3.3. Extraction and Isolation
The materials were totally dried in air under dark. Dried leaves of E. oldhamii Maxim. (10.0 kg) were cut into small pieces and soaked in 70 L of methanol for 7 days. The extraction was repeated three times. The extracts were combined and filtered through filter paper and concentrated under reduced pressure to give a dried extract (801.0 g). The dried extract was suspended in H2O (2 L) and extracted with ethyl acetate (2 L, five times). The resulting ethyl acetate extract was concentrated to yield 302.5 g of a brown-green thick oil. The fraction was further purified on 2.2 kg of silica gel with particle size 0.063–0.200 mm on a column with 10 cm internal diameter and 50 cm length using a gradient of increasing polarity from total n-hexane to total ethyl acetate as mobile phase and was separated into 20 fractions. Fraction 1 (0.52 g) was isolated from the 100% n-hexane mobile phase. Fraction 2 (12.28 g) was isolated from the 1:19 n-hexane/ethyl acetate mobile phase. Fraction 3 (18.95 g) was isolated from the 1:9 n-hexane/ethyl acetate mobile phase. Fractions 4 (10.28 g), 5 (11.41 g), 6 (10.55 g), 7 (15.20 g) and 8 (8.85 g) were isolated from the 8:2 n-hexane/ethyl acetate mobile phase. Fractions 9 (16.69 g) and 10 (13.44 g) were isolated from the 7:3 n-hexane/ethyl acetate mobile phase. Fractions 11 (19.18 g), 12 (11.74 g), 13 (7.84 g) and 14 (11.40 g) were isolated from the 5:5 n-hexane/ethyl acetate mobile phase. Fractions 15 (6.02 g), 16 (14.38 g) and 17 (6.01 g) were isolated from the 3:7 n-hexane/ethyl acetate mobile phase. Fractions 18 (19.91 g), 19 (12.57 g) and 20 (6.13 g) were isolated from the 100% ethyl acetate mobile phase. The ethyl acetate extract remaining on the column was eluted by methanol (28.92 g). The weight of the 20 fractions and the fraction which was eluted by methanol was 261.94 g, so the recovery rate was 86.59%.
Twenty five compounds were isolated in fractions 8, 14 and 15 respectively. Compounds 1 (645.0 mg), 2 (32.1 mg), 3 (22.5 mg), 5 (655.2 mg), 6 (52.0 mg), 7 (15.3 mg), 9 (10.2 mg), 10 (8.5 mg), 11 (11.5 mg), 12 (14.0 mg) and 13 (12.8 mg) were isolated from fraction 8. Compounds 4 (8.8 mg), 8 (13.7 mg), 14 (22.6 mg), 15 (2.7 mg), 16 (11.7 mg), 19 (11.8 mg), 20 (22.0 mg), 21 (45.2 mg), 22 (8.2 mg), 23 (5.8 mg), 24 (14.0 mg) and 25 (8.3 mg) were isolated from fraction 14. Compounds 17 (32.8 mg), and 18 (10.3 mg) were isolated from fraction 15. The isolation procedure of these 25 compounds was described below.
Fraction 8 (8.85 g) was washed with n-hexane and acetone to obtain a mixture of compounds 1 and 5 (total weight equivalent to 1,306.5 mg). These two compounds were separated by semi-preparative RP-HPLC (acetonitrile/0.1% ammonium acetate solution = 9:1, v/v) to afford pure compounds 1 and 5. The remaining fraction 8 (8.14 g) was separated by silica gel column chromatography (n-hexane/ethyl acetate = 8:2, v/v) to give four subfractions (subfraction 8–1 to 8–4). Compounds 2, 3, 6 and 7 were obtained from subfraction 8–1 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v). Compounds 9 and 10 were obtained from subfraction 8–2 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v) and semi-preparative RP-HPLC (acetonitrile/water = 19:1, v/v). Compound 11 was obtained from subfraction 8–3 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v). Compounds 12 and 13 were obtained from subfraction 8–4 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v).
