Dihydroasparagusic acid: Antioxidant and tyrosinase inhibitory activities and improved synthesis

Article abstract of DOI:10.1021/jf401120h

Dihydroasparagusic acid (DHAA) is the reduced form of asparagusic acid, a sulfur-containing flavor component produced by Asparagus plants. In this work, DHAA was synthetically produced by modifying some published protocols, and the synthesized molecule was tested in several in vitro assays (DPPH, ABTS, FRAP-ferrozine, BCB, deoxyribose assays) to evaluate its radical scavenging activity. Results show that DHAA is endowed with a significant in vitro antioxidant activity, comparable to that of Trolox. DHAA was also evaluated for its inhibitory activity toward tyrosinase, an enzyme involved, among others, in melanogenesis and in browning processes of plant-derived foods. DHAA was shown to exert an inhibitory effect on tyrosinase activity, and the inhibitor kinetics, analyzed by a Lineweaver-Burk plot, exhibited a competitive mechanism. Taken together, these results suggest that DHAA may be considered as a potentially active molecule for use in various fields of application, such as pharmaceutical, cosmetics, agronomic and food.

Full text of DOI:10.1021/jf401120h

Article
pubs.acs.org/JAFC
Dihydroasparagusic Acid: Antioxidant and Tyrosinase Inhibitory
Activities and Improved Synthesis
Alessandro Venditti,† Manuela Mandrone,*,§ Anna Maria Serrilli,† Armandodoriano Bianco,†
Carmelina Iannello,§ Ferruccio Poli,§ and Fabiana Antognoni#
†Dipartimento di Chimica, Universita
̀
di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy
di Bologna, Via Irnerio 42, 40126 Bologna, Italy
della Vita, Universita di Bologna, sede di Rimini, Corso Augusto 237, Rimini, Italy
§Dipartimento di Farmacia e Biotecnologie, Universita
̀
#Dipartimento di Scienze per la Qualita
̀
̀
S
* Supporting Information
ABSTRACT: Dihydroasparagusic acid (DHAA) is the reduced form of asparagusic acid, a sulfur-containing flavor component
produced by Asparagus plants. In this work, DHAA was synthetically produced by modifying some published protocols, and the
synthesized molecule was tested in several in vitro assays (DPPH, ABTS, FRAP-ferrozine, BCB, deoxyribose assays) to evaluate
its radical scavenging activity. Results show that DHAA is endowed with a significant in vitro antioxidant activity, comparable to
that of Trolox. DHAA was also evaluated for its inhibitory activity toward tyrosinase, an enzyme involved, among others, in
melanogenesis and in browning processes of plant-derived foods. DHAA was shown to exert an inhibitory effect on tyrosinase
activity, and the inhibitor kinetics, analyzed by a Lineweaver−Burk plot, exhibited a competitive mechanism. Taken together,
these results suggest that DHAA may be considered as a potentially active molecule for use in various fields of application, such
as pharmaceutical, cosmetics, agronomic and food.
KEYWORDS: Asparagus sp., dihydroasparagusic acid, dimercaptanics, in vitro antioxidant activity, polyphenol oxidase,
sulfur-containing compounds, tyrosinase inhibition, Lineweaver−Burk (L-B) plot
Organic sulfur derivatives with sulfhydryl functional groups
(−SH), such as reduced glutathione (GSH) and dihydrolipoic
acid (DHLA), are well-known for their importance as
antioxidants, and reduced levels of thiol compounds are
known to be associated with various disorders such as liver
failure, coronary artery disease, stroke, and neurological
disorders.16,17 Although there are several reports of a wide
array of biological activities and therapeutic properties of LA
and DHLA,18−21 the potential of DHAA has been poorly
explored. Since its discovery, only a few studies have been
carried out on the biological activities of this substance and its
oxidized form (asparagusic acid). In particular, they were
demonstrated to act as growth inhibitors for various plants and
nematodes22,23 while stimulating growth and pyruvate
oxidation in Streptococcus faecalis and asparagus mitochon-
dria;24,25 DHAA was also studied as a chelating ligand for a
series of iron and nickel complexes.26
On the basis of the antioxidant activities displayed by LA and
its reduced form DHLA,27 the latter structurally related to
DHAA, and due to the lack of information about this
compound, our attention was focused first on the determi-
nation of the in vitro antioxidant activity of this dimercaptanic
acid through different methods. In particular, the ABTS and
DPPH tests were used to determine its radical scavenging
capacity, and the FRAP−ferrozine (FRAP-FZ) test was used to
INTRODUCTION
■
Plants of the genus Asparagus, belonging to the Liliaceae, are
widely distributed and cultivated in the Mediterranean area.
Among them, the species Asparagus officinalis is an important
ingredient of the Italian and Mediterranean diet and is largely
used in traditional medicine for its diuretic and depurative
effects. The main secondary metabolites produced include
saponins,1 saccharides,2 and acetylenic and sulfur-containing
compounds.3,4 Studies on A. officinalis extracts have highlighted
how they may have a wide range of biological activities
including antioxidant,5 antidiabetic,6 antitumor,7 antifungal,8
diuretic,9 and immunostimulant.10
Dihydroasparagusic acid (DHAA) is the reduced form of
asparagusic acid, which is produced in several species of
Asparagus,11,12 and has been the first dithiolane isolated from a
natural source. The peculiarity of these compounds is due to
the fact that they are synthesized in intact plant cells, thus
representing an exceptional case of formation of sulfur-
containing flavor components. Indeed, normally, sulfur
compounds in vegetables are formed by enzymatic or chemical
splitting of nonvolatile precursors, like S-alkylcysteine sulfoxides
and glucosinolates during crushing of the plant material. The
highest amount of asparagusic acid is present in the region
around the apexes of the asparagus shoots, the edible part of
the plant,13,14 whereas the reduced form is present only in trace
amounts, due to the fast conversion into other metabolites.12
Among the naturally occurring 1,2-dithiolanes, the most widely
distributed is α-lipoic acid (LA), an essential coenzyme in many
organisms.15
Received: March 12, 2013
Revised: June 7, 2013
Accepted: June 24, 2013
Published: June 24, 2013
© 2013 American Chemical Society
6848
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
and then was kept at 4 °C overnight to improve the crystallization
process. The precipitate was collected by filtration on paper
(Whatman Blue) and the filtrate refluxed again at 115−120 °C. This
procedure was repeated three times, recovering 5.50 g of mixture from
the overall process. The mean composition of the mixture of
iodoacrylic and diiodoisobutanoic acids was 3:1, respectively (mean
molar yield ∼ 57%). 1H NMR, CDCl3, 300 MHz: (II) δ 3.47 (4 H, m,
−CH2I), δ 2.85 (1H, m, CH); (II′) δ 4.08 (2H, s, −CH2I), δ 6.04
(1H, s, CH2a), δ 6.37 (1H, s, CH2b). 13C NMR CDCl3, 75 MHz:
(II) δ 174.9 (−COOH), δ 48.4 (CH), δ 4.2 (−CH2I); (II′) δ 173.8
(−COOH), δ 130.0 (CH2), δ 128.5 (C), δ 5.9 (−CH2I).
