2-(1H-pyrrolyl)carboxylic acids as pigment precursors in garlic greening

Article abstract of DOI:10.1021/jf073025r

Six model compounds having a 2-(1H-pyrrolyl)carboxylic acid moiety and a hydrophobic R group were synthesized to study their effects on garlic greening, the structures of which are similar to that of 2-(3,4-dimethyl-1H-pyrrolyl)-3- methylbutanoic acid (PP

Full text of DOI:10.1021/jf073025r

J. Agric. Food Chem. 2008, 56, 1495–1500 1495
2-(1H-Pyrrolyl)carboxylic Acids as Pigment
Precursors in Garlic Greening
DAN WANG, HUSILE NANDING, NA HAN, FANG CHEN, AND GUANGHUA ZHAO*
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
Six model compounds having a 2-(1H-pyrrolyl)carboxylic acid moiety and a hydrophobic R group
were synthesized to study their effects on garlic greening, the structures of which are similar to that
of 2-(3,4-dimethyl-1H-pyrrolyl)-3-methylbutanoic acid (PP-Val) (a possible pigment precursor for garlic
greening). The puree of freshly harvested garlic bulbs turned green after being soaked in solutions
of all these compounds, and with both increasing concentrations and incubation time the green color
of the puree became deeper. In contrast, neither pyrrole alone nor pyrrole combined with free amino
acids had the ability to discolor the puree. The compounds exhibited a good relationship between
structure and activity of garlic greening, namely, the smaller the size of the R group, the larger the
contribution. Also, it was found that the unidentified yellow species can be produced by reacting the
model compounds with pyruvic acid at room temperature (23–25 °C). Moreover, blue species were
formed by incubation of the model compounds with di(2-propenyl) thiosulfinate at room temperature.
On the basis of these observations, a pathway for garlic greening was proposed.
KEYWORDS: 2-(1H-Pyrrolyl)carboxylic acids; pigment precursor; garlic greening; pyruvic acid; di(2-
propenyl) thiosulfinate; thiosulfinate
INTRODUCTION
solution of laba garlic is proportional to the formation of the
pigments (2). The third step represents the formation of pigment
via a reaction between the pigment precursor(s) and unidentified
compound(s). Recently, Imai et al. prepared 2-(3,4-dimethyl-
1H-pyrrolyl)-3-methylbutanoic acid (PP-Val) and 2-(3,4-di-
methyl-1H-pyrrolyl)propanoic acid (PP-Ala) (Figure 1) by
reacting di(1-propenyl) thiosulfinate with L-valine and L-alanine,
respectively, and found that the PP-Val can react with di(2-
propenyl) thiosulfinate into pigments at 100 °C in vitro (9), so
PP-Val and its derivatives with 2-(1H-pyrrolyl)carboxylic acid
moiety were considered to be the pigment precursor(s). Although
PP-Val and its analogues might act as pigment precursors to
garlic greening, more evidence is needed to verify this idea
because only in vitro results based on model systems do not
yet adequately support the above mechanism.
Garlic greening is a major concern during garlic processing
because it limits commercial utilization and reduces economic
value (1), but it is required and desirable for the preparation of
“laba” garlic, a traditional homemade Chinese food product (2, 3).
Despite having been studied for about 50 years, garlic greening
is still poorly understood. On the basis of previous results, it is
established that the green discoloration of garlic purees occurs
only with garlic which has a high enough content of S-(1-
propenyl)-L-cysteine sulfoxide (1-PeCSO) and is similar to the
pink discoloration of onion, which is a multistep process (4–8).
The first step corresponds to the production of an ether-soluble
organosulfur compound, which is also called “color developer”,
under the action of alliinase on 1-PeCSO. Recent studies showed
that the color developer could be di(1-propenyl) thiosulfinate
(9) or 1-propenyl-containing thiosulfinate (10). Support for this
conclusion comes from previous results showing that the amino
acid 1-PeCSO was necessary for the development of the garlic
green color (11). Also, consistent with this finding, recent results
obtained by Kubec et al. indicated that, at pH 5.5, a dark blue
pigment with a maximal absorbance at 582 nm was formed in
a system containing 1-PeCSO, S-(2-propenyl)-L-cysteine sul-
foxide (2-PeCSO), glycine, and alliinase (8). The second step
corresponds to the formation of pigment precursor(s) by a
reaction between thiosulfinates and amino acids; evidence has
been presented that thiosulfinate consumption in the pickling
Lee et al. tried to isolate and purify a green pigment from a
garlic homogenate, but they were not successful as judged from
the reported 13C NMR spectrum (12). Recent studies from two
other groups suggested that the green pigment(s) responsible
forgarlicgreeningarecomposedofyellowandbluespecies(2,10).
