In vitro antiviral activity of Brazilian Cerrado plant extracts against animal and human herpesviruses

1 Centro de Pesquisa e Desenvolvimento de Sanidade Animal, Instituto Biológico, Av. Cons. Rodrigues Alves, 1252, CEP 04014-900, São Paulo, SP, Brasil. 2 Laboratório de Virologia Animal, Universidade Estadual de Campinas, Campinas, SP, Brasil. 3 Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, São Paulo, Brazil. 4 Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Paulo, Brazil.

herpesvirus is a growing problem, particularly in immunocompromised patients (Vadlapudi et al., 2013).More recently, outbreaks of equine herpesvirus have had an impact on the equine industry, and stimulated interest in antiherpetic interventions (Vissani et al., 2016).The SuHV-1, also known as pseudorabies virus, is the causative agent of Aujeszky"s disease affecting other mammals besides pigs, is still present in wild boar and there are no antiviral drugs approved for veterinary use (Zouharova et al., 2016).Therefore, it is necessary to find new alternative drugs against these diseases that affect both humans and animals (Martinez et al., 2015;Nocchi et al., 2016).Medicinal plants are natural resources, leading to valuable herbal products often used to treat a large number of various diseases in traditional medicine (Katiyar et al., 2012;Wachtel-Galor and Benzie, 2011).
The South American flora represents one of the world"s richest sources of material with pharmacological activity, and Brazil has the biggest vegetal diversity (Dutra et al., 2016).One of them is the "Cerrado" (Brazilian Savannah) covering more than 2 million km 2 and land representing 25% of the area of the country (Sawyer, 2016).This vegetation consists of more than 6,000 vegetal species, having many compounds such as saponins, tannis, steroids, and others (Bessa et al., 2013), with biological activities including antifungal, antibacterial, antiprotozoal and antiviral ones (Alves et al., 2000;Mesquita et al., 2005Mesquita et al., , 2007;;Silva Junior et al., 2009;Brandão et al., 2010).
Many plant extracts have been described for antiviral activity against herpesviruses in vitro such as Conocephalum conicum, Polypodium glycyrrhiza and the water-soluble extract from the narcissus bulb Flos verbasci, showed anti-HSV activity.The aqueous extracts of Helichrysum aureonitens (Asteraceae) shoots inhibited HSV-1 (Chattopadhyay and Khan, 2008).Compounds in green tea extract could also inhibit HSV-1 and black tea extract enriched with theaflavins has the potential to prevent the spread of HSV-1 (Cantatore et al., 2013).Moreover, Lithraea molleiodes, Sebastiana brasiliensis and Sebastiania klotzschiana used against infectious diseases showed antiherpetic activity with no cytotoxicity, as did the aqueous extract of Beta vulgaris (Betancour-Galvis et al., 1999).The aqueous extracts from B. vulgaris and S. brasiliensis also exhibited antiviral activity against swine herpesvirus (Koseki et al., 1990;Simoni et al., 2014) and Cecropia pachystachya, Melochia villosa and P. acuminatum presented the most relevant results against bovine and swine herpesviruses (Simoni et al., 2014).Simoni et al. (2007) reported on the in vitro research for potential antiviral properties of the 16 species collected from the Brazilian Cerrado against bovine herpesvirus (BoHV-1), infectious bursal disease virus (IBDV) and avian reovirus.They found 8 plant extracts active against BoHV-1 including Byrsonima intermedia, Banisteriopsis variabilis, Stryphodendron adstringens and Xylopia Padilla et al. 107 aromatica.Species of Banisteriopsis are described as having antioxidant and antibacterial properties, while also inhibiting the activity of monoamine oxidases.Banisteriopsis caapi is the most studied species of this genus because it is used by some religious groups in Brazil as an ingredient of the drink locally known as ayahuasca.Chemical compounds identified in this plant species include alkaloids, harmine and harmaline (Wang et al., 2010;Santos et al., 2017).B. intermedia belonging to the Malpighiaceae family popularly known as "murici" has been used in folk medicine to treat fever, as diuretic, for skin infections and ulcers.B. intermedia is described mainly as being antimicrobial and as anti-inflammatory (Moreira et al., 2011;Sannomiya et. al., 2007).S. adstringens is one of the most widely used medicinal species by the Brazilian population.The stem bark is usually used to treat leucorrhea, gonorrhea, vulvo-vaginal candidiasis, gastritis, sore throat, diarrhea and bleeding (Costa et al., 2010).Extracts from the leaves present trypanocidal activity associated with the chemical compound tannin (Ishida et al., 2009).Extracts from X. aromatica have been described for antibacterial, antifungal, insecticidal, antimicrobial, and antiparasitic activities.Many compounds in this genus have been identified, but the triterpenoids are present in higher concentrations (Stashenko et al., 2004;Costa et al., 2013).
The aim of this work was continue the search with these same eight positive plant extracts to evaluate the antiviral activity against equine, swine and human herpesviruses together with additional studies of antiviral and virucidal potential.Additionally, the phytochemical profile of the extract from B. intermedia, which displayed higher antiviral activity, was performed by HPLC/MS analysis.

