Antifungal Phenylpropanoid Glycosides from Lippia rubella - Journal

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Antifungal Phenylpropanoid Glycosides from Lippia rubella Gabriel R. Martins,† Thamirys Silva da Fonseca,† Lucero Martínez-Fructuoso,‡ Rosineide Costa Simas,† Fabio T. Silva,† Fatima Regina G. Salimena,§ Daniela S. Alviano,⊥ Celuta Sales Alviano,⊥ Gilda Guimarães Leitão,∥ Rogelio Pereda-Miranda,‡ and Suzana G. Leitão*,†

J. Nat. Prod. Downloaded from pubs.acs.org by WEBSTER UNIV on 02/28/19. For personal use only.

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Faculdade de Farmácia, Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil ‡ Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, 04510 DF, Mexico § ICB, Depto de Botânica, Universidade Federal de Juiz de Fora, 84030-900, Juiz de Fora, MG, Brazil ⊥ Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil ∥ Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil S Supporting Information *

ABSTRACT: Lippia species share various pharmacological activities and are used in traditional cooking and medicine worldwide. Combined chromatographic techniques such as column chromatography, high-performance liquid chromatography, and countercurrent chromatography led to the purification of two new antifungal phenylpropanoid glycosides, lippiarubelloside A (1) and lippiarubelloside B (2), by bioactivity-directed fractionation of an ethanol-soluble extract from Lippia rubella, in addition to the known active related compounds forsythoside A (3), verbascoside (4), isoverbascoside (5), and poliumoside (6). The structures of compounds 1 and 2 were determined by comparison of their NMR spectroscopic data with the prototype active compound 4. Cryptococcus neoformans, which causes opportunistic lung infections, was sensitive to compounds 1−6 in the concentration range of 15−125 μg/mL. A synergistic effect (FICindex = 0.5) between 3 and amphotericin B was demonstrated. The glycosylated flavonoids pectolinarin (7), linarin (8), and siparunoside (9) were also isolated.

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potential of Lippia species,7 the present investigation describes the isolation of phenylpropanoid glycosides with antifungal activity, by bioactivity-directed fractionation of the ethanolsoluble extract of L. rubella against Cryptococcus neoformans, which causes cryptococcocis, an invasive life-threatening opportunistic infection of the lungs in HIV/AIDS patients and organ transplant recipients.8

he genus Lippia (Verbenaceae) comprises ca. 150 species occurring in the tropical and subtropical Americas and Africa. Brazil is one of the Lippia diversity centers, with 98 species occurring with high endemism.1 Members of this genus display various pharmacological activities and are used widely in traditional medicine and as spices in regional gastronomy worldwide.2 The most significant nonvolatile metabolites in the genus are iridoids, phenylpropanoids, naphthoquinones, and flavonoids.3 From the botanical point of view, the related genera Lippia and Lantana are included in the same tribe (Lantaneae Endl.) due to their morphological similarities. After a taxonomic revision of the Lantana species distributed in Brazil, L. rubella Moldenke was excluded from the genus Lantana based on the fruit morphology and transferred to the genus Lippia.4 Lippia rubella (Moldenke) T. Silva & Salimena has been surveyed for its antibacterial, antifungal, antioxidant, and photoprotective activities.5 However, there is only one report on the volatiles from the hexane-soluble fraction of this plant, which were analyzed by GC-MS.6 Further chemical investigation of this species should contribute to uncovering the structural diversity of bioactive metabolites from this group of economically important plants. Thus, motivated by the known antifungal © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The crude ethanol extract was initially screened for its antifungal activity against opportunistic human yeast pathogens (Table 1), showing activity against C. neoformans (MIC 39 μg/mL). Species of the genera Candida and Cryptococcus are responsible for more than 60% of the reported nosocomial fungal infections. However, Cryptococcus is one of the most common causes of meningitis among persons living with HIV/ AIDS, being encountered more frequently than Streptococcus Special Issue: Special Issue in Honor of Drs. Rachel Mata and Barbara Timmermann Received: November 20, 2018

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DOI: 10.1021/acs.jnatprod.8b00975 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. Antifungal Activity of Extracts and Isolated Compounds from L. rubellaa

sample total EtOH extract hexane extract CH2Cl2 extract EtOAc extract n-butanol extract active fraction (F9−F14) 1 2 3 4 5 6 7 8 amphotericin B

Candida albicans (ATCC 10231)

Candida parasilopsis (ATCC 22019)

