Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11.

Genetically modified corn has been approved as an animal feed in several countries, but information about the fate of genetically modified DNA and protein in vivo is insufficient. Genetically modified corn Bt11 is developed by inserting a recombinant DNA sequence encoding insecticidal Cry1Ab protein from Bacillus thuringiensis subsp. kurstaki. We examined the presence of corn intrinsic and recombinant cry1Ab gene by PCR, and the Cry1Ab protein by immunological tests in the gastrointestinal contents of five genetically modified corn Bt11-fed and five nongenetically modified corn-fed pigs. Fragments of corn zein (242 bp), invertase (226 bp) and of ribulose-1,5-bisphosphate carboxylase/ oxygenase genes (1,028 bp) were detected in the gastrointestinal contents of both Bt11 and nongenetically modified corn-fed pigs. Fragments of recombinant cry1Ab gene (110 bp and 437 bp) were detected in the gastrointestinal contents of the Bt11-fed pigs but not in the control pigs. Neither corn intrinsic nor cry1Ab gene fragments were detected in the peripheral blood by PCR. The gastrointestinal contents were positive for Cry1Ab protein by ELISA, immunochromatography, and immunoblot; however, these methods did not work for blood and precluded conclusions about any potential absorption of the protein. These results suggest that ingested corn DNA and Cry1Ab protein were not totally degraded in the gastrointestinal tract, as shown by their presence in a form detectable by PCR or immunological tests.

Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLink CBH351.

We tried to detect DNA fragments derived from maize in the intestinal contents of pigs fed genetically modified (GM) StarLink CBH351 maize (SL) or non-GM maize. Intestinal contents of 8 SL and 8 non-GM maize-fed pigs were collected at slaughter, and the genes of the recombinant cry9C and the maize intrinsic zein (Zel) were assayed by polymerase chain reaction (PCR) 3 times with a total of 4 primer pairs of different expected lengths. The cry9C gene (either 103 or 170 bp) was detected in the rectal contents (with a frequency of 25-37.5%) and in the cecal contents (25-50%) of the pigs fed SL. In a similar fashion, the zein (Zel) gene (either 242 or 329 bp) was detected in the rectal contents (with a frequency of 31.3%) and in the cecal contents (25-37.5%) of pigs fed on SL non-GM maize. These results suggested that ingested DNA was not totally degraded, but is present in a form detectable by PCR.

[A detection method for recombinant DNA from genetically modified maize CBH351].

A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize CBH351, which has not authorized as safe for use in foods and feeds in Japan yet. We analyzed a recombinant DNA (r-DNA) sequence introduced into CBH351 maize and designed specific primer pairs to amplify a segment including part of the r-DNA. The PCR products obtained by using the designed primer pairs are specific for CBH351 and should prevent false positive results caused by other maizes and other main cereal crops. The r-DNA introduced into CBH351 could be detected from maize samples containing 0.05-0.1% CBH351 maize. This sensitivity is theoretically equivalent to a level of several genome copies and so this technique is a very efficient means to detect CBH351 maize.

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Confirmation of the anxiolytic-like effect of dihydrohonokiol following behavioural and biochemical assessments.

Previous studies in this laboratory revealed that dihydrohonokiol-B (DHH-B; 3'-(2 propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol), a partially reduced derivative of honokiol, was an effective anxiolytic-like agent in mice at an oral dose of 0.04 mg kg(-1), and at higher doses, when evaluated by the elevated plus-maze test. The aim of this study was to further confirm the anxiolytic-like effect of DHH-B using an additional behavioural procedure (Vogel's conflict test in mice) and a biochemical assessment (in-vitro determination of muscimol-stimulated 36Cl- uptake into mouse cortical synaptoneurosomes). As in earlier experiments, DHH-B (0.04-1 mg kg(-1), p.o.) was shown to prolong the time spent in the open-sided arms of the elevated plus-maze in a dose-dependent manner. Moreover, in the Vogel's conflict test, DHH-B (5 mg kg(-1), p.o.) significantly increased punished water intake. In tests with mouse cerebral cortical synaptoneurosomes, 10 and 30 microM of DHH-B significantly increased 36Cl- influx in the absence of muscimol. In the presence of 25 microM muscimol, the addition of 1 microM DHH-B led to significant enhancement of 36Cl- uptake, while 30 microM DHH-B was required to further stimulate the 36Cl- uptake induced by 250 microM muscimol. The results of these studies confirm that DHH-B is a potent anxiolytic-like agent and that GABA(A) receptor-gated Cl(-)-channel complex is involved in the anxiolytic-like efficacy of DHH-B.

