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Characteristics and antioxidant of Ulva intestinalis sulphated polysaccharides extracted with different solvents
International Journal of Biological Macromolecules 81 (2015) 912–919
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Characteristics and antioxidant of Ulva intestinalis sulphated polysaccharides extracted with different solvents Napassorn Peasura a , Natta Laohakunjit a,∗ , Orapin Kerdchoechuen a , Sorada Wanlapa b a Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 49 Tientalay 25 Rd., Thakham, Bangkhuntien, Bangkok 10150, Thailand b Thailand Institute of Scientific and Technological Research, 35 Mu 3 Tambon Khlong Ha, Amphoe Khlong Luang, Pathum Thani 12120, Thailand
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Article history: Received 10 June 2015 Received in revised form 7 August 2015 Accepted 17 September 2015 Available online 21 September 2015 Keywords: Ulva intestinalis Sulfated polysaccharide Solvent extraction Antioxidant activity Characterization
a b s t r a c t Ulva intestinalis, a tubular green seaweed, is a rich source of nutrient, especially sulphated polysaccharides. Sulphated polysaccharides from U. intestinalis were extracted with distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h to study the effect of the extraction solvent and time on their chemical composition and antioxidant activity. Different types of solvents and extraction time had a significant influence on the chemical characteristics and antioxidant activity (p < 0.05). Monosaccharide composition and FT-IR spectra analyses revealed that sulphated polysaccharides from all solvent extractions have a typical sugar backbone (glucose, rhamnose, and sulphate attached at C-2 or C-3 of rhamnose). Sulphated polysaccharides extracted with acid exhibited greater antioxidant activity than did those extracted with distilled water and alkali. The results indicated that solvent extraction could be an efficacious method for enhancing antioxidant activity by distinct molecular weight and chemical characteristic of sulphated polysaccharides. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ulva and Enteromorpha species are green seaweeds belonging to the order Ulvales (Chlorophyta). The tubular green seaweed members of the genus Ulva were previously classified as Enteromorpha species [1]. Ulva intestinalis is bright grass-green seaweed, consisting of a tubular frond and unbranched thalli. Most of these seaweeds are used as fertilizer and feedstock. U. intestinalis is still rarely consumed as a food by humans. It is a rich source of vitamins, proteins, carbohydrates, trace minerals, and other bioactive compounds, especially sulphated polysaccharides [2]. Water-soluble sulphated polysaccharides are widespread in the intercellular space and fibrillar wall of the two-cell-layer thick Ulva sp. Thallus [3]. The main sugars of the sulphated polysaccharides of several species include rhamnose, xylose, and glucuronic acid, as well as small amounts of other sugars such as galactose, arabinose, mannose, and glucose [4]. In the cell wall, sulphated polysaccharides are present with -d-glucuronic acid (1→4)-␣l-Rha-3-sulfate or ␣-l-iduronic acid (1→4)-␣-l-Rha-3-sulfate, and the types of glycosidic linkages are highly species-specific
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (N. Laohakunjit). http://dx.doi.org/10.1016/j.ijbiomac.2015.09.030 0141-8130/© 2015 Elsevier B.V. All rights reserved.
[5]. Sulphate groups are also found at the O-3 or O-2 positions of rhamnose and xylose, respectively. In particular, the pattern of iduronic acid and sulphated rhamnose in polysaccharides from U. intestinalis is similar to that of mammalian glycosaminoglycans such as hyaluronan and chondroitin sulphate, but this pattern is not found in terrestrial plants [6]. Sulphated polysaccharides exhibit important biological activities such as anticoagulant, antioxidant, anti-proliferative, antitumour, anti-complementary, anti-inflammatory, anti-viral, and anti-adhesive activities [7]. In general, the desire bioactivity of sulphated polysaccharides depends on the chemical composition, including sulphate content, monosaccharide composition, and molecular weight [8]. The chemical composition of sulphated polysaccharides varies with a range of factors, including plant variety, geographic location [9], and the extraction method employed. Sulphated polysaccharides are usually extracted by water or chemical solvent (acid or alkaline) [10], followed by precipitation with alcohol or quaternary salt [11]. In addition, the temperature and time of the extraction process are important factors that influence the yield and physicochemical characteristics of sulphated polysaccharides [12]. Hot water extraction is the most commonly used method. Water diffusion is increased at high temperature, resulting in mass transfer by causing cell walls to break down, which leads to polysaccharide leakage [13]. Water extraction at high temperature (80–120 ◦ C) for 3 h produces the highest yield of
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sulphated polysaccharides [14], whereas low sulphated polysaccharide yield and high molecular weight (>500 kDa) are obtained with low-temperature extraction (30–40 ◦ C) [11]. Acid and alkaline extraction of sulphated polysaccharides from U. intestinalis has been previously reported. Alkaline extraction (0.1 N NaOH at 60 ◦ C) has been used for removing proteinaceous compounds in Ulva clathrata [15] because the breakage of hydrogen bridges releases proteins. Moreover, the coarse-fibre length in sulphated polysaccharides extracted from U. intestinalis is diminished after alkaline extraction (0.5 M NaOH at room temperature for 2 h), which results in a low molecular weight (46.8 kDa) sulphated polysaccharides with anti-tumour activity [16]. For acid extraction, dilute acetic or hydrochloric acid is used for hydrolysing non-sulphated polysaccharides [17]; however, sulphated polysaccharide linkages from Ulva rotundata can also be hydrolysed, thereby producing a low yield of sulphated polysaccharides. The long extraction time at high temperature used for hydrochloric acid extraction leads to a low sulphate content [18], low molecular weight, and modified sugar structures [19], which enhances antioxidant activity. To the best of our knowledge, no study has compared the effect of different extraction solvents (water, acid, and alkaline solvents) at various extraction times on the chemical composition of sulphated polysaccharides from U. intestinalis. The results from this study will provide valuable information for selecting appropriate extraction solvents to obtain crude sulphated polysaccharides with the desired functionality. 2. Materials and methods 2.1. Materials Mature samples of U. intestinalis were collected in July 2010 from Pattani Bay, Gulf of Thailand, Pattani province, Thailand. The dried seaweeds were ground with a grinder, sieved through 80 mesh screens, and dried at 105 ◦ C for 3 h until the moisture content was 12%. This dried preparation was purified before it was subjected to proximate analysis. All chemical reagents were of analytical grade (Sigma–Aldrich, USA). 2.2. Purification of the dried U. intestinalis preparation Dried seaweed was soaked in 95% ethanol for 24 h at room temperature to remove soluble materials, including free sugars, amino acids, and some phenols [20]. After filtration, samples were dried at 60 ◦ C for 3 h, placed in plastic bags, and placed in a desiccator until further use. 2.3. Proximate analysis of U. intestinalis After removal of impurities, powdered U. intestinalis was analyzed for proximate composition (protein, carbohydrate, lipid, moisture, ash, and fibre) using AOAC [21] methods. Carbohydrate content was estimated using the following formula: Carbohydrate = 100 − (percentage of protein, lipid, moisture, ash, and fibre). All analyses were performed in three replicates. 2.4. Sulphated polysaccharide extraction After removal of impurities, dried samples (20 g) were subjected to extraction with distilled water, 0.1 N NaOH, and 0.1 N HCl for various times for extraction (1, 3, 6, 12, and 24 h). The ratio of seaweed to solvent was 1:20 (w/v), and the extraction temperature was 80 ◦ C. After the treatment, the residue from the extract was separated by filtration through nylon cloth and Whatman No.1 filters. The alkaline extract was neutralized with 1 N HCl, and
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the acid extract was neutralized with 6 N NaOH. The supernatant was evaporated at 40 ◦ C and 483.69 Pa for concentration and then precipitated with 95% ethanol. The precipitant was recovered by centrifugation and washed twice in 95% ethanol. The sulphated polysaccharide sample was dried and kept at room temperature for further analysis. 2.5. Analysis of sulphated polysaccharides 2.5.1. Percentage yield of sulphated polysaccharides After extraction, the %yield of sulphated polysaccharides was calculated using the following equation: % Yield (dry basis) = (weight of extract/weight of dry seaweed) × 100
