Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol.

30
2005 Elsevier B.V. All rights reserved.

79

ANTI-OBESITY EFFECTS OF NATURAL PRODUCTS

HANL-K.\ KIMURAY.^ O K U D A H /
1. Department of Environmental and Symbiotic Sciences, Prefectural
University ofKumamoto, Tsukide 3-1-100, Kumamoto City, Kumamoto
862-8502, Japan.
2. Second Department of Medical Biochemistry School of Medicine,
Ehime University, Shigenobu-cho, Osen-gun, Ehime 791-0295, Japan.
ABSTRACT: Natural products such as tea saponin, Platycodi radix saponin, chitosan
and chondroitin sulfate were found to inhibit the hydrolysis of triolein emulsified with
phosphatidylcholine by pancreatic lipase in vitro. These products reduced the elevation
of the rat plasma triacylglycerol level after an oral administration of a lipid emulsion
containing com oil. Based on these results, experiments were designed to test whether
these products prevented obesity induced by feeding a high fat diet to mice. All these
natural products were proved to prevent the increase in parametrial adipose tissue
weight of mice fed the high fat diet. Such anti-obesity action of these products may be
mediated through delaying the intestinal absorption of dietary fat by inhibiting
pancreatic lipase activity.

INTRODUCTION
It is well known that dietary fat is not absorbed from the intestine unless
it has been subjected to the action of pancreatic lipase [1]. Previously,
we found that basic proteins such as protamines, histones and
purothionine inhibited the hydrolysis of triolein emulsified with
phosphatidylcholine [2]. The inhibition of hydrolysis of dietary fat may
cause a decrease or delay in the intestinal absorption of fat and reduce
blood chylomicron levels, an excess of which is known to induce obesity
[3]. Therefore, there was a possibility that inhibitory substances toward
pancreatic lipase activity may prevent the onset of obesity induced by
feeding a high fat diet to mice. Recently, we found that natural products
such as tea saponin, platycodi radix saponin, chitin-chitosan and
chondroitin sulfate inhibited the pancreatic lipase activity. In the
following section, the anti-obesity effects of these natural products will
be described in detail.

80

Tea saponin
Three kinds of tea, oolong, green and black, have been widely used as
health drinks from ancient times all over the world, especially to prevent
obesity and improve lipid metabolism. Among the three teas, oolong tea
is traditionally reported to have anti-obesity and hypolipidemic actions.
Kimura et al. [4] reported that the three kinds of tea prevented the
elevations of serum and liver lipids (total cholesterol and triglyceride)
and liver injury with an increase in serum transaminases (glutamic
pyruvic transaminase and glutamic oxaloacetic transaminase) in rats fed
peroxidized oil for one week. Furthermore, we found that tea tannins
such as epigallocatechin, epicatechin gallate and epigallocatechin
gallate, strongly inhibited lipid peroxidation in rat liver mitochondria
and microsomes [5]. Recently, we found that obesity was induced by
feeding a high-fat diet containing 40% beef tallow for 10 weeks to
female mice [6]. In the present study, we used a high-fat diet-induced
model of obesity in mice to clarify whether oolong tea prevents obesity.
In addition, we attempted to isolate the anti-obesity effectors from
oolong tea using an inhibitor assay on pancreatic lipase.
Oolong tea produced from Fu-Jian Sheng in China was purchased from
Uchida Wakan-Yak Ind. (Osaka, Japan), and voucher samples are stored
at the 2nd Department of Medical Biochemistry, School of Medicine,
Ehime University. The botanical classification of oolong tea was
identified as Thea sinensis L. by Prof Dr. M. Kubo (Department of
Natural Drug Resources, Faculty of Pharmaceutical Sciences, Kinki
University, Japan) and Prof Dr. Y. Zheng (Chinese Medicine Material
College, Jilin Agricultural University, China). Voucher specimens of
oolong tea were matched with specimens in the botanical repository of
the above two universities in Japan and China.
Female ICR mice (three weeks old) were obtained from CLEA (Osaka,
Japan), and maintained under a 12h/12h light/dark cycle, in a
temperature- and humidity-controlled room. The animals were given
laboratory pellet chow (CLEA Japan Inc; protein 24%, lipid 3.5%),
carbohydrate 60.5%) and water ad libitum. Fifty-four mice were divided
into three groups (n=18 each), with the groups matched for body weight,
after one week of feeding. The control mice were fed laboratory pellet
chow (normal group). The mice in the high-fat diet-fed groups received
the high-fat diet (beef tallow 40%), casein 36%), corn starch 10%), sugar
9%), vitamin mixture l%o and mineral mixture 4%o w/w per 100 g diet)

