Ultrasound and heating treatments improve the antityrosinase ability of polyphenols

Journal Pre-proofs Ultrasound and heating treatments improve the antityrosinase ability of poly‐ phenols Qun Yu, Liuping Fan, Jiajing Duan, Nan Yu, Na Li, Qingqing Zhu, Nana Wang PII: DOI: Reference:

S0308-8146(20)30275-2 https://doi.org/10.1016/j.foodchem.2020.126415 FOCH 126415

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

27 August 2019 3 February 2020 13 February 2020

Please cite this article as: Yu, Q., Fan, L., Duan, J., Yu, N., Li, N., Zhu, Q., Wang, N., Ultrasound and heating treatments improve the antityrosinase ability of polyphenols, Food Chemistry (2020), doi: https://doi.org/ 10.1016/j.foodchem.2020.126415

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Ultrasound and heating treatments improve the antityrosinase ability of

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polyphenols

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Qun Yu, Liuping Fan*, Jiajing Duan, Nan Yu, Na Li, Qingqing Zhu, Nana Wang

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State Key laboratory of Food Science & Technology, Jiangnan University, 1800 Lihu

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Avenue, Wuxi, Jiangsu 214122, China

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*Corresponding author: Dr., Liuping Fan Professor of State Key laboratory of Food

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Science & Technology, Jiangnan University, Wuxi 214122, China

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Tel: 0086-(0) 510-85876799

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E-mail: [email protected]

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ABSTRACT：

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This paper focused on improving antityrosinase ability of quercetin, cinnamic acid,

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and ferulic acid (named Q-CA-FA) from Asparagus by combining heating with

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ultrasound treatments. Fluorescence spectroscopy and UPLC-MS were used to evaluate

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inhibitory mechanisms. Results showed that the impacts of combining heating (150°C

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for 30 min) with ultrasound (600 W for 30 min) treatments was similar to heating

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treatment (150°C for 120 min) alone, and the inhibition rate could reach 98.2% in the

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addition of 5 mM Q-CA-FA. Fluorescence quenching indicated that treated Q-CA-FA-

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tyrosinase complex was more stable, but combining treatments did not change the major

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force between tyrosinase and polyphenols. Thermodynamic analysis illustrated that the

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randomness of compounds was also increased. Interestingly, 2-hydroxy-3-(3-hydroxy-

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4-methoxy-phenyl)-propionic acid 4-(2,3-dihydroxy-propyl)-phenyl ester was newly

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detected, which might be the major reason for enhancing antityrosinase ability. Taken

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together, these results provide a creative insight on increasing antityrosinase activity by

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combining heating with ultrasound treatments.

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Keywords: Antityrosinase activity; Tyrosinase; Ultrasound treatment; Heating

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treatment; Asparagus

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Chemical compounds:

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Quercetin (Pubchem CID: 5280343); Cinnamic acid (Pubchem CID: 637542); Ferulic

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acid (Pubchem CID: 445858)

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1. Introduction

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Asparagus officinalis L. (namely green asparagus) belongs to the family

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Asparagaceae, and it has been used as a herbal medicine for a long time (Zhang et al.,

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2018). The tender stems of Asparagus was generally considered as a delicious and

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nutritional vegetable. Our published studies have proved that the crucial polyphenols

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associated with antityrosinase activity in Asparagus are quercetin, cinnamic acid, and

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ferulic acid (Yu, Fan, & Duan, 2019). In addition, saponins (protodioscin and dioscin)

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were another important bioactive constituents found in Asparagus (Chitrakar, Zhang,

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& Adhikari, 2019). Traditionally, saponins were considered as antinutrients on account

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of their hemolytic activity, but current investigations were focused on its bioactivities,

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such as antitumor, antifungal, antibacterial, anti-inflammatory (Navarro et al., 2018).

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As representative components of Asparagus, polyphenols and saponins were employed

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to evaluate antityrosinase capacity in this paper.

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Although natural polyphenols widely existed in nature, many polyphenols with

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poor hydrophilic polarity led to a relatively poor absorption and limited application.

