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INTRODUCTION
Recently, natural products from plant origin possessing immense ethnopharmacological importance have been given the utmost priority as treatments for inflammation and allergic reactions [1,2]. Agrimonia pilosa Ledeb has been used traditionally for treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema in Korea and other Asian countries since centuries [3]. Pharmacologically, Agrimonia pilosa Ledeb has been reported to possess anti-tumor [4,5], anti-viral [6,7], anti-oxidant [8], anti-microbial [9] and anti-hyperglycemic activity [10]. Reports also indicated that Agrimonia pilosa Ledeb extract inhibited the inflammatory process by suppression of iNOS, ROS and inflammatory cytokine production in microglial cells [11,12].
However the detailed mechanisms of Agrimonia pilosa Ledeb in delivering anti-nociceptive profiles were not fully elucidated. Further, to date no report exists on the anti-nociceptive effect of Agrimonia pilosa Ledeb. Therefore, in this study, we attempted to characterize antinociceptive profiles and mechanisms of Agrimonia pilosa Ledeb extract in various pain models.
METHODS
These experiments were approved by the University of Hallym Animal Care and Use Committee (Registration Number: Hallym 2009-05-01). All procedures were conducted in accordance with the 'Guide for Care and Use of Laboratory Animals' published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain.
Experimental animals
Male ICR mice (MJ Co., Seoul, Korea) weighing 20~25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 22±0.5℃ with an alternating 12 hr light-dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory for at least 2 hr before testing and were only used once. Experiments were performed during the light phase of the cycle (10:00~17:00).
Oral administration, and intraperitoneal (i.p.) and intrathecal (i.t.) injections
Oral administration was performed with gage in a volume of 500 µl/25 g body weight. I.p. injection was conducted to unanesthesized mice with volume of 250 µl. The i.t. administration was performed following the method of Hylden and Wilcox [13,14] using a 30-gauge needle connected to a 25 µl Hamilton syringe with polyethylene tubing. The i.t. injection volume was 5 µl and the injection site was verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the spinal cord. The dye injected i.t. was distributed both rostrally and caudally but with short distance (about 0.5 cm from the injection site) and no dye was found visually in the brain. The success rate for the injections was consistently found to be over 95%, before the experiments were done.
Assessment of antinociception and experimental protocols
All assessments for measuring antinociceptive properties of Agrimonia pilosa Ledeb extract were carried out by blinded observers.
Tail-flick and hot-plate tests
Antinociception was determined by the tail-flick [15] and the hot-plate paw-licking tests [16]. For the measurement of the tail-flick latency, mice were gently held with one hand with the tail positioned in the apparatus (EMDIE Instrument Co., Maidens, VA, USA, Model TF6) and the tail-flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of radiant heat was adjusted so that the animal flicked its tail within 3 to 5 sec. For the hot-plate test, mice were individually placed on the 55℃ hot-plate apparatus (Itic Life Science, Woodland Hills, CA, USA, Model 39 Hot Plate) and then, the reaction time starting from the placement of the mouse on the hotplate to the time of licking the front paw was measured. Basal latency for the hot-plate test was approximately 9 sec. Animals were pretreated orally once with vehicle (control) or Agrimonia pilosa Ledeb extract at 200 mg/kg doses 30 min prior to performing the tail-flick or hot-plate tests.
Acetic acid-induced writhing test
For the writhing test [17], 1% acetic acid was injection i.p. and then, the animals were immediately placed in an acrylic observation chamber (20 cm high, 20 cm diameter). The number of writhes was counted during 30 min after the injection of acetic acid. A writhe was defined as a contraction of the abdominal muscles accompanied by an extension of the forelimbs and elongation of the body. Animals were pretreated orally once with vehicle (control) or Agrimonia pilosa Ledeb extract at 200 mg/kg doses 30 min prior to performing the acetic acid-induced writhing and formalin tests.
Substance P-induced nociceptive behavioral test
Vehicle (control) or 200 mg/kg of Agrimonia pilosa Ledeb extract was pretreated orally 30 min prior to performing i.t. injection of substance P (0.7 µg/5 µl). Immediately after i.t. injection with substance P the mice were placed in an observation chamber (20 cm high, 20 cm diameter) and their nociceptive behavioral responses were recorded during 30 min. The cumulative response time of licking, scratching and biting episodes directed toward the lumbar and caudal region of spinal cord were measured with a stop-watch timer [14].
Pretreatment of antagonists
At first, mice were pretreated i.p. with either saline, naloxone (5 mg/kg), methysergide (5 mg/kg), or yohimbine (5 mg/kg), 10 min before oral administration of vehicle as a control or a fixed dose of Agrimonia pilosa Ledeb extract (200 mg/kg). And then, the writhing response was tested 30 min after the treatment with either vehicle or Agrimonia pilosa Ledeb extract [18-22].