2.5.2. Protein content Protein content was estimated using the Lowry method [22]. Sulphated polysaccharides were dissolved in distilled water (0.03 g/3 mL). A 0.6-mL sample was exposed to 3 mL of Lowry’s solution containing 2% Na2 CO3 in 0.1 M NaOH (reagent A), and 1% CuSO4 and 2% sodium potassium tartrate in water (reagent B). Then, 0.3 mL of 50% Folin–Ciocalteau reagent was added and the mixture was incubated at 35 ◦ C for 30 min. The absorbance of the mixtures was measured at 660 nm using a spectrophotometer (G-10 UV scanning, USA). Protein content was calculated based on a bovine serum albumin standard curve and expressed as grams per 100 g dried weight of sample. 2.5.3. Determination of sulphate content Sulphate content was analyzed using a turbidimetric method [23]. A dried sample (2 mg) was digested with 0.2 mL of 1 N HCl at 95 ◦ C for 5 h. After the samples had been cooled at room temperature, 3.8 mL of 3% trichloroacetic acid was added to mixture. Barium chloride–gelatin reagent (1 mL; prepared as 2 g gelatin and 2 g barium chloride in 400 mL of water) was added and the mixture was left for 20 min at room temperature. The absorbance was then read at 360 nm. The results were calculated based on a sodium sulphate standard curve and expressed as grams per 100 g dried weight of sample. 2.5.4. Determination of total sugar Total sugar was analyzed using a phenol–sulphuric acid method [24]. Sulphated polysaccharide was dissolved in distilled water (0.03 g sample/3 mL). The supernatant (0.5 mL) was prepared by reacting with 0.5 mL of 5% phenol and 2.5 mL of concentrated sulphuric acid for 20 min, and then the absorbance was read at 490 nm. The results were calculated based on a glucose standard curve and expressed as grams per 100 g dried weight of sample. 2.5.5. Determination of monosaccharide composition Sugar composition was determined using high-performance liquid chromatography according to the procedure described by Waffenschmidt and Jaenicke [25]. A sulphated polysaccharide sample (6 mg) was hydrolysed for 4 h in 2 M trifluoroacetic acid (TFA, 0.3 mL) at 105 ◦ C. After the TFA had been removed from the sample solution by a stream of dried nitrogen, the hydrolysed sulphated polysaccharide sample was dissolved in 1 mL of distilled water and filtered through a 0.45-m filter. A 50-L sample was injected into an HPLC machine (Shimadzu LC-3A, Japan) at a flow rate of 1 mL/min through a Prevail Carbohydrate ES column (5 m, 250 mm length × 4.6 mm i.d., Alltech, USA) with acetonitrile (A) and water–acetonitrile (95:5, v/v) (B) as solvents. The ratio of solvent flow was 80 (A)/20 (B). An evaporative light scattering detector
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was used (ELSD, Alltech 3300, USA). The results were compared with authentic standard solutions of glucose, galactose, rhamnose, sucrose, arabinose, and fructose. 2.5.6. Determination of functional groups by FT-IR Samples were prepared following a previously described method [26] for functional group analysis by Fourier transform infrared spectroscopy (FT-IR, Spectrum One, Perkin Elmer, USA). Sulphated polysaccharide (0.2 g) was dissolved in 0.2% phosphate buffer at pH 6. The sample was filtered through 0.45-m filter paper, and the supernatant was analyzed using a Golden-gate Diamond single reflectance ATR in an FTS 7000 FT-IR spectrometer equipped with a DTGS (Digilab, Randolph, MA) detector. The absorbance mode was 4000–400 cm−1 . 2.5.7. Determination of molecular weight The molecular weight of the sulphated polysaccharides was determined by high-performance size exclusion chromatography (Water 600 E, USA). Sulphated polysaccharides (0.2 g) were dissolved in 10% NaCl and filtered through 0.45-m nylon cloth [27]. The supernatant was injected into an Ultrahydrogel linear column (WAT 011545, Waters, USA) at 30 ◦ C (MW resolving range 1–20,000 kDa), equipped with a guard column and a refractive index detector. The column was eluted with 0.05 M sodium bicarbonate buffer (pH 11) at a flow rate of 0.6 mL/min. The column was calibrated with standard dextran (MW: 4.4–401 kDa), and a standard curve was established before sample analysis. 2.6. In vitro antioxidant activity assay of sulphated polysaccharide 2.6.1. 1,1-Diphenyl-2-pycrilhydrazyl (DPPH• ) radical scavenging activity assay The scavenging activity for DPPH• free radicals was measured according to the method of Shimada et al. [28] with a modification. DPPH• solution was prepared at a concentration of 0.1 mM in 95% ethanol. Sulphated polysaccharide solution (2 mL) was mixed with 2 mL of the DPPH• solution. The mixture was incubated in the dark for 30 min at room temperature, prior to measurement of the absorbance at 517 nm. The ability to scavenge DPPH• was calculated as a percentage according to the following equation: % Scavenging activity = [(ADPPH − Asample+DPPH )/ADPPH ] × 100 2.6.2. 2,2 Azino-bis 3-ethyl benzothiazoline-6-sulfuric acid (ABTS•+ ) radical scavenging assay The ABTS radical cation (ABTS•+ ) was produced by reacting 7 mM ABTS•+ aqueous solution with 2.45 mM potassium persulphate at room temperature in the dark for 16 h. The solution was diluted with 95% ethanol to obtain an absorbance of 0.7 ± 0.03 at 734 nm. Then, the diluted solution (4 mL) was mixed with 1 mL of sulphated polysaccharide solution. The decrease in absorbance was recorded at 734 nm after incubation at room temperature for 6 min as described by Ree et al. [29]. Radical scavenging activity (%) was calculated using the following equation: % Scavenging activity = [(AABTS − Asample+ABTS )/ADPPH ] × 100 2.6.3. Hydroxyl radical scavenging assay Deoxyribose (60 mM, 0.2 mL), ferric chloride (1 mM, 0.2 mL), ethylenediaminetetraacetic acid (1.04 mM, 0.2 mL), and sulphated polysaccharide solution (0.2 mL) were mixed in phosphate buffered saline (PBS, 50 mM, pH 7.4, 0.8 mL). The solution was incubated at 37 ◦ C for 20 min; then, 0.2 mL of H2 O2 (0.3%) and 0.2 mL of ascorbic acid (2 mM) were added. The mixture was incubated at 37 ◦ C for
1 h. Subsequently, 2 mL of HCl (25%) and 0.2 mL of thiobarbituric acid (1%, w/v) were added. The mixture was boiled for 15 min and cooled in ice, and absorbance was measured at 532 nm according to a previously described method [30]. The radical scavenging activity was determined according to the following equation: % Scavenging activity = [(1 − Asample )/Acontrol ] × 100
2.7. Statistical analysis All experiments were performed in a 3 × 5 factorial randomized complete block design in triplicate, and the results were the average of three independent experiments. Statistical analysis was performed using Statistical Analysis System (SAS User’s Guide version 6, 4th edn., SAS Institute, Cary, NC, USA, 1990). Duncan’s multiple range test was used to determine significant differences between means (p < 0.05). 3. Results and discussion 3.1. Proximate compositions of U. intestinalis U. intestinalis is good natural source of carbohydrate, protein, and mineral, and contains low levels of lipids. Its chemical content can be influenced by the growing conditions (water temperature, salinity, light, and nutrients) [31]. In this study, U. intestinalis material contained large amounts of carbohydrate (55.43%), followed by ash (19.95%) and protein (16.95%). The high carbohydrate content was consistent with the maximum growth and maturity of seaweed when the materials were collected in July [32]. Seasonal variations in seaweed chemical composition are associated with the particular life stage. Photosynthetic activity increases in periods of maximum growth, which results in higher carbohydrate content and reduced protein content [33]. The inverse relationship between carbohydrate and protein content observed in U. intestinalis resulted from the decrease in nitrogen available for carbohydrate synthesis [33]. The ash content in U. intestinalis was high because of the high sulphate content of seaweed during the maximum growth period, when it was harvested. The high sulphate content probably partly represents ions associated with the charged polysaccharides [34]. 3.2. Effect of water, acid, and alkaline extraction on chemical composition 3.2.1. Sulphated polysaccharide yield The sulphated polysaccharide yields obtained using the different solvents for different times are shown in Fig. 1A. Solvent and extraction time had significant effects on sulphated polysaccharide yield (p < 0.05). At 24 h of extraction, 0.1 N NaOH gave the highest yield, followed by 0.1 N HCl and distilled water. The yields increased gradually as extraction time increased from 1 to 6 h, but did not significantly increase from 6 to 24 h (Fig. 1A). An explanation for the high yield with alkaline extraction was that alkaline extraction caused cellulose to swell, which disrupted the hydrogen bonds between hemicellulose and cellulose in the cell wall, resulting in the solubilization of hemicellulose. Hence, alkaline extraction could effectively release insoluble polysaccharides and convert them into soluble polysaccharides. The alkaline solvent could extract xyloglucan, which was a minor alkaline soluble polysaccharide, and glucuronan [11]. However, protein and minerals associated tightly with the polysaccharides were also extracted. The yield was lower with acid than with alkali at 6–24 h, possibly because of acid hydrolysis. The acid solution hydrolysed the polysaccharide structure, including cellulose, hemicellulose, and lignin, which released the sulphated polysaccharides [35].
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Table 1 Monosaccharide composition of crude sulphated polysaccharides from U. intestinalis extracted by water, acid (0.1 N HCl) and alkaline (0.1 N NaOH) at 80 ◦ C for 24 h. Extraction conditions
Water extraction Acid extraction Alkaline extraction
Monosaccharide compositions (%) Arabinose
Glucose
Nd 0.74 ± 0.11a Nd
11.86 ± 0.12 0.84 ± 0.02b 11.96 ± 0.10a
Rhamnose a
12.7 ± 0.10b 8.29 ± 0.14c 39.24 ± 0.12a
a,b,c Means in the same column with different letters are significantly difference at p < 0.05. Nd = not detected.
