Isolation, Structural Characterization and Macrophage Activation Activity of an Acidic Polysaccharide from Raspberry Pulp

The discovery of safe and effective plant polysaccharides with immunomodulatory effects has become a research hotspot. Raspberry is an essential commercial fruit and is widely distributed, cultivated, and consumed worldwide. In the present study, a homogeneous acidic polysaccharide (RPP-2a), with a weight-average molecular weight (Mw) of 55582 Da, was isolated from the pulp of raspberries through DEAE-Sepharose Fast Flow and Sephadex G-200 chromatography. RPP-2a consisted of rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid, with a molar ratio of 15.4:9.6:7.6:3.2:9.1:54.3:0.8. The results of Fourier transform infrared spectroscopy (FT-IR), gas chromatography-mass spectrometer (GC-MS), 1D-, and 2D-nuclear magnetic resonance (NMR) analyses suggested that the backbone of RPP-2a was primarily composed of →2)-α-L-Rhap-(1→, →2,4)-α-L-Rhap-(1→, →4)-α-D-GalAp-(1→, and →3,4)-α-D-Glcp-(1→ sugar moieties, with side chains of α-L-Araf-(1→, α-L-Arap-(1→, and β-D-Galp-(1→3)-β-D-Galp-(1→ residues linked to the O-4 band of rhamnose and O-3 band of glucose residues. Furthermore, RPP-2a exhibited significant macrophage activation activity by increasing the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and the expression of inducible nitric oxide synthase (iNOS) and cytokines at the transcriptional level in RAW264.7 cells. Overall, the results indicate that RPP-2a can be utilized as a potential natural immune-enhancing agent.


Introduction
Polysaccharides, naturally occurring flexible macromolecular polymers with complex structures, are the primary components of plants, fungi, bacteria, algae, and even animals [1]. Polysaccharides possess numerous pharmacological activities, including antitumor, anti-inflammatory, antioxidant, antimicrobial, antidiabetic, immunomodulatory, etc. [2][3][4]. Their immunoregulatory effects are considered to be the primary activity of polysaccharides. Hence, several polysaccharides are widely utilized as potent immunomodulators in the food and medicine industries [5]. Previous studies have reported that most polysaccharides-induced actions are dependent on macrophage functional ability [6]. Macrophages are the first line of defense, with various activities performed by the multitudinous immune cells, such as phagocytosis, surveillance, chemotaxis, and destruction of targeted organisms [7][8][9]. Research has been proven that macrophage activation is a promising approach to improve host immune capability and strengthen disease resistance [10]. Accumulating evidence suggests that plant-derived polysaccharides, with relatively low toxicity and side effects, possess potent immunomodulatory activity by enhancing or activating the immune responses of macrophages [8,11]. For instance, a polysaccharide from the wall of Sambucus adnate exerts an immunomodulatory effect by activating macrophages and enhancing the host's immune system function [12]. Similarly, the polysaccharides obtained from Radix aconiti significantly promote macrophage phagocytosis and increase the secretion of biological factors [13]. Hence, attempting to discover a safe and effective plant polysaccharide with a potent immunomodulatory effect has become a hot spot in research that is gaining increasing attention worldwide.
Raspberry (Rubus idaeus L.), a perennial shrub belonging to the diverse Rubus genus rank, is an essential commercial fruit that is widely distributed, cultivated, and consumed worldwide [14]. It is usually consumed as fresh fruit or processed into jams, juices, and wines, or served as an ingredient in other products and various foods [15]. In recent years, the raspberry has emerged as the most popular berry due to the presence of numerous bioactive substances, including flavonoids, tannins, phenolic acids, stilbenoids, polysaccharides, vitamins, and minerals [16]. Moreover, the dietary intake of raspberries has been used to treat cardiovascular diseases, obesity, cancer, and degenerative diseases [17]. Polysaccharides are one of the most important bioactive components in raspberries. So far, many bioactive polysaccharides have been isolated from the raspberry. For instance, Yu et al. [18] isolated and purified a heteropolysaccharide from the raspberry fruit, which exhibited excellent antioxidant and antiglycation activities. Xu et al. [19] purified a heteropolysaccharide (RCPI) and obtained a degraded polysaccharide (DRCPI) from the raspberry fruits that displayed high antioxidant activity and thermal stability. Similarly, Ke et al. reported that the raspberry polysaccharides could resist palmitic acid-induced lipotoxicity and ethylcarbamate-induced toxicity [16,20]. Our previous study found that crude raspberry pulp polysaccharides (RPPs) exhibited significant antitumor activity and chemotherapy enhancement effects in vivo by enhancing the cellular immune response of the host organism without any lesions in the liver or kidney tissues [21]. These results indicated that the polysaccharides derived from raspberries could be used as healthcare foods, dietary supplements, or medicines. In this study, an acidic polysaccharide from the pulp of raspberries was isolated and its structure and macrophage activation activity were investigated. This study aims to provide an effective plant polysaccharide for potential application as a natural immune-enhancing agent in functional food supplements or drugs.

