148 E
.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
chromatography on silica gel see Coll and Bowden, 1986. Briefly, the extract was dissolved in a 1:1 mixture of methanol and dichloromethane. A small amount of silica
gel was added to the solution and the solvents were removed using a rotary-evaporator. The dry, even mixture of the extract and silica gel was then loaded onto a silica gel
column for fractionation. Solvent mixtures were used to elute six fractions under vacuum, starting at 5 and finishing with 25 methanol in dichloromethane. The flash
column fraction with the highest antimicrobial activity was prepared for HPLC by loading it onto a C18 Sep Pak Waters Millipore Corporation and eluting it with
solvents of decreasing polarity. The first fraction was eluted with 4:6 methanol distilled water, the second with methanol, the third with 1:1 methanol ethyl acetate, the fourth
with ethyl acetate and the last with hexane. The first two fractions contained antimicrobial activity and were combined for reversed-phase HPLC no antimicrobial
activity was detected in the third and fourth fractions and nothing was eluted with hexane.
Reverse-phase HPLC was carried out on an Alltech Econosil C18 column 10 mm; 250 mm long and 10 mm internal diameter with an isocratic solvent regime nine parts
methanol and one part 5 ammonium acetate in distilled water at a flow-rate of 1.5 ml
21
min . Four major HPLC fractions were collected at the following retention times: 8,
9.5, 10.5 and 13 min. Ammonium acetate was removed from the fractions before they were tested for antimicrobial activity. The fractions were further purified under the same
HPLC conditions before they were characterized by melting point analysis, NMR, UV and mass spectrometry.
3. Results
3.1. Natural concentration of Tubastraea faulkneri extracts Tubastraea faulkneri specimens 15–20 colonies of varying sizes with a total wet
weight of 247.47 g weighed 152.94 g after freeze-drying. Freeze-dried dichloromethane, methanol and distilled water extracts weighed 0.51, 5.40, and 2.23 g, respectively.
Subtracting the weight of the dried skeleton alone gave a wet weight for tissue of 108.63 g 14.1 g dry weight. Therefore the natural concentration of the methanol extract was
21 21
49.7 mg g wet tissue or 383.0 mg g
dry tissue. The natural concentration of the
21 21
distilled water extract was 20.5 mg g wet tissue or 158.2 mg g
dry tissue and that of
21 21
the dichloromethane extract was 4.7 mg g wet tissue or 36.2 mg g
dry tissue. 3.2. Bioactivity of extracts isolated from Tubastraea faulkneri
Bioassay results of the three crude extracts against seven species of microbes Vibrio alginolyticus, V
. harveyi, V. parahaemolyticus, Photobacterium damsela, Alteromonas rubra, Staphylococcus aureus, Synechococcus sp. showed that the methanol extract had
the most bioactivity Table 1. The dichloromethane extract inhibited the growth of
E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
149
Synechococcus sp., but much less intensely than the methanol extract. No inhibitory activity was recorded for the distilled water extract.
3.3. Toxicity of Tubastraea faulkneri extract to scleractinian larvae Tubastraea faulkneri larvae alone among the 12 test species showed no visible toxic
effects from exposure to the extract of T . faulkneri. No conspecific larvae died in any of
the bioassays. In contrast, the larvae of all 11 other species suffered higher mortality at all concentrations of T
. faulkneri extract when compared with solvent controls Figs. 1–3. Mortality of planulae after a 4-h exposure to solvent controls ranged from 0 in
four species Platygyra sinensis, P . daedalea, Goniastrea aspera, Oxypora lacera to
72 in Montipora digitata. There was significant interaction between treatments presence or absence of extract
and concentrations for six planula species Acropora millepora, A . tenuis, Goniastrea
aspera, Platygyra sinensis, P . daedalea, Oxypora lacera; Tables 2–4, meaning that the
effect of increasing concentration differed between larvae treated with solvent plus Tubastraea faulkneri extract and those treated with solvent alone. Acropora millepora
and A . tenuis showed only linear interaction trends implying that response curves for
planulae treated with extract diverged in a consistent manner from response curves for those treated only with solvent Fig. 2, Table 3. For P
. sinensis, G. aspera and O. lacera, the response curves for the treatments diverged, but not in a linear fashion Figs.
1–3, Tables 2–4. In the case of P . sinensis, the nonlinearity was due to total mortality
occurring at relatively low concentrations of extract, so while the mortality increased with increasing concentration of solvent alone, the mortality of larvae treated with T
. faulkneri extract could not increase above 100. For G
. aspera and O. lacera, the nonlinearity resulted from the sharp rise in mortality with increasing concentrations of
extract while mortality remained fairly low and constant as the concentration of solvent alone increased. Platygyra daedalea shows a cubic trend though the consistent decline in
21
mortality at 100 mg ml has no simple explanation Fig. 3, Table 4. There were no
significant trends in interactions in any of the species with non-significant overall interaction terms.
