Identification of a Hydroxymethylfurfural–Lysine Schiff Base and Its

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Food Safety and Toxicology

Identification of a Hydroxymethylfurfural–Lysine Schiff Base and Its Cytotoxicity in Three Cell Lines Ge Wang, Pengzhan Liu, Jun He, Zhao Yin, Shuo Yang, guangwen zhang, Shiyi Ou, Xinquan Yang, and Jie Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04539 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Journal of Agricultural and Food Chemistry



Identification of a Hydroxymethylfurfural–Lysine Schiff



Base and Its Cytotoxicity in Three Cell Lines



Ge Wang , Pengzhan Liu , Jun He , Zhao Yin , Shuo Yang , Guangwen Zhang , Shiyi Ou ,



Xinquan Yang





*,#









*,†

Department of Food Science and Engineering, Jinan University, 510632, Guangzhou, Guangdong,

China.







Guangdong 510641, China.

School of Food Science and Engineering, South China University of Technology, Guangzhou,



Institute of Laboratory Animal Science, Jinan University, Guangzhou 510632, China.

10 

§

11 

China.

12 

§

, Jie Zheng









College of Traditional Chinese Medicine, Jinan University, Guangzhou 510632, Guangdong,



School of Life Sciences, Guangzhou University, Guangzhou 510006, China.

13  14  15  16  17  18  19  20  21  22  23  24  25 

Corresponding Authors

26 

*Tel.: +86 20 85226630; E-mail: [email protected] (J.Z.).

27 

*Tel: +86 20 39366697; E-mail: [email protected] (X.Y.).

28  29 

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ABSTRACT

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5-Hydroxymethylfurfural (HMF) can undergo the Maillard reaction with amino acids.

32 

However, the safety of the products remains unknown. In this study, an HMF–lysine

33 

Schiff base named (E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine (HML)

34 

was identified and detected for the first time in baked foods. HML formation

35 

significantly decreased the cytotoxicity (IC50, mM) of HMF against GES-1 cells

36 

(81.81 vs. 5.02 and 73.76 vs. 2.94 for HML vs. HMF at 24 and 48 h, respectively),

37 

EA.hy926cells (86.05 vs. 4.85 and 77.22 vs. 0.71, respectively), and Caco-2 cells

38 

(155.77 vs. 36.84 and 112.70 vs. 18.51, respectively). Exposure of Caco-2 cells to

39 

HMF at 10.0 mM triggered cell apoptosis of 14.02% (vs. 8.54% in control), whereas

40 

exposure to HML at 10-15 mM hardly increased cell apoptosis. Moreover, the

41 

absorption capacities of HMF and HML by Caco-2 cells were equivalent (p > 0.05) at

42 

7.23%-12.57% after incubation at 2 mM for 30–150 min.

43 

KEYWORDS: 5-Hydroxymethylfurfural, lysine, adduct, Schiff base, cytotoxicity

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INTRODUCTION

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5-Hydroxymethylfurfural (HMF) can be formed through the Maillard reaction,

48 

caramelization, and acidic dehydration of carbohydrates.1,2 It occurs at high amounts

49 

in thermal processed food, such as cookies (0.5–74.5 mg/kg), breads (up to 410

50 

mg/kg), coffee (300–1900 mg/kg), and caramel-containing foods (110–9500 mg/kg),

51 

as well as dried fruits (up to 2200 mg/kg).3,4

52 

HMF has been reported to show beneficial effects on human health, including

53 

anti-oxidative, anti-inflammatory, anti-allergic, antihypoxic, anti-sickling, and

54 

anti-hyperuricemic

55 

5-sulfoxymethyfurfural (SMF) by sulfotransferase in animals and humans.6 In a study

56 

conducted in a Spanish preadolescent population, HMF was found to be effectively

57 

metabolized into SMF in vivo.7 SMF is highly electrophilic and can react with DNA

58 

and other macromolecules; in addition, it has been proved to confer genotoxic,

59 

mutagenic, and carcinogenic effects.5 Its mutagenic effects have been confirmed by

60 

the European Food Safety Authority,8 who established a threshold of concern of 0.54

61 

mg/day for the intake of furan derivatives used as flavoring agents in Europe.9

62 

Whereas, in a study involving 268 Spanish school pupils, the 24 h dietary intake of

63 

HMF of the subjects ranged from 3.14 mg/day to 68.86 mg/day (mean: 13.72 mg/day).

64 

Moreover, SMF was detected in 70 out of 268 subjects with a mean level of 0.33 nM,

65 

which is almost three times higher than that observed in mice 90 min after acute

66 

intake of HMF.7 Thus, the adverse effect of high HMF contents in foods remains a

67 

public concern.

effects.5

However,

HMF

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converted

to

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HMF contains a reactive aldehyde functional group that can react with amino

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acids or polyphenols to form adducts.10‒13 The reaction products, including these

70 

adducts and the other intermediates formed between HMF and amino acids, may bury

71 

HMF exposure level. Previous researches proved that the reaction intermediates

72 

released HMF during the storage and digestion of foods. Michalak et al.  found that

73 

HMF content increased by 28% and 46% in sweetened cacao products and by 11%

74 

and 30% in biscuits for infants after 12 months of storage at 4 °C and 25 °C,

75 

respectively.14

76 

1-dicysteinethioacetal−5-hydroxymethylfurfural (DCH), one of the HMF−cysteine

77 

adducts, released HMF during digestion and metabolism in rats.15 These findings

78 

indicate that the exposure level would be greatly underestimated by the analysis of

79 

HMF content in freshly produced foods.

