Enhancing the antityrosinase activity of saponins and polyphenols from Asparagus by hot air coupled with microwave treatments

Journal Pre-proof Enhancing the antityrosinase activity of saponins and polyphenols from Asparagus by hot air coupled with microwave treatments Qun Yu, Jiajing Duan, Nan Yu, Liuping Fan PII:

S0023-6438(20)30162-6

DOI:

https://doi.org/10.1016/j.lwt.2020.109174

Reference:

YFSTL 109174

To appear in:

LWT - Food Science and Technology

Received Date: 28 October 2019 Revised Date:

15 January 2020

Accepted Date: 14 February 2020

Please cite this article as: Yu, Q., Duan, J., Yu, N., Fan, L., Enhancing the antityrosinase activity of saponins and polyphenols from Asparagus by hot air coupled with microwave treatments, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109174. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Credit author statement Qun Yu: Methodology, Data curation, Writing- Original draft preparation. Jiajing Duan: Methodology, Data curation Nan Yu: Methodology, Data curation Liuping Fan: Conceptualization, Supervision, Validation, Writing- Reviewing and Editing.

1

Enhancing the antityrosinase activity of saponins and polyphenols from

2

Asparagus by hot air coupled with microwave treatments

3

Qun Yu, Jiajing Duan, Nan Yu, Liuping Fan*

4

State Key laboratory of Food Science & Technology, School of Food Science and

5

Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China

6

*Corresponding author: Dr., Liuping Fan Professor of School of Food Science and

7

Technology, Jiangnan University, Wuxi 214122, China

8

Tel: 0086-(0) 510-85876799

9

E-mail: [email protected]

1

10

Abstract

11

The impact of heating treatments on antityrosinase capacities of polyphenols

12

(quercetin, cinnamic acid, ferulic acid) and saponins (protodioscin and dioscin) from

13

Asparagus was evaluated. Upon microwave heating (700 W for 20 min) with hot-air

14

heating (120

15

enhanced the antityrosinase capacities due to the deglycosylation. The analysis of

16

fluorescence spectroscopy showed that combined heating increased the affinity

17

between tyrosinase and inhibitors. Thermodynamic calculation indicated that

18

randomness of molecules in the solution systems increased and hydrophobic force

19

was the major force during interactions. In addition, two ester compounds (Est 1 and

20

Est 2) were newly detected using UPLC-TOF/MS, which might be the major

21

bioactive compounds of enhancing antityrosinase capacities. In particular, the addition

22

of saponins induced the formation of Est 1 and Est 2. Taken together, the

23

deglycosylation of saponins and the new compounds contributed to the enhancement

24

of antityrosinase activity. These findings provide promising insight for increasing the

25

antityrosinase abilities of polyphenols and saponins.

26

Keywords:

27 28

for 30 min), the addition of protodioscin and dioscin significantly

Heating treatment, individual polyphenols, steroid saponins, fluorescence quenching, UPLC-MS

2

29

1. Introduction

30

Asparagus officinalis L. is well known due to high nutrition and function values,

31

such as its antioxidant and antityrosinase activities (Yu, Li, & Fan, 2019; Zhang et al.,

32

2018). The main bioactive constituents of Asparagus include polyphenols, steroid

33

saponins, and dietary fiber (Negi, Singh, Joshi, Rawat, & Bisht, 2010; Zhu, Hao, Li,

34

& Li, 2014), which may possess a potential of skin whitening. Our previous studies

35

have proved that the major bioactive compounds related with tyrosinase in Asparagus

36

were polyphenols and saponins, which included quercetin (Que.), cinnamic acid (Ca.),

37

ferulic acid (Fa.), protodioscin (Pd.), and dioscin (Di.) (Fig. 1) (Yu, Li, & Fan, 2019).

38

Thus, they were chosen as tyrosinase inhibitors for the later experiments in this study.

39

Vegetables-based bioactive constituents are commonly used as tyrosinase

40

inhibitors due to its high polyphenols contents and health safety (Chen et al., 2014;

41

Wang et al., 2011; Wang et al., 2014). Nevertheless, these bioactive substances

42

possess lower inhibition ability in natural state. Recently some appropriate heating

43

treatments such as microwave or hot air heating have been applied to enhance the

44

bioactivities (Kim et al., 2013; Kumar & Karim, 2019; Zhang et al., 2018).

