Article pubs.acs.org/JAFC
Radical-Scavenging Compounds from Olive Tree (Olea europaea L.) Wood Mercedes Pérez-Bonilla,† Sofía Salido,† Teris A. van Beek,‡ and Joaquín Altarejos*,† †
Departamento de Química Inorgánica y Orgánica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus de Excelencia Internacional Agroalimentario, ceiA3, 23071 Jaén, Spain ‡ Laboratory of Organic Chemistry, Natural Products Chemistry Group, Wageningen University, Dreijenplein 8, Wageningen, Huet Brothers 6703, The Netherlands ABSTRACT: The purpose of this study was to complete knowledge on the chemical composition and radical-scavenging activity of olive tree wood. Two new monoterpene glycosides, (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester (6a) and (−)-perillic acid 1′-O-β-D-primeverosyl ester (8), together with the known compounds (−)-oleuropeic acid (1), (−)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), (−)-oleuropeic acid 1′O-β-D-glucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b), and (−)-olivil 4-O-β-D-glucopyranoside (7) were isolated from an ethyl acetate extract. The radical scavengers found (2−4 and 7) were detected and isolated with the help of the online HPLC-DAD-DPPH/ABTS technique. Compounds 2−4 and 7 displayed a higher antioxidative effect against the free radical DPPH than the reference BHT and lower than hydroxytyrosol, whereas compounds 1, 5, 6a, 6b, and 8 showed no activity. KEYWORDS: olive tree wood, Olea europaea, agricultural byproducts, online HPLC-DAD-radical-scavenging technique, (−)-oleuropeic acid, (−)-olivil, aldehydic form of oleuropein aglycone, (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside, (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester, (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester, (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester, (−)-olivil 4-O-β-D-glucopyranoside, (−)-perillic acid 1′-O-β-D-primeverosyl ester
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INTRODUCTION
Olive tree wood constitutes a very important agricultural byproduct since large amounts of biomass from pruning are generated every year in all olive oil producing countries. Just in Spain more than 7 million tonnes per year are produced of this biomass, which consists mainly of wood together with leaves and small stems. Olive tree wood is an untapped resource since nowadays it is basically used for house heating in rural areas. For that reason, alternative uses for olive tree wood are being investigated such as a raw material for production of activated carbon,18 lignin,19 cellulose pulp,20 and bioethanol21 or as a fuel source.22 However, no information is available on the antioxidant activity of the extracts obtained from this part of the olive tree, with the exception of our previous work.14,23−25 With the help of HPLC online assay for antioxidants,26 we detected and isolated the active components of ethyl acetate and ethanol extracts of olive tree wood:14,24,25 oleuropein, ligustroside, hydroxytyrosol, (+)-cycloolivil, oleuropein-3″methyl ether, (7″R)-7″-ethoxyoleuropein, (7″S)-7″-ethoxyoleuropein, (7″S)-7″-hydroxyoleuropein, ligustroside 3′-O-β-Dglucoside, oleuropein 3′-O-β-D-glucoside, jaspolyanoside, jaspolyoside, and isojaspolyoside A (Figure 1). In addition, during the purification work of antioxidants, other compounds which exhibited weak antioxidant activity, such as tyrosol (Figure 1), were also isolated.24 Two of these compounds, oleuropein and (+)-cycloolivil, were included in a study to determine their
Reactive oxygen species (ROS) are widely believed to cause or aggravate several human pathologies such as neurodegenerative diseases, cancer, stroke, and many other ailments.1 ROS are also the main factor responsible for complete spoilage of lipidcontaining foods.2 For both reasons antioxidants are of great importance nowadays in medicine and in the food industry. In the latter field, some concerns have been expressed about the safety of certain synthetic (artificial) antioxidants commonly used to preserve food, such as tert-butylhydroquinone (TBHQ) and tert-butyl-4-hydroxyanisole (BHA),3 which has driven much attention to natural antioxidants and the search of new plant sources containing them. Thus, much work has been done to find interesting sources of potentially safe natural antioxidants in any material of vegetable origin, such as medicinal plants,4 foods, and drinks,5 etc., or even in agroindustrial waste materials.6 The olive tree (Olea europaea L., Oleaceae) is one the most important fruit trees in the Mediterranean countries. The chemical composition of the tree and that of olive oil has been intensively studied for years.7−9 Among the phytochemicals reported in olive tree, the phenolic compounds constitute an interesting group whose presence has been investigated in practically all parts of the plant: fruits,10 leaves,11 stems,12 bark,13 wood,14 stones,15 and roots.16 Due to the high interest of phenolic compounds for the food and pharmacy industries great efforts are also being directed toward their recovery from any byproducts and residues generated in olive oil industrial production.17 © XXXX American Chemical Society
Received: September 20, 2013 Revised: December 7, 2013 Accepted: December 11, 2013
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Figure 1. Compounds previously isolated from olive tree wood.14,24,25,28
antiplatelet aggregation properties.27 More recently, a procedure to obtain D-mannitol and elenolic acid from olive tree wood, along with extracts enriched in antioxidants hydroxytyrosol and oleuropein, have been patented by us.28 Similarly, further work has been reported by others on the composition and applications of fractions obtained from olive tree wood29−32 and also from the whole byproduct obtained during pruning work on olive grooves.33−37 All the work developed on radical scavengers from olive tree wood extracts shows that this agricultural byproduct could become a very interesting source of natural antioxidants, as commercial olive leaf extracts already are,38 i.e., further research is needed. Thus, in this work, an additional activity-guided isolation of antioxidants from olive tree wood and evaluation of their antioxidant activities are reported. To perform the study, the online HPLC-DAD-DPPH/ABTS technique has been used, since it has demonstrated to be a very useful tool for rapid identification of antioxidants in plant extracts, foods, and beverages.39,40 This work complements previous studies from our laboratory to give an overall picture of the chemical constituents present in olive tree wood, a renewable source of natural antioxidants rather unknown until a few years ago.
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MATERIALS AND METHODS
General Experimental Procedures. Optical rotations ([α]D) were recorded in methanol on a Perkin-Elmer 241 automatic polarimeter (Perkin-Elmer Instruments, Norwalk, CT, USA) in a 10 cm 2 mL cell. UV spectra were obtained in methanol on a PerkinElmer Lambda 19 UV/vis/NIR spectrophotometer (Perkin-Elmer Instruments, Norwalk, CT, USA). IR spectra were measured, in liquid films (neat) between KBr plates, on a Perkin-Elmer model 1760X FTIR spectrometer (Perkin-Elmer Instruments, Norwalk, CT, USA). 1 H (400 MHz), 13C (100 MHz), and 2D NMR (500 MHz) spectra were taken on Bruker DPX 400 and Bruker AMX 500 spectrometers (Bruker Daltonik GmbH, Rheinstetten, Germany) using CD3OD as solvent and tetramethylsilane (TMS) as internal reference. When necessary, due to small amounts of samples, NMR spectra were recorded using SHIGEMI tubes (Campro Scientific, The Netherlands) and a Zirconia zircon oxide rotor (Wilmad Labglass, USA). Mass spectra were recorded on two different mass spectrometers: (a) a Hewlett-Packard model HP 5989B mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using the electron impact ionization method (70 eV), ion source temperature of the MS unit = 250 °C, scan range from m/z 35 to m/z 400 and (b) a Finnigan MAT LCQ ion trap mass spectrometer (Waters Integrity System, Milford, MA, USA) with ESI interface used in both modes, with a capillary temperature of 200 °C and spray voltage of −4.5 kV. High-resolution B
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Table 1. 1H and 13C NMR Spectroscopy Data (400 MHz, CD3OD) and HMBC (500 MHz, CD3OD) Correlations of Compounds 6a and 6b (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester (6a) position 1 2 3ax 3eq 4 5ax 5eq 6ax 6eq 7 8 CH3-8 CH3′-8 1′ 2′ 3′ 4′ 5′ 6′a 6′b
