The Effects of Traditional Tonics on Fatigue in Mice Differ from Those of the Antidepressant Imipramine: A Pharmacological and Behavioral Study
Abstract: The present studies were undertaken to investigate the differences between the antidepressant drug, imipramine, and liquid nutritive and tonic drugs (NTDs) that consist of Ginseng radix, Epimedii herba, Holen and an additional eight to twelve crude drugs. After preloading forced swimming, the NTD (applied orally, 0.1 ml/10 g) significantly increased the duration time of swimming and decreased the duration time of immobility, while the administration of imipramine (5, 10 and 20 mg/kg, i.p.) under the same conditions and after the same treatment did not produce these positive effects. After pretreatment with 100 mg/kg tetrabenazine, the NTDs also elicited both the increased locomotor activity and the decreased duration time of immobility. The behavioral effect was similar to treatment with imipramine. The NTDs showed a long lasting effect on swimming behavior in the forced swimming test for 15 min, indicating a prolonged efficacy, not like the short effect of imipramine. The present results indicate that the effect of NTDs on fatigued subjects is different from that of imipramine, probably due to involvement of another factor in addition to the antidepressant effect.
In Japan, the animal or clinical study or liquid nutritive and tonic drug (NTDs) including vitamins, which are classified as medicine, has not been carried out. Although more than 1000 types of these crude liquid drugs are on the market, it remains unknown whether these drugs elicit an anti-fatigue effect or not. This poses a serious problem since NTDs are in great demand. Therefore, we previously undertook a study to clarify the acute anti-fatigue effect of NTD-containing vitamins. After preloading forced swimming or tetrabenazine, a central monoamine depletor, the NTDs caused a significantly marked improvement effect, such as the shortened duration of immobility (Tadano et al., 1992). In contrast, without preloading the forced swimming, NTDs have anti-fatigue effects, but these effects are weaker compared to those after preloading (Table 1). It is difficult to determine the anti-fatigue effect of NTDs in humans since it is difficult to determine the precise criterion of human fatigue, and NTDs including vitamins induce a placebo effect. In contrast to the effect on humans, it seems more appropriate to use rodents with no placebo effect to determine the anti-fatigue effect. However, there are few available animal models in reference to fatigue. Accordingly, we previously studied the effect of NTDs on a fatigue model by examining the immobility induced by forced swimming or by treatment with tetrabenazine. Administration of tetrabenazine, a central monoamine depletor, caused depressive effects such as decreased locomotion and increased duration time of immobility. In this regard, as mentioned above, NTDs including vitamins elicited a positive effect using these animal models. However, it is suggested that the anti-fatigue effect after treatment with the NTDs may primarily be an antidepressant effect since the forced swimming test is used to evaluate the efficacy of antidepressant drugs as shown by Porsolt et al. (1977, 1978, 1981). To study the anti-fatigue mechanism by which NTDs reduce the duration of immobility of mice in the forced swimming test, we investigated the difference in the effect between NTDs and imipramine, a typical antidepressant, on the duration of immobility of mice pretreated with forced swimming and injection of tetrabenazine.
Table 1. Effect of Sample 1, 2 and 3 after Preloading Forced Swimming and Tetrabenazine (TBZ) on the Duration of Immobility and Locomotor Activity
Duration of Immobility (% of Control)
Pretreatment with TBZ
Non-swimming 50 mg/kg 100 mg/kg Load
Sample 1 81% 48% 45% 25%
Sample 2 75% 35% 36% 18%
Sample 3 69% 17% 21% 6%
Locomotor Activity (% of Control)
Pretreatment with TBZ
Non-pretreatment 200 mg/kg
Sample 1 226% 973%
Sample 2 246% 1069%
Sample 3 260% 1154%
The duration of immobility was measured 1 hr after injection of S-1, S-2 and S-3. Each sample and imipramine were given 24 hr after the injection of TBZ.
Materials and Methods
Adult male ddY mice (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing 24~27 gm were used. Animals were housed in cages with free access to food and water under conditions of constant temperature (23 [+ or -] 2 [degrees] C) and humidity (55 [+ or -] 5%) and a 12-hr light/dark cycle (9:00 hr ~ 21:00 hr). Ten mice were used for each treatment. All experiments were performed according to the Guide for Care and Use of Laboratory Animals at Tohoku Pharmaceutical University.
The agents used were three types of prescriptions, which are composed of 11 (sample 1; S-1), 13 (sample 2; S-2) and 15 (sample 3; S-3) crude drugs including vitamin [B.sub.2], vitamin [B.sub.6], taurin and caffeine anhydride (Table 2). Mice in the control group were orally given water (W) at 0.1 ml/10 g mouse body weight.
