Rhamnogalacturonan II from the leaves of Panax ginseng C.A. Meyer as a macrophage Fc receptor expression-enhancing polysaccharide

doi:10.1016/S0008-6215(97)00055-4

Carbohydrate Research

Volume 300, Issue 3, 16 May 1997, Pages 239-249

https://doi.org/10.1016/S0008-6215(97)00055-4 Get rights and content

Abstract

A complex pectic polysaccharide (GL-4IIb2) has been isolated from the leaves of Panax ginseng C.A. Meyer, and shown to be a macrophage Fc receptor expression-enhancing polysaccharide. The primary structure of GL-4IIb2 was elucidated by composition. ¹H NMR, methylation, and oligosaccharide analyses. GL-4IIb2 consisted of 15 different monosaccharides which included rarely observed sugars, such as 2-O-methylfucose, 2-O-methylxylose, apiose, $3-C- carboxy-5-deoxy - l - xylose$ (aceric acid, AceA), 3-deoxy-d-manno-2-octulosonic acid (Kdo), and 3-deoxy-d-lyxo-2-heptulosaric acid (Dha). Methylation analysis indicated that GL-4IIb2 comprised 34 different glycosyl linkages, such as 3,4-linked Fuc, 3- and 2,3,4-linked Rha, and 2-linked GlcA, which are characteristic of rhamnogalacturonan II (RG-II). Sequential degradation using partial acid hydrolysis indicated that GL-4IIb2 contained α-Rhap-(1 → 5)-Kdo and Araf-(1 → 5)-Dha structural elements, an AceA-containing oligosaccharide, and uronic acid-rich oligosaccharide chains in addition to an α-(1 → 4)-galacturono-oligosaccharide chain. FABMS and methylation analyses suggested that the AceA-containing oligosaccharide was a nonasaccharide in which terminal Rha was additionally attached to position 3 of 2-linked Arap of the octasaccharide chain observed in sycamore RG-II. Component sugar and methylation analyses assumed that the uronic acid-rich oligosaccharides possessed a similar structural feature as those in sycamore RG-II. GL-4IIb2 had a larger molecular mass (11,000) than sycamore RG-II (∼5000).

A structure of macrophage Fc receptor expression-enhancing polysaccharide from the leaves of Panax ginseng C.A. Meyer has been characterised to be a rhamnogalacturonan II type polysaccharide containing a characteristic nonasaccharide sequence.