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Taking into consideration of the rise in popularity of green and healthy viewpoint, there

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has been a growing concern surrounding enhancing the antityrosinase ability of natural

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bioactive constituents using physical process. One of the best-known food process

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methods was hot air heating (namely convective drying). Higher temperature led to

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lower solubility of obtained samples, suggesting that heating can change the chemical

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properties of vegetables (Michalska et al., 2017). And ultrasound technology has

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become a potential and efficient enhancement method due to the characteristic of 3

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cavitation in recent years (Zhang et al., 2019). However, most papers relevant to

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polyphenols focused on ultrasound assisted extraction (Gambacorta et al., 2017; Lazar

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et al., 2016), ultrasound assisted purification (Wang et al., 2019), and so on. In terms

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of enzymology, Cheng et al. (2013) found that ultrasound technology was efficient in

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enzyme inactivation. The inactivation rate of enzyme can be further improved by

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combining ultrasound with heating treatments (Cruz et al., 2006; Terefe et al., 2009).

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Nevertheless, there are still few literatures about the effects of combining heating with

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ultrosound technology on increasing the antityrosinase ability of polyphenols.

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Thus, we aimed to study the changes of antityrosinase capacity after the

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recommended heating and ultrasound treatments and understand the changes of

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components. The role of heating treatments (various temperatures and time) and

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combining heating with ultrasound treatments (various powers and time) was firstly

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evaluated in this paper. Based on these results, fluorescence quenching mechanism,

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binding and thermodynamic parameters were calculated to expound the changes caused

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by physical process. Meanwhile, UPLC-MS was employed to analyze the changes of

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chemical constituents after the treatments. This paper might provide a promising insight

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into the further study about tyrosinase inhibitors and reveal the chemical changes

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related with the food process.

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2. Materials and methods

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2.1 Reagents and materials

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Quercetin, cinnamic acid, ferulic acid, and L-DOPA were purchased from the J&K

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Scientific LTD in Shanghai, China. The tyrosinase (EC 1.14.18.1, 128 KDa), dioscin, 4

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and protodioscin were collected from the Yuan Ye Biological Technology Company in

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Shanghai, China. Dimethyl sulfoxide (DMSO) was obtained from the China National

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Pharmaceutical Group (Sinopharm) in Shanghai, China.

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2.2 Heating treatments

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Heating treatments experiments were carried out in a heating oven (Binder,

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Germany), maintaining the temperature at 120 and 150℃ for 30, 60, and 120 min,

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respectively. In these experiments, a mixture of 150 μg/mL quercetin, 80 μg/mL

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cinnamic acid, and 95 μg/mL ferulic acid was added in brown bottle (diameter 10 mm,

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height 40 mm) (Yu et al., 2019). Then these brown bottles were put into the heating

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oven at the set temperature and removed at different time intervals. After the heating

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treatments, the mixture of Q-CA-FA or saponins (protodioscin or dioscin) were

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dissolved into a 2 mL volumetric flask and further treatments will be done. All samples

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were repeated in triplicate.

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2.3 Ultrasound treatments

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The ultrasound treatments was according to published paper with slight revision

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(Cheng et al., 2013). A 1200 W ultrasonic processor (TL-1200Y, Jiangsu Tenlin

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Instrument Co., Ltd., Yancheng, China) with a 15 mm diameter probe tip was employed

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for ultrasound treatments. The working frequency was 20 kHz and the tip was immersed

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to a depth of 10-15 mm in beakers. Firstly, the treatments were carried out 25%, 50%

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of the maximal equipment for 60 min. Then three treatments time (30 min, 60 min, and

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90 min) was investigated. The temperature of samples in the beaker was set 40℃ and

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maintained by ice-bath. In order to facilitate heat dispersal, the time interval was 1 s on 5

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and 1 s off during the ultrasound treatments. All samples were repeated in triplicate.

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2.4 Detemination of tyrosinase relative activity

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The specific method was according to previous paper with minor modification (Yu,

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Fan, & Duan, 2019). In brief, L-DOPA was chosen as a substrate for the tyrosinase

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relative activity experiment. Primarily, different concentrations of polyphenols and

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saponins combinations were prepared using DMSO. Then 2.8 mL of L-DOPA solution

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(2.5 mmol/L) was mixed with 100 μL different samples. The mixture was incubated at

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30℃ for 15 min. Afterwards the tyrosinase relative activity was determined at 475 nm

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by the spectrophotometer (L8, INESA Co., Ltd., Shanghai, China). The relative activity

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of pure tyrosinase was considered as 100%. The relative activity of tyrosinase was

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calculated as followed:

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Relative activity (%) = (B2 - B1) (A2 - A1) × 100

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(1)

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Where A1 is the absorbance of blank at 0 min. B1 is the absorbance of sample at 0

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min. A2 represents that the absorbance of blank after 15 min. B2 represents the

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absorbance of sample after 15 min.

