Till ores et ot BHC Compemertay and Aematve Medne @019)19258 hepato T011852906079 2961 BMC Complementary and Alternative Medicine RESEARCH ARTICLE fey ou aed In vitro and in silico anti-dengue activity of ® eg for compounds obtained from Psidium guajava through bioprospecting Andrea Isabel Trujllo-Correa™, Diane Caroline Quintero-Gil', Fredyc Diaz-Castillo®, Winston Quifiones', Sara M. Robledo® and Marlen Martinez-Gutierrez" Abstract Background: For decades, bioprospecting has proven to be useful forthe identification of compounds with pharmacological potential, Considering the great diversity of Colombian plants and the serious woridwide public health problem of dengue—a disease caused by the dengue virus (DENY)—in the present study, we evaluated the anti-DENV effects of 12 ethanolic extracts derived from plants collected in the Colombian Caribean coast, and 5 fractions and 5 compounds derived from Paicium guajava, Methods: The cytotoxicity and antiviral effect of 12 ethanolic extracts derived from plants collected in the Colombian Caribean coast was evaluated in epithelial VERO ces Five fractions were obtained by open column chromatography from the ethanolic extract with the highest selectivity index (SI) (derived from P. guajava, Sl: 1282). From the Fraction with the highest selectivity (Pg-YP++-22C, Sk 35.5), five compounds were identified by one- and two-dimensional nuclear magnetic resonance spectroscopy. The antiviral effect in vitro of the fractions and compounds was evaluated by different experimental strategies (Pre- and post-treatment) using non-toxic concentrations calculated by MIT method. The DENY inhibition was evaluated by plate focus assay. The results were analyzed by means of statistical analysis using Student's test. Finally the antvial effect in Silico was evaluated by molecular docking, Results: In vitro evaluation of these compounds showed that three of them (galic acid, quercetin, and G2techin) were promising antivirals a5 they inhibit the production of infectious viral particles via different experimental strategies, with the best antiviral being catechin (100% inhibition with a pre-treatment strategy and 91.8% with a post-ireatment strategy). When testing the interactions of these compounds with the viral envelope protein in silico by docking, only naringin and hesperidin had better scores than the theoretical threshold of -7.0 kcal/mol (~80 kcal/mol and—8.2 kcal/mol, respectively). All ligands tested except gallic acid showed higher affinity 10 the NSS protein than the theoretical threshold, Conclusion: Even though bioprospecting has recently been replaced by more targeted tools for identifying compounds with pharmacological potential, our results show It is still useful for this purpose. Additionally, combining in vitro and in slico evaluations allowed us to identify promising antivirals as well as their possible mechanisms of action. Keywords: Dengue virus, Antiviral, Bioprospecting, Psidium guajava, Catechin, Gallic acid, Quercetin, responders arenas ‘Gupo de nvestancion en Cencas Animals GRCA, Facultad de Meena veterinary Zoteci, Unvetsaad Cooper de Coloma, Bucwaranga, Cale 30 #3351, Socstmangn Colombe Ful of auth information fsavaiable athe endo the arte a BMC he Ash 20°9Open Aces This ice tasted urd he er othe Cte Cnr Aston 4B ‘egredcton in ay meer, poise you ge sepopate ct ote igal aur 2 he sou, poe fk Fe eate Crs hea sna exe charges wate Mae The Cee Conor able Danan Deaton ae! (regutzeseecanmensotyeubconaivaea/lay apes oh daa ade albert ls cate SateTrulloCorrea e a BMC Complementary ond Aterative Medicine Introduction Dengue is a growing public health problem worldwide, mainly in tropical and subtropical regions [1]. In the last five decades, on the American continent, the incidence of dengue has increased 30 fold and is the cause of ap. proximately 390 million infections per year, of which 96 million have clinical manifestations [2]. In 2013, in America, the largest number of cases in the history of the disease was reported, with a total of 23 million cases, an alarming figure [3, 4]. In most cases, dengue presents as an asymptomatic disease, however, it can presented with a wide range of clinical manifestations, including fever, headache, pain in various parts of the body, and prostration, among others. Few patients present with serious life-threatening manifestations. This ‘wide clinical variety permits classified dengue in three groups: asymptomatic patients, symptomatic patients (ith or without alarming symptoms) and patients with severe dengue (patients with hemorrhage andor hypo- volemic shock)" [5] The etiological agent that causes this disease is the dengue virus (DENV), a member of the Flaviviridae family, which belongs to the arboviruses (viruses trans: mitted by arthropods). This virus is classified into four serotypes (DENV 1-4) according to genetic and anti genic differences [6]. Although any serotype is equally able to cause dengue, serotype differences have been postulated to lead to differences in pathogenesis [7] such as the case for DENV-2 witich have been related with sever dengue [8]. The DENV genome consists of a strand of positive-sense Ribonucleic Acid (RNA) of ap. proximately 1kb with a m7GpppAmp cap at its 5° end and no poly (A) tail at its 3'-end [9]. Moreover, it has a single reading frame that codes for three structural pro- teins (C, PeM, and E) and seven nonstructural proteins (NS, NS2a, NS2b, NS3, NS4a, NS4b, and NSS), which are mainly involved in viral replication (10, 11] The replication cycle of DENV begins with the E protein binding to receptors on the cell membrane. After this binding, there is a clathrin-mediated endocytosis followed by the formation of an endosome leading to the pH: dependent fusion (Viral envelopejendosome). The acidic pH of the endosome favors the release of the viral RNA in the cytoplasm to be transcribed and translated into ribo. somes associated to the endoplasmic reticulum, resulting in a single viral polyprotein which is cleaved by cellular and viral proteases. The replication complex (formed by RNA, NS5, other nonstructural proteins and cellular fac: tors) is formed in association with intracellular mem: branes, Finally, the assembly of viral proteins with new genomes occurs in the lumen of the endoplasmic reticulum, followed by the passage of the new visions through the Golgi apparatus (where the maturation viral process occurs) to be released the virions by exocytosis (2019) 19298 Page 2 of 16 [12]. Although all steps of the viral replication cycle are likely to be inhibited, most studies have focused on the evaluation of compounds that inhibit the attachment and entry of virus into the cell, among them are heparin (13) and sulfated polysaccharides [13]. Moreover, other com: pounds are able to inhibit the viral genome replication by blocking the synthesis of nucleoside triphosphates. Ribavi rin and mycophenolic acid {14 are two good examples of such inhibitors. Finally, other compounds, including casta nospermine [15] and the Lovastatin [16] inhibit steps in the replication cycle after entry and replication of DENV, possibly affecting