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Cellular Transport of Esculin and its Acylated Derivatives in Caco-2 Cell Monolayers and their Antioxidant Properties in Vitro Mengmeng Zhang, Xuan Xin, Furao Lai, Xiaoyuan Zhang, Xiao-Feng Li, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02525 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Journal of Agricultural and Food Chemistry

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Cellular Transport of Esculin and its Acylated Derivatives in Caco-2

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Cell Monolayers and their Antioxidant Properties in Vitro

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Mengmeng Zhang†, Xuan Xin†, Furao Lai†, Xiaoyuan Zhang§, Xiaofeng Li*#, and Hui Wu*†

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Affiliation

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†College of Food Science and Engineering, South China University of Technology,

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Guangzhou, Guangdong, 510640, China

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§Research Institude of Shaoguan Huagong High-tech Industry, South China

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University of Technology, Guangzhou, Guangdong, 510640, China

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#State Key Laboratory of Pulp and Paper Engineering, South China University of

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Technology, Guangzhou, Guangdong , 510640, China

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Short title: Bioavailability and Antioxidant Properties of Esculin and its Acylated

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Derivatives

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Corresponding authors:

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*Xiaofeng Li, State Key Laboratory of Pulp and Paper Engineering, South China

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University of Technology, Wushan Road 381, Guangzhou, Guangdong, China.

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Tel: (+86)20-22236819; E-mail: [email protected] ; Fax: (+86)20-87112853

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*Hui Wu, College of Food Science and Engineering, South China University of

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Technology, Wushan Road 381, Guangzhou, Guangdong, China. Tel: (+86)20-87112853; E-mail: [email protected]; Fax: (+86)20-87112853 1

ACS Paragon Plus Environment


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ABSTRACT: Esculin has many pharmacological effects, but these are difficult to

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observe after oral administration owing to poor lipid solubility. In our previous study,

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five acylated derivatives with different acyl chain lengths (EA, EP, EO, EL, and EM)

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were synthesized to improve the lipophilicity of esculin. In this study, the

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bioavailability and antioxidant activity of the five derivatives were investigated. The

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logP of esculin, EA, EP, EO, EL, and EM were -1.1±0.1, -0.3±0.14, 0.1±0.17,

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1.6±0.09, 2.4±0.11 and 2.8±0.18, and their Papp were 0.71±0.02, 1.24±0.18,

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1.74±0.11, 11.6±3.6, 4.11±1.03 and 2.64±0.97 ×10−6 cm/s, respectively. Besides, the

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bioavailability of EO, EL, and EM were seriously affected by carboxylesterase. The

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results of ABTS, ORAC, and DPPH assays indicated that the antiradical ability of

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the five derivatives did not exceed that of esculin. However, EA, EP, and EO showed

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more effective inhibition of AAPH-induced oxidative hemolysis than esculin did

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(p

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0.05) before and after the experiment, which indicated that the monolayers retained

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good integrity, and the used exposure times and concentrations of all samples were

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appropriate for the experiment. Table 2 showed the results of the transport experiment.

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It was found that for all samples, the Papp of AP to BL transport was close to that of

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BL to AP transport. This indicated the efficiency of transport from AP and BL sides

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were similar for each compound. Uptake ratio (UR) was defined as the quotient of the

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absorptive permeability and the secretory permeability (Papp AP−BL/Papp BL−AP). A

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value of UR or ER (efflux ratio, Papp BL−AP/Papp AP−BL) higher than 1.5 suggests

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the participation of an active transport mechanism; if the values are close to 1.0, this is

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a passive transport.20 All UR were approximately 1.0 in this result, which indicated

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that passive transport occurred for all samples.

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Propranolol is a high permeability-high solubility lipophilic drug, which can be

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absorbed almost completely after oral consumption. Furosemide is a low

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permeability-high solubility hydrophilic compound with very low bioavailability.19

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On the basis of these two extremes for the estimation of bioavailability, it was

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determined that esculin has a much lower bioavailability than furosemide. The

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bioavailability of EA and EP was better than that of esculin but similar to that of 11



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furosemide. EO had a much better bioavailability than EA and EP did, but it was still

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lower than that of propranolol. These results agreed with the prediction of logP values.

