Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess15190 J. Oleo Sci. 64, (12) 1273-1281 (2015) A Comparative Study of the Effects upon LPS Induced Macrophage RAW264.7 Inflammation in vitro of the Lipids of Hippocampus trimaculatus Leach LiPing Chen1, XuanRi Shen1,＊ , GuoHua Chen2, XianYing Cao1 and Jian Yang3 1 Laboratory of Aquatic Product Processing and Storage, College of Food, Hainan University (No.58, Renmin Avenue, Meilan District, Haikou 570228, CHINA) 2 College of Ocean, Hainan University (No.58, Renmin Avenue, Meilan District, Haikou 570228, CHINA) 3 Hainan Longsheng Bio-tech Development CO., Ltd (No.12, Jinpan Road, Longhua District, Haikou 570100, CHINA) Abstract: The present study attempts to investigate the anti-inflammatory potential of the isolated lipid extracts of three-spot seahorse which is rare marine bony fish. Petroleum ether (PE) extract was obtained from systematic solvent extraction after reflux extraction with 95% ethanol. FrIV was collected after silica gel column chromatography, and neutral lipids (NL), glycolipids (GL), phospholipids (PL) were separated from FrIV. Basic compositions were detected and analyzed via thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR). Anti-inflammatory activities of total lipids (TL), isolated NL, GL, and PL were detected by secretion of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) in murine monocyte macrophage RAW264.7 cells in vitro. The results revealed that lipids of seahorse showed a positive correlation with the in vitro suppression of the release of nitric oxide (NO), interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α potently in a dose dependent manner, and showed cell compatibility. Among the fractions, GL (50 μg/mL) showed the highest capacity to attenuate the generation of pro-inflammatory cytokines which was comparable to that of the positive drug dexamethasone (DX) (20 μg/mL). Collectively, our findings indicated that the lipids from seahorse may be effective in the management of inflammation. Key words: seahorse, lipids, glycolipids, phospholipids, anti-inflammation 1 INTRODUCTUON fatigue7－9）. Up till now, there have been many studies on Inflammation is a protective mechanism by which, upon the lipids including PL, GL of sea cucumber, starfish, sea entry of external materials such as bacteria and viruses urchin and such marine aquatic products benefiting from into the body, inflammatory mediators are secreted1）. One the particular ecological habitat10）. However, there has of the most potent stimuli for macrophage is bacterial en- been no relevant research reported yet exploring the po- dotoxin（LPS）, which activates cells via Toll-like receptor 4 tential anti-inflammation of lipids from seahorse. As a part （TLR4）and induces the production of large amount of pro- of an ongoing research on isolating bioactive metabolites inflammatory cytokines such as TNF-α and IL-1β2－5）. The from three-spot seahorse, lipids were isolated and initially inflammatory response is accompanied by the upregulation identified for composition. This study has focused on analy- of inflammatory cytokines and the release of various in- sis and investigation of anti-inflammatory effects of lipids flammatory mediators including NO, TNF-α6）. from seahorse. Seahorse, a marine teleost fish, is known intimately for its peculiar medicinal composition. Hippocampus trimac- ulatus Leach is used as one of the most famous materials of traditional medicine and has been studied for many 2 EXPERIMENTAL years for its diverse biological activities, including anti-tu- 2.1 Materials and methods mor, appetite enhancement, antioxidant, anti-aging, anti- Three-spot seahorse was purchased from Hainan Long- ＊Correspondence to: XuanRi Shen, Laboratory of Aquatic Product Processing and Storage, College of Food, Hainan University (No.58, Renmin Avenue, Meilan District, Haikou 570228, CHINA) E-mail: [email protected] Accepted September 19, 2015 (received for review August 3, 2015) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1273 L. Chen, X. Shen and G. H. Chen et al. sheng Bio-tech Development CO., Ltd（Haikou, CHN）. under UV detector, among fractions, FrⅣ was mixed with Mouse mono macrophage RAW264.7 cells were obtained chloroform/methanol/H2O in a centrifugal tube in a propor- from Xiehe cell resource center（Beijing, CHN）. Cell culture tion of 8:4:3 by volume, centrifuged under 4000 rpm for 10 basic media DMEM was purchased from gibco life technol- min after vortex mixed for 5 min, the lower layer was col- ogies China（Shanghai, CHN）, penicillin/streptomycin, fetal lected and 0.2 its volume of 0.9％（ m/v）sodium chloride so- bovine serum（FBS）were purchased from gibco life tech- lution was added and vortex mixed for 1 min, and then