Article

A new ganoderic acid from Ganoderma lucidum mycelia and its stability

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Mei-Long Road, Shanghai 200237, China.
Fitoterapia (Impact Factor: 2.35). 11/2012; 84(1). DOI: 10.1016/j.fitote.2012.11.008
Source: PubMed

ABSTRACT

A new ganoderic acid (GA), 3α,22β-diacetoxy-7α-hydroxyl-5α-lanost-8,24E-dien-26-oic acid (1), together with four known compounds GA-Mk (2), -Mc (3), -S (4) and -Mf (5), were isolated and characterized from Ganoderma lucidum mycelia. The structure of compound 1 was elucidated on the basis of interpretation of extensive spectroscopic data (HRMS, IR, UV, 1D and 2D NMR). Due to its apparent degradation during preparation procedures, the stability of compound 1 was assessed in several solvents in a short-term study that demonstrated the optimal stability in aproptic environment. A possible mechanism of acid-catalyzed degradation of compound 1 in methanol was proposed, consisting of a fast protonation, followed by a committed step of hydroxyl group removal. In addition, all isolated compounds were tested in vitro for their cytotoxic activities against 95D and HeLa tumor cell lines, with IC(50) values ranging from14.7 to 38.5μM. The results may improve the understanding of chemical stability of GAs and provide valuable information on their separation, analysis and application.

Full-text

Available from: Jian-Jiang Zhong, May 17, 2015
A new ganoderic acid from Ganoderma lucidum mycelia and its stability
Ying-Bo Li
a
, Ru-Ming Liu
a
, Jian-Jiang Zhong
a,b,
a
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Mei-Long Road, Shanghai 200237, China
b
State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road,
Shanghai 200240, China
article info abstract
Article history:
Received 12 May 2012
Accepted in revised form 8 November 2012
Available online 16 November 2012
A new ganoderic acid (GA), 3α,22β-diacetoxy-7α-hydroxyl-5α-lanost-8,24E-dien-26-oic acid
(1), together with four known compounds GA-Mk (2), -Mc (3), -S (4) and -Mf (5), was isolated
and characterized from Ganoderma lucidum mycelia. The structure of compound 1 was
elucidated on the basis of interpretation of extensive spectroscopic data (HRMS, IR, UV, 1D and
2D NMR). Due to its apparent degradation during preparation procedures, the stability of
compound 1 was assessed in several solvents in a short-term study that demonstrated the
optimal stability in aproptic environment. A possible mechanism of acid-catalyzed degradation
of compound 1 in methanol was proposed, consisting of a fast protonation, followed by a
committed step of hydroxyl group removal. In addition, all isolated compounds were tested in
vitro for their cytotoxic activities against 95D and HeLa tumor cell lines, with IC
50
values
ranging from14.7 to 38.5 μM. The results may improve the understanding of chemical stability
of GAs and provide valuable information on their separation, analysis and application.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Ganoderma lucidum
Ganoderic acid
Cytotoxicity
Stability
1. Introduction
Mushrooms have received increasing attention in recent
years from scientific community not only because of their
great nutritional value, but also their abundant and fascinat-
ing secondary metabolites as a promising potential library for
new drug discovery [1,2]. Ganoderma lucidum (Fr.) Karst
(Ling-zhi, Reishi), a well-known traditional Chinese medic-
inal mushroom, has been used for prevention and treatment
of many kinds of diseases for thousands of years in Mainland
China and other Asian regions [3]. The experience from this
ethnomedicine arouses the interest of scientists around the
world, and the number of publication on the separation,
bioactivity and production of bioactive secondary metabo-
lites of G. lucidum has increased in recent years [4].
