Received: 26 May 2021 | Revised: 22 September 2021 | Accepted: 10 October 2021

DOI: 10.1111/jfbc.13995

ORIGINAL ARTICLE

Effect of ascorbic acid on tyrosinase and its anti-­browning

activity in fresh-­cut Fuji apple

Yi-­Ting Wen | Yu-­Qin Liang | Wei-­Ming Chai | Qi-­Ming Wei | Zi-­Yi Yu |

Lin-­Jun Wang

College of Life Science and Key Laboratory

of Functional Small Organic Molecule, Abstract
Ministry of Education, Jiangxi Normal Tyrosinase (polyphenol oxidase) is the key enzyme of enzymatic browning in fruits
University, Nanchang, China
and vegetables. In this research, the impact of ascorbic acid on tyrosinase and its anti-­
Correspondence browning effect on fresh-­cut Fuji apple were investigated. Ascorbic acid had a dual
Wei-­Ming Chai, College of Life Science and
Key Laboratory of Functional Small Organic effect on tyrosinase with a half inhibitory concentration (IC50) of 13.40 ± 0.05 µM.
Molecule, Ministry of Education, Jiangxi Fluorescence assay demonstrated that ascorbic acid interacted with tyrosinase in
Normal University, Nanchang 330022,
China. a dynamic contaction caused by Förster’s resonance energy transfer (FRET) and
Email: [email protected] induced a conformational change of the enzyme. Thermodynamic analysis, copper
Funding information interaction, and molecular docking further confirmed that ascorbic acid could che-
Natural Science Foundation of Jiangxi late the copper ions located in active center and interact with amino acid residues
province, China, Grant/Award Number:
20192ACBL21028; Graduate Innovation of tyrosinase via hydrophobic interaction. In addition, ascorbic acid prevented the
Fund Project of Jiangxi Normal University, browning of fresh-­cut apples by increasing APX activity and inhibiting PPO and POD
Grant/Award Number: YC2020-­S182;
National Natural Science Foundation of activities which reduce the oxidation of total phenolics and flavonoids.
China, Grant/Award Number: 3216160030
Practical applications
The present study demonstrated that ascorbic acid had a strong inhibitory activity
against tyrosinase (IC50 = 13.40 ± 0.05 µM) and anti-­browning activity against fresh-­
cut Fuji apple. It could delay the browning degree of apple juice, increase APX activ-
ity, inhibit PPO and POD activities, and reduce the oxidation of total phenolics and
flavonoids. These findings provided a basis for the feasible application of ascorbic
acid on the preservation of fruits.

KEYWORDS

anti-­browning, ascorbic acid, fluorescence quenching, inhibition mechanism, molecular

docking, tyrosinase

1 | I NTRO D U C TI O N the enzymatic browning during apples processing reduces their eating
qualities (Bajwa et al., 2015). Hence, it is a top priority to develop safe and
Fuji apple (Malus pumila Mil), with big fruit, good quality and flavor, has feasible methods to control the enzymatic browning. Several physical
expanded around the world (Argentaa et al., 2020). China is the largest and chemical methods including food coating, ultrasonication, high pres-
apple-­producing country in the world. In 2014, Chinese apple produc- sure carbon dioxide processing have been reported for controlling enzy-
tion was 40.6 million tons, which accounted for 48% of the world’s total matic browning and increasing the shelf life of fresh-­cut apples (Ayesha
apple production (Li et al., 2020). Meanwhile, their processed products, et al., 2019; Caroline et al., 2007; Jafari et al., 2018; Xu et al., 2011).
fresh-­cut apple pieces, are popular in food service establishments, with Gil et al. (1998) reported that the reaction between polyphenol ox-
a high nutritional value and economic value (Rico et al., 2007). However, idase (PPO) and phenolic compounds was the reason of browning in

