Phenanthrene derivatives from Cymbidium great flower marie laurencin and their biological activities

Article abstract of DOI:10.1021/np200788u

A new phenanthrendione, ephemeranthoquinone B (1), two phenanthrenes, marylaurencinols A (2) and B (3), and a phenanthrene glucoside, marylaurencinoside A (4), were isolated from the roots of Cymbidium Great Flower Marie Laurencin, along with six known phenanthrenes, 5-10. The structures of these compounds were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analysis and chemical degradation. The compounds were tested for antibacterial activities against Bacillus subtilis and Klebsiella pneumoniae and for cytotoxic activity against the human promyelocytic leukemia (HL-60) cell line. Compounds 1, 3, and 6 showed antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 4.88 to 65.10 μM. Notably, ephemeranthoquinone B (1) had a strong antibacterial effect on B. subtilis. Furthermore, 1 exhibited moderate cytotoxic activity (IC₅₀ 2.8 μM) against HL-60 cells. Compounds 4-9 also showed weak cytotoxic activity against the HL-60 cell line with IC₅₀ values of 19.3-52.4 μM.

Full text of DOI:10.1021/np200788u

Article
pubs.acs.org/jnp
Phenanthrene Derivatives from Cymbidium Great Flower Marie
Laurencin and Their Biological Activities
Kazuko Yoshikawa,*,† Takuya Ito,† Kanako Iseki,† Chihiro Baba,† Hiroshi Imagawa,† Yasuyuki Yagi,†
Hiroshi Morita,‡ Yoshinori Asakawa,† Sachiko Kawano,§ and Toshihiro Hashimoto†
†Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, 770-8514, Japan
‡Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa, Tokyo 142-8501, Japan
§Kawano Mericlone Co., Ltd., Hokushou 562-1, Wakimachi, Mima, Tokushima 779-3604, Japan
S
* Supporting Information
ABSTRACT: A new phenanthrendione, ephemeranthoqui-
none B (1), two phenanthrenes, marylaurencinols A (2) and
B (3), and a phenanthrene glucoside, marylaurencinoside A (4),
were isolated from the roots of Cymbidium Great Flower Marie
Laurencin, along with six known phenanthrenes, 5−10. The
structures of these compounds were established by a combina-
tion of extensive NMR spectroscopy and/or X-ray crystallo-
graphic analysis and chemical degradation. The compounds were tested for antibacterial activities against Bacillus subtilis and Klebsiella
pneumoniae and for cytotoxic activity against the human promyelocytic leukemia (HL-60) cell line. Compounds 1, 3, and 6 showed
antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 4.88 to 65.10 μM. Notably,
ephemeranthoquinone B (1) had a strong antibacterial effect on B. subtilis. Furthermore, 1 exhibited moderate cytotoxic activity (IC50
2.8 μM) against HL-60 cells. Compounds 4−9 also showed weak cytotoxic activity against the HL-60 cell line with IC50 values of
19.3−52.4 μM.
henanthrenes are tricyclic aromatic hydrocarbons, prob-
ably biosynthesized by the oxidative coupling of the
paper describes the isolation, structural elucidation, and
biological activities of these compounds.
P
aromatic rings of stilbene precursors in higher plants.1 A large
number of phenanthrenes have been isolated from the
Orchidaceae family, Cymbidium,2 Dendrobium,3 Bulbophyllum,4
Maxillaria,5 Bletilla,6 Ephemerantha,7 and Eria.8 The Orchid-
aceae family consists of more than 35 000 species in ap-
proximately 750 genera of the flowering plants, which are
widely distributed in temperate and tropical regions. Since
ancient times, the Orchidaceae have been used not only for
ornamental purposes but also as medicinal plants, to treat
paralysis, cholera, diarrhea, and sores.9 Therefore, the Orchid-
aceae may be a prodigious source of potential new drugs.
