Structural characterization and Biological Activity of Sulfolipids from selected Marine Algae

ried from 1.25% (in L. papillose) to 11.82% (in D. fasciola) of the total lipid contents. However, no significant differences in sulfate content (0.13 – 0.21%) were observed among all these algae species. All SLs were characterized by high contents of palmitic acid (C 16:0), which ranged from 30.91% in G. cylindriea to 63.11% in T. atomatia. The main constitutes of algal sulfolipids were identified as sulfoquinovosyl-di-acylglycerol and sulfoquinovosyl acylglycerol. The sulfolipids of different algal species exhibited remarkable antiviral activity against herps simplex virus type 1 (HSV-1) with an IC50 ranging from 18.75 to 70. 2 μg mL . Moreover, algal sulfolipid inhibited the growth of the tumor cells of breast and liver human cancer cells with IC50 values ranging from 0.40 to 0.67 μg mL for human breast adenocarcinoma cells (MCF7).


INTRODUCTION
Glycolipids (GL), sulfolipids (SL) and phospholipids (PL) are usually present in all photosynthetic membranes in plants, algae and various bacteria. However, Sulfolipids (SLs) represent up to 29% of total lipid and have been regarded as predominant lipid components in both prokaryotic and eukaryotic organisms (Norman et al., 1996;Benning and Somerville, 1992). SLs are located in the thylakoid membrane as an important component to preserve the membrane's structure and function. Recently, lipid constituents have been recognized as potentially important factors in the processes of signal transduction and endomembrane transport (Simons and Toomre 2000). However, algae represent valuable sources of a wide spectrum of complex lipids, and their components have promising potential applications especially in the food, cosmetic, and pharmaceutical industries (Hossain et al., 2005). The various biological actions of SLs include: inhibitory effects on DNA polymerase and viral reverse transcriptase, antitumor, anti-inflammatory, and inhibition and promotion of cell growth Liptak et al., 2004;Maeda et al., 2008;Naumann, 2009). Several reports have indicated that SLs compounds isolated from

Structural characterization and Biological Activity of Sulfolipids from some Marine Algae
The sulfolipid classes (SLs) in the total lipids of five species of marine algae, two species of Rhodophyta (Laurencia popillose, Galaxoura cylindriea), one species of Chlorophyta (Ulva fasciata), and two species of Phaeophyta (Dilophys fasciola, Taonia atomaria) were separated and purified on DEAE-cellulose column chromatography. The SLs component was identified by IR, gas chromatography MS/MS and liquid chromatography MS/MS. The level of SLs contents va-

Determination of total sulfate of algal sulfolipids
The sulfate content of algal sulfolipids was determined using a sodium rhodizonate reagent, which in the presence of barium forms a red compound. The reduction of color intensity indicates the quantity of sulfate present in the sample. A standard calibration curve was prepared using sodium sulfate (Na 2 SO 4 ), scaled for 1.0 -12.0 µg sulfate (Terho and Hartiala, 1971).

Identification of algal sulfolipid fatty acids
The algal sulfolipids were subjected to direct transmethylation in 1.5% sulfuric acid:methanol at 95 °C for 2 h (Luddy et al., 1960). Fatty acid methyl esters were analyzed by gas chromatography (Perkin Elmer Autosystem XL) equipped with a flame ionization detector and fused silica capillary column (DB-5 (American) 60 m x 0.32 mm, i.d.) with a film thickness of 0.25/25µm. The column temperature was initially 150 °C and was then gradually increased at rate of 3 °C min -1 up to 250 °C . The injector and detector temperatures were 230 °C and 250 °C, respectively. Helium was used as the carrier gas (at 1mL min -1 ). The split ratio was 1/100. The fatty acids were identified by comparison between the retention times of the samples and those of methyl fatty acid standard mixtures ( Sigma, > 99% purity by GLC).

Identification of function group of marine algal sulfolipids using IR
The IR spectra of algal sulfolipid samples were recorded on a JASCO FT/IR 6100A spectrometer in the wave number range 4000-400 cm -1 , according to the KBr disk method.

