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Mar. Drugs 2013, 11(11), 4176-4192; doi:10.3390/md11114176
Abstract: The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2.
The drug of choice for the prophylaxis and treatment of Herpex Simplex Virus (HSV) infections is acyclovir (ACV), which selectively inhibits HSV DNA replication with low host-cell toxicity. However, the intensive use of antiviral drugs has led to the emergence of resistant viruses [1,2,3]. Recently, De Clercq  described the evolution of antiviral agents against some viral infections, including HSV, confirming that the search for new antiviral agents is still relevant.
Pharmaceutical interest in marine organisms has provided thousands of new and novel compounds that have shown important biological properties, such as anticancer, antiviral, antiprotozoal, and antibacterial activities [2,5,6,7,8]. In this context, marine sponges have been a prolific source of diverse secondary metabolites with complex and unique structures [2,9,10,11,12,13]. Some of them were used as lead compounds to obtain new drugs that are currently used in clinics, such as acyclovir, vidarabine, cytarabine, eribulin mesylate, and others, that are now in clinical stages of evaluation such hemiasterlin [14,15,16]. In addition, several highly active compounds from marine sponges have been reported as new biologically active structures [17,18,19,20,21,22,23,24].
Petromica citrina (Porifera, Demospongie) belongs to a marine sponge genus that occurs only on the Brazilian coast . There are few studies with this species, and most of them describe the evaluation of different pharmacological properties such as antibacterial and antiviral activities for its aqueous extracts [26,27] and n-butanol fraction . Moreover, a restricted number of chemical investigations and a few bioactive constituents have been reported, in particular, a sulfated steroidal compound, identified as halistanol sulfate [29,30].
Recently, our research group described the anti-herpes activity of the n-butanol fraction of P. citrina . Thus, the aim of this investigation was to determine, through a bioguided study, the active compounds responsible for the anti-HSV-1 activity detected.
2. Results and Discussion
2.1. Bioguided Fractionation of the n-Butanol Fraction of P. citrina
In a previous screening of the anti-infective potential of marine invertebrates and seaweeds , we observed a promising activity for the n-butanol fraction (BF) obtained from the ethanolic crude extract of this sponge that led us to perform this study. Our goal was to isolate, through a bioguided study, the anti-herpes bioactive metabolites present in this fraction.
First, the BF fraction was submitted to several Sephadex LH-20 chromatography procedures yielding five fractions (Sep-1 to Sep-5), which were pooled based on thin-layer chromatography (TLC) similarity. Among these fractions, only fraction Sep-5 showed anti HSV-1 activity and was submitted to NMR analysis. The 1H NMR spectrum of Sep-5 displayed characteristic signals of the presence of halistanol sulfates as the major compounds. These major compounds were isolated by C18 column chromatography, yielding compounds 1 and 2 (Figure 1).
The complete structure of compound 1 was determined based on HSQC, HMBC, and COSY spectra, as well as by ESI mass spectrometry and by comparison with literature data [29,30,31,32,33]. The presence of three sulfate groups in the structure could be clearly defined by ESI mass spectrometry (m/z 731 [M − Na]−, m/z 611 [M − NaHSO4], m/z 491 [M − (NaHSO4)2] and m/z 354 [M − (NaHSO4)3]). These sulfate groups was also supported by the IR band (1230 cm−1).