Phenanthrenes exhibit various biological activities such as anti-
inflammatory,9 antiallergic,9 antimicrobial,10 cytotoxic,11,12
antiplatelet aggregation inhibitory,13 phytotoxic,14 antifungal,15
spasmolytic,16 antifibrotic,17 and inhibitory effects on NO
production.9
RESULTS AND DISCUSSION
■
Fresh roots of C. Great Flower Marie Laurencin were extracted
with MeOH at room temperature. The MeOH extract was
partitioned into an EtOAc−H2O mixture to afford EtOAc- and
H2O-soluble portions. The EtOAc extract showed antimicrobial
activity against B. subtilis at 18.75 μg/mL. The EtOAc extract
was subjected to bioassay-guided separation, fractionated by
silica gel column chromatography, and further purified by
RP-HPLC to give a new phenanthrendione, ephemeranthoquinone
B (1), two new phenanthrenes, marylaurencinols A (2) and
B (3), and a phenanthrene glucoside, marylaurencinoside A (4),
together with known phenanthrenes 5−10.
Ephemeranthoquinone B (1) was obtained as red needles.
The EIMS indicated a molecular ion at m/z 256 [M]+, and the
molecular formula was determined as C15H12O4 by HREIMS in
conjunction with NMR data analysis. The IR spectrum of 1
revealed strong absorption bands due to an aromatic ring
(1595, 1452 cm−1), conjugated carbonyl groups (1670, 1639
cm−1), and hydroxy groups (3063 cm−1). The UV spectrum
supported the presence of the aromatic ring (λmax 217.6, 223.4,
242.0, 276.2, 321.2 nm). In the 1H NMR spectrum, four
In the course of our study of bioactive substances from the
Orchidaceae, we started a search for novel antimicrobial
metabolites from Cymbidium Great Flower Marie Laurencin.
Each extract of this plant was tested for antimicrobial activities
against Bacillus subtilis and Klebsiella pneumoniae. From the
bioassay-guided separation of the EtOAc-soluble extract, a new
phenanthrendione, ephemeranthoquinone B (1), two new
phenanthrenes, marylaurencinols A (2) and B (3), and a
phenanthrene glycoside, marylaurencinoside A (4), together
with known phenanthrenes (5−1016−18) were found. This
Received: October 15, 2011
Published: March 9, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
605
dx.doi.org/10.1021/np200788u | J. Nat. Prod. 2012, 75, 605−609

Journal of Natural Products
Article
Figure 2. ORTEP drawing for the X-ray crystal structure of 1.
1094 cm−1, indicating the absence of carbonyl groups
1
comparable to those in 1. The H NMR spectrum showed
the presence of two methoxy groups (δH 3.91, 3.74), two
methylene protons (δH 2.71, 2.64), and four aromatic protons
(δH 7.15, 6.97, 6.84, 6.70). The 13C NMR spectrum revealed
the presence of two methoxy carbons (δC 61.8, 56.2), two
methylene carbons (δC 31.2, 30.4), and 12 aromatic carbons
and the absence of carbonyl carbons. Therefore, 2 was deduced
to have a 9,10-dihydrophenanthrene skeleton. The 1H−1H
COSY and HMQC spectra clarified the presence of two partial
structures and two methoxy groups as shown in Figure 3.
aromatic proton signals at δH 7.23 (t, J = 8.2 Hz), 6.91 (dd, J =
8.2, 1.4 Hz), 6.78 (dd, J = 8.2, 1.4 Hz), and 6.04 (s) were
observed. The last three signals indicated the presence of a
1,2,3-trisubstituted phenyl group. Two methylene signals at δH
2.71 and 2.66 and one methoxy signal at δH 3.90 were also
observed. The 13C NMR spectrum also showed the presence of
two carbonyl carbons (δC 191.5, 180.8), two methylene carbons
(δC 28.5, 21.3), and 10 sp2 hybridized carbons (δC 158.6, 155.3,
143.2, 140.6, 139.0, 132.3, 120.3, 119.3, 117.5, 108.2). The
1H−1H COSY spectrum indicated two partial structures
1
indicated by thick lines in Figure 1. The long-range H−13C
Figure 3. COSY, HMBC, and key NOESY correlations of 2.
The connectivity of these partial structures and the other
carbons was also defined on the basis of the HMBC and
NOESY correlations shown in Figure 3. The structure of 2 was
elucidated to be 3,5-dihydroxy-2,4-dimethoxy-9,10-dihydrophe-
nanthrene.
