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Antifouling and antibacterial

compounds from the gorgonians
Subergorgia suberosa and Scripearia
gracillis
a b a b b
S. H. Qi , S. Zhang , L. H. Yang & P. Y. Qian
a
Guangdong Key Laboratory of Marine Materia Medica, South
China Sea Institute of Oceanology, The Chinese Academy of
Sciences, 164 West Xingang Road, Guangzhou 510301 Guangdong,
P.R. China
b
Department of Biology and Coastal Marine Laboratory, Hong
Kong University of Science and Technology, Clearwater Bay, KLN,
Hong Kong SAR, P.R. China

Available online: 27 Jul 2010

To cite this article: S. H. Qi, S. Zhang, L. H. Yang & P. Y. Qian (2008): Antifouling and antibacterial
compounds from the gorgonians Subergorgia suberosa and Scripearia gracillis , Natural Product
Research, 22:2, 154-166

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 Natural Product Research, Vol. 22, No. 2, 20 January 2008, 154–166

Antifouling and antibacterial compounds from the gorgonians

Subergorgia suberosa and Scripearia gracillis
S. H. QIyz, S. ZHANGy, L. H. YANGz and P. Y. QIAN*z
Downloaded by [CAS Chinese Academy of Sciences] at 02:56 30 June 2011

yGuangdong Key Laboratory of Marine Materia Medica, South China Sea

Institute of Oceanology, The Chinese Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301 Guangdong, P.R. China
zDepartment of Biology and Coastal Marine Laboratory, Hong Kong University of Science
and Technology, Clearwater Bay, KLN, Hong Kong SAR, P.R. China

(Received 29 November 2006; in final form 16 August 2007)

In this study, we investigated the potential antilarval and antibacterial activity of secondary
metabolites of the gorgonians Subergorgia suberosa and Scripearia gracillis from the South
China Sea. Fresh specimens of these two gorgonian corals were collected from a shallow reef in
Sanya Bay of Hainan Island and extracted with different solvents. Antilarval activity of the
chemical extracts and pure compounds was evaluated in settlement inhibition assays with
laboratory-reared Balanus amphitrite and Bugula neritina larvae, while antibacterial activity was
assessed with disc diffusion bioassay on growth inhibition of 15 marine bacterial species. Using
bioassay-guided procedures, we purified and identified nine compounds. The most potent
metabolites produced by these gorgonian corals were subergorgic acid and pregn-4-ene-3,
20-dione extracted from S. suberosa. Our results show that the gorgonian coral S. suberosa and
S. gracillis can produce potent anti-fouling compounds that deserve further exploration.

Keywords: Subergorgia suberosa; Scripearia gracillis; Anti-fouling; Antibacterial; Secondary

metabolites

1. Introduction

Biofouling, the undesirable buildup of sessile marine organisms (such as barnacles,

mussels, tubeworms, and seaweeds) onto man-made surfaces, causes severe problems in
the maritime industry [1], including changes in surface properties [2], speed reduction
and elevated fuel consumption of ships, shiphull corrosion, weight increase of fishing
gears and floats, and distortion of the initial configuration of submerged structures [3].
To control biofouling, anti-fouling coastings have been widely applied over the last
several decades. Among the common anti-foulants, tributyltin (TBT)-based compounds
are the most effective but are also highly toxic, killing marine organisms nonselectively
and harming nontargeted organisms [4,5]. Environmental concerns have led to a total
ban on the production of TBT-based coatings since January 2003 and a complete ban
on the application of TBT-based coatings in January 2008. Therefore, there is a need to

*Corresponding author. Tel.: þ852-2358-7331. Email: boqianpy@ust.hk

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis
http://www.tandf.co.uk/journals
DOI: 10.1080/14786410701642441
 Antifouling and antibacterial compounds 155

develop new environmentally compatible alternatives that would be efficient against

several fouling organisms [6].
In the marine environment, many sessile species such as gorgonians, soft corals,
sponges, and seaweeds effectively defend against predators, competitors, and potential
pathogens [7–12]. For example, gorgonian corals produce chemicals with antimicrobial,
anti-fouling, predator deterrent, and allelopathic properties [13–20].
There are 44 species from six families of gorgonian corals in Chinese waters, mainly
distributed in the South China Sea. We have observed in the field that many of these
species are apparently free of epibiosis, especially Subergorgia suberosa and Scripearia
gracillis, suggesting that the species may possess certain mechanisms to control
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epibiosis. In preliminary experiments, we found that the crude extracts of S. suberosa

