Terpene and lignan glycosides from the twigs and leaves of an endangered conifer, Cathaya argyrophylla

Article abstract of DOI:10.1016/j.phytochem.2012.07.013

Labdane diterpene glycosides cathargyroside A and cathargyroside B, monoterpene glycosides vervenone-10-O-β-d-glucopyranoside and vervenone-10-O-β-d-apiofuranosyl-(1″→6′) -β-d-glucopyranoside, as well as lignan glycosides cedrusinin-4-O-α- l-rhamnopyranoside and (+)-cyclo-olivil-9′-O-β-d-xylopyranoside, along with 39 known compounds, were obtained from the methanol extract of the twigs and leaves of Cathaya argyrophylla. These compounds were identified mainly by analyzing their NMR and MS data. Almost all of these compounds were hitherto unknown in this genus. The isolated compounds were screened against Candida albicans and Staphylococcus aureus for antimicrobial assay, and against K562, HT-29, BEL-7402, SGC-7901, B16, BGC-823, U251 and A549 cancer cell lines for cytotoxic activities. One compound showed antimicrobial activity against C. albicans, and four of them displayed cytotoxicity. Similarity analysis on the chemical constituents of the genera Cathaya, Picea and Pinus supported their close phylogenetic relationships.

Full text of DOI:10.1016/j.phytochem.2012.07.013

Phytochemistry 83 (2012) 63–69
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Terpene and lignan glycosides from the twigs and leaves of an endangered conifer,
Cathaya argyrophylla
Wen-Jun He a,b,1, Zhao-Hui Fu , Guang-Zhi Zeng , Yu-Mei Zhang , Hong-Jin Han , He Yan a,b,
a,1
a
a,
⇑
a
a,b
a
a,
⇑
Chang-Jiu Ji , Hong-Biao Chu , Ning-Hua Tan
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China
Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 March 2011
Received in revised form 11 July 2012
Available online 6 September 2012
Labdane diterpene glycosides cathargyroside A and cathargyroside B, monoterpene glycosides verve-
00
0
none-10-O-b-
as well as lignan glycosides cedrusinin-4-O-
D
-glucopyranoside and vervenone-10-O-b-
D
-apiofuranosyl-(1 ?6 )-b-
D
-glucopyranoside,
0
a-
L
-rhamnopyranoside and (+)-cyclo-olivil-9 -O-b-
D
-xylopy-
ranoside, along with 39 known compounds, were obtained from the methanol extract of the twigs and
leaves of Cathaya argyrophylla. These compounds were identified mainly by analyzing their NMR and
MS data. Almost all of these compounds were hitherto unknown in this genus. The isolated compounds
were screened against Candida albicans and Staphylococcus aureus for antimicrobial assay, and against
K562, HT-29, BEL-7402, SGC-7901, B16, BGC-823, U251 and A549 cancer cell lines for cytotoxic activities.
One compound showed antimicrobial activity against C. albicans, and four of them displayed cytotoxicity.
Similarity analysis on the chemical constituents of the genera Cathaya, Picea and Pinus supported their
close phylogenetic relationships.
Keywords:
Cathaya argyrophylla
Pinaceae
Labdane diterpene glycosides
Lignan glycosides
Cathargyrosides A–B
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
In our program, aimed to characterize new active compounds
in the ITS1 region indicated that the genera Cathaya, Picea and Pi-
nus are closely related (Kan et al., 2007; Wang et al., 1998). Chemo-
taxonomic study of the above genera using serratane-type
triterpenes as a chemotaxonomic marker confirmed further their
close taxonomic phylogenetic relationships (He et al., 1981). There
are only five serratane-type triterpenes (Ma et al., 1981) and two
flavonoids (Ma et al., 1982; Qiu et al., 1981) previously isolated
from C. argyrophylla. In the current study, 45 compounds from
the twigs and leaves of C. argyrophylla were isolated and character-
ized. They were used to screen their cytotoxic and antimicrobial
activities, and to consider the phylogenetic relationships of the
genera Cathaya, Picea and Pinus based on the chemical constituent
analysis.
from gymnosperms, many natural compounds have been isolated
and identified, and some have exhibited inhibitory activities
against cancer cell lines (Fu et al., 2008; He et al., 2011a,b; Xu
et al., 2006a,b; Xu and Tan, 2007; Zhang and Tan, 2007; Zhang
et al., 2004, 2005a,b, 2007, 2009). In this regard, Cathaya argyrophy-
lla Chun et Kuang Gord. (Pinaceae) is a gymnosperm indigenous to
China and classified as a palaeoendemic plant. It has also been
listed as one of the eight most endangered plant species in China
(Wang and Xie, 2004) with the total number of individual plants
being less than 4,000 and trivially named as ‘‘giant panda’’ in the
plant kingdom. The history of its fossil record dates back to the
Cretaceous period (Chun and Kuang, 1962; Wang and Ge, 2006).
It is considered as being important in its conservation and molec-
ular phylogeny (Ge et al., 1998; Kan et al., 2007; Wang and Ge,
2
. Results and discussion
The methanol extract of the twigs and leaves of C. argyrophylla
2
006; Wang et al., 1998, 1997). Nuclear ribosomal DNA (nrDNA)
was subjected to silica gel, MCI, RP-8, Sephadex LH-20 column
chromatographic (CC) and semipreparative HPLC purification
steps to give compounds 1–45. The structures of the new com-
pounds 1–6 (Fig. 1) were elucidated by examining both their spec-
troscopic data and other chemical evidence. In addition to the six
new compounds, two known megastigmane glycosides icariside
has also been used to study phylogenetic relationships at many
taxonomic levels. Phylogenetic relationship studies of GC content
⇑
Corresponding authors. Tel./fax: +86 871 5223800.
E-mail addresses: zymei@mail.kib.ac.cn (Y.-M. Zhang), nhtan@mail.kib.ac.cn
B5 (7) (Matsunami et al., 2010) and dihydrovomifoliol-O-b-
D-glu-
(
N.-H. Tan).
1
These authors have contributed equally to this work.
copyranose (8) (Miyase et al., 1988), six known lignan glycosides
0
031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.07.013

6
4
W.-J. He et al. / Phytochemistry 83 (2012) 63–69
Table 1
Antimicrobial and cytotoxic activities of selected compounds from C. argyrophylla.a
Compounds
IC50
(lM)
IC50 (lM)
C. albicans
HT-29
1
3
3
3
4
7
3
4
9
0
10.74 ± 1.25
–
–
–
–
1.26 ± 0.18
1.12 ± 0.15
1.19 ± 0.06
2.01 ± 0.18
–
Miconazole nitrate salt
Taxol
2.50 ± 0.28
0.006 ± 0.0002
a
HT-29: human colon cancer cell line; ‘‘–’’ means not active; IC50 > 10 lg/mL was
considered to be inactive.
