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ISSN 1068 1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. ... a Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian ...
ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 7, pp. 720–727. © Pleiades Publishing, Ltd., 2013. Original Russian Text © E.A. Martyyas, N.I. Gerasimenko, N.G. Busarova, E.A. Yurchenko, A.V. Skriptsova, M.M. Anisimov, 2012, published in Khimiya Rastitel’nogo Syr’ya, 2012, No. 1, pp. 123–131.

LOWMOLECULARWEIGHT COMPOUNDS

Seasonal Changes in Biological Activity of Lipids and Photosynthetic Pigments of Saccharina cichorioides (Miyabe) (Laminariaceae Family) E. A. Martyyasa, N. I. Gerasimenkoa, 1, N. G. Busarovaa, E. A. Yurchenkoa, A. V. Skriptsovab, and M. M. Anisimova a

Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, pr. 100 let Vladivostoku 159, Vladivostok, 690022 Russia b Zhirmunsky Institute of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, ul. Pal’chevskogo 11, Vladivostok, 690041 Russia Received January 19, 2012

Abstract—Biological (antimicrobial, cytotoxic, embryotoxic, and hemolytic) activity of total lipids, individ ual fractions and classes of lipids, and photosynthetic pigments of Saccharina cichorioides (Miyabe) were studied, together with the effect of season of the alga harvesting and changes in fatty acid composition of lipids on their activity. Antimicrobial activity was more pronounced in total lipids, individual fractions of lipids, and photosynthetic pigments of S. cichorioides collected in March. High antimicrobial activity was also demon strated by total lipids of alga samples collected in September and November and hemolytic activity, in those collected in March and November. Pronounced hemolytic activity was demonstrated by some classes of glyc eroglycolipids of laminaria samples collected in November. Cytotoxic activity toward mouse splenocytes was demonstrated only by total lipids of alga collected in March. Toxic effects toward embryos of sea urchin were demonstrated by lipids of S. cichorioides collected in June and September. None of the lipids or pigments was active against Ehrlich ascites carcinoma. Keywords: brown algae, lipids, glyceroglycolipids, fatty acids, GLC, biological activity DOI: 10.1134/S106816201307008X 1

The work continues a series of our studies on the biological activity of lipids and photosynthesizing pig ments (PSPs) of brown algae of Far Eastern seas [1, 2]. Many studies abroad are focused on the biological activity of various chemical compounds of algae [3– 14]. In our country, as a rule, biological activity of algae polysaccharides is studied [15–18]. Until recently, lipids of algae, as well as PSPs, have been less studied and only in past few years has the biological activity of fucoxanthin [19–22], fatty acids (FAs) [23– 25], and glyceroglycolipids (GL) [26–30] been studied intensively. The aim of the present work was to study antimicro bial, cytotoxic, embryotoxic, and hemolytic activity of total lipids (TL), individual fractions and classes of lipids, as well as PSPs of the S. cichorioides brown alga, the effect of season of alga collection on their activity, and contri bution of FAs to the seasonal activity. RESULTS AND DISCUSSION Table 1 shows that TLs of algae collected in differ ent seasons exerted activity against microorganisms 1 Corresponding author: email: [email protected].

tested in the work. The level of activity depended on the season of collection. Among the bacterial cells, gram positive bacteria S. aureus were the most sensi tive to the inhibitory effect of TLs, E. coli were less sensitive. It is assumed that antibacterial substances are more efficiently transported through dense pepti doglycan layers of gram positive bacteria than through glycolipid layers of gram negative bacteria [5]. The mechanism of the process has not been defined yet. TLs of algae comprise lipids of different structure. The presence of fatty acids is common for all of them. In the work, we studied the effect of FAs on the activity rate of TLs, individual GL classes, as well as the activ ity rate of different fractions of other substances iso lated from TLs. A.P. Desbois and coworkers [24] have demon strated that 16:1 (n7) and 16:3 (n4) FAs of diatomic algae are active against gram positive bacteria. The acid 16:1 (n7) kills bacteria and is highly active against S. aureus, resistant to many drugs. The authors also studied the effect of the double bond on the FA activity. It turned out that 16:0 acid is not active against the bacteria, while the presence of a double bond in FA is critical for its activity. The antimicrobial potential of FAs grows with the increasing number of double bonds

