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International Journal of Environmental Health Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cije20
Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates a
a
b
Norma Patricia Silva-Beltrán , Saul Ruiz-Cruz , Cristobal Chaidez , c
d
José de Jesús Ornelas-Paz , Marco A. López-Mata , Enrique e
a
Márquez-Ríos & Maria Isabel Estrada a
Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, Ciudad Obregón, Mexico b
Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Culiacán, Culiacán, Mexico c
Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Cuauhtémoc, Cuauhtémoc, Mexico d
Departamento de, Ciencias Químicas Biológicas y de Salud, Universidad de Sonora, Ciudad Obregón, Mexico e
Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Mexico Published online: 25 Jul 2014.
To cite this article: Norma Patricia Silva-Beltrán, Saul Ruiz-Cruz, Cristobal Chaidez, José de Jesús Ornelas-Paz, Marco A. López-Mata, Enrique Márquez-Ríos & Maria Isabel Estrada (2014): Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2014.938030 To link to this article: http://dx.doi.org/10.1080/09603123.2014.938030
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International Journal of Environmental Health Research, 2014 http://dx.doi.org/10.1080/09603123.2014.938030
Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates
Downloaded by [York University Libraries] at 22:32 12 August 2014
Norma Patricia Silva-Beltrána, Saul Ruiz-Cruza*, Cristobal Chaidezb, José de Jesús Ornelas-Pazc, Marco A. López-Matad, Enrique Márquez-Ríose and Maria Isabel Estradaa a Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, Ciudad Obregón, Mexico; bCentro de Investigación en Alimentación y Desarrollo A.C. Unidad Culiacán, Culiacán, Mexico; cCentro de Investigación en Alimentación y Desarrollo A.C. Unidad Cuauhtémoc, Cuauhtémoc, Mexico; dDepartamento de, Ciencias Químicas Biológicas y de Salud, Universidad de Sonora, Ciudad Obregón, Mexico; eDepartamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Mexico
(Received 7 January 2014; final version received 15 April 2014) Byproducts of tomato are known to include phenolic compounds but have not been studied in depth. In this study, the phenolic compositions of (stem, leaf, root, and whole plant) of two tomato cultivars, Pitenza and Floradade, were analyzed by HPLC-DAD. In parallel, the antiviral effects of crude extracts on viral surrogates, the bacteriophages MS2 and Av-05 were evaluated. The leaf extracts from the two varieties showed the highest concentration of phenolic compounds. The compounds identified were gallic acid, chlorogenic acid, ferulic acid, cafeic acid, rutin, and quercetin, and they represented 3174.3 and 1057.9 mg/100 g dried weight of the Pitenza and Floradade cultivars, respectively. MS2 and Av-05 titers at 5 mg/mL were reduced by 3.47 and 5.78 log10 PFU/mL and 3.78 and 4.93 log10 PFU/mL by Pitenza and Floradade cultivar leaf extract, respectively. These results show that tomato extracts are natural sources of bioactive substances with antiviral activity. Keywords: extracts; tomato byproducts; bacteriophages; polyphenols
Introduction The properties of tomato fruits (Licopersicum esculentum) have been extensively studied; however, few studies have focused on the whole tomato plant itself. The nutritional properties of the tomato in relation to the feeding of livestock have been reported (Font et al. 2009; Ventura et al. 2009). Tomato plant (leave and stem) are discarded after harvesting because they are considered a waste; however, the contained biologically active substances make an important source of investigation. A recent investigation showed that tomato plant leaves have several active metabolites, such as phenolic compounds (Taveira et al. 2012). Numerous studies have addressed the beneficial effects of phenolic substances derived from plant tissues. These compounds possess antioxidant, antibacterial, and antiviral properties (Chiang et al. 2003; Choi et al. 2009; Quideau et al. 2011). Plant extracts are an abundant source of bioactive substances with antiviral properties. The use of these products is generally accepted among the population because of their natural origin and low cost. In 1996, it was estimated that there were almost *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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500,000 plant species, all containing different concentrations of bioactive components (Li et al. 2013). Plant phenolic metabolites present pharmacological and food preservation properties (Schnitzler et al. 2008; Su & D’Souza 2011). Epidemiological evidence indicates