Our

research

found

that

80 

HMF can also react with amino acids via the Maillard reaction,16,17 but the risk

81 

of the formed products is unknown. Lysine (Lys) is an essential amino acid present in

82 

comparable amount in potato (347.9 μg/g) and cereal products (up to 37 μg/g).18,19

83 

Moreover, it has been widely applied as a nutrient supplement in cereal foods since

84 

195520 and recently as an inhibitor of acrylamide formation in thermal processed

85 

foods.21 The reactive amino group on Lys could react with HMF to form Schiff bases

86 

during the thermal processing or digestion of foods. However, the toxicity of the

87 

HMF–Lys Schiff bases remains unknown, which might increase potential risk for the

88 

consumption of thermal processed foods, especially those containing a high amount of

89 

or enriched with Lys.

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For the first time, we prepared a standard compound corresponding to the Schiff

91 

base formed between HMF and Lys, and found that it occurs in four types of baked

92 

foods. On the basis of this finding, its cytotoxicity against three cell lines was

93 

measured. The apoptosis of Caco-2 cells induced by the treatment of HMF and HMF–

94 

Lys Schiff base was compared. Moreover, the absorption and transformation of the

95 

Schiff base were investigated within Caco-2 cells.

96  97 

MATERIALS AND METHODS

98 

Materials and Chemicals. 5-Hydroxymethyfurfural (98%) and L-lysine (Lys,

99 

99%) were purchased from J & K Scientific Ltd. (Beijing, China). Human colon

100 

(Caco-2) cells (ATCC HTB-37TM) were obtained from the American Type Culture

101 

Collection (Rockville, MD, USA). Human gastric epithelial (GES-1) cells and human

102 

vascular endothelium (EA.hy926) cells were obtained from Shanghai Enzyme

103 

Research Biotechnology Co., Ltd. (Shanghai, China). Penicillin–streptomycin, fetal

104 

bovine serum (FBS), and Dulbecco’s modified Eagle’s medium (DMEM) were

105 

purchased from Thermo Fisher Scientific Inc. (MA, USA). Trypsin–EDTA was

106 

purchased from Biosharp Co., Ltd. (Hefei, China). PBS was obtained from Boster

107 

Biological Technology Co., Ltd. (CA, USA). Propidium iodide staining (PI) and

108 

Annexin V-FITC were purchased from BestBio Co., Ltd. (Shanghai, China). Samples

109 

of food, including biscuits, cookies, cakes, and breads, were bought from the local

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market.

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Effect of pH, Temperature, Reaction Time, and Substrate Concentration on

112 

HMF Elimination. The reactions between HMF and Lys were performed in 25 mL

113 

screw thread stainless steel test tubes. A final volume of 4 mL aqueous-base reaction

114 

mixture was heated at a respective temperature in an oil bath for a respective duration.

115 

Different reaction conditions were investigated individually while keeping the other

116 

conditions constant as follows: concentrations of HMF and Lys, 50 and 100 mM,

117 

respectively; reaction temperature, 160 ℃; reaction duration, 15 min; and pH, 11. To

118 

investigate the effect of pH on the elimination of HMF, we adjusted the pH of the

119 

reaction medium with 0.1% HCl (v/v) and 0.1% NaOH (v/v) to different levels of 1, 3,

120 

5, 7, 9, 11, and 13. The effect of reaction temperature was tested at 80 ℃, 100 ℃,

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120 ℃, 140 ℃, 160 ℃, and 180 ℃, and that of reaction duration at 10, 15, 20, 25,

122 

and 30 min. The concentration ratio of HMF: Lys was set as 2:1, 1:1, 1:2, 1:3, and 1:4,

123 

where equivalent of 1 refers to 50 mM in concentration, to investigate their effects on

124 

the elimination of HMF. After the reaction, the tube was placed immediately in an ice

125 

bath to terminate the reaction. Then, the reaction mixture was diluted to 10 mL with

126 

distilled water and filtered through a 0.45 μm syringe filter for HPLC analysis of

127 

HMF.

