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Author's personal copy Journal of Ethnopharmacology 132 (2010) 200–205
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Olive (Olea europaea L.) leaf extract elicits antinociceptive activity, potentiates morphine analgesia and suppresses morphine hyperalgesia in rats Saeed Esmaeili-Mahani a,∗ , Maryam Rezaeezadeh-Roukerd a , Khadije Esmaeilpour a , Mehdi Abbasnejad a , Bahram Rasoulian b , Vahid Sheibani c , Ayat Kaeidi a,b , Zahra Hajializadeh c a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khoramabad, Iran c Laboratory of Molecular Neuroscience, Kerman Neuroscience Research Center (KNRC), Kerman University of Medical Sciences, Kerman, Iran b
a r t i c l e
i n f o
Article history: Received 4 February 2010 Received in revised form 19 July 2010 Accepted 9 August 2010 Available online 14 August 2010 Keywords: Olive leaf extract Antinociception Tail-flick Hot-plate Formalin test Rats
a b s t r a c t Aim of the study: Olive (Olea europaea) leaves are used as anti-rheumatic, anti-inflammatory, antinociceptive, antipyretic, vasodilatory, hypotensive, antidiuretic and hypoglycemic agents in traditional medicine. Recently, it has been shown that olive leaf extract (OLE) has calcium channel blocker property; however, its influences on nociceptive threshold and morphine effects have not yet been clarified. Materials and methods: All experiments were carried out on male Wistar rats. The tail-flick, hot-plate and formalin tests were used to assess the effect of OLE on nociceptive threshold. To determine the effect of OLE on analgesic and hyperalgesic effects of morphine, OLE (6, 12 and 25 mg/kg i.p.) that had no significant nociceptive effect, was injected concomitant with morphine (5 mg/kg and 1 g/kg i.p., respectively). The tail-flick test was used to assess the effect of OLE on anti- and pro-nociceptive effects of morphine. Results: The data showed that OLE (50–200 mg/kg i.p.) could produce dose-dependent analgesic effect on tail-flick and hot-plate tests. Administration of 200 mg/kg OLE (i.p.) caused significant decrease in pain responses in the first and the second phases of formalin test. In addition, OLE could potentiate the antinociceptive effect of 5 mg/kg morphine and block low-dose morphine-induced hyperalgesia. Conclusion: Our results indicate that olive leaf extract has analgesic property in several models of pain and useful influence on morphine analgesia in rats. Therefore, it can be used for the treatment and/or management of painful conditions. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The management of pain is considered to be a major clinical problem. Opioids have been used for treating moderate to severe pain, but treatment with these drugs leads to the induction of side effects such as analgesic tolerance, physical dependence, emesis, constipation and drowsiness. Therefore, the finding of herbs that have analgesic property without hazardous side effects is essential skills for pain management. The olive tree (Olea europaea L. [Family: Oleaceae]) has been cultivated in the Mediterranean for more than a thousand years. Not only the olive oil, but also the leaves have been used for medi-
∗ Corresponding author at: Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, P.O. Box: 76135-133, Kerman, Iran. Tel.: +98 341 3222032; fax: +98 341 3222032. E-mail address:
[email protected] (S. Esmaeili-Mahani). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.08.013