Triterpenoids, especially ganoderic acids (GAs), are typical
bioactive constituents in this higher fungus and possess many
important bioactivities including anti-cancer [57],anti-
inflammatory [8],anti-HIV[9] and aldose reductase inhibitory
effects [10]. To date, more than 150 triterpeniods have been
separated from Ganoderma spp. [4], and the number of the new
compounds identified from it seems to increase continuously
[11]. Due to their important pharmacological functions,
methods for producing this class of compounds from G. lucidum
have been developed [1214]. A unique two-stage culture
process combining conventional shaking culture (first-stage)
with static culture (second-stage) was developed by our lab
and showed great potential to produce bioactive GAs from
mycelia fermentation of G. lucidum [15]. To search for bioactive
metabolites from this source, several GAs have been identified
and showed anti-tumor activity based on in vitro and in vivo
experiments [1618]. Recently, our continued efforts in finding
the bioactive components from this resource resulted in the
isolation and identification of 7-O-ethyl GA-O with a rare
ethoxyl group at C-7 [19].
In the current work, we described further investigation on
the isolation and structural determination of a new ganoderic
acid (compound 1) and four known compounds GA-Mk
(2), -Mc (3), -S (4) and -Mf (5)(Scheme 1). The structures of
Fitoterapia 84 (2013) 115122
Corresponding author at: State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology, 130 Mei-Long Road,
Shanghai 200237, China. Tel.: +86 21 34206968; fax: +86 21 34204831.
E-mail address: jjzhong@sjtu.edu.cn (J.-J. Zhong).
0367-326X/$ see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.tote.2012.11.008
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Page 1
the known compounds were identified by comparison of the
NMR data with those reported in the literature [2023],while
the structure of compound 1 was elucidated based on its
extensive spectroscopic data including high-resolution mass
spectrometry (HRMS), infra-red (IR) spectrum, and UV, 1D and
2D NMR. Due to its apparent degradation during preparation
procedures and the rare knowledge of chemical stability of
GAs, the stability of compound 1 was investigated under
different conditions of solvents, acidity and temperature. The
stability of the known GAs was also discussed in this work. In
addition, all of the compounds were tested in vitro for their
cytotoxic activities against 95D and HeLa tumor cell lines.
2. Experimental
2.1. General
Optical rotation was recorded on a JASCO automatic
polarimeter. UV spectra on a Thermo Evolution 300 spectrom-
eter and IR spectra on a Bruker Equinox 55 IR spectrometer
were recorded in MeOH and KBr disks, respectively. NMR
spectra were recorded on a Bruker Avance-III spectrometer at
400 MHz (
1
H NMR) and 100 MHz (
13
C NMR) in CDCl
3
with
TMS as internal reference. The compounds were assigned by
1
H,
13
C, distortionless enhancement by polarization transfer
(DEPT), heteronuclear singular quantum correlation (HSQC),
heteronuclear multiple-bond correlation (HMBC),
1
H
1
Hcor-
relation spectroscopy (COSY) and nuclear overhauser enhance-
ment spectroscopy (NOESY). High-resolution ESI-MS were
recorded on a Waters QTOFMS Premier mass spectrometer
(70 eV) using a direct inlet system. Analytical and semi-
preparative HPLC was performed on a Shimadzu (Shimadzu
Ltd., Kyoto, Japan) HPLC coupled with photodiode array (PDA)
detector and a Hypersil C18 column (4.6×250 mm, 5 μm) or
Eclipse XDB-C18 column (10×250 mm, 5 μm). Silica gel (200
300 mesh) was purchased from Qingdao Haiyang Chemical
Group Co. (Shandong Province, China).
2.2. Fungal material
The strain of G. lucidum CGMCC 5.616 from Chinese General
Microbiological Fermentation Collection Center (Beijing, China)
was maintained on potato-dextrose agar slants. A two-stage
culture of G. lucidum by combining conventional shaking
culture (first-stage) with static culture (second-stage) was
applied to obtain the mycelia for ganoderic acid separation and
purification [15].
2.3. Extraction and isolation
G. lucidum mycelia were harvested from a static liquid
culture after 14 d. The mycelia were dried in a dry oven and
then powdered. The dried mycelia powder (300 g) was
extracted with acetyl acetate (3 L) twice, each for 30 min by
ultrasonic-assisted extraction (UAE). The total crude extract
was filtered through an ashless filter paper, and then
evaporated to dryness by rotary evaporator under vacuum
at 30 °C. The resulting extract (25.3 g) was subjected to
chromatography on silica gel (4× 40 cm), eluting with ethyl
ether to afford four crude fractions (AD). Fraction B was
further purified by reversed-phase preparative high perfor-
mance liquid chromatography (RP-pHPLC) eluted with 82.5%
methanol to give 41 mg of compound 4, and 15 mg of
compound 5. Fraction C was re-subjected to chromatography
on silica gel (2.5× 40 cm) and eluted with ethyl ether-
petroleum ether (9:1) to afford three sub-fractions (C-1 to
C-3). Subfractions C-1 and C-3 were crystallized at 20 °C to
yield 95 mg of compound 1 and 37 mg of compound 3.
