Bioactive Dimeric Acylphloroglucinols from the Mexican Fern

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

Bioactive Dimeric Acylphloroglucinols from the Mexican Fern Elaphoglossum paleaceum María Goretti Arvizu-Espinosa,† Gilsane Lino von Poser,‡ Amelia Teresinha Henriques,‡ Aniceto Mendoza-Ruiz,§ Anaberta Cardador-Martínez,⊥ Reinier Gesto-Borroto,† Pablo Noe ́ Núñez-Aragoń ,† María Luisa Villarreal-Ortega,† Ashutosh Sharma,*,⊥ and Alexandre Cardoso-Taketa*,† †

Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca, 62209, Mexico Universidade Federal do Rio Grande do Sul, Porto Alegre, 90040-060, Rio Grande do Sul, Brazil § Departamento de Biología, C.B.S., CDMX, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, 09340, Mexico ⊥ Tecnologico de Monterrey, School of Engineering and Sciences, Campus Querétaro, Avenue Epigmenio González, No. 500, Fracc. San Pablo, Querétaro 76130, Mexico

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‡

S Supporting Information *

ABSTRACT: Two new prenylated acylphloroglucinols, paleacenins A (1) and B (2), were isolated from the rhizome n-hexane and chloroform extracts of the fern Elaphoglossum paleaceum. Both compounds were found to possess the same geranylated filicinic acid moiety but have a different phloroglucinol ring substituent. Their structures were determined using 1H and 13C NMR spectroscopic, HRMS, and ECD analysis. The plant extracts and purified compounds were assayed for inhibition of monoamine oxidase (MAO) activity, and the n-hexane and chloroform extracts displayed 25.0% and 26.5% inhibition of MAO-A, respectively, as well as 42.5% and 23.7% inhibition of MAO-B, respectively. Compounds 1 and 2 exhibited IC50 values of 31.0 (1.3) μM for MAO-A and 4.7 (4.4) μM for MAO-B. Paleacenin A (1) showed a higher selective index (SI) toward MAO-B (SIMAO‑B/MAO‑A 0.1), and paleacenin B (2) exhibited selectivity to MAO-A (SIMAO‑B/MAO‑A, 3.5). The extracts showed cytotoxicity against a panel of prostate, cervix, breast, and colon cancer cell lines (IC50 values between 1.7 and 10.6 μg/mL); the pure compounds were more active against the prostate, cervix, and colon cancer cell lines. Paleacenins A (1) and B (2), with IC50 values of 46 and 41 μM, respectively, inhibited nitric oxide production by the RAW264.7 murine macrophage model.

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described the extraction of unidentified active phloroglucinol derivatives from D. wallichiana and E. erinaceum using supercritical carbon dioxide extraction.5 Phloroglucinol derivatives possess multiple biological activities: antitumor, antiinflammatory, antibacterial, antidepressant, molluscicide, leishmanicidal, and antinociceptive.6−13 Herein are reported the isolation and structure identification of two new acylphloroglucinols (1 and 2), obtained from the rhizome n-hexane and chloroform combined extracts of the fern E. paleaceum, which are both structurally related to compounds described previously from Argentinian ferns (e.g., E. yungense, E. gayanum, E. piloselloides, and E. lindbergii).8,11,12 Furthermore, in vitro biological assays involving monoamine oxidase (MAO) inhibition, cancer cell line cytotoxicity, and murine macrophage nitric oxide (NO) production have been applied.

teridophytes, vascular cryptogams constituting ferns, have been used widely in traditional medicine, mainly by groups or ethnic communities from different parts of the world, including Mexico, to treat a variety of diseases. Pteridophytes encompass between 10 000 and 12 000 species distributed throughout the world; the greatest diversity is in the tropical and subtropical zones.1 The genus Elaphoglossum includes roughly 585 species, of which three-quarters are located in the tropical Americas. Fifty-eight species of this genus are found in Mexico, mainly in the southern part of the country.2,3 Elaphoglossum paleaceum (Hook. & Grev.) Sledge (Figure S1, Supporting Information) has not been described as a medicinal plant in Mexico, and no studies have focused on its chemical or pharmacological properties. Nevertheless, our interest in this fern derives from determining new sources of phloroglucinol derivatives, which have been found in Hypericum species (Hypericaceae) as well as in Pteridophytes of the genera Dryopteris and Elaphoglossum.4,5 We previously © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 11, 2018

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DOI: 10.1021/acs.jnatprod.8b00677 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR (700 MHz) and 13C NMR (175 NMR) Data for Compounds 1 and 2 in Acetone-d6 1 δC

type

1 2 3 4 5 6 7 8 9

187.6 113.3 170.0 49.0 198.2 110.2 15.9 22.4 37.9

C C C C C C CH2 CH3 CH2

10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ OCH3 OH-3 OH-5 OH-2′ OH-6′

