Antiviral Activity of Fridericia formosa (Bureau) L. G. Lohmann (Bignoniaceae) Extracts and Constituents

A phytochemical study of Fridericia formosa (Bignoniaceae) ethanol extracts of leaves, stems, and fruits was guided by in vitro assays against vaccinia virus Western Reserve (VACV-WR), human herpes virus 1 (HSV-1), murine encephalomyocarditis virus (EMCV), and dengue virus type 2 (DENV-2) by the MTT method. All the ethanol extracts were active against DENV-2, HSV-1, and VACV-WR with best results for the fruits extract against DENV-2 (SI > 38.2). For VACV-WR and HSV-1, EC50 values > 200 μg mL−1 were determined, while no inhibition of the cytopathic effect was observed with EMCV. Five compounds were isolated and identified as the C-glucosylxanthones mangiferin (1), 2′-O-trans-caffeoylmangiferin (2), 2′-O-trans-coumaroylmangiferin (3), 2′-O-trans-cinnamoylmangiferin (5), and the flavonoid chrysin (4). The most active compound was 2′-O-trans-coumaroylmangiferin (3) with SI > 121.9 against DENV-2 and 108.7 for HSV-1. These results indicate that mangiferin cinnamoyl esters might be potential antiviral drugs.


Introduction
Viral infections represent a current problem accounting for severe damage to human health and economic losses in livestock [1]. Some viral diseases such as dengue or dengue fever (DF), herpes, smallpox, and encephalomyocarditis have a high impact in public health in the tropical and subtropical regions of the world [1].
Dengue virus belonging to the Flaviviridae family, Flavivirus genus, is responsible for Dengue fever (DF) and is considered the most common arboviral disease of humans. It is estimated that 390 million cases occur every year around the world and it is endemic in more than 100 countries, including the Americas, Southeast Asia, and Western Pacific, regions most seriously affected [1,2]. No effective drug as well as no vaccine is available for human use. The need for a safe and efficient approach either for treatment or prevention of DF has been considered a global priority [1,3].
HSV belongs to the family Herpesviridae and the subfamily Alphaherpesvirinae and is characterized by neurovirulence, latency, and reactivation. The prevalence of HSV infection has increased in recent years, making it a highly relevant public health issue. Early detection and treatment are of paramount importance for disease control [4].
Encephalomyocarditis virus (EMCV) family Picornaviridae, genus Cardiovirus, is a group of closely related virus species with a wide host range. Infections with EMCV are associated with sporadic cases and outbreaks of myocarditis 2 Journal of Tropical Medicine and encephalitis in domestic pigs, in nonhuman primates and other mammalian species. There are few reports of cases of human infection by EMCV [5].
Vaccinia (VACV) is a virus of the genus Orthopoxvirus of the family Poxviridae that, in humans, causes nonlethal, pustular, and localized disease. The vaccinia virus does not have natural hosts, but cases of bovine and human infection by vaccinia virus are reported in Brazil and India, causing economic losses and affecting health services [6,7].
As part of a bioprospecting project, whose main goal is to discover potential antiviral natural products of plants from Brazilian Cerrado and Atlantic Forest biomes, we have screened several species of plants collected in the state of Minas Gerais [1,[8][9][10][11][12]. Among these, Fridericia formosa (Bureau) Sandwith was chosen for bioguided phytochemical investigation due to the good antiviral activity presented by the ethanol extracts of leaves, stems, and fruits.

Preparation of Extracts.
After drying in air circulating oven at 40 ∘ C for 72 h, the plant material-136.3 g of leaves, 461.5 g of stems, and 13.8 g fruits-were ground and extracted by percolation with 96% EtOH at room temperature. The solvent was removed in a rotary evaporator under reduced pressure at 50 ∘ C, leaving dark residues-EEFFL, 34.2 g, EEFFS, 60.0 g, and EEFFF, 2.5 g-for leaves, stems, and fruits, respectively, which were kept in a vacuum desiccator until constant weight.

HPLC Analyses.
In the HPLC analyses, an exploratory gradient elution was used [1,9,10]. Fingerprints were registered by RP-HPLC-DAD on a Waters 2695 apparatus equipped with a UV-DAD detector (Waters 2996). A LiChrospher 100 RP-18 column (5 m, 250 × 4 mm i.d.; Merck, Darmstadt, Germany) was employed at 40 ∘ C, flow rate of 1.0 mL/min, and detection at wavelengths of 220, 280, and 350 nm. To an aliquot 10.0 mg of dried extract/fractions and 1.0 mg of each of the isolated compounds HPLC grade methanol was added and the mixture was dissolved by sonication in an ultrasound bath for 15 min, followed by centrifugation at 10,000 rpm for 10 min. The supernatant was filtered through a Millipore membrane (0.2 m) and injected (10.0 L) onto the equipment. Elution was carried out with a linear gradient of water (a) and acetonitrile (b) (from 5% to 95% of B in 60 min).

