You are on page 1of 7

Phytochemistry 58 (2001) 517523

www.elsevier.com/locate/phytochem

Halogenated metabolites with antibacterial activity from


the Okinawan Laurencia species
Charles Santhanaraju Vairappana,1, Minoru Suzukia,*, Tsuyoshi Abeb, Michio Masudac
a

Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan
b
The Hokkaido University Museum, Sapporo 060-0810, Japan
c
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
Received 4 December 2000; received in revised form 19 April 2001

Abstract
The chemical compositions of ve species of the red algal genus Laurencia from coastal waters of Okinawa Prefecture, Japan, have
been investigated. A halogenated C15 acetogenin, (12E)-lembyne-A, was isolated from L. mariannensis, and a halogenated sesquiterpene,
(6R,9R,10S)-10-bromo-9-hydroxy-chamigra-2,7(14)-diene, was rst found from L. majuscula as a naturally occurring compound.
Laurencia nidica yielded previously known laurinterol and isolaurinterol. Samples of L. cartilaginea and L. concreta aorded no
halogenated metabolites. The structures of these halogenated metabolites as well as their antibacterial activity against some marine
bacteria are reported. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Laurencia; Rhodomelaceae; Red alga; Sesquiterpene; C15 acetogenin; Halogenated compound; Chemotaxonomy; Antibacterial activity

1. Introduction
The red algal genus Laurencia is unique due to its
ability to produce a wide variety of halogenated secondary metabolites with diverse structural features depending on the species and localities. Although the roles of
these halogenated compounds are still a matter of speculation, their importance as chemical defense substances
against marine herbivores is undeniable (Hay et al.,
1987; Kurata et al., 1998).
Some halogenated metabolites have been shown to
possess antibacterial activities against terrestrial bacteria
(Waraszkiewicz and Erickson, 1974; Wratten and Faulkner, 1977; Carter et al., 1978; Caccamese et al., 1980;
Konig and Wright, 1997a). More recently, we reported
on the antibacterial potential of halogenated compounds isolated from the Malaysian Laurencia against
marine bacteria from the algal habitat (Vairappan et al.,
2001), suggesting the possible role played by these
halogenated compounds as the seaweeds chemical
defense substances.
* Corresponding author. Tel.: +81-11-706-2272; fax: +81-11-7064862.
E-mail address: misu@ees.hokudai.ac.jp (M. Suzuki).
1
Present address: Borneo Marine Research Institute, University
Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia.

As part of our continuing study on the chemical compositions of Laurencia species from the Okinawan
waters, we harvested ve species of Laurencia; L. mariannensis Yamada, L. majuscula (Harvey) Lucas, L. nidica J. Agardh, L. cartilaginea Yamada, and L. concreta
Cribb from four dierent sites. Laurencia mariannensis
contained a new C15 acetogenin, (12E)-lembyne-A (1),
along with 2-bromo-3-chloro-5-acetoxy-chamigra-7(14),
9-dien-8-one (2) that was previously isolated from the
Okinawan sea hare Aplysia dactylomela (Sakai et al.,
1986) and from the Okinawan L. majuscula collected at
Taketomi (Masuda et al., 1997). Laurencia majuscula
contained a brominated sesquiterpene, (6R,9R,10S)-10bromo-9-hydroxy-chamigra-2,7(14)-diene (3), together
with previously known (Z)-10,15-dibromo-9-hydroxychamigra-1,3(15),7(14)-triene (4) and (E)-10,15-dibromo9-hydroxy-chamigra-1,3(15),7(14)-triene (5) from this
species collected at Kochi Prefecture, Japan (Suzuki and
Kurosawa, 1978; Suzuki et al., 1979). Metabolite 3 has
not been previously found as a natural source. Furthermore, L. nidica produced laurinterol (6) and isolaurinterol (7), characteristic metabolites of the Japanese
L. okamurae Yamada (Suzuki and Kurosawa, 1979),
while L. cartilaginea and L. concreta produced no halogenated secondary metabolites.
We report herein the chemical compositions of these
Laurencia species, structures of halogenated compounds,

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00260-6

518

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

and their antibacterial activity against marine bacteria


isolated from the algal habitat.

