Isolation of the Molecular Species of Monogalactosyldiacylglycerols

Nov 2, 2014 - Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Center for...
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Isolation of the Molecular Species of Monogalactosyldiacylglycerols from Brown Edible Seaweed Sargassum horneri and Their Inhibitory Effects on Triglyceride Accumulation in 3T3-L1 Adipocytes Ai-Cui Ma,†,‡,§ Zhen Chen,†,‡ Tao Wang,∥ Ni Song,‡ Qian Yan,‡ Yu-Chun Fang,‡ Hua-Shi Guan,‡ and Hong-Bing Liu*,‡ ‡

Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Foods and Drugs, Ocean University of China, Qingdao, Shandong 266003, People’s Republic of China § Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Center for the Toxicology of Fertility and Regulating Drugs, Shanghai 200032, People’s Republic of China ∥ Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin, 300193, People’s Republic of China S Supporting Information *

ABSTRACT: The chemical composition of monogalactosyldiacylglycerols (MGDGs) from brown alga Sargassum horneri and their inhibitory effects on lipid accumulation were investigated in this study. A total of 10 molecular species of MGDGs were identified using nuclear magnetic resonance, alkaline hydrolysis, gas chromatography−flame ionization detector, and highperformance liquid chromatography−tandem mass spectrometry methods. Individual molecular species of MGDGs, including (2S)-1-O-myristoyl-2-O-palmitoleoyl-3-O-β-D-galactopyranosyl-sn-glycerol (1), (2S)-1-O-myristoyl-2-O-linoleyl-3-O-β-D-galactopyranosyl-sn-glycerol (3), (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol (5), (2S)-1-O-myristoyl-2-Ooleyl-3-O-β-D-galactopyranosyl-sn-glycerol (7), (2S)-1-O-palmitoyl-2-O-palmitoleoyl-3-O-β-D-galactopyranosyl-sn-glycerol (8), (2S)-1-O-palmitoyl-2-O-linoleyl-3-O-β-D-galactopyranosyl-sn-glycerol (9), and (2S)-1-O-palmitoyl-2-O-oleyl-3-O-β-D-galactopyranosyl-sn-glycerol (10), were then furnished using semi-preparative high-performance liquid chromatography, and their inhibitory effects on triglyceride (TG) accumulation and free fatty acid (FFA) levels in 3T3-L1 adipocytes were evaluated. Compounds 3 and 9 showed inhibitory effects on TG and FFA accumulation, with TG levels of 1.568 ± 0.2808 and 1.701 ± 0.1460 μmol/L and FFA levels of 0.149 ± 0.0258 and 0.198 ± 0.0229 mequiv/L, respectively, which were more effective than other compounds. The primary structure−activity relationship suggested that linoleyl [18:2(ω-6)] in the sn-2 position played an important role on triglyceride accumulation inhibition. KEYWORDS: Sargassum horneri, monogalactosyldiacylglycerols, triglyceride accumulation inhibition, 3T3-L1 adipocytes

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INTRODUCTION

strong triglyceride (TG) accumulation inhibitory effect on 3T3-L1 adipocytes. MGDGs, digalactosyldiacylglycerols (DGDGs), and sulfoquinovosyldiacylglycerols (SQDGs), are glycoglycerolipids, which are well-known for their various biological activities, such as improving the intestinal environment, and their antitumor activity, anti-inflammatory activity, and protection against cell death.13 However, the action of glycoglycerolipids on lipid metabolism has rarely been reported. Glycoglycerolipids always exist as mixtures because of the chemical diversity of the composition of fatty acids. Therefore, purification, identification, and bioactivity elucidation of glycoglycerolipid molecular species are of interest. In the present study, major molecular species of MGDGs from S. horneri were furnished, and their inhibitory effects on TG and free fatty acid (FFA) accumulation in 3T3-L1 adipocytes were