Isolation of Chemical Components from Stem Extract.
To a portion of EEFFS (10.0 g), cold MeOH was added giving a precipitate which was separated by filtration on sintered glass funnel and washed thoroughly with MeOH yielding 2.43 g. The precipitate was recrystallized out twice from MeOH to give 405.0 mg of compound 1. The mother liquor from the second recrystallization was dried in a rotary evaporator under reduced pressure at 50 ∘ C, leaving a yellow residue (FFSMW2, 1.2 g) that was further subjected to fractionation by HPLC employing Shim-pack PRC-ODS column (20 mm i.d. × 25 cm, flow rate 5.0 ml/min) and a gradient elution with MeOH/H 2 O 45 to 95% for 50 minutes affording compound 4 (511.0 mg).

Isolation of Chemical Components from Fruit Extract.
To a portion of EEFFF (1.0 g), MeOH was added and an insoluble precipitate was separated by filtration on sintered glass funnel and washed thoroughly with methanol. The precipitate was subjected to recrystallization to give compound 1 (80.7 mg.) The filtrate was dried in a rotary evaporator under reduced pressure at 50 ∘ C, leaving a yellow residue (FFFMW, 809.7 mg) that was further purified by HPLC employing Shim-pack PRC-ODS column (20 mm i.d. × 25 cm, flow rate 5.0 ml/min) and a gradient elution with MeOH/H 2 O 45 to 95% for 50 minutes leading to the isolation of compounds 1 (96.3 mg), 2 (13.4 mg), 3 (82.9 mg), and 5 (84.6 mg).

Cytotoxicity
Assay. Vero and LLCMK 2 cells were exposed to different concentrations of extracts/fractions/compounds for 48 and 72 h [1]. After incubation, cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck) assay at a concentration of 2 mg/mL in PBS [1,14]. Each sample was assayed in four replicates for concentrations ranging from 500 to 0.125 g/mL. The cytotoxicity of each sample was expressed as CC 50 , that is, the concentration of sample that inhibited cell growth by 50% [1].
2.9. Antiviral Assays. The antiviral activity (EC 50 ) of ex-tracts\fractions\compounds was evaluated by the MTT assay [15]. Acyclovir (Calbiochem, Merck Brasil, São Paulo, SP, Brazil) and -2a interferon (Bergamo Brasil, São Paulo, SP, Brazil) were used as positive controls [1]. The cell monolayer was infected with viral suspensions with titers of 2.5 × 10 6 ; 1.0 × 10 6 ; 1.0 × 10 6 ; and 1.0 × 10 4 TCID 50 /mL, respectively, for HSV-1, EMCV, VACV-WR, and DENV-2 [1]. Dilutions of the extracts, fractions, and compounds in noncytotoxic concentrations were added to the wells after viral infection. The plates were incubated at 37 ∘ C in humidified 5% CO 2 atmosphere for a period of 48 and/or 72 h [1]. Experiments were carried out with eight different concentrations within the inhibitory range of the samples. The 50% inhibitor concentrations of the viral effect (EC 50 ) for each of the extracts, fractions, and constituents were calculated from concentration-effect-curves after no linear regression analysis [1]. The selectivity index (SI) is defined as CC 50 over EC 50 . Statistical calculations were carried out with the GraphPad prism 5.0 software package (Statistica). Results are expressed as the mean ± SEM of 4 independent experiments. Student's t-test was used for statistical analyses; values > 0.05 were considered to be significant.

Structural Determination.
The compounds isolated were identified on the basis of spectral analyses and comparison with literature data. 1 D and 2 D 1 H and 13 C-NMR spectra such as COSY, HSQC, and HMBC were obtained on a Bruker Avance DRX400 instrument in DMSO-d6 with TMS as internal standard. Chemical shifts are given as (ppm). LC-MS data were obtained by electrospray ionization mass spectrometry (ESI-MS) in an Esquire 3000 Plus Bruker Daltonics equipment, capillary: 4000 V, nebulizer: 27 psi, dry gas: 7.0 L/min, dry temp: 320 ∘ C, and mass flux 100 uL/min, in the Central Analítica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil [1].