117.0 139.6 39.4 26.4 123.9 130.8 24.9 16.7 15.2 202.8 28.3 106.3 164.2 104.2 162.4 93.1 164.3 206.3 45.5 18.0 13.3

CH C CH2 CH2 CH C CH3 CH3 CH3 C CH3 C C C C CH C C CH2 CH2 CH3

position

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RESULTS AND DISCUSSION TLC analysis (Figure S2, Supporting Information) of the nhexane and chloroform extracts of E. paleaceum rhizomes showed yellow to orange spots when visualized with anisaldehyde-sulfuric reagent and heated. HPLC-DAD peaks with maximal ultraviolet absorbance wavelengths at 229 and 291 nm, which are characteristic of phloroglucinol compounds, were observed (Figure S3, Supporting Information).13 The combined n-hexane and chloroform extract was subjected to several open-column chromatographic procedures to obtain an enriched phloroglucinol-containing fraction that afforded the dimeric acylphloroglucinols 1 and 2 after reversed-phase HPLC purification. Both dimeric acylphloroglucinols exhibited a yellow gum appearance. The HREIMS of 1 displayed a molecular ion peak [M]+ at m/z 540.2710, corresponding to the molecular formula C31H40O8 (calcd 540.2723, δ −2.4 ppm) with 12 degrees of unsaturation (Figure S4, Supporting Information). Its 1H NMR spectrum (Figure S5, Supporting Information) exhibited low-field signals at δH 18.56, corresponding to both the chemical shift of an enolizable hydroxy group proton (OH-5), and at δH 9.92 (OH-3), belonging to a quinone moiety of a filicinic acid-type ring (Table 1). Also observed were signals at δH 16.57 (OH-2′), from a second keto−enol system, and at δH 11.13 (OH-6′), both substituted in a phloroglucinol-type ring, for which a dimeric structure has been described by Socolsky et al.12 The 13C NMR data analysis of 1 (Figures S6 and S7, Supporting Information) confirmed the presence of 31 carbons and revealed the occurrence of a filicinic acid moiety based on its oxygenated carbon resonances at δC 198.2 (C-5), 187.6 (C1), and 170.0 (C-3). The phloroglucinol moiety was supported by aromatic oxygen-based carbons occurring at δC 164.3 (C6′), 164.2 (C-2′), and 162.4 (C-4′). Further low-field signals at δC 202.8 and 206.3 were assigned to carbonyl groups of an acetate (C-19) and a butanoate (C-7′) attached to C-6 and C3′, respectively. The location of the acetate group at C-6 was corroborated by an HMBC experiment (Figure S9, Supporting Information), demonstrating long-range 3JHC correlations between the protons of the methyl group at C-20 (δH 2.68) and C-6 (δC 110.2). Furthermore, a long-range correlation between OH-5 (δH 18.56) and C-19 (δC 202.8) indicated the presence of a keto−enol system that involves these atoms, leading to 2JHC coupling, in which the hydroxy group proton is

55.4

CH3

2 δH (J in Hz)

3.53 s 1.52 s 2.60 dd (13.2, 7.2) 2.75 dd (13.2, 8.8) 4.66 t (7.6) 1.56 m 1.63 m 4.88 brt 1.60 s 1.48 s 1.39 s 2.68 s

6.09 s

3.02 m 1.70 m 0.98 t (7.4)

3.91 s 9.92 s 18.56 s 16.57 s 11.13 s

δC

type

187.8 113.0 170.0 49.1 198.2 110.2 16.6 22.2 38.1

C C C C C C CH2 CH3 CH2

117.0 139.6 39.3 26.4 123.9 130.6 24.9 16.7 15.1 202.7 28.2 109.7 159.7 107.6 160.3 116.6 162.4 206.9 43.6 17.9 13.2 22.7 123.4 130.8 17.1 24.9 62.5

CH C CH2 CH2 CH C CH3 CH3 CH3 C CH3 C C C C C C C CH2 CH2 CH3 CH2 CH C CH3 CH3 CH3

δH (J in Hz)

3.58 s 1.54 s 2.58 dd (13.2, 7.2) 2.73 dd (13.2, 8.8) 4.66 t (7.6) 1.59 m 1.65 m 4.86 brt 1.66 s 1.48 s 1.33 s 2.69 s

3.14 1.72 0.97 3.29 5.23

m m t (7.6) m t (6.8)

