Ceramide and cerebrosides from the octocoral sarcophyton ehrenbergi

Article abstract of DOI:10.1021/np800362g

Chemical investigation of the octocoral Sarcophyton ehrenbergi, collected at the Dongsha Islands, Taiwan, has led to the isolation of a known ceramide (1) and two new cerebrosides, sarcoehrenosides A (2)andB(4), along with three known cerebrosides (3, 5,

Full text of DOI:10.1021/np800362g

J. Nat. Prod. 2009, 72, 465–468
465
Notes
Ceramide and Cerebrosides from the Octocoral Sarcophyton ehrenbergi#
Shi-Yie Cheng,† Zhi-Hong Wen,†,o Shu-Fen Chiou,† Chung-Wei Tsai,† Shang-Kewi Wang,‡ Chi-Hsin Hsu,†,o Chang-Feng Dai,
Michael Y. Chiang,| Wei-Hsien Wang,3 and Chang-Yih Duh*,†,o
Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China, Department
of Microbiology, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Institute of Oceanography, National Taiwan
UniVersity, Taipei 106, Taiwan, Republic of China, Department of Chemistry, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan,
Republic of China, National Museum of Marine Biology and Aquarium, Checheng, Pingtung 944, Taiwan, Republic of China, and Center of
Asia-Pacific Marine Research, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China
ReceiVed June 17, 2008
Chemical investigation of the octocoral Sarcophyton ehrenbergi, collected at the Dongsha Islands, Taiwan, has led to
the isolation of a known ceramide (1) and two new cerebrosides, sarcoehrenosides A (2) and B (4), along with three
known cerebrosides (3, 5, and 6). The structures of the new compounds were established by spectroscopic and chemical
methods. Sarcoehrenoside A (2) differs from previously known marine cerebrosides in that it possesses a rare R-glucose
moiety. Compounds 1-6 were evaluated for antimicrobial activity against a small panel of bacteria and for
anti-inflammatory activity using RAW 264.7 macrophages.
Cerebrosides are a large group of molecules containing a
ceramide unit and one or more sugars. The hydrophobic ceramide
moiety includes a sphingoid base and an amide-linked fatty acid
chain. Cerebroside derivatives have been isolated from various
marine invertebrates, such as sea stars,1 soft corals,2 sponges,2a,3
sea anemones,4 and ascidians.5 In recent years several different
types of biological activities have been found for these compounds,
including antifungal, antitumor, antiviral, cytotoxic, and immuno-
modulatory properties.6
In the course of a search for bioactive substances from marine
sources,7 chromatographic separation of an alcyonacean soft coral,
Sarcophyton ehrenbergi Marenzeller (Octocorallia: Alcyonacea),
collected at the Dongsha Islands, Taiwan, has afforded two new
cerebrosides, sarcoehrenosides A (2) and B (4). Also obtained were
a known ceramide (1)8 and three known cerebrosides (3, 5, and
6),3a,9,10 which were isolated from S. ehrenbergi for the first time.
To our knowledge, sarcoehrenoside A (2), in possessing a rare R-D-
glucose moiety at C-1 of the glycerol ether unit, differs from
chain (terminal methyl protons at δ 0.90 and methylene protons at
previous cerebrosides isolated from marine organisms. In this paper,
δ 1.29-1.38), and a sugar (an anomeric proton at δ 4.87) were
we describe the isolation, structural elucidation, and evaluation of
observed, strongly suggesting the glycosphingolipid nature of 2.
the antimicrobial and anti-inflammatory activity of these metabolites.
The R-glucopyranose moiety was indicated by the anomeric proton
Sarcoehrenoside A (2) was isolated as a white, amorphous
at δ 4.87 (1H, d, J ) 4.5 Hz, H-1′′) and the chemical shifts in the
powder and exhibited a [M + Na]+ peak at m/z 774.5490,
1H and 13C NMR spectra (Table 1). The anomeric proton of the
corresponding to a molecular formula of C43H77NO9 in the positive
R-glucose unit exhibited a long-range 1H-13C correlation with C-1
(Figure 1), demonstrating attachment of the glucose moiety at C-1.