β-β′-Ditioacetylisobutyric acid (III). Four grams of the mixture II/
II′ was completely solubilized under magnetic stirring using an 0.8 M
KOH solution, added dropwise. This reaction was carried out in
anhydrous conditions under N2 flux. A CH3COSK/CH3COSH
solution was freshly prepared and added dropwise, maintaining the
temperature at 55 °C. The free thioacetic acid was formed in situ from
thioacetate by adding a stoichiometric quantity of 2 N H2SO4. This
quantity was calculated on the iodomethylacrylic acid amount present
in the mixture. In 4.0 g of mixture (1:3) there were 1.393 g of diiodo
derivative (4.1 × 10−3 mol) and 2.607 g of iodoacrylic derivative (1.23
× 10−2 mol). The quantity of CH3COSK used was 5.94 g (5.4 × 10−2
mol), solubilized in 15 mL of demineralized water, and to this solution
was added 12 mL of 2N H2SO4 to re-form 2.4 × 10−2 mol of free
thioacetic acid. The reaction was monitored over 20 h, and as long as
the acrylic compound was present, it was still possible to readd
thioacetic acid. When the reaction was complete, the reaction mixture
was cooled to room temperature and acidified with 2 M H2SO4. β-β′-
Dithioacetylisobutyric acid, separated as an oil, was extracted with
chloroform (45 mL, three times). The organic solution was dried on
anhydrous sodium sulfate and filtered. Chloroform was eliminated
under reduced pressure to obtain 3.75 g (1.59 × 10−2 mol) of pale
evaluate the reducing power. Moreover, to gain more insight
into the antioxidant capacity of DHAA, two other antioxidant
tests involving the use of biological radicals were carried out:
the deoxyribose assay and the β-carotene bleaching (BCB) test.
Several sulfhydryl compounds, such as reduced glutathione,
L-cysteine, N-acetyl-L-cysteine, and LA, were reported to inhibit
the activity of tyrosinase.28,29 This copper-containing enzyme
(EC 1.14.18.1), also known as polyphenol oxidase (PPO),30 is
widely distributed in nature and catalyzes two distinct reactions
in melanin synthesis, the hydroxylation of a monophenol and
the conversion of an o-diphenol to the corresponding o-
quinone.31 This enzyme is responsible not only for the
melanization process in animals, but also for browning of
plant-derived foods and mushrooms during postharvest
handling. In most fruits, vegetables, and beverages, in fact,
the browning process is due to an enzymatic component, in
which tyrosinase plays a key role by catalyzing the oxidation of
phenolic compounds to the corresponding quinones, and by a
nonenzymatic one, which can be prevented by antioxidants. An
important role in the developmental and defensive functions of
insects has also been reported for tyrosinase, because it is
involved in the insect molting process and adhesion of marine
organisms.32,33 Thus, natural tyrosinase inhibitors represent an
alternative approach for medicinal and cosmetic products, in
agriculture, in the food industry, and in controlling insect
pests.34,35 On the basis of this rationale, the effect of synthetic
DHAA was also tested on in vitro tyrosinase activity, and
inhibition kinetics was also analyzed to understand the
inhibition mechanism.
1
yellow oil (mean molar yield ∼ 97%). H NMR, CDCl3, 300 MHz:
The aim of the present work was to investigate the
antioxidant and in vitro tyrosinase inhibitor activities of
synthetically prepared DHAA. An improved protocol for the
chemical synthesis of this molecule is described. This will
facilitate the production of high amounts of the molecule in
view of its potential uses in various fields of application. The
mechanism of inhibition of tyrosinase by DHAA is discussed.
(III): δ 3.25−3.12 (4H, m, −CH2S−), δ 2.95−2.86 (1H, m, −CH),
δ 2.35 (6H, s, CH3COS−). 13C NMR CDCl3, 75 MHz: (III): δ 194.7
(CH3COS−), δ 177.2 (−COOH), δ 44.8 (−CH2S−), δ 30.1 (CH), δ
29.0 (−SCOCH3).
DHAA. Three grams (1.27 × 10−2 mol) of III was salified with 16
mL of 0.8 M KOH solution (1.27 × 10−2 mol). After complete
dissolution of the oil, 56.5 mL of 0.8 M KOH solution was added
under continuous stirring and under nitrogen atmosphere at 45 °C.
About 7 h was required for the reaction to be complete, then, after
cooling at room temperature, the reaction mixture was acidified with 2
N H2SO4, obtaining an oily suspension. This was extracted with
chloroform (60 mL, three times), and the organic layer was collected,
dried on sodium sulfate, and filtered. The organic solvent was removed
under reduced pressure, giving an oil. This was maintained under
vacuum overnight, to eliminate the remaining acetic acid. After this
step, DHAA crystallized spontaneously when seeded with some
crystals of DHAA from a previous synthesis, recovering 1.82 g of pure
MATERIALS AND METHODS
■
General Experimental Procedures. Solvents, reagents, and
substances were purchased from Sigma-Aldrich (St. Louis, MO,
USA), Acros Organics (Geel, Belgium), ABCR (Karlsruhe, Germany),
and Carlo Erba Reagenti (Milano, Italy) and were used without further
purification.
Thin layer chromatography (TLC) was performed using silica gel
SiF254 (Merck, Darmstadt, Germany). Plates were developed with
chloroform/methanol at various percentages and sprayed with
phosphomolybdic reagent, followed by heating at 120 °C.
NMR spectra were registered on a Varian Mercury 300 MHz and a
Bruker Avance 400 MHz using CDCl3 as deuterated solvents and
TMS as internal standard.