that viruses cause an estimated 59 % of all foodborne illnesses, 26 % of hospitalization, and 11 % of deaths in the United States only (Scallan et al. 2011). Pholyphenols are showed as having good ability to inhibit viral infections (Su et al. 2011; Carvalho et al. 2013). Recent studies have demonstrated antiviral properties of phenolic extracts from plants, including eugenol from clove and extracts from Geranium eugineum and Cistus incanus (Sokmen et al. 2005; Droebner et al. 2007; Elizaquível et al. 2012), and of phenol derivatives, such as isoquercetin, caffeic acid, quinic acid, and chlorogenic acid (Wang et al. 2009; Kim et al. 2010; Xie et al. 2013). In particular, flavonoids have been shown to have anti-enterovirus effects, affecting viruses such as hepatitis A and norovirus (Tsai et al. 2011). Some studies have shown that flavonoids, such as quercetin, inhibit viral polymerase, interfering with the synthesis of nucleic acids and the intracellular stage of the virus replication cycle (Cushnie & Lamb 2005; Kim et al. 2010). Phenolic extracts of grape byproducts from the agro-industry have been studied for control of enteric viruses (Su & D’Souza 2011, 2012). However, antiviral effects of tomato plant byproducts against enteric viruses have not yet been documented. Therefore, this investigation can provide information regarding a possible natural resource to reduce viral spread. Bacteriophages are suitable models for disease-causing viral particles (Dennehy 2009). Phage handling is simple, inexpensive, and does not require specialized personnel or sophisticated facilities. Most intervention studies have been conducted using bacteriophages as models for human enteric viruses. Because extracts derived from tomato plants could have antiviral applications, the objective of this study was to use HPLCDAD to characterize phenolic compounds from two cultivars, Pitenza and Floradade, and determine their antiviral effects using two bacteriophages, MS2 and Av05, as viral surrogates.
Materials and methods Chemicals Water, methanol, formic acid (high-performance liquid chromatography (HPLC) grade), and DMSO (dimethil sulfoxide) were obtained from J.T. Baker (Baker-Mallinckrodt, México). Polyphenol standards, gallic acid, ferulic acid, rutin, quercetin, chlorogenic acid, and caffeic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA).
Bacterial hosts and viruses For phage propagation, E. coli O157:H7 (ATCC 4076) and E. coli (ATCC 15597-B1) were obtained from the Food and Environmental Microbiology Laboratory culture collection of the Centro de Investigación en Alimentación y Desarrollo (CIAD) in Culiacán, Sinaloa, México. Bacteria were grown on CHROMagarTM O157 by streaking and incubating for 24 h at 37 °C. Single colonies were inoculated in 100 mL of tryptic soy broth (TSB) obtained from Bioxón, Becton Dickinson, and were incubated at 37 °C overnight.
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The MS2 bacteriophage was obtained courtesy of Dr. Charles P. Gerba from the culture collection of the Environmental Microbiology Laboratory in the Department of Soil and Water at the University of Arizona. The Av-05 bacteriophage was obtained from the viral collection at CIAD.
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Plant material Tomato plant byproducts (roots, stems, leaves, and whole plants) from the Pitenza and Floradade varieties were obtained from greenhouses in the Yaqui Valley in Sonora, México. The plant materials were washed with distilled water, and plant fractions were dried at 45 °C. The materials were pulverized and passed through a No. 20 sieve (WS Tyler). Extraction process Stem, leaf, root, and whole plant extracts from two tomato cultivars, Pitenza and Floradade, were obtained according to following procedure: 35 g of dried material was mixed with 280 mL of extraction solution, composed of ethanol and 5 % acetic acid (95:5 ratio), and maceration was conducted via constant stirring for 72 h in complete darkness. To obtain the first supernatant, the sample was vacuum-filtered using Whatman No.1 paper. The residue obtained was subjected to 20 min of sonication with 50 mL of extraction solution at a controlled temperature of 20 °C. The sample was filtered to obtain the second supernatant, which was then mixed with the first supernatant. The resulting extracts were evaporated using a rotatory evaporator (Yamato water bath BM500, Yamato rotator evaporator RE301). Finally, the extracts were stored at − 20 °C for subsequent analysis. HPLC analysis Chromatographic analysis was conducted using a modified version of the method proposed by Ćetković et al. (2012), where 20 μL of