Chemical constituents isolated from Antarctic marine-derived Aspergillus sp. SF-5976 and their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells

Article abstract of DOI:10.1016/j.tet.2017.05.060

Three new benzaldehydes (1–3) and two new dioxopiperazine alkaloids (4 and 5), along with 23 known constituents, were isolated from Antarctic marine-derived Aspergillus sp. SF-5976. Their structures were determined by spectroscopic and chemical methods. Among them, 20 compounds showed inhibitory effects toward lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages with IC₅₀ values of 7.3–77.4 μM, while 22 compounds did in BV2 microglia with those of 4.6–72.3 μM. Furthermore, the effects of the newly isolated compounds on LPS-stimulated inducible NO synthase (iNOS) expression were investigated, and 1–3, and 5 inhibited iNOS in RAW 264.7 cells, while 1–5 did in BV2 cells. Also, 1–3 and 5 inhibited LPS-induced prostaglandin E₂ production with IC₅₀ values of 9.1–66.8 μM in RAW 264.7 macrophages by suppressing cyclooxygenase-2 (COX-2), while in BV2 microglia, 1–5 showed activities with those of 2.8–49.4 μM.

Full text of DOI:10.1016/j.tet.2017.05.060

Accepted Manuscript
Chemical constituents isolated from Antarctic marine-derived Aspergillus sp. SF-5976
and their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells
Jaeyoung Kwon, Hyaemin Lee, Wonmin Ko, Dong-Cheol Kim, Kwan-Woo Kim,
Hak Cheol Kwon, Yuanqiang Guo, Jae Hak Sohn, Joung Han Yim, Youn-Chul Kim,
Hyuncheol Oh, Dongho Lee
PII:
S0040-4020(17)30546-X
10.1016/j.tet.2017.05.060
TET 28726
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 11 March 2017
Revised Date: 11 May 2017
Accepted Date: 16 May 2017
Please cite this article as: Kwon J, Lee H, Ko W, Kim D-C, Kim K-W, Kwon HC, Guo Y, Sohn JH, Yim
JH, Kim Y-C, Oh H, Lee D, Chemical constituents isolated from Antarctic marine-derived Aspergillus sp.
SF-5976 and their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells, Tetrahedron
(2017), doi: 10.1016/j.tet.2017.05.060.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Chemical constituents isolated from Antarctic marine-derived Aspergillus sp. SF-5976 and
their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells
a
b
c
c
c
Jaeyoung Kwon , Hyaemin Lee , Wonmin Ko , Dong-Cheol Kim , Kwan-Woo Kim , Hak
a
d
e
f
c
Cheol Kwon , Yuanqiang Guo , Jae Hak Sohn , Joung Han Yim , Youn-Chul Kim ,
c
b, *
Hyuncheol Oh , and Dongho Lee
a
Natural Constituents Research Center, Korea Institute of Science and Technology (KIST)
Gangneung Institute, Gangneung 25451, Republic of Korea
b
Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology,
Korea University, Seoul 02841, Republic of Korea
c
Institute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang
University, Iksan 54538, Republic of Korea
d
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin
Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s
Republic of China
e
College of Medical and Life Sciences, Silla University, Busan 46958, Republic of Korea
f
Korea Polar Research Institute, Incheon 21990, Republic of Korea
*
Corresponding author.
Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail address: dongholee@korea.ac.kr (D.
Lee).
1

ACCEPTED MANUSCRIPT
ABSTRACT
Three new benzaldehydes (1–3) and two new dioxopiperazine alkaloids (4 and 5), along
with 23 known constituents, were isolated from Antarctic marine-derived Aspergillus sp. SF-
5
976. Their structures were determined by spectroscopic and chemical methods. Among
them, 20 compounds showed inhibitory effects toward lipopolysaccharide (LPS)-induced
nitric oxide (NO) production in RAW 264.7 macrophages with IC values of 7.3–77.4 ꢀM,
5
0
while 22 compounds did in BV2 microglia with those of 4.6–72.3 ꢀM. Furthermore, the
effects of the newly isolated compounds on LPS-stimulated inducible NO synthase (iNOS)
expression were investigated, and 1–3, and 5 inhibited iNOS in RAW 264.7 cells, while 1–5
did in BV2 cells. Also, 1–3 and 5 inhibited LPS-induced prostaglandin E production with
2
IC values of 9.1–66.8 ꢀM in RAW 264.7 macrophages by suppressing cyclooxygenase-2
5
0
(COX-2), while in BV2 microglia, 1–5 showed activities with those of 2.8–49.4 ꢀM.
Keywords: Antarctic marine; Aspergillus sp.; Circular dichroism; Anti-inflammation
2

ACCEPTED MANUSCRIPT
1
. Introduction
Inflammation is a valuable defense response against insult or injury, designed to inhibit
1
their detrimental effects. Macrophages and microglia are major inflammatory and immune
effector cells activated by bacterial lipopolysaccharides (LPS), interferon-γ, and pro-
2
inflammatory cytokines. Activated cells regulate the cellular and molecular pathophysiology
of inflammation through the production of inflammatory cytokines and mediators such as
interleukin-1β and -6, tumor necrosis factor-α, nitric oxide (NO), and prostaglandin E2
3
(
PGE ), which are known to be involved in the pathogenesis of chronic disorders such as
2
4
,5
cancer, autoimmune disorders as well as cardiovascular, and neurological diseases.
Therefore, suppression of inflammatory cytokines and mediators can be a therapeutic target
for preventing inflammation-related chronic diseases.
Recently, we reported that some dihydroisocoumarins from Antarctic marine-derived
Aspergillus sp. SF-5976 exhibited anti-inflammatory effects in LPS-stimulated BV2
6
microglia. In this ongoing project, a scale-up fermentation of the aforementioned strain was
conducted to search various types of constituents with anti-inflammatory activity.
Consequently, we isolated 28 constituents including three new benzaldehydes (1–3) and two
dioxopiperazine alkaloids (4 and 5). Described herein are the isolation, structure elucidation,
and the anti-inflammatory effects of the isolated compounds on LPS-stimulated RAW 264.7
macrophages and BV2 microglia.
2
. Results and discussion
2
.1. Isolation and structural elucidation
Compound 1 was isolated as a pale yellow and amorphous solid, and a molecular formula
of C H O was deduced based on HRESIMS data, suggesting eight degrees of unsaturation.
1
9
24
4
3

