ARTICLE
Potential antiviral activity of on the replication of Herpes Simplex Virus type 1

INTRODUCTION

Marine organisms comprise over half a million species. Due to their unusual living environment in comparison with terrestrial organisms, marine organisms produce a variety of substances, which quite often have various unprecedented chemical structures. Issues such as competition for space and predation have originated new biochemical pathways for these organisms, providing essential metabolites for their adaptation¹. Over 40 novel natural product compounds are now commercially available, including antiviral products isolated from various marine organisms, which provide alternative therapeutic drugs^2,3.

A literature survey revealed that Gorgonian corals have proven to be a prolific source of a variety of biologically active compounds with cytotoxic effects on human leukaemia⁴, such as being inhibitors of acetylcholine receptors^5,6,7. The species of corals in tropical or temperate waters of the western North Atlantic are relatively well known, but those found at the south of the Amazon River are not, despite having common elements with the fauna of the Caribbean octocorals⁸. Silva & Pérez⁷, in 2002, reported 59 species of octocorals on the Brazilian coast based on the biosynthetic origin of bioactive terpenes. This classification could provide a system to produce ex situ compounds⁹.

Almost 90% of people worldwide have one or both HSV-1 and HSV-2 viruses. In developed countries, the acquisition of HSV-1 is delayed from early childhood to adolescence or young adulthood¹⁰. Herpes simplex virus (HSV) belongs to Herpesviridae , subfamily Alphaherpesvirinae , and contamination occurs through direct contact with infected secretions, mostly during infancy, with clinical manifestations varying from labial lesions to gingivostomatitis, keratoconjunctivitis, and genital infections¹¹. The virus persists for life in local sensory ganglia and reactivation depends on the status of the patient's immune system. The primary infection or virus reactivation is the cause of herpes encephalitis (HSE), and both demonstrated the need for new drugs due to the increasing resistance to acyclovir, penciclovir, ganciclovir, foscarnet, and cidofovir¹². Resistant viral isolates can be observed especially in immunocompromised patients, in patients with HIV, and in recipients of solid organ or bone marrow transplants who are treated with antivirals for long intervals. Presently, the main focus is to circumvent this problem through the development of broad-spectrum antivirals, in particular those targeting common cellular pathways¹³. For HSV-1, the growing resistance makes the search for innovative antivirals both necessary and urgent^14,15.

This study involved an evaluation of the fractions obtained from Plexaurella regia, Plexaurella grandiflora and Muriceopsis sulphurea, all endemic on the Brazilian coast, as antivirals against HSV-1.

METHODS

RESULTS

The octocorals were collected by scuba divers in the Marine Park of Recife de Fora, Porto Seguro, Bahia, and stored in ethanol to obtain the crude extracts. Specimens were identified at Museu Nacional, UFRJ^20,21. The extracts were separately prepared in organic solvents of different polarities (hexane, dichloromethane (DCM), and ethyl acetate (EtOAc)), and evaporated under reduced pressure except P. grandiflora , which was extracted with DCM and EtOAc (Table 1).

Table 1:

Crude extracts of P. grandiflora, P. regia, and M. sulphurea, extracted successively with hexane, dichloromethane (DCM), and ethyl acetate (EtOAc).

Analysis by thin layer chromatography (TLC) of crude extracts of each coral revealed that hexane and DCM extracts of P. regia and M. sulphurea showed the same chromatographic profile while P. grandiflora corresponded to that observed in other species, i.e., DCM extract provided a separate chromatogram of the EtOAc extract.

For a detailed analysis, each crude extract (15 mg) was filtered on a column by adsorption on silica gel 60-70 to 230 mesh, using DCM (30 mL). After evaporation of solvents, an aliquot of each sample (1 mg/mL) was analyzed by gas chromatography-mass spectrometry (GC-MS), showing that P. regia and M. sulphurea crude extracts have a similar chemical composition with predominance of sterols, fatty acids, and fatty acid esters. While P. grandiflora produces a variety of sesquiterpenes, P. region produces valencene and sesquiterpene. However, for M. sulphurea, such metabolite was not detected in the fractions analyzed by GC-MS (Tables 2, ³ and ⁴).

Table 2:

Secondary metabolites from Plexaurella grandiflora, analysed by gas chromatography coupled with mass spectrometry (GC-MS).

*Molecular ion.

Table 3:

Secondary metabolites obtained from Plexaurella regia, analysed by gas chromatography coupled with mass spectrometry (GC-MS).

Table 4:

Secondary metabolites obtained from Muriceops sulphurea, analysed by gas chromatography coupled with mass spectrometry (GC-MS).

