Bioactive quinone derivatives from marine brown algae Sargassum thunbergii induce anti-adipogenic and pro-osteoblastogenic activities

Jung-Ae Kim

1, 2

, Fatih Karadeniz 2, Byul-Nim Ahn 3, Myeong Sook Kwon 1, Ok-Ju

Mun 1, Min Joo Bae 1, Youngwan Seo

4, 5

, , Mihyang Kim 1, Sang-Hyeon Lee 6, Yuck

Yong Kim 7 and Chang-Suk Kong 1,*

1

Department of Food and Nutrition, College of Medical and Life Science, Silla University, Busan 617-736, Republic of Korea

2

Marine Biotechnology Center for Pharmaceuticals and Foods, Silla University, Busan 617-736, Republic of Korea

3

Department of Organic Material Science and Engineering, Pusan National University, Busan, Republic of Korea

4

Marine Environment and Bioscience, Korea Maritime University, Busan 606-791, Republic of Korea

5

Ocean Science & Technology School, Korea Marine University, Busan 606-791, Republic of Korea

6

Major in Bioscience and Biotechnology, Graduate School, Silla University, Busan 617736, Republic of Korea

7

IS Food Co., Marine Bio-industry Department Center, Busan 619-912, Republic of Korea

*Author to whom correspondence should be addressed; Chang-Suk Kong; E-Mail: [email protected]; Tel.: +82-51-999-5429; Fax: +82-51-999-5457 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7148

This article is protected by copyright. All rights reserved

ABSTRACT Lack of bone formation-related health problems is a major problem for aging population in modern world. As a part of ongoing trend to develop natural substances that attenuate osteoporotic bone loss conditions, the effect of edible brown algae, Sargassum thunbergii and its active contents on adipogenic differentiation in 3T3-L1 fibroblasts and osteoblast differentiation in MC3T3-E1 pre-osteoblasts were evaluated. Sargassum thunbergii treatment significantly reduced the lipid accumulation and the expression of adipogenic differentiation markers such as peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α and sterol regulatory element binding protein 1c. In addition, S. thunbergii successfully enhanced the osteoblast differentiation as indicated by increased alkaline phosphatase activity along raised levels of osteoblastogenesis indicators, namely bone morphogenetic protein-2, osteocalcin and collagen type I. Two compounds, sargaquinoic and sargahydroquinoic acid, were isolated from active extract and shown to be active by means of osteogenesis inducement. In conclusion, S. thunbergii could be a source for functional food ingredients for improved osteoporosis and obesity.

Keywords: osteoporosis; adipogenesis; osteoblastogenesis; Sargassum thunbergii.

This article is protected by copyright. All rights reserved

INTRODUCTION In developed world, diseases of bone related issues are among notable causes of low life quality, morbidity and mortality.

1

For the time being, prolonged life spans exert problems

related to aging, especially in terms of bone health. Osteoporosis which is associated with imbalance in bone mass is one of the most common bone related disorders. Almost half of the patients above 50 suffering from bone fractures are diagnosed with.

2

Osteoporosis is

subjected to treatment with several drug and functional foods, namely estrogen therapy, bisphosphonates, vitamin D analogues and receptor activator of nuclear factor kappa-B ligand (RANKL) inhibition.

3-5

Main idea behind the osteoporosis treatment is to increase bone

tissue by osteoblast differentiation of marrow mesenchymal cells. Elevated differentiation into osteocytes helps to relieve deteriorated bone mass balance. 6 However, close relation of osteoblastogenesis enhancement with obesity and diabetes are major concern for well-being of aged patients.

7,8

Hence, natural sources and bioactive substances are in the center of

attention for novel treatment of osteoporosis with expected fewer side effects. Several researches

already

promoted

marine-based

natural

compounds

that

enhance

osteoblastogenesis in vitro. 9,10 It is also known that, on the course of bone mass loss, decrease in osteoblast differentiation is accompanied by increased adipogenesis. 11 On the other hand, several drugs for diabetic patients, targets bone marrow and cause decrease in osteocytes by inhibiting the osteoblastogenesis, again accompanied by increased amount of adipocytes.

12

Both

osteoblasts and adipocytes in bone tissue are formed by common mesenchymal stem cells relying on related pathways of transcription factors, hormone receptors and their ligands. One of the shared pathways of adipogenic and osteoblast differentiation is peroxisome proliferator activated receptor (PPAR)-γ which inhibits osteoblast differentiation while promoting

This article is protected by copyright. All rights reserved

adipogenesis in the presence of adipogenic differentiation ligands, especially in the cases of age-related obesity and diabetic conditions.

