SUPPLEMENTAL MATERIALS
Extraction optimization and isolation of triterpenoids from Ganoderma lucidum and their
effect on human carcinoma cell growth
Weimei Ruan1, Lim Hiang He Andrew1, Lau Gek Huang1 and David G. Popovich2*
1
2
Department of Chemistry, National University of Singapore, Singapore
Institute of Food, Nutrition and Human Health, College of Health, Massey University,
Palmerston North, New Zealand
*Corresponding author:
Tel: +64 6 350 5904
Fax: +64 6 350 5657
Email address: D.G.Popovich@massey.ac.nz
ABSTRACT
The response surface methodology (RSM) was used to optimize the extraction conditions of
Ganoderma lucidum based on a Box–Behnken design (BBD). A quadratic model sufficiently
simulated the response of ganoderic acid H with a determination coefficient (R2) of 0.98. The
optimal condition for extracting triterpenoids was determined to be 100.00% ethanol at 60.22 °C for
6.00 h, under which the yield of the reference triterpenoid ganoderic acid H increased from 0.88 to
2.09 mg/g powder. Following extraction, triterpenoid enriched fraction was further isolated into 23
fractions and 7 fractions were identified as ganoderic acid A, B, D, G, H and I and ganoderenic acid
D. Of the 7 triterpenoids, ganoderenic acid D was most cytotoxic with LC50s of 0.14±0.01,
0.18±0.02 and 0.26±0.03 mg/mL in Hep G2, Hela and Caco-2 cells respectively. While ganoderic
acid A, G and H were relatively non-cytotoxic. The variation of inhibitory effects for these
triterpenoids was likely related to their chemical structures.
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The molecular weight and structural details of triterpenoids isolated from Ganoderma lucidum
triterpenoid enriched extract are:
Ganoderic acid I
(3.63 mg from 180 mg fraction 2) ESI-MS m/z: 531 [M-H]-,
1063[2M-H]-,513,469,451, 303, 265. 1H NMR δCDCl3 : 3.21 (1H, dd, J=6.0, 11.2Hz, 3-H), 0.87 (1H,
dd, J=1.9, 11.0Hz, 5-H), 4.79 (1H, t, J=8.4Hz, 7-H), 1.14 (3H, s, 18-H), 1.22 (3H, s, 19-H), 1.39
(3H, s, 21-H), 0.85 (3H, s, 31-H), 1.34 (3H, s, 32-H). 13 C NMR δCDCl3 : 34.9, 27.7, 78.3, 38.8, 49.2,
26.6, 66.9, 156.7, 142.5, 38.7, 198.0, 50.6, 45.6, 59.7, 217.7, 36.1, 49.1, 19.0, 18.4, 73.0, 26.6, 52.6,
210.4, 47.5, 34.2, 175.2, 16.9, 28.2, 15.4, 24.8.
Ganoderic acid G (4.41 mg from 180 mg fraction 2) ESI-MS m/z: 531 [M-H]-,
1063[2M-H]-,513,469, 319, 303, 265. 1H NMR δCDCl3 : 3.21 (1H, dd, J=7.6, 8.8Hz, 3-H), 4.77 (1H,
t, J=8.4Hz, 7-H), 4.36 (1H, s, 12-H), 0.79 (3H, s, 18-H), 1.30 (3H, s, 19-H), 1.13 (3H, d, J=6.4Hz,
21-H), 1.21 (3H, d, J=8.0Hz, 26-H), 1.03 (3H, s, 30-H), 0.87 (3H, s, 31-H), 1.44 (3H, s, 32-H). 13 C
NMR δCDCl3 : 34.3, 27.5, 78.3, 38.6, 49.1, 26.7, 66.2, 157.3, 141.9, 38.3, 199.3, 77.8, 51.9, 60.3,
216.8, 38.2, 45.7, 11.9, 18.8, 28.7, 21.3, 48.3, 8.2, 46.2, 34.5, 176.1, 16.9, 28.1, 15.3, 23.0.
