Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.22 no.4 Curitiba July/Aug. 2012 Epub May 15, 2012
Angélica Ribeiro Soares*,I; Marcela C. S. RobainaII; Gabriella S. MendesII; Thalia S. L. SilvaI,III; Lísia M. S. GestinariI; Odinéia S. PamplonaI; Yocie Yoneshigue-ValentinIV; Carlos R. KaiserIII; Maria Teresa Villela RomanosII
INúcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé, Universidade Federal do Rio de Janeiro, Brazil
IIDepartamento de Virologia do Instituto de Microbiologia Prof. Paulo de Góes, Cenctro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Brazil
IIIInstituto de Química, Universidade Federal do Rio de Janeiro, Brazil
IVDepartamento de Botânica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Brazil
Organic extracts of 36 species of marine algae (sixteen species of Rhodophyta, eight species of Ochrophyta and twelve species of Chlorophyta) from seven locations on the Brazilian coast were evaluated for their anti-HSV-1 and anti-HSV-2 activity resistant to Acyclovir (ACV). Activity tests in crude extracts, followed by the identification of the major compounds present, were performed for all species. The chemical profiles of all crude extracts were obtained by 1H-NMR and 13C-NMR spectroscopy. The percentage of extracts with antiviral activity was higher for HSV-1 (86.1%) than for HSV-2 (55.5%). The green algae Ulva fasciata and Codium decorticatum both showed the highest activity (99.9%) against HSV-1, with triacylglycerols and fatty acids as the major components. The red alga Laurencia dendroidea showed good activity against HSV-1 (97.5%) and the halogenated sesquiterpenes obtusol and (-)-elatol were identified as the major components in the extract. Against HSV-2, the green alga Penicillus capitatus (Chlorophyta) and Stypopodium zonale (Ochrophyta) were the most active (96.0 and 95.8%). Atomaric acid, a meroditerpene, was identified as the major secondary metabolite in the S. zonale extract. These results reinforce the role of seaweeds as important sources of compounds with the potential to enter into the pipeline for development of new drugs against herpes simplex.
Keywords: Acyclovir-resistant HSV; marine algae; marine natural products; secondary metabolites
Seaweeds provide a rich source of structurally diverse secondary metabolites. These are mainly terpenes, acetogenins and polyphenols, including many halogenated compounds (Maschek & Baker, 2008). These secondary metabolites provide defense against herbivores (Pereira et al., 2004b; Lima et al., 2008), fouling organisms (Da Gama et al., 2008) and pathogens (Paul & Ritson-Williams, 2008); they also play a role in reproduction (Amsler & Fairhead, 2005), protection from UV radiation (Gomez et al., 1998) and as allelopathic agents (Beach et al., 2003). These compounds have shown some interesting pharmacological activities such as: antitumoral (Barbier et al., 2001), antiparasitic (Davyt et al., 2001), antibacterial (Vairappan, 2003), antiviral (Santos et al., 1999; Pereira et al., 2004a; Soares et al., 2007), antioxidant (Nahas et al., 2007), and antifungal activity (de Oliveira et al., 2008). In particular, antiviral effects of sulfated polysaccharides and terpenes against a variety of enveloped viruses, such as Herpes Simplex Virus type 1 (HSV-1) and 2 (HSV-2), Human Immunodeficiency Virus (HIV), human cytomegalovirus, dengue viruses, respiratory syncytial and influenza viruses have been reported (Laillea et al., 1998; Ghosh et al., 2004; Cirne-Santos et al., 2008; Hidari et al., 2008).
At present, the availability of safe and potent antiviral agents against herpes viruses is far from ideal. Acyclovir (ACV) is the compound chosen for clinical use against HSV-1 and HSV-2 in systemic or topical therapy (Brown et al., 2002). Other ACV-related nucleoside analogs, all targeted against viral DNA synthesis, have recently been approved for human use (De Clercq, 2005). Although these compounds are potent and contribute to the overall reduction of morbidity associated with viral infection, the emergence of viral resistant variants after prolonged treatment in immunocompromised patients still occurs, which justifies the continuous search for novel antiherpetic agents (Jerome, 2005).