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fresh-­cut apple. Chauhan et al. (2011) further confirmed that the rate dimethylsulfoxide (DMSO), and then it was diluted to various con-
and degree of browning in apples were significantly correlated with the centrations with PBS buffer (pH = 7.0, 0.1 M). Both ascorbic acid
browning reactions catalyzed by PPO and peroxidase (POD). Therefore, and DMSO were products of Aladdin Industrial (Shanghai, China).
one of the important ways to maintain the quality and extend the shelf The proportion of DMSO in the reaction system was required to be
life of apples is inhibiting the activity of enzymes related to browning. below 3.33% (v/v) to ensure that it had no impact on the experi-
Tyrosinase (PPO) is widely existed in fruits (Kang et al., 2004). ments (Song et al., 2020). In this study, the proportion of DMSO in
The enzyme is a copper-­
containing oxidase with multi-­
function reaction system was 1%. Polyvinyl pyrrolidone (PVP) was dissolved
such as phenolase, catalase, and catecholase activities (Yamazaki by distilled water and also purchased from Aladdin Industrial.
et al., 2004). It oxidizes monophenols to the corresponding
o-­quinones (Seo et al., 2003; Zhang et al., 2019), which polymerize
spontaneously to form brown or black pigment and cause browning. 2.2 | Enzyme analyses
In brief, tyrosinase is the main culprit of the undesired enzymatic
browning in fruits that adversely affects food quality and economic The activity of enzyme was measured with reference to Chai
value (Chai et al., 2018). Therefore, the identification of novel tyros- et al. (2019). The reaction mixture was mainly composed of L-­DOPA
inase inhibitors is extremely important. (0.3 ml, 2.5 mM) and mushroom tyrosinase solution (0.05 ml, 0.2 mg/
Ascorbic acid (Figure 1) has various biological functions (Du ml). The enzyme was preincubated in a water bath at 30°C for 5 min
et al., 2012). First, it is an effective antioxidant with the capable of before testing. Then ascorbic acid with different concentrations (0, 20,
mopping up free radicals (Patras et al., 2009). Second, this compound 30, 50, 80, 120, 140, 180, 200, 300, and 400 µM) was added into the
can prevent cancer (Hoffer et al., 2015). In addition, it is widely used in cuvette in turn and mixed well. Tyrosinase activity was determined by
whitening products as a kind of natural additive (Balaguer et al., 2008). monitoring the absorbance change per min at 475 nm within 30 s.
Ascorbic acid could stimulate the activity and expression of tyrosinase in
B16F10 cells (Lee et al., 2011). L-­ascorbic acid and 3-­(2,4-­dihydroxyphenyl)
propionic acid had a synergistic effect on the inhibition of tyrosinase 2.3 | UV–­visible spectra assays
(Chen et al., 2016). However, there are few reports about the action
mechanism of ascorbic acid on the tyrosinase. Hence, the aim of this UV–­visible spectra (300–­600 nm scans, 2 nm resolution) were uti-
research was to elucidate the action mechanism of ascorbic acid on the lized to detect the contents of reaction products (Labus et al., 2011).
tyrosinase. In addition, we studied the anti-­browning effect of ascorbic In the presence of different reactants (250 µM L-­DOPA; 5 µM ascor-
acid on fresh-­cut apple. This research offers a theoretical basis for the bic acid), the absorption values of reaction products at 0–­10 min
probable utilization of ascorbic acid in the area of fruit preservation. were scanned by a Beckman DU-­730 spectrophotometer (Backman
Coulter, Brea, CA, USA).

2 | M ATE R I A L S A N D M E TH O DS
2.4 | Oxidation of dopaquinone in the presence of
2.1 | Chemicals ascorbic acid

We selected mushroom tyrosinase and its substrate (L-­DOPA) from The assay was performed according to the previous research (Chai
Sigma-­Aldrich (St. Louis, MO, USA). Ascorbic acid was dissolved with et al., 2020). First, L-­DOPA (0.3 ml, 2.5 mM) was oxidated with so-
dium periodate (NaIO4, 0.3 ml, 2.5 mM) at 30°C, and then ascorbic
acid with different concentrations was added. The UV–­visible spectra
were recorded on the Beckman DU-­730 spectrophotometer (Backman
Coulter, Brea, CA, USA), the wavelength range was set at 230–­550 nm.

2.5 | Fluorescence assays

In order to analyze the interaction between ascorbic acid and ty-

rosinase, fluorescence assays were implemented under the fol-
lowing procedure (Huang et al., 2019). Mainly, sodium phosphate
buffer (2.4 ml, pH = 7.0, 0.1 M), ascorbic acid, mushroom tyrosi-
nase solution (0.3 ml) were added into a cuvette in turn and mixed
evenly. The final volume was kept at 3 ml. The excitation wave-
length was 290 nm and the emission spectrum was set between
FIGURE 1 Chemical structure of ascorbic acid 300 and 500 nm.
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WEN et al. | 3 of 13

Synchronous fluorescence assay was further executed by setting water. Total phenolic content of the fresh-­cut apples was measured
Δλ (λem − λex), and the values of Δλ was 15 and 60 nm, respectively by the Folin–­Ciocalteu method (Chai et al., 2017). The absorbance
(Feng et al., 2014). at 725 nm was recorded by a Beckman DU-­730 spectrophotometer
Three-­
dimensional fluorescence spectroscopy was recorded (Backman Coulter, Brea, CA, USA). Gallic acid was chosen as stand-
to investigate the impact of ascorbic acid on tyrosinase (Ding, Wu, ard. Flavonoid content of the fresh-­cut apples was evaluated accord-
et al., 2018). Both of the excitation and emission wavelengths ranged ing to Lin et al. (2018). The absorbance at 510 nm was measured by a
from 200 to 500 nm. Beckman DU-­730 spectrophotometer (Backman Coulter, Brea, CA,
USA) and catechin was used as standard.

