Chemical characterization of a new sulfated polysaccharide from Gracilaria chouae and its activation effects on RAW264.7 macrophages

Chemical characterization of a new sulfated polysaccharide from Gracilaria chouae and its activation effects on RAW264.7 macrophages 脆江蓠硫酸化多糖的化学表征及其对RAW264.7巨噬细胞的激活作用 1 first-author https://orcid.org/0000-0001-8743-6648 1 2 corresp Kehai LIU, khliu@shou.edu.cn Kehai LIU, khliu@shou.edu.cn khliu@shou.edu.cn https://orcid.org/0000-0003-2505-5553 3 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China Heilongjiang Forest By-product and Speciality Institute, Mudanjiang 157011, China Heilongjiang Forest By-product and Speciality Institute, Mudanjiang 157011, China 目的： 2 脆江蓠是中国沿海广泛分布的重要经济红藻，多糖是其主要生物成分，通过对其多糖分离纯化、化学表征及活性评价，为脆江蓠的开发奠定实验基础。 创新点： 2 首次从脆江蓠中得到均一硫酸化多糖GCP-3A，并发现其多糖对巨噬细胞RAW264.7具有显著激活作用。 方法： 2 以脆江蓠为原料，采用水提醇沉、柱层析等方法分离纯化得到脆江蓠硫酸化多糖GCP-3A；采用凝胶渗透色谱（GPC）、气相色谱-质谱（GC-MS）、傅里叶变换红外光谱（FT-IR）和核磁共振（NMR）探究其化学特征；采用扫描电镜（SEM）和刚果红研究其表面形态；通过噻唑蓝（MTT）和一氧化氮（NO）试验分析其对RAW264.7巨噬细胞的活化作用。 结论： 2 GCP-3A是一种硫酸化酸性多糖，分子量为11.87 kDa，由木糖、半乳糖、葡萄糖和甘露糖组成，四者的摩尔比为3.00:29.28:0.63:0.45，且含有α,β-糖苷键。GCP-3A是一种由不规则片状和棒状交织而成的异质多糖，无三螺旋构象。此外，GCP-3A显著性地促进了RAW264.7巨噬细胞的增殖和NO分泌，具有应用于医药及功能性食品的潜力。 脆江蓠多糖 化学表征 巨噬细胞激活 This study aimed to characterize the chemical composition of a new sulfated polysaccharide from the red alga Gracilaria chouae and evaluate its activation effects on RAW264.7 macrophages. It showed that the obtained G. chouae polysaccharide (GCP-3A) was a sulfated acidic polysaccharide with a molecular weight of 11.87 kDa. GCP-3A was composed of xylose, galactose, glucose, and mannose with a molar ratio of 3.00:29.28:0.63:0.45, and it contained α,β‍-glycosidic linkages. Scanning electron microscopy (SEM) and a Congo red test showed that it was a heterogeneous polysaccharide with irregular interwoven sheets and rods, and did not have a triple-helix conform‍ation. Furthermore, GCP-3A significantly promoted the proliferation of RAW264.7 macrophages and the secretion of nitric oxide (NO) in tests of 3-‍(4,‍5-dimethylthiahiazo-2-yl)‍-2,‍5-diphenytetrazoliumromide(MTT) and NO. G. chouae , an economically important alga, is widely found in the low-tide or subtidal zones along the southeast coast of China ( Chi et al., 2016 Chi et al., 2016 ). In recent years, it has been principally exploited for water quality improvement, abalone culture, and agar extraction; however, it has not been fully developed or utilized ( Torres et al., 2019 Torres et al., 2019 ). Previous research reported that the polysaccharides from G. chouae exerted anti-oxidation and antitumor effects, raising the possibility of applications in functional food and medicine ( Ju et al., 2016 Ju et al., 2016 ; Khan et al., 2019 Khan et al., 2019 ). The biological activity of polysaccharides is closely linked to their structural characteristics, such as monosaccharide composition, molecular weight, substituents, branching, and conformation ( Zhang et al., 2020 Zhang et al., 2020 ). However, there are no clear rules connecting the structure and activity of polysaccharides, and they are all affected by various separation and purification processes. Consequently, more research is required to elucidate polysaccharide structures to define the relationship between structure and activity. We then systematically studied its structural characteristics with gel permeation chromatography (GPC), gas chromatography-mass spectrometry (GC-MS), Fourier transform-infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR). SEM and Congo red were used to explore the surface morphology of the polysaccharides . The activation effects of GCP-3A on RAW264.7 macrophages were studied by MTT and NO assays. We found that GCP-3A was a polysaccharide with relatively uniform molecular weight and charge. It was first purified by cellulose diethylaminoethyl-52 (DEAE‍-52) chromatography column ( Fig. 1 Fig. 1 a) and then by Sephadex G-100 chromatography ( Fig. 1 Fig. 1 b). GCP-3A is a sulfated acid polysaccharide, and its monosaccharide composition is mainly 3,‍6-anhydrogalactose, xylose, and galactose, with minor amounts of glucose and mannose ( Table 1 Table 1 ). The GPC elution profile indicated that GCP-3A had a relatively broad molecular weight distribution, with a weight-average molecular weight and number-average molecular weight of 31.32 kDa and 10.13 kDa, respectively ( Fig. 1 Fig. 1 c). As shown in Fig. 1 Fig. 1 d, FT-IR revealed the typical polysaccharide characteristic peak information for GCP-3A, and confirmed the presence of uronic acid and sulfate groups ( You et al., 2020 You et al., 2020 ). 