Identification of sensory-active phytochemicals in asparagus (Asparagus officinalis L.)

Article abstract of DOI:10.1021/jf3040868

Sensory-directed fractionation of extracts prepared from raw and cooked asparagus (Asparagus officinalis L.), respectively, followed by LC-TOF-MS, LC-MS/MS, and 1D/2D-NMR experiments revealed the chemical structures of nine bitter tasting mono- and bidesm

Full text of DOI:10.1021/jf3040868

Article
pubs.acs.org/JAFC
Identification of Sensory-Active Phytochemicals in Asparagus
(Asparagus officinalis L.)
Corinna Dawid and Thomas Hofmann*
Chair of Food Chemistry and Molecular Sensory Science, Technische Universitat Munchen, Lise-Meitner-Straße 34, D-85354
̈
̈
Freising, Germany
ABSTRACT: Sensory-directed fractionation of extracts prepared from raw and cooked asparagus (Asparagus officinalis L.),
respectively, followed by LC-TOF-MS, LC-MS/MS, and 1D/2D-NMR experiments revealed the chemical structures of nine
bitter tasting mono- and bidesmotic saponins as well as the previously not reported 1,2-dithiolan-4-carboxylic acid 6-D-α/β-
glucopyranose ester exhibiting an interesting buttery mouth-coating effect. Sensory studies showed that the orosensation
imparted by this sulfur compound was reminiscent to that of melting butter and revealed an orosensory recognition threshold of
276.8 μmol/L.
KEYWORDS: asparagus, asparagusic acid, steroidal saponin, taste, taste dilution analysis, bitter, mouth-coating
unknown phytochemicals contribute to the perceived bitterness
of asparagus spears.
INTRODUCTION
■
Raw and cooked spears of white asparagus (Asparagus officinalis
L.) are highly appreciated by the consumers for its pleasant
flavor profile centering around sulfury and buttery notes.
Unfortunately, the palatability of asparagus might be strongly
decreased by a sporadic bitter taste. This off-taste is often the
reason for consumer complaints and, therefore, is causing a
major problem for vegetable processors. Although the so-called
bottom cuts seem to be rich in bitterness,1 the knowledge about
the key molecules imparting the typical bitter off-taste of white
asparagus is rather fragmentary.
In the past, application of the so-called sensomics approach
on various fresh and processed foods enabled the compre-
hensive mapping of several sensory-active substances such as
thermally generated bitter compounds,19,20 cooling compounds
in dark malt,21 bitter off-tastants in carrot products,22 the taste
enhancer alapyridaine in beef bouillon,23 and astringent key
taste compounds in black tea infusions,24 roasted cocoa nibs,25
red current juice,26 and spinach.27
The objective of the present study was to locate the key
molecules contributing to the typical taste profile of raw and
cooked asparagus by means of a sensomics approach, to
determine the chemical structure of the most intense taste-
active phytochemicals by means of LC-MS, 1D/2D-NMR, and
to evaluate their sensory activity by the determination of human
recognition thresholds.
After isolation from asparagus storage roots, the furostanol
saponins 5β-furostane-3β,22,26 triol-3-O-β-D-glucopyranosyl
(1→2)-β-D-glucopyranoside 26-O-β-D-glucopyranoside (offici-
nalidin I) and 5β-furostane-3β,22,26 triol-3-O-β-D-glucopyr-
anosyl (1→2) [β-D-xylopyranoxyl (1→4)]-β-D-glucopyranoside
26-O-β-D-glucopyranoside (officinalisin II), were suggested to
contribute to the bitter off-taste of asparagus, although their
identification was not based on published NMR data.2 In
addition, 25S-furost-5-ene-3β,22,26-triol-3-O-(2,4-di-O-α-L-
rhamnopyranosyl-β-D-glucoyranoside)-26-O-β-D-glucopyrano-
side (ASP-I) was isolated from asparagus bottom cuts and
identified as the bitter tasting (25S)-configumer of the well-
known protodioscin.1,3 As several studies have demonstrated
that the saponin distribution among the organs of a plant varies
considerably and that different organs of asparagus do not
contain the identical saponins, it is unclear whether these
saponins are also the bitter key molecules in the edible spears of
asparagus.2,4 Although treatment with a naringinase preparation
comprising naringinin-α-1,2-rhamnosidase and β-glucosidase
activity induced the decrease of the bitter taste of the juice
prepared from asparagus bottom cuts,2 the exact structures of
the saponins accounting for the bitter off-taste of asparagus
spears are still not known. Furthermore, it is unclear as to
whether besides the saponins identified,1,2,5−9 also flavonoids,10
saccharides,11−13 polyacetylenes,14,15 and the sulfur-containing
asparagusic acid and its esters,16−18 respectively, or previously
MATERIALS AND METHODS
■
Chemicals. The following compounds were obtained commercially
from the sources given in parentheses: formic acid, glucose,
hydrochloric acid (Merck, Darmstadt, Germany); sodium chloride,
L-glutamic acid monohydrate, rhamnose (Fluka, Taufkirchen,
Germany), sodium hydroxide (Riedel-de-Haen, Seelze, Germany).
Deuterated solvents were obtained from Euriso-Top (Gif-Sur-Yvette,
France). Solvents were of HPLC grade (Merck Darmstadt, Germany).
Water for HPLC separation was purified by means of a Milli-Q water
advantage A 10 water system (Millipore, Molsheim, France). For
sensory analysis, bottled water (Evian, low mineralization: 405 mg/L)
was adjusted to pH 5.9 with trace amounts of formic acid prior to use.
Freshly harvested, white asparagus spears (∼21 cm in length) of the
cultivar Grolim were obtained from local producers in Germany,
namely, from Quedlinburg (May/June 2005), Munster (June 2007),
̈
and Schrobenhausen (May 2008), respectively.
Received: September 21, 2012
Revised: November 7, 2012
Accepted: November 8, 2012
Published: November 8, 2012
© 2012 American Chemical Society
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Preparation of Cooked Asparagus. Freshly peeled, white
asparagus spears (2 kg) were divided lengthwise in two equal parts
by means of a kitchen knife. One aliquot was used for the analysis of
the fresh samples, the other one was cooked for 14 min and 95 °C in
water (1.5 L) to simulate domestic processing.