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2.5 Fluorescence quenching analysis

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Fluorescence spectra of tyrosinase in the presence of different samples were

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conducted by a F-7000 fluorescence spectrophotometer (RiLi, Japan) according to a

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published paper with minor revision (Hill et al., 1986). Polyphenols were added into

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DMSO and the ultima concentration is 1 mM. Then, 5 μL of each sample was

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continuously added into a tube containing 2.5 mL enzyme solution (0.2 mg/mL), mixed 6

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intensively, and measured at 298, 303, and 310 K, respectively. The enzyme solution

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was regarded as a control. The intrinsic fluorescence spectra of tyrosinase at different

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quencher were carried out at λex = 280 nm and λem from 290 to 500 nm. The slit width

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were set as 5 nm. All fluorescence intensities needed to be corrected according to the

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following equation (Peng, Ding, Jiang, Sun, & Peng, 2014): 𝐴1 + 𝐴2

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𝐹𝑐 = 𝐹𝑑e

2

(2)

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Where Fc represents the final fluorescence value, Fd represents the detected

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fluorescence value. A1 and A2 denote the absorbance of the polyphenols at excitation

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and emission wavelength, respectively.

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Fluorescence quenching constants are calculated by the Stern-Volmer equation: 𝐹0 𝐹 = 1 + 𝐾𝑞𝜏0[I] = 1 + 𝐾𝑆𝑉[I]

(3)

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Where F0 is the fluorescence value before the addition of the polyphenols; F is the

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fluorescence value in the presence of polyphenols; Kq represents the quenching rate

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constant; 𝜏0=10-8 s (Wang et al., 2014); [I] denotes sample (or inhibitors)

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concentrations; Ksv denotes the quenching constant.

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In some situations, the plot of F0/F vs [samples] showed an upward curve towards

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the vertical coordinate, suggesting that the presence of sphere-of-action or apparent

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static quenching. Thus, the equation describing this mode was as follows(Castanho and

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Prieto, 1998):

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F0 F = (1 + K[I])exp([I]VN 1000)

(4)

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Where V represents the volume of sphere; N refers to Avogadro’s constant. When

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the value of K[I] is small enough, 1 + K[I] can be regarded as exp(K[I]). The value of 7

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exp(K[I]) is equal to exp([I]VN). So the equation (4) was rewritten as the following

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equation (Ferrer-Gallego, Gonçalves, Rivas-Gonzalo, Escribano-Bailón, & de Freitas,

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2012):

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ln(𝐹0 𝐹) = 𝐾𝐹𝑄[𝐼]

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(5)

Where KFQ denotes the modified quenching constant. 2.6 Binding constants calculation

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When the polyphenols independently bind to a series of equal sites on the

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tyrosinase, meanwhile the balance between the free and the bound compounds has been

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reached, the binding constants were calculated obey the following equation (Bi et al.,

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2004):

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lg

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𝐹0 ― 𝐹

(

𝐹

) = nlg𝐾

𝑎

― nlg( [𝐼] ―

1 (𝐹0 ― 𝐹)[𝐸])

(6)

𝐹0

Where Ka denotes the apparent binding constant. And n denotes the number of

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binding sites per tyrosinase. [E] denotes the concentration of enzyme.

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2.7 Thermodynamic parameter calculation

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There are four major interactions roles between the polyphenols and tyrosinase,

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including electrostatic interaction, Van der Waals' force, hydrogen bond, and

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hydrophobic interaction (Zeng et al., 2016). ΔH and ΔS could be obtained obey the Eq.

