Effect of Hochu-ekki-to (TJ-41), a Japanese Herbal Medicine, on Daily Activity in a Murine Model of Chronic Fatigue Syndrome

Abstract

We aimed to evaluate the effect of a Japanese herbal medicine, Hochu-ekki-to (TJ-41), on daily activity in a murine model of chronic fatigue syndrome (CFS). CFS was induced by repeated injection of Brucella abortus (BA) antigen every 2 weeks. TJ-41 was orally administered to mice in a dose of 500 mg/kg/day for 1 week before injecting BA and for 4 weeks thereafter. We evaluated daily running activity in mice receiving TJ-41 as compared with that in untreated mice. Survival of both mouse groups was also monitored during the observation period. Body weight (BW), spleen weight (SW), SW/ BW ratio and expression levels of interleukin-10 (IL-10) mRNA in spleen were determined in both groups at the time of sacrifice. The daily activity was significantly higher in the treated group than in the control. Two mice in the untreated group died 2 days after the second injection of BA, whereas no mice in the group treated with TJ-41 died. The SW and SW/BW ratio were significantly lower in the treated mice than in the control. Suppressed IL-10 mRNA levels were observed in the spleens of the mice treated with TJ-41. Our data suggest that Hochu-ekki-to might possess an inhibitory effect on the marked decrease in running activity following BA injection.

Full text

Introduction
Fatigue is a common symptom observed in general population,
with up to 50% of the respondents reporting fatigue in a survey
(1). It is also presented as a symptom by at least 20% of
individuals seeking medical care (2). This fatigue is usually
transient, self-limiting and explained by prevailing circum-
stances (3). When the fatigue cannot be explained by a medical
condition, it may represent chronic fatigue syndrome (CFS).
Typical presentation of CFS is characterized by sudden onset
of a flu-like illness in which the predominant symptom is severe
and lasting fatigue that greatly reduces activity. The hypotheses
regarding the etiology of CFS propose the involvement of a
specific viral infection and immune dysfunction associated with
the infection, although it remains an important area for research.
Studies in the past have reported an immunologic model of
fatigue following intraperitoneal injection of bacterial antigen;
however, the duration of fatigue evaluated by wheel running
was found to be short (4). Ottenweller et al. (5) have described
the establishment of a potential murine model to study CFS
which could be induced by Brucella abortus (BA) treatment. In
this model, the mice were placed in running wheels and intra-
venously injected with the killed BA antigen, which produces
an acute immune response (5). The mice were found to decrease
their voluntary activity in the running wheel after the injection
of the BA antigen (5). The administration of this antigen has
previously been reported to produce modification in cytokine
gene expression in the lymphocytes of mice (6).
Survey data from the United Kingdom show that herbal
medicine has been tried by about 30% of the British population
(7). Hochu-ekki-to (TJ-41) is one of the herbal medicines pre-
scribed for viral infections, bacterial infections or post-surgical
shock. It has been reported to promote certain biological
Advance Access Publication 4 August 2004 eCAM 2004;1(2)203–206
doi:10.1093/ecam/neh020
© 2004, the authors
Evidenced-based Complementary and Alternative Medicine, Vol. 1, Issue 2 © Oxford University Press 2004; all rights reserved
Brief Communication
Effect of Hochu-ekki-to (TJ-41), a Japanese Herbal
Medicine, on Daily Activity in a Murine Model of
Chronic Fatigue Syndrome
Xin Q Wang,Takashi Takahashi, Shi-jie Zhu, Junji Moriya, Seiichiro Saegusa,
Jun’ichi Yamakawa, Kazuya Kusaka,Tohru Itoh and Tsugiyasu Kanda
Department of General Medicine, Kanazawa Medical University, Ishikawa, Japan
We aimed to evaluate the effect of a Japanese herbal medicine, Hochu-ekki-to (TJ-41), on daily activity
in a murine model of chronic fatigue syndrome (CFS). CFS was induced by repeated injection of
Brucella abortus (BA) antigen every 2 weeks. TJ-41 was orally administered to mice in a dose of
500 mg/kg/day for 1 week before injecting BA and for 4 weeks thereafter. We evaluated daily running
activity in mice receiving TJ-41 as compared with that in untreated mice. Survival of both mouse groups
was also monitored during the observation period. Body weight (BW), spleen weight (SW), SW/ BW
ratio and expression levels of interleukin-10 (IL-10) mRNA in spleen were determined in both groups
at the time of sacrifice. The daily activity was significantly higher in the treated group than in the con-
trol. Two mice in the untreated group died 2 days after the second injection of BA, whereas no mice in
the group treated with TJ-41 died. The SW and SW/BW ratio were significantly lower in the treated
mice than in the control. Suppressed IL-10 mRNA levels were observed in the spleens of the mice
treated with TJ-41. Our data suggest that Hochu-ekki-to might possess an inhibitory effect on the
marked decrease in running activity following BA injection.
