FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 364–366

DOI: 10.1002/ffj.1013

Antibacterial activity of volatile secondary metabolites

from Caribbean soft corals of the genus Gorgonia
Vassilios Roussis,1 Ioanna B. Chinou,1Ł Christina Tsitsimpikou,3 Constantinos Vagias,1 and
Panos V. Petrakis2
1 Divisionof Pharmacognosy, School of Pharmacy, University of Athens, University Campus of Zografou, 157 71 Athens, Greece
2 Hellenic
Ministry of Agriculture and Foresty, Directorate of Informatics, Athens, Greece
3 Doping Control Laboratory of Athens, Olympic Centre, Marousssi, Greece

Received 6 November 2000

Revised 3 April 2001
Accepted 4 April 20001

ABSTRACT: The volatile secondary metabolites of Caribbean corals from three species of the genus Gorgonia
were studied. The main constituents of the gorgonian species G. ventalina, G. flabelum and G. mariae were
identified and 12 metabolites were isolated and identified. The in vitro antibacterial activity of the volatile
constituents was also studied against six Gram-positive or -negative bacteria exhibiting a wide spectrum of
antibacterial activities. Furanotriene, dihydro-˛-santalen-12-one and E-nerolidol showed the most interesting
antibacterial action. Copyright  2001 John Wiley & Sons, Ltd.

KEY WORDS: octocorals; Gorgonia; Gorgonidae; volatiles; terpenes; antibacterial activity

Introduction Aspergillus flavus known to be human, plant and insect

The aim of the present study was the investigation of the
antibacterial activity of the volatile constituents of three
Gorgonia species (Gorgoniidae), Gorgonia ventalina, G.
flabelum and G. mariae,1 from the Bahamian islands
of the Caribbean Sea. Recent studies on the chemical
variability of the genus Gorgonia report the isolation of
several secondary metabolites, mainly marine steroids of
the gorgosterol type, sesquiterpenes hydrocarbons and
oxygenated sesquiterpenes.2 – 4
For the needs of this project, 292 gorgonian colonies,
representing the three above-mentioned species, were
collected and the volatile constituents were identified by
combination of GC–MS and nuclear magnetic resonance
(NMR) methods.
The in vitro antibacterial activity exhibited by the
volatile mixtures of the aforementioned species and the
isolated metabolites was also studied. This is the first
study on the antimicrobial activity of volatile secondary
metabolites from the family Gorgonidae against human
pathogenic bacteria, while current literature reports refer
only to the activity exhibited by other members of
this family against marine bacteria and fungi.5 – 8 In
more recent references, activities against three strains of
*Correspondence to: I. B. Chinou, Division of Pharmacognosy, School
of Pharmacy, University of Athens, University Campus of Zografou,
157 71 Athens, Greece.
pathogens have been studied.9
Experimental
Specimen Collection
Gorgonia specimens were hand-picked by scuba divers
during the 1995 Scripps Institution of Oceanography
expedition in the Caribbean Sea, from nine geographic
localities (Sweetings Cay, Eleuthera Point, Highborne
Cay, Lee Stocking, San Salvador, Long Island, George-
town, Acklins Island and French Wells) extending from
the north to the south Bahamian islands. The collected
taxa were stored at 20 ° C during the cruise. The col-
lection was shipped in dry ice (78 ° C) and was kept
in a deep freeze until analysed. Voucher specimens are
kept at the Laboratory of Pharmacognosy Herbarium,
University of Athens (ATPH/MO/39-48).
Isolation of the Oils
The gorgonian colonies were separately hydrodistilled
for 3 h in a modified Clevenger apparatus with a water-
cooled oil receiver, to reduce hydrodistillation overheat-
ing artifacts. In addition, the head space volatiles of a
number of representative gorgonian specimens were col-
lected and analysed. The chemical composition obtained

Copyright  2001 John Wiley & Sons, Ltd.

