Ultrasonic-promoted enzymatic preparation, identification and multi-active studies of nature-identical phenolic acid glycerol derivatives

Article abstract of DOI:10.1039/c9ra09830e

Phenolic acid glycerols (PAGs) are a group of rare phytochemicals found from potato periderm, which show great potential in the food, cosmetic and pharmaceutical industries. In this study, seven PAGs were enzymatically synthesized via transesterification of ethyl phenates (EPs) with glycerol by ultrasonic promotion. The conversions of 88.1-98.5% could be obtained in 1-9 h. Compared with the conventional stirring methods, the catalytic efficiency was significantly increased 11.0-44.0 folds by ultrasound assistance. The lipid peroxidation inhibition activity increased 8.1-fold and 14.4-fold compared to the parent phenolic acids (PAs). Furthermore, caffeoyl glycerol and feruloyl glycerol exhibited excellent antimicrobial activity against Escherichia coli compared to the corresponding PAs with minimum inhibitory concentration (MIC) decreasing 4-16-fold. The PAGs can also absorb a much wider and higher amount of the harmful UV-B rays than the corresponding PAs. The present strategy for facile synthesis of multifunctional PAGs paves the way for the development and application of natural phytochemicals and novel ingredients.

Full text of DOI:10.1039/c9ra09830e

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Ultrasonic-promoted enzymatic preparation,
identification and multi-active studies of nature-
identical phenolic acid glycerol derivatives†
Cite this: RSC Adv., 2020, 10, 11139
a
Teng Sun,‡a Haiping Zhang,‡a Zhe Dong,a Zengshe Liub and Mingming Zheng
*
Phenolic acid glycerols (PAGs) are a group of rare phytochemicals found from potato periderm, which show
great potential in the food, cosmetic and pharmaceutical industries. In this study, seven PAGs were
enzymatically synthesized via transesterification of ethyl phenates (EPs) with glycerol by ultrasonic
promotion. The conversions of 88.1–98.5% could be obtained in 1–9 h. Compared with the
conventional stirring methods, the catalytic efficiency was significantly increased 11.0–44.0 folds by
ultrasound assistance. The lipid peroxidation inhibition activity increased 8.1-fold and 14.4-fold
compared to the parent phenolic acids (PAs). Furthermore, caffeoyl glycerol and feruloyl glycerol
exhibited excellent antimicrobial activity against Escherichia coli compared to the corresponding PAs
with minimum inhibitory concentration (MIC) decreasing 4–16-fold. The PAGs can also absorb a much
wider and higher amount of the harmful UV-B rays than the corresponding PAs. The present strategy for
facile synthesis of multifunctional PAGs paves the way for the development and application of natural
phytochemicals and novel ingredients.
Received 24th November 2019
Accepted 4th March 2020
DOI: 10.1039/c9ra09830e
rsc.li/rsc-advances
phytochemicals with exceptional free radical scavengers and
peroxyl lipid oxidation inhibitors.5,6 Moreover, the introduce of
Introduction
glycerol rather than longer alkyl chain alcohols would be more
advantageous in the UV lter, which stems from the more
hydrophilic character of glycerol to facilitate the penetration of
the fractional formulation into the skin.4,7 However, the prep-
aration of such natural phytochemicals on a large scale as well
as their structural identication confronts difficulties related to
the trace amount of PAGs in nature (<0.1%).4 Until now, only
feruloyl glycerol, caffeate glycerol and 4-methoxy cinnamoyl
glycerol have been synthesized in the enzymatic method but
other kinds of PAGs with different PAs have rarely been re-
ported.3,8,9 Among the reported references, the studies only
focused on the synthesis technology of PAGs and much less
attention on their structural identication and their functional
properties.10–12 Apart from antioxidant and anti-UV, PAs have
also been reported to have remarkable antibacterial effects
against Gram-positive and Gram-negative bacteria.2,13,14 A recent
study demonstrated that the PA-rich extracts from peanut meal
exhibited a high antibacterial effect, even comparable to
Ampicillin.2,15 However, the antimicrobial performance of the
PAGs has not yet been studied.
Phenolic acids (PAs), widely distributed in the plant kingdom
such as in fruits, grains, vegetables, olive oil, coffee and herbs,
exhibit broad biological and pharmacological activity including
inammatory, antioxidant, anti-carcinogenic and anti-
neuroprotective effects. Due to their multiple benets, PAs
and their derivatives have attracted more attention in recent
year.1–3 However, poor solubility of these compounds in both
water and oil media limits their further application in the food,
cosmetic and pharmaceutical industries.4 Therefore, the
modication of these compounds is essential to enhance their
usefulness and widen their application eld. As an example,
phenolic acid glycerols (PAGs) such as feruloyl glycerol and
caffeoyl glycerol were prepared by phenolic acids/phenolic ethyl
phenates with glycerol in transesterication.
As early as 2000, several PAGs such as 1-mono feruloyl glyc-
erol were found in potato periderm, which were all-natural
aOil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key
Laboratory of Oilseeds Processing, Ministry of Agriculture, Oil Crops and Lipids
Process Technology National
& Local Joint Engineering Laboratory, Hubei Key
Suffering from high enzyme loading but poor catalytic
performance in the enzymatic synthesis of PAGs,16 a facile and
high efficiency strategy to prepare PAGs is still desirable.
