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1 雨生红球藻多糖的提取分离及理化性质研究冯以明 1,2, 李广生 1,2, 吴建东 1,2, 王培培 1,2, 赵峡 1,2 1,2, 于广利 (1. (), ; 2., ) 摘要 : 将雨生红球藻 (Haematococcus pluvialis) 经乙醇 / 乙酸乙酯 (1:2, V/V) 脱脂, 通过冷水热水和 5% 碳酸钠提取, 得到 3 种粗多糖 (HPC HPH 和 HPA), 通过 Q-Sepharose FF 阴离子交换柱进一步分级, 从 HPC 中得到 HPC1 HPC2 和 HPC3 共 3 种多糖组分运用高效液相色谱法 (HPLC) 红外光谱(IR) 等方法对其单糖组成相对分子质量和基本结构特征进行分析比较结果表明, 各组分单糖组成复杂, 主要含有半乳糖 (Gal) 甘露糖(Man) 阿拉伯糖(Ara) 葡萄糖(Glc) 葡萄糖醛酸(GlcUA) 半乳糖醛酸 (GalUA) 木糖(Xyl) 鼠李糖(Rha) 岩藻糖(Fuc) 葡萄糖胺(GlcN) 和半乳糖胺 (GalN), 其中, Gal 含量最高, 大于 20% 多糖 HPC1 HPC2 和 HPC3 的相对峰位分子质量分别为 502.6, 373.2, 和 ku 关键词 : 雨生红球藻 (Haematococcus pluvialis); 多糖 ; 理化性质 ; 单糖组成中图分类号 : Q ; R 文献标识码 : A 文章编号 : (2012) (Haematococcus pluvialis),,,, [1],,,,,, 1 实验材料与方法 1.1 材料与仪器 (Haematococcus pluvialis), Cyanotech ( ku) Shodex ; (Glc, Man, Gal, Fuc, Rha, GlcN, GalN, Xyl, Arb, GlcUA, GalUA) (PMP, 99%) Sigma ;, (Q-Sepharose Fast Flow, 90 μm, GE Healthcare ); (PL aquagel-oh mixed, 30 cm 7.5 mm 8 μm, Perkin Elmer ); Eclipse XDB-C18 (4.6 mm 150 mm, 5 μm, Agilent ); (LC-20AD, ); (Nicolet Nexus 470, Thermo Electron ); (UV-2102 PCS, ( )) 1.2 实验方法 [2] / (1:2, V/V) 3 [3], 40, 30, 3 h 2, 4 95%,, 45, HPC, 30, 85 3 h 2 : ; : : ( ; ); ( ) ; (IRT0944) : (1986-),,,, : ;,,, : , glyu@ouc.edu.cn Marine Sciences / Vol. 36, No. 1 /

2 , 4 95%,, HPH, 20 5%, h 2 4 mol/l ph 7 8, 4 95%,, HPA 3, (MWCO 7kD)2 d,,, 1 HPC Q-Sepharose Fast Flow (XK 3.5/10, V=150 ml) [4] mol/l NaCl 2 ( 3 ml /min, 15mL/ ), [5],,, HPC1 HPC2 HPC3 3 Fig. 1 1 Extraction and separation flow chart of polysaccharides from Haematococcus pluvialis [5] ; - [6] ; Folin- [7] ; [8] [9] : (PL aquagel-oh mixed, 8 μm, 30 cm 7.5 mm); 0.2 mol/l NaNO 3 ( 10 mol/l NaH 2 PO 4, ph 7.0), 0.5 ml/min, 30 ; 10 μl; 18 / 2012 / 36 / 1

3 (lgm W ) (t R ), lgm W = t R, R 2 =0.996 [2, 10] (PMP-HPLC) : 2 mg, 2 mol/l (TFA)1 ml,, h, : Eclipse XDB-C18 (4.6 mm 150 mm, 5 μm); (83:17, V/V), 1.0 ml/min, 25 ; 20 μl; 245 nm %,, 20 min, 10 μl, (0.5 μl), 10 min, : 0.05 mol/l (ph 5.0), 7 ma, 45 min 0.5%(W/V) 20 min, 2% P 2 O 5 48 h, 1 2 mg KBr,, cm 1 2 结果与讨论 2.1 理化性质分析, V A,, [3], (1:2, V/V), 30.7% 3,, 2.74% 1.36% 1.55% HPC (69.8%) (10.9%); HPA (33.2%) (23.5%); 3 (4% ),, 3,, HPC QFF, 2 2, HPC mol/l NaCl, 3 ( HPC1 HPC2 HPC3) HPC1, HPC2 HPC3 HPC1 (84.2%), HPC3 (14.0%) 1 表 1 雨生红球藻中多糖的基本理化性质比较 Tab. 1 The physicochemical characters comparison of polysaccharides (%) (%) (%) (%) HPC HPH HPA HPC HPC HPC 单糖组成分析 [10], PMP HPLC,, 245 nm, PMP-HPLC 3, 2, Fig. 2 2 Q-Sepharose FF Segment elution graph of crude polysaccharide by Q-sepharose fast-flow column Marine Sciences / Vol. 36, No. 1 /

3 3 Fig. 3 The monosaccharides analysis of three kinds of polysaccharides 1. Man 2. GlcN 3. Rha 4. GlcUA 5. GalUA 6. GalN 7. Glc 8. Gal 9. Xyl 10. Ara 11. Fuc 12.unkown 表 2 3 种多糖的主要单糖组成及其相对摩尔比 Tab.

