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\begin{document}

\title{Studies on the enzymatic synthesis and free radical scavenging ability
of phenolic acid glycerols}



\author[1]{Hong Li}%
\author[1]{Yanyan Li}%
\author[2]{Xuezhi Yin}%
\author[1]{Ziyi Li}%
\author[3]{Huiling Mu}%
\author[1]{Ruiling Shen}%
\author[1]{Tao Wei}%
\affil[1]{Zhengzhou University of Light Industry}%
\affil[2]{Central China Publishing and Media Investment Holding Group Co Ltd}%
\affil[3]{University of Copenhagen Section for Geogenetics}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The potentially wide application of Phenolic acids (PAs) in industries
was severely limited by their inadequate solubility and stability in
polar/non-polar media. To overcome these limits, studies on the
enzymatic esterification of PAs with glycerol were carried out to reach
a yield of 95\% of phenolic acid glycerols (PAGs) under the following
reaction conditions: 1:150 molar ratio of caffeic acid (CA) to glycerol;
25\% of Lipozyme 435 relative to the weight of total substrates;
temperature 80 \textsuperscript{o}C, 500 rpm; 86.7 kPa and reaction time
10 h. Three resulting PAGs including caffeoyl glycerol (CG), feruloyl
glycerol (FG) and \emph{p}-hydroxycinnamoyl glycerol ( \emph{p}-HCG)
were confirmed by MS, \textsuperscript{1}H NMR and \textsuperscript{13}C
NMR. Among them, CG showed a comparative free radical scavenging ability
to CA, indicating its potential use as a water-soluble antioxidant
alternative to CA for food and cosmetic applications. Moreover, the
direct esterification of PAs with glycerol decrease the product cost and
increase the feasibility of industrial production compared with the
previous transesterification sythesis methods of PAGs.%
\end{abstract}%



\sloppy


\textbf{Studies on theenzymatic synthesis and free radical scavenging
ability of phenolic acid glycerols}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Journal:\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Journal of the American Oil Chemists Society\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Manuscript Type:\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Original Article\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Date Submitted by the Author:\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
19-Sep-2023\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Complete List of Authors:\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Hong Li; Zhengzhou University of Light Industry Yanyan Li; Zhengzhou
University of Light Industry Xuezhi Yin; Zhongyuan Yuzi Investment
Holding Group Ziyi Li; Zhengzhou University of Light Industry Huiling
Mu; University of Copenhagen Ruiling Shen; Zhengzhou University of Light
Industry Tao Wei; Zhengzhou University of Light Industry\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Author Contributions:\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Hong Li: Conceived, designed and supervised the study. Yanyan Li: Data
acquisition and wrote the first draft of the manuscript. Xuezhi Yin:
Data acquisition and analyzing. Ziyi Li: Data acquisition and evaluation
together with practical supervision. Huiling Mu: Manuscript improving.
Ruiling Shen: Data evaluation. Tao Wei: Project formulation and
manuscript finalizing.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Acknowledgements\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
The authors gratefully acknowledge the funding received from Zhongyuan
Science and Technology Innovation Leading Talent Project {[}Grant
number: 224200510017{]} and ``Three districts'' science and technology
talent support program of Henan Province.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Keywords\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Enzymatic esterification Phenolic acid glycerols Free radical scavenging
ability Antioxidant capacity\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Studies on the enzymatic synthesis and freeradical scavenging
ability of phenolic acid glycerols}

Hong Li \textsuperscript{1*}, Yanyan Li \textsuperscript{1}, Xuezhi
Yin\textsuperscript{2}, Ziyi Li \textsuperscript{1}, Huiling
Mu\textsuperscript{3}, Ruiling Shen \textsuperscript{1}, Tao
Wei\textsuperscript{1}

1 College of Food and Bioengineering, Zhengzhou University of Light
Industry, Zhengzhou, 450000, Henan Province, P. R. China;

