Chemical Structural Elucidation and Immunomodulatory Activity of a New Polysaccharide from Saposhnikoviae Radix

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Materials
• Extraction, isolation, and purification of SRP
• Structural characterization
• Homogeneity and molecular weight parameter analysis
• Uronic acid content analysis
• Monosaccharide composition analysis
• FT-IR analysis
• Methylation analysis
• Partial hydrolysis
• NMR analysis
• SEM and AFM determination
• In vitro immunomodulatory effects of SP4002501
• Cell culture
• Evaluation of proliferative activity
• Evaluation of phagocytic activity
• Evaluation of NO, TNF-α, and IL-6 production
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

The chemical structure and immunomodulatory activity of a new homogeneous polysaccharide, SP4002501, isolated from Saposhnikoviae Radix (SR), were investigated. Purification of SP4002501 was performed by DEAE-Cellulose and Sepharose CL-6B column chromatography. The monosaccharidic constituents were identified as rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), and arabinose (Ara) with a molar ratio of 3.7: 86.6: 2.7: 7.1. According to the methylation analysis, partial hydrolysis, FT-IR, and NMR analysis, SP4002501 had a backbone of polygalacturonic acid units with a small amount of galactose (Gal). Side chains are connected to C-3 of galactose (Gal) and consist of rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), and arabinose (Ara), with arabinose (Ara) as terminal sugar. Biological activity assessment suggests that SP4002501 exhibits immunomodulatory activity through promoting macrophage proliferation and phagocytosis.

Introduction

Saposhnikoviae Radix (SR), derived from the dried root of Saposhnikovia divaricata (Turcz.) Schischk. from the Apiaceae family, has various biological activities such as anti-inflammatory, anti-allergic and immunomodulatory properties [1], [2], [3]. Phytochemical analysis revealed that SR contains chromone, coumarins, polysaccharides, and volatile oil, among which polysaccharides are the predominant chemical components [4]. Polysaccharides, as non-toxic substances, are essential biomolecules, offering a promising resource for the development of drugs and nutraceuticals. Numerous studies have demonstrated that polysaccharides have a variety of health-promoting effects, and their immunomodulatory activity has been extensively recognized [5], [6], [7].

In our preliminary study, Saposhnikoviae Radix polysaccharides (SRP) significantly enhanced the proliferation and phagocytic capacity of RAW264.7 macrophages and promoted the release of NO, TNF-α, IL-1β, and IL-6 [8] in vitro. Further experiments showed that SRP can significantly increase the density of immune cells and the number of macrophages in immunocompromised mice [8], [9]. Accumulating evidences have shown that bioactivity of polysaccharides is related to their structural characteristics such as molecular weight, content of GalA, and number of branches [10], [11], [12], [13]. However, extraction, purification, and structural characterization of homogeneous polysaccharides are technically difficult, which hinders their widespread medicinal use. In an earlier report [14], we overcame the above difficulties and isolated a homogeneous polysaccharide, SP800201, from SR, which laid a reliable experimental basis for our further experiments.

In the present study, a new polysaccharide named SP4002501 was isolated and purified from SR and its homogeneous nature was verified by chromatography with three mobile phase systems. Its basic chemical composition was determined by molecular weight parameter analysis, uronic acid content analysis, and monosaccharide composition analysis. FT-IR, methylation analysis, and partial hydrolysis were then employed to obtain fragment information. With the help of NMR data, the fragment information was pieced together to obtain the full structure of the homogeneous polysaccharide. Finally, a RAW264.7 cell model was selected to study the immunomodulatory activity [15].

Results and Discussion

SP4002501, obtained by DEAE-Cellulose and Sepharose CL-6B chromatography, showed a uniform symmetrical peak on high-performance size-exclusion chromatography under three chromatographic conditions (Fig. 1S, Supporting Information). Weight-average molecular weight (M_w ), number-average molecular weight (M_n ), and peak molecular weight (M_p ) of SP4002501 were greater than 1.1 × 10⁶ g/mol, indicating that SP4002501 may be composed of thousands of sugar residues, similarly to LDP-1 extracted from Lactarius deliciosus [16]. The uronic acid content of SP4002501 was 88.7%. The monosaccharidic components of SP4002501 were Rha, GalA, Gal, and Ara (Fig. 2SA and B, Supporting Information) with a molar ratio of 3.7: 86.6: 2.7: 7.1. Uronic acid content and monosaccharidic components were mutually corroborating. The uronic acid content of SP4002501 was clearly higher than that of most polysaccharides found so far [17], [18], [19], [20], [21], meaning that SP4002501 may have a unique structural backbone. It is worth noting that some reports about the structure–activity relationship indicate that polysaccharides containing a high content of uronic acid show significant biological activities [22], [23], [24].

The IR spectrum of SP4002501 (Fig. 3S, Supporting Information) showed a broad and strong absorption band at 3600 – 3200 cm⁻¹ assigned to the -OH stretching vibration, while bands at 2930 cm⁻¹ and 1746 cm⁻¹ were assigned to the C – H stretching vibration and C=O stretching vibration, respectively [25]. Characteristic peaks at 1409 cm⁻¹ and 1235 cm⁻¹ were due to the C – H variable angle vibration, and the peak at 1101 cm⁻¹ was assigned to the C – O stretching vibration. The peak at 919 cm⁻¹ was characteristic for β-glycosidic bonds, while the peak at 831 cm⁻¹ was typical for α-glycosidic bonds [26].

Information on glycosidic linkages of polysaccharides can be obtained by methylation analysis. Based on the retention time (Fig. 4S, Supporting Information), standard data in Complex Carbohydrate Structure Database and literature reports [27], [28], the proportions of the methylated alditol acetates of SP4002501 were determined as reported in [Table 1]. Results suggested that Ara was present at the end of the sugar chain in the pyran and furan forms. GalA was present as (1 → 2)-, (1 → 3)-, and (1 → 4)-linked residues, while Rha was part of (1 → 3)- linked residues. Gal was present as (1 → 2)- and (1 → 2,3)-linked residues, indicating that there were branched chains in the polysaccharide structure and that Gal may be at the branching points.

The preparation of polysaccharide oligomers through partial hydrolysis provides the fragment information of complex polysaccharides and is an important step for polysaccharide characterization. We previously applied partial acid hydrolysis to identify the fragments of SRP [29]. In this study, partial acid hydrolysis and enzymatic hydrolysis were performed to infer the terminal composition of SP4002501 and analyze its fragment structure. Acid hydrolysis under three different conditions showed that best results were obtained with 0.55 M TFA at 85℃ (Fig. 5S, Supporting Information). By analysis of the hydrolysate outside the dialysis bag (Figs. 6S-8S, Supporting Information), Ara was detected, suggesting that Ara was on the branched chain, which was consistent with the results of the methylation analysis.

After hydrolysis by pectinase and cellulase, six fragments with a polymerization degree of 2 to 3 (m/z 339.1, m/z 341.1, m/z 355.1, m/z 369.1, m/z 383.1, and m/z 559.1) were detected in the ESI-MS/MS spectra of the enzymatic hydrolysate ([Table 2]).

