine was active only in aorta with endothelium.
Table 3 shows the IC50 values and maximum effect for the lyophilized leaf decoction, crude oleuropeoside and oleuropeoside against phenylephrine (10-i M) and vasopressin (10-i M) induced contractions in isolated rat aorta with endothelium.
Discussion
Lyophilized olive leaf showed a marked hypotensive activity when administered intravenously to normotensive rats. This effect may be due to the relaxation of the vascular smooth muscle.
water such that volumes of less than 0.1 ml were added to the organ
chamber. Concentrations of drugs were expressed as the final molar concentration in the bath solution, except for the lyophilized decoction, as its molecular weight is unknown.
Results
The vascular endothelium has been shown by Furchgott and Zawadzki to be of crucial importance in the relaxation of blood vessels in response to acetylcholine and some other naturally occurring vasodilator substances (8, 9). It has likewise been shown that some antihypertensive vasodbators such as hydralazine also require integrity of the endothelium to show activity (13). The results of the
Hypotensive effect
Table 1 summarizes the decreases in arterial pressure recorded after the administration of increasing doses of lyophilized olive leaf decoction. The hypotensive effect appeared from a dose of 2.5 mg/kg, and produced a maximum reduction of approximately 30mm Hg at doses
present study indicate that the relaxant activity of lyophilized decoction of olive leaf on smooth vascular mus-
Table
cle is independent of the integrity of the vascular en-
Hypotensive effect of the lyophilized decoction; each value represents the mean S.E.M. obtained from 5 rats. 1
dothelium, as we found no statistically significant difference between the IC50 values or maximum effects obtained in the presence and in the absence of endothelium. This contrasts with the findings for the relaxant action of acetylcholine, which required an intact endothelium. Our findings thus rule out a possible role of choline esters in the
Decrease in Arterial Pressure (mm Hg)
Dose (mg/kg)
0 1.1±0.84
0.25 2.5 12.5 25.0
15.8±3.13 29.4 3.27 30.8 2.86
50.0
hypotensive action of olive leaL in contrast to earlier reports (14, 15).
With Drug Decoction olive Acetylcholine Sodium Nitroprusside
IC55
1.12 4.09 1.22
Ema.
0.37 (mg/mI)
0.41 (x io M) 0.33 (x 10_B M)
70.0 75.5 100.0
IC55 (mg/mi)
Lyophilized decoction Crude oleuropeoside Pure oleuropeoside
1.12 1.07
0.55
0.37 0.22 0.09
Em..
IC55
5.2 1.16 0.16 (mg/mI) — 4.2 0.0 1.51 0.23 (x i05
En.. 70.00 78.29
80.10
IC55 (mg/mi)
5.20 5.39 8.20
2.29 0.87
0.51
0.13
0.10 0.03
sents the mean
S.E.M. obtained from 6 rats.
79.4 9.8 —
M( 100.0 0.0
Table 3 IC55 values and maximum effect of
Vasopressin
Phenylephrine
Drug
Table 2 Relaxant effect in the rat thoracic aorta with and without endothelium; each value repre-
Without
lyophilized decoction of O/ea europaea leaf, crude Em.,
77.70 77.82
5.42 6.19
68.35 8.14
oleuropeoside, and pure oleuropeoside against phenylephrine )10 M) and vasopressin )10 M). Each value represents the mean S.E.M. obtained from 6 rats.
Downloaded by: University of Arizona Library. Copyrighted material.
Calculations
Planta Med. 57(1991) 419
Vasodilator Effect of Olive Leaf
References
The values of IC50 for lyophilized leaf decoc-
1 Leclerc, H., Decaux, F., Valery-Leclerc, H. (1954) Rev. de Phytother. 18, 7. 2 Capretti, G. (1948) Giorn di Gun. Med. 29, 394.
Capretti, G. (1948) Giorn di Clin. Med. 29, 491. Panizzi, L., Scarpati, M. L., Oriente, G. (1960) Gazz. Chim. hal. 90,
least one other principle is to be found in the olive leaf which
is either a vasodilator itself or else potentiates the relaxant effect of oleuropeoside. If oleuropeoside were the only active principle in the lyophilized decoction, no differentiation should occur when the IC50 values obtained against different agonists (phenylephrine and vasopressin) are compared. The results obtained show that the IC50 ratio between lyophilized decoction and pure oleuropeoside are different against phenylephrine (2.03) and vasopressin (22.9). So, these results confirm the presence, at least, of another component.
