Structural Characteristics, Antioxidant, and Immunostimulatory Activities of an Acidic Polysaccharide from Raspberry Pulp

The extraction and characterization of new bioactive plant-derived polysaccharides with the potential for use as functional foods and medicine have attracted much attention. In the present study, A novel acidic polysaccharide (RPP-3a) with a weight-average molecular weight (Mw) of 88,997 Da was isolated from the raspberry pulp. RPP-3a was composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid at a molar ratio of 13.1:28.6:16.8:1.4:6.2:33.9. Structural analysis suggested that the RPP-3a backbone was composed of repeating units of →4)-β-Galp-(1→3,4)-α-Rhap-(1→[4)-α-GalAp-(1→4)-α-GalAp-(1→]n with branches at the C-4 position of rhamnose. The side chain of RPP-3a, containing two branch levels, was comprised of α-Araf-(1→, →5)-α-Araf-(1→, →3,5)-α-Araf-(1→, →3)-β-Galp-(1→, →3,6)-β-Galp-(1→, →4)-β-Glcp-(1→, and →2,6)-α-Manp-1→ residues. RPP-3a exhibited moderate reducing power and strong hydroxyl and superoxide anion radical scavenging abilities. RPP-3a significantly promoted the viability of RAW264.7 macrophages by increasing the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) at both the expression and transcriptional levels. In summary, the immunostimulatory and antioxidant activities make RPP-3a a viable candidate as a health-beneficial functional dietary supplement.


Introduction
As a member of the berry family, raspberry (Rubus idaeus L.) is widely distributed and cultivated worldwide [1]. Its fruit, sub-globose and bright red, is also known as "fu-pen-zi" in China and is commonly used in traditional Chinese medicine to treat urination frequency, kidney enuresis, and other illnesses [2]. Presently, raspberry is considered a green fruit and has gained more acceptance due to its high nutritional value. Raspberry fruit contains numerous bioactive compounds, including flavonoids, tannins, phenolic acids, stilbenoids, polysaccharides, vitamins, and minerals [3], and dietary intake of them has the advantage of treating cardiovascular diseases, obesity, cancer, and degenerative diseases [4].
Polysaccharides, a class of carbohydrates, consist of numerous monosaccharides joined by glycosidic bonds in branched or unbranched chains. They are usually considered the primary and active components in plants, animals, and microorganisms [5]. Accumulating evidence suggests that polysaccharides extracted from plants, with relatively low toxicity and side effects, exert diverse biological activity, such as antitumor, anti-inflammatory, antioxidant, antimicrobial, antidiabetic effect, and immunomodulatory effects [6][7][8]. Hence, the extraction and characterization of new bioactive plant-derived polysaccharides with the potential for use as functional foods and medicine have attracted much attention. Coincidentally, polysaccharides are one of the most important bioactive components in effects [6][7][8]. Hence, the extraction and characterization of new bioactive plant-derived polysaccharides with the potential for use as functional foods and medicine have attracted much attention. Coincidentally, polysaccharides are one of the most important bioactive components in raspberries. In recent years, the biological activities of polysaccharides derived from raspberry fruit have been reported in several studies. The crude polysaccharides from raspberry fruit possessed antioxidant, anti-inflammatory, anticancer activity, and even detoxification effect [9,10]. Our previous study found that crude raspberry pulp polysaccharides (RPPs) exhibited significant antitumor activity and chemotherapy enhancement in vivo by increasing the cellular immune response of the host organism without any lesions in the liver or kidney tissues [11]. Along with continuous research, more and more homogeneous bioactive polysaccharides have been separated from raspberries. Ke et al. (2019) reported that a heteropolysaccharide separated from raspberry fruit afforded protection against palmitic acid-induced lipotoxicity in human hepatocytes [3]. A heteropolysaccharide (RCP I) and a degraded polysaccharide (DRCP I) were obtained from raspberry fruits, both of which displayed high antioxidant activity [12]. In our recent study, a novel acidic polysaccharide named RPP-2a was isolated from raspberry pulp and exhibited significant macrophage activation activity [13]. These results indicated that the polysaccharides derived from raspberries have great potential for in-depth development and utilization. In the present work, another acidic polysaccharide was isolated from raspberry pulp, and its structural information was systematically characterized. Its antioxidant and immunoregulatory activities were also investigated.