It has been well-characterized that 2 mol of S-alk(en)yl-L-
cysteine sulfoxides was catalyzed by alliinase into 1 mol of
corresponding thiosulfinates, 2 mol of pyruvic acid, and 2 mol
of ammonia (13), demonstrating that pyruvic acid was produced
accompanied by the formation of thiosulfinates. Therefore,
during the formation of PP-Val from the reaction of di(1-
propenyl) thiosulfinate with L-valine (9), a certain amount of
pyruvic acid must be produced. This opens the question of
* Corresponding author (telephone +86-10-62737645; fax +86-10-
62737434-11; e-mail gzhao318@yahoo.com.cn).
10.1021/jf073025r CCC: $40.75
2008 American Chemical Society
Published on Web 01/16/2008

1496 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Wang et al.
as a solvent with tetramethylsilane (TMS) as an internal standard. Mass
spectra were obtained by using LC-MS/MS (Alliance2695/Quattro
Micro API, Waters Co.), and detection was performed in the negative
mode.
Plant Materials. Freshly harvested (May 2007) garlic bulbs were
obtained from a local market at China Agriculture University, stored
at room temperature, and used immediately for the following
experiments.
Preparation of Garlic Purees and Measurement of Their UV-Vis
Absorption or Spectra. After the garlic bulbs had been cracked,
shriveled, damaged, and small cloves were discarded; the remaining
cloves were peeled and rinsed with distilled water three times. Garlic
(420 cloves) was homogenized in a blender. The resulting materials
were equally divided into 14 parts. Eight parts were soaked in 20 mL
of 2.5 mM acetic acid solution, 2.5 mM pyrrole solution, and 2.5 mM
pyrrole combined with six free amino acids (glycine, alanine, valine,
leucine, isoleucine, and phenylalanine, 2.5 mM) solution as controls,
respectively. The remaining six parts were immersed in 20 mL of P-Gly,
P-Ala, P-Val, P-Ile, P-Leu, and P-Phe, respectively, at four different
concentrations (1.0, 2.5, 5.0, and 10.0 mM) for up to 15 days of
incubation at room temperature (23–25 °C). For P-Ile, P-Leu, and P-Phe,
only two different concentrations, 1.0 and 2.5 mM, were used to pickle
garlic puree due to limited solubility in water. The final pH for all
samples was in the range of 4.0-5.0. All experiments were performed
in triplicate.
Figure 1. Structures of pigment precursors 2-(3,4-dimethyl-1H-pyrrolyl)-3-
methylbutanoic acid (PP-Val) and 2-(3,4-dimethyl-1H-pyrrolyl)propanoic acid
(PP-Ala) and their six model compounds, 2-(1H-pyrrolyl)carboxylic acids.
whether pyruvic acid could react with PP-Val or its analogues
to produce pigment(s).
In this study, a series of pyrrole derivatives similar to PP-
Val and PP-Ala in structure were prepared to obtain insights
into the mechanism of garlic greening. At the same time, the
possible pathways of the formation of yellow and blue species
in which these pyrrole derivatives were involved were also
explored. It was found that all of them can facilitate garlic
greening but to different degrees upon their addition to garlic
puree prepared from freshly harvested garlic. Moreover, these
model compounds can react with pyruvic acid and di(2-
propenyl) thiosulfinate to form unidentified yellow and blue
pigments, respectively.
After 1 day of incubation, all resultant mixtures were centrifuged at
10000g for 10 min at 4 °C. The residues produced were kept for further
use, and the resulting supernatant was collected, 1 mL of which was
placed into a quartz cuvette for UV-vis spectral measurement. After
UV–vis measurement, the 1 mL aliquot was recombined with the
supernatant and the residual for further incubation. The same procedure
was repeated every day at the same time as above until the 16th
day.
