neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the

Article Cite This: J. Nat. Prod. 2017, 80, 3003-3009

pubs.acs.org/jnp

neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the Expression of Extracellular Matrix Components in Human Dermal Fibroblasts Elihú Bautista,*,† Naytzé Ortiz-Pastrana,§ Guillermo Pastor-Palacios,† Angélica Montoya-Contreras,‡ Rubén A. Toscano,§ Jesús Morales-Jiménez,† Luis A. Salazar-Olivo,‡ and Alfredo Ortega§ †

CONACYT-CIIDZA and ‡División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A. C., Camino a la Presa San José 2055, Lomas 4a Sección, 78216 San Luis Potosí, México § Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, México City, México S Supporting Information *

ABSTRACT: Eleven neo-clerodane diterpenoids (1−11) including the new analogues 1, 2, and 10, and 3′,5,6,7tetrahydroxy-4′-methoxyflavone (12) were isolated from the aerial parts of Salvia polystachya. Polystachyne G (1) and 15epi-polystachyne G (2) were isolated as an epimeric mixture, containing a 5-hydroxyfuran-2(5H)-one unit in the side chain at C-12 of the neo-clerodane framework. Polystachyne H (10) contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy group. The structures of these compounds were established by analysis of their NMR spectroscopic and MS spectrometric data. The absolute configurations of compounds 3, 4, and 10 were determined through single-crystal X-ray diffraction analysis. The antibacterial, antifungal, and phytotoxic activities of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of nine of the isolates (1−8 and 11) was assayed. Compounds 1−4, 8, and 11 increased the expression of the genes codifying for type I, type III, and type V collagens and for elastin.

addition, other neo-clerodane diterpenoids have been isolated from the aerial parts including polystachynes A−F, salvifaricin, and dehydrokerlin.12 Herein, the isolation and structural elucidation of three new diterpenoids, as well as their antimicrobial and phytotoxic properties, and their capability to modulate the expression of extracellular matrix components in human dermal fibroblasts are discussed.

neo-Clerodane diterpenoids constitute a group of secondary metabolites of pharmacological interest due to their structural diversity, lipophilic properties, and capability to interact with molecular targets, which makes this class of compounds ideal molecules for their bioprospecting in several pharmacological models.1 These compounds have shown anti-inflammatory2 and neuroprotective activities3 and have the potential to be used to treat Alzheimer’s disease,4 colitis,5 and obesity.6 Other bioactivities of neo-clerodane diterpenoids are their affinity for the κ-opioid receptor7 and their cytotoxicity against different human tumor cell lines.8 The broad spectrum of pharmacological activities attributed to neo-clerodane diterpenoids prompted an investigation into the phytochemical analysis of the aerial parts of Salvia polystachya. Salvia polystachya (Lamiaceae, subgenera Calosphace) is a shrub endemic to Mexico.9 This plant is known in folk medicine as “chı ́a”, and together with 17 other species of the genera (including S. hispanica), make up the “chı ́a” complex of medicinal plants.10 The aerial parts are used as a purgative and diuretic and to treat dysentery. A previous bioguided study led to the isolation of the diterpenoid linearolactone as the active principle responsible for the antiamoebic and antigiardial effects of the acetone-soluble extract of S. polystachya leaves.11 In © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The phytochemical reinvestigation of an acetone-soluble extract of the leaves of S. polystachya afforded 11 neo-clerodane diterpenoids (1−11) and 3′,5,6,7-tetrahydroxy-4′-methoxyflavone (12).13 Three of the diterpenoids, 1, 2, and 10, are new and are accompanied by the known diterpenoids salvifilines A (3) and C (5), polystachynes A (7), B (8), and E (9), and linearolactone (11). Polystachyne G (1) and 15-epi-polystachyne G (2) were isolated as colorless crystals and occurred as a 1:1 epimeric mixture. Its molecular formula, C20H22O8, was deduced from its protonated molecular ion at m/z 391.13979 [M + H]+ by Received: July 13, 2017 Published: November 14, 2017 3003