Isolation and Purification of Homogeneous Acidic Polysaccharide RPP-3a
Crude polysaccharide RPPs were separated by DEAE-Sepharose fast flow chromatography according to different polarities of polysaccharides. As depicted in Figure 1a, three fractions were collected from DEAE-Sepharose fast flow chromatography, named RPP-1, RPP-2, and RPP-3, which were eluted with deionized water, 0.2 M, and 0.4 M NaCl solutions, respectively. RPP-3 was further purified by a Sephadex G-200 column according to the molecular weight (Figure 1b), and two peaks (RPP-3a and RPP-3b) were observed. However, further purification work was needed due to the heterogeneity of RPP-3b. Thus, RPP-3a was concentrated, dialyzed, and lyophilized for further analysis.

Homogeneity and Molecular Weight of RPP-3a
A single symmetrical peak was obtained from high-performance gel permeation chromatography (HPGPC), suggesting the molecular weight homogeneity and the purity of RPP-3a ( Figure 2). The weight-average molecular weight (Mw), number-average molecular weight (Mn), and peak molecular weight (Mp) of RPP-3a were 88,997 Da, 59,284

Homogeneity and Molecular Weight of RPP-3a
A single symmetrical peak was obtained from high-performance gel permeation chromatography (HPGPC), suggesting the molecular weight homogeneity and the purity of RPP-3a ( Figure 2). The weight-average molecular weight (Mw), number-average molecular weight (Mn), and peak molecular weight (Mp) of RPP-3a were 88,997 Da, 59,284 Da, and 70,366 Da, respectively. Mw/Mn is a measure of the width of the molecular weight distribution [14], and the Mw/Mn value of RPP-3a was 1.501. Da, and 70,366 Da, respectively. Mw/Mn is a measure of the width of the molecular weight distribution [14], and the Mw/Mn value of RPP-3a was 1.501.

Fourier Transform Infrared Spectrophotometer Spectrum (FT-IR) of RPP-3a
The FT-IR spectrum of RPP-3a is illustrated in Figure 3. RPP-3a displayed the typical absorption peaks of polysaccharides at 3426.89 cm −1 , 2927.41 cm −1 , and 1421.28 cm −1 [15]. The band in the region of 3426.89 cm −1 indicated the characteristic absorption of -OH stretching vibration [16,17]. The absorption peaks near 2927.41 cm −1 and 1421.28 cm −1 were caused by the stretching vibration of C-H [18]. The peaks at 1741.41 and 1616.06 cm −1 corresponded to symmetric and asymmetric C=O stretching vibrations [19]. The absorption band at 765.60 cm −1 suggested the presence of a pyranose structure [20]. The peaks at 1103.08 cm −1 and 1020.18 cm −1 were assigned to C-O and O-H stretching vibrations, respectively [21].

Fourier Transform Infrared Spectrophotometer Spectrum (FT-IR) of RPP-3a
The FT-IR spectrum of RPP-3a is illustrated in Figure 3. RPP-3a displayed the typical absorption peaks of polysaccharides at 3426.89 cm −1 , 2927.41 cm −1 , and 1421.28 cm −1 [15]. The band in the region of 3426.89 cm −1 indicated the characteristic absorption of -OH stretching vibration [16,17]. The absorption peaks near 2927.41 cm −1 and 1421.28 cm −1 were caused by the stretching vibration of C-H [18]. The peaks at 1741.41 and 1616.06 cm −1 corresponded to symmetric and asymmetric C=O stretching vibrations [19]. The absorption band at 765.60 cm −1 suggested the presence of a pyranose structure [20]. The peaks at 1103.08 cm −1 and 1020.18 cm −1 were assigned to C-O and O-H stretching vibrations, respectively [21]. Da, and 70,366 Da, respectively. Mw/Mn is a measure of the width of the molecular weight distribution [14], and the Mw/Mn value of RPP-3a was 1.501.