Viability and antiviral activities of the octocorals fractions were identified according to the species and the solvent used for purification. Thus, P-1 corresponds to Plexaurella regia , P-2 to Plexaurella grandiflora, M-1 to Muriceopsis sulphurea and each one adds 1 for hexane, 2 for dichloromethane and 3 for ethyl acetate.

Tests of cell viability performed in Vero cells from kidneys of African green monkeys (Cercopithecus aethiops ) showed that all extracts from Muriceopsis sulphurea provided very low CC₅₀ values and consequently have not been investigated in antiviral assays (Figure 1). The same was observed for hexane and dichloromethane extracts from P. grandiflora . Moreover, the results obtained with the ethyl acetate were the most promising, reaching a CC₅₀ value similar to the reference, the LCA²², reaching 132 µg/ mL (Figure 1). From analyzing the results separately for each fraction, we identified the fractions P-1.3, P-2.2 and P-2.3 as a dose dependent toxicity ensuring that the toxicity of the substance is in accordance to the administered dose.

Figure 1:

Effect of coral extracts on Vero cell viability. The cytotoxic concentration that causes 50% lysis and cell death (CC₅₀) were obtained by linear regression of measurements obtained from the average of triplicates of four different concentrations (25, 50, 75, and 100 µg/ml). P-1 refers to Plexaurella regia , P-2 to Plexaurella grandiflora, and M-1 to Muriceopsis sulphurea . According to the solvent used for purification, 1 - hexane, 2 - dichloromethane and 3 - ethyl acetate.

The antiviral assay was performed with the fractions that showed the highest values of CC50. It has been also identified the fraction P-1.3 as the compound with antiviral activity (Figure 2). Concentrations were 100% effective in inhibiting viral production, even at a concentration of 2.5 µg /mL (Figure 2). These data were used for the determination of TI (Therapeutic Index), which indicates the safety of the substance as an antiviral drug. TI is determined by the ratio CC₅₀/ EC₅₀ (Table 5).

Figure 2:

Concentrations of coral fractions that inhibit 50 % of viral production. The antiviral effect that causes 50 % of inhibition (EC₅₀) was obtained by plaque assay with 2,5, 5, 10, 12, 15 and 20 µg/ml of hexane fraction from Plexaurella regia (P1.3), dichloromethane (P 2.2), and ethyl acetate (P 2.3) from Plexaurella grandiflora in Vero cells infected with HSV-1 (MOI 1).

Table 5:

Antiviral activities of extracts from Plexaurella regia, Plexaurella grandiflora, and Muriceopsis sulphurea on HSV-1 replication.

*Acyclovir; **data obtained by Castro¹⁷; ***CC50=100mg/mL; a: In this assay, Vero cells were cultured in the presence of the extracts for 72 hours at 37°C; b: Antiviral assay that determined the concentration of the extract that reduces the titer of HSV-1 by 50% in Vero cell culture; c: Selectivity Index means how safe some fractions could be as antivirals as reference EC₅₀ = 2.5 µg/mL.

DISCUSSION

Recently, the great challenge for HSV infections lies in the search for drugs that could control the development of resistance and latency, especially in AIDS patients and individuals after hematopoietic stem cell transplantation (HSCT)²³. Resistance to ACV is mediated in 95% of the cases by mutations in the TK gene and in 5% of the cases by mutations in the DNA pol gene, resulting in the alteration of enzyme activity^15,24.

Genotyping findings confirmed that the UL23 TK gene of HSV-1 has an uncommonly high polymorphism^15,25. The recently discovered inhibitors of the HSV helicase-primase are the most potent development candidates today, but they depend on long-term studies²⁶.

Marine sponges are considered notable sources of bioactive compounds found in the marine environment. The most important antiviral reported so far is the nucleoside Ara-A (vidarabine), isolated from the sponge Tethya crypta. It inhibits viral DNA polymerase and DNA synthesis of herpes, vaccinia, and varicella zoster viruses²⁷. In our study we analysed crude extracts, believing that natural products have great relevance in pharmacology due to their high chemical diversity. The purification of P. regia and P. grandiflora extracts revealed fatty acid esters as common substances for both algae. Additionally, it is consistent with previous antiviral studies that showed that fatty acid esters, specially C14 and C15 isoforms, are able to inactivate enveloped viruses like herpes²⁸. These bioactive molecules are often secondary metabolites, whose main function is to enable and/or modulate cellular communication and defence.

CONCLUSION

The highest antiviral activity for HSV-1 was obtained by P. regia extract isolated by ethyl acetate. The majority compound is valencene and its molecular formula is C15H24. In conclusion, we suggest Plexaurella spp as a source for anti-HSV compounds.