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J Nutr

Anti-Tumor

Activity

Sci

Vitaminol,

1997,

43, 455-461

of Squid Ink

Jin-ichi SASAKI,1Kunio ISHITA,1Yoshiaki TAKAYA,2


Hidemitsu UCHISAWA2and Hajime MATSUE2
1 Departmentof Bacteriology,Hirosaki UniversitySchoolof Medicine,
Hirosaki 036, Japan
2AomoriAdvancedIndustrial TechnologyCenter,
Aomori030-01, Japan
(Received December 19, 1996)
Summary

The

isolated

from

anti-tumor

squid

ink

tumor

from

BALB/c

tained

the

peptidoglycan

transplanted
the

mice

acetone
in

the

the

treated

served.
a

similar

the

cure

mented
Key
phage

to
was

or

rate

for

the

activation,

squid

ink,
lipid

injections

a prolongation

of

at

100

of

tumor.

The

cells.

delipidated
in

ink

to

was

10 min

also

direct
it

was

may

mainly

of
the

observed

did

not

affect

being
brought

delipidated
no

tumor

of

potentiality
ink

A
con

One-fifth

survival

its
the

Meth

which

(1mg/head)

for

ink,

Hence

of

Words

tumor

activity

immunity

with

for

acetone,

activity.

but

in

peptidoglycan

64%

administered

macrophages

Meth

of
of

was

fraction
A

type
rate

anti-tumor

of

the

cellular

(w/w),

delipidated

Meth

new

a cure

delipidated

treatment
the

have

the

cured

ink

of

0.1%

examine

Heat

activity

for

anti-tumor

ink

acetone-extractable

phagocytic

observed

The

squid

activity

The

mice.

as

animals.

anti-tumor

to

mice

delipidated

of

shown

at

so

tumor-bearing

activity

was

ink

enhanced

cytotoxicity
be
due

pre
about

said
to

was
that
the

the
aug

vivo.

delipidated

fraction,

anti-tumor

activity,

macro

fraction

Squid ink has been traditionally used from the past to preserve raw squid in
Japan. Japanese have already noticed, through experience, the anti-bacterial
potency of the ink and have used it as an additive to foodstuffs. However, most
squid ink in the fish processing industry is discarded at present. It would be
beneficial if we could find recycling methods for the ink.
On the other hand, extensive work has been conducted to find novel anti-tumor
compounds from natural resources (1-6). Recently, we have succeeded in the
isolation of a new type of peptidoglycan from squid ink and demonstrated its
anti-tumor activity with a 60-70% cure rate against Meth A fibrosarcoma in
BALB/c mice (7).
However, it remains questionable whether or not squid ink as a whole elicits
anti-tumor activity, which has never been investigated. If anti-tumor or another
455

456

J SASAKI

biological

activity

becomes

well

can

a new

In

this

as

its

be

material

paper,
lipid

demonstrated

for

we

by

re-usage

describe

as

Meth

anti-tumor

ink

was

for

powder

about

of

saline

cell

to
The

was

was
the

the

The

mice

and

were

incubated
a

medium

the

mice

model

of

mice
of

the

ink

on

days

were

each

2, 4 and
Mice

fed

10
was

with

saline.

mice

in

for
IP

treated

non-containing

Tumor-free

rinsed
rpm

After

IP

delipidated

a manner

were
1,500

HBSS.

were

acetone
examined

in

mice
at

in

(n=10)

with

The
and

mice

from

to

acetone-soluble
ink.

BALB/c

2~106/mL

1mg

wet

centrifuged

acetone

evaporated

and

saline

obtained
and

to

or

initial

and

The

further
ink

the

transported
at -30

filtration.

physiological

cells

treated

rapid
were

of
in

7.2)

tumor.

in

saline

solution

anti-tumor
of

5%
for

to

of

lipids

a reduced

mice

was

heated

activity
on

on
(n=5)

days

was

days

of

2,

2, 4

at

the

4 and

exudated
10%
air

for

100

were

at

for

heat-treated

6 (n=5

of
cells

of
yeast

least

10 min

to

delipidated

each)

particles

a test

make

after

tumor

of

a thin

with

tumor

delipidated

ink

in

The

the

of

homo

transplantation.

was
4

days
USA)

cells

were

activity

(8).

