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Allelopathic potential of alkylphenols from Dactylis glomerata subsp. hispanica (Roth) Nyman
Phytochemistry Letters 5 (2012) 206–210
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Allelopathic potential of alkylphenols from Dactylis glomerata subsp. hispanica (Roth) Nyman Monica Scognamiglio a, Vittorio Fiumano a, Brigida D’Abrosca a, Severina Pacifico a, Anna Messere b, Assunta Esposito a, Antonio Fiorentino a,* a b
Dipartimento di Scienze della Vita – Laboratorio di Fitochimica – Seconda Universita` degli Studi di Napoli - via Vivaldi 43, I-81100 Caserta, Italy Dipartimento di Scienze Ambientali – Seconda Universita` degli Studi di Napoli - via Vivaldi 43, I-81100 Caserta, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2011 Received in revised form 13 December 2011 Accepted 16 December 2011 Available online 27 December 2011
Eleven alkylphenols were isolated from the aerial parts of Dactylis glomerata subsp. hispanica, six of them described for the first time. The structural characterization of these compounds has been elucidated by 1D and 2D NMR techniques. The fragmentation patterns of the metabolites obtained by GC–MS analysis allowed the side chain to be elucidated. The allelopathic potential of three alkylphenols, representative of each homologous series of alkylphenols from D. glomerata subsp. hispanica, has been assayed on D. glomerata subsp. hispanica and an herbaceous coexisting species, Phleum subulatum. The bioassay results showed a high auto-stimulation values of germination and root and shoot elongation for D. glomerata subsp. hispanica at high concentrations. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Dactylis glomerata subsp. hispanica Poaceae Alkylphenols Spectroscopic analysis Allelopathy
1. Introduction Allelopathy is one of the most sophisticated mechanisms of plant defenses. It could be understood as the ability of plants to inhibit or stimulate growth of other organisms in the environment by releasing chemicals, named allelochemicals. These compounds, mainly secondary metabolites, introduced in the environment by exudation, decomposition, leaching, and volatilization, may be toxic or stimulatory to the releaser, other plant species, and soil microorganisms (Papavizas, 1966; Grodzinsky, 1992; Rice, 1995; Yu, 1999). Allelopathic plants get some benefit from the release of allelochemicals by inhibiting coexisting competitors for nutrients and/or by promoting their development and propagation. As a consequence, allelopathy has been discussed as one of the main factors affecting plant community composition, structures and productivity (Rice, 1984, Chaves and Escudero, 1997). Among the several aspects influencing allelopathic effects (e.g. active concentration of allelochemicals in the soil; biological and chemical characteristics of the soil, plant density, etc.), the life cycle of the allelopathic plant represents one of the most important characters influencing plant community structure. Perennial plant species are likely to release and add allelochemicals to the soil over more than
* Corresponding author. Tel.: +39 0823 274576; fax: +39 0823 274571. E-mail addresses: antonio.fi[email protected], antonio.fi[email protected] (A. Fiorentino).
one season. This is probably the reason why most reports concerning the ability of a plant to invade or dominate in an ecosystem, has involved allelopathic perennials (Chaves and Escudero, 1997; Mallik, 2000). For annual plant species the allelopathic activity has been suggest to be part of their success as weeds with several species showing growth pattern characterized by several life cycles completed within one year with the aim to maintain the pool of allelochemicals (Inderjit and Dakshini, 1998). Moreover perennial plants exert allelopathy also against itself mainly by inhibiting the seed germination. This autotoxic effects occur when a plant species inhibit or delay germination and growth of the same plant species (Putnam and Tang, 1986; Singh et al., 1999). On the contrary, annual plants, to make sure their survival, seem to affect positively the germination of their seeds and negatively that of other species (Lovett and Jackson, 1980). Allelopathic effects of both perennials and annual plants, might be intensified in natural communities characterized by harsh environmental conditions and other constraints (Rice, 1984). This is particularly true in Mediterranean region where, to face the biotic and abiotic stress, the biodiversity of plants present in this area, produces a large variety of secondary metabolites causing stimulating or inhibiting effects on coexisting organisms and regulating, in such away, the equilibrium within the ecosystem (Thorpe et al., 2009; Xuana et al., 2005; Bonanomi et al., 2006; Esposito et al., 2008). In the aim to investigate on the allelopathic interactions among plants of Mediterranean macchia vegetation,
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.12.009
M. Scognamiglio et al. / Phytochemistry Letters 5 (2012) 206–210
we undertook the chemical and biological study of several herbaceous and shrub species present in the Natural Reserve of Castel Volturno (D’Abrosca et al., 2005, 2010; Fiorentino et al., 2007, 2008b, 2011; Pacifico et al., 2009), a flat coastal area located in the north of Naples (Southern Italy). These studies led to the isolation of a number of secondary metabolites, able to stimulate or inhibit seed germination and seedling growth of coexisting species (Fiorentino et al., 2008a, 2009a,b, 2010). Continuing the phytochemical investigation of this area, we undertook the chemical study of Dactylis glomerata subsp. hispanica (Roth) Nyman (Poaceae), a perennial steno-Mediterranean grass with a frequent dominance cover in herbaceous community.
HO
R OH
207
HO
1
5
1'
O
R
1
5
3
3
OH
OH
1'
R
1 R=(CH2)19CH3
3 R=(CH2)15CH3
9
2 R=(CH2)21CH3
4 R=(CH2)17CH3
10 R=(CH2)17CH3 11 R=(CH2)19CH3
5 R=(CH2)18CH3
R=(CH2)15CH3
6 R=(CH2)19CH3 7 R=(CH2)21CH3 8 R=(CH2)23CH3 Fig. 1. Chemical structures of alkylphenols from Dactylis glomerata subsp. hispanica.