HPLC Analyses, Isolation, and Identification of Compounds from Fridericia formosa.
HPLC-DAD analyses allowed identifying xanthones as major constituents in all the extracts, as inferred from their UV spectra that were registered online (Figure 1). UV spectra of 1,3,6,7-tetraoxygenated xanthones are characterized by the presence of three or more absorption bands of decreasing intensity [18]. A compound with retention time (RT) of 7.8 min was detected in all the ethanol extracts as the major constituent in extracts of stems and fruits.
Bioguided fractionation of F. formosa ethanol extracts led to the isolation of five compounds (Figure 2) which were identified by comparison with literature spectroscopic data ( 1 H and 13 C NMR, DEPT-135 experiment, COSY, HMQC, HMBC, IR, and MS). From the EtOH extract of leaves three compounds (1-3) were obtained. Compound 1 was identified as mangiferin, a C-glucosylxanthone ( max 239, 258, 316, and 365 nm, HPLC-DAD online). Its identification was confirmed by HRMS and comparison of 1 H and 13 C NMR spectra with literature data [19]. Additionally, mangiferin (1) was also isolated from stems (EEAFS) and fruit (EEAFF) extracts. Compounds 2 and 3 were purified from leaves and fruits by Sephadex LH-20 gel filtration and preparative HPLC. The presence of the mangiferin chromophore for both compounds was indicated by their UV spectra. The protonated molecular ions [M + H] + for 2 and 3 (m/z 585.1084 and m/z 569.1144, resp.) were determined by accurate positive HRESI-MS. A comparative analysis of fragment ions detected in experiments by HPLC/ESI-MS\MS showed that the only noticeable differences were associated with the cinnamic acid moieties attached to the mangiferin unity, caffeic acid in 2, and p-coumaric acid in 3. The isolation of mangiferin cinnamic esters from Fridericia samydoides and F. patellifera was previously reported [20,21] and a comparative analysis of these NMR data confirmed the identification of compounds 2 and 3 as 2 -O-trans-caffeoylmangiferin and 2 -Otrans-coumaroylmangiferin, respectively. The position of the cinnamic ester group in the glucose unity was proved by longrange HMBC correlation between the ester carbonyl group and the glucosyl hydrogen: C 165.2 and H-2 H 5.65, in compound 2, and C 165.8 and H-2 H 5.56, in compound 3.
Additional quantity of mangiferin (1) was obtained as an unsoluble fraction when methanol was added to the dried stem ethanol extract. Fractionation of the filtrate (AFSMW2) by preparative RP-HPLC afforded compound 4 whose spectral data (UV, IR, 1 H and 13 C NMR) and comparison with literature data [22] allowed its identification as the flavonoid chrysin (4).
Finally, column chromatographic fractionation of the ethanol fruit extract afforded four C-glucosylxanthones which were shown to be identical to those obtained from the leaves extract, mangiferin (1), caffeoyl mangiferin (2), and coumaroyl mangiferin (3), besides compound 5, and MM 542 Da, which might correspond to a mangiferin cinnamic ester, was confirmed by NMR data. Two regioisomers, 2 -O-trans-cinnamoylmangiferin and 3 -O-transcinnamoylmangiferin, have been previously isolated from stems and leaves extracts of F. samydoides and F. patellifera, respectively [20,21]. A comparison of 13 C and 1 H NMR data, including two-dimensional COSY, HSQC, and HMBC, with those previously reported [20] allowed the identification of compound 5 as 2 -O-trans-cinnamoylmangiferin.

