Anti-inflammatory principles from Cordyceps sinensis

Article abstract of DOI:10.1021/np100902f

In order to explore the anti-inflammatory principles of the mycelia of Cordyceps sinensis, the crude extract and partially purified fractions were examined for their inhibition of superoxide anion generation and elastase release. Further chemical investigation of the bioactive fractions has resulted in the identification of 50 compounds, including five constituents, cordysinins A-E (1-5), reported from a natural source for the first time. In addition, compounds were examined for their anti-inflammatory activity. 1-(5-Hydroxymethyl-2-furyl)-β-carboline displayed the most significant inhibition of superoxide anion generation and elastase release with IC ₅₀ values of 0.45 ± 0.15 and 1.68 ± 0.32 μM, respectively.

Full text of DOI:10.1021/np100902f

NOTE
pubs.acs.org/jnp
Anti-inflammatory Principles from Cordyceps sinensis
Mei-Lin Yang,†,§ Ping-Chung Kuo,‡,§ Tsong-Long Hwang,^ and Tian-Shung Wu*,†,||
†Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, Republic of China
‡Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, Republic of China
^Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, Republic of China
||Department of Pharmacy and Chinese Medicine Research and Development Center, China Medical University, Taichung 401, Taiwan,
Republic of China
S Supporting Information
b
ABSTRACT:
In order to explore the anti-inflammatory principles of the mycelia of Cordyceps sinensis, the crude extract and partially purified
fractions were examined for their inhibition of superoxide anion generation and elastase release. Further chemical investigation of
the bioactive fractions has resulted in the identification of 50 compounds, including five constituents, cordysinins AꢀE (1ꢀ5),
reported from a natural source for the first time. In addition, compounds were examined for their anti-inflammatory activity. 1-(5-
Hydroxymethyl-2-furyl)-β-carboline displayed the most significant inhibition of superoxide anion generation and elastase release
with IC50 values of 0.45 ( 0.15 and 1.68 ( 0.32 μM, respectively.
he Cordyceps genus, including C. sinensis, C. militaris,
C. pruinosa, and C. ophioglossoides and more than 700 other
and water-soluble fractions, respectively. The n-hexane fraction
was partitioned between n-hexane and MeOHꢀH2O (70:30)
to yield a n-hexane/n-hexane fraction (CSEHH) and a n-hexane/
MeOHꢀH2O fraction (CSEHM). The water fraction was
further extracted with ethyl acetate to an afford an ethyl acetate
fraction (CSEE) and a water-soluble fraction (CSEW), respec-
tively. The crude extract and the partially purified fractions were
evaluated for their effects on superoxide anion generation
and elastase release. At the tested concentration (10 μg/mL),
CSEHH, CSEHM, and CSEE fractions displayed significant
inhibition of superoxide anion generation and elastase release
(44.02% and 70.76%; 94.99% and 97.10%; 82.73% and 97.49%,
respectively). Thus these fractions were subjected to further
purification to afford five compounds, cordysinins AꢀE (1ꢀ5),
reported from the natural source for the first time, along with 45
known compounds, 6ꢀ50. The structures of these constituents
were established on the basis of 1D and 2D NMR and mass
spectroscopic analyses and also by chemical transformation.
Compound 1 was isolated as an optically active, colorless
powder and by HR-FABMS showed a pseudomolecular formula
of C11H19O3N2. The IR absorption bands at 3402, 3236, and
T
species, constitutes a valuable fungal medicinal source in tradi-
tional Chinese medicine.1ꢀ3 Among these species, Cordyceps
sinensis is a parasitic fungus growing on the larvae of Lepidoptera,
and it has been reported to exhibit various pharmacological
activities.4 It has been used for treating coughs, for disorders of
the lungs, kidneys, and stomach, and also as a tonic food for
invigoration or nutritional supplementation.5 Previous research
results provided evidence that Cordyceps species possess antioxidant,6
anti-inflammatory,7,8 antitumor,9,10 immunomodulatory,1,11 and
kidney-protective bioactivities.12 However, until now there have
been no reports of the anti-inflammatory compounds from C.