Table 2. Components of Samples 1 (S-l), 2 (S-2) and 3 (S-3)
Crude drugs S-1 (mg/50ml) S-3
Ginseng Radix 600 600 600
Astragali Radix 600 300
Polygonati Rhizoma 600 300
Glycyrrhizae Radix 150 150
Zizyphi Fructus 300
Hoelen 300 300
Discoreae Rhizoma 300
Angelicae Radix 50 50
Rehmanniae Radix 300 300
Lycii Fructus 300
Cistanchis Herba 500
Epimedii Herba 1000 1000 1000
Cervi Parvum Cornu 300 300
Cnidii Monnieris Fructus 300
Cuscutae Semen 300
Eucommiae Cortex 300
Phocae Testis Et Penis 100
Corni Fructus 500
Schisandrae Fructus 300
Agkistrodon Japonicae 250 250 250
Muirapuama 300 300 300
Bupleuri Radix 150
Cinnamomi Cortex 150
Zingiberis Rhizoma 75
Vitamin B2 5 5 5
Vitamin B6 5 5 5
Taurin 500 500 500
Caffeine anhydride 50 50 50
Chemicals were obtained from the following sources: imipramine hydrochloride from Chiba-Geigy Pharmaceutical Company, Ltd., (Tokyo, Japan); tetrabenazine from INC Pharmaceuticals Inc. (Switzerland).
Forced Swimming Test
Before the experiments, mice were individually placed in a vertical glass cylinder (height 20 cm; diameter 10 cm) which contained 8 cm of water maintained at 25~26 [degrees] C for 5 min. They were removed and allowed to dry off in a drying room. One hr after the preloading forced swimming, S-1, S-2 or S-3 was administered orally. They were again put into the glass cylinders, and the total duration of immobility, struggle and swimming was measured during a 5-min or 15-min period.
Measurement of Locomotor Activity
The locomotor activity was measured with a 12-channel Animex-auto system (Animex, Muromachi Kikai Co., Tokyo, Japan). The measurement of activity was conducted between 9:00 and 15:00, during the light phase. Mice were placed in the apparatus and adapted for 15 min on the day before the experiment.
Measurement of Locomotor Activity and Forced Swimming after Pretreatment with Tetrabenazine
Twenty-four hr after injection of tetrabenazine (100 mg/kg or 200 mg/kg, i.p.), the NTD sample or imipramine was administered. The measurements of forced swimming and locomotor activity were carried out 1 hr after the administration of each sample or imipramine.
Experimental data are expressed as mean [+ or -] SEM. Turkey’s test was used to analyze the data, and critical differences in the means were calculated by Dunnett’s test.
Effects of NTD and Imipramine on Locomotor Activity and Forced Swimming
After pretreatment with tetrabenazine (100 mg/kg, i.p.), each sample (S-1, 973%; S-2, 1069%; S-3, 1154%) and imipramine (5 mg/kg, 213%; 10 mg/kg, 384%; 20 mg/kg, 565%) showed an increase in the locomotor activity. The effect of imipramine, however, was significantly less than that induced by each tonic drug. After preloading by forced swimming for 5 min, all of the tonics (S-1, 25%; S-2, 18%; S-3, 6%) significantly reduced the duration time of immobility, compared to the non-preloaded mice (S-1, 81%; S-2, 75%; S-3, 69%) (Tables 1 and 3). In addition, without the preloading of forced swimming or the pretreatment with tetrabenazine, the efficacy of each sample (S-1, 226%; S-2, 246%; S-3, 260%) on locomotor activity was weak, compared to mice who had preloading. Tetrabenazine (100 mg/kg, i.p.) given to mice 24 hr before treatment with water also caused a significant increase in the duration of immobility as compared to those not given tetrabenazine. The increased duration time of immobility induced by the administration of tetrabenazine was reduce significantly by each tonic (S-1, 45%; S-2, 36%; S-3, 2%). In contrast, after preloading by forced swimming for 5 min, imipramine did not show any change in the duration time of immobility (5 mg/kg, 117%; 10 mg/kg, 114%; 20 mg/kg, 99%), but that was not the case in the mice who received pretreatment with tetrabenazine (Table 3).
Table 3. Effects of S-1, -2, -3 and Imipramine (IMP) after Pretreatment with Tetrabenazine (TBZ) or Preloading Forced Swimming on Locomotor Activity and the Duration of Immobility
Locomotor Activity (% of Control)
Imipramine (mg/kg) Sample
5 10 20 1 2 3
Non-pretreatment 105% 113% 111% 226% 246% 260%
Pretreatment with 213% 384% 565% 973% 1069% 1154%
TBZ (100 mg/kg)
Duration of Immobility (% of Control)
Imipramine (mg/kg) Sample
5 10 20 1 2 3
Swimming Load 117% 114% 99% 25% 18% 6%
Pretreatment with 69% 50% 33% 45% 36% 21%
TBZ (100 mg/kg)
The duration of immobility was measured 1 hr after injection of S-1, S-2, S-3 or IMP. S (1-3) and IMP was given 24 hr after the injection of tetrabenazine (TBZ).
