Immunostimulatory activity and structural characterization of polysaccharides from Astragalus membranaceus = 황기 （Astragalus membranaceus） 다당류의 면역 자극 활성 및 구조적 특성 규명

다국어 초록 (Multilingual Abstract)

Polysaccharides obtained from natural sources have attracted considerable interest because of their structural diversity and broad range of biological activities. Astragalus polysaccharides （APS） derived from Astragalus membranaceus （AM） have been extensively investigated as functional bioactive materials. However, only a limited number of systematic structural studies on APS, based on activity-guided fractionation using enzymatic digestion and column chromatography, have been conducted to date. Therefore, in this study, activity-guided fractionation was used to isolate macrophage-activating polysaccharides and identify their structural characteristics. First, the active macrophage-stimulating subfraction （AME-A-III-EP-I） was purified using sequential enzymatic digestion and column chromatography. Macrophages were activated by AME-A-III-EP-I through signaling pathways mediated by mitogen-activated protein kinases and nuclear factor kappa B, leading to transcriptional upregulation of immune-related genes in macrophages and subsequent cytokine secretion. Structural characterization revealed that AME-A-III-EP-I is a rhamnogalacturonan-I （RG-I） -rich polysaccharide fraction with a molecular weight of 85.9 kDa. These findings indicate that macrophage-activating polysaccharides purified from AM by enzymatic digestion and chromatographic fractionation are rich in RG-I structures, providing valuable structural and mechanistic insights into the immunostimulatory activity of APS.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 4
• 2.1. Preparation of the enzymatic digest AME-A 4
• 2.2. RAW 264.7 cell culture and cytokine production 6
• 2.3. Anion-exchange chromatographic fractionation 7
• 2.4. Molecular weight distribution 8
• 2.5. Monosaccharide composition 9
• 2.6. Enzymatic digestion and size-exclusion chromatography 11
• 2.7. Phagocytic activity of RAW 264.7 cells 12
• 2.8. Analysis of intracellular signaling pathways 13
• 2.8.1. Gene expression analysis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 13
• 2.8.2. Immunoblotting analysis 15
• 2.8.3. Immunofluorescence analysis 16
• 2.9. Structural characterization of the active moiety 17
• 2.9.1. Congo red assay 17
• 2.9.2. Fourier-transform infrared (FT-IR) spectroscopy 18
• 2.9.3. Glycosidic linkage composition analysis 19
• 2.10. Statistical analysis 21
• Ⅲ. Results 22
• 3.1. Identification of the active moiety through activity-guided fractionation 22
• 3.1.1. Macrophage activation by AME-A 22
• 3.1.2. Fractionation of AME-A 24
• 3.1.3. Enzymatic digestion and size -exclusion chromatography of AME-A-III 29
• 3.1.4. Phagocytic activity 33
• 3.2. Intracellular mechanism of macrophage activation 35
• 3.2.1. mRNA expression analysis 35
• 3.2.2. MAPK and NF-κB signaling pathways 37
• 3.3. Structural and compositional characterization 41
• 3.3.1. General and monosaccharide composition 41
• 3.3.2. Conformational characteristics 43
• 3.3.3. Major functional groups 44
• 3.3.4. Glycosidic linkage composition 45
• Ⅳ. Discussion 48
• References 53