Chemistry and Antiviral Activity of Arrabidaea pulchra (Bignoniaceae)

The aim of the present work was to carry out a bioguided isolation of antiviral chemical constituents from an ethanol extract of leaves from Arrabidaea pulchra (Cham.) Sandwith (EEAPL) that had shown in vitro activity in a previous screening using DNA and RNA viruses. The activity of EEPAL was evaluated against the DNA viruses Human herpesvirus 1 (HSV-1) and Vaccinia virus Western Reserve (VACV-WR) as well as against the RNA viruses Murine encephalomyocarditis virus (EMCV), and Dengue virus 2 (DENV-2) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cytotoxicity was determined in LLCMK2 and Vero cells and the Selectivity Indexes (SI) were calculated. The most potent effect was observed against DENV-2 (EC50 46.8 ± 1.6 µg mL−1; SI 2.7). For HSV-1 and VACV-WR EC50 values > 200 µg mL−1 were determined, while no inhibition of the cytopathic effect was observed with EMCV. Bioguided fractionation of EEAPL by partition between immiscible solvents followed by chromatography over a Sephadex LH20 column afforded two arylpropanoid glycosides, verbascoside (AP 1) and caffeoylcalleryanin (AP 2), along with a terpenoid, ursolic acid (AP 3). AP 1 and AP 3 exhibited similar anti-DENV-2 profiles, with SI values of 3.8 and 3.1, respectively, while AP 2 was the most effective anti-DENV-2 constituent, with a SI of 20.0. Our results show that A. pulchra leaves ethanol extract (EEAPL) affords compounds with antiviral activity, mainly against DENV-2.


Results
Preliminary fractionation of EEAPL by sequential extractions of its aqueous methanol solution with immiscible solvents (dichloromethane, ethyl acetate and n-butanol) led to four fractions: dichloromethane fraction (APDL), ethyl acetate fraction (APEL), n-butanol fraction (APBL) and aqueous fraction (APAL). APDL showed good activity against HSV-1 and DENV-2, with EC 50 values of 18.6 ± 0.9 µg/mL and 15.4 ± 2.1 µg/mL, respectively. APEL was weakly active against HSV-1 (EC50 121.9 ± 9.8 µg/mL) and disclosed good activity against VACV (EC 50 18.4 ± 1.9 μg/mL) and DENV-2 (EC 50 12.2 ± 1.6 µg/mL), with SI values > 10 in these two last viruses. Chromatography of APEL over a Sephadex LH20 column led to the isolation of verbascoside (AP 1, 33 mg) and caffeoylcalleryanin (AP 2, 23 mg). APDL (1.4 g) was exhaustively extracted with diethyl ether affording ursolic acid (AP 3, 203 mg). Identification of the isolated compounds was based on spectrometric analyses (UV, IR, 1 H-and 13 C-NMR and MS) and comparison with literature data [11,12]. The chemical structures are shown in Figure 1 and antiviral activity in Table 1. EEAPL, APDL and APEL were characterized by their RP-HPLC-DAD fingerprints. Peaks at RT 11.3 and 13.6 min in the chromatograms of EEAPL and APEL, with detection at 350 nm (Figure 2A,C), correspond to AP 1 and AP 2, respectively, and both of them showed UV spectra characteristic of cinnamic acid esters. Ursolic acid (AP 3) was related to the peak with RT 46.1 min in the chromatographic fingerprint of APDL with detection at 220 nm and the corresponding online UV spectrum is typical of a non-conjugated alkene ( Figure 2B). Figure 2 shows that AP 1 and AP 2 are the major constituents of APEL. Fractions APSE2, APSE3 and APSE4, which were obtained from APEL by chromatography over a Sephadex LH20 column, have quite distinct fingerprints ( Figure 3). AP-1 is concentrated in APSE2, AP 2 is the major component in APSE4 and APSE3 contains similar concentrations of these two arylpropanoid glycosides. EEAPL, APDL, APEL fractions derived from the Sephadex LH20 column chromatography of APEL (APSE2 to APSE7), and the isolated compounds (AP 1, AP 2 and AP 3) were assayed against HSV-1, VACV-WR, EMCV and DENV-2 (Table 1).
Compounds AP 1, 2 and 3 were not active towards VAC-WR and EMCV, and AP 3 showed a low activity towards HSV-1. The three compounds disclosed good activity against DENV-2, although the SI values are low for AP 1 and AP 3, as a consequence of their toxicity to LLCMK 2 cells. AP 2 was the most potent one, with SI > 10 (EC 50 2.8 ± 0.4 μg/mL, SI 20.0, Table 1). Dose-response curves for the active samples are shown in Figure 4. Figure 4A shows that EEAPL and APEL were very active against HSV-1 (virus inhibition > 75%) at concentrations higher than 150 μg/mL, while AP 3 was active at concentrations as low 2 μg/mL, but with only 50% of virus inhibition. EEAPL was more active against VACV-WR at concentrations of 240 μg/mL, inhibiting 50% of the virus, while the fraction APEL and the Sephadex LH20 fractions APSE2-4, derived from it, have shown enhanced antiviral effects, with inhibition > 75% at concentrations of approximately 40 μg/mL. The strongest EEAPL antiviral effect was towards DENV-2 with 80% inhibition at 100 μg/mL. APEL and APSE2-4 caused approximately 100% inhibition at concentrations of 40 μg/mL. Finally, the highest potency of AP 2 against DENV-2 was demonstrated by the dose-response curves of Figure  4D, in which 100% of virus inhibition was observed at 10 μg/mL.