2. Results and discussion


2.1. Laurencia mariannensis
A sample collected from Teruma, Yonashiro, was
extracted with methanol. The methanol extract was fractionated by CC over silica gel with a stepped gradient
(hexane and EtOAc). The fraction eluted with hexane
EtOAc (9:1) was further subjected to prep. TLC with
hexaneEtOAc (3:1) to give 2-bromo-3-chloro-5-acetoxy-chamigra-7(14),9-dien-8-one (2) (Sakai et al., 1986;
Masuda et al., 1997) in 14% yield based on the weight
of the extracts. The fraction eluted with hexaneEtOAc
(4:1) was also separated using prep. TLC with hexane
EtOAc (3:1) to give compound 1 (6%).

Compound 1, oil, 24
D +42 (CHCl3), was assigned the
molecular formula C15H16BrClO2 (HRFDMS). Its IR
spectrum revealed absorptions due to a terminal acetylenic group (vmax 3310 cm1), a vinyl ether stretching
(vmax 1680 cm1), and CO bands (vmax 1110 and 1050
cm1). In addition, since there were no hydroxyl or
carbonyl absorptions, both oxygens were suggested to
be involved in ether links. The presence of a terminal
conjugated enyne moiety, which is frequently encountered in Laurencia C15 acetogenins, was evident from
the 1H NMR spectrum (Table 1) [H 3.31 (1H, br s),
5.58 (1H, dd, J=10.7, 1.5 Hz) and 6.06 (1H, dd,
J=10.7, 10.7 Hz)]. Interpretation of the 1H1H COSY
spectrum showed the presence of a partial structure 1a
Table 1
13
C NMR (100 MHz, DEPT), 1H NMR (400 MHz), and HMBC
spectral dataa for 1
Cb

13
C
()

1
H
()

Multiplicity,
J (Hz)

1
2
3
4
5
6
7
8

78.7
85.8
110.9
142.2
54.9
54.2
77.9
36.8

3.31

br s

9
10
11
12
13
14

81.5
84.2
45.2

5.58
6.06
4.72
2.74
4.29
1.92
1.71
4.67
5.13
3.46

dd, J=10.7, 1.5


dd, J=10.7, 10.7
dd, J=10.7, 10.7
ddd, J=10.7, 9.3, 4.4
dd, J=4.4, 4.4
d, J=13.6
m
dd, J=6.8, 4.9
dd, J=4.9, 4.9
dd, J=9.3, 4.9

105.8
28.1

15

13.8

2.64
2.38
1.10

dq, J=14.6, 7.3


dq, J=14.6, 7.3
dd, J=7.3, 7.3

a
b

HMBC
correlations

H-1, H-4,
H-3, H-5
H-3, H-4,
H-4, H-5,
H-6, H-9,
H-6, H-7,

H-5
H-7
H-7, H-10, H-11
H-10, H-11
H-10

H-7, H-10, H-11


H-7, H-9, H-11
H-5, H-6, H-7, H-9, H-10
H2-14, H3-15
H3-15
H2-14

Measured in chloroform-d1.
Assignments were made with the aid of the HSQC spectrum.

Fig. 1. Partial and planar structures for 1.

