Effects of epigallocatechin gallate on the stability of epicatechin in a photolytic process

Article abstract of DOI:10.3390/molecules24040787

Catechins belonging to polyhydroxylated polyphenols are the primary compounds found in green tea. They are associated with many physiological properties. Epicatechin (EC) is a non-gallate-type catechin with four phenolic hydroxyl groups attached. The changes in EC treated with color light illumination in an alkaline condition were investigated by chromatographic and mass analyses in this study. In particular, the superoxide anion radical (O₂?^?) was investigated during the EC photolytic process. EC is unstable under blue light illumination in an alkaline solution. When EC was treated with blue light illumination in an alkaline solution, O₂?^? was found to occur via a photosensitive redox reaction. In addition, the generation of monomeric, dimeric, and trimeric compounds is investigated. On the other hand, epigallocatechin gallate (EGCG), which is a gallate-type catechin, is stable under blue light illumination in an alkaline solution. Adding EGCG, during the blue light illumination treatment of EC decreased photolytic formation, suggesting that gallate-type catechins can suppress the photosensitive oxidation of EC. Gallate-type catechins are formed via the esterification of non-gallate-type catechins and gallic acid (GA). The carbonyl group on the gallate moiety of gallate-type catechins appears to exhibit its effect on the stability against the photosensitive oxidation caused by blue light illumination.

Full text of DOI:10.3390/molecules24040787

molecules
Article
Effects of Epigallocatechin Gallate on the Stability of
Epicatechin in a Photolytic Process
Shiuh-Tsuen Huang 1, Yi-An Hung 2, Meei-Ju Yang 3, Iou-Zen Chen 4, Jeu-Ming P. Yuann 2,
*
and Ji-Yuan Liang 2,
*
1
Department of Science Education and Application, National Taichung University of Education,
Taichung 40306, Taiwan; hstsuen@mail.ntcu.edu.tw
Department of Biotechnology, Ming-Chuan University, Gui-Shan 33343, Taiwan;
amyhung840809@gmail.com
Tea Research and Extension Station, Taoyuan 32654, Taiwan; 762204@gmail.com
Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei 10617, Taiwan;
chenyo@ntu.edu.tw
2
3
4
*
Correspondence: jyuann@mail.mcu.edu.tw (J.-M.P.Y.); liang121@mail.mcu.edu.tw (J.-Y.L.);
Tel.: +886-3-3507001 (ext. 3555) (J.-M.P.Y.); +886-3-3507001 (ext. 3774) (J.-Y.L.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Eduardo Sobarzo-Sánchez
Received: 14 January 2019; Accepted: 19 February 2019; Published: 22 February 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Catechins belonging to polyhydroxylated polyphenols are the primary compounds found
in green tea. They are associated with many physiological properties. Epicatechin (EC) is a
non-gallate-type catechin with four phenolic hydroxyl groups attached. The changes in EC treated
with color light illumination in an alkaline condition were investigated by chromatographic and
mass analyses in this study. In particular, the superoxide anion radical (O2
−) was investigated
•
during the EC photolytic process. EC is unstable under blue light illumination in−an alkaline
solution. When EC was treated with blue light illumination in an alkaline solution, O2
•
was found
to occur via a photosensitive redox reaction. In addition, the generation of monomeric, dimeric,
and trimeric compounds is investigated. On the other hand, epigallocatechin gallate (EGCG), which
is a gallate-type catechin, is stable under blue light illumination in an alkaline solution. Adding
EGCG, during the blue light illumination treatment of EC decreased photolytic formation, suggesting
that gallate-type catechins can suppress the photosensitive oxidation of EC. Gallate-type catechins are
formed via the esterification of non-gallate-type catechins and gallic acid (GA). The carbonyl group
on the gallate moiety of gallate-type catechins appears to exhibit its effect on the stability against the
photosensitive oxidation caused by blue light illumination.
Keywords: blue light; catechin; epicatechin; EGCG; gallic acid
1. Introduction
Polyphenols are polyhydroxylated compounds and secondary metabolites in plant products.
These phenolic compounds have one or more hydroxyl groups attached to an aromatic ring and the
structure may consist of a simple phenolic compound to a complex polymer [1]. Polyphenols also have
beneficial characteristics, including anti-oxidative activity, as well as anti-radiation and anti-microbial
activities [2].
Catechins are a class of polyphenolic compounds that are most commonly found as a natural
product in foods and beverages, such as cacao, cacao products, grapes, red wine, green tea, and so forth.
According to the gallic acid (GA) ester at the C3 position of the catechins, two major catechin types
comprise about 80% of the polyphenolic compounds found in green tea: non-gallate-type catechins,
such as epicatechin (EC) and catechin; gallate-type catechins, such as epicatechin gallate (ECG) and
Molecules 2019, 24, 787; doi:10.3390/molecules24040787
www.mdpi.com/journal/molecules