The anxiolytic effect of Sho-ju-sen, a Japanese herbal medicine, assessed by an elevated plus-maze test in mice.

Sho-ju-sen (SK), a Japanese herbal medicine with a nourishing tonic action, is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) (SS), and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots (Panax ginseng C. A. Meyer) (PX) in the ratio 8:1:1. In this study, an elevated plus-maze test in mice was carried out to assess whether SK had an anxiolytic effect. No significant change was observed in either the plus-maze or activity test following a single administration of SK (10 and 20 mL/kg p.o.). However, mice allowed a free intake of SK (10% solution) for 5 days and longer showed a significant prolongation of the time spent in the open arms (an anxiolytic effect), as long as that caused by the benzodiazepine anxiolytic diazepam (1 mg/kg p.o.). SK (1%, 3% and 30% solutions for 7 days) tended to develop the anxiolytic effect. Of the constituents of SK, SS (8% solution), but not PN (1% solution) or PX (1% solution), resulted in the anxiolytic effect. Except for a slight acceleration in the motor activity by PN (1% solution), no significant change in the motor activity was produced by any treatment with SK, SS or PX. The combined treatment of SK (10% solution) or SS (8% solution) with 1 mg/kg diazepam enhanced the anxiolytic effect. Flumazenil (0.1 mg/kg s.c.), a benzodiazepine receptor antagonist, alone did not change the time spent in the open arms. However, it completely reversed the anxiolytic effect of SK, SS and diazepam. The present results suggest that: (1) long-term treatment with SK develops an anxiolytic effect, (2) SS is the main constituent for the anxiolytic effect of SK, and (3) benzodiazepine receptors are involved in the anxiolytic effect of SK and SS.

Effects of drugs acting as histamine releasers or histamine receptor blockers on an experimental anxiety model in mice.

Experimental anxiety in mice was evaluated using a light/dark test at 60 min after injection of various histaminergics. Thioperamide, a histamine H(3) receptor inhibitor (5-20 mg/kg, intraperitoneal [IP]), Compound 48/80, a mast cell degranulator (0.1-10 microg/2 microl, intracerebroventricularly [ICV]), mepyramine, a histamine H(1) receptor antagonist (0.1-10 microg/2 microl, ICV) or cimetidine, a histamine H(2) receptor antagonist (0.1-10 microg/2 microl, ICV) alone did not affect the locomotive activity, the time spent in the light zone, and number of shuttle crossings in the light/dark test. However, the time spent in the light zone and the number of shuttle crossings significantly decreased only when cimetidine (0.1-10 microg/2 microl, ICV) was co-treated with either thioperamide (10 mg/10 ml/kg, IP) or Compound 48/80 (1.0 microg/2 microl, ICV). The decrease in these behavioral parameters suggests induced experimental anxiety in mice. The experimental anxiety was antagonized by mepyramine (10 microg/2 microl, ICV). These results suggest that not only neuronal histamine release induced by thioperamide but also non-neuronal (mast cells) histamine release induced by Compound 48/80 play an important role in inducing experimental anxiety via post-synaptic H(1) and H(2) receptors. In addition, it is likely that the anxiety may be mediated by the stimulation of H(1) receptors, while H(2) receptors may inhibit the anxiety produced by the activation of H(1) receptors.

Does dihydrohonokiol, a potent anxiolytic compound, result in the development of benzodiazepine-like side effects?