and S. gracillis inhibited growth of marine bacteria and settlement of barnacle larvae. In
this study, we aimed to 1) isolate anti-fouling compounds from the gorgonian corals of
S. suberosa and S. gracillis using a bioassay-guided fractionation and purification
process; 2) examine the antibacterial activity of pure compounds against common
marine pathogenic bacteria and bacterial strains isolated from marine natural biofilms,
which could induce larval settlement of the polychaete Hydroides elegans; and 3)
investigate the anti-fouling effect of those compounds against larval settlement of the
barnacle Balanus amphitrite and the bryozoan Bugular neritina.

2. Materials and methods

2.1. Sample collection

Specimens of S. suberosa and S. gracillis were collected by SCUBA divers from a coral
reef at a depth of 10–20 m in Sanya Bay (18
110 N, 109
250 E) in the South China Sea,
Hainan province, P.R. China in October 2004. Coral samples were brought to the
surface in plastic bags, immediately placed in a cooler, and then taken to the nearby
Sanya Tropical Marine Ecology Station, Chinese Academy of Sciences where they were
stored in the freezer (20
C) until being treated with chemical solvents. The species
were identified through a voucher by Prof. R. L. Zou of the South China Sea Institute
of Oceanology, Chinese Academy of Sciences.

2.2. Extraction and isolation of bioactive compounds

The coral samples were extracted three times with a mixture of EtOH : CH2Cl2 (2 : 1 in
volume) at room temperature. The resulting extracts were combined and concentrated
in vacuo at 35
C. The residue of S. suberosa was suspended in H2O and extracted three
times with each of CHCl3 and n-BuOH. For S. gracillis, the residue was suspended in
H2O and extracted three times with each of petroleum ether and EtOAc. The extracts
were evaporated in vacuo at 35
C. The CHCl3 extract from S. suberosa was subjected to
column chromatography using silica gel and a step-wise gradient solvent system
(CHCl3 : Me2CO, from 10 : 0 to 0 : 10 in volume) until pure compounds were obtained.
The petroleum ether and EtOAc extracts were also subjected to column chromato-
graphy using silica gel with gradient systems of petroleum ether : EtOAc until pure
compounds were obtained.
 156 S. H. Qi et al.

2.3. Compound structural identification

The structures of the resulting pure compounds were elucidated by using a Bruker
AV-500 MHz NMR spectrometer in CDCl3 at 500.13 MHz and 125.77 MHz at 298 K
with Tetramethylsilan (TMS) as a reference. The structures were further confirmed
using an LCQDECA XP HPLC/MSn spectrometer for ESIMS. Structural elucidation of
the pure compounds was based on interpretation of their spectral data (NMR, MS) and
comparison with published values.
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2.4. Antibacterial assays

Antibacterial activities of nine pure compounds 1–9 and the petroleum ether and
EtOAc extracts of S. gracillis (10, 11) were tested against 15 strains of bacteria using a
standard disc diffusion assay [21]. Among the 15 bacterial strains, eight induce larval
settlement: Loktanella hongkongensis (strain designation UST950701-009), Micrococcus
luteus (UST950701-006), Pseudoalteromonas sp. (UST010723-006), Rhodovulum sp.
(UST950701-012), Ruegeria sp. (UST010723-008), Staphylococcus haemolyticus
(UST950701-004), Vibrio halioticoli (UST010723-002), Vibrio vulnificus (UST001201-
001) [22] and seven are marine pathogenic bacteria: Moraxella phenylpyruvica
(UST000621-002), Pseudoalteromonas piscida (UST010620-005), Shewanella
algae (UST010723-014), Staphylococcus aureus (UST950701-005), Vibrio alginolyticus
(UST981130-062), Vibrio furnissii (UST010723-010), and Vibratos harveyi (UST020129-
010). All bacterial cultures were first grown to stationary phase in broth (2–3 days,
30
C); 200 mL aliquots were then spread onto seawater-based nutrient agar (0.3% yeast
extract, Oxoid, 0.5% peptone, Oxoid, 1.5% agar, Oxoid). Sterile 6 mm diameter
circular discs of filter paper (Whatman No. 1, disc volume ¼ 20 mm3) were loaded with
50 mg of pure compound or 200 mg of extract dissolved in methanol, evaporated to
dryness, and then placed onto the bacterial lawn. An additional set of discs with
10 mg mL1 of streptomycin was used as the positive controls. The agar plates were
incubated for 24 h at 30
C until bacteria had developed in a confluent film. The
inhibition zones between the disc and the bacterial lawn (inhibition radius) were
measured to the nearest 0.5 mm. The experiment was run in three replicates.