the assembly process. Despite the large number of possible antiviral candi dates [17], to date, only a few have been tested in clinical trials, such as balapiravir, chloroquine, lovastatin, pred: nisolone, and celgosivir [18]. However, none of these compounds is being used as an effective anti-dengue therapy. Thus, there remains an important need to iden- tify effective and tolerable medications for treatment of DENV- infected patients both in the early phase, to pre- vent progression to fatal outcomes, and to minimize deaths after patients develop severe complications [19]. For this reason, the agenda of research priorities pro- posed by the World Health Organization proposed, a decade ago, that included searching for antivirals (ether second-use drugs or natural product derivatives), is still valid today [20]. Moreover, other of the main reasons for the high incidence of the DENV worldwide is that thus far, only one licensed vaccine exists for use in a few countries [21]. This vaccine was produced by Sanofi Pas- teur (CYD-TDV) is composed of four attenuated vac: cines (CYD-1-4) isa life recombinant vaccine, based on a yellow fever vaccine 17D (FV 17D) backbone [22 Plants have been used as medicinal sources to treat many diseases for thousands of years. Ancestral commu: nities could empirically identify plants to fight infections, passing many of these findings from generation to gen- eration until today [23]. Ethnobotany has proven useful for preserving such knowledge in communities that use plants to treat diseases has encouraged bioprospecting studies, which allow the identification of compounds with pharmacological potential [24]. However, is import: ant highlight that in some cases the communities, with: out scientific knowledge, use plants included in the IUCN Red List of Threatened Species [25] to treat dis eases, and for these reason is important encourage to preserve, protect and promote the traditional knowledge but with scientific support. Due to the diversity of chemical compounds present in plants, they are an important source of pharmaco: logical candidates, Several studies have identified po- tential plant derived candidates compounds as a potential of new drugs candidates that inhibit the ac- tivity of viruses such as herpes virus [26], hepatitis CTrulloCorrea e a BMC Complementary ond Aterative Medicine virus, [27], human immunodeficiency virus type I [28], rotavirus [29], influenza virus, [30], chikungunya virus 31), and DENV [32]. Specifically, for DENV, the anti viral effects of some compounds has been demon- strated, inhibiting both, infectivity and/or viral spread in vitro and in vivo, such as glabranine (derived from ‘Tephrosia madrensis) (33), panduratin (derived from Boesenbergia rotunda 1.) [34], and castanospermine (derived from Castanospermum australe) 15]. Re- cently, inhibitory activity against DENV-2 infection has been identified in ethanolic extracts of Colombian plants such as Cassia grandis and Tabernaemontana exmosa (T. cymosa), with 99.9% inhibition observed for the 7: cymosa extract [35]. Moreover, several com: pounds derived from Mammea americana (couma: rins) and 7. eymosa (lupeol and voacangine) that can inhibit infection in vitro at percentages greater than 50% have been reported [36] Given the great variety of Colombian plants, the present study evaluated the anti-DENV effect of 12 etha- nolic extracts obtained from selected plants based on an ethnobotanical survey conducted in the city of Cartagena (in the Colombian Caribbean coast). The ethanolic ex: tract with the highest selectivity (derived from Psidivm ‘uajava) was fractionated, and the anti-DENV effect of each fraction was tested. Moreover the results of in vitro assay were contrasted with the in silico assays to postu: late promising antivirals. Methodology Plant selection Vegetal material was selected based on data obtained from an ethnobotanical survey conducted in the city of Cartagena (Colombia) in 2009 (unpublished results) and from a literature search on plant extracts with reported antiviral activity against viruses that cause febrile illness. In total twelve plants were included: Ambrosia cuma nnensis Kunt (A. cumanensis), Cavanillesia platanifolia Bonpl (C. platanifolia), Chenopodium ambrosioides L. (C. ambrosioides), Chrysobalanus icaco L. (C. icaco), Croton malambo Karst (C. matambo), Cymbopogon citratus Stapf (C. citratus), Diospyros inconstans Jacq (D. inconstans), Mammea americana L. (M. americana), Momordica charantia L. (M. charantia), Psidium gua: Java L. (P. guajava), Sarcostemma clausum Jacq (S. claw sum), and Trichilia hirta 1. (I hirta), that were collected between 2009 and 2016 in Cartagena city (Colombia, South America). The collection of most plants was done with the permission of the CARDIQUE (Corporacion Autonoma Regional del Canal del Dique, resolution 0751. June 27/2014). Moreover, the com: pounds derived from Psidium guajava are subject of the contract for access to genetic resources and derived products No, 130 of 2016 (RGEOI76) signed with the (2019) 19298 Page 3 of 16 Ministry of Environment and Sustainable Development of the Republic of Colombia, Obtaining extracts and fractions Weighed amounts (1000) of each plant material were collected for testing (phytochemical and biological exami: nations) and for identification at the herbarium of the Universidad de Antioquia (HUA) (Medellin, Colombia) and at the herbarium of the Universidad Nacional de Colombia (COL) (Bogoti, Colombia). Moreover, the ma terial was identified by authorized personnel atthe Jardin Botanico Guillermo Piferes in Cartagena ()BQ), Colombia (woucher number JBC 1208). The collected plant material was dried at room temperature, weighed, ground, and ‘macerated with 95% ethanol for 72h. Each sample was then filtered and concentrated in a rotary evaporator. Each dried extract was suspended in a mixture of ethanol distilled water and subjected to liquid-liquid partitioning \ith solvents of increasing polarity in the following order: dichloromethane, ethyl acetate, and butanol, as shown in Fig. 1. For the preliminary phytochemical screening of each extract, identification tests were performed for differ: ent secondary metabolites according to the method de- scribed in [37 Isolation and purification of the compounds present in the active fractions The fractions were subjected to several column chromato- graphic procedures using silica gel and Sephadex and to normal-phase preparative thin-layer chromatography (PTL, depending on their polarities, molecular size, and complexity. Final purification was performed using normal or reverse-phase high-performance liquid chromatography (HPLC) as necessary. The structures of the compounds present in the most active fraction of P. guajava (Pg-YP-L 22C) were elucidated using standard analytical methods, in tluding melting point determination, co-chromatography with reference compounds, and 'H nuclear magnetic reson- ance (NMR) and "C NMR spectroscopy techniques in one and two dimensions, such as the attached proton test (APT), distortionless enhancement of polarization transfer (DEPT, correlation