Effects of Puerarin on Scopolamine-, Mecamylamine-, p-Chloroamphetamine- and Dizocilpine-Induced Inhibitory Avoidance Performance Impairment in Rats

• Abstract
• Introduction
• Materials and Methods
• Animals
• Chemicals
• Inhibitory avoidance task
• Inhibitory avoidance behavior with a brief shock
• Effect of puerarin on cholinergic antagonists-, PCA-, and
  MK-801-induced amnesia
• Inhibitory avoidance behavior with no shock
• Statistics
• Results
• Discussion
• Acknowledgements
• References

Abstract

Puerarin at 10 - 50 mg/kg attenuated the mecamylamine- but not scopolamine-induced acquisition impairment of inhibitory avoidance performance in an inverse U-shape manner. p-Chloroamphetamine- and dizocilpine-induced acquisition impairment were reversed by puerarin at 25 - 50 mg/kg. Both piracetam, and tacrine attenuated impairment of inhibitory avoidance performance induced by all used drugs. Furthermore, puerarin, piracetam and tacrine alone did not alter step-through latency in the training trail but puerarin at 50 mg/kg and tacrine plus mecamylamine prolonged it in comparison with mecamylamine alone. From these results, we suggest that puerarin attenuated the deficits of inhibitory avoidance performance induced by mecamylamine, p-chloroamphetamine, and dizocilpine, the effects were related to increasing cholinergic activity via nicotinic but not muscarinic receptors, activating NMDA receptors, and decreasing serotonergic neuronal activity.

Introduction

Pueraria lobata (Willd.) Ohwi, a traditional Chinese medicine, was used for the treatment of headache and cardiovascular disease by physicians. Early reports suggested that the roots of P. lobata possessed anti-arrhythmia and vasodilative activities [1]. Recent researchers suggested the radix of P. lobata after chronic administration reversed the impairment of step-down performance induced by scopolamine, sodium nitrite and 40 % ethanol, and its total isoflavonoids also reversed memory deficits induced by scopolamine, ethanol, D-galactose, and middle cerebral artery obstruction [2], [3]. Puerarin whose chemical structure is shown in Fig. [1] was a special ingredient of total isoflavonoids isolated from P. lobata [4]. According to the cholinergic theory on memory function that cholinergic muscarinic and nicotinic receptors play important roles in learning and memory [5] and earlier reports related to the attenuating effects of P. lobata and its total isoflavonoids on muscarinic receptor antagonist scopolamine-induced impairment [2], [3], our preliminary data showed that impure puerarin attenuated memory deficits in rats induced by nicotinic receptor antagonist mecamylamine.

Secondly, much evidence suggests that 5-HT neurons participate in learning and memory in humans and animals [6]. Because serotonergic system mainly inhibits the acetylcholine release in hippocampal slices [7], recent studies show a consistent trend in which the central 5-HT system plays an inhibitory role in avoidance and aversive conditioning tasks [8]. Therefore, p-chloroamphetamine, a serotonin releaser, is also used as a model in the screening of anti-amnestic drugs [8]. Finally, NMDA receptors play an important role in learning acquisition, long-term potentiation and synaptic plasticity [9]. Dizocilpine, a NMDA receptor antagonist, induced learning acquisition impairment in inhibitory avoidance performance and water maze in rodents when administered before the training trial [10]. Therefore, the present study again investigated the attenuating effect of pure puerarin (98 %) on scopolamine-, mecamylamine-, p-chloroamphetamine-, and dizocilpine-induced impairment of inhibitory avoidance performance in rats with piracetam and tacrine as positive controls.

Materials and Methods

Animals

Male Sprague-Dawley rats, weighed 200 - 250 g, were obtained from the Animal Center of China Medical College. All rats were used in the experiments below according to the Guiding Principles for the Care and Use of Laboratory Animals. They were randomly housed six per wire-mesh cage (39 × 26 × 21 cm) for at least 1 week before starting experiments in a temperature-(23±1 °C) and humidity-(60 %) regulated environment with free access to standard food in pellets (supplied and designed by Fwusow Industry Co. Ltd., Taiwan) and tap water, and a 12 h - 12 h light/dark cycle (light phase: 08 : 00 to 20 : 00 h) was maintained. The rats were randomly assigned into groups for the following experiments. The number of subjects in each group is depicted in the figures. Then, the drugs below were administered and the behavioral assays were operated in a double-blind method.

