Antioxidant activities of chlorogenic acid derivatives with different acyl donor chain lengths and their stabilities during in vitro simulated gastrointestinal digestion

Article abstract of DOI:10.1016/j.foodchem.2021.129904

In this study, chlorogenic acid (CA) was acylated with vinyl esters of different carbon chain lengths under the action of the lipase Lipozyme RM. Five CA derivatives (C2-CA, C4-CA, C6-CA, C8-CA, and C12-CA) with different lipophilicities were obtained, and their digestive stabilities and antioxidant activities were evaluated. The lipophilicities were positively correlated with the digestive stabilities of CA derivatives. The antioxidant activities of CA derivatives did not change with the reduction of phenolic hydroxyl groups, and their capacity to scavenge 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺?) and 1,1-diphenyl-2-picrylhydrazyl (DPPH?) were similar to those of CA. In cellular antioxidant activity (CAA) tests, it was found that the capacity of these derivates to cross cell membranes were enhanced upon enhancing lipophilicity, and their antioxidant activities were improved. C12-CA showed the best antioxidant activity with a median effective dose (EC₅₀) of 9.40 μg/mL, which was significantly lower than that of CA (i.e., 29.08 μg/mL).

Full text of DOI:10.1016/j.foodchem.2021.129904

Food Chemistry 357 (2021) 129904
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Antioxidant activities of chlorogenic acid derivatives with different acyl
donor chain lengths and their stabilities during in vitro simulated
gastrointestinal digestion
a
,
b
a,
c
d
a
e
a
Shan Wang , Yue Li , Xiangyong Meng , Shangwei Chen , Dejian Huang , Yongmei Xia ,
Song Zhu
a,b,*
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
College of Life Science, Anhui Normal University, Wuhu, Anhui 241000, China
b
c
d
e
Department of Food Science and Technology, National University of Singapore, Singapore 117543, Singapore
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, chlorogenic acid (CA) was acylated with vinyl esters of different carbon chain lengths under the
action of the lipase Lipozyme RM. Five CA derivatives (C2-CA, C4-CA, C6-CA, C8-CA, and C12-CA) with different
lipophilicities were obtained, and their digestive stabilities and antioxidant activities were evaluated. The lip-
ophilicities were positively correlated with the digestive stabilities of CA derivatives. The antioxidant activities of
CA derivatives did not change with the reduction of phenolic hydroxyl groups, and their capacity to scavenge
Chlorogenic acid
Acylated chlorogenic acid
Antioxidant activity
Digestive stability
Lipophilization
′
+
2
,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •) and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) were
similar to those of CA. In cellular antioxidant activity (CAA) tests, it was found that the capacity of these der-
ivates to cross cell membranes were enhanced upon enhancing lipophilicity, and their antioxidant activities were
improved. C12-CA showed the best antioxidant activity with a median effective dose (EC50) of 9.40
which was significantly lower than that of CA (i.e., 29.08 g/mL).
μ
g/mL,
μ
1
. Introduction
The structural differences between CA and its lipophilic derivatives
result in different biological activities (Tang et al., 2016). Lopez-Giraldo
́
Chlorogenic acid (CA) is widely found in honeysuckle, coffee beans,
et al. (2009) reported that the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
free radical scavenging pathways observed for CA were different from
those of CA esters obtained by the reaction of CA with different fatty
alcohols. These researchers confirmed that during the first two minutes
of the reaction, only one unstable H atom could be transferred to the
DPPH radical by the CA esters. While there were two unstable H atoms
could be transferred to the DPPH radical by CA. Three new acylated
quercetin analogs were also synthesized by the acylation of quercetin
with cinnamic acid, whereby the acylated quercetins were more lipo-
philic than quercetin (Saik, Lim, Stanslas, & Choo, 2016). In addition,
Nardi et al. (2017) reported that lipophilic oleuropein aglycone de-
rivatives displayed higher lipophilicities than oleuropein, and their
radical scavenging powers in organic media and biological environ-
ments were also strengthened.
and other plants, and is known to exhibit various positive biological
activities (Araújo, de Paulo Farias, Neri-Numa, & Pastore, 2021; Sato
et al., 2011). For example, CA exhibits good antioxidant activity owing
to its polyphenolic structure (Zhong & Shahidi, 2011). However, the CA
polar nature renders it relatively insoluble in a lipid matrix (Hernandez,
Chen, Chang, & Huang, 2009). Moreover, its polyphenolic structure
leads to its instability and poor penetration across the lipophilic mem-
brane barrier, limiting its absolute bioavailability in the human body
(
Chen et al., 2017). This indicates that the biological activity of CA is
closely related to its chemical structure. Lipophilic reactions were used
to alter the molecular structure of CA and improve its hydrophobicity
́
Lopez-Giraldo et al., 2009). One of these lipophilic reactions is acyla-
(
tion, which can effectively improve the hydrophobicity of CA by
reducing the number of hydroxyl groups.
The antioxidant activities determined by spectrophotometric
*
Corresponding author.
