Structural characterization and immunostimulatory activity of a novel polysaccharide isolated with subcritical water from Sagittaria sagittifolia L.

International Journal of Biological Macromolecules 133 (2019) 11–20

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Structural characterization and immunostimulatory activity of a novel polysaccharide isolated with subcritical water from Sagittaria sagittifolia L. Jixian Zhang a, Meng Chen a, Chaoting Wen a, Jie Zhou a, Jinyan Gu a, Yuqing Duan a,b,⁎, Haihui Zhang a,b,⁎, Xiaofeng Ren a, Haile Ma a,b a b

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China Institute of Food Physical Processing, Jiangsu University, Zhenjiang 212013, China

a r t i c l e

i n f o

Article history: Received 30 January 2019 Received in revised form 5 April 2019 Accepted 11 April 2019 Available online 12 April 2019 Keywords: Pressurized hot water Arrowhead Isolation and purification Structural elucidation Immunoactivity

a b s t r a c t In the present study, we obtained polysaccharides from Sagittaria sagittifolia L. (SSP) with subcritical water extraction (SWE). Two water-soluble polysaccharides (SSP-W1 and SSP-S1) from the acquired SSP were isolated with DEAE-52 and Sephadex G-100. Besides, the structural characteristics and immunostimulatory activity were also investigated. The results showed that both SSP-W1 and SSP-S1 were homogeneous polysaccharides and the molecular weight was 62.03 KDa and 15.2 KDa, respectively. In addition, both SSP-W1 and SSP-S1 are heteropolysaccharides. Moreover, FT-IR analysis showed that SSP-W1 was α-pyranose polysaccharide, while SSP-S1 was a typical β-pyranose polysaccharide. Congo red staining showed that there was no triple helix structure in both SSP-W1 and SSP-S1. Furthermore, both SSP-W1 and SSP-S1 could promote the proliferation, production of NO, and secretion of TNF-α and IL-10 of macrophages RAW 264.7, significantly. Therefore, the polysaccharides extracted from Sagittaria sagittifolia L. with SWE have the potential to be used as immunoreactive agent in medicine and functional foods. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Arrowhead (Sagittaria sagittifolia L.) is an aquatic flowering plant of the family Alismataceae, which is widely cultivated in North America, Europe and Asia [1]. The round tuber of arrowhead is rich in carbohydrates (54.60 g/100 g), protein (16.4 g/100 g), and fat (0.47 g/100 g), among others [2]. Especially, polysaccharides are one of the most important biologically active ingredients in Sagittaria sagittifolia L. and have attracted considerable attention. Recent studies have shown that polysaccharides extracted from natural products, without toxic and side effects, not only serve as biological response modifiers, but also have a variety of biological activities, including antitumor activity [3], immunological activity [4], antibacterial activity [5], among others. Especially, antitumor activity and immunological activity have captured more and more attention in biochemical and medical fields. More importantly, polysaccharides are not only contributed to improve the host's defense against pathogens, but also regulate adaptive immunity [6]. Furthermore, these biological activities were reported to be associated with solubility, monosaccharide composition, molecular weight, and advanced structure [7]. In addition to ⁎ Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China. E-mail addresses: [email protected] (Y. Duan), [email protected] (H. Zhang).

https://doi.org/10.1016/j.ijbiomac.2019.04.077 0141-8130/© 2019 Elsevier B.V. All rights reserved.

these factors, the biological activity of polysaccharides is also affected by the extraction methods. Therefore, the choice of polysaccharides extraction method is also very noteworthy. Traditionally, the polysaccharides is mainly extracted by hot water, polyethylene glycol, and alkali solution, among others [8]. However, these extraction methods have some disadvantages such as time consuming, low extraction efficiency, and environmental pollution [9]. Based on above situations, some new innovative green and promising methods have emerged, including SWE [10–12], supercritical fluid extraction (SFE) [13,14], ultrasonic extraction (UE) [15–17], and ultrahigh pressure extraction (UPE) [18–20], among others. Especially, there are increasing reports on the extraction of polysaccharides by using SWE in recent years due to its energy saving, high efficiency, no pollution [21]. For example, our previous study has showed that the polysaccharides from Lentinus edodes extracted with SWE had better antioxidant activity and antitumor activity than hot water extraction [22,23]. Thus, the SWE is considered as a novel technique to apply in polysaccharides extraction fields. In addition, it is very important to study the relationship between subcritical water properties and polysaccharides extraction. Subcritical water is defined as hot water at sufficient pressure to maintain the liquid state at critical temperature between 100 °C (the boiling point of water) and 374 °C (the critical point of water) under the critical pressure (1–22.1 MPa) [24,25]. In the subcritical state, the