δH mult. (J, Hz) 6.97−7.00, 1.92−2.03, 2.26−2.33, 1.45−1.53, 1.13−1.21, 1.92−2.03, 2.04−2.14, 2.43−2.50,
m m m m m m m m
1.13, s 1.13, s 5.03, d (3.7) 3.27−3.35, m 3.63, t (9.3) 3.27−3.35, m 3.94, ddd (1.9, 5.1, 9.3) 4.34, dd (1.9, 11.3) 4.21, dd (5.1, 11.3)
δC, mult. 131.2, qC 141.4, CH 28.5, CH2 45.5, CH 24.5, CH2 26.3, CH2 168.9, 72.8, 26.4, 27.0, 94.0, 73.8, 74.8, 72.0, 70.8, 64.8,
qC qC CH3 CH3 CH CH CH CH CH CH2
(−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b) δH mult. (J, Hz)
HMBC 3, 1, 1, 3, 1, 1, 1, 1,
4, 2, 2, 5, 3, 2, 3 2,
6, 4, 4, 6, 4, 3,
7 6, 5 8, 6, 4,
8, CH3-8 CH3-8, CH3′-8, 8 8, CH3-8
4, 5, 7
3, 4, 5, 8, CH3′-8 3, 4, 5, 8, CH3-8 3′, 5′ 3′, 5′, 6′ 1′, 2′, 4′ 5′, 6′ 3′, 4′ 7, 4′, 5′ 7, 4′, 5′
6.97−7.00, 1.92−2.03, 2.26−2.33, 1.45−1.53, 1.13−1.21, 1.92−2.03, 2.04−2.14, 2.43−2.50,
m m m m m m m m
1.13, s 1.13, s 4.44, d (7.8) 3.27−3.35, m 3.09, t (8.5) 3.27−3.35, m 3.44−3.49, m 4.40, dd (1.7, 12.1) 4.19, dd (5.8, 12.1)
mass spectra (HRESIMS) were recorded on a Waters Micromass AutoSpec NT spectrometer (Waters Integrity System, Milford, MA, USA). Silica gel 60 (Merck), particle size 40−63 μm, was used for column chromatography. Silica gel 60 F254 precoated aluminum sheets (0.25 mm, Merck) were employed for thin-layer chromatography (TLC). High-performance liquid chromatography (HPLC) analyses were performed by an analytical RP-HPLC (Spherisorb ODS-2 column, 250 mm × 3 mm i.d., 5 μm, Waters Chromatography Division, Milford, MA, USA) on a Waters 600E instrument (Waters Chromatography Division, Milford, MA, USA) equipped with a diode array detector, scan range = 190−800 nm (Waters CapLC 2996 Photodiode Array Detector, Waters Chromatography Division, Milford, MA, USA), and operating at 30 °C. Samples of the extracts were prepared in MeOH at a concentration of 10 mg/mL, and the injection volume was 10 μL. The best separation was obtained with H2O:CH3COOH, 99.8:0.2 v/v (solvent A), and CH3OH:CH3COOH, 99.8:0.2, v/v (solvent B), at a flow rate of 0.7 mL/min: linear gradient from 20% to 70% B for 55 min and another 10 min to return to the initial conditions. Preparative HPLC separations were performed with a Alltima C18 column (250 mm × 22 mm i.d., 5 μm, Alltech Associates Inc., Deerfield, IL, USA) on a Shimadzu preparative HPLC instrument (Shimadzu, Kyoto, Japan) equipped with a diode array detector, scan range = 190−600 nm (SPD-M10Ap Photodiode Array Detector, Shimadzu, Kyoto, Japan), and a sample collector FRC-10A (Shimadzu, Kyoto, Japan), operating at 30 °C and a flow rate of 12 mL min−1. Chemicals. Solvents used for extraction and chromatographic separation (dichloromethane, ethyl acetate, ethanol, and chloroform) were glass distilled prior to use. Methanol used for radical-scavenging activity assays was of HPLC grade. Deuterated methanol was used to prepare solutions of purified compounds for NMR analysis. The following reagents were used for radical-scavenging assays: 2,2diphenyl-1-picrylhydrazyl radical (DPPH•) (95%, Sigma-Aldrich Chemie, Steinheim, Germany), 2,2′-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS) (95%, Fluka Chemie, Buchs, Switzerland), potassium persulfate (99%, Sigma-Aldrich Chemie, Steinheim, Germany), NaCl (99%, Panreac, Barcelona, Spain), KH2PO4 (99.5%, Riedel de Haën, Seeize, Germany), Na2HPO4 (99.5%, Riedel de Haën, Seeize, Germany), KCl (99%, Panreac, Barcelona, Spain), hydroxytyrosol (Extrasynthese, Genay,
δC, mult. 131.2, qC 141.4, CH 28.5, CH2 45.5, CH 24.5, CH2 26.3, CH2 168.9, 72.8, 26.4, 27.0, 98.2, 77.9, 76.2, 71.7, 75.4, 64.8,
qC qC CH3 CH3 CH CH CH CH CH CH2
HMBC 3, 1, 1, 3, 1, 1, 1, 1,
4, 2, 2, 5, 3, 2, 3 2,
6, 4, 4, 6, 4, 3,
7 6, 5 8, 6, 4,
8, CH3-8 CH3-8, CH3′-8, 8 8, CH3-8
4, 5, 7
3, 4, 5, 8, CH3′-8 3, 4, 5, 8, CH3-8 2′, 3′, 5′ 4′ 1′, 2′ 2′, 3′, 5′, 6′ 3′, 4′ 7, 4′, 5′ 7, 5′
France), and 2,6-di-tert-butyl-hydroxytoluene (BHT) (99%, SigmaAldrich Chemie, Steinheim, Germany). Plant Material. The wood sample of O. europaea L. (Picual variety) used in this study was collected from the pruning of a tree growing near the town of Fuensanta (province of Jaén, Spain) in April 2003 and consisted of a single piece of 25 cm diameter and 70 cm length. The sample was stored for 3 months in a dry and dark place at room temperature with passive ventilation prior to extraction. Just before starting the extraction process, the wood piece was scraped in a local sawmill (wood shavings length 3−5 cm, thickness 0.1−0.3 mm). Extraction and Prefractionation of Olive Wood Extract. Nine individual but identical batches of 250 g of wood chips (9 × 250 g) were extracted successively by maceration during 24 h at room temperature with dichloromethane (9 × 3.5 L) and then for 2 h at reflux with ethyl acetate (9 × 3.5 L). Solvents were evaporated under reduced pressure to give the corresponding dichloromethane (9.2 g) and ethyl acetate (31.2 g) extracts. An aliquot of the ethyl acetate extract (23.4 g) was chromatographed by open column chromatography on silica gel 60 (230 g, 40−63 μm) using CHCl3:EtOH mixtures of increasing polarity. Fractions of 350 mL were collected, monitored by TLC, pooled, and evaporated to give 11 major fractions: C1 (0.38 g), C2 (2.75 g), C3 (3.35 g), C4 (3.17 g), C5 (4.98 g), C6 (4.00 g), C7 (1.09 g), C8 (1.17 g), C9 (0.60 g), C10 (1.27 g), and C11 (0.20 g). Activity-Guided Chromatographic Isolation of Antioxidants. Fractions C1−C11 were assessed for their ability to scavenge the DPPH radical by the offline spectrophotometric DPPH assay (calculated as radical-scavenging percentage, RSP) according to the procedure described below. Among all these fractions, the most active ones were C3, C5, C4, and C6, with RSP values of 91.8%, 90.4%, 76.9%, and 55.6%, respectively. These active fractions were further monitored by online HPLC-DAD-DPPH/ABTS assays to detect the active components present in each fraction according to the procedure described below. Thus, analysis of fraction C5 revealed that the active constituent was the previously isolated antioxidant oleuropein (Figure 1),14 and analyses of fractions C3, C4, and C6 showed the presence of some further minor active constituents not reported yet. Accordingly, fraction C3 (3.3 g) was subjected to silica gel 60 (235 g, 40−63 μm) column chromatography using CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 16 fractions (C3-I−C3-XVI). These latter fractions were again monitored by the online assays, which allowed focusing attention C
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Table 2. 1H and 13C NMR Spectroscopy Data (400 MHz, CD3OD) and HMBC (500 MHz, CD3OD) Correlations of Compound 8
on fractions C3-VII and C3-X. Thus, an aliquot of fraction C3-VII (101 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with H2O:CH3COOH, 99.8:0.2 v/v (solvent A), and CH3OH:CH3COOH, 99.8:0.2 v/v (solvent B): isocratic conditions of 25% B for 10 min and then a linear gradient from 25% to 30% B in 10 min yielded pure compound 1: (−)-oleuropeic acid (10 mg). An aliquot of fraction C3-X (104 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with isocratic conditions of 30% B for 25 min yielding pure compound 2: (−)-olivil (17.3 mg). Fraction C4 (3.0 g) was subjected to silica gel 60 (177 g, 63−200 μm) column chromatography using CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 20 fractions (C4-I−C4-XX). These latter fractions were again monitored by online assays. This allowed focusing attention on fraction C4-III (10.4 mg), which was identified as pure compound 3: the aldehydic form of oleuropein aglycone. Fraction C6 (4.0 g) was fractionated by column chromatography over silica gel 60 (180 g, 40−63 μm) and eluted with CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 13 fractions (C6-I− C6-XIII). These latter fractions were again monitored by online assays. This allowed focusing attention on fractions C6-VIII, C6-X, and C6-XII. An aliquot of fraction C6-VIII (237 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with a linear gradient from 25% to 40% B in 40 min yielding pure compound 4: (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (5.1 mg). An aliquot of fraction C6-X (207.2 mg) was further separated by preparative RP-HPLC. Separation was carried out with H 2 O:CH 3 OH:CH 3 COOH, 79.8:20:0.2 v/v (solvent C), and CH3OH:CH3COOH, 99.8:0.2 v/v (solvent B); isocratic conditions of 15% B for 5 min and then a linear gradient from 15% to 100% B in 30 min to yield pure compound 5: (−)-oleuropeic acid 1′-O-β-Dglucopyranosyl ester (4.5 mg). Similarly, another aliquot of fraction C6-X (31.4 mg) was