E-mail address: zhusong@jiangnan.edu.cn (S. Zhu).
https://doi.org/10.1016/j.foodchem.2021.129904
Received 16 September 2020; Received in revised form 24 March 2021; Accepted 28 March 2021
Available online 20 April 2021
0
308-8146/© 2021 Elsevier Ltd. All rights reserved.

S. Wang et al.
Food Chemistry 357 (2021) 129904
methods strongly depends on the experimental conditions (Tomac,
penicillin, and streptomycin were obtained from Thermo Fisher Scien-
tific (Waltham, MA, USA). The water used in all experiments was puri-
fied using a Milli-Q purification system.
ˇ
Seruga, & Labuda, 2020). Various methods for determining antioxidant
activities, based on different mechanisms, have been established to
comprehensively evaluate the antioxidant activities of the substances.
For example, 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
2
.2. Preparation of the acylated CA derivatives
(
ABTS) scavenging is employed to determine the antioxidant activity of
substances, whereas the ferric reducing power is mainly used to deter-
mine the reducing ability (Oh & Shahidi, 2017; Tomac et al., 2020).
However, the radical scavenging effects observed in a chemical matrix
are rarely observed in the human body (Schaich, Tian, & Xie, 2015). An
antioxidant’s effectiveness mainly depends on its molecular structure,
hydrophobicity, and cellular uptake (Liao, Brock, Jackson, Greenspan, &
Pegg, 2020). Notably, upon ingestion, an antioxidant initially undergoes
digestion in the gastrointestinal tract, which is a complex system
wherein many factors (e.g., pH and presence of enzymes) affect the
stability of the antioxidant. In the context of this study, CA is known to
be unstable and is easily affected by many factors, such as temperature
and pH. Therefore, the digestive stabilities of acylated CA derivatives
were examined in an in vitro simulated digestive system (Limwachiranon
et al., 2020). Furthermore, the celluar antioxidant activity (CAA)
experiment, established by Wolfe and Liu (2007), was employed to
reflect the absorption and antioxidant capacities of the antioxidants at
the cellular level.
Five acylated CA derivatives (Fig. 1), namely 4-O-acetyl-chlorogenic
acid (C2-CA), 4-O-butyryl-chlorogenic acid (C4-CA), 4-O-hexanoyl-
chlorogenic acid (C6-CA), 4-O-octanoyl-chlorogenic acid (C8-CA), and
4
-O-lauroyl-chlorogenic acid (C12-CA), were synthesized using Lip-
ozyme RM, as reported previously (Zhu, Wang, Chen, Xia, & Li, 2020).
CA (100 mg), Lipozyme RM (110 mg), MTBE (10 mL), and acyl donors
(
vinyl acetate, vinyl butyrate, ethenyl hexanoate, vinyl octanoate, and
◦
vinyl laurate; CA-to-acyl donor molar ratio = 1:10) were mixed at 55 C
and 400 rpm for 7 d. The acylated CA derivatives were then separated
and purified using a Waters 2545 preparatory HPLC instrument (Waters,
Milford, MA, USA) and subsequently identified according to the method
reported by Zhu et al. (2020).
2
.3. Analysis of the CA derivatives
The method used to analyze CA and its derivatives was adapted from
a previously reported method with some modifications (Zhu et al.,
Although several studies have been conducted on the antioxidative
and biological activities of CA (Naveed et al., 2018; Nikpayam et al.,
2
1
020). CA and its derivatives were monitored at 320 nm using an Agilent
260 HPLC system (Santa Clara, CA, USA) equipped with a C18 column
2
020), related studies on the acylated derivatives of CA were limited,
(
250 mm × 4.6 mm; i.d., 5
μ
m, Waters, Milford, MA, USA) at 320 nm and
especially in terms of the structural effects of different chain lengths at
the same substitution site. Since the chemical structure of an antioxidant
determines its mode of action, the differences in the chemical structures
of CA and its derivatives would lead to different antioxidant effects.
However, the antioxidant results obtained using chemical media have
been questioned in terms of their relevance in the context of an in vivo
environment (Liao et al., 2020).
◦
3
0 C. The flow rate was 0.8 mL/min, and the injection volume was 10
μ
L. Mobile phases A and B comprised water:methanol:phosphoric acid in
ratios of 90:10:0.05 and 20:80:0.05 (v/v/v), respectively. The gradient
elution process was as follows: 0.00–20.00 min, 20–100% B;
0.00–30.00 min, 100% B, 30.00–35.00 min, 100–20% B, and
5.00–45.00 min, 20% B.
2
3
In this study, given the advantages of using a lipase, including good
substrate specificity and mild reaction conditions (Villeneuve, 2007), in
addition to the observed enhancement in the hydrophobicity of CA
following acylation, the lipase Lipozyme RM was used to catalyze the
acylation of CA to acquire CA derivatives. The CA derivatives were
prepared from CA and vinyl esters with different acyl donor chain
lengths using Lipozyme RM as the catalyst. The lipophilicities, digestive
stabilities, and antioxidant properties (DPPH and ABTS radical scav-
enging, ferric reducing power, and CAA tests) of CA and its derivatives
were then investigated.
2
.4. Octanol-water partition coefficient (log P)
To evaluate the lipophilicities of CA and its acylated derivatives, the
log P values were determined using a method previously reported by
Yang, Kortesniemi, Yang, and Zheng (2018), with some modifications.