min, 100% B; 55 min, 0% B; 68 min, 0% B. The effluent was separated
into 19 subfractions, namely, III-B-1 up to II-B-19, which were
collected individually in several runs. After addition of water (10 mL),
the solvent was removed in a vacuum. Each fraction was finally
separated from buffer by means of solid phase extraction using C-18 E
cartridges (1 g/6 mL; Phenomenex, Aschaffenburg, Germany) which
were preconditionated with methanol (3 × 10 mL), followed by water
(3 × 10 mL). After sample application, the cartridges were rinsed with
water (6 mL), followed by methanol (10 mL) to obtain the target
compounds, which were separated from solvent in a vacuum at 38 °C,
followed by lyophilization. Fraction III-B-12 and III-B-14, judged with
the highest sensory impact, were further purified by rechromatography
by means of semipreparative RP-HPLC using a 0.5 mL sample loop
and a 250 × 10 mm i.d., 5 μm, HyperClone ODS C18 column
(Phenomenex, Aschaffenburg, Germany) equipped with a guard
column of the same type. Using 1% formic acid in water (v/v) as
solvent A and 1% formic acid in acetonitrile (v/v) as solvent B, the
following solvent gradient (3 mL/min) was used for chromatography:
0 min, 0% B; 5 min, 0% B; 10 min, 10% B; 15 min, 10% B; 25 min,
100% B; 30 min, 100% B; 35 min, 0% B; 40 min, 0% B. Before each
injection the column was equilibrated for 10 min at the starting
conditions. The effluents of the chromatographic separation of
fractions III-B-12 and III-B-14 were collected individually in several
runs and the corresponding fractions were combined. After removing
the solvent in vacuum, each HPLC fraction was again separated from
buffer by means of solid phase extraction as described above. After
freeze-drying twice, the structures of taste compound 1 (III-B-12) and
2 (III-B-14) were determined as 1,2-dithiolan-4-carboxylic acid 6-D-α-
glucopyranoseester and 1,2-dithiolan-4-carboxylic acid 6-D-β-glucopyr-
anoseester, respectively, by means of UV−vis, LC-MS/MS, TOF-MS,
1D/2D NMR, and hydrolytic degradation experiments.
Sequential Solvent Extraction. Cooked and fresh white
asparagus spears were cut into 2 cm pieces with a kitchen knife and
finely ground in a laboratory blender (Retsch, Haan, Germany) at
3500 U/min for 60 s. Methanol (2.0 L) was added to the puree (∼1
kg) of raw and cooked asparagus, respectively, and the mixtures were
vigorously stirred for 60 min at room temperature under an
atmosphere of argon, followed by filtration (Schleicher & Schuell
filter, 24 cm). The filtrates were collected and the residues were
extracted three times (2 L each) with methanol/water (70/30, v/v)
adjusted to pH 5.9 with aqueous formic acid (1% in water). After
filtration, the combined filtrates were separated from methanol in a
vacuum at 38 °C, followed by freeze-drying. The lyophilized material
obtained from cooked and raw asparagus, respectively, was taken up in
water (1 L) and, then, sequentially extracted with n-pentane (4 × 0.5
L), dichloromethane (4 × 0.5 L), followed by ethyl acetate (4 × 0.5
L). The corresponding extracts were combined accordingly and
separated from solvent in a vacuum at 38 °C, followed by
lyophilization to obtain the pentane extractables (fraction I), the
dichloromethane extractables (fraction II), the ethyl acetate extract-
ables (fraction III), and the water-solubles (fraction IV), respectively
(Table 1). The fully extracted asparagus residues were freeze-dried to
give the insoluble materials which did not exhibit any taste activity.
Table 1. Yields of Total Extract and of Fractions I−IV
Isolated from Raw and Cooked Peeled White Asparagus,
Respectively
1,2-Dithiolan-4-Carboxylic acid 6-D-α/ß-Glucopyranoseester, 1/
2, Figure 1. LC-MS (ESI−): m/z (%) 357.7 (100, [M + HCOO]−),
311.0 (21, [M − H]−), 347.6 (15, [M + Cl]−); LC-MS/MS (DP =
−60 V): m/z (%) 311 (6), 144 (5), 131 (100), 112 (22), 104 (10);
LC-MS-TOF: m/z 311.0273 (measured), m/z 311.0254 (calcd. for
b
yield (g/100 g) from
a
sample
raw white asparagus
cooked white asparagus
total extract
fraction I
3.95
0.20
0.21
0.09
3.38
1.86
0.44
0.06
0.03
1.22
1
[C10H16O7S2−H+]−); H and 13C NMR data are given in Table 2.
Alkaline Hydrolysis of 1 and 2 and Identification of
Asparagusic Acid. Aliquots (∼0.5 mg) of compounds 1 and 2,
respectively, were dissolved in aqueous sodium hydroxide (2 mol/L; 1
mL) and heated at 110 °C for 120 min in a closed vial. After cooling,
water (50 mL) was added and the pH value was adjusted to 5.0 with
an aqueous hydrogen chloride solution (4 mol/L). High-performance
ion chromatographic analysis of a minor aliquot (1 mL) of this
hydrolysate and cochromatography with reference carbohydrates
revealed glucose as the carbohydrate moiety in 1 and 2, respectively.
The major aliquot (50 mL) of the hydrolysate was extracted three
times with ethyl acetate (30 mL), and the pooled organic extracts were
separated from solvent in vacuum to afford asparagusic acid (3) as a
pale yellow powder in a purity of >98% (HPLC/ELSD).
fraction II
fraction III
fraction IV
a
Total extract obtained by extracting minced asparagus with methanol
and methanol/water and individual fractions (I−IV) obtained by
b
sequential solvent extraction of total extract. Yields were determined
by weight; based on fresh weight.
Separation of Fraction III by Means of Solid Phase
Extraction. An aliquot (600 mg) of lyophilized fraction III isolated
from fresh asparagus was dissolved in methanol/water (30/70, v/v; 10
mL) and applied onto the top of a Strata C18-E SPE cartridge (10 g/
60 mL; Phenomenex, Aschaffenburg, Germany) preconditioned with
methanol (60 mL), methanol/water (50/50, v/v; 100 mL), followed
by water (100 mL) by using a vacuum extraction box (J. T. Baker,
Philipsburgh, NJ, USA). Separation was performed by flushing the
cartridge with a sequence of methanol/water mixtures (100 mL each)
to give fraction III-A (0/100, v/v; yield: 57.7%), fraction III-B (30/70,
v/v; yield: 5.0%), fraction III-C (70/30, v/v; yield: 6.9%), and finally
fraction III-D (100/0, v/v; yield: 10.0%), respectively. Each fraction
was collected, separated from solvent in a vacuum, and the residues
were taken up in water, freeze-dried twice, and were then kept at −20
°C until used for sensory and chemical analysis, respectively.
Identification of Key Taste Compounds in Fraction III-B.
Fraction III-B was dissolved in acetonitrile/water (10/90, v/v; 2 mL/
50 mg) and, after membrane filtration, was injected onto a 250 × 21.2
mm i.d., 5 μm, Microsorb-MV C18 column (Varian, Darmstadt,
Germany) equipped with a guard column of the same type operated
with a flow rate of 18 mL/min. Using 0.1% formic acid in water (v/v)
as solvent A and 0.1% formic acid in acetonitrile (v/v) as solvent B,
chromatography was performed with the following gradient: 0 min, 0%
B; 5 min, 0% B; 10 min, 10% B; 27 min, 10% B; 40 min, 100% B; 50
Asparagusic Acid, 3, Figure 1. LC-MS (ESI−): m/z (%) 149 (100,
[M − H]−), 299 (87, [2M − H]−), 195 (80, [M + HCOO]−), 185
(14, [M + Cl]−); LC-MS/MS (DP = −60 V): m/z (%) 104 (20), 63
(100); LC-MS-TOF: m/z 148.9744 (measured), m/z 148.9731 (calcd.
