FTIR characterization and antioxidant activity of water soluble crude polysaccharides of Sri Lankan marine algae

Materials

Polysaccharide standards (alginic acid from brown algae, A7003; fucoidan from Fucus vesiculosus, F5631; ι-carrageenan, C1138; agar, 56763) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All organic solvents used during the sample preparation were of analytical grade. Chang liver cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) was purchased from Gibco Inc. (Grand Island, NY, USA). Potassium bromide (Fourier transform infrared [FTIR] grade) was purchased from Sigma-Aldrich.

Collection of algal samples

Chaetomorpha antennina (SLGS1), Halimeda discoidea (SLGS2), Halimeda gracilis (SLGS3), Gracilaria corticata var. ramalinoides (SLRS5), Gracilaria foliifera (SLRS6), Ahnfeltiopsis pygmaea (SLRS7), Gracilaria corticata (SLRS8), and Jania adhaerens (SLRS9) were collected from the cost of Galle, Sri Lanka (6°4′54.19″ N/80°8′51.78″ E). Caulerpa racemosa var. racemosa f. remota (SLGS4) was collected from the cost of Hikkaduwa, Sri Lanka (6°4′54.19″ N/80°8′51.78″ E) and Chnoospora minima (SLBS11), and Gracilaria edulis (SLRS10) were collected from the coast of Kalpitiya, Sri Lanka (6°4′54.19″ N/80°8′51.78″ E). Samples were identified by Dr. Chandrika Nanayakkara, a specialist in algal identification based on morphological and anatomical characters. Repositories were stored in the herbarium at the University of Colombo. Samples were washed thoroughly to remove any attached epiphytes and debris. Subsequently, samples were lyophilized, ground into a fine powder, and stored at −20°C until further use.

Extraction of crude polysaccharides (CPs)

Algae powders (5.0 g each) were depigmented with acetone and extracted twice using distilled water at 90–95°C under continuous shaking for 3–4 h. Extracts were vacuum filtered and concentrated to one-fourth of the original volume. CPs were precipitated from extracts by adding three volumes of 95% ethanol bringing it up to the original volume. Mixtures were allowed to stand for 8 h at 4°C. Precipitated CPs were separated by centrifugation (12,000 rpm) at 4°C. Hereafter the precipitate will be referred to as CP fraction.

Chemical analysis

The proximate composition of the 11 algal samples was analyzed according to the Association of Official Analytical Chemists (AOAC) 2005 methods. Accordingly, the protein content was determined with the standard Kjeldahl method, the lipid content with the Soxhlet method, and the ash content by dry ashing in a furnace at 550°C for 6 h (Horwitz and Latimer 2005). The total polysaccharide contents were analyzed using the phenol–sulfuric acid method as described by DuBois et al. (1956). The total polyphenolic content was analyzed according to the method described by Chandler and Dodds (1983) The protein content of the CPs was analyzed using the Thermo Scientific Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). The sulfate content was measured with the BaCl₂ gelation method (Dodgson and Price 1962).

Evaluation of antioxidant activities

The antioxidant activity of each CP fraction was analyzed as the measurement of DPPH, alkyl, and hydroxyl free radical scavenging activities. The analysis was done using an electron spin resonance spectrometer (JES-FA200; Jeol, Tokyo, Japan) at 298 K. The DPPH radicle scavenging activity was analysed according to the method described by Nanjo et al. method (Nanjo et al. 1996). The alkyl radical scavenging activity was analyzed according to the method described by Hiramoto et al.’s method (Hiramoto et al. 1993). The hydroxyl radical scavenging activity was evaluated according to the method described by Finkelstein et al. (1980).

Cell culture

“Chang” liver cells were maintained in DMEM supplemented with 1% antibiotic (100 μg mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin) and 10% FBS. Cell cultures were maintained in incubators provided with a humidified atmosphere at 37°C with 5% CO₂. Cells were subcultured within each 2 days and the cells under exponential growth were seeded for experiments. Experiments were carried out using Chang cells seeded in 96 well culture plates following the same methods as described by Wijesinghe et al. (2011). Cytotoxicity of the CPs was evaluated as a measurement of cell viability using MTT colorimetric assay. Readings were obtained using a synergy HT multi-detection microplate reader (BioTek Inc., Winooski, VT, USA).