Cryptococcus neoformans T1444

Cryptococcus neoformans 24067

>1250

>1250

39

b

39 78 19.5 1250 62.5

b

b

b

1250

312.5

b

b

b

b

>125

62.5

>125 >125 >125 >125

125 >125 >125 125

b

b

>125 >125 >125 0.250

>125 >125 >125 0.125

15.6 125 31.25 62.5 >125 62.5 >125 125 0.125

b b b

62.5 b b

31.25 62.5 b b b b

0.125

Minimum inhibitory concentrations (MIC) in μg/mL. bNot tested.

a

pneumoniae or Neisseria meningitides.8c Thus, the results of this preliminary screening procedure were the starting point to explore the potential as antifungal agents of the isolated phenylpropanoid glucosides from L. rubella, since the therapeutic options for the treatment of cryptococcocis are extremely limited. All of the organic solvent-soluble fractions of L. rubella obtained by liquid−liquid partition with water were further assayed against this microorganism. The ethyl acetate-soluble fraction was found to concentrate the activity (MIC 19.5 μg/ mL) and so was selected for further fractionation. Combined chromatographic techniques including silica gel or Sephadex CC, HPLC, and high-speed countercurrent chromatography (HSCCC) allowed the antifungal activity to be traced to a mixture of phenylpropanoid glycosides. Two new dicaffeoyl phenylpropanoid glycosides, which were given the trivial names lippiarubellosides A (1, MIC 15.6 μg/mL) and B (2, MIC 125 μg/mL), were isolated in addition to the active mixture (F9−F14, MIC 62.5 μg/mL) of forsythoside A (3, MIC 31.25 μg/mL), verbascoside (4, MIC 62.5 μg/mL), isoverbascoside (5, MIC >125 μg/mL), and poliumoside (6, MIC 62.5 μg/mL). The known and inactive flavonoids pectolinarin (7), linarin (8), and siparunoside (9) were also identified. Compounds 1 and 2 showed [M − H]− ions at m/z 785.22736 (mass accuracy −3.1 ppm) and 769.23248 (mass accuracy −3.2 ppm), respectively, accounting for a difference of one oxygen (16 amu) in their molecular formulas (C38H42O18 and C38H42O17, respectively). The difference of 162 Da in relation to verbascoside (4), as well as the presence in the 1H NMR spectrum of two sets of α,β-unsaturated carbonyl groups at δ 5.84−6.28 (d, J = 16.0 Hz, 2H) and δ 7.37−7.65 (d, J = 16.0 Hz, 2H), suggested the presence of an additional caffeoyl group in 1 (Figure S3, Supporting Information). In compound 1, nine aromatic protons from three aromatic rings were identified, and all of these presented typical HH coupling constants for AMX systems of 1,2,4trisubstituted benzene rings. For example, the aromatic signals

for the caffeoyl group esterifying rhamnose H-4″ were observed as a doublet at δ 6.65 (3JAM = 8.2 Hz) for H-8‴′, a doublet of doublets at δ 6.93 (3,4JAM,MX = 8.2, 2.1 Hz) for H9‴′, and a second doublet at δ 7.06 (4JMX = 2.1 Hz) for H-5‴′. The signals for the aromatic ring of the phenylethyl alcohol moiety were assigned as follows: a doublet of doublets at δ 6.57 (3,4JAM,MX = 8.2, 2.1 Hz) for H-6, a doublet at δ 6.68 (3JAM = 8.2 Hz) for H-5, and doublet at δ 6.70 (4JMX = 2.1 Hz) for H-2. Finally, three signals were part of the second caffeoyl moiety at δ 7.31 (dd, 3,4JAM,MX = 7.0, 2.8 Hz) for H-9‴ and one broad signal for overlapped H-5‴ and H-8‴ at δ 7.38 (Figure S3, Supporting Information). For compound 2, the difference of 146 Da when compared to compound 4 indicated the presence of a coumaroyl residue in addition to the caffeoyl unit also present in 1. The A2X2 system, readily recognizable from its symmetry, helped identify the p-disubstituted benzene ring of the coumaroyl moiety in 2. These signals were recognized through the two doublet signals centered at δ 7.09 and 6.69 (J5‴/9″′,6′′′/8′′′ = 8.60), with each one integrating for two protons (Figure S4, Supporting Information). The 1H and 13C NMR data of these new phenylpropanoids were closely related to those of 6′-caffeoyl-verbascoside, previously isolated from Pedicularis kansuensis, a member of the family Scrophulariaceae,9 which also displays an extra caffeoyl group, attached to glucose C-6. In the 13C NMR spectra of 1 and 2 (Table 2), 12 carbons were clearly identified that corresponded to the disaccharide core formed by B