Molecules 2019, 24, 787
2 of 13
epigallocatechin gallate (EGCG) [3]. The structures of EC, EGCG, pyrogallol (PG), and GA are shown
in Figure 1.
Figure 1. The molecular structures of (
A) epicatechin (EC), (B) epigallocatechin gallate (EGCG),
(C) pyrogallol (PG), and (D) gallic acid (GA).
One of the significant disadvantages of catechins is their instability [
oxidized, owing to the loss of hydrogen atoms in solution, with the generation of quinone-oxidized
products and semiquinone radical species occurring via oxidation processes [ ]. Catechin and EC are
isomers that are structurally similar. It has been reported that catechin is stable in acidic conditions, but
the color of catechin solution at pH 7.4 was altered after 96 h incubation [ ]. Catechin is not stable in
4]. Catechins are easily
5,6
7
neutral and alkaline conditions. The catechin epimerization (i.e., the reaction with bicarboxylic acid via
cross-linkage) can be achieved in neutral or alkaline conditions under hydrothermal treatment [2,8].
Catechins are unstable under UV light illumination. It was previously reported that EC
and catechin were ultraviolet B (UVB)-sensitive and that yellow photoproducts were produced
by the treatment with UVB radiation, whereas other catechins (ECG and EGCG) were relatively
UVB-insensitive [
heterocyclic ring and the epimerization of catechin is achieved via UV light illumination [10
Catechin is also unstable under visible light illumination. Catechin is sensitive under blue light
illumination in an alkaline solution with the superoxide anion radical (O2
−), and a chromogenic
catechin dimer being generated during catechin photolysis via an electron-transfer mechanism [12 13].
9]. The photolysis of EC and catechin cleaves the ether bond of the flavan-3-ols
,
11].
•
,
EGCG is a gallate-type catechin compound and the most abundant one among tea catechins [14]. It is
uncertain whether the peroxidation response exists or whether the free radicals are produced during
the photolysis of the two major types of catechins treated with blue light illumination.
Catechin is unstable under blue light illumination and the catechin dimer can be suppressed in the
presence of ascorbic acid during catechin photolysis in an alkaline solution, suggesting that ascorbic
acid can inhibit catechin oxidation caused by photolysis [13]. Acting as antioxidants that scavenge
diffusible free radical species, tea polyphenols are considered one of the main healthy components
of tea beverages. It would be of interest to study how anti-oxidative reagents, such as gallate-type
catechins (EGCG), decrease the photolytic oxidation of non-gallate-type catechins (EC) under blue
light illumination in an alkaline solution.
The structural difference between epigallocatechin (EGC) and EC is an additional hydroxyl group
in EGC at the 50-position of the B ring. EGCG is formed via the esterification of EGC, comprising an
ester derivative at the 30-hydroxyl position on the C ring with a moiety gallate [15]. It would be of

Molecules 2019, 24, 787
3 of 13
interest to find out whether attaching to a gallate group, by esterification, can enhance the stability of
EGCG under blue light illumination in an alkaline solution.
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), the hydroxyl radical (
the superoxide anion radical (O2 ), are reactive in general [16].
−), and the peroxyl radical (ROO
The O2
−, mostly formed as an intermediate, occurs via the reduction of oxygen, leading to harm to
•
OH),
•
•
•
the organized system, inflammation, and atherosclerosis in addition to aging cells [17,18]. Previously,
Liang et al. reported that the ROS from blue light-excited riboflavin or riboflavin-50-phosphate (FMN)
can cleave the plasmid DNA in integrity assays and inactivate Escherichia col−i or methicillin-resistant
Staphylococcus aureus by ROS formation [19
–
21]. It has been reported that O2
•
can be generated from
catechin hydrate via blue light photolysis and can cause the inactivation of Acinetobacter baumannii
(A. baumannii) [13]. EC is a non-gallate-type catechin with four phenolic hydroxyl groups. Further
research will be aimed at investigating the ROS generated from EC photolysis.
Catechins are the largest fraction of polyphenols in green tea. The photolysis of two types of
catechins (non-gallate- and gallate-type catechins) treated with blue light illumination was investigated
in this study. This study was directed towards understanding the effects of blue light illumination
on the changes in EC and the influence that EGCG or GA exerts on EC in a photoreaction system by
−
chromatography and mass spectrometry techniques. The production of O2
and the structural changes in EC treated with blue light were also examined.
•
from light-excited EC
2. Results
2.1. Effects of Blue, Green, and Red Lights on EC Photolysis
Firstly, 1 mM EC in 0.1 M phosphate buffer solution (ECPB), treated with color light illumination,
was examined. Figure 2A shows the spectra (250–750 nm) of ECPB at pH 8.0 metered by blue, green,
and red light illumination. As observed, there was one absorption peak at 275 nm for EC in the dark.
The absorbance of EC at 438 nm was significantly increased by blue light illumination, showing the
highest efficiency in EC photolysis. The green and red lights exhibited little influence as the changes
in the spectra at 438 nm were of no significance. Figure 2B,C show the effects of the illumination
time of blue light on EC photolysis. EC, when in an alkaline solution, turned yellow under blue light
illumination. As observed, the photochemical reaction of EC increased with the illumination time.
Figure 2. The absorption spectra of (
illuminated by blue, green, and red lights at 2.0 mW/cm2 for 1 h, (
blue light illumination at 2.0 mW/cm2 for 1, 2, and 3 h, and (
A
) 1 mM EC in 0.1 M phosphate buffer solution (ECPB) at pH 8,
) ECPB treated with or without
) the color changes in ECPB treated with
B
C
or without blue light illumination at 2.0 mW/cm2 for 1, 2, and 3 h. The absorbance of the reaction
solutions was measured within the spectral range from 250 to 750 nm.