1
compound (1.19 × 10−2 mol, yield ∼ 94%). H NMR, CDCl3, 300
MHz: (DHAA) δ 3.00−2.80 (5H, m, overlapped signals), δ 1.52 (2H,
t, J 8.5 Hz, −CH2−SH). 13C NMR CDCl3, 75 MHz: (DHAA) δ 178.3
(−COOH), δ 50.8 (CH), δ 24.0 (−CH2SH).
DPPH and ABTS Assays. The DPPH assay was performed
according to the method of Brand-Williams et al.36 with some
modifications. Stock solutions of DHAA were prepared in water to
obtain different final concentrations (from 5 to 40 μM in the assay) to
calculate the IC50 value. One and a half milliliters of a 0.05 mM DPPH
methanol solution was added to different concentrations of DHAA
and allowed to react at room temperature. The assay was performed in
a final volume of 2 mL. After 20 min, the absorbance (Abs) values
were measured at 517 nm and converted into percentage antioxidant
activity using the following formula:
MS spectra were performed on a Q-TOFMICRO spectrometer
(Micromass, now Waters, Manchester, UK) equipped with an ESI
source, in the negative ion mode. The rate of sample infusion was 10
μL/min with 100 acquisitions per spectrum. Data were analyzed using
the MassLynx software developed by Waters.
Biological assays were performed using a microplate reader, Victor
X3 Perkin-Elmer (Perkin-Elmer Inc., Boston, MA, USA) and data
analyzed with the software Work Out 2.5 or in a Jasco V-530
spectrophotometer (Jasco Europe, Cremella, Italy).
Synthetic Procedure. β-β′-Diiodoisobutyric Acid (II)/Iodome-
thylacrylic Acid (II′). Diethyl bis(dihydroxymethilmalonate) (I; 8.80 g
(4 × 10−2 mol) was refluxed at 115−120 °C with 37.0 mL of HI 57%.
During the first 45−60 min, the reaction was carried out with a
continuous air flux to eliminate the volatile materials, which interfere
with the desired reaction, and then the mixture was refluxed for 6 h.
The reaction mixture crystallized spontaneously at room temperature
scavenging capacity% = [1 − (Abssample/Abscontrol) × 100]
The DPPH solution plus methanol was used as a negative control,
whereas TR, AA, and BHA at different concentrations (from 5 to 40
μM) were used as reference antioxidant compounds. The IC50 values
6849
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
were calculated by logarithmic regression of plots, where the x-axis
represents DHAA or reference compounds concentrations and the y-
axis the average percentage of scavenging capacity from three
independent experiments with duplicate samples.
Gutteridge et al.40 with some modifications. The reaction mixture,
containing 64 μL of EDTA (5 mM), 64 μL of FeCl3 (5 mM), and 64
μL of H2O2 (2.5 mM), was incubated at 37 °C for 15 min. Then, H2O
and a blend of deoxyribose (140 mM) and ascorbic acid (5 mM) was
added to obtain a final volume of 0.64 mL. Different concentrations of
DHAA (from 0.1 to 2 mM) or reference compounds (from 0.01 to 0.5
mM) were added, and the mixture incubated at 37 °C for 1 h. After
the addition of TBA and TCA, the mixture was incubated for 15 min
at 90 °C and the Abs was read at 490 nm in a microplate reader. The
IC50 and TEAC were calculated as described above.
Tyrosinase Assay. The tyrosinase inhibition assay was performed
according to the method of Masamoto et al.41 Mushroom tyrosinase
(EC 1.14.18.1) was purchased from Sigma-Aldrich Co (St. Louis, MO,
USA). Nine units of enzyme (1 unit being defined by the producer as
the ΔA280 of 0.001 min−1 at pH 6.5 at 25 °C in 3 mL of reaction mix
containing L-tyrosine) and DHAA at different concentrations (30−200
μM) were incubated for 5 min in 0.1 M sodium phosphate buffer, pH
6.8, in 0.1 mL final volume. The specific activity of the enzyme is 1715
U/mg. L-DOPA (final concentration = 3 mM) was added to start the
reaction, and the formation of dopachrome was immediately
monitored for 5 min at 490 nm in a microplate reader under a
constant temperature of 30 °C. ΔAbs values were calculated in the first
240 s and referred to 1 min.
The ABTS assay was performed according to the method of Arnao
et al.37 with some modifications. ABTS•+ radical was generated by
mixing a 2 mM ABTS solution with 7 mM K2S2O8 and incubating in
the dark for 24 h at room temperature. Before usage, the ABTS•+
solution was diluted (1−25 mL methanol) to obtain an Abs value of
0.7 at 734 nm. Upon addition of 1 mL of the diluted ABTS•+ solution
to 10 μL of reference compounds or DHAA stock solutions (from 5 to
40 μM), the Abs at 734 nm was recorded after 1 min. The final TEAC
value of DHAA was calculated by comparing ABTS•+ decolorization
with that of Trolox. The IC50 value was calculated as described above.
FRAP−Ferrozine Assay. The colorimetric method used to
determine the capacity of DHAA to reduce ferric ions (Fe3+) to
ferrous ions (Fe2+) was obtained according to the method of Berker et
al.38 with some modifications.
A calibration curve was created for the Fe2+/ferrozine (FZ) complex
using increasing concentrations of FeCl2 (between 10 and 100 μM)
and 0.5 mM FZ (in distilled water) in a final volume of 1 mL. A blank
was prepared with FZ only. An amount of 0.2 mL of a previously
prepared mixture containing FeCl3 (1 mM) and FZ (5 mM) was
added to 0.1 mL of reference compound at different concentrations
(from 0.05 to 20 μM) or DHAA at different concentrations (from 15
to 65 μM), and the volume was brought to 1 mL with distilled water.
The Abs was read using the microplate reader at 570 nm after 5 min of
incubation at room temperature. The Abs of the FeCl3/FZ mixture
was subtracted from that obtained with reference compounds or
DHAA. To build a dose−response curve, the amount of Fe2+ produced
by different compound concentrations was calculated from the
calibration curve. Results are expressed in micromolar FRAP,
according to the original method, which represents the Fe2+
micromolar produced by 0.1 mM of the substance under investigation.
On the basis of the dose−response curve of TR, TEAC values were
calculated. The assay was performed in duplicate and repeated three
times.
