Mechanisms of Ageing and Development
122 (2001) 341– 352
www.elsevier.com/locate/mechagedev

Immunological restoration and anti-tumor
effect by Japanese herbal medicine in aged mice
Masanori Utsuyama a,b, Heinrich Seidlar a,
Masanobu Kitagawa a, Katsuiku Hirokawa a,*
a
Department of Pathology and Immunology, Aging and De6elopmental Sciences,
Tokyo Medical and Dental Uni6ersity Graduate School, 1 -5 -45, Yushima, Bunkyo-ku,
Tokyo 113 -8519, Japan
b
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35 -2,
Sakaecho, Itabashi-ku, Tokyo 173 -0015, Japan

Received 3 July 2000; received in revised form 14 November 2000; accepted 15 November 2000

Abstract
We examined the effect of two Japanese herbal medicines (Kampo-Hozai) on immunological functions and anti-tumor activity in old mice. Hochu-ekki-to (TJ-41) was remarkably
effective in the restoration of impaired immune functions of old mice, in terms of number of
T cells and NK cells, and anti-SRBC antibody response, while it was not effective in
enhancing immune functions of young mice. Juzen-taiho-to was also effective in increasing
the number of T cells, remarkably, and NK cells, slightly, in the aged mice. While a
signiﬁcant increase was not observed in young mice. NK activity increased both in young
and old mice with the treatment of TJ-48. A signiﬁcant decrease was observed in metastatic
pulmonary colonies of B16 melanoma cells both in young and old mice treated with
Juzen-taiho-to for 16 weeks. These results suggested that some of Japanese herbal medicines
were useful in restoration of impaired immune functions of old mice and could be
recommended for human elderly. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Japanese herbal medicines; Kampo-Hozai; Immune functions; Aging; NK activity; Anti-tumor effect

* Corresponding author. Tel.: 81-3-58035173; fax: +81-3-38131790.
E-mail address: hirokawa.pth2@med.tmd.ac.jp (K. Hirokawa).
0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 4 9 - 9

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1. Introduction
A group of Japanese herbal medicine, so-called ‘Kampo-Hozai’ have been used
to improve the physical condition of various patients. Among various KampoHozais, we focused on Hochu-ekki-to (TJ-41) and Juzen-taiho-to (TJ-48). TJ-41 is
known as a useful drug to recover immune function and was reported to be useful
in infection (Mori et al., 1999; Yamaoka et al., 2000), oncostatics-induced leukopenia (Kaneko et al., 1999), and allergy (Suzuki et al., 1999). TJ-48 is useful not only
for the recovery of immune function (Abe et al., 1998), but also for the enhancement of anti-tumor effect (Onishi et al., 1998; Saiki et al., 1999).
We have been interested in methods reversing and restoring immune functions.
There are about four methods for this purpose: (1) transplantation of cells or
tissues, (2) dietary manipulations, (3) chemicals or drugs such as anti-oxidants, (4)
endocrine manipulations (Hirokawa, 1997). In a previous communication, we
reported that vitamin E was effective in enhancing immune vigor of young, but not
old mice (Wakikawa et al., 1999). On the contrary, cyclophosphamide, a well
known anti-tumor drug was effective in restoring immune functions of old mice,
but not young ones (Ishiyama et al., 1999).
In the present study, we examined efﬁcacy of Kampo-Hozai in terms of immunological restoration and inhibition of tumor metastasis and compared the effects
between young and old mice.

2. Material and methods

2.1. Mice
C57BL/6 mice were purchased from SLC (Shizuoka, Japan) and maintained in a
SPF colony of Tokyo Metropolitan Institute of Gerontology. In this study, young
mice were 3 months of age and old ones were 18–24 months of age.

2.2. Japanese herbal medicine (Kampo-Hozai)
Hochu-ekki-to (TJ-41) was prepared as a spray-dried powder of hot-water extract
obtained from ten medical plants in the ratio of Astragali Radix (4.0), Atractylodis
Lanceae Rhizoma (4.0), Ginseng Radix (4.0), Angelicae Radix (3.0), Bupleuri
Radix (2.0), Zizyphi Fructus (2.0), Auranti Nobilis Pericarpium (2.0), Glycyrrhizae
Radix (1.5), Cimicifugae Rhizoma (1.0) and Gingiberis Rhizoma (0.5). Juzen-taihoto (TJ-48) was prepared in the same way from ten medical plants in the ratio of
Astragali Radix (3.0), Cinamomi Cortex (3.0), Rehmanniae Radix (3.0), Paeoniae
Radix (3.0), Cnidii Rhizoma (3.0), Angelicae Radix (3.0), Ginseng Radix (3.0),
Hoelen (3.0), Glycyrrhizae Radix (1.5) and Atractylodis Lanceae Rhizoma (3.0).
An-chu-san (TJ-5) was prepared from six medical plants in the ratio of Cinamomi
Cortex (4.0), Corydalis Tuber (3.0), Ostreae Testa (4.0), Foeniculi Fructus (1.5),
Glycyrrhizae Radix (1.5), Amomi Semen (1.0) and Alpinae Ofﬁc Rhizoma (0.5).