Molecules 2019, 24, 787
4 of 13
2.2. Detection of O2•− in EC Photolysis
−
The production of O2
•
from catechin hydrate or tetracycline under blue light illumination was
investigated using the nitro blue tetrazolium (NBT) reduction method [13,22]. The reductions in the
EC/NBT system upon blue light illumination at 2.0 mW/cm2 for 10, 20, 30, 40, 50, and 60 min are
shown in Figure 3. Figure 3 reveals that the photochemical effect of NBT reduction in EC was found to
−
increase in a time-dependent manner under blue treatment. O2
•
was generated from EC under blue
light illumination in phosphate buffer solution (PBS) at pH 7.8, suggesting that EC photolysis can be
applied as a photosensitive oxidation process using generated O2•− in the EC/NBT system.
Figure 3. Effects of 1 mM EC on nitro blue tetrazolium (NBT) reduction by blue light irradiation at
2.0 mW/cm2 for 10–60 min. The results are represented by mean
Statistically significant differences (p < 0.05) between each treatment are indicated by the different
±
standard deviation, where n = 3.
letters above each bar.
2.3. Effects of Blue Light on EC, EGCG, GA, and PG Photolysis
Unlike EC, which is a non-gallate-type catechin, EGCG is a gallate-type catechin. PG is a
trihydroxybenzene, while GA is a trihydroxybenzoic acid. The effects of blue light on EC, EGCG, GA,
and PG photolysis were examined herein. The spectra of GA and EGCG in PBS at pH 8.0, treated with
or without blue light illumination at 2.0 mW/cm2 for 1 h, were unchanged, as shown in Figure 4A,B.
However, when EC and PG in PBS were treated with blue light illumination at 2.0 mW/cm2 for 1 h,
the spectra were significantly altered, as shown in Figure 4C,D.
2.4. Effects of EGCG and GA on the EC Photolysis
The effects of EGCG and GA on the spectral changes in ECPB at pH 8.0 under blue light
illumination were studied. The absorption spectra of 1 mM ECPB treated with the same concentration
of EGCG or GA, under blue light illumination at 2.0 mW/cm2 for 1, 2, and 3 h, achieve different levels
of photolysis, as shown in Figure 5. It is also observed that ECPB under blue light illumination shows
two peaks at 275 and 438 nm, as shown in Figure 5A,B. However, when ECPB was treated with 1 mM
EGCG, under blue light illumination at 2.0 mW/cm2 for 1 and 2 h, the absorbance at 438 nm was
suppressed in Figure 5A. By quantifying the absorbance at 438 nm, the extent of the ECPB reduction
percentage upon blue light illumination at 2.0 mW/cm2 for 3 h was 57.9% in the presence of EGCG,
suggesting that the addition of EGCG can inhibit the photosensitized oxidation of EC under blue
light illumination.
The effect of GA on ECPB photolysis was similar to that on EGCG, as shown in Figure 5B. For the
absorbance at 438 nm, the extent of the ECPB reduction percentage upon blue light illumination at
2.0 mW/cm2 for 3 h was 64.5% for the GA treatment under the same condition. The photolytic process
of EC under blue light illumination can be inhibited by EGCG and GA.

Molecules 2019, 24, 787
5 of 13
Figure 4. The absorption spectra of (A) GA, (B) EGCG, (C) PG, and (D) EC in phosphate buffer solution
at pH 8.0, illuminated by blue light at 2.0 mW/cm2 for 1 h. The absorbance of the reaction solutions
was measured within the spectral range from 250 to 750 nm.
Figure 5. The absorption spectra of (
A
) ECPB at pH 8.0 treated with EGCG with illumination by blue
) ECPB treated with GA and illuminated by blue light
light at 2.0 mW/cm2 for 1, 2, and 3 h; and (
B
at 2.0 mW/cm2 for 1, 2, and 3 h. The absorbance of the reaction solutions was measured within the
spectral range from 250 to 750 nm.
2.5. Molecular Identification by LC–MS/MS Analysis
EC solutions with basified and photochemical products were examined via an LC–MS/MS
analysis. As shown in Figure 6, the total ion chromatogram of the PBS at pH 8.0 was observed at
2.84 min. In Figure 6A, the total ion chromatogram of the ECPB at pH 8.0 without photolysis processing
was observed at 14.6 min (EC), and four signals were observed at 8.82 (Poly-A), 12.79 (C), 13.27 (Poly-B),
and 14.6 min (EC) after blue light illumination in Figure 6C. As shown in Figure 2, for 1.0 mM ECPB at
pH 8.0, treated with blue light illumination for 2 h, the absorption peak at 438 nm was significantly
increased. An EC reduction percentage of 58.6% was observed under the same conditions by HPLC
analyses in Figure 6. In Figure 6B, the total ion chromatogram of the ECPB treated with GA without
photolysis processing was observed at 8.47 (GA) and 14.6 min (EC), whereas three signals could be
observed at 8.47 (GA), 12.79 (C), and 14.6 min (EC) after blue light illumination, as shown in Figure 6D.

Molecules 2019, 24, 787
6 of 13
Figure 6. Total ion chromatogram of an HPLC–MS analysis of (
A) ECPB at pH 8.0 in the dark, (B) ECPB
treated with GA in the dark, (
C,D
) ECPB treated with blue light illumination at 2.0 mW/cm2 for 2 h in
the absence and presence of GA, respectively.
Figure 7 shows the electron ionization mass spectra after analyses from ECPB photolysis.
As shown in Figure 7A, EC was observed at 14.6 min, with the major ion fragment being m/z
289.1. Poly-A and Poly-B were the photolysis products that were observed at 8.82 and 13.27 min,
respectively, with their major ion fragments being m/z 863.1 and 577.1, respectively, as shown in
Figure 7B,C. The emerging peak C was observed at 12.79 min, very similar to catechin eluted at the
same retention time (data not shown). In addition, the compound of peak C, one of the EC photolytic
products, was verified by its mass spectrum, with the major ion being m/z 289.1, in Figure 7D and thus
−
identified as catechin. The signals were attributed to the quasi-molecular ions [M–H] in mass spectra.
The molecular weights of Poly-A and Poly-B were supposedly 863.1 and 577.1 Da, respectively.
2.6. HPLC–DAD Analysis of EGCG Under Blue Light Illumination
As shown in Figure 8, the chromatograms of EGCG after blue light illumination were analyzed by
an HPLC–photodiode-array detector (HPLC–DAD) at 280 nm. The pH of 1 mM EGCG in pure water
was 6.2 and pH of EC was 6.9. EGCG dissolved in H2O was observed at 14.9 min and was determined
to be stable, as shown in Figure 8A. For EGCG in PBS at pH 8, many other chromatographic signals
were also found, as shown in Figure 8B. After blue light illumination, the photoreaction products of
EGCG in PBS were observed in Figure 8C, with GA being confirmed at 8.40 min by its mass spectrum
of m/z 169.1, as shown in Figure 8D.