The relative configuration of the R-glucopyranose moiety was
HRESIMS. The IR absorptions at 1638 and 1547 cm-1 and 13C
NMR signals at δ 54.6 (C-2) and 175.4 (C-1′) suggested a secondary
1
amide in the molecule. In the H and 13C NMR spectra of 2, the
1
determined by a NOESY experiment (Figure 2) and from the H
characteristic signals of an amide linkage (a nitrogenated methane
NMR J values. In the 1H NMR spectrum, a small coupling constant
(J ) 4.5 Hz)11 was observed for H-1′′, which required an equatorial-
axial relationship between the anomeric proton (H-1′′) and H-2′′.
The large coupling constants (J ) 7.5 Hz) between H-2′′ and H-3′′,
H-3′′ and H-4′′, and H-4′′ and H-5′′ as well as the NOESY
correlations of H-2′′/H-1′′, H-4′′ and H-3′′/H-5′′ suggested that
H-2′′, H-3′′, H-4′′, and H-5′′ are all axial. Thus, the relative
configuration of the R-glucose moiety at the C-1 glycerol ether could
be established. Methanolysis of 2 gave a mixture of R- and ꢀ-methyl
glucopyranoside. The specific rotation of the methyl glucoside
proton at δ 3.93 and an amide carbonyl carbon at δ 175.4), a long
# Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute
and State University for his pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: 886-7-5252000,
ext. 5036. Fax: 886-7-5255020. E-mail: yihduh@nsysu.edu.tw.
† Department of Marine Biotechnology and Resources, National Sun Yat-
sen University.
‡ Department of Microbiology, Kaohsiung Medical University.
Institute of Oceanography, National Taiwan University.
| Department of Chemistry, National Sun Yat-sen University.
3 National Museum of Marine Biology and Aquarium.
o Center of Asia-Pacific Marine Research.
mixture, [R]24 +74.3 (c 0.2, MeOH), was close to that of an
D
10.1021/np800362g CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/12/2008

466 Journal of Natural Products, 2009, Vol. 72, No. 3
Notes
Table 1. 1H and 13C NMR Spectroscopic Data of Compounds 2 and 4a
2
4
C/H
13C
68.8 tb
1H
13C
69.9 tb
1H
lipid base unit
lipid base unit
1
a
b
3.88 dd (10.5, 4.5)c
3.83 dd (10.5, 4.5)
3.93 dd (8.0, 4.5)
4.14 t (8.0)
5.47 dd (15.5, 8.0)
5.72 dd (15.5, 6.5)
2.08 td (7.5, 6.5)
2.20 td (7.5, 7.0)
5.35 t (7.0)
a
b
4.12 dd (10.5, 3.5)c
3.71 dd (10.5, 3.5)
3.99 dd (7.5, 3.5)
4.13 t (7.5)
5.48 dd (15.5, 7.5)
5.74 dd (15.5, 6.0)
2.06 m
2
3
4
5
6
7
8
9
54.6 d
72.9 d
131.6 d
134.3 d
33.7 t
54.7 d
73.0 d
131.3 d
134.8 d
34.0 t
29.0 t
28.8 t
2.07 m
5.14 t (6.5)
130.6 d
135.3 s
136.3 d
128.7 d
34.1 t
125.0 d
136.9 s
40.9 t
10
11
12
13
6.02 d (15.5)
5.55 dt (15.5, 7.0)
2.08 td (7.5, 7.0)
1.38 m
1.97 t (7.5)
1.39 m
29.3 t
30.4 t
12-15
14, 15
16
17
18
30.5-30.9 t
1.29 m
30.4-31.1 t
33.2 t
23.9 t
14.6 q
12.9 q
1.29 m
1.29 m
1.29 m
0.90 t (6.0)
1.71 s
33.2 t
23.9 t
14.6 q
16.3 q
1.29 m
1.29 m
0.90 t (6.5)
1.59 s
19
N-acyl unit
N-acyl unit
1′
2′
3′
4′
175.4 s
74.3 d
129.1 d
135.0 d
33.6 t
177.4 s
73.2 d
36.0 t
26.3 t
4.45 d (6.0)
3.99 dd (8.0, 4.0)
1.70 m; 1.55 m
1.41 m
5.50 dd (15.5, 6.0)
5.83 dd (15.5, 6.5)
2.03 td (7.5, 6.5)
5′
5′-19′
6′
30.5-30.9 t
1.29 m
30.5 t
1.38 m
7′-15′
16′
17′
18′
20′
21′
22′
30.4-31.1 t
1.29 m
1.29 m
1.29 m
0.90 t (6.0)
33.2 t
23.9 t
14.6 q
23.9 t
14.6 q
1.29 m
0.90 t (6.5)
R-D-glucose unit
ꢀ-D-glucose unit
1′′
2′′
3′′
4′′
5′′
6′′
103.6 d
79.1 d
76.3 d
74.4 d
83.6 d
64.3 t
4.87 d (4.5)
3.96 dd (7.5, 4.5)
4.10 t (7.5)
3.63 m
3.69 m
3.63 m
104.9 d
75.1 d
78.1 d
71.7 d
78.1 d
62.8 t
4.27 d (8.0)
3.19 dd (9.5, 8.0)
3.34 m
3.30 m
3.28 m
a
b
a
b
3.87 dd (12.0, 1.5)
3.67 dd (12.0, 5.0)
3.56 m
a Spectra were measured in CD3OD (1H, 500 MHz; 13C, 125 MHz). b Multiplicities were deduced by HSQC and DEPT experiments. c J values (in
Hz) are in parentheses.
