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Variability of a diterpene with potential anti-HIV activity
isolated from the Brazilian brown alga Dictyota menstrualis
Diana Negrão Cavalcanti & Meire-Anne Rezende de Oliveira & Joel Campos De-Paula &
Leandro Silva Barbosa & Tamara Fogel & Marcelo Alves Pinto &
Izabel Christina Nunes de Palmer Paixão & Valéria Laneuville Teixeira
Received: 20 July 2010 /Revised and accepted: 22 September 2010 /Published online: 9 October 2010
# Springer Science+Business Media B.V. 2010
Abstract This study evaluated the variability in the
production of an HIV-1 antiviral diterpene in individuals
of Dictyota menstrualis throughout reproductive stages.
The brown alga Dictyota has an isomorphic biphasic life
cycle. The quantification of the active principle by GC–FID
indicated a greater production of the diterpene in the female
gametophytic phase (42.11 ppm). However, these individ-
uals had the highest variation between individuals (standard
deviation of 68.20 ppm in the range from 0.39 to 227 ppm).
Sporophytic individuals showed less variability. This
variability in the production of the antiviral diterpene is
important to the development of future antiviral drugs.
Keywords Dictyota menstrualis . Antiviral against HIV.
Diterpene . Variability
Introduction
Although the advent of antiviral therapy has transformed
HIV infection from a fulminant and fatal disease to, in
many cases, a long-term chronic disease, the therapy is not
perfect. The costs, the toxicities, the unknown long-term
effects, and the emergence of strains which are resistant to
current drug regimens are problematic (Williams 2009).
The development of new antiretroviral drugs is a dynamic
process that is continuously fueled by identification of new
molecular targets and new compounds for known targets.
The high number of citations of a previous review on
antiviral compounds from plants (Cos et al. 2008) indicates
the importance of natural product research.
The use of natural products as potential drugs has been
widely recognized. A number of marine natural products
have been demonstrated to have pharmacological activities
against a wide range of pathogens, including viruses (e.g.,
De Clercq 2000; Newman and Cragg 2004; Queiroz et al.
2008; Smit 2004). Natural products isolated from the alga
Dictyota have shown potential antiviral activity. The main
compounds isolated from the brown alga Dictyota menstru-
alis, identified as 6-hydroxydichotoma-3,14-dien-1,17-dial
and its acetate derivative 6-acetoxydichotoma-3,14-dieno-
1,17-dial (Fig. 1, compounds 1 and 2, respectively), exhibit
inhibitory activities against HIV-1 replication since they act
in the recombinant HIV-1 reverse transcriptase (RT) activity
of dose-dependent form (Pereira et al. 2004, 2005). The
dolabellane diterpene isolated from Dictyota pfaffii inhibits
the HIV-1 infection in human primary cells and tumor cells
lines and inhibits the activity of a purified HIV-1 enzyme
RT in a dose-dependent manner (Barbosa et al. 2004;
Cirne-Santos et al. 2006, 2008). Recently, the diterpenes
isolated from D. pfaffii and D. menstrualis were shown to
inhibit HSV-1 infection in Vero cells (Abrantes et al. 2009).
D. N. Cavalcanti :M.-A. R. de Oliveira :V. L. Teixeira (*)
Programa de Pós-Graduação em Biologia Marinha,
Departamento de Biologia Marinha, Instituto de Biologia,
Universidade Federal Fluminense,
P.O. Box 100.644, CEP 24001-970 Niterói, RJ, Brazil
e-mail: valerialaneuville@gmail.com
J. C. De-Paula
Instituto de Biociências,
Universidade Federal do Estado do Rio de Janeiro,
Avenida Pasteur 458, Urca,
22290-240 Rio de Janeiro, Rio de Janeiro, Brazil
L. S. Barbosa : T. Fogel :M. A. Pinto
Fundação Oswaldo Cruz,
Av. Brasil, 4365—Manguinhos,
21040-360 Rio de Janeiro, Rio de Janeiro, Brazil
I. C. N. de Palmer Paixão
Departamento de Biologia Celular e Molecular,
Instituto de Biologia, Universidade Federal Fluminense,
CEP 24020-150,
Niterói, Rio de Janeiro, Brazil
J Appl Phycol (2011) 23:873–876
DOI 10.1007/s10811-010-9601-z
Dictyota menstrualis is distributed worldwide, including
the North, South, and Central America, the Atlantic, and
Caribbean Islands. The male and female gametophytes and
the sporophyte are identical in appearance. In our previous
studies conducted along the Brazilian littoral, it was
observed in the field that different haploid–diploid ratios
of the alternating life stages of D. menstrualis can be found
at the same time in the field throughout the year (Cavalcanti
et al. 2006; Ortiz-Ramirez et al. 2008; Pereira et al. 2000;
Teixeira et al. 2001).