Isolation and Purification of Homogeneous Acidic Polysaccharide (RPP-2a)
The crude RPPs were purified by DEAE-Sepharose Fast Flow chromatography and a Sephadex G-200 column. As depicted in Figure 1a, two fractions designated RPP-1 and RPP-2 were eluted with 0 M and 0.2 M NaCl solutions and collected from the DEAE-Sepharose Fast Flow chromatography system. In our previous study, the properties of RPP-1 were extensively studied. The present study focused on RPP-2 only. RPP-2 was further purified by a Sephadex G-200 column. As illustrated in Figure 1b, two peaks designated RPP-2a and RPP-2b were observed. Compared to RPP-2b, RPP-2a exhibited a larger peak area, suggesting that it was the major ingredient in RPP-2. Thus, RPP-2a was concentrated, dialyzed, and lyophilized for further analysis.

Fourier Transform Infrared Spectrophotometer (FT-IR) Spectrum of RPP-2a
The FT-IR spectrum of RPP-2a is illustrated in Figure 3. RPP-2a displayed the characteristic absorption peaks of polysaccharides at 3401.82 cm −1 , 2927.41 cm −1 , and 1421.28 cm −1 [22]. The band that appeared at 3401.82 cm −1 corresponded to -OH stretching vibration [23,24]. The absorption peaks at 2927.41 cm −1 and 1421.28 cm −1 were related to the stretching vibration of CH [25]. The intense peaks at 1745 and 1616 cm −1 corresponded to the symmetric and asymmetric C=O stretching vibration [26], and the absorption bands observed at 910 and 757 cm −1 suggested that RPP-2a was comprised of a pyranose structure [27]. The absorbance peaks at 1200-1000 cm −1 were assigned to the contribution of C-OH or C-O-C stretching vibration [28].

The Molecular Weight of RPP-2a
A single and symmetrical peak was obtained for RPP-2a from the HPGPC, suggesting that the purified polysaccharide was homogeneous ( Figure 4). The weight-average molecular weight (Mw), number-average molecular weight (Mn), and peak molecular weight (Mp) of RPP-2a were 55582 Da, 38824 Da, and 45471 Da, respectively. Mw/Mn is a measure of the width of the molecular weight distribution [29]. The Mw/Mn value of RPP-2a was 1.432.