Briefly, a saturated mixture of n-octanol and water (1:1, v/v) was pre-
◦
pared in a shaking water bath at 100 rpm and 37 C for over 24 h. To
separate the water-saturated n-octanol from the n-octanol-saturated
water, the mixture was allowed to stand overnight. Subsequently, the CA
and acylated CA samples (2 mg) were dissolved in water-saturated n-
octanol (5 mL). An aliquot of each sample (0.4 mg/mL, 1 mL) and n-
octanol-saturated water (1 mL) were then mixed in a shaking water bath
2
. Materials and methods
◦
at 100 rpm and 37 C for 24 h. After allowing standing overnight, the
2
.1. Materials
sample solution (200 L) was drawn from the upper (n-octanol) and
μ
lower (water) layers and subsequently measured using an Agilent 1260
HPLC system (Santa Clara, CA, USA). The CA and CA derivative contents
were quantified from the HPLC peak area using the method described in
section 2.3. The log P values were calculated using the following
equation:
CA (purity > 98%), quercetin, and ABTS were purchased from
Macklin Biochemical Technology Ltd. (Shanghai, China). Vinyl acetate
>99%), vinyl butyrate (>99%), vinyl hexanoate (>99%), vinyl octa-
(
noate (>99%), and vinyl laurate (>99%) were purchased from Tokyo
Chemical Industry Ltd. (Tokyo, Japan). Pepsin, trypsin, DPPH, 3-(4,5-
′
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2 -
LogP = log A1
A
2
(1)
azobis (2-amidinopropane) dihydrochloride (ABAP), and 2ʹ,7ʹ-dichlor-
ofluorescin diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Lipozyme Rhizomucor miehei (RM) was supplied by
Novozymes (Bagsværd, Denmark). Analytical grade absolute ethanol,
methyl tert-butyl ether (MTBE), potassium persulfate, dibasic sodium
phosphate, sodium chloride, sodium dihydrogen phosphate, potassium
ferricyanide, trichloroacetic acid solution, iron(III) chloride hexahy-
drate, octanol, hydrochloric acid, sodium bicarbonate, methanol,
vitamin C (Vc), tert-butylhydroquinone (TBHQ), and dimethyl sulfoxide
where A
1
and A
2
are the peak areas of the sample in n-octanol and water,
respectively.
2.5. In vitro simulated digestion
The digestive stabilities of CA and its acylated derivatives were
evaluated by an in vitro simulated digestion method that involved a two-
stage process, as adapted from a previous study with some modifications
(Celep, Charehsaz, Akyüz, Acar, & Yesilada, 2015). More specifically, in
a conical flask with a stopper, an aqueous NaCl solution (4 mL, 9 mg/
(
(
(
DMSO) were provided by the Sinopharm Group Chemical Reagent Ltd.
Shanghai, China). Finally, Dulbecco’s modified Eagle’s medium
DMEM), phosphate buffer solution (PBS), fetal bovine serum (FBS),
2

S. Wang et al.
Food Chemistry 357 (2021) 129904
Fig. 1. Structures of the acylated CA derivatives.
mL) was mixed with a pepsin solution (4 mL, 1 mg/mL), prepared using
a 0.1 mol/L aqueous HCl solution. The pH of the resulting solution was
adjusted to 2.0–2.5 using 0.1 mol/L HCl to afford the simulated stomach
solution. Solutions of CA and the acylated CA samples (1 mL, 3 mg/mL)
prepared with anhydrous ethanol and the simulated stomach solution
acylated derivatives according to previously reported methods (Liu &
Yan, 2019; Sun et al., 2020) with some modifications. Vc and TBHQ
were used as the controls.
The samples (CA and its acylated derivatives, 1 mL), with derivative
concentrations ranging from 0 to 300 mol/L, were mixed well with an
μ
◦
were then incubated in a shaking water bath at 100 rpm and 37 C for 2
ethanolic solution of DPPH (1 mL, 0.2 mmol/L) and incubated in the
◦
h. Aliquots of the mixture (200
and 2 h.
μL) were drawn from the conical flask at
dark for 30 min at 30 C. Subsequently, the absorbance, A, of the sample
0
was determined at 517 nm using a UV-1206 spectrophotometer (Shi-
madzu Corporation, Kyoto, Japan). The DPPH radical scavenging ac-
tivities of CA and its acylated derivatives were then calculated using the
following equation:
The pH of the remaining solution was then adjusted to 6.5–7.0 using
◦
a 1 mol/L aqueous NaHCO
3
solution. After shaking at 100 rpm and 37 C
for 45 min, a trypsin solution (1 mL, 0.9 mg/mL) prepared using 0.1
mol/L NaHCO was added, and the pH of the mixture solution was
adjusted to 7.0–7.5 using 1 mol/L NaHCO to afford the simulated in-
testinal solution. This latter solution was shaken for 3 h to simulate in-
3
A
0
ꢀ (A
1
ꢀ A )
2
3
DPPH radical scavenging activity (%) =
where A , A , and A
× 100
(3)
A
0
testinal digestion. Aliquots of the mixture (200
the conical flask at 0 and 3 h.