Marylaurencinol B (3) was obtained as brown needles. The
EIMS showed an [M]+ ion at m/z 288, and the molecular
formula was determined as C16H16O5 by HREIMS in
conjunction with NMR analysis. The IR spectrum of 3
contained bands for hydroxy groups (3232 cm−1). The H
1
and 13C NMR spectra showed differences in the C-1−4, 4a,
10, and 10a resonances compared to those of 2 (Table 2).
Figure 1. COSY and HMBC correlations of 1.
1
correlations were analyzed via an HMBC spectrum that showed
correlations of H-3 to C-1, C-2, C-4, and C-4a; H-6 to C-4b,
C-5, and C-8; H-7 to C-5 and C-8a; H-8 to C-4b, C-6, and C-
8a; H-9 to C-4b, C-8, C-8a, and C-10a; H-10 to C-1, C-4a, C-
8a, and C-10a; and 2-OCH3 to C-2 (Figure 1). Furthermore,
NOE correlations were observed between H-3 (δH 6.04) and
2-OCH3 (δH 3.90) and between H-8 (δH 6.78) and H-9 (δH 2.71).
On the basis of these results, the structure of 1 was determined as
5-hydroxy-2-methoxy-9,10- dihydrophenanthrene-1,4-dione. X-ray
crystallographic analysis confirmed the structure of 1 (Figure 2).
Marylaurencinol A (2) was obtained as a white, amorphous
powder. The EIMS revealed a molecular ion at m/z 272 [M]+,
and the molecular formula was determined as C16H16O4 by
HREIMS. The IR spectrum of 2 exhibited strong absorption
bands at 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, and
Analysis of the H NMR spectrum showed the presence of
two methoxy groups (δH 3.75, 3.51), three aromatic protons
(δH 7.11, 6.83, 6.81), and two methylene protons (δH 2.57,
2.51). The 13C NMR and HMQC spectra further revealed
the presence of two methoxy carbons (δC 61.8, 60.1), two
methylene carbons (δC 30.3, 21.7), and nine qua-
ternary aromatic carbons. However, the connectivity
between these partial structures, as shown in Figure 4,
1
could not be established on the basis of the H−1H COSY
and HMBC correlations. Therefore, a NOESY experiment in
DMSO-d6 was conducted. The NOE correlations between
2-OCH3 and 1-OH, 3-OH; 4-OCH3 and 3-OH, 5-OH; and
5-OH and 4-OCH3, H-6 confirmed the connectivities of the
hydroxy, methoxy groups, and aromatic protons (Figure 4).
On the basis of the above evidence, the structure of 3 was
606
dx.doi.org/10.1021/np200788u | J. Nat. Prod. 2012, 75, 605−609

Journal of Natural Products
Article
1
Table 1. H (600 MHz) and 13C NMR (150 MHz)
Spectroscopic Data for Compounds 1 and 2 in CDCl3
1
2
position
δC mult.
δH (J in Hz)
δC mult.
δH (J in Hz)
1
180.8, qC
107.8, CH
146.3, qC
137.1, qC
142.5, qC
118.9, qC
119.9, qC
153.4, qC
6.70, s
2
158.6, qC
3
108.2, CH
6.04, s
4
191.5, qC
4a
4b
5
139.0, qC
117.5, qC
Figure 4. COSY, HMBC, and key NOESY correlations of 3.
155.3, qC
6
119.3, CH
6.91, dd (8.2, 1.4) 117.9, CH
7.23, t (8.2) 128.2, CH
6.97, d (8.0)
7.15, t (8.0)
6.84, d (8.0)
7
132.3, CH
8
120.3, CH
6.78, dd (8.2, 1.4) 119.8, CH
140.5, qC
8a
9
140.6, qC
28.5, CH2 2.71, m
2.71, m
31.2, CH2 2.71, m
2.71, m
10
21.3, CH2 2.66, m
2.66, m
30.4, CH2 2.64, m
2.64, m
10a
143.2, qC
132.0, qC
2-OCH3
4-OCH3
5-OH
56.6, CH3 3.90, s
56.2, CH3 3.91, s
61.8, CH3 3.74, s
8.67, s
Figure 5. ORTEP drawing for the X-ray crystal structure of 3.
established as 1,3,5-trihydroxy-2,4-dimethoxy-9,10-dihydro-
phenanthrene. The structure was confirmed by X-ray
crystallographic analysis as shown in Figure 5.