1
for [C4H5O2−H+]−); H NMR (500 MHz, DMSO-d6; COSY): δ/
ppm: 3.30 [dd, 2H, J = 4.2 Hz, J = 11.4 Hz, H−C(3a, 5a)]; 3.40 [dd,
2H, J = 6.1 Hz, 11.4 Hz, H−C(3b, 5b)]; 3.49−3.54 [m, 1H, H−C(4)];
12.8 [s, 1H, COOH]; 13C NMR (125 MHz, DMSO-d6; HMQC,
HMBC): 41.5 [C(3, 5)]; 50.7 [C(4)]; 173.4 [C(6)].
Identification of Key Taste Compounds in Fraction III-C. A
solution (100 mg/10 mL) of an aliquot of fraction III-C in methanol/
water (50/50, v/v) was separated by preparative HPLC using a 1 mL
injection loop and a 250 × 21.2 mm i.d., 5 μm, HyperClone ODS 120
column (Phenomenex, Aschaffenburg, Germany) with a binary
gradient using 0.1% formic acid in water as solvent A and 0.1%
formic acid in acetonitrile as solvent B (flow rate 20.0 mL/min): 0
min, 5% B; 5 min, 5% B; 10 min, 30% B; 25 min, 30% B; 30 min, 35%
B; 40 min, 40% B; 45 min, 40% B; 45 min, 100% B; 50 min, 100% B;
55 min, 5% B; 60 min, 5% B. Prior to injection, the column was
equilibrated for 10 min using the starting conditions. The effluent was
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Figure 1. Chemical structures of 1,2-dithiolan-4-carboxylic acid 6-D-α-glucopyranose ester (1), 1,2-dithiolan-4-carboxylic acid 6-D-β-glucopyranose
ester (2), asparagusic acid (3), 3-O-[α-L-rhamnopyranosyl-(1→2)-{α-L-rhamnopyranosyl-(1→4)}-β-D-glucopyranosyl]-26-O-[β-D-glucopyranosyl]-
(25R)-22-hydroxyfurost-5-ene-3β,26-diol (4a; R1 = CH3, R2 = H), 3-O-[α-L-rhamnopyranosyl-(1→2)-{α-L-rhamnopyranosyl-(1→4)}-β-D-
glucopyranosyl]-26-O-[β-D-glucopyranosyl]-(25S)-22-hydroxyfurost-5-ene-3β,26-diol (4b; R1 = H, R2 = CH3), (25R)-furost-5-en-3β,22,26-triol-3-
O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-glucopyranoside (5a; R1 = CH3, R2 = H), (25S)-furost-5-en-3β,22,26-triol-3-O-[α-
L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-glucopyranoside (5b; R1 = H, R2 = CH3), (25R)-furostane-3β,22,26-triol-3-O-[α-L-
rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-glucopyranoside (6a; R1 = CH3, R2 = H), (25S)-furostane-3β,22,26-triol-3-O-[α-L-
rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-glucopyranoside (6b; R1 = H, R2 = CH3), and 3-O-[β-D-glucopyranosyl-(1→2)-β-D-
xylopyranosyl-(1→4)-β-D-glucopyranosyl]-(25S)-5β-spirostan-3β-ole (7), 3-O-[{α-L-rhamnopyranosyl-(1→2)}{α-L-rhamnopyranosyl-(1→4)}-β-D-
glucopyranosyl]-(25S)-spirost-5-ene-3β-ol (8a; R1 = CH3, R2 = H), and 3-O-[{α-L-rhamnopyranosyl-(1→2)}{α-L-rhamnopyranosyl-(1→4)}-β-D-
glucopyranosyl]-(25R)-spirost-5-ene-3β-ol (8b; R1 = H, R2 = CH3).
Table 2. Assignment of 1H NMR (400 MHz, DMSO-d6/D2O; 95/5, v/v) and 13C NMR Signals (100 MHz, DMSO-d6/D2O; 95/
5, v/v) of 1,2-Dithiolan-4-Carboxylic Acid 6-D-α-Glucopyranose Ester (1) and 1,2-Dithiolan-4-Carboxylic acid 6-D-β-
Glucopyranose Ester (2)
ab
,
ab
,
1 (α)
2 (β)
position
3
δC / ppm
HSQC
[CH2]
δH / ppm
M [J Hz]
δC / ppm
HSQC
[CH2]
δH / ppm
M [J Hz]
41.5
3.27−3.34
3.35−3.45
3.56−3.64
3.27−3.34
3.35−3.45
m
41.5
3.27−3.34
3.35−3.45
3.56−3.64
3.27−3.34
3.35−3.45
m
m
m
m
m
m
m
m
m
4
5
50.2
41.5
[CH
50.2
41.5
[CH]
[CH2]
[CH2]
6
171.9
92.5
72.4
73.5
70.7
69.3
65.0
[C]
171.9
97.2
74.9
76.5
70.2
73.0
65.0
[C]
1′
2′
3′
4′
5′
6′
[CH]
[CH]
[CH]
[CH]
[CH]
[CH2]
4.87
d [J = 3.6]
[CH]
[CH]
[CH]
[CH]
[CH]
[CH2]
4.27
d [J = 7.9]
3.02−3.14
3.35−3.43
3.02−3.14
3.73−3.77
4.02−4.10
4.25−4.32
m
m
m
m
m
2.89
t [J = 8.4]
3.02−3.14
3.02−3.14
3.27−3.45
4.02−4.10
4.25−4.32
m
m
m
m
m
m
b
a
Arbitrary numbering according to structures 1 and 2 in Figure 5. Assignments confirmed by HSQC, HMBC, DEPT, and COSY experiments.
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separated into 16 subfractions, namely, III-C-1 to III-C-16, which were
collected individually in several runs. After combining the correspond-
ing fractions, the fractions were separated from solvent in vacuum,
freeze-dried twice, and then dissolved in water containing 2% ethanol
in their “natural” concentration ratios to be used for taste dilution
analysis. The bitter tasting fractions III-C-5, III-C-6, and III-C-7 were
further separated by means of semipreparative RP-HPLC using a 0.5
mL sample loop and a 250 × 10 mm i.d., 5 μm, Hyperclone ODS 120
column (Phenomenex, Aschaffenburg, Germany) equipped with a
guard column of the same type. Chromatography was performed using
the following gradient at a flow rate of 3.5 mL/min: 0 min, 60% B; 20
min, 60% B; 25 min, 100% B; 30 min, 100% B; 40 min, 60% B; 65
min, 60% B. Prior to injection, the column was equilibrated for 10 min
using the starting conditions. The peaks were collected individually in
several runs, the corresponding fractions were combined, and the
purity (≥98%) of each fraction was checked by means of analytical
HPLC/ELSD as well as HPLC-MS. After separating the solvent in
vacuum, the isolated materials were freeze-dried twice and used for
sensory analysis as well as structure determination by means of LC-
MS/MS, UPLC-TOF-MS, 1D/2D NMR, and hydrolysis experiments.
