Phytochemistry Letters 4 (2011) 118–121 Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol Lignans from Phillyrea angustifolia L. Marina DellaGreca *, Anna Mancino, Lucio Previtera, Armando Zarrelli, Simona Zuppolini Dipartimento di Chimica Organica e Biochimica, Università Federico II, via Cinthia 4, I-80126 Napoli, Italy A R T I C L E I N F O A B S T R A C T Article history: Received 7 October 2010 Received in revised form 22 December 2010 Accepted 23 December 2010 Available online 6 January 2011 A new lignan epoxide together with the seven known lignans: pinoresinol, pinoresinol-O-b-Dglucopyranoside, pinoresinol monomethyl ether-O-b-D-glucopyranoside, lariciresinol, lariciresinol-4-Ob-D-glucopyranoside, lariciresinol-40 -O-b-D-glucopyranoside, and syringaresinol monoglucopyranoside were isolated from the hydroalcoholic and organic extracts of the whole plant of Phillyrea angustifolia L. (Oleaceae). The structure of the new constituent was elucidated by spectroscopic methods (UV, IR, and 1D- and 2D-NMR) and by mass spectrometry (HR-ESI-MS), mainly using 2D-NMR techniques. The effects of these compounds on germination and growth of dicotyledon Lactuca sativa L. (lettuce) were studied in the 10 4 to 10 7 M concentration range. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Keywords: Phillyrea angustifolia Lactuca sativa Phytotoxic activity Spectroscopic analysis Lignans Epoxylignan 1. Introduction Phillyrea angustifolia L. is a wind-pollinated shrub and one of the handful of species reported to be androecium. P. angustifolia is a native Mediterranean species, which has recently been considered to be suitable for landscaping purposes (De Marco et al., 2005). Polyphenolic compounds, flavonoids, oleuropein derivatives and carbohydrates in Phillyrea leaves were identified (Romani et al., 1996) and carbohydrate distribution may be linked to the evolution pattern of this species which usually grows in severely stressed environments (Vitale et al., 2008). The chemical composition of extracts of P. angustifolia has been investigated, and resulted in the isolation and structure elucidation of a novel epoxylignan and seven known lignans. 2. Results and discussion Fresh plants of P. angustifolia were powdered and infused with CH3OH:H2O (1:9), for seven days, and successively with CH3OH:CH2Cl2 (1:9), for two weeks. The hydroalcoholic solution, without proteinaceous materials which were removed by centrifugation, was partitioned between EtOAc and H2O. The EtOAc extract and the crude dichloromethane-methanolic infusion were fractionated by column chromatography, and the fractions purified by preparative thin-layer chromatography and HPLC, yielding pure lignans. * Corresponding author. Tel.: +39 081 674162; fax: +39 081 674393. E-mail address: dellagre@unina.it (M. DellaGreca). The lignans were identified as a new lignan (1), pinoresinol (2) (Pelter et al., 1982), pinoresinol-4-O-b-D-glucopyranoside (3) (Chiba et al., 1980), pinoresinol monomethyl ether-4-O-b-Dglucopyranoside (4) (Kitagawa et al., 1988), lariciresinol (5) (Davin et al., 1992), lariciresinol-4-O-b-D-glucopyranoside (6) (Sugiyama and Kikuchi, 1993), lariciresinol-40 -O-b-D-glucopyranoside (7) (Sugiyama and Kikuchi, 1993), syringaresinol monoglucopyranoside (8) (Rao and Wu, 1978), by comparison of their spectral data with those reported. The EIMS of compound 1 had a molecular peak at m/z 356 consistent with a molecular formula C20H20O6 and a prominent peak at m/z 135 [C8H7O2]+. The