Salt stress enhancement of antioxidant and antiviral efficiency of Spirulina platensis

Cultivation of Spirulina platensis under salt stress conditions (0.02 M as control), 0.04 and 0.08 M NaCl led to a remarkable alteration of algal metabolism as well as an enhancement or induction of biologically active compounds. Concerning algal growth, salt stress caused a decrease in dry weight, chlorophyll a content as well as certain xanthophylls (neoxanthin and violaxanthin) while β-carotene production was stimulated especially at higher salt concentrations. Biochemical analysis of salt stressed algal revealed that lipid content was slightly increased together with certain saturated and unsaturated fatty acids especially the polyunsaturated ones (γ-inolenic acid, omega 3 fatty acid). Electrophoretic analysis of soluble protein pointed out that certain high molecular weight protein bands were not detected comparing with the protein marker. Five new protein bands of molecular weights 190, 158, 113, 77 and 28 KDa were recorded, in addition to an increase in the intensity of 6 already existing bands. Phosphate buffer and water extracts of the algal exhibited antiviral activities against both Hepatitis-A-virus-type-MBB (HAV-MBB strain, RNA virus) and Herpes simplex-virus-type-1 (HSV-1, DNA virus). Water extracts were found to be more effective than phosphate buffer extracts in inducing antiviral activities (98%) especially against HSV-1 virus. The same water extract of the salt stressed algal demonstrated higher anticoagulating activity compared with those of heparin and the positive control measured by clotting time assay. Antioxidant activity of the algal successive extracts against 2, 2 diphenyl-1-picrylhydrazyl and 2,2'azino-bis (ethylbenzthiazoline-6sulfonic acid) radical methods revealed moderate antioxidant activity of the non-polar algal extracts (petroleum ether) which were doubled with increasing extract concentration. The lowest activity was recorded by the partially polar (ethyl acetate) algal extract of both concentrations at all salinity levels. While the polar extracts (ethanol and water) showed higher antioxidant activities which were doubled with increasing extract concentration. Ethanolic algal extract (100 μg/ ml at 0.08 M NaCl) exhibited the highest antioxidant activity compared with those of the synthetic antioxidant butylated hydroxy anisol as standard (85.0, 89.9 and 86.0, 91.8% respectively).


Introduction
Many cyanobacteria and microalgae were considered as a natural source of various biologically and pharmacologically active compounds with structurally complex molecules which are difficult or impossible to be produced by chemical synthesis (Smith and Doan, 1999).
Genus Spirulina has gained an importance and international demand for its high phytonutrients value and pigments which have applications in healthy foods, animal feed, therapeutics and diagnostics (Becker, 1994;Vonshak and Tomaselli, 2000). Spirulina has been used as food and nutritional supplements since long time (Dillon et al., 1995). It is generally a rich source of protein, vitamins, essential amino acids, minerals, essential fatty acids such as γ-linolenic acid and sulfolipid (Mendes et al., 2003). Moreover in addition to ω-3 and ω-6-poly unsaturated fatty acids,it has also phycocyanin and other phytochemicals . Some Spirulina species exhibit antibacterial (Ozdemir et al., 2004), antiplatelet (Hsiao et al., 2005), antihepatoxic (Mohan et al., 2006) and antiviral activities (Hernandez-Corona et al., 2002). Spirulina as many other cyanobacteria species have the potential to produce a large number of antimicrobial substances, so they are considered as suitable candidates for exploitation as biocontrol agents of plant pathogenic bacteria and fungi (Kulik, 1995).
Salinity represents one of the most important factors exerting stress injury on the growth and metabolism of plants.
Salt stress causes an imbalance of the cellular ions resulting in ion toxicity and osmotic stress, leading to retardation of growth either directly by salt or indirectly by oxidative stress induced by reactive oxygen species (ROS).
Salinity can cause significant accumulation of compatible solutes which acts as enzyme producers, stabilizing the structure of macromolecules and organelles (Dahlich et al., 1983). Salinity stress may alter the metabolic pathways of stressed organism(s) leading to either enhancement or induction of biologically active compounds.
The present work aimed to investigate the different biological activities of Spirulina platensis and the relations with its biochemical composition, pigments and different constituents which may vary with salt stress culture conditions.