Subfraction C-2 was further purified by RP-pHPLC eluted
with 80% methanol to give 15 mg of compound 2.
3α,22β-diacetoxy-7α-hydroxyl-5α-lanost-8,24E-dien-26-
oic acid (compound 1): white amorphous powder. α½
25
D
9
(c=0.1, MeOH); UV (MeOH) λ
max
(logε): 222 (4.05) nm; IR
(KBr film) ν
max
: 3501, 3172, 2960, 1724, 1652, 1379, 1269,
1198, 1156, 1024 cm
1
;
1
Hand
13
C NMR spectral data, see
Table 1; high-resolution electrospray ionization mass spec-
trometry (HR-ESI-MS) (positive mode): m/z 595.3623
[M+Na]
+
(calc. for C
34
H
52
O
7
Na
+
, 595.3611). HR-ESI-MS
(negative mode): m/z 571.3627 [MH]
+
(calc. for C
34
H
51
O
7
,
571.3635).
2.4. Stability studies
2.4.1. Stability of compound 1 in different solvents
Samples of compound 1 (0.6 mg/mL) were prepared in
different solvents, in screw-cap vials (Agilent). The solvents
were 100% chloroform, 100% ethyl acetate, 100% acetonitrile,
acetonitrile/water 80/20 (v/v), 100% methanol, methanol/water
(1)R
1
= α−OH, β−H; R
2
=H
2
,R
3
=OAc
(3)R
1
= α−OAc, R
2
= α−OH, R
3
= OAc
(2)R
1
= α−OAc, β−H; R
2
= α−OH,β−H; R
3
=OAc
(4)R
1
= α−OH, β−H; R
2
=H
2
,R
3
= OAc
(5)R
1
= α−OAc, β−H; R
2
= α−OH, β−H; R
3
=H
2
Scheme 1. Chemical structures of compounds 15.
116 Y.-B. Li et al. / Fitoterapia 84 (2013) 115122
Page 2
80/20 (v/v), 100% methanol containing 1 mM NaOH, 100%
methanol containing 1 mM acetic acid, and 100% methanol
containing 1 mM HCl. The samples were heated at 40 °C in
thermostatic water bath for 2 h and analyzed by LC and
LC-HRESI-MS. All experiments were performed in triplicate.
2.4.2. Stability of compound 1 under thermal treatment
The effect of temperature was investigated in methanol.
Kinetic studies were done under the temperatures of 55, 65
and 75 °C. The initial concentration was 0.6 mg/mL for all.
The kinetic parameters in each temperature, i.e., reaction
order and rate constants (k), were obtained using the integral
method [24]. This method uses a trial-and-error procedure to
find reaction order, and the result showing the best
correlation coefficient was selected. The absolute tempera-
ture T dependence of the degradation rate constant was
determined by the Arrhenius equation:
k ¼ k
0
exp E
a
R
1
T
1

ð1Þ
which was arranged to
ln k ¼ ln k
ref
þ E
a
R
1
T
ref
1
T
1

ð2Þ
where k
ref
is the degradation rate constant at reference
temperature T
ref
, which was chosen as the average of all the
temperatures investigated in these cases, and R is the
universal gas constant (8.314 J/mol
1
K
1
). E
a
is the activa-
tion energy, and was calculated using linear regression
analysis by plotting the natural logarithm of k values as a
function of the reciprocal of the absolute temperature T.