Chemicals

Puerarin whose purity was above 98 % was purchased from Wako Pure Chemical Industries. Ltd. (Japan) and dissolved with 0.02 % ethanol. Other used drugs were purchased from Sigma Chemical Co. (USA), except for dizocilpine (MK-801) and 9-amino-1,2,3,4-tetrahydroacridine hydrochloride (tacrine, THA) purchased from Research Biochemical Inc. (USA). The vehicle-treated rats received the same volume (0.1 ml/100 g body weight). Piracetam, tacrine, scopolamine hydrobromide (SCOP), mecamylamine (MECA), p-chloroamphetamine (PCA), and MK-801 were dissolved in saline. Finally, all drugs were intraperitoneally administered 30 min before the training trial.

Inhibitory avoidance task

The apparatus consisted of two compartments with a steel-rod grid floor (36 parallel steel rods, 0.3 cm in diameter set 1.5 cm apart). One light compartment (48 × 20 × 30 cm) with a 20W lamp centrally 30 cm above it, and one dark compartment of the same size, were connected through a guillotine door (5 × 5 cm). The room was dark during the experimental sessions that were conducted between 09 : 00 and 12 : 00 hours.

Inhibitory avoidance behavior with a brief shock

On the training trial, the guillotine door between the light and dark compartment was closed. When each rat was placed in the light compartment with its back to the guillotine door, the door was opened and simultaneously the time (step-through latency, STL) was measured with a stopwatch until the rat entered the dark compartment. After the rat entered the dark compartment, the door was closed. An inescapable scrambled footshock (1 mA for 2 s) was delivered through the grid floor. The rat was removed from the dark compartment 5 s after the shock. Then the rat was put back into the home cage until the retention trial. Twenty-four hours later, the retention trial was carried out. The rat was again placed in the light compartment, as on the training trial, the guillotine door was opened and the step-through latency was recorded and used as a measure of retention [11]. An upper cut-off time of 300 sec was set.

Effect of puerarin on cholinergic antagonists-, PCA-, and
MK-801-induced amnesia

All rats were separated into five groups induced by saline, SCOP, MECA, PCA, and MK-801. SCOP (0.5, 1 mg/kg), MECA (10 mg/kg), PCA (5 mg/kg), and MK-801 (0.1 mg/kg) were intraperitoneally given to rats 30 min before the training trial respectively [8], [11], [12]. Each group, except for saline, was intraperitoneally coadministered with vehicle or puerarin (10, 25 and 50 mg/kg), and then the training trial was carried out 30 min after the treatment. Piracetam (50 mg/kg) and tacrine (3 mg/kg) as positive controls were given intraperitoneally to rats 30 min before the training trial [13].

Inhibitory avoidance behavior with no shock

Experimental steps were the same as described in the above section, but without the 1.0 mA footshock. Twenty-four hours later, the retention trial was carried out. The rat was again placed in the light compartment, as on the training trial, the guillotine door was opened and the step-through latency was recorded and used as a measure of retention [11].

Statistics

All data of the inhibitory avoidance behavior with a brief shock were expressed with medians and interquartile ranges and analyzed using a Kruskal-Wallis non-parametric one-way analysis of variance, followed by Mann-Whitney’s U-test because the data were derived from a cut-off procedure. All data of the inhibitory avoidance behavior with no shock were expressed in terms of mean and standard errors, and further analyzed with one-way analysis of variance (ANOVA) followed with Scheffe multiple range test because the data were represented by means with a normal distribution. The criterion for statistical significance was p < 0.05 in all statistical evaluations.