Reactions between 2-(1H-Pyrrolyl)carboxylic Acids and Pyruvic
Acid. Both pyruvic acid (0.284 mM) and P-Gly (0.284 mM) in
methanol were mixed thoroughly with a volumetric ratio of 1 to 1.
The resulting mixture was allowed to stand at room temperature (23–25
°C) for 5 min, and 1 mL of the reaction solution was put into a quartz
cuvette for UV-vis spectral measurement. The reactions of other
compounds containing P-Ala, P-Val, P-Ile, P-Leu, or P-Phe with pyruvic
acid were carried out with the same procedure used for P-Gly. All of
the chemicals had good solubility in methanol.
MATERIALS AND METHODS
Chemicals. L-Glycine, L-alanine, L-valine, L-leucine, L-isoleucine,
L-phenylalanine, and pyrrole were purchased from Xinjingke Biotech-
nological Co. (Beijing, China). 2,5-Dimethoxytetrathydrofuran and
diallyl disulfide were obtained from Fluka (Beijing, China). Methanol,
hydrochloric acid, acetic acid, sodium acetate anhydrous, ethyl acetate,
sodium sulfate anhydrous, ethanol, and potassium hydroxide were
purchased from Beijing Chemistry Co. (Beijing, China). Pyruvic acid
was obtained from Sinopharm Chemical Reagent Co. (Beijing, China).
All solvents/chemicals used were of analytical grade or purer.
All 2-(1H-pyrrolyl)carboxylic acids containing 2-(1H-pyrrolyl)acetic
acid (P-Gly), 2-(1H-pyrrolyl)propanoic acid (P-Ala), 2-(1H-pyrrolyl)-
3-methylbutanoic acid (P-Val), 2-(1H-pyrrolyl)-4-methylvaleric acid
(P-Leu), 2-(1H-pyrrolyl)-3-methylvaleric acid (P-Ile), and 2-(1H-
pyrrolyl)-3-phenylpropanoic acid (P-Phe) were synthesized by reacting
corresponding amino acids with 2,5-dimethoxytetrahydrofuran as
previously described (14, 15) except for purification procedures. Briefly,
sodium acetate anhydrous (2.0 g) and corresponding amino acids (27.2
mmol) were dissolved in 50 mL of glacial acetic acid. 2,5-Dimethoxy-
tetrahydrofuran (27.2 mmol) was added to the above solution. The
mixture was stirred under reflux for 40 min and then was poured into
300 mL of ice–water. The resultant solution was extracted with ethyl
acetate. After the solvent was removed by rotary evaporation, the resulting
residual was dissolved in water, and its pH was adjusted to 7.0. The
resulting solution was acidified by hydrochloric acid (2.0 M) to pH in the
rage of 1.0-2.0. The resulting precipitate or oil was collected for 1H NMR
and MS measurements. The identity and purity of the synthesized
compounds were checked by 1H NMR and MS. The di(2-propenyl)
thiosulfinate was synthesized as previously described (16).
Reactions between 2-(1H-Pyrrolyl)carboxylic Acids and Di(2-
propenyl) Thiosulfinate. Di(2-propenyl) thiosulfinate (0.246 mM) and
P-Gly (0.246 mM) in methanol were mixed with a volumetric ratio of
1 to 1 followed by standing at room temperature (23–25 °C) for 5 min.
Then, 1 mL of the reaction solution was placed into a quartz cuvette
for UV-vis spectral measurement. Other reactions between other
compounds containing P-Ala, P-Val, P-Ile, P-Leu, P-Phe, and di(2-
propenyl) thiosulfinate were carried according to the same procedure
as used for P-Gly. All compounds are soluble in methanol.