sinensis. In the present paper the crude extracts and partial puri-
fied fractions of mycelia of C. sinensis were examined for their
inhibitory effects on superoxide anion generation and elastase
release by human neutrophils in response to N-formyl-methio-
nyl-leucyl phenylalanine/cytochalasin B (FMLP/CB). Several
fractions exhibited significant inhibition of superoxide anion
generation and elastase release, and these fractions were inves-
tigated for their chemical constituents and bioactivities.
Dried mycelia of C. sinensis were extracted with ethanol, and
the concentrated extracts (CSE) were suspended in H2O and
partitioned with n-hexane to afford n-hexane-soluble (CSEH)
Received: June 23, 2010
Published: August 17, 2011
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np100902f J. Nat. Prod. 2011, 74, 1996–2000
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
Figure 1. Chemical structures and significant NOESY and HMBC correlations of 1ꢀ5.
1658 cmꢀ1 indicated the presences of the amine, hydroxyl, and
amide carbonyl groups. In the H NMR spectrum, the proton
bands at 3268, 1620, and 1424 cmꢀ1 assignable to hydroxyl,
carbonꢀcarbon double-bond, and carbonꢀnitrogen double-
bond functionalities suggested a mixture of enatiomers of a
β-carboline derivative. In the 1H NMR spectrum, two mutually
coupled doublets with a small coupling constant (J = 5.4 Hz) at
δ 8.20 and 7.99 were indicative of H-4 and H-3 protons of a
β-carboline alkaloid skeleton. A set of four mutually coupled
aromatic protons at δ 8.17, 7.64, 7.54, and 7.25 suggested that the
aromatic ring A of β-carboline was unsubstituted. In addition,
two mutually coupled proton signals at δ 5.33 and 1.65 indicated
the presence of a CH3CHꢀ fragment. On the basis of the above
spectroscopic evidence, this enantiomeric mixture was con-
cluded to be 1-(9H-β-carbolin-1-yl)ethanol, which had been
previously reported as a synthetic product,18 but no enantiomeric
purity was determined. Thus with the aid of HPLC equipped
with a chiral column the relative composition of this mixture was
analyzed and the enantiomers were purified as cordysinins C (3)
and D (4), in which the area percentages of 3 and 4 were 49.5%
and 50.5%, respectively. To determine the stereochemistry at
C-10, Mosher’s reagents were utilized to prepare the (S)- and (R)-
MTPA esters of 3 and 4.14 The absolute configuration at C-10 in 3
was concluded as R, and that of 4 was determined as S (Figure 1).
The molecular formula of cordysinin E (5) was determined as
C14H14N2O2. The UV and IR spectra of 5 exhibited character-
istic absorption maxima and bands of a β-carboline chromo-
phore. In the 1H NMR spectrum, two ortho-coupled doublets at
δ 8.46 and 8.30 with a small coupling constant (J = 5.0 Hz) and a
set of four mutually coupled symmetrical-type aromatic protons
were similar to those of β-carboline. Moreover, five mutually
coupled protons at δ 3.75 (1H, m), 3.55 (2H, m), 2.06 (1H, dd,
J = 13.5, 4.8 Hz), and 1.87 (1H, dd, J = 13.5, 8.6 Hz) in the 1H
NMR spectrum along with the carbon signals at δ 72.8, 67.4, and
29.0 in the 13C NMR spectrum suggested the presence of
a ꢀCH2CHOHCH2OH substituent. In the HMBC experi-
1
signals of 1 were very similar with those of cyclo(L-Leu-L-Pro).13
The only spectral differences were the appearance of one
oxygenated methine group at δ 4.44 (1H) in 1 and the disap-
pearance of one methylene group. This indicated that the basic
skeleton of 1 was the same as cyclo(L-Leu-L-Pro) and one of
the methylene carbons in 1 was hydroxylated. In addition, the
HMBC experiment also displayed long-range correlations be-
tween δ 3.38 (H-4) and 68.8 (C-3) and between δ 2.20 (H-5)
and 68.8 (C-3), which suggested the C-3 was hydroxylated. The
absolute configuration at C-3 was further confirmed as R by
Mosher’s method.14 Therefore the structure of 1 was established
as cyclo(L-leucine-L-hydroxyproline) (Figure 1). It is a new
compound and named trivially as cordysinin A.
