versión impresa ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.4 Curitiba jul./agosto 2011 Epub 03-Jun-2011
Izabella T. SilvaI; Thiago CaonI; Débora D. LückemeyerI; Freddy A. RamosII; Edisson TelloII; Catalina Arévalo-FerroIII; Eloir P. SchenkelIV; Carmenza DuqueII; Cláudia M. O. SimõesI,*
ILaboratório de Virologia Aplicada, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Brazil
IIDepartamento de Química, Universidad Nacional de Colombia, Colombia
IIIDepartamento de Biología, Universidad Nacional de Colombia, Colombia
IVDepartamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Brazil
The exploration of marine environment represents a promising strategy in the search for new active antiviral compounds. The isolation and characterization of the nucleosides spongothymidine and spongouridine from the sponge Cryptotethia crypta used as models for the synthesis of ara-A (vidarabine), that has been used therapeutically against herpetic encephalitis, was the most important contribution since the late 1970s. This paper describes the in vitro antiviral evaluation of 26 organic extracts obtained from eleven octocoral species and fifteen marine sponges. Cytotoxicity was evaluated on Vero cells by MTT assay and the antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by plaque number reduction assay. Results were expressed as 50% cytotoxic (CC50) and 50% inhibitory (IC50) concentrations, respectively, in order to calculate the selectivity index (SI= CC50/IC50) of each extract. Among the tested marine octocoral species, only three extracts showed antiviral activity, but with low selectivity indices (<3.0). Among the tested marine sponges, eight extracts showed SI values higher than 2.0, and three can be considered promising (Aka cachacrouense, Niphates erecta and Dragmacidon reticulatum) with SI values of 5.0, 8.0 and 11.7, respectively, meriting complementary studies in order to identify the bioactive components of these sponge extracts, which are in course now.
Keywords: antiherpes, marine organisms, marine sponges, octocoral species, screening
Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) are a worldwide occurring human pathogens. There is an urgent need to discover and develop new alternative agents for the management of HSV infection. They are frequently responsible for infections on skin and mucosa of different locations including oral and genital regions. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in neonates and immunocompromised patients. Both types 1 and 2 were the first human herpesviruses to be discovered and are among the most intensively investigated due to their ability to cause a variety of infections, the capacity to remain latent in their host for life, and the possibility to reactivate, causing lesions at or near the site of initial infection. (Roizman et al., 2007).
Currently, there is no cure for the chronic infection, and prolonged therapy with the available antiherpes drugs has resulted in some undesirable effects and induced the emergence of drug-resistant virus strains. Moreover, HSV has been described as a risk factor for HIV infection. This scenario has triggered the search for new antiherpetic agents, especially those with different mechanism of action from that of acyclovir (Van de Perre et al., 2008).
Although the diversity of life in the terrestrial environment is extraordinary, the greatest biodiversity is in the world's oceans, with 34 of the 36 phyla of all globe, approximately 300,000 described species of plants and animals, such as sponges, corals, tunicates, shellfish, bacteria, seaweeds, which represent only a small percentage of the total number of species have yet to be discovered. Marine organisms are increasingly being examined as possible sources of unusual bioactive compounds, therefore the marine environment represents a treasure of useful products awaiting discovery for the treatment of infectious diseases. Ecological pressures, including competition for space, the fouling of the surface, predation, and successfully reproducing have led to the evolution of unique secondary metabolites with various biological activities. The importance that these secondary metabolites play in the control of infectious and parasitic organisms was for many years largely overlooked (Da Silva et al., 2006; Laport et al., 2009; Yasuhara-Bell & Lu, 2010).
Viruses have remained resistant to treatment or prophylaxis longer than any other infectious organism. The search for viral chemotherapeutic agents from marine sources has yielded several promising therapeutic leads reported to display notable antiviral activity. Perhaps the most important antiviral lead of marine origin reported thus far is the nucleoside ara-A. Ara-A (vidarabine) is a semisynthetic compound based on the arabinosyl nucleosides isolated from the sponge Cryptotethia crypta that has been used therapeutically against herpetic encephalitis since the late 1970s. Other examples of drugs obtained from semisynthetic modifications of these nucleosides are ara-C (cytarabine), acyclovir and azidothymidine (zidovudine), which are nowadays in clinical use (Donia & Hamann, 2003; Molinski et al., 2009; Yasuhara-Bell & Lu, 2010).
In this study, was evaluated the inhibitory activity against Herpes Simplex Virus type 1 (KOS, strain) of 26 organic extracts (CH2Cl2-MeOH 1:1) obtained from eleven octocoral species and fifteen marine sponges.
