Antiviral Activity of Geopropolis Extract from Scaptotrigona Aff. Postica against Rubella Virus

The search for functional foods, which possess bioactive substances, is a new trend for the obtention of alternative and more effective treatments of many diseases with fewer side effects. Geopropolis, elaborated by stingless bees, is a mixture of plant resin sources, wax and soil. In the geopropolis from Scaptotrigona affinis postica (Latreille, 1807), (Hymenoptera, Apidae, Meliponini) was not observed the presence of soil. In a previous study, the extract of geopropolis provided by the beekeeper, from S. postica of Barra do Corda, Maranhão State, exhibited potent antiviral activity against herpes simplex virus. In this study, the propolis extract was prepared experimentally and characterized by RP-HPLC-DAD-ESI-MS/MS. The objective of this study was to evaluate the antiviral activity of an experimentally prepared geopropolis extract from S. postica against Rubella Virus infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Rubella virus infection of susceptible women during the first trimester of pregnancy, often results in a combination of birth defects in newborns. There is not an effective treatment for rubella virus infection. Different protocols were carried out to evaluate, the antiviral effect of geopropolis extract on the viral replication of infectious RV. Cell viability and cell proliferation assays indicated that this geopropolis was not toxic to cultured SIRC cells. In the viral binding assay, antiviral assay, real-time PCR, and transmission electron microscopy, was observed that different concentrations of geopropolis (17, 34 and 68 μg/mL) was able to inhibit the binding of virions to the cell receptor and the production of infectious RV particles in post treated and pre treated infected SIRC cells. The antiviral activity could to be attributed to the high contents of the apigenin derivatives, vicenin-2 and schaftoside. As far as we know, this is the first report about the antiviral activity of geopropolis from Scaptotrigona postica against a Togaviridae virus.