further separated by preparative RP-HPLC with isocratic conditions of 10% B for 60 min to yield a mixture of compounds 6a ((−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester) and 6b ((−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester) (8.3 mg). Fraction C6-XII (1.1 g) was chromatographed on silica gel 60 (172 g, 40−63 μm), eluting with a 90:10 EtOAc:EtOH mixture. Fractions of 30 mL were collected and combined on the basis of TLC into 13 fractions (C6-XIIa−C6-XIIm). These latter fractions were again monitored by online assays. This allowed focusing attention on fraction C6-XIIj. Thus, an aliquot of fraction C6-XIIj (214.1 mg) was further separated by preparative RP-HPLC with isocratic conditions of 15% B for 10 min and then a linear gradient from 15% to 100% B in 20 min to yield pure compound 7: (−)-olivil 4-O-β-D-glucopyranoside (7.5 mg). Finally, another aliquot of fraction C6-XIIj (91.8 mg) was further separated by preparative RP-HPLC with a linear gradient from 20% to 70% B in 55 min to yield pure compound 8: (−)-perillic acid 1′-O-β-D-primeverosyl ester (3.5 mg). Structural Elucidation of New Natural Products. (−)-Oleuropeic Acid 6′-O-α/β-D-Glucopyranosyl Esters (6a and 6b). Yellowish syrup; UV (MeOH) λmax (log ε) 218.8 (3.8) nm; IR (neat) νmax 3384, 2925, 1701, 1649, 1384, 1258, 1058 cm−1; ESIMS m/z 369.2 ([M + Na]+), 345.2 ([M − H]−); HRESIMS m/z 347.1701 [M + H]+ (calcd for C16H27O8, 347.3856), 391.1594 [M + HCOO]− (calcd for C17H27O10, 391.3954); 1H and 13C NMR, see Table 1. (−)-Perillic Acid 1′-O-β-D-Primeverosyl Ester (8). Yellowish syrup; [α]25 D −41° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 222 (3.6), 302.8 (2.9) nm; IR (neat) νmax 3356, 1702, 1654, 1070 cm−1; ESIMS m/z 483.2 ([M + Na]+), 942.7 [2M + Na]+, 459.1 [M − H]−, 919.0 [2M − H]−; HRESIMS m/z 483.1831 [M + Na]+ (calcd for C21H32O11Na, 483.4683), 505.1927 [M + HCOO]− (calcd for C22H33O13, 505.4962); 1H and 13C NMR, see Table 2. Offline Spectrophotometric DPPH Assay. Radical-scavenging activity of chromatographic fractions, purified compounds, and reference compounds was determined spectrophotometrically with the stable DPPH radical.41 Methanolic solutions (2.4 mL) of DPPH• (∼7 × 10−5 M) with an absorbance at 515 nm of 0.80 ± 0.03 AU were mixed with methanolic solutions (1.2 mL) of samples at different
(−)-perillic acid 1′-O-β-D-primeverosyl ester (8) position 1 2 3eq 3ax 4 5eq 5ax 6eq 6ax 7 8 9a 9b 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″a 5″b a
δH mult. (J in Hz) 7.14, bs 2.34−2.49, 2.16−2.27, 2.34−2.49, 1.85−1.97, 1.44−1.56, 2.34−2.49, 2.16−2.27,
m m m m m m m
4.76, s 4.73, s 1.75, s 5.49, d (7.5) 3.35−3.50, m 3.50−3.56, m 3.35−3.50, m 3.35−3.50, m 4.08, bd (11.2) 3.70, dd (4.8, 11.2) 4.27, d (7.5) 3.12−3.21, m 3.35−3.50, m 3.35−3.50, m 3.83, dd (5.8, 11.3) 3.12−3.21, m
δC, mult. 130.51, qC 142.50, CH 32.27, CH2
HMBC 6, 7 2
41.36, CH 28.18, CH2 3, 4 25.41, CH2 167.26, qC 150.02, qC 109.79, CH2 20.87, CH3 95.74, CH 73.92, CH 77.75,a CH 70.96,b CH 77.88,a CH 69.42, CH2 105.14, CH 74.87, CH 77.70,a CH 71.16,b CH 66.91, CH2
4, 10 4, 10 4, 8, 9 7 1′, 4′ 2′
3′, 4′, 1″ 6′, 5″ 1″, 4″ 5″ 1″, 3″, 4″ 1″
Interchangeable signals. bInterchangeable signals.
concentrations by dissolving the dry samples in methanol. The experiment was carried out in triplicate. Samples were shaken and kept in the dark for 15 min at room temperature, and then the decrease of absorbance was measured at 515 nm. Radical-scavenging activity of chromatographic fractions, using a concentration of 50 μg/mL, is expressed as radical-scavenging percentage (RSP) and was calculated by the following formula41
⎡A − A ⎤ A RSP = ⎢ B ⎥ × 100 ⎣ AB ⎦ where AB is the absorbance of the blank (t = 0 min) and AA is the absorbance of tested sample solution (t = 15 min). Radical-scavenging activity of purified and reference compounds, using different concentrations (4−100 μg/mL), is expressed in terms of the antioxidant concentration (μM) required to decrease the initial DPPH• concentration by 50% (efficient concentration, EC50). The percentage of DPPH• remaining, calculated by the following equation42
%DPPH rem =
[DPPH] × 100 [DPPH]0
where [DPPH] is the concentration of DPPH• at the time measured (t = 15 min) and [DPPH]0 is the initial concentration of DPPH• (t = 0 min), was plotted against the sample concentration (μg/mL), a linear or logarithmic regression curve being established in order to calculate the EC50. Online HPLC-DAD-DPPH/ABTS Assays. Online HPLC radicalscavenging assays of the fractions from the olive tree wood ethyl acetate extract were performed to detect active compounds in complex mixtures.39 The setup for online assessment of radical-scavenging D
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activity consisted of an eluent pump, a photodiode array detector, and a Agilent 1100 Series controller, equipped with a C18 Waters Spherisorb S5 ODS2 (250 × 3 mm, 5 μm) analytical column. Methanolic solutions of fractions were prepared by dilution in methanol at a concentration of 10 mg/mL, and the injection volume was 10 μL. Separation was performed by a linear gradient from 20% to 70% B in 55 min, isocratic hold at 100% B for 5 min, linear gradient from 100% to 20% B in 10 min, and 10 min isocratic hold at 20% B for re-equilibration of the column. After HPLC separation and UV detection, the flow was mixed with a methanolic DPPH• solution or an ABTS•+ solution in PBS.43 Radical solutions were delivered using a Gilson model 302 pump at 0.5 mL/min for both DPPH• and ABTS•+ solutions. The reaction between separated compounds and radicals took place in a 3.5 m × 0.25 mm i.d. PEEK reaction coil. The decrease in absorbance was measured at 515 nm for DPPH• and at 414 nm for ABTS•+ in an Applied Biosystems UV/vis 759A spectrophotometer equipped with a tungsten lamp.
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RESULTS AND DISCUSSION Isolation of Compounds from the Olive Wood Extract. Olive wood chips were sequentially extracted with dichloromethane and ethyl acetate at room temperature and under reflux conditions, respectively. Dichloromethane extraction (0.4% yield) allowed removing the nonpolar components, which showed low antioxidant activity.25 The resulting ethyl acetate extract (1.4% yield) was prefractionated by open column chromatography on silica gel, yielding 11 major fractions (C1−C11). These fractions were assessed for their ability to scavenge the DPPH radical by offline spectrophotometric measurements. This approach was chosen because it is generally known that lipid oxidation is initiated by a free radical attack and due to their simplicity and worldwide acceptance for comparative purposes. Among all these fractions, in terms of DPPH radical-scavenging percentages (RSP), the most active fractions were, in this order, C3 (14.3% yield), C5 (21.3% yield), C4 (13.5% yield), and C6 (17.1% yield). These active fractions were further monitored by online HPLC-DADDPPH/ABTS assays to detect the active components present in each fraction.14 After a combination of conventional silica gel column chromatography and preparative RP-HPLC the following antioxidants were finally isolated: (−)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-Dglucopyranoside (7) (Figure 2). In addition, during the purification work of antioxidants, several nonactive compounds, such as (−)-oleuropeic acid (1), (−)-oleuropeic acid 1′-O-β-Dglucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-α-Dglucopyranosyl ester (6a), (−)-oleuropeic acid 6′-O-β-Dglucopyranosyl ester (6b), and (−)-perillic acid 1′-O-β-Dprimeverosyl ester (8) (Figure 2), were also isolated. Structure Elucidation of the Isolated Compounds. Compounds 1−8 were characterized and identified by UV, IR, MS, HRMS, 1H NMR, 13C NMR, 2D NMR, and specific optical rotation measurements. Spectral and physical data of compounds 1−5, 6b, and 7 are in agreement with earlier published data: (−)-oleuropeic acid (1),44 (−)-olivil (2),45 the aldehydic form of oleuropein aglycone (3),46 (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4),13 (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester (5),44 (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b),44 and (−)-olivil 4-O-β-Dglucopyranoside (7) (Figure 2).47 Compounds 2−4 and 7 have been reported before from other parts of the olive tree. Compounds 1, 5, and 6b have been previously found in the leaves of Cunila spicata L.44 esterified with acylated glucose,
Figure 2. Compounds isolated in this work from an olive tree wood EtOAc extract.
