J. Agric. Food Chem. 2009, 57, 11557–11563
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DOI:10.1021/jf901786p
Phytochemical Profile and Anticholinesterase and Antimicrobial Activities of Supercritical versus Conventional Extracts of Satureja montana FILIPA V. M. SILVA,†,‡ ALICE MARTINS,† JOANA SALTA,† NUNO R. NENG,† JOSE M. F. NOGUEIRA,† DELFINA MIRA,‡ NATALIA GASPAR,‡ JORGE JUSTINO,‡ CLARA GROSSO,§ JOSE S. URIETA,# ANTONIO M. S. PALAVRA,§ AND AMELIA P. RAUTER*,† †
Faculdade de Ci^encias da Universidade de Lisboa, Centro de Quı´ mica e Bioquı´ mica/Departamento de Quı´ mica e Bioquı´ mica, Ed. C8, Campo Grande, 1749-016 Lisboa, Portugal, ‡Instituto Politecnico de Santarem, Escola Superior Agraria de Santarem, Quinta do Galinheiro, 2001-904 Santarem, Portugal, § Instituto Superior Tecnico, Departamento de Engenharia Quı´ mica e Biol ogica, Universidade Tecnica de Lisboa, Avenida Rovisco Pais 1, 1049-001 Lisboa, Portugal, and #Quı´ mica Organica y Quı´ mica Fı´ sica, Universidad Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain
Winter savory Satureja montana is a medicinal herb used in traditional gastronomy for seasoning meats and salads. This study reports a comparison between conventional (hydrodistillation, HD, and Soxhlet extraction, SE) and alternative (supercritical fluid extraction, SFE) extraction methods to assess the best option to obtain bioactive compounds. Two different types of extracts were tested, the volatile (SFE-90 bar, second separator vs HD) and the nonvolatile fractions (SFE-250 bar, first and second separator vs SE). The inhibitory activity over acetyl- and butyrylcholinesterase by S. montana extracts was assessed as a potential indicator for the control of Alzheimer’s disease. The supercritical nonvolatile fractions, which showed the highest content of (þ)-catechin, chlorogenic, vanillic, and protocatechuic acids, also inhibited selectively and significantly butyrylcholinesterase, whereas the nonvolatile conventional extract did not affect this enzyme. Microbial susceptibility tests revealed the great potential of S. montana volatile supercritical fluid extract for the growth control and inactivation of Bacillus subtilis and Bacillus cereus, showing some activity against Botrytis spp. and Pyricularia oryzae. Although some studies were carried out on S. montana, the phytochemical analysis together with the biological properties, namely, the anticholinesterase and antimicrobial activities of the plant nonvolatile and volatile supercritical fluid extracts, are described herein for the first time. KEYWORDS: Satureja montana; supercritical fluid extraction; HPLC-DAD; anticholinesterase activity; Alzheimer’s disease; antimicrobial activity
*Corresponding author (e-mail
[email protected]; telephone þ351 21 7500952; fax þ351 21 7500088).
which contained R-terpineol as the major constituent (3). The antimicrobial activity of S. montana essential oils is well documented (4-7). In addition, Cetkovic et al. reported that various S. montana L. subsp. kitaibelii extracts expressed inhibitory activity against both Gram-positive and Gram-negative bacteria (8). The Local Food Nutraceuticals Consortium, which aimed to study traditional foods known to have potential against agingrelated pathologies and to develop new products to be used as high-quality food supplements, studied a wide number of plant species including S. montana, which revealed a high activity on antioxidant, anti-inflammatory, and mood-disorder related assays as well as on xanthine oxidase (XO) and myeloperoxidasecatalyzed guaiacol oxidation (G-OH) enzyme inhibition assays (9). More recently, Cetkovic et al. reported on the antioxidant activity of various S. montana L. subsp. kitaibelii extracts (8), and S. montana phenolic constituents thymol and carvacrol, present in the essential oil (55% in total), were considered by Cavar et al.
