Characterization and biological properties of sulfated polysaccharides of Corallina officinalis and Pterocladia capillacea

Red seaweed possess various sulfated polysaccharides (SPs) that could potentially be exploited as bioactive agents for medical and industrial applications. Crude polysaccharides from the red algae Corallina officinalis (SP1) and Pterocladia capillacea (SP2) were extracted and characterized according to their chemical content and their antioxidant, anti-inflammatory, anticoagulant, antibacterial, antifungal, and antifouling activities. The isolated polysaccharides contained low levels of protein and high levels of carbohydrate and sulfate. The extracted SPs were characterized by Fourier–transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectral data and revealed that SP1 is composed of carrageenan, while SP2 is composed of polysaccharides containing sulfated galactans plus κ – and ι –carrageenan. Both isolated SPs exhibited all the tested biological activities but those of SP2 were superior. These results reflect the beneficial effects that red algal polysaccharides have as a natural renewable bio–product and that there is a significant relationship between polysaccharide structure, sulfate content and their biological properties. Further studies should be undertaken on the fractionation and characterization of polysaccharides extracted from species of red seaweed in addition to experiments to verify the efficiency of the extracted SPs for food and medical uses in vivo .


Introduction
Macroalgae are one of the largest biomass producers in the marine ecosystem and have many bioactive metabolites with valuable applications in the nutritional and pharmaceutical industry (Ismail et al. 2016;Tanna & Mishra 2019).Also, they are renewable, easily cultivated, non-toxic and without side effects (Ismail & El-Sheekh 2017).
Among the different bioactive compounds, sulfated polysaccharides (SPs) represent the main biochemical structure relevant to the algal taxonomic position.Diversity in the chemical composition of algal polysaccharides varied according to phylum, species, different habitats and harvest time (Li et al. 2008).Whereas, SPs are complex and heterogeneous anionic macromolecules and present at high concentrations up to 4 -76 % of macroalgal dry weight (Paniagua-Michel et al. 2014).The macroalgae cell wall is characterized by a high amount of polysaccharides that are majorly substituted by sulfate, which are not present in terrestrial plants (Mourao 2007).
Red seaweed dietary fibers are mostly composed of sulfated polysaccharides galactans (a polymer of galactose), e.g.agar or carrageenan (Fonseca et al. 2008;Cunha & Grenha 2016).Carrageenans are used mainly in the nutrition manufacture due to their gelling, suspension, thickening or water-holding properties (Norziah et al. 2006).The structures of polysaccharides and their sulfate contents markedly varied between species (Amorim et al. 2011).This variation has gained the scientist attention as this contributes to the various facets of their pharmacological ability (Manlusoc et al. 2019).
The crude SPs from the red algae "Corallina sp. and Pterocladia capillacea" have different biological activities such as antimicrobial, antioxidant and anticoagulant properties (Sebaaly et al. 2012;2014;Abou Zeid et al. 2014).The SPs from C. officinalis have shown its relevance as natural antioxidants in many economical applications (Benattouche et al. 2017).The high antioxidant activity of Pt. capillacea may be attributed to galactose and mannose sugars in the polysaccharide chain besides its high content of phenolic compounds (Fleita et al. 2015).In addition, Pt. capillacea polysaccharide fraction revealed anticoagulant activity by different anticoagulant analyses (Abou Zeid et al. 2014).However, there are few systematically studied reports on the biological abilities of polysaccharides from Egyptian seaweed.Hence, this research aims to characterize the crude polysaccharides extracted from the tested seaweed "C.officinalis and Pt.capillacea" as well as screens their antioxidant, anti-inflammatory, anticoagulant, antimicrobial, and antifouling efficiencies.

Collection and identification of the selected red algae
The red seaweed Corallina officinalis Linnaeus and Pterocladia capillacea (S.G.Gmelin) Bornet were freshly collected during summer season 2019 from Sidi Kirayr coast, Mediterranean sea, Egypt (Longitude 29°65' to 29°85' E and Latitude 31°3' to 31°9' N), and then were washed with distal water to remove epiphytes and debris.On the same day of collection, some of the seaweed samples were prepared as herbarium and other complete thalli were preserved in 5 % formalin in seawater for taxonomical identification according to Aleem (1993); Jha et al. (2009); Kanaan & Belous (2016).The names of the species were used according to Guiry & Guiry (2019).The other part was air-dried at room temperature on absorbent paper.The dried algae samples were crushed to a fine powder and stock up at -20 ºC.

