東京医療保健大学紀要

原 著

第6巻

第1号

2012. 3. 1

Effect of Pufferﬁsh liver on the ability of mice inlearning and memorizing
マウスにおけるフグ肝の記憶学習能力効果

大貫和江
Kazue ONUKI,

島田誠

Makoto SHIMADA,

柴真悟

Shingo SHIBA,

野口玉雄

Tamao NOGUCHI

〈 原著論文 〉

Effect of Pufferfish liver on the ability of mice in learning and memorizing
マウスにおけるフグ肝の記憶学習能力効果

大貫和恵

1

柴

真悟

1

島田

誠

野口玉雄

2

1

1

Division of Medical Nutrition, Faculty of Healthcare, Tokyo Healthcare University
2

Division of Medical Nutrition, Tokyo Healthcare University graduate school

1

Kazue ONUKI

東京医療保健大学

1

2

Shingo SHIBA

医療保健学部

Tomo SHIMADA

1

Tamao NOGUCHI

医療栄養学科1, 東京医療保健大学大学院

医療保健学研究科

A b s t r a c t ：It is clearly proved now that nontoxic pufferfish can be produced if they arecultivated in a net at sea or an aquarium in the land and fed a TTX-free diet. It was
found that they contain functional IPA and DHA, especially in their liver. DHA isrelated to learning and memorizing. From the effect of the pufferfish liver on theability in learning and memorizing by the passive avoidance test using the mice,
improvement in their ability was shown.
Mice were divided into two groups, “Control”（n=16）whose food contained no-IPA
and no-DHA and “the Pufferfish Liver”（n=16）whose food contained a lot of IPA
and DHA. After electric stimulation, the mice in the “pufferfish liver group” seemed
to be watching the dark compartment but those in the “control group” moved to thedark compartment immediately（p ＜ 0.05）. Moreover, after electric stimulation, theLT of “pufferfish liver group” tended to be longer than that of the “control group” andshowed a significantly different behavior（p ＜ 0.05）.
Therefore, it might be proven from the result of the passive avoidance test in themice that “pufferfish liver” including a lot of functional components, IPA and DHA
showed an effective improvement in the learning and memorizing ability of the mice.
It could be a promising functional food for the near future.
Keywords：pufferﬁsh liver, functional component, DHA （docosahexaenoic acid）, passive avoidance test, learningand memory ability

キーワード：フグ肝、 機能性成分、ドコサヘキサエン酸、 受動回避試験、 記憶学習能力
受付 : 2011 年 3 月 30 日

受理 :2011 年 9 月 16 日

and further make it a valuable processed dish.

Introduction

Consequently, it was found that it contains rich and

The Japanese Ministry of Health and Welfare
prohibited the serving of all kinds of pufferfish liverfunctional IPA（icosapentaenoic acid）and DHA（docosahexaenoic acid）.

1）

as a food in 1983 . Since then, such dishes have

These components are involved in the effective

not been traditional food. But it has been alreadyprevention of cardiovascular disease ） and78）, dementia ） ,proved that nontoxic pufferfish can be produced if

improvements in visual function

they are cultivated in a net at sea or an aquarium inmental stabilization ）, the development, learning andthe land and fed a TTX-free diet

2-3）

10

11-12）. Physiochemical
4-6）

9memory of the brain

13-14）and others

.

have

There are a lot of animal tests carried out tobeen investigated to revive it as a traditional dishevaluate their functionality as follows: passive

properties of the non-toxic pufferfish liver̶1̶

東京医療保健大学 紀要

第1号

Kazue ONUKI

2010 年

avoidance, active avoidance, maze tests（water maze,Tomo SHIMADA

Tamao NOGUCHI

Although for the lipid of mice food in AIN-

15）

redial maze, Y-maze）

Shingo SHIBA.

93G is the soybean oil used, it is not suitable for

Improvements in learning and memorizing from

evaluating the effect of the functional components

the effect of pufferfish liver were clearly shown byof the pufferfish liver because it includes a lot of n-3

the passive avoidance test. This passive avoidance

polyunsaturated fatty acid（linoleic acid（50 g%）） ）.

test was found to be a practically available tool for the

Therefore, lard（SHIGMA-ALDRICH）was usedevaluation of functionality in the pufferfish liver.

instead of the soybean oil in this study.