The molecular formula of compound 2 was established as
C11H15N5O4 by the HR-ESIMS analysis. The UV absorption
maxima at 262 and 207 nm and the IR absorption bands at 3337,
2929, 1645, and 1600 cmꢀ1 corresponded to the presence of
an adenosine derivative.15 The 1H NMR and 13C NMR spectra
suggested the presence of adenosine,15 and the only difference
between adenosine and 2 is one more methoxyl singlet at δ 3.42
3
(3H), which displayed J-HMBC correlation with the carbon
signal at δ 84.6 (C-20), indicating that the methoxyl substitution
was located at C-20. The stereochemistry of the sugar moiety of 2
was determined by the coupling constants and NOESY correla-
tion spectrum. Thus the chemical structure of 2 was established
as shown in Figure 1. Although this compound had already been
reported in the previous synthesis work,16 this is the first report
from a natural source, and it was trivially named cordysinin B (2).
The mixture of enatiomers 3 and 4 was purified through
the assistance of reversed-phase HPLC, and the molecular
formula was established as C13H12N2O using HR-ESIMS. The
UV absorption maxima characteristic of a β-carboline chromo-
phore at 349, 302, 289, and 251 nm17 and the infrared absorption
2
3
ment, the J- and J-correlations constructed the 1-substituted
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dx.doi.org/10.1021/np100902f |J. Nat. Prod. 2011, 74, 1996–2000

Journal of Natural Products
NOTE
β-carboline basic skeleton. The absolute configuration at C-20
was determined by comparison of the sign of optical rotation
and the CD spectrum of 5 with those of cordysinin C (3) and
cordysinin D (4). Compound5displayed a positive optical rotation
that was the same sign as that of cordysinin D (4). In addition, the
CD measurement of 3 displayed a positive Cotton effect in the
360ꢀ380 nm regions, and in contrast compound 4 exhibited a
negative Cotton effect in the same regions. Comparatively, the
CD spectrum of 5 showed a negative Cotton effect, which
suggested that the spatial arrangement of the functional group
at C-20 was the same as that of cordysinin D (4).19 Therefore,
the chemical structure of new compound 5 was characterized
as shown.
Forty-five known compounds were identified by comparison
of their spectral and physical data (see Supporting Information).
Those purified compounds isolated in sufficient quantity were
evaluated for inhibition of O2•ꢀ generation and elastase release
by human neutrophils in response to FMLP/CB (Table 1).20
Compounds ergosteryl-3-O-β-D-glucopyranoside (39) and per-
lolyrine (43) inhibited superoxide anion generation and elastase
release in FMLP/CB-activated human neutrophils in a concen-
tration-dependent manner. The compound perlolyrine demon-
strated the most significant inhibition toward superoxide anion
generation and elastase release with IC50 values of 0.45 ( 0.15
and 1.68 ( 0.32 μM, respectively, compared with the reference
compound indomethacin (IC50 of 38.32 ( 5.38 and 31.98 (
6.49 μM, respectively). The formation of O2•ꢀ in neutrophils can
be inhibited by modulating cellular signaling pathways, but also
by direct radical scavenging. Therefore the purified constituents
were also subjected to the DPPH radical scavenging assay in cell-
free systems (data not shown).21 Among the tested compounds,
only 30,40,7-trihydroxyisoflavone displayed significant scavenging
of DPPH free radicals with IC50 values of 31.97 μM, respectively,
compared with the reference compound vitamin C (IC50 of
38.63 μM). None of the other constituents significantly inhibited
free radical scavenging at 500 μM.