© 2009 American Chemical Society
Published on Web 11/20/2009
INTRODUCTION
Satureja montana, commonly called winter or mountain savory, is a perennial flowering plant, evergreen shrub belonging to the Lamiaceae family, which is native to warm temperate regions of the Mediterranean. This aromatic herb, found in nature and also cultivated, has been used for hundreds of years as a food condiment, as a tea, and also to garnish salads. In addition, various medicinal benefits have been reported for this plant, in particular, upon the whole digestive system. With respect to the biological properties and economic value, European and international patents were recently registered for S. montana extracts, claiming their anti-infertility properties (1). Other bioactivities described for S. montana include potent anti-HIV-1 activity of its aqueous extracts (2) and antiproliferative activity on human erythroleukemic K562 cells of the herb essential oil,
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to be responsible for the overall reactivity of the savory oil toward DPPH (4). However, the antiradical activity of a savory essential oil from Slovenia, with respect to DPPH, was greater than that determined for its major components carvacrol (41.5%) and thymol (8.6%) (10). Antioxidants may be applied as neuroprotective agents in the early stage of Alzheimer’s disease (AD) (11). Although the origin of AD remains unknown, the depletion of neurotransmitters such as acetylcholine caused by the enzymes acetylcholinesterase (AChE) is clearly related to this disease (12). The mechanism of action of the available drugs for treating AD is based mainly on the inhibition of the enzyme AChE. Although a variety of natural AChE inhibitors are already known (13), the screening of plants used to treat memory dysfunction for AChE inhibitory activity continues to be pursued (14, 15). Recently, butyrylcholinesterase (BChE) received particular attention because it is a coregulator of cholinergic neurotransmission and its activity is increased in AD and associated with all neuropathological lesions in this disease (16). Although selective BChE inhibitors of synthetic origin based on quinazolinimines and lipoic acid have been reported (11), the search for natural substances, more efficient and less expensive than those currently in use for patient’s palliative care and treatment, is a mandatory research branch. The promising bioactivities described for Satureja species encouraged us to further investigate S. montana and to compare various extraction methods focusing on the enrichment of active principles in the extract. Hydrodistillation (HD) and Soxhlet extraction (SE) are traditional techniques to recover compounds from aromatic plants. However, some drawbacks can be attributed to them, namely, hydrolysis and thermal degradation for HD and solvent contamination for SE. To overcome these limitations, supercritical fluid extraction (SFE) was used as an alternative method. SFE with CO2 is a versatile technique, very suitable to obtain different plant extracts, because the manipulation of process parameters such as temperature and pressure of the supercritical fluid can change its solvent power (17, 18). Hence, the main objectives of this work are as follows: (i) to produce S. montana extracts by SFE and by conventional methods and to characterize the chemical profile of each extract; (ii) to study the effect of the extraction method on the bioactivities of the extracts; (iii) to investigate both AChE and BChE inhibition and give new perspectives for the plant extracts as potential nutraceuticals against neurodegenerative disorders; (iv) to evaluate the influence of the extraction methodologies over the microbial susceptibility of different bacteria and fungi strains. MATERIALS AND METHODS