45

Microwave heating is widely used in industrial and domestic applications and the

46

major characteristics supporting its applications are high quality, low energy

47

consumption, and easy operation (Guo, Sun, Cheng, & Han, 2017; Joyner, Jones, &

48

Rasco, 2016; Kumar & Karim, 2019). Since the major drawbacks of hot air heating

49

are longer heating time, even high temperatures. A novel heating treatment of

50

combining microwave with hot air treatment was recently employed to improve the 3

51

performance (Kumar, Millar, & Karim, 2015). Fava et al. (2013) found that hot air –

52

microwave drying could preserve the quality of fruit and cereal products. Talens et al.

53

(2017) also concluded that microwave power coupled with hot air drying could

54

enhance the process efficiency. The main strategies used to improve the functionality

55

of food is to increase the particle size and swelling capacity of bioactive substances

56

(Fava et al., 2013; Talens et al., 2017).

57

Although combined heating was recognized as a promising physical process, its

58

effects on tyrosinase inhibitors have not been investigated. This paper aims at

59

investigating the effects of combining microwave with hot air treatment on the major

60

polyphenols and saponins from Asparagus. Fluorescence quenching mechanism,

61

binding and thermodynamic parameters were determined to provide a theoretical basis

62

for the practical application of antityrosinase compounds. In addition, there is a

63

limited amount of papers on the characterization of chemical constituents from the

64

inhibitors after combined heating treatments. Thus, polyphenols and saponins with

65

antityrosinase capacities and their action mechanisms were also investigated in this

66

paper to provide a new insight for the further utilization of Asparagus.

67

2. Materials and methods

68

2.1.Reagents

69

Quercetin,

cinnamic

acid,

ferulic

acid,

kojic

acid,

and

3,

70

4-dihydroxy-L-phenyl-alanine (L-DOPA) were obtained from J&K Scientific Limited

71

Company (Beijing, China). In addition, tyrosinase (EC 1.14.18.1, 128 KDa),

72

protodioscin, and dioscin were obtained from the Shanghai Yuanye Biological 4

73

Technology Co., Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained

74

from the China National Pharmaceutical Foreign Trade Corporation (Shanghai,

75

China).

76

2.2.Preparation of Asparagus stem extract

77

The fresh Asparagus was purchased from Jiangsu Province in November 2018,

78

which were immediately washed with distilled water, freeze-dried, and smashed. 20

79

mL ethanol (90%, v/v) was used to extract the bioactive substances from asparagus

80

powder (1 g). During the extraction process, the solutions were stirred for 40 min at

81

75 . The extraction steps were repeated three times. Then all samples were

82

evaporated to dryness at 50

83

-20

84

2.3.Polyphenols and saponins measurement

and dissolved 10 mL. The samples should be stored at

until analysis (Vázquez-Castilla et al., 2013).

85

The polyphenols contents that might be related with antityrosinase capacities in

86

asparagus were determined by the methods of Yu et al. (2019) with minor

87

modifications. The HPLC system was conducted to measure the individual

88

polyphenols. Solvent A was water with 0.1% formic acid, and solvent B was

89

acetonitrile with 0.1% formic acid. The separation process was as follows: 0-5 min,

90

30% B; 5-25 min, 60% B; 25-30 min, 100% B; 30-35 min, 7% B. The sample

91

injection volume was 20 µL.

92

The Waters Maldi Synapt quadrupole time of flight (Q-TOF) mass spectrometer

93

(Massachusetts, USA) equipped with an ESI ionization source was used to detect the

94

saponins (Yang et al., 2016). Mobile phase A was acetonitrile and mobile phase B was 5

95

0.1% formic acid. The gradient elution was carried out for 15 min and the flow rate

96

was 0.3 mL/min. The gradient eluting system was applied: 0.1 min 5% A, 95% B; 8

97

min 15% A, 85% B; 12 min 21% A, 79% B; 14 min 60% A, 40% B. 15 min 90% A,

98

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

99

system. The mass spectrum conditions were as follows: negative ion modes; capillary

100

voltage, 3.0 kV; source temperature, 100 ; desolvation temperature, 400 ; cone gas

101

flow, 50 L/h; desolvation gas flow, 700 L/h; and cone voltage, 30 V. Data acquisition

102

and analysis were performed with MassLynx 4.1 version.

