Article

Cite This: J. Agric. Food Chem. 2020, 68, 340−350 pubs.acs.org/JAFC

Mechanism for Inhibition of Folic Acid Photodecomposition by

Various Antioxidants
Wusigale,†,‡ Lyulin Hu,†,‡ Hao Cheng,†,‡ Yahui Gao,‡ and Li Liang*,†,‡
†
State Key Laboratory of Food Science and Technology and ‡School of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu, China
*
S Supporting Information

ABSTRACT: Folic acid, a synthetic form of folate, is a water-soluble vitamin that is essential during periods of rapid cell
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division and growth. However, it decomposes upon ultraviolet irradiation to form inactive photoproducts. In this study, the
protective effect and mechanisms of antioxidants, including cinnamic acids, flavonoids, catechol and its derivatives, stilbenes, p-
benzoquinone and its derivatives, isoprenoids, curcumin, oleic acid, and linoleic acid, against folic acid photodecomposition
were investigated by using fluorescence and absorbance spectroscopy, high-performance liquid chromatography, and
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antioxidant assay. It was found that antioxidants could inhibit or delay the folic acid decomposition in varying degrees, among
which caffeic acid was the most effective. The increase in its remarkable antioxidant efficiency and absorbance in the UVA
region during irradiation contributed to its effective protection. This finding could be useful for the protection of photolabile
components in food and other uses.
KEYWORDS: folic acid, antioxidant, photodecomposition, inhibition

1. INTRODUCTION antioxidant, provided better protection against the photo-

Fruits and vegetables are rich in folates and represent >35% of
folates’ intake within the diet of adults in France.1 The generic
term folate is a water-soluble compound belonging to the B
group of vitamins. Folate is composed of a pterin, a p-
aminobenzoic acid, and a reduced mono- and polyglutamate
moiety. It is essential for one-carbon transfer reactions in
metabolism and biosynthesis of nucleic acids and proteins.
Folate deficiency reduces the division rate of all cells in the
body and results in megaloblastic anemia, neural tube defects,
cardiovascular diseases, and cancers.2 The term folic acid (FA),
which is composed of a 6-methylpterin residue, a p-
aminobenzoic acid residue, and a L-glutamic acid residue,
refers to the fully oxidized, more stable, easily absorbable, and
synthetic monoglutamate.2 Folic acid is usually used for food
fortification and in pharmaceutical preparations.
Folic acid is sensitive to light. Under ultraviolet (UV)
radiation, folic acid decomposed by cleavage on the C9−N10
bridge into 6-formylpterin (FPT) and p-aminobenzoylgluta-
mate (PGA); both underwent further alterations, such as the
formation of 6-carboxypterin (PCA) or cleavage of PGA
leading to p-aminobenzoic acid (PABA) and glutamic acid
(GA).3 The photodecomposition of folic acid has many effects
on physiological processes, including the oxidation of DNA in
mammalian cells and the color evolution of human skin.3 The
photodecomposition also reduced the content of folic acid in
juices stored in transparent bottles.4
The photostability of folic acid could be improved by
interacting with proteins5,6 or β-cyclodextrin7 to form
complexes or by encapsulation using biopolymers through
spray drying or the electrospraying technique.8 Moreover, the
presence of low-molecular-weight antioxidants, such as
ascorbic acid or catechins, results in only a limited extent of
folate loss in fruits and vegetables.9,10 Resveratrol, a phenolic
decomposition of folic acid than did β-lactoglobulin, a major
bovine milk whey protein.11 Transglycosylated rutin could also
inhibit the folic acid photodecomposition by its antioxidant
effect.12 Ascorbic acid, dithiothreitol (DTT), β-mercaptoetha-
nol, and 2,3-dimercaptoethanol have been used as antioxidants
to reduce the loss of folates during extraction and purification
from food products.9
Photodecomposition of folic acid is generally assumed to be
an oxidation, since folic acid is photostable in the absence of
oxygen in aqueous solution.2 Cinnamic acids, flavonoids,
stilbenes, curcumin, salicylic acid, guaiacol, p-benzoquinone
and its derivatives, and isoprenoids (Figure 1) show
antioxidant ability and are thus used for food preserva-
tion.10,13−16 Although many studies have investigated the
chemical identity and antioxidant ability of antioxidants, the
influence of photoinduced change in the antioxidants on their
protective effectiveness has not been evaluated yet.
The aim of this study was to investigate the protective effect
of these antioxidants against photodecomposition of folic acid
by using fluorescence spectroscopy and high-performance
liquid chromatography (HPLC). The protective mechanisms
were explored by absorbance spectroscopy and antioxidant
activity as determined by DPPH and ABTS assay. The
structure−activity relationship was also discussed systemati-
cally.
Received: October 5, 2019
Revised: November 26, 2019
Accepted: December 12, 2019
Published: December 12, 2019

