Bioguided isolation of an antiviral compound from Xylophragma myrianthum (Cham.) Sprague (Bignoniaceae Juss.)

Xylophragma Sprague species (family Bignoniaceae Juss.) are climbing plants belonging to the tribe Bignonieae Juss. and some species have a wide spectrum of traditional medicinal uses including remedies for the treatment of infections. This paper reports the bioguided fractionation of an ethanol extract of X. myrianthum (Cham.) Sprague stems (EEXMS) for antiviral effects against Human herpesvirus 1 (HSV-1), Dengue virus 2 (DENV2), murine Encephalomyocarditis virus (EMCV) and Vaccinia virus (VACV) that afforded XM-1 as an active compound. Spectroscopic analyses allowed the identification of XM-1 as arjunic acid whose occurrence in the Bignoniaceae and anti-DENV-2 activities are reported for the first time. X. myrianthum is revealed herein as a source of an antiviral compound and fractions.


Introduction
Xylophragma Sprague species are climbing plants belonging to the family Bignoniaceae Juss.(tribe Bignonieae Dumort.).Several representatives of the Bignoniaceae have a wide spectrum of traditional medicinal uses including remedies for the treatment of infections.The family Bignoniaceae comprises about 82 genera and 827 species that are distributed mainly in tropical regions around the world (Gentry, 1992;Lohmann, 2006).This botanical family consists of trees, lianas, and more rarely shrubs and herbs.Brazil is an important center of diversity of the Bignoniaceae family with the occurrence of 32 genera and about 390 species (Lohmann, 2014).Ten species are recorded for the Xylophragma genus according to The Plant List ( The Plant List, 2014).Of the six species occurring in Brazil, three are endemic, among them Xylophragma myrianthum (Cham.)Sprague.No report was found on medicinal uses of Xylophragma species.The scarcity of data on this genus has motivated its inclusion in our project on the antiviral activity of Bignoniaceae.
Plants belonging to the family Bignoniaceae are used as timbers and ornamentals.Some species have a traditional history of use as astringents and in the treatment of inflammation, syphilis, intestinal cramps, diarrhea, leucorrhea, anemia, leukemia, skin disorders and gonorrhea in different countries (Brandão et al., 2010a).
Several Bignoniaceae species have been investigated for their medicinal value including the treatment of symptoms possibly related to viral infections (Brandão et al., 2010a).Evaluations of the anticancer activity have revealed the presence of highly cytotoxic naphthoquinones, mainly in the genus Tabebuia Gomes ex DC. (Hussain et al., 2007).
Plants belonging to the family Bignoniaceae have been investigated for antiviral activity.The naturally occurring naphthoquinone β-lapachone and the semi-synthetic derivative succinamidyl-β-norlapachone have shown significant inhibition of echovirus type 19 (Pinto et al., 1987).Ethanol extracts of Markhamia lutea (Benth.)K. Schum., a plant used in folk medicine in Rwanda, showed prominent activity against herpes simplex virus and moderate activities against Coxsakie virus and polio virus (Vlientinck et al., 1995); phenylpropanoid glycosides isolated from this species were active against sincial respiratory viruses (RSV) (Kernan et al., 1998).
Biologically active compounds from natural sources have always been of great interest as sources of potentially useful drugs against infectious diseases.Viral infections are a current problem of industrialized and developing countries, accounting for severe damages in human health and economic losses in livestock.The limited number of antiviral drugs in clinical use explains the search for new drugs and/or templates, and the plant chemical diversity might represent a source of novelty (Chattopadhyay and Naik, 2007).Within this context, the aim of the present study was the bioguided fractionation of X. myrianthum extracts and constituents against Human herpesvirus 1 (HSV-1), Dengue virus 2 (DENV-2), murine Encephalomyocarditis virus (EMCV) and Vaccinia virus (VACV).The antiviral activity of X. myrianthum against VACV-WR was first reported recently along with the evaluation of the ethanol extract of eight other species of the Bignoniaceae family (Brandão et al., 2010b).
The four viral samples used in the present investigation represent virus of human and veterinary clinical interest.HSV-1, an RNA virus, is a highly prevalent pathogen causing primary infections which present clinically as herpes labialis or as primary herpetic gingivostomatitis.About 12% of primary HSV-1 infections are associated with symptoms, e.g.epidermal lesions inside and around the mouth.Acyclovir remains as the main antiherpetic drug although drug resistant strains frequently develop following therapeutic treatment of herpes virus (Whitley and Roizman, 2001).
VACV is a poxvirus (family Poxviridae), with a DNA genome that can infect invertebrates and vertebrates including humans as natural hosts.Re-emergence of infections by human vaccinia virus (VACV) besides exanthematic VACV outbreaks have affected dairy cattle and rural workers in Brazil and Asia causing economic losses and affecting health services (Assis et al., 2013).
EMCV (Picornaviridae family) is associated to sporadic miocarditis and encephalitis in domestic swines, several non-human primates and other mammals.The infection is frequently fatal with sudden death.Outbreaks of this virus have been recorded in captive livestock (Oberste et al., 2009).
Dengue virus is an arbovirus of the Flaviviridae family with a RNA genome.With more than one-third of the world's population living in areas at risk for infection, dengue virus is a leading cause of illness and death in the tropics and subtropics.As many as 400 million people are infected yearly.No vaccine or specific antiviral therapy currently exists to address the growing threat of dengue.In Brazil, dengue is the fastest growing disease with an increasing number of dengue hemorrhagic fever cases (Teixeira, 2012).