Fraction 14 (11.40 g) was separated by Sephadex LH 20 column chromatography (chloroform/methanol = 3:7, v/v) to give six subfractions (subfraction 14–1 to 14–6). Compounds 4, 8, 23 and 25 were obtained from subfraction 14–2 through semi-preparative NP-HPLC (n-hexane/acetone = 1:1, v/v) and semi-preparative RP-HPLC (acetonitrile/water = 8:2, v/v). Compound 19 was obtained from subfraction 14–2 through semi-preparative NP-HPLC (n-hexane/acetone = 1:1, v/v) and NP-HPLC (n-hexane/ethyl acetate = 1:1, v/v). Compound 22 was obtained from subfraction 14–3 through semi-preparative NP-HPLC (n-hexane/acetone = 1:1, v/v). Compound 24 was obtained from subfraction 14–3 through semi-preparative NP-HPLC (n-hexane/acetone = 1:1, v/v) and semi-preparative RP-HPLC (n-hexane/ethyl acetate = 1:1, v/v). Compound 20 was obtained from subfraction 14–4 through semi-preparative NP-HPLC (n-hexane/acetone = 3:2, v/v) and NP-HPLC (n-hexane/ethyl acetate = 52:48, v/v). Compounds 14, 15, and 21 were obtained from subfraction 14–5 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v). Compound 16 was obtained from subfraction 14–5 through semi-preparative NP-HPLC (n-hexane/acetone = 8:2, v/v) and semi-preparative NP-HPLC (n-hexane/ethyl acetate = 2:3, v/v). Fraction 15 (6.02 g) was separated by Sephadex LH 20 column chromatography (chloroform/methanol = 3:7, v/v) to give four subfractions (subfraction 15–1 to 15–4). Compounds 17 and 18 were obtained from subfraction 15–3 through semi-preparative NP-HPLC (n-hexane/acetone = 2:3, v/v) and semi-preparative NP-HPLC (n-hexane/acetone = 1:2, v/v).
3.4. Spectroscopic Data
Oleanolic acid (
1). White powder,
1H-NMR (500 MHz, C
5D
5N): δ 5.52 (1H,
br s, H-12), δ 3.46 (1H,
dd,
J = 10.5, 5.5 Hz, H-3), δ 3.33 (1H,
dd,
J = 13.8, 3.7 Hz, H-18), δ 1.30 (3H,
s), δ 1.26 (3H,
s), δ 1.04 (6H,
s), δ 1.03 (3H,
s), δ 0.97 (3H,
s), δ 0.91 (3H,
s), δ 0.88 (1H,
d,
J = 12.0 Hz, H-5). The above data were identical to the literature data [
32].
3-O-(Z)-Coumaroyl oleanolic acid (
2). White powder,
1H-NMR (500 MHz, C
5D
5N): δ 8.12 (2H,
d,
J = 8.6 Hz, H-5' and H-9'), δ 7.21 (2H,
d,
J = 8.6 Hz, H-6' and H-8'), δ 7.01 (1H,
d,
J = 12.9 Hz, H-3'), δ 6.05 (1H,
d,
J = 12.9 Hz, H-2'), δ 5.50 (1H,
br s, H-12), δ 4.82 (1H,
dd,
J = 11.8 , 4.6 Hz, H-3), δ 1.28 (3H,
s), δ 1.03 (3H,
s), δ 1.00 (3H,
s), δ 0.98 (3H,
s), δ 0.97 (3H,
s), δ 0.90 (3H,
s), δ 0.85 (3H,
s). The above data were identical to the literature data [
33].