13

Hence, regulation of adipose and osteoblast

tissue balance is a main target for controlling and treatment of osteoporosis. Moreover, relation between adipogenic and osteoblast differentiation remains significant field for researches that target to promote new ways to treat age-related bone and obesity disorders. Taken together, inhibiting adipogenic differentiation in both bone and adipose tissues while elevating osteoblast differentiation is considered to be target for treating patients with age related obesity and osteoporosis. Up to now, treatment costs and side effects of drugs are a major problem for patients’ quality of life. Expectedly, researches towards discovering and developing a natural substance that acts as adipogenesis inhibitor and osteoblastogenesis promoter at the same time, are of main interest. Recent trend towards marine organisms produced various highly bioactive natural compounds. In this context, seaweeds are considered to be major sources of a numerous substances such as phenol derivatives, fatty acids, chromenes and polysaccharides which are all reported to possess a number of health beneficial effects. 14-16 Several brown algae species are researched to be found to contain bioactive tannins, quinones and polysaccharides.

17,18

Studies evidenced some of these substances act as adipogenesis inhibitor as well. Sargassum thunbergii is a brown algae species and widely distributed and consumed as a delicacy in coasts between Korea and Japan. Researches exhibited S. thunbergii to contain natural compounds that show in vitro and in vivo bioactivities against oxidative stress, inflammation and allergy. 20,21 Extracts of S. thunbergii also have been shown to possess antitumor activity, due its high content of fucoidan. In this study, extract of S. thunbergii is extensively tested for its anti-adipogenesis effect. In addition, ability of the extract on osteoblast differentiation also studied in order to evaluate the potential of S. thunbergii extract as a source for substances against obesity and osteoporosis. Moreover, two quinones were isolated as active ingredients

This article is protected by copyright. All rights reserved

which are supposed to be responsible for S. thunbergii extract’s bioactivity of promoting osteoblastogenesis.

MATERIALS AND METHODS

Materials. The S. thunbergii was air-dried, ground to a powder and extracted with MeOH for 3 times. The crude extracts were concentrated under reduced pressure via rotary evaporator (RV 10 Series, IKA, NC, USA). Cell culture mediums and materials were purchased from GibcoBRL (Gaithersburg, MD, USA).

Cell culture and adipocyte/osteoblast differentiation. Murine 3T3-L1 pre-adipocytes (ATCC CL-173) were seeded in 6-well plates at a density of 2 x 105 cells/well and grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. At 1 day postconfluence (designated “day 0”), cell differentiation was induced with a mixture of 3isobutyl-1-methylxanthine (0.5 mM), dexamethasone (0.25 M) and insulin (5 µg/ml) in DMEM containing 10% FBS. After 48 h (day 2), the induction medium was removed and replaced with DMEM containing 10% FBS supplemented with insulin (5 µg/ml). This medium was changed every 2 days. S. thunbergii extract was administered to the culture medium from day 0 to day 8. Murine osteoblast-like MC3T3-E1 cells (ATCC CRL-2594) were seeded in 6-well plates at a density of 1 x 105 cells/well and grown to confluence in α-Modified minimal essential medium (αMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 100 units/L penicillin and 100 mg/L streptomycin at 37°C in a humidified

This article is protected by copyright. All rights reserved

atmosphere of 5% CO2. The cells were cultured for osteoblast differentiation in the presence of ascorbic acid and β-glycerophosphate for 5 days. At confluence, the cell differentiation was initiated with culture medium containing 50 μg/ml ascorbic acid and 10 mM β glycerophosphate for 3 days. Then, the induction medium was removed and the cell monolayer was washed twice with phosphate buffered saline (PBS). S. thunbergii extract was administered to the culture medium and further incubated for 48 h.

Determination of lipid accumulation. For Oil-Red O staining

22

, cells were fixed with 10% fresh formaldehyde in PBS for 1 h at

room temperature and stained with filtered Oil-red O solution (60% isopropanol and 40% water) for at least 1 h. After staining, the Oil-red O staining solution was removed and the plates were washed with the distilled water or PBS and dried. Images of lipid droplets in 3T3L1 adipocytes were collected by an Olympus microscope (Tokyo, Japan). Finally, the dye retained in the cells was eluted with isopropyl alcohol and quantified by measuring optical absorbance at 500 nm using a microplate reader (Bio-Tec Instruments, USA).

Cellular ALP activity. Cellular ALP activity was measured in cells treated with S. thunbergii extract and in control cells incubated for 7 days. The cell monolayer was gently washed twice with PBS and lysed using 0.1% Triton X-100 and 25 mM Carbonate buffer. The lysates were centrifuged at 4°C 12,000 ×g for 15 min. The supernatants were used to measure the ALP activity using enzyme assay buffer (15 mM ρ-nitrophenyl phosphate, 1.5 mM MgCl2 and 200 mM carbonate buffer). The absorbance of reactive solution was measured at 405 nm.