All sulphated polysaccharide extracts were contaminated with protein because of the ester sulphate moieties, which can form strong anions and attract positively charged proteins. However, the amount of protein was low. It was possible that solvent extraction could reduce protein contamination in sulphated polysaccharides. The protein content of sulphated polysaccharides with distilled water (0.48–0.63%) was lower than that with acid (0.9–2.96%) and alkaline (3.53–3.97%) extraction. There was a significant difference (p < 0.05) in the protein content among water, alkaline, and acid extraction. An increase in the extraction time from 1 to 24 h also had a significant effect (p < 0.05) on the protein content. Sulphated polysaccharides extracted with 0.1 N NaOH for 24 h had the highest protein content because of release of proteins by the breakage of hydrogen bridges in the alkaline solution.
Fig. 1. (A) Sulphated polysaccharide yield; (B) total sugar; (C) sulphate content of sulphated polysaccharides extracted from U. intestinalis with water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h. Values presented as mean ± SD (n = 3). a,b,c,. . . Means above the bars of each parameter with different letters are significantly different at p < 0.05.
However, the sulphated polysaccharides could also be hydrolysed by acid, and as they were mainly short-chain polysaccharides, this would decrease the final recovery. The lowest yield was obtained from water extraction, as water dissolves only long-chain soluble polysaccharides. Extraction time also influenced the yield of sulphated polysaccharides extracted from U. intestinalis. This finding may be explained by the early release of sulphated polysaccharides in the extraction process. In addition, the yield from acid extraction at 1–3 h was higher than that with alkaline extraction. The acid solution degraded the carbohydrate chain backbone structure of the sulphated polysaccharides when the duration of extraction increased. This result was in agreement with [18], who reported that a longer acid extraction time for polysaccharides from brown seaweed led to a decreased yield of sulphated polysaccharides. 3.2.2. Protein content Proteins have been described as potential contaminants of cell wall polysaccharides, mostly because they form part of the structure of cell walls and were closely associated with polysaccharides.
3.2.3. Sulphate content Sulphate in seaweed cell walls plays an important part in the structural strength, enabling the plant to survive in an environment with high kinetic forces such as sea water. The sulphate content of sulphated polysaccharides extracted with distilled water, acid, and alkali initially increased with increasing extraction time, but did not further increase between 6 and 24 h (p > 0.05). The lowest sulphate content was found when using acid, ranging from 36.72% to 38.35% with extraction for 1 to 24 h (Fig. 1C). Acid can eliminate the sulphate group attached to the polysaccharides [36], degrading the substituent sulphate group in the sulphated polysaccharide chain. Sulphated polysaccharides extracted with the alkaline solution had the highest sulphate content because of ester linkages. These chemical bonds between the polysaccharide chain and the sulphate group were not easily cleaved by alkaline extraction [37]. The sulphate content from distilled water extraction was higher than that from acid extraction, possibly because water extraction at high temperature may break down the ester bonds in the sulphated polysaccharide chain [38]. 3.2.4. Total sugar and monosaccharide compositions Total sugar content was determined using a phenol–sulphuric acid method as described in Section 2 [25]. Sulphated polysaccharides extracted at 80 ◦ C for 24 h with acid had the highest total sugar (38.35%), followed by those extracted using alkali (37.43%) and distilled water (11.01%). However, after 6 h of extraction, the total sugar content of sulphated polysaccharides extracted with distilled water, acid, and alkaline extraction was not significantly different (p > 0.05) (Fig. 1B). Sulphated polysaccharides extracted with acid had the highest total sugar content because the glycosidic bond in the polysaccharide chain could be hydrolysed by acid more readily than by alkali and distilled water. Moreover, the total sugar content of sulphated polysaccharides was higher with acid extraction than with alkaline extraction, and this was not related to the percentage yield of the extracts. This finding indicated that the alkaline process could extract a mixture of biopolymers such as proteins from U. intestinalis at a higher yield than acid extraction.
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Fig. 2. FT-IR spectrum of sulphated polysaccharides from U. intestinalis extracted with (A) distilled water; (B) 0.1 N HCl; (C) 0.1 N NaOH at 80 ◦ C for 24 h.
The monosaccharide profiles of sulphated polysaccharides extracted from U. intestinalis using distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 24 h were identified using HPLC (Table 1). Sulphated polysaccharides extracted with distilled water mainly consisted of rhamnose (12.70%), followed by glucose (11.86%). For acid extraction, the major monosaccharide was rhamnose (8.29%), followed by glucose (0.84%), with a trace amount of arabinose (0.74%). The low amount of arabinose acid extraction resulted from hydrolysis of arabinosyl linkages, which were probably present as side chains of the sulphated polysaccharides. Rhamnose was the predominant monosaccharide in the sulphated polysaccharides extracted with alkali and water (39.24% and 12.70%, respectively). The highest level of glucose was found with alkaline extraction. This glucose may originate from other non-sulphated polysaccharides such as cellulose or hemicellulose [39]. Generally, sulphated polysaccharides extracted from Ulva and Enteromorpha species are a group of heteropolysaccharides mainly composed of rhamnose, xylose, glucose, and sulphate, with low amounts of mannoses, arabinose, and galactose [40]. Glucuronic acid may be found in sulphated polysaccharides because carbon 6 of glucose was oxidized to a carboxyl group in the extraction process [41]. The difference in monosaccharide composition probably arose from differences in extraction procedures.
3.2.5. Functional groups FT-IR spectroscopy can be used for approximate identification of polysaccharides in plant materials when combined with chemical analyses. The FT-IR spectra of the sulphated polysaccharides extracted with distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 24 h are illustrated in Fig. 2. The wide absorption at 3437 cm−1 resulted from the hydroxyl stretching vibrations and the absorption bands at 2938 cm−1 originated from C H stretching of a methyl group. The intense peaks at approximately 1625 cm−1 were attributed to C O asymmetric stretching vibration in a carbonyl group. The absorbance at 1427 cm−1 was associated with
hemicellulose and represents the C H bending or stretching frequencies. The band at 850 cm−1 was ascribed to C O S stretching of axial sulphate groups related to a C O SO3 group. The characteristic of absorptive peaks confirmed that the sulphated group had been associated with polysaccharides to form a sulphate ester [42]. The low intensity at 1126 cm−1 of sulphated polysaccharides extracted with acid indicated the presence of arabinosyl side chains. The intensity of the absorbance at 1259 cm−1 in sulphated polysaccharides extracted with distilled water was attributed to the stretching of S O. It was assumed that the main sulphate groups occupy positions C-2 or C-3, and less of the sulphate was located at C-4 of the side chain [43]. In addition, sulphated polysaccharides extracted with distilled water and alkali presented a high absorption band at approximately 1056 cm−1 , probably as the result of C O stretching from rhamnose. The FT-IR spectra of distilled water, acid, and alkali extracts showed a similar pattern in accordance with their monosaccharide composition. However, a carboxyl group from glucuronic acid was not shown in the FTIR profile of all sulphated polysaccharides, which implied that distilled water, acid, and alkaline extraction may not modify the sugar structure. This observation confirmed the results from the monosaccharide composition analysis.
3.2.6. Molecular weight Fig. 3 shows the weight-average molecular weight (Mw ) of three crude extracts of sulphated polysaccharides. Based on the calibration with standard dextran, the molecular weights of sulphated polysaccharides extracted with distilled water, 0.1 N NaOH, and 0.1 N HCl at 80 ◦ C for 24 h were 300 kDa, 110 kDa, and 88 kDa, respectively. All three extracts were mostly composed of one to two peaks. Differences in the elution profiles reflect differences in molecular size. Sulphated polysaccharides extracted with distilled water produced a unique wide peak that mainly contained high molecular mass. The molecular weight distribution of these sulphated polysaccharides was associated with polysaccharides with
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Fig. 3. High pressure size exclusion chromatographic profile of sulphated polysaccharides from U. intestinalis extracted by (A) distilled water; (B) 0.1 N HCl; (C) 0.1 N NaOH at 80 ◦ C for 24 h.
sulphate. In contrast, the profiles of acid and alkaline extraction (Fig. 3B and C) presented two peaks because of contamination by linear xyloglucan and co-elution of glucuronan with proteins [12]. The molecular weight of sulphated polysaccharides extracted with acid and alkali was obviously lower than that of sulphated polysaccharides extracted with distilled water, indicating that the polysaccharide chains may have been disrupted and degraded during the acid and alkaline extractions. 3.3. Effect of water, acid, and alkaline extraction on antioxidant activity 3.3.1. DPPH• radical scavenging of sulphated polysaccharides DPPH• is a free-radical compound that has been widely used to determine the free radical scavenging ability of antioxidants. DPPH• is a stable free radical, and the reduction of DPPH• to DPPH-H in the presence of a proton-donating substance can be used as an indicator of antioxidant activity. At a concentration of 3 mg/mL, sulphated polysaccharides extracted with distilled water, acid, and alkali showed scavenging abilities higher than 50% for DPPH• radicals (Fig. 4A). The presence of the sulphate group likely decreases the bond energy of the C H in the vicinity of the glycosidic bond, and then increases the hydrogen atom donation capability [44]. Although sulphate content and molecular weight were low after extraction with 0.1 N HCl for 24 h, these sulphated polysaccharides had significantly higher (p < 0.05) DPPH• scavenging abilities than sulphated polysaccharides extracted with alkali (55.97%) or distilled water (56.18%). These results indicated that the molecular weight of polysaccharides plays an important role in their bioactivity, and a relatively low molecular weight appears to increase the antioxidant activity. Similar results have been found for polysaccharides from other plants [45], and it was believed that low-molecular-weight polysaccharide molecules have more reductive OH terminals available for reacting with radical species. According to Shao et al. [46], the lowest molecular weight of sulphated polysaccharide from brown seaweed had the highest DPPH• radical scavenging activity. In addition, monosaccharide property indicated that arabinose of the side chain in the sulphated polysaccharide extracted with acid was related to the scavenging effect on DPPH• radicals [15]. DPPH• radical scavenging was not significantly different (p > 0.05) for sulphated polysaccharides extracted with alkali and distilled water. Although alkaline-extracted sulphated
Fig. 4. (A) DPPH• radical scavenging (%); (B) ABTS•+ radical scavenging (%); (C) hydroxyl radical scavenging (%) of sulphated polysaccharides extracted from U. intestinalis with water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h. Values presented as mean ± SD (n = 3). a,b,c,. . . Means above the bars of each parameter with different letters are significantly different at p < 0.05.