while the methyl ester of compound 1 has been also isolated from Eucalyptus cypellocarpa leaves.48 Compounds 6a and 6b were assigned a molecular formula of C16H26O8 by HRESIMS. They are anomeric glucosides, and their 1 H NMR spectrum showed, apart from signals corresponding to two monoterpene aglycone units, similar to that of compound 1, a set of signals assignable to two glucose moieties; one anomeric proton is centered at 4.44 ppm (1H, d, 7.8 Hz), consistent with the configuration for a β-Dglucopyranosyl group, while the other is centered at 5.03 ppm (1H, d, 3.7 Hz), consistent with the configuration for an α-D-glucopyranosyl group (Table 1). In the HMBC spectrum, cross peaks between the proton signals H-6′a and H-6′b and the carbon C-7 indicate the linkage of the monoterpene residues and the glucose units. Further cross peaks between the proton signal at δ 5.03 (H-1′), from the α anomer, and the carbons at δ 74.8 (C-3′) and 70.8 (C-5′) and between the proton signal at δ 4.44 (H-1′), from the β anomer, and the carbons at δ 77.9 (C-2′), 76.2 (C-3′), and 75.4 (C-5′) allowed us to assign 13C peaks for each anomer. Accordingly, the structure of compound 6a was determined to be (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester, while 6b was determined to be (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (Figure 2). Integration of anomeric protons in the 1H NMR spectrum indicated an equilibrium mixture of anomers, 6a and 6b, with a ratio of 1:1. Compound 8 was assigned a molecular formula of C21H32O11 by HRESIMS. The 1H NMR spectrum (Table 2) showed signals of an isopropenyl moiety [δ 1.75 (3H, s), 4.73 (1H, s), and 4.76 (1H, s)], an olefinic proton [δ 7.14 (1H, br s)], three methylene groups [δ 1.44−2.49 (6H, m)], and a methyne group [δ 2.34−2.49 (1H, m)], corresponding to a monoE
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than those observed for other secoiridoids, such as jaspolyoside (55 μM), oleuropein 3′-O-β-D-glucoside (66 μM), isojaspolyoside A (108 μM), 7″S-hydroxyoleuropein (113 μM), oleuropein-3″-methyl ether (170 μM), ligustroside (233 μM), ligustroside 3′-O-β-D-glucoside (290 μM), and jaspolyanoside (847 μM), and iridoids, such as 7-deoxyloganic acid (742 μM).14,27 The lignans (−)-olivil (2), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-D-glucopyranoside (7) showed EC50 values of 176, 164, and 134 μM, respectively. These activities are lower than that observed for lignan (+)-cycloolivil (90 μM).27 Monoterpenoids 1, 5, 6a, 6b, and 8 did not show any antioxidant activity. In summay, this work completes previous studies on the composition of an ethyl acetate extract from olive tree wood,14,24,25 as well as their antioxidant activity. On the basis of their chemical structures, the 25 compounds isolated from olive tree wood can be classified into various phytochemical subclasses including simple phenols [hydroxytyrosol, tyrosol], monoterpenoids [(−)-oleuropeic acid (1), (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-αD-glucopyranosyl ester (6a), (−)-oleuropeic acid 6′-O-β-Dglucopyranosyl ester (6b), and (−)-perillic acid 1′-O-β-Dprimeverosyl ester (8)], lignans [(+)-cycloolivil, (−)-olivil (2), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-D-glucopyranoside (7)], iridoids [7-deoxyloganic acid], secoiridoids [ligustroside, oleuropein, the aldehydic form of oleuropein aglycone (3), oleuropein-3″-methyl ether, (7″R)-7″-ethoxyoleuropein, (7″S)-7″-ethoxyoleuropein, (7″S)7″-hydroxyoleuropein, ligustroside 3′-O-β-D-glucoside, oleuropein 3′-O-β-D-glucoside, jaspolyanoside, jaspolyoside, and isojaspolyoside A], and a sugar alcohol [D-mannitol]. The results obtained here complete the current knowledge of olive tree wood composition and confirm that this agricultural byproduct contains diverse phytochemicals, among which secoiridoids predominate, which in turn could offer interesting potential applications in the food, cosmetic, and pharmaceutical industries.
terpene aglycone unit. Additionally, a set of signals assignable to two sugar moieties was observed; one anomeric proton is centered at 4.27 ppm (1H, d, 7.5 Hz), while the other is centered at 5.49 ppm (1H, d, 7.5 Hz), indicating the β configuration for both sugar units. The 13C NMR spectrum showed the presence of an isopropenyl group (a methyl [δ 20.87 (C-10)], a terminal methylene [δ 109.79 (C-9)], and a quaternary carbon [δ 150.02 (C-8)]), three methylene groups [δ 25.41 (C-6), 28.18 (C-5), and 32.27 (C-3)], a methyne carbon [δ 41.36 (C-4)], two olefinic carbons [δ 130.51 (C-1) and 142.50 (C-2)], and a quaternary carbon of an α,βunsaturated ester [167.26 (C-7)]. The 13C NMR spectrum also showed 11 signals in the sugar region, two of them being anomeric carbons [δ 95.74 (C-1′) and 105.14 (C-1″)]. The two sugars were identified as glucose and xylose. In the HMBC spectrum, cross peaks between the proton signals at δ 5.49 (H1′) and the carbon at δ 167.26 (C-7) indicate the linkage between the monoterpene residue and the glucose unit. Similarly, cross peaks between the proton signal at δ 4.27 (H-1″) and the carbon at δ 69.42 (C-6′) and between the protons at δ 4.08 (H-6′a) and δ 3.70 (H-6′b) with the carbon at δ 105.14 (C-1″) showed the linkage between the xylose and the glucose units. A glucose and xylose moiety linked via C-6′ → C-1″ is known as the disaccharide primeverose. This linkage explained the upfield shifts observed for the glucose carbons C1′, C-2′, and C-5′ and the downfield shifts for carbon C-6′. Accordingly, the structure of compound 8 was determined to be (−)-perillic acid 1′-O-β-D-primeverosyl ester, which is a new natural product (Figure 2). Radical-Scavenging Activity of the Isolated Antioxidants. The antioxidant activity of the isolated compounds from olive tree wood was determined with the DPPH radicalscavenging assay and expressed in terms of the antioxidant concentration (μM) required to reduce the initial DPPH• concentration after 15 min by 50% (efficient concentration, EC50). In Table 3, EC50 values for the active compounds 2−4
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Table 3. DPPH Radical-Scavenging Activity of the Isolated Active Compounds from Olive Wood Ethyl Acetate Extract, Expressed as Efficient Concentration (EC50) compounds
EC50 (μM)a
hydroxytyrosol aldehydic form of oleuropein aglycone (3) (−)-olivil 4-O-β-D-glucopyranoside (7) (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4) (−)-olivil (2) BHTb
39 45 134 164 176 505
AUTHOR INFORMATION
Corresponding Author
*Tel.: +34 953 212743. Fax: +34 953 211876. E-mail: jaltare@ ujaen.es. Funding
This study was supported by the Spanish Ministerio de Educación y Ciencia (R+D Project CTQ2005-07005/PPQ; partial financial support from the FEDER funds of the European Union). M.P.-B. was the recipient of a predoctoral fellowship granted by the Andalusian Government Junta de Andalucia.́ Part of the work was supported by the Centro de ́ Instrumentación Cientifico-Té cnica of the University of Jaén.
a
EC50 values are means of three replicates, and the RSD is less than 1%. b2,6-Di-tert-butyl-4-methylphenol.
and 7 and the reference compounds BHT and hydroxytyrosol are presented as the average of three replicates. The activities of compounds 2−4 and 7 were higher than that of reference BHT (505 μM) and lower than that of hydroxytyrosol (39 μM), which was the most active component identified in olive tree wood together to oleuropein (32 μM).27 Hydroxytyrosol is more active than tyrosol (826 μM),24 which could be related to the stabilization by delocalization of the o-diphenolic moiety present in hydroxytyrosol, since o-quinone formation is favored. Thus, the aldehydic form of oleuropein aglycone (3) exhibited marked activity; its efficient concentration (45 μM) was just somewhat lower than that of oleuropein (32 μM) and higher
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank Dr. P. de Waard for his technical assistance in recording NMR spectra.
