key: cord-0729034-q9ohezi0 authors: dos Santos Oliveira, Mônika Bezerra; Valentim, Iara Barros; dos Santos Rocha, Tauane; Santos, Jaqueline Correia; Nobre Pires, Keyla Silva; Lira Tanabe, Eloiza Lopes; Cruz Borbely, Karen Steponavicius; Borbely, Alexandre Urban; Fonseca Goulart, Marília Oliveira title: Schinus terebenthifolius Raddi extracts: from sunscreen activity toward protection of the placenta to Zika virus infection, new uses for a well-known medicinal plant date: 2020-04-28 journal: Ind Crops Prod DOI: 10.1016/j.indcrop.2020.112503 sha: 0d81dfed14616501feaeb0a3e075f5c417905e2c doc_id: 729034 cord_uid: q9ohezi0 Abstract Schinus terebinthifolius Raddi is a well-known medicinal plant native of South America. This species has demonstrated important biological activities such as antihypertensive and vasodilator, antimicrobial, anti-inflammatory and antioxidant. However, no studies have been, so far, reported with the fruits of S. terebinthifolius as a protector of the placenta against Zika virus infection and as sunscreen agents. The present study aimed to investigate new uses for the ethanolic fruit extracts of S. terebinthifolius, from fruits’peel (STPE) and from the whole fruits (STWFE). Zika virus (ZIKV) has been linked to several fetal malformations, such as microcephaly and other central nervous system abnormalities. Thus, the potential of these natural extracts against ZIKV infection was evaluated, using an in vitro method. The photoprotective potential, determined by spectrometry, along with phenolic content, antioxidant capacity, and chemical composition of both extracts were also evaluated. The chemical composition of the extracts was evaluated by HPLC-UV / vis. The cytotoxicity of peel and whole fruit extracts in vero E6 cell lines, in placental cell lines and placental explant cultures were evaluated by the MTT assay. The infectivity of placental cells and explants was evaluated by qRT-PCR and the effects of extracts on ZIKV infection were investigated using HTR-8/SVneo cells, pre-treated with 100 μg mL-1 of STWFE for 1 h, and infected with MR766 (AD) or PE243 (EH) ZIKV strains. STFE and STWFE were well-tolerated by both placental-derived trophoblast cell line HTR-8/SVneo as well as by term placental chorionic villi explants, which indicate absence of cytotoxicity in all analysed concentrations. Two strains of ZIKV were tested to access if pre-treatment of trophoblast cells with the STWFE would protect them against infection. Flow cytometry analysis revealed that STWFE extract greatly reduced ZIKV infection. The extracts were also photoprotective with SPF values equivalent to the standard, benzophenone-3. The formulations prepared in different concentrations of the extracts (5-10%) had shown maximum SPF values of 32.21. STWFE represents a potential natural mixture to be used in pregnancy in order to restrain placental infection by ZIKV and might potentially protect fetus against ZIKV-related malformations. The extracts exhibited photoprotective activity and some of the phenolic compounds, mainly resveratrol, catechin and epicatechin, are active ingredients in all assayed activities. The development of biotechnological/medical products, giving extra value to products from family farming, is expected, with strong prospects for success. Herbs and spices are of great commercial importance. In particular, Schinus terebinthifolius Raddi (Anacardiaceae), a South America-native plant, widely found on the Brazilian coast and popularly known as "pimenta-rosa" or "aroeira-vermelha". This plant provides a fruit, used as a refined spice in world cuisine, due to its soft taste and good appearance (Pagani et al., 2014; . Re-examination of safe medicinal natural products toward new targets is strategic. Medicines (RENISUS) (Brazilian Ministry of Health, 2017) , in which there are 71 medicinal plants that present the potential to generate products of interest to the Brazilian Unified Health System (SUS) (DiCiaula et al., 2014; Torres et al., 2016) . This species is present in the Phytotherapeutic Form of the Brazilian Pharmacopoeia (2011) (Anvisa, 2011) . Important biological activities, such as antihypertensive and vasodilatory (Glória et al., 2017) , antimicrobial (Degaspari et al., 2005; ; anti-allergic (Cavalher-Machado et al., 2008) , antioxidant, antiproliferative and anti-inflammatory ; antioxidant and protective against doxorubicin-induced cardiotoxicity (Rocha et al., 2018) and, more recently, against multidrug-resistant strains of hospital origin (Gomes et al., 2019) were already reported. Zika virus (ZIKV) and dengue virus (DENV), viral infections transmitted by vectors, are threats to health and reasons for international concern, for instance, microcephaly among newborns and incidences of Guillain-Barré syndrome among adults. As such, we decided to evaluate the ability