The aims of this study were to assess whether dihydrohonokiol, 3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol (DHH-B), a potent anxiolytic compound, developed benzodiazepine-like side effects. A 1 mg kg(-1) dose of diazepam, almost equivalent to the minimum dose for the anxiolytic effect, disrupted the traction performance, potentiated hexobarbital-induced sleeping and impaired learning and memory performance. DHH-B, even at a dose of 1 mg kg(-1) (i.e. five times higher than the minimum dose for significant anxiolytic effect) neither developed diazepam-like side effects nor enhanced the side effects of diazepam. Rather, the potentiation by diazepam of hexobarbital-induced sleeping was reduced by 1 mg kg(-1) DHH-B. Furthermore, mice treated with 10 daily administrations of 1 and 5 mg kg(-1) diazepam, but not 0.2-5 mg kg(-1) DHH-B, showed precipitated withdrawal symptoms characterized by hyper-reactivity, tremor and tail-flick reaction when they were challenged with flumazenil (10 mg kg(-1) i.p.). These results suggest that, unlike the benzodiazepine anxiolytic diazepam, DHH-B is less likely to induce motor dysfunction, central depression, amnesia or physical dependence at the effective dose required for the anxiolytic effect.

Long-term clomipramine treatment upregulates forebrain acetylcholine muscarinic receptors, and reduces behavioural sensitivity to scopolamine in mice.

We have investigated the effects of long-term treatment with clomipramine, a tricyclic antidepressant, on central muscarinic acetylcholine receptors (mAChR) in mice. Repeated clomipramine administration resulted in an increase in the forebrain receptor density value (Bmax) for [3H]quinuclidinyl benzilate, a muscarinic ligand (P < 0.05), that was dependent on dose per administration (saline or 5, 10, or 20 mg kg(-1) once a day for 7 days) and number of days treated (20 mg kg(-1) for 1, 3, 5, or 7 days). No change in apparent affinity (defined as the reciprocal of the dissociation constant) (KD) occurred. Seven daily treatments with clomipramine (saline or 5, 10, or 20 mg kg(-1)) reduced hyperlocomotion induced by scopolamine (0.5 mg kg(-1), s.c.) dose-dependently, and the effect of 20 mg kg(-1) clomipramine was significant (P < 0.05). These results suggest that an upregulation of mAChR is produced by repeated clomipramine administration, and such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine.

Learning and memory in mice treated with choline oxidase, a hydrolytic enzyme for choline.

Learning and maintenance of memory in mice intraperitoneally (IP) injected with choline oxidase (ChO, 6 units/g), a hydrolytic enzyme for choline (Ch), were assessed by means of a step-through passive-avoidance task. The ChO treatment induced a hydrolysis of free Ch in plasma, which in turn, induced a decrease in cerebral acetylcholine (ACh) release. In the learning test, the ChO-treated mice showed significant inhibition to learn the avoidance from electric shock. In the retention test, the impairment of the memory once established was not produced by posttreated ChO. We concluded that the decreased cerebral cholinergic neurotransmission induced by ChO retarded acquisition of passive-avoidance learning more readily than the maintenance of memory.

Comparative assessment of the anxiolytic-like activities of honokiol and derivatives.

Honokiol has previously been shown to be an effective anxiolytic-like agent in mice when administered for 7 days at 0.2 mg/kg/day prior to evaluation in an elevated plus-maze, while 20 mg/kg is required for efficacy as a single oral dose. The aim of this study was to find analogs of honokiol that are more effective for acute administration. Among the eight analogs evaluated, one partially reduced derivative of honokiol [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol] exhibited significant anxiolytic-like activity at 0.04 mg/kg. Following oral administration of 1 mg/kg of this analog, anxiolytic-like activity was clearly evident at 1 h, peaked at 3 h, and remained significant for longer than 4 h after treatment. Combined administration of the derivative with diazepam led to enhanced anxiolytic-like efficacy. Moreover, as with diazepam, the anxiolytic-like effect of the analog was reduced by flumazenil. In contrast, bicuculline, a GABA(A) antagonist, had no effect on the activity of the derivative. Taken together, these results suggest that this analog of honokiol acts at the benzodiazepine recognition site of the GABA(A)-benzodiazepine receptor complex.

