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Structural characteristics of a red ginseng acidic polysaccharide rhamnogalacturonan I with immunostimulating activity from red ginseng

Background Many researchers reported that the various immune activities of red ginseng are due to acid polysaccharides. But, the exact structural characteristics of the acidic polysaccharide in red ginseng have not been fully elucidated. Therefore, we isolated the acidic polysaccharide from red ginseng and characterized the structural property of the active moiety of this polysaccharide, which contributes to the immunostimulatory activity of red ginseng. 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-complementory and macrophage stimulatory activity. The structure of RGP-AP-I was characterized by HPLC, sugar composition, β-glucosyl Yariv reagent and methylation analysis. Results Peritoneal macrophages stimulated using RGP-AP-I significantly augmented the production of various cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α. The primary 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 β–glucosyl Yariv reagent, revealing the presence of arabino-β-3,6-galactan. Methylation analysis indicated that RGP-AP-I comprises 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-β-3,6-galactan, which branch at the C(O)4 positions of Rha residues in the main chain of RGP-AP-I.


Introduction
Representative herbal medicine Korean ginseng (the root of Panax ginseng Meyer) is ethnopharmacologically used in the East Asian countries such as Korea and China [1]. They have been considered to be a supplementary herbal medicine that exerts modulatory activities in numerous diseases such as cardiovascular diseases [2], cancer [3], diabetes [4], and hypertension [5]. Active constituents of ginseng have been reported to be ginsenosides, polyphenols, flavonoids, and polysaccharides [6]. Among these, ginsenosides have been recognized as the most active ingredients, having various pharmacological activities [6]. In contrast, the pharmacological efficacies of ginseng polysaccharides have not been as actively investigated as those of ginsenosides.
Meanwhile, fresh ginseng is easily degraded by several physical factors. Considering its characteristics, red ginseng (RG) is frequently processed from fresh ginseng via simple or continuous steaming/drying process. In general, the processed RG has significantly higher biological activity and fewer side effects than the fresh and white ginsengs [7,8]. Recently, many researchers reported that the various immune activities of RG are due to acid polysaccharides [1,9e11]. But the exact structural characteristics of the acidic polysaccharide in RG have not been fully elucidated because of their complexity and heterogeneity.
At present, an analytical technique for the structural characterization of natural polysaccharides is necessary; however, owing to their diversity and structural complexity, this characterization seems difficult. Appropriate characterization involves the determination of sugar compositions, configurations, anomerizations, ring forms, glycosyl linkages, and sequences of the polysaccharides [12]. Methylation is widely used to determine the linkage of complex carbohydrates; it involves the hydrolysis of a polysaccharide to a partially methylated alditol acetate (PMAA) and is carried out via GCeMS [13].
In this study, we isolated the acidic polysaccharide from RG and characterized the structural property of the active moiety of this polysaccharide, which contributes to the immunostimulatory activity of RG.

Material and animals
RG concentrates (15 brix Female Balb/c mice (6 weeks old) were purchased from Saer-onBio Inc. (Uiwang, Gyeonggi, the Republic of Korea). They were housed in a specific pathogenefree room maintained at a constant temperature (20 AE 1 C) and humidity (55%) in a clean rack with 12 h of light and dark cycles. Water and pellet diet were supplied ad libitum. All animal experiments were carried out according to the instructions by the Ethics Committee for Use of Experimental Animals at Kyonggi University (admission number: 2017-007).

Isolation and purification of polysaccharide from RG concentrates
Isolation and purification of polysaccharides from RG concentrate were carried out via ethanol precipitation, enzyme hydrolysis, and size exclusion chromatography (Fig. 1). Briefly, 95% cold ethanol (EtOH) was added to the RG concentrate, and precipitate was collected and redissolved with water, followed by dialysis for 3 days using a dialysis membrane (cutoff: 12,000e14,000; Sigma). The solution was lyophilized to obtain a crude polysaccharide (RGP-0) from RG concentrate. To remove the starch-like materials, the lyophilized RGP-0 was suspended in the solution (pH 5.2), aamylase (3000 units) and amyloglucosidase (300 units) were added, and hydrolysis was performed at 50 C for 48 h. After heating for 15 min, the enzyme reaction was quenched, and four volumes of 95% EtOH were then added to precipitate the polysaccharide fraction from the enzyme digest of RGP-0. Dried polysaccharide, which was named as RGP-A, was prepared after dialysis and lyophilization as mentioned previously. To remove homogalacturonan (HG), RGP-A was treated with pectinesterase for 48 h (pH 7.5; 30 C) and was then hydrolyzed with polygalacturonase for 24 h (pH 5.5; 40 C). The enzymatic hydrolysates were eventually prepared as dried RGP-AP by the same method as mentioned previously. RGP-AP was applied to a column (3 Â 120 cm) packed with Sephadex G-100, and elution was carried out with 50 mM ammonium formate buffer (pH 5.5). Four major polysaccharides subfractions (RGP-AP-I, RGP-AP-II, RGP-AP-III, and RGP-AP-IV) were obtained followed by desalination using dialysis membrane and lyophilization ( Fig. 2 (A)).

