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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. References Abrantes JL, Barbosa J, Cavalcanti D, Pereira RC, Fontes CFL, Teixeira VL, Souza TML, Paixão ICP (2009) The effects of the diterpenes isolated from the Brazilian brown algae Dictyota pfaffii and Dictyota menstrualis against the herpes simplex type-1 replicative cycle. 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Mol Ther 17:209–210 876 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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