Anxiolytic-Like Effects of Gastrodia elata and Its Phenolic Constituents in Mice

Transcription

1 February 2006 Biol. Pharm. Bull. 29(2) (2006) 261 Anxiolytic-Like Effects of Gastrodia elata and Its Phenolic Constituents in Mice Ji Wook JUNG, a Byung Hoon YOON, a Hye Rim OH, a Jae-Hyeong AHN, a Sun Yeou KIM, b Sang-Yong PARK, a and Jong Hoon RYU*,a a Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University; and b Department of Herbal Pharmacology, Graduate School of East-West Medical Science, Kyung Hee University; 1 Hoeki-dong, Dongdeamoon-ku, Seoul , Korea. Received August 4, 2005; accepted November 18, 2005 The purpose of this study was to characterize the putative anxiolytic-like effects of the aqueous extract of the rhizome of Gastrodia elata along with its phenolic constituents, 4-hydroxybenzyl alcohol (HA) and 4-hyroxybenzaldehyde (HD), using an elevated plus maze (EPM) in mice. The mice were administered either the aqueous G. elata extract orally or received an intraperitoneal injection of the phenolic constituents, 1 h before the behavioral evaluation in the EPM. A single treatment of the aqueous G. elata extract significantly increased the percentage of time spent and arm entries into the open arms of the EPM versus the saline controls. Among the phenolic constituents of G. elata, HA and HD significantly increased the percentage of time spent and arm entries into the open arms of the EPM versus saline controls (p 0.05). Moreover, there were no changes in the locomotor activity and myorelaxant effects in any group compared with the saline controls. In addition, the anxiolytic-like effects of G. elata extract were blocked by both WAY (0.3 mg/kg, i.p.), a 5-HT 1A receptor antagonist, and flumazenil (10 mg/kg, i.p.), a GABA A receptor antagonist. The anxiolytic-like effects of HA were inhibited by WAY and the effects of HD were antagonized by flumazenil. These results indicate that G. elata is an effective anxiolytic agent, and suggests that the anxiolytic-like effects of G. elata via the serotonergic nervous system depends on HA and those effects of G. elata via the GABAergic nervous system depends on HD. Key words anxiety; Gastrodia elata; 4-hydroxybenzyl alcohol; 4-hyroxybenzaldehyde; WAY ; flumazenil Anxiety affects one eighth of the total population and has become a very important area of research in psychopharmacology in the current decade. Since the introduction of benzodiazepines in the 1960s, they have remained the most commonly prescribed treatment for anxiety. Although these compounds are the mainstay of drug treatment for anxiety disorders, they have many side-effects such as sedation, myorelaxation, ataxia, amnesia, and pharmacological dependence. 1) Recently, research has been conducted to identify safer, more specific, and perhaps lower cost therapies. 2) Gastrodia rhizome, the steamed and dried tuber of Gastrodia elata (G. elata) BLUME. (Orchidaceae) is a very important traditional Chinese herbal medicine used to treat headache, migraine, paralysis, and other nervous disorders. 3) Phytochemical studies of this plant have revealed the presence of several phenolic compounds, including 4-hydroxybenzyl alcohol (HA), 4-hyroxybenzaldehyde (HD), vanillin, vanillyl acohol, b-sitosterol, and gastrodin. 4) Recently Hsieh et al. reported the anticonvulsive effect of G. elata in kainic acidtreated rats. 5) In addition, the ether fraction of the methanol extracts of G. elata attenuates the decrease in the GABA level and the increase in the glutamate content showing anticonvulsant effects on pentylenetetrazole-induced seizures. 