structure of compound 1 was established using 1H NMR and 13C NMR including COSY, NOESY, HSQC, and HMBC experiments. The 1H NMR spectrum (Table 1) showed six aromatic protons as four doublets at d 6.67, 6.75, 6.84 and 6.87, and two double doublets at d 6.63 and 6.70, which were typical of 1,2,4-trisubstituted aromatic rings. The 1H NMR spectrum and the 1H–1H COSY spectrum allowed identification of the H2-7 protons as a double doublet at d 2.84 (J = 10.7, 2.9 Hz) and 2.65 (overlapped), the H-8 proton at d 2.62 (overlapped) and the H2-9 protons as double doublets at d 4.26 and 3.72 (J = 9.8, 8.8, 6.7 Hz), additionally the H-70 singlet at d 4.89 and the H2-90 protons as doublets at d 2.89 and 2.46 (J = 4.9 Hz). In the 1H NMR spectrum a methoxyl group at d 3.90 and a singlet methylene at d 5.95 were also present. The 13C NMR spectrum showed 19 carbon signals (Table 1). The DEPT experiment defined the carbons as a methyl, four methylenes and eight methines, which were correlated to the corresponding protons by an HSQC experiment. Long-range correlations between the H2-7 and the C-2 and C-6 methine carbons, and the C-1 and C-80 quaternary carbons, the H2-9 and the 1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2010.12.006 M. DellaGreca et al. / Phytochemistry Letters 4 (2011) 118–121 119 Table 1 NMR spectral data of compound 1 in CDCl3. dHa Position 1 2 3 4 5 6 7 8 9a9b 10 20 30 40 50 60 70 b 80 90 a 90 b OCH2O– 30 -OCH3 J (Hz) 6.67 d 2.0 7 6.75 6.63 2.84 2.62 4.26 8.5 8.5, 2.0 10.7, 2.9 7 2, 6, 9, 70 9.8, 8.8, 6.7 9.8, 8.8, 6.7 7, 70 6.87 d 1.9 70 , 30 -OCH3 6.84 d 6.70 dd 4.89 s 8.9 8.9, 1.9 2.89 d, 2.46 d 5.95 s 3.90 s 4.9 d dd dd, 2.65 ovd ov dd, 3.72 dd 70 7, 9, 20 , 60 , 90 70 20 HMBCb dC NOESY 132.6 108.8 147.8 147.8 108.3 121.4 36.4 44.8 71.5 127.5 110.2 146.4 147.8 113.5 120.8 81.3 67.5 46.1 100.9 55.9 c (q) (t) (q) (q) (t) (t) (s) (t) (s) (q) (t) (q) (q) (t) (t) (t) (q) (s) (s) (p) 4, 6, 7 1, 2, 1, 1, 7, 3 4, 7 2, 6, 8, 9, 80 90 8, 70 , 80 40 , 60 , 70 10 , 30 20 , 40 , 70 10 , 20 , 60 , 90 8, 70 3, 4 30 a 1 b c d H chemical shift values (d ppm from SiMe4) followed by multiplicity and then the coupling constants (J in Hz). HMBC correlations from H to C. Letters, p, s, t and q, in parentheses indicate, respectively, the primary, secondary, tertiary and quaternary carbons, assigned by DEPT. Overlapped. concentration tested, while compound 7 showed an activity of 35 and 45%, respectively. The results reported in Fig. 2B and C showed greater phytotoxic activities by compounds 1 and 4. Related to radicle shoot, compounds 1 and 4 showed at higher concentration a phytotoxicity estimated at 75 and 85%, respectively. However, even at lower concentration they retained inhibitory activity of 30 and 40%, respectively. For shoot length, the compounds revealed approximately 85% inhibition at a concentration of 10 4 M and, however, greater than 55% at a lower concentration (10 7 M). It is noteworthy that glycosilated compounds exhibited low phytotoxic activity (Fiorentino et al., 2007) as compounds 3, 6–8. Differently, the compound 4 exhibited high activity. C-7 methylene carbon, C-8 and C-70 methine carbons, and C-80 , H70 and C-20 and C-60 methine carbons, and C-90 methylene carbon in the HMBC spectrum (Table 1) allowed definition of the planar structure of lignan 1. According to the structure, the analysis of the NOESY spectrum showed NOEs of H-2 and H-6 with H2-7 protons, H-20 and H-60 with the H-70 proton, and the methoxyl with the H-20 proton. The