http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1056312
ORIGINAL ARTICLE
Antinociceptive and anti-inflammatory effects of the aerial parts of Artemisia dracunculus in mice Akram Eidi1, Shahrbanoo Oryan2, Jalal Zaringhalam3, and Mitra Rad1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2Biological Science Faculty, Kharazmi University, Tehran, Iran, and 3Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Abstract
Keywords
Context: Tarragon (Artemisia dracunculus L., Asteraceae) is an ancient herb, which is widely used as a medicine, flavoring, or fragrance. Objective: To determine the antinociceptive and anti-inflammatory effects of aerial parts of tarragon, we investigated the effects of ethanolic extract of the plant in adult male Balb/c mice. Materials and methods: Antinociceptive activity was determined using formalin, hot-plate, and writhing tests. The effect of the ethanolic extract on acute inflammation was evaluated by xylene-induced ear edema in mice. The ethanolic extract was administered at doses of 5, 10, 50, and 100 mg/kg, i.p. The control group received saline as vehicle of ethanolic extract. Results: Our results showed that the ethanolic extract (50 and 100 mg/kg) decreased both phases of pain in the formalin test (ED50 ¼ 109.66 and 87.13 mg/kg, respectively). In the hot-plate test, the extract (50 and 100 mg/kg) increased pain threshold during 60 min (ED50 ¼ 81.03 mg/kg). The extract (50 and 100 mg/kg) exhibited antinociceptive activity against acetic acid-induced writhing (ED50 ¼ 66.99 mg/kg). The extract (50 and 100 mg/kg) showed significant activity in the xylene ear edema test (ED50 ¼ 78.20 mg/kg). Pretreatment of the animals with naloxone decreased the analgesia induced by the extract in hot-plate and formalin tests; therefore, opioid receptors may be involved, at least partly, in the analgesic effect of tarragon extract. Discussion and conclusion: The results suggested that tarragon have significant analgesic and anti-inflammatory effects in mice, and, therefore, further studies are required to evaluate these effects and additional potential of the plant.
Analgesia, inflammation, tarragon
Introduction Artemisia dracunculus L. (Asteraceae) or tarragon is a small shrubby perennial herb. The species has been under cultivation for a long time for its aromatic value in seasoning salads, edibles, and also for medicinal purposes. Different alkamide, coumarin (Saadali et al., 2001) and isocoumarin (Engelmeier et al., 2004; Lutz-Kutschera et al., 2003) compounds have been isolated from various parts of tarragon. Tarragon is safe to use as dietary supplements or in functional foods (Ribnicky et al., 2004). It has been established that aerial parts of tarragon produce a volatile essential oil that has medicinal value (Aglarova et al., 2008). It has antibacterial (Deans & Svobada, 1988), antifungal, antitumor (Meepagala et al., 2002; Zani et al., 1991), antidiabetic (Swanston et al., 1991), anticoagulant, antihyperlipidemic (Yazdanparast & Saee, 1999), and antioxidative (Parejo et al., 2002; Saadali et al., 2001) activities. In Iranian traditional medicine, dried aerial Correspondence: Akram Eidi. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel: +9821 44865939. Fax: +9821 44865939. E-mail: [email protected], [email protected]
History Received 31 October 2014 Accepted 25 May 2015 Published online 16 June 2015
parts of tarragon have been used orally for the treatment of epilepsy (Aqili Khorasani, 1992). The species is also used as a sleep aid and mild sedative (Chevallier, 1996). It was reported that monoterpenes present in tarragon essential oil are responsible for its anticonvulsant and sedative effects (Sayyah et al., 2004). Classical analgesics are classified into two main groups: opioid drugs and non-steroidal anti-inflammatory drugs (NSAIDs). The first group activates opioid receptors as main action mechanisms, while the second group inhibits prostaglandin synthesis by inhibition of cyclooxygenase enzyme. Opioids are considered to be the primary drugs for the treatment of moderate to severe acute, chronic, and cancer pain. Despite their effectiveness, they have several clinically significant adverse effects, such as sedation (cognitive dysfunction), nausea, vomiting, respiratory depression, cardiovascular depression, constipation, and hyperalgesia (Ballantine & Mao, 2003). Medical doctors have been commonly prescribing NSAIDs and immunosuppressants for the treatment of inflammatory and arthritic diseases; however, some of them can cause serious side-effects, including gastric mucosal damage, water and salt retention, and even carcinomas (Su¨leyman &
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Bu¨yu¨kokurog˘lu, 2001). Therefore, alternative agents with less severe side-effects are needed, and botanical products can be important candidates (Verpoorte, 1998). Based on traditional use of the plant for pain relief, we decided to investigate the effects of ethanolic extract of aerial parts of tarragon on different nociceptive models induced by thermal or chemical stimuli in Balb/c mice.