Preparation of crude extracts
Crude aqueous extracts were prepared by grinding dried leaves with de-ionized distilled water (10%, w/v) in a mixer and maintenance at 4ºC overnight.The aqueous extracts were filtered on Whatman-1 filter paper and freeze-dried in Flex-dry MP FTS systems.Lyophilized extracts were dissolved in equal parts of sterile de-ionized distilled water and Eagle minimum essential medium (MEM 2x) at a final concentration of 10,000, 4,000 or 2,000 µg/mL.The extracts were centrifuged at 2,500 g/10 min in a Hettich Zentrifugen model Rotina R and sterilized by filtration (0.22 µm filter).

HPLC/MS analysis
Crude aqueous extract from B. intermedia was filtered on a Sep-Pak column using MeOH as eluent.Samples containing 1 ml of the crude extract was analyzed by HPLC using a Luna C-18 (Phenomenex) column (5 mm, 250 X 4 mm), with a gradient from MeOH:H2O:HCCOH 1% 75:25 (0 min) to MeOH 100% (30 min), flow rate 1.0 ml/min and detection at 254 nm.HPLC/MS (negative form) were acquired on a Bruker micrOTOF-QII coupled to an Apollo ion source set as follows: dry temperature at 180°C and voltage at 4.5 kV.The mass/charge ratios were detected in scan (m/z 100-1200 Da) and product ion scan (m/z 50-1200 Da) modes, using the same chromatographic method described above.

Cytotoxicity assay
The assays were performed using 96-well microtiter plates with 30,000 cell/well.After 24 h of incubation at 37ºC in a humidified 5% CO2 atmosphere, each cell type was exposed to decreasing concentrations of plant extract in triplicate.Any cell morphology alteration was observed at light microscopy during the next 3 days to determine the maximum non-cytotoxic concentration (MNCC).Monolayers of cells incubated only with MEM were used as a control.

Antiviral assay
For the assays, MDBK or Vero cells were prepared in 96-well microplates at density of 3 × 10 4 cells per well and incubated for 24 h at 37ºC with 5% CO2.After 24 h of incubation, the medium was poured off and 100 µL of extracts at dilutions corresponding at MNCC were added.The cells were incubated for 1 h and after this period, 50 µL of logarithmic dilutions of viruses were inoculated for 96 h.MDBK cells were infected with SuHV-1 and Vero cells were infected with EHV-1 or HSV-1.Controls consisted of untreated infected (virus titer), treated non-infected (extract control), untreated non-infected (cell control) cells.In addition, to verify if the antiviral activity of the extract remained below its MNCC, a dose-response experiment was carried out using it at range of 250 until 1.9 µg/mL.

Direct anti-herpes assay
The assay was described previously by Simoni et al. (1996).Briefly, 100 µL of each extract at MNCC was mixed with 100 µL of logarithmic dilutions of each virus, incubated at 37°C in 5% CO2 for 1 h.Then, 50 µL of mixture was inoculated at the monolayers in each well of a 96-well plate.In addition, to verify if the direct antiherpes effect of the extract remained below its MNCC, a doseresponse experiment was carried out using it at range of 250 until 1.9 µg/mL.