In ESI-MS/MS spectra, oligosaccharides produce different fragment ions through glycosidic bond and cross-ring cleavages [30]. A fragment ion at m/z 163.0 [Rha-H]⁻ (C₆H₁₁O₅) was produced by the precursor ion at m/z 339.1 [GalA-Rha-H]⁻ (C₁₂H₁₉O₁₁), indicating that SP4002501 contains GalA-Rha units (Figs. 9Sa and 10Sa, Supporting Information). The precursor ion at m/z 341.1 [Gal-Gal-H]⁻ (C₁₂H₂₁O₁₁) produced a [Gal-O-H]⁻ ion at m/z 163.3 (C₆H₁₁O₅), indicating that SP4002501 contains Gal-Gal fragments (Fig. 9Sb and 10Sb, Supporting Information). The parent ion at m/z 355.1 [GalA-Gal-H]- (C₁₂H₁₉O₁₂) yielded the fragment ions at m/z 179.1 [Gal-H]⁻ (C₆H₁₁O₆) and m/z 337.2 [GalA-Gal-H₂O-H]⁻ (C₁₂H₁₇O₁₁), while the ion at m/z 311.1 was produced by cross-ring cleavages of ^4,5 A₁, indicating that SP4002501 contains GalA-Gal fragments (Figs. 9Sc and 10Sc, Supporting Information). Comprehensive analysis showed that the ion at m/z 369.1 (Fig. 9Sd and 10Sd, Supporting Information) may be related to the GalAOMe-Gal or GalA-GalA fragments. The precursor ion at m/z 369.1 [GalAOMe-Gal-H]⁻ (C₁₂H₁₇O₁₃) produced a [GalA – H]⁻ ion at m/z 193.2 (C₆H₉O₇). The ion at m/z 308.9 was produced by cross-ring cleavages of ^2,4 X₁, ^1,3 X₂, or ^0,2 A₂, indicating that SP4002501 contains GalAOMe-Gal fragments. An ion at m/z 193.2 [GalA – H]⁻ (C₆H₉O₇) produced by the precursor ion at m/z 369.1 [GalA-GalA – H]⁻ (C₁₂H₁₇O₁₃) was also detected, and intracyclic cleavage produced ions at m/z 308.9 and m/z 235.0, suggesting that SP4002501 also may contain GalA-GalA structural fragments. The ion at m/z 383.1 may come from GalAOMe-GalA or GalA-GalAOMe fragments (Figs. 9Se and 10Se, Supporting Information). The parent ion at m/z 383.1 [GalAOMe-GalA – H]⁻ (C₁₃H₁₉O₁₃) produced fragment ions at m/z 192.8 [GalA – H]⁻ (C₆H₉O₇) by glycosidic bond breaking, while ions at m/z 322.6 and m/z 234.7 were produced by cross-ring cleavages of ^2,4 X₂ or ^2,4 A₂ and ^0,2 X₂, respectively, indicating that the ion at m/z 383.1 comes from a GalAOMe-GalA fragment. The GalA-GalAOMe fragment also produced ions at m/z 192.8 [GalA – H]⁻ (C₆H₉O₇), m/z 322.6, and m/z 234.7. Similarly, the ion at m/z 559.1 may result from GalAOMe-GalA-GalA or GalA-GalA-GalAOMe fragments based on the ions observed at m/z 383.2, m/z 193.2, m/z 499.1, and m/z 322.6 (Figs. 9Sf and 10Sf, Supporting Information).

Information from 1D and 2D NMR analysis, in combination with previously reported literature data, was used to characterize the detailed structure of SP4002501. In the ¹H NMR spectrum ([Fig. 1 a]), peaks in the range of δH 4.50 to 5.50 corresponded to the H-1 (anomeric proton) signals of the sugar residues, while signals between δH 3.30 and 4.50 were attributed to the non-anomeric hydrogen atoms on the sugar rings. The signal at δH 3.93 was assigned to methoxy groups. Resonance peaks at δH 1.42 and 1.36 were characteristic of the methyl signal of Rha residues. In the ¹³C NMR spectrum ([Fig. 1 b]), peaks in the range of δC 100.0 to 115.0 were assigned to the C-1 signals of the sugar residues, and peaks at δC 62.0 – 90.0 were attributed to non-anomeric carbon signals [31]. Peaks in the range of δC 174.0 to 178.1 were assigned to the C-6 signals of the uronic acid residues and the signal at δC 56.0 corresponded to the signal of methoxy groups [32]. In the DEPT-135 spectrum ([Fig. 1 c]), downfield resonances signals corresponding to -CH₂- residues were attributed to C-5 of Ara and C-6 of Gal. Peaks at 79.8 ppm and 69.6 ppm were assigned to C-5 of Araf and Arap, respectively. Due to the severe overlap of some proton signals, it was not possible to assign all signals based solely on the information from the 1D NMR spectrum. However, by integrating information from 2D NMR spectra, such as ¹H-¹H COSY and HSQC, we could assign and identify most signals.

The methylation results showed that SP4002501 contains eight types of glycosidic fragments. By combining the above results and literature data [33], [34], [35], [36], we could further assign the carbon and proto signals in the NMR spectra through the analysis of the ¹H-¹H COSY and HSQC spectra of SP4002501. ¹H NMR signals at δH 5.22, 5.28, 5.27, 5.28, 4.59, and 5.26 were attributed to H-1 of Araf, Arap, [→ 2,3)-β-Galp-(1→], [→ 2)-α-Galp-(1→], [→ 3)-β-Rhap-(1→], and [→ 4)-α-GalAp-(1→], while signals at δH 5.08 were attributed to H-1 in [→ 2)-α-GalAp-(1→] or [→ 3)-α-GalAp-(1→]. ¹³C NMR signals at δC 110.2, δC 103.5, 102.6, 102.3, 103.6, 102.6, and 106.2 were assigned to C-1 of Araf, Arap, [→ 2)-α-GalAp-(1→], [→ 2)-α-Galp-(1→], [→ 2,3)-β-Galp-(1→], [→ 3)-α-GalAp-(1→], [→ 4)-α-GalAp-(1→], and [→ 3)-β-Rhap-(1→], while resonances at δC 19.9 were assigned to methyl C-6 of Rha.

In the ¹H-¹H COSY spectrum ([Fig. 2 a]), we focused on the assignment of signals related to the anomeric hydrogen atoms. The cross-peak at δH/H 5.08/4.58 was assigned to H-1/H-2 of [→ 2)-α-GalAp-(1→], while δH/H 5.28/4.55 and 5.27/4.50 were attributed to H-1/H-2 overlapping signal of [→ 2)-α-Galp-(1→] and [→ 2,3)-β-Galp-(1→], respectively. The cross-peaks at δH/H 5.22/4.10, 5.28/4.08, 5.26/3.86, and 5.08/3.85 were assigned to H-1/H-2 of [α-Arap-(1→], [α-Araf-(1→], [→ 4)-α-GalAp-(1→], and [→ 3)-α-GalAp-(1→], respectively. The cross-peaks at δH/H 3.85/4.60 and 4.50/4.14 were due to H-2/H-3 of [→ 3)-α-GalAp-(1→] and [→ 2,3)-β-Galp-(1→]. In the HSQC spectrum ([Fig. 2 b]), the cross-peaks at δH/C 5.28/110.2 and 5.22/110.7 were assigned to H-1/C-1 of [α-Araf-(1→] and [α-Arap-(1→], while δH/C 5.08/103.6 and 5.08/103.5 were attributed to the H-1/C-1 overlapping signal of [→ 3)-α-GalAp-(1→] and [→ 2)-α-GalAp-(1→]. The cross-peaks at δH/C 5.27/102.3, 5.28/102.6, 5.26/102.6, and 4.59/106.2 were assigned to H-1/C-1 of [→ 2,3)-β-Galp-(1→], [→ 2)-α-Galp-(1→], [→ 4)-α-GalAp-(1→], and [→ 3)-β-Rhap-(1→]. The assignment of further carbon and proton signals is presented in [Table 3].