The bitter compounds 4a/b−6a/b (Figure 1) could be successfully
identified in the following HPLC fractions: 3-O-[α-L-rhamnopyrano-
syl-(1→2)-{α-L-rhamnopyranosyl-(1→4)}-β-D-glucopyranosyl]-26-O-
[β-D-glucopyranosyl]-(25R)-22-hydroxyfurost-5-ene-3β,26-diol (4a)
and 3-O-[α-L-rhamnopyranosyl-(1→2)-{α-L-rhamnopyranosyl-(1→
4)}-β-D-glucopyranosyl]-26-O-[β-D-glucopyranosyl]-(25S)-22-hydrox-
yfurost-5-ene-3β,26-diol (4b) in fraction III-C-5, (25R)-furost-5-en-
3β,22,26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyrano-
side]-26-O-β-D-glucopyranoside (5a) and (25S)-furost-5-en-3β,22,26-
triol-3-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-
D-glucopyranoside (5b) in fraction III-C-6, and (25R)-furostane-
3β,22,26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyrano-
side]-26-O-β-D-glucopyranoside (6a) and (25S)-furostane-3β,22,26-
triol-3-O-[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-
D-glucopyranoside (6b) in fraction III-C-7. The analytical details of the
structure determination, including the comprehensive set of MS and
1D/2D-NMR data are published in a companion paper.28
Identification of Key Taste Compounds from Fraction III-D.
Aliquots (50 mg) of SPE fraction III-D were separated by preparative
HPLC using a 2.0 mL sample loop and a 250 × 21.2 mm i.d., 5 μm,
Microsorb-MV 100-5 C18 column (Varian, Darmstadt, Germany)
with gradient elution at a flow rate of 20 mL/min. Using 0.1% formic
acid in water (v/v) as solvent A and 0.1% formic acid in acetonitrile
(v/v) as solvent B, chromatography was performed as follows: 0 min,
20% B; 5 min, 50% B; 10 min, 50% B; 20 min, 65% B; 25 min, 100%
B; 30 min, 100% B; 35 min 20% B; 40 min, 20% B. Prior to injection,
the column was equilibrated for 10 min using the starting conditions.
The effluent containing the target taste compound was collected from
several separate HPLC runs, combined, freed from solvent in a
vacuum, freeze-dried twice, and the residues obtained were used for
sensory and instrumental analysis. HPLC-MS/MS and 1D/2D-NMR
analysis led to the unequivocal identification of 3-O-[{β-D-glucopyr-
anosyl-(1→2)}{β-D-xylopyranosyl-(1→4)} β-D-glucopyranosyl]
(25S),5β-spirostan-3β-ol (7, Figure 1) as well as 3-O-[{α-L-
rhamnopyranosyl-(1→2)}{α-L-rhamnopyranosyl-(1→4)}-β-D-gluco-
pyranosyl]-(25-S)-spirost-5-ene-3β-ol (8a, Figure 1) and 3-O-[{α-L-
rhamnopyranosyl-(1→2)}{α-L-rhamnopyranosyl-(1→4)}-β-D-gluco-
pyranosyl]-(25R)-spirost-5-ene-3β-ol (8b, Figure 1) in fraction III-D-
4. The details of the isolation and full spectroscopic structure
determination demonstrating the chemical identity of these bitter
saponins 4a/b−8a/b are published separately in a companion paper.28
High-Performance Ion Chromatography. Anion exchange
chromatography was performed using an ICS-2500 ion chromatog-
raphy system (Dionex, Idstein, Germany) consisting of a GS 50
gradient pump, an AS 50 autosampler, an AS 50 thermal compartment,
and an ED 50 electrochemical detector operating in pulsed
amperometric detection mode. The detector was equipped with a
gold working electrode operating with a standard carbohydrate
quadrupole waveform supplied by manufacturer. Data acquisition
and instrumental control was completed with the Chromeleon
software (version 6.80, Dionex). Chromatographic separation was
performed at 30 °C on a 150 × 3 mm CarboPac PA-20 column
(Dionex) connected with a 30 × 3 mm CarboPac PA-20 guard column
(Dionex) using an isocratic gradient of sodium hydroxide solution (2.5
mM) for 20 min. After each sample, the column was washed with a
sodium hydroxide solution (200 mM) and equilibrated with sodium
hydroxide solution (2.5 mM) for 10 min prior to injection.
Chromatography was performed with an injection volume of 10 μL
and a flow rate of 0.5 mL/min. For qualitative analysis, glycosidically
bound carbohydrates were identified by comparison of retention times
and cochromatography with reference monosaccharides.
Sensory Analyses. Training of the Sensory Panel. Seven female
and five male panelists (25−40 years in age), who had given the
informed consent to participate in the sensory tests of the present
investigation and had no history of known taste disorders, participated
in this study. Each panelist took place in a weekly training session for
at least two years in order to get familiarized with the taste language
and methodologies used. Aqueous solutions (2.0 mL; pH 5.9) of the
following reference compounds were used for sensory training: sucrose
(50 mmol/L) for sweet taste, L-lactic acid (20 mmol/L) for sour taste,
NaCl (20 mmol/L) for salty taste, caffeine (1 mmol/L) for bitter taste,
monosodium L-glutamate (3 mmol/L) for umami taste. For
astringency the panel was trained by using tannic acid (0.05%) and
quercetin-3-O-β-D-glucopyranoside (0.01 mmol/L), respectively, using
the so-called half-tongue test.24,25 Sensory analyses were performed by
means of the sip-and-spit method in an air-conditioned room at 22−25
°C in three independent sessions. To prevent cross-model interactions
with odorants reported in asparagus,16,17 nose clips were used by the
assessors.
Precaution Taken for Sensory Analysis of Food Fractions and
Taste Compounds. To remove solvent traces and buffer compounds
from all fractions and compounds isolated from asparagus, the
individual fractions were suspended in water, and, after the volatiles
1
were removed in high vacuum (<5 mPa), were freeze-dried twice. H
NMR chromatographic, GC/MS, and high-performance ion chroma-
tographic analysis of an aliquot revealed that fractions treated by that
procedure are essentially free of solvents and buffer compounds used.
