Silica gel 60 aluminium sheets

The extraction procedure described in Nyaba et al. [16 (link)] was followed. Briefly, the powdered bark of W. salutaris (1 kg) was extracted with dichloromethane (DCM) (1:5 w/v) for three days successively. Whatman (No.1) filter paper was used to filter the crude extract, followed by sample concentration under a reduced pressure (45 °C) to a minimum volume using a Heidolph rotary evaporator (Heidolph Instruments GmbH & CO. KG, Schwabach, Germany). This crude extract was allowed to dry in the fume hood. A portion of the DCM extract was subjected to silica gel column chromatography (60 × 1000 mm; Merck silica gel, 60:0.063–0.200 mm). The eluent system used was hexane: ethyl acetate (8:2), which yielded 35 fractions. The fractions were monitored by Thin Layer Chromatography (silica gel 60 aluminium sheets, F254—Merck, Whitehouse Station, NJ, USA), profiling the fractions into seven combined fractions. The TLC spots were fixed with a solution made of 20% H₂SO₄ in methanol, heated for colour development, and visualized under UV light (254 nm). The fractions were left overnight to until desiccation was achieved. A white powder (NN-01) was obtained from the combined fractions 2–4.

The fine stem bark powder (1 kg) was subjected to maceration with dichloromethane (1:5 w/v) for 3 days with periodic shaking. The maceration extract was filtered through Whatman (No. 1) filter paper and concentrated under reduced pressure at 45 °C. Silica gel column chromatography (60 × 1000 mm; Merck silica gel, 60: 0.063–0.200 mm) was used to isolate the compound from the DCM extract. The column was eluted with hexane: ethyl acetate gradient and the ratio 8:2 yielded 35 fractions. The fractions were grouped according to the thin layer chromatography (silica gel 60 aluminium sheets, F 254—Merck, Whitehouse Station, New Jersey, USA) profiles, into seven combined fractions (Cfrs). The spots were fixed on the TLC plates with a 20% H₂SO₄ in methanol mixture and viewed under UV light (254 nm). The combined fractions were then dried under a fume-hood overnight; Cfrs 2–4 produced a white amorphous powder (NN-01). NN-01 was recrystallized from methanol to give white needles (mpt. 95–98 °C); [α]_D = − 11(0.1M in CHCl₃); HREI-MS: MF = C₁₇H₂₄O₅, [M+Na]⁺ ion at m/z 331.1619 (calcd: 331.1521).