Ganoderic acid B
(3.42 mg from 180 mg fraction 2) ESI-MS m/z: 515 [M-H]-, 1031[2M-H]-,
497, 453 , 303, 287, 249. 1H NMR δCDCl3 : 3.21 (1H, dd, J=6.0, 11.2Hz, 3-H), 0.87 (1H, dd, J=3.6,
8.3Hz,5-H), 4.79(1H, t, J=8.4Hz, 7-H), 1.00 (3H, s, 18-H), 1.21 (3H, s, 19-H), 0.98 (3H, d, J=6.0Hz,
21-H), 1.24 (3H, d, J=6.4Hz, 26-H), 1.03 (3H, s, 31-H), 1.34 (3H, s, 32-H). 13 C NMR δCDCl3 : 34.3,
28.1, 78.3, 38.8, 45.3, 27.6, 66.8, 156.8, 142.7, 45.5, 197.8, 50.3, 38.6, 59.4, 207.6, 40.9, 49, 16.9,
17.4, 31.9, 19.6, 49.1, 217.5, 46.5, 34.8, 26.6, 179.5, 15.4, 24.4, 18.4.
Ganoderic acid A
(5.70 mg from 180 mg fraction 2) ESI-MS m/z: 515 [M-H]-, 1031[2M-H]-,
497, 435, 405, 301, 285. 1H NMR δCDCl3 : 4.62(1H, t, J=8.0Hz, 7-H), 4.79 (1H, t, J=8.0Hz,15-H),
0.98 (3H, s, 18-H), 1.25 (3H, s, 19-H), 0.88 (3H, d, J=6.0Hz, 21-H), 1.24 (3H, d, J=7.3Hz, 26-H), 1.1
(3H, s, 30-H), 1.12 (3H, s, 31-H), 1.28 (3H, s, 32-H). 13 C NMR δCDCl3 : 35.6, 34.3, 208.5, 46.8, 48.9,
29.2, 68.9, 159.0, 140.6, 46.6, 200.2, 51.7, 38.0, 54.0, 72.5, 36.3, 47.9, 17.3, 19.5, 32.6, 19.3, 49.5,
216.9, 46.4, 34.5, 27.3, 179.8, 16.9, 20.7, 19.7.
Ganoderic acid H (4.12 mg mg from 180 mg fraction 2) ESI-MS m/z: 571 [M-H]-,
1143[2M-H]-, 553, 511,467, 437, 319, 303, 301. 1H NMR δCDCl3 : 3.25 (1H, dd, J=7.6, 8.8Hz, 3-H),
5.63 (1H, s, 12-H), 0.82 (3H, s, 18-H), 1.33 (3H, s, 19-H), 0.99 (3H, d, J=6.4Hz, 21-H), 1.21 (3H, d,
J=8.0Hz, 26-H), 1.03 (3H, s, 30-H), 0.88 (3H, s, 31-H), 1.73 (3H, s, 32-H). 13 C NMR δCDCl3 : 33.2,
2
27.3, 77.2, 40.4, 51.4, 36.6, 198.8, 151.7, 145.7, 39.1, 193.9, 79.2, 47.9, 58.4, 205.7, 37.9, 44.7,
12.2, 17.9, 29.4, 21.6, 48.3, 207.4, 46.4, 34.3, 176, 16.9, 27.9, 15.5, 21.3
Ganoderenic acid D
(2.49 mg mg from 180 mg fraction 2) ESI-MS m/z: 511 [M-H]-,
1023[2M-H]-, 493, 449, 301, 285, 247, 149. 1H NMR δCDCl3 : 4.87 (1H, dd, J=7.6, 9.2Hz,7-H), 0.89
(3H, s, 18-H), 1.24 (3H, s, 19-H), 2.18 (3H, s, 21-H), 6.05 (1H, s, 22-H), 1.23 (3H, d, J=5.6Hz, 26-H),
1.13 (3H, s, 30-H), 1.11 (3H, s, 31-H), 1.40 (3H, s, 32-H). 13 C NMR δCDCl3 : 35.6, 34.3, 216.5, 46.7,
48.8, 27.6, 66.3, 157.4, 141.4, 38.2, 197.8, 48.9, 45.9, 58.6, 216.4, 37.8, 49.7, 19.0, 18.1, 153.8,
21.0, 124.6, 196.1, 47.5, 34.6, 180.6, 16.9, 27.0, 20.8, 24.7.