2.6 | Copper interaction

2.8.3 | Extraction and determination of PPO and
The interaction between ascorbic acid and copper ions was inves- POD activities
tigated by measuring the UV–­visible spectra (200–­4 00 nm) of the
ascorbic acid in the absence and presence of Cu2+ with a Beckman PPO was extracted according to a previous report (Lin et al., 2018).
DU-­730 spectrophotometer (Backman Coulter, Brea, CA, USA) (Chai Apple grinding liquid was composed of Na2HPO 4-­NaH2PO 4 buffer
et al., 2013). The mixtures, consist of 150 ml ascorbic acid solution, (pH 7.0, 0.1 M) and 0.2% (w/v) PVP. Apple homogenates were centri-
100 ml CuSO 4 solution, and 2.75 ml of sodium phosphate buffer so- fuged to collect supernatant after grinding. The supernatant (1 ml),
lution, were incubated at 30°C for 10 min before testing. Na2HPO 4-­NaH2PO 4 buffer (pH 7.0, 0.1 M, 1 ml), and pyrocatechol
(50 mM, 1 ml) were added in order and mixed well. The absorbance
at 420 nm was recorded every 1 min and detected continuously for
2.7 | Molecular docking 5 min. The PPO activity was calculated by the following equation:

ΔA420 × V
Molecular docking was performed by using the MOE software. The PPO activity (U/g FW ⋅ min) = (1)
0.01t × VS × m
3D structure of tyrosinase (pdb entry 1wx2) was selected as the ini-
tial protein model for docking. The 3D structure of ascorbic acid was in this equation, ΔA420 represents the absorbance changes of the mix-
drawn by ChemBioDraw Ultra 8.0. The docked conformation with tures at 420 nm. V and VS stand the total volume and volume of sample
lowest energy value was selected to judge the binding mechanism. extract, respectively. Besides, m is the sample quality, t is reaction time.
POD was extracted according to a previous report (Pasquariello
et al., 2015) with slight modifications. After ground by Na2HPO4-­
2.8 | Evaluation of anti-­browning effect of ascorbic NaH2PO4 buffer (pH 7.0), apple homogenates were centrifuged at 4°C,
acid on fresh-­cut Fuji apples and then the supernatant was collected. The substrate mixture was
prepared on the grounds of the method of Lin et al. (2013). In brief, 30
2.8.1 | Apples treatment µl of guaiacol and 25 ml of Na2HPO4-­NaH2PO4 buffer (pH 7.0, 0.1 M)
were mixed by 78HW-­1 magnetic stirrer (Changzhou, China), then 20
Fuji apples (Malus pumila Mil) with uniform shape, maturity, and size µl of 30% (v/v) H2O2 was added. The supernatant and the reaction mix-
were purchased from China Resources Vanguard in October 2019. ture were mixed at a volumetric ratio of 1:2 (v/v). The absorbance of
Then apples were peeled and cut into equal blocks. The blocks were mixture at 470 nm was continuously detected for 5 min and recorded
immersed in ascorbic acid solutions or distilled water (control) for every 1 min. The POD activity was computed by the following equation:
20 min. Then these naturally dried fresh-­cut apples were packed in
ΔA470 × V
polyethylene bags and stored in PQX-­280A-­3H(L) artificial climate POD activity (U/g FW ⋅ min) = (2)
0.01t × VS × m
box (Ningbo, China) at 15°C, 80% relative humidity. Following indi-
cators related to browning, the contents of total phenols and flavo- where ΔA470 is the absorbance changes of the mixtures at 470 nm.
noids, the PPO, POD, and APX activities, and the browning degree V and VS stand the total volume and volume of sample extract, respec-
were measured (Lin et al., 2018). tively. In addition, m is the sample quality, t is reaction time.

