Samples of S. cichorioides (Miybei) C. E. Lane, C. Mayes, Druehl et G.W. Saunders, C. costata (Turner) Saunders, C. filum (Linnaeus) Stackhouse and E. fistulosa (order Laminariales); D. divaricata (Okamura) Okamura (order Dictyotales); D. chordaria Areschoug (order Ectocarpales); S. babingtonii (Harvey) E. A. Serrao, T. O. Cho, S. M. Boo et Brawley and F. evanescens C. Agardh (order Fucales) were collected in Trinity Bay (42˚38'N and 131˚06'E) Peter the Great Gulf of the Sea of Japan on Marine Experimental Station. Algal samples were cleaned from sand particles and epiphytes and rinsed in running water. Samples were dried out with filter paper and weighed. Substances extracted of immediately. Algal thalli crushed in blender, mixed carefully. 200 - 300 g of sample was used for homogenization with EtOH (0.5 L) and the mixture of EtOH/CHCl₃ (1:1, 0.7 L × 2). Extracts were combined, and distilled water was added to form a biphasic system. Water layer was separated and extract evaporated to dryness and weighed. It was stored in sealed flasks at −25˚C. Substances content was determined by the gravimetric method and as percentage of the algae wet weight.

GLs content was determined according to the sulfuric-orcinol procedure [38] with slight modification. Briefly, TLC of the GLs carried out on the 12 × 12 cm plates covered with silica gel 60_F254 (Merck, Germany) using mobile phase (CH₃)₂СO/C₆H₆/H₂O (91:30:8, v/v/v). Spots corresponding to several classes GLs were scraped off the plate in a tubes and sulfuric-orcinol reagent was added. The test tubes were warmed to 80˚C for 20 min. After centrifugation, the absorbance was measured at 505 nm. MGDG, DGDG and SQDG were using for constructed of calibration curves. Phospholipids (PL) were quantified based on the content of phosphorus using molybdate reagent [39] . Neutral lipids (NL) were analyzed by TLC on the 12 × 12 cm plates covered with silica gel 60_F254 in solvent system C₆H₁₄/(C₂H₅)₂O/CH₃COOH (80:20:1, v/v/v). The content of TAG was estimated by gas chromatography (GC), using 17:0 FA as an internal standard [40] .

The content of carotenoids and chlorophylls were determined after separation extracts on the plates 10 × 15 cm with silica gel 60_F254 (Merck, Germany) using of mobile phase C₆H₁₄/(CH₃)₂CO/(C₂H₅)₂O (50:20:4, v/v/v). Carotenoids were identified by comparison with authentic standards of fucoxanthin and b-carotene (Sigma-Al- drich). Major carotenoids and chlorophylls bands were scraped off the plate and eluted with CHCl₃. Absorption maxima (λ max, nm) were measured with using the SF 2000 spectrophotometer (Spektr, St. Petersburg, Russia). Quantitative estimates of carotenoids were determined using the following extinction coefficients of E1% (1 cm path) in ethanol: 2500 for a mixture of pigments at λ max 450 nm [41] [42] and 1280 at λ max 448 nm for fucoxanthin [43] . The total chlorophylls content was determined using of E1% 840 (in acetone) at λ max 663 nm [41] .

All extracts of algae were separated with use by one scheme. Extracts (300 - 500 mg) was suspended in a small volume of n-hexane and applied to the top of a column (12 - 15 × 2.0 - 2.5 cm) filled with silica gel 40/100 μm (Chemapol, Lachema, Czech Republic) in n-hexane. Neutral lipids and photosynthetic pigments were eluted consequently with n-hexane, C₆H₁₄/(C₂H₅)₂O (95:5 → 50:50, v/v) and then with CHCl₃ (fractions fucoxanthin and free FA). Fractions of fucoxanthin were monitored by TLC using C₆H₁₄/(CH₃)₂CO (70:30, v/v) as mobile phase and standard fucoxanthin (Sigma-Aldrich).GLs were eluted with mixtures of CHCl₃/(CH₃)₂СO (90:10 → 50:50, v/v, fractions 1 - 3). The fractions of GLs were monitored by TLC using solvent system (CH₃)₂СO/ C₆H₆/H₂O (91:30:8, v/v/v) with authentic standards of GLs. In additional C₆H₁₄/(CH₃)₂CO (70:30, v/v) mobile phase and standard chlorophyll a were used. The fractions containing identical substances were combined, dried in vacuum and dissolved in CHCl₃. Fractions 1 consisted MGDG and a little of carotenoids and chlorophylls and free fatty acids; fraction 2-DGDG with a little amount of phospholipids and chlorophylls, fractions 3-SQDG with chlorophylls and phospholipids. These fractions were purified again on additional columns of silica gel (5 - 6 × 1.5 cm).Substances were eluted with CHCl₃ and CHCl₃/(CH₃)₂СO as described above. Fractions were collected with a volume of 5 ml and purity of substances monitored by TLC. Fucoxanthin, MGDG, DGDG and SQDG were dried in vacuum and stored at −25˚C in sealed flasks before analysis.