Table 1. Inhibitory Effects of Purified Samples on Superoxide
Anion Generation and Elastase Release by Human Neutro-
phils in Response to FMLP/CB
IC50 (μg/mL)a or (Inh %)b
compound
superoxide anion generation
elastase release
1
(11.34 ( 4.95)
(28.25 ( 2.73)d
6.19 ( 1.25
(13.02 ( 3.20)
(41.59 ( 6.25)d
(24.22 ( 6.57)c
(7.03 ( 3.76)
(2.79 ( 3.64)
(10.37 ( 0.55)
(11.72 ( 5.92)
(11.06 ( 6.14)
5.62 ( 0.37
16
17
25
(5.59 ( 3.26)
(6.59 ( 2.11)
(12.83 ( 6.94)
(5.65 ( 1.64)
(9.61 ( 5.51)
5.42 ( 0.50
28
30
31
32
39
40
(21.62 ( 6.28)
0.45 ( 0.15
(7.13 ( 5.55)
1.68 ( 0.32
43
48
(6.43 ( 3.72)
38.32 ( 5.38
(6.83 ( 2.46)
31.98 ( 6.49
indomethacin
a Concentration necessary for 50% inhibition. b Percentage of inhibition
(Inh %) at 10 μg/mL concentration. Results are presented as mean (
SEM (n = 3ꢀ4). c p < 0.05 compared with the control value. d p < 0.001
compared with the control value.
n-hexane/n-hexane fraction (650 g, CSEHH), a n-hexane/MeOHꢀH2O
fraction (150 g, CSEHM), an ethyl acetate fraction (110 g, CSEE), and a
water-soluble fraction (1090 g, CSEW), respectively.
The CSEHH fraction was purified with silica gel column chromatog-
raphy eluted by a step gradient of n-hexane/ethyl acetate (100:1 to 1:1)
to afford seven subfractions. Only subfractions 5 and 6 displayed
significant spots, so they were further purified. Subfraction 5 (15.2 g)
was column chromatographed over silica gel using a stepwise gradient of
n-hexane/EtOAc (10:1 to 1:1) to afford 6 (120.0 mg), 7 (4.2 mg), 8
(3.1 mg), 9 (4.0 mg), and 10 (3.0 mg), respectively. Further purification
of subfraction 6 (35.0 g) by repeated column chromatography with
n-hexane/EtOAc gradient mixtures (from 5:1 to 1:1) and preparative
TLC purification gave a mixture of 11 and 12 (360.4 mg), a mixture of
13 and 14 (3.8 mg), 15 (45.0 mg), and 16 (10.5 mg), respectively. The
CSEHM fraction was separated by column chromatography over silica
gel with gradient mixtures of chloroform and methanol (100:1, 75:1,
50:1, 25:1, 10:1, 5:1, 3:1, 1:1, and 0:1) to yield eight subfractions. The
second subfraction (4.3 g) was purified with silica gel column chroma-
tography eluted by chloroform/methanol (50:1) and recrystallized with
methanol to yield 17 (254.2 mg). Subraction 3 (5.6 g) was further
isolated by repeated column chromatography with gradient mixtures of
chloroform/methanol (from 50:1 to 1:1) and preparative TLC purifica-
tion with solvent mixtures of chloroform/methanol (10:1) to afford 18
(100.9 mg) and 19 (8.2 mg). Silica gel column chromatography of
subfraction 4 (3.5 g) by a mixture of chloroform/methanol (20:1) and
preparative TLC purification with the solvent pair of chloroform/
methanol (10:1) yielded 20 (4.0 mg). The fifth subfraction (10.2 g)
rechromatographed by a silica gel open column using step gradient
mixtures of chloroform/methanol (from 20:1 to 1:1) and further
recrystallization with chloroform/acetone afforded 21 (632.5 mg). Re-
peated silica gel column chromatography of subfraction 6 (13.2 g) with
solvent mixtures of chloroform/methanol (10:1) and further recrystalliza-
tion with chloroform/acetone gave 22 (15.3 mg) and 23 (524.0 mg),
respectively.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were deter-
mined using a Yanagimoto MP-S3 apparatus. Optical rotations were