Plant material
Aerial parts of X. myrianthum were collected in Caratinga, state of Minas Gerais, Brazil.The species was identified by Dr. J. A. Lombardi, Department of Botany, Institute of Biosciences, UNESP, Rio Claro, Brazil.A voucher specimen is deposited in the BHCB/ UFMG, Belo Horizonte, Minas Gerais, Brazil, under the number 24760.

Extraction, isolation and chromatographic analyses
After drying at 40 °C for 72 h, plant leaves (133.8 g) and stems (268.4 g) were ground and exhaustively extracted by percolation with 96% EtOH at room temperature.The solvent was removed in a rotatory evaporator under vacuum at 50 ºC, giving a dark residue (EEXML, 28.9 g and EEXMS, 18.1 g), which was kept in a disseccator until constant weight.A portion of EEXMS (10.0 g) underwent filtration on a column of silica gel eluted successively with 1:1 n-hexane-CH 2 Cl 2 , CH 2 Cl 2 , 1:1 CH 2 Cl 2 -EtOAc, EtOAc, 1:1 EtOAc-MeOH, MeOH and 8:2 MeOH-H 2 O.The elution was monitored by TLC observing the plates under UV (254 and 365 nm) and visible light, before and after spraying with sulfuric p-anisaldehyde.
Partial concentration of the ethyl acetate fraction afforded a white solid which was recrystallized in ethanol, giving XM-1 (52.8 mg).

Structure determination
Structure determination was accomplished by spectral analysis and comparison with literature data. 1 H NMR, 13 C NMR, NOESY, TOCSY, HSQC, and HMBC spectra were obtained in DMSO-d 6 with TMS as internal standard and were recorded on a Bruker Advance DPX400 equipment.Chemical shifts are given as d (ppm).LC-MS were obtained by electrospray ionization mass spectrometry (ESI-MS) on an UPLC Acquity (Waters) with Argon as the collision gas, and the collision energy was set at 40 eV.Analysis was performed on a quadrupole instrument fitted with an electrospray source in the positive mode.Ion spray voltage: -4 kV: orifice voltage -60 V.

Conditions
A LiChrospher 100 RP-18 column (5 μm, 250 × 4 mm i.d.) (Merck) was employed at a temperature of 40 o C, flow rate of 1.0 mL/min and detection at wavelengths of 220, 280 and 350 nm.The injection volume was 10.0 μL.Elution was carried out with a linear gradient of water (A) and acetonitrile (B) (from 5% to 95% of B in 60 min).

Sample preparation
To an aliquot (10.0 mg) of dried EEXMS, HPLC grade MeOH was added.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 μm) before injection.

