Abstract: Gastrodia elata B1. (GE) is a traditional Chinese herb that is commonly used in Chinese communities to treat convulsive disorders such as epilepsy. The purpose of the present study was to determine the anticonvulsive and free radical activities of GE in rats. In vitro studies were conducted by using brain tissue from 6 male Sprague-Dawley (SD) rats treated with 120 [micro]g/ml of kainic acid (KA), with or without the addition of various concentrations of GE. In vivo studies were conducted in a total of 30 male SD rats divided into 5 groups of 6 rats which were treated as follows: 1) the normal group received an intraperitoneal injection (i.p.) of PBS (Phosphate buffer saline, 1 ml/kg); 2) the control group received KA (12 mg/kg) i.p.; 3) the GE 1.0 group received oral administration of GE 1.0 g/kg 30 min prior to KA administration; 4) the GE 0.5 group received oral administration of GE 0.5 g/kg 30 min prior to KA administration; 5) the PH group received oral administration of phenytoin 20 mg/kg 30 min prior to KA administration. Seizures were verified by behavioral observations, electroencephalograph (EEG) and electromyography (EMG). Lipid peroxide levels in the rat brain, luminol chemiluminescence (CL) and lucigenin-CL in the peripheral blood were measured simultaneously after behavioral observations. The results indicate that GE administration significantly reduced KA-induced lipid peroxide levels in vitro. Oral administration of GE 1.0 g/kg and phenytoin 20 mg/kg significantly reduced counts of wet dog shakes (WDS), paw tremor (PT) and facial myoclonia (FM) in KA-treated rats. In addition, oral administration of GE 1.0 g/kg significantly delayed the onset of WDS, from 30 min in the control group to 46 min in the 0.5 g/kg group, and 63 min in the GE 1.0 g/kg group. A significantly reduced level of lipid peroxides in the rat brain was found in the GE 1.0 g/kg, 0.5 g/kg, and phenytoin 20 mg/kg groups, The GE 1.0 g/kg group showed significant reduction of luminol-CL and lucigenin-CL counts in the peripheral blood compared to the control group. The results of the present study demonstrate that GE has anticonvulsive and free radical scavenging activities. Further studies are needed to determine the clinical effectiveness of GE as an anticonvulsant in humans.
Gastrodia elata B1. (GE) is a traditional Chinese herbal drug used in the treatment of dizziness and convulsive disorders. A previous study found that GE can inhibit the increase of lipid peroxide levels evoked by ferric chloride in rats, and also that GE has free radical scavenging effects (Liu and Mori, 1992). These effects of GE were considered to result mainly from the activity of its active components, including vanillin and p-hydroxybenzyl alcohol (Liu and Mori, 1992, 1993).
Kainic acid (KA) is a neurotic analogue of glutamate, which has a potent neuroexcitatory action. Several studies have demonstrated that intracerebral or intraperitoneal injection (i.p.) of KA can induce limbic motor seizures in rats, with behaviors mainly including wet dog shakes (WDS), paw tremor (PT) and facial myoclonia (FM) (Schwob et al., 1980; Tremblay et al., 1984; Nitecka et al., 1984). The behavioral manifestations of KA-treated rats are similar to those of temporal lobe seizures in humans, and are thought to result from neuronal damage mainly in the hippocampus and amygdala complex of the central nervous system (Schwob et al., 1980; Tremblay et al., 1984; Nitecka et al., 1984; Wuerthele et al., 1978; Ben-Ari, 1985; Tanaka et al., 1988, 1990, 1992). Our previous studies showed that kainic acid i.p. can induce the increase of lipid peroxide levels in the rat brain and evoke the generation of oxygen free radicals in the peripheral blood (Hsieh et al., 1999a). We also found that the time course of WDS correlated positively with brain lipid peroxide levels, and luminol chemiluminescence (CL) and lucigenin-CL counts in the whole blood in KA-treated rats (Hsieh et al., 1999b). Previous studies have demonstrated that KA may cause pathological changes similar to those of brain infarction (Jorgensen et al., 1991; Liu et al., 1996), and also induce increases of lipid peroxide levels, suggesting that KA-induced neuronal damage is possibly mediated by oxygen free radicals (Bruce and Baudry 1995). Oxidative stress can be measured from the whole blood using an ultra-sensitive chemiluminescence analyzer and lucigenin amplifier without leukocyte isolation as previously reported (Sun et al., 1996, 1998).
Although GE is used as an anticonvulsant in Chinese communities, the mechanism of its anticonvulsive actions remains unclear. The aim of the present study was to investigate the anticonvulsive and free radical scavenging activities of GE. First, we studied the effect of GE on KA-induced lipid peroxidation in rat brain in vitro. Second, we determined the anticonvulsive effect of GE on KA-induced seizures. Finally, we measured the level of oxygen free radicals in KA-treated rats with or without GE pretreatment. Seizures were verified by behavioral observations, EEG and EMG recordings. The counts of WDS, PT, and FM, and the onset time of WDS were used as indicators of the anticonvulsive effects. Lipid peroxide levels in rat brains and luminol-CL and lucigenin-CL counts in the peripheral whole blood were measured simultaneously.