Extraction and chemical analysis of the tested crude polysaccharides
Macroalgal polysaccharides were extracted by Imbs et al. (2009) methods.Total sugars were measured by the phenol-H 2 SO 4 reaction using D-glucose as a standard (Dubois 1956).Polysaccharides sulfate contents were estimated turbidimetrically (Hach 2100A) after acid hydrolysis of the polysaccharides (HCl 6 mol/L, 100 ºC, 4 h) as indicated by the gelatin-barium method (Lloyd et al. 1961), sodium sulfate was used as standard.The contaminant protein content was estimated by Bradford assay (1976), using bovine serum albumin as standard.

Characterization of the extracted polysaccharides
Fourier transform infrared spectra were recorded on a DRS-800 spectrometer (FTIR).Data were collected in the range of 4000 -400 cm -1 at a resolution of 4 cm -1 .The two extracted SPs were prepared for measurement in the form of KBr pellets.Also, the extracted sulfated polysaccharides (2 -3 mg) were dissolved in 0.5 ml of 99 % D 2 O and analyzed using Nuclear Magnetic Resonance spectra (NMR) (JEOL ECA 500), at the Central Labs, Mansoura University, Egypt, with a frequency of 300 MHZ, an acquisition time of 5.29 s and duration of impulse of 11 μs at room temperature.

Biological activities of the extracted polysaccharides Antioxidant activity
The ability of both isolated polysaccharides methanolic extract to scavenge DPPH free radical was estimated according to Ye et al. (2009) method.Briefly, a 0.1 mM of methanolic DPPH solution was prepared, to give the initial absorbance value of 0.993 at 517 nm.The different concentration of samples (in 0.1 ml) of each sample (with appropriate dilution if necessary) was added to 3.0 ml of ethanolic DPPH solution.After incubation for 30 min in the dark, the absorbance was measured at 517 nm.The percentage of DPPH scavenging activity which was scavenged was calculated using the following formula: About 1 ml of methanolic extract of both tested polysaccharides was mixed with 3 ml of TAC reagent solution.The tubes were capped and incubated at 95 °C for 90 min.After cooling, the absorbance of each sample was measured at 695 nm and ascorbic acid was used as standard (Prieto et al. 1999).

Anti-inflammatory potential
Anti-inf lammator y potential of different concentrations of both polysaccharides, in comparison to standard drug sodium diclofenac was in vitro estimated, using the method suggested by Rahman et al. (2015).The absorbance was measured using a UV visible spectrophotometer at 255 nm.

Anticoagulant activity
Blood was collected by venous puncture from 3 individual healthy donors with no history of bleeding or thrombosis and carefully mixed with 3.2 % sodium citrate at a proportion of 9:1, and then the blood was centrifuged at 1000× g for 10 min at ambient temperature.After centrifugation, the supernatant was removed and stored in siliconized tubes that representing the citrated pool of plasma.Activated partial thromboplastin time (APTT) and prothrombin time (PT) of the plasma pool were mixed with different concentrations of the tested polysaccharides (25, 50, 75 and 100 μg/ml) as described by Hassan et al. (2009).

