Abstract
The ethanol extract of Achillea nobilis. L. subsp. neilreichii. (Kerner) Formanék (Asteraceae) flower heads was investigated for its antinociceptive and anti-inflammatory activities and for acute toxicity in mice and rats. While the extract exhibited an antinociceptive effect during the late phase of the formalin test (100, 200, and 400 mg/kg, i.p.) and an anti-inflammatory effect in the paw edema test (100 and 200 mg/kg, i.p.), it did not exert any significant antinociceptive effect in the tail-flick test. Furthermore, administration of 400 mg/kg extract increased the latency to hot-plate test at 60 and 90 min. No significant change was detected in sensory motor performance. The acute LC50 value of the extract was 4456 mg/kg (i.p.) in mice. The current results demonstrate that an ethanol extract of A. nobilis. L. subsp. neilreichii. exerts anti-inflammatory activity.
Introduction
Achillea. L. (Asteraceae) species, primarily indigenous to Europe and the Middle East, are used as herbal remedies for their anti-inflammatory, spasmolytic, hemostatic, digestive, and cholagogue effects in traditional medicine (Kastner et al., Citation1995a). Achillea nobilis. L. subsp. neilreichii. (Kerner) Formanék (ANN), commonly known as “ayvadana,” is a perennial medicinal plant with a height of 30–60 cm, and is widespread in Turkey. The dried flower heads are sold in local markets and used as a diuretic and an emmenagogue, in wound healing, for abdominal pain, and against diarrhea and flatulence in Turkey (Baytop, Citation1999).
As the most characteristic secondary metabolites in ANN, flavonoids (luteolin and quercetin derivatives, orientin, isoorientin, vitexin, isoschaftoside, and 5-hydroxy-3,6,7,4′-tetramethoxyflavone) (Valant-Vetschara & Karin, Citation1986; Kastner et al., Citation1995b; Marchart et al., Citation2003; Krenn et al., Citation2003) and sesquiterpene lactones of various types (chrysartemin A, canin, anolide, chrysanthemol derivatives, and tanaparthin-beta-peroxide) have been reported (Turdybekov et al., Citation1994; Kastner et al., Citation1995b; Turmukhambetoy et al., Citation1999).
The antibacterial and antifungal potential of ANN have been investigated (Abbasoglu & Kusmenoglu, 1999; Palic et al., Citation2003). It was shown that the total extract of A. nobilis. subsp. sipylea. exhibited antispasmodic activity on rat duodenum (Karamenderes & Apaydın, Citation2003). Infusions prepared from ANN had antioxidant capacity, which was consistent with their total flavonoid and phenol contents (Konyalıoğlu & Karamenderes, Citation2004). The protective effect of ANN against H2O2-induced oxidative damage in human erythrocytes and leukocytes was also examined (Konyalıoğlu & Karamenderes, Citation2005). The aim of the current study was to investigate for the first time the antinociceptive and anti-inflammatory activities and acute toxicity of the ethanol. extract of ANN in mice and rats.
Materials and Methods
Plant material
ANN was collected from Denizli, Turkey, in June 2000. The plant was identified by Prof. Dr. Ozcan Secmen, from the Section of Botany, Department of Biology, Faculty of Science, Ege University, and a voucher specimen was deposited in the herbarium of Ege University, Faculty of Pharmacy, Izmir, Turkey (IZEF 5510).
Preparation of extract
Flower heads of the plant were selected, completely dried under shade, and powdered finely. Crude material (50 g) was extracted with EtOH (70%) in a Soxhlet extractor for 3 days and evaporated to dryness under reduced pressure on a rotary evaporator and lyophilized (yield 28.66%). The extract was administered to mice and rats immediately after suspension in saline. All doses are expressed as w/v of the plant extract.