The HMBC spectrum ([Fig. 2 c] and [d]) was used to further explore and identify the linkages between different sugar residues. The cross-peak at δH/C 5.26/80.1 was assigned to the correlation between C-4 and H-1 of [→ 4)-α-GalAp-(1→], showing that there were repeating [→ 4)-α-GalAp-(1 → 4)-α-GalAp-(1→] units. The cross-peak at δH/C 5.22/80.1 was due to the correlation of C-4 of [→ 4)-α-GalAp-(1→] with H-1 of [α-Arap-(1→], revealing repeating [α-Arap-(1 → 4)-α-GalAp-(1→] units. The cross-peak at δH/C 4.07/110.2 revealed that C-1 of [α-Araf-(1→] was linked to H-4 of [→ 4)-α-GalAp-(1→], suggesting that there were repeating [α-Araf-(1 → 4)-GalAp-(1→] units. The cross-peak at δH/C 5.08/71.7 showed that C-3 of [→ 2,3)-β-Galp-(1→] was linked to H-1 of [→ 2)-α-GalAp-(1→], indicating repeating [→ 2)-α-GalAp-(1 → 3)-β-Galp(2)-(1→] units. Similarly, the cross-peak at δH/C 5.08/81.9 showed that C-2 of [→ 2,3)-β-Galp-(1→] was linked to H-1 of [→ 2)-α-GalAp-(1→], thus revealing repeating [→ 2)-α-GalAp-(1 → 2)-Galp(3)-(1→] units. Thus, most of the repeating units have been determined ([Table 4]).

In summary, the amount of sugar residue and the number of interglycosidic connectivities were determined from methylation data. Partial hydrolysis, FT-IR analysis, and 1D NMR and 2D NMR spectra further enabled us to identify the residue structure, and the fragments were assembled as repeating units from 2D NMR correlations. Based on the above analysis, a putative structure could be deduced for SP4002501 ([Fig. 3]). The backbone of SP4002501 consists of a GalA chain containing a small amount of Gal. Side chains are connected to C-3 of Gal and consist of Rha, GalA, Gal, and Ara, with Ara as terminal sugar moiety. Some of the GalA carboxylic groups are present as methyl esters.

The surface morphology of SP4002501 was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SP4002501 had a lamellar appearance ([Fig. 4 a]–d) with jungle bumps on its surface ([Fig. 4 e]–f).

Macrophages, comprising a diverse class of innate immune cells, patrol their host for unwanted objects and then ingest them by phagocytosis and digest them [37]. Thus, the number and phagocytic capacity of macrophages is an important indicator of immunomodulatory activity in vitro. In addition, macrophages could mediate the interaction between cells and regulate immune response by releasing NO or numerous cytokines such as TNF-α and IL-6 [38]. In this study, RAW 264.7 cells were selected to evaluate the potential immunomodulatory activity of the purified polysaccharides.

SP40 and SP4002501 induced the proliferation of RAW 264.7 cells, indicating that macrophages were activated by SP40 and SP4002501 ([Fig. 5 a]). Compared with the blank control group, SP40 stimulated the proliferation of macrophages in the concentration range of 200 – 800 µg/mL (p < 0.05) in a dose-dependent manner. The homogeneous polysaccharide SP4002501 obtained after further purification induced the proliferation of macrophages in the concentration range of 6.25 – 100 µg/mL (p < 0.05) ([Fig. 5 c]) in a dose-dependent manner, indicating that the minimum effective concentration of polysaccharides was reduced after purification.

Compared with the blank control group, SP40 enhanced phagocytosis of macrophages significantly at a concentration of 200 – 800 µg/mL (p < 0.05), while the homogeneous polysaccharide SP4002501 increased the phagocytic activity of macrophages in the concentration range of 6.25 – 25 µg/mL (p < 0.05) ([Fig. 5 b] and [d]). The phagocytic effects were promoted in a dose-dependent manner by both polysaccharides.

In view of the proliferative and phagocytic-promoting activity of SP40 and SP4002501, NO, TNF-α, and IL-6 production was further investigated. When compared with the blank group, SP4002501 significantly increased the production of TNF-α in the concentration range of 6.25 – 25 µg/mL (p < 0.05) ([Fig. 5 f]). In contrast, SP4002501 did not significantly promote the production of NO and IL-6 ([Fig. 5 e] and [g]).

In our previous study, a homogeneous polysaccharide named SP800201 was also isolated and purified [14]. Compared with SP800201, the phagocytic promoting activity of SP4002501 was stronger, but its effects on promoting the release of NO and cytokines were weaker than those of SP800201. When comparing the basic chemical composition of SP4002501 and SP800201, SP4002501 has higher molecular weight and uronic acid content. GalA is the prominent monosaccharide in SP4002501, while Gal is the most abundant monosaccharide in SP800201. Both of them have α-1,3, α-1,4, β-1,2, and β-1,3 glycosidic bonds, but SP4002501 mainly contains α-glycosidic linkages, while SP800201 has more β-glycosidic linkages. In addition, SP800201 has more branches, but the branched structure of SP4002501 is more complex. Thus, differences in the content of uronic acid and the structures of backbone and side chain may play important roles for the differences observed in the immunomodulatory activity between SP4002501 and SP800201. SP4002501 was non-toxic to macrophages within the tested concentrations and could be a potential immunomodulatory agent.

In conclusion, polysaccharides have attracted much attention due to their safety characteristics and biological activities. However, research on polysaccharides remains challenging due to difficulties in obtaining a homogeneous polysaccharide and elucidating the polysaccharide structure. In the present study, a new homogeneous polysaccharide named SP4002501 was isolated and purified from SRP by water extraction, alcohol precipitation, and column chromatography. A combination of molecular weight parameter analysis, uronic acid content analysis, monosaccharide composition analysis, methylation analysis, partial hydrolysis, FT-IR, and NMR was used to elucidate its structure. In addition, the surface morphology of SP4002501 was investigated by SEM and AFM.

SP4002501 has a high galacturonic acid content (88.7%). Its backbone consists of GalA and a small amount of Gal. Side chains are connected to C-3 of Gal and include Rha, GalA, Gal, and Ara, with Ara as the terminal sugar. Immunomodulatory activity assessment showed that both SP40 and SP4002501 are non-toxic to macrophages and enhance proliferative and phagocytic activity. These data suggest that SP4002501 is worthy of being explored deeper in view of a potential application as an immunomodulatory agent. Furthermore, given the structure and activity differences between SP4002501 and other SRP obtained in our previous study, the structure–activity relationships and action mechanism deserve further study.