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dielectric constant of water is similar to that of organic solvents. Besides, the polarity of subcritical water decreases gradually with the increase of temperature [26]. Therefore, the polar, bipolar, and nonpolar compounds can be extracted by controlling temperature [27,28]. Additionally, compared with SFE, the operation of SWE device is simple and easy, and the pressure is lower. Therefore, SWE has been applied not only to the extraction of large-scale raw materials, but also in the extraction of bioactive polysaccharides and begun to show great potential. For example, some polysaccharides were successfully extracted with SWE from Grifola frondosa [29], Lycium barbarum L. [11,30], and apple pomace [31], among others. Interestingly, polysaccharides extracted with subcritical water have higher yield and stronger biological activities than other traditional extraction method. At the same time, it is worth noting that subcritical water also has a modified function on the molecular structure of the polysaccharides, which makes it exhibit stronger activity [32]. At present, most of literatures were reported on the crude polysaccharides treated by SWE [11,31]. However, no study was reported on purified Sagittaria sagittifolia L. polysaccharides. In addition, our group has optimized the SWE extraction process of SSP by orthogonal experiment design. The results showed that pH, extraction temperature, extraction time, and liquid-to-solid ratio were the major variables which influenced the yields of polysaccharides. The optimum extraction conditions were pH 7, extraction temperature 170 °C, extraction time 16 min, and liquid to solid ratio 30:1 (mL/g), respectively. However, the structure and the biological activities of purified SSP are still unclear and need further study. Therefore, it is necessary to investigate the purification, characterization, and immunostimulatory activity of SSP obtained from Sagittaria sagittifolia L. with SWE. It is expected that all these results could contribute to the development and utilization of arrowhead resources and the application of SWE in related polysaccharides. 2. Materials and methods 2.1. Materials and reagents Arrowhead (Sagittaria sagittifolia L.) was purchased from JiangDa Farmer's market (Zhen Jiang, Jiangsu province, China). RAW 264.7 cell line was obtained from the Institute of Biochemistry and Cell Biology (SIBS, CAS; Beijing, China). Standard monosaccharides (L-rhamnose, D-arabinose, D-xylose, D-mannose, D-glucose, and D-galactose), lipolysaccharide (LPS), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma (MO, USA). DEAE-52 was purchased from Whatman International Ltd. (Maidstone, Kent, UK);

Sephadex G-100 was purchased from Pharmacia Co. (Sweden). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Hylone (Logan, USA). All other reagents were analytical reagent grade. 2.2. Extraction of polysaccharides with SWE The schematic diagram of the experiment was shown in Fig. 1. Subcritical water extractor was purchased from Nantong Huaan Scientific Research Devices Co., Ltd. (Jiangsu, China). Sagittaria sagittifolia L. was dried in an oven at 50 °C and then extracted under a pressure of 1 MPa with subcritical water. After extraction, the solution was centrifuged at 4500 g for 20 min, and the supernatant was concentrated under reduced pressure. Finally, four times of 95% ethanol was added to the concentration of supernatant at 4 °C overnight to precipitate the polysaccharides. The polysaccharides were collected and dialyzed against ultrapure water (cut-off 3500 Da). After concentrated and lyophilized, the polysaccharides were stored at 4 °C for further analysis. The polysaccharides yield was measured by phenol sulfuric acid method by using D-glucose as standard. The SWE-SSP yield (%) was calculated by the following formula: y (%) = c/w 100%, where c represents the weight of polysaccharides and w represents the dried extracts weight. 2.3. Isolation and purification of crude polysaccharides Before purification, it is necessary to remove the impurities from the crude polysaccharides, mainly including proteins and pigments. The polysaccharides solution was deproteinized by the Sevage method [33] about six times, and treated with 30% H2O2 to decolorize [34]. Then the polysaccharides solution was freeze-dried for further analysis. The crude polysaccharides (500 mg) were dissolved in distilled water (30 mL), and then were centrifuged at the speed of 5000 rpm for 15 min. The polysaccharides supernatant was loaded onto the DEAE52 cellulose column (2.6 × 60 cm) which were pre-equilibrated overnight, and then eluted with distilled water and different concentration of NaCl solutions (0.1, 0.2, 0.3, and 0.5 M) at the flow rate of 2 mL/min. Two fractions (SSP-W and SSP-S) were separated and considered to be the main component of SWE-SSP. Then, SSP-W and SSP-S were purified by size-exclusion chromatography on a Sephadex G-100 column (1.6 × 90 cm), eluted with distilled water at the speed of 0.25 mL/min. Two further purified polysaccharides (SSP-W1 and SSPS1) were concentrated, dialyzed (3500 Da), and freeze-dried for further analysis.

Fig. 1. Schematic diagram of the experiment.

J. Zhang et al. / International Journal of Biological Macromolecules 133 (2019) 11–20

2.4. Carbohydrate, uronic acid and protein contents The phenol-sulfuric acid method was used to measure the content of carbohydrate with D-Glucose as standard [35]. The m-hydroxybiphenyl colorimetric procedure was used to measure the content of uronic acid by using D-glucuronic acid as standard [36]. The content of protein was determined by Bradford method by using bovine serum albumin (BSA) as standard [37]. 2.5. Characterization of purified polysaccharides

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alcohol was hydrolyzed, derived, and analyzed by same GC procedure described in Section 2.5.1. 2.5.5. FT-IR analysis FT-IR spectra can be used to analyze monosaccharide types, functional groups, and glucosidic bonds [42], and investigate the change of dipole moment in the process of molecular vibration or rotation and the vibration of the polar bonds between different atoms. SSP-W1 and SSP-S1 (40 mg) were mixed with spectroscopic KBr powder (400 mg) and identified by using FT-IR spectra (Nicolet™ IS™ 50, New York, USA).

2.5.1. Monosaccharide composition Monosaccharides composition of SSP-W1 and SSP-S1 were measured by GC procedure according to the previous method with minor modification [38]. Specifically, both SSP-W1 and SSP-S1 (10 mg) were degraded with sulfuric acid (3 mol/L, 5 mL) at 100 °C for 6 h. After hydrolysis, BaCO3 was used to neutralize sulfuric acid. Then, the supernatant was concentrated, and dried by rotary evaporation under reduced pressure. Hydroxylamine hydrochloride (10 mg) and pyridine (0.5 mL) were added to above products and reacted at 90 °C for 30 min. After that, acetic anhydride (0.5 mL) was added to react at 90 °C for 30 min. The final products were analyzed by GC with a HP-5 fused silica capillary column (30 m × 0.32 mm × 0.25 μm) and equipped with a flame-ionization detector (FID). The GC procedure was as follows: N2 (30 mL/min), H2 (38 mL/min) and air (370 mL/min). The temperature of the detector and injector were 300 °C and 280 °C, respectively. The heating procedure is to keep the temperature at 130 °C for 5 min, and then rise to 240 °C at a speed of 4 °C/min. The standard monosaccharides were analyzed by the same GC procedure.