81

and water for 10 weeks ad libitum. The mice in the experimental group
received the high-fat diet containing 5% oolong tea powder (raw plant
dry leaf) for 10 weeks. The body weight of each mouse was estimated
once a week. The total amount of food intake by each mouse was
recorded at least three times every week. Following overnight starvation
they were sacrificed after anaesthetization with diethyl ether for about 2
min, and the liver and parametrial white adipose tissues were quickly
removed. The liver tissues were stored at - 80C until analysis was
performed. The liver triglyceride content was estimated as follows; a
portion (0.5 g) of the liver tissue was homogenized in Krebs Ringer
phosphate buffer (pH 7.4, 4.5 ml), and the homogenate (0.2 ml) was
extracted with chloroform-methanol (2:1, v/v, 4 ml). The extract was
concentrated under a nitrogen stream, and the residue was analyzed
using a Triglyceride E-Test kit.
Fig. (1) shows the changes in body weights of the g roups during the
experiment. Feeding a high-fat diet containing 40% beef tallow for 10
weeks produced significant increases in body weight and parametrial
white adipose tissue weight compared to laboratory chow-fed mice.
Furthermore, the high-fat diet also induced fatty liver, with the
accumulation of triglyceride when compared to the normal group (Table
1). Feeding a high-fat diet containing 5% oolong tea powder reduced the
increases in body weight and final parametrial adipose tissue weight, and
the accumulation of liver triglyceride compared to the high-fat diet
group (Fig. (1) and Table 1).
Table 1 Effects of oolong tea on parametrial adipose tissue weight, liver weight and hepatic triyglyceride in
mice fed a high fat diet for 10 weeks.
Mean
Group

Parametrial adipose tissue (g)

Liver (g)

Hepatic triglyceride (mg/g)

Lean control group

0.89

1.27

63.3

High-fat diet-treated group

1.39

2.24

116.5

High-fat diet plus 5 % oolong tea powdergroup

0.67

2.12

49.8

82

50

lean control group

high-fat diet-treated group
high-fat diet plus 5 % oolong tea-treated group

45

40
43

:^5
O
OQ

30

25

20

5
6
Weeks

10

Fig. (1). Effects of oolong tea on body weight in mice fed a high-fat diet for 10 weeks. Values are expressed
as mean + s.e.m. of 18 mice in each group. Significantly different from the high-fat diet-treated group;
*p<0.05.

The rate of reduction in body weight corresponded with that in

parametrial adipose tissue weight The mean food consumption per week
per mouse during the whole experimental period was significantly
different between the laboratory chow and high-fat diet groups, being
98.52.13 kcal in the laboratory chow group and 147.87.8 kcal in the
high-fat diet group. There was no significant difference in food
consumption between the high-fat diet group and high-fat plus oolong
tea diet (161.58.9 kcal per mouse per week). These results suggested
that the prevention of high-fat diet-induced obesity by oolong tea may be
due to a decrease in the intestinal absorption of lipid. Then, we
attempted to isolate an anti-obesity effector from oolong tea using an
inhibitory assay for pancreatic lipase. It has been clinically reported that

83

a pancreatic lipase inhibitor orlistat (Ro 18-0647) prevented obesity and

hyperlipidaemia after treatment for 12 weeks through the inhibition of
fat absorption [7-10].
0.25

0.20

0.15

OH

0.10
0

500

1000

1500

2000

Oolong tea extract (\i g/mL)

Fig. (2). Effects of the water extract of oolong tea on pancreatic lipase activity. Values are expressed as mean
+ s.e.m. of four experiments. FFA=free fatty acid.

In the present study, we found that the water extract of oolong tea
inhibited pancreatic lipase at the concentrations of 0.5-2 mg/ml, in a
dose-dependent manner, Fig. (2). Although caffeine was identified as an
activator of noradrenaline-induced lipolysis, it failed to inhibit the
pancreatic lipase activity (data not shown). Tea tannins such as
epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin
gallate also had no effect on the pancreatic lipase activity (data not
shown). These results suggest that the inhibition of pancreatic lipase
activity may be caused by tea saponin fractions in oolong tea that differ
from caffeine and tannins. In this experiment, we found that tea saponin
(a mixture of theasaponins Ei and E2) isolated from oolong tea inhibited

84

pancreatic lipase in vitro, Fig. (3). Furthermore, an experiment was

designed to clarify whether or not tea saponin prevented obesity induced
by long-term feeding of a high-fat diet containing 40% beef tallow.
Female ICR mice (three weeks old) were divided into three groups, with
each group matched for body weight, after one week of being fed
laboratory pellet chow ad libitum. The control group (n=10) continued to
be fed laboratory pellet chow ad libitum. The high-fat diet group (n=14)
was given the high-fat diet containing 40% beef tallow, 36%) casein,
10%) corn starch, 9% sugar, 1% vitamin mixture and 4%) mineral
mixture. The high-fat diet plus tea saponin group (n=12) was given the
high-fat diet containing 40% beef tallow, 35.5%) casein, 10% corn starch,
9% sugar, \% vitamin mixture and 4%) mineral mixture with tea saponin
mixed in at a concentration of 0.5%. The mean food consumption per
week per mouse was significantly different between the control group
and high-fat diet groups, being 43012 kJ in the control group and
48717 kJ in the high-fat diet group, but not significantly different
between the high-fat and high-fat plus 0.5% tea saponin diet groups,
being 48717 kJ (high-fat diet) and 58259 kJ (high-fat plus 0.5%) tea
saponin). Feeding a high-fat diet plus tea saponin had no effect on stool
frequency and content, but significantly increased the triacylglycerol
contents in feces compared to feeding a high-fat diet (triacylglycerol
contents in feces: control group, 4.30.2; high-fat diet group, 37.26.7;
high-fat plus 0.5%) tea saponin diet group, 81.926.4 mmol/g). Fig. (4)
shows the changes in body weights of the groups during the
experiments. Feeding a high-fat diet for 11 weeks caused significant
increases in body weights at 5 to 11 weeks compared to the control
group (laboratory pellet chow). Feeding a high-fat diet with 0.5%) tea
saponin significantly suppressed the increase in body weight compared
to the high-fat diet during the treatment period.