Materials and Methods

Materials

Saposhnikoviae Radix was purchased from Huacaosheng Traditional Chinese Medicine Co. in Anguo Materia Medica Market and identified by Professor Zhang Yuan, Beijing University of Chinese Medicine. A voucher specimen (SD201801) is stored at Laboratory 211, College of Biological Engineering, Beijing Vocational College of Electronic Science and Technology. RAW 264.7 cells were obtained from the National Experimental Cell Resource Sharing Platform (Beijing headquarters).

Extraction, isolation, and purification of SRP

Saposhnikoviae Radix (3 kg) was pulverized and passed through a sieve with an inner diameter of 180 µm and was then extracted by boiling with 30 L of water for 2.5 h. The filtrate was concentrated by heating and mixed with 95% ethanol to a final concentration of 80% to form the precipitate [39]. After deproteinization by the Sevag method [32], the total polysaccharide solution was mixed with 95% ethanol to a final concentration of 40% to give a precipitate, which was repeatedly washed sequentially three times with ethanol (400 mL), acetone (400 mL), and diethyl ether (400 mL) to obtain the crude polysaccharide named SP40.

Anion exchange chromatography with DEAE-Cellulose was used for the separation of SP40 with water and linear-gradient sodium chloride solution (from 0 to 0.6 M) to obtain SP40025. SP40025 was further purified using a Sepharose CL-6B column with a sodium chloride solution (0.01%) and was then desalinated by Sephadex G-15 with purified water to obtain a highly purified polysaccharide named SP4002501.

Structural characterization

Homogeneity and molecular weight parameter analysis

High-performance size-exclusion chromatography was used to determine the homogeneity and molecular weight parameters of SP4002501 [40]. Three mobile phases (0.01% of sodium chloride, 0.025 M of borate buffer, and 0.02 M of phosphate) were used on a G5000PWXL column (300 mm × 7.8 mm) and a G3000PWXL column (300 mm × 7.8 mm) connected in series to confirm homogeneity. The molecular weight of SP4002501 was evaluated by a set of standard dextrans (Dextran 1000, 5000, 12 000, 80 000, 270 000, 410 000, and 1 100 000, Sigma Corporation) and then calculated by GPC software.

Uronic acid content analysis

The uronic acid content of SP4002501 was measured using the sulfuric acid-m-hydroxydiphenyl method [41]. A sodium tetraborate–concentrated sulfuric acid solution (0.0125 M, 3 mL) was added to each tube of a series of solutions of GalA. After vortexing, the mixture was incubated in a boiling water bath for 5 min and then cooled to room temperature. An m-hydroxydiphenyl solution (0.15%, w/v, 0.05 mL) was added to the mixture and then the absorbance was measured at 520 nm. SP4002501 (1 mg) was processed using the procedure above to calculate the content of uronic acid in the SP4002501.

Monosaccharide composition analysis

High-performance liquid chromatography (HPLC) and 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivatization were used to confirm the monosaccharide composition of SP4002501 [42]. SP4002501 (2 mg) was completely hydrolyzed with 1.0 mL of 2.25 M trifluoroacetic acid (TFA) at 115℃ for 6 h. After TFA was removed via a rotary evaporator under vacuum, 0.4 mL of NaOH (0.3 M) and PMP (0.3 M) solution were added to react for 1 h at 70℃. Standard monosaccharides (mannose, glucose, galactose, xylose, rhamnose, glucuronic acid, galacturonic acid, fucose, and arabinose) were derivatized by PMP in the same manner. Then, 0.4 mL of HCl (0.3 M) was added and the mixture was extracted three times with 600 µL chloroform. Samples were analyzed on an Agilent Eclipse XDB-C18 column (5 µm, 4.6 × 250 mm) connected to an Agilent 1200 high-performance liquid chromatography system with UV detector. Detection was at 245 nm.

FT-IR analysis

SP4002501 (2 mg) was ground with dried KBr and pressed into a disk. The IR spectrum was determined on a Spectrum Two FT-IR spectrometer (PerkinElmer) in the wavelength range of 4000 – 400 cm⁻¹.

Methylation analysis

The methylation of SP4002501 was performed according to our previously reported method [14]. After determination of the methylation degree of SP4002501 by FT-IR spectroscopy, methylated products were hydrolyzed, reduced, and derivatized to get the corresponding partially methylated alditol acetates (PMAAs). PMAAs were analyzed on a SCION TQ GC-MS chromatographic system connected to an HP-5 fused-silica capillary column (0.25 µm × 0.25 mm × 30 m, Agilent) and were identified based on their fragments, published data, and online databases (Complex Carbohydrate Structure Database created by the Complex Carbohydrate Research Center of the University of Georgia, https://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html). The molar ratio of each sugar residue was calculated according to the peak areas.

Partial hydrolysis

Partial acid hydrolysis: SP4002501 (6.7 mg) was partially hydrolyzed with TFA at different concentrations and temperatures (0.055 M TFA, 55℃; 0.55 M TFA, 65℃; 0.55 M TFA, 85℃) for 2 h. After hydrolysis, the solution was dialyzed using a dialysis bag (3.5 – 5.0 kDa). Then, the molecular weight distribution and monosaccharidic constituents of the hydrolysate inside and outside the dialysis bag were analyzed.

Partial enzymatic hydrolysis: SP4002501 (1 mg) was partially hydrolyzed with 1 mL of pectinase (50 µg/mL) solution or cellulase solution for 0.5 h at 40℃. The hydrolysate was mixed with 0.9 mL of methanol for 10 min and centrifuged to obtain the supernatant (10 000 r/min, 10 min). Samples were analyzed by ESI-MS/MS in the negative ion mode.

NMR analysis

SP4002501 (30 mg) was dissolved in 0.6 mL of D₂O. NMR spectra were recorded at 318 K on a Bruker Avance III HD700 MHz spectrometer (Bruker). Then, 4,4-dimethyl-4-silapentane-1-sulfonic acid (DDS) was used as the internal standard.

SEM and AFM determination

An appropriate amount of freeze-dried polysaccharide powder was fixed on the double-sided adhesive tape mounted on SEM tubs and treated with gold spray in a vacuum-plating apparatus for 120 s. The morphology of SP4002501 was observed under a scanning electron microscope (JSM-7001F). Then, 10 µL of polysaccharide solution (10 µg/mL) was placed on the surface of freshly peeled mica tablets and dried overnight at room temperature. The image of SP4002501 was then reconfirmed by atomic force microscopy (AFM).

In vitro immunomodulatory effects of SP4002501

Cell culture

RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. Passaging was performed when cell growth density reached about 80%. Cells were maintained at 37℃ in a 5% CO₂ incubator.

Evaluation of proliferative activity

RAW264.7 macrophages in logarithmic growth phase were blown into a cell suspension with a density of 5 × 10⁴ cells/mL, which were seeded into a 96-well cell plate (100 µL per well). After adherent growth for 24 h, the same volume of complete medium was added to the blank control group and test-substance-containing medium (SP40: 50, 100, 200, 400, and 800 µg/mL; SP4002501: 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 µg/mL) was added to the test groups. After treating for 24 h, the culture medium was discarded, and 100 µL of complete medium containing 10% CCK-8 solution was added to each well. The OD value was measured after 1 h at 450 nm.