cal purposes, and were introduced recently into the Pharmacopoea PhEur 5. In many countries, they are known as a folk remedy for hypertension and diabetes (Cherif et al., 1996). It has been traditionally used to cure rheumatic and neuralgic diseases in Lebenan (El Beyrouthy et al., 2008) and also to alleviate muscle and joint pain in some regions of Iran. Olive leaf contains the active iridoid constituent oleuropein (chief constituent). Other secoiridoids include 11-demethyloleuropein, 7,11-dimethyl ester of oleoside, ligustroside, oleuroside, and un-conjugated secoiridoid aldehydes. Triterpenes and flavonoids, including luteolin, apigenin, rutin, and diosmetin, are also present. Oleasterol, leine, and glycoside oleoside have also found in the leaves (Briante et al., 2002). It has been documented that olive leaf extract had Ca2+ channelblocking activity (Gilani et al., 2005; Scheffler et al., 2008). Several investigators have reported that calcium ion has a physiological role in the regulation of pain sensitivity, and inhibition of calcium movement contributes to antinociception (Galeotti et al., 2004;
Author's personal copy S. Esmaeili-Mahani et al. / Journal of Ethnopharmacology 132 (2010) 200–205
Weiss and De Waard, 2006). Furthermore, L-type Ca2+ channel antagonists produce analgesia after peripheral and central administration (Miranda et al., 1993; Esmaeili-Mahani et al., 2006). Due to the fact that calcium influx blockade is essential for normal opioid receptor signaling, Ca2+ channel antagonists have been shown to elevate antinociceptive effect of morphine (Fukuizumi et al., 2003; Esmaeili-Mahani et al., 2005) and suppress morphineinduced hyperalgesia (Esmaeili-Mahani et al., 2008). Based on the facts that calcium influx is essential for pain perception and its blockade is necessity for normal opioid receptor signaling, the present study was designed to test the hypothesis that olive leaf extract could exert antinociceptive effects on chemical and thermal models of pain and influence morphine analgesic/hyperalgesic properties in rats. 2. Materials and methods 2.1. Animals All experiments were carried out on male Wistar rats, weighing 200–250 g, that were housed four per cage under a 12 h light/dark cycle in a room with controlled temperature (22 ± 1 ◦ C). Food and water were available ad libitum. Animals were handled daily (between 9:00 and 10:00 A.M) for 3 days, before the experiment day in order to adapt them to manipulation and minimize nonspecific stress responses. Rats were divided randomly into several experimental groups, each comprising 6–8 animals. All experiments followed the guidelines on ethical standards for investigation of experimental pain in animals (Zimmermann, 1983), and were approved by the Animal Experimentation Ethic Committee of Kerman Neuroscience Research Center (EC/KNRC/89-101-2). 2.2. Preparation of olive leaf extract An ethanolic olive (Olea europaea; variety of Sevillano) leaf dry extract was prepared in Razi Herbal Medicines Research Center (Lorestan, Iran). There are different varieties of olive trees in some parts of Iran but the variety of Sevillano has the maximum oleuropein level (Hashemi et al., 2010). Olive leaves of Sevillano variety were collected from the Khoramabad Agricultural Research Orchard, Lorestan province, Iran, in August 2009. A sample was deposited at the herbarium of Lorestan Agricultural and Natural Resources Research Center with reference number 11505. Two hundred grams of the air-dried leaves was grinded into fine powder. The powder was extracted twice, on each occasion with 1 l of 80% ethyl alcohol. The collective ethanol extract was filtered, and the filtrate was concentrated to dryness under reduced pressure in a rotary evaporator and the resulting ethanol extract was freeze-dried. Quantification of some identified compounds of the extract using high performance liquid chromatography (HPLC) by our colleagues showed that oleuropein (356 mg/g), tyrosol (3.73), hydroxy tyrosol (4.89) and caffeic acid (49.41) were the main compositions of the olive leaf extract (Mohagheghi et al., 2010). The same extract was also used in this study. 2.3. Drugs Aliquot portions of the crude olive leaf extract were weighed and dissolved in physiological saline plus dimethyl sulfoxide (DMSO) for use on each day of our experiments. The percentages of DMSO and saline in the final volume were 2% and 98%, respectively. Morphine hydrochloride (TEMAD, Iran) was dissolved in physiological saline. These drugs were given in the volume of 1 ml/kg (i.p.) and in a total volume of 10 l (i.t.).
