Identification of intracellular activation mechanism of rhamnogalacturonan-I type polysaccharide purified from Panax ginseng leaves in macrophages and roles of component sugar chains on activity.

This study aimed to investigate the mechanisms underlying intracellular signaling pathways in macrophages in relation to the structural features of rhamnogalacturonan (RG) I-type polysaccharide (PGEP-I) purified from Panax ginseng leaves. For this investigation, we used several specific inhibitors and antibodies against mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and pattern recognition receptors (PRRs). Furthermore, we investigated the roles of component sugar chains on immunostimulating activity through a sequential enzymatic and chemical degradation steps. We found that PGEP-I effectively induced the phosphorylation of several MAPK- and NF-κB-related proteins, such as p38, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p65. Particularly, immunocytochemistry analysis confirmed the PGEP-I-induced translocation of p65 into the nucleus. Furthermore, the breakdown of PGEP-I side chains and main chain during sequential enzymatic and chemical degradation reduced the PGEP-I-induced macrophage cytokine secretion activity. IL-6, TNF-α, and NO secreted by macrophages are associated with several signaling pathway proteins such as ERK, JNK, and NF-κB and several PRRs such as dectin-1, CD11b, CD14, TLR2, TLR4, and SR. Thus, these findings suggest that PGEP-I exerts potent macrophage-activating effects, which can be attributed to its typical RG-I structure comprising arabinan, type II arabinogalactan, and rhamnose-galacturonic acid repeating units in the main chain.

Tumor inhibitory effect via immunostimulating activities of a rhamnogalacturonan-I-rich polysaccharide isolated from turmeric (Curcuma longa L.).

In this study, a turmeric polysaccharide (TP-0) was isolated through hot water extraction and ethanol precipitation to produce a novel active polysaccharide from turmeric other than curcuminoids. TP-0 was found to be primarily composed of eight different monosaccharides, such as galactose (15.9%), galacturonic acid (15.2%), arabinose (11.4%), and rhamnose (9.7%), which are typical rhamnogalacturonan (RG)-I sugars. When stimulated with TP-0, peritoneal macrophages secreted a variety of immunostimulatory cytokines. In addition, intravenous and oral administration of TP-0 significantly enhanced the natural killer (NK) cells and cytotoxic T lymphocyte (CTL)-mediated cytotoxicity against tumor cells. In an assay for lung cancer induced by Colon26-M3.1 carcinoma, prophylactic intravenous and oral administration of TP-0 effectively inhibited lung cancer. These findings reveal that TP-0, a typical RG-I-type polysaccharide that is isolated from turmeric, has potent anti-metastatic activities, and these activities are linked to various immunological factors such as macrophages, NK cells, and CTL. PRACTICAL APPLICATIONS: Many studies related with turmeric have only focused that a curcuminoid of turmeric has beneficial effects on human health system. Nevertheless, in this study, it was confirmed that polysaccharide isolated from turmeric showed potent anti-cancer effects via activities of various immunological factors such as macrophages, NK cells, and CTL. These results suggest the high potential for development value of turmeric as a new candidate for immunostimulating-related health functional food ingredients.

Immunostimulating and intracellular signaling pathways mechanism on macrophage of rhamnogalacturonan-I type polysaccharide purified from radish leaves.

In this study, the intracellular signaling pathways involved in macrophage activation through the RG-I-type polysaccharide (REP-I) purified from radish leaves were elucidated. The gene expression and secretion of immune-related factors such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nitrogen oxide (NO) from macrophages were enhanced by the addition of REP-I. Moreover, immunoblotting and immunocytochemistry analyses indicated that REP-I dose-dependently phosphorylated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways. An investigation using different inhibitors revealed that the effect of REP-I on NO secretion was mostly promoted by c-Jun N-terminal kinase (JNK) and NF-κB. Furthermore, the secretion of IL-6 was mostly induced via extracellular-signal-regulated kinase (ERK), JNK, and NF-κB. TNF-α secretion was mostly induced via NF-κB. In contrast, an investigation using anti-pattern recognition receptor (PRR) antibodies revealed that the effect of REP-I on the secretion of NO was mostly related with dectin-1, scavenger receptor (SR), toll-like receptor (TLR)2, TLR4, CD14, and CD11b. Furthermore, the secretion of IL-6 was mostly involved with SR, and the secretion of TNF-α was mostly relevance to TLR2. In conclusion, it is affirmed that immunostimulatory activation of macrophage of REP-I purified from radish leaves was deeply associated with several PRR and phosphorylating MAPK and NF-κB.

Clarification of the structural features of Rhamnogalacturonan-I type polysaccharide purified from radish leaves.

Determining the structure of REPI, an immunostimulatory polysaccharide fraction from radish leaves, is an important health objective. Herein, we show that REP-I contains nine different monosaccharides, including GalA (22.2%), Gal (32.6%), Ara (27.5%), and Rha (10.2%) as main sugars. REP-I was also reacted with ß-glucosyl Yariv reagent (29.8%), suggesting the presence of the arabino-ß-3,6-galactan. Furthermore, methylated-product analysis revealed that REP-I contains 13 different glycosyl linkages, including 4-linked GalpA (21.0%), 2,4-linked Rhap (7.0%), 4-linked Galp (5.8%), 5-linked Araf (10.1%), and 3,6-linked Galp (7.9%), which are characteristic of RG-I. Microstructural information was obtained by sequential degradation using four linkage-specific glycosylases and ß-elimination, with fragments analyzed on the basis of sugar composition, methylation, and MS/MS spectra. The results show that the immunostimulatory activity of REP-I is possibly due to the structure of RG-I, which is composed of a main chain with repeating [→2)-Rhap-(1 â†’ 4)-GalpA-(1→] linkage units and three side-chains: a branched α(1 â†’ 5)arabinan, a ß(1 â†’ 4)galactan, and arabino-ß-3,6-galactan, which are branched at the C(O)4 position of each Rha residue in the REP-I main chain.

Characterization, prebiotic and immune-enhancing activities of rhamnogalacturonan-I-rich polysaccharide fraction from molokhia leaves.

Plant-derived polysaccharides possess potential health benefits that improve intestinal health and the immune system. Molokhia leaves have a large amount of mucilage polysaccharide; in the present study, crude polysaccharide extract was prepared from molokhia leaves. The molecular weight of molokhia leaf polysaccharide fraction (MPF) was estimated to be 51.2 × 103 Da. Polysaccharide was methylated and the structure of MPF was mainly composed of rhamnogalacturonan-I structure with side chains, such as galactans and linear glucan (starch), as shown by GC-MS analysis. To study the biofunctional effects of MPF, its prebiotic and intestinal immune-enhancing activities were assayed in vitro. MPF exhibited good prebiotic activity, as shown by its high prebiotic scores, and increased contents of total short-chain fatty acids on five probiotic strains. In addition, MPF showed immune-enhancing activity on Peyer's patches, as revealed by the high bone marrow cell proliferating activity and production of immunoglobulin A and cytokines. These results demonstrate that MPF may be a potential beneficial prebiotic and intestinal immune-enhancer, which may have wide implications in the food industry.