2.5. Analyses
2.5.1. Quantitative analysis of compound 1 by HPLC
A Shimadzu (Shimadzu Ltd., Kyoto, Japan) HPLC coupled
with PDA detector was used. Separation was performed at
25 °C using a Hypersil C18 column (4.6× 250 mm). The mobile
phases were 0.01% acetic acid (glacial, ACS certified, Fischer
Scientific, Pittsburgh, PA) in water (solvent A) and acetonitrile
(solvent B) (Merck, Germany), and isocratic gradient of 95% B
was adopted. Samples of 20 μL were injected and a flow rate of
1.000 mL/min was used. Detection wavelength was set at
222 nm. The standard curve of compound 1 was prepared in
the range of 0.010.84 mg/mL. The regression correlation (r
2
)
of the standard curve in this study was 0.999. Quantification of
compound 1 was done using the response factors calculated
from the standard curves.
The relative concentration of compound 1 (C
R
, %) is the
remaining concentration (C) of compound 1 as a percentage
of the initial concentration (C
0
), as shown in Eq. (3):
C
R
ð %Þ¼
C
C
0
100: ð3Þ
2.5.2. Qualitative analysis of compound 1 and its degraded
products by HPLCHR-ESI-MS
Samples were analyzed on a Waters ACQUITY UPLC system
equipped with a binary solvent delivery manager and a sample
manager, coupled with a Waters Micromass Q-TOF Premier
Mass Spectrometer equipped with an electrospray interface
(Waters Corporation, Milford, MA) at the Instrumental Analyt-
ical Center of Shanghai Jiao Tong University (Shanghai, China).
An Acquity BEH C18 column (100× 2.1 mm id., 1.7 μm; Waters,
Milford, USA) was used. The column was maintained at 45 °C
and eluted with gradient solvent from A:B (0:100)to A:B
(10:90) in 10 min at a flow rate of 0.40 mL/min, where A was
acetonitrile (0.1% (v/v) formic acid) and B was aqueous formic
acid (0.1% (v/v) formic acid). The MS was run with the ESI
probe in both the positive and negative modes. The optimized
parameters in the negative/positive ion mode were as follows:
capillary voltage, 3.0 kV; sampling cone, 35 V; collision energy,
4 eV; source temperature, 100 °C; desolvation temperature,
300 °C; and desolvation gas, 500 L/h. A full scan mass spectrum
was obtained over the range of 501000 m/z.
2.5.3. Preparation of degraded products of compound 1 in
methanol
Seventy-five milligrams of compound 1 was dissolved in
20 mL methanol and heated at 55 °C for 24 h, then dried by
rotary evaporator at reduced vacuum. Then concentrated
Table 1
NMR spectral data of compound 1 in CDCl
3
(δ in ppm).
Position δ
H
(J, Hz) δ
C
a
HMBC (H C)
1α 1.50(1H, overlapping) 30.3t C-3, C-28
1β 1.87(1H, overlapping) C-3, C-29
2α 1.89(1H, overlapping) 23.3t C-4, C-19
2β 1.66(1H, overlapping) C-4
3β 4.69(1H, br. s) 77.6d C-2 C-4, C-5, C-29, 3-Ac-Me
4 36.3s
5 1.76 (1H, m) 40.2d C-3, C-28
6α 1.75 (1H, overlapping) 28.9t C-5, C-7, C-28
6β 1.66 (1H, overlapping) C-5, C-8, C29
7β 4.18 (1H, br. s) 67.0t C-5, C-8, C-9
8 135.6s
9 141.3s
10 38.2s
11α 2.03 (1H, overlapping) 21.0t C-9, C-12
11β 2.10 (1H, overlapping) C-9, C12
12α 1.78 (overlapping) 31.0t C9, C10
12β 1.64 (1H,br s) C-10, C-11
13 45.0s
14 49.7s
15α 1.81 (1H, dd, J =9.6, 5.8) 29.8d C-14, C-17
15β 1.73 (1H, dd, J =7.6, 4.2)
16α 1.35 (1H, br.d, J= 5.0) 27.9t C-12, C-14
16β 2.06 (1H, overlapping) C-12, C-13, C-20
17 1.66(1H, overlapping) 47.1s C-11, C-13, C-18
18 0.61 (3H, s) 15.9q C-12, C-13, C-14, C-17
19 0.96 (3H, s) 17.3q C-1, C-9, C10
20 1.52 (1H, overlapping) 39.7d C-16, C-21
21 0.99 (3H, d, J= 10.8) 12.8q C-17, C-20, C-22
22 5.11 (1H, t, J= 7.2) 74.7d C-17. C-21, 22-AcMe, C-24
23α 2.41 (1H, m) 31.8t C-22, C-24, C-25
23β 2.60 (1H, m) C-22, C-24, C-25
24 6.83 (1H, dd, J =7.2,6.8) 139.4d C-26, C-27
25 129.2s
26
172.4s
27
1.86 (3H, s) 12.3q C-24, C-25, C-26
28 0.89 (3H, s) 27.4q C-3, C-4, C-5, C-29
29 0.93 (3H, s) 22.0q C-3, C-4, C-5, C-10, C-28
30 1.05 (3H, s) 26.3q C-8, C-13, C-14, C-15
C_O 170.9
C_O 170.7
AcMe 2.05 (3H,s) 21.4 C-3, 3-AcMe
AcMe 2.07 (3H,s) 21.0 C-24, 22-AcMe
a
Multiplicity derived from DEPT measurements.