DOI: 10.1021/acs.jnatprod.7b00591 J. Nat. Prod. 2017, 80, 3003−3009

Journal of Natural Products

Article

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of Compounds 1/2 and 10 (in CDCl3−DMSO-d6, 9:1) 1/2a,b δH mult. (J in Hz)

δC

1 2

3.11 br dd (6.0, 3.2) 3.30 dd (5.6, 3.6)

55.1, CH 50.4, CH

3α 3β 4 5 6α

2.31 mb 1.82 dd (16.0, 16.0) 2.31 mb

18.9, CH2

6β

1.49 br d (14.0)

7α 7β

4.51 d (4.0)

position

8 9 10 11α 11β 12 13

HRMS (DART-TOF+) and C NMR data, indicating 10 indices of hydrogen deficiency. The IR spectrum of 1/2 showed absorption bands at 3362 and 1767 (broad) cm−1, indicating the presence of hydroxy and γ-lactone groups. The presence of the γ-lactone moiety was confirmed by the signals observed in the 1H and 13C NMR spectra (δC 174.2, C-18; 79.7, C-19; δH 4.82, dd, J = 7.6, 2.4 Hz, H-19β; 4.02, dd, J = 8.0 Hz, H-19α). In addition, the 13C NMR and DEPT spectra displayed 20 pairs of signals; these signals corresponded to a pair of methyls, four pairs of methylenes, 10 pairs of methines, and five pairs of nonprotonated carbons (Table 1). The above information and analysis of their 1D and 2D NMR data showed that 1/2 possess a neo-clerodane framework.14 In addition, comparison of these NMR data with those of diterpenoids derived from S. polystachya revealed that 1/2 have structures closely similar to polystachyne B (8),12a differing only in the C-12 side chain moieties. The 1H NMR signals (Table 1) at δH 7.03 (d, J = 6.5 Hz) and 6.14 (dd, J = 8.0, 6.5 Hz) correlated in the COSY spectrum and showed HMBC correlations with a signal at δC 169.7/169.6 (C-16). The above data suggested the presence of a 5-hydroxyfuran-2(5H)-one unit at C-12 of the neo-clerodane framework, and these signals were assigned to H-14 (δC 145.2/ 144.6, C-14) and H-15 (δC 97.1, C-15), respectively. This assignment was supported by the HMBC correlations of H-14 and H-15 with C-13 (δC 137.9/137.6) and of H-12 (δH 5.08, dd, J = 8.0, 7.6 Hz; δC 75.2, C-12) with C-14 and C-16. In the 1 H NMR spectrum of 1/2, the signals at δH 5.33/5.31 (s) and 4.51 (d, J = 4.0 Hz) were assigned to the acetalic protons H-20 (δC 110.6/110.4, C-20) and the oxymethine H-7 (δC 87.8, C7), respectively. This assignment was based on the HMBC correlations of H-20 with C-8 (δC 38.1), C-9 (δC 59.0), and C10 (δC 46.2/46.1) and of H-7 with C-5 (δC 43.0), C-9, and C17 (δC 14.0). Additional signals at δH 3.30 (dd, J = 5.6, 3.6 Hz) and 3.11 (br dd, J = 6.0, 3.2 Hz) were assigned to the protons of the oxirane moiety, H-2 (δC 50.4, C-2) and H-1(δC 55.1, C1), respectively. These spectroscopic data established the structure of compounds 1/2 as depicted.

10c,d

13 14 15

1.71 dd (13.6, 4.4)

δH mult. (J in Hz) 6.33 d (6.0) 6.37 dd (9.0, 6.0) 5.51 d (9.0)

45.7, CH 43.0, C 30.5, CH2

1.24 br d (14.5) 1.64 br dd (14.5, 12.5) 2.14 m 2.24 dd (14.0, 3.0) 2.67 m

87.8, CH

2.95 dq (6.8, 4.0) 2.37 br s 2.04 dd (13.6, 8.0)/ 2.00 dd (13.6, 8.0) 2.74 dd (13.2, 7.6)/ 2.71 dd (13.2, 7.6) 5.08 dd (8.0, 7.6)

