Isolation and Purification of Bioactive Compounds from Coelogyne fuscescens

Dried whole plants of C. fuscescens Lindl. var.brunnea (2.3 kg) were macerated with ethanol (EtOH) (5 × 10 L). The
ethanolic extract (530 g) was mixed with water and subjected to partitioning
with ethyl acetate (EtOAc). This process yielded an ethyl acetate
extract (99.2 g) and an aqueous extract (428.2 g).
The ethyl
acetate extract was then separated by vacuum liquid chromatography
(silica gel, EtOAc-hexane, gradient) to yield nine fractions (F1–F9).
F4 (7.8 g) was subjected to further fractionation on a silica gel
column (EtOAc-hexane, gradient) to yield compound 5 (50
mg). Fraction F6 (5 g) was separated on a silica gel column (EtOAc-hexane,
gradient), resulting in three fractions (F6A–F6C), and fraction
F6B (1 g) was then subjected to CC using an EtOAc-hexane gradient
to yield three fractions (F6BA–F6BC). F6BA (150 mg) was separated
again on CC (silica gel, EtOAc-hexane, gradient) to give compounds 6 (7.8 mg) and 7 (2.8 mg). F6BB (250 mg) was
separated by CC (silica gel, EtOAc-hexane, and gradient) to produce
compounds 8 (29.3 mg) and 9 (14 mg). F6BC
(500 mg) was subjected to CC (silica gel, EtOAc-hexane, and gradient)
to produce compounds 10 (37.1 mg) and 11 (120.9 mg). Fraction F7 (35 g) was fractionated on a silica gel
column (EtOAc-hexane, gradient), and four fractions (F7A–F7D)
were obtained. Fraction F7D (155 mg) was purified again by Sephadex
LH-20 (acetone) to give three fractions (F7D1–F7D3). Fraction
F7D3 (34 mg) was then separated on silica gel (EtOH-toluene, 0.5:9.5)
to furnish compound 12 (14 mg). Fraction F8 (5 g) was
then separated by CC on silica gel with a gradient of EtOH–dichloromethane
to yield four fractions (F8A–F8D). F8B (800 mg) was separated
again by Sephadex LH-20 (acetone) to yield three fractions (F8B1–F8B3).
F8B2 (300 mg) was further separated on a silica gel column (EtOH-dichloromethane,1:9),
and three fractions (F8B2A–F8B2C) were obtained. F8B2A (50
mg) was separated again by HPLC using the reverse phase (MeOH–H₂O, 1:1) to yield compound 1 (2.3 mg). F8C (450
mg) was isolated again by Sephadex LH-20 (methanol) to give four fractions
(F8C1–F8C4). F8C2 (100 mg) is separated again with a silica
gel column (EtOH-dichloromethane,1:9) to yield four fractions (F8C2A–F8C2D).
Compound 2 (1.9 mg) was obtained from the fraction F8C2C
(30 mg) by Sephadex LH-20 (acetone). F8C3 (100 mg) was separated on
CC (silica gel, EtOH-dichloromethane,1:9) to produce four fractions
(F8C3A–F8C3D). F8C3B (50 mg) was separated by Sephadex LH-20
(MeOH), and compound 4 (17.7 mg) was obtained. Separation
of fraction F8D (899 mg) was done by reverse-phase column chromatography
(RP-18), with gradient elution of MeOH (0–100%) in H₂O to give four fractions (F8D1–F8D4). F8D2 was separated again
by Sephadex LH-20 (MeOH) to produce three fractions (F8D2A–F8D2C).
F8D2B (8.5 mg) was separated by HPLC using reverse-phase (MeOH–H₂O, 1:1) and normal-phase preparative thin-layer chromatography
using mobile phase (EtOH-dichloromethane, 2:8) to furnish compound 13 (3.5 mg). F8D4 (20.7 mg) was separated by CC (silica gel,
EtOH-dichloromethane, and gradient) to produce three fractions (F8D4A–F8D4C).
Compound 14 (3.5 mg) was obtained by purification of
F8D4B (6 mg) with Sephadex LH-20 (MeOH).
The aqueous extract
(428.2 mg) was separated with Diaion HP-20
(MeOH–H₂O, gradient) to give five fractions (AA–AE).
Fraction AB (2 g) is separated again on a silica gel column (EtOH-dichloromethane,
3:7), and five fractions (ABA–ABE) were obtained. Compound 15 (68.4 mg) was produced by the purification of the ABE fraction
(200 mg) with Sephadex LH-20 (MeOH). Fraction AC (1 g) was purified
again by Sephadex LH-20 (MeOH) to give four fractions (ACA–ACD).
ACB (200 mg) was separated by CC (silica gel, EtOH-dichloromethane,
3:7) to produce three fractions (ACB1–ACB3). ACB2 (36.8 mg)
was subjected to semipreparative HPLC (silica gel) using the mobile
phase (MeOH-dichloromethane, 1:9), and compound 3 (4.3
mg) was furnished.