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However, EL and EM had a much lower bioavailability than EO did, which was

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similar to that of EA and EP. It is very different from the prediction of logP values.

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This difference was not caused by the molecular weight. Although the molecular

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weights of EL and EM were greater than 500 g/mol, they were only 56 and 85 g/mol

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more than that of than EO (Mw = 466 g/mol), respectively. Thus, there must be other

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explanations for the huge difference.

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The results in Table 2 showed that the recovery of esculin, EA, and EP was

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greater than 90%. However, EO, EL, and EM had a very low recovery. A recovery

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of >80% gives an acceptable approximation of the Papp value. A lower recovery will

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result in the underestimation of the Papp values;20 thus, the practical Papp values of EO,

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EL, and EM were greater than the measured values. The reason may be that the

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compound was retained inside the cells or that the compound was metabolized.20 Then,

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the cells were lysed and the cellular concentrations of these esters were determined.

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As shown in Table 3, these esters were detected in the lysate. However, compared

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with the initial concentrations (100 nmol), the concentration of the esters in cells was

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so low that the loss could not be offset. This means that EO, EL, and EM were

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metabolized. In fact, vast amounts of esculin were detected in the samples of the AP

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and BL sides of EO, EL, and EM (Table 3). This indicated that the ester bonds of EO,

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EL, and EM were broken, and EO, EL, and EM were metabolized back to esculin. In

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addition, almost no esculin was detected in the samples of EA and EP. This may have 12


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occurred because the enzyme breaking the ester bond was mainly present in the

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cytoplasm. The poor permeability of EA and VP would make it hard for these

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compounds to enter the cytoplasm. Even if EA and VP were hydrolyzed, the

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concentration of the product was too low to be detected. Interestingly, for EO, EL,

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and EM, the amount of esculin molecules on the AP side was 2-3 times higher than

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that on the BL side, which appeared to indicate that the enzyme was mainly situated

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in the cytomembrane of the AP side.

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Transport of Esculin and its Esters Across Caco-2 Cell Monolayers without

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CES-mediated Hydrolysis CES are members of the α/β hydrolase fold family and

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show ubiquitous tissue expression profiles with high levels in the liver, small intestine

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and lung. They are the most important hydrolases for ester-containing drugs.21 Caco-2

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cells were reported to have sufficient CES.21, 33 Thus, it was speculated that CES

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hydrolyzed EO, EL, and EM. To demonstrate this speculation, BNPP, a specific CES

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inhibitor was used to inhibit CES-mediated hydrolysis. After pretreatment with BNPP,

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the Papp of EA and EP was unchanged, whereas the Papp of EO, EL, and EM increased

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significantly (Table 4); especially, the Papp of EO was close to that of propranolol.

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Additionally, the recoveries of EO, EL, and EM were greater than 90% (Table 4).

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These results demonstrated that CES led to the main hydrolysis of EO, EL, and EM.

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However, a small amount of esculin was detected (data not shown), which indicated

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that other esterases existed.17 CES is predominantly located in the endoplasmic

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reticulum membrane,34 which explained the low levels of hydrolysis of EA and EP.

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However, it is difficult to determine why the mole number of esculin in the AP side 13



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was much greater than that in the BL side. In fact, the efflux of drugs was dependent

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on the relative size of the AP and BL membranes in Caco-2 cells. It is already

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accepted that the brush border membrane has a larger surface area than the BL

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membrane owing to the presence of microvilli.21 Thus, after the hydrolysis, the

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produced esculin would be apt to flow to the AP side. The process of transport is

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shown in Figure 3, and the amounts of each part are shown in Table 3. In addition, the

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Papp of EO was close to that of EL and EM after the treatment with BNPP, but the Papp

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of EO was higher than that of EL and EM without BNPP treatment. These results

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suggested that the esters with a longer acyl chain were more easily hydrolyzed by

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CES; this may be another explanation for the low levels of hydrolysis of EA and EP.