MTT assay
The MTT assay was performed to confirm qualitatively the MNCC and the antiviral activity of the extracts that presented PI% more than 97%.Cells in a 96-well microplate were incubated with 100 μL/well of increasing concentrations of crude extracts in quadruplicate with at least 5 different concentrations.Thereafter, 100 TCID of virus was added.After incubation for 72 h, the medium was removed and added on cell monolayer 50 µL of MTT (1 mg/mL) to each well.The microplate was then incubated at 37ºC for 4 h.The supernatant was then removed from each well without disturbing the cell clusters containing formazan crystals.To solubilize the formazan crystal, 100 µL of SDS was added and the microplate was incubated again overnight.The absorbance of the wells was read in a computer-controlled microplate reader (Spectra Max Plus 384) at 540 nm wavelengths.The 50% cytotoxic concentration (CC50) of the test compound was defined as the concentration that reduced the absorbance of mock-infected cells by 50% of that of the control.The 50% antiviral effective concentration (IC50) was expressed as the concentration that achieved 50% protection of virus-infected cells from the HSV-1, EHV-1 and SuHV-1 induced destruction (Takeuchi et al., 1991).

Data and statistical analysis
The antiviral and virucidal activities were based on reduction of viral titers using CPE criteria.Values were expressed as titer (TCID 50 µL) (Reed and Muench, 1938) and viral inhibition index (VII) calculated as the difference of virus titer between treated and untreated infected control cultures.The VII was considered positive when ≥ 1.5 (Barros et al., 2012).
The 50% cytotoxic (CC 50 ) and 50% inhibition (IC 50 ) concentrations were calculated from concentration-effect curves obtained from nonlinear regression analysis of concentration-effect curves by the GraphPad Prism 5 Demo program.The results were obtained from triplicate independent assays.The percentage of cytotoxicity was calculated as [(A -B)/A] × 100, where A and B are the OD540 of untreated and of treated cells, respectively.The percentages of protection were calculated as [(A − B) × 100/(C − B)], where A, B and C indicate the absorbance of the extracts/fractions, virus and cell controls, respectively.Each obtained EC 50 value was defined as the effective concentration that reduced the absorbance of infected cells to 50% when compared with cell and virus controls.The selectivity index (SI) was determined by the ratio of CC 50 to EC 50 and expressed as mean ± s.e.m.The statistically different effects of tested extracts on the inhibition of virus replication were compared with the control group using the Student"s t-test with p≤0.05 for significant result.

Cytotoxicity
The MNCCs of the extracts were determined in two different cell lines, MDBK and Vero and the concentrations used in bioassays are presented in Table 1.In Vero cells, the CC 50 was lower than that one observed in MDBK cells.X. aromatica presented the lowest cytotoxic to Vero and MDBK cells with MNCC of 1250 and 625 µg/mL, respectively.The extract from O. pulchella was the most cytotoxic for both cell lines with

Direct anti-herpes effect
Assays to study the direct anti-herpes effect were done only with the extracts that were effective for at least two of the viruses tested in the antiviral assays.These results are presented in Table 2.The extracts of B. variabilis and B. intermedia, presented an inhibition against the three viruses with VII greater or equal to 3.0.X. aromatica was only effective for SuHV-1and HSV-1, while the extracts from S. adstringens had action only on HSV-1.

Additional studies with Byrsonima intermedia
B. intermedia was selected for additional studies and to characterize its antiviral properties because it showed the highest values of VII against all herpesviruses.
Table 3 shows the results of the cytotoxic, antiviral activities and selectivity index (SI) obtained from B. intermedia extracts.The MNCC of B. intermedia extracts that did not cause alterations in the morphology of the cells visible under the optical microscope (Table 2) were lower than that obtained with CC 50 of 141.1 for Vero cells and 782.1 for MDBK cells.B. intermedia presented SI values of 41.76 ± 0.04; 4.12 ± 0.1 and 193.97 ± 0.09 respectively against HSV-1, EqHV-1 and SuHV-1.
Figure 1 shows the antiviral activity of extract from B. intermedia at 8 set concentrations against the three viruses showing that treatment with B. intermedia extract resulted in reduced viral titers in dose-response curves.At 15.6 µg/mL, the initial reduction occurred firstly with SuHV-1 and then with EHV-1 and HSV-1 at 62.5 µg/mL.At 250 µg/mL, the greater VII was also obtained with a: 50% cytotoxic concentration (μg/mL); b: 50% inhibitory concentration (μg/mL); c: SI -Seletivity Index -ratio of CC50 to IC50.
SuHV-1 followed by EHV-1 and HSV.Likewise, Figure 2 shows the direct anti-herpes effect against SuHV-1, EHV-1 and HSV-1 showing that treatment of virions with this various concentrations of extracts for one hour resulted in reduced viral titers.The extracts were active at the concentrations range 250 to 31.2 µg/mL.At 3.9 µg/mL, the reduction occurred firstly with SuHV-1 and then with EHV-1 and HSV-1 at 7.8 µg/mL.At 250 µg/mL, the greater VII was obtained for SuHV-1 and EHV-1 followed by HSV.