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2.4. Intrathecal catheter implantation and drug delivery Animals were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) i.p. An intrathecal catheter (PE-10) was implanted in each rat according to a previously published method (Yaksh and Rudy, 1976). Animals that exhibited neurological deficits (e.g., paralysis) after the catheter implantation or during drug delivery were excluded from the experiments. 2.5. Tail-flick test Antinociception was assessed by tail-flick test (D’Amour and Smith, 1941). The tail-flick latency for each rat was determined three times and the mean was designated as baseline latency before drug injection. The intensity of the beam was adjusted to produce mean control reaction time between 2 and 4 s. The cut-off time was fixed at 10 s in order to avoid any damage to the tail. After determination of baseline latencies, rats received intraperitoneal injection of drugs, and the reaction latency was determined in different times after injection. The tail-flick latencies were converted to the percentage of antinociception according to the following formula: %Antinociception (%MPE) =
reaction time of test − basal reaction time . cut-off time − basal reaction time
Hyperalgesia was assessed by a small modification in tail-flick test (Esmaeili-Mahani et al., 2007). The intensity of the beam was adjusted to produce a mean control reaction time between 4 and 6 s and the cut-off time was fixed at 15 s. In this manner, we were able to reveal potential, subtle alternations that may occur in basal thermal nociception. Experimentally induced decreases in control tail-flick latency provide an indication of hyperalgesic effect. 2.6. Hot-plate test Rats were individually placed on a hot-plate maintained at 55 ± 0.2 ◦ C and the time of licking of the hind paws or attempt to jump out of the beaker was recorded as the latency period. The cut-off time was 60 s to avoid tissue damage. Before drug administration, baseline latency was examined. The paw withdrawal latency was tested after drug administration. The maximum possible effect (MPE) was calculated as: MPE% = (latency after drug administration − baseline latency)/(60 − baseline latency) × 100. 2.7. Formalin test The formalin test was carried out as described by Dubuisson and Dennis (1977). 10 l of a 5% formalin solution in saline was injected in the dorsal surface of the left hind paw using a tuberculin syringe. Each animal was then placed in an observation chamber and monitored for 1 h. Severity of pain responses was recorded based on the following scale: (0) rats walked or stood firmly on injected paw; (1) the injected paw was favored or partially elevated; (2) the injected paw was clearly lifted off the floor; (3) the rats licked, chewed or shook the injected paw. This method of scoring allows a graded determination of responses thus showing finer degrees of antinociception as opposed to the method in which only the time the animal spent licking the injected paw is recorded. Antinociceptive effect was determined in two phases, an early acute phase and a late or tonic phase (0–5 and 15–60 min after formalin injection, respectively).
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Table 1 Antinociceptive effects of intraperitoneal (i.p.) and intrathecal (i.t.) olive leaf extract (OLE) on tail-flick test in rats. Experimental groups
Antinociception (%MPE) in different times after injection 15 min
30 min
45 min
60 min
Vehicle (i.p.) OLE (50 mg/kg i.p.) OLE (100 mg/kg i.p.) OLE (200 mg/kg i.p.)
1.9 18.02 24.73 32.5
± ± ± ±
2.2 5.9* 1.54*** 1.9***,++
1.2 0.89 11.89 40.92
± ± ± ±
1.66 0.92 3.1 2.5***,+++,###
2.6 1.5 9.61 18.61
± ± ± ±
1.6 0.91 1.9 2.2**,++
Vehicle (i.t.) OLE (100 g i.t.) OLE (200 g i.t.) OLE (400 g i.t.)