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(7):

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∆𝐻

∆𝑆

lg𝐾𝑎 = ― 2.303𝑅𝑇 + 2.303𝑅

(7)

R is equal to 8.314 J mol-1 K-1. And T is the absolute temperature during the experiment. Ka represents the binding constants at 298, 303, and 310 K, respectively. 8

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Moreover, ΔG can be obtained from the Eq. (8): ∆G = ∆H - T∆S

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(8) 2.8 UPLC-MS analysis

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The samples for UPLC-MS analysis contained 150 μg/mL quercetin, 80 μg/mL

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cinnamic acid, and 95 μg/mL ferulic acid after different treatments conditions (Yu, Fan,

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& Duan, 2019), including heating treatment (150℃ for 30 min), ultrasound treatment

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(600 W for 30 min), and combining heating (150℃ for 30 min) with ultrasound

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treatments (600 W for 30 min). The samples were determined by the Waters Maldi

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Synapt quadrupole time of flight (Q-TOF) mass spectrometer (Massachusetts, USA)

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equipped with an ESI ionisation source. The analysis conditions for LC were as follows:

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mobile phase A is acetonitrile and mobile phase B is 0.1% methanoic acid; 0.1 min 5%

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A, 95% B; 8 min 15% A, 85% B; 12 min 21% A, 79% B; 14 min 60% A, 40% B. 15

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min 90% A, 10% B; 15.10 min 5% A, 100% B. 10 μL sampes was injected into the

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analysis system. The gradient elution was carried out for 15 min and the flow rate is 0.3

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mL/min.

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2.9 Statistical analysis

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Each experiment step was repeated three times. MassLynx (4.1 version) was used

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to acquisite the UPLC-MS data. The results were exhibited as mean values ± SD for

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each measurement. The experimental data was conducted using SPSS 20.0 software

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(IBM, Chicago, USA). All figures were accomplished by Origin 9.0 software.

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3. Results and discussion 9

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3.1 Effect of heating temperatures and time on antityrosinase ability of polyphenols or

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saponins

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Q-CA-FA (KI = 0.239 mM) possessed synergistic effect on tyrosinase, which

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possessed a higher inhibition ability than quercetin (KI = 0.361 mM) (Yu, Fan, & Duan,

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2019). Based on the results of previous studies, experiments were designed to study the

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detailed impact of heating treatments on their antityrosinase ability, and their structures

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were shown in supplementary Fig. F1. Different combinations of polyphenols and

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saponins were heated at 120, 150℃ for 30, 60, 120 min, respectively. Fig. 1A and B

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indicated that the antityrosinase ability of Q-CA-FA increased with the increasing of

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heating time at 120℃ and 150℃. Heating for 30 min at 120℃ did not cause a

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significant enhancement of antityrosinase activity. However, the antityrosinase activity

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enhanced rapidly above 30 min. For instance, the tyrosinase inhibition rate of untreated

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Q-CA-FA was 17.61%. However, the antityrosinase capacity of Q-CA-FA were

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increased by approximately 38.2% and 65.3% at 120℃ for 60 min and 120℃ for 90

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min, respectively (Fig. 1A). Furthermore, residual tyrosinase activities in the addition

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of Q-CA-FA after heating at 150℃ for 30 min and 60 min were only about 16.9% and

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9.1%, respectively. The result indicated that higher temperature (150℃) was more

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beneficial for increasing the antityrosinase ability of polyphenols. At the same time, the

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minimum relative activity of tyrosinase was 2.2%. The data suggested that the

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tyrosinase inhibition rate was 97.8% in the addition of Q-CA-FA after heating at 150℃

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for 120 min. It might attribute to the fact that there were some newly detected

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compounds in the samples, indicating that polyphenols were significantly influenced 10

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by heating treatments (Lee and Lee, 2012). The heating treatments could enhance other

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biological activity was well reported, nevertheless, there was significant difference in

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the published literatures. Park et al. (2019) found that the antioxidant activity enhanced

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with the increasing of temperatures and time, but heating at 90℃ for 48 h could lead to

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curcuminoid degradation. Sun et al. (2017) treated sweet potato leaf polyphenols at

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different temperatures and observed that high temperature and long-term heat

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treatments were bad for the antioxidant activity.

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The antityrosinase capacity of the mixture of Q-CA-FA and protodioscin (or

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dioscin) after heating treatments were exhibited in Fig. 1C-F. In general, the relative

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activity in the addition of Q-CA-FA and protodioscin (or dioscin) was similar to that in

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the addition of Q-CA-FA. Increasing the temperature from 120℃ to 150℃ can further

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decrease the relative activity of tyrosinase. When the mixture of Q-CA-FA and

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protodioscin or the mixture of Q-CA-FA and dioscin was heated at 150℃ for 30 min,

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the relative activity of tyrosinase was 37.2% and 15.4%, respectively. The reason for

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this difference was the structure characterization difference of protodioscin and dioscin,

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which referred to the numbers of sugar units attached at C22 (supplementary Fig. F1).