Materials and methods
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Animals In this study, adult male Balb/c mice (25–30 g) were used. The mice were housed in a room maintained at 22–24 C and 50–55% humidity on a 12 h light/dark cycle. All experiments were carried out according to the guidelines set forth by the International Association for the Study of Pain (Zimmermann, 1983). Each animal was tested only once. Nine mice were used in each experiment.
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the formalin-injected paw. Nociceptive behavior was quantified as the numbers of flinches of the injected paw during 1min periods every 5 min, up to 45 min after injection (Wheeler-Aceto & Cowan, 1991). Formalin-induced flinching was biphasic. The initial acute phase (0–5 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–45 min). A modified Dubuisson method (Dubuisson & Dennis, 1977) was used to assess the pain response. The scoring was as follows: 0: walking as usual; 1: limping, not moving with the injected paw at the floor; 2: raising the injected paw; and 3: paw licking or gnawing. The duration of the behaviors described above was recorded separately (Hunskaar et al., 1985; Moore et al., 1991). Ethanolic extract of tarragon (10, 50, and 100 mg/kg), morphine (10 mg/kg), or saline (control group) was administered intraperitoneally to different groups of mice 30 min before formalin injection. Hot-plate test
Chemicals Materials used in this study included acetic acid (Merck, Gernsheim, Germany), formalin (Merck, Gernsheim, Germany), and xylene (Merck, Gernsheim, Germany). The drugs included morphine sulfate (Temad, Tehran, Iran), indomethacin (Sigma, Poole, UK), and dexamethasone (Sigma, Poole, UK). All the drugs were dissolved in saline. All other chemicals used were of good quality and of analytical grade. Preparation of extract Aerial parts of tarragon were purchased from a retail food store (Tehran, Iran) in June 2013 and identified by Dr. Ali Mazooji from the Department of Botany, Islamic Azad University (Voucher number: 04128, deposited in: I.A.U. Herbarium). The dried aerial parts (200 g) were grounded (500 mm) and extracted three times (48 h) with ethanol:water (4:6) solution (1200 ml) and filtered with a glass filter funnel. The extracts were gathered and the ethanol was evaporated under reduced pressure at 40 C in a rotavapor, and the remaining mixed and homogenized. Phytochemical screening The standard phytochemical screening test was used to screen the extracts. The extracts were analyzed qualitatively to detect saponins, alkaloids, and terpenoids. Flavonoids were detected using aluminum chloride as a spray reagent (Logendra et al., 2006).
The hot-plate test was carried out at a fixed temperature of 55 ± 1 C (Hu et al., 2008). The reaction consisted of hind leg flinching, paw licking, and jumping. The time between reaching the platform and the reaction provoked was recorded as the response latency. Reaction times were recorded before (0 min) and at 15, 30, 45, and 60 min after intraperitoneal administration of tarragon extract (10, 50, and 100 mg/kg) to different groups of mice. Morphine (10 mg/kg) was used as a reference drug. The control group was administered saline as the vehicle of ethanolic extract. Acetic acid-induced writhing response Antinociceptive activities of tarragon ethanolic extract were assessed by measuring the response to an intraperitoneal injection of acetic acid solution (1.0%, 10 ml/kg body wt), which presented as writhes (contraction of the abdominal muscles and full extension of both hind paws) (Fischer et al., 2008). Mice (nine per group) were intraperitoneally pretreated with the tarragon ethanolic extract (10, 50, and 100 mg/kg) or indomethacin (10 mg/kg) 30 min before acetic acid injection. Control animals received a similar volume of saline. After 5 min, the number of writhes was recorded during the subsequent 20-min period. The number of abdominal writhes was cumulatively counted every 5 min over a period of 20 min immediately after the acetic acid injection. The antinociceptive activity was expressed as the inhibition percentage of abdominal writhes. ED50 determination
Analgesic activity Formalin test Mice were placed in an open plexiglas observation chamber (30 12 13 cm3) for 30 min to acclimate to their surroundings; then, they were removed and gently restrained while the dorsum of the hind paw was injected with 50 ll of diluted formalin (2.5%) using a 30-gauge needle. The animals were returned to the chamber and the nociceptive behavior was observed immediately after formalin injection. A mirror was placed in each chamber to enable unhindered observation of
In all the above tests, ED50 values (the concentration which suppresses the analgesic response by 50%) were determined by regression analysis of concentration–response curve. Involvement of opioid receptors The possible involvement of the opioid system in the antinociceptive effect caused by tarragon ethanolic extract was investigated using a method similar to the formalininduced paw licking and hot-plate tests. The animals were pretreated with tarragon ethanolic extract (100 mg/kg),
Antinociceptive and anti-inflammatory effects of tarragon
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morphine (10 mg/kg), or saline 30 min before formalin injection or before they were placed on the hot-plate. A non-selective opioid receptor antagonist, naloxone (2 mg/kg, i.p.), was injected 15 min beforehand against the antinociceptive effect induced by both morphine and tarragon extract (Sulaiman et al., 2010).