measured using a JASCO DIP-370 polarimeter. The UV spectra were
obtained on a Hitachi UV-3210 spectrophotometer, and IR spectra were
recorded on a Shimadzu FTIR-8501 spectrophotometer. NMR spectra
were obtained on Bruker AMX-400 and AV-500 NMR spectrometers,
with tetramethylsilane (TMS) as internal standard, and the chemical
shifts are reported in δ values (ppm). The low- and high-resolution FAB
and ESI mass spectra were obtained on JEOL JMS-700 and Bruker
APEX II mass spectrometers, respectively. High-performance liquid
chromatography (HPLC) was performed on a Shimadzu LC-10ATVP
series pumping system equipped with a Shimadzu SPD-M10AVP
diode array detector. CD spectra were recorded with a JASCO J-720
spectropolarimeter.
Mycelia. C. sinensis mycelia from fermentation in germinated
soybean were provided by Taiwan Sugar Company (TSC) on January
2002. A voucher specimen (TSWu_TSC_200201) was deposited in the
Department of Chemistry, National Cheng Kung University, Tainan,
Taiwan.
Extraction and Isolation. The mycelia of C. sinensis were obtained
from the medium by centrifugation and dried at room temperature.
Mycelia of C. sinensis (10 kg) were extracted with ethanol (7 ꢁ 50 L)
under reflux for 8 h and concentrated to give a brown syrup (2.0 kg,
CSE). The extract was suspended in H2O and partitioned to afford a
The ethyl acetate-soluble CSEE fraction was subjected to silica gel
column chromatography with step gradient mixtures of chloroform
and methanol (20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1), yielding 10
1998
dx.doi.org/10.1021/np100902f |J. Nat. Prod. 2011, 74, 1996–2000

Journal of Natural Products
NOTE
subfractions. Subfraction 2 (3.6 g) was purified with silica gel column
chromatography using a stepwise gradient of chloroform/methanol
(from 20:1 to 1:1) and successive preparative TLC purification to
obtain 24 (5.4 mg), 25 (110.0 mg), 26 (5.1 mg), and 27 (15.3 mg). The
third subfraction (3.4 g) was rechromatographed with silica gel by
mixing eluents of chloroform/methanol (20:1), and then the resulting
fractions were recrystallized with chloroform/methanol to afford 28
(152.5 mg), 29 (5.7 mg), and 30 (70.6 mg), respectively. Column
chromatography over silica gel of the subfraction 4 (5.1 g) by a stepwise
gradient of chloroform/methanol (from 10:1 to 1:1) and recrystalliza-
tion of the afforded fractions with chloroform/methanol yielded 31
(220.2 mg) and 32 (65.6 mg), respectively. Further preparative TLC
purification of the minor fractions eluting with chloroform/methanol
(10:1) afforded 33 (6.4 mg), 34 (3.1 mg), and 35 (4.6 mg), respectively.
The fifth subfraction (8.4 g) was purified with open silica gel column
chromatography eluting by step gradient mixtures of chloroformꢀ
methanol (from 5:1 to 1:1) to afford six minor fractions (5.1ꢀ5.6).
Fraction 5.1 was recrystallized with acetone to yield 1 (120.0 mg), and
preparative TLC purification of the residues gave 36 (4.3 mg). Rechro-
matography of fraction 5.2 and recrystallization of the resulting minor
fractions afforded 37 (48.7 mg) and 38 (35.1 mg). Silica gel column
chromatography of fraction 5.3 and recrystallization with chloroform/
methanol yielded a white solid, 39 (134.7 mg), and further preparative
TLC purification gave the minor constituents 40 (5.2 mg) and 41 (5.0 mg).