2.5. Larval culture

The barnacle B. amphitrite (Cirripedia) and the bryozoan B. neritina (Bryozoa) were
used to test the antilarval settlement activity of the pure compounds and crude extracts.
Adults of B. amphitrite (Cirripedia) were collected in March 2006 from the intertidal
zone in Pak Sha Wan, Hong Kong (22
190 N, 114
160 E). After 12 h of exposure to air,
several hundred adults were placed in a container filled with filtered seawater (FSW) (30
ppt salinity) to induce the release of larvae. Larval culture was maintained according to
the method described in Thiyagarajan et al. [23]. Briefly, nauplii obtained from several
adults were mass-reared to cyprid stage at a density of two larvae mL1 in 0.22-mm-
filtered seawater at 30 ppt salinity (FSW) using Chaetoceros gracilis Schutt as the only
food source and a rearing temperature of 28
C. Newly transformed cyprids were used
for the experiment.
 Antifouling and antibacterial compounds 157

Colonies of B. neritina containing mature and developing embryos in their ovicells

were collected in March 2006 from a fish farm near Wong Shek, Hong Kong (22
250 N,
114
200 E). Adult colonies were transferred to a small glass aquarium and kept overnight
in the dark in nonaerated seawater. To induce release of larvae, the water in the
aquarium was changed and the colonies were exposed for about 30 min to the overhead
room light, as well as to a supplementary light installed close to the aquarium [24]. The
positively phototactic larvae that were attracted to the beam of the supplementary light
source were then transferred into a clean beaker by using a wide-mouthed pipette [25]
and were immediately used for bioassay.
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2.6. Larval settlement bioassays

Larval settlement bioassays were performed using sterile 24-well polystyrene plates
(; ¼ 48 mm; FALCON #1006, Becton Dickinson, USA). In this study, first, we tested
the antilarval activities of the petroleum ether and EtOAc extracts of S. gracillis
(10, 11), and the CHCl3 extract of S. suberosa (12) against B. amphitrite larvae at
100 mg mL1. Then we tested the antilarval activities of pure compounds 1–5, 7, 9
against B. amphitrite larvae at 50 mg mL1. Later, because the amount of tested samples
was limited, we tested the antilarval activity of only active compounds (1, 2)
against B. neritina larvae to define their EC50 and LC50 values (see below).
Pure compounds (1 and 2) in DMSO were dissolved to the concentrations ranging
from 0.1 to 200 mg mL1 in autoclaved FSW. Other compounds (3–7, 9) were prepared
in a concentration of 50 mg mL1. About 20 competent larvae were added to each well in
1 mL of the test solution. The experiment was repeated twice with four replicates each
time. Wells containing only FSW with DMSO served as the controls. The plates were
incubated at 27
C for 1 h for B. neritina and for 24 h for B. amphitrite. Percentage of
larval settlement was determined by counting the settled, live individuals under a
dissecting microscope and expressing the result as a proportion of the total number of
larvae in the well. Statistical calculations were performed with the SPSS Version 11
software package. Differences in the larval settlement between the experimental
treatments and controls were determined by one-way ANOVA followed by a Dunnett
test. EC50 (inhibits 50% of settlement of B. amphitrite larvae in comparison with the
control) and LC50 (the concentration at which 50% of larvae were dead in comparison
with the control) levels of pure compounds were calculated by using the Probit software
program.

3. Results

3.1. Isolation and identification of bioactive compounds

Figure 1 shows six compounds (1–6) obtained from S. suberosa, namely, subergorgic
acid (1) [26], pregn-4-ene-3, 20-dione (progesterone) (2) [27], 5-pregn-3, 20-dione (3)
[27], 3-pregn-5-ene-20-one-3-ol (4) [27], and 3, 5-pregn-20-one-3-ol (5) [27] and
stigma-7, 22-dien-3, 5, 6-triol (6) [28], and three compounds isolated from
S. gracillis, that is, 3,7,9-tri-Me-6, 8-purinediol (7) [29], 1,2,3,4-tetrahydro--carboline
(8) [30] and batyl alcohol (9) [31]. General chemical structure characteristics of these
 158 S. H. Qi et al.