2.66 37.87 55.14 63.04
± ± ± ±
3.7 5.86*** 4.1*** 2.2***,++
3.1 9.8 47.25 92.42
± ± ± ±
1.67 5.4 6.0***,+++ 5.4***,+++,##
4 1.01 26.57 76.38
± ± ± ±
1.6 0.69 5.1*,+ 8.8***,+++,###
120 min
1 1.1 8.63 4.64
± ± ± ±
2.6 1.07 0.96 3.1
0.5 1.33 0.85 1.1
± ± ± ±
1.9 0.71 0.55 2.57
2.66 0.72 6.97 36.11
± ± ± ±
2.8 0.28 4.5 3.1***,+++,###
2.5 0.85 5.75 4.1
± ± ± ±
1.95 0.55 0.49 2.45
Values represent mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01 and ***P < 0.001 significantly different versus vehicle-treated group at the same time. + P < 0.05, ++ P < 0.01 and +++ P < 0.001 significantly different versus 50 mg/kg OLE (in i.p. injected groups) and versus 100 g OLE (in i.t. injected groups) at the same time. # P < 0.05, ## P < 0.01 and ### P < 0.001 significantly different versus 100 mg/kg OLE (in i.p. injected) and versus 200 g OLE (in i.t. injected) at the same time. Table 2 Antinociceptive effect of intraperitonealy (i.p.) olive leaf extract (OLE) on hot-plate test in rats. Experimental groups
Antinociception (%MPE) in different times after injection 15 min
Vehicle (i.p.) OLE (50 mg/kg i.p.) OLE (100 mg/kg i.p.) OLE (200 mg/kg i.p.)
2.1 20.43 23.52 30.31
± ± ± ±
30 min 1.13 5.08* 3.46** 2.82**
1.2 4.28 0.92 45.18
± ± ± ±
45 min 1.66 0.29 0.79 4.9***,+++,###
1.95 0.57 1.25 53.02
± ± ± ±
60 min 0.94 0.29 0.91 5.51***,+++,###
1.12 1 1.83 29.14
± ± ± ±
120 min 0.68 0.57 0.91 3.5***,+++,###
Values represent mean ± SEM (n = 6–8). *P < 0.05, **P < 0.01 and ***P < 0.001 significantly different versus vehicle-treated group at the same time. different versus 50 mg/kg OLE at the same time. ### P < 0.001 significantly different versus 100 mg/kg OLE at the same time.
Mean of pain responses were converted to percentage of control as follows: % of control =
mean number with drug in phase 1 or 2 × 100. mean number of control in phase 1 or 2
2.8. Measurement of locomotor activity in open field The locomotor activity in open field was carried out in an activity monitoring apparatus. The activity chambers (27 cm × 30 cm × 26 cm, width × length × height) were placed in soundproof boxes with dim illumination and a fan. Horizontal locomotor activity was recorded by breaking the infrared beams. After 5-min acclimatization, the locomotor activity was recorded for 60 min. 2.9. Statistical analysis
+++
1.09 1.75 0.9 3.38
± ± ± ±
0.98 0.37 1.9 1.32
P < 0.001 significantly
400 g OLE was greater than 100 and 200 g at 30, 45 and 60 min after (Table 1). 3.2. The analgesic effect of olive leaf extract (OLE) on hot-plate test Hot-plate data showed that OLE (200 mg/kg i.p.) could elicit an antinociceptive effect which appeared 15 min after the injection, reaching a peak at 45 min and persisting significant up to 60 min. In addition, 50 and 100 mg/kg OLE induced a moderate analgesia 15 min after the injection (P < 0.05 and P < 0.01, respectively). Administration of vehicle did not show any nociceptive response (Table 2). Comparison of analgesic effects of 200 mg/kg (i.p.) OLE 15, 30, 45 and 60 min after injection on tail-flick and hot-plate tests showed that OLE at 45 and 60 min after injection had greater effects in hotplate than in tail-flick test (Fig. 1).
The results are expressed as mean ± SEM. The difference in MPE% (antinociception) in and between groups over the time course of study was determined by one or two-way analysis of variance (ANOVA), respectively, followed by the Newman–Keuls test with 5% level of significance (P < 0.05). 3. Results 3.1. The analgesic effect of olive leaf extract (OLE) on tail-flick test There were no significant differences in baseline tail-flick latency in all experimental groups. Administration of vehicle had no significant effect on nociceptive threshold. OLE at doses of 50 and 100 mg/kg i.p. showed analgesia at 15 min after injection (P < 0.05 and P < 0.001, respectively). Olive leaf extract in dose 200 mg/kg exerted an antinociceptive activity [F(5,36) = 363.63, P = 0.0001] which appeared 15 min after injection and picked at 30 min and significantly persisted up to 45 min (Table 1). In addition, intrathecal administration of 100, 200 and 400 g OLE also exerted significant antinociceptive activity. The analgesic effect of
Fig. 1. Comparison of the analgesic effects of 200 mg/kg olive leaf extract 15, 30, 45 and 60 min after injection on tail-flick and hot-plate tests. Values represent mean ± SEM (n = 6–8). ***P < 0.001 significantly different versus tail-flick values at the same time.