Discussion
Phytochemical investigation of EtOH extracts of leaves, stems, and fruits from F. formosa led to the isolation of five compounds. Four of them were identified as Cglucosylxanthones, namely, mangiferin (1), along with three cinnamoyl esters of mangiferin (2, 3, and 5), and one flavonoid, chrysin (4) (Figure 2). Chrysin (4) was isolated from the stems extract and showed low antiviral activity against VACV-WR and HSV-1 with EC 50 > 100 g/ml and did not inhibit the replication cycle of DENV-2 and EMCV. Good activity of this flavonoid against HSV-1 with an EC 50 2.5 M was previously reported [23] and our negative result might be related to difference in susceptibility of strains. This flavonoid is present in other species of the Bignoniaceae family, such as F. samydoides [24]. Previously isolated from Oroxylum indicum (Bignoniaceae), it is reported as disclosing in vitro anti-inflammatory and anticancer effects [25][26][27]. Marketed as a bodybuilding supplement it is claimed to increase testosterone levels or stimulate testosterone production; however, clinical tests have shown no effect on testosterone levels in men [28]. As shown in Figure 2, the four xanthones isolated from the extracts of leaves, stems, and fruits of A. formosa were identified as mangiferin (1), 2 -O-trans-caffeoylmangiferin (2), 2 -O-trans-coumaroylmangiferin (3), and 2 -O-transcinnamoylmangiferin (5). The antiviral effect of these compounds was evaluated against DENV-2, EMCV, HSV-1, and VACV-WR (Figure 3). Excepting mangiferin (1), the other three xanthones showed good antiviral effects and inhibited the replication cycle of DENV-2, HSV-1, and VACV-WR (Table 1 and Figure 4).
Mangiferin (1) is mainly obtained from mango tree (Mangifera indica) and preclinical studies showed that it exhibits antidiabetic, antioxidant, antiviral, cardiotonic, hypotensive, and anti-inflammatory properties [29]. The biological activities of mangiferin have been attributed to modulating expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, inflammation, and various autoimmune diseases [30]. Furthermore mangiferin disclosed low cytotoxicity and good inhibitory activity on HIV-1 replication in a dose dependent manner [30,31]. Mechanism studies revealed that mangiferin might inhibit the HIV-1 protease and is, therefore, a novel nonpeptide protease inhibitor of HIV protease [30,31].
Recent data on biological activity of mangiferin cinnamoyl esters showed that they are antioxidant agents similar to mangiferin and are antiplasmodial with moderate activity in vitro against Plasmodium falciparum 3D7 clone, which is chloroquine-sensitive (IC 50 18.1 to 26.5 M) [21]. Recently, a total of twenty xanthones were isolated from Swertia mussotii (Gentianaceae) and their antiviral activity was evaluated [32]. Eight of these xanthones exhibited significant activity against hepatitis B virus inhibiting DNA replication with EC 50 values from 0.01 mM to 0.13 mM [32]. Additionally, the xanthones norbellidifolin, 1,5,8-trihydroxy-3-methoxyxanthone, and 2-C--Dglucopyranosyl-1,3,7-trihydroxyxanthone showed remarkable activity with EC 50 values of 0.77, > 0.98, and 0.21 mM for hepatitis B surface antigen (HBsAg) and <0.62, 0.35, and 0.04 mM for hepatitis B antigen (HBeAg), respectively [32]. Besides, euxanthone, from Garcinia oblongifolia (Clusiaceae) leaves extract, disclosed significant activity in vitro against Enterovirus 71, with EC 50 value of 12.2 M [33]. In addition, the selectivity index of this compound was 3.0 in relation to the cytotoxicity to Vero cells (CC 50 36.6 M) [33]. Molecular docking studies of 272 xanthones for interactions with a group of seven fungal and two viral enzymes showed that prenylated xanthones are important hits for inhibition of the selected enzymes [34]. In general, prenylated xanthones were able to establish significantly stronger complexes with the tested enzymes [34]. Some compounds were pointed out as potential inhibitors for those enzymes, including nigrolineaxanthone and latisxanthone D as probably potent inhibitors of HIV-1 reverse transcriptase [34]. Xanthones have important advantages as potential antiviral agents because of their availability as natural compounds and the possibility of being easily synthesized and also for the demonstrated interaction with some important microorganisms targets [34].

Conclusions
Our results reveal that F. formosa is a rich source of mangiferin (1) (≅47 g⋅kg −1 of dry leaves), a C-glucosyl xanthone with several therapeutic and cosmetic uses [30]. Its content in F. formosa is higher than in mango tree (Mangifera indica), its usual source (≅1.7 g⋅kg −1 of dry peel) [35]. However, mangiferin was practically inactive against the virus assayed. On the other hand, minor constituents, represented by mangiferin cinnamoyl esters, seem to be the main responsible constituents for the antiviral activity previously reported for extracts of different botanical parts of this species [8]. Special attention is called for the IS of these xanthone derivatives, particularly for the caffeoyl (2) and cinnamoyl (5) esters, with IS > 100. Our findings are the first report on the chemical and antiviral activity of F. formosa constituents. Our results are in line with the traditional use of Fridericia species as antiinfectious agents in different South American countries [1,9] and might be of interest for the development of standardized antiviral phytomedicines.

Conflicts of Interest
The authors declare no conflicts of interest.