LC-MS-MS analysis of algal sulfolipids
An aliquot of sulfolipid fraction was analyzed by LC-MS-MS (LCQ Advantage Max, Thermo Finnegan, USA) using a triple mass spectrometer operating in positive electro spray ionization (ESI). The heated capillary and voltage were maintained at 255 °C and 4.5 KeV, respectively. The full scans of mass spectra of the sulfolipids were carried out from m/z 500 to 2000 using 500 ms for the collection of ion in the trap. MS/MS was used to break down the most abundant [M+H] + ion from MS with depended collection induced dissociation (CID) (Pons et al., 2002).
The aim of the present work is to characterize the chemical structure and evaluate the biological activity of the sulfolipids of some marine algae collected from the Red and Mediterranean seas in Egypt.

Collection of marine algal samples
Samples of Laurencia popillose and Galaxoura cylindriea were collected from the Red Sea (Faied and Ein Al-Sokhna at Suez gulf canal). Ulva fasciata and Taonia atomaria were collected from the Mediterranean Sea at (Abu-Qir city) Alexandria Governmate and Dilophys fasciola from (Matroh city) Marsa Matrouh Government. All algal samples were washed several times with tap water and then left to air dry. Samples of 500 g were ground and stored in brown glass containers at room temperature for further analysis.

Identification of marine algae species
After preparation of herbarium specimens of the algae species, they were identified by Dr. Rauhaiya Abdul-Latif, Professor of Botany, Department of Botany, Faculty of Science, Al-Azhar University.

Extraction and determination of total lipid
The total lipids of marine algae (10 g) were extracted with 100 mL of the mixture: methanol: chloroform (2:1, v/v) (Roughan and Bratt, 1968). After filtration, the mixture was evaporated at 40 °C to a minimum volume (15 mL) and dried under N 2 . Then, the total lipid contents were determined by weight.

Antimicrobial activity of algal sulfolipids
The antimicrobial activities were determined by the conventional agar diffusion assay (Greenwood, 1983) using one gram positive (Bacillus subtilis NRRL B-94) one gram negative (Escherichia coli NRRL B-3703) bacteria, fungi (Aspergillus niger NRRL 313), and yeast (Candida albicans NRRL 477). The microbial growth inhibition zone was measured after incubation at 30 • C by the appearance of a clear, microbial free inhibition zone, beginning within 24 h for yeast, 24-48 h for bacteria and 72-96 h for fungus. The most active sulfolipid fractions were tested for their MIC according to Hammer et al., (1999). MIC was determined as the lowest concentration of sulfolipid fractions inhibiting the visible growth of each organism on the agar plate.

Statistical analysis
Data were statistically analyzed through the analysis of variance (ANOVA) and Duncan's test and the P> 0.01 probability level was applied (Gomes and Gomes, 1984)

Marine algal total lipid contents
The total lipid contents (TL) of marine algae ranged from 0.09 to 2.35% (Table 1). U. fasciata (2.55%) had the highest TL content followed by D. fasciola (1.11%), whereas the lowest level was found in G. cylindriea (0.09%) followed by T. atomaria (0.66%) and L. popillose (0.88%). However, the concentrations of TL in the tested algae species were within the ranges (1.0 -5.0%) reported in the literature for several algae species (Matanjun et al., 2009;Manivannan et al., 2008). For instance, the TL contents in 12 species of marine algae belong to 3 families from with a 0.25 µm film phase and fused CP-Sil5 CB Low bleed-MS capillary column (25 m x 0.32 mm). The temperature of the injector was 280 °C. Sulfolipids were analyzed using the following temperature program: 90 °C for 3 min then gradually increased at the ratio of 5 °C min -1 until 260 °C. The analyses were performed in the EI mode (ionization energy 70 eV; source temperature 150 °C) (Pons et al., 2002) 2.6. Biological evaluation of marine algal sulfolipids 2.6.1. Antiviral activity of algal sulfolipids Preparation of the algal extract for bioassay. A stock solution of algal SLs was freshly prepared by dissolving 100 mg of sulfolipid fraction in 10 mL of dimethyl sulfoxide (DMSO) in water (9:1, v/v) and kept at 4 °C until use; appropriate dilutions of the solution were used in each assay. All the tests were carried out in three independent assays, and the means were applied.
Antiviral screening of algal sulfolipids. Algal SLs were evaluated for antiviral activity against herps simplex virus type-1 (HSV-1). The virus was obtained from the Virology Laboratory, Water Pollution Research Dept., National Research Center (NRC), Egypt. The virus was propagated in viro cell cultures. The Inhibition % of the virus was calculated as plaque reduction as a result of being subjected to a given extract (Tebas et al., 1995).