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Table 1. Antimicrobial activity of TLs of S. cichorioides Microorganism Alga collection season

S. aureus

E. coli

C. albicans

A. niger

F. oxysporum

V. alginolyticus

Lysis zone, mm from well edge March: mature juvenile May* June* July* September* October* November* Nitrofungin

6.8 ± 0.2 4.3 ± 0.2 4.0 ± 0.3 3.0 ± 0 – 6.7 ± 0.3 5.3 ± 0.2 3.4 ± 0.2 14.0 ± 0

2.3 ± 0.2 8.8 ± 0.2 1.3 ± 0.2 1.0 ± 0 1.0 ± 0 3.7 ± 0.2 2.5 ± 0 – 3.8 ± 0.2

0.8 ± 0.2 1.0 ± 0 – 1.0 ± 0 – 3.0 ± 0 2.0 ± 0 – 6.0 ± 0

2.2 ± 0.2 … 3.0 ± 0.2 4.0 ± 0 – 4.0 ± 0 4.8 ± 0.2 – 12.0 ± 0.2

3.8 ± 0.2 – 0.8 ± 0.2 3.0 ± 0 – 9.8 ± 0.2 7.5 ± 0.5 2.0 ± 0 3.8 ± 0.2

– … – – … 4.0 ± 0 2.0 ± 0 – 6.0 ± 0

*, mature algae.

in acids having the same number of carbon atoms in the chain. The double bond position in a FA also affects the level of antimicrobial activity. The exact mechanism of antimicrobial effect of FAs is not known, although it is assumed that they initiate pro cesses of peroxidation and inhibit FA synthesis in bac teria; otherwise, FAs may interact with membranes inhibiting cell respiration. In their review [25], Des bois and Smith provide an extensive list of FAs exhib iting various biological activities, including antibacte rial activity. Lipids of algae contain many of the FAs from the list. However, TLs of S. cichorioides were not always active and the rate of activity in different sea sons of collection differed considerably. For example, TLs of S. cichorioides samples collected in March, September, and October suppressed bacteria growth more actively (Table 1). We analyzed FA composition of these TLs to determine the FAs that influence TL activity (Table 2). Lipids of algae collected in March contained 18:1 (n9), 18:2 (n6), 18:4 (n3), 20:4 (n6), and 20:5 (n3) in considerable amounts. In September and October the levels of 16:1 (n7) and 18:1 (n9) were high. In March, polyunsaturated fatty acids (PUFAs) made 43% to the total FAs, and in September and October their fraction was approximately 3.5 times lower (Table 2). The share of monoene and saturated FAs was higher than in March. Antibacterial activity of TLs from samples of algae collected in May and June was somewhat lower (Table 1). In that time of the year, lev els of 18:1 (n9), 18:2 (n6), 18:4 (n3), 20:4 (n6), and 20:5 (n3) were high and the level of 16:1 (n7) was higher than in March. The share of PUFAs in total FAs was comparable to that in March. In July, as well as in March, May, and June, levels of the same FAs were high. However, TLs isolated from algae collected in July were inactive against S. aureus and demon RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

strated weak activity against E. coli (Table 1). TLs from algae collected in November were active only against S. aureus. The level of PUFAs in them was the highest of all studied months, and the level of saturated FAs was the lowest (Table 2). It should be noted that 16:0 acid was the dominant FA in the lipids from March to November with the level within the range 15.6 to 40.4%. Apparently, there is no distinct connection between the level of TL activity and the FA species. Probably, for manifestation of activity, the ratio between molec ular species of FAs and the presence of other compo nents in TLs are important. We compared the activity of TLs of mature and juvenile samples of algae (col lected in March). Composition of FA in lipids did not change with aging, but the ratio between certain FA content changed. For example, in young algae the content of 20:4 (n6) and 16:1 (n7) acids was higher, that is 17.2 and 4.9%, respectively, against 13.1 and 3.8% in mature algae. Content of 18:4 (n3) in young algae was lower, namely 6.8%, against 8.1% in mature algae. TLs from juvenile algae were more active against E. coli than against S. aureus and in mature algae, the other way round (Table 1). In the work, we also studied the effect of other components of TLs on their activity. Individual frac tions comprising TLs were isolated from algae col lected in March and November. Fractions isolated from TLs collected in March (Table 3), similar to TLs of S. cichorioides, were more noticeably active against bacteria than fractions of TLs from algae collected in November (Table 4). Fraction activity depended on fraction composition. The fraction comprising tria cylglycerols (TAGs), free sterols (fS), and Chl (chlo rophyll), the fraction of free S and Chl, and fractions of Chl and fucoxanthin were the most efficient ones against S. aureus. The fraction of glyceroglycolipids Vol. 39