relative configuration of the furan ring (Fig. 1) was deduced from a detailed analysis of the NOEs observed in the NOESY experiments. The NOEs of the H-70 b proton with both H2-7, H-90 b at d 2.46 and H-9b at d 3.72 protons indicated that the phenyl and the epoxidic oxygen were on the same side of the tetrahydrofuran ring and the benzyl and the phenyl were in a trans-orientation. These data led to the structure of (3R,4R,7S) 7(3,4-methylenedioxybenzyl)-4-(4-hydroxy-3-methoxyphenyl)1,5-dioxaspiro[2.4]heptene (1) or its enantiomer, isolated for the first time. 2 7 O H and 13C NMR spectra were run on a Varian INOVA 500 NMR spectrometer at 500 and 125 MHz, respectively, in CDCl3 at 25 8C. MS spectra were obtained with a HP 6890 spectrometer equipped O O 8' 3.1. General experimental procedures 1 9 8 O 3. Experimental [()TD$FIG] 7' 9' OMe 5' OH The phytotoxicity of the lignans 2 and 5 on the seeds of Lactuca sativa was previously reported (Cutillo et al., 2003). Compounds 1, 3, 4, 6–8 were tested for their activities on the seeds of L. sativa. Their aqueous solutions, ranging from 10 4 to 10 7 M, were used to evaluate the inhibitory or stimulatory effects on germination, shoot length, and root length of treated lettuce seeds. They were investigated in accordance with the procedures optimized by Macias et al. and the results are shown in Fig. 2A–C. All compounds showed a low inhibition of germination, with rates of up to 15% at the highest concentration. Moreover, all showed a good correlation between the activities shown and the concentrations tested. With regard to the radicle and shoot inhibitions, compounds 3, 6 and 8 showed a variable behaviour within 25–30% at higher Fig. 1. Selected NOEs of compound 1. [()TD$FIG] 120 M. DellaGreca et al. / Phytochemistry Letters 4 (2011) 118–121 3.3. Extraction and isolation Fig. 2. (A) Effect of compounds 1, 3, 4, 6–8 on germination of Lactuca sativa L. Value presented as percentage differences from control and are not significantly different with P > 0.05 for Student’s t-test. (a) P < 0.01; (b) 0.01 < P < 0.05. (B) Effect of compounds 1, 3, 4, 6–8 on root length of Lactuca sativa L. Value presented as percentage differences from control and are not significantly different with P > 0.05 for Student’s t-test. (a) P < 0.01; (b) 0.01 < P < 0.05. (C) Effect of compounds 1, 3, 4, 6–8 on shoot length of Lactuca sativa L. Value presented as percentage differences from control and are not significantly different with P > 0.05 for Student’s t-test. (a) P < 0.01; (b) 0.01 < P < 0.05. with a MS 5973 N detector. HR-ESI-MS/MS: Q-TRAP model API2000 LC–MS/MS system equipped with a heated nebulizer source and using the Analyst software of Applied Biosystem; in m/z. IR spectra were recorded on a Jasco FT:IR-430 instrument. HPLC was performed on an Agilent 1100 by using an UV detector. Preparative HPLC was performed using RP-18 (LiChrospher 10 mm, 250 mm  10 mm i.d., Merck) column. Silica gel 60 (230–400 mesh, Merck) was used for CC, and preparative TLC was performed on silica gel (UV-254 precoated) plates with 0.5 and 1.0 mm thickness (Merck). 