polysaccharides had higher sulphate content, the molecular weight was lower than that for water-extracted sulphated polysaccharides. This result indicated that not only sulphate content but also other factors such as the position of sulphate groups and monosaccharide content contribute to the antioxidant activity. Moreover, a higher glucose level was found in sulphated polysaccharides extracted with alkali and distilled water than in those extracted with acid. These findings suggested that high glucose content affects DPPH• radical scavenging activity. The scavenging ability of sulphated polysaccharides extracted with alkali and distilled water may be attributed to the presence of hydrogen atoms from the specific monosaccharide compositions and their side chain linkages. These results agreed with those of Lo et al. [47] who reported that the monosaccharide composition (arabinose, mannose, and glucose) of polysaccharides was markedly associated with the free-radical scavenging ability. 3.3.2. ABTS•+ radical scavenging The antioxidant capacity of the sulphated polysaccharides with respect to an electron transfer reaction was assayed by using the
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ABTS•+ radical cation, which presents a strong absorbance in the visible region. The absorbance of the ABTS•+ solution decreased when an electron was donated by an antioxidant to quench the free-radical. Sulphated polysaccharides extracted with acid had the highest radical scavenging activity (71.87%), followed by those extracted with distilled water (68.06%) and alkali (61.01%) (Fig. 4B). The polysaccharides extracted with alkali with the high sulphate content presented the lowest activity. This finding suggested that the chemical structure of the polysaccharides plays a role in the H abstraction reaction by the ABTS•+ cation radical [44]. The results agreed with a previous study that observed a decrease in the activity towards the radical cation with an increase in sulphate content for commercial -, -, and -carrageenans [44]. Sulphated polysaccharides extracted with distilled water with a high molecular weight (300 kDa) and the lowest total sugar/protein ratio (10.08%) had the highest ABTS•+ radical scavenging. It was found that antioxidant activities of sulphated polysaccharide might depend on the amount of bound protein present in the form of polysaccharide–protein complexes [15]. According to Gómez˜ Ordónez et al. [48] the highest MW (1248–1425 kDa) galactan fraction with a low content of sulphate groups (3.9–1.9%) showed significant scavenging activity towards ABTS•+ . The results in this study and previous reports indicated that the reaction of sulphated polysaccharides with ABTS•+ radicals is complex. Moreover, no relationship was found between sulphate content and antioxidant capacity. Structural features such as monosaccharide composition, molecular weight, and position of sulphate groups were involved in antioxidant activity. 3.3.3. Hydroxyl radical scavenging The hydroxyl radical scavenging activity of sulphated polysaccharides extracted from U. intestinalis was investigated by measuring their ability to prevent oxidative degradation of deoxyribose substrates (Fig. 4C). A similar trend to the DPPH• radical scavenging results was observed, with the highest activity (54.15%) found in the sulphated polysaccharides extracted with acid for 24 h. Although sulphated polysaccharides extracted with distilled water and alkali had higher sulphate content than those extracted with acid, their hydroxyl scavenging ability was weaker. Hydroxyl radical scavenging activity of sulphated polysaccharide in this study was different from red seaweed, in that high sulphate content had greater hydroxyl radical scavenging activity than low sulphate content [49]. Interestingly, hydroxyl radical scavenging activities of sulphated polysaccharide from green seaweed related to its low molecular weight [50]. Previous studies suggested that the scavenging activity for hydroxyl radicals resulted from inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu+ [51]. The sulphate molecules could chelate ions in a Fenton reaction, which may be responsible for the inhibition of deoxyribose oxidation. The hydroxyl radical scavenging activity was probably related to high sulphate content (specific chelating groups) within the molecule because of their high nucleophilic character [52]. However, the sulphated polysaccharides with low sulphate and low molecular weight extracted with acid had the highest hydroxyl radical scavenging activity, possibly because of the molecular weight, which has a direct influence on solubility and viscosity in solution, thus improving the antioxidant activity. Most of sulphated polysaccharide from U. intestinalis had the strongest scavenging activity against ABTS•+ radicals based on electron transfer mechanism [53] of negative charge of the sulphate groups [54], which was slightly higher from that of DPPH• and hydroxyl radical scavenging activity. Moreover, the molecular weight of sulphated polysaccharides is a more important factor for scavenging free radicals than sulphate content [54]. This response might be due to non-compact structure of low-molecular weight sulphated polysaccharides and consequently the more potentially
available electron transport reacting with free radicals [55]. This observation was consistent with antioxidant activity of sulphated polysaccharide from Ulva fasciata which had a prominent ABTS•+ radical scavenging activity [46]. A similar trend in higher scavenging activity in ABTS•+ than DPPH• radical was also found in sulphated polysaccharide from Sargassum horneri [46], which sulphated polysaccharide could donate hydrogen react with free radical by hydrogen transfer reaction as possible antioxidant mechanisms [56]. According to Sun et al. [50], sulphated polysaccharides from Porphyridium cruentum exhibited the strongest DPPH• radical scavenging activity. The results obtained in this study clearly demonstrate that antioxidant activities of polysaccharides were not a function of a single factor, but a combination of several factors, such as, degree of sulfating, molecular weight, type of the major sugar, and glycosidic branching [57,58]. 4. Conclusions The chemical and antioxidant characteristics of sulphated polysaccharides were affected by the solvents and extraction time. Distilled water, acid, and alkaline extraction did not change the main structure of the sulphated polysaccharides, resulting in monosaccharide ratio variation. The sugar backbone of the sulphated polysaccharides from all extractions was composed of rhamnose and glucose, containing sulphate at the C2 or C3 positions of rhamnose. Although sulphated polysaccharides extracted from U. intestinalis with three solvents also exhibited potent antioxidant activities, sulphated polysaccharide extracted with 0.1 N HCl at 80 ◦ C for 24 h had distinctly different both in molecular weight and chemical characterization from sulphated polysaccharide extracted with distilled water and alkaline which enhanced the antioxidant activity. It was clear that their antioxidant activities were dependent on their fine structural features, which will certainly present a good feasibility to elucidate the antioxidant activity of sulphated polysaccharide. This information is important for the regional development of value-added products, particularly as ingredients in functional foods and nutraceutical products. Acknowledgments This work was supported by the Thailand Institute of Scientific and Technological Research, National Research University (NRU) Project of Thailand, and King Mongkut’s University of Technology Thonburi, Thailand. References [1] H.S. Hayden, J. Blomster, C.A. Maggs, P.C. Silva, M.J. Stanhope, J.R. Waaland, Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera, Eur. J. Phycol. 38 (2003) 277–294. [2] C.S. Kumar, P. Ganesan, P.V. Suresh, N. Bhaskar, Seaweeds as a source of nutritionally beneficial compounds: a review, J. Food Sci. Technol. 45 (2008) 1–13. [3] L. Wang, X. Wang, H. Wu, R. Liu, Overview on biological activities and molecular characteristics of sulfated polysaccharides from marine green algae in recent years, Mar. Drugs 12 (2014) 4984–5020. [4] B. Ray, Polysaccharides from Enteromorpha compressa: isolation, purification and structural features, Carbohydr. Polym. 66 (2006) 408–416. [5] V. Ivanova, M.K. Rousevar, J. Serkedjieva, R. Rachev, N. Maolova, Isolation of a polysaccharide with antiviral effect from Ulva lactuca, Prep. Biochem. Biotechnol. 242 (1994) 83–97. [6] F. Chiellini, A. Morelli, A versatile platform of biomaterials from renewable resources, in: R. Pignatello (Ed.), Biomaterials – Physics and Chemistry, InTech, Rijeka, 2011, pp. 75–98. [7] L.S. Costa, G.P. Fidelis, S.L. Cordeiro, R.M. Oliveira, D.A. Sabry, R.B.G. Camara, T.D.B. Nobre, M.S.S.P. Costa, J. Almeida-Lima, E.H.C. Farias, E.L. Leite, H.A.O. Rocha, Biological activities of sulfated polysaccharides from tropical seaweeds, Biomed. Pharmacother. 64 (2010) 21–28. [8] Y. Wang, M. Zhang, D. Ruan, A.S. Shashkov, M. Kilcoyne, A.V. Savage, L. Zhang, Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos, Carbohydr. Res. 339 (2) (2004) 327–334.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Characteristics and antioxidant of Ulva intestinalis sulphated polysaccharides extracted with different solvents Napassorn Peasura a , Natta Laohakunjit a,∗ , Orapin Kerdchoechuen a , Sorada Wanlapa b a Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 49 Tientalay 25 Rd., Thakham, Bangkhuntien, Bangkok 10150, Thailand b Thailand Institute of Scientific and Technological Research, 35 Mu 3 Tambon Khlong Ha, Amphoe Khlong Luang, Pathum Thani 12120, Thailand