Molecules 2019, 24, 787
7 of 13
Figure 7. Electron ionization mass spectra of EC and the photoreaction products from ECPB,
illuminated by blue light for 2 h. Product ion spectra of [M–H]− for a blue-light-treated ECPB at
pH 8.0. The precursor ion selected is m/z 289.1 and the proposed fragments are shown.
Figure 8. Chromatograms of HPLC–photodiode-array detector (HPLC–DAD) analyses of (
H2O, ( ) EGCG in phosphate buffer solution (pH 8), ( ) EGCG in phosphate buffer solution (pH 8)
treated with blue light illumination at 2.0 mW/cm2 for 2 h, and (
) electrospray ionization mass
spectra of the photoreaction products from the EGCG in (C) at 8.4 min.
A) EGCG in
B
C
D

Molecules 2019, 24, 787
8 of 13
3. Discussion
Yang et al. reported that the ROS from blue light-excited catechin hydrate can lead to the
inactivation of A. baumannii−by ROS formation [13]. In this study, EC at pH 7.8, under blue light
illumination, generates O2
•
by aerobic photo-oxidative processes. The slope of the NBT reaction
curve for the photolytic time (min) of EC can be applied as an index to show the production of ROS
with respect to the standard. The slope of the NBT reaction curve of ROS production for EC was similar
to that of catechin hydrate (0.018) photolysis under blue light illumination in the same conditions (data
not shown). Under blue illumination, EC photolysis reactions can also inactivate A. baumannii (data
not shown). Therefore, EC can be considered as a potential photosensitizer for microbial inactivation
by EC photosensitized reactions.
The effects of EGCG on the photo-oxidation stability of EC photolysis were examined in this study.
Green tea infusion turning brown is an important issue for the shelf life of tea drinks in the tea industry
and was shown to be due to the oxidation of polyphenols [13]. Shi et al. reported that catechin or
an EC solution under UVB illumination can generate several new photoproducts via photo-induced
electron transfer [9]. Yang et al. reported that catechin hydrate at pH 8.0 under blue light illumination
can generate a chromogenic catechin dimer (proanthocyanidin) via photosensitized oxidation and
photo-induced electron transfer. However, addition of ascorbic acid during the photochemical process
can suppress the photosensitive oxidation of catechin [13]. Upon the addition of EGCG or GA,
the inhibition of a photosensitized oxidation process of EC under blue light illumination was observed,
as evidenced by the disappearance of the absorbance of EC at 438 nm.
EC (a non-gallate-type catechin) is sensitive to blue light illumination, while EGCG (a gallate-type
catechin) in PBS at pH 8 is insensitive to change by the spectrophotometric method. GA comprises
a PG moiety attached to a carboxylic acid group. It is shown in this study that GA in PBS at pH 8.0,
treated with or without blue light illumination, was insignificantly changed by the spectrophotometric
method, but PG at pH 8.0 is sensitive to blue light illumination. GA and PG contain a benzene
ring attached to three hydroxyl groups, both showing a strong UV absorption capacity due to the
π
electrons of the benzene ring. PG in basified solution is not stable during photo-oxidation caused
by blue light illumination. The stability of GA can be at least partly explained by its carboxylic acid
group, an electron-withdrawing group that makes the three hydroxyl groups less likely to undergo
photo-oxidation as it occurs in PG. In this study, EC and PG at pH 8.0 are sensitive to blue light
illumination. Belonging to gallate-type catechins, EGCG is formed via the esterification of EGC
(containing a B ring with a moiety PG) with a moiety of gallate attached to the C ring by ester linkage.
At pH 8.0 and under blue light illumination, EGCG can generate GA, as shown in this study. By making
benzene rings less electron-rich, the carbonyl group on the gallate moiety of EGCG exhibits its effect
on the spectrophotometric change caused by blue light illumination.
EC is stable in water and does not change the pH of its aqueous solution. However, in this study,
EC at pH 8.0 was not stable under blue light illumination and the addition of EGCG or GA can inhibit
the photosensitized oxidation process of EC. Moreover, monomeric, dimeric, and trimeric compounds
were observed when ECPB was treated with photolysis processing at pH 8.0, but the product of ECPB
treated with an anti-oxidative reagent (GA) under blue light illumination is only a monomeric one.
Based on the results of this study, an EC photoreaction mechanism under the influence of EGCG and
GA is proposed, as shown in Figure 9.
EC is a diastereomer of catechin and, unsurprisingly, they exhibit similar photochemical properties.
For the photo-oxidation of flavan-3-ols, two plausible pathways are proposed (i.e., a single oxygen
molecule reacts with the substrate), which results in the generation of radicals [13,23]. Additionally,
by losing hydrogen atoms, the auto-oxidation of EC arises with the formation of quinone-oxidized
−
products via semiquinone radical intermediates (Figure 9I), and O2
•
in the reaction mixture [5,6,13].
The irradiation of EC under blue light leads to the one-electron oxidation of the B ring, as shown by the
. The –OH bond of the heterocyclic ring was excited via photosensitized oxidation,
so as to form an o-quinone compound, as shown in Figure 9G. Owing to its low bond dissociation
−
appearance of O2
•