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There are five subfamilies of CES: CES1–CES5. The major CES isozyme in

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human intestine is human CES2 isozyme (hCE2), whereas the human CES1 isozyme

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(hCE1) is predominant in Caco-2 cells. The substrate specificities of hCE1 and hCE2

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are different, with hCE2 mainly hydrolyzing drugs in which an alcohol group of the

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pharmacologically active parent drug has been modified with a simple acyl group. In

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contrast, drugs in which the carboxyl group of the pharmacologically active drug is

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modified with small alcohol groups are preferentially hydrolyzed by hCE1. hCE1 is

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also capable of hydrolyzing substrates of hCE2 at low levels of activity, whereas

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hCE2 is unable to hydrolyze substrates of hCE1.21 Esculin esters appeared to be

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substrate of hCE2, which suggested that they may undergo more extensive hydrolysis

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in the human intestine. Besides CES, there are other enzymes or proteins affecting the

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bioavailability of esters in the human intestine, such as, UGT, cytochrome P450, 14


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multidrug resistance-associated protein 2, breast cancer resistance protein.35 The

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colonic microflora also can hydrolyze the ester drugs.36 Even food microstructure

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affects the bioavailability.37 Thus, for the bioavailability of esculin and its esters in

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body, more research is needed.

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Antiradical Ability of Esculin and its Esters Scavenging free radicals is an

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essential ability for most antioxidants. The antioxidant activities of esculin and its

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esters were measured by the ABTS, ORAC, and DPPH assays. Like most of the

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acylated derivatives and their parent compounds,14,

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esculin esters was similar to or lower than that of esculin (Table 5 and Figure 4).

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However, one report indicates that the antiradical ability of acylated derivatives was

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higher than that of parent compounds.40 That study may be explained by the location

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at which the acylation occurred, which was the phenolic hydroxyl group. The

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phenolic hydroxyl group of polyphenols is closely related to their antioxidant

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activity.41 The incorporated fatty acid chains caused electronic and steric effects in the

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benzene ring and improved the antioxidant activity.40 In our study, the acylation

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occurred at the stable glycosidic moiety and had little effect on the active site of

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esculin. Thus, the antiradical ability of the acylated derivatives could not exceed that

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of esculin. As shown in Table 5 and Figure 4, in the ABTS and ORAC assays, the

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antiradical ability of esculin esters decreased with an increase in the acyl chain length.

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The values for EA and EP were close to those for esculin, but those of EO, EL, and

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EM were much lower than that of esculin. The cause of these differences is shown in

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Figure 5. At the same concentration, the distribution of antioxidants in Figure 5A

38, 39

15


the antiradical ability of


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provided more contact with free radicals than that in Figure 5B. Therefore, the

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antioxidants in Figure 5A would exhibit more effective antiradical ability than those

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in Figure 5B. The logP values of EA and EP indicated a relatively high hydrophilicity.

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The ABTS and ORAC assays occur in a hydrophilic environment. Thus, EA and EP

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could be evenly distributed in the system, whereas EO, EL, and EM had higher

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lipophilicity and would form micelles. This structure will decrease the contact

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between the active site of esculin and free radicals. In the DPPH assay, the antioxidant

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activity of EO was similar to that of esculin, because that the solvent in the reaction

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system was methanol. EO is more soluble in methanol and a homogeneous

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distribution can be achieved. This result was in agreement with some previous

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studies.39, 42 However, EL and EM had a lower DPPH free radical scavenging ability.