Discussion
In this study, an acidic homogeneous polysaccharide RPP-2a was isolated from the pulp of raspberries. The result of monosaccharide composition, methylation analysis,  [16]. The monosaccharide composition of another raspberry polysaccharide was galacturonic acid, rhamnose, arabinose, xylose, mannose, glucose, and galactose, with a molar ratio of 1.00:0.15:0.65:0.26:0.11:0.10:0.46 [19]. Although the types of monosaccharide in RPP-2a varied between these two raspberry polysaccharides, both were consistent in that galacturonic acid had the highest concentration. Thus, RPP-2a was inferred to be a novel polysaccharide isolated from the raspberry fruits. The variation in the structural features of polysaccharides might be due to the differences in geographical environment and climatic conditions, cultivars, or the extraction and purification procedures [43]. However, the differences in polysaccharides from different raspberry parts could be due to another reason.
The relationship between the structure and immunomodulatory activity is difficult to estimate due to the structural heterogeneity and complex composition of polysaccharides [53]. It is noteworthy that the immunomodulatory activity of a polysaccharide is affected by its conformation, monosaccharide composition, molecular weight, branching degree and functional groups [54]. RPP-2a was composed of rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid, with the highest content being galacturonic acid. Moreover, the monosaccharide composition of polysaccharides influences their biological activities. It is reported that a high percentage of uronic acid might contribute to the immunomodulatory effects of polysaccharides [55]. For instance, Ketha and Gudipati reported that the carboxyl group of uronic acid in mung bean non-starch polysaccharides was involved in the activation of macrophages [56]. Similarly, another in vitro study indicated that galacturonic acid played important roles in the macrophage's proliferative activity [57]. Thus, RPP-2a might exhibit a strong immunomodulatory activity due to its high galacturonic acid content. Besides monosaccharide composition, the molecular weight might also contribute to the activity of polysaccharides. Lower and higher molecular weight polysaccharides show distinct immune regulation effects in different substances. The lower molecular weight polysaccharides have simpler structural conformation, allowing then to pass through the cell barrier with less hinderance [58]. In contrast, some high molecular weight polysaccharides also have strong immune regulation effects due to the presence of more receptors [59]. The Mw of RPP-2a was 55582 Da, which was not very large, but might be favorable to its immunomodulatory activity. It has been reported that the immunomodulatory activity of pectic polysaccharides is mainly associated with the flexible chain conformations and branching degrees and the removal of branching regions might diminish their immunostimulatory activity [60]. RPP-2a was composed of a branched heteropolysaccharide with ramified chains of α-L-Araf-(1→, α-L-Arap-(1→, and β-D-Galp-(1→3)-β-D-Galp-(1→ moieties, contributing to its immunostimulatory activity. Overall, it was speculated that RPP-2a might exhibit a strong immunomodulatory activity.
Macrophages are unique cells in the immune system [61]. After being activated, the macrophages produce NO and release various cytokines, including tumor necrosis factors and interleukins. These cytokines act as the mediators of immune responses to modulate immunity and participate in proinflammatory and anti-inflammatory actions [40]. Moreover, these effectors positively promote the macrophage's functions [62]. NO is a gaseous molecule that enhances the lysis and phagocytosis of macrophages and is an excellent biomarker for evaluating macrophage activation [63]. TNF-α is the earliest mediator of inflammatory reactions and can promote the activity of macrophages [64]. IL-1β plays a vital role in macrophage activation and acts with TNF-α in inflammation [65]. IL-6 can regulate both the cellular and humoral immunity and participate in phagocytosis, antigen-presenting, and inflammatory regulation [66]. Thus, macrophages are utilized as the potential cell models to evaluate the immunomodulatory activities of bioactive compounds [67]. As a member of the macrophage family, the murine RAW264.7 cells have been widely used for immune activity studies [68]. In this study, RPP-2a significantly promoted the viability of macrophages and increased the levels and mRNA expressions of NO, TNF-α, IL-6, and IL-1β in RAW246.7 cells. these results confirmed that RPP-2a possessed significant macrophage activation activity and exhibited a strong potential to be utilized as a natural immune-enhancing agent. However, the underlying mechanism of the macrophage activation activity of RPP-2a should be investigated in future studies.