© 2019 American Chemical Society 340 DOI: 10.1021/acs.jafc.9b06263

J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of cinnamic acids, catechol and derivatives, flavonoids, stilbenes, p-benzoquinone (p-BQ) and derivatives (5-
hydroxy-p-naphthoquinone (5-H-p-NQ) and kojic acid), curcumin, isoprenoids (β-ionone and retinol), oleic acid, and linoleic acid.
2. MATERIALS AND METHODS
Materials. Folic acid (purity ≈ 98%), p-aminobenzoylglutamate
(PGA, >98%), 6-carboxypterin (PCA, >98%), resveratrol (trans-
isomer, >99%), (−)-epigallocatechin-3-gallate (EGCG, >95%),
flavone (∼99%), quercetin (>95%), p-coumaric acid (>98%), caffeic
acid (>98%), ferulic acid (>99%), curcumin (>80%), retinol (>90%),
β-ionone (>96%), trans-stilbene (>96%), p-benzoquinone (p-BQ,
>98%), 5-hydroxy-p-naphthoquinone (5-H-p-NQ, >97%), kojic acid
(>99%), oleic acid (>99%), linoleic acid (>99%), and DPPH [2,2-
diphenyl-1-picrylhydrazyl] were purchased from Sigma-Aldrich Co.
(St. Louis, MO, USA). 6-Formylpterin (FPT) was obtained from
Schircks Laboratories (Jona, Switzerland). Salicylic acid (>98%),
acetylsalicylic acid (>98%), guaiacol (>98%), and catechol (>98%)
were obtained from Sangon Biotech Co., Ltd. (Shanghai, China).
ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammo-
nium salt)] was purchased from Aladdin Bio-Chem Technology Co.
Ltd. (Shanghai, China). Acetic acid and methanol of HPLC grade
were purchased from Fisher Scientific Co. (Shanghai, China). Other
reagents of analytical reagent grade were purchased from SinoPharm
CNCM Ltd. (Shanghai, China). Deionized water was produced by
using a Milli-Q water purification system (Millipore, Billerica, MA,
USA).
Sample Preparation. Stock solutions of folic acid and EGCG at a
concentration of 200 μM were prepared by dissolving in 10 mM
phosphate buffer at pH 7.4. A stock solution of trans-resveratrol at
200 μM was prepared by dissolving in 70% ethanol at 2 mM followed
by diluting with phosphate buffer. A stock solution of cis-resveratrol
341
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was prepared by irradiating the trans-isomer solution under UV light
(peak λ = 365 nm) for 60 min.17 Stock solutions of other antioxidants
(cinnamic acids, flavonoids, trans-stilbene, curcumin, catechol and its
derivatives, p-benzoquinone and its derivatives, isoprenoids, oleic acid,
and linoleic acid) at 200 μM were prepared by dissolving in absolute
ethanol at a concentration of 2 mM followed by diluting with
phosphate buffer. Mixtures of folic acid and antioxidants were
prepared by diluting folic acid stock solution with phosphate buffer
and then adding stock solutions of antioxidants into diluted folic acid
solutions in varying proportions. All samples were covered with
aluminum foil and incubated for about 1 h at room temperature
before measurement.
Irradiation Procedure. Samples in 1 cm quartz cuvettes were
exposed to UVA light (peak λ = 365 nm) using a ZF-7A ultraviolet
lamp (Guang Hao Analytical Instruments, Shanghai, China) with a
power of 4 W and were placed 9 cm away from the light source.
Samples were analyzed every 20 min for up to 240 min.
Fluorescence Measurement. Steady-state fluorescence was
recorded on a FluoroMax-4 fluorescence spectrophotometer (Horiba
Jobin Yvon Inc., Edison, NJ). Fluorescence emission spectra of folic
acid were measured at an excitation wavelength of 360 nm using a
quartz cell with a path length of 1 cm. Fluorescence intensity at an
emission maximum (λmax) of around 445 nm was recorded in the
absence and presence of antioxidants under UV irradiation. The
spectral resolution for both excitation and emission was 1.5 nm.
Backgrounds of antioxidants were subtracted from the raw spectra.
DOI: 10.1021/acs.jafc.9b06263
J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry Article

Fluorescence intensity was normalized relative to that of 10 μM folic 3. RESULTS AND DISCUSSION
acid after 240 min of UV irradiation at λmax.
High-Performance Liquid Chromatography. The contents of Effect of Antioxidants on Folic Acid Photodecompo-
folic acid and its photoproducts were determined using a Waters sition. Fluorescence. Fluorescence quantum yields of pterin
HPLC system (Waters, Milford, MA, USA) equipped with a 1525 and its derivatives are in the range from 0.07 to 0.33 in
Binary Pump, a 2489 UV/Visible detector, and a Waters Symmetry aqueous solutions at pH 5.5−10.5,20 while folic acid has a very
C18 column (4 μm, 250 × 4.6 mm). The column temperature was 25 low fluorescence quantum yield (<0.005) due to internal
°C. The injection volume was 10 μL. A mobile phase consisting of a
mixture of 40% (v/v) methanol and 60% (v/v) water containing
quenching.20 Fluorescence intensities of folic acid at λmax in the
0.17% acetic acid was developed. UV detection was used at 280 nm. absence and presence of antioxidants as a function of
The elution times of FPT, PGA, folic acid and PCA were 3.0, 4.5, 5.2, irradiation time are shown in Figure 2. When folic acid at 10
and 6.3 min, respectively. μM was exposed to UV radiation, the fluorescence intensity
Antioxidant Activity by DPPH Assay. DPPH assay was increased quickly until 100 min and then increased slowly,
measured according to the method of Brand-Williams et al.18 A reaching a maximum at 200 min, after which it kept constant.
stock solution of DPPH• at 60 μM was prepared in methanol and
This is attributed to the photodecomposition of folic acid to
stored at −20 °C for use. Samples were diluted with methanol to
various concentrations, 0.1 mL of which was then mixed with 3.9 mL
of 60 μM DPPH solution. The decrease in the absorbance at 515 nm
was recorded after 60 min of reaction by using an AOE UV−vis
spectrophotometer (AOE Instruments, Shanghai, China). The
concentration of DPPH• (CDPPH•) and DPPH• scavenging percentage
were calculated using eqs 1 (standard curve) and 2. EC50 values
(amount of antioxidants necessary to reduce 50% of DPPH•) were
calculated from the curve of scavenging percentage of DPPH• as a
function of the molar ratio of antioxidants and DPPH•. For the
compounds that could not scavenge 50% of the initial DPPH•,
samples at 100 mM reacted with 60 μM DPPH•, except for the
saturated solution trans-stilbene was used. DPPH• scavenging
percentage was calculated using eq 2