Antimicrobial activities
The antibacterial property of each isolated polysaccharide was determined by the standard disk diffusion technique (5 mm) (Kirby Bauer test) against six pathogenic bacterial species (Bacillus subtilis 6633, Escherichia coli 19404, Enterococcus faecalis 29212, Klebsiella pneumonia 13883, Pseudomonas aeruginosa 15442 and Staphylococcus aureus 25923) which were kindly taken from the Microbiology Laboratory at the National Institute of Oceanography and Fisheries "NIOF", Alexandria, Egypt.After incubation at 37 ºC overnight, the radius of the inhibition zone around each disc was measured in mm.Piperacillin (30 mg/ml) was used as control.
Four fungi species (Aspergillus niger, Fusarium solani, Penicillium decumbens, and Rhizoctonia solani) were obtained from the Microbiology Laboratory at NIOF, Alexandria, Egypt.About 1 mg of each polysaccharide was added to 100 ml of a modified Czapek Yeast Extract Agar (CYEA) medium then poured in sterile Petri dishes (9 cm).By using cork borer, pre-activated pathogenic fungi (5 mm diameter) were inoculated in the center of solidified plates, while the negative control (un-amended (CYEA) plates).The commercial antifungal miconazole was used as a positive control.Then all plates were incubated at 28 °C for a week.Miconazole is used.The inhibition ratio was calculated by using the following equation (APHA 1995): Antifouling activity About 1 ml seawater was mixed with nutrient broth medium (20 ml) in 50 ml conical flask containing cover glass and incubated overnight at 28 ºC.The tested polysaccharides "200 μl" were added into the flask (as an antifouling agent), then stained with 0.4 % crystal violet solution for 10 minutes.The cover glass was washed by water, dried at room temperature and examined under the microscope (Kumaran et al. 2011).Another flask without polysaccharides was used as a control for comparison.

Polysaccharides chemical characterization
The yield of the polysaccharide extracted from C. officinalis and Pt.capillacea, obtained from aqueous extraction, under heating represented 36.57and 42.19 % of the seaweed dry weight, respectively (Tab.1).The estimated yield for both seaweed was similar that obtained for G. gracilis (36.8 -46.6 %) (Skriptsova & Nabivailo 2009).The sulfate content of the tested polysaccharides was 3.2 and 1.5 % corresponding to C. officinalis and Pt.capillacea.These ratios were higher than those obtained for G. birdiae 1.0 % (Barros et al. 2013) but they were lower than determined from G. domingensis (7.6 %) and G. mammillaris (8.9 %) (Valiente et al. 1992).The results in (Tab. 1) showed the tested seaweed contained high levels of polysaccharides (48.73 -61.75 %).On the other hand, no protein was detected in the isolated polysaccharides.Data are mean of three replicates (±SD).

Molecular structure
The molecular structures of the polysaccharides are shown by using FTIR and NMR techniques.
FTIR analyses show the most functional groups and similarities between compounds (Fig. 2).Broad bands are assigned at 3437-3435 cm −1 for SP1 (Fig. 2A) and SP2 (Fig. 2B) that are interpreted as being due to the stretching vibration of O-H (Sekkal & Legrand 1993).The small band at 2934 cm -1 may be related to the C-H stretching vibration.The signals at 1647 cm −1 for SP1 and 1639 cm −1 for SP2 correlated to the carboxyl group of uronic acid (Silva et al. 2005).
The regions at 1416.81 cm -1 (SP1) and 1424.51cm -1 (SP2) may be assigned to the C-OH bending vibration with the contribution of carboxyl group O-C-O (Mathlouthi et al. 2001).FTIR of the SP1 and SP2 showed absorption at 1240 and 1249 cm -1 corresponding to S-O stretching vibration and suggesting the presence of ester sulfate.The Weak bands at 1152.77 and 1157.45cm -1 are due to the stretching vibration of sulfate esters, ɣ (C-O-C), or ɣ (C-C).The FTIR of SP2 polysaccharides had a small signal at 770 cm -1 which is characteristic of the agarocolloids of the red seaweed compound (Fonseca et al. 2008).
The band around 1072.13 for SP 1 and 1069.81cm _1 for SP2 was equivalent to the skeleton of galactans and stretching vibration of sulfate group SO (Chopin et al. 1999).Whereas FT-IR spectrum of SP2 showed a weak peak located at 877.14 cm -1 correspond to a specific agar band (Souza et al. 2012).The spectrum appeared the band at 933.29 cm -1 (SP1) and 933.09 cm -1 (SP2) has been assigned to 3,6anhydrobridge which is common in κ-and ι-carrageenan not in λ-carrageenan (Silva et al. 2010).The FTIR of SP2 polysaccharides had a small signal at 770 cm -1 which characteristic bands of agarocolloids of the red seaweed compound (Fonseca et al. 2008).These compounds are mainly galactans consisting entirely of galactose or modified galactose units.