The anxiolytic effect of two oriental herbal drugs in Japan attributed to honokiol from magnolia bark.

An improved elevated plus-maze test in mice revealed that seven daily treatments with two different traditional Chinese medicines, known as Kampo medicines in Japan, Hange-koboku-to (composed of extracts of 5 plants) and Saiboku-to (composed of extracts of 10 plants), produced an anxiolytic effect, and the effect was mainly due to the presence of honokiol derived from magnolia. This study was carried out to evaluate the anxiolytic potential of honokiol, Hange-koboku-to and Saiboku-to, which were prescribed with two different magnolia samples: Kara-koboku (Magnoliae officinalis) (KA) or Wa-koboku (Magnoliae obovata) (WA). The doses of test samples were adjusted to ensure a constant dose of honokiol at 0.2 mg kg(-1). Although the doses of magnolol (an isomer of honokiol), as well as those of undetermined chemicals, varied among samples, the seven daily treatments with 9 out of 10 test samples produced an anxiolytic effect almost equivalent to that produced by 0.2 mg kg(-1) honokiol. The only exception was the sample containing the lowest amount of honokiol. Magnolia-free preparations of Hange-koboku-to or Saiboku-to did not have any anxiolytic effect. These results confirm that honokiol derived from magnolia is the causal chemical of the anxiolytic effect of Hange-koboku-to and Saiboku-to.

Application of the elevated plus-maze test in mice for evaluation of the content of honokiol in water extracts of magnolia.

In our previous study using an improved elevated plus-maze in mice, the oriental herbal medicine Saiboku-to prolonged the time spent in open arms, showing an anxiolytic effect, and the effect was mainly caused by honokiol derived from magnolia. This study was carried out to compare the anxiolytic potentials of honokiol and water extracts of three magnolia samples; two being Kara-koboku (Magnolia officinalis) (KA: from Zhejiang-sheng, China; honokiol 0.25% and magnolol 1.16%, and KB: from Sichuan-sheng, China; honokiol 1.72% and magnolol 1.71%), and one being Wa-koboku (Magnolia obovata) (WA: from Iwate-ken, Japan; honokiol 0.32% and magnolol 0.81%). Seven daily treatments with 0.1-1 mg/kg honokiol, but not 0.2 and 1 mg/kg magnolol, revealed an anxiolytic effect with the peak potential at 0. 2 mg/kg. The anxiolytic potentials of 40 and 80 mg/kg KA, which contained the highest amount of magnolol, were almost equivalent to those of 0.1 and 0.2 mg/kg honokiol, respectively. KB, at 11.6 mg/kg, and 62.5 mg/kg WA resulted in almost the same anxiolytic potential as that of 0.2 mg/kg honokiol. No significant change in the ambulatory activity was produced by any drug treatment. These results suggest that honokiol is the chemical responsible for the anxiolytic effect of the water extract of magnolia and that the other chemicals including magnolol in magnolia scarcely influence the effect of honokiol. It is also considered that the elevated plus-maze test is applicable for evaluation of the content of honokiol in magnolia.

Honokiol, a putative anxiolytic agent extracted from magnolia bark, has no diazepam-like side-effects in mice.