128 

Separation, Purification, and Identification of HMF–Lys Schiff Base. HMF–

129 

Lys Schiff base was prepared for food analysis and cellular experiments by reacting

130 

HMF and Lys under optimized reaction conditions. A mixture of 50 mM HMF and

131 

100 mM Lys in distilled water with pH 11 was kept at 160 °C for 25 min to form the

132 

HMF–Lys Schiff base. The reaction was then terminated with an ice bath. The

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mixture was concentrated with a rotary evaporator at 40 °C and then purified

134 

successively with macroporous resin HP-20 (Sinopharm Chemical Reagent Co., Ltd.,

135 

Shanghai, China) using distilled water as eluent and Octadecylsilyl-A-HG (YMC Co.,

136 

Ltd., Tokyo, Japan) using 2% methanol in water as eluent. The purity of the eluted

137 

adduct was analyzed by HPLC, and those with purity over 98% were collected and

138 

lyophilized using a Scientz-10N vacuum freeze-dryer (SCIENTZ Biotechnology Co.,

139 

Ltd., Ningbo, China). The purified and lyophilized HMF–Lys Schiff base was

140 

identified by high-resolution mass spectrometry (HRMS, X500R QTOF mass

141 

spectrometer, AB Sciex, CA) and nuclear magnetic resonance (NMR, AVANCE III

142 

600 MHz spectrometer, Bruker, Fällanden, Switzerland).

143 

Cell Culture. The cytotoxicity of the HMF–Lys Schiff base was investigated in

144 

the cell lines of GES-1, Caco-2, and EA.hy926. GES-1 and EA.hy926 cells were

145 

cultivated in DMEM with 10% FBS, and Caco-2 cells were cultivated in DMEM with

146 

20% FBS in a humidified incubator containing 5% CO2 and 95% air at 37 °C. The

147 

densities of cells were maintained as recommended, and 0.25% trypsin was used for

148 

detaching adherent cells from plates.

149 

Measurement of Cell Viability. The cell counting kit-8 (CCK-8, Biosharp,

150 

Hefei, China) was used to assess the growth inhibition of cells under different

151 

treatments of HMF and HMF–Lys Schiff base for 24 and 48 h as previously described

152 

by Zhao et al.22 Different levels of HMF and HMF–Lys Schiff base were set for the

153 

treatments of different cells. In detail, HMF showed high cytotoxicity on GES-1 and

154 

EA.hy926 cells. Therefore, concentrations of 4, 8, 12, 24, 28, and 32 mM were

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selected for the HMF treatments of GES-1 and EA.hy926 cells. Meanwhile, the

156 

HMF–Lys Schiff base showed comparably low cytotoxicity on these two cells, and

157 

the concentrations of the HMF–Lys Schiff base were set as 8, 16, 32, 64, 90, and 128

158 

mM. For the treatment of Caco-2 cells, the same concentration levels of 2, 4, 8, 16, 32,

159 

64, 128, 192, and 256 mM were set for both HMF and HMF–Lys Schiff base.

160 

Analysis of Apoptosis by Flow Cytometry. Caco-2 cells (3×104 cells/well)

161 

were treated with different concentrations (2.5, 5.0, 10.0, and 15.0 mM) of HMF and

162 

HMF–Lys Schiff base for 48 h in 6-well culture plates. The treated cells were

163 

harvested by EDTA-free trypsinization and washed once with PBS. Cells at 1–5×105

164 

were collected and then Annexin V-FITC (5 μL, BestBio, Shang Hai, China) was

165 

added to the binding buffer (50 μL, BestBio, Shang Hai, China). After mixing to a

166 

homogenous cell suspension, 450 μL of the binding buffer and 1 μL of PI were added.

167 

Flow cytometric analysis using a NovoCyte Flow cytometer (ACEA Biosciences,

168 

Hang Zhou, China) was then performed within 1 h.

169 

Absorption of HMF and HMF–Lys Schiff Base by Caco-2 Cells. The

170 

absorption of HMF and HMF–Lys Schiff base by Caco-2 cells was determined in

171 

accordance with the method applied by Sampath et al. with some modifications.23

172 

Cells (3×104 cells/well) were seeded in 24-well culture plates and then incubated in a

173 

humidified incubator containing 5% CO2 and 95% air at 37 °C. The cells were treated

174 

with 2 mM HMF and HMF–Lys Schiff base for 30, 60, 90, 120, and 150 min. At the

175 

end of incubation, the medium was collected as the extracellular fluid. After washing

176 

thrice with 100 μL of PBS, the cells were trypsinized and collected. RIPA cell lysis

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buffer (50 μL) was then added, and the cells were allowed to lyse in an ice bath for 30

178 

min with vigorous mixing every 5 min. After cells were completely lysed, the

179 

intracellular fluid was collected after centrifugation at 12,000×g for 15 min at 4 °C.

180 

All samples were filtered through a 0.22 μm syringe filter into an autosampler vial

181 

prior to the HPLC analysis of HMF and HML.

182 

HPLC-DAD Analysis of HMF and HMF–Lys Schiff Base in Reaction

183 

Mixture, Cells, and Cell Cultures. For the determination of HMF and HMF–Lys

184 

Schiff base, a 10 μL sample was injected into an LC-20AT HPLC equipped with an

185 

SPD-M20AVP DAD linked to a CBM-20A controller (Shimadzu Corporation, Kyoto,

186 

Japan). The sample was separated on an Agilent Zorbax SBAq C18 column (4.6 ×

187 

250 mm, 5 μm) (Agilent Technologies, CA, USA) with the mobile phase of 5%

188 

methanol in MQ-water at the column temperature of 40 °C. The flow rate was kept

189 

constant at 0.6 mL/min. HMF and HMF–Lys Schiff base were detected at 284 and

190 

253 nm, respectively. The contents of HMF and HMF–Lys Schiff base were

191 

quantified using the calibration curves of the commercial standard and the standard (>

192 

98%) prepared by our laboratory, respectively, as external standards.

