Extraction and isolation

One hundred and eight grams of powdered fruit of M. leucadendron was extracted with MeOH (3 × 400 mL, reflux, 2 h), and the MeOH solution was filtered and evaporated under reduced pressure to provide a MeOH extract (8.6 g). Following this, the MeOH extract was successively dissolved with ethyl acetate (EtOAc) and CHCl₃ (3 × 250 mL each) to yield an EtOAc-soluble fraction (3.1 g), a CHCl₃-soluble fraction (2.1 g), and a residue (2.2 g), respectively. The EtOAc-soluble fraction was rechromatographed by MPLC on a YMC silica gel column (4 cm × 30 cm, flow rate 20 mL/min) using a step gradient of CHCl₃ − n-hexane (5:5, 5:4, 5:3, 5:2, 5:1, each 1.5 L, subfraction collection was 100 mL to afford 32 subfractions). Subfractions 12–15 were collected and concentrated under reduced pressure purposely to result in 640 mg of the fraction. The fraction was subsequently purified on Sephadex LH-20 (30 cm × 3 cm, collecting the volume of 50 mL) to result in 310 mg ursolic acid (2). While fraction 16–30 was rich in carbohydrates.

CHCl₃-soluble fraction was separated on the same MPLC and eluted with CHCl₃-MeOH (5:1, 5:2, 5:3, 5:4, 5:5, each 1.5 L) solvent systems to afford 25 subfractions. Subfractions 9–16 were collected to obtain 980 mg dry fraction. The fraction was purified on the Sephadex LH-20 column with MeOH as a solvent to afford betulinic acid (568 mg) and a mixture of ursolic acid and betulinic acid (258 mg). Subfractions 1–8 had so low quantity to separate, while subfractions 17–25 was rich in fatty acids.

Betulinic acid (1) - ¹H NMR (CDCl₃, 400 MHz) δ4.7 (1H, dd, J = 10.2; 5.5, H-3) 3.0 (t, J = 10.2; 5.6, H-19), 0.82 (s, H-23), 0.91 (s, H-24), 0.81 (s, H-25), 0.95 (s, H-26), and 0.93 (s, H-27); ¹³C NMR (CDCl₃, 100 MHz) δ37.8 (C-1), 27.9 (C-2), 79.01 (C-3), 38.71 (C-4), 55.5 (C-5), 18.32 (C-6), 34.3 (C-7), 40.93 (C-8), 50.51 (C-9), 37.2 (C-10), 20.8 (C-11), 25.22 (C-12), 38.4 (C-13), 42.4 (C-14), 30.6 (C-15), 31.1 (C-16), 56.3 (C-17), 46.8 (C-18), 49.2 (C-19), 140.4 (C-20), 29.6 (C-21), 34.09 (C-22), 27.99 (C-23), 15.3 (C-24), 16.0 (C-25), 16.1 (C-26), 14.77 (C-27), 180.3 (C-28), 109.6 (C-29), and 19.4 (C-30).

Ursolic acid (1) - ¹H NMR (CDCl₃, 400 MHz) δ 3.43 (1H, br, H-3), 5.42 (1H, br, H-10), 2.52 (d, J = 10.2, H-18), 1.24 (s, H-23), 1.02 (s, H-24), 0.93 (s, H-25), 1.05 (s, H-26), 1.22 (s, H-27), 0.97 (s, H-29), and 0.99 (d, J = 6.1, H-30); ¹³C NMR (CDCl₃, 100 MHz) δ 38.4 (C-1), 28.1 (C-2), 78.1 (C-3), 38.4 (C-4), 55.8 (C-5), 18.8 (C-6), 33.6 (C-7), 40.0 (C-8), 48.3 (C-9), 37.4 (C-10), 23.6 (C-11), 125.6 (C-12), 139.7 (C-13), 42.5 (C-14), 28.7 (C-15), 24.9 (C-16), 48.0 (C-17), 53.7 (C-18), 39.5 (C-19), 39.1 (C-20), 31.1 (C-21), 37.3 (C-22), 28.8 (C-23), 15.7 (C-24), 16.6 (C-25), 17.4 (C-26), 23.9 (C-27), 180.0 (C-28), 17.5 (C-29), and 21.4 (C-30).