MALDI-HRMS Imaging Maps the Localization of Skyrin, the Precursor of Hypericin, and Pathway Intermediates in Leaves of Hypericum Species

Hypericum perforatum and related species (Hypericaceae) are a reservoir of pharmacologically important secondary metabolites, including the well-known naphthodianthrone hypericin. However, the exact biosynthetic steps in the hypericin biosynthetic pathway, vis-à-vis the essential precursors and their localization in plants, remain unestablished. Recently, we proposed a novel biosynthetic pathway of hypericin, not through emodin and emodin anthrone, but skyrin. However, the localization of skyrin and its precursors in Hypericum plants, as well as the correlation between their spatial distribution with the hypericin pathway intermediates and the produced naphthodianthrones, are not known. Herein, we report the spatial distribution of skyrin and its precursors in leaves of five in vitro cultivated Hypericum plant species concomitant to hypericin, its analogs, as well as its previously proposed precursors emodin and emodin anthrone, using MALDI-HRMS imaging. Firstly, we employed HPLC-HRMS to confirm the presence of skyrin in all analyzed species, namely H. humifusum, H. bupleuroides, H. annulatum, H. tetrapterum, and H. rumeliacum. Thereafter, MALDI-HRMS imaging of the skyrin-containing leaves revealed a species-specific distribution and localization pattern of skyrin. Skyrin is localized in the dark glands in H. humifusum and H. tetrapterum leaves together with hypericin but remains scattered throughout the leaves in H. annulatum, H. bupleuroides, and H. rumeliacum. The distribution and localization of related compounds were also mapped and are discussed concomitant to the incidence of skyrin. Taken together, our study establishes and correlates for the first time, the high spatial distribution of skyrin and its precursors, as well as of hypericin, its analogs, and previously proposed precursors emodin and emodin anthrone in the leaves of Hypericum plants

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

Xanthones from roots, hairy roots and cell suspension cultures of selected Hypericum species and their antifungal activity against Candida albicans

Abstract Key message Highest xanthone contents were found in Hypericum pulchrum and H. annulatum untransformed roots. The best anti- Candida activity was obtained for hairy roots extracts of H. tetrapterum clone 2 ATCC 15834. Abstract Extracts of root cultures, hairy roots and cell suspensions of selected Hypericum spp. were screened for the presence of xanthones and tested for their antifungal activity 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 or mangiferin was identified in methanolic extracts of the untransformed root cultures. The highest total xanthone content, with five xanthones, was found in untransformed H. pulchrum and H. annulatum root cultures. Hairy roots and the controls of H. tetrapterum contained 1,7-dihydroxyxanthone, while hairy root cultures and the corresponding controls of H. tomentosum contained toxyloxanthone B, 1,3,6,7- and 1,3,5,6-tetrahydroxyxanthone. Two xanthones, cadensin G and paxanthone, were identified in cell suspension cultures of H. perforatum. Their content increased about two-fold following elicitation with salicylic acid. The anti-Candida activity of the obtained extracts ranged from MIC 64 to >256 µg ml(-1). Among the extracts of Hypericum untransformed roots, the best antifungal activity was obtained for extracts of H. annulatum grown under CD conditions. Extracts of hairy roots clones A4 and 7 ATCC15834 of H. tomentosum and clone 2 ATCC15834 of H. tetrapterum displayed inhibition of 90 % of Candida growth with 256 μg ml(-1). Extracts from chitosan-elicitated cells did not show antifungal activity