7.03 d (6.5) 6.14 d (6.5)/6.10 d (6.8)

38.1, CH 59.0, C 46.2/46.1, CH 34.6/34.4, CH2

2.03 dd (14.5, 2.5) 2.63 dd (14.5, 12.5) 5.36 dd (12.5, 2.0)

75.2, CH 137.9/137.6, C 145.2/144.6, CH 97.1, CH

16 17 18 19α

4.02 d (8.0)

169.7/169.6, C 14.0, CH3 174.2, C 79.7, CH2

19β 20

4.82 dd (7.6, 2.4) 5.33 s/5.31 s

110.6/110.4, CH

1.27 d (6.8)

6.62 br s 7.68 br s 7.75 br s

3.39 dd (8.5, 1.0) 4.58 d (9.0) 1.18 s

δC 118.8, CH 126.8, CH 120.5, CH 74.7, C 47.2, C 21.8, CH2

18.2, CH2

46.8, CH 37.3, C 142.2, C 39.4, CH2

70.3, CH 125.1, C 108.9, CH 143.8, CH 140.4, CH 171.5, C 177.2, C 72.7, CH2

31.9, CH3

OH-15 signal at δH 7.66 (t, 4.8). bOverlapped. cIn DMSO-d6 at 500 MHz (1H) and 125 MHz (13C). dOH-4 signal at δH 5.94 (s). a

A previous phytochemical study of neo-clerodane diterpenoids from S. f ilipes reported the isolation and structural elucidation of salvifilines A−D; salvifilines A (3) and B as well as salvifilines C (5) and D are C-4 epimers. In the present work, only the C-15 epimeric mixtures of 1/2 and 3/4 and the C-16 epimeric mixture of 5/6 were isolated. Comparison of the 1D and 2D NMR data of 1/2 with those of salvifiline A (3) indicated that 1/2 differed from 3/4 by the presence of a 1,2oxirane moiety.15 A single-crystal X-ray diffraction study of salvifiline A/15-epi-salvifiline A (3/4) permitted the assignment of their (4S,5S,7R,8S,9S,10R,12R,15R/15S,20R) absolute configurations [Flack parameter = 0.05(7)]16 and confirmed their epimeric relationship (Figure 1). In addition, the concomitant occurrence of 1/2 with 3/4 and the analysis of their NOESY spectra established the relative configuration of 1/2. The crosspeaks of H-19α with H-10 and H-4 suggested a cis-fused decalin ring and a trans-fused 18,19-γ-lactone moiety (Figure 2). The orientation of the oxirane ring was established as β by the correlations of H-1 and H-2 with H-10 and H-3α. The NOE correlation of H-19β and H-10 with H-20 determined an αdisposition of the oxymethine. The orientation of the 5hydroxyfuran-2(5H)-one unit also was determined as α, based on the correlations of H-12 with H-11β and H3-17. In addition, 3004



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additionally supported by the HMBC correlations of H-1 with C-10 (δC 142.2) and C-9 (δC 37.3) and of H-3 with C-4 (δC 74.7), C-5 (δC 47.2), and C-18 (δC 177.2). In the 1H NMR spectrum a signal at δH 5.94 (s) did not show correlation in the HSQC spectrum, suggesting the presence a tertiary hydroxy group.16 Comparison of the NMR data of 10 with those of 1,10-dehydrosalviarin14a indicated that 10 possessed one more oxygen atom. The C-4 location of the hydroxy group was indicated by the HMBC correlations of HO-4 with C-3, C-4, C5, and C-18. In addition, the relative configuration of 10 was established by the analysis of the NOESY spectrum (Figure 4), Figure 1. ORTEP drawing of compounds 3/4.