3-O-(E)-Coumaroyl oleanolic acid (
3). White powder,
1H-NMR (500 MHz, C
5D
5N): δ 8.03 (1H,
d,
J = 15.9 Hz, H-3'), δ 7.68 (2H,
d,
J = 8.6 Hz, H-5' and H-9'), δ 7.19 (2H,
d,
J = 8.6 Hz, H-6' and H-8'), δ 6.71 (1H,
d,
J = 15.9 Hz, H-2'), δ 5.49 (1H,
br s, H-12), δ 4.90 (1H,
br d,
J = 9.8 Hz, H-3), δ 1.29 (3H,
s), δ 1.03 (3H,
s), δ 1.02 (3H,
s), δ 0.98 (6H,
s), δ 0.97 (3H,
s), δ 0.87 (3H,
s). The above data were identical to the literature data [
33].
3-O-Caffeoyl oleanolic acid (
4). White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.53 (1H,
d,
J = 15.8 Hz, H-3'), δ 7.03 (1H,
s, H-5'), δ 6.93 (1H,
d,
J = 8.1 Hz, H-9'), δ 6.77 (1H,
d,
J = 8.1 Hz, H-8'), δ 6.23 (1H,
d,
J = 15.8 Hz, H-2'), δ 5.24 (1H,
br s, H-12), δ 4.56 (1H,
br d,
J = 10.0 Hz, H-3), δ 1.17 (3H,
s), δ 1.00 (3H,
s), δ 0.96 (3H,
s), δ 0.94 (3H,
s), δ 0.90 (6H,
s), δ 0.83 (3H,
s). The above data were identical to the literature data [
34].
Ursolic acid (
5). White powder,
1H-NMR (500 MHz, C
5D
5N): δ 5.49 (1H,
br s, H-12), δ 3.45 (1H,
dd,
J = 9.7, 6.4 Hz, H-3), δ 2.63 (1H,
d,
J = 11.4 Hz, H-18), 1.24 (3H,
s), 1.23 (3H,
s), 1.05(3H,
s), δ 1.02 (3H,
s), δ 1.00 (1H,
d,
J = 6.5 Hz), δ 0.96 (1H,
d,
J = 6.2 Hz), δ 0.89 (3H,
s), δ 0.86 (1H,
d,
J = 11.5 Hz, H-5). The above data were identical to the literature data [
32].
3-O-(Z)-coumaroyl ursolic acid (
6) White powder,
1H-NMR (500 MHz, CDCl
3): δ 7.59 (2H,
d,
J = 8.1 Hz, H-5' and H-9'), δ 6.76 (2H,
d,
J = 8.1 Hz, H-6' and H-8'), δ 6.84 (1H,
d,
J = 12.8 Hz, H-3'), δ 5.83 (1H,
d,
J = 12.8 Hz, H-2'), δ 5.23 (1H,
br s, H-12), δ 4.56 (1H,
br d,
J = 9.3 Hz, H-3), δ 1.08 (3H,
s), δ 0.95 (6H,
s), δ 0.87 (3H,
s), δ 0.86 (3H,
s), δ 0.82 (3H,
s), δ 0.76 (3H,
s). The above data were identical to the literature data [
35].
3-O-(E)-Coumaroyl ursolic acid (
7). White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.62 (1H,
d,
J = 15.9 Hz, H-3'), δ 7.45 (2H,
d,
J = 8.6 Hz, H-5' and H-9'), δ 6.80 (2H,
d,
J = 8.6 Hz, H-6' and H-8'), δ 6.38 (1H,
d,
J = 15.9 Hz, H-2'), δ 5.24 (1H,
br s, H-12), δ 4.63 (1H,
br d,
J = 9.9 Hz, H-3), δ 1.14 (3H,
s), δ 1.06 (3H,
s), δ 0.97 (3H,
s), δ 0.94 (3H,
s), δ 0.89 (3H,
s), δ 0.88 (3H,
s), δ 0.86 (3H,
s). The above data were identical to the literature data [
35].
