Anti-Obesity and Hypotriglyceridemic Properties of Coffee Bean Extract in SD Rats

Abstract

Coffee bean extract (CBE) was prepared from raw green coffee beans and contained 10.0% caffeine and 27.0% chlorogenic acid. Male Sprague-Dawley rats were fed a diet containing 1% CBE for 4 weeks. Although there was no difference in food intake between rats fed the control diet without CBE and those fed the CBE-containing diet, body weight gain and white adipose tissue weight were significantly decreased in CBE-fed rats than in control rats. The CBE-fed group exerted a significant and extreme reduction in serum and liver triglyceride concentrations compared to the control group. Also, in the CBE-fed group, activities of fatty acid synthetic enzymes in the hepatic cytosol were significantly decreased, while that of fatty acid oxidative enzymes in the hepatic mitochondria was significantly increased. Our results suggest that CBE has potent anti-obesity and hypotriglyceridemic properties, and there is a possibility that these effects are exerted at least in part by the suppression of lipogenesis and the acceleration of lipolysis.

Full text

Food Sci. Technol. Res., 15 (2), 147 – 152, 2009
Anti-Obesity and Hypotriglyceridemic Properties of Coffee Bean Extract in SD Rats
Kazunari TaNaka1*, Shoko NishizoNo1,2, Shizuka TaMaru1, Mihoko koNdo1, Hiroshi shiModa3, Junji TaNaka3 and
Tadashi okada3
1 Graduate School of Human Health Science, University of Nagasaki, Siebold, 1-1-1 Manabino, Nagayo-cho, Nishisonogi-gun, Nagasaki
851-2195, Japan
2 Cooperative Research Center, University of Miyazaki, 1-1 Gakuen, Kibanadai-nishi, Miyazaki-shi, Miyazaki 889-2191, Japan
3 Research & Development Division, Oryza Oil & Fat Chemical Co., Ltd., 1 Numata Kitagata-cho, Ichinomiya-shi, Aichi 493-8001,
Japan
Received September 22, 2008; Accepted December 2, 2008
Coffee bean extract (CBE) was prepared from raw green coffee beans and contained 10.0% caffeine
and 27.0% chlorogenic acid. Male Sprague-Dawley rats were fed a diet containing 1% CBE for 4 weeks.
Although there was no difference in food intake between rats fed the control diet without CBE and those
fed the CBE-containing diet, body weight gain and white adipose tissue weight were significantly de-
creased in CBE-fed rats than in control rats. The CBE-fed group exerted a signicant and extreme reduc-
tion in serum and liver triglyceride concentrations compared to the control group. Also, in the CBE-fed
group, activities of fatty acid synthetic enzymes in the hepatic cytosol were signicantly decreased, while
that of fatty acid oxidative enzymes in the hepatic mitochondria was signicantly increased. Our results
suggest that CBE has potent anti-obesity and hypotriglyceridemic properties, and there is a possibility
that these effects are exerted at least in part by the suppression of lipogenesis and the acceleration of li-
polysis.
Keywords: coffee bean extract, anti-obesity, hypotriglyceridemic activity, lipogenesis, lipolysis
*To whom correspondence should be addressed.
E-mail: katanaka@sun.ac.jp
Introduction
Coffee is among the most widely consumed beverages
in the world. Raw green coffee beans, which are materi-
als of coffee, are rich in caffeine, chlorogenic acid, and its
related components, such as quinic acid, caffeic acid, and
p-coumaric acid. Caffeine has been reported to promote li-
polysis in adipocytes of experimental animals and humans
(Hasegawa and Mori, 2000; Zheng et al., 2004; Lopez-
Garcia et al., 2006). Kobayashi-Hattori et al. (2005) has ob-
served that caffeine reduced body fat mass and body fat per-
centage in a dose-dependent manner in rats fed high-fat diets.