Fraction 5.4 was rechromatographed with silica gel and further recry-
stallized with acetone to afford 42 (36.3 mg). The residual soluble was
purified by reversed-phase HPLC with a Supelco Discovery HS C-18
(250 ꢁ 4.6 mm, 5 μm) column eluted at 0.5 mL/min with MeOH/H2O
(70:30) to give 5 (4.1 mg). Fraction 5.5 was purified by repeated column
chromatography and preparative TLC to yield 43 (8.3 mg) and 44
(5.1 mg). Fraction 5.6 was subjected to column chromatography eluted
with a gradient of chloroform/methanol (from 50:1 to 5:1) to yield 45
(8.1 mg). Subfraction 7 (3.6 g) of CSEE was divided into five fractions
(7.1ꢀ7.5) by silica gel column chromatography using a stepwise gradient
of chloroform/methanol (3:1 to 1:1). Fractions 7.1 and 7.2 were
subjected to preparative TLC to afford 46 (3.5 mg) and 47 (5.2 mg),
and 48 (10.1 mg), respectively. Fraction 7.3 was rechromatographed
with silica gel open column and preparative TLC to give 49 (3.4 mg).
Fraction 7.4 was purified by repeated column chromatography and
preparative TLC with chloroform/methanol (5:1) to afford 2 (5.2 mg).
With the aid of preparative TLC by mixing eluents of chloroform and
methanol (5:1) the enatiomeric mixture of 3 and 4 was isolated from
fraction 7.5, and further resolution of this mixture was achieved by
reversed-phase HPLC equipped with Chiralcel OD-H (250 ꢁ 4.6 mm,
5 μm) at 0.5 mL/min with hexanes/2-propanol (90:10) as eluents to
yield 3 (2.6 mg, tR 14.8 min) and 4 (2.4 mg, tR 21.4 min), respectively.
The subfraction 8 (5.2 g) was rechromatographed with silica gel using a
stepwise gradient of chloroformꢀmethanol (2:1 to 1:1), and the result-
ing fractions were recrystallized with methanol to give 50 (12.3 mg).
Cordysinin A (1): colorless powder; mp 179ꢀ181 ꢀC (acetone);
[R]25D ꢀ37.8 (c 0.05, MeOH); IR (neat) νmax 3402, 3236, 1658, 1429,
1308, 1217, 1101 cmꢀ1; 1H NMR (acetone-d6, 500 MHz) δ 6.91 (1H, br
s, D2O exchangeable, NH-8), 4.46 (1H, m, H-6), 4.44 (1H, m, H-3),
4.21 (1H, br s, D2O exchangeable, OH-3), 4.11 (1H, m, H-9), 3.62 (1H,
dd, J = 12.4, 4.4 Hz, H-4), 3.38 (1H, d, J = 12.4 Hz, H-4), 2.20 (1H, m,
H-5), 2.08 (1H, m, H-5), 1.97 (2H, m, H-10 and -11), 1.49 (1H, qd,
J = 8.7, 8.7 Hz, H-10), 0.95 (6H, m, CH3-12 and -13); 13C NMR
(acetone-d6, 125 MHz): δ 171.3 (C-7), 167.3 (C-1), 68.8 (C-3), 58.2
(C-6), 54.8 (C-4), 54.0 (C-9), 39.4 (C-10), 38.2 (C-5), 25.4 (C-11),
23.3 (C-13), 22.2 (C-12); FABMS m/z 227 ([M + H]+); HR-FABMS
m/z 227.1395 ([M + H]+, calcd for C11H19O3N2, 227.1396).
