Isolation and Purification of Bioactive Compounds from Hypericum sampsonii

Leaves of four-month-old plants (10 g dry weight) were lyophilised and homogenised (M20 Universal mill; IKA, Staufen, Germany). Eight aliquots of the resulting powder were transferred to 50 mL conical reaction tubes and extracted three times with 50 mL dichloromethane. The combined extract was centrifuged, the supernatant filtrated through filter paper, and the solvent evaporated under N₂ gas. The residue (1 g) was fractionated using an Isolera One Flash Purification Chromatography system (Biotage, Uppsala, Sweden), equipped with a Chromabond Flash BT cartridge (SiOH, 40–63 µm, 40 g; Macherey-Nagel, Düren, Germany). Separation was stretched over 23 column volumes (CV), using n-hexane (A) and ethyl acetate (B) as mobile phase (gradient: 2 CV 0% B, 6 CV 0–10% B, 15 CV 10–60% B). A total of 30 fractions a 45 mL were collected, automatically controlled by detection of UV signals in the λ-all mode. After solvent evaporation under N₂ gas, the fractions were passed through a Chromabond HR-X SPE column (6 mL, 55–60 µm; Macherey-Nagel) to remove chlorophyll. Lipophilic target compounds were isolated by semi-preparative HPLC on a normal-phase EC NUCLEODUR column (100-5 silica, 4.6×250 mm, 5 µm; Macherey-Nagel) with a flow rate of 3 mL min⁻¹. The mobile phase consisting of n-hexane (A) and ethyl acetate (B) was used for isocratic elution at 3% B for 16 min. Separation of fractions 16 and 17 yielded pure nemosampsone 4 (7 mg), while fraction 18 yielded 7-epi-nemorosone 3 (6 mg), compound 4 (4 mg), and 7-epi-clusianone 5 (3 mg). More polar target compounds were purified on a reversed-phase EC NUCLEODUR column (π², 10 × 250 mm, 5 µm; Macherey-Nagel) at a flow rate of 3 mL min⁻¹ using 0.1% formic acid in water (A) and acetonitrile (B) as mobile phase (80–92% B from 0–15 min). Separation of fractions 23–28 resulted in the isolation of grandone 1 (5 mg) and kolanone 2 (2 mg). 7-epi-secohyperforin 6 (0.28 mg) was isolated on the above normal-phase HPLC system from fresh H. sampsonii roots used for RNA isolation.