Keywords: Hochu-ekki-to – herbal medicine – daily activity – chronic fatigue syndrome
For reprints and all correspondence: Tsugiyasu Kanda, Department of
General Medicine, Kanazawa Medical University, 1–1 Daigaku,
Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan.
Fax: 81-76-286-2702. E-mail: kandat@kanazawa-med.ac.jp
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access
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activities including enhancement of NK cells (8) and
macrophage (9) activity. It has also been indicated that
Hochu-ekki-to has an inhibitory effect on influenza virus
infection via enhancement of host immune responses in virus-
infected mice (10).
In this paper, we describe the effect of a Japanese herbal
medicine, Hochu-ekki-to (TJ-41), on daily activity in a mouse
model of CFS.
Subjects and Methods
Mice and Experimental Conditions
Female BALB/c mice, 8 weeks of age, were obtained from
Charles River (Kanagawa, Japan), and housed singly in cages
(230  100  100 mm) including running wheels (230 mm in
diameter), counters showing running wheel activity and water
taps, which were obtained from Natsume Seisakusho Co., Ltd.
(Tokyo, Japan). These cages were maintained under a light-
dark photoperiod (10 h vs 14 h) provided by fluorescent bulbs
fitted in the cage floor. We fed all the mice (n  20) every day
during the course of the experiment. Environmental air tem-
perature was maintained at 24–25C. The daily running activ-
ity of mice was defined as the number of wheel turns per 24 h.
The running activity was measured at 9 o’clock when the
environmental lighting was turned on. Approval for this exper-
iment was obtained from the animal experiment committee in
Kanazawa Medical University.
Induction of CFS by BA
Fixed killed whole BA ring test antigen was obtained from the
National Veterinary Services Laboratories in the United States
Department of Agriculture. CFS was induced by repeated
twice injection of original BA antigen solution (0.2 ml per
mouse) via the tail vein every 2 weeks. In the pilot experiment,
we found that the BA-injected mice showed less running
activity for 2–3 weeks after injection.
Treatment of Mice with Hochu-ekki-to
We obtained Hochu-ekki-to (TJ-41) that contains a mixture of
spray-dried hot water extracts of 10 medicinal plants from the
Ibaraki Plant of Tsumura Co., Ltd. (Tokyo, Japan). The 10 med-
ical plants are Astragali radix (16.7%), Atractyloclis lanceae
rhizoma (16.7%), Ginseng radix (16.7%), Angelicase radix
(12.5%), Bupleuri radix (8.3%), Zizyphi fructus (8.3%), Aurantii
nobilis pericarpium (8.3%), Glycyrrhizae radix (6.3%),
Cimicifugae rhizoma (4.2%) and Zingiberis rhizoma (2.0%).
