New Odorant Compounds Identified in Cress Extracts
J. Agric. Food Chem., Vol. 55, No. 5, 2007 1933
The ethereal fraction (F2) was washed three times with 50 mL of
saturated K2CO3 (Aldrich, 209619) solution and gave, after drying over
MgSO4, 19.90 g of neutral fraction F2.3. The aqueous phase (pH 10)
was acidified with HCl 1 N until pH 2 and extracted with ether and
gave, after drying over MgSO4, 0.04 g of (acid) fraction F2.4
.
Silica gel column chromatography (open column, 150 g of silica
gel 60, 40-63 µm; Merck, 109385) of 4.51 g of fraction F2.3 was
realized to isolate the unknown major compound (gradient from 100%
n-pentane to 100% diethyl ether). The unknown compound was isolated
after further silica gel column fractionation of the elution fraction 50%
n-pentane/50% diethyl ether (gradient from 100% pentane to 100%
diethyl ether) by using 80% n-pentane/20% diethyl ether. All obtained
fractions were analyzed by GC-MS and GC-FID (Scheme 1).
Analytical Studies. GC-MS. GC-MS analysis was carried out using
a HP 5890 series II GC coupled to a HP 5971A MS (Hewlett-Packard,
Massy, France). Samples were analyzed on a fused-silica capillary
column HP-1 (polydimethylsiloxane, 50 m × 0.20 mm i.d. × film
thickness ) 0.50 µm; Interchim, Montluc¸on, France) and HP-20M
(polyethyleneglycol, 50 m × 0.20 mm i.d. × film thickness ) 0.33
µm; Interchim): carrier gas, helium; constant pressure, 220 kPa; injector
temperature, 230 °C; split ratio, 1:100; temperature program, raised
from 60 to 250 °C at 2 °C/min and then held isothermal (120 min) at
250 °C (apolar column) or 220 °C (polar column); ion source
temperature, 230 °C; transfer line temperature, 280 °C (apolar column)
or 230 °C (polar column); ionization energy, 70 eV; electron ionization
mass spectra were acquired over the mass range of 35-400 amu.
Component Identification. Identification of the constituents was based
on computer matching against commercial libraries (Wiley, MassFinder
2.1 Library, NIST98), laboratory mass spectra libraries built from pure
substances, and MS literature data (11-14) combined with comparison
of GC retention indices (RI) on apolar and polar columns. RIs were
calculated with the help of a series of linear alkanes C6-C26 and C21-
C40 on apolar and polar columns (HP-1 and HP-Innowax). Compounds
available in the laboratory were confirmed by external standard
compound co-injection.
Figure 1. Benzyl glucosinolate (glucotropaeolin) and phenethyl glucosi-
nolate (gluconasturtiin) (5).
up to an approximative height of 40 cm. Its main glucosinolate
is glucotropaeolin (benzyl glucosinolate) (5). The two main
glucosinolates of the two cress species are represented in
Figure 1.
The aim of this work is the identification of molecules with
interesting organoleptic properties in cress extracts with focus
on organonitrogen and organosulfur compounds.
MATERIALS AND METHODS
Chemicals and Extracts. Botanical Source and Plant Material.
Commercial Indian cress absolute was provided by Robertet S.A.,
Grasse, France. It had been produced in Rustenburg, South Africa, by
extraction of the fresh plant with n-hexane followed by treatment with
ethanol.
Watercress was purchased from Cresson de Provence, Hye`res,
France, in April 2006. A voucher specimen was deposited at the
Herbarium of the Botanical Garden of Nice (voucher B-9298).
Commercial watercress (Florette, Lessay, L’Isle-sur-Sorgue, France)
stored under plastic packaging was purchased at a supermarket in Nice,
France, in June 2005.
NMR. NMR was carried out on a Bruker Avance AC200 (200 MHz)
and DRX500 (500 MHz) (Bruker, Wissembourg, France). The solvent
was CDCl3 (Eurisotop, D007H); chemical shifts (δ) are given in parts
per million (ppm), and coupling constants (J) are given in hertz.
Thiocarbamates present two rotamere forms in NMR.
Quantification. Quantification was performed on an Agilent HP
6890N GC (Agilent, Massy, France) equipped with a fused-silica
capillary column HP-1 (polydimethylsiloxane, 50 m × 0.20 mm i.d.
× film thickness ) 0.33 µm; Interchim) and a FID: carrier gas, helium;
constant flow, 1 mL/min; injector and detector temperatures, 250 °C;
split ratio, 1/10; temperature program, raised from 60 to 230 °C at 2
°C/min and then held isothermal (120 min).