Effects of NTD (S-3) and Imipramine on the Swimming Pattern Induced by Forced Swimming
As shown in Table 3, after preloading stress the effect of S-3 was different from that induced by imipramine. To clarify this difference between NTD and imipramine, the duration time of immobility, struggle and swimming after pretreatment with tetrabenazine (200 mg/kg, i.p.) were observed for 15 min using forced swimming. During the first 0~5 min after the tetrabenazine injection, the duration time of each swimming pattern did not show a significant change as compared to that induced by imipramine. At 5~10 min and 10~15 min after the tetrabenazine injections, the duration time of swimming (6~10 sec) and straggle (0~5 sec) were short with imipramine (swimming, 5~10 min: 26 sec; 10~15 min, 5 sec). In contrast, S-3 showed a prolonged effect on the duration of swimming at 5~10 min (99 sec) and 10~15 min (73 sec) after the tetrabenazine injection (Figure 1).
[Figure 1 ILLUSTRATION OMITTED]
There are many NTDs including crude drags in the prescriptions of traditional Chinese medicine, which are classified as over-the-counter drags and are generally termed “Drinkzai,” in Japan. Many Japanese people use NTD for relief of physical and psychological fatigue. However, the pharmacological and clinical effects of NTD to suppress fatigue remain unclarified. It is, therefore, of great interest to study the anti-fatigue effect of NTD. In a previous study we reported that NTD exhibits an anti-fatigue effect (Tadano et al., 1992). Here, after preloading by forced swimming or pretreatment with tetrabenazine, the test samples (S-1, S-2 and S-3) given orally showed an anti-fatigue effect based on the fact that these samples significantly decreased the duration time of immobility in the forced swimming test and they increased the locomotor activity (Table 1). However, without the preloading of forced swimming or the pretreatment with tetrabenazine, the efficacy of NTDs on the swimming test and on locomotor activity was less than that in mice after preloading. Porsolt et al. (1977, 1978, 1981) reported that the forced swimming test is useful to research the effect of antidepressant drugs. They suggested that this characteristic behavioral immobility is a state of despair in which the mouse has learned that escape is impossible and it resigns itself to the experimental condition. They thus proposed that the immobility represents a despair model in mice and rats (Porsolt et al., 1977, 1981). For these reports it is assumed that the duration of immobility reduced by NTDs is included in the effect of anti-depressants but it is not a tonic effect. Therefore, to clarify this problem, in the present study the difference between NTD and imipramine on the forced swimming test was examined.
In the present study, imipramine reduced the duration of immobility and increased the locomotor activity in mice treated with tetrabenazine. NTD after pretreatment with tetrabenazine also caused decreased immobility and increased locomotor activity. This inhibitory effect of imipramine combined with tetrabenazine was assumed to be related to the antidepressant activity. It has been reported that tetrabenazine depletes the supply of brain serotonin and norepinephrine in mice and rabbits (Pletscher, 1957; Quinn et al., 1959). Regarding the effect of tetrabenazine on the contents of brain serotonin and norepinephrine, it was reported that at 24 hr after the injection of tetrabenazine (50 or 100 mg/kg), the serotonin content almost returns to the normal level (Pletscher, 1957), but the brain norepinephrine content is markedly decreased (Kimura et al., 1980). Based on these findings, it is assumed that the increased immobility at 24 hr after the administration of tetrabenazine may be due to the brain norepinephrine level but not to the serotonin level (Araki et al., 1985; Borsini et al., 1985). Taken together, the inhibition of immobility found in this study indicates that both drugs might have a stimulating action on the release of norepinephrine from either the stored or the newly synthesized granules in the brain. In contrast, after preloading by forced swimming, imipramine did not show a significant change in the duration of immobility. Considering these findings, imipramine may not cause tonic effects, but rather a “native” effect with physical work. However, NTDs were shown to be effective on mice fatigued with physical stress, such as forced swimming, since the immobility behavior induced by both types of preloading (tetrabenazine and forced swimming) was signficantly reduced by NTDs. Thus, the present findings support that the effects induced by NTDs differ from those caused by imipramine.
Armario et al. (1988) have reported that an additional swimming pattern, struggle and swimming, is a more reliable measure of antidepressant action than immobility alone in the forced swimming test. Based on this hypothesis, further experiments were carried out upon the effect of NTDs on swimming, struggle and immobility in order to discriminate between the effects of NTDs and imipramine. In these experiments, after pretreatment with tetrabenazine, NTDs and imipramine in the first 5-min period showed a similar effect on each swimming pattern. However, NTDs in the second period (5~10 min) and third period (10~15 min) showed a more prolonged effect of swimming. In contrast, imipramine returned to almost the normal level in the second and third periods.
In conclusion, the present results suggest that the anti-fatigue effect of NTDs not only shows the anti-depressant effect of imipramine, but also is related to some central action.
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Takeshi Tadano, Osamu Nakagawasai, Fukie Niijima Koichi Tan-No and Kensuke Kisara
Department of Pharmacology, Tohoku Pharmaceutical University 4-4-1, Komatsushima, Aoba-ku, Sendai, 981-8558 Japan
(Accepted for publication September 15, 1999)
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