Saline-induced

macrophages

with

the

ink-induced

delipidated

inside

volumes
of

ink

saline

(Gibco,

phagocytic

film

1,000

cells)

collected
1640

lipid

acetone

only.

the

non-adherent

and

aliquots

(2~106
saline

were

tube

with

treated

with

RPMI

macrophages.

phagocytosis

after

(PEC)

and

to

were

delipidated

FBS-containing

into

homogenized

treated

atmosphere,

separation
using

mice
and

poured

condition

property

solution

in

was

thoroughly

similarly

activating

C02

acetone

Five

were

1mg/mL

compare

in

pressure

film

liposomes.

peritoneal

examined

a control

(2~106),

The

lipid

macrophage
Each

under

cells

treatment

under
Then

prepare

mice

follows.

adjusted

dig

BALB/c

acetone

delipidated

tumor

pH

IP

in

argentinus)

absolute

filtrate
of

dissolved

was

held

solution

(1mL/head)

Control

as

(2~106).

tube.
to

ink

recurrence.
ink

by

the

(Illex

of
before

tumor

200

stability.

1mg/mL

genate

was

no

heat

evacuated

saline

squid

maintained

squid

respectively,

(HBSS,

of

(IP)

yields

Meth

were

death

measured

test

discarded

METHODS

frozen

brief,

containing

transplantation
A

In

(n=10)

confirm

its

delipidated

volumes

was

the

tumor

delipidated

examine

the

temperature

3%,

solution

the

mice
until

months

of

20

and

ink

number

solution

observed

activities

is

industry.

experiments.

The

and

(7).

into

Control

same

with

salt

the

transplanted

ink

20

activity

and

the

pressure.

were

balanced

min,

mL

at

described

Hanks

that

food

from

with

delipidated

anti-tumor

for

peptidoglycan

the

previously

ink

the

intraperitoneally

at 4

reduced

(lipids)

powder

6.

24h

under

material

the

in

AND

provided

homogenized

containing

dryness

for

and

separated

Argentina,

stirred

was

intervals

The
from

ink,

additive

fraction.

fibrosarcoma

two-week

intact

an

MATERIALS

at

et al

was

IP

tested

injected

later.

The

for

2h

washed
of

as
into

PEC

at

37
out

in

macrophages
were

used

as

macrophages.
J Nutr

Sci

Vitaminol

Anti-Tumor

A
in

cytotoxicity

vitro.

FBS

test

Meth

containing

CO2-

air

trypan

RPMI

atmosphere.
blue

exclusion

Statistical
Student's

of

the

tumor

Activity

ink

cells

was

of Squid

Ink

performed

(1~107/mL)

against

were

1640

medium

(1mg/mL)

The

viability

of

tumor

between

the

test

457

Meth

mixed

and

incubated

cells

was

with

tumor

the

at

37

counted,

as

groups

was

cells

ink

in

10%

under

a 5%

usual,

by

the

method.

significance

and

control

evaluated

by

t-test.

RESULTS
The

chemical

ink

are

quoted

of

7.8%

peptide,

saccharide

had

analyses

of

a major

from

our

previous

here

57%
a

unique

From

peptide-chain.
Table

The

there

was

no
A

reduced

in

Table

was

2.

not

due

The

Fig.

The

Table

1.

This

table

present

A:

Vol

of

was

three

the

delipidated
composition

kinds

nor

poly
acid,

iduronic

proposed

of

peptidoglycan

The
glucuronic

acid

acid

to

sugar

contain

bound

against

It

thus

to

by

likely

quoted

experiment.

times

43, No 4, 1997

the

to

mouse

heated

into
on

days

2,

Fr.

