Anti-HIV-1 activity in human primary cells and Anti-HIV-1 RT inhibitory activity of extracts from the red seaweed Acanthophora spicifera

1 Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, P. O. Box 100.644, 24001970, Niterói, RJ, Brazil. 2 Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, 24020-141, Niterói, RJ, Brazil. 3 Laboratório de Imunologia Clínica, Instituto Oswaldo Cruz, FIOCRUZ, 21.040-900, Rio de Janeiro, RJ, Brazil. 4 Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, FIOCRUZ, 21.040-900, Rio de Janeiro, RJ, Brazil.


INTRODUCTION
Since the discovery of the human immunodeficiency virus, many drugs have been developed in an attempt to inhibit its replication.However, HIV is resistant to treatment with known drugs (Kuritzkes, 2007 al., 2008;Manhanzva et al., 2015).This is due to the high mutation rate of HIV and does not have an effective mechanism for error correction during replication.One of the strategies adopted was the combination of two or more drugs known as Highly Active Antiretroviral Therapy (HAART) (Marrazzo et al., 2014).Such treatment may reduce viral load to undetectable levels in the blood and provide long-lasting clinical benefit.However, some patients do not respond to this treatment, making the search for new molecules with anti-HIV activity an urgent need.Seaweeds are a source of many bioactive compounds.Several extracts, fractions, and natural products isolated from seaweeds have demonstrated effective anti-HIV activity (Vo and Kim, 2010), making it an interesting base from which to develop new medicines.Of the seaweeds, red seaweeds produce natural products such as acetogenins (Gutiérrez-Cepeda et al., 2011), sesquiterpenes (Chen et al., 2016), monoterpenes (Silva et al., 2015), bromophenols (Popplewell and Northcote, 2009), and sulfated polysaccharides (Coura, 2012) that can be used for anti-HIV drug development.
Acanthophora spicifera (Rhodophyta) is an excellent model for studies of biological activity in Brazil because it has natural banks on the coast of Rio de Janeiro, is easily identified (Perrone et al., 2006), has an experimental field cultivation described in the literature (Kaliaperumal et al., 1986), and is a part of the food chain to other species, indicating a low toxicity (Cruz-Rivera and Villareal, 2005).Furthermore, fractions rich in sulfated polysaccharides from the red seaweed Acanthophora are an effective antiviral against HSV-1 and HSV-2 strains (Duarte et al., 2004).Therefore, the objective of this study was to evaluate the antiviral effect of extracts from A. spicifera regarding HIV-1 replication in human primary cells and their ability to inhibit the enzyme reverse transcriptase.

Cell and virus
Peripheral blood mononuclear cells (PBMCs) from healthy human donors (confidential information) were obtained through density centrifugation over Ficoll-Hypaque (Sigma) as described by Yeap et al. (2007).Cells were re-suspended in a RPMI 1640 medium supplemented with 10% fetal bovine serum and stimulated with 5 g/mL −1 of phytohemagglutinin (PHA, Sigma) for three days and further maintained in culture medium containing 5 U/mL −1 of recombinant human interleukin-2 (Sigma).The viral strain HIV Ba-L (R5-tropic) Virus type 1 was donated by the National Institutes of Health (NIH, USA) and kept in storage at -80°C.

Cytotoxicity assay
The cytotoxicity of extracts from the red seaweed Acanthophora spicifera was assessed by monitoring the conversion of MTT 3-(4,5dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide (Sigma-Aldrich) to formazan as previously described (Mosmann 1983) with some modification.Peripheral blood mononuclear cells were maintained in 96-well plates containing 2x10 5 cells per well and treated with increasing concentrations of the extracts (6.75, 12.5, 25, and 50 µg/mL -1 in DMSO) for one week at 37°C under a 5% CO2 humidified atmosphere in triplicate.The DMSO concentration in the final volume of the well was less than 0.5%.After seven days of incubation, the supernatant was removed, and the MTT (20 μL of 5 mg/mL −1 in medium) was added to each well.Plates were incubated for two hours at 37°C with a 5% CO2 atmosphere.The 96 well plates were then centrifuged at 100 X g for ten minutes, the supernatant was discarded, and 100 µl of DMSO was added to each well.Finally, the optical density was measured at 545 nm on a microplate reader.The result was expressed as the 50% loss of viable cells concentration (CC50).The compound concentration required to reduce the optical density of MTT in relation to not treated cells was calculated using linear regression.