Cytotoxicity assay
Vero and LLCMK 2 cell monolayers were trypsinized, washed with culture medium and plated in a 96-well flat-bottomed plate with 6.0 × 10 4 cells per well.After 24 h incubation, the diluted extracts, fractions and compound (500 -0.125 μg/mL) were added to the wells and the plates were further incubated for 48 h and 72 h at 37 ºC in a humidified incubator with 5% CO 2 .The supernatants were removed from the wells and 28 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Merck, 2 mg/mL solution in PBS) were added to each well.The plates were incubated for 1.5 h at 37 ºC and DMSO (130 μL) was added to the wells to dissolve the resulting formazan crystals.The plates were placed on a shaker for 15 min and the optical density was determined at 492 nm (OD 492 ) on a multi-well spectrophotometer (Stat Fax 2100) (Kumar and Das, 1996).The results were obtained from four replicates with at least four concentrations of each sample.Cytotoxicity (percentage inhibition of cell growth) was calculated using the equation 100×(A − B)/A, where A and B are the OD 492 values of untreated and treated cells, respectively.
The antiviral activity of the extracts and isolated compounds were evaluated by the MTT colorimetric assay (Betancur-Galvis et al., 1999).Vero cell monolayers were grown in 96 well microtiter plates.Dilutions of the extracts and compounds in non-cytotoxic concentrations were added to the wells after viral infection.The plates were incubated at 37 ºC in humidified 5% CO 2 atmosphere for a period of 48 and/or 72 h.Controls consisted of untreated infected, treated non-infected and untreated non-infected cells.Positive controls (acyclovir, Calbiochem, USA; α-2a interferon, Bergamo, Brazil) were also employed in each assay.Cell viability was evaluated by the MTT colorimetric method as described above for the cytotoxicity assay.
The 50% cytotoxic concentration (CC 50 ) of the assayed samples is defined as the concentration that reduces the OD 492 value of treated uninfected cells to 50% of that of untreated uninfected cells.The 50% antiviral effective concentration (EC 50 ) is expressed as the concentration that achieves 50% protection of treated infected cells from the cytophatic effect of the virus.The percentage of protection is estimated by the equation [(A − B) / (C − B)] × 100, where A, B and C are the OD 492 values of treated infected, untreated infected and untreated uninfected cells, respectively.CC 50 and EC 50 values for each sample were obtained from dose-effect curves and are the average of four assays carried out with four different concentrations within the inhibitory range of the samples.The selectivity index (SI) is defined as CC 50 /EC 50 .
XM-1 was isolated as a white solid, melting point 278.6 -280.4 o C, showing one purple spot in TLC when sprayed with the Liebermann-Burchard reagent that is a positive result for triterpenes (Wagner, Bladt and Zgainsky, 1984).The IR spectrum disclosed a wide band for O-H stretching (3386 cm -1 ), an intense band for C=O stretching (1688 cm -1 ); bands at 1031-1048 cm -1 are related to C-O in alcohols and no signals were registered at 1600 and 1500 cm -1 confirming the aliphatic character of XM-1 (Siverstein and Webster, 2000).
The 1 H-NMR spectrum (400 MHz, DMSO-d 6 , TABLE 2) was typical of a triterpene exhibiting more intense signals in the range of d 0.6 to 1.6 with seven singlets for seven methyl groups linked to quaternary carbons.A multiple at d 5.23 was assigned to an olefinic hydrogen.The signals at d 3.11 (dd, J = 8.0 and 4.0 Hz, 1H) and d 2.74 (d, J = 8.0 Hz, 1H) should be related to two carbinolic hydrogens located at C-3 and C-2, respectively.Considering that H-3 is in an axial position, as it is in most of the oleanenes, H-2 must be also in an axial position and, therefore, the hydroxy groups at C-2 and C-3 are in a trans-diequatorial relationship, as is confirmed by the coupling constant between these hydrogens (JH 2 H 3 = 8.0 Hz).The hydroxy groups exhibited signals at d 5.14 (m, 1H) and d 4.47 (d, J = 4.0 Hz, 1H).The multiplicity of these signals is explained by the use of DMSO-d 6 as   3.
The spectroscopic data allowed the identification of XM-1 as arjunic acid by comparison (TABLE 2) with previously reported 13 C NMR data (Delgado et al., 1984).Some discrepancies are certainly due to the use of different solvents: DMSO-d 6 and C 5 D 5 N. Up to now, arjunic acid is described mainly in the family Combretaceae and discloses a wide spectrum of biological activities (Eldeen et al., 2008).
XM-1 is responsible for the peak at Rt 33.2 min in the HPLC-DAD profile of EEXMS (FIGURE 2) and its UV spectrum registered online shows only a terminal absorption curve that is coherent with the structure of arjunic acid.Importantly, EEXMS exhibited moderate activity against VACV (EC 50 36.4± 3.7 mg/ml, IS = 13.7) but was inactive against DENV-2.This result is an indication of the low content of XM-1 in EEXMS.
The AcOEt fraction was more potent than XM-1 disclosing an EC 50 of 12.5 μg/mL and IS > 5 against DENV-2 that may be related to possible synergism with other compounds present in this fraction.The presence of other antiviral compounds in the AcOEt fraction can be inferred from its activity against VACV and HSV-1 while XM-1 has shown no effect against these viruses (TABLE 1).