B:

80,000,

of

of

our

Meth

Mice
4

and
Fr.

the

the

the

tumor

100
of

previous
tumor

after
C:

was
against

for

is

tumor

40,000.

of
of

ink

(7)
(2~106)

treated

with

transplantation.

the

to

squid

compare

delipidated

were

ink

immunity.
its

heat

illustrated

in

despite

heat

ink.

with

results

of

intraperitoneally

200g/mL/head
Molecular

1).

is shown

examine
are

not
ink

(Fig.

cells

preserved

from

to

(data

cellular

results
was

fractionated

report

tumor

same

delipidated

prolonged

the

The

the

However,

dosage
the

greatly

10 min

view.

cells
IP

this

activity

delipidated

were
6

rate
ink

with

shots).

of

enhancement

peptidoglycan

from

mice.

at

tested

three
with

anti-tumor

to

point

activity

first

1mg/mL/head

survival

but

activity

Anti-tumor

100,000,

that

was

to

delipidated

anti-tumorigenic

anti-tumor

was

the

the

cytotoxicity

was

obtained

200g

and
of

ink

an

efficiency

20%

seems

direct

ink

(200g/mL/head,

from

cytotoxicity

transplanted
three

delipidated

therapeutic
increase

growth
of

from
2.

of

the
the

(melanin).

ratios

peptidoglycan

the

of

peptidoglycan

delipidated

stability

the

of

the

apparent

lack

pigment

mannuronic

activity

activity
of

dosage

tumor
The

30%

from

revealed

equimolar

consisting

anti-tumor

dosage

shown).

analyses,

They

1.

anti-tumor

therapeutic

Neither

chains

The

in

fucose.

those

polysaccharide

shown

with

isolated

(7).

and

structure
and

detected.

data

polysaccharide

unique

N-acetylgalactosamine
was

peptidoglycan

the
(IP)

peptidoglycan,
weight

of

Fr.

458

J SAAI

Fig.

1.

Anti4umor

were

activity

1m/mL/head

Table

2.

Meth

ink,

treatment,

and

survived
The

treatment

with

Fig. 3 Deli
the

control.

difference

per

of

The
cell

between

ink

6 after

Meth

delipidated

5%

tumor

cells

were

tumor

against

with

under

cured

lipid

fraction

(1mg/head)
without
of

(2~106)

treated

with

transplantation.

tumor

squid

CO2-air.

cells.

ink

(1mg/mL)

Cytokilling

tumor.

Another

in

activity

was

animal

had

ink4nduced

macrophages

both

of

groups

ink

of

15

is shown

in

Table

tumorbearin

macrophages

Such

yeasts

delipidated

the
3

3.

mice,

recurrence.

ink.

the

from
cured

intraperitoneal

delipidated

number
for

squid

37

was

the

fraction

activity
the

4 and

Mice

4 weeks.

2 months

idated

1.0}007
the

over

haocytic

2,

incubated

at

mice

activity
the

days

Meth

mice.

ink.

five

rate of

of

were

medium

delipidated

of

nti4uor

injections

which

of

one

survival

The
Three

the

on

ink.

into

delipidated

(1~107)

1640
in

of

squid

transplanted

times

test

cells

observed

prolonged

three

Cytotoxicity

A tumor

delipidated

(IP)

10% FBS RPMI


not

of

intraperitoneally

et al

spread

ingested
ink
was

was

photographic

and

significant

by

examples
well

as

a macrophage

saline-induced

enhanced
are

compared

with

was

8.2}1.3

groups,

by

shown

in
those
and

respectively;

at p<0.005.
J Nutr

Sci Vitaminol

Anti-Tumor

Fig.

2.

