Male ddy mice (Japan SLC Inc., Shizuoka, Japan) were used for the experiments. The animals were housed at 23°C ± 1°C and were fed standard non-purified diet (CE-2, Clea Japan Inc., Shizuoka, Japan) and tap water ad libitum. The experiments were conducted in accordance with the Guidelines for Animal Experimentation (Japan Association for Laboratory Animal Science, 1987). All experimental designs were approved by the Ethical Committee for Use of Experimental Animals.
Preparation and determination of GCBE
GCBE (yield: 13.7%) was extracted from green coffee (coffea canephora
) beans at 70°C for 2 h using 70% ethanol. Caffeine, chlorogenic acid and its related compounds were analysed by HPLC using anhydrous caffeine (Kishida Chemical Co., Ltd., Osaka, Japan) and chlorogenic acid (Sigma-Aldrich Co., Ltd., St. Louis, MO, USA) as standards (Figure ). HPLC equipped with a Capcellpack C18 (4.6
× 250 mm, Shiseido, Tokyo, Japan) and a photodiode array detector (SPD-10 Avp, Shimadzu, Kyoto, Japan) was used. The solvent used included (A) 2 mM H3
and (B) CH3
CN; a linear gradient of (A) was changed to (B) after 35 min. The flow rate was maintained at 1.0 mL/min. The amounts of caffeine and chlorogenic acid detected were 10.0% and 27.0%. As shown in Figure , GCBE contains chlorogenic acid and other related compounds, namely, 3-caffeoylquinic acid (neochlorogenic acid) and a mixture of feruloylquinic acids. These compounds were isolated and identified from GCBE [14
]. The amounts of 3-caffeoylquinic acid and a mixture of feruloylquinic acids using standard chlorogenic acid were 5.5% and 16.0%. The content of 4,5-dicaffeoylquinic acid was 5.2% using an authentic sample which was isolated from Japanese butterbur.
Three dimensional HPLC chromatogram of GCBE.
Chemical structures of constituents of GCBE.
Sesamin (Industrial Research, Ltd., Wellington, New Zealand) and orlistat (Hoffman-La Roche Ltd., Basel, Switzerland) were used as reference compounds. EDTA was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan), while palmitoyl CoA and L-carnitine were obtained from Sigma-Aldrich Co., Ltd. (MA, USA). Calcium and magnesium-free phosphate buffered saline [PBS (-)], Triglyceride E-test Wako, olive oil, sucrose, Triton X-100 and 5,5'-dithiobis(2-nitrobenzoate) (DTNB) were purchased from Wako Pure Chemicals Industries, Ltd., Osaka, Japan.
Measurements of mice body weight and visceral fats
Six week-old mice were given free access to nonpurified diet (CE-2) containing 0.5% or 1% GCBE for 14 days. Caffeine (0.05% and 0.1%) and chlorogenic acid (0.15% and 0.3%) were added to the diet. Mice were weighed every 2 days. Mice epididymal and perirenal fats were removed and weighed at the end of the experiment. Orlistat, a medicine prescribed for obesity, was used as a positive control.
Measurements of hepatic triglyceride level in mice
Acacia gum (5%) suspension containing GCBE (100 and 200 mg/kg· day), caffeine (10 and 20 mg/kg· day) and chlorogenic acid (30 and 60 mg/kg· day) was administered daily to 5 week-old mice for 13 days. On day 14, the livers were removed and approximately 200 mg of the liver was homogenized in 1 mL of PBS (-). This suspension was then centrifuged (2200 × g, 10 min), followed by the extraction of hepatic triglyceride (TG) using a mixture of chloroform and methanol (2:1). The extract was evaporated and resuspended in 1 mL PBS (-). The TG content was measured using Triglyceride E-test Wako. Sesamin was used as a positive control.
Determination of serum TG in olive oil-loaded mice
The experiments were carried out as described by Shimoda et al
]. Initial blood samples were collected from infraorbital venous plexus of mice (aged 5 to 6 weeks) after fasting for 20–22 h. The mice were administered with an acacia gum (5%) suspension containing GCBE (200 and 400 mg/kg), caffeine (20 and 40 mg/kg) and chlorogenic acid (60 and 120 mg/kg). After 30 min, olive oil (5 mL/kg) was administered to the mice. Blood samples were collected at intervals of 2 h after loading with olive oil. The samples were centrifuged and serum TG was measured using Triglyceride E-test Wako. Orlistat was used as a positive control.
Measurements of hepatic carnitine palmitoyltransferase activity in mice
Six week-old mice were fed a diet (CE-2) containing GCBE (0.5% and 1%), caffeine (0.05% and 0.1%), chlorogenic acid (0.15% and 0.3%), neochlorogenic acid (0.028% and 0.055%) and a mixture of feruloylquinic acids (0.081%) for 6 days. Mice livers were removed and homogenized in Tris-HCl buffer (pH 7.4) weighing 6- fold that of liver weight containing 0.25 M sucrose and 1 mM EDTA. The homogenate was centrifuged (880 × g, 4°C, 10 min) and the supernatant was collected; this was followed by a second centrifugation (11,770 × g, 4°C) to obtain mitochondrial fraction. The precipitate of the mitochondrial fraction was flushed and suspended in buffer to obtain a protein concentration of 6 mg/mL. CPT activity was measured according to the method described by Markwell et al
]. Tris buffer (58 mM, pH 8.0, 1 mL) containing 1.25 mM EDTA, 0.1% Triton X-100 and 0.25 mM DTNB, 37.5 μM palmitoyl CoA (20 μL) and 6 mg/mL mitochondrial fraction (20 μL) were mixed, and the optical density (OD) was recorded at 412 nm for 5 min. Subsequently, 1.25 mM L-carnitine (20 μL) was added, and the OD was recorded for 5 min. The CPT activity was calculated as follows:
ΔODa: Linearity of optical density change for 1 min after addition of L-carnitine solution
ΔODb: Linearity of optical density change for 1 min at baseline
c: Volume of mitochondrial fraction (μL)
d: Molar extinction coefficient of palmitoyl-CoA
e: Protein contents in mitochondrial fraction (mg protein/mL)
The results were expressed as mean ± SE. Significance of the differences was examined using the one-way ANOVA method, followed by Dunnett's test. Results with p < 0.05 were considered significant.