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Table 2. Composition of fatty acids (% to total) of lipids in S. cichorioides Months of alga collection (March through November) Fatty acids 3*

5*

6*

7*

9*

10

11*

14:0 15:0 16:0 16:1 n7 16:2 n6 18:0 18:1 n9 18:1 n7 18:2 n6 18:3 n6 18:3 n3 18:4 n3 20:0 20:3

8.1 0.4 23.0 3.8 traces 1.2 17.9 1.1 9.4 – 2.1 8.1 traces 0.5

9.5 0.4 18.8 7.2 0.9 1.4 15.9 0.9 8.8 0.2 5.5 7.8 0.2 –

6.0 0.4 20.1 14.6 – 1.4 13.6 1.4 10.1 – 3.7 6.2 traces

⬘⬘

6.5 1.4 19.0 9.4 1.9 0.9 15.3 1.1 8.5 – 8.3 5.5 – –

12.9 1.9 40.4 14.2 0.9 2.1 16.8 – 6.1 – 2.5 – – –

9.7 1.1 37.3 7.8 0.6 3.0 26.0 2.0 6.6 – 2.0 0.9 1.1 –

3.0 0.2 15.6 4.0 0.5 2.0 15.9 1.3 8.6 0.6 5.3 5.5 1.3 1.5

20:4 n6 20:4 n3 20:5 n3

13.1 0.5 9.2

11.1 0.5 9.9

8.5 – 13.6

9.8 – 11.7

1.3 – 0.5

1.6 – 0.3

15.5 0.8 15.2

32.7 22.8 42.9 22.5 19.9

30.3 24.4 44.3 21.3 23.7

27.9 29.6 42.1 18.6 23.5

27.8 25.8 46.4 20.9 29.5

57.3 31.0 11.7 8.3 3.0

52.2 35.8 12.0 8.8 3.2

22.1 21.2 53.5 25.2 26.8

Σ saturated Σ monoene Σ polyene Σ PUFA (n–6) Σ PUFA (n–3)

*, sample contains nonidentified FAs. Data are presented as mean values calculated form triplicate analyses. Standard deviations typi cally were ± 0.2–1.0% and are not displayed in the table.

(GLs) exhibited low activity against bacteria (see Table 3). The composition of fractions isolated from TLs of algae collected in November was less diverse, and, similar to TLs; individual fractions of TLs were less active against bacteria (Table 4). Only fractions of fS, monogalactosyl diacylglycerols (MGDGs), and fucoxanthin exhibited measurable activity against S. aureus (Table 4). Comparison of fractions contain ing TAGs (Tables 3 and 4) evidences that increase in TAG content in a fraction results in a loss in the activ ity. Inclusion of fS increases their activity against S. aureus mainly. Fractions containing only fS are the most active against S. aureus. Chlorophylls did not exhibit any activity (Table 4), but fractions with Chls were active (Table 3). In the case of fucoxanthin, most probably its vary ing activity depended on the ratio between oxidized and nonoxidized forms of the pigment and purity of the fraction. Therefore, fraction activity depends on the composi tion and relative content of components, and the compo nents’ activity is subject to synergism and antagonism.

