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In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1
Research in Veterinary Science 91 (2011) e158–e162
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1 H.D. Gravina a,b, N.F. Tafuri a,b, A. Silva Júnior a,b, J.L.R. Fietto a, T.T. Oliveira c, M.A.N. Diaz d, M.R. Almeida a,⇑ a
Molecular Animal Infectology Laboratory, Applied Biotechnology Institute (BIOAGRO), Federal University of Vicosa, Vicosa 36570-000, Brazil Animal Virology Laboratory, Department of Veterinary Sciences, Federal University of Vicosa, Vicosa 36570-000, Brazil c Biofarmacos Laboratory, Department of Biochemistry and Molecular Biology, Federal University of Vicosa, Vicosa 36570-000, Brazil d Biomolecular Chemistry Laboratory, Department of Biochemistry and Molecular Biology, Federal University of Vicosa, Vicosa 36570-000, Brazil b
a r t i c l e
i n f o
Article history: Received 4 May 2010 Accepted 22 November 2010
Keywords: Antiviral activity Flavonoids EHV-1
a b s t r a c t The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at 1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Although effective antivirals have been utilized over the past 60 years (Felipe et al., 2006), viral diseases continue to be a problem, due to the toxicity of new antivirals and the development of resistant viruses. Various secondary plant metabolites, including flavonoids, tannins, saponins and phenolic acids, exhibit promising antiviral activity (Muller et al., 2007). In particular, flavonoids have been reported to have inhibitory effects on several viruses (Choi et al., 2009; Mucsi and Pragai, 1985). Quercetin is a water-soluble flavonoid that has been reported to have many biological actions, including antiviral activity against several viruses (Mucsi and Pragai, 1985; Douglas et al., 2003; Davis et al., 2008). Morin is a flavonoid that displays a variety of biological actions, such as antioxidant (Kitagawa et al., 2004), anti-inflammatory (Galvez et al., 2001), anti-allergic (Kim et al., 2009) and anti-mutagenic activities (Bhattacharya and Firozi, 1988). However, antiviral activities have not been reported. Trans-cinnamic acid occurs naturally in nature, being the precursor of flavonoids. It has great potential for a therapeutic role, showing antimicrobial, antifungal and antitumour activity (Rastogi
et al., 1998; Said et al., 2004; Neves et al., 2005; Qian et al., 2010). A compound derived from trans-cinnamic acid, p-sulphoxy-cinnamic acid, showed antiviral activity against dengue virus, suggesting a possible antiviral potential of trans-cinnamic acid (Rees et al., 2008). Equid herpesvirus 1 (EHV-1), a DNA virus member of the family Herpesviridae (Ostlund, 1993), is responsible for respiratory disease, abortion, neonatal death and nervous system disorders in horses (Patel and Heldens, 2005). Despite the existence and frequent use of vaccines against EHV-1, outbreaks still occur, with significant economic impact on the equine industry, because existing vaccines are not sufficiently protective (Reed and Toribio, 2004). In addition, infection control may be difficult due to the establishment of life-long latency after primary infection (Field et al., 2006). Due to the importance of EHV-1 as a ubiquitous pathogen of horses and the absence of an antiviral treatment for the clinical disease, this study aimed to investigate the antiviral potential of trans-cinnamic acid and the flavonoids quercetin and morin on EHV-1 in vitro. 2. Materials and methods 2.1. Cells and virus
⇑ Corresponding author. Address: Molecular Animal Infectology Laboratory, Applied Biotechnology Institute (BIOAGRO), Federal University of Vicosa, Vicosa 36570-000, MG, Brazil. Tel.: +55 31 3899 29 11; fax: +55 31 3899 23 74. E-mail addresses: [email protected], [email protected] (M.R. Almeida). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.11.010
VERO cells were kept in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (SBF), penicillin (1.6 mg/L) and streptomycin (0.4 mg/L). EHV-1 was derived from
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a standard A4/72 sample courtesy of the University of Santa Maria, RS, Brazil, and was titrated by the tissue culture infectious dose 50 (TCID50) method, as described by Reed and Muench (1938). 2.2. Preparation of compounds The three compounds (quercetin, morin and trans-cinnamic acid) were purchased from Sigma–Aldrich (Deisenhofen, Germany). The compounds were initially dissolved in dimethylsulphoxide (DMSO) at a stock concentration of 100 mg/mL at 4 °C. At the time of use, dilutions in MEM were performed so that the final concentration of DMSO did not exceed 1.2%. 