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These data indicated that the glycosylation of saponins might be adverse to its

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antityrosinase activity. The relative activity of tyrosinase in addition of protodioscin

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and dioscin were 1.2% and 2.8% when the heating time extended to 120 min, deducing

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that the deglycosylation might occur upon heating processing. The studies of Kim et al.

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(2013) were in agreement with this phenomenon, and they found that the

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deglycosylation of ginsenosides Rd contributed to increase anticancer activity of 11

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ginseng by heating process. In summary, Q-CA-FA played the major role in

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antityrosinase activity, so polyphenols were used to conduct the later experiments.

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3.2 Effect of ultrasound time on antityrosinase ability of polyphenols

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Ultrasound treatments have attracted researchers interests due to its low energy

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consumption since 1970s (Awad et al., 2012). Moreover, ultrasound was used to purify

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the polyphenols by adsorption and desorption on the microporous resins. The

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adsorption capacity of total polyphenols after ultrasound treatments (120 W) at 25 °C

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was 3.95 mg/g, which was two times than that obtained after shaking at 120 rpm (Yu,

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Fan, & Li, 2020). Nevertheless, in this paper, combining ultrasound with heating

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treatments was an efficient method to increase the antityrosinase capacity of

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polyphenols. In Section 3.1, the Q-CA-FA after heating at 150℃ for 120 min possessed

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the best antityrosinase ability, but long heating time led to high energy consumption.

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As a result, ultrasound was further investigated in this paper. In Fig. 2A, the relative

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activity of tyrosinase was well inhibited in addition of Q-CA-FA after combining

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heating treatments (150℃ for 30 min) and ultrasound treatments (600 W for 30 min),

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and the tyrosinase inhibition rate can reach 94.9%. The major role induced by

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ultrasound was attributed to the mechanical and chemical impact (Leong et al., 2014;

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Paniwnyk, 2017). However, when ultrasound was applied at 600 W for ultrasound time

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of 60 min and 90 min, inhibition rate of tyrosinase were 94.6% and 93.8%, respectively.

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These data indicated that the relative activity of tyrosinase was almost same under

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different treatment time at 600 W. As the ultrasound time increased, tyrosinase

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inhibition rate did not increase. Thus, ultrasound time of 30 min was considered an 12

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efficient time on Q-CA-FA.

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3.3 Effect of ultrasound power on antityrosinase ability of polyphenols and saponins

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A synergistic effect of heating and ultrasound on enzyme has also been reported

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in other literatures, such as tomato peroxidase (Ercan & Soysal, 2011), polyphenol-

254

oxidase in pear, apple (Sulaiman et al., 2015). Nevertheless, few literatures have been

255

published about the investigation of antityrosinase activity changes after different

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ultrasound power. Based on the results of heating treatments, different concentrations

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of Q-CA-FA treated by various ultrasound power were investigated and exhibited in

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Fig. 2B. The relative activity of tyrosinase in the addition of 5 mM control was 37.2%,

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and the relative activity of tyrosinase decreased to 15.9% and 1.8% under ultrasound

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power of 300 W and 600 W for 30 min, respectively. Interestingly, the Q-CA-FA

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treated at 600 W possessed the highest antityrosinase capacity, indicating that

262

ultrasound power could effectively enhance the antityrosinase ability of polyphenols.

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On the one hand, the temperature of the inhibitors increased with the increasing of

264

ultrasound power, which meant ultrasound could create extreme heat. When the sample

265

was exposed to ultrasound at 600 W for 30 min, the temperature of the solutions was

266

increased to 82℃. In heating treatment experiment, we found that temperature was an

267

important factor associated with antityrosinase ability of polyphenols, so there could be

268

a synergistic effect of temperature and ultrasound on Q-CA-FA. On the other hand, the

269

low and high pressure could be created due to cavitation bubbles of sonication (Chemat

270

et al., 2011; Paniwnyk, 2017). Thus, Q-CA-FA after combining heating (150℃ for 30

271

min) with ultrasound (600 W for 30 min) treatments could enhance the antityrosinase 13

272

ability of polyphenols, and the increasing effect was close to a more intense heating

273

treatment (150℃ for 120 min).

