PHYTOTHERAPY RESEARCH Phytother. Res. 28: 1701–1709 (2014) Published online 15 June 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5186
Gelidium elegans, an Edible Red Seaweed, and Hesperidin Inhibit Lipid Accumulation and Production of Reactive Oxygen Species and Reactive Nitrogen Species in 3T3-L1 and RAW264.7 Cells Hui-Jeon Jeon,1† Min-Jung Seo,1† Hyeon-Son Choi,1 Ok-Hwan Lee2 and Boo-Yong Lee1* 1
Department of Food Science and Biotechnology, CHA University, Kyonggi 463-836, South Korea Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, South Korea
2
Gelidium elegans is an edible red alga native to the intertidal area of northeastern Asia. We investigated the effect of G. elegans extract and its main flavonoids, rutin and hesperidin, on lipid accumulation and the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in 3T3-L1 and RAW264.7 cells. Our data show that G. elegans extract decreased lipid accumulation and ROS/RNS production in a dose-dependent manner. The extract also inhibited the mRNA expression of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha, while enhancing the protein expression of the antioxidant enzymes superoxide dismutases 1 and 2, glutathione peroxidase, and glutathione reductase compared with controls. In addition, lipopolysaccharide-induced nitric oxide production was significantly reduced in G. elegans extract-treated RAW264.7 cells. In analysis of the effects of G. elegans flavonoids on lipid accumulation and ROS/RNS production, only hesperidin showed an inhibitory effect on lipid accumulation and ROS production; rutin did not affect adipogenesis and ROS status. The antiadipogenic effect of hesperidin was evidenced by the downregulation of peroxisome proliferator-activated receptor gamma, CCAAT/ enhancer-binding protein alpha, and fatty acid binding protein 4 gene expression. Collectively, our data suggest that G. elegans is a potential food source containing antiobesity and antioxidant constituents. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Gelidium elegans; hesperin; rutin; lipid accumulation; 3T3-L1; ROS production.
INTRODUCTION Seaweed has been used as a source of commercial products such as agar and carrageenans in the food industry and in biotechnology for a long time (Stein and Borden, 1984). It has been appreciated as a good source of various components that are beneficial to health (Stein and Borden, 1984; Pangestuti and Kim, 2011). Seaweed contains various kinds of active compounds that show biological activities in humans, animals, and plants (Chojnacka et al., 2012). They include polyphenols, polysaccharides, proteins, pigments, polyunsaturated fatty acids, minerals, and plant growth hormones (Chojnacka et al., 2012). One of the most studied seaweed compounds is fucoidan. Fucoidan is a sulphated polysaccharide, usually obtained from brown algae such as Fucus evanescens that shows various biological activities including inhibition of adipogenesis (Kuznetsova et al., 2003; Kim et al., 2010). Like terrestrial plants, seaweed also produces polyphenols such as phenolic acids, flavonoids, cinnamic acid, benzoic acid, and quercetin, * Correspondence to: Boo-Yong Lee, Department of Food Science and Biotechnology, CHA University, 222 Yatap, Bundang, Seongnam, Kyonggi 463-836, South Korea. E-mail: [email protected] † These authors contributed equally and are listed in alphabetical order.
Copyright © 2014 John Wiley & Sons, Ltd.