Figure 4. Key NOESY correlations for compound 10. Figure 2. Key NOESY correlations for compounds 1/2.

taking into account the same criteria as for 1 and 2. The NOE correlations of the HO-4 with H-6β suggested a β-disposition of the hydroxy group, and this correlation together with those of H-19β with H3-20 indicated the presence of a cis-fused γlactone moiety. The cross-peak of H3-20 with H-8 established the presence of a cis-fused δ-lactone, and the cross-peaks of H14 and H-15 with H-11α suggested an α-oriented furan ring. The structure and the (4S,5R,8S,9S,12R) absolute configuration of 10 was confirmed by a single-crystal X-ray diffraction study [Figure 5, Flack parameter = 0.01(3)].

a single-crystal X-ray crystallographic study confirmed the structures and the relative configuration (Figure 3) proposed for 1/2 based on the analysis of their NMR data.

Figure 3. ORTEP drawing of compounds 1/2.

Polystachyne H (10) was isolated as colorless needles from EtOAc−hexanes. Its HRMS (DART-TOF+) spectra showed an ammonium adduct ion at m/z 374.15957 [M + NH4]+, indicating a molecular formula of C20H24N1O6 (calcd for 374.16036). The IR spectrum indicated the presence of OH (3334 cm−1), γ- and δ-lactone (1775 and 1705 cm−1), and furan (873 cm−1) functionalities. In the 13C NMR spectrum 20 signals were observed, which were classified by the DEPT experiment as a methyl, four methylenes, eight methines, and seven nonprotonated carbons (Table 1). As with compounds 1 and 2, the above data indicated that 10 was also a neo-clerodane diterpenoid. In the 1H NMR spectrum of 10 (Table 1), the signals at δH 7.75 (br s), 7.68 (br s), and 6.62 (br s) were assigned, respectively, to H-16 (δC 140.4), H-15 (δC 143.8), and H-14 (δC 108.9) of the furan moiety. The signals of the olefinic protons at δH 6.37 (1H, dd, J = 9.0, 6.0 Hz), 6.33 (1H, d, J = 6.0 Hz), and 5.51 (1H, d, J = 9.0 Hz) indicated the presence of a 1(10),2-diene moiety in the ring A of the neoclerodane backbone,14a which displayed cross-peaks in the COSY experiment, and were assigned to H-2 (δC 126.8, C-2), H-1 (δC 118.8, C-1), and H-3 (δC 120.5, C-3). This was

Figure 5. ORTEP drawing of compound 10.

Previous research showed that naturally occurring diterpenoids modulate the expression of genes codifying for extracellular matrix proteins in several types of animal cells. Oridonin, purified from Rabdosia rubescens (Lamiaceae), markedly decreased expression of the gene codifying for collagen type I, in hepatic stellate cells.17 On the contrary, kirenol, a diterpenoid present in Siegesbeckia orientalis (Asteraceae), increased the expression of the type I collagen gene in MC3T3-E1 cells.18 Genkwadaphnin, a daphnane-type diterpenoid from the flower buds of Daphne genkwa (Thymelaeaceae), markedly induced the expression of genes codifying for type I, type II, and type X collagens in ATDC5 chondroprogenitor cells.19 In the same manner, tanshinone IIA 3005



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Figure 6. Effect of compounds 1/2, 3/4, 5/6, 7, 8, and 11 on the expression of extracellular matrix proteins in human dermal fibroblasts. The expression of genes codifying for collagen type I (COL1A1) and collagen type III (COL3A3) was analyzed by RT-PCR in total mRNA obtained from proliferative normal human dermal fibroblasts exposed for 48 h to described treatments. Expression of GAPDH was used as a housekeeping gene. Results are presented as mean ± SD. Statistical significance of treatments with respect to the control was determined by one-way ANOVA. P < 0.05 was considered significant (*).

fold), by 1/2 at 25.6 μM (1.9-fold), and by 5/6 at 26.7 μM (1.7-fold) (Figure 7A). In the same manner, 1/2 stimulated the expression of the elastin gene by 1.7- and 2.4-fold with respect to the MB control, exhibiting an effect comparable to the AA control (Figure 7B). Moreover, elastin gene expression was induced between 2.7and 3.2-fold by 8 and between 2.1- and 3.4-fold by 11. All the remaining compounds discretely induced the expression of this gene, and none inhibited the expression of the elastin gene (Figure 7B). Compounds 1−4, 8, and 11 were biologically evaluated in an antimicrobial assay, and compounds 1−6 for their phytotoxicity; however, none of them showed detectable activity.

