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Antiviral activity of Faramea bahiensis leaves on dengue virus type-2 and characterization of a new antiviral flavanone glycoside
Phytochemistry Letters 19 (2017) 220–225
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Short communication
Antiviral activity of Faramea bahiensis leaves on dengue virus type-2 and characterization of a new antiviral flavanone glycoside$ Adriana C. Nascimentoa , Ligia M.M. Valentea,* , Mário Gomesb , Rodolfo S. Barbozaa , Thiago Wolffa , Rômulo L.S. Nerisc, Camila M. Figueiredoc, Iranaia Assunção-Mirandac,* a Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos 149, Centro de Tecnologia, Bl. A, Cidade Universitária, 21941-909, Rio de Janeiro, RJ, Brazil b Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, R. Jardim Botânico 1008, 22470-180, Rio de Janeiro, RJ, Brazil c Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bl. I, Cidade Universitária, 21941-970, Rio de Janeiro, RJ, Brazil
A R T I C L E I N F O
Article history: Received 29 September 2016 Received in revised form 8 January 2017 Accepted 25 January 2017 Available online xxx Keywords: Faramea bahiensis Rubiaceae Flavanone Anti-dengue Antiviral Dengue virus
A B S T R A C T
Dengue virus infection is a neglected disease prevalent in most tropical and subtropical areas. Currently there is no any antiviral drug indicated for the routine treatment of dengue patients. As part of our ongoing search for potential anti-dengue virus agents we have investigated the methanol extract of Faramea bahiensis (Rubiaceae) leaves. This species is endemic in Brazil, and there are no reports on its chemical composition or therapeutic potential. Its crude MeOH extract showed in vitro non-cytotoxicity and anti-dengue virus serotype 2 (DENV-2) activity in human hepatocarcinoma cell lineage (HepG2). A marked reduction on viral load (100%) was observed. Sequential fractionation of the bioactive crude extract led to the isolation of a bioactive new flavanone glycoside: 5-hydroxy-40 -methoxy-flavanone-7O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside, the known 5,40 -dihydroxy-flavanone-7-O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside and a diateroisomeric epimer pair of the known 5,30 ,50 trihydroxy-flavanone-7-O-ß-D-glucopyranoside. The treatment of DENV-2 infected HepG2 cells with the new flavanone was able to control viral replication promoting a reduction of the number of infected cells (12%), together with a decrease of infectious particles in the culture supernatant (97%) and of the number of RNA copies of DENV-2 in HepG2 cells (67%). Structural determinations were made by NMR techniques in one and two dimensions (1H NMR, 13C NMR, COSY, HSQC and HMBC), HRMS, UV, OR and CD. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Dengue fever is a mosquito-borne viral neglected disease prevalent in most tropical and subtropical areas and its incidence has dramatically increased in the last decades (WHO, Updated in 2016). According to the World Health Organization (WHO), only in the Americas in 2015 2.35 million cases were reported, of which 10,200 were diagnosed as the severe form of the disease (also known as dengue hemorrhagic fever), causing around 1000 deaths (WHO, Updated in 2016). In Brazil, it is placed among one of the most serious public health issues registering about 1.6 million cases in 2015 (Brazilian-Health-Ministry, 2016). The dengue
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Faramea bahiensis was formerly identified as Faramea marambaiae. * Corresponding authors. E-mail addresses: [email protected] (L.M.M. Valente), [email protected] (I. Assunção-Miranda).
symptoms usually start with high fever that can be accompanied by severe headache, retro-orbital pain, muscle and joint pains, nausea, vomiting, swollen glands or rash. In severe cases, vascular permeability leading to hypotension may evolve to shock and hemorrhagic manifestations may occur as well. Currently there is no drug indicated for the routine treatment of dengue patients. Thus, the discovery of drugs that can exert antiviral action against dengue virus (DENV), inhibiting the virus replication cycle without being toxic to the host cell and thus mitigating the symptoms, is highly desirable. The genus Faramea Aubl. (Rubiaceae) is native, but nonendemic to Brazil and contains species in form of shrubs, subshrubs or trees. Despite the diversity of the species, few reports can be found on their chemical composition and on their pharmacological potential. Previous phytochemical investigations of Faramea species have resulted in the isolation and/or characterization of anthraquinones and naphthopyrans (Ferrari et al., 1985), flavans
http://dx.doi.org/10.1016/j.phytol.2017.01.013 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
A.C. Nascimento et al. / Phytochemistry Letters 19 (2017) 220–225
(Sauvain et al., 1994), methyl salicylate, hydroxyl acetophenone derivatives and phenylpropanoids (Ribeiro et al., 2003). The species Faramea bahiensis Mull. Arg. is endemic in Brazil, and to our knowledge no studies have been reported on it. As part of an ongoing search for potential anti-dengue agents the antiviral and immunomodulatory activities of the species Uncaria tomentosa (Rubiaceae) on human monocytes infected with DENV serotype 2 (DENV-2) have been reported (Lima-Junior et al., 2013; Reis et al., 2008). Clinical and experimental evidences point out that hepatocytes are target for DENV replication and are involved in the pathogenesis during infection (Lee et al., 2012; Parkash et al., 2010; Suksanpaisan et al., 2007). Some hepatocytederived cell lines have been used as targets for in vitro DENV infection models (Assunção-Miranda et al., 2016; Samsa et al., 2009; Suksanpaisan et al., 2007). Continuing our search, the antiDENV activity of extracts from other Rubiaceae species was screened in hepatocarcinoma cell lineage (HepG2), revealing the significant antiviral effect of the MeOH extract of F. bahiensis leaves. In this paper, we report the isolation of a new antiviral flavanone apiofuranoside (1), a known flavanone apiofuranoside (2) and a diasteroisomeric epimer pair of the known flavanone glucoside (3a and 3b) from its bioactive extract. 2. Results and discussion The crude MeOH extract of the leaves of F. bahiensis was assayed for in vitro cytotoxic and anti-DENV-2 effects in HepG2. After 48 h of infection (hpi) DENV-2 replication promoted a reduction of about 15% of HepG2 viability, that was not observed when cells
Fig. 1. In vitro cytotoxicity and cytoprotection effect of: (A) MeOH extract of Faramea bahiensis leaves; (B) flavonoid-rich MeOH/H2O 1:1 fraction. Control: HepG2 cells; DENV: HepG2 cells infected with DENV-2 16681.
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were infected and treated with the F. bahiensis extract (Fig. 1A). Non-cytotoxicity or proliferative effect was evident. In addition, DENV-2 particles were not detected by plaque assay in the culture supernatant after treatment with F. bahiensis extract, representing a 100% reduction in viral load (data not shown). The liquid-liquid partition of the extract yielded a flavonoid-rich MeOH/H2O 1:1 fraction (monitored by TLC) which showed similar behavior in the MTT assays (Fig. 1B). These results indicate that compounds present in the MeOH extract of F. bahiensis leaves and preserved in its derived hydromethanolic fraction have an efficient antiviral activity against DENV-2. Sequential fractionation of the flavonoidrich fraction involved RP-CC and RP-HPLC to obtain the new compound 1 and the known compounds 2-3a/3b (Fig. 2), of which 1 was found to be bioactive. Compound (1) showed molecular formula C27H32O14 (HRESITOF, m/z 579.1727 [MH], C27H31O14 requires 579.1713). The analysis of its 1H NMR spectrum together with the 1H-1H correlations in the COSY spectrum showed an ABX-system [dH 2.69 (1H, dd, J = 17.2 and 2.9 Hz), 3.08 (1H, dd, J = 17.2 and 13.0 Hz) and 5.36 (1H, dd, J = 13.0 and 2.9 Hz)]; protons at dH 4.86 (1H, d, J = 7.4 Hz) and 4.89 (1H, d, J = 2.4 Hz) coupled to a signal set between dH 3.3-4.0 ppm suggesting two sugar units; four protons at dH 7.35 (2H, d, J = 8.7 Hz) and 6.90 (2H, d, J = 8.7 Hz) showing a pdisubstituted aromatic ring; two protons at dH 6.15 brs and a methoxy group at dH 3.74 ppm (Table 1). The 13C NMR spectrum and the 1H-13C correlations in the HSQC spectrum showed, among others, a ketone carbonyl (dC 198.39), four downfield quaternary carbons (dC 161.45-166.89) and a methylene carbon at dC 44.14 (Table 1). The UV maximum absorptions at 282 (log e 4.19) and 328 nm (log e 3.52) which suffered a bathochromic shift to 306 and 383 nm respectively, after treatment with AlCl3 (Mabry et al., 1970), together with the analysis of the long-range 1H-13C correlations in the HMBC spectrum and biogenetic arguments pointed out compound 1 as a 5-hydroxy-flavanone with a pmethoxy substituted B ring in equatorial position (Fig. 3). The correlation in the HMBC spectrum between the anomeric proton at dH 4.86 (1H, d, J = 7.4 Hz) and the carbon at dC 166.89 revealed a
Fig. 2. Chemical structures of compounds 1-3a/3b.
