A polysaccharide fraction from Achillea millefolium increases cytokine secretion and reduces activation of Akt, ERK and NF-κB in THP-1 monocytes
Introduction
Achillea millefolium, commonly known as yarrow, is native in most of Europe, North America and Asia where it grows in dry banks and fields. The medicinal properties of A. millefolium are recognized worldwide and the plant is included in the national Pharmacopoeias of several countries. It has been used to treat gastrointestinal disorders, loss of appetite, menstrual problems, skin inflammation, wounds and external bleeding (Applequist and Moerman, 2011, Nemeth and Bernath, 2008, Saeidnia et al., 2011).
Several studies have investigated various bioactivities of aqueous extracts of A. millefolium. The ones that have examined the effects of aqueous extracts on inflammation related activities have shown increased circulating levels of transforming growth factor-β and inhibition of the development of symptoms and complications in experimental autoimmune encephalitis in mice (Vazirinejad et al., 2014); inhibition of human neutrophil respiratory burst activity (Choudhary et al., 2007); inhibition of prostaglandin formation and platelet activating factor-induced exocytosis (Tunon, Olavsdotter, & Bohlin, 1995); hepatoprotective effects against lipopolysaccharide induced hepatitis (Yaeesh, Jamal, Khan, & Gilani, 2006); antimicrobial properties (Frey & Meyers, 2010) and in our previous study we showed reduced capacity of dendritic cells (DCs) to induce a Th17 response (Jonsdottir, Omarsdottir, Vikingsson, Hardardottir, & Freysdottir, 2011). In none of these studies were the effects linked to particular compounds in the aqueous extracts. Other bioactivities of A. millefolium have been associated with choline (spasmogenic effect on gastric antrum) (Borrelli et al., 2012), sesquiterpene lactones (reduction of the glycemia rate in rats) (Cavalcanti et al., 2006) or phenols (antioxidative effect) (Konyalioglu & Karamenderes, 2005). Studies examining the bioactivities of extracts from A. millefolium have mostly used the aerial part of the plant, with (Frey and Meyers, 2010, Jonsdottir et al., 2011, Tunon et al., 1995, Vazirinejad et al., 2014, Yaeesh et al., 2006) or without the flowers (Cavalcanti et al., 2006) or only the flowering tops (Borrelli et al., 2012, Choudhary et al., 2007, Konyalioglu and Karamenderes, 2005) or the leaves (Baggio et al., 2016) with no apparent association between the parts used and the bioactivities observed.
Polysaccharides are the most prominent constituents of water-soluble extracts from plants. Polysaccharides isolated from botanical sources have attracted a great deal of attention in the biomedical arena because of their broad spectrum of therapeutic properties and relatively low toxicity (Schepetkin & Quinn, 2006). In studies investigating the immunomodulatory effects of fungal, bacterial and plant polysaccharides, the predominant finding is that they have immunostimulatory effects (Giavasis, 2014, Schepetkin and Quinn, 2006, Wack and Gallorini, 2008, Wong et al., 1994).
The major non-starch polysaccharides in plants are cellulose, microfibrils, hemicelluloses and pectins. Pectins are acidic plant cell wall heteropolysaccharides, which have been shown to possess immunomodulatory effects (Popov & Ovodov, 2013). Pectins are structurally characterized by their linear 1,4 linked galacturonan region and rhamnogalacturonan (RG) region (Waldron & Faulds, 2007). The ramified RGs are further classified based on their side chains. On one hand the side chains of RG-I are mainly composed of arabinogalactans, arabinan and galactan. On the other hand RG-II have low molecular weight and are mainly composed of homogalacturonanan and contain unusual monosaccharide residues (Perez, Rodriguez-Carvajal, & Doco, 2003). The majority of pectins contain RG-I although their detailed structural characteristic differs (Popov & Ovodov, 2013).
The aqueous extract from A. millefolium that we used in our previous study most likely contained polysaccharides, but the effect of the extract was not immunostimulatory but modulated the immune function of DCs. Therefore, the aim of this study was to use bioguided fractionation to isolate the active polysaccharide fraction from the aqueous extract of A. millefolium, to test its immunomodulatory effects on THP-1 monocytes and determine how it affects intracellular signaling in the cells.
Section snippets
Isolation of Am-25-d
A. millefolium was collected in Iceland in 2008, dried at room temperature and stored in a dry, dark place until used (Jonsdottir et al., 2011). Aqueous extract was prepared by grinding the whole, dried plant and shaking 300 g of the plant material in 4.5 l of 85 °C distilled water for 2 h, followed by centrifugation, dialyzation of the supernatant for 4 days (cut-off MW 6–8 kDa) and lyophilization. The lyophilized extract (300 mg) was dissolved in 50 ml of water and added to a 2.6 × 65 cm DEAE Sepharose
Characterization of the polysaccharide fraction Am-25-d
The polysaccharide fraction obtained from an aqueous extract of A. millefolium using anion exchange chromatography, dialysis and desalting was named Am-25-d. It showed a homogeneity and apparent molecular weight of 270 kDa. The monosaccharide composition analysis indicated that Am-25-d contained GalA, Gal, Ara, Xyl, Rha in molar ratio of 28:26:23:9:7 (Fig. S1, Table S1). The sugar content of the polysaccharide fraction was 94% and the protein content 2.1%.
Am-25-d did not affect viability of the THP-1 cells and had little effect on their appearance
Am-25-d (10, 25, 50 and 100 μg/ml), when
Discussion
We have isolated a bioactive polysaccharide fraction from the aqueous extract of A. millefolium and shown it to have a molecular weight around 270 kDa and a relatively high molar ratio of GalA, Gal and Ara. The monosaccharide analysis of the polysaccharide fraction Am-25-d is similar to the monosaccharide content of pectins (Ho et al., 2016, Popov and Ovodov, 2013). The content of galacturonic acid residues is, however, lower (28%) than usually seen in plant pectins. The molar ratio of Gal and
Conflict of interest statement
The authors have no conflict of interest.
Acknowledgements
The authors would like to thank Mr. Maonian Xu for performing the monosaccharide analysis and Ms. Hildur Sigurgrimsdottir for technical assistance. This work was supported by the Icelandic Research Fund (110418021), the University of Iceland Research Fund and the Landspitali University Hospital Research Fund. We thank Gudrun Tryggvadottir for providing the image of Yarrow used in the graphical abstract.
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These authors contributed equally to this work.