Marylaurencinoside A (4) was obtained as a white,
amorphous powder. The FABMS showed a quasimolecular
ion at m/z 419 [M − H]−. The molecular formula was
determined as C21H24O9 by HRFABMS. The IR spectrum
revealed strong absorption bands due to hydroxy groups (3230
1
Table 2. H (600 MHz) and 13C NMR (150 MHz)
Spectroscopic Data for Compounds 3 and 4
a
b
3
4
1
cm−1). The H and 13C NMR data were similar to those
position
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
for 2 and 3 except for the signals assignable to a sugar moiety
(Table 2). The 1H NMR spectrum of 4 showed the presence of
a methoxy group (δH 3.86), four aromatic protons (δH 7.07,
6.84, 6.82, 6.54), four aliphatic protons (δH 2.89, 2.81, 2.63, 2.56),
an anomeric proton (δH 4.69), two oxymethylene protons (δH
3.75, 3.63), and four oxymethine protons (δH 3.46, 3.42, 3.37,
3.15). Analyses of the 13C NMR and HMQC spectra revealed
the presence of a methoxy carbon (δC 56.3), four aromatic
carbons (δC 128.5, 121.0, 116.8, 101.4), two methylene carbons
(δC 35.4, 31.5), an anomeric carbon (δC 106.0), an oxy-
methylene carbon (δC 62.8), four oxymethine carbons (δC 78.1,
78.0, 75.8, 71.6), and eight quaternary aromatic carbons.
Analysis of the 1H−1H COSY spectrum revealed a sugar moiety
and two partial structures indicated by thick lines in Figure 6.
1
143.4, qC
136.1, qC
141.5, qC
137.1, qC
120.7, qC
120.0, qC
153.3, qC
117.1, CH
128.1, CH
119.4, CH
140.8, qC
30.3, CH2
101.4, CH
153.1, qC
151.6, qC
137.2, qC
115.3, qC
122.3, qC
153.2, qC
6.54, s
2
3
4
4a
4b
5
6
6.81, d (8.1) 116.8, CH
7.11, t (8.1) 128.5, CH
6.82, dd (8.1, 1.2)
7.07, t (8.0)
7
8
6.83, d (8.1) 121.0, CH
142.7, qC
6.84, dd (8.1, 1.2)
8a
9
2.51, m
2.51, m
2.66, m
2.66, m
31.5, CH2 2.63, m
2.56, m
10
21.7, CH2
116.8, qC
25.4, CH2 2.89, m
2.81, m
10a
137.3, qC
56.3, CH3 3.86, s
2-OCH3
4-OCH3
1-OH
3-OH
5-OH
Glc-1′
Glc-2′
Glc-3′
Glc-4′
Glc-5′
Glc-6′
60.1, CH3 3.75, s
61.8, CH3
3.51, s
8.68, s
8.96, s
8.86, s
106.0, CH
75.8, CH
78.0, CH
71.6, CH
78.1, CH
4.69, d (7.7)
3.46, dd (9.1, 7.7)
3.42, dd (9.1, 8.8)
3.37, dd (9.3, 8.8)
3.15, m
62.8, CH2 3.75, dd (11.8, 2.3)
3.63, dd (11.8, 5.2)
a
b
Recorded in DMSO-d6. Recorded in methanol-d4.
Figure 6. COSY, key HMBC, and key ROESY correlations of 4.