β-Carotene Bleaching Test. Prevention of the autoxidation of
emulsified linoleic acid was determined by modifying the method of
Mikami et al.39 Briefly, 20 μL of different DHAA or reference
compounds stock solutions (50, 100, 150, and 200 μM) was added to
the microplate wells (Costar 3599) in duplicate. Then, 10 μL of
linoleic acid, 47 μL of Tween 40, and 2.5 mL of β-carotene (2 mg/mL
in chloroform) were placed in a flask. After removal of chloroform
with nitrogen gas, 22.5 mL of distilled hot water (50 °C) and 2.5 mL
of 0.1 M sodium phosphate buffer (pH 6.8) were added to the same
flask and shaken well. An amount of 0.2 mL of the linoleic acid−β-
carotene emulsion were transferred rapidly to each well, kept under
constant temperature (50 °C), and the Abs at 490 nm monitored for
60 min. H2O (20 μL) and reference compounds (20 μL) at different
concentrations (0.25, 0.5, 1, 1.5, and 2.5 mM) were used as negative
and positive controls, respectively.
The percentage inhibition of enzyme activity was calculated by the
following formula:
%inhibition = [1 − (ΔAbs/minsample /ΔAbs/minnegative control
× 100]
)
IC50 (concentration necessary for 50% inhibition of enzyme
activity) was calculated by constructing a linear regression curve
showing DHAA concentrations on the x-axis and percentage inhibition
on the y-axis. A negative control was obtained by adding water instead
of DHAA. A positive control was performed using kojic acid, a well-
known tyrosinase inhibitor.
A Lineweaver−Burk (L-B) plot was constructed to calculate the
kinetic parameters (Km expressed in mM and Vmax in μkat) of the
enzymatic reaction without and with DHAA at the IC50 concentration
(0.13 mM). Different L-DOPA concentrations were used in the range
from 0.095 to 6 mM; the rate of the enzymatic reaction, expressed in
microkatals, was calculated from ΔAbs 120 to 240 s to avoid the lag
phase, considering dopachrome ε at 490 nm = 3.6201 mM−1 cm−1 and
a light path length of 0.8 cm. Kiapp was calculated by the equation
⎡
⎡ ⎤
⎤
⎦
Km app = Km 1 + I /K
(
)
i app
⎣
⎣ ⎦
where Km app is the apparent Km in the presence of DHAA.
Fe2+ Chelating Assay. The ferrous ion-chelating potential was
investigated according to the method of Decker and Welch42 with
some modifications, wherein the Fe2+-chelating ability was calculated
by measuring the ferrous iron−FZ complex at 560 nm in a microplate
reader. Briefly, the reaction mixture, containing different concen-
trations of DHAA (from 10 μM to 5 mM), FeCl2 (0.2 mM), and FZ
(0.4 mM), was adjusted to a total volume of 0.2 mL with H2O, shaken
well, and incubated for 10 min at room temperature. The absorbance
of the mixture was measured at 560 nm against a blank. EDTA (from
10 μM to 5 mM) was used as positive control. The ability of the test
compound to chelate ferrous ions was calculated using the following
equation:
The difference in Abs at 50 and 5 min (Δ = Abs50 min − Abs5 min
)
was calculated. Results are expressed in terms of percentage bleaching
inhibition of the initial linoleic acid−β-carotene emulsion by the test
samples according to the following equation:
%bleaching inhibition
= [1 − (ΔAbssample/ΔAbsnegative control) × 100]
IC50 values were calculated by logarithmic regression in terms of
micromolar. The antioxidant activity was also expressed as TEAC, by
comparing IC50 values obtained for DHAA, AA, and BHA with that
TR.
chelating activity (%)
= [1 − (ΔAbssample/ΔAbsnegativecontrol) × 100]
Deoxyribose Assay. 2-Deoxy-D-ribose is degraded when exposed
to hydroxyl radicals generated by the Fenton reaction. The
degradation can be detected by heating the products with 2-
thiobarbituric acid (TBA-0.5% in 1 N NaOH, w/v), under acidic
conditions using trichloroacetic acid (TCA, 2.5%, w/v). The effects of
DHAA on deoxyribose oxidation were assessed as described by
Statistical Analysis. Values are expressed as the mean SD of
three independent experiments with samples in triplicate. Statistical
analysis was performed using Graph Pad Prism 4 software (La Jolla,
CA, USA) by one-way analysis of variance (ANOVA), considering
significant differences at P values of <0.05.
6850
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
Figure 1. Projected scheme for DHAA synthesis.
1
bond. The reaction can be easily monitored by H NMR
spectroscopy. In fact, by observing the spectrum, one can add
the required amount of thioacetic acid and potassium
thioacetate, because the integrals relative to a single olefinic
proton belonging to the acrylic compound and of the single
methine proton belonging to the dihalogenated derivate reflect
their relative abundance (see the Supporting Information,
Figure S1). Therefore, the analysis of NMR data allows one to
check the progression of the reaction and, on this basis, to add
more reagents if required to displace the reaction and thus
maximize product yield (see the Supporting Information,
Figure S2). In particular, it was often necessary to readd
thioacetic acid when the acrylic compound was still present,
after several hours of reaction, especially with large amounts of
starting material. This is probably due to the instability of
thioacetic acid in an aqueous medium and its transformation
into acetic acid (which does not get added to the double bond).
This synthetic procedure was also transferred to gram scale,
resulting in a quasi-quantitative yield from the last two steps.
Indeed, yields of 97 and 94% were obtained from
thioacetylation and hydrolysis reactions, respectively. In
particular, the hydrolysis reaction gave an unexpectedly good
result because, as previously reported by Yanagawa,43 when the
hydrolysis reaction was performed following Schotte’s method,
no satisfactory yield was often obtained due to the instability of
the final product in alkaline medium.44 On the contrary, we
obtained an average yield of 94%, and this result is probably
due to the mild conditions used during the reaction, namely, a
low-concentration alkaline solution, and a nitrogen atmosphere,
which avoids oxidation (see the Supporting Information,
Figures S3 and S4).