2.8.2 | Determination of total phenol and 2.8.4 | Extraction and determination of APX activity
flavonoid contents
APX was extracted according to the method of Ren et al. (2012).
Fresh-­cut apples (5 g) were homogenized and then ultrasonically di- Fresh-­cut apples (3 g) was homogenized with Na2HPO 4-­NaH2PO 4
gested three times with 5 ml of 70% (v/v) acetone for 15 min. The su- buffer (pH 7.5, 0.1 M) containing 1 mM ethylene diamine tetraacetic
pernatant was collected into a volumetric flask after centrifuging the acid (EDTA). Then apple homogenates were centrifuged at 4°C to
mixture for 10 min at 10,000 r/min and diluted to 25 ml with distilled collect supernatant. APX activity was measured according to a
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4 of 13 | WEN et al.

previous report (Cao et al., 2010) with some modifications. Briefly, in test tubes and mixed fully. These test tubes were stored in PQX-­
the supernatant (0.2 ml), 0.1 M Na2HPO 4-­NaH2PO 4 buffer (pH 7.5, 280A-­3H(L) artificial climate box (Ningbo, China) at 15°C, 80% rela-
2 ml), 30% (v/v) H2O2 (0.5 ml), and 3 mM ascorbic acid (0.8 ml) were tive humidity. The absorbance of the mixture was determined at
added into a cuvette and mixed well. The absorbance of mixture at 420 nm every day (Hariklia et al., 2009).
290 nm was continuously detected for 5 min and recorded every
1 min. The APX activity was calculated by the following equation:
2.9 | Statistical analysis
ΔA290 × V
APX activity (U/g FW ⋅ min) = (3)
0.01t × VS × m
All the experiments were carried out three times under the same con-
in this equation, ΔA 290 is the absorbance changes of the mixtures ditions. The results were expressed as mean ± standard deviation. The
at 290 nm. significance analyses were performed with IBM SPSS Statistics (v.25).

2.8.5 | Determination of browning degree of 3 | R E S U LT S A N D D I S CU S S I O N

apple juice
3.1 | Dual effect of ascorbic acid on tyrosinase
The apples were washed and peeled for juice extraction. The apple activity
juice was centrifuged for 10 min at 4°C, and the supernatant was
collected. The apple juice supernatant (3 ml) and ascorbic acid (1 ml) Tyrosinase was mixed with L-­DOPA (Figure 2a) and ascorbic acid
with different concentrations (0, 0.06, 0.3, and 1.5 mM) were added (Figure 2b), respectively, and then the mixture was determined by

F I G U R E 2 Assay of tyrosinase activity. Wavelength scanning of the oxidation product of L-­DOPA catalyzed by tyrosinase (a); wavelength
scanning of the mixture of ascorbic acid and tyrosinase (b); the effect of ascorbic acid on the activity of tyrosinase (c); UV-­Vis spectra for the
oxidation of L-­DOPA, C(L-­DOPA) = 250 µM, C(NaIO 4) = 250 µM (d). AA represents ascorbic acid
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WEN et al. | 5 of 13