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Table 2. 1H and 13C NMR (500 and 125 MHz, respectively, in MeOH-d4, δ in ppm, J in Hz) Spectroscopic Data of Lippiarubellosides A (1) and B (2) 1 moieties phenylethyl alcohol

position 1 2 3 4 5 6 α β

glucose

rhamnose

coumaroyl

caffeoyl

caffeoyl

1′ 2′ 3′ 4′ 5′ 6a′ 6b′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 7⁗ 8⁗ 9⁗

2

1

13

H

C

6.70 (1H, d, 1.9)

6.68 (1H, d, 8.0) 6.57 (1H, dd, 8.0, 1.9) 2.81 (1H, ddd, 14.0, 9.0, 7.0) 2.79 (1H, ddd, 14.0, 9.0, 7.0) 3.62 (1H, ddd, 10.0, 9.0, 7.0) 3.95 (1H, ddd, 10.0, 9.0, 7.0) 4.39 (1H d, 7.9) 3.46 (1H dd, 9.2, 7.9) 3.93 (1H dd, 9.2) 4.99 (1H dd, 9.9, 9.9) 3.57 (1H ddd, 9.9, 5.7, 1.8) 3.64 (1H dd, 10.0, 1.8) 3.54 (1H dd, 10.0, 5.7) 5.46 (1H d, 1.7) 3.93 (1H dd, 3.3, 1.7) 3.77 (1H dd, 9.3, 3.3) 4.99 (1H dd, 9.3, 9.3) 3.88 (1H dd, 9.3, 6.3) 1.11 (1H d, 6.3)

131.4 117.1 144.6 146.1 116.3 121.2 36.5 72.3 104.2 76.6 72.4 70.3 75.9 62.3 101.5 75.6 70.2 75.5 68.0 18.1

1

13

H

C

6.72 (1H, d, 1.9)

6.69 (1H, d, 8.0) 6.58 (1H, dd, 8.0, 1.9) 2.82 (1H, ddd, 14.0, 8.5, 5.8) 2.80 (1H, ddd, 14.0, 8.5, 5.8) 3.75 (1H, ddd, 10.0, 8.5, 5.8) 4.06 (1H, ddd, 10.0, 8.5, 5.8) 4.41 (1H d, 7.9) 3.47 (1H dd, 9.2, 7.9) 3.93 (1H dd, 9.2) 4.98 (1H dd, 9.9, 9.9) 3.58 (1H ddd, 9.9, 5.7, 1.8) 3.66 (1H dd, 11.1, 1.8) 3.56 (1H dd, 11.1, 5.7) 5.46 (1H d, 1.7) 3.95 (1H dd, 3.3, 1.7) 4.99 (1H dd, 9.3, 3.3) 3.77 (1H dd, 9.3, 9.3) 3.87 (1H dd, 9.3, 6.3) 1.11 (1H d, 6.3) 5.95 (1H d, 15.9) 7.49 (1H d, 15.9) 6.81 (2H d, 8.7) 7.21 (2H d, 8.7) 7.21 (2H d, 8.7) 6.81 (2H d, 8.7)

168.4 118.3 146.7 135.4 130.0 148.3 146.7 131.4 129.2 168.2 114.3 148.3 127.4 114.9 147.0 150.0 116.6 123.2

6.12 (1H d, 16.0) 7.54 (1H d, 16.0) 7.38 (1H d, 2.8)

7.38 (1H, d, 7.0) 7.31 (1H dd, 7.0, 2.8) 6.29 (1H d, 16.0) 7.65 (1H d, 16.0) 7.06 (1H d, 2.1)

6.65 (1H d, 8.3) 6.93 (1H, dd 8.3, 2.1)

6.30 (1H d, 15.9) 7.66 (1H d, 15.9) 7.07 (1H d, 2.1)

6.69 (1H d, 8.2) 6.94 (1H dd 8.2, 2.1)

131.1 117.1 144.6 146.1 116.5 121.2 36.2 72.3 104.1 76.6 78.8 75.2 75.9 62.3 101.5 72.3 70.3 70.2 67.9 18.1 169.1 114.7 146.9 127.0 116.8 131.2 161.1 131.2 116.8

168.1 114.3 148.2 127.4 115.0 146.9 150.0 116.3 123.2

the main core of 1 was comparable to that of 4 through the observed 3JCH cross-peaks for C-1‴ of one caffeoyl moiety (δ 168.4) with glucose H-4′ (δ 4.99) and the CH2-β of the phenylethanoid moiety (δ 72.3) with glucose H-1′ (δ 4.39). In compound 1, the second caffeoyl group is attached to C-4″ of the rhamnose moiety (Figure S13, Supporting Information), as confirmed by the HMBC correlation of C-1⁗ of the second

glucopyranosyl and rhamnopyranosyl units with a Rha-1→Glu3 linkage, as confirmed by the observed HMBC correlation (3JCH) of C-3′ of glucose with H-1″ of rhamnose in both compounds. The measured chemical shift values for glucose C6 at δ 62.3 in both compounds 1 and 2 indicated that this position was not substituted, as described for 6′-caffeoylverbascoside (δ 60.9).9 HMBC experiments established that C