Phytochemical profile of Byrsonima intermedia
As follow-up, the B. intermedia extract was subjected in the current study to HPLC/MS analysis (Figure 3).The peak at R t = 10.2 min corresponds to a [M -H] -quasimolecular ion peak at m/z 169, suggesting the occurrence of gallic acid (A).The second peak, detected at Rt = 13.6 min corresponds to a [M -H] -quasimolecular ion peak at m/z 301, suggesting the presence of flavonoid quercetin (B).

Cytotoxicity
The cytotoxic assay at the first stage of screening is very important to determine the maximum concentration of the aqueous extract that did not induce changes in cell morphology.This step is critical because the virus is an intracellular parasite and uses the cell machinery to replicate, so it must be ensured that the virus have the ideal conditions for their growth (Cos et al., 2006).
The study of cytotoxicity in different cell lines is important because one of the inherent drawbacks of in vitro antiviral testing is the environmental sensitivity of animal cells in culture, although the in vitro methodology is faster and less costly (McCutcheon et al., 1995).

Antiviral activity
Brazilian Cerrado plants may lead to the study of a broad source of new natural compounds; however, most are being developed for use as antibiotics more than antiviral drugs.This is because the viruses present certain characteristics among others of replicating within the cell and persisting in the host.Their combat must be more rational by the choice of species of plants that have already been used by the population to treat the diseases.
Many Brazilian Cerrado species have already been described as having antiviral activity.Chattopadhyay et al. (2015) showed that Byrsonima verbascifolia had strong anti-HSV activities, whereas Maldini et al. (2011) found that B. crassifolia used as anti-inflammatory has been described as having compounds that also display antiviral activity against HSV-1 and HIV.
In the present study, eight extracts reduced at least one of the viral titers, as compared to untreated control cells.B. variabilis, B. intermedia and X. aromatica species were active against all these herpesviruses.Furthermore, a strong inhibition was obtained from B. variabilis and B. intermedia.S. adstringens extract also showed an effective inhibition against SuHV-1 and HSV-1.Extract from X. aromatica presented the highest values of VII against SuHV-1.Antiviral activity was also observed for extracts from C. xanthocarpa and O. pulchella only against SuHV-1.Frias et al. (2012) isolated from metanolic extract of B. variabilis, the flavonoids quercetin, rutin, and apigenin.Quercetin is found in a wide variety of plants and studies showed that it could reduce infectivity of target cells and replication against herpesviruses.The results of our work indicated that the extracts of B. variablis showed inhibitory effect against the three herpesviruses and probably this inhibitory effect is due to presence of these flavonoids.Felipe et al. (2006) described the inhibition BoHV-1 replication of aqueous extracts from S. adstringens stem bark.Simoni et al. (2007) showed that the leaves presented moderate activity against BoHV-1.In the present work, leaves extracts from S. adstringens also presented moderate activity against EHV-1 but strong inhibition against HSV-1 and SuHV-1.
Antiviral drugs have been reported for herpetic infections for both human and animal herpesviruses.Although the regular use of vaccines helps in improvement of animal herds and avoids viral shedding, treatment of herpesvirus infections is not completely prevented by immunization (Gulati et al., 2016;Maxwell, 2017).Thus, antiviral agents can be useful not only as preventives but also as therapeutic in herpesvirus infections which leads to a great interest in discovering new effective and safe drugs.The results of this present work suggest the importance of searching for new compounds for veterinary and human medicine in Brazilian Cerrado plants because they have shown potential for developing new antivirals.
Several plant extracts with promising results have been described for their antiviral potential against diverse herpes viruses.For example, Cardamine angulata, Conocephalum conicum and Polypodium glycyrrhiza showed activity against bovine herpesvirus and HSV-1 (McCutcheon et al., 1995).Aqueous extract from Guettarda angelica seeds exhibited strong inhibition for three animal herpesviruses including bovine, swine and equine herpesviruses 1 (Barros et al., 2012).
The present results were promising especially for B. intermedia and indicating that materials derived from their leaves exhibited antiviral activity and selectivity toward three herpesviruses HSV-1, EqHV-1 and SuHV-1.In 2007, Simoni et al. (2007) described the antiviral activity of the B. intermedia against bovine herpesvirus (BoHV-1) and against avian reovirus indicating a broad spectrum of action of this plant species.