extract was injected onto a 0.45 μm nylon membrane for HPLC resolution using a Waters 2690 diode array detector equipped with an automatic injector and automatic degassing system. A 4.6 mm ID × 250 mm Zorbax SB-C18 column with a 5 μm particle size was used with a mobile-phase mixture of 1 % formic acid in water v/v (A) and 100 % methanol (B) at a flow of 1 mL/min. The method consisted of a step gradient starting at 0–40 min of 20 % (B) and 80 % (A) to 65 % (B) and 35 % (A); 92 % (B) and 8 % (A) 45 min and 100 % (B) 0 % (A) at 50 min. Finally, the column was reconditioned. The absorbance was recorded at 254 and 324 nm. The compounds were identified through comparisons of their UV spectrum data with an established database of reference standards. The phenolic content was calculated using standard curves and was expressed as mg/100 g dry weight (DW). Propagation of MS2 (RNA) and Av-05 (DNA) bacteriophages For propagation of the bacteriophages Av-05 and MS2 were used as the hosts the bacterium E. coli O157:H7 (ATCC 4076) and E. coli (ATCC 15597-B1), respectively. Single-host bacterial colonies were cultured in 25 mL of TSB at 37 °C for 24 h under aerobic conditions until they reached the exponential growth phase. After incubation, 1 mL of bacteria was mixed with 100 μL of bacteriophages and 3 mL of
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TSB containing 0.4 % agarose, as described by Jamalludeen et al. (2009). This mixture was deposited onto 1.5 % TSA Petri dishes and incubated for 24 h at 37 °C. After incubation, 6 mL of SM buffer solution (50 mM Tris–HCl pH 7.5, 8 mM MgSO47H2O, 100 mM NaCl, and 0.002 % gelatin w/v) was added and the Petri dishes were oscillated in the multi-purpose rotator for 3 h. The surface layer of each dish was recovered by removal with a bacteriological loop, and the resulting suspensions were placed in sterile tubes. The samples were centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatants were filtered through a cellulose acetate membrane (Whatman, USA) with a pore size of 0.2 μm using a method adapted from López-Cuevas et al. (2012). The obtained suspensions were centrifuged at 40,000 × g for 2 h at 4 °C and the pellets were resuspended in SM buffer. The viral titers were determined according to Carey-Smith et al. (2006). Where, 1 mL decimal dilutions were completed up 1012. In tubes containing 3 mL of TSB supplemented with 0.4 % w/v agar (in a liquid state and at a temperature below 45 °C), 100 μL of each dilution was added to the mixture, and 1 mL of bacteria was added when appropriate to the bacteriophage. The mixtures were stirred gently and deposited into Petri dishes containing 1.5 % TSA in a solid state, which resulted in a homogeneously distributed, solidified layer. The Petri dishes were incubated at 37 °C for 24 h and the PFU/mL (plaque-forming units per unit volume) was measured.
Virucidal evaluation of extracts The antiviral effects of Pitenza cultivar leaf extract (PCLE) and Floradade cultivar leaf extract (FCLE) against bacteriophages Av-05 (DNA) and MS2 (RNA) were measured. The extracts were dissolved in 5 % DMSO and sterilized through a 0.45 μm membrane filter. The concentrations of extracts tested were 1 and 5 mg/mL. An aliquot of 100 μL of bacteriophages was added to 3 mL of the extract. We used four contact times: 0, 6, 12, and 24 h. At each time point, a neutralizing solution was added according to De Siqueira et al. (2006) and SCFI (1999), and the samples were mixed. Then, the following 1 mL decimal dilutions were completed up 1010 and the PFU/mL for each sample was measured using a double-layer agar technique previously described. The effects on MS2 and Av-05 were determined separately. A control was included that consisted of 100 μL of bacteriophages and 3 mL of 0.1 M phosphate buffer (PBS). We also evaluated the effect of 5 % DMSO.
Statistical analysis The principal phenolic components of the extracts were analyzed using Statgraphic for Windows® v. 4.0. Differences between groups were analyzed by analysis of variance (ANOVA) performed and Tukey comparisons applied. The confidence limits were 95 % (p < 0.05). The statistical analysis used to evaluate the survival of phages was random and considered three factors. The factors were the extract, either PCLE or FCLE; the concentration, either 1 or 5 mg/mL, and the contact time that is, 0, 6, 12, or 24 h. Considering the variability of the response, the reduction was scored as log10 phage titer. The treatments were performed in duplicate with two replicates each. We used Statgraphic for Windows® v.4.0 to perform the ANOVA and test for significance with Tukey post hoc tests and α = 0.05.