Chlorogenic acid, another main component of raw coffee
beans, has been found to reduce cholesterol and triglyceride
concentrations in serum and liver (Rodriguez de Sotillo and
Hadley, 2002). Although there are several reports that coffee
intake reduces body fat accumulation (Thom, 2007) and tri-
glyceride levels (Carson et al., 1994), moderate coffee intake
does not seem to easily induce the suppression of obesity
and the improvement of lipid proles (Acheson et al., 1980;
Greenberg et al., 2006). In general, a relatively large amount
of caffeine is required to reduce body fat. While roasting raw
coffee beans develops a mild and desirable aroma, it also re-
duces the caffeine and chlorogenic acid contents (del Castillo
et al., 2002). Therefore, to substantially enhance anti-obesity
and lipid-lowering activities, a large intake of roasted coffee
for a long period would be needed to reduce body fat and the
hypolipidemic effect. On the other hand, coffee bean extract
(CBE) is characterized to be rich in caffeine and chlorogenic
acid. In the present study, we investigated the effects of CBE
on body fat and lipid metabolism in rats.
Materials and Methods
Preparation and determination of CBE CBE was ob-
tained by extracting raw green coffee (coffea canephora)
beans with 70% ethanol at 70℃ for 2 h. The chemical com-
position of CBE preparation was determined. The crude pro-
tein and lipid contents, which were assayed by the Kjeldahl

method and Soxhlet method, were 29.2% and 0.3%, respec-
tively. Moisture, which was determined as the loss in weight
after drying 105℃ for 24 h, was 2.2%. CBE contained
10.2% ash, as measured by the direct ignition method (540℃,
overnight). Caffeine, chlorogenic acid, and its related com-
ponents were assayed by HPLC with a Capcellpack C18
(4.6 × 250 mm, Shiseido, Tokyo, Japan) and a photodiode
array detector (SPD-10 Avp Shimadzu, Kyoto, Japan), us-
ing anhydrous caffeine (Kishida Chemical Co., Ltd., Osaka,
Japan) and chlorogenic acid (Sigma-Aldrich Co., Ltd., St.
Louis, MO, USA) as standards. The solvents included either
2 mM H3PO4 (A) or CH3CN (B), and a linear gradient of
solvent A was changed to solvent B after 35 min. The ow
rate was maintained at 1.0 mL/min. The amounts of caffeine
and chlorogenic acid were 10.0% and 27.0%, respectively.
The CBE included chlorogenic acid related components,
3-caffeoylquinic acid, a mixture of feruloylquinic acids, and
4,5-dicaffeoylquinic acid accounting for 5.5%, 16.0%, and
5.2%, respectively.
Animals and diets Male, 4-week-old Sprague-Dawley
rats (Japan SLC, Inc., Hamamatsu, Japan) were housed
individually in stainless-steel cages under a controlled at-
mosphere (temperature, 22 ± 1℃; humidity, 55 ± 5%; light
cycle, 8:00-20:00). Rats were given a commercial pellet
diet (Type CE-2, Clea, Tokyo, Japan) for 5 days and then
divided into two groups of equal body weight. The control
diet was prepared according to the formula recommended
by the American Institute of Nutrition (Reeves et al., 1993)
(Table 1). Experimental diets contained 0.5% cholesterol and
0.125% sodium cholate and supplemented with 1% CBE,
at the expense of cornstarch as in the control diets. Rats had
free access to the diets and water for 4 weeks. Food intake
and body weight of the rats were recorded daily. After the
rats were fasted for 6 h, their blood was collected with de-
capitation, and perirenal and epididymal white adipose tis-
sues and liver were immediately excised and weighed.
All animal studies were carried out under the guidelines
for animal experiments at University of Nagasaki, Siebold
(Nagasaki, Japan), and under Law No. 105 and Notication
No. 6 of the Government of Japan.
Preparation of hepatic subcellular fractions A sample
of freshly excised liver was homogenized in 6 volumes of
0.25 M sucrose solution containing 1 mM EDTA in a 10
mM Tris-HCl buffer (pH 7.4). After precipitating the nuclei
fraction, the supernatant was centrifuged at 100,000 × g for
60 min to precipitate microsomes, with the remaining super-
natant being used as the cytosol fraction. The mitochondrial
and microsomal pellets were resuspended in the same 0.25
M sucrose solution.