A515 − 0.0017
C DPPH (μ M) =
0.0105 (1)

C0 − Cs
DPPH ·scavenging percentage (%) = × 100
C0 (2)

where A515 is the absorbance at 515 nm and C0 and Cs are the

concentrations of DPPH• plus methanol and of DPPH• plus samples
after 60 min of reaction, respectively, which is calculated using eq 1.
Antioxidant Activity by ABTS Assay. ABTS assay of
antioxidants under irradiation was measured according to a previous
method.19 Briefly, ABTS•+ solution was prepared by reacting 7.4 mM
ABTS and 2.6 mM K2S2O8 in equal amounts and kept in the dark for
12 h. The ABTS•+ solution was further diluted 50 times with 10 mM
phosphate buffer at pH 7.4. The absorbance at 729 nm was recorded
for 6 min after mixing 0.5 mL of the diluted ABTS•+ solution with 0.3
mL of buffer or samples using an AOE UV−vis spectrophotometer
(AOE Instruments, Shanghai, China). An initial concentration of 10
or 500 μM was tested for compounds that are active free radical
scavengers or therelatively inactive compounds, respectively. ABTS•+
scavenging capacity was calculated using eq 3

A 0 − As
ABTS•+ scavenging capacity, % = × 100
As (3)

where A0 and As are the absorbance of ABTS radical plus buffer and of
ABTS radical plus samples, respectively.
Absorbance Measurement. Absorbance spectra were measured
with a path length of 1 cm on a CARY 50 UV−vis spectrophotometer
(Agilent Technologies, Santa Clara, CA, USA). Samples of
antioxidants were diluted with 10 mM phosphate buffer at pH 7.4
to an absorbance between 0.2 and 1.0 at the absorption peak. The
spectra of antioxidants were recorded from 210 to 590 nm.
Statistical Analysis. All of the experiments were conducted in
triplicate with data reported as the mean ± standard deviation. The
mean values were compared by one-way analysis of variance
(ANOVA) using Duncan’s multiple-range test to determine Figure 2. Fluorescence intensities of 10 μM folic acid at λmax in the
significant differences (P < 0.05). Pearson’s correlation test was absence and presence of 1 (A), 0.1 (B), and 10 (C) μM antioxidants
performed using the SPSS 20.0 package (IBM, Armonk, NY, USA). as a function of irradiation time.