117Y.-B. Li et al. / Fitoterapia 84 (2013) 115122
Page 3
sample was dissolved in 10 mL methanol and purification was
done by using HPLC (Eclipse XDB-C18 column: 10×250 mm,
5 μm) with isocratic gradient method. The mobile phase was
95% acetonitrile. The flow rate was 2.5 mL/min and the
detecting wavelength was set at 222 nm. Fifteen milligrams
of compound 6 and 20 mg of compound 7 were obtained. The
chemical structures of both compounds were confirmed by MS
and NMR.
2.6. Cytotoxicity assay
Cytotoxicity of compounds 15 and 7 against the human
highly metastatic lung tumor cell line 95-D and human cervical
cancer cell line HeLa (Cell Bank of Chinese Academy of
Sciences, Shanghai, China) was evaluated by using the MTT
(methyl thiazole tetrazolium) method as described previously
[17]. Hydroxycamptothecine was used as a positive control
(IC
50
: 29.5 μM against HeLa and 21.3 μM against 95D).
3. Results and discussion
The molecular formula of compound 1 was determined as
C
34
H
52
O
7
by the HR-ESI-MS (m/z 571.3627 [MH]
(calc. for
C
34
H
51
O
7
, 571.3635) and m/z 595.3623 [M +Na]
+
(calc. for
C
34
H
52
O
7
Na
+
, 595.3611)). According to its positive HR-ESI-
MS, the fragment ion peaks at m/z 555.3702 [MH
2
O+H]
+
suggested that it had one hydroxyl group. The fragment ion
peaks at m/z 495.3467 [MH
2
OHOAc+ H]
+
and 435.3265
[MH
2
O2HOAc+ H]
+
further revealed that compound 1
possessed two acetyl groups. This compound showed
maximum absorption at 222 nm in the UV spectrum.
Hydroxyl (3501 cm
1
) and α, β-unsaturated carbonyl
(1724 cm
1
) were seen in the IR spectrum. The
1
H NMR
spectrum of compound 1 showed the presence of seven
methyl singlets at δ
H
0.61, 0.89, 0.93, 0.99, 1.05, 2.05, and
2.07, a methyl doublet at δ
H
0.96, a vinyl methyl singlet at δ
H
1.86, three O-bearing methine signals at δ
H
4.18, 4.69, and
5.09, and an olefinic proton double of doublets at δ
H
6.81. The
13
C NMR spectrum and DEPT measurement of compound 1
(Table 1) showed the presence of 9 methyls, 8 methylenes, 7
methines, 3 sp
3
quaternary carbons, 4 olefinic carbons, and
3carbonyl carbons. The
1
H NMR spectrum of compound 1
was very close to that of 3α,22β-diacetoxy-7α-methoxy-
5α-lanosta-8,24E-dien-26-oic acid (also called GA-Md) [20]
except for the loss of the methoxy methyl proton signal at δ
H
3.29 and the down-field shift (δ
H
4.18) of H-7 signal in the
spectrum of GA-Md. The
13
C NMR spectrum of compound 1
was also similar to that of GA-Md except for the loss of the
methoxy methyl carbon signal at δ
C
55.6 and the up-field
shift (δ
C
67.0) of methoxy methane carbon signal in the
spectrum of GA-Md. These data suggested that compound 1
had a hydroxyl group instead of a methoxyl group attached
to C-7. The attachment of the hydroxyl group to C-7 was also
confirmed by the HMBC cross-peaks between the proton
signal at δ
H
4.18 and the carbon resonances at δ
C
141.7 (C-9),
δ
C
135.6 (C-8), and δ
C
40.2 (C-5). The relative configuration of
compound 1 was determined by analyzing the NOESY spectrum.