2.5.6. Congo red analysis Congo red assay was carried out by the previous method with minor modification [43]. SSP-W1 and SSP-S1 (1 mg/mL, 2 mL) mixed with the Congo red reagent (91 μmol/L, 2 mL). The NaOH concentration in the solution gradually increased from 0 to 0.5, and the maximum absorption wavelength at each NaOH concentration was recorded in the process with an ultraviolet-visible spectrophotometer (Shimadzu Co., Japan). The Congo red solution without polysaccharides was used as control.

2.5.2. Molecular weight and homogeneity determination The method of molecular weight analysis was slightly modified according to the previous method [39]. The molecular weight of SSP-W1 and SSP-S1 were measured by using a high performance sizeexclusion chromatograph (HPSEC), which equipped with a differential Refractive Index detector (Agilent G 1362 A, USA) and a Multi-Angle Laser Light Scattering detector (MALLs, DAWN HELEOS II, Wyatt Technology, USA). Two columns (shodex OHpak SB-805 and shodex OHpak SB-806) connected in series were used to elute polysaccharides with 0.1 M NaCl solution. The loading concentration of polysaccharide is 1 mg/mL. The refractive index increment (dn/dc) was 0.138 mL/g. Data acquisition was analyzed by ASTRA software (Wyatt Technology, USA).

2.6.1. Proliferation activity assay of macrophage RAW 264.7 in vitro Macrophage RAW 264.7 proliferation activity assay was performed with previous described method [44]. The RAW 264.7 cells were plated in 96 well plates (2 × 105 cells/mL) and treated with different concentrations of SWE-SSP, SSP-W1 and SSP-S1 (50, 100, and 200 μg/mL) which were dissolved in DEME medium. LSP (1 μg/mL) was used as a positive control. After 24 h incubation, 100 μL/well of MTT solution (1 mg/mL) was added. After 4 h incubation, cell supernatant was removed and 100 μL of DMSO were added for cell lysis and crystal solubilization. Absorbance was recorded at 540 nm. Cell proliferation index was calculated as the following formula:

2.5.7. Atomic force microscopy (AFM) analysis SSP-W1 and SSP-S1 were dissolved with distilled water to a final concentration of 5 μg/mL. Polysaccharides solution (10 μL) was added dropwise to the freshly stripped mica and dried overnight. The atomic force microscopy (Multimode 8 instrument, USA) was used to obtain the image of the polysaccharides. The software of NanoScope was used to process the image or data. 2.6. Determination of immunostimulatory activity

Cell proliferation index ¼ A1 =A0 2.5.3. Partial acid hydrolysis assay In order to investigate the structure of SSP-W1 and SSP-S1, partial acid hydrolysis assay also need to be down [40]. SSP-W1 and SSP-S1 (25 mg) were hydrolyzed by trifluoroacetic acid (0.02 M) at 110 °C for 3 h. After that, the temperature is cooled down to room temperature, and then the residual trifluoroacetic acid is removed by rotating evaporation. The above products were dialyzed against distilled water (3500 Da) to separate oligosaccharides and monosaccharides, which were collected and freeze-dried. The final products were analyzed by same GC procedure described in Section 2.5.1. 2.5.4. Periodate oxidation and smith degradation Periodate oxidation and smith degradation can be used to determine the type of polysaccharides glycosidic bond [41]. SSP-W1 and SSP-S1 (40 mg) were dissolved in 40 mL NaIO4 solution and stored it in darkness. The absorbance of polysaccharides solution was measured at 223 nm every 4 h until it is basically stable. The HCOOH content was calculated by titration of standard NaOH solution (0.01 M). After periodate oxidation, the residual periodate was reduced by adding ethylene glycol (4 mL). Then the oxidation product was dialyzed (3500 Da) against distilled water for 36 h. After that, the pH was adjusted to 5.5, and then the product was dialyzed (3500 Da) against distilled water for 72 h, and lyophilized to obtain the multi-sugar alcohol. Finally, the multi-sugar

ð1Þ

where A1 is the absorbance of sample; A0 is the absorbance of the control. 2.6.2. Macrophage RAW 264.7 phagocytosis assay in vitro The method of Macrophage RAW 264.7 phagocytosis assay was according to the previous method [45] with minor modification. Macrophage RAW 264.7 cells were seeded in 96-well plates at a density of 2 × 105 cells/mL, and treated with various final concentrations of SWESSP, SSP-W1 and SSP-S1 (50, 100, and 200 μg/mL). In addition, LSP (1 μg/mL) was used as a positive control. After 24 h incubation in the condition of 37 °C and 5% CO2, the medium was discarded and 0.1% neutral red dye solution was added to each well (100 μL). After 4 h incubation, the plate was washed two times with PBS. Each well was mixed with 100 μL of cell lysates (ethanol and acetic acid mixed in equal volume), shaken for 10 min, and fully blended. The absorbance was measured at 540 nm by using a microplate reader. The phagocytic index was calculated by the following formula: Phagocytic index ¼ A1 =A0

ð2Þ

where A1 is the absorbance of sample; A0 is the absorbance of the control.