Preparation of Green Garlic Pickling Solution and Separation
of Yellow and Blue Species. Freshly harvested garlic bulbs were stored
at 4 °C for 2 months. After the garlic bulbs had been cracked, garlic
cloves were peeled, rinsed with tap water, and then triple rinsed with
distilled water three times. The peeled cloves (500 g) were pickled in
500 mL of acetic acid solutions (5%, v/v) at pH 2.0 for 4 days. The
resulting green pickling solution was filtered and passed through an
Amberlite CG-50 cation exchange column (2.6 × 50 cm) eluted with
ethanol/acidic water (1:9 v/v) at 5 mL/min. Eluent (70–80 mL) with a
green color was collected and concentrated to 5 mL by ultrafilitration.
The resulting solution was subjected to an LH-20 column (1.6 × 75
cm), which was eluted with methanol at 0.3 mL/min. The resulting
yellow and blue fractions were collected and concentrated to 2 mL,
respectively, for UV-vis spectral measurements.
Statistical Analysis. Statistics on a completely randomized design
were determined using SAS 9.0 for Windows. Duncan’s multiple-range
test (p < 0.05) was used to determine significance of differences
between means.
Instrumentals. UV-visible spectra were recorded with a Cary 50
UV–vis spectrophotometer (Varian Co.). 1H NMR spectra were obtained
on a dpx-300 MHz NMR spectrometer (Brucker Co.). DMSO was used

Garlic Greening and 2-(1H-Pyrrolyl)carboxylic Acids
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1497
Figure 2. (A) Photographs and (B) UV–vis spectra of green color formation
of puree of freshly harvested garlic cloves immersed in 2-(1H-pyrrolyl)acetic
acid (P-Gly) for 7 days at various concentrations (b, 1 mM; c, 2.5 mM; d,
5 mM; e, 10 mM). No color was formed when the freshly harvested garlic
was soaked in 2.5 mM acetic acid solution, 2.5 mM pyrrole, or 2.5 mM
pyrrole plus 2.5 mM six free amino acids for 7 days (a), respectively.
Figure 3. Effects of P-Gly at four different concentrations (1.0, 2.5, 5.0,
and 10.0 mM) on the green discoloration of puree made from freshly
harvested garlic. The garlic greening was detected at both 590 nm (A)
and 440 nm (B). Each point represents a separate sample, and its value
is the average of three independent measurements. Vertical bars represent
the standard deviation.
RESULTS
On the basis of their 1H NMR spectra and MS data
(Supporting Information), the structures of six model compounds
were identified as 2-(1H-pyrrolyl)carboxylic acids. Melting
points of P-Gly and P-Phe were 84–87 and 90–93 °C,
respectively, which are nearly identical to those (P-Gly, 85–88
°C; P-Phe, 90–93 °C) reported in the literature (14, 15),
confirming the purity of these synthesized compounds. All of
them have a structure similar to that of PP-Val (Figure 1) and
just lack two methyl groups located on the pyrrole moiety as
compared to PP-Val. In addition, these model compounds are
soluble in water and methanol, but P-Leu, P-Ile, and P-Phe have
relatively lower solubility in water with respect to P-Gly, P-Ala,
and P-Val. Therefore, only two concentrations, 1.0 and 2.5 mM,
were used for making P-Leu, P-Ile, and P-Phe samples
throughout this study.
PP-Val and their derivatives with 2-(1H-pyrrolyl)carboxylic
acid moiety could be pigment precursors during the discoloration
of onion and garlic (9). If PP-Val were a pigment precursor,
freshly harvested garlic puree would also turn green upon
addition of these model compounds. First, P-Gly was chosen
to add to puree of freshly harvested garlic because it has the
simplest structure among the six compounds. As expected, the
garlic purees became green after being immersed in P-Gly
solution at four different concentrations (1.0, 2.5, 5.0, and 10.0
mM), and with increasing P-Gly concentration, the green color
became deeper and deeper after 7 days incubation (Figure 2A).
Their corresponding UV–vis spectra are displayed in Figure
2B. It was observed that there are two maximum absorptions
at ca. 440 and 600 nm, which are in good agreement with the
UV–vis absorptions (440 and 590 nm) of pigment(s) responsible
for garlic greening reported in the literature (2, 8, 10). Similarly,
puree of freshly harvested garlic also became pronouncedly
green when P-Ala and P-Val were used instead of P-Gly. In
contrast, the degree of garlic greening became less when the
other three compounds were used instead under the same
experimental conditions (data not shown). However, neither
pyrrole alone nor the mixture of pyrrole and free amino acids
had the ability to discolor the puree under the same conditions
(data not shown), indicating that both pyrrole and carboxylic
moieties were necessary for the structure of the pigment
precursor(s).
