Chemical structures of novel maillard reaction products under hyperglycemic conditions

Article abstract of DOI:10.1248/cpb.c17-00809

Two novel and two known compounds, 4-quinolylaldoxime and indole-3-aldehyde, were isolated from a reaction mixture consisting of D-glucose and L-tryptophan at physiological temperature and pH. The chemical structures of the two novel compounds were elucidated by spectroscopic analysis such as X-ray crystallography. One of the novel compound and the indole-3-aldehyde showed mutagenicity toward Salmonella typhimurium YG1024 with S9 mix. Furthermore, 4-quinolylaldoxime was detected from streptozotocin-induced diabetic rat plasma by LC-MS/MS analysis; however, the isolated compounds were not detected in rat diet extracts. To our knowledge, this is the first report in which 4-quinolylaldoxime was detected in rat plasma. These results suggest that amino-carbonyl reaction products may be formed in diabetic condition and induce genetic damage.

Full text of DOI:10.1248/cpb.c17-00809

Vol. 66, No. 4
Chem. Pharm. Bull. 66, 363–367 (2018)
363
Regular Article
Chemical Structures of Novel Maillard Reaction Products under
Hyperglycemic Conditions
Daisuke Imahori, Takahiro Matsumoto, Naoto Kojima, Tomohiro Hasei, Megumi Sumii,
Taishi Sumida, Masayuki Yamashita, and Tetsushi Watanabe*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received October 7, 2017; accepted January 19, 2018
Two novel and two known compounds, 4-quinolylaldoxime and indole-3-aldehyde, were isolated from a
reaction mixture consisting of D-glucose and L-tryptophan at physiological temperature and pH. The chemi-
cal structures of the two novel compounds were elucidated by spectroscopic analysis such as X-ray crystal-
lography. One of the novel compound and the indole-3-aldehyde showed mutagenicity toward Salmonella ty-
phimurium YG1024 with S9 mix. Furthermore, 4-quinolylaldoxime was detected from streptozotocin-induced
diabetic rat plasma by LC-MS/MS analysis; however, the isolated compounds were not detected in rat diet
extracts. To our knowledge, this is the first report in which 4-quinolylaldoxime was detected in rat plasma.
These results suggest that amino-carbonyl reaction products may be formed in diabetic condition and induce
genetic damage.
Key words Maillard reaction; structure elucidation; mutagenicity; LC-MS/MS analysis
10)
11)
The Maillard reaction comprises of a series of complex 4-quinolylaldoxime (3) and indole-3-aldehyde (4) (Fig. 1).
non-enzymatic reactions between carbonyl groups of a re- Compound 1 was isolated as yellow crystals with nega-
ducing sugar and amino groups of amino acids, peptides, or tiveꢀspecificꢀrotationsꢀ([α] −51.2ꢀinꢀCHCl ). For the electron
2
5
D
3
1–3)
+
proteins.
reaction that occur between reducing sugars and amino acids observed at m/z 291 and the molecular formula C H N O
2
In diabetic patients, accelerated amino-carbonyl ionization (EI)-MS of 1, a molecular ion peak [M] was
1
7
13
3
to form reaction products is manifested as hyperglycemia and was determined by high resolution (HR)-MS measurements of
1
13
isꢀ proposedꢀ toꢀ playꢀ aꢀ significantꢀ roleꢀ inꢀ theꢀ complicationsꢀ ofꢀ the molecular ion peak. The H-NMR (CDCl ) and C-NMR
3
diabetes. Epidemiological studies have shown that diabetic pa- (Table 1) spectra of 1, which were assigned by various NMR
tients have increased incidence of cancer in certain organs in- experiments, showed signals associated with two aryl groups
4
–6)
cluding the liver, pancreas and kidney.