Figure 1. Key 1H-1H COSY ()) and HMBC (f) correlations of
2 and 4.
Figure 2. Selected NOESY correlations of 2 and 4.
authentic sample ([R]25D +77.3).1f Therefore, the absolute config-
uration of the R-glucose moiety was deduced to be the D-isomer.
The large vicinal coupling constants (J ) 15.5 Hz) between H-4
and H-5, H-10 and H-11, and H-3′ and H-4′ clearly indicated E
geometry for ∆4,10,3′ in 2. The key HMBC correlations from H-3
to C-4, from H-2′ to C-3′, and from H3-19 to C-8, C-9, C-10, and
C-11 helped locate the positions of the double bonds (Figure 1).
The UV absorption maximum at 224 nm indicated the presence of
a conjugated diene moiety.1f Furthermore, the stereochemistry of
the conjugated diene moiety was assigned as S-trans from NOE
correlations (Figure 2) from Me-19 to H-7 and H-11. The H-1H
1
COSY correlations observed between H-2 and both H-1 and H-3
were used to define the 2-amino-1,3-dioxygenated fragment. The
relative configurations of C-2 (δ 54.6) and C-3 (δ 72.9) were
predicted to be the D-erythro stereochemistry at C-2 and C-3, which

Notes
Journal of Natural Products, 2009, Vol. 72, No. 3 467
Figure 3. Effect of compounds 1-6 at 10 µM on the LPS-induced pro-inflammatory iNOS and on COX-2 protein expression of RAW
264.7 macrophage cells by immunoblot analysis. (A) Immunoblot of iNOS. (B) Immunoblot of COX-2. (C) Immunoblot of ꢀ-actin. A and
B values are mean ( SEM (n ) 5). The relative intensity of the LPS alone stimulated group was taken as 100%. *Significantly different
from LPS-stimulated (control) group (*P < 0.05).
was consistent with those reported for other (2S,3R,2′R) sphingosine
moieties.12,13 The stereochemistry of 2 (2S,3R), as shown in its
structure, was confirmed after determination of the stereochemistry
of 1 on the basis of biosynthetic reasoning. Methanolysis of 2
yielded methyl (2′R,3′E)-2′-hydroxyoctadec-3′-enoate (2a), for
which the molecular formula C19H36O3 was determined by ESIMS-
MS (m/z 335.0 [M + Na]+) as C19H36O3. On the basis of the
aforementioned observations, sarcoehrenoside A (2) was character-
ized unambiguously as 1-O-(R-D-glucopyranosyl)-(2S,3R,4E,8E,10E)-
2-[(2′R,3′E)-2′-hydroxyoctadec-3′-enoylamino]-9-methyloctadeca-
4,8,10-triene-1,3-diol.
scens (ATCC25419), Salmonella enteritidis (ATCC13076), Yersinia
enterocolitica (ATCC23715), and Shigella sonnei (ATCC11060).
None of these compounds exhibited any antibacterial activity at a
concentration of 100 µg/disk.
A previous study has reported that glycosphingolipids and
sphingolipids possess cyclooxygenase-2 inhibition,14 which prompted
us to evaluate the anti-inflammatory effect of our isolated metabo-
lites. As shown in Figure 3, the in vitro anti-inflammatory activity
of compounds 1-6 was tested using LPS-stimulated cells. Stimula-
tion of RAW 264.7 cells with LPS resulted in up-regulation of the
pro-inflammatory iNOS and COX-2 proteins. Both compounds 1
and 6 reduced the levels of iNOS to 46.9 ( 9.7% and 20.3 ( 6.8%,
respectively, and of COX-2 to 77.2 ( 9.9% and 64.3 ( 8.6%,
respectively, in comparison with those of the control groups.
