Antiviral, cytotoxic, antioxidant and anticholinesterase activities of polysaccharides isolated from microalgae Spirulina platensis, Scenedesmus obliquus, and Dunaliella salina

Quantitative estimation of vegetative and stress forms of Spirulina platensis and Scenedesmus obliquus, as well as a vegetative form of Dunaliella salina, revealed that S. obliquus constituted the highest polysaccharide content than other tested microalgae. The isolated polysaccharides characterized as heterogeneous polysaccharides bounded protein by FT-IR, GLC, and Elemental Microanalysis. These polysaccharides constituted of 47-66% of sugar and 14.88-41.06% of protein contents whereas galactose, mannose, glucose, and rhamnose were represented as predominant sugar in isolated polysaccharides. The isolated polysaccharides were evaluated in vitro as antiviral, cytotoxic, antioxidant and anti-cholinesterase properties. The non-toxic dose of isolated polysaccharides on Huh 7.5, MA104, BGM, and Vero cell lines were determined. The S. platensis (CEM and HEM) polysaccharides have promising antiviral, which reduced replication up to 50 – 87.6% of HCV genotype 4a replicon, coxsackievirus B4, rotavirus and herpes simplex type 1 virus at nontoxic doses 1.8 and 1.5 mg/ml, respectively. Furthermore, the isolated polysaccharides were assessed for in-vitro cytotoxic activity against MCF-7, HepG2, and HCT116 cell lines. The cytotoxic activity revealed that D. salina HEM polysaccharide show potent cytotoxic activity against HCT 116 cell line with IC50 64.2 μg/mL. Additionally, the isolated polysaccharides showed DPPH• scavenging activity in a dose-dependent relationship and D. salina HEM and S. obliquus CEM showed the significantly highest activity (308.16 and 308.69%, respectively) at 100 μg/mL. Furthermore, S. obliquus CEM and HEM polysaccharides exhibited the significant highest cholinesterase % inhibition activity. Microalgal polysaccharides have great therapeutically potential in drug development used as antiviral, antitumor, antioxidant and anticholinesterase agents in near future.


INTRODUCTION
Microalgae are still paid attention as a valuable source of various bioproducts in spite of their required for growth only inorganic compounds and light as energy sources [1]. Among these bioproducts were polysaccharides which were diverse, abundant and exhibited numerous biological properties as well as they had great potential applications for pharmaceutical and medicine industries [1]. Microalgae are easy to grow and cultivate economically and enable the production of polysaccharides without depending on the climate or season [1]. The interest in microalgal polysaccharides is growing increasingly; especially they possess several biological applications with various health benefits such as antiviral agents, antioxidants, anti-inflammatory, immunomodulatory and lubricants for bone joints [2]. Intracellular polysaccharides and exopolysaccharides were isolated from different Spirulina species, which showed broad-spectrum antiviral activity [2]. S. platensis produced a polysaccharide known as calcium spirulan that exhibited antiviral and anticoagulant effects and prevented pulmonary metastasis in addition treated spinal cord injuries [3,4]. In addition, a water-soluble polysaccharide isolated from S. platensis reduced Hepatitis C replicon to 50% and displayed antioxidant activity as well as cytotoxic activity against hepatocarcinoma [5]. Ishaq et al. (2016) reported that water-soluble polysaccharide from S. obliquus had oxidative stability [6]. The crude polysaccharide isolated from D. salina containing glucose, galactose, xylose, mannose, and rhamnose [7]. Furthermore, Dai et al., (2010) identified acidic heteropolysaccharide, glucans, sulfated polysaccharides and polysaccharide linked with nucleic acids by covalent bonds in fractions yielded from crude polysaccharide isolated D. salina [8]. Whereas, Zhang et al. 2009 found that sulfated polysaccharides fraction inhibit influenza virus FM1 and strengthen immune function more than de-sulfated polysaccharides [9]. On the other hand, extracellular polysaccharides isolated from D.
salina possess cytotoxic and immunomodulatory activities [10]. In addition, polysaccharides derived from Spirulina platensis, showed protective effects against neuronal damage [11]. Considering the importance of inflammation and oxidative stress in Parkinson's disease (PD), Spirulina had a neuroprotective potential recommending its use as an alternative treatment for Parkinson's disease [12].
Therefore, our study aims to investigate the chemical characterization of the polysaccharides isolated from microalgae Spirulina platensis, Scenedesmus obliquus, and Dunaliella salina and evaluates their antiviral activity against (HCV, Rotavirus, Herpes simplex & Coxsackievirus) as well as cytotoxic activity on liver, breast and colon cell lines. In addition, DPPH • scavenging efficacy and cholinesterase property of isolated polysaccharides were evaluated.