Key NOESY correlations were observed between H-7 and H-18,
H-19, and H-29 indicating that H-7 was on the β-orientation
same as H-18, H-19 and H-29. Therefore, the structure of the
compound 1 was elucidated as 3α,22β-diacetoxy-7α-hydroxyl-
5α-lanost-8,24E-dien-26-oic acid (Fig. 1).
Compound 1 was observed to degrade during separation,
suggesting its instability. Stability of bioactive compounds
was a key problem regarding its application and analysis.
Therefore, the stability of compound 1 was studied in different
solvents. As shown in Fig. 2, exposed to aproptic solvents like
chloroform, ethyl acetate and acetonitrile, compound 1 was
stable. In the solvents with proptic property such as acetonitrile/
water, methanol, methanol/water, methanol containing 1 mM
acetic acid or HCl, compound 1 showed different degrees of
degradation. At 1 mM HCl of methanol, compound 1 was
degraded completely only in 5 min. However, at 1 mM acetic
acid of methanol, the degradation percentage of compound 1
was less than 50%. This difference was consistent with the
difference of the acidity of the acids studied. On the contrary,
the effect of alkali on its stability was weak. Dissolved in basic
condition, the degradation extent was almost identical to that
Fig. 1. Key HMBC (H C) and NOESY (H H) correlations of compound 1.
118 Y.-B. Li et al. / Fitoterapia 84 (2013) 115122
Page 4
in methanol. These results clearly implied that solvents
with proptic property rather than polarity affect the stability
of compound 1 and the degradation of compound 1 is
acid-catalyzed.
Temperature is another important factor affecting the
stability of organic compounds. The effect of temperature on
the stability of compound 1 was therefore studied at three
different temperatures. As expected, the increasing temper-
ature destroyed compound 1 to a higher degree (Fig. S11 in
supplementary data). To analyze the results for the thermal
stability studies, first-order reaction model and Arrhenius
equation were found to be suitable to describe the degrada-
tion kinetics of compound 1 and to obtain the activation
energy (Ea), respectively. Data in Fig. S11 were fitted, and the
resulting kinetic parameters were shown in Table 2. The
evaluated apparent activation energy of the compound 1
degradation was 139.9 kJ/mol in methanol.
For better understanding of the degradation mechanism of
compound 1, degradation products of compound 1 were
purification and identified by spectral analysis (supplementary
data). In methanol, significant decrease of compound 1 and
apparent increase of two degraded products (compounds 6
and 7) were observed in HPLC chromatogram (Fig. 3A).
Compared to the mass spectrum of compound 1 (Fig. 3B), the
molecular ion peaks at m/z 585.3748 ([MH]
)(Fig. 3B) of
compound 6 showed an increase of 14 amu, suggesting that
compound 6 may be a methyl derivative of compound 1.In
addition, they had identical UV spectra, indicating that
compound 6 possesses one double bond at C-8 (9) like
compound 1. When compared to compound 1 (Fig. 3B),
compound 7 (Fig. 3D) showed a loss of 18 amu, suggesting
that compound 7 may be formed by a loss of H
2
Ofrom
compound 1. Moreover, compound 7 showed UV absorption
bands at 252, 243, and 235 nm indicating that this compound
possessed heteroannular conjugated diene in the skeleton [25],
which is different from those of compounds 1 and 6. Pure
compounds 6 and 7 were obtained by preparative HPLC and
determined by NMR (refer to the supplementary data). Their
1
Hand
13
C NMR spectra were identical to those of GA-Md and
GA-R that were previously reported [21,23]. Thus, compounds
6 and 7 were elucidated as 3α,22β-diacetoxy-7α-methoxy-
5α-lanosta-8,24E-dien-26-oic acid (named GA-Md) and
3α,22β-diacetoxy-5α-lanosta-7,9(11),24E-triien-26-oic acid,
respectively. Based on these results, possible mechanism of
acid-catalyzed compound 1 was proposed as shown in Fig. 4.