The above results strongly suggested that a compound
including the 2-(1H-pyrrolyl)carboxylic acid unit is involved
in garlic greening. This finding encouraged further investigation
into the relationship between these pyrrole derivatives and garlic
greening. Therefore, the kinetics of garlic greening was studied
using UV–vis spectrophotometry upon addition of these com-
pounds at four different concentrations (1.0, 2.5, 5.0, and 10.0
mM). Consistent with the above observation, garlic greening
became stronger and stronger with time upon addition of P-Gly
to garlic puree. In addition, it was evident that the degree of
garlic greening is a function of its concentration (Figure 3). A
similar profile was observed with P-Ala and P-Val, whereas
the other compounds, P-Ile, P-Leu, and P-Phe, turned garlic
puree green to a lesser extent (data not shown). These results
suggest that these compounds might exhibit a different effect
on garlic greening.
To clarify their contributions to garlic greening, garlic puree
was immersed in a series of solutions containing 2.5 mM
concentrations of the six model compounds, respectively (Figure

1498 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Wang et al.
Figure 6. UV–vis difference spectra of the formation of blue species by a
reaction between three synthesized derivative compounds (P-Gly, P-Ala, and
P-Val) and di(2-propenyl) thiosulfinate. The concentrations of both the six
compounds and di(2-propenyl) thiosulfinate are 0.246 mM in methanol.
Figure 4. Effects of six 2-(1H-pyrrolyl)carboxylic acids (2.5 mM) on the
greening discoloration of puree made from freshly harvested garlic. Garlic
greening was detected at both 590 nm (A) and 440 nm (B). Each point
represents a separate sample, and its value is the average of three
independent measurements. Vertical bars represent the standard deviation.
Figure 7. (A) Photograph of two solutions containing blue (a) and yellow (c)
species separated from green pickling solution (b) of “Laba” garlic. (B) UV-vis
spectra of the three solutions (a, b, and c). Green garlic-pickling solution (b)
was diluted 10 times prior to UV-vis measurement.
discoloration. A similar result was also found with the absorption
at 440 nm, which characterized yellow species also related to
garlic greening (2) except that P-Val and P-Gly switched their
positions in the ability to make garlic puree yellow (Figure 4B).
Thus, among the compounds, P-Gly makes the largest contribu-
tion to the formation of the blue species, whereas P-Val has
the largest effect on the production of the yellow species.
Recent studies have shown that green pigments responsible for
garlic greening might consist of yellow and blue species (2, 10).
To explore the pathway of the formation of yellow pigment(s),
reactions between these PP-Val model compounds and pyruvic
acid [a product accompanied with the formation of di(1-
propenyl) thiosulfinate from 1-PeCSO by alliinase] were studied.
It was found that these compounds can react with pyruvic acid
to produce unidentified yellow species with a maximum UV–vis
absorption in the range of 418-460 nm. The order of UV–vis
absorption intensity of the yellow species at 440 nm is as
Figure 5. UV–vis difference spectra of the formation of yellow species
by a reaction between six synthesized compounds (P-Gly, P-Ala, P-Val,
P-Ile, P-Leu, and P-Phe) and pyruvic acid. The concentrations of both
the six compounds and pyruvic acid equal 0.284 mM in methanol.
4). Because pigments related to garlic greening have two
maximum absorptions at 440 and 590 nm, the two absorptions
were used as detection wavelengths (2, 8). Interestingly, there
is a good relationship between garlic greening and structure,
namely, garlic greening correlates with molecular size (Figure
1). With increasing size of the R group from H- (P-Gly), CH3-
(P-Ala), (CH3)2CH- (P-Val), CH3CH2CH(CH3)- (P-Ile), or
(CH3)2CHCH2- (P-Leu) to benzyl (P-Phe), the absorption at
590 nm generally decreased (Figure 4A), indicating that the
compounds with larger R groups made less of a contribution to
the production of blue pigments, which are involved in garlic

Garlic Greening and 2-(1H-Pyrrolyl)carboxylic Acids
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1499
Figure 8. Proposed six-step pathway responsible for garlic greening.