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Anti-inflammatory activity – xylene-induced ear edema Edema was expressed as the percentage of increase in ear weight due to inflammatory challenge. Thirty minutes after intraperitoneal injection of the ethanolic extract (10, 50, and 100 mg/kg) or dexamethasone (10 mg/kg), 0.03 ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. Two hours after xylene application, mice were sacrificed and both ears were removed. To evaluate the ear weight, 7-mm diameter ear punch biopsies were collected using a metal punch and individually weighed. The increase in ear weight caused by the irritant was measured by subtracting the weight of the right ear (inflamed) from that of the left ear (non-inflamed). The mean percentage edema inhibition (%) was calculated by comparing with the negative control group. The control group received saline as vehicle of ethanolic extract (Saraiva et al., 2011). Statistical analysis Statistical analysis was carried out using SPSS for Windows statistical package (version 12.0) (SPSS Inc., Chicago, IL). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. The criterion for statistical significance was set at p50.05. All values were expressed as mean values ± SEM.
Results
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Analgesic effect of tarragon ethanolic extract in formalin test Administration of vehicle had no significant effect on nociceptive responses in the first and second phases of the formalin test (Table 1). Intraperitoneal injection of tarragon ethanolic extract (50 and 100 mg/kg) reduced formalininduced nociceptive responses during both phases. Morphine (10 mg/kg), as a reference analgesic drug, produced significant analgesic effects in the first and second phases of the test. The mechanism of action of tarragon extract against nociception was investigated using naloxone. Pretreatment of animals with naloxone reversed the analgesic effect of tarragon extract (100 mg/kg) in the both phases. The analgesic effect of morphine (10 mg/kg) was also significantly suppressed in the presence of naloxone (2 mg/kg) in the both phases of formalin test. The ED50 values were equal to 109.66 and 87.13 mg/kg for the first and second phases of formalin test, respectively. The analgesic effect of tarragon ethanolic extract on hot-plate test As presented in Figure 1, tarragon ethanolic extract at doses of 50 and 100 mg/kg, elicited a significant analgesic activity in the thermal nociceptive test. The response was shown by the increase in latency time in seconds. The increase in latency time was found to be dose dependent, and was measured at 0, 15, 30, 45, and 60 min after the administration of saline, tarragon extract, and standard drug morphine. The involvement of opioid receptor was assessed by the administration of naloxone (2 mg/kg), 15 min prior to the administration of test samples. The administration of naloxone caused a significant attenuation in antinociceptive activity of tarragon ethanolic extract (Figure 1), which established the participation of opioid receptor in the central analgesic activity. Moreover, morphine-induced analgesia was completely abolished by the presence of naloxone. The ED50 value was 81.03 mg/kg.
Phytochemical analysis Phytochemical screening of the ethanolic extract of the aerial parts of tarragon afforded essential oils (3.1% v/w), coumarins (41% v/w), flavonoids, phenolcarbonic acids, and alkamides.
Analgesic effect of tarragon ethanolic extract on acetic acid-induced writhing response The cumulative amount of abdominal stretching was correlated with the level of acetic acid-induced pain (Table 2).
Table 1. Effect of tarragon ethanolic extract in formalin test in mice.a Response scores Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Morphine (10 mg/kg) Naloxone (2 mg/kg) Naloxone (2 mg/kg) + Tarragon extract (100 mg/kg) Naloxone (2 mg/kg) + Morphine (10 mg/kg) a
% Inhibition
Early phase (0–5 min)
Late phase (15–45 min)
Early phase (0–5 min)
Late phase (15–45 min)
2.70 ± 0.15 2.54 ± 0.11 2.48 ± 0.13 1.76 ± 0.12* 1.56 ± 0.13** 1.45 ± 0.04*** 2.53 ± 0.14 2.40 ± 0.11 2.47 ± 0.13
2.21 ± 0.05 1.98 ± 0.04 1.77 ± 0.06 1.52 ± 0.08* 0.96 ± 0.03*** 0.92 ± 0.04*** 2.13 ± 0.06 2.04 ± 0.05 2.09 ± 0.07
– 5.93 8.14 34.81 42.22 46.29 6.29 11.11 8.51
– 10.40 19.90 31.22 56.56 58.37 3.61 7.69 5.42
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. *p50.05; **p50.01; ***p50.001 different from the control group.
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Figure 1. Effect of the intraperitoneally administration of tarragon ethanolic extract, morphine (Mor.) and naloxone (Nal.) on the hot-plate test. Thermal antinociceptive latency was measured before, and at 15, 30, 45, and 60 min after the treatment. Each column represents mean ± SEM for nine mice. The control group was treated with saline as a vehicle. **p50.01, ***p50.001 different from the control group.