which have antioxidant properties because of their reactive oxygen-scavenging properties (Gupta and Abu-Ghannam, 2011b; Keyrouz et al., 2011). Specifically, Yoshie-Stark et al. (2003) reported their analysis of seven kinds of flavonoids, including hesperidin and rutin, from a variety of seaweeds. Flavonoids are a group of polyphenols found in vegetables, fruits, tea, wine, nuts, and cereals (Nijveldt et al., 2001). They are known to have various health-promoting properties (Hung et al., 2004; Middleton, 1998; Romano et al., 2013). A study by Knekt et al. (1996) suggested that intake of flavonoids reduces the mortality rate of coronary disease. The most well-known property of flavonoids is their antioxidant effect, which stems from their reactive oxygen species (ROS)-scavenging action (Nijveldt et al., 2001). Many studies have shown that flavonoids also have other pharmacological effects, such as antiinflammatory, antiallergic, anticarcinogenic, antiviral, and antiatherosclerotic effects (Kaul et al., 1985; Hertzog et al., 1995; Middleton, 1998; Stefani et al., 1999). Also, hesperidin exerts antiinflammatory (Moon and Kim, 2012) action and has effects in the central nervous (Wasowski et al., 2012) and in the cardiovascular systems (Ikemura et al., 2012). Recent studies have shown the effects of flavonoids on adipocyte differentiation (Hsu and Yen, 2007; Kim et al., 2012). Kim et al. (2012) showed that aurantium, a citrus flavonoid, inhibited adipogenesis via Akt signaling. Received 19 November 2013 Revised 14 May 2014 Accepted 15 May 2014
1702
H.-J. JEON ET AL.
Obesity is a serious metabolic syndrome, which causes other chronic diseases such type 2 diabetes, cancer, and cardiovascular diseases (Moller and Flier, 1991). It is caused by excessive accumulation of white adipose tissue. Adipose tissue plays an important role as an energy source and is an endocrine organ involved in metabolism (Scherer, 2006). Transcription factors such as CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) govern adipogenesis and lipogenic expression by regulating various genes that stimulate lipid uptake and glucose metabolism in adipocytes (Pearson et al., 1996). Thus, they have been standard markers to analyze adipogenic regulation (Pearson et al., 1996). Reactive oxygen species levels in the cell affect signaling molecules, thereby modulating physiological processes including cell growth and proliferation (Adachi and Toishi, 2009). The ROS are also produced by adipocytes during adipogenesis. Recent studies have shown that ROS generation is associated with the increased prevalence of obesity and diabetes (Furukawa and Fujita, 2004). Moreover, ROS tend to work with reactive nitrogen species (RNS) to affect cell functions (Bashan et al., 2009). Nitric oxide (NO) is an RNS produced by macrophages that plays an important role in inflammation (Bashan et al., 2009). Overexpression of ROS/RNS level is regulated by antioxidant enzymes such as copper–zinc superoxide dismutase (Cu/Zn-SOD, SOD1), manganese SOD (Mn-SOD, SOD2), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR; McCord and Fridovich, 1988). Thus, controlling ROS and RNS levels is important to protect against obesity and inflammation. Gelidium elegans is a red alga distributed across Korea, Japan, and Russia (Takamatsu et al., 2003). It usually grows in clean areas in which the tidal current flows smoothly (Takamatsu et al., 2003) and is one of the most prevalent sources of agar (Thomas, 2002). Although it has been consumed as an edible seaweed for a long time, the study of its biological functions has been limited. We previously showed that G. elegans extract has antioxidant and antiinflammatory properties in in vitro system (Jeon et al., 2012). In this study, we examined the effect of G. elegans on adipogenesis and ROS/RNS scavenging in 3T3-L1 and RAW264.7 cells. Our results show that G. elegans extract inhibits lipid accumulation by suppressing adipogenic transcription factors and reduces ROS production by controlling antioxidant enzymes during adipogenesis in 3T3-L1 cells; it also decreases NO production induced by lipopolysaccharide (LPS) in RAW264.7 cells. We also examined rutin and hesperidin, the compounds known to be the main flavonoids in G. elegans.
MATERIALS AND METHODS Materials. Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum, fetal bovine serum (FBS), penicillin–streptomycin (P/S), phosphate-buffered saline (PBS), and trypsin–ethylenediaminetetraacetic acid (trypsin-EDTA) were purchased from Gibco (Gaithersburg, MD, USA). Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, Oil Red O, nitroblue tetrazolium (NBT), LPS, Copyright © 2014 John Wiley & Sons, Ltd.
