Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages
Abstract
:1. Introduction
2. Results
2.1. Polysaccharide Purification
2.2. FTIR Analysis of GP2a
2.3. Molecular Weight and Monosaccharide Composition of GP2a
2.4. Linkage Composition of GP2a
2.5. Micromorphology Structure of GP2a
2.6. NMR Spectrum Analysis of GP2a
2.7. Effect of GP2a on RAW 264.7 Cell Viability
2.8. Effects of GP2a on NO and Cytokine Production in RAW 264.7 Cells
3. Discussion
4. Materials and Methods
4.1. GP2a Preparation
4.2. GP2a Identification
4.2.1. FTIR Scanning
4.2.2. Molecular Weight Determination
4.2.3. Monosaccharide Composition Analysis
4.2.4. Methylation Analysis
4.2.5. SEM Observation
4.2.6. NMR Spectroscopy
4.3. Immunomodulatory Effect of GP2a on RAW 264.7 Cells
4.3.1. Cell Culture
4.3.2. Cell Viability Assay
4.3.3. Determination of NO and Cytokine Production in RAW 264.7 Cells
4.4. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Monosaccharide | Relative Percentage * (%) |
---|---|
GalA | 63.1 ± 1.7 |
Ara | 11.1 ± 0.6 |
Gal | 10.9 ± 0.5 |
Glc | 7.4 ± 0.3 |
Rha | 2.7 ± 0.1 |
Man | 2.2 ± 0.2 |
GlcA | 1.2 ± 0.0 |
Xyl | 0.8 ± 0.1 |
Fuc | 0.2 ± 0.0 |
Linkage Pattern | Methylated Sugar | Molecular Weight (Da) | Relative Molar Percentage (%) | RT (min) |
---|---|---|---|---|
t-Araf | 1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol | 279 | 3.1 | 5.940 |
5-Araf | 1,4,5-tri-O-acetyl-2,3-di-O-methyl arabinitol | 307 | 3.0 | 10.537 |
t-Galp | 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol | 323 | 3.4 | 9.888 |
3-Galp | 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl galactitol | 351 | 1.4 | 12.812 |
3,4-Galp | 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol | 379 | 0.7 | 15.723 |
3,6-Galp | 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl galactitol | 379 | 1.9 | 18.881 |
t-GalpA | 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol (6,6′-dideuterio) | 325 | 3.4 | 9.888 |
4-GalpA | 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol (6,6′-dideuterio) | 353 | 74.8 | 13.814 |
3,4-GalpA | 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol (6,6′-dideuterio) | 381 | 0.7 | 15.723 |
4,6-GalpA | 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl galactitol (6,6′-dideuterio) | 381 | 2.0 | 18.698 |
4-Glcp | 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol | 351 | 4.3 | 14.043 |
2,4-GlcpA | 1,2,4,5-tetra-O-acetyl-3,6-di-O-methyl glucitol (6,6′-dideuterio) | 381 | 1.3 | 16.921 |
Sugar Residue | Chemical Shift (ppm) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6a | 6b | CH3 of OMe | |||
A | →4)-α-D-GalpA-6-OMe-(1→ | H | 4.86 | 3.62 | 3.89 | 4.35 | 5.05/4.98 | 3.70 | ||
C | 101.90 | 69.44 | 69.59 | 80.68 | 72.27 | 172.30 | 54.42 | |||
B | →4)-α-D-GalpA-(1→ | H | 4.94 | 3.70 | 3.90 | 4.27 | n.d. | |||
C | 101.74 | 69.59 | 69.80 | 80.68 | n.d. | 176.64 | ||||
C | →2)-α-L-Rhap-(1→ | H | 5.03 | 4.06 | 3.84 | 3.66 | 3.90 | 1.15 | ||
C | 100.78 | 77.30 | 74.29 | 74.13 | 69.78 | 15.64 | ||||
D | →2,4)-α-L-Rhap-(1→ | H | 5.12 | 4.09 | 3.84 | 3.65 | 3.90 | 1.15 | ||
C | 100.83 | 77.05 | 74.29 | 81.72 | 69.78 | 15.64 | ||||
E | →3)-β-D-Galp-(1→ | H | 4.57 | 3.68 | 3.75 | 4.10 | 3.61 | 3.64 | 3.74 | |
C | 105.59 | 71.81 | 83.57 | 69.75 | 76.08 | 62.30 | ||||
F | →3,6)-β-D-Galp-(1→ | H | 4.41 | 3.56 | 3.61 | 4.02 | 3.82 | 3.80 | 3.94 | |
C | 104.52 | 71.59 | 81.88 | 70.12 | 74.81 | 71.07 | ||||
G | β-D-Galp-(1→ | H | 4.50 | 3.50 | 3.66 | 3.91 | 3.61 | 3.64 | 3.74 | |
C | 105.84 | 72.54 | 73.65 | 69.96 | 76.66 | 62.30 | ||||
H | α-L-Araf-(1→ | H | 5.13 | 4.12 | 3.89 | 4.02 | 3.69/3.74 | |||
C | 110.85 | 83.57 | 78.14 | 85.26 | 64.89 | |||||
K | →5)-α-L-Araf-(1→ | H | 4.98 | 4.03 | 3.90 | 4.01 | 3.69/3.78 | |||
C | 109.38 | 82.46 | 78.14 | 85.47 | 68.43 |
Groups | Cell Viability (%) | |||
---|---|---|---|---|
24 h | 48 h | 72 h | ||
Blank control | 100.0 ± 0.0 b | 100.00 ± 0.0 b | 100.0 ± 0.0 b | |
Samples | GP2a 7.5 (µg/mL) | 99.4 ± 2.4 b | 103.6 ± 1.3 ab* | 104.8 ± 1.6 ab* |
GP2a 15 (µg/mL) | 101.3 ± 1.4 b | 103.1 ± 1.5 b | 103.5 ± 2.4 b | |
GP2a 30 (µg/mL) | 105.3 ± 1.5 ab | 102.2 ± 2.5 b | 104.0 ± 1.4 b | |
GP2a 60 (µg/mL) | 106.2 ± 1.1 a | 104.9 ± 2.3 ab | 102.1 ± 2.4 b | |
GP2a 120 (µg/mL) | 103.3 ± 1.5 b | 107.9 ± 2.2 ab* | 105.6 ± 2.4 ab | |
GP2a 240 (µg/mL) | 102.5 ± 1.9 b | 110.2 ± 1.8 ab* | 110.3 ± 3.55 ab* | |
GP2a 300 (µg/mL) | 104.3 ± 1.2 ab | 115.1 ± 2.8 ab* | 108.0 ± 3.0 ab | |
Positive control (2 µg/mL LPS) | 109.8 ± 5.5 a | 118.6 ± 2.1 a* | 45.9 ± 3.2 a* |
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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. https://doi.org/10.3390/ijms231911279
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. International Journal of Molecular Sciences. 2022; 23(19):11279. https://doi.org/10.3390/ijms231911279
Chicago/Turabian StyleLin, Pingdong, Lifei Chen, Xiaojing Huang, Fangnan Xiao, Lei Fu, Dingding Jing, Jingjing Wang, Hong Zhang, Lifang Sun, and Yunkun Wu. 2022. "Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages" International Journal of Molecular Sciences 23, no. 19: 11279. https://doi.org/10.3390/ijms231911279