Evaluation of intracellular ROS scavenging activities

The 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay was adopted to evaluate the ROS scavenging ability of the CPs as described by Engelmann et al. (2005). Chang cells pre-seeded in 96-well plates at 1.0 × 10⁵ cells mL⁻¹ were treated with different sample concentrations. After 1 h of incubation, H₂O₂ (1 mM) or 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH; 10 mM) were added to each well except the control. After 1 h of incubation, DCFH-DA (25 μg mL⁻¹) was added to each well following a 10 min incubation period. Fluorescence readings were obtained with a synergy HT multi-detection microplate reader at a 485 nm excitation and 530 nm emission wavelengths.

Characterization of CPs by Fourier transform infrared spectroscopy and neutral sugar analysis

FTIR spectra of the CPs and standard fucoidan, agar, lambda-carrageenan, and alginic acid in KBr pellets were analyzed using an FTIR spectrometer (Nicolet 6700; Thermo Scientific). To analyze neutral sugars CPs were hydrolyzed using 4 M of trifluoroacetic acid in sealed glass tubes. Analysis was carried out using the method described by Kang et al. (2015).

Computational calculations

Infrared (IR) vibrational wave numbers for designed dimeric units of polysaccharides were calculated using the Gaussian 09 package. Initial optimization of the molecular geometry was performed using the PM6 semi-empirical method. Geometry optimization and harmonic vibrational frequencies were calculated using time-dependent density functional theory (DFT) quantum-chemical calculations at the RB3LYP level using the 6-31G(d,p) basis set as described by Cardenas-Jiron et al. (2011). The calculated vibrational spectra were scaled with 0.9645, 0.9799, 0.9819, 0.8625, 0.8719, and 0.9319 for alginic acid, fucoidan, sulfated galactan, mannan, agar, and lambda carrageenan, respectively.

Statistical analysis

All the data values are expressed as mean ± SD based on at least three independent experiments. Statistical analysis for comparing the data was performed using IBM SPSS Statistics 20 software (IBM Corp., Armonk, NY, USA) using one-way ANOVA by Duncan’s multiple range test. p-values less than 0.05 (p < 0.05) were considered as significant.

Proximate composition

As shown in Table 1, J. adhaerens showed the highest ash content followed by the two Halimeda species. The thallus of the aforementioned three algae species was highly calcified. The highest protein content was displayed by C. racemosa var. racemosa f. remota. The highest lipid content was shown by the two G. corticata species. The brown alga C. minima had the highest carbohydrate content followed by the red algae of the three Gracilaria species and A. pygmaea.

Table 1

Proximate composition of the 11 Sri Lankan algae samples

Chemical composition

The highest carbohydrate content was shown by SLRS10P with a percentage of 93.83 ± 1.07% followed by SLGS1P with a percentage of 91.45 ± 1.02% (Table 2). The protein content was higher in SLBS11P and SLRS8P. The highest phenolic and sulfate contents were observed in SLBS11P (Table 2).

Table 2

The chemical composition of the crude polysaccharide fraction

Radical scavenging activities of CPs

The highest DPPH and alkyl radical scavenging activities were observed in SLBS11P with IC₅₀ values of 89.51 ± 17.00 and 106.80 ± 0.66 μg mL⁻¹, respectively (Table 3). The highest hydroxyl radical scavenging activity was observed in SLGS1P followed by SLBS11P. The antioxidant activity of standard ascorbic acid was measured using each respective radical scavenging assay. Accordingly, IC₅₀ values were 23.22 ± 0.52 μg mL⁻¹ for the DPPH radical scavenging activity, 248.35 ± 0.52 μg mL⁻¹ for the hydroxyl radical scavenging activity and 35.62 ± 0.41 μg mL⁻¹ for the alkyl radical scavenging activity.