Griess reagent for nitrite, and N-acetyl cysteine were obtained from Sigma (St Louis, MO, USA). Rutin (purity, ≥98%) was purchased from Pure Chemistry Scientific Inc. (Sugerland, TX, USA), and hesperidin (Purity, ≥95%) was obtained from LKT Laboratories Inc. (St Paul, MN, USA). Primary antibodies specific for GAPDH, Cu/ZnSOD, GPx, and GR were acquired from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased from Sigma.
Preparation of the G. elegans extract. Specimens were collected from Jeju Island in Korea. Identification of seaweed species was performed by Dr K. P. Song at Jeju Biodiversity Research Institute (Jeju, Korea). Collected G. elegans samples were gently washed with distilled water and dried by hot-air drying in an HB-501VL vacuum drying oven (Hanbaek, Kyonggi, Korea) for 4 h at 60 °C. Dried G. elegans (100 g) was extracted with ten volumes of 70% ethanol (1 L) at room temperature for 24 h. The extracts were filtered through filter paper (no. 2; Whatman Ltd., Maidstone, Kent, UK), concentrated with a vacuum evaporator (Rotavapor R-200; BUCHI Korea Inc., Seoul, Korea) and completely dried with a DC1316 freeze drier (IlSHIN Lab Co., Ltd., Seoul, Korea). G. elegans extract was expressed as percentage (w/w) of samples on a dry weight basis (weight of freeze-dried sample/hot air-dried original seaweed). Crude powder was weighed and used as samples for experiments.
Cell culture. 3T3-L1 preadipocyes obtained from the American Type Culture Collection (CL-173; ATCC, Manassas, VA, USA) were cultured, maintained, and differentiated (Lee et al., 2011). Briefly, cells were plated and grown in DMEM containing 3.7 g/L sodium bicarbonate, 1% P/S, and 10% bovine calf serum and incubated at 37 °C in a humidified 5% CO2/90% air atmosphere. Adipocyte differentiation was induced by treatment of 2-day postconfluent cells with 10% FBS and a hormonal mixture (MDI) consisting of 0.5 mM 3-isobutyl-1-methylxanthine, 1.0 μM dexamethasone, and 1.67 μM insulin. Two days after the initiation of differentiation, the culture medium was replaced with DMEM supplemented with only 1.67 μM insulin and 10% FBS. For treatments, 2-day postconfluent cells were differentiated with MDI in the presence of 10 mM N-acetyl cysteine and G. elegans 70% ethanol extract (1, 10, 20, 40, 80, and 100 μg/mL; Jejuhidi, Jeju, Korea). This medium was then replenished with the samples every other day. For RAW264.7 cell culture, RAW264.7 cells (TIB-71; ATCC, Manassas, VA, USA), a murine macrophage cell line, were incubated in DMEM containing 3.7 g/L sodium bicarbonate, 1% P/S, and 10% FBS at 37 °C in a humidified 5% CO2/90% air atmosphere. Cells were plated in a 96-well plate at a concentration of 1 × 105 cells/well and incubated for 24 h. Cells were pretreated with the G. elegans samples (20, 40, 80, and 100 μg/mL) for 1 h and then treated with LPS (1 μg/mL) for an additional 24 h. After incubation, the media were used for NO assay.
Determination of NO production from RAW264.7 cells. The RAW264.7 cells culture medium were mixed with an equal volume of Griess reagent for nitrite (100 μL) Phytother. Res. 28: 1701–1709 (2014)
G. ELEGANS AND HESPERIDIN INHIBIT ADIPOGENESIS AND ROS PRODUCTION
and incubated at room temperature for 10 min. The amount of NO was measured at 540 nm (VICTOR3 multilabel counter, PerkinElmer, Seoul, South Korea). Cytotoxicity. 3T3-L1 preadipocytes (~1 × 104 cells/well) in 96-well plates were treated with the G. elegans extract, rutin and hesperidin for 8 days. RAW264.7 cells (~1 × 105 cells/well) were treated with the G. elegans extract for 24 h. 2,3-Bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium5-carboxanilide (XTT) reagent was added to the culture after incubation. The cytotoxicity of the samples was monitored every 2 h at wavelengths of 450 and 690 nm.