The synthetic procedure represents a great advantage for
molecules such as DHAA. In fact, according to Tressl et al.,12
who investigated the biosynthesis of sulfur-containing acids in
Asparagus, DHAA was found only in the enzyme-inhibited
aroma extracts of the plant, thus suggesting that it is formed in
the intact plant cells and might be one possible intermediate in
the biosynthesis of asparagusic acid. Similar results were
reported by Yanagawa et al.,22 who isolated trace amounts of
DHAA and other sulfur compounds during an investigation of
plant growth regulating substances from extracts of A.
officinalis. Moreover, the yield obtained with the chemical
synthesis described here was much higher than that reported by
Jansen,11 who prepared the molecule by chemical reduction of
asparagusic acid extracted from its natural source (and obtained
RESULTS AND DISCUSSION
■
Synthetic DHAA was obtained, after revisiting some pre-
existent protocols, by following the reaction by 1H NMR
spectroscopy. In particular, the method reported by Yanagawa
was adopted to obtain the first intermediate,43 β-β′-
diiodoisobutyric acid (II) from diethyl bis-
(dihydroxymethylmalonate) (I, Figure 1). Subsequently, as
reported by Schotte and Storm,44 intermediate II was subjected
to a nucleophilic substitution by thioacetate, leading to the
formation of the dithioacetylated derivative (III), which, after
hydrolysis, formed DHAA (Figure 1).
During the first step of this synthetic procedure, the
formation of an acrylic byproduct (II′) took place (Figure 2),
̈
Figure 2. Formation of iodoacrylic byproduct.
which involves a drastic reduction of the final yield. In
Yanagawa’s paper this aspect was not considered,43 but in a
previous study on the effects of mineral acids on diethyl
bis(dihydroxymethylmalonate) (I), the consistent formation of
the iodoacrylic derivative was reported.45 In the same paper, the
instability of these halogenated derivatives (II and II′) was
pointed out, so they were used in the following reactions as
soon as possible. Although the formation of this byproduct
could not be avoided, it may be at least minimized (7:3 ratio)
by modulating some conditions, such as temperature and
reaction time; a mean yield of about 57% of the mixture was
obtained using the conditions reported under Materials and
Methods.
Therefore, to increase the yield, we considered another
synthetic procedure based on the use of this acrylic byproduct
as intermediate, to whose double-bond thioacetic acid can be
added.46 On this basis, we modified the reaction conditions to
exploit the byproduct as a reaction intermediate, avoiding any
preliminary purification of the mixture obtained from the first
step (Figure 3).
The presence of both thioacetate ion and thioacetic acid in
the reaction mixture leads to the simultaneous substitution of
halogen atoms and the addition of thioacetic acid to the double
Figure 3. Modified synthesis of DHAA.
6851
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
only 15.21 g from 1400 kg of asparagus butts, yield = 1.1 ×
10−5%). Another advantage derived from the chemical synthesis
is to overcome the problem arising from the variability in
endogenous content observed among different varieties of
asparagus.12
With regard to the biological activities, DHAA showed a
good radical scavenging capacity, comparable to that of well-
known antioxidant compounds, such as AA, BHA, and TR.
With regard to DPPH and ABTS tests, a dose−response
curve for DHAA is shown in Figure 4. IC50 values for DHAA
measuring antioxidant activity, they may not always be the best
way to get information about some types of antioxidants. In
particular, for S−S type antioxidants such as glutathione
(GSSG), LA, and HCYSS, the antioxidant capacity was
undetectable or very low using these tests, thus not reflecting
their action in vivo.48,49
Consequently, the FRAP-FZ test was also performed. This
test was modified with respect to the original as concerns the
pH value, and a physiological pH was used instead of an acidic
one.50 In fact, the reducing capacity of some antioxidant
compounds, such as DHAA, may be suppressed under acidic
conditions due to their protonation, and this may indeed
explain the generally low antioxidant capacity found by
Grungor et al. with the FRAP test.49 Conversely, with the
̈
FRAP-FZ test used in this work, DHAA showed a strong redox
reactivity toward Fe3+/Fe2+, revealing itself, also in this assay, as
potent as antioxidant reference compounds, in particular, BHA
and TR (TEAC = 0.99). Results obtained with the BCB test
revealed that DHAA was also able to prevent lipid peroxidation
through a chain-breaker mechanism, showing an IC50 value
similar to that of the other reference antioxidant compounds
(Table 1).
Finally, DHAA was tested using the deoxyribose assay for
evaluating the scavenging capacity toward the hydroxyl radical
produced by the H2O2/[Fe-EDTA]3+/AA system and detected
by deoxyribose degradation. Unlike all of the other tests,
DHAA showed a very low activity (IC50 = 1.37 0.04 mM;
TEAC = 0.02, data not shown), whereas TR turned out to be
rather active (IC50 = 23.51 2.23 μM). This result is in line
with that reported by Scott et al.,51 who observed a prooxidant
effect of DHLA on deoxyribose degradation induced by H2O2/
FeC13/AA. A low scavenging activity of dithiol compounds
toward the hydroxyl radical was also found by Mazor et al.,48
whereas Suzuki reported a positive result for DHLA as hydroxyl
scavenger, even though a different detection method was
used.52 With regard to DHAA’s ability to chelate free Fe2+ ions,
it was evaluated spectrophotometrically by the Fe2+-chelating
assay, but no such activity was found (data not shown).
In vitro tyrosinase activity was evaluated using L-DOPA as
substrate and following its oxidation to dopachrome
spectrophotometrically. Using this method, DHAA showed a
particularly high inhibitory activity, and a reaction kinetics
typical of thiolic compounds was observed (Figure 5).53
Indeed, when DHAA was added, there was a lag before any
increase in absorbance, and this lag period was longer with
higher concentrations of DHAA, whereas addition of kojic acid,
used as a positive control, did not show a similar trend (Figure
5). As reported by Negishi and Ozawa,54 the lag time observed
when tyrosinase activity was followed in the presence of thiol
compounds is probably due to a nonenzymatic reaction in
which the o-quinone radicals produced from L-DOPA by the
Figure 4. Dose−response curve of DHAA for ABTS (A) and DPPH
(B) assays. IC50 values were calculated from the following equation: y
= ax + b, where a = 2.6033, b = −6.5437, R2 = 0.9955 for ABTS; a =
2.1835, b = 4.670, R2 = 0.9877 for DPPH. Data are the means of three
independent determinations.
obtained in these assays were 20.76 and 21.72 μM, respectively,
which are very close to those of AA and TR (Table 1). In fact,
TEAC values, defined as the millimolar concentration of a TR
solution having activity equivalent to that of a 1 mM solution of
the substance under investigation,47 turned out to be very close
to 1. It is noteworthy to observe that the TEAC value of DHAA
in the ABTS assay was similar to that reported for other SH-like
antioxidants, such as 1,4-dithioerythritol, N-acetylcysteine,
GSH, cysteine, and homocysteine (HCYSS), assayed by other
authors using the same test and higher than that of DHLA.48,49
Even if the ABTS and DPPH in vitro assays are often used for
a
Table 1. Antioxidant Activity of DHAA Assayed by DPPH, ABTS, BCB, and FRAP-FZ Tests
DPPH
ABTS
BCB
FRAP-FZ
μM FRAP
IC50
TEAC
IC50
TEAC
IC50
TEAC
.