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Results Phenolic profiling Different fractions (stems, leaves, roots, and whole plant) of two cultivars, Pitenza and Floradade, were used to prepare extracts of tomato byproducts. The phenolic compounds present in these extracts were identified and quantified by analytical HPLC-DAD. Figure 1 shows the typical chromatogram of the tomato plant extracts. The phenolic compounds identified were divided into three groups: hydroxycinnamic acids, benzoic acid, and flavonols. The first group detected at 324 nm corresponds to chlorogenic, caffeic, and ferulic acids (peaks 2, 3, and 4); in the second gallic acid (peak 1), and in the third rutin (peak 5) and quercetin (peak 6) both groups were detected at 254 nm. All the results were compared with the retention times and absorption spectra of authentic standards. The concentrations of phenolic compounds identified in the different extracts of tomato products are shown in Figure 2. The contents of the individual constituents showed significant differences (p < 0.05). The major component in the Pitenza extract was gallic acid, with values of 104.6–463.41 mg/100 g DW, followed by chlorogenic acid, rutin, caffeic acid, quercetin, and ferulic acid, with levels of only 0.0195–8.09 mg/100 g DW (Figure 2(A)). In cultivate extracts derived from Floradade, the major component gallic acid was present at 9.97–229.05 mg/100 g DW. Rutin was the second most abundant constituent, with values of 7.9583–126.483 mg/100 g DW, followed by chlorogenic acid, caffeic acid, ferulic acid, and quercetin (Figure 2(B)). The analysis obtained of different extract types showed that roots extracts presented low phenolic content. In contrast, leaf extracts contained the majority of the phenolic constituents found in the tomato plants evaluated in this study; consequently, these results suggest that tomato leaves are important reservoir of phenolic compounds. Inhibitory effects against the MS2 and Av-05 bacteriophages The leaf extracts of the tomato plant PCLE and FCLE were applied to two bacteriophages at room temperature, to determine the virus survival rate and the effective dose and the time required for a reduction in virus level.
Figure 1. HPLC chromatogram of phenolic compounds present in tomato plant extracts. Peaks: (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid, (4) ferulic acid, (5) rutin, and (6) quercetin.
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Figure 2. Phenolic composition of tomato plant extracts. (A) Individual composition of the Pitenza cultivar. (B) Individual composition of the Floradade cultivar. The data shown are the means of at least three HPLC experiments at 254 and 324 nm; the error bar represents the standard deviation (SD).
Figures 3 and 4 show the log10 reduction of the MS2 and Av-05 bacteriophages in PFU/mL. The biological activity of the extracts on the infectivity of the viral surrogates after 0, 6, 12, and 24 h also were evaluated. The results showed that the concentration and contact time of the extracts significantly affected the viral titer when compared to the control (p < 0.05). In addition, the figures show that the antiviral effect of PCLE and FCLE was found to be dose dependent with increasing concentrations of extracts resulting in increased antiviral characteristic. The highest reduction observed of MS2 and Av-05 was in response to 5 mg/mL of PCLE and a contact time longer than 12 h; a reduction of 3.8 and 5 log10 PFU/mL was recorded for MS2 and Av-05, respectively. FCLE (Figures 3(B) and 4(B)) showed minimal antiviral activity at 1 mg/mL, and an incubation time of 0 min achieved a reduction of 0.57 and 1.6 log10 PFU/mL by MS2 and Av-05, respectively. The control condition, MS2 and Av-05 in PBS, did not show a significant reduction in titer over 24 h. Our results also indicated that 5 % DMSO has no antiviral activity in tested bacteriophages
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Figure 3. The reduction (log10 PFU/mL) of MS2 after treatment with PCLE (A) and FCLE (B) for various contact times (0, 6, 12 and 24 h) at two concentrations (1–5 mg/mL). The vertical error bars represent the standard deviation (SD).
