Food Sci. Biotechnol. 24(1): 23-30 (2015) DOI 10.1007/s10068-015-0004-4
RESEARCH ARTICLE
Isolation, Structural Characterization, and Lymphopoiesis Stimulant Activity of a Polysaccharide from the Abalone Gonad Jing-feng Yang, Yue-hui Li, Jun Zhao, Peng-fei Li, Ce Zhu, Ye-han Song, Lan-yi Zhang, and Bei-wei Zhu
Received March 26, 2014; revised July 24, 2014; accepted July 29, 2014; published online February 28, 2015 © KoSFoST and Springer 2015
Abstract A novel polysaccharide (AGP-32) from the gonad of Haliotis discus hannai Ino was isolated using a protease-assisted process and successive ion-exchange and gel-filtration chromatography. The backbone of AGP-32 was determined using hydrolysis with trifluoroacetic acid. FTIR, NMR, and methylation analysis, and periodate oxidation and Smith degradation analysis revealed that the AGP-32 backbone mainly consisted of (1→6)-linked mannose, (1→3)-linked galactose, and (1→3)-linked glucose in a proportion of 2:3:1. An in vitro cell assay indicated that AGP-32 promoted mice splenic lymphocyte proliferation by 26% at a concentration of 50 µg/mL. AGP-32 had an effect on immune protection and is a candidate for consideration as a functional food. Keywords: abalone, gonad, polysaccharide, backbone, lymphocyte
Introduction Haliotis discus hannai Ino, a large, single-shelled abalone and a marine mollusk of the genus Haliotis, is widely cultured in eastern Asia and is considered to be a health food in China. Many bioactive compounds isolated from abalone have been reported with biological activities, including antiviral agents (1), antidepressant substances (2) and antioxidant peptides (3). Jing-feng Yang, Yue-hui Li, Jun Zhao, Peng-fei Li, Ce Zhu, Ye-han Song, Lan-yi Zhang, Bei-wei Zhu () School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian 116034, China Tel, Fax: +86-411-86323262 E-mail:
[email protected] Jun Zhao School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
The crescent shaped gonad of the pacific abalone extends around the pleopod with a grey-green color for females and a cream color for males. This abalone gonad, comprising 30% of the body weight, is an edible organ that is enriched in a polysaccharide, determined to be 18% of the dry tissue weight. The polysaccharide from the gonad was reported to have antithrombotic and antifatigue activities and could resolve blood clots in vitro with rates of 62.9 and 67.7% for clot-resolution reported at 50 and 200 µg/mL, respectively (4). The abalone gonad polysaccharide is a sulfate polysaccharide. Sulfate polysaccharide commonly had the immuno-stimulation activities and these activities are important factors for cancer therapy and other anticancer properties (5). During factory abalone processing, gonad was usually discarded as a by-product. Analysis of the gonad polysaccharide will contribute to exploitation of the abalone gonad for deep processing. However, no detailed structural or immuno-functional information about the abalone gonad polysaccharide has been published. This study aimed to provide information about the structure and the immuno-stimulation activities of the polysaccharide from H. discus hannai Ino. An homogeneous polysaccharide was isolated from the abalone gonad and the backbone structure of this polymer was elucidated using chemical methods, IR spectroscopy, and NMR spectroscopy. The biological activity of the polysaccharide was also investigated.