85

Table 2. Effects of tea saponin on parametrial adipose weight and the diameter of
adipose cells of mice fed a high-fat diet for 11 weeks.

Group

Mean
Cell diameter (\im)

Parametrial adipose (g)

Control

0.98

90.4

High-fat diet

2.08

117.6

High-fat diet+0.5% teasaponin

1.04

83.5

The final parametrial adipose tissue weights of the groups are shown in
Table 2. The final parametrial adipose tissue weight was significantly
increased by feeding a high-fat diet compared to the control group. That
in animals with a high-fat diet containing 0.5% tea saponin was
significantly reduced compared to the high-fat diet group. As shown in
Table 2, the diameter of fat cells was significantly greater in the high-fat
diet group than in the control group, and tea saponin completely
prevented the high-fat diet-induced increase in cell diameter.

86

Triolein emulsified with lecithin

Triolein emulsified with gum arable
Triolein emulsified with triton X-100

a
o
O

OH

1.0

1.5

2.0

Concentrations (mg / mL)

Fig. (3)EtTects of tea saponin on pancreatic lipase activity. Results are expressed as
means s.e.m. of four experiments.

It was demonstrated that the anti-obesity effects of oolong tea in high-fat

diet-treated mice might be partly due to the inhibitory actions of the
saponin fraction in oolong tea on pancreatic lipase activity. In this study,
tea saponin prevented the high-fat diet-induced increases in body and
parametrial adipose tissue weights by inhibiting the intestinal absorption
of dietary fat via inhibition of pancreatic lipase activity, Fig. (5).

87

413

o
PQ

Control
High-fat diet
High-fat diet+0.5%Tea saponin

10
0

11
Weeks

Fig. (4) Effects tea saponin on body weight in mice fed a high-fat diet for 11 weeks. Results are expressed as
mean + s.e.m. of 10-14. *p<0.05, Significantly different from high-fat diet group.

88

300 r

225

150

075

000
300

Time (min)
Fig. (5) Effects of tea saponin on rat plasma triacylglycerol levels after oral administration of a lipid emulsion.
Each point represents the mean is.e.m. of four rats. *p<0.05, significantly different from lipid emulsion onlytreated group.

Platycodi radix saponin

Platycodi radix, the roots of Platycodon grandiflorum (Jacq.) A.DC, has
been used traditionally as an expectorant and a remedy for bronchits,
tonsillitis, laryngitis and suppurative dermatitis in China, Korea and
Japan. In China and Korea, the fresh roots of P. grandiflorum have been
eaten as pickles for preventing obesity. Although it has been thought
recently that Platycodi radix pickles have anti-obesity activity, only
hearsay evidence exists. It has been reported that Platycodi radix
prevented hypercholesterolaemia and hyperlipidaemia [11]. However,
the mechanism of the anti-obesity and -hyperlipidaemic effects of
Platycodi radix remain to be clarified. In preliminary experiments, we
examined the effects of the aqueous extract of Platycodi radix on
pancreatic lipase activity in vitro and on the elevation of plasma
triacylglycerol levels caused by the oral administration of a lipid
emulsion to rats.

89

The aqueous extract of Platycodi radix inhibited the pancreatic lipase

activity in a dose-dependent manner in the assay system using triolein
emulsified with phosphatidylcholine, Fig. (6). At 2, 3 and 4 h after the
administration of the aqueous extract of Platycodi radix, the plasma
triacylglycerol concentration was significantly lower in the treated rats
than in the control group. Fig. (7).
Table 3. Effects of the aqueous extract of Platycodi radix on liver weight, liver triacylglycerol and total
cholesterol in mice fed the high-fat diet for 8 weeks.
Mean
Liver weight
(g/lOOg body weight)

Triacylglycerol
C^tnol/g liver)

Total cholesterol
(\^ mol/g liver)

High-fat diet

7.21

142.49

13.6

High-fat plus 2 % aqueous extract of Platycodi radix

6.35

118.43

13.8

High-fat plus 5% aqueous extract Platycodi radix

6.17

109.02

14.1

Group

On the basis of these results, the principal study was designed to

clarify whether the aqueous extract of Platycodi radix prevented obesity
induced by feeding a high-fat diet for 8 weeks. Energy consumption at
4-8 weeks was not significantly different among the high-fat diet and the
high-fat diet plus aqueous extract of Platycodi radix (2 or 5%) group as
follows: 753.033.0 kJ/week/mouse in the high-fat diet group;
712.735.2 kJ/week/mouse in the high-fat diet plus 2% aqueous extract
of Platycodi radix group; 682.845.8 kJ/week/mouse in the high-fat diet

90

120
Inulin
Aqueous extract of Platvcodi radix

12

16

Concentration (g/L)
Fig. (6) Effects of the aqueous extract of Platycodi radix and inulin isolated from the aqueous extract of
Platycodi radix on pancreatic lipase activity in vitro. Results are expressed as mean + s.e.m. of four
experiments.

plus 5% aqueous extract of Platycodi radix group. Consumption of the

high-fat diet for 8 weeks caused significant increases in body, parametrial

91

I
O

t
6

Lioid emulsion
' Lioid emulsion olus the aaueous extract Platvcodiradix

0.0

60

120

180

240

Time (min)
Fig. (7) Effect of the aqueous extract of Platycodi radix on rat plasma triacylglycerol level after oral
administration of a lipid emulsion. Each point represents the mean + s.e.m. of three experiments. *p<0.05,
significantly different from the group treated with lipid emulsion alone at the same time.