Introduction
Rubella virus is classified as the only member of the genus Rubivirus belonging to the family Togaviridae.Rubella virus (RV) is a positive-sense, single-stranded RNA virus, hemagglutinin-containing surface projections.Chikungunya and Mayaro virus belong to the same family.Rubella, known more popularly as German measles, is a childhood disease, possessing a worldwide distribution (Parkman, 1996).Rubella virus is formed by the structural polypeptides, the membrane glycoproteins E1 and E2 and a single nonglycosylated RNA-associated capsid protein C (Lee & Bowden, 2000).In a recent research, five genotypes of RV, 1E, 2B, 1J, 1I, and 1a were identified (Martí nez-Torres et al., 2016).The vaccine is a live attenuated preparation of the virus (RA 27/3), which induces immunity by producing a modified rubella infection (Parkman, 1996).The vaccines produced by attenuated rubella virus are effective, however possess some side effects and are uneffective for pregnant women and immunodeficiency people (Petrova et al., 2016).
Postnatal rubella infection causes mild febrile illness accompanied by maculopapular rash and lymphadenopathy, while maternal infections during the first trimester of pregnancy result in a combination of birth defects in newborns, known as congenital rubella syndrome (Plotkin, 2011).RV can establish persistent infection in the developing fetus.Beside this, its replication can induce multiple pathological changes (Curti et al., 2013).It is estimated that more than 100,000 cases of congenital rubella syndrome occur in developing countries every year, representing a considerable social and economic burden (-WHO | World Health Organization,‖ 2017).In a study carried out among 2012 and 2013, 68,968 rubella cases were registered in 28 countries of the WHO European Region (Muscat et al., 2014).The treatment for this virus infection is limited, since the commonly used antiviral drugs, acyclovir or immunoglobulin, are inefficient in the elimination of RV from chronically infected hosts (Gualberto et al., 2013).
Propolis is produced by Apis mellifera (Apidae) from resin of the leaf buds of numerous tree species, like birch, poplar, conifers, pine, alder, willow, palm, Baccharis dracunculifolia and Dalbergia ecastaphyllum (Huang et al., 2014;Li et al., 2016).Meliponinae is genera of Hymenoptera, known as stingless bees, which is highly social organisms that occur in tropical and subtropical areas throughout the world, including Brazil.Propolis produced by stingless bees, is a mixture containing plant resin source, wax and clay or soil particles (Massaro et al., 2014, Carneiro, et al., 2016).A study demonstrated that Corymbia torelliana is the resin source for the elaboration of geopropolis from Australian Tetragonula carbonaria, as was evidenced by the great similarity in their methylated flavanone profiles by HPLC analyses of their respective extracts (Massaro et al., 2014).Geopropolis from stingless bee Scaptotrigona affinis postica (Latreille, 1807), (Hymenoptera, Apidae, Meliponini), analysed in this study, contains no soil.Propolis and geopropolis possess the same chemical class of compounds, which are extracted from its respective resin source.Flavonoids, terpenes, phenylpropanoids, triterpenoids, catechins and caffeoylquinic acid derivatives were detected in geopropolis (Silva et al., 2014;Dutra et al., 2014;Batista et al., 2016;Sawaya et al. 2009;Ferreira et al., 2017).A pyrrolizidine alkaloid derived from retronecine was detected in geopropolis from stingless bee S. postica (Coelho et al., 2015).However, the chemical composition of propolis from Apis mellifera is qualitatively the same in the geographic region where it was produced.As for example, the resin source for European propolis is poplar species, for Brazilian green propolis is Baccharis dracunculifolia and for red South American propolis is Dalbergia ecastophyllum (Osé s et al., 2015;Huang et al., 2014;Valenzuela-Barra et al., 2015).
On the other hand, in general, geopropolis show a wide variation even among samples from the same region, since stingless bee collect material from plants near their hives.Different chemical profile was observed among geopropolis samples from Melipona fasciculata Smith harvested in municipalities of Maranhã o State, northeastern Brazil.Cycloartane, ursane and oleanane derivatives and phenolic acids (protocatechuic acid and gallic acid) were detected in geopropolis harvested in Palmeirâ ndia, while gallic and ellagic acid were the main constituents detected in geopropolis harvested in Fernando Falcã o (Batista et al., 2016).Phenolic acids and hydrolyzable tannins (gallotannins and ellagitannins) were detected in geopropolis from Melipona fasciculata harvested in Baixada Maranhense, also in Maranhã o State (Dutra et al., 2014).However, samples of geopropolis from stingless bee Tetragonisca angustula, independently of their geographic origin, presented a similar composition to the flowers extracts of Schinus terebinthifolius Raddi (Anacardiaceae), their possible resins source (Carneiro et al, 2016).
There are more studies for Melipona than Scaptotrigona stinglees bee species (Santos et al., 2017).Sawaya et al. (2009) analysed geopropolis extract from three species of Scaptotrigona harvested monthly from two distinct regions in Brazil.Geopropolis from Scaptotrigona ssp. was harvested in the state of Maranhã o, Northeastern region of Brazil, while geopropolis from Scaptotrigona aff.depillis and Scaptotrigona bipunctata was harvested in the state of Sã o Paulo, South eastern region of Brazil.Diterpenes acid derivatives were found as the main constituents.However, was observed that, the chemical profile obtained for geopropolis from Scaptotrigona ssp., harvested in Maranhã o State, was different for that obtained for the Scaptotrigona species harvested in Sã o Paulo State (Sawaya et al. 2009).Flavonols, such as quercetin methyl ethers, and methoxy chalcones were detected in geopropolis from Scaptotrigona aff.depillis, harvested in the state of Rio Grande do Norte, Northeast region of Brazil (Ferreira et al., 2017).
The search for functional foods, that possess bioactive substances, is a new trend, which can provide more effective treatments of diseases with fewer side effects.The numerous bioactive compounds collected by honeybees from exudates and buds of plants, are utilized in the elaboration of propolis that exert a defensive barrier against microorganism (Saeed et al., 2016;Salas et al., 2016).It is extensively used for centuries, in foodstuffs and beverages to improve health related disorders.Propolis and geopropolis exhibited a wide variety of pharmacological properties, such as, anti-inflammatory, antioxidant, antitumor, antiulcer and for treatment of respiratory diseases (Berretta et al., 2017;Montenegro & Mejí as, 2013;Pippi et al., 2015;Nina et al., 2015).The antiviral activity of propolis from different geographic regions is known, since ancient times.Propolis has been pointed out as an alternative for the treatment of disease caused by virus, since its antiviral properties has been evidenced in different steps of viral replication (Silva-Carvalho et al., 2015;Saeed et al., 2016;Salas et al., 2016).Propolis exerted antiviral activity against influenza virus A and B, herpes, Vaccinia Virus, Hepatitis B Virus, Calicivirus, Newcastle disease virus, Avian reo virus, Bursal disease virus and human immunodeficiency virus (HIV) (Silva-Carvalho et al., 2015;Oldoni et al., 2015).The green propolis and its resin source Baccharis drancunculifolia exhibited antiviral activity on poliovirus type 2 (Búfalo et al., 2009).Propolis extracts exhibited high anti-herpetic activity against Herpes virus type I and II, by different mechanism of action (Nolkemper et al., 2010;Schnitzler et al., 2010), and anti-influenza virus activity against influenza infection in mice (Shimizu et al., 2008).The hydroalcoholic extract from Brazilian brown propolis promoted protective effect on herpes infected mice, acting on inflammatory and oxidative processes (Sartori et al. 2012).Hatay propolis samples exhibited antiviral effects against Herpes virus type I and II (Yildirim et al., 2016).Propolis extract collected in a Canadian region, rich in poplar trees, exhibited high virucidal effect against herpes simplex viruses type 1 and type 2, due to its interference in virus adsorption (Bankova et al., 2014).The geopropolis from S. postica, that contain high contents of vicenin-2 and schaftoside, exhibited high antiviral activity against herpes virus (Coelho et al. 2015).The results obtained in different studies had shown that propolis with different chemical profile, harvested in different geographic region exhibited antiviral activity against herpes simplex viruses and other types of virus (Coelho et al. 2015, Bankova et al., 2014, Yildirim et al., 2016).Attachment to cellular receptors and entry into the host cell are the first steps in viral infection (Rasbach et al., 2013).It is known that flavonoids can prevent the virus binding to host cell receptor and penetration within cells, exerting an inhibitory effect on the early stage of virus infection (Ahmad et al., 2015;Kai et al., 2014).
The aim of this study was to evidence the effectiveness of an experimentally prepared extract of geopropolis (HMEG) from S. postica, harvested in Barra do Corda, Maranhã o State, against Rubella virus infected Statens Serum Institut Rabbit Cornea (SIRC) cells.In the present study, viral binding and penetration assays were included, to determine if treatment of RV with an extract rich in flavones-6,8-di-C-glycosides could disrupt virions from binding to the SIRC receptor of the cell membrane and its penetration into the cell.