Author's personal copy S. Esmaeili-Mahani et al. / Journal of Ethnopharmacology 132 (2010) 200–205 Table 3 The effects of different doses of olive leaf extract (OLE) and morphine on nociceptive responses in different phases of formalin test. Experimental groups
Control Vehicle (i.p.) OLE (50 mg/kg i.p.) OLE (100 mg/kg i.p.) OLE (200 mg/kg i.p.) Morphine (5 mg/kg i.p.)
This analgesic activity reached a peak 30 min after injection and lasted for about 120 min. Concomitant administration of morphine (5 mg/kg) with OLE (12 mg/kg) produced significantly antinociceptive effect 15, 30, 60 and 120 min following administration (Table 4). In addition, 25 mg/kg OLE significantly enhanced the antinociception elicited by injection of 5 mg/kg morphine reaching a peak at 30 min and persisting significant up to 180 min. As is shown in Table 5, morphine (1 g/kg i.p.) produced a hyperalgesic response in control animals. The maximum hyperalgesic effect was reached at 30 min after injection and persisted almost unchanged up to 180 min and then diminished. Administration of vehicle, and OLE (6 or 12 mg/kg i.p.) did not show a nociceptive response. Co-administration of 6 or 12 mg/kg OLE completely blocked the hyperalgesic effect of 1 g/kg morphine, while vehicle had no effect on 1 g/kg morphine-induced hyperalgesia (Table 5).
Inhibition of pain response (%Control) First phase 100 93.44 83.37 74.95 61.83 55.11
± ± ± ± ± ±
Second phase
7.94 5.28 3.44 6.94 6.52**,++ 5.54***,+++
100 101.07 96.66 100.25 85.89 72.05
± ± ± ± ± ±
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2.84 3.35 3.41 4.94 1.15*,+ 6.31***,+++
Values represent mean ± SEM (n = 7–8). *P < 0.05 and **P < 0.01 significantly different versus control group and + P < 0.05 and ++ P < 0.01 as compared with vehicle-treated animals.
3.3. The analgesic effect of olive leaf extract (OLE) on formalin test Administration of vehicle had no significant effect on nociceptive responses in first and second phases of formalin test (Table 3). Intraperitoneal injection of OLE at doses of 50 and 100 mg/kg did not reduce formalin-induced nociceptive responses during both phases. There was a significant reduction in pain response with 200 mg/kg OLE during phases 1 (P < 0.01) and 2 (P < 0.05) in the formalin test when compared to the control and vehicle-treated animals. Morphine (5 mg/kg i.p), as a reference analgesic drug, produced significant analgesic effects in the first and second phases of test (P < 0.001).
3.5. The effect of olive leaf extract (OLE) on locomotor activity In order to determine whether the thermal antinociception following the injection of OLE could be attributed to a diminished sensory and/or motor performance in the tested animals, we performed sensorimotor testing at 60 min post-treatment using 50, 100 and 200 mg/kg OLE. Injection of vehicle or different doses of OLE did not significantly affect walk performance and distance traveling compared to control animals [F(5,38) = 2.089, P = 0.0881] suggesting that their sensory and motor capabilities remained intact (Table 6).