17

Table1. 1.
Proximatecomposition
feed for a mouse
Table
Proximatecompositionof feed forof
a mouseMaterial and Methods

Composition (g/100 g)

1. Material

Corn StarchControlPufferfish liver

00052.9486

00052.9286

Casein20.00019.816

Sucrose

10.0

10.0

in Yobuko, Saga Prefecture, Japan in 2009 were

Cellulose

05.0

05.0

submitted as material.

Lard

07.0

0004.168－

04.0

AIN-93 mineral mixture

03.5

03.5

AIN-93 vitamin mixture

01.0

01.0

L-Cystine

00.3

00.3

fatty acid（IPA and DHA）and vitamins were

Choline Hydrogen Tartrate

000.25000.25tert-Butyl Hydroquinone

00000.0014

00000.0014

analyzed according to the standard table of foodTotal

100.000100.964＊2

composition ）.

＊1: Crude fat components lard (4.168 g) and pufferfish liver (4 g) .
Net fat in pufferfish liver is 2.832 g/4 g.

The livers dissected from the non-toxic pufferfish
reared in a circular aquarium with an open system

Pufferfish liver

Their toxicity was examined by an intraperitoneal
injection using male mice of ddY strain（18 － 20g）according to the Japanese official method ） for16

determination of TTX. Their proximate composition,

17＊1＊2: Moisture in total is 0.964 g/100 g.

2. Methods
2.1 Animals

Table
Fatty
compositions
feed for a mouse
Table
2. 2.
Fatty
acid acidcompositionsof feed forof
a mouseFor the passive avoidance test, five-weeks-old male

Composition (%)

mice of the ddY strain, weighing 26 – 30 g wereused. They were housed in individual cages in an
air-conditioned room with a controlled temperature（23 ± 2℃）and humidity of 50 ～ 60% under 12 hours

2.2 Mouse food

The mice were divided into two groups, of“Control”（n=16）
whose food contained no-IPA and noDHA and ”Pufferfish Liver group”（n=16）whose foodcontained a lot of IPA and DHA. The food of the micein each group was adjusted according to the feed
mixture of AIN（American Institute of Nutrition）-93G ）.
18

Polyunsaturated fatty acids

The vitamin A content in the pufferfish liver

Monounsaturated fatty
acids

Saturated fatty acids

light-dark cycle. Water and food ad libitum until used.

Saturated fatty acids
Unsaturated fatty acids
Monoenoic acid
Dinoeic acid
Trienoic acid
Tetraenoic acidPentaenoic acid
Hexaenoic acid
14:0
15:0
16:0
17:0
18:0
20:0
14:1
16:1
17:1
18:1
20:1
22:1
24:1
16:2
16:3
16:4
n-6
18:2
n-3
18:3
n-3
18:4
n-6
20:2
n-3
20:4
n-6
20:4
n-3
20:5
n-3
21:5
n-6
22:4
n-3
22:5
n-6
22:5
n-3
22:6

analyzed was 9910 µg%. The tolerable upper intake
level（UL）of vitamin A for the human by Dietary
Reference Intakes for Japanese, 2010 is 2700 µgRE/
day（above 18 years old） ）. Taking account of the
19

above, AIN-93G was feasibly modified for the material
used and its content as follows.
̶2̶

ControlPufferfish liver

27.7
70.8
47.9
21.4
01.3
00.2
00.0
00.0
00.6
00.0
21.7
00.0
05.4
00.0
0.2
06.4
00.0
41.0
00.3
00.0
00.0
00.0
00.0
00.0
21.2
01.3
00.0
00.2
00.0
00.2
00.0
00.0
00.0
00.0
00.0
00.0

23.7
70.6
38.4
02.1
01.0
03.2
13.6
12.3
02.9
00.3
16.1
00.5
03.7
00.2
0.0
08.6
00.4
20.3
05.4
03.0
00.7
00.5
00.3
00.3
01.3
00.7
01.5
00.3
00.6
00.6
07.4
00.5
00.2
05.4
00.3
12.3

東京医療保健大学 紀要

Effect of Pufferﬁsh liver on the ability of mice in learning and memorizing

Finally, the feed for the mice was fixed as shown第1号

2010 年

continuously carried out for two weeks.

in Table 1-2, and the pufferfish liver content was
adjusted to 4 g%.