spectroscopy (COSY), nuclear Overhau: ser effect spectroscopy (NOESY), heteronuclear multiple quantum correlation spectroscopy (HMQQ), and heteronue: lear multiple-bond correlation spectroscopy (HMBC) Virus and cell maintenance Epithelial VERO cells (Cercopithecus aethiops) were ob: tained from the American Type Culture Collection (ATCC), and Co/36HT cells (from Aedes albopictus mos: quito larvae) were donated by Dr. Guadalupe Guzman, Department of Virology, Pedro Kouri Institute (Havana, Cuba). C6/36HT cells were used to produce the viralTrujllo Correa e a BMC Complementary ond Atematve Medicine (2019) 19298, Page 4 of 16 2) Cerna yas (ay emp °C 10905834 ma eno, cio ron se 8 esr stn 701 a) aed “Poca yl nn he deat carmen lec Fig. 1 Fractionation ofthe ethanoc extract of P.quaiaua bark. Ope rmatogrephe fatlonation ofthe acive Faction A-YPS22C ‘obtained rom the total ethane extract off. guaanabark ung Sephac fo G10 aba sationaryphove stocks and the VERO cells were used to made the antiviral assays. The cells and the virus strain were maintained accord: ing to the protocols described previously [38] . Briefly, the VERO cells were maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco") supplemented with 2% fetal calf serum (FCS, Gibco") and were incubated at 37°C in a 5% CO; atm. The C6/36HT cells were main- tained in DMEM (Gibco") supplemented with 10% FCS (Gibcot) at 34°C. The DENV 2/NG strain, which was used for all biological assays, was donated by Dr. Jorge Osorio, Department of Pathobiological Sciences, Univer: sity of Wisconsin (Madison, W1, United States). Determination of the selectivity index (1) The Sl of each ethanolic extract, fraction, or com- pound was calculated from the relationship between the cytotoxic concentration 50% (Css) and effective concentration 50% (EC:a) [39]. The CCjo was deter: mined via the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe- nyl-2H-tetrazolium bromide) (MTT, Sigma-Aldrich) method. For this, 2.5 x 10* VERO cells were seeded in 96-well plates for 24h. Subsequently, serial dilutions of the ethanolic extracts (7.8 g/mL. to 1000 ug/ml), fractions (6.25 to 400 g/mL}, and characterized com: pounds (6.25 to 400 ug/ml.) were performed and incu- bated with the cells for 48h. After the incubation period, an MTT solution (0.5 mg/mL) was added to the cultures, which were then incubated for an add. itional 3h at 37°C. Finally, dimethyl sulfoxide was added, and the absorbance was read at 450nm in a Bio-Rad Benchmark" microplate reader. Cultures with. out extract, fractions, or compounds were processed as positive controls for viability. The CCso was calcu: lated as the extract concentration that reduced cell viability by 50% using regression analysis (Probit soft ‘wate). Each experimental condition was tested in trip licate in two independent experiments (n=6). To calculate the ECio, 25 x 10* VERO cells were seeded in 96-well plates for 24h. At 24h, serial dilutions of each ethanolic extract (7.8gimL to 1000 ug/ml), fraction (6.25 to 400ugimL.), and characterized com: pound (6.25 to 400ug/mL) were mixed with the DENV-2/NG strain (at a multiplicity of infection (MOL=1) and added to the cell monolayers for 2h. At 2h post-inoculation (hpi), the mixtures were removed, and the same serial dilutions of the extracts, fractions, or compounds were added again and incubated for 24 h, Finally, the supernatants were collected and stored at ~ 70°C until performing the multiwell plate focus assay [40]. Multiwell plate focus assay A total of 25%10* cells/well were seeded in 96-well plates. The cells were inoculated the following day with serial dilutions (10° to 10-®) of the supernatants from the ECso experiments for 2h. Next, the inoculum were re moved, and 1.5% carboxymethylcellulose (Sigma-Aldrich) prepared in DMEM (Gibco") supplemented with 2% ECS (Gidcot) was added, followed by incubation at 37°C in a 5% CO 2 atmosphere. On the third day, the cells were fixed with a methanol-acetone (1:1) solution for 10 min, ‘washed three times with PBS, and permeabilized with 0.1% Triton X-100 for 30 min, after which nonspecific site blocking was performed with 10% FBS in PBS. Then, DENV anti-envelope monoclonal antibody (mAb) was di luted 1:500 with PBS containing 10% FCS (Gibco") was added, followed by incubation for 1h at 37°C. After wash ing with PBS, the plates were incubated for 30 min with an anti-mouse IgG secondary antibody conjugated toTrulloCorrea e a BMC Complementary ond Aterative Medicine peroxidase, Finally, the reactions were stained with 3. amino-9-ethylearbazole (AEC, Sigma-Aldrich), and the ‘number of foci in each well were counted. Determining the antiviral effects ofthe fractions and compounds on virus cell entry A total of 25x 10* VERO cells were seeded in 24-well plates for 24h, and the fractions (100 ygml.) oF charac terized compounds (50 ug/ml} were then added and in cubated with the cells for an additional 24h. (pre treatment strategy) [16]. Subsequently, treatment was withdrawn, and a viral inoculum (DENV-2/NG at an MOI=1) was added and incubated for 2h. Subse quently, the virus was removed, and fresh medium was added for an additional 48, after which the superna tants were collected and stored at ~ 70°C until further processing via the mulwell plate focus asay. For each fraction and compound, two independent experiments ‘were performed each with two replicates (n = 4), Heparin (Sigma-Aldrich) served as the positive control for veal inhibition [41]. Determining the antiviral effects of the fractions and compounds after virus cell entry A total of 25% 10" VERO cells were seeded in 24-well plates for 24h, and a viral inoculum (DENV-2/NG at an MOI = 1) was then added and incubated for 2h. Subse: quently, the inoculum was removed, and the fractions (100 pgimL) or characterized compounds (50 ygimL) ‘were added and incubated with the cells for an add. itional 48 h (post-treatment strategy) [16]. Next, the su- pernatants were collected and stored at ~70°C until further processing via the multiwell plate focus assay. For each fraction or compound, two independent experi ments were performed each with two replicates (n= 4) Suramin served as the positive control for viral inhib: ition [41]. Viral inhibition assay in VERO cells A total of 25% 10" VERO cells were seeded in 24-well plates, and a viral inoculum was added at 24h, followed by incubation for 2h. Subsequently, the inoculum was removed, and the cells were treated with a single noncy- totoxic concentration of each of the five fractions and compounds obtained from the fraction with the highest selectivity. At 48h, the supernatants were removed and stored at ~ 70°C until further processing via the multi wall plate focus assay. Suramin served as the positive control for viral inhibition [41]. Indirect immunofluorescence Some cell monolayers were washed with Phosphate Buff ered Saline pH7.4 (PBS, Gibco" and fixed with 3.8% paraformaldehyde (PEA, Sigma-Aldrich) in PBS at 37°C (2019) 19298 Page 5 of 16 for 30min. The cells were then treated with 50mM. NH.C1 for 10min at room temperature and perme: abilized with 0.3% Triton X-100, after which the