Table 3

IC₅₀ values for the radical scavenging activities of crude polysaccharide fractions

Cytotoxicity and protective effects of CPs

None of the CP fractions showed a considerable cytotoxic effect on Chang cells at the concentrations tested (Fig. 1A). Cell viabilities were above 80% even in the presence of the highest concentration (200 μg mL⁻¹). Results of the DCFH-DA assay indicate a reduction in the intracellular ROS levels of Chang cells, induced with H₂O₂ or AAPH in a dose-dependent manner compared to the respective positive control (Fig. 1B & C). SLBS11P displayed the strongest scavenging effects. In addition to SLBS11P, a robust intracellular H₂O₂ scavenging activity was observed in SLGS1P (p < 0.001).

Fig. 1

Evaluation of sample toxicity and intracellular reactive oxygen species (ROS) scavenging activities of the samples against H₂O₂ and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) induced oxidative stress in “Chang” cells. (A) Sample toxicity. (B) AAPH induced intracellular ROS scavenging activity. (C) H₂O₂ induced intracellular ROS scavenging activity. Results represent the percentage (%) of cell viability and intracellular ROS levels. Values were obtained from three independent experiments and represented as means ± standard deviation. ^ap < 0.05 and ^bp < 0.001 were considered as significant compared to the control (sample toxicity) and positive control (ROS scavenging).

Structural analysis

The assignment and characterization of IR vibrational spectra were done based on computational calculations performed on pre-designed monomeric and dimeric units of polysaccharides using the DFT method at the RB3LYP/6-31G(d,p) level and based on relevant literature (Tul’chinsky et al. 1976, Christiaen and Bodard 1983, Mathlouthi and Koenig 1987, Mollet et al. 1998, Roberts and Quemener 1999, Marais and Joseleau 2001, Pereira et al. 2003, 2013, Chandía et al. 2004, Praiboon et al. 2006, Leal et al. 2008, Alves et al. 2010, Ji et al. 2013, Xia et al. 2014). Fig. 2 shows the structures of the constructed dimeric units and their corresponding energy values.

Fig. 2

Optimized molecular geometries of the modeled dimeric units of polysaccharide residues. (A) Fucoidan. (B) Alginic acid. (C) Sulfated galactan. (D) Mannan. (E) Agar. (F) Lambda carrageenan. Computational calculations were performed using density functional theory method at RB3LYP/6-31G(d,p) level. G_f^ø represents the “Sum of electronic and thermal Free energies” (Gibbs free energy) of the molecule in kJ mol⁻¹. Color code for spheres: yellow, sulfur; red, oxygen; blue, nitrogen; grey, carbon; white, hydrogen.

The calculated FTIR spectra are shown in Fig. 3. The IR spectra within the 500 cm⁻¹ to 2,000 cm⁻¹ wavenumber region (fingerprint region for polysaccharides) were used for data analysis. Table 4 summarizes some of the major IR vibrational modes of polysaccharides (Mathlouthi and Koenig 1987, Mollet et al. 1998, Alves et al. 2010, Pereira et al. 2013). All FTIR spectra identify a basic polysaccharide backbone with an intense peak centering 1,035 cm⁻¹ representing the stretching vibrations of the glycoside bridge (C─O─C) (Pereira et al. 2013). This intense peak is broadened (1,010–1,090 cm⁻¹) due to the overlap with of other peaks (Pereira et al. 2003, Xia et al. 2014).

Fig. 3

Fourier transform infrared analysis of the crude polysaccharide fractions. (A) Brown algae crude polysaccharides. (B) Green algae crude polysaccharides. (C) Red algae crude polysaccharides. Experimental infrared (IR) spectra of the crude polysaccharide samples have been compared with IR spectra of standard polysaccharides and calculated IR spectra of constructed dimeric units of polysaccharides. Computational calculations were performed using density functional theory at RB3LYP/6-31G(d,p) level.