193 

Detection of the HMF–Lys Schiff Base in Foods. For determination of HMF–

194 

Lys Schiff base content in foods, about 5 g of fully grinded sample was mixed with 50

195 

mL of n-hexane and stirred occasionally for 24 h to remove fat-soluble components.

196 

The supernatant was discarded after centrifugation at 10,000×g for 20 min at 4 °C,

197 

and the residues were extracted twice with 30 mL of MQ-water. The supernatants

198 

were collected and combined after centrifugation at 10,000×g for 20 min at 4 °C. The

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sample was evaporated to dryness with a rotary evaporator under 40 °C, re-dissolved

200 

with 4 mL of MQ-water, and then filtered through a 0.45 μm syringe filter. A sample

201 

of 10 μL was injected into an Agilent 1100 Series LC (Agilent Technologies, CA,

202 

USA) interfaced to an AB 4000 Q-Trap mass spectrometer (AB Sciex, MA, USA).

203 

The sample was separated on an Agilent Zorbax SBAq C18 column (4.6 × 250 mm, 5

204 

μm) (Agilent Technologies, CA, USA) with the same elution conditions described in

205 

the HPLC-DAD analysis. The HMF–Lys Schiff base was detected with an

206 

electrospray ionization mass spectrometer operated in the positive mode. The

207 

capillary voltage was set at 5.5 kV, and the temperature was set at 500 °C. The

208 

pressure of nebulizer gas and drying gas were 55 and 50 psi, respectively. The

209 

qualification and quantification of the HMF–Lys Schiff base were achieved by

210 

multiple reaction monitoring (MRM) with the transitions of m/z 255.1→130.0 and

211 

m/z 255.1→84.1, respectively. The declustering potential and collision energy were

212 

set at 50 and 19 V, respectively. The content of the HMF–Lys Schiff base was

213 

quantified based on the calibration curve of the standard prepared by our laboratory.

214 

pH Measurement of Foods. For pH determination, 5 mL of distilled water was

215 

added to 5 g of food sample and then ground thoroughly. The pH of the slurry was

216 

then measured using a STARTER300 pH meter incorporated with a ST 270 pH probe

217 

(Ohaus Corporation, NJ, USA).

218 

Statistical analysis. All experiments were conducted in triplicate. Statistical

219 

analyses were performed using SPSS 16.0.1 (SPSS, Inc., Chicago, IL). Differences

220 

between the effects of different concentrations and incubation times of HMF and

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HMF–Lys Schiff base on cell viability, and differences in values of pH, HMF and

222 

HML between different food samples were investigated by one-way ANOVA.

223 

Duncan’s multiple-range test (p < 0.05) was further applied to carry out the multiple

224 

comparisons between different samples. Comparison between IC50 values of HMF

225 

and HMF–Lys Schiff base treatments of various cell lines was conducted by an

226 

independent-sample t test.

227 

RESULTS AND DISCUSSION

228 

Effect of Different Reaction Parameters on HMF Elimination. In previous

229 

studies, HMF is largely eliminated by some amino acids through the Maillard reaction

230 

and Michael addition reactions.11,12,17,24 In order to obtain sufficient amount of

231 

HMF-Lys Schiff base standard, the effects of pH, temperature, reaction time, and

232 

HMF-to-Lys concentration ratio on HMF elimination were investigated.

233 

Results showed that the elimination of HMF by Lys was greatly influenced by

234 

pH. At pH 1–5, HMF was only depleted by 6.14%–10.13%. As the pH was increased

235 

to 9, HMF elimination sharply increased to 65.46% and then moderately increased

236 

afterward (Figure 1A). Deprotonation of the amino group in amino acid is the

237 

premise for the Maillard reaction. The pKa value of Lys is 10.4;25 as a result, higher

238 

pH favors the Maillard reaction as a complete deprotonation of the amino group in

239 

Lys would occur after pH 10.4. Although Lys could deplete 75.30% of HMF at

240 

reaction pH of 13, pH 11 (70.21% HMF depletion) was chosen for the preparation of

241 

the HMF–Lys Schiff base because of the least production of impurities.

242 

Elevation of temperature increased the elimination of HMF. About 68.67% of

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HMF was depleted when the temperature was increased to 160 ℃. A further increase

244 

in the temperature showed limited impact on the elimination of HMF (Figure 1B),

245 

whereas the production of impurities became apparent. Therefore, 160 ℃ was used

246 

for the preparation of targeted HMF–Lys Schiff base.

247 

Reaction time also influenced HMF elimination, but its impact was not as

248 

remarkable as those of pH and temperature (Figure 1C). The elimination percentage

249 

increased from 51.70% to 85.03% as the reaction time was prolonged from 10 min to

250 

30 min. Thus, the reaction time of 25 min was finally chosen for the maximum

251 

preparation of the HMF–Lys Schiff base and the minimum production of impurities.