Algae material
Algae strains were obtained from algal biotechnology unit, National Research Centre. The used strains were Spirulina platensis belonging to Cyanophyta, Scenedesmus obliquus and Dunaliella salina belonging to Chlorophyta. All of them were originally varied due to their salinity margin. Spirulina platensis was laboratory grown using Zarrouk medium [13] while Scenedesmus obliquus was laboratory grown using BG-II medium [14] and artificial seawater medium was used for growing Dunaliella salina [15]. Stress growth for each alga was achieved basically by increasing salinity concentration to 2.0% sodium chloride, 45 mM organic carbon as sodium acetate and 125 ppm iron as ferrous sulfate [16]. Vegetative and stressgrowth was performed within a 200-L vertical sheet photo bioreactor [17]. Growth conditions were varied based on the growth site (in and outdoor). Indoor cultivation was performed as early described by El-Sayed (2007) and El-Sayed et al., (2015) [18,17]. Fully transparent plexicolumns containing 2L growth medium for each alga separately were used. The light was provided from one side light bank (6x40 watt white cool lamps), free oil, compressed air supported aeration and turbulence from the lower end of the growth column. When growth reached the maximum, the obtained biomass was used for growth scaling up until the desired volume. Outdoor growth was performed as described by El-Sayed et al, (2001) [19]. The microalgae have been harvested by settling and centrifugation at 3000 g in room temperature (Runne Heidelberg RSV-20, Germany) and were dried in an oven (Heraeus, Germany) at 45 °C overnight then ground to a fine powder [18]. Purification of the obtained biomass was performed by a series of precipitation of the microalgae and washings using distilled tape water and a cooling centrifuge.

Extraction and purification of watersoluble polysaccharide
The dried powder of three microalgae S. platensis, S. obliquus, and D. salina was subjected to cold and hot water extraction methods after defatting using petroleum ether 40-60 °C and chloroform. From defatted dried powder, the isolation of polysaccharides was performed as described in Matloub et al, 2015 [20] and then kept in the refrigerator for chemical and biological evaluations.

Chemical characterization
The phenol-sulfuric method was used for quantification of total polysaccharide and sugar content in dried algal samples and isolated polysaccharides, respectively [21]. The content of carbon, hydrogen, nitrogen, and sulfur was determined in the isolated polysaccharides and fractions by Elemental Microanalysis (Elementary Vario EL) [22]. Protein content and the degree of substitution (DS) were calculated as mentioned in Matloub et al, 2015 [20]. IR spectra (using KBr pellets) ranging between 400 and 4000 cm -1 were recorded with an FT/IR-6100 (JASCO, Japan). The polysaccharide extracts were hydrolyzed with 4N hydrochloric acid and the hydrolysate was analyzed by GLC analyses (HP 6890, USA), after derivatization using the trimethylsilylation reagent (Merk), under the following condition: ZB-1701 capillary column, 30 m in length, 0.25mm i.d; 0.25 μm film thickness, carrier gas, helium at a flow rate at 1.2 ml/min, temperature programmed 150-200 °C at a rate of 7 °C/min, flame ionization detector. Sugar identification was done by comparison with reference sugars (arabinose, fructose, fucose, glucose, galactose, mannose, mannitol, rhamnose, ribose, sorbitol, and xylose).