Acknowledgements We thank Ms Karen Shashok for translating pans of the original manuscript into English.
1449. Esdorn, J. (1954) Planta Med. 5, 145. 6
10
12 13 14
Esdorn,J.(1955)ActaPhytother. 1,10,13. Schwarz, F. K. T. (1954) Medizinische 41, 1387. Stegmann, K. (1955) Landarzt 15, 375. Furchgott, H. F., Vanhoutte, P. M. (1989) FASEB 3, 2007. Farmacopea Española IX Ed. (1954) Real Academia Nacional de Medicina p. 396. De Mey, J. G., Venhoutte, P.M. (1981) J. Physiol. 316, 347. Katusic, Z. S., Shepherd, J. T., Vanhootte, P. M. (1984) Circulation lIes. 55, 575. Spokas, E. G., Folco, G., Quilley, J., Chander, P., McGiff, J. C. (1983) Hypertension 5, 1-107. Samuelsson, G. (1964) in Piante Medicinali, Vol. 2, (Benigni, B., Capra, C., Cattorini, P. E., eds.), p. 1019, Inverni Della Beffa, Milano. Janku, J., Raskova, H., Moth, 0., Blazek, Z. (1957) Arch. intern. pharmacodyn. ther. 112, 342.
Downloaded by: University of Arizona Library. Copyrighted material.
tion and crude oleuropeoside against phenylephrine (1.12 0.33 mg/mi and 1.074 0.22 mg/mi, respectively) were found to be similar. As crude oleuropeoside makes up only 1.2% of the lyophilized decoction, it seems likely that at
Vasodilator Effect of Olive Leaf A. Zarzuelo1, .1 Duarfe1', J. Jiménez', M. Gonzalez', andM. P. Utrilla' 'Department of Pharmacology, School of Pharmacy, University of Granada, E-18071 Granada, Spain 2 Address for correspondence Received: July 13, 1990
We studied the importance of the smooth vascular muscle endothelium in the vasodilator action of the decoction of olive (Olea europaea) leaf The decoction caused relaxation of isolated rat aorta preparations both in the presence (IC50 1.12 0.33 mg/ml) and in the absence (ICse 1.67 0.16 mg/mi) of endothelium. The results indicate that the relaxant activity of the lyophilized decoction is independent of the integrity of the vascular
Materials and Methods Plant material The plant material consisted of leaves of Olea europaea L. (Oleaceae) collected in November1988 in Martos (province of Jadn, Spain). The sample, upon arrival in the laboratory, was powdered and frozen at —10°C until use.
Preparation of extracts A portion of the powdered leaves was used to prepare a decoction (10), which was subsequently lyophilized
endothelium. We also showed that oleuropeoside is a
(yield 39.7%) and refrigerated at 4°C until use.
component responsible for vasodilator activity but, from the results, it seems likely that at least one other principle is to be found in the olive leaf which is either a vaso dilator
crude oleuropeoside (4) (yield 1.19%). We determined the
itself or else potentiates the relaxant effect of oleuropeoside.
Key words Olea europaea, vasodilatation, oleuropeoside, aorta preparations.
Introduction
The other portion of the leaves was used to obtain
oleuropeoside content in the crude residue (yield 48%) by HPLC with a Waters apparatus, using an RP-18 column (25cm x 2.5 cm), a Perkin-Elmer LC-235 diode array detector at 230 nm, and MeOHH20 (7: 3) as the mobile phase at a flow rate of 1 mI/mm, using an
internal standard, pure oleuropeoside.
Hypotensive activity assay Male Wistar rats weighing approximately 200 g were anesthetized with ether. The right jugular vein and the left carotid artery were cannulated for sample administration and blood pressure recording respectively. The latter was recorded continuously by means of a Bell and Howell pressure transducer coupled to a Beckman Dynograph type R41 1 recorder. Twentyfour
The hypotensive action of olive leaf (Olea europaea) has been used for sometime, both empirically as well as in experimental and clinical studies (1, 2, 3). At the
beginning of the 20th century a substance named
oleuropeoside (4) was isolated from olive leaves and credited with the hypotensive action of the leaf decoction (5, 6, 7, 8).
Vasodilator agents are known to act both directly on smooth vascular muscle or to facilitate the release of relaxant factors, upon acting on the endothelium of
hours later, the decrease in diastolic pressure was determined after the i.e. administration of the different doses of the lyophilized decoction. In all cases 0.05 ml fluidIlOO g body weight was injected.