Biological activities Antioxidant activity
The DPPH free-radical scavenging efficiency demonstrated that the isolated crude polysaccharides had a moderate impact on preventing the formation of these radicals.These results are in agreement with those of Souza et al. (2012) who detected that the aqueous extracted SP from Gracilaria birdiae exhibited moderate antioxidant potency as estimated by DPPH scavenging effect.Two SP fractions "galactose and xylose" from C. officinalis had considerable antioxidant capacities (Yang et al. 2011).The SPs from C. officinalis showed a high antioxidant property (Costa et al. 2010).Table 2 shows that the TAC of both isolated SPs varied by algal species and their sulfate concentrations.The antioxidant efficiency of SP2 appeared higher TAC than SP1; this might be related to its sulfate concentration.Whereas there is a positive correlation between the antioxidant ability of algal polysaccharides and their sulfate content (Zhong et al. 2019).

Anti-inflammatory activity
The SP2 exhibited a higher capacity for anti-inflammatory than SP1 (Tab.2).Besides, the extracted polysaccharides had the highest anti-inflammatory activity compared to standard drug 'Diclofenac'.As documented by many studies, the anti-inflammatory ability of the isolated polysaccharides extracted from Gelidium pacificum (Cui et al. 2019); Hypnea musciformis (Brito et al. 2013).Gracillaria verrucosa had anti-inflammatory potency by their inhibitory impacts on the pro-inflammatory mediator's production (NO, IL-6, and TNFα) (Dang et al. 2008).

Anticoagulant activity
The anticoagulant activity of two SP depended on algal species, their structure and concentrations.In agreement with Shanmugam & Mody (2000) who demonstrated the anticoagulant potency of algal polysaccharides is attributed to their composition, sulfate content and molecular weight.Also, they may be related to the similarity between heparin and SPs from marine algae, while red seaweed SPs had 4.8 times more activity than heparin (Güven et al. 2019).
Sulfated galactans from red seaweed have being associated to anticoagulant, fibrinolytic and platelet aggregation activities (Pereira et al. 2002).
The activated partial thromboplastin time "APTT" and prothrombin time "PT" analysis are common tests that characterize blood coagula ion, while APTT estimates the influence of compounds under intrinsic and common coagulation pathways (Silva et al. 2005).On the base of the standard range of clotting time APTT (28-38 s) which may vary depending on the reagents used and the laboratory.Both tested polysaccharides showed anticoagulant potency, while SP2 had more anticoagulant ability (40-51 Sec.) than SP1 (37-46 Sec.).The increases in anticoagulant activity were contributed to the increases in polysaccharide and sulfate concentrations.This variation may be due to the polysaccharides types, structure, content and position of sulfate group (Suwan et al. 2009).The polysaccharides such as agar, galactan, carrageenan, porphyran from red seaweed contained -O-SO 3 H group which played a critical role in blood clotting inhibition (Güven et al. 2019).Moreover, carrageenans extended the clotting time via inactivation of thrombin and antithrombin III (Kindness et al. 1979).In this connection, Sebaaly et al. (2014) detected the carrageenans more pronounced anticoagulant effect than galactan isolated from the same species Corallina.On the other hand, galactan from Pt. capillacea was higher APTT than carrageenan (Sebaaly et al. 2012).
According to the APTT / APTT control ratio, the compounds that have a ratio greater than 1.2 acts as a reactive anticoagulant agent (Karaki et al. 2013) so all tested polysaccharides (1.23 -1.7 %) are recommended as safe anticoagulant compounds.
PT test estimates the influence under extrinsic and common coagulation pathways (Silva et al. 2005).SP2 had more PT activity than SP1 which increases with the increase in the polysaccharides concentrations (Tab.3).Data are mean of three replicates (± SD).Ratio was calculated by the formula: Ratio = APTT measured / APTT control "30 second".
Results of our study showed that SP1 and SP2 had an anticoagulant impact that prolonging the PT and APTT.The prolongation of PT indicates that the extrinsic pathway of coagulation was inhibited, whereas the prolongation of APTT suggests the inhibition of the intrinsic and/or common pathway (Liu et al. 2018).
There is a significant relationship between anticoagulant potency (APTT and PT) of the SPs and their sulfate content.Carrageenan with a high level of sulfate content displayed an anticoagulant efficiency higher than that of low sulfate content (Shanmugam & Mody 2000).