Sargassum is a genus of approximately 250 species in Sargassaceae and is geographically widespread in all tropical and temperate oceans.1 Sargassum horneri (Turner) C. Agardh is a planktonic species in this genus and is mainly distributed in the coast of the north Pacific Ocean, with an abundant biomass in China. As an important economic seaweed, S. horneri has been used in the phycocolloid industry as well as for nutritional food and herbal medicine for treating hyperlipidemia, hypertension, heart disease, inflammatory diseases, and cancer in humans.2−4 In recent years, most of the phytochemical studies have focused on water extracts from S. horneri, especially on polysaccharides with bioactivities of anticancer,5−7 antivirus,8 anticoagulative,9 antioxidant,10 and anabolic effects on bone components.11,12 It is worth noting that S. horneri has a long medicinal history of treating cardiovascular diseases (CVDs).3,4 However, the anti-CVD functional components in S. horneri have not been systematically investigated. In our studies on bioactive chemical constituents of this seaweed, we have isolated a mixture of monogalactosyldiacylglycerols (MGDGs), which showed a © 2014 American Chemical Society

Received: Revised: Accepted: Published: 11157

June 27, 2014 October 29, 2014 November 2, 2014 November 2, 2014 dx.doi.org/10.1021/jf503068n | J. Agric. Food Chem. 2014, 62, 11157−11162

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H2O, dried over anhydrous Na2SO4, and evaporated to yield a mixture of fatty acid methyl esters (FAMEs). The MeOH layer was neutralized with 2 N HCl/MeOH and evaporated to give a solid residue, which was purified using flash silica gel column chromatography with CH2Cl2/MeOH (16:9) to obtain monogalactosylglycerol. The optical rotation of the monogalactosylglycerol was then measured to resolve the absolute configuration of glycerol. Gas Chromatography−Flame Ionization Detector (GC−FID) Analysis. GC analysis was performed using a HP 5890 series II chromatograph (Hewlett-Packard, Avondale, PA) equipped with a flame ionization detector (FID), and a SGE 30QC3/AC 20-0.5 capillary column (30 m × 0.32 mm, 0.5 μm, SGE Analytical Science, Ringwood, Australia) was used for separation. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL/min. The oven temperature was held at 150 °C for 1 min, increased by 15 °C/min to 200 °C, and then increased by 2 °C/min to 250 °C. The injector temperature was set at 270 °C, and the detector temperature was 270 °C. The injection volume was 0.6 μL. Identification of the Molecular Species in MGDGs. Highperformance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) analysis was performed using a Waters ACQUITY liquid chromatography system (Waters Corp., Milford, MA) coupled with a diode array detector (DAD) and an Amazon SL mass