1.77 s 1.59 s 3.75 s 9.99 s 18.57 s 15.75 s 11.39 s

shared among the oxygen atoms at C-5 and C-19. The C-5 hydroxy group proton also displayed a strong 2JHC correlation with C-5 (δC 198.2) and a 3JHC correlation with C-6 (δC 110.2). A similar observation has been described for elaphogayanin A, a compound isolated from E. gayannum.11 The enolizable atoms at C-5 and C-19 and the carbonyl group at C-1 constitute a β-triketone system. The keto−enol tautomerism at C-5 and C-19 drives the deshielding effects on the nuclei that compose the acetate group at C-19 (δC 202.8), C-20 (δC 28.3), and H-20 (δH 2.68), for which their resonances, if not chelated, typically should be close to δC ∼170, δC ∼20, and δH ∼2.0, respectively; the dramatic lowfield shifts resulted from the strong intramolecular H-bonded enol forms of the ortho-ketophenol interaction. B



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In addition, the 13C NMR spectrum of 1 (Table 1) exhibited a signal at δC 15.9 for a secondary carbon (C-7) (Figure S7, Supporting Information), serving as a link between the filicinic acid-type ring and the phloroglucinol-type ring. The protons of a CH2-7 bridge displayed a singlet centered at δH 3.53 and showed long-range correlations with carbons of the filicinic acid-type ring at C-1 (δC 187.6), C-2 (δC 113.3), C-3 (δC 170.0), C-1′ (δC 106.3), C-2′ (δC 164.2), and C-6′ (δC 164.3) from the phloroglucinol ring. A 3JHC cross-peak between the methoxy group at δH 3.91 and the aromatic carbon δC 162.4 established its position at C-4′ in the phloroglucinol moiety. The 1D (1H, 13C, DEPTs 90, and 135) and 2D NMR (HSQC and HMBC) data of the acylphloroglucinol 1 indicated the presence of a prenylated chain attached at C-4, consistent with the filicinic acid aglycone of the yungesins reported for E. yungenese.8 The two olefinic protons at δH 4.66 (H-10, t, 7.6 Hz) and δH 4.88 (H-14, brt) were assigned to prenyl units of a geranyl side chain attached to C-4. This assertion was supported by the ion fragment at m/z 137 [C10H17]+ of a geranyl radical and a peak at m/z 404 [M + H − 137 (C10H17)]+, consistent with the loss of the prenyl group from the filicinic acid moiety in FABMS (Figure S4B, Supporting Information). The base peak at m/z 223 [M − 317 (C19H25O4)]+ was a result of a fragmentation β to the methylene bridge, corresponding to the phloroglucinol ion (C12H15O4), and an α rupture to the filicinic ring containing the prenyl group (C19H25O4, 317 amu) (Figure 1). The

The HREIMS of paleacenin B (2) displayed a molecular ion peak [M]+ at m/z 608.3331 that was identical to the molecular formula, C36H48O8 (calcd 608.3349, δ −3.0 ppm), with 13 degrees of unsaturation (Figure S10, Supporting Information). The 1H NMR spectrum of 2 (Figure S11, Supporting Information) also displayed two enolizable protons at very low field at δH 18.57 (OH-5) and δH 15.75 (OH-2′), as well as signals at δH 11.39 (OH-6′) and δH 9.99 (OH-3). These observations supported the presence of a dimeric structure composed of a filicinic acid-type ring and a phloroglucinol-type ring, as described for 1. The 1H and 13C NMR spectra of 2 resembled those of 1 and differed structurally by the presence of an isoprenyl group in 2, which is consistent with the differences of five carbons and one unsaturation. The region of the olefinic protons displayed a set of three signals, with two of them from the geranyl chain noted above at δH 4.66 (H-10) and δH 4.86 (H-14). The isoprenyl unit displayed NMR signals of olefinic atoms at δH 5.23 and δC 123.4 (CH-12′), and δC 130.8 (C-13′), methyl group resonances at δH 1.77 (3H) and δC 17.1 (CH3-14′), and δH 1.59 (3H) and δC 24.9 (CH3-15′), and methylene resonances at δH 3.29 (2H) and δC 22.7 (CH211′) (Figures S11−S14, Supporting Information). The available evidence was used to locate unequivocally this prenyl side chain at C-5′ in the acylphloroglucinol-type ring; namely, (a) the HMBC experiment on 2 (Figure S16, Supporting Information) revealed a correlation between C-5′ (δC 116.6) and protons at δH 3.29 (2H-11′) that displayed further 2,3JCH couplings to the olefinic carbons C-12′ and C-13′, confirming the assignments to this isoprenyl unit; (b) the only aromatic proton signal at δH 6.09 (H) in the 1H NMR spectrum of paleacenin A (1) was absent in the spectrum of paleacenin B (2), in which the isoprenyl group is attached at C-5′; and (c) the linkage of the isoprenyl group at C-5′ caused a deshielding effect at 23.5 ppm on this carbon in relation to the same position in the structure of 1, where the isoprenyl group was lacking. The long-range 3JHC cross-peaks between protons of the methoxy group at δH 3.75 (3H, s) and δC 160.3 supported its substitution at C-4′ of the aromatic-phloroglucinol moiety of 2; this same observation was made for 1. The low-resolution FABMS of paleacenin B (2) (Figure S10B, Supporting Information) displayed a base peak at m/z 291 produced by a fragmentation β to the benzene ring, which was consistent with the location of the isoprenyl, butanoyl, and methoxy groups in the phloroglucinol-type aglycone. The ECD spectrum of 2 in EtOH was similar to that of 1 and exhibited strong positive Cotton effects at Δε298 +11.0 and Δε226 +30.9 and negative Cotton effects at Δε266 −21.5 and Δε203 −31.5, indicative of the S configuration to the stereogenic center at C4 (Figure S17, Supporting Information). The structure of paleacenin B (2) was very similar to the prenylated acylphloroglucinol yungensin D isolated from E. yungense,8 which possesses a hydroxy group attached at C-4′ instead of a methoxy group in 2. On the basis of the evidence obtained, the structure of paleacenin B (2) was proposed as 2-{[2,6dihydroxy-4-methoxy-5-(3-methyl-2-butenyl)-3butanoylphenyl]methyl}-(4S)-3,5-dihydroxy-4-methyl-4-(3,7dimethyl-2,6-octadienyl)-6-acetyl-2,5-cyclohexadiene-1-one. The n-hexane and chloroform extracts from the fronds, petioles, and rhizomes of E. paleaceum, as well as compounds 1 and 2, were assayed in vitro for MAO inhibitory activity, obtained from rat brain mitochondria (Table 2). Organic extracts of the fronds displayed no inhibition of MAO-A and very low percentage inhibition values of MAO-B (2.0% to n-