In this context, metabolites from algae represent interesting types of compounds to assay as promising antiviral agents. This study presents the in vitro antiherpetic properties and the chemical profiles of most active crude extracts of seaweeds from the Brazilian coast.
Materials and Methods
Thirty-six species of macroalgae, belonging to three algal divisions (Rhodophyta, Ochrophyta and Chlorophyta), were collected from six sites in Rio de Janeiro state, on the southeastern Brazilian coast: Forno beach (22º44'31.70"S, 41º52'35.97"O), Rasa beach (22º44'3.15"S, 41º57'30.15"O), Francês Island (22º24'6.46"S, 41º41'37.16"O), Tatagiba beach (21º23'31.11"S, 40º59'9.64"O), Cavaleiros beach (22º24'18.50"S, 41º47'42.48"O) and Cabo Frio Island (23º0'10.02"S, 42 0'24.43"O), between February, 2006, and March, 2007. Five species of Chlorophyta were collected in Bahia state, on the northeast coast (17º6'38.86"S, 39º10'54.95" W), in March, 2009 (Table 1). All algae were collected by A. R. Soares and identified by L. M. S. Gestinari and Y. Yoneshigue-Valentin. The algae were washed in seawater to eliminate associated organisms and air-dried. Voucher specimens were deposited at RFA (Thiers, 2008).
The air-dried algal material was powdered and extracted three times with dichloromethane:methanol (1:1) at room temperature, except the material collected in Bahia state, which was extracted three times with dichloromethane. After the evaporation of the solvent, all the crude extracts were analyzed by 1H-NMR (Nuclear Magnetic Resonance) (300 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) spectroscopy (Bruker Avance spectrometer using tetramethylsilane (TMS) as internal standard) and thin layer chromatography (Silica gel GF254 TLC plates, Merck) with 2% Ce(SO4)2 in sulphuric acid as the spray detection reagent and heating the TLC plates at 100ºC. The crude extracts were used to perform antiherpetic activity evaluation.
Cells and viruses
Vero cells were grown in Eagle's minimum essential medium (MEM) supplemented with 2 mM L-glutamine, 50 μg/mL gentamicin, 2.5 μg/mL fungizon, plus 10% of heat-inactivated fetal bovine serum (FBS) (Schmidt, 1979) and kept at 37 ºC in an atmosphere of 5% CO2. Acyclovir-resistant HSV-1 and HSV-2 strains isolated from typical oral and genital lesions, respectively, were used. The isolates were typed by the polymerase chain reaction (PCR) using specific primers to identify HSV-1 and HSV-2 (Markoulatos et al., 2001) and evaluated with regard to sensibility to Acyclovir (De La Iglesia et al., 1998).
The algal extracts were solubilized in dimethylsulfoxide (final concentration 1%) and diluted in water to a concentration of 400 μg/mL, sterilized by filtration through a Millipore membrane filter (0.22 μm) and frozen at -20 ºC until use. The cytotoxicity assay was performed by incubating, in triplicate, Vero cell monolayers cultivated in 96-well microplates with two-fold serial dilutions of the extracts for 48 h at 37 ºC. Morphological alterations of the treated cells were observed in an inverted optical microscope (Leitz-Germany 633456), and the maximum non-toxic concentrations (MNTC) were determined (Walker et al., 1972). Cellular viability was further evaluated by the neutral red dye-uptake method (Neyndorff et al., 1990). The 50% cytotoxic concentration (CC50) was defined as the dilution that caused a reduction of 50% in the number of viable cells.
Anti-HSV activity was evaluated by reduction of the virus titers using TCID50 (50% tissue culture infective dose) determinations. Vero cell monolayers cultivated in 96-well microplates were treated with the algal extracts at the MNTC. Immediately after, logarithmical dilutions of HSV-1 and HSV-2 suspensions were added to treated and untreated cell cultures and incubated in a 5% CO2 atmosphere for 48 h at 37 ºC. Following incubation, the virus titers were calculated using the Reed & Muench (1938) statistical method and expressed as TCID50 values. Results of the antiviral activity were expressed as Percentage of Inhibition (PI) (Nishimura et al., 1977) using antilogarithmic values of the TCID50 values as follows: PI=[1-(antilogarithm of the test value/antilogarithm of the control value)]x100.