Recently, an effective enzymatic synthesis strategy with ultra-
sound pretreatment was developed and has been applied in the
preparation of phytosterol esters, avonoid esters and feruloyl
glycerol with superior performance.17,18 Known as an
Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China. E-mail:
zhengmingming@caas.cn
bBio-Oils Research Unit, United States Department of Agriculture, Agricultural
Research Service, National Center for Agricultural Utilization Research, 1815 N.
University St., Peoria, Illinois 61604, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra09830e
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 11139–11147 | 11139

View Article Online
RSC Advances
Paper
outstanding process pretreatment technique, ultrasound can
signicantly shorten reaction time and remarkably increase
conversion.19–21 Thus, it would be wise to introduce ultrasound
pretreatment in PAGs synthesis.
In the present study, seven PAGs were synthesized by enzy-
matic transesterication of glycerol with seven different ethyl
PAs by ultrasound promotion. The structures of seven PAGs
were identied precisely by HPLC-MS and nuclear magnetic
resonance (NMR). The antioxidant capacity of these PAGs was
evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), uorescence
recovery aer photobleaching (FRAP) and b-carotene/linoleic
acid methods. Furthermore, the antimicrobial and anti-UV-B
activities were also evaluated for the rst time, which opens
new opportunities for developing PAGs as a group of promising
multifunctional and nature-identical ingredients.
Fig. 1 Scheme for enzymatic synthesis of PAGs.
Immobilized lipase was removed from the mixture aer
reaction by centrifuge (5000 rpm). The crude product was
extracted by solvent mixture (water : ethyl acetate ¼ 2 : 1, v/v) for
three times, the excess EPs dissolved in ethyl acetate layer and the
excess glycerol dissolved in water layer. Meanwhile, the white
PAGs powder precipitated in water layer during each extraction.
Then, the puried PAGs (white powder) could be obtain aer
centrifugal separation from the water layer and freeze-dried. The
obtained PAGs were subject to ELSD-HPLC and analysed for
purity.
Experimental section
Chemicals and reagents
Analysis and structural identication of PAGs
Ethyl ferulate, ethyl caffeate, ferulic acid, caffeic acid, cinnamic
acid, p-hydroxycinnamic acid, m-hydroxycinnamic acid,
p-methoxycinnamic acid and 2-methoxycinnamic acid (>99%) were
purchased from Aladdin Reagent (Shanghai, China). Ethyl cinna-
mate, ethyl p-hydroxycinnamate, ethyl m-hydroxycinnamate, ethyl
p-methoxycinnamate and ethyl 2-methoxycinnamate (>95%) were
obtained from Shanghai Drum Hill Biotechnology (Shanghai,
The products were monitored by HPLC with the detector
photodiode array (PDA) and evaporative light-scattering
detector (ELSD). The detecting condition was set on as
following: elution was composed of methanol (A) and 0.5%
acetic acid (B) at a ow rate of 1 mL minꢁ1 with 10 mL injection
volume. The elution sequence was from 58% B (v/v) to 100% B
(v/v) in 20 min consecutively in a linear gradient, followed by
100% B for 2 min at 40 ꢂC. The elusion was analyzed at 280 nm
and 325 nm. The EꢂLSD conditions were: gain, 6; gas pressure, 60
psi; dri tube, 40 C.
The identication of PAGs was performed on a Shimadzu
MS-8050 mass spectrometer (Tokyo, Japan), with an APCI
interface in positive mode. The other MS parameters was
selected as following: isospray voltage was set at 4 kV, DL
temperature 250 ꢂC, heat block temperature 400 ꢂC, nebulizing
gas 2 L minꢁ1 and drying gas 10 L minꢁ1, heating gas 10
L minꢁ1, interface temperature 300 ꢂC; collision energy was set
at 35 V. The mass range was from 100 to 1000 m/z. The PAGs
were further characterized by 1H NMR spectroscopy (Bruker
Avance II HD, Faellanden, Switzerland) with D6-DMSO (0.01%
TMS) and CDCl3 (0.01% TMS) as solvent at 400 MHz frequency.
ꢀ
China). Glycerol (99%, dehydrated using an activated 3 A molecular
sieve before use) and ethyl acetate were purchased from Tianjin
Guoyao Chemical (Tianjin, China). The catalyst is CALB
immoPlus™, immobilized lipase from Candida Antarctica, ob-
tained from Purolite (Wales, UK). The mobile phase of HPLC was
made of glacial acetic acid and methanol (HPLC grade), purchased
from Merck Chemical Technology (Shanghai, China). All other
regents were analytical grade. The Inertsil ODS-SP column (5 mm,
250 mm ꢀ 4.6 mm) was purchased from GL Science Inc. (Tokyo,
Japan). UV-vis spectrophotometer was DU800 (Beckman Coulter,
Brea, CA).
Enzymatic synthesis and purication of PAGs
The PAGs were synthesis by enzymatic alcoholysis of phenolic
acid ethyl esters with glycerol in solvent free system (Fig. 1).