Serum and liver lipid analyses Serum lipids were as-
sayed enzymatically using commercial kits (Cholesterol
E-Test, Triglyceride E-Test, Phospholipid C-Test, Wako Pure
Chemical Industries, Osaka, Japan; and HDL-C, 2-Daiichi,
Daiichi Chemicals, Tokyo, Japan). Lipid peroxide in serum
was measured by a hemoglobin methylene blue assay with
Determiner LPO kits (Kyowa Medex Co., Ltd., Tokyo, Ja-
pan). Liver lipids were extracted by the method of Folch et
al. (1957). The concentrations of cholesterol, triglyceride,
and phospholipid were measured by the methods of Sperry
and Webb (1950), Fletcher (1968), and Rouser et al. (1966),
respectively.
Measurement of hepatic enzyme activities The activities
of cytosolic fatty acid synthase (FAS) (Kelly et al., 1986),
glucose 6-phosphate dehydrogenase (G6PDH) (Kessy and
k. TaNaka et al.
tcartxenaebeeffoClortnoC
teidfogk/g
002002)eerf-nimativ(niesaC
0505lionroC
Mineral mixture (AIN-93G-MX) 35 35
Vitamin mixture (AIN-93-VX) 10 10
0505redwopesolulleC
22etartratibenilohC
tert 410.0410.0enoniuqordyhlyhtuB-
33enitsyC-L
001001esorcuS
α231231hcratsnroC-
55loretselohC
52.152.1etalohcmuidoS
010tcartxenaebeeffoC
0001ot0001othcratsnroC
Table 1. The composition of experimental diets.
148

Kletzien, 1984), malic enzyme (Ochoa, 1955), microsomal
phosphatidic acid phosphohydrolase (PAP) (Walton and Pos-
smayer, 1985), and mitochondrial carnitine palmitoyltrans-
ferase (CPT) (Markwell et al., 1973) were determined in
the liver. Protein was assayed by the method of Lowry et al.
(1951), using bovine serum albumin as a standard.
Statistical analysis Data are reported as means ± SEM.
Data were inspected by the Student’s t-test. Values of p < 0.05
were considered statistically signicant.
Results
Although there was no difference in food intake between
rats fed the control diet and in those fed the CBE diet, body
weight gain was signicantly lower in CBE-fed rats than in
control rats (Table 2). Relative liver weight was comparable
between the two groups, but perirenal and epididymal white
adipose tissue weights were significantly low in the CBE
group.
The serum triglyceride level was extremely low in the
CBE group, compared with the control group (Table 3).
Feeding on the CBE diet induced the mild, but not signi-
cant, increase of the serum cholesterol concentration com-
pared to feeding on the control diet, but it did not modulate
the high density lipoprotein (HDL)-cholesterol concentra-
tion, consequently resulting in a lower HDL-cholesterol/total
cholesterol ratio. The level of phospholipids in the serum
was identical between both diets. The lipid peroxide concen-
tration in the serum was signicantly low in the CBE group.
Anti-Obese Effect of Coffee Bean Extract
tcartxenaebeeffoClortnoC
Body weight (g)
3±2412±341laitinI
*41±30301±683laniF
*21±1619±442niaG
0.1±5.125.0±7.22)yad/g(ekatnidooF
Tissue weight (g / 100g of body weight)
82.0±49.531.0±33.6reviL
White adipose tissue
*90.0±56.002.0±47.1lanerireP
*60.0±28.011.0±82.1lamydidipE
Perirenal + epididymal 3.03 ± 0.30 1.47 ± 0.13*
Each value is the mean ± SEM of 6 rats.