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2.3. Experimental protocol
2.3.1. Experiment 1
TJ-41 was suspended in water and administered orally using gavage. Young
and old mice were separated into three groups, respectively; high dose (2000
mg/kg per day), low dose (500 mg/kg per day) and saline as control. Each group
was composed of six mice. TJ-41 was given once a day for 14 days and mice
were sacriﬁced 1 day after the last administration. Sheep red blood cells (SRBC)
were i.p. given 4.5 days before sacriﬁce for the assessment of anti-SRBC antibody (Fig. 1a). The total amount of TJ-41 per mouse was 940 mg in young high
dose, 226 mg in young low dose, 860 mg in old high dose and 222 mg in old
low dose.
2.3.2. Experiment 2
Young or old mice were separated into three groups, respectively, and fed the
original diet without Kampo-Hozai or the diet containing TJ-48 or TJ-5 as
mentioned below. Each group was composed of ten mice. The diets were given
for 16 weeks and mice were sacriﬁced for various assessments. B 16 melanoma
cells (F10, 5×104) were intravenously injected 3 weeks before the sacriﬁce (Fig.
1b).
2.4. Diet
Regular diet, CE-2 (Clea, Japan) was mixed with 1.6% of Anchu-san (TJ-5
diet) or Juzen-taiho-to (TJ-48 diet). Young and old mice were separated into
three groups, respectively and given either regular diet (control), TJ-5 diet or
TJ-48 diet for 16 weeks. The amount of diet taken per mouse was approximately
3.6 g/day in both young and old mice. Accordingly, the amount of KampoHozai taken per mouse for 16 weeks was estimated at approximately 6.5 g in
total.

2.5. Flow cytometry
Cell suspension was prepared from spleen. The percentage of lymphocyte subsets was assessed by FACScan (Becton Dickinson, Mountain View, CA). Monoclonal antibodies employed were anti-CD3, anti-NK1.1, anti-CD4, anti-CD8 and
anti-B220 (PhaMingen, San Diego, CA).

2.6. Anti-SRBC antibody response
Mice were injected intraperitoneally (i.p.) with 0.5 ml of 2% sheep red blood
cells (SRBC) in saline and sacriﬁced 4.5 days later. The spleen cells suspensions
were prepared and used for the assessment of direct plaque-forming cells (PFC)
as described previously (Wakabayashi et al., 1999).

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Fig. 1. Experimental protocol. (a) Oral administration of TJ-41 by gavage for 14 days. Sheep red blood
cells (SRBC) was intraperitoneally injected 4.5 days before the sacriﬁce. Control mice were given saline
in the same way. (b) Feeding of diet containing TJ-48 or TJ-5 for 16 weeks, ad libitum. Mouse
melanoma cells, B-16 (F10), was intravenously injected 3 weeks before the sacriﬁce. Control mice were
fed the regular diet without drug.

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2.7. NK acti6ity
A ﬁxed number of 51Cr-labeled YAC-1 target cell (4× 104) was mixed with either
0.5, 1, 2 or 4×106 spleen cells in a total volume of 0.2 ml in microplate with round
bottomed wells. The plate was incubated for 5 h, centrifuged and the radioactivity
of the supernatant was counted by a gamma-counter (ARC-380, Aloka, Japan).

2.8. Assessment of lung metastasis of B16 melanoma
B16 melanoma F10 cells (established by ten repetitions of an intravenous
injection and recovery of cells from a lung metastasis in young C57BL/6 mice) were
frozen and kept at −80°C. The number of 5× 104 of F10 cells was intravenously
injected into the tail vein of the young and old mice after feeding of the diet with
or without Kampo-Hozai mentioned above. The mice were sacriﬁced 3 weeks later
and lungs were perfused with buffered formalin, then transferred to Bouin’s
solution. Metastatic nodules on the surface of the lungs were traced, and the
numbers were measured.