1 D NMR ( 1 H, 13 C) spectra indicated that GCP-3A had α‍- and β‍-configuration (Figs. 1e 1e and 1f 1f ). In addition, GCP-3A contains several complex sugar residues, and exhibits substitution of methyl, acetyl, and sulfate groups ( Liu et al., 2018 Liu et al., 2018 ). 1;2; Data are expressed as mean±standard deviation ( n =3). GCP-3A: Gracilaria chouae polysaccharide; Xyl: xylose; Gal: galactose; Glc: glucose; Man: mannose. Data about chemical composition content of GCP-3A were adapted with permission from Li and Liu (2021) Li and Liu (2021) , copyright 2021, J Shandong Agric Univ (Nat Sci Ed) . 2 Name 5 Content (%, mass fraction) 2 Neutral sugar (Xyl:Gal:Glc:Man) Total sugar Uronic acid Protein Sulfate group 3,6-Anhydrogalactose GCP-3A 63.80±0.05 10.23±0.12 0.11±0.06 7.37±0.63 15.75±0.49 3.00:29.28:0.63:0.45 SEM analysis showed the surface morphology of GCP-3A at 350× and 1500× magnifications (Figs. 2a 2a and 2b 2b ). GCP-3A was predominantly interwoven by irregular sheets and rods, and the surface was granular. A Congo red experiment suggested that GCP-3A did not have a triple-helical conformation in solution ( Fig. 2 Fig. 2 c). An MTT assay indicated that GCP-3A promoted the proliferation of RAW264.7 cells at concentrations of 25‍–300 μg/mL ( Fig. 3 Fig. 3 a). GCP-3A did not exhibit any detectable cytotoxicity. In fact, it significantly promoted the proliferation of RAW264.7 cells compared with the control group ( P < 0.05). In the NO assay, GCP-3A significantly stimulated NO secretion in RAW264.7 cells compared with the control group, and this stimulation was in a dose-dependent manner in the range of 25–200 μg/mL ( Fig. 3 Fig. 3 b). The production of NO reached the highest level (6.64 μmol/L) at 200 μg/mL. In this study, the total sulfate content of GCP-3A was 7.37% (mass fraction), which was similar to previously reported levels ( Sudharsan et al., 2015 Sudharsan et al., 2015 ; Imjongjairak et al., 2016 Imjongjairak et al., 2016 ). In addition, the results showed that the galactose content was much higher than that of other monosaccharides in GCP-3A, which was consistent with other polysaccharides from red algae. However, the types and proportions of monosaccharides varied, which may be related to species, growth environment, and treatment methods ( de Oliveira et al., 2020 de Oliveira et al., 2020 ; Han et al., 2020 Han et al., 2020 ; Li et al., 2020 Li et al., 2020 ). We found that there were granular substances on the surface of GCP-3A, which was also observed previously in G. chouae ( Khan et al., 2019 Khan et al., 2019 ). According to previous reports ( Gu et al., 2020 Gu et al., 2020 ; Zhao et al., 2021 Zhao et al., 2021 ; Zhu et al., 2021 Zhu et al., 2021 ), the activated macrophages can directly kill pathogens and release effector molecules such as NO, tumor necrosis factor, and interleukin, which provide resistance to microbial infection and pathogen invasion. In addition, NO participated in various physiological and pathological reactions and is considered an important indicator of immune cell activation ( Huang et al., 2017 Huang et al., 2017 ; Lan et al., 2021 Lan et al., 2021 ). Here, GCP-3A promoted the proliferation of macrophages and the release of NO, which suggested that RAW264.7 macrophages were activated. In conclusion, GCP-3A is a potential bioactive ingredient for the pharmaceutical and health product industries. In the future, more studies should be carried out to clarify the activation effects of GCP-3A on RAW264.7 macrophages. Because of the complexity of this polysaccharide, the sequence of glycosidic bonds should be elucidated to provide a reference for the structure-activity relationship of polysaccharides in general. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References Chi S , Feng YJ , Liu T , 2016 . Molecular cloning, characterization, and comparison of UDP-glucose pyrophosphorylase from Gracilaria chouae and Saccharina japonica . J Appl Phycol , 28 ( 3 ): 2051 - 2059 . https://doi.org/10.1007/s10811-015-0738-7 https://doi.org/10.1007/s10811-015-0738-7 Molecular cloning, characterization, and comparison of UDP-glucose pyrophosphorylase from Gracilaria chouae and Saccharina japonica de Oliveira FBB , Bingana RD , Morais PAF , et al. , 2020 . Sulfated polysaccharide from Gracilaria caudata reduces hypernociception and inflammatory response during arthritis in rodents . Int J Biol Macromol , 161 : 1061 - 1069 . https://doi.org/10.1016/j.ijbiomac.2020.06.060 https://doi.org/10.1016/j.ijbiomac.2020.06.060 Sulfated polysaccharide from Gracilaria caudata reduces hypernociception and inflammatory response during arthritis in rodents Gu JY , Zhang HH , Zhang JX , et al. , 2020 . Optimization, characterization, rheological study and immune activities of polysaccharide from Sagittaria sagittifolia L . Carbohyd Polym , 246 : 116595 . https://doi.org/10.1016/j.carbpol.2020.116595 https://doi.org/10.1016/j.carbpol.2020.116595 Optimization, characterization, rheological study and immune activities of polysaccharide from Sagittaria sagittifolia L Han R , Pang DR , Wen LR , et al. , 2020 . In vitro digestibility and prebiotic activities of a sulfated polysaccharide from Gracilaria lemaneiformis . J Funct Foods , 64 : 103652 . https://doi.org/10.1016/j.jff.2019.103652 https://doi.org/10.1016/j.jff.2019.103652 In vitro digestibility and prebiotic activities of a sulfated polysaccharide from Gracilaria lemaneiformis Huang JQ , Qi RT , Pang MR , et al. , 2017 . Isolation, chemical characterization, and immunomodulatory activity of naturally acetylated hemicelluloses from bamboo shavings . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 18 ( 2 ): 138 - 151 . https://doi.org/10.1631/jzus.B1500274 https://doi.org/10.1631/jzus.B1500274 Isolation, chemical characterization, and immunomodulatory activity of naturally acetylated hemicelluloses from bamboo shavings Imjongjairak S , Ratanakhanokchai K , Laohakunjit N , et al. , 2016 . Biochemical characteristics and antioxidant activity of crude and purified sulfated polysaccharides from Gracilaria fisheri . Biosci Biotechnol Biochem , 80 ( 3 ): 524 - 532 . https://doi.org/10.1080/09168451.2015.1101334 https://doi.org/10.1080/09168451.2015.1101334 Biochemical characteristics and antioxidant activity of crude and purified sulfated polysaccharides from Gracilaria fisheri Ju YY , Ye TW , Xie F , et al. , 2016 . Antitumor activity in vivo and mechanism of action of Gracilaria chouae polysaccharides . Food Sci , 37 ( 5 ): 208 - 213 (in Chinese) . Antitumor activity in vivo and mechanism of action of Gracilaria chouae polysaccharides Khan BM , Qiu HM , Wang XF , et al. , 2019 . Physicochemical characterization of Gracilaria chouae sulfated polysaccharides and their antioxidant potential . Int J Biol Macromol , 134 : 255 - 261 . https://doi.org/10.1016/j.ijbiomac.2019.05.055 https://doi.org/10.1016/j.ijbiomac.2019.05.055 Physicochemical characterization of Gracilaria chouae sulfated polysaccharides and their antioxidant potential Lan HB , Cheng YX , Mu JJ , et al. , 2021 . Glucose-rich polysaccharide from dried 'Shixia' longan activates macrophages through Ca 2+ and CR3-mediated MAPKs and PI3K-AKT pathways . Int J Biol Macromol , 167 : 845 - 853 . https://doi.org/10.1016/j.ijbiomac.2020.11.040 https://doi.org/10.1016/j.ijbiomac.2020.11.040 Glucose-rich polysaccharide from dried 'Shixia' longan activates macrophages through Ca2+ and CR3-mediated MAPKs and PI3K-AKT pathways Li FF , Liu KH . 