Determination of vasodilator capacity in the rat thoracic aorta with and without endothelium The vasodilator action of the sample was determined in isolated vessels from adult male Wistar rats. Thoracic aortas were excised and fat and connective tissue were removed taking special care to leave the endothelium intact. Spiral (0.3 2cm) strips were cut and suspended in a standard muscle bath
smooth muscle. Among these latter agents are included acetylcholine and its derivatives, which through their ac- chamber in physiologic salt solution of the following composition mM): NaC1 118, KC1 4.7, KH,P04 1.18, Mg504. 7 H,0 1.17, tion on the endothelium favor the release of relaxant fac- (in CaC1,- 2 H,0 1.6, NaHCO, 25.0, and glucose 11.1. In some spiral tors, which in turn stimulate guanylcyclase, and thus in- strips the endothelium was mechanically removed by gently rubcrease the level of cGMP, the agent ultimately responsible for vascular relaxation (9).
bing the intimal surface (11, 12). The strips were maintained at 37°C and aerated with 95% 02, 5% CO2. Vascular strips were attached to a force transducer (Letigraph 2000, Letica) to measure
In this study we set out to confirm the
isometric contraction force. The strips were maintained at a resting tension of 2 g throughout the experiments. After a 1 h equilibration
hypotensive action of olive leaf, and to determine the role of the endothelium in the vascular muscle relaxation caused by this drug. We also aimed to determine whether the relaxant action was related with that of oleuropeoside.
the strips were precontracted with iO M phenylephrine. When the contraction reached a plateau, cumulative concentration-response curves were generated for the sample.
Downloaded by: University of Arizona Library. Copyrighted material.
Abstract
418 Planta Med. 57(7991)
A. Zarzuelo et at
The presence or absence of the endothelium was tested by the ability of acetyicholine to elicit relaxation, as well as by nitroprusside staining at the end of the experiment in selected tissues (13).
of 25—50mg/kg (basal diastolic blood pressure: 89 6mm Hg).
Relaxant effect in isolated aorta
The decrease in tension in response to these
Table 2 presents the IC50 values and
agents was based on the percent of maximal contraction obtained with phenylephrine.
maximum effect obtained with acetylcholine, nitroprusside and lyophilized decoction against phenylephrine-induced contractions (107M) in the presence and in the absence of endothelium. Both nitroprusside and lyophilized olive leaf
The IC55 values were calculated for each strip by
hnear interpolation between the two concentration evoking responses just above and just below 50% of the maximum. The data are expressed as mean S.E.M. obtained from 'n" rats. Statistical
comparisons of data, with and without endothelium were made using Student's t test for paired comparisons. P values less than 0.05 were considered statistically significant.
Drugs The following agents were used: lyophilized de-
coction, acetyicholine chloride (Merck), sodium nitroprusside (Merck), phenylephrine chloride (Landerland), oleuropeoside (Sigma), vasopressin (Sigma). Drugs were dissolved in distilled
were active in aorta with and without endothelium,
whereas acetylcholine was active only in aorta with endothelium.
Table 3 shows the IC50 values and maximum effect for the lyophilized leaf decoction, crude oleuropeoside and oleuropeoside against phenylephrine (10-i M) and vasopressin (10-i M) induced contractions in isolated rat aorta with endothelium.
Discussion
Lyophilized olive leaf showed a marked hypotensive activity when administered intravenously to normotensive rats. This effect may be due to the relaxation of the vascular smooth muscle.
water such that volumes of less than 0.1 ml were added to the organ
chamber. Concentrations of drugs were expressed as the final molar concentration in the bath solution, except for the lyophilized decoction, as its molecular weight is unknown.
Results
The vascular endothelium has been shown by Furchgott and Zawadzki to be of crucial importance in the relaxation of blood vessels in response to acetylcholine and some other naturally occurring vasodilator substances (8, 9). It has likewise been shown that some antihypertensive vasodbators such as hydralazine also require integrity of the endothelium to show activity (13). The results of the
Hypotensive effect
Table 1 summarizes the decreases in arterial pressure recorded after the administration of increasing doses of lyophilized olive leaf decoction. The hypotensive effect appeared from a dose of 2.5 mg/kg, and produced a maximum reduction of approximately 30mm Hg at doses
present study indicate that the relaxant activity of lyophilized decoction of olive leaf on smooth vascular mus-
Table
cle is independent of the integrity of the vascular en-
Hypotensive effect of the lyophilized decoction; each value represents the mean S.E.M. obtained from 5 rats. 1
dothelium, as we found no statistically significant difference between the IC50 values or maximum effects obtained in the presence and in the absence of endothelium. This contrasts with the findings for the relaxant action of acetylcholine, which required an intact endothelium. Our findings thus rule out a possible role of choline esters in the
Decrease in Arterial Pressure (mm Hg)
Dose (mg/kg)
0 1.1±0.84
0.25 2.5 12.5 25.0
15.8±3.13 29.4 3.27 30.8 2.86
50.0
hypotensive action of olive leaL in contrast to earlier reports (14, 15).