3.2. Plant material Aerial parts of P. angustifolia were collected in Castelvolturno reserve Caserta (Italy) in the spring of 2008 and identified by Professor Anna De Marco of the Dipartimento di Biologia Strutturale e Funzionale of University of Naples. Voucher specimens (HERBNAQA650) are deposited at the Dipartimento di Biologia Strutturale e Funzionale of University Federico II of Naples. Fresh leaves and twigs (23.0 kg) of the plant were frozen at 80 8C, powdered, and infused in the darkness at room temperature with CH3OH:H2O (1:9), for seven days, and successively with CH3OH:CH2Cl2 (1:9). The hydroalcoholic solution, after the evaporation of the CH3OH, was concentrated in vacuo (850 ml), cold CH3COCH3 was added (1.0 l) and the mixture was placed on a stir plate overnight in a cold room. The CH3COCH3 addition produced heavy precipitation consisting mostly of proteinaceous materials which was removed by centrifugation. The CH3COCH3 was removed by evaporation and the clear aqueous extract was partitioned between EtOAc and H2O. After removal of the solvent, the crude residue of the organic fraction was chromatographed on Amberlite XAD-2, with H2O, CH3OH, and CH3COCH3 to give three fractions. The fraction eluted with CH3OH (55.0 g) was rechromatographed on Sephadex LH-20 by using H2O and successively increasing the CH3OH concentration by 25, 50 and 100% in CH3OH. Fractions of 10 ml were collected and fractions with similar TLC profiles were combined. The sixth fraction eluted with H2O:CH3OH (1:1) (600 mg) was rechromatographed on a silica gel column to give fractions A–P. Fraction C (200 mg), eluted with CH3OH:CH2Cl2 (1:99), was rechromatographed on a silica gel column to give fractions C1–C10. Fraction C8, eluted with EtOAc:CH2Cl2 (1:4) contained pure 2 (28 mg). Fraction H (10 mg), eluted with CH3OH:CH2Cl2 (1:4), was purified by analytical TLC with CH3OH:CH2Cl2 (3:17), to yield pure 4 (3 mg). Fraction I (216 mg), eluted with CH3OH:CH2Cl2 (1:4), was purified by analytical TLC with CH3OH:EtOAc (1:9), to yield pure 3 (5 mg). Fraction L (22 mg), eluted with CH3OH:CH2Cl2 (1:3), was purified by C-18 HPLC with CH3OH:CH3CN:H2O (1:1:3), to give 8 (3 mg). Fraction N (16 mg), eluted with CH3OH:CH2Cl2 (1:1), was purified by C-18 HPLC with CH3OH:CH3CN:H2O (4:1:5), to give 6 and 7 (2 mg, each). The organic infusion was dried (Na2SO4) and concentrated in vacuo to yield 150 g of crude residue. It was stored at 80 8C until purification on silica gel column chromatography, by using CH2Cl2 and successively increasing the EtOAc concentration by 5, 10, 20, 30, 50, and 100% in CH2Cl2, to give twenty fractions. The fourteenth fraction eluted with EtOAc (4.0 g) was rechromatographed on silica gel flash chromatography with mixtures of CH2Cl2, EtOAc, CH3COCH3, and CH3OH, gave fractions A–T. Fraction G (15 mg), eluted with EtOAc:CH2Cl2 (1:9), was purified by analytical TLC with EtOAc:CH2Cl2 (1:9), to yield a pure sample of compounds 5 and 1 (5 and 2 mg, respectively). Compound 1: [a]25D 22.0 (c 0.12, CH3OH); IR (CHCl3) nmax 3595, 3058, 2929, 1603, 1250,1034 cm 1; 1H NMR and 13C NMR data, see Table 1; EIMS m/z 356 [M]+ (40), 325 [M-OCH3]+ (20), 135 [C8H7O2]+ (70); HREIMS m/z 356.1257 (calcd for C20H20O6, 356.1260). 3.4. Bioassays Seeds of L. sativa L. (cv Napoli V. F.) collected during 2003, were obtained from Ingegnoli S.p.a. All undersized or damaged seeds were discarded and the assay seeds were selected for uniformity. Bioassays used Petri dishes (50 mm diameter) with one sheet of Whatman No. 1 filter paper as support. In four replicate experiments, germination and growth were conducted in aqueous solutions at controlled pH, using MES (2-[N-morpholino]ethanesulfonic acid, 10 mM, pH 6). Test solutions (10 4 M) were prepared in MES and the rest (10 5 to 10 7 M) were obtained by dilution. Parallel controls were performed. After adding 25 seeds and 5 ml test