of this medicinal plant to inhibit ZIKV infection, especially in placenta. The placenta is a unique immunological site, responsible for maternal tolerance to the fetus and for maternal and fetal defense against possible pathogens. Maternal decidual J o u r n a l P r e -p r o o f cells and several leukocytes in the basal decidua of the placenta are involved in local and systemic immunomodulation, but trophoblast cells also express different receptors from the innate immune response, which are known to bind to a plethora of pathogens, one of them the Zika virus (ZIKV) (Tabata et al., 2016; Aagard et al., 2017) . ZIKV belongs to the Flaviviridae family and was first isolated in Nigeria, in 1954. Since then, several outbreaks were reported mainly between 2007 and 2015, when it lastly hitted the Americas, causing a worrying number of newborns with brain malformations (Rodriguez-Morales et al., 2018) . During pregnancy, the ZIKV can be vertically transmitted, and infects the fetus, which may develop the congenital Zika syndrome, characterized by stillbirth/miscarriage, fetal growth restriction, microcephaly, ocular abnormalities, ventriculomegaly and other brain malformations (França et al., 2016) . Diverse studies using mice models were published in recent years (Miner et al., 2016; Adibi et al., 2016; Aliota et al., 2016; Cao et al., 2017) . The animal models have successfully demonstrated that ZIKV infects the placenta and fetal brain, during early pregnancy, with human placentas' comparable histopathological findings, represented by poor pregnancy outcomes, as fetal growth restriction, abortions and fetal demise (Miner et al., 2016; Adibi et al., 2016; Aliota et al., 2016) . Nevertheless, the placenta is morphologically and physiologically different between rodents and humans, and results should be carefully analysed (Chaouat and Clarke, 2015) . Furthermore, experimental infection models using in vitro human placental explants, and bi-and tri-dimensional culture models, have also demonstrated ZIKV infection and replication in the placenta. Mainly, Hofbauer cells, mesenchymal cells, fibroblasts and cytotrophoblast cells (both villous and extravillous) are prone to be infected and act as virus reservoirs, as wells as decidual cells and decidual macrophages in the maternal compartment (Tabata et al., 2016; Aagard et al., 2017) . As such, J o u r n a l P r e -p r o o f trophoblast cells that compose the placental barrier can be infected and, besides primary cultures, several trophoblast cell lines were also successfully proven to be infected by ZIKV, as Swan 71, JEG-3, BeWo and HTR-8/SVneo cells. As such, they provide good models to test ZIKV molecular interactions and potential interveners (Aldo et al., 2016; Arumugasaamy et al., 2018; Luo et al., 2018; Cao et al., 2017) . In this context, inhibiting ZIKV infection of trophoblast cells and its crossing of the placental barrier would be important to prevent ZIKV deleterious effects to the developing fetus, and several natural products have been investigated, due to their antiviral effects on viral entrance and cellular replication (Batista et al., 2019) . Additionally, phytochemical studies have reported the presence of phenolic compounds as the main constituents of extracts of Schinus terebinthifolius Raddi Pagani et al., 2014; Feuereisen et al., 2014; Feuereisen et al., 2017; Gomes et al., 2019) . They exert important biological activities as antioxidant and photoprotective compounds, due to their capacity to fight against reactive oxygen and nitrogen species and through absorbing ultraviolet (UV) radiation (Saewan et al., 2013; Agati et al., 2013; Souza et al., 2015) . Ultraviolet A (320-400 nm) and B (290-320 nm) radiations are associated with a number of irreversible damages to cutaneous tissue, such as photoaging and skin cancer, while UVB radiation is related with immune dysfunction, erythema and sunburn, triggering oxidative stress and DNA damage (Afaq, 2011; Gregoris et al., 2011) . One of the preventive measures against these radiations is the use of sunscreens. There is a renewed interest in the incorporation of natural extracts with antioxidant properties, in a sunscreen. The reasons for that are the perspective of reducing oxidative damage induced by UV rays, increasing physical and chemical stabilities of the sunscreens, and complementing the photoprotective action (Scalia et al., 2010; Prá et al., 2016) , as recently demonstrated (Afonso et al., 2014; Działo et al., 2016; Oliveira-Júnior et al., J o u r n a l P r e -p r o o f 2017). Protective formulations prepared with the crude extract of the leaves of S. terebinthifolius showed absorption on the UVB photoprotection area (Bulla et al., 2015) . Hitherto, up to our knowledge, there are no studies with the peel and fruits of S. terebinthifolius, as placenta protector