With Drug Decoction olive Acetylcholine Sodium Nitroprusside
IC55
1.12 4.09 1.22
Ema.
0.37 (mg/mI)
0.41 (x io M) 0.33 (x 10_B M)
70.0 75.5 100.0
IC55 (mg/mi)
Lyophilized decoction Crude oleuropeoside Pure oleuropeoside
1.12 1.07
0.55
0.37 0.22 0.09
Em..
IC55
5.2 1.16 0.16 (mg/mI) — 4.2 0.0 1.51 0.23 (x i05
En.. 70.00 78.29
80.10
IC55 (mg/mi)
5.20 5.39 8.20
2.29 0.87
0.51
0.13
0.10 0.03
sents the mean
S.E.M. obtained from 6 rats.
79.4 9.8 —
M( 100.0 0.0
Table 3 IC55 values and maximum effect of
Vasopressin
Phenylephrine
Drug
Table 2 Relaxant effect in the rat thoracic aorta with and without endothelium; each value repre-
Without
lyophilized decoction of O/ea europaea leaf, crude Em.,
77.70 77.82
5.42 6.19
68.35 8.14
oleuropeoside, and pure oleuropeoside against phenylephrine )10 M) and vasopressin )10 M). Each value represents the mean S.E.M. obtained from 6 rats.
Downloaded by: University of Arizona Library. Copyrighted material.
Calculations
Planta Med. 57(1991) 419
Vasodilator Effect of Olive Leaf
References
The values of IC50 for lyophilized leaf decoc-
1 Leclerc, H., Decaux, F., Valery-Leclerc, H. (1954) Rev. de Phytother. 18, 7. 2 Capretti, G. (1948) Giorn di Gun. Med. 29, 394.
Capretti, G. (1948) Giorn di Clin. Med. 29, 491. Panizzi, L., Scarpati, M. L., Oriente, G. (1960) Gazz. Chim. hal. 90,
least one other principle is to be found in the olive leaf which
is either a vasodilator itself or else potentiates the relaxant effect of oleuropeoside. If oleuropeoside were the only active principle in the lyophilized decoction, no differentiation should occur when the IC50 values obtained against different agonists (phenylephrine and vasopressin) are compared. The results obtained show that the IC50 ratio between lyophilized decoction and pure oleuropeoside are different against phenylephrine (2.03) and vasopressin (22.9). So, these results confirm the presence, at least, of another component.
Acknowledgements We thank Ms Karen Shashok for translating pans of the original manuscript into English.
1449. Esdorn, J. (1954) Planta Med. 5, 145. 6
10
12 13 14
Esdorn,J.(1955)ActaPhytother. 1,10,13. Schwarz, F. K. T. (1954) Medizinische 41, 1387. Stegmann, K. (1955) Landarzt 15, 375. Furchgott, H. F., Vanhoutte, P. M. (1989) FASEB 3, 2007. Farmacopea Española IX Ed. (1954) Real Academia Nacional de Medicina p. 396. De Mey, J. G., Venhoutte, P.M. (1981) J. Physiol. 316, 347. Katusic, Z. S., Shepherd, J. T., Vanhootte, P. M. (1984) Circulation lIes. 55, 575. Spokas, E. G., Folco, G., Quilley, J., Chander, P., McGiff, J. C. (1983) Hypertension 5, 1-107. Samuelsson, G. (1964) in Piante Medicinali, Vol. 2, (Benigni, B., Capra, C., Cattorini, P. E., eds.), p. 1019, Inverni Della Beffa, Milano. Janku, J., Raskova, H., Moth, 0., Blazek, Z. (1957) Arch. intern. pharmacodyn. ther. 112, 342.
Downloaded by: University of Arizona Library. Copyrighted material.
tion and crude oleuropeoside against phenylephrine (1.12 0.33 mg/mi and 1.074 0.22 mg/mi, respectively) were found to be similar. As crude oleuropeoside makes up only 1.2% of the lyophilized decoction, it seems likely that at