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3.4 Fluorescence quenching of Q-CA-FA after heating and ultrasound treatments

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Fluorescence spectroscopy was applied to study the changes of binding reactions

276

caused by Q-CA-FA after heating and ultrasound treatments and to obtain information

277

about their conformational changes in this paper. Untreated and treated Q-CA-FA after

278

combining heating (150℃ for 30 min) with ultrasound (600 W for 30 min) treatments

279

were further investigated. The excitation wavelength was set as 280 nm, and the

280

fluorescence emission spectroscopy of tyrosinase with different concentrations of

281

untreated and treated Q-CA-FA were exhibited in Fig. 3A and B. Tyrosinase had the

282

strongest fluorescence emission peak at 337 nm, while untreated and treated Q-CA-FA

283

solutions showed little fluorescence absorption (line m and n in Fig. 3A and B). In

284

addition, with increasing concentrations of Q-CA-FA, the fluorescence intensities of

285

tyrosinase decreased gradually, suggesting that the polyphenols played an efficient

286

quenching role in the fluorescence of tyrosinase. This discovery would be direct

287

evidence for the binding reaction between the polyphenols and tyrosinase. At the same

288

time, a red shift of tyrosinase from 335 to 340 nm was found, indicating that inhibitors

289

could interact with tyrosinase and alter the microenvironment around the chromophore

290

tryptophan residues, meanwhile the polarity of enzyme increased but its hydrophobicity

291

decreased (Zhang and Ma, 2013; Zhang et al., 2017).

292

Fluorescence quenching might result from a series of binding reaction, including

293

three major types: dynamic quenching, static quenching, and a combination of them. 14

294

Among them, the collision between the enzyme and inhibitors was the main causes of

295

dynamic quenching (Pushpam et al., 2013). For static quenching, the formation of

296

fluorophore-quencher complex could result in the reduction of emission intensity.

297

Before heating and ultrasound treatments, Q-CA-FA showed a linear Stern-Volmer plot,

298

meaning a single class of fluorophores in tyrosinase (Fig. 3a), which could be equally

299

accessible to the inhibitors (Bose, 2016). The values of KSV at 298, 303, and 310 K

300

were 0.72 × 105, 0.64 × 105, and 0.30 × 105 L mol-1, respectively. This phenomenon

301

also indicated that only static quenching mechanism existed in the binding reaction

302

between untreated Q-CA-FA and tyrosinase rather than a dynamic collision quenching.

303

However, a different formation were observed in the addition of treated Q-CA-FA. The

304

Stern-Volmer plot showed an upward tendency (Fig. 3b) at high concentrations,

305

meanwhile the values of R12 were below 0.99 (Table 1), which could mean that there

306

was a complex binding process between tyrosinase and polyphenols. Two major

307

situations were used to explain the phenomenon. On the one hand, the upward tendency

308

suggested that the fluorophore of tyrosinase could be quenched by both static and

309

dynamic mechanisms (Sun et al., 2016; Zu et al., 2017). On the other hand, the upward

310

tendency indicated the existence of a sphere-of-action (Bose, 2016). It assumed the

311

presence of a sphere of volume around a fluorophore of tyrosinase which a polyphenol

312

could cause quenching a probability of equality. Fluorescence quenching of tyrosinase

313

occurred when the polyphenols were adjacent to the fluorophore. This type of apparent

314

static quenching was called the model “sphere of action”. Based on these results, the

315

modified Stern-Volmer equation (Eq. (5)) was employed to analyze the fluorescence 15

316

quenching mechanism of Q-CA-FA after combining heating and ultrasound treatments.

317

The modified KFQ values for treated Q-CA-FA at 298, 303, and 310 K were 6.52 × 104,

318

6.89 × 104, and 6.93 × 104 L mol-1, respectively (Table 1). The modified Eq. (5) was

319

more suitable for calculating the quenching constants because that all R22 values of

320

treated Q-CA-FA were greater than 0.99. Furthermore, the quenching form could be

321

different due to their dependence on temperature. The Ka values decreased with

322

increasing temperature represented static quenching, and the opposite effect would be

323

found in dynamic quenching, concluding that heating and ultrasound treatments

324

changed the quenching form to some extent. Hence, the KFQ of latter being larger than

325

that of former suggested that treated Q-CA-FA possessed more potential of

326

antityrosinase capacity than untreated Q-CA-FA.