103

2.4.Relative activity measurement

104

The relative activity of tyrosinase was determined by a method of Yu et al. (2019)

105

with minor modifications. The positive control was kojic acid. The relative activity of

106

the pure tyrosinase was regarded as 100%. The percent relative activity of tyrosinase

107

was analyzed as followed:

108 109

Relative activity % = B2 -B1

A2 -A1 ×100

(1)

Where A1, A2 denote that the absorbance of the blank at 0, 10 min, respectively.

110

B1, B2 denote that the absorbance of the samples at 0, 10 min, respectively.

111

2.5.Combined heating processing model experiment

112

Our previous study has proved that the combination of 5 mM quercetin, ferulic

113

acid, cinnamic acid (named Group 11) presented strong effects on tyrosinase activity

114

(Yu, Fan, & Duan, 2019). In addition, we have found that saponin was a crucial and

115

representative constituent in Asparagus (Yu, Li, & Fan, 2019). Thus, they were

116

chosen for the later experiments and different combinations of polyphenols and 6

117

saponins were shown in Table 1. Hot air treatments were carried out in a drying oven

118

(Binder, Germany), heating at 120

119

heating, 200 µL samples were poured in brown bottle (diameter 10 mm, height 40

120

mm). Then the samples were put into the microwave oven (700 W for 20 min).

121

Solutions were transferred into a 2 mL volumetric flask with DMSO and stored at 4

122

for further analysis.

123

2.6. Fluorescence measurements

124

for 30, 60, and 120 min, respectively. After

In order to investigate the affinity between tyrosinase and bioactive constituents

125

(polyphenols

126

spectrophotometer (RiLi, Japan) was employed to determine the fluorescence

127

intensity, which is equipped with a 450 W Xe lamp and a heated water bath. The

128

excitation wavelength was set as 280 nm, and the bath was set as three temperatures

129

(298, 303 and 310 K) with a scanning wavelength change from 290 to 500 nm. All of

130

the fluorescence values need to be corrected because of the presence of inner-filter

131

effect, and the related equation is as follows (Peng, Ding, Jiang, Sun, & Peng, 2014):

132

and

saponins)

from

asparagus,

an

F-7000

fluorescence

Fc =Fm e(A1 +A2 )⁄2

(2)

133

Where Fc denotes the corrected fluorescence intensity and Fm denotes the

134

detected fluorescence intensity. A1 is the absorbance of the inhibitor at excitation

135

wavelength and A2 denotes the absorbance of the inhibitors at emission wavelength.

136

The Stern-Volmer equation was used to analyze the values of KSV and Kq:

137 138

⁄ =1+KSV Q =1+Kq τ0 [Q]

(3)

Where Ksv denotes the quenching constant, Kq denotes the quenching rate 7

139

constant,

140

concentrations of the inhibitors.

141 142

The Stern-Volmer equation suitable for this mode is as follows: ⁄ = 1+

143 144 145

=10-8 s(Wang, Zhang, Yan, & Gong, 2014), and [Q] denotes the

(4)

Where V denotes the volume of the sphere, and N denotes the Avogadro’s constant. When the K[Q] is about zero, the Eq. (4) becomes the another equation: ⁄ =

(5)

146

Where KFQ denotes the apparent static quenching constant.

147

The double logarithm regression curve was employed to obtain the binding

148

constant (KA) and binding sites (n) (Nan et al., 2019).

149

log

150

− ⁄

=nlogKa +nlog

(6)

Where Ka means the apparent binding constant. n denotes the binding sites. [P]

151

denotes the concentration of tyrosinase.

152

2.7. Thermodynamic parameters

153

The binding reaction of inhibitors with enzyme is related to the temperature, thus

154

the thermodynamic parameters were calculated to account for this interaction (Fan et

155

al., 2013). Eq. (7) and Eq. (8) were used to investigate the binding forces between

156

tyrosinase and inhibitors:

157

∆G=∆H-T∆S

158

lg

159 160

∆# ! =- $.& &'(

(7) ∆)

+ $.&

&'

(8)

Where R is equal to 8.314 J/ (mol·K); T denotes the temperature, K. 2.8. Statistical analysis 8

161

The experimental results were repeated three times except special explanation.

162

The values were presented as mean values ± SD for each measurement. The analysis

163

was accomplished with SPSS software (SPSS 20.0; IBM, Chicago, USA).