Preparation and Phytochemical Analysis of Experimentally Prepared Extract of Geopropolis (HMEG) from S. Postica using Reversed Phase HPLC-DAD-ESI-MS/MS.
Geopropolis sample (15 g) from S. postica harvested in the region of Barra do Corda, Maranhã o state, Brazil, (5º 30'S, 45º 14'O) was treated with solvents of increasing polarity (hexane, chloroform, ethyl acetate and methanol) in Sohxlet apparatus.The obtained fractions were concentrated and stored at freezer until sample workup.The yield of methanolic extract was 15.08 % by dry weight.For antiviral tests, the dry methanolic fraction, was dissolved in water and denominated HMEG.This extract rich in hydrosoluble compounds was analyzed by HPLC-DAD-ESI-MS/MS.The analysis was conducted on DADSPD-M10AVP Shimadzu system equipped with a photodiode array detector coupled to Amazon speed ETD, Bruker Daltonics, as previously described by Coelho et al. (2015).The identification of constituents was established on the basis of their UV and mass spectral (MS) data, which were compared with MS data reported by Coelho et al. (2015), Negri et al. (2018), Mihajlovic et al. (2015) and the chemical databases Phenol-Explorer (www.phenol-explorer.eu),Chem.Spider (http://www.chemspider.com),METLIN (http://metlin.scripps.edu)and HMDB (www.hmdb.ca).

Determination of the Virus Infectious Dose
The determination of the virus infectious dose was carried out using the methodology reported by Coelho et al. (2015) with modifications.The confluent monolayers were dispersed with 0.2% trypsin and 0.02% versene, resuspended in Dulbecco's minimum Eagle essential growth medium (DMEM) with 100 IU/ml penicillin G and 100 mg/ml streptomycin.The SIRC cell suspension was diluted to 2.0x10 4 cells/ml and placed into 96-well plate.Plates were seeded with 200 µL of cell suspension and incubated at 37º C in a humidified 5% CO 2 atmosphere.RA 27/3 strain (Meruvax II, Merck, Sharp and Dohme) stock virus was quantified by medium tissue, using cell culture infections with 0.01 MOI (multiplicity of infection).HMEG was added to the cells at 3 h prior of the virus infections (pre treatment) and 1 h after virus infection (post treatment).The antiviral screenings were repeated three times with different concentrations of HMEG (0.6, 2.4, 8.6, 17, 34 and 68 µg/mL).