μL) were also drawn from
0
1
2
are the absorbance values of the ethanol/DPPH,
the sample/DPPH, and the sample/ethanol mixtures, respectively.
Subsequently, the ABTS radical solution was prepared using an
aqueous ABTS solution (7 mmol/L) and an equal volume of aqueous
The CA and CA derivative contents in the overall system before and
after digestion were quantified from the HPLC peak area using the
method described in section 2.3. The recovery index (RI; digested-to-
nondigested sample concentration ratio) values, which were used to
evaluate the stability of the samples, were calculated using the following
equation:
◦
potassium persulfate solution (2.45 mmol/L). After incubation at 30 C
in the dark for 12–16 h, the mixture was diluted using anhydrous
ethanol to form the final ABTS radical test solution. This test solution
(
1.9 mL) was then mixed with different concentrations of the sample
◦
RI(%) = A1 × 100
(100
μL, 0–100
μmol/L) at 30 C for 6 min, after which the absorbance
(2)
was determined at 734 nm. The ABTS radical scavenging activities of CA
and its acylated derivatives were calculated using the following
equation:
A
2
where A
1
and A
2
are the peak areas of the digested and nondigested
samples, respectively.
A
0
ꢀ (A
1
ꢀ A )
2
ABTS
​
radical scavenging activity(%) =
× 100
(4)
A
0
2
.6. Antioxidant capacity
where A
0 1 2
, A , and A are the absorbance values of the ethanol/ABTS
The DPPH and ABTS radical scavenging activities and ferric reducing
radical test solutions, sample/ABTS radical test solutions, and sample/
capacities were used to evaluate the antioxidant capacities of CA and its
3

S. Wang et al.
Food Chemistry 357 (2021) 129904
ethanol solutions, respectively.
rescence intensity versus time curve and the control fluorescence in-
Finally, the sample (CA and its acylated derivatives, 100
μ
L, 0–100
tensity versus time curve, respectively. The median effective dose (EC50)
μ
mol/L), phosphate buffer solution (2.5 mL, 2 mol/L, pH 6.6), and po-
was calculated using a previously described method (Wolfe & Liu,
◦
tassium ferricyanide (2.5 mL, 1%, w/w) were incubated at 50 C for 20
min. After rapid cooling of the mixture, trichloroacetic acid (2.5 mL,
2007).
1
0%, w/w) was added, and each mixture was deposited for 10 min.
2.10. Statistical analysis
Subsequently, the supernatant (2.5 mL), water (2.5 mL), and ferric
◦
chloride (0.5 mL, 0.5%, w/w) were mixed at 30 C for 10 min. Using an
All data obtained in this study were analyzed using IBM SPSS Sta-
tistics 21.0 (SPSS Inc., Chicago, IL, USA) and Origin 8.6.0 software
(OriginLab Corp., Northampton, MA, USA). The results are presented as
the mean ± standard deviation (SD), and differences of p < 0.05 were
considered significant.
absorbance wavelength of 700 nm, the ferric reducing capacities of CA
and its acylated derivatives were calculated using the following
equation:
Reducing activity = A
t
ꢀ A
0
(5)
3
. Results and discussion
where A and A are the absorbance of the blank and sample,
0
t
respectively.
.7. Cell culture
HepG2 cells were obtained from the American Type Culture Collec-
3
.1. Lipophilicity
2
The log P value, which was used to evaluate lipophilicity, was
positively correlated with lipophilicity (Yang et al., 2018). According to
Fig. 2(A), the lipophilicity of the acylated CA (p < 0.05) increased with
the increasing carbon chain length of the acyl donor. More specifically,
the log P value was ꢀ 0.25 ± 0.04 for CA and 1.44 ± 0.05 for C12-CA.
The lipophilicities of resveratrol derivatives were also reported to in-
crease with increasing chain length (Oh & Shahidi, 2017). However,
according to the report of Oh and Shahidi (2017), when unsaturated
fatty acids were used to acylate resveratrol, the lipophilicity of resver-
atrol decreased, regardless of the extension in the chain length. This was
attributed to the properties of the carbon–carbon double bond. There-
fore, we assumed that in addition to the chain length, the properties of
the nonpolar groups also increase the lipophilicity of CA. Moreover, the
lipophilicities of acylated epigallocatechin gallate, hydrophilic cyanidin,
and delphinidin rutinosides were reported to be higher than those of
their nonacylated equivalents (Wang, Zhang, Zhong, Perera, & Shahidi,
2016; Yang, Kortesniemi, Ma, Zheng, & Yang, 2019), thereby expanding
the range of applications of such compounds. Viskupicova, Danihelova,
Ondrejovic, Liptaj, and Sturdik (2010) also reported that more lipophilic
of rutin derivatives, the superior antioxidant effect of it in an oil matrix.
tion (Rockville, MD, USA) and cultured in a growth medium prepared
with DMEM, FBS (10%, v/v), and antimycotic solution (1%, v/v, 100 U/
◦
mL penicillin, and 100
μ
g/mL streptomycin) at 37 C and 5% CO
2
, as
previously reported (Wolfe & Liu, 2007). Cells between passages 27 and
3
6 were used in this study.