Algal species and culture conditions
Spirulina platensis was obtained from the lab of phycology In Botany Department, Faculty of Science, Cairo University, Egypt. The alga was cultivated on liquid Zarrouk medium (Zarrouk 1966

Phytochemical analysis Extraction and determination of algal pigments
Determination of chlorophyll was carried out according to Holden (1965) method, where the fresh sample (0.5g) was grinded in a mortar with acetone in presence of calcium carbonate then filtered. The absorbance of extracts was measured at 663 and 645 nm in 1cm quartz cell against 80% aqueous acetone as blank.
Extraction and determination of water soluble pigments (phycobiliprotein): The water soluble phycobiliproteins pigments including allophycocyanin (APC), phycocyanin (PC) and C-phycoerytherine (C-PC) were extracted from algal cells (1g) with 10 ml phosphate buffer (0.05 M, pH 6.8) according to the method described by Bryantl,. 1979. The absorbance (A) of the extracts was recorded at the following wave lengths: 650nm; 620nm and 565nm.

Electrophoretic fractionation of soluble proteins
Polyacrylamide gel electrophoreses in the presence of Sodium Dodecyl Sulphate (SDS-PAGE) was used for determining the molecular weight of protein fractions (Water soluble protein) according to method of Laemmli (1970). Standard molecular weight proteins marker was obtained from Sigma, this marker content proteins at varied molecular weight: 119 (β-galactosidase), 98 (Bovin serum albumin), 52 (Ovalbumin), 36 (Carbonic anhydrase) and 30 (Soybean trypsin inhibitor) kDa. Extraction and determination of total lipids: Lipids were extracted by the modified method described by Xu et al. (1998).

Separation and identification of fatty acids
Fatty acids methyl esters (FAME) were analyzed by gas liquid chromatography (GLC) according to Farag et al. (1986) under specific conditions of column. The separated fatty acids were identified by comparing their retention times with those of standard fatty acid methyl ester (purity 99% by GLC, sigma Co.). Also, Co-chromatography and GC/MS methods were used for verification of the peaks identity and position of double bond in fatty acid molecules.

Separation and identification of unsaponified matter
The unsaponifiable compounds were identified by GLC using an instrument equipped with a flame ionization detector (FID). The unsaponifiable compounds (hydrocarbon and sterols) were identified by comparing their retention times with those of standard hydrocarbons from C8 to C36 and some authentic sterols (Cholesterol, stigmasterol and βsitosterol).
Biological Activities Antiviral activity of algal extracts Preparation of samples for antiviral bioassay Extracts were dissolved as 100mg each in 1ml of 0.1 M phosphate buffer (pH 7). The final concentration was 100 μg/μl (Stock solution)

Viruses used
Herpes simplex virus type 1 (HSV-1) and Hepatitis A virus (cell culture adapted strain MBB). The two viruses were obtained from virology laboratory, NRC. Viruses were propagated and titrated on Vero cell (HSV-1) and HepG2 for HAV-MBB strain.

Cytotoxicity assay
Double fold dilution of each sample was prepared in deionized water (1:2 to 1:256) and diluted samples were inoculated in 96 well. Twice culture plate containg confluent monolayer of Vero cell and another plate containing HepG2. Cells were incubated at 37 ºC overnight and examined microscopically for cytopathic effect (CPE). The lower dilutions, which showed no morphological changes on cell cultures, were selected for antivirus bioassay (Abad et al., 2000).

Plaque infectivity reduction assay
The method described by Silva et al. (1997) was used where, a 6-well plate was cultivated with Vero cell (HSV) and another plate containing HepG2 (HAV) culture (10 5 cell/ml) and incubated for 2 days at 37 0 C. virus was diluted to give 10 7 PFU/ml as final concentrations and mixed with the algal extract at the previous concentration and incubated overnight at 4 0 C. Growth medium was removed from the multiwell plate and virus-compound mixture was inoculated (100µl/well). After contact time, inocula were aspirated, agarose were overlaid, and plates were left to solidify and incubated until the development of virus plaques. Cell sheets were fixed in 10% formalin and stained with crystal violet stain. Virus plaques were counted and the percentage of reduction was calculated.