Previously, we have [δ 8.24 (d, J=6.8ꢀHz,ꢀ H-1),ꢀ 7.45ꢀ (t,ꢀ J=6.8Hz, H-2), 7.73 (t,
identifiedꢀaꢀnovelꢀmutagen,ꢀ5-amino-6-hydroxy-8H-benzo[6,7]- J=6.8Hz, H-3), 7.60 (d, J=6.8Hz, H-4), 6.66 (d, J=8.3Hz,
azepino[5,4,3-de]quinolin-7-one (ABAQ), that was a Mail- H-16), 7.02 (t, J=8.3Hz, H-17), 6.86 (t, J=8.3Hz, H-18), and
lard reaction product between D-glucose and L-tryptophan at 7.42 (d, J=8.3Hz, H-19)], methine groups bearing two ni-
7
)
physiological temperature and pH. We reported that ABAQ trogen functions [δꢀ 5.97ꢀ (s,ꢀ H-13)],ꢀ andꢀ aꢀ methyleneꢀ groupꢀ [δ
showed genotoxicity in vitro and in vivo and tumor-initiating 3.70 (d, J=14.5ꢀHz,ꢀ H-11)ꢀ andꢀ 3.76ꢀ (d,ꢀ J=14.5ꢀHz,ꢀ H-11)].ꢀ Asꢀ
activity in mouse colon. The mutagenicity of ABAQ was ac- shownꢀinꢀFig.ꢀ2,ꢀtheꢀꢀdoubleꢀquantumꢀfilteredꢀ(DQF)ꢀcorrela-
7
–9)
counting for 18% of the reaction mixture.
Therefore, to tion spectroscopy (COSY) and heteronuclear multiple bond
determine whether other mutagens are formed under physi- connectivity (HMBC) experiments of 1 indicated the presence
ological conditions, we continued to isolate various products ofꢀcertainꢀstructures.ꢀSpecifically,ꢀlong-rangeꢀcorrelationsꢀwereꢀ
from the reaction mixture. This paper reports the chemical observed between the following proton and carbon pairs: H-1
structure, the mutagenic activities, and the detection of reac- andꢀ C-5,ꢀ 7,ꢀ H-11ꢀ andꢀ C-9,ꢀ 12,ꢀ 13,ꢀ 20,ꢀ H-13ꢀ andꢀ C-9,ꢀ 20,ꢀ 15,ꢀ
tion products from diabetic rat plasma.
and H-19 and C-12. Since single crystals of 1 were obtained
from an EtOH–H O solution, X-ray diffraction was performed
2
Results and Discussion
toꢀdetermineꢀtheꢀrelativeꢀconfigurationꢀofꢀ1 (Fig. 3). The rela-
To identify mutagens that are formed under physiological tiveꢀ configurationꢀ wasꢀ determinedꢀ toꢀ beꢀ 12R*, 13S*. Accord-
conditions, we isolated reaction products from previously re- ing to the results from various spectroscopic analysis, includ-
portedꢀreactionꢀmixtures.ꢀBriefly,ꢀ D-glucose and L-tryptophan ing X-ray crystallographic analysis diffraction, the chemical
were dissolved in phosphate buffer, and FeSO and H O (Fen- structure of compound 1ꢀwasꢀclarifiedꢀasꢀshownꢀinꢀFig.ꢀ1.
4
2
2
ton reagent) was subsequently added to the mixture. In previ-
Compound 2 was also isolated as yellow crystals. For the
ous study, we described the mutagenicity of reaction mixture EI-MS of 2, a molecular ion peak [M]+ was observed at m/z
with and without Fenton reagent, and we found Fenton reagent 170 and the molecular formula C H N O was determined
10
6
2
induce eight times stronger mutagenicity. Then, the mixture by HR-MS measurements of the molecular ion peak. The
1
13
was incubated at 37°C for 3d and extracted with CHCl . The
H-NMR (CDCl ) and C-NMR (Table 1) spectra of 2, which
3
3
CHCl soluble fraction was subjected to normal- and reversed- were assigned by various NMR experiments, showed sig-
3
phase silica-gel column chromatography and repeated HPLC. nals associated with a quinoline group [δ 9.24 (d, J=4.1Hz,
From the CHCl -soluble fraction, two new compounds (1 H-2),ꢀ7.95ꢀ(d,ꢀJ=4.1Hz, H-3), 7.04 (d, J=6.9Hz, H-7), 7.71 (t,
3
and 2) were isolated together with two known compounds J=6.9Hz, H-8), 7.67 (d, J=6.9Hz, H-9)]. As shown in Fig. 2,
*
To whom correspondence should be addressed. e-mail: watanabe@mb.kyoto-phu.ac.jp
©
2018 The Pharmaceutical Society of Japan

3
64
Chem. Pharm. Bull.