164

3. Results and discussion

165

3.1.Changes of the antityrosinase activities induced by combined heating

166

Three individual polyphenols and two saponins in Asparagus were screened due

167

to their antityrosinase capacities (Fig. 1). Our previous studies indicated that obvious

168

antityrosinase ability was found in the mixtures of quercetin, ferulic acid, cinnamic

169

acid, protodioscin, and dioscin (Yu, Fan, & Duan, 2019; Yu, Li, & Fan, 2019).

170

Nevertheless, protodioscin and dioscin showed little antityrosinase ability when they

171

were added alone. Fig. 2 showed the relative activities of tyrosinase in the addition of

172

various samples after combined heating treatment. Without combined heating

173

treatments, the relative activities of tyrosinase were 70-80%; however, lower activities

174

of tyrosinase were observed after combined heating treatment. More specifically, the

175

addition of heat-treated protodioscin and dioscin at 500 µg/mL decreased the relative

176

activity of tyrosinase by 24.5% and 35.5%, respectively (Fig. 2B and C). These data

177

suggested that dioscin and protodioscin might be the key bioactive components of the

178

antityrosinase activities. The structural difference between protodioscin and dioscin

179

was less glucopyranoside on the dioscin (Dawid & Hofmann, 2012), and the

180

antityrosinase of dioscin was stronger than that of protodioscin, indicating that the

181

glucopyranoside was a crucial structure in its antityrosinase capacity. We speculated

182

that the presence of glucopyranoside group should have an adverse effect on 9

183

antityrosinase activity of saponins. Furthermore, we found that deglycosylation would

184

be favorable to the antityrosinase capacity. That is to say, combined heating

185

treatments might lead to the deglycosylation of saponins.

186

3.2.Fluorescence quenching

187

As shown in Fig. 3A-C, with increasing the tested polyphenols and saponins

188

concentrations, the fluorescence intensity of tyrosinase decreased regularly,

189

suggesting that they could react with tyrosinase. The maximum emission wavelength

190

of tyrosinase was 334 nm, while the addition of polyphenols and saponins induced a

191

red shift of maximum emission wavelength (from 334 to 339 nm). The process that

192

fluorophores were replaced from the less-polar interior of the tyrosinase to

193

solution-exposed regions upon unfolding was the major reason for red shift. The

194

above phenomenon was also found in α-amylases. Tea polyphenols could induce

195

red-shift of UV absorbance and fluorescence quenching of α-amylase, indicating

196

possible changes in the structure of α-amylase (Fei et al., 2014). A comparable

197

process in fluorescence emission spectra was discovered in samples after combined

198

heating treatments (Fig. 3D-F). The addition of heat-treated polyphenols and saponins

199

exhibited λmax around 342 nm, which denoted much larger red shifts. The

200

new-produced constituents most probably explained this feature. Strong interactions

201

between the tyrosinase and inhibitors (polyphenols and saponins) and the relevant

202

changes in the microenvironment would be expected.

203

The decrease of fluorescence intensities induced by enzyme interactions with

204

quencher molecules was called fluorescence quenching (Zu et al., 2017). Static 10

205

quenching is the most common fluorescence quenching mechanism, in addition,

206

dynamic quenching and combined quenching of dynamic and static quenching are

207

also found in some published papers (Nan et al., 2019; Sun et al., 2016). Nan et al.

208

(2019) discovered that the binding mechanisms between bovine serum albumin and

209

graphene oxide was static quenching combined with dynamic quenching. In some

210

situations, the figure of F0/F vs [Q] exhibits a curve towards the vertical coordinates,

211

meaning that the tyrosinase might be quenched either by dynamic and static

212

quenching or by sphere-of-action (Castanho & Prieto, 1998; Nan, Hao, Ye, Feng, &

213

Sun, 2019; Sun et al., 2016). The static quenching denotes the fluorescence intensity

214

decrease due to the generation of weak-fluorescence compound. Dynamic quenching

215

means that the collision between tyrosinase and inhibitor results in the decrease of

216

fluorescence intensity, and this process will cause the loss of excitation energy, and

217

make tyrosinase return to the ground state from the excited state. Generally, the Eq. (3)

218

was used to calculate the quenching constant (KSV). The linear plot of F0/F vs

219

[Samples] at room temperature was shown in Fig. 3a, and KSV was recorded in Table

220

2. We can know that the values of the samples 4-6 (1.474, 2.090, 1.906×105 L/mol)

221

were higher than samples 1-3 (1.453, 1.389, 1.228 ×105 L/mol), implying that

222

heat-treated polyphenols and saponins possessed stronger quenching. The values of

223

R12 were ≤ 0.99, which suggested the Stern-Volmer plot was not linear at higher

224

concentrations and have a tendency toward the vertical axis. Thus, tyrosinase might

225

be quenched by dynamic and static modes, or a sphere-of-action might be conducted.