Anti-HIV-1 activity in human primary cells
PBMCs were maintained in 96-well plates containing 2x10 5 cells per well and stimulated with IL-2.PBMCs were infected with 5 ng/mL -1 of the HIV-1 Ba-L strain and incubated for two hours at 37°C under a 5% CO2 humidified atmosphere.Then, the 96 well plate was centrifuged at 100 X g for ten minutes, its supernatant was removed, new medium containing 50 µg/mL -1 of extracts diluted in DMSO was added, and it was incubated at 37°C with a 5% CO2 atmosphere for seven days.After incubation, the supernatant was collected, and the production of the p24 antigen was evaluated using the immunoassay ELISA (Zeptometrix).The absorbance was measured in a spectrophotometer at 450 nm.Each value is expressed as mean±SEM in triplicate experiments.The average value of absorbance was used to determine the concentration of p24 in the samples treated by comparison to a standard curve (Trinchero et al., 2009).

Anti-HIV-1 RT inhibitory activity
E. coli strain BL21 (DE3) was used as a recipient for DNA transformations.Overnight, the E. coli cells transformed with the plasmid containing RTp66 and RTp51 HIV-1 genes were cultured in Luria-Bertani (LB) containing ampicillin (100 µg/ mL -1 ) under shaking at 220 rpm at 37°C.These overnight cultures were used as the inoculum for one liter of LB medium containing 100 µg/mL of ampicillin.Cells were grown for six hours at 37°C with vigorous shaking and then induced with isopropyl-b-D-thiogalactopyranoside (IPTG) (1 mM) for two hours.Cells were harvested by centrifugation (5000 x g, 15 min), and bacterial lysates were prepared using a lysis buffer (50 mM Tris-HCl (pH 7.9 at 4°C), 60 mM NaCl, 1 mM EDTA, and lysozyme/DNAse I treatment.Clarified lysates were used for the isolation of the p51/p66 heterodimeric RT.The active RT heterodimer was purified using the MagneHis™ Protein Purification System according to the manufacturer's instructions.Data are expressed as mean±S.E. of three independent experiments.The CC50 of each extract was calculated using regression line.The ability to inhibit the enzyme HIV-1 reverse transcriptase was evaluated using a fluorescence RT assay kit (EnzChek® Molecular Probes) according to the manufacturer's protocol.Briefly, 20 μL of Nogueira et al. 623 reaction mixture containing a poly(A) ribonucleotide template/oligo d(T)16 primer and dTTP was added to the wells of a microtiter plate and mixed with 5 μL of increasing concentrations of the EtOAc extract.Finally, one μL of the enzyme (15−80 ng/mL) in reaction buffer was addedand incubated at 37ºC for one hour.2 μL of 200 mM EDTA was added to stop the reaction.Fluorescence intensity was measured using a microplate reader (Spectramax-M4 Molecular Devices) (ex.480 nm, em.520 nm) after the addition of 173 μL of fluorescent PicoGreen® reagent, which selectively binds to dsDNA or DNA-RNA heteroduplexes over single-stranded nucleic acids or free nucleotides.Efavirenz was used as a positive control.The IC50 values were determined using Prism5 (GraphPad Software).All assays were performed in triplicate.

Chemical profile of extract
To analyze the chemical profile, a 5 mg aliquot of EtOAc extract was removed and analyzed by proton nuclear magnetic resonance (NMR-H).The NMR spectra were obtained on a Varian Unity Plus 300 at 300 MHz.The internal reference standard was TMS, and the samples were dissolved in CDCl3.

