Isochromans and related constituents from the endophytic fungus Annulohypoxylon truncatum of Zizania caduciflora and their anti- inflammatory effects

Article abstract of DOI:10.1021/acs.jnatprod.6b00698

Six new isochroman derivatives (annulohypoxylomans A-C, 1-3; annulohypoxylomanols A and B, 6 and 7; and annulohypoxyloside, 8), an isocoumarin (annulohypoxylomarin A, 4), and an azaphilone derivative (xylariphilone, 5) were isolated from an ethyl acetate extract derived from cultures of the endophytic fungus JS540 found in the leaves of Zizania caducif lora. The JS540 strain was identified as Annulohypoxylon truncatum. The structures of the isolated compounds were elucidated by one- and two-dimensional nuclear magnetic resonance and mass spectrometry and by comparison with related compounds from the literature. The anti-inflammatory activities of the isolated compounds were evaluated in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells. Xylariphilone (5) exhibited significant inhibitory effects on LPS-induced interleukin (IL)-6, IL-12 p40, and tumor necrosis factor (TNF)-α production with IC₅₀ values of 5.3, 19.4, and 37.6 μM, respectively. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jnatprod.6b00698

Note
pubs.acs.org/jnp
Isochromans and Related Constituents from the Endophytic Fungus
Annulohypoxylon truncatum of Zizania caduciflora and Their Anti-
Inflammatory Effects
Wei Li,† Changyeol Lee,‡ Sung Hee Bang,‡ Jin Yeul Ma,† Soonok Kim,§ Young-Sang Koh,∥
and Sang Hee Shim*,‡
†KM Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Republic of Korea
‡College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea
§National Institute of Biological Resources, Incheon 22689, Republic of Korea
∥School of Medicine and Brain Korea 21 PLUS Program and Institute of Medical Science, Jeju National University, Jeju 690-756,
Republic of Korea
S
* Supporting Information
ABSTRACT: Six new isochroman derivatives (annulohypoxylomans A−
C, 1−3; annulohypoxylomanols A and B, 6 and 7; and annulohypoxylo-
side, 8), an isocoumarin (annulohypoxylomarin A, 4), and an azaphilone
derivative (xylariphilone, 5) were isolated from an ethyl acetate extract
derived from cultures of the endophytic fungus JS540 found in the leaves
of Zizania caduciflora. The JS540 strain was identified as Annulohypoxylon
truncatum. The structures of the isolated compounds were elucidated by
one- and two-dimensional nuclear magnetic resonance and mass
spectrometry and by comparison with related compounds from the
literature. The anti-inflammatory activities of the isolated compounds were
evaluated in lipopolysaccharide (LPS)-stimulated bone marrow-derived
dendritic cells. Xylariphilone (5) exhibited significant inhibitory effects on
LPS-induced interleukin (IL)-6, IL-12 p40, and tumor necrosis factor
(TNF)-α production with IC50 values of 5.3, 19.4, and 37.6 μM, respectively.
ndophytes are microbes that colonize living, internal
tissues of plants without causing any immediate negative
investigating novel bioactive constituents, eight secondary
metabolites were isolated from the ethyl acetate extract of
A. truncatum (JS540). The anti-inflammatory effects of the
isolated compounds on lipopolysaccharide (LPS)-induced
expression of the pro-inflammatory cytokines interleukin
(IL)-6, IL-12 p40, and tumor necrosis factor (TNF)-α in
bone marrow derived dendritic cells (BMDCs) were
investigated. To our knowledge, this is the first report on the
isochroman components of A. truncatum and their anti-
inflammatory activity.
Eight secondary metabolites were isolated from an ethyl
acetate extract derived the endophytic fungus A. truncatum.
Their structures were identified as annulohypoxyloman A (1),
annulohypoxyloman B (2), annulohypoxyloman C (3),
annulohypoxylomarin A (4), xylariphilone (5),10 annulohypox-
ylomanol A (6), annulohypoxylomanol B (7), and annulohy-
poxyloside (8). Their structures were elucidated by one- and
two-dimensional NMR as well as mass spectrometry. Among
them, compounds 1−3 and 6−8 are new compounds, and
compound 4 was isolated from nature for the first time.