Molecules 2019, 24, 787
9 of 13
energy, the ether bond of the heterocyclic ring (ring C) of EC can be readily cleaved with the ring
opened by photolysis, resulting in the generation of a free radical intermediate at m/z 289, as shown
in Figure 9B [
9
,
13]. The neutral radicals can also be ionized in a polar solvent via a photo-induced
24].
electron transfer reaction, with the product being a carbocation of EC, as shown in Figure 9C [13
,
In Figure 9J, the formation of a B-type proanthocyanidin dimer at m/z 577 can be achieved from the
condensation of the o-quinone compound and a carbocation intermediate of EC [13,25–27].
Figure 9. Proposed scheme for the mechanism of the EC photoreaction and the interactions of the
intermediates with EGCG.
The constitutive building blocks of the B-type proanthocyanidin dimer, usually flavan-3-ols
(e.g., (+)-catechin and (
linkages [28]. It has been reported that an initial oxidative removal of hydride ion results in the
oxidative transformation of B-type procyanidins into A-type procyanidins [29 30]. As shown in
−
)-epicatechin) can be linked together by a single bond through interflavan
,
Figure 9H, an A-type procyanidin dimer derived from the B-type procyanidin dimer is observed at
m/z 575. As shown in Figure 9D, further intramolecular oxidation causes the transformation of the
A-type proanthocyanidin dimer into an A-type proanthocyanidin dimer o-quinone at m/z 573 [31].
The A-type proanthocyanidin trimer (m/z 863) can be generated from the condensation of the A-type
proanthocyanidin dimer o-quinone and a carbocation intermediate of EC, as shown in Figure 9K.
In Figure 9L, the o-quinone intermediate was generated from semiquinone radical intermediates
(Figure 9I) via a photo-induced electron transformation. The o-quinone intermediate, unstable in
alkaline conditions, can be reduced to generate EC at m/z 289 (Figure 9A) or catechin at m/z 289
(Figure 9M) [13,32].
GA and EGCG are used as the antioxidants because of their strong reducing power. EGCG
is also used for the treatment of superficial bladder cancer or to alleviate the symptoms of Down
syndrome [33,34]. In this study, EGCG treated with blue light illumination at pH 8.0 can generate
GA. GA is a trihydroxybenzoic acid and exhibits two pKa values, 4.1 and 8.38, for the carboxyl
and hydroxyl groups, respectively [35]. It was reported that GA in an alkaline condition can
be rapidly oxidized through the three hydroxyl groups attached to the aromatic ring that are
prone to oxidation [36], resulting in a two-electron, two-proton, and anion gallate free radical
oxidation scheme [37]. In Figure 9J,K, the disconnection within proanthocyanidins is achieved by
the cleavage of C–C or C–O–C bonds between the B-type proanthocyanidin dimer and the A-type
proanthocyanidin trimer, through the protons or quinone methide cleavage via GA in an alkaline
condition [13,26,36,38,39]. Ultimately, a depolymerization process promotes the monomeric catechin
(Figure 9M) and/or EC (Figure 9A) generation.

Molecules 2019, 24, 787
10 of 13
4. Materials and Methods
4.1. Chemicals
GA, EGCG, methanol, and PG were obtained from Sigma-Aldrich (St. Louis, MO, USA). NBT
was obtained from Bio Basic, Inc. (Markham, ON, Canada). ( )-Epicatechin (EC) was purchased from
−
Tokyo Chemical Industry Co. (Tokyo, Japan). Ultra-pure water by the Milli-Q system (Merck Millipore,
Burlington, MA, USA) was used as a solvent throughout this study.
4.2. Illumination System
The illumination system comprises an opaque illumination chamber (height, 80 mm; diameter,
70 mm), and a power supply (YP30-3-2, Chinatech Co., New Taipei City, Taiwan) as described
previously [13,22]. Six blue, green, or red LED lamps (DC 12 V 5050, vitaLED Technologies Co.,
Tainan, Taiwan) were applied to surround the inner walls of the chamber. The response solution was
left within a glass test tube and maintained on the upper level of the chamber. The power supply and
a solar power meter (TM-207, Tenmars Electronics Co., Taipei, Taiwan) were used to provide light
illumination. The temperature of the photoreaction system was kept at 25
of the emitted maxima of the blue, green, and red lights were 463, 529, and 632 nm, with their W1/2
(spectral width at half height) being 23, 31, and 14 nm, respectively, as described previously [40].
±
3 ◦C. The wavelengths
4.3. Effects of Blue, Green, and Red Lights on EC Photolysis
The effects of blue, green, and red lights on EC photolysis were investigated by a UV/Vis
spectrometer (Lambda35, Perkin-Elmer, Waltham, MA, USA). Briefly, (A) 1 mM EC in 0.1 M phosphate
buffer solution (ECPB) at pH 8.0 in the dark was used as a standard control solution, (B) 1 mM ECPB
was illuminated by blue, green, and red lights at 2.0 mW/cm2 for 1 h, and (C) 1 mM ECPB was
illuminated by blue light at 2.0 mW/cm2 for 1, 2, and 3 h. The absorbance of the ECPB was recorded
within the range of 250–750 nm by a UV/Vis spectrometer.
4.4. O2•− Determination
−
A direct or an indirect method can be utilized for the detection of O2
•
. Using the direct method
requires a special apparatus, such as an electron paramagnetic resonance (EPR) spectrometer, whereas
indirect assays are more broadly practiced in analyses [13]. The nitro blue tetrazolium (NBT) reduction
−
method can be applied to detect O2
•
activity and is a widely used indirect method for the detection
of O2•− levels [40].
−
The effects of EC photolysis on O2
•
production were studied. The chemicals were freshly
prepared prior to the NBT reduction method. The reactants comprised EC, methionine, and NBT of
1, 9, and 0.15 mM concentrations in 0.1 M phosphate buffer solution (PBS) at pH 7.8, respectively.
The reactants were then treated with blue light illumination at 2.0 mW/cm2 for 10, 20, 30, 40, 50, and
generated from EC photolysis was observed when NBT undergoes
reduction to form blue formazan, which showed absorbance at 560 nm.
−
60 min. The occurrence of O2
•
4.5. Effects of Blue Light on the Photolysis of EC, EGCG, GA, and PG
The effects of blue light on the photolysis of catechins studied herein were investigated via UV/Vis
spectrophotometry. In brief, (A) 1 mM EC, EGCG, GA, or PG in 0.1 M PBS at pH 8.0 in the dark was
used as a standard control solution, and (B) EC, EGCG, GA, or PG in 0.1 M PBS was illuminated by
blue light at 2.0 mW/cm2 for 1 h. The absorbance of the reaction solutions was recorded within the
range of 250–750 nm by a UV/Vis spectrometer.
4.6. Effects of EGCG or GA on EC Under Blue Light Illumination
The effects of EGCG or GA on EC under blue light illumination were examined
spectrophotometrically. In brief, (A) 1 mM ECPB at pH 8.0 treated with either 1 mM EGCG or