These effects are pronounced in TLs. The extent of the effects depends on the season of alga collection, since qualitative and quantitative compositions of the alga are differ from season to season [31, 33]. Glyceroglycolipids (GLs) produce an important fraction of algae and, as is demonstrated above, GL fractions isolated from TLs of S. cichorioides collected in March were active against the bacteria (Table 3). GL fraction contains MGDG, digalatosyl diacylglyc erol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). We studied the activity of each class of GLs. MGDG were the most active, but only against S. aureus. Neither of the classes was active against E. coli. These classes of GLs isolated from algae col lected in November behaved the same way. We studied FA composition of these classes (Table 5). In MGDG (March and November) we noted high level of PUFAs, and in DGDG and SQDG, high level of sat urated FAs. Apparently, domination of polyene FAs in MGDG leads to its higher activity against a gram pos itive bacteria S. aureus, but not against E. coli. High level of saturation of DGDG and SQDG, apparently,

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Table 3. Antimicrobial activity of lipids and PSPs of S. cichorioides collected in March Microorganism Fraction1

S. aureus

E. coli

C. albicans

A.niger

F. oxysporum

V. alginolyticus

– 3.8 ± 0.3 4.7 ± 0.3 2.0 ± 0 4.5 ± 0.2 3.0 ± 0 – – –

– 3.7 ± 0.2 – – 4.8 ± 0.2 3.0 ± 0 – – –

1.0 ± 0 – – – 1.0 ± 0 – – – –

12.0 ± 0.2

3.8 ± 0.2

6.0 ± 0

Lysis zone, mm from well edge TAG, free FA, Chl2 TAG, fS, Chl3 fS and Chl4 Fucoxanthin5 Chl GL MGDG DGDG SQDG Nitrofungin

2.8 ± 0.2 6.2 ± 0.3 5.7 ± 0.3 8.3 ± 0.3 8.0 ± 0 1.3 ± 0.3 3.0 ± 0 – – 14.0 ± 0

2.0 ± 0 1.7 ± 0.2 1.3 ± 0.2 2.3 ± 0.2 2.0 ± 0 1.8 ± 0.2 – – –

0.8 ± 0.2 0.8 ± 0.2 1.0 ± 0 1.0 ± 0 1.7 ± 0.2 – – – –

3.8 ± 0.2

6.0 ± 0

1, % content in fractions: 2, 85 : 5 : 10; 3, 10 : 45 : 45; 4, 70 : 30; 5, 90 : 10.

Table 4. Antimicrobial activity of lipids and PSPs of S. cichorioides collected in November Microorganism Fraction

S. aureus

E. coli

C. albicans

A. niger

F. oxysporum

V. alginolyticus

1.5 ± 0.2 4.7 ± 0.3 4.0 ± 0 6.0 ± 0 2.5 ± 0.2 – 1.0 ± 0 3.8 ± 0.2

– 2.0 ± 0 2.0 ± 0 1.7 ± 0 – – – 6.0 ± 0

Lysis zone, mm from well edge TAG fS Chl Fucoxanthin MGDG DGDG SQDG Nitrofungin

– 3.6 ± 0.3 – 1.5 ± 0 2.0 ± 0 – – 14.0 ± 0

– – – – – – – 3.8 ± 0.2

– – – – – – – 6.0 ± 0

influences activity of these classes making them inac tive. Most probably, when in a mixture, the effects of individual GLs are added making the combined frac tion active against both S. aureus and E. coli. FAs possess both antibacterial and antifungal activ ity [25]. We studied the activity of TLs against dimor phic yeast cells of C. albocans that cause many diseases in humans, from skin infections to deep mycoses. Sup pression of growth was moderately pronounced only in TLs of algae collected in September and October (Table 1). As it has been shown by Tibane and co workers [23], lipids isolated from marine organisms are rich in PUFAs and with the acids, such as 18:4 (n 3), 20:5 (n3), 22:5 (n3), and 22:6 (n3), they inhibit mitochondrial metabolism of C. albicans efficiently. TLs of S. cichorioides collected in September and October and exhibiting high activity against C. albicans contained a high share of 16:0, 16:1 (n7), and 18:1 (n9) (Table 2). The level of PUFAs was not high (12% total) and only 18:2 (n6) was represented RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

– – – – – – – 12.0 ± 0.2

measurably. We studied the activity of the fractions isolated from samples of algae collected in March (Table 3) and November (Table 4). TLs of March algae, as well as the purified fraction isolated thereof, exerted low activity (Table 3). Only in the GL fraction and individual GL classes, no activity against C. albi cans was observed. Also, TLs of November samples of algae and their individual components were not active against C. albicans (Table 4). In both March and November, TLs of algae contained high level of PUFAs (Table 2), while only the former ones were active. As in the case with bacteria, TLs exert activity through joined action of individual constituents. Con tent of individual substances is subject to seasonal vari ations. A. niger is an opportunistic pathogen, however under conditions of compromised immunity it adopts pathogenic properties. It is especially dangerous at the stage of fructification, when it forms black mold on walls in damp rooms. To destroy it, chemical agents, Vol. 39