(Fig. 1) together with an isolated ethyl group. The Jvalue (10.7 Hz) between H-3 and H-4 as well as the
chemical shift value (H 3.31) of the acetylenic proton
indicated the geometry of the double bond at C-3 to be
Z (Suzuki and Kurosawa, 1987).
The 13C NMR spectrum (Table 1) revealed the presence of 14 carbon atoms, 12 of which were hydrogenbearing carbons. Among 14 carbon atoms, three oxygen-bearing carbons were demonstrated by the signals
at C 77.9, 81.5 and 84.2 and further attributed to C-7,
C-9 and C-10, respectively, by HSQC spectrum. Furthermore, based on the chemical shift values of H-5 (H
4.72) and C-5 (C 54.9), a halogen atom was deduced to
be attached to C-5 position. The planar formula for
compound 1 could be assigned by the HMBC spectrum
whose results are summarized in Table 1. In the HMBC
spectrum, cross peaks of C-7/H-10 and C-10/H-7
established the ether ring closure between C-7 and C-10.
In addition, a cross peak between the terminal methyl
group and the vinyl carbon at C 105.8 indicated that
the ethyl group was attached to the tetrasubstituted
double bond. The chemical shift value (C 105.8) of this
vinyl carbon atom suggested that another substituent is
a halogen atom (Suzuki et al., 1983). Therefore, this
double bond had to be inserted between C-9 and C-11 to
give a planar formula 1b or 1c. Facile loss of a chlorine
atom on the FDMS is evident from the fragment ion of m/
z 309, 307 [M-Cl]+ which strongly suggested the presence
of a vinyl bromide moiety in 1. Hence the chlorine atom
was linked to C-5 and the bromine atom to C-13.
The 1H and 13C NMR spectral data of 1 were very
similar to those of lembyne-A (10 ), which has recently
been isolated from the Malaysian Laurencia sp. (Vairappan et al., 2001). The chemical shifts and coupling
constants of the protons from C-1 to C-10 in the 1H NMR
spectrum of 1 are almost identical to those of 10 . However,

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

distinct dierences are observed in the chemical shift


values of H-11 and H2-14. The low-eld chemical shift
of the H-11 in 1 is due to the deshielding being caused
by the bromine atom at C-13 that is situated close to the
H-11. Furthermore, the large magnetic nonequivalence of
the H2-14 in 1 is attributed to the electronic inuence of
the oxygen atom that is cis to the ethyl group. Thus the
stereochemistry of the vinyl ether moiety in 1 is established as E-conguration. In consequence, the structure of
compound 1 must be represented by formula 1, (12E)lembyne-A, including the same relative stereochemistry,
5S*, 6R*, 7R*, 9R*, 10R*, and 11S*, as lembyne-A (10 ).
(12E)-Lembyne-A (1) and lembyne-A (10 ) have structures very similar to cis-maneonene-C (100 ) and (12Z)cis-maneonene-C (1000 ), respectively, the latter of which
was obtained from cis-maneonene-C by treatment with
p-toluenesulfonic acid (Waraszkiewicz et al., 1978).
However, there are some obvious dierences between our
1
H and 13C NMR spectral data (in C6D6) and the published data (Waraszkiewicz et al., 1978) along with their
optical rotations (1: [a]D +42 ; 10 : [a]D +198 , 1b00 : [a]D
+336 , 1000 : [a]D +137  C). Thus cis-maneonene-C and
(12Z)-cis-maneonene-C may be a stereoisomer at C-5 of
(12E)-lembyne-A and lembyne-A, respectively.

519

2.2. Laurencia majuscula


The methanol extract of a sample collected from
Yagachi, Nago, was subjected to a combination of column and thick-layer chromatography to give 10-bromo9-hydroxy-chamigra-2,7(14)-diene (3) (9%) along with
previously known (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (4) (3%) and (E)-10,15-dibromo9-hydroxy-chamigra-1,3(15),7(14)-triene (5) (4%), isolated from this species which had been collected at
Kochi Prefecture, Japan (Suzuki and Kurosawa, 1978;
Suzuki et al., 1979).