Table 2
1
3C NMR spectroscopic data for compounds 1–4 in CD
3
OD.
No.
1
2
3
4a,b
C
d , mult.
d
C
, mult.
d
C
, mult.
d
C
, mult.
1
2
3
4
5
6
7
8
9
1
1
1
13
1
39.2 t
19.9 t
39.9 t
44.7 s
65.4 d
200.7 s
128.4 d
164.8 s
58.6 d
45.4 s
22.5 t
45.7 t
74.2 s
146.2 d
112.4 t
27.8 q
22.8 q
29.6 q
175.9 s
15.5 q
95.8 d
74.2 d
78.1 d
71.1 d
79.0 d
62.5 t
39.1 t
19.9 t
39.9 t
44.7 s
65.3 d
200.6 s
128.5 d
164.4 s
57.4 d
45.4 s
22.7 t
42.7 t
139.1 s
125.9 d
59.4 t
16.3 q
22.8 q
29.6 q
175.8 s
15.6 q
95.8 d
74.2 d
78.2 d
71.1 d
79.0 d
62.5 t
59.5 d
206.6 s
119.5 d
172.3 s
46.7 d
55.7 s
42.2 t
26.8 q
22.4 q
70.4 t
58.5 d
202.6 s
119.4 d
169.0 s
45.4 d
53.5 s
40.7 t
26.2 q
22.0 q
69.8 t
Fig. 1. Structures of compounds 1–6 isolated from twigs and leaves of C.
argyrophylla.
massonianoside B (9) (Bi et al., 2001), (2S,3R)-2,3-dihydro-7-hy-
0
0
droxy-2-(4 -hydroxy-3 -methoxyphenyl)-3-hydroxymethyl-5-ben-
0
zofuranpropanol 4 -O-b-
002), (2S,3R)-2,3-dihydro-7-methoxy-2-(4 -hydroxy-3 -methoxy-
phenyl)-3-hydroxymethyl-5-benzofuranpropanol 4 -O-b-
D
-glucopyranoside (10) (Machida et al.,
0
0
2
0
1
2
0
D-gluco-
pyranoside (11) (Machida et al., 2002), (ꢀ)-(7R,8R)-threo-
0
0
0
7
,9,3 ,9 -tetrahydroxy-3-methoxy-8-4 -oxyneolignan-4-O-b-
D
-
4
xylopyranoside (12) (Lundgren et al., 1981; Pan and Lundgren,
⁄
15
1
995, 1996; Thomas and Olof, 1977) and a mixture of (ꢀ)-(7S ,8-
1
1
6
7
⁄
0
0
0
R )-erythro-7,9,3 ,9 -tetrahydroxy-3-methoxy-8-4 -oxyneolignan-
⁄
⁄
0
0
4
-O-b-
D
-xylopyranoside (13) and (ꢀ)-(7R ,8S )-erythro-7,9,3 ,9 -
18
19
0
tetrahydroxy-3-methoxy-8-4 -oxyneolignan-4-O-b-
D
-xylopyrano-
2
1
2
3
4
5
6
0
side (14) (Lundgren et al., 1981; Pan and Lundgren, 1995, 1996;
Thomas and Olof, 1977), three known phenylpropanoid glycosides
0
104.0 d
75.0 d
78.0 d
71.6 d
78.1 d
62.7 t
104.3 d
75.1 d
78.5 d
71.7 d
77.3 d
69.0 t
0
0
0
0
0
dihydroconiferyl alcohol 4-O-b-
D
-glucopyranoside (15) (Higuchi
0
0
et al., 1977), 2-O-[4 -(a-hydroxypropyl)-2 -methoxyphenyl]-1-O-
b-
D-glucopyranosyl glycerol (16) (Pan and Lundgren, 1996) and
b-hydroxypropiovanillone 3-O-b-
D
-glucopyranoside (17) (Xiang
a
13C NMR data (d
et al., 2004), one known flavonoid glycoside quercitrin (18) (Yin
C
) were measured in C
5 5
D N for 4.
b
00
00
0
Data of the apiofuranosyl unit of 4, d 111.2 (d, C-1 ), 77.8 (d, C-2 ), 80.5 (s, C-
et al., 2002), three known aromatic glycosides 1 -O-benzyl-
a
-L
-
C
0
0
00
00
00
0
3 ), 75.0 (t, C-4 ), 65.5 (t, C-5 ).
rhamnopyranosyl-(1 ?6 )-b-
D
-glucopyranoside (19) (Hamerski
-glucopyranoside (20)
Pan and Lundgren, 1995) and tachioside (21) (Pan and Lundgren,
et al., 2005), 3,4-dimethoxyphenyl-b-
D
(
illin (43) (Fu et al., 2008) and benzoic acid (44), and b-sitosterol
(45) (Fu et al., 2008) were also isolated from C. argyrophylla. Their
structures were confirmed by comparison of their H NMR,
1
995), eight known lignans (+)-berchemol-9-acetate (22) (Ando
1
13
et al., 2007), (ꢀ)-berchemol (23) (Ando et al., 2007), (ꢀ)-secoisol-
ariciresinol (24) (Achenbach et al., 1983), (ꢀ)-carinol (25) (Achen-
bach et al., 1983), (+)-isolariciresinol (26) (Fonseca et al., 1978),
C
NMR, [ and MS data with the corresponding authentic sample
a]
D
or literature data. Using HPLC, compound 22 was not detected in
the crude extract and should thus be an artifact during isolation.
All compounds were tested against Candida albicans and Staphylo-
coccus aureus for their antimicrobial activities, and against K562,
HT-29, BEL-7402, SGC-7901, B16, BGC-823, U251 and A549 cancer
cell lines for cytotoxic activities. Compound 17 showed antimicro-
(
+)-cyclo-olivil (27) (Kanchanapoom et al., 2006), (+)-dihydrode-
hydrodiconiferyl alcohol (28) (Fu et al., 2008) and (+)-1-hydrox-
ypinoresinol (29) (Tsukamoto et al., 1984), three known
diterpenes 12,13E-ozic acid (30) (Deng et al., 1997), 13-epicupres-
sic acid methyl ester (31) (Su et al., 1994) and 13-hydroxy-
8
,11,13-podocarpatrien-18-oic acid (32) (Cheung et al., 1993),
bial activity against C. albicans with the IC50 value of 10.74 lM.