2. Results and discussion Eleven alkylphenols were isolated from leaves of D. glomerata subsp. hispanica. Compounds 3, 5–8 and 11 were isolated for the first time (Fig. 1). To elucidate the structures, NMR and GC–MS analyses were carried out. 1D and 2D-NMR spectra were useful to characterize the aromatic group. Three different substitution patterns for the aromatic ring were identified, and different homologous of each series did not show differences in the NMR data. In fact, the 1H NMR spectrum of compounds 1 and 2 was consistent with a 1,3,5 substitution, since two signals at d 6.14 (brs, H-2) and 6.19 (brs, H4, H-6) which can be integrated, respectively, for one and two protons, were evident. In the HMBC experiment for these compounds, correlations were observed between the proton H-2 and the carbons at d 107.9 (C-4, C-6), and between the proton H-4 (and H-6) and the carbons at d 100.2 (C-2) and 157.2 (C-1, C-3). Moreover, the proton H-4 (and H-6) showed correlation with the carbon at d 36.1 (C-10 ), and the benzylic proton at d 2.45 (2H, J = 7.5 Hz, H-10 ) with C-4 and C-6. Thus, compounds 1 and 2 are 5alkylresorcinols, having reference to the aromatic A skeleton. The 1H NMR spectrum of compounds 3–8 contained a pattern distinctive of a tetrasubstituted aromatic ring, showing signal at d 6.21 (2H, s) assignable to both H-4 and H-6 protons and d 2.08 (3H, s, C-2–CH3). The HMBC experiment for these compounds showed correlations between the aromatic methyl proton and the carbons at d 107.9 (C-4 and C-6) and 154.7 (C-1 and C-3), and between the proton H-4 (and H-6) and C-1, C-3, C-6 (and C-4). Furthermore, correlations between the proton H-4 (and H-6) and the carbon at 35.7 (C-10 ), and vice versa the benzylic proton at d 2.43 (2H, t, J = 7.5 Hz, H-10 ) and the carbon at d 141.9 (C-5), C-4 and C-6 were observed. Thus, compounds 3–8 are identified to be 5-alkyl-2methylresorcinols, referring to the aromatic B skeleton. The 1H NMR spectrum of compounds 9–11 revealed a doublet of doublet at d 6.21 (1H, H-2), two broad singlets at d 6.19 (1H, H-4)
and 6.26 (1H, H-6) and a singlet at d 3.76 (3H, C-1–OCH3), diagnostic of an aromatic methoxyl. In the HMBC experiment for these compounds, correlations between the proton H-2 and the carbons at d 55.2 (C-1–OCH3), 106.8 (C-4) and 158.1 (C-3), between the aromatic methoxyl protons and the carbon at d 98.6 (C-2), and between both protons H-4 and H-6 and the carbon at d 145.8 (C-5) were revealed. Once again, correlations between the proton H-4 and the carbon at d 36.0 (C-10 ), and between the proton at d 2.44 (2H, H-10 ) and C-4 were evident. Thus, compounds 9–11 are determined to be 5-alkylresorcinol-3-methyl ethers, being ascribable to the aromatic C skeleton. No further information about structural features of the alkylic chains could be deduced by NMR experiments. Underivatized compounds were analyzed by GC–MS and specific ion fragmentation patterns allowed the aromatic skeletons to be confirmed and the side chain structures elucidated. A base peak in the EIMS spectrum at m/z 124 confirmed the presence of the resorcinol moiety in compounds 1–2, while a base peak at m/z 138 supported the presence of a 2-methylresorcinol, or a resorcinol methyl ether, or a 2,5-dihydro-1,4-benzochinon residue in compounds 3–8, 9–11, respectively. Molecular weights and spectral characteristics (Table 1) suggested the presence of all unbranched, saturated, odd numbered hydrocarbon chains from C17 to C25, except for compound 5, which is 5-eicosanoyl-2methylresorcinol. Both metabolites 1 and 2 are reported as constituents of Poaceae species (Kno¨dler et al., 2010). Compound 4, known as polygonocinol, has been previously isolated from Polygonum maritimum (Kazantzoglou et al., 2009). 3-Heptadecyl-5-methoxyphenol (9) was described as metabolite from Oxalis erythrorhiza active towards Leishmania amazonensis and Leishmania donovani promastigotes (Feresin et al., 2003). Finally, compound 10 was isolated from the aerial parts of Artemisia annua (Brown, 1992).
Table 1 GC–MS data of the alkylphenols in D. glomerata subsp. hispanica. Comp.
RT (min)
[M]+. m/z (% rel. abundance)
Major fragment ions m/z (% relative abundance)
1 2 3 4 5 6 7 8 9
21:46 26:27 16:52 19:16 20:47 22:50 27:53 35:44 15:49
404 432 362 390 404 418 446 474 362
10
17:39
390 (3.4)
11
20:21
418 (3.0)
362 222 320 348 218 376 207 221 320 180 348 125 207
(2.1) (2.0) (4.2) (3.5) (2.5) (3.5) (3.8) (2.0) (3.5)
(0.1), (0.2), (0.2), (0.2), (6.6), (0.1), (9.2), (0.6), (0.2), (3.0), (0.1), (1.9), (7.5),
250 180 236 306 203 334 193 207 306 165 306 107 193
(0.1), (0.5), (0.2), (0.1), (3.6), (0.1), (1.3), (6.9), (0.1), (0.7), (0.1), (2.0), (0.9),
236 (0.1), 222 (0.1), 194 (0.2), 180 (0.5), 166 (3.6), 151 (0.7), 137 (8.5), 124 (100) 166 (3.8), 151 (0.7), 137 (8.4), 124 (100) 222 (0.4), 207 (1.1), 194 (0.5), 180 (3.3), 165 (0.8), 151 (10.0), 138 (100), 123 (3.4) 236 (0.1), 222 (0.3), 207 (0.3), 180 (3.0), 165 (0.6), 151 (9.4), 138 (100), 123 (3.2) 151 (9.3), 138 (100), 123 (5.0), 109 (5.2) 207 (0.5), 194 (0.4), 180 (3.4), 166 (0.6), 151 (9.3), 138 (100), 123 (2.9), 109 (1.0) 180 (3.5), 151 (8.9), 138 (100), 123 (3.8) 180 (3.3), 166 (0.8), 152 (1.7), 138 (100), 123 (3.5) 292 (0.1), 278 (0.1), 264 (0.1), 250 (0.1), 236 (0.2), 222 (0.3), 207 (0.6), 194 (0.4), 151 (11.2), 138 (100), 125 (1.9), 107 (2.1), 91 (1.3) 264 (0.1), 222 (0.3), 207 (1.2), 194 (0.4), 180 (3.0), 165 (0.7), 151 (10.6), 138 (100), 91 (1.1) 180 (3.2), 166 (0.8), 151 (12.3), 138 (100), 125 (2.1), 107 (2.8)
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208
Table 2 Phytotoxicity effects of compound 1, 4 and 9 at three different concentration (H, high = 103 M; M, medium = 106 M; L, low = 109 M). The values are referred as % from control with: 0 = no effect (range value: 5 to 5); ++, high stimulation (range value: up to 25); +, low stimulation (range value: 5–25); , low inhibition (range value: 5 to 25). Compound
1
Concentration
H
M
L
H
M
L
M
L
++ 0
0 0
0 0
++ +
+ +
0
0 0
+ 0
++ ++
+ +
0 0
+ ++
+ +
0
0 0
0 +
++ +
+ +
+ 0
++ 0
+ 0
+
+ +
+ 0
Germination Dactylis glomerata Phleum subulatum Root elongation Dactylis glomerata Phleum subulatum Shoot elongation Dactylis glomerata Phleum subulatum
4
