Biotechnol. J. 2010, 5, 961–969

DOI 10.1002/biot.201000215

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Research Article

Fucoxanthin-rich seaweed extract suppresses body weight gain and improves lipid metabolism in high-fat-fed C57BL/6J mice Seon-Min Jeon1*, Hye-Jin Kim2*, Myoung-Nam Woo1, Mi-Kyung Lee3, Young Chul Shin4, Yong Bok Park5, Myung-Sook Choi1 1 Department

of Food Science and Nutrition, Kyungpook National University, Daegu, Republic of Korea R&D, CJ CheilJedang Corp., Seoul, Republic of Korea 3 Department of Food and Nutrition, Sunchon National University, Suncheon, Republic of Korea 4 Amicogen, Inc., Jinsung, Jinju, Republic of Korea 5 School of Life Science and Biotechnology, Kyungpook National University, Daegu, Republic of Korea 2 Foods

An ethanol extract of fucoxanthin-rich seaweed was examined for its effectiveness as a nutraceutical for body fat-lowering agent and for an antiobese effect based on mode of actions in C57BL/6J mice. Animals were randomized to receive a semi-purified high-fat diet (20% dietary fat, 10% corn oil and 10% lard) supplemented with 0.2% conjugated linoleic acid (CLA) as the positive control, 1.43% or 5.72% fucoxanthin-rich seaweed ethanol extract (Fx-SEE), equivalent to 0.05% or 0.2% dietary fucoxanthin for six weeks. Results showed that supplementation with both doses of Fx-SEE significantly reduced body and abdominal white adipose tissue (WAT) weights, plasma and hepatic triglyceride (TG), and/or cholesterol concentrations compared to the high-fat control group. Activities of adipocytic fatty acid (FA) synthesis, hepatic FA and TG synthesis, and cholesterol-regulating enzyme were also lowered by Fx-SEE supplement. Concentrations of plasma high-density lipoprotein-cholesterol, fecal TG and cholesterol, as well as FA oxidation enzyme activity and UCP1 mRNA expression in epididymal WAT were significantly higher in the Fx-SEE groups than in the high-fat control group. CLA treatment reduced the body weight gain and plasma TG concentration. Overall, these results indicate that Fx-SEE affects the plasma and hepatic lipid profile, fecal lipids and body fat mass, and alters hepatic cholesterol metabolism, FA synthesis and lipid absorption.

Received 23 June 2010 Revised 12 July 2010 Accepted 14 July 2010

Keywords: Antiobesity · Cholesterol synthesis · Fatty acid synthesis · Fucoxanthin rich seaweed ethanol extract · Lipid absorption

1 Introduction The worldwide prevalence of obesity continues to increase, with devastating implications for the Correspondence: Dr. Myung-Sook Choi, Department of Food Science and Nutrition, Kyungpook National University, 1370 Sankyuk -Dong, Buk-gu, Daegu 702-701, Republic of Korea E-mail: [email protected] Fax.: +82-53-950-6229 Abbreviations: CLA, conjugated linoleic acid; CPT, carnitine palmitoyltransferase; FA, fatty acid; FAS, FA synthase; FFA, free FA; Fx-SEE, Fucoxanthinrich seaweed ethanol extract; G6PD, glucose-6-phosphate dehydrogenase; HDL-C, high-density lipoprotein-cholesterol; HFC, high-fat control; ME, malic enzyme; PAP, phosphatidate phosphohydrolase; TG, triglyceride; UCP, uncoupling protein; WAT, white adipose tissue

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overall health of those afflicted. Obesity is characterized by an enlarged fat mass and elevated lipid concentration in the blood [1]. De novo fat synthesis includes the synthetic processes of fatty acids (FAs) and subsequent triglycerides (TGs) in the liver and the adipose tissue. Lipogenesis is regulated by numerous factors, including nutritional, hormonal, and genetic elements. The amount of fat mass is increased when the number and/or size of adipocytes multiply by proliferation and differentiation. Differentiated adipocytes store FAs in the form of TGs in cytoplasm, with the involvement of various enzymes such as malic enzyme (ME), glu-

* These authors contributed equally.