Compound 3, oil, C15H23OBr, 25
D 110 (CHCl3),
was readily assigned the planar structure by its 1D and
2D (1H1H COSY, HSQC, and HMBC) NMR spectra.
The relative conguration was determined by the
NOESY spectrum. A NOE between H-10 and Hb-5
showed that the H-10 adopts an axial conguration and
hence the bromine atom is equatorial. Since based on
the J-value (J9,10=2.9 Hz), the vicinal bromine and
hydroxyl groups are in cis-conguration as in the case
of compounds 4 and 5, the C-9 hydroxyl group is axial.
Furthermore, NOEs between Ha-14/Ha-1 and H3-13
(one of gem-dimethyl)/Ha-4 also conrmed the stereochemistry of the spiro carbon at C-6. Thus the relative
stereochemistry of 3 would be represented by formula
3a as shown in Fig. 2.
A literature survey indicated that the 1H and 13C
NMR spectral data of 3 were almost identical with
those of deschloroelatol (30 ), which has previously
been isolated from Laurencia obtusa (Kennedy et al.,
1988) and L. rigida (Konig and Wright, 1997b). However, judging from the signs of the optical rotations,
compound 3 must be an enantiomer of deschloroelatol.
Compound 3 had previously been obtained from
obtusol as the partially dehalogenated compound by
treatment with lithium aluminum hydride in ether
(Gonzalez et al., 1976). Co-occurrence of compound 3
with halochamigrenes 4 and 5 in the same alga also
suggested that the absolute congurations of chiral
centers at C-6, C-9, and C-10 are the same as those of 4
and 5.

Fig. 2. Stereochemistry of 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (3).

520

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

sample of L. cartilaginea collected at Sakurajima,


Kagoshima Prefecture in Japan, close to the type locality,
Moji, Fukuoka Prefecture, also showed the absence of
halogenated compounds in its extract. Both Laurencia
species have no corps en cerise (our unpublished
observations). Corps en cerise are considered to be
the sites of synthesis and/or storage for the halogenated
secondary metabolites of Laurencia (Young et al.,
1980). Although the Hawaiian L. cartilaginea was
reported to produce halogenated chamigrane-type sesquiterpenes (Juagdan et al., 1997), the identication of
their material seems to be questionable.
2.5. Antibacterial activity

Accordingly, the structure of compound 3 should be


(6R,9R,10S) - 10 - bromo - 9 - hydroxy - chamigra - 2,7(14) diene, which has not previously been found as a naturally
occurring compound.
The dierence of the chemical compositions in the
samples from Yagachi, Nago (present study) and Taketomi island (Masuda et al., 1997) suggests that chemical
races (Abe et al., 1999) are also present in Laurencia
majuscula.

The antibacterial activities of the isolated halogenated


metabolites of Okinawan Laurencia were tested against
eight species of marine bacteria. These were Alcaligenes
aquamarinus, Alteromonas sp., Azomonas agilis, Azotobacter beijerinckii, Erwinia amylovora, Escherichia coli,
Halobacterium sp., and Halococcus sp. All these bacteria
were Gram-negative strains. The results of the paper
disc diusion assays are shown in Table 2. Prominent
antibacterial activities were seen in compounds 1, 3, 6
and 7, while 4 and 5 only showed very marginal activity.
Compound 2 was inactive towards the bacteria tested.
Compound 1 showed activity against Alcaligenes aquamarinus, Azomonas agilis, Erwinia amylovora, and
Escherichia coli. Their minimum inhibitory concentration (MIC) values are in the range of 2030 mg/disc as
shown in Table 3. Compound 3 showed antibacterial
activity against ve species of bacteria, Alcaligenes aquamarinus, Azomonas agilis, Azotobacter beijerinckii, Erwinia amylovora, and Escherichia coli. Its MIC values were
in the range of 1030 mg/disc (Table 3). Compounds 6
and 7 showed better antibacterial activity than compounds 1 and 3. Their antibacterial activities and respective MIC values are shown in Tables 2 and 3, respectively.

2.3. Laurencia nidica


The methanol extract of a sample collected from
Toyohara, Nago, gave laurinterol (6) (22%) and isolaurinterol (7) (4%) (Suzuki and Kurosawa, 1979). The
chemical composition of Laurencia nidica collected at
two sites (Teruma and Toyohara) is dierent from that
of a sample harvested from Goza, Mie Prefecture, Japan
(Shizuri et al., 1984); it is, however, rather similar to that
of a Hawaiian sample (a clumpy pink variety) from Oahu
island (Waraszkiewicz and Erickson, 1974, 1975).
2.4. Laurencia cartilaginea and Laurencia concreta
No halogenated secondary metabolites were found in
both extracts of Laurencia cartilaginea and L. concreta.
As reported in a previous paper (Suzuki et al., 1987), a