E
effects.1 The ecological roles and chemical interactions of
endophytes are currently under investigation, particularly in
relation to their host plants. This is changing rapidly, however,
as it is now evident that plants have intricate relationships with
an array of fungi, which can lead to an increase in plant vigor,
growth, and development as well as changes in plant
metabolism.2,3 Therefore, such beneficial endophytic fungi
may be very valuable for the development of more productive
and sustainable agricultural practices or for the production of
chemicals for pharmacological purposes.4,5 Moreover, endo-
phytes are a rich and reliable source of genetic diversity and
may include previously undescribed species.6,7 Novel microbes
(as defined at the morphological and/or molecular level) are
often associated with novel natural products. In an effort to
investigate natural compounds with intriguing structures in
endophytic fungi, we selected plants from distinct environ-
mental settings. Several endophytes were isolated from the
leaves and stems of aquatic reed plants. Among them, a strain of
endophyte, Annulohypoxylon truncatum (JS540), was isolated
from the leaves of Zizania caduciflora. A previous report
showed that the metabolites of A. truncatum, such as
truncaquinones, display antibacterial activity.8 In the course of
Received: July 27, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00698
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
a
1
Table 1. H NMR Data of Compounds 1−4 and 6−8 (600 MHz)
b
c
c
d
b
b
c
1
2
3
4
6
7
8
1
5.02, d (14.8)
5.44, d (14.8)
3.80, m
4.52, d (14.8)
4.79, d (14.8)
3.31, m
4.55, d (15.0)
4.80, d (15.0)
3.71, m
5.08, d (15.1)
5.39, d (15.1)
3.93, m
5.16, d (15.0)
5.49, d (15.0)
3.98, m
4.52, d (14.9)
4.79, d (14.9)
3.70, m
3
4
4.60, m
2.62, dd (16.1, 10.5)
2.70, dd (16.1, 2.7)
6.64, br s
2.45, dd (16.1, 10.5)
2.50, dd (16.1, 2.8)
6.10, s
2.52, dd (16.1, 10.6)
2.61, dd (16.1, 3.0)
6.24, s
2.63, dd (16.6, 11.6)
2.86, dd (16.6, 3.3)
6.72, d (8.5)
4.81, t (8.0)
4.83, t (8.0)
2.40, dd (16.1, 10.5)
2.63, dd (16.1, 2.8)
6.11, s
5
7.26, s
7.29, s
6
7.20, d (8.5)
7
6.83, br s
11
1.32, d (6.1)
1.25, d (6.1)
3.72, s
1.28, d (6.2)
3.78, s
1.47, d (6.3)
2.11, s
1.69, d (6.1)
3.75, s
1.68, d (6.1)
3.72, s
1.26, d (6.2)
6-OMe
7-OMe
7-Me
8-OMe
1′
2′
3′
4′
5′
3.73, s
3.83, s
3.58, s
5.59, d (4.2)
4.21, d (5.5)
4.10, dd (5.8, 1.6)
4.21, d (4.2)
3.56, d (4.2)
a
b
c
Assignments were done by HMQC and HMBC experiments; J values (Hz) are in parentheses. Measured in pyridine-d5. measured in methanol-
d
d4. measured in chloroform-d.
Compound 1 was isolated as a yellow, amorphous powder.
an oxygenated methine carbon at δC 71.1 (C-3), an oxygenated
methylene carbon at δC 65.7 (C-1), a methylene carbon at δC
36.9 (C-4), and a methyl carbon at δC 22.3 (C-11). Thus, both
1H and 13C NMR data demonstrated that 1 is an isochroman
derivative. Key HMBC correlations between H-5 (δH 6.64)/C-
9 (δC 114.1) and C-10 (δC 136.7) and between H-7 (δH 6.64)
and C-9 (δC 114.1) indicated two hydroxy groups located at C-
6 and C-8 (Figure 2), which was similar to (3S)-6-hydroxy-8-
methoxy-3-methylisochroman.11 The obvious difference be-
tween these compounds was the configuration of C-3. This was
further confirmed by comparing the [α]D values for both
compounds, which were found to be +45 for (3S)-6-hydroxy-8-
methoxy-3-methylisochroman and −72.6 for 1. Thus, R was
deduced as the absolute configuration of C-3. Therefore,
compound 1 was named (3R)-6,8-dihydroxy-3-methylisochro-
man (annulohypoxyloman A).