Evaluation of phagocytic activity

RAW 264.7 macrophages in logarithmic growth phase were blown into a cell suspension with density of 1 × 10⁵ cells/mL, which were seeded into a 96-well cell plate (100 µL per well). RAW264.7 macrophages were treated with SP40 (100, 200, 400, and 800 µg/mL), SP4002501 (6.25, 12.5, and 25 µg/mL) and LPS (1.5 µg/mL) for 24 h respectively. Then culture medium was discarded, and 100 µL of 0.1% neutral red solution was added to each well. Cells were cultured at 37℃ in a 5% CO₂ incubator for 20 min and were washed three times with PBS. Finally, 200 µL of acetic acid ethanol lysis solution was added to lyse cells for 1 h. The OD value was determined at 540 nm.

Evaluation of NO, TNF-α, and IL-6 production

The level of NO was determined using the Griess reagent kit (BN27106, Bairuiji), while levels of TNF-α and IL-6 were determined using ELISA kits (BN50578, BN50553, Bairuiji). Briefly, RAW264.7 macrophages were treated with SP4002501 (6.25, 12.5, and 25 µg/mL) and LPS (1.5 µg/mL) for 24 h. Cell supernatants were collected, and the levels of NO, TNF-α, and IL-6 were measured.

Statistical analysis

Data were presented as the mean ± SD. Groups were compared by one-way analysis of variance (ANOVA), and p < 0.05 was considered statistically significant. Each experiment was repeated at least three times.

Contributorsʼ Statement

Conception and design of the work: Yanyan Jiang, Bin Liu; data collection: Yifang Cui, Haitao Fan, Meng Sun; analysis and interpretation of the data: Yifang Cui, Haitao Fan; drafting the manuscript: Yifang Cui, Haitao Fan, Meng Sun, Xinyang He, Jie Li; critical revision of the manuscript: Guangzhong Tu, Yanyan Jiang, Bin Liu.

Conflict of Interest

The authors declare that they have no conflict of interest.

