Extraction, Isolation and Structural Characterization of ... - Springer Link

J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-010-0193-7 ISSN 1672-5182, 2010 9 (2): 193-197 http://www.ouc.edu.cn/xbywb/ E-mail:[email protected]

Extraction, Isolation and Structural Characterization of Polysaccharides from a Red Alga Gloiopeltis furcata YU Guangli*, HU Yannan, YANG Bo, ZHAO Xia, WANG Peipei, JI Guoli, WU Jiandong, and GUAN Huashi Laboratory of Glycoscience and Glycoengineering, School of Medicine and Pharmaceutics, Ocean University of China, Qingdao 266003, P. R. China (Received January 7, 2010; revised January 18, 2010; accepted January 31, 2010) © Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2010 Abstract Three kinds of polysaccharides: GFW, GFH and GFA, were sequentially extracted from a red alga Gloiopeltis furcata with 25℃ and 85℃ water, and 60℃ 4% NaOH water solution. Based on the defatted alga, the yields of the polysaccharide were 57.9%, 2.5% and 2.6%, respectively. Their monosaccharide compositions, average molecular weights and structural characters were determined by gas chromatography (GC), high performance liquid chromatography (HPLC), fourier transform infrared spectroscopy (FTIR) or 13C-NMR spectroscopy. The results showed that GFW, GFH and GFA were all composed of D-galactose (Gal) and 3,6-anhydro-L-galactose (AnG), and particularly GFA also contained xylose (Xyl). The average molecular weights of GFW, GFH and GFA were 22.6 kD, 26.5 kD and 49.8 kD, respectively, with the respective sulfate content 31.2%, 25.1% and 22.7%. The data of FTIR and 13C-NMR confirmed the sulfate ester location at C6 of galactose. It is concluded that all the three polysaccharides extracted from Gloiopeltis furcata were sulfated galactans, two being sulfated-agarose, and one being xylose-containing sulfated galactan. Key words

Gloiopeltis furcata; sulfated galactan; extraction; structural characterization

1 Introduction

2 Materials and Methods

Gloiopeltis furcata is an annual red alga and belongs to the genus Gloiopeltis of the family of Endocladiaceae. The water-soluble extract from this important marine algae was used as pulp of textile as early as in the Song Dynasty, and used as traditional medicine to treat dysentery and colitis in Guangdong, P. R. China; It was also approved as a safe food thickener in Japan. Polysaccharides with different activities (i.e. antitumor, antiviral, anti-inflammation) were found in water and ethanol extracts from G. furcata (Niu et al., 2003; Yin et al., 1988; Song and Yung, 2007). Moreover, the polysaccharide from G. furcata was confirmed to be agar by Hirase and Araki (1958) and Izumi (1973). Ji and Gu (1987) also found sulphated agarose from G. furcata. However, the polysaccharide structure of G. furcata originating from different area, has not been studied in detail. In this paper, three types of polysaccharides were obtained from G. furcata with different extraction methods and their structural characters were analyzed and compared based on the physicochemical studies. These data are useful for future studies on G. furcata polysaccharide and its structure-activity relationship.

2.1 Materials The dried red alga Gloiopeltis furcata (originating from Fujian, P. R. China) was purchased from Kunshan Yihong Seaweed Co. Ltd, (Jiangsu, P. R. China) and identified by Professor Gong Xiangzhong in College of Marine Life Sciences, Ocean University of China. Rhamnose, mannose, arabinose, fucose, xylose, galactose, glucose, 3,6-anhydro-galactose, methylmorpholineborane (MMB) and BSA were purchased from Sigma Company (Shanghai, China). Dextran standards (788 kD, 404 kD, 212 kD, 112 kD, 47.3 kD, 22.8 kD, 11.8 kD, 5.9 kD) were purchased from Shodex Company (Tokyo, Japan). 1,1-Diethoxyethane was purchased from J&K Chemical (Beijing, China). All the reagents were of analytical reagent grade. PL aquagel-OH column (30 cm×7.5 mm, 8 μm) was from Perkin Elmer Company (Massachusetts, USA). Fused Silica Capillary Column DB-225 (30 m×0.32 mm, 0.25 μm) was purchased from J&W Scientific (Folsom, CA, USA). Other equipment used included HPLC (LC-20AD, Shimadzu Company, Japan), GC (Agilent HP5890Ⅱ, USA), FTIR spectrometer (Nicolet Nexus 470, Thermo Electron, USA), NMR Spectrometer (JNM-ECP 600, Jeol, Japan), Spectrophotometer (UV-2102 PCS,

* Corresponding author. Tel: 0086-0532-82031560 E-mail: [email protected]

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YU et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2010 9: 193-197

Unico, Japan), and Freeze-dryer (EZ50, FTS company, USA).