3.4. The effect of olive leaf extract (OLE) on analgesic and hyperalgesic effects of morphine
4. Discussion As it is shown in Table 4, morphine (5 mg/kg) produced moderate analgesic responses 15, 30 and 60 min after injection [F(7,48) = 16.849, P = 0.0001]. OLE (6, 12 or 25 mg/kg i.p.) had no significant effects on nociceptive threshold. Co-administration of 6 mg/kg OLE with morphine produced potent antinociceptive effects which were grater than those in morphine-treated group.
Despite the number of papers published on olive leaf and the effects of its constituents, none has focused on its influence on nociceptive threshold and its analgesic activity. Our results showed that olive leaf extract had analgesic property in chemical and thermal nociceptive tests in rats.
Table 4 The effect of morphine and morphine concomitant with 6, 12 or 25 mg/kg olive leaf extract (OLE) on nociceptive threshold in rats. The tail-flick test was used to assess the effect of drugs. Experimental groups
Antinociception (%MPE) in different times after injection 15 min
OLE (6 mg/kg i.p.) OLE (12 mg/kg i.p.) OLE (25 mg/kg i.p.) Mor (5 mg/kg i.p.) Mor + OLE (5 + 6 mg/kg i.p.) Mor + OLE (5 + 12 mg/kg i.p.) Mor + OLE (5 + 25 mg/kg i.p.)
2.2 2.91 7.07 56.22 62.06 64.61 70.98
± ± ± ± ± ± ±
30 min 3.2 3.9 1.12 9.17 6.37+++ 4.49### 8.11$$$
1.8 1.8 5.41 50.57 82.65 95.30 96.07
± ± ± ± ± ± ±
60 min 1.71 3.11 1.19 6.29 5.16**,+++ 0.99***,### 3.08***,$$$
1.31 2.1 4.6 29.19 64.04 71.34 81.04
120 min
± ± ± ± ± ± ±
1.8 1.51 0.44 11.64 7.92***,+++ 2.91***,### 7.11***,$$$
1.55 3.1 3.9 7.03 14.23 33.69 52.56
± ± ± ± ± ± ±
180 min 1.62 2.64 0.43 4.02 7.07 4.28*** 5.48***,$$$
0.0 2.01 1.64 1.92 1.45 5.56 22.52
± ± ± ± ± ± ±
0.44 0.43 0.72 0.96 1.05 3.41 4.39*,$
Values represent mean ± SEM (n = 7–8). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with morphine-treated group at the same time. +++ P < 0.001 significantly different versus 6 mg/kg OLE at the same time. ### P < 0.001 significantly different versus 12 mg/kg OLE at the same time. $ P < 0.05 and $$$ P < 0.001 significantly different versus 25 mg/kg OLE at the same time. Table 5 The effect of morphine (1 g/kg i.p.) and morphine concomitant with 6 or 12 mg/kg OLE on nociceptive threshold in rats. Experimental groups
Tail-flick latency before and at different times after administration Pre
Vehicle-treated OLE (6 mg/kg) OLE (12 mg/kg) Mor (1 g/kg) Mor + Veh (1 g/kg + 0) Mor + OLE (1 g/kg + 6 mg/kg) Mor + OLE (1 g/kg + 12 mg/kg)
5.12 4.93 4.89 4.76 5.2 4.73 4.81
30 min ± ± ± ± ± ± ±
0.16 0.18 0.14 0.11 0.16 0.16 0.19
4.9 4.79 5.27 2.96 3.07 5.6 5.19
± ± ± ± ± ± ±
60 min 0.19 0.16 0.19 0.1+++ 0.43+++ 0.23 0.2
4.85 4.9 5.14 2.9 3.05 5.1 5.02
± ± ± ± ± ± ±
120 min 0.14 0.15 0.1 0.08+++ 0.47+++ 0.47 0.12
5.02 4.82 5.09 3.11 2.85 5.3 4.81
± ± ± ± ± ± ±
0.11 0.11 0.13 0.11+++ 0.31+++ 0.19 0.21
180 min 4.93 4.89 4.97 3.28 2.92 4.9 4.95
± ± ± ± ± ± ±
0.15 0.15 0.16 0.21+++ 0.19+++ 0.19 0.11
Values represent mean ± SEM (n = 7–8). + P < 0.05, ++ P < 0.01 and +++ P < 0.001 significantly different versus before (Pre) drug administration.