Results

SCOP at 0.5 or 1 mg/kg, administered to rats 30 min before the training trial, significantly shortened the step-through latency in the retention trial compared to the vehicle-treated rats. Puerarin at 10 - 50 mg/kg did not reverse the shorter step-through latency induced by SCOP, but piracetam and tacrine did (Fig. [2]). The step-through latency in the retention trial of the MECA-treated group was also significantly shorter than that of the vehicle-treated group. The step-through latency shortened by MECA was significantly reversed by puerarin at 10 - 50 mg/kg, piracetam and tacrine (Fig. [3]). The step-through latency in the retention trial of the PCA-treated group was significantly shorter than that of the vehicle-treated group. The shorter step-through latency induced by PCA was also significantly reversed by puerarin at 25 - 50 mg/kg, piracetam and tacrine (Fig. [3]). MK-801 at 0.1 mg/kg, administered to rats 30 min before the training trial, significantly shortened the step-through latency in the retention trial compared to the vehicle-treated rats. Puerarin at 25 - 50 mg/kg, piracetam and tacrine reversed the shorter step-through latency induced by MK-801 (Fig. [4]).

The step-through latency in the training trial of rats receiving puerarin, piracetam and tacrine was not different from that of the vehicle-treated rats (Table [1]). The step-through latency in the training trial of rats receiving SCOP, MECA, PCA, or MK-801 alone was not different from that of the vehicle-treated rats. Neither rats receiving puerarin at 10 - 50 mg/kg plus SCOP, MECA, PCA or MK-801 nor rats receiving piracetam plus the above drugs were different from rats receiving SCOP, MECA, PCA or MK-801 alone with regard to the step-through latency in the training trial, in addition to the step-through latency of rats treated with puerarin at 50 mg/kg plus MECA was longer than that of MECA-treated rats (Table [2]). Rats receiving tacrine plus SCOP, PCA or MK-801 were not different from rats receiving SCOP, PCA or MK-801 with regard to the step-through latency in the training trial, but the step-through latency of tacrine-treated rats plus MECA was longer than that of MECA-treated rats (Table [2]).

Discussion

Our previous report and other reports suggested that SCOP at 0.5 - 1 but not at 0.3 mg/kg significantly shortened the step-through latency in the retention trial [11], [12]. The present data showed that puerarin at any dose after acute administration did not reverse the impairment of inhibitory avoidance performance induced by SCOP at 0.5 or 1 mg/kg and these results were inconsistent with those of the total isoflavonoids of P. lobata after chronic administration [2], [3]. Because total isoflavonoids at 0.5 - 1 g/kg after chronic administration reversed the step-through latency shortened by SCOP and the yield of puerarin isolated from total isoflavonoids of P. lobata was about 2.3 %, the dosage of puerarin used in the present study might be too low to attenuate memory deficits induced by SCOP. As for the effects of puerarin after chronic administration on memory deficits, thus must remain to be further evaluated. Two positive controls, piracetam and tacrine, reversed the step-through latency shortened by SCOP and such findings are consistent with a previous report [13]. Then we might suggest that puerarin itself has no effect on cholinergic muscarinic receptors after acute administration in contrast to piracetam and tacrine. According to a recent review, cholinergic nicotinic receptors also play important roles in learning and memory [5]. The present data, similar to an early report [10], revealed that MECA at 10 mg/kg significantly shortened the step-through latency in the retention trial. Puerarin at 10 - 50 mg/kg reversed the impairment of inhibitory avoidance performance induced by MECA in an inverse U-shaped manner, and its effect was best at 25 mg/kg. Two positive controls, piracetam and tacrine, also reversed the step-through latency shortened by MECA. The potency of puerarin-induced reversal at 25 mg/kg was equal to that of tacrine at 3 mg/kg. A previous report pointed out that muscarinic and nicotinic receptors have the differential effects in learning and memory [14]. Therefore, the reversal caused by puerarin from the impairment might be related to activating the cholinergic neuronal system via nicotinic but not muscarinic receptors.