Experimental procedures and chemicals
Experiments were performed on male Balb-C albino mice (20–25 g, each) and Wistar rats (125–150 g, each) The protocol was approved by the Animal Ethics Committee of the Faculty of Medicine, Ege University (20.06.2003, no. 2003/28). Animals were housed in a room maintained at 22±1°C with an alternating 12 h light-dark cycle. Food and water were available ad libitum.. The animals were transported to a quiet laboratory at least 1 h before the experiment. All experiments assessing the analgesic activity were carried out between 09:00 and 12:00 in normal room light and temperature (22±1°C). Formalin, carrageenan, DMSO, morphine, naloxone, and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
The antinociceptive activity was measured in 15 h fasted male Balb-C albino mice by the formalin test, and male Wistar rats by light tail-flick and hot-plate methods. All experiments conformed with ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann, Citation1983). The number of animals and the intensity of noxious stimuli were the minimum possible with which to demonstrate reliable effects of the agents tested.
Antinociceptive activity
Formalin test
Formalin (2.5% v/v, 20 µL) was injected in the subplantar region of the right hind paw of mice, and the duration of paw licking (an index of nociception) was monitored between 0 and 5 min (early phase) and 15 and 30 min (late phase) (Handy et al., Citation1995). The early-phase response is believed to represent a direct irritant effect of formalin on sensory C fibers, while the late-phase response is most likely secondary to the development of an inflammatory response and the release of algesic mediators (Hunskaar & Hole, Citation1987). Extract (100, 200, 400 mg/kg; n = 10 for each group) or vehicle (saline; n = 10) was administered 30 min before the formalin injection. The formalin test was repeated with 10 mg/kg morphine administration.
Tail flick test
The tail-flick test was evoked by a source of radiant heat, which was focused on the dorsal surface of the tail. Rats were examined for latency to withdraw their tails from a noxious thermal stimulus using a tail-flick meter (MAY-TF 0703, Turkey; n = 10 for each group). Each rat was tested twice before the administration of the extract, and the reaction times were averaged to obtain a baseline. The intensity of heat stimulus was adjusted to achieve a mean tail-flick latency of 3–4 s in control animals. Each rat was then tested 30, 45, 60, 75, 90, 105, and 120 min after the i.p. administration of 100, 200, 400 mg/kg extract. Control rats received saline instead of the extract. The light tail-flick test was repeated with 10 mg/kg morphine administration. Treatments were terminated if the animals did not respond within 15 s in order to avoid tissue damage (Rupniak et al., Citation1993).
Hot-plate test
Rats were placed on an aluminum hot plate (MAY-AHP 0603, Ankara, Turkey) kept at a temperature of 55±0.5°C for a maximum time of 30 s (Morteza-Semnani et al., Citation2002). Reaction time was recorded (when the animals licked their fore and hind paws and jumped) before and 15, 30, 45, 60, and 90 min after i.p. administration of 100, 200, 400 mg/kg extract (n = 10 for each groups). Morphine (10 mg/kg body weight) was used as the reference drug, and the test was repeated with 10 mg/kg morphine + 2 mg/kg naloxone and 400 mg/kg extract + 2 mg/kg naloxone administration.
Anti-inflammatory activity
Carrageenan-induced paw edema in rats
The anti-inflammatory activity was evaluated by the carrageenan-induced paw edema test in the rat (Schapoval et al., Citation1998). Male Wistar rats were deprived of food overnight and treated by intraperitoneal route with saline (as control group) and extract (100, 200, 400 mg/kg; n = 10 for each group), 30 min before 0.1 mL carrageenan in saline (1%) was injected subplantarly into the left hind paw. The contralateral paw was injected with 0.1 mL saline and used as a control. Paw volume was measured by water plethysmometer (Lettica, LE 7500, Barcelona, Spain) before and 1, 2, 3, 4, 5, and 6 h after the injection of carrageenan into the plantar region of the left hind paw. The anti-inflammatory test was repeated with 10 mg/kg indomethacin administration.