(data not shown). Additionally, Figures 3 and 4 also indicate that FCLE and PCLE in concentrations of 1 and 5 mg/mL showed a minimal reduction (
International Journal of Environmental Health Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cije20
Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates a
a
b
Norma Patricia Silva-Beltrán , Saul Ruiz-Cruz , Cristobal Chaidez , c
d
José de Jesús Ornelas-Paz , Marco A. López-Mata , Enrique e
a
Márquez-Ríos & Maria Isabel Estrada a
Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, Ciudad Obregón, Mexico b
Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Culiacán, Culiacán, Mexico c
Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Cuauhtémoc, Cuauhtémoc, Mexico d
Departamento de, Ciencias Químicas Biológicas y de Salud, Universidad de Sonora, Ciudad Obregón, Mexico e
Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Mexico Published online: 25 Jul 2014.
To cite this article: Norma Patricia Silva-Beltrán, Saul Ruiz-Cruz, Cristobal Chaidez, José de Jesús Ornelas-Paz, Marco A. López-Mata, Enrique Márquez-Ríos & Maria Isabel Estrada (2014): Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates, International Journal of Environmental Health Research, DOI: 10.1080/09603123.2014.938030 To link to this article: http://dx.doi.org/10.1080/09603123.2014.938030
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
International Journal of Environmental Health Research, 2014 http://dx.doi.org/10.1080/09603123.2014.938030
Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates
Downloaded by [York University Libraries] at 22:32 12 August 2014
Norma Patricia Silva-Beltrána, Saul Ruiz-Cruza*, Cristobal Chaidezb, José de Jesús Ornelas-Pazc, Marco A. López-Matad, Enrique Márquez-Ríose and Maria Isabel Estradaa a Instituto Tecnológico de Sonora, Departamento de Biotecnología y Ciencias Alimentarias, Ciudad Obregón, Mexico; bCentro de Investigación en Alimentación y Desarrollo A.C. Unidad Culiacán, Culiacán, Mexico; cCentro de Investigación en Alimentación y Desarrollo A.C. Unidad Cuauhtémoc, Cuauhtémoc, Mexico; dDepartamento de, Ciencias Químicas Biológicas y de Salud, Universidad de Sonora, Ciudad Obregón, Mexico; eDepartamento de Investigación y Posgrado en Alimentos, Universidad de Sonora, Hermosillo, Mexico
(Received 7 January 2014; final version received 15 April 2014) Byproducts of tomato are known to include phenolic compounds but have not been studied in depth. In this study, the phenolic compositions of (stem, leaf, root, and whole plant) of two tomato cultivars, Pitenza and Floradade, were analyzed by HPLC-DAD. In parallel, the antiviral effects of crude extracts on viral surrogates, the bacteriophages MS2 and Av-05 were evaluated. The leaf extracts from the two varieties showed the highest concentration of phenolic compounds. The compounds identified were gallic acid, chlorogenic acid, ferulic acid, cafeic acid, rutin, and quercetin, and they represented 3174.3 and 1057.9 mg/100 g dried weight of the Pitenza and Floradade cultivars, respectively. MS2 and Av-05 titers at 5 mg/mL were reduced by 3.47 and 5.78 log10 PFU/mL and 3.78 and 4.93 log10 PFU/mL by Pitenza and Floradade cultivar leaf extract, respectively. These results show that tomato extracts are natural sources of bioactive substances with antiviral activity. Keywords: extracts; tomato byproducts; bacteriophages; polyphenols
Introduction The properties of tomato fruits (Licopersicum esculentum) have been extensively studied; however, few studies have focused on the whole tomato plant itself. The nutritional properties of the tomato in relation to the feeding of livestock have been reported (Font et al. 2009; Ventura et al. 2009). Tomato plant (leave and stem) are discarded after harvesting because they are considered a waste; however, the contained biologically active substances make an important source of investigation. A recent investigation showed that tomato plant leaves have several active metabolites, such as phenolic compounds (Taveira et al. 2012). Numerous studies have addressed the beneficial effects of phenolic substances derived from plant tissues. These compounds possess antioxidant, antibacterial, and antiviral properties (Chiang et al. 2003; Choi et al. 2009; Quideau et al. 2011). Plant extracts are an abundant source of bioactive substances with antiviral properties. The use of these products is generally accepted among the population because of their natural origin and low cost. In 1996, it was estimated that there were almost *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Downloaded by [York University Libraries] at 22:32 12 August 2014
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N.P. Silva-Beltrán et al.