spectrometer (Bruker Daltonics, Bremen, Germany). The HPLC separation used a YMC ODS Pack-A C18 column (250 × 4.6 mm inner diameter, 5 μm, Tokyo, Japan). The mobile phase was a mixture of water (A) and methanol (B) as follows: 0−40 min, 96 → 100% B; and 40−50 min, 100% B. A flow rate of 1.0 mL/min with detection at 203 nm was used. The injection volume was 10 μL, and injections were performed using the autosampler. Electrospray ionization−quadrupole ion trap mass spectrometry (ESI−QITMS) in positive-ion mode was used. The parameters of the ESI interface were as follows: high-voltage (HV) capillary, 4500 V; HV end plate offset, −500 V; MS/MS isolation width, 4.0 units; and collision gas, helium. All skimmer collisionally activated dissociation (CAD) and MS/MS spectra represent averages of five scans. In the MS/MS experiment, the data-dependent MS/MS scan mode was used. The scan mass range was m/z 100−1000. Preparation of Major MGDG Molecular Species. Semipreparative HPLC was performed on a Shimadzu LC-6AD (Tokyo, Japan) coupled to a photodiode array detector (SPD-M20A). The semi-preparative HPLC column (250 × 10 mm inner diameter, 5 μm) was prefilled with C18 (YMC ODS Pack-A, Tokyo, Japan), and a linear gradient of H2O (A) and MeOH (B) was used at a flow rate of 4.0 mL/min. The following elution program was used for separation: 0−40 min, 96 → 100% B; 40−50 min, 100% B; and 50−55 min, 100 → 96% B. The detection wavelength was 203 nm. This preparation yielded compounds 1 (3.1 mg), 3 (1.7 mg), 5 (3.4 mg), 9 (6.4 mg), and 10 (26.2 mg) as well as a mixture of compounds 7 and 8 (12.1 mg). Bioassay. The inhibitory effect on lipid accumulation was assessed in 3T3-L1 adipocytes by measuring the intracellular contents of TG and FFAs. 3T3-L1 preadipocytes were cultured and induced to differentiate into adipocytes as previously described.18−20 Briefly, confluent 3T3-L1 preadipocytes after 1 day were treated with 1 μmol/ L dexamethasone, 0.5 mmol/L 3-isobutyl-methylxanthine, and 10 μg/ mL (270 munits/mL) insulin for 3 days in high-glucose DMEM containing 10% FBS. At the same time, cells of compound-treated groups were treated with 10 μM MGDGs 1, 3, 5, 7 + 8, 9, or 10 dissolved in DMSO (final DMSO concentration of 0.5%, v/v), respectively. While for the control group, cells were cultured without compounds. Cells were then switched to 10% FBS/DMEM containing only 5 μg/mL (135 munits/mL) insulin for 3 more days and then switched to 10% FBS/DMEM without insulin. In a normal group, cells were cultured without induction medium or any compounds. At 14 days after induction of differentiation, 3T3-L1 cells in 48-well plates were lysed and analyzed using the TG kit and the NEFA-C kit according to the protocols provided by the manufacturers. The TG and NEFA values were corrected by their protein content.