Fourier Transform Infrared Spectrophotometer Spectrum (FT-IR) of RPP-3a
The FT-IR spectrum of RPP-3a is illustrated in Figure 3. RPP-3a displayed the typical absorption peaks of polysaccharides at 3426.89 cm −1 , 2927.41 cm −1 , and 1421.28 cm −1 [15]. The band in the region of 3426.89 cm −1 indicated the characteristic absorption of -OH stretching vibration [16,17]. The absorption peaks near 2927.41 cm −1 and 1421.28 cm −1 were caused by the stretching vibration of C-H [18]. The peaks at 1741.41 and 1616.06 cm −1 corresponded to symmetric and asymmetric C=O stretching vibrations [19]. The absorption band at 765.60 cm −1 suggested the presence of a pyranose structure [20]. The peaks at 1103.08 cm −1 and 1020.18 cm −1 were assigned to C-O and O-H stretching vibrations, respectively [21].
The comprehensive analysis of the monosaccharide composition, methylation analysis, and 1D and 2D-NMR data confirmed that the main backbone of RPP-3a was composed of

Antioxidant Activity of RPP-3a
As depicted in Figure 7a, the reducing power of RPP-3a significantly increased with increasing concentrations and reached 0.489 at 3.2 mg/mL, which was approximately half that of vitamin C (VC) at the same concentration. Figure 7b,c depict a comparison between the hydroxyl and superoxide anion radical scavenging ability of RPP-3a with VC. Both the hydroxyl and superoxide anion radical scavenging rates of RPP-3a were concentration-dependent, and the maximal scavenging rate of RPP-3a of hydroxyl and superoxide anion radicals was 81.79% and 63.09%, respectively. However, the superoxide anion radical scavenging rate of RPP-3a was stronger than that of VC at concentrations of 0.05, 0.1, and 0.15 mg/mL. The above results indicate that RPP-3a had a moderate reducing power and strong hydroxyl and superoxide anion radical scavenging ability. the hydroxyl and superoxide anion radical scavenging ability of RPP-3a with VC. Both the hydroxyl and superoxide anion radical scavenging rates of RPP-3a were concentration-dependent, and the maximal scavenging rate of RPP-3a of hydroxyl and superoxide anion radicals was 81.79% and 63.09%, respectively. However, the superoxide anion radical scavenging rate of RPP-3a was stronger than that of VC at concentrations of 0.05, 0.1, and 0.15 mg/mL. The above results indicate that RPP-3a had a moderate reducing power and strong hydroxyl and superoxide anion radical scavenging ability. As shown in Figure 8, the viability of the macrophages in the RPP-3a-treated groups was significantly or extremely significantly different compared to the control group (* p < 0.05, ** p < 0.01), indicating that RPP-3a significantly promoted the viability of RAW264.7 macrophages in a concentration range of 20-160 μg/mL. As shown in Figure 8, the viability of the macrophages in the RPP-3a-treated groups was significantly or extremely significantly different compared to the control group (* p < 0.05, ** p < 0.01), indicating that RPP-3a significantly promoted the viability of RAW264.7 macrophages in a concentration range of 20-160 µg/mL.

The Effect of RPP-3a on RAW264.7 Macrophage Viability
As shown in Figure 8, the viability of the macrophages in the RPP-3a-treated groups was significantly or extremely significantly different compared to the control group (* p < 0.05, ** p < 0.01), indicating that RPP-3a significantly promoted the viability of RAW264.7 macrophages in a concentration range of 20-160 μg/mL.

Effect of RPP-3a on Nitric oxide (NO), Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β) Production in RAW264.7 Macrophages
As depicted in Figure 9, RPP-3a significantly increased the concentration of NO, TNF-α, IL-6, and IL-1β in the RAW246.7 cells at a concentration range of 20-160 µg/mL (** p < 0.01 vs. the control group). The level of NO was significantly increased in a dosedependent manner (Figure 9a). However, the opposite phenomenon was observed for TNF-α (Figure 9b As depicted in Figure 9, RPP-3a significantly increased the concentration of NO, TNF-α, IL-6, and IL-1β in the RAW246.7 cells at a concentration range of 20-160 μg/mL (** p < 0.01 vs. the control group). The level of NO was significantly increased in a dosedependent manner (Figure 9a). However, the opposite phenomenon was observed for TNF-α (Figure 9b  . Enhanced effect of RPP-3a on NO and pro-inflammatory cytokine production. NO (a), TNF-α (b), IL-6 (c), and IL-1β (d). The concentration of NO was determined using Griess reagent, and the concentrations of TNF-α, IL-6, and IL-1β were determined by ELISA assay kits. Data are presented as the mean ± SD; ** p < 0.01 vs. the control group.