 VOLATILES AND ANTIBACTERIAL ACTIVITY OF GORGONIA
by the two techniques was qualitatively identical and the
study was safely carried out using the essential oils.
Gas Chromatography
Analysis was carried out on a Perkin-Elmer 8500 gas
chromatograph with FID, fitted with a Supelcowax-10,
fused silica capillary column (30 m ð 0.32 mm i.d.: film
thickness, 0.25 µm). The column temperature was pro-
grammed from 75 ° C to 200 ° C at a rate of 2.5 ° C/min.
The injector and detector temperatures were programmed
at 230 ° C and 300 ° C, respectively.
Gas Chromatography–Mass Spectrometry
The GC–MS analyses were carried out using a
Hewlett Packard 5973–6890 GC–MS system operating
on EI mode (equipped with a HP 5MS 30 m ð
0.25 mm ð 0.25 µm film thickness capillary column).
Helium (2 ml/min) was used as carrier gas. The initial
temperature of the column was 60 ° C and then was
heated to 280 ° C at a rate of 3 ° C/min.
Antibacterial Activity
Bacteriostatic activity against two Gram-positive bacte-
ria, Staphylococcus aureus (ATCC 25923) and Staphy-
lococcus epidermidis (ATCC 12228), and four Gram-
negative bacteria, Escherichia coli (ATCC 25922), En-
terobacter cloacae (ATCC 13047), Klebsiella pneu-
moniae (ATCC 13883) and Pseudomonas aeruginosa
(ATCC 227853), were determined using the disc dif-
fusion method. Standard antibiotics (netilmicin, amoxy-
cillin and clavulanic acid) were used to compare the
effectiveness of the mixtures and isolates tested. Tech-
nical data have been described elsewhere.10,11
Isolation–Identification of Components
The organic extracts (CH2 Cl2 /MeOH) of several gor-
gonians were chromatographed on silica gel with mix-
tures of EtOAc in cyclohexane. The non-polar fractions
containing the constituents of interest were further puri-
fied on normal phase HPLC with cyclohexane/EtOAc,
and were isolated and identified by spectroscopic means
and according the literature as: the oxygenated cyclic
sesquiterpene 5,10-epoxymuurolane; the furanosesquiter-
penes furanotriene, trans, trans-germacrone, cis, cis-ger-
macrone and cis-C3,C4-trans-C7,C8-germacrone; and
12,13-epoxy-˛-santalene, along with ˛-santalene deriva-
tives; isosericenine and E-nerolidol.
The identification of the chemical constituents was
based on comparison of their Kováts indices (KI), reten-
tion times (RT) and mass spectra with those obtained
Copyright  2001 John Wiley & Sons, Ltd.
365
from authentic samples and/or the NIST/NBS, Wiley
Library spectra and the literature,12 1-D and 2-D NMR
spectra were recorded using Bruker AC 200 and a Bruker
DRX 400 spectrometers.
Results and Discussion
The collected colonies exhibited a noticeable variation in
colour and odour. All specimens were recognized, on the
basis of the conventional taxonomic criteria, (morpho-
anatomical characteristics as well as colour and odour),
as either G. ventalina (78 colonies), G. flabellum (197
colonies) or G. mariae (seven colonies). G. ventalina
and G. flabellum appear to be chemically very similar
and both species possess the same metabolites, with
several quantitative differences, such as in the 5,10-
epoxymuurolane, 12,13-epoxy-˛-santalene and isoseri-
cenine contribution. G. mariae colonies were charac-
terized by the absence of several metabolites, such as
the three germacrone isomers, ledol and 5,10-epoxymu-
urolene.
From the 62 identified volatile metabolites of the
Caribbean gorgonians, 12 were isolated as pure com-
pounds and characterized by spectroscopic techniques
(NMR, MS). The oxygenated cyclic sesquiterpene, 5,10-
epoxymuurolane, is a novel compound found only in
G. ventalina and G. flabellum.13 Only two other cyclic
ethers other than epoxides, 1,4-epoxycadinane and 4,10-
epoxymuurolane, have been isolated from marine organ-
isms.14,15 The furanosesquiterpene furanotriene, which
represents one of the most abundant volatile metabolites
in the three gorgonian species, was originally reported
from the coral Pseudopterogorgia americana.16 A few
years later, the isolation of isofuranotriene was published
from the same organism.17 The compound trans, trans-
Germacrone was identified in the Indian and African
medicinal plant Fagara chalybea.18 The other two geo-
metric isomers, cis, cis-germacrone and cis-C3,C4-trans-
C7,C8-germacrone, have been chemically synthesized.19
12, 13-Epoxy-˛-santalene, found in all three gorgonian
species in significant amounts, was isolated and iden-
tified from the terrestrial root-bark of Severina buxifo-
lia, along with the other ˛-santalene derivatives found
in the gorgonian specimens.20 Sericenine and isoserice-
nine isolated previously from two Muricea species are
also common in the genus Gorgonia.2 E-nerolidol was
found to be very common among terrestrial and marine
organisms.12
Among the microorganisms tested, only the two
Gram-positive bacteria were found to be resistant to
the assayed metabolites and mixtures. In particular,
furanotriene exhibited a wide spectrum of activity, as
well as the highest one, against all the bacteria tested.
cis-C3-C4, trans-C7-C8 Germacrone exhibited good
activity only against E. cloaceae and K. pneumoniae,
Flavour Fragr. J. 2001; 16: 364–366
366 V. ROUSSIS ET AL.
while dihydro-˛-santalen-12-one and E-nerolidol were
the only metabolites, apart from furanotriene, highly
active against E. coli. Isosericenine and the examined
santalene derivatives (˛-santalene, 12,13-epoxy-˛-santa-
lene, 13,14 -iso-˛-santalol) had similar levels of activity
against P. aeruginosa, E. cloacae and K. pneumoniae.
In conclusion, the chemical analysis of the three mor-
phologically similar gorgonian species revealed the pres-
ence of 62 volatile constituents, mainly sesquiterpenes,
which formed three related but distinct chemical pro-
files. Furanotriene, the most active among the isolated
metabolites, exhibited significant levels of antibacterial
activity.
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