1222, 1094 cmꢀ1; 1H NMR (CD3OD, 500 MHz) δ 8.33 (1H, s, H-2),
8.19 (1H, s, H-8), 6.06 (1H, d, J = 6.1 Hz, H-10), 4.49 (1H, dd, J = 5.0, 2.8
Hz, H-30), 4.43 (1H, dd, J = 6.1, 5.0 Hz, H-20), 4.16 (1H, ddd, J = 2.8, 2.8,
2.5 Hz, H-40), 3.89 (1H, dd, J = 12.5, 2.5 Hz, H-50), 3.76 (1H, dd,
J = 12.5, 2.8 Hz, H-50), 3.42 (3H, s, OCH3-20); 13C NMR (CD3OD, 125
MHz) δ 157.6 (C-5), 153.7 (C-8), 150.1 (C-4), 141.8 (C-2), 121.0
(C-6), 89.2 (C-10), 88.4 (C-40), 84.6 (C-20), 70.9 (C-30), 63.2 (C-50),
58.8 (OCH3-20); ESIMS m/z 304 ([M + Na]+, 100), 283 (14), 282
(20); HR-ESIMS m/z 304.1023 ([M + Na]+, calcd for C11H15N5O4Na,
304.1022).
Cordysinin C (3): light yellow powder; mp 169ꢀ171 ꢀC (EtOAc/
MeOH); [R]25D ꢀ57.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 349
(3.76), 302 (4.09), 289 (4.29), 251 (4.57, sh), 241 (4.63), 236 (4.63),
208 (4.48) nm; IR (neat) νmax 3268, 3072, 2828, 1620, 1578, 1489,
1424, 1310, 1229 cmꢀ1; 1H NMR (CD3OD, 400 MHz) δ 8.20 (1H, d,
J = 5.4 Hz, H-4), 8.17 (1H, d, J = 8.0 Hz, H-5), 7.99 (1H, d, J = 5.4 Hz,
H-3), 7.64 (1H, dd, J = 8.2, 0.9 Hz, H-8), 7.54 (1H, ddd, J = 8.2, 8.0, 0.9
Hz, H-7), 7.25 (1H, t, J = 8.0 Hz, H-6), 5.33 (1H, q, J = 6.6 Hz, H-10),
1.65 (3H, d, J = 6.6 Hz, CH3-20); ESIMS m/z 213 ([M + H]+, 86), 195
(100); HR-ESIMS m/z 213.1029 [M + H]+ (calcd for C13H13N2O,
213.1028); CD (MeOH, c 0.00157) [θ]378 +47, [θ ]356 ꢀ93, [θ]336 +25,
[θ]318 +20, [θ]306 ꢀ20, [θ]284 +451, [θ]226 +395.
Cordysinin D (4): yellow powder (EtOAc/MeOH); mp 168ꢀ170 ꢀC
(EtOAc/MeOH); [R]25 +60.4 (c 0.05, MeOH); UV (MeOH) λmax
D
(log ε) 350 (3.98), 303 (4.23), 289 (4.49), 251 (4.74, sh), 241 (4.78),
236 (4.82), 209 (4.66) nm; IR (neat) νmax 3268, 3072, 2828, 1620, 1578,
1489, 1424, 1310, 1229 cmꢀ1; 1H NMR (CD3OD, 400 MHz) δ 8.20
(1H, d, J = 5.4 Hz, H-4), 8.16 (1H, d, J = 7.7 Hz, H-5), 7.99 (1H, d, J = 5.4
Hz, H-3), 7.64 (1H, dd, J = 8.2, 0.9 Hz, H-8), 7.54 (1H, ddd, J = 8.2, 7.8,
0.9 Hz, H-7), 7.25 (1H, t, J = 7.7 Hz, H-6), 5.33 (1H, q, J = 6.6 Hz, H-10),
1.65 (3H, d, J = 6.6 Hz, CH3-20); ESIMS m/z 213 ([M + H]+, 100), 195
(96); HR-ESIMS m/z 213.1029 ([M + H]+, calcd for C13H13N2O,
213.1028); CD (MeOH, c 0.00236) [θ]383 +23, [θ]349 +226, [θ]312 +129,
[θ]301 ꢀ34, [θ]281 +434, [θ]252 +286, [θ]217 +228.