Table 2. Effect of tarragon ethanolic extract in the writhing test in mice.a Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Morphine (10 mg/kg) Indomethacin (10 mg/kg)
Number of writhing
% Inhibition
75.4 ± 4.6 70.8 ± 3.9 69.1 ± 4.3 38.4 ± 4.7** 24.6 ± 3.5*** 19.8 ± 2.7*** 22.1 ± 2.5***
– 6.10 8.35 49.07 67.37 73.74 70.68
Table 3. Effect of tarragon ethanolic extract in xylene-induced ear edema in mice.a Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Dexamethasone (10 mg/kg)
Edema weight (mg)
% Inhibition
10.1 ± 0.9 9.7 ± 0.6 9.2 ± 0.8 6.8 ± 0.9** 3.7 ± 0.4*** 3.4 ± 0.5***
– 3.96 8.91 32.67 63.36 66.33
a
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. **p50.01, ***p50.001 different from the control group.
a
Tarragon ethanolic extract (50 and 100 mg/kg), in a dosedependent manner, significantly inhibited the number of writhes compared with normal controls. Non-narcotic analgesic drug, indomethacin (10 mg/kg), also suppressed the pain induced by acetic acid. The ED50 value was 66.99 mg/kg.
Our results showed that tarragon ethanolic extract in a dose-dependent manner inhibited both phases of formalininduced pain. This study confirms that administration of formalin in mice induces a behavioral response consisting of a typical biphasic response, as seen in all formalin assays (Miranda et al., 2009; Raboisson & Dallel, 2004). Phase I results from direct stimulation of nociceptors, while phase II involves a period of sensitization, during which inflammatory phenomena occur through peripheral mechanisms (Le Bars et al., 2001). Naloxone (a non-selective opioid receptor antagonist) prevented the antinociceptive effect observed with tarragon ethanolic extract in both phases of the formalin test, suggesting participation of opioid receptors in this effect. Our results indicated that tarragon ethanolic extract significantly increased the nociceptive threshold measured by the increased latencies in the hot-plate test. Animals treated with the extract presented a longer latency time in the hot-plate test compared with the control group. The hot-plate test is commonly used to assess narcotic analgesics (Vaz et al., 1997). It has been suggested that tarragon ethanolic extract has a central analgesic effect, as evidenced by the
Effects of tarragon ethanolic extract on xylene-induced ear edema in mice The results obtained from xylene-induced mice ear edema are shown in Table 3. The administration of tarragon ethanolic extract (50 and 100 mg/kg) and dexamethasone (10 mg/kg) significantly reduced the ear edema induced by xylene. The ED50 value was equal to 78.20 mg/kg.
Discussion The present study was performed to determine the possible antinociceptive and anti-inflammatory activities of tarragon ethanolic extract using formalin, hot-plate, writhing, and ear edema tests.
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. **p50.01; ***p50.001 different from the control group.
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increase in reaction time of mice in the hot-plate test. The paw-licking hot-plate response is a more complex supraspinally organized behavior (Chapman et al., 1985). Moreover, in this study, the antinociceptive action of morphine and tarragon extract was abolished by naloxone. Reduction in acetic acid-induced writhing responses (a pain model) may be related to reduced synthesis of inflammatory mediators by inhibiting cyclooxygenases and/or lipoxygenases (Franzotti et al., 2000; Ojewole, 2006). Our results showed that acetic acid injection induced a characteristic writhing response in the mice. The tarragon ethanolic extract had a significant analgesic effect on the number of writhes induced by acetic acid, and, therefore, exhibited significant antinociceptive effects. Our results suggest that tarragon extract may play a role in the inhibition of prostaglandins synthesis. In addition to prostaglandins, several other inflammatory mediators, including sympathomimetic amines, tumor necrosis factor-a, interleukin-1b and interleukin-8, have been reported to be associated with the nociceptive response to acetic acid in mice (Duarte et al., 1988; Ferreira et al., 1988, 1993a,1993b; Ribeiro et al., 2000a). It has been claimed that writhing response induced by acetic acid is highly dependent on both peritoneal macrophages and mast cells (Ribeiro et al., 2000b). Xylene-induced neurogenous swelling was partially associated with substance P, as a common inflammatory model for evaluating vascular permeability (Luber-Narod et al., 1997). Our results showed that tarragon extract significantly inhibited xylene-induced ear swelling in mice, and thus it might reduce the release of substance P or antagonize its action. Xylene could increase capillary permeability and leukocyte infiltration in mice. Our results showed that tarragon extract dose dependently inhibited capillary permeability. Our results suggested that tarragon extract has a significant anti-inflammatory effect. The present study supports the claims of Iranian traditional medicine showing that tarragon ethanolic extract has an analgesic effect. The mechanism involved in the antinociception produced by tarragon extract still remains unclear, but seems to involve, at least in part, an interaction with the opioid system. These observations are substantiated by the fact that, like morphine, the antinociception induced by the tarragon extract was almost completely prevented by the nonselective opioid antagonist naloxone. However, further investigations are required to evaluate the efficacy and safety of this herbal medication in man as well as its mechanism of action in animal trials.
Declaration of interest The authors report no conflicts of interest.