nonspe- cific sites were blocked with 5% FBS, To detect the E protein, the monolayers were incubated sequentially ‘with an anti-DENV-2 envelope primary mAb and an anti-mouse secondary antibody conjugated to Alexa 488. Finally, the plates were examined using a NIKON Eclipse T'S100 microscope. Molecular docking For molecular docking, the structures of the isolated compounds and that of suramin were downloaded from the DrugBank database or were constructed from SMILES strings using CHIMERA [42]. The three: dimensional structures of two viral proteins, the struc: tural envelope protein (E, PDB: 3UZV) and the non. structural protein 5 (NSS, PDB: 2)7U), were obtained by using the Protein Data Bank (PDB) database. Struc: tures with a resolution equal to or less than 2.5.A were considered. The 3D models of interest were prepared for docking using the Autodock tools package, remov ing both the water molecules and co-crystallized mole cules not part of the target protein and adding Gasteiger charges and nonpolar hydrogens. To deter mine the active sites or binding sites of the protein molecules, the CASTp tool was used [43]. According to this, a grid box coordinates were defined as follows: center x = ~ 23,889, center y= ~ 4.278, and center 2 30.778 for E and center x= 22.029, center y = 68.945, and center 2 = 22.667 for NS5, with both boxes having 1A spacing and a number of points in xyz = 30 and be ing evaluated with an exhaustiveness of 10. Finally, Autodock Vina software (Version 1.1.2) was used to de. termine the best interactions between the viral proteins and compounds [44], For Autodock Vina running, a configuration file including the name of the protein with the extension *pdbqt, the ligand name with the same extension, the geid box center and the exhaustive ness, was prepared as a CONF file. Best interations ‘were identified based on a score from 0 to ~7.0Kcal/ mol [43], with scores equal to or less than ~ 7.0 kcal! rol corresponding to the best affinities. The generated interactions were analyzed using LigPlot+ v1.45 [45] Statistical analysis To determine the Cay and ECjo, a regression analysis ‘was performed with Probit Software. The SI of each molecule was determined from the relationship between the CCiy and ECso, using the formula SI=CCsa/ECso, Yo compare the number of infectious viral patticles re leased between the cells treated with each fraction or compound (for both the pre- and post-treatment strat: egies) and the untreated cells, Student's ttest was used.TrulloCorrea e a BMC Complementary ond Aterative Medicine Al statistical analyses were performed using the Prism" 7.01 for Windows” package (GraphPad Software, San Diego, CA), considering differences statistically signif cant at p < 0,05 in all cases. Each experimental condition ‘was tested in triplicate in two independent experiments (w= 6) to guaranty the statistical analysis and the figures show the average of those experiments. Results Antiviral effects of the ethanolic extracts Overall, the cytotoxicity of the extracts was low (all had (CC values greater than 100 pg/ml) The extract with the highest CCsp was derived from the bark of P. guajava (CCyo= 1000 pgimL), and the extract with the lowest CCzp was from the leaves of A. cumanensis (CCso 1124 ygimL}. Considering antiviral effectiveness, we found a wide range of effective concentration values. ‘The extract with the highest ECio was derived from the bark of D. inconstans (ECiq = 538.6 ug/ml), and the extract with the lowest Eso was from the bark of P. guajava (ECso 7.8ugimL). Finally, the extracts were classified into four groups based on their SI values (calculated according to the CCi/ECso ratio). The first group contained nonscec: tive extracts (S1<2.0), including those derived from the leaves of C. citratus (S1=0.3), the leaves of S. clauswm (S1=12), the bark of D. inconstans (SI= 14), and the seeds of C: icaco (SI= 1.7). The second group comprised extracts with low selectivity (S12 20 and <5), including those from the seeds of 7: hirta (SI = 34), the bark of C ‘malambo Karst (St = 4.1), and the leaves of A. cumanensis Kant (SI =42), The third group contained extracts with moderate selectivity (S125 and<10), including those from the leaves of C. ambrosioides (S1= 53), the fruit of C platanifola Bonpl (St=5.8), the seeds of M. charantia (S179), and the leaves of M. americana (SI =84). Fi nally, the group with high selectivity (S12 10) included the extract from the bark of P. guajava (SI = 1282). Based on these results (Table 1), the bark extract of P. guajava was further investigated via fractionation. Preliminary phytochemical screening of the Psidium ‘guajava bark fractions From the bark extract of P. guajava, five fractions were obtained: Pg-YP-1-22A, Pg-YP-1-22B, Pg-YP-1-22C, Pg-YP- 1-22D, and Pg-YP-1-22E. Phytochemical characterization showed that alkaloids and tannins were present in all frac tions except for Pg-YP-1-22A. Flavonoids were found in Pg-¥P-1-22C, Pg-YP-1-22D, and Pg-YP-1-22E; glycosides in PpYP-1-22C and Pg-YP-L-22D; triterpenes in Pg-YP-1 22A and Py YP-1-22B; and sterols in Pg-YP-1-22A, Pe-YP- 1-228, and Pg-YP-1-22C. Finally, coumarins were detected only in Pg-YP-1-22D, and saponins or quinones were not detected in any of the fractions, Table 2 shows these results (2019) 19298 Page 6 of 16 Antiviral effects ofthe fractions obtained from the P. ‘quajava extract Overall, the cytotoxicity ofthe fractions was low with Cay values greater than 100 giml,the least toxic fraction was PEYP-L2C (CCig = 6257 ugimL). Considering anti-DENV effectiveness, we found that most fractions were effective at low concentrations (EC3p values of less than 100 ug/ml.) ex: cept for PpYP-22A (ECjy=1344yg/mL). Finally, SL values were calculated for the fractions, which were then classified into the same four groups as those for the extracts Accordingly, Pg-YP-1-22A was nonselective (SI = 1.0), Pg YPA-22D had low selectivity (S1=32), Py-YP-1-22E was moderately selective (SI = 6.1), and Pg-YP-1-22B and Pg-YP. 1-22 were highly selective (Sl= 11.7 and 354, respectively) Table 1 shows these results. Considering the cytotoxicity re sults, a nontoxic concentration (100 pg/mL) was chosen to evaluate the antiviral effects of said fractions via an inkib- ition test, which examined the production of infectious viral particles (Fig. 2). Only Py-YP-1-22C and Pg-YP-1-22D signif cantly inhibited infection compared to the control without treatment (p<005; the inhibition rates were 782 and 637%, respectively). After evaluating the SI values and virus replication inhibition percentages, Pg-YP-1-22C was chosen for further testing as its selectivity was high (SI: 354) and viral replication inhibition rate of 78.2%. Identification of the compounds in the Pg-YP-122C fraction Compounds 22CKOO1, 22CKO02, 22CKO03, 22CKOo4, and 22CKO0S were isolated from the Pg-YP-1-22C frac: tion. Table 3 shows the results of the open column chro: matography experiments. Next, the compounds were identified by one- and two-dimensional NMR spectros: copy (1D and 2D NMR) techniques and through com: parisons with data reported in the literature (Fig. 3) ‘Compound 