UV–­visible wavelength scanning. It was observed that the product acid (curves 2–­5). The maximum fluorescence intensity of the en-
accumulated only in the mixture of tyrosinase and L-­DOPA, the zyme decreased to 45.2 ± 2.1% when the concentration of ascorbic
results showed that ascorbic acid could not act as a substrate of acid reached to 200 µM (Figure 3b). The result proved that ascorbic
tyrosinase. As shown in Figure 2c, tyrosinase activity was slightly acid effectively quenched the intrinsic fluorescence of tyrosinase,
activated in less than 7.5 µM ascorbic acid, and then inactivated indicating a strong interaction between the ascorbic acid and the
until the tyrosinase activity completely abolished at 40 µM ascor- enzyme. Besides, a discernible red shift was detected (Figure 3a),
bic acid. Ascorbic acid acted as an activator of tyrosinase at the which uncovered structure change of tyrosinase in the presence of
lower concentration (less than 7.5 µM) and an inhibitor at the higher ascorbic acid.
concentration. It had a dual effect on tyrosinase activity when it The quenching mechanisms can be classified as dynamic quench-
was coordinated with L-­DOPA. The inhibitory concentration (IC 50) ing and static quenching. Dynamic quenching is caused by molec-
of ascorbic acid on the tyrosinase activity was calculated to be ular collision, and static quenching refers to fluorophore-­quencher
13.40 ± 0.05 µM. Compared with kojic acid (Xie et al., 2017) (IC 50 complex formation (Zhang et al., 2013). For the dynamic quenching,
= 20.00 ± 1.08 µM, p = .019) and luteolin (Zhang et al., 2017) (IC 50 the quenching constants increase with the increasing temperature,
= 266.67 ± 4.28 µM, p < .001), the IC50 of ascorbic acid was sig- because there is a positive correlation between the rise in tempera-
nificantly lower than kojic acid and luteolin (p < .05). The results tures and the increases of diffusion coefficients, which is opposite in
testified that ascorbic acid was a more efficient tyrosinase inhibitor. the static quenching (Hu et al., 2004).
Chai et al. (2013) found that hawthorn proanthocyanidins activated The Stern–­
Volmer equation was employed to investigate the
tyrosinase activity in a low concentration which caused by the pres- quenching mechanism and parameters of ascorbic acid on tyrosinase:
ence of substrate analogues (catechin/epicatechin). However, there
was not product accumulated in tyrosinase and ascorbic acid, which F0
(4)
[ ] [ ]
= 1 + Kq 𝜏 0 Q = 1 + KSV Q
meant that there was another activation mechanism between ty- F
rosinase and ascorbic acid. According to the report (Krupyanko
et al., 2017), there was a positive cooperativity between two where F0 and F indicate the fluorescence intensities of tyrosinase in
L-­lysine α-­
oxidase’s subunits which participate the formation of the absence and presence of ascorbic acid, respectively. [Q], KSV, and
enzyme-­substrate complex, so the binding of the substrate to the Kq mean the quencher (ascorbic acid) concentration, Stern–­Volmer
allosteric enzyme could promote the further binding of the sub- quenching constant, and biomolecular quenching rate constant,
strate molecule to other subunits of the enzyme. We speculated respectively. τ0 (10−8 s) was the lifetime of fluorophore without
that ascorbic acid binding to the substrate binding sites of the en- quencher.
zyme could also promote further binding of L-­DOPA to other subu- Figure 3c showed the plot of F0/F against ascorbic acid, and the
nits of the tyrosinase, so that low concentrations of ascorbic acid results from Figure 3c and Equation (4) were listed in Table 1. The
could activate tyrosinase. KSV values gradually increased with the rising temperatures, indicat-
ing that there was a dynamic quenching of tyrosinase by ascorbic
acid. In addition, the values of Kq were respectively calculated to be
3.2 | Interaction of the ascorbic acid with 4.99 × 1011, 5.41 × 1011, and 5.73 × 1011 L mol−1 S−1 at 290, 300,
o-­quinones and 310 K, which were larger than the maximum scattering collision
quenching constants of biological molecules (2.0 × 1010 L mol−1 S−1).
To illustrate whether the ascorbic acid could form a redox reaction Therefore, it was speculated that there was another interaction be-
with o-­quinones, L-­DOPA was oxidized by NaIO 4 in the absence or tween tyrosinase and ascorbic acid. In the light of previous report
presence of ascorbic acid and the absorbance of oxidative product (Van de Weert & Stella, 2011), there were several other interactions
was recorded (Figure 2d, green). The result showed that the absorb- for dynamic quenching, such as Förster’s resonance energy transfer
ance slightly reduced along with the addition of ascorbic acid at 10 (FRET) or photo-­induced electron transfer.
and 40 mM (Figure 2d, purple and yellow). Thus, the ascorbic acid The binding constant (K A) and the number of binding site (n) were
might prevent the formation of o-­quinones and influence the inhibi- evaluated by using the following equation (Laskar et al., 2016).
tory effect of ascorbic acid on tyrosinase. However, this influence
was limited in our test concentration. F0 − F
(5)
[ ]
log = nlog Q + logKA
F

3.3 | Fluorescence quenching analysis As shown in Figure 3d and Table 1, the K A gradually increased
along with increasing temperature, indicating a dynamic quenching
As shown in Figure 3a, tyrosinase showed a maximum fluorescence mechanism of ascorbic acid on tyrosinase. Besides, n was approxi-
intensity around 328 nm (curve 1), and the fluorescence intensity mately equal to 1 (Table 1), indicating only one site for ascorbic acid
of the enzyme reduced progressively with the addition of ascorbic binding to the enzyme.
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6 of 13 | WEN et al.

F I G U R E 3 Fluorescence spectra of tyrosinase in the presence of ascorbic acid at different concentrations (a). Curve a is fluorescence
intensity of ascorbic acid at 200 µM, the concentration of ascorbic acid for curves 1–­5 were 0, 50, 100, 150 and 200 µM, respectively;
Relative intensity of tyrosinase at different concentrations of ascorbic acid (b); The Stem-­Volmer plots for the fluorescence quenching
of tyrosinase by ascorbic acid (c); Plots of log [(F0‒­F )/F] against log [Q] at 290, 300, 310 K respectively (d), F0 and F are the fluorescence
intensities of tyrosinase in the absence and presence of ascorbic acid, [Q] represents the concentration of ascorbic acid; The spectral overlap
(e) of ascorbic acid absorption (a) and tyrosinase fluorescence (b)

