Antioxidant Properties of Heterocyclic Intermediates of the Maillard Reaction and Structurally Related Compounds

Article abstract of DOI:10.1021/acs.jafc.6b03398

It is well established that a wide range of reductones is formed in the course of the Maillard reaction and that these substances contribute to the oxidative stability of food. The aim of this study was to analyze 12 important heterocyclic intermediates with and without reductone structure as well as structurally related substances under equal conditions to compare their antioxidant properties in detail. For this purpose, five methods were selected including photometrical methods such as the trolox equivalent antioxidant capacity assay and an electron paramagnetic resonance spectroscopic method. Reductones with furan-3-one structure and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one were reducing in all assays, whereas isomaltol and maltol did not react in assays based on the reduction of metal ions because of their complexing abilities. The introduction of protecting groups to the free hydroxyl functions of selected reductones could nearly eliminate their reducing abilities. In addition, the oxidation products of the different reductive heterocycles were compared after treatment with iodine. Mainly short-chained organic acids such as lactic, glycolic, and glyceric acid are formed as result of the degradation, which indicates 1,3-dicarbonyl cleavage reactions of corresponding tricarbonyl compounds as intermediates of the oxidation.

Full text of DOI:10.1021/acs.jafc.6b03398

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Article
Antioxidant Properties of Heterocyclic Intermediates of
the Maillard Reaction and Structural Related Compounds
Clemens Kanzler, Paul Timo Haase, Helena Schestkowa, and Lothar W. Kroh
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03398 • Publication Date (Web): 02 Oct 2016
Downloaded from http://pubs.acs.org on October 9, 2016
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Page 1 of 38
Journal of Agricultural and Food Chemistry
Antioxidant Properties of Heterocyclic Intermediates of the Maillard
Reaction and Structural Related Compounds
Clemens Kanzlera,*, Paul T. Haasea, Helena Schestkowaa, and Lothar W. Kroha
a Berlin Institute of Technology, Department of Food Chemistry and Food Analysis, Gustav-
Meyer-Allee 25, TIB 4/3-1, D-13355 Berlin, Germany
* Address of correspondence: Clemens Kanzler, Berlin Institute of Technology, Department
of Food Chemistry and Food Analysis, GustavꢀMeyerꢀAllee 25, Dꢀ13355 Berlin, Germany.
Telephone: +49ꢀ30ꢀ31472404; Fax: +49ꢀ30ꢀ31472585; eꢀMail: clemens.kanzler@tuꢀberlin.de
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ABSTRACT
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It is well established that a wide range of reductones is formed in the course of the
Maillard reaction and that these substances contribute to the oxidative stability of food. The
aim of this study was to analyze twelve important heterocyclic intermediates with and without
reductone structure as well as structural related substances under equal conditions to compare
their antioxidant properties in detail. For this purpose, five methods were selected including
photometrical methods like the trolox equivalent antioxidant capacity assay and an electron
paramagnetic resonance spectroscopic method. Reductones with furanꢀ3ꢀone structure and
2,3ꢀdihydroꢀ3,5ꢀdihydroxyꢀ6ꢀmethylꢀ4Hꢀpyranꢀ4ꢀone were reducing in all assays while
isomaltol and maltol did not react in assays based on the reduction of metal ions because of
their complexing abilities. The introduction of protecting groups to the free hydroxyl
functions of selected reductones could nearly eliminate their reducing abilities. In addition,
the oxidation products of the different reductive heterocycles were compared after treatment
with iodine. Mainly shortꢀchained organic acids like lactic, glycolic and glyceric acid are
formed as result of the degradation which indicate 1,3ꢀdicarbonyl cleavage reactions of
corresponding tricarbonyl compounds as intermediates of the oxidation.
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18 KEYWORDS
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Maillard reaction; antioxidant; prooxidant; reductone; heterocyclic compound
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20 INTRODUCTION
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Most foods are treated with heat during their production or preservation, which leads to the
thermal degradation of reducing carbohydrates in the corresponding food matrices, especially
in the presence of amino compounds like amino acids or proteins.1–3 The multitude of
reactions taking place under the described circumstances is commonly summarized under the
term Maillard reaction. Products of this complex reaction cascade exhibit color, flavor, taste
and texture impressions that the consumer typically associates with certain foods.
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Beside their significance for organoleptic characteristics Maillard reaction products are
known for their functional abilities such as their complexing4–8 or reducing9–14 properties,
which can take influence on the oxidative stability of different foods. In roasting processes
during the production of beer,15 coffee16; 17 and cacao18; 19 antioxidants and prooxidants are
formed through degradation of carbohydrates to a relevant extent. In literature, the reductive
potential of Maillard reaction mixtures is mostly traced back to a substance class called
reductones, a term either used without structural relation or to describe a structure with an
enediol next to a carbonyl (structure 1 in Figure 1). The latter can react analogous to ascorbic
acid and consequently protect sensitive substrates from oxidation agents or radicals through
an oxidation reaction (A) to the corresponding tricarbonyl compound 2, which defines them
as antioxidants. But again in analogy to ascorbic acid they are capable of reducing metal ions
such as copper(II) or iron(III) (B). This behavior may eliminate these ions as oxidizers,
however, unlike other oxidizing agents their reduced form is not inactive, but generates
reactive oxygen species (ROS) under aerobe conditions in a Fenton like reaction (C).20–22
Thus, reductones can create a prooxidative milieu in presence of metal ions, but like indicated
in Figure 1, they are capable of compensating their selfꢀinduced ROS generation at the
expense of their antioxidant capacity.