O O

CO2H

O O
1 H
2 3
O O
Downloaded by [CAS Chinese Academy of Sciences] at 02:56 30 June 2011

HO HO
H
4 HO
5 OH
OH 6
O

N N CH2OH
NH CHOH
N O N
H CH2O CH2 (CH2)16CH3
N
8 9
7
Figure 1. Chemical structures of the gorgonian metabolites.

compounds are as follows:

Subergorgic acid (1): White powder; C15H20O3; 1H-NMR (500 MHz, CDCl3) H: 2.02
(dd, J ¼ 16.7, 12.5 Hz, H-3), 2.35 (dd, J ¼ 16.7, 6.7 Hz, H-3), 1.64 (m, H-4),
2.08 (m, H-5), 1.64 (m, H-6), 1.64 (m, H-6), 1.62 (m, H-7), 1.80 (m, H-7), 6.41
(s, H-9), 3.00 (q, J ¼ 7.1 Hz, H-11), 1.10 (d, J ¼ 6.4 Hz, H-12), 1.20 (s, H-13), 1.11
(d, J ¼ 6.4 Hz, H-15); 13C-NMR (125 MHz, CDCl3) C: 217.8 (s, C-2), 169.4 (s, C-14),
152.3 (d, C-9), 136.7 (s, C-10), 68.6 (s, C-1), 62.8 (d, C-5), 61.8 (s, C-8), 51.7 (d, C-11),
49.9 (t, C-3), 38.3 (t, C-7), 33.4 (d, C-4), 28.4 (t, C-6), 23.4 (q, C-13), 19.9 (q, C-12), 17.7
(q, C-15); Negative-ion ESI-MS m/z: 247 [M  1]1.
Pregn-4-ene-3, 20-dione (2): White powder; C21H30O2; 1H-NMR (500 MHz, CDCl3)
H: 5.73 (1H, s, H-4), 0.62 (3H, s, H-18), 1.01 (3H, s, H-19), 2.10 (3H, s, H-21);
13
C-NMR (125 MHz, CDCl3) C: 13.3 (t, C-1), 34.0 (t, C-2), 199.2 (s, C-3), 124.0 (d,
C-4), 170.7 (s, C-5), 32.0 (t, C-6), 32.8 (t, C-7), 35.6 (d, C-8), 53.8 (d, C-9), 38.6 (s, C-10),
21.1 (t, C-11), 38.7 (t, C-12), 43.9 (s, C-13), 56.1 (d, C-14), 24.4 (t, C-15), 23.0 (t, C-16),
63.6 (d, C-17), 13.3 (q, C-18), 17.4 (q, C-19), 209.0 (s, C-20), 31.4 (q, C-21);
Negative-ion ESI-MS m/z: 313 [M  1]1.
5-Pregn-3, 20-dione (3): White powder; C21H32O2; 1H-NMR (500 MHz, CDCl3)
H: 0.63 (3H, s, H-18), 1.02 (3H, s, H-19), 2.15 (3H, s, H-21); 13C-NMR (125 MHz,
CDCl3) C: 37.0 (t, C-1), 39.0 (t, C-2), 212.7 (s, C-3), 42.3 (t, C-4), 40.9 (d, C-5), 25.8 (t,
C-6), 26.6 (t, C-7), 35.6 (d, C-8), 44.2 (d, C-9), 35.0 (s, C-10), 21.3 (t, C-11), 37.1 (t, C-12),
44.4 (s, C-13), 56.7 (d, C-14), 24.4 (t, C-15), 23.0 (t, C-16), 63.8 (d, C-17), 13.4 (q, C-18),
22.6 (q, C-19), 209.1 (s, C-20), 31.4 (q, C-21); Negative-ion ESI-MS m/z: 315 [M  1]1.
3-Pregn-5-ene-20-one-3-ol (4): White powder; C21H32O2; 1H-NMR (500 MHz,
CDCl3) H: 5.36 (1H, br s, H-6), 0.60 (3H, s, H-18), 1.02 (3H, s, H-19), 2.12 (3H, s,
 Antifouling and antibacterial compounds 159