6,7) However, there were no reports on the anxiolytic effects of G. elata. The purpose of this study was to characterize the anxiolytic-like activity of the aqueous extract of G. elata (AEGE) and to show which constituent of G. elata exhibits such activity. Its anxiolytic and myorelaxant effects were examined using the elevated plus-maze (EPM) and a horizontal wire test in mice, respectively. In addition, this study also investigated which nervous systems are involved in the anxiolyticlike effects of the AEGE through the co-administration of AEGE and either flumazenil or WAY MATERIALS AND METHODS Animals Male ICR mice, weighing g, were purchased from the Orient Co., Ltd. of the Charles River branch (Seoul, Korea). The animals were housed 5 or 6 per cage, allowed access to water and food ad libitum, and maintained under a constant temperature (23 1 C) and humidity (60 10%) under a 12-h light/dark cycle (light on h). Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No , revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Korea. Materials Buspirone, HA, HD, WAY , and flumazenil were purchased from the Sigma Chemical Co. (U.S.A.). The rhizome of G. elata was obtained from a herbal supplier in Seoul, Korea, and voucher specimens (KHUOPS ) were deposited at the herbarium of the College of Pharmacy, Kyung Hee University (Seoul, Korea). The material was authenticated by Prof. C.S. Yook of the Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University. All the other materials were of the highest grade commercially available. Sample Preparation and Drug Administration The G. elata extract was prepared by boiling in 10 volumes of water for 120 min. The aqueous solution obtained was filtered, concentrated in a water bath under vacuum, frozen and lyophilized (Eyela, model FDU-2000, Japan) to yield the aqueous extract (yield 18.1%), which was then stored at 20 C until required. The AEGE was standardized as the contents of HA and HD using high performance liquid chromatography (HPLC) analysis. The AEGE was hydrolyzed with 10% of HCl in the MeOH/EtOH ( 7:3) under reflux for 30 min. The HPLC system used a Waters 510 pump (U.S.A.) equipped with a Waters 2487 UV detector set at To whom correspondence should be addressed. jhryu63@khu.ac.kr 2006 Pharmaceutical Society of Japan

2 262 Vol. 29, No nm. The column was a Capcell Pak C18 UG120 (4.6 mm 250 mm, 5 mm, Shiseido, Tokyo, Japan) with a flow rate of 1 ml/min. The injection volume was 100 ml. The mobile phase was a 30% acetonitrile with 1% acetic acid. HA and HD contents in the AEGE were % and %, respectively. AEGE was freshly dissolved in saline and orally administered. HD and flumazenil were suspended with a 10% aqueous solution of Tween-80 for the intraperitoneal injection. Buspirone, HA, and WAY were dissolved in saline, respectively. Spontaneous Behavior in the Open Field Test Testing was carried out in clear black Plexiglas boxes ( cm) equipped with the video-based Ethovision System (Noldus, Wageningen, The Netherlands). The mice were placed in the center of the apparatus to evaluate horizontal locomotor activity 1 h after being treated with AEGE (50, 100, 200, 400 mg/kg), HA (5, 10, 25, 50, 100 mg/kg), or HD (5, 10, 25, 50, 100 mg/kg) and video-recorded for 5 min. The horizontal locomotor activity is expressed in terms of the total ambulatory distance and the frequency of rearing. Elevated Plus-Maze Test The EPM for mice consisted of two perpendicular open arms (30 7 cm) and two enclosed arms (30 7 cm) with 20 cm high walls, extending from the central platform (7 7 cm). The open and closed arms were connected by a central square, 7 7 cm, to give an apparatus of a plus sign appearance. The floor and walls of the maze were constructed from the dark opaque polyvinylplastic. The maze was raised to a height of 50 cm above the floor level in a dimly lit room (20 lux) and a video camera was suspended above the maze to record the movements for analysis. 