In addition, the 1H NMR spectrum of compound 1 showed carbinol signals at δH 4.83 (sl), δH 4.76 (sl; J = 1.8 Hz), and δH 4.20 (dt; J = 11.0; 4.4 Hz), corresponding in the HSQC spectrum to the signals at δc 75.6 (CH-2 and CH-3), and δc 78.8 (CH-6), respectively. These data, together with characteristic signals of two methyl singlets at δ 0.70 (CH3-18) and δ 1.07 (CH3-19), suggested a sulfated sterol nucleus. The structure of the side chain of compound 1 was elucidated by analysis of 2D NMR data. The NMR spectra showed the presence of a side chain containing two secondary methyls at δ 0.95 (d; J = 6.4 Hz) and δ 0.84 (d; J = 6.8 Hz) attributed to positions C21 and C28, also based on HMBC data. The 1H NMR spectra revealed a singlet at δ 0.86 (9H), which was connected to carbon at δ 27.9, suggesting a t-butyl group on the side chain. HMBC correlations of carbons at δ 27.9 (C26, C27 and C29), δ 34.2 (C25), and δ 45.5 (C24) to the proton at δ 0.86 confirmed that C26, C27, and C29 were connected to C25. Therefore, compound 1 was identified as halistanol sulfate, a steroid previously reported for marine sponges such as Halichondria cf. , Epipolasis sp. , Petromica ciocalyptoides , Haliclona sp. , and Petromica citrina .
Halistanol sulfate (HS) was first reported in 1981 by Fusetani et al.  and, in that work, the authors only showed the 13C NMR data of HS. New compounds of the halistanol sulfate series (halistanol sulfates A to H) were isolated in the subsequent years [32,34], but the nomenclature and the chemical shift values in the 1H NMR spectra of the side chain are still not completely defined [29,32]. Therefore, it is important that the details of the structural elucidation of compounds 1 and 2 are also presented.
Compound 2 also showed the same halistanol steroidal nucleus signals, but with a shorter side chain, which was inferred by NMR data together with the information of the ESI mass spectrum. Moreover, the ESI/MS spectrum showed the presence of three sulfate groups (m/z 703 [M − Na]−; m/z 583[M − NaHSO4]; m/z 463 [M − (NaHSO4)2] and m/z 340 [M − (NaHSO4)3]) in the structure. As well as for compound 1 the presence of sulfate groups in the structure was also supported by the IR band (1226 cm−1). Although the 1H-NMR spectra of compound 1 displayed two methyl doublets at δ 0.95 and δ 0.84 on the side chain, corresponding to C21 and C28, respectively, the 1H NMR of compound 2 only one doublet signal at δ 0.94 (d; J = 6.6 Hz), corresponding to the C21 methyl group. In addition, the 1H NMR data did not show a t-butyl group at the end side of the chain. Furthermore, two new methyl signals at δ 0.87 (d; J = 6.6 Hz) and δ 0.89 (d; J = 6.6 Hz) were identified. Considering the J values of these protons, we could suggest the presence of an isopropyl on the side chain. Thus, based on the data obtained, compound 2 was identified as halistanol sulfate C, a steroid previously reported for Pseudoaxinissa digitata  and Epilopasis sp. . As far as we are aware, this is the first report of halistanol sulfate C for Petromica citrina.
Sulfated sterols have been described from a wide variety of marine organisms, such as sponges and echinoderms. Several of these sterols have a great structural diversity and broad spectrum of biological activities [35,36,37,38,39].
The first reported compound of the halistanol family was halistanol sulfate, isolated from the marine sponge Halichondria cf. moorei Bergquist . Important biological activities have been reported for this steroid sulfate, such as anti-HIV effects , cytotoxic activity against human hepatoma cells (QGY-7701), and chronic myelogenous leukemia cells (K562) . Afterwards, the same compound was isolated from Petromica ciocalyptoides and Topsentia ophiraphidites, showing inhibitory activity of Leishmania tarentola  and a wide spectrum of activity against resistant bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum, and Neisseria gonorrheae .
Thus far, eight sulfated sterols have been described with this fundamental nucleus and named as halistanol sulfates A to H (Figure 2). All of them are characterized by the same 2β, 3α, 6α-trisulfoxy functionalities, differing only in their side chains [32,34,35]. The most promising pharmacological activities described for these compounds were the anti-HIV-1 and anti-HIV-2 effects for halistanol sulfates F and G .
In addition, there are many other reports about different members of the halistanol series that have shown important pharmacological properties. One of the first reported members of this series was ibisterol sulfate, isolated from Topsentia sp., which showed anti-HIV activity . Other examples of halistanol-type compounds with antiviral activity are weinbersterol disulfates A and B isolated from the sponge Petrosia weinbergi which exhibited activity against leukemia virus (FeLV), mouse influenza virus (PR8), and mouse coronavirus (A59) replication .