Materials and Methods
Extraction of GE
The GE used in this study originated from the Sichuan province of China, and was collected in crude form in Taiwan, and authenticated by the Graduate Institute of Chinese Pharmaceutical Sciences, China Medical College, Taichung, Taiwan. Two kg of crude GE were extracted 4 times with 3 liters of 50% alcohol. The extracts were filtered, frozen-dried, and then stored in a drier box. The total yield was 147 g (7.35%).
Adult male SD rats weighing 200-250 g were housed in groups of 3 rats in iron cages and maintained on a 12-hr, light-dark cycle at 25 [degrees] C. The present study was divided into two experiments as follows: 1) measurement of lipid peroxide levels in vitro; and 2) behavioral observation, electroencephalograph (EEG) and electromyograph (EMG) recordings and measurement of free radical levels in vivo.
Measurement of Lipid Peroxide Levels in vitro
Six rats were killed under pentobarbital anesthesia (50 mg/kg i.p.). The brain of each rat was removed after transcardiac perfusion of 10 ml iced normal saline, and the cerebral cortex was then separated from the brain. The cerebral cortex was thawed and homogenized in Tris-HCl solution (pH 7.4), and the homogenates were centrifuged at 3000 rpm for 15 min. The supernatant was then used to determine the lipid peroxide level by measuring the concentration of malondialdehyde (MDA). Either GE 10 mg/ml, GE 1 mg/ml, GE 100 [micro]g/ml, GE 10 [micro]g/ml or vitamin E 10 mM was added, respectively, to the samples after the addition of KA (King Dom Co., Taiwan, 120 [micro]g/ml). The samples were incubated for 2 hrs at 37 [degrees] C, followed by the addition of 8.1% SDS and TBAR (2-thiobarbituric acid + 20% acetic acid). The samples were then incubated again at 95 [degrees] C for 1 hr and cooled to room temperature, followed by the addition of 5 ml n-butanol and centrifugation at 3000 rpm for 15 min. The MDA level was read at 532 nm (Spectrophotometer, Beckman Instruments Inc., California, USA). A standard curve was established by using known amount of MDA equivalent under the same assay conditions, and results were calculated as n mol/g tissue.
Behavioral Observations and EEG and EMG Recordings
A total of 30 rats were divided into 5 different treatment groups of 6 rats as follows: 1) the normal group received PBS (phosphate buffer saline, Sigma Co., USA) 1 ml/kg i.p.; 2) the control group received KA (12 mg/kg) i.p.; 3) the GE 1.0 group received oral administration of GE 1.0 g/kg 30 min prior to KA administration; 4) the GE 0.5 group received oral administration of GE 0.5 g/kg 30 min prior to KA administration; 5) the PH group received oral administration phenytoin (Sigma Co., USA) 20 mg/kg 30 min prior to KA administration. One week prior to the experiment, recording electrodes were placed under pentobarbital (50 mg/kg, i.p.) anesthesia by fixing the head of each rat in a stereotactic apparatus. The skull was exposed and stainless steel screws were implanted on the dura over the bilateral sensorimotor cortices. A reference electrode was placed in the frontal sinus. Bipolar electrical wires passing through the subcutaneous tissue were placed on the neck muscle for EMG recording. These electrodes were plugged into a connector, and then connected to an EEG and EMG-recorder machine (MP 100WSW, BIOPAC System, Inc., California, USA). Behavioral observations, and EEG and EMG recordings were performed continuously from 30 min prior to drug administration to 3 hrs after KA administration. The anticonvulsive effects of GE were evaluated after KA administration using the total counts of WDS, PT, and FM and the onset time of WDS as indicators. The type of seizure was verified by behavioral observations and the presence of epileptiform activity on EEG and EMG recordings.
Measurement of Free Radicals In Vivo
Lipid peroxide levels in rat brain and luminol-CL and lucigenin-CL counts in the peripheral whole blood were measured simultaneously after behavioral observations and EEG and EMG recordings. Whole blood samples (2 ml) were obtained by transcardiac puncture with heparinized plastic syringes under pentobarbital anesthesia in each rat. Blood samples were immediately wrapped with aluminum foil to prevent light exposure and kept in an ice box until testing for CL, which in general was measured within 2 hr. The tube of 1 ml blood with EDTA was used for counting white blood cells (WBC). After transcardiac perfusion with 10 ml iced saline, the frontal cortex, amygdala, and hippocampus were removed from the brains, thawed, and homogenized in Tris-HCl pH 7.4 solution. The homogenates were then centrifuged at 3000 rpm for 15 min and the supernatants were used for determination of the lipid peroxide level via the MDA method, as described above.