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Our previous investigations14 showed that early Maillard intermediates with potential
reductone isomers such as the 1,2ꢀdicarbonyl compounds 1ꢀdeoxyglucosone and glucosone
exhibit reducing and radical scavenging properties, but the heterocyclic intermediate maltol
was reacted considerably faster. Therefore, the aim of the present work was to investigate the
antioxidant properties of important heterocyclic Maillard reaction products of different
substance classes in comparison to structural related compounds. An overview of the
examined substances is given in Figure 2.
Most of the used Maillard intermediates are successors of 1,2ꢀdicarbonyls and some carry
over the reductone structure of their precursors. In these particular compounds, one of the
hydroxyl functions of the reductone is involved in an ether bridge, for this reason they are
called reductone ethers.23 1ꢀdeoxyglucosone is known to form a wide range of different
heterocyclic degradation products, for example furans like 2ꢀacetylfuran 524 and isomaltol,25
furanꢀ3ꢀones like norfuraneol 1026 and furaneol 11a24 or pyranꢀ4ꢀones like 2,3ꢀdihydroꢀ3,5ꢀ
26
dihydroxyꢀ6ꢀmethylꢀ4Hꢀpyranꢀ4ꢀone (DHHM) 16.2;
The corresponding 1ꢀdeoxyosone
derivatives of maltose and lactose degrade additionally to the pyranꢀ4ꢀone maltol 13a27 and
the furan Oꢀgalactosylisomaltol 6a.28; 29 But the main degradation products of 1ꢀdeoxyosones
derivatives of monoꢀ and disaccharides are the pyranꢀ4ꢀones 16 and 13a, respectively. In
contrast, osones and 3ꢀdeoxyosones form a smaller product range and the main degradation
pathways lead to furans in both cases: furfural 3 and HMF 4, respectively.2 2ꢀacetylpyrrole 7
as the only pyrrole structure in this selection is formed by 3ꢀdeoxyosones.24 Several of the
named compounds are also formed through aldol reactions of shortꢀchained degradation
products of C6ꢀ1,2ꢀdicarbonyls, for example 4 from methylglyoxal and glyceric aldehyde30 or
11a from methylglyoxal.31 The formation of sotolon 8 and abhexon 9, which show strong
structural similarities to 11a and 12, is described in literature32; 33 via aldol reaction or
condensation from different acids and aldehydes.
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Besides these Maillard reaction products, structural related substances were analyzed in
comparison, like 12 and 14 as ethyl derivatives of 11a and 13a or acetyl protected derivatives
of different reductone ethers like 6b, 11b and 13b. Kojic acid 15 and deferiprone 17 were
included for their structural similarities to 13a.
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Up to now, the majority of studies on antioxidant Maillard reaction intermediates did focus
on reaction mixtures or fractions of these and only a small amount did analyze isolated
substances. The results of these publications are hard to compare and consequently, the
antioxidant properties of many important compounds are only partially described. Therefore,
we analyzed all the substances mentioned above under identical conditions with an array of
five methods in comparison to ascorbic acid to overcome the described shortcomings.
Additionally, we investigated the oxidation products of selected reductone ethers and
postulated an according reaction pathway. These results might help in understanding the
effects of Maillard reaction products on the oxidative stability of food.
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82 MATERIALS AND METHODS
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Chemicals. 2ꢀacetylfuran, 5ꢀhydroxymethylfurfural, acetyl chloride, deuterium oxide,
dichloromethaneꢀd2, ethylmaltol, furaneol, glycolic acid, ꢀascorbic acid, piperidine and
L
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propionic acid were purchased from Acros Organics (Geel, Belgium); aqueous hydrogen
chloride solution (1 mol/L) and hydrogen chloride (37 %) were purchased from BerndꢀKraftꢀ
GmbH (Duisburg, Germany); methanolꢀd4 was purchased from Eurisoꢀtop (Paris, France);
ammonium acetate and petroleum ether were purchased from Fisher Scientific (Hagen,
Germany); aqueous sodium hydroxid solution (1 mol/L), formic acid, iron(III) nitrate
nonahydrate, lactose monohydrate, potassium dihydrogen phosphate, potassium hydrogen
phosphate, silica gel 60, sodium carbonate and sodium chloride were purchased from Merck
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(Darmstadt, Germany); acetic acid, aqueous iodine reference solution (0,05 mol/L), Dꢀ
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glucose, hexane and sodium sulfate were purchased from Roth (Karlsruhe, Germany); 1,10ꢀ
phenanthroline, 2ꢀacetylpyrrole, abhexone, ammonium 2,2'ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ
6ꢀsulphonate), acetic anhydride, copper(II) sulfate pentahydrate, deferiprone, ethylfuraneol,
FolinꢀCiocalteu reagent (2 mol/L), furfural, hemicalcium glycerate dehydrate, kojic acid,
maltol, mesifuran, trimethylsilyl chloride/N,Oꢀbis(trimethylsilyl)trifluoroacetamide (1:99,
v/v), norfuraneol, N-tertꢀbutylꢀαꢀphenylnitrone, 6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromanꢀ2ꢀ
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carboxylic acid,
potassium
nitrosodisulfonate,
potassium
persulfate, sodium
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bathocuproinsulfonate, sodium lactate, sotolone and βꢀgalactosidase (13,4 units/mg) were
purchased from SigmaꢀAldrich (Steinheim, Germany); dodecyl chloroformate was purchased
from TCI chemicals (Eschborn, Germany); 2ꢀpropanol, ethanol, ethyl acetate, methanol and
trichloromethane were purchased from VWR chemicals (Darmstadt, Germany).