2.3. Analysis of cytotoxicity The cytotoxicity of the compounds was microscopically assessed via changes in cellular morphology and was confirmed and measured by the colorimetric method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma–Aldrich, Deisenhofen, Germany) by mitochondrial enzymes (Mosmann, 1983). Cells were distributed in 96-well microplates and after 3 h of incubation at 37 °C in an atmosphere containing 5% CO2 the medium was removed and the cells further incubated with different concentrations of each test compound and DMSO at 37 °C in an atmosphere containing 5% CO2 for 72 h. After 72 h incubation, the effects on cell morphology (loss of monolayer, granulation, vacuolization in the cytoplasm, elongation and narrowing of the extension, and darkening of cell limits) were observed microscopically. The plates were then washed twice with phosphate-buffered saline (PBS), pH 7.2, and further incubated at 37 °C with 10% MTT for 4 h. Subsequently, the salt that had formed was dissolved by the addition of isopropanol–HCl 0.04 N and the absorbances read on a multiwall spectrophotometer (Bio-TekÒ, Elx800) at 550 nm. The percentage of viable treated cells was calculated in relation to the untreated control (% of control cells: optical density test (OD) test/optical density control cell (OD) control cell 100). The maximum non-toxic concentration (MNTC) of the compounds was determined. 2.4. Determination of virucidal activity In order to assess the virucidal activity of the compounds, EHV1 serial dilutions (10–107 TCID50/mL) were incubated for 1 h with quercetin (15, 30 and 60 lg/mL), morin (30, 60 and 90 lg/mL) or trans-cinnamic acid (25, 50 and 100 lg/mL) at temperatures ranging from 35 to 37 °C. At specific time intervals aliquots of each virus/compound suspension were added to the already adherent and plated cells and incubated at 37 °C and 5% CO2. After 72 h, the viral titre was calculated using the method described by Reed and Muench (1938) and compared with the control titre (untreated virus).
containing the compounds for 1 h (1 h: effect of penetration). Two hours after viral infection another aliquot of infected cells was treated with the compounds diluted in MEM (2 h: effect after virus infection). The antiviral activity of all phases was assessed by measuring the reduction in viral titre after 72 h and calculated using the method of Reed and Muench (1938). 2.6. DMSO assays All antiviral assays were assessed under the same conditions with DMSO to evaluate the possible interference of this solvent. Two concentrations were used: 1.2%, which corresponded to the highest percentage of DMSO found in the dilutions of compounds, and 2.4%, which was used to assess with greater accuracy whether it acts as an inert compound. 2.7. Statistical analysis Statistical analysis was performed using the Statistical Analysis System program (SAEG – Version 9.1/2007, Federal University of Viçosa, Brazil). One-way ANOVA and two-way ANOVA repeated measures were used for cytotoxicity and timing of addition assays, respectively. The Tukey test was used to compare the means (P < 0.05). For the results of the virucidal activity test, linear regression and non-linear fit one-phase decay was determined using the GraphPad Prism software (California, USA). 3. Results The cytotoxic effects of the compounds were deduced from their antiviral activity by determining the MNTC of each compound using microscopic analyses and the MTT colorimetric test. Microscopically, it was observed that as the compound concentration increased, more morphological changes were visible in the cells (loss of monolayer, granulation, vacuolization in the cytoplasm, darkening of cell boundaries). Table 1 shows the MNTC of each compound
Table 1 Maximum non-toxic concentrations of quercetin, morin, trans-cinnamic acid and DMSO for VERO cells, assessed by optical microscopy (MNTC/OM) and assessed by MTT test (MNTC/MTT). [A1], [A2], [A3] are concentrations used in the antiviral tests. Compounds
MNTC/OM (lg/mL)
MNTC/MTT (lg/mL)
[A1] (lg/mL)
[A2] (lg/mL)
[A3] (lg/mL)
Quercetin Morin Trans-cinnamic acid DMSO
70 100 110 3.0 (%)
75.1 101.3 109.3 3.1 (%)
15 30 25 1.2 (%)
30 60 50 2.4 (%)
60 90 100
2.5. Time of addition studies To investigate the mechanism by which the compounds inhibit the replication cycle of EHV-1, a study was conducted following previous methods with some modifications (Serkedjieva and Ivancheva, 1999). Cell monolayers in 96-well plates were incubated with solutions of quercetin (15, 30 and 60 lg/mL), morin (30, 60 and 90 lg/mL) or trans-cinnamic-acid (25, 50 and 100 lg/mL) for 1 h (1 h: pre-treatment) and then washed twice with PBS and challenged with 10–107 TCID50/mL. Other cell monolayers in 96-well plates were infected with EHV-1 at 10–107 TCID50/mL and, during adsorption for 1 h at 4 °C, were treated with the compounds to prevent viral internalization (0 h: effect of adsorption). One fraction of infected non-treated cells was incubated at 37 °C with MEM
Fig. 1. Virucidal activity of DMSO [1.2% and 2.4%] tested at different times.