Antitumor activity of algal sulfolipids
The potential antitumor activity of algal sulfolipids was tested using the method of Skehan et al. (1990). Human hepato cellular carcinoma cells (Hep G2) and breast adenocarcinoma cells (MCF-7) were plated on 96 multi-well plates for 24 h before treatment with the algal sulfolipid to allow for the attachment of cells to the well of the plate. Sulfolipids and antitumor reference drugs (Novantron) were added at serial concentrations to cell monolayers. After incubation for 48 h at 37 °C in an atmosphere of 5% CO 2 , the cytotoxicity was determined spectrophotometrically by measuring

Sulfate content of marine algal sulfolipids (SLs)
The sulfate content among the algae sulfolipids ranged between 0.13 and 0.21%. The highest sulfate content was found in D. fasciola (0.21%) followed by G. cylindriea (0.17%), T. atomaria (0.16%), L. papillose (0.16%) and U.fasciata (0.13%). These results agree with those obtained by Sanina et al. (2004) in which the sulfolipid contents of the total lipids in A. tobuchiensis (Rodophyta), L. japonica and S. pallidum (Phaeophyta), U. fenestrata (Chlorophyta) and Z. marina (Embriophyta) were 10.0%, 15.0%, 7.1%, 9.7% and 6.2%, respectively. In addition, these results are in accordance with those of Gerasimenko et al. (2010), who stated that the sulfolipid contents of juvenile and adult brown  fig. 4b). Furthermore, this structure was provided by the GC-MS, and LC-MS/ MS fragmentation pattern and was similar to that reported by Keusgen et al., (1997). The SQDG of the Chondria armata fraction was consistent with the sodiated molecular ion [M + Na] + at m/z 629, which have a mass difference corresponding to a likely loss of sulfonic acid (SO 3 H) and sodium salt (SO 3 Na), respectively. The product ion observed at m/z 345 appears to have originated by the loss of a fatty acyl side chain as corresponding acid (palmitic acid, C 16:0 ).

Antiviral activity
The antiviral activity of algal sulfolipids was evaluated according to the plaque reduction method and the results are summarized in Table 3. Algal sulfolipids showed high antiviral inhibitions against HSV-1, which ranged from 18.75 to 70.12 %. All algal sulfolipids had low virus inhibition at the concentration of 10.0 µg, while the highest inhibition HSV1 was observed at 20 µg mL -1 for D. fasciola (70.12%). Thus, the sulfolipid fraction had dose-dependant antiviral activity. Among several antiviral available, acyclovir was used as a positive control in this study. The inhibitory effect of algae SLs (IC 50 ranged from 15.0 to 25.0 µg mL -1 ) was shown to be quite a bit more potent than that of acyclovir (IC 50 5.5 µg mL -1 ). These results are in agreement with the results obtained by Chirasuwan et al., (2009) who found that sulphoquinovosyl-di-acylglycerol compounds extracted from Spriulina platensis have antiviral activity (HSV-1) in virus cells. Ohta et al., (1998) found that the new sulfolipid, 6-sulfo-alpha-D-quinovopyranosyl-1',2'diacylglycerol (SQDG) isolated from G. tenella (marine red alga) contains a potent inhibitor of eukaryotic DNA fatty acids except for SQDG from Chlorophyta (Z. marina), which contained the highest contents of C 18: 2n-6 and C 18 : 3n-3 acids (Khotimchenko, 2003). Hossain et al. (2005) found that the major fatty acids of SQDG from S. horneri (brown algae) were C 16:0 , C 18:1 , C 18:4 , C 20:1 , C 20:4 , and C 20:5