Culture cells for in vitro antiviral
Human hepatocyte (Huh 7.5), MA104, BGM and Vero cell lines (obtained from the Holding Company for Biological Products & Vaccines VACSERA, Egypt) were used for growth HCV genotype 4a[ED-43/SG-Feo (VYG) replicon], rotavirus Wa, coxsackievirus B4, and HSV1, respectively. They were cultured using specific growth media Dulbecco΄s Modified Eagle Medium (DMEM) and will be kept in a CO 2 incubator. The cells were seeded in 96-well tissue culture plates (Greiner Bio-One, Germany) and incubated at 37 °C in a humidified atmosphere of 5% (v/v) CO 2 . After 24 h incubation, the medium was discarded from confluent cells monolayers and replenished with 100 µL of bi-fold dilutions of different samples tested prepared in DMEM (GIBCO BRL which stated that 0.1mM solution of 1, 1-diphenyl-2picryl-hydrazyl (DPPH . ) was prepared in 100 ml absolute methanol and 1 mL of this solution was added to 1 mL of each polysaccharide sample and ascorbic acid (reference drug) at three concentrations (1, 10 and 100 μg/mL). Discoloration was measured at 517 nm after incubation for 30 min. Measurements were taken at least in triplicate. The scavenging ability of DPPH • was calculated using the following equation: Scavenging effect (%) = 0− 1 0×100 Where A0 is the absorbance of DPPH • solution (without the tested polysaccharides) and A1 is the absorbance of the tested polysaccharides with DPPH • solution.

Assay of acetyl cholinesterase (AChE) enzyme activity by the spectrophotometric method
AChE activity was measured by using spectrophotometer based on Ellman's method [29]. The enzyme hydrolyzes the substrate acetylthiocholine resulting in the product thiocholine which reacted with Ellman's reagent (DTNB) to produce 2-nitrobenzoate-5mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 412 nm. In test tube 1710 μL of 50 mM Tris-HCl buffer pH 8.0 and 250 μL of polysaccharide samples of tested microalgae and standard drug at three concentrations of 1, 10 and 100 μg/ mL, 10 μL 6.67 UmL-1 AChE and 20 μL of 10 mM of DTNB (5,5'-dithio-bis[2-nitrobenzoic acid]) in buffer were added. Positive control namely galanthamine was prepared in serial concentration as same as tested samples by dissolving in 50 mM Tris-HCl buffer pH 8.0. The mixture was incubated for 15 min at 37 ºC.
Then, 10 μL of acetylthiocholine iodide (200 mM) in buffer was added to the mixture and the absorbance was measured at 412 nm every 10 sec for 3 min. For a blank, the buffer instead of enzyme solution was used. The enzyme inhibition (%) was calculated from the rate of absorbance change with time (V= Abs/Δt) according to calculation as follows: Inhibition (%) = 100 -Change of sample absorbance X 100 change of blank absorbance The experiment was done in triplicate for each concentration of the tested samples that inhibit the hydrolysis of the substrate (acetylcholine). The percent of acetylcholinesterase inhibition was calculated as follows: % Inhibition = 100 − [Absorbance of the test polysaccharides/Absorbance of the control] × 100.

Statistical analysis
Data of cytotoxic activity were analyzed by one way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) program, version 14 (IBM software, NY, USA). The difference was considered significant where P<0.05. In addition, a probit analysis was carried for IC 50 and IC 90 determination using SPSS 11 program. While statistical analysis for antioxidant and anticholinestrase is carried out using two ways ANOVA coupled with CO-state computer program.

Chemical characterization of isolated polysaccharides from Spirulina platensis, Scenedesmus obliquus, and Dunaliella salina:
The phenol-sulfuric estimation of the carbohydrate content of the vegetative and stress forms of S. platensis and S. obliquus as well as the vegetative form of D. salina revealed that total carbohydrate of the vegetative forms of S. The IR spectrum data of vegetative and stress forms of S. platensis, S. obliquus, and D salina were illustrated in Figs. (1-3) Table  (2). Eight monosaccharides were detected in CEM and HEM of S. platensis and S. obliquus, as well as ten and seven monosaccharides, were identified in CEM & HEM of D. salina, respectively.     The integration of GC, FT-IR and microelement analysis revealed that the isolated polysaccharides from tested microalgae were heterogeneous and bounded with protein.