H-21); 13C-NMR (125 MHz, CDCl3) C: 37.3 (t, C-1), 31.6 (t, C-2), 71.7 (s, C-3), 38.2
(t, C-4), 44.9 (d, C-5), 28.6 (t, C-6), 32.0 (t, C-7), 35.5 (d, C-8), 54.3 (d, C-9), 36.5
(s, C-10), 21.1 (t, C-11), 38.9 (t, C-12), 44.3 (s, C-13), 56.9 (d, C-14), 24.4 (t, C-15), 22.8
(t, C-16), 63.9 (d, C-17), 13.2 (q, C-18), 12.3 (q, C-19), 209.6 (s, C-20), 31.5 (q, C-21);
Negative-ion ESI-MS m/z: 315 [M  1]1.
3, 5-Pregn-20-one-3-ol (5): White powder; C21H34O2; 1H-NMR (500 MHz, CDCl3)
H: 4.70 (1H, m, H-3), 0.63 (3H, s, H-18), 1.02 (3H, s, H-19), 2.18 (3H, s, H-21);
13
C-NMR (125 MHz, CDCl3) C: 36.5 (t, C-1), 26.5 (t, C-2), 71.8 (s, C-3), 34.6 (t, C-4),
40.6 (d, C-5), 27.1 (t, C-6), 30.6 (t, C-7), 35.9 (d, C-8), 42.1 (d, C-9), 34.7 (s, C-10), 20.9
(t, C-11), 39.3 (t, C-12), 44.4 (s, C-13), 56.8 (d, C-14), 24.5 (t, C-15), 23.0 (t, C-16), 63.9
Downloaded by [CAS Chinese Academy of Sciences] at 02:56 30 June 2011

(d, C-17), 13.4 (q, C-18), 23.3 (q, C-19), 209.4 (s, C-20), 31.5 (q, C-21); Negative-ion
ESI-MS m/z: 317 [M  1]1.
Stigma-7, 22-dien-3, 5, 6-triol (6): White powder; C28H46O3; 1H-NMR (500 MHz,
C5D5N): 5.73 (1H, d, J ¼ 2.5 Hz, H-7), 5.23 (1H, m, H-22), 5.15 (1H, m, H-23), 4.80
(1H, m, H-3), 4.31 (1H, br s, H-6), 1.08 (3H, s, H-19), 1.07 (3H, d, J ¼ 7.0 Hz, H-21),
1.01 (3H, d, J ¼ 7.0 Hz, H-28), 0.96 (3H, d, J ¼ 7.0 Hz, H-26), 0.89 (3H, d, J ¼ 7.0 Hz,
H-27), 0.68 (3H, s, H-18); 13C-NMR (125 MHz, C5D5N) : 32.6 (t, C-1), 33.8 (t, C-2),
67.5 (d, C-3), 40.0 (t, C-4), 76.1 (s, C-5), 74.2 (d, C-6), 120.4 (d, C-7), 141.5 (d, C-8), 43.8
(d, C-9), 38.0 (s, C-10), 23.5 (t, C-11), 40.9 (t, C-12), 43.9 (s, C-13), 55.2 (d, C-14), 22.4
(t, C-15), 28.6 (t, C-16), 56.2 (d, C-17), 12.4 (q, C-18), 18.7 (q, C-19), 41.9 (s, C-20), 21.4
(q, C-21), 136.3 (d, C-22), 132.2 (d, C-23), 43.7 (t, C-24), 33.4 (d, C-25), 20.2 (q, C-26),
19.8 (q, C-27), 18.1 (q, C-28); Negative-ion ESI-MS m/z: 429 [M  1]1.
3,7,9-Tri-Me-6, 8-purinediol (7): Yellow powder, C8H10N4O2; 1H-NMR (500 MHz,
CDCl3) H: 3.99(3H, s), 3.59 (3H, s), 3.41 (3H, s); 13C-NMR (125 MHz, CDCl3) C: 41.0
(t, C-1), 42.2 (t, C-3), 19.5 (t, C-4), 106.9 (s, C-4a), 127.3 (s, C-4b), 118.5 (d, C-5), 119.7
(d, C-6), 122.3 (d, C-7), 111.9 (d, C-8), 137.7 (s, C-8a); Positive-ion ESI-MS m/z: 195
[M þ 1]þ1.
1,2,3,4-Tetrahydro--carboline (8): Yellow powder; C11H12N2; 1H-NMR (500 MHz,
CDCl3) H: 3.08, 3.62 (each 1H, s, H-1), 3.99 (1H, s, H-2), 3.67 (1H, t, J ¼ 5.95 Hz, H-3),
3.12 (1H, t, H-4), 7.38 (1H, d, J ¼ 7.60 Hz, H-5), 7.26 (1H, t, J ¼ 7.5 Hz, H-6), 7.31 (1H,
t, J ¼ 7.5 Hz, H-7), 7.65 (1H, d, J ¼ 7.7 Hz, H-8), 12.2 (1H, s, H-9); 13C-NMR
(125 MHz, CDCl3) C: 41.0 (t, C-1), 42.2 (t, C-3), 19.5 (t, C-4), 106.9 (s, C-4a), 127.3
(s, C-4b), 118.5 (d, C-5), 119.7 (d, C-6), 122.3 (d, C-7), 111.9 (d, C-8), 137.7 (s, C-8a),
128.0 (s, C-9a); Positive-ion ESI-MS m/z: 173 [M þ 1]þ1.
Batyl alcohol (9): White powder; C21H44O3; 1H NMR (500 MHz, CDCl3) H: 3.86
(1H, m), 3.72 (1H, dd, J ¼ 3.5, 11.5 Hz), 3.65 (1H, dd, J ¼ 5.5, 11.5 Hz), 3.55 (1H, m),
3.52 (1H, m), 3.46 (2H, m), 1.58 (2H, m), 1.26 (30 H, br s), 0.88 (3H, t, J ¼ 7.0 Hz);
13
C-NMR (125 MHz, CDCl3) C: 72.0 (t, C-1), 72.5 (t, C-2), 64.4 (t, C-3), 71.0 (t, C-10 ),
32.0 (t, C-20 ), 26.4 (t, C-30 ), 29.1-29.7 (t, C-50 to C-150 ), 22.6 (t, C-160 ), 31.8 (t, C-170 ),
14.0 (t, C-180 ); Negative-ion ESI-MS m/z: 343 [M  1]1.