Use of the elevated plus-maze experiment and activity and traction tests in mice have revealed that seven daily treatments with 0.2 mg kg(-1) and higher doses of honokiol, a neolignane derivative extracted from Magnolia bark, had an anxiolytic effect without change in motor activity or muscle tone. Diazepam, 1 mg kg(-1), had the same anxiolytic potential as 0.2 mg kg(-1) honokiol but induced muscle relaxation. The aim of this study was to determine whether honokiol had diazepam-like side-effects. Mice treated with 1-10 mg kg(-1) diazepam, but not those treated with 0.1-2 mg kg(-1) honokiol, for 12 days showed withdrawal symptoms characterized by hyperactivity and running-fit when they were challenge-administered intraperitoneal flumazenil (10 mg kg(-1)) 24 h after the last treatment with diazepam. Oral diazepam (0.5-2 mg kg(-1), 10 min before) dose-dependently prolonged hexobarbital (100 mg kg(-1), i.p.)-induced sleeping, disrupted learning and memory, and inhibited (+)-bicuculline (40 mg kg(-1), i.p.)-induced death. Honokiol (0.2-20 mg kg(-1), p.o., 3 h before) had no such effects. The prolongation by diazepam (1 mg kg(-1)) of hexobarbital-induced sleeping was not modified by honokiol (0.2-20 mg kg(-1)). These results suggest that honokiol is less likely than diazepam to induce physical dependence, central depression and amnesia at doses eliciting the anxiolytic effect. It is also considered that honokiol might have no therapeutic effect in the treatment of convulsion.

Does nicotine modify the psychotoxic effect of methamphetamine? Assessment in terms of locomotor sensitization in mice.

In this study, effects of nicotine on locomotor sensitization to methamphetamine in mice were investigated to assess whether nicotine modified induction and expression of psychotoxic action of methamphetamine. Although nicotine (0.03-1 mg/kg s.c.) had no effect at first administration, 5-time nicotine administrations at 3-day intervals progressively developed a significant locomotor stimulant effect, and caused an enhanced sensitivity (cross-sensitization) to methamphetamine (2 mg/kg s.c.). Five-time administrations of methamphetamine (2 mg/kg) at 3-day intervals produced not only a locomotor sensitization to methamphetamine itself, but also a cross-sensitization to nicotine (0.1-1 mg/kg). Nicotine (0.03-1 mg/kg) did not affect the locomotor stimulant effect of methamphetamine (2 mg/kg) in the drug-naive mice. However, nicotine acted dose-dependently to reduce the progressive enhancement of the locomotor stimulant effect of methamphetamine during 5-time repeated administrations. Mice treated with coadministration of methamphetamine with nicotine (1 mg/kg) showed less sensitization to methamphetamine than mice treated with methamphetamine alone. In addition, nicotine (1 mg/kg) inhibited the locomotor stimulant effect of methamphetamine in mice sensitized to methamphetamine. These results suggest that methamphetamine and nicotine produce a symmetrical cross-sensitization, although nicotine may act to inhibit the induction and expression of locomotor sensitization to methamphetamine in mice.

[Evaluation of transition zone biopsy in systematic prostate biopsy].

We evaluated the systematic biopsies performed on 83 patients suspected of having prostate cancer. In the systematic biopsy, 6 cores were from the peripheral zone and 2 cores from the transition zone. Cancer was detected in 25 patients (30.1%). The percentage of patients who had abnormal digital rectal examination and transrectal echo findings, average PSA and PSA density, and the number of examinations which suggested cancer were higher in the cancer group than in the non-cancer group, although the mean prostate volume was smaller. Cancer was more frequently detected in the peripheral zone than in the transition zone. Cancer was detected only in the transition zone in only 1 of the 25 cancer patients. We conclude that biopsy of the transition zone to all the patients is not always needed in systematic biopsy.

[Prostate specific antigen doubling time in prostate cancer before treatment and in refractory status].

Prostate-specific antigen(PSA) increases exponentially in prostate cancer patients before treatment and in refractory status. PSA increases in 68-86% of prostate cancer patients before treatment, and that of the remaining 14-32% of the patients is stable. Those patients with a higher pre-treatment PSA level are more likely to have a shorter PSA-doubling time(PSA-DT). The relationship between pre-treatment stage, grade and PSA-DT is controversial. PSA-DT in biochemical failure patients predicts the risk of clinical recurrence. PSA-DT was correlated well with time to clinical recurrence after biochemical failure. Distant recurrence was associated with short PSA-DT. Higher clinical stage and lower differentiation before treatment correlated with shorter PSA-DT in recurrent cancer patients. PSA-DT is an important parameter for judging malignant potential of each cancer.