Determination of lipid accumulation and reactive oxygen species production. The extent of differentiation, reflected by lipid accumulation and ROS production at 8 days, was determined by Oil Red O staining and NBT assay, respectively. Briefly, cells for Oil Red O staining were fixed in 10% formaldehyde in PBS for 1 h, washed with distilled water, and completely dried. Cells were stained with 0.5% Oil Red O solution in 60:40 (v/v) isopropanol:H2O for 30 min at room temperature, washed four times with water, and dried. Differentiation was also monitored under a microscope and quantified by Oil Red O stain elution with isopropanol and measurement of the optical density (OD) at 490 nm (Lee et al., 2011). Cells for the NBT assay were incubated for 90 min in PBS containing 0.2% NBT. Formazan was dissolved in 50% acetic acid, and the absorbance was determined at 570 nm (Furukawa and Fujita, 2004)
Determination of total polyphenol and flavonoid contents of G. elegans extracts. The total polyphenol content was determined according to the modified Folin–Ciocalteu method (Lee et al., 2011). G. elegans (100 μg) was placed in a test tube with distilled water (7 mL) and Folin–Ciocalteu reagent (0.5 mL), saturated with sodium carbonate solution (1 mL), and allowed to stand for 30 min. The reaction was monitored by measuring the absorbance at 715 nm. Gallic acid (G7384; Sigma) was used as a standard marker for total phenols. For the total flavonoid contents (Kang et al., 2011) of G. elegans extracts, 5 mL of 50% methanol solution, 1 mL of sample, and 10 mL of diethylene glycol were mixed with 1 mL of 1 N NaOH solution and incubated for 1 h at 37 °C. The reaction color was examined by measuring the absorbance at 420 nm. Naringin (Sigma) was used as a standard marker for total flavonoids.
RNA extraction and semiquantitative reverse transcription-polymerase chain reaction. Total RNA was extracted from 3T3-L1 adipocytes with TRIzol
1703
reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. RNA samples with OD260/OD280 ratios higher than 2.0 were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RNA treated with DNase was used for the production of cDNA using a RT-PCR system. The oligonucleotide primer sequences were listed in Table 1. The PCR products were then run on 1.5% (v/v) agarose gels, stained with ethidium bromide, and photographed. The expression levels were quantified using densitometry by scanning with a gel documentation system (Geneflash transilluminator; Syngene, Frederick, MD, USA) and analysis with IMAGEJ software (NIH, Bethesda, MD, USA).
Western blot analysis. Cells were harvested in lysis buffer (pH 7.4) containing protease inhibitor (100 mM benzamidine, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 0.25 mg/mL pepstatin, and 100 mM sodium orthovanadate) and using cell scraper. Cell lysates were centrifuged by 12 000 rpm at 4 °C to obtain supernatant as protein extract. Protein extracts (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then blocked and immunoblotted with primary antibodies specific for GAPDH, Cu/Zn-SOD, Mn-SOD, GPx, and GR. Secondary antibodies conjugated to horseradish peroxidase (dilution 1:1000) were applied for 1 h. The protein bands were visualized by enhanced chemiluminescence and detected with luminescent image analyzer LAS4000 system (FujiFilm, Tokyo, Japan).
Quantification of triglyceride. Accumulated triglycerides were measured using a total triglyceride assay kit provided from Zen-Bio, Inc. Cells were washed with PBS to remove residual medium, and the cells were lysed with lysis buffer. Triglycerides were digested with Reagent B (Zen-Bio, Inc.) containing lipase for 2 h to release hydrolyzed glycerols into the buffer. Diluted hydrolysates were incubated with reagent A (Zen-Bio, Inc.) containing peroxidase to produce quinoneimine dye, which shows an absorbance maximum in spectrophotometric detection at 540 nm. The absorbance at 540 nm of the samples was linear over a standard glycerol concentration range (0–200 μM).