Compound 2 was obtained as a yellow, amorphous powder.
Its molecular formula was established as C11H14O4 using
HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 1
and 2) of compound 2 were similar to those 1, except for a
substituent group (methoxy group) at C-7. Its HMBC
spectrum displayed key correlations between 7-OMe (δH
3.72) and C-7 (δC 149.8), suggesting that the methoxy group
is located at C-7 (Figure 2). Compound 2 was identified as
(3R)-6,8-dihydroxy-7-methoxy-3-methylisochroman (annulo-
hypoxyloman B).
The molecular formula was determined to be C10H12O3 by
1
HRESIMS. The H NMR data of 1 (Table 1) revealed two
aromatic proton signals at δH 6.64 (br s, H-5) and 6.83 (br s, H-
7), an oxygenated methylene signal at δH 5.02 (d, J = 14.8 Hz,
H-1a) and 5.44 (d, J = 14.8 Hz, H-1b), an oxygenated methine
signal at δH 3.80 (m, H-3), a methylene signal at δH 2.62 (dd, J
= 16.1, 10.5 Hz, H-4a) and 2.70 (dd, J = 16.1, 2.7 Hz, H-4b),
and a methyl signal at δH 1.32 (d, J = 6.1 Hz, H-11).
Correspondingly, the 13C NMR spectrum (Table 2) showed
the signals of six aromatic carbons at δC 101.7 (C-7), 107.4 (C-
5), 114.1 (C-9), 136.7 (C-10), 155.9 (C-8), and 158.7 (C-6),
Table 2. 13C NMR Data of Compounds 1−4 and 6−8 (150
a
MHz)
b
c
c
d
b
b
c
1
2
3
4
6
7
8
1
65.7
71.1
65.4
72.1
65.9
72.1
170.2
75.3
65.4
76.9
65.7
77.2
64.1
70.9
3
4
36.9
36.3
37.1
31.8
71.5
71.6
35.1
5
107.4
158.7
101.7
155.9
114.1
136.7
22.3
107.8
149.8
134.8
147.0
114.2
130.3
21.6
104.7
153.4
135.9
147.5
116.2
130.7
22.1
115.5
137.8
124.8
160.3
107.9
136.9
20.8
102.3
153.0
136.2
147.1
117.3
135.4
19.6
102.1
148.8
134.4
143.0
117.4
130.3
19.6
106.3
148.6
128.6
146.5
112.5
129.4
20.4
6
7
8
9
10
Compound 3 was isolated as a yellow, amorphous powder,
and its molecular formula was determined to be C12H16O4
based on a pseudomolecular ion peak in the HRESIMS
11
6-OMe
7-OMe
7-Me
8-OMe
1′
2′
3′
4′
5′
56.7
56.1
56.1
60.9
61.6
60.8
1
spectrum. The H and 13C NMR spectroscopic data (Tables 1
17.9
and 2) closely resemble those of 1 and 2, the only difference
being that 3 possesses a methoxy group at C-6 instead of an
OH group. There were key HMBC correlations between 6-
OMe (δH 3.78) and C-6 (δC 153.4) and between 7-OMe (δH
3.73) and C-7 (δC 135.9), suggesting that two methoxy groups
are located at C-6 and C-7 (Figure 2). Thus, compound 3 was
identified as (3R)-6,7-dimethoxy-8-hydroxy-3-methylisochro-
man (annulohypoxyloman C).
62.9
102.5
72.8
70.6
87.2
61.9
a
Assignments were done by HMQC and HMBC experiments; J values
b
c
The synthesis of compound 4 has been reported, but its
NMR spectroscopic data have not been assigned. For the first
(Hz) are in parentheses. Measured in pyridine-d5. Measured in
d
methanol-d4. Measured in chloroform-d.
B
DOI: 10.1021/acs.jnatprod.6b00698
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Journal of Natural Products
Note
Figure 1. Structures of compounds 1−8 from A. truncatum.