500,000 plant species, all containing different concentrations of bioactive components (Li et al. 2013). Plant phenolic metabolites present pharmacological and food preservation properties (Schnitzler et al. 2008; Su & D’Souza 2011). Epidemiological evidence indicates that viruses cause an estimated 59 % of all foodborne illnesses, 26 % of hospitalization, and 11 % of deaths in the United States only (Scallan et al. 2011). Pholyphenols are showed as having good ability to inhibit viral infections (Su et al. 2011; Carvalho et al. 2013). Recent studies have demonstrated antiviral properties of phenolic extracts from plants, including eugenol from clove and extracts from Geranium eugineum and Cistus incanus (Sokmen et al. 2005; Droebner et al. 2007; Elizaquível et al. 2012), and of phenol derivatives, such as isoquercetin, caffeic acid, quinic acid, and chlorogenic acid (Wang et al. 2009; Kim et al. 2010; Xie et al. 2013). In particular, flavonoids have been shown to have anti-enterovirus effects, affecting viruses such as hepatitis A and norovirus (Tsai et al. 2011). Some studies have shown that flavonoids, such as quercetin, inhibit viral polymerase, interfering with the synthesis of nucleic acids and the intracellular stage of the virus replication cycle (Cushnie & Lamb 2005; Kim et al. 2010). Phenolic extracts of grape byproducts from the agro-industry have been studied for control of enteric viruses (Su & D’Souza 2011, 2012). However, antiviral effects of tomato plant byproducts against enteric viruses have not yet been documented. Therefore, this investigation can provide information regarding a possible natural resource to reduce viral spread. Bacteriophages are suitable models for disease-causing viral particles (Dennehy 2009). Phage handling is simple, inexpensive, and does not require specialized personnel or sophisticated facilities. Most intervention studies have been conducted using bacteriophages as models for human enteric viruses. Because extracts derived from tomato plants could have antiviral applications, the objective of this study was to use HPLCDAD to characterize phenolic compounds from two cultivars, Pitenza and Floradade, and determine their antiviral effects using two bacteriophages, MS2 and Av05, as viral surrogates.
Materials and methods Chemicals Water, methanol, formic acid (high-performance liquid chromatography (HPLC) grade), and DMSO (dimethil sulfoxide) were obtained from J.T. Baker (Baker-Mallinckrodt, México). Polyphenol standards, gallic acid, ferulic acid, rutin, quercetin, chlorogenic acid, and caffeic acid were obtained from Sigma–Aldrich (St. Louis, MO, USA).
Bacterial hosts and viruses For phage propagation, E. coli O157:H7 (ATCC 4076) and E. coli (ATCC 15597-B1) were obtained from the Food and Environmental Microbiology Laboratory culture collection of the Centro de Investigación en Alimentación y Desarrollo (CIAD) in Culiacán, Sinaloa, México. Bacteria were grown on CHROMagarTM O157 by streaking and incubating for 24 h at 37 °C. Single colonies were inoculated in 100 mL of tryptic soy broth (TSB) obtained from Bioxón, Becton Dickinson, and were incubated at 37 °C overnight.
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The MS2 bacteriophage was obtained courtesy of Dr. Charles P. Gerba from the culture collection of the Environmental Microbiology Laboratory in the Department of Soil and Water at the University of Arizona. The Av-05 bacteriophage was obtained from the viral collection at CIAD.