evaluated. The primary structure−activity relationship of the MGDGs was also discussed.

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MATERIALS AND METHODS

Chemicals. Spectral-grade solvents for spectroscopic measurements were obtained from Sigma-Aldrich. High-performance liquid chromatography (HPLC)-grade solvents were obtained from Merck, Inc. (Merck KgaA, Darmstadt, Germany). All of the other solvents were of analytical grade (Yuwang Reagent Company, Shandong, China). Cell culture materials, such as Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin, glutamine, and penicillin/streptomycin (PES), were obtained from Gibco. Dexamethasone, 3-isobutyl-methylxanthine, insulin, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. The TG kit was purchased from BioSino Biotechnology and Science, Inc., China, and the NEFA-C kit was purchased from NEFA C-test Wako, Japan. General Experimental Procedures. Optical rotation was measured on a PerkinElmer model 241 polarimeter (Norwalk, CT). 1 H and 13C nuclear magnetic resonance (NMR) spectra were recorded on JEOL JNM-ECP 600 spectrometers using tetramethylsilane (TMS) as the internal standard, and chemical shifts were recorded as δ values. Silica gel (100−200 and 200−300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare BioSciences AB, Uppsala, Sweden) were used for column chromatography. Thin-layer chromatography (TLC) silica gel GF254 plates (Yantai Zi Fu Chemical Co., Ltd., Yantai, China) were used for TLC analysis. Plant Material. Fronds of S. horneri (Turner) C. Agardh were collected from Changdao in Shandong, China, in October 2010. The original seaweed was identified by Prof. Xiaoqi Zeng (College of Fisheries, Ocean University of China), and a voucher specimen was deposited at the School of Medicine and Pharmacy, Ocean University of China. The alga was washed using water after its collection, dried thoroughly, and stored at room temperature. Extraction and Isolation. S. horneri (1170.8 g) was powdered and repeatedly refluxed 3 times with 70% alcohol to obtain a hydroalcoholic solution. The solution obtained was concentrated under reduced pressure, and the remaining aqueous suspension was extracted with the same volume of ethyl acetate (EtOAc) to obtain an EtOAc extract. The EtOAc extract (4.25 g) was fractionated using vacuum liquid chromatography (VLC) over silica gel by gradient elution using CH2Cl2/MeOH. The sixth fraction (95:5 CH2Cl2/MeOH, 853.6 mg) was subsequently separated over Sephadex LH-20 with CH2Cl2/ MeOH (1:1), followed by flash silica gel column chromatography with CH2Cl2/MeOH (96:4) to obtain a mixture of MGDGs (white powder, 153.4 mg). The purity of the MGDGs was analyzed using TLC developed with three solvent systems (I, 93:7 CH2Cl2/MeOH; II, 1:2 CH2Cl2/EtOAc; and III, 1:1 CH2Cl2/Me2CO). Only one purplish pink spot was visualized using a 5% methanolic sulfuric acid spray, followed by heating at 115 °C. Structure Elucidation of MGDGs. White amorphous powder. 1H NMR (600 MHz, CDCl3) δ: 4.39 (1H, dd, J = 3.1 and 12.0 Hz, H-1), 5.29 (1H, m, H-2), 3.91 (2H, dd, J = 5.4 and 11.1 Hz, H-3), 4.26 (1H, d, J = 7.6 Hz, H-1′), 3.66 (1H, m, H-2′), 3.59 (1H, m, H-3′), 3.69 (1H, m, H-4′), 3.54 (1H, t, J = 5.3 Hz, H-5′), 3.84 (1H, dd, J = 4.4 and 11.8 Hz, H-6′), 3.71 (1H, m, H-6′), 1.25−1.35 [brs, (CH2)n], 0.87 (6H, t, J = 7.0 Hz, CH3). 13C NMR (150 MHz, CDCl3) δ: 63.0 (C-1), 70.3 (C-2), 68.2 (C-3), 104.2 (C-1′), 71.4 (C-2′), 73.6 (C-3′), 69.2 (C-4′), 74.7 (C-5′), 62.0 (C-6′), 174.0 and 173.6 (−CO−), 27.3−34.4 [(CH2)n], 14.3 (CH3). This powder was identified to be 1,2-O-diacyl3-O-β-D-galactopyranosylglycerol (MGDG) by comparing NMR data to the literature.14 Alkaline Hydrolysis.15−17 A solution of the total MGDGs (35 mg) in MeOH (3.5 mL) was treated with 5% NaOMe/MeOH (3.5 mL) under stirring at room temperature for 10 min. The mixture was extracted with hexane 3 times. The hexane layer was washed with 11158

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Statistical Analysis. Values are expressed as the mean ± standard deviation (SD). Statistical analysis was performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA), and Tukey’s studentized range test was used for post-hoc evaluations. p < 0.05 was considered to indicate statistical significance.