Table 2
Antibacterial activity of the halogenated compounds against marine
bacteria isolated from algal habitats in the Japanese coastal waters
Test bacteria

Alcaligenes aquamarinus
Alteromonas sp.
Azomonas agilis
Azobacter beijerinckii
Erwinia amylovora
Escherichia coli
Halobacterium sp.
Halococcus sp.
a

Compoundsa
1

++

++

++
++

++

++
++
++
++

++
+++
++
+++
++

+++
+++

+++
+++

Inhibition zone diameter; +++: 1924 mm, ++: 1218 mm, +:


712 mm, : no inhibition. Concentration: 90 mg/disc.

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523


Table 3
Minimal inhibitory concentration (MICa) of the halogenated compounds against marine bacteria isolated from algal habitats in the
Japanese coastal waters
Test bacteria

Alcaligenes aquamarinus
Alteromonas sp.
Azomonas agilis
Azobacter beijerinckii
Erwinia amylovora
Escherichia coli
Halobacterium sp.
Halococcus sp.
a

Compoundsa
1

20

30

30
30

20

20
15
15
10

5
5
15
5
5

10
5

5
10

Concentration: mg/disc. : no inhibition.

521

3.3. Extraction and separation of Laurencia mariannensis


The partially dried alga (4 g) was soaked in methanol
for one week. The MeOH soln was concentrated in vacuo
and partitioned between Et2O and H2O. The Et2O soln.
was washed with water, dried over anhydrous Na2SO4,
and evaporated to leave a dark green oil (65 mg). The
extract was fractionated by Si gel column chromatography with a stepped gradient (hexane and EtOAc).
The fraction eluted with hexaneEtOAc (9:1) was further submitted to prep. TLC with hexaneEtOAc (3:1)
to give 2-bromo-3-chloro-5-acetoxy-chamigra-7(14),9dien-8-one (2) (14%). Further separation of the fraction
eluated with hexaneEtOAc (4:1) under the abovementioned TLC yielded compound 1 (6%).
3.4. Compound 1

The possibility that these halogenated compounds are


involved in protecting these Laurencia species against
bacterial intrusion is real when we look at the distribution
of these compounds. Basically, halogenated compounds
are reported to be contained in corps en cercise (Young
et al., 1980), an unusually swollen refractile inclusion,
that are located in the outer cortical layer and trichoblast cells of Laurencia. Corps en cercise are absent in
the inner cortical layers, and their specic distribution
pattern could be suggestive of their importance as barricades to withhold any bacterial intrusion.

3. Experimental


1
Oil; 24
D +42 (CHCl3; c 0.02); IR vmax (neat) cm :
1
13
3310, 1680, 1110, 1050, 930, 880; H and C NMR
spectra, Table 1; 1H NMR spectrum (400 MHz, C6D6),
 1.21 (3H, dd, J=7.3, 7.3 Hz, H3-15), 1.28 (1H, m,
Ha-8), 1.78 (1H, d, J=13.6 Hz, Hb-8), 2.41 (1H, ddd,
J=10.7, 9.3, 4.4 Hz, H-6), 2.50 (1H, dq, J=14.6, 7.3 Hz,
Ha-14), 2.71 (1H, dd, J=2.4, 0.9 Hz, H-1), 2.84 (1H, dq,
J=14.6, 7.3 Hz, Hb-14), 3.39 (1H, dd, J=9.3, 4.9 Hz,
H-11), 3.85 (1H, dd, J=4.9, 4.4 Hz, H-7), 4.18 (1H, dd,
J=6.8, 4.9 Hz, H-9), 4.52 (1H, dd, J=4.9, 4.9 Hz, H10), 4.86 (1H, dd, J=10.7, 10.7 Hz, H-5), 5.02 (1H, dd,
J=10.7, 2.4 Hz, H-3), 5.43 (1H, ddd, J=10.7, 10.7, 0.9
Hz, H-4); LR-FDMS m/z (rel. int.): 346, 344, 342
(30:90:76) [M]+, 309, 307 (11:10) [MCl]+; HRFDMS
m/z: 342.0013. Calc. for C15H1679Br35ClO2, 342.0022 [M].