stimulates the synthesis and deposition of elastin in cultures of human cardiac fibroblasts.20 To initiate the characterization of the biological effects of the diterpenoids described here, their roles in the expression of genes codifying for the type I collagen, COL1A1, type III collagen, COL3A3, type V collagen, COL5A1, and elastin, ELN, in human dermal fibroblasts (Figures 6 and 7) were evaluated. As shown in Figure 6A, compounds 5/6 and 11 did not affect the transcription of COL1A1, while 1/2 induced only moderate increases in the expression of this gene. Compounds 3/4, 7, and 8 induced COL1A1 transcription by 1.6-fold (7 at 29.2 μM and 8 at 0.28 μM) or 1.8-fold (3/4 at 0.27 μM) with respect to MB control. The effect of 3/4 on COL1A1 expression was higher than that observed for the positive control, ascorbic acid (AA) at 100 μM. Genes codifying for other proteins of dermal extracellular matrix were also clearly induced by the diterpenoids isolated from S. polystachya (Figures 6 and 7). Compounds 1/2 and 5/6 induced expression of the COL3A3 gene between 1.5- and 2.2fold, while 8 stimulated the expression of this gene by 2.1- and 2.5-fold at the assayed concentrations, and 11 2.5-fold at 0.29 μM and 3.4-fold at 2.93 μM (Figure 6B). The expression of COL5A1, the gene codifying for type V collagen, was strongly induced by 11 at 2.93 μM (2.5-fold), by 8 at 0.28 μM (2.2-

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 343 polarimeter. UV spectra were recorded on a Shimadzu UV 160U spectrophotometer. IR spectra were obtained on a Bruker Tensor 27 spectrometer. NMR experiments were performed on a Bruker Avance III 400 MHz or on a Varian Unity Plus 500. Chemical shifts were relative to tetramethylsilane, and J values are given in Hz. X-ray crystallographic data were obtained on a Bruker D8 Venture κ-geometry diffractometer with Cu Kα radiation (λ = 1.541 78 Å). HRDARTMS data were recorded on a JEOL AccuTOF JMS-T100LC mass spectrometer. 3006



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Figure 7. Effect of compounds 1/2, 3/4, 5/6, 7, 8, and 11 on the expression of extracellular matrix proteins in human dermal fibroblasts. The expression of genes codifying for collagen type V (COL5A1) and elastin (ELN) was analyzed by RT-PCR in total mRNA obtained from proliferative normal human dermal fibroblasts exposed for 48 h to described treatments. Expression of GAPDH was used as a housekeeping gene. Results are presented as means ± SD. Statistical significance of treatments with respect to the control was determined by one-way ANOVA. P < 0.05 was considered significant (*). hexanes. Fr. B was subjected to flash CC (4.0 × 9.0 cm, 100 mL) using mixtures of CHCl3−acetone. The fractions were analyzed by TLC in the following manner: Fr. B1 (frs. 1−16, eluted with CHCl3−acetone, 95:5), Fr. B2 (frs. 17−23, eluted with CHCl3−acetone, 90:10), and Fr. B3 (frs. 24−31, eluted with CHCl3−acetone, 80:20). From Fr. B2 compounds 3/4 (387 mg) were obtained and purified by crystallization from EtOAc. The mother liquors were subjected to silica gel CC (1.7 × 8.5 cm, 20 mL), eluted with a mixture of hexanes− EtOAc (1:1), to yield from frs. 8−9 compounds 5/6 (25.8 mg) and from frs. 16−18 compounds 3/4 (39.1 mg). Fr. B3 was resubjected to silica gel CC (1.7 × 9.0 cm, 15 mL), eluted with a mixture of hexanes− CHCl3−MeOH, 45:50:5, to give compounds 1/2 (25.3 mg). Fr. C was purified by silica gel CC (4.5 × 8.0 cm, 100 mL) using mixtures of hexanes−EtOAc in increasing polarity to give 7 (100 mg). Compound 8 (6.5 g) from fr. D was obtained by multiple crystallizations from acetone−hexanes. The mother liquors were subjected to several silica gel CC using as eluents mixtures of hexanes−EtOAc, CHCl3−acetone, and CH2Cl2−EtOAc to afford compounds 11 (1.2 g), 9 (7.1 mg), and 10 (6.0 mg). Polystachyne G (1)/15-epi-Polystachyne G (2): colorless crystals, mp 240−242 °C (EtOAc); [α]25D +2 (c 0.1, Me2CO); UV (MeOH) λmax (log ε) 205 (2.62); IR (KBr) νmax 1767, 873 cm−1; 1H and 13C NMR (CDCl3−DMSO-d6) see Table 1; HRMS (DART-TOF+) m/z 391.13979 [M + H]+ (calcd for C20H23O8, 391.13929). X-ray crystallographic analysis of polystachyne G (1)/15-epi-polystachyne G (2): formula C20H22O8, MW = 390.38, monoclinic, space group P21,