Compounds 33, 34, 39 and 40 displayed cytotoxicity against the
HT-29 cell line with the IC50 values of 1.26, 1.12, 1.19, and
eight known triterpenes 3 -methoxyserrat-14-en-21b-ol (33) (Ta-
a
naka et al., 1994a), 3b,21b-dihydroxyserrat-14-ene (34) (Tanaka
et al., 1994b), 21b-methoxyserrat-14-en-3b-ol (35) (Conner
et al., 1980; Rowe et al., 1972), 3b-hydroxyserrat-14-en-21-one
2.01
Cathargyroside A (1) was obtained as colorless solid, and its
molecular formula was deduced to be C26 by negative ion
HRESIMS (calcd for C26 495.2594; found 495.2595), requiring
7° of unsaturation. The IR spectrum suggested the presence of hy-
lM, respectively (Table 1).
(36) (Chernenko et al., 1993), serratenediol-3b-acetate (37) (Pei
40 9
H O
et al., 2004), 21b-hydroxyserrat-14-en-3-one (38) (Tanaka et al.,
39 9
H O
1
1
994b), 21b-methoxyserrat-14-en-3-one (39) (Tanaka et al.,
994a) and serrat-14-en-3,21-dione (40) (Tanaka et al., 1997),
ꢀ
1
ꢀ1
droxy (3375 cm ), ester (1749 cm ) and a,b-unsaturated ketone
ꢀ1
1
13
two known flavonols kaempferol (41) (Fu et al., 2008) and querce-
tin (42) (Xu and Tan, 2007), two known aromatic compounds van-
(1677 cm ) groups. Observed in the H and C NMR spectra (Ta-
bles 2 and 3) were four tertiary methyl groups [d 0.96 (3H, s), 1.28
H

W.-J. He et al. / Phytochemistry 83 (2012) 63–69
65
Table 3
1
3
H NMR spectroscopic data for compounds 1–4 in CD OD.
No.
1
1
2
3
4a,b
H
d (J in Hz)
d
H
(J in Hz)
1.88 overlapped
.34 overlapped
d
H
(J in Hz)
d
H
(J in Hz)
1.87 m
1.34 overlapped
1.51 m
2.60 m
2.64 m
1
2
3
1.49 overlapped
2.22 overlapped
2.23 m
6.06 bs
6.44 bs
1
.34 overlapped
1.34 overlapped
2.45 s
5
2.43 s
2.53 m
2.33 t (5.7)
7
7
8
9
1
a
b
5.82 bs
5.85 bs
2.92 dt (9.3, 5.5)
2.11 d (9.3)
1.53 s
1.01 s
4.53 dd (17.6, 1.7)
2.56 dt (9.0, 5.7)
1.89 d (9.0)
1.21 s
0.85 s
4.97 dd (17.1, 1.7)
4.51 dd (17.1, 1.7)
2.21 overlapped
1.57 overlapped
2.29 m
0
1
2
4.39 dd (17.6, 1.7)
1
1
1.66 m
1.59 m
2.35 m
2.13 m
1
.55 overlapped
1.86 overlapped
.62 overlapped
1
1
1
1
1
1
1
2
1
2
3
4
5
6
6
4
5.93 dd (17.4, 10.8)
5.22 dd (17.4, 1.4)
5.06 dd (10.8, 1.4)
1.28 s
2.01 s
1.43 s
5.42 m
4.10 d (6.8)
5a
5b
6
7
8
1.72 s
2.04 s
1.43 s
0.96 s
5.40 d (8.2)
3.30 overlapped
3.41 m
3.32 overlapped
3.34 overlapped
3.85 dd (12.1, 1.5)
3.67 dd (12.1, 4.8)
0
0.96 s
5.40 d (8.2)
0
4.33 d (7.7)
3.45 m
3.30 m
3.50 overlapped
3.50 overlapped
3.70 m
4.88 d (7.7)
0
0
0
0
3.30 overlapped
3.41 m
3.32 overlapped
3.34 overlapped
3.85 dd (12.1, 1.6)
3.67 dd (12.1, 4.8)
4.08 overlapped
4.21 overlapped
4.07 overlapped
4.10 overlapped
4.74 d (12.0)
0
0
a
b
3.65 m
4.17 overlapped
a
1H NMR data (d
H
) were measured in C
Data of the apiofuranosyl unit of 4, d
5 5
D N for 4.
b
00
00
00
00
00
H
5.78 (1H, d, 2.5, H-1 ), 4.75 (1H, bs, H-2 ), 4.59 (1H, d, 9.3, H-4 a), 4.34 (1H, d, 9.3, H-4 b), 4.14 (2H, s, H-5 ).
(
(
(
d
(
3H, s), 1.43 (3H, s), 2.01 (3H, s), d
q)], an ,b-unsaturated ketone [d
s), 200.7 (s)], an ABM coupling system of three olefinic protons at
5.06 (1H, dd, J = 10.8, 1.4 Hz), 5.22 (1H, dd, J = 17.4, 1.4 Hz), 5.93
1H, dd, J = 17.4, 10.8 Hz), d 146.2 (d), 112.4 (t), an ester carbonyl
group [d 175.9 (s)], and an oxygenated carbon [d 74.2 (s)]. Thus,
C
15.5 (q), 22.8 (q), 27.8 (q), 29.6
C-16 carbons were similar to those of the corresponding carbons
in the co-occurring 13-epicupressic acid methyl ester (31) and
7b,13S-dihydroxylabda-8(17),14-dien-19-oic acid (Wang et al.,
2008), indicating that the configuration of the C-13 position is S.
Therefore, structure 1 was established as 13S-hydroxy-labda-
a
H
5.82 (1H, bs), d 128.4 (d), 164.8
C
H
C
C
C
7,14-dien-6-oxo-19-oic acid b-
D-glucopyranoside.