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Table 5. Composition of fatty acids (% to total) of GLs in the S. cichorioides brown alga MGDG

DGDG

SQDG

Fatty acids 14:0 15:0 16:0 16:1 n7 16:1 n5 17:0 18:0 18:1 n9 18:1 n7 18:2 n6 18:3 n6 n3 18:4 n3 20:0 20:3 20:4 n6 20:4 n3 20:5 n3 Σ saturated Σ monoene Σ polyene

March

November

March

November

March

November

16.9 – 16.7 – 2.5 – 1.8 9.6 – 11.7 4.5 13.6 – – 6.6 9.2 6.9

6.5 – 21.4 7.1 – – 4.2 19.8 – 9.8 14.1 7.6 – – 2.6 – 6.9

– – 66.7 – – – 6.0 11.6 – 9.9 – – – 5.8 – – –

2.0 8.8 29.8 2.1 – 1.4 22.9 7.0 11.7 3.8 2.4 4.1 – – – – 4.0

3.5 0.6 44.4 1.2 3.5 – 3.1 28.4 0.8 9.4 – 1.4 1.5 – 0.8 – 1.4

1.3 – 48.3 7.9 – – 2.0 31.7 0.8 8.0 – – – – – – –

35.4 12.1 52.5

32.1 26.9 41.0

72.7 11.6 15.7

64.9 20.8 14.3

53.1 33.9 13.0

51.6 40.4 8.0

ultrasound, or ultraviolet irradiation are used typically. TLs of laminaria exert higher activity against A. niger, than against C. albicans (Table 1). Activity against A. niger was more pronounced in TLs from S. cichori oides collected in June, September, and October. TLs from alga samples collected in July and November showed no activity (Table 1). Individual fractions iso lated from alga samples collected in March demonstrated higher activity than that of TL fraction (Table 3). Frac tions containing fS and Chls, Chl, or GLs exhibited the most pronounced activity (Table 2). It should be noted that individual classes of GLs were not active. Fractions of lipids and PSPs of S. cichorioides col lected in November were not active against A. niger (Table 4). F. oxysporum is a pathogenic fungus causing dis eases of many cultured plants. TLs of algae collected in September, October, and March exerted high activ ity against the fungus (Table 1). Individual fractions of lipids—TAGs, fS, and Chls; Chls; the fraction of fucoxanthin; and GLs—isolated from laminaria col lected in March exhibited high activity (Table 3). Fractions of lipids and PSPs from algae collected in November also demonstrated a high level of activity against F. oxysporum (Table 4). TAGs exhibited activ ity against the fungus, however, within fractions (March) they were less active or their activity increased in combination with fS and Chl. MGDG

and SQDG were active against F. oxysporum, while MGDG, DGDG, and SQDG isolated from March algae were not active against the fungus (Table 3). In November, all classes of GLs contained 16:1 (n7), while in March it was absent from these classes of GLs or its level was very low (Table 5). In November, sam ples of MGDG, DGDG, and SQDG, monoene FA lev els were higher than in the March ones, while polyene FA levels decreased. Most likely, changes in the ratio between monoene and polyene FAs influence the activity of indi vidual classes of GLs against F. oxysporum. Only TLs of algae collected in September and October were active against a marine vibrion V. algi nolyticus pathogenic for human and fish. In other months, TLs did not exert any activity (Table 1). The fraction comprising TAGs, free FAs, and Chl, and the Chl fraction (collected in March) exerted weak activ ity against the pathogen (Table 3). Chlorophyll, fucox anthin, and fS of November algae exhibited activity against V. alginolyticus while TAGs and individual classes of GLs were not active (Table 4). Hemolytic activity of the extracted compounds of marine algae is connected with their toxic effects [34]. Many authors explain this activity by the presence of glyceroglycolipids [35–37]. Hemolytic activity of TLs also depended on the S. cichorioides collection season (Table 6). TLs of algae collected in March and November were the most hemolytic. They also exhib