Identification of the MGDG Molecular Species. The composition of molecular species of MGDGs in S. horneri was clarified through HPLC−MS/MS. A total of 10 MGDGs were found under the optimized HPLC condition (Figure 2), and their identities are shown in Table 1 (MS/MS spectrum; see the Supporting Information). The molecular weight of each MGDG was determined by [M + Na]+ and [M + NH4]+ ions in positive ESI−MS. In the following MS/MS, the precursor [M + NH4]+ ions produced fragment ions, namely, [M + H − 180]+ and [M + H − 162]+, because of the loss of a galactosyl moiety, and the precursor also produced [RxCO + 74]+ ions because of the loss of the neutral fatty acid. In addition, the precursor [M + Na]+ ions yielded a pair of abundant fragment ions, namely, [M + Na−RxCOOH]+, which were derived from the neutral loss of free fatty acid from the sn-1 (x = 1) or sn-2 (x = 2) position. These two ions were easy to distinguish in MS/MS spectra because of their high peak intensity. Most importantly, the peak intensity of [M + Na−R1COOH]+ was always greater than that of [M + Na−R2COOH]+,22−24 which was used to determine the fatty acyl attachments in each MGDG. For example, MGDG 10 (Figure 3) had a molecular formula of C43H80O10 and was assigned on the basis of m/z 779 (100%, [M + Na]+) and 774 (35%, [M + NH4]+) as well as relevant ions m/z 595 (11%, [M + H − 162]+) and 577 (19%, [M + H − 180]+). The base peak at m/z 523 (100%, [M + Na− R1COOH]+) and the minor peak at m/z 497 (37%, [M + Na− R2COOH]+) in the MS/MS spectra indicated that hexadecanoyl (palmityl, 16:0) was linked to the sn-1 position and that octadecadienoyl (18:1) was linked to the sn-2 position. In combination with the GC analysis results, the 18:1 fatty acyl was determined as oleoyl [18:1(ω-9)]. On the basis of the above findings, 10 molecular species of MGDGs were identified (Figure 1 and Table 1). Preparation of Major MGDG Molecular Species. The major MGDGs (1, 3, 5, 9, and 10 as well as a mixture of 7 and 8) were subsequently obtained using semi-preparative HPLC. This study was the first report on the MGDG molecular species 3, 5, 9 and 10 isolated from S. horneri. Effect of MGDG Molecular Species on TG and FFA Levels in Mature 3T3-L1 Adipocytes. 3T3-L1 preadipocytes were treated with compounds 1, 3, 5, 9, and 10 as well as the mixture of compounds 7 and 8 at a concentration of 10 μM.

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RESULTS Isolation and Structure Elucidation of MGDGs. The EtOAc extract of S. horneri was chromatographed repeatedly over silica gel and Sephadex LH-20 to obtain a mixture of MGDGs as white powder (Figure 1). The 1H NMR spectrum

Figure 1. Structure of MGDGs.

showed the following characteristic signal pattern because of glycoglycerolipid: a triplet at δH 0.87 (6H, t, J = 7.0 Hz, terminal methyl in fatty acyl), a broad signal centered at δH 1.27 (methylene in the fatty acyl), a mass of signals between δH 3.5 and 5.3 (12H, sugar and glycerol moiety), and a doublet signal at δH 4.26 (1H, d, J = 7.6 Hz, anomeric proton). The 13C NMR spectrum revealed one anomeric carbon at δC 104.2 and two carbonyl carbons at δC 174.0 and 173.6. The spectrum was identified as MGDGs by comparison of the NMR spectra to previously reported data.14 To determine the absolute configuration of C-2 in the glycerol moiety, an alkaline hydrolysis experiment was performed. The monogalactosylglycerol from the MeOH layer was identified as (2R)-1-O-β-D15 galactopyranosylglycerol with [α]25 D 7.7° (c 0.8, H2O) (9.0°, 16 21 10.1°, or 7.9° ). The optical rotation of (2S)-1-O-β-Dgalactopyranosylglycerol was reported to be +6.2°.17 The composition of fatty acids was identified through GC− FID analysis for FAMEs, which was derived from an alkaline hydrolysis experiment. The following composition was identified: a mixture of myristate (14:0), palmitate (16:0), palmitoleate [16:1(ω-7)], oleate [18:1(ω-9)], linoleate [18:2(ω-6)], and linolenate [18:3(ω-3)] in the ratio of 21.1:29.3:8.0:9.4:12.5:19.6.