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2.6.3. Nitric oxide (NO) production assay in vitro Nitric oxide (NO) production was determined by previous method [46] with minor modification. Briefly, Macrophage RAW 264.7 cells were seeded in 48 well-plates at a density of 2 × 105 cells/mL, and treated with different concentrations of SWE-SSP, SSP-W1, and SSP-S1 (50, 100, and 200 μg/mL) for 48 h as described in Section 2.6.2. LSP (1 μg/mL) was used as a positive control. Then the medium was collected and mixed with an equal volume of Griess reagent (1% sulfanilamide in dd H2O, 0.1% N (1-naphthyl) ethylenediamide in 5% phosphoric acid). After 10 min, the absorbance was measured by using a microplate reader at 540 nm. The standard curve was formed with sodium nitrite.

were measured by enhanced chemiluminescence (ECL) detection kit and results were analyzed by Quantity One software. 2.7. Statistical analysis The data was expressed as the Mean ± SD. The significance of the difference was evaluated by multiple comparison analysis with Duncan's tests (SPSS 18.0 software). A value of p b 0.05 was considered to define as statistically significant. 3. Results and discussion 3.1. Extraction and purification of SWE-SSP

2.6.4. Western blotting analysis The method of western blotting analysis was according to our previous method with minor modification [47]. After treated with different concentrations of SWE-SSP, SSP-W1 and SSP-S1 (50, 100, and 200 μg/mL), the RAW 264.7 cells were seeded in 6 well-plates at a density of 2 × 105 cells/mL and pre-incubated for 24 h. LSP (1 μg/mL) was used as a positive control. After that, RAW 264.7 cells were collected, cleaned twice by PBS, and centrifuged. Subsequently, cell lysate (50 μL) was lysed at 4 °C for 30 min and then centrifuged at 4 °C for 5 min (10,000 rpm). Protein content in supernatant was measured by using enhanced bicinchoninic acid assay (BCA) kit (Beyotime, Jiangsu, China). The equal amount of protein was loaded onto 15% SDS-PAGE. Protein was blotted onto polyvinylidene-difluoride (PVDF) membrane at 200 mA for 90 min at 4 °C. The membrane was sealed with TBST containing 5% skimmed milk powder for 1.5 h and washed with TBST for three times (5 min each time). After that, the membrane was incubated primary antibody (1:1000) overnight at 4 °C with 5% non-fat milk/TBST. After washing three times with TBST (5 min each time), the membrane was incubated in the appropriate HRP-conjugated secondary antibody (1:3000) for 1.5 h at room temperature. Finally, the target proteins

The yield of SWE-SSP obtained from Sagittaria sagittifolia L. was approximately (24.57 ± 0.49) %. To better understand the immunostimulatory activity of SWE-SSP, the further purification on its major fractions is necessary. The absorbance of each eluent was determined by phenol sulfuric acid method at 490 nm. At this study, the SWE-SSP was purified by using DEAE-52 cellulose chromatography column as shown in Fig. 2A. Two peaks of SWE-SSP were eluted successfully by using distilled water and 0.1 M NaCl, and named SSP-W and SSP-S, respectively. Therefore, SSP-W was a neutral polysaccharide, while SSP-S was an acidic polysaccharide. The rate of recovery of SSPW and SSP-S were 18.67% and 8.33%, respectively. However, the content of carbohydrates of SSP-W and SSP-S were only 86.09% and 48.75% (based on the weight of SWE-SSP), respectively. Therefore, each peak need be further purified by using size exclusion chromatography (Sephadex G-100). SSP-W and SSP-S were eluted by distilled water to obtain one single peak, respectively. Finally, after concentration, dialysis (3500 Da) and freeze-drying, we obtained SSP-W1 and SSP-S1 (Fig. 2B and C), and their recovery rate were 36.68% and 18.59%, respectively. The carbohydrates content of SSP-W1 and SSP-S1 were 94.61% and

Fig. 2. Separation and Purification of SSP. (A) elution curve of SSP on the DEAE-52 cellulose chromatography column and elution curve of SSP-W (B) and SSP-S (C) on Sephadex G-100.

J. Zhang et al. / International Journal of Biological Macromolecules 133 (2019) 11–20

88.82%, respectively. The uronic acid content of SSP-S1 was 2.18%. Both of SSW-W1 and SSP-S1 were protein free. 3.2. Structural analysis of the polysaccharide fractions 3.2.1. Monosaccharide composition analysis The monosaccharide composition of purified polysaccharides was shown in Fig. 3(A–C). SSP-W1 was composed of arabinose, glucose, and galactose with the molar ratio of 1:341.126:28.689. While SSP-S1 was composed of rhamnose, arabinose, glucose, and galactose with the molar ratio of 1:2.883:59.812:6.795. The results indicated that both SSP-W1 and SSP-S1 were heteropolysaccharides, which also demonstrated that rhamnose, arabinose, glucose, and galactose are the primary monosaccharides of Sagittaria sagittifolia L. polysaccharides and glucose is the major monosaccharide component. Interestingly, the monosaccharide composition of SSP-W1 and SSP-S1 showed obvious differences, not only in proportion but also in type. Zhu et al. reported that the polysaccharides obtained from Cordyceps gunnii mycelia with ultrasound extraction were composed of D-mannose, D-glucose, and D-galactose [48]. In addition, the monosaccharide of polysaccharides extracted from Giant African snail (Achatina fulica) with pressurized hot water extraction (PHWE) was composed of only one mark corresponding to glucose [49]. Two novel polysaccharides (MRP 5 and MRP 5A) were extracted from the root of Panax notoginseng by using column chromatography. Their results showed that, MRP5 consisted of