607
dx.doi.org/10.1021/np200788u | J. Nat. Prod. 2012, 75, 605−609

Journal of Natural Products
Article
Analysis of the HMBC spectrum of 4 further clarified the
presence of a hexose skeleton (based on the correlations
between H-1′ and C-3′, C-5′; H-2′ and C-1′, C-3′, C-4′) and a
9,10-dihydrophenanthrene aglycone skeleton (based on the
correlations between H-1 and C-2, C-3, C-4a, C-10a; 2-OCH3
and C-2; H-6 and C-4b, C-5; H-7 and C-5, C-8a; H-8 and
C-4b, C8a, C-9; H-9 and C4b, C8a, C10, C10a), respectively
(Figure 6). Furthermore, the HMBC correlation between C-4
(δC 137.2) of the A-ring and H-1′ (δH 4.69) of the hexose
moiety indicated that the sugar moiety was linked to the
4-hydroxy group of the A-ring. On the basis of this evidence,
the molecular structure of marylaurencinoside A (4) was
determined as shown. The relative configuration of the hexose
moiety was elucidated by analysis of the 1H NMR and ROESY
spectra of 4. The coupling constant of 7.7 Hz between H-1′ and
H-2′ indicated a diaxial relationship between these two protons.
In the ROESY spectrum of the hexose moiety, correlations
between H-1′ and H-3′, H-5′; H-3′ and H-1′, H-5′; and H-2′ and
H-4′ (Figure 6) indicated the presence of a β-glucopyranose
unit. Hydrolysis of 4 in 2.5% H2SO4 gave D-glucose, which was
identified by HPLC using an optical rotation detector.19 Thus,
the structure of 4 was determined as 3,4,5-trihydroxy-2-
methoxy-9,10-dihydrophenanthrene-4-O-β-D-glucopyranoside.
The isolated phenanthrene derivatives (1−10) were tested
for antimicrobial activity against B. subtilis and K. pneumoniae.
Phenanthrenes 1, 3, and 6 exhibited moderate to insignificant
antimicrobial activity against B. subtilis with MIC values of
4.88, 65.10, and 52.82 μM, respectively. Notably, the new
phenanthrendione, ephemeranthoquinone B (1), showed
moderate antibacterial activity against B. subtilis, with an MIC
value of 4.88 μM. However, none of them were active against
K. pneumoniae. The cytotoxic activities of pure compounds 1−10
were evaluated against a HL-60 cell line. The results revealed
weak activity of 4−9 against the HL-60 cell line, with IC50
values of 52.4, 31.8, 19.3, 52.4, 44.6, and 34.2 μM, respectively.
Table 4. Cytotoxic Activity of Compounds 1−10 against
HL-60 Cells
a
a
compd
IC50
compd
IC50
1
2
3
4
5
2.8
96.8
173.5
52.4
31.8
19.3
7
52.4
44.6
34.2
103.3
0.1
8
9
10
b
MMC
6
a
b
IC50 values, in μM. MMC is mitomycin C.
rotation was measured using a JASCO DIP-1000 polarimeter. The UV
spectra were recorded using a Shimadzu UV-6000 spectrophotometer.
IR spectra were recorded on a JASCO FT/IR-5300. NMR spectra
were recorded on a Varian Unity 600 spectrometer. NMR experiments
included COSY, HMQC, HMBC, NOESY, and ROESY. Coupling
constants (J values) are given in Hz. HREIMS and HRFABMS were
measured on a JEOL JMS-700 MS station. HPLC separation was
performed on a JASCO PU1580 pump with a JASCO UV-970
detector. For HPLC, COSMOSIL 5C18-AR-II (Nacalai Tesque, Japan,
20 mm i.d. × 250 mm) was used. TLC was performed on precoated
silica gel 60F254. Spots were detected by examining plates sprayed with
the p-anisaldehyde/H2SO4/MeOH reagent followed by heating on a hot
plate.
Plant Material. Fresh roots of C. Great Flower Marie Laurencin
(Ministry of Agriculture, Forestry and Fisheries of Japan, seed
registration no. 2841) were cultivated and harvested in November
2008 at Kawano Mericlone Co., Ltd. (Tokushima Prefecture, Japan)
and were identified by one of the authors (S.K.). A voucher specimen
(TB 5430) has been deposited in the Herbarium of Faculty of
Pharmaceutical Sciences, Tokushima Bunri University, Tokushima,
Japan.
Extraction and Isolation. The fresh roots (8.0 kg) were extracted
with MeOH at room temperature for one month and concentrated in
vacuo. The MeOH extract was partitioned into an EtOAc−H2O
mixture to afford EtOAc- and H2O-soluble portions. The EtOAc
extract (42 g) was subjected to Si gel CC with n-hexane−EtOAc−
MeOH (10:1:0−0:1:10) to afford eight fractions (Fr. 1−8). Fr. 2 (0.58 g)
was further purified using RP-HPLC (70% aq MeOH) to give 5 (8 mg).
