Isolation, purity analysis and stability of hyperforin as a standard material from Hypericum perforatum L.

Abstract

In 1996 131.5 million daily doses of preparations containing extracts of Hypericum perforatum L. were prescribed in Germany for treating mild to moderately severe depressive disorders. New pharmacological and clinical results focus on hyperforin as the main active ingredient of the drug. Hyperforin (C35H52O4) is one of the main components (2-4%) of the dried herb Hypericum perforatum L. It was isolated after six consecutive steps: extraction of deep-frozen blossoms (-20 degrees C) with n-hexane by means of an Ultra Turrax at room temperature; separation of lipophilic substances on a silica gel column; purification of the relevant fraction by preparative HPLC; evaporation of the mobile phase under reduced pressure; removal of the remaining water by freeze-drying; and storage of hyperforin at -20 degrees C under nitrogen. The identity and purity of the isolated substance were determined by high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) with diode-array and ultraviolet detection (DAD and UV), Fourier-transformed infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and liquid chromatography coupled with positive-ion electrospray-ionization tandem mass spectrometry (LC-ESI(+)-MS-MS). By use of these methods the purity of hyperforin was shown to be >99.9%. Peroxides present at each step of the isolation were detected by titration and by means of Merckoquant analytical peroxide test-strips. Elimination of the peroxides and stabilization of hyperforin was achieved by consistent protection from oxidation-the mobile phases were protected by use of ascorbic acid; evaporation and freeze-drying were performed under nitrogen; and the mobile phase used for preparative HPLC was sparged with helium. Stability testing was performed by HPLC-the samples were stored at -30 degrees C in a normal atmosphere and at -20, 4, and 20 degrees C in a normal atmosphere or under nitrogen. Results were compared with those obtained after storage under liquid nitrogen (-196 degrees C). Because of its high sensitivity to oxidation, hyperforin was more stable under nitrogen under all test conditions. There was no statistically significant difference between results obtained after 8 months at -20 degrees C under nitrogen or at -30 degrees C under a normal atmosphere and those from the reference sample stored under liquid nitrogen (-196 degrees C). Despite this, because of the tendency of hyperforin to degrade, long-term storage at -70 degrees C under nitrogen is recommended.