nologies（AUS）, Griess reagent and LPS of Escherichia centrifuged under 4000 rpm for 5 min, then the lower coli O111:B4 were purchased from Sigma Chemicals, MTT organic layer was collected and concentrated in rotary reagent was purchased from Invitrogen Molecular Probes under 45℃11）. After the wash process, it was dissolved in （Eugene, Oregon, USA）, ELISA kits were purchased from small volume of chloroform and applied to the silica gel Elabscience（Wuhan, CHN）, BCA kit was purchased from column eluting by chloroform, acetone and methanol, re- Beyotime Biotechnology（Shanghai, CHN）, TLC plates spectively. Crude NL, GL and PL were obtained12, 13（） Fig. （Silica MF-254, Agela Technologies, CHN）were used in an- 1）. alytical TLC. Other chemicals and reagents used were of analytical grade available commercially. 2.3 TLC analysis Different elution parts were developed with a solvent 2.2 Preparation of lipids system consisting of a mixture of hexane/diethyl ether/ 2.2.1 Extraction of PE extract methanol/acetic acid（90:20:5:2, v/v/v/v）on TLC plate, Three-spot seahorse was washed with tap water to iodine vapor was used to indicate the distribution of lipids. remove organs and sediment, then put in the constant SubFrⅡ was developed with CHCl3/methanol/acetic acid temperature drying box and allowed to be dried for 24 h （65:15:2, v/v/v）on TLC plate and the plate was immersed under 60℃ and through 20 mesh sieve after crashing. Ana- in Molish reagent（15％ α-naphthol in ethanol/sulfuric acid/ lytical 95％ ethanol was used for reflux extraction under ethanol/water, 10.5:6.5:40.5:4, v/v/v/v）for a few seconds to 80℃ by 1:10（m/v）and lasted for 1.5 h（×3）. The obtained detect GL14）, the purple red spot could be visualized after crude ethanol extract was filtrated through buchner heating in oven under 105℃ for 10 min. SubFrⅢ was de- funnel, the residue was re-extracted（×2）, resulted extract veloped with CHCl3/methanol/H2O（65:25:4, v/v/v）on TLC was concentrated by rotary evaporator at 45℃. The extract plate and Vaskovosky reagent was sprayed to detect PL15）, was suspended by 1:1（m/v）of raw material and deionized the blue spot could be observed after heating in oven water in a separating funnel. Solvent extraction with equal under 110℃ for 5 min. volume of PE, the upper organic phase（×3）was collected and combined before concentrated by rotary evaporator at 2.4 FTIR analysis of lipids 45℃. A spectral range of 4000-500 cm－1 was collected using 2.2.2 Silica gel column chromatography an FTIR spectrometer（Tensor27, Bruker Optics, PE extract was applied to column packaging silica gel, Germany）. Briefly, each around 2 mg lyophilized fraction and eluted by PE and ethanol and detected and monitored was mixed and grinded with potassium bromide, interval Fig. 1 Three-spot seahorse extraction of lipids. 1274 J. Oleo Sci. 64, (12) 1273-1281 (2015) A comparative study of seahorse lipids upon LPS induced inflammation scanning was conducted with the wavenumbers ranging cells were pre-incubated overnight in 96-well plates using from 4000 to 500 cm－1 and determined the infrared ab- DMEM without phenol red at a density of 1×104 cells per sorption spectrum, the functional group and molecular well, followed by the treatment of lipids（5, 25 and 50 μg/ structure were inferred in accordance to infrared absorp- mL）. NO production was stimulated by incubating with tion spectrum absorption peak and wavenumber. LPS（1 μg/mL final concentration）for 48 h. Each was carried out in quadruplicate. Fifty microliter of culture su- 2.5 Fatty acids analysis pernatant from each sample was collected and mixed with Characterization of the fatty acids was conducted by equal volume of Griess reagent and allowed to incubate for conversion to the corresponding fatty acid methyl esters 15 min at 25℃. Absorbance values were read at 540 nm on （FAME）followed by GC-MS analysis. Briefly, the lyophi- an ELISA microplate reader（Synery HT, Gene Company lized TL, NL, GL, PL（around 20 mg）was transmethylated Limited）. The nitrite（stable oxidation product of NO）levels with 2.5 mL of methanol containing 2％（ v/v）sulfuric acid of the media were calculated from regression analysis using at 70℃ for 2 h16）. After the suspension cooled, 1 mL known concentrations of sodium nitrite to deliver a stan- hexane and 1 mL saturated sodium chloride solution were dard curve（y＝0.0124x＋0.0013, R2＝0.996, liner range added to form separated layers in the tube. The upper （3.12-100 μM））. hexane layer containing FAME was removed for analysis by GC-MS（7890B/7000B, Agilent, US）. Samples were injected 2.8 Enzyme immuno assay of TNF-α, IL-1β, IL-6 into a 30 m long with 0.25 mm inner diameter and 0.25 μm The inhibitory effects of lipids on the production of pro- film thickness HP-5MS capillary column.