327

3.5 Changes of binding constants and thermodynamic parameters

328

Binding parameters Ka and n were crucial in the study of tyrosinase inhibitors,

329

which were obtained from the slope and intercept of the plots of lg(F0-F)/F vs lg(1/([I]-

330

(F0-F)[E])/F0), and the values were exhibited in Table 1. All the values of lgKa were

331

relative with the number of n with high correlation coefficient (R32 > 0.99), which

332

verified that the mathematical model used in this study was suitable to investigate the

333

interaction of Q-CA-FA with tyrosinase (Liu et al., 2015). The Ka values of treated Q-

334

CA-FA at 298, 303, and 310 K were 1.05 × 105, 1.22 × 105, and 1.13 × 105 L mol-1,

335

respectively, which were larger than that of untreated Q-CA-FA (0.72 × 105, 0.67 ×

336

105, and 0.61 × 105 L mol-1), meaning that a higher affinity existed between treated Q-

337

CA-FA and tyrosinase. Moreover, the Ka values of untreated Q-CA-FA showed a 16

338

decreasing tendency along with the increase of temperature, demonstrating that the

339

stability of polyphenols-tyrosinase complex decreased during the studied temperatures

340

range. Nevertheless, the treated Q-CA-FA-tyrosinase complex was more stable at 303

341

K. When the temperature was 310 K, the treated Q-CA-FA-tyrosinase complex would

342

also be partly decomposed, and then lead to the reduction of Ka values. These data also

343

proved that the fluorescence quenching of untreated Q-CA-FA was a static quenching.

344

The values of n at the studied concentrations were approximately equal to one,

345

indicating that there was one category binding site for polyphenols on enzyme and the

346

inhibitors molecule formed 1 : 1 complex with enzyme molecule (Liu et al., 2015).

347

We also calculate the intermolecular forces and the thermodynamic parameters.

348

The ΔH and ΔS values were obtained from the plots of lgKa vs 1/T (Table 1). These

349

values showed that ΔH did not change significantly within the investigated

350

temperatures range, so it could be regarded as a constant. The positive ΔS values

351

suggested that the complexation had a favorable change of entropy, and an increased

352

randomness occurred in the binding reaction of Q-CA-FA with tyrosinase (Seraj and

353

Rouhani, 2017). Besides, positive ΔS values (109.76 and 127.71 J mol-1 K-1) were

354

frequently considered as typical evidence for hydrophobic interaction. From these

355

results, combining heating and ultrasound treatments of Q-CA-FA did not change the

356

major force between enzyme and inhibitors, but some new compounds might be

357

produced in this solution due to the increased ΔS value (127.71 J mol-1 K-1). The related

358

discussion would be presented in Section 3.6. All ΔG values were negative, indicating

359

that the binding process between tyrosinase and inhibitors was spontaneous. 17

360

3.6 UPLC-MS analysis

361

The UPLC-MS chromatograms of Q-CA-FA upon different treatments conditions

362

were shown in Fig. 4. The chromatograms were recorded 15 min. Peaks were named as

363

Ca (cinnamic acid), Fa (ferulic acid), Que (quercetin), and Co (new detected compound),

364

respectively. The retention time, UV absorption wavelength, parent ions and product

365

ions of the major polyphenols were listed in Table 2. Among them, three polyphenols

366

corresponding to negative molecular ions at m/z 163, 193, and 301 were assigned as

367

cinnamic acid, ferulic acid, and quercetin, respectively. Quercetin was the dominant

368

polyphenols in Q-CA-FA, meanwhile it exhibited the fragmentation patterns at m/z 179

369

and 151 due to the characteristic of quercetin aglycone (Maulidiani et al., 2019).