2
.8. Cytotoxicity analysis
3
HepG2 cells were seeded on 96-well plates (7 × 10 /well) in a
◦
growth medium (100
μ
L) and cultured at 37 C and 5% CO
2
for 24 h. The
growth medium was then removed, and the cells were washed once with
PBS. The CA and acylated CA derivatives were then dissolved in DMSO
and diluted to different concentrations (0–100
μ
g/mL) using DMEM
containing 1% antimycotic solution before their addition to the wells.
After 24 h, the DMEM was removed, and the wells were fortified with 5
mg/mL MTT solution (50 L) and incubated for 4 h. Subsequently, the
μ
MTT was removed, and the produced formazan was dissolved in DMSO
with shaking for 15 min. The absorbance of the plate was measured at
5
70 nm using a Multiskan GO spectrophotometer (Thermo Fisher Sci-
3.2. In vitro simulated digestion analysis
entific, Waltham, MA, USA). The cell survival rate, which represents the
sample cytotoxicity, was calculated using the equation:
As a member of the phenol family, CA is also unstable and poorly
absorbed in the small intestine. However, the metabolism and
bioavailability of this compound in the body directly influence its bio-
logical activities. Therefore, improvement in the stability and effec-
tiveness of CA, previously achieved by the addition of wheat gluten
hydrolysate to simulated intestinal juice (He et al., 2020), is considered
highly important. In this study, we aimed to improve the stability of CA
by forming CA derivatives. Therefore, the digestive stabilities of CA and
its acylated derivatives were evaluated, and the effect of the chain length
on the stability of CA was also examined by in vitro stimulated digestion.
As shown in Fig. 2(B), in the gastric digestion simulation stage, CA
was relatively stable, and its RI approached 100%. Although most of the
CA derivatives remained relatively stable, their RI values decreased. The
RI values of C2-CA, C4-CA, C6-CA, and C8-CA were ˃90%. In addition,
with an increase in the chain length of the acyl donor, the RI value
initially increased and then decreased after C4-CA (p < 0.05). Notably,
C4-CA showed the highest RI value among the acylated CA derivatives,
which was closed to that of CA (p > 0.05). On the other hand, in the
intestinal digestion stage, the RI value of CA was minimal and only
slightly higher than that of C2-CA (p > 0.05). The regularity of the CA
derivative chain length and RI trend was unclear during the intestinal
digestion stage. However, the RI of C4-CA was higher than that of the
other acylated CA derivatives and 2.11 times higher than that of CA.
Previous studies have also shown that the amount of CA remained
relatively unchanged during the gastric digestion stage and decreased
significantly during the intestinal digestion (Bermúdez-Soto, Tom a´ s-
A
A
sample ꢀ Ablank
control ꢀ Ablank
Cell survival rate(%) =
× 100
(6)
where A is the absorbance at 570 nm. The concentrations of CA and its
acylated derivatives with absorbance values > 90% compared to that of
the control were considered nontoxic.
2
.9. Cellular antioxidant activity (CAA)
4
HepG2 cells were seeded on 96-well black plates (6 × 10 /well) in a
◦
growth medium (100
μ
L) and cultured at 37 C and 5% CO
2
for 24 h.
Then, the growth medium was removed, and the cells were washed once
with PBS. DMEM (100 L) containing 1% antimycotic solution, the
sample, and DCFH-DA (25 mol/L) was used to treat the wells for 1 h.
Subsequently, ABAP (100 L, 600 mol/L) was applied to the cells after
μ
μ
μ
μ
either a PBS wash or no PBS wash. The 96-well black plate was measured
using a Thermo Scientific Fluoroskan Ascent FL (Thermo Fisher Scien-
tific, Waltham, MA, USA) every 5 min for 1 h with an emission of 538 nm
and excitation of 485 nm. The CAA value was calculated using the
equation:
(
∫
)
SA
CA
CAA unit = 1 ꢀ ∫
× 100
(7)
∫
∫
where SA and CA equal the integrated areas under the sample fluo-
4

S. Wang et al.
Food Chemistry 357 (2021) 129904
Fig. 2. Lipophilicities and simulated digestion
stabilities of CA and its acylated derivatives:
(
A): Lipophilicities of CA and its acylated de-
rivatives; lipid-water partition coefficient (log
P). Significant differences (p < 0.05) between
the samples are marked as a, b, c, d, e, and f.
(
B): Simulated digestion stabilities of CA and its
acylated derivatives; gastric digestion at 2 h,
and intestinal digestion at 3 h. Significant dif-
ferences (p < 0.05) between the samples in the
gastric digestion experiments are marked as a,
b, c, d, e, and f, and in the intestinal digestion
experiments these are marked as A, B, C, D, E,
and F.
Barber a´ n, & García-Conesa, 2007; Narita & Inouye, 2013). It indicated
that during gastrointestinal digestion, the stability of CA improved after
acylation. Under neutral or alkaline conditions, CA is easily digested by
mucosal esterases or oxidized to other substances (Xiong et al., 2020).