8,9) Each mouse was placed at the center of the platform, its head facing an open arm. The animals were tested individually and only once for 5 min. The maze was cleaned after each trial so as to remove any residue or odors. The following measurements were taken and analyzed using the videobased Ethovision System: the number of entries into the open or closed arms, the time spent in each arm, and the total distance moved in the EPM. All the experiments were carried out between 10:00 and 16:00 o clock. One hour after the AEGE treatment (50, 100, 200, 400 mg/kg, p.o.), the mice were placed in the EPM. For the phenolic constituents of G. elata, the mice were administered HA and HD (5, 10, 25, 50, 100 mg/kg, i.p.). The mice in the control group were given the vehicle solvent only, and the animals were tested individually once only for 5 min. In a separate antagonism study, the mice were subjected to the coadministration of AEGE (400 mg/kg, p.o.) and either WAY (0.3 mg/kg, i.p.) or flumazenil (10 mg/kg, i.p.) 1 h and 30 min prior to testing, respectively. In addition, 30 min after the HA or HD (100 mg/kg, i.p.) treatment, the mice were injected with either WAY (0.3 mg/kg, i.p.) or flumazenil (10 mg/kg, i.p.). The mice were treated with buspirone (2 mg/kg, i.p.) 1 h before EPM test and used as the positive controls. Horizontal Wire Test A horizontal wire test was carried out by treating the mice with AEGE (50, 100, 200, 400 mg/kg, p.o.), HA (5, 10, 25, 50, 100 mg/kg, i.p.), or HD (5, 10, 25, 50, 100 mg/kg, i.p.) according to a slight modification of the method reported by Bonetti et al. 10) Briefly, the mice were lifted by the tail and allowed to grasp a horizontally strung wire (1 mm diameter, 15 cm long and placed 20 cm above the table) with their forepaws, after which they were then released. The number of mice from each treatment group that did not grasp the wire with their forepaws within a 10 s period was recorded. A myorelaxant drug would impair the ability of the mice to grasp the wire, and muscle relaxation is commonly associated with sedation. Statistics The values are expressed as means S.E.M. The percentage of time spent in the open arms was calculated using the formula as follows: time spent in the open arms/ (time spent in the open arms time spent in the closed arms) 100. The percentage of the number of entries into the open arms was calculated by the same method. The data was analyzed by a one-way analysis of variance (ANOVA) followed by Student-Newman Keuls test for the multiple comparisons. Statistical significance was set at p RESULTS Effect of AEGE and Its Constituents Treatment in the EPM The mice in the saline-treated group typically avoided spending time on or entering into the open arms. The vehicle-treated mice remained for s in the open arms. The percentage of time spent in the open arms was significantly increased in the AEGE-treated mice (400 mg/kg) compared with saline treated group (Fig. 1; p 0.05). In addition, there was also significantly increased in the percentage of open arm entries in the AEGE-treated mice (400 mg/kg) compared with saline treated group (Fig. 1; p 0.05). However, no significant change was observed in terms of the percentage of time spent or the open arm entries at doses of 50, 100 and 200 mg/kg of AEGE. Among the G. elata constituents, HA (50, 100 mg/kg) and HD (100 mg/kg) significantly increased the percentage of time spent and arm entries into the open arms (Figs. 2, 3; p 0.05). In the buspirone-treated (2 mg/kg) group, as a positive control, the percentages of time spent and arm entries into the open arms were significantly increased compared with the saline-treated Fig. 1. Effect of a Single Treatment of the Aqueous Extract of Gastrodia elata BLUME. (G. elata) on the Percentage of the Time Spent in and the Number of Entries into the Open Arms of the Elevated Plus-Maze over a 5 min Test Period in the Mice given saline orally. p values for the group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 as compared with the saline-treated control group).