Effects of RPP-3a on the Expression of Inducible Nitric Oxide Synthase (iNOS) and Cytokines in RAW264.7 Macrophages
The production of NO is firmly regulated by iNOS [33]. Thus, to further verify the effect of RPP-3a on NO and cytokine secretion in RAW264.7 cells, the mRNA expression of iNOS, IL-1β, TNF-α, and IL-6 was determined by RT-qPCR. As depicted in Figure 10a, RPP-3a promoted the mRNA expression of iNOS in a concentration range of 40-160 Figure 9. Enhanced effect of RPP-3a on NO and pro-inflammatory cytokine production. NO (a), TNF-α (b), IL-6 (c), and IL-1β (d). The concentration of NO was determined using Griess reagent, and the concentrations of TNF-α, IL-6, and IL-1β were determined by ELISA assay kits. Data are presented as the mean ± SD; ** p < 0.01 vs. the control group.

Effects of RPP-3a on the Expression of Inducible Nitric Oxide Synthase (iNOS) and Cytokines in RAW264.7 Macrophages
The production of NO is firmly regulated by iNOS [33]. Thus, to further verify the effect of RPP-3a on NO and cytokine secretion in RAW264.7 cells, the mRNA expression of iNOS, IL-1β, TNF-α, and IL-6 was determined by RT-qPCR. As depicted in Figure 10a, RPP-3a promoted the mRNA expression of iNOS in a concentration range of 40-160 µg/mL (** p < 0.01 vs. the control group), indicating that RPP-3a enhanced NO production by inducing iNOS expression at the mRNA level. Compared to the control group, the mRNA expression of TNF-α (Figure 10b), IL-6 (Figure 10c), and IL-1β (Figure 10d) was significantly stimulated by RPP-3a (** p < 0.01 vs. the control group), suggesting that RPP-3a increased the production of these cytokines in RAW264.7 macrophages at the transcriptional level.

Discussion
In the present study, an acidic homogeneous polysaccharide RPP-3a, with an Mw of 88,997 Da, was isolated and purified from crude raspberry pulp polysaccharides ( Figures  1 and 2). RPP-3a was composed of Rha, Ara, Gal, Glu, Man, and GalA at a molar ratio of 13.1:28.  Table 2). Compared to the previously reported polysaccharides obtained from raspberries, RPP-3a differed in monosaccharide composition, molecular weight, and glycosidic linkages. Ke et al. reported that the polysaccharide separated from Rubus chingii Hu, with a molecular weight of 837,820 Da, was composed of Man, Rha, GlcA, GalA, Glc, Gal, Ara, and Fuc, of which GalA and Ara were the major monosaccharides [3]. Another raspberry polysaccharide called RCPI was composed of GalA, Rha, Ara, Xyl, Man, Glc, and Gal at a molar ratio of 1.  [14]. The variation in the structural features of the polysaccharides derived from raspberries might be due to differences in the geographical environment and climatic conditions, cultivars, extraction and purification procedures, and the different parts of the raspberries used for the extractions [23]. Thus, RPP-3a was inferred to be a novel acid polysaccharide isolated from raspberries.