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Radical-Scavenging Compounds from Olive Tree (Olea europaea L.) Wood Mercedes Pérez-Bonilla,† Sofía Salido,† Teris A. van Beek,‡ and Joaquín Altarejos*,† †
Departamento de Química Inorgánica y Orgánica, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus de Excelencia Internacional Agroalimentario, ceiA3, 23071 Jaén, Spain ‡ Laboratory of Organic Chemistry, Natural Products Chemistry Group, Wageningen University, Dreijenplein 8, Wageningen, Huet Brothers 6703, The Netherlands ABSTRACT: The purpose of this study was to complete knowledge on the chemical composition and radical-scavenging activity of olive tree wood. Two new monoterpene glycosides, (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester (6a) and (−)-perillic acid 1′-O-β-D-primeverosyl ester (8), together with the known compounds (−)-oleuropeic acid (1), (−)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), (−)-oleuropeic acid 1′O-β-D-glucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b), and (−)-olivil 4-O-β-D-glucopyranoside (7) were isolated from an ethyl acetate extract. The radical scavengers found (2−4 and 7) were detected and isolated with the help of the online HPLC-DAD-DPPH/ABTS technique. Compounds 2−4 and 7 displayed a higher antioxidative effect against the free radical DPPH than the reference BHT and lower than hydroxytyrosol, whereas compounds 1, 5, 6a, 6b, and 8 showed no activity. KEYWORDS: olive tree wood, Olea europaea, agricultural byproducts, online HPLC-DAD-radical-scavenging technique, (−)-oleuropeic acid, (−)-olivil, aldehydic form of oleuropein aglycone, (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside, (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester, (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester, (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester, (−)-olivil 4-O-β-D-glucopyranoside, (−)-perillic acid 1′-O-β-D-primeverosyl ester
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INTRODUCTION
Olive tree wood constitutes a very important agricultural byproduct since large amounts of biomass from pruning are generated every year in all olive oil producing countries. Just in Spain more than 7 million tonnes per year are produced of this biomass, which consists mainly of wood together with leaves and small stems. Olive tree wood is an untapped resource since nowadays it is basically used for house heating in rural areas. For that reason, alternative uses for olive tree wood are being investigated such as a raw material for production of activated carbon,18 lignin,19 cellulose pulp,20 and bioethanol21 or as a fuel source.22 However, no information is available on the antioxidant activity of the extracts obtained from this part of the olive tree, with the exception of our previous work.14,23−25 With the help of HPLC online assay for antioxidants,26 we detected and isolated the active components of ethyl acetate and ethanol extracts of olive tree wood:14,24,25 oleuropein, ligustroside, hydroxytyrosol, (+)-cycloolivil, oleuropein-3″methyl ether, (7″R)-7″-ethoxyoleuropein, (7″S)-7″-ethoxyoleuropein, (7″S)-7″-hydroxyoleuropein, ligustroside 3′-O-β-Dglucoside, oleuropein 3′-O-β-D-glucoside, jaspolyanoside, jaspolyoside, and isojaspolyoside A (Figure 1). In addition, during the purification work of antioxidants, other compounds which exhibited weak antioxidant activity, such as tyrosol (Figure 1), were also isolated.24 Two of these compounds, oleuropein and (+)-cycloolivil, were included in a study to determine their
Reactive oxygen species (ROS) are widely believed to cause or aggravate several human pathologies such as neurodegenerative diseases, cancer, stroke, and many other ailments.1 ROS are also the main factor responsible for complete spoilage of lipidcontaining foods.2 For both reasons antioxidants are of great importance nowadays in medicine and in the food industry. In the latter field, some concerns have been expressed about the safety of certain synthetic (artificial) antioxidants commonly used to preserve food, such as tert-butylhydroquinone (TBHQ) and tert-butyl-4-hydroxyanisole (BHA),3 which has driven much attention to natural antioxidants and the search of new plant sources containing them. Thus, much work has been done to find interesting sources of potentially safe natural antioxidants in any material of vegetable origin, such as medicinal plants,4 foods, and drinks,5 etc., or even in agroindustrial waste materials.6 The olive tree (Olea europaea L., Oleaceae) is one the most important fruit trees in the Mediterranean countries. The chemical composition of the tree and that of olive oil has been intensively studied for years.7−9 Among the phytochemicals reported in olive tree, the phenolic compounds constitute an interesting group whose presence has been investigated in practically all parts of the plant: fruits,10 leaves,11 stems,12 bark,13 wood,14 stones,15 and roots.16 Due to the high interest of phenolic compounds for the food and pharmacy industries great efforts are also being directed toward their recovery from any byproducts and residues generated in olive oil industrial production.17 © XXXX American Chemical Society
Received: September 20, 2013 Revised: December 7, 2013 Accepted: December 11, 2013
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Figure 1. Compounds previously isolated from olive tree wood.14,24,25,28
antiplatelet aggregation properties.27 More recently, a procedure to obtain D-mannitol and elenolic acid from olive tree wood, along with extracts enriched in antioxidants hydroxytyrosol and oleuropein, have been patented by us.28 Similarly, further work has been reported by others on the composition and applications of fractions obtained from olive tree wood29−32 and also from the whole byproduct obtained during pruning work on olive grooves.33−37 All the work developed on radical scavengers from olive tree wood extracts shows that this agricultural byproduct could become a very interesting source of natural antioxidants, as commercial olive leaf extracts already are,38 i.e., further research is needed. Thus, in this work, an additional activity-guided isolation of antioxidants from olive tree wood and evaluation of their antioxidant activities are reported. To perform the study, the online HPLC-DAD-DPPH/ABTS technique has been used, since it has demonstrated to be a very useful tool for rapid identification of antioxidants in plant extracts, foods, and beverages.39,40 This work complements previous studies from our laboratory to give an overall picture of the chemical constituents present in olive tree wood, a renewable source of natural antioxidants rather unknown until a few years ago.
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MATERIALS AND METHODS
General Experimental Procedures. Optical rotations ([α]D) were recorded in methanol on a Perkin-Elmer 241 automatic polarimeter (Perkin-Elmer Instruments, Norwalk, CT, USA) in a 10 cm 2 mL cell. UV spectra were obtained in methanol on a PerkinElmer Lambda 19 UV/vis/NIR spectrophotometer (Perkin-Elmer Instruments, Norwalk, CT, USA). IR spectra were measured, in liquid films (neat) between KBr plates, on a Perkin-Elmer model 1760X FTIR spectrometer (Perkin-Elmer Instruments, Norwalk, CT, USA). 1 H (400 MHz), 13C (100 MHz), and 2D NMR (500 MHz) spectra were taken on Bruker DPX 400 and Bruker AMX 500 spectrometers (Bruker Daltonik GmbH, Rheinstetten, Germany) using CD3OD as solvent and tetramethylsilane (TMS) as internal reference. When necessary, due to small amounts of samples, NMR spectra were recorded using SHIGEMI tubes (Campro Scientific, The Netherlands) and a Zirconia zircon oxide rotor (Wilmad Labglass, USA). Mass spectra were recorded on two different mass spectrometers: (a) a Hewlett-Packard model HP 5989B mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using the electron impact ionization method (70 eV), ion source temperature of the MS unit = 250 °C, scan range from m/z 35 to m/z 400 and (b) a Finnigan MAT LCQ ion trap mass spectrometer (Waters Integrity System, Milford, MA, USA) with ESI interface used in both modes, with a capillary temperature of 200 °C and spray voltage of −4.5 kV. High-resolution B
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Table 1. 1H and 13C NMR Spectroscopy Data (400 MHz, CD3OD) and HMBC (500 MHz, CD3OD) Correlations of Compounds 6a and 6b (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester (6a) position 1 2 3ax 3eq 4 5ax 5eq 6ax 6eq 7 8 CH3-8 CH3′-8 1′ 2′ 3′ 4′ 5′ 6′a 6′b