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ORIGINAL ARTICLE
Antinociceptive and anti-inflammatory effects of the aerial parts of Artemisia dracunculus in mice Akram Eidi1, Shahrbanoo Oryan2, Jalal Zaringhalam3, and Mitra Rad1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran, 2Biological Science Faculty, Kharazmi University, Tehran, Iran, and 3Neurophysiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Abstract
Keywords
Context: Tarragon (Artemisia dracunculus L., Asteraceae) is an ancient herb, which is widely used as a medicine, flavoring, or fragrance. Objective: To determine the antinociceptive and anti-inflammatory effects of aerial parts of tarragon, we investigated the effects of ethanolic extract of the plant in adult male Balb/c mice. Materials and methods: Antinociceptive activity was determined using formalin, hot-plate, and writhing tests. The effect of the ethanolic extract on acute inflammation was evaluated by xylene-induced ear edema in mice. The ethanolic extract was administered at doses of 5, 10, 50, and 100 mg/kg, i.p. The control group received saline as vehicle of ethanolic extract. Results: Our results showed that the ethanolic extract (50 and 100 mg/kg) decreased both phases of pain in the formalin test (ED50 ¼ 109.66 and 87.13 mg/kg, respectively). In the hot-plate test, the extract (50 and 100 mg/kg) increased pain threshold during 60 min (ED50 ¼ 81.03 mg/kg). The extract (50 and 100 mg/kg) exhibited antinociceptive activity against acetic acid-induced writhing (ED50 ¼ 66.99 mg/kg). The extract (50 and 100 mg/kg) showed significant activity in the xylene ear edema test (ED50 ¼ 78.20 mg/kg). Pretreatment of the animals with naloxone decreased the analgesia induced by the extract in hot-plate and formalin tests; therefore, opioid receptors may be involved, at least partly, in the analgesic effect of tarragon extract. Discussion and conclusion: The results suggested that tarragon have significant analgesic and anti-inflammatory effects in mice, and, therefore, further studies are required to evaluate these effects and additional potential of the plant.
Analgesia, inflammation, tarragon
Introduction Artemisia dracunculus L. (Asteraceae) or tarragon is a small shrubby perennial herb. The species has been under cultivation for a long time for its aromatic value in seasoning salads, edibles, and also for medicinal purposes. Different alkamide, coumarin (Saadali et al., 2001) and isocoumarin (Engelmeier et al., 2004; Lutz-Kutschera et al., 2003) compounds have been isolated from various parts of tarragon. Tarragon is safe to use as dietary supplements or in functional foods (Ribnicky et al., 2004). It has been established that aerial parts of tarragon produce a volatile essential oil that has medicinal value (Aglarova et al., 2008). It has antibacterial (Deans & Svobada, 1988), antifungal, antitumor (Meepagala et al., 2002; Zani et al., 1991), antidiabetic (Swanston et al., 1991), anticoagulant, antihyperlipidemic (Yazdanparast & Saee, 1999), and antioxidative (Parejo et al., 2002; Saadali et al., 2001) activities. In Iranian traditional medicine, dried aerial Correspondence: Akram Eidi. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel: +9821 44865939. Fax: +9821 44865939. E-mail: [email protected], [email protected]
History Received 31 October 2014 Accepted 25 May 2015 Published online 16 June 2015
parts of tarragon have been used orally for the treatment of epilepsy (Aqili Khorasani, 1992). The species is also used as a sleep aid and mild sedative (Chevallier, 1996). It was reported that monoterpenes present in tarragon essential oil are responsible for its anticonvulsant and sedative effects (Sayyah et al., 2004). Classical analgesics are classified into two main groups: opioid drugs and non-steroidal anti-inflammatory drugs (NSAIDs). The first group activates opioid receptors as main action mechanisms, while the second group inhibits prostaglandin synthesis by inhibition of cyclooxygenase enzyme. Opioids are considered to be the primary drugs for the treatment of moderate to severe acute, chronic, and cancer pain. Despite their effectiveness, they have several clinically significant adverse effects, such as sedation (cognitive dysfunction), nausea, vomiting, respiratory depression, cardiovascular depression, constipation, and hyperalgesia (Ballantine & Mao, 2003). Medical doctors have been commonly prescribing NSAIDs and immunosuppressants for the treatment of inflammatory and arthritic diseases; however, some of them can cause serious side-effects, including gastric mucosal damage, water and salt retention, and even carcinomas (Su¨leyman &
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Bu¨yu¨kokurog˘lu, 2001). Therefore, alternative agents with less severe side-effects are needed, and botanical products can be important candidates (Verpoorte, 1998). Based on traditional use of the plant for pain relief, we decided to investigate the effects of ethanolic extract of aerial parts of tarragon on different nociceptive models induced by thermal or chemical stimuli in Balb/c mice.
Materials and methods
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Animals In this study, adult male Balb/c mice (25–30 g) were used. The mice were housed in a room maintained at 22–24 C and 50–55% humidity on a 12 h light/dark cycle. All experiments were carried out according to the guidelines set forth by the International Association for the Study of Pain (Zimmermann, 1983). Each animal was tested only once. Nine mice were used in each experiment.