against Zika virus infection and as sunscreen agents. Thus, the objectives of this work are to reveal new and relevant properties of the ethanolic extracts of the peel and whole fruit of S. terebinthifolius and to contribute insights into the development of ZIKV therapeutics and photoaging, adding potential value for this product from family farming. The analytical grade compounds were purchased from Sigma-Aldrich (St. Louis, USA): DPPH (α,α-diphenyl-picrylhydrazyl radical), TPTZ (2,4,6-tripyridyl-s-triazine), Folin-Ciocalteau reagent (FC), quercetin, catechin, resveratrol, gallic, coumaric and acetic acids, DMEM/F12 culture medium, L-15 medium, Hank's balanced salt solution (HBSS), phosphate buffered saline (PBS) and trypsin-EDTA. Fetal bovine serum (FBS) and L-glutamine were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Benzophenone-3 was purchased from Pharma Nostra (São Paulo, Brazil). Chloroquine was provided by Farmanguinhos (Fundação Oswaldo Cruz, Rio de Janeiro, Brazil). The Lanette base used as a vehicle for the preparation of the formulations was purchased from local merchandising pharmacy. Acetonitrile and ethanol were acquired from Merck (Germany). All the reagents were of analytical grade and the stock solutions and buffers were prepared with Milli-Q purified water. J o u r n a l P r e -p r o o f The HTR-8/SVneo (ATCC ® CRL-3271 ™ ) was kindly donated by Prof. Estela Bevilacqua from University of Sao Paulo (USP, São Paulo, Brazil) . It is a placenta cell line derived from first trimester extravillous trophoblast cells, routinely cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM Lglutamine and kept in humid incubator at 37 C and 5% CO2. Cells were subcultured every 5 days with 80% confluence for HTR-8/SVneo cells and the medium is replenished every 2 days. The use of placentas was approved by the ethical committee from Federal University of Alagoas, throughout the national Plataforma Brasil unified system (CAEE 57828616.3.0000.5013), and with signed informed consent from all patients according to Brazilian Health Ministry guidelines. Placentas derived from term pregnancies (37-40 weeks of pregnancy) were obtained at the Obstetrics Service from Prof. Alberto Antunes University Hospital of the Federal University of Alagoas, Brazil (HUPAA/UFAL) by elective cesarean section. Three term placentas from healthy pregnant women were collected from women without detectable infections, hypertensive disorders, chronic diseases or other conditions. After placenta collection, fresh samples from the chorionic villi were extensively washed in 0.1 mol L -1 PBS and in HBSS, followed by the separation of terminal chorionic villi for J o u r n a l P r e -p r o o f explants culture in 24-well plates with DMEM/F12 supplemented medium, and kept in humid incubator at 37 C and 5% CO2, where they can be cultured until 12 days, without loosing significant viability, as described previously (Miller et al., 2005) and routinely used worldwide. The fruit peels and whole fruits from S. terebinthifolius The crude extracts of the whole fruits (STWFE) and the fruit's peel of S. terebinthifolius (STPE) were prepared using ethanol, in a Soxhlet apparatus, heated for a period of 6 hours. The solvent was removed in a rotary evaporator, to obtain STPE and STWFE. The extracts were stored in amber glass, under refrigeration (5º C). The total phenolic contents (TPC) of the ethanolic extracts were determined using FC reagent, as described by Cicco et al., (2009) with the following modifications: aliquots (120 µL) of ethanolic solutions of dried extract (10 µg mL -1 ), were placed in test tubes, followed by the addition of 180 µL of water. Then, 300 µL of FC reagent were added to each tube. After 2 min, 2.4 mL of a 5% (w/v) sodium carbonate solution were added. The J o u r n a l P r e -p r o o f mixture was shaken and heated, at 40 ºC, in a water bath, for 20 min. The tubes were, then, rapidly cooled and the developed color analysed at 760 nm, in a MultiSpec-1501 UV-vis spectrophotometer (Shimadzu, Japan). The same procedure was performed, using 120 µL of ethanol as a blank. The concentration of phenolic compounds was estimated using a calibration curve traced with gallic acid (GA) in ethanol (1.5 -13 x 10 -4 mol L -1 ) as a polyphenol reference, in triplicate. The results are expressed as mg of GA equivalents g -1 of dry extract (mg GAE g -1 dry extract). The antioxidant capacities of the extracts were measured in terms of their radical The assay was performed according to the method described by Benzie and Strain (1996) , with some modifications. Briefly, the FRAP reagent is prepared by mixing 2.5 mL of FeCl3 (20 mmol L -1 ), 2.5 mL of a solution of TPTZ (10 mmol L -1 ) in 40 mmol L -1 HCl and 25 mL of 0.30 mol L -1 acetate buffer (pH 3.6). Sample aliquots (90 µL) were mixed with 270 µL of