Extraction and isolation
Big, young leaves were powdered (165.5 g) and extracted with hot MeOH five times. The solvent was
concentrated under re- duced pressure to give a crude extract (53.5 g), a part of which was
dissolved in 10 % aqueous acetic acid. The insoluble material was removed by filtration through
Celite to give the AcOH-inso- luble fraction solution (AcOH-insoluble fraction, 50.3 g). The
aqueous layer was basified with Na2CO3 at 0 °C and extracted with CHCl3. The organic layer was
washed with water, dried
over MgSO4, and then evaporated to give the crude base fraction (2.43 g). The aqueous layer was
further extracted with n-BuOH, which was concentrated under reduced pressure to yield the n- BuOH
fraction (4.77 g). A part of the residual aqueous solution
(10 mL) was lyophilized to give a hygroscopic solid (ca. 2g), which was isolated with ethanol
using a Soxhlet extractor in or- der to remove the inorganic materials. The ethanol extract was
evaporated to give a residue containing the water-soluble organ- ic materials (water-soluble
fraction, 1.08 g). The inhibitory activ- ity of each fraction (at 100 !lg/mL) on
electrically-induced twitch contraction in guinea-pig ileum was as follows: 37.0 ± 5.2% (crude
extract); 7.3 ± 4.5 % (AcOH-insoluble fraction); 72.5 ±
8.1 % (crude base fraction); -5.7 ± 3.7 % (n-BuOH fraction); -3.8
± 4.1 % (water-soluble fraction).
The crude base fraction (2.0 g), which exhibited an opioid agonis- tic effect in the guinea-pig
ileum, was purified by SiO2 column chromatography (6 x 17 cm) using CHCl3/AcOEt (9 : 1, 370 mL;
fraction A), CHCl3/AcOEt (4 : 1, 240 mL; fraction B), CHCl3/AcOEt
(1 : 1, 320 mL; fraction C), AcOEt (80 mL; fraction D), MeOH/
AcOEt (1 : 19, 120 mL; fraction E), MeOH/AcOEt (1 : 4, 160 mL; fraction F), MeOH/AcOEt (1 : 1, 80
mL; fraction G), and MeOH (150 mL; fraction H). The combined fractions C and D were fur- ther
purified by SiO2 column chromatography (3 x 17 cm) using
an n-hexane/AcOEt (3 : 2, 1 : 1, 1 : 5, 30 mL each) gradient that af-
forded 24 fractions. Fractions 2 - 8 contained mitragynine
{1343 mg, 66 % based on the crude base, [a]D : -126° (c 1.2, CHCl3)} and fractions 18 - 22
yielded 7-hydroxymitragynine
{40 mg, 2% based on the crude base, [a]D : + 47.9° (c 0.55, CHCl3)}. From fraction E,
paynantheine {178 mg, 8.9 % based on the crude base, [a]D : + 29.4° (c 1.2, CHCl3)} was obtained.
Frac- tion F afforded speciogynine {132mg, 6.6 % based on the crude
base, [a]D : + 26.8° (c 0.85, CHCl3)}. Fraction G was subjected to MPLC (SiO2, 2.5 x 10 cm) with
MeOH/CHCl3 (1 : 9, 3 mL/min) to provide speciociliatine {tR: 18 min, 15 mg, 0.8 % based on the
crude base, [a]D : -10.5° (c 1.2, CHCl3)}. The isolated compounds were identified by direct
comparison with the corresponding au-
thentic samples. The purity (> 99 %) of the above compounds was
checked by HPLC and 1H-NMR (500 MHz) analyses.