Experiments were carried out in a 25 mL glass bottle with
magnetic stirring (200 rpm) (IKA, Staufen, Germany) in the
Antioxidant evaluation
presence or absence of ultrasound assistance irradiated from 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay:
the microtip probe (diameter of 3 mm) of an ultrasonic 0.5 mL of PAGs sample (1.0 mM in methanol) was mixed with
homogenizer (Scientz-IID, Zhejiang, China). The ultrasonic- 2.5 mL of 0.1 mM DPPH in methanol. The reaction mixture was
promoted enzymatic reaction device was shown in Fig. S1.† incubated in the dark at room temperature for 30 min. The
The enzymatic reaction was conducted by combining 8 mmol mixture was put into the quartz cell for ultraviolet detection
of ethyl phenates (EPs) and 80 mmol glycerol and lipase radical at 517 nm to calculate the scavenging activity.22
(6% of the total weight of the substrates). The ultrasonic
b-Carotene/linoleic acid assay: b-carotene was dissolved in
parameters were set as following: the ultrasonic parameters chloroform solution with the concentration 0.1 mg mLꢁ1. 40 mg
were set as following: ultrasonic output (28 W mLꢁ1), ultra- linoleic acid was added in 4 mL of above liquid, followed by
sonic intermittent ratio (3 s/9 s, working/waiting) and ultra- 400 mg Tween 40, then chloroform was distilled off under
sonic time (1–9 h). The composition of the crude products reduced pressure. 100 mL distilled water was added to the above
was monitored by HPLC (LC-M20A, SHIMADZU, Kyoto, residue and dissolved as reaction solution. 0.2 mL PAGs sample
Japan).
(1.0 mM in methanol) was added into 4 mL reaction solution,
11140 | RSC Adv., 2020, 10, 11139–11147
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
then the mixture solution was measured at 450 nm against pure
methanol (blank) using a UV-vis spectrophotometer in a 1 cm
quartz cell for 0 min and 180 min. The antioxidant activity (AA)
was calculated using the following equation:
Determination of the minimum inhibitory concentration
(MIC)
The 48-well plate was sterilized for use. Before use, the 48-well
plate was irradiated with UV light for at least 20 min. To each
well, 0.5 mL medium containing sample was added at
a concentration of 10, 5, 2.5, 1.25, 0.63 and 0.32 mg mLꢁ1. Then
10 mL Escherichia coli, Staphylococcus aureus, Bacillus subtilis and
yeast were added to the 48-well plates. Bacteria was cultured at
37 ꢂC for 24 h (yeast at 28 ꢂC for 48 h), and the turbidity of the
bacterial liquid was observed. The MIC of the control group
(10% DMSO in distilled water) was determined by the degree of
clarication.
% AA ¼ [1 ꢁ (Asample 0 ꢁ Asample 180)/(Acontrol 0 ꢁ Acontrol 180)]
ꢀ 100.
Ferric reducing antioxidant power (FRAP) assay: the FRAP
solvent was comprised of 5 mL of a 10 mmol Lꢁ1 2,4,6-tris(2-
pyridyl)-s-triazine (TPTZ) solution (156 mg TPTZ dissolve in
50 mL 40 mmol Lꢁ1 HCl), 5 mL of 20 mmol Lꢁ1 FeCl3$6H2O and
50 mL of 0.1 mol Lꢁ1 acetate buffer (pH 3.6). The FRAP solvent
was incubated at 37 ꢂC for 10 min. Then, 2 mL of the above
liquid were taken out into a 10 mL colorimetric tube, followed
by 0.5 mL of 1 mmol Lꢁ1 PAGs sample and distilled water to
lled in the whole 10 mL. The solutions were kept at room
temperature for 20 min and then were analysed on UV-vis at
593 nm.22 The result was shown by mmol TE/L using Trolox as
the reference.
UV absorption test
PAs and PAGs of 1 mmol Lꢁ1 were dissolved in methanol, and
the absorption spectrum in the range of 200–800 nm was
measured against a blank (methanol).
Result and discussion
Effect of ultrasound irradiation on the conversion of PAGs
Activated bacteria and preparation
Under the optimized conditions, a comparable study was per-
Escherichia coli DH5a, Bacillus subtilis 168, Staphylococcus aureus formed on the transesterication of glycerol with different EPs
SI-27, and Saccharomyces cerevisiae were purchased from Wuhan under stirring with ultrasound assistance. The reaction time
University, China Center for Type Culture Collection (Wuhan, and conversion under ultrasonic pretreatment were 4.0–14.0
China). Bacteria: the bacteria required for the experiment on the times shorter and 1.4–7.5 times higher than that of mechanical
LB agar medium is yeast extract 5 g, NaCl 5 g, peptone 10 g, stirring, respectively (Table 1). In this study, the turnover
distilled water volume to 1000 mL. Agar addition amount is 1.8%, number (TON) dened as the molar amount of converted
incubated at 37 ꢂC, in a constant temperature incubator for 24 h. substrate per gram per hour effective enzyme loading was
Saccharomyces cerevisiae: the experiment required bacteria inoc- applied to evaluate the catalytic efficiency. The TON of ultra-
ulated into YPD Agar medium (yeast extract 10 g, peptone 20 g, sound assistance was 12.0–45.0 times higher than under
glucose 20 g, distilled water volume to 1000 mL). Agar addition mechanical stirring, which was the best performance in the
amount is 1.8%, incubated at 28 ꢂC for 48 h. All experiments were enzymatic synthesis of PAGs. However, by extending the reac-
ꢂ
aseptic and stored at 4 C for use. Bacteria was inoculated into tion time during stirring, the conversion of EPs decreased. This
Luria broth (LB) at 37 ꢂC, 220 rpm for 12 h and made with sterile phenomenon may be ascribed to the excess ethanol accumu-
saline at a certain concentration (E. coli, Staphylococcus aureus 108 lated during the reaction reverses the transesterication reac-
cfu mLꢁ1, Bacillus subtilis 106 cfu mLꢁ1). Saccharomyces cerevisiae tion. On the contrary, the transient high temperature generated
ꢂ
was inoculated into YPD at 28 C, 180 rpm for 12 h and made by ultrasound could promote the volatilization of ethanol,
with sterile saline at 106 cfu mLꢁ1
.