Compounds 1a, 2, 4, and 5 reduced iNOS protein expression (55.6
( 2.9%, 47.3 ( 7.1%, 46.5 ( 5.3%, and 25.8 ( 6.2%, respec-
tively), but did not inhibit COX-2 protein expression. All com-
pounds did not affect ꢀ-actin protein expression at a 10 µM
concentration. Under the same experimental conditions, 10 µM
CAPE (caffeic acid phenylthyl ester) reduced the levels of iNOS
and COX-2 protein to 1.5 ( 2.1% and 70.2 ( 11.5%, respectively,
relative to the control cells stimulated with LPS.
Sarcoehrenoside B (4) was found to have a molecular formula
of C46H87NO9, as indicated by HRESIMS. In addition to the
pseudomolecular ion peak at m/z 820.6 [M + Na]+, the ESIMS-
MS of 4 also exhibited an intense fragment peak at m/z 496.7,
produced by elimination of the N-acyl unit from the molecular ion.
The spectroscopic data for 4 (Table 1) exhibited the presence of
an amide linkage, a long chain, and a sugar, consistent with the
C-9 methyl cerebroside nature of 4. In the 1H and 13C NMR spectra
of 4, an anomeric proton appeared at δ 4.27 (1H, d, J ) 8.0 Hz,
H-1′′) and 13C NMR signals resonated at δ 104.9 (H-1′′), 75.1 (H-
2′′), 78.1 (H-3′′), 71.7 (H-4′′), 78.1 (H-5′′), and 62.8 (H-6′′),
supporting the presence of a ꢀ-glucopyranose moiety.3b Metha-
nolysis of 4 also yielded an R- and ꢀ-glucopyranosyl mixture, the
Experimental Section
specific rotation ([R]24 +71.4, MeOH) of which was used to
D
General Experimental Procedures. Optical rotations were deter-
mined on a JASCO P1020 polarimeter. UV spectra were obtained on
a Hitachi U-3210 spectrophotometer, and IR spectra were recorded on
a JASCO FT/IR-4100 spectrophotometer. The NMR spectra were
recorded on a Bruker Avance 300 NMR spectrometer at 300 MHz for
1H and 75 MHz for 13C, or on a Varian MR 400 NMR spectrometer at
identify ꢀ-glucose as a D-isomer. The glycerol ether linkage between
the anomeric proton (δ 4.27, H-1′′) and C-1 was confirmed by
HMBC correlations (Figure 1) of H-1′′ with C-1 and H-1 with C-1′′.
The coupling constant (15.5 Hz) indicated an E geometry for ∆4.
A NOE correlation from Me-19 to H-7 supported the presence of
an (E)-8,9 double bond. Therefore, 3 was defined as a C-9 methyl
4E,8E-sphingadiene-type cerebroside. The connectivity between the
acyl moiety and the NH of the sphingosine base was confirmed by
HMBC correlations of H-2 with C-1′ and H-2′ with C-1′. Metha-
nolysis of 4 yielded methyl (2′R)-2′-hydroxyhenicosanoate (4a),
for which the molecular formula, C22H44O3, was identified by
ESIMS-MS analysis (m/z 379.3 [M + Na]+). The specific rotation
value ([R]24D -3.8) of 4a indicated a 2′R absolute configuration in
4.1f The relative configuration at C-2 and C-3 was determined as
2S,3R (erythro) based on their 13C NMR chemical shifts and the
specific rotation of this compound.1f Consequently, sarcoehrenoside
B (4) was fully assigned as 1-O-(ꢀ-D-glucopyranosyl)-(2S,3R,4E,8E)-
2-[(2′R)-2′-hydroxyhenicosanoylamino]-9-methyl-4,8-octadecadi-
ene-1,3-diol.