Antiviral Effect of Geopropolis on Infected SIRC Cells
The antiviral effect was evaluated according to the method described by Carvalho et al. (2017) and Coelho et al. (2015) with modifications.SIRC cells were grown to approximately 90% confluence in 96 well plates in DMEM, supplemented with 2 mM of L-glutamine and 10% phosphate buffered saline (PBS).Plates were incubated at 37°C in a humidified 5% CO 2 atmosphere.The confluent cells were infected with RA 27/3 (MOI=0.1)and monitored for cytopathic effects, for 3 days.The extract was added to the cells at 3 h prior of the virus infections (pre treatment) and 1 h after virus infection (post treatment).The antiviral screenings were independently repeated, three times with different concentrations of HMEG (0.6, 2.4, 8.6, 17, 34 and 68 µg/mL).After this, the determination of the HMEG effect on the infected cells was carried out using Real-Time quantitative polymerase chain reaction (qPCR).

Binding-penetration Assays
The aim of binding-penetration assays is to measure interactions between virus and cells.These assays were carried out according to the method described by Carvalho et al. (2017) with modifications.The binding assay was carried out at 4°C, a temperature that allowed the binding of RV to cell receptors.In this temperature RV cannot penetrate within cells.The penetration occurs most efficiently at 37°C.SIRC cells were placed in 24-well plate and allowed to reach confluency.The cells were infected with RV (MOI=0.1)dilutions and treated with different concentrations of HMEG (17, 34 and 68µg/mL).The infected SIRC cells treated or untreated were allowed to adsorb for 1 h at 4 °C.Unabsorbed virions were then aspirated, and the cells were washed twice with PBS and were again treated with different concentrations of HMEG (17, 34 and 68µg/mL) for 1h at 37°C and 5% CO 2 .RV virions penetrated within cells at 37°C to complete its life cycle.Unabsorbed virions were then aspirated, and the cells washed with PBS twice.After this, cells were incubated for 72 h at 37°C and 5% CO 2 .The results were analysed using qPCR.

Quantitative Real-time PCR Assay -qPCR
Quantitative real-time PCR (qPCR) is used for the quantification of viral nucleic acids, being a reliable method for measure gene expression (Carvalho et al. 2017).The total RNA for evaluation of antiviral activity of HMEG in pre treatment, post treatment, binding and penetration assay was measured by qPCR.The quantification was carried out according to the method described by Coelho et al., (2015) and Carvalho et al. (2017) with slight modification.The extraction of total RNA from homogeneous cell group, were carried out using the MagNA Pure extractor (Roche, Basel, Switzerland).To amplify the RV genomic sequence, Real-Time quantitative polimerase chain reaction (RT-PCR) was performed using the Superscript III Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations.The set of primers used was described by CDC/USA (Abernathy et al. 2009).The assay was performed in triplicate with 25µl reactions mixtures containing reaction buffer (Invitrogen, Carlsbad, CA, USA), 0.5 U of a Superscript-Taq enzyme mixture, 0.2µM of each primer, 0.1µM of the labeled probe (Invitrogen, Carlsbad, CA, USA) and 5µl of RNA.The assay was carried out also including a no-template control.The thermal cycling was carried out with an Applied Biosystems 7500 thermal cycle with the following procedure: 50 C for 10 min; 95 C for 2 min; and 40 cycles of 95 C for 15 s and 60 C for 1 min.The presence of intact RNA in the samples was confirmed with primers specific for RNase P RNA.Standard curves were prepared by qPCR using serial dilutions of known copy numbers of the purified amplification product for RV.A reaction mixture containing water as the template was run on each plate as negative control.The percentage of reduction was defined as follows: [copy no. of infected cells -copy no. of treated cells/copy no. of infected cells X 100].The data were analysed with SDS software (version 2.1; Applied Biosystems, Grand Island, NY, USA).