which pushed the reaction forward.19 Beneting from the
Table 1 The transesterification of ethyl phenates (EPs) with glycerol under ultrasound assistance and mechanical stirringa
Ultrasound assistance
Mechanical stirring
Conversion
Time (h) (%)
EPs
Time (h) Conversion (%) TONu (mmol gꢁ1 hꢁ1
)
TONs (mmol gꢁ1 hꢁ1
)
TONu/TONs
Ethyl cinnamate
1
3
6
6
6
9
3
93.9
89.6
89.4
88.1
88.6
90.1
98.5
14.3
4.5
2.1
2.0
2.2
1.5
4.8
24
24
24
24
24
24
24
61.3
17.5
17.3
12.7
17.1
12.1
71.7
0.4
0.1
0.1
0.1
0.1
0.1
0.4
35.8
45.0
21.0
20.0
22.0
15.0
12.0
Ethyl p-hydroxycinna-mate
Ethyl m-hydroxycinna-mate
Ethyl p-methoxycinna-mate
Ethyl 2-methoxycinna-mate
Ethyl caffeate
Ethyl ferulate
a
TON: turnover number was evaluated on the basis of the molar amount of converted substrate per gram per hour effective enzyme loading.
Reaction condition: ethyl phenate/glycerol 1 : 10 (mol molꢁ1), reaction temperature 65 ꢂC, enzyme loading 6%, ultrasonic power 280 W,
ultrasonic intermittent ratio: 3 s/9 s (working/waiting).
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 11139–11147 | 11141

View Article Online
RSC Advances
Paper
Fig. 2 Effect of ultrasound pretreatment or assistance on the conversion of EPs. Reaction condition: (a) was ethyl cinnamate/glycerol (1 : 10, mol
molꢁ1), reaction time 1 h; (b) was ethyl ferulate/glycerol (1 : 10, mol molꢁ1); reaction time 3 h; other reaction condition: enzyme loading 6%,
reaction temperature 65 ꢂC, ultrasound pretreatment 1 h or continuous ultrasound assistance, ultrasonic power: 28 W mLꢁ1, ultrasonic inter-
mittent ratio: 3 s/9 s (working/waiting).
cavitation effect of ultrasound, the contact probabilities of methyl cinnamate. The cinnamoyl daughter ion was at 131.1 m/
substrate and enzyme could be increased, thereby further z [M + H–C3H8O3]+. The MS of peak #2 (Fig. 4b) revealed an
accelerating the reaction.18,23
abundant addition of 261.1 m/z [M + Na]+ and an molecular ion
In order to evaluate the effect of ultrasound irradiation on of 239.2 m/z [M + H]+, daughter ion of 179.1 m/z [M + H–
enzyme activity and substrate solubility, ultrasound pretreat- C2H6O2]+, which was the daughter for methyl hydrox-
ment was applied to the lipase and substrate individually. Two ycinnamate. The other daughter ion at 147.1 m/z [M + H–
different ultrasound pretreatment patterns were designed: one C3H8O3]+ belonged to hydroxycinnamoyl. The MS of peak #3
was continuous ultrasound irradiation throughout the reaction and #4 displayed in Fig. 4c and d, respectively, showing that
and the other was only pretreatment at the beginning of reac- higher intensity of m/z 275.2 [M + Na]+ and the daughter ion at
tion for a certain time.24,25 The controlled trial was performed by m/z 193.2 [M + H–C2H6O2]+ corresponded to methyl methox-
ultrasound pre-irradiation (1 h) of only lipase and in the ycinnamate, and daughter ion of m/z 161.1 [M + H–C3H8O3]+
absence of lipase aer which the lipase was added in. As shown was for methoxy cinnamoyl. The MS of peak #5 (Fig. 4e)
in Fig. 2, there was no signicant difference in conversion produced the abundant addition of m/z 277.2 [M + Na]+ and
among stirring and ultrasonic pretreatment of only the daughter ions at m/z 195.1 [M + H–C2H6O2]+ ascribe to methyl
substrate (without the lipase). In contrast, in ultrasound methoxy caffeate, and daughter ion of m/z 161.1 [M + H–
pretreatment of lipase for 1 h, the conversion increased sharply. C3H8O3]+ ascribe to caffeoyl. The MS of peak #6 (Fig. 4f) dis-
This result may stem from an alteration in the statement of the played an daughter ion of m/z 291.1 [M + Na]+ and an addition
lipase ‘lid’ from closed to open aer being activated by ultra- ion of m/z 269.1 [M + H]+, daughter ion at m/z 209.1 [M + H–
sound pretreatment, which contributed to the trans- C2H6O2]+ ascribe to methyl ferulate, and daughter ion of m/z
esterication process.20,26 Continuous ultrasound irradiation 177.1 [M + H–C3H8O3]+ ascribe to ferulyl. It is worth mentioning
showed the highest conversion among the different ultrasound that the monoglycerol with m-hydroxycinnamic acid and 2-
modes. Considering the conversion and reaction efficiency, methoxy cinnamic acid were synthesized and identied for the
ultrasound irradiation throughout the reaction was selected in rst time.
1
this study.
The structures of PAG were further determined by H NMR
(Table S1†). Chemical shis were given on a d (ppm) scale and
the corresponding carbon number was shown in Fig. 4.