Figure 1. Fragmentation patterns (m/z values) observed for compounds 1 and 2 by LRFABMS experiments, demonstrating the presence of the filicinic acid-type and phloroglucinol-type moieties.

phloroglucinol moiety proposed for 1 is identical to a moiety described for both elaphopilosin D and lindbergin A from E. piloselloides11 and E. lindbergii,9 respectively. The depicted structure presents only one chiral center at C-4; for acylphloroglucinols R or S absolute configurations are possible. The electron circular dichroism (ECD) spectrum of 1 in EtOH revealed strong positive Cotton effects at Δε298 +8.2 and Δε219 +16.5 and negative Cotton effects at Δε266 −10.2 and Δε200 −22.9 (Figure S17, Supporting Information). This pattern is similar to the bands observed for lindbergins, isolated from E. lindbergii, which possess an S configuration. Accordingly, the structure of paleacenin A (1) was established as 2-{[2,6dihydroxy-4-methoxy-3-butanoylphenyl]methyl}-(4S)-3,5-dihydroxy-4-methyl-4-(3,7-dimethyl-2,6-octadienyl)-6-acetyl2,5-cyclohexadiene-1-one. C



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search of natural compounds with MAO inhibition, especially due to their selective action toward both MAO isoforms. It is well known that the lack of selectivity drives several undesirable side effects, e.g., inhibition of cytochrome P450.16 For decades, plant constituents containing MAO-A and MAO-B inhibitors have been associated with the treatment of depression and Parkinson diseases, respectively.17 The anti-inflammatory effects of compounds 1 and 2 as assessed using a murine macrophage cell line were investigated. Both compounds were cytotoxic to RAW264.7 cells at 185 and 139 μM (1) and 164 and 123 μM (2), killing 100% of the cells (Figure 2). Acylphloroglucinol 1 remained 100% toxic at 92 μM, and 2 exhibited 50% cell viability at 82 μM. A lower concentration of 1 and 2 at 46 and 41 μM, respectively, did not affect the viability of the cells (Figure 2A). Paleacenins A (1) and B (2) inhibited NO production at a test concentration of 25 μg/mL (46 μM for 1 and 41 μM for 2), suggesting that both compounds might have an anti-inflammatory effect. Additional studies must be performed to support this conclusion (e.g., inhibition of interleukins, interferon-inducible protein (IP)-10, keratinocyte-derived chemokines, and vascular endothelial growth factors, which are important proinflammatory mediators). NO is essential for a variety of processes in organisms of all kingdoms; it is a versatile signal molecule that acts as a cellular target via redox or additive reactions, and it has a broad influence on human physiopathology.18 The crude n-hexane and chloroform extracts of the fronds and petioles of E. paleaceum did not show cytotoxicity on the breast (MCF7), cervix (SiHa), colon (HF6), or prostate (PC3) human cancer cell lines. Nevertheless, the rhizome nhexane extract was active and displayed IC50 values between 1.7 and 4.4 μg/mL for all cell lines tested. With the exception of the MCF7 cell line, the rhizome chloroform extract was also active against the panel of cancer cells assayed (IC50 value range: 4.3−10.6 μg/mL). Both compounds 1 and 2 were not cytotoxic to the MCF7 cell line, exhibiting IC50 values of >10 μM. However, they were active toward the other cell lines used, especially PC3 cells, with IC50 values of 1.7 and 2.9 μM for 1 and 2, respectively. By contrast, both compounds at 46 and 41 μM, respectively, exhibited no cytotoxic effect to macrophage cells. Acylphloroglucinols 1 and 2 were very cytotoxic to normal fibroblast cells HFS-30 (IC50 0.009 and 0.08 μM, respectively), comparable to the controls utilized,