Antiviral activities
The determination of nontoxic dose of the isolated polysaccharide of stress form of S. platensis and S. obliquus as well as vegetative form of D. salina against Huh 7.5, MA104, BGM and Vero cell lines showed the same toxicity for each isolated polysaccharide and their nontoxic concentration was ranged from 1.1 to 1.8 mg/mL ( Table 3). The antiviral activity of the isolated polysaccharide against HCV, rotavirus, coxsakievirus and HSV1 was compiled in Table  (

Cytotoxic activity
Assessment of cytotoxic activity of the CEM and HEM of stress form of S. platensis and S. obliquus as well as a vegetative form of D. salina in vitro on HepG2, MCF7 and HCT116 human cell lines comparing with doxorubicin as reference drug was illustrated in Figs. 4-6. The cytotoxic activity revealed that the polysaccharide D. salina HEM with an IC 50 value 64.2 μg/mL exhibited significantly a potent cytotoxicity effect on HCT116 human cell line than other tested isolated polysaccharides. The percentage inhibition of different tested cell lines at the maximum concentration tested (100 μg/mL) were compiled in Table 4. The IC 50 values of other polysaccharides could not be determined even at the maximum concentration (100 μg/mL).   Several studies evaluated the antiviral activity of polysaccharides isolated from microalgae particular Spirulina platensis against the pathogenic human virus. These studied revealed that the polysaccharides had broad antiviral spectrum against enveloped viruses such as the herpes simplex virus type 1 (HSV-1), the human immunodeficiency virus type 1 (HIV-1) or influenza virus type A (IFV-A). The mode of antiviral action of polysaccharides is still not recognized but it may be attributed to inhibition viral adsorption, the penetration, or the replication in the host cells [33-35]. In the current study noted that both polysaccharides isolated from S. platensis were bounded with protein and had nearly the same chemical composition, constituted highest Gal/Glu and Man/Glu ratios in double folds (i.e galactose and mannose contents are 2 folds to glucose content) than other tested polysaccharides. On the other hand, these polysaccharides exhibited potent antiviral activity than other tested polysaccharides. this result was agreed with Matloub et al., 2017 [5] where the polysaccharides isolated from a vegetative form of S. platensis reduced replication of HCV genotype 4a replicon to 50% which composed of Gal/Glu and Man/Glu ratios in 0.5 fold (i.e galactose and mannose 0.5 fold to glucose). While the polysaccharides isolated from stress form reduced replication of HCV genotype 4a replicon to 85%. Galctose and mannose to glucose ratios may be played a key role in their antiviral activity.  On the other hand, the anticholinesterase activity of Scenedesmus sp was estimated for the first time in the present study and demonstrated promising inhibiting activity at a high concentration of polysaccharides. In addition, marked anticholinesterase activity of Dunaliella salina was observed in the current study, which is run in parallel with the findings of Aly et al.

CONCLUSION
Among edible microalgae, S. platensis, S. obliquus, and D. salina are paid attention because of their nutritional value for human and aquatic animals beside their medicinal applications. The polysaccharides isolated from microalgae have still attracted to scientist because of their special physicochemical properties and varied biological activities. They are crucial sources of structurally diverse bioactive polysaccharides and remain largely unexploited in nutraceutical and pharmaceutical areas. Fortunately, the possibility for optimization of these biopolymer productions by manipulating growth conditions is economically easy for biomedical and pharmaceutical industries. Our investigation emphasized these microalgal biopolymers have great therapeutically potential in drug development used as broad spectrum antiviral especially enveloped virus, antitumor, antioxidant and anticholinestrae agents in near future.