distilled water and 2.7 mL of FRAP reagent and incubated at 37 ºC for 30 min, resulting in a final concentration of 10 µg mL -1 of extract. The absorbance of the reaction mixture was measured at 595 nm and a calibration curve was obtained, using Trolox (0.15 -30 μmol L -1 ). The results are expressed as Trolox Equivalent Antioxidant Capacities (TEACFRAP), in µmol of Trolox g -1 of dry extract. The photoprotective capacity of the ethanolic extracts was determined according to the method described by Mansur et al. (1986) . Aliquots of the stock solution of the crude extract (0.1 g mL -1 ) were used in the preparation of the following concentrations: 0.2, 2, 5, 10 and 15 mg mL -1 . These solutions were read in triplicate, in a spectrophotometer (MultiSpec-1501 UV-Vis -Shimadzu, Japan), at the wavelength range of 290-320 nm with 5 nm increments. Ethanol was used as a blank. The SPF values of the ethanol extracts of S. terebinthifolius (STWFE and STPE) were calculated using Eq. 1. The identification of phenolic compounds was carried out, using a liquid chromatograph Shimadzu (VP series, Kyoto, Japan), a system controller ( STPE and STWFE extracts were dissolved in the mobile phase (5 mg mL -1 ) and filtered through a 0.45 µm nylon membrane, prior to HPLC injection. The injection volume was 20 µL, using a detection wavelength of 280 nm. The phenolic compounds present in the extracts were identified by comparing the retention times (Rt) of the standards, as well as by co-injection of the samples with standards. The standards were dissolved in the mobile phase to reach a concentration of 0.5 mg mL -1 . The phenolic standards were analysed using the same conditions of the extract. (STWFE incorporated to lanette cream in 5%), CSTWFE10 (STWFE incorporated to lanette cream in 10%) and CB5 (benzophenone incorporated to lanette cream in 5%). Aliquots of the stock solution of the formulations (0.1 g mL -1 ) were used in the preparation of the following concentrations: 0.2, 2, 5, 10 and 15 mg mL -1 . The methodology used for the SPF measurements of formulations was the same as described for the crude extract (section 2.6.4). The Lanette base was used as a blank. The analyses were performed in triplicate. The MTT assay was used to verify possible cytotoxicity of the peel and the whole fruit extracts, on both placenta cell lines and placenta explants cultures. As such, placenta explants were weighted and plated in equal proportion to each well, and Vero E6 cells or HTR-8/SVneo cells were plated at 2.5 × 10 5 cells and treated with the peel and the whole fruit extracts diluted in DMEM/F12 medium in different concentrations (0.01, 0.1, 1, 10, and 100 g mL -1 ), and further cultured for 24 h. Since chloroquine diphosphate has been J o u r n a l P r e -p r o o f described as a potent anti-ZIKV drug (Delvecchio et al., 2016; Schiryaev et al., 2017) , we tested if it would affect cell lines or explants viability in different concentrations based on Delvecchio and coworkers (2016) (6.25, 12.5, 25, 50 and 100 g mL -1 ), in order to use it, as a positive anti-ZIKV control, added 1 h after ZIKV incubation. The control group had no addition of extracts. The medium was replaced with a fresh culture medium containing 5 mg/mL of MTT and the supernatant was discarded after a 4 h incubation period at 37 °C, followed by the addition of 150 L of DMSO. The absorbance of the dissolved MTT formazan product was spectrophotometrically measured at 540 nm. The viability percentage was determined in relation to the controls [(absorbance of treated cells/absorbance of untreated cells) × 100]. violet crystal staining. Viral titration of MR766 strain was stocked at 5 x 10 5 PFU/mL and PE243 at 3 x 10 5 PFU/mL. Cells were plated at 3 × 10 5 cells in 24 well plates for 24 h and received 100 g After ZIKV incubation and treatment, pictures were taken in an inverted optical microscope coupled with camera (Olympus, Japan) at 0 h, 2 h, 24 h and 48 h (200  magnification), and images were analysed for morphological cytopathic effects. All culture groups were fixed with 4% paraformaldehyde in PBS and were visualized with a fluorescence microscope Nikon DS-Ri1 (Nikon, Japan) and images were acquired using the DP2-BSW software (Nikon). To The assays were performed in triplicate for each sample and the results were expressed as mean and standard deviations. The statistical evaluation was determined by analysis of variance ANOVA with Geisser-Greenhouse correction, followed by Dunnett post test, at the level of significance of 95%, using the software Graphpad Prism 8.3.0. All results are depicted as Mean ± SEM. HPLC technique has been widely used for the detection of phenolic compounds in plant extracts, due to their versatility and accuracy. In general, the phenolic compounds are analysed in an HPLC instrument, using reverse phase C18 