Statistical analysis. All experiments were repeated three times. The results were analyzed statistically by analysis of variance and Duncan’s multiple range test. A p-value of
Gelidium elegans, an Edible Red Seaweed, and Hesperidin Inhibit Lipid Accumulation and Production of Reactive Oxygen Species and Reactive Nitrogen Species in 3T3-L1 and RAW264.7 Cells Hui-Jeon Jeon,1† Min-Jung Seo,1† Hyeon-Son Choi,1 Ok-Hwan Lee2 and Boo-Yong Lee1* 1
Department of Food Science and Biotechnology, CHA University, Kyonggi 463-836, South Korea Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, South Korea
2
Gelidium elegans is an edible red alga native to the intertidal area of northeastern Asia. We investigated the effect of G. elegans extract and its main flavonoids, rutin and hesperidin, on lipid accumulation and the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in 3T3-L1 and RAW264.7 cells. Our data show that G. elegans extract decreased lipid accumulation and ROS/RNS production in a dose-dependent manner. The extract also inhibited the mRNA expression of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha, while enhancing the protein expression of the antioxidant enzymes superoxide dismutases 1 and 2, glutathione peroxidase, and glutathione reductase compared with controls. In addition, lipopolysaccharide-induced nitric oxide production was significantly reduced in G. elegans extract-treated RAW264.7 cells. In analysis of the effects of G. elegans flavonoids on lipid accumulation and ROS/RNS production, only hesperidin showed an inhibitory effect on lipid accumulation and ROS production; rutin did not affect adipogenesis and ROS status. The antiadipogenic effect of hesperidin was evidenced by the downregulation of peroxisome proliferator-activated receptor gamma, CCAAT/ enhancer-binding protein alpha, and fatty acid binding protein 4 gene expression. Collectively, our data suggest that G. elegans is a potential food source containing antiobesity and antioxidant constituents. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Gelidium elegans; hesperin; rutin; lipid accumulation; 3T3-L1; ROS production.
INTRODUCTION Seaweed has been used as a source of commercial products such as agar and carrageenans in the food industry and in biotechnology for a long time (Stein and Borden, 1984). It has been appreciated as a good source of various components that are beneficial to health (Stein and Borden, 1984; Pangestuti and Kim, 2011). Seaweed contains various kinds of active compounds that show biological activities in humans, animals, and plants (Chojnacka et al., 2012). They include polyphenols, polysaccharides, proteins, pigments, polyunsaturated fatty acids, minerals, and plant growth hormones (Chojnacka et al., 2012). One of the most studied seaweed compounds is fucoidan. Fucoidan is a sulphated polysaccharide, usually obtained from brown algae such as Fucus evanescens that shows various biological activities including inhibition of adipogenesis (Kuznetsova et al., 2003; Kim et al., 2010). Like terrestrial plants, seaweed also produces polyphenols such as phenolic acids, flavonoids, cinnamic acid, benzoic acid, and quercetin, * Correspondence to: Boo-Yong Lee, Department of Food Science and Biotechnology, CHA University, 222 Yatap, Bundang, Seongnam, Kyonggi 463-836, South Korea. E-mail: [email protected] † These authors contributed equally and are listed in alphabetical order.
Copyright © 2014 John Wiley & Sons, Ltd.
