Phytosterols inhibit the formation of 3-monochloropropane-1,2-diol esters in model reactions and the possible mechanism

Abstract

The effects of three phytosterols (β-sitosterol, stigmasterol, and campesterol) on the formation of 3-monochloropropane-1,2-diol (3-MCPD) esters and the possible mechanism were investigated in this study. Model reaction with 1,2-dipalmitoyl-sn-glycerol (DPG) and sodium chloride as the reacting precursors was adopted to investigate effects of three phytosterols on the formation of 3-MCPD esters. The results showed that three phytosterols exhibited inhibiting activity on the formation of 3-MCPD esters in the model reactions. ESR signal detection indicated that the phytosterols suppressed the formation of free radical in the model reactions. In addtion, 3-chloro-Δ5-cholestenes were detected in the reaction models by using UPLC-APCI-MS. It suggested that phytosterols reacted with chloride and transformed to 3-chloro-Δ5-cholestenes. Therefore, the possible mechanism of phytosterols inhibiting the formation of 3-MCPD esters could be proposed that phytosterols suppressed formation of free radical intermediates in model reactions and competed with DPG for chloride ion to reduce the formation of 3-MCPD esters.

Introduction

3-MCPD esters are a group of food contaminants. 3-MCPD esters can be detected in a wide range of foods, such as edible oil (Craft et al., 2013; Li et al., 2015a; Zelinkova et al., 2006), bread (Dolezal et al., 2009), potato products (Ilko et al., 2011; Zelinkova et al., 2009), meat products (Ilko and Dolezal, 2013), cereal (Hamlet and Sadd, 2004), coffee (Dolezal et al., 2005), malts (Divinova et al., 2007), infant and baby foods (Zelinkova et al., 2008) and goat's milk (Cerbulis et al., 1984). 3-MCPD esters can be metabolized to release free 3-MCPD in the intestinal tract (Abraham et al., 2013; Buhrke et al., 2011). 3-MCPD was categorized as a “possible human carcinogen” — category 2B by International Agency for Research on Cancer (IARC) (IARC, 2012). Several studies had demonstrated the oral toxicity and cytotoxicity of 3-MCPD mono- and di-fatty acid esters using in vitro and in vivo approaches (Barocelli et al., 2011; Liu et al., 2012; Tee et al., 2011). Therefore, 3-MCPD esters had raised wide concern in recent years.

3-MCPD esters were mainly formed in edible oils during deodorization (Hrncirik and van Duijn, 2011; Li et al., 2016). Triacylglycerol, diacylglycerol and monoacylglycerol were regarded as the main precursors of 3-MCPD esters. During heat process, glycerides and chlorine donors were induced to form radical intermediates respectively at high temperature and then through complex reactions to form 3-MCPD esters finally (Yue et al., 2016; Zhang et al., 2013; Zhang et al., 2015). Q-TOF MS and FT-IR spectroscopy analyses confirmed that CAFR could transform from monoglyceride and diglyceride during heating at high temperature (Yue et al., 2016; Zhang et al., 2013). Based on these findings, effects of antioxidants on the formation of 3-MCPD esters were investigated and the results consequently confirmed that adding antioxidants could reduce the formation of 3-MCPD eaters in edible oil during heating (Li et al., 2015b; Zhang et al., 2016).

Except for glycerides, the main constituents of edible oil, there are also some minor components existing in the unsaponifiable matter. Phytosterols are the components of the unsaponifiable fraction of plant lipids (Wanasundara et al., 1997). β-Sitosterol, stigmasterol, and campesterol are the major phytosterols found in plant lipids (Gordon and Magos, 1983; Singh, 2013), which differ from one another mainly in their degree of unsaturation and type of substitution of the side chain on C17. Their impacts on the thermal and oxidative stability of edible oils had been reported (Gordon et al., 1983; Singh, 2013). In the previous study, the antioxidant effects of phytosterols (including β-sitosterol, stigmasterol, and campesterol) against lipid peroxidation were examined, and the results showed that phytosterol chemically acted as an antioxidant and a radical scavenger (Yoshida and Niki, 2003). Also, it had been reported that antioxidants could inhibit the formation of 3-MCPD esters in edible oil during heating (Li et al., 2015b; Zhang et al., 2016). However, there are no any reports of effects of phytosterols on the formation of 3-MCPD esters.