Figure 2. HPLC−MS analysis of MGDGs from S. horneri: (a) high-performance liquid chromatography−diode array detection (HPLC−DAD) chromatogram at 203 nm and (b) total ion chromatogram (TIC). 11159

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Table 1. HPLC−MS/MS Analysis of MGDGs 1−10 MGDG

sn-1/sn-2

molecular formula

molecular weight

a

14:0/16:1 16:0/14:1 14:0/18:2 16:0/16:2 16:0/18:3 14:0/16:0 14:0/18:1 16:0/16:1 16:0/18:2 16:0/18:1

C39H72O10 C39H72O10 C41H74O10 C41H74O10 C43H76O10 C39H74O10 C41H76O10 C41H76O10 C43H78O10 C43H80O10

700 700 726 726 752 702 728 728 754 756

1 2 3a 4 5a 6 7a,b 8a,b 9a 10a a

MS data [M + Na]+ 723 723 749 749 775 725 751 751 777 779

MS/MS data [M + Na−RCOOH]+

(100) (100) (100) (100) (100) (100) (100) (100) (100) (100)

495 467 521 493 519 497 523 495 521 523

(100), (100), (100), (100), (100), (100), (100), (100), (100), (100),

469 497 469 497 497 469 469 497 497 497

(38) (41) (42) (39) (40) (52) (41) (32) (42) (36)

retention time (min) 17.9 18.4 19.5 20.0 21.4 22.8 23.3 23.9 25.4 30.5

Obtained using semi-preparative HPLC. bObtained as a mixture of compounds 7 and 8.

Figure 3. (a) Full-scan LC/ESI−MS spectrum of MGDG 10 in positive-ion mode, (b) MS2 spectrum of the trapped [M + Na]+ ion at m/z 779, and (c) MS2 spectrum of the trapped [M + NH4]+ ion at m/z 774.

Figure 4. Effects of MGDGs on TG and FFA levels in 3T3-L1 adipocytes. The following groups were analyzed: N, normal groups, cells were cultured without induction medium or any samples; C, control groups, cells were cultured with induction medium for 14 days; compound-treated groups, cells were cultured with induction medium and treated with 10 μM MGDGs 1, 3, 5, 7 + 8, 9, and 10 for 14 days. At 14 days after induction of differentiation, the cells were lysed using an Omni Ruptor and analyzed using the TG and NEFA kits according to the protocols provided by the manufacturers. Data represent the means ± SD of six determinations. (###) p < 0.001 versus the normal group. (∗∗) p < 0.01 and (∗∗∗) p < 0.001 versus the control group.

At this concentration, there were no treatment-related changes in cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method; data not shown]. In comparison to untreated cells (Figure 4), compounds 3 [sn-1/sn-2 14:0/18:2(ω-6)] and 9 [sn-1/sn-2 16:0/18:2(ω-6)] significantly suppressed the accumulation of TGs in mature 3T3-L1 cells with TG levels of 1.568 ± 0.2808 and 1.701 ± 0.1460 μmol/L, respectively. In contrast, compounds 1 [sn-1/ sn-2 14:0/16:1(ω-7)], 7 [sn-1/sn-2 14:0/18:1(ω-9)] and 8 [sn1/sn-2 16:0/16:1(ω-7)], and 10 [sn-1/sn-2 16:0/18:1(ω-9)]

did not effectively suppress the accumulation of TG levels, with TG levels of 2.484 ± 0.0460, 2.395 ± 0.2148, and 2.084 ± 0.2015 μmol/L, respectively (Figure 4a). The FFA content in mature 3T3-L1 cells was also determined. Compounds 3, 5, and 9 showed inhibitory effects on FFA accumulation, with FFA levels of 0.149 ± 0.0258, 0.195 ± 0.0079, and 0.198 ± 0.0229 mequiv/L, respectively (Figure 4b). As a result, the pattern of inhibitory effects on FFA levels was similar to that on TG accumulation. These results indicated that some MGDG molecular species from S. horneri reduced the TG and FFA 11160

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levels in lipid metabolism, and these results also indicated that linoleyl [18:2(ω-6)] at the sn-2 position was an essential group for these activities.

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ASSOCIATED CONTENT

S Supporting Information *

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MS data of MGDGs 1−10. This material is available free of charge via the Internet at http://pubs.acs.org.

