How does purslane polysaccharide enhance the human immune regulation activity?

Introduction

Purslane polysaccharides (PP), the primary chemical component of purslane, exhibit diverse biological activities, including hypoglycemic, immunomodulatory, neuroprotective, and antitumor activities. To identify polysaccharides with immunomodulatory activity, two polysaccharides, POL-1 and POL-2, were extracted and purified from purslane and their structures characterized. Furthermore, their immunomodulatory activities were evaluated using the RAW 264.7 macrophage model. This study provides a foundation for further understanding the structure and biological activities of purslane polysaccharides and offers valuable information for the application of purslane as an immune adjuvant and immunopotentiator.

Results and Discussion

Purification and Molecular Weight Determination

The crude polysaccharide was separated into six fractions (POL40, POL60, POL40d, POL60d, POL40q, and POL60q) by quaternary ammonium salt precipitation. Of the six crude polysaccharides, POL40d and POL60q were selected for further purification due to the presence of single peaks observed in HPGPC analysis. POL-1 and POL-2 were ultimately obtained. Based on the HPGPC calibration curve of dextran standards, the weight-average molecular weights (Mw) of POL-1 and POL-2 were determined to be 64,100 and 21,000 Da, respectively, with polydispersities (Mw/Mn) of 6.1 and 1.9. As shown in Figure 1, both POL-1 and POL-2 exhibited symmetrical peaks, indicating a well-uniform molecular weight distribution.

Figure 1 HPGPC Spectra and Molecular Weight Distribution of POL-1 and POL-2

UV and FT-IR Spectroscopic Analysis

The UV spectra of POL-1 and POL-2 are shown in Figure 2A. No significant characteristic absorption patterns were observed at wavelengths of 260 nm and 280 nm. This indicates the absence of nucleic acids and proteins in the polysaccharides POL-1 or POL-2. Figure 2B shows the FT-IR spectra of POL-1 and POL-2. The broad and intense peaks at 3430 cm−1 and 3417 cm−1 for POL-1 and POL-2, respectively, are attributed to O-H stretching vibrations. The characteristic absorption patterns at 1739 cm−1 and 1614 cm−1 indicate the presence of ester and carbonyl functional groups in POL-1. It can be inferred that the carboxyl groups on the sugar rings are partially methyl-esterified.

Figure 2 UV (A) and FT-IR spectra (B) of polysaccharides POL-1 and POL-2

Monosaccharide Composition Analysis

The monosaccharide composition of POL-1 and POL-2 was investigated using PMP pre-column derivatization. Figure 3 shows the HPLC chromatograms of a mixed standard of eight monosaccharides, including Man, Rha, GlcA, GalA, Glc, Gal, Xyl, and Ara. The monosaccharide composition of the sample was determined by comparing the retention times of the standard monosaccharides, and the molar ratio of the monosaccharides in the polysaccharide was calculated using the standard curves for each monosaccharide. POL-1 is composed of GalA, the most common monosaccharide in pectin. POL-2 is composed of Glc and Gal in a molar ratio of approximately 1.0:3.3.

Figure 3 Monosaccharide composition profiles of standard monosaccharides, polysaccharides POL-1, and POL-2

Methylation Analysis

The total ion chromatograms (TICs) of partially methylated alditol acetates (PMAA) of POL-1 and POL-2 are shown in Figure 4. However, the low reduction rate of the carboxyl group results in a low abundance of the methylated product (Figure 4A). The corresponding glycosidic bonds and their molar ratios for POL-1 and POL-2 are summarized in Table 1.