2021 . Physicochemical analysis and their antioxidant evaluation of polysaccharides from Gracilaria chouae . J Shandong Agric Univ (Nat Sci Ed) , 52 ( 05 ): 746 - 752 (in Chinese) . Physicochemical analysis and their antioxidant evaluation of polysaccharides from Gracilaria chouae Li X , Huang SM , Chen X , et al. , 2020 . Structural characteristic of a sulfated polysaccharide from Gracilaria lemaneiformis and its lipid metabolism regulation effect . Food Funct , 11 ( 12 ): 10876 - 10885 . https://doi.org/10.1039/D0FO02575E https://doi.org/10.1039/D0FO02575E Structural characteristic of a sulfated polysaccharide from Gracilaria lemaneiformis and its lipid metabolism regulation effect Liu YF , Su PF , Xu JF , et al. , 2018 . Structural characterization of a bioactive water-soluble heteropolysaccharide from Nostoc sphaeroids kütz . Carbohyd Polym , 200 : 552 - 559 . https://doi.org/10.1016/j.carbpol.2018.08.034 https://doi.org/10.1016/j.carbpol.2018.08.034 Structural characterization of a bioactive water-soluble heteropolysaccharide from Nostoc sphaeroids kütz Sudharsan S , Subhapradha N , Seedevi P , et al. , 2015 . Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis (Forsskal) . Int J Biol Macromol , 81 : 1031 - 1038 . https://doi.org/10.1016/j.ijbiomac.2015.09.046 https://doi.org/10.1016/j.ijbiomac.2015.09.046 Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis (Forsskal) Torres P , Santos JP , Chow F , et al. , 2019 . A comprehensive review of traditional uses, bioactivity potential, and chemical diversity of the genus Gracilaria (Gracilariales, Rhodophyta) . Algal Res , 37 : 288 - 306 . https://doi.org/10.1016/j.algal.2018.12.009 https://doi.org/10.1016/j.algal.2018.12.009 A comprehensive review of traditional uses, bioactivity potential, and chemical diversity of the genus Gracilaria (Gracilariales, Rhodophyta) You X , Li Z , Ma K , et al. , 2020 . Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5 . Carbohyd Polym , 235 : 115977 . https://doi.org/10.1016/j.carbpol.2020.115977 https://doi.org/10.1016/j.carbpol.2020.115977 Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5 Zhang R , Zhang XX , Tang YX , et al. , 2020 . Composition, isolation, purification and biological activities of Sargassum fusiforme polysaccharides: a review . Carbohyd Polym , 228 : 115381 . https://doi.org/10.1016/j.carbpol.2019.115381 https://doi.org/10.1016/j.carbpol.2019.115381 Composition, isolation, purification and biological activities of Sargassum fusiforme polysaccharides: a review Zhao Y , Zhang BZ , Zhang QQ , et al. , 2021 . Tumor-associated macrophages in osteosarcoma . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 11 ): 885 - 892 . https://doi.org/10.1631/jzus.B2100029 https://doi.org/10.1631/jzus.B2100029 Tumor-associated macrophages in osteosarcoma Zhu HY , Yan SC , Wu JS , et al. , 2021 . Serum macrophage migration inhibitory factor as a potential biomarker to evaluate therapeutic response in patients with allergic asthma: an exploratory study . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 6 ): 512 - 520 . https://doi.org/10.1631/jzus.B2000555 https://doi.org/10.1631/jzus.B2000555 Serum macrophage migration inhibitory factor as a potential biomarker to evaluate therapeutic response in patients with allergic asthma: an exploratory study Feifei LI performed the experimental research and data analysis, and wrote and edited the manuscript. Kehai LIU contributed to the study design, data analysis, and editing of the manuscript. Kewu LIU contributed to the study design and editing of the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. 81572989) and the International Academic Cooperation of Science and Technology Committee of Shanghai, China (No. 18430721100). 10.1631/jzus.B2100508 1673-1581（2022）01-0084-05 This work was supported by the National Natural Science Foundation of China (No. 81572989) and the International Academic Cooperation of Science and Technology Committee of Shanghai, China (No. 18430721100). 2021-06-10 2021-08-23 2022-01-15 2022-04-19 2022-01-07 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2022 浙江大学出版社 Feifei LI, Kehai LIU, and Kewu LIU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Supplementary information 1 Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 23