However, the RI value of CA increased when CA was acylated, especially
when acylation was conducted using an acyl donor containing four
carbon atoms.
rutinoside were significantly lower than the corresponding values for
unmodified anthocyanins. Chlorogenic laurate, a lipophilized CA de-
rivative, exhibits relatively strong antioxidant activity, which is higher
than that of CA in the DPPH assay (Xiang & Ning, 2008); however, the
DPPH radical scavenging activities of the acylated CA prepared using
palmitoyl chloride were 70–80% that of CA (Lorentz et al., 2010). These
contradictory results were attributed to the different numbers of carbon
atoms in the substances used to improve the polyphenol lipophilicities.
In this study, the antioxidant activities of CA and its derivatives prepared
using vinyl esters of different chain lengths were evaluated using DPPH
and ABTS radical scavenging and ferric reducing capacity assays. These
assays are based on the same electron transfer mechanism, and thus, the
relationship between lipophilicity and antioxidant activity based on
electron transfer is easier to elucidate. However, there were also some
differences between the three methods. Indeed, the medium of the DPPH
radical scavenging test comprised an organic system, the ABTS radical
scavenging test comprised a mixed system of ethanol and water, and the
ferric reducing capacity assay comprised an aqueous solution.
3
.3. Antioxidant assay
The biological activities of polyphenols were reported to be posi-
tively correlated with the number of hydroxyl groups in their molecular
structures. However, when the hydroxyl group was replaced with an
acyl group, the stability and lipophilicity improved, which was condu-
cive to the dissolution of such compounds in lipid substances. In the
DPPH and ABTS radical scavenging experiments, most lipophilized
EGCG derivatives possessed better scavenging capacities than EGCG
(
Wang et al., 2016). On the other hand, the scavenging capacities of the
In the DPPH assay, CA exhibited a weaker antioxidant capacity than
the controls (hydrophilic Vc and lipophilic TBHQ; p < 0.05) but a su-
perior capacity than its acylated derivatives, except C4-CA (Fig. 3(A)
and Table 1). Indeed, C4-CA may have a greater affinity for the lipo-
philic DPPH radical than CA (p < 0.05), owing to its superior
resveratrol derivatives were lower than that of resveratrol (Oh & Sha-
hidi, 2017), while those of the EGCG derivatives acylated with palmitoyl
chloride slightly decreased in the ABTS assay (Liu & Yan, 2019). Yang
et al. (2019) also reported that the reducing powers of acylated del-
phinidin-3-O-glucoside, cyanidin-3-O-glucoside, and cyanidin-3-O-
5

S. Wang et al.
Food Chemistry 357 (2021) 129904
Fig. 3. Antioxidant activities of CA, its acylated derivatives, Vc, and TBHQ in chemical media: (A): DPPH radical scavenging capacity, (B): ABTS radical scavenging
capacity, and (C): ferric reducing capacity.
derivatives initially increased (p < 0.05) before decreasing again after
Table 1
C4-CA (p > 0.05). This was attributed to the lower lipophilicity of the
IC50 values for of samples in DPPH and ABTS radical scavenging tests and the
ABTS radical. Thus, upon increasing the derivative lipophilicity, the
EC50 values of samples in CAA test.
affinity to the ABTS radical decreased, resulting in reduced ABTS radical
Samples
IC50
,
μ
mol/L
EC50
, g/mL
μ
scavenging abilities for the various derivatives (Wang et al., 2016). It
was also observed that the scavenging effects of CA and its acylated
derivatives on the ABTS radical were higher than the equivalent effects
on the DPPH radical. In the ferric reducing capacity test, the ferric ion in
potassium ferricyanide was reduced to the ferrous ion by CA and its
acylated derivatives. Prussian blue, which exhibits a strong absorption
at 700 nm, was formed by the reaction of the ferrous ions produced from
the ferric ion in ferric chloride. These results indicated that a greater
absorbance corresponded to a superior reducing capacity. Fig. 3(C) il-
lustrates that the reducing capacities of the acylated CA derivatives were
slightly lower than those of CA but higher than those of Vc and TBHQ.
DPPH
ABTS
CAA unit
No wash
12.05 ±
Wash
b
eB
gB
fB
CA
45.61 ± 2.30
44.55 ±
29.08 ± 0.30
b
dA
1
.35
0.31
c
fA
C2-CA
C4-CA
C6-CA
C8-CA
C12-CA
110.67 ±
53.21 ± 0.70
20.79 ± 0.40
33.12 ± 1.40
30.92 ± 0.65
25.96 ± 0.40
24.28 ± 0.73
d
2
.30
b
c
c
c
eA
cA
cA
43.80 ± 0.90
51.73 ± 0.68
53.13 ± 0.66
52.87 ± 2.63
42.09 ±
15.44 ± 0.24
11.38 ± 0.31
11.28 ± 0.56
10.04 ±
b
1
.14
dB
cB
43.31 ±
b
1
.27
44.11 ±
b
1
.88
Using a sample concentration of 100
μmol/L, C6-CA exhibited the
bA
45.38 ±
9.40 ± 0.96
highest reducing capacity, followed by C4-CA. The reducing ability,
therefore, decreased in the following order: CA > C6-CA > C4-CA > C8-
CA > C2-CA > C12-CA > TBHQ > Vc, thereby indicating that the ferric
reducing capacity test was more sensitive to CA and its derivatives than
to Vc and TBHQ.
b
bA
1
.43
0.40
a
a
a
a
Vc
36.48 ± 0.50
38.97 ± 0.18
–
38.27 ± 1.42
39.32 ± 1.03
–
–
–
–
–
TBHQ
Quercetin
aA
aB
8.03 ± 0.30
6.86 ± 0.34
In this study, the CA derivatives acylated with medium-length chains
presented stronger antioxidant capacities in all tests employed
compared to the capacities observed for the other CA derivatives.