Figure 4 Total ion chromatogram (TIC) spectra of partially methylated alditol acetates (PMAA) from the polysaccharides POL-1 (A) and POL-2 (B)

Table 1 GC-MS signal assignments of methylated products from POL-1 and POL-2

NMR Analysis

The 1D (1H and 13C) NMR spectra of POL-1 and the 1D NMR, HMBC, and HSQC-TOCSY spectra of POL-2 are shown in Figures 5 and 6. In the 1D NMR spectrum of POL-1, chemical shifts at δ 5.43 and 99.4 confirm the presence of the α-configuration of GalA. The methyl groups of the O-methyl and O-acetyl groups produce proton signals at δ 3.71 and 1.97, respectively, which are consistent with the results of FT-IR analysis. Crossovers in the HMBC spectrum provided no clues to the glycosidic linkage of POL-1, while methylation results indicated that 1,4-linked GalpA was the primary bonding mode in POL-1. The 1H and 13C signals of POL-2 are listed in Table 2. In the 1D NMR spectrum, the α-configuration of the Glc residue was inferred from the shared chemical shifts of its anomeric carbon (δC 98.6–99.6) and protons (δH 4.98–5.42) downfield. The anomeric resonances at δ 4.65 and 4.60 were assigned to residues A and B, respectively. The coupling constant (JH1-H2) between A-1 and A-2 of residue A was 7.80 Hz, indicating a β-configuration of the Gal residue. Based on 1H-1H COSY, coupled signals observed at δ 4.65/3.69 (A-1/A-2), 3.69/3.79 (A-2/A-3), and 3.79/4.18 (A3/A-4) belong to residue A. Similarly, other related peaks were identified at δ 5.42/3.65 (D1/D2), 3.65/3.97 (D2/D3) and were assigned to the D2 and D3 protons of residue D. Overall, the proposed structures of POL-1 and POL-2 are shown in Figures 7A-B using DrawGlycanSNFG (version 2.0.3) software.

Figure 5 NMR spectrum of POL-1

Figure 6 NMR spectrum of POL-2

Figure 7 Proposed structures of POL-1 (A) and POL-2 (B) from Portulaca oleracea

Table 2 1H and 13C NMR chemical shifts of the polysaccharides POL-1 and POL-2

TEM analysis

TEM images of POL-1 and POL-2 are shown in Figure 8. SDS was used as a surfactant to disperse the polysaccharides and the mixture was heated to 80°C. After 100 °C, dispersed polysaccharides were observed. As shown in Figures 8A-B, TEM images of POL-1 in its fully extended state show an unbranched and dispersed conformation. By comparison, POL-2 molecules tend to form entangled chains with side chains (Figures 8C-D), which is consistent with the results of GC-MS and NMR analyses. At the same magnification, the molecular size of POL-1 is larger than that of POL-2.

Figure 8 TEM images of POL-1 and POL-2 in sodium dodecyl sulfate solution

Evaluation of Immunomodulatory Activity

1. Effects of POL-1 and POL-2 on RAW 264.7 Cell Viability

In this study, RAW 264.7 macrophages were used to evaluate the immunomodulatory activity of purified Portulaca oleracea polysaccharides. First, the MTT assay was used to examine the effects of POL-1 and POL-2 on RAW 264.7 cell viability. As shown in Figures 9A-B, POL-1 exhibited a significantly increased immunomodulatory activity compared to the control at 12.5–400 °C. POL-1 had no inhibitory effect on cell viability within the concentration range of 1 μg/mL, while POL-2 significantly inhibited cell proliferation at 400 μg/mL (P < 0.01). Therefore, concentrations of 50, 100, and 200 μg/mL were selected for the following experiments.

Figure 9 Effects of POL-1 on RAW 264.7 cell viability (A); Effects of POL-2 on RAW 264.7 cell viability (B); Effects of POL-1 on phagocytic activity of RAW 264.7 cells (C); Effects of POL-2 on phagocytic activity of RAW 264.7 cells (D); Effects of POL-1 on NO production in RAW 264.7 cells (E); Effects of POL-2 on NO (F), TNF-α (G), and IL-6 (H) production in RAW 264.7 cells

2. Effects of POL-1 and POL-2 on phagocytic activity

Compared with the control group, LPS-treated RAW The phagocytic function of RAW264.7 cells was significantly enhanced. Similarly, POL-2 significantly promoted cellular uptake of neutral red within the concentration range of 50 to 200 μg/mL (Figure 9D), indicating enhanced phagocytic capacity. However, no significant effect on phagocytosis was observed when treated with different concentrations of POL-1 (50, 100, and 200 μg/mL) (Figure 9C).