Discussion
In the present study, an acidic homogeneous polysaccharide RPP-3a, with an Mw of 88,997 Da, was isolated and purified from crude raspberry pulp polysaccharides (Figures 1 and 2). RPP-3a was composed of Rha, Ara, Gal, Glu, Man, and GalA at a molar ratio of 13.1:28.  Table 2). Compared to the previously reported polysaccharides obtained from raspberries, RPP-3a differed in monosaccharide composition, molecular weight, and glycosidic linkages. Ke et al. reported that the polysaccharide separated from Rubus chingii Hu, with a molecular weight of 837,820 Da, was composed of Man, Rha, GlcA, GalA, Glc, Gal, Ara, and Fuc, of which GalA and Ara were the major monosaccharides [3]. Another raspberry polysaccharide called RCPI was composed of GalA, Rha, Ara, Xyl, Man, Glc, and Gal at a molar ratio of 1.  [14]. The variation in the structural features of the polysaccha-rides derived from raspberries might be due to differences in the geographical environment and climatic conditions, cultivars, extraction and purification procedures, and the different parts of the raspberries used for the extractions [23]. Thus, RPP-3a was inferred to be a novel acid polysaccharide isolated from raspberries.
An increasing number of studies have shown that the biological activity of polysaccharides was closely related to their structural features, including conformation, monosaccharide composition, molecular weight, glycosidic bonds, and degree of branching [28,34]. The high percentage of uronic acid could contribute to the immunomodulatory and antioxidative effects of polysaccharides [35,36]. Ketha reported that the polysaccharides isolated from mung beans were shown to activate macrophages due to the high uronic acid content [37]. Similarly, another in vitro study proved that galacturonic acid played a significant role in the proliferative activity of macrophages [38]. Li reported that the level of uronic acids was positively correlated with the in vitro antioxidant activity of polysaccharides, higher uronic acid content, and higher protective effect against H 2 O 2 -induced oxidative damage in HepG2 cells [7]. Wang et al. also reported that the uronic acid content of polysaccharides exerted significant effects on hydroxyl radical scavenging [39]. Polysaccharides with Rha and Ara exhibited better antioxidant activity than those without these monosaccharides [40]. In this study, GalA, Rha, and Ara were the major monosaccharides of RPP-3a ( Figure 4). Thus, RPP-3a might exhibit strong immunomodulatory and antioxidant activities. Besides the monosaccharide composition, the molecular weight might also contribute to the bioactivity of the polysaccharides. Studies have proved that the polysaccharides with lower molecular weight may have significant immunoregulatory effects, as their simpler structural conformation allows them to pass through the cell barrier easier [41]. Interestingly, some high molecular weight polysaccharides also have strong immune regulatory effects due to the presence of more receptors [42]. As for the antioxidative effect, the polysaccharide with a lower molecular weight had better antioxidant activity due to more reductive hydroxyl group terminals [43]. In the present study, the Mw of RPP-3a was 88,997 Da (Figure 2), which was not very large but still might contribute to its immunomodulatory and antioxidant activities. Another factor that affects the biological activity of polysaccharides is the degree of branching. A previous study revealed that the branching degree was closely related to the immunomodulatory activity of pectic polysaccharides, and the immunomodulatory activity could be diminished by removing the branching regions [44]. According to the structural analysis, the side chain of RPP-3a had two levels of branches ( Figure 6). Such a complex branching degree with a large amount of reductive hydroxyl group terminals may significantly contribute to its immunostimulatory and antioxidant effects. Overall, it was speculated that RPP-3a might exhibit strong immunomodulatory and antioxidant activity.
Free radicals are by-products produced during metabolic processes in organisms. Excessive free radicals induce various harmful effects in the human body, such as cancer, diabetes, liver injury, skin damage, aging, and fatigue [45]. Hydroxyl radicals, also known as the most reactive free radicals, can react with almost all of the biomacromolecules functioning in living cells [46]. Superoxide anions are a precursor of active free radicals and are relatively weak oxidants. They can generate strong reactive oxidative species and cause oxidant damage and various diseases upon interacting with singlet oxygen or hydroxyl radicals [47]. Fe (III) reduction, reflecting a vital mechanism of polysaccharide antioxidation, could be used as an indicator of electron-donating activity [48]. In the present study, RPP-3a exhibited a moderate reducing power and strong hydroxyl and superoxide anion radical scavenging ability (Figure 7). The presence of large amounts of branched chains and high uronic acid content in RPP-3a may contribute to its antioxidant activity, providing hydrogen or an electron through its hydroxyl groups to free radicals for transformation to a harmless substance and radical chain reaction inhibition [28].
Of the multitude of immune cells, macrophages are the first line of defense with various activities, such as phagocytosis, surveillance, chemotaxis, and the destruction of targeted organisms [26,49]. Research has shown that a macrophage activation is a promising approach to improving host immune capability and strengthening disease resistance [50]. Activated macrophages induced the production of NO and various immunomodulatory cytokines, such as tumor necrosis factors and interleukins, which act as mediators of immune responses to modulate immunity and participate in pro-inflammatory and antiinflammatory activities [51]. NO, a short-lived gaseous molecule produced by macrophages, is crucial for enhancing the lysis and phagocytosis of macrophages. Thus, it is an excellent biomarker for evaluating macrophage activation [52]. TNF-α is the earliest and a potent pro-inflammatory mediator secreted by activated macrophages, which exerts a variety of biological effects, such as cell proliferation, differentiation, and multiple pro-inflammatory effects [53]. IL-1β acts with TNF-α in inflammatory processes and plays a vital role in the activation of macrophages [54]. IL-6 is a vital bioactive molecule, which can participate in phagocytosis, antigen-presentation, and inflammation regulation, regulating both cellular and humoral immunity [55]. Most polysaccharide-induced immune-enhancing effects are dependent upon the macrophage function [56]. Thus, macrophages and effectors including NO, TNF-α, IL-1β, and IL-6 were utilized to evaluate the immunomodulatory activity of RPP-3a in the present work. The results showed that RPP-3a could significantly promote the viability of macrophages ( Figure 8) and increase the production of NO, TNF-α, IL-6, and IL-1β at both the expression and transcriptional levels in RAW246.7 cells (Figures 9 and 10). These results strongly confirmed that RPP-3a possessed significant immune-enhancing activity. However, studies should be undertaken to reveal the underlying immune-promoting mechanisms.