2 Zhongyuan Yuzi Investment Holding Group, Zhengzhou, 450000, Henan
Province, P. R. China;

3 Department of Pharmacy, University of Copenhagen, Copenhagen, 2100,
Denmark;

\textbf{To whom all correspondence should be addressed}

\textbf{* Corresponding author. \emph{Phone}: +86-371-86609631}

\emph{E-mail}\textbf{: 2007025@email.zzuli.edu.cn}

\textbf{Abstract:} The potentially wide application of Phenolic acids
(PAs) in industries was severely limited by their inadequate solubility
and stability in polar/non-polar media. To overcome these limits,
studies on the enzymatic esterification of PAs with glycerol were
carried out to reach a yield of 95\% of phenolic acid glycerols (PAGs)
under the following reaction conditions: 1:150 molar ratio of caffeic
acid (CA) to glycerol; 25\% of Lipozyme 435 relative to the weight of
total substrates; temperature 80\textsuperscript{o}C, 500 rpm; 86.7 kPa
and reaction time 10 h. Three resulting PAGs including caffeoyl glycerol
(CG), feruloyl glycerol (FG) and \emph{p} -hydroxycinnamoyl glycerol
(\emph{p} -HCG) were confirmed by MS, \textsuperscript{1}H NMR and
\textsuperscript{13}C NMR. Among them, CG showed a comparative free
radical scavenging ability to CA, indicating its potential use as a
water-soluble antioxidant alternative to CA for food and cosmetic
applications. Moreover, the direct esterification of PAs with glycerol
decrease the product cost and increase the feasibility of industrial
production compared with the previous transesterification sythesis
methods of PAGs.

\textbf{Keywords} Enzymatic esterification; Phenolic acid glycerols;
Free radical scavenging ability; Antioxidant capacity

\textbf{Introduction}

Phenolic acids (PAs) are secondary plant metabolites widely existed in
many agricultural products such as fruits, vegetables and cereals
(Crozier et al., 2009 ; Xu et al., 2018; Varga et al., 2018). In recent
years, PAs have attracted considerable attention due to their biological
and pharmacological activities, for example antioxidative activities,
free radical scavenging, chelating metal ions, antimutation, boost
immunity and neuroprotection (Balakrishna et al., 2017; Jo \& Kim 2016;
Kaki et al., 2015; Pei et al., 2015; Pontiki et al., 2014; Kikugawa et
al., 2017). However, the unsatisfactory solubility and stability of PAs
in polar/non-polar media limit their further application in water/oil
systems (Sun et al., 2020; Weng et al., 2019). Therefore, the structural
modifications of PAs are necessary to widen their application in food,
pharmaceutical, and cosmetics industries.

Many studies have shown that the esterified derivatives of PAs could not
only change their surface activity, but also significantly increase the
physiological activity of the parent compound, indicating that
esterification is a very effective method to improve the functional
properties of PAs (Balakrishna et al., 2017; Weng et al., 2019; Yesiltas
et al., 2019; Wang et al., 2020; Faggiano et al., 2022; Weng et al.,
2020). As an example, phenolic acid glycerols (PAGs) such as caffeoyl
glycerol (CG), feruloyl glycerol (FG) and \emph{p} -hydroxycinnamoyl
glycerol (\emph{p} -HCG) have been prepared by PAs/ phenolic acid esters
with glycerol to improve their hydrophilicity (Cumming \& Marshall,
2021; Sun \& Hu, 2017; Sun et al., 2017; Sun \& Hou, 2019; Yao \& Sun,
2020). Chemical (Molinero et al., 2014), chemo-enzymatic (Meng et al.,
2018; Hollande et al., 2018) and enzymatic methods (Compton et al.,
2012; Zhang et al., 2021; Sankar \& Achary, 2017) in organic solvent
(Cumming \& Marshall, 2021), solvent-free (Sun \& Hu, 2017) or ionic
liquids (Sun et al., 2017) have been developed for the preparation of
PAGs. Compared with chemical catalysts, enzyme was popular catalyst for
the preparation of PAGs due to their mild reaction conditions (Weng et
al., 2020). To improve the enzyme performance, ultrasound was used in
enzymatic synthesis of PAGs (Xu et al,, 2018; Sun et al., 2020).
Furthermore, high vacuum was usually indispensable to remove the
byproducts including ethanol or water during the whole reaction course,
which could increase the yield of product (Sun \& Hu, 2017). However,
until now the reported enzymatic synthesis of PAGs was rarely by the
esterification of PAs with glycerol, but mostly the transesterification
of phenolic acid esters, such as phenolic acid methyl (Meng et al.,
2018), ethyl (Sun et al., 2020;Weng et al., 2019) or other esters (Meng
et al., 2018) with glycerol in organic solvent (Cumming \& Marshall,
2021; Compton et al., 2012), solvent free (Sun \& Hu, 2017) or ionic
liquids (Sun et al., 2017). Compared with PAs as raw material of the
synthesis of PAGs, the use of phenolic acid esters synthesized by the
esterification of PAs with alkanol would reduce the final reaction
efficiency and increase the cost.