3.2. Antibacterial activity

Antibacterial activities of nine pure compounds 1–9 and extracts (10–11) against the 15
bacteria are showed in table 1. The samples (1, 6–11) inhibited the growth of certain
marine bacterial species with inhibition zones ranging from 1.5 to 13 mm (table 1),
 Downloaded by [CAS Chinese Academy of Sciences] at 02:56 30 June 2011

160

Table 1. Antibacterial activity of pure compounds (1, 6–9) at 50 mg disc1 and extracts (10–11) at 200 mg disc1 was determined by standard disc-diffusion assays.

Testing bacteria (UST number) Streptomycin 1 6 7 8 9 10 11

Pseudoalteromonas piscida (UST010620-005) 4.10  1.04 – 3.20  1.22 3.04  1.26 5.0  1.02 4.02  0.89 2.10  0.26
Rhodovulum sp. (UST950701-012)* 4.05  0.93 2.00  0.81 – – – – – 3.06  1.00
Ruegeria sp. (UST010723-008)* 5.02  1.23 – 2.06  1.06 – – 2.15  1.03 – –
Shewanella algae (UST010723-014) 2.89  0.65 – – – 2.20  0.73 – – –
Staphylococcus aureua (UST950701-005) 2.01  0.34 – – – – – 3.03  0.56(kill) –
Staphylococcus haemolyticus (UST950701-004)* 1.00  0.26 – – – – – 1.50  0.38(kill) –
Vibrio alginolyticus (UST981130-062) 2.51  0.76 – – – – 6.20  2.00 4.02  1.30 7.18  2.63
S. H. Qi et al.

Vibrio furnissii (UST010723-010) 1.00  0.24 – – – – 3.00  1.35 – –

Vibratos harveyi (UST020129-010) 2.52  0.78 – – 2.13  1.08 – 5.10  2.03 13.15  2.57 3.02  0.65

The target bacterial species are commonly found in biofilms and have shown inductive effects on larval settlement of H. elegans (with *) or common pathogens (without *) isolated from
Hong Kong waters [21]. Controls were 10 mg streptomycin per disc. Data presented are the mean radius of the clear zone (mm)  SD of three replicates indicating complete growth inhibition.
 Antifouling and antibacterial compounds 161

while compounds 2–5 did not show any inhibitive effect on the growth of any of the
bacteria tested (data not shown).