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This may be explained by the hydrophobicity of EL and EM, which was much higher

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than that of methanol and still led to a low solubility in methanol.43

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Attenuation of Erythrocyte Hemolysis by Esculin and its Esters Although

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many reports have indicated that the antiradical ability of acylated derivatives was

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lower than that of their parent compounds, these new compounds exhibited more

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remarkable antioxidant capacities when tested in food matrices, such as oils and

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oil-in-water emulsions.25 This phenomenon can be explained by the polar paradox

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theory, whcih can be applied to cells.9, 25 However, there are only a few studies that

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use cells to evaluate the antioxidant activity of these acylated derivatives.39, 42 The cell

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lines used in those studies may possess esterases. As previously mentioned, the esters

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may be hydrolyzed by certain esterases in cells. Thus, to exclude the effect of 16


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esterases on these esters, the erythrocytes were used as a model to explore the

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antioxidant activities of esculin and its esters, because the metabolic networks in

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erythrocytes are relatively simple. The esterases are not included in metabolic

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networks of erythrocytes.16 The hemolysis of erythrocytes has been used extensively

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as an ex vivo model for the study of the antioxidant activity. In the erythrocyte

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hemolysis assay, free radicals induced by AAPH can cause lipid peroxidation and loss

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of erythrocyte membrane integrity, which can ultimately lead to hemolysis.16

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As shown in Figure 6A, erythrocyte hemolysis was effectively attenuated by

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esculin and its esters. Their activity was in the order EO > EP ≈ EA > esculin > EL ≈

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EM. The samples without AAPH supplementation did not induce hemolysis (data not

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shown). AAPH-induced ROS generation can cause membrane lipid peroxidation and

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result in the release of MDA. As seen in Figure 6B, the level of MDA in erythrocytes

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was in accordance with the result of the hemolysis.

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The structure of an erythrocyte is similar to a micro-balloon with a lipid bilayer

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surface. Therefore, its interaction with water is similar to an oil-in-water emulsion. An

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oil-in-water emulsion generally consists of three essential parts: lipid droplets, the

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continuous aqueous phase, and the oil-water interface. According to the polar paradox

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theory, nonpolar antioxidants are more effective than their polar homologs in

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oil-in-water emulsions.44 Nonpolar antioxidants are mainly concentrated at the

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oil-water interface, the optimum location for shielding the oil droplets from oxidation;

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therefore, they would inhibit lipid oxidation more efficiently.25 In fact, lipophilicity

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has been viewed as an important factor with respect to the effectiveness of the 17



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anti-hemolytic substances in hemolytic studies.16,

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hydrophilicity of esculin was high and it was mainly distributed in the aqueous phase.

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The mechanism of the inhibitory effect on hemolysis is only dependent on the ability

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of scavenging free radicals produced by AAPH. For EA, EP, and EO which have a

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relatively higher lipophilicity; the mechanism is not only dependent on the antiradical

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ability, but also the location on the “oil-water interface” (Figure 7B). The increased

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lipophilicity of the esters made it easier to form a shield around the cells; thus, the

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inhibition of hemolysis occurred in the order: EO > EP ≈ EA > esculin

As shown in Figure 7A, the

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However, the polar paradox theory has faced some challenges in recent years. One

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such challenge is the “cutoff effect”, which means that a nonlinear relationship

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existed between polarity and the antioxidant efficacy in emulsions for antioxidants.

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There is a threshold for the antioxidant activity conferred by the increased alkyl

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chain lengths; with further chain length extension, the activity will drastically

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decrease. Several studies have revealed that short-medium-chain lipophilic esters

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were able to improve the efficacy of antioxidants in emulsions better than long-chain

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esters.9 This phenomenon also was observed in this study. The reason may be that if

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the lipophilicity of the antioxidants was too high, they would most likely be placed at

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the interior of the emulsion dissolved in the oil droplet, or aggregate readily in the

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aqueous phase rather than orienting themselves at the interfacial layer.9,

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indicated that the low inhibitory effects of EL and EM on hemolysis may be a result

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of the ease of entering the cell and forming micelles (Figure 7C).