Vol. 66, No. 4 (2018)
the DQF COSY and HMBC experiments of 2 indicated the troaromatic compounds. Accordingly, compound 1 and indole-
presenceꢀofꢀcertainꢀstructures.ꢀSpecifically,ꢀlong-rangeꢀcorrela- 3-aldehyde (4) showed mutagenicity toward YG1024 in a
tions were observed between the following proton and carbon dose-dependentꢀmannerꢀwithꢀS9ꢀmixꢀ(Tableꢀ2).ꢀThisꢀisꢀtheꢀfirstꢀ
pairs:ꢀH-2ꢀandꢀC-4,ꢀ10,ꢀH-3ꢀandꢀC-5,ꢀ11,ꢀH-7ꢀandꢀC-5,ꢀ9,ꢀandꢀ report to evaluate the mutagenic effect of compound 1. The
H-8ꢀ andꢀ C-6,ꢀ 10.ꢀ However,ꢀ weꢀ couldꢀ notꢀ confirmꢀ theꢀ cor- mutagenic effect of indole-3-aldehyde (4) in the Ames test was
13)
relation between the C-6 position and amide moiety by two- reported in previous report.
dimensional NMR (2D-NMR). Therefore, X-ray diffraction
Identification of the Isolated Compounds (1–4) in Dia-
wasꢀ performedꢀ toꢀ confirmꢀ theꢀ chemicalꢀ structureꢀ ofꢀ 2 (Fig. betic Ratsꢀ ꢀT oꢀ confirmꢀ thatꢀ theꢀ isolatedꢀ reactionꢀ productsꢀ
). Based on the results from X-ray diffraction, the chemi- 1–4 are formed in vivo, we analyzed rat plasma samples by
4
cal structure of compound 2ꢀwasꢀclarifiedꢀasꢀshownꢀinꢀFig.ꢀ1.ꢀ LC-MS/MS. For analysis, a Multiple Reaction Monitoring
The compounds with this three rings skeleton were previously (MRM) method was established. The daughter ions were
isolated as the cell cycle modulators reported from a marine- selected for MRM analysis by product ion scan using m/z
1
2)
derived Streptomyces species.
274.00, 101.00, 129.00, and 91.00 for 1–4, respectively. These
+
Mutagenicity of the Isolated Compounds (1–4) The mu- ions were formed from protonated molecular ions [M+H]
tagenic effects of the isolated compounds (1–4) were tested on of 1–4. The chromatographic run time for the samples was
Salmonella typhimurium YG1024 with S9 mix. S. typhimuri- 75ꢀmin.ꢀ Aꢀ mobileꢀ phaseꢀ consistingꢀ ofꢀ methanolꢀ withꢀ 0.1%ꢀ
um YG1024 is sensitive to mutagens that require metabolic formic acid and water with 0.1% formic acid was used in a
activation by O-acetyltransferase, such as amino- and/or ni-
Fig. 2. Important 2D-NMR Correlations of Compounds 1 and 2
Fig. 1. Chemical Structures of Isolated Compounds
1
13
Table 1. H- and C-NMR Spectroscopic Data for Compounds 1 and 2
1
2
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
7
8
9
126.4
126.7
134.8
127.1
149.1
121.3
161.5
8.24 (d, 6.8)
7.45ꢀ(t,ꢀ6.8)
7.73 (t, 6.8)
7.60 (d, 6.8)
153.8
117.6
134.8
120.2
139.6
107.3
133.4
120.8
146.1
168.8
9.24 (d, 4.1)
Fig. 3. X-Ray Crystal Structure of 1
7.95ꢀ(d,ꢀ4.1)
7.04 (d, 6.9)
7.71 (t, 6.9)
7.67 (d, 6.9)
156.2
1
1
0
1
44.0
3.70ꢀ(d,ꢀ14.5)
3
.76ꢀ(d,ꢀ14.5)
12
13
14
15
16
17
18
19
20
82.8
83.4
5.97ꢀ(s)
148.2
110.7
131.5
120.4
124.3
128.9
6.66 (d, 8.3)
7.02 (t, 8.3)
6.86 (t, 8.3)
7.42 (d, 8.3)
Measured in CDCl3.