A B
High-performance size-exclusion chromatography of the polysaccharide sample was carried out using an Agilent 1260 Infinity LC system (Agilent Technologies Co., Ltd., Palo Alto, CA, USA) according to the method described by Lee et al. [20].

Anticomplementary activity
The anticomplementary activity was measured by the method reported by Mayer [21]. In brief, sample was mixed with normal  2 Monosaccharides were analyzed using alditol acetates. 3 Mole % was calculated from the detected total carbohydrate.
A B D C human serum (complement sauce) with gelatin veronal buffer (pH 7.4) and activated at 37 C. After 30 min, IgM from hemolysinsensitized sheep erythrocytes (EA cells, 1 Â 10 8 cells/mL) were added to the mixture and incubated at 37 C for 1 h. Two and a half milliliters of phosphate-buffered saline was added to terminate the reaction and centrifuged at 800 Âg for 10 min. Optical density of the supernatant was measured at 412 nm. Polysaccharide K (PSK) from Coriolus versicolor and CVT-E002 (sold commercially as Cold-FX, Valeant Pharmaceuticals International, Canada) from Panax quinquefolium were used positive controls [22,23]. The anticomplementary activity has been expressed as follows: Total hemolytic complement (TCH 50 ) (%) ¼ TCH 50 (control) À TCH 50 (treated with sample)/TCH 50 (control)

Assay of cytokines on murine peritoneal macrophages
To determine the immunostimulatory activity of polysaccharide samples, peritoneal macrophages were isolated from Balb/c mice injected with 5% thioglycollate medium, as previously described [24]. The collected macrophages were suspended in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific) plated at a density of 2.5 Â 10 6 cells/mL into 96-well culture plates (100 mL). After 2 h, polysaccharide samples (100 mL) were added to the cells and incubated at 37 C for 24 h. The concentration of cytokines [IL-6, IL-12, and tumor necrosis factor (TNF)-a] accumulated in the culture medium was determined using enzyme-linked immonusorbent assay kits (BD Biosciences, San Diego, CA, USA; eBioscience, San Diego, CA, USA ) according to the manufacturer's protocols.

Glycosidic linkage analysis
To determine the glycosidic linkage of the polysaccharide, methylation was carried out according to the methods described by Hakomori [26] and Pettolino et al [13], with slight modifications. In brief, polysaccharide fraction was dissolved in dimethyl sulfoxide, and methylsulfinyl carbanion with iodomethane (Sigma) was added to prepare the methylated polysaccharide. The unmethylated parts were removed using a Sep-pak C18 cartridge (Waters, Dublin, Ireland), and the methylated sample was hydrolyzed by 1 M trifluoroacetic acid (Sigma) for 90 min at 121 C. Reduction was carried out with sodium borodeuteride (Sigma) for 240 min, and then acetylation was carried out with acetic anhydride for 180 min at 121 C. The resulting partially methylated alditol acetates (PMAAs) were analyzed by GCeMS (Agilent, Santa Clara, CA, USA), and the glycosidic linkage of the polysaccharide fraction was calculated based on the method of Lee et al [27].

Statistical analysis
All statistical analyses evaluated were analyzed by a one-way analysis of variance and Duncan's multiple range test. All data are presented as the mean AE standard deviation (SD).

Preparation of polysaccharide fraction from an RG concentrate
Initially, RGP-0 was isolated from an RG concentrate with a yield of 25.21 w/w%. As mentioned in Table 1 enzymes such as aeamylase and amyloglucosidase was carried out to yield a starch-deprived polysaccharide fraction (RGP-A, yield 21.29% against RGP-0). The Glc content (9.0%) was dramatically decreased in RGP-A, whereas the GalA (46.3%), galactose (Gal; 21.3%), and arabinose (Ara; 16.4%) contents were enriched via the catalytic action of starch-depriving enzymes (Table 1). Large amounts of GalA, Gal, and Ara strongly suggested that RGP-A consists primarily of pectic polysaccharide, which comprises three major groups such as HG, rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II).
To appropriately characterize the ginseng polysaccharide structure, RGP-A was further treated with pectinesterase, which catalyzes the deesterification of the methyl-esterified HG region of the pectic polysaccharide into pectate and methanol [28]. As a result of the pectinesterase digestion, RGP-AP (8.2% yield against RGP-A) was prepared ( Table 1). The chemical component and sugar analyses suggested that Gal (23.2%) and Ara (22.0%) contents were slightly increased in RGP-AP, whereas Glc (2.3%) content was greatly diminished compared with that of RGP-A. Interestingly, GalA content was rarely affected by pectinesterase digestion.