Table 2. IC50 of Compounds 1 and 2 from E. paleaceum, Selective Controls on MAO-A and MAO-B Inhibition, and Selective Index (SI) Values compound 1 2 clorgyline pargyline

IC50 of MAO-A (μM)a

IC50 of MAO-B (μM)a

SI IC50 MAO‑B/IC50 MAO‑A

31.0 ± 0.13 1.3 ± 0.27 0.008 ± 0.0009

4.7 ± 0.23 4.4 ± 0.30

0.1 3.5

0.22 ± 0.0125

Results are expressed as means ± SD.

a

hexane and 5.4% to chloroform at 100 μg/mL). The petiole extracts displayed increased but still low activity on MAO-A and -B (inhibition % range: 6.8−17.1). However, significant inhibition percentages were found for the rhizome extracts (range 23.7−42.5%). Taking into account that phloroglucinol derivatives with antidepressant-like activity have been isolated from Elaphoglossum and Hypericum species, e.g., crassipin A (E. crassipes)10 and uliginosin B (H. polyanthemum),14 the present study demonstrated the presence of active acylphloroglucinols [1 (56.7% MAO-A and 71.7% MAO-B inhibition) and 2 (85.9% MAO-A and 82.5% MAO-B inhibition) at 100 μg/mL] as being responsible for MAO activity of E. paleaceum rhizome extracts (Table S1, Supporting Information). The less polar compound 2 exhibited higher inhibitory concentration 50 (IC50) values of 1.3 μM for MAO-A and 4.4 μM for MAO-B than 1 (IC50 of 31.0 μM for MAO-A and 4.7 μM for MAO-B), indicating that compound 1 is more selective to MAO-B (SI 0.1) and compound 2 is more selective to the MAO isoform A (SI 3.5) (Table 2). Isocarboxazid, phenelzine, and tranylcypromine currently are available as irreversible inhibitors, but these monoamine oxidase inhibitors (MAOIs) lack specificity for any subtype of MAO enzymes. Their use is also limited due to hypertensive crises associated with foods containing tyramine. There is a need to develop new MAOIs that are more selective for specific subtypes of the MAO enzyme and with fewer side effects compared to the classic MAOIs.15 Paleacenin B (2) was shown to be more active for MAO-A than the naturally occurring compounds reported in Hypericum perforatum, such as hypericin (IC50 35.5 μM) and quercetin (IC50 11.1 μM). The present study has revealed the interesting potential of paleacenins A (1) and B (2) as drug leads in the

Figure 2. (A) Viability of RAW264.7 macrophages under the presence of DMEM-F12/FBS medium and cells, LPS (1 μg/mL), Tween 80 (0.08%), aminoguanidine at 226, 452, 679, and 905 μM (+ control), and paleacenins A (1) at 46, 92, 139, and 185 μM and B (2) at 41, 82, 123, and 164 μM. (B) Effect of compounds 1 and 2 and + control on nitrite (NO) production in LPS-stimulated macrophages. Values are the means ± SD of three independent experiments (p < 0.05); **** showed no differences between groups. D



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Table 3. Cytotoxicity of Compounds 1 and 2 of E. paleaceum against a Panel of Human Tumor Cell Linesa IC50 (μM)b cell lines compound

MCF7

1 2 podophyllotoxin paclitaxel

>10 >10 0.005 ± 0.02 0.05 ± 0.01

SiHA

HF6

PC3

± ± ± ±

>10 6.4 ± 0.06 1.0 ± 0.02 1.6 ± 0.01

1.7 ± 0.04 2.9 ± 0.08 0.2 ± 0.05 0.15 ± 0.01

6.0 5.6 0.1 1.4

0.02 0.03 0.02 0.01

HFS-30 0.009 0.08 0.002 0.002

± ± ± ±

0.0001 0.001 0.0002 0.0001

a

MCF7 (breast), SiHa (cervix), HF6 (colon), and PC3 (prostate) human cancer cells lines and HFS-30 (fibroblast) normal human cells. bResults are expressed as means ± SD.

podophyllotoxin and paclitaxel (IC50 0.002 μM for both controls) (Table 3). Compound 2 exhibited better selective indices than 1 in all cancer cell lines in which it was tested (Table 4).