Indeed, as previously reported, the antioxidant capacity of a sample is
related not only to the number of hydroxyl groups, but also to the lip-
ophilicity and electronic distribution (Wang et al., 2016). Thus, the
lower reducing capacity of the acylated derivatives, compared to that of
CA itself was likely due to the loss of a hydroxyl group, and the number
of hydroxyl groups was related to the establishment of stable antioxidant
properties (Naveed et al., 2018). Furthermore, the lipophilicities of the
CA derivatives were superior to that of CA where the ferric reducing
capacity assay was carried out in an aqueous solution. Thus, because the
lipophilicity to that of CA. Moreover, the hydrogen atom donation
capability of CA may be affected by its electron density, which in turn
may be altered by acylation (Zhong & Shahidi, 2011). Among these
derivatives, the DPPH radical scavenging ability of C2-CA was the
poorest, and its half-maximal inhibitory concentration (IC50) was 2.43
times that of CA (p > 0.05). In the ABTS test (Fig. 3(B) and Table 1), the
antioxidant abilities of CA and its derivatives were also weaker than
those of Vc and TBHQ (p < 0.05). In addition, the IC50 value of C2-CA
was 1.18 times higher than that of CA, while the radical scavenging
ability of C4-CA was superior to that of CA. Moreover, with increasing
acyl donor chain length, the ABTS radical scavenging abilities of the CA
6

S. Wang et al.
Food Chemistry 357 (2021) 129904
solubilities of the CA derivatives were lower than that of CA in the ferric
reducing capacity assay medium (i.e., water), a decrease in the reducing
capacity was observed for the CA derivatives.
antioxidant activity in HepG2 cells (p < 0.05), decreased with increasing
chain length. Moreover, the cellular antioxidant activities of C6-CA, C8-
CA, and C12-CA were higher than that of CA (p < 0.05), regardless of
whether a PBS wash was carried out. The results also indicated that the
antioxidant activities of the CA derivatives were enhanced with
increasing chain length in the order C12-CA > C8-CA > C6-CA > CA >
C4-CA > C2-CA. Thus, upon increasing the chain length, the lip-
ophilicity advantage of the CA derivatives gradually appeared and
compensated for the loss of one hydroxyl group.
3
.4. Caa
Despite the wide usage of DPPH and ABTS radical scavenging and
ferric reducing capacity assays, their accuracy in predicting in vivo
antioxidant activities has been questioned owing to their simplistic
media (Kellett, Greenspan, & Pegg, 2018). Biological systems are more
complex than chemical systems, and antioxidants that enter an organism
are affected by a range of mechanisms, such as uptake, distribution, and
metabolism. In addition, because the results obtained from the different
antioxidant tests were different, it was necessary to study the antioxi-
dant activities of CA and its acylated derivatives by CAA testing to reflect
the complexity of the biological medium.
It has been previously reported that the surface ultrastructure of the
cell membrane affects the entry of antioxidants into cells (Lu et al.,
2
020). Thus, the antioxidant activities of the samples with and without a
PBS wash were compared to evaluate the absorption of the antioxidants
and the binding degree between the membrane and the antioxidants
(
Wolfe & Liu, 2007). More specifically, when the cells were washed with
PBS, the EC50 values of CA and its derivatives, except C12-CA, were
almost twice those recorded in the absence of a PBS wash. On the other
hand, similar to quercetin, C12-CA did not exhibit any significant dif-
ferences in its EC50 values. Following cell washing with PBS, the anti-
oxidants present on the cell surface that were either not bound or were
loosely bound to the cell membrane were removed, and the antioxidants
that were absorbed by the cells or tightly bound to the cell membrane
were retained. As a result, the CAA unit decreased when PBS washing
was employed. We also observed that an increase in the CA derivative
hydrophobicity resulted in a decrease in the EC50 value. However, in the
case of C12-CA, no significant differences in the EC50 values for the two
test methods were observed. This suggests that C12-CA effectively enters
or combines with the cell membrane to improve its antioxidant capacity,
thereby playing a more important role than that of CA in vivo.