Fr. 3 (5.69 g) was chromatographed further over Si gel using n-hexane−
EtOAc (7:3) to afford five subfractions (Fr. 3-A−E). Fr. 3-C was purified
by RP-HPLC (60% aq MeOH) to yield 2 (390 mg) and 6 (80 mg). Fr. 3-D
was chromatographed further over Si gel using n-hexane−EtOAc (6:4)
and purified by RP-HPLC (40-65% aq MeOH) to afford 1 (13.6 mg),
3 (23 mg), 7 (38 mg), and 8 (52 mg). Fr. 4 (4.49 g) was further
subjected to Si gel CC with n-hexane−EtOAc (6:4) and reseparated
using RP-HPLC (85% aq MeOH) to furnish 1 (80 mg), 9 (70 mg),
and 10 (117 mg). Fr. 7 (0.78 g) was also purified using RP-HPLC (45% aq
MeOH) to give 4 (6.5 mg).
Table 3. Antibacterial Activity of Isolated Compounds
compd
Bacillus subtilis
Klebsiella pneumoniae
a
b
extract
18.75
c
1
4.88
c
3
65.10
c
6
52.82
c
c
7
185.19
185.19
c
9
185.19
c
10
250.00
c
c
ampicillin
0.36
107.45
a
Ephemeranthoquinone B (1): red needles (MeOH); mp 148−
150 °C; UV (MeOH) λmax (log ε) 217.6 (3.67), 223.4 (3.67), 242 (3.46),
276.2 (3.38), 321.2 (3.38) nm; FT-IR (film) νmax 3063, 2993, 1670, 1639,
1595, 1452, 1246 cm−1; 1H and 13C NMR data, see Table 1; EIMS m/z
256 [M]+ (100), 213 (61), 171 (46.5), 127 (38), 115 (81), 69 (33);
HREIMS m/z 256.0709 (calcd for C15H12O4, 256.0736).
EtOAc-soluble portion of MeOH extract of roots of Cymbidium
b
c
Great Flower Marie Laurencin. MIC values, in μg/mL. MIC values,
in μM.
In contrast, ephemeranthoquinone B (1) show weak cytotoxic
activity against HL-60 cells with an IC50 value of 2.8 μM (Table 4).
A large number of phenanthrene-producing plants have been used
in traditional medicine throughout the world, and phenan-
threnes have been identified as their active constituents from
phytochemical−pharmacological investigations. According to
our studies, ephemeranthoquinone B (1) is a promising natural
bioactive product.
Marylaurencinol A (2): white, amorphous powder; UV (MeOH)
λmax (log ε) 225.4 (3.89), 273.4 (3.80), 306.2 (3.81) nm; FT-IR (film)
1
νmax 3348, 3215, 1616, 1582, 1501, 1452, 1329, 1236, 1094 cm−1; H
and 13C NMR data, see Table 1; EIMS m/z 272 [M]+ (100), 257 (32),
225 (32), 197 (74), 169 (42), 157 (31), 141 (29), 139 (40), 128 (32),
115 (33); HREIMS m/z 272.1040 (calcd for C16H16O4, 272.1049).
Marylaurencinol B (3): brown needles (MeOH); mp 195.8−
196.8 °C; UV (MeOH) λmax (log ε) 227 (3.99), 270 (3.99), 297 (3.88)
nm; FT-IR (film) νmax 3232, 1610, 1458, 1252, 1097, 1047 cm−1; 1H and
13C NMR data, see Table 2; EIMS m/z 288 [M]+ (100), 273 (52), 241
(9.8), 213 (9.2), 185 (5.4), 127 (6.4), 115 (6.1), 70 (3.3), 43 (2.3);
HREIMS m/z 288.1003 (calcd for C16H16O5[M]+, 288.0998).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined with a Yanagimoto micro melting point apparatus. Optical
608
dx.doi.org/10.1021/np200788u | J. Nat. Prod. 2012, 75, 605−609

Journal of Natural Products
Article
Marylaurencinoside A (4): brown, amorphous powder; [α]D +6.3
(c 0.7, MeOH); UV (MeOH) λmax (log ε) 218 (4.11), 268 (3.89), 301
(3.75) nm; FT-IR (film) νmax 3230, 1610, 1450, 1221, 1188, 1095
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra of compounds 1−4 are available free of charge via
1
cm−1; H and 13C NMR data, see Table 2; HRFABMS m/z 419.1346
the Internet at http://pubs.acs.org.