(Milford, MA, USA) scales using HPLC-grade solvents. Chemicals for bioassays were purchased from Sigma-Aldrich (St. Louis, MO, USA). Plant Material. E. paleaceum was collected near the Teotitlán Flores Magón-Huautla route (Oaxaca, Mexico) in April 2013. A voucher specimen (No. 19003) was authenticated by one of the authors (A.M.-R.) and deposited at the HUMO Herbarium-UAEM. Extraction and Isolation. Air-dried fronds, petioles, and rhizomes of E. paleaceum (45.5 g) were dried at room temperature and each ground to a fine powder using an electric mill. A 50 mL amount of n-hexane was added, and the material sonicated for 15 min in an 80 W ultrasonic power apparatus at 20 °C and then filtered using Whatman No. 2 filter paper to complete a series of seven consecutive extractions. All extracts were combined and evaporated in a rotavapor at 40 °C until they were dry. Next, the material was extracted with chloroform following the same steps to finally yield crude extracts for the rhizomes (387 mg of n-hexane and 397 mg of chloroform crude extracts), fronds (395 mg of n-hexane and 295 mg of chloroform crude extracts), and petioles (309 mg of n-hexane and 300 mg of chloroform crude extracts). After TLC and HPLC analysis, 90% of the rhizome n-hexane and chloroform extracts were combined for isolation purposes, and the other part was used for biological assays. The resulting reddish gum product was then fractionated by column chromatography using silica gel as adsorbent and n-hexane− EtOAc (3:1) as eluent to yield 113 eluates of 1.5 mL each. Fractions 27−41, yielding 199 mg, were individually analyzed using C18-phase HPLC (CH3CN−H2O, 98:2, 0.001% trifluoroacetic acid, 1 mL/min) to indicate the presence of 1 (tR 13.0 min) and 2 (tR 22.1 min). The final purifications of 1 (7.2 mg) and 2 (7.8 mg) were achieved by preparative-scale HPLC on a C18 column (7 μm, 19 × 300 mm) with the same isocratic phase used for the analytical separation and a flow rate of 8 mL/min. Paleacenin A (1): yellow gum; [α]22D +18.2 (c 0.7, EtOH); ECD (EtOH) λmax nm (Δε) 298 (+8.2), 266 (−10.2), 219 (+16.5), 200 (−22.9); 1H NMR data (700 MHz, acetone-d6), see Table 1; 13C NMR data (175 MHz, acetone-d6), see Table 1; LRFABMS m/z 541 (13), 471 (2), 404 (20), 223 (100), 137 (8), 69 (31), 43 (4); HREIMS m/z 540.2710 (calcd for C31H40O8, 540.2723, δ −2.4 ppm). Paleacenin B (2): yellow gum; [α]22D +44.1 (c 0.6, EtOH); ECD (EtOH) λmax nm (Δε) 298 (+11.0), 266 (−21.5), 226 (+30.9), 203 (−31.5); 1H NMR data (700 MHz, acetone-d6), see Table 1; 13C NMR data (175 MHz, acetone-d6), see Table 1; LRFABMS m/z 609 (30), 539 (2), 472 (18), 291 (100), 137 (8), 69 (38), 43 (6); HREIMS m/z 608.3331 (calcd for C36H48O8, 608.3349, δ −3.0 ppm). MAO Inhibition Assay. The assay was performed using the in vitro fluorometric method of Krajl,25 with some modifications. A sample of the MAO enzyme was obtained from the mitochondria of the brains of five adult male Wistar rats (180−220 g). Rats were housed at 21 ± 2 °C with a 12/12 h light−dark cycle. To obtain the tissue homogenates, the brains were removed after sacrifice by decapitation and dissected without a cerebellum. The resulting tissue was homogenized in a glass-Teflon homogenizer in 10 nM HEPES buffer, pH 7.4, containing 68 mM sucrose, 10 mM KCl, and 200 mM mannitol, and centrifuged at 5000 rpm for 10 min. The supernatant was further centrifuged at 11 500 rpm for 15 min to obtain a mitochondrial pellet. All procedures were carried out at 4 °C, and the material was stored at −75 °C. To determine the concentration of the

Table 4. Selectivity Index (SI) of Compounds 1 and 2 and Controlsa compound

MCF7

SiHA

HF6

PC3

1 2 podophyllotoxin paclitaxel

0.0002 0.0025 0.450 0.050

0.002 0.015 0.020 0.002

0.0005 0.013 0.002 0.001

0.005 0.029 0.110 0.015

a

MCF7 (breast), SiHa (cervix), HF6 (colon), and PC3 (prostate) human cancer cells lines and HFS-30 (fibroblast) normal human cells. SI = IC50 normal fibroblast cell (HFS-30)/IC50 cancer cell line.