As compounds with sulfated groups are described to have antiviral properties [34,38,43,44,45,46], and due to the anti-herpetic activity shown by the BF fraction, we decided to verify the anti-HSV-1 activity of compounds 1 and 2 and the TSH fraction and to elucidate their mode of action.
3. Experimental Section
3.1. General Experimental Procedures
General 1D and 2D NMR experiments were performed on a Bruker Avance 2 (500 MHz) instrument at 500 MHz for 1H and 125 MHz for 13C. All spectra were recorded in CD3OD using the signals of residual non-deuterated solvent as internal reference. Mass spectrometric analyses were performed using a Bruker micrOTOF-Q II mass spectrometer (Bruker® Daltonics, Billerica, MA, USA), equipped with ESI. Multi-point mass calibration was carried out using a mixture of sodium formate from m/z 50 to 900. Data acquisition and processing were carried out using the Bruker Compass Data Analysis version 4.0 software supplied with the instrument. All the analytical solutions (0.5 mg/mL) were prepared using methanol LCMS grade. Compounds were infused into the source using a KDS 100 syringe pump (KD Scientific, Holliston, MA, USA) at a flow rate of 180 mL/min. General MS conditions: Capillary 3.5 kV (negative ion mode), dry heater 180 °C, nebulizer 0.4 bar, dry gas (N2), 4 L/min (UMYMFOR/UBA). Silica gel 60 (70–230 mesh) Merck®, RP18 (Fluka®, Buchs, Switzerland), Sephadex LH-20 (GE healthcare®, Chalfont St Giles, UK), TLC analysis was performed on Silica gel F254 and RP18 plates (Sigma-Aldrich®, St. Louis, MO, USA).
3.2. Sponge Collection
Petromica citrina was collected from January to July 2010 at Xavier Island (27°36′39′′ S; 48°23′32′′ W), Santa Catarina State, Brazil, at a depth of 9–17 m, and immediately frozen. The material was identified by Dr. João Luís Carraro and voucher specimens were deposited in the Porifera collection of the Museu de Ciências Naturais da Fundação Zoobotânica do Rio Grande do Sul, Brazil (MCNPOR 8777, 8778, 8779, 8780, 8781).
3.3. Extraction and Isolation of Compounds 1 and 2
The frozen sponge (1700 g, wet) was exhaustively extracted with ethanol for three days at room temperature. The crude ethanolic extract (CHE) was filtered, the ethanol was eliminated under reduced pressure, and the gummy residue was suspended in H2O before being extracted successively with ethyl acetate (EtOAc) and n-butanol (n-BuOH) (3 × 500 mL) yielding three fractions: EtOAc (EAF), n-BuOH (BF) and aqueous residue (AR), respectively. Next, the BF fraction (2.0 g) was subjected to Sephadex LH-20 column chromatography (790 mm × 25 mm) using methanol (MeOH) as eluent. A total of 180 tubes (20 mL) were collected and combined into five fractions (Sep-1, 650 mg; Sep-2, 450 mg; Sep-3, 350 mg; Sep-4, 250 mg; and Sep-5, 300 mg) based on Silica gel thin-layer chromatography (TLC) similarity. Because the fraction Sep-5 (named TSH fraction) showed only one spot by TLC analysis, this fraction was forwarded to 1HNMR analysis and proved to be a mixture of halistanol sulfate (Compound 1) and halistanol sulfate C (Compound 2) as the major compounds.
The TSH fraction (200 mg) was then dissolved in methanol and submitted to a reversed-phase column chromatography (300 mm × 20 mm) packed with RP18 as stationary phase and MeOH:H2O (1:1 v/v) as mobile phase. This procedure resulted in two isolated compounds: halistanol sulfate (30 mg, compound 1) and halistanol sulfate C (12 mg, compound 2). Figure 4 shows the steps of purification.
