Fig 1.
Structure of betulin, lupeol and betulinic acid.
Fig 2.
Extraction and purification process.
Table 1.
Amount of betulin, betulinic acid, lupeol and other triterpenes in fractions.
Fig 3.
GC-MS chromatograms of four important fractions.
B0—original extract, A1—by-product enriched with betulinic acid, L2—by-product enriched with lupeol, B5—pure betulin; BA—betulinic acid; relative detector response (Y-axis) against retention time in minutes (X-axis).
Fig 4.
Images of crystals of betulin, fraction B5.
A—microphotograph of crystalline fraction; B—large crystals obtained by slow cooling; C and D—macro- and microphotographs of crystals after free evaporation of ethanol.
Fig 5.
Crystals of fraction B5 during heating.
Fig 6.
A—bright-field microscopy, B—fluorescence microscopy of outer bark stained by nile red.
Fig 7.
Antiproliferative/cytotoxic effect.
B0—original extract, B3—fraction 3 without betulinic acid and lupeol, B4—amorphous fraction of pure betulin, B5—crystalline fraction of pure betulin, B-S—pure betulin obtained from Sigma-Aldrich. The results are given as relative values to the untreated control in percent. Cells treated with topoisomerase II inhibitor doxorubicin at 1 and 2 μM were used as positive control for decreased cell survival. Bars indicate SD in three independent experiments. *Significantly different from control (P ≤ 0.001).