2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DHHM 16). The synthesis of 16
was carried out as described by Kim et al.25, Voigt et al.34, Davidek et al.35 with minor
modifications. 36.03 g
D
ꢀglucose (0.2 mol) and 19.8 mL piperidine (0.2 mol) were solved in
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150 mL ethanol and stirred for 90 min at 90 °C. A mixture of 11.4 mL glacial acetic acid
(0.2 mol) and 30 mL ethanol was added dropwise through the reflux condenser. The reaction
mixture was stirred for 22 h under reflux, concentrated under reduce pressure to one third of
the initial volume, filtered and the residue was washed with 150 mL 2ꢀpropanol. The solvent
of the filtrate was removed in vacuum and the residue was taken up in 200 mL water. The pH
value was adjusted to 4 after addition of 60 g sodium chloride. The solution was extracted
overnight with 400 mL ethyl acetate in a rotational perforator at room temperature. The
organic layer was dried over sodium sulfate and the solvent was removed under reduced
pressure. The crude product was cleaned up with column chromatography (silica gel 60; ethyl
acetate/petroleum ether, 1:1, v/v) and vacuum distillation (0.1 mbar; oil bath at 180 °C). The
product fraction between 120 and 127 °C was taken up in 50 mL diethyl ether and stored at –
20 °C. The product crystallized overnight, was filtered off and again taken up in 20 mL
diethyl ether. After storage at –20 °C 1.993 mg (13.4 mmol; 7 %) of the product in form of
beige crystals were obtained overnight. 1HꢀNMR (500 MHz, methanolꢀd4): δ (ppm) = 2.03 (s,
3H); 4.07 (dd, J = 11.15 Hz; J = 9.60 Hz, 1H); 4.17 (dd, J = 9.63 Hz; J = 4.68 Hz, 1H); 4.32
(dd, J = 11.18 Hz; J = 4.68 Hz, 1H). 13CꢀNMR (125 MHz, methanolꢀd4): δ (ppm) = 15.5;
69.0; 72.7; 132.8; 161.1; 189.2. GCꢀMS: tR = 24.30 min; m/z = 144 [M+, 57 %], 101 (49), 73
(31), 55 (24), 43 (100).
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O-galactosylisomaltol (6c). 6c was synthesized according to Fox et al.36 1HꢀNMR
(500 MHz, deuterium oxide): δ (ppm) = 2.35 (s, 3H) 6.54 (d, J = 2.12 Hz, 1H); 3.71–3.74 (m,
3H); 3.81–3.86 (m, 2H); 3.96 (d, J = 3.35 Hz, 1H); 5.06 (d, J = 7.80 Hz, 1H); 6.62 (d,
J = 2.05, 1H) 7.62 (d, J = 2.05 Hz, 1H). 13CꢀNMR (125 MHz, deuterium oxide): δ (ppm) =
26.1; 60.7; 68.3; 70.2; 72.5; 75.9; 101.9; 104.4; 137.5; 148.7; 154.1; 189.2. ESIꢀMS:
m/z = 289 [M+H, 2 %], 127 [isomaltol+H, 100 %], 311 [M+Na, 91 %], 327 [M+K, 28 %],
306 [M+NH4, 3 %].
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Isomaltol (6a). 6a was synthesized from 6c according to Fox et al.36 βꢀgalactosidase was
1
used for the cleavage of 6c instead of defatted almond meal. HꢀNMR (500 MHz,
dichlormethaneꢀd2): δ (ppm) = 2.42 (s, 3H); 6.34 (d, J = 2.04 Hz, 1H); 7.36 (d, J = 2.03 Hz,
13
1H). CꢀNMR (125 MHz, dichlormethaneꢀd2): δ (ppm) = 24.4; 104.7; 137.0; 146.2; 156.7;
189.8. GCꢀMS: tR = 18.50 min; m/z = 126 [M+, 68 %], 111 (100), 83 (8), 55 (23), 43 (28).
Acetylation of Reductones. All acetylation reactions were performed as described by
Sasaki et al.37 for furaneol. Esterifications with acetic anhydride were carried out at 80 °C for
6 h and esterifications with acetyl chloride at room temperature for 3 h.
O-Acetylisomaltol (6b). Acetyl chloride was used as acetylation reagent and furaneol 11a
was replaced by 6a. Purification of the crude product was done by column chromatography
1
(silica gel 60; ethylacetate/petroleum ether, 1:1, v/v). HꢀNMR (500 MHz, dichlormethaneꢀ
d2): δ (ppm) = 2.35 (s, 3H); 2.45 (s, 3H); 6.67 (d, J = 2.00 Hz, 1H); 7.52 (d, J = 2.00 Hz, 1H).
13CꢀNMR (125 MHz, dichlormethaneꢀd2): δ (ppm) = 20.6; 26.8; 108.6; 140.8; 143.1; 144.5;
167.2; 185.4. GCꢀMS: tR = 26.02 min; m/z = 168 [M+, 7 %], 126 (54), 111 (77), 43 (100), 42
(5).
O-Acetylfuraneol (11b). Acetyl chloride was used as acetylation reagent. Purification of
the crude product was done by column chromatography (silica gel 60; ethylacetate/petroleum
1
ether, 3:7, v/v). HꢀNMR (500 MHz, deuterium oxide): δ (ppm) = 1.44 (d, J = 7.25 Hz, 3H);
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2.22 (s, 3H); 2.27 (s, 3H); 4.83 (q, J = 7.10 Hz, 1H). CꢀNMR (125 MHz, deuterium oxide):
δ (ppm) = 13.6; 15.3; 19.5; 82.8; 128.2; 171.0; 185.5; 199.2. GCꢀMS: tR = 25.88 min;
m/z = 170 [M+, 5 %], 128 (32), 85 (38), 72 (10), 57 (28), 43 (100).