which have antioxidant properties because of their reactive oxygen-scavenging properties (Gupta and Abu-Ghannam, 2011b; Keyrouz et al., 2011). Specifically, Yoshie-Stark et al. (2003) reported their analysis of seven kinds of flavonoids, including hesperidin and rutin, from a variety of seaweeds. Flavonoids are a group of polyphenols found in vegetables, fruits, tea, wine, nuts, and cereals (Nijveldt et al., 2001). They are known to have various health-promoting properties (Hung et al., 2004; Middleton, 1998; Romano et al., 2013). A study by Knekt et al. (1996) suggested that intake of flavonoids reduces the mortality rate of coronary disease. The most well-known property of flavonoids is their antioxidant effect, which stems from their reactive oxygen species (ROS)-scavenging action (Nijveldt et al., 2001). Many studies have shown that flavonoids also have other pharmacological effects, such as antiinflammatory, antiallergic, anticarcinogenic, antiviral, and antiatherosclerotic effects (Kaul et al., 1985; Hertzog et al., 1995; Middleton, 1998; Stefani et al., 1999). Also, hesperidin exerts antiinflammatory (Moon and Kim, 2012) action and has effects in the central nervous (Wasowski et al., 2012) and in the cardiovascular systems (Ikemura et al., 2012). Recent studies have shown the effects of flavonoids on adipocyte differentiation (Hsu and Yen, 2007; Kim et al., 2012). Kim et al. (2012) showed that aurantium, a citrus flavonoid, inhibited adipogenesis via Akt signaling. Received 19 November 2013 Revised 14 May 2014 Accepted 15 May 2014
1702
H.-J. JEON ET AL.
Obesity is a serious metabolic syndrome, which causes other chronic diseases such type 2 diabetes, cancer, and cardiovascular diseases (Moller and Flier, 1991). It is caused by excessive accumulation of white adipose tissue. Adipose tissue plays an important role as an energy source and is an endocrine organ involved in metabolism (Scherer, 2006). Transcription factors such as CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) govern adipogenesis and lipogenic expression by regulating various genes that stimulate lipid uptake and glucose metabolism in adipocytes (Pearson et al., 1996). Thus, they have been standard markers to analyze adipogenic regulation (Pearson et al., 1996). Reactive oxygen species levels in the cell affect signaling molecules, thereby modulating physiological processes including cell growth and proliferation (Adachi and Toishi, 2009). The ROS are also produced by adipocytes during adipogenesis. Recent studies have shown that ROS generation is associated with the increased prevalence of obesity and diabetes (Furukawa and Fujita, 2004). Moreover, ROS tend to work with reactive nitrogen species (RNS) to affect cell functions (Bashan et al., 2009). Nitric oxide (NO) is an RNS produced by macrophages that plays an important role in inflammation (Bashan et al., 2009). Overexpression of ROS/RNS level is regulated by antioxidant enzymes such as copper–zinc superoxide dismutase (Cu/Zn-SOD, SOD1), manganese SOD (Mn-SOD, SOD2), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR; McCord and Fridovich, 1988). Thus, controlling ROS and RNS levels is important to protect against obesity and inflammation. Gelidium elegans is a red alga distributed across Korea, Japan, and Russia (Takamatsu et al., 2003). It usually grows in clean areas in which the tidal current flows smoothly (Takamatsu et al., 2003) and is one of the most prevalent sources of agar (Thomas, 2002). Although it has been consumed as an edible seaweed for a long time, the study of its biological functions has been limited. We previously showed that G. elegans extract has antioxidant and antiinflammatory properties in in vitro system (Jeon et al., 2012). In this study, we examined the effect of G. elegans on adipogenesis and ROS/RNS scavenging in 3T3-L1 and RAW264.7 cells. Our results show that G. elegans extract inhibits lipid accumulation by suppressing adipogenic transcription factors and reduces ROS production by controlling antioxidant enzymes during adipogenesis in 3T3-L1 cells; it also decreases NO production induced by lipopolysaccharide (LPS) in RAW264.7 cells. We also examined rutin and hesperidin, the compounds known to be the main flavonoids in G. elegans.
MATERIALS AND METHODS Materials. Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum, fetal bovine serum (FBS), penicillin–streptomycin (P/S), phosphate-buffered saline (PBS), and trypsin–ethylenediaminetetraacetic acid (trypsin-EDTA) were purchased from Gibco (Gaithersburg, MD, USA). Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, Oil Red O, nitroblue tetrazolium (NBT), LPS, Copyright © 2014 John Wiley & Sons, Ltd.
Griess reagent for nitrite, and N-acetyl cysteine were obtained from Sigma (St Louis, MO, USA). Rutin (purity, ≥98%) was purchased from Pure Chemistry Scientific Inc. (Sugerland, TX, USA), and hesperidin (Purity, ≥95%) was obtained from LKT Laboratories Inc. (St Paul, MN, USA). Primary antibodies specific for GAPDH, Cu/ZnSOD, GPx, and GR were acquired from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased from Sigma.