(calcd for C21H23O9, 419.1343).
X-ray Crystallographic Data for Ephemeranthoquinone B (1)
and Marylaurencinol B (3) (ref 20). Single crystals of 1, obtained by
slow evaporation of MeOH, were selected, fitted onto a glass fiber, and
measured at −173 °C with a Bruker Apex II Ultra diffractometer using
Mo Kα radiation. Data correction and reduction were performed with
the crystallographic package Apex II. The structures were solved by
direct methods using SHELXS-97 (Sheldrick, 1990) and refined using
full matrix least-squares based on F2 with SHELXL-97 (Sheldrick,
1997). All non-hydrogen atoms were refined anisotropically, and
hydrogen atoms were positioned geometrically. A total of 205
parameters were considered. Final disagreement indices were R1 =
0.0770 and wR2 = 0.1830 (I > 2σ(I)). The ORTEP plot was obtained
by the program PLATON (A. L. Spek, 2009). Crystal data: C15H12O4,
MW = 256.25, orthorhombic, space group P212121, Z = 4, a =
7.0616(16) Å, b = 8.2285(19) Å, c = 19.953(5) Å, V = 1159.4(5) Å3.
X-ray analysis of 3 was conducted in the same way as described for 1.
A total of 195 parameters were considered. Final disagreement indices
were R1 = 0.0284 and wR2 = 0.0789 (I > 2σ(I)). The ORTEP plot
was obtained with the program PLATON (A. L. Spek, 2009). Crystal
data: C16H14O5, MW = 286.27, monoclinic, space group Pn, Z = 2, a =
7.4378(11) Å, b = 9.5159(14) Å, c = 10.0687(15) Å, β = 95.705(2)°.
V = 709.18(18) Å3.
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-88-602-8439. Fax: +81-88-655-3051. E-mail:
yosikawa@ph.bunri-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Vicinelli, V.; Ceroni, P.; Maestri, M.; Lazzari, M.; Balzani, V.; Lee,
S.-K.; Heyst, J.; Vogtle, F. Org. Biomol. Chem. 2004, 2, 2207−2213.
̈
(2) Majumder, P. L.; Sen, R. C. Phytochemistry 1991, 30, 2432−2434.
(3) Honda, C.; Yamaki, M. J. Nat. Med. 2001, 55, 68−70.
(4) Leong, Y.-W.; Kang, C.-C.; Harrison, L. J.; Powell, A. D.
Phytochemistry 1997, 44, 157−165.
(5) Estrada, S.; Toscano, R. A.; Mata, R. J. Nat. Prod. 1999, 62,
1175−1178.
(6) Lin, Y.-L.; Chen, W.-P.; Macabalang, A. D. Chem. Pharm. Bull.
2005, 53, 1111−1113.
(7) Tezuka, Y.; Hirano, H.; Kikuchi, T.; Xu, G.-J. Chem. Pharm. Bull.
1991, 39, 593−598.
(8) Majumder, P. L.; Banerjee, S. Phytochemistry 1990, 29, 3052−
3055.
Acid Hydrolysis of Marylaurencinoside A (4). A solution of 4
(1 mg) in 5% H2SO4−dioxane (1:1) was heated at 100 °C for 2 h. The
reaction mixture was diluted with H2O, neutralized with Amberlite
IRA-35, and evaporated dry in vacuo. The identification of
D-(+)-glucose was determined using RI detection (Shimadzu RID-
10A) and chiral detection (Shodex OR-1) by HPLC (Shodex RSpak
NH2P-50 4D, CH3CN−H2O−H3PO4, 95:5:1, 1 mL/min, 47 °C), by
comparison with an authentic D-glucose sample. The sugar portion
gave a peak of D-(+)-glucose at 20.7 min.