O-Diacetylfuraneol (11c). Acetic anhydride was used as acetylation reagent. Purification
of the crude product was done by column chromatography (silica gel 60;
ethylacetate/petroleum ether, 1:1, v/v). 1HꢀNMR (500 MHz, dichlormethaneꢀd2):
δ (ppm) = 2.16 (s, 3H); 2.24 (s, 3H). 13CꢀNMR (125 MHz, dichlormethaneꢀd2):
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δ (ppm) = 10.9; 20.0; 128.0; 138.0; 167.8. GCꢀMS: tR = 27.81 min; m/z = 212 [M+, 17 %],
170 (35), 128 (99), 127 (26), (31), 43 (100).
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O-Acetylmaltol (13b). Acetic anhydride was used as acetylation reagent and furaneol 11a
was replaced by maltol 13a. Purification of the crude product was done by column
chromatography (silica gel 60; ethylacetate). 1HꢀNMR (500 MHz, deuterium oxide):
δ (ppm) = 2.31 (s, 3H); 2.34 (s, 3H); 6.53 (d, J = 5.65 Hz, 1H); 8.06 (d, J = 5.65 Hz, 1H). 13Cꢀ
NMR (125 MHz, deuterium oxide): δ (ppm) = 14.5; 19.6; 115.5; 137.3; 157.5; 163.4; 171.0;
175.0. GCꢀMS: tR = 28.33 min; m/z = 168 [M+, 4 %], 126 (100), 71 (33), 55 (17), 43 (87).
Oxidative Degradation of Selected Reductone Ethers. To 10 mL of aqueous solutions of
norfuraneol 10, furaneol 11a, ethylfuraneol 12, maltol 13a, ethylmaltol 14 or DHHM 16
(20 mmol/L) 4 mL of aqueous iodine reference solution (50 mmol/L iodine/potassium iodid)
were added dropwise. After every 1 mL of added iodine solution the pH value of the reaction
mixture was adjusted to 5 and after addition of all 4 mL to 7.
GC-MS. For all GCꢀMS analyses the following system was used: autosampler, Shimadzu
AOCꢀ20i; gas chromatograph, Shimadzu GCꢀ2010; detector, Shimadzu GCMSꢀQP2010 Plus
(Duisburg, Germany); column, Supelco SLB™ꢀ5ms 60 m x 0.25 mm x 0.25 µm (Bellefonte,
USA).
GC-MS Analysis of Heterocyclic Compounds. The following settings were used: carrier
gas, helium; flow, 2.00 mL/min; split, 1:5; injection volume, 1 µL; injection temperature,
200 °C; interface temperature, 270 °C; ion source temperature, 200 °C; ionization energy,
70 eV; solvent cut time, 12.50 min; mass scan, 35–250 m/z; temperature gradient, 30 °C for
3 min, 5 °C/min to 120 °C, 13 °C/min to 180 °C, 20 °C/min to 300 °C, 300 °C for 5 min. The
samples were dissolved in ethyl acetate (1 mmol/L) for analysis.
GC-MS Analysis of Organic Acids as Trimethylsilyl Derivatives. The following
settings were used: carrier gas, helium; flow, 1.00 mL/min; split, 1:10; injection volume,
1 µL; injection temperature, 250 °C; interface temperature, 300 °C; ion source temperature,
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200 °C; ionization energy, 70 eV; solvent cut time, 8.00 min; mass scan, 28–800 m/z;
temperature gradient, 80 °C for 1 min, 6 °C/min to 240 °C, 240 °C for 20 min, 10 °C/min to
320 °C, 320 °C for 4 min. The solvent of 200 µL sample was removed under a nitrogen
stream and the residue taken up in 50 µL pyridine and 50 µL silylation reagent (trimethylsilyl
chloride/N,Oꢀbis(trimethylsilyl)trifluoroacetamide, 1:99, v/v). After derivatization over night
at room temperature the samples were diluted with 150 µL ethyl acetate and subjected to GCꢀ
MS analysis.
GC-MS Analysis of Acetic and Formic Acid as Dodecyl Esters. The following settings
were used: carrier gas, helium; flow, 1.64 mL/min; split, 1:30; injection volume, 1 µL;
injection temperature, 220 °C; interface temperature, 270 °C; ion source temperature, 200 °C;
ionization energy, 70 eV; solvent cut time, 7.10 min; mass scan, 25–450 m/z; temperature
gradient, 100 °C for 0 min, 50 °C/min to 200 °C, 10 °C/min to 270 °C, 270 °C for 2 min,
5 °C/min to 300 °C, 300 °C for 20 min. 100 µL sample and 50 µL pyridine were mixed with
50 µL dodecyl chloroformate for derivatization. After 5 min the samples were extracted with
200 µL hexane, 100 µL of the organic layer were taken off and diluted with 200 µL hexane
before subjected to GCꢀMS analysis.
ESI-MS. A Thermo Scientific TSQ Vantage System mass spectrometer (Darmstadt,
Germany) equipped with an Ion Max Source (HꢀESI II probe) ion source was used for direct
injection. The ESIꢀMS was controlled via Thermo Xcalibur v2.1.0 and the settings were as
following: ionization voltage, 3000 V; ionization temperature, 450 °C; capillary temperature,
270 °C; flow, 100 µL/min. The samples were solved in methanol/water mixtures with
50 mmol/L ammonium acetate as modifier.