22CKOO1 exhibited the following physical and spectral properties: white solid. MP 251°C. "H NMR (300 MHz, MeOD) 8 7.07 (2H, s, H-2/H-6). PC NMR (75 MHz, MeOD) 8 170.40 (s, COOH), 146.38 (4, C-2/C-6), 139.58 (s, C-1), 121.94 (s. C-4), 110.30 (5, C3/ C3). This compound was identified as gallic acid (Fig. 3a) by comparing its "H-NMR and °C-NMR physicochemi- cal and spectral data (Fig. 3b and ¢, respectively) with those reported in the literature. ‘Compound 22CK002 exhibited the following physical and spectral properties: amorphous solid. MP 85°C. "H NMR: 5 1.29 (3H, d, J= 6.0 Hz, 5”'-CHs), 2.74 (UH, dd, J= 180, 30 He, H-3ax), 3.14 (IH, dd, J= 15.0, 30 Ha, H- 3eq), 305-319 (2H, 313 (@d, J= 103, 102H2), 3.12 (dd, J= 103, 10.2 Ha)), 324-334 (2H, 3.26 (dd, J= 35, 2.9H2), 331 (dd, J= 102, 35 H2)), 335-350 (3H, 341 (at, J=103, 65 Ha), 342 (t J= 102 He), 345 (ad, J 103, 10.2H)), 380-384 (2H, 382 (4, J= 65 Hz), 382 (4, J=65H2)), 403 (1H, dq, J= 103, 6.7 H2), 5.10 (1H,Trullo-Corea etal BMC Complementary and Atematve Medicine (2019)19298, Page 7 of 16 Table 1 Cytotoxic concentration S0% (CC effective concentration 50% (ECsa, and selectivity index (3) values of the evaluated ethanolic extracts, fractions, and compounds in VERO cells infected with ENV 2/NG Typecteompard _Scentienane Tami VouchernumbePantpat Cty isy—ST Farol eaacts Cymbopogon ovatos Sof Poaceae WBC 01S Teas ir saat 05 Sarcosterma cowsum Jacq Asclpiedaceae’ ——J8C 2502 Leaves ss? 58712 Dospyosinccrstanslacq _—_benacene WiC 188 fark mi site Orpobalous aco L Crysobolaraccae BC 534 Seeds sae mse 17 Tren na eiaceoe 8c 917 seeds 13662934 Coton mambo Kast Euphorbiaceae JBC 12008 bar mama ‘Arbvoso cumanenss kunt Aseacene 01 538808 Lees 1A 6742 Chenopodium ambroscies| Chenopaviacese JBC 405 Leowes 16 B83 CvanitesapetarfokaBonp! —_Borssacacese 5c 47576 Amend 2506S Momeacca cova Coaubitacoe JBC 788 Seeds m3 15979 Mammen amencano | Calophylacese ——8C.457 Leas 40723 ae Pum guoiova L Nytaceae JC 1208 fark ocoo 781282 Fractons Psu guclovot - Hu 14093 PoP 1805 13k fovea = 3oae 26s poveioxe 6571774 FoveimD 779,561.32 PoveiozeE 1022167 Compounds Pyvp2ac - - Galles 584 5B DN Ce Queen e892 Gatechn 83 Hessen 4138 25818 ‘Gap ho wc 80 ‘Coup 8: Law sec, 1220 and <5 ‘Group C Moderate ety Si and-<10 The plats Cymbopogon cvs Stat Saosterma cous Joa, Doss incanstans cg Chysobaans coo L. Tha ha and Can malambo Karst ‘henopodum emesis |. Cronies pstntole Som Mamarde carat Mamma amercona au guava were ere 3 the ain Bot ‘de catagea J8C) The plan too comanens Kt wae eed tthe Herbarium o he Unveiaa Naciorl de Colm (CO The pln snd to oan the acon am Pau guava wa ented the Herbaram fe Unive de Aoguia HUAI 4, J= 6 Hz, HI’), 525 (1H, bs, H-2"), 537 (1H, dd, J 120, 30Hz), 613 (1H, d, J=3.0Hz, H-8), 6.15 (1H, d, J = 3.0 Hz, H-6), 6.83 (2H, d,. 0 Hz, H-3"/ H-5’ (QH, d, J=9.0Hz, H-2") 1-6"). °C NMR (75 MHz, Table 2 Phyrachemical screening ofthe P.quoiava bark MeOD) 5 19855 (s, C-4), 16658 (5, C7), 166.50, 165.00, fractions 164.95, 16466, 16463, 159.12, 13076, 129.18, 116.34, estore EPDA TPE PAPC TAEDD PTE 10489, 10258, 10251, 99.36, 99.31, 97.85, 9674, 80.72, Wook ar a 79:17, 79.02, 78.95, 78.11, 73.90, 72.15, 71.21, 69.98, tee we 64.73, 6225, 4395 (¢, C-3), 1822 (q, C6) This com. a Ses pound was identified as naringin (Fig. 3d) by comparing 8 its "H-NMR and "°C-NMR physicochemical and spectral ‘Gcosides — - * ” - data (Fig. 3e and f, respectively) with those reported in Trepens HO - - the literature. secs He - Compound 22CK003 exhibited the following physical counees ~ oo oe and spectral properties: yellow needles (fom methanol. Sons : - MP 316°C. "HNMR [300 MHz, CD,OD, 6 (ppm)i: 'H ae 5 . - NMR (300 MHz, MeOD) 8 7.73 (d, J=30H2, H-2'), 761 (dd, OH, 9.0 Hz, H-6"), 6.88 (4, J= 84 Hz, HeTrulloCorrea e a BMC Complementary ond Aterative Medicine (2019) 19298 Page 8 of 16 180 Infeetion (%) CONTROL SURAMIN Po-YPHA22APq:P-t Fig. 2 Innibcon of infectious pale production by fhe fv fractions 100 yin). The error bars coresoord to the SEM. Student's tet 9 005) in lation to the 2 P.quaowa Facto -22B Pg-YP-L20C PaYP-1-220 PgYP--22E Treatment Culsres were infected (MOI= 1) and hen rested for 48h with each The asters n ate canes wth statisti sr 5°), 638 (d, J= 30 Hz, H-8), 618 (2, J= 3.0 Hz, H-6). 8C NMR (75 MHz, CD,OD) 8 177.27 (C4); 16351 (C-7), 162.45 (C-5), 15817 (C2), 4871 (C-4’), 147.93 (C3), 13718 (C-3), 12410 (C.6'), 121.64 (C-1)), 116.18 (C2, 11395 (C5), 10448 (C-10), 99.19 (C-6), 9437 (C8). This compound was identified as quercetin (Fig. 3g) by comparing its 'H-NMR and "C-NMR physicochemical and spectral data (Fig. 3h and i, respectively) with those reported in the literature. Compound 22CKO04 exhibited the following physical and spectral properties: Orange amorphous solid. 213°C. 1H NMR (300 MHz, CD,OD) 5 459 (4, J= 6.0 Hz, H.2), 4001 (daa, J = 9.0, 60, 6.0 Hz, H-3), 252 (dd, J= 15.0, 60 Ha, H-4b), 2.84 (dd, J =15.0, 60 Hz, H-da), 593 (4, J 30H, H-6), 585 (4, = 30 Hz, H-8), 685 (4, J= 15 Ha, 1.2, 679 (4, J= 90 He, H-5'), 672 (dd, J= 15, 9.0H2, 1.6), °C NMR (75 MHz, CD,OD) 6 8285 (C-2), 6881 (C3), 2852 (C-4), 157.58 (C-5), 96.26 (C-6), 157.83 (C. 7), 95.48 (C-8), 15691 (C-8}, 100.80 (C-10), 132.20 (C. ‘Table 3 Open column chromatographic fractionation of the 3 guojava bark 1), 11524 (C-2'), 146.24 (C3), 14622 (C-4"), 116.07 (C-5°), 120.04 (C-6"). This compound was identified as catechin (Fig. 3)) by comparing its "H-NMR and °C: NMR physicochemical and spectral data (Fig. 3k and 1, respectively) with those reported in the literature. ‘Compound 22CK005 exhibited the following physical and spectral properties: amorphous solid. MP 250. 253°C. "HNMR (DMSO-46, 300 MHz) 6 12.02 (1H, br 5, 5-OH), 6.93 (1H, d, J= 2.0 Hz, H-2'), 686 (1H, J= 8.0 Hz, H-5’), 683 (1H, dd, J= 80, 2.0 Hz, H-6'), 6.13 (IH, 4, J=20Hz, H-8), 6.1 (1H, d J= 20H, H-6), 5.43 (IH, da, J= 11.0, 50 Hz, H-2), 4.96 (1H, d, J=7.2 He, He 1"), 454 (1H, be s, H-1), 3.80 (GH, s, 4-OCH3), 320. 3.63 (6H, m, H-2" to H-6"), 320-3.63 (3H, m, H-2 to 1-6), 308 (1H, dd, J = 17.0, 11.0 Ha, H-3a), 2.74 (1H, dd, J=17.0, 50 Ha, H-3b), 2.48 (1H, d, = 6.0 Hz, H-5), 1.06 (BH, d, J =6.0 Ha, H-6); °C NMR (DMSO-d6, 75 MHz) 8 1974 (s, C-4), 165.2 (s, C-7}, 1630 (5, C5), 1627 (5 C9), 1478 (, C4"), 1462 (5, C3’), 1310 C1), 22C obtained from the ethan« ive fraction Po-YP- relma Fraction code Tote phe 2x00 MeOheAceic acid 0.1% (1585) 2ackoos MeottAcetic a 0.19% (2080) zicKoo8 MeOheAcetic acd 01% 2575)TrulloCorrea e a BMC Complementary ond Aterative Medicine (2019) 19298 Page 9 of 16 B c E H Ls i lls fil K veer Lila LL techniques an throug ech compound € (41 1179 (s, C6"), 1142 (4, C-2'), 1120 (4, C-5°), 108.1 (5, C10), 1007 (4, C-1), 993 (4, C-1"), 96.1 (4, C-6), 954 (4 C8), 783 (4, C-2), 764 (4, C5"), 754 C3"), 730 (d, C-4), 723 (4, C-2"), 71.02 (4, C-4”), 703 (4, C 3), 693 (€, C-2), 686 (4, C5), 664 (t, C-6"), 55.5 (q, # OCHS), 424 (t, C-3), 1812 (q C-6). This compound ‘was identified as hesperidin (Fig. 3m) by comparing its "H-NMR and C-NMR physicochemical and spectral data (Fig. 3n and o) with those reported in the literature. Anti-DENV effects of the compounds obtained from the P9.