1
Figure 2. H−1H COSY and key HMBC correlations of compounds 1−4 and 6−8.
1
time, we report H and 13C NMR data for annulohypox-
spectroscopic data of 6 were very similar to those of 3, which
was coisolated with 6. The only difference is that 6 possesses an
oxymethine group (δH 4.83; δC 71.5) instead of a geminal
coupling at C-4, which is supported by key HMBC correlations
between H-5 (δH 7.26) and C-4 (δC 71.5) and between H-4
(δH 4.81) and C-9 (δC 117.3) (Figure 2). NOE correlations
were observed between H-11 (δH 1.69) and H-4 (δH 4.81),
indicating their cofacial position. The large coupling constant
(8.0 Hz) between H-3 and H-4 is indicative of an axial−axial
coupling. Chiral center C-3 is assumed to have an R
configuration, as found in its coisolated compound (annulohy-
poxyloman C, 3), and the absolute configuration at C-4 was
deduced to be S.12 Thus, 6 was identified as (3R,4S)-6,7-
dimethoxy-8-hydroxy-3-methylisochromanol (annulohypoxylo-
manol A). In a similar manner, compound 7 was indentified as
(3R,4S)-6-methoxy-7,8-dihydroxy-3-methylisochromanol (an-
ylomarin A (4) (Tables 1 and 2).
The molecular formula of compound 6 was determined to be
C12H16O5 based on a pseudomolecular ion peak from
1
HRESIMS. The H NMR data of 6 (Table 1) showed an
aromatic proton signal at δH 7.26 (s, H-5), an oxygenated
methylene signal at δH 5.08 (d, J = 15.1 Hz, H-1a) and 5.39 (d,
J = 15.1 Hz, H-1b), two oxygenated methine signals at δH 3.93
(m, H-3) and 4.81 (t, J = 8.0 Hz, H-4), two methoxy signals at
δH 3.75 (s, 6-OMe) and 3.83 (s, 7-OMe), and a methyl signal at
δH 1.69 (d, J = 6.1 Hz, H-11). The 13C NMR data (Table 1)
showed signals for six aromatic carbons at δC 102.3 (C-5),
117.3 (C-9), 135.4 (C-10), 136.2 (C-7), 147.1 (C-8), and 153.0
(C-6), two oxygenated methine carbons at δC 71.5 (C-4) and
76.9 (C-3), an oxygenated methylene carbon at δC 65.4 (C-1),
two methoxy carbons at δC 56.1 (6-OMe) and 60.8 (7-OMe),
and a methyl carbon at δC 19.6 (C-11). The 1H and 13C NMR
C
DOI: 10.1021/acs.jnatprod.6b00698
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Journal of Natural Products
Note
nulohypoxylomanol B) via comparison of its HRESIMS and 1H
and 13C NMR data with those of compound 6.
(TLC) was performed using precoated silica gel 60 F254 and RP-18
F254S plates (both 0.25 mm, Merck, Darmstadt, Germany).
Fungal Material. JS540 was isolated from the leaves of
Z. caduciflora, collected from a swamp at Suncheon, South Korea.
The fungal strain (GenBank accession No. FJ478107.1) was identified
by one of the authors (S.K.). Parts of the material were deposited in
the Wildlife Genetic Resources Bank at NIBR. Leaf tissues were cut
into small pieces (0.5 × 0.5 cm), and surfaces were sterilized with 2%
sodium hypochlorite for 1 min and 70% ethanol for 1 min and then
washed with sterilized distilled water. Fungal strains were grown out
from plant tissues after about 7 days’ incubation on malt extract agar
(Difco) added to 50 ppm kanamycin, 50 ppm chloramphenicol, and
50 ppm Rose Bengal at 22 °C. Fungal strains were cultured by
transferring actively growing edges to a new potato dextrose agar
(Difco) and were stored as 20% glycerol stocks in a liquid nitrogen
tank.