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were obtained for C. neoformans with MIC values ranging from 15.6 to 125 μg/mL. Of the eight compounds tested, two were inactive: the phenylpropanoid glucoside isoverbascoside (5) and the flavonoid pectolinarin (7), with MIC values higher than 125 μg/mL, while linarin (8) and lippiarubelloside B (2) showed moderate antifungal activity against C. neoformans (MIC 125 μg/mL). Among the other phenylpropanoid glucosides, lippiarubelloside A (1, MIC 15.6 μg/mL) was the most potent compound and was responsible for most of the activity displayed by the ethyl acetate-soluble extract (MIC 19.5 μg/mL), followed by forsythoside A (3, MIC 31.25 μg/ mL). Their antifungal activities closely resembled that of magnoloside A, a phenylpropanoid glucoside found to act specifically on the calcineurin pathway of C. neoformans.14 These results showed that the flavonoids investigated do not contribute greatly to the antifungal activity of the EtOAcsoluble extract. Also noteworthy is the fact that the substantial difference in the antifungal activity between lippiarubellosides A (1) and B (2), with a range of 102 in potency, indicates that the transposition of the esterification from C-4″ to C-3″ in the rhamnose moiety had an effect on the antifungal activity. The presence of a catechol group in the second esterifying moiety instead of a phenol also influenced the resultant antifungal activity. In a previously reported investigation on the antifungal activity of six Lippia species,7 verbascoside and asebogenin represented the most active compounds against C. neoformans (90012) growth, showing a MIC value of 15.6 μg/mL for both compounds. Since the present results also provided a lower MIC value for verbascoside (4), this substance, in addition to forsythoside A (3) and the active fraction (F9−F14), was tested against another strain of C. neoformans (24067), for which similar results were obtained (Table 1). It is interesting to note that although the n-butanol extract of L. rubella did not display any antifungal activity in the screening (MIC 1250 μg/mL), poliumoside (6) displayed good antifungal activity (MIC 62.5 μg/mL) against C. neoformans (T1444). This lack of detected activity could be due to a dilution effect of this compound in the sugar-rich nbutanol-soluble extract. The main components in the ethyl acetate-soluble extract were present in the active F9−F14 fraction (79.5% w/w), formed mainly by forsythoside A (3, 55.3%) and verbascoside (4, 38.6%) (Figure S19, Supporting Information). Since they are the main compounds in the whole extract, a synergistic effect between both isolated compounds in the ethyl acetate extract was considered to explain its MIC value (19.5 μg/mL). Numerous publications have demonstrated that combinations of different antifungal agents or natural products with standard clinical drugs can reduce their distant MIC values,15 especially to overcome acquired antifungal multidrug resistance by lowering current effective therapeutic doses, extending the fungicidal coverage, and decreasing toxicity. The sum of the fractional inhibitory concentration index (∑FIC) assay is widely accepted to evaluate synergistic antimicrobial interactions16 with natural products, especially for essential oils.15a Thus, a factorial dose matrix was used to sample mixtures of serially diluted agents, either the active fraction (F9−F14) or 2 or 3, with amphotericin B (Tables S4− S7, Supporting Information). The tested combinations produced FICindex values ranging from 0.25 to 1.0 (Table 3). A synergistic effect was observed with the combination of amphotericin B and the active fraction (F9−F14), leading to a reduction of their MIC values from 0.125 to 0.015 μg/mL and