Sensorimotor performance
Because sedation can affect the reaction to noxious stimuli, the integrity of motor coordination was assessed with a rotarod apparatus, at a rotating speed of 16 rpm. A preliminary selection of mice and rats was made on the day of the experiment to exclude those that did not remain on the rotarod bar for two consecutive periods of 45 s each (Pieretti et al., Citation1999). Sensorimotor performance was assessed 30 min after the injection of saline and 100, 200, 400 mg/kg of extract (n = 10 for each group) in mice and rats separately. Results are expressed as percentage of animals that succeeded in remaining on the rod for 45 s, which was the cutoff time.
Acute toxicity
In acute toxicity tests, male albino mice weighing 20–25 g were used (n = 10 for each group). The number of animals that died during this period was expressed as a percentage, and LC50 was determined by a Probit test (Morteza-Semnani et al., Citation2002) using 50% death within 24 h after i.p. administration of saline and the extract at different doses (400, 800, 1600, and 3200 mg/kg).
Statistical analysis
Results are reported as means±SEM (n), with n indicating the number of animals. Data were analyzed using the Student-Newman-Keuls test and ANOVA (SPSS for Windows, release 11.0) and were considered significant at p < 0.01 and p < 0.05.
Results
Antinociceptive activity
ANN extract was not effective during the first phase of the formalin test. However, ANN extract caused a significant inhibition of licking and biting of the hind-paw responses during the late phases (100, 200, and 400 mg/kg body weight) of formalin-induced hind-paw licking (p < 0.01). When morphine responses were compared with that of control, a significant inhibition of early and late phases was shown (p < 0.01) ().
Figure 1 Antinociceptive effect of ANN extract (mg/kg, i.p.) determined as the effect on the early phase (dark columns) and late phase (light columns) formalin-induced hind-paw licking behavior in mice. ANN extract (100, 200, 400 mg/kg), saline (control), and morphine were administered before the formalin injection. M = 10 mg/kg morphine, *p < 0.01 according to control animals (n = 10 for each group).
ANN extract administration did not increase the tail-flick latencies when compared with control rats (p > 0.05). When morphine responses were compared with that of control, it increased the latency significantly at 30, 45, 60, 75 (p < 0.01) and 90 min (p < 0.05) ().
Figure 2 Latency to withdraw the tail from noxious thermal stimulation in rats after treatment with saline (control) and 100, 200, and 400 mg/kg i.p. ANN extract injection. M = 10 mg/kg, i.p. morphine, *p < 0.01, #p < 0.05 according to control animals (n = 10 for each group).
Administration of 400 mg/kg ANN extract increased the latency to hot-plate test at 60 (p < 0.05) and 90 min (p < 0.01). Morphine administration increased the latency significantly at 15 min (p < 0.05) and at 30, 45, 60, and 90 min (p < 0.01). Naloxone reversed the morphine responses at 30, 45, and 60 min (p < 0.01) and at 90 min (p < 0.05). When the effect of 400 mg/kg extract + naloxone administration was compared with 400 mg/kg ANN extract alone, a significant increase in latency was observed at 45 (p < 0.05) and 60 min (p < 0.01) ( ).
Table 1.. Effect of ANN extraction the latency time of rats subjected to the hot-plate test.
Anti-inflammatory activity
The intraplantar injection of carrageenan caused a time-dependent paw edema in the rat, although saline injection caused no swelling (data not shown). Intraperitoneal administration of 100 and 200 mg/kg ANN extract inhibited paw swelling at 2, 3, 4, 5 (p < 0.01) and 6 h (p < 0.05). Similarly, a significant inhibition after 2, 3, 4, 5 (p < 0.01) and 6 h (p < 0.05) was obtained by indomethacin administration (). Percent increment in paw swelling was calculated by using the values before carrageenan injection.
Figure 3 Percent increase in carrageenan-induced paw edema in control rats treated with control (saline), in rats treated with 100, 200, and 400 mg/kg i.p. ANN extract, and 10 mg/kg i.p. indomethacin. Percent increment in paw swelling was calculated by using the values before carrageenan injection (n = 10 for each group). *p < 0.05, #p < 0.01 versus control.