Fermentation, Extraction, and Isolation. The fermentation was
performed in Erlenmeyer flasks (20 × 500 mL) on solid rice medium
containing 80 g of rice, 2.0 g of sea salt, and 80 mL of demineralized
water. After autoclaving at 121 °C for 20 min and then cooling to
room temperature, each flask was inoculated and then incubated at 28
°C under static conditions. After 30 days, the fermentation was
stopped by adding 500 mL of EtOAc to each flask. The extraction was
completed after the flasks had been shaken on a laboratory shaker at
150 rpm for 2 h.
Compound 8 was obtained as a yellow, amorphous solid. Its
molecular formula, C16H22O8, was determined based on
1
positive HRESIMS. Comparison of the H and 13C NMR
data (Tables 1 and 2) with those of compounds 1−3 suggested
that they have the same isochroman skeleton. Further
comparison of their 1D NMR data with several ribofuranosides
indicated that 8 is a ribofuranoside.13,14 The connection
between the sugar moiety and the skeleton via the O bond
was established by the key HMBC correlation between H-1′
(δH 5.59) and C-7 (δC 128.6). D-Ribose was identified as the
sugar moiety by measurement of its optical rotation following
acid hydrolysis ([α]2D5 −21.2 (c 0.095, H2O)).15,16 The sugar
moiety was further determined as α-D-ribofuranose through
comparison of the J1′,2′ value (4.2 Hz) with those of methyl-α-
D-ribofuranoside (J1,2 = 4.3 Hz) and methyl-β-D-ribofuranoside
(J1,2 = 1.2 Hz).17 Thus, compound 8 was identified as (3R)-6-
hydroxy-8-methoxy-3-methylisochroman 7-O-α-D-ribofuranose
(annulohypoxyloside).
To investigate the active anti-inflammatory constituents in
the ethyl acetate extract from A. truncatum, the production of
IL-12 p40, IL-6, and TNF-α in LPS-stimulated BMDCs was
evaluated. The cytotoxicity of compounds 1−8 (at a
concentration of 50 μM) against BMDCs was evaluated using
the MTT assay (Sigma, St. Louis, MO, USA). The results
showed that these compounds were inactive at the concen-
trations evaluated. To examine the effects of compounds 1−8
on the secretion of cytokines, their ability to inhibit the
production of IL-6, IL-12 p40, and TNF-α at a concentration of
50 μM was assessed. Xylariphilone (5) significantly decreased
the production of IL-6, IL-12 p40, and TNF-α; several of the
isolated compounds (1−4 and 6−8) had no effect at the
concentrations evaluated (IC50 > 100 μM). The effect of
compound 5 on the production of IL-6, IL-12 p40, and TNF-α
at various concentrations (2, 5, 10, 25, and 50 μM) was then
determined. SB203580, 4-[4-(4-fluorophenyl)-2-(4-methylsulfi-
nylphenyl)-1H-imidazol-5-yl]pyridine, an inhibitor of p38
kinase, which inhibits IL-6, IL-12 p40, and TNF-α production
with IC50 values of 3.5, 5.0, and 7.2 μM, respectively, was used
as a positive control.18 The results showed that compound 5
significantly inhibited the production of IL-6, IL-12 p40, and
TNF-α, with IC50 values of 5.3 0.8, 19.4 0.5, and 37.6
0.9 μM, respectively. Moreover, the observed anti-inflammatory
activities and structural features of compounds 1−8 provide
information regarding structure−activity relationships. Xylar-
iphilone (5) contains a ketone group (C-8), showed strong
activity. This suggests that the ketone group (C-8) of
isochroman derivatives plays an important role in the anti-
inflammatory activity.