1
400 MHz for H and 100 MHz for 13C, or on a Varian Unity INOVA
500 FT-NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C.
Chemical shifts are expressed in δ (ppm) referring to the solvent peaks
δH 3.30 and δC 49.0 for CD3OD and δH 7.265 and δC 77.0 for CDCl3,
and coupling constants are expressed in Hz. Electrospray ionization
MS/MS analysis was recorded using a positive-mode API 4000 tandem
mass spectrometer, with a capillary voltage of 5500 V applied, and
the spectra were acquired at m/z 400-1000 for parent ion spectra and
m/z 50-1000 for fragment ion spectra. ESIMS were recorded by ESI
FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck,
230-400 mesh) was used for column chromatography; precoated Si
gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC
analysis. C18 reversed-phase silica gel (230-400 mesh, Merck) was
also used for column chromatography. High-performance liquid chro-
matography (HPLC) was performed on a Hitachi L-7420 UV detector
L-7100 pump apparatus equipped with a Merck Hibar RP-18e column
(250 × 10 mm, 5 µm).
Compounds 1-6 were tested against five bacterial strains,
comprising Enterobacter aerogenes (ATCC13048), Serratia marce-

468 Journal of Natural Products, 2009, Vol. 72, No. 3
Notes
Animal Material. The octocoral S. ehrenbergi was collected by hand
using scuba at Dongsha Islands, Taiwan, in April 2007, at a depth of
10 m, and was stored in a freezer for 5 weeks until extraction. This
soft coral was identified by one of the authors (C.-F.D.). A voucher
specimen (TS-07) was deposited in the Department of Marine Bio-
technology and Resources, National Sun Yat-sen University.
In Vitro Anti-inflammatory Assay. The anti-inflammatory assay
was modified from Ho et al.16 and Park et al.17 Murine RAW 264.7
macrophages were obtained from the American Type Culture Collection
(ATCC, No. TIB-71). The cells were activated by incubation in medium
containing Escherichia coli LPS (0.01 µg/mL; Sigma) for 16 h in the
presence or absence of various compounds. Then, cells were washed
with ice-cold PBS, lysed in ice cold lysis buffer, and centrifuged at
20000g for 30 min at 4 °C. The supernatant was decanted from the
pellet and retained for Western blot analysis. Protein concentrations
were determined by the DC protein assay kit (Bio-Rad) modified by
the method of Lowry et al.18 Samples containing equal quantities of
protein were subjected to SDS-polyacrylamide gel electrophoresis, and
the separated proteins were electrophoretically transferred to polyvi-
nylidene difluoride membranes (PVDF; Immobilon-P, Millipore, 0.45
µm pore size). The resultant PVDF membranes were incubated with
blocking solution and incubated for 180 min with antibody against
inducible nitric oxide synthase (iNOS; 1:1000 dilution; Transduction
Laboratories) and cyclooxygenase-2 (COX-2; 1:1000 dilution; Cayman
Chemical) protein. The blots were detected using ECL detection
reagents (Perkin-Elmer, Western Blot Chemiluminescence Reagent
Plus) according to the manufacturer’s instructions.
Extraction and Isolation. Soft coral extracts were prepared by
percolating aliquots of 50 g of freeze-dried S. ehrenbergi with acetone
for 24 h at room temperature. The combined acetone extracts were
concentrated to a brown gum, which was partitioned between H2O and
EtOAc. The dried EtOAc-soluble (20.0 g) partition was chromato-
graphed over a silica column using n-hexane, n-hexane-EtOAc, and
EtOAc-MeOH mixtures of increasing polarity to obtain fractions 1-40.
Fraction 28 (0.5 g), eluted with EtOAc-MeOH (1:1), was further
subjected to RP-18 gravity column chromatography by eluting with
80% MeOH in H2O, 90% MeOH in H2O, and 100% MeOH. Altogether,
six fractions were obtained, of which fraction 1 (60 mg) was purified
further by RP-18 HPLC column chromatography (5% CH3CN in
MeOH, flow rate 5.0 mL/min) to afford 1 (3 mg), 2 (2 mg), 3 (2 mg),
4 (3 mg), 5 (2 mg), and 6 (6 mg). The retention time for each metabolite
was as follows: 1 (26.5 min), 2 (35.6 min), 3 (48.6 min), 4 (44.2 min),
5 (41.0 min), and 6 (33.5 min).
Sarcoehrenoside A (2): white, amorphous powder; [R]23 +77.0
Acknowledgment. Financial support was provided by Ministry of
Education (96C031703) and National Science Council of Taiwan
(NSC96-2320-B-110-003-MY3) awarded to C.Y.D.
D
(c 0.2, MeOH); IR (KBr) 3387, 2951, 2859, 1638, 1547, 1456, 1387,
1241, 1131, 1035, 737 cm-1; UV λmax (MeOH) (log ε) 224 (3.97) nm;
1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 774.5490
[M + Na]+ (calcd for C43H77NO9Na, 744.5496).