2.2 Extraction and Fractionation Extraction of polysaccharides was carried out by the procedure previously described (Yu et al., 2007). The red alga was dried at 40℃, and then pulverized and passed through a 40 mesh sieve. The treated alga (100 g) was extracted two times with 85% ethanol at 80℃ and the defatted alga powder (87.1 g) was obtained. The defatted alga was extracted three times with distilled water at 25℃ for 3 h and centrifuged. The supernatant was rotaryevaporated, concentrated and precipitated with 4 volumes of ethanol. The precipitated polysaccharide was freezedried and named as GFW. The algal residue was further extracted three times with hot distilled water at 85℃ for 3 h and centrifuged; the supernatant was treated with the same method as the above mentioned to obtain the polysaccharide, namely GFH. The algal residue was then soaked in 4% NaOH solution and extracted two times at 60℃ for 2 h. The supernatant was neutralized, dialyzed and precipitated with 4 volumes of ethanol. The precipitated polysaccharide was freeze-dried and named as GFA. 2.3 Determination of Average Molecular Weight The average molecular weight (Mw) of the three kinds of polysaccharides, following the methods of Wei and Fang (1989), was determined by HPLC with a PL aquagel-OH column (30 cm×7.5 mm, 8 μm, Perkin Elmer Company, USA), eluted with 0.1 molL-1 Na2SO4 (pH 7.0) at a flow rate of 1.0 mL min-1 at 35℃, and detected with RI detector. The column was calibrated with Dextran standards, and the regression equation was logMW =14.8636−0.6837tR, where tR is retention time; r2 = 0.998.

2.4 Total Sugar and Protein Content Analysis Total sugar content was determined by phenol-sulfuric acid method using galactose as standard (Dubois et al., 1956). The content of galactose was determined by anthrone-sulfuric acid method. The content of 3, 6-anhydrogalactose (AnG) was determined by resorcinol method using fructose as standard (Yaphe and Arsenault., 1965). The Xylose content was determined by orcinol method. Sulfate content was determined by BaCl2-Gelatin method (Dodgson and Price, 1962). The content of crude protein was determined by Lowry method (Lowry et al., 1951). 2.5 Monosaccharide Composition Analysis As the traditional acid hydrolysis method causes damage to AnG, a two-step reductive hydrolysis method was used in this study, with MMB used as a reducing agent (Yu et al., 2007; Stevenson and Furneaux, 1991). The sugar alditol was acetylated with a mixture of 500 μL EtOAc, 1.5 mL Ac2O, and 50 μL HClO4. The mixture was sonicated at room temperature for 10 min and standed for

another 15 min. After addition of 5 mL distillated water, the water phase was extracted with 2 mL dichloromethane (DCM). The DCM fraction, which contained the alditol acetates, was extracted with 5 mL water for three times, and the DCM layer was used for GC analysis. The alditol acetates were analyzed by GC using a Fused Silica Capillary Column DB-225. The sample was detected with flame-ionization detector (FID) at 250℃, with the injector and the oven temperatures set at 250℃ and 210℃, respectively. For GC analysis, composition and content of monosaccharide were determined by retention time and peak area, in comparison with standard sugars.

2.6 FTIR and 13C-NMR Spectroscopy Analysis For FTIR analysis, the samples (1−2 mg) were kept in P2O5 desiccator for 48 h, mixed with 100 mg KBr and then pressed under 7 kg cm-2 to make a transparent film (Yu et al., 2007). The film was put into the FTIR instrument and scanned from 400 to 4000 cm–1. For 13C-NMR analysis, the sample (10 mg) was dissolved in 0.5 mL D2O and freeze-dried twice to replace all exchangeable protons with deuterium. The 13C spectrum was recorded on a JNM-ECP 600 spectrometer, with acetone-d6 serving as an internal reference.

3 Results and Discussion 3.1 Polysaccharides Extraction and Fractionation This method eliminated not only lipids but also impurities, including amino acids，fatty acids, salts, etc. Three types of polysaccharides, GFW, GFH and GFA, were extracted from G. furcata. Based on the defatted alga, the yield (57.9%) of GFW of defatted alga was highest, followed by GFH (2.5%) and GFA (2.6%). The total yield of the polysaccharides was 63%, of which the GFW accounted for up to 92%. 3.2 Basic Physicochemical Analysis In order to compare the physicochemical properties of polysaccharides extracted by different methods, the total sugar, sulfate, AnG, galactose and crude protein content were analyzed, and their average molecular weights were also determined by HPLC. As shown in Table 1, GFW had the highest sulfate (31.2%) and the lowest crude protein (2.8%); GFH had the highest crude protein (6.9%); GFA had the highest AnG (34.6%) and the lowest sulfate (22.7%). The average molecular weights of GFW, GFH and GFA were 22.6 kD, 26.5 kD and 49.8 kD, respectively. These polysaccharides had different properties, but all were sulfated galactan. The monosaccharide composition analysis of polysaccharide is very important. Owning to the instability of AnG in acid environment, the reductive hydrolysis is needed to avoid AnG being changed to 5-Hydroxymethylfurfural (5-HMF) and other products under acid hydrolysis condition (Quemener and Lahaye, 1998; Yang et al., 2009). The monosaccharide composition of the three kinds of polysaccharides were hydrolyzed with the reduc-

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tive hydrolysis and determined by gas chromatography (GC). Compared with Gal and AnG standard, GFW was composed of AnG and Gal in a molar ratio of 1:1.2, GFH

was composed of AnG and Gal in a molar ratio of 1:1.6, and GFA was composed of AnG and Gal in a molar ratio of 1:0.8 but with a small amount of xylose (0.2) (Table 1).