This class of secondary metabolites, characterized by a long aliphatic chain bonding a phenolic moiety, is reported to have different biological activities, since they have been identified as responsible for antioxidant activity in cashew (Anacardium occidentale) (Trevisan et al., 2006), allelophatic potential (Dayan et al., 2010), cytotoxicity against various tumour cell lines (Kubo et al., 1993), and antinematodal properties (Alen et al., 2000). Synthetic alkylphenols have been used as non-ionic surfactants in a variety of industrial and household applications; their breakdown products, octyl- and nonylphenols have been proposed to function as oestrogens and have been attributed a role in endocrine disruption (Soares et al., 2008). Compounds 1, 4 and 9, representative of each homologous series of alkylphenols from D. glomerata subsp. hispanica, have been assayed on D. glomerata subsp. hispanica and Phleum subulatum to evaluate their effects on seed germination and plant growth. The results, reported in Table 2, showed high auto-stimulation values of germination and root and shoot elongation for D. glomerata subsp. hispanica at high concentration of compounds 1 and 4, while low or no effects have been evidenced by the compound 9. As regards Phleum subulatum, compounds 1 and 4 showed significative stimulating effects only for the root elongation and at the highest concentration, while low or no effect, respect to the control, was found at the lowest concentrations. No significative effects for both the test species were observed for compound 9. Despite the wide evidences of the auto-inhibiting effects of allelochemicals (Putnam and Tang, 1986; Singh et al., 1999; Fernandez et al., 1999), few data report the role of allelochemicals in terms of positive effects of autostimulation on root (Lovett and Jackson, 1980) and shoot growth (Tomita-Yokotani et al., 2003) as observed in this work. Although the nature of the stimulatory allelochemical remains to be defined, we can assume that the observed positive effects on germination and growth produced by allelochemicals of D. glomerata subsp. hispanica could represent an adaptative strategy to make sure the survival, by enhancing the competitive capacity of the species in high harsh environmental conditions through the accumulation of allelochemicals in the soil until the following vegetative period in the spring season (unpublished results). 3. Experimental 3.1. General experiment procedures TLC was performed on Kieselgel 60 F254 (Merck) or RP-8 F254 (Merck) plates with 0.2, 0.5 or 1 mm film thickness. Column chromatography (CC) was performed on Kieselgel 60 (70–240 mesh, Merck), RP-18 (230–400 mesh, Baker), NH2 (40–63 mm, Merck) or on Sephadex LH-20 (Pharmacia). NMR spectra were recorded at 300 (1H) and 75 MHz (13C) on a Varian 300 FT-NMR spectrometer in CDCl3 solns, at 25 8C; d (chemical shift) in ppm, J (coupling constant)
9
in Hz. Proton-detected heteronuclear correlations were measured by gradient heteronuclear single-quantum coherence (HSQC) optimized for 1J(H,C) = 140 Hz, by gradient heteronuclear multiplebond coherence (HMBC) optimized for nJ(H,C) = 8 Hz. GC/MS analyses were carried out using a HP 6890 GC instrument (Zebron ZB-5MS column, He flow 1.0 ml/min) coupled with a 5973 N mass spectrometer, equipped with an electron ionization source (EIMS). Underivatized samples were analyzed in the following conditions: initial temperature 45 8C (2 min isothermal), from 45 to 300 8C at 25.5 8C/min, and then isothermal for 25 min. 3.2. Plant material Plants of Dactylis glomerata subsp. hispanica (Roth) Nyman (syn. Dactylis hispanica Roth) were collected in field at vegetative state, in June 2010, in ‘‘Castel Volturno’’ Nature Reserve (Southern Italy). A voucher specimen (CE0115) has been deposited at the Herbarium of the Dipartimento di Scienze della Vita of SUN. 3.3. Extraction and isolation Dried aerial parts of D. glomerata subsp. hispanica (1744.0 g) were infused in petroleum ether (PE) for 3 days in a refrigerated chamber at 4 8C in the dark. After distillation of the solvent in vacuum we obtained PE crude extract (12.3 g). The PE extract was chromatographed on SiO2 to obtain three fractions, A, B and C. The first one, eluted with PE/CHCl3 (1:1), was chromatographed on Sephadex LH-20 eluting with Ex/CHCl3/MeOH (6:1:1) and collecting fractions of 10 mL. Fractions 7–9 were chromatographed by NH2 CC eluting with PE/EtOAc solns. The fraction eluted with PE/ EtOAc (19:1) was chromatographed by SiO2 TLC eluting with CHCl3/MeOH (19:1) to give a fraction containing compounds 9, 10 and 11 definitively purified by RP-18 HPLC [MeOH–H2O (4:1)]. Fraction B, eluted with PE/CHCl3 (1:1), was chromatographed on Sephadex LH-20 eluting with Ex/CHCl3/MeOH (5:1:1) and collecting fractions of 10 mL. Fractions 13–16 were chromatographed by SiO2 TLC eluting with EtOAc/CHCl3 (19:1) to give two fractions. Both the fractions were definitively purified by RP-18 HPLC [MeOH–H2O (4:1)]. The first fraction furnished compounds 4, 5, 6, 7 and 8; the second one gave pure compounds 3, 4 and 5. Fraction C, eluted with pure CHCl3 was chromatographed on Sephadex LH20 eluting with Ex/CHCl3/MeOH (5:1:1) and collecting fractions of 10 mL. Fractions 8–12 were chromatographed by NH2 CC eluting with PE/EtOAc/MeOH solns. The fraction eluted with pure MeOH contained compounds 1 and 2. 3.4. 5-Heptadecyl-2-methylbenzene-1,3-diol 3 Colourless oil, UV lmax (log e) (MeOH) 280 (3.79) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29
M. Scognamiglio et al. / Phytochemistry Letters 5 (2012) 206–210
(overlapped, 28H, H-30 –H-160 ), 0.86 (t, J = 7.2 Hz, 3H, H-170 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-150 ), 22.9 (C-160 ), 14.4 (C-170 ), 7.9 (CH3); Anal. Calcd for C24H42O2: C, 79.50; H, 11.68. Found: C, 79.59; H, 11.97. 3.5. 5-Icosyl-2-methylbenzene-1,3-diol 5 Colourless oil, UV lmax (log e) (MeOH) 280 (3.76) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 34H, H-30 –H-190 ), 0.86 (t, J = 7.2 Hz, 3H, H-200 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-180 ), 22.9 (C-190 ), 14.4 (C-200 ), 7.9 (CH3); Anal. Calcd for C27H48O2: C, 80.14; H, 11.96. Found: C, 79.99; H, 11.86. 3.6. 5-Henicosyl-2-methylbenzene-1,3-diol 6 Colourless oil, UV lmax (log e) (MeOH) 280 (3.24) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 36H, H-30 –H-200 ), 0.86 (t, J = 7.2 Hz, 3H, H-210 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-190 ), 22.9 (C-200 ), 14.4 (C-210 ), 7.9 (CH3); Anal. Calcd for C28H50O2: C, 80.32; H, 12.04. Found: C, 80.41; H, 11.99. 3.7. 2-Methyl-5-tricosylbenzene-1,3-diol 7 Colourless oil, UV lmax (log e) (MeOH) 280 (3.64) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 40H, H-30 –H-220 ), 0.86 (t, J = 7.2 Hz, 3H, H-230 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-210 ), 22.9 (C-220 ), 14.4 (C-230 ), 7.9 (CH3); Anal. Calcd for C30H54O2: C, 80.65; H, 12.18. Found: C, 80.57; H, 12.09. 3.8. 2-Methyl-5-pentacosylbenzene-1,3-diol 8 Colourless oil, UV lmax (log e) (MeOH) 279 (3.64) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 44H, H-30 –H-240 ), 0.86 (t, J = 7.2 Hz, 3H, H-250 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-230 ), 22.9 (C-240 ), 14.4 (C-250 ), 7.9 (CH3); Anal. Calcd for C32H58O2: C, 80.95; H, 12.31. Found: C, 80.78; H, 13.23. 3.9. 3-Heptadecyl-5-methoxyphenol 9 Colourless oil, UV lmax (log e) (MeOH) 278 (3.07) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 28H, H-30 –H-160 ), 0.81 (t, J = 6.6 Hz, 3H, H-170 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C-3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-150 ), 22.7 (C-160 ), 14.1 (C-170 ); Anal. Calcd for C24H42O2: C, 79.50; H, 11.68. Found: C, 79.45; H, 11.71. 3.10. 3-Nonadecyl-5-methoxyphenol 10 Colourless oil, UV lmax (log e) (MeOH) 278 (3.45) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers
209
to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H-10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 32H, H-30 –H-180 ), 0.81 (t, J = 6.6 Hz, 3H, H-190 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-170 ), 22.7 (C-180 ), 14.1 (C-190 ); Anal. Calcd for C26H46O2: C, 79.94; H, 11.87. Found: C, 79.90; H, 11.81. 3.11. 3-Henicosyl-5-methoxyphenol 11 Colourless oil, UV lmax (log e) (MeOH) 278 (3.86) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H-10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 36H, H-30 –H-200 ), 0.81 (t, J = 6.6 Hz, 3H, H-210 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-190 ), 22.7 (C-200 ), 14.1 (C-210 ); Anal. Calcd for C28H50O2: C, 80.32; H, 12.04. Found: C, 80.26; H, 11.95. 3.12. Phytotoxicity test Seeds were selected for uniformity observing them under a binocular microscope in order to discard the undersized and damaged ones. Furthermore, a preliminary test to evaluate the germinability of the selected test species was carried out in a growth chamber KBW Binder 240 at 27 8C in the dark. The observed percentage of germinability for D. glomerata subsp. hispanica and P. subulatum was of 90%. The bioassays were conducted as described by Fiorentino et al. (2009a). For each metabolite, three different concentrations were tested: 103 M, 106 M, and 109 M (high, medium and low concentration, respectively). 50 mm diameter Petri dishes and 1 mL test solutions were used. The dishes were placed in a growth chamber KBW Binder 240 at 27 8C in the dark for six days (no more germination occurred after this time). Seedlings were frozen at 20 8C to avoid further growth up to the root and shoot elongation measurements. Germination rate as well as root and shoot lengths were recorder using a Fitomedß system (Castellano et al., 2001). Data are reported as percentage differences with respect to control. Hence positive values indicate stimulation, while negative values represent inhibition of the studied parameters. Statistical analysis was carried out on the data set. References Alen, Y., Nakajima, S., Nitoda, T., Baba, N., Kanzaki, H., Kawazu, K., 2000. Antinematodal activity of some tropical rainforest plants against the pinewood nematode, Bursaphelenchus xylophilus. Z. Naturforsch. C. 55, 300–303. Bonanomi, G., Sicurezza, M.G., Caporaso, S., Esposito, A., Mazzoleni, S., 2006. Phytotoxicity dynamics of decaying plant materials. New Phytol. 169, 571–578. Brown, G.D., 1992. Two new compounds from Artemisia annua. J. Nat. Prod. 55, 1756–1760. Castellano, D., Macı´as, F.A., Castellano, M., Cambronero, R., June 2001. Inventor. University of Ca´diz, assignee. FITOMED (Automated system for the measurement of variable lengths). Spain Patent No. P9901565. Chaves, N., Escudero, J.C., 1997. Allelopathic effect of Cistus ladanifer on seed germination. Funct. Ecol. 11, 432–440. D’Abrosca, B., Fiorentino, A., Golino, A., Monaco, P., Oriano, P., Pacifico, S., 2005. Carexanes: prenyl stilbenoid derivatives from Carex distachya. Tetrahedron Lett. 46, 5269–5272. D’Abrosca, B., Fiorentino, A., Ricci, A., Scognamiglio, M., Pacifico, S., Piccolella, S., Monaco, P., 2010. Structural characterization and radical scavenging activity of monomeric and dimeric cinnamoyl glucose esters from Petrorhagia velutina leaves. Phytochem. Lett. 3, 38–44. Dayan, F.E., Rimando, A.M., Pan, Z., Baerson, S.R., Gimsing, A.L., Duke, S.O., 2010. Sorgoleone. Phytochemistry 71, 1032–1039. Esposito, A., Fiorentino, A., D’Abrosca, B., Izzo, A., Cefarelli, G., Golino, A., Monaco, P., 2008. Potential allelopathic interferences of Melilotus neapolitana metabolites
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Kno¨dler, M., Most, M., Schieber, A., Carle, R., 2010. A novel approach to authenticity control of whole grain durum wheat (Triticum durum Desf.) flour and pasta, based on analysis of alkylresorcinol composition. Food Chem. 118, 177–181. Kubo, I., Ochi, M., Vieira, P.C., Komatsu, S., 1993. Antitumor agents from the cashew (Anacardium occidentale) apple juice. J. Agric. Food Chem. 41, 1012–1015. Lovett, J.V., Jackson, H.F, 1980. Allelopathic activity of Camelina sativa (L.) Crantz in relation to its phyllosphere bacteria. New Phytol. 86, 273–277. Mallik, A.U., 2000. Challenges and opportunities in allelopathy research: a brief review. J. Chem. Ecol. 26, 2007–2009. Pacifico, S., D’Abrosca, B., Pascarella, M.T., Letizia, M., Uzzo, P., Piscopo, V., Fiorentino, A., 2009. Antioxidant efficacy of iridoid and phenylethanoid glycosides from the medicinal plant Teucrium chamaedrys in cell-free systems. Bioorg. Med. Chem. 17, 6173–6179. Papavizas, G.C., 1966. Suppression of Aphanomyces root rot of peas by cruciferous soil amendment. Phytopathology 56, 1071–1075. Putnam, A., Tang, C.S., 1986. The Science of Allelopathy. John Wiley and Sons Inc., New York, pp. 67–72. Rice, E.L., 1984. Allelopathy, 2nd ed. Academic Press, Orlando, FL, pp. 351–352. Rice, E.L., 1995. Biological Control of Weeds and Plant Diseases: Advances in Applied Allelopathy. University of Oklahoma Press, Oklahoma. Singh, H.P., Batish, R.D., Kohli, K.R., 1999. Autotoxicity: concept, organisms and ecological significance. Crit. Rev. Plant Sci. 18, 757–772. Soares, A., Guieysse, B., Jefferson, B., Cartmell, E., Lester, J.N., 2008. Nonylphenol in the environment: a critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ. Int. 34, 1033–1049. Thorpe, A.S., Thelen, G.C., Diaconu, A., Callaway, R.M., 2009. Root exudate is allelopathic in invaded community but not in native community: field evidence for the novel weapons hypothesis. J. Ecol. 97, 641–645. Tomita-Yokotani, K., Kato, T., Parvez, M.M., Mori, Y., Goto, N., Hasegawa, K., 2003. Approach of allelopathy study with Arabidopsis thaliana (L.) Hevnh. and Neurospora crassa. Weed Biol. Manage. 3, 93–97. Trevisan, M.T.S., Pfundstein, B., Haubner, R., Wurtele, G., Spiegelhalder, B., Bartsch, H., Owen, R.W., 2006. Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity. Food Chem. Toxicol. 48, 188–197. Xuana, T.D., Shinkichi, T., Khanh, T.D., Min, C.I., 2005. Biological control of weeds and plant pathogens in paddy rice by exploiting plant allelopathy: an overview. Crop. Prot. 24, 197–206. Yu, J.Q., 1999. Allelopathic suppression of Pseudomonas solanacearum infection of tomato (Lycopersicon esculentum) in a tomato-chinese chive (Allium tuberosum) intercropping system. J. Chem. Ecol. 25, 2409–2414.
