US 20020147318A1

(19) United States

(12) Patent Application Publication (10) Pub. No.: US 2002/0147318 A1
(43) Pub. Date:

Cho et al.
(54) PREPARATION METHOD OF WATER
SOLUBLE CARBOXYLMETHYL CHITOSAN
HAVING ANTI-LIPID PEROXIDATION
ABILITY

Oct. 10, 2002

Publication Classi?cation
(51)

Int. Cl? .

(52)

Us. 01. .............................................................. .. 536/20

C08B 37/08

(76) Inventors: Fu Chuan Cho, Taipei (TW);

Fung-Jou Lu, Taipei (TW)

Correspondence Address:
ROSENBERG, KLEIN & LEE
3458 ELLICOTT CENTER DRIVE-SUITE 101

ELLICOTT CITY, MD 21043 (US)

(57)

ABSTRACT

A preparation method of Water soluble carboxylrnethyl

chitosan (CM chitosan) is proposed to provide anti-lipid

peroXidation ability. The Water soluble CM chitosan is

prepared by special chemical methods from both shrirnps

(21) Appl. No.:

09/834,610

and crabs shells and the molecular Weight thereof is

betWeen 220-250 Kda and the deacetylated degree thereof is

(22) Filed:

Apr. 16, 2001

about 92%. The tWo kinds of CM chitosan derived from

shrirnps and crabs shells are powerful antioxidants to

(30)

Foreign Application Priority Data

prevent lipid peroXidation of rat liver rnicrosorne. Their

antioXidative abilities are as powerful as that of Water

Apr. 9, 2001

(CN) .................................... .. 01110671.X

soluble form of TroleX (Water soluble vitarnin

Patent Application Publication

Oct. 10, 2002 Sheet 1 0f 3

US 2002/0147318 A1

1-A

P 00

alM5atbs3iBogrpnAhtmion
2
I
PA

0.0

0. 000

0. 002

0. 004

0. 006

0. 008

concentratioMmg/ml)

FIG. 1A
100

1-B
(I) O

ipnehrcbto(%)

CD 0

.p. O

I050 =6.1Ug [01

IOI

0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009

concentration(mg/ml)

FIG. 1B

Patent Application Publication

O (O

P 00

Q N

Q 0)

P 01

US 2002/0147318 A1

2-A

Q o

lMab5atisD3ogrAphntimo

Oct. 10, 2002 Sheet 2 0f 3

0. 000

0. 002

0.004

0.006

0.000

concentration (mg/ml)

FIG. 2A
O: Q

inpherbcto(%)

2-B
1

NGOA01Q0CD
|

L C)

O
0.001

0.002 0.003 0.004 0.005 0.006 0.007 0.000 0.009

concentrati0n(mg/ml)

FIG.2B

Patent Application Publication

Oct. 10, 2002 Sheet 3 0f 3

US 2002/0147318 A1

A o

3-A
O no

5ata3blMsoirnBpgmtihAotn

CD 0)

O A

0.0

0.000

0.003

0.006

0.000

0.012

ooncentration(mg/ml)
100

IC50 =4.96ug /fnl

80-

peinrhcbt(o%)

3-B

AO

0.000

0.003

0.000

0.009

concentration(mg/ml)

FIG.3B

0.012

Oct. 10, 2002

US 2002/0147318 A1

PREPARATION METHOD OF WATER SOLUBLE

CARBOXYLMETHYL CHITOSAN HAVING
ANTI-LIPID PEROXIDATION ABILITY

solution, for example 5% NaOH (sodium hydroxide), With

ratio 1:20 (W/V) at room temperature for 20 hours to remove

FIELD OF THE INVENTION

protein from the shrimp crusts. The resulting material is

Washed by Water several times and then dipped in Weak acid
solution, for example 5% HCl, With ratio 1:20
at

[0001] The present invention relates to a preparation

method of Water soluble carboxylrnethyl chitosan having

ing material is Washed by Water and then dried to obtain

anti-lipid peroxidation ability.

chitin raw material.

BACKGROUND OF THE INVENTION

[0002] The chitosan has been found in Wide scope of

natural source and is only less abounding to the cellulose.
The chitosan can be abstracted from shrimp or crab crusts

and has the advantages of biological decornposability,

recycled usage, physiological and biological effects. More
over, the chitosan does not cause environrnent pollution. The

National Science Council, Republic of China had conducted

research of deriving chitosan and chitin from the exoskel

room temperature for 10 hours to dernineraliZe. The result

[0013] (2) The preparation of deacetylated chitosan

[0014] The thus obtained chitin raw material is pulveriZed
by 0.2 mm rnesh then sifted by 40-60 rneshes. The sifted
chitin raw material is mixed With 50% NaOH by the ratio
1:15 (W/V) at 100 C. for 6 hours with mechanical stirring
at 250 rpm. The deacetylated chitosan is Washed by Water
until the deacetylated chitosan is test to be neutraliZed by

cobaltous chloride (Whatrnan, USA). The deacetylated chi

eton of insects and marine invertebrates since 1996.

tosan is Washed by distill Water for three times and then

dried at 50 C.

