Melo´n de Olor Aroma Studies
J. Agric. Food Chem., Vol. 48, No. 12, 2000 6201
The eluate was concentrated to dryness under reduced pres-
sure, redissolved in 50 mL of 0.2 M citrate-phosphate (pH 5.0),
and extracted with diethyl ether to remove remaining volatiles.
En zym a tic Hyd r olysis. A nonselective pectinase (300 µL
of Rohapect D5L, Ro¨hm, Darmstadt, Germany) was added to
the glycosidic extract together with phenyl â-D-glucopyranoside
as internal standard (800 µg/kg), and the mixture was incu-
bated at 37 °C overnight. The liberated aglycones were
extracted with diethyl ether, and the organic phase was dried
over anhydrous sodium sulfate, concentrated (Vigreux column,
36 °C) to 0.2 mL, and subjected to HRGC, HRGC-MS, and
HRGC-O analyses.
Ca p illa r y Ga s Ch r om a togr a p h y (HRGC). Experiments
were performed on a Carlo Erba Fractovap 4160 Gas Chro-
matograph (Milano, Italy), equipped with a split-splitless
injector and a flame ionization detector (FID), both operating
at 250 °C. A J &W fused-silica DB-Wax capillary column (30
m × 0.25 mm i.d.; film thickness 0.25 µm) was used with the
following temperature program: 3 min isothermal at 50 °C,
then raised to 240 °C at 4 °C/min, and finally held at 240 °C
for 10 min. The flow rate for the carrier gas was 1.6 mL/min
He. Volumes of 1 µL were injected with a split ratio of 1:50.
Temperature programmed retention indices were estimated
using normal paraffins (C9-C32) as standards. Quantitative
data for free and bound volatiles were obtained by the internal
standard method using 2-undecanol and phenol (released by
enzymic hydrolysis of phenyl â-D-glucopyranoside), respec-
tively, as reference substances without considering calibration
factors, i.e., F ) 1.00 for all compounds.
a flow rate of 5.0 mL/min, to yield 29 mg of semipure
glucoconjugate 2. The semipure compound 2 was further
acetylated (Ac2O/pyridine) and purified by silica gel flash
chromatography (SiO2 60 Merck, 0.032-0.063 µm) with ethyl
ether as mobile phase, to finally yield 4 mg of pure peracety-
lated glycoconjugate 2a .
4-(â-D-glucopyranosyloxy)benzyl alcohol 1: UV (MeOH) λmax
1
272 nm; H and 13C NMR data were in good agreement with
those published by Taguchi et al. (1981).
Peracetylated 4-(â-D-glucopyranosyloxy)benzyl alcohol 1a :
UV (MeOH) λmax 270 nm; FAB-MS, m/z (%) 519 (8, [M+Na]+),
1
497 (1, [M+H]+), 331 (86), 271 (4), 169 (64); H and 13C NMR
data were in good agreement with those published by Taguchi
et al. (1981).
Peracetylated [4-(â-D-glucopyranosyloxy)benzyl] 2,3-dihy-
droxy-3-methylbutanoate 2a : UV (MeOH) λmax 270 nm; HR-
FAB-MS, m/z 613.2139 [M+H]+ (C28H37O15 requires 613.2132);
FAB-MS, m/z (%) 635 (26, [M+Na]+), 613 (27, [M+H]+), 331
(52), 271 (39), 211 (41), 169 (100);1H and 13C NMR, see Table
2 below.
Biom im etic Rea ction s. Compounds 1 and 2 were submit-
ted to enzymatic hydrolysis with â-glucosidase in the same
conditions as described for Rohapect D5L enzyme (Ro¨hm,
Darmstadt, Germany). The liberated aglycones were extracted
with diethyl ether and the organic phase was dried over
anhydrous sodium sulfate, concentrated (Vigreux column, 36
°C) to 0.2 mL, and subjected to HRGC and HRGC-MS.
RESULTS AND DISCUSSION
Ca p illa r y Ga s Ch r om a togr a p h y-Ma ss Sp ectr om etr y
(HRGC-MS). A Varian Aerograph 1440 gas chromatograph
(Palo Alto, CA) directly coupled to a Finnigan MAT 44 mass
spectrometer (Bremen, Germany) was used with the same type
of column and temperature conditions as mentioned above for
HRGC; electron energy 70 eV; mass range 30-300.
Results of qualitative analyses (mass spectral data studies)
were verified by comparing the retention indices and mass
spectral data with those of authentic reference substances and/
or with other published spectra (EPA/NIH mass spectral
library).