Table 1 Comparison of basic physicochemical characters of three kinds of polysaccharides Sample Appearance Yield of defatted alga (%) Total sugar (%) Galactose (%) 3,6-Anhydrogalactose (%) Xylose (%) Sulfate (%) Crude protein (%) Monosaccharides molar ratio Relative molecular weight ( kD)

GFWa

GFHb

GFAc

White, powder 57.9 66.3 37.9 28.4 − 31.2 2.8 AnG:Gal 1:1.2 22.6

Light brown, powder 2.5 67.1 42.9 24.2 − 25.1 6.9 AnG:Gal 1:1.6 26.5

Brown, powder 2.6 71.9 30.8 34.6 8.5 22.7 4.3 AnG:Gal:Xyl 1:0.8:0.2 49.8

Notes: GFWa: The polysaccharide was extracted from a red alga Gloiopeltis furcata by water (25℃). GFHb: The polysaccharide was extracted from a red alga Gloiopeltis furcata by 85℃ water. GFAc: The polysaccharide was extracted from a red alga Gloiopeltis furcata by 4% NaOH at 60℃ water solution.

3.3 FTIR Analysis The characters of the three kinds of polysaccharides were also compared by FTIR. The IR spectroscopy (Fig.1) of GFW, GFH and GFA all showed peaks at 930, 1250 and 820 cm-1, but the absorption strength was different.

The peaks at 1250 and 820 cm-1 indicated the presence of sulfate groups. The peaks at 930 and 820 cm-1 suggested the presence of AnG and axial sulfate ester at C6 of D-galactopyranose (G6S), respectively. These characteristics indicated all these polysaccharides were sulfated galactan.

Fig.1 The FTIR spectra of GFW (a), GFH (b) and GFA (c).

3.4 13C-NMR Analysis The red alga polysaccharides are usually classified as agar and carrageenan, and the major difference lies in the configuration of AnG residue. The AnG in carrageenan is D-form, but in agar it is L-form. The anomeric signal of L-AnG is at (95−100)×10-6, while D-AnG is at (90−95) ×10-6. As shown in Fig.2, the three kinds of polysaccha-

rides in this study were classified as agaran, since all C-1 signals of AnG were about 98×10-6. The unsubstituted C-6 signal of D-Gal, shifting from 61×10-6 to 67×10-6, indicated the substitution of sulfate groups at the C-6 position, and this result was consistent with the IR data. The signal of 58.73×10-6 is C6-O-Me of Gal. The C-5 signal of xylose was found at 58×10-6, and all the other carbon signals (61.12−102.31)×10-6 were shown in Table 2.

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Fig.2 The 13C-NMR spectra of GFW (a), GFH (b) and GFA (c). Table 2 The 13C-NMR signals assignment of GFW, GFH and GFA Sample

Residues

Signals (×10-6)

GFW

Gal AnG†† G6S†††

C1 102.31 98.10 102.56

GFH

Gal AnG G6S

102.30 98.08 102.52

69.87 69.49 70.10

81.98 79.90 83.46

68.21 77.34 68.09

75.40 75.61 72.73

61.13 69.17 67.31

GFA

Gal AnG G6S Xyl

102.30 98.32 102.47 93.49

69.84 69.49 70.06 72.13

81.98 79.87 83.38 72.99

68.61 77.66 68.21 79.63

75.40 75.61 72.85 61.31

61.23 69.18 67.31 −

†

C2 69.69 69.30 70.49

C3 81.77 79.70 83.19

C4 68.48 77.39 68.04

C5 75.36 75.21 72.55

C6 61.12 69.00 67.12

Note: †: D-Galactose; ††: 3,6–anhydro-L-Galactose; †††: 6-sulfated-D-Galactose.


In summary, the three kinds of polysaccharides, i.e. GFW, GFH and GFA, were extracted from a red alga Gloiopeltis furcata by successive extractions from 25℃ and 85℃ water, and 60℃ 4% NaOH solution. They were all identified as sulfated polysaccharides by chemical methods, using GC, HPLC, FTIR or 13C-NMR spectroscopy. GFW and GFH were composed of D-galactose and 3,6-anhydro-L-galactose. Apart from Gal and AnG, a small amount of xylose was found to exist in GFA. These results offered scientific evidence for the development of Gloiopeltis furcata polysaccharides and their structure-activity relationship.

Acknowledgements This study was supported in part by the International Science and Technology Cooperation Program of China (2007DFA30980), the National High Technology Research and Development Program (2007AA09Z445), and the National Natural Science Foundation of China (30870506).

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