Zika Virus Activity of the Leaf and Branch Extracts of Tontelea micrantha and Its Hexane Extracts Phytochemical Study

The new triterpene friedelan-1,3,21-trione, the known compounds friedelan-3-one, 3β-friedelinol, 3,4-seco-friedelan-3-oic acid, 28-hydroxyfriedelan-3-one, friedelan-3-oxo28-al, friedelan-3,21-dione, 30-hydroxyfriedelan-3-one, a mixture of 30-hydroxyfriedelan3-one/21α-hydroxyfriedelan-3-one, 21β-hydroxyfriedelan-3-one, gutta-percha, squalene, and a mixture of palmitic/stearic/oleic acids were isolated from the hexane extracts of leaves and branches of T. micrantha. Their chemical structures were established by Fourier transform infrared spectroscopy (FTIR), gas chromatography (GC), 1D/2D nuclear magnetic resonance (NMR) and comparison with the literature data. All compounds were described for T. micrantha and the genus Tontelea for the first time. The branch and leaf extracts displayed anti-Zika virus activity at the lowest tested concentration of 15.6 μg mL, mainly virucidal effect, and presented no cytotoxicity to Vero cells. Furthermore, the ethyl acetate and methanolic leaf extracts demonstrated the best activities at the concentration of 31.2 and 15.6 μg mL at the viral adsorption and penetration stages, respectively. These results showed that these extracts may be promising candidates for the Zika virus treatment.


Introduction
The Celastraceae family comprises about 106 genera with 1300 species. 1,2Many Brazilian species of this family have been studied due to their use in traditional medicine and pharmacological properties. 2 Tontelea micrantha (Mart.)A.C.Sm. is a species of the Celastraceae family popularly known as "rufão" and is found in the Brazilian Cerrado, mainly in the north of Minas Gerais State.][5] Furthermore, its fruits are edible, suggesting low or even absent toxicity.Even though the literature 3 also describes the histochemical and pharmacognostic profile of the aerial and underground parts of T. micrantha, there are no reports of detailed chemical studies of this species or some other member of the Tontelea genus.
Phytochemical studies of the Celastraceae family led to the isolation of many bioactive secondary metabolites such as flavonoids, steroids and different classes of pentacyclic triterpenes.Also, many reports describe pharmacological properties of triterpenes like anti-inflammatory, 6,7 antiulcerogenic, 8 analgesic, 9 antibacterial, antifungal, antiviral, antiparasitic, antioxidant, hepatoprotective, neuroprotective, insecticidal and others. 10Moreover, some species are already employed in the treatments of gastric ulcers, presenting anti-inflammatory and analgesic activities such as Maytenus ilicifolia Mart.ex Reiss.and M. aquifolium Reiss. 11Additionally, the hydroalcoholic leaf extract from M. ilicifolia was active against bovine herpesvirus type 5 and avian metapneumovirus. 12iral infections represent an important target to the pharmacological research, especially aiming virus such as the Zika virus (ZIKV).Infections by ZIKV can be very serious and dangerous for pregnant women because it may induce microcephaly, a brain anomaly that causes a malformation of the brain and head of newborns, loss of pregnancy, stillbirth and other congenital disabilities. 13,144][15] Unfortunately, there is no treatment or specific drugs against ZIKV until this moment.For these reasons, researches leading to potential antiviral substances are essential. 16,17In this context, the Celastraceae family represents a source of high diversity for bioprospecting new substances with antiviral properties.
In the present work, the effect of leaf and branch extracts of T. micrantha against ZIKV was evaluated.Furthermore, the phytochemical study of the hexane extract of leaves and branches led to the identification of fourteen known compounds and the novel triterpene friedelan-1,3,21-trione (Figure 1), herein described for the first time.All compounds were characterized using spectroscopic analysis and comparison with the literature data.

Plant material
Leaves and branches of T. micrantha were collected in Montes Claros, Minas Gerais State, Brazil.The plant was identified by the botanist Dr Maria Olívia Mercadante-Simões and a voucher specimen (No.BHCB 144.623) was deposited in the Herbarium of Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil.

