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.
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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)
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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 - 80°C 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.
Group
Lean control group
High-fat diet-treated group
High-fat diet plus 5% oolong tea powder-group
Parametrial adipose tissue (g)
0.89
1.39
0.67
Mean
Liver (g)
1.27
2.24
2.12
Hepatic triglyceride (mg/g)
63.3
116.5
49.8
82
43
O OQ
50
45
40
: 5
30
25
20
lean control group
high-fat diet-treated group
high-fat diet plus 5 % oolong tea-treated group
J L
5 6 Weeks
9 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 98.5±2.13 kcal in the laboratory chow group and 147.8±7.8 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 (161.5±8.9 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
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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
I
O H
0.20
0.15
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
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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 430±12 kJ in the control group and 487±17 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 487±17 kJ (high-fat diet) and 582±59 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, 4.3±0.2; high-fat diet group, 37.2±6.7; high-fat plus 0.5%) tea saponin diet group, 81.9±26.4 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.
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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
Control
High-fat diet
High-fat diet+0.5% teasaponin
Mean
Parametrial adipose (g)
0.98
2.08
1.04
Cell diameter (\im)
90.4
117.6
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.
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a o O
O H
Triolein emulsified with lecithin
Triolein emulsified with gum arable
Triolein emulsified with triton X-100
1.0 1.5
Concentrations (mg / mL)
2.0
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
10
Control
High-fat diet
High-fat diet+0.5%Tea saponin
0 1 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.
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300 r
O
6 M
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 only-treated 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.
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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.
Group
High-fat diet
High-fat plus 2% aqueous extract of Platycodi radix
High-fat plus 5% aqueous extract Platycodi radix
Liver weight (g/lOOg body weight)
7.21
6.35
6.17
Mean
Triacylglycerol C^tnol/g liver)
142.49
118.43
109.02
Total cholesterol (\^ mol/g liver)
13.6
13.8
14.1
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: 753.0±33.0 kJ/week/mouse in the high-fat diet group; 712.7±35.2 kJ/week/mouse in the high-fat diet plus 2% aqueous extract of Platycodi radix group; 682.8±45.8 kJ/week/mouse in the high-fat diet
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120
Inulin
Aqueous extract of Platvcodi radix
8 12
Concentration (g/L)
16
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
o
I O
t 6
0.0
• Lioid emulsion
' Lioid emulsion olus the aaueous extract Platvcodiradix
60 120
Time (min)
180 240
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.
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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 high-fat 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
I/)
<D
O OH
2.0L
X 1 1.5L
•Z3
O CI.
2.0W
1 ^ 1.51 i
X
X 1.0|i
S3
1.0|i
ooi H+2 H+5
0.51
O.Oi
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.
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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 chitin-chitosan 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 lecithin-emulsified triolein were 6.06 |Lig/ml and 8.7 nmol/ml/min, respectively. The Ki value of chitin-chitosan on the lipase activity in lecithin-emulsified 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.
Day
Day 1 Feces (g)
ExcretionTG (\i mol/g) Day 2 Feces (g) ExcretionTG (\i mol/g)
Day 3 Feces (g)
ExcretionTG (n mol/g)
Control group
1.40
4.86
3.02
2.56
1.64
3.26
HF-treated group
0.27
15.7
0.25 19.9
0.18
41.8
Mean
HF plus 3% chhin-chtitosan-treated group
0.43
15.2
0.54 40.4
0.35
50.2
HF plus 7% chitin-chtitosan-treatedgroup
0.56
28.6
2.15 46.2
1.18
55.3
HFpluslS%chitin-chtitosan-treated group
0.44
17.1
1.16 49.1
1.04
59.3
a T 3
00 02 06 01
Chitin-Chitosan (^ig/mL) 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'
X
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% chitin-chitosan-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 high-fat 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
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Table 5. Effect of chitin-chitosan on liver weight, hepatic triacylglycerol and total cholesterol in mice fed i
high-fat (HF) diet.
Liver weight (g/1 OOg body weight)
Triacylglycerol (M- mol/g)
Total cholesterol (^ mol/g)
Control group
4.01
11.47
6.33
HF-treated group
5.864
108.73
12.37
Mean
HFplus3% chitin-chtitosan-treated group
5.21
72.54
9.74
HFplu$7%chitin. chtitosan-treated group
5.21
59.44
8.68
HFplus 15% chttln-chtitosan-treated group
4.93
52.61
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 long-term (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.
Triacylglycerols(mM)
Total cholesterol(raM)
Free fatty acid(mEqa)
Control group
1.07
1.79
0.75
HF-treated group
1.65
3.02
0.98
Mean
HFplus 3% chitln-chtitosan-treated grou p
0.88
2.69
0.88
HFplu$7%chitin-chtitosan-treated group
0.87
2.32
0.90
HFplus 15% chttin-chtltosan-treated group
0.93
2.23
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 532.3±53.2 kJ in the laboratory chow and 862.5±68.2 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 862.5±68.2 kJ (high-fat diet), 927.4±29.5 kJ (3% chitin-chitosan diet), 850.1±43.4 kJ (7% chitin-chitosan diet) and 892.4±44.3 kJ (15% chitin-chitosan diet). This indicates that chitin-chitosan 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 high-fat 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).
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Substrate :triolein emulsified
with gum arabic
> 1
5 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
6
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 2-monooleoylglycerol (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 533.9±19.1 kJ/week/mouse in the control group and 1026.0±38.0 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 1026.0±38.0 kJ/week/mouse (high-fat diet group), 1074.0±19.7 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 1017.0±20.3 kJ/week/mouse (high-fat diet plus 13%) chondroitin sulfate group).
105
Lipid emulsion alone
6,
B
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
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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
o 03
45r
3or
15*
- 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
4
Weeks
8
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.
1.8|
1.5
12
o.9r
0.6h
0.3
0.0'
1
•b
X
*ab
X X
HF-0 HF-3 HF-7 HP-13
ICT
8
1 ^ > 1 ^ ^
1 >
2
b
*a
*b •b
T *ab
T •ab 1
HF-0 HF-3 HF-7 HF-13
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
U
i 60f
2or
C 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.
Table 8. Effects of chondroitin sulfate on hepatic triacyiglycerol and total cholesterol in mice fed a high-fat diet (HP) for 8 weeks.
Control
HFerouD
HFDIUS 3% chondroitin sulfate-treated
HFDIUS 7% chondroitin sulfate-treated
HFplus 13% chondroitin sulfate-treated
Mean
Triacyiglycerol (\i mol/g)
21.24
124.25
94.18
72.66
68.65
Total cholesterol (jl mol/g)
8.35
11.74
8.83
8.28
7.58
REFERENCES [1] Verger, R. Pancreatic lipase; Borgstrom, B. and Brockman HL. Ed.; Lipase.
Elsevier: Amsterdam, 1984; pp. 83-150. [2] Tsujita, T.; Matsuura, Y.; Okuda, H.; J. Lipid Res., 1996, 37, 1481-1487.
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[3] Hill, JO.; Peters, JC; Lin, D.; Yakubu, F.; Greene, H.; Swift, L.; Int. J. Obes., 1993, 17, 223-236.