Direct anti-herpes effect
To verify if the mechanism of action of extracts is also due to inhibition on the viruses in extracellular conditions, the selected extracts showing antiviral activity were studied.
Many plants may exhibit different mechanisms of viral inhibition acting on more than one target during viral replication.The Opuntia streptacantha inhibited virus replication and inactivated extracellular virus, such as HSV, equine herpes virus, pseudorabies virus (Jassim and Naji, 2003).Bidens pilosa extract was effective against HSV-1 and HSV-2 with potent virucidal activity (Vadlapudi et al., 2013).Virucidal activity has also been described in crude hydroethanolic extract from the stem bark of S. terebinthifolia and was effective against HVS-1 in the attachment and penetration stages (Nocchi et al., 2016).Moreover, hot water extract of B. pilosa showed potent inhibitory activity against HSV-1 and HSV-2 in Vero cells.The authors suggest multiple targets of activity because this extract showed virucidal activity to block the binding of virus to host cells and viral cell penetration.The B. pilosa extract also inhibits virus adsorption to cells and affects some intracellular steps of viral replication (Nakama et al., 2012).These findings are in agreement with those observed in this study.

Additional studies with Byrsonima intermedia
The extracts from B. intermedia were tested in their maximum non-toxic concentrations in which no alteration of normal cell morphology was observed, and the CC 50 for both MDBK and Vero cells was obtained.Therefore, the extract showed no toxicity even when used at high concentrations in the reduction of viral titers assay and direct anti-herpes assay.Further, B. intermedia extracts showed anti-herpes activity in a concentration-dependent manner in both assays, suggesting that this effect may have occurred either acting directly on the viral particle or in other stages of the replicative cycle.
Additional studies should be done to study the mechanism of action of extracts of B. intermedia against those herpesviruses indicating at which stage the extract has activity against viruses.

Phytochemical profile
Using dereplication procedures by HPLC/MS, the occurrence of gallic acid and quercetin in the active extracts from B. intermedia was identified.As previously reported, these compounds were detected in the MeOH leaves extract from B. intermedia which displayed mutagenic activity (Sannomiya et al., 2007).
Gallic acid is a phenolic compound present in many plants, fruits and vegetables.Studies have demonstrated that gallic acid exhibits a strong inhibitory effect against several viruses such as the herpes simplex virus, hepatite C virus, HIV and enterovirus 71.Therefore, the potential activity observed by GA suggests that this activity is due to the hydrophobic interaction between the functional group, (hydroxyl) and virion components of enveloped herpesvirus.In addition, gallic acid showed an inhibitory effect in a time-dependent manner, notably similar to the current study (Govea-Salas et al., 2016).Quercetin, a flavonoid found in several plant species, including fruits and edible vegetables, displayed several biological potentials (Sharma et al., 2018), including antiviral against HSV-1 ( Garrett et al., 2012).Additionally, Choi et al. (2009) described that quercetin-3-rhamnoside from Houttynia cordata extract inhibit influenza A replication in the initial stage of virus infection by indirect interaction with virus particles.
New studies are needed to bring the active substances present in B. intermedia extract and to identify in detail the ways in which they act.The other extracts that can be considered promising must also pass through the processes of fractionation and isolation of substances in order to verify the potential of this activity.All of them here have shown their potential to lead to the development of an alternative therapy for the treatment of such important infections worldwide.

Conclusion
This study has demonstrated a broad antiviral activity of Brazilian Cerrado plants and indicated that they can be Padilla et al. 113 effective potential candidates for the development of new strategies to treat viral infections.B. intermedia was chosen for further research as well as gallic acid and quercetin all necessary to elucidate the mechanism of action of the extract or compounds both on herpesviruses replication and on direct antiviral effect.Bartheldyova E, Miller AD, Ruzek D, Turaneka J (2016).Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1) Vet.Microbiol.184(29):84-93.

Figure 3 .
Figure 3. HPLC analysis of crude aqueous extract from B. intermedia leaves and identified compounds gallic acid (A) and quercetin (B).

Table 2 .
Direct anti-herpes effect of extracts at MNCC against herpes simplex virus, equine herpesvirus and swine herpesvirus in cell cultures.

Table 3 .
Selectivity index, cytotoxic and antiviral activities from B. intermedia aqueous extract against swine herpesvirus, equine herpesvirus and herpes simplex virus by the MTT assay.