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and of DMSO and the three lowest concentrations ([A1], [A2], [A3]) used in the antiviral tests. Figs. 1 and 2 show that there was no significant difference between the titres of DMSO, in any assay evaluating antiviral activity. These results show that DMSO, in the concentrations used to dissolve quercetin, morin and trans-cinnamic acid, does not have antiviral activity against EHV-1, nor does it interfere with the subsequent antiviral analysis. Quercetin, morin and trans-cinnamic acid showed antiviral activity in the tests determining virucidal capacity and/or in the time of addition tests. In the virucidal capacity test, we studied the ability of the compounds to produce a direct virus-inactivating
effect. The three concentrations of quercetin tested reduced the viral titre by similar amounts, with a maximum reduction of 99.9% (Fig. 3). The results of the morin virucidal capacity test showed a reduction in viral titre at the three tested concentrations, with a maximum reduction of 99.9% (Fig. 4). In contrast, trans-cinnamic acid showed no virucidal action at the tested concentrations, as shown in Fig. 5. To investigate the effect of the compounds on the different steps of viral replication the preparation was added at various times relative to viral infection. Quercetin at 30 and 60 lg/mL showed antiviral action at 0 and 1 h, as shown in Fig. 6. Virus
Fig. 5. Virucidal activity of trans-cinnamic acid [15, 30 and 60 lg/mL] tested at different times.
Fig. 2. Inhibitory effects of the addition of DMSO at different times on the viral replication cycle. Two different concentrations of DMSO [1.2% and 2.4%] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
Fig. 6. Inhibitory effects of the addition of quercetin at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [15, 30 and 60 lg/ mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
Fig. 3. Virucidal activity of quercetin [15, 30 and 60 lg/mL] tested at different times.
Fig. 4. Virucidal activity of morin [30, 60 and 90 lg/mL] tested at different times.
Fig. 7. Inhibitory effects of the addition of morin at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [30, 60 and 90 lg/ mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
H.D. Gravina et al. / Research in Veterinary Science 91 (2011) e158–e162
Fig. 8. Inhibitory effects of the addition of trans-cinnamic acid at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [25, 50 and 100 lg/mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
replication was slightly reduced when quercetin was added at the time of adsorption and penetration. Fig. 7 shows the high antiviral action of morin at adsorption (0 h), regardless of the concentration, which significantly reduced the viral titre. With trans-cinnamic acid, the pre-treatment of cells (1 h) as well as addition at adsorption (0 h) resulted in a reduction in virus infectivity (Fig. 8).