3.1. General
3.5. Extraction and separation of Laurencia majuscula
1

H (400 MHz) and 13C NMR (100 MHz); CDCl3 or


C6D6, TMS as int. standard (coupling constant, J in Hz);
low and high resolution MS: 70 eV; optical rotations:
CHCl3; CC: silica gel (Merck, Kieselgel 60, 70230 mesh);
prep. TLC: silica gel 60 F254 (Merck). All known metabolites were identied by comparison of the spectral data
with those of the authentic specimens. Yields are based
on the weights of the extracts.
3.2. Collection
Five Laurencia samples were collected from four sites in
Okinawa Prefecture, Japan: L. mariannensis Yamada at
Teruma, Yonashiro, on 4 June 1997; L. majuscula (Harvey) Lucas at Yagachi, Nago, on 6 June 1997; L. nidica
J. Agardh at Teruma,Yonashiro, on 4 June 1997, and
Toyohara, Nago, on 5 June, 1997; L. cartilaginea Yamada
at Yagachi, Nago, on 6 June 1997; L. concreta Cribb at
Hedomisaki, Kunigami, on 6 June 1997. The voucher
specimens are deposited in the Herbarium (SAP) of the
Graduate School of Science, Hokkaido University.

Extraction of partially dried alga (8 g) was carried out


by conventional methods described above to yield a
brownish green extract (95 mg). A combination of column
and thick-layer chromatography of the MeOH extract
gave three halochamigrenes, 10-bromo-9-hydroxychamigra-2,7(14)-diene (3) (9%), (Z)-10,15-dibromo-9hydroxy-chamigra-1,3(15),7(14)-triene (4) (3%), and
(E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)triene (5) (4%).
3.6. 10-Bromo-9-hydroxy-chamigra-2,7(14)-diene (3)

1
Oil; 25
D 110 (CHCl3; c 0.20); IR vmax (neat) cm :
3550, 3450, 1640, 1395, 1365, 1220, 1200, 1180, 1085,
1070, 1035, 895, 830, 810; 1H NMR (400 MHz, CDCl3),
 1.10 (6H, s, H3-12 and H313), 1.50 (3H, br s, H3-15),
1.67 (1H, m, Hb-4), 1.75 (1H, m, Hb-5), 1.80 (1H, m,
Ha-5), 1.84 (1H, m, Ha-4), 2.19 (1H, m, Hb-1), 2.20
(1H, m, Ha-1), 2.48 (1H, dd, J=14.2, 2.9 Hz, Hb-8),
2.71 (1H, dddd, J=14.2, 2.9, 2.9, 1.5 Hz, Ha-8), 4.16