Extraction and isolation
After dried at room temperature, samples of leaves and branches of T. micrantha were fragmented in a knife mill resulting in powders (720.0 g for leaves; 2.6 kg for branches) that were macerated with organic solvents further removed using a rotatory evaporator.The following extracts were obtained for the leaves: hexane (43.5 g), ethyl acetate (ELE, 45.0 g), and methanolic (MLE, 38.3 g).The following extracts were obtained for the branches: hexane (25.7 g), chloroform (CBE, 61.1 g) and ethyl acetate (EBE, 9.75 g).During the hexane removal, white solids precipitated from both extracts and were separated by filtration (WLS, 0.87 g from leaves, and WBS, 5.3 g from branches).These solids were further chromatographed.
TLC analysis of the hexane extracts from leaves and branches showed that both have an expressive amount of the polymer trans-1,4-polyisoprene (gutta-percha), which is very common in the Celastraceae family. 18odrigues et al. 19 described an efficient method for guttapercha removal from the extract, which consists in a silica gel CC exhaustively eluted with methanol followed by chloroform.The first elution using MeOH removes all compounds except gutta-percha that remains retained at the top of the column.Then, this polymer is isolated by eluting the column with chloroform.
Employing the methodology described by Rodrigues et al., 19 the hexane extracts from the leaves (43.5 g) and branches (25.7 g) were chromatographed on silica gel CC, firstly with MeOH until the TLC analysis showed no more eluted compounds, and then with CHCl 3 .The chloroform fractions from leaves and branches furnished gutta-percha (11) (20.3 and 1.5 g, respectively).The methanolic fraction from the leaves, after solvent removal with a rotatory evaporator, yielded 15.0 g of a greenish solid (HLE).Differently, the initial methanolic fraction from the branches presented a yellow color and it was reduced with a rotatory evaporator furnishing 2.2 g of a yellowish solid (YBS).Continuing the elution with MeOH, a further fraction was then separated until the TLC analysis showed no more eluted compounds, leading to a slightly greenish solid (HBE, 20.4 g) after solvent removal.All solids were further chromatographed.

Cytotoxicity assay
Prior to the antiviral assays, the 50% cytotoxic concentration (CC 50 ) of the crude extracts of T. micrantha was established for the Vero cell lineage (ATCC CCL-81™).Vero cells were distributed into 96-wells microplates (4 × 10 5 cells per 100 µL per well) and incubated at 37 °C for 24 h.Then, 200 µL well -1 of DMEM (Dulbecco's minimum essential medium) with 5% FBS (fetal bovine serum, v v -1 ) and the stock solution of the extracts dissolved in DMSO 20% in water (final DMSO concentration per well of 0.2%) were added starting with 1000 µg mL -1 in successive serial dilutions to 7.8 µg mL -1 .The employed solvent was used as a control (DMSO 0.2%), and the assays were performed in triplicate.After the 48 h incubation period, 25 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (2 mg mL -1 in PBS) was added, and the plates were incubated at 37 °C for 90 min.Then, 130 µL of the solvent (DMSO) was added to each well to dissolve the MTT-formazan crystals, and the cultures were kept under stirring at 150 rpm for 15 min.The CC 50 was calculated using the absorbance (λ = 492 nm) in the wells, determined on a plate reader, as B/A × 100 (A = untreated cells; B = treated cells).The data presented are the means obtained from at least three experiments with internal triplicates.