ControlPufferfish liver（The acquisition trial）
for 2 weeks

n=16（system of step-through2.3 System of passive-avoidance
test）

n=16

（The electrical stimuli trial）

Mice learning and memory ability was evaluatedelectric stimulation

using a step-through passive avoidance test.

n=8

n=8

n=8

n=8

（The learning and memory trial）

A step-through apparatus（Muromachi Kikai）was

3 days after electric stimulation

used for the passive avoidance test and the system
consists of a shock generator（SGS-003DX）for the

7 days after electric stimulationelectrical stimuli trial and a step-through cage（STC-CA

001M）for the acquisition trial（Fig. 1）.

Fig. 2. Experiment schedule of step – through test for mouse.
Fig.
2. Experiment schedule of step ‒ through test for mouse.

CB

LA

LB

3.2 The electrical stimuli trial

d)
b)
c)

e)

In order to learn and memorize the pain sufferedin the dark compartment, the following trials werea)given to them.

g)
a)
f)

h)

i)

j)
k)

For electrical stimuli, the mice were placed in the

Right :
Shock generator (SGS-003DX)
f) Powerg) Shock intensity (mA)h) Timer Remotei) Timer (min)j) Indicator (Shock rump)k) Manual operate button

lighted compartment, facing away from the dark

Fig.1.AAstep
step‒– throughthrough test
forfora mouse.
Fig.1.
testsystemsystema mouse.

was closed, and the LT to enter was recorded. Two

Left :
Mouse cage (STC-001M)a) Excreta trayb) Dark compartmentc) Lighted compartmentd) Guillotine doore) Grid part

one and allowed to explore for 30 seconds. After 30
seconds, the guillotine door was opened. When themice entered the dark one with all four paws, its door
seconds after the door was closed, a shock（0.4 mA）

In addition, the cage consists of two compartments,

was administered to their feet. Thirty seconds after

a lighted compartment（W90 × D115 × H150 mm）

the electric stimulation the mice were returned to theand a dark compartment（W145× D175× H150 mm）.

cage again.

They are separated by a guillotine door（W3 × H3.5cm）
. The dark compartment has a series of stainless-

3.3 The learning and memory trial

steel rods through which a constant electrical current

To evaluate whether the mice could learn andmemorize the danger of the dark compartment or

is delivered.

not, the following trials were given.
3. Step-through passive avoidance test（Fig. 2）

Three days（control group: CA, pufferfish liver

3.1 The acquisition trial

group: LA）and 7 days（control group: CA/CB,

To learn and memorize the dark compartment as

pufferfish liver group: LA/LB）after electricalbeing safe for mice, the following trials were carriedstimulation, the LT that the mice had moved from theout.

lighted compartment to the dark one was measured.

The mice were placed in the light compartment

Further, in order to evaluate fear suffered by micefacing away from guillotine door and allowed toin the dark compartment, their behavior before andexplore the room for 30 seconds. The door wasafter the electric stimulation was observed.

opened, and as soon as mouse entered the dark
compartment with all four paws, the latency time

4. The comparison of cholesterol content in the blood（LT）was immediately recorded. Thereafter theserum between “Control group” and “Pufferfish Liver”

mice were kept inside for 30 seconds before being

group

returned to their cages. The maximum observation

4.1 Preparation of blood serum

period in this test was 300 seconds. This trial was

Thirty-six mice of each group were dissected to̶3̶東京医療保健大学 紀要

第1号

Kazue ONUKI

2010 年

Shingo SHIBA

Tomo SHIMADA

Tamao NOGUCHI

collect their blood having fasting for 18 hours after

follows: Some kept in the lighted compartment, andexperiment. Sample was centrifuged at 3500 rpm

others wandered there, sometime peeping at the darkfor 10 min at 4℃ to get the serum which was themcompartment and stretching their bodies, including

preserved at -80℃ until use.

fear. Especially, 7 days after electric stimulation, all
the mice of LA showed negative-movements towards

4.2 The total cholesterol content in each serum

the dark compartment and the significant difference

Total cholesterol（TC）of each serum wasfrom those of CA was shown（p<0.01）.

measured by the enzyme method using E-TEST
WAKO of clinical chemical kit（Wako Pre Chemical

2. The LT of the step-through passive avoidance test

Result of passive avoidance test by the mice was

Industries Ltd.）according to the instructions of theshowed in Fig. 3.

manufacturer.
5. Statistical analyses

＊

300

＊

The data is presented as the mean ± SEM.
Latency time (sec.)