Extraction of Crude Polysaccharides
Crude RPPs were prepared according to the method described in our earlier study [11]. In brief, the frozen raspberries were dried, ground, and sieved to remove the seeds. The raspberry pulp powder was defatted with petroleum ether (boiling point, 60 • C) at room temperature for 24 h with continuous stirring and then extracted with 80% ethanol at 60 • C for 2 h to remove the ethanol-soluble constituents. With the assistance of ultrasound, the residue was extracted in distilled water at 60 • C for 2 h. The concentrated portion was deproteinized using Sevage solution. Thereafter, a 4-fold volume of 95% ethanol was added to precipitate the polysaccharides at 4 • C overnight. The crude polysaccharides were collected and freeze-dried for further analyses.

Isolation of RPP-3a
The further purification of RPPs was carried out using DEAE Sepharose fast flow chromatography. The column (7.5 cm × 60 cm) was eluted with deionized water, 0.2 M, and 0.4 M NaCl solution at a flow rate of 15 mL/min, respectively. The eluate was monitored using the phenol-sulfuric acid method [16]. Fractions containing the main elution peak were lyophilized and further purified on a Sephadex G-200 column (2.6 cm × 60 cm) according to the molecular weight, using distilled water as the eluent at a flow rate of 0.5 mL/min. The eluates were monitored and combined using highperformance liquid chromatography (HPLC; RID-10A FRC-10A, Shimadzu, Tokyo, Japan) online detection system equipped with a refractive index detector (RI-502, Shodex, Tokyo, Japan). The refined polysaccharide fractions were concentrated, dialyzed, and freeze-dried for further analyses.

Homogeneity and Molecular Weight Determination
The molecular weight and purity of RPP-3a were determined by the HPGPC method using an LC-10A HPLC system (Shimadzu, Tokyo, Japan) equipped with a BRT105-104-102 series column (8 mm × 300 mm) and a refractive index detector. The experimental settings were as follows: the temperature was maintained at 40 • C, and the mobile phase was 0.05 M NaCl solution with a flow rate of 0.6 mL/min. Dextran of different molecular weights (5000; 11,600; 23,800; 48,600; 80,900; 148,000; 273,000; 409,800, and 670,000 Da) was used as the standards.

Infrared Spectrum Analysis
RPP-3 (2 mg) and 200 mg of dried KBr were ground and pressed into a pellet. Then, the absorbance curves were recorded using an FT-IR spectrophotometer (FT-IR650, Tianjin Gangdong Co., Tianjin, China) in the wavelength range of 4000-400 cm −1 [30].

Monosaccharide Composition Analysis
The acetylated derivatives of RPP-3a and monosaccharide standards were converted and analyzed using high-performance anion-exchange chromatography (HPAEC) according to a previously reported method [57,58]. A Dionex ICS-5000 system (Thermo Scientific Co., Waltham, MA, USA) equipped with a CarboPac TM PA-20 analytical column (3 mm × 150 mm) and a pulsed amperometric detector was employed in this analysis. The column was eluted with an isocratic NaOH solution (250 mM) for 10 min, followed by NaOAc (500 mM) containing 50 mM of NaOH for another 30 min. The elution temperature, injection volume, and flow rate were set to 30 • C, 5 µL, and 0.3 mL/min, respectively. The reducing power was tested by the ferric-reducing antioxidant power (FRAP) assay [60]. Approximately 2.5 mL of RPP-3a at different concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg/mL) was mixed with potassium phosphate buffer (2.5 mL, 0.2 M) at pH 6.6 and potassium ferricyanide solution (2.5 mL, 1% (w/v)). The obtained mixture was kept at 50 • C for 20 min, supplemented with trichloroacetic acid (2.5 mL, 10% (w/v)), and then centrifuged at 3000 for 10 min. The upper layer was recovered, of which 2.5 mL was mixed with 2.5 mL of distilled water and ferric chloride solution (0.5 mL, 0.1% (w/v)). After 10 min of incubation, the absorbance was recorded at 700 nm. VC served as the positive control. An equivalent amount of distilled water was used as the control group instead of a polysaccharide solution.