solutions, Petri dishes were sealed with Parafilm1 to ensure closed-system models. Seeds were placed in a growth chamber KBW Binder 240 at 258 in the dark. Germination percentage was determined daily for five days (no more germination occurred after M. DellaGreca et al. / Phytochemistry Letters 4 (2011) 118–121 this time). After growth, plants were frozen at 20 8C to avoid subsequent growth until the measurement process. Data are reported as percentage differences from control in the graphics and tables. Thus, zero represents the control; positive values represent stimulation of the control; positive values represent stimulation of the parameter studied and negative values represent inhibition. 3.5. Statistical treatment The statistical significance of differences between groups was determined by a Student’s t-test, calculating mean values for every parameter (germination average, shoot and root elongation) and their population variance within a Petri dish. The level of significance was set at P < 0.05. Acknowledgment NMR experiments have been performed at Centro Interdipartimentale di Metodologie Chimico-Fisiche of University Federico II of Naples on a 500 MHz spectrometer of Consortium INCA Lab. References Chiba, M., Hisada, S., Nishibe, S., Thieme, H., 1980. 13C NMR analysis of symplocosin and (+)-epipinoresinol glucoside. Phytochemistry 19, 335–336. 121 Cutillo, F., D’Abrosca, B., DellaGreca, M., Fiorentino, A., Zarrelli, A., 2003. Lignans and neolignans from Brassica fruticulosa: effects on seed germination and plant growth. J. Agric. Food Chem. 52, 6165–6172. Davin, L.B., Bedgar, D.L., Katayama, T., Lewis, N.G., 1992. On the stereoselective synthesis of (+)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol. Phytochemistry 31, 3869–3874. De Marco, A., Gentile, A.E., Arena, C., Virzo De Santo, A., 2005. Organic matter, nutrient content and biological activity in burned and unburned soils of a Mediterranean maquis area of southern Italy. Int. J. Wildland Fire 14, 365– 377. Fiorentino, A., DellaGreca, M., D’Abrosca, B., Oriano, P., Golino, A., Izzo, A., Zarrelli, A., Monaco, P., 2007. Lignans, neolignans and sesquilignans from Cestrum parqui L’Herr. Biochem. Syst. Ecol. 35, 392–396. Kitagawa, S., Nishibe, S., Benecke, R., Thieme, H., 1988. Phenolic compounds from Forsythia leaves. II. Chem. Pharm. Bull. 36, 3667–3670. Macias, F.A., Castellano, D., Molinillo, J.M.G., 2000. Search for a standard phytotoxic bioassay for allelochemicals. Selection of standard target species. J. Agric. Food Chem. 48, 2512–2521. Pelter, A., Ward, R.S., Watson, D.J., Collins, P., Kay, I.T.J., 1982. Synthesis of 2,6-diaryl4,8-dihydroxy-3,7-dioxabicyclo[3.3.0]octanes. J. Chem. Soc., Perkin Trans. 1: Org. Biorg. Chem. (1972–1999) 1, 175–181. Rao, K.V., Wu, W.-N., 1978. Glycosides of magnolia III: structural elucidation of magnolenin C. Lloydia 41, 56–62. Romani, A., Baldi, A., Mulinacci, N., Vincieri, F.F., Tattini, M., 1996. Extraction and identification procedures of polyphenolic compounds and carbohydrates in Phillyrea (Phillyrea angustifolia L.) leaves. Chromatographia 42, 571–577. Sugiyama, M., Kikuchi, M., 1993. Characterization of lariciresinol glucosides from Osmanthus asiaticus. Heterocycles 36, 117–121. Vitale, L., Arena, C., Virzo De Santo, A., D’Ambrosio, N., 2008. Effects of heat stress on gas exchange and photosystem II (PSII) photochemical activity of Phillyrea angustifolia exposed to elevated CO2 and subsaturating irradiance. Can. J. Bot. 86, 435–441.