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Table 1 1 H and 13C NMR (500 and 125 MHz respectively) data (d in ppm) of 1 in CD3OD. Position
dH mult.(J in Hz)
dC
2 3eq 3ax 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 1’’ 2’’ 3’’ 4’’ 5’’ 6’’
5.36 dd (13.0, 2.9) 2.69 dd (17.2, 2.9) 3.08 dd (17.3, 13.0) – – 6.15 brs – 6.15 brs –
80.43 44.14
1’’’ 2’’’ 3’’’ 4’’’ 5’’’ OCH3
– 7.35 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 7.35 d (8.7) 4.86 d (7.4) 3.36 m 3.51 m 3.22 dt (3.2, 1.6) 3.35 m 3.51 m 3.90 d (10.8) 4.89 d (2.4) 3.75 d (2.4) – 3.64 d (9.8) 3.88 d (9.8) 3.40 brs (2H) 3.74 s
198.39 164.87 97.05 166.89 98.11 164.44 104.93 132.06 129.08 115.04 161.45 115.04 129.08 101.15 75.13 77.78 71.48 78.19 68.83 111.05 78.21 80.46 75.13 65.94 55.79
glycosylation at C-7 (Fig. 3). The site of glycosylation was confirmed by the observed downfield effect on the H-6 and H-8 chemical shifts when compared with 7-OH flavanones (Hashmi et al., 2014; Moco et al., 2006). The complete analyses of the NMR spectra and the comparison with previously reported flavanone glycoside data (Takahashi et al., 2001; Zhang et al., 2003), allowed identifying (1) as a flavanone with an 7-O-b-D-apiose-(1 ! 6)-b-Dglucose sugar unit (Table 1,Fig. 3). The absolute stereochemistry on C-2 was derived from the analyses of its OR and CD data when compared to similar data previously described for naringin (Gaffield, 1970). The OR datum ([a] = 88) and CD spectrum profile that showed a positive Cotton effect at 336 nm (u = +741) (n ! p*) and negative at 287 nm (u = 11,481) (p ! p*) (Fig. 4), led to establish 1 as the new flavanone 2S-5-hydroxy-40 -methoxyflavanone-7-O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside. Compound (2) revealed NMR spectra very similar to those of 1 without the signal corresponding to the methoxy group and with some differences in the chemical shifts of the B ring aromatic
Fig. 3. Main 1H-1H correlations in the COSY, 1H-13C in the HSQC and 1H-13C longdistance in the HMBC spectra of 1. (Color figure available online only).
Fig. 4. Circular dichroism spectra of compounds 1 and 3a/3b (in MeOH). (Color figure available online only).
protons. The UV maximum absorptions at 283 (log e 4.2) and 328 (log e 3.7) nm that suffered a similar bathochromic shift after treatment with AlCl3 together with the analysis of the long-range 1 H-13C correlations in the HMBC spectrum showed 2 also as a 5hydroxy-7-O-glycosylflavanone with a p-hydroxy substituted B ring in equatorial position. The complete analyses of its NMR spectra showed that the structure of 2 was similar to that of the previously described Pyrroside B (Masuoka et al., 2003). The OR datum ([a] = 88) allowed suggesting the absolute stereochemistry on C-2 as 2S as revealed for 1. Compound (3) showed molecular formula C21H22O11 (HRESITOF, m/z 449.1088 [MH], C21H21O11 requires 449.1083) with a fragment ion at m/z 287.0563 corresponding to [M-H-162.0526] suggesting the loss of a glycopiranosyl unit. The 1H NMR spectrum and the 1H-1H and 1H-13C correlations in the COSY and HSQC spectra respectively showed some similarities with those of compounds 1 and 2. However, some signals appeared duplicated (with the same intensity) and/or with some differences in their multiplicities, such as the signals corresponding to the C ring of flavanones: dH 2.75 (dt, J = 17.2 and 3.3 Hz), dH 3.12 (dd, J = 17.2 and 12.7 Hz)/dC 44.18/44.34 and dH 5.32 (dt, J = 12.6 and 2.9 Hz)/dC 80.64/80.81. The long-range 1H-13C correlations in the HMBC spectrum between the anomeric proton signals at dH 4.97/4.98 and the carbon signals at dC 167.09/167.16 in addition to the chemical shift values for H-8 and H-6 at dH 6.18 (d, J = 2.0 Hz) and 6.21 (d, J = 2.0 Hz) respectively, indicated a glycosylation at C-7. On the other hand, there is no correlation between these anomeric proton signals with any carbon at the B ring which could indicate a mixture of different compounds. The UV spectra (before and after the AlCl3 treatment) and the detailed analysis of the NMR spectra allowed suggesting 3 as a 5,30 ,50 -trihydroxy-flavanone-7-O-glucopyranoside, with the B ring in equatorial position (Chen et al., 2009; Sun et al., 2007). The biosynthesis of flavanones involves an intramolecular Michael-type nucleophilic cyclization of chalcones mediated by the enzyme chalcone isomerase producing almost exclusively the (2S)-isomer although it may potentially lead to the existence of two stereoisomeric forms. Subsequent enzymatic glycosylation steps, lead to flavanone glycosides. The possible resulting changes of configuration at their stereogenic centers determine that flavanone glycosides may exist as mixtures of diastereoisomers (Maltese et al., 2009). In fact, mixtures of (2R)and (2S)- flavanone glycosides have been detected in natural matrices such as for naringin and hesperidin in mature citrus fruits and for two isomeric tetrahydroxyflavanone-7-O-b-D-glucopyranoside in the aerial parts of Lippia salviaefolia (Funari et al., 2011;
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Gaffield et al., 1975; Khan et al., 2014; Maltese et al., 2009). The process probably occurs via C ring opening. The intermediate chalcones thus formed would rapidly recyclize due to their instability, in a nonstereospecific way, leading to the corresponding 2S and 2R flavanones (Gaffield et al., 1975; Maltese et al., 2009). The CD spectrum profile with a negative Cotton effect at 334 nm (u = 800) (n ! p*) and negative at 286 nm (u = 3.133) (p ! p*) (Fig. 4) together with the NMR evidences indicated 3 as a diasteroisomeric epimer pair, 2R + 2S of the known flavanone 5,30 ,50 -trihydroxy-flavanone-7-O-ß-D-glucopyranoside. Compound 1 showed non-cytotoxicity and a cytoprotective action without proliferative effect when assayed in the DENVinfected HepG2 cells at different doses (Fig. 5A). Moreover, it was possible to demonstrate a dose-dependent reduction in viral load in the culture supernatant (Fig. 5B). The dose of 1 required to inhibit 50% of viral replication (IC50) was 13.1 mg/mL. The dose of 50 mg/mL was chosen to evaluate the effect of 1 in the progression of DENV-2 infection in HepG2 by presenting the best doseresponse relationship. The treatment of infected HepG2 with 1 promoted a reduction in the number of infected cells of approximately 12% when compared with untreated HepG2 (Fig. 6A). Quantification of DENV-2 particles in the supernatant by plaque assay demonstrated that treatment with 1 reduced by approximately 97% the number of infectious particles in the culture supernatant when compared with untreated condition (Fig. 6B). Moreover, quantifying the number of RNA copies of DENV-2 by real time PCR in HepG2 extract showed that after treatment with 1 the copy number of DENV RNA was reduced by about 67% (Fig. 6C). Taken together, these results indicate that 1 is able to control DENV-2 replication. To confirm that this effect was not associated with a virucidal activity of 1 against DENV-2, a preincubation of DENV-2 with 1 at 37 C for 1 h was also performed and the infectious particles remained after incubation were quantified by plaque assay. No difference in viral title was observed after incubation (Fig. 6D). This contributed to demonstrate the ability of 1 to control DENV-2 replication in some step of its replication in target cells. In this study, we report for the first time the isolation of flavanones in a Faramea species and reveal the potential anti-
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Fig. 6. HepG2 infected with DENV2 at low MOI (0.5) and treated or not with 1. (A) % of infected HepG2 assessed by flow cytometry 48 hpi; (B) viral load in the supernatant determined by plaque assay; (C) quantification of number of copies of DENV RNA obtained by real time-PCR analysis; (D) virucidal activity by incubating DENV with 1 or vehicle for 1 h at 37 C. Results are expressed as the mean S.E.M. of three independent experiments. *P < 0.05.