References and Notes
Sarcoehrenoside B (4): white, amorphous powder; [R]23 +51.3
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D
(c 0.3, MeOH); IR (KBr) 3396, 2951, 2857, 1638, 1546, 1457, 1385,
-1
1
1242, 1178, 1131, 1036, 736 cm ; H NMR and 13C NMR data, see
Table 1; HRESIMS m/z 820.6274 [M + Na]+ (calcd for C46H87NO9Na,
820.6278).
Methanolysis of 2 and 4. An aliquot (1 mg) of 2 and 4 in a mixture
of 1 N aqueous HCl (0.1 mL) and methanol (0.9 mL) was refluxed for
18 h on a magnetic stirrer. The reaction mixture was neutralized with
NaHCO3 and diluted with H2O (1.5 mL). The aqueous solution was
extracted with n-hexane three times, and the organic phase was dried
with anhydrous MgSO4. After removal of solvent and purification by
silica gel column chromatography, methyl (2′R,3′E)-2′-hydroxyoctadec-
3′-enoate (2a, 0.3 mg) and methyl (2′R)-2′-hydroxyhenicosanoate (4a,
0.4 mg) were obtained and identified by ESIMS-MS peaks at m/z 335.0
and 379.3 [M + Na]+, respectively. In addition, the aqueous layer was
removed and purified on a C18 reversed-phase column, eluted with
MeOH-H2O (4:1), to give 2b [0.2 mg, [R]24 +74.3 (c 0.2, MeOH)]
D
and 4b [0.2 mg, [R]24 + 72.5 (c 0.2, MeOH)].
D
Methyl (2′R,3′E)-2′-hydroxyoctadec-3′-enoate (2a): white, amor-
1
phous powder; [R]24 -17.5 (c 0.3, CHCl3); H NMR (CDCl3, 400
D
MHz) δ 5.90 (1H, dt, J ) 15.6, 6.4 Hz, H-4′), 5.50 (1H, dd, J ) 15.6,
6.4 Hz, H-3′), 4.61 (1H, d, J ) 6.4 Hz, H-2′), 3.80 (3H, s, COOCH3),
2.07 (2H, td, J ) 7.6, 6.4 Hz, H-5′), 1.39 (2H, m, H-6′), 1.26 (22H,
brs, H-7′-H-17′), 0.88 (3H, t, J ) 6.4 Hz, H-18′); ESIMS-MS m/z 335.0
[M + Na]+.
Methyl (2′R)-2′-hydroxyhenicosanoate (4a): white, amorphous
1
powder; [R]24 -3.8 (c 0.4, CHCl3); H NMR (CDCl3, 400 MHz) δ
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D
4.20 (1H, dd, J ) 7.6, 4.4 Hz, H-2′), 3.79 (3H, s, COOCH3), 1.76 (1H,
m, H-3a′), 1.58 (1H, m, H-3b′), 1.45 (2H, m, H-4′), 1.26 (32H, brs,
H-5′-H-20′), 0.88 (3H, t, J ) 6.4 Hz, H-21′); ESIMS-MS m/z 379.3
[M + Na]+.
In Vitro Antimicrobial Activity. Bacterial strains were grown in
LB (Luria-Bertani) broth medium for 24 h at 37 °C. Then, 17 mL of
LB hard agar (1.5% agar) was poured into sterile Petri dishes (9 cm)
and allowed to set. Next, 2.7 mL of molten LB soft agar (0.7% agar,
45 °C) was inoculated with 0.3 mL of broth culture of the test organism
and poured over the base hard agar plates, forming a homogeneous
top layer. Sterile paper disks (Advantec, 8 mm) were placed onto the
top layer of the LB agar plates. Ten microliters (2 µg/µL) of the tested
compounds was applied onto each filter paper disk. Ampicillin (5 µg/
µL) and the same solvents served as positive and negative controls.
All plates were incubated at 37 °C, 24 h prior to antibacterial activity
evaluation. The antimicrobial activity of compounds 1-6 was tested
up to 100 µg/mL against E. aerogenes (ATCC13048), S. marcescens
(ATCC25419),S.enteritidis(ATCC13076),Y.enterocolitica(ATCC23715),
and S. sonnei (ATCC11060). All bacterial strains were obtained from
the American Type Culture Collection. The antibiotic activity evaluation
method was conducted on the basis of previous reports.15
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NP800362G