a r t i c l e
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Article history: Received 10 June 2015 Received in revised form 7 August 2015 Accepted 17 September 2015 Available online 21 September 2015 Keywords: Ulva intestinalis Sulfated polysaccharide Solvent extraction Antioxidant activity Characterization
a b s t r a c t Ulva intestinalis, a tubular green seaweed, is a rich source of nutrient, especially sulphated polysaccharides. Sulphated polysaccharides from U. intestinalis were extracted with distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h to study the effect of the extraction solvent and time on their chemical composition and antioxidant activity. Different types of solvents and extraction time had a significant influence on the chemical characteristics and antioxidant activity (p < 0.05). Monosaccharide composition and FT-IR spectra analyses revealed that sulphated polysaccharides from all solvent extractions have a typical sugar backbone (glucose, rhamnose, and sulphate attached at C-2 or C-3 of rhamnose). Sulphated polysaccharides extracted with acid exhibited greater antioxidant activity than did those extracted with distilled water and alkali. The results indicated that solvent extraction could be an efficacious method for enhancing antioxidant activity by distinct molecular weight and chemical characteristic of sulphated polysaccharides. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Ulva and Enteromorpha species are green seaweeds belonging to the order Ulvales (Chlorophyta). The tubular green seaweed members of the genus Ulva were previously classified as Enteromorpha species [1]. Ulva intestinalis is bright grass-green seaweed, consisting of a tubular frond and unbranched thalli. Most of these seaweeds are used as fertilizer and feedstock. U. intestinalis is still rarely consumed as a food by humans. It is a rich source of vitamins, proteins, carbohydrates, trace minerals, and other bioactive compounds, especially sulphated polysaccharides [2]. Water-soluble sulphated polysaccharides are widespread in the intercellular space and fibrillar wall of the two-cell-layer thick Ulva sp. Thallus [3]. The main sugars of the sulphated polysaccharides of several species include rhamnose, xylose, and glucuronic acid, as well as small amounts of other sugars such as galactose, arabinose, mannose, and glucose [4]. In the cell wall, sulphated polysaccharides are present with -d-glucuronic acid (1→4)-␣l-Rha-3-sulfate or ␣-l-iduronic acid (1→4)-␣-l-Rha-3-sulfate, and the types of glycosidic linkages are highly species-specific
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (N. Laohakunjit). http://dx.doi.org/10.1016/j.ijbiomac.2015.09.030 0141-8130/© 2015 Elsevier B.V. All rights reserved.
[5]. Sulphate groups are also found at the O-3 or O-2 positions of rhamnose and xylose, respectively. In particular, the pattern of iduronic acid and sulphated rhamnose in polysaccharides from U. intestinalis is similar to that of mammalian glycosaminoglycans such as hyaluronan and chondroitin sulphate, but this pattern is not found in terrestrial plants [6]. Sulphated polysaccharides exhibit important biological activities such as anticoagulant, antioxidant, anti-proliferative, antitumour, anti-complementary, anti-inflammatory, anti-viral, and anti-adhesive activities [7]. In general, the desire bioactivity of sulphated polysaccharides depends on the chemical composition, including sulphate content, monosaccharide composition, and molecular weight [8]. The chemical composition of sulphated polysaccharides varies with a range of factors, including plant variety, geographic location [9], and the extraction method employed. Sulphated polysaccharides are usually extracted by water or chemical solvent (acid or alkaline) [10], followed by precipitation with alcohol or quaternary salt [11]. In addition, the temperature and time of the extraction process are important factors that influence the yield and physicochemical characteristics of sulphated polysaccharides [12]. Hot water extraction is the most commonly used method. Water diffusion is increased at high temperature, resulting in mass transfer by causing cell walls to break down, which leads to polysaccharide leakage [13]. Water extraction at high temperature (80–120 ◦ C) for 3 h produces the highest yield of
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sulphated polysaccharides [14], whereas low sulphated polysaccharide yield and high molecular weight (>500 kDa) are obtained with low-temperature extraction (30–40 ◦ C) [11]. Acid and alkaline extraction of sulphated polysaccharides from U. intestinalis has been previously reported. Alkaline extraction (0.1 N NaOH at 60 ◦ C) has been used for removing proteinaceous compounds in Ulva clathrata [15] because the breakage of hydrogen bridges releases proteins. Moreover, the coarse-fibre length in sulphated polysaccharides extracted from U. intestinalis is diminished after alkaline extraction (0.5 M NaOH at room temperature for 2 h), which results in a low molecular weight (46.8 kDa) sulphated polysaccharides with anti-tumour activity [16]. For acid extraction, dilute acetic or hydrochloric acid is used for hydrolysing non-sulphated polysaccharides [17]; however, sulphated polysaccharide linkages from Ulva rotundata can also be hydrolysed, thereby producing a low yield of sulphated polysaccharides. The long extraction time at high temperature used for hydrochloric acid extraction leads to a low sulphate content [18], low molecular weight, and modified sugar structures [19], which enhances antioxidant activity. To the best of our knowledge, no study has compared the effect of different extraction solvents (water, acid, and alkaline solvents) at various extraction times on the chemical composition of sulphated polysaccharides from U. intestinalis. The results from this study will provide valuable information for selecting appropriate extraction solvents to obtain crude sulphated polysaccharides with the desired functionality. 2. Materials and methods 2.1. Materials Mature samples of U. intestinalis were collected in July 2010 from Pattani Bay, Gulf of Thailand, Pattani province, Thailand. The dried seaweeds were ground with a grinder, sieved through 80 mesh screens, and dried at 105 ◦ C for 3 h until the moisture content was 12%. This dried preparation was purified before it was subjected to proximate analysis. All chemical reagents were of analytical grade (Sigma–Aldrich, USA). 2.2. Purification of the dried U. intestinalis preparation Dried seaweed was soaked in 95% ethanol for 24 h at room temperature to remove soluble materials, including free sugars, amino acids, and some phenols [20]. After filtration, samples were dried at 60 ◦ C for 3 h, placed in plastic bags, and placed in a desiccator until further use. 2.3. Proximate analysis of U. intestinalis After removal of impurities, powdered U. intestinalis was analyzed for proximate composition (protein, carbohydrate, lipid, moisture, ash, and fibre) using AOAC [21] methods. Carbohydrate content was estimated using the following formula: Carbohydrate = 100 − (percentage of protein, lipid, moisture, ash, and fibre). All analyses were performed in three replicates. 2.4. Sulphated polysaccharide extraction After removal of impurities, dried samples (20 g) were subjected to extraction with distilled water, 0.1 N NaOH, and 0.1 N HCl for various times for extraction (1, 3, 6, 12, and 24 h). The ratio of seaweed to solvent was 1:20 (w/v), and the extraction temperature was 80 ◦ C. After the treatment, the residue from the extract was separated by filtration through nylon cloth and Whatman No.1 filters. The alkaline extract was neutralized with 1 N HCl, and
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the acid extract was neutralized with 6 N NaOH. The supernatant was evaporated at 40 ◦ C and 483.69 Pa for concentration and then precipitated with 95% ethanol. The precipitant was recovered by centrifugation and washed twice in 95% ethanol. The sulphated polysaccharide sample was dried and kept at room temperature for further analysis. 2.5. Analysis of sulphated polysaccharides 2.5.1. Percentage yield of sulphated polysaccharides After extraction, the %yield of sulphated polysaccharides was calculated using the following equation: % Yield (dry basis) = (weight of extract/weight of dry seaweed) × 100