3-O-Caffeoyl ursolic acid (
8). White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.53 (1H,
d,
J = 15.9 Hz, H-3'), δ 7.16 (1H,
s, H-5'), δ 7.03 (1H,
d,
J = 8.2 Hz, H-9'), δ 6.85 (1H,
d,
J = 8.2 Hz, H-8'), δ 6.29 (1H,
d,
J = 15.9 Hz, H-2'), δ 5.25 (1H,
br s, H-12), δ 4.75 (1H,
dd,
J = 11.4 , 4.5 Hz, H-3), δ 1.20 (3H,
s), δ 1.00 (3H,
s), δ 0.95 (3H,
s), δ 0.94 (3H,
s), δ 0.92 (3H,
s), δ 0.90 (3H,
s), δ 0.82 (3H,
s). The above data were identical to the literature data [
36].
3β, 13β-Dihydroxyolean-11-en-28-oic acid (
9). White powder,
1H-NMR (500 MHz, CDCl
3): δ 6.04 (1H,
d,
J = 10.2 Hz, H-12), δ 5.41 (1H,
dd,
J = 10.2, 2.4 Hz, H-11), δ 3.22 (1H,
dd,
J = 11.4, 4.3 Hz, H-3), δ 1.25 (3H,
s), δ 1.06 (3H,
s), δ 0.98 (3H,
s), δ 0.97 (3H,
s), δ 0.91 (3H,
s), δ 0.88 (3H,
s), δ 0.78 (3H,
s). The above data were identical to the literature data [
37].
3β, 13β-Dihydroxyurs-11-en-28-oic acid (
10). White powder,
1H-NMR (500 MHz, CDCl
3): δ 5.96 (1H,
d,
J = 10.2 Hz, H-12), δ 5.25 (1H,
br d,
J = 10.2, H-11), δ 3.22 (1H,
dd,
J = 11.6, 4.6 Hz, H-3), δ 1.16 (3H,
s), δ 1.00 (3H,
d,
J = 6.3), δ 0.99 (3H,
s), δ 0.94 (3H,
s), δ 0.94 (3H,
d,
J = 6.3), δ 0.91 (3H,
s), δ 0.78 (3H,
s).The above data were identical to the literature data [
38].
Uvaol (
11). White powder,
1H-NMR (500 MHz, CDCl
3): δ 5.12 (1H,
t,
J = 3.5 Hz, H-12), δ 3.54 (1H,
d,
J = 11.0 Hz, H-28β), δ 3.20 (1H,
dd,
J = 11.2, 5.0 Hz, H-3), δ 3.18 (1H,
d,
J = 11.0 Hz, H-28α), δ 1.09 (3H,
s), δ 0.99 (3H,
s), δ 0.94 (3H,
s), δ 0.93 (3H,
d,
J = 5.8 Hz), δ 0.80 (3H,
d,
J = 5.8 Hz), δ 0.78 (3H,
s), δ 0.72 (3H,
d,
J = 11.2 Hz, H-5). The above data were identical to the literature data [
39].
Betulin (
12). White powder,
1H-NMR (500 MHz, CDCl
3): δ 4.68 (1H,
br s, H-29β), δ 4.58 (1H,
br s, H-29α), δ 3.79 (1H,
d,
J = 10.8 Hz, H-28β), δ 3.32 (1H,
d,
J = 10.8 Hz, H-28α), δ 3.20 (1H,
dd,
J = 11.4, 4.6 Hz, H-3), δ 1.67 (3H,
s), δ 1.01 (6H,
s), δ 0.98 (3H,
s), δ 0.96 (3H,
s), δ 0.82 (3H,
s), δ 0.75 (3H,
s). The above data were identical to the literature data [
40].
Lupeol (
13). White powder,
1H-NMR (500 MHz, CDCl
3): δ 4.68 (1H,
br s, H-29β), δ 4.56 (1H,
br s, H-29α), δ 3.18 (1H,
dd,
J = 11.4, 4.9 Hz, H-3), δ 1.68 (3H,
s), δ 1.02 (3H,
s), δ 0.96 (3H,
s), δ 0.94 (3H,
s), δ 0.83 (3H,
s), δ 0.79 (3H,
s), δ 0.76 (3H,
s). The above data were identical to the literature data [
41].