F r ee a n d Glycosid ica lly Bou n d Vola tiles. Either
the free volatile extract or the volatiles generated by
enzymatic hydrolysis from the glycosidic fraction ob-
tained from melo´n de olor (Sicana odorifera) fruit pulp
showed aroma notes resembling the flavor of fresh fruit,
described as fruity-watery-green-fatty-sweet and watery-
fatty-sweet, respectively.
Table 1 shows the free and bound volatile compounds
identified in the above-mentioned extracts by HRGC
and HRGC-MS analyses, the retention indices found
experimentally for each compound and those obtained
for authentic reference substances or those reported in
the chemical literature, as well as the concentration of
each aroma compound calculated on the basis of the
standard added and the odor description of each GC-
separated volatile.
As can be seen in Table 1, 37 compounds (94.8% of
total extract) were identified as free volatiles with
3-methyl-2-butanol, 3-hydroxy-2-butanone, ethyl 3-hy-
droxybutanoate, and (Z)-3-hexenol being the major
components. Aliphatic alcohols (61.1%) predominated in
the melo´n de olor free volatiles profile, followed by hy-
droxyketones (14.6%), aliphatic acids (7.5%), hydroxy-
esters (4.8%), terpenes (2.7%), aromatic compounds
(2.6%), aldehydes (1.5%), and unidentified compounds
(5.2%). The free volatile profile found in melo´n de olor
is quite different from that reported for melo´n (Cucumis
melo) (Homatidou et al., 1992), its close relative which
also belongs to the Cucurbitaceae family. In melo´n fruit,
sulfur compounds constitute the major part of volatiles
compared with the flavor of other tropical fruits (Shiba-
moto and Tang, 1990) such us banana, mango, papaya,
passion fruit, and guava. In contrast, the aroma spec-
trum here reported for melo´n de olor is unique.
Table 1 also shows 22 aglycones released by enzy-
matic hydrolysis which were identified for the first time
as bound aroma constituents in melo´n de olor fruit. The
identified glycosidically bound compounds mainly con-
sisted of compounds having aromatic structure (87.3%),
and these were followed by aliphatic acids (5.0%),
Capillar y Gas Ch r om atogr aph y-Olfactom etr y (HRGC-
O). HRGC with a simultaneous flame ionization detector (FID)
and an odor evaluation port was carried out using a HP 5890
gas chromatograph (Palo Alto, CA) operated under the same
conditions as mentioned above for HRGC. Approximately 50%
of the effluent was diverted through a heated, glass-lined
capillary (60 °C) to a sniffing mask, where it was mixed with
humidified air. Each sample was sniffed three times by people
trained in that technique. FID was used as monitor detector.
Isola tion of Glycocon ju ga tes. To isolate the intact con-
jugate derivatives of 4-hydroxybenzyl alcohol, 18 g of glycosidic
extract was obtained by adsorption chromatography on XAD-2
resin as described above but using 19 kg of fruit pulp. Then,
portions of 1 g of glycosidic extract were fractionated by
multilayer coil countercurrent chromatography (MLCCC) with
an Ito multilayer coil separator-extractor (PC Inc., Potomac,
MD) equipped with a 75 m × 2.6 mm i.d. PTFE tubing for
separation of glycosides. The instrument was operated using
CHC13/MeOH/H2O (7:13:8, v/v/v) as solvent system at a flow
rate of 1.0 mL/min, and at a rotational speed of 800 rpm.
Elution mode head to tail was used. Fifty fractions of 5 mL
each were collected. Combined fractions (6-7) containing a
precursor of 4-hydroxybenzyl alcohol as revealed by enzymatic
hydrolysis were refractionated by MLCCC as described above
and finally purified by RP-HPLC (Eurospher 100 C-18 column,
5 µm, 250 × 46 mm) with MeOH/H2O (1:3, v/v) as mobile phase
at a flow rate of 5.0 mL/min, to obtain 15 mg of glycoconjugate
1. Additionally, 5 mg of compound 1 was acetylated (Ac2O/
pyridine) and further purified by silica gel flash chromatog-
raphy to obtain peracetylated glycoconjugate 1a .
In addition, combined fractions (9-10) from the first ML-
CCC were refractionated by this chromatographic technique
using the same conditions as described above and finally
purified using RP-HPLC (Eurospher 100 C-18 column, 5 µm,
250 × 46 mm) with MeOH/H2O (1:1, v/v) as a mobile phase at
Parada, Fabian