Discussion
According to our previously published results [10], the antiviral profile of the ethanol extract of the leaves of A. pulchra (EEAPL) is inactive towards EMCV, shows low activity against HSV-1 (235.3 ± 9.7 μg/mL, SI > 2.21) and VACV-WR (245.2 ± 13.4 μg/mL, SI > 2.0) and good activity towards DENV-2 (EC 50 46.8 ± 1.6 μg/mL), although with a low SI (2.7, against LLCMK 2 cells). (Table 1). Based on the EC 50 value lower than 100 μg/mL as determined for EEAPL, the limit we established for a plant extract to be considered of interest for a bioguided fractionation, this extract was initially submitted to a liquid-liquid partition aiming to promote the separation of the EEPAL constituents according to their polarity/solubility. The dicloromethane fraction (APDL) inhibited the viral replication of VACV-WR and DENV-2, showing EC 50 values of 18.6 ± 0.9 μg/mL (VACV-WR) On the other hand, ursolic acid (AP 3), which was isolated from APDL, was inactive against VACV-WR, but has shown activity against HSV-1 and DENV-2 (EC 50 6.2 ± 0.1 μg/mL and 3.2 + 0.6 μg/mL, respectively) although with low SI values (1.6 and 3.1, respectively), due to its cytotoxicity to Vero and LLCMk2 cells (8.1 ± 0.9 and 9.9 ± 1.3 μg/mL, respectively). APEL showed a better profile than APDL for both VACV and DENV-2, with good antiviral activity (EC 50 < 20 μg/mL) and SI > 10 and, therefore, it was submitted to chromatography over a Sephadex LH20 column aiming at separation of its active constituents. Three fractions (APSE2-4) out of the seven fractions obtained have shown good activity against VACV-WR and DENV-2 with SI > 10, for both of these viruses, and particularly against DENV-2 (SI > 22.7). APSE 2 led to the isolation of verbascoside (AP 1) and caffeoylcallerianin (AP 2) from APSE4. AP 1 and AP 2 have inhibited the replication of DENV-2 with EC 50 values of 3.4 μg/mL and 2.8 μg/mL, respectively, but AP 2 was less cytotoxic to LLCMk2 cells, thus resulting in a more favorable SI (20.0), while for AP 1 the SI value (3.8) is approximately five times lower. Indeed, the cytotoxicity of ursolic acid is recognized. It is reported to be involved in endoplasmic reticulum stress pathway [13] targeting apoptosis and its potent cancer-preventive activity and great cancer therapeutic potential have been pointed out [14]. None of the tested samples inhibited the multiplication of EMCV. EEAPL, fractions derived from it and isolated compounds have generally shown low cytotoxicity, except APDL and AP 1 with CC 50 in the range of 19.6 μg/mL for LLCMK 2 cells and 25.9 μg/mL for Vero cells. Concerning the effects against DENV-2, it is interesting to note that APEL, which contains similar contents of AP 1 and AP 2 (Figure 2), has shown an enhancement of the antiviral activity (SI > 16.3) in relation to the crude ethanol extract (EEAPL, SI 2.7) while APSE4 (SI > 22.7), containing predominantly AP-2 (Figure 3), was closer to the value for this compound (SI 20.0). Caffeoylcalleryanin (AP 2) was first isolated in 1968 from Pyrus calleryana (Rosaceae) [12], however this is the first report on its isolation from a Bignoniaceous species, as well as on its antiviral activity.
Verbascoside (AP 1, synonyms: acteoside, kusaginin) is a phenylethanoid glycoside isolated from various plant species that displays an interesting spectrum of biological activities [15]. In the genus Arrabidaea, verbascoside was isolated from A. harleyi [11] and its occurrence has been reported in some other genera of the family. It was also isolated from Markhamia lutea (Bignoniaceae) and has shown a potent antiviral effect against respiratory syncytial virus (RSV, EC 50 0.80 µg/mL) [16]. Recently the activity against HSV-1 (EC 50 58.0 µg/mL) and HSV-2 (EC 50 8.9 µg/mL) was reported for this compound [17]. However, in the present work, no effect was observed for verbascoside against this virus. Our result is in agreement with a previous report in which this compound had not shown anti-herpes activity either [16] and these contradictory results might be related to different degrees of virulence of the strains used in the assays.
Ursolic acid (AP 3), a pentacyclictriterpene acid, is frequently found in several plant taxa and has shown a very wide biological activity profile [18]. We have recently described its activity against the same strain of HSV-1 when investigating the antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae) [19]. Terpenoids possess a broad spectrum of antiviral activity against diverse virus families, as reported in a review on antivirals of ethnomedicinal origin that unfortunately does not report the cytotoxicity of the compounds. Ursolic acid, related triterpenoids and their derivatives isolated from many plants inhibit HIV-1 protease and the stability of the gp120/gp41 complex.
The mechanisms of antiviral action of this group of triterpenoids have been shown to involve inhibition of virus entry, of protease and of replication [1].
Verbascoside (AP 1) and caffeoylcalleryanin (AP 2) belong to the group of naturally occurring polyphenols that are known as phenylethanoid glycosides, supporting each one a moiety of glucose esterified by caffeic acid and bonded to an ethylcatechol unity by an ether linkage. The polyphenols often showed virucidal effects in several viral systems by attaching to the proteins and/or host cell surfaces, resulting in reduction or prevention of viral adsorption; inhibition of reverse transcriptase (RTase) of HIV-1 and RNA polymerase of influenza virus. The ortho-dihydroxy systems are probably responsible for polyphenols' antiviral activity [1].
In light of our results, further investigations are needed to understand the mechanisms of the antiviral activity, especially of caffeoylcalleryanin, which shows the lowest toxicity in LLCMK 2 cells of the tested compounds and was active only in DENV-2. Further assays are also needed to investigate virucidal activity and targets in the viral replication cycle.