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cose-6-phosphate dehydrogenase (G6PD) and fatty acid synthase (FAS). This overall lipid synthetic process or the sequence of reactions involved in the formation of lipids, is known as lipogenesis. It occurs in the cytoplasm in contrast to the degradation (oxidation), which occurs in the mitochondria. In contrast to lipogenesis, lipolysis is the metabolic breakdown of TG into free fatty acids (FFAs) within cells. The FFAs are then broken down for energy production via ß-oxidation. In this lipolytic process, several factors are involved, such as carnitine palmitoyltransferase-1 (CPT-1) and ß-oxidation. Lipid homeostasis is maintained by the finetuning of lipogenesis and lipolysis, which is regulated by the cooperative action of various enzymes in the metabolic organs such as adipose tissue, liver and muscle. Undaria pinnatifida, an edible brown alga, is rich in carotenoids in nature, such as fucoxanthin [2]. Fucoxanthin has been revealed to have anticarcinogenic [3] and anti-inflammatory effects [4] as well as apoptotic effects in cancer cells [5] and radical scavenging activity [6]. Furthermore, Maeda et al. [2] reported that fucoxanthin supplementation significantly reduces white adipose tissue (WAT) in rats and mice with an increased expression of uncoupling protein-1 (UCP1) mRNA. C57BL/6J mice represents an obese-prone animal model that can develop severe obesity, hyperglycemia, and hyperinsulinemia when consuming a high-fat and/or high-sucrose diet [7, 8]. The objective of the current study was, therefore, to further explore the biological effects of an ethanol extract of the fucoxanthin-rich seaweed (FX-SEE), particularly in relation to FA and TG synthesis and hepatic cholesterol metabolism.

2 Materials and methods 2.1

Preparation of Fx-SEE

Dried brown algae, U. pinnatifida, were harvested from a clean region of the South Sea in Wando County, Jeonnam, Korea. To obtain the seaweed lipid extracts containing fucoxanthin, an extraction process utilizing ethanol (1:6) was carried out with 50 kg of the commercial dried seaweed in the dark for 6 h. The extract was recovered by centrifugation and subsequently filtered through a filter-pad (pore size 0.4 µm, 300 mm × 300 mm). The supernatants were condensed and subsequently freeze-dried for 48 h. The content of the fucoxanthin in the seaweed ethanol extract was 3.5% and general composition of Fx-SEE is as shown in Table 1.

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Biotechnol. J. 2010, 5, 961–969

Table 1. General composition of seaweed ethanol extract (SEE) and fucoxanthin yield (%)

Component Fat (g/100 g Fx-SEE) Protein (g/100 g Fx-SEE) Carbohydrate (g/100 g Fx-SEE) Sodium (g/100 g Fx-SEE) Energy (kcal/100 g Fx-SEE) Fucoxanthin yield (%)

2.2

Content 70.6 4.4 4.3 6.0 670.2 3.5

Animals and diets

Forty 4-week-old C57BL/6J male mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The animals were individually housed in a room with controlled temperature (20–23°C) and lighting (12-h light/dark cycles). They were fed an unpurified commercial pellet diet for 1 week after arrival, and then the mice were randomly divided into four groups with ten mice per group.The highfat control (HFC) group was fed a high-fat diet (20 g fat/100 g diet) based on AIN-76 semi-purified diet [9], while the other three groups were fed a highfat diet with conjugated linoleic acid (CLA, Konkuk Dairy-Konkuk Ham, Seoul, Korea; 0.2 g/100 g diet), low dose Fx-SEE (1.43 g/100 g diet, equivalent to 0.05% dietary fucoxanthin, 0.05Fx-SEE) or high dose Fx-SEE (5.72 g/100 g diet, equivalent to 0.2% dietary fucoxanthin, 0.2Fx-SEE) for 6 weeks (Table 2). All diets were isoenergetic and isonitrogenous. The CLA was used as the positive control for body fat-lowering functional food.The mice were allowed free access to food and water. Food consumption was measured daily and body weight weekly. At the end of the experimental period, the mice were anesthetized with Ketamin-HCl after fasting for 12 h. Blood samples were taken from the inferior vena cava to determine the plasma biomarkers. The liver and adipose tissues were removed and rinsed with a physiological saline. The feces were collected over the last 3 days of the experimental periods to measure the fecal TG and cholesterol contents. All samples were stored at –70°C until analysis. All data were derived from triplicate measurements for all parameters. The current study protocol was approved by the Ethics Committee at Kyungpook National University for animal studies.