Ganoderic acid D (2.71 mg from 180 mg fraction 2) ESI-MS m/z: 513 [M-H]-, 1027[2M-H]-,
495, 451, 301, 285, 247, 149. 1H NMR δCDCl3 : 4.80 (1H, t, J=8.8Hz, 7-H), 0.99 (3H, s, 18-H), 1.20
(3H, s, 19-H), 0.94 (3H, d, J=7.6Hz, 21-H), 1.21(3H, d, J=7.6Hz, 26-H), 1.06 (3H, s, 30-H), 1.03
(3H, s, 31-H), 1.30 (3H, s, 32-H). 13 C NMR δCDCl3 : 35.6, 34.3, 217.6, 46.8, 48.9, 27.6, 66.3, 157.8,
141.2, 38.2, 197.6, 50.1, 45.0, 59.3, 216.7, 41.0, 45.6, 17.6, 18.1, 32.0, 19.6, 48.9, 207.6, 46.6, 34.3,
179.4, 16.9, 27.0, 20.8, 24.7.
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Table S1. Regression coefficients of predicted quadratic polynomial models for the yields of total
extract and ganoderic acid H.
Source
X1
Sum of Squares
total
ganoderic
extract
acid H
7.61
0.037
df
1
Mean Square
total
ganoderic
extract
acid H
7.61
0.037
F Value
total
ganoderic
extract
acid H
40.20
8.33
p-value (Prob > F)
total
ganoderic
extract
acid H
0.0014
0.034
X2
1.21
0.39
1
1.21
0.39
6.42
88.62
0.052
0.0002
X3
1.17
0.024
1
1.17
0.024
6.17
5.43
0.056
0.067
X1 X2
6.53
0.13
1
6.53
0.13
34.52
29.54
0.0020
0.0029
X1 X3
0.37
0.039
1
0.37
0.039
1.93
8.81
0.22
0.031
X2 X3
10.08
0.22
1
10.08
0.22
53.28
49.42
0.0008
0.0009
X1
2
14.76
0.15
1
14.76
0.15
77.99
33.05
0.0003
0.0022
X2
2
0.071
0.049
1
0.071
0.049
0.37
11.20
0.57
0.020
X3
2
13.49
0.20
1
13.49
0.20
71.28
46.72
0.0004
0.0010
Model
Pure
Error
57.64
1.26
9
6.40
0.14
33.85
31.92
0.0006
0.0007
0.41
0.00071
2
0.20
0.00036
Total
58.59
1.28
14
X1 refers to extraction time; X2 refers to the ethanol ratio; X3 refers to the extraction temperature.
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Table S2. ANOVA for the fitted quadratic polynomial model of extraction of total extract and
ganoderic acid H.
Total extract
Ganoderic acid H
Mean
8.79
1.40
Std. Dev.
0.44
0.066
R-Squared
0.98
0.98
Adj R-Squared
0.95
0.95
C.V. %
4.95
4.73
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Figure S1. (a) Representative HPLC chromatogram of extraction of Ganoderma lucidum under
Box–Behnken factorial design (BBD) conditions. Ganoderic acid H was calibrated using an
external standard. (b) HPLC profile of triterpenoid enriched fraction (fraction 2) by
semi-preparative HPLC isolation. Peak numbers refer to the collected fractions: 8, ganoderic acid I;
12, ganoderic acid G; 14, ganoderic acid B; 17, ganoderic acid A; 18, ganoderic acid H; 20,
ganoderenic acid D; 21, ganoderic acid D. (c) Chemical structures of identified triterpenoids from
Ganoderma lucidum.:”-“ refers to no attachment in this position.
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Figure S2. Dose-response relationship of ganoderic acid A, B, D, G, H and I and ganoderenic acid
D in (a) HeLa cells, (b) Caco-2 cells and (c) Hep G2 cells after 72 h treatment measured by MTT
assay. Values are expressed as mean ± SD.
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