342 DOI: 10.1021/acs.jafc.9b06263

J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry
yield PGA and FPT, followed by the transformation of FPT to
PCA (Scheme 1).2 Addition of 1 μM antioxidants to 10 μM
Scheme 1. Protective Mechanism of Antioxidants against
the Photodecomposition of Folic Acida
a
Notes: FA, folic acid; 3FA*, triplet excited state of folic acid; FA•−,
folic acid radical; ISC, intersystem crossing; AH, antioxidants; AH•+,
antioxidants radical; AH-ox, oxidized products of antioxidants.
folic acid resulted in the broad categorization into three
classes: (a) caffeic acid, ferulic acid, p-coumaric acid, quercetin,
EGCG, curcumin, trans- and cis-resveratrol, catechol, and p-BQ
could completely inhibit the decomposition of folic acid; (b)
guaiacol could significantly delay the decomposition of folic
acid; (c) 5-H-p-NQ, kojic acid, salicylic acid, retinol, β-ionone,
acetylsalicylic acid, trans-stilbene, flavone, cinnamic acid,
linoleic acid, and oleic acid slightly slowed down or had no
influence on the decomposition of folic acid (Figure 2A). The
concentration of a-group antioxidants was reduced to 0.1 μM
(Figure 2B), and the concentration of b- and c-group
antioxidants was increased to 10 μM (Figure 2C) to
differentiate their protective effect.
Cinnamic acid at 10 M had no influence on the
photodecomposition of folic acid (Figure 2C). Hydroxycin-
namic acids at 0.1 μM could significantly delay the vitamin
decomposition, which ranked in order caffeic acid > ferulic
acid ≈ p-coumaric acid (Figure 2B). The protective effect of
cinnamic acid and hydroxycinnamic acids was positively
correlated with the number of phenolic hydroxyl groups.
Similar results with hydroxycinnamic acids were previously
reported for inhibition of the oxidation of human low-density
lipoprotein, oils, and biodiesels.21−23 Catechol at 0.1 μM could
also significantly delay the vitamin decomposition, and its
protective effect was slightly less than that of caffeic acid
(Figure 2B). Guaiacol at 10 μM could inhibit the
decomposition of folic acid, salicylic acid significantly delayed
the vitamin decomposition, but acetylsalicylic acid had no
influence on the decomposition (Figure 2C). The protection
of catechol and its derivatives was also positively correlated
with the number of phenolic hydroxyl groups, but their
effectiveness was weaker than that of cinnamic acid derivatives
with the same number of phenolic hydroxyl groups.
Curcumin has a heptadienone linkage between the two
methoxyphenol rings.14 Its protective effect on folic acid was
weaker than that of 0.1 μM ferulic acid (Figure 2B), which is
one of the degradation products of curcumin. However,
curcumin and its degradation products (such as ferulic acid
and vanillin) had a similar beneficial effect against oxidative
stress and a reduced risk of the incidence of related diseases.24
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trans-Stilbene at 10 μM delayed the decomposition of folic
acid slightly (Figure 2C), while both trans- and cis-resveratrol,
which contain three phenolic hydroxyl groups at the 3-, 5-, and
4′-positions of stilbene, at 0.1 μM showed a similar protective
effect to curcumin (Figure 2B). Flavone at 10 μM had no
influence on the decomposition of folic acid (Figure 2C), while
quercetin containing five phenolic hydroxyl groups showed a
better protective effect on folic acid than it did by EGCG
containing eight phenolic hydroxyl groups (Figure 2B).
Quercetin reportedly exhibited a higher efficiency on inhibition
of low-density lipoprotein oxidation than EGCG25 but a
similar effect on the inhibition of DNA damage with EGCG.26
p-Benzoquinone at 0.1 μM could delay the decomposition of
folic acid, with the protective effect being weaker than that of
curcumin (Figure 2B), while 5-H-p-NQ showed a slightly
weaker protective effect than did kojic acid at 10 μM (Figure
2C). Moreover, oleic acid at 10 μM had no influence on the
decomposition of folic acid, while linoleic acid showed weak
protection comparable to trans-stilbene (Figure 2C). At 10
μM, β-ionone with the isoprene-conjugated double-bond
structure provided a stronger protective effect than did retinol,
which contains five conjugated double bonds and differs from
β-ionone by the length of the isoprenoid chain, and their
protective effect was greater than that of linoleic acid.
From fluorescence analysis, the protective effect of various
antioxidants against photodecomposition of folic acid ranked
in order of effectiveness caffeic acid > catechol > quercetin >
ferulic acid ≈ p-coumaric acid > EGCG > resveratrol >
curcumin > p-benzoquinone > guaiacol > kojic acid > 5-H-p-
NQ > salicylic acid > β-ionone > retinol > linoleic acid > trans-
stilbene. Since both FPT and PCA could emit fluorescence
when exited at 360 nm, the photoprotective effect on folic acid
was further validated by analyzing the content of folic acid and
its photodecomposition products using HPLC.
HPLC. When folic acid alone was exposed to UV radiation,
its content decreased quickly until 100 min and loss was
complete after 200 min (Figure 3A and 3B), consistent with
fluorescence results (Figure 2). The content of PGA increased
rapidly to 4.0 μM until 100 min and then decreased to a value
of about 3.2 μM after 240 min (Figure 3C and 3D), possibly
due to the degradation of PGA to yield PABA and GA
(Scheme 1).3 The content of FPT increased rapidly to about
2.3 μM after 100 min and then began to decrease (Figure 3E
and 3F), due to the transformation of FPT to PCA,2 and the
loss was complete after 200 min. The content of PCA
increased to about 3.9 μM after 160 min and then basically
kept constant (P = 0.356, Figure 3G and H). The
decomposition of folic acid into PGA and FPT (Figure 3A,
3C, and 3E) and the transformation from FPT to PCA (Figure
3G) was delayed in the presence of the antioxidants at 0.1 μM,
with the effectiveness ranked in the order caffeic acid >
catechol > quercetin > p-coumaric acid > trans-resveratrol ≈
cis-resveratrol. The protective effect was consistent with that
obtained using fluorescence (Figure 2B). Isomerization of
resveratrol was of negligible importance for inhibition of folic
acid photodecomposition and the transformation from FPT
and PCA (Figure 3A, 3C, 3E, and 3G). The maximal content
of FPT (Figure 3E) was greater in the presence of trans- and
cis-resveratrol than in their absence, suggesting that resveratrol
could also inhibit the transformation of FPT to PCA. Both β-
ionone and retinol at 10 μM delayed the photodecomposition
of folic acid and the transformation from FPT to PCA with β-
ionone being a better protector (Figure 3B, 3D, 3F, and 3H),
DOI: 10.1021/acs.jafc.9b06263
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Figure 3. Concentration of folic acid (A and B), p-aminobenzoylglutamate (PGA, C and D), 6-formylpterin (FPT, E and F), and 6-carboxypterin
(PCA, G and H) under UV irradiation in the absence and presence of antioxidants at 0.1 (A, C, E, G) and 10 μM (B, D, F, H).