The present study was therefore aimed to develop a direct enzymatic
esterification of PAs with glycerol for the preparation of PAGs (Scheme
1), which would have higher reaction efficiency and lower product cost
than those of reported enzymatic transesterification of phenolic acid
esters with glycerol. The effects of reaction variables, such as enzyme
types and load, molar ratio, reaction temperature and reaction time on
the esterification of PAs with glycerol were studied. High performance
liquid chromatography (HPLC) was used to monitor the esterification.
Three kinds of PAGs (CG, FG and\emph{p} -HCG) were synthesized and
identified by HPLC, MS and NMR. Subsequently, the antioxidant capacities
of PAGs were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH).

\textbf{Scheme 1.} Lipase-catalyzed esterification of PAs with glycerol

\textbf{Materials and methods}

\textbf{Materials}

Lipase Novozyme 435, Lipozyme 435, Lipozyme TLIM, Lipozyme RMIM, donated
by Aarhus University, Denmark. Chemical reagents, including glycerol,
caffeic acid (CA), ferulic acid (FA), \emph{p} -hydroxycinnamic acid
(\emph{p} -HCA), ethyl acetate, chloroform, methanol, toluene, glacial
acetic acid, anhydrous ethanol and DPPH were purchased from Aladdin
reagent (Shanghai, China). All solvents for HPLC analysis were purchased
from Sigma-Aldrich.

\textbf{Enzymatic esterification}

To optimize the esterification conditions of PAs (CA, FA or\emph{p}
-HCA) with glycerol, CA was used as the material in the model of the
reaction. The enzymatic esterification of 0.25 mmol CA with different
mole of glycerol (12.5-37.5 mmol) was carried out in a 10 mL schlenk
flask. Reaction mixtures were incubated at various temperatures (70-90
\textsuperscript{o}C) using heating block with a magnetic stirrer at 500
rpm under 86.7 kpa. Then a certain amount of lipase (3-30 \% of the
total weight of the substrates) was added to the reaction mixture. At
regular time intervals, samples (20 \selectlanguage{greek}μ\selectlanguage{english}L) were withdrawn and diluted in
980 \selectlanguage{greek}μ\selectlanguage{english}L of mobile phase for quantitative analysis using HPLC as described
in Section 2.3.

\textbf{Quantitative analysis of synthesized CG}

Quantitative analysis of synthesized CG was carried out by HPLC (Thermo
Fisher Scientific Inc.) equipped with a C18 column (Kinetex 5 mm,
150\selectlanguage{ngerman}×4.6 mm) fitted with an ultraviolet-visible detector (UVD) at
25\textsuperscript{o}C, and eluted with a binary solvent system
consisting of solvent A (pH 3 water with
H\textsubscript{3}PO\textsubscript{4}) and solvent B (acetonitrile). The
chromatographic separation was carried out using 9.8\% B as an isocratic
elution at a flow rate of 1 mL/min. The eluate was monitored at 325 nm.
The retention time of CG was approximately 7 min. Purified CG was used
to construct standard curve, which was used to calculate the yield of CG
using the Eq (1). Each analysis was performed in triplicate and reported
as mean values.

\selectlanguage{english}(1\selectlanguage{english})

Where \emph{Y\textsubscript{CG}} is the yield of
CG;\emph{C\textsubscript{CG}} is the concentration of CG measured by
HPLC;\emph{V} is the volume of the sample solution,
mL;\emph{m\textsubscript{CG}} is the theoretical mass of CG, mg.