Halistanol sulfate (1): White amorphous powder; IR (KBr)νmax 3442, 2953, 1643, 1392, 1230, 1068 cm−1; 1H NMR (CD3OD, 500 MHz) δ 4.83 (1H, sl, H-2), 4.76 (1H, sl, J = 1.8 Hz, H-3), 4.20 (1H, dt, J = 4.4, 10.9 Hz, H-6), 2.37 (1H, dl, J = 10.2 Hz, H-7a), 2.30 (1H, dl, J = 14.5 Hz; H-4a), 2.11 (1H, dl, J = 14.3 Hz, H-1a), 2.01 (1H, dl, J = 12.4 Hz, H-12a), 1.86 (1H, m, H-16a), 1.81 (1H, tl, J = 14.4 Hz, H-4b), 1.69 (1H, m, H-16b), 1.63 (1H, m, H-5), 1.64 (1H, m, H-15a), 1.58 (1H, m, H-22a), 1.55 (1H, m, H-11a), 1.54 (1H, m, H-8), 1.49 (1H, dd, J = 4.0, 14.3 Hz, H-1b), 1.38 (1H, m, H-20), 1.33 (1H, m, H-11b), 1.29 (1H, m, H-12b), 1.15 (1H, m, H-14), 1.12 (1H, m, H-15b), 1.10 (1H, m, H-17), 1.07 (3H, s, H-19), 1.02 (1H, m, H-7b), 0.99 (2H, m, H-24), 0.95 (3H, d, J = 7.4 Hz, H-21), 0.90 (1H, m, H-22b), 0.78 (2H, m, H-23), 0.86 (9H, sl, H-26, 27, 29), 0.84 (3H, d, H-28) 0.78 (1H, m, H-9), 0.70 (3H, sl, H-18); 13C NMR (CD3OD, 125 MHz): δ 78.8 (CH, C-6), 75.6 (CH, C-3), 75.6 (CH, C-2), 57.7 (CH, C-17), 57.6 (CH, C-14), 55.8 (CH, C-9), 45.5 (CH, C-24), 45.4 (CH, C-5), 43.8 (C, C-13), 41.3 (CH2, C-12), 40.1 (CH2, C-7), 39.2 (CH2, C-1), 37.7 (CH2, C-22), 37.7 (CH, C-20), 37.7 (C, C-10), 35.2 (CH, C-8), 34.1 (C, C-25), 29.2 (CH2, C-16), 27.9, (CH3, C-26), 27.9 (CH3, C-27), 27.9 (CH3, C-29), 25.2 (CH2, C-15), 25.1 (CH2, C-4), 21.9 (CH2, C-11), 22.03 (CH2, C-23), 19.6 (CH3, C-21), 15.3 (CH3, C-19), 15.0 (CH3, C-28), 12.5 (CH3, C-18). ESI-MS m/z 731.2198 [M − Na]− (calcd for C29H49Na3O12, 754.2100).