dengue activity of the MeOH extract of F. bahiensis leaves. In addition, the new flavanone apiofuranoside (1) isolated from this bioactive extract showed to be effective to protect HepG2 cells from DENV-2 infection and to control DENV-2 replication. 3. Materials and methods 3.1. General experimental procedures The HPLC analyses were performed using a Perkin-Elmer Series 200 equipment with quaternary pump, autosampler and diodearray detector. The NMR spectra were recorded in CD3OD (Cambridge Isotope Laboratories Inc.) on Bruker DRX 400 or on Varian System 500 spectrometers using the solvent as internal standard. UV spectra were performed on a Varian Cary 1E (UVVIS) spectrophotometer. Optical rotations (OR) were obtained on a Perkin-Elmer 341 LC polarimeter. Circular dichroism (CD) spectra were carried out on a Jasco J-715 spectropolarimeter. HRESITOF analyses were made with a Bruker Daltonics micrOTOF instrument. RP-C18 (40–63 mm Merck) (4.0 cm e.d. 15.0 cm phase) was used for column chromatography (CC). The TLC analyses were performed in pre-coated silica gel 60 F254 (Merck). MeOH, CH3CN (both HPLC grade), CHCl3, EtOAc, EtOH, and Hexane (all p.a., ACS grade) were purchase from Tedia. Milli-Q grade water was used for HPLC. 3.2. Plant material
Fig. 5. Dose-response curve of compound 1 (cpd1) in DENV-infected HepG2 cells. HepG2 cells were infected or not with DENV2, treated with indicated concentrations of compound 1 and analyzed 48 h post infection. (A) cell viability determined by MTT assay; (B) viral load in cell culture determined by plaque assay. DENV: HepG2 cells infected with DENV-2 16681. Result of at least three independent experiments.
The species Faramea bahiensis was collected at the Restinga de Marambaia, Rio de Janeiro, Brazil in March 2010, and a voucher specimen was deposited at the Herbarium of the Instituto de Biologia of the Universidade Federal do Rio de Janeiro, RJ, Brazil, under number RFA 37489. The collection had previous permission from SISBIO-ICMBio-MMA-Brazil under number 46504-2.
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3.3. Extraction and isolation The leaves were dried at 40 C for 24 h and the dried and sieved ( 335 mm) material (151.3 g) was exhaustively and sonically extracted with MeOH. The MeOH was removed under low pressure to yield 7.8 g of crude extract. Part of the crude MeOH extract (6.36 g) was dissolved in 500 mL of MeOH/H2O 9:1 and partitioned with n-hexane (500 mL 24). Next, more H2O was added (400 mL) to the hydromethanolic fraction until MeOH/H2O 1:1 and then partitioned with CHCl3 (500 mL 3) yielding 1.35 g, 0.58 g and 3.90 g of the hexane, CHCl3 and MeOH/H2O fractions respectively. The fractions were submitted to TLC in different mobile phases and stain reagent systems aiming at screening for the presence of terpenoids, alkaloids and flavonoids, classes of natural products usually found in Rubiaceae spp. The tests showed the presence of flavonoids in the MeOH/H2O 1:1 fraction by using EtOAc/HCOOH/ AcOH/H2O 100:11:11:27 followed by derivatization with 1% NP reagent in MeOH/UV irradiation at 365 nm (Wagner and Bladt, 1996). Part of the MeOH/H2O fraction (3.0 g) was submitted to RPCC using a solvent gradient from MeOH/H2O 3:7 ! 7:3, MeOH, EtOH, EtOAc and CHCl3 yielding 31 fractions that were pooled together by TLC similarities. Some of the yielded fractions were then submitted to RP-HPLC-DAD in analytical and semi-preparative scales. Compound (1) (10.8 mg) was isolated from sub-fraction 14 (67.8 mg, eluted with MeOH) by semi-Prep HPLC using a LiChrocart LiChrospher RP-18 column (250 6.4 mm, 5 mm) (Merck) and as mobile phases: CH3CN as solvent A and H2O with 0.1% HCOOH, pH 3 as solvent B. Elutions were performed in a linear gradient: 22% of A for 15 min, 22–25% of A for 30 min, 25% of A, for 3 min, then 95% of B for 12 min. The flow rate was 1.7 mL/min, the column temperature 40 C and the injection volume 120 mL at c = 25 mg/mL. Compounds (2) (14.2 mg) and (3) (36.1 mg) were isolated from sub-fractions 8–10 (303.4 mg eluted with MeOH/H2O 4:6) by semi-Prep HPLC using a Zorbax Eclipse XDB C18 column (250 9.4 mm; 5 mm) (Agilent) and as mobile phases: CH3CN as solvent A and H2O with 0.1% HCOOH, pH 3 as solvent B. Elutions were performed in a linear gradient: 5–25% of A for 35 min, 25–95% of A for 5 min, then 95% of B for 10 min. The flow rate was 2.5 mL/ min, the column temperature 40 C and the injection volume 100 mL at c = 100 mg/mL. In both cases the diode-array detector was set at an acquisition range of 190–400 nm and flavonoid monitoring was performed at 280 and 320 nm.
77.99 (C-3’’), 71.67 (C-4’’), 77.29 (C-5’’), 68.97 (C-6’’), 111.25 (C-1’’’), 78.35 (C-2’’’), 80.62 (C-3’’’), 75.30 (C-4’’’), 66.10 (C-5’’’). 3.3.3. Compound (3a/3b) oil: [a]D (20 C) 40,0 (c = 0.203, MeOH); UV lMAX nm (log e): 288 (4.07), 330 (3.35), lMAX + AlCl3: 303, 384, lMAX + AlCl3 + HCl: 304, 380; CD (c = 400 mM, MeOH) Cotton effect at 334 nm (u = 800) (n ! p*) and at 287 nm (u = 3.133) (p ! p*); HRESITOF m/z 449.1088 [MH] (C21H21O11 requires 449.1083); 1H NMR (400 MHz in CD3OD, d in ppm): dH 5.32 (1H, dt, J = 12.6, 2.9 Hz, H2), 2.75 (1H, dt, J = 17.2, 3.3 Hz, H-3eq), 3.12 (1H, dd, J = 17.2, 12.7 Hz, H-3ax), 6.21 (1H, d, J = 2.0 Hz, H-6), 6.18 (1H, d, J = 2.0 Hz, H-8), 6.79 (2H, brs, H-20 and H-60 ), 6.92 (1H, brs, H-40 ), 4.97/4.98 (d, J = 7.3 Hz, H-1’’), 3.46 (1H, m, H-2’’), 3.45 (1H, m, H-3’’), 3.40 (1H, m, H-4’’), 3.46 (1H, m, H-5’’), 3.69 (1H, ddd, J = 13.8, 5.0, 2.2 Hz, H-6a”), 3.88 (1H, dt, J = 13.8, 1.7 Hz, H-6b”); 13C NMR (100.57 MHz in CD3OD, d in ppm): dC 80.64/80.81 (C-2), 44.18/44.34 (C-3), 198.67 (C-4), 165.04/ 165.08 (C-5), 97.10/97.18 (C-6), 167.09/167.16 (C-7), 98.14 (C-8), 164.70 (C-9), 105.08/105.10 (C-10), 131.65/131.68 (C-1’), 116.42 (C2’), 147.10 (C-3’), 119.48/119.50 (C-4’), 146.66 (C-5’), 114.87/114.94 (C-6’), 101.38/101.40 (C-1’’), 74.80 (C-2’’), 77.96 (C-3’’), 71.31 (C-4’’), 78.40 (C-5’’), 62.50 (C-6’’). 3.4. HepG2 infection and treatment Human hepatocarcinoma cell lineage (HepG2) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (LGC Biotecnologia) supplemented with 10% fetal bovine serum (FBS), at 37 C, in an atmosphere of 5% CO2. HepG2 cells were infected with DENV-2 (strain 16681) in M.O.I. of 1 or 0.5 for 1 h at 37 C in 5% CO2. After infection, the medium was replaced by fresh medium (DMEM with 5% FBS) with or without 50 mg/mL (in DMSO) of the MeOH extract of F. bahiensis leaves, the hydromethanolic fraction or different doses of 1 (as indicated in Fig. 5), and cultured at 37 C in 5% CO2. The samples (stock 100 mg/mL) were added to the medium (DMEM with 5% FBS) to obtain the desired concentration. The final concentration of DMSO in HepG2 culture was 0.05%, which was also added to the infected and untreated condition. After 48 h of infection, the culture medium was collected for virus titration. Cellular extracts were used to determine cell viability, real-time PCR and for flow cytometry assays (as described below). 3.5. Cell viability assay