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Allelopathic potential of alkylphenols from Dactylis glomerata subsp. hispanica (Roth) Nyman Monica Scognamiglio a, Vittorio Fiumano a, Brigida D’Abrosca a, Severina Pacifico a, Anna Messere b, Assunta Esposito a, Antonio Fiorentino a,* a b
Dipartimento di Scienze della Vita – Laboratorio di Fitochimica – Seconda Universita` degli Studi di Napoli - via Vivaldi 43, I-81100 Caserta, Italy Dipartimento di Scienze Ambientali – Seconda Universita` degli Studi di Napoli - via Vivaldi 43, I-81100 Caserta, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 August 2011 Received in revised form 13 December 2011 Accepted 16 December 2011 Available online 27 December 2011
Eleven alkylphenols were isolated from the aerial parts of Dactylis glomerata subsp. hispanica, six of them described for the first time. The structural characterization of these compounds has been elucidated by 1D and 2D NMR techniques. The fragmentation patterns of the metabolites obtained by GC–MS analysis allowed the side chain to be elucidated. The allelopathic potential of three alkylphenols, representative of each homologous series of alkylphenols from D. glomerata subsp. hispanica, has been assayed on D. glomerata subsp. hispanica and an herbaceous coexisting species, Phleum subulatum. The bioassay results showed a high auto-stimulation values of germination and root and shoot elongation for D. glomerata subsp. hispanica at high concentrations. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Dactylis glomerata subsp. hispanica Poaceae Alkylphenols Spectroscopic analysis Allelopathy
1. Introduction Allelopathy is one of the most sophisticated mechanisms of plant defenses. It could be understood as the ability of plants to inhibit or stimulate growth of other organisms in the environment by releasing chemicals, named allelochemicals. These compounds, mainly secondary metabolites, introduced in the environment by exudation, decomposition, leaching, and volatilization, may be toxic or stimulatory to the releaser, other plant species, and soil microorganisms (Papavizas, 1966; Grodzinsky, 1992; Rice, 1995; Yu, 1999). Allelopathic plants get some benefit from the release of allelochemicals by inhibiting coexisting competitors for nutrients and/or by promoting their development and propagation. As a consequence, allelopathy has been discussed as one of the main factors affecting plant community composition, structures and productivity (Rice, 1984, Chaves and Escudero, 1997). Among the several aspects influencing allelopathic effects (e.g. active concentration of allelochemicals in the soil; biological and chemical characteristics of the soil, plant density, etc.), the life cycle of the allelopathic plant represents one of the most important characters influencing plant community structure. Perennial plant species are likely to release and add allelochemicals to the soil over more than
* Corresponding author. Tel.: +39 0823 274576; fax: +39 0823 274571. E-mail addresses: antonio.fi[email protected], antonio.fi[email protected] (A. Fiorentino).
one season. This is probably the reason why most reports concerning the ability of a plant to invade or dominate in an ecosystem, has involved allelopathic perennials (Chaves and Escudero, 1997; Mallik, 2000). For annual plant species the allelopathic activity has been suggest to be part of their success as weeds with several species showing growth pattern characterized by several life cycles completed within one year with the aim to maintain the pool of allelochemicals (Inderjit and Dakshini, 1998). Moreover perennial plants exert allelopathy also against itself mainly by inhibiting the seed germination. This autotoxic effects occur when a plant species inhibit or delay germination and growth of the same plant species (Putnam and Tang, 1986; Singh et al., 1999). On the contrary, annual plants, to make sure their survival, seem to affect positively the germination of their seeds and negatively that of other species (Lovett and Jackson, 1980). Allelopathic effects of both perennials and annual plants, might be intensified in natural communities characterized by harsh environmental conditions and other constraints (Rice, 1984). This is particularly true in Mediterranean region where, to face the biotic and abiotic stress, the biodiversity of plants present in this area, produces a large variety of secondary metabolites causing stimulating or inhibiting effects on coexisting organisms and regulating, in such away, the equilibrium within the ecosystem (Thorpe et al., 2009; Xuana et al., 2005; Bonanomi et al., 2006; Esposito et al., 2008). In the aim to investigate on the allelopathic interactions among plants of Mediterranean macchia vegetation,
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.12.009
M. Scognamiglio et al. / Phytochemistry Letters 5 (2012) 206–210
we undertook the chemical and biological study of several herbaceous and shrub species present in the Natural Reserve of Castel Volturno (D’Abrosca et al., 2005, 2010; Fiorentino et al., 2007, 2008b, 2011; Pacifico et al., 2009), a flat coastal area located in the north of Naples (Southern Italy). These studies led to the isolation of a number of secondary metabolites, able to stimulate or inhibit seed germination and seedling growth of coexisting species (Fiorentino et al., 2008a, 2009a,b, 2010). Continuing the phytochemical investigation of this area, we undertook the chemical study of Dactylis glomerata subsp. hispanica (Roth) Nyman (Poaceae), a perennial steno-Mediterranean grass with a frequent dominance cover in herbaceous community.
HO
R OH
207
HO
1
5
1'
O
R
1
5
3
3
OH
OH
1'
R
1 R=(CH2)19CH3
3 R=(CH2)15CH3
9
2 R=(CH2)21CH3
4 R=(CH2)17CH3
10 R=(CH2)17CH3 11 R=(CH2)19CH3
5 R=(CH2)18CH3
R=(CH2)15CH3
6 R=(CH2)19CH3 7 R=(CH2)21CH3 8 R=(CH2)23CH3 Fig. 1. Chemical structures of alkylphenols from Dactylis glomerata subsp. hispanica.