[0003] The present invention is intended to provide a

preparation method of Water soluble carboxylrnethyl chito

ethyl chitosan (CM chitosan)

san (CM chitosan) having anti-lipid peroxidation ability. The

CM chitosan has excellent antioxidant effect and can be used

as food, biornedical material and cosmetic ingredient, and

can prevent the liver from the damage of lipid peroxidation.
SUMMARY OF THE INVENTION

[0004] It is the object of the present invention to provide

a preparation method of Water soluble carboxylrnethyl chi

tosan having anti-lipid peroxidation ability.

[0005] The Water soluble CM chitosan is prepared by
special chemical methods from both shrirnps and crabs
shells and the molecular Weight thereof is betWeen 220-250
Kilodalton (Kda). The tWo kinds of CM chitosan derived
from shrirnps and crabs shells are poWerful antioxidants to

prevent lipid peroxidation of rat liver rnicrosorne. Their

antioxidative abilities are as poWerful as that of Water

soluble form of Trolex (Water soluble vitarnin

[0015] (3) The preparation of Water soluble carboxylrn

[0016] The deacetylated chitosan (20 g) is suspended in

400 ml of isopropyl alcohol With stirring, then treated With
50.4 ml of 10M NaOH and stirred by 45 minutes. The
resulting solution is added With 24 g rnonochloracetic acid
poWder at 50 C. for 6 hours. The mixture is poured into
about 35 ml of Water. The pH of the resulting solution is
adjusted to 7 With glacial acetic acid, and the precipitate is
collected by ?ltration. The obtained solid is Washed With 500
ml of 70% methanol and then Washed again with methanol.
After that, it Was dried at 60 C. to give 30 g of the product

Water soluble carboxylrnethyl chitosan (CM chitosan).

[0017]

The molecular Weight of thus obtained Water

soluble CM chitosan is measure to be 220-250 Kda and the

deacetylated degree thereof is about 92%. The Water soluble

CM chitosan made from shrirnp crusts has satisfactory

anti-lipid peroxidation effect.

[0006] The various objects and advantages of the present

EXAMPLE 2

invention Will be more readily understood from the folloW

ing detailed description When read in conjunction With the

appended draWing, in Which:
BRIEF DESCRIPTION OF DRAWING

[0007] FIG. 1 shoWs the anti-lipid peroxidation effect of

CM chitosan frorn shrirnp according to the present inven

tion;
[0008] FIG. 2 shoWs the anti-lipid peroxidation effect of
CM chitosan frorn crab according to the present invention;
[0009] FIG. 3 shoWs the anti-lipid peroxidation effect of

Trolox;
DESCRIPTION OF THE INVENTION
EXAMPLE 1

[0010]

The Preparation Method of Water Soluble CM

Chitosan

[0018] Antioxidative Effect on Lipid of Rat Micorsornes

by Water soluble CM chitosan made from shrirnp crusts.
[0019]

In the present, the rat rnicrosornes is used to

provide lipid source. The product of lipid peroxidation

induced by NADPH With heat treatment (deconstructing
rnicrosorne enzyme) is referred as MDA (rnalondialdehyde).
The MDA is added With TBA (thiobarbituric acid) to form
MDA-TBA compound with pink ?uorescence. The MDA
TBA compound has absorption spectrum at 535 nrn (or 532
nrn) Wavelength. The content of MDA can be manifested by

the absorption degree of light at those Wavelengths. There

fore, the degree of lipid peroxidation can also be knoWn. If
the content of MDA is decreased after addition of Water
soluble CM chitosan, the reduction of MDA can be attrib
uted to the Water soluble CM chitosan. It means that the

Water soluble CM chitosan has the ability of anti-lipid

peroxidation. In the present invention, the anti-lipid peroxi

[0011] (1) The preparation of chitin raw material

dation ability of the Water soluble CM chitosan is compared

With that of the Trolex (Water soluble vitarnin

[0012] The chitin is isolated from shrirnp crusts. The

shrirnp crusts are ?rstly dried and then dipped in Weak basic

[0020] Result 1: FIG. 1 shoWs the experimental result for

antioxidant ability of Water soluble CM chitosan in the

Oct. 10, 2002

US 2002/0147318 A1

biological lipid peroxidation system using rat liver

microsomes, wherein the Water soluble CM chitosan is

derived from shrimps.

