Phytochemical Profile and Phototoxicity of Eleven Hypericum Species Extracts

The genus Hypericum (family Hypericaceae) contains 484 species, one of which, Hypericum perforatum, is largely used in medicine as antidepressant, as well as antinflammatory, and antimicrobial agent. Hypericin, together with the other naphthodianthrone derivatives named hypericins (the red pigments recognized as partially responsible for biological activities of this plant), has been identified largely in more than 300 Hypericum species (Skalkos et al., 2006). Particularly, hypericins are one of the most potent naturally occurring photodynamic agents; indeed, upon light irradiation, they very effectively induce apoptosis and/or necrosis of cancer cells (Karioti et al., 2010) and thus might be valued for the photodynamic therapy of cancer. The aim of this study was to evaluate the in vitro phototoxic activity and antioxidant efficacy of 11 different (wild or cultivated) Hypericum species (perforatum L., aegypticum L., androsaemum L., calycinum L., hircinum L., hirsutum L., montanum L., patulum Thunb., perfoliatum L., pubescens Boiss., tetrapterum Fr.) also in correlation with their phytochemical profiles. Plant samples were extracted with ethanol in dark conditions and analyzed by HPLC-DAD-MS in order to determine the content of the main polyphenols, phloroglucinols, and naphtodiantrones. The extracts were also evaluated for their photocytotoxicity using murine fibroblasts NIH/3T3 exposed to either white light (5.2 J/cm2) or dark conditions. The photocytotoxic effects of the Hypericum extracts were determined by means of the sulforhodamine B assay and compared to hypericin and pseudohypericin as positive control. Moreover, the antioxidant/radical scavenging capacity of the Hypericum ethanolic extracts was investigated by means of 3 redox-based assays (Folin-Ciocalteau assay, DPPH assay, and ORAC-FL assay). We found that H. perforatum and H. perfoliatum are the two species with the highest content of hypericin and its biosynthetic precursors, being significant amounts of these compounds present also in H. montanum, H. pubescens and H. tetrapterum. Five flavonoids (quercetin-3-O-glucoside, quercetina-3-O-galattoside, quercitrin, quercetin and biapigenin) due their presence in all samples are possible genus markers, while myricitrin (found only in H. perfoliatum) and quercetin-3-O-rutinoside (recovered only in H. calycinum and H. aegypticum) are possible species chemotaxonomic markers. Furthermore we demonstrated a relevant dose-dependent photocytotoxicity of Hypericum extracts under light exposure conditions and the activity was significantly stronger for the species with the higher amount of naphtodianthrones such as H. perfoliatum and H. tetrapterum. Finally, all Hypericum extracts exhibited a good antioxidant radical scavenger activity. Our study represents a further contribute to the knowledge of the chemical composition and biological activity of Hypericum species. Results show that, in addition to the well known H. perforatum, at least other three species (H. tetrapterum, H. pubescens and H. montanum) are potential sources of biologically active compounds, and at least other two (H. perfoliatum and H. tetrapterum), due their phototoxicity, may be good candidates for their application in photodynamic therapy.