DHAA
TR
20.76 4.0 a
22.25 2.5 a
17.87 2.3 ab
13.60 1.5 b
1.07
1
21.72 3.0 a
20.19 1.9 ab
22.35 2.8 a
14.49 2.8 a
0.93
1
14.50 2.7 abc
21.05 3.0 b
13.04 2.5 ac
15.45 2.8 abc
1.45
1
236.41 12 a
239.36 20 a
175.48 14 b
226.65 15 a
0.99
1
AA
1.25
1.64
0.90
1.39
1.61
1.36
0.73
0.95
BHA
a
Data are expressed as IC50 values (μM) for DPPH, ABTS, and BCB assays, and as μM FRAP values for FRAP-FZ assay. Different letters, within the
same column, indicate significant differences at p < 0.05. Results are means SD of three independent experiments with three replicates.
6852
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
□
■
▲
Figure 5. Inhibitory effect on the rate of the oxidation of L-DOPA by mushroom tyrosinase in control ( ), kojic acid ( ), and DHAA ( ). The
enzyme and DHAA were incubated for 5 min, and assay was performed with 3 mM L-DOPA concentration.
enzyme are converted back to the original structure. After this
lag phase, a reaction occurs between quinones and the chemical
species derived from DHAA during the previous reaction,
leading to the formation of colorless conjugates. A similar result
was also reported by Tsuji-Naito et al., who demonstrated that
the sulfhydryl groups of DHLA covalently react with DOPA-
quinone to form lipoyl DOPA conjugates.55 Thus, thiol
compounds were able to prevent the polymerization of
phenolics, which results in browning, through this non-
enzymatic mechanism.
Using a 3 mM concentration of L-DOPA, the IC50 value of
DHAA, calculated from the dose−response curve (Figure 6),
Figure 7. Lineaweaver−Burk plots of mushroom tyrosinase and L-
■
▲
DOPA without ( ) and with ( ) 0.13 mM DHAA. The enzyme and
DHAA were incubated for 5 min, and assay was performed with an L-
DOPA concentration range from 0.095 to 6.0 mM.
Vmax remained unchanged, thus suggesting that DHAA
inhibited tyrosinase activity competitively, without the
inactivation of the enzyme. A similar L-B plot was obtained
for a water-soluble LA derivative.57 The kinetic parameters of
the enzyme assay in the presence or absence of DHAA are
shown in Table 2. To confirm the lack of irreversible
Table 2. Kinetic Inhibition Parameters of DHAA on
Mushroom Tyrosinase
Figure 6. Inhibition of in vitro tyrosinase activity in the presence of
increasing DHAA concentrations. Enzyme assay was performed using
3 mM L-DOPA as substrate. Data represent the mean SD of three
independent experiments with samples in triplicate.
enzyme
enzyme−DHAA
IC50
Km
0.13 mM
0.81 mM
4.23 mM
Vmax
0.198 ΔA/min
(0.228 μkat)
0.193 ΔA/min
(0.223 μkat)
0.275 mM
was 0.13 mM. This value turned out to be very close to that
reported for L-cysteine by Kermasha et al.53 (IC50 = 0.15 mM),
even though the assay conditions used by these authors were
slightly different. The inhibitory activity of DHAA, expressed in
terms of IC50 values, was about 9 times lower than the one
Ki app
inactivation of the enzyme by DHAA, the assay was performed
by increasing the incubation time from 5 to 30 min, and results
were compared with a nonincubation assay. No differences in
the inhibitory activities were observed among 0, 5, and 30 min
of incubation (data not shown), thus suggesting that no
irreversible inactivation occurs. With regard to kojic acid, a
mixed mechanism of inhibition was found (data not shown),
confirming previously reported results.56
obtained with kojic acid (IC50 = 14.7
2.0 μM), but it is
important to consider that these two molecules act through
different mechanisms.56 To investigate in more detail this
aspect, a L-B plot was built by monitoring the reaction in the
presence of different L-DOPA concentrations, keeping a
DHAA concentration equivalent to the IC50. The L-B plot
(Figure 7) shows that only the Km value was modified by the
presence of the inhibitor, increasing from 0.81 mM (without
inhibitor) to 4.23 mM in the presence of DHAA, whereas the
In conclusion, the protocol for the synthesis of DHAA
developed in this work results in higher yields compared to the
6853
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
(8) Wang, H.; Ng, T. B. Isolation of a novel deoxyribonuclease with
antifungal activity from Asparagus officinalis seeds. Biochem. Biophys.
Res. Commun. 2001, 23, 120−124.
(9) Balansard, S.; Rayband, M. Diuretic action of Asparagus
officinalis. Crit. Rev. Soc. Biol. 1987, 126, 954−956.
(10) Thatte, U. M.; Dahanukar, S. A. Comparative study of
immunomodulating activity of Indian medicinal plants, lithium
carbonate and glucan. Methods Find. Exp. Clin. Pharmacol. 1988, 10,
639−644.
(11) Jansen, E. F. The isolation and identification of 2,2′-
dithiolisobutyric acid from Asparagus. J. Biol. Chem. 1948, 176, 657−
664.
(12) Tressl, R.; Holzer, M.; Apetz, M. Formation of flavor
components in asparagus. 1. Biosynthesis of sulfur-containing acids
in Asparagus. J. Agric. Food Chem. 1977, 25, 455−459.
(13) Kitahara, Y.; Yanagawa, H.; Kato, T.; Takahashi, N. Asparagusic
acid, a new plant growth inhibitor in etiolated young asparagus shoots.
Plant Cell Physiol. 1972, 13, 923−925.
previously reported protocols. Results obtained from antiox-
idant tests demonstrate that this molecule may be considered as
a new potentially useful ingredient or preservative in the
preparation of functional foods, food supplements, and
nutraceuticals by virtue of its considerable antioxidant power.