DISCUSSION
follows: P-Ala > P-Val > P-Gly > P-Ile > P-Leu > P-Phe
(Figure 5), generally consistent with the order of these
compounds indicative of their contributions to the absorption
at 440 nm related to garlic greening (Figure 4B). This result
supports the proposed pathway of the yellow species production.
Likewise, the possible pathway correlated with the formation
of the blue species was also studied. The UV–vis spectra of the
reaction solution of the six compounds and di(2-propenyl)
thiosulfinate are shown in Figure 6. It was found that a reaction
solution of P-Gly and di(2-propenyl) thiosulfinate exhibits the
deepest blue color, the maximum absorption of which is at 608
nm (Figure 6). A reaction solution of di(2-propenyl) thiosul-
finate and P-Ala also showed a blue color with a maximal
UV–vis absorption at 602 nm, whereas the mixture of di(2-
propenyl) thiosulfinate and P-Val had a very weak blue color
with an absorption at about 594 nm (Figure 6). In contrast, no
color change was observed with P-Ile, P-Leu, or P-Phe upon
incubation with di(2-propenyl) thiosulfinate under the same
conditions. These results suggested that only P-Gly, P-Ala, and
P-Val can react with di(2-propenyl) thiosulfinate to form the
blue species under this condition.
A large body of work demonstrated that garlic greening
occurs only with aged garlic, which is typically stored at low
temperature (10 °C or lower) for more than 1 month (1–3, 11, 17).
Later, it was found that this kind of storage can markedly increase
the amount of 1-PeCSO depending on storage time. Moreover,
1-PeCSO was necessary for the development of garlic greening
on the basis of the observation that the degree of garlic greening
was proportional to the concentration of 1-PeCSO (11, 17). Support
for this conclusion came from a recent paper showing that a
dark blue pigment with a maximal absorbance was formed in a
system containing 1-PeCSO, 2-PeCSO, glycine, and alliinase;
pink and magenta pigments were produced, respectively, in a
model system consisting of 1-PeCSO and alliinase and in
another system of 1-PeCSO, glycine, and alliinase (8). Only
1-propenyl-containing thiosulfinates can react with amino acids
to form pigments (10). All of these results emphasize the
importance of 1-PeCSO for garlic greening. However, the
pathways in which 1-PeCSO participates in the formation of
green pigment(s) are still poorly understood.
Di(1-propenyl) thiosulfinate, a product from the catalytic conver-
sion of 1-PeCSO by alliinase, can react with L-valine or L-alanine
in vitro to produce 2-(3,4-dimethyl-1H-pyrrolyl)-3-methylbutanoic
acid (PP-Val) and 2-(3,4-dimethyl-1H-pyrrolyl)propanoic acid (PP-
Ala) (Figure 1), and PP-Val can react with di(2-propenyl)
thiosulfinate, another thiosulfinate, to form pigment(s) (9). This
finding raises the possibility that a compound containing the 2-(1H-
pyrrolyl)carboxylic acid moiety could act as a precursor to the
pigment(s) involved in garlic greening.
Recent studies suggested that green pigments are a mixture
consisting of yellow and blue species (2, 10). Agreeing with
this idea, yellow and blue pigments were obtained from the
green pickling solution of “laba” garlic (2) as shown in Figure
7. A photograph of solutions containing yellow pigment(s) and
blue pigment(s) separated from green garlic pickling solution
is displayed in Figure 7A. Corresponding UV-vis spectra of
the three different solutions are given in Figure 7B. It was
observed that a solution containing yellow species only have
one visible absorption at about 440 nm in the visible region,
whereas a solution containing blue species exhibited a visible
absorption at about 590 nm, demonstrating that green pigments
responsible for garlic greening are a mixture of yellow and blue
species (2, 10).