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rhamnose (3.6%), arabinose (14.5%), glucose (51.2%) and galactose (29.7%), and MRP 5A was composed of rhamnose (7.7%), arabinose (6.6%), glucose (69.8%) and galactose (15.9%) [50]. Therefore, the monosaccharide composition and molar ratio may depend on their raw materials, separation and purification methods. 3.2.2. Molecular weight and homogeneity Previous reports have shown that the biological activities of polysaccharides is closely related to the molecular weight [51]. In this work, the weight-average molecular weights (Mw), number-average molecular weights (Mn) and the polydispersity of the SSP fractions were determined by SEC-MALLS-RI combined technology. The HPSEC chromatograms of SSP-W1 and SSP-S1 were shown in Fig. 3(D, E). The results indicated that there was a single and symmetrical peak of both SSPW1 and SSP-S1 on RI chromatograms. Meanwhile, the LS signal was in general agreement with that of RI signal, which suggested that both SSP-W1 and SSP-S1 are homogeneous polysaccharides. The main peaks of LS and IR spectra were analyzed by ASTRA software. It can be concluded that, the Mw of SSP-W1 and SSP-S1 were 62.03 KDa and 15.2 KDa, and the Mn were 52.5 KDa and 13.35 KDa, respectively. In addition, the molecular weight distribution coefficients (Mw/Mn) were 1.182 and 1.139, respectively. The low distribution coefficients (Mw/ Mnb2) showed that molecules disperse lesser in aqueous solution and do not form large aggregates [52]. Our previous study also found that the polysaccharides extracted with subcritical water from Lentinus

Fig. 3. Gas chromatography of Standard monosaccharides (A), SSP-W1 (B), and SSP-S1 (C). Peaks: (1) L-Rhamnose; (2) D-Arabinose; (3) D-xylose; (4) D-Mannose; (5) D-Glucose; (6) D-Galactose; HPSCE chromatograms of SSP-W1 (D) and SSP-S1 (E).

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edodes has a low polydispersity coefficient, and it does not form agglomeration and has good solubility [22,23]. 3.2.3. Partial acid hydrolysis The principle of partial acid hydrolysis is that some glycosidic bonds are more stable to acids than other glycosidic bonds [51]. The sugar alcohol acetate derivative of each component was subjected to GC analysis, and the results were shown in Table 1. The results indicated that DArabinose, D-Glucose, D-Galactose may be the backbone and branched structure of SSP-W1 with a molar ratio of 1: 129.75:18.17 and 1:23.99:1.13, respectively. In addition, L-Rhamnose, D-Arabinose, DGlucose, and D-Galactose may be the backbone structure of SSP-S1 with a molar ratio of 1:3.15:27.93:2.51, and L-Rhamnose, D-Glucose, and D-Galactose may be the branched structure of SSP-S1 with a molar ratio of 1.39:8.41:1. In addition, other studies have also utilized partial acid hydrolysis to determine the monosaccharide composition of the backbone and side chains. The partial acid hydrolysis of a water-soluble polysaccharide (ABPS-21) from Acanthopanax brachypus was determined, which showed that the backbone of ABPS-21 may be composed of galactose, glucose and rhamnose in a molar ratio of 3.0:1.0:1.0, and the side chain was composed of glucose and rhamnose [53]. Li et al. reported a novel water-soluble polysaccharide (GCP50–1) from Ganoderma capense, the partial acid hydrolysis showed that both backbone and branch fractions of GCP50–1 were composed of glucose [54]. Therefore, these results indicated that partial acid hydrolysis can be widely applied to the characterization of the monosaccharide composition of the main and side chain of the polysaccharides. 3.2.4. Periodate oxidation and smith degradation Periodate oxidation and smith degradation assay are mainly used to determine the type of glycoside bonds of polysaccharides [55]. The results of periodate oxidation were shown in Table 2. The formic acid was produced in SSP-W1 and SSP-S1, which indicating that both of them contain 1 → and 1 → 6 linkages [56] with the molar number of 0.288 and 0.185, respectively. In addition, the consumption of HIO4 in both SSP-W1 and SSP-S1 was twice higher than formic acid production, which suggesting that both SSP-W1 and SSP-S1 contain 1 → 2, 1 → 2,6, 1 → 4, and 1 → 4,6 linkages [57] with the molar number of 0.253 and 0.387, respectively. Furthermore, the consumption of HIO4 was lower than the molar mass of the sample, there were 1 → 3 type linkages [53] in both SSP-W1 and SSP-S1 which was not oxidized by periodic acid, and the molar number was 0.459 and 0.428, respectively. There have also been other studies on the attachment of polysaccharides glycosidic bonds by periodate oxidation. For example, the results of periodate oxidation on polysaccharides extracted from Cedrus deodara demonstrated that 1 mol of polysaccharide residue consumed 0.68 mol of periodate and produced 0.13 mol formic acid, which indicated that some monosaccharides were linked by 1 → or 1 → 6 linkage. In addition, the consumption of periodate was more than consumption of formic acid (0.13 mol × 2), which demonstrated that there were other linkage types that could not produce formic acid, such as 1 → 3, 1 → 3,6 [58]. The smith degradation products from SSP-W1 and SSP-S1 were analyzed by GC. The production of glycerol showed that there were 1→, 1 → 2, 1 → 6, and 1 → 2,6 linkages [59], the production of erythritol showed that it contained 1 → 4 and 1 → 4,6 linkages [60], and the