consistent with fluorescence results (Figure 2C). However,
trans-stilbene and flavone had a negligible effect on folic acid
photodecomposition (Figure 3D). The transformation from
FPT to PCA was delayed in the presence of trans-stilbene and
flavone (Figure 3F and 3H), possibly contributing to the
delayed increase in the fluorescence intensity in Figure 2C.
344
Antioxidant Activity. The photodecomposition of folic
acid was a complex process including the initial excitation of
the pterin ring, the formation of the corresponding radical ions
through an electron transfer (Type I) rather than a 1O2-
mediated reaction from p-aminobenzoic acid residue to the
excited triplet pterin, and the trapping of folic acid cation
DOI: 10.1021/acs.jafc.9b06263
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radical by O2, since folic acid behaves as a poor 1O2 sensitizer consistent with their protective effect on folic acid. Overall, the
with a quantum yield of 1O2 production of <0.02.2,27 The protective effect and antioxidant activity of phenolic com-
degree of folic acid photodecomposition is influenced by pounds were higher than those of the compounds with olefin
antioxidant levels.9 The activity of antioxidants during double bonds, mainly due to the fact that (1) the phenolic
irradiation was assessed using DPPH and ABTS assays having hydroxyl groups are able to reduce free radicals through one-
electron-transfer and hydrogen-atom-transfer mechanisms.28 electron donation and (2) the aromatic structures allow
DPPH• Scavenging Ability. The smaller the EC50, the more stabilization by resonance of the resultant aroxyl radicals.32
efficient the antioxidant is. The DPPH• scavenging capacity of Two o-methoxyphenolic groups and the conjugated
cinnamic acids ranked in the order of caffeic acid ≈ ferulic acid heptadienone chain contribute to antioxidant activity of
> p-coumaric acid > cinnamic acid and of catechol, and its curcumin.14 Curcumin had comparable DPPH• scavenging
derivatives ranked catechol > guaiacol > salicylic acid > ability to ferulic acid (Table 1), but its protective effect on folic
acetylsalicylic acid (Table 1). Similar results were observed by acid was on the contrary weaker than that of ferulic acid
(Figure 2). Three hydroxyl groups in the B ring and the
Table 1. EC50 (μM/μM DPPH•) Values or DPPH• conjugation of the gallic acid portion contribute to the
Scavenging Percentage (%) of Antioxidantsa antioxidant activity of EGCG, while an o-dihydroxyl group in
the B ring and the double bond in the C ring in conjugation
EC50 DPPH• scavenging percentage
antioxidant (μM/μM DPPH•) (%)b with the 4-oxo group contribute to the antioxidant activity of
quercetin.30 Their DPPH• scavenging ability was similar
caffeic acid 0.151 ± 0.003ab
ferulic acid 0.443 ± 0.005bc
(Table 1), but quercetin showed a better protective effect on
p-coumaric acid 114.465 ± 4.266g
folic acid than it did by EGCG (Figure 2). The conjugated
cinnamic acid 1.78 ± 0.16a
double bond is also responsible for scavenging active radicals.30
EGCG 0.047 ± 0.001a β-Ionone could scavenge DPPH radical (Table 1) owing to an
quercetin 0.104 ± 0.002ab
isoprene-conjugated double-bond structure.15 The DPPH•
flavone 4.72 ± 0.38b
scavenging capacity and protective effective on folic acid of
catechol 0.113 ± 0.001ab β-ionone and retinol was opposite. These results suggest that
guaiacol 0.602 ± 0.000c
more factors than the antioxidant activity of the compounds
salicylic acid 10.16 ± 0.10c
affect their protective effect against folic acid photodecompo-
acetylsalicylic 8.42 ± 0.16d sition.
acid ABTS•+ Scavenging Ability. The ABTS•+ scavenging ability
curcumin 0.272 ± 0.009abc of the antioxidants ranked in order ferulic acid ≈ quercetin ≈
p-benzoquinone 4.898 ± 0.243e EGCG > p-coumaric acid ≈ resveratrol > curcumin > kojic
5-H-p-NQ 7.847 ± 0.211f acid > guaiacol ∼ caffeic acid > catechol > p-benzoquinone >
kojic acid 49.40 ± 0.26e 5-H-p-NQ > salicylic acid > retinol > linoleic acid >
resveratrol 0.529 ± 0.003c
acetylsalicylic acid ≈ flavone ≈ trans-stilbene ≈ β-ionone >
trans-stilbene 0.00 ± 0.00 cinnamic acid ≈ oleic acid (Table 2). This finding was in line
retinol 3.233 ± 0.241d with those reported for EGCG, quercetin, and ferulic, caffeic,
β-ionone 879.050 ± 11.038h and p-coumaric acids by Apak et al. (2007)33 and also for
linoleic acid 5.11 ± 0.24f curcumin and cinnamic acid reported by Lee, Oh, Cho, and
oleic acid 1.22 ± 0.08g Ma (2015).34 The radical scavenging ability of caffeic acid,
a
Notes: Values with the same letter were not significantly different (P ferulic acid, and p-coumaric acid, of catechol and guaiacol, of p-
< 0.05) in each column. bScavenging percentage of 60 μM DPPH• at benzoquinone and kojic acid, and of retinol and β-ionone
100 mM, except for saturated solution of trans-stilbene. (Table 2) was not consistent with their protection on folic acid
(Figures 2 and 3). These results support that their protective
Brand-Williams et al. (1995) for caffeic acid, ferulic aicd, p- effect on folic acid is dependent on more factors than the
coumaric acid, and guaiacol and by Ordoudi et al. (2006) for antioxidant activity of the compounds themselves.
caffeic acid, catechol, and guaiacol.18,29 Caffeic acid and The ABTS•+ scavenging ability of antioxidants was further
catechol with an o-dihydroxy phenolic structure showed a high investigated under irradiation. The ABTS•+ scavenging ability
antioxidant ability (Table 1), partially due to the hydrogen of EGCG, quercetin, p-coumaric acid, resveratrol, curcumin,
bonds between hydroxyl groups next to each other to stabilize guaiacol, and catechol at 10 μM and 5-H-p-NQ, retinol,
the aryloxyl radical.16,18 Methoxy substitution in the ortho cinnamic acid, flavone, trans-stilbene, linoleic acid, and oleic
position to the phenolic group increased the antioxidant acid at 500 μM remained constant during irradiation (Table 2
activity of monophenols substantially,30 such as ferulic acid and and Figure S1). Different from our findings, Song and others
guaiacol (Table 1). Their DPPH• scavenging abilities (Table (2016) reported that the ABTS scavenging ability of curcumin,
1) were consistent with the protective effectiveness against resveratrol, quercetin, and p-coumaric acid significantly
folic acid’s decomposition (Figure 2 and 3). The ability of decreased upon photosensitization by riboflavin.35 It has
resveratrol to scavenge DPPH• and to protect folic acid was been reported that both quercetin and EGCG retained
also consistent. It is known that radicals interact with quinones constant DPPH radical-scavenging activity before and after
to form stabilized radicals,31 allowing some quinones to be heating, even though their content significantly decreased.36
inhibitors in free radical chain reactions. The DPPH • Quercetin, catechin, and their related flavonoids also largely
scavenging capacity of p-benzoquinone was higher than that conserved the oxygen radical absorbance capacity despite
of 5-H-p-NQ and kojic acid (Table 1), in agreement with their undergoing alkli or enzymatic oxidation.37 Resveratrol retained
protective effect on folic acid (Figure 2). The DPPH• antioxidant activity during the phototransformation proc-
scavenging capacity of linoleic acid and oleic acid was also ess.17,38 However, there was a decrease from about 98% to
345 DOI: 10.1021/acs.jafc.9b06263
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Journal of Agricultural and Food Chemistry Article