Transmission Electron Microscopy (TEM)
The transmission electron microscopy was carried out according to the methodology reported by Coelho et al., (2015) and Carvalho et al. (2017) with slight modification.SIRC cells were cultivated on Aclar film seeded in 24-well plates and incubated for 48 h at 37 o C in a humidified atmosphere with 5% CO 2 .RVs (MOI = 0.1) were treated with 68 µg/mL of HMEG for 1 h at 37 o C prior to cell infection.DMSO used as the negative control, did not exhibited any noticeable effects on the cell lines.The cells inoculated with RV, treated and untreated with HMEG, and allowed to adsorb RV for 1 h at 37 o C in 5% CO 2 .After 48 h, the cells were fixed with 2.5% glutaraldehyde (Sigma, St. Louis, MO, USA) in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 hours at 4°C.After rinsing with cacodylate buffer, the cultures were post-fixed in a solution containing 1% osmium tetroxide, washed in 0.1 M sodium-cacodylate buffer, dehydrated in graded acetone, and embedded in epoxy resin.Ultrathin sections stained with uranyl acetate and lead citrate were examined under a Jeol Transmission Electron Microscope at 80 kV.Images were recorded under a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan).

Statistical Analysis
Statistical analysis was performed using Exstat software.For analyses of the cell viability data and the antiviral activity by mRNA quantification, the Student's T test was used with the p value corrected by the Bonferroni-Sidak method.

Antiviral Effect of HMEG on SIRC Infected Cells
Results of cell viability and cell proliferation assays indicated that HMEG was not toxic to cultured SIRC cells.SIRC infected cells were treated with different concentrations of HMEG (0.6, 2.4, 8.6, 17, 34 and 68 µg/mL) prior to RV infection (pre treated cells) and after infection (post treated cells).In the MTT assay was not observed cytotoxic effect on SIRC cells treated with HMEG at concentration of 68 ug/mL, as can be seen in Figure 1.The 50% cytotoxic concentration (CC50) of HMEG was 150 ug/mL.As can be seen in Figure 2, the post treatment and pre treatment with HMEG reduced the number of copies of RV in the cell lysates, reducing the viral load, which was dose dependent.In the pre treatment with HMEG, the reduction of viral load ranged from 20% to 90%.On the other hand, was observed a reduction of 80% of viral load, after post treatment with 0.6 µg of HMEG.The post treatment carried out with 68 µg/mL of HMEG resulted in 98% of inhibition of the viral replication (see Figure 2).The viral binding assay was performed at 4°C, to determine if treatment of RV with HMEG could disrupt virions from binding to the SIRC cellular receptor.In this temperature did not occur the penetration of RV within SIRC cells, which occur after the increase of temperature at 37° C, completing its life cycle.The cytopathic effect was observed in the cells infected and untreated and in cells DMSO-treated RVs, however was not observed with RV infected SIRC cells treated with HMEG.In the viral binding assay was observed that the treatment of infected SIRC cells with different concentrations of HMEG (17, 34 and 68µg/mL), was efficient to block the binding the virus on cell receptor, inhibiting the infection of SIRC cells, as can be seen in Figure 3. Thus, the results (repeated in triplicates) indicate that HMEG was able to inhibit the binding of virions to the SIRC cells receptor.
Virus binding to cellular receptors leads to the direct penetration into cells.Beside this, the results obtained by penetration assays (Figure 4), indicated that HMEG efficiently prevented viral penetration and replication.The reduction of viral load was observed by qPCR, which indicated a decrease of the RNA copy number of RV.These results were corroborated by TEM assay.As can be seen in Figure 5, in the electron micrographs, in the cytoplasm of infected SIRC cells treated with HMEG, were not observed the rearrangement of organelles and the presence of RV-like particles.
Figure 5.The SIRC cells were cultivated on Aclar film and after 48 hours were inoculated with binding sample and processed by TEM.A-SIRC cells inoculated with RV.Note the presence of a typical particle viral.B-SIRC cells inoculated with binding sample.Note Golgi complex (GC), Vesicles (V) and Mitochondria (M).C-SIRC cells inoculated with virucida sample.It is important to note that RV like particles is not found.The Golgi complex (GC), Vesicles (V) and Mitochondria (M) are marked in the cells.
Compound 20 was tentatively identified as 6-C-fucosyl luteolin, since in its MS/MS spectrum was observed the loss of water (Table 1), which is representative of C-6-isomers (Elliger et al., 1980).The presence of