Compared to other sulfhydryl compounds, such as LA and
DHLA, DHAA has the advantage of being soluble in aqueous
solutions: this makes it more readily exploitable in different
applications. The inhibitory activity exerted on PPO
(tyrosinase) activity and thus on the polymerization of
quinones occurring during browning represents an added
value for this molecule and increases its potential in the food
industry and for medicinal, agronomic, and cosmetic uses.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
(14) Yanagawa, H. Evidence for asparagusate and asparagusate
dehydrogenase localized near the apices of asparagus shoots. Plant Cell
Physiol. 1976, 17, 932−937.
(15) Reed, L. J. Multienzyme complex. Acc. Chem. Res. 1974, 7, 40−
46.
AUTHOR INFORMATION
■
̈
̈
Corresponding Author
(16) Yardim-Akaydin, S.; Ozkan, Y.; Ozkan, E.; Torun, M.; Simsek,
B. The role of plasma thiol compounds and antioxidant vitamins in
patients with cardiovascular disease. Clin. Chim. Acta 2003, 338, 99−
105.
*(M.M.) Phone: +390512091294. Fax +39051242576. E-mail:
manuela.mandrone2@unibo.it.
Notes
(17) Bilska, A.; Wodek, L. Lipoic acid − the drug of the future?
Pharmacol. Rep. 2005, 57, 270−577.
The authors declare no competing financial interest.
(18) Navari-Izzo, F.; Quartacci, M. F.; Sgherri, C. Lipoic acid: a
unique antioxidant in the detoxification of activated oxygen species.
Plant Physiol. Biochem. 2002, 40, 463−470.
ACKNOWLEDGMENTS
■
We thank Prof. Stefania Biondi for the English revision of the
manuscript.
(19) Ho, Y. S.; Lai, C. S; Liu, H. I.; Ho, S. Y.; Tai, C.; Pan, M. H.;
Wang, Y. J. Dihydrolipoic acid inhibits skin tumor promotion through
anti-inflammation and anti-oxidation. Biochem. Pharmacol. 2007, 73,
1786−1795.
ABBREVIATIONS USED
■
(20) Wollin, S. D.; Jones, P. J. H. α-Lipoic acid and cardiovascular
disease. J. Nutr. 2003, 113, 3327−3330.
AA, ascorbic acid; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-
6-sulfonic acid); BCB, β-carotene bleaching; BHA, tert-butyl-4-
hydroxyanisole; DHAA, dihydroasparagusic acid; DHLA,
dihydrolypoic acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl;
FRAP, ferric reducing antioxidant power; FZ, ferrozine; GSH,
glutathione; HCYSS, homocysteine; LA, α-lipoic acid; TEAC,
Trolox equivalent antioxidant capacity; TR, Trolox
(21) Lai, Y. S.; Shih, C. Y.; Huang, Y. F.; Chou, T. C. Antiplatelet
activity of α-lipoic acid. J. Agric. Food Chem. 2010, 58, 8596−8603.
(22) Yanagawa, Y.; Kato, T.; Kitahara, Y. Asparagusic acid,
dihidroasparagusic acid and S-acetyldihydroasparagusic acid, a new
plant growth inhibitor in etiolated young asparagus shoots.
Tetrahedron Lett. 1972, 25, 2549−2552.
(23) Takasugi, M.; Yachida, Y.; Anetai, M.; Masamune, T.; Kegasawa,
K. Identification of asparagusic acid as a nematicide occurring naturally
in the roots of asparagus. Chem. Lett. 1975, 4, 43−44.
(24) Yanagawa, H.; Kato, T.; Kitahara, Y.; Takahashi, N. Stimulation
of growth and pyruvate oxidation in Streptococcus faecalis by
asparagusic acid and its derivatives. Plant Cell Physiol. 1973, 14,
791−795.
REFERENCES
■
(1) Pant, G.; Panwar, M. S.; Negi, D. S.; Rawat, M. S. M.; Morris, G.
A. Spirostanol glycoside from fruits of Asparagus officinalis.
Phytochemistry 1988, 27, 3324−3325.
(2) Shiomi, N.; Yamada, J.; Izawa, M. A novel pentasaccharide in the
roots of asparagus (Asparagus officinalis L.). Agric. Biol. Chem. 1979,
43, 1375−1377.
(3) Terada, K.; Honda, C.; Suwa, K.; Takeyama, S.; Oku, H.;
Kamisako, W. Acetylenic compounds isolated from cultured cells of
Asparagus officinalis. Chem. Pharm. Bull. 1995, 43, 564−566.
(4) Matsubayashi, Y.; Sakagami, Y. Phytosulfokine, sulfated peptides
that induce the proliferation of single mesophyll cells of Asparagus
officinalis L. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7623−7627.
(5) Rodriguez, R.; Jaramillo, S.; Rodriguez, G.; Espejo, J. A.; Guilln,
R.; Fernandez, I.; Heredia, A.; Jimnez, A. Antioxidant activity of
ethanolic extracts from several asparagus cultivars. J. Agric. Food Chem.
2005, 29, 5212−5217.
(25) Yanagawa, H.; Kato, T.; Kitahara, Y. Stimulation of pyruvate
oxidation in asparagus mitochondria by asparagusic acid. Plant Cell
Physiol. 1973, 14, 1213−1216.
(26) Volkers, P. I.; Rauchfuss, T. B.; Wilson, S. R. Coordination
chemistry of 3-mercapto-2-(mercaptomethyl) propanoic acid (dihy-
droasparagusic acid) with iron and nickel. Eur. J. Inorg. Chem. 2006, 23,
4793−4799.
(27) Packer, L.; Witt, E. H.; Tritschler, H. J. α-Lipoic acid as a
biological antioxidant. Free Radical Biol. Med. 1995, 19, 227−250.
(28) Matheis, G.; Whitaker, J. R. Modification of proteins by
polyphenol oxidase and peroxidase and their products. J. Food Biochem.
1984, 8, 137−162.
(29) Mayer, A. M. Polyphenol oxidases in plants: recent progress.
Phytochemistry 1987, 26, 11−20.
(30) Whitaker, J. R. Polyphenol oxidase. In Food Enzymes, Structure
and Mechanism; Wong, D. W. S., Ed.; Chapman and Hall: New York,
1995; pp 271−307.
(6) Said, O.; Khalil, K.; Fulder, S.; Azaizeh, H. Ethnopharmacological
survey of medicinal herbs in Israel, the Golan Heights and the West
Bank region. J. Ethnopharmacol. 2002, 83, 251−265.