In this study, we evaluated the changes in the concen-
tration of the antiviral active diterpene in different life
stages using gas chromatography–flame ionization detector
(GC–FID) and whether the antiviral activity found for the
active principle was maintained when using the crude
extract.
Material and methods
Specimens of Dictyota menstrualis (Hoyt) Schnetter,
Hörnig, and Weber- Peukert were collected at Praia Rasa,
Armação dos Búzios, RJ (N 22° 44′ 00″; W 41° 57′ 25″),
on 7 May 2004, and sporophytes and male and female
gametophytes were separated. The material was stored in a
freezer at −20°C for 23 months, and subsequently it was
dried at room temperature for 5 days. Each one of the 15
individuals of each reproductive state was extracted with
acetone twice, separately. The solvent was evaporated
under reduced pressure. Qualitative analyses were moni-
tored by thin-layer chromatography (silica gel, n-hexane/
ethyl acetate, 7:3). Spots of diterpenes were detected after
inspection at ultraviolet light (254 and 365 nm) and
spraying with 2% ceric sulfate in sulfuric acid, followed
by heating the plates at 100°C for 3 min.
Specimens of D. menstrualis were collected in July of
2007 by snorkeling at depth of 2–5 m, at Praia Rasa (N 22°
44′ 00″; W 41° 57′ 25″) and Enseada do Forno (N 22° 45′
48″, W 41° 52′ 26″), Armação dos Búzios, RJ, Brazil.
Identification of different reproductive stages (sporophytes
and female and male gametophytes) was made afterwards
by one of us (JCP), and voucher specimens were deposited
at the Herbarium of the Universidade do Estado do Rio de
Janeiro (HB 84815). The air-dried algae (86.28 g) were
extracted with CH2Cl2 (3×50 mL), and the crude extract
(5.75 g) was partitioned between n-hexane and MeOH/H2O
(1:1), for three times. The hexane fraction (4.22 g) was
purified by silica gel 60 Merck (0.063–0.200 μm) column
chromatography, eluted with n-hexane/ethyl acetate (7:3).
Fraction 14 containing the crude diterpene 6-
hydroxydichotoma-3,14-dien-1,17-dial (1), which was pu-
rified by new silica gel column chromatography, eluted
with n-hexane/ethyl acetate (7:3), giving pure diterpene
(9.20 mg). The purity and structure chemistry elucidation
was conducted using GC, GC–mass spectrometry, and
NMR analysis based on comparison with literature data
(Cavalcanti et al. 2006; Teixeira et al. 2001).
GC analysis was carried out using an HP 6890 GC
equipped with an HP-1 capillary column (30 m×0.25 mm;
film thickness of 0.25 μm) using an FID detector. The
temperature program was kept at 160°C and then
programmed to 250°C at a rate of 4°C min−1, for 2.5 min,
and finally raised to 290°C at a rate of 10°Cmin−1 for
3 min. Helium was the carrier gas at a flow rate of 2 mL
min−1. Injector and detector temperatures were set at 270°
C. Diluted samples were injected in the split mode (1/10).
Purified diterpene 1 was used as an external standard,
and quantifications were made using analytical curves
calculated from five different concentrations of a standard
solution in dichloromethane (75.6, 50.4, 25.2, 10.1, 5.0,
and 1.0 ppm).
The sample solutions (2 μL each) were taken, and each
one of them was applied on the GC equipment in triplicate.
The experimental parameters were identical for the above
analysis.
The extract antiviral effect was tested on HSV-1
replication. Vero cells were infected with an acyclovir-
resistant HSV strain (AR-29) (Lagrota et al. 1994) and
treated with 1 μg mL−1 extract. After 72 h at 37°C, the
monolayerswere fixed with 10% formaldehyde in PBS and
stained with a 0.1% solution of crystal violet in 70%
methanol, and the virus inhibited was calculated by scoring
the plaque-forming units.
Statistical analysis was conducted using one-way
ANOVA. P values >0.05 were considered to be significant.
Results and discussion
The antiviral diterpene 1 showed a linear response with
increasing concentrations of the product (Fig. 2). The
retention time (tR) of compound 1 was 16.7 min, and the
limits of detection and quantification were 0.10 and
H
H
OHC
OHC OH
1 
H
H
OHC
OHC OAc
2
Fig. 1 Antiviral diterpenes 1
(6-hydroxydichotoma-3,14-
dien-1,17-dial) and 2 (6-acetox-
ydichotoma-3,14-dieno-1,17-di-
al) isolated from Brazilian D.
menstrualis
874 J Appl Phycol (2011) 23:873–876
0.33 ppm, respectively. Representative GC chromatographs
of D. menstrualis in different phases of the reproductive
cycle are presented in Fig. 3.