Preparation and Purification of Polysaccharides from Raspberry Pulp
The crude raspberry pulp polysaccharides (RPPs) were extracted according to the previously reported methods [21]. Briefly, the dried raspberry pulp powder was defatted with petroleum ether (boiling point, 60 • C) at room temperature for 24 h, and then continuously stirred. Finally, it was extracted with 80% ethanol at 60 • C for 2 h to remove the oligosaccharides, colored contaminants, and monosaccharides. The residue was extracted by distilled water at 60 • C for 2 h with ultrasonic assistance. The concentrated portion was subjected to deproteination by the Sevage method three times. Thereafter, a 4-fold volume of 95% ethanol was added to precipitate the polysaccharide at 4 • C overnight. The crude polysaccharides were then collected and freeze-dried for further analyses.
The RPPs were further purified through the DEAE Sepharose Fast Flow chromatography column (7.5 cm × 60 cm) using an NaCl gradient solution (0-0.2 M) as the eluent at a flow rate of 15 mL/min. The eluates were collected automatically, and the polysaccharide content was monitored using the phenol-sulfuric acid method at 490 nm [23]. The fractions were collected, lyophilized, and further purified through a Sephadex G-200 gel permeation column (2.6 cm × 60 cm), and eluted with distilled water at a flow rate of 0.5 mL/min. The fractions were monitored and combined using an 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 homogeneity and molecular weight of RPP-2a were estimated by a High-Performance Gel Permeation Chromatography (HPGPC) system. The process was performed on an LC-10A HPLC system (Shimadzu, Tokyo, Japan) equipped with a BRT105-104-102 series column (8 mm × 300 mm) maintained at a temperature of 40 • C, and a refractive index detector. The mobile phase was 0.05 M NaCl solution, and the flow rate was 0.6 mL/min. Dextrans with different molecular weights (5000, 11,600, 23,800, 48,600, 80,900, 148,000, 273,000, 409,800, 670,000 Da) were used as the standards.

Infrared Spectrum Analysis
The IR spectrum of RPP-2a was analyzed by a Fourier transform infrared spectrophotometer (FT-IR650, Tianjin Gangdong CO., Hebei, China) based on the potassium bromide disk method within the frequency range of 4000-400 cm −1 [36].

Monosaccharide Composition Analysis
The monosaccharide composition of RPP-2a was analyzed by high-performance anion exchange chromatography (HPAEC) [69]. The monosaccharide standards and RPP-2a were converted to their acetylated derivatives according to a previously reported method [70]. 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. Different isocratic schemes with various concentrations of NaOH and sodium acetate (NaOAc) were used to analyze the acidic sugars, with isocratic NaOH (250 mM) for 10 min, followed by NaOAc (500 mM) containing a fixed 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.

Methylation and GC-MS Analysis
RPP-2a was methylated, hydrolyzed, reduced, and acetylated, following the previously reported methods [71]. Acetylates were examined using a GC-MS instrument (GCMS-QP-2010, Shimadzu, Tokyo, Japan) equipped with an RXI-5 SIL MS chromatographic column (30 m × 0.25 mm × 0.25 µm). The temperature program was set as follows: the initial column temperature was maintained at 120°C, increased to 250 • C at 3 • C/min and maintained for 5 min. The flow rate of H 2 was 1mL/min and the detector temperature was 250 • C.

Determination of NO, TNF-α, IL-6 and IL-1β
The cell seeding and treatment processes were the same as described above in Section 4.9. The NO level in the cell supernatants was assessed using the Griess reagent, and the concentrations of TNF-α, IL-6 and IL-1β in the cell culture medium were determined by the ELISA assay kits according to the manufacturer's instructions.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
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 a series of concentrations of RPP-2a or LPS for 24 h. The total RNAs were extracted using the RNAsimple Total RNA Kit (TIANGEN, Beijing, China), and then were reverse-transcribed to cDNAs using a FastKing cDNA Dispelling RT SuperMix Kit (TIANGEN, Beijing, China). The RT-qPCR assay was performed on a LightCycler 96 PCR System (Roche, Basel, Switzerland) with SYBR Green (TIANGEN, Beijing, China). The relative expression levels of the target genes were calculated based on 2 − Ct . The GADPH gene was used as a housekeeping gene. The primers used in this study are listed in Table 3. Table 3. PCR primers used in the measurement of mRNA expression.

Statistical Analysis
The data were analyzed by a 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 < 0.05 (*) was considered as a significant difference and p < 0.01 (**) was considered as a highly significant difference.

Conclusions
In this study, a homogeneous acidic polysaccharide, named RPP-2a, was purified from the pulp of raspberries. RPP-2a consisted of rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid, among which the galacturonic acid content was the highest.