Finally, the cytotoxicity of the isolated GAs against human
HeLa cervical cancer cells was tested and summarized in
Table 3. Among the compounds examined, 3α,22-diacetoxy-
7α-hydroxyl-5α-lanost-8,24E-dien-26-oic acid (1)showed
the best cytotoxic activities against 95D and HeLa human
tumor cell lines with IC
50
values of 23.0 and 14.7 μM,
respectively. The other compounds showed similar activities
against the two tumor cell lines.
To date, although over 150 triterpenoids have been
isolated from Ganoderma spp., little is known about the
chemical stability of these compounds. For the first time, our
very recent work systematically described the stability of
7-O-ethyl GA-O in various conditions [26]. We deduced that
unstable property of 7-O-ethyl GA-O was ascribed to the
ether bond at C-7. Indeed, GA-Md that had a methoxy group
at C-7 was reported to be converted into GA-R by treatment
of H
2
SO
4
[20]. However, compound 1 in this work possesses
hydroxyl group instead of oxyalkyl at C-7 and was also prone
to be degraded in proptic environment. Based on the analysis
of its degraded products and kinetics, this compound showed
Table 2
Rate constant, half life and activation energy obtained from the degradation
of compound 1 at methanol.
Temperature (°C)
(correlation coefficient)
K
10
3
h
1
)
Half-life
(h)
Activation energy
(KJ/mol)
55(r
2
=0.966) 0.005 38.51 139.9
65(r
2
=0.927) 0.03 6.42
75(r
2
=0.999) 0.095 2.03
Fig. 2. Stability of compound 1 in different solvents at 40 °C after 2 h. AcN: acetonitrile, 80% AcN: 80% aqueous acetonitrile, CHCl
3
: chloroform, EtOAc: acetic
acetate, MeOH: methanol, 80% MeOH: 80% aqueous methanol, MeOH-Na: methanol containing 1 mM NaOH, MeOH-Ac: methanol containing 1 mM acetic acid,
and MeOHHCl: methanol containing 1 mM HCl. * denoted that compound 1 was completely degraded in the MeOHHCl solution.
119Y.-B. Li et al. / Fitoterapia 84 (2013) 115122
Page 5
C
D
B
A
6
1
7
24 hours
0 hour
Fig. 3. HPLC chromatograms of compound 1 in methanol at 0 and 24 h (A) and mass spectra of (B) compound 1, (C) 6, and (D) 7.
120 Y.-B. Li et al. / Fitoterapia 84 (2013) 115122
Page 6
a similar chemical reaction mechanism to that of 7-O-ethyl
GA-O [26]. Interestingly, we also found that GA-Mc (com-
pound 3) that contains an acetyl group at the position of C-7
was readily converted to GA-Mk (compound 2) at proptic
conditions (Fig. S12 in the supplementary data). The stability
of other GAs (2, 4, 5 and 7) that possess two pairs of double
bonds at the positions of C-7 and 9 (11) was also studied.
Even at the HClMeOH solution, these compounds were
shown rather stable (Fig. S12 in the supplementary data).
These results suggested that O-bearing group at the position
of C-7 and the double bonds at the position of C-8 (9) were
responsible for the inherent instable property of these
compounds. The triterpenoids isolated from Ganoderma spp.
were a class of highly oxygenated compounds, with hydroxyl,
carbonyl, acetyl or oxyalkyl groups at positions 3, 7, 11, 12
and 15, and often double bonds at the position of C-8 (9)
[2023,27,28]. As indicated in this work, it is interesting to
focus on the stability of these compounds, especially the ones
with hydroxyl group at C-7 and C-11. Since triterpenoids
were the principal constituents of Ganoderma spp. and
recommended for the quality evaluation of GA, attention
should be paid to the chemical stability of these compounds.