252 

Further prolongation of the reaction time (30 min) hardly influenced the formation of

253 

the HMF–Lys Schiff base but increased the side-products, which might be attributed

254 

to further reactions of the HMF–Lys Schiff base.

255 

Molar ratio of HMF to Lys showed comparable impact on HMF elimination.

256 

Higher ratio of Lys/HMF promoted the elimination of HMF. Figure 1D shows that

257 

the elimination of HMF increased remarkably by two times as the molar ratio of HMF

258 

to Lys changed from 2:1 to 1:2. The elimination of HMF increased but moderately as

259 

the molar ratio further increased to 1:4. Although the highest elimination percentage

260 

(70.60%) was reached at the molar ratio of 1:4 (HMF: Lys), the molar ratio of 1:2 was

261 

chosen for the preparation of Schiff base to remove less Lys during the purification of

262 

the HMF–Lys Schiff base.

263 

On the basis of the results mentioned above, the preparation of the HMF–Lys

264 

Schiff base was optimized as follows: 50 mM HMF and 100 mM Lys reacted at pH 11

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and 160 ℃ for 25 min. Under this condition, the elimination of HMF was 74.70%,

266 

and the HMF–Lys Schiff base appeared as the predominant peak in the HPLC

267 

chromatogram (Supplementary Figure S1).

268 

Identification of HMF–Lys Schiff base. The HMF–Lys Schiff base was

269 

produced from HMF and Lys under the optimized conditions obtained in the present

270 

research. After successive purification using macroporous resin HP-20 and

271 

Octadecylsilyl-A-HG, the target product was obtained as odorless white amorphous

272 

powder with high hygroscopy, being soluble in water while insoluble in methanol.

273 

The product with purity over 98% underwent identification analyses by HRMS and

274 

NMR. The molecular weight of the product was 254.1 according to the molecular ion

275 

detected at m/z 255.1331 in the positive mode of HRMS analysis (Supplementary

276 

Figure S2), which was indicated as a Schiff base. The exact structure of the HMF–

277 

Lys Schiff base was further elucidated by 1H and 13C NMR data and the correlations

278 

from COSY and HMBC experiments (Figure 2). The downfield shift of the

279 

corresponding chemical shifts for proton and carbon indicated that the Schiff base

280 

formed between the ε-NH2 group in Lys and the aldehyde group of HMF. It was

281 

assigned as (E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine (HML). The

282 

molecular formula is C12H18N2O4, with the theoretical molecular weight of 254.29,

283 

which is consistent with the m/z value of 255.1331 [M + H]+ obtained by mass

284 

spectrometric analysis.

285 

Detection of HML in foods. In the current study, four types of bakery foods,

286 

including biscuits, cookies, cakes, and breads, were analyzed and proved to contain

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the HMF–Lys Schiff base, i.e., HML (Table 1). Among them, biscuits displayed the

288 

highest amount of HML (p < 0.05), followed by cookies and breads, and cakes being

289 

the lowest. The biscuit displayed the highest pH in all the samples, although the

290 

difference between it and other samples was small but significant. A combined but

291 

complex impact of rapid heat conduction and comparable alkaline condition of the

292 

biscuit dough might contribute to the formation of high-level HML. Moreover, all the

293 

samples tested are common bakery products. An enrichment of lysine as nutrient

294 

supplements may further increase the formation of HML in some special nutritional

295 

fortified foods.

296 

Impact of HMF and HML on the Cell Viability of GES-1, Caco-2, and

297 

EA.hy926 Cells. Concerning the digestion and absorption of food components in

298 

humans, epithelial cells in the gastrointestinal tract (GES-1 and Caco-2 cells) and

299 

endothelial cells in the blood vascular system (EA.hy926 cells) were chosen to

300 

investigate the effect of HML on cell viability, using its precursor HMF as a

301 

control.22,26‒28

302 

As shown in Figure 3, HML and HMF displayed distinguishing potent

303 

cytotoxicity in the different cell lines. HMF caused significant injury on GES-1 and

304 

EA.hy926 cells at low concentrations (below 32 mM). Over 95% of GES-1 cells died

305 

after exposure to 28 and 24 mM HMF for 24 and 48 h, respectively; the same

306 

situation occurred in EA.hy926 cells exposed to 24 and 12 mM HMF, respectively

307 

(Figure 3). Based on the concentration test, the IC50 values of HMF were calculated

308 

as 5.02 and 2.94 mM against GES-1 cells after incubation of 24 and 48 h, respectively,

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and 4.85 and 0.71 mM against EA.hy926 cells, respectively. By contrast, Caco-2 cells

310 

showed higher resistance against the exposure to HMF than GES-1 and EA.hy926

311 

cells. The IC50 values of HMF against Caco-2 cells were 36.84 and 18.51 mM after 24

312 

and 48 h incubation, respectively, which were 7.2 and 6.1 times higher than those

313 

against GES-1 cells and 7.6 and 26.1 times higher than those against EA.hy926 cells.