Cinnamoyl glycerol (CDCl3, 400 MHz): d (ppm) 7.74 (1H, d, J
Structure characterization of PAGs
The enzymatic synthesis of PAGs was analysed by HPLC-ELSD. ¼ 16.0), 7.54 (2H, m), 7.41 (3H, m), 6.48 (1H, d, J ¼ 16.0), 4.33
The chromatograms of raw materials including glycerol, EPs (1H, dd, J ¼ 11.7, 5.4), 4.02 (1H, dd, J ¼ 4.8, 4.3), 3.93 (1H, d, J ¼
and corresponding PAGs obtained aer purication are shown 4.7), 3.73 (1H, dd, J ¼ 3.5, 11.6).
in Fig. 3. The result from HPLC chromatogram indicated that
Coumaroyl glycerol (DMSO, 400 MHz): d (ppm) 10.03 (1H, s),
the purication of PAGs was successful with a highly purity 7.57 (2H, d, J ¼ 8.5), 6.79 (2H, m), 6.39 (1H, dd, J ¼ 15.9, 6.0),
(>95%) and no residual ethyl PAs or glyceride were detected by 4.01 (1H, dd, J ¼ 11.2, 6.5), 3.71 (1H, tq, J ¼ 10.0, 5.6, 4.8), 3.53
HPLC.
(1H, dd, J ¼ 11.2, 5.4), 3.35 (1H, dd, J ¼ 11.2, 5.6).
The synthesized PAGs were identied by HPLC/APCI-MS.
m-Hydroxycinnamoyl glycerol (DMSO, 400 MHz): d (ppm)
The mass spectrograms of PAGs are shown in Fig. 4. The MS 9.65 (1H, d, J ¼ 3.3), 7.57 (1H, dd, J ¼ 16.0, 10.2), 7.15 (1H, m),
of peak #1 (Fig. 4a) showed an molecular ion of 223.1 m/z [M + 6.97 (1H, m), 6.84 (1H, dd, J ¼ 8.2, 2.2), 6.51 (1H, dd, J ¼ 16.0,
H]+, daughter ion of 161.3 m/z [M + H–C2H6O2]+ ascribe to
11142 | RSC Adv., 2020, 10, 11139–11147
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 3 The HPLC-ELSD chromatograms of the raw material and the final products after purification. Peak: (1) glycerol, (2) cinnamoyl glycerol, (3)
ethyl cinnamate, (4) p-hydroxy cinnamoyl glycerol, (5) ethyl p-hydroxycinnamate, (6) m-hydroxycinnamoyl glycerol, (7) ethyl m-hydrox-
ycinnamate, (8) p-methoxycinnamoyl glycerol, (9) ethyl p-methoxycinnamate; (10) 2-methoxycinnamoyl glycerol, (11) ethyl 2-methox-
ycinnamate, (12) caffeoyl glycerol, (13) ethyl caffeate, (14) feruloyl glycerol, (15) ethyl ferulate.
2.4), 4.97 (1H, d, J ¼ 5.3), 4.03 (1H, dd, J ¼ 11.2, 6.5), 3.71 (1H, 4.9), 4.41 (1H, s), 3.50 (1H, d, J ¼ 1.7), 3.43 (1H, m), 3.28 (1H, dd,
m), 3.35 (1H, m).
J ¼ 10.8, 5.6).
p-Methoxy cinnamoyl glycerol (CDCl3, 400 MHz): d (ppm)
Feruloyl glycerol (DMSO, 400 MHz): d (ppm) 9.63 (1H, s), 7.56
7.69 (1H, m), 7.49 (2H, m), 6.92 (2H, m), 6.33 (1H, m), 4.32 (2H, (1H, d, J ¼ 15.9), 7.33 (1H, d, J ¼ 2.0), 7.12 (1H, dd, J ¼ 8.2, 2.0),
qd, J ¼ 11.7, 5.3), 4.02 (1H, d, J ¼ 6.6), 3.75 (1H, dd, J ¼ 11.6, 4.0), 6.79 (1H, d, J ¼ 8.1), 4.15 (2H, dd, J ¼ 11.2, 4.1), 4.01 (1H, dd, J ¼
3.67 (1H, dd, J ¼ 11.6, 5.6).
11.2, 6.5), 3.82 (3H, s), 3.70 (2H, m).
2-Methoxy cinnamoyl glycerol (CDCl3, 400 MHz): d (ppm)
7.95 (1H, d, J ¼ 17.4), 7.77 (1H, s), 7.49 (1H, s), 7.17 (1H, s), 7.06
(1H, s), 6.69 (1H, d, J ¼ 16.2), 4.25 (1H, d, J ¼ 11.0), 3.90 (1H, s),
3.73 (3H, h, J ¼ 4.6), 3.67 (1H, s).
Antioxidant evaluation
The antioxidant activity of PAs has been reported in several
literatures.11,27 However, the antioxidant activity of PAGs only
focused on a single category.6,16,21 Based on the synthesis and
identication of seven PAGs mentioned above, the antioxidant
Caffeoyl glycerol (DMSO, 400 MHz): d (ppm) 8.32 (1H, s), 7.22
(1H, q, J ¼ 4.5, 3.8), 7.02 (1H, m), 6.68 (1H, s), 5.32 (2H, t, J ¼
Fig. 4 APCI-MS analysis of the synthesized PAGs. (a) Cinnamoyl glycerol, (b) hydroxycinnamoyl glycerol, (c) p-methoxy cinnamoyl glycerol, (d)
2-methoxy cinnamoyl glycerol, (e) caffeoyl glycerol (f) feruloyl glycerol.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 11139–11147 | 11143

View Article Online
RSC Advances
Paper
activities of PAs and PAGs were evaluated using DPPH radical Therefore, the hydrogen radical of the hydroxyl group was more
scavenging, FRAP and b-carotene/linoleic acid assay, active and thus exhibited stronger antioxidant activity than the
respectively.