Sensory motor performance
The sensorimotor performance of control mice and rats were found as 98.6 ± 3.2 and 99.2 ± 2.8%, respectively. No significant change was observed with the administration of the extract in any of the doses tested (p > 0.05). In fact, in the treatment groups, the percentage of animals with success in the rotarod test ranged between 97.8 ± 2.9% and 99.6 ± 3.1% for mice and 98.8 ± 2.7% and 99.8 ± 4.5% for rats (n = 10 for each group).
Acute toxicity
No lethality was observed among mice treated with i.p. doses of ANN extract. Acute LC50 value of this extract after i.p. administration in mice was 4456 mg/kg body weight in 24 h.
Discussion
Considering the active constituent(s) might only be accumulated in the flower heads as it was used in folkloric practice, ethanol extracts of dried flower heads of ANN were prepared and administered to animals. It was demonstrated that ANN extract caused a significant inhibition of licking and biting of the hind-paw responses during the late phases of formalin-induced hind-paw licking. The formalin test is a valid and reliable model of nociception, and it is sensitive for various classes of analgesic drugs. The test produces a biphasic response: early phase is thought to result from direct chemical activation of nociceptive afferent fibers, and peripheral inflammatory processes seem to be responsible for the late phase (Tjolsen et al., Citation1992). Drugs that act primarily as central analgesics inhibit both phases while peripherally acting drugs inhibit only the late phase (Morteza-Semnani et al., Citation2002). Centrally acting drugs such as opioids exert an inhibition in both phases (Shibata et al., Citation1989) as it is clearly consistent with our morphine results in the formalin test, whereas peripherally acting drugs such as indomethacin, aspirin, and hydrocortisone only inhibit the late phase, which seems to be a result of an anti-inflammatory response (Elisabetsky et al., Citation1995). In our study, ethanol extract of ANN produced a marked reduction of the duration of the licking in the late phase, consistent with the inflammatory reaction (Hunskaar & Hole, Citation1987). Inhibition of only the second phase of the formalin test is a typical characteristic of cyclooxygenase inhibitors (Rosland et al., Citation1990) suggesting a peripheral analgesic activity of ANN.
The hot-plate test is commonly used to assess narcotic analgesia. Administration of 400 mg/kg ANN extract did not increase the tail-flick latencies when compared with control rats and increased the latency to the hot-plate test at 60 (p < 0.05) and 90 min (p < 0.01). Hence, it is assumed that ANN does not exert a central analgesic effect, and this effect may probably be due to its peripheral analgesic effect. The different results obtained among the different types of nociceptive tests might reflect the differential processing of noxious stimuli with different physical and temporal properties and which trigger different motor responses. According to our knowledge, it is not possible to discriminate the specific modulation of these responses by different molecules (Rojas-Corrales et al., Citation2003).
Morphine was used as a positive control for all three antinociceptive tests we have performed. In all of them, a significant antinociceptive effect was obtained with morphine, and this effect was successfully reversed with the administration of naloxone, which is a specific opioid receptor antagonist. Similarly, the effect of 400 mg/kg, (i.p.) ANN extract + naloxone administration was compared with the effect of 400 mg/kg ANN extract administered alone in the hot-plate test in order to evaluate the possible opioid-like mechanism of the analgesic effect of the extract. While no significant difference was observed in the tail-flick test, a significant increase in latency was observed for different time points in the hot-plate test between 400 mg/kg ANN extract + naloxone and 400 mg/kg ANN extract administered groups. This increase in latency may be due to the possible partial opioid agonistic effect of ANN extract while it has clearly been shown that the analgesic effects of κ partial agonists like nalbuphine, pentazocine, and butorphanol may be enhanced by low-dose naloxone administration (Gear et al., Citation2003). Therefore, the possible partial opioid agonistic effect of the components of the extract requires further investigation.