Materials and Methods
Collection of marine organism
The marine invertebrates (33) were collected in different locations at the Santa Marta bay (11º15´01´´ N and 74º13´50´´ W, approximately) and at the Rosario islands (40 km south west from Cartagena Bay (10º10´27´´N and 75º48´40´´ W, approximately), by means of scuba dive, between September 2005 and March 2006. The samples were identified by Dr. Sven Zea and Dr. Mónica Puyana. Vouchers of each one of the studied species were deposited at the Instituto de Ciencias Naturales-Universidad Nacional de Colombia and at the Instituto de Investigaciones Marinas y Costeras "José Benito Vives De Andréis" Invemar Collections. General information about these marine organisms is shown in Table 1.
Organic extracts of the tested marine organisms were prepared according to standard procedures (Tello et al., 2009). Briefly, once the organisms were collected, and rinsed with sterile sea water to remove associated debris, they were frozen until extraction. The samples were cut into small pieces and extracted by maceration in CH2Cl2-MeOH (1:1) for 24 h, three times. The extract obtained after filtration was then concentrated to dryness under vacuum at 40 ºC, and stored at 4 ºC before use. All extracts were dissolved in DMSO 1% (Merck) and culture medium to a final concentration of 1000 µg/mL, aseptically filtered (Millipore 0.22 µm), and stored at -20 ºC until tested.
Virus and cell line
Vero cells (ATCC CCL 81, Rockville, MD, USA) were grown in Eagle's Minimum Essential Medium (MEM; Cultilab, Brazil) and supplemented with 10% fetal bovine serum (FBS; Gibco, Brazil), penicillin G (100 U/mL), streptomycin (100 µg/mL) and amphotericin B (25 µg/mL, Cultilab). The cells were maintained at 37 ºC in a humidified atmosphere of 5% CO2 in air. The Herpes Simplex Virus type 1 (HSV-1, KOS strain) (Faculty of Pharmacy, University of Rennes, France) was propagated in Vero cells, titrated on the basis of plaque forming units (PFU) count by plaque assay as previously described (Burleson et al., 1992) and stored at -80 ºC until the experiments.
Evaluation of cytotoxicity
It was performed by MTT [3-(4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide] assay, with minor modifications (Mosmann, 1983). To assess the cytotoxic effects of the samples on uninfected Vero cells (2.5 x 104 cells per well) seeded onto 96-well culture plates, 200 µL of their dilutions ranging from 0 to 1000 µg/mL (ratio 1:2) were added to confluent cell monolayers. As cell controls, only 200 µL of MEM were added to the cells. After 72 h at 37 ºC, the medium was removed and 50 µL of MTT solution prepared in MEM (1 mg/mL; Sigma) were added to each well and the plates incubated for 4 h. The MTT solution was removed, 100 µL of DMSO (Nuclear, Brazil) were added to each well to dissolve formazan crystals, and the plates have been gently shaken, whereby crystals were completely dissolved. The absorbances were read on a multiwell spectrophotometer (Bio-Tek®, Elx 800) at 540 nm. The 50% cytotoxic concentration (CC50) was defined as the concentration of the extracts that reduced cell viability by 50% when compared to untreated controls.
Viral plaque formation number reduction assay
This assay followed the procedures previously described (Silva et al., 2010b), with minor modifications. Vero cells (2.5 x 105 cells per well) were seeded onto 24-well culture plates, and after 24 h, the cells were infected with 100 PFU of HSV-1. After 1h adsorption at 37 ºC, the plates were washed and overlaid with MEM plus 1.5% carboxymethylcellulose (CMC, Sigma) containing or not different concentrations of the extracts. After 72 h, the cells were fixed and stained with naphtol blue-black (Sigma) and viral plaque formation were counted. The 50% inhibitory concentration (IC50) was defined as the concentration that inhibited 50% of viral plaque formation when compared to untreated controls. Acyclovir (Sigma®) was used as a positive control (10 µg/mL), since it completely inhibited the viral replication. Its stock solution was prepared in DMSO (Merck) at the final concentration of 1000 µg/mL.
Results were expressed as 50% cytotoxic (CC50) and 50% inhibitory (IC50) concentrations, respectively, in order to calculate the selectivity index (SI= CC50/IC50) of each extract (Cos et al., 2006).
The mean±standard deviations are representative of three independent experiments. For the determination of CC50 and IC50 values non-linear regressions of concentration-response curves were used.
Results and Discussion
Over the past 50 years, marine organisms have provided key structures and compounds that proved their potential for industrial development as cosmetics, nutritional supplements, fine chemicals, agrochemicals and therapeutic agents for a variety of diseases. During the past 30 years, thousands of novel compounds with diverse biological activities ranging from anticancer to antiviral have been isolated from various marine sources (Yasuhara-Bell & Lu, 2010).