92

adipose tissue and liver weights. Body weight at 3 to 8 weeks, Fig. (8)
and final parametrial adipose tissue, Fig. (9) and liver

.top

o
OQ

Fig. (8) Effects of the aqueous extract of Platycodi radix (A) and inulin isolated from the aqueous extract of
Platycodi radix (B) on body weight in mice fed a high-fat diet for 8 weeks. Results are expressed as means +
s.e.m., n=14. *p<0.05, significantly different from high-fat diet goup.

weights (Table 3) were significantly reduced by consumption of the

high-fat diet containing 5% aqueous extract of Platycodi radix compared
with feeding the high-fat diet alone. Feeding the high-fat diet containing
2% aqueous extract of Platycodi radix did not reduce the body or
parametrial adipose tissue weights. On the other hand, feeding the highfat diet containing 2% aqueous extract of Platycodi radix significantly
reduced liver weight. Feeding the high-fat diet caused fatty liver with
accumulation of triacylglycerol and total cholesterol (Table 3).

93

2.5r

2.5f

2.0L

2.0W

I/)

<D
O
OH

O
CI.

1.5L

X
1.0|i

Z3

1.51

S3
1.0|i

0.51

ooi

O.Oi

H+2
H+5
H+0.5
H+1
Fig. (9) Effects of the aqueous extract of Platycodi radix (A) and inulin isolated from the aqueous extract of
Platycodi radix (B) on parametrial adipose tissue weight in mice fed a high-fat diet for 8 weeks. H: high-fat
diet; H+2: high-fat diet plus 2% aqueous extract of Platycodi radix; H+5: high-fat diet plus 5%aqueous
extract of platycodi radix; H+0.5: high-fat diet plus 0.5% inulin; H+1: high-fat diet plus 1% inulin. Results
are expressed as means + s.e.m., n=14. *p<0.05, significantly different from high-fat diet group.

The accumulation of hepatic triacylglycerol caused by the high-fat diet

was significantly decreased by feeding the high-fat diet containing 5%
aqueous extract of Platycodi radix compared with feeding the high-fat
diet alone. However, consumption of the high-fat diet plus the aqueous
extract of Platycodi radix did not affect the hepatic total cholesterol
concentration. To clarify the active substances in Platycodi radix, we
examined the effects of the major components of Platycodi radix, the
inulin and saponin fractions, on pancreatic lipase activity. Inulin had no
effect on the pancreatic lipase activity, and high-fat diet-induced obesity
in mice was not prevented by the administration of inulin.

94

a
o
U
(^
o

Crude saponin (g/L)

Fig. (10) Effect of crude saponin fraction isolated from the aqueous extract of Platycodi radix
on pancreatic lipase activity in vitro Results are expressed as mean s.e.m. of four experiments.

It has been reported that various saponins isolated from foodstuffs have
anti-obesity or hypolipidaemic actions [12-16]. We found that the crude
saponin fractions isolated from Platycodi radix strongly inhibited the
pancreatic lipase activity, Fig. (10).
Therefore, it seems likely that the anti-obesity, anti-hypolipidaemic,
anti-fatty liver actions of the aqueous extract of Platycodi radix may be
attributed in part to its crude saponin fractions.

95

Chitin-chitosan
Chitin-chitosan is a mixture of chitin (20%) and chitosan (80%). Chitin
and chitosan are polymers containing more than 5,000
acetylglucosamine and glucosamine units, respectively, and their
molecular weights are over one million Daltons. Although chitin is
widely distributed in natural products such as the protective cuticles of
crustaceans and insects, and the cell walls of some fungi and microorganisms, it is usually prepared from shells of crabs and shrimp.
Alkaline hydrolysis (45% NaOH, lOO'^C) of chitin converts it to
chitosan. The alkaline hydrolysate, however, contains chitosan (80%))
and non-reactive chitin (20%)) and is usually called 'chitin-chitosan'.
Inclusion of chitin-chitosan in the diet, reduced total plasma cholesterol
and inhibited the absorption of cholesterol and triacylglycerol from
lymph in animal models [17-20]. Previously, we reported that chitinchitosan reduced blood pressure elevated by NaCl intake, and
augmented the cytolytic activity of mouse lymphocytes [21-22]. In the
course of those experiments, we found that chitin-chitosan might inhibit
the intestinal absorption of dietary fat by inhibiting hydrolysis of the fat
by pancreatic lipase. To test this possibility, we studied the effect of
chitin-chitosan on pancreatic lipase activity in vitro and measured the fat
balance by determination of fat excretion in the feces of the mice fed a
high-fat diet or high-fat diet plus chitin-chitosan for three days.
Moreover, the present investigation was designed to clarify whether or
not chitin-chitosan prevented obesity induced by feeding a high-fat diet
for a longer term (9 weeks).
As shown in Fig. (Ua), chitin-chitosan inhibited the pancreatic lipase
activity dose-dependently between the concentrations of 6.25 |Lig/ml and
200 |Lig/ml in the assay system, using triolein emulsified with lecithin.
For characterization of the mechanism involved in the inhibition of
pancreatic lipase by chitin-chitosan, the enzyme activity was assayed at
various concentrations of lecithin-emulsified triolein and in the presence
of increasing concentrations of chitin-chitosan. A Lineweaver-Burk plot
of the data in Fig. (11 b) shows that chitin-chitosan was a competitive
inhibitor. The Km and Vmax values of the lipase activity for lecithinemulsified triolein were 6.06 |Lig/ml and 8.7 nmol/ml/min, respectively.
The Ki value of chitin-chitosan on the lipase activity in lecithinemulsified triolein was 17.6 |Lig/ml. When triolein was emulsified with

