Plants of the genus Hypericum are widely known for their therapeutic properties. The most biologically active compounds of this genus are naphtodianthrones and phloroglucinols. Indirect desorption electrospray ionization mass spectrometry (DESI-MS) imaging allows visualization and localization of secondary metabolites in different plant tissues. This study is focused on localization of major secondary compounds in the leaves of 17 different in vitro cultured Hypericum species classified in 11 sections. Generally, all identified naphtodianthrones, protohypericin, hypericin, protopseudohypericin and pseudohypericin were co-localized in the dark glands of eight hypericin producing species at the site of their accumulation. The known phloroglucinols, hyperforin, adhyperforin, hyperfirin and some new phloroglucinols with m/z [M - H](-) 495 and 569 were localized in the translucent and pale cavities within the leaf in the majority of studied species. The comparison of different Hypericum species revealed an interspecific variation in the distribution of the dark and translucent glands corresponding with the localization of hypericins and phloroglucinols. Moreover, similarities in the localization and composition of the phloroglucinols were observed in the species belonging to the same section. Adding to various quantitative studies focused on the detection of secondary metabolites, this work using indirect DESI-MSI offers additional valuable information about localization of the above-mentioned compounds.

A plethora of plants belonging to the genus Hypericum have been investigated so far owing to the biological efficacies of pharmacologically important secondary metabolites produced by several Hypericum species. However, there is currently a dearth of information about the localization (accumulation) of these compounds in the plants in situ. In particular, the biosynthetic and ecological consequence of acclimatization of in vitro cultured Hypericum spp. to outdoor conditions is not fully known. Herein, we report an application of matrix-assisted laser desorption/ionization high-resolution mass spectrometry (MALDI-HRMS) to reveal the distribution of major naphthodianthrones hypericin, pseudohypericin, protohypericin, and their proposed precursor emodin as well as emodin anthrone, along with the phloroglucinol derivative hyperforin, the flavonoids quercetin, quercitrin, rutin and hyperoside (and/or isoquercitrin), and chlorogenic acid in Hypericum leaves. Plants encompassing seventeen Hypericum species classified into eleven sections, which were first cultured in vitro and later acclimatized to outdoor conditions, were studied. We focused both on the secretory (dark and translucent glands, other types of glands, and glandular-like structures) as well as the non-secretory leaf tissues. We comparatively analyzed and interpreted the occurrence and accumulation of our target compounds in different leaf tissues of the seventeen species to get an intra-sectional as well as inter-sectional perspective. The naphthodianthrones, along with emodin, were present in all species containing the dark glands. In selected species, hypericin and pseudohypericin accumulated not only in the dark glands, but also in translucent glands and non-secretory leaf tissues. Although hyperforin was localized mainly in translucent glands, it was present sporadically in the dark glands in selected species. The flavonoids quercetin, quercitrin, and hyperoside (and/or isoquercitrin) were distributed throughout the leaves. Rutin was present only within sections Hypericum, Adenosepalum, Ascyreia, and Psorophytum. Our study provides insights into the prospects and challenges of using in vitro cultured Hypericum plants, further adapted to field conditions, for commercial purposes.