314 

The formation of HML significantly decreased the toxicity of HMF toward all the

315 

three cell lines. After HML incubation for 24 and 48 h, its IC50 value increased by

316 

16.3- and 25.1-fold in GES-1 cells, 17.7- and 108.8-fold in EA.hy926 cells, and 4.2-

317 

and 6.1-fold in Caco-2 cells, respectively, compared with those of HMF (Figure 3).

318 

HML showed 1.5–2-fold lower toxicity toward Caco-2 cells than GES-1 and

319 

EA.hy926 cells. Moreover, the concentration impact of HML on cell viability differed

320 

between Caco-2 cells and the other two lines of cells. The viability of GES-1 and

321 

EA.hy926 cells remained almost constant as that of the blank until the HML exposure

322 

level reached 32 mM and then started to decrease sharply afterward in a

323 

dose-dependent manner until almost total death of the cells occurred at the treatment

324 

level of 128 mM. By contrast, only 40.62% and 60.85% of the Caco-2 cells died

325 

under HML treatment at 128 mM for 24 and 48 h, respectively. The injury of Caco-2

326 

cells occurred significantly at very low concentrations of HML and then increased

327 

mildly as the treatment concentration was increased (Figure 3).

328 

In our previous study,22 we reported the HMF–cysteine adduct DCH and

329 

evaluated its cytotoxicity against Caco-2 cells. The IC50 value of DCH against Caco-2

330 

cells was assessed to be 31.26 ± 1.50 mM, whereas that of HMF was 14.95 ± 1.09

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331 

mM, after 72 h of incubation. Therefore, lysine rather than cysteine might contribute

332 

better in detoxification of HMF against Caco-2 cells.

333 

Effects of HMF and HML on Apoptosis of Caco-2 Cells. In the current study,

334 

flow cytometry was applied to evaluate cell apoptosis induced by HMF and HML.

335 

Figure 4 shows that exposure to HML at 2.5-15.0 mM hardly induced the apoptosis

336 

of Caco-2 cells, whereas exposure to HMF at 10-15 mM resulted in considerable cell

337 

apoptosis. Approximately 8.54% of the normal cells were apoptotic; the population of

338 

apoptotic cells remained almost unchanged below 9.68% after exposure to HML at

339 

2.5-15.0 mM and HMF at 2.5-5.0 mM for 48 h; however, exposure to HMF at 10.0

340 

and 15.0 mM for 48 h increased the percentage of apoptotic cells from 8.54% (control)

341 

to 14.02% and 14.68%, respectively. These results further proved that the formation

342 

of HML decreased the cytotoxicity of HMF against Caco-2 cells in terms of cell

343 

apoptosis induction.

344 

Absorption of HMF and HML by Caco-2 cells. In the current study, we

345 

incubated Caco-2 cells with HMF and HML at 2 mM for various periods from 30 min

346 

to 150 min to determine the absorption of these compounds by Caco-2 cells. Table 2

347 

shows that both HMF and HML could be absorbed by Caco-2 cells from 7.23% to

348 

12.57%. Prolongation of incubation period hardly influenced the absorption of both

349 

HMF and HML. Moreover, the absorption of the two compounds always remained at

350 

the same statistical level (p > 0.05) after incubation of a same period, which indicated

351 

that Caco-2 cells possess the same absorption affinity toward HMF and HML.

352 

However, in our previous study, the HMF–cysteine adduct DCH22 showed less

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353 

absorption by Caco-2 cells compared with HMF after incubation for 4–24 h. About

354 

4.51% DCH was absorbed by Caco-2 cells after 24 h treatment with 2 mM DCH,

355 

whereas 36.5% HMF was absorbed after HMF treatment. Moreover, 91% of the

356 

absorbed DCH disappeared upon absorption by Caco-2 cells and suggested to be

357 

metabolized, of which 1% was transformed to HMF in intracellular fluids. Meanwhile,

358 

HMF was detected neither in the extracellular fluid nor in the intracellular fluids in

359 

the current study, indicating that HMF was not released from HML in Caco-2 cells

360 

under the incubation conditions applied. However, the intracellular HML levels

361 

(varied from undetectable level to 0.70 nmol/well) were considerably lower than the

362 

absorbed amounts (180–250 nmol/well). Thus, we hypothesized that HML in Caco-2

363 

cells was converted to other compounds. Further investigations of the in vitro and in

364 

vivo metabolic pathways of these HMF–amino acid adducts should be conducted to

365 

completely understand the metabolic route of these novel compounds and their safety.

366 

HMF, a potential food-borne hazard, can easily form adducts with various amino

367 

acids in food materials during the thermal processing or gastrointestinal digestion of

368 

foods. Despite previous investigation on the HMF–cysteine adduct DCH, the safety of

369 

the HMF–amino acid adducts and their absorption in the gastrointestinal tract was still

370 

hardly evaluated. The present research evaluated the cytotoxicity of the HMF–lysine

371 

Schiff base adduct HML against representative epithelial cells in the gastrointestinal

372 

tract and endothelial cells in the blood vascular system. Results indicated that the

373 

formation of HML remarkably lowered the toxicity of HMF against all the tested cell

374 

lines. Moreover, HMF and HML were equally absorbed by Caoco-2 cells and largely

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Page 18 of 31

375 

metabolized to other compounds upon absorption. Further investigations on other

376 

HMF–amino acid adducts and their absorption and metabolism in the digestion tract

377 

and related target organs should be carried out to completely understand the potential

378 

safety risks of these adducts.