3. Results

3.1. Effect of Hochu-ekki-to (TJ-41) on the immunological functions of young and
old mice, by oral administration using ga6age
TJ-41, at high and low doses, was orally given to young and old mice for 14 days
and the effect on the immune system was examined.
Number of T cells (CD3+ cells) in the spleen did not change in young mice, but
signiﬁcantly increased in old mice by both high and low dose treatments (Fig. 2a).
The ratio of CD4/CD8 in T cells did not change greatly in young mice, but
signiﬁcantly increased in old mice at dose dependent manner, approaching to the
level of young mice (Fig. 2b).
Number of NK1.1+ cells slightly increased in young mice at dose dependent
manner. In old mice, number of NK1.1+ cells was very low in control mice, but it
signiﬁcantly increased by treatment with TJ-41, although the level was still lower as
compared with the young level (Fig. 2c).
Anti-SRBC antibody response did not change greatly by administration of TJ-41
in young mice. In old mice however, the low level of anti-SRBC antibody response
signiﬁcantly increased at dose dependent manner (Fig. 3a).
As reﬂected with a slight increase of NK1.1+ cells, NK activity also slightly
increased in young mice. In old mice however, NK activity was very low in control
mice and did not change after the treatment (Fig. 3b). We thought that the low NK
activity in old mice was caused by stress of ﬁrm grasp during oral administration of
herbal medicine through gavage. Therefore, in the next experiment, we employed
the feeding of diet containing herbal medicine to avoid the stress.

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3.2. Effect of Juzen-taiho-to (TJ-48) on the immunological functions of young and
old mice, by dietary feeding
In the 2nd experiment, mice were fed ad limitum the diet containing Juzen-taihoto (TJ-48) for 16 weeks. TJ-48, rather than TJ-41, was employed for its stronger
capacity to restore anti-tumor activity. Number of T cells (CD3+ cells) in the spleen
did not change in young mice fed TJ-48 diet. In old mice, the level of T cell number
was low in old mice fed control diet or TJ-5 diet, but signiﬁcantly increased in the
group fed TJ-48 diet (Fig. 4a).
Number of NK1.1+ cells in the spleen did not change greatly in young mice fed
TJ-48 diet, as compared with other groups. It was of interest to note that number
of NK1.1+ in old control mice was slightly higher than in young mice (Fig. 4b) and
signiﬁcantly higher than in old control mice of the ﬁrst experiment (Fig. 2c). In the

Fig. 2. Effect of oral administration of TJ-41 by gavage on immune system of young and aged mice. (a)
Number of CD3+ cells in spleen. (b) The ratio of CD4+/CD8+ T cells in spleen. (c) Number of NK1.1+
cells in spleen. Open columns, control. Gray columns, low dose of TJ-41. Solid columns, high dose of
TJ-41. Each group, six mice. Vertical bars, S.E.M.

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347

Fig. 3. Effect of oral administration of TJ-41 by gavage on ant-SRBC antibody response (a) and NK
activity (b) of young and aged mice. Open columns, control. Gray coloumns, low dose of TJ-41. Solid
columns, high dose of TJ-41. Each group, six mice. Vertical bars, S.E.M.

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Fig. 4. Effect of feeding of TJ-5 diet and TJ-48 diet on the immune system of young and aged mice. (a)
Number of CD3+ cells in spleen. (b) Number of NK1.1+ cells in spleen. Open columns, control.
Hatched columns, TJ-5. Gray columns, TJ-48. Each group, ten mice. Vertical bars, S.E.M.

2nd experiment, a trend of increase was observed in the old group fed TJ-48 diet
(Fig. 4b).
NK activity signiﬁcantly increased in young mice fed TJ-48 diet as compared
with control (Fig. 5). The level in control group was slightly higher in young than
in old mice. It signiﬁcantly increased in old mice fed TJ-48 diet and approached to
the young level (Fig. 5). It was important to note that the level of NK activity in
old control mice of the 2nd experiment (Fig. 5) was signiﬁcantly higher than in old
control mice of the ﬁrst experiment (Fig. 3b).
Number of metastatic colony in lungs signiﬁcantly low in young mice fed either
TJ-5 diet or TJ-48 diet as compared with control (Fig. 6). A signiﬁcant decrease of
colony number was also observed in old mice fed TJ-48 diet (Fig. 6). Fig. 7 shows
examples of lung metastatic colonies indicating that a signiﬁcant decrease was
observed in both young and old mice fed TJ-48 diet.

4. Discussion
One major problem in the elderly people is susceptibility to infection mainly due
to immunological insufﬁciencies (Hirokawa, 1998). Elucidation of mechanism of
immunological insufﬁciencies in the elderly is a very important ﬁeld. But at the
same time, it would be urgent to develop methods to mending immunological
insufﬁciencies of the elderly. Thus, we have been testing various methods to reverse
and restore impaired immune functions of old mice.