Preparation of the G. elegans extract. Specimens were collected from Jeju Island in Korea. Identification of seaweed species was performed by Dr K. P. Song at Jeju Biodiversity Research Institute (Jeju, Korea). Collected G. elegans samples were gently washed with distilled water and dried by hot-air drying in an HB-501VL vacuum drying oven (Hanbaek, Kyonggi, Korea) for 4 h at 60 °C. Dried G. elegans (100 g) was extracted with ten volumes of 70% ethanol (1 L) at room temperature for 24 h. The extracts were filtered through filter paper (no. 2; Whatman Ltd., Maidstone, Kent, UK), concentrated with a vacuum evaporator (Rotavapor R-200; BUCHI Korea Inc., Seoul, Korea) and completely dried with a DC1316 freeze drier (IlSHIN Lab Co., Ltd., Seoul, Korea). G. elegans extract was expressed as percentage (w/w) of samples on a dry weight basis (weight of freeze-dried sample/hot air-dried original seaweed). Crude powder was weighed and used as samples for experiments.
Cell culture. 3T3-L1 preadipocyes obtained from the American Type Culture Collection (CL-173; ATCC, Manassas, VA, USA) were cultured, maintained, and differentiated (Lee et al., 2011). Briefly, cells were plated and grown in DMEM containing 3.7 g/L sodium bicarbonate, 1% P/S, and 10% bovine calf serum and incubated at 37 °C in a humidified 5% CO2/90% air atmosphere. Adipocyte differentiation was induced by treatment of 2-day postconfluent cells with 10% FBS and a hormonal mixture (MDI) consisting of 0.5 mM 3-isobutyl-1-methylxanthine, 1.0 μM dexamethasone, and 1.67 μM insulin. Two days after the initiation of differentiation, the culture medium was replaced with DMEM supplemented with only 1.67 μM insulin and 10% FBS. For treatments, 2-day postconfluent cells were differentiated with MDI in the presence of 10 mM N-acetyl cysteine and G. elegans 70% ethanol extract (1, 10, 20, 40, 80, and 100 μg/mL; Jejuhidi, Jeju, Korea). This medium was then replenished with the samples every other day. For RAW264.7 cell culture, RAW264.7 cells (TIB-71; ATCC, Manassas, VA, USA), a murine macrophage cell line, were incubated in DMEM containing 3.7 g/L sodium bicarbonate, 1% P/S, and 10% FBS at 37 °C in a humidified 5% CO2/90% air atmosphere. Cells were plated in a 96-well plate at a concentration of 1 × 105 cells/well and incubated for 24 h. Cells were pretreated with the G. elegans samples (20, 40, 80, and 100 μg/mL) for 1 h and then treated with LPS (1 μg/mL) for an additional 24 h. After incubation, the media were used for NO assay.
Determination of NO production from RAW264.7 cells. The RAW264.7 cells culture medium were mixed with an equal volume of Griess reagent for nitrite (100 μL) Phytother. Res. 28: 1701–1709 (2014)
G. ELEGANS AND HESPERIDIN INHIBIT ADIPOGENESIS AND ROS PRODUCTION
and incubated at room temperature for 10 min. The amount of NO was measured at 540 nm (VICTOR3 multilabel counter, PerkinElmer, Seoul, South Korea). Cytotoxicity. 3T3-L1 preadipocytes (~1 × 104 cells/well) in 96-well plates were treated with the G. elegans extract, rutin and hesperidin for 8 days. RAW264.7 cells (~1 × 105 cells/well) were treated with the G. elegans extract for 24 h. 2,3-Bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium5-carboxanilide (XTT) reagent was added to the culture after incubation. The cytotoxicity of the samples was monitored every 2 h at wavelengths of 450 and 690 nm.