*Significantly different from the control group at p< 0.05.
tcartxenaebeeffoClortnoC
Serum lipids
Triglyceride (mmol/L) 2.04 ± 0.36 0.75 ± 0.13*
Total cholesterol (mmol/L) 4.35 ± 0.37 6.50 ± 0.51
HDL-cholesterol (mmol/L) 0.67 ± 0.07 0.62 ± 0.04
HDL-cholesterol/ Total cholesterol ratio (%)
05.1±98.91.2±9.51
Phospholipid (mmol/L) 2.36 ± 0.16 2.49 ± 0.11
Lipid peroxide (nmol/mL) 15.6 ± 1.0 10.5 ± 1.2*
Liver lipids (µ )g/lom
Triglyceride 85.4 ± 7.5 49.7 ± 3.6*
71±2816±971loretselohC
Phospholipid 35.7 ± 0.7 40.2 ± 1.3*
Each value is the mean ± SEM of 6 rats.
*Significantly different from the control group at p< 0.05.
Table 2. Effects of dietary coffee bean extract on growth parameters in rats.
Table 3. Effects of dietary coffee bean extract on serum and liver lipid concentrations in rats.
149

The level of triglyceride in the liver was significantly
lower in the CBE group than in the control group, whereas
that of cholesterol was comparable between both groups
(Table 3). Moreover, the hepatic phospholipid concentration
increased in the CBE group compared to the control.
The activities of enzymes related to fatty acid synthesis,
such as FAS, G6PDH, and malic enzyme, in the hepatic cy-
tosol were signicantly lower in the CBE group than in the
control group (Table 4). There was no difference in the activ-
ity of PAP, the rate-limiting enzyme of triglyceride synthesis,
in the hepatic microsomes between the control and CBE
groups. The activity of hepatic mitochondrial CPT, the rate-
limiting enzyme of mitochondrial β-oxidation, was signifi-
cantly higher in rats fed the CBE diet than in those fed the
control diet.
Discussion
Obesity is one of the risk factors for several lifestyle-
related diseases, including coronary heart diseases, diabetes
mellitus, hyperlipidemia, and is characterized by fat stor-
age in the adipose tissues. The intake of certain beverages
and functional foods has been shown to be effective at the
suppression or reduction of body fat accumulation (Maki et
al., 2002; Nosaka et al., 2003; Ikeda et al., 2005). Rats who
were fed CBE, which contains 10.0% caffeine and 27.0%
chlorogenic acid as the principal constituents, showed sup-
pressed body weight gain irrespective of food and energy
intake compared to those fed the control diet not containing
CBE (CBE group: 82.3 ± 3.6 kcal/day; control group: 86.8
± 2.0 kcal/day). The perirenal and epididymal adipose tissue
weights in CBE-fed rats were markedly lower than control
rats, strongly suggesting that slower body weight gain in the
CBE group is exerted by the suppression of visceral fat ac-
cumulation.
Caffeine has been demonstrated to reduce the weight
of adipose tissues in experimental animals (Hasegawa and
Mori, 2000; Zheng et al., 2004). Kobayashi-Hattori et al.
(2005) have shown that the intake of caffeine elevated the
serum level of catecholamine in rats fed a high fat diet, and
they presumed that the enhancement of the production of
catecholamine accelerated lipolysis. Caffeine of CBE is
thought to encourage the degradation of fat in adipose tissues
by stimulating catecholamine secretion. The portion of fatty
acids that released from adipose tissues is transferred to the
liver and is then oxidized. Therefore, the decreasing deposi-
tion of visceral fat may be in part related to the enhanced
oxidation of fatty acids in the liver. In the present study, CBE
increased the activity of mitochondrial CPT in the liver. This
enhanced activity is considered to be responsible for the re-
duction of adipose tissue weight and the suppression of body
weight gain.
Another reason for the anti-obesity activity of CBE may
be the suppression of postprandial hypertriglyceridemia.
Han et al. (1999, 2001) have pointed out that slower absorp-
tion of dietary fat decreased the deposition of visceral fat.