226

To discuss the relationship between ln (F0/F) vs [Samples] more accurately, corrected 11

227

plot was shown in Fig. 3b, and the values of KFQ were summarized in Table 2. The

228

quenching mechanism shows that the quenching is due to the interactions between

229

tyrosinase and quenchers (Cai, Yu, Xu, Liu, & Yang, 2015; Sun, Gidley, & Warren,

230

2018). The calculated KFQ values of samples 4-6 (6.458, 7.69, 7.79×104 L/mol) were

231

much greater than that of samples 1-3 (6.538, 6.154, 5.866×104 L/mol), suggesting

232

that heating treatment can enhance the inhibitory ability of polyphenols and saponins,

233

which was consistent with our previous findings.

234

3.3.Binding parameters

235

Another corrected constant Ka was determined based on Eq. (6), which possesses

236

similar implication with the binding constant and can be an index of the affinity

237

between tyrosinase and inhibitors. As shown in Table 2, the Ka values of tyrosinase

238

with the polyphenols and saponins were in the range of 1.09 to 2.11 × 105 L/mol.

239

Considering that the samples were consisted of polyphenols and saponins, these data

240

were higher than that reported by other researchers. For example, previous work

241

indicated that cardanols had high binding affinity to tyrosinase (Ka was equal to

242

1584.9 L/mol) (Yu et al., 2016). And the value of Ka for glabridin-tyrosinase

243

interactions was 2.56 × 104 L/mol (Chen, Yu, & Huang, 2016). These observations

244

indicated that the affinity of the groups were stronger than that of a single constituent

245

inhibitor. In samples 4-6, binding constant Ka exhibited 1.30, 1.63, and 1.20 × 105

246

L/mol, respectively. These data were higher than those calculated in samples 1-3 (1.20,

247

1.22, and 1.09 × 105 L/mol), indicating that combined heating treatments could cause

248

an enhancement in the tyrosinase binding with inhibitors. Moreover, the values of Ka 12

249

showed a decrease with increasing temperatures, indicating the higher temperature

250

might affect the microenvironment of binding sites. The amino acid residues of

251

binding sites might be exposed at 310 K (Silva, Cortez, & Louro, 2004). Therefore,

252

the interaction of tyrosinase and polyphenols (or saponins) induced the

253

conformational changes in enzyme, which affected the secondary and tertiary

254

structure of tyrosinase. The values of n were about equal to one, which suggested that

255

there was only one category binding site in tyrosinase for inhibitors (Liu et al., 2015).

256

It was accounted for that tyrosinase contained multiple binding regions, which

257

possessed the same binding site.

258

3.4.Thermodynamic calculation

259

For affirming the binding forces between tyrosinase and inhibitors, the Eq. (7)

260

and Eq. (8) were established to calculate the values of ∆H, ∆S, and ∆G. Based on the

261

thermodynamic parameters, the binding mode can be determined. In order to clarify

262

the binding between the tyrosinase and the polyphenols (or saponins), the interaction

263

process were carried out at three temperatures (298, 303, and 310 K). ∆H was

264

regarded as a constant because that it does not vary significantly under the tested

265

temperatures. The thermodynamic parameters for the interaction were included in

266

Table 2. All values of ∆G were negative, suggesting that the reaction processes

267

between tyrosinase and samples were spontaneous. ∆S represented the randomness of

268

molecules in the solution systems. Significant variations of ∆S values were observed

269

in samples 4-6 than samples 1-3, indicating that there might be richer components in

270

samples 4-6. Some further results would be discussed in Section 3.5. All values of ∆H 13

271

were positive, meaning that the major interaction force of tyrosinase and samples was

272

hydrophobic force (Arroyo-Maya, Campos-Terán, Hernández-Arana, & McClements,

273

2016). The ∆H values of samples 5-6 were 21.06 and 15.41 KJ/mol, which were

274

larger than that of samples 1-4, suggesting that an additional contribution to the

275

enthalpy change was due to the addtion of heat-treated protodioscin and dioscin.

