Typha latifolia L. fruit polysaccharides induce the differentiation and stimulate the proliferation of human keratinocytes in vitro
Graphical abstract
Highlights
► Typha latifolia fruits were used for skin treatment. ► We isolated and characterized the water-soluble polysaccharides and tested their bioactivity on human skin cells. ► Five heterodisperse polysaccharide fractions were obtained differing in their monosaccharide composition. ► Effects on fibroblasts were not observed but keratinocyte proliferation was increased by three fractions while the fraction with high beta-D-glucose content induced the differentiation.
Introduction
Typha latifolia L., Typhaceae (bulrush, cattail or reed mace) is a commelinid monocotyledon. In Northern America the fruits (achene and perigone hairs) were used as wound dressing (Moermann, 1998) to absorb the ichors. In the Dr. Dukes Phytochemical and Ethnobotanical Database Typha latifolia is one plant pronounced to be traditionally used for wound healing (Duke, 2006). Medicinal use is also reported for roots and the sticky juice between young leaves (Carman, 2006). The plant is herbaceous, rhizomatous and perennial with long sword-like leaves from the bare stalk which terminates in a cylindrical inflorescence of female flowers immediately below the male flowers, erupting as fluff when matured. The habitat includes shallow water of lake and pond edges, marshes, and ditches at elevations lower than 2000 m. Phytochemical analysis of Typha latifolia was done so far on leaves, roots, rhizomes and pollen. Alkanolamides (Peru et al., 2004) and flavonol glucosides (Sick Woo et al., 1983) were found in leaves. Contents of carbohydrates and sugars were investigated in overwintering parts (Kausch et al., 1981, Biesboer, 1984) and the stem (Pollard, 1982). Carotinoid-like compounds and allelopathic sterols of the whole plants were also characterized (Dell Greca et al., 1990a, Dell Greca et al., 1990b) as the lipid fraction of Typha latifolia pollen (Caffrey et al., 1987).
Polysaccharides, tannins and glycosides are the most plausible phytochemicals that would be extracted in a wet wound atmosphere from plant derived wound dressings. A direct influence on the cell physiology of epidermal and connective tissue cell is possible since such effects have been described for other carbohydrates like alginates (Edwards et al., 2003), glycosaminoglycans (Kirker et al., 2002) and β-glucan (Delatte et al., 2001). Recent reports also indicate that such polysaccharide-mediated effects are strongly dependent on the structural features of the polymers. (Kirker et al., 2002, Ni et al., 2004).
Wound healing is a dynamic and complex regenerative process in which different cell species of the skin and the immune system working together in three distinct phases: the inflammatory phase, the reepitheliazation phase and the remodeling phase. During reepitheliazation keratinocyte proliferate at the wound edge, consequently differentiate to corneocytes and form a thin new epidermis. This process is followed by migration of fibroblasts to the wound edge subsequently proliferating and producing extracellular matrix. During the remodeling phase fibroblasts transform into myofibroblasts and deposit collagen (Werner and Grose, 2003). While each cell type has its own function their cross talk by growth factors and cytokines is essential for wound closure (Werner et al., 2007). The wound healing process is mostly investigated by in vivo models but for ethical and financial reasons these models are for screening usually changed to in vitro bioassays. The established in vitro cell culture based models cover the different wound healing processes like proliferation, metabolic activity and differentiation of human skin fibroblasts and keratinocytes.
The aim of this study was to isolate the polysaccharides of an aqueous extract of Typha latifolia fruit and to investigate their effect on human skin cells.
Section snippets
General
All chemicals used were of analytical quality and were purchased from Diagonal (Münster, Germany). Cell culture media supplements are from PAA (Coelbe, Germany) as not mentioned otherwise.
Isolation, fractionation and characterization of polysaccharide
Typha latifolia L. fruits were collected from a private pond in the north western part of Germany (Münster, Westfalen) in late autumn 2006. A reference specimen (TL561/D) is placed at the Institute of Pharmaceutical Biology and Phytochemistry in Münster. The fruits (215 g), including the trichomes were grinded
Polysaccharides from Typha latifolia fruits
Water-soluble polysaccharides were extracted from the fruits by aqueous extraction at room temperature, followed by ethanol precipitation of the raw polysaccharides (RPS) at a 1% yield and containing about 10% of carbohydrates, beside residual polyphenolic compounds and proteins. RPS was separated into five subfractions by anion exchange chromatography using a step gradient with increasing ionic strength of buffer. The main fraction TL3 was obtained at a buffer concentration of 0.25 mol/L, while
Discussion
The activity of the tested polysaccharides on human skin cells showed that the impure RPS reduced significantly cell proliferation and enzymatic activity pointing to a pro-apoptotic potential since the test for necrotic activity was negative. This effect is maybe reduced to the impurities, since it is known that some phenols are cytotoxic or pro-apoptotic and because of this effect was not shown in case of the AEC fractions (Ugartondo et al., 2007, Koppikar et al., 2010). Contrary to TL4, which
Conclusions
Regarding these results Typha latifolia fruit polysaccharides have even though less effects on reepitheliazation and remodeling, two phases of the wound healing process. In particular more information must be collected to find out the signal pathway responsible for the cells reactivity on these different polysaccharides and their structure.
Acknowledgements
The authors thank Cathleen Possemeyer for technical assistance and Dr. Lohse, Department of Paediatric Surgery, University of Münster for support with skin grafts. Financial support for the study was given by A. Hensel.
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