Halistanol sulfate C (2): White amorphous solid; IR (KBr)νmax 3442, 2949, 1625, 1384, 1226, 1070 cm−1; 1H NMR (CD3OD, 500 MHz) δ 4.83(1H, m, H-2), 4.76 (1H, q, J = 2.7 Hz, H-3), 4H-2), 4.20 (1H, td, J = 11.1, 4.4 Hz, H-6), 2.37 (1H, dt, J = 12.3, 4.4 Hz, H-7a), 2.30 (1H, dt, J = 15.0, 2.8, 1.1 Hz; H-4a), 2.11 (1H, dd, J = 14.7, 1.7 Hz, H-1a), 2.01 (1H, dl, J = 12.7, 3.5 Hz, H-12a), 1.86 (1H, m, H-16a), 1.81 (1H, ddd, J = 15.0, 13.2, 2.8 Hz, H-4b), 1.63 (1H, m, H-5), 1.61 (1H, m, H-15a), 1.53 (1H, m, H-8), 1.52 (2H, m, H-8, H-25), 1.48 (1H, dd, J = 14.7, 3.9 Hz, H-1b), 1.38 (1H, m, H-20), 1.31 (1H, qd, J = 13.1, 3.5 Hz, H-11), 1.29 (1H, m, H-16b), 1.14 (1H, m, H-12b), 1.12 (3H, m, H-14, H-15a, H-17), 1.07 (3H, sl, H-19), 1.02 (1H, m, H-7b), 0.94 (3H, d, J = 6.5 Hz, H-21), 0.9–1.4 (6H, m, H2-22, H2-23, H2-24), 0.87 (3H, d, J = 6.6 Hz, H-26), 0.89(3H, d, J = 6.6 Hz, H-27), 0.78 (1H, m, H-9), 0.69 (3H, sl, H-18). 13C NMR (CD3OD, 125 MHz): δ 78.7 (CH, C-6), 75.5 (CH, C-2), 75.5 (CH, C-3), 57.6 (CH, C-17), 57.5 (CH, C-14), 55.8 (CH, C-9), 45.3 (CH, C-5), 43.8 (C, C-13), 41.2 (CH2, C-12), 40.8 (CH2, C-24), 40.0 (CH2, C-7), 40.0 (CH2, C-7), 39.2 (CH2, C-1), 37.6 (C, C-10), 37.3 (CH2, C-22), 37.0 (CH, C-20), 35.1 (CH, C-8), 29.2 (CH2, C-16), 29.1 (CH, C-25), 25.1 (CH2, C-4), 25.0 (CH2, C-23), 24.9 (CH2, C-15), 23.1 (CH3, C-26), 22.9 (CH3, C-27), 21.8 (CH2, C-11), 19.1 (CH3, C-21), 15.2 (CH3, C-19), 12.5 (CH3, C-18). ESI-MS m/z 703.2032 [M − Na]− (calcd for C27H45Na3O12S3, 726.1800).
3.4. Anti-HSV-1 Activity
3.4.1. Virus and Cell Line
HSV-1 (KOS strain, Faculty of Pharmacy, University of Rennes, France) was propagated in Vero cells. Viral stocks were stored at −80 °C and titrated based on plaque forming units (PFU) counted by plaque assay as previously described .
Vero (ATCC: CCL 81) cells were grown in Eagle’s minimum essential medium (MEM; Cultilab®, Campinas, Brazil) supplemented with 10% fetal bovine serum (FBS; Gibco®, Carlsbad, CA, USA), 100 U/mL penicillin G, 100 µg/mL streptomycin, and 25 µg/mL amphotericin B (Cultilab®), and maintained at 37 °C in humidified 5% CO2.
3.4.2. Cytotoxicity Assay
Vero cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide—Sigma-Aldrich®, St. Louis, MO, USA) . Briefly, confluent Vero cells were exposed to different concentrations of samples for 72 h, and after incubation, the 50% cytotoxic concentration (CC50) of each one was calculated as the concentration that reduces cell viability by 50%, when compared to untreated controls.
In summary, these results suggest that the TSH fraction and compounds 1 and 2 present antiherpes activity through the reduction of viral infectivity, inhibition of virus entry into the cells, and by the impairment of levels of ICP27 and gD proteins of HSV-1.
The relevant selectivity index of 15.33 of TSH fraction and its content (compounds 1 and 2) as well as the strong synergism effects observed suggest that the detected anti-HSV activity could be explained by the synergic effects of these major compounds present in the TSH fraction.
We would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq, MCTI (Grants 151561/2008-7, 306917/2009-2 and 471307/2011-4)], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [PNPD, CAPES, MEC (Grant 2207/2009)], Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC/BIODIVERSIDADE, Grant 14170/2010) for their financial support. The authors are also grateful to CNPq and CAPES for their research fellowships.
Heteronuclear Multiple Bond Correlation
Heteronuclear Single Quantum Correlation
Herpes Simplex Virus
Minimal Essential Medium
Nuclear Magnetic Resonance
Plaque Forming Units
Conflict of Interest
The authors declare no conflict of interest.
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