A three-step culture system to increase the xanthone production and antifungal activity of Hypericum perforatum subsp. angustifolium in vitro roots

Hypericum perforatum is a well-known medicinal plant. Among all secondary metabolites produced by this species, xanthones are very interesting for their antifungal activity. In the present study, with the aim to improve xanthone production and antifungal activity of H. perforatum subsp. angustifolium (sin. Fröhlich) Borkh in vitro roots, a new methodology consisting of a three-step culture system, has been developed. Regenerated roots of H. perforatum were cultured in a three-step culture system: in the first step, to increase biomass, the roots were cultured in half-strength liquid Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) indole butyric acid (IBA) and 1.5% sucrose. In the second and third steps, to stimulate secondary metabolism, the roots were cultured with 1.1 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.215 mg L(-1) kinetin (KIN), and 0.186 mg L(-1) 1-naphthalenacetic acid (NAA). In the third step, some of the roots were treated with chitosan. Xanthone production increased 2.7 times following the three-step method. The mean minimal inhibitory concentration (MIC) values were of 36.9, 26.7, and 65 μg mL(-1), against Candida species, Cryptococcus neoformans and dermatophytes, respectively. A positive correlation between xanthone accumulation and antifungal activity has been shown.