Shimoda et al. (2006) have shown that CBE and caffeine,
but not chlorogenic acid, suppress the elevation of the serum
triglyceride level after oral oil administration to mice. Thus,
the caffeine in CBE might suppress body fat accumulation
via suppressing postprandial hypertriglyceridemia. Since
we did not measure fecal fat excretion, it is unclear whether
CBE suppressed dietary fat absorption in the intestine. How-
ever, CBE intake effectively decreased both liver and serum
triglyceride concentrations. If CBE induces the inhibition of
intestinal fat absorption, the activities of hepatic lipogenic
enzymes may increase to compensate for the reduction in the
k. TaNaka et al.
tcartxenaebeeffoClortnoC
nietorpgm/nim/lomn
Lipogenic enzymes
Cytosol
Fatty acid synthase 5.29 ± 0.90 1.90 ± 0.78*
Glucose 6-phosphate dehydrogenase 17.2 ± 2.2 11.0 ± 1.0*
*94.1±8.3148.0±3.91emyznecilaM
semosorciM
Phosphatidic acid phosphohydrolase 4.46 ± 0.19 5.29 ± 0.40
Lipolytic enzyme
Mitochondria
Carnitine palmitoyltransferase 3.93 ± 0.38 5.05 ± 0.31*
Each value is the mean ± SEM of 6 rats.
*Significantly different from the control group at p< 0.05.
Table 4. Effects of dietary coffee bean extract on hepatic lipogenic and lipolytic enzyme activities in rats.
150

triglyceride level in the body. In the present study, these en-
zyme activities were signicantly suppressed by CBE intake,
indicating that dietary fat absorption in the intestine was
not suppressed. More detailed experiments are necessary to
clarify this effect.
CBE effectively lowered serum and hepatic triglyceride
concentrations. The activities of cytosolic FAS, malic en-
zyme, and G6PDH in the liver were decreased, whereas that
of mitochondrial CPT in the liver was increased in CBE-
fed rats. The reduction in the serum and hepatic triglyceride
levels in CBE-fed rats is thought to be induced by both the
suppression of fatty acid synthesis in hepatic cytosol and the
acceleration of fatty acid oxidation in hepatic mitochondria.
Chlorogenic acid has been shown to inhibit FAS activity (Li
et al., 2006), while there are few reports that caffeine affects
the activities of fatty acid synthetic enzymes. Therefore,
chlorogenic acid in CBE may be responsible for the suppres-
sion of fatty acid synthesis in the liver. Shimoda et al. (2006)
have observed that caffeine and chlorogenic acid alone have
no effect on CPT activity in the liver mitochondria of mice.
The combination of caffeine and chlorogenic acid or other
components of CBE may induce the enhancement of CPT
activity. Kobayashi-Hattori et al. (2005) has reported that
caffeine intake elevates the activity of acyl-CoA oxidase in
the liver. Caffeine in CBE may therefore accelerate hepatic
lipolysis by increasing acyl-CoA oxidase activity but not
CPT activity.
The serum lipid peroxide level in the CBE group was
two-thirds of that in the control group. Since chlorogenic
acid has an antioxidant property (Rodriguez de Sotillo et al.,
2002), it is presumed to contribute to the reduction in the
lipid peroxide level. The antioxidant activity of CBE is ex-
pected to reduce the risk of cardiovascular diseases by sup-
pressing oxidation of low-density lipoprotein cholesterol and
total cholesterol.
Raw green coffee bean contains cafestol, which is a di-
terpene, and potently increases serum cholesterol level in
humans and experimental animals (Urgert and Katan, 1997;
Post et al., 2000). CBE prepared from raw green coffee
beans contains cafestol. However, CBE-fed rats showed no
signicant increases in the serum cholesterol concentration
compared to a control diet. Also, the level in the liver was
the same between the control and CBE groups. Since CBE
contains a relatively large amount of chlorogenic acid, which
decreases low density lipoprotein cholesterol and total cho-
lesterol concentrations (Rodriguez de Sotillo and Hadley,
2002), it might not increase the serum cholesterol level.
In conclusion, CBE appears to effectively suppress body
fat and serum triglyceride levels through at least in part the
decrease in fatty acid synthesis and the acceleration of fatty
acid oxidation, showing that CBE may be a novel functional
food material for suppressing fat deposition.
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