96

gum arable instead of lecithin, chltin-chitosan did not inhibit its

hydrolysis. In addition, we examined the effects of chitin-chitosan on the
lipase activity using neutral polymer Triton X-100 as emulsifier. When
triolein was emulsified with Triton X-100 (final concentration: 0.25
mg/ml) instead of gum arable, chitin-chitosan did not inhibit the
hydrolysis. Fig. (11 a).
Table 4. Effect of chitin-chitosan on fat excretion into feces of mice fed a high-fat (HF) diet for three days.
Mean
Day
Day 1
Feces (g)

Control
group

HF-treated
group

HF plus 3% chhinchtitosan-treated group

HF plus 7% chitinchtitosan-treatedgroup

HFpluslS%chitinchtitosan-treated group

1.40

0.27

0.43

0.56

0.44

4.86

15.7

15.2

28.6

17.1

3.02
2.56

0.25
19.9

0.54
40.4

2.15
46.2

1.16
49.1

Day 3
Feces (g)

1.64

0.18

0.35

1.18

1.04

ExcretionTG (n mol/g)

3.26

41.8

50.2

55.3

59.3

ExcretionTG (\i mol/g)

Day 2
Feces (g)
ExcretionTG (\i mol/g)

a
T3

00

Chitin-Chitosan (^ig/mL)

02

06

01

l/[Triolein, mg/mL]

Fig. (11) Effects of chitin-chitosan on pancreatic Hpase activity, a: Results are expressed as means s.e.m. of
four experiments, b: Lineweaver-Burk plots of the released oleic acid with lecithin-emulsified triolein as
substrate in the presence of various concentrations of chitin-chitosan.

These results suggest that the inhibitory effects of chitin-chitosan on

pancreatic lipase activity may be mediated by the interaction between
substrate and enzyme through lecithin. Moreover, as shown in Table 4,

97

the feeding of a high-fat diet plus 7% or 15% chitin-chitosan enhanced

fat excretion in feces and inhibited absorption of the ingested fat in the
balance study. Fig. (12) shows the changes in body weights of the

O Chitin-chitosan 0 p, g/ ml
# Chitin-chitosan 6.25 jLi g/ ml
91 Chitin-chitosan 12.5 |a g/ ml

groups during the experiments. The final parametrial adipose tissue and
liver weights of the groups are shown in Fig. (13) and Table 5,
respectively. Feeding a high-fat diet containing 40% beef tallow for nine
weeks caused significant increases in body weight at 4-9 weeks and in
final liver and parametrial adipose tissue weights, compared to the normal
diet group (laboratory pellet chow), (Fig. (12), Fig. (13) and Table 5).

98

50

Control
High fat diet
High-fat + 3% chitosan diet

'""""f]2""'"

High-fat + 7% chitosan diet

:-:i^>

High-fat + 15% chitosan diet

10
4

Weeks
Fig. (12) Effect of chitin-chitosan on body weight in mice fed a high-fat diet for 9 weeks. Values
are means s.e.m. of 13 mice.*p<0.05, significantly different from high-fat diet-treated group.

Feeding a high-fat diet containing 3%, 7% or 15% chitin-chitosan

significantly reduced the increase in body weight at 4-9 weeks, Fig. (12),
and the final parametrial adipose tissue weight compared to feeding a
high-fat diet, Fig. (13). In the experiments, it was found that feeding a
high-fat diet caused hyperlipidaemia, with elevations of serum

99

2.0

1.5

1.0

0.5

0.0'

1
Fig. (13) Effects of chitin-chitosan on parametrial adipose tissue weight in mice fed a high-fat diet for 9 weeks. l=Control
group; 2=High-fat diet-treated group; 3=High-fat diet plus 3% chitin-chitosan treated groups; 4=High-fat diet plus 7% chitinchitosan-treated group; 5=High-fat diet plus 15% chitin-chitosan-treated group. Values are means s.e.m. of 13 mice.
p<0.05, significantly different from high-fat diet-treated group.

triacylglycerol and total cholesterol, and caused fatty liver with

accumulation of triacylglycerol and total cholesterol in the liver. Serum
triacylglycerol was significantly reduced by feeding a high-fat diet
containing 3%, 7% or 15% chitin-chitosan compared to feeding a highfat diet (Table 6). Serum total cholesterol was also reduced by feeding
the 7% and 15% chitin-chitosan diets (Table 6). Feeding the 3% and
15% chitin-chitosan diets reduced the serum free fatty acid level
compared to feeding the high-fat diet (Table 6). The oral administration