A total of 36 macroalgae species from seven sites along the Brazilian coast were tested against acyclovir-resistant HSV-1 and HSV-2. Of these, sixteen species were Rhodophyta (44.4%), eight species were Ochrophyta (22.2%) and twelve species were Chlorophyta (33.3%, Table 1). The results of the antiviral activity were expressed as PI. All results are reported in Table 2.
Out of all the crude extracts, 31 (86.1%) showed some activity against HSV-1 (PI values ranging from 20.6 to 99.9%) and 20 (55.5%) some activity against HSV-2 (PI values ranging from 20.6 to 96.0%). Among the three Phyla, the Ochrophyta showed the highest percentages of active extracts against HSV-1, with 100% of the extracts exhibiting activity. The Chlorophyta and Rhodophyta represented 91.6 and 75.0%, respectively of the active extracts. However, against HSV-2, Chlorophyta exhibited the highest percentage of active extracts (66.7%), followed by Ochrophyta (62.5%) and Rhodophyta (43.7%). A strong anti-herpetic activity was considered for extracts with PI>90%. Eleven species (30.5%), listed in Table 2, showed anti-HSV-1 activity with a PI superior to 90%. Among these, L. dendroidea, S. zonale, S. cymosum, U. fasciata and C. decorticatu showed very high activities (97.5, 96.8, 98.2, 99.9 and 99.9% respectively). On the other hand, strong anti-HSV-2 activity (PI>90%) was observed in only four species (11.1%), i.e., S. zonale, S. cymosum, C. acicularis and P. capitatus, with P. capitatus being the most active (96.0%). All algal extracts with strong anti-HSV activity (PI>90%) presented no toxicity to Vero cells (CC50>200 μg/mL), except the extract from Laurencia dendroidea (CC50 48.2 μg/mL).
All the crude extracts were analyzed by 1H-NMR and 13C-NMR spectroscopy. The major constituents of the most active extracts (L. dendroidea, S. zonale, S. cymosum, U. fasciata, C. decorticatum and P. capitatus) were identified. Characteristic signals for terpenoids were observed in the crude extracts from the algae L. dendroidea and S. zonale. Comparison of the spectroscopic data with previously reported data allowed the identification of the halogenated sesquiterpenes obtusol (1) and (-)-elatol (2) from L. dendroidea and the meroditerpenoid atomaric acid (3) from S. zonale (González et al., 1979; Wessels et al., 1999; Soares et al., 2003; Machado et al., 2011). The phenolic composition of the S. cymosum extract was suggested by the signals in the 1H NMR spectrum at δ 6.54 (bs), 6.51 (d, J=3.0 Hz), 6.48 (d, J=3.0 Hz) and 6.45 (bs), characteristic to two coupled aromatic protons meta to each other, and a group of the signals at 160.0-120.0 ppm in the 13C NMR spectra, characteristic of a phenolic moiety. The presence of triacylglycerols and fatty acids as the major components from the algae U. fasciata, C. decorticatum and P. capitatus is indicated by the strong 1H NMR signals at δ 4.29 ppm (dd, J=7.4; 14.6 Hz), 4.16 ppm (dd, J=6.0; 12.4 Hz) and 5.36 ppm (m), characteristic of triacylglycerols. The strong signal from the terminal methyl groups of the fatty acid esters were clearly observed at δ 0.88 ppm (t, J=7.2 Hz). The signals at δ 2.31ppm (t, J=7.4 Hz) and 1.60 ppm (m) correspond to the methylene protons α- and β- to the carbonyl groups, respectively. Peaks at δ 2.80 ppm and 2.02 ppm are attributed to methylene protons adjacent to double bonds. A strong peak for the internal methylene groups of the long chain of the fatty acid esters was observed at δ 1.26 ppm (bs). The 13C NMR spectra of the extracts showed resonances of fatty acid ester carboxyl groups and the signals at 60-70 ppm indicated the presence of the glycerol moiety. All of the NMR data are shown in Table 3.