¥P+-22C fraction The toxicity of the compounds was very low with CCsg values greater than 400 ug/ml; catechin was the least toxic (Cap = 8333 ygimL). For antiviral effectiveness, ‘we found that four of the five compounds were effective at concentrations lower than 100 pg/mL, with the most effective being quercetin (EC3y = 19.2 ugiml.); hesperidin was effective only at a higher concentration (ECs 225.8 ygimL). Finally, the SI was calculated for each compound. Hesperidin was the only compound not con- sidered selective, as it had an SI value less than 2.0 (SI 1.8); all of the other compounds were considered highly selective, with quercetin being the most selective (SI 34.3). Table 1 shows these results Ant-DENV effects of the compounds on some steps of the DENV replication cycle Using noncytotoxic concentrations of each compound, two different experimental strategies were performedTrulloCorrea e a BMC Complementary ond Aterative Medicine (pre- and post-treatment strategies) to evaluate their effects on processes before or after viral entry. In the pretreatment strategy, only gallic acid, quercetin, and catechin decreased infection in a statistically signifi cant way (with viral inhibition percentages of 52.6, 50.0, and 100%, respectively). Conversely, in the post: treatment strategy, all the compounds except for hes- peridin inhibited infection in a statistically significant ‘way; the viral inhibition percentage was highest in the cultures treated with quercetin (100.0%), followed by catechin (91.8%), naringin (64.5%), and gallic acid (67.3%) (Fig. 4). These results were confirmed by de- creases in viral antigen in the infected cultures treated with each compound (Fig. 5) In silico analysis of binding between the compounds and viral proteins The docking energy of the five favonoids and suramin ‘was examined using Autodock Vina ln this study, domain LL of the E protein and the polymerase domain of the NSS protein were used. All ligands showed favorable bind. ing energies, and the interactions with the E protein were mediated by hydrogen bonds in addition to at east one hydrophilic amino acid (Ser192 and/or Lys361); only two of the five compounds, naringin (~ 80 kcal/mol) and hes: peridin (-82kcal/mol), had better scores than the theor etical threshold of -7.0kcal/mol with the E protein (Table 4). The control compound suramin interacted to the E protein with a binding energy of ~7.9kcal/mo, (2019) 19298 Page 10 of 16 forming four hydrogen bonds with Val2, Lys8, Tyr106, 1Lys361, and to the NSS protein with a binding energy of 120 kcal/mol forming six hydrogen bonds (Fig. 6) all i gands tested except for gallic acd (-5.3kcal/mol) had docking energies above the theoretical threshold when interacting with the NS5 protein (Table 4), and ther inter: actions were mediated by 1 to 7 hydrogen bonds. Among. the compounds, although the hesperidin-NS5 interaction had the highest number of hydrogen bonds, the shortest distance to a hydrogen bond between the ligand and target protein appeared in the catechin-NSS interaction (273 A) The amino acids Tsp477 and Gln601 participated in the interaction with gallc acid, navingin, and quercetin, while the amino acids Asn608, Asp663, and His798 were in. volved in the interaction with catechin and hesperidin, as ‘well as suramin (Table 4 Fig 6). Discussion At present, due to the lack of a specific antiviral drug, different strategies are being used to control symptoms in patients with dengue, such as the administration of fluids and corticosteroids and the transfusion of blood desivatives [46]. Considering the agenda of research pi orities proposed by WHO as well as the biodiversity of plants in Colombia, we searched for antivirals derived from natural products that could be evaluated in preclin icalclinical studies in the medium/long term, This study frst determined the selectivity of 12 ethanolic cotracts derived from plants collected in the Colombian ry ‘0 -° z 3 ie « * * [e » $ ° * * * CONTRGL—SURAMN—GALLEAGO NARNGNN CLERCETN CATEOIN ESPERO Tsien Fig. 4 Arisa f ‘veted with the compounds fr #8 andthe tars Inaction, ndeperdent cell cukures wer infec rica by det bas) 08) inl the compounds derive frm the i= YPA-22C fected with OLNY WO) th DENY (WO he ero: bars covespond to the SEM. The asters inal faction on some steps ofthe DENV replication oj, The cls were )-thisinfecton strategy (pe-veanrent i the cark and then treated wth the compounds (posttreatment stategy es with statistical signa ferences Students tesTrujllo Correa e a BMC Complementary ond Atematve Medicine (2019) 19298, Page 11 of 16 Fig. $Immunoderecon of viel antigen (F Protein) cures of VERO cals, Representative Images of cues cath ofthe compounes slate fom the Pg 220 Nat hespeiin fected and ater ested wth 4 Catechin.€ Ou Caribbean coast. According to the SI values, the extracts The differences in the results could be due to the serotype were clasified into four groups (Table 1). The group of tested as wells the low specificity ofthe technique used in nonselective extracts (S1<2.0) comprised thvee plants. In the previous report (a qualitative technique based on the contrast to our results, extracts of C. eltratus have been re- cytopathic effects produced by DENV-1). An inhibition of ported to inhibit DENV infection by less than 50% [47], less than 50% could indicate low selectivity, which would Table 4 Inds and De ng scares for the Interaction letween P. guaiava con and NSS proteins Tage Compound Bindng — -yarogen Minimum Residues foming H Residues pariogsing in hySiophabic ineactons an 73 7 a, ya Ty ue, POTTS ys, LBaTBD, GTS, SEHD, ASDSSD cuuc 49 27 Tyr06,Ser82Lys361.AatOSLyst81, Guts, e194, A262 Aco NARNGIN 808 an Lys3.1)106, e107, AlN10, Gina, PoI79, PreT8D Lye, e194, ASHE yal, Gute, Ser, QUERCETIN 662 288 lys36 lyst CATECHN = 4 270 ro 79, Pro, uN, AlN, Ty, Leta serge 82 283 1ys3, las, Tpt07, 0, ys), ut, Aap 29 Sern, ys, S33, Ns SURAMIN 1207 20 ys36,THs39 Asn, 2354, VaB68, la, AspS28, ns, Sees, ASS553, S798 9598, Ser600, TOS, TS, CyB, L889, C09, TroATY, LysS7R Gye ValKSO, Arg, LyaS5, V6, Val? G 508, NARNGIN 845 20 Trpt77, lns97, C360, Phe, Vale, Vala, Arye, Asp, cleo? \als7, Vas, arg308, Se QUERCETN = 78 292 Tet 53, Phes5t VoB88, AES, Lys577, VSP, Vals, 6599, Gy, Glee? Rn 6 an Ser, Ty, Asn608, Tr, Gye e797 S681, ASO, 5798 7 285 Asp533,AspS38, Asoo, 682, ASA, C9709, S710, Hs71,Ta754, AS08653, y8689, Axg729, Ser7965, 79Trujllo Correa e a BMC Complementary ond Atematve Medicine (2019) 19298, Page 12 of 16 GALLIC ACID Fig. 6 Protin interactions with the compounds slated fom the Fg72-226 faction. nteraction of val Protein L withthe comaounds. rteracions of vil Protein NSS withthe compounds. Hycrogen bard interactions are represented by dashes nes in green and thet dist ae ind cated (A, Hyrophot rteroctions oe presente 3s red eyes, The eames and num’ inthe eslbuescoespon to the target proteins. rages obtained using Ligh 1.45, be consistent with our results. Antiviral activity has not been reported for the other plants in Group A, but antioxi: dant activity has been reported for extracts from D. incon: stans [48], and antitumor [49] and antifungal activites [50] have