276

3.5. Changes in the chemical composition induced by combined heating

277

The HPLC chromatograms were shown in Fig. 4, and the UV and MS

278

information was listed in Table 3. Three polyphenols, two saponins, and two newly

279

compounds

280

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

281

cinnamic acid, ferulic acid, and quercetin, respectively. Two saponins corresponding

282

to negative molecular ions at m/z 1047 and 867 were assigned as protodioscin and

283

dioscin. Dioscin, a steroid saponin, was too stable to produce the MS ions during the

284

fragmentation, which led to the unchanged fragment at m/z 867. Que., Ca., Fa., Pd.,

285

and Di. were detected at 7.71, 4.84, 5.49, 7.75, 11.57 min, respectively. After

286

combined heating, Est1 and Est2 were newly detected (Fig. 4B-D), which showed a

287

negative molecular ion at m/z 601 and 361. Interestingly, cinnamic acid contained in

288

sample 4 decreased but the contents of ferulic acid increased (Fig. 4B). The peaks of

289

Est1 and Est2 from sample 5 and 6 greatly increased than that of sample 4. That is to

290

say, saponins play a crucial role in the reaction. Related reports were not published

291

except ginseng, suggesting that deglycosylation of ginsenoside Rd contributes to

292

enhanced anticancer activity by heat processing (Kim et al., 2013). In addition, the

were

tentatively

identified.

Among

them,

three

polyphenols

14

293

concentration of cinnamic acid decreased by 0.3 mg/mL in sample 4 and the

294

concentration of ferulic acid decreased by 0.11 and 0.27 mg/mL in sample 5 and 6,

295

but quercetin presented higher thermal stability than other polyphenols and remained

296

the similar contents after combined heating (Table 3). These results indicated that

297

newly detected substances (Est1 and Est2) might be the product of esterification of

298

cinnamic acid and ferulic acid. Similar phenomenon was also found in onion (Juániz

299

et al., 2016). Quercetin derivates possessed higher thermal stability than isorhamnetin

300

derivates. These findings indicated that heating treatment provided polyphenols and

301

saponins from Asparagus stronger antityrosinase activity and changes in the chemical

302

compositions.

303

3.6.Changes in the chemical structure induced by combined heating

304

We have designed combined heating model experiments using polyphenols and

305

saponins to testify the changes in the structures of each bioactive substances during

306

the heating process and their contributions to the increased antityrosinase activity,

307

which have not been illustrated until now. The mass spectrum was conducted to

308

characterize the newly detected compounds. Fig. 5 exhibited the fragmentation

309

pathway and tentative structures of Est1 and Est2. MS fragmentation suggested that

310

they might belong to the same structural family (Li et al., 2017). The MS spectra of

311

Est1 exhibited ions at m/z 601 and 299 for C30H18O14 (Table 3), suggesting that Est1

312

might be a compound produced by esterification reaction. The strong UV absorption

313

peak was at 249 nm and the small peak of lower intensity was at 307 nm, indicating

314

that the Est1 might be flavanone or dihydroflavonol (Zhong et al., 2019). Thus, one 15

315

potential candidate compound could correspond to Est1 and the structure was

316

exhibited in Fig. 5a. Peak Est2 had a λmax at 254 nm, and parent ion was at m/z 361,

317

accompanied by product ions at m/z 300 for C17H14O9 (Fig. 5b). The new compounds

318

might be the characteristic constituents contributing to the increased antityrosinase

319

ability. These constituents were not contained in Asparagus in general but abundantly

320

contained in the samples after combined heating. Interestingly, our results firstly

321

discovered that heating could improve the antityrosinase ability of polyphenols and

322

saponins, and this phenomenon might be related with Est1 and Est2, which needed

323

further investigation.

324

4. Conclusions

325

Combined heating tended to enhance the antityrosinase capacity of polyphenols

326

and saponins from Asparagus. The polyphenols and saponins after heating treatments

327

had stronger quenching effects on the intrinsic fluorescence of tyrosinase in static and

328

dynamic quenching manner. The major interaction force of tyrosinase and samples

329

was hydrophobic force. UPLC-MS suggested that there are some new compounds

330

after heating. Besides, the deglycosylation of saponins contributed to enhance

331

antityrosinase ability of polyphenols and saponins. In brief, polymers Est 1 and Est 2

332

were the major bioactive compounds of increasing antityrosinase ability. This study

333

probed a novel method of increasing the antityrosinase capacities of polyphenols and

334

saponins, which might provide the theoretical basis for the activity screening and

335

structural modification of Asparagus as efficient tyrosinase inhibitors and be helpful

336

to promote the applications of polyphenols and saponins in food preservation and 16

337

dietary adjuvant treatment of pigmentation disorders.

