Drosera rotundifolia and Drosera tokaiensis suppress the activation of HMC-1 human mast cells

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Abstract

Ethnopharmacological relevance

Several Northern Hemisphere Drosera species have been used in the therapy of respiratory tract infections as the traditional medicine Droserae Herba.

Aim of the study

To determine the anti-inflammatory effects of Drosera species and to investigate a substitute material for Droserae Herba, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 human mast cells.

Materials and methods

Drosera rotundifolia, Drosera spatulata and Drosera tokaiensis were collected in Japan. Herbs were extracted with 80% EtOH, and subsequently applied to OASIS HLB column. HMC-1 cells were treated with each Drosera column-adsorbed fraction for 15 min, and subsequently added to aTc-m and incubated for 16 h. Inflammatory gene and protein expressions were determined by DNA microarray, RT-PCR and Western blotting.

Results

Drosera rotundifolia and Drosera tokaiensis fractions, but not the Drosera spatulata fraction, suppressed inflammatory gene expression induced by aTc-m in HMC-1 cells.

Conclusions

Drosera rotundifolia and Drosera tokaiensis suppressed activation of HMC-1 cells induced by aTc-m. Since the Drosera tokaiensis fraction was more effective than the traditionally used Drosera rotundifolia, Drosera tokaiensis is a likely substitute as a source of Droserae Herba.

Introduction

Nearly 150 species of the carnivorous genus Drosera (Droseraceae) are distributed mainly in Australia, Africa and South America, with some Northern Hemisphere species (Rivadavia et al., 2003). Several Northern Hemisphere species, including Drosera rotundifolia L., Drosera intermedia Hayne and Drosera anglica Huds., have been used as traditional medicine in the therapy of respiratory tract infections. Droserae Herba, which is comprised chiefly of Drosera rotundifolia, has been commonly used in the treatment of convulsive or whooping cough since the 17th century (Paper et al., 2005). Faced with imminent extinction, some European species are now protected by law, and African or Asian Drosera species (e.g. Drosera madagascariensis DC. and Drosera peltata Smith) have recently been used as substitutes for medicinal purposes (Krenn et al., 1995). Drosera tokaiensis (Komiya and C. Shibata) T. Nakamura and Ueda, which is identified in Japan, was suggested to be an amphidiploid between Drosera rotundifolia and Drosera spatulata Labill. Using molecular cluster analysis, we recently indicated that Drosera tokaiensis was an allopolyploid (2n = 60, hexaploid) with hybrid origin between Drosera rotundifolia (2n = 20, diploid) as paternal ancestor and Drosera spatulata (2n = 40, tetraploid) as maternal ancestor (Hoshi et al., 2008). Drosera tokaiensis, which is closely related to Drosera rotundifolia at the genome level, is a promising substitute source for the herbal medicine used in the treatment of respiratory tract infections.

Drosera species contain physiologically active compounds such as flavonoids, ellagic acid and naphthoquinones (Budzianowski, 1996, Krenn et al., 1998, Paper et al., 2005). Flavonoids and naphthoquinones have been reported to possess antimicrobial and anti-inflammatory properties, which are efficacious in the treatment of oral infectious diseases. Some anti-inflammatory agents counteract the effect of neutrophil elastase. The 70% ethanol extract of Drosera madagascariensis and the flavonoids quercetin, hyperoside and isoquercitrin inhibited the human neutrophil elastase (Melzig et al., 2001). Droserae Herba generally comprises dried aerial parts of Drosera plants and is usually applied as aqueous or ethanol extract in therapy. Both aqueous and ethanol extracts of Drosera rotundifolia inhibited the human neutrophil elastase and induced antispasmodic effect in guinea-pig ileum (Krenn et al., 2004). Similarly, the extract of Drosera peltata and the naphthoquinone, plumbagin, exhibited antimicrobial activity against oral bacteria (Didry et al., 1998). Thus the aqueous or ethanol extracts and the constituents of Drosea species have been shown to possess the anti-inflammatory properties. In this study, we examined the anti-inflammatory effect of 80% ethanol extracts of Drorea species because the flavonoids and other physiologically active polyphenols are efficiently extracted with 70–80% ethanol.

Mast cells play an important role in allergic and inflammatory reactions. Mast cell-mediated allergic inflammation is involved in many diseases, including asthma, consequently releasing proinflammatory cytokine, proteases, histamine and heparin (Brill et al., 2004, Min et al., 2007). Mast cells reside in close physical proximity with T cells in inflamed allergic tissues and at the site of parasitic infections (Bhattacharyya et al., 1998). Degranulation of HMC-1 human mast cells was shown to be induced by the proinflammatory cytokine TNF, produced by activated T cells or T cell membranes (Bhattacharyya et al., 1998).

To determine the anti-inflammatory effect of Drosera species, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 cells. Flavonoid contents of the three species of Drosera were also determined, since it has been suggested that flavonoids are the active anti-inflammatory components.

Section snippets

Materials

Seeds of Drosera rotundifolia were collected in Hiroshima, Japan. Seeds of Drosera spatulata and Drosera tokaiensis were collected in Shizuoka, Japan. Plant species were morphologically and cytologically identified. Surface of the seeds was sterilized with 1% benzalkonium chlorite solution for 5 min and with 1% sodium hypochlorite solution for 5 min. The seeds were then rinsed with sterilized water and aseptically germinated on half-strength Murashige-Skoog (MS) basal medium (Murashige and Skoog,

Results

Drosera fractions suppressed the morphological change of HMC-1 human mast cells activated by PMA activated T cell membrane (aTc-m).

PMA activated T cell membrane (aTc-m) induced the expression of inflammatory cytokines and chemokines, and the adhering and spreading in HMC-1 cells after 16 h incubation (Nagai et al., submitted for publication). Drosera OASIS HLB column-absorbed fractions (Drosera fractions) were added to the medium of HMC-1 cells and then activated by aTc-m for 16 h. Activated

Discussion and conclusions

Suppression of aTc-m-induced morphological changes in HMC-1 cells suggests that Drosera fractions suppress the aTc-m-induced activation of HMC-1 cells. To elucidate the suppressive effect of Drosera species on aTc-m-induced activation of HMC-1 cells, inflammatory gene expression in aTc-m activated HMC-1 cells, treated with Drosera fractions, was determined using a DNA microarray bearing genes related to allergic inflammation. Activated T cell membrane induced the expression of 35 genes >3-fold

Acknowledgements

We wish to thank Professor J.H. Butterfield for supplying HMC-1 cells and for technical advice. We also thank Yokogawa Electric Co. for the loan of a DNA microarray reader and technical advice. This work was financially supported in part by grants from the Ministry of Agriculture, Forestry and Fisheries.

References (21)

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