2. Results and discussion Eleven alkylphenols were isolated from leaves of D. glomerata subsp. hispanica. Compounds 3, 5–8 and 11 were isolated for the first time (Fig. 1). To elucidate the structures, NMR and GC–MS analyses were carried out. 1D and 2D-NMR spectra were useful to characterize the aromatic group. Three different substitution patterns for the aromatic ring were identified, and different homologous of each series did not show differences in the NMR data. In fact, the 1H NMR spectrum of compounds 1 and 2 was consistent with a 1,3,5 substitution, since two signals at d 6.14 (brs, H-2) and 6.19 (brs, H4, H-6) which can be integrated, respectively, for one and two protons, were evident. In the HMBC experiment for these compounds, correlations were observed between the proton H-2 and the carbons at d 107.9 (C-4, C-6), and between the proton H-4 (and H-6) and the carbons at d 100.2 (C-2) and 157.2 (C-1, C-3). Moreover, the proton H-4 (and H-6) showed correlation with the carbon at d 36.1 (C-10 ), and the benzylic proton at d 2.45 (2H, J = 7.5 Hz, H-10 ) with C-4 and C-6. Thus, compounds 1 and 2 are 5alkylresorcinols, having reference to the aromatic A skeleton. The 1H NMR spectrum of compounds 3–8 contained a pattern distinctive of a tetrasubstituted aromatic ring, showing signal at d 6.21 (2H, s) assignable to both H-4 and H-6 protons and d 2.08 (3H, s, C-2–CH3). The HMBC experiment for these compounds showed correlations between the aromatic methyl proton and the carbons at d 107.9 (C-4 and C-6) and 154.7 (C-1 and C-3), and between the proton H-4 (and H-6) and C-1, C-3, C-6 (and C-4). Furthermore, correlations between the proton H-4 (and H-6) and the carbon at 35.7 (C-10 ), and vice versa the benzylic proton at d 2.43 (2H, t, J = 7.5 Hz, H-10 ) and the carbon at d 141.9 (C-5), C-4 and C-6 were observed. Thus, compounds 3–8 are identified to be 5-alkyl-2methylresorcinols, referring to the aromatic B skeleton. The 1H NMR spectrum of compounds 9–11 revealed a doublet of doublet at d 6.21 (1H, H-2), two broad singlets at d 6.19 (1H, H-4)
and 6.26 (1H, H-6) and a singlet at d 3.76 (3H, C-1–OCH3), diagnostic of an aromatic methoxyl. In the HMBC experiment for these compounds, correlations between the proton H-2 and the carbons at d 55.2 (C-1–OCH3), 106.8 (C-4) and 158.1 (C-3), between the aromatic methoxyl protons and the carbon at d 98.6 (C-2), and between both protons H-4 and H-6 and the carbon at d 145.8 (C-5) were revealed. Once again, correlations between the proton H-4 and the carbon at d 36.0 (C-10 ), and between the proton at d 2.44 (2H, H-10 ) and C-4 were evident. Thus, compounds 9–11 are determined to be 5-alkylresorcinol-3-methyl ethers, being ascribable to the aromatic C skeleton. No further information about structural features of the alkylic chains could be deduced by NMR experiments. Underivatized compounds were analyzed by GC–MS and specific ion fragmentation patterns allowed the aromatic skeletons to be confirmed and the side chain structures elucidated. A base peak in the EIMS spectrum at m/z 124 confirmed the presence of the resorcinol moiety in compounds 1–2, while a base peak at m/z 138 supported the presence of a 2-methylresorcinol, or a resorcinol methyl ether, or a 2,5-dihydro-1,4-benzochinon residue in compounds 3–8, 9–11, respectively. Molecular weights and spectral characteristics (Table 1) suggested the presence of all unbranched, saturated, odd numbered hydrocarbon chains from C17 to C25, except for compound 5, which is 5-eicosanoyl-2methylresorcinol. Both metabolites 1 and 2 are reported as constituents of Poaceae species (Kno¨dler et al., 2010). Compound 4, known as polygonocinol, has been previously isolated from Polygonum maritimum (Kazantzoglou et al., 2009). 3-Heptadecyl-5-methoxyphenol (9) was described as metabolite from Oxalis erythrorhiza active towards Leishmania amazonensis and Leishmania donovani promastigotes (Feresin et al., 2003). Finally, compound 10 was isolated from the aerial parts of Artemisia annua (Brown, 1992).
Table 1 GC–MS data of the alkylphenols in D. glomerata subsp. hispanica. Comp.
RT (min)
[M]+. m/z (% rel. abundance)
Major fragment ions m/z (% relative abundance)
1 2 3 4 5 6 7 8 9
21:46 26:27 16:52 19:16 20:47 22:50 27:53 35:44 15:49
404 432 362 390 404 418 446 474 362
10
17:39
390 (3.4)
11
20:21
418 (3.0)
362 222 320 348 218 376 207 221 320 180 348 125 207
(2.1) (2.0) (4.2) (3.5) (2.5) (3.5) (3.8) (2.0) (3.5)
(0.1), (0.2), (0.2), (0.2), (6.6), (0.1), (9.2), (0.6), (0.2), (3.0), (0.1), (1.9), (7.5),
250 180 236 306 203 334 193 207 306 165 306 107 193
(0.1), (0.5), (0.2), (0.1), (3.6), (0.1), (1.3), (6.9), (0.1), (0.7), (0.1), (2.0), (0.9),
236 (0.1), 222 (0.1), 194 (0.2), 180 (0.5), 166 (3.6), 151 (0.7), 137 (8.5), 124 (100) 166 (3.8), 151 (0.7), 137 (8.4), 124 (100) 222 (0.4), 207 (1.1), 194 (0.5), 180 (3.3), 165 (0.8), 151 (10.0), 138 (100), 123 (3.4) 236 (0.1), 222 (0.3), 207 (0.3), 180 (3.0), 165 (0.6), 151 (9.4), 138 (100), 123 (3.2) 151 (9.3), 138 (100), 123 (5.0), 109 (5.2) 207 (0.5), 194 (0.4), 180 (3.4), 166 (0.6), 151 (9.3), 138 (100), 123 (2.9), 109 (1.0) 180 (3.5), 151 (8.9), 138 (100), 123 (3.8) 180 (3.3), 166 (0.8), 152 (1.7), 138 (100), 123 (3.5) 292 (0.1), 278 (0.1), 264 (0.1), 250 (0.1), 236 (0.2), 222 (0.3), 207 (0.6), 194 (0.4), 151 (11.2), 138 (100), 125 (1.9), 107 (2.1), 91 (1.3) 264 (0.1), 222 (0.3), 207 (1.2), 194 (0.4), 180 (3.0), 165 (0.7), 151 (10.6), 138 (100), 91 (1.1) 180 (3.2), 166 (0.8), 151 (12.3), 138 (100), 125 (2.1), 107 (2.8)
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208
Table 2 Phytotoxicity effects of compound 1, 4 and 9 at three different concentration (H, high = 103 M; M, medium = 106 M; L, low = 109 M). The values are referred as % from control with: 0 = no effect (range value: 5 to 5); ++, high stimulation (range value: up to 25); +, low stimulation (range value: 5–25); , low inhibition (range value: 5 to 25). Compound
1
Concentration
H
M
L
H
M
L
M
L
++ 0
0 0
0 0
++ +
+ +
0
0 0
+ 0
++ ++
+ +
0 0
+ ++
+ +
0
0 0
0 +
++ +
+ +
+ 0
++ 0
+ 0
+
+ +
+ 0