The EtOAc solution was then evaporated under reduced pressure at
45 °C to give an EtOAc extract (28.5 g). The EtOAc extract (26.0 g)
was subjected to silica gel (5 × 30 cm) column chromatography with a
gradient of hexane−EtOAc−MeOH (30:1:0, 10:1:0, 4:1:0, 2:1:0;
1.5:1:0.12, 1:1:0.2, 0:5:1, 0:0:1; 1.5 L for each step) to give eight
fractions (Fr. 1A−1H). Fraction 1B (3.3 g) was separated using YMC
(2.0 × 80 cm) column chromatography with a MeOH−acetone−H2O
(0.2:0.2:1, 0.5:0.5:1, 1:1:1, 2:2:1, 4:4:1, 8:8:1; 1.0 L for each step)
elution solvent to give 16 fractions (Fr. 1B1−1B16). Fraction 1B2
(50.0 mg) was separated using silica gel (1 × 80 cm) column
chromatography with a hexane−EtOAc (2.5:1; 650 mL) elution
solvent to give compound 4 (39.0 mg). Fraction 1B4 (103.0 mg) was
separated using silica gel (1 × 80 cm) column chromatography with a
hexane−acetone−MeOH (6:1:0.1; 1.0 L) elution solvent to give
compound 5 (12.0 mg). Fraction 1B5 (110.0 mg) was separated using
silica gel (1 × 80 cm) column chromatography with a hexane−
acetone−MeOH (6:1:0.1; 1.0 L) elution solvent to give compound 7
(21.0 mg). Fraction 1B7 (22.0 mg) was separated using silica gel (1 ×
80 cm) column chromatography with a hexane−acetone (2:1; 800
mL) elution solvent to give compound 3 (16.0 mg). Fraction 1D (0.6
g) was separated using silica gel (1 × 80 cm) column chromatography
with a gradient of hexane−EtOAc−MeOH (6:1:0, 5:1:0, 3.5:1:0,
2:1:0.1; 550 mL for each step) elution solvent to give compounds 2
(71.0 mg) and 6 (14.0 mg) and three fractions (Fr. 1D1−1D3).
Fraction 1D1 (18.0 mg) was separated using silica gel (1 × 80 cm)
column chromatography with a hexane−acetone (5:1; 800 mL)
elution solvent to give compound 1 (2.5 mg). Fraction 1D3 (70.0 mg)
was separated using silica gel (1 × 80 cm) column chromatography
with a hexane−acetone−MeOH (6:1:0.1; 1.0 L) elution solvent to
give compound 8 (3.6 mg).
Annulohypoxyloman A (1): yellow, amorphous powder; [α]D25
−72.6 (c 0.1, MeOH); UV (MeOH) 204, 280 nm; 1H NMR
(pyridine-d5, 600 MHz) and 13C NMR data (pyridine-d5, 150 MHz),
see Tables 1 and 2; HRESIMS m/z 203.0678 [M + Na]+ (calcd for
203.0679, C10H12NaO3).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
determined using a Jasco DIP-370 automatic polarimeter. UV spectra
were recorded using a Beckman Du-650 UV−vis recording
spectrometer. The NMR spectra were recorded using a JEOL ECA
600 spectrometer (1H, 600 MHz; 13C, 150 MHz). The LCQ
Advantage trap mass spectrometer (Thermo Finnigan, San Jose, CA,
USA) was equipped with an electrospray ionization (ESI) source, and
high-resolution electrospray ionization mass spectra (HRESIMS) were
obtained using an Agilent 6530 Accurate-Mass Q-TOF LC/MS
system. Column chromatography was performed using silica gel
(Kieselgel 60, 70−230 and 230−400 mesh, Merck, Darmstadt,
Germany) and YMC RP-18 resins, and thin-layer chromatography
Annulohypoxyloman B (2): yellow, amorphous powder; [α]D25
−55.8 (c 0.1, MeOH); UV (MeOH): 205, 280 nm; 1H NMR
(methanol-d4, 600 MHz) and 13C NMR data (methanol-d4, 150
MHz), see Tables 1 and 2; HRESIMS m/z 233.0786 [M + Na]+ (calcd
for 233.0784, C11H14NaO4).
Annulohypoxyloman C (3): yellow, amorphous powder; [α]D25
−63.1 (c 0.1, MeOH); UV (MeOH) 204, 280 nm; 1H NMR
(methanol-d4, 600 MHz) and 13C NMR data (methanol-d4, 150
MHz), see Tables 1 and 2; HRESIMS m/z 247.0946 [M + Na]+ (calcd
for 247.0941, C12H16NaO4).
D
DOI: 10.1021/acs.jnatprod.6b00698
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Journal of Natural Products
Note
Annulohypoxylomarin A (4): orange solid; [α]2D5 −39.2 (c 0.1,
AUTHOR INFORMATION
Corresponding Author
*E-mail (S. H. Shim): sangheeshim@duksung.ac.kr. Tel: +82 2
901 8774. Fax: +82 2 901 8386.