Anti-tumor

for
planted

Meth

into

delipidated

Table

activity

10 min,

3.

mice.
ink,

Mice

were

tumor

IP

were

times

on

cells
treated

of

2,

isolated

were

ink

Ink

tumor

squid

lipid,

(IP)
three

at

100

(IP)
of

trans

heat4reated

transplantation.

ink.

intraperitoneally

1mg/mL/head

treatment

1m/mL/head

6 after

from

heat

intraperitoneally

with

4 and

459

after

were

treated

days

(2~106)
with

(2~106)
IP

lipid

of Squid

squid

cells

Mice

activity

tumor

delipidated

tumor

three

Anti4umor

Meth

of

Activity

times

transplanted
on

days

into
2,

and

mice
6

after

transplantation.

* Survival

day

was

described

for

nonmcured

mice.

DISCUSSION
Extensive
efforts
aspects using surgical,

have been made for the eradication


radio-, chemo- and iuno-therapeutical

two are the main strategies


supplemental
ones followed
With
potential
potentiatin
aiming at

respect
anti4umor

of therapies
and the latter two are considered
by surgical removal of cancer foci.

to immunotherapy
agents

without

for cancer,
any serious

agents in therapy
are usually used
preventing
the regro
the of remaining

Vol 43, No 4, 1997

of cancer from various


methods.
The first

it is indispensable

sidepefects,

because

to

to employ
such immuno

longterm after surgical operation,


or metastatic
cancer cells.

460

J SASAKI

et al

Fig. 3. Augmentation of phagocytic activity of macrophages by delipidated squid


ink. A: Saline-induced macrophages. B: Delipidated ink-induced macrophages.
Increasing phagocytic activity was observed in the ink-induced macrophages
(B).
We
have

have

been

recently

demonstrates
for

the

content

Meth

the

activity

at

Meth

A tumor.

longer

when

ink

to

was

the

mouse

without

less

in

peptidoglycan
in

than

concern

true

the
for

activate

and

natural

showing
in

squid

models

any
the

directed

resources,

from
tumor

0.1%
was

the

(7).

and

and

ink

which

The

side-effects.

ink

toward

20%
as

regrowth

enhancement

cure

However,

it

is

not

anti-tumor

effect

macrophages

of

the

shown

easy

to

activity

anti-tumor

lipid

survival

their

Fig.

and

at

two

The

of

exhibited
of

general,

may

cured

much

of

activity

tumor
of

the

10 min.
phago

delipidated
A similar

ink

in

might

explanation

phospholipids

participate

the

were

increasing

the

in vivo.
In

against

rates

mice

for

anti-tumor

days)

Anti-tumor
100

immunity

thereby

1.

tumor.

action

fraction.

showed

and
in

the

ink
of

treatment

cellular

(8)

each,

the

intervals

macrophages

the
of

of

at

of

heat

ink-induced
that

fraction

times

control,

against

speculated

the

lipid

were

without
stable

the
(three

rates

delipidated
we

by

to

60%

1mg/head

2 months

mediated

potency

our

cure

compared

ink

hold

of

Their

activity,

may

about

reason,

compounds
of

activity

is

delipidated

over

Since

be

was

this

a dosage

delipidated

cytic

type

ink.

Both

survived

anti-tumor

peptidoglycan

For

squid

find
novel

anti-tumor

A tumor
of

it.

intact

to
a

potential

rate

separate

trying

separated

have
tumor

the

eradica

J Nutr Sci Vitaminol

Anti-TumorActivityof SquidInk

461

tion. Taking into consideration other reports on biological activities of the ink ,
such as regulation of gastric juice secretion (9) or anti-ulceration activity (10), it is
very advantageous to utilize squid ink as a foodstuff or an additive for health
improvement. Incidentally, no toxicity of the squid ink is supported by its use as a
traditional food additive in Japan for over 100 years.
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1)

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investigation of the antitumor peptidoglycan fraction from squid ink. Biol Pharm Bull
17: 846-849.
8) Sasaki J, Kitagawa M, Satoh K. 1987. Macrophage activating property of Bean
lecithin. Nutr Res 7: 865-870.
9) Mimura T, Maeda K, Hariyama H, Aonuma S, Satake M, Fujita T. 1982. Studies on
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rats. Chem Pharm Bull 30: 1381-1386.
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Vol

43, No 4, 1997