Mode of action
The inhibition mechanism of virus by crude algal extracts was studied using two methods: Viral replication according to Amoros et al. (1994) and Viral adsorption according to Zhang et al. (1995).

Determination of anticoagulation activity:
The anticoagulating activity of water algal extracts were investigated using the method of USA pharmacopia (1985) as follow: Each tubes, 0.8 ml of extract solution (1 %), 0.8 ml of standard heparin sodium solution (0.5 U.S.P unit/0.8 ml), or 0.8 ml saline solution was added. Then, 1 ml plasma and 0.2 ml of calcium chloride solution (1%) were added in each tube. The tubes were stopped immediately, and inverted three times in such a way to mixed the contents that the entire inner surface of the tube became wet. The time required for clotting was determined.

Determination of Antioxidant activity: a-DPPH method
The 2, 2 diphenyl-1-picrylhydrazyl (DPPH) test was carried out as described by Burits and Bucar (2000). One ml of Spirulina extract (Hexane, chloroform, ethyl acetate, ethanol and water extracts) at different concentration was mixed with 1ml DPPH reagent (0.002% (w/v) /methanol water solution). After an incubation period, the absorbance was measured at 517nm. BHA was used as positive control and extracts concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentages against extract concentration. %Antioxidant activity =Ac-At / Ac x 100 where: At was the absorbance of the algal extract samples and Ac the absorbance of methanolic DPPH solution.

b-ABTS method
This assay was based on the ability of different substances to scavenge 2,2'-azino-bis (ethylbenzthiazoline-6-sulfonic acid (ABTS.+) radical cation in comparison to the standard BHA (50 and 100 μg/ml). According to the method of Re et al. (1999), the antioxidant activity of the tested samples was calculated by determined the decrease in absorbance at different concentrations (50 and 100 μg/ml) by using the following equation: Antioxidant activity= ((Ac-At)/ Ac) x 100, where: At and Ac are the respective absorbances of tested samples and ABTS .+ .

Statistical analysis
Data were subjected to an analysis of variance, and the means were compared using the "Least Significant Difference (LSD)" test at the 0.05 and 0.01 levels, as recommended by Snedecor and Cochran (1982).