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the formalin-injected paw. Nociceptive behavior was quantified as the numbers of flinches of the injected paw during 1min periods every 5 min, up to 45 min after injection (Wheeler-Aceto & Cowan, 1991). Formalin-induced flinching was biphasic. The initial acute phase (0–5 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–45 min). A modified Dubuisson method (Dubuisson & Dennis, 1977) was used to assess the pain response. The scoring was as follows: 0: walking as usual; 1: limping, not moving with the injected paw at the floor; 2: raising the injected paw; and 3: paw licking or gnawing. The duration of the behaviors described above was recorded separately (Hunskaar et al., 1985; Moore et al., 1991). Ethanolic extract of tarragon (10, 50, and 100 mg/kg), morphine (10 mg/kg), or saline (control group) was administered intraperitoneally to different groups of mice 30 min before formalin injection. Hot-plate test
Chemicals Materials used in this study included acetic acid (Merck, Gernsheim, Germany), formalin (Merck, Gernsheim, Germany), and xylene (Merck, Gernsheim, Germany). The drugs included morphine sulfate (Temad, Tehran, Iran), indomethacin (Sigma, Poole, UK), and dexamethasone (Sigma, Poole, UK). All the drugs were dissolved in saline. All other chemicals used were of good quality and of analytical grade. Preparation of extract Aerial parts of tarragon were purchased from a retail food store (Tehran, Iran) in June 2013 and identified by Dr. Ali Mazooji from the Department of Botany, Islamic Azad University (Voucher number: 04128, deposited in: I.A.U. Herbarium). The dried aerial parts (200 g) were grounded (500 mm) and extracted three times (48 h) with ethanol:water (4:6) solution (1200 ml) and filtered with a glass filter funnel. The extracts were gathered and the ethanol was evaporated under reduced pressure at 40 C in a rotavapor, and the remaining mixed and homogenized. Phytochemical screening The standard phytochemical screening test was used to screen the extracts. The extracts were analyzed qualitatively to detect saponins, alkaloids, and terpenoids. Flavonoids were detected using aluminum chloride as a spray reagent (Logendra et al., 2006).
The hot-plate test was carried out at a fixed temperature of 55 ± 1 C (Hu et al., 2008). The reaction consisted of hind leg flinching, paw licking, and jumping. The time between reaching the platform and the reaction provoked was recorded as the response latency. Reaction times were recorded before (0 min) and at 15, 30, 45, and 60 min after intraperitoneal administration of tarragon extract (10, 50, and 100 mg/kg) to different groups of mice. Morphine (10 mg/kg) was used as a reference drug. The control group was administered saline as the vehicle of ethanolic extract. Acetic acid-induced writhing response Antinociceptive activities of tarragon ethanolic extract were assessed by measuring the response to an intraperitoneal injection of acetic acid solution (1.0%, 10 ml/kg body wt), which presented as writhes (contraction of the abdominal muscles and full extension of both hind paws) (Fischer et al., 2008). Mice (nine per group) were intraperitoneally pretreated with the tarragon ethanolic extract (10, 50, and 100 mg/kg) or indomethacin (10 mg/kg) 30 min before acetic acid injection. Control animals received a similar volume of saline. After 5 min, the number of writhes was recorded during the subsequent 20-min period. The number of abdominal writhes was cumulatively counted every 5 min over a period of 20 min immediately after the acetic acid injection. The antinociceptive activity was expressed as the inhibition percentage of abdominal writhes. ED50 determination
Analgesic activity Formalin test Mice were placed in an open plexiglas observation chamber (30 12 13 cm3) for 30 min to acclimate to their surroundings; then, they were removed and gently restrained while the dorsum of the hind paw was injected with 50 ll of diluted formalin (2.5%) using a 30-gauge needle. The animals were returned to the chamber and the nociceptive behavior was observed immediately after formalin injection. A mirror was placed in each chamber to enable unhindered observation of
In all the above tests, ED50 values (the concentration which suppresses the analgesic response by 50%) were determined by regression analysis of concentration–response curve. Involvement of opioid receptors The possible involvement of the opioid system in the antinociceptive effect caused by tarragon ethanolic extract was investigated using a method similar to the formalininduced paw licking and hot-plate tests. The animals were pretreated with tarragon ethanolic extract (100 mg/kg),
Antinociceptive and anti-inflammatory effects of tarragon
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morphine (10 mg/kg), or saline 30 min before formalin injection or before they were placed on the hot-plate. A non-selective opioid receptor antagonist, naloxone (2 mg/kg, i.p.), was injected 15 min beforehand against the antinociceptive effect induced by both morphine and tarragon extract (Sulaiman et al., 2010).