Hydroxyl Radical Scavenging Assay
The scavenging ability of hydroxyl radicals was determined by the previously reported Fenton method [53]. Briefly, 1 mL of polysaccharide solution was mixed with 1 mL of ferrous sulfate solution (9 mM), salicylic acid-ethanol solution (9 mM), and H 2 O 2 solution (8.8 mM) at various concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg/mL). The mixed solution was incubated in a water bath at 37 • C for 30 min, and then its absorbance was detected at 510 nm. VC served as the positive control. The hydroxyl radical scavenging capacity was calculated using the following equation: where A 0 is the absorbance of the blank (distilled water instead of the sample), A 1 is the absorbance of the sample, and A 2 is the absorbance of the control (distilled water instead of H 2 O 2 ).

Superoxide Anion Radical Scavenging Assay
The superoxide anion radical scavenging assay was performed according to a previously reported method [16]. Tris-HCl buffer (2 mL of 50 mM, pH 8.2) was mixed with 1 mL of RPP-3a solution at various concentrations (0.05, 0.1, 0.15, 0.25, 0.3, 0.6, and 1.2 mg/mL). After the addition of 0.2 mL of pyrogallol (6 mM), the mixture was shaken vigorously and reacted at 25 • C for 5 min. Finally, 0.5 mL of 0.1M HCl was added to terminate the reaction. The absorbance of the samples was determined at 320 nm using VC as the positive control. The superoxide anion radical scavenging ability was calculated using the following equation: where A 0 is the auto-oxidation rate of the control group, and A 1 is the auto-oxidation rate of the sample.

Cell Culture
RAW264.7 macrophages were maintained in a DMEM medium supplemented with 10% FBS and 1% P/S in a 5% CO 2 incubator at 37 • C and humidified atmosphere [61].

Determination of NO, TNF-α, IL-6, and IL-1β Levels
The cell seeding and treatment processes were the same as those described in Section 2.10. NO levels in the cell supernatants were assessed using Griess reagent, and the concentration of TNF-α, IL-6, and IL-1β in the cell culture medium was determined by ELISA assay kits according to the manufacturer's instructions.

Real-Time Quantitative Polymerase Chain Reaction
RAW264.7 cells (2 × 10 6 cells/well) were seeded into 6-well plates and incubated overnight at 37 • C. The cells were subsequently exposed to RPP-3a (20,40,80, and 160 µg/mL) or LPS (5 µg/mL) for 24 h. The total RNA in the RAW264.7 cells was extracted using the RNAsimple Total RNA Kit (TIANGEN, Beijing, China) and reverse-transcribed into cDNA using the FastKing cDNA Dispelling RT SuperMix Kit (TIANGEN, Beijing, China). The real-time quantitative polymerase chain reaction (RT-qPCR) assay was performed on a LightCycler 96 PCR System (Roche, Basel, Switzerland) with SYBR Green (TIANGEN, Beijing, China). The primer sequences are listed in Table 3. The amplification conditions were as follows: 95 • C of initial denaturation for 15 min, followed by 40 cycles of denaturation at 95 • C for 10 s, annealing at 60 • C for 30 s, and extension at 72 • C for 30 s. The GADPH gene was used as the internal reference gene. The relative expression of the target genes was calculated using the 2 −∆∆Ct method. Table 3. Primers used in RT-qPCR.

Statistical Analysis
The data were analyzed by one-way ANOVA, followed by Tukey's post hoc test using SPSS version 19.0 software (SPSS, Chicago, IL, USA) and expressed as mean ± standard deviation (SD.). A p-value of <0.05 (* p < 0.05 in the figure) indicated a significant difference, and p-value of <0.01 (** p < 0.01 in the figure) indicated a highly significant difference.
Author Contributions: Conceptualization, funding acquisition, writing-original draft preparation, and project administration, Y.Y.; methodology, investigation, validation, and data curation, X.Y.; supervision and writing-review and editing, D.Z.; writing-review and editing and data curation, B.Z.; data curation and formal analysis, J.L.; data curation, X.W. All authors have read and agreed to the published version of the manuscript.