Table 2. ABTS•+ Scavenging Capacities of Antioxidants at an Initial Concentration of 10 μM and 500 μM before or after UV
Irradiation for 240 mina
ABTS•+ scavenging at 10 μM (%) ABTS•+ scavenging at 500 μM (%)
antioxidant before irradiation after irradiation before irradiation after irradiation
caffeic acid 41.06 ± 0.89fA 99.26 ± 0.08aB
ferulic acid 98.45 ± 0.76aA 89.82 ± 1.62bB
p-coumaric acid 85.49 ± 4.74bA 83.99 ± 2.70cA
cinnamic acid 1.73 ± 0.56fA 4.37 ± 1.73gA
EGCG 98.32 ± 1.12aA 97.36 ± 0.18aA
quercetin 99.37 ± 0.36aA 98.26 ± 1.49aA
flavone 4.96 ± 0.67efA 5.11 ± 3.50gA
catechol 22.07 ± 0.91gA 21.20 ± 0.97hA
guaiacol 42.16 ± 2.26fA 48.19 ± 4.43eA
salicylic acid 74.71 ± 0.78bA 68.74 ± 0.29cB
acetylsalicylic acid 8.29 ± 1.96eA 13.59 ± 0.07fB
curcumin 76.53 ± 1.17dA 75.95 ± 2.66dA
p-benzoquinone 11.20 ± 2.94hA 37.69 ± 9.06fB
5-H-p-NQ 89.21 ± 2.86aA 90.99 ± 0.90bA
kojic acid 71.20 ± 2.90 eA
31.75 ± 1.62 gB

resveratrol 80.55 ± 0.50cA 79.44 ± 0.17cdA

trans-stilbene 8.43 ± 7.27eA 1.73 ± 0.53ghA
retinol 33.98 ± 2.14cA 30.80 ± 3.53dA
β-ionone 4.82 ± 5.29efA 94.89 ± 2.69aB
linoleic acid 22.49 ± 0.73dA 21.45 ± 0.93eA
oleic acid 0.09 ± 0.16fA −0.53 ± 1.75hA
a
Note: Values with the same superscript letter (upper case for same row, lower case for same column) were not significantly different (P < 0.05).

Table 3. Pearson Correlation Coefficient between Fluorescence Intensity of Folic Acid in the Presence of Antioxidants at 180
min of Irradiation and ABTS•+ Scavenging Capacities of Antioxidants before and after Irradiation for 240 min
fluorescence at 0.1 μM vs ABTS at 10 μM fluorescence at 0.1 μM vs ABTS at 10 μM fluorescence at 10 μM vs ABTS at 500 μM (n
(n = 8)a (n = 9)b = 10)
parameter before irradiation after irradiation before irradiation after irradiation before irradiation after irradiation
r −0.096 −0.721c 0.168 −0.076 −0.876d −0.894d
P 0.821 0.044 0.665 0.845 0.001 0.000
a
Catechol, guaiacol, and kojic acid were not included. bGuaiacol and kojic acid were not included since their concentration was 10 μM for
fluorescence in Figure 2C. cCorrelation is significant at P < 0.05. dCorrelation is significant at P < 0.01.