Germination Dactylis glomerata Phleum subulatum Root elongation Dactylis glomerata Phleum subulatum Shoot elongation Dactylis glomerata Phleum subulatum
4
This class of secondary metabolites, characterized by a long aliphatic chain bonding a phenolic moiety, is reported to have different biological activities, since they have been identified as responsible for antioxidant activity in cashew (Anacardium occidentale) (Trevisan et al., 2006), allelophatic potential (Dayan et al., 2010), cytotoxicity against various tumour cell lines (Kubo et al., 1993), and antinematodal properties (Alen et al., 2000). Synthetic alkylphenols have been used as non-ionic surfactants in a variety of industrial and household applications; their breakdown products, octyl- and nonylphenols have been proposed to function as oestrogens and have been attributed a role in endocrine disruption (Soares et al., 2008). Compounds 1, 4 and 9, representative of each homologous series of alkylphenols from D. glomerata subsp. hispanica, have been assayed on D. glomerata subsp. hispanica and Phleum subulatum to evaluate their effects on seed germination and plant growth. The results, reported in Table 2, showed high auto-stimulation values of germination and root and shoot elongation for D. glomerata subsp. hispanica at high concentration of compounds 1 and 4, while low or no effects have been evidenced by the compound 9. As regards Phleum subulatum, compounds 1 and 4 showed significative stimulating effects only for the root elongation and at the highest concentration, while low or no effect, respect to the control, was found at the lowest concentrations. No significative effects for both the test species were observed for compound 9. Despite the wide evidences of the auto-inhibiting effects of allelochemicals (Putnam and Tang, 1986; Singh et al., 1999; Fernandez et al., 1999), few data report the role of allelochemicals in terms of positive effects of autostimulation on root (Lovett and Jackson, 1980) and shoot growth (Tomita-Yokotani et al., 2003) as observed in this work. Although the nature of the stimulatory allelochemical remains to be defined, we can assume that the observed positive effects on germination and growth produced by allelochemicals of D. glomerata subsp. hispanica could represent an adaptative strategy to make sure the survival, by enhancing the competitive capacity of the species in high harsh environmental conditions through the accumulation of allelochemicals in the soil until the following vegetative period in the spring season (unpublished results). 3. Experimental 3.1. General experiment procedures TLC was performed on Kieselgel 60 F254 (Merck) or RP-8 F254 (Merck) plates with 0.2, 0.5 or 1 mm film thickness. Column chromatography (CC) was performed on Kieselgel 60 (70–240 mesh, Merck), RP-18 (230–400 mesh, Baker), NH2 (40–63 mm, Merck) or on Sephadex LH-20 (Pharmacia). NMR spectra were recorded at 300 (1H) and 75 MHz (13C) on a Varian 300 FT-NMR spectrometer in CDCl3 solns, at 25 8C; d (chemical shift) in ppm, J (coupling constant)
9
in Hz. Proton-detected heteronuclear correlations were measured by gradient heteronuclear single-quantum coherence (HSQC) optimized for 1J(H,C) = 140 Hz, by gradient heteronuclear multiplebond coherence (HMBC) optimized for nJ(H,C) = 8 Hz. GC/MS analyses were carried out using a HP 6890 GC instrument (Zebron ZB-5MS column, He flow 1.0 ml/min) coupled with a 5973 N mass spectrometer, equipped with an electron ionization source (EIMS). Underivatized samples were analyzed in the following conditions: initial temperature 45 8C (2 min isothermal), from 45 to 300 8C at 25.5 8C/min, and then isothermal for 25 min. 3.2. Plant material Plants of Dactylis glomerata subsp. hispanica (Roth) Nyman (syn. Dactylis hispanica Roth) were collected in field at vegetative state, in June 2010, in ‘‘Castel Volturno’’ Nature Reserve (Southern Italy). A voucher specimen (CE0115) has been deposited at the Herbarium of the Dipartimento di Scienze della Vita of SUN. 3.3. Extraction and isolation Dried aerial parts of D. glomerata subsp. hispanica (1744.0 g) were infused in petroleum ether (PE) for 3 days in a refrigerated chamber at 4 8C in the dark. After distillation of the solvent in vacuum we obtained PE crude extract (12.3 g). The PE extract was chromatographed on SiO2 to obtain three fractions, A, B and C. The first one, eluted with PE/CHCl3 (1:1), was chromatographed on Sephadex LH-20 eluting with Ex/CHCl3/MeOH (6:1:1) and collecting fractions of 10 mL. Fractions 7–9 were chromatographed by NH2 CC eluting with PE/EtOAc solns. The fraction eluted with PE/ EtOAc (19:1) was chromatographed by SiO2 TLC eluting with CHCl3/MeOH (19:1) to give a fraction containing compounds 9, 10 and 11 definitively purified by RP-18 HPLC [MeOH–H2O (4:1)]. Fraction B, eluted with PE/CHCl3 (1:1), was chromatographed on Sephadex LH-20 eluting with Ex/CHCl3/MeOH (5:1:1) and collecting fractions of 10 mL. Fractions 13–16 were chromatographed by SiO2 TLC eluting with EtOAc/CHCl3 (19:1) to give two fractions. Both the fractions were definitively purified by RP-18 HPLC [MeOH–H2O (4:1)]. The first fraction furnished compounds 4, 5, 6, 7 and 8; the second one gave pure compounds 3, 4 and 5. Fraction C, eluted with pure CHCl3 was chromatographed on Sephadex LH20 eluting with Ex/CHCl3/MeOH (5:1:1) and collecting fractions of 10 mL. Fractions 8–12 were chromatographed by NH2 CC eluting with PE/EtOAc/MeOH solns. The fraction eluted with pure MeOH contained compounds 1 and 2. 3.4. 5-Heptadecyl-2-methylbenzene-1,3-diol 3 Colourless oil, UV lmax (log e) (MeOH) 280 (3.79) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29
M. Scognamiglio et al. / Phytochemistry Letters 5 (2012) 206–210