columns, a diode arrangement detector (PDA), and acidified polar organic solvents. The composition profile of the phenolic compounds in S. terebinthifolius was analysed using HPLC. In Firstly, the dry STWFE and STPE extracts were dissolved in aqueous solution and analysed in HTR-8/SVneo trophoblast cell line. Regarding the whole fruit aqueous extract, none of the concentrations altered cell viability (Fig. 1B) , the same occurring to chloroquine diphosphate-treated cells (Fig. 1A) . The aqueous peel extract slightly reduced cell viability, only at concentrations of 0.01 μg mL -1 and 100 μg mL -1 (0.01 μg mL -1 , cell viability = 83.72% ± 5.58%, p < 0.05; 100 μg mL -1 cell viability = 83.4 ± 6.64, p < 0.05, when compared to Control group that was cultured only with DMEM/F12), whereas at 0.1 μg mL -1 , 1 μg mL -1 and 10 μg mL -1 , the extract unchanged cell viability ( Figure 1C ). Afterwards, we tested STWFE and STPE extracts in Vero E6 cell line, and no changes in cell viability were observed whatsoever ( Figure 1D -E) Cytopathic effects are morphological/structural changes observed in host cells caused by viral invasion. These effects can range from cytoplasmic inclusion bodies to total cell destruction. Herein, we observed cell morphology from 0 h to 48 h after ZIKV MR766 infection and with association to the treatments with chloroquine diphospate, and STWFE and STPE extracts ( Figure 3A ). After 2 h we observed the formation of scarce J o u r n a l P r e -p r o o f syncytium in the MR766 group, while none was observed in all the treament groups. Also in the MR766 group, from 24 to 48 h, we observed an incidence in cell destruction and possible apoptotic bodies, more present at 48 h . Nevertheless, we also observed the same effects in all other groups, although in lesser amounts ( Figure 3A ). To evaluate cellular alterations with more detail and to analyse how these trophoblast cells were affected morphologically, we stained cells for phalloidin to observe and, when cells were infected, but still preserved their structures, NS1 protein was more located perinuclearly, close to the endoplasmic reticulum ( Figure 3B ). Also important to highlight the increased amount of cells without cytoplasm or with scarce cytoplasm residues, greatly stained for NS1 ZIKV protein ( Figure 3B inset), indicating that in only 24 h, the PE243 strain at MOI 1 induced more pronounced cytopathic effects in comparison to the MR766 strain. HTR-8/SVneo cells were plated at 1 x 10 6 cells per well and treated as described before, with only 1 h of ZIKV incubation, culture medium removal and PBS washes with the addition of new medium. After 24 h, the MR766 strain had an average viral load of 0.929 x 10 5 PFU/μL (± 0.399 x 10 5 PFU/μL), and both treatments were not statistically significant in reducing the viral load, although a clear tendency could be observed, as treatment with STWFE reduced to 0.234 x 10 5 PFU/μL (± 0.086 x 10 5 PFU/μL), and with STPE to 0.223 x 10 5 PFU/μL (± 0.033 x 10 5 PFU/μL) ( Figure 4A ). Nevertheless, the PE243 strain had an average of 7.901 x 10 5 PFU/μL (± 4.505 x 10 5 PFU/μL), and a remarkable reduction of viral load, as treatment with STWFE reduced to 0.519 x 10 5 PFU/μL (± 0.052 x 10 5 PFU/μL, p = 0.0074), and with STPE to 0.579 x 10 5 PFU/μL (± 0.025 x 10 5 PFU/μL; p < 0.05) ( Figure 4B ). As such, both treatments seem to consistently reduce the viral load at least for the PE243 strain. Some natural products have already been described to inhibit ZIKV infection or its replication in different cell types. In addition to the promising extracts from S. terebinthifolius Raddi, we have used the antimalarial chloroquine diphosphate as a J o u r n a l P r e -p r o o f positive control of antiviral activity, as it has been described by different sources (Delvecchio et al., 2016; Shiryaev et al., 2017; Kao et al., 2018) . Other active plants have been already described, such as Doratoxylon apetalum, an indigenous medicinal plant from Mascarene Island, which inhibited Zika and Dengue virus infection in leukocytes (Haddad et al., 2019) . One polyphenol present in large amounts in the green tea, (−)-epigallocatechin gallate (EGCG), has been also described as a potent antiviral molecule, able to inhibit ZIKV entry in Vero E6 cells (Carneiro et al., 2016) . Delphinidin Curcumin and suramin can also inhibit ZIKV infection on different cells (Wah et al., 2017; Mounce et al., 2017) and other compounds such as GSK126, nanchangmycin, obatoclax, pentagalloylglucose, saliphenylhalamide (SaliPhe) and 25-hydroxycholesterol have been also described to inhibit ZIKV endocytosis or entry in different models . Nevertheless, it is important to highlight that our study is pioneer in showing antiviral candidates against ZIKV infection with action in trophoblast cells, without cellular toxicity. Phenolic