δH mult. (J, Hz) 6.97−7.00, 1.92−2.03, 2.26−2.33, 1.45−1.53, 1.13−1.21, 1.92−2.03, 2.04−2.14, 2.43−2.50,
m m m m m m m m
1.13, s 1.13, s 5.03, d (3.7) 3.27−3.35, m 3.63, t (9.3) 3.27−3.35, m 3.94, ddd (1.9, 5.1, 9.3) 4.34, dd (1.9, 11.3) 4.21, dd (5.1, 11.3)
δC, mult. 131.2, qC 141.4, CH 28.5, CH2 45.5, CH 24.5, CH2 26.3, CH2 168.9, 72.8, 26.4, 27.0, 94.0, 73.8, 74.8, 72.0, 70.8, 64.8,
qC qC CH3 CH3 CH CH CH CH CH CH2
(−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b) δH mult. (J, Hz)
HMBC 3, 1, 1, 3, 1, 1, 1, 1,
4, 2, 2, 5, 3, 2, 3 2,
6, 4, 4, 6, 4, 3,
7 6, 5 8, 6, 4,
8, CH3-8 CH3-8, CH3′-8, 8 8, CH3-8
4, 5, 7
3, 4, 5, 8, CH3′-8 3, 4, 5, 8, CH3-8 3′, 5′ 3′, 5′, 6′ 1′, 2′, 4′ 5′, 6′ 3′, 4′ 7, 4′, 5′ 7, 4′, 5′
6.97−7.00, 1.92−2.03, 2.26−2.33, 1.45−1.53, 1.13−1.21, 1.92−2.03, 2.04−2.14, 2.43−2.50,
m m m m m m m m
1.13, s 1.13, s 4.44, d (7.8) 3.27−3.35, m 3.09, t (8.5) 3.27−3.35, m 3.44−3.49, m 4.40, dd (1.7, 12.1) 4.19, dd (5.8, 12.1)
mass spectra (HRESIMS) were recorded on a Waters Micromass AutoSpec NT spectrometer (Waters Integrity System, Milford, MA, USA). Silica gel 60 (Merck), particle size 40−63 μm, was used for column chromatography. Silica gel 60 F254 precoated aluminum sheets (0.25 mm, Merck) were employed for thin-layer chromatography (TLC). High-performance liquid chromatography (HPLC) analyses were performed by an analytical RP-HPLC (Spherisorb ODS-2 column, 250 mm × 3 mm i.d., 5 μm, Waters Chromatography Division, Milford, MA, USA) on a Waters 600E instrument (Waters Chromatography Division, Milford, MA, USA) equipped with a diode array detector, scan range = 190−800 nm (Waters CapLC 2996 Photodiode Array Detector, Waters Chromatography Division, Milford, MA, USA), and operating at 30 °C. Samples of the extracts were prepared in MeOH at a concentration of 10 mg/mL, and the injection volume was 10 μL. The best separation was obtained with H2O:CH3COOH, 99.8:0.2 v/v (solvent A), and CH3OH:CH3COOH, 99.8:0.2, v/v (solvent B), at a flow rate of 0.7 mL/min: linear gradient from 20% to 70% B for 55 min and another 10 min to return to the initial conditions. Preparative HPLC separations were performed with a Alltima C18 column (250 mm × 22 mm i.d., 5 μm, Alltech Associates Inc., Deerfield, IL, USA) on a Shimadzu preparative HPLC instrument (Shimadzu, Kyoto, Japan) equipped with a diode array detector, scan range = 190−600 nm (SPD-M10Ap Photodiode Array Detector, Shimadzu, Kyoto, Japan), and a sample collector FRC-10A (Shimadzu, Kyoto, Japan), operating at 30 °C and a flow rate of 12 mL min−1. Chemicals. Solvents used for extraction and chromatographic separation (dichloromethane, ethyl acetate, ethanol, and chloroform) were glass distilled prior to use. Methanol used for radical-scavenging activity assays was of HPLC grade. Deuterated methanol was used to prepare solutions of purified compounds for NMR analysis. The following reagents were used for radical-scavenging assays: 2,2diphenyl-1-picrylhydrazyl radical (DPPH•) (95%, Sigma-Aldrich Chemie, Steinheim, Germany), 2,2′-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS) (95%, Fluka Chemie, Buchs, Switzerland), potassium persulfate (99%, Sigma-Aldrich Chemie, Steinheim, Germany), NaCl (99%, Panreac, Barcelona, Spain), KH2PO4 (99.5%, Riedel de Haën, Seeize, Germany), Na2HPO4 (99.5%, Riedel de Haën, Seeize, Germany), KCl (99%, Panreac, Barcelona, Spain), hydroxytyrosol (Extrasynthese, Genay,
δC, mult. 131.2, qC 141.4, CH 28.5, CH2 45.5, CH 24.5, CH2 26.3, CH2 168.9, 72.8, 26.4, 27.0, 98.2, 77.9, 76.2, 71.7, 75.4, 64.8,
qC qC CH3 CH3 CH CH CH CH CH CH2
HMBC 3, 1, 1, 3, 1, 1, 1, 1,
4, 2, 2, 5, 3, 2, 3 2,
6, 4, 4, 6, 4, 3,
7 6, 5 8, 6, 4,
8, CH3-8 CH3-8, CH3′-8, 8 8, CH3-8
4, 5, 7
3, 4, 5, 8, CH3′-8 3, 4, 5, 8, CH3-8 2′, 3′, 5′ 4′ 1′, 2′ 2′, 3′, 5′, 6′ 3′, 4′ 7, 4′, 5′ 7, 5′
France), and 2,6-di-tert-butyl-hydroxytoluene (BHT) (99%, SigmaAldrich Chemie, Steinheim, Germany). Plant Material. The wood sample of O. europaea L. (Picual variety) used in this study was collected from the pruning of a tree growing near the town of Fuensanta (province of Jaén, Spain) in April 2003 and consisted of a single piece of 25 cm diameter and 70 cm length. The sample was stored for 3 months in a dry and dark place at room temperature with passive ventilation prior to extraction. Just before starting the extraction process, the wood piece was scraped in a local sawmill (wood shavings length 3−5 cm, thickness 0.1−0.3 mm). Extraction and Prefractionation of Olive Wood Extract. Nine individual but identical batches of 250 g of wood chips (9 × 250 g) were extracted successively by maceration during 24 h at room temperature with dichloromethane (9 × 3.5 L) and then for 2 h at reflux with ethyl acetate (9 × 3.5 L). Solvents were evaporated under reduced pressure to give the corresponding dichloromethane (9.2 g) and ethyl acetate (31.2 g) extracts. An aliquot of the ethyl acetate extract (23.4 g) was chromatographed by open column chromatography on silica gel 60 (230 g, 40−63 μm) using CHCl3:EtOH mixtures of increasing polarity. Fractions of 350 mL were collected, monitored by TLC, pooled, and evaporated to give 11 major fractions: C1 (0.38 g), C2 (2.75 g), C3 (3.35 g), C4 (3.17 g), C5 (4.98 g), C6 (4.00 g), C7 (1.09 g), C8 (1.17 g), C9 (0.60 g), C10 (1.27 g), and C11 (0.20 g). Activity-Guided Chromatographic Isolation of Antioxidants. Fractions C1−C11 were assessed for their ability to scavenge the DPPH radical by the offline spectrophotometric DPPH assay (calculated as radical-scavenging percentage, RSP) according to the procedure described below. Among all these fractions, the most active ones were C3, C5, C4, and C6, with RSP values of 91.8%, 90.4%, 76.9%, and 55.6%, respectively. These active fractions were further monitored by online HPLC-DAD-DPPH/ABTS assays to detect the active components present in each fraction according to the procedure described below. Thus, analysis of fraction C5 revealed that the active constituent was the previously isolated antioxidant oleuropein (Figure 1),14 and analyses of fractions C3, C4, and C6 showed the presence of some further minor active constituents not reported yet. Accordingly, fraction C3 (3.3 g) was subjected to silica gel 60 (235 g, 40−63 μm) column chromatography using CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 16 fractions (C3-I−C3-XVI). These latter fractions were again monitored by the online assays, which allowed focusing attention C
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Table 2. 1H and 13C NMR Spectroscopy Data (400 MHz, CD3OD) and HMBC (500 MHz, CD3OD) Correlations of Compound 8
on fractions C3-VII and C3-X. Thus, an aliquot of fraction C3-VII (101 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with H2O:CH3COOH, 99.8:0.2 v/v (solvent A), and CH3OH:CH3COOH, 99.8:0.2 v/v (solvent B): isocratic conditions of 25% B for 10 min and then a linear gradient from 25% to 30% B in 10 min yielded pure compound 1: (−)-oleuropeic acid (10 mg). An aliquot of fraction C3-X (104 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with isocratic conditions of 30% B for 25 min yielding pure compound 2: (−)-olivil (17.3 mg). Fraction C4 (3.0 g) was subjected to silica gel 60 (177 g, 63−200 μm) column chromatography using CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 20 fractions (C4-I−C4-XX). These latter fractions were again monitored by online assays. This allowed focusing attention on fraction C4-III (10.4 mg), which was identified as pure compound 3: the aldehydic form of oleuropein aglycone. Fraction C6 (4.0 g) was fractionated by column chromatography over silica gel 60 (180 g, 40−63 μm) and eluted with CHCl3:EtOH mixtures of increasing polarity. Fractions of 50 mL were collected and combined on the basis of TLC into 13 fractions (C6-I− C6-XIII). These latter fractions were again monitored by online assays. This allowed focusing attention on fractions C6-VIII, C6-X, and C6-XII. An aliquot of fraction C6-VIII (237 mg) was further chromatographed by preparative RP-HPLC. Separation was carried out with a linear gradient from 25% to 40% B in 40 min yielding pure compound 4: (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (5.1 mg). An aliquot of fraction C6-X (207.2 mg) was further separated by preparative RP-HPLC. Separation was carried out with H 2 O:CH 3 OH:CH 3 COOH, 79.8:20:0.2 v/v (solvent C), and CH3OH:CH3COOH, 99.8:0.2 v/v (solvent B); isocratic conditions of 15% B for 5 min and then a linear gradient from 15% to 100% B in 30 min to yield pure compound 5: (−)-oleuropeic acid 1′-O-β-Dglucopyranosyl ester (4.5 mg). Similarly, another