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DISCUSSION The present study showed that palmitic acid (16:0) was the most abundant fatty acid (FA) in the MGDGs of S. horneri. This result agreed with Terasaki et al.25 and Airanthi et al.,26 who reported that 16:0 is the major FA in the total lipids of the same species. The major unsaturated fatty acids (UFAs) found in our study were C18 UFAs [including 18:3(ω-3), 18:2(ω-6), and 18:1(ω-9)], which differed from the study by Hossain et al.,27 who reported that C20 UFAs are the major UFAs in this species. The FA composition is influenced by species28,29 and environmental factors.29−31 Seasonal variation is an important factor for the difference in polyunsaturated fatty acid (PUFA) species. Sanina et al.30 reported that partial substitution of C20 by C18 PUFAs in glycolipids occurs from summer to winter in marine macrophytes. Our results supported this view because the seaweed used in the present study was collected in October, but C20 UFAs were not detected. This result was due to the extraction and isolation process, which is often used for studies on chemical constituents of traditional Chinese medicine (TCM). Procedures using classic lipid extraction methods32,33 as well as anti-enzyme and antioxidant measures should be used in future studies. MGDGs identified in the present study contained mainly C14 and C16 saturated FAs at the sn-1 position as well as C18 and C16 unsaturated FAs at the sn-2 position of the glycerol backbone. The eukaryotic pathway consists of two reactions involving glycoglycerolipid synthetase in the plastid and endoplasmic reticulum (ER). The synthetase in the plastid mainly generates glycoglycerolipids containing 16 carbon atoms at the sn-2 position, and the synthetase in the ER generates glycoglycerolipids containing 18 carbon atoms at the sn-2 position.34,35 Our data suggested that the glycoglycerolipid synthetase in S. horneri exists in both the plastid and ER. Compounds 3 and 9 with linoleyl [18:2(ω-6)] at the sn-2 position of the glycerol backbone showed stronger inhibitory effects on TG and FFA accumulation than the other compounds. Saturated fatty acyl (14:0 and 16:0) at the sn-1 position contributed to the inhibitory effect. As a well-known essential fatty acid, linoleic acid [18:2(ω-6)] functions as a precursor of arachidonic acid [20:4(ω-6)] and a series of proinflammatory eicosanoids. Linoleic acid can regulate lowdensity lipoprotein cholesterol (LDL-C) metabolism by downregulating LDL-C production and enhancing its clearance.36 Studies have demonstrated that linoleic acid also suppresses sterol regulatory element-binding protein 1c (SREBP-1c) expression37 and activates peroxisome proliferator-activated receptor (PPAR) α and γ,38,39 thus leading to lipid degradation. Metabolic disorders, such as obesity, type-2 diabetes, and metabolic syndrome, can occur when TG and FFA accumulate in adipocytes.19 Our results introduced a new chemical structure type of MGDGs with linoleyl in the sn-2 position that can reduce the levels of TG and FFA in adipocytes, which might be beneficial for lipid metabolism improvement. Because lipid metabolism disorders are an important risk factor of CVDs, our findings also indicated that MGDGs in S. horneri might be important effective components associated with the anti-CVD function of this edible seaweed.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-532-82031823. Fax: +86-532-82033054. Email: [email protected]. Author Contributions †

Ai-Cui Ma and Zhen Chen contributed equally to this work.

Funding

Financial support by the National Natural Science Foundation of China (NSFC, 31271845) to Hong-Bing Liu is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors thank Prof. Yuan-hong Wang (Laboratory of Chemical Analysis, Ocean University of China) for providing standards and assisting in FAME identification.

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ABBREVIATIONS USED MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; TG, triglyceride; FFA, free fatty acid; FA, fatty acid; FAME, fatty acid methyl ester; CVD, cardiovascular disease; DMEM, Dulbecco’s modified Eagle’s medium; TCM, traditional Chinese medicine; ER, endoplasmic reticulum; LDL-C, low-density lipoprotein cholesterol; SREBP-1c, sterol regulatory element-binding protein 1c; PPAR, peroxisome proliferator-activated receptor

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