Fig. 4. X-Ray Crystal Structure of 2

Vol. 66, No. 4 (2018)ꢀ
Chem. Pharm. Bull.
365
Table 2. Mutagenicity of the Isolated Reaction Products toward S. typhimurium YG1024 with S9 Mix
Revertants (plate)
Dose (µg/plate)
Control
25
50
100
200
400
1
2
3
4
91± 2.5
83± 2.5
83± 2.5
83± 2.5
138± 15.5
142± 0.5
101± 0.5
108± 12.0
115± 13.5
173± 1.5
96± 8.5
103± 2.5
128± 1.5
243± 21.0
111± 13.5
125± 8.0
183± 10
96± 2.0
138± 0.5
249± 2.0
Control
0.5
1
2
Benzo[a]pyrene
64± 1.5
106± 15.5
133± 9.5
193± 12.0
COSMOSILꢀ 5C18-AR-IIꢀ (5.0 ꢀµ m,ꢀ 250×4.6mm i.d.) column. raphy: normal-phase silica gel column chromatography (CC),
To prepare the rat plasma samples, the blood samples were silica gel 60-N (Kanto Chemical Co., Inc. (Tokyo, Japan),
deproteinizedꢀ byꢀ centrifugingꢀ atꢀ 5000ꢀrpmꢀ forꢀ 10ꢀminꢀ afterꢀ 163–210 µm);ꢀ reversed-phaseꢀ silicaꢀ gelꢀ CC,ꢀ Cosmosilꢀ 75C -
18
adding MeOH. The plasma samples were subsequently stored OPNꢀ (Nacalaiꢀ Tesqueꢀ Inc.ꢀ (Kyoto,ꢀ Japan),ꢀ 75–140ꢀµm); TLC,
at −20°C until LC-MS/MS analysis. By using this method, pre-coated TLC plates with silica gel 60F254ꢀ(Merck,ꢀ0.25ꢀmm)ꢀ
1–4 in the plasma samples from diabetic rats yielded by in- (ordinary phase) and silica gel RP-18 F254Sꢀ (Merck,ꢀ 0.25ꢀmm)ꢀ
traperitoneal injection of streptozotocin (STZ) (Sigma, St. (reversed phase); reversed-phase high performance thin-layer
Louis, MO, U.S.A.), healthy rat and the CHCl extract of rat chromatography (HPTLC), pre-coated TLC plates with silica
3
diet were analyzed. As the result, compound 3 was success- gel RP-18 WF254Sꢀ(Merck,ꢀ0.25ꢀmm).
fully detected from three independent diabetic rat plasma and
Preparation of the Reaction Products Mixture D-
healthy rat plasma (Supplementary materials). The concentra- Glucoseꢀ (12.5ꢀmmol),ꢀ L-tryptophanꢀ (25ꢀmmol),ꢀ andꢀ FeSO4
tion of compound 3 in diabetic rat plasma samples were cali- (2.5ꢀmmol)ꢀ wereꢀ dissolvedꢀ inꢀ 0.5ꢀM phosphate buffer (pH 7.4,
culatedꢀasꢀ3.56± 1.24ng/mL using caliburation curb (n=3). On 500ꢀmL),ꢀ andꢀ 30%ꢀ H O (10mL) was added to the solution.