• References

• 1 Jiang Y, Zhong S, Tan H, Fu Y, Lai J, Liu L, Weng J, Chen H, He S. Study on the mechanism of action of Saposhnikovia divaricata and its key phytochemical on rheumatoid arthritis based on network pharmacology and bioinformatics. J Ethnopharmacol 2024; 322: 117586
  PubMedSearch in Google Scholar
• 2 Liang X, Li X, Sun S, Zhang H, Wang B, Xu F, Zhang Y, Liu Z. Effects and potential mechanisms of Saposhnikovia divaricata (Turcz.) Schischk. On type I allergy and pseudoallergic reactions in vitro and in vivo. J Ethnopharmacol 2024; 318: 116942
  PubMedSearch in Google Scholar
• 3 Erdenebileg S, Son YJ, Kim M, Oidovsambuu S, Cha KH, Kwon J, Jung DS, Nho CW. Saposhnikovia divaricata root and its major components ameliorate inflammation and altered gut microbial diversity and compositions in DSS-induced colitis. Integr Med Res 2023; 12: 100998
  PubMedSearch in Google Scholar
• 4 Xu X, Yan S, Zhang Y, Cao L, Chen T, Yang X, Liu G, Meng J, Ren S, Wang D, Liu X, Pan Y. Comparison of the chemical constituents of Saposhnikoviae radix associated with three different growth patterns and its therapeutic effect against atopic dermatitis. J Ethnopharmacol 2024; 333: 118417
  PubMedSearch in Google Scholar
• 5 Hu Y, He Y, Niu Z, Shen T, Zhang J, Wang X, Hu W, Cho JY. A review of the immunomodulatory activities of polysaccharides isolated from Panax species. J Ginseng Res 2022; 46: 23-32
  PubMedSearch in Google Scholar
• 6 Wu Y, Zhou H, Wei K, Zhang T, Che Y, Nguyẽn AD, Pandita S, Wan X, Cui X, Zhou B, Li C, Hao P, Lei H, Wang L, Yang X, Liang Y, Liu J, Wu Y. Structure of a new glycyrrhiza polysaccharide and its immunomodulatory activity. Front Immunol 2022; 13: 1007186
  PubMedSearch in Google Scholar
• 7 Sun Y, Sun T, Wang F, Zhang J, Li C, Chen X, Li Q, Sun S. A polysaccharide from the fungi of Huaier exhibits anti-tumor potential and immunomodulatory effects. Carbohydr Polym 2013; 92: 577-582
  PubMedSearch in Google Scholar
• 8 Sun M, Fan HT, Jiang YY, Liu B. Study on the evaluation of immunoregulatory activity of polysaccharide from Saposhnikovia divaricata in vitro and in vivo. Chinese Arch Tradit Chin Med 2024; 42: 88-91 + 278–279
  PubMedSearch in Google Scholar
• 9 Sun M, Wang WD, Li Y, Liu KC, Xia Q, Jiang YY, Liu B. Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model. Zhongguo Zhong Yao Za Zhi 2023; 48: 1916-1926
  PubMedSearch in Google Scholar
• 10 Chen SK, Wang X, Guo YQ, Song XX, Yin JY, Nie SP. Exploring the partial degradation of polysaccharides: Structure, mechanism, bioactivities, and perspectives. Compr Rev Food Sci Food Saf 2023; 22: 4831-4870
  PubMedSearch in Google Scholar
• 11 Xiong C, Li P, Luo Q, Yan J, Zhang J, Jin X, Huang W. Effect of γ-irradiation on the structure and antioxidant activity of polysaccharide isolated from the fruiting bodies of Morchella sextelata. Biosci Rep 2020; 40: BSR20194522
  PubMedSearch in Google Scholar
• 12 Dong YH, Chen C, Huang Q, Fu X. Study on a novel spherical polysaccharide from fructus mori with good antioxidant activity. Carbohydr Polym 2021; 256: 117516
  PubMedSearch in Google Scholar
• 13 Chen N, Jiang T, Xu J, Xi W, Shang E, Xiao P, Duan JA. The relationship between polysaccharide structure and its antioxidant activity needs to be systematically elucidated. Int J Biol Macromol 2024; 270: 132391
  PubMedSearch in Google Scholar
• 14 Fan H, Sun M, Li J, Zhang S, Tu G, Liu K, Xia Q, Jiang Y, Liu B. Structure characterization and immunomodulatory activity of a polysaccharide from Saposhnikoviae radix. Int J Biol Macromol 2023; 233: 123502
  PubMedSearch in Google Scholar
• 15 Lin P, Chen L, Huang X, Xiao F, Fu L, Jing D, Wang J, Zhang H, Sun L, Wu Y. Structural characteristics of polysaccharide GP2a in Gardenia jasminoides and its immunomodulatory effect on macrophages. Int J Mol Sci 2022; 23: 11279
  PubMedSearch in Google Scholar
• 16 Cheng XD, Wu QX, Zhao J, Su T, Lu YM, Zhang WN, Wang Y, Chen Y. Immunomodulatory effect of a polysaccharide fraction on RAW 264.7 macrophages extracted from the wild Lactarius deliciosus. Int J Biol Macromol 2019; 128: 732-739
  PubMedSearch in Google Scholar
• 17 Peng Y, Zhang Z, Chen W, Zhao S, Pi Y, Yue X. Structural characterization, α-glucosidase inhibitory activity and antioxidant activity of neutral polysaccharide from apricot (Armeniaca Sibirica L. Lam) kernels. Int J Biol Macromol 2023; 238: 124109
  PubMedSearch in Google Scholar
• 18 Meng Y, Yi L, Chen L, Hao J, Li DX, Xue J, Xu NY, Zhang ZQ. Purification, structure characterization and antioxidant activity of polysaccharides from Saposhnikovia divaricata. Chin J Nat Med 2019; 17: 792-800
  PubMedSearch in Google Scholar
• 19 Li YR, Liu ST, Gan Q, Zhang J, Chen N, Han CF, Geng WJ, Wang BX, Han N, Jia SR, Han PP. Four polysaccharides isolated from Poria cocos mycelium and fermentation broth supernatant possess different activities on regulating immune response. Int J Biol Macromol 2023; 226: 935-945
  PubMedSearch in Google Scholar
• 20 Dore CM, das C Faustino Alves MG, Will LS, Costa TG, Sabry DA, de Souza Rêgo LA, Accardo CM, Rocha HA, Filgueira LG, Leite EL. A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects. Carbohydr Polym 2013; 91: 467-475
  PubMedSearch in Google Scholar
• 21 Zhang T, Ye J, Xue C, Wang Y, Liao W, Mao L, Yuan M, Lian S. Structural characteristics and bioactive properties of a novel polysaccharide from Flammulina velutipes. Carbohydr Polym 2018; 197: 147-156
  PubMedSearch in Google Scholar
• 22 Wu S, Li F, Jia S, Ren H, Gong G, Wang Y, Lv Z, Liu Y. Drying effects on the antioxidant properties of polysaccharides obtained from Agaricus blazei Murrill. Carbohydr Polym 2014; 103: 414-417
  PubMedSearch in Google Scholar
• 23 He P, Zhang A, Zhang F, Linhardt RJ, Sun P. Structure and bioactivity of a polysaccharide containing uronic acid from Polyporus umbellatus sclerotia. Carbohydr Polym 2016; 152: 222-230
  PubMedSearch in Google Scholar
• 24 Ji X, Yan Y, Hou C, Shi M, Liu Y. Structural characterization of a galacturonic acid-rich polysaccharide from Ziziphus Jujuba cv. Muzao. Int J Biol Macromol 2020; 147: 844-852
  PubMedSearch in Google Scholar
• 25 Cui L, Guan X, Ding W, Luo Y, Wang W, Bu W, Song J, Tan X, Sun E, Ning Q, Liu G, Jia X, Feng L. Scutellaria baicalensis Georgi polysaccharide ameliorates DSS-induced ulcerative colitis by improving intestinal barrier function and modulating gut microbiota. Int J Biol Macromol 2021; 166: 1035-1045
  PubMedSearch in Google Scholar
• 26 Mitić Ž, Nikolić GM, Cakić M, Nikolić GS, Živanović S, Mitić S, Najman S. Synthesis, spectroscopic and structural characterization of Co(II)-pullulan complexes by UV-Vis, ATR-FTIR, MALDI-TOF/TOF MS and XRD. Carbohydr Polym 2018; 200: 25-34
  PubMedSearch in Google Scholar
• 27 Bi Z, Zhao Y, Hu J, Ding J, Yang P, Liu Y, Lu Y, Jin Y, Tang H, Liu Y, Zhang Y. A novel polysaccharide from Lonicerae japonicae caulis: Characterization and effects on the function of fibroblast-like synoviocytes. Carbohydr Polym 2022; 292: 119674
  PubMedSearch in Google Scholar
• 28 OʼNeill MA, Black I, Urbanowicz B, Bharadwaj V, Crowley M, Koj S, Peña MJ. Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide Rhamnogalacturonan II. SLAS Technol 2020; 25: 329-344
  PubMedSearch in Google Scholar
• 29 Li J, Sun M, Xu C, Zhou C, Jing SJ, Jiang YY, Liu B. An integrated strategy for rapid discovery and identification of the potential effective fragments of polysaccharides from Saposhnikoviae radix. J Ethnopharmacol 2024; 319: 117099
  PubMedSearch in Google Scholar
• 30 Xia YG, Yu LS, Liang J, Yang BY, Kuang HX. Chromatography and mass spectrometry-based approaches for perception of polysaccharides in wild and cultured fruit bodies of Auricularia auricular-judae. Int J Biol Macromol 2019; 137: 1232-1244
  PubMedSearch in Google Scholar
• 31 Zhu R, Zhang X, Wang Y, Zhang L, Zhao J, Chen G, Fan J, Jia Y, Yan F, Ning C. Characterization of polysaccharide fractions from fruit of Actinidia arguta and assessment of their antioxidant and antiglycated activities. Carbohydr Polym 2019; 210: 73-84
  PubMedSearch in Google Scholar
• 32 Wang S, Yang Y, Wang Q, Wu Z, Liu X, Chen S, Zhou A. Structural characterization and immunomodulatory activity of a polysaccharide from finger citron extracted by continuous phase-transition extraction. Int J Biol Macromol 2023; 240: 124491
  PubMedSearch in Google Scholar
• 33 Barbieri SF, da Costa Amaral S, Mazepa E, Filho APS, Sassaki GL, Silveira JLM. Isolation, NMR characterization and bioactivity of a (4-O-methyl-α-D-glucurono)-β-D-xylan from Campomanesia xanthocarpa berg fruits. Int J Biol Macromol 2022; 207: 893-904
  PubMedSearch in Google Scholar
• 34 Zhang J, Huo J, Zhao Z, Lu Y, Hong Z, Li H, Chen D. An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation. Phytomedicine 2022; 107: 154453
  CrossrefPubMedSearch in Google Scholar
• 35 Liu J, Shang F, Yang Z, Wu M, Zhao J. Structural analysis of a homogeneous polysaccharide from Achatina fulica. Int J Biol Macromol 2017; 98: 786-792
  PubMedSearch in Google Scholar
• 36 Yang WW, Wang LM, Gong LL, Lu YM, Pan WJ, Wang Y, Zhang WN, Chen Y. Structural characterization and antioxidant activities of a novel polysaccharide fraction from the fruiting bodies of Craterellus cornucopioides. Int J Biol Macromol 2018; 117: 473-482
  PubMedSearch in Google Scholar
• 37 Joffe AM, Bakalar MH, Fletcher DA. Macrophage phagocytosis assay with reconstituted target particles. Nat Protoc 2020; 15: 2230-2246
  PubMedSearch in Google Scholar
• 38 Yu Y, Shen M, Wang Z, Wang Y, Xie M, Xie J. Sulfated polysaccharide from Cyclocarya paliurus enhances the immunomodulatory activity of macrophages. Carbohydr Polym 2017; 174: 669-676
  PubMedSearch in Google Scholar
• 39 Dong CX, Liu L, Wang CY, Fu Z, Zhang Y, Hou X, Peng C, Ran RX, Yao Z. Structural characterization of polysaccharides from Saposhnikovia divaricata and their antagonistic effects against the immunosuppression by the culture supernatants of melanoma cells on RAW264.7 macrophages. Int J Biol Macromol 2018; 113: 748-756
  PubMedSearch in Google Scholar
• 40 Yu S, Dong X, Ma R, Ji H, Yu J, Liu A. Characterization of a polysaccharide from Polygala tenuifolia willd. with immune activity via activation MAPKs pathway. Bioorg Chem 2023; 130: 106214
  PubMedSearch in Google Scholar
• 41 Zhang W, Xiang Q, Zhao J, Mao G, Feng W, Chen Y, Li Q, Wu X, Yang L, Zhao T. Purification, structural elucidation and physicochemical properties of a polysaccharide from Abelmoschus esculentus L (okra) flowers. Int J Biol Macromol 2020; 155: 740-750
  PubMedSearch in Google Scholar
• 42 Wu M, Xu L, Zhao L, Xiao C, Gao N, Luo L, Yang L, Li Z, Chen L, Zhao J. Structural analysis and anticoagulant activities of the novel sulfated fucan possessing a regular well-defined repeating unit from sea cucumber. Mar Drugs 2015; 13: 2063-2084
  PubMedSearch in Google Scholar