An external standardization method was used. Linearity of the
calibration curve was found for concentrations between 0.124 and 0.962
mg/g. For each compound, five solutions of different equidistant
concentrations were prepared. For concentrations close to the lower
and to the upper limit, 10 GC-FID injections were realized, 2 for the
others. Homogeneity of variances was verified by statistic bilateral
Fisher test by taking homogeneity of variances as Ho (15).
Elementary Analysis. Compounds with no known CAS Registry
Number (CAS RN) were submitted to elementary analysis at the Service
Central d’Analyse du CNRS, Service Central d’Analyse, Vernaison,
France.
Melting Point. The melting point of solid compounds was determined
on an Electrothermal 9100 apparatus and is given in degrees centigrade.
Chemicals. All chemicals were purchased from Sigma-Aldrich (St.
Quentin Fallavier, France), Merck (Fontenay-sous-Bois, France), VWR
Prolabo (Fontenay-sous-Bois, France), or Eurisotop (Saint Aubin,
France): allyl isothiocyanate (Aldrich, 377430), benzyl isothiocyanate
(Aldrich, W510548), ethyl isothiocyanate (Aldrich, E33904), 3-(me-
thylthio)propyl isothiocyanate (Aldrich, W331201), phenethyl isothio-
cyanate (Aldrich, W401404), absolute ethanol (Merck, 100983),
1-propanol (Fluka, 82092), methanol (Riedel-de Hae¨n, 34860), diethyl
ether (Riedel-de Hae¨n, 24004), methylene chloride (Riedel-de Hae¨n,
24233), pentane (Riedel-de Hae¨n, 32288), petroleum ether (Riedel-de
Hae¨n, 24587), hexane (Riedel-de Hae¨n, 15671), cyclohexane (Riedel-
de Hae¨n, 15329), and ethanol 96% (APC, 200-578-6). Solvents were
distilled prior to use.
Linearity of the calibration function was tested by comparison of
the calibration function of first order to the calibration function of
second order to prove that the diminution of the residual variance was
significant. The nonsignificance of residual variance was taken as Ho
(16).
Specific Detection. Detection of organosulfur analytes was carried
out on a HP 5990 series II GC equipped with a FID and a FPD. Fused-
silica capillary column: HP-1 (polydimethylsiloxane, 50 m × 0.20 mm
i.d. × film thickness ) 0.33 µm; Agilent, Massy, France); carrier gas,
nitrogen; constant pressure, 220 kPa; detector gas (flow), nitrogen (1.08
mL/min), hydrogen (75 mL/min), air (100 mL/min); injector temper-
ature, 230 °C; detector temperature, 250 °C; splitless mode; temperature
program, raised from 60 to 250 °C at 2 °C/min and then held isothermal
(120 min).
GC-Olfactometry. GC-O analysis was performed on a Shimadzu
GC-2010 GC (Shimadzu, Champs-sur-Marne, France) equipped with
an automatic injector type AOC-20i, a FID and an ATAS olfactory
port OP275 with a glass nasal cone (ATAS, Veldhoven, The Nether-
lands): column, Equity-5 (30 m × 0.25 mm i.d. × film thickness )
0.25 µm; Supelco, L’Isle d’Abeau, France); carrier gas, nitrogen;
constant flow, 0.8 mL/min; injector temperature, 250 °C; detector
temperature, 250 °C; split ratio, 1:100; transfer line for nearly
Plant Extraction. Fresh watercress (2300 g; leaves and stems, cut
into pieces; origin, Hye`res) were extracted with 3.6 L of n-hexane during
6 h (agitation, T ) 62-63 °C) to give, after filtration, treatment
with MgSO4 (VWR, 25162.361), and solvent evaporation, 2.48 g
(yield ) 0.11%) of concrete. Extraction was carried out on the same
day as the harvest.
Watercress (89.60 g; leaves and stems, cut into pieces, from a
supermarket) were extracted in a Soxhlet apparatus with 400 mL of
cyclohexane during 4 h to give, after filtration, treatment with MgSO4,
and solvent evaporation, 0.05 g (yield ) 0.06%) of concrete.
Fractionation. Fractionation was performed according to the litera-
ture (9, 10). Indian cress absolute (20.03 g) was solubilized in 100 mL
of diethyl ether. The ethereal solution was washed three times with 50
mL of HCl 1 N (Riedel-de Hae¨n, 30721). The aqueous phases were
united (F1), extracted with ether (F2), and treated with NaOH (Sigma-
Aldrich, 221465) 1 N solution until pH 9. After ethereal extraction
and drying over MgSO4, 0.09 g of a (basic) fraction F1.1 was obtained.