aliquot of fraction C6-X (31.4 mg) was further separated by preparative RP-HPLC with isocratic conditions of 10% B for 60 min to yield a mixture of compounds 6a ((−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester) and 6b ((−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester) (8.3 mg). Fraction C6-XII (1.1 g) was chromatographed on silica gel 60 (172 g, 40−63 μm), eluting with a 90:10 EtOAc:EtOH mixture. Fractions of 30 mL were collected and combined on the basis of TLC into 13 fractions (C6-XIIa−C6-XIIm). These latter fractions were again monitored by online assays. This allowed focusing attention on fraction C6-XIIj. Thus, an aliquot of fraction C6-XIIj (214.1 mg) was further separated by preparative RP-HPLC with isocratic conditions of 15% B for 10 min and then a linear gradient from 15% to 100% B in 20 min to yield pure compound 7: (−)-olivil 4-O-β-D-glucopyranoside (7.5 mg). Finally, another aliquot of fraction C6-XIIj (91.8 mg) was further separated by preparative RP-HPLC with a linear gradient from 20% to 70% B in 55 min to yield pure compound 8: (−)-perillic acid 1′-O-β-D-primeverosyl ester (3.5 mg). Structural Elucidation of New Natural Products. (−)-Oleuropeic Acid 6′-O-α/β-D-Glucopyranosyl Esters (6a and 6b). Yellowish syrup; UV (MeOH) λmax (log ε) 218.8 (3.8) nm; IR (neat) νmax 3384, 2925, 1701, 1649, 1384, 1258, 1058 cm−1; ESIMS m/z 369.2 ([M + Na]+), 345.2 ([M − H]−); HRESIMS m/z 347.1701 [M + H]+ (calcd for C16H27O8, 347.3856), 391.1594 [M + HCOO]− (calcd for C17H27O10, 391.3954); 1H and 13C NMR, see Table 1. (−)-Perillic Acid 1′-O-β-D-Primeverosyl Ester (8). Yellowish syrup; [α]25 D −41° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 222 (3.6), 302.8 (2.9) nm; IR (neat) νmax 3356, 1702, 1654, 1070 cm−1; ESIMS m/z 483.2 ([M + Na]+), 942.7 [2M + Na]+, 459.1 [M − H]−, 919.0 [2M − H]−; HRESIMS m/z 483.1831 [M + Na]+ (calcd for C21H32O11Na, 483.4683), 505.1927 [M + HCOO]− (calcd for C22H33O13, 505.4962); 1H and 13C NMR, see Table 2. Offline Spectrophotometric DPPH Assay. Radical-scavenging activity of chromatographic fractions, purified compounds, and reference compounds was determined spectrophotometrically with the stable DPPH radical.41 Methanolic solutions (2.4 mL) of DPPH• (∼7 × 10−5 M) with an absorbance at 515 nm of 0.80 ± 0.03 AU were mixed with methanolic solutions (1.2 mL) of samples at different
(−)-perillic acid 1′-O-β-D-primeverosyl ester (8) position 1 2 3eq 3ax 4 5eq 5ax 6eq 6ax 7 8 9a 9b 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″a 5″b a
δH mult. (J in Hz) 7.14, bs 2.34−2.49, 2.16−2.27, 2.34−2.49, 1.85−1.97, 1.44−1.56, 2.34−2.49, 2.16−2.27,
m m m m m m m
4.76, s 4.73, s 1.75, s 5.49, d (7.5) 3.35−3.50, m 3.50−3.56, m 3.35−3.50, m 3.35−3.50, m 4.08, bd (11.2) 3.70, dd (4.8, 11.2) 4.27, d (7.5) 3.12−3.21, m 3.35−3.50, m 3.35−3.50, m 3.83, dd (5.8, 11.3) 3.12−3.21, m
δC, mult. 130.51, qC 142.50, CH 32.27, CH2
HMBC 6, 7 2
41.36, CH 28.18, CH2 3, 4 25.41, CH2 167.26, qC 150.02, qC 109.79, CH2 20.87, CH3 95.74, CH 73.92, CH 77.75,a CH 70.96,b CH 77.88,a CH 69.42, CH2 105.14, CH 74.87, CH 77.70,a CH 71.16,b CH 66.91, CH2
4, 10 4, 10 4, 8, 9 7 1′, 4′ 2′
3′, 4′, 1″ 6′, 5″ 1″, 4″ 5″ 1″, 3″, 4″ 1″
Interchangeable signals. bInterchangeable signals.
concentrations by dissolving the dry samples in methanol. The experiment was carried out in triplicate. Samples were shaken and kept in the dark for 15 min at room temperature, and then the decrease of absorbance was measured at 515 nm. Radical-scavenging activity of chromatographic fractions, using a concentration of 50 μg/mL, is expressed as radical-scavenging percentage (RSP) and was calculated by the following formula41
⎡A − A ⎤ A RSP = ⎢ B ⎥ × 100 ⎣ AB ⎦ where AB is the absorbance of the blank (t = 0 min) and AA is the absorbance of tested sample solution (t = 15 min). Radical-scavenging activity of purified and reference compounds, using different concentrations (4−100 μg/mL), is expressed in terms of the antioxidant concentration (μM) required to decrease the initial DPPH• concentration by 50% (efficient concentration, EC50). The percentage of DPPH• remaining, calculated by the following equation42
%DPPH rem =
[DPPH] × 100 [DPPH]0
where [DPPH] is the concentration of DPPH• at the time measured (t = 15 min) and [DPPH]0 is the initial concentration of DPPH• (t = 0 min), was plotted against the sample concentration (μg/mL), a linear or logarithmic regression curve being established in order to calculate the EC50. Online HPLC-DAD-DPPH/ABTS Assays. Online HPLC radicalscavenging assays of the fractions from the olive tree wood ethyl acetate extract were performed to detect active compounds in complex mixtures.39 The setup for online assessment of radical-scavenging D
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activity consisted of an eluent pump, a photodiode array detector, and a Agilent 1100 Series controller, equipped with a C18 Waters Spherisorb S5 ODS2 (250 × 3 mm, 5 μm) analytical column. Methanolic solutions of fractions were prepared by dilution in methanol at a concentration of 10 mg/mL, and the injection volume was 10 μL. Separation was performed by a linear gradient from 20% to 70% B in 55 min, isocratic hold at 100% B for 5 min, linear gradient from 100% to 20% B in 10 min, and 10 min isocratic hold at 20% B for re-equilibration of the column. After HPLC separation and UV detection, the flow was mixed with a methanolic DPPH• solution or an ABTS•+ solution in PBS.43 Radical solutions were delivered using a Gilson model 302 pump at 0.5 mL/min for both DPPH• and ABTS•+ solutions. The reaction between separated compounds and radicals took place in a 3.5 m × 0.25 mm i.d. PEEK reaction coil. The decrease in absorbance was measured at 515 nm for DPPH• and at 414 nm for ABTS•+ in an Applied Biosystems UV/vis 759A spectrophotometer equipped with a tungsten lamp.
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RESULTS AND DISCUSSION Isolation of Compounds from the Olive Wood Extract. Olive wood chips were sequentially extracted with dichloromethane and ethyl acetate at room temperature and under reflux conditions, respectively. Dichloromethane extraction (0.4% yield) allowed removing the nonpolar components, which showed low antioxidant activity.25 The resulting ethyl acetate extract (1.4% yield) was prefractionated by open column chromatography on silica gel, yielding 11 major fractions (C1−C11). These fractions were assessed for their ability to scavenge the DPPH radical by offline spectrophotometric measurements. This approach was chosen because it is generally known that lipid oxidation is initiated by a free radical attack and due to their simplicity and worldwide acceptance for comparative purposes. Among all these fractions, in terms of DPPH radical-scavenging percentages (RSP), the most active fractions were, in this order, C3 (14.3% yield), C5 (21.3% yield), C4 (13.5% yield), and C6 (17.1% yield). These active fractions were further monitored by online HPLC-DADDPPH/ABTS assays to detect the active components present in each fraction.14 After a combination of conventional silica gel column chromatography and preparative RP-HPLC the following antioxidants were finally isolated: (−)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-Dglucopyranoside (7) (Figure 2). In addition, during the purification work of antioxidants, several nonactive compounds, such as (−)-oleuropeic acid (1), (−)-oleuropeic acid 1′-O-β-Dglucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-α-Dglucopyranosyl ester (6a), (−)-oleuropeic acid 6′-O-β-Dglucopyranosyl ester (6b), and (−)-perillic acid 1′-O-β-Dprimeverosyl ester (8) (Figure 2), were also isolated. Structure Elucidation of the Isolated Compounds. Compounds 1−8 were characterized and identified by UV, IR, MS, HRMS, 1H NMR, 13C NMR, 2D NMR, and specific optical rotation measurements. Spectral and physical data of compounds 1−5, 6b, and 7 are in agreement with earlier published data: (−)-oleuropeic acid (1),44 (−)-olivil (2),45 the aldehydic form of oleuropein aglycone (3),46 (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4),13 (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester (5),44 (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (6b),44 and (−)-olivil 4-O-β-Dglucopyranoside (7) (Figure 2).47 Compounds 2−4 and 7 have been reported before from other parts of the olive tree. Compounds 1, 5, and 6b have been previously found in the leaves of Cunila spicata L.44 esterified with acylated glucose,
Figure 2. Compounds isolated in this work from an olive tree wood EtOAc extract.