Plant Material. Aerial parts (leaves and flowers) of S. montana (cultivated at Centro de Investigacion y Tecnologı´ a Agroalimentaria, Ejea de los Caballeros, Zaragoza, Spain, 2005) were frozen with liquid N2 and ground with a commercial mill. Different particle sizes were obtained and separated with a series of sieves (1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1 mm). After this process, the fractions were kept at -20 °C in dark bottles. The initial moisture content of the plant material was 11.8%. SFE Apparatus. The SFE apparatus used in this work was described in detail by Reis-Vasco et al. (19). Briefly, it is composed by a diaphragm pump, an extraction vessel (1 L), and two separators (0.27 L), operating in series. A back-pressure regulator is used to control the pressure, which is measured with a Bourdon type manometer, and the total volume of CO2 is determined with a dry test meter. A preset temperature in the extraction vessel is reached with the aid of a water jacket. The CO2 (99.995% purity) used in the studies was supplied by Air Liquid (Lisbon, Portugal). Extraction of Essential and Volatile Oils. The essential oil was isolated by HD in a Clevenger apparatus, for 4 h, using 40 g (dw) of plant material with a mean particle size of 0.6 mm (extract C). For volatile oil isolation by SFE, plant material (100 g dw) was placed inside the extractor
Silva et al. between two layers of glass wool, and the extraction conditions were as follows: pressure of 90 bar, temperature of 40 °C, mean particle size of 0.6 mm, and flow rate of 1.1 kg/h. To obtain a pure volatile oil free of waxes in the second separator (extract A), a pressure of 80 bar and a temperature of -8 °C, in the first separator (to allow waxes precipitation), and a pressure of 20 bar and a temperature of -15 °C, in the second one (to allow the volatile oil precipitation), were selected. These were the experimental conditions that represented the best compromise between yield and volatile oil composition. Moreover, the amounts of waxes and volatile oil were determined gravimetrically (w/w). Extraction of the Nonvolatile Fraction. After HD, the plant residue (5 g) was dried and subjected to a Soxhlet extraction with acetone (SE), for 5 h, to obtain the nonvolatile components (extract D). After SFE at 90 bar, 40 °C, 0.6 mm particle size, and 1.1 kg/h of CO2 flow rate, the same plant matrix was submitted to an extraction, changing only the pressure to 250 bar, for 4 h (SFE extracts B1 and B2, obtained in the first and second separators, respectively, operated at 90 bar/40 °C and 20 bar/20 °C).
Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of the Essential Oil. GC quantitative analyses were performed in a Hewlett-Packard 5890 gas chromatograph (HP, Waldbronn, Germany), equipped with a flame ionization detector (FID) and a fused-silica DB-5 capillary column (J&W; 30 m 0.25 mm i.d., film thickness = 0.25 μm; Folsom, CA). Oven temperature was programmed isothermally to 40 °C, during 2 min, then was raised, at 3 °C/min to 230 °C, and finally increased at 5 °C/min to 310 °C, and held at this temperature for 15 min. The injector and detector were set at the same temperature, 310 °C. Helium was used as carrier gas at a flow rate of 24 cm/s, and the split ratio was 1:50. The percentage composition of the oils was computed by the normalization method from the GC peak areas without using response factors. The GC-MS system consisted of a Perkin-Elmer Autosystem XL gas chromatograph (Perkin-Elmer, Shelton, CT) equipped with a DB-1 fusedsilica column (30 m 0.25 mm i.d., film thickness 0.25 μm; J&W Scientific Inc., Agilent Technologies, Santa Clara, CA), and interfaced with a Perkin-Elmer Turbomass mass spectrometer (software version 4.1; PerkinElmer, Shelton, CT). Oven temperature was programmed from 45 to 175 °C, at 3 °C/min, subsequently at 15 °C/min to 300 °C, and then held isothermal for 10 min; injector and detector temperatures, 280 and 290 °C, respectively; transfer line temperature, 280 °C; ion trap temperature, 220 °C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; ionization current, 60 μA; scan range, 40-300 u; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C9-C18 n-alkanes indices (Supelco, Bellefonte, PA) and GC-MS spectra from a homemade library, constructed on the basis of the analyses of reference oils, laboratorysynthesized components, and commercial available standards. HPLC-DAD Analysis of the Nonvolatile Fraction. For HPLC purposes, the following chemicals and standards were purchased: acetonitrile (ACN), (99.9%) and