240 min 4.81 4.95 5.2 3.72 3.32 4.6 4.9
± ± ± ± ± ± ±
0.14 0.13 0.25 0.32+ 0.42++ 0.32 0.22
300 min 4.95 4.81 5.16 4.7 4.4 4.7 5.12
± ± ± ± ± ± ±
0.12 0.17 0.12 0.24 0.26 0.21 0.09
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Distance traveling (cm)
Control Vehicle OLE (25 mg/kg) OLE (50 mg/kg) OLE (100 mg/kg) OLE (200 mg/kg)
46.05 45.3 44.83 42.75 49.2 42.45
± ± ± ± ± ±
1.928 1.061 0.750 1.096 2.121 3.001
The data are expressed as mean ± SEM of distance traveled during 60 min test session (n = 6–8).
It is well known that oleuropein, hydroxytyrosol, tyrosol and caffeic acid are the main constituents of olive leaves, which is thought to be responsible for their pharmacological effects. Furthermore, olive leaves contain p-coumaric acid, vanillic acid, vanillin, luteolin, diosmetin, rutin, luteolin-7-glucoside, apigenin7-glucoside, and diosmetin-7-glucoside (Bianco and Uccella, 2000; Ryan et al., 2002). It has been documented that olive leaf extract had Ca2+ channelblocking activity (Gilani et al., 2005). This observed effect of Olea europaea leaf extract has been attributed to the main component oleuropein (Scheffler et al., 2008). HPLC analysis of olive leaf extract by our colleagues showed that the major constituent of our extract is also oleuropein, comprising 35.6% of the extract (Mohagheghi et al., 2010). Several lines of evidence indicate that nociception is related to the intraneuronal Ca2+ level. The lowering of the neuronal Ca2+ induces analgesia (Del Pozo et al., 1990; Dolatshahi-Somehsofla et al., 2009). Not surprisingly, herb such as olive leaf extract which reduces Ca2+ availability could exert analgesic effect. Recently, Gong et al. (2009) showed that hydroxytyrosol, a simple flavonic compound, had anti-inflammatory and antinociceptive effects and also inhibited IL-1 and TNF-␣ expression. In addition, Pathak et al. (1991) reported antinociceptive and antiinflammatory activities for luteolin. Therefore, it is possible that the luteolin and hydroxytyrosol present in the extract may account, at least in part, for the antinociceptive effects observed in the present study. Recent findings have demonstrated that administration of vanilloid compounds through alternative routes, such as application of capsaicinoids onto peripheral nerves, or injecting the agent into the epidural or subarachnoid space produced highly effective antinociception (Nagy et al., 2004). Wu et al. (2009) have found that vanillin produced anti-inflammatory effect by reducing the expressions of proinflammatory cytokines interleukin-1beta, interleukin-6, interferon-gamma, and tumor necrosis factor-alpha and by stimulating the expression of anti-inflammatory cytokine (IL-4). In addition, vanillin has an alleviating effect on mechanical allodynia in rat model of neurophatic pain (Beaudry et al., 2010). Therefore, vanilloid compounds of olive leaf extract also serve to explain the observed analgesic effect of the extract in this study. Caffeic acid and its derivatives are widely distributed in medicinal plant e.g. fruits, vegetables, wine and olive (Bianco and Uccella, 2000). There is a report indicating that caffeic acid and all of its derivatives with aliphatic chain elicit significant antinociceptive effect in experimental models of pain (de Campos Buzzi et al., 2009). It may be worth that further investigation and elucidation regarding the mechanism(s) of olive leaf extract-induced analgesia to be performed. In this study three experimental models of pain were used to assess the analgesic property of the ethanolic extract of Olea europaea leaves. The methods were selected such that both centrally and peripherally mediated effects were investigated. The tail-flick and hot-plate tests revealed central activity, while the formalin test investigated both central and peripheral effects. Our