3.3. Antilarval settlement activity

The CHCl3 extract of S. suberosa, petroleum ether and EtOAc extracts of S. gracillis led
to 0% larval settlement and 0% mortality at 100.0 mg mL1; subergorgic acid (1) and
pregn-4-ene-3, 20-dione (2) effectively inhibited larval settlement of both B. amphitrite
and B. neritina (table 2). The EC50 values of compounds 1 and 2 against B. amphitrite
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larvae were 1.2 mg mL1 and 16.7 mg mL1, respectively (figures 2 and 3), while the LC50
of both compounds exceeded 200 mg mL1. The EC50 values of compounds 1 and 2
against B. neritina larvae were 3.2 mg mL1 and 13.0 mg mL1, and LC50 values

Table 2. Antilarval settlement activities of compounds (1–3) and extracts (10–12) against Balanus amphitrite
and Bugula neritina.

Balanus amphitrite Bugula neritina

EC50 C50 Settlement Mortality EC50 LC50

Tested sample (mg ml1) (mg mL1) (%) (%) (mg mL1) (mg mL1)
1 1.2 >200 3.2 >200
2 16.7 >200 13.0 33.7
3 (50 mg mL1) 0 9.8
10 (100 mg mL1) 0 0
11 (100 mg mL1) 0 0
12 (100 mg mL1) 0 0

120
Batch 1
100 Batch 2
% Larval settlement

80

60

40

20

** ** **
0
Control 0.1 1 10 50 100
Concentration (mg mL−1)
Figure 2. Effect of subergorgic acid on the percent cyprid settlement of B. amphitrite. Data plotted are
means þ SD for four replicates. Data significantly different according to a one-way ANOVA followed by a
posthoc Dunnett’s test (p < 0.05) are indicated by an asterisc above the bars.
 162 S. H. Qi et al.

120

100 Batch 1
Batch 2

% Larval settlement 80

60

40
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20
** **
0
Control 0.1 1 10 50 100
Concentration (mg mL−1)
Figure 3. Effect of pregn-4-ene-3, 20-dione on the percent cyprid settlement of B. amphitrite. Data plotted
are means þ SD for four replicates. Data significantly different according to a one-way ANOVA followed by
a posthoc Dunnett’s test (p < 0.05) are indicated by an asterisc above the bars.

120

Batch 1
100
Batch 2
% Larval settlement

80

60

40

20

** **
0
Control 0.1 1 10 50 100 150 200
Concentration (mg mL−1)
Figure 4. Effect of subergorgic acid on the percent larval settlement of B. neritina. Data plotted are
means þ SD for four replicates. Data significantly different according to a one-way ANOVA followed by a
posthoc Dunnett’s test (p < 0.05) are indicated by an asterisc above the bars.

were >200 mg mL1 and 33.7 mg mL1, respectively (figures 4 and 5). Compound 3
inhibited larval settlement of both B. amphitrite and B. neritina and caused 9.8% larval
mortality at 50.0 mg mL1, but its EC50 and LC50 values were not obtained because
there were not sufficient pure compounds for the bioassay. Compounds 4, 5, 7, 9
showed no effect on larval settlement (data not shown).
 Antifouling and antibacterial compounds 163

120
Batch 1
100 Batch 2

% Larval settlement 80

60

40
Downloaded by [CAS Chinese Academy of Sciences] at 02:56 30 June 2011

20

0
Control1 1 5 15
Concentration (mg mL−1)

120

100 Batch 1
Batch 2
80
% Mortality

60

40

20
** * *
0
Control1 15 50 100 200
Concentration (mg mL−1)
Figure 5. Effect of pregn-4-ene-3,20-dione on the percent larval settlement and percent larval mortality of
B. neritina. Data plotted are means þ SD for four replicates. Data significantly different according to a one-
way ANOVA followed by a posthoc Dunnett’s test (p < 0.05) are indicated by an asterisc above the bars.

Overall, the EC50 values of compounds 1 and 2 were lower than the standard
requirement of an EC50 of 25 mg mL1 established by the US Navy program as an
efficacy level for natural anti-foulants, while their LC50 exceeded 200 mg mL1,
indicating that compounds 1 and 2 are potential nontoxic anti-fouling agents.