compound 1 had a bicyclic-labdane diterpenoid skeleton. Compar-
ison of the NMR spectroscopic data of 1 with those of 6-oxo-isom-
anol (Zdero et al., 1991) established that they were quite similar,
except for a moiety on ring A. The presence of the partial structures
HRESIMS of 2 gave a molecular formula of C26H O , the same
as compound 1. Its H and C NMR spectroscopic data were sim-
ilar to those of 1, with the significant difference being the absence
40 9
1
13
of the partial structure –C(CH
and the presence of the partial structure –C(CH
3
)(OH)–CH@CH
2
(C-13 to C-16) in 2
)@CHCH (OH)
–
CH
2
CH
2
CH
2
– (C-1 to C-3) and –CH(C-9)–CH
2
(C-11)–CH
2
(C-12)–
3
2
1
1
was inferred from the H– H COSY and HSQC spectra of 1. Through
careful analysis of the HMBC spectrum (Figure S1, Supplementary
information), the partial structures were correlated to constitute a
(C-13 to C-16) which was supported by the measurement of 2D
NMR spectroscopic data in 2 as follows. Observations of HMBC
cross-peaks of H-15 (d
and of H-16 (d 1.72, 3H, s) with C-12, C-13 and C-14 supported
this assignment. Chemical shifts at C-13, C-14, C-15, and C-16 in
2 were similar to those of 7 -hydroxyisocupressic acid (Lin and
Rosazza, 1998), further supporting the presence of the partial
structure –C(CH )@CHCH (OH) (C-13 to C-16) and the E-geometry
H
4.10, 2H, d, J = 6.8 Hz) with C-13, C-14,
labdane on the basis of the cross-peaks of: H-5 (d
C-4, C-6, C-9, C-10, C -18, C -19 and C -20; H-14 (d
J = 17.4, 10.8 Hz) with C-12, C-13, C-16; H-17 (d 2.01, 3H, s) with
C-7, C-8, C-9; H-18 (d 1.43, 3H, s) with C-3, C-4, C-5 and C-19; H-
0 (d 0.96, 3H, s) with C-1, C-5, C-9 and C-10, respectively. The
signals at d 95.8 (d), 79.0 (d), 78.1 (d), 74.2 (d), 71.1 (d) and
2.5 (t) in the C NMR spectra of 1, established that the sugar
H
2.43, 1H, s) with
H
H
5.93, 1H, dd,
H
a
H
2
H
3
2
C
of the double bond at C-13. Comprehensive 2D NMR (HSQC, HMBC
and ROESY) correlation analysis further established the full struc-
1
3
6
was a glucose moiety, with the b-configuration of the glucose
ture 2 as 15-hydroxy-labda-7,13E-dien-6-oxo-19-oic acid b-D-
determined from the coupling constant (8.2 Hz) of the anomeric
proton signal in the H NMR spectrum. The configuration of the su-
glucopyranoside, named cathargyroside B.
1
The IR spectrum of vervenone-10-O-b-D-glucopyranoside (3)
ꢀ1
gar unit was confirmed as
D-glucose, after acid hydrolysis of 1 by
indicated the presence of hydroxy (3426 cm ) and an a,b-unsatu-
ꢀ1
analysis of its R
f
and its specific rotation. The correlation between
rated ketone (1672 cm ) which was confirmed by analysis of the
UV spectrum with the absorption maximum at 251 nm. Compound
3 exhibited a quasi-molecular ion peak at m/z 329 in the positive
FABMS. Its positive HRESIMS had the same molecular formula as
0
H-1 of the glucose and C-19 in the HMBC spectrum suggested that
the b-
correlations of Me-20 to H-11, but not to H-5 and from H-5 to H-
, Me-18, but not to Me-20 indicated a trans fusion of the A and
D-glucopyranosyl moiety was located at C-19. The ROESY
9
that of vervenone-8-O-b-
D
-glucopyranoside (Shikishima et al.,
2001). The H and C NMR spectroscopic data of 3 were also sim-
ilar to vervenone-8-O-b- -glucopyranoside, the difference being in
the location of the b- -glucopyranose which was determined as
1
13
B rings, a trans orientation of H-9 and Me-20, as well as a cis orien-
tation of H-5 and Me-18 (Figure S1, Supplementary information).
The shifts of the resonances for the C-12, C-13, C-14, C-15, and
D
D

6
6
W.-J. He et al. / Phytochemistry 83 (2012) 63–69
follows. Long-range correlations were observed from H-10a, 10b
32
A molecular formula of C25H O11 was established for 6 through
0
0
(d
H
4.53, 4.39) to C-1 of glc (d
C
104.0) as well as H-1 (d
H
4.33) to
high-resolution ESI-TOF-MS measurement of the ion at m/z
ꢀ
C-10 (d 70.4) in the HMBC experiment (Figure S1, Supplementary
C
507.1858 [M ꢀ H] (calcd: 507.1866). The existence of a sugar unit
information), which indicated the point of attachment of the b-glu-
copyranose to C-10. ROESY (Figure S1, Supplementary information)
correlations of H-8 to H-1, H-5, and of H-9 to H-7 implied that C-7
H
was indicated by the observation of an anomeric proton at d 4.27
(1H, d, J = 7.5 Hz) and the corresponding anomeric carbon at d
C
1
3
105.6. The C NMR spectroscopic data of the sugar unit were con-
sistent with those of xylose. The large coupling constant for the
anomeric proton indicated a b-configuration for the xylopyranosyl
moiety. Acid hydrolysis of 6, with 2 N HCl at 70 °C for 6 h, produced
possessed b-orientation. Acid hydrolysis of 3 released
D
-glucose,
O). Full 1H and C NMR spectro-
scopic assignments of 3 were carried out by detailed 2D analyses
1
8
13
with an ½
aꢁ
þ 45:3 (c 0.21, H
2
D
1
D
8
(
HSQC, HMBC and ROESY) (Tables 2 and 3).
D-xylose (½
a
ꢁ
þ 21:3 (c 0.10, H
2
O)). The chemical shifts of the agly-
Compound 4 was obtained as colorless solid, and its molecular
cone moiety were similar to those of (+)-cyclo-olivil (27)
(Sugiyama et al., 1993), suggesting that compound 6 was an aryl-
naphthalene lignan glycoside. This coincided with the fact that
the molecular weight of 6 was 132 amu more than that of 27. This
was supported by acidic hydrolysis of 6, providing compound 27
formula was confirmed to be C21
H
32
O
11 from the sodiated ion peak
+
at m/z 483.1851 [M + Na] (calcd for C21 11Na, 483.1842) on the
32
H O
1
13
basis of positive HRESIMS. The H and C NMR spectra of the agly-
cone portion of 4 were similar to those of 3, with compound 4 hav-
ing five carbon atoms more than 3. The molecular weight of 4 was
3 3 f
which was identified by co-TLC (CHCl :CH OH, 9:1, R 0.5), co-HPLC
1
32 amu more than that of 3. A consideration of the NMR signals of
[MeCN–H O (2:8)] with t of 25.3 min with corresponding authen-
2
R
the five carbon atoms of 4 and its molecular weight indicated that
the five carbon atom portion of 4 was a sugar. Comparison of chem-
ical shifts of the sugars of 4 with those of plucheoside C (Uchiyama
et al., 1991) indicated that 4 had the same sugar moieties. HMBC
experiment showed the presence of a long-range correlation be-
tic sample. Also, the relative configuration was corroborated by
ROESY experiment as discussed below. In the ROESY spectrum,
the correlations between H-8 and H-2, H-6, H-9b were observed,
but there was no correlation from H-8 to H-7. The observation of
long-range correlations in the HMBC spectrum between d
H
4.27
00
0
00
0
tween H-1 and C-6 , which indicated that the sugar moiety of 4
(Xyl 1 ) and d
C
76.8 (Agly 9 ) proved the sugar unit to be attached
00
0
0
was b-
D
-apiofuranosyl(1 ?6 )-b-
D-glucopyranose. The relative
at C-9 of aglycone. This attachment was confirmed by observation
0
configuration of the aglycone was the same as that of 3 in the ROESY
spectrum. Finally, structure 4 was elucidated as vervenone-10-O-b-
of a reverse correlation between d
H
3.61, 4.10 (Agly 9 ) and d
C
105.6
0
0
(Xyl 1 ). Accordingly, structure 6 was determined as (+)-cyclo-oli-
00
0
0
D
-apiofuranosyl-(1 ?6 )-b-
Compound 5 was established to have a molecular formula of
by a HRESIMS analysis in the positive ion mode. A consid-
D
-glucopyranoside.