3.4 | Thermodynamic analysis interaction can be regarded as a constant (Ding et al., 2012; Zeng
et al., 2016). Thermodynamic parameters were calculated accord-
When the temperature rage is not too wide, the influence of ing to the van’t Hoff equation to understand the driving force of
the temperature is negligible, the enthalpy changes (ΔH°) of the the interaction between ascorbic acid and tyrosinase. The values
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WEN et al. | 7 of 13

TA B L E 1 The quenching constants KSV, the binding constants K A , the number of binding sites n, and relative thermodynamic parameters
(ΔH°, ΔG°, and ΔS°) of the interaction between ascorbic acid and tyrosinase at three different temperatures

KSV (103 Kq (1011 K A (103 ΔH° ΔS°

T (K) L mol−1) Ra L mol−1 S−1) L mol−1) n Rb (kJ mol−1) ΔG° (kJ mol−1) (J mol−1 K−1)

290 4.99 ± 0.13b 0.9979 4.99 ± 0.13b 6.09 ± 0.2c 1.02 ± 0.03 0.9972 31.69 ± 1.3 −20.82 ± 1.4a 181.08 ± 1.6
300 5.41 ± 0.17a 0.9969 5.41 ± 0.17a 7.39 ± 0.4b 1.04 ± 0.02 0.9986 31.69 ± 1.3 −22.64 ± 1.2ab 181.08 ± 1.6
310 5.73 ± 0.18a 0.9968 5.73 ± 0.18a 14.31 ± 0.6a 1.11 ± 0.05 0.9934 31.69 ± 1.3 −24.45 ± 1.0b 181.08 ± 1.6
a b
Note: R is the correlation coefficient for the KSV values. R is the correlation coefficient for the K A values. Values with letters a, b, and c show
significantly different (p < .05).

TA B L E 2 The overlapping integral J, the transmission efficiency

E, the critical distance R0, the distance r between tyrosinase and
ascorbic acid at three different temperatures

T (K) J (cm3 L mol−1) E (%) R0 (nm) r (nm)

−16
290 4.75 × 10 c 2.40c 1.66b 3.08a
300 5.03 × 10 −16b 4.41b 1.68ab 2.8b
310 6.75 × 10 −16a 8.09a 1.76a 2.64c

Note: Values with letters a, b, and c show significantly different (p < .05).

of enthalpy changes (ΔH°), entropy changes (ΔS°), and free energy

changes (ΔG°) were calculated on the basis of equations as follows
(Zhang et al., 2013).

ΔH◦ ΔS ◦ (6)
logKA = +
2.303RT 2.303R

ΔG◦ = ΔH◦ − TΔS ◦ (7)

in the Equation (6), R (gas constant) is 8.314 J mol−1 K−1.

The mechanism of action of ascorbic acid on tyrosinase can be
judged by thermodynamic parameters. As illustrated in Table 1, ΔG°
< 0 showed the binding was a spontaneous process. In addition,
the values of positive ΔH° indicated that the interaction of ascorbic
acid and tyrosinase was an exothermic reaction (Laskar et al., 2016).
Furthermore, both positive values of ΔH° and ΔS° manifested that
hydrophobic interaction was main driving force for the binding of
ascorbic acid on the enzyme (Roy et al., 2017).

F I G U R E 4 Synchronous fluorescence spectra of tyrosinase in

3.5 | Energy transfer between tyrosinase and
the absence and presence of ascorbic acid. (a) Δλ = 15 nm; (b) Δλ =
ascorbic acid 60 nm. The concentration of ascorbic acid for curves 1–­6 are 0, 15,
45, 60, 75, and 100 µM, respectively
Through non-­radioactive dipole–­dipole coupling, the fluorescence
of the excited-­state donor fluorophore was absorbed by the accep-
F(𝜆) 𝜀(𝜆) 𝜆4 Δ𝜆
∑
tor, which was accompanied by the FRET happened between donor J= ∑ (10)
F(𝜆) Δ𝜆
and acceptor (Hao et al., 2017). In the light of Förster’s theory, the
binding distance (r) between ascorbic acid and tyrosinase was calcu-
lated according to the following formulas (Zhang et al., 2008). Here, E, R0, J, and ε(λ) represent energy efficiency, critical dis-
tance, overlapping integral of donor fluorescence spectrum and
R60 F0 − F
E= = (8) receptor ultraviolet absorption spectrum, and molar absorption
R60 + r6 F0
coefficient of acceptor, respectively. K 2 (dipole spatial orienta-
tion factor) is 2/3. N (1.336) represents the refractive index of the
R60 = 8.79 × 10−25 N−4 K 2 J𝜑 (9)
medium.
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8 of 13 | WEN et al.