ACCEPTED MANUSCRIPT
-1
The IR spectrum revealed the presence of a carbonyl group (1641 cm ), an aromatic ring
-1
-1
1
(
1666 and 2920 cm ), and some hydroxy groups (3426 cm ). The H NMR data (Table 1)
indicated the presence of a chelated hydroxy proton [δ 11.99 (1H, s, 6-OH)] and an aldehyde
H
proton [δ 10.22 (1H, s, H-7)] in the lower field. Furthermore, an aromatic proton [δ 6.97
H
H
(
1H, s, H-4)] and three olefinic protons [δ 5.88 (1H, dq, J = 15.0, 6.5 Hz, H-6′), 5.56 (1H,
H
ddd, J = 15.0, 7.0, 1.5 Hz, H-5′), and 5.29 (1H, t, J = 7.5 Hz, H-2′′)] were observed in the
aromatic and olefinic regions, respectively. Moreover, two oxygenated methine protons [δH
4
.13 (1H, dd, J = 8.0, 7.0 Hz, H-4′) and 3.81 (1H, ddd, J = 8.0, 7.5, 2.0 Hz, H-3′)], three
methylene protons [δ 3.29 (2H, d, J = 7.5 Hz, H-1′′), 3.16 (1H, m, H-1′a), 3.00 (1H, m, H-
H
1
′b), 2.06 (1H, m, H-2′′a), and 1.74 (1H, m, H-2′′b)], and three methyl protons [δ 1.77 (3H,
H
1
3
d, J = 6.0 Hz, H-7′), 1.76 (3H, s, H-4′′), and 1.70 (3H, s, H-5′′)] were observed. The C NMR
data (Table 1) exhibited 19 carbon signals attributable to three methyl, three methylene, six
methine, two oxygenated tertiary, one carbonyl, and four quaternary carbons. Detailed
1
13
analysis of the H and C NMR data revealed that 1 has a benzaldehyde framework with a 2-
(1-hydroxy-but-2-enyl)-dihydropyran and a 3-methylbut-2-enyl (prenyl) groups, similarly to
7
chaetopyranin (10), except that the hydroxy group is present at C-4′ and the double bond is
1
3
at C-5′ and C-6′, as evidenced by the C NMR chemical shifts [δ 75.1 (C-4′), 128.8 (C-5′),
C
and 130.9 (C-6′)] and the 2D NMR studies (Fig. 2). The positions of the remaining
substituents were deduced by the 2D NMR experiments including COSY and HMBC
correlations. Therefore, the new compound 1 was elucidated as shown, and was given the
trivial name of asperanin A.
4

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Fig. 1. Structures for compounds 1–5.
Compound 2 showed the same molecular formula of C H O , as deduced by HRESIMS
1
9
24
4
1
13
data, and its H and C NMR spectroscopic data (Table 1) were very similar to those of 1.
1
Despite differences in the H NMR data of 2 [δ 4.34 (1H, dd, J = 7.0, 3.5 Hz, H-4′) and 3.92
H
(
1H, ddd, J = 7.5, 3.5, 1.5 Hz, H-3′)] compared to those of 1, the 2D NMR experiments of 2
asperanin B) suggested that both compounds 1 and 2 have the same planar structure.
The relative configurations of 1 and 2 were found to be threo- and erythro-isomers,
(
respectively, as deduced from the coupling constants between H-3′ and H-4′ (J = 8.0 Hz in 1
8
and J = 3.5 Hz in 2). The absolute configurations of 1 and 2 were determined by comparing
experimental and theoretical ECD spectra using time-dependent density functional theory
9
,10
(TDDFT) method as reported previously.
Conformational searches were performed under
11
the Merk Molecular Force Field (MMFF) using the Spartan′14 software, and geometry
optimization of the obtained conformers was subsequently carried out at the B3LYP/6-
1
2
3
1+G(d,p) level with the Gaussian 09 package. The calculated ECD spectra were produced
at the CAM-B3LYP/TZVP level with a conductor-like polarizable continuum solvent model
(CPCM) in MeCN. They were in agreement with the experimental results, suggesting the
5

ACCEPTED MANUSCRIPT
absolute configuration of 1 as 3′R and 4′S, and that of 2 as 3′R and 4′R (Fig. 3).
Compound 3 had a molecular formula of C H O , as determined by HRESIMS analysis.
1
5
18
4
1
13
The H and C NMR data (Table 1) were similar to those of 1 and 2, except for the
replacement of a 1-hydroxy-but-2-enyl group at C-3′ by a hydroxy group, as indicated by the
absence of signals derived from C-4′ to C-7′ and the downfield shift of H-3′ [δ 5.61 (1H, t, J
H
=
3.0, H-3′)]. Further analysis of the 2D NMR spectra suggested the structure of the new
compound 3 (asperanin C). The absolute configuration of 3 was determined based on the
comparison between experimental and calculated ECD spectra using the TDDFT method, and
resulted in a 3′R configuration (Fig. 3).
Fig. 2. HMBC and COSY correlations of compounds 1 and 4.
6

ACCEPTED MANUSCRIPT
Table 1
NMR spectroscopic data of compounds 1–3 in CDCl3
Position
1
δC
2
δC
3
δC
a
a
a
δH (mult, J)
HMBC
δH (mult, J)
HMBC
δH (mult, J)
HMBC
1
2
3
4
5
6
7
116.4
119.6
146.2
127.3 6.97 (s)
130.2
116.5
119.7
146.7
127.3 6.96 (s)
129.7
116.3
119.8
143.5
127.3 6.92 (s)
130.2
2, 3, 6, 1′′
2, 3, 6, 1′′
2, 3, 6, 1′′
156.4
194.4 10.22 (s)
156.3
194.4 10.22 (s)
156.5
194.4 10.27 (s)
1, 6
1, 6
1, 6
1
′
′
20.3
23.2
3.16 (m)
.00 (m)
2.06,(m)
.74 (m)
3.81 (ddd, 8.0, 7.5, 2.0)
4.13 (dd, 8.0, 7.0)
2, 3, 2′, 3′
2, 2′
2, 1′
20.5
21.3
3.20 (m)
3.00 (m)
2.06 (m)
1.86 (m)
3.92 (ddd, 7.5, 3.5, 1.5)
4.34 (dd, 7.0, 3.5)
2, 3, 2′, 3′
2, 2′
2, 1′
16.1
26.3
90.2
3.15 (m)
3.09 (m)
2.12 (m)
2.00 (m)
5.61 (t, 3.0)
3
2
1
1′, 3′
1′, 3′
3
4
5
6
7
1
2
3
4
5
6
′
′
′
′
′
′′
′′
′′
78.1
75.1
78.1
74.1
3′, 5′, 6′
128.8 5.56 (ddd, 15.0, 7.0, 1.5) 7′
130.9 5.88 (dq, 15.0, 6.5) 4′, 7′
18.0
27.0
121.0 5.29 (t, 7.5)
133.9
25.8
17.8
128.5 5.60 (ddd, 15.0, 7.0, 1.5) 7′
130.0 5.84 (dq, 15.0, 6.5) 4′, 7′
18.0
27.0
121.1 5.29 (t, 7.5)
133.8
25.8
17.8
1.77 (d, 6.0)
3.29 (d, 7.5)
5′, 6′
4, 5, 6, 2′′, 3′′
4′′, 5′′
1.76 (d, 6.0)
3.29 (d, 7.5)
5′, 6′
4, 5, 6, 2′′,
4′′, 5′′
27.0
3.29 (d, 7.5)
4, 5, 6, 2′′, 3′′
121.0 5.28 (t, 7.0)
133.9
25.8
17.8
′′
′′
1.76 (s)
1.70 (s)
11.99 (s)
2′′, 3′′, 5′′
2′′, 3′′, 4′′
1, 5, 6
1.76 (s)
1.70 (s)
11.98 (s)
2′′, 3′′, 5′′
2′′, 3′′, 4′′
1, 5, 6
1.75 (s)
1.69 (s)
11.98 (s)
2′′, 3′′, 5′′
2′′, 3′′, 4′′
1, 5, 6
-OH
a
HMBC correlations, optimized for 8 Hz.
7

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Fig. 3. Calculated and experimental ECD spectra of compounds 1–3 (A–C), and 4 and 5 (D).
8

ACCEPTED MANUSCRIPT
Table 2
NMR spectroscopic data of compounds 4 and 5 in CDCl3
Position
4
δC
5
δC
a
a
δH (mult, J)
HMBC
δH (mult, J)
HMBC
2
3
142.3
104.0
129.2
115.8 7.18, s
133.6
123.7 6.76, s
122.0
132.7
142.4
103.6
129.1
115.5 7.16 (s)
133.3
123.5 6.73 (s)
123.3
132.8
3
4
5
6
7
7
8
a
a
3, 3a, 6, 7a,
4, 7a, 21, 26
3, 6, 7a, 21
4, 7a, 21, 26
29.4
3.65 (dd, 14.5, 3.5)
.19 (dd, 14.5, 11.5) 2, 3, 3a, 9
29.4
3.65 (dd, 15.0, 3.5)
3.19 (dd, 14.5, 11.5) 3, 3a
3
9
1
1
1
1
1
1
54.6
168.4
50.8
167.8
39.0
4.40 (dd, 11.5, 3.5)
54.6
168.4
50.8
167.7
39.0
4.41 (dd, 11.5, 3.5)
0
2
3
5
6
7
4.09 (q, 7.0)
13, 20
4.09 (q, 7.0)
10, 20
145.8 6.12 (dd, 17.5, 10.5) 15
112.1 5.15 (d, 17.5) 15, 16
.14 (d, 10.5)
146.0 6.12 (dd, 17.5, 10.5) 15
111.9 5.13 (d, 17.5)
5.11 (d, 10.5)
15
15
5
15, 16
1
1
2
2
2
2
2
2
2
8
9
0
1
2
3
4
5
6
28.0
27.9
19.9
34.5
1.55 (s)
1.53 (s)
1.53 (d, 7.0)
3.39 (m)
2, 15, 16, 19
2, 15, 16, 18
12, 13
28.0
27.9
19.9
34.6
1.54 (s)
1.52 (s)
1.53 (d, 7.0)
3.39 (m)
2, 15, 16, 19
2, 15, 16, 18
12, 13
5, 22, 23
124.4 5.34 (t, 7.0)
131.7
17.9
25.8
35.6
124.6 5.35 (t, 7.0)
131.6
17.9
25.8
36.1
1.74 (s)
1.73 (s)
3.08 (d, 14.0)
22, 23, 25
22, 23, 24
6, 7, 7a, 28
6, 7, 7a, 27
7
1.74 (s)
1.73 (s)
2.92 (dd, 14.5, 8.5)
2.84 (d, 14.5)
3.79 (d, 9.0)
22, 23, 25
22, 23, 24
6, 27
2
.92 (dd, 14.5, 9.0)
6, 7a
2
2
2
3
1
7
8
9
0
80.6
75.7
28.4
26.9
3.91 (d, 8.5)
78.1
77.6
20.3
18.5
1.74 (s)
1.68 (s)
9.22 (s)
5.69 (s)
6.07 (s)
27, 28, 30
27, 28, 29
2, 3, 3a
10, 12
1.28 (s)
1.24 (s)
9.84 (s)
5.67 (s)
5.85 (s)
3.27 (s)
27, 28, 30
27, 28, 29
3, 3a
-NH
11-NH
1
2
4-NH
8-OCH3
9, 13
49.2
28
a
HMBC correlations, optimized for 8 Hz.
Compound 4 was isolated as a colorless amorphous solid. The molecular formula of
C H ClN O was deduced from HRESIMS analysis, and suggested 11 degrees of
29
40
3
3
unsaturation. Particularly, the presence of a chlorine group was inferred by an isotopic cluster
9

ACCEPTED MANUSCRIPT
–
–
at m/z 512 [M – H] and 514 [M + 2 – H] with a ratio of 3:1 (Fig. S28, Supporting
-1
Information). The IR spectrum revealed the presence of amide carbonyl groups (1677 cm ),
-1
-1
1
an aromatic ring (2923 cm ), and a hydroxy group (3357 cm ). The H NMR spectroscopic
data (Table 2) showed signals for three amino protons [δ 9.22 (1H, s, 1-NH), 6.07 (1H, s,
H
1
4-NH), and 5.69 (1H, s, 11-NH)], two aromatic protons [δ 7.18 (1H, s, H-4) and 6.76 (1H,
H
s, H-6)], and four olefinic protons [δ 6.12 (1H, dd, J = 17.5, 10.5 Hz, H-16), 5.34 (1H, t, J =
H
7
.0 Hz, H-22), 5.15 (1H, d, J = 17.5 Hz, H-17a), and 5.14 (1H, d, J = 10.5 Hz, H-17b)].
Furthermore, three methine protons including one oxygenated [δ 4.40 (1H, dd, J = 11.5, 3.5
H
Hz, H-9), 4.09 (1H, q, J = 7.0 Hz, H-12), and 3.91 (1H, d, J = 8.5 Hz, H-27)], three
methylene protons [δ 3.65 (1H, dd, J = 14.5, 3.5 Hz, H-8a), 3.39 (2H, m, H-21), 3.19 (1H,
H
dd, J = 14.5, 11.5 Hz, H-8b), 3.08 (1H, d, J = 14.0 Hz, H-26a), and 2.92 (1H, dd, J = 14.5,
9
.0 Hz, H-26b)], and seven methyl protons [δ 1.74 (3H, s, H-24), 1.74 (3H, s, H-29), 1.73
H
(3H, s, H-25), 1.68 (3H, s, H-30), 1.55 (3H, s, H-18), 1.53 (3H, d, J = 7.0 Hz, H-20), and 1.53
1
3
(
3H, s, H-19)] were observed. The C NMR spectroscopic data (Table 2) exhibited 29 carbon
signals that were assigned to seven methyl, four methylene, seven methine, one chlorine-
containing and two nitrogen-containing tertiary, two amide carbonyl, and six quaternary
carbons. The 1D NMR spectroscopic data revealed that 4 is an dioxopiperazine alkaloid
1
3
similar to variecolorin F, except for the presence of a prenyl group at C-5, as evidenced by
the HMBC cross peaks of H-21/C-5 (δ 133.6) and H-4/C-21 (δ 34.5), and the absence of
C
C
1
3
the double bond at C-8 and C-9, as shown by the C NMR chemical shifts [δ 54.6 (C-9) and
C
2
9.4 (C-8)]. The gross structure of 4 (5-prenyl-dihydrovariecolorin F) was assigned by
detailed analysis of its COSY and HMBC data (Fig. 2). The absolute configuration at C-27
1
14
was assigned by the modified Mosher’s method with COSY and H NMR data (Fig. 4), and
that at C-9 and C-12 was determined from both the NOESY cross peak of H-9/H-12 and the
1
0

ACCEPTED MANUSCRIPT
1
5
advanced Marfey’s method with 1-fluoro-2,4-dinitrophenyl-5- -leucinamide ( -FDLA). The
L
L
retention time of Ala- -FDLA product in the acid hydrolysate of 4 was identical to that of -
L
D
Ala- -FDLA derivative (25.3 min), whereas that of -Ala- -FDLA was 23.7 min (Fig. S30,
L
L
L
Supporting Information). In addition, comparison of the experimental and theoretical ECD
spectra using the TDDFT method was carried out to reconfirm the absolute configuration of 4,
exhibiting good result (Fig. 3). Above results suggested the absolute configuration of 4 as 9R,
1
2R, and 27S.
The molecular formula of compound 5 was deduced as C H N O from HRESIMS
3
0
43
3
4
1
13
analysis. Its H and C NMR spectroscopic data (Table 2) were similar to those of 4, except
for the replacement of the chlorine group at C-28 by a methoxy group, as indicated by the
HMBC cross peak of 28-OCH /C-28 (δ 77.6). Further analysis of the 2D NMR
3
C
spectroscopic data identified the structure of 5 (5-prenyl-dihydrorubrumazine A). The
Mosher’s method was used to determine a 27S configuration (Fig. 4). The NOESY
correlation of H-9/H-12 and the close similarity of the experimental ECD curve between 4
and 5 suggested the absolute configuration at C-9 and C-12 as 9R and 12R (Fig. 3).
Fig. 4. ꢀδ values (δ − δ ) in ppm obtained for the two MTPA esters derived from 4 and 5 in
S
R
pyridine-d5.
1
1

ACCEPTED MANUSCRIPT
Additionally,
the
previously
reported
compounds
were
identified
as
1
6
tetrahydroauroglaucin
(6),
2-(2′,3-epoxy-1′,3′-heptadienyl)-6-hydroxy-5-(3-methyl-2-
7
16
7
butenyl)benzaldehyde (7), dihydroauroglaucin (8),
chaetopyranin (9), 6'-oxo-
1
7
18
19
chaetopyranin (10), isotetrahydroauroglaucin (11), eurotiumide A (12), variecolorins G
1
3
20
21
and H (13 and 14), neoechinulin B (15), isoechinulins A and B (16 and 17),
2
2
23
24
dehydroechinulin (18), echinulin (19), variecolortide C (20), 6′-hydroxy-4-O-methyl-
2
5
26
27
cladosporin (21), 7′-hydroxy-4-O-methyl-cladosporin (22), evariquinone (23), 6-
2
8
29
farnesyl-5,7-dihydroxy-4-methylphthalide (24), isocladosporin, 7′-hydroxy-4-O-methyl-
2
5
30
31
cladosporin, physcion, and erythroglaucin, by comparing their spectroscopic data with
those published in the literature.
2
.2. Anti-inflammatory activity
All compounds were examined to assess their anti-inflammatory activity measured as
inhibition of LPS-induced NO production in the murine macrophage-like cell line RAW
64.7, and in the immortalized murine brain macrophages BV2 cell (Table 3). These cells are
2
appropriate models for evaluating inflammatory responses triggered by activation of
inducible NO synthase (iNOS) and consequent iNOS-derived NO production, as well as
activation of cyclooxygenase-2 (COX-2) and consequent COX-2-derived PGE production,
2
3
2,33
both prompted by LPS.
Among the isolated compounds, dihydroisocoumarins 21 and 22
not only had structural similarity with those of our previous report, but exhibited similar
biological activities regarding to the inhibitory effects of NO production, and consequently
this indicated the compounds identified in this study are assumed to be more potent than
6
those of our previous report (Table 3). Particularly, compounds 10 (benzaldehyde) and 24
(phthalide) exhibited the most potent activity for inhibition of NO production in Raw 264.7
1
2

ACCEPTED MANUSCRIPT
cells, and 19 (dioxopiperazine alkaloids) as well as 24 did in BV2 cells, respectively.
However, despite the analysis of the structural characteristics and bioassay results of the
isolated compounds, clear structure-activity relationship could not be determined.
As part of further study, the new compounds 1–5 were selected to evaluate the effects on
PGE production as well as on iNOS and COX-2 protein expression by ELISA and western
2
blot analysis, respectively. In the case of LPS-stimulated RAW 264.7 cells, 1–3, and 5
decreased PGE production in a concentration-dependent manner, while 1–5 inhibited the
2
production of PGE in LPS-stimulated BV2 cells (Table 4). Furthermore, 1–3, and 5 inhibited
2
iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 cells (Fig. 5), while 1–5
suppressed the expression of iNOS and COX-2 proteins in LPS-stimulated BV2 cells (Fig. 6).
1
3

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Table 3
Inhibitory effects of isolated compounds on LPS-stimulated NO production in RAW 264.7
and BV2 cells
a
Compound
IC (ꢀM)
50
RAW 264.7
BV2
1
2
3
4
5
6
7
8
9
20.2
28.2
25.3
> 80.0
51.7
28.8
17.2
39.5
32.5
12.4
18.7
15.7
22.0
20.5
69.8
16.7
30.8
22.4
> 40.0
38.0
13.5
17.3
20.0
25.4
72.3
23.6
24.3
24.6
11.4
4.6
b
1
0
1
1
c
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
> 30.0
> 80.0
> 80.0
26.1
34.1
30.8
74.8
77.4
26.9
55.6
52.0
68.5
7.3
22.5
45.3
21.4
> 80.0
7.1
a
IC50 is expressed as a mean ± SD of triplicate
b
determinations. Compound 8 showed toxic effect above
4
c
0 ꢀM. Compound 12 showed toxic effect above 30
ꢀ
M.
1
4

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Table 4
Inhibitory effects of isolated new compounds 1–5 on LPS-stimulated PGE production in
2
RAW 264.7 and BV2 cells
a
Compound
IC (ꢀM)
50
RAW 264.7
9.1
BV2
2.8
1
2
3
4
5
12.1
16.8
> 80.0
66.8
4.2
9.4
18.0
49.4
a
IC is expressed as a mean ± SD of triplicate determinations.
5
0
Fig. 5. Effects of compounds 1−5 on iNOS and COX-2 protein expression in LPS-stimulated
RAW 264.7 macrophages. Cells were pretreated for 3 h with the indicated concentrations of
1
−5, and then further stimulated for 24 h with LPS (1 ꢀg/mL). Western blot analysis (A−F)
was performed as described in the Experimental Section. Band intensity was quantified by
densitometric analysis and normalized to β-actin. The values are presented at the bottom of
each band.
1
5

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Fig. 6. Effects of compounds 1−5 on iNOS and COX-2 protein expression in LPS-stimulated
BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of 1−5, and
then stimulated for 24 h with LPS (1 ꢀg/mL). Western blot analysis (A−F) was performed as
described in the Experimental Section. Band intensity was quantified by densitometric
analysis and normalized to β-actin. The values are presented at the bottom of each band.
3
. Conclusion
It has been previously reported that several marine fungi-derived compounds exhibited
anti-inflammatory effects in RAW 264.7 or BV2 cells, and their mechanism of action was
3
4-36
further investigated.
The present study describes characterization of several constituents
isolated from the scale-up fermentation of the marine-derived fungus Aspergillus sp. SF-5976.
Moreover, some of these components were found to have in vitro anti-inflammatory effects
through the inhibition of iNOS and COX-2 protein expression, and production of their
respective products such as NO and PGE . These findings demonstrate that these compounds
2
might be useful bioactive molecules for the inhibition of inflammation, and should therefore
be further studied.
1
6

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4
. Experimental
4
.1. General experimental procedures
Optical rotations were measured on a JASCO P-2000 (JASCO, Tokyo, Japan). UV
spectra were performed on an Optizen Pop UV-Vis spectrophotometer (Mecasys, Daejeon,
Korea). IR spectra were obtained on a Varian 640 FT-IR spectrometer (Varian, Palo Alto, CA,
USA). ECD spectra were recorded on a JASCO J-1100 spectropolarimeter. NMR spectra
were recorded on a Varian 500 MHz NMR spectrometer. ESITOFMS data were measured on
a Waters Q-TOF micromass spectrometer (Waters, Manchester, UK). HPLC-MS data was
recorded on an Agilent 1200 system connected with 6120 quadrupole MSD (Agilent, Santa
Clara, CA, USA) equipped with a Phenomenex Luna C18 (2) column (5 ꢀm, 150 × 4.6 mm
i.d., Phenomenex, Torrance, CA, USA). HPLC was performed on a Waters system (Waters,
Millford, MA, USA) using a 515 pump and a 2996 PDA detector equipped with an YMC
Pack ODS-A column (5 ꢀm, 250 × 20 mm i.d., YMC, Kyoto, Japan). MPLC was carried out
on a Biotage Isolera One system with a SNAP cartridge KP-Sil 100 g (Biotage AB, Uppsala,
Sweden). TLC was performed on pre-coated silica gel plates (0.25 mm, Merck, Darmstadt,
Germany).
4
.2. Fungus cultivation
Aspergillus sp. SF-5976 was obtained from an unidentified marine organism collected in
the Ross Sea (N 76°06.256′, E 169°12.675′), and identified based on rRNA sequence analysis.
Although a GenBank search with the 28S rRNA gene of SF-5976 (GenBank accession
number KR150444) suggested A. pseudoglaucus (FR839678), A. cibarius (FR848828), and A.
glaucus (JN938918), with 100% sequence identities, a specific species were not definitively
identified. The fungal strain SF-5976 was cultivated on potato dextrose agar medium
1
7

ACCEPTED MANUSCRIPT
supplemented with 3% NaCl on Petri dishes (150 mm × 2 cm × 150 plates) at 25 °C for 20
days.
4
.3. Extraction and Isolation
The fungal medium were extracted with MeOH (3 × 2.0 L) at room temperature and
evaporated in vacuo at 30 °C. The residue was suspended in H O (1.0 L) and partitioned with
2
EtOAc (3 × 1.0 L) to obtain an EtOAc-soluble extract (2.6 g). The extract was further
partitioned on a silica gel by MPLC (n-hexane–CHCl –acetone, 1:1:0 to 0:0:1, flow rate 35.0
3
mL/min) to give 16 fractions (F1 to F16). F2 (112.0 mg) was isolated by preparative HPLC
(
MeCN–H O, 4:1 to 1:0, flow rate 8.0 mL/min) to afford 2-(2′,3-epoxy-1′,3′-heptadienyl)-6-
2
hydroxy-5-(3-methyl-2-butenyl)benzaldehyde (7, 3.2 mg). F3 (143.4 mg) was purified by
preparative HPLC (MeCN–H O, 7:3 to 1:0, flow rate 8.0 mL/min) to obtain
2
dihydroauroglaucin (8, 5.5 mg), tetrahydroauroglaucin (6, 43.7 mg), physcion (2.3 mg), and
erythroglaucin (1.8 mg). F4 (76.6 mg) was isolated by preparative HPLC (MeCN–H O, 7:3
2
to 1:0, flow rate 8.0 mL/min) to afford 6′-oxo-chaetopyranin (10, 2.0 mg). F5 (68.3 mg) was
purified by preparative HPLC (MeCN–H O, 1:1 to 1:0, flow rate 8.0 mL/min) to give
2
chaetopyranin (9, 12.0 mg), isotetrahydroauroglaucin (11, 2.4 mg), asperanin A (1, 1.4 mg),
asperanin B (2, 2.9 mg), asperanin C (3, 1.0 mg), and 6-farnesyl-5,7-dihydroxy-4-
methylphthalide (24, 1.2 mg). F7 (134.4 mg) was isolated by preparative HPLC (MeCN–H O,
2
2
:3 to 1:0, flow rate 8.0 mL/min) to obtain eurotiumide A (12, 1.6 mg). F8 (112.2 mg) was
purified by preparative HPLC (MeCN–H O, 3:7 to 1:0, flow rate 8.0 mL/min) to give
2
isocladosporin (0.5 mg), evariquinone (23, 0.7 mg), variecolorin H (14, 3.4 mg), neoechinulin
B (15, 3.6 mg), and isoechinulin B (17, 1.3 mg). F9 (26.8 mg) was isolated by preparative
HPLC (MeCN–H O, 3:7 to 1:0, flow rate 8.0 mL/min) to afford isoechinulin A (16, 3.9 mg),
2
1
8

ACCEPTED MANUSCRIPT
variecolorin G (13, 2.9 mg), dehydroechinulin (18, 2.6 mg), and echinulin (19, 2.5 mg). F12
(
147.0 mg) was purified by preparative HPLC (MeCN–H O, 1:4 to 1:0, flow rate 8.0 mL/min)
2
to obtain 6′-hydroxy-4-O-methyl-cladosporin (21, 1.4 mg), 7′-hydroxy-4-O-methyl-
cladosporin (22, 0.8 mg), 5-prenyl-dihydrovariecolorin F (4, 3.4 mg), and 5-prenyl-
dihydrorubrumazine A (5, 1.7 mg). F13 (84.8 mg) was isolated by preparative HPLC
(
MeCN–H O, 1:4 to 17:3, flow rate 8.0 mL/min) to give 7′-hydroxy-4-O-methyl-cladosporin
2
(5.3 mg) and variecolortide C (20, 1.8 mg).
4
.3.1. Asperanin A (1)
Pale yellow amorphous solid; [α] –7.0 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 208
22
D
(4.0), 236 (3.7), 277 (3.6), 386 (3.3) nm; IR νmax (ATR) 3426, 2920, 1641, 1436, 1297, 1195
-1
1
13
cm ; ECD (c 0.5 mM, MeCN) ꢀε –0.9 (219), +1.1 (232), +0.7 (240); H and C NMR
–
(
CDCl , 500 and 125 MHz), see Table 1; ESIMS (negative) m/z 315 [M – H] ; HRESIMS m/z
3
–
3
15.1607 [M – H] (calcd for C H O , 315.1596).
1
9
23
4
4
.3.2. Asperanin B (2)
22
Pale yellow amorphous solid; [α] –5.8 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 206
D
(3.9), 233 (3.6), 277 (3.5), 386 (3.1) nm; IR νmax (ATR) 3427, 2920, 1640, 1434, 1296, 1194
-1
1
13
cm ; ECD (c 0.5 mM, MeCN) ꢀε +0.8 (212), +1.7 (220), +0.7 (231), +1.2 (241); H and C
–
NMR (CDCl , 500 and 125 MHz), see Table 1; ESIMS (negative) m/z 315 [M – H] ;
3
–
HRESIMS m/z 315.1598 [M – H] (calcd for C H O , 315.1596).
1
9
23
4
4
.3.3. Asperanin C (3)
Pale yellow amorphous solid; [α] –3.2 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 208
22
D
1
9

ACCEPTED MANUSCRIPT
-1
(
3.9), 234 (3.7) 276 (3.6) nm; IR νmax (ATR) 3408, 2925, 1680, 1640, 1435, 1295, 1210 cm ;
1
13
ECD (c 0.5 mM, MeCN) ꢀε –1.6 (206), +0.3 (218), –0.1 (225), +0.9 (244); H and C NMR
–
(
CDCl , 500 and 125 MHz), see Table 1; ESIMS (negative) m/z 261 [M – H] ; HRESIMS m/z
3
–
2
4
2
61.1136 [M – H] (calcd for C H O , 261.1127).
1
5
17
4
.3.4. 5-Prenyl-dihydrovariecolorin F (4)
2
4
White amorphous solid; [α] –41.5 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 208 (3.7),
D
-1
30 (3.8), 288 (3.2) nm; IR νmax (ATR) 3347, 2925, 1674, 1457, 1373, 1327 cm ; ECD (c 0.5
1
mM, MeCN) ꢀε –3.2 (210), +6.2 (225), –1.1 (240), +2.9 (252), +3.9 (272), –0.4 (301); H
1
3
+
and C NMR (CDCl , 500 and 125 MHz), see Table 2; ESIMS (positive) m/z 514 [M + H] ;
3
–
–
ESIMS (negative) m/z 512 [M – H] , 514 [M + 2 – H]; HRESIMS m/z 512.2665 [M – H]
(
calcd for C H ClN O , 512.2680).
29 39 3 3
4
.3.5. 5-Prenyl-dihydrorubrumazine A (5)
2
3
White amorphous solid; [α] –23.0 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 207 (3.7),
D
-1
2
28 (3.7), 279 (3.2) nm; IR νmax (ATR) 3357, 2923, 1677, 1442, 1378, 1209 cm ; ECD (c 0.5
1
13
mM, MeCN) ꢀε +0.5 (208), +3.9 (228), –2.8 (243), +1.7 (278), –1.4 (300); H and C NMR
+
(
CDCl , 500 and 125 MHz), see Table 2; ESIMS (positive) m/z 510 [M + H] ; ESIMS
3
–
–
(
negative) m/z 508 [M – H] ; HRESIMS m/z 508.3171 [M – H] (calcd for C H N O ,
30 42 3 4
5
08.3175).
4
.4. Preparation of (S)- and (R)-MTPA ester derivatives of 5-prenyl-dihydrovariecolorin F (4)
and 5-prenyl-dihydrorubrumazine A (5)
.2 mg of compounds 4 and 5 in 600 µL of pyridine-d were divided into two parts and
1
5
2
0

ACCEPTED MANUSCRIPT
transferred into clean tubes. Afterward, (R)-(–)-α-methoxy-α-(trifluoromethyl)phenylacetyl
(MTPA) chloride (20 ꢀL) (Sigma-Aldrich, St. Louis, MO, USA) was added to NMR tubes
under a N gas stream, and the solution was then carefully mixed. The tubes were left at room
2
temperature for 24 h, and the reactions were completed to yield the mono (S)-MTPA ester
derivative of 4 (4a). The (R)-MTPA ester derivative of 4 (4b) was obtained from the
treatment of (S)-(+)-α-MTPA chloride. The same procedure was performed to yield the (S)-
1
1
and (R)-MTPA esters of 5 (5a and 5b). The H NMR chemical shifts were obtained from H
and COSY NMR spectra.
4
.4.1. (S)-MTPA ester of 4 (4a)
1
H NMR (pyridine-d , 500 MHz) δ 6.20 (1H, m, H-27), 3.62 (1H, overlapped, H-26a),
5
H
3
7
.43 (1H, overlapped, H-26b), 1.54 (3H, s, H-29), 1.51 (3H, s, H-30); ESIMS (positive) m/z
+
30 [M + H] .
4
.4.2. (R)-MTPA ester of 4 (4b)
1
H NMR (pyridine-d , 500 MHz) δ 6.16 (1H, m, H-27), 3.65 (1H, overlapped, H-26a),
5
H
3
7
.47 (1H, overlapped, H-26b), 1.49 (3H, s, H-29), 1.46 (3H, s, H-30); ESIMS (positive) m/z
+
30 [M + H] .
4
.4.3. (S)-MTPA ester of 5 (5a)
1
H NMR (pyridine-d , 500 MHz) δ 5.95 (1H, m, H-27), 3.45 (1H, overlapped, H-26a),
5
H
3
7
.18 (1H, overlapped, H-26b), 1.21 (3H, s, H-29), 1.19 (3H, s, H-30); ESIMS (positive) m/z
+
26 [M + H] .
2
1

ACCEPTED MANUSCRIPT
4
.4.4. (R)-MTPA ester of 5 (5b)
1
H NMR (pyridine-d , 500 MHz) δ 5.93 (1H, m, H-27), 3.49 (1H, overlapped, H-26a),
5
H
3
7
.25 (1H, overlapped, H-26b), 1.07 (3H, s, H-29), 1.00 (3H, s, H-30); ESIMS (positive) m/z
+
26 [M + H] .
4
.5. Advanced Marfey’s analysis of 5-prenyl-dihydrovariecolorin F (4)
0
.6 mg of compound 4 was hydrolyzed with 0.5 mL of 6 N HCl at 110 °C for 2 h, and
sequentially evaporated with distilled water to remove tracing HCl. The - and -Alanine
D
L
(
Sigma-Aldrich) and the hydrolysate were treated with 100 ꢀL of 1 N NaHCO and 50 ꢀL of
3
1
% -FDLA (Tokyo Chemical Industry, Tokyo, Japan) in acetone, followed by stirring at
L
8
0 °C for 15 min. The reacted mixtures were neutralized by 50 ꢀL of 2 N HCl, and diluted
with 800 ꢀL of MeOH. The samples were analyzed by HPLC-MS in a linear gradient to
determine the chirality of the amino acids.
4
.6. Computational methods
The 3D models of 1–4 were built from Chem3D modeling, and the conformational
11
searches were performed by MMFF force field as implemented in the Spartan′14 software.
Geometry optimization of the obtained conformers were carried out at the B3LYP/6-
1+G(d.p) level. TDDFT ECD calculations of the optimized conformers were performed at
the CAM-B3LYP/TZVP or SVP levels with a CPCM solvent model in MeCN, by using the
3
1
2
Gaussian 09 software. The calculated ECD spectra were simulated with half bandwidth of
3
7
0
.2–0.4 eV. The ECD spectra were extracted with the SpecDis 1.64 software, and weighted
by Boltzmann distribution after UV correction.
2
2

ACCEPTED MANUSCRIPT
4
.7. Cell culture and viability assay
Raw 264.7 and BV2 cells were plated at a density of 5 × 10 cells/mL in DMEM medium
5
with supplements according to the method as previously described, and cell viability was
6
determined from MTT assay.
4
.8. Nitrite production determination
The nitrite concentration in the medium was determined by the Griess reaction as
indicator of NO production. The 100 µL of each supernatant was mixed with the same
volume of Griess reagent (Solution A: 222488; Solution B: S438081, Sigma-Aldrich), and the
absorbance was measured at 525 nm using an ELISA plate reader.
4
.9. PGE assay
2
RAW 264.7 and BV2 cells were plated in 24-well plates. Pre-incubation for 3 h with
various concentrations of compound 1−5 was followed by stimulation for 24 h with LPS. The
00 µL of each supernatant from the culture was collected to measure the PGE concentration
1
2
using an ELISA kit (R & D Systems, Minneapolis, MN, USA).
4
.10. Western blot analysis
RAW 264.7 and BV2 cells were collected and pelleted by centrifugation at 16,000 rpm
for 15 min. The cells were washed by PBS and lysed with 20 mM Tris-HCl buffer (pH 7.4)
consisting of a protease inhibitor mixture such as 0.1 mM PMSF, 5 mg/mL aprotinin, 5
mg/mL pepstatin A, and 1 mg/mL chymostatin. The protein concentration was measured by a
Lowry protein assay kit (P5626, Sigma-Aldrich). The same amount of proteins was resolved
by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a
2
3

ACCEPTED MANUSCRIPT
Hybond enhanced chemiluminescence nitrocellulose membrane (Bio-Rad, Hercules, CA,
USA) blocked with 5% skimmed milk. The membrane was then maintained with the primary
antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and horseradish peroxidase-
coupled secondary antibody followed by ECL detection (Amersham Pharmacia Biotech,
Piscataway, NJ, USA).
Acknowledgements
This research was supported by a grant from the National Research Foundation of Korea
(NRF-2015R1D1A1A01060321) and the Korea Polar Research Institute (PM15050).
Appendix A. Supplementary data
Information regarding the spectroscopic data of compounds 1–5 as well as the structures
1
and H NMR data of compounds 6–24 can be found at http://dx.doi.org/
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Highlights
5 previously unknown and 23 known compounds were isolated from Antarctic marine-
derived Aspergillus sp. SF-5976.
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Structures were elucidated by spectroscopic and chemical methods.
Absolute configurations were determined by chemical reactions and calculations of ECD
spectra.
Several compounds show anti-inflammatory activities in LPS-stimulated RAW 264.7 and
BV2 cells.
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