Table 1 Monosaccharide mole ratio of backbone and branched structure of polysaccharide. Polysaccharides

Backbone

Branch

SSP-W1

Ara:Glu:Gal 1:129.75:18.17 Rha:Ara:Glu:Gal 1:3.15:27.93:2.51

Ara:Glu:Gal 1:23.99:1.13 Rha:Glu:Gal 1.39:8.41:1

SSP-S1

Rha: L-Rhamnose; Ara: D-Arabinose; Glu: D-Glucose; Gal: D-Galactose.

detection of monosaccharides indicated the presence of linkages which is not oxidized by periodate, such as 1 → 3, 1 → 3,4, and 1 → 3,6 [58]. As shown in Table 2, there were a large amount of glycerol and erythritol, and a small amount of glucose in SSP-W1. In combination with partial acid hydrolysis, we surmised that the glucose in the backbone and branch of SSP-W1 mainly could be connected with 1 → 3, 1 → 2,3, 1 → 2,4, 1 → 3,4, 1 → 3,6, 1 → 2,3,4, 1 → 2,3,6 or 1 → 3,4,6 linkages which not be oxidated by periodate, and arabinose and galactose were linked by 1→, 1 → 2, 1 → 6, 1 → 2,6, 1 → 4 and 1 → 4,6 linkages in the backbone and branch. In addition, GC analysis of smith degradation products of SSP-S1 showed (Table 2) that there existed glycerol, erythritol, arabinose, glucose and galactose. The results indicated that rhamnose was connected by 1→, 1 → 2, 1 → 6, 1 → 2,6, 1 → 4, and 1 → 4,6 linkage which can be oxidized. In addition, the presence of arabinose, glucose and galactose showed that these monosaccharides may be connected by 1 → 3, 1 → 2,3, 1 → 2,4, 1 → 3,4, 1 → 3,6, 1 → 2,3,4, 1 → 2,3,6, or 1 → 3,4,6 linkages which cannot be oxidized by periodate [53]. Other reports have also resulted in the similar smith degradation results. For example, during smith degradation of HSP-III extracted from Hirsutella sinensis, the production of saccharides showed that there existed the 1 → 3 type linkages in HSP-III. The production of glycerol demonstrated that the main chain or branch was linked by 1 → 2 or 1 → 6 linkage. The production of erythritol suggested the abundance of the 1 → 4 linkage [60]. However, methylation and nuclear magnetic resonance (NMR) analysis of SSP should be carried out to further refine the structure. 3.2.5. FT-IR analysis FT-IR analysis showed that SSP-W1 and SSP-S1 contain the characteristic absorption peaks of polysaccharides (Fig. 4A). The absorption peak at 3400 cm−1 is caused by O\\H stretching vibrations. The weak and narrow absorption peak at 2800–3000 cm−1 is due to the stretching vibration of C\\H. The bands in the 1650 cm−1 are due to the associated water. The peaks at 1400 cm−1 are the bending vibration absorption peaks of C\\H. The absorptions in the range of 1200–1500 cm−1 are mainly due to the C\\H deformation vibrations and C-OH bending vibrations. The bands in the range of 1000–1200 cm−1 are reflected by the stretching vibrations of C\\C and C\\O [61]. Furthermore, the characteristic peak at around 850.45 cm−1 and 892.88 cm−1 was found in SSP-W1 and SSP-S1, which indicating the existence of α-type glycosidic linkage and β-type glycosidic linkage, respectively [62,63]. In addition, the absorption at around 1025 cm−1 indicates a pyranose form of polysaccharides [64]. Therefore, it is indicated that SSP-W1 may be αconfiguration of pyranose, and SSP-S1 may be β-configuration of pyranose. These results are similar to the conclusion reported by Wang et al. [65]. 3.2.6. Analysis of triple helix Congo red can form a complex with a polysaccharide which having a triple helix structure. In addition, the maximum absorption wavelength of the complex is red-shifted compared to Congo red [66,67]. The results of Congo red experiments were showed in Fig. 4B. It is obvious that both SSP-W1 and SSP-S1 have no red shifts of maximum wavelength in the concentration range of 0.1–0.25 M. Therefore, there are no triple helix structure existed in both SSP-W1 and SSP-S1. In our previous reports, it has been reported that polysaccharides extracted from Lentinus edodes had a red shift of the maximum absorption wavelength under different concentration range of NaOH, and it is confirmed that lentinan has a triple helix structure [22]. In addition, previous studies have also shown that only polysaccharides with a molecular weight N 90 KDa can form a triple helix structure, which is consistent with molecular weight analysis of our result [68]. Interestingly, the polysaccharides with triple helix structure appear to have better antitumor activity [7]. Therefore, the antitumor activity of the Sagittaria sagittifolia L. polysaccharides also need further study.

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Table 2 Analysis of Periodate oxidation and Smith degradation of SSP-W1 and SSP-W2. Periodate oxidation

SSP-W1

SSP-S1

Smith degradation

SSP-W1

SSP-S1

Consumption of HIO4 (mmol) Consumption of HIO4/hexose (mol/mol Amount of formic acid (mmol) Amount of formic acid/hexose (mol/mol)

0.184 0.829 0.064 0.288

0.168 0.757 0.041 0.185

glycerin erythritol Rhamnose Arabinose Glucose Galactose

+a + -b + -

+ + + + +

a b

Detectable on GC. Undetectable on GC.