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Plant material Tomato plant byproducts (roots, stems, leaves, and whole plants) from the Pitenza and Floradade varieties were obtained from greenhouses in the Yaqui Valley in Sonora, México. The plant materials were washed with distilled water, and plant fractions were dried at 45 °C. The materials were pulverized and passed through a No. 20 sieve (WS Tyler). Extraction process Stem, leaf, root, and whole plant extracts from two tomato cultivars, Pitenza and Floradade, were obtained according to following procedure: 35 g of dried material was mixed with 280 mL of extraction solution, composed of ethanol and 5 % acetic acid (95:5 ratio), and maceration was conducted via constant stirring for 72 h in complete darkness. To obtain the first supernatant, the sample was vacuum-filtered using Whatman No.1 paper. The residue obtained was subjected to 20 min of sonication with 50 mL of extraction solution at a controlled temperature of 20 °C. The sample was filtered to obtain the second supernatant, which was then mixed with the first supernatant. The resulting extracts were evaporated using a rotatory evaporator (Yamato water bath BM500, Yamato rotator evaporator RE301). Finally, the extracts were stored at − 20 °C for subsequent analysis. HPLC analysis Chromatographic analysis was conducted using a modified version of the method proposed by Ćetković et al. (2012), where 20 μL of extract was injected onto a 0.45 μm nylon membrane for HPLC resolution using a Waters 2690 diode array detector equipped with an automatic injector and automatic degassing system. A 4.6 mm ID × 250 mm Zorbax SB-C18 column with a 5 μm particle size was used with a mobile-phase mixture of 1 % formic acid in water v/v (A) and 100 % methanol (B) at a flow of 1 mL/min. The method consisted of a step gradient starting at 0–40 min of 20 % (B) and 80 % (A) to 65 % (B) and 35 % (A); 92 % (B) and 8 % (A) 45 min and 100 % (B) 0 % (A) at 50 min. Finally, the column was reconditioned. The absorbance was recorded at 254 and 324 nm. The compounds were identified through comparisons of their UV spectrum data with an established database of reference standards. The phenolic content was calculated using standard curves and was expressed as mg/100 g dry weight (DW). Propagation of MS2 (RNA) and Av-05 (DNA) bacteriophages For propagation of the bacteriophages Av-05 and MS2 were used as the hosts the bacterium E. coli O157:H7 (ATCC 4076) and E. coli (ATCC 15597-B1), respectively. Single-host bacterial colonies were cultured in 25 mL of TSB at 37 °C for 24 h under aerobic conditions until they reached the exponential growth phase. After incubation, 1 mL of bacteria was mixed with 100 μL of bacteriophages and 3 mL of
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TSB containing 0.4 % agarose, as described by Jamalludeen et al. (2009). This mixture was deposited onto 1.5 % TSA Petri dishes and incubated for 24 h at 37 °C. After incubation, 6 mL of SM buffer solution (50 mM Tris–HCl pH 7.5, 8 mM MgSO47H2O, 100 mM NaCl, and 0.002 % gelatin w/v) was added and the Petri dishes were oscillated in the multi-purpose rotator for 3 h. The surface layer of each dish was recovered by removal with a bacteriological loop, and the resulting suspensions were placed in sterile tubes. The samples were centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatants were filtered through a cellulose acetate membrane (Whatman, USA) with a pore size of 0.2 μm using a method adapted from López-Cuevas et al. (2012). The obtained suspensions were centrifuged at 40,000 × g for 2 h at 4 °C and the pellets were resuspended in SM buffer. The viral titers were determined according to Carey-Smith et al. (2006). Where, 1 mL decimal dilutions were completed up 1012. In tubes containing 3 mL of TSB supplemented with 0.4 % w/v agar (in a liquid state and at a temperature below 45 °C), 100 μL of each dilution was added to the mixture, and 1 mL of bacteria was added when appropriate to the bacteriophage. The mixtures were stirred gently and deposited into Petri dishes containing 1.5 % TSA in a solid state, which resulted in a homogeneously distributed, solidified layer. The Petri dishes were incubated at 37 °C for 24 h and the PFU/mL (plaque-forming units per unit volume) was measured.
Virucidal evaluation of extracts The antiviral effects of Pitenza cultivar leaf extract (PCLE) and Floradade cultivar leaf extract (FCLE) against bacteriophages Av-05 (DNA) and MS2 (RNA) were measured. The extracts were dissolved in 5 % DMSO and sterilized through a 0.45 μm membrane filter. The concentrations of extracts tested were 1 and 5 mg/mL. An aliquot of 100 μL of bacteriophages was added to 3 mL of the extract. We used four contact times: 0, 6, 12, and 24 h. At each time point, a neutralizing solution was added according to De Siqueira et al. (2006) and SCFI (1999), and the samples were mixed. Then, the following 1 mL decimal dilutions were completed up 1010 and the PFU/mL for each sample was measured using a double-layer agar technique previously described. The effects on MS2 and Av-05 were determined separately. A control was included that consisted of 100 μL of bacteriophages and 3 mL of 0.1 M phosphate buffer (PBS). We also evaluated the effect of 5 % DMSO.
Statistical analysis The principal phenolic components of the extracts were analyzed using Statgraphic for Windows® v. 4.0. Differences between groups were analyzed by analysis of variance (ANOVA) performed and Tukey comparisons applied. The confidence limits were 95 % (p < 0.05). The statistical analysis used to evaluate the survival of phages was random and considered three factors. The factors were the extract, either PCLE or FCLE; the concentration, either 1 or 5 mg/mL, and the contact time that is, 0, 6, 12, or 24 h. Considering the variability of the response, the reduction was scored as log10 phage titer. The treatments were performed in duplicate with two replicates each. We used Statgraphic for Windows® v.4.0 to perform the ANOVA and test for significance with Tukey post hoc tests and α = 0.05.