result suggests the antinociceptive effect of OLE at the spinal and supraspinal levels using tail-flick and hot-plate tests. However, the pattern of antinociceptive effects on hot-plate and tail-flick tests was different so that the stronger effect was shown in hot-plate test (Fig. 1). Nociceptive information is processed and integrated peripherally as well as at spinal and supraspinal levels within the central nervous system. Tail-flick test is a spinally integrated nociceptive reflex, while hot-plate test is a complex response which is supraspinally integrated. Thus, the observed differences indicate that the supraspinal mechanisms are more significant in antinociceptive effects of OLE than the spinal mechanisms. In the formalin test there is a distinctive biphasic nociceptive response termed early and late phase. Drugs that act mostly on the central nervous system inhibit both phases while peripherally acting drugs inhibit the late phase. Attenuation of both phases of pain as observed with 200 mg/kg OLE in this study also lends strong evidence to the presence of central effects. In conclusion, this study shows that the ethanolic extract of olive leaves has significant antinociceptive effects in rats. The analgesic property of this plant might be related with the significant phytochemicals such as oleuropein, triterpenes, flavonoids, and sterols reported in the leaf extract which needs further investigation. Our results showed that olive leaf extract could potentiate the antinociceptive property of morphine sub-effective dose and suppress low-dose morphine hyperalgesia. Opioids in ordinary (analgesic) doses have an inhibitory effect on calcium channels, but could activate these channels in very low doses which elicit a hyperalgesic property (Esmaeili-Mahani et al., 2008). Therefore, it seems logical that olive leaf extract with calcium channel blockade activity could block morphine-induced hyperalgesia and also potentiate morphine analgesia. In addition, there is a report indicating that the VR1 agonist and the mu-receptor agonist, when co-administered into the ventrolateral-PAG at non-analgesic doses, produce antinociceptive effect in nociceptive thermal tests (Maione et al., 2009). We assume that vanilloid compounds of OLE can be involved in the effect of OLE on morphine analgesia. However, this issue needs to be clarified by further studies. In conclusion, our results indicate that olive leaf extract has analgesic property in several models of pain and has helpful influence on morphine analgesia in rats. Therefore, it can be used for the treatment and/or management of painful conditions. Acknowledgment This work was supported by funds from Shahid Bahonar University of Kerman. References Beaudry, F., Ross, A., Lema, P.P., Vachon, P., 2010. Pharmacokinetics of vanillin and its effects on mechanical hypersensitivity in a rat model of neuropathic pain. Phytotherapy Research 24, 525–530. Briante, R., Patumi, M., Terenziani, S., Bismuto, E., Febbraio, F., Nucci, R., 2002. Olea europaea L. leaf extract and derivatives: antioxidant properties. Journal of Agricultural and Food Chemistry 50, 4934–4940. Bianco, A., Uccella, N., 2000. Biophenolic components of olives. Food Research International 33, 475–485. Cherif, S., Rahal, N., Haouala, M., Hizaoui, B., Dargouth, F., Gueddiche, M., et al., 1996. A clinical trial of a titrated Olea extract in the treatment of essential arterial hypertension. Journal de Pharmacie de Belgique 51, 69–71. D’Amour, F.E., Smith, D.L., 1941. A method of determining loss of pain sensation. Journal Pharmacology and Experimental Therapeutic 27, 74–79. de Campos Buzzi, F., Franzoi, C.L., Antonini, G., Fracasso, M., Filho, V.C., Yunes, R.A., et al., 2009. Antinociceptive properties of caffeic acid derivatives in mice. European Journal of Medicinal Chemistry 44, 4596–4602. Del Pozo, E., Rouiz Garcia, C., Bayens, J.M., 1990. Analgesic effect of diltiazem and verapamil after central and peripheral administration in the hot-plate test. General Pharmacology 21, 681–685.
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