100

Table 5. Effect of chitin-chitosan on liver weight, hepatic triacylglycerol and total cholesterol in mice fed i
high-fat (HF) diet.
Mean
Control
group

HF-treated
group

4.01

5.864

Liver weight
(g/1 OOg body weight)

HFplus3% chitinHFplus 15% chttlnHFplu$7%chitin.

chtitosan-treated group chtitosan-treated group chtitosan-treated group
5.21

5.21

4.93

Triacylglycerol (M- mol/g)

11.47

108.73

72.54

59.44

52.61

Total cholesterol (^ mol/g)

6.33

12.37

9.74

8.68

8.53

of 3%, 7% or 15% chitin-chitosan significantly reduced the liver weight,

and prevented the accumulation of liver triacylglycerol and total
cholesterol caused by a high-fat diet (Table 5). In addition, in the longterm (9 weeks) experiment, chitin-chitosan significantly reduced both
Table 6. Effect of chitin-chitosan on serum triacylglycerol, total cholesterol and free fatty acid in mice fed a
high-fat (HF) diet.
Mean
Control
group

HF-treated
group

HFplus 3% chitlnchtitosan-treated grou p

HFplu$7%chitinchtitosan-treated group

HFplus 15% chttinchtltosan-treated group

Triacylglycerols(mM)

1.07

1.65

0.88

0.87

0.93

Total cholesterol(raM)

1.79

3.02

2.69

2.32

2.23

Free fatty acid(mEqa)

0.75

0.98

0.88

0.90

0.67

body and parametrial adipose tissue weights at doses of 3%, 7% and

15%, whereas no differences in the energy consumed were found among
the experimental groups (except the lab chow group). The mean food
consumption per week per mouse during the whole experimental period
was significantly (p<0.05) different between the laboratory chow and
high-fat diet groups, being 532.353.2 kJ in the laboratory chow and
862.568.2 kJ in the high-fat diet group, but not significantly different
between the high-fat and high-fat plus 3%, 7% or 15% chitin-chitosan
diet groups, being 862.568.2 kJ (high-fat diet), 927.429.5 kJ (3%
chitin-chitosan diet), 850.143.4 kJ (7% chitin-chitosan diet) and
892.444.3 kJ (15% chitin-chitosan diet). This indicates that chitinchitosan prevents the high-fat diet-induced increase in body weight by
affecting food absorption.
It was concluded that the anti-obesity effects of chitin-chitosan in high-

101

fat diet-treated mice might be partly due to the inhibition of intestinal

absorption of dietary fat. Consequently, chitin-chtosan might cause an
improvement in the fatty liver and hyperlipidaemia in mice fed a highfat diet through inhibiting the intestinal absorption of dietary fat.
Chondroitin sulfate
Chondroitin sulfate consists of repeating D-glucuronic acid and D-A^acetylgalactosamine units, and the A^-acetylgalactosamine is substituted
with a sulfate at either its 4'or 6' position, with approximately one sulfate
being present per disaccharide unit. Chondroitin sulfate is present in the
cartilage, bone and cornea of animals. Previously, we reported that
chondroitin sulfate prepared from the nasal cartilage of salmon inhibited
glucose uptake into the brush border membrane vesicles prepared from
rat jejunum [23]. Based on the inhibitory action of chondroitin sulfate on
glucose uptake in the small intestine, we studied the effects of
chondroitin sulfate on pancreatic lipase activity and fatty acid uptake
into the brush border membrane vesicles in vitro. In addition, we
examined the effects of chondroitin sulfate on the plasma triacylglycerol
levels after oral administration of lipid emulsion. Moreover, the present
investigation was designed to clarify whether or not chondroitin sulfate
prevented obesity induced by longer-term feeding of a high-fat diet (8
weeks).

102

Substrate :triolein emulsified

with gum arabic

>
1

10

15

Chondroitin sulfate (mg/mL)

Fig. (14) Effects of chondroitin sulfate on pancreatic lipase activity. Results are expressed
as means s.e.m. of four experiments. *p<0.05, significantly different from control.

As shown in Fig. (14), chondroitin sulfate inhibited the pancreatic

lipase activity dose-dependently at the concentrations of 1-20 mg/ml in
the assay system using triolein emulsified with phosphatidylcholine; at
10 mg/ml it inhibited triolein hydrolysis by about 60%. On the other

103

hand, when triolein was emulsified with gum arable instead of

phospatidylcholine, chondroitin sulfate only slightly inhibited its
hydrolysis at concentrations of over 10 mg/ml; at 10 mg/ml it inhibited
by about 20%.
120 r

1001

Chondroitin sulfate (m^mL")

Fig. (15) Efifects of chondroitin sulfate on palmitic acid absorption by brush border membrane vesicles. The
rate of absorption is expressed as the percentage of the activity in the absence of chondroitin sulfate. Results
are expressed as means + s.e.m. of four experiments. **p<0.01, significantly different from control.