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Results Phenolic profiling Different fractions (stems, leaves, roots, and whole plant) of two cultivars, Pitenza and Floradade, were used to prepare extracts of tomato byproducts. The phenolic compounds present in these extracts were identified and quantified by analytical HPLC-DAD. Figure 1 shows the typical chromatogram of the tomato plant extracts. The phenolic compounds identified were divided into three groups: hydroxycinnamic acids, benzoic acid, and flavonols. The first group detected at 324 nm corresponds to chlorogenic, caffeic, and ferulic acids (peaks 2, 3, and 4); in the second gallic acid (peak 1), and in the third rutin (peak 5) and quercetin (peak 6) both groups were detected at 254 nm. All the results were compared with the retention times and absorption spectra of authentic standards. The concentrations of phenolic compounds identified in the different extracts of tomato products are shown in Figure 2. The contents of the individual constituents showed significant differences (p < 0.05). The major component in the Pitenza extract was gallic acid, with values of 104.6–463.41 mg/100 g DW, followed by chlorogenic acid, rutin, caffeic acid, quercetin, and ferulic acid, with levels of only 0.0195–8.09 mg/100 g DW (Figure 2(A)). In cultivate extracts derived from Floradade, the major component gallic acid was present at 9.97–229.05 mg/100 g DW. Rutin was the second most abundant constituent, with values of 7.9583–126.483 mg/100 g DW, followed by chlorogenic acid, caffeic acid, ferulic acid, and quercetin (Figure 2(B)). The analysis obtained of different extract types showed that roots extracts presented low phenolic content. In contrast, leaf extracts contained the majority of the phenolic constituents found in the tomato plants evaluated in this study; consequently, these results suggest that tomato leaves are important reservoir of phenolic compounds. Inhibitory effects against the MS2 and Av-05 bacteriophages The leaf extracts of the tomato plant PCLE and FCLE were applied to two bacteriophages at room temperature, to determine the virus survival rate and the effective dose and the time required for a reduction in virus level.
Figure 1. HPLC chromatogram of phenolic compounds present in tomato plant extracts. Peaks: (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid, (4) ferulic acid, (5) rutin, and (6) quercetin.
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Figure 2. Phenolic composition of tomato plant extracts. (A) Individual composition of the Pitenza cultivar. (B) Individual composition of the Floradade cultivar. The data shown are the means of at least three HPLC experiments at 254 and 324 nm; the error bar represents the standard deviation (SD).
Figures 3 and 4 show the log10 reduction of the MS2 and Av-05 bacteriophages in PFU/mL. The biological activity of the extracts on the infectivity of the viral surrogates after 0, 6, 12, and 24 h also were evaluated. The results showed that the concentration and contact time of the extracts significantly affected the viral titer when compared to the control (p < 0.05). In addition, the figures show that the antiviral effect of PCLE and FCLE was found to be dose dependent with increasing concentrations of extracts resulting in increased antiviral characteristic. The highest reduction observed of MS2 and Av-05 was in response to 5 mg/mL of PCLE and a contact time longer than 12 h; a reduction of 3.8 and 5 log10 PFU/mL was recorded for MS2 and Av-05, respectively. FCLE (Figures 3(B) and 4(B)) showed minimal antiviral activity at 1 mg/mL, and an incubation time of 0 min achieved a reduction of 0.57 and 1.6 log10 PFU/mL by MS2 and Av-05, respectively. The control condition, MS2 and Av-05 in PBS, did not show a significant reduction in titer over 24 h. Our results also indicated that 5 % DMSO has no antiviral activity in tested bacteriophages
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Figure 3. The reduction (log10 PFU/mL) of MS2 after treatment with PCLE (A) and FCLE (B) for various contact times (0, 6, 12 and 24 h) at two concentrations (1–5 mg/mL). The vertical error bars represent the standard deviation (SD).
(data not shown). Additionally, Figures 3 and 4 also indicate that FCLE and PCLE in concentrations of 1 and 5 mg/mL showed a minimal reduction (