Plant Material
The leaves of A. pulchra were collected in Belo Horizonte, Minas Gerais, Brazil. The plants were taxonomically identified by Dr. J.A. Lombardi, Departamento de Botânica, Instituto de Biociências, UNESP, Rio Claro, Brazil. A voucher specimen was deposited at the BHCB/UFMG, Belo Horizonte, Minas Gerais, Brazil, under the number 21573.

Extraction and Isolation
The leaves (129.6 g) were dried in a ventilated oven at 40 °C, for 72 h, ground and exhaustively extracted by percolation with 96% ethanol (5 × 250 mL) at room temperature for 48 h each time. A crude dark green ethanol extract residue (EEAPL, 30.8 g) was obtained in a rotatory evaporator under reduced pressure at 50 °C. A portion of EEAPL (10.0 g) was dissolved in methanol-H 2 O (7:3) and this solution was submitted to successive extractions with immiscible solvents affording the corresponding dichloromethane (APDL, 1.4 g), ethyl acetate (APEL, 1.5 g), n-butanol (APBL, 1.3 g) fractions and the final aqueous fraction (APAL, 2.9 g). Chromatography of APEL (1.2 g) through a Sephadex LH 20 ® column eluted with methanol, led to 66 fractions of 10 mL each, which were combined according to their TLC profiles, affording a total of seven fractions (APSE1-7). Recrystallization of fractions APSE2 and APSE4 from methanol yielded AP 1 (33 mg) and AP 2 (23 mg), respectively. APDL was exhaustively extracted with diethyl ether that was concentrated to a residue (430 mg) which was recrystallized from ethanol to give ursolic acid (AP 3, 203 mg). The fractionation process was undertaken as shown in Figure 5.

Cytotoxicity Assay
Vero and LLCMK 2 cells were exposed to different concentrations of extracts/fractions/compounds for 48 and 72 h. After incubation, cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Merck) assay at a concentration of 2 mg mL −1 in PBS [23]. Each sample was assayed in four replicates for concentrations ranging from 500 to 0.125 µg mL −1 . The cytotoxicity of each sample was expressed as CC 50 , i.e. the concentration of sample that inhibited cell growth by 50%.

Antiviral Assays
The antiviral activity (EC 50 ) was evaluated by the MTT assay [23]. Acyclovir (Calbiochem, Merck Brasil, São Paulo, SP, Brazil) and α-2a interferon (Bergamo Brasil, São Paulo, SP, Brazil) were used as positive controls. Experiments were carried out with eight different concentrations within the inhibitory range of the samples. The 50% inhibitor concentration of the viral effect (EC 50 ) for each extract, fractions, and constituents were calculated from concentration-effect-curves after no linear regression analysis ( Figure 5). The selective index (SI) is defined as CC 50 over EC 50 .

Conclusions
Our results reveal the ethanol extract of A. pulchra leaves (EEAPL) to be a promising source of the anti-dengue arylpropanoid glycoside derivatives verbascoside and caffeoylcallerianin, which might represent the main responsible constituents for its activity against DENV-2. Our findings are in line with the traditional use of Arrabidaea species as anti-infectious agents in different South American countries [9] and are of interest for the development of standardized phytomedicines [24].