Column chromatography (CC) assisted with vacuum was performed on silica gel 60 (Merck G), unless otherwise stated. Silica gel 230−400 mesh (Macherey−Nagel) was used for flash chromatography. TLC was carried out on precoated Macherey−Nagel Sil G/UV254 plates of 0.25 thickness, and spots were visualized by UV light at 254 nm and then spraying with 3% CeSO4 in 2 N H2SO4, followed by heating. Plant Material. The leaves and flowers of S. polystachya were collected in Huitzilac, Morelos, México, in September 2011. A voucher specimen was deposited (MEXU-573762) at the National Herbarium, Instituto de Biologia,́ Universidad Nacional Autónoma de México. Extraction and Isolation. The powdered plant material (4.25 kg) was defatted with hexanes (16 L) and extracted by percolation with Me2CO (12 L) to obtain a dried extract (247 g), which was dissolved in a mixture of MeOH−H2O (4:1, 1.5 L) at 50 °C and partitioned with hexanes (3 × 1 L). The MeOH was evaporated under reduced pressure, water (0.5 L) was added, and the mixture was partitioned again with EtOAc (4 × 0.3 L) to give 68 g of residue. The EtOAc fraction was subjected to silica gel 60 G CC (10.5 × 12.0 cm, 500 mL) using hexanes−EtOAc mixtures as eluents. The 57 fractions obtained were analyzed by TLC and grouped as follows: fraction A (frs. 12−17, 7.0 g, eluted with hexanes−EtOAc, 7:3), fraction B (frs. 18−21, 4.81 g, eluted with hexanes−EtOAc, 7:3), fraction C (frs. 22−24, 4.40 g, eluted with hexanes−EtOAc 3:2), and fraction D (frs. 25−38, 27.0 g, eluted with hexanes−EtOAc, 1:1). Fraction A was subjected to several silica gel CC eluted with hexanes−EtOAc in increasing polarity to give 259 mg of 7, which was purified by crystallization with acetone− 3007