Cordysinin E (5): light yellow powder; mp 210 ꢀC (dec) (MeOH);
[R]25D +45.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 379 (2.79), 312
(2.84), 283 (3.22), 217 (3.62) nm; IR (neat) νmax 3286, 2911, 1593,
1369, 1265, 1201 cmꢀ1; 1H NMR (CD3OD, 500 MHz) δ 8.46 (1H, d,
J = 5.0 Hz, H-4), 8.30 (1H, d, J = 5.0 Hz, H-3), 8.22 (1H, d, J = 7.9 Hz,
H-5), 7.71 (1H, d, J = 8.0 Hz, H-8), 7.59 (1H, t, J = 8.0 Hz, H-7), 7.31
(1H, t, J = 7.9 Hz, H-6), 3.75 (1H, m, H-20), 3.55 (2H, m, H-30), 2.06
(1H, dd, J = 13.5, 4.8 Hz, H-10), 1.87 (1H, dd, J = 13.5, 8.6 Hz, H-10); 13C
NMR (CD3OD, 125 MHz) δ 143.5 (C-13), 138.6 (C-4), 137.5 (C-10),
136.9 (C-1), 133.3 (C-11), 130.3 (C-7), 122.7 (C-5), 121.7 (C-12),
121.6 (C-6), 120.1 (C-3), 113.5 (C-8), 72.8 (C-20), 67.4 (C-30), 29.0 (C-
10); FABMS m/z 243 ([M + H]+, 1), 217 (3), 192 (8), 176 (9), 154
(100); HR-FABMS m/z 243.1135 [M + H]+ (calcd for C14H15N2O2,
243.1134); CD (MeOH, c 0.00206) [θ]380 +24, [θ]338 +65, [θ]318 ꢀ42,
[θ]290 ꢀ37, [θ]267 +8, [θ]245 ꢀ37, [θ]226 ꢀ58, [θ]206 +122.
•ꢀ
Measurement of O2 Generation and Elastase Release.
•ꢀ
The O2 generation and elastase release assays were based on the
reported method.20
Diphenyl Picrylhydrazyl (DPPH) Assay. The DPPH free radical
scavenging assay was based on the reported method.21
Statistical Analysis. Results were expressed as mean ( SEM.
Computation of 50% inhibitory concentration (IC50) was computer-
assisted(PHARM/PCSv.4.2). Statisticalcomparisonsweremadebetween
groups using Student’s t test. Values of p less than 0.05 were considered to
be statistically significant.
’ ASSOCIATED CONTENT
Cordysinin B (2): light yellow needles; mp 220 ꢀC (dec) (MeOH);
[R]25D ꢀ47.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 262 (4.01),
207 (4.18) nm; IR (neat) νmax 3337, 2929, 1645, 1600, 1464, 1321,
S
Supporting Information. Extraction and partial purifica-
b
tion of the mycelia of C. sinensis, the known compounds, and their
1999
dx.doi.org/10.1021/np100902f |J. Nat. Prod. 2011, 74, 1996–2000

Journal of Natural Products
NOTE
references, and the preparation of MTPA esters of 1, 3, and 4,
along with the NMR spectra for 1ꢀ5 are provided. This
information is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 886-6-2757575, ext 65333. Fax: 886-6-2740552. E-mail:
tswu@mail.ncku.edu.tw.
Author Contributions
§Both authors provided equal contributions to this work.
’ ACKNOWLEDGMENT
The authors are thankful for the financial support from the
National Science Council, Taiwan, ROC, awarded to T.S.W. This
study is supported in part by Taiwan Department of Health
Clinical Trial and Research Center of Excellence (DOH 100-TD-
B-11-004).
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