3.2.7. AFM analysis AFM is a powerful technology to determine soft biological surfaces, has been widely used to analyze the structure of biological macromolecules [69]. In recent years, AFM has become a valuable tool to characterize the surface structure of polysaccharides [70]. The AFM images of SSP-W1 and SSP-S1 indicated that they were spherical shape with the mean diameter about 65 nm and 28 nm, respectively. As shown in Fig. 4(C, D), the height of SSP-W1 was higher than the theoretical diameter of single chain of polysaccharides (about 0.1–1 nm), which indicated that the structure of SSP-W1 agglomerated due to the intertwining of molecular chains and intermolecular and intramolecular hydrogen bonding interactions [71]. However, the height of SSP-S1 was close to the theoretical value of the single chain of polysaccharide. Therefore, SSP-S1 can be regarded as being in a single-chain form, and dispersed uniformly. In fact, polysaccharides may exhibit a variety of different conformations in solution, such as single helix [72], double helix [73], triple helix [74], aggregates [75], random coil [76], rod-like structure [77], and sphere-like structure [78]. The biological activity of polysaccharides is strongly dependent on the conformation of polysaccharides. 3.3. Immunostimulatory activities of SWE-SSP fractions 3.3.1. Effects on macrophage RAW 264.7 proliferation The effects of SWE-SSP, SSP-W1 and SSP-S1 on the proliferation of macrophages RAW 264.7 were shown in Fig. 5A. It can be seen that

SWE-SSP, SSP-W1 and SSP-S1 can stimulate the proliferation of macrophages RAW 264.7 in a dose-dependent manner. The proliferation of macrophages RAW264.7 in SWE-SSP, SSP-W1 and SSP-S1 groups was significantly different from the normal group (p b 0.01) in the concentration range of 50–200 μg/mL. The effects of SWE-SSP, SSP-W1 and SSP-S1 on the proliferation of macrophages RAW 264.7 were not significantly different in the concentration of 50 μg/mL. Interestingly, SWESSP had the best proliferation of macrophages RAW 264.7 in the concentration of 100 μg/mL, and SSP-S1 had the best proliferative effect on macrophages RAW 264.7 in the concentration of 200 μg/mL. Therefore, the polysaccharides obtained from Sagittaria sagittifolia L. with SWE have a good effect on promoting macrophage RAW 264.7 proliferation. Some other studies have also shown that polysaccharides have a significant proliferative effect on macrophages RAW264.7 [79–81].

3.3.2. Effects on macrophage RAW 264.7 phagocytosis The SWE-SSP and each purified fraction (SSP-W1 and SSP-S1) on the phagocytosis and the NO production of macrophages RAW 264.7 were investigated in vitro. Phagocytosis is the first step in the immune response of macrophages to exogenous substances, which activates adaptive immune responses [82]. As shown in Fig. 5B, compared with control, SWE-SSP, SSP-W1, and SSP-S1 can promote the phagocytosis of macrophages RAW 264.7 in the concentration range of 50–200 μg/mL, and with significant difference in

Fig. 4. FT-IR analysis of SSP-W1 and SSP-S1 (A); Maximum absorption λmax of the Congo red–polysaccharide complex at various concentrations of NaOH (B); AFM image of SSP-W1 (C) and SSP-S1 (D).

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Fig. 5. Proliferative activity of macrophages RAW 264.7 cells (A); Effects on NO production by macrophages RAW 264.7 in vitro (B); Effects on macrophage RAW 264.7 phagocytosis (C); Effects of SWE-SSP (D), SSP-W1 (E), and SSP-S1 (F) on the relative expression content of IL-10 and TNF-α in macrophage RAW 264.7.

the concentration of 200 μg/mL (p b 0.01). The extent of stimulation followed the order: SSP-S1NSWE-SSPNSSP-W1. Activated macrophages can produce a variety of immune responses, while releasing NO and cellular immune factors, thereby promoting their induction of tumor cell growth and apoptosis [83]. Therefore, the yield of NO is also a key index for judging whether the SSP can enhance the immunological activity of macrophages RAW 264.7. In addition, NO is one of the most important molecules of macrophages RAW 264.7 against tumor cells, and it has the dual effects of inhibiting the proliferation of tumor cells and antiinflammatory response [84]. Fig. 5C showed that SWE-SSP, SSP-W1, and SSP-S1 can stimulate the NO production of macrophages RAW 264.7 in a dose-dependent (pb0.01). NO production in SWE-SSP group was higher than the other two groups (SSP-W1 and SSP-S1) in the concentration of 50 and 100 μg/mL. Interestingly, the NO production in the SSP-S1 group was higher than the other two groups (SWE-SSP and SSP-W1) in the concentration of 200 μg/mL. The amount of NO production stimulated by each fraction followed the order: SSP-S1NSWE-SSPNSSP-W1, which was consistent with the levels of phagocytic activity observed upon treatment with these fractions. Our conclusion was consistent with the study of Yu et al. [44], who highlighted that the CPS (extracted from American ginseng) and each purified fraction had enhanced macrophage

phagocytosis to various degree in the dose range of 50–200 μg/mL, the NO production was stimulated by CPS and each purified fraction.