RNA extraction and reverse transcription-polymerase chain reaction analysis.

This article is protected by copyright. All rights reserved

Total RNA was isolated from 3T3-L1 adipocytes and MC3T3-E1 osteoblasts treated with/without S. thunbergii extracts using Trizol reagent (Invitrogen Co., CA, USA). For synthesis of cDNA, RNA (2 μg) was added to RNase-free water and oligo (dT), denaturated at 70°C for 5 min and cooled immediately. RNA was reverse transcribed in a master mix containing 1X RT buffer, 1mM dNTPs, 500 ng oligo (dT), 140 U M-MLV reserve transcriptase and 40 U RNase inhibitor at 42°C for 60 min and at 72°C for 5 min using an automatic T100 Thermo Cycler (Bio-Rad, UK). The target cDNA was amplified using the following sense and antisense primers: forward 5'-TTT-TCA-AGG-GTG-CCA-GTT-TC-3' and reverse 5'-AAT-CCT-TGG-CCC-TCT-GAG-AT-3' for PPARγ; forward 5'-TGT-TGGCAT-CCT-GCT-ATC-TG-3' and reverse 5'-AGG-GAA-AGC-TTT-GGG-GTC-TA-3' for SREBP1c; forward 5'-TTA-CAA-CAG-GCC-AGG-TTT-CC-3' and reverse 5'-GGC-TGGCGA-CAT-ACA-GTA-CA-3' for C/EBPα; forward 5'-GGA-TCA-GGT-TTT-GTG-GTGCT-3' and reverse 5'-TTG-TGG-CCC-ATA-AAG-TCC-TC-3' for Leptin; forward 5'-CCAGCA-GGT-TTC-TCT-CTT-GG-3' and reverse 5'-CTG-GGA-GTC-TCA-TCC-TGA-GC-3' for ALP; forward 5'-GGA-CCC-GCT-GTC-TTC-TAG-TG-3' and reverse 5'-GCC-TGCGGT-ACA-GAT-CTA-GC-3' for BMP-2; forward 5'-GCT-GTG-TTG-GAA-ACG-GAG-TT3' and reverse 5'-CAT-GTG-GGT-TCT-GAC-TGG-TG-3' for Osteocalcin; forward 5'-GAGCGG-AGA-GTA-CTG-GAT-CG-3' and reverse 5'-TAC-TCG-AAC-GGG-AAT-CCA-TC-3' for Collagen I; forward 5'-CCA-CAG-CTG-AGA-GGG-AAA-TC-3' and reverse 5'-AAGGAA-GGC-TGG-AAA-AGA-GC-3' for β-actin. The amplification cycles were carried out at 95°C for 45 sec, 60°C for 1 min and 72°C for 45 sec. After 30 cycles, the PCR products were separated by electrophoresis on 1.5% agarose gel for 30 min at 100 V. Gels were then stained with 1 mg/ml ethidium bromide visualized by UV light using Davinch-Chemi imagerTM (CAS-400SM, Seoul, Korea).

This article is protected by copyright. All rights reserved

Western blot analysis. Western blotting was performed according to standard procedures. Briefly, cells were lysed in RIPA lysis buffer (Sigma-Aldrich Corp., St. Louis, USA) at 4°C for 30 min. Cell lysates (35 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech., England, UK), blocked with 5% skim milk and hybridized with primary antibodies (diluted 1:1000) against PPARγ, SREBP1c, C/EBPα, RXRα and RXRβ, LXRα and LXRβ and ALP. After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescece ECL assay kit (Amersham Pharmacia Biosciences, England, UK) according to the manufacturer's instructions. Western blot bands were visualized using a Davinch-Chemi imagerTM (CAS-400SM, Seoul, Korea).

Structural Identification. The 1H and 13C NMR spectra were recorded on a Varian NMR 300 spectrometer (300 MHz for 1H and 75.5 MHz for 13C). Chemical shifts (δ in ppm) were referenced to the residual solvent peak. The used solvent was CD3OD (Cambridge Isotope Laboratories, Inc., USA, deuterium degree 99.95%). The heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectra were recorded using pulsed field gradients. The fast atom bombardment mass spectrometry (FAB-MS) spectra were obtained with a Concept-1S mass spectrometer (Kratos Co.).

Statistical Analysis. The data were presented as mean ± SD. Differences between the means of the individual groups were analyzed using the analysis of variance (ANOVA) procedure of Statistical

This article is protected by copyright. All rights reserved

Analysis System, SAS v9.1 (SAS Institute, Cary, NC, USA) with Duncan’s multiple range tests. The significance of differences was defined at the p