The method for measuring luminol-CL was similar to that described previously (Sun et al., 1998). Briefly, 0.2 ml of whole blood was mixed with 0.1 ml of PBS (pH 7.4) in a special chamber unit (Model CLD-110, Tohoku Electronic Indust. Co., Sendai Japan). The chemiluminescence was then measured in an absolutely dark chamber of the Chemiluminescence Analyzing System (Tohoku). This system includes a photon detector (Model CLD-110), a chemiluminescence counter (Model CLC-10), a water circulator (Model CH-201), and a 32-bit IBM personal computer. After 200 seconds, 1.0 ml of 25 [micro]M luminol (Sigma Co., USA) in PBS was injected into the stainless steel cell and the chemiluminescence of the blood sample was measured continuously. After 600 seconds, 0.2 ml of Zymosan-A (Sigma Co., USA) was added, and the chemiluminescence of the blood sample was measured continuously for a total of 1020 seconds. The total chemiluminescence count was calculated by integrating the area under the curve and subtracting it from the background level. The production of CL per WBC (white blood cell) was calculated by dividing the blood CL levels by the WBC count, and expressed as CL/1000 WBC.
The method for measuring lucigenin-CL was similar to that described previously (Sun et al., 1996, 1998; Chen et al., 1997). Briefly, 0.1 ml PBS (pH 7.4) was added to 0.2 ml of blood sample. The chemiluminescence count was then measured in an absolutely dark chamber of the CL Analyzing System, as described above. After 200 seconds, 1.0 ml 0.01 mM Lucigenin (Sigma Co., USA) in PBS was injected into the stainless steel cell and the chemiluminescence of the blood sample was measured continuously. After 600 seconds, 0.2 mi Zymosan-A was added by the same method, and the chemiluminescence of the blood sample was measured continuously for a total of 1020 seconds. The total CL and the production of CL per WBC were calculated as described above.
The data are reported as mean [+ or -] SD. One way analysis of variance (ANOVA) followed by Scheffe's test was used for comparisons among groups. A p-value [is less than] 0.05 was considered statistically significant.
Effect of GE on KA-induced Lipid Peroxidation In Vitro
The level of lipid peroxides increased after KA administration in vitro (P [is less than] 0.001, Figure 1). This increase was inhibited by GE 10 mg/ml, GE 1 mg/ml, GE 100 [micro]g/ml, GE 10 [micro]g/ml and vitamin E 10 mM/ml (P [is less than] 0.001, Figure 1). GE 10 mg/ml and GE 1 mg/ml showed the strongest inhibition, followed by GE 100 [micro]g/ml and GE 10 [micro]g/ml (Figure 1). Vitamin E (10 mM/ml) had a significantly greater inhibitory effect than GE 1 mg/kg, GE 100 [micro]g/ml and GE 10 [micro]g/ml (all P [is less than] 0.01, Figure 1).
Effects of GE on KA-induced Seizures
All 24 SD rats developed epileptic seizures after i.p. administration of KA (12 mg/kg). The types of seizure included WDS, PT, and FM. Each type of seizure had its own characteristic electrophysiological pattern (Figure 2).
Oral administration of both GE 1 g/kg and phenytoin 20 mg/kg significantly reduced the counts of WDS (P [is less than] 0.001, Figure 3), PT (P [is less than] 0.001) and FM (P [is less than] 0.05, Figure 3) in KA treated rats, but no similar effect on FM was found in rats pretreatment with GE 0.5 g/kg (P [is greater than] 0.05, Figure 3). The time to onset of WDS after KA administration increased from 30 min in the control group, to 46 and 63 min in the GE 0.5 group and in the GE 1.0 group (P [is less than]0.001, Figure 3), but the phenytoin 20 mg/kg group showed no significant delay in the onset of WDS (P [is greater than] 0.05, Figure 3).
Effects of GE on Free Radical Scavenging Activity in KA-treated Rats In Vivo
The lipid peroxide levels in the frontal cortex, amygdala, and hippocampus regions of rat brain increased significantly after KA administration in vivo (P [is less than] 0.001, Figure 4). These increases were significantly suppressed by oral administration of GE 1.0 g/kg, 0.5 g/kg or phenytoin 20 mg/kg (all P [is less than] 0.001, Figure 4).
The luminol-CL counts in the whole blood increased after KA administration (P [is less than] 0.001, Figure 5), and this increase was significantly inhibited by oral administration of GE 1.0 g/kg (P [is less than] 0.001, Figure 5), but no significant inhibition was observed in the GE 0.5 g/kg group and the phenytoin 20 mg/kg group (P [is less than] 0.05, Figure 5).