Antiviral evaluation
Only T. micrantha extracts that presented a CC 50 higher than 100 µg mL -1 were subjected to in vitro antiviral assays.Vero cells were distributed in a 96-wells microplate (4 × 10 5 cells per 100 µL per well) and incubated at 37 °C for 24 h.Then, the culture medium was removed, and 100 µL serial extract dilutions (250 to 15.6 µg mL -1 ) were added to the wells.In a second 96-wells microplate, the same serial extract dilutions were mixed with the virus inoculums with a multiplicity of infection (moi), which corresponds to the average number of virus particles infecting each cell, of 0.1 virus cell -1 .Both 96-wells microplates were incubated at 5% CO 2 atmosphere at 37 °C for 30 min.Then, the suspension of the second microplate was transferred to the first microplate containing cells and incubated for 48 h at 37 °C.This procedure was done to ensure an adequate evaluation of the antiviral effect, investigating the extracts action in the virus and the cells to be infected throughout the multiplicative cycle of the virus.After 48 h of infection, the methodology for the cytotoxicity assay was used again to establish the median antiviral effective concentration (EC 50 ), which is the concentration that induced 50% protection of treated cells from viral infection.The EC 50 was calculated using the absorbance (λ = 492 nm) in the wells, determined on a plate reader, as [(A -B)/(C -B)] × 100 (A = treated and infected cells; B = untreated and infected cells; C = untreated and noninfected cells).The data presented are the means obtained from three experiments with internal triplicates.

Evaluation of the different stages in the viral infection cycle
In order to establish the antiviral effect, the cells and virus were incubated with active extracts at different stages of the viral infection cycle.At the adsorption stage, the cells were pretreated with the extracts before viral infection.At the penetration stage, the cells were infected with the virus before the addition of the extracts.The virucidal effect was also evaluated to verify if the extracts were capable of interacting with the virus at a stage prior to the viral infection of host cells.Then, the virus was incubated with the extracts before infecting the cells.The data presented are the means obtained from three experiments with internal triplicates.

Antiviral activity at the adsorption stage
The extracts in concentration ranging from 250.0 to 15.6 µg mL -1 (serial dilutions) were added to a monolayer cell (24-wells microplate, 5 × 10 5 cells well -1 ) and maintained at 37 °C for 1 h in 5% CO 2 atmosphere.Then, the cells were washed and infected with ZIKV (moi = 0.1 virus cell -1 ).
After one hour of viral adsorption, the non-adsorbed viral inoculum was removed and the cells were washed with PBS before addition of 1 mL of semi-solid medium 199 containing 2% FBS, 2% CMC (carboxymethylcellulose, m v -1 ).After 48 h, the cells were fixed with PBS/10% formaldehyde (v v -1 ) for 30 min, washed and stained with 5% crystal violet solution (m v -1 ) for 15 min.The viral lysis plaques were observed and compared with untreated and non-infected cells (cell control), cells treated with DMSO 0.2% and infected (vehicle control), cells treated with ribavirin 200 µg mL -1 and infected (drug control) and only infected cells (ZIKV control).Ribavirin (Sigma-Aldrich) was used as a negative control since it can inhibit the viral multiplication in the intracellular phase, but does not prevent viral adsorption.

Antiviral activity at the penetration stage
Vero cells were distributed in 24-wells microplates (5 × 10 5 cells per 100 µL per well) 24 h before the assay, then the cells were infected with ZIKV (moi = 0.1 virus cell -1 ) without pretreatment and incubated at 4 °C for one hour.Then, the viral inoculum was removed and the cells were washed with PBS.The extracts in concentration ranging from 250.0 to 15.6 µg mL -1 (serial dilutions) were added to the infected cells and maintained at 37 °C for one hour in 5% CO 2 atmosphere.The extracts were removed, and the cells were washed with citrate buffer (pH 3) for 1 min.After, a semi-solid medium 199 containing 2% FBS, 2% CMC (m v -1 ) and the antibiotic mixture were added to the plate, which was incubated at 37 °C for 48 h in 5% CO 2 atmosphere.The revelation methodology and control groups were the same as described for the adsorption assays.

Virucidal effect
The extracts were added to the viral inoculum at 37 °C for 1 h before cell infection.Then, they were placed in a 24-wells microplate containing Vero cells and incubated at 37 °C for 1 h in 5% CO 2 .In the sequence, the viral inoculum was removed, a semi-solid medium 199 containing 2% FBS and 2% CMC (m v -1 ) was added and the microplate was maintained at 37 °C for 48 h in 5% CO 2 atmosphere.For the visualization of viral plaques, the cells were treated 48 h later by fixation with PBS/10% formaldehyde (v v -1 ) and stained with 1% crystal violet solution (m v -1 ) for 15 min.The control groups were the same as described for the adsorption assays.