This agent was dissolved in distilled water and diluted with water
to the appropriate concentration. TJ-41 solution was adminis-
tered orally in a dose of 500 mg/kg once daily through a feeding
needle inserted down the throat of the mice (n  10) for 1 week
before the induction of CFS and for 4 weeks thereafter. The dose
of the herbal medicine was determined on the basis of findings
of previous reports (11). Untreated mice (n  10) were given
saline during the same period. The mice enrolled into the exper-
iment were randomly assigned to the treated or the control group.
Parameters Determined for Evaluation of
Hochu-ekki-to
We started to examine the running activity (2 weeks) at base-
line 2 weeks after the mice were housed, since the activity was
stabilized after 2–3 weeks of housing (5). Daily activity during
2 weeks after each injection of BA was evaluated in the mice
receiving TJ-41 as compared with that in the untreated mice.
Survival in both groups was also monitored during the obser-
vation period. The mice in both groups were sacrificed by cer-
vical dislocation 4 weeks after the first BA injection. Ratios of
spleen weight (SW) (mg) to body weight (BW) (g) (SW/BW),
thymus weight (TW) (mg) to BW (TW/BW), heart weight
(HW) (mg) to BW (HW/BW) and lungs weight (LW) (mg) to
BW (LW/BW) as well as the weights of the organs and the
body were assessed between both groups at the time of sacri-
fice. Expression levels of interleukin-10 (IL-10) mRNA in
spleens from both groups were determined by using real-time
quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) as described previously (5). Optimum number of
cycles within the RT-PCR was examined for the IL-10 mRNA
levels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene was used as an endogenous internal standard, and was
amplified with specific primers for the number of cycles. The
IL-10 mRNA levels were calculated as comparative values,
which were normalized to the cytokine mRNA in the spleen
from normal female BALB/c mouse (value  1).
Statistical Analysis
Data are expressed as mean values  SD. Data differences
between the mice treated with Hochu-ekki-to and the control
were analyzed by the unpaired Student’s t-test. A P value
of 0.05 was considered to be statistically significant.
Results
Daily Running Activity in Mice
The daily running activity of both mouse groups before
and after the induction of CFS is presented in Fig . 1. Baseline
levels of daily activity were not significantly different between
both the groups. However, the daily activity was significantly
higher in the group treated with herbal medicine than in the
control during the 2 weeks after each BA injection (8157.6 
3287.6 vs 5884.3  2977.5 and 5584.9  2897.4 vs 2758.5 
2612.1 during the 2 weeks after the 1st and 2nd injection,
respectively, P  0.05). Two mice in the untreated group died
2 days after the 2nd injection of BA, whereas no mice in the
group treated with TJ-41 died.
Organ Weights and Expression Levels of
IL-10 mRNA in Spleen
BW, SW, TW, HW and LW in both groups are shown in
Table 1. Significant decrease in SW was observed in the mice
treated with TJ-41 as compared with that in the control (546 
37 mg vs 693  107 mg, respectively, P  0.05), while there
204 Effect of herbal medicine on chronic fatigue

were no differences in BW and the weights of other organs
between both the groups (Table 1). SW/BW ratio was signifi-
cantly lower in the group treated with TJ-41 than in the
untreated group at the time of sacrifice (24.2  1.4 vs 31.8 
6.3, respectively, P  0.05), whereas TW/BW, HW/BW and
LW/BW ratios were not significantly different between both
the groups. IL-10 mRNA levels in spleens of the treated mice
(n  6) were significantly lower than those in the control
(n  4) (5.47  1.81 vs 11.39  3.99, respectively, P  0.05).
Discussion
Running wheel activity is a comparatively sensitive indicator
to measure the severity of CFS. It has been reported that
blinded observers could not distinguish BA-injected mice from
saline-injected mice 11 days after the injection on the basis of
poor grooming (5). In practice, treatment of CFS, whether
pharmacological or non-pharmacological, has generally been
directed toward relieving symptoms and improving the
impaired functions (3). Therefore, the therapeutic effect of this
herbal medicine on the running behavior is considered to be a
more robust evidence than the effect on grooming.