vil-9 -O-b-
D
-xylopyranoside.
In the Supplementary information, a comparison of the chemi-
cal constituents of the genera Cathaya, Picea and Pinus is provided.
This analysis indicated that serratane-type triterpenes exist only in
Cathaya (33–40), Picea (33–40) and Pinus (34–36) of the Pinaceae.
Lignans (9–14, 23–29), phenylpropanoids (15–17), aromatic com-
pounds (20–21), diterpenoids (30–32) and flavonoids (18, 41–42)
isolated from Cathaya had been also isolated from Picea or Pinus
species or both. Therefore, the chemical constituents of these three
genera are very similar, which confirmed that they are closely re-
lated in taxonomy and phylogeny.
C
25 32 9
H O
eration of the NMR spectra of 5 as discussed below and comparison
with the co-occurring massonianoside B (9) (Bi et al., 2001) indi-
cated that its aglycone portion was that of a dihydrobenzofuranoid
lignan. Two pairs of ABX protons in the aromatic region were con-
1
3
firmed by the C NMR spectra, which gave clear evidence of the
presence of two 1,2,4-substituted benzene rings. The furthest up-
field signals at d
ylene carbon (C-7 ) and the central methylene carbon (C-8 ) of the
n-propanol chain, respectively. The methine resonance at d 55.3
was ascribed to the C-8 benzofuran methine. The downfield signal
at d 88.0 was assigned to the aryl-substituted benzofuranmethine
C-7). The upfield resonances at d
C
32.6 and 35.9 were assigned to the benzylic meth-
0
0
C
3
. Concluding remarks
C
(
C
65.3 and 62.2 were ascribed to
There is only one species in Cathaya, i.e., C. argyrophylla. Herein
0
the two hydroxymethylene (C-9, C-9 ). 2D NMR studies confirmed
that the aglycone was dihydrobenzofuranoid lignan. In addition,
one methoxy group was clearly recognized from its signal at
a systematic chemical study on C. argyrophylla led to 45 com-
pounds isolated, including six new compounds and five biologi-
cally active compounds, which extends the compound diversity
of this plant. The results of careful comparison of chemical constit-
uents of the genera Cathaya, Picea and Pinus of Pinaceae indicated
that they are closely related in taxonomy and phylogeny, which is
consistent with the results of molecular phylogeny.
3
.79 ppm. This methoxy group correlated with the aromatic quater-
nary carbon at d 152.1 (C-3) in the HMBC spectrum which indi-
C
cated the position of this group to be at C-3. This connection was
confirmed by a ROESY correlation of H-2 to OMe-3. The chemical
shifts of the sugar portion matched the literature values for the su-
gar portion of 9 which was confirmed by basic hydrolysis of 5 yield-
1
D
8
ing
L
-rhamnose with ½
aꢁ
þ 12:5 (c 0.10, H
2
O). The
L
-rhamnose was
4. Experimental
linked to the 4-position of the aglycone from a strong HMBC corre-
lation of H-1 to C-4. This connection was also confirmed by a
ROESY correlation of H-1 to H-5. Strong NOE correlations were ob-
served between protons H-7 and H-9a, but only weak correlations
between H-7 and H-8. The value of the coupling constant J7,8
00
4.1. General experimental procedures
0
0
IR spectra were performed on a Bio-Rad FTS-135 spectropho-
tometer (KBr pellets), whereas UV spectra were obtained using a
Shimadzu 2401PC spectrophotometer. Optical rotations were re-
corded on a Horbia SEAP-300 polarimeter. MS spectra were carried
out on VG Autospec-3000 spectrometer and API Qstar Pulsar
instrument. NMR spectra were measured with Bruker AM-400
and DRX-500 spectrometers (TMS, internal standard). Semiprepar-
ative HPLC was carried out on an Agilent 1100 apparatus with a UV
detector at a flow rate of 2 mL/min. Samples were loaded on a
YMC-Pack ODS-A (YMC, 10 mm ꢂ 15 cm) column. Sephedex LH-
(
5.8 Hz) was also in agreement with those for trans-substituted
dihydrobenzofuranoid lignan glycosides, 9 (Bi et al., 2001), 10
0
(
Machida et al., 2002), 11 (Machida et al., 2002), (2S,3R)-3,3 -dide-
methoxydihydrodehydrodiconiferyl alcohol-4-O-b-D-glucopyrano-
side (Xu et al., 2006a), i.e., 5.8, 5.7, 5.8 and 5.6 Hz. On the basis of
the above evidence, the orientation of the substituents at C-7, 8
was trans. Thus, structure 5 was assigned as the new compound
cedrusinin-4-O-a-L-rhamnopyranoside.
The negative ion FABMS of 6 displayed a quasi-molecular ion
20 (25–100
l
m, Pharmacia Fine Chemical Co., Ltd., Sweden), silica
ꢀ
peak at m/z 507 [M ꢀ H] , indicating a molecular weight of 508.
gel (10–40
lm, 100–200 mesh and 200–300 mesh, Qingdao Marine

W.-J. He et al. / Phytochemistry 83 (2012) 63–69
67
Chemical, Inc., China), Lichroprep RP-8 (40–63
l
m, Merck, Darms-
column (MeOH) and then purified by semipreparative HPLC
[MeCN–H O (24:76)] to yield compound 20 (8 mg). Fraction 1.3
was applied to an MCI column, eluting with a MeOH–H O gradient,
to yield 4 fractions 1.31–1.34. Fraction 1.31 (0.8 g) was submitted
to RP-8 using a H O–MeOH gradient (100:0 ? 0:100), yielding two
fractions 1.311–1.312. Fraction 1.311 was purified by semiprepar-
ative HPLC [MeCN–H O (11:89)] to give 17 (30 mg), a mixture of 4
and 18 (67 mg). Compounds 4 and 18 were further purified by
semipreparative HPLC using a H O–MeOH gradient (20:80 ? 50:50)
to afford 4 (5 mg) and 18 (8 mg). Fraction 1.312 was purified by
semipreparative HPLC [MeCN–H O (13:87)] to yield compounds
tadt, Germany), and MCI gel (75–150 m, Mitsubishi Chemical
l
2
Corporation, Japan) were used for column chromatography (CC).