Chemistry
The phytochemical study of hexane leaf and branch extracts of T. micrantha yielded ten friedelane triterpenes (1 to 10), the natural polymer gutta-percha (11), squalene (12), and a mixture of long-chain fatty acids (13) (Figure 1).All compounds are herein described for the first time as constituents of T. micrantha, as well as of the genus Tontelea.The chemical structures of these compounds were characterized by IR, GC, 1 H and 13 C NMR and through comparison with literature data.
The triterpene 1 was isolated as a white amorphous solid material with [α] D 21 +86.0 (CHCl 3 ) and mp 224-226 °C.Its molecular formula, C 30 H 46 0 3 , was established by HR-ESI-MS (m/z: 477.3221 [M + Na] + , calcd.477.3339).The IR spectrum showed a large band at 1716 cm -1 , which was attributed to carbonyl groups.In this spectrum, the bands at 3548 and 3412 cm -1 were attributed to hydroxyl groups, probably due to the keto-enolic equilibrium.The 1 H NMR spectrum showed signals at d H 0.71, 1.05, 1.07, 1.09, 1.14, 1.16, 1.18 and 1.22, associated to eight methyl groups.Signals at d H 2.39 (1H, s), 2.58-2.61(2H, m), 3.24 (1H, d, J 16.0 Hz) and 3.44 (1H, d, J 16.0 Hz) were correlated as α-carbonyl protons according to heteronuclear multiple bond correlation (HMBC) and heteronuclear single quantum correlation (HSQC) analyses.The 13 C NMR spectrum presented 30 signals that, with the aid of the distortionless enhancement by polarization transfer (DEPT)-135, were classified as 8 primary, 9 secondary, 4 tertiary and 9 quaternary carbon atoms, suggesting a friedelane skeleton. 20Among the quaternary carbon atoms, three were characterized as carbonyl groups due to the chemical shifts at d C 202.73, 203.96 and 218.75.The hydroxyl groups suggested by the IR spectrum were absent in the NMR spectrum since no signals were observed for carbinolic carbon atoms.Probably, the hydroxyl bands were related to a keto-enolic equilibrium indicating triterpene 1 as a beta di-ketonic compound.In the HSQC spectrum, the signal at d C 7.  2.
After detailed analyses of 2D experiments, compound 1 was identified as friedelan-1,3,21-trione and its complete NMR spectral data are shown in Table 1.The known friedelane triterpenes 2 to 10 were isolated as white amorphous solid materials, pure or as mixtures.The structures of these compounds were determined comparing their respective NMR data with those previously published.Compounds 2-3 and 5-10 showed the duplet of methyl H-23, at the range of d H 0.8-0.9,confirming the presence of 5 rings in the friedelane skeleton.Compound 2 was identified as friedelan-3-one and 3 as 3β-friedelinol, mainly due to the carbonyl group at d C 213.20 and the hydroxyl group at d C 72.38, respectively. 20,21The 1 H NMR spectrum of 5 presented seven signals of methyl groups and a singlet at d H 3.63 (2H), relative to a hydroxylated methylene carbon.Its 13 C NMR spectrum showed signals at d C 212.99 (C=O) and 68.08, which were attributed to C-3 and C-28, respectively, characterizing the compound as 28-hydroxyfriedelan-3-one. 20 Compound 6 was identified as friedelan-3-oxo-28-al due to the aldehyde signal at d C 208.97 (HC=O) attributed to C-28. 22Compound 7 showed two carbonyl groups at d C 212.85 and 218.74 in the 13 C NMR spectrum and was identified as friedelan-3,21-dione. 23 The 13 C NMR spectrum of 8 (Table 1) showed signals at d C 213.21 (C=O)/71.98(CH 2 OH) that were similar as the signals of 30-hydroxyfriedelan-3-one published by Magalhães et al. 24 However, the shifts of C12, C16, C19, C22, C26 and C27 were uncorrectly attributed in the literature, and after a detailed analysis of the 2D NMR the assignments were corrected and are presented in Table S1 (Supplementary Information).Based mainly on the signals at d C 213.07 (C=O) and 74.32 (CHOH), compound 9 was identified as 21α-hydroxyfriedelan-3-one. 25For compound 10, the signals at d C 213.17 (C=O) and 75.82 (CHOH) were coherent with the structure of 21β-hydroxyfriedelan-3-one. 26ompound 4 was identified as 3,4-seco-friedelan-3-oic acid due to the triplets at d H 0.79 (t, J 8.43 Hz, H-23) and 2.38 (t, J 8.50 Hz, H-2) together with the signal at d C 179.05, which was attributed to a carboxyl group. 27The 1 H NMR spectrum of compound 11 showed an olefinic proton at d H 5.12 (t, 1H, J 6.4 Hz) and methylene groups at d H 1.98 (2H, m)/2.06 (2H, m).Also, the 13 C NMR spectrum presented signals associated to unsaturated carbon atoms at d C 124.25 and 134.93, allowing the identification of this compound as gutta-percha. 28The 1 H NMR spectrum of compound 12 showed signals between d H 5.10-5.15(6H, m), associated to protons bonded to C=C, and between d H 1.60-1.68,attributed to eight methyl groups, according to integrations.Moreover, the 13 C NMR spectrum showed signals at d C 124.30, 124.43, 131.25 and 135.12, which were attributed to unsaturated carbon atoms, identifying compound 12 as squalene. 29he 1 H NMR spectrum of mixture 13 indicated a longchain fatty acid due to the signals of α-carboxylic protons (d H 2.34, 2H, t, J 7.2 Hz), β-carboxylic protons (d H 1.63, 2H, qt, J 8.0 Hz) and terminal methyl protons (d H 0.88, 3H, t, J 6.4 Hz).This was confirmed by the 13 C NMR spectrum because of the signals at d C 179.77 (C=O) and 14.24 (CH 3 ). 30GC analysis revealed that the mixture 13 was mainly composed by 66% palmitic acid, 12% stearic acid and 11% oleic acid.