Molecules 2019, 24, 787
11 of 13
1 mM GA in the dark was used as a standard control solution, (B) 1 mM ECPB was illuminated by
blue light at 2.0 mW/cm2 for 1, 2, and 3 h, and (C) 1 mM ECPB was treated with 1 mM EGCG or 1 mM
GA under blue light illumination at 2.0 mW/cm2 for 1, 2, and 3 h. The absorbance of the ECPB was
recorded within the range of 250–750 nm by a UV/Vis spectrometer.
4.7. LC–MS/MS Analysis of EC Treated with GA Under Blue Light Illumination
ECPB and its related photolysis products subjected to an LC/MS/MS analysis were analyzed by
an Agilent 6410B triple quadrupole LC/MS/MS and Agilent 1200 HPLC (Agilent Technologies, Palo
Alto, CA, USA) connected with an electrospray ionization (ESI) source, as described previously [13].
The negative ion mode of the ESI technique was used in this study. Data were obtained by the Agilent
Mass Hunter Workstation software version B.06.00.
The reaction solutions were eluted using an Agilent Poroshell 120 EC-C18 column (2.7µm,
4.6 × 150 mm, Agilent Technologies, Palo Alto, CA, USA). The effects of GA on EC under blue light
illumination were examined via LC–MS/MS analysis. In brief, (A) 1 mM ECPB at pH 8.0 in the dark
was used as a standard control solution, (B) 1 mM ECPB was illuminated by blue light at 2.0 mW/cm2
for 2 h, (C) 1 mM ECPB was treated with 1 mM GA in 0.1 M PBS at pH 8.0 in the dark, and (D) 1 mM
ECPB was treated in the presence of 1 mM GA under blue light illumination at 2.0 mW/cm2 for 2 h.
The sample solution was filtered through a 0.45 µm filter (Millipore, Burlington, MA, USA) prior to
its use. In the beginning, the mobile phase comprised 0.1% formic acid as solvent (A) and acetonitrile as
solvent (B) with the gradient profile set as described previously [13]. The linear gradient started with 0–3
min, 1%–10% solvent B; 3–10 min, 10%–20% solvent B; 10–16.5 min, 20%–25% solvent B; and 16.6–20 min,
50%–100% solvent B; and returned to the original ratio at 23 min (i.e., 1% solvent B). Each sample of 10
µL was injected with a flow rate at 500 µL/min and the detection wavelength was set at 280 nm.
4.8. HPLC–DAD Analysis of EGCG Under Blue Light Illumination
EGCG and its relevant photoproducts were investigated in this study. In brief, (A) 1 mM EGCG
in 0.1 M PBS at pH 8.0 in the dark was used as a standard control solution, and (B) 1 mM EGCG in
0.1 M PBS was illuminated by blue light at 2.0 mW/cm2 for 2 h. Both the HPLC–DAD and ESI mass
spectral analyses of EGCG photolysis were examined as described in Section 4.7.
4.9. Statistics
The experiments were performed at least in triplicate. Data are expressed as the mean
±
standard
deviation of every single test. A one-way analysis of variance (ANOVA) was performed to identify
the significant differences in more than three groups. When statistically significant differences were
indicated, an unpaired Student’s t-test was used for further analysis. The difference of two groups of
measurements was considered statistically significant when p < 0.05.
5. Conclusions
Chromatographic and mass analyses were applied to investigate the effects of photolysis on
the conformational changes in epicatechin (EC) in an alkaline condition. EC is a non-gallate-type
catechin and is sensitive to blue light illumination. Blue light illumination can; therefore, be efficient
in terms of studying EC photosensitized oxidation. The epimerization of EC was achieved by
epi-configuration with the dimeric and trimeric compounds being formed from the electron transfer
of EC under blue light treatment in an alkaline condition. EGCG is a gallate-type catechin and is
formed via the esterification of non-gallate-type catechins and GA. Addition of EGCG can inhibit the
photosensitized redox reaction, such as ROS formation, and suppress the oxidation of EC caused by
blue light illumination.
Author Contributions: Formal analysis, S.-T.H. and Y.-A.H.; investigation, J.-Y.L.; resources, M.-J.Y. and I.-Z.C.;
supervision, S.-T.H. and J.-M.P.Y.; writing—original draft, J.-Y.L.; writing—review and editing, J.-M.P.Y. and J.-Y.L.