Materials and Methods Materials The gonads of abalone H. discus hannai Ino obtained from Dalian Zhangzidao Group Co. (Dalian, China) were collected from the Yellow Sea. The fresh gonad was vacuum freeze-dried by freezer dryer LG-1.0 (Xinyang Co., Ltd., Shenyang, China) and smashed with a grinder DFT-250A (Baidian Co., Ltd., Shanghai, China)
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before subjection to extraction. Alkaline protease and pepsin were purchased from Sanland Chemicals Co. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Fluka Chemie Co. (Buchs, Switzerland). Trifluoroacetic acid (TFA), standard monosaccharides, standard molecular weight (Mw) dextran and MD-25 dialysis tubes (Mw cutoff=7 kg/mol) were purchased from Pharmacia Co. (Uppsala, Sweden). Cellulose DEAE-52 and Sepharose CL-6B were purchased from Amersham Co. (Uppsala, Sweden). All chemicals used in this study were of analytical reagent grade or better. The Kunming species rats were obtained from Dalian Medical University (China). General methods 1H and 13C NMR spectra were recorded using D2O on a Bruker AV-500 spectrometer (Bremen, Germany) operating at 500.13 and 125.75 MHz. The polysaccharide was dissolved in D2O, and chemical shifts were referenced to Me4Si. IR spectroscopy was recorded on a Spectrum One-B FTIR Spectrometer (Perkin Elmer, Waltham, MA, USA) using KBr pellets. The protein content was measured following the Lowry method (6) with BSA as a standard. The polysaccharide content was determined following the phenol-acid method using glucose as a standard (7). The uronic acid content was measured according to a meta-hydroxydiphenyl colorimetric method (8). The sulfate content was assessed following the barium chloride method using potassium sulfate as a standard (9). All chromatographic assays were conducted following the phenol-acid method (7). The centrifugation experiments were all performed by centrifugal apparatus CF16RXII (Hitachi, Tokyo, Japan). All dialysis in this experiment was carried out by putting sample solution to MD-25 dialysis tubes (Mw cut-off=7 kg/mol) and dialyzed against water. All lyophilized experiments were performed by putting frozen sample to a freeze drying equipment OENT7 (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, Zejiang, China). Isolation and purification of the polysaccharide The smashed abalone gonad was dissolved in a 25× tap-water (v/v), adjust to pH 10 by NaOH (6 M) and adding 2 wt% alkaline protease. After incubation at 45oC for 3 h, the mixture was boiled for 10 min, adjusted to a neutral pH value by HCl (6 M), and centrifuged at 1,520×g for 10 min. The centrifugal supernatant was precipitated using 3 volumes of 95% ethanol at 4oC for 12 h. The precipitate was redissolved by tap water and dialyzed against tap water and lyophilized to dry, resulting in an abalone gonad polysaccharide residue. For further removal of conjugated proteins, the entire extraction procedure was repeated with pepsin incubated at pH 2.0. Dissociative proteins were then removed following the method of Staub (10). The deproteinated sample was frozen with BD-226 refrigerator
Yang et al.
(Hair, Qingdao, China) at −20oC for 24 h. After thawing at room temperature, the solution was centrifuged at 9,500×g for 20 min. The supernatant was collected and lyophilized to obtain the crude polysaccharide. The crude polysaccharide was dissolved in deionized water to a concentration of 100 mg/mL, then applied to a DEAE-Cellulose-52 column (4 cm diameter×40 cm). Elution was performed at a flow rate of 40 mL/h using deionized water (120 mL), followed by a 0.15 M gradient of a sodium chloride solution (120 mL), and finally with a 0.5 M sodium chloride solution (120 mL). Polysaccharide positive fractions were combined, concentrated to 5% (w/w) by water bath evaporation HH-4 (ZBR, Changzhou, China), then further purified using gel permeation chromatography carried out on a Sepharose CL-6B column (1.6 cm diameter ×70 cm). Elution was performed using a 0.15 M NaCl aqueous solution at a flow rate of 40 mL/h. Obtained fractions were combined according to the total carbohydrate content assayed (7), and lyophilized. The fractions eluted from the Sepharose CL-6B column were collected for structural analysis. Homogeneity analysis and molecular weight determination The homogeneity of the purified polysaccharide was determined using a LC-10Avp Plus HPLC system (Shimadzu, Kyoto, Japan) equipped with a TSK-gel G4000PWXL (7.8 diameter×300 mm; Tosoh, Tokyo, Japan) column and a 2000ES evaporative light scattering detector (Alltech, Ames, IA, USA). An amount of 20 µL of sample solution (1 mg/mL) was injected in each run with distilled water as the eluent at a flow rate of 0.2 mL/min. The Mw values of samples were determined using a Sephorose CL-6B column (1.2 cm diameter×60 cm) eluted with 0.15 M NaCl at a flow rate of 11 mL/h. The column was calibrated