Antiviral activity
The cytotoxicity to Vero cells and global antiviral activity against ZIKV of the extracts of T. micrantha were evaluated by the cytotoxic concentration (CC 50 ), effective concentration (EC 50 ) and selective index (SI) values presented in Table 2. CC 50 values higher than 100 µg mL -1 indicate a non-toxic extract. 31On the other hand, lower values of EC 50 display an effective antiviral activity.Moreover, the selective index (SI) was calculated by the ratio of the concentration of the extract that reduced cell viability to 50% (CC 50 ) to the concentration needed to inhibit the cytopathic effect to 50% (EC 50 ).
All leaf and branch extracts were non-toxic to Vero cells, once their values of CC 50 were higher than 100 µg mL -1 .The values of EC 50 varied from 38.53 to 83.05 µg mL -1 indicating that the extracts presented antiviral activity.The methanolic leaf extract (MLE) together with the hexane and chloroform branch extracts (HBE and CBE) showed the highest SI displaying a promising viral inhibitory effect.All extracts with an SI higher than three were evaluated at different stages of infection (adsorption and penetration of viral particle) and also for their virucidal effect.
At the ZIKV adsorption stage, the Vero cells were pretreated with the crude extracts prior to the infection to verify interactions with cellular receptors.The ELE and MLE showed a more pronounced activity than the other extracts with a protective concentration of the cells higher than 50% at 31.2 µg mL -1 (Figure 3).These concentration values of the leaf extracts are similar to those reported by Tan et al. 32 for the anti-parasitic drug suramin, which induced a ZIKV adsorption blockade of 80% at a concentration of 50.0 µg mL -1 .The CBE also showed a significant antiviral activity at the concentration of 125.0 µg mL -1 , displaying lysis plates in smaller amounts when compared to the non-treated viral control.The HBE showed no activity at this stage.
In order to evaluate the penetration stage, the Vero cells were infected with ZIKV and incubated at 4 °C for one hour.This step was necessary because lower temperatures deactivate the enzymes responsible for the viral penetration, causing only viral adsorption on the cells.Then, the extracts were added to the microplates in serial dilutions and the temperature was raised to 37 °C to reactivate the enzymes.Both leaf extracts (ELE and MLE) protected the cell, even at the lowest concentration of 15.6 µg mL -1 (Figure 4) when compared to the viral control Ribavarin, an antiviral nucleoside analogue.The CBE partially inhibited the penetration of the virus at the concentration of 62.5 µg mL -1 .The HBE promoted a partial protection of the cells only at the highest tested concentration (250 µg mL -1 ).
The ability to inhibit the viral multiplication cycle prior to the cell infection (virucidal effect) was assessed by adding serial dilutions of the extracts to the viral suspension (moi = 0.1 virus cell -1 ) before incubation with Vero cells.All tested extracts inhibited 100% of the viral infection even at the lowest tested concentration (15.6 µg mL -1 ) since no virus plaques were observed in the microplates (Figure 5).
This assay demonstrated that all extracts act mainly on the viral particle.In general, an efficient antiviral agent should inhibit viral multiplication without interfering directly in the host cell, enabling a cellular infection recovery and metabolic maintenance.However, the antiviral activity was also detected for the extracts at the adsorption and penetration stage, implying the possibility of a cellular component on the mechanism as well.In fact, antiviral effects have been reported to some Celastraceae family species.Kanyara and Njagi 33 demonstrated that Maytenus buchanani and M. senegalensis extracts were able to block HIV-10 viral infection, and Kohn et al. 12 showed bovine herpesvirus inhibition by extracts of M. ilicifolia.Nonetheless, this is the first time that the antiviral potential was reported for the Tontelea genus.Further studies concerning the antiviral activity of the isolated compounds and the phytochemical investigation of more polar extracts  are currently being performed in our laboratory, and the results will be reported in due course.