90% for ferulic acid at 10 μM during irradiation (Table 2 and
Figure S1). The ABTS•+ scavenging ability of 10 μM kojic acid
declined linearly from about 71% to 32% as the irradiation
progressed (Table 2 and Figure S1). The ABTS•+ scavenging
ability of 500 μM salicylic acid slightly decreased from 75% to
69% (Table 2 and Figure S1). It was interesting to note a
significant increase in the scavenging ability of 10 μM caffeic
acid, 10 μM p-BQ, 500 μM acetylsalicylic acid, and 500 μM β-
ionone during UV irradiation, presumably due to the higher
radical scavenging ability of their photoproducts.39 Esculetin
with a mass-to-charge ratio of 177 was identified as a
photoproduct of caffeic acid using ESI(−)-LC-MS (Figure
S2). The content of esculetin after 240 min irradiation
increased to 18% (Figure S3) due to the photoisomerization of
caffeic acid followed by a cyclization to form esculetin.40
Esculetin has been reported to possess higher antioxidant
activity relative to caffeic acid.41 After irradiation for 240 min,
the ABTS•+ scavenging ability of the antioxidants ranked in
order caffeic acid ≈ quercetin ≈ EGCG > ferulic acid > p-
coumaric acid > resveratrol > curcumin > guaiacol > p-
benzoquinone > kojic acid > catechol > β-ionone > 5-H-p-NQ
> salicylic acid > retinol > linoleic acid > acetylsalicylic acid >
cinnamic acid ≈ flavone ≈ trans-stilbene > oleic acid. The
after-irradiation scavenging ability showed a significant
346
negative correlation with fluorescence intensity (r = −0.721,
P = 0.044, Table 3), suggesting that the ability was basically
consistent with their protective effect on folic acid. In the case
of exceptional catechol and guaiacol, their DPPH• scavenging
capacity was in agreement with their protective effect on folic
acid.
Absorption Spectra of Antioxidants during UV Irradi-
ation. Photolabile agents can be protected from light using
light absorbers by partially or wholly absorbing visible or UV
light.42 UVA light has higher transmittance through colorless
glass and plastic bottles than UVB light. The absorbance in the
UVA region of the antioxidants is very important for their
effective protection. The UV−vis absorbance evolution of
antioxidants during irradiation is depicted in Figure 4. Caffeic
acid displayed broad-band absorption spanning the UVA and
UVB regions. When caffeic acid was exposed to UV radiation, a
significant decrease in the absorbance in the UVB region along
with a new absorption band around 370 nm appeared. The
new absorption might be attributed to its photoproduct
esculetin.40 In the case of ferulic acid, only a decrease in the
absorbance of the UVB region was observed (Figure 4).
Ferulic acid had similar or even stronger antioxidant activity
(Tables 1 and 2), but caffeic acid had a better photostabilizing
effect (Figure 2). Therefore, it is suggested that the increasing
DOI: 10.1021/acs.jafc.9b06263
J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry Article

Figure 4. Absorption spectra of hydroxycinnamic acids (caffeic acid and ferulic acid), flavonoids [(−)-epigallocatechin-3-gallate (EGCG) and
quercetin], curcumin (C), and p-benzoquinone (p-BQ) and its derivative [5-hydroxy-p-naphthoquinone (5-H-p-NQ)] under UV irradiation.