Correspondence

Publication History

Received: 12 September 2024

Accepted after revision: 02 April 2025

Accepted Manuscript online:
03 April 2025

Article published online:
08 May 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

• References

• 1 Jiang Y, Zhong S, Tan H, Fu Y, Lai J, Liu L, Weng J, Chen H, He S. Study on the mechanism of action of Saposhnikovia divaricata and its key phytochemical on rheumatoid arthritis based on network pharmacology and bioinformatics. J Ethnopharmacol 2024; 322: 117586
  PubMedSearch in Google Scholar
• 2 Liang X, Li X, Sun S, Zhang H, Wang B, Xu F, Zhang Y, Liu Z. Effects and potential mechanisms of Saposhnikovia divaricata (Turcz.) Schischk. On type I allergy and pseudoallergic reactions in vitro and in vivo. J Ethnopharmacol 2024; 318: 116942
  PubMedSearch in Google Scholar
• 3 Erdenebileg S, Son YJ, Kim M, Oidovsambuu S, Cha KH, Kwon J, Jung DS, Nho CW. Saposhnikovia divaricata root and its major components ameliorate inflammation and altered gut microbial diversity and compositions in DSS-induced colitis. Integr Med Res 2023; 12: 100998
  PubMedSearch in Google Scholar
• 4 Xu X, Yan S, Zhang Y, Cao L, Chen T, Yang X, Liu G, Meng J, Ren S, Wang D, Liu X, Pan Y. Comparison of the chemical constituents of Saposhnikoviae radix associated with three different growth patterns and its therapeutic effect against atopic dermatitis. J Ethnopharmacol 2024; 333: 118417
  PubMedSearch in Google Scholar
• 5 Hu Y, He Y, Niu Z, Shen T, Zhang J, Wang X, Hu W, Cho JY. A review of the immunomodulatory activities of polysaccharides isolated from Panax species. J Ginseng Res 2022; 46: 23-32
  PubMedSearch in Google Scholar
• 6 Wu Y, Zhou H, Wei K, Zhang T, Che Y, Nguyẽn AD, Pandita S, Wan X, Cui X, Zhou B, Li C, Hao P, Lei H, Wang L, Yang X, Liang Y, Liu J, Wu Y. Structure of a new glycyrrhiza polysaccharide and its immunomodulatory activity. Front Immunol 2022; 13: 1007186
  PubMedSearch in Google Scholar
• 7 Sun Y, Sun T, Wang F, Zhang J, Li C, Chen X, Li Q, Sun S. A polysaccharide from the fungi of Huaier exhibits anti-tumor potential and immunomodulatory effects. Carbohydr Polym 2013; 92: 577-582
  PubMedSearch in Google Scholar
• 8 Sun M, Fan HT, Jiang YY, Liu B. Study on the evaluation of immunoregulatory activity of polysaccharide from Saposhnikovia divaricata in vitro and in vivo. Chinese Arch Tradit Chin Med 2024; 42: 88-91 + 278–279
  PubMedSearch in Google Scholar
• 9 Sun M, Wang WD, Li Y, Liu KC, Xia Q, Jiang YY, Liu B. Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model. Zhongguo Zhong Yao Za Zhi 2023; 48: 1916-1926
  PubMedSearch in Google Scholar
• 10 Chen SK, Wang X, Guo YQ, Song XX, Yin JY, Nie SP. Exploring the partial degradation of polysaccharides: Structure, mechanism, bioactivities, and perspectives. Compr Rev Food Sci Food Saf 2023; 22: 4831-4870
  PubMedSearch in Google Scholar
• 11 Xiong C, Li P, Luo Q, Yan J, Zhang J, Jin X, Huang W. Effect of γ-irradiation on the structure and antioxidant activity of polysaccharide isolated from the fruiting bodies of Morchella sextelata. Biosci Rep 2020; 40: BSR20194522
  PubMedSearch in Google Scholar
• 12 Dong YH, Chen C, Huang Q, Fu X. Study on a novel spherical polysaccharide from fructus mori with good antioxidant activity. Carbohydr Polym 2021; 256: 117516
  PubMedSearch in Google Scholar
• 13 Chen N, Jiang T, Xu J, Xi W, Shang E, Xiao P, Duan JA. The relationship between polysaccharide structure and its antioxidant activity needs to be systematically elucidated. Int J Biol Macromol 2024; 270: 132391
  PubMedSearch in Google Scholar
• 14 Fan H, Sun M, Li J, Zhang S, Tu G, Liu K, Xia Q, Jiang Y, Liu B. Structure characterization and immunomodulatory activity of a polysaccharide from Saposhnikoviae radix. Int J Biol Macromol 2023; 233: 123502
  PubMedSearch in Google Scholar
• 15 Lin P, Chen L, Huang X, Xiao F, Fu L, Jing D, Wang J, Zhang H, Sun L, Wu Y. Structural characteristics of polysaccharide GP2a in Gardenia jasminoides and its immunomodulatory effect on macrophages. Int J Mol Sci 2022; 23: 11279
  PubMedSearch in Google Scholar
• 16 Cheng XD, Wu QX, Zhao J, Su T, Lu YM, Zhang WN, Wang Y, Chen Y. Immunomodulatory effect of a polysaccharide fraction on RAW 264.7 macrophages extracted from the wild Lactarius deliciosus. Int J Biol Macromol 2019; 128: 732-739
  PubMedSearch in Google Scholar
• 17 Peng Y, Zhang Z, Chen W, Zhao S, Pi Y, Yue X. Structural characterization, α-glucosidase inhibitory activity and antioxidant activity of neutral polysaccharide from apricot (Armeniaca Sibirica L. Lam) kernels. Int J Biol Macromol 2023; 238: 124109
  PubMedSearch in Google Scholar
• 18 Meng Y, Yi L, Chen L, Hao J, Li DX, Xue J, Xu NY, Zhang ZQ. Purification, structure characterization and antioxidant activity of polysaccharides from Saposhnikovia divaricata. Chin J Nat Med 2019; 17: 792-800
  PubMedSearch in Google Scholar
• 19 Li YR, Liu ST, Gan Q, Zhang J, Chen N, Han CF, Geng WJ, Wang BX, Han N, Jia SR, Han PP. Four polysaccharides isolated from Poria cocos mycelium and fermentation broth supernatant possess different activities on regulating immune response. Int J Biol Macromol 2023; 226: 935-945
  PubMedSearch in Google Scholar
• 20 Dore CM, das C Faustino Alves MG, Will LS, Costa TG, Sabry DA, de Souza Rêgo LA, Accardo CM, Rocha HA, Filgueira LG, Leite EL. A sulfated polysaccharide, fucans, isolated from brown algae Sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects. Carbohydr Polym 2013; 91: 467-475
  PubMedSearch in Google Scholar