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Anti-inflammatory activity – xylene-induced ear edema Edema was expressed as the percentage of increase in ear weight due to inflammatory challenge. Thirty minutes after intraperitoneal injection of the ethanolic extract (10, 50, and 100 mg/kg) or dexamethasone (10 mg/kg), 0.03 ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. Two hours after xylene application, mice were sacrificed and both ears were removed. To evaluate the ear weight, 7-mm diameter ear punch biopsies were collected using a metal punch and individually weighed. The increase in ear weight caused by the irritant was measured by subtracting the weight of the right ear (inflamed) from that of the left ear (non-inflamed). The mean percentage edema inhibition (%) was calculated by comparing with the negative control group. The control group received saline as vehicle of ethanolic extract (Saraiva et al., 2011). Statistical analysis Statistical analysis was carried out using SPSS for Windows statistical package (version 12.0) (SPSS Inc., Chicago, IL). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. The criterion for statistical significance was set at p50.05. All values were expressed as mean values ± SEM.
Results
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Analgesic effect of tarragon ethanolic extract in formalin test Administration of vehicle had no significant effect on nociceptive responses in the first and second phases of the formalin test (Table 1). Intraperitoneal injection of tarragon ethanolic extract (50 and 100 mg/kg) reduced formalininduced nociceptive responses during both phases. Morphine (10 mg/kg), as a reference analgesic drug, produced significant analgesic effects in the first and second phases of the test. The mechanism of action of tarragon extract against nociception was investigated using naloxone. Pretreatment of animals with naloxone reversed the analgesic effect of tarragon extract (100 mg/kg) in the both phases. The analgesic effect of morphine (10 mg/kg) was also significantly suppressed in the presence of naloxone (2 mg/kg) in the both phases of formalin test. The ED50 values were equal to 109.66 and 87.13 mg/kg for the first and second phases of formalin test, respectively. The analgesic effect of tarragon ethanolic extract on hot-plate test As presented in Figure 1, tarragon ethanolic extract at doses of 50 and 100 mg/kg, elicited a significant analgesic activity in the thermal nociceptive test. The response was shown by the increase in latency time in seconds. The increase in latency time was found to be dose dependent, and was measured at 0, 15, 30, 45, and 60 min after the administration of saline, tarragon extract, and standard drug morphine. The involvement of opioid receptor was assessed by the administration of naloxone (2 mg/kg), 15 min prior to the administration of test samples. The administration of naloxone caused a significant attenuation in antinociceptive activity of tarragon ethanolic extract (Figure 1), which established the participation of opioid receptor in the central analgesic activity. Moreover, morphine-induced analgesia was completely abolished by the presence of naloxone. The ED50 value was 81.03 mg/kg.
Phytochemical analysis Phytochemical screening of the ethanolic extract of the aerial parts of tarragon afforded essential oils (3.1% v/w), coumarins (41% v/w), flavonoids, phenolcarbonic acids, and alkamides.
Analgesic effect of tarragon ethanolic extract on acetic acid-induced writhing response The cumulative amount of abdominal stretching was correlated with the level of acetic acid-induced pain (Table 2).
Table 1. Effect of tarragon ethanolic extract in formalin test in mice.a Response scores Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Morphine (10 mg/kg) Naloxone (2 mg/kg) Naloxone (2 mg/kg) + Tarragon extract (100 mg/kg) Naloxone (2 mg/kg) + Morphine (10 mg/kg) a
% Inhibition
Early phase (0–5 min)
Late phase (15–45 min)
Early phase (0–5 min)
Late phase (15–45 min)
2.70 ± 0.15 2.54 ± 0.11 2.48 ± 0.13 1.76 ± 0.12* 1.56 ± 0.13** 1.45 ± 0.04*** 2.53 ± 0.14 2.40 ± 0.11 2.47 ± 0.13
2.21 ± 0.05 1.98 ± 0.04 1.77 ± 0.06 1.52 ± 0.08* 0.96 ± 0.03*** 0.92 ± 0.04*** 2.13 ± 0.06 2.04 ± 0.05 2.09 ± 0.07
– 5.93 8.14 34.81 42.22 46.29 6.29 11.11 8.51
– 10.40 19.90 31.22 56.56 58.37 3.61 7.69 5.42
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. *p50.05; **p50.01; ***p50.001 different from the control group.
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Figure 1. Effect of the intraperitoneally administration of tarragon ethanolic extract, morphine (Mor.) and naloxone (Nal.) on the hot-plate test. Thermal antinociceptive latency was measured before, and at 15, 30, 45, and 60 min after the treatment. Each column represents mean ± SEM for nine mice. The control group was treated with saline as a vehicle. **p50.01, ***p50.001 different from the control group.
