university of kwazulu-natal faculty of science and agriculture school ...

Fraction 67-71 was purified with 20% ethyl acetate in dichloromethane on a 2 cm
diameter column collecting 20 ml fractions where fraction 25-42 contained the pure
compound III.
The dichloromethane, ethyl acetate and methanol extracts were chromatographed with
step gradients from hexane through to methanol but did not result in the isolation of any
compounds that could be identified.
Separation of the stem extract
The hexane extract (3.96g) was eluted with dichloromethane:hexane (1:4; 2:3; 3:2; 4:1;
1:0), ethyl acetate:dichloromethane (1:4; 2:3; 3:2; 4:1; 1:0) and ethyl acetate:methanol
(4:1; 1:1; 0:1). Two fractions of interest were obtained; fractions 13-16 and 17-23.
Fraction 13-16 was purified further on a 2 cm diameter column with 20%
dichloromethane in hexane collecting 20 ml fractions. Fraction 18-20 of this column
was further purified with 20% ethyl acetate in hexane as above where fraction 6
contained more stigmasterol (V).
Fractions 17-23 of the crude column was purified on a 3cm column with
dichloromethane:hexane (3:7 for fractions 1-20 and 7:3 for fractions 21-40), where
fractions 27-30 was purified with 30% ethyl acetate in hexane and then fraction 6 of this
column was purified with 20% ethyl acetate in hexane where fraction 10-11 resulted in
the pure compound II and fraction 12 contained lupeol (VI). The purifications were done
in 2cm columns, collecting 20 ml fractions. Lupeol was identified from its 1 H NMR
spectrum and in comparison with an authentic sample contained in the laboratory.
The dichloromethane extract (1.39g) was eluted with dichloromethane:hexane (2:3; 3:2;
4:1; 1:0), ethyl acetate:dichloromethane (1:4; 2:3; 3:2; 4:1; 1:0) and ethyl
acetate:methanol (4:1; 1:1; 0:1) on a 3 cm column collecting 100 ml fractions at each
stage. Fraction 53-60 was purified with ethyl acetate:dichloromethane (1:1) on a 3cm
column collecting 50 ml fractions, where fractions 4-6 yielded compound IV.
101