2
2
the other hand, the concentration of compound 3 in healthy rat The mixture was incubated at 37°C for 3d. The mixture was
plasma samples was caliculated as 2.20ng/mL (n=1). In addi- extractedꢀ withꢀ 500ꢀmLꢀ ofꢀ CHCl . Evaporation of the organic
3
tion, compound 3 was not detected in the CHCl extract of rat layer under reduced pressure was due to a CHCl soluble frac-
3
3
diet.ꢀForꢀtheꢀquantitativeꢀanalysisꢀofꢀtheꢀcompoundsꢀidentifiedꢀ tion (20.0g).
in this study, further study is necessary.
Isolation of the Reaction Products A CHCl3 sol-
uble fraction (20.0g) was subjected to normal phase
Conclusion
silica gel CC [600g, n-hexane:CHCl3 (1:1→1:3→1ꢀ:ꢀ5ꢀ
In this study, we determined two novel and two known v/v)→CHCl →CHCl ꢀ:ꢀMeOHꢀ (50ꢀ:ꢀ1→20:1→10:1→2:1→1:1→
3
3
compounds, 4-quinolylaldoxime and indole-3-aldehyde, pro- 1:2→1:4 v/v)→MeOH] to give eight fractions [fr. 1, fr. 2,
duced from a reaction of D-glucose and L-tryptophan at physi- fr.ꢀ 3,ꢀ fr.ꢀ 4ꢀ (4.5ꢀg),ꢀ fr.ꢀ 5ꢀ (3.6ꢀg),ꢀ fr.ꢀ 6,ꢀ andꢀ fr.ꢀ 7].ꢀ Fractionꢀ 4ꢀ
ological temperature and pH. The chemical structures of the was separated by HPLC {mobile phase: H O–MeCN (80:20,
2
novelꢀ compoundsꢀ wereꢀ clarifiedꢀ byꢀ spectroscopicꢀ analysisꢀ v/v)ꢀ [COSMOSILꢀ 5C18-MS-IIꢀ (250×10mm i.d.)]} to give 4
such as X-ray crystallography. Compounds 1 and 3 showed (1167ꢀmg).ꢀFractionꢀ5ꢀwasꢀfurtherꢀseparatedꢀbyꢀreversedꢀphaseꢀ
mutagenicity toward YG1024 in a dose-dependent manner silica gel CC [100g, MeOH:H O (2:8→3:7→4:6→5ꢀ:ꢀ5→
2
with S9 mix. In addition, we detected one of the isolated reac- 6:4→7:3→8:2→9:1 v/v)→MeOH] to give twelve fractions [fr.
tion products 3 in rat plasma. These results suggest that iso- 5-1,ꢀfr.ꢀ5-2ꢀ(348ꢀmg),ꢀfr.ꢀ5-3,ꢀfr.ꢀ5-4,ꢀfr.ꢀ5-5,ꢀfr.ꢀ5-6ꢀ(802ꢀmg),ꢀfr.ꢀ
lated compounds may be formed in plasma and induce genetic 5-7,ꢀfr.ꢀ5-8,ꢀfr.ꢀ5-9,ꢀfr.ꢀ5-10,ꢀfr.ꢀ5-11,ꢀandꢀfr.ꢀ5-12].ꢀFractionꢀ5-2ꢀ
damage. Further studies regarding the biological activities of was separated by HPLC {mobile phase: H Oꢀ:ꢀMeCNꢀ (85ꢀ:ꢀ15,ꢀ
2
reaction products, such as genotocixity in vivo, and the quan- v/v)ꢀ [COSMOSILꢀ 5C18-MS-IIꢀ (250×20mm i.d.)]} to give 2
tificationꢀ ofꢀ isolatedꢀ compoundsꢀ fromꢀ biologicalꢀ samplesꢀ ofꢀ (12.7mg), and 3ꢀ(2.9ꢀmg).ꢀFractionꢀ5-6ꢀwasꢀseparatedꢀbyꢀHPLCꢀ
diabetic individuals are important to estimate the risk of these {mobile phase: H Oꢀ:ꢀMeCNꢀ (70ꢀ:ꢀ30,ꢀ v/v)ꢀ [COSMOSILꢀ 5C18-
2
products.