■
1
MeOH); UV (MeOH) 215, 268 nm; H NMR (chloroform-d, 600
MHz) and 13C NMR data (chloroform-d, 150 MHz), see Tables 1 and
2; HRESIMS m/z 215.0684 [M + Na]+ (calcd for 215.0679,
C11H12NaO3).
Annulohypoxylomanol A (6): brown, amorphous powder; [α]D25
−22.8 (c 0.1, MeOH); UV (MeOH) 203, 290 nm; 1H NMR
(pyridine-d5, 600 MHz) and 13C NMR data (pyridine-d5, 150 MHz),
see Tables 1 and 2; HRESIMS m/z 263.0896 [M + Na]+ (calcd for
263.0890, C12H16NaO5).
ORCID
Wei Li: 0000-0001-7272-7290
Sung Hee Bang: 0000-0001-6085-193X
Notes
Annulohypoxylomanol B (7): brown, amorphous powder; [α]D25
−27.6 (c 0.1, MeOH); UV (MeOH) 205, 290 nm; 1H NMR
(pyridine-d5, 600 MHz) and 13C NMR data (pyridine-d5, 150 MHz),
see Tables 1 and 2; HRESIMS m/z 249.0738 [M + Na]+ (calcd for
249.0733, C11H14NaO5).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a grant (K16281) awarded to
the Korean Institute of Oriental Medicine by the Ministry of
Education, Science and Technology (MEST); Priority Research
Centers Program through the National Research Foundation
(NRF) funded by the Ministry of Education, Science, and
Technology (NRF-2016R1A6A1A03007648); and also Basic
Science Research Programs through NRF funded by the
M i n i s t r y o f E d u c a t i o n ( G r a n t N o . N R F -
2015R1D1A1A01057914).
Annulohypoxyloside A (8): yellow, amorphous solid; [α]2D5 −17.9 (c
0.1, MeOH); UV (MeOH) 245, 280 nm; 1H NMR (methanol-d4, 600
MHz) and 13C NMR data (methanol-d4, 150 MHz), see Tables 1 and
2; HRESIMS m/z 365.1209 [M + Na]+ (calcd for 365.1207,
C16H22NaO8).
Acid Hydrolysis. A solution of compound 8 (1.5 mg) in 1 M
hydrochloric acid (HCl) (1 mL) was reacted for 3 h at 90 °C. The
reaction mixture was extracted with EtOAc repeatedly to remove the
aglycone fraction. The water layer was then concentrated to furnish
the sugar residue (0.7 mg). The rotation recorded for the ribose was
[α]2D5 −21.2 (c 0.095, H2O), which closely matched that for D-ribose
(lit. −20).9
REFERENCES
■
(1) Strobel, G.; Daisy, B.; Castillo, U.; Harper, J. J. Nat. Prod. 2014,
67, 257−268.
Cell Culture. BMDCs were grown from wild-type C57BL/6 mice
(Orient Bio Inc., South Korea) as previously described. All animal
procedures were approved and performed according to the guidelines
of the Institutional Animal Care and Use Committee of Jeju National
University (#2010-0028). Briefly, the mouse tibia and femur were
obtained by flushing with Dulbecco’s modified Eagle medium to yield
bone marrow cells. The cells were cultured in RPMI 1640 medium
containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, NY,
USA), 50 μM β-ME, and 2 mM glutamine supplemented with 3%
J558L hybridoma cell culture supernatant containing granulocyte-
macrophage colony stimulating factor. The culture medium was
replaced with fresh medium every second day. At day 6 of culture,
nonadherent cells and loosely adherent DC aggregates were harvested,
washed, and resuspended in RPMI 1640 supplemented with 5% FBS.
Cell Toxicity Assay. Cell-Counting Kit (CCK)-8 (Dojindo,
Kumamoto, Japan) was used to analyze the effect of compounds on
cell toxicity according to the manufacturer’s instructions. Cells were
cultured overnight in a 96-well plate (∼1 × 104 cells/well). Cell
toxicity was assessed after the addition of compounds in a dose-
dependent manner. After 24 h of treatment, 10 μL of the CCK-8
solution was added to triplicate wells and incubated for 1 h.