Electron Paramagnetic Resonance (EPR) Spectroscopy. EPR measurements were
performed with a Magnettech MiniScope MS 100 spectrometer (Berlin, Germany).
Recording of EPR Radical Scavenging Kinetics. Potassium nitrosodisulfonate (Fremy’s
salt) was used as stabilized radical. The following settings were used: magnetic flux density,
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338.8 mT; sweep, 7.0 mT; sweep time, 30 s; modulation, 0.150 mT; microwave attenuation,
10 dB; gain, 6; passes, 1; measurement interval, 60 sꢀ1; measurement time, 30 min.
Approximately 300 mg of Fremy’s salt were dissolved in 100 mL phosphate buffer
(50 mmol/L, pH 7.4). This solution was diluted until a 1:1 mixture with a trolox solution
(0.25 mmol/L) showed a radical degradation of 50 % after 10 min.
100 µL diluted Fremy’s salt solution (1 mmol/L) and 100 µL sample (diluted with water)
were mixed for 10 s and measured in comparison to a blank sample that consisted of 100 µL
diluted Fremy’s salt solution (1 mmol/L) and 100 µL phosphate buffer (pH 7.4). For the
calculation of the antioxidant capacity, the measurement point at 30 min was used.
Recording of EPR Spectra of PBN radical adducts. The following settings were used:
magnetic flux density, 338.6 mT; sweep, 7.0 mT; sweep time, 30 s; modulation, 0.100 mT;
microwave attenuation, 10 dB; gain, 500; passes, 16.
100 µL PBN solution (100 mmol/L) in phosphate buffer (50 mmol/L, pH 7.4) were mixed
with 100 µL sample (20 mmol/L) and incubated at 50 °C for 2 h before measurement.
Addition of metal ions was not necessary because of trace amounts of copper(II) and iron(III)
in all used chemicals and the membrane filtrated water.
Recording of EPR Spectra of Copper(II) Complexes. The following settings were used:
magnetic flux density, 312.0 mT; sweep, 150.0 mT; sweep time, 30 s; modulation, 1.000 mT;
microwave attenuation, 10 dB; gain, 100; passes, 16.
Complex solutions with final concentration of 10 mmol/L copper(II) and 20 mmol/L
coordinator were prepared in membrane filtrated water. 250 µL complex solution were mixed
with 750 µL membrane filtrated water or phosphate buffer (50 mmol/L, pH 7.4).
UV-Vis Photometric Assays. For the TEAC and the FolinꢀCiocalteu assay, a BioꢀTek
Instruments UVIKON XL photospectrometer was used. Semiꢀmicro disposable cuvettes were
purchased from VWR (Darmstadt, Germany).
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TEAC Assay. Six trolox standards (0.05–0.50 mmol/L) were used for calibration.
Solutions of ABTS (0.5 mmol/L) and potassium persulfate (10 mmol/L) were prepared in
PBS buffer (5 mmol/L, pH 7.2–7.4). For the measurement 750 µL ABTS solution and 150 µL
sample were mixed in a 1.5 mL reaction tube. The reaction was started with addition of
300 µL potassium persulfate solution. The mixture was homogenized and after 6 min of
incubation the absorption at 734 nm was measured in comparison to an empty cuvette.
Folin–Ciocalteu Assay. The calibration was performed with five trolox standards (0.20–
2.00 mmol/L). 150 µL sample, 750 µL Folin & Ciocalteus phenol reagent (diluted 1:10 with
membrane filtrated water) and 600 µL sodium carbonate solution (7.5 wt%) were mixed for
the measurement. This mixture was incubated for 15 min at 35 °C and measured at 736 nm
compared to a blank sample (membrane filtrated water instead of sample).
Microplate Assays. For the CUPRAC and phenanthroline assay, a Tecan Infinite M200
microplate reader was used. Micro plates with 96 wells were purchased from TPP
(Trasadingen, Switzerland).
CUPRAC Assay. The calibration was performed with five trolox standards (0.050–
0.100 mmol/L). 50 µL sample, 50 µL membrane filtrated water and 100 µL BCS solution
(0.63 mmol/L) were mixed for the initial measurement at 490 nm. After addition of 50 µL
copper(II) solution (0.25 mmol/L) and 30 min incubation time the absorbance at 490 nm was
measured again.
Phenanthroline Assay. The calibration was performed with five trolox standards (0.050–
0.100 mmol/L). 50 µL sample, 160 µL membrane filtrated water and 40 µL phenanthroline
solution (6.66 mmol/L) were mixed for the initial measurement at 505 nm. After addition of
40 µL iron(III) solution (1.875 mmol/L) and 180 min incubation time the absorbance at
505 nm was measured again.
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Antioxidant Capacity. For the determination of antioxidant properties, it is important to
consider different antioxidant assays, because of the different reaction mechanisms of each
assay and antioxidant.38–40 Therefore, five assays with trolox as reference were used: the
trolox equivalent antioxidant capacity (TEAC) assay to measure the inhibition of ABTS
radical cation formation, the Folin–Ciocalteu reagent (FCR) assay to measure the reduction of
an inorganic oxidizer, an electron paramagnetic resonance (EPR) spectroscopic method to
determine the radical scavenging abilities, the cupric ion reducing antioxidant capacity
(CUPRAC) assay to measure the reduction of copper(II) ions and the phenanthroline assay to
measure the reduction of iron(III) ions.
