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Journal of Ginseng Research J Ginseng Res 44 (2020) 570e579 Contents lists available at ScienceDirect Journal of Ginseng Research journal homepage: http://www.ginsengres.org Research Article Structural characteristics of a red ginseng acidic polysaccharide rhamnogalacturonan I with immunostimulating activity from red ginseng Sue Jung Lee 1,GyoIn2, Sung-Tai Han 2, Mi-Hyang Lee 2, Jong-Won Lee 2, Kwang-Soon Shin 1,* 1 Department of Food Science and Biotechnology, Kyonggi University, Suwon, Republic of Korea 2 Korea Ginseng Research Institute, Korea Ginseng Corporation, Daejeon, Republic of Korea article info abstract Article history: Background: Many researchers reported that the various immune activities of red ginseng are due to acid Received 15 February 2019 polysaccharides. But, the exact structural characteristics of the acidic polysaccharide in red ginseng have Received in Revised form not been fully elucidated. Therefore, we isolated the acidic polysaccharide from red ginseng and char- 2 April 2019 acterized the structural property of the active moiety of this polysaccharide, which contributes to the Accepted 7 May 2019 immunostimulatory activity of red ginseng. Available online 14 May 2019 Methods: A polysaccharide (RGP-AP-I) was purified from red ginseng via size-exclusion chromatography using Sephadex G-100. Immunostimulatary activity of RGP-AP-I was investigated via anti-com- Keywords: Immunostimulatory activity plementory and macrophage stimulatory activity. The structure of RGP-AP-I was characterized by HPLC, b Pectin sugar composition, -glucosyl Yariv reagent and methylation analysis. Red ginseng polysaccharide Results: Peritoneal macrophages stimulated using RGP-AP-I significantly augmented the production of Rhamnogalacturonan-I various cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-a. The primary Structural characterization structure of RGP-AP-I was elucidated by assessing its sugar composition and methylation analysis. RGP- AP-I is a 96 kDa acidic polysaccharide, and comprises nine different monosaccharides, which mainly include sugars such as rhamnose (Rha, 9.5%), galacturonic acid (GalA, 18.4%), galactose (Gal, 30.4%), and arabinose (Ara, 35.0%). RGP-AP-I exhibited an considerable reaction with the beglucosyl Yariv reagent, revealing the presence of arabino-b-3,6-galactan. Methylation analysis indicated that RGP-AP-I com- prises 21 different glycosyl linkages, such as 3-, 4-, 6- and 3,6-linked Galp; 5-linked Araf; 2,4-linked Rhap; and 4-linked GalAp, which are characteristics of rhamnogalacturonan I (RG-I). Conclusion: we assumed that the immunostimulatory activity of RGP-AP-I may be due to the RG-I structure, which comprises a main chain with a repeating linkage unit, [/2)-Rhap-(1/4)-GalAp-(1/] and three groups of side chains such as (1/5)-linked arabinan, (1/4)-linked galactan, and arabino-b- 3,6-galactan, which branch at the C(O)4 positions of Rha residues in the main chain of RGP-AP-I. Ó 2019 The Korean Society of Ginseng. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction polyphenols, flavonoids, and polysaccharides [6]. Among these, ginsenosides have been recognized as the most active ingredients, Representative herbal medicine Korean ginseng (the root of having various pharmacological activities [6]. In contrast, the Panax ginseng Meyer) is ethnopharmacologically used in the East pharmacological efficacies of ginseng polysaccharides have not Asian countries such as Korea and China [1]. They have been been as actively investigated as those of ginsenosides. considered to be a supplementary herbal medicine that exerts Meanwhile, fresh ginseng is easily degraded by several physical modulatory activities in numerous diseases such as cardiovascular factors. Considering its characteristics, red ginseng (RG) is diseases [2], cancer [3], diabetes [4], and hypertension [5]. Active frequently processed from fresh ginseng via simple or continuous constituents of ginseng have been reported to be ginsenosides, steaming/drying process. In general, the processed RG has * Corresponding author. Department of Food Science and Biotechnology, Kyonggi University, Suwon, Gyeonggi, 16227, Republic of Korea. E-mail address: [email protected] (K.-S. Shin). https://doi.org/10.1016/j.jgr.2019.05.002 p1226-8453 e2093-4947/$ e see front matter Ó 2019 The Korean Society of Ginseng. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S.J. Lee et al / Structural characteristics of red ginseng acidic polysaccharide 571 A B C D Fig. 1. Purification scheme of active polysaccharides from red ginseng concentrate. (A) Purification scheme of polysaccharides with different molecular weights from enzyme digests of red ginseng concentrate. Elution pattern of (B) RGP-0, (C) RGP-A, and (D) RGP-AP on high-performance size-exclusion chromatography (HPSEC) linked with Superdex 75 GL column. significantly higher biological activity and fewer side effects than supplements were purchased from Thermo Fisher Scientific(Wal- the fresh and white ginsengs [7,8]. Recently, many researchers re- tham, MA, USA). All other analytical reagents were purchased from ported that the various immune activities of RG are due to acid Sigma (USA). polysaccharides [1,9e11]. But the exact structural characteristics of Female Balb/c mice (6 weeks old) were purchased from Saer- the acidic polysaccharide in RG have not been fully onBio Inc. (Uiwang, Gyeonggi, the Republic of Korea). They were elucidated because of their complexity and heterogeneity. housed in a specific pathogenefree room maintained at a constant At present, an analytical technique for the structural character- temperature (20 Æ 1C) and humidity (55%) in a clean rack with 12 ization of natural polysaccharides is necessary; however, owing to h of light and dark cycles. Water and pellet diet were supplied ad their diversity and structural complexity, this characterization seems libitum. All animal experiments were carried out according to the difficult. Appropriate characterization involves the determination instructions by the Ethics Committee for Use of Experimental An- of sugar compositions, configurations, anomerizations, ring forms, imals at Kyonggi University (admission number: 2017-007). glycosyl linkages, and sequences of the polysaccharides [12]. Methylation is widely used to determine the linkage of complex car- 2.2. Isolation and purification of polysaccharide from RG bohydrates; it involves the hydrolysis of a polysaccharide to a partially concentrates methylated alditol acetate (PMAA) and is carried out via GCeMS [13]. In this study, we isolated the acidic polysaccharide from RG and Isolation and purification of polysaccharides from RG concen- characterized the structural property of the active moiety of this trate were carried out via ethanol precipitation, enzyme hydrolysis, polysaccharide, which contributes to the immunostimulatory ac- and size exclusion chromatography (Fig. 1). Briefly, 95% cold tivity of RG. ethanol (EtOH) was added to the RG concentrate, and precipitate was collected and redissolved with water, followed by dialysis for 3 2. Materials and methods days using a dialysis membrane (cutoff: 12,000e14,000; Sigma). The solution was lyophilized to obtain a crude polysaccharide (RGP- 2.1. Material and animals 0) from RG concentrate. To remove the starch-like materials, the lyophilized RGP-0 was suspended in the solution (pH 5.2), a- RG concentrates (15 brix) were supplied by the Korean Ginseng amylase (3000 units) and amyloglucosidase (300 units) were Corporation (KGC Co., Daejeon, Republic of Korea). Carbohydrate- added, and hydrolysis was performed at 50C for 48 h. After heating digesting enzymes, such as a-amylase from Aspergillus oryzae,amy- for 15 min, the enzyme reaction was quenched, and four volumes of loglucosidase from Aspergillus niger, and pectinesterase from orange 95% EtOH were then added to precipitate the polysaccharide frac- peel were all obtained from Sigma (St. Louis, MO, USA), whereas pol- tion from the enzyme digest of RGP-0. Dried polysaccharide, which ygalacturonase from Aspergillus aculeatus was purchased from Meg- was named as RGP-A, was prepared after dialysis and lyophilization azyme (Bray, Ireland). Sephadex G-100 was obtained from GE as mentioned previously. To remove homogalacturonan (HG), RGP- Healthcare (Uppsala, Sweden). Sheep red blood cells were obtained A was treated with pectinesterase for 48 h (pH 7.5; 30C) and was fromInnovativeResearch,Inc.(Novi,MI,USA).Dulbecco’sModified then hydrolyzed with polygalacturonase for 24 h (pH 5.5; 40C). Eagle’s Medium, fetal bovine serum, and other cell culture The enzymatic hydrolysates were eventually prepared as dried 572 J Ginseng Res 2020;44:570e579 RGP-AP by the same method as mentioned previously. RGP-AP was [14], m-hydroxybiphenyl method [15],modified thiobarbituric acid applied to a column (3 Â 120 cm) packed with Sephadex G-100, and method [16], and Bradford method [17] with protein assay dye (Bio- elution was carried out with 50 mM ammonium formate buffer (pH Rad laboratories, Hercules, CA, USA), respectively. Sugar composi- 5.5). Four major polysaccharides subfractions (RGP-AP-I, RGP-AP-II, tions were determined by the 1-phenyl-3-methyl-5-pyrazolone RGP-AP-III, and RGP-AP-IV) were obtained followed by desalination derivatives method [18,19] by HPLC (Shimadzu Corporation, Kyoto, using dialysis membrane and lyophilization (Fig. 2 (A)). Japan) equipped with SPD-20A UV/VIS detector and Acclaim 120 C18 column (Thermo Fisher Scientific). The optimal analytical condition 2.3. General analysis of the column was maintained at 30 C temperature. The 1-phenyl- 3-methyl-5-pyrazolone derivative elution was carried out with a The contents of total carbohydrate, uronic acid, 2-keto-3-deoxy- mixture of 82% phosphate buffer (0.1 M, pH 6.7) and 18% acetonitrile fl D-manno-octulosonic acidelike materials, and protein in the poly- at a ow rate of 1 mL/min, and UV absorbance of the eluate was saccharide samples were measured by phenolesulfuric acid method monitored at 245 nm. A B Fig.
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