As shown in Fig. 5a, cinnamic acid (2.3%) and cinnamic amount of hydroxyl groups may have a dominant inuence on
glycerol (5.9%) showed weak radical scavenging ability. Among their radical scavenging ability. Therefore, as bis-
mono-hydroxycinnamic acid derivatives.28 In addition, the
a
the methoxy-containing cinnamic acid derivatives, ferulic acid hydroxycinnamic acid derivative, caffeic acid, showed the
showed stronger antioxidant activity (93.8%). One reason may highest antioxidant capacity (95.0%).29,30 The radical-scavenging
be –OCH3 at C-3 had electron-withdrawing ability; the other ability of PAGs was similar to the parent PAs, indicating that
reason could be the hydrogen bond between –OH and –OCH3 transesterication modication does not show a negative
oxygen weakens the O–H bond, facilitating radical formation. inuence on the antioxidant properties of PAs. The radical-
scavenging capacity order of PAGs and usual antioxidants was
L-ascorbic acid > L-ascorbyl palmitate > a-tocopherol > caffeoyl
glycerol > feruloyl glycerol > TBHQ > BHT > p-hydroxycinnamoyl
glycerol > p-methoxy cinnamoyl glycerol > 2-methoxy cinnamoyl
glycerol > m-hydroxy cinnamoyl glycerol > cinnamoyl glycerol.
Caffeoyl glycerol (94.6%) and feruloyl glycerol (92.6%) exhibited
better radical scavenging capability than BHT and were equal to
TBHQ.
The total antioxidant capacity was evaluated by FRAP assay.
As shown in Fig. 5b, although the antioxidant ability of feruloyl
glycerol was 16% less than the ferulic acid, it was still higher
than the conventional antioxidants, such as L-ascorbic acid, L-
ascorbyl palmitate, a-tocopherol, BHT and TBHQ. Under the
experimental conditions (pH 3.6), PAs of 4-OH were observed to
obviously reduce iron ability, however, the antioxidant ability of
corresponding PAGs decreased obviously. The reason could be
that esterication affected the interaction of –OH and –COOH at
low pH, and thus decreased the total antioxidant capacity. The
antioxidant capacity of ferulic acid was less affected by trans-
esterication than that of caffeic acid, which only retained
63.2% of the initial antioxidant ability. This phenomenon could
be explained in that the dihydroxy PAs were more susceptible to
esterication modication than monohydroxy PAs. Further-
more, the –OCH3 group in ferulic acid might promote the
activity of –OH radicals.
The peroxidation inhibitory ability was measured by the b-
carotene/linoleic acid emulsion oxidation method. As shown
in Fig. 5c, all PAs showed weak peroxidation inhibitory ability
(<20%), which might be ascribed to the poor solubility of PAs in
the emulsied system. Compared with the corresponding PAs,
the peroxidation inhibitory ability of caffeoyl glycerol (68.2%)
and feruloyl glycerol (74.8%) increased 8.1-fold and 14.4-fold,
respectively, which showed much better peroxidation inhibitory
ability than BHT and L-ascorbyl palmitate. This phenomenon
could be explained in that the transesterication of EPs and
glycerol increase the amphipathic nature of PAs, which
improves the dispersibility of PAGs in the emulsication
system, and thus greatly enhanced the lipid peroxidation
Fig. 5 Antioxidant capacity evaluation of phenolic acids (PAs) and
inhibitory ability of PAs.
PAGs in DPPH (a), FRAP (b) and b-carotene/linoleic acid (c) methods.
The concentration of PAs and PAGs was kept at 1 mmol Lꢁ1. (1-1)
Cinnamic acid, (1-2) cinnamoyl glycerol, (2-1) p-hydroxycinnamic
Antimicrobial activity
acid, (2-2) p-hydroxycinnamoyl glycerol, (3-1) m-hydroxycinnamic
acid, (3-2) m-hydroxycinnamoyl glycerol, (4-1) methoxy cinnamic
acid, (4-2) p-methoxy cinnamoyl glycerol, (5-1) 2-methoxy cinnamic
acid, (5-2) 2-methoxy cinnamoyl glycerol, (6-1) caffeic acid, (6-2)
caffeoyl glycerol, (7-1) ferulic acid, (7-2) feruloyl glycerol, (Ctrl-1) a-
tocopherol, (Ctrl-2) TBHQ, (Ctrl-3) BHT, (Ctrl-4) L-ascorbyl palmitate,
(Ctrl-5) L-ascorbic acid.
The development of a natural and safe bacteriostatic has been
gaining popularity. In recent years, the wide antibacterial
activities of PAs have been reported.8,31 However, the antibac-
terial activity of PAGs has never been reported in any literature.