Prior to CAA testing, cell cytotoxicity was analyzed to determine the
sample concentrations that had no effect on cell viability (i.e., cell sur-
vival rate > 90%). As shown in Fig. 4, with a sample concentration of ≤
5
0 g/mL, the cell survival rate was > 90%. However, with a sample
μ
concentration of 100
μ
g/mL, only C8-CA and C12-CA met the exper-
g/mL were used in
imental requirements. Thus, concentrations of ≤ 50
μ
the subsequent experiments. It was also found that the cytotoxicity of CA
and its derivatives was reduced upon increasing the chain length. This
was attributed to the loss of a hydroxyl group in the CA structure, which
led to reduced biological activity. Moreover, the increase in the lip-
ophilicity of the CA derivatives allowed them to enter the cells more
easily and play a protective role, which is consistent with the CAA test
results.
As shown in Fig. 5, the CAA unit of the samples and that of quercetin
increased in a dose-dependent manner. More specifically, when the
4
. Conclusions
sample concentration was 50 g/mL, the CAA unit of C12-CA was > 85,
μ
In this study, we evaluated the differences in the digestive stabilities
which well exceeds the corresponding values of CA and the other CA
derivatives. The EC50 value was also calculated to evaluate the antiox-
idant capacities of CA and its acylated derivatives (Table 1). Interest-
ingly, we observed that the EC50 value, which corresponds to stronger
and antioxidant activities of CA and its derivatives, which were syn-
thesized using different vinyl esters. The relationship between lip-
ophilicity and digestive stability as well as antioxidant capacity was
Fig. 4. Cytotoxicity of CA and its acylated derivatives toward HepG2 cells.
7

S. Wang et al.
Food Chemistry 357 (2021) 129904
Fig. 5. Dose-response curves for inhibition by CA, its acylated derivatives and quercetin: (A): No PBS wash between the antioxidant and ABAP treatments, and (B):
PBS wash between the antioxidant and ABAP treatments.
8

S. Wang et al.
Food Chemistry 357 (2021) 129904
investigated. The results showed that the lipophilicity of CA was
improved by acylation using CA and vinyl esters (vinyl acetate, vinyl
butyrate, vinyl hexanoate, vinyl octanoate, and vinyl laurate). Following
acylation, the antioxidant activity was found to be retained among the
CA derivatives (with the exception of C2-CA), and their digestive sta-
bilities were found to be improved. In the CAA test, the lipophilicities of
the CA derivatives were found to be positively correlated with their
antioxidant activities. Furthermore, the EC50 value of C12-CA, which
possessed the longest-chain length of the derivatives examined herein,
was significantly lower than those of CA and the other derivatives. These
results indicate that lipophilic CA derivatives with good antioxidant
activities exhibit great potential for application in lipid-based foods.
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(
2017). Synthesis and antioxidant evaluation of lipophilic oleuropein aglycone
derivatives. Food and Function, 8(12), 4684–4692.
Shan Wang: Formal analysis, Methodology, Writing - original draft.
Yue Li: Formal analysis, Methodology. Xiangyong Meng: Data cura-
tion, Formal analysis, Methodology. Shangwei Chen: Data curation,
Formal analysis, Methodology. Dejian Huang: Writing - review &
editing. Yongmei Xia: Conceptualization, Formal analysis, Methodol-
ogy. Song Zhu: Conceptualization, Data curation, Investigation,
Resources.
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values and effects of ascorbic acid and epigallocatechin gallate on its stability under
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Declaration of Competing Interest
Oh, W. Y., & Shahidi, F. (2017). Lipophilization of resveratrol and effects on antioxidant
activities. Journal of Agricultural and Food Chemistry, 65(39), 8617–8625.
Saik, A. Y. H., Lim, Y. Y., Stanslas, J., & Choo, W. S. (2016). Lipase-catalyzed acylation of
quercetin with cinnamic acid. Biocatalysis and Biotransformation, 34(1), 33–43.
Sato, Y., Itagaki, S., Kurokawa, T., Ogura, J., Kobayashi, M., Hirano, T., Sugawara, M., &
Iseki, K. (2011). In vitro and in vivo antioxidant properties of chlorogenic acid and
caffeic acid. International Journal of Pharmaceutics, 403(1-2), 136–138.
Schaich, K. M., Tian, X., & Xie, J. (2015). Hurdles and pitfalls in measuring antioxidant
efficacy: a critical evaluation of ABTS, DPPH, and ORAC assays. Journal of Functional
Foods, 14, 111–125.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Sun, J., Jing, H., Liu, T., Dong, S., Obadi, M., & Xu, B. (2020). Evaluation of antioxidant
modification on the functional and structural properties of EWP conjugates. RSC
Advances, 10(18), 10666–10672.
This work was supported financially by the National Natural Science
Foundation of China (No. 31871794, 31701663), an Innovative Project
of the State Key Laboratory of Food Science and Technology (SKLF-ZZB-
Tang, B., Huang, Y., Ma, X., Liao, X., Wang, Q., Xiong, X., & Li, H. (2016).
Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to
human serum albumin: effects of esteryl position on affinity. Food Chemistry, 212,
434–442.
2
01809), the Natural Science Foundation of Anhui Province
(
1808085MC70), and Fundamental Research Funds for the Central
ˇ
Tomac, I., Seruga, M., & Labuda, J. (2020). Evaluation of antioxidant activity of
Universities (JUSRP21905).
chlorogenic acids and coffee extracts by an electrochemical DNA-based biosensor.
Food Chemistry, 325, 126787.
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