3.3.1. Compound (1) oil; [a]D (20 C) 88 (c = 1, MeOH); UV lMAX nm (log e): 282 (4.19), 328 (3.52), lMAX + AlCl3: 306, 383, lMAX + AlCl3 + HCl: 305, 380; CD (c = 400 mM, MeOH) Cotton effect at 336 nm (u = +741) (n ! p*) and negative at 287 nm (u = 11,481) (p ! p*); HRESITOF m/z 579.1727 [MH] (C27H31O14 requires 579.1713); 1H and 13C NMR (500 and 125 MHz respectively in CD3OD) data, see Table 1. 3.3.2. Compound (2) oil; [a]D (20 C) 88 (c = 1, MeOH); UV lMAX nm (log e): 283 (4.2), 328 (3.7), lMAX + AlCl3: 306, 382, lMAX + AlCl3 + HCl: 307, 383; 1 H NMR (400 MHz in CD3OD, d in ppm): dH 5.42 (1H, dt, J = 12.8, 2.7 Hz, H-2), 2.76 (1H, dt, J = 17.2, 2.7 Hz, H-3eq), 3.18 (1H, dd, J = 17.2, 12.8 Hz, H-3ax), 6.22 (2H, brs, H-6 and H-8), 6.84 (2H, d, J = 8.4 Hz, H-3’and H-50 ), 7.35 (2H, d, J = 8.4 Hz, H-2’ and H-6’), 4.94 (1H, d, J = 7.2 Hz, H-1’’), 3.46 (2H, m, H-2’’ and H-3’’), 3.35 (1H, m, H-4’’, 3.63 (1H, m, H-500 ), 3.63 (1H, m, H-6a”), 4.02 (1H, m, H-6b”), 4.96 (1H, d, J = 2.4 Hz, H-1’’’), 3.87 (1H, d, J = 2.4 Hz, H-200 ’), 3.75 (1H, d, J = 9.7 Hz, H-4a”’), 4.00 (1H, d, J = 9.7 Hz, H-4b”’), 3.52 (2H, s, H-5’’’); 13 C NMR (100.57 MHz in CD3OD, d in ppm): dC 80.82 (C-2), 44.32 (C-3), 198.43 (C-4), 165.09 (C-5), 97.19/98.21 (C-6 or C-8), 167.10 (C7), 164.72 (C-9), 105.11 (C-10), 131.05 (C-1’), 129.32 (C-2’ and C-6’), 116.56 (C-3’ and C-5’), 159.25 (C-4’), 101.34 (C-1’’), 74.79 (C-2’’),
The effect of the MeOH extract of F. bahiensis leaves, hydromethanolic fraction or 1 in infected HepG2 cell viability was determined by measuring the metabolization of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT metabolization assay) by the cells. Cells seeded in a 24-well plate were infected with DENV-2 and treated as previously described. Cytotoxicity and/or proliferative effects were assessed treating uninfected HepG2 cells in the same conditions. Forty-eight hpi cells were washed with balanced salt solution (BSS) prior to the addition of 500 mL of 0.5 mg/mL MTT (Sigma-Aldrich Co.) in BSS to each well. After 1 h, MTT solution was discarded and the formazan crystals formed were solubilized in each well using 500 mL of 0.04 M HCl solution in i-PrOH. The optical density (OD) of the samples was read at 570 nm and 650 nm for background correction. 3.6. Viral quantification The virus titer in the culture medium of infected HepG2 cells was quantified by plaque assay in Baby Hamster Kidney cells (BHK21 cells). Briefly, BHK-21 cells were grown in Minimum Essential Medium (MEM) a (Invitrogen) supplemented with 10% FSB and
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seeded in 24-well plates and cultured overnight at 37 C with 5% CO2. Ten-fold serial dilutions of the samples were performed using a-MEM and used to infect BHK-21 cells at 37 C for 1 h. After this period, 1% carboxymethyl cellulose in a-MEM with 2% FBS was added and the cells were kept in culture at 37 C with 5% CO2 for five days. Then, the cells were fixed with formaldehyde 4% and the plaque was visualized by staining with crystal violet (1% crystal violet powder (w/v), 20% MeOH and H2O). To determine the dose required to inhibit 50% of viral replication (IC50), viral title after treatment with different doses of 1 (0, 1, 5, 10, 25, 50, 100 and 200 mg/mL) was used to obtain a non-linear regression analysis with a sigmoidal and variable slope profile using the software GraphPad Prism 6 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. For virucidal analysis, 106 plaque forming unit (PFU) of DENV-2 was incubated with 50 mg/mL of 1 in DMSO or only with the same proportion of DMSO for 1 h at 37 C. Infectious particles were then quantified by plaque assay. 3.7. Quantification of DENV2 infected cells by flow cytometry Flow cytometry analysis was performed for the quantification of DENV-2 infected cells by detecting intracellular viral antigens. After 48 h of infection, cells were washed with PBS, harvested and fixed in 4% formaldehyde in PBS at room temperature for 15 min. After washing, the cells were permeabilized with 0.1% saponin in PBS and incubated with blocking solution (PBS supplemented with 2% FBS and 0.1% bovine serum albumin) for 30 min, at room temperature. Then, the cells were incubated for 1 h with antiDengue Virus Complex, clone D3-2H2-9-21 antibody (1:500, Millipore) in 0.1% BSA in PBS. The cells were washed and incubated with Alexa Fluor1 488 Goat Anti-Mouse IgG (H + L) antibody (1:1000, Invitrogen) in 0.1% BSA in PBS for 30 min. The percentage of infected cells was analyzed by FACScan Flow Cytometer and CellQuest software (Bectan Dickinson). 3.8. Quantification of viral RNA by real time PCR analysis Total RNA from HepG2 cells was isolated using Trizol1 (Invitrogen) 48 hpi after treatment of culture with (1). 4 mg of total RNA were reverse-transcribed using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and the amount of DENV RNA of each sample was determined by real-time PCR analysis using TaqMan reagents (Applied Biosystems, Foster City, CA, USA). Primers, probe and DENV RNA quantification method was performed as previously described (Lima-Junior et al., 2013). 3.9. Statistical analysis Statistical calculations were carried out with the GraphPad Prism 5 software. Results are expressed as the mean S.E.M. of 3 independent experiments. Student’s t-test was used for statistical analyses; P values >0.05 were considered to be significant. Acknowledgments The authors thank the NMR Labs of the Instituto de Tecnologia em Fármacos Fundação Oswaldo Cruz/RJ and of the Instituto de Pesquisa em Produtos Naturais, Universidade Federal do Rio de Janeiro for the NMR spectra, the MS Lab of the Instituto de Pesquisa em Produtos Naturais for the HRESIMS and FAPERJ-Brazil for grant support (Proc. No E-26/111.373/2014). A.C.N. thanks CNPq-Brazil and T.W. CAPES-Brazil for fellowships.