522

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523

(1H, dd, J=2.9, 2.9 Hz, H-9), 4.68 (1H, d, J=2.9 Hz,
H-10), 4.80 (1H, br s, Hb-14), 5.10 (1H, dd, J=1.5, 1.5
Hz, Ha-14), 5.29 (1H, br s, H-2); 13C NMR (100 MHz,
CDCl3), CH3:  21.5 (C-13), 23.8 (C-15), 25.0 (C-12), CH2:
 26.6 (C-5), 28.5 (C-4), 30.9 (C-1), 38.8 (C-8), 116.5 (C14), CH:  72.6 (C-10), 73.2 (C-9), 120.2 (C-2), C:  43.9
(C-11), 47.8 (C-6), 133.4 (C-3), 141.9 (C-7); LREIMS
m/z (rel.int.): 300, 298 (14:15) [M]+, 285, 283 (12:12) [MCH3]+, 219 (21) [MBr]+, 217 (32) [MBrH2]+, 201
(100) [MBrH2O]+, 173 (27), 119 (64), 105 (54), 69 (52),
55 (45), 41 (44); HREIMS m/z: 298.0927. Calc. for
C15H2379BrO, 298.0933 [M]. The 1H and 13C NMR
spectral data were almost identical with those reported
in the literature for deschloroelatol (Konig and Wright,
1997b).
3.7. Extraction and separation of Laurencia nidica
Extraction of partially dried alga (10 g) was carried
out by conventional methods as described above to give
a green oily extract (110 mg). A combination of column
and thick-layer chromatography of the MeOH extracts
gave laurinterol (6) (22%) and isolaurinterol (7) (4%).
3.8. Extraction and separation of Laurencia cartilaginea
and Laurencia concreta
The MeOH extracts of both samples contained no
halogenated compounds.
3.9. Antibacterial bioassay
Antibacterial bioassay was carried out using eight species of marine bacteria isolated from algal surfaces collected from the Japanese waters. These bacteria are
Alcaligenes aquamarinus, Alteromonas sp., Azomonas agilis, Azotobacter beijerinckii, Erwinia amylovora, Escherichia coli, Halobacterium sp., and Halococcus sp. One
loopful of each organism was precultured in 20 ml of
peptone water (3% NaCl) overnight. The turbidity of
the culture was adjusted to an optical density (OD)
McFarland 0.5 (Sonnerwirth and Jarett, 1980; Hindler
et al., 1990). Then 0.1 ml of the precultured bacterial
suspension was used to seed Nutrient Agar plates (3%
NaCl). Paper discs (Whatman, 6 mm) impregnated with
various amounts of the respective pure compounds were
placed on the seeded agar plates and the diameters of
the inhibitory zones were measured after the plates were
incubated at 28  C for 24 h.

Acknowledgements
This study was supported in part by a Grant-in Aid for
Scientic Research (No. 09839001) from the Ministry of
Education, Science, Sports and Culture of Japan and

also by the Sasakawa Scientic Research Grant from


the Japan Science Society (No. 12-300).
References
Abe, T., Masuda, M., Suzuki, T., Suzuki, M., 1999. Chemical races in
the red alga Laurencia nipponica (Rhodomelaceae, Ceramiales).
Phycological Research 47, 8795.
Caccamese, S., Azzolina, R., Duesler, E.N., Paul, I.C., Rinehart Jr.,
K.L., 1980. Laurencienyne, a new acetylenic cyclic ether from the
marine red alga Laurencia obtusa. Tetrahedron Letters 21, 22992302.
Carter, G.T., Rinehart Jr., K.L., Li, L.H., Kuentzel, S.L., Connor,
J.L., 1978. Brominated indoles from Laurencia brongniartii. Tetrahedron Letters, 44794482.
Gonzalez, A.G., Darias, J., Daz, A., Fourneron, J.D., Martn, J.D.,
Perez, C., 1976. Evidence for the biogenesis of halogenated chamigrenes from the red alga Laurencia obtusa. Tetrahedron Letters, 3051
3054.
Hay, M.E., Fenical, W., Gustafson, K., 1987. Chemical defense
against diverse coral-reef herbivores. Ecology 68, 15811591.
Hindler, J.A., Gonzalez, A.H., Drake, T.A., 1990. Stability of viablebacterium counts in liquid media used for preparation of inocula
and subsequent impact on antimicrobial susceptibility test results.
Journal of Clinical Microbiology 28, 12711275.
Juagdan, E.G., Kalidindi, R., Scheuer, P., 1997. Two new chamigranes from an Hawaiian red alga, Laurencia cartilaginea. Tetrahedron 53, 521528.
Kennedy, D.J., Selby, I.A., Thomson, R.H., 1988. Chamigrane metabolites from Laurencia obtusa and L. scoparia. Phytochemistry 27,
17611766.
Konig, G.M., Wright, A.D., 1997a. Sesquiterpene content of the antibacterial dichloromethane extract of the marine alga Laurencia
obtusa. Planta Medica 63, 186187.
Konig, G.M., Wright, A.D., 1997b. Laurencia rigida: chemical investigations of its antifouling dichloromethane extract. Journal of
Natural Products 60, 967970.
Kurata, K., Taniguchi, K., Agatsuma, Y., Suzuki, M., 1998. Diterpenoid
feeding-deterrents from Laurencia saitoi. Phytochemistry 47, 363369.
Masuda, M., Itoh, T., Matsuo, Y., Suzuki, M., 1997. Sesquiterpenoids
of Laurencia majuscula (Ceramiales, Rhodophyta) from the Ryukyu
Islands, Japan. Phycological Research 45, 5964.
Sakai, R., Higa, T., Jeord, C.W., Bernardinelli, G., 1986. The absolute congurations and biogenesis of some new halogenated chamigrenes from the sea hare Aplysia dactylomela. Helvetica Chimica
Acta 69, 91105.
Shizuri, Y., Yamada, A., Yamada, K., 1984. Laurequinone, a cyclolaurane sesquiterpene from the red alga Laurencia nidica. Phytochemistry 23, 26722673.
Sonnerwirth, C.A., Jarett, L., 1980. Gradwohls Clinical Laboratory
Methods and Diagnosis, 8th Edition. The C. V. Mosby Company,
St. Louis, pp. 19591970.
Suzuki, M., Furusaki, A., Hashiba, N., Kurosawa, E. 1979. The
structures and absolute stereochemistry of two halogenated chamigrenes from the red alga Laurencia majuscula Harvey. Tetrahedron
Letters 879882.
Suzuki, M., Kurosawa, E., 1978. Two new halogenated sesquiterpenes
from the red alga Laurencia majuscula Harvey. Tetrahedron Letters,
48054808.
Suzuki, M., Kurosawa, E., 1979. Halogenated and non-halogenated
aromatic sesquiterpenes from the red algae Laurencia okamurai
Yamada. Bulletin of the Chemical Society of Japan 52, 33523354.
Suzuki, M., Kurosawa, E., 1987. (3E)-Laureatin and (3E)-isolaureatin,
halogenated C-15 non-terpenoid compounds from the red alga
Laurencia nipponica Yamada. Bulletin of the Chemical Society of
Japan 60, 37913792.