In Figure 3e, the overlapping part of ultraviolet spectrum of ascor-

bic acid (a) and fluorescence spectrum of enzyme (b) is indicated by
gray. Based on previous report (Zhang et al., 2008), the φ equal to 0.24.
Based on Equations (8)–­(10), the values of J, R0, E, and r were calculated
and listed in Table 2. The r < 8 nm demonstrated that the energy trans-
fer from tyrosinase to ascorbic acid with high probability. The result
confirmed FRET was the main mechanism of dynamic quenching.

3.6 | Synchronous fluorescence analysis

The measurement of synchronous fluorescence spectrum aimed to

clarify the interaction between ascorbic acid and tyrosinase. It can
offer microenvironment changes of the chromophore molecules (ty-
rosine and tryptophan residues) (Pacheco & Bruzzone, 2013).
The fluorescence intensity of the tyrosinase quenched gradu-
ally along with the increasing concentration of ascorbic acid, sug-
gesting that ascorbic acid can interact with tyrosinase (Figure 4a,b).
Meanwhile, there were red shifts both in the fluorescence spectra
of tyrosine (Δλ = 15 nm) and tryptophan (Δλ = 60 nm) residues, sug-
gesting that ascorbic acid altered the hydrophobicity of them and
caused the conformation change of tyrosinase.

3.7 | 3D fluorescence spectroscopy assay

In Figure 5a,b and Table 3, peak a is the Rayleigh scattering peak (λex =
λem) and peak b denotes the second-­order scattering peak (λem = 2λex)
(Ding, Hu, et al., 2018). Peak 1 (λex = 225 nm, λem = 340 nm) reflects
the fluorescence spectra of the polypeptide backbone structure of
tyrosinase caused by the n → π* transition. Peak 2 (λex = 280 nm, λem
= 340 nm) denotes the spectral behavior of tyrosine and tryptophan
residues. After adding ascorbic acid to tyrosinase solution, peak 1 de-
creased from 172.5 to 151.5, and that of peak 2 decreased from 160
to 103.6. In addition, red shifts in the emission wavelength of peak
F I G U R E 5 Three-­dimensional fluorescence spectra of 1 and excitation wavelength of peak 2 were found. These phenom-
tyrosinase in the absence (a) and presence of ascorbic acid (b).
ena revealed that ascorbic acid changed the structure of polypeptide
(A):C(tyrosinase) = 0.2 mg/ml; (B):C(tyrosinase) = 0.2 mg/ml,
C(ascorbic acid) = 50 µM backbone as well as tyrosine and tryptophan residues.

TA B L E 3 Three-­dimensional fluorescence spectral characteristics (peak a, peak 1, peak 2, and peak b) of tyrosinase in the absence or
presence of ascorbic acid

Tyrosinase Tyrosinase with ascorbic acid

Peak position Stokes Peak position Stokes

Fluorescence
peaks λex/λem (nm/nm) Δλ (nm) Intensity F λex/λem (nm/nm) Δλ (nm) Intensity F

Peak a 200/200 → 600/600 0 23.35 → 281.8 200/200 → 600/600 0 24.54 → 339.7

Peak 1 225/335 110 172.5 225/340 115 151.5
Peak 2 280/340 60 160 285/340 55 103.6
Peak b 240/480 240 96.11 240/480 240 60.36

Note: λex is the excitation wavelength, λem is emission wavelength, Δλ is the difference between λem and λex.
 17454514, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13995 by Quoc-Duy Nguyen - Rmit University Library , Wiley Online Library on [06/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WEN et al. | 9 of 13

3.8 | Copper ion chelating ability of Cu2+ (Figure 6), indicating the binding of ascorbic acid and Cu2+
(Zhu et al., 2013).
Interactions between ascorbic acid and Cu2+ were studied by UV–­
visible spectra analysis. The result showed that the absorbance val-
ues of ascorbic acid at 265 nm decreased distinctly with the increase 3.9 | Molecular docking

Molecular docking was further carried out to better understand the

action mechanism of ascorbic acid on tyrosinase (Figure 7). The re-
sult revealed that the ascorbic acid could insert the active site of
tyrosinase and chelate with copper ion, which was consist with the
result of the above copper ion chelating ability analysis. Besides,
ascorbic acid was encircled by amino acid residues His244, Val248,
Asn260, His263, Phe264, and Val283, which probably caused by
the hydrophobic interaction between ascorbic acid and tyrosinase.
These results illustrated that the binding of ascorbic acid and tyrosi-
nase was driven by hydrophobic interaction.