3.3.3. Effects on the expression of TNF-α and IL-10 The macrophages stimulated by polysaccharides have two trends of polarization (M1 and M2). M1 can produce TNF-α IL-1 IL-6 IL-12, and M2 can produce IL-10, etc. [84]. The effect of SWE-SSP, SSP-W1, and SSP-S1 on the secretion of TNF-α and IL-10 by macrophages RAW264.7 was shown in Fig. 5 (D, E, and F). The results showed that, compared with control group, SWE-SSP, SSP-W1 and SSP-S1 can significantly up-regulate the expression of IL-10 and TNF-α (pb0.01), respectively. The expression of TNF-α and IL-10 was in a dose-dependent manner with the increase of the concentration. When the concentration of SWE-SSP, SSP-W1, and SSP-S1 was 200 μg/mL, the relative expression content of TNF-α was 1.95, 1.51, and 1.62 times higher than that of control group, and the relative expression content of IL-10 protein was 1.97, 1.49, and 1.56 times higher than that of control group, respectively. Interestingly, TNF-α and IL-10 could promote the proliferation and differentiation of T and B cells during the process of acquired immune response [85]. Besides, similar result was in agreement with the report of Jozˇica et al., who highlighted that TNF-α and IL-10 plays an important role in the immunomodulation [86]. Therefore, SWE-SSP, SSP-W1

J. Zhang et al. / International Journal of Biological Macromolecules 133 (2019) 11–20

and SSP-S1 can promote the secretion of TNF-α and IL-10, thereby can promote the immune function of macrophages RAW 264.7. 4. Conclusion In the present study, the novel polysaccharides were obtained from Sagittaria sagittifolia L. The molecular weights of SSP-W1 and SSP-S1 were 62.03 KDa and 15.2 KDa, respectively. SSP-W1 was composed of arabinose, glucose, and galactose with the molar ratio of 1:34.126:28.69. SSP-S1 was composed of rhamnose, arabinose, glucose, and galactose with the molar ratio of 1:2.88:59.81:6.80. In addition, FTIR analysis suggested these two purified polysaccharide fractions were composed of pyranose form sugar. The potency of their immunostimulatory effects can be ordered SSP-S1 N SWE-SSP N SSPW1 in the concentration of 200 μg/mL. These results showed that SSP has high immunostimulatory activity and can be utilized as an immunoadjuvant or a functional food. These findings provided a basis for further investigation of the high-efficiency and specific immunobioactive constituents of the SSP. However, the repeating units, sugar sequence, glycosidic linkage and the degree of branching of the potent component need further research. The details on the mechanisms and structure-activity relationship would be the subject of the continuing study. Acknowledgement This work was funded by National Key R & D Program, China (2016YFD0400303); Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX17_1799); Key Research and Development Plan of Jiangsu Province (BE2016350, BE2017353); The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] A.A. Wani, I.A. Wani, P.R. Hussain, A. Gani, T.A. Wani, F.A. Masoodi, Physicochemical properties of native and γ-irradiated wild arrowhead (Sagittaria sagittifolia L.) tuber starch, Int. J. Biol. Macromol. 77 (2015) 360–368. [2] I.A. Wani, A.A. Wani, A. Gani, S. Muzzaffar, M.K. Gul, F.A. Masoodi, T.A. Wani, Effect of gamma-irradiation on physico-chemical and functional properties of arrowhead (Sagittaria sagittifolia L.) tuber flour, Food Biosci. 11 (2015) 23–32. [3] M. Zhang, L. Zhang, P.C.K. Cheung, V.E.C. Ooi, Molecular weight and anti-tumor activity of the water-soluble polysaccharides isolated by hot water and ultrasonic treatment from the sclerotia and mycelia of Pleurotus tuber-regium, Carbohydr. Polym. 56 (2) (2004) 123–128. [4] G. Zhao, J. Kan, Z. Li, Z. Chen, Structural features and immunological activity of a polysaccharide from Dioscorea opposita Thunb roots, Carbohydr. Polym. 61 (2) (2005) 125–131. [5] W. Xie, P. Xu, W. Wang, Q. Liu, Preparation and antibacterial activity of a watersoluble chitosan derivative, Carbohydr. Polym. 50 (1) (2002) 35–40. [6] J. Vera, J. Castro, A. Gonzalez, A. Moenne, Seaweed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants, Mar. Drugs 9 (12) (2011) 2514–2525. [7] M. Zhang, S. Cui, P. Cheung, Q. Wang, Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity, Trends Food Sci. Technol. 18 (1) (2007) 4–19. [8] Y. Zhang, S. Li, X. Wang, L. Zhang, P.C. Cheung, Advances in lentinan: isolation, structure, chain conformation and bioactivities, Food Hydrocoll. 25 (2) (2011) 196–206. [9] Z. Hromadkova, A. Ebringerova, P. Valachovič, Ultrasound-assisted extraction of water-soluble polysaccharides from the roots of valerian (Valeriana officinalis L.), Ultrason. Sonochem. 9 (1) (2002) 37–44. [10] X. Wang, Q. Chen, X. Lü, Pectin extracted from apple pomace and citrus peel by subcritical water, Food Hydrocoll. 38 (2014) 129–137. [11] C. Zhao, R. Yang, T. Qiu, Ultrasound-enhanced subcritical water extraction of polysaccharides from Lycium barbarum L, Sep. Purif. Technol. 120 (2013) 141–147. [12] J. Zhang, C. Wen, H. Zhang, M. Zandile, X. Luo, Y. Duan, H. Ma, Structure of the zein protein as treated with subcritical water, Int. J. Food Prop. 21 (1) (2018) 128–138. [13] K. Ghafoor, J. Park, Y.-H. Choi, Optimization of supercritical fluid extraction of bioactive compounds from grape (Vitis labrusca B.) peel by using response surface methodology, Innovative Food Sci. Emerg. Technol. 11 (3) (2010) 485–490.

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