There are no significant differences among the spor-
ophytes (10.66±15.31 ppm or 0.30% of the crude extract)
and female (42.11±68.20 ppm or 0.84% of the crude
extract) and male gametophytes (25.25±35.22 ppm or
0.64% of the crude extract) (Fig. 4). On the other hand,
we observed large variability among individuals of the
same stage. In female gametophytes, the diterpene 1
concentration showed the greatest and the smallest values
(0.39 and 227.47 ppm), whereas in the sporophytes the
values are more homogeneous.
In previous ecological studies, the different reproductive
stages of some red algae (macroalgae with complex life
histories and multiple distinct phases) have shown differ-
ential production of natural products or chemical defenses
(e.g., Plouguerne et al. 2007; Vergés et al. 2008). However,
studies on the variation of potential drugs in different
reproductive stages are rare. Moreover, less accurate
techniques have been used, and the studies were conducted
on red algae (e.g., Centeno and Ballantibe 1999). The
stages of heteromorphic seaweeds often differ considerably
Fig. 4 Diterpene concentrations and standard deviation of male
gametophytes (n=14), female gametophytes (n=15), and sporophytes
(n=13)
Fig. 3 Sample GC–FID chromatograms of individuals of D.
menstrualis for the different reproductive stages. Compound 1
corresponds to antiviral diterpene. a Male individual; b sporophyte
individual, and c female individual
Fig. 2 Standard curve of antiviral diterpene 1 in GC–FID
0
2
4
6
8
10
12
14
16
1 3 5 6 7 8 9 10 11 12 13 14 15
female gametophyte individuals
cr
u
de
 ex
tra
ct
 (m
g)
0
2
4
6
8
10
12
14
16
cr
u
de
 ex
tra
ct
 (m
g)
cr
u
de
 ex
tra
ct
 (m
g)
0
50
100
150
200
250
ac
tiv
e d
ite
rp
en
e (
pp
m)
 
0
50
100
150
200
250
ac
tiv
e d
ite
rp
en
e (
pp
m)
 
0
50
100
150
200
250
ac
tiv
e d
ite
rp
en
e (
pp
m)
 
0
5
10
15
20
25
sporophyte individuals
42
1 3 5 6 7 8 9 10 11 12 13 14 15
male gametophyte individuals
42
1 3 5 6 7 8 9 10 11 12 13 14 1542
Fig. 5 The crude extract yield and diterpene 1 concentrations of D.
menstrualis for the different reproductive stages: female gametophyte
individuals (n=15), male gametophyte individuals (n=14), and sporo-
phyte individuals (n=13). Black square, crude extract concentrations;
and white square, active diterpene concentrations
J Appl Phycol (2011) 23:873–876 875
in their physiological and ecological properties. However,
Dictyota has a life history with isomorphic alternation of
generation. In the brown algae, Cronin and Hay (1996)
obtained the same concentrations of chemical defenses
(diterpenes) in the different life stages of Dictyota ciliolata.
The individuals used in our experiments were homoge-
neous with respect to dry weight (106.4±31.7 mg) and
weight of the extract obtained (9.9±3.3 mg). In general, the
correlation among dry weight, extract, and the diterpene
concentration values was maintained, except for the female
gametophytes (Fig. 5).
In order to evaluate the activity of the extract containing
diterpene, an antiviral assay was developed. An extract pool
from different stages (sporophytes and gametophytes) was
tested, showing a medium diterpene concentration. In Vero
cells, the replication of a HSV-1-resistant acyclovir strain
was 43% inhibited by 1 μg mL−1 of extract. In a previous
study, the pure diterpene 1 shows 51% of inhibition in
HSV-1 replication with 1.6 μM, representing 0.7 ppm of
the diterpene in the solution (Pereira et al. 2005). According
to our results, in a 1 1 μg mL−1 of extract, there is a
diterpene average concentration of 0.59 ppm, and they
showed almost the same antiviral activity in HSV-1
replication than the diterpene alone. Probably, other
diterpenes present in the extract also has some antiviral
activity, increasing the extract activity even in a lower
concentration. These results are promising for a future
herbal development.
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876 J Appl Phycol (2011) 23:873–876
	Variability of a diterpene with potential anti-HIV activity isolated from the Brazilian brown alga Dictyota menstrualis
	Abstract
	Introduction
	Material and methods
	Results and discussion
	References

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