Antimicrobial Assay. B. subtilis NBRC 3134 and K. pneumoniae
NBRC 3512 were used for testing antimicrobial activity. These strains
were tested by using microdilution assays, and MIC values were
determined. Bacterial strains were inoculated on YP agar plates [1%
polypeptone (Nihon Pharmaceutical, Japan), 0.2% yeast extract
(Difco, USA), 0.1% MgSO4−7H2O, and 6% agar (Nacalai Tesque,
Japan)] and incubated at 37 °C (B. subtilis) and 27 °C (K. pneumoniae)
for 12 h. A stock solution of crude extracts or samples was prepared at
10 mg/mL in DMSO and further diluted to varying concentrations in
96-well plates that contained microbial strains incubated in YP medium for
the bacterial strains. Each plate was further incubated at 37 °C overnight,
and ampicillin (Nacalai Tesque, Japan) was used as a reference reagent for
the bacterial strains.
Cytotoxic Assay. The antiproliferative potential of the test
compounds was evaluated by the WST-8 assay (Promega, USA)21
against the HL-60 cell line (DS Pharma Biomedical, Japan). HL-60
cells were maintained in RPMI-1640 medium (Nacalai Tesque, Japan)
supplemented with 10% fetal bovine serum (Invitrogen, USA) at
37 °C in a humidified atmosphere of 5% CO2. Cell growth was quanti-
fied by the ability of living cells to reduce WST-8 (2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
monosodium) to a highly water-soluble formazan. Briefly, cells were
seeded in 96-well plates at a density of 5 × 103 cells/well (100 μL/
well) and incubated for 2 h. Then, 100 μL of medium containing test
compounds was added, and the cells were incubated for 72 h. After
incubation, 20 μL of WST-8 solution was added and the cells were
incubated for an additional 3 h. The optical density at 450 nm was
measured by using a microplate reader (SpectraMax 340PC, Molecular
Devices, Japan).
(9) Kovac
1084−1110.
́
s, A.; Vasas, A.; Hohmonn, J. Phytochemistry 2008, 69,
(10) Takagi, S.; Yamaki, M.; Inoue, K. Phytochemistry 1983, 22,
1011−1015.
(11) Lee, Y. H.; Park, J. D.; Beak, N. I.; Kim, S. I.; Ahn, B. Z. Planta
Med. 1995, 61, 178−180.
(12) Xue, Z.; Li, S.; Wang, S.; Wang, Y.; Yang, Y.; Shi, J.; He, L.
J. Nat. Prod. 2006, 69, 907−913.
(13) Fan, C.; Wang, W.; Wang, Y.; Qin, G.; Zhao, W. Phytochemistry
2001, 57, 1255−1258.
(14) DellaGreca, M.; Fiorentino, A.; Isidori, M.; Lavorgna, M.;
Monaco, P.; Previtera, L.; Zarrelli, A. Phytochemistry 2002, 60, 633−
638.
(15) Coxon, D. T.; Ogundana, S. K.; Dennis, C. Phytochemistry 1982,
21, 1389−1392.
(16) Hernandez-Romero, Y.; Rojas, J.-I.; Castillo, R.; Rojas, A.; Mata,
́
R. J. Nat. Prod. 2004, 67, 160−167.
(17) Yang, H.; Sung, S. H.; Kim, Y. C. J. Nat. Prod. 2007, 70, 1925−
1929.
(18) Tezuka, Y.; Hirano, H.; Kikuchi, T.; Xu, G.-Y. Chem. Pharm.
Bull. 1991, 39, 593−598.
(19) Yoshikawa, K.; Yagi, C.; Hama, H.; Tanaka, M.; Arihara, S.;
Hashimoto, T. J. Nat. Prod. 2010, 73, 1763−1766.
(20) Crystallographic data (excluding structural factors) for the
structures of 1 and 3 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication numbers
CCDC 848411 and CCDC 848412, respectively. Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ UK (fax: +44(0)-1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk.
(21) Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.;
Hamamoto, T.; Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47.
609
dx.doi.org/10.1021/np200788u | J. Nat. Prod. 2012, 75, 605−609