Based on the analysis above, the present study was proposed to investigate the effects and the possible mechanism of campesterol, β-sitosterol and stigmasterol on the formation of 3-MCPD esters in the model reactions. Model reaction is a simplified system and has been developed for investigating the effects of antioxidants on the formation of 3-MCPD esters in previous researches (Li et al., 2015b; Zhang et al., 2016). In this study, DPG was selected as a model reacting precursor with sodium chloride as chlorine donor. The effects of phytosterol dosage and heating time on the formation of 3-MCPD esters were investigated by monitoring the content of 3-MCPD esters in model reactions. The effects of phytosterols and their concentration on suppression of free radical formation were monitored using an ESR spectrometer. The changes and transformation of phytosterols were monitored by mass spectra.

Materials and Methods

Chemicals and reagents    DPG (≥ 99%), β-sitosterol (≥ 90%), stigmasterol (≥ 95%) campesterol (≥ 95%) and PBN were purchased from Sigma-Aldrich (St. Louis, MO, USA). D5-3-MCPD-1,2-bis-palmitoyl ester and 3-MCPD- 1,2-bispalmitoyl ester were from Toronto Research Chemicals (Toronto, ON, Canada). Toluene, tert-butyl methyl ether, methanol, iso-hexane, ethyl acetate, diethyl ether isooctane, sulfuric acid, and sodium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium methoxide, sodium sulfate anhydrous, sodium bromide, phenylboronic acid, n-hexane, pentadecane and acetic acid were bought from Aladdin Industrial Inc. (Shanghai, China). Acetonitrile (ACN) (LC-MS grade) was obtained from Merck (Darmstadt, Germany).

Effects of phytosterols on the formation of 3-MCPD esters in reaction models    In this study, 1,2-dipalmitoyl-sn-glycerol (DPG) was selected as the precursor of 3-MCPD ester and sodium chloride was taken as chlorine donor. The glyceride solution was prepared by dissolving DPG in pentadecane at a final concentration of 3.0 mg/mL. Sodium chloride was dissolved in ultrapure water at a concentration of 10.0 mg/mL. Stigmasterol, β-sitosterol and campesterol were respectively dissolved in n-hexane at a concentration of 0.05 mg/mL. In the model reaction, 50 µL sodium chloride solution and 10 mL DPG solution were adopted as the fundamental reactants.

In order to investigate the dosage effect of β-sitosterol, campesterol and stigmasterol on the formation of 3-MCPD esters, different volume (20, 40, 60, 80, 100, 120 and 140 µL) of sterols were added into the reaction models. The model reactions were performed at 230 °C for 45 min in sand bath.

To investigate the effects of periods of heating time on the inhibiting activity of phytosterols, 60 µL sterol solution were added and mixed thoroughly with the fundamental reactants. The mixtures were heated at 230 °C for 30, 45, 60, 75, 90, 105, 120, 135 and 150 min in sand bath.

The model reaction was performed in a sealed pressure tube. All experiments were repeated three times. The content of 3-MCPD ester in the final reaction system was determined by GC-MS.

Determination of 3-MCPD esters by GC-MS    The determination of 3-MCPD esters was performed using the method described in our previous publication (Li et al., 2015c). Briefly, free 3-MCPD was released from its ester through basic hydrolysis by using sodium methoxide, phenylboronic acid (PBA) was used as the derivatizing reagent, and d5-3-MCPD-1,2-bis-palmitoyl ester was served as the internal standard. A quantitative analysis of the 3-MCPD derivatives was carried out on an Agilent 6890A/7000 system (Agilent, USA) equipped with a capillary column HP-5-MS (30 m × 0.32 mm × 0.25 µm).

Analysis of free radicals by ESR    The free radical signals were monitored by Electron Spin Resonance (ESR) (JES FA200, JEOL). The central magnetic field of ESR was 323.171 mT. The scanning time was 1 min. The scanning range was 200 G. The microwave frequency was 9056.237 MHz. The microwave power was 0.998 mW, and the modulation frequency was 100.00 kHz. N-tert-butyl-α-phenylnitrone (PBN) was used as spin trapping reagent and added into samples before thermal treatment. The final concentration of PBN in the sample was 0.1 mg/g. The samples were heated online at 150 °C for 30 min or more time and their ESR signals were measured.