2.3

Fat pad weights

Fat pads from each animal, including the epididymal, perirenal and interscapular WAT, were dissected according to defined anatomical landmarks

Biotechnol. J. 2010, 5, 961–969

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Table 2. Compositions of diets (%)a)

Component

HFC

CLA

0.05Fx-SEE

0.2Fx-SEE

Casein D,L-Methionine Sucrose Cellulose powder Corn oil Lard Choline bitartrate AIN-mineralb) AIN-vitaminc) CLA Fx-SEE

20.0 0.3 50.0 5.0 10.0 10.0 0.2 3.5 1.0 _ _

20.0 0.3 49.8 5.0 10.0 10.0 0.2 3.5 1.0 0.2 _

20.0 0.3 48.57 5.0 10.0 10.0 0.2 3.5 1.0 – 1.43

20.0 0.3 44.28 5.0 10.0 10.0 0.2 3.5 1.0 – 5.72

Total

100

100

100

100

a) CLA, conjugated linoleic acid; Fx-SEE, fucoxanthin-rich seaweed ethanol extract; HFC, high-fat control; CLA, HFC diet with 0.2% CLA; 0.05Fx-SEE, HFC diet with 0.05% Fx-SEE; 0.2Fx-SEE, HFC diet with 0.2% Fx-SEE. b) AIN-76 mineral mixture (Harlan Teklad Co. USA). c) AIN-76 vitamin mixture (Harlan Teklad Co. USA).

and their weights were measured. The fat pads were then immediately frozen in liquid nitrogen and stored at –70°C.

2.4

Blood analyses

The plasma TG, total-cholesterol, high-density lipoprotein-cholesterol (HDL-C) and FFA (non-esterified fatty acid, NEFA) concentrations were measured using commercial assay kits (Asan Co. Seoul, Korea, and NEFA-Wako; Wako Pure Chemical Industries, Osaka, Japan). Activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were also measured using a commercially available kit (Asan Co.).

2.5

Hepatic and fecal lipids measurement

The hepatic and fecal lipids were extracted using the procedure developed by Folch et al. [10]. The dried lipid residues were dissolved in 1 mL ethanol for cholesterol and TG assays. Triton X-100 and a sodium cholate solution (in distilled H2O) were added to 200 μL of the dissolved lipid solution to produce a final concentration of 5 g/L and 3 mmol/L, respectively. The cholesterol and TG contents in the liver and feces were analyzed with the same enzymatic kit that was used in the plasma analysis.

2.6

Enzyme analyses

The liver and WAT were prepared according to Hulcher [11] with slight modifications to measure the FAS, ME, G6PD, phosphatidate phosphohydro-

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lase (PAP), CPT and FA ß-oxidation activities. The FAS activity was measured according to Nepokroeff’s method [12] by monitoring the malonylCoA-dependent oxidation of NADPH at 340 nm, where the activity was represented by nmol oxidized NADPH/min/mg protein. ME activity was measured according to Ochoa’s method [13] by monitoring the production of NADPH at 340 nm, where the activity was represented by the formation of nmol NADPH/min/mg protein. G6PD activity was assayed by spectrophotometric methods according to the procedures described by Pitkanen [14], where the activity was expressed as the nmol reduced NADPH/min/mg protein. PAP activity was determined using Walton’s method [15]. CPT activity was analyzed using Markwell’s method [16]. The results were expressed as nmol/ min/mg protein. The FA ß-oxidation was determined using Lazarow’s method [17] by monitoring the reduction of NAD to NADH at 340 nm, where the activity was expressed as the reduced nmol NAD/min/mg protein. The 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was determined as described by Shapiro et al. [18] and this activity was defined as mevalonate synthesized⋅in pmol/min/ mg protein. The acyl-CoA:cholesterol acyltransferase (ACAT) activity was determined according to the method of Gillies et al. [19] and ACAT activity was defined as cholesteryl oleate synthesized·in pmol/min/mg protein. The protein concentrations were measured according Bradford’s method [20] using BSA as the standard.