4. Discussion Many natural products are being tested as antiviral agents because the control of viral infections remains a challenge for researchers. These products are considered to be antiviral agents since interfere with one or more processes during viral biosynthesis, or inactivate extracellular virus. Consequently, these products are candidates for clinical use (Cos et al., 2006). Virucidal effects are directly dependent on the presence and concentration of different substances such as flavonoids, phenolic acids and tannins, among others (Serkedjieva and Ivancheva, 1999). Among these natural compounds, flavonoids have an antiviral activity that has been widely studied (Andres et al., 2009; Hudson, 1990; Selway, 1986). Anti herpesvirus activity has been demonstrated for many plant extracts (Ozcelik et al., 2005, 2009). Some antivirals, although synthetic, have already been tested in vitro against EHV-1, for example, ganciclovir, 20 -fluoropyrimidine nucleosides, (S)-9-[3-hydroxy-2-phosphonylmethoxypropyl]adenine or HPMPA and the HPMPA analogue cidofovir, as reviewed by Garré et al. (2007). In this study, three different compounds, two flavonoids and one flavonoid precursor, were investigated for their inhibitory effect against EHV-1. All compounds tested showed some activity. In this context, the MNTC of each compound was initially measured in order to determine the lowest concentrations with antiviral and virucidal activity. Another important factor was the use of the solvent DMSO in the dilution of substances at concentrations below their MNTC. DMSO had no cytotoxic effect on the cells and did not interfere in the results of later antiviral analyses. The non-antiviral activity of DMSO was confirmed by the virucidal activity and time of addition assay. In preliminary studies by Formica and Regelson (1995), the antiviral activity of quercetin was found to be related to its ability to bind to the envelope glycoproteins or viral capsid, interfering with the binding and penetration of the virus in the cell, in addition to interference with DNA synthesis. Other studies assessing the antiviral activity of extracts of Acanthospermum hispidum against suid herpesvirus 1 (SuHV-1) and bovine herpesvirus 1 (BoHV-1), suggested that viral inhibition was related to binding of the aqueous extract to structures in the viral envelope, inhibiting viral penetra-
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tion (Summerfield et al., 1997). Corroborating these findings, the positive results in the present study for quercetin in the virucidal capacity and time of addition tests suggest that it irreversibly binds to the viral particles or destabilizes essential structures in the virus, such as the glycoprotein envelope, or even acts on specific steps in the replication cycle, especially during its early stages (effects on adsorption and penetration). Consequently, in addition to its virucidal activity, quercetin may interfere with the binding of the virus to its cellular receptor and the internalization process of EHV-1 in cells, reducing the number of newly infected cells and therefore containing viral propagation and the formation of progeny. Studies have shown that changes in the chemical structure of flavonoids affect their biological action. Si et al. (2009) showed that the different capacity of flavonoids to interact with cytochrome P450 are due to structural differences in these flavonoid molecules. Another study reported that the chemical modifications achieved by the bromination of flavonoids leads to changes in their structures and interferes with their antioxidant activity (Justino et al., 2009). So, although quercetin and morin have a similar chemical structure, the position of the hydroxyl carbon C30 (the ortho position) in quercetin and C20 (the para position) in morin (Justino et al., 2009) may have enabled morin to act mainly upon viral adsorption (0 h), with non-significant results at 1 and 2 h (effects on penetration and after virus infection), whereas quercetin influenced viral adsorption (0 h) and penetration (1 h). The antiviral properties of p-sulphoxy-cinnamic acid, zosteric acid and five other compounds derived from modified trans-cinnamic acid have also been demonstrated against dengue virus (Rees et al., 2008). In our study, trans-cinnamic acid produced negative results in the virucidal activity test, indicating that it had no effect on the viral particles. However, the reduction in the viral titre at 1 and 0 h in the time of addition test suggests that this compound has an effect on cell receptors, destabilizing them or acting as a competitive inhibitor to the virus, and possibly also an effect at the viral adsorption stage. The trans-cinnamic acid compound, being one of the precursors of flavonoids that show antiviral activity, demonstrated a positive link between the phenolic structure of the compounds and antiviral activity, as represented in this research by its antiviral activity against EHV-1. This relationship between polyphenols and antiviral activity seems to be related to the ability of these compounds to bind to proteins and form unstable complexes (Selway, 1986). The anti-herpes effect of aqueous and ethanol extracts of Salvia officinalis has been reported, mainly based on the quantity and composition of the particular phenolic compounds of this species (Schnitzler et al., 2008). In addition, enveloped viruses, such as EHV-1, may be more vulnerable to action of polyphenols as these compounds are able to connect easily to viral envelope glycoproteins (Haslam, 1996; Serkedjieva and Ivancheva, 1999). 5. Conclusion Further studies are necessary to determine the future medical use of flavonoids, their precursors and products such as herbal medicines with antiviral activities. This study showed the potential of the flavonoids as candidates for antiviral agents against EHV-1 and other viruses of great importance in veterinary medicine and public health. Acknowledgements The authors acknowledge the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support. Humberto Gravina was a master’s student, funded by the Coordenação
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de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank Dr. Eduardo Furtado Flores, Department of Preventive Veterinary Medicine, Federal University of Santa Maria, for providing the isolated sample of EHV-1.