The lucigenin-CL counts in the whole blood also increased after KA administration (P [is less than] 0.001, Figure 5), and this increase was significantly inhibited by oral administration of either GE 1.0 g/kg, GE 0.5 g/kg or phenytoin 20 mg/kg (all P [is less than] 0.001, Figure 5).
GE Has Anticonvulsive Effects in KA-treated Rats
Our results showed that rats developed seizures, including WDS, PT and FM after KA administration. These findings are consistent with our previous study and other previous studies of the effect of KA administration in rats (Schwob et al., 1980; Tremblay et al., 1984; Nitecka et al., 1984; Hsieh et al., 1999a). Previous authors have considered the generation of these seizures to result mainly from KA-induced cerebral damage in the hippocampus and amygdala complex regions. The results of the present study indicate that oral administration of GE 1.0 g/kg and phenytoin 20 mg/kg 30 min prior to KA administration significantly reduced the frequency of WDS, PT and FM. In addition, GE 1.0 g/kg delayed the onset of WDS from 30 min in the control group to 63 min, suggesting that GE has anticonvulsant effects in KA-treated rats. These results may be explained by a previous study which demonstrated that binding of KA to glutamate receptors can be inhibited by a water extract of GE (Andersson et al., 1995). Lanthorn and Isaacson (1978) demonstrated that the development of WDS has a close relation to the glutamate receptor because it can be inhibited with a glutamate receptor blocking agent, such as glutamate acid diethylester (GDEE).
GE Inhibits KA-induced Lipid Peroxidation In Vitro
We found that GE has a suppressive effect on KA-induced lipid peroxidation in rat brain in vitro. Melchiorri et al. (1995) reported that oxidative damage in different regions of rat brain may be induced by KA administration, therefore, suggesting GE has a free radical scavenging effect in ICA treated rat in vitro. This finding is also similar to a previous study indicating that GE inhibits the increase of lipid peroxide levels in ferric chloride-treated rats in vitro (Liu and Mori 1992).
GE Exhibits Free Radical Scavenging Activity
Our results indicate that lipid peroxide levels increased in the frontal cortex, amygdala and hippocampus regions after KA administration. These results are in good agreement with two previous studies (Bruce and Baudry, 1995; Melchiorri et al., 1995). Several studies have considered KA-induced neuronal damage to be mediated via the formation of free radicals (Bruce and Baudry, 1995; Kim et al., 1997). In the present study, we found that the increase of lipid peroxide levels in KA-treated rats was significantly inhibited by oral administration of GE 1.0 g/kg. GE 0.5 g/kg or phenytoin 20 mg/kg. This effect of GE is very similar to previous findings that GE and its active components, including p-hydroxybenzyl alcohol and vanillin, have a suppressive effect on ferric chloride-induced lipid peroxidation in rat brains (Liu and Mori, 1992, 1993).
Our results demonstrate that GE 1.0 g/kg significantly reduced the increase of luminol-CL and lucigenin-CL counts in the peripheral blood evoked by KA administration, suggesting that GE exhibits free radical scavenging activity since luminol-CL levels are considered to be a sensitive indicator of the production of reactive oxygen species, including superoxide anion, hydroxyl radicals, hydrogen peroxide and hypochlorous acid (Kaever et al., 1992; Sun et al., 1996). Lucigenin-CL levels can be generated by NADP(H) oxidase in leucocytes, and specifically represents superoxide anion (Gyllenhammar, 1987; Lu et al., 1996; Chen et al., 1997).
In conclusion, the results of the present study demonstrate that GE has anticonvulsive and free radical scavenging activities. Further studies are needed to determine the clinical effectiveness of GE as an anticonvulsant in humans.
This study was supported by a grant from the Committee on Chinese Medicine and Pharmacy, Department of the Health, Executive Yuan, R.O.C., CCMP88-RD-020.
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Ching-Liang Hsieh(1)(*), Su-Yin Chiang(2), Ken-Sheng Cheng(3), Yu-Hsien Lin(4), Nou-Ying Tang(2), Chia-Jung Lee(2), Chu-Zong Pon(2) and Ching-Tou Hsieh(5)
(1) Chang Gung Traditional Chinese Medicine Hospital and Chang Gung University, Graduate Institute of Traditional Chinese Medicine, 5, Fu-Shing Street, Kwei-Shan, Taoyuan, Taiwan (2) School of Chinese Medicine and Institute of Chinese Medical Science, China Medical College, Taichung, Taiwan (3) Division of Gastroenterology, Department of Internal Medicine, China Medical College Hospital, Taichung, Taiwan (4) Department of Life Science, School of Life Science, National Yang-Ming University, Taipei, Taiwan (5) Department of Internal Medicine, Jen-Ai Hospital, Taichung, Taiwan
(*) Corresponding author
(Accepted for publication August 28, 2000)
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