Table 2. Effect of tarragon ethanolic extract in the writhing test in mice.a Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Morphine (10 mg/kg) Indomethacin (10 mg/kg)
Number of writhing
% Inhibition
75.4 ± 4.6 70.8 ± 3.9 69.1 ± 4.3 38.4 ± 4.7** 24.6 ± 3.5*** 19.8 ± 2.7*** 22.1 ± 2.5***
– 6.10 8.35 49.07 67.37 73.74 70.68
Table 3. Effect of tarragon ethanolic extract in xylene-induced ear edema in mice.a Groups Control Tarragon extract (5 mg/kg) Tarragon extract (10 mg/kg) Tarragon extract (50 mg/kg) Tarragon extract (100 mg/kg) Dexamethasone (10 mg/kg)
Edema weight (mg)
% Inhibition
10.1 ± 0.9 9.7 ± 0.6 9.2 ± 0.8 6.8 ± 0.9** 3.7 ± 0.4*** 3.4 ± 0.5***
– 3.96 8.91 32.67 63.36 66.33
a
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. **p50.01, ***p50.001 different from the control group.
a
Tarragon ethanolic extract (50 and 100 mg/kg), in a dosedependent manner, significantly inhibited the number of writhes compared with normal controls. Non-narcotic analgesic drug, indomethacin (10 mg/kg), also suppressed the pain induced by acetic acid. The ED50 value was 66.99 mg/kg.
Our results showed that tarragon ethanolic extract in a dose-dependent manner inhibited both phases of formalininduced pain. This study confirms that administration of formalin in mice induces a behavioral response consisting of a typical biphasic response, as seen in all formalin assays (Miranda et al., 2009; Raboisson & Dallel, 2004). Phase I results from direct stimulation of nociceptors, while phase II involves a period of sensitization, during which inflammatory phenomena occur through peripheral mechanisms (Le Bars et al., 2001). Naloxone (a non-selective opioid receptor antagonist) prevented the antinociceptive effect observed with tarragon ethanolic extract in both phases of the formalin test, suggesting participation of opioid receptors in this effect. Our results indicated that tarragon ethanolic extract significantly increased the nociceptive threshold measured by the increased latencies in the hot-plate test. Animals treated with the extract presented a longer latency time in the hot-plate test compared with the control group. The hot-plate test is commonly used to assess narcotic analgesics (Vaz et al., 1997). It has been suggested that tarragon ethanolic extract has a central analgesic effect, as evidenced by the
Effects of tarragon ethanolic extract on xylene-induced ear edema in mice The results obtained from xylene-induced mice ear edema are shown in Table 3. The administration of tarragon ethanolic extract (50 and 100 mg/kg) and dexamethasone (10 mg/kg) significantly reduced the ear edema induced by xylene. The ED50 value was equal to 78.20 mg/kg.
Discussion The present study was performed to determine the possible antinociceptive and anti-inflammatory activities of tarragon ethanolic extract using formalin, hot-plate, writhing, and ear edema tests.
The values are the mean ± SEM for nine mice. The control group was administered with saline as a vehicle. Statistical differences from the control were determined by ANOVA followed the Tukey test. **p50.01; ***p50.001 different from the control group.
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increase in reaction time of mice in the hot-plate test. The paw-licking hot-plate response is a more complex supraspinally organized behavior (Chapman et al., 1985). Moreover, in this study, the antinociceptive action of morphine and tarragon extract was abolished by naloxone. Reduction in acetic acid-induced writhing responses (a pain model) may be related to reduced synthesis of inflammatory mediators by inhibiting cyclooxygenases and/or lipoxygenases (Franzotti et al., 2000; Ojewole, 2006). Our results showed that acetic acid injection induced a characteristic writhing response in the mice. The tarragon ethanolic extract had a significant analgesic effect on the number of writhes induced by acetic acid, and, therefore, exhibited significant antinociceptive effects. Our results suggest that tarragon extract may play a role in the inhibition of prostaglandins synthesis. In addition to prostaglandins, several other inflammatory mediators, including sympathomimetic amines, tumor necrosis factor-a, interleukin-1b and interleukin-8, have been reported to be associated with the nociceptive response to acetic acid in mice (Duarte et al., 1988; Ferreira et al., 1988, 1993a,1993b; Ribeiro et al., 2000a). It has been claimed that writhing response induced by acetic acid is highly dependent on both peritoneal macrophages and mast cells (Ribeiro et al., 2000b). Xylene-induced neurogenous swelling was partially associated with substance P, as a common inflammatory model for evaluating vascular permeability (Luber-Narod et al., 1997). Our results showed that tarragon extract significantly inhibited xylene-induced ear swelling in mice, and thus it might reduce the release of substance P or antagonize its action. Xylene could increase capillary permeability and leukocyte infiltration in mice. Our results showed that tarragon extract dose dependently inhibited capillary permeability. Our results suggested that tarragon extract has a significant anti-inflammatory effect. The present study supports the claims of Iranian traditional medicine showing that tarragon ethanolic extract has an analgesic effect. The mechanism involved in the antinociception produced by tarragon extract still remains unclear, but seems to involve, at least in part, an interaction with the opioid system. These observations are substantiated by the fact that, like morphine, the antinociception induced by the tarragon extract was almost completely prevented by the nonselective opioid antagonist naloxone. However, further investigations are required to evaluate the efficacy and safety of this herbal medication in man as well as its mechanism of action in animal trials.
Declaration of interest The authors report no conflicts of interest.
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