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The recovery of pectic polysaccharides with high rhamnogalacturonan I (RG-I) branches from citrus canning processing water was achieved in a previous study aimed at reducing chemical oxygen demand and benefiting both process economics and the environment. However, the large molecular size and poor in vivo bioavailability of these polysaccharides limit the application of these pectic polysaccharides in functional foods. We report the development of an ultrafast and green approach to depolymerize pectic polysaccharides using an ultrasound-accelerated metal-free Fenton chemistry, relying on H₂O₂/ascorbic acid. The results show that ultrasound enhances the efficiency of H₂O₂/ascorbic acid system to degrade pectin into 7.9 kDa pectic fragments within 30 min through both chemical effects (increasing the amount of hydroxyl radicals and lowering activation energy of H₂O₂ decomposition) and mechanical effects (disaggregating polysaccharide clusters). The backbones of the resulting fragments mainly correspond to RG-I patterns (molar ratio galacturonic acid (GalA): rhamnose (Rha) ∼ 1.06:1) with a high degree of rhamnose branching. Free radicals preferentially act on the GalA backbone in the HG region and maintain the RG-I region. Antitumor activities, assessed using human breast cancer cells (MCF-7), suggest that the resulting fragments significantly inhibit cancer cell growth and that activity increases with decreasing molecular weight. The resulting ultralow molecular weight pectic fragments have potential application for the development of functional foods and antitumor drugs.
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Ginseng marc is a major by-product of the ginseng industry currently used as animal feed or fertilizer. This fibrous, insoluble waste stream is rich in cell wall polysaccharides and therefore a potential source of ingredients for functional food with health-promoting properties. However, the extraction of these polysaccharides has proved problematic and their exact composition remains unknown. Here we have analysed the composition, structure and biological activity of polysaccharides from ginseng root, stem and leaf marc fractionated using a chelator and alkali solutions. The pectic fraction has been extracted from root marc in high abundance and can activate the production of interleukine-1α and the hematopoietic growth factor by RAW 264.7 murine macrophage cells, which are important immune regulators of T-cells during inflammatory responses and infection processes. Our study reveals the potential to increase the value of ginseng marc by generating carbohydrate-based products with a higher value than animal feed.
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Due to the covalently linkage of RG-II, the content of GalA in HG fractions of ginseng flower buds was lower than that in ginseng roots, and the DM for these fractions was from 20.0% to 40.0%. Although pectin from ginseng leaves has not been characterized comprehensively like that from roots and flower buds, it also comprises of HG, RG-I, and RG-II domains with RG-II more abundant in leaves [35–37]. Different compositions and structures of polysaccharides in distinct organs of ginseng might be related to their various physiological functions in the plant.
Water-soluble pectic polysaccharides isolated from Panax ginseng flower buds (WGFPA) were completely fractionated into six homogeneous fractions (WGFPA-1a, WGFPA-2a, WGFPA-3a, WGFPA-1b, WGFPA-2b and WGFPA-3b) by a combination of ion-exchange and size exclusion chromatographies. Monosaccharide composition, enzymatic hydrolysis and ¹³C nuclear magnetic resonance (NMR) spectra analysis were combined to characterize their structural features. Furthermore, the interactions between these polysaccharides and galectin-3 were evaluated by biolayer interferometry assay. The results showed that WGFPA-1a, WGFPA-2a and WGFPA-3a were rhamnogalacturonan I (RG-I) type pectin with abundant side chains, including α-L-1,5-arabinan, β-D-1,4-galactan, arabinogalactan I (AG-I) and arabinogalactan II (AG-II), exhibiting strong binding activities to galectin-3 with apparent KD values 4.9 μM, 0.71 μM and 0.24 μM, respectively. WGFPA-1b, WGFPA-2b and WGFPA-3b were homogalacturonan (HG) type pectin covalently linked with different ratios of rhamnogalacturonan II (RG-II) domains, showing weaker or no interactions with galectin-3. This study provides useful structural information for further investigation on the structure-activity relationship of ginseng flower buds pectin.
Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer
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Citation Excerpt :
Therefore, ginseng RG-I domain is composed of both low-branched region with short side chains and high-branched region with long and various side chains. Several RG-II domains (GL-4IIb2, GL-RI, GL-RII and GL-RIII) had been extracted from ginseng leaves and structurally analyzed (Shin, Kiyohara, Matsumoto, & Yamada, 1997). However, there is no study concerning RG-II domains from ginseng roots.
Rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II) domains were isolated from ginseng pectin by alkali saponification and endo-polygalacturonase hydrolysis, then purified by anion-exchange and size-exclusion chromatography. Monoclonal antibody detection indicated that ginseng RG-I contained →4)-α-GalpA-(1→2)-α-Rhap-(1→ repeating units as backbone, with arabinan, galactan and type II arabinogalactan (AG-II) as side chains. The use of galactose- and arabinose-releasing enzymes, mass spectrometry analysis of the oligosaccharides generated by rhamnogalacturonan hydrolase, and glycosidic linkage analyses provided evidence that ginseng RG-I contains both single galactose-branched subunits and highly branched subunits with arabinan and AG-II side chains. RG-II was analyzed by sequential acid hydrolysis followed by mass spectrometry. Ginseng RG-II contains 9 galacturonic acid units as backbone. Side chain A is an octasaccharide with 0 ∼ 1 methyl ether group. Side chain B is a nonasaccharide with 0 ∼ 1 acetyl group. These results provide useful information for further investigation of structure-activity relationship of ginseng pectin.
Molecular mechanisms of immunomodulatory activity by polysaccharide isolated from the peels of Citrus unshiu
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The extracts of the Citrus unshiu fruit induce numerous biological activities such as anticancer, anti-adipogenic, and antimicrobial effects. Furthermore, its peel has been used as a Chinese medicine for centuries and is consumed as a tea in China, Japan, and Korea. We investigated the effects of the polysaccharide isolated from C. unshiu peel (CPE-II) on cytokine and inflammatory mediator production in RAW 264.7 macrophages and performed signal transduction experiments to identify the pathways involved in its actions. CPE-II exhibited macrophage-stimulatory activity by inducing the production of interleukin (IL)-6, tumor necrosis factor (TNF)-α and nitric oxide (NO) in RAW 264.7 macrophages. Signal transduction experiments showed that CPE-II phosphorylated mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in RAW 264.7 cells in a concentration-dependent manner. The effects of CPE-II on IL-6/IL-12 and TNF-α production were completely suppressed by a specific inhibitor of c-Jun N-terminal kinase (JNK). Moreover, experiments with neutralizing antibodies showed that Toll-like receptor 2 (TLR2) and TLR4 were involved in the stimulation of IL-6 and NO production by CPE-II in RAW 264.7 cells. Collectively, these results suggest that the TLR2/4 and JNK pathways are essential for the CPE-II immune activity in RAW 264.7 cells.
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Our previous study showed polysaccharide (GS-P) isolated from the leaves of Panax ginseng C.A. Meyer possessed anti-tumor metastatic activity in mouse model. In this study, we evaluated the immunoadjuvant effect of GS-P on the induction of humoral and cellular immune responses against ovalbumin (OVA) in mice. When mice were immunized subcutaneously with OVA admixed with or without GS-P, the OVA + GS-P group showed significantly higher antibody production than the group immunized with OVA alone. This suggests that GS-P has the ability to enhance the adaptive immune response. In addition, the OVA + GS-P + FIA (Freund’s incomplete adjuvant) group induced higher levels of antigen-specific IgG1 and IgG2b antibodies than the OVA + FIA group. The culture supernatant obtained from the splenocytes of mice immunized with OVA + GS-P + FIA showed higher levels of OVA-specific Th1-type (IL-2, IFN-γ, GM-CSF) and Th2-type (IL-10) cytokines. Following in vitro analysis of T cell proliferation, the splenocytes of mice treated with OVA + GS-P + FIA showed significantly more proliferation than those treated with OVA + FIA. Further, the production of IgE antibody was dramatically reduced when OVA + GS-P + FIA was used to immunize mice rather than OVA + FIA or OVA + FCA (Freund’s complete adjuvant). Collectively, these results suggest that GS-P may possess adjuvant activity that potentially enhances humoral as well as cellular immune responses.

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¹: Present address: Department of Food Technology, Kyonggi University, Suwon, Kyonggi-do 442-760, Korea.

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Regular paperRhamnogalacturonan II from the leaves of Panax ginseng C.A. Meyer as a macrophage Fc receptor expression-enhancing polysaccharide

Abstract

Anal. Biochem.

J. Biol. Chem.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Int. J. Immunopharmacol.

Carbohydr. Res.

J. Biol. Chem.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Phytochemistry

Carbohydr. Res.

Carbohydr. Res.

Carbohydr. Res.

Regular paper
Rhamnogalacturonan II from the leaves of Panax ginseng C.A. Meyer as a macrophage Fc receptor expression-enhancing polysaccharide