In the present work, Escherichia coli, Staphylococcus aureus,
11144 | RSC Adv., 2020, 10, 11139–11147
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Table 2 The MIC results of seven PAGs and the corresponding phenolic acids
Escherichia
Bacillus
Staphylococcus
Saccharomyces
Sample
coli (mg mLꢁ1
)
subtilis (mg mLꢁ1
)
aureus (mg mLꢁ1
)
cerevisiae (mg mLꢁ1
)
Cinnamic acid
Cinnamoyl glycerol
2.5
10
2.5
10
2.5
10
5
0.32
10
10
2.5
0.32
2.5
0.63
5
10
2.5
10
2.5
10
2.5
10
10
2.5
1.25
2.5
2.5
1.25
5
2.5
10
5
10
2.5
5
2.5
5
2.5
5
5
p-Hydroxycinnamic acid
p-Hydroxycinnamoyl glycerol
m-Hydroxycinnamic acid
m-Hydroxycinnamoyl glycerol
p-Methoxy cinnamic acid
p-Methoxy cinnamoyl glycerol
2-Methoxycinnamic acid
2-Methoxycinnamoyl glycerol
Caffeic acid
5
10
>10
2.5
10
10
10
2.5
10
5
5
2.5
2.5
2.5
1.25
Caffeoyl glycerol
Ferulic acid
Feruloyl glycerol
10
Bacillus subtilis and Saccharomyces cerevisiae, which belong to value of feruloyl glycerol decreased 2-fold compared with ferulic
the Gram-negative bacteria, Gram-positive bacteria and fungus acid. However, it failed to show any signicant advantage of
families were used to evaluate the antimicrobial activity of PAGs over PAs in antifungal activity. Taking the antibacterial
PAGs. The samples concentration was kept in the range of 0.32– capacity and range into account, p-methoxy cinnamoyl glycerol,
10 mg mLꢁ1 for antimicrobial ability evaluation using the MIC 2-methoxy cinnamoyl glycerol, caffeoyl glycerol and feruloyl
method. As shown in Table 2, the prepared PAGs exhibited glycerol exhibited superb application prospects in bacterio-
better antimicrobial ability for Gram-negative bacteria than for static. One could deduce that the existence of methoxy cinna-
Gram-positive bacteria. Compared with p-methoxy cinnamic moyl might remarkably increase the antimicrobial activity of
acid, caffeic acid and ferulic acid, the corresponding PAGs PAGs, and the increase in the hydroxyl number might also have
showed much superior anti-E. coli activities, with MIC values a positive effect on the antimicrobial ability.32
decreasing 16.0-fold, 8-fold and 4-fold, respectively. The MIC
values of 2-methoxy cinnamoyl glycerol and feruloyl glycerol
were 2.5 mg mLꢁ1 and 1.25 mg mLꢁ1, respectively, which
decreased 4-fold and 2-fold than their PAs for anti-Bacillus
subtilis, respectively. In anti-Staphylococcus aureus, the MIC
UV absorption evaluation
UV light can be classied into three categories according to the
wavelength including UV-A (315–400 nm), UV-B (280–315 nm)
and UV-C (100–280 nm). Excessive exposure of UV-A and UV-B
Fig. 6 UV absorption evaluation of phenolic acids (PAs) and PAGs in the range of 200–800 nm. The concentration of PAs and PAGs was kept at
1.0 mmol Lꢁ1
.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 11139–11147 | 11145

View Article Online
RSC Advances
Paper
could result in free radical, toxic elements into skin cells and
even skin cancer. Some natural compounds include typically
single or multiple aromatic structures conjugated or unconju-
gated with carbon–carbon double bonds and thus could absorb
light in both UV-A and UV-B wavelength regions. The UV
absorption ability of PAs and PAGs are shown in Fig. 6 with the
concentration at 1 mmol Lꢁ1. The UV spectra of the maximum
absorption wavelength (lmax) of PAGs increased by 10–30 nm
compared with the parent PAs. The result may be explained that
the cinnamoyl structure has a conjugate bond thus maximizing
the electron cloud density around the p bond. The reaction,
making glycerol combine with the phenolic skeleton instead of
H, increased the electron cloud density around the p bond to
cause lmax red-shi and the UV absorption bands become
wider.33 Although both PAs and PAGs could absorb UV-A and
UV-B, the UV absorption values of PAGs were 0.8–26.2% higher
and 6.7–55.5% wider than those of PAs. The reason may be the
transesterication modication increased the amphiphilic
nature of PAs, thus increasing the UV absorption intensity and
2 O. Ogah, C. S. Watkins, B. E. Ubi and N. C. Oraguzie, J. Agric.
Food Chem., 2014, 62, 9369–9386.
3 B. Reis, M. Martins, B. Barreto, N. Milhazes, E. M. Garrido,
P. Silva, J. Garrido and F. Borges, J. Agric. Food Chem.,
2010, 58, 6986–6993.
4 M. C. Figueroaespinoza and P. Villeneuve, J. Agric. Food
Chem., 2005, 53, 2779–2787.
5 J. Graca and H. Pereira, J. Agric. Food Chem., 2000, 48, 5476–
5483.
6 D. L. Compton, J. A. Laszlo and K. O. Evans, Ind. Crops Prod.,
2012, 36, 217–221.
7 A. C. D. Camargo, G. B. Rasera, L. D. P. Silva, V. O. Alvarenga,
A. S. Sant'Ana and F. Shahidi, Food Chem., 2017, 237, 538–
544.
8 M. Balakrishna, S. S. Kaki, M. S. Karuna, S. Sarada,
C. G. Kumar and R. B. Prasad, Food Chem., 2017, 221, 664–
672.
9 D. Patil, B. Dev and A. Nag, J. Mol. Catal. B: Enzym., 2011, 73,
5–8.
range.16,34 The study provided the important basic data support 10 C. M. Verdasco-Martın, E. Garcia-Verdugo, R. Porcar,
´
for the application of PAGs in sunscreen products.
R. Fernandez-Lafuente and C. Otero, Food Chem., 2017,
245, 39–46.