been documented for extracts from Chk, icaco, For the three plants in Group B (low selectivity, $12 20 and <5), this study is the first to report anti-DENV activity. How- ever, antitumor activity has been reported for C. malantbo 51) and A. cumanensis [52], and lymphocyte proliferative activity has been documented for extracts from T: hhirta 53]. The third group included plants whose extracts had moderate selectivity (S125 and<10) and included Ch. ‘ambrosioides, C. plataniflia, M. charantia, and M. amer- cana, Previous reports of antibacterial and insecticidal [54], antieishmanial [55], and antimalarial activities have been described for C. ambrosioides L. [56]. Extracts ofthis plant have also been reported to increase nitric oxide production 57], which would favor its possible antiviral activity as in creases in nitric oxide are known to lead to a decrease in DENV-2 replication [58]. For M. charantia, anti-DENV-1 activity has been reported for both extracts [47] and MAP30 protein derived from the plant [59]. Anti-DENV-2 activity has been described for two coumarins derived from ‘M. americana [36]. This report isthe first to document bo- activity for C. platanifolia, Its important to clarify that we decide to evaluate extracts from this plant, because the re sults from an ethnobotanical survey conducted in the city of Cartagena (Colombia) in 2009, Finally, the fourth group included the P. guajava extract, which was the only one with high selectivity (S12 10). P. guajava (which belongs to the Myrtaceae family and Myrtoideae subfamily) is a tree that grows in tropical ‘America and tropical & subtropical regions of Asia. It is recognized mainly for its fruit, the guava [60]. The bio activities of some parts of the P. guajava have been de- scribed for several viral infections. For example, extract from P. guajava leaves inhibits approximately 50% of hu: ‘man and simian rotavirus infections in vitro [61], andTrulloCorrea e a BMC Complementary ond Aterative Medicine aqueous and methanol extracts can inhibit murine leukemia virus retrotranscriptase at percentages greater than 50% [62]. In our study, the extract of this plant proved to be highly selective (SI = 128.2) with a percent: age of inhibition greater than 90% (data not shown). The inhibitory effect of leave extracts from P.guajava in cul ture infected with DENV has been reported previously showed a IS of 153.18 ug/ml. (very similar to our results) 63]. By other hand, some studies have shown that ex- tracts derived from P. guajava are effective against dia betes mellitus due to its antihypergiycemic activity via inhibition the enzyme a-glucosidase [64] ‘Accordingly, the anti-DENV effect of the P. guaiava ex: tract could be linked to the inhibition of this enzyme, as it has been described as essential forthe correct folding of vial elycoproteins and for virion assembly [65] during the repli Cation cycle. Moreover, it has been reported that other frac: tions derived from P. guajava can have benefits in patients infected with Dengue. For example, in culture of HepG2 cells the trombinol, a bioactive fraction of Psidivun guajava, induced the thrombopoietin production, and the authors postulate that this production could be considered as an al temative treatment in patients infected with dengue [6] In this study, five fractions were isolated from an ethanolic extract of P. guajava bark, which showed a high content of tannins, alkaloids, and flavonoids (Table 2), in accord with the phytochemical compos: ition described by other authors for this plant [67 Only four of the five fractions showed selectivity, with the most selective being Pg-YP-I-22C (SI = 35.4). This value was almost four times lower than that obtained with the crude ethanolicic extract (SI = 1282), which could indicate that the greater effectiveness of the ex: tract may be due to synergy. From the most selective fraction (Pg-YP-1-22C), five compounds were isolated and identified (Table 3 and Fig. 3). The first of these, gallic acd, is a polyphenolic compound [68] with diverse biological activity. Our results showed that gallic acd significantly inhibited viral activity via both the pre- and post-treatment strategies (percent ages of inhibition greater than 50%). Previously, a cocktail (containing several compounds including gallic acid) de- rived from plants in the Phyllanthaceae family was shown to have an antiviral effect against DENV [68], unfortu: nately our results cannot be compared with those previ ously reported, as the effect of one compound alone is not comparable to that produced by the synergy of several compounds. However, recently has been reported that the isobutyl gallate (a gallic acid derivative) is an antiviral against DENV-2, with a SI=25.6 in Huh 7 cells quite similar to our results in VERO cells (SI = 21.1) [70]. The second compound, naringin, is also a flavonoid with di verse biological activity [71] In our study, naringin signif cantly inhibited DENV-2 infection only it was added to (2019) 19298 Page 13 of 16 cells after viral inoculation (post-treatment). The anti DENV activity of naringin has been previously demon- strated [72], but such inhibition occurred only when viral inoculation of the cells was performed in the presence of the compound (a method we did not use, thus making the results incomparable). This previous report did not inves tigate viral inhibition when the treatment was performed after inoculation, in contrast with our results. However it is important to note that the concentrations we used were higher than those previously reported without being cyto toxic and that our SI (SI = 13.5) was ten times higher than that previously reported (SI= 1.3). The third compound, quercetin, is another flavonoid with diverse biological ac: tivity [73], including anti-DENV activity [72]. Although our results agree with those previously reported, we emphasize that the previously demonstrated selectivity (S1=75) is much lower than that reported by us (Sl 34.3), which may be because we tested a higher concentra tion than that used previously. Quercetin has been postu: lated to directly inhibit the viral NS3 protein (a protein ‘with multiple roles in DENV replication) [74], and it could interrupt virus entry by inhibiting fusion [75], making this compound a very promising antiviral. Catechin, the fourth compound isolated, is a polyphenol that is mainly derived from green tea [76]. Of the five compounds identified in this study, catechin isthe one that induced the best viral Inhibition when added before (100% inhibition) or after Inoculation (91.8%). Recent molecular docking studies have shown that catechin has a high binding affinity for the NS4B protein of DENV [77], which is an important protein in the formation of the viral replication complex (together with NS4A) in the endoplasmic reticulum of host cells [78]. Finally, the fifth compound, hesperidin is a flavonoid [79] of which no antiviral effect has been re: ported, agreeing with our results. It was the only nonseec- tive compound (SI = 1.8) and did not inhibit infection in any ofthe experimental strategies used. In addition to the in vitro