379  380 

ABBREVIATIONS USED

381 

ANOVA, analysis of variance; ATCC, American type culture collection; CCK-8, cell

382 

counting kit-8; Lys, cysteine; DCH, 1-dicysteinethioacetal-5-hydroxymehtylfurfural;

383 

DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylene diamine tetraacetic

384 

acid;

385 

(E)-N6-((5’-(hydroxymethyl)furan-2’-yl)methylene)lysine;

386 

performance liquid chromatography-diode array detection; HRMS, high-resolution

387 

mass spectrometry; IC50, half maximal inhibitory concentration; MRM, multiple

388 

reaction monitoring; NMR, nuclear magnetic resonance; PBS, phosphate buffered

389 

saline; PI, propidium iodide; RIPA, radio-immunoprecipitation assay; SMF,

390 

5-sulfoxymethyfurfural.

391  392 

AUTHOR INFORMATION

393 

Corresponding Authors

394 

*Tel.: +86 20 85226630; E-mail: [email protected] (J.Z.).

395 

*Tel: +86 20 39366697; E-mail: [email protected] (X.Y.).

396 

ORCID

397 

Zheng Jie: 0000-0001-9755-5595

398 

Shiyi Ou: 0000-0002-6779-0858

FBS,

fetal

bovine

serum;

HMF,

5-hydroxymethylfurfural;

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HPLC-DAD,

HML, high

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Journal of Agricultural and Food Chemistry

399 

Funding

400 

This work was financially supported by the National Natural Science Foundation of

401 

China (31671957; 81603165; 31972180), and Department of Science and Technology

402 

of Guangdong Province (2018B050502008).

403 

Notes

404  405 

The authors declare no competing financial interest.

406 

SUPPORTING INFORMATION

407 

HPLC chromatogram detected at 205 nm of reaction mixture of 50 mM HMF and 100

408 

mM lysine reacted at 160 ℃ for 25 min under pH 11 (Figure S1); High-resolution

409 

full scan mass spectra of HMF–Lys Schiff base  detected in positive mode (Figure

410 

S2).

411  412 

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413 

REFERENCE

414 

(1) Ames, J. M. The Maillard reaction. In Biochemistry of Food Proteins; Hudson, B.

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J. F., Ed.; Springer: Boston, MA, 1992; pp. 99−153.

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review on metabolism, toxicity, occurrence in food and mitigation strategies.

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bees and human health. Chem. Cent. J. 2018, 12, 35.

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adducts formed between hydroxymethylfurfural and selected amino acids.; and their

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release in simulated gastric model. Int. J. Food Sci. Technol. 2016, 51, 1002−1009.

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(13) Qi, Y. J.; Zhang, H.; Zhang, H.; Wu, G. C.; Wang, L.; Qian H. F.; Qi, X. G.

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Epicatechin adducting with 5‑hydroxymethylfurfural as an inhibitory mechanism

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storage on acrylamide and 5-hydroxymethylfurfural contents in selected processed

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plant products with long shelf-life. Plant Food. Hum. Nutr. 2016, 71, 115−122.

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Zhang, Q. R.; Ou, S. Y.; Zhou, H.; Wang, Y.; Bai, W. B.; Li, Y. Q. Chlorogenic acid

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3-aminopropionamide deamination. J. Hazard. Mater. 2014, 268, 1−5.

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role of 5-hydroxymethyl-2-furfural in acrylamide formation from asparagine. Food

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Chem. 2012, 132, 168−174.

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Measurement of acrylamide and its precursors in potato, wheat, and rye model

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systems. J. Agric. Food Chem. 2005, 53, 1286–1293.

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acids in cereal products. Food Chem. 2007, 105, 317–324.

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Nutr. 1993, 12, 486−500.

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(21) Friedman, M.; Levin, C. E. Review of Methods for the Reduction of Dietary

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Content and Toxicity of Acrylamide. J. Agric. Food Chem. 2008, 56, 6113–6140.

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(22) Zhao, Q. Z.; Zou, Y. Y.; Huang, C. H.; Lan, P.; Zheng, J.; Ou, S. Y. Formation of a

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hydroxymethylfurfural−cysteine adduct and its absorption and cytotoxicity in Caco‑2

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apple, tea, and ginger protect against dicarbonyl induced stress in cultured human

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retinal epithelial cells. Phytomedicine 2016, 23, 200−213.

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of glycine. J. Agric. Food Chem. 2011, 59, 10104–10113.

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lysine and arginine side chains by indirect

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Application to apo calmodulin. J. Am. Chem. Soc. 2007, 129, 15805−15813

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Water-soluble Ru(II)-anethole compounds with promising cytotoxicity toward the

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human gastric cancer cell line AGS. Life Sci. 2019, 217, 193−201.