Then, we attempted to examine the effects of chondroitin sulfate on

palmitic acid and 2-monooleoylglycerol uptake into the brush border
membrane vesicles of the rat small intestine in vitro. As shown in Fig.
(15), chondroitin sulfate inhibited the incorporation of palmitic acid into

104

the brush border membrane vesicles dose-dependently at concentrations

of 1-10 mg/ml; at 10 mg/ml it inhibited palmitic acid incorporation by
about 80%. However, it did not affect the incorporation of 2monooleoylglycerol (data not shown). Fig. (16) shows the time course of
the plasma triacylglycerol concentrations when lipid emulsion with or
without chondroitin sulfate was administered orally to rats. Two hours
after chondroitin sulfate administration the plasma triacylglycerol
concentration decreased significantly compared with control. No
elevation of the plasma total cholesterol or free fatty acid level was
caused by oral administration of the lipid emulsion (data not shown).
Based on these results, we examined the effects of chondroitin sulfate on
food consumption, and body, parametrial adipose tissue and liver
weights in mice fed a high-fat diet for 8 weeks. Since chondroitin sulfate
could not be absorbed by oral administration [24], the amount of
chondroitin sulfate intake was not entered in the energy intake
calculations. The mean food consumption per week per mouse at 4-8
weeks was significantly (p<0.05) different between the control group
and high-fat diet groups, being 533.919.1 kJ/week/mouse in the control
group and 1026.038.0 kJ/week/mouse in the high-fat diet group, but not
significantly different among the high-fat plus chondroitin sulfate (3%,
7% or 13%) groups, being 1026.038.0 kJ/week/mouse (high-fat diet
group), 1074.019.7 kJ/week/mouse (high-fat diet plus 3% chondroitin
sulfate group), 990.5 16.0 kJ/week/mouse (high-fat diet plus 7%
chondroitin sulfate group) and 1017.020.3 kJ/week/mouse (high-fat
diet plus 13%) chondroitin sulfate group).

105

Lipid emulsion alone

6,

Fig. (16) Effects of chondroitin sulfate on rat plasma triacylglycerol levels after oral administration of lipid
emulsion. Each point represents the means + s.e.m. of 10 rats. *p<0.05, significantly different from lipid
emulsion alone-treated group.

Fig. (17) and Fig. (18) show the changes in body weights, liver and
parametria! adipose tissue weights of the groups during the experiment.
Feeding a high-fat diet for 8 weeks caused significant increases in body
weight at 2-8 weeks, and in the liver and parametria! adipose tissue
weights, compared to the control diet group (laboratory pellet chow).
The body weight at 3-8 weeks and parametria! adipose tissue and liver

106

weights were significantly reduced by feeding a high-fat diet containing

7% or 13% chondroitin sulfate compared to feeding a high-fat diet alone.
Feeding a high-fat diet containing 3% chondroitin sulfate slightly, but
not significantly reduced the body weight. However, feeding a high-fat
diet containing 3% chondroitin sulfate significantly reduced the
parametrial adipose tissue weight. As shown in Fig. (19), the diameter of
fat cells was significantly greater in the high-fat diet group than in the
control group, and chondroitin sulfate completely prevented the high-fat
diet-induced increase in cell diameter. Moreover, as shown in Tables 7
and 8, feeding a high-fat diet caused hyperlipidaemia with elevations of
plasma triacylglycerol (2.3-fold), total cholesterol (1.4-fold) and free
fatty acid (1.5-fold), and caused fatty liver with accumulation of
triacylglycerol (5.8-fold) and total cholesterol (1.4-fold). Plasma
triacylglycerol and total cholesterol were significantly reduced by
feeding a high-fat diet containing 3%, 7% or 13% chondroitin sulfate
compared to feeding a high-fat diet alone: 68%, 70% or 62% for
triacylglycerol and 80%, 79% or 70% for total cholesterol at 3%, 7% or
13%) chondroitin sulfate diet, respectively (Table 7). Plasma free fatty
acid was also significantly reduced by treatment with \3% chondroitin
sulfate compared to feeding a high-fat diet alone (about 84%)).
Chondroitin sulfate at 3%), 7%) and 13%) also prevented the accumulation
of hepatic triacylglycerol and total cholesterol caused by a high-fat diet:

107

45r

3or
o
03

- O " Control
#

High-fat

""" High-fat diet plus 3% chondroitin

'""VST^ High-fat diet plus 7% chondroitin
..%vQ:..v.. High-fat diet plus 13% chondroitin
15*

Weeks
Fig. (17) EtYects of chondroitin sulfate on body weight in mice fed a high-fat diet for 8 weeks. Results are
expressed as means + s.e.m. of 10-15 mice. *p<0.05, significantly different from high-fat diet-treated group.

76%, 58% or 55% for triacylglycerol and 75%, 71% or 65%) for total
cholesterol, respectively, compared to feeding a high-fat diet alone
(Table 8).
Summarizing all these results, the reduction of fat storage and the
antihyperlipidaemic action of chondroitin sulfate might be due to the
inhibition of small intestinal absorption of dietary fat through the

108

inhibition of pancreatic lipase activity and fatty acid uptake through the
brush border membranes.
ICT

1.8|

X
1.5

*b

*ab

12

1>^ ^1
o.9r

0.6h

*ab

ab

HF-7

HF-13

*a

^
^

1
>
2

0.3

0.0'

HF-0

HF-3

HF-7

HP-13

HF-0

HF-3

Fig. (18) Effects of chondroitin sulfate on parametria! adipose tissue (a) and liver (b) weights in mice fed a high-fat diet for 8
weeks. C, control; HG-0, high-fat diet; HF-3, high-fat diet plus 3% chondroitin sulfate; HF-7, high-fat plus 7% chondroitin
sulfate; HF-13, high-fat diet plus 13% chondroitin sulfate; Results are expressed as means s.e.m. of 10-15 mice. a:p<0.05,
significantly different from high-fat diet-ftreated group. *b: p<0.05, significantly different from control group.