TLC was carried out using precoated silica gel GF254 glass plates,
with plates visualized using UV light or by spraying with 10%
2
2
2 4
H SO in 95% EtOH and heating.
2
4.2. Material
2
Twigs and leaves of C. argyrophylla were collected from the Na-
tional Nature Reserve of Huaping, Guangxi Zhuang Autonomous
Region, China, in June 2006. Authenticity of the plant was con-
firmed by Prof. Zhong-Shu Yue (Kunming Institute of Botany, Chi-
nese Academy of Sciences). A voucher sample (No. 0010442) is
held at the herbarium of Kunming Institute of Botany, Chinese
Academy of Sciences. Cancer cell lines, i.e., K562, HT-29, BEL-
2
10 (4 mg), 11 (1 mg) and 15 (3 mg).
The EtOAc extract (130 g) was subjected to silica gel CC eluting
with a gradient of CHCl
tion 3 was submitted to silica gel CC using a petroleum ether and
Me CO gradient, to yield five fractions. Fraction 3.2 was passed
through a Sephadex LH-20 column (MeOH) and then loaded on
an RP-8 column using a H O–MeOH gradient (100:0 ? 0:100) to
give 22 (33 mg) and 25 (15 mg). Fraction 4 was subjected to silica
gel CC eluting with a gradient of CHCl and MeOH to obtain six frac-
tions. Fraction 4.3 was passed through a Sephadex LH-20 column
MeOH) to afford four fractions. Fraction 4.33 was purified by semi-
preparative HPLC using a MeCN–H O gradient (30:70 ? 90:10) to
afford 23 (20 mg), 24 (15 mg) and 26 (10 mg). Fraction 4.34 was
submitted to an RP-8 column using a H O–MeOH gradient
(100:0 ? 0:100) and then purified by semipreparative HPLC using
a MeCN–H O gradient (30:70 ? 90:10) to afford 28 (16 mg), 29
(5 mg) and 41 (20 mg). Fraction 5 was submitted to semiprepara-
tive HPLC using a H O–MeOH gradient (70:30 ? 0:100) to obtain
3
and MeOH to afford eight fractions. Frac-
2
7
402, SGC-7901, B16, BGC-823, U251 and A549, were bought from
the Cell Culture Center of Institute of Basic Medical, Chinese Acad-
emy of Medical Sciences, Beijing, China. Bacteria S. aureus and C.
albicans were purchased from the China General Microbiological
Culture Collection Center, Beijing, China.
2
3
(
4.3. Extraction and isolation
2
Air-dried twigs and leaves (5 kg) were powdered and extracted
2
with MeOH (3 ꢂ 10 L, each 7 days) at room-temperature, and a res-
idue (650 g) was obtained. The latter was suspended in H O and
2
2
partitioned in sequence with petroleum ether (60–80 °C, unless
otherwise noted), EtOAc, and n-BuOH to afford a petroleum
ether-soluble fraction (100 g), an EtOAc-soluble fraction (130 g),
2
27 (60 mg) and 42 (60 mg).
a n-BuOH-soluble fraction (210 g), and a H
120 g).
The n-BuOH extract (210 g) was subjected to polyamide CC
eluting with H O to obtain Fraction 1 (20 g). Fraction 1 was sub-
jected to silica gel CC eluting with a gradient of CHCl and MeOH,
and three fractions were obtained. Fraction 1.1 was submitted to
RP-8 using a H O–MeOH gradient (100:0 ? 0:100), yielding three
fractions 1.11–1.13. Fraction 1.11 was repurified on MCI, eluting
with a MeOH–H O gradient, to yield nine fractions 1.111–1.119.
Fraction 1.111 was passed through a Sephadex LH-20 column
2
O-soluble fraction
The petroleum ether extract (100 g) was subjected to silica gel
CC eluting with petroleum ether–EtOAc (100:0; 10:1; 5:1; 1:1;
1:2; 0:100) to obtain nine fractions. Fraction 2 was subjected to sil-
ica gel CC using a petroleum ether and EtOAc gradient, to yield 30
(210 mg) and 33 (70 mg). Fraction 3 was applied to silica gel CC
using a gradient of petroleum ether and EtOAc and then passed
(
2
3
2
3
through a Sephadex LH-20 column [CHCl –MeOH (1:1)] to obtain
37 (10 mg) and 39 (40 mg). Fraction 5 was subjected to silica gel
CC eluting with a gradient of petroleum ether and EtOAc to give
32 (10 mg) and 45 (20 mg). Fraction 6 was applied to silica gel
CC using a gradient of petroleum ether and EtOAc and then passed
2
(
MeOH) and then loaded on semipreparative HPLC [MeOH–H
2
O
(
18:82)] to give 21 (6 mg). Fraction 1.116 was passed through a
through a Sephadex LH-20 column [CHCl
35 (60 mg), 38 (500 mg) and 40 (620 mg). Fraction 7 was submit-
ted to silica gel CC using a petroleum ether and Me CO gradient
to obtain four fractions. Fraction 7.2 was passed through a Sepha-
dex LH-20 column [CHCl –MeOH (1:1)] to obtain 44 (50 mg). Frac-
3
–MeOH (1:1)] to obtain
Sephadex LH-20 column (MeOH) and then purified by semiprepar-
ative HPLC [MeCN–H O (16:84)] to give 7 (12 mg) and 3 (9 mg).
Fraction 1.12 was purified by semipreparative HPLC [MeOH–H
2
2
2
O
(
40:60)] to give 5 (2 mg). Fraction 1.13 was subjected to semipre-
3
parative HPLC [MeOH–H
2
O (57:43)] to give 1 (24 mg) and 2 (2 mg).
tion 7.3 was subjected to silica gel CC using a gradient of petroleum
ether and EtOAc to give 31 (230 mg) and 34 (120 mg). Fraction 8
was applied to silica gel CC eluting with a gradient of petroleum
ether and EtOAc and then passed through a Sephadex LH-20 col-
Fraction 1.2 was applied to an MCI column, eluting with a MeOH–
O gradient, to yield six fractions 1.21–1.26. Fraction 1.21 was
passed through a Sephadex LH-20 column (MeOH) and then loaded
on a semipreparative HPLC [MeOH–H O (24:76)] to yield com-
pound 8 (9 mg). Fraction 1.22 was submitted to RP-8 using a
O–MeOH gradient (100:0 ? 0:100), yielding four fractions
H
2
2
3
umn [CHCl –MeOH (1:1)] to obtain 36 (1 g) and 43 (30 mg).