Taste Profile Analysis. Aliquots of the puree prepared from cooked
or fresh white asparagus spears, respectively, were pressed through a
tea towel and, then, filtered (Schleicher & Schuell filter, 24 cm) to
obtain a juice which was judged by the trained sensory panelists in the
intensity of sweet, sour, umami, salty, bitter, astringent, and buttery
mouth-coating on a linear scale from 0 (not detectable) up to 5 (very
intense).
Comparative Taste Profile Analysis. Aliquots of the lyophilized
fractions isolated from raw and cooked asparagus, respectively, were
dissolved in their “natural” concentrations (corresponding to 25 g of
fresh asparagus) in bottled water (Evian, low mineralization: 405 mg/
L) containing 2% ethanol and were then adjusted to pH 5.9 with trace
amounts of aqueous formic acid. These solutions were then presented
to the sensory panel, which was asked to rate the intensity of the
individual taste qualities on a linear scale from 0 to 5 in comparison to
the raw or cooked asparagus juice, respectively, as the control.
Taste Dilution Analysis (TDA). Aliquots of the HPLC fractions
were dissolved in “natural” ratios in 5.0 mL of bottled water (pH 5.9,
1% ethanol) and, then, sequentially diluted 1:1 with bottled water (pH
5.9, 1% ethanol). The serial dilutions of each of these fractions were
then presented in order of increasing concentration to the trained
sensory panel, which was asked to evaluate each dilution step by means
of the recently developed half-tongue test.24,25 As previously reported,
the dilution at which a taste difference between the diluted extract and
the blank (control) could just be detected was defined as taste dilution
(TD) factor.18 The TD factors for each HPLC-fraction evaluated by
four different assessors in three different sessions were averaged. The
TD factors between individuals and separate sessions did not differ by
more than plus/minus one dilution step.
Half-Tongue Test. Taste dilution factors and oral recognition
thresholds for astringent and buttery mouth-coating fractions and
compounds, respectively, were determined by means of the recently
developed half-tongue test24,25 in order to overcome carry-over effects.
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Figure 2. Taste profile analysis of fresh pressed juice () and methanol extractables (- - -) of (A) raw and (B) cooked white asparagus (14 min at
95 °C).
Therefore, serial 1:1 dilutions of the samples using bottled water
adjusted to pH 5.9 with trace amounts of aqueous formic acid (1% in
water) as the solvent were presented in order of increasing
concentrations to a trained panel of 12 subjects in three different
sessions using the sip-and-spit method and an interstimulus interval
length of 2 min. In case of a correct selection by the panelist, the same
concentration was presented again besides one blank as a proof for the
correctness of the data. The geometric mean of the last and the second
last concentration was calculated and taken as the individual
recognition threshold. The values between individuals and between
three separate sessions differed by not more than plus or minus one
dilution step, which means a threshold value of 276.8 μmol/L for the
1,2-dithiolan-4-carboxylic acid 6-D-α/β-glucopyranoseesters represents
a range from 138.4 to 553.6 μmol/L.
High Performance Liquid Chromatography (HPLC). Prepara-
tive analyses of fraction III-B was performed on a HPLC apparatus
consisting of two S 1122 pumps (Sykam, Eresing, Germany), a Sedex
LT-ELSD detector Model 85 (Sedere, Alfortville, France), and a Rh
7725i type Rheodyne injection valve (Rheodyne, Bensheim,
Germany). Chromatography was done using a preparative 250 ×
21.2 mm i.d., 5 μm, Microsorb column (Varian, Darmstadt, Germany)
operated with a flow rate of 18.0 mL/min, respectively. The split ratio
was set to 1.0 mL/min for the ELSD detector. Data acquisition was
done by means of Chromstar V. 6.2 (SCPA, Weyhe, Germany).
Semipreparative analyses of fraction III-B was performed on a
HPLC apparatus (Shimadzu, Duisburg, Germany) consisting of a LC-
20AT pump, a SIL-20A autosampler, a DGU-20A3 degaser, and a
Sedex LT-ELSD detector Model 75 (Sedere, Alfortville, France).
Chromatography was done using a semipreparative 250 × 10 mm i.d.,
5 μm, HyperClone ODS 120 column (Phenomenex, Aschaffenburg,
Germany) operated with a flow rate of 3.5 mL/min, respectively. The
split ratio was set to 0.8 mL/min for the ELSD detector. Data
acquisition was done by means of LabSolutions LCsolutions V. 1.21
(Shimadzu, Duisburg, Germany).
LC/Time-of-Flight Mass Spectrometry (LC/TOF-MS). High-
resolution mass spectra were measured on a Bruker Micro-TOF-Q
mass spectrometer (Bruker Daltronics, Bremen, Germany) with flow
injection referenced on sodium formate (5 mmol). Data acquisition
was performed by using Daltonics DataAnalysis software (version 3.4;
Bruker).
High-Performance Liquid Chromatography/Tandem Mass
Spectrometry (HPLC-MS/MS). For compound identification, mass
and product ion spectra were acquired on an API 4000 Q Trap triple
quadrupole/linear ion trap mass spectrometer (ABSciex, Darmstadt,
Germany). The isolated fractions were dissolved in a mixture of
methanol/water (50/50, v/v) and directly introduced into the mass
spectrometer by flow infusion using a syringe pump. For electrospray
ionization, the ion spray voltage was set at −4500 V in the negative
mode and at 5500 V in the positive mode. Both quadrupoles operated
at unit mass resolution and nitrogen served as curtain gas (25 psi) and
as turbo gas (425 °C). Fragmentation of the pseudo molecular ions
[M + H]+ or [M − H]− into specific product ions was induced by
collision with nitrogen (4.5 × 10−5 Torr). Data acquisition and
instrumental control was performed with Analyst 1.4.2 software
(ABSciex, Darmstadt, Germany).
HPLC-MS/MS analysis of the bitter compounds 4a/b−8a/b in
asparagus fractions was performed on a 150 × 2.1 mm i.d., 5 μm,
Zorbax Eclipse XDB-C 18 column (Agilent, Waldbronn, Germany)
connected to the mass spectrometer operating in the multiple reaction
monitoring (MRM) mode with negative electrospray ionization (spray
voltage: −4500 V). The quadrupoles operated at unit mass resolution
and nitrogen was served as curtain gas (20 psi), nebulizer gas (50 psi),
and as turbo gas (425 °C). Fragmentation of the pseudo molecular
ions [M − H]− into specific product ions was induced by collision
with nitrogen (4 × 10−5 Torr).