109

nor

locr

a
60f
U

2or

HF-0

HF-3

HF-7

HF-

Fig. (19) EtYects of chondroitin sulfate on the diameter of adipose cells of mice fed a high-fat diet for 8 weeks. C, control;
HF-0, high-fat diet; HF-3, high-fat diet plus 3% chondroitin sulfate; HF-7, high-fat diet plus 7% chondroitin sulfate; HF-13,
high-fat diet plus 13% chondroitin sulfate; Each column represents the means + s.e.m. of four to six separate assays, a:
p<0.05, significantly different from high-fat diet-treated group. *b: p<0.05, significantly different from control group.

T a b l e 8. Effects of chondroitin sulfate on hepatic triacyiglycerol and total cholesterol in mice fed a high-fat
diet (HP) for 8 weeks.
Mean
Triacyiglycerol (\i mol/g)

Total cholesterol (jl mol/g)

Control

21.24

8.35

HFerouD

124.25

11.74

HFDIUS 3% chondroitin sulfate-treated

94.18

8.83

HFDIUS 7% chondroitin sulfate-treated

72.66

8.28

HFplus 13% chondroitin sulfate-treated

68.65

7.58

REFERENCES
[1]
[2]

Verger, R. Pancreatic lipase; Borgstrom, B. and Brockman HL. Ed.; Lipase.

Elsevier: Amsterdam, 1984; pp. 83-150.
Tsujita, T.; Matsuura, Y.; Okuda, H.; J. Lipid Res., 1996, 37, 1481-1487.

110

[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]

Hill, JO.; Peters, JC; Lin, D.; Yakubu, F.; Greene, H.; Swift, L.; Int. J. Obes.,
1993, 17, 223-236.
Kimura, Y.; Okuda, H.; Mori, K.; Okuda, T.; Arichi, S.; J Japan Soc Nutri Food
Sci, 1984, 37, 223-232.
Okuda, T.; Kimura, Y.; Yoshida, T.; Hatano, T.; Okuda, H.; Arichi, S.; Chem.
Pharm. Bull., 19S3, 31,1625-1631.
Han, L-K.; Li, J.; Sumiyoshi, M.; Tsujita, T.; Kimura, Y.; Okuda, H.; J. Trad
Med, 1999, 16,66-11.
Drent, ML.; Popp, SC; Ader, HJ.; Jansen, JB.; van der Veen, EA.; Obes Res,,
1995,3,573-581.
Dren, ML.; van der Veen, EA.; Obes Res., 1995, 3 (Suppl), 623s-625s.
Dren, ML.; Larsson, I.; William, OT.; Quaade, F.; Czubayko, F.; von
Bergmann, K.; Strobel, W.; van der Veen, EA.; Int. J. Obes., 1995,19, 221-226.
Hauptman, JR.; Jeunet, FS.; Hartmann, D.; Am J din Nutr, 1992, 55 (Suppl 1),
309s-313s.
Kim, K.-S.; Ezaki, O.; Ikemoto, S.; Itakura, H.; J. Nutr. Sci. Vitaminol, 1995,
41,485-491.
Kawano-Takahashi, Y.; Ohminami, H.; Okuda, H.; Kitagawa, I.; Yoshikawa,
M.; Arichi, S.; Hayashi, T.; Int. J. Obes., 1986,10, 293-302.
Kimura, Y.; Okuda, H.; Arichi, S.; Takemoto, T.; Shoyakugaku Zasshi, 1983,
37, 272-275.
Oakenfull, D. G.; Fenwick, D. E.; Hood, R. L.; Topping, D. L.; lilman, R. L.;
Storer, G. B.; Br. J. Nutr., 1979, 42, 209-216.
Sirtori, C. R.; Agradi, E.; Conti, F.; Mantero, O.; Gatti, E.; Lancet, 1977, 1, 275277.
Yamamoto, M.; Kumagai, A.; Yamamura, Y.; Arzneim.-Forsch.(DTUg
Research), 1975, 25, 1240-1243.
Ikeda, I.; Tomari, Y.; Sugano, M.; J. Nutr., 1989, 119, 1383-1387.
Sugano, M.; Watanabe, S.; Kishi, A.; Izume, M.; Ohtakara, A.; Lipids, 1988, 23,
187-191.
Ebihara, K.; Schneeman, BO.; J.Nutr., 1989, 119,1100-1106.
Razdan, A.; Petterson, D.; Br. J. Nutr., 1994,72, 277-288.
Zhou, A.; Matsuura, Y.; Okuda, H.; J. Trad Med, 1994,11, 62-64.
Kato, H.; Taguchi. T.; Okuda, H.; Kondo, M.; Takara, M.; J. Trad Med, 1994,
11, 198-205.
Takeda, T.; Majima, M.; Okuda, H.; J. Jpn. Soc. Nutr. Food sci., 1998, 51, 213217.
Andermann, G.; Dietz, M.; Eur. J. Drug. Metab. Pharmacokinet, 1982, 7, 11-16.