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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1 H.D. Gravina a,b, N.F. Tafuri a,b, A. Silva Júnior a,b, J.L.R. Fietto a, T.T. Oliveira c, M.A.N. Diaz d, M.R. Almeida a,⇑ a
Molecular Animal Infectology Laboratory, Applied Biotechnology Institute (BIOAGRO), Federal University of Vicosa, Vicosa 36570-000, Brazil Animal Virology Laboratory, Department of Veterinary Sciences, Federal University of Vicosa, Vicosa 36570-000, Brazil c Biofarmacos Laboratory, Department of Biochemistry and Molecular Biology, Federal University of Vicosa, Vicosa 36570-000, Brazil d Biomolecular Chemistry Laboratory, Department of Biochemistry and Molecular Biology, Federal University of Vicosa, Vicosa 36570-000, Brazil b
a r t i c l e
i n f o
Article history: Received 4 May 2010 Accepted 22 November 2010
Keywords: Antiviral activity Flavonoids EHV-1
a b s t r a c t The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at 1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Although effective antivirals have been utilized over the past 60 years (Felipe et al., 2006), viral diseases continue to be a problem, due to the toxicity of new antivirals and the development of resistant viruses. Various secondary plant metabolites, including flavonoids, tannins, saponins and phenolic acids, exhibit promising antiviral activity (Muller et al., 2007). In particular, flavonoids have been reported to have inhibitory effects on several viruses (Choi et al., 2009; Mucsi and Pragai, 1985). Quercetin is a water-soluble flavonoid that has been reported to have many biological actions, including antiviral activity against several viruses (Mucsi and Pragai, 1985; Douglas et al., 2003; Davis et al., 2008). Morin is a flavonoid that displays a variety of biological actions, such as antioxidant (Kitagawa et al., 2004), anti-inflammatory (Galvez et al., 2001), anti-allergic (Kim et al., 2009) and anti-mutagenic activities (Bhattacharya and Firozi, 1988). However, antiviral activities have not been reported. Trans-cinnamic acid occurs naturally in nature, being the precursor of flavonoids. It has great potential for a therapeutic role, showing antimicrobial, antifungal and antitumour activity (Rastogi
et al., 1998; Said et al., 2004; Neves et al., 2005; Qian et al., 2010). A compound derived from trans-cinnamic acid, p-sulphoxy-cinnamic acid, showed antiviral activity against dengue virus, suggesting a possible antiviral potential of trans-cinnamic acid (Rees et al., 2008). Equid herpesvirus 1 (EHV-1), a DNA virus member of the family Herpesviridae (Ostlund, 1993), is responsible for respiratory disease, abortion, neonatal death and nervous system disorders in horses (Patel and Heldens, 2005). Despite the existence and frequent use of vaccines against EHV-1, outbreaks still occur, with significant economic impact on the equine industry, because existing vaccines are not sufficiently protective (Reed and Toribio, 2004). In addition, infection control may be difficult due to the establishment of life-long latency after primary infection (Field et al., 2006). Due to the importance of EHV-1 as a ubiquitous pathogen of horses and the absence of an antiviral treatment for the clinical disease, this study aimed to investigate the antiviral potential of trans-cinnamic acid and the flavonoids quercetin and morin on EHV-1 in vitro. 2. Materials and methods 2.1. Cells and virus
⇑ Corresponding author. Address: Molecular Animal Infectology Laboratory, Applied Biotechnology Institute (BIOAGRO), Federal University of Vicosa, Vicosa 36570-000, MG, Brazil. Tel.: +55 31 3899 29 11; fax: +55 31 3899 23 74. E-mail addresses: [email protected], [email protected] (M.R. Almeida). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.11.010
VERO cells were kept in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (SBF), penicillin (1.6 mg/L) and streptomycin (0.4 mg/L). EHV-1 was derived from
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a standard A4/72 sample courtesy of the University of Santa Maria, RS, Brazil, and was titrated by the tissue culture infectious dose 50 (TCID50) method, as described by Reed and Muench (1938). 2.2. Preparation of compounds The three compounds (quercetin, morin and trans-cinnamic acid) were purchased from Sigma–Aldrich (Deisenhofen, Germany). The compounds were initially dissolved in dimethylsulphoxide (DMSO) at a stock concentration of 100 mg/mL at 4 °C. At the time of use, dilutions in MEM were performed so that the final concentration of DMSO did not exceed 1.2%. 