Secondly, puerarin at 25 - 50 mg/kg, piracetam, and tacrine reversed the impairment of inhibitory avoidance performance induced by PCA and MK-801. The potency of puerarin (25 mg/kg)-induced reversal of PCA-induced impairment was equal to that of piracetam at 50 mg/kg. The potency of the puerarin (50 mg/kg)-induced reversal from MK-801-induced impairment was equal to those of tacrine at 3 mg/kg and piracetam at 50 mg/kg. Gandolfi et al. reported that the NMDA receptor antagonist APV impaired the passive avoidance retention via 5-HT-stimulated phosphatidylinositol metabolism [15]. Other researchers pointed out that MK-801 increased the number of 5-HT_1A serotonin receptors in rat brain [16]. Moreover, the 5-HT_1A receptor antagonist WAY 100 635 prevented the impairment of spatial learning induced by an NMDA receptor antagonist [17]. Yin and Zeng suggested that puerarin inhibited 5-HT release on platelet aggregation [18]. Therefore, the attenuated effects of puerarin on PCA- and MK-801-induced impairment might be related to modulating the central serotonergic activity. On the other hand, the serotonergic system mainly inhibits acetylcholine release in hippocampal slices [7] and nicotinic acetylcholine receptor mediated modulation of evoked excitatory amino acid release in rat [19]. Therefore, the relationship among excitatory amino acids, serotonin and nicotinic receptors on the reversal caused by puerarin from the impairment shall be further investigated and demonstrated.

On the other hand, the relationship between motor alteration and performance in rats receiving puerarin, piracetam, tacrine and tested drugs were demonstrated because an alteration in the motivational or motor systems during training can affect acquisition of the avoidance response. Although recent reports indicated that total isoflavonoids shortened sleeping time in mice [20] and that puerarin inhibited [³ H]-flunitrazepam binding to rat brain membrane [21], puerarin alone and the tested drugs did not alter motor activities. However, the step-through latency of rats given puerarin at 50 mg/kg plus MECA was longer than that of rats treated with MECA alone. Therefore, the data demonstrated that the beneficial effects of puerarin in rats might be related to memory processes but not to motor activity and attention. Since the effect of puerarin at 50 mg/kg on MECA-induced impairment was worse, it might be related to decreasing attention.

In summary, the results of the present study proved that puerarin attenuated the deficits of inhibitory avoidance performance induced by drugs, and that the effects were different from those of piracetam and tacrine. The reversal induced by puerarin from the acquisition impairment was related to activating the central cholinergic neuronal system via muscarinic and nicotinic receptors. However, the role of other neurotransmitters on the reversal caused by puerarin from the impairment must be further studied.

Acknowledgements

We are thankful to the National Sciences Council for the financial support of this manuscript under countract No. NSC88-2314-B-039-002, NSC89-2320-B-039-049 and NSC89-2745-P-039-002, and Chin Medical College CMC89-CPS-02 and CMC90-CPS-05.

References

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Assistant Professor Dr. Chi-Rei Wu

Institute of Chinese Pharmaceutical Sciences

China Medical College

91, Hsieh Shih Road

40421, Taichung, Taiwan, R.O.C.