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Table 6. Cytotoxic activity of TLs and individual compounds of S. cichorioides. All values are calculated as EF50 and expressed in μg/mL Months of alga collection/Substances

March May June September October November TAG, free FA, Chl2 TAG, fS, Chl3 fS and Chl4 Fucoxanthin Chl GL MGDG DGDG SQDG TAG fS MGDG DGDG SQDG Chlorophylls Fucoxanthin Saponin

Hemolytic activity pH 6.0 2.3 … 15.2 40.6 26.0 2.42 4.03 >100.0 >100.0 >100.0 >100.0 >100.0 93.7 >100.0 >100.0 10.8 9.8 5.8 2.3 5.8 10.3 12.8 7.5

pH 7.4 TLs 3.2 43.2 25.0 36.6 24.0 5.8 Substances (March): 7.7 >100.0 >100.0 >100.0 >100.0 >100.0 97.8 >100.0 >100.0 Substances (November): >100.0 19.6 14.3 45.3 47.3 45.8 18.8 40.0

ited relatively high cytotoxic activity against murine splenocytes. TLs of algae collected in May, June, and October induced lysis of erythrocytes at rather high concentrations (Table 6). Enhancement of hemolysis occurred upon pH change from 7.4 to 6.0. As it has been demonstrated in the review (Desbois and Smith, [25]), hemolytic activity is exerted by such FAs as 20:4 (n6), 20:5 (n6), and acids with 18 carbon atoms. TLs of S. cichorioides contained these FAs, however their relative content in TLs, similar to the relative content of different classes of lipids and pig ments, depended on season. Fractions isolated form TLs of algae collected in March did not exhibit hemolytic activity with the exception of a single fraction comprising TAGs, free FAs, and Chl, that induced erythrocyte lysis at rather low concentrations (Table 6). Higher activity was demonstrated by MGDG, DGDG, and SQDG, lower activity, by TAGs, fS, Chls, and fucoxanthins. Enhancement of hemolysis under the effect of these substances occurred upon pH decrease from 7.4 to 6.0. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

Cytotoxic activity

Embryotoxic activity

8.6 >100.0 >100.0 >100.0 63.0 15.8

75.3 72.8 36.0 20.3 68.4 …

>100.0 >100.0 >100.0 >100.0 >100.0 >100.0 >100.0 >100.0 >100.0

4.5 2.9 >100.0 4.3 3.9 >100.0 … … …

>100.0 32.0 >100.0 >100.0 >100.0 26.2 35.3 …

… … … … … … … …

Hemolytic activity of GL classes of S. cichorioides col lected in different seasons showed significant differ ences. One may not exclude that the ability of GLs to destroy the membrane is determined by monoene FAs, in particular, 16:1 (n7) acid. MGDG, DGDG, SQDG, and TAGs isolated form TLs of S. cichorioides (November) were not toxic against murine spleno cytes. Only pigments and fS exerted some toxicity (Table 6). All fractions isolated from March algae were not toxic against murine splenocytes. TLs and their fractions were not active toward Ehrlich ascyte carci noma. TLs of algae collected in June and September were most toxic against embryos of marine urchin Strongylocentrotus intermedius. The reasons for activity can hardly be explained only by FA composition, since they differed considerably during these months. TLs of March algae exerted weak embryotoxicity, and frac tions (TAGs, free S, and Chl), (TAGs, free FAs, and Chl), Chl, and fucoxanthin, on the contrary, exhibited high activity (2.9, 4.5, 3.9, and 4.3 µg/mL, respec tively). Vol. 39