\textbf{Purification of PAGs}

To verify the universality of the enzymatic esterification method, the
optimal conditions of synthesis of CG obtained above were used to
synthesize FG and \emph{p} -HCG. The optimal conditions were as follows:
0.01 mol PAs (CA, FA, or\emph{p} -HCA) was reacted with 1.5 mol of
glycerol catalyzed by 25 \% of Lipozyme 435 (relative to the weight of
total substrates) at 80\textsuperscript{o}C under 86.7 kPa for 10 h.
Then the enzyme was filtered off. The filtrate was extracted with ethyl
acetate (reaction mixture/ethyl acetate, 1:1, V/V) for three times. The
extracted organic phase was combined and concentrated using a rotary
evaporator at 50\textsuperscript{o}C. When the liquid change to be
turbid, the liquid was crystallized at ambient temperature for 24 h. The
solid was carefully separated after centrifugation (3,000 rpm) for 10
min. Then washed with distilled water and subsequently freezing dried.
The liquid was concentrated and separated by silica gel column
chromatography using chloroform/ toluene/ methanol/ acetate acid (CG,
32:20:10:1; FG and\emph{p} -HCG, 32:20:1.5:1) as mobile phase. After
vacuum evaporation, the white solid was obtained. Combined with the
freeze dried solid, 2.16 g of CG (85.0 \% of isolated yield), 2.20 g of
FG (82.1 \% of isolated yield) and 1.92 g of \emph{p} -HCG (80.7\% of
isolated yield) were obtained respectively. The purity of isolated PAGs
were reconfirmed by TLC and HPLC.

\textbf{Structure identification of PAGs}

The structures of CG, FG and \emph{p} -HCG were identified
by\textsuperscript{1}H NMR, \textsuperscript{13}C NMR, MS and melting
point. NMR investigations were carried out at 14.1 T on a Bruker Avance
III spectrometer in an inverse TXI
(\textsuperscript{1}H,\textsuperscript{13}C) 1.7 mm probe with a
z-gradient at 300 K. The isolated PAGs were dissolved in D6-DMSO (0.01\%
TMS). Mass spectroscopic analysis were performed using Triple TOF 6600
LC-MS/MS with an ESI interface in positive mode. The operating
conditions were as follows: capillary voltage of 3.88 kV, ion source
temperature of 120 \selectlanguage{english}, cone voltage of 30 V, desolvation temperature of
280 \selectlanguage{english}, photo- multiplier voltage of 680 V, vacuum degree of
2.6e\textsuperscript{-5} mBar and gas flow rate of 4.2 L/h. The melting
point was determined by JHY-50 melting point apparatus.

Caffeoly glycerol (CG). White solid, m.p. 124.1-124.8
\textsuperscript{o}C; \textsuperscript{1}H NMR (DMSO, 600 MHz): d (ppm)
7.50 (d, \emph{J} = 15.9 Hz, 1H), 7.05 (d, \emph{J} = 1.9 Hz, 1H), 7.00
(dd, \emph{J} = 8.2, 2.0 Hz, 1H), 6.76 (d, \emph{J} = 8.1 Hz, 1H), 6.26
(d, \emph{J} = 15.9 Hz, 1H), 4.15 (dd, \emph{J} = 11.2, 4.1 Hz, 1H),
4.01 (dd, \emph{J} = 11.2, 6.6 Hz, 1H), 3.71 (dt,\emph{J} = 11.8, 6.1
Hz, 1H), 3.39 (td, \emph{J} = 11.6, 5.6 Hz, 2H);\textsuperscript{13}C
NMR (DMSO, 151 MHz): d (ppm) 167.11, 149.04, 146.11, 145.61, 125.87,
121.85, 116.21, 115.17, 114.35, 69.87, 66.07, 63.52; HREIMS m/z 277.0681
{[}M+Na{]}\textsuperscript{+}.

Feruloyl glycerol (FG). White solid, m.p.
121.2-121.9\textsuperscript{o}C; \textsuperscript{1}H NMR (DMSO, 600
MHz): d (ppm) , 7.57 (d, \emph{J} = 15.9 Hz, 1H), 7.33 (d, \emph{J} =
1.8 Hz, 1H), 7.12 (dd, \emph{J} = 8.2, 1.8 Hz, 1H), 6.80 (d, \emph{J} =
8.1 Hz, 1H), 6.49 (d, \emph{J} = 15.9 Hz, 1H), 4.16 (dd, \emph{J} =
11.2, 4.1 Hz, 1H), 4.03 (dd, \emph{J} = 11.2, 6.5 Hz, 1H), 3.83 (s, 3H),
3.74 - 3.70 (m, 1H), 3.44 - 3.38 (m, 2H); \textsuperscript{13}C NMR
(DMSO, 151 MHz): d (ppm) 167.19, 149.78, 148.41, 145.47, 126.09, 123.59,
115.98, 115.02, 111.60, 69.93, 66.06 , 63.18, 56.15; HREIMS m/z 291.0829
{[}M+Na{]}\textsuperscript{+}.