4 3 3 Fig. 3 The monosaccharides analysis of three kinds of polysaccharides 1. Man 2. GlcN 3. Rha 4. GlcUA 5. GalUA 6. GalN 7. Glc 8. Gal 9. Xyl 10. Ara 11. Fuc 12.unkown 表 2 3 种多糖的主要单糖组成及其相对摩尔比 Tab. 2 The monosaccharides composition and relative molar ratio analysis of three polysaccharides (%) Gal Man Ara Glc GlcUA GalUA Xyl Rha Fuc GlcN GalN (ku) HPC , HPC HPC , Gal Man Glc Ara Xyl, GlcUA GalUA, Rha Fuc, GlcN GalN HPC1 Man Ara 20%, HPC2 HPC3 Glc 18%HPC3 (GlcUA 8.4% GalUA 1.7%) Fuc (11.4%), QFF HPC1 HPC2 Man ( 12), 2.3 醋酸纤维素薄膜电泳纯度分析 HPC HPC1 HPC2 HPC3 ( 4) HPC1,,, IR ; HPC2 HPC, ; HPC3 3, 2.4 红外光谱分析 5, HPC1 HPC2 HPC3, 3600~ 3200 cm cm 1 ; 1640 cm cm 1 C O,,, HPC3 ; 1240 cm 1 S O, HPC1, HPC2, HPC3, 1 Fig HPC 1. HPC1 2. HPC2 3. HPC3 4. CSA 4 Cellulose acetate membrane electrophoresis graph of different polysaccharides 20 / 2012 / 36 / 1

5 Fig The IR spectrum comparison of three kinds of polysaccharides 51(4): [2] YU Guangli, YANG Bo, ZHAO Xia, et al. A comparative analysis of four kinds of polysaccharides purified from Furcellaria lumbricalis [J]. Journal of Ocean University of China, 2007, 6(1): [3] Yuan J P, Chen F. Chromatographic separation and purification of trans-astaxanthin from the extracts of Haematococcus pluvialis [J]. Journal of Agricultural and Food Chemistry, 1998, 46(8): [4],,,. [J]., (13): [5] DuBois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination of sugar and related substances [J]. Analytical Biochemistry, 1956, 28(3): 结论 (Haematococcus pluvialis), 3, HPC 3,, [10],, : [1] Margalith PZ. Production of ketocarotenoids by microalgae [J]. Applied Microbiology Biotechnology, 1999, [6] Therho T T, Hartiala K. Method for determination of the sulfate content of glycosamino glycans [J]. Analytical Biochemistry, 1971, 41(2): [7] Lowry O H, Rosebrough N J, Farr A L, et al. Protein measurement with the Folin Phenol reaction [J]. The Journal of Biological Chemistry, 1951, 193: [8],. 9- [J]., 1962, 1: [9],,,. [J]., 2007, 28(2): [10],,,. [J]., 2008, 27(4): Marine Sciences / Vol. 36, No. 1 /

6 Extraction and isolation of polysaccharides from Haematococcus pluvialis and their physicochemical characters study FENG Yi-ming 1, 2, LI Guang-sheng 1, 2, WU Jian-dong 1, 2, WANG Pei-pei 1, 2, ZHAO Xia 1, 2, YU Guang-li 1, 2 (1. Key Laboratory of Marine Drugs (Ocean University of China), Ministry of Education, Qingdao , China; 2. Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering, Qingdao , China) Received: Jan., 27, 2011 Key words: Haematococcus pluvialis; polysaccharide; Extraction; isolation; monosaccharide composition Abstract: Three kinds of crude polysaccharides HPC, HPH and HPA were obtained from aqueous extracts of the green alga Haematococcus pluvialis with cold water, hot water and 5% Na 2 CO 3 water solution. The crude extracts were fractionated by ion-exchange chromatography of Q-Sepharose Fast Flow column. The purified extracts were named HPC1, HPC2 and HPC3. The monosaccharide composition, relative molecular mass (Mw) and structural feature of the polysaccharides were analyzed and compared by high-performance liquid chromatography (HPLC) and Fourier Transform Infrared spectroscopy (FTIR), respectively. The results showed that the monosaccharide composition was complicated and the main components included Galactose, Mannose, Arabinose, Glucose, Glucuronic acid, Galacturonic acid, Xylose, Rhamnose, Fucose, Glucosamine and Galactosamine. In particular, the content of Galactose was the highest and it s higher than 20%. The relative molecular masses of polysaccharides HPC1, HPC2 and HPC3 were 502.6, 373.2, and ku, respectively. ( 本文编辑 : 康亦兼 ) 海洋科学 2012 年征订启事,,, : ( ), 15, 16, 96, 30, 360 ( : ISSN ; : CN /P; : 2-655; : M6666), 20,,,, : 7, : pxzhang@qdio.ac.cn 22 / 2012 / 36 / 1

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0.

DOI /j.issn Food Research And Development UPLC-MS/MS 5 0. 118 217 5 38 1 DOI 1.3969/j.issn.5-6521.217.1.28 Food Research And Development 1 2 1 2 1 2 1 2 1 2 1 2 1. 731 2. 731 - UPLC-MS/MS 5. 1 mol/l ProElut PXC 5 % - Thermo Hypersil Gold mm 2.1 mm 1.9 μm ESI