absorbance in the UVA region under irradiation (Figure 4) is
also a factor for caffeic acid to be the most efficient
photoprotector (Figure 2).
Most flavonoids absorb UV radiation with the B ring in the
300−400 nm range and the A ring in the 240−285 nm range.43
UVA filtration by flavonoids has been proposed as a protective
347
mechanism in Rosa and Fuchsia plants.44 Of the flavonoids
tested, quercetin showed stronger and broader absorbance in
the UVA region than EGCG, even though a decrease in the
absorbance was observed for quercetin upon UV exposure
(Figure 4). Quercetin had similar antioxidant activity to EGCG
(Tables 1 and 2) but showed more effective protection on folic
DOI: 10.1021/acs.jafc.9b06263
J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry
acid than it did by EGCG (Figure 2). This confirms that one
plausible photoprotective mechanism would be direct
absorption of UV radiation by quercetin.
Curcumin had a strong and broad absorption in UV and
visible light region with a sharp peak at 263 nm, a broad peak
at 425 nm, and a shoulder around 365 nm (Figure 4). Under
UV irradiation, a decline in absorbance around 425 nm and a
relatively slower decrease in absorbance around 365 nm were
observed. It has been reported that a photosensitive drug,
nifedipine, was well protected by curcumin, since the
absorption spectrum of nifedipine in the long-wavelength
region could be almost overlapped by curcumin.45 Curcumin
had a stronger and broader absorption in the UV region
(Figure 4) but less ABTS•+ scavenging ability (Table 2) than it
did by ferulic acid. Moreover, curcumin is reported to be an
active photosensitizer, while ferulic acid does not show
photosensitizing activity.46 Therefore, the protective effect of
curcumin on folic acid was weaker than that of ferulic acid
(Figure 1).
The absorption spectrum of p-benzoquinone showed a peak
at 245 nm (Figure 4). Under UV exposure, the 245 nm
absorbance decreased with a slight red shift, while a broad
band around 290 nm increased, mainly because of the
formation of hydroquinone as a photoproduct.47 5-H-p-NQ
had a broad absorption in the UV and visible region centered,
respectively, at 252 and 425 nm and a shoulder around 340 nm
(Figure 4). There was no significant change in the absorbance
when irradiated with UV light, in agreement with the
unchanged radical scavenging ability (Table 2). As the
absorbance spectrum of 5-H-p-NQ could overlap that of folic
acid, 5-H-p-NQ could act as an effective photoprotector.
Mechanism. A high decrease in folic acid concentration
during UV irradiation by cleavage of the C9−N10 bridge into
FPT and PGA and then further formation of PCA and cleavage
of PGA to PABA and GA via an electron-transfer reaction were
found (Scheme 1).2 The photodecomposition is complete after
200 min (Figure 3). Following UV light absorption, folic acid
reaches its singlet excited state (1FA*) and then converts into
the triplet state (3FA*) by intersystem crossing (ISC).
Chemical quenching of singlet-excited compounds is consid-
ered to be less relevant due to the short lifetime (∼5 ns) and
highly efficient intersystem crossing to the longer-lived triplet
state.43 Usually a quencher with a concentration of higher than
30 mM, which is unacceptably high, would be required to
prevent intersystem crossing.43 3FA* could be deactivated
through three reaction pathways: intersystem crossing to the
singlet ground state, energy transfer to molecular oxygen
leading to the formation of 1O2, and electron-transfer reaction
from biomolecules to the triplet-excited molecule to form the
corresponding radicals. As reported, at higher oxygen
concentrations, the reaction shifts to 1O2 formation (type-II
reaction).43 However, in aqueous solutions, oxygen is less
soluble and the rate of the electron-transfer reaction from
biomolecules to triplet-excited molecule to form the
corresponding radicals that subsequently react with molecular
oxygen to generate oxidized products is significant.48−50
Therefore, 3FA* can react with antioxidants (AH) through
an electron-transfer (Type-I) reaction, forming antioxidants
radical (AH•+) and folic acid radical (FA•−) that subsequently
react with molecular oxygen to generate oxidized products and
reactive oxygen species (Scheme 1).51
Several antioxidants interact directly with light to protect
against photoinduced damage. Flavonoids such as quercetin
348
Article
are able to convert absorbed photon energy into heat (Scheme
1). After further exposure, photodecomposition of quercetin
may occur with formation of 2,4,6-trihydroxybenzoic acid and
3,4-dihydroxybenzoic acid.52 In the case of epigallocatechin,
the gallo group converts into the quinoid structure under UV
irradiation.53 Most of the previous reports and our study
confirmed that UVA radiation induces the photoisomerization
of trans-caffeic acid to cis-caffeic acid which then undergoes a
cyclization to form the esculetin (Figures S2 and S3).40 UVA
irradiation causes the photoisomerization of trans-resveratrol
to cis-resveratrol.17 Curcumin upon UV irradiation produces
vanillin and ferulic acid.54 It is worth mentioning that the
above-mentioned antioxidants could retain their antioxidant
abilities despite undergoing photodecomposition (Table 2).
Therefore, it is indicated that the photoproducts of
antioxidants also play an important role in protecting against
the photodecomposition of folic acid (Scheme 1). Further-
more, it has been made clear that the photoantioxidant ability
is dependent on the maximum absorption wavelength (λmax)
rather than the extinction coefficient (ε), for example, caffeic
acid vs ferulic acid and quercetin vs EGCG (Figure 4), because
the photoinduced reaction speeds up with an increase in the
wavelength.55
In conclusion, the photostability of folic acid increased by
supplementation with antioxidants. The effective protectors
were hydroxycinnamic acids (caffeic acid, ferulic acid, p-
coumaric acid), flavonoids (quercetin, EGCG), curcumin,
resveratrol, and p-benzoquinone, among which caffeic acid was
the most effective. The protective effect and antioxidant
activity of phenolic compounds were higher than those of the
compounds with olefin double bonds. The protective effect of
antioxidants on folic acid was more dependent on their activity
after irradiation than the initial activity. The absorbance in the
UVA region of antioxidants under UVA irradiation also
contributed to effective protection. These findings should be
useful for the protection of food and beverages against
undesired effects of light exposure to prevent premature quality
loss and for the coencapsulation of folic acid with these
effective antioxidants as an effective way to provide protection.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.9b06263.
ABTS•+ scavenging capacities of compounds at an initial
concentration of 10 and 500 μM under UV irradiation;
negative-ion mass spectrum of photoproduct of caffeic
acid after 240 min of irradiation; contents of trans-caffeic
acid, cis-caffeic acid, and esculetin as a function of
irradiation time (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +86(510)8519-7367; E-mail: liliang@jiangnan.edu.
cn.
ORCID
Li Liang: 0000-0001-9584-6778
Notes
The authors declare no competing financial interest.
DOI: 10.1021/acs.jafc.9b06263
J. Agric. Food Chem. 2020, 68, 340−350
Journal of Agricultural and Food Chemistry
■ ACKNOWLEDGMENTS
This work received support from the National Natural Science
Foundation of China (NSFC Project 31571781), the
Fundamental Research Funds for the Central Universities
(JUSRP51711B), and the 2018 Postgraduate Research &
Practice Innovation Program of Jiangsu Province
(KYCX18_1768).
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350 DOI: 10.1021/acs.jafc.9b06263

J. Agric. Food Chem. 2020, 68, 340−350