The present in vivo study confirms the above proposed
pathways of the pigment precursor formation, namely, that
1-PeCSO was first converted into di(1-propenyl) thiosulfinate
by alliinase and the produced thiosulfinate subsequently reacted
with amino acids in the absence of enzyme to form a compound

1500 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Wang et al.
containing a 2-(1H-3,4-dimethyl-1H-pyrrolyl)carboxylic acid
unit called the pigment precursor (PP). If a compound containing
the 2-(1H-pyrrolyl)carboxylic acid moiety were not PP, it would
not turn puree (which was made from freshly harvested garlic
bulbs) green. This was not the case, and the addition of the
synthesized model compounds to the garlic puree, from freshly
harvested garlic bulbs, resulted in garlic greening to a different
extent depending on their structures. It seems there is a good
relationship between the structure and activity of these com-
pounds, that is, the smaller the side chain size of the model
compounds, the stronger the garlic greening (Figures 3 and 4).
This result suggests that the synthesized compound with a
smaller size side chain could contribute more to the formation
of the final pigment.
Supporting Information Available: 1H NMR spectra and MS
data of six compounds including P-Gly, P-Ala, P-Val, P-Leu,
P-Ile, and P-Phe. This material is available free of charge via
the Internet at http://pubs.acs.org.
LITERATURE CITED
(1) Adams, J. B.; Brown, H. M. Discoloration in raw and processed fruits
and vegetables. Crit. ReV. Food Sci. Nutr. 2007, 47, 319–333.
(2) Bai, B.; Chen, F.; Wang, Z.; Liao, X.; Zhao, G.; Hu, X.
Mechanism of the greening color formation of “Laba” garlic, a
traditional homemade Chinese food product. J. Agric. Food Chem.
2005, 53, 7103–7107.
(3) Bai, B.; Li, L.; Hu, X.; Wang, Z.; Zhao, G. Increase in the
permeability of tonoplast of garlic (Allium satiVum) by monocar-
boxylic acids. J. Agric. Food Chem. 2006, 54, 8103–8107.
(4) Joslyn, M. A.; Sano, T. The formation and decomposition of green
pigment in crushed garlic tissue. Food Res. 1956, 21, 170–183.
(5) Shannon, S.; Yamaguchi, M.; Howard, F. D. Precursors involved
in the formation of pink pigments in onion purees. J. Agric. Food
Chem. 1967, 15, 423–426.
(6) Shannon, S.; Yamaguchi, M.; Howard, F. D. Reactions involved
in formation of a pink pigment in onion purees. J. Agric. Food
Chem. 1967, 15, 417–422.
(7) Imai, S.; Akita, K.; Tomotake, M.; Sawada, H. Model studies on
precursor system generating blue pigment in onion and garlic. J.
Agric. Food Chem. 2006, 54, 848–852.
(8) Kubec, R.; Hrbácˇová, M.; Musah, R. A.; Velíše, J. Allium
discoloration: precursors involved in onion pinking and garlic
greening. J. Agric. Food Chem. 2004, 52, 5089–5094.
(9) Imai, S.; Akita, K.; Tomotake, M.; Sawada, H. Identification of
two novel pigment precursors and a reddish-purple pigment
involved in the blue-green discoloration of onion and garlic. J.
Agric. Food Chem. 2006, 54, 843–847.
(10) Kubec, R.; Velíšek, J. Allium discoloration: the color-forming
potential of individual thiosulfinates and amino acids: structural
requirements for the color-developing precursors. J. Agric. Food
Chem. 2007, 55, 3491–3497.
Because of the formation of the pigment precursor by the
reaction of di(1-propenyl) thiosulfinate with amino acids (9), the
present study also provided some information on the contribution
of these amino acids to garlic greening. The amino acids with a
smaller size side chain such as glycine, alanine, and valine make
a bigger contribution to garlic greening as compared with other
analogues with a larger size side chain including leucine, isoleu-
cine, and phenylalanine. Agreeing with this observation, glycine,
alanine, and valine have been found to be involved in the formation
of blue and red pigments (8, 9). Further support comes from recent
studies showing that a species produced from reacting glycine or
alanine or valine with 1-propenyl-containing thiosulfinate has a
stronger color than that from a reaction of leucine or isoleucine or
phenylalanine with 1-propenyl-containing thiosulfinate (10). How-
ever, it is possible that blue pigment might be produced by reaction
between P-Ile, P-Leu, and P-Phe and di(2-propenyl) thiosulfinate
under other conditions including increasing the incubation time or
the temperature.