Chao et al. (4) have previously reported that splenic enlarge-
ment (mg/g body weight) was found on day 12 post-inocula-
tion in a murine model of immunologically mediated fatigue
(24.1  1.8 in the Corynebacterium parvum group and 20.2 
1.6 in the Toxoplasma gondii group vs 5.3  0.2 in the saline
group). Marked elevation of SW/BW ratio after the repeated
BA injection was also observed in the present experiment, and
a significant reduction in SW/BW ratio was found in the mice
treated with the herbal medicine, suggesting that the measure-
ment of splenic enlargement might be useful to monitor the
improvement in immune stimulation during treatment.
We found a significant decrease in IL-10 mRNA levels in the
spleens of the mice treated with TJ-41 as compared with those
from the untreated mice. Abnormal expression of various
cytokine (IL-10, IL-4, IL-2 and interferon-) genes in the
spleen has been demonstrated in previous reports (5,6).
Circulating concentrations of these cytokines also need to be
measured for the evaluation of therapeutic effects in this
mouse model, since the measurement of circulating cytokine
levels will potentially be applied to future clinical studies.
CFS has been described to possess significant symptom over-
lap and comorbidity with psychiatric disorders (3). Effects of
herbal medicines including Hochu-ekki-to on behavioral
despair and acetic acid-induced writhing in mice has been
reported earlier, suggesting that Kampo medicines may have
anti-depressive and anti-nociceptive properties (12). Results of
our study may also be associated with the psychiatric profile of
Hochu-ekki-to.
eCAM 2004;1(2) 205
Table 1. Effect of Hochu-ekki-to on body weight and weight of organs including spleen, thymus, heart and lungs at the time of sacrifice
BW (g) SW (mg) TW (mg) HW (mg) LW (mg)
Treated mice (n  10) 22.6  2.3 546  37* 43  12 118  19 149  11
Untreated mice (n  8) 21.8  1.1 693  107 47  25 110  22 154  21
Data are expressed as means  SD. BW, body weight; SW, spleen weight; TW, thymus weight; HW, heart weight; LW, lungs weight. *P  0.05 compared with
spleen weight in the untreated mice.
Treated mice
Untreated mice
∗
∗
Figure 1. Effect of Hochu-ekki-to on the
daily running activity in a mouse model
of chronic fatigue syndrome. Data are
expressed as means  SD. BA, Brucella
abortus. Daily running activity was
defined as the number of wheel turns per
24 h. *P  0.05 compared with the daily
activity in the untreated mice. † Two mice
in the untreated group died 2 days after
the second injection of BA, while no mice
in the treated group died.

There are few studies demonstrating the effectiveness of alter-
native and complementary approaches including herbal therapies
for patients with CFS (3). Daily running activity in a mouse
model of CFS was significantly higher in the group treated with
Hochu-ekki-to (500 mg/kg/day) than in the control. SW/BW ratio
was significantly lower in the treated mice than in the control at
the time of sacrifice. Our results suggest that Hochu-ekki-to treat-
ment might have an inhibitory effect on the marked decrease in
running activity following the BA injection via modulation of
host immune responses, although the optimum dose and the
appropriate time of initiating the treatment of this herbal medicine
have not been established in this study. The difference between
the effectiveness of this agent and a positive control drug such as
a western medicine should also be assessed. Intraperitoneal treat-
ment with potent antioxidants such as carvedilol (5 mg/kg) and
melatonin (5 mg/kg) has been shown to produce a significant
decrease in the immobility period in a mouse model of CFS (13).
Further studies are required to elucidate these issues.
Acknowledgment
This study was supported in part by the Science Research
Promotion Fund of the Promotion and Mutual Aid Corporation
for Private Schools of Japan (Dr. T. Kanda).
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Received December 5, 2003; accepted March 26, 2004
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