C.S. Vairappan et al. / Phytochemistry 58 (2001) 517523


Suzuki, M., Kurosawa, E., Furusaki, A., Matsumoto, T., 1983. The
structures of (3Z)-epoxyvenustin, (3Z)-venustin, and (3Z)-venustinene, new halogenated C15-nonterpenoids from the red alga Laurencia venusta Yamada. Chemistry Letters, 779782.
Suzuki, M., Kurosawa, E., Kurata, K., 1987. (E)-Tridecyl-2-heptadecenal, an unusual metabolites from the red alga Laurencia species.
Bulletin of the Chemical Society of Japan 60, 37933794.
Vairappan, C.S., Daitoh, M., Suzuki, M., Abe, T., Masuda, M., 2001.
Antibacterial halogenated metabolites from the Malaysian Laurencia species. Phytochemistry, in press.
Waraszkiewicz, S.M., Erickson, K.L., 1974. Halogenated sesquiterpenoids from the Hawaiian marine alga Laurencia nidica: nidicene
and nididiene. Tetrahedron Letters, 20032006.

523

Waraszkiewicz, S.M., Erickson, K.L., 1975. Halogenated sesquiterpenoids from the Hawaiian marine alga Laurencia nidica. II. Nididienol. Tetrahedron Letters, 281284.
Waraszkiewicz, S.M., Sun, H.H., Erickson, K.L., Finer, J., Clardy, J.,
1978. C15 halogenated compounds from the Hawaiian marine alga
Laurencia nidica. Maneonenes and isomaneonenes. The Journal of
Organic Chemistry 43, 31943204.
Wratten, S.J., Faulkner, D.J., 1977. Metabolites of the red alga
Laurencia subopposita. The Journal of Organic Chemistry 42, 3343
3349.
Young, D.N., Howard, B.M., Fenical, W., 1980. Subcellular localization of brominated secondary metabolites in the red alga Laurencia
snyderae. Journal of Phycology 16, 182185.