The following mass transitions were recorded for the taste
compounds using the declustering potential (DP), collision energy
(CE), and collision cell exit potential (CXP) given in parentheses: 4a/
b, m/z 1047.5 → 901.6 (DP/CE/CXP: −180/−60/−25) and m/z
1047.7 → 755.4 (DP/CE/CXP: −180/−70/−27); 5a/b, m/z 903.6
→ 757.5 (DP/CE/CXP: −195/−58/−21) and m/z 903.6 → 59.0
(DP/CE/CXP: −195/−128/−7); 6a/b, m/z 901.6 → 755.5 (DP/
CE/CXP: −190/−52/−21) and m/z 901.6 → 101.0 (DP/CE/CXP:
−185/−82/−15); 7, m/z 871.5 → 739.6 (DP/CE/CXP: −195/−52/
−11); 8a/b, m/z 867.5 → 721.4 (DP/CE/CXP: −200/−46/−43) and
m/z 867.5 → 59.0 (DP/CE/CXP: −160/−110/−1). Using
acetonitrile containing 0.1% formic acid as solvent A and 0.1% formic
acid in water as solvent B, chromatography was performed using the
following gradient at a flow rate of 0.2 mL/min: 0 min, 5% A; 5 min,
5% A; 10 min, 25% A; 25 min, 30% A; 30 min, 0% A; 38 min, 0% A;
42 min, 5% A; 50 min, 5% A.
Analytical and (semi)preparative analyses of fractions III-C and III-
D were performed on a HPLC apparatus (Jasco, Groß-Umstadt,
Germany) consisting of two PU-2087 Plus pumps, an AS-2055 Plus
autosampler, a Sedex LT-ELSD detector Model 85 (Sedere, Alfortville,
France), and a Rh 7725i type Rheodyne injection valve (Rheodyne,
Bensheim, Germany). Chromatography was done using an analytical
250 × 4.6 mm i.d., a semipreparative 250 × 10 mm i.d., and a
preparative 250 × 21.2 mm i.d., 5 μm, HyperClone ODS 120 column
(Phenomenex, Aschaffenburg, Germany) operated with a flow rate of
1.0 mL/min, 3.5 mL/min, and 20.0 mL/min, respectively. The split
ratio was set to 1.0 mL/min for the ELSD detector. Data acquisition
was done by means of Chrompass 1.8.6.1 (Jasco, Groß-Umstadt,
Germany).
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Figure 3. Taste profile of fractions I−IV isolated from (A) raw and (B) cooked white asparagus (14 min at 95 °C). Sensory data are given as the
means of triplicates; error bars indicate the 95% confidence interval of the arithmetical mean.
Nuclear Magnetic Resonance Spectroscopy (NMR). One- and
descriptors were evaluated with lower scores in the cooked
asparagus as in the fresh sample.
1
two-dimensional H and 13C NMR spectra were acquired on a 500
MHz Bruker Avance III and a 400 MHz DRX spectrometer (Bruker,
Rheinstetten, Germany). Samples were analyzed in a mixture (95/5, v/
v) of DMSO-d6 and D2O. Chemical shifts are reported in parts per
In order to isolate the nonvolatile taste compounds, raw and
cooked asparagus spears were comprehensively extracted with
methanol and methanol/water. After separating the solvent in
vacuum and freeze-drying, the extracts prepared from both
asparagus samples were solubilized in bottled water in
concentrations of 3.95 (raw) and 1.86 g/100 mL (cooked) to
match the concentration in asparagus. After pH adjustment
(pH 5.9) with trace amounts of formic acid, the solutions were
presented to the sensory panelists, who were asked to evaluate
the intensities of given taste descriptors on a linear 5-point scale
(Figure 2). The methanol extractables from raw (+0.2) and
cooked asparagus (+0.3) showed somewhat higher bitter
intensities, but a decrease in the buttery mouth-coating
sensation (−0.8/−0.5). The lower intensities of this buttery
mouth-coating sensation might be explained by the instability
of the respective chemosensates during sample workup or by
mixture suppression due to the increased intensity of the
perceived bitterness.29−34 The following experiments were
focused on the identification of the molecules imparting the
bitterness and the typical buttery asparagus-like orosensation.
Solvent Fractionation of Raw and Cooked Asparagus.
To gain first insight into the hydrophobicity of the taste-active
molecules, the freeze-dried methanol extractables isolated from
fresh and cooked asparagus, respectively, were taken up in
water and separated by sequential solvent extraction affording
the pentane extractables (fraction I), the dichloromethane
extractables (fraction II), the ethyl acetate extractables (fraction
III), and the remaining water-solubles (fraction IV), respec-
tively. The highest yields were found for fraction IV accounting
for 86 and 66% of the fresh weight of raw and cooked asparagus
(Table 1). Fractions I, II, and III were obtained in
comparatively low yields of less than 2%.
1
million relative to the solvent signal (DMSO-d6: H: 2.49 ppm; 13C:
39.7 ppm). For structure elucidation and NMR signal assignment 2-D
NMR experiments, like COSY-, J-RESOLVE-, HMQC/HSQC-,
DEPT-, and HMBC-spectroscopy were carried out using the pulse
sequences taken from the Bruker software library. Data processing was
performed by using XWin-NMR software (version 3.5; Bruker,
Rheinstetten, Germany) and (Mestrelab Research, La Coruna,
̃
Spain), respectively.
RESULTS AND DISCUSSION
■
Preliminary sensory analysis of the fresh juice prepared from
white asparagus by mincing and pressing revealed a taste profile
centering around sweet, bitter, and astringent taste qualities and
was complemented by another orosensation described by the
trained panel as a typical white asparagus-like, buttery mouth-
coating effect, reminiscent to melting butter on the tongue.
Taste Profile Analysis of Raw and Cooked Asparagus.
In order to gain a first insight into the influence of cooking on
the taste profile of white asparagus, juices prepared from raw
and cooked peeled spears, respectively, were presented to the
trained sensory panel who were asked to rate the intensity of
the taste modalities astringent, bitter, sour, salty, sweet, umami,
and buttery mouth-coating on a linear 5-point intensity scale.
The asparagus-like, buttery mouth-coating sensation was
evaluated with highest scores of 2.8 and 2.2 in the raw and
cooked sample, respectively, and was judged as a lingering and
long-lasting orosensation (Figure 2), followed by sweetness
(2.5/1.8) and bitterness (1.9/1.0) with a somewhat lower taste
impact. In comparison, umami taste (0.7/0.5), saltiness (0.5/
0.4), and sourness (both 0.3) were rated with rather low
intensities. It is interesting to notice that the individual
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Figure 4. Taste profile of fractions III-A to III-D isolated from raw white asparagus. Sensory data are given as the means of triplicates; error bars
indicate the 95% confidence interval of the arithmetical mean.
Figure 5. RP-HPLC chromatograms and taste dilution (TD)-factors of SPE-fractions III-B (A), III-C (B), and III-D (C) prepared from white
asparagus.
To evaluate their taste impact, fractions I−IV isolated from
fresh and cooked asparagus, respectively, were taken up in
water (pH 5.9) containing 2% aqueous ethanol and were then
comparatively analyzed by means of taste profile analysis.