(overlapped, 28H, H-30 –H-160 ), 0.86 (t, J = 7.2 Hz, 3H, H-170 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-150 ), 22.9 (C-160 ), 14.4 (C-170 ), 7.9 (CH3); Anal. Calcd for C24H42O2: C, 79.50; H, 11.68. Found: C, 79.59; H, 11.97. 3.5. 5-Icosyl-2-methylbenzene-1,3-diol 5 Colourless oil, UV lmax (log e) (MeOH) 280 (3.76) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 34H, H-30 –H-190 ), 0.86 (t, J = 7.2 Hz, 3H, H-200 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-180 ), 22.9 (C-190 ), 14.4 (C-200 ), 7.9 (CH3); Anal. Calcd for C27H48O2: C, 80.14; H, 11.96. Found: C, 79.99; H, 11.86. 3.6. 5-Henicosyl-2-methylbenzene-1,3-diol 6 Colourless oil, UV lmax (log e) (MeOH) 280 (3.24) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 36H, H-30 –H-200 ), 0.86 (t, J = 7.2 Hz, 3H, H-210 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-190 ), 22.9 (C-200 ), 14.4 (C-210 ), 7.9 (CH3); Anal. Calcd for C28H50O2: C, 80.32; H, 12.04. Found: C, 80.41; H, 11.99. 3.7. 2-Methyl-5-tricosylbenzene-1,3-diol 7 Colourless oil, UV lmax (log e) (MeOH) 280 (3.64) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 40H, H-30 –H-220 ), 0.86 (t, J = 7.2 Hz, 3H, H-230 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-210 ), 22.9 (C-220 ), 14.4 (C-230 ), 7.9 (CH3); Anal. Calcd for C30H54O2: C, 80.65; H, 12.18. Found: C, 80.57; H, 12.09. 3.8. 2-Methyl-5-pentacosylbenzene-1,3-diol 8 Colourless oil, UV lmax (log e) (MeOH) 279 (3.64) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3) d 6.21 (s, 2H, H-2 and H-6), 2.43 (t, J = 7.5 Hz, 2H, H-10 ), 2.08 (s, 3H, Me), 1.50 (m, 2H, H-20 ), 1.29 (overlapped, 44H, H-30 –H-240 ), 0.86 (t, J = 7.2 Hz, 3H, H-250 ); 13C NMR (75 MHz, CDCl3) d 154.7 (C-1 and C-3), 142.1, (C-5), 135.8 (C2), 107.9 (C-4 and C-6), 35.7 (C-10 ), 32.1 (C-20 ), 29.5 (C-30 –C-230 ), 22.9 (C-240 ), 14.4 (C-250 ), 7.9 (CH3); Anal. Calcd for C32H58O2: C, 80.95; H, 12.31. Found: C, 80.78; H, 13.23. 3.9. 3-Heptadecyl-5-methoxyphenol 9 Colourless oil, UV lmax (log e) (MeOH) 278 (3.07) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 28H, H-30 –H-160 ), 0.81 (t, J = 6.6 Hz, 3H, H-170 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C-3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-150 ), 22.7 (C-160 ), 14.1 (C-170 ); Anal. Calcd for C24H42O2: C, 79.50; H, 11.68. Found: C, 79.45; H, 11.71. 3.10. 3-Nonadecyl-5-methoxyphenol 10 Colourless oil, UV lmax (log e) (MeOH) 278 (3.45) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers
209
to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H-10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 32H, H-30 –H-180 ), 0.81 (t, J = 6.6 Hz, 3H, H-190 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-170 ), 22.7 (C-180 ), 14.1 (C-190 ); Anal. Calcd for C26H46O2: C, 79.94; H, 11.87. Found: C, 79.90; H, 11.81. 3.11. 3-Henicosyl-5-methoxyphenol 11 Colourless oil, UV lmax (log e) (MeOH) 278 (3.86) nm; EI-MS: see Table 1; 1H NMR (300 MHz, CDCl3, the atom numbering refers to Fig. 1) d 6.26 (brs, 1H, H-6), 6.19 (brs, 1H, H-4), 6.16 (dd, J = 2.4 and 2.2 Hz, 1H, H-2), 3.75 (s, 3H, OMe), 2.44 (dd, J = 7.8 and 7.5 Hz, 2H, H-10 ), 1.51 (m, 2H, H-20 ), 1.19 (overlapped, 36H, H-30 –H-200 ), 0.81 (t, J = 6.6 Hz, 3H, H-210 ); 13C NMR (75 MHz, CDCl3) d 158.1 (C3), 156.5 (C-1), 145.8, (C-5), 107.8 (C-6), 106.8 (C-4) 98.6 (C-2), 55.2 (OCH3), 36.0 (C-10 ), 31.9 (C-20 ), 29.5 (C-30 –C-190 ), 22.7 (C-200 ), 14.1 (C-210 ); Anal. Calcd for C28H50O2: C, 80.32; H, 12.04. Found: C, 80.26; H, 11.95. 3.12. Phytotoxicity test Seeds were selected for uniformity observing them under a binocular microscope in order to discard the undersized and damaged ones. Furthermore, a preliminary test to evaluate the germinability of the selected test species was carried out in a growth chamber KBW Binder 240 at 27 8C in the dark. The observed percentage of germinability for D. glomerata subsp. hispanica and P. subulatum was of 90%. The bioassays were conducted as described by Fiorentino et al. (2009a). For each metabolite, three different concentrations were tested: 103 M, 106 M, and 109 M (high, medium and low concentration, respectively). 50 mm diameter Petri dishes and 1 mL test solutions were used. The dishes were placed in a growth chamber KBW Binder 240 at 27 8C in the dark for six days (no more germination occurred after this time). Seedlings were frozen at 20 8C to avoid further growth up to the root and shoot elongation measurements. Germination rate as well as root and shoot lengths were recorder using a Fitomedß system (Castellano et al., 2001). Data are reported as percentage differences with respect to control. Hence positive values indicate stimulation, while negative values represent inhibition of the studied parameters. Statistical analysis was carried out on the data set. References Alen, Y., Nakajima, S., Nitoda, T., Baba, N., Kanzaki, H., Kawazu, K., 2000. Antinematodal activity of some tropical rainforest plants against the pinewood nematode, Bursaphelenchus xylophilus. Z. Naturforsch. C. 55, 300–303. Bonanomi, G., Sicurezza, M.G., Caporaso, S., Esposito, A., Mazzoleni, S., 2006. Phytotoxicity dynamics of decaying plant materials. New Phytol. 169, 571–578. Brown, G.D., 1992. Two new compounds from Artemisia annua. J. Nat. Prod. 55, 1756–1760. Castellano, D., Macı´as, F.A., Castellano, M., Cambronero, R., June 2001. Inventor. University of Ca´diz, assignee. FITOMED (Automated system for the measurement of variable lengths). Spain Patent No. P9901565. Chaves, N., Escudero, J.C., 1997. Allelopathic effect of Cistus ladanifer on seed germination. Funct. Ecol. 11, 432–440. D’Abrosca, B., Fiorentino, A., Golino, A., Monaco, P., Oriano, P., Pacifico, S., 2005. Carexanes: prenyl stilbenoid derivatives from Carex distachya. Tetrahedron Lett. 46, 5269–5272. D’Abrosca, B., Fiorentino, A., Ricci, A., Scognamiglio, M., Pacifico, S., Piccolella, S., Monaco, P., 2010. Structural characterization and radical scavenging activity of monomeric and dimeric cinnamoyl glucose esters from Petrorhagia velutina leaves. Phytochem. Lett. 3, 38–44. Dayan, F.E., Rimando, A.M., Pan, Z., Baerson, S.R., Gimsing, A.L., Duke, S.O., 2010. Sorgoleone. Phytochemistry 71, 1032–1039. Esposito, A., Fiorentino, A., D’Abrosca, B., Izzo, A., Cefarelli, G., Golino, A., Monaco, P., 2008. Potential allelopathic interferences of Melilotus neapolitana metabolites
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