Therefore, this work may be useful to the further studies on
their purification, analysis and biological application.
Acknowledgments
Financial support from the National Basic Research
Program of China (973 program no. 2012CB721006), the
National Natural Science Foundation of China (NNSFC project
no. 30821005) and the National High Technology R&D
Program (863 project no. 2012AA021701) is gratefully
acknowledged. The authors also express their appreciation
to Dr. Jie-Li Wu (Analytical Center of Shanghai Jiao Tong
University) and Dr. Feng Lei (Analytical Center of Shanghai
Jiao Tong University) for their kind help in spectrum analysis.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2012.11.008.
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  • Source
    • "This mushroom was utilized by traditional Chinese medication for the treatment and prevention of human infections, namely gastric cancer, hypertension , hepatitis, chronic bronchitis, and hypercholesterolemia (Paterson, 2006). Also, it is the chosen organism for creating useful products (supplements, antimicrobial products, and biofilms) (Paterson, 2006), with significant interest in the literature due to its bioactive properties (Li et al., 2013; Liu et al., 2012; Ruan and Popovich, 2012). However, isolating EPS from the fungus is costly and the production rate is insufficient to meet the current market demand. "
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    • "54 7-Oxo-ganoderic acid Z G. lucidum, Li et al. (2006); G. resinaceum Peng et al. (2013) 55 7-Oxo-ganoderic acid Z 2 G. resinaceum Peng et al. (2013) 56 7-Oxo-ganoderic acid Z 3 G. resinaceum Peng et al. (2013) 57 Ganorbiformin B G. orbiforme Isaka et al. (2013) 58 Ganorbiformin C G. orbiforme Isaka et al. (2013) 59 Ganorbiformin D G. orbiforme Isaka et al. (2013) 60 Ganorbiformin E G. orbiforme Isaka et al. (2013) 61 Ganorbiformin F G. orbiforme Isaka et al. (2013) 62 3a,22b-Diacetoxy-7a-hydroxyl-5a-lanost-8,24E-dien-26-oic acid G. lucidum Li et al. (2013c) 63 3b,15a-Diacetoxy lanosta-8,24-dien-26-oic acid G. lucidum Lin et al. (1988a) 64 11a-Hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-26-oic acid G. lucidum Cheng et al. (2010) 65 11b-Hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-26-oic acid G. lucidum Cheng et al. (2010) 66 Ganoderic acid P G. lucidum, "
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  • Source
    • " whose extracts showed anti - invasive ef - fects on hepatoma cells , owing to extraordinary highs level of lucidenic acids ( Weng et al . 2007 ) . Moreover , a new ganoderic acid named 3α , 22β - diacetoxy - 7α - hydroxy - 5α - lanosta - 8 , 24E - dien - 26 - oic acid ( 18 ) isolated from G . lucidum mycelia with considerable cytotoxic activity ( Li et al . 2013 ) ."
    [Show abstract] [Hide abstract] ABSTRACT: Exploration of natural sources for novel bioactive compounds has been an emerging field of medicine over the past decades, providing drugs or lead compounds of considerable therapeutic potential. This research has provided exciting evidence on the isolation of microbe-derived metabolites having prospective biological activities. Mushrooms have been valued as traditional sources of natural bioactive compounds for many centuries and have been targeted as promising therapeutic agents. Many novel biologically active compounds have been reported as a result of research on medicinal mushrooms. In this review, we compile the information on bioactive structure-elucidated metabolites from macrofungi discovered over the last decade and highlight their unique chemical diversity and potential benefits to novel drug discovery. The main emphasis is on their anti-Alzheimer, antidiabetic, anti-malarial, anti-microbial, anti-oxidant, antitumor, anti-viral and hypocholesterolemic activities which are important medicinal targets in terms of drug discovery today. Moreover, the reader’s attention is brought to focus on mushroom products and food supplements available in the market with claimed biological activities and potential human health benefits. Keywords: Medicinal mushrooms. Anti-oxidant . Anti-tumor . Anti-HIV . Anti-microbial . Anti-viral . Hypocholesterolemic . Anti-diabetic . Anti-Alzheimer . Anti-malarial . Food supplements
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