4. Discussion

In our study, we found that the CHCl3 extract of S. suberosa, the petroleum ether and
EtOAc extracts of S. gracillis inhibited larval settlement of B. amphitrite at
100.0 mg mL1 without killing larvae. Furthermore, these extracts inhibited the
growth of certain marine bacteria isolated from sponge surfaces or natural biofilm.
 164 S. H. Qi et al.

Bioassay-guided fractionation led to identification of three anti-fouling compounds

(subergorgic acid, progesterone, and 5-pregn-3, 20-dione) and five antibacterial
compounds (subergorgic acid, stigma-7, 22-dien-3, 5, 6-triol, 3,7,9-tri-Me-6,
8-purinediol, 1,2,3,4-Tetrahydro--carboline, and batyl alcohol). Our results confirmed
the findings made by Wilsanand et al. [32,33] that gorgonian coral extracts displayed
high incidence of attachment inhibitory activity, implying the presence of potent broad
spectrum antifouling compounds.
Previous studies on the chemical constituents of S. suberosa have led to the isolation
of several steroids [34,35], and sesquiterpenes [36–38]. Koh et al. [39] found that
S. suberosa from reefs in Singapore inhibited growth of several fungal species found on
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gorgonians in concentrations lower than their natural concentration. Subrahmanyam

et al. [40] also reported that subergorgic acid, N-hexadecanoyl-2-amino-1, 3-
dihydroxyoctadec-4-ene, and one pregnane mixture obtained from S. suberosa collected
from the Mandapam coast, Tamil Nadu, exhibited antibacterial and antifungal
activities. In fact, subergorgic acid was regarded as a near-perfect agent for chemical
self-protection available to Pacific corals, when it was first isolated from the South Sea
gorgonian S. suberosa in 1982; it was also considered an unusually powerful cardiotoxic
agent being capable of inhibiting neuromuscular transmission at levels below
0.20 mg mL1 [26]. In addition, subergorgic acid had anticholinesterase activity [41],
activity against ‘‘Soman’’ toxicity in mice [42], and acted on cholinesterase with a
reversible inhibition. Because of its novel structure and potent bioactivities, there have
been many studies about its total synthesis [43,44]. In this study, we observed that when
larva was placed into a dish containing subergorgic acid solution, the larvae were
paralyzed immediately. This reaction may be caused by the anti-cholinesterase activity
of subergorgic acid. The surprising relationship between subergorgic acid’s concentra-
tion and settlement inhibition against B. neritina (figure 4) could be caused by its
reversible inhibition on cholinesterase. Overall, our findings present the first laboratory
evidence of the inhibitive activity of subergorgic acid on larval settlement of major
fouling organisms as well as against a broad spectrum of marine bacterial species,
extending the list of potential natural functions for this compound.
In this study, we found potent antilarval activity of progesterone, a hormone in
mammalia against B. amphitrite and B. neritina. The mode-of-action of this
compound against marine larvae remains to be explored. On the other hand, 3,7,9-
Tri-Me-6, 8-purinediol was first found in terrestrial plants, such as coffee and tea. It
was interesting to find this compound in gorgonian coral and to see its weak
antibacterial activity against V. harveyi (table 1). 1,2,3,4-Tetrahydro--carboline was,
for the first time, isolated from a natural source and showed some antibacterial
activity in this study. A series of 1-substituted 1,2,3,4-tetrahydro--carboline
derivatives were synthesized for the discovery of natural -carboline metabolites as
potent anti-tumor and antiviral agents [45, 46]. Also, batyl alcohol was first obtained
from gorgonian coral in 1958 and showed potent erythropoietic simulatory activity
and hemopoietic activity [47]. It has been used as a medicine for increasing human
leucocyte in clinic. In this study, we found for the first time its potent activity against
five marine bacterial species. In conclusion, the gorgonian coral S. suberosa and
S. gracillis produce diverse compounds that have a range of bioactivities, including
anti-fouling activities (as shown in this study), which deserve further exploration as
nontoxic natural anti-fouling compounds.
 Antifouling and antibacterial compounds 165

Acknowledgements

This work was supported by grants from National Science Foundation of China
(20502027), a grant from China Ocean Mineral Resources Research & Development
Association, CAS-Hong Kong Croucher Foundation (CAS-CF04/05.SC01), and the
Knowledge Innovation Program of Chinese Academy of Science (KZCX3–SW–216).

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