Several molecules extracted from marine algae possess a broad spectrum of antiviral activity. Chemical classes for these compounds include sterols, terpenes, acetogenins, polyssacharides, fatty acids and polyphenols (Pereira et al., 2004a; Maschek & Baker, 2008; Hidari et al., 2008). In this study, we investigated the anti-herpetic activity against acyclovir-resistant HSV-1 and HSV-2 of lipophilic extracts from 36 Brazilian seaweeds. Of all the crude extracts, 31 (86.1%) showed some activity against HSV-1 and 20 (55.5%) some activity against HSV-2. The most active anti-HSV extracts were obtained from the species L. dendroidea, U. fasciata, C. decorticatum, S. zonale, S. cymosum, C. acicularis and P. capitatus.
Red algae of the genus Laurencia are found in tropical and subtropical regions throughout the world and are an extremely rich source of secondary metabolites with diverse structural features, mainly halogenated terpenes and C15-acetogenins, with a broad spectrum of biological activity (Machado et al., 2010). The halogenated sesquiterpenes obtusol (1) and (-)-elatol (2) were identified as the major compounds of L. dendroidea.
The green algae U. fasciata, C. decorticatum and P. capitatus showed high activity against HSV-1. These genera had high concentrations of polysaccharides and fatty acids (Pope et al., 1996). These compounds may be responsible for the observed activity. Fatty acid-treated cells are resistant to infection by a variety of lipid-enveloped viruses, including herpes viruses (Pope et al., 1998). The chemical profiles of the crude extracts, obtained by the use of 1H-NMR and 13C-NMR spectroscopy, showed the presence of triacylglycerols and a mixture of fatty acids as the major components in these extracts.
The brown algae of the genera Stypopodium (Dictyotales) and Sargassum (Fucales) are abundantly found along the Brazilian coast. Both genera are known to produce meroditerpenes (mixed biogenesis diterpenes). Other metabolites of structural classes such as glycerides (Tang et al., 2002a), steroids (Tang et al., 2002b), dipeptides (Liu et al., 2009) and flavonoids (Liu et al., 2009) were isolated from the genus Sargassum. The species S. cymosum together with S. zonale were the only species that were highly active against both the viruses HSV-1 and HSV-2. The meroditerpenoid atomaric acid (3) was identified as the major secondary metabolite in the S. zonale extract. Meroditerpenes from S. zonale are known to have diverse biological activities (Wessels et al., 1999; Sabry et al., 2005), including anti-HSV-1 activity (Soares et al., 2007). The 1H-NMR spectroscopic data of the crude extract of S. cymosum showed signals characteristic of phenolic compounds as the major constituents. Phenolic compounds have received considerable attention because of their therapeutic effects and their favorable antiviral activity (Quideau et al., 2004; Likhitwitayawuid, 2005; Tareq et al., 2007).
Although it is not possible to determine whether only one or a combination of several molecules are responsible for the observed anti-HSV-1 and anti-HSV-2 activity of the extracts, the presence of terpenes, fatty acids and phenolic compounds is consistent with the observed anti-herpetic activity since these types of metabolites, isolated from marine and terrestrial sources, have already been shown to have anti-herpetic activity (Khan et al., 2005; Hayashi et al., 2008).
The results of the present study indicate that different crude extracts from marine algae exhibit high anti-herpetic activity. The present findings provide a basis for further experiments on the identification and characterization of specific compounds with high anti-herpetic activities.
The authors thank Soluza dos Santos Gonçalves for technical assistance. This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), FINEP (Number of process: 3175/06) and Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil. We thank Heitor Monteiro Duarte and Tatiana Konno for valuable comments.
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Angélica R. Soares
Grupo de Produtos Naturais de Organismos Aquáticos, Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé, Universidade Federal do Rio de Janeiro
Rua Rotary Club, s/n, São José do Barreto, Post Office Box 119331, 27910-970 Macaé-RJ, Brazil.
Received 14 Oct 2011
Accepted 6 Nov 2011