9530 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Taveira et al.
respectively. The antibacterial and antifungal activity of both
L. esculentum seeds extracts and the determined chemical com-
ponents were evaluated by broth dilution assays.
and those resulting from the alkaline hydrolysis were derivatized to their
methyl ester forms with BF3 methanolic solution (10%), at 90 °C for
10 min. The methyl ester derivatives were extracted with isooctane, and
anhydrous sodium sulfate was added to ensure the total absence of water.
The resulting extract was evaporated to dryness under a stream of nitrogen
and redissolved in isooctane. All the assays were performed in triplicate.
Gas Chromatography/Mass Spectrometry Conditions. Standard mixture/
sample extracts (1 μL) were analyzed using a Varian CP-3800 gas
chromatograph (USA) equipped with a VARIAN Saturn 4000 mass
selective detector (USA) and a Saturn GC/MS workstation, software
version 6.8. AVF-5 ms (30 m ꢀ 0.25 mm ꢀ0.25 μm) column (VARIAN)
was used. The injector port was heated to 250 °C. Injections were per-
formed in split mode, with a ratio of 1/40. The carrier gas was helium C-60
(Gasin, Portugal), at a constant flow of 1 mL/min. The oven temperature
was set at 40 °C for 1 min, then increasing 5 °C/min to 250 °C, 3 °C/min to
300 °C and held for 15 min. All mass spectra were acquired in the electron
impact (EI) mode. Ionization was maintained off during the first 4 min, to
avoid solvent overloading. The ion trap detector was set as follows:
transfer line, manifold and trap temperatures were 280, 50, and 180 °C,
respectively. The mass ranged from 50 to 600 m/z, with a scan rate of
6 scan/s. The emission current was 50 μA, and the electron multiplier was
set in relative mode to auto tune procedure. The maximum ionization time
was 25,000 μs, with an ionization storage level of 35 m/z. The analysis was
performed in full scan mode. Identification of compounds was achieved
by comparison of their retention time and mass spectra with those from
pure standards injected under the same conditions, and from NIST 05 MS
Library Database. The amount of fatty acid methyl esters (FAME) present
in the extract samples was achieved from the calibration curve of the
respective FAME standards. The FAME values were then converted into
their respective fatty acid contents.
MATERIALS AND METHODS
Standards and Reagents. All chemicals used were of analytical grade.
Reference compounds were purchased from various suppliers: quercetin-
3-O-rutinoside (rutin), kaempferol-3-O-rutinoside, and isorhamnetin-3-
O-rutinoside were from Extrasynthese (Genay, France). The studied
´
organic acids were obtained from the following source: oxalic, citric,
fumaric, malic, acetic, aconitic and pyruvic acids from Sigma-Aldrich
(Steinheim, Germany). Pentadecanoic, palmitic, palmitoleic, heptadeca-
noic, stearic, oleic and linoleic acids methyl esters, and boron trifluoride
(BF3) 10% methanolic solutions were purchased from Sigma-Aldrich
(Steinheim, Germany). Ciprofloxacin, fluconazole, methanol, chloridric,
acetic and sulfuric acids were obtained from Merck (Darmstadt, Germany).
Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) media
were purchased from Liofilchem (Teramo, Italy) and Saboraud dextrose
ꢀ
agar (SDA) and Sabouraud dextrose broth (SDB) from Bio-Merieux
(Marcy L’Etoile, France). The water was treated in a Milli-Q water
purification system (Millipore, Bedford, MA).
Plant Material. Seeds of L. esculentum Mill. from “Bull’s heart” (RJS,
Portugal) and “Cherry” (Galassi Sementi, Italy) varieties were purchased
from local commerce. These two cultivars correspond to the most con-
sumed ones in Portugal. No antibacterial or antifungal agents were added
by the producer for conservation. Seeds were washed with water, then
surface sterilized with 10% sodium hypochlorite solution, rinsed with
sterile distilled water and were air-dried at room temperature. The samples
were ground into a fine powder.
Antimicrobial Activity. Microorganisms. Nine bacterial species
were used for the experiment: Staphylococcus aureus (ATCC 20231),
Staphylococcus epidermidis (ATCC 20044), Salmonella typhimurium
(ATCC 43971), Proteus mirabilis (ATCC 4479), Escherichia coli (ATCC
30083), Pseudomonas aeruginosa (ATCC 50071), Bacillus cereus (ATCC
31), Enterococcus faecalis (ATCC 20477) and Micrococcus luteus (ATCC
20030). Cultures were obtained from the Department of Microbiology,
Faculty of Pharmacy, Porto University, Portugal. The same organisms
were also tested against the reference antibacterial drug ciprofloxacin.
Stock cultures were maintained on MHA at 4 °C.