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MATERIALS AND METHODS Saccharina cichorioides (Miyabe) C.E. Lane, C. Mayers, Druehl & G.W. Sauders (syn. Laminaria cichorioides (Miyabe)) algae were collected March through November, 2010, in Troitsa gulf of the Peter the Great bay, the Sea of Japan, on a marine experi mental station of the Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences (Primorskii krai, Khasan region) at a depth of 8 m. Each collection of algae contained 2–3 thal lomes. Extraction of TLs (comprising PSPs, which are extracted together with lipids) was performed as described previously [31]. The amount of TLs was determined gravimetrically. Fatty acid methyl esters (FAMEs) were obtained by reesterification of lipids according to Carreau and Dubacq [32]. FAMEs were analyzed by GLC as described previously [31]. Fractions of lipids and PSPs were separated on a 40–100 µm silica gel column (Chemapol, Czech Republic). To elute neutral lipids (NLs), hexane and a gradient of diethyl ether in hexane were used progres sively and acetone–ethanol 7 : 3 (by vol) mixture was used to elute GLs. Elution of the compounds from column was controlled by TLC in the presence of standard solutions of Spinacia oleracea lipids: triacyl glycerols (TAGs), free FAs, cholesterol, fucoxanthin, chlorophyll (Chl), and GLs. In the NL and GL frac tions eluted from the column, component content was calculated using GLC [31]. Free sterol (fS) content was determined via the Liebermann–Burchard test measuring absorption at 656 nm [31]. PSP content was determined as described previously [31]. Monogalac tosyl diacylglycerols (MGDGs), digalactosyl diacyl glycerols (DGDGs), sulfoquinovosyl diacylglycerols (SGQGs), fucoxanthin, Chl, TAGs, and fS were iso lated from fractions containing the compounds using additional silica gel (20–40 µm) columns. Antimicrobial activity was studied against the fol lowing microorganisms: Vibrio alginolyticus KMM 644, Staphylococcus aureus ATCC 21027, Escherichia coli ATCC 15034, Candida albicans KMM 453, Fusar ium oxysporum KMM 4639, and Aspergillus niger KMM 4634 from the collection of microorganisms of the Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences. Anti microbial activity was determined using the method of agar diffusion as described previously [2]. To culture V. alginolyticus, marine watercontaining medium (0.5% peptone, 0.02% K2HPO4, 0.005% MgSO4, 0.1% glucose, 0.1% yeast extract, 0.01% NaOH, 1.8% agar, and a mixture of marine water–distilled water, 1 : 1) was used; to culture bacteria a medium without marine water was used. C. albicans, F. oxysporum, and A. niger were cultured on a Saburo nutrition medium (3% glucose, 3% peptone, 1.8% agar, and distilled water). Nitrofungin® (IVAX, Czech Republic) (chlor nitrophenol), 1 mg/mL, was used as a positive control, and DMSO, as a negative control.

Hemolytic activity was determined at two pH val ues (6.0 and 7.4) using erythrocyte suspensions of white outbred mice according to a technique described earlier [2]. Saponin (Fluka, Germany) was used as control. To determine cytotoxicity, splenocytes and tumor cells of white outbred mice of both genders (18–20 g) were used as test subjects. Splenocytes were isolated from spleen homogenates and tumor cells, from ascitic fluid of animals, by triple centrifugation for 5 min at 450 µg in saline followed by resuspension in saline. The final concentration of cells in culture medium was (3–5) × 106 cells/mL. Ten microliters of preparation under study and 190 µL erythrocyte suspension were placed in wells of a 96well plate. Plates were shaken and incubated in thermostat at 37°C for 1 h. After incubation, a 10 µL cell suspension was mixed with 10 µL 0.4% trypan blue solution in balanced salt solution (DiaM, Russia) and transferred onto a cover slide. After 1–5 min the number of live cells was counted using an Imager A1 (Carl Zeiss, Germany) micro scope and an AxioVision (Carl Zeiss) software, and cytotoxic activity of the fractions (ratio of the number of dead cells over the number of live cells) was calcu lated. Then, effective dose was determined using the Statistica 6.0 software and expressed as ED50. To determine embryotoxic activity, germ cells of marine urchin Strongylocentrotus intermedius were used. Germ cells were washed three times with sterile marine water and fertilized. A suspension of fertilized oocytes was poured into 24well plates with tested compounds and incubated at 24°C for 3 h. The process of embryo development without the addition of tested compounds and with the maximum amount of DMSO (1%) was used as control. Embryotoxic activity was expressed through inhibitory concentration IC50. CONCLUSION Seasonal changes in biochemical composition and biological activity of a brown alga S. cichorioides were recorded. In S. cichorioides, we observed considerable differences in FA content in TLs and individual GLs and variations in individual lipid classes, as well as in PSP content, that are in connection to season and life cycle of the alga. Manifestation of antimicrobial activ ity of TLs and their fractions, apparently, is species specific and is determined by the structure of compo nents interacting with target microorganisms. Both the degree of FA saturation and FA ratio are important for the manifestation of lipid activity against bacteria. PSPs of S. cichorioides (March) demonstrated rather high antimicrobial activity while exhibiting no toxicity against animal cells. PSPs of S. cichorioides (Novem ber) did not possess pronounced antimicrobial activ ity; however, they induced erythrocyte lysis at rather low concentrations. Apparently, this seasonal rise of activity occurs due to changes in relative content of molecular species of chlorophylls or carotenoids in the