Results and Discussion
Concerning the biochemical analysis of the salt stressed S. platensis, the algal growth was slightly affected by low salt concentration (0.02M NaCl), increasing salinity (0.04 and 0.08 M) led to a marked and progressive inhibition of growth translated as a decrease in algal dry weight (Fig. 1). Our results were in accordance with the results recorded by Fodorpataki  The inhibition of growth under salt stress conditions was certainly due to alteration of algal metabolism which might be directed towards the production of substances which have a role in algal salt tolerance or defense mechanism. Chlorophyll content of S. platensis (Table 1) was affected by salt stress conditions where chlorophyll a, neoxanthin, violaxanthin were recorded at low salinity and faintly detected at higher once (using TLC) while the reverse was shown by β-Carotene. The water soluble phycobiliprotein (Table 2) composed of phycocyanin (CPC), phycoerythrin (PE) and allophycocyanin (APC). Increasing salt conc. enhanced the production of both phycocyanin and phycoerythrin while allophycocyanin (APC) production was inhibited leading to a marked decrease in total phycobiliprotein content. Our results went parallel with those of many investigators (Cifferi 1983, Piorreck et al., 1984, Becker 1994and Rogel-Yogui et al., 2004 where different salt and nitrogen concentrations induced changes of chlorophyll and phycobiliprotein pigment contents of Spirulina species. It seems that under severe salt stress, the algal defense mechanisms do not allowed to spend too much energy for the synthesis of many new chlorophyll molecules and binding proteins which may explain the decrease in chlorophyll and allophycocyanin contents in our results (Fodorpataki and Bartha .,2004). Moreover, these were confirmed by the absence of 47 kDa chlorophyll protein and 94 kDa protein linking phycobilisomes to thylakoid from Spirulina platensis SDS-electropheritic analysis (Garnia et al., 1994;Fodorpataki and Bartha, 2004).
Salt stress conditions not only affected algal growth, pigment content but also protein and lipid production of the stressed alga. Analysis of soluble proteins (by SDS electrophorsis) of S. platensis cultivated under different salt concentrations and recorded in Table (3) and Fig. (2), revealed that, no protein bands of high molecular weight , were recorded at the highest NaCl conc. used (0.08 M). While two new highly intensive protein bands of molecular wts, 113, 77 were recorded only at higher NaCl conc. Also certain bands were present at low and moderate salt conc. (0.02 and 0.04 M) but absent (not detected) at higher ones (0.08 M). Moreover six protein bands were detected at low and/or moderate salt conc. but their intensities were highly increased at higher salt stress conditions (of M.wts 106, 90, 82, 67, 35 and 30). Absence of either new protein bands or an increase in the intensity of 42 and 37 KDa bands confirmed the obtained results concerning the decrease in total phycobiliprotein pigments under salt stress conditions. The obtained results concerning protein analysis of salt stressed S. platensis was comparable to those of S. maxima cultivated under nitrogen stress condition (Shalaby 2004). Both Spirulina species have two specific new protein bands of molecular weight 113 and 76 in addition to a highly intensive band at M.wt 103. Higher numbers of new protein bands were recorded in S. maxima at different nitrogen conc. and not equivalent to similar bands (of the same M.wt) produced by S. platensis under salinity stress conditions. These differences may be due to variable metabolic processes in both species and to the availability of nitrogen (essential for protein synthesis) in study of S. maxima and present only as normal medium constituent in experiments of S. platensis.    Generally, exposure of microalgae to any deleterious environmental change responds in many different ways, one of which is the modification of lipid composition in order to maintain the critical degree of membrane fluidity (Romano et al., 2000). The relative percentage of fatty acids in stressed S. platensis lipids illustrated in Table (  Relative percentage of hydrocarbons was also affected by salinity stress where C12, C15 and C36 were not detected at all NaCl conc, while C18 was only produced with moderate percentage at 0.04 M then markedly decreased at higher salinity level. C20 was only recorded at lower salt conc. on the other hand C21, C22, C26, C28, C29 and C30 were present at all salinity levels but with different relative percentages, C21 was highly increased at moderate salinity conc. (53.7%) then dropped to 2.5% at high salt conc., while C22 markedly increased at higher NaCl conc. (28.3%). Also C29 its relative % at low NaCl conc. (22.3%) was doubled at higher salt stress condition to reach 43.9%. C32 was markedly decreased with increasing NaCl concentration from 0.02 to 0.04 (18, 1.2%) then completely disappeared at higher salinity level. The obtained biochemical analysis of S. platensis encouraged the investigation of various biological activities.
Concerning the antiviral activity, Table (5) showed that algal water extract (50 µg/ml) of low salt concentration exhibited (0.02 M) relatively higher (60.0%) antihepatitis A virus-type MBB more than phosphate buffer extract (9.0%) of the same concentration and the activity of the latter extract increased (56.0-58.0%) at moderate salt concentration (0.04 M) using 20 and 50 µg/ml extract concentration respectively. On the other hand, the antiviral activity against herpes simplex virus -type 1 showed a comparable activity by both water and phosphate buffer extracts of both concentrations at all salinity levels with maximum antiviral activity (98.0%) at 50 ug/ml extract concentration.  Each value is presented as mean of triplet treatments, means within each row with different letters (a-c) differ significantly at P # 0.05 according to Duncan's multiple range test,

