Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures.

Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1-5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05-1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionization-mass spectrometry (ESI-MS) with electrospray in negative ion mode [M-H] . Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25-50 microg/g dry mass (DM)) and pseudohypericin (170-350 microg/g DM) in shoots. In callus cultures, BA (4.0-5.0 mg/l) did not changed hypericin contents (15-20 microg/g DM) but influenced pseudohypericin productions (120-180 microg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30-100 microg/g DM) and pseudohypericin (120-400 microg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.