\emph{p} -Hydroxycinnamoyl glycerol (\emph{p} -HCG).White solid, m.p.
119.6-120.2 \textsuperscript{o}C; \textsuperscript{1}H NMR (DMSO, 600
MHz): 7.58 (d, \emph{J} = 16.0 Hz, 1H), 7.56 (d, \emph{J} = 8.6 Hz, 2H),
6.80 (d, \emph{J} = 8.6 Hz, 2H), 6.40 (d, \emph{J} = 15.9 Hz, 1H), 4.16
(dd, \emph{J} = 11.2, 4.1 Hz, 1H), 4.02 (dd, \emph{J} = 11.2, 6.6 Hz,
1H), 3.74 - 3.69 (m, 1H), 3.42 - 3.38 (m, 2H); \textsuperscript{13}C NMR
(DMSO, 151 MHz): d (ppm) 167.14, 160.36, 145.16, 130.76, 125.54, 116.26,
114.67, 69.90, 66.09, 63.17; HREIMS m/z 261.0730
{[}M+Na{]}\textsuperscript{+}.

\textbf{DPPH radicals scavenging ability detection}

The assay was performed according to the previous study (Weng et al.,
2019) with some modifications. Samples of CA, FA, \emph{p} -HCA, CG, FG
and \emph{p} -HCG were dissolved in anhydrous ethanol at different
concentrations (1.0, 0.5, 0.25, 0.125, 0.0625 mM). DPPH was dissolved in
anhydrous ethanol to make a solution with a concentration of 1.0 mM.
Equal amounts of DPPH and sample solutions were mixed and then monitored
spectrophotometrically at 517 nm using the UV-Vis spectrophotometer
after standing for 30 min. Instead of antioxidant solution, anhydrous
ethanol was used in the control groups. All the samples and reagents
were freshly prepared daily. The corresponding inhibition percentages
were calculated according to the following Eq.(2):\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

(2)

where \emph{A\textsubscript{c}} and \emph{A\textsubscript{s}} are the
absorbance values of the controls and tested samples, respectively.

\textbf{Statistical Analysis}

All experiments were performed at least in triplicate. Results were
expressed as mean values \selectlanguage{ngerman}± standard deviation (SD). The response
variable was subjected to analysis of variance (ANOVA) with 2-way
interactions. Statistical significance was considered at
\emph{p}\textless{} 0.05.

\textbf{Results and Discussion}

\textbf{Structural characterization of PAGs}

CG was synthesized via lipase-catalyzed esterification of CA with
glycerol in a ``solvent-free'' reaction (Scheme 1). Products of the
reaction system were monitored and quantified by HPLC-UV at 325 nm (Fig.
1). Peak 3 in fig 1 was confirmed to be CG by the mass spectrometry
(Fig. 2a) with a fragmentation cation of \emph{m/z} 277.0681
representing {[}CG+Na{]}\textsuperscript{+}. Similarly, FG and\emph{p}
-HCG were synthesized via lipase-catalyzed esterification of FA and
\emph{p} -HCA with glycerol and their identities were confirmed by mass
spectrometry (Fig. 2b and 2c), with the fragmentation cations
of\emph{m/z} 291.0829 and 261.0730 representing
{[}FG+Na{]}\textsuperscript{+} and {[}\emph{p}
-HCG+Na{]}\textsuperscript{+}, respectively. In order to further verify
the structures of PAGs, \textsuperscript{1}H NMR
and\textsuperscript{13}C NMR analyses of isolated CG, FG and \emph{p}
-HCG were also performed. Their chemical shifts were consistent with the
previous studies (Sun et al., 2020; Weng et al., 2019), indicating the
successful synthesis of PAGs.

Hereafter, CG was used as a model to optimize reaction conditions to
achieve the highest yield.

Fig. 1. HPLC chromatogram of lipase-catalyzed esterification mixture of
CA with glycerol at 325 nm. Reaction conditions are as follows: CA :
glycerol = 1:150 (mol : mol), Lipozyme 435 load 25\% (relative to the
weight of total substrates), 80 \textsuperscript{o}C, 86.7 kPa, 12 h.

Fig. 2. ESI-MS analysis of PAGs. (a) CG, (b) FG, (c) \emph{p} -HCG.