The results of the antioxidant assays expressed as trolox equivalents (TE) are shown in
Figure 3. Compounds reaching 1000 mmol TE per mol of substance (mmol TE/mol) exhibit
the same antioxidant capacity as trolox. In addition, the weighted average antioxidant capacity
(WAAOC) as suggested by Tabart et al.38 was calculated for the metal based and nonꢀmetal
based assays separately to present the results in a more comprehensive way (Figure 4). The
reductone ascorbic acid (asc in Figure 3 and 4) was measured in comparison to the reductone
ethers and did not perform equally in all assay in respect to trolox. While the metal based
assays give nearly identical antioxidant capacities with (1012 ± 49) mmol TE/mol (CUPRAC)
and (1061 ± 102) mmol TE/mol (phenanthroline), ascorbic acid shows varying capacities of
(272 ± 73) mmol TE/mol
(TEAC),
(1745 ± 206) mmol TE/mol
(FCR)
and
(939 ± 19) mmol TE/mol (EPR) in the reducing and radical scavenging assays. Nevertheless,
both WAAOCs are close to the reference trolox with 0,88 and 1,03. Similar observations
could be made for the reductones 10, 11a, 12, 13 and 16. Their WAAOCs are on comparable
level, but the results of the single assays are diverse especially for the maltol derivatives 13a,
14, 15, 16 in the nonꢀmetal based assays. This underlines the need to use a range of several
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assays instead of single methods to produce representative results for the measurement of
antioxidant capacities.
Based on the results of the used assays the tested compounds can be divided into three
different groups (compare Table 1). The first group does not exhibit reducing abilities at all.
Under the conditions of the used testing systems this includes all compounds lacking a
reductone structure like furfural 3, HMF 4, 2ꢀacetylfuran 5 or 2ꢀacetylpyrrole 7 as well as
compounds with protected reductone functions like the acetylated derivatives 6b, 11b, 11c
and 13b. The second group shows reducing abilities in all assays, whereas the third and last
group is nonꢀreductive in the CUPRAC and phenanthroline assay but in the rest of the assays.
Although all substances of the second and third group are reductone ethers only the furaneol
derivatives 10, 11a and 12 as well as DHHM 16 are reducing under all conditions. Isomaltol
6a and the maltol derivatives 13a, 14, 15, 17 exhibit complexing abilities in presence of metal
ions, which are used as oxidizers in the CUPRAC and phenanthroline assay, respectively.
This behavior is well described in literature4–8 and explains the missing reducing abilities of
the latter substances in both assays.
Reducing Abilities and Radical Scavenging. The furaneol derivatives 10, 11a, 12, maltol
13a and DHHM 16 exhibit WAAOCs between 70 and 80 % of ascorbic acid. While the effect
of the alkyl sidechains of 10 and 12 on the antioxidant behavior in comparison to 11a is
negligible, the ethyl derivative of maltol 14 shows a significant lower antioxidant capacity in
the TEAC and the FCR assay than maltol 13a resulting in a lower WAAOC as well. The
maltol isomer 6a exhibits even weaker reducing abilities than 14. In contrast, the structural
related compounds kojic acid 15 and deferiprone 17 show higher performances in single
assays (Figure 3) than 13a causing higher WAAOCs for these substances (Figure 4). In the
case of 17 the WAAOC is twofold of its Oꢀanalogue 13a. In general, the antioxidant
capacities of the furaneol derivatives are more homogeneous in the single assays than the
capacities of the substances structural related to maltol. The introduction of protecting groups
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nearly eliminates the reducing abilities of all tested reductone ethers and even the weak
reducing abilities of 6a get lowered. Consequently, the precursor of 6a, Oꢀgalactosylisomaltol
6c, does not exhibit a relevant WAAOC as well.
The antioxidant properties of sotolon 8 and abhexon 9, which are isomers of 11a and 12,
are comparable to 6a although both compounds do not possess a reductone structure.
Reduction of Metal Ions. Even though the CUPRAC and the phenanthroline assay are
commonly used to measure antioxidant capacities, the reduction of metal ions might cause
prooxidative effects due to radical generation as mentioned in the introduction. Therefore,
substances that do not show reducing abilities in these assays are not necessarily weak
antioxidants.
Likewise, as for the reducing and radical scavenging assays, the WAAOCs of the furaneol
derivatives 10, 11a, 12 as well as the WAAOC of DHHM 16 are on an equal level between 70
and 80 % of ascorbic acid (Figure 4). The maltol derivatives 13a, 14, 15, 17 and isomaltol 6a
are known to form sTable complexes with metal ions involving their carbonyl and hydroxyl
functions4–8 and consequently do not exhibit reducing abilities in both metal based assays.
The different behavior of selected reductones in presence of copper(II) ions was additionally
analyzed with EPR spectroscopy (Supporting Information A). While 6a and 13a show strong
complex signals at pH 7.4 11a and 16 do not give signals, indicating that they are not capable
of complex formation under the given conditions. Considering, that 11a and 16 share relevant
structural features with the complexing agents 6a and 13a, the different reactivity has to be
caused by other factors. The most obvious distinction of these substances is their conjugated
doubleꢀbond system. 11a and 16 are only partially sp2ꢀhybridized and might be more prone to
oxidation then the aromatic compounds 6a and 13a which also show a considerably slower
radical scavenging behavior in the EPR assay (Figure 3) in support of this hypothesis.
Furthermore, the additional hydroxyl function of 16 could possibly interfere during the
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complex formation and the known enolization reactions of 11a could affect its complexing
abilities negatively.