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Caco-2 characteristics and particle uptake. Int. J. Pharmacol. 2010, 387, 7−18.

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P.

S.;

Y.;

Sarria

Yaylayan,

B.;

Mateos

V.

R.;

A.

15

Thermal

N and

Goya,

499 

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13

L.;

decomposition

of

C NMR spectroscopy: 

Bravo-Clemente,

L.

Journal of Agricultural and Food Chemistry

500 

Figure captions

501 

Figure 1. Effects of pH (A), temperature (B), reaction time (C), and concentration

502 

ratio of HMF:Lys (D) on the elimination of HMF. Error bars represent the standard

503 

deviations of the mean of three replicates (n = 3). For the concentration ratio,

504 

equivalent of 1 refers to 50 mM in concentration.

505 

Figure 2. Chemical structure and 1H (500 MHz) and 13C (125 MHz) NMR data of the

506 

HMF–Lys Schiff base in D2O.

507 

Figure 3. Effect of different concentrations of HMF and HML on the viability of

508 

GES-1, Caco-2, and EA.hy926 cells, respectively, after incubation for 24 and 48 h.

509 

Different letters indicate significant differences (p < 0.05) between different

510 

treatments. * indicates significant differences (p < 0.05) between IC50 values of HMF

511 

and HML treatments at 24 and 48 h, respectively.

512 

Figure 4. Apoptosis in Caco-2 cells after 48 h treatment with various concentrations

513 

of HMF and HML.

514  515 

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516  517 

518  519 

Table 1. Contents of HMF and HML in different bakery foods Sample

pH

HMF (mg/kg)

HML (μg/kg)

Biscuit

7.24 ± 0.14d

8.98 ± 0.02a

1.9 ± 0.1c

Bread

5.66 ± 0.03a

9.94 ± 0.90ab

0.3 ± 0.1ab

Cookie 1

6.99 ± 0.07c

10.76 ± 0.21b

0.7 ± 0.2b

Cookie 2

6.29 ± 0.03b

12.54 ± 1.10c

0.2 ± 0.0ab

Cake

7.13 ± 0.09cd

9.54 ± 0.19ab

0.1 ± 0.0a

Different letters in upper cases indicate significant differences (p < 0.05) between different samples.

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Table 2. Extracellular and intracellular levels of HMF and HML in Caco-2 cells after different incubation time Treatment

HMFcontent (μmoL/well)

HML content (μmoL/well)

Incubation time (min)

Extracellular

Intracellular (×10 )

Extracellular

Intracellular (×10-3)

2 mM HMF

30 60 90 120 150

1.84 ± 0.02bc 1.86 ± 0.04c 1.78 ± 0.08ab 1.79 ± 0.01abc 1.75 ± 0.06a

ND ND 0.06 ± 0.05 0.07 ± 0.05 0.11 ± 0.07

— — — — —

— — — — —

2 mM HML

30 60 90 120 150

— — — — —

— — — — —

1.82 ± 0.05abc 1.77 ± 0.04ab 1.75 ± 0.01a 1.75 ± 0.04a 1.75 ± 0.02a

ND ND 0.40 ± 0.00 0.70 ± 0.00 0.50 ± 0.10

-3

Different letters in upper cases indicate significant differences (p < 0.05) between different treatments.

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60 40 20 0 2

4

6 pH

8

10

12

80 60 40 20 0 70 80 90 100 110 120 130 140 150 160 170 180 190 Temperature (ºC)

14

C

100 Eliminaton of HMF (%)

Elimination of HMF (%)

80

0

B

100

A

100

Elimination of HMF (%)

Elimination of HMF (%)

Page 27 of 31

80 60 40 20

100

D

80 60 40 20 0

0 5

10

15

20 Time (min)

25

30

35

Figure 1.

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2:1

1:1 1:2 1:3 1:4 Concentration ratio of HMF: Lys

Journal of Agricultural and Food Chemistry

Position 1 2 3 4 5 6 1’ 2’ 3’ 4’ 5’ 5’-CH2

δC (ppm) 175.21 s 54.59 d 30.24 t 21.52 t 30.26 t 56.29 t 135.31 d 137.67 s 134.53 d 128.70 d 165.76 s 58.85 t

δH (ppm)

1

H-1HCOSY

3.60 (t, J = 6.36 Hz) 1.79 (m) 1.39 (m) 1.90 (m) 4.30 (t, J = 7.74 Hz) 7.67 (d, J = 2.76 Hz)

H-3 H-2, 4 H-3, 5 H-4, 6 H-5

7.32 (dd, J = 8.94, 2.76 Hz) 7.47 (d, J = 8.94 Hz)

H-4’ H-3’

4.70 (m)

Figure 2.

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HMBC H-2 H-3 H-2, 5 H-2, 6 H-6 H-1’ H-6 H-6, 1’, 3’, 4’, 5’-CH2 H-1’ H-3’, 5’-CH2 H-4’ H-4’

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Journal of Agricultural and Food Chemistry

Figu ure 3.

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F Figure 4.

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Tab ble of Conteents

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