methanol (MeOH, 99.9%) (Panreac, Barcelone, Spain); acetic acid (99.8%) (Sigma-Aldrich, Buchs, Switzerland); ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid, 99%), caffeic acid (3,4-dihydroxycinnamic acid, 97% predominantly trans), syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid, 98%), gallic acid (3,4,5-trihydroxybenzoic acid, 97%), gentisic acid (2,5-dihydroxybenzoic acid, 98%), coumaric acid (trans-4-hydroxycinnamic acid, 98%), protocatechuic acid (3,4-dihydroxybenzoic acid, 97%), vanillic acid (4-hydroxy-3-methoxybenzoic acid, 97%), and (þ)-catechin hydrate ((2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7triol, 98%) (Sigma-Aldrich, Buchs, Switzerland); chlorogenic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4-dihydroxycinnamate), g95%) (Extrasynthese, Genay, France) and (-)-epicatechin green tea ((2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol, g98%) (Sigma-Aldrich, Buchs, Switzerland). Ultrapure water was obtained from Milli-Q water purification systems (Millipore, Bedford, MA). The stock solutions of individual standards (1.00 mg/L) and samples were prepared in HPLC grade MeOH and used to prepare the working standard mixtures at the desired concentration. To prevent photodegradation, after experimental procedure, the stock solutions and working standards were wrapped in aluminum foil and stored at -4 °C. HPLC-DAD analyses were carried out on an Agilent 1100 series LC system (Agilent Technologies, Waldbronn, Germany), composed by the
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following modules: vacuum degasser (G1322A), quaternary pump (G1311A), autosampler (G1313A), thermostated column compartment (G1316A), and diode array detector (G1315B). Data acquisition and instrumental control were performed by the software LC3D ChemStation (version rev. A. 10.02[1757]; Agilent Technologies). Analyses were performed on a Tracer excel 120 ODS-A column, 150 mm 4.0 mm, 5 μm particle size (Teknokroma, Barcelone, Spain). The mobile phase consists of a mixture of acetonitrile (solvent A) and acetic acid aqueous solution, pH 2.8 (solvent B). The applied gradient was 0-40 min, 1-32.2% A; 40-45 min, 32.2-40% A; 45-50 min, 40-1% A; and hold at 1% A for 10 min. The flow rate was 1.0 mL/min, the analyses were performed at 26 °C, and the injection volume was 10 μL with a draw speed of 200 μL/min. The detector was set at 280 nm. For identification purposes, a standard addition method was used by spiking the samples with the pure standards, as well as by comparing the retention parameters and UV-visible spectral reference data. The composition was determined through the normalized peak areas at 280 nm. Anticholinesterase Assays. Ellman’s assay (20) was used to screen the in vitro anticholinesterase activities of five S. montana extracts. Absorbency of the colored ion was detected at 410 nm on a double-beam spectrophotometer Shimadzu equipped with thermostatic cell holders operating on the kinetic mode. Appropriate disposable plastic Plastibrand cuvettes were used in the kinetic experiments, and absorbance data were acquired by means of UV Probe software. Enzyme activity (percent) and enzyme inhibition (percent) were calculated from the rate of absorbance change with time (V = ΔAbs/Δt) data as follows: enzyme inhibition ð%Þ ¼ 100 - enzyme activity ð%Þ enzyme activity ð%Þ ¼ 100 V =V max Maximum rates (Vmax) are obtained when no inhibitor is used, whereas V is the rate obtained in the presence of the inhibitor. The following pa reagents were purchased from Sigma-Aldrich (Germany): AChE from bovine erythrocytes, BChE from human serum, acetylthiocholine iodide (ATchI), S-butyrylthiocholine iodide (BTchI), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), KH2PO4, KOH, and NaHCO3. Deionized/sterilized water was used to prepare the buffer (pH 8.0), substrate, and DTNB solutions. The extracts of S. montana and the controls (rivastigmine and donepezil) were initially dissolved in DMSO and diluted with distilled water until concentrations decrease to values between 0.044 and 4.4 mg/mL, to yield the final concentrations for the enzymatic test between 1 and 100 μg/mL. No inhibition was detected by residual DMSO (