caffeoyl moiety (δ 168.2) and rhamnose H-4″ (δ 4.99). For compound 2, H-4′ of the glucose (δ 4.98) correlated with C1‴ of the coumaroyl moiety (δ 169.1), and C-1⁗ of the caffeoyl group (δ 168.1) interacted with H-3″ (δ 4.99) of the rhamnose moiety (Figure S20, Supporting Information), and the CH2-β of the phenylethanoid moiety (δ 72.3) is attached to glucose H-1′ (δ 4.41). The complete assignment of all resonance values in the disaccharide core was accomplished by COSY, HSQC, and HMBC (Table 2). Spectroscopic simulation10 was used to duplicate conclusively the measured 1H NMR data and thus permit the correct chemical shift assignments and coupling constants of all overlaid signals and tightly coupled protons in compounds 1 and 2 (Table S3, Supporting Information). This methodology used the MestReNova program (Figures S1−4, Supporting Information). For example, in compound 2, the signal for glucose H-2′ occurred as a triplet-like signal at 3.46 ppm (Figure S2, Supporting Information), and its multiplicity pattern was evident by visual inspection as a double of doublets (J = 9.2, 7.9 Hz). In other cases, such as the multiplet centered at ca. 4.86 ppm, a double of doublets for rhamnose H-3″ and one overlapping H-3 doublet of doublets for glucose were uncovered by careful analysis. For second-order analysis, where coupling constants could not be estimated directly from the spectrum, rhamnose and glucose coupling constants were used as initial estimates for the simulation.11 The parameters were varied by a nonlinear fit of the spectra to experimental 1H NMR parameters until an optimal agreement between measured and calculated spectra was achieved, as shown in Figures S1−S4 (Supporting Information) for compounds 1 and 2, respectively. Simulation was applied where a visual NMR analysis was impossible, as for example for the multiplet signal centered at 3.45−3.48 ppm, where H-5′ and one H-6 of glucose overlapped and produced a non-first-order resolution. The chemical shifts and coupling constants used to calculate the final spectra are summarized in Table S3 (Supporting Information). Amphotericin B was used as positive control in the screening procedure utilized for in vitro activity against a broad spectrum of clinically relevant fungal isolates. This antibiotic drug has a low minimum inhibitory concentration resistance pattern across most Candida species in adults with candidemia or invasive candidiasis and remains the drug of choice in neonatal candidiasis in the U.S.12 Amphotericin B is the first drug option to treat patients with several other fungal infections, including cryptococcosis, despite its known nephrotoxicity and hepatotoxicity. Renal function usually returns to normal after interruption of amphotericin B treatment, although nephrotoxicity is common in older patients but is better tolerated by infants.12b It still provides the most reliable alternative in combination antifungal therapy for serious infections such as aspergillosis, blastomycosis, coccidioidomycosis, and cryptococcosis.13 However, it would be of great importance to find alternative antifungal substances in order to substitute or reduce the amounts of amphotericin B used to treat patients in combination therapy aiming to overcome the side effects of this conventional antifungal drug. The antifungal activity of the isolated compounds is summarized in Table 1. From the column chromatographic fraction (F9−F14) containing a mixture of compounds 3−6 (MIC 62.5 μg/mL against Candida parapsiloides), all isolated compounds exhibited MIC values higher than 125 μg/mL against Candida species (Table 1). However, promising results D