Acropora formosa, A . pulchra, Montipora digitata, Fungia fungites, Favia pallida
and Oxypora lacera at high concentrations all had significantly higher mortality in the presence of Tubastraea faulkneri extract Tables 2–4, Figs. 1–3. Included in this group
are the only two species, M . digitata and O. lacera tested at high concentrations
showing unequal variances among treatment groups; these are associated with main effect ‘Treatment’. The inequality comes about because all the larvae died when exposed
to extract of T . faulkneri at experimental concentrations Figs. 2, 3. As a result, these
treatment groups had zero variance in mortality while control groups showed less mortality and some variance. The differences in mortality are highly significant by both
ANOVA and the randomization tests Tables 3, 4. The effect of concentration was also statistically significant for two of these species:
planulae of Fungia fungites and Favia pallida showed linear increases in mortality with increasing dosage of extract and of solvent Fig. 2, Table 3. This implies that
150 E
.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
Fig. 2. Toxic effects of Tubastraea faulkneri extract on seven species of scleractinian planulae. Error bars are 61 sample standard error [solid symbols 5 proportion planulae dead after 4 h in T. faulkneri extract; open
symbols 5 proportion planulae dead after 4 h in solvent controls].
E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
151
Fig. 3. Toxic effects of Tubastraea faulkneri extract on planulae of Oxypora lacera and Platygyra daedalea. The bioassays were initially conducted at three extract concentrations and repeated at a later date at seven
extract concentrations see Materials and methods. Error bars are 61 sample standard error [solid symbols 5 proportion planulae dead after 4 h in T
. faulkneri extract; open symbols 5 proportion planulae dead after 4 h in solvent controls].
experimental concentrations of the solvent were toxic to planulae of these two species, but in each case the presence of Tubastraea faulkneri extract caused significant
additional mortality Fig. 2, Table 3.
3.4. Isolation and purification of active components Flash column separation of the methanol extract yielded six fractions with the most
activity in fraction 3. Subsequent separation of this fraction by reversed-phase HPLC produced four major fractions that were yellow in color and not soluble in water. These
HPLC fractions showed some antimicrobial activity when tested at the approximate proportions in which they occurred in the crude extract Table 5. Activity was not
restricted to a single fraction; all the fractions were active against Synechococcus sp. and two were active against Staphylococcus aureus. After further purification by HPLC,
spectral and melting point analyses of the fractions identified them as aplysinopsin, 6-bromoaplysinopsin, 6-bromo-29-de-N-methylaplysinopsin and its dimer Table 6.
These four compounds accounted for 72 of the methanol extract.
152 E
.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160 Table 3
a
Toxicity of Tubastraea faulkneri extract to seven species of scleractinian planulae Planula species
Source df
S.S. M.S.
F P
RP Acropora millepora
Treatment 1
208.0 208.0
189.1 , 0.001
n a Concentration
2 20.6
10.3 9.4
0.001 Treat. 3 Conc.
2 11.3
5.6 5.1
0.014 Trend: Linear
1 10.1
10.1 9.1
0.006 Quadratic
1 1.2
1.2 1.1
0.305 Error
24 26.4
1.1 Acropora tenuis
Treatment 1
76.8 76.8
38.1 , 0.001
n a Concentration
2 122.6
61.3 30.4
, 0.001 Treat. 3 Conc.
2 30.2
15.1 7.5
0.003 Trend: Linear
1 23.3
23.3 11.6
0.002 Quadratic
1 6.9
6.9 3.4
0.078 Error
24 48.4
2.0 Goniastrea aspera
Treatment 1
229.6 229.6
265.0 , 0.001
n a Concentration
2 106.1
53.0 61.2
, 0.001 Treat. 3 Conc.
2 88.5
44.2 51.0
, 0.001 Trend: Linear
1 83.3
83.3 95.7
0.000 Quadratic
1 5.2
5.2 6.0
0.022 Error
24 20.8
0.9 Acropora pulchra
Treatment 1
83.3 83.3
42.4 , 0.001
n a Concentration
2 7.8
3.9 2.0
0.160 Treat. 3 Conc.
2 6.9
3.4 1.8
0.196 Error
24 47.2
2.0 Montipora digitata
Treatment 1
100.8 100.8
28.4 , 0.001
, 0.001 Concentration
2 4.1
2.0 0.6
0.572 0.752
Treat. 3 Conc. 2
4.1 2.0
0.6 0.572
0.776 Error
24 85.2
3.6 Fungia fungites
Treatment 1
240.8 240.8
62.3 , 0.001
n a Concentration
2 51.7
25.8 6.7
0.005 Trend: Linear
1 50.1
50.1 12.9
0.001 Quadratic
1 1.6
1.6 0.4
0.525 Treat. 3 Conc.
2 6.1
3.0 0.8
0.468 Error
24 92.8
3.9 Favia pallida
Treatment 1
563.3 563.3
1024.2 , 0.001
n a Concentration
2 5.1
2.5 4.6
0.020 Trend: Linear
1 4.6
4.6 8.4
0.008 Quadratic
1 0.5
0.5 0.8
0.371 Treat. 3 Conc.
2 1.9
0.9 1.7
0.205 Error
24 13.2
0.6
a
Factor ‘Treatment’ refers to presence or absence of extract in test solution. Factor ‘Concentration’ refers to concentrations of extract and corresponding solvent controls. Shows significant P values , 0.05. Variances
were homogeneous among treatment groups for interaction and main effects for all species except Montipora digitata. RP values are probabilities from randomization tests; these supported the ANOVA results. Trends
refer to partitioning of the significant concentration or interaction terms using polynomial contrasts.
E .G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160
153 Table 4
a
Toxicity of Tubastraea faulkneri extract to planulae of Oxypora lacera and Platygyra daedalea Planula species
Source df
S.S. M.S.
F P
RP Oxypora lacera
Treatment 1
440.8 440.8
389.0 , 0.001
, 0.001 Concentration
2 2.1
1.0 0.9
0.415 0.933
Treat. 3 Conc. 2
0.9 0.4
0.4 0.686
0.973 Error
24 27.2
1.1 Treatment
1 1251.7
1251.7 3650.7
, 0.001 n a
Concentration 6
19.5 3.3
9.5 , 0.001
Treat. 3 Conc. 6
21.1 3.5
10.3 , 0.001
Trend: Linear 1
3.9 3.9
11.3 0.001
Quadratic 1
14.3 14.3
42.0 , 0.001
Other 4
3.0 0.8
2.2 0.079
Error 56
19.2 0.3
Platygyra daedalea Treatment
1 529.2
529.2 588.0
, 0.001 n a
Concentration 2
11.5 5.7
6.4 0.006
Treat. 3 Conc. 2
10.4 5.2
5.8 0.009
Trend: Linear 1
0.1 0.1
0.1 0.760
Quadratic 1
10.3 10.3
11.5 0.002
Error 24
21.6 0.9
Treatment 1
1010.8 1010.8
879.0 , 0.001
n a Concentration
6 51.1
8.5 7.4
, 0.001 Treat. 3 Conc.
6 41.6
6.9 6.0
, 0.001 Trend: Linear
1 0.3
0.3 0.3
0.614 Quadratic
1 2.0
2.0 1.7
0.195 Other
4 39.3
9.8 8.5
0.000 Error
56 64.4
1.2
a
Factor ‘Treatment’ refers to presence or absence of extract in test solution. Factor ‘Concentration’ refers to concentrations of extract and corresponding solvent controls. The bioassays were repeated at lower extract
concentrations see Materials and methods. Shows significant P values , 0.05. Variances were homoge- neous among treatment groups for interaction and main effects, except for Oxypora lacera when tested at
higher concentrations. RP values are probabilities from randomization tests; these supported the ANOVA results. Trends refer to partitioning of the significant interaction term using polynomial contrasts.
Table 5 Antimicrobial bioassay of HPLC fractions of the methanol-soluble extract from Tubastraea faulkneri, tested at
a
concentrations proportionate to levels found in 500 mg of extract Bioassay organism
Inhibition zone of fractions mm HPLC 3
HPLC 4 HPLC 5
HPLC 6
21 21
21 21
[96 mg disc ]
[107 mg disc ]
[146 mg disc ]
[11 mg disc ]
Vibrio alginolyticus Vibrio harveyi
Not tested Photobacterium damsela
Alteromonas rubra Staphylococcus aureus
1.0 1.5
Synechococcus sp. 4.5
15.0 8.5
6.0
a
N 5 5 for each treatment combination. Solvent controls had no activity. Bioassay concentrations of each fractions are indicated in square brackets.
154 E
.G.L. Koh, H. Sweatman J. Exp. Mar. Biol. Ecol. 251 2000 141 –160 Table 6
Characterization of bioactive chemicals isolated from Tubastraea faulkneri HPLC 3
HPLC 4 HPLC 5
HPLC 6 Chemical identity
Dimer of Aplysinopsin
6-Bromo-29- 6-Bromo-
6-bromo-29- de-N-methyl-
aplysinopsin de-N-methyl-
aplysinopsin aplysinopsin
Retention time min 8.0
9.5 10.5
13.0 UV max. nm
378.8 370.0
383.2 379.2
Melting point 8C 246
221–224 238–240
254 Molecular weight
636 254
318 332
Concentration in wet 9.5
10.6 14.5
1.1
21
tissue mg g Concentration in dry
73.5 82.0
111.8 8.4
21
tissue mg g
4. Discussion