using standard dextran (4.1×105, 2.7×105, 8×104, and 1.2×104 g/ mol; Sigma). Monosaccharide composition The monosaccharide composition of samples was determined based on quantitative analysis of the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivative using liquid chromatography. An amount of 5 mg of the polysaccharide was dissolved in 1 mL of trifluoroacetic acid (TFA, 2 M) and subsequently hydrolyzed by a thermostatic drying chamber DHG-9015A (Shanghai Yiheng Co., Ltd., Shanghai, China) at 121oC for 2 h. The hydrolysate was fully converted to PMP derivatives (11). The monosaccharide composition was then quantitatively analyzed based on liquid chromatography (12). Partial acid hydrolysis and product purification AGP32 (300 mg) was dissolved in 12 mL of a 0.05 M TFA solution. After incubation at 95oC for 16 h under nitrogen, the mixture was centrifuged at 9,600×g for 3 min and a
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Structure of Abalone Gonad Polysaccharide
precipitate was obtained. After residual TFA was removed by water bath evaporation (ZBR), this precipitate was lyophilized, then dissolved in water and purified using a Sephorose CL-6B column (1.2 cm diameter×60 cm). The main hydrolysate fractions were combined, concentrated by water bath evaporation (ZBR), desalted by dialysis, and lyophilized to obtain a backbone of AGP-32 (BAGP-32). Methylation analysis BAGP-32 was methylated following the method of Needs and Selvendran (13). Completion of methylation was indicated by the disappearance of the hydroxyl absorption peak in the IR spectrum (Perkin Elmer) at 3,400/cm. The methylated product was depolymerized by hydrolysis with 85% formic acid for 4 h (100oC), then further hydrolyzed with 2 M TFA for 6 h at 100oC. Hydrolysates were then reduced and acetylated. Products were quantitatively analyzed using the GC-MS method described by Yang et al. (14). Exact types (glycosidic bond type) of methylated alditol acetates and molar ratios were obtained by GC-MS result. Periodate oxidation and Smith degradation The precipitate product from partial acid hydrolysis of AGP-32 (25 mg) was dissolved in a 0.015 M NaIO4 aqueous solution (25 mL), kept in the dark, and monitored every 2 h at an absorbance value of 223 nm using a spectrophotometer UV2800 (SDPTOP, Shanghai, China). The reaction was judged to be finished when the absorbance values did not decrease. Then, excess of NaIO4 was decomposed with addition of ethylene glycol (0.2 mL). The reaction mixture was dialyzed in running tap-water (24 h) and distilled water (24 h), and the retentate was reduced using NaBH4 (25 mg, 12 h). After adjustment of the pH to 6 using acetic acid (50%), the retentate was dialyzed against tap water (24 h) and distilled water (24 h). The retentate was hydrolyzed using addition of an equivalent volume of 1 M H2SO4 (25oC, 40 h). The pH was adjusted to 6 using BaCO3, then the solution was passed through filter paper and the filtrate was dialyzed against distilled water (48 h). The retentate was precipitated using 3 volumns of 95% ethanol and the residue was collected for monosaccharide compositional analysis. Reduction of AGP-32 The glucuronic acid in AGP-32 was reduced to corresponding sugar residues following the method of Yang et al. (14). AGP-32 (30 mg) was dissolved in 30 mL of water. After addition of 750 mg of 4-[2-[(cyclohexylcarbonimidoyl)amino]-ethyl]-4-methyl-morpholiniu salt with 4-methylbenzenesulfonic acid 1-cyclohexyl-3-(2morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMC), the reaction solution was adjusted to pH 4.75 using HCl (0.01 M) and kept for 3 h. Then, 15 mL of KBH4 (2 M) was added and the solution was adjusted to
pH 7.0 using 4 M HCl. The solution was dialyzed in running tap water for 3 days, then the retentate was lyophilized. The process was repeated until there was no glucuronic acid detected during monosaccharide analysis. The reduced product was assayed for 13C NMR analysis. Assay for lymphocyte proliferation activity Spleens were aseptically removed from 3 Kunming species healthy mice. Spleen cells were obtained as described as Sun et al. (15) and the splenocyte density was adjusted to 5×106 cells/mL. Cell viability was more than 98% determined using the trypan blue staining method. Proliferation of spleen lymphocytes was determined using an MTT assay (16). An aliquot of 100 µL of splenocytes was seeded onto a 96-well microplate, then AGP-32 was added at concentrations of 5, 10, and 50 µg/mL. The assay was performed following the method of Sun et al. (15). The absorbance at 450 nm was measured on an ELISA reader (model 680; Bio-Rad, Hercules, CA, USA) to benchmark the proliferation activity. All measurements were conducted in quintuplicate. Statistical analysis Bioassay data was statistically evaluated using the Student’s t-test function of the SASS software package (SASS Institute, Cary, NC, USA) and p