studies, we used computa: tional tools to explore the possible mechanisms involved in viral inhibition, For this, the E and NSS proteins were selected as targets for the compounds tested, as in vitro Inhibition was found for pre- and post-entry steps and their three-dimensional structures were available for in silico tests. Using computational methods, new antiviral molecules targeting the NS3 protease have already been reported that, when evaluated in vitro, inhibited DENV- 2 infection up to 1 log PFU/mL. [80], demonstrating the ower of these methods in facilitating the search for new antiviral molecules. In our study, for the E protein, the structure used corresponded to domain Ill of DENV-2; however, the crystal structure probably corresponds to the mature protein, as it was co-crystallized with the variable por: tion of monoclonal antibody 4£11 [81]. Nonetheless,TrulloCorrea e a BMC Complementary ond Aterative Medicine based on the in vitro experiments, the molecules tested might influence the immature protein, possibly affecting the interaction by molecular docking. For the NSS protein, even though the crystal structure corre: sponds to the catalytic domain [82], the binding pocket was defined using computational tools; thus, the docking energy values could vary. However, the negative docking energy values suggest that all the in. teractions were favorable [83]. Notably, the interaction of suramin with both proteins was favorable, but the best docking energy was found for its interaction with NS5 (-12.0kcal/mol), which was mediated by six hydrogen bonds (Fig. 6). This result is consistent with the data from the in vitro experiments in which better inhibition was observed for this compound via the post-treatment strategy (Fig. 4), suggesting that this compound acts on viral polymerase. The docking energy values depend not only on the bind ing site but also on the virus type, target protein, and lig. and source. A consensus model of the NSS protein from four DENV serotypes was used previously against natural compounds obtained from the PubChem database22 and the SuperNatural I! database2, yielding binding energies of < ~105kcal/mol (84%; conversely, the compounds tested herein were obtained by conventional bioprospect: ing methods, The hydrogen bonds and hydrophobic inter: actions between the target protein and ligands determine the molecular docking score [85], and the distance be tween the atoms that are part of the bond is of great bio logical importance, especially if they range between 25 and 35 A, as those with small distances are more relevant 83]. Such is the case of the hesperidin-E interaction, ‘which has 11 theoretical hydrogen bonds compared to the four theoretical hydrogen bonds formed in the catechin-E interaction (Table 4) However, the shortest distance be- tween the atoms forming a hydrogen bond in the protein ligand pairs was obtained in the catechin-E interaction (270A). The distance between atoms was also the lowest (273A) in the catechin-NSS interaction compared to those of the other interactions evaluated, Similar studies have reported that catechin can interact with other viral proteins such as NSAB from DENV-2 with negative dock ing scores (~ 4.06 kcal/mol), which suggests that this inter action would be viable [77]. Other factors that can affect the docking score are ligand size and the number of atoms forming bonds. Consequently, smaller ligands such as gallic acid, quercetin, and catechin have lower docking scores and lower numbers of hydrophobic interactions compared to those of naringin and hesperidin (Table 4). In spite of the importance of ENV and NSS proteins for viral replication, well-conserved non-structural. proteins such as NS1, NS3/NS2B have been also evaluated against phytochemicals reported in literature, and the evaluated in teractions were favorable with scores ranging from ~ 671 (2019) 19298 Page 14 0f 16 to ~3224 Keallmol according to MOE Dock tool [86 Similarly, NS3 was evaluated against the compounds de rived from the medicinal plant Vetveria zizanioids, and one compound showed favorable interactions according to the scores obtained with Surflex-dock, mediated by Hydro gen bonds wth 272A being the lowest distance found me dating this interactions (87. ‘Although bioprospecting has been replaced by more targeted tools, it i still useful for identifying antiviral compounds, as we have shown in this study. Addition ally, combining in vitro and in silico tests allowed us not only to identify promising antivirals but also to suggest their posible mechanisms of action Conclusions (Our results showed that four compounds derived from P. guajava highly selectively inhibited DENV-2 replica: tion. Catechin is the most promising compound with Viral inhibition percentages of greater than 90% in the two different experimental strategies, Studies are in pro: gress that will allow us to elucidate the antiviral mecha- nisms of these compounds. Abbreviations ‘Acknowledgments Authors! contributions MU concaed the uy. ATC, DCOG and MEG were ved In al a aleson, cata anys se cafing an eng te manuserp, FOC and (WO were oh nal aspects of tha uy related wen the phytochemical 2s St was Ive n ating ana cetng the manus Al urs Funding THs woth was funded by the DeparamenceAminsrato de Cen, ivesigacgn — COLCINCAS [Admistatve Departnent Crue Cloyne ses enacts Mi ac 1p Hed 273K} 9S, WartnesEetancur ¥, Varn ls Marines Guten Wnfecton of epithe ial cas wih dengue vs promotes the expression of poten “voring the repication of carta vial rains Mee Wo, 2016868 ast Cheng ef nF Yang CM Wang KE, Lin. Mecha of con of ‘nesses of herpes sen wins fe 2 repens a Merees nfo 20048,76-44 Taio TV, Borst M, Lo 2, Zed Hu Beth SA Ctinon ar witaten oa page ection nevrazaton tet forthe {etecton ef avaaing anscooes to teehee of ng us ed super of dengue vacene deve opment. rn op Med Hygiene 203 sas}9e-70, 122 Foy Mt YourgN Feaman ¢ L295 M. Atlee othe rear sulfite mimetic, PBR aga dengue and ecephae aves rst es. 20068501318 Derren, Coder TD, Huang C, Cove S, reed OM, Meng erin UCSE Cintas valet sen fet evsotoy eth rd ars Comput Crem. 2082531160512 Dunas | yang 2. Teng Shows Tupaz, Lang | CAST: ete as of saecetpoaratyof rons wth statu) ard topographical nappg of untonaly annoated resus MC Ads Hes 206246up0 2107168 “oO, on A) AuzoDack Vi iproving the speed and acuay of Godkng mt 3 new seonng fenton ice option ane musieteadig.! Compu Chem 20103124556 Maloce A, Licks RA, Thorton JM LGPLOT a regan te gene seramate dagams of przinigarsneactens Petar Eng Des Sl sma (Chan Cy 0el = Dengue an update on test options. Fre erol 2050121203. “ng Lng AP Kab RY, Ce 9 Voor KG Seren of nega city in mettanae extrac of medina pants. 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SA Canpatatona sovering of rescina lan ahytocernca's to dicover pater por serve ns aps ere ‘eS fap. 2019011832 |, “avy. arala Aotaraty A. ng 4 metyhany 4 genenate ‘om Yee davai habs dengue NS2-NSS pres ad reer a sey 9 computations mae roms os Sock My Cel Biochem Sophy. 201674313375, Publisher's Note Seige Nature emai neal wis tga 0 jrslcional dams a Publishes maps and statera tos,