Molecules 2019, 24, 787
12 of 13
Funding: This research received no external funding.
Acknowledgments: The authors appreciate Chun-Yi Wu for his assistance in performing the experimental work.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Balasundram, N.; Sundram, K.; Samman, S. Phenolic compounds in plants and agri-industrial by-products:
Antioxidant activity, occurrence, and potential uses. Food Chem. 2006, 99, 191–203. [CrossRef]
Chen, L.Y.; Cheng, C.W.; Liang, J.Y. Effect of esterification condensation on the Folin-Ciocalteu method for
the quantitative measurement of total phenols. Food Chem. 2015, 170, 10–15. [CrossRef] [PubMed]
Chen, G.H.; Yang, C.Y.; Lee, S.J.; Wu, C.C.; Tzen, J.T. Catechin content and the degree of its galloylation in
oolong tea are inversely correlated with cultivation altitude. J. Food Drug Anal. 2014, 22, 303–309. [CrossRef]
Dube, A.; Ng, K.; Nicolazzo, J.A.; Larson, I. Effective use of reducing agents and nanoparticle encapsulation
in stabilizing catechins in alkaline solution. Food Chem. 2010, 122, 662–667. [CrossRef]
Janeiro, P.; Oliveira Brett, A.M. Catechin electrochemical oxidation mechanisms. Anal. Chim. Acta 2004, 518,
109–115. [CrossRef]
Mochizuki, M.; Yamazaki, S.; Kano, K.; Ikeda, T. Kinetic analysis and mechanistic aspects of autoxidation of
catechins. Biochim. Biophys. Acta 2002, 1569, 35–44. [CrossRef]
Chobot, V.; Huber, C.; Trettenhahn, G.; Hadacek, F. (+/ )-catechin: Chemical weapon, antioxidant, or stress
−
regulator? J. Chem. Ecol. 2009, 35, 980–996. [CrossRef] [PubMed]
Chen, L.Y.; Wu, J.Y.; Liang, J.Y. Using chromatography and mass spectrometry to monitor isomerization of
catechin in alkaline aqueous with thermal processing. J. Food Process. Preserv. 2018, 42, e13365. [CrossRef]
Shi, M.; Nie, Y.; Zheng, X.Q.; Lu, J.L.; Liang, Y.R.; Ye, J.H. Ultraviolet B (UVB) photosensitivities of tea
catechins and the relevant chemical conversions. Molecules 2016, 21, 1345. [CrossRef]
10. Forest, K.; Wan, P.; Preston, C.M. Catechin and hydroxybenzhydrols as models for the environmental
photochemistry of tannins and lignins. Photochem. Photobiol. Sci. 2004, 3, 463–472. [CrossRef]
11. Wilhelm-Mouton, A.; Bonnet, S.L.; Ding, Y.; Li, X.C.; Ferreira, D.; van der Westhuizen, J.H. Photochemistry
synthesis. Part 2: Enantiomerically pure polyhydroxy-1, 1, 3-triarylpropan-2-ols. J. Photochem. Photobiol.
A Chem. 2012, 227, 18–24. [CrossRef]
12. Liang, J.Y.; Wu, J.Y.; Yang, M.Y.; Hu, A.; Chen, L.Y. Photo-catalytic polymerization of catechin molecules in
alkaline aqueous. J. Photochem. Photobiol. B Biol. 2016, 165, 115–120. [CrossRef] [PubMed]
13. Yang, M.J.; Hung, Y.A.; Wong, T.W.; Lee, N.Y.; Yuann, J.P.; Huang, S.T.; Wu, C.Y.; Chen, I.Z.; Liang, J.Y.
Effects of blue-light-induced free radical formation from catechin hydrate on the inactivation of Acinetobacter
baumannii, including a carbapenem-resistant strain. Molecules 2018, 23, 1631. [CrossRef]
14. Shi, M.; Shi, Y.L.; Li, X.M.; Yang, R.; Cai, Z.Y.; Li, Q.S.; Ma, S.C.; Ye, J.H.; Lu, J.L.; Liang, Y.R.; et al. Food-grade
encapsulation systems for (−)-epigallocatechin gallate. Molecules 2018, 23, 445. [CrossRef] [PubMed]
15. Braicu, C.; Pilecki, V.; Balacescu, O.; Irimie, A.; Neagoe, I.B. The relationships between biological activities
and structure of flavan-3-ols. Int. J. Mol. Sci. 2011, 12, 9342–9353. [CrossRef]
16. Yuann, J.M.P.; Wang, J.S.; Jian, H.L.; Lin, C.C.; Liang, J.Y. Effects of Clinacanthus nutans (Burm. f) Lindau leaf
extracts on protection of plasmid DNA from riboflavin photoreaction. MC-Trans. Biotech. 2012, 4, 45–59.
17. Halliwell, B.; Gutteridge, J.M. Role of free radicals and catalytic metal ions in human disease: An overview.
Methods Enzymol. 1990, 186, 1–85.
18. Juen, J.W.; Jian, H.L.; Liang, J.Y. The effect of illuminance on light induced reduction of nitro blue tetrazolium.
MC-Trans. Biotech. 2010, 2, e2.
19. Liang, J.Y.; Yuann, J.M.; Cheng, C.W.; Jian, H.L.; Lin, C.C.; Chen, L.Y. Blue light induced free radicals from
riboflavin on E. coli DNA damage. J. Photochem. Photobiol. B Biol. 2013, 119, 60–64. [CrossRef]
20. Liang, J.Y.; Cheng, C.W.; Yu, C.H.; Chen, L.Y. Investigations of blue light-induced reactive oxygen species
from flavin mononucleotide on inactivation of E. coli. J. Photochem. Photobiol. B Biol. 2015, 143, 82–88.
[CrossRef]
21. Wong, T.W.; Cheng, C.W.; Hsieh, 0Z.J.; Liang, J.Y. Effects of blue or violet light on the inactivation of
Staphylococcus aureus by riboflavin-5 -phosphate photolysis. J. Photochem. Photobiol. B Biol. 2017, 173, 672–680.
[CrossRef] [PubMed]