Triterpenes as antivirals
There are few reports on the antiviral activity of

Triterpenes as antivirals
There are few reports on the antiviral activity of triterpenes.Recently, Brandão and Collaborators (2013) reported the antiviral activity of ursolic acid against HSV-1 and dengue virus 2 (DENV-2) with EC 50 values of 6.2 and 3.2 μg/mL, respectively.
Arjunic acid was firstly isolated from Terminalia arjuna (Roxb.ex DC.) Wight & Arn. and occurs in several other plant species (Verma et al., 2012).Triterpenes are a class of natural products that exhibit a broad spectrum of biological activities including anti-inflammatory, antimicrobial, anti-cancer, antimalarial and other activities (Lucetti et al., 2010;Manzano et al., 2013;Wu et al., 2013).Several triterpenes have shown antiviral activity (Cos et al., 2004;Jassim and Naji, 2003;Khan et al., 2005).The highly oxygenated triterpenes ganoderiol F and ganodermanontriol have been isolated from the fruits of Ganoderma lucidum and are active against HIV-1 (Et-Mekkawy et al., 1998).Another triterpene with activity against HIV-1 is lancilactone C that inhibited the replication of this virus with an EC 50 value of 1.4 μg/mL and a therapeutic index greater than 71.4 (Chen et al., 1999).Chiang and Collaborattors (2005) reported the broad spectrum antiviral activity of Ocimum basilicum L., the sweet basil of Indian and Chinese medicine, against diverse virus families.Aqueous and ethanolic extracts afforded ursolic acid that showed strong activity against HSV-1, adenovirus 8 (ADV-8), CVB1 and enterovirus 71 (EV71) with EC 50 values of 6.6, 4.2, 0.4 and 0.5 μg/mL, respectively.The antiviral activity of ursolic acid against CVB1 and EV71 is evident during the infection process and the replication phase, indicating that ursolic acid can be a potential candidate against these RNA viruses, what deserves further investigation (Chiang et al. 2005).
Ursolic, oleanolic and betulinic acids and their derivatives occur frequently and sometimes abundantly in many plants and inhibit HIV-1 protease and the stability of the gp120/gp41 complex (Matthe'e; Wright and Konig, 1999;Labrosse, Treboute and Alizon, 2000;Cos et al., 2003;Yogeeswari and Sriram, 2005).Betulinic acid is more active with an EC 50 value of 1.4 μM (Ikeda et al, 2005;Chattopadhyay and Naik, 2007).Dihydrobetulinic acid has an EC 50 of 0.9 μM, while the esterification at C-3 hydroxyl of those acids resulted in the more potent antiviral compound 3-O-(3,3'-dimethylsuccinyl) betulinic acid (DSB) with an EC 50 < 3.5 × 10 -4 μM.DSB can block a key step in the processing of a viral core capsid protein (Kashiwada et al. 1996) and is very active against drug-resistant virus, effective in an animal model of HIV infection and suiTABLE for use in combination therapy and is under phase II clinical trial.Pavlova and Collaborators (2003) reported antiviral properties of betulin, betulinic and betulonic acids in cell cultures infected with HSV-1, influenza FPV/ Rostock and ECHO 6 viruses.All the evaluated triterpenes were active against HSV-1.Betulin, and especially betulinic acid, also suppressed ECHO 6 virus replication.

Conclusion
The presently reported results identified the ethanol extract of X. myrianthum stems as a source of antidengue and anti-vaccinia virus compounds.Finally, our findings are in line with the traditional use of Bignoniaceae species as antiviral agents in different South American countries.To the best of our knowledge, the occurrence of arjunic acid in Bignoniaceae and the anti-DENV-2 activity are reported here for the first time.