3. Effects of POL-1 and POL-2 on NO Production

As an important mediator of immune responses, NO secretion can be determined as a quantitative indicator of macrophage activation. Both LPS and POL-2 (50, 100, and 200 μg/mL) significantly increased NO production in RAW264.7 cells (P < 0.001). However, treatment with POL-1 (50, 100, and 200 μg/mL) significantly increased NO production in RAW264.7 cells (P < 0.001). Treatment with POL-2 (50, 100, and 200 μg/mL) had no effect on NO secretion (Figures 9E-F).

4. Effect of POL-2 on Cytokine Secretion

To further demonstrate the potential immunomodulatory activity of POL-2, the effect of POL-2 on cytokine secretion (IL-6 and TNF-α) was evaluated. LPS stimulation increased the production of IL-6 and TNF-α. Treatment with POL-2 (50, 100, and 200 μg/mL) significantly promoted TNF-α secretion in a dose-dependent manner (Figure 9G). However, IL-6 production remained unchanged at 50 and 100 μg/mL. No significant differences were observed between the POL-2-treated groups (Figure 9H).

5. Effect of POL-2 on NF-κB Pathway Activation

The effect of POL-2 on the expression and phosphorylation levels of P65 and IκB-α in macrophages was examined to investigate whether the NF-κB signaling pathway is involved in POL-2-induced macrophage activation. As shown in Figure 10, compared with the control group, no significant changes were observed in the expression of P65 and IκB-α, whereas the POL-2-treated groups (50-200 μg/mL) increased the expression levels of phosphorylated P65 and IκB-α. This suggests that POL-2 activates the NF-κB signaling pathway by enhancing the phosphorylation levels of P65 and IκB-α in RAW 264.7 cells.

Figure 10 Effect of POL-2 on the Expression of Proteins Related to the NF-κB Signaling Pathway

Conclusion

This study isolated and purified two polysaccharides from water-soluble polysaccharides using graded alcohol precipitation and DEAE anion exchange chromatography. These polysaccharides were designated POL-1 (molecular weight 64,100 Da) and POL-2 (molecular weight 21,000 Da). Da). The physicochemical properties and structures of POL-1 and POL-2 were studied using UV, IR, GC-MS, and NMR techniques. Monosaccharide composition analysis showed that POL-1 was composed of GalA, while the main components of POL-2 were Glc and Gal, with a molar ratio of 1.0:3.3. Combined with infrared spectroscopy, methylation analysis, and nuclear magnetic resonance data, it was speculated that POL-1 was composed of →4)-GalpA-(1→. POL-1 was partially esterified with a methylation degree of 9.71% and an acetylation degree of 0.34%. POL-2 contained a 1,4-linked β-Galp skeleton, whose short side chains were →4)-α-Glcp-(1→ and →6)-α-Glcp-(1→ →4)-α-Glcp-(1→ is connected to the O-6 position of →4)-β-Galp-(1→. The TEM image of POL-2 showed that POL-1 was in an unbranched and dispersed conformation, while the TEM image of POL-2 showed that the side chains of POL-2 were entangled with each other. In addition, the evaluation of immunomodulatory activity was studied using the RAW264.7 macrobiological model. The results showed that POL-2 activated the NFκB signaling pathway by promoting the phosphorylation of P65 and IκBα, significantly increasing the phagocytic function of macrophages and the secretion of NO, TNF-α and IL-6. The results of this study suggest that Portulaca oleracea polysaccharide POL-2 can be used as an immune enhancer and dietary supplement.

anna@hihealthbio.com