338

Acknowledgements

339

This research was subsidized by the Jiangsu Agriculture Science and Technology

340

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

341

Program of Jiangsu Province (KYCX-1821), College Students' innovation project of

342

Jiangnan University, which has enabled us to accomplish this study.

343

References

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373

Arroyo-Maya, I. J., Campos-Terán, J., Hernández-Arana, A., & McClements, D. J. (2016). Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins. Food Chemistry, 213, 431-439. https://doi.org/10.1016/j. foodchem.2016.06.105. Cai, X., Yu, J., Xu, L., Liu, R., & Yang, J. (2015). The mechanism study in the interactions of sorghum procyanidins trimer with porcine pancreatic α-amylase. Food Chemistry, 174, 291-298. https://doi.org/10.1016/j.foodchem.2014.10.131. Castanho, M. A. R. B., & Prieto, M. J. E. (1998). Fluorescence quenching data interpretation in biological systems: The use of microscopic models for data analysis and interpretation of complex systems. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1373(1), 1-16. https://doi.org/10. 1016/S0005-2736(98)00081-9. Chen, J., Sun, H., Tao, X., Wang, S., & Sun, A. (2014). Inhibitory Mechanism and Kinetics Study of Apple Polyphenols on the Activity of Tyrosinase. International Journal of Food Properties, 17(8), 1694-1701. https://doi:10.1080/10942912.2012.675606. Chen, J., Yu, X., & Huang, Y. (2016). Inhibitory mechanisms of glabridin on tyrosinase. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 168, 111-117. https://doi.org/10. 1016/j.saa.2016.06.008. Dawid, C., & Hofmann, T. (2012). Structural and Sensory Characterization of Bitter Tasting Steroidal Saponins from Asparagus Spears (Asparagus officinalis L.). Journal of Agricultural and Food Chemistry, 60(48), 11889-11900. https://doi:10.1021/jf304085j. Fan, M., Zhang, G., Pan, J., & Gong, D. (2017). An inhibition mechanism of dihydromyricetin on tyrosinase and the joint effects of vitamins B6, D3 or E. Food & Function, 8(7), 2601-2610. https://doi:10.1039/C7FO00236J. Fan, Y., Zhang, S., Wang, Q., Li, J., Fan, H., & Shan, D. (2013). Interaction of an amino-functionalized ionic liquid with enzymes: A fluorescence spectroscopy study. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 105, 297-303. https://doi.org/10.1016/j.saa.2012. 12.038. Fava, F., Zanaroli, G., Vannini, L., Guerzoni, E., Bordoni, A., Viaggi, D,...Brendle, H. G. (2013). New advances in the integrated management of food processing by-products in Europe: sustainable exploitation of fruit and cereal processing by-products with the production of new food 17

374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417

products. New Biotechnology, 30, 647-655. https://doi.org/10.1016/j.nbt.2013.05.001. Fei, Q., Gao, Y., Zhang, X., Sun, Y., Hu, B., Zhou, L., Zeng, X. (2014). Effects of Oolong Tea Polyphenols, EGCG, and EGCG3″Me on Pancreatic α-Amylase Activity in Vitro. Journal of Agricultural and Food Chemistry, 62(39), 9507-9514. https://doi:10.1021/jf5032907. Guo, Q., Sun, D. W., Cheng, J. H., & Han, Z. (2017). Microwave processing techniques and their recent applications in the food industry. Trends in Food Science & Technology, 67, 236-247. https://doi.org/10.1016/j.tifs.2017.07.007. Joyner, H. S., Jones, K. E., & Rasco, B. A. (2016). Microwave Pasteurization of Cooked Pasta: Effect of Process Parameters on Texture and Quality for Heat-and-Eat and Ready-to-Eat Meals. Journal

of

Food

Science

(John

Wiley

&

Sons,

Inc.),

81(6),

E1447-E1456.

https://doi:10.1111/1750-3841.13334. Juániz, I., Ludwig, I. A., Huarte, E., Pereira-Caro, G., Moreno-Rojas, J. M., Cid, C., & De Peña, M.-P. (2016). