Neritinaceramides A-E, new ceramides from the marine bryozoan Bugula neritina inhabiting South China Sea and their cytotoxicity

Article abstract of DOI:10.3390/md12041987

Five new ceramides, neritinaceramides A (1), B (2), C (3), D (4) and E (5), together with six known ceramides (6-11), two known alkyl glycerylethers (12 and 13) and a known nucleoside (14), were isolated from marine bryozoan Bugula neritina, which inhabits the South China Sea. The structures of the new compounds were elucidated as (2S,3R,3′S,4E,8E,10E)-2-(hexadecanoylamino)- 4,8,10-octadecatriene-l,3,3′-triol (1), (2S,3R,2′R,4E,8E,10E)-2- (hexadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol (2), (2S,3R,2′R,4E,8E,10E)-2-(octadecanoylamino)-4,8,10-octadecatriene-l,3, 2′-triol (3), (2S,3R,3′S,4E,8E)-2-(hexadecanoylamino)-4,8- octadecadiene-l,3,3′-triol (4) and (2S,3R,3′S,4E)-2- (hexadecanoylamino)-4-octadecene-l,3,3′-triol (5) on the basis of extensive spectral analysis and chemical evidences. The characteristic C-3′S hydroxyl group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structural feature of ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1-3, was found from marine bryozoans for the first time. The new ceramides 1-5 were evaluated for their cytotoxicity against HepG2, NCI-H460 and SGC7901 tumor cell lines, and all of them exhibited selective cytotoxicity against HepG2 and SGC7901 cells with a range of IC ₅₀ values from 47.3 μM to 58.1 μM. These chemical and cytotoxic studies on the new neritinaceramides A-E (1-5) added to the chemical diversity of B. neritina and expanded our knowledge of the chemical modifications and biological activity of ceramides.

Full text of DOI:10.3390/md12041987

Mar. Drugs 2014, 12, 1987-2003; doi:10.3390/md12041987
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Neritinaceramides A–E, New Ceramides from the Marine
Bryozoan Bugula neritina Inhabiting South China Sea and
Their Cytotoxicity
Xiang-Rong Tian 1, Hai-Feng Tang 2,3,*, Jun-Tao Feng 1, Yu-Shan Li 4, Hou-Wen Lin 5,
Xiao-Pei Fan 1 and Xing Zhang 1,*
1
Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest
A&F University, Yangling 712100, China; E-Mails: tianxiangrong@163.com (X.-R.T.);
fengjt@nwsuaf.edu.cn (J.-T.F.); ting2008ting2008@163.com (X.-P.F.)
2
Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University,
Xi’an 710032, China
3
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University,
Xi’an 710032, China
4
School of Traditional Chinese Medicines, Shenyang Pharmaceutical University, Shenyang 110016,
China; E-Mail: liyushan8888@yahoo.com.cn
5
Department of Pharmacy, Renji Hospital, Affiliated to School of Medicine, Shanghai Jiao-Tong
University, Shanghai 200127, China; E-Mail: franklin67@126.com
* Authors to whom correspondence should be addressed;
E-Mails: tanghaifeng71@163.com (H.-F.T.); zhxing1952@126.com (X.Z.);
Tel.: +86-29-84774748 (H.-F.T.); Fax: +86-29-84774748 (H.-F.T.).
Received: 23 January 2014; in revised form: 2 March 2014 / Accepted: 13 March 2014 /
Published: 2 April 2014
Abstract: Five new ceramides, neritinaceramides A (1), B (2), C (3), D (4) and E (5),
together with six known ceramides (6–11), two known alkyl glycerylethers (12 and 13) and
a known nucleoside (14), were isolated from marine bryozoan Bugula neritina, which
inhabits the South China Sea. The structures of the new compounds were elucidated as
(2S,3R,3′S,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,3′-triol
(2S,3R,2′R,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol
(2S,3R,2′R,4E,8E,10E)-2-(octadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol
(1),
(2),
(3),
and
(2S,3R,3′S,4E,8E)-2-(hexadecanoylamino)-4,8-octadecadiene-l,3,3′-triol
(4)
(2S,3R,3′S,4E)-2-(hexadecanoylamino)-4-octadecene-l,3,3′-triol (5) on the basis of
extensive spectral analysis and chemical evidences. The characteristic C-3′S hydroxyl

Mar. Drugs 2014, 12
group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structural feature of
1988
ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1–3,
was found from marine bryozoans for the first time. The new ceramides 1–5 were
evaluated for their cytotoxicity against HepG2, NCI-H460 and SGC7901 tumor cell lines,
and all of them exhibited selective cytotoxicity against HepG2 and SGC7901 cells with a
range of IC50 values from 47.3 μM to 58.1 μM. These chemical and cytotoxic studies on
the new neritinaceramides A–E (1–5) added to the chemical diversity of B. neritina and
expanded our knowledge of the chemical modifications and biological activity of ceramides.
Keywords: marine bryozoan; Bugula neritina; ceramide; neritinaceramide; cytotoxicity
1. Introduction
Ceramides, a family of sphingolipids, are important components of a wide variety of tissues and
organs in biological systems [1]. Chemically, ceramides are composed of a sphingoid long chain
base (LCB) and an amide-linked long-chain fatty acid base (FAB) [2]. Ceramides and their
glycosylated-ceramides (cerebrosides), exhibiting a wide variety of biological effects including
cytotoxic, antifungal, immunostimulating and immunosuppressive activities [3], have been isolated
from a number of marine invertebrates, including starfishes [4], sea anemones [5,6], sponges [7],
corals [8], tunicates [9] and bryozoans [10], as well as terrestrial plants [11]. Marine bryozoans are
sedentary, colonial invertebrates that are widely distributed throughout the marine environment,
but are less common in freshwater. They have been proved to be an important marine drug sources
due to their affluent bioactive secondary metabolites, including macrolide, alkaloids, sterols and
heteratom-containing compounds, etc., [12]. In particular, a highly oxygenated macrolide, bryostatin 1,
isolated previously from marine bryozoan Bugula neritina, has been focused on preclinical and clinical
studies [13]. In our previous studies focused on marine bryozoans, a new terminal branched ceramide
and its analogues [14], cytotoxic sterols [15,16], and bromized alkaloids [17], have been reported from
B. neritina and Cryptosula pallasiana. As part of our ongoing investigations toward the discovery of
bioactive secondary metabolites from marine bryozoan B. neritina, five new ceramides,
neritinaceramides A–E (1–5), six known ceramides (6–11), two known alkyl glycerylethers (12 and 13),
and a known nucleoside (14) were isolated. The characteristic C-3′S hydroxyl group in the fatty acid
moiety in compounds 1, 4 and 5, was a novel structure feature of ceramides. The rare 4E,8E,10E-triene
structure in the sphingoid base of compounds 1–3, was found from marine bryozoans for the first time.
Herein, we report the isolation, structure identification and cytotoxicity evaluation of those new ceramides.
2. Results and Discussion
The marine bryozoan Bugula neritina was extracted with 95% EtOH, condensed, and suspended in
water, subsequently, and then was further extracted with EtOAc. The EtOAc extract was subjected to
column chromatography (CC) over normal silica gel and Sephadex LH-20, respectively, and then was
further purified by reverse semi-preparative HPLC to yield compounds 1–14 (Figure 1).

Mar. Drugs 2014, 12
1989
Figure 1. Chemical structures of compounds 1–14 from the marine bryozoan
Bugula neritina.
Neritinaceramide A (1) was isolated as a white amorphous powder. The positive ion mode HR-ESI-MS
showed a pseudomolecular ion peak at m/z 572.4658 [M + Na]+ (calcd. for C34H63NO4Na, 572.4655),
which, together with the pseudomolecular ion peak at m/z 584 [M + Cl]− in the negative mode
ESI-MS, enabled the determination of the molecular formula as C34H63NO4, with the help of NMR
1
spectral data. A close scrutiny of the H-NMR and 13C-NMR spectra of 1 (Table 1) by DEPT and
HSQC experiments revealed the presence of an amide group [δ 173.0 (C-1′), δ 6.65 (1H, d,
J = 8.0 Hz, NH)], a nitrogen-linked methine [δ 54.4 (C-2), δ 3.92 (1H, m, H-2)], a hydroxylated
methylene [δ 62.0 (C-1), δ 3.72 (1H, br d, J = 8.3 Hz, H-1a), 3.90 (1H, br d, J = 8.3 Hz, H-1b)] and
two hydroxylated methines [δ 74.2 (C-3), δ 4.31 (1H, br s, H-3); δ 68.9 (C-3′), δ 4.00 (1H, m, H-3′)],
which together with the long aliphatic carbon signals at 29.2–29.7, demonstrated a trihydroxyl
1
ceramide structure of compound 1. The observation of H-1H COSY correlations from H2-1 to H-2,
and in turn, from H-2 to NH and H-3, from H-3′ to H-2a′ (δ 2.30), H-2b′ (δ 2.43), H-4a′ (δ 1.43) and
H-4b′ (δ 1.51), and the HMBC correlations from NH to C-1′, and from H2-2′ to C-1′ and C-3′,
indicated the three hydroxyl groups were located at C-1, C-3 an C-3′, respectively. The 1H-, 13C-NMR
and HSQC spectra of 1 suggested the presence of three double bonds (C4=C5, C8=C9 and C10=C11)
on the basis of six proton signals at δ 5.54 (1H, dd, J = 15.2, 6.2 Hz, H-4), 5.77 (1H, dt, J = 15.2,
6.3 Hz, H-5), 5.59 (1H, m, H-8), 5.99 (1H, dd, J = 15.3, 5.0 Hz, H-9), 6.00 (1H, dd, J = 15.3, 5.0 Hz,
H-10) and 5.52 (1H, m, H-11), as well as the corresponding carbons at δ 129.3 (C-4), 133.2 (C-5),
133.3 (C-8), 130.0 (C-9), 131.2 (C-10) and 130.7 (C-11). The positions of the three double bonds were
1
confirmed to be C-4, C-8 and C-10 on the basis of H-1H COSY correlations of H-3/H-4, H-4/H-5,
H-5/H2-6, H2-6/H2-7, H2-7/H-8, H-8/H-9, H-9/H-10 and H-10/H-11, and HMBC correlations of

Mar. Drugs 2014, 12
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H-4/C-3, H-5/C-3, H-5/C-6, H2-6/C-4, H2-6/C-5, H2-6/C-7, H2-7/C-8, H2-7/C-9, H-8/C-10, H-11/C-9
and H-11/C-12 (Figure 2A). The lengths of the sphingoid long chain base (LCB) and the
amide-linked long-chain fatty acid base (FAB) were determined to be composed of 18 and 16 carbons,
respectively, based on the negative ESI-MS fragment ions at m/z 165, 227, 255, 283, 297, 311 and 336
(Figure 2B). Based on the above evidences, the planar structure and the key connectivities of ceramide
1 were established.
Table 1. 1H and 13C-NMR data of compounds 1 and 2 (500MHz for 1H-NMR, 125MHz for
13C-NMR) a.
1
2
No.
δC, mult. b
62.0 (t)
δH (int., mult., J in Hz) b
3.72 (1H, br d, 8.3),
3.90 (1H, br d, 8.3)
3.92 (1H, m)
δC, mult. b
62.0 (t)
δC, mult. c
61.9 (t)
δH (int., mult., J in Hz) c
4.22 (1H, dd, 10.8, 4.2),
4.46 (1H, dd, 10.8, 4.5)
4.67 (1H, m)
1
2
3
54.4 (d)
74.2 (d)
129.3 (d)
133.2 (d)
31.9 (t)
32.1 (t)
133.3 (d)
130.0 (d)
131.2 (d)
130.7 (d)
32.1 (t)
32.6 (t)
29.4 (t)
29.2 (t)
31.9 (t)
22.7 (t)
14.1 (q)
173.0 (s)
43.1 (t)
68.9 (d)
37.1 (t)
25.6 (t)
54.4 (d)
74.2 (d)
129.1 (d)
133.3 (d)
31.9 (t)
32.1 (t)
133.5 (d)
130.0 (d)
131.2 (d)
130.7 (d)
32.1 (t)
32.6 (t)
29.4 (t)
29.2 (t)
31.9 (t)
22.7 (t)
14.1 (q)
175.0 (s)
72.4 (d)
34.8 (t)
31.8 (t)
25.1 (t)
56.1 (d)
73.0 (d)
131.4 (d)
132.6 (d)
32.7 (t)
32.9 (t)
132.8 (d)
131.0 (d)
131.6 (d)
131.5 (d)
32.7 (t)
32.1 (t)
29.4 (t)
29.3 (t)
32.1 (t)
22.9 (t)
14.2 (q)
175.4 (s)
72.5 (d)
35.8 (t)
32.0 (t)
25.9 (t)
4.31 (1H, br s)
4.83 (1H, br t, 6.1)
6.13 (1H, m)
4
5.54 (1H, dd, 15.2, 6.2)
5.77 (1H, dt, 15.2, 6.3)
2.16 (2H, m)
5
6.05 (1H, m)
6
2.18 (1H, m)
7
2.04 (2H, dt, 14.1, 6.9)
5.59 (1H, m)
2.04 (1H, m)
8
5.64 (1H, m)
9
5.99 (1H, dd, 15.3, 5.0)
6.00 (1H, dd, 15.3, 5.0)
5.52 (1H, m)
6.11 (1H, m)
10
11
12
13
14
15
16
17
18
1′
2′
3′
4′
5′
5.65 (1H, m)
5.99 (1H, m)
2.16 (2H, m)
2.18 (2H, br s)
1.23 (2H, br s) d
1.33 (2H, m) d
1.33 (2H, m) d
1.23 (2H, br s) d
1.23 (2H, br s) d
0.84 (3H, t, 7.1)
-
2.04 (2H, dt, 14.1, 6.9)
1.36 (2H, m) d
1.36 (2H, m) d
1.26 (2H, br s) d
1.26 (2H, br s) d
0.88 (3H, t, 7.1)
-
a 2.30 (1H, m), b 2.43 (1H, d, 2.2)
4.00 (1H, m)
4.59 (1H, m)
2.22 (1H, m), 2.03 (1H, m)
1.23 (2H, br s) d
1.64 (2H, m)
a 1.43 (1H, m), b 1.51 (1H, m)
1.37 (2H, m)
6′~13′ 29.2–29.7 (t)
1.26 (16H, br s) d
1.26 (2H, br s) d
1.26 (2H, br s) d
0.88 (3H, t, 7.1)
6.65 (1H, d, 8.0)
29.2–29.7 (t) 29.3–30.0 (t)
1.23 (16H, br s) d
1.23 (2H, br s) d
1.23 (2H, br s) d
0.84 (3H, t, 7.1)
8.34 (1H, d, 8.7)
14′
15′
16′
NH
31.9 (t)
22.7 (t)
14.1 (q)
-
31.9 (t)
22.7 (t)
14.1 (q)
-
32.1 (t)
22.9 (t)
14.2 (q)
-
a
b
Assignments aided by the DEPT, COSY, HSQC, HMBC, and NOESY experiments; data were obtained in CDCl3;
c data were obtained in C5D5N; d overlapped with other signals.

Mar. Drugs 2014, 12
1991
1
Figure 2. Key H–1H COSY and HMBC correlations (A), together with negative ESI-MS
fragments (B) of compound 1.
The geomertry of the C-4/C-5, C-8/C-9 and C-10/C-11 alkenyl bonds was confirmed to be trans
based on the vicinal coupling constants of J4,5 (15.2 Hz), J8,9 (15.3 Hz) and J9,10 (15.3 Hz). Moreover,
the trans configuration (E) for those alkenyl bonds was further confirmed by the chemical shifts of the
allylic carbons C-6 (δ 31.9), C-7 (δ 32.1) and C-12 (δ 32.1), since allylic carbon signals of trans- and
cis-isomers were observed at δ 32–33 and 27–28, respectively [18]. The relative stereochemistry at C-2
and C-3 in LCB of 1 were found to be same as that of D-sphingosine (D-erythro), as evidenced by
13C-NMR chemical shifts of C-2 (δ 54.4) and C-3 (δ 74.2), which are consistent with those reported for
natural product N-palmitoyl-D-erythro-(2S,3R)-octadecasphinga-4(E)-ene (δ 54.5 and 74.7) [19].
Furthermore, the specific rotation ([α]D = −2.8°) of LCB 1 obtained by methanolysis of ceramide 1,
was in good accordance with D-erythro-sphingosine, (2S,3R)-(E)-2-aminooctadec-4-ene-1,3-diol
([α]D = −3.0°) [20], while different from L-erythro-sphingosine, (2R,3S)-(+)-erythro-N-lauroyldoco
sasphinga-4,8-dienine ([α]D = +2.94°) [21]. This further confirmed the 2S,3R configuration for
ceramide 1. Similarly, the 3′S-configuration was proved by the methanolysis of 1 and comparison of
the specific rotation of the resulting methyl ester of the fatty acid with known compounds. In details,
methanolysis of 1 by hydrochloric acid/methanol gave fatty acid methyl ester (FAME 1) as the major
product (Figure 3). The specific rotation of FAME 1 ([α]D +12.5°) was in good agreement with the
synthetic (S)-methyl 3-hydroxyhexadecanoate ([α]D = +13.8°), while the specific rotation [α]D for
(R)-methyl 3-hydroxyhexadecanoate was −16.6° [22]. Thus, the structure of neritinaceramide A
was unambiguously assigned as (2S,3R,3′S,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadeca
triene-l,3,3′-triol.
Neritinaceramide B (2) was isolated as an isomer of 1, due to the same molecular formula of
C34H63NO4 from ESI-MS at m/z 572 [M + Na]+ (positive mode) and 584 [M + Cl]− (negative mode),
and the HR-ESI-MS pseudomolecular ion peak at m/z 572.4652 [M + Na]+ (calcd. for C34H63NO4Na,
572.4655). By comparison of the 13C-NMR data of 2 with those of 1, it could be confirmed that 2 and
1 shared the same LCB structural skeleton, while the hydroxyl group at C-3′ in ceramide 1 was
replaced to be at C-2′ in 2, as 2 showed different chemical shifts at C-1′ (δ 175.4), C-2′ (δ 72.5) and
C-3′ (δ 35.8) in its FAB. The methene protons H2-3′ (δ 2.03 and 2.22) showed coupling relationships
1
with H2-4′ (δ 1.23) and H-2′ (δ 4.59) in the H-1H COSY spectrum, which together with HMBC
correlations from NH (δ 8.34, d, J = 8.7 Hz) to C-1′, from H-2′ to C-1′ and from H2-3′ to C-2′,
confirmed that the hydroxyl group was attached at C-2′ (Figure 4A). Fragment peaks at m/z 166, 227,
255, 283 and 311 indicated that the number of carbons in the sphingoid long chain base was 18, and in
the fatty acid base was 16 (Figure 4B). The stereochemistry at C-2, C-3 and C-2′ were proposed as 2S,
3R and 2′R on the basis of the chemical shifts of C-2, C-3, and C-2′ in CDCl3 (δ 54.4, 74.2 and 72.4,

Mar. Drugs 2014, 12
1992
respectively) and C5D5N (δ 56.1, 73.0 and 72.5, respectively), which were close to analogue
(2S,3R,4E,2′R)-2-(2-hydroxyhexadecanoylamino)-16-methyl-4-octadecene-1,3-diol [δ 54.6 (C-2), 74.3
(C-3) and 72.5 (C-2′) in CDCl3, and δ 56.1 (C-2), 73.1(C-3) and 72.5 (C-2′) in C5D5N.] [23].
Moreover, the configurations were confirmed by the specific rotation of ceramide 2 ([α]D = +9.8°)
which was consistent with that of the synthetic ceramide with the 2S,3R,2′R-configuration
([α]D = +7.4°) [24], and this method has been applied to determine the stereochemistry for ceramide
with the core structure of 2,3,2′ chiral centre [25,26]. Besides, methanolysis of 2 with the same method
of 1 gave fatty acid methyl ester (FAME 2), its specific rotation ([α]D = –6.7°) was similar to
(R)-methyl 2-hydroxyhexadecanoate ([α]D = –5.7°) [26], while was different with (S)-methyl
2-hydroxyhexadecanoate ([α]D = +1.15°) [27], which also confirmed the 2′R-configuration for
ceramide 2. Accordingly, the structure of neritinaceramide
B
(2) was deduced as
(2S,3R,2′R,4E,8E,10E)-2-(hexadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol.
Figure 3. Methanolysis of compound 1.
Figure 4. Key 1H-1H COSY and HMBC correlations (A), together with ESI-MS fragments
(B) of compound 2 (a positive fragment ions; b negative fragment ions).
Neritinaceramide C (3) was found to have a molecular formula C36H67NO4 according to the positive
ESI-MS pseudomolecular ion peak at m/z 600 [M + Na]+, and negative ESI-MS pseudomolecular ion
peak at m/z 576 [M − H]−, with the help of NMR spectral data (Table 2). The NMR spectra of 3
showed a close resemblance to those of ceramide 2, except for the carbon number in the FAB. The

Mar. Drugs 2014, 12
1993
negative ESI-MS fragment ions at m/z 324, 298, 283, 255 and 225 indicated that the numbers of
carbons for both of the FAB and LCB were 18 (Figure 5). The carbon number of the FAB was further
validated through EI-MS molecular ion at m/z 314 (M+) for the methyl ester obtained after
methanolysis of 3 with 5% mehtanolic HCl. Therefore, neritinaceramide C (3) was determined to be
(2S,3R,2′R,4E,8E,10E)-2-(octadecanoylamino)-4,8,10-octadecatriene-l,3,2′-triol.
1
1
Table 2. H and 13C-NMR data of compounds 3–5 (CDCl3, 500MHz for H-NMR,
125MHz for 13C-NMR) a.
3
4
5
No.
δH (int., mult.,
δH (int., mult.,
J in Hz)
δH (int., mult.,
J in Hz)
δC, mult.
62.2 (t)
δC, mult.
61.8 (t)
δC, mult.
J in Hz)
3.71
3.75 (1H, m),
3.89
(1H, d, 11.0),
3.86
3.74 (1H, m),
3.87 (1H, m)
1
61.9 (t)
(1H, br d, 10.0)
(1H, d, 11.2)
3.91
2
3
4
54.4 (d)
74.2 (d)
129.2 (d)
54.5 (d)
74.1 (d)
128.9 (d)
3.92 (1H, m)
56.1 (d)
74.1 (d)
128.6 (d)
3.93 (1H, m)
(1H, br d, 10.0)
4.28 (1H, m)
4.28 (1H, br s)
4.28 (1H, m)
5.51
5.53
5.54 (1H, m)
5.77 (1H, m)
(1H, dd, 15.5, 6.0)
(1H, br d, 14.0)
5.65
5
133.3 (d)
133.5 (d)
5.77 (1H, m)
134.3 (d)
(1H, dt, 14.3, 6.5)
1.96
6
7
8
31.9 (t)
32.1 (t)
2.16 (2H, t, 2.9)
32.6 (t)
32.4 (t)
32.3 (t)
2.05 (2H, m)
1.26 (2H, br s) b
1.26 (2H, br s) b
(2H, q-like, 6.2)
2.11 (2H, m)
2.04 (2H, q, 7.1)
5.59
29.2–29.7 (t)
29.2–29.7 (t)
133.5 (d)
131.3 (d)
5.41 (1H, m)
(1H, dt, 15.0, 6.0)
5.98 (1H, m)
6.00 (1H, m)
5.52 (1H, m)
2.16 (2H, t, 2.9)
2.04 (2H, q, 7.1)
1.36 (2H, m)
1.36 (2H, m)
1.26 (2H, br s) b
1.26 (2H, br s) b
0.87 (3H, t, 7.1)
-
9
130.0 (d)
131.2 (d)
130.7 (d)
32.1 (t)
32.6 (t)
29.4 (t)
29.2 (t)
31.9 (t)
22.7 (t)
14.1 (q)
175.1 (s)
129.0 (d)
32.2 (t)
5.38 (1H, m)
2.07 (2H, m)
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
0.88 (3H, t, 7.0)
-
29.2–29.7 (t)
29.2–29.7 (t)
29.2–29.7 (t)
29.2–29.7 (t)
29.2–29.7 (t)
29.2–29.7 (t)
29.2–29.7 (t)
32.0 (t)
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
0.88 (3H, t, 7.1)
-
10
11
12
13
14
15
16
17
18
1′
29.3–29.7 (t)
29.3–29.7 (t)
29.3–29.7 (t)
29.3–29.7 (t)
29.3–29.7 (t)
32.0 (t)
22.7 (t)
22.7 (t)
14.1 (q)
14.1 (q)
173.0 (s)
173.0 (s)
a 2.29
4.12
(1H, dd, 14.5, 9.5),
b 2.41
a 2.31 (1H, m),
b 2.41 (1H, m)
2′
72.4 (d)
43.4 (t)
43.3 (t)
(1H, dd, 7.8, 3.4)
(1H, d, 14.3)
4.00 (1H, m)
a 1.43 (1H, m),
b 1.51 (1H, m)
3′
4′
34.8 (t)
31.8 (t)
2.16 (2H, t, 2.9)
1.26 (2H, br s) b
68.9 (d)
37.2 (t)
68.9 (d)
37.1 (t)
4.00 (1H, m)
a 1.43 (1H, m),
b 1.51 (1H, m)

Mar. Drugs 2014, 12
1994
Table 2. Cont.
a 1.26 (1H, br s) b,
a 1.30 (1H, m),
b 1.43 (1H, m)
29.2–29.7 (t) 1.26 (16H, br s) b
5′
25.1 (t)
1.62 (2H, m)
25.6 (t)
25.6 (t)
b 1.43 (1H, m)
1.26 (16H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
0.88 (3H, t, 7.0)
-
6′~13′ 29.1–29.7 (t) 1.26 (16H, br s) b 29.3–29.7 (t)
14′
15′
16′
17′
18′
NH
29.1–29.7 (t)
29.1–29.7 (t)
31.9 (t)
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
1.26 (2H, br s) b
0.87 (3H, t, 7.1)
7.20 (1H, d, 7.9)
32.0 (t)
32.0 (t)
1.26 (2H, br s) b
1.26 (2H, br s) b
22.7 (t)
22.7 (t)
14.1 (q)
14.1 (q)
0.88 (3H, t, 7.1)
22.7 (t)
-
-
-
-
-
-
-
14.1 (q)
-
-
-
6.76 (1H, d, 7.8)
6.77 (1H, d, 7.8)
a Assignments aided by DEPT, 1H-1H COSY, HSQC, HMBC and NOESY experiments; b overlapped with other signals.
b
Figure 5. Key ESI-MS fragments of compound 3 (a positive fragment ions; negative
fragment ions).
The molecular formula of neritinaceramide D (4) was assigned as C34H65NO4 on the basis of
HR-ESI-MS pseudomolecular ion peak at m/z 574.4815 [M + Na]+ (calcd. for C34H65NO4Na,
1
13
1
574.4811) and the results of H and C NMR spectral interpretations (Table 2). Again, the H- and
13C-NMR spectra of 4 were found to be nearly identical to those of 1 except for the missing of two
olefinic carbons at C-10 and C-11 in the LCB. The positions of the four reminder olefinic carbons δ
133.5 (C-5), 131.3 (C-8), 129.0 (C-9) and 128.9 (C-4) were determined to be C-4 and C-8 mainly by
1H-1H COSY correlations from H-3 (δ 4.28) to H-4 (δ 5.51), and in turn, from H-4 to H-5 (δ 5.77), H-5
to H2-6 (δ 1.96), H2-6 to H2-7 (δ 2.11), H2-7 to H-8 (δ 5.41), H-8 to H-9 (δ 5.38) and H-9 to H2-10
(δ 2.07), and further confirmed by HMBC correlations from H-3 to C-4 and C-5, H-5 to C-6 (δ 32.6),
H2-6 to C-4 and C-5, H-8 to C-6, C-7 (δ 32.4) and C-9, and H-9 to C-10 (δ 32.2) (Figure 6A).
Ceramide 4 possessed the similar negative ESI-MS fragment ions as compared with 1, while the
fragment ion m/z 338 of 4 replaced the fragment ion m/z 336 of 1 (Figure 6B). This also confirmed the
missing of two olefinic carbons in 4, and further deduced that 1 and 4 possessed the same carbon
number in their long aliphatic chain. The stereochemistry of 4 was determined by the same method
with those of 1. Finally, neritinaceramide D (4) was elucidated as (2S,3R,3′S,4E,8E)-2-(hexa
decanoylamino)-4,8-octadecadiene-l,3,3′-triol.
Neritinaceramide E (5) was obtained as a white amorphous powder with a molecular formula of
C34H67NO3, determined by positive HR-ESI-MS m/z 576.4967 [M + Na]+ (calcd. for C34H67NO3Na,
576.4968), and the negative ESI-MS m/z 552 [M − H]−, with the help of NMR data. Comparison of the
NMR spectra of 5 with 4 indicated that they possessed the same ceramide skeleton, except for the
absence of a double bond between C-8 and C-9 of 5. The fragment ions of 5 were almost identical with
those of 4 in the negative ESI-MS, but the fragmet ion m/z 338 in 4 was replaced by fragmet ion m/z

Mar. Drugs 2014, 12
1995
340 in 5 (Figure 7). This indicated that the numbers of carbons of the FAB and LCB were also
16 and 18, respectively, and allowed the assignment of structure 5 to be (2S,3R,3′S,4E)-2-(hexa
decanoylamino)-4-octadecene-l,3,3′-triol.
Figure 6. Key 1H–1H COSY and HMBC correlations (A), and negative ESI-MS fragments
(B) of compound 4.
Figure 7. Key negative ESI-MS fragments of compound 5.
The remainder six known ceramides were established as (2S,3R,2′R,4E)-2-(hexadecanoylamino)-4-
octadecene-l,3,2′-triol (6) [28], (2S,3R,2′R,4E,8E)-2-(hexadecanoylamino)-4,8-octadecadiene-l,3,
2′-triol (7) [29], (2S,3R,4E)-2-(hexadecanoylamino)-4-octadecene-l,3-diol (8) [30], (2S,3R)-2-(hexa
decanoylamino)-4-octadecane-l,3-diol (9) [14], (2S,3R,4E)-2-(hexadecanoylamino)-4-nonadecene-l,
3-diol (10) [14], and (2S,3R,4E)-2-(hexadecanoylamino)-4-eicosene-l,3-diol (11) [14], and a known
nucleoside was elucidated as adenine riboside (14) [31], by comparing their spectroscopic data with
those published in the literatures. The 1H- and 13C-NMR spectra of 12 and 13 showed a resemblance to
those identified ceramides, since they shared the similar long chain and oxygen-bearing signals.
However, the structural type of 12 and 13 was determined as alkyl glycerylether, since they lack a
carbonyl carbon signal at δ 173.0–175.1 in the 13C-NMR spectra, and the loss of NH signal at δ 6.65–8.34
1
in the H-NMR spectra. The 13C-NMR and DEPT spectra of 12 showed an oxygenated methine at
δ 64.5 (C-1) and three oxygenated methylenes at δ 64.5 (C-1), 72.0 (C-3) and 72.7 (C-16′), indicating
the terminal substituted alkyl glycerylether for 12. The pseudomolecular ion peak at m/z 339 [M + Na]+
in the positive ESI-MS, along with the analysis of NMR spectral data enabled the determination of the
molecular formula of 12 as C19H40O3, and 16 carbons for the alkyl long chain accordingly. Thus, the
structure of 12 was determined as 1-O-palmityl glycerin ether [32]. Similarly, alkyl glycerylether 13,
also known as batiolum, was elucidated as 1-O-octadecyl glycerin ether based on the pseudomolecular
ion peak at m/z 367 [M + Na]+ in the positive ESI-MS, with the help of NMR spectral data [33].
Ceramides 9–11, alkyl glycerylethers 12 and 13, and nucleoside (14) were isolated from marine
bryozoans for the first time. Besides, batiolum (13) has been developed as medicine to increase the
level of leukocyte [34].

Mar. Drugs 2014, 12
1996
The new ceramides 1–5 were evaluated for their cytotoxicity against human tumor cell lines
HepG2, NCI-H460 and SGC7901 in vitro with a method of MTT assay. The results of their
cytotoxicity were shown in Table 3. Ceramides 1–5 exhibited selective cytotoxicity against HepG2 and
SGC7901 cells with IC50 values range from 47.3 μM to 58.1 μM, while showed relative weak activity
to NCI-H460 cell line with IC50 values more than 84.4 μM. The above information suggested a good
selectivity of those ceramides against different tumor cell lines. Besides, ceramides 2 and 3
characterized at a 2′R hydroxyl group, showed more activity than ceramides 1, 4 and 5 with a 3′S
hydroxyl group. This indicated that the 2′R hydroxyl group in FAB is important for their
cytotoxicity [35]. However, all of the test ceramides appeared lower cytotoxicity compared with
corresponding glycosylated-ceramides, cerebrosides, which suggested that the sugar moiety is critical
to cytotoxic activity [36]. In fact, multiple related studies indicated that the structure-activity
relationships (SAR) about the antitumor activity of ceramides and cerebrosides were rendered by the
presence of the trans double bond between C-4 and C-5 in the vicinity of their polar head [37,38], the
category of the sugar moieties at C-1 in the LCB [36,39], and the additional hydroxyl group at position
C-2′ or C-4 [3,35]. The SAR studies of those ceramides provided potential chemical modification
values to search for approach to expand their biological properties.
Table 3. Cytotoxicities of compounds 1–5 against three tumor cell lines in vitro
(IC50, μM) a.
Compound
HepG2
52.1
NCI-H460
>100
84.4
SGC7901
57.7
1
2
47.3
52.2
3
47.8
85.1
52.9
4
53.7
>100
>100
0.13
57.9
5
54.6
58.1
Adriamycin b
0.14
0.17
a
IC50 values are means from three independent experiments in which each compound concentration was
tested in three replicate wells; b Adriamycin as positive control.
Among the marine invertebrates, the primary ceramides and cerebrosides were often found as those
derived from the doubly unsaturated LCB with 4E-sphinganine and 4E,8E-sphingadiene [19], as well
as from the saturated phytosphingosine, 4-hydroxysphingosine [40]. To date, the 4E,8E,10E-sphingatriene
LCB in ceramides and cerebrosides were rare encountered in natural, and just isolated from the star
fish Ophidiaster ophidiamus [2], ascidian Phallusia fumigate [41] and gorgonian Acabaria undulate [19].
Ceramides (1−3) characterized at a 4E,8E,10E-sphingatriene LCB were isolated from marine
bryozoans for the first time. Interestingly, to the best of our knowledge, naturally occurring FAB in
ceramides included no hydroxyl substituted form [19] and 2′(R)-hydroxyl substituted form [3]. The
3′(S)-hydroxyl substituted FAB in compounds 1, 4 and 5 was a novel structural feature in ceramides.
Thus, the discovery of those five new ceramides, neritinaceramides A−E (1−5), from Bugula neritina
was a typical example to illustrate the chemical diversity of the title species.

Mar. Drugs 2014, 12
1997
3. Experimental Section
3.1. General Experimental Procedures
Melting points were determined on an XT5-XMT apparatus and uncorrected. Specific rotation [α]D
was obtained on a Perkin-Elmer 343 polarimeter. 1D and 2D NMR spectral experiments were
measured in CDCl3 or pyridine-d5 on a Bruker AVANCE-500 spectrometer with TMS as an internal
standard. Chemical shifts (δ) were expressed in ppm and coupling constants (J) in Hz. EI-MS spectra
were obtained on a Finnigan MAT 212 mass spectrometer. ESI-MS and HR-ESI-MS spectra were
carried out on a Micromass Quattro Micro mass spectrometer. Column chromatography (CC) was
performed on silica gel H (10−40 μm, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex
LH-20 (GE-Healthcare, Sweden). HPLC was carried out on a Dionex P680 liquid chromatograph
equipped with a UV 170 UV/Vis detector at 206 nm using a YMC-Pack R & D ODS-A column
(250 × 20 mm i.d., 5 μm, YMC, Kyoto, Japan) for semi-preparation and a Thermo ODS-2 column
(250 × 4.6 mm i.d., 5 μm, Thermo Hypersi-Keystone Inc., Bellefonte, USA) for analysis. TLC
detection was achieved by spraying the silica gel plates (Qingdao Marine Chemical Inc., Qingdao,
China) with 10% H2SO4 in EtOH, followed by heating for 3 min.
3.2. Animal Material
Marine bryozoan Bugula neritina was collected in March 2008 in Daya Bay, Shenzhen, China. The
specimen identification was conducted by Prof. H.-W. Lin. A voucher specimen numbered 20080312,
was deposited in the Herbarium of Pharmacy, Changzheng Hospital, Second Military Medical
University, Shanghai, China.
3.3. Extraction and Isolation
The marine bryozoan B. neritina (about 100 kg, dry weight) was extracted with 95% EtOH (4 × 500 L)
at room temperature with each extraction taking one week. The concentrated EtOH extract (2.0 kg)
was suspended in water, and then partitioned successively with EtOAc to obtained EtOAc extract
(600 g). One third of the EtOAc extract was subjected to CC over silica gel (1200 g, 10−40 μm)
eluting with the CHCl3-MeOH (100:1, 50:1, 20:1, 10:1, 1:1, 0:1) gradient to afford nine major
fractions (A−I) based on TLC analysis. Fr. E (15.0 g) was subject to CC on silica gel (150 g,
10−40 μm) eluting with petroleum ether-EtOAc (10:1 to 1:1) gradient to give four major fractions
(Fr. E1−E4). The Fr. E3 (2.5 g) was recrystallisated with CH3COCH3 to obtained a crude white
powder (900 mg), and then further purified by semi-preparative reverse HPLC with MeOH-H2O (97:3)
as the mobile phase at a flow rate of 8.0 mL/min to afford five new ceramides 1 (3.7 mg,
tR = 32.9 min), 2 (11.5 mg, tR = 39.9 min), 3 (19.7 mg, tR = 38.3 min), 4 (50.3 mg, tR = 45.5 min) and
5 (8.5 mg, tR = 57.9 min). Fr. E4 (1.3 g) was eluted with CHCl3-MeOH (1:1) on Sephadex LH-20 to
remove pigments, and then further purified by semi-preparative reverse HPLC using MeOH-H2O
(97:3) as mobile phase at a flow rate of 8.0 mL/min to afford ceramides 6 (11.4 mg, tR = 41.5 min) and
7 (8.7 mg, tR = 43.7 min). Similarly, the remainder four known ceramides 8 (34.0 mg, tR = 65.4 min),
9 (14.4 mg, tR = 75.3 min), 10 (10.0 mg, tR = 63.2 min) and 11 (12.6 mg, tR = 94.4 min) were obtained

Mar. Drugs 2014, 12
1998
from Fr. E2 (176.5 mg) by the same method with Fr. E4. The two known alkyl glycerylethers
12 (13.0 mg, tR = 37.2 min) and 13 (18.7 mg, tR = 39.5 min) were isolated from Fr. D via CC on silica
gel column with 5:1 ether-EtOAc as eluting solvent, and finally further purified by semi-preparative
reverse HPLC using MeOH-H2O (97:3) as mobile phase at a flow rate of 8.0 mL/min. The known
nucleoside 14 (9.0 mg, tR = 24.7 min) was purfied from Fr. H via CC over repeated sephadex LH-20
with CHCl3-MeOH (1:1) as eluting solvent, and then further purified by semi-preparative HPLC with
MeOH-H2O (90:10) as mobile phase at a flow rate of 7.0 mL/min.
3.3.1. Neritinaceramide A (1)
1
White amorphous powder, mp 109110 °C; [α]20 −4.5° (c 0.05, CHCl3); H-NMR (500 MHz,
D
CDCl3) and 13C-NMR (125 MHz, CDCl3) data see Table 1; Positive ESI-MS m/z 572 [M + Na]+, 437,
409, 330, 312, 303, 284, 275, 256; Negative ESI-MS m/z 548 [M − H]−, 584 [M + Cl]−, 336,
325, 311, 297, 283, 255, 227, 165, 113; Positive HR-ESI-MS m/z 572.4658 [M + Na]+ (calcd. for
C34H63NO4Na, 572.4655).
3.3.2. Neritinaceramide B (2)
1
White amorphous powder, mp 111112 °C; [α]20 +9.8° (c 0.14, CHCl3); H-NMR (500 MHz,
D
C5D5N) and 13C-NMR (125 MHz, C5D5N) data see Table 1; Positive ESI-MS m/z 572 [M + Na]+, 437,
409, 312, 284, 274, 256, 166; Negative ESI-MS m/z 548 [M − H]−, 584 [M + Cl]−, 283, 255, 227, 113;
Positive HR-ESI-MS m/z 572.4652 [M + Na]+ (calcd. for C34H63NO4Na, 572.4655).
3.3.3. Neritinaceramide C (3)
1
White amorphous powder, mp 105106 °C; [α]20 +10.1° (c 0.10, CHCl3); H-NMR (500 MHz,
D
CDCl3) and 13C-NMR (125 MHz, CDCl3) data see Table 2; Positive ESI-MS m/z 600 [M + Na]+, 437,
409, 330, 311, 283, 274, 264, 256, 219, 166; Negative ESI-MS (−) m/z 576 [M − H]−, 340, 324, 312,
298, 283, 271, 255, 253, 237, 225, 113.
3.3.4. Neritinaceramide D (4)
1
White amorphous powder, mp 104105 °C; [α]20 −4.3° (c 0.15, CHCl3); H-NMR (500 MHz,
D
CDCl3) and 13C-NMR (125 MHz, CDCl3) data see Table 2; Positive ESI-MS m/z 574 [M + Na]+, 1125
[2M + Na]+, 437, 409, 274, 262, 256; Negative ESI-MS m/z 550 [M − H]−, 586 [M + Cl]−, 338, 283,
255, 228, 113; Positive HR-ESI-MS m/z 574.4815 [M + Na]+ (calcd. for C34H65NO4Na, 574.4811).
3.3.5. Neritinaceramide E (5)
1
White amorphous powder, mp 101102 °C; [α]20 −4.1° (c 0.10, CHCl3); H-NMR (500 MHz,
D
CDCl3) and 13C-NMR (125 MHz, CDCl3) data see Table 2; Positive ESI-MS m/z 576 [M + Na]+, 437,
409, 311, 300, 283, 274, 264, 256, 228; Negative ESI-MS m/z 552 [M − H]−, 588 [M + Cl]−, 340, 283,
255, 227, 113; Positive HR-ESI-MS m/z 576.4967 [M + Na]+ (calcd. for C34H67NO3Na, 576.4968).
The copies of the original spectra of the new compounds 15 are available in
Supplementary Information.

Mar. Drugs 2014, 12
1999
3.3.6. 1-O-Palmityl Glycerin Ether (12)
1
White amorphous powder, mp 60–62 C. H-NMR (500 MHz, CDCl3) δ 0.88 (3H, t, J = 6.5 Hz,
H3-1′), 1.26 (26H, br s, H2-2′ to H2-14′), 1.58 (2H, t, J = 7.0 Hz, H2-15′), 3.52 (4H, m, H2-3 and
H2-16′), 3.65 (1H, dd, J = 11.0, 5.0 Hz, Ha-1), 3.72 (1H, dd, J = 11.5, 3.5 Hz, Hb-1), 3.86 (1H, m, H-2);
13C-NMR (125 MHz, CDCl3) δ 64.5 (C-1), 70.6 (C-2), 72.0 (C-3), 14.3 (C-1′), 23.1 (C-2′), 32.1 (C-3′),
29.1-29.8 (C-4′~13′), 26.2 (C-14′), 29.8 (C-15′), 72.7 (C-16′); Positive ESI-MS m/z 339 [M + Na]+,
655 [2M + Na]+; Negative ESI-MS m/z 631 [2M − H], 351 [M + Cl], 361 [M + COOH].
3.3.7. 1-O-Octadecyl Glycerin Ether (13)
1
White amorphous powder, mp 62–63 C. H-NMR (500 MHz, CDCl3) δ 0.86 (3H, t, J = 7.0 Hz,
H3-1′), 1.27 (30H, br s, H2-2′ to H2-16′), 1.57 (2H, t, J = 7.0 Hz, H2-17′), 3.49 (4H, m, H2-3 and
H2-18′), 3.63 (1H, dd, J = 11.5, 5.5 Hz, Ha-1), 3.72 (1H, dd, J = 11.5, 4.0 Hz, Hb-1), 3.86 (1H, m, H-2);
13C-NMR (125 MHz, CDCl3) δ 64.4 (C-1), 70.6 (C-2), 72.0 (C-3), 14.3 (C-1′), 22.8 (C-2′), 32.1 (C-3′),
29.5-29.9 (C-4′~15′), 26.2 (C-16′), 29.9 (C-17′), 72.6 (C-18′); Positive ESI-MS m/z 367 [M + Na]+,
711 [2M + Na]+.
3.4. Methanolysis of Ceramides 15
Each ceramide (about 2 mg) was dissolved in 5% HCl-MeOH (2 mL) and refluxed for 10 h at
80 °C . The reaction mixture was extracted with n-hexane for three times. The n-hexane layer was
washed with H2O and concentrated in vacuo to yield the corresponding fatty acid methyl ester (FAME
15) [14]. The remainder H2O layer was neutralized with NH4OH, and then further concentrated
in vacuo to yield the corresponding long chain base (LCB 1–5) with specific rotation [α]20 about
D
−2.8° (c 0.10, CHCl3).
(S)-Methyl 3-hydroxylhexadecanoate (FAME 1, 4 and 5): [α]20D +12.5° (c 0.11, CHCl3); EI-MS m/z
286 [M]+ (4), 268 [M − H2O]+ (9), 236 (6), 211 (4), 194 (7), 111 (9), 103 (100), 83 (10), 74 (22), 55 (19).
(R)-Methyl 2-hydroxylhexadecanoate (FAME 2): [α]20 −6.7° (c 0.15, CHCl3); EI-MS m/z 286
D
[M]+ (9), 254 [M − OCH3 − H]+ (4), 227 (67), 208 (6), 145 (13), 111 (50), 103 (21), 97 (96), 83 (100).
(R)-Methyl 2-hydroxyloctadecanoate (FAME 3): [α]20D −6.5° (c 0.10, CHCl3); EI-MS m/z 314 [M]+
(26), 296 [M − H2O]+ (4), 282 [M − OCH3 − H]+ (6), 269 (5), 255 (76), 236 (6), 145 (14), 111 (52),
103 (19), 97 (93), 83 (85), 57 (100).
3.5. MTT Cytotoxicity Assays
Neritinaceramides AE (15) were evaluated for their cytotoxic activity against human hepatoma
HepG2, pulmonary carcinoma NCI-H460 and gastric carcinoma SGC7901 tumor cells by microculture
tetrazolium (MTT) assay. The cells were obtained from American Type Culture Collection (ATCC),
and were seeded to RPMI-1640 medium with 10% fetal bovine serum and 100 U/mL benzyl
penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing in 5% CO2/air for
24 h. Then the test samples were added and incubated at 37 °C for another 72 h. The detailed

Mar. Drugs 2014, 12
2000
experiments procedure has been described in our previous published literature [16]. The inhibition was
expressed as IC50 value with adriamycin as positive control.
4. Conclusions
The present chemical study of the marine bryozoan B. neritina resulted in the isolation and
characterization of five new ceramides, neritinaceramides AE (15), six known ceramides (6–11),
two known alkyl glycerylethers (12 and 13) and a known nucleoside (14). The characteristic C-3′S
hydroxyl group in the fatty acid moiety in compounds 1, 4 and 5, was a novel structure feature in
ceramides. The rare 4E,8E,10E-triene structure in the sphingoid base of compounds 1–3, was found
from marine bryozoans for the first time. Bioacitve studies indicated that those new ceramides (15)
exhibited selective cytotoxicity against HepG2 and SGC7901 cells with IC50 values range from
47.3 μM to 58.1 μM. These chemical and cytotoxic studies on the new neritinaceramides A–E (1–5)
added to the chemical diversity of B. neritina and expanded our knowledge of the chemical
modifications and biological activity of ceramides.
Acknowledgments
This work was financially supported by National Natural Science Foundation of China
(No. 31201551) and National High-Tech Research and Development Project (863 Project,
No. 2007AA09Z401). The authors are grateful to Hui-Min Wang, Mass Measurement Center,
Shanghai Institute of Pharmaceutical Industry, for the MS measurements, Min-Chang Wang, Nuclear
Magnetic Resonance Center, Xi’an Modern Chemistry Research Institute, for the NMR measurements,
and Ping-Hu Zhang, Jiangsu Center for Drug Screening & National Drug Screening Laboratory, China
Pharmaceutical University, for the evaluation of cytotoxicity.
Author Contributions
The listed authors contributed to this work as described in the following. Xiang-Rong Tian, Jun-Tao
Feng and Xiao-Pei Fan carried out the extraction and isolation. Xiang-Rong Tian also carried out the
methanolysis of ceramides 1–5 and participated in the structural elucidation. Jun-Tao Feng also
conducted the MTT cytotoxicity assays and helped interpreting the results. Yu-Shan Li participated in
the structural elucidation. Hou-Wen Lin collected and identified the samples of Bugula neritina, and
participated in the structural elucidation. As corresponding authors Hai-Feng Tang and Xing Zhang
organized the study and participated in the structural elucidation. All authors helped preparing the
manuscript and approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.
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