2.3. Analysis of cytotoxicity The cytotoxicity of the compounds was microscopically assessed via changes in cellular morphology and was confirmed and measured by the colorimetric method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma–Aldrich, Deisenhofen, Germany) by mitochondrial enzymes (Mosmann, 1983). Cells were distributed in 96-well microplates and after 3 h of incubation at 37 °C in an atmosphere containing 5% CO2 the medium was removed and the cells further incubated with different concentrations of each test compound and DMSO at 37 °C in an atmosphere containing 5% CO2 for 72 h. After 72 h incubation, the effects on cell morphology (loss of monolayer, granulation, vacuolization in the cytoplasm, elongation and narrowing of the extension, and darkening of cell limits) were observed microscopically. The plates were then washed twice with phosphate-buffered saline (PBS), pH 7.2, and further incubated at 37 °C with 10% MTT for 4 h. Subsequently, the salt that had formed was dissolved by the addition of isopropanol–HCl 0.04 N and the absorbances read on a multiwall spectrophotometer (Bio-TekÒ, Elx800) at 550 nm. The percentage of viable treated cells was calculated in relation to the untreated control (% of control cells: optical density test (OD) test/optical density control cell (OD) control cell 100). The maximum non-toxic concentration (MNTC) of the compounds was determined. 2.4. Determination of virucidal activity In order to assess the virucidal activity of the compounds, EHV1 serial dilutions (10–107 TCID50/mL) were incubated for 1 h with quercetin (15, 30 and 60 lg/mL), morin (30, 60 and 90 lg/mL) or trans-cinnamic acid (25, 50 and 100 lg/mL) at temperatures ranging from 35 to 37 °C. At specific time intervals aliquots of each virus/compound suspension were added to the already adherent and plated cells and incubated at 37 °C and 5% CO2. After 72 h, the viral titre was calculated using the method described by Reed and Muench (1938) and compared with the control titre (untreated virus).
containing the compounds for 1 h (1 h: effect of penetration). Two hours after viral infection another aliquot of infected cells was treated with the compounds diluted in MEM (2 h: effect after virus infection). The antiviral activity of all phases was assessed by measuring the reduction in viral titre after 72 h and calculated using the method of Reed and Muench (1938). 2.6. DMSO assays All antiviral assays were assessed under the same conditions with DMSO to evaluate the possible interference of this solvent. Two concentrations were used: 1.2%, which corresponded to the highest percentage of DMSO found in the dilutions of compounds, and 2.4%, which was used to assess with greater accuracy whether it acts as an inert compound. 2.7. Statistical analysis Statistical analysis was performed using the Statistical Analysis System program (SAEG – Version 9.1/2007, Federal University of Viçosa, Brazil). One-way ANOVA and two-way ANOVA repeated measures were used for cytotoxicity and timing of addition assays, respectively. The Tukey test was used to compare the means (P < 0.05). For the results of the virucidal activity test, linear regression and non-linear fit one-phase decay was determined using the GraphPad Prism software (California, USA). 3. Results The cytotoxic effects of the compounds were deduced from their antiviral activity by determining the MNTC of each compound using microscopic analyses and the MTT colorimetric test. Microscopically, it was observed that as the compound concentration increased, more morphological changes were visible in the cells (loss of monolayer, granulation, vacuolization in the cytoplasm, darkening of cell boundaries). Table 1 shows the MNTC of each compound
Table 1 Maximum non-toxic concentrations of quercetin, morin, trans-cinnamic acid and DMSO for VERO cells, assessed by optical microscopy (MNTC/OM) and assessed by MTT test (MNTC/MTT). [A1], [A2], [A3] are concentrations used in the antiviral tests. Compounds
MNTC/OM (lg/mL)
MNTC/MTT (lg/mL)
[A1] (lg/mL)
[A2] (lg/mL)
[A3] (lg/mL)
Quercetin Morin Trans-cinnamic acid DMSO
70 100 110 3.0 (%)
75.1 101.3 109.3 3.1 (%)
15 30 25 1.2 (%)
30 60 50 2.4 (%)
60 90 100
2.5. Time of addition studies To investigate the mechanism by which the compounds inhibit the replication cycle of EHV-1, a study was conducted following previous methods with some modifications (Serkedjieva and Ivancheva, 1999). Cell monolayers in 96-well plates were incubated with solutions of quercetin (15, 30 and 60 lg/mL), morin (30, 60 and 90 lg/mL) or trans-cinnamic-acid (25, 50 and 100 lg/mL) for 1 h (1 h: pre-treatment) and then washed twice with PBS and challenged with 10–107 TCID50/mL. Other cell monolayers in 96-well plates were infected with EHV-1 at 10–107 TCID50/mL and, during adsorption for 1 h at 4 °C, were treated with the compounds to prevent viral internalization (0 h: effect of adsorption). One fraction of infected non-treated cells was incubated at 37 °C with MEM
Fig. 1. Virucidal activity of DMSO [1.2% and 2.4%] tested at different times.