Conclusions
In this work, Tontelea micrantha was phytochemically studied for the first time.The new triterpene friedelan-1,3,21-trione (1), eleven known compounds and a mixture of long-chain fatty acids were isolated and chemically characterized.This is also the first report of these compounds for the Tontelea genus.Branch and leaf extracts were also tested for their activity against ZIKV in the early stages of viral infection and virucidal effect.They presented a virucidal effect, strongly acting on the viral particle, and inhibited the infection at the adsorption and penetration stages, except for the hexane branch extract.These results demonstrate that these extracts may be promising candidates for the ZIKV treatment.

Figure 1 .
Figure 1.Chemical structures of compounds from hexane leaf and branch extracts of Tontelea micrantha.

Figure 4 .
Figure 4. Effect of the extracts on the Zika virus cellular penetration stage.CC (cell control) = untreated and non-infected cells; VC (vehicle control) = infected cells treated with DMSO 0.2%, DC (drug control) = infected cells treated with ribavirin (200 µg mL -1 ); ZC (Zika virus control) = cells infected by the virus, but non-treated.Ribavirin  was used as negative control due to its inhibition property of the viral multiplication in the intracellular phase.Tontelea micrantha samples: ELE = leaf ethyl acetate extract; MLE = leaf methanolic extract; HBE = branches hexane extract; CBE = branches chloroform extract.

Figure 3 .
Figure 3.Effect of the extracts on cellular adsorption stage of the Zika virus.Decrease in cell damage by viral infection due to the increase in ELE, MLE, HBE and CBE extracts concentration.CC (cell control) = untreated and non-infected cells; VC (vehicle control) = infected cells treated with DMSO 0.2%; DC (drug control) = infected cells treated with ribavirin (200 µg mL -1 ); ZC (Zika virus control) = cells infected by the virus, but non-treated.Ribavirin  was used as negative control due to its inhibition property of the viral multiplication in the intracellular phase.Tontelea micrantha samples: ELE = leaf ethyl acetate extract; MLE = leaf methanolic extract; HBE = branches hexane extract; CBE = branches chloroform extract.

Table 2 .
Cytotoxic concentration (CC 50 ) to Vero cells, effective concentration (EC 50 ) against ZIKV and selectivity index (SI) of the extracts of T. micrantha