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Interconverting flavanone glucosides and other phenolic compounds in Lippia salviaefolia Cham. ethanol extracts. Phytochemistry 72, 2052–2061. Gaffield, W., Lundin, R.E., Gentili, B., Horowitz, R.M., 1975. C-2 stereochemistry of naringin and its relation to taste and biosynthesis in mature grapefruit. Bioorg. Chem. 4, 259–269. Gaffield, W., 1970. Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides. Tetrahedron 26, 4093–4108. Hashmi, M.A., Khan, A., Ayub, K., Farooq, U., 2014. Spectroscopic and density functional theory studies of 5,7,3',5'-tetrahydroxyflavanone from the leaves of Olea ferruginea. Spectrochim. Acta Part A 128, 225–230. Khan, M.K., Huma, Z.-E., Dangles, O., 2014. A comprehensive review on flavanones, the major citrus polyphenols. J. Food Compos. Anal. 33, 85–104. Lee, L.K., Gan, V.C., Lee, V.J., Tan, A.S., Leo, Y.S., Lye, D.C., 2012. Clinical relevance and discriminatory value of elevated liver aminotransferase levels for dengue severity. PLoS Negl. Trop. Dis. 6, e1676. Lima-Junior, R.S., Mello, C.S., Siani, A.C., Valente, L.M.M., Kubelka, C.F., 2013. Uncaria tomentosa alkaloidal fraction reduces paracellular permeability, IL-8 and NS1 production on human microvascular endothelial cells infected with dengue virus. Nat. Prod. Commun. 8, 1547–1550. Mabry, M., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, Berlin. Maltese, F., Erkelens, C., Kooy, F., Choi, Y.H., Verpoorte, R., 2009. Identification of natural epimeric flavanone glycosides by NMR spectroscopy. Food Chem. 116, 575–579. Masuoka, C., Ono, M., Yasuyuki, I., Nohara, T., 2003. Antioxidative, antiyaluronidase and antityrosinase activities od some constituents from the aerial part of Piper elongatum Vahl. Food Sci. Technol. Res. 9, 197–201. Moco, S., Tseng, L.-H., Spraul, M., Chen, Z., Vervoort, J., 2006. Building-up a comprehensive database of flavonoids based on Nuclear Magnetic Resonance data. Chromatographia 64, 503–508. Parkash, O., Almas, A., Jafri, S.M., Hamid, S., Akhtar, J., Alishah, H., 2010. Severity of acute hepatitis and its outcome in patients with dengue fever in a tertiary care hospital Karachi, Pakistan (South Asia). BMC Gastroenterol. 7 (10), 43. Reis, S.R., Valente, L.M.M., Sampaio, A.L., Siani, A.C., Gandini, M., Azeredo, E.L., D'Avila, L.A., Mazzei, J.L., Henriques, M., Kubelka, C.F., 2008. Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2. Int. Immunopharmacol. 8, 468–476. Ribeiro, A.F., Almeida, S.S., Andrade, E.H.A., Zoghbi, M.G.B., 2003. A influência de fatores ambientais nos óleos essenciais de Faramea anisocalyx Poepp. & Endl.: O experimento seca floresta, CBO-013 – Estação Científica Ferreira Penna – dez anos de pesquisa na Amazônia. Museu Paraense Emílio Goeldi, Belém, PA. Samsa, M.M., Mondotte, J.A., Iglesias, N.G., Assunção-Miranda, I., Barbosa-Lima, G., Poian, A.T., Bozza, P.T., Gamarnik, A.V., 2009. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 5, e1000632. Sauvain, M., Dedet, J.P., Kunesch, N., Poisson, J., 1994. Isolation of flavans from the Amazonian shrub Faramea guianesis. J. Nat. Prod. 57, 403–406. Suksanpaisan, L., Cabrera-Hernandez, A., Smith, D.R., 2007. Infection of human primary hepatocytes with dengue virus serotype 2. J. Med. Virol. 79, 300–307. Sun, J.-M., Yang, J.-S., Zhang, H., 2007. Two new flavanone glycosides of Jasminum lanceolarium and their anti-oxidant activities. Chem. Pharm. Bull. 55, 474–476. Takahashi, H., Hirata, S., Minami, H., Fukuyama, Y., 2001. Triterpene and flavanone glycoside from Rhododendron simsii. Phytochemistry 56, 875–879. WHO, Updated in 2016. Dengue and severe dengue, Fact sheet 117. Wagner, H., Bladt, S., 1996. 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Short communication
Antiviral activity of Faramea bahiensis leaves on dengue virus type-2 and characterization of a new antiviral flavanone glycoside$ Adriana C. Nascimentoa , Ligia M.M. Valentea,* , Mário Gomesb , Rodolfo S. Barbozaa , Thiago Wolffa , Rômulo L.S. Nerisc, Camila M. Figueiredoc, Iranaia Assunção-Mirandac,* a Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos 149, Centro de Tecnologia, Bl. A, Cidade Universitária, 21941-909, Rio de Janeiro, RJ, Brazil b Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, R. Jardim Botânico 1008, 22470-180, Rio de Janeiro, RJ, Brazil c Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, Centro de Ciências da Saúde, Bl. I, Cidade Universitária, 21941-970, Rio de Janeiro, RJ, Brazil
A R T I C L E I N F O
Article history: Received 29 September 2016 Received in revised form 8 January 2017 Accepted 25 January 2017 Available online xxx Keywords: Faramea bahiensis Rubiaceae Flavanone Anti-dengue Antiviral Dengue virus
A B S T R A C T
Dengue virus infection is a neglected disease prevalent in most tropical and subtropical areas. Currently there is no any antiviral drug indicated for the routine treatment of dengue patients. As part of our ongoing search for potential anti-dengue virus agents we have investigated the methanol extract of Faramea bahiensis (Rubiaceae) leaves. This species is endemic in Brazil, and there are no reports on its chemical composition or therapeutic potential. Its crude MeOH extract showed in vitro non-cytotoxicity and anti-dengue virus serotype 2 (DENV-2) activity in human hepatocarcinoma cell lineage (HepG2). A marked reduction on viral load (100%) was observed. Sequential fractionation of the bioactive crude extract led to the isolation of a bioactive new flavanone glycoside: 5-hydroxy-40 -methoxy-flavanone-7O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside, the known 5,40 -dihydroxy-flavanone-7-O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside and a diateroisomeric epimer pair of the known 5,30 ,50 trihydroxy-flavanone-7-O-ß-D-glucopyranoside. The treatment of DENV-2 infected HepG2 cells with the new flavanone was able to control viral replication promoting a reduction of the number of infected cells (12%), together with a decrease of infectious particles in the culture supernatant (97%) and of the number of RNA copies of DENV-2 in HepG2 cells (67%). Structural determinations were made by NMR techniques in one and two dimensions (1H NMR, 13C NMR, COSY, HSQC and HMBC), HRMS, UV, OR and CD. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Dengue fever is a mosquito-borne viral neglected disease prevalent in most tropical and subtropical areas and its incidence has dramatically increased in the last decades (WHO, Updated in 2016). According to the World Health Organization (WHO), only in the Americas in 2015 2.35 million cases were reported, of which 10,200 were diagnosed as the severe form of the disease (also known as dengue hemorrhagic fever), causing around 1000 deaths (WHO, Updated in 2016). In Brazil, it is placed among one of the most serious public health issues registering about 1.6 million cases in 2015 (Brazilian-Health-Ministry, 2016). The dengue
$
Faramea bahiensis was formerly identified as Faramea marambaiae. * Corresponding authors. E-mail addresses: [email protected] (L.M.M. Valente), [email protected] (I. Assunção-Miranda).
symptoms usually start with high fever that can be accompanied by severe headache, retro-orbital pain, muscle and joint pains, nausea, vomiting, swollen glands or rash. In severe cases, vascular permeability leading to hypotension may evolve to shock and hemorrhagic manifestations may occur as well. Currently there is no drug indicated for the routine treatment of dengue patients. Thus, the discovery of drugs that can exert antiviral action against dengue virus (DENV), inhibiting the virus replication cycle without being toxic to the host cell and thus mitigating the symptoms, is highly desirable. The genus Faramea Aubl. (Rubiaceae) is native, but nonendemic to Brazil and contains species in form of shrubs, subshrubs or trees. Despite the diversity of the species, few reports can be found on their chemical composition and on their pharmacological potential. Previous phytochemical investigations of Faramea species have resulted in the isolation and/or characterization of anthraquinones and naphthopyrans (Ferrari et al., 1985), flavans
http://dx.doi.org/10.1016/j.phytol.2017.01.013 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
A.C. Nascimento et al. / Phytochemistry Letters 19 (2017) 220–225
(Sauvain et al., 1994), methyl salicylate, hydroxyl acetophenone derivatives and phenylpropanoids (Ribeiro et al., 2003). The species Faramea bahiensis Mull. Arg. is endemic in Brazil, and to our knowledge no studies have been reported on it. As part of an ongoing search for potential anti-dengue agents the antiviral and immunomodulatory activities of the species Uncaria tomentosa (Rubiaceae) on human monocytes infected with DENV serotype 2 (DENV-2) have been reported (Lima-Junior et al., 2013; Reis et al., 2008). Clinical and experimental evidences point out that hepatocytes are target for DENV replication and are involved in the pathogenesis during infection (Lee et al., 2012; Parkash et al., 2010; Suksanpaisan et al., 2007). Some hepatocytederived cell lines have been used as targets for in vitro DENV infection models (Assunção-Miranda et al., 2016; Samsa et al., 2009; Suksanpaisan et al., 2007). Continuing our search, the antiDENV activity of extracts from other Rubiaceae species was screened in hepatocarcinoma cell lineage (HepG2), revealing the significant antiviral effect of the MeOH extract of F. bahiensis leaves. In this paper, we report the isolation of a new antiviral flavanone apiofuranoside (1), a known flavanone apiofuranoside (2) and a diasteroisomeric epimer pair of the known flavanone glucoside (3a and 3b) from its bioactive extract. 2. Results and discussion The crude MeOH extract of the leaves of F. bahiensis was assayed for in vitro cytotoxic and anti-DENV-2 effects in HepG2. After 48 h of infection (hpi) DENV-2 replication promoted a reduction of about 15% of HepG2 viability, that was not observed when cells
Fig. 1. In vitro cytotoxicity and cytoprotection effect of: (A) MeOH extract of Faramea bahiensis leaves; (B) flavonoid-rich MeOH/H2O 1:1 fraction. Control: HepG2 cells; DENV: HepG2 cells infected with DENV-2 16681.