Fatty acid methyl esters (FAME) prepared according to the method of Prevot and Mordret [44] with slight modification. Briefly, 2 ml n-hexane and 0.4 ml 2 N KOH in MeOH was added to an aliquot of lipids, vortexed for 30 s and incubated at 50˚C up to 2 min. Then 0.4 ml 2 N HCl in MeOH was added to the solution and vortexed up to 3 min at room temperature. The mixture was left for1-2 min. After that, the upper hexane layer containing FAME was recover and crude FAME were purified by TLC in C₆H₁₄/C₆H₆ (7:3, v/v) as mobile phase and analyzed by GC using Shimadzu 2010 Plus gas chromathograph (Japan) with a flame ionization detector. Supelcowax 10 column (30 m × 0.25 mm i.d.; 0.25 μm film, Supelco, USA) was used (isotherm, 210˚C; He-1 ml/min; He-linear velocity, 40 cm/sec). FAME was identified by equivalent chain length values (ECL) [45] . Pirrolidide derivatives (N-acyl pirrolidides) were used for determination of double bond positions. These derivatives were prepared by direct treatment of FAME with C₄H₉N/CH₃COOH (10:1, v/v) at 80˚C for 45 min [46] and purified by TLC in mixture C₆H₁₄/(C₂H₅)₂O (2:1, v/v). The pyrrolidides were analyzed by gas chromatography/mass spectrometry (GC/MS) using Agilent 6890 gas chromathograph with quadrupole mass selective detector (MSD) HP 5973 (ionization energy was 70 eV) and HP-5ms column (30 m × 0.25 mm i.d.; 0.25 μm film, Agilent, USA). Temperature regime was programmed: 205˚C―5 min, 5˚C/min to 240˚C, 20˚C―30 min; He― 1.3 ml/min. The solvent delay for pirrolidide derivatives was 3 min.

The murine macrophage Raw 264.7 cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Sigma-Aldrich), at 37˚C under a humidified 5% CO₂ atmosphere in incubator (MCO- 18AIC, Sanyo, Japan).

The cell viability assay was evaluated according to the MTT method [47] with slight modification. Briefly, RAW 264.7 murine macrophages (5 × 10⁵/well) were seeded in a 96-well microplate and cultured at 37˚C for 2 h. After cell adhesion, the cell monolayer was washed with phosphate-buffered saline (PBS) and incubated with fresh medium containing various concentrations of test compounds (10; 20; 40; 60; and 100 μg/ml) for 24 h. Subsequently, 10 μL of MTT (Sigma) stock solution (5 mg/ml) was added to each well, and the microplate was incubated for 4 h. After that 100 μL of SDS-0.01 M HCl was added to each well followed by incubation for 18 h. Absorbance of converted dye formazan, was measured using a Multiskan FC microplate photometer (Thermo Scientific) at 570 nm with background subtraction at 630 - 690 nm.

The cells Raw 264.7 macrophages were plated into 96-well microplates (Costar^®, Corning, NY) and incubated at 37˚C with 5% CO₂ for 24 h. After adhesion, cells were incubated with tested compounds at concentrations 10 μg/ml and LPS (1.0 μg/ml) for another 24 h. To study ROS formation, 20 μl of 2,7-dichlorodihydrorofluo- rescein diacetate (H₂DCF-DA) solution (Molecular Probes, final concentration 10 μM) was added to each well and the microplate was incubated for an additional 10 min at 37˚C. Prior to fluorescence registration, the cells were washed three times with PBS and then bathed in 200 μl/well of PBS. The intensity of dichlorofluorescein fluorescence was measured at λex = 485 nm, and λem = 518 nm [48] . In each experiment, LPS from E. сoli serotype 055:В5 (Sigma) were used as a positive control. Fluorescent intensity was measured using plate reader PHERAstar FS (BMG Labtech, Germany).

All assays were performed at least in triplicate. The results are expressed as the mean values (MV) ± standard deviation (SD). A Student’s t-test was used to evaluate the data with the significance level of p < 0.05. Figures were built using Microsoft Office Excel.