Monitoring the changes of phytosterols using UPLC-APCI-MS    The changes of the phytosterols added in the reaction models were monitored by UPLC-APCI-MS. The method was adapted from the previous publication (Lerma-Garcia et al., 2010). The UPLC was coupled to the APCI ion source of an SQD mass spectrometer (Waters Corporation, USA), which was used as detection system. Separation was carried out on an AcQuity UPLC BEH C18 column (50×2.1 mm, 1.7 µm, Waters, USA). Mobile phases were prepared by mixing ACN:acetic acid (100:0.01, v/v) (phase A) with water:acetic acid (100:0.01, v/v) (phase B). Elution was performed using a linear gradient from 80 to 100% A for 0.5 min followed by an isocratic elution with 100% A for 4.5 more min. The column temperature was kept at 10 °C, and the flow rate was 0.8 mL/min. The injection volume was 15 µL. Ionization was performed in APCI positive ion mode. The ionization source parameters were as follows: corona, 4 kV; source temperature, 120 °C; desolvation gas temperature, 400 °C; with a flow rate of 750 L/h. Nitrogen was used as desolvation gas. The software used was Mass Lynx 4.1 (Waters, USA). The reaction mixture was washed with 5 times of ultrapure water and the aqueous phase was discarded. The residue organic phase was diluted 100-fold in ACN. Phytosterols and chlorated phytosterols (3-chloro-Δ5-cholestene) were measured in the selected ion recording (SIR) mode.

Statistical analysis    The results were analyzed by one-way analysis of variance and Duncan's multiple tests to identify significant differences in comparison of means. P values ≤ 0.05 were considered significant. All analyses were performed using SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results and Discussion

Effects of phytosterols on the formation of 3-MCPD esters in model reactions    As reported in previous studies (Ermacora and Hrncirik, 2014; Freudenstein et al., 2013; Li et al., 2015b; Shimizu et al., 2012; Zhang et al., 2013; Zhang et al., 2015), the glycerides and chlorine donors were the main precursors of 3-MCPD esters. In this study, DPG were mixed with sodium chloride for generating 3-MCPD esters. Different doses of β-sitosterol, stigmasterol and campesterol with concentration of 0.05 mg/mL, were added into each model reaction respectively and their effects on the formation of 3-MCPD esters were examined. As shown in Fig. 1, with the dosage of phytosterol increasing from 0 to 100 µL, the content of 3-MCPD esters in the reaction systems decreased gradually. When the dose of phytosterol increased from 100 to 140 µL, there were no significant difference (p ≤ 0.05) in the content of 3-MCPD esters detected. In addition, among three phytosterols, their inhibiting rates exhibited no significant difference (p ≤ 0.05) at the same dosage.

Fig. 1.

Effects of three phytosterols on the formation of 3-MCPD ester at different doses in model reactions (at 230 °C for 45 min)

To investigate the effects of heating time on the formation of 3-MCPD esters in the reaction models with or without phytosterol, the reaction mixtures were heated at 230 °C for different periods of time. As shown in Fig. 2, phytosterols exhibited inhibiting activity in different degrees at set heating temperatures. With the increase in heating time, the content of 3-MCPD esters increased in the model without phytosterol (blank) until 90 min, while in the models with phytosterols, the content of 3-MCPD esters increased until 75 min. As can be seen from Fig. 1, the content of 3-MCPD esters tended to level off (without significant difference, p ≤ 0.05), as the heating time increased continually to 150 min, whether in the blank or in the phytosterol models. In addition, among three phytosterols, their inhibiting rates exhibited no significant difference (p ≤ 0.05) at the same heating time. It suggested that the heating time had no significant effect on the inhibiting rates.

Fig. 2.

Effects of three phytosterols on the contents of 3-MCPD esters in the model systems at 230 °C for different periods of time (30, 45, 60, 75, 90, 105, 120, 135 and 150 min)

Monitoring of free radical formation in DPG models with or without the addition of phytosterols using ESR    The previous publication reported the free radical mechanism of 3-MCPD esters formation (Zhang et al., 2013). The free radicals generated in the reaction models with and without phytosterols were measured by ESR spectrometer for the effects of phytosterols on the formation of 3-MCPD esters. Spectra in Fig. 3 show the ESR signal of DPG heated at 150 °C for 30 min with blank, β-sitosterol, campesterol and stigmasterol, respectively. It can be seen from Fig. 3, the ESR signals decreased in the reactions with phytosterols, which indicated that the phytosterols suppressed the formation of free radical in the reaction models. The ESR signal intensity was not found with significant difference (p > 0.05) among reaction models with phytosterol. The ESR signals in the reaction models without or with different doses of phytosterol were also monitored. From spectra in Fig. 4, we can find that the higher the dosage of stigmasterol, the better the suppression efficiency becames. β-Sitosterol and campesterol exhibited similar effects on the formation of free radicals in model reaction with stigmasterol, the spectra were not shown. It suggested that the dosage of phytosterol played an important effect in the suppression of free radical formation. The appropriate hyperfine coupling constants (HFCCs) (mT) for nitrogen (a_N) and hydrogen (a_H) were obtained by analysis based on the measured spectra.

Fig. 3.