Sensory evaluation of the individual fractions revealed higher
intensities of the individual taste descriptors in raw than in
cooked asparagus (Figure 3). Independent on the thermal
treatment of asparagus, the typical buttery, asparagus-like
orosensation as well as the bitterness could be located primarily
in fractions III and IV, e.g., fractions III and IV exhibited
bitterness with an intensity of 1.7/0.8 and 1.5/1.1 for raw/
cooked asparagus while fractions I and II exhibited just a very
low bitterness (<0.7). Therefore the following experiments
were focused on the identification of the bitter and buttery
mouth-coating compounds in the taste-active fractions III and
IV isolated from raw asparagus.
Sensory-Directed Separation of Raw Asparagus
Fraction III. To sort out the bitter and mouth-coating
compounds from the bulk of less taste-active or tasteless
substances present in fraction III, the ethyl acetate extractables
isolated from raw asparagus were separated by means of solid
phase extraction on RP-18 material using methanol/water
mixtures to give fraction III-A (0/100, v/v), fraction III-B (30/
70, v/v), fraction III-C (70/30, v/v) and fraction III-D (100/0,
v/v), respectively.
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Figure 6. Excerpt of HMBC spectrum (400 MHz, DMSO) of 1,2-dithiolan-4-carboxylic acid 6-D-α-glucopyranose ester (1, A) and 1,2-dithiolan-4-
carboxylic acid 6-D-β-glucopyranose ester (2, B).
After separating the solvent in a vacuum, the individual
fractions were taken up in water in their natural concentration
ratios and were presented to the trained sensory panel which
was asked to rate the intensity of different taste modalities
astringent, bitter, sour, salty, sweet, umami and buttery mouth-
coating on a linear 5-point intensity scale (Figure 4). Fractions
III-C and III-D showed the highest bitter intensities of 4.0 and
2.5, respectively, whereas fractions III-A and III-B exhibited just
a rather faint bitterness. In comparison, the buttery mouth-
coating orosensation was detectable in all the fractions III-A to
III-D, among which the early eluting fractions III-A and III-B
were judged with the highest scores between 3.0 and 2.2
(Figure 4). As preliminary HPLC analysis revealed fractions III-
A and III-B to be substantially equivalent with higher levels of
polar carbohydrates in III-A, the following investigations were
focused on the less complex fraction III-B.
Taste Dilution Analysis of Fraction III-B. To trace the
compounds imparting the buttery mouth-coating effect, a taste
dilution analysis was applied on fraction III-B. To achieve this,
fraction III-B was separated by means of RP-HPLC coupled to
an evaporative light scattering detector (ELSD) to give a total
of 19 HPLC fractions, which were collected individually and
freeze-dried (Figure 5A). The lyophilizates of these fractions
were taken up with the same amount of bottled water (pH 5.9),
stepwise diluted 1:1 with water (pH 5.9), and then presented in
order of ascending concentrations to the trained sensory
panelists who were asked to determine their taste dilution
(TD)-factors by means of an half-tongue procedure. Fractions
III-B-12 and III-B-14 were identified with the highest TD-factor
of 1024 and were, therefore, considered as key contributors to
the buttery mouth-coating oral sensation of white asparagus.
After purifying the taste-active fractions III-B-12 and III-B-14
by means of semipreparative RP-HPLC, the chemical structures
of the respective chemosensates 1 and 2 were determined by
means of LC-MS/MS and 1D/2D-NMR experiments. It is
interesting to notice that isolation of the target compound 1 or
2 always led to a mixture of compounds 1 and 2 eluting in
fraction III-B-12 and III-B-14, thus indicating the existence of
two isomers. LC-TOF-MS analysis confirmed the target
compounds 1 and 2 to have the same molecular formula of
1
C10H16O7S2. The H NMR spectrum of this isomeric mixture
displayed two anomeric protons of a glycoside resonating at
4.27 and 4.87 ppm. On the basis of the coupling constants
between each anomeric and vicinal protons, one of the isomers
was identified as the α-anomer (4.87 ppm; 3.8 Hz), while the
other sugar indicated a β-configuration (4.27 ppm) with a
larger coupling constant of 7.9 Hz. The assignment of the sugar
moiety as well as the linkage of the sugar to the aglycone was
performed by COSY- and HMBC-experiments. For example,
the HMBC spectrum allowed the determination of the linkage
of the low-field shift protons H−C(6′) resonating at 4.02−4.10
ppm and 4.25−3.32 ppm to the carboxy carbon C(6) of
asparagusic acid by a long-range coupling (Figure 6). The
unequivocal identification of glucose as the glycosidically bound
carbohydrate was further confirmed by high-performance ion
chromatography after alkaline hydrolysis. Ethyl acetate
extraction of the alkaline hydrolysate of 1 and 2, followed by
solvent removal in vacuum revealed asparagusic acid (3; Figure
1) as the aglycone in glucosides 1 and 2 by means of LC-MS
and NMR analysis. LC-TOF-MS exhibiting m/z 148.974427
[C4H5O2−H+]− as the pseudomolecular ion ([M + H]−) and
1H/13C NMR spectra showing a total of five protons and four
carbon atoms was well in line with the elemental formula of
C4H6O2S2 for the aglycone. 1D/2D NMR data unequivocally
validated the structure of the aglycone as asparagusic acid (3),
which has been previously reported as a phytochemical in
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Figure 7. RP-HPLC-MS/MS (ESI−) analysis of purified saponin references 4a/b (A), 5a/b (B), 6a/b (C) and 7 (D), 8a/b (E), and fractions III (F)
and IV (G) isolated from asparagus.
asparagus16,17,35−37 (Figure 1). Taking all these data into
consideration, the taste active compounds 1 and 2 were
unequivocally identified as 1,2-dithiolan-4-carboxylic acid 6-D-
α-glucopyranoseester and 1,2-dithiolan-4-carboxylic acid 6-D-β-
glucopyranoseester (Figure 1), which to the best of our
knowledge have not been previously reported.
Taste Dilution Analysis of Fractions III-C and III-D. To
locate the major bitter compounds in raw asparagus fraction III,
the SPE fraction III-C and III-D were further separated by
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means of RP-HPLC/ELSD to give a total of 16 and 12
subfractions from III-C and III-D, respectively, which were used
for taste dilution analysis (Figure 5B/C). The highest TD-
factor of 1024 was found for the bitter fractions III-C-5 and III-
D-4, followed by fractions III-C-4, III-C-7, and III-C-10
exhibiting bitterness up to a dilution of 1:512. Next to their
bitter taste, fractions III-C-4 to III-C-6 exhibited oral
astringency with TD-factors between 32 and 512. Aimed at
discovering the molecules imparting the typical bitter taste of
white asparagus, the following identification experiments were
focused on the taste compounds located in fractions III-C-5 to
III-C-7 and III-D-4.