In addition, the present study proposed a possible pathway for
garlic greening, which likely contains six steps as depicted in
Figure 8. Step 1 was described in a previous paper and corresponds
to the catalytic conversion of 1-PeCSO under the action of alliinase
into di(1-propenyl) thiosulfinate (9, 13, 16). Step 2 represents the
formation of the pigment precursors by the reaction of the produced
di(1-propenyl) thiosulfinate with amino acids. The role of the
pigment precursors was proven by the fact that 2-(1H-pyrroly)car-
boxylic acids can turn garlic puree (which was prepared from
freshly harvested garlic bulbs) green. Step 3 is a reaction similar
to step 1, corresponding to the production of di(2-propenyl)
thiosulfinate from 2-PeCSO catalyzed by alliinase. The yellow
species was produced in step 4 by the reaction of the pigment
precursor and pyruvic acid, another product from step 1. In parallel,
a reaction between the pigment precursor and di(2-propenyl)
thiosulfinate representing step 5 also occurs accompanying step 4
to form unidentified blue pigment(s). Step 6 corresponds to another
possible pathway related to the production of yellow pigment(s)
(2). Different from the yellow species produced in step 4, yellow
species formed through step 6 stem from the degradation of blue
species produced in step 5 (2). The mixing of both yellow and
blue pigments finally leads to garlic greening. In accordance with
this idea, yellow and blue pigments were obtained from the green
pickling solution of laba garlic (Figure 7). This conclusion is also
in accordance with recent observations (2, 10). Although the above
six-step pathway was proposed, other pathways associated with
garlic greening also probably coexist because garlic greening is a
very complex process and many compounds occurring in garlic
may attend this process in a different way (8, 10). However, it is
likely that there is a predominant pathway related to garlic greening
which needs to be determined in future study.
(11) Lukes, T. M. Factors governing the greening of garlic puree. J.
Food Sci. 1986, 51, 1581–1582.
(12) Lee, E. J.; Cho, J. E.; Kim, J. H.; Lee, S. K. Green pigment in
crushed garlic (Allium satiVum L.) cloves: purification and partial
characterization. Food Chem. 2007, 101, 1677–1686.
(13) Imai, S.; Tsuge, N.; Tomotake, M.; Nagatome, Y.; Sawada, H.;
Nagata, T.; Kumagai, H. An onion enzyme that makes the eyes
water. A flavoursome, user-friendly bulb would give no cause
for tears when chopped up. Nature 2002, 419, 685.
(14) Gloede, J.; Poduška, K.; Gross, H.; Rudinger, J. Amino acids and
peptides. LXXIX. R-Pyrrolo analogues of R-amino acids. Collect.
Czech. Chem. Commun. 1968, 33, 13071314..
(15) Sircar, I.; Winters, R. T.; Quin J., III; Lu, G. H.; Major, T. C.;
Panek, R. L. Nonpeptide angiotensin II receptor antagonists. 1.
Synthesis and in vitro structure-activity relationships of 4-[[[(1H-
pyrrol-1-ylacetyl)amino]phenyl]methyl] imidazole derivatives as
angiotensin II receptor antagonists. J. Med. Chem. 1993, 36, 1735–
1745.
(16) Shen, C.; Parkin, K. L. In Vitro biogeneration of pure thiosulfinates and
propanethial-S-oxide. J. Agric. Food Chem. 2000, 48, 6254–6260.
(17) Ichikawa, M.; Ide, N.; Ono, K. Changes in organosulfur com-
pounds in garlic cloves during storage. J. Agric. Food Chem. 2006,
54, 4849–4854.
Received for review October 13, 2007. Revised manuscript received
December 10, 2007. Accepted December 15, 2007. This project was
supported by the National Natural Science Foundation of China
(30570181), the Ministry of Education of the People’s Republic of
China, Specialized Research Fund for the Doctoral Program of
Higher Education (20070019004), and the National Key Technology
R&D Program (2006BAD06B02).
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