The extracts from eight brown algae were prepared with using EtOH/CHCl₃ mixture. These extracts have demonstrated anti-inflammatory activity, which vary considerably in different species of brown algae. Such differences could be explained by the composition of the extracts and by the ratio of the components therein. All extracts have included lipids and PSP (Table 1). Their total content was 0.27% - 1.30% of wet weight algae. Lipid moiety was represented neutral lipids (NL), primarily triacylglycerol (TAG), GLs, phospholipids (PL). GLs were the main polar lipids, the contents of which vary over a wide range (Table 1). In PSP were dominated of chlorophylls, but carotenoids were much in some algae also (Table 1). The content of MGDG, DGDG, SQDG,

Data presented ^* as % of wet weight alga (SD < 0.04), ^** as % of total content substances in extracts. ^*** shown only TAG content and only fucoxanthin. The content of substances are presented as MV ± SD of three replicate assays.

and carotenoids showed species-specific differences and has depended on the month of collecting the algae (Table 1).

The anti-inflammatory activity of the algal extracts and substances was tested in RAW 264.7 macrophage cells. Preliminarily cell viability was measured using MTT assay [47] . The RAW 264.7 murine macrophage cells were treated with varying concentrations (10.0, 20.0, 40.0, 60 and 100 μg/ml) extracts. The results showed that algal extracts did not affect cell viability within this a concentrations range (Figure not shown) and 10 μg/ml as minimal concentration of extracts was chosen as for investigations.

Algal extracts, for exception of C. costata and E. fistulosa extracts, showed barely noticeable increase in ROS formation in RAW 264.7 macrophage cells compared with pro-inflammatory endotoxin lipopolysaccharide from E. coli, which generated a large amount of ROS in macrophages (Figure 1).

Further RAW 264.7 macrophage cells were stimulating with LPS from E. coli, which generated a large amount of ROS in them. These LPS-stimulated macrophage cells were treated by algal extracts. Extracts showed different abilities to suppress of ROS in LPS-stimulated cells (Figure 2). Among of tested extracts, extracts of D. divaricata, D. chordaria, C. filum and S. cichorioides, S. babingtonii and F. evanescens showed strongest inhibitory effect. They reduced the level of ROS at the 30% - 55% relatively positive control of LPS (Figure 2). Extracts S. cichorioides (collected in August), C. costata and E. fistulosa were weaker. They lowered the ROS level only by 12% - 27% (Figure 2). Algae S. cichorioides and D. divaricata were collected in different months and this affected the degree of suppression of the ROS. Extracts of S. cichorioides, which were collected in July and November decreased ROS level by 30% - 31% and the samples collected in August reduced him on 27%. Extract of D. divaricata collected in July reduced the level of ROS by 55%, while the extract of this alga that was collected in September lowered him on 33%. Obviously, the species of algae and month of collection have an impact on the degree of suppression of ROS.

As shown earlier, algae produce many glyceroglycolipids [49] [50] and fucoxanthin among carotenoids of brown algae is the main component [51] - [54] . As indicated above, some species of brown algae of the Sea of

Japan contain large quantities of GLs and fucoxanthin (Table 1). Polar lipids [31] [32] [55] and fucoxanthin [28] [29] are of interest as anti-inflammatory agents. Therefore GLs and fucoxanthin were isolated by column chromatography for investigation their ability to suppress of ROS. All GLs showed no cytotoxic effect and not stimulated ROS formation in RAW 264.7 cells. On the contrary, they are suppressed ROS in LPS-stimulated RAW 264.7 macrophages.

MGDG from three species algae: E. fistulosa, D. divaricata, and C. filum (first group) have high activity, decreasing ROS level in LPS-stimulated RAW 264.7 macrophages at 52% - 58% (Figure 3) relatively of LPS. MGDG of D. chordaria, F. evanescens and S. babingtonii have middle activity (second group) and they lowered the ROS level by 33% - 35%, but MGDG S. cichorioides and C. costata (third group) reduce ROS level by 17% -

31% (Figure 3). Month of collecting the algae has influenced on suppression of ROS. So, MGDG from seaweed S. cichorioides, which were collected in August, weaker inhibited ROS than MGDG algae collected in July and November. MGDG D. divaricata that were collected in July and September decreased ROS levels in equal measure (Figure 3).

DGDG can be divided into two groups according to the degree of inhibition. The first group consists of the DGDG with a low degree of inhibition, which were isolated from S. cichorioides, C. costata, C. filum, F. evanescens and S. babingtonii. They suppressed of ROS by 20% - 30% (Figure 3). The second group were of DGDG of E. fistulosa, D. divaricata, and D. сhordaria that had high activity. They lowered the level ROS by 38% - 50% relatively of LPS (Figure 3). Among these brown algae, DGDG of D. divaricata was the strongest a suppressor (Figure 3).