MS-IIꢀ(250×10mm i.d.)]} to give 1ꢀ(25.5ꢀmg).
25
Compound 1: Yellow crystals; [α] −51.2ꢀ (c 0.84, CHCl );
D
3
1
13
Experimental
For H-NMR (CDCl , 600MHz,) and C-NMRꢀ (150ꢀMHz)ꢀ
3
General Experimental Procedures The following in- spectroscopic data, see Table 1; UV (MeOH) λ
240.8nm
max
+
strumentsꢀ wereꢀ usedꢀ toꢀ obtainꢀ theꢀ physicalꢀ data:ꢀ specificꢀ (logε 4.29); EI-MS: m/z 291 [M] ; HR-EI-MS m/z 291.1013
+
rotations, JASCO P-2200 polarimeter; EI-MS, JEOL JMS- (Calcd for C H N O [M] : m/z 291.1008).
17
13
3
2
1
1
GC MATE mass spectrometer; H-NMR spectra, JNM-ECA
Compound 2: Yellow crystals; For H-NMR (CDCl3,
00K (600MHz,) spectrometers; C-NMR spectra, JNM- 600MHz,) and C-NMRꢀ (150ꢀMHz)ꢀ spectroscopicꢀ data,ꢀ seeꢀ
13
13
6
ECAꢀ 600ꢀKꢀ (150ꢀMHz,)ꢀ spectrometers;ꢀ X-rayꢀ crystallographyꢀ Table 1; UV (MeOH) λmax 249.8nm (logε 4.07); EI-MS: m/z
+
+
RIGAKU R-AXIS RAPID diffractometer; HPLC, a Shimadzu 170 [M] HR-EI-MS m/z 170.0477 (Calcd for C H N O [M] :
10
6
2
(
Kyoto, Japan) SPD-M10AVP UV-VIS detector. COSMOSIL m/z 170.0480).
5
C18-MS-IIꢀ (250×4.6ꢀmmꢀ i.d.ꢀ andꢀ 250×10mm i.d.) columns
X-Ray Diffraction Analysis of Compound
1
The
were used for analytical and preparative procedures. The yellow crystals of C H N O ·H O had approximate di-
1
7
13
3
2
2
following experimental materials were used for chromatog- mensionsꢀ ofꢀ 0.250×0.100×0.050ꢀmmꢀ andꢀ wasꢀ mountedꢀ

3
66
Chem. Pharm. Bull.
Vol. 66, No. 4 (2018)
onꢀ aꢀ glassꢀ fiber.ꢀ Allꢀ measurementsꢀ wereꢀ performedꢀ usingꢀ was evaporated until dry, and MeOH (1mL) was added to the
a Rigaku R-AXIS RAPID diffractometer using graphite residue. The experiments were conducted according to the
monochromated Mo-Kα radiation. Monoclinic, space group “Guidelines for Animal Experiments at Kyoto Pharmaceutical
P2 /c (#14), a=8.33540ꢀÅ,ꢀ b=23.22490ꢀÅ,ꢀ c=14.79704ꢀÅ,ꢀ University,” and the animal studies were approved by the Ex-
1
3
3
β=91.34600°, V=2863.75127ꢀÅ , Z=8, Dcalcd=1.435ꢀg /cm , perimental Animal Research Committee.
μ(Mo-K )=1.008 cm , F(000)=1296.00,ꢀ No.ꢀ ofꢀ reflectionsꢀ
−1
LC-MS/MS Analysis of Compounds (1–4) in Rat
α
measured:ꢀ total,ꢀ 27452;ꢀ unique,ꢀ 6514ꢀ (R =0.2175).ꢀ Forꢀ otherꢀ Plasma LC-MS/MS analysis were performed using a Shi-
int
crystallographic data regarding the compound structure, see madzu LC-20 AD Nexera Liquid Chromatograph, a Shimadzu
Supplementary materials.