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were infected and treated with the F. bahiensis extract (Fig. 1A). Non-cytotoxicity or proliferative effect was evident. In addition, DENV-2 particles were not detected by plaque assay in the culture supernatant after treatment with F. bahiensis extract, representing a 100% reduction in viral load (data not shown). The liquid-liquid partition of the extract yielded a flavonoid-rich MeOH/H2O 1:1 fraction (monitored by TLC) which showed similar behavior in the MTT assays (Fig. 1B). These results indicate that compounds present in the MeOH extract of F. bahiensis leaves and preserved in its derived hydromethanolic fraction have an efficient antiviral activity against DENV-2. Sequential fractionation of the flavonoidrich fraction involved RP-CC and RP-HPLC to obtain the new compound 1 and the known compounds 2-3a/3b (Fig. 2), of which 1 was found to be bioactive. Compound (1) showed molecular formula C27H32O14 (HRESITOF, m/z 579.1727 [MH], C27H31O14 requires 579.1713). The analysis of its 1H NMR spectrum together with the 1H-1H correlations in the COSY spectrum showed an ABX-system [dH 2.69 (1H, dd, J = 17.2 and 2.9 Hz), 3.08 (1H, dd, J = 17.2 and 13.0 Hz) and 5.36 (1H, dd, J = 13.0 and 2.9 Hz)]; protons at dH 4.86 (1H, d, J = 7.4 Hz) and 4.89 (1H, d, J = 2.4 Hz) coupled to a signal set between dH 3.3-4.0 ppm suggesting two sugar units; four protons at dH 7.35 (2H, d, J = 8.7 Hz) and 6.90 (2H, d, J = 8.7 Hz) showing a pdisubstituted aromatic ring; two protons at dH 6.15 brs and a methoxy group at dH 3.74 ppm (Table 1). The 13C NMR spectrum and the 1H-13C correlations in the HSQC spectrum showed, among others, a ketone carbonyl (dC 198.39), four downfield quaternary carbons (dC 161.45-166.89) and a methylene carbon at dC 44.14 (Table 1). The UV maximum absorptions at 282 (log e 4.19) and 328 nm (log e 3.52) which suffered a bathochromic shift to 306 and 383 nm respectively, after treatment with AlCl3 (Mabry et al., 1970), together with the analysis of the long-range 1H-13C correlations in the HMBC spectrum and biogenetic arguments pointed out compound 1 as a 5-hydroxy-flavanone with a pmethoxy substituted B ring in equatorial position (Fig. 3). The correlation in the HMBC spectrum between the anomeric proton at dH 4.86 (1H, d, J = 7.4 Hz) and the carbon at dC 166.89 revealed a
Fig. 2. Chemical structures of compounds 1-3a/3b.
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Table 1 1 H and 13C NMR (500 and 125 MHz respectively) data (d in ppm) of 1 in CD3OD. Position
dH mult.(J in Hz)
dC
2 3eq 3ax 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 1’’ 2’’ 3’’ 4’’ 5’’ 6’’
5.36 dd (13.0, 2.9) 2.69 dd (17.2, 2.9) 3.08 dd (17.3, 13.0) – – 6.15 brs – 6.15 brs –
80.43 44.14
1’’’ 2’’’ 3’’’ 4’’’ 5’’’ OCH3
– 7.35 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 7.35 d (8.7) 4.86 d (7.4) 3.36 m 3.51 m 3.22 dt (3.2, 1.6) 3.35 m 3.51 m 3.90 d (10.8) 4.89 d (2.4) 3.75 d (2.4) – 3.64 d (9.8) 3.88 d (9.8) 3.40 brs (2H) 3.74 s
198.39 164.87 97.05 166.89 98.11 164.44 104.93 132.06 129.08 115.04 161.45 115.04 129.08 101.15 75.13 77.78 71.48 78.19 68.83 111.05 78.21 80.46 75.13 65.94 55.79
glycosylation at C-7 (Fig. 3). The site of glycosylation was confirmed by the observed downfield effect on the H-6 and H-8 chemical shifts when compared with 7-OH flavanones (Hashmi et al., 2014; Moco et al., 2006). The complete analyses of the NMR spectra and the comparison with previously reported flavanone glycoside data (Takahashi et al., 2001; Zhang et al., 2003), allowed identifying (1) as a flavanone with an 7-O-b-D-apiose-(1 ! 6)-b-Dglucose sugar unit (Table 1,Fig. 3). The absolute stereochemistry on C-2 was derived from the analyses of its OR and CD data when compared to similar data previously described for naringin (Gaffield, 1970). The OR datum ([a] = 88) and CD spectrum profile that showed a positive Cotton effect at 336 nm (u = +741) (n ! p*) and negative at 287 nm (u = 11,481) (p ! p*) (Fig. 4), led to establish 1 as the new flavanone 2S-5-hydroxy-40 -methoxyflavanone-7-O-ß-D-apiofuranosyl-(1 ! 6)-ß-D-glucopyranoside. Compound (2) revealed NMR spectra very similar to those of 1 without the signal corresponding to the methoxy group and with some differences in the chemical shifts of the B ring aromatic
Fig. 3. Main 1H-1H correlations in the COSY, 1H-13C in the HSQC and 1H-13C longdistance in the HMBC spectra of 1. (Color figure available online only).
Fig. 4. Circular dichroism spectra of compounds 1 and 3a/3b (in MeOH). (Color figure available online only).
protons. The UV maximum absorptions at 283 (log e 4.2) and 328 (log e 3.7) nm that suffered a similar bathochromic shift after treatment with AlCl3 together with the analysis of the long-range 1 H-13C correlations in the HMBC spectrum showed 2 also as a 5hydroxy-7-O-glycosylflavanone with a p-hydroxy substituted B ring in equatorial position. The complete analyses of its NMR spectra showed that the structure of 2 was similar to that of the previously described Pyrroside B (Masuoka et al., 2003). The OR datum ([a] = 88) allowed suggesting the absolute stereochemistry on C-2 as 2S as revealed for 1. Compound (3) showed molecular formula C21H22O11 (HRESITOF, m/z 449.1088 [MH], C21H21O11 requires 449.1083) with a fragment ion at m/z 287.0563 corresponding to [M-H-162.0526] suggesting the loss of a glycopiranosyl unit. The 1H NMR spectrum and the 1H-1H and 1H-13C correlations in the COSY and HSQC spectra respectively showed some similarities with those of compounds 1 and 2. However, some signals appeared duplicated (with the same intensity) and/or with some differences in their multiplicities, such as the signals corresponding to the C ring of flavanones: dH 2.75 (dt, J = 17.2 and 3.3 Hz), dH 3.12 (dd, J = 17.2 and 12.7 Hz)/dC 44.18/44.34 and dH 5.32 (dt, J = 12.6 and 2.9 Hz)/dC 80.64/80.81. The long-range 1H-13C correlations in the HMBC spectrum between the anomeric proton signals at dH 4.97/4.98 and the carbon signals at dC 167.09/167.16 in addition to the chemical shift values for H-8 and H-6 at dH 6.18 (d, J = 2.0 Hz) and 6.21 (d, J = 2.0 Hz) respectively, indicated a glycosylation at C-7. On the other hand, there is no correlation between these anomeric proton signals with any carbon at the B ring which could indicate a mixture of different compounds. The UV spectra (before and after the AlCl3 treatment) and the detailed analysis of the NMR spectra allowed suggesting 3 as a 5,30 ,50 -trihydroxy-flavanone-7-O-glucopyranoside, with the B ring in equatorial position (Chen et al., 2009; Sun et al., 2007). The biosynthesis of flavanones involves an intramolecular Michael-type nucleophilic cyclization of chalcones mediated by the enzyme chalcone isomerase producing almost exclusively the (2S)-isomer although it may potentially lead to the existence of two stereoisomeric forms. Subsequent enzymatic glycosylation steps, lead to flavanone glycosides. The possible resulting changes of configuration at their stereogenic centers determine that flavanone glycosides may exist as mixtures of diastereoisomers (Maltese et al., 2009). In fact, mixtures of (2R)and (2S)- flavanone glycosides have been detected in natural matrices such as for naringin and hesperidin in mature citrus fruits and for two isomeric tetrahydroxyflavanone-7-O-b-D-glucopyranoside in the aerial parts of Lippia salviaefolia (Funari et al., 2011;
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Gaffield et al., 1975; Khan et al., 2014; Maltese et al., 2009). The process probably occurs via C ring opening. The intermediate chalcones thus formed would rapidly recyclize due to their instability, in a nonstereospecific way, leading to the corresponding 2S and 2R flavanones (Gaffield et al., 1975; Maltese et al., 2009). The CD spectrum profile with a negative Cotton effect at 334 nm (u = 800) (n ! p*) and negative at 286 nm (u = 3.133) (p ! p*) (Fig. 4) together with the NMR evidences indicated 3 as a diasteroisomeric epimer pair, 2R + 2S of the known flavanone 5,30 ,50 -trihydroxy-flavanone-7-O-ß-D-glucopyranoside. Compound 1 showed non-cytotoxicity and a cytoprotective action without proliferative effect when assayed in the DENVinfected HepG2 cells at different doses (Fig. 5A). Moreover, it was possible to demonstrate a dose-dependent reduction in viral load in the culture supernatant (Fig. 5B). The dose of 1 required to inhibit 50% of viral replication (IC50) was 13.1 mg/mL. The dose of 50 mg/mL was chosen to evaluate the effect of 1 in the progression of DENV-2 infection in HepG2 by presenting the best doseresponse relationship. The treatment of infected HepG2 with 1 promoted a reduction in the number of infected cells of approximately 12% when compared with untreated HepG2 (Fig. 6A). Quantification of DENV-2 particles in the supernatant by plaque assay demonstrated that treatment with 1 reduced by approximately 97% the number of infectious particles in the culture supernatant when compared with untreated condition (Fig. 6B). Moreover, quantifying the number of RNA copies of DENV-2 by real time PCR in HepG2 extract showed that after treatment with 1 the copy number of DENV RNA was reduced by about 67% (Fig. 6C). Taken together, these results indicate that 1 is able to control DENV-2 replication. To confirm that this effect was not associated with a virucidal activity of 1 against DENV-2, a preincubation of DENV-2 with 1 at 37 C for 1 h was also performed and the infectious particles remained after incubation were quantified by plaque assay. No difference in viral title was observed after incubation (Fig. 6D). This contributed to demonstrate the ability of 1 to control DENV-2 replication in some step of its replication in target cells. In this study, we report for the first time the isolation of flavanones in a Faramea species and reveal the potential anti-
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Fig. 6. HepG2 infected with DENV2 at low MOI (0.5) and treated or not with 1. (A) % of infected HepG2 assessed by flow cytometry 48 hpi; (B) viral load in the supernatant determined by plaque assay; (C) quantification of number of copies of DENV RNA obtained by real time-PCR analysis; (D) virucidal activity by incubating DENV with 1 or vehicle for 1 h at 37 C. Results are expressed as the mean S.E.M. of three independent experiments. *P < 0.05.