SQDG were isolated from extracts of S. cichorioides that were collected in July, August, and November, and also F. evanescens. All SQDG had weaker activity than galactolipids MGDG and DGDG (Figure 3).

As shown, fucoxanthin well suppresses the production of inflammatory mediators [28] [29] . He was isolated by column chromatography from extracts several algae as C. filum, D. chordaria and D. divaricata for comparison his activity. Purity of carotenoids was examinated by TLC. Fucoxanthin from C. filum contained small amount chlorophyll a and fucoxanthinol and from D. chordaria he was pure. Fucoxanthin of D. divaricata, which was collected in July and in September, was separate on two fractions: pure of fucoxanthin and fucoxanthin together with its metaboliteas fucoxanthinol. Pure fucoxanthin from different algae suppressed the ROS a greater degree, by 55% - 60%, whereas fucoxanthin with impurity other pigments showed low level of suppression of ROS that was in range by 20% - 50% (Figure 4). Chlorophyll c not was active, but chlorophyll c₁ lowered level of ROS by 23% (Figure 4). These pigments did not affected cell viability and they not stimulated ROS formation in RAW 264.7 cells.

Thus, GLs and pigments demonstrated ROS-inhibitory activity. Extracts of algae are complex mixtures of lipids and PSP and synergistic effect manifested as collective influence of their on the activity. In addition, quantitative content of components in the extracts could influence the level of their activity. This is especially noticeable for algae E. fistulosa, which included very few polar lipids and many pigments (Table 1) and C. costata with high content pro-inflammatory fatty acids (Table 2).

As shown in several studies, the activity of the lipid extracts depends on the composition and ratio of fatty acids

in them [23] [34] . It is believe, that a low n − 6/n − 3 ratio may be one of the factors influencing their anti-inflammatory properties [23] . These assumptions were based on experimental studies, which showed that incorporation of n − 3 PUFA modifies inflammatory and immune reactions, making n − 3 PUFA potential therapeutic agents for inflammatory and autoimmune diseases. The ratio n − 6/n − 3 as 1 − 4/1 rather than the ratio of 20 − 16/1 from diets is more physiologic [56] [57] .

Research FAs of EtOH/CHCl₃ extracts showed that they had a high proportion of polyunsaturated fatty acids (PUFA), which was of the order of 36.6% - 59.0%, but the level saturated FAs (SFA) and monounsaturated (MUFA) FAs was also relatively high (Table 2). In extracts with high levels of suppression of ROS had greater of PUFA. As considered, fatty acids 18:3n − 3 (ALA), 18:4n− 3 and 20:5n − 3 (EPA) has anti-inflammatory function [58] - [60] , while 18:2n − 6 (LA) and 20:4n − 6 (AA) showed of pro-inflammatory properties [59] [60] . The content 18:3n − 3, 18:4n − 3 and 20:5n − 3 varied in broad range in extracts of different algae. However, n − 3 PUFA were dominant PUFA in extracts with high activity. The exception was an extract of D. divaricata (September), which had an equal ratio n − 3 and n − 6 PUFA (Table 2). As a rule, all extracts, for exception extract of D. divaricata, have a low ratios n − 6/n − 3 PUFA and LA + AA/ALA + EPA. Ratio n − 6/n − 3 was in range 0.38 - 0.99 and ratio LA + AA/ALA + EPA was 0.51 - 0.98. In less active extracts S. cichorioides (collected in August) and C. costata there were many of PUFA. However, PUFA content in this extract of S. cichorioides was lowest, but the proportions of n − 6 and n − 3 PUFA were close, and in the extract C. costata, which had a high amount of PUFA dominated of n − 6 PUFA. Ratio n − 6/n − 3 was in range 0.98 - 1.11 and ratio LA + AA/ALA + EPA was 1.39 - 1.42. Extracts of S. cichorioides and D. divaricata that showed changes in activity depending on the month of collecting the algae had appreciable differences in the amount of SFA, MUFA and PUFA and in the ratios of n − 6/n − 3 and LA + AA/ALA + EPA (Table 2). It should be noted that in the extract of E. fistulosa PUFA was greater than 54%, and in them dominated of n − 3 PUFA, but the activity of this extract was the lowest. As can be seen, algae extracts are complex mixtures (Table 1). In extract of E. fistulosa was dominated by the pigments (above 63%), mainly chlorophylls, and there were not much of polar lipids (24.7%), and TAG (8.4%). Inhibition of ROS is likely, occurred due to the synergistic action of the various components including FAs of lipids. Probably that low suppression of the ROS was due to high content of PSP and the lower percentage of lipids in extract of E. fistulosa.