3 February Fig. 2. Effect of 4-Hyroxybenzyl Alcohol on the Percentage of the Time Spent in and the Number of Entries into the Open Arms of the Elevated Plus-Maze over a 5 min Test Period in the Mice given saline intraperitoneally. p values for group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 as compared with the saline-treated control group). Fig. 4. Anxiolytic-Like Effects of Gastrodia elata BLUME. (G. elata) Were Both Blocked by WAY and Flumazenil and the number of entries into the open arms of the elevated plus-maze, 1 h after the oral administration of the water extract of Gastrodia elata BLUME. (G. elata) (400 mg/kg), the water extract of G. elata (400 mg/kg) WAY (0.3 mg/kg) or flumazenil (10 mg/kg) (30 min prior testing, i.p.), or vehicle solvent; n mice per group. p values for the group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 versus the saline-treated control, # p 0.05 as compared with the AEGE-treated group). Fig. 3. Effect of 4-Hyroxybenzaldehyde on the Percentage of the Time Spent in and the Number of Entries into the Open Arms of the Elevated Plus-Maze over a 5 min Test Period in the Mice given solvent intraperitoneally. p values for group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 as compared with the vehicle-treated control group). Fig. 5. Anxiolytic-Like Effects of 4-Hydroxybenzyl Alcohol (HA) Were Blocked by WAY But Not by Flumazenil group (p 0.05). Effect of WAY and Flumazenil on the Anxiolytic-Like Activity of AEGE, HA, or HD In order to determine if the anxiolytic effect of AEGE is exerted via the serotonergic or GABAergic nervous system, the AEGE (400 mg/kg) treated mice were subjected to a co-treatment with either WAY , a 5-HT 1A receptor antagonist, or flumazenil, a GABA A receptor antagonist. As shown in Fig. 4, the anxiolytic-like effects of AEGE were antagonized by both WAY (0.3 mg/kg) and flumazenil (10 mg/kg). Among the constituents of G. elata, the anxiolytic-like effects of HA (100 mg/kg) were blocked by WAY (0.3 mg/kg) but not by flumazenil (10 mg/kg). In contrast, the anxiolytic-like effects of HD (100 mg/kg) were inhibited by flumazenil but not by WAY (Figs. 5, 6; p 0.05). Effect of AEGE, HA, or HD on the Locomotor Activity and the number of entries into the open arms of the elevated plus-maze, 1 h after the intraperitoneally injection of HA (100 mg/kg), HA (100 mg/kg) WAY (0.3 mg/kg) or flumazenil (10 mg/kg) (30 min prior testing, i.p.), or vehicle solvent; n mice per group. p values for the group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 versus the salinetreated control, # p 0.05 as compared with the HA-treated group). Test and Horizontal Wire Test A locomotor activity test was performed to differentiate between the possible stimulatory effects of the tested drugs on the modulation of exploratory behavior. AEGE (50, 100, 200, 400 mg/kg), HA (5, 10, 25, 50, 100 mg/kg), or HD (5, 10, 25, 50, 100 mg/kg) caused no significant changes in either the total ambulatory distances or rearing frequencies compared with the saline control group (data not shown). Moreover, AEGE (50, 100, 200, 400 mg/kg), HA (5, 10, 25, 50, 100 mg/kg), or HD (5, 10, 25, 50, 100 mg/kg) did not

4 264 Vol. 29, No. 2 Fig. 6. Anxiolytic-Like Effects of 4-Hydroxybenzaldehyde Were Blocked by Flumazenil But Not by WAY and the number of entries into the open arms of the elevated plus-maze, 1 h after the intraperitoneally injection of HD (100 mg/kg), HD (100 mg/kg) WAY (0.3 mg/kg) or flumazenil (10 mg/kg) (30 min prior testing, i.p.), or vehicle solvent; n mice per group. p values for the group comparisons were obtained by one way ANOVA followed by Student Newman Keuls test ( p 0.05 versus the vehicletreated control, # p 0.05 as compared with the HD-treated group). compromise the mice grasping the wire compared with saline control group, indicating a lack of myorelaxation at these doses. DISCUSSION AEGE significantly increased the percentage of time spent in the open arms and the percentage of the open arm entries in a mouse using the EPM test, and these effects were antagonized by both WAY and flumazenil. This demonstrated the involvement of both the 5-HT 1A and GABA A receptors. Moreover, the anxiolytic-like effects of HA and HD, which are the main ingredients of AEGE, were blocked by WAY and flumazenil, respectively. Therefore, both HA and HD play roles in the anxiolytic-like effects of AEGE. However, there were no changes in locomotor activities or myorelaxant effects. Accordingly, AEGE has an anxiolytic-like effect that is mediated by 5-HT 1A and GABA A receptor activation, which is dependent on the HA and HD, and has no adverse effect, such as myorelaxant effects. The dried rhizome of G. elata is used in traditional Chinese, Japanese, and Korean medicine as an anticonvulsant, an analgesic, and a sedative against general paralysis, epilepsy, vertigo, and tetanus. G. elata is always included as an essential ingredient in prescriptions issued by traditional Chinese medicine practitioners for the treatment of convulsion, epilepsy, and cerebral apoplexy. 