Carrageenan paw edema test is a suitable test for the evaluation of anti-inflammatory drugs and has frequently been used to assess the antiedematous effects of natural products (Segura et al., Citation1998). Recent investigations demonstrated that carrageenan edema was effectively decreased by cyclooxygenase inhibitors (Dimartino et al., Citation1987). We have observed that the ANN extract significantly inhibited carrageenan-induced paw edema at 100 and 200 mg/kg, i.p. doses. It may be assumed that the anti-edema effects of the extract might be due to possible inhibition of the cyclooxygenase pathway.
In a previous study, the antioxidant capacity of infusions prepared from ANN was evaluated by using different methods (total antioxidant capacity, free radical and OH⋅ radical scavenging capacity, H2O2 reducing power), and the results clearly demonstrated that ANN infusion had an antioxidant activity with 8.09 mM α-tocopherol/100-mL (Konyalıoğlu & Karamenderes, 2004). Total flavonoid (0.14%) and total phenol contents (119.4 mg/L) were also determined by using the aluminum-chloride method and modified colorimetric method using the Folin-Ciocalteu reagent, respectively (Konyalıoğlu & Karamenderes, 2004). Recently, infusion of ANN was studied for its protective effect against H2O2-induced oxidative damage in human erythrocytes and leukocytes, and it was found that they show evidence of a significant antioxidant activity (Konyalıoğlu & Karamenderes, Citation2005). The role of oxygen-derived free radicals, such as OH⋅ radical and superoxide radical, in the inflammatory process is well-known (Fantone & Ward, Citation1982). It is also generally assumed that most of the antioxidants possess anti-inflammatory effects (Jang et al., Citation1997). A number of plant polyphenols have been shown to have antioxidant and anti-inflammatory properties (Rao et al., Citation1982).
Sesquiterpene lactones were demonstrated to possess anti-inflammatory properties in vivo. (Hall et al., Citation1979), and they inhibit neutrophil migration and chemotaxis in vitro. (Hall et al., Citation1980). The mechanisms by which sesquiterpene lactones inhibit inflammatory processes are not completely understood, but recent data demonstrated that these compounds are potent inhibitors of the proinflammatory transcription factor NF-κ.B (Hehner et al., Citation1998).
There is much evidence accumulated that flavonoids possess important effects on various biological systems, which may explain their widespread therapeutic uses (Di Carlo et al., Citation1999). Flavonoids demonstrate various pharmacological activities in vitro. and in vivo., including anticancer, anti-inflammatory, and antiallergic activities. Among these actions, the anti-inflammatory activity of flavonoids may be mediated by the inhibition of the AA-metabolizing enymes, COX/LOX, as well as by their antioxidative properties (Chi et al., Citation2001). Moreover, phenolic compounds decrease inflammatory mediator production in human whole blood cultures (Miles et al., Citation2005).
Flavonoids (Valant-Vetschara & Karin, Citation1986; Kastner et al., Citation1996; Marchart et al., Citation2003; Krenn et al., Citation2003) and sesquiterpenes lactones of various types (Turdybekov et al., Citation1994; Kastner et al., Citation1995b; Turmukhambetoy et al., Citation1999) were previously isolated from A. nobilis.. We suppose that the anti-inflammatory effect of the ethanol. extract of ANN could be related to radical scavenging activity and that it depends on a synergic action of all the components of the ethanol. extract.
The acute LC50 value of the extract of ANN after i.p. administration in mice was 4456 mg/kg in 24 h. Because the anti-inflammatory effect of ANN extract was induced at doses far below the LD50 value, this effect is of potential therapeutic use, and this extract deserves further pharmacological investigation.
In conclusion, it was clearly demonstrated in our study that ethanol extract of ANN exhibits anti-inflammatory activity in rats. These findings suggested initiation of a detailed evalution of the anti-inflammatory effect of the extract of ANN with a view to possible development of a superior anti-inflammatory drug.
Acknowledgments
We thank Prof. Dr. Ozcan Secmen for his help in identification of plant material. This study was designed and performed at Ege University, Center for Drug R&D and Pharmacokinetic Applications, Izmir, Turkey.
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