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and of DMSO and the three lowest concentrations ([A1], [A2], [A3]) used in the antiviral tests. Figs. 1 and 2 show that there was no significant difference between the titres of DMSO, in any assay evaluating antiviral activity. These results show that DMSO, in the concentrations used to dissolve quercetin, morin and trans-cinnamic acid, does not have antiviral activity against EHV-1, nor does it interfere with the subsequent antiviral analysis. Quercetin, morin and trans-cinnamic acid showed antiviral activity in the tests determining virucidal capacity and/or in the time of addition tests. In the virucidal capacity test, we studied the ability of the compounds to produce a direct virus-inactivating
effect. The three concentrations of quercetin tested reduced the viral titre by similar amounts, with a maximum reduction of 99.9% (Fig. 3). The results of the morin virucidal capacity test showed a reduction in viral titre at the three tested concentrations, with a maximum reduction of 99.9% (Fig. 4). In contrast, trans-cinnamic acid showed no virucidal action at the tested concentrations, as shown in Fig. 5. To investigate the effect of the compounds on the different steps of viral replication the preparation was added at various times relative to viral infection. Quercetin at 30 and 60 lg/mL showed antiviral action at 0 and 1 h, as shown in Fig. 6. Virus
Fig. 5. Virucidal activity of trans-cinnamic acid [15, 30 and 60 lg/mL] tested at different times.
Fig. 2. Inhibitory effects of the addition of DMSO at different times on the viral replication cycle. Two different concentrations of DMSO [1.2% and 2.4%] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
Fig. 6. Inhibitory effects of the addition of quercetin at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [15, 30 and 60 lg/ mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
Fig. 3. Virucidal activity of quercetin [15, 30 and 60 lg/mL] tested at different times.
Fig. 4. Virucidal activity of morin [30, 60 and 90 lg/mL] tested at different times.
Fig. 7. Inhibitory effects of the addition of morin at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [30, 60 and 90 lg/ mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
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Fig. 8. Inhibitory effects of the addition of trans-cinnamic acid at different times on the viral replication cycle. Different concentrations of trans-cinnamic acid [25, 50 and 100 lg/mL] were added at various times pre-treatment (1 h), adsorption (0 h), penetration (1 h) and after virus infection (2 h). The asterisk indicates a significant difference between test and control (P < 0.05).
replication was slightly reduced when quercetin was added at the time of adsorption and penetration. Fig. 7 shows the high antiviral action of morin at adsorption (0 h), regardless of the concentration, which significantly reduced the viral titre. With trans-cinnamic acid, the pre-treatment of cells (1 h) as well as addition at adsorption (0 h) resulted in a reduction in virus infectivity (Fig. 8).