Preparation of Extracts. Seeds were extracted using five different
solvents: methanol, chloroform, ethyl acetate, hexane and sulfuric acid
0.005 M. Eight grams of powdered seeds was extracted with 200 mL of
each solvent with sonication (5 min), followed by 24 h of extraction at
room temperature with mechanic stirrer (200 rpm). The resulting extracts
were filtered through a Buchner funnel and evaporated to dryness under
reduced pressure using a rotary evaporator, at 40 °C. The extracts were
redissolved in 4 mL of methanol and preserved at -20 °C until analysis.
HPLC/DAD Phenolic Compounds Analysis. For identification
and quantification of phenolic compounds, 20 μL of redissolved extract
was analyzed using a HPLC/DAD unit (Gilson) and a Spherisorb ODS2
(25.0 ꢀ 0.46 cm; 5 μm particle size) column. The mobile phase consisted
of two solvents: water/acetic acid (1%) (A) and methanol (B), starting
with 5% B and using a gradient to obtain 50% at 30 min and 80% at
37 min. Spectral data from all peaks were accumulated in the range of
200-400 nm, and chromatograms were recorded at 350 nm. The different
phenolic compounds were identified by comparing their chromatographic
behavior and UV-vis spectra in the 200-400 nm range with authentic
standards and with published data (6). Data were processed on Unipoint
system software (Gilson Medical Electronics, Villiers le Bel, France). Peak
purity was checked by the software contrast facilities. Phenolic compounds
quantification was achieved by the absorbance recorded in the chromato-
grams relative to external standards. Because standards of all identified
compounds were not commercially available, kaempferol derivatives were
quantified as kaempferol-3-O-rutinoside, quercetin derivatives as querce-
tin-3-O-rutinoside, and isorhamnetin derivatives as isorhamnetin-3-O-
rutinoside. Kaempferol-3-O-sophoroside plus isorhamnetin-3-O-sophoro-
side and kaempferol-3-O-(2-sophorosyl)glucoside plus quercetin-3-O-(2-
pentosyl)rutinoside were quantified together as kaempferol-3-O-rutinoside.
HPLC/UV Organic Acids Analysis. Twenty microliters of redis-
solved extract was analyzed on an analytical HPLC unit (Gilson), using an
ion exclusion column Nucleogel Ion 300 OA (300 ꢀ 7.7 mm), in conjunc-
tion with a column heating device set at 30 °C. Elution (70 min) was carried
out at a solvent flow rate of 0.2 mL/min, isocratically, with sulfuric acid
0.005 M as the mobile phase. Detection was performed with a Gilson UV
detector at 214 nm. The organic acids quantification was achieved by the
absorbance recorded in the chromatograms relative to external standards.
GC/MS Fatty Acid Analysis. Derivatization. Fatty acid derivati-
zation was performed according to a described procedure (10). Briefly,
sterified fatty acids in the extracts were hydrolyzed with KOH methanolic
solution (11g/L), at 90 °C for 10 min. The free fatty acidsoriginally present
Antifungal activity was checked against Candida albicans (ATCC
10231), Aspergillus fumigatus (ATCC 46645) and Trichophyton rubrum
(CECT 2794). The same organisms were also tested against the reference
antifungal drugs fluconazole (C. albicans) and voriconazole (A. fumigatus
and T. rubrum) for comparison of results. Candida parapsilosis (ATCC
22019) and Candida krusei (ATCC 6258) were used for quality control. All
strains were stored in SDB with 20% glycerol at -70 °C and subcultured in
SDA before each test, to ensure optimal growth conditions and purity.
Antibacterial Effect. Bacterial inocula were prepared by growing cells
in MHB for 24 h, at 37 °C. Cell suspensions were diluted in sterile MHB
to provide initial cell counts of about 106 colony-forming unit per mL
(CFU/mL). The minimum inhibitory concentration (MIC) of seeds
extracts, organic acids and rutin were determined by 2-fold serial dilution
method, in 96-well plates. All tests were performed in MHB. The initial
concentration was 20 mg/mL for the seed extracts and all organic acids,
with the exceptions of aconitic (10 mg/mL) and fumaric (2.5 mg/mL) acids.
Rutin was tested at a maximum concentration of 69 μg/mL. The final
concentration of methanol did not exceed 1%(v/v). Briefly, 90μL of MHB
and 10 μL of a suspension containing 106 CFU/mL were added in each
well, which contained 100 μL of extract/compound. Negative controls in
MBH alone and with 1% methanol (v/v) and sterility and growth controls
were included. A positive control with ciprofloxacin was also performed.
Plates were incubated for 24 h, at 37 °C, and then examined by a binocular
microscope. The MIC for bacteria was determined as the lowest concen-
tration of seed extracts or standard compounds (organic acids or rutin)
inhibiting the visual growth of the test culture on the microplate. The
experiments were performed in duplicate and repeated independently three
times, yielding essentially the same results (a range of values is presented
when different results were obtained).
Antifungal Effect. Broth microdilution methods based on the Clinical
and Laboratory Standards Institute (CLSI, formerly NCCLS) reference