Bioactive xanthones from roots, hairy roots and cell suspension cultures of some Hypericum species and antifungal activity against Candida albicans

Members of the genus Hypericum L. are distributed worldwide but predominantly in the temperate zones of the Earth. Up to the present, almost 500 species of the genus were documented (Nürk et al., 2013, Mol Phylogenet Evol 66:1-16) but at least 40% of them have not been phytochemically characterized yet. Species of the genus Hypericum, a natural sources of bioactive compounds display several roles in multiple bioassays (reviewed by Wolfle et al. 2014, Planta Med 80:109-120). Among them, xanthones are attaining interest because of their powerful antioxidant, antibacterial, antitumor and antifungal activities (reviewed by Fotie & Bohle 2006, Anti-Infect Agents Med Chem 5: 15-31; El-Seedi et al., 2010, Curr Med Chem 17: 854–901). Xanthones are mostly accumulated in the roots. In vitro grown root cultures of Hypericum perforatum represent a suitable biotechnological tool for production of standardized extracts with high content of xanthones (Tocci et al., 2011, Appl Microbiol Biotechnol 91: 977-987; Tocci et al. 2012, Plant Physiol Biochem 57: 54-58). In addition to standard root cultures, Tusevski et al. (2013, Cent Eur J Biol 8: 1010-1022) determined significant quantities of xanthones in hairy roots of H. perforatum obtained by Agrobacterium rhizogenes transformation. The aim of this study was (i) to determine xanthone profile in root culture extracts of several Hypericum species not studied from this view point so far; (ii) to evaluate the capacity for xanthone production by H. tetrapterum Fries. and H. tomentosum L. hairy roots; (iii) to ascertain the effect of elicitation with chitosan and salicylic acid on xanthone content in cell suspension culture of H. perforatum L. and finally, (iv) to assess antifungal activity of these extracts against Candida albicans strain ATCC 10231. At least one of the following xanthones, 5-methoxy-2-deprenylrheediaxanthone; 1,3,6,7-tetrahydroxyxanthone; 1,3,5,6-tetrahydroxyxanthone; paxanthone; kielcorin and mangiferin, was present in methanolic extracts of the root cultures. The highest total xanthone content with five identified xanthones was determined in untransformed H. pulchrum and H. annulatum root cultures. The composition of xanthones in hairy roots was species-specific. While the hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, hairy root cultures and controls of H. tomentosum contained toxyloxanthone B; 1,3,6,7 and 1,3,5,6 tetrahydroxyxanthone. Two xantnones, cadensin G and paxanthone, were found in cell suspension cultures of H. perforatum. Their content increased about twofold after elicitation with salicylic acid. Among untrasformed roots, inhibition of C. albicans ATCC 10231 growth was observed by methanolic extracts of H. annulatum roots cultured in the dark with MIC50 of 128 μg ml-1. Regarding hairy roots extracts, H. tomentosum (clone A4 and clone 7 ATCC 15834) and H. tetrapterum (clone 2 ATCC15834 and clone 2 A4), showed anti- Candida activity with the MIC50 range from μg.ml-1 and from 64 to 128 μg.ml-1, respectively. Extracts obtained by chitosan-elicitated cells did not show antifungal activity against C. albicans