2.5.2. Protein content Protein content was estimated using the Lowry method [22]. Sulphated polysaccharides were dissolved in distilled water (0.03 g/3 mL). A 0.6-mL sample was exposed to 3 mL of Lowry’s solution containing 2% Na2 CO3 in 0.1 M NaOH (reagent A), and 1% CuSO4 and 2% sodium potassium tartrate in water (reagent B). Then, 0.3 mL of 50% Folin–Ciocalteau reagent was added and the mixture was incubated at 35 ◦ C for 30 min. The absorbance of the mixtures was measured at 660 nm using a spectrophotometer (G-10 UV scanning, USA). Protein content was calculated based on a bovine serum albumin standard curve and expressed as grams per 100 g dried weight of sample. 2.5.3. Determination of sulphate content Sulphate content was analyzed using a turbidimetric method [23]. A dried sample (2 mg) was digested with 0.2 mL of 1 N HCl at 95 ◦ C for 5 h. After the samples had been cooled at room temperature, 3.8 mL of 3% trichloroacetic acid was added to mixture. Barium chloride–gelatin reagent (1 mL; prepared as 2 g gelatin and 2 g barium chloride in 400 mL of water) was added and the mixture was left for 20 min at room temperature. The absorbance was then read at 360 nm. The results were calculated based on a sodium sulphate standard curve and expressed as grams per 100 g dried weight of sample. 2.5.4. Determination of total sugar Total sugar was analyzed using a phenol–sulphuric acid method [24]. Sulphated polysaccharide was dissolved in distilled water (0.03 g sample/3 mL). The supernatant (0.5 mL) was prepared by reacting with 0.5 mL of 5% phenol and 2.5 mL of concentrated sulphuric acid for 20 min, and then the absorbance was read at 490 nm. The results were calculated based on a glucose standard curve and expressed as grams per 100 g dried weight of sample. 2.5.5. Determination of monosaccharide composition Sugar composition was determined using high-performance liquid chromatography according to the procedure described by Waffenschmidt and Jaenicke [25]. A sulphated polysaccharide sample (6 mg) was hydrolysed for 4 h in 2 M trifluoroacetic acid (TFA, 0.3 mL) at 105 ◦ C. After the TFA had been removed from the sample solution by a stream of dried nitrogen, the hydrolysed sulphated polysaccharide sample was dissolved in 1 mL of distilled water and filtered through a 0.45-m filter. A 50-L sample was injected into an HPLC machine (Shimadzu LC-3A, Japan) at a flow rate of 1 mL/min through a Prevail Carbohydrate ES column (5 m, 250 mm length × 4.6 mm i.d., Alltech, USA) with acetonitrile (A) and water–acetonitrile (95:5, v/v) (B) as solvents. The ratio of solvent flow was 80 (A)/20 (B). An evaporative light scattering detector
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was used (ELSD, Alltech 3300, USA). The results were compared with authentic standard solutions of glucose, galactose, rhamnose, sucrose, arabinose, and fructose. 2.5.6. Determination of functional groups by FT-IR Samples were prepared following a previously described method [26] for functional group analysis by Fourier transform infrared spectroscopy (FT-IR, Spectrum One, Perkin Elmer, USA). Sulphated polysaccharide (0.2 g) was dissolved in 0.2% phosphate buffer at pH 6. The sample was filtered through 0.45-m filter paper, and the supernatant was analyzed using a Golden-gate Diamond single reflectance ATR in an FTS 7000 FT-IR spectrometer equipped with a DTGS (Digilab, Randolph, MA) detector. The absorbance mode was 4000–400 cm−1 . 2.5.7. Determination of molecular weight The molecular weight of the sulphated polysaccharides was determined by high-performance size exclusion chromatography (Water 600 E, USA). Sulphated polysaccharides (0.2 g) were dissolved in 10% NaCl and filtered through 0.45-m nylon cloth [27]. The supernatant was injected into an Ultrahydrogel linear column (WAT 011545, Waters, USA) at 30 ◦ C (MW resolving range 1–20,000 kDa), equipped with a guard column and a refractive index detector. The column was eluted with 0.05 M sodium bicarbonate buffer (pH 11) at a flow rate of 0.6 mL/min. The column was calibrated with standard dextran (MW: 4.4–401 kDa), and a standard curve was established before sample analysis. 2.6. In vitro antioxidant activity assay of sulphated polysaccharide 2.6.1. 1,1-Diphenyl-2-pycrilhydrazyl (DPPH• ) radical scavenging activity assay The scavenging activity for DPPH• free radicals was measured according to the method of Shimada et al. [28] with a modification. DPPH• solution was prepared at a concentration of 0.1 mM in 95% ethanol. Sulphated polysaccharide solution (2 mL) was mixed with 2 mL of the DPPH• solution. The mixture was incubated in the dark for 30 min at room temperature, prior to measurement of the absorbance at 517 nm. The ability to scavenge DPPH• was calculated as a percentage according to the following equation: % Scavenging activity = [(ADPPH − Asample+DPPH )/ADPPH ] × 100 2.6.2. 2,2 Azino-bis 3-ethyl benzothiazoline-6-sulfuric acid (ABTS•+ ) radical scavenging assay The ABTS radical cation (ABTS•+ ) was produced by reacting 7 mM ABTS•+ aqueous solution with 2.45 mM potassium persulphate at room temperature in the dark for 16 h. The solution was diluted with 95% ethanol to obtain an absorbance of 0.7 ± 0.03 at 734 nm. Then, the diluted solution (4 mL) was mixed with 1 mL of sulphated polysaccharide solution. The decrease in absorbance was recorded at 734 nm after incubation at room temperature for 6 min as described by Ree et al. [29]. Radical scavenging activity (%) was calculated using the following equation: % Scavenging activity = [(AABTS − Asample+ABTS )/ADPPH ] × 100 2.6.3. Hydroxyl radical scavenging assay Deoxyribose (60 mM, 0.2 mL), ferric chloride (1 mM, 0.2 mL), ethylenediaminetetraacetic acid (1.04 mM, 0.2 mL), and sulphated polysaccharide solution (0.2 mL) were mixed in phosphate buffered saline (PBS, 50 mM, pH 7.4, 0.8 mL). The solution was incubated at 37 ◦ C for 20 min; then, 0.2 mL of H2 O2 (0.3%) and 0.2 mL of ascorbic acid (2 mM) were added. The mixture was incubated at 37 ◦ C for
1 h. Subsequently, 2 mL of HCl (25%) and 0.2 mL of thiobarbituric acid (1%, w/v) were added. The mixture was boiled for 15 min and cooled in ice, and absorbance was measured at 532 nm according to a previously described method [30]. The radical scavenging activity was determined according to the following equation: % Scavenging activity = [(1 − Asample )/Acontrol ] × 100
2.7. Statistical analysis All experiments were performed in a 3 × 5 factorial randomized complete block design in triplicate, and the results were the average of three independent experiments. Statistical analysis was performed using Statistical Analysis System (SAS User’s Guide version 6, 4th edn., SAS Institute, Cary, NC, USA, 1990). Duncan’s multiple range test was used to determine significant differences between means (p < 0.05). 3. Results and discussion 3.1. Proximate compositions of U. intestinalis U. intestinalis is good natural source of carbohydrate, protein, and mineral, and contains low levels of lipids. Its chemical content can be influenced by the growing conditions (water temperature, salinity, light, and nutrients) [31]. In this study, U. intestinalis material contained large amounts of carbohydrate (55.43%), followed by ash (19.95%) and protein (16.95%). The high carbohydrate content was consistent with the maximum growth and maturity of seaweed when the materials were collected in July [32]. Seasonal variations in seaweed chemical composition are associated with the particular life stage. Photosynthetic activity increases in periods of maximum growth, which results in higher carbohydrate content and reduced protein content [33]. The inverse relationship between carbohydrate and protein content observed in U. intestinalis resulted from the decrease in nitrogen available for carbohydrate synthesis [33]. The ash content in U. intestinalis was high because of the high sulphate content of seaweed during the maximum growth period, when it was harvested. The high sulphate content probably partly represents ions associated with the charged polysaccharides [34]. 3.2. Effect of water, acid, and alkaline extraction on chemical composition 3.2.1. Sulphated polysaccharide yield The sulphated polysaccharide yields obtained using the different solvents for different times are shown in Fig. 1A. Solvent and extraction time had significant effects on sulphated polysaccharide yield (p < 0.05). At 24 h of extraction, 0.1 N NaOH gave the highest yield, followed by 0.1 N HCl and distilled water. The yields increased gradually as extraction time increased from 1 to 6 h, but did not significantly increase from 6 to 24 h (Fig. 1A). An explanation for the high yield with alkaline extraction was that alkaline extraction caused cellulose to swell, which disrupted the hydrogen bonds between hemicellulose and cellulose in the cell wall, resulting in the solubilization of hemicellulose. Hence, alkaline extraction could effectively release insoluble polysaccharides and convert them into soluble polysaccharides. The alkaline solvent could extract xyloglucan, which was a minor alkaline soluble polysaccharide, and glucuronan [11]. However, protein and minerals associated tightly with the polysaccharides were also extracted. The yield was lower with acid than with alkali at 6–24 h, possibly because of acid hydrolysis. The acid solution hydrolysed the polysaccharide structure, including cellulose, hemicellulose, and lignin, which released the sulphated polysaccharides [35].
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Table 1 Monosaccharide composition of crude sulphated polysaccharides from U. intestinalis extracted by water, acid (0.1 N HCl) and alkaline (0.1 N NaOH) at 80 ◦ C for 24 h. Extraction conditions
Water extraction Acid extraction Alkaline extraction
Monosaccharide compositions (%) Arabinose
Glucose
Nd 0.74 ± 0.11a Nd
11.86 ± 0.12 0.84 ± 0.02b 11.96 ± 0.10a
Rhamnose a
12.7 ± 0.10b 8.29 ± 0.14c 39.24 ± 0.12a
a,b,c Means in the same column with different letters are significantly difference at p < 0.05. Nd = not detected.
All sulphated polysaccharide extracts were contaminated with protein because of the ester sulphate moieties, which can form strong anions and attract positively charged proteins. However, the amount of protein was low. It was possible that solvent extraction could reduce protein contamination in sulphated polysaccharides. The protein content of sulphated polysaccharides with distilled water (0.48–0.63%) was lower than that with acid (0.9–2.96%) and alkaline (3.53–3.97%) extraction. There was a significant difference (p < 0.05) in the protein content among water, alkaline, and acid extraction. An increase in the extraction time from 1 to 24 h also had a significant effect (p < 0.05) on the protein content. Sulphated polysaccharides extracted with 0.1 N NaOH for 24 h had the highest protein content because of release of proteins by the breakage of hydrogen bridges in the alkaline solution.