370

Qualitative analysis revealed variation in phytochemical constituents after combining

371

heating and ultrasound treatments. We found that cinnamic acid and ferulic acid

372

contained in Q-CA-FA decreased (Table 2), deducing that the newly detected

373

constituents might be consisted of cinnamic acid and ferulic acid by esterification

374

reaction. Similar phenomenon was also found in onion (Juániz et al., 2016), which

375

indicated that the antityrosinase activity of samples were enhanced after heating

376

treatments. Co, called as 2-hydroxy-3-(3-hydroxy-4 methoxy-phenyl)-propionic acid

377

4-(2,3-dihydroxy-propyl)-phenyl ester, a newly detected compound, was produced

378

after combining heating and ultrasound treatments, which led to the fragment at m/z

379

361. Peak Co had a λmax at 257 and 375 nm, and parent ion at m/z 361, accompanied by

380

product ions at m/z 300, 249, and 197 for C17H14O9, and the structure was shown in

381

supplementary Fig. F2. Previous studies reported that cinnamic acid ester derivatives 18

382

had the potential in antityrosinase capacity (Sheng et al., 2018). In addition, an efficient

383

method to modify the natural inhibitors was designed by the esterification of cinnamic

384

acid. The new compound might be the characteristic constituents contributing to the

385

increased antityrosinase ability. In particular, this constituent was almost not contained

386

in untreated samples in general but abundantly contained in the samples after heating

387

treatments (150℃ for 30 min), ultrasound treatments (600 W for 30 min), and

388

combining treatments (Fig. 4a-d). Taken together, ultrasound treatments also played an

389

indispensable role in formation of Co. Interestingly, our results firstly discovered that

390

combining treatments could improve the antityrosinase ability, and this phenomenon

391

might be related with Co, which would be further investigated.

392

4. Conclusions

393

In this pioneering study, a potential method to improve the antityrosinase ability

394

of polyphenols from Asparagus was evaluated. It notably increased the antityrosinase

395

activity by combining ultrasound (600 W for 30 min) with heating treatments (150℃

396

for 30 min), and the increasing effect of which was close to longer time heating

397

treatments (150℃ for 120 min). The decrease of fluorescence intensities and the

398

improvement of binding constant, as well as the result of increased randomness,

399

explained the enhancing effect mechanism induced by combining ultrasound and

400

heating treatments. In addition, UPLC-MS analysis indicated that ultrasound treatments

401

also played an important role in the formation of 2-hydroxy-3-(3-hydroxy-4-methoxy-

402

phenyl)-propionic acid 4-(2,3-dihydroxy-propyl)-phenyl ester, which was newly

403

detected and might be the major bioactive substances associated with the increasing 19

404

antityrosinase ability.

405

Acknowledgements

406

This research was subsidized by the Jiangsu Agriculture Science and Technology

407

Innovation Fund (CX(18)3070), the Postgraduate Research & Practice Innovation

408

Program of Jiangsu Province (KYCX-1821), which has enabled us to accomplish this

409

study.

410

References

411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440

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22

526

Figure Captions

527

Fig. 1 Influences of heating temperatures and time on Q-CA-FA (A) (B), the mixture of Q-CA-FA

528

and protodioscin (C) (D), the mixture of Q-CA-FA and dioscin (E) (F).

529

Fig.2 Effect of ultrasound time (A) and ultrasound power (B) on antityrosinase activity of Q-CA-

530

FA.

531

Fig. 3 (A) Flurescence spectra of tyrosinase in addition of Q-CA-FA. (B) Flurescence spectra of

532

tyrosinase in addition of Q-CA-FA after heating (150℃ 30 min) and ultrasound (600 W 30 min)

533

treatments. c (samples) = 0, 2, 4, 6, 8, 10, 12, 14, 16 uM for curves a→i, respectively. Curve m, n

534

are flurescence spectra of samples only (a) (b) The Stern-Volmer plots of F0/F vs [Q-CA-FA] and

535

[Treated Q-CA-FA], respectively.

536

Fig. 4 (A) (B) (C) (D) UPLC-MS chromatograms of Q-CA-FA, Q-CA-FA after heating treatment

537

(150℃ for 30 min), Q-CA-FA after ultrasound treatment (600 W for 30 min), and Q-CA-FA after

538

combining treatments, respectively. (a) (b) (c) (d) UPLC-MS chromatograms of m/z 361 under

539

untreated condition, heating treatment (150℃ for 30 min), ultrasound treatment (600 W for 30 min),

540

and combining treatments, respectively.

23