compounds are known as naturally occurring antioxidants and are widely distributed in plants. Several assays have been applied to evaluate antioxidant activity of the fruit, peel, seed and pulp extracts of different plants and agroindustrial residues (Oliveira et al., 2009 ). In the present work, the total phenolic content (TPC) was determined using the FC reagent and antioxidant capacity by DPPH • and FRAP methods. Some works report that fruit peels and seeds present higher amounts of total phenols and antioxidant activity compared to the edible portions (Omena et al., 2012) . Concerning Punica granatum, Li et al. (2006) found 249.4 mg GAE g -1 dry extract for peel and 24.7 mg GAE g -1 dry extract for pulp. Several factors such as climatic conditions, cultivation regions and extraction methods may influence the results of TPC (Oliveira et al., 2009) . Costa et al. (2015) reported the effects of the extraction processes (Soxhlet extraction and maceration) on TPC for fruit extracts of S. terebinthifolius, with a variation of 5-110 mg GAE g -1 dry extract. According to the studies of Tlili et al. (2018) , extracts of the fruits of S. terebenthifolius from two localities in Tunisia were analysed by the FC reagent method and presented 35.23 and 32.39 mg GAE g -1 dry extract, being these values much lower than the values obtained in the present work. For the DPPH • method, STWFE and STPE extracts exhibited RSA of 24.1 and 78.4%, respectively ( In summary, STPE extract has shown the best results in all antioxidant assays. The extracts of S. terebinthifolius and respective formulations presented photoprotective potential, evaluated through the method developed by Mansur et al., (1986) . SPF values ( (Ribeiro, 2006) . The extracts and J o u r n a l P r e -p r o o f formulations had shown satisfactory SPF values, being higher than the value established by ANVISA. In addition, they can be compared with the SPF values of benzophenone-3. By statistical analysis, it was possible to observe that CSTPE5 and CSTPE10 showed values very close to the benzophenone-3 at 5 mg mL -1 dilution, similarly to CSTWFE10 at 10 mg mL -1 dilution. In this study, it was possible to observe STPE and its formulations presented values of SPF higher than the values found for STWFE, with saturation levels of UVB radiation absorption at around 2 mg mL -1 . The potential photoprotection exhibited by the extracts can be attributed to the presence of phenolic compounds in the samples, as shown in topic 3.6. Recent studies have demonstrated the photoprotective potential of natural active principles such as plant extracts, which, due to their chemical composition, most often present bioactive constituents such as phenolic compounds, in particular flavonoids, which exert antioxidant action, neutralizing the actions of free radicals and inhibiting oxidation processes. In addition, these compounds possess the ability to absorb UV radiation (Saewan et al., 2013; Oliveira-Junior et al. 2017) . Extracts of leaves of S. terebinthifolius have been studied in relation to their photoprotective potential. Bulla et al. (2015) , analysed the in vitro photoprotective potential and the in vivo percutaneous penetration of the hydroethanolic extracts and leaf formulations of S. terebinthifolius. The methanolic solutions of extracts at 10 and 25% (w/v) presented UV absorption with UVB photoprotective potential, with SPF values of 2.40 and 6.89, respectively. Spectroscopic measurements confirmed absorption in the UV region and the topical application of the formulations did not cause histological changes in the skin of the mice. Other plant species also showed photoprotective action. Nunes et al. (2018) demonstrated that the ethanolic extracts of the leaves and barks of Amburana cearenses Table 3 ), and the ethanolic extract of Camellia sinensis (Kaur et al., 2011) , which presented a value of SPF 18.10 ± 0.05 at the same concentration . Silva et al. (2016) investigated the crude peptone extract of Spondias purpurea at 10 mg mL -1 and obtained SPF values of the formulations prepared, with the extracts in 10, 20 and 30% of 4.13, 7.82 and 13.99, respectively, inferior to the present data (Table 3) . Considering tables 1, 2 and 3 and the results against ZIKV infection, we searched biological activities of the chemical constituents of both extracts. The results are presented in Table 4 , with special emphasis on antiviral and anti-aging activities. The investigation of the mechanism of antiviral action is very complex (Jassim and Naii, 2003 , De Clercq, 2002 , Rodriguez et al., 2019 , is under way and is not possible to define, at the time being. Antiviral drug analysis has as main targets viral or cellular proteins or the search of broader activity spectrum, with less chance of resistance development. Among the chemical constituents analysed, resveratrol