Natural GLs always exist as mixtures molecular species because of the diversity composition of FAs. We studied influence of FAs natural GLs on suppression ROS, although recent works have investigated anti-in- flammatory properties only their molecular species [31] [32] [61] [62] . Variations of FAs content of GLs brown algae shown in Table 3.

In MGDG of the first group, which included C. filum, E. fistulosa, and D. divaricata, the amount of SFA,

MUFA and PUFA was varied in a wide range but n − 3 PUFA prevailed among PUFA (Table 3). MGDG E. fistulosa, which had highest activity, was most noticeable in the content of n − 3 PUFA, which was about 73%. Less all n − 3 PUFA was in MGDG C. filum (about 20%) and there was many SFA and MUFA. In MGDG D. divaricata collected in July also had a lot of n − 3 PUFA (about 42%), but in MGDG of September algae n − 3 PUFA was comparatively little (29%). At the same time, in these MGDG had enough many n − 6 PUFA, regardless of the month of collection. Ratio n − 6/n − 3 for algae of the first group was in range 0.12 - 0.59 and ratios LA + AA/ALA + EPA were 0.28 - 0.81. The lowest ratio of n − 6/n − 3 had MGDG E. fistulosa, which was 0.12. In this alga had also very little of n − 6 PUFA. MGDG from D. chordaria, F. evanescens and S. babingtonii, which showed middle activity possessed also pretty low ratio n − 6/n − 3 that was 0.56 - 0.82, but ratio LA + AA/ALA + EPA was >1. In MGDG were many PUFA and n − 3 PUFA there are predominated. MGDG with a

low activity from algae of S. cichorioides and C. costata had high percentages of SFA and MUFA. MGDG S. cichorioides that was collected in August contained only about 2% PUFA namely n − 6 PUFA and activity this MGDG was very low (Figure 3). MGDG from alga collected in July as well as MGDG C. costata contained little n − 6 PUFA and very little n − 3 PUFA and they were slightly more active, than the MGDG of August of algae. At the same time, in MGDG November algae had many PUFA, and among them n − 6 PUFA were prevailed. This MGDG stronger than others in third group inhibited ROS. In this group of algae, n − 6/n − 3 ratio was 0 - 3.96 and LA + AA/ALA + EPA ratio was 0 - 4.25.

In most cases, MGDG larger inhibit of ROS, than DGDG (Figure 3). In DGDG of all the algae had a lot SFA. In DGDG of the first group with low level of ROS inhibition had a lot of 18:2n − 6, but 20:4n − 6 was less and the content of 18:3n − 3 and 20:5n − 3 was varied in a wide range. Portion n − 6 PUFA was high or they predominated in PUFA DGDG. In general, the ratio of n − 6/n − 3 ranged from 0.69 to 1.27, but ratio LA + AA/ALA + EPA was greater than or equal to 1 (Table 3). DGDG S. cichorioides also showed differences in the composition of the FAs depending on the months of collecting the alga. Content 18:2n − 6 was close in different months, and the contents of 20:4n − 6, 18:3n − 3, 18:4n − 3, 20:5n − 3 varies considerably. At the same time, the ratios of n − 6/n − 3 and LA + AA/ALA + EPA are slightly different (Table 3). In DGDG second group algae predominated of n − 3 PUFA. The ratio n − 6/n − 3 was lower, than in the DGDG algae the first group and was 0.67 - 0.73. The ratio LA + AA/ALA + EPA was also smaller than in the first group. DGDG from D. divaricata collected in July and September had close amounts of both n − 3 PUFA and n − 6 PUFA. However, n − 3 PUFA predominated in both months. The ratio n − 6/n − 3 and LA + AA/ALA + EPA in DGDG of these algae were very close (Table 3).

SQDG inhibit ROS weakly. SQDG S. cichorioides had high levels of SFA and MUFA and small amount of PUFA in boths month of collecting algae (Table 3). In PUFA SQDG S. cichorioides was greater n − 6 PUFA, and in SQDG of F. evanescens the content n − 6 and n − 3 PUFA were pretty close.

As can be seen, GLs of these algae demonstrated differences in the ratio of FAs. In general, the species of algae, the month of their collection, FAs of lipids, and polar heads of GLs affected the ability to suppress ROS.