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In animal experiments, the level of immune functions of old mice can be restored
to a level approaching that of young adult mice by grafting both newborn thymus
and bone marrow cells of young donor mice (Hirokawa et al., 1976). However this
method is not easy for human application, because it is almost impossible to ﬁnd
donors of newborn thymus of the same MHC type.
Effect of dietary caloric or energy restriction in rodents are pronounced in terms
of elongation of life span and immunological restoration (Effros et al., 1990). This
method appears to be useful for middle-aged adult in preventing immunological
decline in the rest of life, but not applicable to the elderly with immune deﬁciencies.
Various thymic peptides such as thymosin, THF, thymopentin and thymulin
(Dardenne and Bach, 1988) are possible candidates for substances to restore
immune functions, but reports are still conﬂicting (Hirokawa, 1997).
Solid effect of anti-oxidants or free radical scavengers may be expected in the
restoration of immune deﬁciencies of the elderly. Vitamin E is believed to be useful
for immunological restoration (Bendich, 1996). In our experiment using mouse
model, however, vitamin E is effective in young, but not in old mice (Wakikawa et
al., 1999). There are still many anti-oxidants and research in this area would be
promising.

Fig. 5. Effect of feeding of TJ-5 diet and TJ-48 diet on NK activity of young and aged mice. Open
columns, control. Hatched columns, TJ-5. Gray columns, TJ-48. Each group, ten mice. Vertical bars,
S.E.M.

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Fig. 6. Effect of feeding of TJ-5 diet and TJ-48 diet on the number of metastatic colonies of B16
melanomas in lungs. Open columns, control. Hatched columns, TJ-5. Gray columns, TJ-48. Each group,
ten mice. Vertical bars, S.E.M.

Japanese herbal medicine (Kampo-Hozai) is a mixture of many plant materials,
and thus the mechanism of effect appears to be complicated. We selected two
Kampo-Hozais, TJ-41 and TJ-48, to test the capacity to restore the immune
function and anti-tumor activity, respectively. There have been numerous reports
describing the beneﬁcial effect of TJ-41 on immune functions (e.g. Kaneko et al.,
1999; Mori et al., 1999; Yamaoka et al., 2000). TJ-48 is known to enhance
anti-tumor activity (Onishi et al., 1998; Saiki et al., 1999), in addition to the activity
to restore immune functions (Abe et al., 1998). In these experiments, the effect of
Kampo-Hozais were tested in young animals in which immune deﬁciencies were
experimentally induced by either stress, anti-tumor drugs or infection.
The present study is one of a few studies employing old mice having deﬁnitely
depressed immune function. Iijima et al. also employed old mice to see the effect of
Juzen-taiho-to on the modulation of type 1 and type 2 immune response in old mice
(Iijima et al., 1999). We tested the effect of Kampo-Hozai in these old mice in terms
of immunological restoration and anti-tumor effect. In the ﬁrst experiment, TJ-41
was administered orally using gavage by ﬁrmly grasping mouse, every day, for 14
days. Although the restoration of T cell number and anti-SRBC response were
observed, the number and activity of NK cells in old mice was very low even in
control. We thought that this was caused by stress of ﬁrm grasp during oral
administration of herbal medicine through gavage. In this respect, we obtained the

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351

Fig. 7. Effect of feeding of diet containing TJ-41 and TJ-48 on the gross appearance of pulmonary
metastases in young and old mice. Randomly selected ﬁve lobes of the lungs are shown for control diet,
TJ-5 diet and TJ-48 diet.

similar results by the experiment to see the effect of restraint stress on immune
system of old mice (Kanno et al., 1997).
Therefore in the next experiment to see the effect of TJ-48, mice were fed ad
libitum the diet containing TJ-48 for 16 weeks. In this experiment, a signiﬁcant
increase in the number of T cells was again observed in old experimental group and
besides the number of NK cells was well preserved in old control mice and slightly
enhanced in old experimental mice. NK activity was enhanced deﬁnitely in old mice
and slightly in young mice. Reﬂecting the increase of NK activity, number of
metastatic colonies in lungs was signiﬁcantly decreased in both young and old mice.
The results indicated that NK cells of old mice were susceptible to stress. Oral
administration of foods or drug through gavage must be avoided in experiments to
see data, which are susceptible to stress. Both TJ-41 and TJ-48 are useful to restore
some of immunological indices of old mice. The magnitude of enhancement is more
pronounced in old mice than in young mice. TJ-41 and TJ-48 are already used in
patients suffering from cancers or infections to activate immune functions. Considering the present study, they may be also recommended for elderly people to restore
the impaired immune functions.

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