was shown to exhibit direct virucidal activity against ZIKV, with anti-ZIKV replication properties (Mohd et al., 2019) . Up to our knowledge, no studies have been so far conducted showing a natural product with absent or low cytotoxicity to the placenta and with such a good potential of inhibition of ZIKV entrance in trophoblast cells. As such, our study is pioneer as we show not only STPE and STWFE extracts are well-tolerated by trophoblast cells and placental tissue explants, but also both extracts successfully inhibited ZIKV entry in trophoblast cells, presenting a potential early antiviral effect. Since the extracts presented such similar response to inhibiting ZIKV infection, we must now search for molecules present in both extracts to identify which one(s) would be responsible for such promising effect. As such, present data represent a potential hope in a scarce therapeutical field of such sensible and neglected populations of pregnant women. Additionally, it is also the first to report the photoprotective potential of the peel and whole fruit of this species. The formulations incorporated with low concentrations of the extracts presented values of SPF equivalent to the standard benzophenone-3, used in the production of commercial sunscreens. The present activities may be related to the presence of the phenolic compounds gallic acid, catechin, epicatechin, p-coumaric acid and resveratrol, the last one, being described for the first time in this species. As such, STPE and STWFE can be considered active ingredients in the preparation of photoprotective formulations. Further studies are required, including biological tests in vivo, definition of the mechanism of antiviral activities, among others, to ensure a cosmetic/medicinal application with quality, safety and efficacy. 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(Oleacaceae) Optimization of solvent mixtures for extraction from bark of Schinus terebinthifolius by a statistical mixturedesign technique and development of a UV-VIS spectrophotometric method for analysis of total polyphenols in the extract In Vitro Anti-oxidant, Anti-fungal and Antistaphylococcal Activity of Resveratrol-Producing Endophytic Fungi The potential of plant phenolics in prevention and therapy of skin disorders Potential application of herbs and spices and their effects in functional dairy products Antibacterial efficacy of epicatechin and rutin from Acacia catechu leaf extract against Enterococcus faecalis and Streptococcus mutans -An in vitro study Resveratrol suppresses nuclear factor-B in herpes simplex virus infected cells Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening Characterization of phenolic compounds in Brazilian pepper (Schinus terebinthifolius Raddi) exocarp Differentiation of Brazilian peppertree (Schinus terebinthifolius Raddi) and Peruvian peppertree (Schinus molle L.) fruits by UHPLC-UV-MS analysis of their anthocyanin and biflavonoid profiles Congenital zika virus syndrome in Brazil: a case series of the first 1501 livebirths with complete investigation Phenolic compounds present in Schinus terebinthifolius raddi influence the lowering of blood pressure in rats Residues from the Brazilian pepper tree (Schinus terebinthifolia Raddi) processing industry: Chemical profile and antimicrobial activity of extracts against hospital bacteria Propolis as potential cosmeceutical sunscreen agent for its combined photoprotective and antioxidant properties Doratoxylon apetalum, an indigenous medicinal plant from Mascarene Islands, is a potent inhibitor of Zika and Dengue Virus infection in human cells Evaluation of the anti-diabetic effects of epicatechin and/or gallic acid in STZ/NA-induced diabetic Wister rats Novel antiviral agents: a medicinal plant perspective Pharmacological effects of gallic acid in health and diseases: A mechanistic review The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR Photochemoprotective Activity of Alcoholic Extract of Camellia sinensis Genetic and serologic properties of Zika virus associated with and Epidemic, Yap State, Micronesia Antiviral Effects of Black Raspberry (Rubus coreanus) Seed and Its Gallic Acid against Influenza Virus Infection Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract Chloroquine, a FDA-approved Drug, Preventes Zika Virus Infection and its Associated Congenital Microcephaly in Mice Effective inhibition of MERS-CoV infection by resveratrol Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells Determinacão do fator de proteção solar por espectrofotometria Human placental explants in culture: approaches and assessments Zika virus infection during pregnancy in mice causes placental damage and fetal demise Resveratrol effects Zika virus replication in vitro Curcumin inhibits Zika and chikungunya virus infection by inhibiting cell binding The