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VNMRSYS-500 (Agilent, USA) and on a Bruker AV-600 (Bruker, Germany) with tetramethylsilane as internal reference, using CD3OD as solvent, δ in ppm, and J in Hz. The exact mass of the molecules was obtained by direct infusion on an LTQ-Orbitrap Hybrid mass spectrometer (Thermo Scientific, Milan, Italy) equipped with an Electrospray Finnigan Ion Max source. For sample preparation, 1 mg of the sample was diluted to 1000 μL with MeOH−H2O (90:10, v/v). Negative-ion spectra were obtained by the addition of 0.1% NH4OH to the analyte solution to help deprotonation and increase sensitivity. Samples were infused into the mass spectrometer at a flow rate of 5 μL·min−1. Each sample was analyzed in the full-scan mode using a m/ z 100−1000 mass range, under the following instrumental conditions: AGC target 5 × 104, 500 ms maximum inject time, 1 microscan, scan time 1.9 s, resolving power 100.000, capillary temperature 270 °C; spray voltage applied to the needle 3.5 kV, capillary voltage 37 V, nebulizer gas (nitrogen) flow rate set at 5 (a.u.), acquisition time 1 min. Direct infusion ESIMS spectra were deconvoluted by Xcalibur for Qual Browser v. 2.2. SP1 2.2 (Thermo Fischer Scientific, Milan, Italy). TLC plates were silica gel F254 from Merck (Germany), and spots were visualized by UV light and/or by spraying with vanillin and H2SO4−EtOH. Column chromatography (CC) was performed using silica gel (70−230 mesh; Merck, Germany), and an ODS Kromasil C18 column (250 mm × 4.6 mm i.d., 5 μm) and a Luna C18 column (250 mm × 21.2 mm i.d., 10 μm) were employed as packing materials for analytical and semipreparative HPLC. Semipreparative HPLC analysis was performed using a Dionex Ultimate 3000 apparatus (Thermo Scientific, Milan, Italy), and analytical HPLC analysis was performed using a Hitachi Elite LaChrom apparatus (Hitachi High Technologies America, San Jose, CA, USA). Countercurrent chromatography separations were performed on a PC Inc. (Potomac, MD, USA) apparatus equipped with a triple polytetrafluoroethylene multilayer coil (15 mL + 80 mL + 280 mL, 1.6 mm i.d.) equilibrated by a counterweight. The rotation speed was adjustable from 0 to 1000 rpm. The 80 mL coil was used in all experiments. The solvents were pumped with a Waters HPLC solvent delivery system model M-45 (Milford, MA, USA), and the fractions were collected in a Super Fraction Collector SF-2120 Advantec (MFS Inc., Tokyo, Japan). Solvent system testing was performed as follows: small amounts of a sample extract were dissolved in a test tube containing a two-phase solvent system. After shaking and allowing compounds to partition between the two phases, equal aliquots of each phase were spotted individually on silica gel TLC plates in order to determine the distribution coefficients (KD) by visual inspection. Solvent systems used in all separations by CCC were prepared in a separatory funnel at room temperature. After equilibration, the two phases were separated and degassed by sonication for 5 min. In each run, a CCC column was first filled with the stationary phase, and, after setting the rotation, the mobile phase was pumped in. Samples were dissolved in equal volumes of phases and were injected after the hydrodynamic equilibrium inside the column was reached. Plant Material. Lippia rubella was collected in April 2015 from cultivated specimens at the Plant Experimental Area at the Federal University of Juiz de Fora, Minas Gerais, Brazil. Dr. Fátima Regina Gonçalves Salimena identified the plant material. A voucher specimen was deposited in the Herbarium Leopoldo Krieger, Universidade Federal de Juiz de Fora, under the accession number CESJ 56942. Extraction and Isolation. The aerial parts (1152 g) were extracted exhaustively by percolation with ethanol (95%, 8 times, 1:5 w/v). The resulting extract was filtered and concentrated under reduced pressure at 40 °C, using a vacuum rotary evaporator, to yield 180 g of a brownish, oily extract. Upon concentration of the last percolations (fourth and fifth), an abundant yellow solid precipitated. This precipitate (17.13 g) was separated from the crude extract and treated separately. Part of the ethanol extract (90 g) was suspended with water and then submitted to liquid−liquid partition with organic solvents to afford hexane (5.9 g), dichloromethane (1.8 g), ethyl acetate (21.2 g), and n-butanol (29.1 g) extracts. Part of the ethyl acetate extract (4.0 g) was initially fractionated by column chromatography over silica gel eluted with EtOAc−acetone−H2O (25:8:2, organic layer) to afford 124 fractions, which were combined

Table 3. Evaluation of the Interactions Resulting from the Combination of Active Fraction (F9−F14) and the Test Compounds 3 and 4 with Amphotericin B by FICindex Determination (Checkerboard Technique)a MIC (μg/mL) sample


3 31.25 4 62.5 active fraction (F9−F14) 62.5 amphotericin B 0.125 MIC in Combination (μg/mL) sample


3 amphotericin B FICindex 4 amphotericin B FICindex 3 4 FICindex active fraction (F9−F14) amphotericin B FICindex

7.8 0.03 0.50 (S) 15.6 0.06 0.72 (A) 30.95 0.97 1.0 (A) 7.8 0.015 0.250 (S)

a

Abbreviations: (S) synergistic; (A) additive.

from 62.5 to 7.8 μg/mL, respectively. This association produced a 12-fold MIC reduction for both samples. Forsythoside A (3) and amphotericin B also displayed a synergistic effect (FICindex = 0.5), with a 24-fold MIC reduction for both samples. Additive effects were also detected for the combinations of verbascoside (4) and amphotericin B (FICindex = 0.72), as well as for the combination of compounds 3 and 4 (FICindex = 1.0). These results are in contrast to those previously found by Ali and co-workers, who investigated the interactions between amphotericin B and 4, showing that the combination displayed an effective synergism with a potent and extended antifungal effect against pathogenic fungi.11 The synergistic and additive effects16 between amphotericin B and the phenylpropanoid glycosides tested open up new possibilities for lowering the current effective therapeutic doses of this antibiotic and improve therapeutic options, particularly in light of evolving antifungal resistance. The hemolytic activities of compounds 3 and 4, the major components in the ethyl acetate extract, were evaluated. The mechanical stability of the erythrocytic membrane is a suitable guide for cytotoxicity and is dependent on the physical and structural properties of the compounds tested. 14 The compounds evaluated did not display hemolytic activity in the same concentration range of their MIC values and only exhibited toxic concentrations of ≥50% for the erythrocytes at a concentration of 1000 μg/μL. These results demonstrate that both compounds have a very low toxicity toward red blood cells and show the potential for drug development of plant phenylpropanoid glycosides due to their antifungal activity against drug-resistant strains of Cryptococcus.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired on a JASCO P-2000 digital polarimeter (Easton, MD, USA). 1H (500 and 600 MHz), 13C (125 and 150 MHz), and 2D NMR spectra were obtained using standard programs on a E