\textbf{Effects of different enzymes on the yield of CG}

Enzyme is a critical factor on the esterification of CA with glycerol.
Four kinds of enzyme including NOVOzyme 435, Lipozyme 435, Lipozyme TLIM
and Lipozyme RMIM were used as the catalyst (loaded at 3 \% (relative to
the weight of total substrates)) in the esterification of CA with
glycerol. In this experiment, the molar ratio of CA to glycerol was
fixed at 1:150. Fig. 3 showed the yield of CG at 80 \textsuperscript{o}C
after the reaction time of 24 h. The yield of CG achieved with Lipozyme
435 was slightly higher than that of Novozyme 435, while much higher
than those of Lipozyme TLIM and Lipozyme RMIM. Hence, Lipozyme 435 was
chosen as the suitable catalyst.

\textbf{Fig. 3.} Effect of enzyme type on the yield of CG. Reaction
conditions: the molar ratio of CA to glycerol 1:150, reaction
temperature 80 \textsuperscript{o}C, enzyme load 3 \% (relative to the
weight of total substrates), 86.7 kpa, 24 h.

\textbf{Effect of enzyme load on the yield of CG}

The influence of the enzyme load on the product yield was investigated
as shown in Fig. 4. The enzyme dosage was varied from 5 \% to 30 \% of
the total substrate weight. It was found that the yield of CG gradually
increased with the increase of enzyme dosage, likely due to more active
sites, a higher probability of enzyme-substrate collision, and a higher
reaction rate (Yadav \& Devendran, 2012). However, when enzyme load was
above 25 \%, the yield of CG was almost the same with that of 25 \%,
likely due to biocatalyst agglomeration and possible diffusional
problems of substrates at higher enzyme load (Sun \& Hu, 2017). So, the
optimal enzyme dosage was selected as 25 \%.

\textbf{Fig. 4.} Effect of enzyme load on the yield of CG. Reaction
conditions: the molar ratio of CA to glycerol 1:150, reaction
temperature 80 \textsuperscript{o}C, 86.7 kpa.

\textbf{Effect of substrate molar ratio on the yield of CG}

The effect of molar ratio of CA to glycerol on the yield of CG was
evaluated in the range from 1:75 to 1:175. As shown in Fig. 5, as the
molar ratio changed from 1:75 to 1:125, the yields of CG increased,
while they were all lower than 80\% at 12 h. When the molar ratio of CA
to glycerol varied from 1:150 to 1:175, the yields were similar with
each other and over 95 \% after 10 h, which could be attributed to the
presence of excessive glycerol as reactant and reaction medium for the
esterification. Therefore, the substrate molar ratio of 1:150 was chosen
for the esterification in the following experiments.

\textbf{Fig. 5.} Effect of substrate molar ratio on the yield of CG.
Reaction conditions: 25\% of Lipozyme 435 (relative to the weight of
total substrates), 86.7 kpa, 80 \textsuperscript{o}C.

\textbf{Effect of reaction temperature on the yield of CG}

The conversion, solubility of the PAs and the stability as well as the
activity of the lipase are strongly related to the reaction temperature.
The effect of reaction temperature on esterification of CA with glycerol
in lipase-catalyzed reaction was investigated at different temperatures
from 70 \selectlanguage{english} to 90 \selectlanguage{english}. As shown in Fig. 6, the yields of CG increased with
the rising of the temperature from 70 \selectlanguage{english} to 80 \selectlanguage{english} and reached 94.4 \% at
80 \selectlanguage{english} after 10 h, which were mainly attributed to the decrease of the
reaction system viscosity and fast mass transfer rate of caffeoyl to
glycerol at a high temperature. However, the yields of CG decreased when
the temperature was over 80 degC, which could due to be the thermal enzyme
deactivation at high temperature. Similar phenomenon could also be found
in other studies (Sun \& Hu, 2017; Sun \& Chen, 2015). Therefore, the
optimal reaction temperature was chosen to be 80 degC.

\textbf{Fig. 6.} Effect of reaction temperature on the yield of CG.
Reaction conditions: molar ratio of CA to glycerol 1:150, 25\% of
Lipozyme435 (relative to the weight of total substrates), 86.7 kpa.

\textbf{Optimum conditions and verification}

The optimum reaction variables for CG synthesis were as follows: load of
Lipozyme 435 25 \% (relative to the weight of total substrates), molar
ratio of CA to glycerol 1:150, 80 \textsuperscript{o}C and 10 h. Under
the optimized conditions, CG yield was 95.0 +- 1.0 \%, which was similar
with the yield reported in the literature (Sun \& Hu, 2017). However, CG
was in the past synthesized by the transesterification of ethyl caffeate
with glycerol, with the disadvantage of reducing the final reaction
efficiency and increasing cost.