Fig. 1. (A) Sulphated polysaccharide yield; (B) total sugar; (C) sulphate content of sulphated polysaccharides extracted from U. intestinalis with water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h. Values presented as mean ± SD (n = 3). a,b,c,. . . Means above the bars of each parameter with different letters are significantly different at p < 0.05.
However, the sulphated polysaccharides could also be hydrolysed by acid, and as they were mainly short-chain polysaccharides, this would decrease the final recovery. The lowest yield was obtained from water extraction, as water dissolves only long-chain soluble polysaccharides. Extraction time also influenced the yield of sulphated polysaccharides extracted from U. intestinalis. This finding may be explained by the early release of sulphated polysaccharides in the extraction process. In addition, the yield from acid extraction at 1–3 h was higher than that with alkaline extraction. The acid solution degraded the carbohydrate chain backbone structure of the sulphated polysaccharides when the duration of extraction increased. This result was in agreement with [18], who reported that a longer acid extraction time for polysaccharides from brown seaweed led to a decreased yield of sulphated polysaccharides. 3.2.2. Protein content Proteins have been described as potential contaminants of cell wall polysaccharides, mostly because they form part of the structure of cell walls and were closely associated with polysaccharides.
3.2.3. Sulphate content Sulphate in seaweed cell walls plays an important part in the structural strength, enabling the plant to survive in an environment with high kinetic forces such as sea water. The sulphate content of sulphated polysaccharides extracted with distilled water, acid, and alkali initially increased with increasing extraction time, but did not further increase between 6 and 24 h (p > 0.05). The lowest sulphate content was found when using acid, ranging from 36.72% to 38.35% with extraction for 1 to 24 h (Fig. 1C). Acid can eliminate the sulphate group attached to the polysaccharides [36], degrading the substituent sulphate group in the sulphated polysaccharide chain. Sulphated polysaccharides extracted with the alkaline solution had the highest sulphate content because of ester linkages. These chemical bonds between the polysaccharide chain and the sulphate group were not easily cleaved by alkaline extraction [37]. The sulphate content from distilled water extraction was higher than that from acid extraction, possibly because water extraction at high temperature may break down the ester bonds in the sulphated polysaccharide chain [38]. 3.2.4. Total sugar and monosaccharide compositions Total sugar content was determined using a phenol–sulphuric acid method as described in Section 2 [25]. Sulphated polysaccharides extracted at 80 ◦ C for 24 h with acid had the highest total sugar (38.35%), followed by those extracted using alkali (37.43%) and distilled water (11.01%). However, after 6 h of extraction, the total sugar content of sulphated polysaccharides extracted with distilled water, acid, and alkaline extraction was not significantly different (p > 0.05) (Fig. 1B). Sulphated polysaccharides extracted with acid had the highest total sugar content because the glycosidic bond in the polysaccharide chain could be hydrolysed by acid more readily than by alkali and distilled water. Moreover, the total sugar content of sulphated polysaccharides was higher with acid extraction than with alkaline extraction, and this was not related to the percentage yield of the extracts. This finding indicated that the alkaline process could extract a mixture of biopolymers such as proteins from U. intestinalis at a higher yield than acid extraction.
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Fig. 2. FT-IR spectrum of sulphated polysaccharides from U. intestinalis extracted with (A) distilled water; (B) 0.1 N HCl; (C) 0.1 N NaOH at 80 ◦ C for 24 h.
The monosaccharide profiles of sulphated polysaccharides extracted from U. intestinalis using distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 24 h were identified using HPLC (Table 1). Sulphated polysaccharides extracted with distilled water mainly consisted of rhamnose (12.70%), followed by glucose (11.86%). For acid extraction, the major monosaccharide was rhamnose (8.29%), followed by glucose (0.84%), with a trace amount of arabinose (0.74%). The low amount of arabinose acid extraction resulted from hydrolysis of arabinosyl linkages, which were probably present as side chains of the sulphated polysaccharides. Rhamnose was the predominant monosaccharide in the sulphated polysaccharides extracted with alkali and water (39.24% and 12.70%, respectively). The highest level of glucose was found with alkaline extraction. This glucose may originate from other non-sulphated polysaccharides such as cellulose or hemicellulose [39]. Generally, sulphated polysaccharides extracted from Ulva and Enteromorpha species are a group of heteropolysaccharides mainly composed of rhamnose, xylose, glucose, and sulphate, with low amounts of mannoses, arabinose, and galactose [40]. Glucuronic acid may be found in sulphated polysaccharides because carbon 6 of glucose was oxidized to a carboxyl group in the extraction process [41]. The difference in monosaccharide composition probably arose from differences in extraction procedures.
3.2.5. Functional groups FT-IR spectroscopy can be used for approximate identification of polysaccharides in plant materials when combined with chemical analyses. The FT-IR spectra of the sulphated polysaccharides extracted with distilled water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 24 h are illustrated in Fig. 2. The wide absorption at 3437 cm−1 resulted from the hydroxyl stretching vibrations and the absorption bands at 2938 cm−1 originated from C H stretching of a methyl group. The intense peaks at approximately 1625 cm−1 were attributed to C O asymmetric stretching vibration in a carbonyl group. The absorbance at 1427 cm−1 was associated with
hemicellulose and represents the C H bending or stretching frequencies. The band at 850 cm−1 was ascribed to C O S stretching of axial sulphate groups related to a C O SO3 group. The characteristic of absorptive peaks confirmed that the sulphated group had been associated with polysaccharides to form a sulphate ester [42]. The low intensity at 1126 cm−1 of sulphated polysaccharides extracted with acid indicated the presence of arabinosyl side chains. The intensity of the absorbance at 1259 cm−1 in sulphated polysaccharides extracted with distilled water was attributed to the stretching of S O. It was assumed that the main sulphate groups occupy positions C-2 or C-3, and less of the sulphate was located at C-4 of the side chain [43]. In addition, sulphated polysaccharides extracted with distilled water and alkali presented a high absorption band at approximately 1056 cm−1 , probably as the result of C O stretching from rhamnose. The FT-IR spectra of distilled water, acid, and alkali extracts showed a similar pattern in accordance with their monosaccharide composition. However, a carboxyl group from glucuronic acid was not shown in the FTIR profile of all sulphated polysaccharides, which implied that distilled water, acid, and alkaline extraction may not modify the sugar structure. This observation confirmed the results from the monosaccharide composition analysis.
3.2.6. Molecular weight Fig. 3 shows the weight-average molecular weight (Mw ) of three crude extracts of sulphated polysaccharides. Based on the calibration with standard dextran, the molecular weights of sulphated polysaccharides extracted with distilled water, 0.1 N NaOH, and 0.1 N HCl at 80 ◦ C for 24 h were 300 kDa, 110 kDa, and 88 kDa, respectively. All three extracts were mostly composed of one to two peaks. Differences in the elution profiles reflect differences in molecular size. Sulphated polysaccharides extracted with distilled water produced a unique wide peak that mainly contained high molecular mass. The molecular weight distribution of these sulphated polysaccharides was associated with polysaccharides with
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Fig. 3. High pressure size exclusion chromatographic profile of sulphated polysaccharides from U. intestinalis extracted by (A) distilled water; (B) 0.1 N HCl; (C) 0.1 N NaOH at 80 ◦ C for 24 h.
sulphate. In contrast, the profiles of acid and alkaline extraction (Fig. 3B and C) presented two peaks because of contamination by linear xyloglucan and co-elution of glucuronan with proteins [12]. The molecular weight of sulphated polysaccharides extracted with acid and alkali was obviously lower than that of sulphated polysaccharides extracted with distilled water, indicating that the polysaccharide chains may have been disrupted and degraded during the acid and alkaline extractions. 3.3. Effect of water, acid, and alkaline extraction on antioxidant activity 3.3.1. DPPH• radical scavenging of sulphated polysaccharides DPPH• is a free-radical compound that has been widely used to determine the free radical scavenging ability of antioxidants. DPPH• is a stable free radical, and the reduction of DPPH• to DPPH-H in the presence of a proton-donating substance can be used as an indicator of antioxidant activity. At a concentration of 3 mg/mL, sulphated polysaccharides extracted with distilled water, acid, and alkali showed scavenging abilities higher than 50% for DPPH• radicals (Fig. 4A). The presence of the sulphate group likely decreases the bond energy of the C H in the vicinity of the glycosidic bond, and then increases the hydrogen atom donation capability [44]. Although sulphate content and molecular weight were low after extraction with 0.1 N HCl for 24 h, these sulphated polysaccharides had significantly higher (p < 0.05) DPPH• scavenging abilities than sulphated polysaccharides extracted with alkali (55.97%) or distilled water (56.18%). These results indicated that the molecular weight of polysaccharides plays an important role in their bioactivity, and a relatively low molecular weight appears to increase the antioxidant activity. Similar results have been found for polysaccharides from other plants [45], and it was believed that low-molecular-weight polysaccharide molecules have more reductive OH terminals available for reacting with radical species. According to Shao et al. [46], the lowest molecular weight of sulphated polysaccharide from brown seaweed had the highest DPPH• radical scavenging activity. In addition, monosaccharide property indicated that arabinose of the side chain in the sulphated polysaccharide extracted with acid was related to the scavenging effect on DPPH• radicals [15]. DPPH• radical scavenging was not significantly different (p > 0.05) for sulphated polysaccharides extracted with alkali and distilled water. Although alkaline-extracted sulphated
Fig. 4. (A) DPPH• radical scavenging (%); (B) ABTS•+ radical scavenging (%); (C) hydroxyl radical scavenging (%) of sulphated polysaccharides extracted from U. intestinalis with water, 0.1 N HCl, and 0.1 N NaOH at 80 ◦ C for 1, 3, 6, 12, and 24 h. Values presented as mean ± SD (n = 3). a,b,c,. . . Means above the bars of each parameter with different letters are significantly different at p < 0.05.