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the spectroscopic parameters with the original recorded spectra (600 MHz) using MestReNova.10 Antifungal Activity. Different pathogenic fungi were used: Candida albicans (ATCC 10231), Candida parapsilosis (ATCC 22019), and two strains of Cryptococcus neoformans, T1-444 (isolated from clinical cases at Universidade Federal de São Paulo, UNIFESP) and ATCC 24067. The minimum inhibitory concentration (MIC) was determined using the microdilution broth method according to the Clinical Laboratory Standards Institute (CLSI M27-A).24 The fungi (106 cells/mL) were incubated in fresh BHI medium in the presence of several concentrations (1 pg/mL to 1 mg/mL) of active total extracts, fractions, and isolated compounds at 37 °C for 48 h. Cell growth was determined daily by visible turbidity to evaluate the results. The lowest concentration of each active sample that prevented fungal growth was used to determine the MIC. Amphotericin B was used as positive control. Growth inhibition was confirmed after the addition of 30 μL of resazurin solution (5 mg/100 mL of phosphate buffer saline, PBS; pH 7.2) and further incubation at 37 °C for 3 h, as previously described.25 Synergism Assay. The synergistic effect of forsythoside A (3), verbascoside (4), and the active fraction (F9−F14) with amphotericin B was performed as previously described.26 Amphotericin B was combined separately with compounds 3 and 4 and the active fraction at lower concentrations than their individual MIC values in 96-well microplates. Each plate was inoculated with 103 cells/mL and incubated at room temperature for 48 h. The results were based on visual growth, which was confirmed with the addition of resazurin as described above. Fractional inhibitory concentrations for each substance and their combinations with amphotericin B were calculated as follows: FICx = concentration that inhibited 100% of growth in combination/concentration that inhibited 100% of growth alone. The FICindex was calculated by adding both FIC values. The interaction was classified as synergistic when FICindex values < 0.5, additive when 0.5 < FICindex ≤ 1; indifferent when 1 < FICindex > 4, and antagonistic when FICindex > 4.27 Hemolytic Activity. The hemolytic activity was investigated as previously described28 with modifications. First, 20 μL of 2000 to 15.6 μg/mL solutions of compounds 3 and 4, as well as with the active fraction (F9−F14), were incubated with 80 μL of a 4% red blood cell (human O+) suspension in PBS for 1 h at 37 °C in a 96-well microplate. After an incubation period, 200 μL of PBS was added to reduce the reaction; cell lysis was determined spectrophotometrically at 590 nm. For the negative control, 20 μL of PBS solution was added instead of the sample, and for the positive control, Milli-Q water was supplemented instead of PBS solution.