Email: crw@mail.cmc.edu.tw

Fax: 886-0943806654

References

• 1 Tsui S C, Peng H L. Recent researches on pharmacological effects of Pueraria lobata .  Shizhen J Trad Chin Med Res. 1998;  9 279-80
  PubMedSearch in Google Scholar
• 2 Wang H, Hsu T. Effects of Pueraria lobata and Cindium officinale on cognitive function in rodents.  J China-Japan Friendship Hosp. 1995;  9 191-4
  PubMedSearch in Google Scholar
• 3 Yu C L, Chang K C, Chao H C. Effects of total flavonoids isolated from Pueraria lobata on memory behavior in mice.  J China Pharm Univ. 1997;  28 350-3
  PubMedSearch in Google Scholar
• 4 Chen M H. Studies on the chemical constituents of Pueraria lobata .  Bull Chin Mat Med. 1985;  10 34-6
  PubMedSearch in Google Scholar
• 5 Levin E D, Simon B B. Nicotinic acetylcholine involvement in cognitive function in animals.  Psychopharmacology. 1998;  138 217-30
  CrossrefPubMedSearch in Google Scholar
• 6 McEntee W J, Crook T H. Serotonin, memory and the aging brain.  Psychopharmacology. 1991;  103 143-9
  PubMedSearch in Google Scholar
• 7 Muramastu M, Tamaki-Ohashi J, Usuki C, Araki H, Aihara H. Serotonin-2 receptor-mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat.  Neuropharmacology. 1988;  27 603-9
  CrossrefPubMedSearch in Google Scholar
• 8 Ogren S O. Evidence of a role of brain serotonergic neurotransmission in avoidance learning.  Acta Physiol Scand Suppl. 1985;  544 1-71
  PubMedSearch in Google Scholar
• 9 Cain D P. Testing the NMDA, long-term potentiation, and cholinergic hypotheses of spatial learning.  Neurosci Biobehav Rev. 1998;  22 181-93
  CrossrefPubMedSearch in Google Scholar
• 10 Hiramatsu M, Murasawa H, Nabeshima T, Kameyama T. Effects of U-50,488H on scopolamine-, mecamylamine- and dizocilpine-induced learning and memory impairment in rats.  J Pharmacol Exp Ther. 1998;  284 858-67
  PubMedSearch in Google Scholar
• 11 Wu C R, Hsieh M T, Liao J. p-Hydroxybenzyl alcohol attenuates learning deficits in the inhibitory avoidance task: involvement of serotonergic and dopaminergic systems.  Chin J Physiol. 1996;  39 265-73
  PubMedSearch in Google Scholar
• 12 Rush D K. Scopolamine amnesia of passive avoidance: a deficit of information acquisition.  Behav Neural Biol. 1988;  50 255-74
  CrossrefPubMedSearch in Google Scholar
• 13 Chopin P, Briley M. Effects of four non-cholinergic cognitive enhancers in comparison with tacrine and galanthamine on scopolamine-induced amnesia in rats.  Psychopharmacology. 1992;  106 26-30
  PubMedSearch in Google Scholar
• 14 Glick S D, Greenstein S. Differential effects of scopolamine and mecamylamine on passive avoidance behavior.  Life Sci. 1972;  11 169-79
  PubMedSearch in Google Scholar
• 15 Gandolfi O, Dall’Olio R, Roncada P, Montanaro N. NMDA antagonists interact with 5-HT-stimulated phosphatidylinositol metabolism and impair passive avoidance retention in the rat.  Neurosci Lett. 1990;  113 304-8
  CrossrefPubMedSearch in Google Scholar
• 16 Wedzony K, Mackowiak M, Czyrak A, Fijal K, Michalska B. Single doses of MK-801, a non-competitive antagonist of NMDA receptors, increase the number of 5-HT1A serotonin receptors in the rat brain.  Brain Res. 1997;  756 84-91
  CrossrefPubMedSearch in Google Scholar
• 17 Carli M, Silva S, Balducci C, Samanin R. WAY 100 635, a 5-HT1A receptor antagonist, prevents the impairment of spatial learning caused by blockade of hippocampal NMDA receptors.  Neuropharmacology. 1999;  38 1165-73
  CrossrefPubMedSearch in Google Scholar
• 18 Yin Z Z, Zeng G Y. Pharmacology of puerarin V. Effects of puerarin on platelet aggregation and release of 5-HT from platelets.  Acta Acad Med Sin. 1981;  3 44-7
  PubMedSearch in Google Scholar
• 19 Ashworth-Preece M A, Jarrott B, Lawrence A J. Nicotinic acetylcholine receptor mediated modulation of evoked excitatory amino acid release in the nucleus tractus solitarius of the rat: evidence from in vivo microdialysis.  Brain Res. 1998;  806 287-91
  CrossrefPubMedSearch in Google Scholar
• 20 Wang C D, Chiang C H, Sun W H, Hsia W. Total isoflavonoids isolated from Pueraria lobata suppress alcohol-induced inhibition of central nervous system in mice.  Henan J Med Univ. 1998;  33 117-9
  PubMedSearch in Google Scholar
• 21 Shen X L, Witt M R, Nielsen M, Sterner O. Inhibition of [³ H]-flunitrazepam binding to rat brain membranes in vitro by puerarin and daidzein.  Acta Pharm Sin. 1996;  31 59-62
  PubMedSearch in Google Scholar

Assistant Professor Dr. Chi-Rei Wu

Institute of Chinese Pharmaceutical Sciences

China Medical College

91, Hsieh Shih Road

40421, Taichung, Taiwan, R.O.C.

Email: crw@mail.cmc.edu.tw

Fax: 886-0943806654