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25

This

Some reports have deemed that a good antioxidant for oil-in-water emulsions 18


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should be an effective surfactant. At certain hydrophilic-lipophilic balance (HLB)

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values (between 8 and 11), the lipophilic antioxidants can be located in the oil-water

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interface. The cutoff effect appears when HLB values are lower than 8.25 However, all

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HLB values of esculin esters were greater than 11 (EA, 16.9; EP, 16.3; EO, 13.9; EL,

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12.3; EM, 11.7), and the cutoff effect appeared at 12.3. The explanation for the

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different cutoff chain lengths may be that an effective surfactant depends on different

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parameters that may vary for each specific series of phenolipids, such as the optimum

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HLB value or the specific polarity and geometry of the polar head.25 Although

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erythrocytes could be approximately regarded as the emulsion or oil droplets, the

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structure of the cytomembrane is quite different from that of oil droplets.

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The Role of “Oil-Water Interface” in Inhibiting Hemolysis A “shield” around

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the cells was supposed to exist according to the polar paradox theory. The existence of

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the “shield” was dependent on the “oil-water interface”. To further demonstrate the

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role of “oil-water interface”, erythrocyte hemolysis was performed in cold ultrapure

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water. The water was then treated with AAPH and esculin or esculin esters according

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to the method described in the assay for erythrocyte hemolysis. The level of MDA

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showed that esculin was a potent inhibitor of membrane lipid peroxidation. The

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activity of the esters was in the order, EA ≈ EP > EO > EL ≈ EM (Figure 8), which

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was similar to the order of the antiradical abilities. This indicated that the inhibitory

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effect of the esters on membrane lipid peroxidation was dependent on the intact closed

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structure of the cytomembrane. The structure of cytomembrane was destroyed and, as

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there was not the “oil-water interface”, the esters could not aggregate. Thus the 19



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“shield” cannot be formed, and the esters cannot show an improved inhibition of lipid

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peroxidation.

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In conclusion, this study investigated the bioavailability and antioxidant activity of

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esculin and its acylated derivatives. It was found that all derivatives had a better

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bioavailability than esculin. However, the bioavailability of derivatives with short or

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long alkyl chain still was low. The derivative with medium alkyl chain had a much

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better bioavailability. Besides, derivatives with medium and long alkyl chain were

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hydrolyzed by CES, which significantly decreased the bioavailability of esters.

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These results hinted that the acylation was an effectual method to improve the

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bioavailability of polyphenols with poor lipophilicity. However, certain factors should

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be considered in choosing acyl donors, such as the suitability between the lipotropism

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of the acyl donor and hydrophilicity of these polyphenols and relationship between

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the structure of the acyl donor and the substrate specificity of CES. The antioxidant

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activity analysis showed that although the antiradical ability of the acylated esculin

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was not improved, the protective effect on cells under the oxidative stress conditions

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was enhanced. These results indicated that the antioxidant activity of these acylated

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derivatives was dependent on not only the antioxidant properties of their parent

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compound, but also their distribution in the reaction system of the antioxidant activity

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assays. Compared with other research, our study proposed that acyl donors should be

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chosen according to the purpose of acylation. For example, if the acylated derivatives

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will be used as drug or prodrug, the acyl donor should help the derivatives cross the

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intestinal wall and vascular wall. If the derivatives were used to protect the fatty or 20


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oily foods from oxidation, the acyl donor should help the derivatives locate in the

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oil-water interface; thus, this study provides the guidance for the acylation of

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polyphenols with poor lipophilicity.

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Funding This work was financially supported by the National Natural Science

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Foundation of China (Nos. 31270636, 21676105), Self Determined Research Fund

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of SCUT from the College Basic Research and Operation of MOE (No. 2015ZZ111),

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and Science and Technology Planning Project of Guangdong Province (Nos.

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2016A040402020, 2016B010121014).

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Supporting Information Standard curves, limit of quantification and limit of

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detection; Intra- and inter-day accuracy and precision; HPLC chromatograms of

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blank HBSS and HBSS spiked with standard compounds.

21



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FIGURE CAPTIONS

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Figure 1 Chemical structure of esculin and its esters.

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Figure 2 LogP values of esculin and its esters. Different letters indicate significant

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differences (p

Cellular Transport of Esculin and Its Acylated ... - ACS Publications

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