11 J. Wei, X. Huo, Z. Yu, X. Tian, S. Deng, C. Sun, L. Feng,
C. Wang, X. Ma and J. Jia, Fitoterapia, 2017, 121, 129–135.
Conclusions
In this study, seven PAGs were enzymatically synthesized by 12 Z. Yang, Z. Guo and X. Xu, Food Chem., 2012, 132, 1311–1315.
ultrasonic assistance with the highest catalytic efficiency among 13 J. A. Laszlo, K. O. Evans, K. E. Vermillion and M. Appell, J.
the reported methods. Among the active derivatives, caffeoyl
glycerol and feruloyl glycerol exhibited excellent antioxidant 14 S. Gholivand, O. Lasekan, C. P. Tan, F. Abas and L. S. Wei,
activity either in DPPH, FRAP or b-carotene/linoleic acid assay, Food Chem., 2017, 224, 365–371.
Agric. Food Chem., 2010, 58, 5842–5850.
even comparable or better than the chemical antioxidants such as 15 S. Sun and B. X. Hu, Food Chem., 2017, 214, 192–198.
BHT and TBHQ. Several PAGs such as p-methoxycinnamic acid 16 D. L. Compton, J. A. Laszlo and T. A. Isbell, J. Am. Oil Chem.
glycerol, caffeoyl glycerol and feruloyl glycerol, showed much
better antimicrobial activities than their parent PAs, with MIC 17 S. Caillet, J. Cote, J. F. Sylvain and M. Lacroix, Food Control,
values decreasing 2–16-fold. Compared with PAs, PAGs can absorb
2012, 23, 419–428.
a much wider and higher intensity of the harmful UV-A and UV-B 18 M. A. Polewski, C. G. Krueger, J. D. Reed and G. Leyer, J.
rays. Taking advantage of their multifunction including antioxi- Funct. Foods, 2016, 25, 123–134.
dant, antimicrobial and anti-UV, this study not only paves the way 19 S. R. Bansode and V. K. Rathod, Ultrason. Sonochem., 2017,
to develop the resultant PA derivatives into the eld of multi- 38, 503–529.
functional ingredients, but also opens a new opportunity for 20 M. M. Zheng, L. Wang, F. H. Huang, L. Dong, P. M. Guo,
Soc., 2004, 81, 945–951.
ˆ ´
advancing the process of phytochemical modication.
Q. C. Deng, W. L. Li and C. Zheng, Ultrason. Sonochem.,
2012, 19, 1015–1020.
21 C. Xu, H. Zhang, J. Shi, M. Zheng, X. Xiang, F. Huang and
J. Xiao, Ultrason. Sonochem., 2018, 41, 120–126.
Conflicts of interest
´
There are no conicts to declare.
22 A. Szydłowskaczerniak, I. Bartkowiakbroda, I. Karlovic,
G. Karlovits and E. Szłyk, Food Chem., 2011, 127, 556–563.
23 M. M. Zheng, Q. Huang, F. H. Huang, P. M. Guo, X. Xiang,
Q. C. Deng, W. L. Li, C. Y. Wan and C. Zheng, J. Agric. Food
Chem., 2014, 62, 5142–5148.
Acknowledgements
This study was supported by the National Natural Science Foun-
dation of China (31671820, 31972038), the Applied Basic Frontiers 24 K. S. Jaiswal and V. K. Rathod, Ultrason. Sonochem., 2018, 40,
Program of Wuhan City (2019020701011474) and the Agricultural
727.
Science and Technology Innovation Project of the Chinese 25 X. Ma, D. Wang, M. Yin, J. Lucente, W. Wang, T. Ding, X. Ye
Academy of Agricultural Sciences (CAAS-ASTIP-2013-OCRI).
and D. Liu, Ultrason. Sonochem., 2017, 36, 1–10.
26 R. Fernandez-Lafuente, J. Mol. Catal. B: Enzym., 2010, 62,
197–212.
Notes and references
27 M. A. Raza and D. Shahwar, J. Chem., 2015, 2015, 1–8.
1 S. A. Heleno, A. Martins, M. J. Queiroz and I. C. Ferreira, Food 28 H. Kikuzaki, M. Hisamoto, K. Hirose, A. Kayo Akiyama and
Chem., 2015, 173, 501–513.
H. Taniguchi, J. Agric. Food Chem., 2002, 50, 2161–2168.
11146 | RSC Adv., 2020, 10, 11139–11147
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
´
´
´
¨
29 A. Kurekgorecka, A. Rzepeckastojko, M. Gorecki, J. Stojko, 32 A. F. Sanchez-Maldonado, A. Schieber and M. G. Ganzle, J.
´
M. Sosada and G. Swierczekzi˛eba, Molecules, 2013, 19, 78–
101.
Appl. Microbiol., 2011, 111, 1176–1184.
33 J. L. Song, L. M. Gong, S. A. Feng, J. H. Zhao, J. F. Zheng and
Z. P. Zhu, Spectrochim. Acta, Part A, 2009, 74, 1084–1089.
34 M. Stasiuk and A. Kozubek, Cell. Mol. Life Sci., 2010, 67, 841–
860.
30 M. Leja, A. Mareczek, G. Wy˙zgolik, J. Klepacz-Baniak and
´
K. Czekonska, Food Chem., 2007, 100, 237–240.
31 L. Y. Chen, C. W. Cheng and J. Y. Liang, Food Chem., 2015,
170, 10–15.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 11139–11147 | 11147