Antimicrobial and Antiviral Activity of Polyphenols from Almond Photoprotective potential of medicinal plants from Cerrado biome (Brazil) in relation to phenolic content and antioxidant activity Total phenolic content and free radical scavenging activities of methanolic extract powders of tropical fruit residues Development and Evaluation of Photoprotective O / W Emulsions Containing Hydroalcoholic Extract of Neoglaziovia variegata (Bromeliaceae) Antioxidant, antiacetylcholinesterase and cytotoxic activities of ethanol extracts of peel, pulp and seed of exotic Brazilian fruits Cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids Quantification of bioactive compounds of pink pepper (Schinus terebinthifolius Raddi) p-Coumaric acid and its conjugates: dietary sources, pharmacokinetic properties and biological activities Photoprotective activity of resveratrol analogues Ultrasound-assisted extraction of bioactive compounds from palm pressed fiber with high antioxidant and photoprotective activities Biological functions of epicatechin: Plant cell to human cell health Plants and phytoconstituents having sunscreen activity Cosmetologia aplicada a dermoestética, second ed. Pharmabooks, São Paulo Antioxidant and Protective Effects of Schinus terebinthifolius Raddi Against Doxorubicin-Induced Toxicity Molecular characteristics and replication mechanism of dengue, zika and chikungunya arboviruses, and their treatments with natural extracts from plants: an updated review Diagnosis and outcomes of pregnant women with Zika virus infection in two municipalities of Risaralda, Colombia: second report of the ZIKERNCOL study Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents p-Coumaric acid ameliorates ethanol-induced kidney injury by inhibiting inflammatory cytokine production and NF-κB signaling in rats Photoprotection of natural flavonoids Potential Adverse Effects of Resveratrol: A Literature Review Essential oils from Schinus terebinthifolius leaves chemical composition and in vitro cytotoxicity evaluation Comparison of in vivo and in vitro testing of sunscreening formulas Photostabilization effect of quercetin on the UV filter combination, butyl methoxydibenzoylmethane-octyl methoxycinnamate Protective effects of p-coumaric acid against oxidant and hyperlipidemia-an in vitro and in vivo evaluation Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis Effects of Different Drying Conditions on Key Quality Parameters of Pink Peppercorns (Schinus terebinthifolius Raddi) In vitro photoprotective activity of the Spondias purpurea L. peel crude extract and its incorporation in a pharmaceutical formulation Schinus terebinthifolius: phenolic constituents and in vitro antioxidant, antiproliferative and in vitro antiinflammatory activities A review of the ongoing research on Zika virus treatment Anti-infective potential of catechins and their derivatives against viral hepatitis Identification of flavonol glycosides and in vitro photoprotective and antioxidant activities of Triplaris gardneriana Wedd Photoprotective characteristics of natural antioxidant polyphenols Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission Schinus terebinthifolius vs Schinus molle: A comparative study of the effect of species and location on the phytochemical content of fruits Activity of the aqueous extract of Schinus terebinthifolius Raddi on strains of the Candida genus A Systematic Review on Anti-diabetic and Cardioprotective Potential of Gallic Acid: A widespread dietary phytoconstituent Effect of Gallic acid and Myricetin on ovarian cancer models: a possible alternative antitumoral treatment Antiviral properties of the natural polyphenols delphinidin and epigallocatechin gallate against the flaviviruses west nile virus, zika virus, and dengue virus Spices as functional foods Polysulfonate suramin inhibits Zika virus infection Antioxidant and Anti-Aging Assays of Oryza Sativa Extracts, Vanillin and Coumaric Acid Antipandemic influenza A (H1N1) virus potential of catechin and gallic acid Resveratrol protects HaCaT cells from ultraviolet B-induced photoaging via upregulation of HSP27 and modulation of mitochondrial caspase-dependent apoptotic pathway Epicatechin Antinociceptive and anti-inflammatory Antiviral (exerted antiviral effects against Zika virus (ZIKV) replication in a dosedependent manner) UV protective (inhibited UVB-induced apoptosis by promoting HSP27 expression The authors gratefully acknowledge the financial support of the Antimicrobial and antiviral (evaluated against HSV-1 viruses as a representative of DNA viruses and PI-3 as a representative of RNA viruses, with significant activity) Antiviral (fraction of Rubus coreanus seed extract and its gallic acid derivative were evaluated against strains of influenza A and B. Both showed potential and broad antiviral activity. ( Lin et al., 2017)