4. Discussion Many natural products are being tested as antiviral agents because the control of viral infections remains a challenge for researchers. These products are considered to be antiviral agents since interfere with one or more processes during viral biosynthesis, or inactivate extracellular virus. Consequently, these products are candidates for clinical use (Cos et al., 2006). Virucidal effects are directly dependent on the presence and concentration of different substances such as flavonoids, phenolic acids and tannins, among others (Serkedjieva and Ivancheva, 1999). Among these natural compounds, flavonoids have an antiviral activity that has been widely studied (Andres et al., 2009; Hudson, 1990; Selway, 1986). Anti herpesvirus activity has been demonstrated for many plant extracts (Ozcelik et al., 2005, 2009). Some antivirals, although synthetic, have already been tested in vitro against EHV-1, for example, ganciclovir, 20 -fluoropyrimidine nucleosides, (S)-9-[3-hydroxy-2-phosphonylmethoxypropyl]adenine or HPMPA and the HPMPA analogue cidofovir, as reviewed by Garré et al. (2007). In this study, three different compounds, two flavonoids and one flavonoid precursor, were investigated for their inhibitory effect against EHV-1. All compounds tested showed some activity. In this context, the MNTC of each compound was initially measured in order to determine the lowest concentrations with antiviral and virucidal activity. Another important factor was the use of the solvent DMSO in the dilution of substances at concentrations below their MNTC. DMSO had no cytotoxic effect on the cells and did not interfere in the results of later antiviral analyses. The non-antiviral activity of DMSO was confirmed by the virucidal activity and time of addition assay. In preliminary studies by Formica and Regelson (1995), the antiviral activity of quercetin was found to be related to its ability to bind to the envelope glycoproteins or viral capsid, interfering with the binding and penetration of the virus in the cell, in addition to interference with DNA synthesis. Other studies assessing the antiviral activity of extracts of Acanthospermum hispidum against suid herpesvirus 1 (SuHV-1) and bovine herpesvirus 1 (BoHV-1), suggested that viral inhibition was related to binding of the aqueous extract to structures in the viral envelope, inhibiting viral penetra-
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tion (Summerfield et al., 1997). Corroborating these findings, the positive results in the present study for quercetin in the virucidal capacity and time of addition tests suggest that it irreversibly binds to the viral particles or destabilizes essential structures in the virus, such as the glycoprotein envelope, or even acts on specific steps in the replication cycle, especially during its early stages (effects on adsorption and penetration). Consequently, in addition to its virucidal activity, quercetin may interfere with the binding of the virus to its cellular receptor and the internalization process of EHV-1 in cells, reducing the number of newly infected cells and therefore containing viral propagation and the formation of progeny. Studies have shown that changes in the chemical structure of flavonoids affect their biological action. Si et al. (2009) showed that the different capacity of flavonoids to interact with cytochrome P450 are due to structural differences in these flavonoid molecules. Another study reported that the chemical modifications achieved by the bromination of flavonoids leads to changes in their structures and interferes with their antioxidant activity (Justino et al., 2009). So, although quercetin and morin have a similar chemical structure, the position of the hydroxyl carbon C30 (the ortho position) in quercetin and C20 (the para position) in morin (Justino et al., 2009) may have enabled morin to act mainly upon viral adsorption (0 h), with non-significant results at 1 and 2 h (effects on penetration and after virus infection), whereas quercetin influenced viral adsorption (0 h) and penetration (1 h). The antiviral properties of p-sulphoxy-cinnamic acid, zosteric acid and five other compounds derived from modified trans-cinnamic acid have also been demonstrated against dengue virus (Rees et al., 2008). In our study, trans-cinnamic acid produced negative results in the virucidal activity test, indicating that it had no effect on the viral particles. However, the reduction in the viral titre at 1 and 0 h in the time of addition test suggests that this compound has an effect on cell receptors, destabilizing them or acting as a competitive inhibitor to the virus, and possibly also an effect at the viral adsorption stage. The trans-cinnamic acid compound, being one of the precursors of flavonoids that show antiviral activity, demonstrated a positive link between the phenolic structure of the compounds and antiviral activity, as represented in this research by its antiviral activity against EHV-1. This relationship between polyphenols and antiviral activity seems to be related to the ability of these compounds to bind to proteins and form unstable complexes (Selway, 1986). The anti-herpes effect of aqueous and ethanol extracts of Salvia officinalis has been reported, mainly based on the quantity and composition of the particular phenolic compounds of this species (Schnitzler et al., 2008). In addition, enveloped viruses, such as EHV-1, may be more vulnerable to action of polyphenols as these compounds are able to connect easily to viral envelope glycoproteins (Haslam, 1996; Serkedjieva and Ivancheva, 1999). 5. Conclusion Further studies are necessary to determine the future medical use of flavonoids, their precursors and products such as herbal medicines with antiviral activities. This study showed the potential of the flavonoids as candidates for antiviral agents against EHV-1 and other viruses of great importance in veterinary medicine and public health. Acknowledgements The authors acknowledge the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support. Humberto Gravina was a master’s student, funded by the Coordenação
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de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank Dr. Eduardo Furtado Flores, Department of Preventive Veterinary Medicine, Federal University of Santa Maria, for providing the isolated sample of EHV-1.
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