Even though the sotolon derivatives 8 and 9 did show antioxidant capacities in the FCR
and the TEAC assay, they are not reducing in presence of copper(II) or iron(III). EPR
experiments confirmed that this is not due to their complexing abilities (data not shown).
Prooxidative Behavior. The radical generation via Fenton reaction (Figure 1) by
reductone ethers is already reported for DHHM 1641 and the furaneol derivatives 1042, 11a43;
44, 1245 and 4ꢀhydroxyꢀ2ꢀ(hydroxymethyl)ꢀ5ꢀmethylfuranꢀ3(2H)ꢀone.46 Consequently, for
most of these substances effects like DNA strand breaking and mutagenicity could also be
observed by the respective authors. These results are in compliance with the behavior of 10,
11a, 12 and 16 in the CUPRAC and the phenanthroline assay where they did show reducing
abilities (Figure 3 and 4). To compare the radical generation of the reducing substances used
in this study under identical conditions EPR spin trap experiments were conducted with
selected reductone ethers as well as their acetylated derivatives. As expected, samples of the
furaneol derivatives 10, 11a and 12 give a PBN signal due to radical generation while the
complexing agents 13a and 14 do not (selected spectra are in Supporting Information B). The
signal of the protected furaneol derivative 11b is significant smaller than the signal of 11a.
Surprisingly, 6a and 6b give comparable small signals even though 6a has complexing
abilities and 6b carries a protecting group. This observation might be caused by thermal
degradation of both compounds under the chosen incubation conditions (5 h at 50 °C)
because, unlike all other samples the solutions of 6a and 6b turned yellow after incubation. 16
induces a smaller PBN signal than 10, 11a and 12 corresponding to its weaker metal reducing
abilities found in the CUPRAC and the phenanthroline assay (Figure 3 and 4).
Oxidation Mechanism. Our results show that especially the furaneol derivatives 10, 11a,
12 and DHHM 16 exhibit antioxidant capacities that are comparable to the reductone ascorbic
acid, even though one of the hydroxyl functions is etherified in the respective heterocycles. It
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could be assumed that the free hydroxyl function of the reductone ethers is of importance for
the oxidation mechanism, what could be verified with the introduction of protecting groups.
The monoꢀ and diesterification of 11a, which leads to 11b and 11c, did additionally show the
significance of the carbonyl function adjacent to the enediol. 11a exhibits ketoꢀenolic
tautomerism in aqueous solutions, which is evident in NMR spectra recorded in deuterium
oxide (data not shown), because of the hydrogenꢀdeuterium exchange. Theoretically, ketoꢀ
enolic tautomerism is still possible for the monoester 11b and consequently the enediol could
still be oxidized, but 11b did exhibit less than 20 % of the WAAOC of 11a indicating a slow
enolization. This is supported by NMR data that show only a slow H/D exchange (the
corresponding signal of the exchanged proton decreased by approximately 75 % within 4
weeks relative to the signals of the remaining protons). Consequently, the diester 11c did not
show relevant reducing abilities because of the inability to form an enediolic structure. These
results confirm that enolization reactions which form oxidizable enediols only occur next to
carbonyls, which stabilize them, at least at near neutral pH values.
It seems likely that the oxidation of 11a is initiated by the abstraction of a proton and an
electron from the free hydroxyl function (Figure 5). In more recent publications43; 44 the
authors suggest a ring opening through hydrolysis before the actual oxidation, but 11a is
sTable in neutral aqueous or phosphate buffered (pH 7.4) solutions over a longer time frame
(data not shown) and able to reduce iodine, ABTS radical cations, Fremy’s salt and metal ions
under these conditions. Therefore, we propose that the hydrolysis is induced by the oxidation
and occurs in the course of the reaction rather than beforehand. The radical 18 resulting from
the first electron transfer could be resonanceꢀstabilized by the πꢀsystem of the molecule.
Lacking a second free hydroxyl function, the reductone ether 11a cannot transfer an
additional proton and electron in its original form. Thus, the addition of water in the course of
the second electron transfer may lead to the tricarbonyl compound acetylformoin 19. This
compound is also suggested as an intermediate in literature.43; 44 Both isomers of 19, the openꢀ
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chained form as well as the cyclic hemiacetal, are reductones but an oxidation to the
corresponding tetracarbonyl compound as suggested by Yamashita et al.44 seems unlikely.
Even though 19 is considered to be a reductone (ether) it is a highly insTable tricarbonyl
compound at the same time. Following this mechanism furaneol 11a could be oxidized two
times in a row and should consequently show the twofold antioxidant capacity of ascorbic
acid what could not be observed. Additionally, the identified oxidation products do not
indicate the tetracarbonyl as possible intermediate. For these reasons, 19 is more probable to
be cleaved after the first oxidation than to be oxidized a second time.
The oxidation of 11a yielded mainly lactic acid 20 while methylglyoxal 21 was formed as
byproduct in trace amounts (Figure 5). Both compounds are typical products of dicarbonyl
cleavage reactions25; 47–50 and this particular combination could be derived from hydrolytic
1,3ꢀdicarbonyl cleavage of 19 between Cꢀ3 and Cꢀ4. The formation of an acid and a ketene
structure from a tricarbonyl compound was suggested before by Voigt et al.34 The highly
reactive ketene could easily be hydrolyzed to lactic acid 20 or rearrange to methylglyoxal 21
via ketoꢀenolic tautomerism. The formation of a ketene structure seems unlikely, but the
nearly exclusive formation of 20 supports the described reaction sequence. The corresponding
cleavage between Cꢀ2 and Cꢀ3 would yield acetic acid as well as 3ꢀhydroxyꢀ2ꢀoxobutanal or
2,3ꢀdihydroxybutanoic acid, respectively. This pathway seems to be less favored considering
that acetic acid is only found in trace amounts. The cleavage of the tetracarbonyl as
hypothetical oxidation product of 19 would produce lactic 20 and pyruvic acid but the latter
cannot be detected.