HAV-MBB virus (RNA virus) HSV-1 virus (DNA virus
The antiviral activity against HSV-1 (DNA virus) was markedly pronounced (98.0%) than that against HAV-MBB (60.0%) which is an RNA virus. These activities which were shown to be controlled by both type and concentration of algal extract (water or phosphate buffer, at 20 and 50 µg/ml) may be induced by the sulphated polysaccharide and tannins in S. platensis extracts (Witvrouw and De clereq 1997). These antivirus substances may interfere at one or more of viral stages, either at the stage of virus attachment or penetration to the host cell, or at the virus replication or the virus maturity and release stages. The obtained results concerning these activities against HAV-MBB and HSV-1 viruses were in agreement with the data obtained by Hayashi et al. (1996) who found that water extract of S.platensis inhibited the replication in vitro of herpes simplex virus type 1 in Hela cell within the concentration range 80-50 ug/ml. our results were also in accordance with those reported by Witvrouw and De clercq 1997, who emphasized that sulphated polysaccharides were found to be potent and selective inhibitors of HIV-1 replication in cell culture. Moreover, Ayehunine et al. (1998) reported that an aqueous extract of S. platensis inhibited HIV-1 replication in Human T-cell lines and langerhans cells and their antiviral activity was found in polysaccharides fraction. Our results were confirmed by and coincided with the results reported by Shalaby (2004), where phosphate buffer and water extracts of S. maxima cultivated under different N-Conc. Exhibited weak antiviral activity against HAV-MBB and highly pronounced activity against HSV-1 viruses. He added that the sulphated polysaccharides produced from fractionation of water extract (called Ca-Spirulina) caused the inhibition of virus penetration into the host cells.
Experiments carried out with dextran sulphate revealed that the antiviral activity increased with increasing molecular weight and degree of sulfation of the sulphated polysaccharides. Many microalgal polysaccharides significantly inhibit the infection of Vero cell by HSV-1, HSV-2 and VZV viruses, and these compounds did not show any cytotoxic effect even at greater dose concentration (Huleih et al., 2001).
The activity of polar extract of Spirulina platensis (at concentration 20 and 50 μg/ml) against HSV-1 and the clinical strain were evaluated by the plaque reduction assay. the algal extract did not induce any effect on virus replication The effect of algal extract on virus adsorption In the second set of experiment, the inhibitory effect of extract of Spirulina sp (at concentration 20 and 50 μg/ml) on virus adsorption to host cell was measured by monitoring the attachment of infectious HSV virions on to host cells in the presence of their extract. The results indicated that algal extracts completely inhibited (99.99%) the cell-associated infectivity. as compared with that in control levels. These results were agreement with these obtained by Boyed et al. (1997) who found that the antiviral activity of cyanovirin-N (CV-N) isolated from Nostoc sp against HIV-2 is due, at least in part, to unique, highaffinity interaction of CV-N with the viral surface envelope glycoprotein gp120.
The mode of action of S. platensis extracts (at concentration 20 and 50 μg/ml) against both viruses were evaluated by the plaque reduction assay. The results reported that the microalgae extract did not induce any effect on virus replication algal extracts completely inhibited (100%) the cellassociated infectivity as compared with that in control levels.
Regarding the anticoagulation activity of the hot water extract of salt stressed S. platensis, the obtained results (Fig.3) showed that great anticoagulating efficiency (expressed by clotting time assay) compared with that of the standard anticoagulatant heparin (sulfate glucouronic acid) to be 13, 17 min respectively. our results were in agreement with those recorded by Shalaby (2004) on S.maxima where the clotting times were inversely proportional to nitrate concentration in the growth media (11,12,13 at 410, 205, 102.5 ppm N compared with 16 min in case of heparin). This activity was reported to have a close relation with the water extract containing sulfated polysaccharides and phenolic compounds and depend upon the molecular size, type of sugar and sulphate content and position of the active components (Shanmugam and Mody 2000). Therefore, in future algal sulphated polysaccharides water extracts can be used as anticoagulant/antithrombitic agent, in medical purposes, replacing the known heparin which was extracted from internal organs of higher animals and exhibited haemorrhagic like side effects.
Extracts of S. platensis by organic solvents of different polarities and concentrations showed that the polar solvents (ethanol and water) extracts at higher concentration (100 ug/ml) exhibited higher antioxidant activity (85.0, 89.9% by DPPH and ABTS respectively) comparable to the standard antioxidant, BHA (86.5 and 91.8 %). This is followed in the second order by the non-polar (pet. ether) extract at high concentration (100 µg/ml) of both low and moderate salinity levels (55.0, 62.3% and 60.4, 66.8% by DPPH and ABTS respectively) while the partially polar ethyl acetate extracts demonstrated the lowest antioxidant activities at all salt concentrations (ranged from 0.0 to 23.6%) as recorded in Tables 6 and7.
The obtained results revealed that polar antioxidant substances might be present in the polar Spirulina extract to which attributed the antioxidant activity. These polar substances were found in extracts of different red, brown and green seaweeds   2.825 Each value is presented as mean of triplet treatments, means within each row with different letters (a-p) differ significantly at P # 0.05 according to Duncan's multiple range test.