RESULTS AND DISCUSSION
The CC 50 values of the extracts are reported in Table 1.
The methanol and ethyl acetate extracts showed lower cytotoxicity in comparison with the extracts prepared in dichloromethane and acetone.
The percentages of p24 in the supernatants of each extract can be seen in Figure 1.When the culture supernatant infected with the Ba-L strain of HIV-1 was treated with 50 μg/mL -1 of the extract obtained by organic solvent ethyl acetate, it was possible to observe approximately 60% reduction in the p24 levels.Treatment of infected cells with ethyl acetate extract resulted in a reduction of p24 level compared to infected untreated cells.Therefore, the extract obtained with solvent ethyl acetate was used to evaluate the ability to inhibit the activity of reverse transcriptase enzyme, an important step of HIV replication cycle.
The inhibitory activity of the EtOAc extract against HIV-1 RT is shown in Table 2.The chemical profile of ethyl acetate extract from A. spicifera was analyzed with NMR.The ethyl acetate extract showed signs in the chemical shift region from 7 ppm, which is characteristic of aromatic compounds (doublet in 7.53 ppm, triplet in 7.36 ppm and double doublet in 7.13 ppm).
Cytotoxicity is critical in drug development (Putnam et al., 2002).Natural seaweed products have demonstrated low levels of cytotoxicity (Karadeniz et al., 2014) even lower than commercial drugs such as AZT (Barbosa et al., 2004).The toxicity of A. spicifera extracts has already been studied in mice (Naqvi et al., 1980) and Vero cell line (Duarte et al., 2004).However, the present article reports for the first time the cytotoxicity of extracts from A. spicifera in human cells.
Compared with the data found in this article, treatment using AcOEt extract from A, spicifera showed more efficient results.The partition in ethyl acetate from brown seaweed Ecklonia cava had anti-HIV-1 activity, and this activity was confirmed by the presence of phenolic compounds.Other phenolic compounds such as flavonoids also showed effective anti-HIV activity (Casano et al., 2010;Wang et al., 2014).We evaluated the presence of phenolic compounds in the extracts obtained in increasing polarity from red seaweed A. spicifera by 1 H-NMR.Only the extract obtained with ethyl acetate showed phenolic compounds.These data corroborate the results obtained by Zeng et al. (2001), which showed the isolation of two phenolic compounds from the extract of A. spicifera obtained in ethyl acetate.Therefore, we believe that the anti-HIV effect of the EtOAc extract of red seaweed A. spicifera is due to the presence of phenolic compounds.
In a Korean study, 26 extracts from red seaweeds were tested to evaluate the inhibition of reverse transcriptase (Ahn et al., 2002).Most of the extracts were not able to inhibit more than 65% of the enzyme activity when tested at a concentration of 200 µg/mL.These data indicate that our results interesting, since the AcOEt extract was able to inhibit 80% of enzyme activity at a lower concentration.Ahn et al. (2004) demonstrated that the inhibitory effect of brown seaweed E. cava was caudal by the presence of phenolic compounds.In conclusion, the AcOEt extract from red seaweed A. spicifera presents an efficient activity against HIV-1 virus.Finally, in accordance with the observed data, the inhibitory activity of the ethyl acetate extract in HIV reverse transcriptase-1 may be due to presence of aromatic compounds.
; Hirsch et *Corresponding author.E-mail: valerialaneuville@gmail.com Tel: +55 (21) 2629-2296.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

Figure 1 .
Figure 1.Histograms showing p24 levels found in the supernatant of untreated culture (Virus Control -CV) and treated with the different extracts in dichloromethane (CH2Cl2), ethyl acetate (AcOEt), acetone (Me2CO) and methanol (MeOH).Each value is expressed as mean±SEM in triplicate experiments.*p<0.05,significant value as analysed by Tukey's multiple comparison test.

Table 2 .
Inhibitory Effects of ethyl acetate extract from Red Seaweed Acanthophora spicifera and Efavirenz on HIV-1 Reverse Transcriptase.Data are expressed as mean±S.E. of three independent experiments.The IC50 of each extract was calculated using regression line.
conducted used partitions of CH 2 Cl 2 /MeOH, hexane, CH 2 Cl 2 , and CH 2 Cl 2 /EtOAc with brown seaweed Dictyota menstrualis.This group showed an inhibition in p24 levels of approximately 40% when tested at a concentration of 50 g/mL -1 of the extract prepared in ethyl acetate, there was approximately 60% decrease in p24 levels.A similar study was