dengue activity of the MeOH extract of F. bahiensis leaves. In addition, the new flavanone apiofuranoside (1) isolated from this bioactive extract showed to be effective to protect HepG2 cells from DENV-2 infection and to control DENV-2 replication. 3. Materials and methods 3.1. General experimental procedures The HPLC analyses were performed using a Perkin-Elmer Series 200 equipment with quaternary pump, autosampler and diodearray detector. The NMR spectra were recorded in CD3OD (Cambridge Isotope Laboratories Inc.) on Bruker DRX 400 or on Varian System 500 spectrometers using the solvent as internal standard. UV spectra were performed on a Varian Cary 1E (UVVIS) spectrophotometer. Optical rotations (OR) were obtained on a Perkin-Elmer 341 LC polarimeter. Circular dichroism (CD) spectra were carried out on a Jasco J-715 spectropolarimeter. HRESITOF analyses were made with a Bruker Daltonics micrOTOF instrument. RP-C18 (40–63 mm Merck) (4.0 cm e.d. 15.0 cm phase) was used for column chromatography (CC). The TLC analyses were performed in pre-coated silica gel 60 F254 (Merck). MeOH, CH3CN (both HPLC grade), CHCl3, EtOAc, EtOH, and Hexane (all p.a., ACS grade) were purchase from Tedia. Milli-Q grade water was used for HPLC. 3.2. Plant material
Fig. 5. Dose-response curve of compound 1 (cpd1) in DENV-infected HepG2 cells. HepG2 cells were infected or not with DENV2, treated with indicated concentrations of compound 1 and analyzed 48 h post infection. (A) cell viability determined by MTT assay; (B) viral load in cell culture determined by plaque assay. DENV: HepG2 cells infected with DENV-2 16681. Result of at least three independent experiments.
The species Faramea bahiensis was collected at the Restinga de Marambaia, Rio de Janeiro, Brazil in March 2010, and a voucher specimen was deposited at the Herbarium of the Instituto de Biologia of the Universidade Federal do Rio de Janeiro, RJ, Brazil, under number RFA 37489. The collection had previous permission from SISBIO-ICMBio-MMA-Brazil under number 46504-2.
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3.3. Extraction and isolation The leaves were dried at 40 C for 24 h and the dried and sieved ( 335 mm) material (151.3 g) was exhaustively and sonically extracted with MeOH. The MeOH was removed under low pressure to yield 7.8 g of crude extract. Part of the crude MeOH extract (6.36 g) was dissolved in 500 mL of MeOH/H2O 9:1 and partitioned with n-hexane (500 mL 24). Next, more H2O was added (400 mL) to the hydromethanolic fraction until MeOH/H2O 1:1 and then partitioned with CHCl3 (500 mL 3) yielding 1.35 g, 0.58 g and 3.90 g of the hexane, CHCl3 and MeOH/H2O fractions respectively. The fractions were submitted to TLC in different mobile phases and stain reagent systems aiming at screening for the presence of terpenoids, alkaloids and flavonoids, classes of natural products usually found in Rubiaceae spp. The tests showed the presence of flavonoids in the MeOH/H2O 1:1 fraction by using EtOAc/HCOOH/ AcOH/H2O 100:11:11:27 followed by derivatization with 1% NP reagent in MeOH/UV irradiation at 365 nm (Wagner and Bladt, 1996). Part of the MeOH/H2O fraction (3.0 g) was submitted to RPCC using a solvent gradient from MeOH/H2O 3:7 ! 7:3, MeOH, EtOH, EtOAc and CHCl3 yielding 31 fractions that were pooled together by TLC similarities. Some of the yielded fractions were then submitted to RP-HPLC-DAD in analytical and semi-preparative scales. Compound (1) (10.8 mg) was isolated from sub-fraction 14 (67.8 mg, eluted with MeOH) by semi-Prep HPLC using a LiChrocart LiChrospher RP-18 column (250 6.4 mm, 5 mm) (Merck) and as mobile phases: CH3CN as solvent A and H2O with 0.1% HCOOH, pH 3 as solvent B. Elutions were performed in a linear gradient: 22% of A for 15 min, 22–25% of A for 30 min, 25% of A, for 3 min, then 95% of B for 12 min. The flow rate was 1.7 mL/min, the column temperature 40 C and the injection volume 120 mL at c = 25 mg/mL. Compounds (2) (14.2 mg) and (3) (36.1 mg) were isolated from sub-fractions 8–10 (303.4 mg eluted with MeOH/H2O 4:6) by semi-Prep HPLC using a Zorbax Eclipse XDB C18 column (250 9.4 mm; 5 mm) (Agilent) and as mobile phases: CH3CN as solvent A and H2O with 0.1% HCOOH, pH 3 as solvent B. Elutions were performed in a linear gradient: 5–25% of A for 35 min, 25–95% of A for 5 min, then 95% of B for 10 min. The flow rate was 2.5 mL/ min, the column temperature 40 C and the injection volume 100 mL at c = 100 mg/mL. In both cases the diode-array detector was set at an acquisition range of 190–400 nm and flavonoid monitoring was performed at 280 and 320 nm.