IR analysis
As illustrated in Figure 1, the infrared spectra of all algal sulfolipid fractions showed two characteristic absorption bands for sulpher-containing compounds. The first one appeared at 927cm -1 , indicating the presence of a strong dehydration of SO 3 and the second was at 771cm -1 , indicating a symmetrical C-O-S associated with a C-O-SO 3 group (Ermanno et al., 1994 andRanjaniv andSteven 1995). Regarding other absorption bands, there were large amounts of -OH stretching at 3400 cm -1 , symmetric CH 3 bending at 1380 cm -1 and C-H stretching at 2930 cm -1 .

Identification of marine algal sulfolipid compounds by GG-MS and LC-MS/MS
The proposed chemical structure of the active constituents of the algal sulfolipids was determined using GC-MS and LC-MS-MS. Several compounds of sulfolipids were separated from algae and two of them were identified by EI-MS and ESI/ MS fragmentations. The ESI/ MS and EI/ MS of the major component (compound 1) of sulfolipids consisted of molecular ion [M + H] + at m/z= 820 corresponding to the molecular formula of C 43 H 78 O 12 S ( Fig. 2 and  4a). The main fragmentations of compound 1 were the peak at m/z = 564.3 (Fig. 2) and was due to the loss of fatty acyl (plamitic acid C 16:0 , m/z 255.3). The peak at m/z = 329 was due to the loss of linoleic acid (C 18:2 ), which is characteristic for SQDG. The peak at m/z= 243 was due to the loss of glycerol (m/z= 87). The fragmentation of compound 1 may result in sulfoquinovosyl-di-acylglycerol (Figure 4a).
The LC/MS/MS data revealed that the minor second compound was consistent with the polymerases and HIV-reverse transcriptase type 1 (Gustafson et al. 1998). The SQDG fraction of S. hofmanii contains (C18:2/C16:0) and showed a higher antiviral activity against HCM-virus, with an IC 50 of 19.0 µg mL -1 (Naumann, 2009).

Antitumor activity
Antitumor activity against human breast carcinoma (MCF-7). The cytotoxic activities of five algal sulfolipid fractions were tested against MCF-7 and the results are illustrated in Table 4. All algal sulfolipid fractions showed high inhibition percentages (%) toward the MCF7 cell, which ranged from 66.24 to 94.19%. L. popillose presented the highest antitumoral activity against the MCF-7 at all concentrations followed by T. atomaria, G. cylindriea and U. fasciata. In addition,    (Table 5). These results are in agreement with those obtained by all algal sulfolipid fractions showed high potential activities with an IC 50 of 0.40-0.67 µg mL -1 against the MCF-7 cell (Table 5). In addition all algal sulfolipids have a significant antitumor activity compared to the reference antitumor drug novantron (IC 50 = 1.40 µg mL -1 ).
Antitumor activity against human hepato carcinoma (Hep G2). Table 4 illustrates that the  (Maeda et al., 2005). Sahara et al. (2002) and (Mizushina et al., 2003) found that the synthetic sulfoquinovosyl-mono-acylglycerols had a highly significant effect in suppressing the growth of solid tumors (human lung adenocarcinoma A-549 cells), and showed a potent inhibition of DNA polymerase and potent antineoplastic agents against the gastric cancer cell line (NUGC3).
In conclusion, the GC/MS and LC/Ms identified many compounds from extracted algal sulfolipids and two major compounds were identified by ESI/MS fragmentation. The ESI/MS of the major component of algal sulfolipids (SQDM) was a consistent molecular ion [M + H] + at m/z= 820 corresponding to the molecular formula of