SIL-30 AC Nexera autosampler, and Shimadzu CTO-20 AC
X-Ray Diffraction Analysis of Compound 2 The yel- Prominence Column Oven that were connected to a Shi-
low crystals of C H N O had approximate dimensions of madzu LCMS-8030 Tandem Quadrupole LC-MS/MS system
10
6
2
0.300×0.300×0.100ꢀmmꢀ andꢀ wereꢀ mountedꢀ onꢀ aꢀ glassꢀ fiber.ꢀ (Shimadzu). Coupling was through the electrospray ioniza-
All measurements were performed using a Rigaku R-AXIS tion (ESI) probe that was equipped with the instrument. A
RAPID diffractometer with graphite monochromated Mo-Kα COSMOSILꢀ 5C18-AR-IIꢀ (5.0 ꢀµ m,ꢀ 250×4.6mm i.d.) column
radiation. The crystal-to-detector distance was 127.40mm. maintained at 40°C was used. The mobile phases were water
Molecularꢀ weightꢀ (MW)ꢀ 170.05,ꢀ monoclinic,ꢀ spaceꢀ groupꢀ withꢀ 0.1%ꢀ formicꢀ acidꢀ (A)ꢀ andꢀ MeOHꢀ (B),ꢀ andꢀ theꢀ flowꢀ rateꢀ
P2 /c (#14), a=5.7523(7)Å,ꢀ b=15.5021(17)Å,ꢀ c=8.7414(10) was 1mL/min. The gradient elution was performed with the
1
o
3
3
Å,ꢀ β=92.320(3) , V=778.85(16)Å , Z=2, D =0.726g/cm , followingꢀ program:ꢀ 15%ꢀ B,ꢀ increasingꢀ toꢀ 100%ꢀ Bꢀ atꢀ 45ꢀmin,ꢀ
μ(Mo-K )=0.489cm , F(000)=176.00,ꢀ No.ꢀ ofꢀ reflectionsꢀ maintaining this condition for 10min, ramping back down
calcd
−1
α
measured:ꢀ total,ꢀ 7502;ꢀ unique,ꢀ 1775ꢀ (R =0.0322). For other toꢀ15%ꢀBꢀatꢀ5ꢀminꢀandꢀholdingꢀatꢀthisꢀconditionꢀforꢀaꢀfurtherꢀ
int
crystallographic data regarding the compound structure, see 15ꢀminꢀforꢀaꢀtotalꢀruntimeꢀofꢀ75ꢀmin.ꢀTheꢀcolumnꢀovenꢀandꢀtheꢀ
Supplementary materials.
autosamplerꢀtemperaturesꢀwereꢀkeptꢀconstantꢀatꢀ40ꢀandꢀ15°C,ꢀ
Mutagenicity Assay The mutagenicity of the Mail- respectively,ꢀandꢀtheꢀsampleꢀinjectionꢀvolumeꢀwasꢀ50ꢀµL. Mass
lard reaction products was assayed by the Ames test using spectrometry was performed by positive mode electrospray
S. typhimurium YG1024 with S9 mix. Each sample was ionization. The conditions of the interface were: DL tempera-
dissolved in dimethyl sulfoxide. S. typhimurium YG1024, ture,ꢀ280°C;ꢀnebulizingꢀgasꢀflow,ꢀ3ꢀL/min;ꢀheatꢀblockꢀtempera-
which was kindly provided by Dr. Nohmi of the National ture,ꢀ400°C;ꢀdryingꢀgasꢀflow,ꢀ15ꢀL/min;ꢀandꢀInterfaceꢀVoltage,ꢀ
Institute of Health, is an O-acetyltransferase-overproducing 4.5ꢀkV.ꢀMRMꢀwasꢀused;ꢀtheꢀdwellꢀtimeꢀforꢀeachꢀtransitionꢀwasꢀ
derivative of S. typhimurium TA98 and is sensitive to the 100ꢀmsꢀ andꢀ theꢀ collisionꢀ energyꢀ wasꢀ 35ꢀV.ꢀ Theꢀ MRMꢀ transi-
mutagenicity of Maillard reaction products. A mammalian tions were 146.00/91.00, 171.00/101.00, 173.00/129.00, and
metabolic system (S9 mix) was prepared using the livers of 292.00/274.00 (m/z) for 1–4, respectively. The MS parameters
male Sprague-Dawley rat (SLC Inc., Shizuoka, Japan) treated consisted of: collision-induced dissociation (CID) gas pres-