Table 4

A list of infrared (IR) vibrational modes characteristic to polysaccharides

Considering the polysaccharides found in brown algae, except for the characteristic IR peaks shared by polysaccharides, the band at 1,135 cm⁻¹ (Fig. 3A) in SLBS11P and fucoidan standard indicates stretching vibrations of the glycosidic C─O group of fucoidan. The broadened peak between 1,120–1,270 cm⁻¹ indicate sulfate groups (S═O stretching) branching off from fucoidan or alginic acid residues. The IR band at 845 cm⁻¹, shows the C─O─S bending vibration and further confirms the presence of a sulfate group. The peak at 1,616 cm⁻¹ (Fig. 3A) is originated from the asymmetric stretching vibrations of the carboxylate O─C─O bond. The intense peak at 1,710 cm⁻¹ in the commercial alginic acid indicates a stretching vibration of the carbonyl group in carboxylic acid groups (C═O), and the two peaks at 1,705 and 1,715 cm⁻¹ in calculated alginic acid spectra confirms this feature (Jeon et al. 2002). The 1,710 cm⁻¹ peak was not observed in SLBS11P, since alginic acid is clearly absent from SLBS11P. The absence of IR bands at 808 cm⁻¹ (C¹─H deformation vibration of guluronic acid residues), the bands at 808 and 822 cm⁻¹ (guluronic acid residues), and the peak centered at 940 cm⁻¹ (stretching vibration of C─O in uronic acid residues) further confirm this scenario (Chandía et al. 2001, 2004, Leal et al. 2008).

Considering the polysaccharides found in green algae, seaweed galactans have IR peaks at 930, 845, 820, and 805 cm⁻¹ (Fig. 3B) associated with 3,6-anhydrogalactose and the sulfation of C-4, C-6 of galactose units and of C-2 of 3,6-anhydrogalactose (Matsuhiro 1996). The broadened IR peak between 1,120 and 1,270 cm⁻¹ is indicative of the S═O stretching vibration of sulfate groups, which was observed in all the green algal polysaccharide samples. The peak near 1,380 cm⁻¹ is also indicative of sulfate substitution (Fenoradosoa et al. 2009). Peaks at 1,146 and 1,390 cm⁻¹ are due to the presence of mannans (Dunn et al. 2007).

For red algal polysaccharides, the characteristic peak at 930 cm⁻¹ (Fig. 3C) represents the C─O─C vibration of 3,6-anhydro-L-galactose and 3,6-anhydro-D-galactose residues found in both agar and carrageenan. The peak near 1,375 cm⁻¹ indicates the presence of sulfate groups. Peaks at 740 and 716 cm⁻¹ are associated with the C─O─C bending vibration separately in glycosidic linkages. The peak at 890 cm⁻¹ represents the stretching vibration of the anomeric C-H of unsulfated β–galactopyranosyl residues. Based on their degree interaction with sulfates, carrageenans are categorized into several types (Roberts and Quemener 1999). The peak between 1,210 and 1,270 cm⁻¹ is associated with stretching vibration of the S═O bond in sulfate groups, which is generally observable in all carrageenan types. This feature was observed in commercial lambda-carrageenan, SLRS6P, SLRS7P, SLRS8P, and SLRS9P but not SARS10P. According to Roberts and Quemener’s characteristic spectral features can be observed in different carrageenan types (Roberts and Quemener 1999): peaks at 840–850 cm⁻¹ (D-galactose-4-sulfate), at 820–830 cm⁻¹ (D-galactose-2-sulfate), and between 800 and 805 cm⁻¹ (3,6-anhydro-D-galactose-2-sulfate). The weak band observed at 770 cm⁻¹ shows the skeletal bending of galactose rings (Pereira et al. 2003).

Monosaccharide composition of CP fractions

The monosaccharide analysis revealed relatively higher glucose levels in green algae (Table 5), except for SLGS1P that was mainly composed of galactose. Red algae had higher levels of galactose and glucose. Except for SLRS9P, red algae had higher galactose levels than glucose levels. All green and red algal CPs indicated negligible amounts of fucose, whereas the highest reported level of fucose was from the CPs of the SLBS11P brown algae.

Table 5

Monosaccharide composition of the eleven crude algal polysaccharide fractions