Elaphoglossum (Dryopteridaceae) is one of the most representative Pteridophyta genera.19,20 A large number of studies have been performed with South American Elaphoglossum, especially those from Argentina and Brazil.7−12 A series of acylated-phloroglucinol derivatives have been isolated and tested toward different pharmacological models. Plants containing phloroglucinols may be an alternative to treat neuroinflammatory diseases. In fact, hyperforin, an acylphloroglucinol-type structure substituted with four lipophilic prenyl units, is the principal bioactive molecule in Hypericum perforatum (St. John’s wort), which is well known for its antidepressant action due to modulation in the neuronal ionic conductance21 and ability to inhibit serotonin reuptake.22 Phloroglucinol has exhibited therapeutic effects in cellular and animal models of Parkinson’s disease, a progressive neurodegenerative disorder that causes motor dysfunctions;23 its derivatives have exhibited anti-neuroinflammatory activity.24 This first investigation on the chemistry and pharmacology of E. paleaceum has demonstrated the cytotoxic and antiinflammatory properties for the new dimeric prenylated acylphloroglucinols paleacenins A (1) and B (2).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a PerkinElmer 341 polarimeter. ECD spectra were obtained on a JASCO CD-1500 spectropolarimeter (Easton, MA, USA) using EtOH as solvent. 1D and 2D NMR spectra were obtained on a Varian VNMRS 700 MHz apparatus using acetone-d6 as solvent. Mass spectra were measured on a JMS700-JEOL (Tokyo, Japan) instrument using FAB(+) ionization. All of the solvents for column chromatography were analytical reagent grade. TLC analysis was carried out using precoated Si gel 60 F254 aluminum sheets. Silica gel (40−60 μm) was used for open column chromatography. Symmetry C18 columns were used for HPLC analysis on analytical (Waters; 5 μm, 4.6 × 250 mm) and preparative (Waters; 7 μm, 19 × 300 mm) E



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enzyme, the Lowry protein quantification method was used. Prior to the assay, the enzyme homogenate (not purified) was adjusted to a concentration of 2500 μg/mL of protein using a phosphate buffer solution (PBS). The extracts and pure compounds were dissolved in 10% dimethyl sulfoxide (DMSO) to obtain a concentration of 1 mg/ mL. A curve was also prepared for the pure compounds from the initial solution to obtain different concentrations (1, 5, 10, 25, 50, 75, 100 μg/mL). Kynuramine (1 mM) was used as a substrate, as well as clorgyline (10 μM) and pargyline (10 μM), as selective inhibitors of the MAO-A and MAO-B isoforms, respectively. The reaction was prepared in 2 mL Eppendorf tubes by combining 575 μL of PBS (pH 7.4), 25 μL of inhibitor, 100 μL of extract (1 mg/mL), and 200 μL of enzyme. Next, 190 μL of the previously incubated solutions were placed in each of the wells of the plate, which were incubated for 15 min at 37 °C. A 10 μL amount of kynuramine was then added, and the solutions were incubated again for 15 min at 37 °C. The reaction was stopped with 75 μL of 2 M NaOH. The control experiments were conducted without an inhibitor, and the targets were performed without any mitochondrial suspension. The product of the enzymatic reaction (4-hydroxyquinoline) was measured at an excitation wavelength of 315 nm and an emission wavelength of 380 nm. The percentage of inhibition for both MAO-A and MAO-B was calculated according to the following equation: % inhibition = [(fluorescence control − sample fluorescence)/fluorescence control] × 100. The extracts and compounds that showed an inhibition greater than 50% for some of the MAO isoforms were evaluated over a range of 0.1− 100 μg/mL to determine their IC50 values. The IC50 values were calculated using Graph Pad Prism software version 4.0 (San Diego, CA, USA) and were expressed as mean values ± standard error (Table 2). All of the assays were performed in triplicate. In vivo experiments were conducted in rats according to the Mexican Guidelines for Animal Welfare NOM-062-ZOO-1999 and were approved by the Ethics and Security Committee (CBEA 01-12-17) from the Centro de Investigación en Biotecnologia,́ UAEM, Mexico. Cytotoxic Assays. The n-hexane and chloroform fronds, petioles, and rhizome extracts as well as compounds 1 and 2 were subjected to cytotoxic evaluations using breast (MCF7), cervix (SiHa), colon (HF6), and prostate (PC3) human cancer cell lines from ATCC (American Type Culture Collection, USA), along with a normal human fibroblast cell line (HSF-30), via the sulforhodamine B (SRB) method. Cell cultures were grown in RPMI-1640 medium (SigmaAldrich) supplemented with 10% fetal bovine serum (FBS, Invitrogen), and 7.5% NaHCO3. They were cultivated in 96-well plates (104 cells/mL) at 37 °C in a 5% CO2 atmosphere and humidity at 100%. Normal human fibroblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% FBS. During the log phase of the cellular growth cycle, using the criteria of 70% confluence, they were treated in triplicate with various concentrations of the test samples (0.16−20 μg/mL), dissolved in DMSO (Sigma-Aldrich) and incubated for 72 h, under the conditions described above. The results were determined as the dose that inhibited 50% control growth after the incubation period (IC50). The values were expressed on a log 10 plot of the drug concentration versus the percentage of viable cells. Podophyllotoxin and paclitaxel (Sigma-Aldrich) were used as controls. Extracts with IC50 ≤ 20 μg/ mL and compounds with IC50 ≤ 4 μg/mL were considered active according to NCI guidelines.26 The specificity of cytotoxic activity was obtained by an assay against the normal skin fibroblast cell line (HFS-30); IC50 values were used in the following equation to determine the selective index (SI): IC50 normal cell line HFS-30/IC50 cancer cell line. The experiments were performed in triplicate, and one-way ANOVA analysis was used to compare the mean values, with p values less than 0.05 presumed to indicate statistical significance. Assay for Nitric Oxide Inhibitory Effect Using RAW264.7 Cells. The suppression of lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)-activated murine macrophage RAW264.7 cells was performed using a modified form of the method reported by Min et al.27 Briefly, RAW264.7 (ATCC) murine macrophage cells were grown in DMEM/Nutrient Mixture F-12 (DMEM-F12) from Biowest and supplemented with 10% heat-