Antimicrobial activities
The marine algal polysaccharides have antimicrobial potency against different pathogenic microbe (Jun et al. 2018).Antibacterial potency of both SPs toward three Grampositive and three Gram-negative strains are illustrated in Figure 4.There are significant variations in antibacterial potency of the isolated SPs, which may be related to the used pathogenic bacteria species and polysaccharides structure and seaweed species.Both SPs had no impact on the growth of all bacterial Gram-negative, except E. coli growth was inhibited by SP1 "11±0.5 mm".Both SPs showed antibacterial toward gram-positive bacteria "Bacillus cereus "10±1.8mm; 12±1.2 mm, respectively "and Staphylococcus aureus "8±1.3 mm; 9.5±0.7 mm, respectively".repulsion between the sulfated groups and bacterial cell wall (Rostand & Esko 1997).
Algal polysaccharides enhance plant defense responses and protection by activating salicylic acid, jasmonic acid, and/or ethylene signaling pathways at a systemic level against various pathogenic fungi (Vera et al. 2011).The antifungal mechanisms of carrageenans from Chondracathus teedei are depended on alterations of the cell walls of A. fumigatus and A. infectoria (Soares et al. 2016).
The antifungal ability of the isolated sulfated polysaccharides against four pathogenic fungi is cleared in Figure 5.These variations ranged from 30 % to 100 % according to algal species and pathogenic fungi species.Generally, the average of antifungal inhibition of SP2 exhibited the maximum value "74 %" comparing with SP1 "35 %" and miconazole "65 %" toward all the tested pathogenic species.
Algal polysaccharides enhance plant defense responses and protection by activating salicylic acid, jasmonic acid and/ or ethylene signaling pathways at a systemic level against different pathogenic fungi (Vera et al. 2011).The antifungal mechanisms of carrageenans from Chondracathus teedei depending on alterations of the cell walls of A. fumigatus and A. infectoria (Soares et al. 2016).

Antifouling activity
Marine fouling is the main problem faced by mankind in its oceanic activities.Seaweed and their extracts are natural, renewable and safe antifouling agents for epibiosis inhibition, in addition to corals, ascidians, and many invertebrates species (Da-Gama & Pereira 1995).Figure 6 explains the antagonistic effect of SP1 (Fig. 6A) and SP2 (Fig. 6B) on biofilm formation compared with the control (biofilm formed without the addition of the polysaccharides) (Fig. 6C).This demonstrated the potential antifouling effect of both extracted polysaccharide which decreased the bacterial density due to their antibacterial activity.In this context, Carvalho et al. (2016) observed the antifouling potency of Pt. capillacea against bacterial quorum sensing (QS).Pérez et al. (2016) detected the antifouling activity of the aqueous extracts of 30 marine algal species against 35 isolates of marine bacteria in vitro.

Conclusion
The results of this study indicate that the crude polysaccharides from Pterocladia capillacea have promising antioxidant, anticoagulant, antibacterial and antifungal capabilities that require more investigation to be integrated into nutritional and/or medical uses.Moreover, they can be used as a natural antifouling agent against the bacterial biofilm which is the base layer of the fouling process.The main extracted polysaccharides with various biological activities were identified as κ-and ι-carrageenan in SP2.The present results serve as a starting point for further studies on the isolation, purification, and molecular identification of polysaccharide compounds, which could contribute to the production of innovative natural bioactive compounds in the field of medical and anti-fouling materials required on a large scale.
Inhibition growth = Diameter of fungal growth on control (m mm) -Diameter on treatment plates (mm) Diameter of fungal growth on control plate (mm) x100

Figure 4 .
Figure 4.The antibacterial activity of SP1 and SP2 (mm) (The data are given as means ± SD).

Figure 5 .
Figure 5.The antifungal inhibition ratio % of SP1 and SP2 (The data are given as means ± SD).
Table 2 clarifies significant DPPH inhibitory potency and TAC of the isolated crude polysaccharides.

Table 3 .
Anticoagulant properties of SP1 and SP2