Molecules 2019, 24, 787
13 of 13
22. Huang, S.T.; Wu, C.Y.; Lee, N.Y.; Cheng, C.W.; Yang, M.J.; Hung, Y.A.; Wong, T.W.; Liang, J.Y. Effects of
462 nm light-emitting diode on the inactivation of Escherichia coli and a multidrug-resistant by tetracycline
photoreaction. J. Clin. Med. 2018, 7, 278. [CrossRef] [PubMed]
23. Fourie, T.G.; Ferreira, D.; Roux, D.G. Flavonoid synthesis based on photolysis of flavan-3-ols,
3-hydroxyflavanones, and 2-benzylbenzofuranones. J. Chem. Soc. Perkin 1 1977, 125–133. [CrossRef]
24. Faria, J.; Steenken, S. Photoionization (lambda.= 248 or 308 nm) of triphenylmethyl radical in aqueous
solution. Formation of triphenylmethyl carbocation. J. Am. Chem. Soc. 1990, 112, 1277–1279. [CrossRef]
25. Dixon, R.A.; Xie, D.Y.; Sharma, S.B. Proanthocyanidins-a final frontier in flavonoid research? New Phytol.
2005, 165, 9–28. [CrossRef] [PubMed]
26. Jiang, X.; Liu, Y.; Wu, Y.; Tan, H.; Meng, F.; Wang, Y.S.; Li, M.; Zhao, L.; Liu, L.; Qian, Y.; et al. Analysis of
accumulation patterns and preliminary study on the condensation mechanism of proanthocyanidins in the
tea plant [Camellia sinensis]. Sci. Rep. 2015, 5, 8742. [CrossRef] [PubMed]
27. Ojwang, L.O.; Yang, L.; Dykes, L.; Awika, J. Proanthocyanidin profile of cowpea (Vigna unguiculata) reveals
catechin-O-glucoside as the dominant compound. Food Chem. 2013, 139, 35–43. [CrossRef]
28. Huvaere, K.; Sinnaeve, B.; Van Bocxlaer, J.; Skibsted, L.H. Flavonoid deactivation of excited state flavins:
Reaction monitoring by mass spectrometry. J. Agric. Food Chem. 2012, 60, 9261–9272. [CrossRef]
29. Burger, J.F.; Kolodziej, H.; Hemingway, R.W.; Steynberg, J.P.; Young, D.A.; Ferreira, D. Oligomeric
flavanoids. Part W. base-catalyzed pyran rearrangements of procyanidin B-2, and evidence for the oxidative
transformation of B-to A-type procyanidins. Tetrahedron 1990, 46, 5733–5740. [CrossRef]
30. He, F.; Pan, Q.H.; Shi, Y.; Duan, C.Q. Chemical synthesis of proanthocyanidins in vitro and their reactions in
aging wines. Molecules 2008, 13, 3007–3032. [CrossRef]
31. de Souza, L.M.; Cipriani, T.R.; Iacomini, M.; Gorin, P.A.; Sassaki, G.L. HPLC/ESI-MS and NMR analysis of
flavonoids and tannins in bioactive extract from leaves of Maytenus ilicifolia. J. Pharm. Biomed. Anal. 2008
47, 59–67. [CrossRef] [PubMed]
,
32. Zhang, M.; Vervoort, L.; Moalin, M.; Mommers, A.; Douny, C.; den Hartog, G.J.M.; Haenen, G. The chemical
reactivity of ( )-epicatechin quinone mainly resides in its B-ring. Free Radic. Biol. Med. 2018, 124, 31–39.
−
[CrossRef] [PubMed]
33. Dettlaff, K.; Stawny, M.; Ogrodowczyk, M.; Jelinska, A.; Bednarski, W.; Watrobska-Swietlikowska, D.;
Keck, R.W.; Khan, O.A.; Mostafa, I.H.; Jankun, J. Formulation and characterization of EGCG for the treatment
of superficial bladder cancer. Int. J. Mol. Med. 2017, 40, 329–336. [CrossRef] [PubMed]
34. Wyganowska-Swiatkowska, M.; Matthews-Kozanecka, M.; Matthews-Brzozowska, T.; Skrzypczak-Jankun, E.;
Jankun, J. Can EGCG alleviate symptoms of Down syndrome by altering proteolytic activity? Int. J. Mol. Sci.
2018, 19, 248. [CrossRef] [PubMed]
35. Naz, S.; Khaskheli, A.R.; Aljabour, A.; Kara, H.; Talpur, F.N.; Sherazi, S.T.H.; Khaskheli, A.A.; Jawaid, S.
Synthesis of highly stable cobalt nanomaterial using gallic acid and its application in catalysis. Adv. Chem.
2014. [CrossRef]
36. Badhani, B.; Sharma, N.; Kakkar, R. Gallic acid: A versatile antioxidant with promising therapeutic and
industrial applications. RSC Adv. 2015, 5, 27540–27557. [CrossRef]
37. Eslami, A.C.; Pasanphan, W.; Wagner, B.A.; Buettner, G.R. Free radicals produced by the oxidation of gallic
acid: An electron paramagnetic resonance study. Chem. Cent. J. 2010, 4, 15. [CrossRef]
38. Gunckel, S.; Santander, P.; Cordano, G.; Ferreira, J.; Munoz, S.; Nunez-Vergara, L.J.; Squella, J.A. Antioxidant
activity of gallates: An electrochemical study in aqueous media. Chem. Biol. Interact. 1998, 114, 45–59. [CrossRef]
39. Liu, H.; Zou, T.; Gao, J.M.; Gu, L. Depolymerization of cranberry procyanidins using (+)-catechin,
(
−
)-epicatechin, and ( )-epigallocatechin gallate as chain breakers. Food Chem. 2013, 141, 488–494. [CrossRef]
−
40. Cheng, C.W.; Chen, L.Y.; Chou, C.W.; Liang, J.Y. Investigations of riboflavin photolysis via coloured light in
the nitro blue tetrazolium assay for superoxide dismutase activity. J. Photochem. Photobiol. B Biol. 2015, 148,
262–267. [CrossRef]
Sample Availability: Samples of the compounds (epicatechin) are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).