Effect of enzymatic digestion on immunostimulatory activity of RG polysaccharide
The anticomplementary activity and production activities of immunostimulatory cytokines in polysaccharide samples isolated through the enzymatic digestion of the RG concentrate were estimated and compared. As presented in Fig. 3A, the anticomplementary activities of RGP-0, RGP-A, and RGP-AP were 22.1%, 31.1%, and 35.8%, respectively; whereas, those of PSK and CVT, which were used as positive controls, were 60.0% and 45.2%, respectively. In addition, the effects of the three polysaccharide samples on the production of macrophage-stimulating cytokines, such as IL-6, IL-12, and TNF-a were quantified and expressed in Fig. 3BeD. Considering the same concentration range, RGP-A increased the production of the three cytokines more than RGP-0, suggesting that the removal of starch could presumably enhance the macrophage activity of RGP. Meanwhile, although RGP-0 and RGP-A induced the production of the three cytokines in high concentration ranges (above 62.5 mg/mL), RGP-AP exhibited greater production of the three cytokines in low concentration ranges (0.06w0.98 mg/mL). Interestingly, the production of the three cytokines peaked when RGP-AP stimulus was provided to RAW 264.7 cells at a concentration of 0.98 mg/mL; however, it decreased at higher doses. Collectively, these results indicated that enzymatic digestion is a useful method for enhancing the anticomplementary activity of RGP. Considering the association analysis with sugar composition, as presented in Table 1, the small amounts of Glc in RGP-AP suggest that glucans, including starch, are not important moieties for the immunostimulatory activity of RGP-AP. Consequently, the amylase digestion of the RG concentrate is a useful method for significantly enhancing its immunostimulatory activity.

Chemical property and immunostimulatory activity of subfractions retrieved from RGP-AP
As the immunostimulatory polysaccharide (RGP-AP) exhibited a relatively higher activity than RGP-0 and RGP-A, it was further fractionated into four subfractions via gel permeation chromatography using Sephadex G-100 ( Fig. 2A), that is, RGP-AP-I (yield; 2.9% against RG concentrate) was eluted in a void volume, RGP-AP-II (1.9%) and RGP-AP-III (1.3%) were eluted in intermediate volumes, and RGP-AP-IV (2.7%) was obtained in an inner volume. To compare the immunostimulatory activity of the subfractions, the anticomplementary and macrophage stimulatory activities were reestimated. As presented in Fig. 4A, RGP-AP-I increased the anticomplementary activity to similar level with the positive control (PSK and CVT), whereas the other fractions exhibited decreased activity compared with that of RGP-AP. The results of the production of macrophage stimulatory cytokines such as IL-6, IL-12, and TNF-a were illustrated in Fig. 4BeD, respectively. Among four fractions, only the highest-molecular-weight fraction, RGP-AP-I showed significantly increased production of IL-6, IL-12, and TNFa compared with those of RGP-AP, at doses above 1 mg/mL. Interestingly, the production of IL-12 and TNF-a showed decreased tendency at doses above 10 mg/mL in both RGP-AP and RGP-AP-I, suggesting that treatment of them to RAW 264.7 cells was more suitable at dose of 1 mg/mL. Other fractions beside RGP-AP-I did not show enhanced production activity of three cytokines compared with those of RGP-AP. Therefore, we preferred RGP-AP-I as an immunostimulatory polysaccharide and carried out further analysis to identify its appropriate structure. Table 1 lists the chemical composition of subfractions isolated from RGP-AP. The maximum high-molecular-weight fraction, RGP-AP-I, comprised a large proportion of neutral sugars (67.7%), consisting mainly of Ara (35.0%), Gal (30.4%), and GalA (18.4%). In comparison with RGP-AP, the proportion of neutral sugars such as Rha, Ara, and Gal was considerably enriched in RGP-AP-I; however, the content of uronic acid was decreased by more than 15% in RGP-AP-I compared with that in RGP-AP, and the major uronic acid was confirmed as being GalA (18.4%) rather than glucuronic acid (0.9%). The high proportion of Ara and Gal suggests that RGP-AP-I presumably possesses few side chains, such as arabinan, galactan, or arabinogalactan (AG), which are attached to the backbone of RG-I [29]. Meanwhile, the determination of the molecular weight distribution of the RGP-AP-I fraction via HPLC is illustrated in Fig. 2 (B). RGP-AP-I appeared as a symmetrical, single peak, and its molecular weight was estimated to be 96 kDa according to the calibration curve prepared using a dextran reference at various concentrations.