Behavioural pharmacological characteristics of honokiol, an anxiolytic agent present in extracts of Magnolia bark, evaluated by an elevated plus-maze test in mice.

Honokiol, a neolignane derivative of Magnolia bark, has central depressant action and, at much lower doses, anxiolytic activity. We have investigated the characteristics of the behavioural effects of honokiol by means of an elevated plus-maze test. In the plus-maze test a single oral dose of 20 mg kg(-1) honokiol significantly prolonged the time spent in the open arms of the maze, suggesting anxiolytic effect. Moreover, when honokiol was administered daily for seven days and the plus-maze test was conducted 3 or 24 h after the last administration, significant prolongation of the time in the open arms was manifested even for doses of 0.2 mg kg(-1). The maximum effect was observed for doses of 0.5 mg kg(-1). Honokiol at any dose in both single and repeated administration schedules caused neither change in motor activity nor disruption of traction performance. Orally administered diazepam, 0.5-2 mg kg(-1), caused dose-dependent prolongation of the time spent in the open arms of the maze with a significant increase in motor activity at 1 mg kg(-1), and dose-dependent disruption of traction performance. The changes in the plus-maze performance after treatment for seven days with 0.2 mg kg(-1) honokiol and after a single treatment with 1 mg kg(-1) diazepam were almost equivalent. The effect of honokiol (0.2 mg kg(-1), treatment for seven days) was inhibited by subcutaneous flumazenil (0.3 mg kg(-1)) and (+)-bicuculline (0.1 mg kg(-1)) and by intraperitoneal CCK-4 (50 microg kg(-1)) and caffeine (30 mg kg(-1)). The anxiolytic effect of diazepam (1 mg kg(-1)) was also inhibited by flumazenil and bicuculline. However, the combined administration of diazepam with caffeine enhanced the effect, and diazepam completely reversed the effect of CCK-4. These results suggest that, in contrast with diazepam, honokiol selectively induces an anxiolytic effect with less liability of eliciting motor dysfunction and sedation or disinhibition. The combined effects of the drug also revealed that the mechanism of anxiolytic effect of honokiol is partially different from that of diazepam.

Importance of initial environments in the development of ambulatory sensitization to methamphetamine and cocaine in mice.

Repeated administration of CNS stimulants such as amphetamines and cocaine induces behavioural sensitization which can be influenced by the animal's environment. This study has evaluated the effect of restraint on the development and maintenance of ambulatory sensitization to methamphetamine and cocaine in mice. Subcutaneous administration of the CNS stimulants methamphetamine (2 mg kg(-1)) and cocaine (20 mg kg(-1)) seven times at three-day intervals resulted in ambulatory sensitization when the mice were placed in 20-cm diameter activity cages after each dose of the drug. However, if methamphetamine or cocaine was administered when the mice were in small jars (6-cm diameter) in which expression of ambulation, but not of circling and rearing, was completely restricted, the development of ambulatory sensitization was retarded or inhibited, with circling behaviour concurrently increased, when subsequent repeated doses of the drug were administered in the activity cage. Subsequent repeated treatment of ambulatory-sensitized mice with the drug or saline when the mice were in the jars did not change the levels of the ambulatory sensitization or the circling behaviour. These results suggest that the mice are sensitized to the behavioural effect of CNS stimulants which can be expressed in the environment in which the drug is administered. It is also considered that the established sensitization is strongly retained and is responsible for retardation or suppression of the development of sensitization to other behavioural stimulant effects.

Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine.

The principal active anxiolytic components in Saiboku-to, an Oriental herbal medicine, have been isolated and identified as magnolol (5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol) and honokiol (3',5-di-2-propenyl-1,1'-biphenyl-2,4'-diol). Evaluation by means of an elevated plus-maze test showed that honokiol was at least 5000 times more potent than Saiboku-to when mice were treated orally for 7 days.