(7) Shao, Y.; Chin, C. K.; Ho, C. T.; Ma, W.; Garrison, S. A.; Huang,
M.-T. Anti-tumor activity of the crude saponins obtained from
asparagus. Cancer Lett. 1996, 24, 31−36.
6854
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855

Journal of Agricultural and Food Chemistry
Article
(31) Robb, D. A. Tyrosinase. In Copper Proteins and Copper Enzymes;
Lontie, R., Ed.; CRC Press: Boca Raton, FL, 1984; Vol. 2, pp 207−
241.
(32) Andersen, S. O. Sclerotization of insect cuticle. In Molting and
Metamorphosis; Ohnishi, E., Ishizaki, H., Eds.; Japan Science Society
Press: Tokyo, Japan, 1990; pp 133−155.
(33) Shiino, M.; Watanabe, Y.; Umezawa, K. Synthesis of N-
substituted N-nitrosohydroxylamines as inhibitors of mushroom
tyrosinase. Bioorg. Med. Chem. 2001, 9, 1233−1240.
(34) Kubo, I.; Quing-Xi, C. Molecular design of antibrowning agents:
antioxidative tyrosinase inhibitors. Food Chem. 2003, 81, 241−247.
(35) Friedman, M. Food browning and its prevention: an overview. J.
Agric. Food Chem. 1996, 44, 631−653.
(54) Negishi, O.; Ozawa, T. Inhibition of enzymatic browning and
protection of sulfhydryl enzymes by thiol compounds. Phytochemistry
2000, 54, 481−487.
(55) Tsuji-Naito, K.; Hatania, T.; Okadab, T.; Teharaa, T. Evidence
for covalent lipoyl adduction with dopaquinone following tyrosinase-
catalysed oxidation. Biochem. Biophys. Res. Commun. 2006, 15−20.
(56) Chen, J. S.; Wie, C. I.; Marshall, M. R. Inhibition mechanism of
kojic acid on polyphenol oxidase. J. Agric. Food Chem. 1991, 39, 1897−
1901.
(57) Tsuji-Naito, K.; Hatania, T.; Okadab, T.; Tehara, T. Modulating
effects of a novel skin-lightening agent, α-lipoic acid derivative, on
melanin production by the formation of DOPA conjugate products.
Bioorg. Med. Chem. 2007, 15, 1967−1975.
(36) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free
radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol.
1995, 28, 25−30.
(37) Arnao, M. B.; Cano, A.; Alcolea, J. F.; Acosta, M. Estimation of
free radical-quenching activity of leaf pigment extracts. Phytochem.
Anal. 2001, 12, 138−143.
(38) Berker, K. I.; Gucļ u, K.; Demirata, B.; Apak, R. A novel
̈ ̈
antioxidant assay of reducing capacity measured using ferrozine as the
colour forming complexation reagent. Anal. Methods 2010, 2, 1770−
1778.
(39) Mikami, I.; Yamaguchi, M.; Shinmoto, H.; Tsushida, T.
Development and validation of a microplate-based β-carotene
bleaching assay and comparison of antioxidant activity (AOA) in
several crops measured by β-carotene bleaching, DPPH and ORAC
assays. Food Sci. Technol. Res. 2009, 15, 171−178.
(40) Gutteridge, J. M. C.; Halliwell, B. The deoxyribose assay: an
assay both for ‘free’ hydroxyl radical and for site-specific hydroxyl
radical production. Biochem. J. 1988, 253, 931−933.
(41) Masamoto, Y.; Iida, S.; Kubo, M. Inhibitory effect of chinese
crude drugs on tyrosinase. Planta Med. 1980, 40, 361−365.
(42) Decker, E. A.; Welch, B. Role of ferritin as a lipid oxidation
catalyst in muscle food. J. Agric. Food Chem. 1990, 38, 674−677.
(43) Yanagawa, H.; Kato, T.; Sagami, H.; Kitahara, Y. Convenient
procedure for the synthesis of asparagusic acid. Synthesis 1973, 10,
607−608.
(44) Schotte, L.; Strom, H. The preparation of 1,2-ditholane-4-
̈
carboxylic acid. Acta Chem. Scand. 1956, 10, 687−688.
(45) Ferris, A. F. The action of mineral acid on diethyl
bis(hydroxymethyl)malonate. J. Org. Chem. 1955, 20, 780−787.
(46) Singh, R.; Whitesides, G. M. Comparison of rate constant for
thiolate-disulfide interchange in water and in polar aprotic solvents
1
using dynamic H NMR line shape analysis. J. Am. Chem. Soc. 1990,
112, 1190−1197.
(47) Rahman, I.; Macnee, W. Role of oxidants/antioxidants in
smoking-induced lung diseases. Free Radical Biol. Med. 1996, 21, 669−
681.
(48) Mazor, D.; Greenberg, L.; Shamir, D.; Meyerstein, D.;
Meyerstein, N. Antioxidant properties of bucillamine: possible mode
of action. Biochem. Biophys. Res. Commun. 2006, 349, 1171−1175.
̈
(49) Gungor, N.; Ozyurek, M.; Gucļ u, K.; Çekic,̧ S. D.; Apak, R.
̈ ̈
̈
̈
̈
Comparative evaluation of antioxidant capacities of thiol-based
antioxidants measured by different in vitro methods. Talanta 2011,
83, 1650−1658.
(50) Benzie, I. F. F.; Strain, J. J. The ferric reducing ability of plasma
(FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal.
Biochem. 1996, 239, 70−76.
(51) Scott, B. C.; Aruoma, O. I.; Evans, P. J.; O’Neill, C.; Van
DerVliet, A.; Cross, C. E.; Tritschler, H.; Halliwell, B. Lipoic and
dihydrolipoic acid as antioxidants: a critical evaluation. Free Radical
Res. 1994, 20, 119−133.
(52) Suzuki, Y. J.; Tsuchiya, M.; Packer, L. Thioctic acid and
dihydrolipoic acid are novel antioxidants which interact with reactive
oxygen species. Free Radical Res. Commun. 1991, 15, 255−263.
(53) Kermasha, S.; Goetghebeur, M.; Monfette, A.; Metche, M.;
Rovel, B. Inhibitory effects of cysteine and aromatic acids on tyrosinase
activity. Phytochemistry 1993, 34, 349−353.
6855
dx.doi.org/10.1021/jf401120h | J. Agric. Food Chem. 2013, 61, 6848−6855