Determination of lipid accumulation and reactive oxygen species production. The extent of differentiation, reflected by lipid accumulation and ROS production at 8 days, was determined by Oil Red O staining and NBT assay, respectively. Briefly, cells for Oil Red O staining were fixed in 10% formaldehyde in PBS for 1 h, washed with distilled water, and completely dried. Cells were stained with 0.5% Oil Red O solution in 60:40 (v/v) isopropanol:H2O for 30 min at room temperature, washed four times with water, and dried. Differentiation was also monitored under a microscope and quantified by Oil Red O stain elution with isopropanol and measurement of the optical density (OD) at 490 nm (Lee et al., 2011). Cells for the NBT assay were incubated for 90 min in PBS containing 0.2% NBT. Formazan was dissolved in 50% acetic acid, and the absorbance was determined at 570 nm (Furukawa and Fujita, 2004)
Determination of total polyphenol and flavonoid contents of G. elegans extracts. The total polyphenol content was determined according to the modified Folin–Ciocalteu method (Lee et al., 2011). G. elegans (100 μg) was placed in a test tube with distilled water (7 mL) and Folin–Ciocalteu reagent (0.5 mL), saturated with sodium carbonate solution (1 mL), and allowed to stand for 30 min. The reaction was monitored by measuring the absorbance at 715 nm. Gallic acid (G7384; Sigma) was used as a standard marker for total phenols. For the total flavonoid contents (Kang et al., 2011) of G. elegans extracts, 5 mL of 50% methanol solution, 1 mL of sample, and 10 mL of diethylene glycol were mixed with 1 mL of 1 N NaOH solution and incubated for 1 h at 37 °C. The reaction color was examined by measuring the absorbance at 420 nm. Naringin (Sigma) was used as a standard marker for total flavonoids.
RNA extraction and semiquantitative reverse transcription-polymerase chain reaction. Total RNA was extracted from 3T3-L1 adipocytes with TRIzol
1703
reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. RNA samples with OD260/OD280 ratios higher than 2.0 were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RNA treated with DNase was used for the production of cDNA using a RT-PCR system. The oligonucleotide primer sequences were listed in Table 1. The PCR products were then run on 1.5% (v/v) agarose gels, stained with ethidium bromide, and photographed. The expression levels were quantified using densitometry by scanning with a gel documentation system (Geneflash transilluminator; Syngene, Frederick, MD, USA) and analysis with IMAGEJ software (NIH, Bethesda, MD, USA).
Western blot analysis. Cells were harvested in lysis buffer (pH 7.4) containing protease inhibitor (100 mM benzamidine, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 0.25 mg/mL pepstatin, and 100 mM sodium orthovanadate) and using cell scraper. Cell lysates were centrifuged by 12 000 rpm at 4 °C to obtain supernatant as protein extract. Protein extracts (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then blocked and immunoblotted with primary antibodies specific for GAPDH, Cu/Zn-SOD, Mn-SOD, GPx, and GR. Secondary antibodies conjugated to horseradish peroxidase (dilution 1:1000) were applied for 1 h. The protein bands were visualized by enhanced chemiluminescence and detected with luminescent image analyzer LAS4000 system (FujiFilm, Tokyo, Japan).
Quantification of triglyceride. Accumulated triglycerides were measured using a total triglyceride assay kit provided from Zen-Bio, Inc. Cells were washed with PBS to remove residual medium, and the cells were lysed with lysis buffer. Triglycerides were digested with Reagent B (Zen-Bio, Inc.) containing lipase for 2 h to release hydrolyzed glycerols into the buffer. Diluted hydrolysates were incubated with reagent A (Zen-Bio, Inc.) containing peroxidase to produce quinoneimine dye, which shows an absorbance maximum in spectrophotometric detection at 540 nm. The absorbance at 540 nm of the samples was linear over a standard glycerol concentration range (0–200 μM).
Statistical analysis. All experiments were repeated three times. The results were analyzed statistically by analysis of variance and Duncan’s multiple range test. A p-value of