with phenobarbital and β-naphthoflavone.ꢀBenzo[a]pyrene and sure, 230kPa; conversion dynode, −6kV; detector voltage,
−3
2
-acetylaminofluoreneꢀ wereꢀ usedꢀ asꢀ positiveꢀ controls.ꢀ Theꢀ −1.9kV; PG vacuum, 8.2e; and IG vacuum, 1.7e . The data
slope of the dose–response curve obtained with four doses and generatedꢀwasꢀprocessedꢀusingꢀLabSolutionsꢀversionꢀ5.6ꢀ(Shi-
duplicate plates at each dose was adopted as the mutagenic madzu). For quantitative analysis, the calibration graph was
potency. Samples were considered positive when they induced drawn from 0.1, 1, and 10ng/mL of compound 3. The blood
twofold increases compared to the average of spontaneous sugarꢀlevelsꢀofꢀdiabeticꢀmiceꢀwereꢀmoreꢀthanꢀ500ꢀmg/dL,ꢀwhileꢀ
revertants and showed well-behaved concentration–response those of normal mice were less than 200mg/dL.
patterns.
Animals Used for the Identification of Compounds (1–4)
Acknowledgment This research was supported in part
Diabetes was induced by intraperitoneal injections of freshly by a Grant-in-Aid for Young Scientists (B) from the Japan
prepared STZ (90mg/kg of body mass) in 0.36mL saline in Society for the Promotion of Science (JSPS) Grant Number
5
ꢀ healthyꢀ 6-week-oldꢀ maleꢀ albinoꢀ ratsꢀ thatꢀ weighedꢀ betweenꢀ JP17K15473.
60–180g. Wistar rat were purchased from Japan SLC. The
animals were housed in a temperature- and light-controlled
1
Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
room (12h light/dark cycles) and were given food and water interest.
ad libitum (MF, Oriental Yeast, Tokyo, Japan). Blood samples
for the daily analysis of glucose levels were collected from
Supplementary Materials The online version of this ar-
the tail veins of the diabetic rats, and the blood glucose lev- ticle contains supplementary materials.
1
13
els were measured using a One-Touch glucometer (Johnson
Experimental details: H- and C-NMR spectrums of com-
&
Johnson, U.S.A.). The intake of solid food and water for pounds 1 and 2,ꢀLC-MS/MSꢀchromatogramꢀforꢀtheꢀidentifica-
each mouse was monitored daily throughout the experiment. tion of compound 3 in diabetic rat plasma, X-ray experimental
Diabetic rats with blood glucose levels ≥500ꢀmg/dLꢀ(27.8ꢀmM) details of compounds 1 and 2.
11ꢀ weeksꢀ afterꢀ theꢀ firstꢀ STZꢀ administrationꢀ wereꢀ usedꢀ inꢀ theꢀ
experiments. Blood samples were collected by heart puncture References
from the rats anaesthetized with ether and were subsequently
centrifugedꢀatꢀ5000ꢀrpmꢀforꢀ10ꢀminꢀatꢀ4°C.ꢀFromꢀtheꢀresultingꢀ
ratꢀplasmaꢀsamples,ꢀ500ꢀµL aliquots were subsequently deprot-
einized by adding MeOH (2mL) followed by mixing for 1min
andꢀ centrifugationꢀ atꢀ 5400ꢀrpmꢀ forꢀ 15ꢀmin.ꢀ Theꢀ supernatantꢀ
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