H
2
4.3.1. Cathargyroside A (1)
2
D
7
1
2
.221–1.224. Fraction 1.221 was passed through a Sephadex LH-
0 column (MeOH) and then purified by semipreparative HPLC
Colorless solid; ½
a
ꢁ
ꢀ 3:9 (c 0.60, MeOH); UV kmax (MeOH) nm
KBr
max
ꢀ1
(log ) 240 (3.75); IR
e
m
cm : 3375, 3265, 2974, 2940, 1749,
1
[
1
MeCN–H
2
O (15:85)] to give 16 (30 mg) and 19 (73 mg). Fraction
.223 was passed through a Sephadex LH-20 column (MeOH)
O (10:90)]
1677, 1467, 1434, 1400, 1208, 1138, 1079, 840, 802, 724; For
H
1
3
NMR (CD
3
OD, 400 MHz) and C NMR (CD
3
OD, 100 MHz) spectro-
and then loaded on semipreparative HPLC [MeCN–H
2
scopic data, see Tables 2 and 3; FABMS (negative) m/z (%): 495
ꢀ
ꢀ
to give 6 (7 mg), 9 (37 mg) and a mixture of compounds 12, 13
and 14 (100 mg). Compounds 12, 13 and 14 were further purified
[M ꢀ H] (20); HRESIMS (negative) m/z 495.2595 [M ꢀ H] , (calcd
for C26 , 495.2594).
39 9
H O
2
by semipreparative HPLC [MeOH–H O (25:75)] using a ZORBAX
Eclipse XDB C18 (Agilent, 9.4 mm ꢂ 25.0 cm) column at a flow rate
4.3.2. Cathargyroside B (2)
2
5
of 2 mL/min to afford 12 (8 mg) and a mixture of 13 and 14
Colorless solid; ½
a
ꢁ
ꢀ 23:8 (c 0.04, MeOH); UV kmax (MeOH) nm
D
KBr
max
ꢀ1
(
52 mg). Fraction 1.23 was submitted to RP-8 using a H
2
O–MeOH
(log ) 201 (3.82), 230 (3.67) nm; IR
e
m
cm : 3440, 1679, 1639,
1
gradient (100:0 ? 0:100), yielding two fractions 1.231–1.232.
Fraction 1.231 (0.8 g) was passed through a Sephadex LH-20
1511, 1437, 1206, 1137, 1075, 1036, 840, 802, 723; For H NMR
1
3
3 3
(CD OD, 400 MHz) and C NMR (CD OD, 100 MHz) spectroscopic

6
8
W.-J. He et al. / Phytochemistry 83 (2012) 63–69
1513, 1449, 1430, 1375, 1275, 1255, 1042; For 1H NMR (CD
OD,
3
Table 4
1
H NMR and 13C NMR spectroscopic data for compounds 5–6 in CD
3
OD.
13
4
00 MHz) and C NMR (CD
3
OD, 100 MHz) spectroscopic data,
ꢀ
No.
5
6
5
6
see Table 4; FABMS (negative) m/z (%) 507 [M ꢀ H] (8); HRESIMS
ꢀ
d
H
(J in Hz)
d
H
(J in Hz)
d
C
d
C
(negative) m/z 507.1858 [M ꢀ H] , (calcd for C25
31
H O11, 507.1866).
1
2
3
4
5
6
7
8
139.1 s
111.2 d
152.1 s
146.5 s
119.6 d
119.1 d
88.0 d
138.3 s
114.1 d
149.1 s
146.1 s
116.0 d
123.6 d
44.7 d
7.01 overlapped
6.70 d (1.5)
4
.4. Acid hydrolysis of 1–4, 6
7.07 d (8.5)
6.90 dd (8.5, 1.8)
5.51 d (5.8)
3.44 m
3.86 m
3.75 m
6.75 d (8.0)
6.66 dd (8.0, 1.5)
4.07 d (10.1)
2.02 m
3.85 overlapped
3.51 overlapped
3.78 s
A solution of fraction 1.13 (1 and 2) (20 mg) and fraction 1.116
(3 and 4) (20 mg) in 2 N HCl (3 mL) was individually incubated at
0 °C for 6 h. After cooling, NaHCO was added to neutralize the
reaction mixture. The reaction mixture was extracted with CHCl
55.3 d
65.3 t
47.6 d
60.8 t
7
3
9
9
3
1
2
3
4
5
6
7
7
8
9
9
3
1
2
3
4
5
5
6
a
b
3
.
D-Glucose was confirmed in the aqueous layer by TLC comparison
-OMe
3.79 s
56.4 q
56.4 q
0
3
with an authentic sample [CHCl –MeOH (8:2)]. The aqueous solu-
tion was dried and subjected to silica gel chromatography [CHCl ,
3
CHCl
4
135.8 s
125.9 d
128.5 s
159.4 s
109.8 d
129.8 d
32.6 t
126.5 s
112.9 d
147.5 s
145.2 s
117.3 d
133.5 s
40.0 t
0
0
0
0
0
0
0
0
0
7.08 bs
6.62 s
6.18 s
18
D
3
–MeOH (8:2)] to give (3.8 mg, ½
a
ꢁ
þ 43:5 (c 0.30, H
2
O);
18
D
.1 mg, ½
aꢁ
þ 45:3 (c 0.21, H
2
O)) respectively. The glucose was
6.74 d (8.2)
7.02 overlapped
2.62 t (7.7)
identified on the basis of their R
Similar treatment to 6 (5 mg) gave
c 0.10, H O)).
f
and specific rotation.
1
D
8
a
b
3.26 d (16.8)
2.76 d (16.8)
D
-xylose (0.7 mg, ½
a
ꢁ
þ 21:3
(
2
1.80 m
3.56 t (6.5)
35.9 t
62.2 t
74.8 s
76.8 t
a
b
4.10 d (9.4)
3.61 d (9.4)
3.80 s
4.27 d (7.5)
3.20 overlapped
3.34 m
3.51 overlapped
3.87 overlapped
3.22 overlapped
0
0
-OMe
56.4 q
105.6 d
75.0 d
77.8 d
71.2 d
67.1 t
4.5. Basic hydrolysis of 5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5.33 d (1.2)
3.86 m
4.05 m
3.44 m
3.72 m
101.4 d
72.2 d
72.1 d
73.8 d
70.8 d
A solution of compound 5 (1.5 mg) in 3% NaOH (5 mL) was incu-
bated at room temperature for 2 h. To the resulting solution, it was
adjusted to pH 7 by 2 N HCl, and extracted with CHCl
was confirmed in the aqueous layer by comparison with an
authentic sample using CHCl –MeOH (8:2) as a developing system.