Drugs
Acetic acid, substance P, naloxone, methysergide and yohimbine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agrimonia pilosa Ledeb (300 g) was dissolved in 80% ethanol (1,500 ml) and extracted as refluxing for 3 hours, and then the extract was filtered for obtaining A. This process was repeated again once to obtain B from residue. A and B were mixed. This mixture was decompressed and dried for using as Agrimonia pilosa Ledeb extract. Agrimonia pilosa Ledeb extract, naloxone, methysergide and yohimbine were dissolved in saline. All drugs were prepared just before use.
Statistical analysis
Data were presented as the mean±SEM. The statistical significance of differences between groups was assessed with one-way ANOVA with Bonferroni's post-hoc test using GraphPad Prism version 4.0 for Windows Vista (GraphPad Software, San Diego, CA, USA); p<0.05 was considered significant.
RESULTS
Effect of Agrimonia pilosa Ledeb extract on the tail-flick and hot-plate paw-licking responses
As revealed in Fig. 1, oral treatment of Agrimonia pilosa Ledeb extract at the dose of 200 mg/kg increased latencies of the tail-flick and hot-plate paw-licking responses compare to the control group of mice. The sedative effect was manifested, when the mice were treated with Agrimonia pilosa Ledeb extract orally at the dose of 200 mg/kg. However, there were no paralysis and motor changes.
Effect of Agrimonia pilosa Ledeb extract on the nociceptive behavior induced by acetic acid and substance P
Agrimonia pilosa Ledeb extract attenuated the acetic acid-induced writhing numbers (Fig. 2A). Treatment with Agrimonia pilosa Ledeb extract at the dose of 200 mg/kg led to 68% decrease in the acetic acid-induced writhing response compare to the control group of mice. In vehicle-treated control mice, i.t. injection of substance P (0.7 µg) caused acute, immediate behavioral responses, i.e., licking, scratching and biting the lumbar or caudal region, which lasted about 30 min. As shown in Fig. 2B, cumulative nociceptive response times for i.t. administration of substance P was significantly diminished by 85%.
Effect of opioidergic, serotonergic and adrenergic system on the inhibition of writhing response induced by Agrimonia pilosa Ledeb extract
We examined the possible involvement of opioidergic, serotonergic and adrenergic system in the Agrimonia pilosa Ledeb extract-induced antinociception. The pretreatment with naloxone (opioid receptor antagonist, Fig. 3A) or methysergide (serotonergic receptor antagonist, Fig. 3B) did not affect Agrimonia pilosa Ledeb extract-induced antinociception. However, the blockade of α2-adrenergic receptor with systemic pre-administration of yohimbine abolished the Agrimonia pilosa Ledeb extract-induced inhibition of the writhing response (Fig. 3C). The treatment of naloxone, methysergide or yohimbine itself did not affect the writhing response (Fig. 3).
DISCUSSION
In the present study, we found that Agrimonia pilosa Ledeb extract administered orally produces antinociception in various pain models. The tail-flick response is believed to be a spinally mediated reflex and the paw-licking hotplate response is a more complex supraspinally organized behavior (for review, see [23]). Moreover, Grumbach [24] has shown that the effectiveness of analgesic agents in the tail-flick pain model is highly correlated with relief of human pain. Our results demonstrate that Agrimonia pilosa Ledeb extract causes to prolong the tailflick and hot-plate response latencies, indicating the increase of nociceptive threshold.
We also examined the effect of Agrimonia pilosa Ledeb extract on the acetic acid-induced writhing test. I.p. injection of acetic acid can produce the peritoneal inflammation (acute peritonitis), which cause a response characterized by contraction of the abdominal muscles accompanying an extension of the forelimbs and elongation of the body. This writhing response is considered as a visceral inflammatory pain model [17 for review, see 25]. In the present study, we clearly showed the antinociceptive effect of Agrimonia pilosa Ledeb extract in an acetic acid-induced writhing test. Furthermore, it has been reported that i.t. injection of substance P in mice can also elicit nociceptive responses, consisting of biting, scratching and licking the caudal parts of the body [14,26]. We found in the present study that Agrimonia pilosa Ledeb extract was also effective in attenuating substance P-induced nociceptive responses. These results suggest furthermore that Agrimonia pilosa Ledeb extract may exert their antinociceptive effect via the central sites, possibly spinally mediated mechanisms.
The roles of opioid, serotonergic and adrenergic receptors in the regulation of modulation of nociceptive processing have been demonstrated in many previous studies. For example, it is well known that opioid receptors are involved in the antinociception [27-29]. Also, it has been reported that blockade of the spinal serotonergic or noradrenergic receptors by spinal injection of methysergide or yohimbine antagonize the antinociception induced by morphine administered supraspinally [28,30,31]. We observed in the present study that α2-adrenergic receptor, but not opioidergic and serotonergic receptors, appear to be involved in orally administered Agrimonia pilosa Ledeb extract-induced antinociception.
In conclusion, our results suggest that Agrimonia pilosa Ledeb extract shows an antinociceptive property in various pain models. Furthermore, this antinociceptive effect of Agrimonia pilosa Ledeb extract may be mediated by α2-adrenergic receptor, but not opioidergic and serotonergic receptors.
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