4
J.-L. SONG ET AL.
Plant, Shanghai, People’s Republic of China). Methanol used for HPLC was of HPLC-grade
(J&K, Beijing, People’s Republic of China). Silica gel (200–300 mesh) (Qingdao Haiyang
Chemical Plant, Qingdao, People’s Republic of China), C18 reversed-phase silica gel (150–200
mesh, Merck), MCI gel (CHP20P, 75–150 μM, Mitsubishi Chemical Industries Ltd.) and
Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography. TLC
spots were visualised under UV light and by dipping into 5% H2SO4 in alcohol followed by
heating. Purification by means of HPLC was conducted using Alltima C18 column
(250 mm × 10 mm, 5 μM, Tong Heng Innovation Technology Co., Ltd., Beijing, People’s
Republic of China).
3.2. Plant material
The stems of E. chinensis were collected in Nankang County, Jiangxi Province, China, in June
2013 and identified by prof. Fu-Wu Xing, South China Botanical Garden. A voucher specimen
(No. 201309) has been deposited at the Laboratory of Natural Product Chemistry Biology
(SCBG), South China Botanical Garden.
3.3. Extraction and isolation
Dried-powdered stems of E. chinensis (50.0 kg) were extracted by 95% ethanol at room tem-
perate and filtered, then the filtrate was concentrated under vacuum to give a crude residue
(2.1 kg). The residue (2.1 kg) was suspended in H2O and extracted with n-hexane and EtOAc.
The EtOAc-soluble fraction (1.2 kg) was subjected to column chromatography (CC) over silica
gel eluting with n-hexane/acetone in a stepwise manner to give six fractions (E1–E6). Fraction
E4 (237 g) was then subjected to CC over silica gel eluting with CH2Cl2/EtOAc in a stepwise
manner, affording four fractions (E4A–E4F). Fraction E4C was subjected to CC over MCI gel
(MeOH/H2O, 30–70%) to give five subfractions (E4C1–E4C5). Subfraction E4C2 was further
subjected to CC over silica gel eluting with CH2Cl2/EtOAc in a stepwise manner to yield 2
(70 mg). Fraction E5 (98.3 g) was subjected to CC over MCI gel (MeOH/H2O, 30–70%) to give
10 fractions (E5A–E5 J). E5H (2.6 g) was subjected to CC over Sephadex LH-20 (MeOH) to
afford five subfractions (E5H1–E5H5). E5H3 (78 mg) was subjected to CC over silica gel eluting
with CH2Cl2/EtOAc in a stepwise manner to yield 1 (12 mg). E5H4 (213 mg) was subjected
to CC over silica gel eluting with CH2Cl2/EtOAc in a stepwise manner to yield 4 (60 mg) and
yield 5 (93 mg). E5I (3.8 g) was subjected to CC over Sephadex LH-20 (MeOH) to afford six
subfractions (E5I1–E5I6). E5I3 (240 mg) was subjected to CC over silica gel eluting with
CH2Cl2/EtOAc in a stepwise manner to yield 3 (6 mg).
Ent-16α,17-[4-hydroxy-4-(2-hydroxyethyl)-cyclohexylidenedioxy]-(–)-19-oic acid (1): Amorphous
powder; [ꢀ]2D5−28.8 (c 1.70; CH OH); CD Δε (c 1.03, CH OH) −0.18 (224.3 nm); IR (KBr) ν
3
3
max
3409, 2929, 2853, 1699, 1449, 1371, 1263, 1106, 1030, 957 cm−1; HR-ESI-MS [M − H]− at m/z
475.3027 (Calcd for C28H44O6–H, 475. 3060); 1H NMR (500 MHz, CD3OD): δH 1.88 (1H, m, H-1a),
0.84 (1H, m, H-1b), 1.93 (1H, m, H-2a), 1.40 (1H, m, H-2b), 2.15 (1H, m, H-3a), 1.03 (1H, m,
H-3b), 1.08 (1H, m, H-5), 1.85 (2H, m, H-6), 1.56 (1H, overlapped, H-7a), 1.47 (1H, m, H-7b),
1.01 (1H, brs, H-9), 1.65 (1H, overlapped, H-11a), 1.40 (1H, overlapped, H-11b), 1.56
(2H, overlapped, H-12), 2.07 (1H, m, H-13), 2.01 (1H, overlapped, H-14a), 1.52 (1H, m, H-14b),
1.74 (1H, d, J = 4.2 Hz, H-15a), 1.79 (1H, d, J = 4.2 Hz, H-15b), 4.10 (1H, d, J = 8.6 Hz, H-17a),
3.96 (1H, d, J = 8.6 Hz, H-17b), 1.20 (3H, s, H-18), 0.99 (3H, s, H-20), 1.68 (2H, overlapped, H-2′),