Statistical analyses used SPSS（ver. 11.5）
and the data
was performed by Wilcoxon rank-sum test for nonparametric systems.

200
＊

＊

139

150

56

0

0

1. Behavioral changes in the mice

42

41

4

3

7

0

7

0

CB
Control

7

6

3

CA

The effect of the pufferfish liver in passive

124

124

100
50

Result＊＊＊250

Passage day after shock

3

7

0

LA

7

(day)

LB
Pufferfish liver

n=8＊：p＜0.05, ＊＊ ：p＜0.01

Fig. 3. Effect of pufferfish liver in the step-through passive avoidance test.

Fig. 3. Effect of pufferﬁsh liver in the step-through passive avoidance test.

avoidance learning and memory was investigatedthrough the behavioral changes in the mice
（Table3）.

Each mean LT to enter the dark compartment in

Before the electric stimulation, each mouse（n=32）

CA and CB was 4 seconds, 3 seconds respectively

in CA and LA moved to the dark compartment

and in LA and LB was 6 seconds, 7 seconds. The

immediately when both groups were put in the

LT to enter the dark compartment was not different

lighted compartment.

among the groups. On three days after electric

T hree days after electric stimulation was

stimulation, the LT in CA showed a significant

administered to the mice that moved to the darkroom,

difference from that before electric stimulation

half of the mice（n=4）in CA moved but none in LA.

though extended from 4 to 56 seconds. In addition,

There was a significant difference between CA and

7 days after electric stimulation, each LT of CA and

LA in comparison to before the electric stimulation

CB showed a significantly different acquisition trial

was administered（p ＜ 0.05）. 7 days after electric（p ＜ 0.05）, in comparison with that before electric

stimulation, the same result was also obtained（p ＜stimulation.
On the other hand, 3 days after electric stimulation,0.05）.
Next, the mice in LA and LB tended to watch

each LT of LA extended from 6 to 139 seconds,

the dark compartment after electric stimulation as

and showed a significant difference（p ＜ 0.05）, incomparison with that before electric stimulation. In
addition, 7 days after electric stimulation, the LT of

Table 3.
of aofmouse
Table
3.Behavior
Behaviormodification
modiﬁcationa mouseControl

Passage day after shock0

LA and LB was the same as 124 seconds for each

Pufferfish liver

CA (n=8)

CB (n=8)

3 days 7 days

0

7 days0

LA (n=8)

LB (n=8)

3 days 7 days

0

7 daysother and showed a significantly different acquisitiontrial（p ＜0.05）, in comparison with that before electric

It moves to the dark
compartment at once.

8

4

6

8

6

8

0

0

8

3

It peeps into the dark
compartment.

0

5

2

0

2

0

7

8

0

5

It is wandering in thelighted compartment.

0

2

1

0

1

0

3

1

0

0

and 139 seconds, respectively. The LT in LA was

It doesn't move at all.

0

0

0

0

0

0

2

2

0

1

obviously longer than and significantly different from,stimulation.
Next, each LT of CA and LA on 3 days was 56

̶4̶

東京医療保健大学 紀要

Effect of Pufferﬁsh liver on the ability of mice in learning and memorizing

第1号

2010 年

that of CA resulting in effective acquisition（p ＜0.05）.

Mr. T. Nagai, Miss N, Nakagaki, Miss A. Fukumoto,On the other hand, 7 days after electric stimulation,

Mr. N. Hosaka, Mr. R. Masuda, Miss T. Obara, Miss N.

each LT both 124 seconds of LA and LB tended to

Kobayashi, Mr. Y. Kondo, Mr. Y. Sakai, Miss S. Hihara,

be very long in comparison with those in CA（41Miss A. Minagawa, who cooperated in this study. In

seconds）and CB（42 seconds）but not significantly

addition, we wish to express their gratitude to Manbodifferent from the letter（p ＜ 0.05）. The effective

Ltd. that kindly offered the samples.

acquisition in this trial seems not continue for 7 days.

Moreover, a part of this study was supported by

Further trials will be done to confirm the time tothe special research expense in 2006 from the Tokyo

keep effective acquisition.

Healthcare University and a subsidiary of Manbo Ltd.

3. The total cholesterol level of the mice

References

TC level of control and the pufferfish liver groupwere 148±28 mg/dl and 125±17 mg/dl respectively.