3.10 | Effect of ascorbic acid on the preservation of

fresh-­cut Fuji apples

As shown in Figure 8a,b, the total phenolics and flavonoids

F I G U R E 6 UV absorption spectra of ascorbic acid in the contents in fresh-­
c ut apples decreased during storage time.
absence and presence of Cu2+ It was observed that total phenolics content and flavonoids

FIGURE 7 The docked binding mode of ascorbic acid and tyrosinase

 17454514, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13995 by Quoc-Duy Nguyen - Rmit University Library , Wiley Online Library on [06/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
10 of 13 | WEN et al.

content treated with ascorbic acid were markedly higher than As the crucial enzymes of enzymatic browning, PPO and POD
that of control group (p < .05) according to statistical analysis. catalyze the oxidation of phenolic compounds (Zhou et al., 2015).
These results showed that ascorbic acid could prominently pre- In Figure 8c, PPO activity in fresh-­cut apples showed an increasing
vent the degradation of total phenolic and flavonoids in fresh-­ trend during the whole storage period. Compared with the control
cut apple. group, the PPO activity declined with the increasing concentration

F I G U R E 8 Anti-­browning activity of ascorbic acid on fresh-­cut Fuji apple. The content of total phenolics (a), the content of flavonoid (b),
the polyphenol oxidase and peroxidase activities (c, d) of fresh-­cut apples after treateding with ascorbic acid at different concentrations.
The fresh-­cut apples were stored at 15°C for 8 days, FW represents the fresh weight. The APX activity (e) of fresh-­cut apples after treated
with ascorbic acid at different concentrations. The fresh-­cut apples were stored at 15°C for 6 days, FW represents the fresh weight. The
browning degree (f) of apple juice by treating with ascorbic acid at different concentrations. The apple juice was stored at 15°C for 7 days.
The detection data of the last day were selected for significant analysis. Values with different letters (a, b, c, and d) denote significantly
different (p < .05)
 17454514, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jfbc.13995 by Quoc-Duy Nguyen - Rmit University Library , Wiley Online Library on [06/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
WEN et al. | 11 of 13

of ascorbic acid. The PPO activity of fresh-­cut apples treated with of total phenolics and flavonoids. These findings provide a theoreti-
ascorbic acid was substantially lower than that of controls. In conclu- cal basis for the feasible application of ascorbic acid on the preserva-
sion, ascorbic acid can remarkably inhibit PPO activities. tion of fruits.
The changes of activities of POD were showed in Figure 8d.
These results showed that POD activity increased over the study AC K N OW L E D G M E N T S
period, by contrast, reduced with the increase of ascorbic acid The work was supported by National Natural Science Foundation of
concentrations. It was observed that POD activity of the control China (No. 3216160030) the Natural Science Foundation of Jiangxi
group was higher than that treated with various concentrations of province, China (No. 20192ACBL21028), Graduate Innovation Fund
ascorbic acid. These results showed ascorbic acid can remarkably Project of Jiangxi Normal University (No. YC2020-­S182).
inhibit POD activities. Therefore, it can be inferred that ascorbic
acid prevent the browning of fresh-­cut apples by inhibiting PPO C O N FL I C T O F I N T E R E S T
and POD activities and reducing the oxidation of total phenolics The author declares that there is no conflict of interest that could be
and flavonoids. perceived as prejudicing the impartiality of the research reported.
APX is one of the important antioxidant enzymes in plant. It
can eliminate H2O2 and protect plants from oxidative damage AU T H O R C O N T R I B U T I O N S
(Mittler, 2002). The changes of APX activities of fresh-­cut apples Yi-­Ting Wen: Methodology; Resources; Software; Writing-­original
were shown in Figure 8e. It was observed that the APX activity draft. Yu-­Qin Liang: Data curation. Wei-­Ming Chai: Funding acqui-
of the fresh-­cut apples in the control group decreased during sition; Project administration; Supervision; Validation; Visualization.
the whole storage period. However, ascorbic acid enhanced the Qi-­Ming Wei: Funding acquisition; Methodology; Resources;
APX activity of fresh-­cut apple. Meanwhile, the APX activity of review & editing. Zi-­Yi Yu: Funding acquisition. Lin-­Jun
Writing-­
fresh-­cut apples treated with ascorbic acid was significantly higher Wang: Software.
(p < .05) than the control group according to statistical analysis.
These results showed that ascorbic acid could increase the APX DATA AVA I L A B I L I T Y S TAT E M E N T
activity, thereby alleviating oxidative damage and delaying the The data that support the findings of this study are available from
browning of fresh-­cut apples. the corresponding author upon reasonable request.
The changes of browning degree of apple juice were shown in
Figure 8f. The results confirmed that the browning degree of apple ORCID
juice showed an increasing trend with the extension of storage time. Wei-­Ming Chai https://orcid.org/0000-0001-9163-8138
However, compared with the control, the browning degree of apple
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