Kaempferol (
14). Yellowish powder,
1H-NMR (500 MHz, CD
3OD): δ 8.06 (2H,
d,
J = 8.8 Hz, H-2' and H-6'), δ 6.88 (2H,
d,
J = 8.8 Hz, H-3' and H-5'), δ 6.37 (1H,
d,
J = 1.7 Hz, H-8), δ 6.16 (1H,
d,
J = 1.7 Hz, H-6). The above data were identical to the literature data [
42].
Aromadendrin (
15). White powder,
1H-NMR (500 MHz, CD
3OD): δ 11.71 (1H,
br s, OH), δ 7.41 (2H,
d,
J = 8.5 Hz, H-2' and H-6'), δ 6.89 (2H,
d,
J = 8.5 Hz, H-3' and H-5'), δ 5.99 (1H,
d,
J = 1.9 Hz, H-8), δ 5.94 (1H,
d,
J = 1.9 Hz, H-6), δ 5.08 (1H,
d,
J = 11.5 Hz, H-2), δ 5.65 (1H,
d,
J = 11.5 Hz, H-3). The above data were identical to the literature data [
43].
Epigallocatechin (
16). White powder,
1H-NMR (500 MHz, CD
3OD): δ 8.17 (1H,
br s, OH), δ 8.01 (1H,
br s, OH), δ 7.92 (2H,
br s, OH), δ 6.57 (2H,
s, H-3' and H-5'), δ 6.01 (1H,
d,
J = 2.3 Hz, H-6), δ 5.91 (1H,
d,
J = 2.3 Hz, H-8), δ 4.81 (1H,
s, H-2'), δ 4.18 (1H,
d,
J = 3.5 Hz, H-3), δ 3.52 (1H,
d,
J = 5.1 Hz, OH), δ 2.74 (1H,
dd,
J = 16.5, 4.6 Hz, H-4α), δ 2.71 (1H,
dd,
J = 16.5, 3.4 Hz, H-4β). The above data were identical to the literature data [
44].
cis-Tiliroside (
17). Yellowish powder,
1H-NMR (500 MHz, CD
3COCD
3): δ 12.37 (1H,
br s, OH), δ 8.09 (2H,
d,
J = 8.8 Hz, H-2' and H-6'), δ 7.68 (2H,
d,
J = 8.6 Hz, H-2''' and H-6'''), δ 6.92 (2H,
d,
J = 8.8 Hz, H-3' and H-5'), δ 6.78 (2H,
d,
J = 8.6 Hz, H-3''' and H-5'''), δ 6.78 (1H,
d,
J = 12.9 Hz, H-7'''), δ 6.47 (1H,
br s, H-8), δ 6.27 (1H,
br s, H-6), δ 5.62 (1H,
d,
J = 12.9 Hz, H-8'''), δ 5.17 (1H,
d,
J = 7.3 Hz, H-1''), δ 4.28 (1H,
dd,
J = 11.9, 1.8 Hz, H-6β''), δ 4.17 (1H,
dd,
J = 11.9, 6.0 Hz, H-6α''), δ 3.55 (1H,
m, H-2''), δ 3.55 (1H,
m, H-5''), δ 3.49 (1H,
t,
J = 8.9 Hz, H-4''), δ 3.41 (1H,
d,
J = 9.3 Hz, H-3''). The above data were identical to the literature data [
45].