The aqueous solution was dried and subjected to silica gel CC to
3
. L-Rhamnose
a
b
1.22 d (6.2)
18.0 q
3
1
8
give
L
-rhamnose (0.3 mg, ½
a
ꢁ
þ 12:5 (c 0.10, H
2
O)). The
L-rham-
D
nose was established using their R
f
and specific rotation.
ꢀ
data, see Tables 2 and 3; FABMS (negative) m/z (%): 495 [M ꢀ H]
ꢀ
(
C
3); HRESIMS (negative) m/z 495.2588 [M ꢀ H] , (calcd for
26
H
39
O
9
, 495.2594).
4.6. Cytotoxic assay
4
.3.3. Vervenone-10-O-b- -glucopyranoside (3)
D
Cytotoxic activities of the compounds were measured with the
sulphorhodamine B (SRB) assay (Han et al., 2008). Aliquots (90 L)
of cells cultured in RPMI 1640 medium (Sigma) were seeded in 96-
2
8
Colorless solid; ½
aꢁ
ꢀ 94:0 (c 0.25, MeOH); UV kmax (MeOH) nm
D
l
KBr
max
ꢀ1
(
log ) 202 (3.44), 251 (3.59) nm; IR
e
m
cm : 3426, 2925, 2877,
1
1
4
672, 1204, 1078, 1036, 896, 869; For
3
H NMR (CD OD,
well flat-bottomed microtiter plates (Greiner) at a density of
1
3
00 MHz) and C NMR (CD
3
OD, 100 MHz) spectroscopic data,
4
3
.3–7.7 ꢂ 10 cells/mL. Twenty-four hours later, 10
l
L compounds
g/mL.
for another 48 h, and
COOH. Cells were maintained at
°C for 1 h. Dye was removed, and SRB was added in 10 mM Tris
+
see Tables 2 and 3; FABMS (positive) m/z (%) 329 [M + H] (100);
HRESIMS (positive) m/z 329.1595 [M + H] , (calcd for C16
3
were added in triplicate with a final concentration of 10
Cells were maintained at 37 °C in 5% CO
fixed by adding 50% ice-cold CCl
l
+
25 7
H O ,
2
29.1600).
3
4
00
0
4
.3.4. Vervenone-10-O-b-D-apiofuranosyl-(1 ?6 )-b-D-
buffer (100 lL), and the absorbance was read with a Plate Reader
glucopyranoside (4)
Colorless solid; ½
at 560 nm (Molecular Devices, SPECTRA MAX 340) after plates
were air-dried. IC50 values (50% inhibitory concentration) were
used for cell growth inhibition, which were measured by dose–re-
sponse curves with serial fivefold compounds dilutions.
2
D
0
a
ꢁ
ꢀ 72:0 (c 0.25, MeOH); UV kmax (MeOH) nm
KBr
max
ꢀ1
(
log
e
) 202 (3.93), 252 (3.6) nm; IR
419, 1062, 875, 592; For H NMR (C
m
cm : 3428, 2925, 1645,
1
13
1
5
D
5
N, 400 MHz) and
C
NMR (C
5 5
D N, 100 MHz) spectroscopic data, see Tables 2 and 3;
+
ESIMS (positive) m/z (%) 483 [M + Na] (100); HRESIMS (positive)
m/z 483.1851 [M + Na] , (calcd for C21
+
32
H O11Na, 483.1842).
4.7. Antibacterial assay
4
.3.5. Cedrusinin-4-O- -rhamnopyranoside (5)
a
-
L
The turbidimetric method has been used with some modifica-
2
3
Colorless solid; ½
aꢁ
ꢀ 50:0 (c 0.10, MeOH); UV kmax (MeOH) nm
tion for antimicrobial experiment (Joslyn and Galbraith, 1950). S.
aureus and C. albicans were maintained in media Mueller–Hinton
Broth (Oxiod, CM0405, Hampshire, England) and Potato Dextrose
D
KBr
ꢀ1
(
log ) 207 (3.87), 225 (4.06), 283 (3.50) nm; IR
e
m
cm : 3424,
max
2
8
1
939, 1680, 1513, 1489, 1401, 1207, 1139, 1066, 1026, 982, 838,
1
13
03, 724; For H NMR (CD
3
OD, 500 MHz) and C NMR (CD
3
OD,
Broth (PDB, lab made), respectively to McFarland 0.5 and diluted
6
00 MHz) spectroscopic data, see Table 4; FABMS (positive) m/z
with the media at a density of 1 ꢂ 10 CFU/mL. Aliquots of 90
lL
(
[
%): 477 [M + H]+ (15); HRESIMS (positive) m/z 499.1933
were filled in 96-well U-bottomed microplate. The isolated com-
pounds, dissolved in DMSO and diluted with the medium (10 L),
were dispensed in triplicate in the wells at a final density of
10 g/mL. Absorbance was read at 620 nm with the microplate
+
M + Na] , (calcd for C25
H
32
9
O Na, 499.1944).
l
0
0
D
4
.3.6. (+)-Cyclo-olivil-9 -O-b-
D
-xylopyranoside (6)
l
2
Colorless solid; ½
a
ꢁ
þ 26:7 (c 0.10, MeOH); UV kmax (MeOH) nm
reader mentioned above after 24 h. Inhibition was measured as
the percentage of maximum absorbance (0.04% DMSO control).
KBr
ꢀ1
(
log ) 203 (4.80), 284 (3.85) nm; IR
e
m
cm : 3426, 2925, 1618,
max

W.-J. He et al. / Phytochemistry 83 (2012) 63–69
69
Ma, Z.W., He, G.F., Yin, W.F., Chen, M.L., 1981. Study of triterpenoid components in
Acknowledgements
trunk of Cathaya argyrophylla Chun et Kuang native in China. J. Integr. Plant Biol.
2
3, 316–321.
This research was supported by the National Natural Science
Foundation of China (U1032602, 91013002, 30725048, 20972168),
the National New Drug Innovation Major Project of China
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(
(
(
2011ZX09307-002-02), National Basic Research Program of China
2009CB522300) and the Fund of Chinese Academy of Sciences
KSCX2-YW-R-177). We thank the analytical group from the State
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B . Chem.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2012.
0
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