[0021] As can be seen from FIG. 1, the addition of the
Water soluble CM chitosan derived from shrimps can sup

press the lipid peroxidation in rat liver microsomes. The

Water soluble CM chitosan derived from shrimps has high

antioxidant ability.
[0022] More particularly, FIG. 1A shoWs that MDA, the
product of lipid peroxidation, is monotonically decreased as
the added amount of Water soluble CM chitosan derived

from shrimps is increased.

[0023] FIG. 1B shoWs that the IC5O (the concentration

required to suppress 50% lipid peroxidation) of Water
soluble CM chitosan derived from shrimps is 6.1 (by taking
average over at least three data).

[0024] Result 2: FIG. 2 shoWs the experimental result for

antioxidant ability of Water soluble CM chitosan in the

biological lipid peroxidation system using rat liver

microsomes, Wherein the Water soluble CM chitosan is
derived from crabs.
[0025]

[0032] As can be seen from above experimental results,

the Water soluble CM chitosan derived from shrimps or

crabs has high antioxidant ability, Which is compatible With

that of Trolex (Water soluble vitamin

Their effects are

summarized as folloWs:

[0033] (a) The IC5O of Trolex (Water soluble vitamin

E) is 4.96 pig/ml.
[0034] (b) The IC5O of Water soluble CM chitosan
derived from shrimps is 6.1 pig/ml.
[0035] (c) The IC5O of Water soluble CM chitosan
derived from crabs is 7.9 pig/ml.

[0036] Although the present invention has been described

With reference to the preferred embodiment thereof, it Will
be understood that the invention is not limited to the details
thereof. Various substitutions and modi?cations have sug

gested in the foregoing description, and other Will occur to

those of ordinary skill in the art. Therefore, all such substi
tutions and modi?cations are intended to be embraced

Within the scope of the invention as de?ned in the appended

claims.

As can be seen from FIG. 2, the addition of the

Water soluble CM chitosan derived from crabs can suppress

the lipid peroxidation in rat liver microsomes. The Water

soluble CM chitosan derived from crabs has high antioxi

dant ability.
[0026] More particularly, FIG. 2A shoWs that MDA, the
product of lipid peroxidation, is monotonically decreased as

I claim:

1. Apreparation method of Water soluble carboxylmethyl

chitosan having anti-lipid peroxidation ability, comprising

folloWing steps:

the added amount of Water soluble CM chitosan derived

from crabs is increased.

(1) the shrimp or crab crusts are treated by basic solution

to remove protein therefrom, the resulting material is
treated by acid solution to demineraliZe, thus obtaining

[0027] FIG. 2B shoWs that the IC5O (the concentration

required to suppress 50% lipid peroxidation) of Water
soluble CM chitosan derived from crabs is 7.9 (by taking

(2) the chitin raW material is treated in basic solution at

80-100 C. to obtain deacetylated chitosan.

average over at least three data).

[0028] Result 3: FIG. 3 shoWs the experimental result for

antioxidant ability of Trolex (Water soluble vitamin E) in the

biological lipid peroxidation system using rat liver

microsomes.
[0029]

As can be seen from FIG. 3, the addition of Trolex

(Water soluble vitamin E) can suppress the lipid peroxidation

chitin raW material;

(3) the deacetylated chitosan is dissolved in alcohol to

carboxylmethylate, the resulting solution is then dried

to obtain carboxylmethyl chitosan (CM chitosan).

2. The preparation method of Water soluble carboxylm
ethyl chitosan as in claim 1, Wherein the basic solution in
step (1) or step (2) can be selected from the group consisting
of NaOH and KOH.

E) has high antioxidant ability.

3. The preparation method of Water soluble carboxylm

ethyl chitosan as in claim 1, Wherein the acid solution in step
(1) can be selected from the group consisting of hydrochlo

[0030] More particularly, FIG. 3A shoWs that MDA, the

product of lipid peroxidation, is monotonically decreased as

ric acid, acetic acid, nitric acid, phosphoric acid, oxalic acid,
and butyric acid.

the added amount 0 Trolex (Water soluble vitamin E) is

increased.

4. The preparation method of Water soluble carboxylm

ethyl chitosan as in claim 1, Wherein the alcohol in step (3)
can be selected from the group consisting of isopropyl
alcohol and isobutyl alcohol.

in rat liver microsomes. The Trolex (Water soluble vitamin

[0031] FIG. 3B shoWs that the IC5O (the concentration

required to suppress 50% lipid peroxidation) of Trolex
(Water soluble vitamin E) is 4.96 (by taking average over at
least three data).