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fractions. Activity of individual classes of glycerogly colipids also depended on season of algae collection. Manifestation of antimicrobial and hemolytic activity of lipids and individual fractions and classes of lipids is of a complex nature. Probably, both relative content of TL components and FA distribution over classes of lipids, degree of saturation of FAs, and position of double bonds in FAs are important for activity mani festation. The data on the periods of accumulation of indi vidual species of FAs and PSPs in S. cichorioides are of industrial interest since many of them have high bio logical potential. REFERENCES 1. Gerasimenko, N.I., Chaikina, E.L., Busarova, N.G., and Anisimov, M.M., Appl. Biochem. Microbiol., 2010, vol. 46, pp. 426–430. 2. Anisimov, M.M., Martyyas, E.A., Chaikina, E.P., and Gerasimenko, N.I., Khim. Rastit. Syr’ya, 2010, no. 4, pp. 125–130. 3. da Matta, C.B.B., de Souza, E.T., de Queiroz, A.C., de Lira, D.P., de Araujo, M.V., CavalcanteSilva, L.H.A., de Miranda, G.E., de AraujoJunior, J.X., Barbosa Filho, J.M., de Oliveira Santos, B.V, and Alexandre Moreira, M.S., Mar. Drugs, 2011, vol. 9, pp. 307–318. 4. Folmer, F., Jaspars, M., Dicato, M., and Diederich, M., Phytochem. Rev., 2010, vol. 9, pp. 557–579. 5. Fenical, W. and Hugles, C.C., Chem.Eur. J., 2010, vol. 16, pp. 12512–12525. 6. El Gamal, A.A., Saudi Pharm. J., 2010, vol. 18, pp. 1–25. 7. Ben Aoun, Z., Ben Said, R., and Farhat, F., Bot. Mar., 2010, vol. 53, no. 3, pp. 259–264. 8. Tierney, M.S., Croft, A.K., and Hayes, M., Bot. Mar., 2010, vol. 53, no. 5, pp. 387–408. 9. Kamenarska, Z., Serkedjieva, J., Najdenski, H., Ste fanov, K., Tsvetkova, I., DimitrovaKonaklieva, S., and Popov, S., Bot. Mar., 2009, vol. 52, pp. 80–86. 10. Demirel, Z., YilmazKoz, F.F., KarabayYavasoglu, U.N., Ozdemir, G., and Sukatar, A., J. Serbian Chem. Soc., 2009, vol. 74, no. 6, pp. 619–628. 11. Manial, A., Sujith, S., Selvin, J., Shakir, C., and Kiran, G.S., Phyton—Int. J. Exp. Bot., 2009, vol. 76, pp. 161–166. 12. Lategan, C., Kellerman, T., Afolayan, A.F., Mann, M.G., Antunes, E.M., Smith, P.J., Bolton, J.J., and Beukes, D.R., Pharm. Biol., 2009, vol. 47, no. 5, pp. 408–413. 13. Ibtissam, C., Hassane, R., Jose, M.L., Francisco, D.S.J., Antonio, G.V.J., Hassan, B., and Mohamed, K., Afri can J. Biotech., 2009, vol. 8, no. 7, pp. 1258–1262. 14. Kandhasamy, M. and Arunachalam, K.D., African J. Biotech., 2008, vol. 7, no. 12, pp. 1958–1961. 15. Kuznetsova, T.A., Bull. Exp. Biol. Med., 2009, vol. 147, no. 1, pp. 66–69. 16. Lapicova, E.S., Drozd, N.N., Tolstenkov, A.S., Makarov, V.A., Zvyagintseva, T.N., Shevchenko, N.M., Bacunina, I.U., Besednova, N.N., and Kuznetsova, T.A., Bull. Exp. Biol. Med., 2008, vol. 146, no. 3, pp. 328– 333. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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