Article

(fw: 5′-AAGTCAAGGAGAAAGTGGTCG-3′, rv: 5′CTCGTTCTCCATTCTTACCAGG-3′), collagen type V (fw: 5′CGGAACCTTGACGAGAACTAC-3′, rv: 5′-TCTCCCTTTTGGCCTTTCTC-3′), elastin (fw: 5′-CCTGGCTTCGGATTGTCTC3′, rv: 5′-CAAAGGGTTTACATTCTCCACC-3′), and GAPDH (fw: 5′-GAAGGTGGTGAAGCAGGCGT-3′, rv: 5′-ATGTGGGCCATGAGGTCCACCA-3′). After electrophoresis on 1.5% agarose gel, each band was quantified by ImageLab software (Bio-Rad). Antimicrobial Activities. Qualitative antibacterial tests against Escherichia coli and Bacillus sp. of compounds 1−4, 8, and 11 were carried out in vitro by the agar diffusion method. The medium used was Bioxon nutrient agar for both bacterial strains (pH 6.8). Bacterial cells were suspended in saline solution at a density of 108 CFU mL−1, and 0.1 mL was inoculated in nutrient agar plates. Paper filter disks with 8, 4, 2, 1, and 0.5 μg of each compound were placed onto inoculated plates. Plates were incubated at 37 °C for 18 h under aerobic conditions. Filter disks with ampicillin and DMSO were used as positive and negative controls. All compounds were dissolved in DMSO. The zone of growth inhibition was measured manually using a Vernier caliper.22 The qualitative antifungal tests against Candida albicans ATCC 10235 and C. glabrata CBS138 were carried out in the same way as the antibacterial test. However, the medium was Bioxon potato dextrose agar, the fungal inocula were prepared at a final concentration of 105 CFU mL−1, and the plates were incubated at 37 °C for 24 h under aerobic conditions. Filter disks with amphotericin B and DMSO were used as positive and negative controls.23 Phytotoxic Activity. The phytotoxic activity was evaluated using a germination inhibition bioassay.24 Lycopersicum esculentum, Capsicum annuum, and Lactuca sativa seeds were used for the tests. The surface seeds were sterilized in 2% NaOCl for 10 min, rinsed three times in sterile distilled water, and air-dried on filter paper. A layer of Whatman No. 1 filter paper was placed in 90 mm diameter glass Petri dishes. Compounds 1−6 were dissolved in acetone and water containing 0.1% DMSO in a 1:1 proportion at a concentration of 10 mM. Corresponding dilutions were made to obtain the assayed concentrations (100, 10, and 1 μM). In each dish, 30 seeds were placed equidistantly, and 2.5 mL of a solution of each compound was added. The Petri dishes were sealed with Parafilm to prevent solvent evaporation and incubated for 6 days in the dark inside a growth chamber at 25 ± 2 °C. A similar treatment with distilled water served as a control. Starting from the second day of the experiment, germinated seeds were counted daily. A seed with 2 mm of radicle was considered germinated. The percentage of germination in each case was calculated and compared to the control (pendimethalin), and the data were analyzed by ANOVA (p < 0.05).