inactivated FBS from Gibco, without antibiotics. Macrophages were cultured in a 25 cm2 flask in a humidified incubator at 37 °C and a 5% CO2 atmosphere. For the assay, 100 μL of RAW264.7 cells (2 × 105 cells/mL) was incubated for 1 h in 96-well culture plates in 100 μL of DMEM-F12 and FBS. Next, the macrophages were incubated with paleacenins A (1) and B (2) at four concentrations (25, 50, 75, 100 μg/mL) in the vehicle Tween 80 (0.8% v/v). As a positive control, aminoguanidine hydrochloride was used at the same concentrations (μg/mL) as the evaluated compounds. Then, the cells were stimulated by LPS (1 μg/mL) and incubated for 24 h. As nitrite is a stable product of NO oxidation, it was used as an indicator of the NO production in the culture medium based on the Griess reaction. A 100 μL amount of the cell culture medium was then mixed with 100 μL of the Griess reagent [50 μL of 1% sulfanilamide and 50 μL of 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid] and incubated at room temperature for 10 min. Then, the absorbance at 540 nm was measured using a microplate reader (SpectraMax iD3). The amount of nitrite in the samples was calculated using a NaNO2 standard curve obtained in fresh medium culture. The assay was performed in triplicate, and the data were expressed as the means ± standard error of the mean. Cell viability was measured using a fluorometric assay based on the reduction of resazurin (blue dye, weakly fluorescent) into pink resorufin (highly fluorescent) due to the activity of mitochondrial and cytosolic enzymes in live cells. After the nitrite determination, 180 μL of fresh DMEM-F12 medium supplemented with 10% heat-inactivated FBS and 20 μL of resazurin (2 mM) was added to each well. Then, RAW264.7 macrophages were incubated for another 10 h in a humidified incubator at 37 °C and a 5% CO2 atmosphere. Following the incubation, the fluorescence was read on a microplate reader (SpectraMax iD3); excitation was measured at 544 nm, and emission was measured at 590 nm.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00677. Plant photograph, TLC and HPLC analysis of the extracts, as well as EIMS, FABMS, NMR, and ECD materials of paleacenins A (1) and B (2) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +52 442 238322. Fax: +52 442 2383220. E-mail: [email protected]. *Tel: +52 777 3297000. Fax: +52 777 3297030. E-mail: [email protected]. ORCID

Anaberta Cardador-Martínez: 0000-0003-2942-1186 Alexandre Cardoso-Taketa: 0000-0002-7055-0062 Author Contributions

Results were taken from the Ph.D. thesis of M. G. ArvizuEspinosa (Doctorado en Ciencias Naturales, UAEM). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to acknowledge the pioneering role of Dr. Rachel Mata, of the National Autonomous University of Mexico, Mexico City, Mexico, in the study of bioactive principles of Mexican medicinal plants. This research was supported by CONACyT (Nos. 156276 and 222714 and a bilateral project Mexico-Brazil) developed at the Centro de F



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Investigación en Biotecnologı ́a of the Universidad Autónoma del Estado de Morelos and at the Departamento de Bioingenierıá del Tecnológico de Monterrey, Campus Querétaro, Mexico. Thanks are due to A. Zenil for support in the HPLC analysis. M.G.A.E. acknowledges the receipt of a Ph.D. fellowship No. 274158 from CONACYT.

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(27) Min, H. Y.; Kim, M. S.; Jang, D. S.; Park, E. J.; Seo, E. K.; Lee, S. K. Int. Immunopharmacol. 2009, 9, 844−849.

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Bioactive Dimeric Acylphloroglucinols from the Mexican Fern

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