• 21 Zhang T, Ye J, Xue C, Wang Y, Liao W, Mao L, Yuan M, Lian S. Structural characteristics and bioactive properties of a novel polysaccharide from Flammulina velutipes. Carbohydr Polym 2018; 197: 147-156
  PubMedSearch in Google Scholar
• 22 Wu S, Li F, Jia S, Ren H, Gong G, Wang Y, Lv Z, Liu Y. Drying effects on the antioxidant properties of polysaccharides obtained from Agaricus blazei Murrill. Carbohydr Polym 2014; 103: 414-417
  PubMedSearch in Google Scholar
• 23 He P, Zhang A, Zhang F, Linhardt RJ, Sun P. Structure and bioactivity of a polysaccharide containing uronic acid from Polyporus umbellatus sclerotia. Carbohydr Polym 2016; 152: 222-230
  PubMedSearch in Google Scholar
• 24 Ji X, Yan Y, Hou C, Shi M, Liu Y. Structural characterization of a galacturonic acid-rich polysaccharide from Ziziphus Jujuba cv. Muzao. Int J Biol Macromol 2020; 147: 844-852
  PubMedSearch in Google Scholar
• 25 Cui L, Guan X, Ding W, Luo Y, Wang W, Bu W, Song J, Tan X, Sun E, Ning Q, Liu G, Jia X, Feng L. Scutellaria baicalensis Georgi polysaccharide ameliorates DSS-induced ulcerative colitis by improving intestinal barrier function and modulating gut microbiota. Int J Biol Macromol 2021; 166: 1035-1045
  PubMedSearch in Google Scholar
• 26 Mitić Ž, Nikolić GM, Cakić M, Nikolić GS, Živanović S, Mitić S, Najman S. Synthesis, spectroscopic and structural characterization of Co(II)-pullulan complexes by UV-Vis, ATR-FTIR, MALDI-TOF/TOF MS and XRD. Carbohydr Polym 2018; 200: 25-34
  PubMedSearch in Google Scholar
• 27 Bi Z, Zhao Y, Hu J, Ding J, Yang P, Liu Y, Lu Y, Jin Y, Tang H, Liu Y, Zhang Y. A novel polysaccharide from Lonicerae japonicae caulis: Characterization and effects on the function of fibroblast-like synoviocytes. Carbohydr Polym 2022; 292: 119674
  PubMedSearch in Google Scholar
• 28 OʼNeill MA, Black I, Urbanowicz B, Bharadwaj V, Crowley M, Koj S, Peña MJ. Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide Rhamnogalacturonan II. SLAS Technol 2020; 25: 329-344
  PubMedSearch in Google Scholar
• 29 Li J, Sun M, Xu C, Zhou C, Jing SJ, Jiang YY, Liu B. An integrated strategy for rapid discovery and identification of the potential effective fragments of polysaccharides from Saposhnikoviae radix. J Ethnopharmacol 2024; 319: 117099
  PubMedSearch in Google Scholar
• 30 Xia YG, Yu LS, Liang J, Yang BY, Kuang HX. Chromatography and mass spectrometry-based approaches for perception of polysaccharides in wild and cultured fruit bodies of Auricularia auricular-judae. Int J Biol Macromol 2019; 137: 1232-1244
  PubMedSearch in Google Scholar
• 31 Zhu R, Zhang X, Wang Y, Zhang L, Zhao J, Chen G, Fan J, Jia Y, Yan F, Ning C. Characterization of polysaccharide fractions from fruit of Actinidia arguta and assessment of their antioxidant and antiglycated activities. Carbohydr Polym 2019; 210: 73-84
  PubMedSearch in Google Scholar
• 32 Wang S, Yang Y, Wang Q, Wu Z, Liu X, Chen S, Zhou A. Structural characterization and immunomodulatory activity of a polysaccharide from finger citron extracted by continuous phase-transition extraction. Int J Biol Macromol 2023; 240: 124491
  PubMedSearch in Google Scholar
• 33 Barbieri SF, da Costa Amaral S, Mazepa E, Filho APS, Sassaki GL, Silveira JLM. Isolation, NMR characterization and bioactivity of a (4-O-methyl-α-D-glucurono)-β-D-xylan from Campomanesia xanthocarpa berg fruits. Int J Biol Macromol 2022; 207: 893-904
  PubMedSearch in Google Scholar
• 34 Zhang J, Huo J, Zhao Z, Lu Y, Hong Z, Li H, Chen D. An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation. Phytomedicine 2022; 107: 154453
  CrossrefPubMedSearch in Google Scholar
• 35 Liu J, Shang F, Yang Z, Wu M, Zhao J. Structural analysis of a homogeneous polysaccharide from Achatina fulica. Int J Biol Macromol 2017; 98: 786-792
  PubMedSearch in Google Scholar
• 36 Yang WW, Wang LM, Gong LL, Lu YM, Pan WJ, Wang Y, Zhang WN, Chen Y. Structural characterization and antioxidant activities of a novel polysaccharide fraction from the fruiting bodies of Craterellus cornucopioides. Int J Biol Macromol 2018; 117: 473-482
  PubMedSearch in Google Scholar
• 37 Joffe AM, Bakalar MH, Fletcher DA. Macrophage phagocytosis assay with reconstituted target particles. Nat Protoc 2020; 15: 2230-2246
  PubMedSearch in Google Scholar
• 38 Yu Y, Shen M, Wang Z, Wang Y, Xie M, Xie J. Sulfated polysaccharide from Cyclocarya paliurus enhances the immunomodulatory activity of macrophages. Carbohydr Polym 2017; 174: 669-676
  PubMedSearch in Google Scholar
• 39 Dong CX, Liu L, Wang CY, Fu Z, Zhang Y, Hou X, Peng C, Ran RX, Yao Z. Structural characterization of polysaccharides from Saposhnikovia divaricata and their antagonistic effects against the immunosuppression by the culture supernatants of melanoma cells on RAW264.7 macrophages. Int J Biol Macromol 2018; 113: 748-756
  PubMedSearch in Google Scholar
• 40 Yu S, Dong X, Ma R, Ji H, Yu J, Liu A. Characterization of a polysaccharide from Polygala tenuifolia willd. with immune activity via activation MAPKs pathway. Bioorg Chem 2023; 130: 106214
  PubMedSearch in Google Scholar
• 41 Zhang W, Xiang Q, Zhao J, Mao G, Feng W, Chen Y, Li Q, Wu X, Yang L, Zhao T. Purification, structural elucidation and physicochemical properties of a polysaccharide from Abelmoschus esculentus L (okra) flowers. Int J Biol Macromol 2020; 155: 740-750
  PubMedSearch in Google Scholar
• 42 Wu M, Xu L, Zhao L, Xiao C, Gao N, Luo L, Yang L, Li Z, Chen L, Zhao J. Structural analysis and anticoagulant activities of the novel sulfated fucan possessing a regular well-defined repeating unit from sea cucumber. Mar Drugs 2015; 13: 2063-2084
  PubMedSearch in Google Scholar

• Supplementary Material