Absorbance was measured at 450 nm to determine viable cell numbers
in wells.
Cytokine Production Measurements. BMDCs were incubated
in 48-well plates in 0.5 mL containing 1 × 105 cells per well and then
treated with the isolated compounds 1−8 at the indicated
concentration for 1 h before stimulation with 10 ng/mL LPS from
Salmonella minnesota (Alexis, NY, USA). Supernatants were harvested
18 h after stimulation. Concentrations of murine TNF-α, IL-6, and IL-
12 p40 in the culture supernatants were determined by ELISA (BD
PharMingen, CA, USA) according to the manufacturer’s instructions.
The data are presented as means plus SD of at least three independent
experiments performed in triplicate.
(2) Aly, A. H.; Debbab, A.; Proksch, P. Appl. Microbiol. Biotechnol.
2011, 90, 1829−1845.
(3) Ludwig-Muller, J. Biotechnol. Lett. 2015, 37, 1325−1334.
̈
(4) Sikora, R. A. Annu. Rev. Phytopathol. 1992, 30, 245−270.
(5) Schouten, A. Annu. Rev. Phytopathol. 2016, 54, 121.
(6) Smith, S. A.; Tank, D. C.; Boulanger, L. A.; Bascom-Slack, C. A.;
Eisenman, K.; Kingery, D.; Babbs, B.; Fenn, K.; Greene, J. S.; Hann, B.
D.; Keehner, J.; Kelley-Swift, E. G.; Kembaiyan, V.; Lee, S. J.; Li, P.;
Light, D. Y.; Lin, E. H.; Ma, C.; Moore, E.; Schorn, M. A.; Vekhter, D.;
Nunez, P. V.; Strobel, G. A.; Donoghue, M. J.; Strobel, S. A. PLoS One
2008, 3, e3052.
(7) Newman, D. J.; Cragg, G. M. Front. Chem. 2015, 3, 34.
(8) Surup, F.; Wiebach, V.; Kuhnert, E.; Stadler, M. Tetrahedron Lett.
2016, 57, 2183−2185.
(9) Lin, C. N.; Lu, C. M.; Lin, H. C.; Ko, F. N.; Teng, C. M. J. Nat.
Prod. 1995, 58, 1934−1940.
(10) Arunpanichlert, J.; Rukachaisirikul, V.; Phongpaichit, S.;
Supaphon, O.; Sakayaroj, J. Nat. Prod. Res. 2016, 30, 46−51.
(11) Orfali, R. S.; Aly, A. H.; Ebrahim, W.; Rudiyansyah; Proksch, P.
Phytochem. Lett. 2015, 13, 234−238.
(12) Glauser, G.; Gindro, K.; Fringeli, J.; De Joffrey, J. P.; Rudaz, S.;
Wolfender, J. L. J. Agric. Food Chem. 2009, 57, 1127−1134.
(13) Thomas, E. W.; Harry, P. C. H.; Terry, E. N.; Nick, A. M.
Biochemistry 1974, 13, 2650−2655.
(14) Li, Y.; Li, X.; Lee, U.; Kang, J. S.; Choi, H. D.; Sona, B. W.
Chem. Pharm. Bull. 2006, 54, 882−883.
(15) Robert, K. N.; Harry, W. D.; Hewitt, G. F., Jr. J. Am. Chem. Soc.
1954, 76, 763−767.
(16) Du, L.; Zhu, T.; Liu, H.; Fang, Y.; Zhu, W.; Gu, Q. J. Nat. Prod.
2008, 71, 1837−1842.
(17) Anthony, S. S.; Robert, B. J. Org. Chem. 1984, 49, 3292−3300.
(18) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.;
Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.; Heys, J. R.;
Landvatter, S. W. Nature 1994, 372, 739−746.
ASSOCIATED CONTENT
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S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00698.
1H and 13C NMR, HMQC, HMBC, COSY, NOESY, and
HRESIMS spectra of compounds 1−4 and 6−8 (PDF)
E
DOI: 10.1021/acs.jnatprod.6b00698
J. Nat. Prod. XXXX, XXX, XXX−XXX