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2.7 RNA extraction and messenger RNA expression analysis Total RNA was extracted from epididymal WAT using a TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA samples were quantified spectrophotometrically. Complementary DNA was synthesized using a Moloney murine leukemia virus reverse transcriptase (Fermentas, Burlington, ON, Canada), random hexamers, deoxyribonucleoside triphosphates, and 5 µg total RNA. After first-strand complementary DNA synthesis, the RNA expression was quantified by a real-time quantitative PCR using a SYBR green polymerase as the chain reaction reagents (Applied Biosystems, Foster City, CA, USA) and the SDS7000 sequence-detection system (Applied Biosystems). Gene-specific mouse primers were used for uncoupling protein-1 (UCP-1), 5’-tcaggattggcctctacgac-3’ (forward) and 5’-tgcattctgaccttcacgac-3’ (reverse) and glyceraldehyde-3phosphate dehydrogenase (GAPDH), 5’-accacagtccatgccatcac-3’ (forward) and 5’-tccaccaccctgttgctgta-3’ (reverse). The relative quantitation values were calculated by analyzing the changes in the SYBR green fluorescence during the PCR, according to the manufacturer’s instructions. The Ct values obtained were the threshold cycles at which a statistically significant increase in SYBR green emission intensity occurred. Using the 2-ΔΔCt method, the fold changes were calculated; transcripts of the GAPDH were also amplified from the samples to assure normalized real-time quantitative RT-PCR detection.

2.8

Statistical analysis

All data are presented as the mean ± SEM. Significant differences among the groups were deter-

Biotechnol. J. 2010, 5, 961–969

mined by a one-way ANOVA using SPSS. Duncan’s multiple-range test was performed if differences were identified among groups at p < 0.05.

3

Results

3.1 Effects of Fx-SEE supplementation on body weight, liver and adipose tissue weights and food intake Body weight gain of the 0.2Fx-SEE group was significantly suppressed from the week 5 after starting the experimental diet compared to the HFC and CLA groups. At week 6, the body weights of both Fx-SEE groups were significantly lower than the HFC group (Fig. 1). Within adipose tissues from different sites, weights of each WAT, including the epididymal, perirenal, interscapular and total dissected WAT, were significantly lower in the CLA group and both of the Fx-SEE groups (Fig. 2). The 0.2Fx-SEE supplementation was more effective at reducing body weight and WAT weights than 0.05Fx-SEE. Food intake, food efficiency ratio (FER) and liver weight were not significantly different among the groups (Table 3).

3.2 Effects of Fx-SEE supplementation on parameters related to obesity in plasma, liver and feces Plasma TG concentration was significantly lower in the 0.2Fx-SEE group than in the HFC group, while the plasma FFA concentration was not significantly different among the groups (Table 4). The plasma total cholesterol concentration was significantly lower in the CLA and the two Fx-SEE groups than in HFC group, and the plasma HDL-C

Figure 1. Weekly changes of body weight in C57BL/6J mice supplemented fucoxanthin rich seaweed ethanol extract. Mean ± SEM (n = 10). abc Means not sharing a common letter are significantly different among groups at p < 0.05 as determined by a one-way ANOVA test. HFC: high-fat control, CLA: HFC diet with 0.2% CLA, 0.05Fx-SEE: HFC diet with 0.05% fucoxanthin rich seaweed ethanol extract, 0.2Fx-SEE: HFC diet with 0.2% fucoxanthin rich seaweed ethanol extract

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Biotechnol. J. 2010, 5, 961–969

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Figure 2. Effects of Fx-SEE on adipose tissue weights in high-fatfed C57BL/6J mice. Mean ± SEM (n = 10). abc Means not sharing a common letter are significantly different among groups at p