trans-
Tiliroside (
18). Yellowish powder,
1H-NMR (500 MHz, CD
3OD): δ 7.96 (2H,
d,
J = 8.8 Hz, H-2' and H-6'), δ 7.38 (1H,
d,
J = 16.5 Hz, H-7'''), δ 7.28 (2H,
d,
J = 8.5 Hz, H-2''' and H-6'''), δ 6.80 (2H,
d,
J = 8.8 Hz, H-3' and H-5'), δ 6.78 (2H,
d,
J = 8.5 Hz, H-3''' and H-5'''), δ 6.27 (1H,
br s, H-8), δ 6.11 (1H,
br s, H-6), δ 6.05 (1H,
d,
J = 16.5 Hz, H-8'''), δ 5.23 (1H,
d,
J = 7.4 Hz, H-1''), δ 4.31 (1H,
dd,
J = 11.8, 1.9 Hz, H-6β''), δ 4.18 (1H,
dd,
J = 11.8, 6.6 Hz, H-6α''), δ 3.55 (1H,
m, H-5''), δ 3.48 (1H,
m, H-3''), δ 3.47 (1H,
m, H-2''), δ 3.46 (1H,
m, H-4''). The above data were identical to the literature data [
45].
Isoamericanol B (
19). Yellowish oil,
1H-NMR (500 MHz, CD
3COCD
3): δ 7.69 (1H,
s, OH), δ 7.06 (1H,
d,
J = 1.6 Hz, H-2), δ 6.92 (1H,
dd,
J = 8.2, 1.6 Hz, H-6), δ 6.87 (1H,
d,
J = 8.2 Hz, H-5), δ 6.87 (1H,
d,
J = 1.8 Hz, H-2'), δ 6.86 (1H,
d,
J = 8.2 Hz, H-5'), δ 6.79 (1H,
dd,
J = 8.2, 1.8 Hz, H-6'), δ 6.38 (1H,
d,
J = 11.8 Hz, H-7'), δ 5.76 (1H,
m, H-8'), δ 5.18 (1H,
d,
J = 2.6 Hz, H-7), δ 4.60 (1H,
d,
J = 6.6, 2.6 Hz, H-8), δ 4.38 (2H,
d,
J = 5.0 Hz, H-9'), δ 3.83 (3H,
s, OCH
3), δ 1.68 (1H,
br s, OH), δ 1.08 (3H,
d,
J = 6.6 Hz, H-9). The above data were identical to the literature data [
46].
trans-
p-
Coumaric acid (
20) White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.58 (1H,
d,
J = 15.9 Hz, H-7), δ 7.43 (2H,
d,
J = 8.6 Hz, H-2 and H-6), δ 6.79 (2H,
d,
J = 8.6 Hz, H-3 and H-5), δ 6.27 (1H,
d,
J = 15.9 Hz, H-8). The above data were identical to the literature data [
47].
Protocatechuic acid (
21) White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.52 (1H,
d,
J = 1.9 Hz, H-2), δ 7.47 (1H,
dd,
J = 8.3, 1.9 Hz, H-6), δ 6.89 (1H,
d,
J = 1.9 Hz, H-5). The above data were identical to the literature data [
48].
Salicylic acid (
22) White powder,
1H-NMR (500 MHz, CD
3COCD
3): δ 7.84 (1H,
d,
J = 7.8 Hz, H-6), δ 7.52 (1H,
t,
J = 7.8 Hz, H-4), δ 7.89 (1H,
d,
J = 8.3 Hz, H-3), δ 6.87 (1H,
t,
J = 8.5 Hz, H-5). The above data were identical to the literature data [
49].
trans-
Ferulic acid (
23). White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.58 (1H,
d,
J = 15.9 Hz, H-7), δ 7.17 (1H,
br s, H-2), δ 7.05 (1H,
br d,
J = 8.1 Hz, H-6), δ 6.80 (1H,
d,
J = 8.1 Hz, H-5), δ 6.30 (1H,
d,
J = 15.9 Hz, H-8), δ 3.88 (3H,
s, OCH
3). The above data were identical to the literature data [
50].
Syringic acid (
24). White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.31 (2H,
s, H-2 and H-6), δ 3.87 (6H,
s, OCH
3). The above data were identical to the literature data [
51].
3-O-Methylgallic acid (
25) White powder,
1H-NMR (500 MHz, CD
3OD): δ 7.17 (2H,
s, H-2 and H-6), δ 3.86 (3H,
s, OCH
3). The above data were identical to the literature data [
52].