unit cell dimensions a = 10.9433(12) Å, b = 7.8994(9) Å, c = 11.2708(17) Å, α = 90°, β = 118.237(7)°, γ = 90°, V = 858.36(19) Å3, Z = 2, Dc = 1.510 g/cm3, F(000) = 412. A total of 3289 unique reflections were collected, with 1891 reflections greater than I ≥ 2σ(I) (Rint = 0.2394). The structure was solved by direct methods and refined by full-matrix least-squares on F2, with anisotropic displacement parameters for non-hydrogen atoms at final R indices [I > 2σ(I)], R1 = 0.1321, wR2 = 0.3358; R indices (all data), R1 = 0.1919, wR2 = 0.3676. Flack parameter = 0.5(4). Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1561999) and can be obtained free of charge via http://www.ccdc.ac.uk./data_request/cif. Polystachyne H (10): colorless crystals, mp 234−236 °C (acetone− hexanes); [α]25D −26 (c 0.2, Me2CO); UV (MeOH) λmax (log ε) 205 (3.76), 268 (3.53); IR (KBr) νmax 3334, 1775, 1705, 873 cm−1; 1H and 13 C NMR (CDCl3−DMSO-d6) see Table 1; HRMS (DART-TOF+) m/z 374.15957 [M + NH4]+ (calcd for C20H24N1O6, 374.16036). Xray crystallographic analysis of polystachyne H (10): formula C20H20O6, MW = 356.36, orthorhombic, space group P212121, unit cell dimensions a = 7.7431(7) Å, b = 14.6879(14) Å, c = 14.9959(14) Å, V = 1705.5(3) Å3, Z = 4, Dc = 1.388 g/cm3, F(000) = 752. A total of 3479 unique reflections were collected, with 3435 reflections greater than I ≥ 2σ(I) (Rint = 0.0261). The structure was solved by direct methods and refined by full-matrix least-squares on F2, with anisotropic displacement parameters for non-hydrogen atoms at final R indices [I > 2σ(I)], R1 = 0.0284, wR2 = 0.0750; R indices (all data), R1 = 0.0287, wR2 = 0.0753. Flack parameter = 0.01(3). Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1562001) and can be obtained free of charge via http://www.ccdc.ac.uk./data_request/cif. X-ray Crystallographic Analysis of Salvifiline A (3)/15-episalvifiline A (4): moiety formula C20H22O7, MW = 374.38, monoclinic, space group P21, unit cell dimensions a = 10.9006(6) Å, b = 7.8767(5) Å, c = 11.1971(6) Å, α = 90°, β = 118.648(2)°, γ = 90°, V = 843.70(9) Å3, Z = 2, Dc = 1.474 g/cm3, F(000) = 396. A total of 3395 unique reflections were collected, with 3260 reflections greater than I ≥ 2σ(I) (Rint = 0.0514). The structure was solved by direct methods and refined by full-matrix least-squares on F2, with anisotropic displacement parameters for non-hydrogen atoms at final R indices [I > 2σ(I)], R1 = 0.0303, wR2 = 0.0724; R indices (all data), R1 = 0.0321, wR2 = 0.0737. Flack parameter = 0.05(7). Crystallographic data have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1562000) and can be obtained free of charge via http://www.ccdc.ac.uk./data_request/cif. Expression of the Extracellular Matrix Components. Cell Culture. Normal human dermal fibroblasts (HDFs) were derived from biopsies of patients subjeted to plastic surgery procedures who gave their informed consent for the use of discarded tissues. Collagenasedissagregated HDFs were cultured in L15 Leibovitz medium added with 10% fetal bovine serum (HyClone, Logan UT) and antibiotics (penicillin 80 U/mL, streptomycin 80 μg/mL) (basal medium; BM) under a conventional humidified atmosphere at 37 °C. HDFs at 70% confluence were refed with BM added with 0.26−0.29, 2.56−2.93, or 25.6−29.3 μM of compounds 1/2, 3/4, 5/6, 7, 8, or 11. Control cultures were maintained in BM or BM added with 100 μM AA.21 RT-PCR Analysis. Total RNA from HDF maintained for 48 h under the aforementioned conditions was extracted with TRIzol and subjected to reverse transcription. cDNA was synthesized from 1 μg of total RNA using 200 U of reverse transcriptase (M-MLV RT) and 0.5 μg of oligo dT. PCR was performed in a final volume of 15 μL reaction mix containing 300 ng of the RT reaction mixture, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 μM of each primer, and 1.25 U of Taq DNA polymerase. Thermal cycling over 30 cycles consisted in an initial denaturation at 95 °C for 4 min, then 95 °C for 30 s, 56.5 °C for 1 min for collagen type I, III, and V and elastin, and 62 °C for 1 min for GAPDH and then 72 °C for 1 min. It was terminated by a final extension at 72 °C for 5 min. GAPDH mRNA level was used for sample standardization. The following oligonucleotides were used as primers: collagen type I (fw: 5′-CCCCTGGAAAGAATGGAGATG3′, rv: 5′-TCCAAACCACTGAAACCTCTG-3′), collagen type III

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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.7b00591.

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NMR spectra for compounds 1, 2, and 10 (PDF) CIF files of 1−4 and 10 (CIF) (CIF) (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +52 444 834 2000. Fax: +52 444 834 2010. E-mail: [email protected] (E. Bautista). ORCID

Elihú Bautista: 0000-0002-7050-9182 Notes

The authors declare no competing financial interest. 3008



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Article

(24) Julio, L. F.; Barrero, A. F.; Herrador-Del Pino, M. M.; Arteaga, J. F.; Burillo, J.; Andres, M. F.; Díaz, C. E.; González-Coloma, A. J. Nat. Prod. 2016, 79, 261−266.

ACKNOWLEDGMENTS We acknowledge the technical assistance of R. Gaviño, H. Rios, I. Chávez, E. Huerta, A. Peña, B. Quiroz, C. Márquez, E. Garcia,́ ́ L. Martinez, and R. Patiño. E.B. is grateful to CONACYT for a Research Fellowship.

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REFERENCES

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