into 22 fractions (F1 to F22) according to their chromatographic similarity. Fraction F3 (80.3 mg, dissolved in 5 mL of the biphasic solvent system) was fractionated by countercurrent chromatography with the solvent system hexane−EtOAc−MeOH−H2O (1:3:1:3; v/ v); flow rate, 2 mL/min; organic phase as mobile phase; 71.25% stationary phase retention (Sf), 850 rpm, to afford 9.3 mg of lippiarubelloside A (1, subfractions 74−86) and 15.8 mg of lippiarubelloside B (2, subfractions 94−101). Subfractions 74/75 and 94/95 were used for the antifungal screening (Table 1; Figure S21, Supporting Information), and their purity was confirmed by 1H NMR (Figures S5 and S14, Supporting Information). Fractions F9−F14 (991 mg), in which the antifungal activity was concentrated, were purified by semipreparative HPLC: sample (80 mg/mL, total volume 2.5 mL), Luna C18 column (250 mm × 21.20 mm i.d., 10 μm), with a gradient elution by CH3CN−H2O−MeOH (5% CH3CN in 5 min, 40% CH3CN in 60 min, purge 100% MeOH in 65 min, and 5% CH3CN in 175 min), a flow rate of 10 mL/min, injection volume of 500 μL, detection at 320 nm, to afford forsythoside A (3, tR 8.69 min),17 verbascoside (4, tR 8.95 min),18 and isoverbascoside (5, tR 9.43 min).19 From fractions F16 (77 mg) and F18 (35 mg), yellow solids precipitated (30 and 7 mg, respectively), which were identified as pectolinarin (7)20 and linarin (8).21 Part of the solid precipitate (500 mg) from the concentration of the ethanol extract was submitted to acetylation with Ac2O and pyridine (1:1, v/v) overnight. The resulting mixture was worked up as usual to obtain a residue (613 mg), which was submitted to silica gel CC eluted with hexane−EtOAc−MeOH (4:2:0.5), yielding peracetylated linarin (8a, 10.9 mg), pectolinarin (7a, 217 mg), and siparunoside (9a, 4 mg). Purified peracetyl siparunoside (9a) was saponified (KOH(aq) 5%) under reflux at 95 °C for 3 h. The reactions mixture was acidified with HCl to pH 5 and extracted with n-butanol to afford compound 9 (2.1 mg).22 Finally, part of the n-butanol extract (520 mg), dissolved in 5 mL of the biphasic solvent system, was fractionated by countercurrent chromatography with the solvent system EtOAc−n-butanol−H2O (4:1:5; v/v), 2 mL/min, organic as mobile phase, 66.25% stationary phase retention (Sf), 850 rpm, to afford 80 fractions (4 mL). Fractions 12−16 afforded a mixture of forsythoside A (3), verbascoside (4), and pectolinarin (7). Fractions 28−39 (78 mg) were further submitted to CC with Sephadex LH-20 to afford 3.9 mg of poliumoside (6).23 Lippiarubelloside A (1): mp 125 °C; ORD (c 0.1 mg/mL, MeOHd4); [α]D +3.1; 1H NMR (600 MHz, MeOD4) and 13C NMR (150 MHz, MeOH-d4) data (Figures S6 and S17, Supporting Information), see Table 2; HRESIMS m/z 785.22736 [M − H]− (calcd for C38H41O18 requires 785.22983, δ −3.1 ppm). Lippiarubelloside B (2): mp 200 °C; ORD (c 0.1 mg/mL, MeOHd4); [α]D +9.3; 1H NMR (600 MHz, MeOD4) and 13C NMR (150 MHz, MeOH-d 4 ) data (Figures S14 and S15, Supporting Information), see Tables 2; HRESIMS m/z 769.23248 [M − H]− (calcd for C38H41O17 requires 769.234923, δ = −3.2 ppm). Forsythoside A (3): 1H NMR (400 MHz, MeOH-d4) and 13C NMR (100 MHz, MeOH-d4) data, see Table S1, Supporting Information. Verbascoside (4): 1H NMR (400 MHz, MeOH-d4) and 13C NMR (100 MHz, MeOH-d4) data, see Table S1, Supporting Information. Isoverbascoside (5): 1H NMR (400 MHz, MeOH-d4) and 13C NMR (100 MHz, MeOH-d4) data, see Table S1, Supporting Information. Poliumoside (6): 1H NMR (500 MHz, MeOH-d4) and 13C NMR (125 MHz, MeOH-d4) data, see Table S1, Supporting Information. Pectolinarin (7): 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table S2, Supporting Information. Linarin (8): 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table S2, Supporting Information. Peracetyl Siparunoside (9a): 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) data, see Table S2, Supporting Information. Spectroscopic Simulations. 1H NMR chemical shifts and 3JH,H coupling constants were simulated through nonlinear adjustment of

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00975. 1

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H and 13C NMR spectra, in addition to COSY, HSQC, and HMBC spectra, for compounds 1 and 2; 1H and 13C NMR data of known compounds 3−9; HPLC chromatograms of the ethyl acetate extract, active fraction, and test compounds; FICx for the tested combinations (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +55 21 3938 6413. Fax: +55 21 3938 6522. E-mail: [email protected]. ORCID

Rogelio Pereda-Miranda: 0000-0002-0542-0085 Suzana G. Leitão: 0000-0001-7445-074X F



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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was provided by CNPq and FAPERJ. We are indebted to Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas and to LAMAR (Laboratório Multiusuário de Análises por RMN) at the Federal University of Rio de Janeiro for access to NMR facilities and to Instituto Nacional de Tecnologia (INT, Rio de Janeiro) for HRMS. G.R.M. is grateful to CAPES for a postdoctoral scholarship. We are indebted to Fatima Regina de Vasconcelos Goulart for performing the antimicrobial assays. R.P.-M. was a Visiting Research Scientist at Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, with partial financial support from the Dirección General de Asuntos del Personal Académico, UNAM.

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DEDICATION Dedicated to Dr. Rachel Mata, National Autonomous University of Mexico, Mexico City, Mexico, and Dr. Barbara N. Timmermann, University of Kansas, for their pioneering work on bioactive natural products.

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