while the methyl ester of compound 1 has been also isolated from Eucalyptus cypellocarpa leaves.48 Compounds 6a and 6b were assigned a molecular formula of C16H26O8 by HRESIMS. They are anomeric glucosides, and their 1 H NMR spectrum showed, apart from signals corresponding to two monoterpene aglycone units, similar to that of compound 1, a set of signals assignable to two glucose moieties; one anomeric proton is centered at 4.44 ppm (1H, d, 7.8 Hz), consistent with the configuration for a β-Dglucopyranosyl group, while the other is centered at 5.03 ppm (1H, d, 3.7 Hz), consistent with the configuration for an α-D-glucopyranosyl group (Table 1). In the HMBC spectrum, cross peaks between the proton signals H-6′a and H-6′b and the carbon C-7 indicate the linkage of the monoterpene residues and the glucose units. Further cross peaks between the proton signal at δ 5.03 (H-1′), from the α anomer, and the carbons at δ 74.8 (C-3′) and 70.8 (C-5′) and between the proton signal at δ 4.44 (H-1′), from the β anomer, and the carbons at δ 77.9 (C-2′), 76.2 (C-3′), and 75.4 (C-5′) allowed us to assign 13C peaks for each anomer. Accordingly, the structure of compound 6a was determined to be (−)-oleuropeic acid 6′-O-α-D-glucopyranosyl ester, while 6b was determined to be (−)-oleuropeic acid 6′-O-β-D-glucopyranosyl ester (Figure 2). Integration of anomeric protons in the 1H NMR spectrum indicated an equilibrium mixture of anomers, 6a and 6b, with a ratio of 1:1. Compound 8 was assigned a molecular formula of C21H32O11 by HRESIMS. The 1H NMR spectrum (Table 2) showed signals of an isopropenyl moiety [δ 1.75 (3H, s), 4.73 (1H, s), and 4.76 (1H, s)], an olefinic proton [δ 7.14 (1H, br s)], three methylene groups [δ 1.44−2.49 (6H, m)], and a methyne group [δ 2.34−2.49 (1H, m)], corresponding to a monoE
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than those observed for other secoiridoids, such as jaspolyoside (55 μM), oleuropein 3′-O-β-D-glucoside (66 μM), isojaspolyoside A (108 μM), 7″S-hydroxyoleuropein (113 μM), oleuropein-3″-methyl ether (170 μM), ligustroside (233 μM), ligustroside 3′-O-β-D-glucoside (290 μM), and jaspolyanoside (847 μM), and iridoids, such as 7-deoxyloganic acid (742 μM).14,27 The lignans (−)-olivil (2), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-D-glucopyranoside (7) showed EC50 values of 176, 164, and 134 μM, respectively. These activities are lower than that observed for lignan (+)-cycloolivil (90 μM).27 Monoterpenoids 1, 5, 6a, 6b, and 8 did not show any antioxidant activity. In summay, this work completes previous studies on the composition of an ethyl acetate extract from olive tree wood,14,24,25 as well as their antioxidant activity. On the basis of their chemical structures, the 25 compounds isolated from olive tree wood can be classified into various phytochemical subclasses including simple phenols [hydroxytyrosol, tyrosol], monoterpenoids [(−)-oleuropeic acid (1), (−)-oleuropeic acid 1′-O-β-D-glucopyranosyl ester (5), (−)-oleuropeic acid 6′-O-αD-glucopyranosyl ester (6a), (−)-oleuropeic acid 6′-O-β-Dglucopyranosyl ester (6b), and (−)-perillic acid 1′-O-β-Dprimeverosyl ester (8)], lignans [(+)-cycloolivil, (−)-olivil (2), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), and (−)-olivil 4-O-β-D-glucopyranoside (7)], iridoids [7-deoxyloganic acid], secoiridoids [ligustroside, oleuropein, the aldehydic form of oleuropein aglycone (3), oleuropein-3″-methyl ether, (7″R)-7″-ethoxyoleuropein, (7″S)-7″-ethoxyoleuropein, (7″S)7″-hydroxyoleuropein, ligustroside 3′-O-β-D-glucoside, oleuropein 3′-O-β-D-glucoside, jaspolyanoside, jaspolyoside, and isojaspolyoside A], and a sugar alcohol [D-mannitol]. The results obtained here complete the current knowledge of olive tree wood composition and confirm that this agricultural byproduct contains diverse phytochemicals, among which secoiridoids predominate, which in turn could offer interesting potential applications in the food, cosmetic, and pharmaceutical industries.
terpene aglycone unit. Additionally, a set of signals assignable to two sugar moieties was observed; one anomeric proton is centered at 4.27 ppm (1H, d, 7.5 Hz), while the other is centered at 5.49 ppm (1H, d, 7.5 Hz), indicating the β configuration for both sugar units. The 13C NMR spectrum showed the presence of an isopropenyl group (a methyl [δ 20.87 (C-10)], a terminal methylene [δ 109.79 (C-9)], and a quaternary carbon [δ 150.02 (C-8)]), three methylene groups [δ 25.41 (C-6), 28.18 (C-5), and 32.27 (C-3)], a methyne carbon [δ 41.36 (C-4)], two olefinic carbons [δ 130.51 (C-1) and 142.50 (C-2)], and a quaternary carbon of an α,βunsaturated ester [167.26 (C-7)]. The 13C NMR spectrum also showed 11 signals in the sugar region, two of them being anomeric carbons [δ 95.74 (C-1′) and 105.14 (C-1″)]. The two sugars were identified as glucose and xylose. In the HMBC spectrum, cross peaks between the proton signals at δ 5.49 (H1′) and the carbon at δ 167.26 (C-7) indicate the linkage between the monoterpene residue and the glucose unit. Similarly, cross peaks between the proton signal at δ 4.27 (H-1″) and the carbon at δ 69.42 (C-6′) and between the protons at δ 4.08 (H-6′a) and δ 3.70 (H-6′b) with the carbon at δ 105.14 (C-1″) showed the linkage between the xylose and the glucose units. A glucose and xylose moiety linked via C-6′ → C-1″ is known as the disaccharide primeverose. This linkage explained the upfield shifts observed for the glucose carbons C1′, C-2′, and C-5′ and the downfield shifts for carbon C-6′. Accordingly, the structure of compound 8 was determined to be (−)-perillic acid 1′-O-β-D-primeverosyl ester, which is a new natural product (Figure 2). Radical-Scavenging Activity of the Isolated Antioxidants. The antioxidant activity of the isolated compounds from olive tree wood was determined with the DPPH radicalscavenging assay and expressed in terms of the antioxidant concentration (μM) required to reduce the initial DPPH• concentration after 15 min by 50% (efficient concentration, EC50). In Table 3, EC50 values for the active compounds 2−4
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Table 3. DPPH Radical-Scavenging Activity of the Isolated Active Compounds from Olive Wood Ethyl Acetate Extract, Expressed as Efficient Concentration (EC50) compounds
EC50 (μM)a
hydroxytyrosol aldehydic form of oleuropein aglycone (3) (−)-olivil 4-O-β-D-glucopyranoside (7) (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4) (−)-olivil (2) BHTb
39 45 134 164 176 505
AUTHOR INFORMATION
Corresponding Author
*Tel.: +34 953 212743. Fax: +34 953 211876. E-mail: jaltare@ ujaen.es. Funding
This study was supported by the Spanish Ministerio de Educación y Ciencia (R+D Project CTQ2005-07005/PPQ; partial financial support from the FEDER funds of the European Union). M.P.-B. was the recipient of a predoctoral fellowship granted by the Andalusian Government Junta de Andalucia.́ Part of the work was supported by the Centro de ́ Instrumentación Cientifico-Té cnica of the University of Jaén.
a
EC50 values are means of three replicates, and the RSD is less than 1%. b2,6-Di-tert-butyl-4-methylphenol.
and 7 and the reference compounds BHT and hydroxytyrosol are presented as the average of three replicates. The activities of compounds 2−4 and 7 were higher than that of reference BHT (505 μM) and lower than that of hydroxytyrosol (39 μM), which was the most active component identified in olive tree wood together to oleuropein (32 μM).27 Hydroxytyrosol is more active than tyrosol (826 μM),24 which could be related to the stabilization by delocalization of the o-diphenolic moiety present in hydroxytyrosol, since o-quinone formation is favored. Thus, the aldehydic form of oleuropein aglycone (3) exhibited marked activity; its efficient concentration (45 μM) was just somewhat lower than that of oleuropein (32 μM) and higher
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank Dr. P. de Waard for his technical assistance in recording NMR spectra.
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