polysaccharides had higher sulphate content, the molecular weight was lower than that for water-extracted sulphated polysaccharides. This result indicated that not only sulphate content but also other factors such as the position of sulphate groups and monosaccharide content contribute to the antioxidant activity. Moreover, a higher glucose level was found in sulphated polysaccharides extracted with alkali and distilled water than in those extracted with acid. These findings suggested that high glucose content affects DPPH• radical scavenging activity. The scavenging ability of sulphated polysaccharides extracted with alkali and distilled water may be attributed to the presence of hydrogen atoms from the specific monosaccharide compositions and their side chain linkages. These results agreed with those of Lo et al. [47] who reported that the monosaccharide composition (arabinose, mannose, and glucose) of polysaccharides was markedly associated with the free-radical scavenging ability. 3.3.2. ABTS•+ radical scavenging The antioxidant capacity of the sulphated polysaccharides with respect to an electron transfer reaction was assayed by using the
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ABTS•+ radical cation, which presents a strong absorbance in the visible region. The absorbance of the ABTS•+ solution decreased when an electron was donated by an antioxidant to quench the free-radical. Sulphated polysaccharides extracted with acid had the highest radical scavenging activity (71.87%), followed by those extracted with distilled water (68.06%) and alkali (61.01%) (Fig. 4B). The polysaccharides extracted with alkali with the high sulphate content presented the lowest activity. This finding suggested that the chemical structure of the polysaccharides plays a role in the H abstraction reaction by the ABTS•+ cation radical [44]. The results agreed with a previous study that observed a decrease in the activity towards the radical cation with an increase in sulphate content for commercial -, -, and -carrageenans [44]. Sulphated polysaccharides extracted with distilled water with a high molecular weight (300 kDa) and the lowest total sugar/protein ratio (10.08%) had the highest ABTS•+ radical scavenging. It was found that antioxidant activities of sulphated polysaccharide might depend on the amount of bound protein present in the form of polysaccharide–protein complexes [15]. According to Gómez˜ Ordónez et al. [48] the highest MW (1248–1425 kDa) galactan fraction with a low content of sulphate groups (3.9–1.9%) showed significant scavenging activity towards ABTS•+ . The results in this study and previous reports indicated that the reaction of sulphated polysaccharides with ABTS•+ radicals is complex. Moreover, no relationship was found between sulphate content and antioxidant capacity. Structural features such as monosaccharide composition, molecular weight, and position of sulphate groups were involved in antioxidant activity. 3.3.3. Hydroxyl radical scavenging The hydroxyl radical scavenging activity of sulphated polysaccharides extracted from U. intestinalis was investigated by measuring their ability to prevent oxidative degradation of deoxyribose substrates (Fig. 4C). A similar trend to the DPPH• radical scavenging results was observed, with the highest activity (54.15%) found in the sulphated polysaccharides extracted with acid for 24 h. Although sulphated polysaccharides extracted with distilled water and alkali had higher sulphate content than those extracted with acid, their hydroxyl scavenging ability was weaker. Hydroxyl radical scavenging activity of sulphated polysaccharide in this study was different from red seaweed, in that high sulphate content had greater hydroxyl radical scavenging activity than low sulphate content [49]. Interestingly, hydroxyl radical scavenging activities of sulphated polysaccharide from green seaweed related to its low molecular weight [50]. Previous studies suggested that the scavenging activity for hydroxyl radicals resulted from inhibition of hydroxyl radical generation by chelating ions such as Fe2+ and Cu+ [51]. The sulphate molecules could chelate ions in a Fenton reaction, which may be responsible for the inhibition of deoxyribose oxidation. The hydroxyl radical scavenging activity was probably related to high sulphate content (specific chelating groups) within the molecule because of their high nucleophilic character [52]. However, the sulphated polysaccharides with low sulphate and low molecular weight extracted with acid had the highest hydroxyl radical scavenging activity, possibly because of the molecular weight, which has a direct influence on solubility and viscosity in solution, thus improving the antioxidant activity. Most of sulphated polysaccharide from U. intestinalis had the strongest scavenging activity against ABTS•+ radicals based on electron transfer mechanism [53] of negative charge of the sulphate groups [54], which was slightly higher from that of DPPH• and hydroxyl radical scavenging activity. Moreover, the molecular weight of sulphated polysaccharides is a more important factor for scavenging free radicals than sulphate content [54]. This response might be due to non-compact structure of low-molecular weight sulphated polysaccharides and consequently the more potentially
available electron transport reacting with free radicals [55]. This observation was consistent with antioxidant activity of sulphated polysaccharide from Ulva fasciata which had a prominent ABTS•+ radical scavenging activity [46]. A similar trend in higher scavenging activity in ABTS•+ than DPPH• radical was also found in sulphated polysaccharide from Sargassum horneri [46], which sulphated polysaccharide could donate hydrogen react with free radical by hydrogen transfer reaction as possible antioxidant mechanisms [56]. According to Sun et al. [50], sulphated polysaccharides from Porphyridium cruentum exhibited the strongest DPPH• radical scavenging activity. The results obtained in this study clearly demonstrate that antioxidant activities of polysaccharides were not a function of a single factor, but a combination of several factors, such as, degree of sulfating, molecular weight, type of the major sugar, and glycosidic branching [57,58]. 4. Conclusions The chemical and antioxidant characteristics of sulphated polysaccharides were affected by the solvents and extraction time. Distilled water, acid, and alkaline extraction did not change the main structure of the sulphated polysaccharides, resulting in monosaccharide ratio variation. The sugar backbone of the sulphated polysaccharides from all extractions was composed of rhamnose and glucose, containing sulphate at the C2 or C3 positions of rhamnose. Although sulphated polysaccharides extracted from U. intestinalis with three solvents also exhibited potent antioxidant activities, sulphated polysaccharide extracted with 0.1 N HCl at 80 ◦ C for 24 h had distinctly different both in molecular weight and chemical characterization from sulphated polysaccharide extracted with distilled water and alkaline which enhanced the antioxidant activity. It was clear that their antioxidant activities were dependent on their fine structural features, which will certainly present a good feasibility to elucidate the antioxidant activity of sulphated polysaccharide. This information is important for the regional development of value-added products, particularly as ingredients in functional foods and nutraceutical products. Acknowledgments This work was supported by the Thailand Institute of Scientific and Technological Research, National Research University (NRU) Project of Thailand, and King Mongkut’s University of Technology Thonburi, Thailand. References [1] H.S. Hayden, J. Blomster, C.A. Maggs, P.C. Silva, M.J. Stanhope, J.R. Waaland, Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera, Eur. J. Phycol. 38 (2003) 277–294. [2] C.S. Kumar, P. Ganesan, P.V. Suresh, N. Bhaskar, Seaweeds as a source of nutritionally beneficial compounds: a review, J. Food Sci. Technol. 45 (2008) 1–13. [3] L. Wang, X. Wang, H. Wu, R. Liu, Overview on biological activities and molecular characteristics of sulfated polysaccharides from marine green algae in recent years, Mar. Drugs 12 (2014) 4984–5020. [4] B. Ray, Polysaccharides from Enteromorpha compressa: isolation, purification and structural features, Carbohydr. Polym. 66 (2006) 408–416. [5] V. Ivanova, M.K. Rousevar, J. Serkedjieva, R. Rachev, N. Maolova, Isolation of a polysaccharide with antiviral effect from Ulva lactuca, Prep. Biochem. Biotechnol. 242 (1994) 83–97. [6] F. Chiellini, A. Morelli, A versatile platform of biomaterials from renewable resources, in: R. Pignatello (Ed.), Biomaterials – Physics and Chemistry, InTech, Rijeka, 2011, pp. 75–98. [7] L.S. Costa, G.P. Fidelis, S.L. Cordeiro, R.M. Oliveira, D.A. Sabry, R.B.G. Camara, T.D.B. Nobre, M.S.S.P. Costa, J. Almeida-Lima, E.H.C. Farias, E.L. Leite, H.A.O. Rocha, Biological activities of sulfated polysaccharides from tropical seaweeds, Biomed. Pharmacother. 64 (2010) 21–28. [8] Y. Wang, M. Zhang, D. Ruan, A.S. Shashkov, M. Kilcoyne, A.V. Savage, L. Zhang, Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos, Carbohydr. Res. 339 (2) (2004) 327–334.
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