Yariv reagent assay of polysaccharide isolated from RG concentrate
After estimating that all polysaccharide fractions contained different proportions of Ara and Gal, we analyzed single radial gel diffusion assay with b-glucosyl Yariv reagent for the identification and quantification of AG-II. The Yariv reagent has the ability to precipitate polysaccharides containing AG-II. Depending on the length of the Gal chain, a precipitate will form if the Gal units contain branching at positions 3 and 6 [25,30]. As presented in Fig. 5, a positive correlation was revealed between the Yariv reagents in the standard reference gum arabic, and the area of precipitation measured by the square of the diameter of the halo formed in the radial diffusion assay. The amount of AG-II in a sample was determined by a calibration curve based on the square value of the halo formed using gum arabic [29]. Although RGP-0 rarely possessed AG-II moiety in their structure, AG-II contents were enriched in its enzyme-digested fractions, RGP-A and RGP-AP (3.9% and 6.3%, respectively). Furthermore, the immunostimulatory subfraction, RGP-AP-I, exhibited higher content of AG-II (11.3%) among all polysaccharide samples tested. This result suggested that AG-II moiety is important for expressing the immunostimulatory activity in RGP. The amount of AG-II in RGP-II was calculated to be a 5.6%, whereas AG-II moiety is not included both in RGP-III and RGP-IV.

Methylation analysis of RGP-AP-I
The structural characterization of polysaccharides requires the identification of sugar compositions, anomeric configurations, ring forms, glycosyl linkages, and sequences. Glycosyl linkages are confirmed by NMR, FT-IR, and methylation analysis. In particular, the methylation-based method is well established and is widely used for determining the structure of the polysaccharide [12]. In this method, all the free hydroxyl groups of the sample (polysaccharide) are methylated, and the methylated polysaccharide is hydrolyzed using trifluoroacetic acid. The partially methylated monosaccharide is reduced by NaBH 4 and then acetylated to PMAAs [13]. This method follows the same basic procedures as summarized in Fig. 6.
The immunostimulatory subfraction RGP-AP-I was analyzed by GCeMS to identify the active structure of RG. RGP-AP-I comprises 20 types of neutral glycosyl linkages ( Fig. 7 and Table 2), consisting mainly of galactopyranosyl (Galp), arabinofuranosyl (Araf), and rhamnopyranosyl (Rhap) linkages (30.4, 26.9, and 8.2 mole %, respectively), in addition to small amounts of glucopyranosyl (Glcp)  and xylopyranosyl (Xylp) linkages (2.0 and 0.3 mole %, respectively). Unfortunately, the linkage confirmation of GalA was not carried out in this study because they are not detectable as alditol acetate unless they are prereduced to their neutral sugar counterparts [13]. Nevertheless, the result of sugar composition (Table 1)  Based on the aforementioned results, we assumed that the RGP-AP-I purified from RGP comprises a typical RG-I structure, which includes several side chains such as galactan, highly branched arabinan, and arabino-b-3,6-galactan. The proposed structure of RGP-AP-I is illustrated in Fig. 8.

Conclusion
Many researchers have reported pharmacological activities such as radioprotective [34], antifatigue [35], anticancer [36], and immunological activities [37] of P. ginseng polysaccharides, and they investigated active structure of polysaccharides [38]. Tomoda et al [37] reported that the active polysaccharide fraction (S-IIA) isolated from the root of P. ginseng is mainly comprised of L-Ara, D-Gal, D-Glc, and D-GalA and has a 100-kDa molecular weight and a relatively high content of a-3,5-barached L-Ara and b-1,4-linked D-Gal residues. Moreover, Zhang et al [39] reported that water-soluble polysaccharides isolated from P. ginseng include pectic arabinogalactans and pectins enriched in RG-I, HG, and starch-like glucans. In addition, several studies have demonstrated that the immunostimulatory functions of RG could be due to the presence of an acidic polysaccharide. Nevertheless, the exact structural features of these acidic polysaccharides have not been fully elucidated. Furthermore, the structure of the RG polysaccharide has rarely been studied in comparison with that of wild ginseng. Therefore, in this study, we isolated an acidic polysaccharide with enhanced immunostimulatory activity and elucidated the structure of the key moiety of the polysaccharide, contributing to its immunostimulatory activity. RGP-AP-I was purely isolated via starch and homogalacturonan elimination and gel filtration chromatography, which indicated enhanced macrophage-stimulatory and anticomplementary activities than the other fractions. RGP-AP-I was estimated as having a molecular weight of 96 kDa and mainly comprised Ara, Gal, and GalA. As a result of methylation analysis, RGP-AP-I was found to comprise a pectic RG-I backbone highly substituted with arabinan, linear galactan, and arabino-b-3,6-galactan branches. Based on these results, the active immunostimulatory structure of RGP was suggested, and this may be the first research of its kind on RG polysaccharide.

Conflicts of interest
The authors claim no conflicts of interest.