11) Ha et al. reported that the ether fraction of the methanol extracts of G. elata inhibited the GABA transaminase activity and on the radioligands to the GABA A receptor complexes of rat cerebral cortices. 12) Wu et al. reported that HA can act by suppressing the dopaminergic and serotonergic activities thereby improving learning. 13) However, to our knowledge no study has examined the anxiolytic-like effects of AEGE and its constituents or determined what neuronal system is primarily involved in. The EPM is considered to be an etiologically valid animal model of anxiety because it uses natural stimuli, such as a fear of a new, brightly lit open space and a fear of balancing on a relatively narrow raised surface. 14) An anxiolytic agent increases the frequency of entries into the open arms and increases the time spent in open arms of the EPM. In this study, the buspirone treatment prolonged the percentage of time spent in the open arms and the percentage of open arm entries (Fig. 1). The AEGE treatment also prolonged the percentage of time spent in the open arms as well as the percentage of open arm entries without altering the spontaneous behavior at the chosen dose regimen. The total distances of movement on the EPM were also unchanged by the AEGE treatment versus the saline controls (data not shown). In the horizontal wire test, no significant myorelaxant effect was observed after administering the AEGE. These observations suggest that the anxiolytic-like effect of AEGE is selective, and not the result of either a general stimulation of the locomotor activity or an exploratory behavior consequent to exposure to a novel environment. In addition, after treating the mice with AEGE at 400 mg/kg, the anxiolytic-like effects were antagonized by both WAY , a 5-HT 1A receptor antagonist and flumazenil, a GABA A receptor antagonist (Fig. 4). Therefore, it is believed that the 5-HT 1A and GABA A receptors primarily mediate the anxiolytic-like effects of AEGE. HA and HD are major phenolic compounds isolated from G. elata. Previously, it was reported that HD and its derivative, 4-hydroxy-3-methoxybenzaldehyde inhibited the [ 3 H]Ro and [ 3 H]flunitrazepam binding to the benzodiazepine receptor on the GABA A receptor complex without a concentration response relationship. 6) The present results showed that HD (100 mg/kg) has anxiolytic-like effects and, probably works via the activation of the benzodiazepine site of the GABA A receptors in the central nervous system because those effects were blocked by flumazenil (Fig. 6). Many clinical and preclinical studies have confirmed the anxiolytic properties of the 5-HT 1A receptor agonists, such as buspirone. 15) EPM test showed that the HA (50, 100 mg/kg) treatment produced good anxiolytic-like activities (Fig. 2). Furthermore, these anxiolytic-like behaviors were completely blocked by WAY , a specific 5-HT 1A receptor antagonist (Fig. 5). Therefore, the anxiolytic-like activity of HA may be mediated via the activation of the 5- HT 1A receptor. However, vanillin which is also a main ingredient of G. elata did not exhibit any anxiolytic-like effects (data not shown). Collectively, these results suggested that both HA and HD are responsible for the anxiolytic-like effect of AEGE because the anxiolytic-like effect of AEGE is mediated by the activation of the both 5-HT 1A and GABA A receptors. However, considering the contents of HA and HD from AEGE as described in the Materials and Methods part (400 mg of AEGE contained 38 mg of HA and 14 mg of HD), they are not the only compounds showing the anxiolytic-like effect because these effects were observed at slightly high doses (50 or 100 mg/kg). It is speculative that the other components may co-act with HA and HD on the anxiolytic-like effects of AEGE. Otherwise, the other unidentified compounds may have the anxiolytic properties. Further studies are required to elucidate a major compound of AEGE.

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