77.99 (C-3’’), 71.67 (C-4’’), 77.29 (C-5’’), 68.97 (C-6’’), 111.25 (C-1’’’), 78.35 (C-2’’’), 80.62 (C-3’’’), 75.30 (C-4’’’), 66.10 (C-5’’’). 3.3.3. Compound (3a/3b) oil: [a]D (20 C) 40,0 (c = 0.203, MeOH); UV lMAX nm (log e): 288 (4.07), 330 (3.35), lMAX + AlCl3: 303, 384, lMAX + AlCl3 + HCl: 304, 380; CD (c = 400 mM, MeOH) Cotton effect at 334 nm (u = 800) (n ! p*) and at 287 nm (u = 3.133) (p ! p*); HRESITOF m/z 449.1088 [MH] (C21H21O11 requires 449.1083); 1H NMR (400 MHz in CD3OD, d in ppm): dH 5.32 (1H, dt, J = 12.6, 2.9 Hz, H2), 2.75 (1H, dt, J = 17.2, 3.3 Hz, H-3eq), 3.12 (1H, dd, J = 17.2, 12.7 Hz, H-3ax), 6.21 (1H, d, J = 2.0 Hz, H-6), 6.18 (1H, d, J = 2.0 Hz, H-8), 6.79 (2H, brs, H-20 and H-60 ), 6.92 (1H, brs, H-40 ), 4.97/4.98 (d, J = 7.3 Hz, H-1’’), 3.46 (1H, m, H-2’’), 3.45 (1H, m, H-3’’), 3.40 (1H, m, H-4’’), 3.46 (1H, m, H-5’’), 3.69 (1H, ddd, J = 13.8, 5.0, 2.2 Hz, H-6a”), 3.88 (1H, dt, J = 13.8, 1.7 Hz, H-6b”); 13C NMR (100.57 MHz in CD3OD, d in ppm): dC 80.64/80.81 (C-2), 44.18/44.34 (C-3), 198.67 (C-4), 165.04/ 165.08 (C-5), 97.10/97.18 (C-6), 167.09/167.16 (C-7), 98.14 (C-8), 164.70 (C-9), 105.08/105.10 (C-10), 131.65/131.68 (C-1’), 116.42 (C2’), 147.10 (C-3’), 119.48/119.50 (C-4’), 146.66 (C-5’), 114.87/114.94 (C-6’), 101.38/101.40 (C-1’’), 74.80 (C-2’’), 77.96 (C-3’’), 71.31 (C-4’’), 78.40 (C-5’’), 62.50 (C-6’’). 3.4. HepG2 infection and treatment Human hepatocarcinoma cell lineage (HepG2) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (LGC Biotecnologia) supplemented with 10% fetal bovine serum (FBS), at 37 C, in an atmosphere of 5% CO2. HepG2 cells were infected with DENV-2 (strain 16681) in M.O.I. of 1 or 0.5 for 1 h at 37 C in 5% CO2. After infection, the medium was replaced by fresh medium (DMEM with 5% FBS) with or without 50 mg/mL (in DMSO) of the MeOH extract of F. bahiensis leaves, the hydromethanolic fraction or different doses of 1 (as indicated in Fig. 5), and cultured at 37 C in 5% CO2. The samples (stock 100 mg/mL) were added to the medium (DMEM with 5% FBS) to obtain the desired concentration. The final concentration of DMSO in HepG2 culture was 0.05%, which was also added to the infected and untreated condition. After 48 h of infection, the culture medium was collected for virus titration. Cellular extracts were used to determine cell viability, real-time PCR and for flow cytometry assays (as described below). 3.5. Cell viability assay
3.3.1. Compound (1) oil; [a]D (20 C) 88 (c = 1, MeOH); UV lMAX nm (log e): 282 (4.19), 328 (3.52), lMAX + AlCl3: 306, 383, lMAX + AlCl3 + HCl: 305, 380; CD (c = 400 mM, MeOH) Cotton effect at 336 nm (u = +741) (n ! p*) and negative at 287 nm (u = 11,481) (p ! p*); HRESITOF m/z 579.1727 [MH] (C27H31O14 requires 579.1713); 1H and 13C NMR (500 and 125 MHz respectively in CD3OD) data, see Table 1. 3.3.2. Compound (2) oil; [a]D (20 C) 88 (c = 1, MeOH); UV lMAX nm (log e): 283 (4.2), 328 (3.7), lMAX + AlCl3: 306, 382, lMAX + AlCl3 + HCl: 307, 383; 1 H NMR (400 MHz in CD3OD, d in ppm): dH 5.42 (1H, dt, J = 12.8, 2.7 Hz, H-2), 2.76 (1H, dt, J = 17.2, 2.7 Hz, H-3eq), 3.18 (1H, dd, J = 17.2, 12.8 Hz, H-3ax), 6.22 (2H, brs, H-6 and H-8), 6.84 (2H, d, J = 8.4 Hz, H-3’and H-50 ), 7.35 (2H, d, J = 8.4 Hz, H-2’ and H-6’), 4.94 (1H, d, J = 7.2 Hz, H-1’’), 3.46 (2H, m, H-2’’ and H-3’’), 3.35 (1H, m, H-4’’, 3.63 (1H, m, H-500 ), 3.63 (1H, m, H-6a”), 4.02 (1H, m, H-6b”), 4.96 (1H, d, J = 2.4 Hz, H-1’’’), 3.87 (1H, d, J = 2.4 Hz, H-200 ’), 3.75 (1H, d, J = 9.7 Hz, H-4a”’), 4.00 (1H, d, J = 9.7 Hz, H-4b”’), 3.52 (2H, s, H-5’’’); 13 C NMR (100.57 MHz in CD3OD, d in ppm): dC 80.82 (C-2), 44.32 (C-3), 198.43 (C-4), 165.09 (C-5), 97.19/98.21 (C-6 or C-8), 167.10 (C7), 164.72 (C-9), 105.11 (C-10), 131.05 (C-1’), 129.32 (C-2’ and C-6’), 116.56 (C-3’ and C-5’), 159.25 (C-4’), 101.34 (C-1’’), 74.79 (C-2’’),
The effect of the MeOH extract of F. bahiensis leaves, hydromethanolic fraction or 1 in infected HepG2 cell viability was determined by measuring the metabolization of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT metabolization assay) by the cells. Cells seeded in a 24-well plate were infected with DENV-2 and treated as previously described. Cytotoxicity and/or proliferative effects were assessed treating uninfected HepG2 cells in the same conditions. Forty-eight hpi cells were washed with balanced salt solution (BSS) prior to the addition of 500 mL of 0.5 mg/mL MTT (Sigma-Aldrich Co.) in BSS to each well. After 1 h, MTT solution was discarded and the formazan crystals formed were solubilized in each well using 500 mL of 0.04 M HCl solution in i-PrOH. The optical density (OD) of the samples was read at 570 nm and 650 nm for background correction. 3.6. Viral quantification The virus titer in the culture medium of infected HepG2 cells was quantified by plaque assay in Baby Hamster Kidney cells (BHK21 cells). Briefly, BHK-21 cells were grown in Minimum Essential Medium (MEM) a (Invitrogen) supplemented with 10% FSB and
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seeded in 24-well plates and cultured overnight at 37 C with 5% CO2. Ten-fold serial dilutions of the samples were performed using a-MEM and used to infect BHK-21 cells at 37 C for 1 h. After this period, 1% carboxymethyl cellulose in a-MEM with 2% FBS was added and the cells were kept in culture at 37 C with 5% CO2 for five days. Then, the cells were fixed with formaldehyde 4% and the plaque was visualized by staining with crystal violet (1% crystal violet powder (w/v), 20% MeOH and H2O). To determine the dose required to inhibit 50% of viral replication (IC50), viral title after treatment with different doses of 1 (0, 1, 5, 10, 25, 50, 100 and 200 mg/mL) was used to obtain a non-linear regression analysis with a sigmoidal and variable slope profile using the software GraphPad Prism 6 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. For virucidal analysis, 106 plaque forming unit (PFU) of DENV-2 was incubated with 50 mg/mL of 1 in DMSO or only with the same proportion of DMSO for 1 h at 37 C. Infectious particles were then quantified by plaque assay. 3.7. Quantification of DENV2 infected cells by flow cytometry Flow cytometry analysis was performed for the quantification of DENV-2 infected cells by detecting intracellular viral antigens. After 48 h of infection, cells were washed with PBS, harvested and fixed in 4% formaldehyde in PBS at room temperature for 15 min. After washing, the cells were permeabilized with 0.1% saponin in PBS and incubated with blocking solution (PBS supplemented with 2% FBS and 0.1% bovine serum albumin) for 30 min, at room temperature. Then, the cells were incubated for 1 h with antiDengue Virus Complex, clone D3-2H2-9-21 antibody (1:500, Millipore) in 0.1% BSA in PBS. The cells were washed and incubated with Alexa Fluor1 488 Goat Anti-Mouse IgG (H + L) antibody (1:1000, Invitrogen) in 0.1% BSA in PBS for 30 min. The percentage of infected cells was analyzed by FACScan Flow Cytometer and CellQuest software (Bectan Dickinson). 3.8. Quantification of viral RNA by real time PCR analysis Total RNA from HepG2 cells was isolated using Trizol1 (Invitrogen) 48 hpi after treatment of culture with (1). 4 mg of total RNA were reverse-transcribed using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and the amount of DENV RNA of each sample was determined by real-time PCR analysis using TaqMan reagents (Applied Biosystems, Foster City, CA, USA). Primers, probe and DENV RNA quantification method was performed as previously described (Lima-Junior et al., 2013). 3.9. Statistical analysis Statistical calculations were carried out with the GraphPad Prism 5 software. Results are expressed as the mean S.E.M. of 3 independent experiments. Student’s t-test was used for statistical analyses; P values >0.05 were considered to be significant. Acknowledgments The authors thank the NMR Labs of the Instituto de Tecnologia em Fármacos Fundação Oswaldo Cruz/RJ and of the Instituto de Pesquisa em Produtos Naturais, Universidade Federal do Rio de Janeiro for the NMR spectra, the MS Lab of the Instituto de Pesquisa em Produtos Naturais for the HRESIMS and FAPERJ-Brazil for grant support (Proc. No E-26/111.373/2014). A.C.N. thanks CNPq-Brazil and T.W. CAPES-Brazil for fellowships.
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