1） Pufferfishes available in Japan -an illustrated

That of the pufferfish liver group was lower thanguide to their identification -secondsond edition.

that of the control group and showed a significant

Environmental Health Bureau, Ministry of Health anddifference（p ＜ 0.01）, in comparison with that of the

Welfare. Chuo-hokishuppan, Tokyo 1984; 56-79.

control group.

2） Noguchi, T., Arakawa, O., Takatani, T. Toxiciy of

pufferfish Takifugu rubripes cultured in netcages at
sea or aquaria on land. C.B.P. Part D 1 2006; 153-

Conclusion

157.

For behavior of mice after electric stimulation,3） Noguchi, T., Arakawa, O., Takatani, T. TTX

mice in LA and LB seemed to be watching the dark

accumulation in pufferfish. C. B. P. Part D 1 2006;

compartment though those in CA and CB moved to145-152.

the dark compartment. It was clearly shown that4） Onuki K., Noguchi, T., Arakawa, O. Confirmation

mice in LA and LB did the behavioral learning andof non-toxicity of the pufferfish cultivated inmemorized fear of electric stimulation, different fromopen system of circular aquarium and functional

the mice in CA and CB.

compartments in the lipid J. J. of Food Chemistry

Since, the LT in LA and LB was much longer thanand Safety. 2009; 16（3）: 157-162.

that in CA and CB, it was strongly postulated that5） Onuki K., Noguchi, T., Arakawa, O. Nutrition andthe fear of electric stimulation was maintained for one

functional components of processed pufferfish livers

week. The effective improvement of the learning andwith safety. J. J. of Food Chemistry and Safety 2010;

memory ability on the aversive stimulus seems to17（1）: 6-11.

reflect on the score of LA and LB.

6） Mineki, M., Onuki, K., Sawasaki, E., et all. The

Therefore, it was clearly proven from the result

utilization, chemical composition, and fatty acidof the passive avoidance test in the mice thatcomposition of non-toxic livers of pufferfish（Takifugu

the pufferfish liver including a lot of functional

rubripes）cultured at aquaria on land. Japanesecomponents, IPA and DHA showed the effective

Journal of Sensory Evaluation 2009; 13: 23-30.

improvement of the learning and memory ability.

7） Hirai, A., Hamazaki, T., Terano, T., Nishikawa, T.,

In addition, it was found that they were effective in

et all. Eicosapentanoic acid and platelet function in

reduced in cholesterol. It will be promising functional

Japanese. Lancet 1980; 2: 1132-1133.

food for the near future.

8） Hoffman, DR., Birch, EE., Birch, DG., et all. Impactof early dietary intake and blood lipid composition of
long-chain polyunsaturated fatty acids on later visual

Acknowledgements

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O. Arakawa, Dr S. Taniyama and seminar students of9） Joshi, H., Kaur, N., Jyotibala, C. Evaluation ofthis University: Miss S. Okimura, Miss Y. Takamizu,

nootropic effect of argyreia speciosa in mice. J̶5̶

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第1号

Kazue ONUKI

2010 年

Health Sci. 2007; 53: 382-388.

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Tamao NOGUCHI

Folia pharmacologica japonica 2007; 130: 112-116．

10）Hamazaki, T., Sawazaki, S., Itomura, M., et all. The

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analyses for learning and memory in mice and rats.
要

旨：最近，フグ毒保有の餌を遮断した環境で養殖すれば無毒のフグが生産され，その肝も無毒であることが分かった。そのフグ肝には，機能性成分 IPA と DHA が非常に多く含まれ，DHA に記憶学習の維持向上効果が認められている。そこで本研究によりフグ肝は食品として記憶学習能力の向上に大きな効果をもたらすことを受動回避試験によりマウスを用いて検討した。
マウスの餌には，IPA および DHA を多量に含むフグ肝投与群（n=16）とこれら成分を含まないコントロール群（n=16）に分けて受動回避試験により記憶学習能力への効果を調べた。電気ショック後のフグ肝投与群は，コントロール群より明室内に留まったり，徘徊したり，暗室になかば身をのばし，有意に多く暗室を警戒した行動を起こし（p＜ 0.05），さらに，電気ショック後のマウスの反応潜時（LT）は，フグ肝投与群がコン
トロール群より明らかに長く，有意な差が認められた（p ＜ 0.05）。
これら受動回避試験の結果から，フグ肝には記憶学習能力の向上効果の可能性が認められた。将来，フグ肝は機能性を持った優れた食品になることが期待される。

̶6̶