Experiments with derivatives of furaneol show that the mechanism described in Figure 5
can be transferred to other reductone ethers as well. The organic acids that derive from
oxidation of norfuraneol 10 and ethylfuraneol 12 comply with the substitution patterns of their
precursors (Figure 6). While 11a yields two identical Cꢀ3 bodies as cleavage products the
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unsymmetrical substituted derivatives degrade to equal amounts of lactic 20 and glycolic acid
22 or 20 and 2ꢀhydroxybutanoic acid 23.
Comparable cleavage patterns can be observed for maltol derivatives (Figure 7). The
common cleavage product of 13a and 14 is 3ꢀhydroxyacrylic acid 25 that was not available as
reference standard but the mass and corresponding fragments of its trimethylsilyl derivative
can be found with GCꢀMS. Additionally, based on the substitution lactic 20 or 2ꢀ
hydroxybutanoic acid 23 are formed. The oxidation of DHHM 16 yields glyceric acid 24 and
20. Both compounds were previously found by Beck et al.51 which were also able to detect an
ester of 20 and 24 as possible intermediate indicating that the missing link in the reaction
sequence could indeed be a tricarbonyl compound analogous to 19 (Figure 5). But the
postulated tricarbonyl derived from 16 could perform a 1,3ꢀdicarbonyl cleavage in its cyclic
form while 19 only could be cleaved in its openꢀchained form.
Connection between Heterocyclic Intermediates and Summarized Antioxidant
Properties of Maillard Reaction Mixtures. In our previous work,14 we could show that there
is a correlation between browning and antioxidant activity in Maillard reaction mixtures
derived from Dꢀglucose or Dꢀfructose with Lꢀalanine addition suggesting that in all stages of
the reaction antioxidant products are formed. But even though 1ꢀdesoxyglucosone and
glucosone could be identified as reductones, the antioxidant properties of these early
intermediates were not sufficient to explain the measured antioxidant activities of the reaction
mixtures. The heterocyclic substances investigated in the present work represent compounds
of the soꢀcalled intermediate stage,1 which are more stable than their 1,2ꢀdicarbonyl
successors. As expected, all tested reductone ethers showed reducing abilities based on their
stabilized endiol structure incorporated in the corresponding heterocycles (Supporting
Information C) and are likely to make a relevant contribution to the summarized antioxidant
properties of Maillard reaction mixtures not only under laboratory condition but also in food.
However, because of the prooxidative behavior of compounds like the furaneol derivatives
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10, 11a and DHHM 16 analogue to ascorbic acid mixtures of Maillard reaction products bear
the risk to lower the oxidative stability dependent on the content of redox active metal ions.
On the other hand, intermediates like isomaltol 6a and maltol 13a are capable to form
complexes with metal ions (Supporting Information C). Further investigations have to show
the behavior of isolated reductones in complex mixtures and how different reductones may
interact in a positive or negative way. Moreover, it remains an open question in which way
the Maillard reaction might be controlled during food technological processes in order to
increase the oxidative stability of resulting products.
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445 ABBREVIATIONS USED
446
447
448
449
450
451
452
453
454
ABTS, ammonium salt of 2,2'ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ6ꢀsulphonic acid); asc,
ascorbic acid; BCS, sodium salt of bathocuproine sulfate; com, complexing; CUPRAC,
cupric ion reducing antioxidant capacity; DHHM, 2,3ꢀdihydroꢀ3,5ꢀdihydroxyꢀ6ꢀmethylꢀ4Hꢀ
pyranꢀ4ꢀone; EPR, electron paramagnetic resonance; ESI, electron spray ionization FCR,
Folin–Ciocalteu Reagent; GC, gas chromatography; HPLC, high performance liquid
chromatography; HMF, 5ꢀhydroxymethylfurfural; in, inactive; MS, mass spectrospcopy;
PBN, N-tertꢀbutylꢀαꢀphenylnitrone; red, reducing; ROS, reactive oxygen species; TE, trolox
equivalents; TEAC, trolox equivalent antioxidant capacity; WAAOC, weighted average
antioxidant capacity.
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455 SUPPORTING INFORMATION
456
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458
EPR spectra of copper(II) complexes of selected reductone ethers; EPR spectra of PBN
spin adducts after incubation of selected reductone ethers and PBN; structures of complexing
and reducing reductone ethers.
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599 FIGURE CAPTIONS
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Figure 1: Reductones can act as antioxidants by reduction of oxidizers and radicals (A) or
as prooxidants by reduction of metal ions such as iron(III) or copper(II) (B) initiating a redox
cycling of these via Fenton reaction (C) what causes generation of reactive oxygen species
(ROS).
Figure 2: Overview of the analyzed Maillard reaction products and structural related
substances. Substitution patterns and according names are given in Table 1.
Figure 3: Antioxidant capacities for all substances in Figure 2 and ascorbic acid (asc) as
reference.
Figure 4: WAAOCs for all substances in Figure 2 and ascorbic acid (asc) as reference.
Figure 5: Possible oxidation pathway of furaneol 11a.
Figure 6: Oxidation products of norfuraneol 10, furaneol 11a and ethylfuraneol 12.
Figure 7: Oxidation products of DHHM 16, maltol 13a and ethylmaltol 14.
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