ESR spectra of DPG heated at 150 °C for 30 min with blank, β-sitosterol, campesterol and stigmasterol, respectively. The concentration of phytosterol was 0.016% (molar percentage). PBN was added before ESR measurement.

Fig. 4.

ESR spectra of DPG heated at 150 °C for 30 min with 0, 40, 80 and 120 µL stigmasterol, respectively. PBN was added before ESR measurement.

Monitoring the changes of phytosterols using UPLC-APCI-MS    It has been reported that phytosterol could transform to 3-chloro-Δ5-cholestene under the heating circumstance with hydrogen ion and chloride ion (Velisek et al., 1986). DPG could release fatty acid during heating. Fatty acid could be a source of hydrogen ion. In this context, phytosterol was speculated to react with chloride ion as a competitor of DPG. To verify the speculation, the possible products of the selected phytosterols, 3-chloro-Δ5-cholestene, were examined in the selected ion recording (SIR) mode by UPLC-APCI-MS. [M+Na]⁺ with m/z 456.142, m/z 454.126 and 442.115 were selected as the ions for identifying three chlorated phytosterols (Fig. 5). It indicated that phytosterols can transform to chlorated phytosterols by reacting with chloride ion during heating at high temperature. The peaks of 3-chloro-Δ5-cholestenes can be all observed in the reaction models (spectra A, B and C in Fig. 5). In addition, after heating for different periods of time, the residue of phytosterols in reaction models were also monitored using UPLC-APCI-MS in SIR mode. Phytosterol is difficult to take charge in the ESI ionization source. APCI source by high voltage discharge can make some neutral molecules in the air ionization. These ions ionize sterol molecules through ion-molecule reaction, and generate positive ions by proton transfer and charge exchange. The mass spectra obtained by APCI ionization source have few fractional ions, mainly excimer ions.The hydroxyl group in the phytosterol molecule is ionized with protons to form oxonium ion, which is easy to lose H₂O and form stable allyl ion. In APCI positive ion mode, phytosterol has good responsiveness with [M+H-H₂O]⁺. Monitored by using UPLC-APCI-MS, it was found that the residual phytosterols in the model reactions decreased with the increase of heating time (30, 60, 90 and 120 min) (Fig. 6). The decrease rate of phytosterols in the model reactions were shown in Table 1. These findings indicated that phytosterol played double roles in inhibiting the formation of 3-MCPD esters, which were the scavenger of free radical intermediates and the competitor with DPG for chloride ion, respectively.

Fig. 5.

Chloro-phytosterols in reaction models determined with UPLC-APCI-MS in the selected ion recording (SIR) mode.

Fig. 6.

SIRs of three phytosterols in reaction models heated for different periods of time (30, 60, 90 and 120 min) at 230 °C.

Table 1. Decrease rates of phytosterols in model reactions heated for different time (30, 60, 90 and 120 min)

Heating time (min)	Decrease rates(%)
	campesterol	stigmasterol	β-sitosterol
30	35.2±2.6a	37.5±3.1a	34.7±2.3a
60	50.6±3.9b	56.3±4.1b	51.9±3.8b
90	77.0±5.3c	81.7±6.2c	76.6±5.0c
120	89.2±4.8d	90±5.5d	88.3±6.4d

Note: The letters a–d represent significant differences among different heating time (p ≤ 0.05).

Conclusion

Phytosterols could inhibit the formation of 3-MCPD esters in the model reactions. Within low concentration range, the amount of phytosterol has a negative effect on the formation of 3-MCPD esters. The heating time has no significant effect on the inhibiting rates at same temperature. Phytosterols could play double roles during inhibiting the formation of 3-MCPD esters in model reactions, which are scanvenging free radical intermediates and competing chloride ion with DPG.

Acknowledgements    This research was financially supported by the National Natural Science Foundation of China (No. 31760443), National Key Research and Development Program of China (2017YFC1600405, 2019YFE0106000) and the Natural Science Foundation of Jiangxi Province, China (Grant. 20171BAB204030).

Conflict of interest    There are no conflicts of interest to declare.

Abbreviations

ACN

acetonitrile

CAFR

cyclic acyloxonium free radicals

DPG 1

2-dipalmitoyl-sn-glycerol

ESR

electron spin resonance

FT-IR

Fourier transform infrared spectroscopy

GC-MS

gas chromatography-mass spectrometry

HFCCs

hyperfine coupling constants

3-MCPD

3-monochloropropane-1,2-diol

PBN

N-tert-butyl-α-phenylnitrone

Q-TOF

tandem quadrupole-time-of-flight

SIR

selected ion recording

UPLC-APCI-MS

ultra-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry

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