In this study, 26 organic extracts obtained from eleven octocoral species and fifteen marine sponges were investigated for their antiviral activity against Herpes Simplex Virus type 1 (HSV-1, KOS strain). Before the evaluation of the antiviral activity, the cytotoxic effects of the selected samples were investigated on VERO cells by MTT assay. For each tested sample, a CC50 value was calculated. This assay has several advantages: it is easy to perform, the evaluations are objective, it can be automated using a personal computer and the cytotoxicity evaluation can be made in parallel with antiviral activity evaluation (Andrighetti-Frohner et al., 2003; Müller et al., 2007; Takeuchi et al., 1991). The results of the cytotoxicity evaluation and the antiherpes activity of the tested extracts are shown in Tables 2 and 3.
Among the octocoral tested extracts, only Eunicea succinea (STA2), Eunicea fusca and Pseudopterogorgia elisabethae (SAN ANDRES) showed antiviral activity. Their IC50 values ranged from 50 to 62.5 µg/mL, and their selectivity index values were 1.2, 2.2 and 3.0, respectively. The extracts of Eunicea species pointed to recognize them as a rich source of cembranoid and fuscoside diterpenes, and some sesquiterpenes (Rodríguez, 1995). Several biological activities as antimicrobial, cytotoxic, and acetyl choline inhibitors were reported for compounds isolated from soft octocorals belonging to this genus (Berrue & Kerr, 2009). Several cembranolides exhibiting cytotoxic and antibacterial activity have been isolated from E. succinea (Rodríguez, 1995). E. fusca contains fuscosides A-D, glycosydicaly bound diterpenes with lobane and eudesmane related diterpene skeleton that presented interesting anti-inflammatory activity (Shin & Fenical, 1991; Jacobson & Jacobs, 1992). On the other hand, P elisabethae has been widely studied by the presence of diterpenes, mainly pseudopterosins and related compounds that present proved activity as anti-inflammatory compounds (Correa et al., 2009). In Colombia, two chemotypes with a different and characteristic contents of pseudopterosins and secopseudopterosins has been reported as part of our research (Puyana et al., 2004) as well as several new compounds have been isolated (Duque et al., 2004, Duque et al., 2006).
The natural products chemistry of tropical marine sponges has been well investigated (Faulkner, 1998), and many sponge secondary metabolites have been isolated and characterized, including some with potent antiviral activity (Waddell & Pawlik, 2000).
For organic extract from the Niphates erecta, a moderate antiviral activity (SI=8) was shown (Cos et al., 2006). It has been reported that an anti-human immunodeficiency virus (HIV)-bioassay-guided fractionation of aqueous extracts of this caribbean sponge, led to isolation of a novel anti-HIV protein, named niphatevirin (O'Keefe et al., 1997). Therefore, the antiviral effect observed for Niphates erecta could be associated with the presence of this compound (Kleymann, 2005; Kucze et al., 2010).
Dragmacidon reticulatum was the extract that showed higher activity against HSV-1 (SI=11.7). For this genus, the occurrence of fosfolipids (Barnathan et al., 1992) and sterols (Sjöstrand et al., 1981) has been described and related to the antiviral activity detected for this extract (Hudson & Towers, 1999). For the Aka cachacrouense, a modest activity against the replication of the HSV-1 (SI=5.0) was found. The others organic extracts from marine sponges presented lower anti-HSV-1 activity (SI values ranged between 2.0 and 2.5).
Currently, more than 200 natural products with promising levels of antiviral activity have been isolated from aqueous or organic extracts of marine organisms. Continuous searching for and testing of natural compounds for their antiviral potential will likely lead to the discovery and development of a new generation of antiviral agents that can effectively control viral diseases in humans, as well as in other valuable animal species (Yasuhara-Bell & Lu, 2010).
Considering the results obtained, it can be stated that the tested extracts protect against viral infection, but the mechanism of their antiviral action and the active substances have not identified yet. Further studies are needed in order to verify which compounds could be responsible for this activity and how they exert their antiviral effects. Studies with these goals are under development in our laboratory.
This work was conducted as part of a bilateral (Brazil and Colombia) cooperative project financially supported by CNPq/MCT/Brazil (grant number 490151/2007-8) and COLCIENCIAS/Colombia (358-2007). CMO Simões and EP Schenkel are grateful to CNPq for their research fellowships. IT Silva, DD Lückemeyer and T Caon also acknowledged CAPES/MEC/Brazil and CNPq for their graduate fellowships.
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Received 28 Jul 2010
Accepted 16 Dec 2010
* Correspondence Cláudia M. O. Simões, Laboratório de Virologia Aplicada, Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina Campus Universitário Trindade, Florianópolis, 88040-900, SC, Brazil email@example.com, Tel.: +55 48 3721 5207, Fax: +55 48 3721 9258