Discussion
The potent antiviral activity of the extract of geopropolis from S. postica provided by beekeeper, against herpes simplex virus was reported previously (Coelho et al., 2015;Silva-Carvalho et al., 2015).The present study evaluated the effect of an experimentally prepared extract of geopropolis from S. postica (HMEG) on RV infected SIRC cells.Rubella was described in 1866, by Henry Veale, a British Army surgeon (Muscat et al., 2014).Generally, cells cultivated with RV strains cause cytopathic effects or morphological changes in the host cell (Carvalho et al. 2017).In this study, SIRC cells cultivated with RA 27/3 exhibited clear growth of the RV and readily detectable cytopathic effects.The replication of RV was observed on untreated SIRC cells cultivated with RA 27/3 after 48 h, as described by Figueiredo et al., (2000).The growth of the RV, with the arrangement of organelles was not observed in cells infected and treated with HMEG.The binding-penetration assays indicated that HMEG inhibited RV entry into SIRC cells by interfering with the binding/ adsorption of the virions to the cellular receptor, and consequently caused not only a reduction of viral load but also a decrease of cytopathic effects and viral protein synthesis.Thus, vicenin-2 and schaftoside was been able to block the binding of RV with receptors on SIRC plasma membrane and prevent the penetration within cells, affecting the steps of viral cycle replication into SIRC cells or lead to the DNA degradation, before the virus entry into cells.
Endoplasmic reticulum, membranous networks of the cell, is a crucial organelle used for viral entry and viral replication.Rubella virus possesses the ability to rearrange cellular membranes to facilitate its viral replication (Lee & Bowden, 2000, Petrova et al., 2016).The endoplasmic reticulum, Golgi complex, and mitochondria are often closely arranged around the virus replication complex, in RV infected SIRC cells (Lee & Bowden, 2000).The results measured by qPCR and visualized by transmission electron microscopy (TEM) demonstrated a reduction in infectivity on the RV infected SIRC cells treated with HMEG.In TEM assay, was not observed the rearrangement of organelles, the typical replication complex, rubella virions and RV-like particles on infected SIRC cells treated with HMEG in concentrations of 0.6-68 ug/mL.In qPCR assays was observed that the inhibition of the cytopathic effect and viral replication on infected and treated SIRC cells, was dose dependent.Thus, was observed that pre treatment of SIRC cells with HMEG, carried out 3 h before of the virus infection and post treatment 1 h after of virus infection, inhibited the viral replication.The post treatment exhibited the best antiviral activity.The results indicated that HMEG inhibited RV entry into target cells interfering with the binding/adsorption of the virions to the cellular receptor.
The antiviral activity of caffeolyquinic acids, catechins and hydroxycinnamic acid amide derivatives was also known.An aqueous extract of Brazilian green propolis, rich in caffeoylquinic acids derivatives, exhibited anti-influenza activity (Takemura et al., 2012;Urushisaki et al., 2011).Catechins inhibited RNA replication of influenza virus (Song, Lee, & Seong, 2005), and the process of fusion of HIV virus with the cell receptor (Liu et al., 2005).Hydroxycinnamic acid amide derivatives or triacylated spermidines derivatives exhibited antimicrobial activity against viruses, bacteria and fungi (Mihajlovic et al., 2015).The geopropolis from S.
Postica is used by the population of Maranhã o State, in the treatment of wounds (Coelho et al., 2015;Souza et al., 2015).Pyrrolizidine alkaloids exhibited antimicrobial activity and are promising prototypes for new drugs, especially for topical use (Silva Negreiros Neto et al., 2016).

Conclusion
There is not an effective treatment for rubella virus infection.This study indicated that geopropolis from Scaptotrigona postica of Barra do Corda, Maranhã o State, possess potent antiviral activity against Rubella, a Togaviridae virus.HMEG at low concentrations, was able to inhibit the replication of Rubella virus.The best antiviral activity was observed in the post treatment with HMEG.Results of cell viability and cell proliferation assays indicated that HMEG was not toxic to cultured SIRC cells.The results obtained by viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy demonstrate that HMEG can be able to inhibit the production of infectious RV particles.This activity could be attributed to the high content of vicenin-2 and schaftoside, which probably acted blocking the RV binding to the receptor of SIRC cell membrane, and the penetration within the cell, preventing the viral replication.

Figure 1 .Figure 2 .
Figure 1.Cell viability of SIRC cells treated with different concentrations of HMEG.The number represents the mean of three replicates

Figure 3 .
Figure 3. Binding assay of untreated virus and RV treated with HMEG (17, 34 and 68ug/mL) on receptor of SIRC cells.Infectivity was determined by qPCR.The numbers represent the mean triplicate trials

Table 1 .
Compounds 1-22 detected in experimentally prepared extract of geopropolis from S. postica (HMEG) of Barra do Corda, Maranhã o State, by HPLC-DAD-ESI-MS/MS analyses Constituents previously reported for geopropolis from S. postica of Barra do Corda, Maranhã o State