HPLC-MS/MS and 1D/2D-NMR analysis of the bitter
compounds in fraction III-C-5 led to the identification of an
epimeric mixture as 3-O-[α-L-rhamnopyranosyl-(1→2)-{α-L-
rhamnopyranosyl-(1→4)}-β-D-glucopyranosyl]-26-O-[β-D-glu-
copyranosyl]-(25R)-22-hydroxyfurost-5-ene-3β,26-diol (4a,
Figure 1), earlier reported as protodioscin in asparagus seed,3
and 3-O-[α-L-rhamnopyranosyl-(1→2)-{α-L-rhamnopyranosyl-
(1→4)}-β-D-glucopyranosyl]-26-O-[β-D-glucopyranosyl]-
(25S)-22-hydroxyfurost-5-ene-3β,26-diol (4b, Figure 1), which
has been coined protoneodioscin, AS-P2-I, or ASP-I,
respectively, and has been reported in the bottom cuts as
well as in the edible shoots of Asparagus officinalis L.1,3,38 Both
saponins, protodioscin (4a) and protoneodiscin (4b), were
earlier isolated also from the fresh stems of Dracaena
cochinchinensis.39
LC-MS and NMR analysis of the key bitter compounds in
fraction III-C-6, evaluated with a TD factor of 64, led to the
identification of the epimeric mixture of the bidesmosidic
saponin (25R/S)-furost-5-en-3β,22,26-triol-3-O-[α-L-rhamno-
pyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-glucopyrano-
side (5a/b) (Figure 1). Although the (25R)-epimer 5a was
reported earlier to be released from protodioscin upon
treatment with an enzyme from Curvularia lunata, to the best
of our knowledge has this saponin not yet been reported as a
bitter compound in asparagus. In contrast, its (25S)-epimer
(5b) was previously postulated in asparagus.1,40 Moreover, the
bitter compounds 6a and 6b isolated from HPLC fraction III-
C-7 could be identified as (25R)-furostane-3β,22,26-triol-3-O-
[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-
glucopyranoside (6a) and (25S)-furostane-3β,22,26-triol-3-O-
[α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside]-26-O-β-D-
glucopyranoside (6b, Figure 1).
also present in the bitter tasting aqueous fraction IV (Figure 3),
fractions prepared from raw and cooked asparagus, respectively,
were screened for the bitter compounds 4a/b−8a/b by means
of RP-HPLC-MS/MS (ESI−) operating in the multiple reaction
monitoring (MRM) mode. As postfragmentation in the ion
source of compound 4a/b was found to deliver the same
daughter ions as analytes 5a/b and 6a/b (Figure 7), HPLC
conditions were optimized to achieve full baseline separation of
the saponins 4a/b−8a/b. This helped also to overcome
problems regarding the spectral-overlap of the mass transitions
901.6 → 755.5 and 903.5 → 757.4 of the precursor ions of 5a/
b and 6a/b, respectively, showing only a 2 Da difference in
mass/charge ratio. Comparison of retention times and
characteristic mass transitions, followed by cochromatography
with the purified compounds isolated from fraction III led to
the unequivocal identification of the polar saponins 4a/b−6a/b
in the bitter fraction IV of asparagus, while the less polar,
monodesmotic saponins 7 and 8a/b could not be detected
(Figure 7). To the best of our knowledge, the saponins 4a/b−
8a/b have not been previously reported as key bitter taste
compounds in white asparagus spears.
Oral Threshold Concentrations of Asparagusic Acid
Derivatives. Prior to sensory analysis, the purity of the test
compounds was checked by HPLC-ELSD as well as HPLC-MS
to be more than 98%. To determine the recognition threshold
concentration for the buttery mouth-coating and astringent oral
sensation of the glucosides 1/2 and the aglycone asparagusic
acid (3), aqueous solutions (pH 5.9) of the target compounds
were evaluated by means of the half-tongue test.24,25 The
mixture of 1,2-dithiolan-4-carboxylic acid 6-D-glucopyranose
esters 1 and 2 in bottled water was found to exhibit a low
recognition threshold of 276.8 μmol/L (Table 3) and imparted
Table 3. Human Recognition Taste Thresholds of
Compounds 1−3
threshold concentrations [μmol/L] of
a
compound
1/2
3
b
buttery mouth-coating
astringent
276.8
b
132.9
a
b
Structures of compounds are given in Figure 1. Taste threshold
concentrations were determined in aqueous solution.
In addition to the saponins 4a/b−6a/b, the bitter taste of the
comparatively hydrophobic fraction III-D-4 was found to be
due to 3-O-[β-D-glucopyranosyl-(1→2)-β-D-xylopyranosyl-(1→
4)-β-D-glucopyranosyl]-(25S)-5β-spirostan-3β-ole (AS-1) (7,
Figure 1) and the epimeric mixture of 3-O-[α-L-rhamnopyr-
anosyl-(1→2)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyrano-
syl]-(25R/S)-spirost-5-en-3β-ol (8a/b, Figure 1). While com-
pound 7 was previously isolated from asparagus bottom
cuts,38,41 to the best of our knowledge this saponin has not
been reported as a bitter compound in asparagus spears.
Although 8a was recently isolated from asparagus bottom
cuts,12 the epimeric dioscin (8b) has just been reported in
other plant families such as, e.g., Dioscorea.38 The details of the
isolation and full spectroscopic structure determination
demonstrating the chemical identity of these bitter saponins
4a/b−8a/b are published separately in a companion paper.28
LC-MS/MS Screening of Bitter Saponins 4a/b−8a/b in
Asparagus Fractions. In order to answer the question as to
whether the saponins 4a/b−8a/b identified in fraction III are
a lingering orosensation with described as a buttery, typical
asparagus-like mouth-coating effect, reminiscent of the
perception of melting butter on the tongue. Interestingly, a
solution of asparagusic acid (3) in bottled water (pH 5.9) was
described as astringent only with a recognition threshold
concentration of 132.9 μmol/L, implying the prime importance
of the glucosylation of aspargusic acid for the buttery mouth-
coating activity of 1 and 2.
In conclusion, sensory-guided fractionation of asparagus
extracts led to the identification of two previously not reported
1,2-dithiolan-4-carboxylic acid 6-D-glucopyranose esters 1 and 2
as chemosensates imparting a buttery mouth-coating orosensa-
tion. Furthermore a series of saponins (4a/b−8a/b) were
found to impart the bitter taste of white asparagus. Aimed at
demonstrating their contribution to the taste of raw and cooked
asparagus, quantitative studies are currently in progress and will
be published elsewhere.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-8161/71-2902. Fax: +49-8161/71-2949. E-mail:
thomas.hofmann@tum.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
̈
We are grateful to the Julius Kuhn-Institut, Institut fur
̈
̈
Okologische Chemie, Pflanzenanalytik und Vorratsschutz
(Quedlinburg, Germany) for providing asparagus samples.
We further thank H. Luftmann, I. Otte and S. Kaviani-Nejad for
the exact mass measurements.
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