4.3.1. Preparation of the Birch Bark and Isolation of Betulin from the Birch Bark

The bark was boiled in a 1% aqueous KOH solution for 2 h. The bark was then filtered on a Büchner funnel and washed with hot water. The bark was dried in a vacuum dryer at 80 ℃ for 2 days.

The isolation of betulin was carried out from the shredder birch bark of the Betula verrusoca tree. An amount of 100 g of dried bark was first extracted with hexane (500 mL) in a Soxhlet apparatus, for 24 h (2 × 12 h), then with methylene chloride (500 mL) for 24 h (2 × 12 h). After evaporation to dryness, the hexane extract yielded 3.25 g of dry extract, while the methylene chloride extract yielded 11.17 g of dry extract.

The 11.17 g of dry extract obtained was heated under a reflux condenser in methanolic KOH 2% solution (100 mL) for 10 h. The flask was allowed to stand overnight at room temperature. The precipitate was drained on a Büchner funnel and washed copiously with hot water. The obtained impure betulin (8.4 g) was dried in a vacuum oven at 80 °C overnight. The betulin was then purified on a chromatography column in the system hexane:ethyl acetate (H:OAc) 40:1→5:1 v/v, monitoring the extraction with TLC (H:OAc 5:1). Finally 7.23 g of pure betulin was obtained (1).