The above optimal reaction conditions were also used to synthesize FG
and \emph{p} -HCG \emph{via} esterification of FA and \emph{p} -HCA with
glycerol, resulting in isolated yields of 82.1 \% and 80.7 \%
respectively, validating the reaction model in the synthesis of PAGs.

\textbf{Free radical scavenging ability}

The free radical scavenging abilities of PAs and PAGs in ethanol system
were evaluated using DPPH assay. As shown in Fig. 7, the DPPH inhibition
decreased with the reduction of sample concentration. The radical
scavenging ability towards DPPH depends on structural features, such as
the dissociation energy of hydrogen atoms from hydroxyl groups,
resonance delocalization of the phenol radical (PheO*) and steric
hindrance (Shahidi \& Naczk, 1995). The amount of phenolic hydroxyl
groups may have a dominant influence on the free radical scavenging
ability (Sun et al., 2020), enabling CA (94.9 \% at 1 mM) to have the
highest radical scavenging ability because of its bis-hydroxyl
structure. Although FA and \emph{p} -HCA had the same number of phenolic
hydroxyl groups, FA (80.5 \% at 1 mM) exhibited higher antioxidant
activity than \emph{p} -HCA (13.1 \% at 1 mM) because of the resonance
delocalization of the phenol radical (PheO*) induced by methoxy group in
the structure of FA. There was a similar variation rule in the radical
scavenging ability of CG (91.5 \% at 1 mM), FG (66.1 \% at 1 mM)
and\emph{p} -HCG (5.74 \% at 1 mM) with the parent PAs. The glycerol
moiety of PAGs may have caused steric hindrance making antioxidants
inaccessible to the DPPH radical, but also increased the hydrophilicity
of PAGs causing antioxidants more available for the DPPH radicals in
polar systems, so that the radical scavenging ability of PAGs was not
significantly different to that of the parent PAs (Zhang \& Akoh, 2020).
Fig. 7 showed that the radical scavenging ability of PAGs was slightly
lower than that of PAs, indicating that the accessibility of the
molecule had greater effect on the scavenging activity in the DPPH
assay. Therefore, the radical scavenging capacity order of PAs and PAGs
was CA \textgreater{} CG \textgreater{} FA \textgreater{} FG
\textgreater{} \emph{p} -HCA \textgreater{} \emph{p} -HCG.

Fig. 7 DPPH inhibition capacity of PAs and PAGs at various
concentrations

\textbf{Conclusion}

In this study, an efficient enzymatic route for PAGs synthesis by the
esterification of PAs with glycerol under solvent-free condition was
developed. The optimal process conditions for the synthesis of PAGs
were: 25 \% Lipozyme435, molar ratio of PAs to glycerol 1:150, reaction
temperature 80 \selectlanguage{english}, and reaction time 10 h. Under the optimal conditions,
three kinds of PAGs including CG, FG and \emph{p} -HCG were synthesized
and identified by MS, \textsuperscript{1}H NMR and \textsuperscript{13}C
NMR with the isolated yield of 85.0 \%, 82.1 \% and 80.7 \%,
respectively. Among them, CG showed a comparative free radical
scavenging ability to CA, indicating that CG may be used as a
water-soluble antioxidant alternative to CA for food and cosmetic
applications.

\textbf{AUTHOR CONTRIBUTIONS}

Hong Li: Conceived, designed and supervised the study. Yanyan Li: Data
acquisition and wrote the first draft of the manuscript. Xuezhi Yin:
Data acquisition and analyzing. Ziyi Li: Data acquisition and evaluation
together with practical supervision. Huiling Mu: Manuscript improving.
Ruiling Shen: Data evaluation. Tao Wei: Project formulation and
manuscript finalizing.

\textbf{Acknowledgements}

The authors gratefully acknowledge the funding received from Zhongyuan
Science and Technology Innovation Leading Talent Project {[}Grant
number: 224200510017{]} and ``Three districts'' science and technology
talent support program of Henan Province.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare that they have no conflict of interest.

\textbf{ETHICS STATEMENT}

This research falls outside of human or animal studies and institutional
ethical approval was not required.

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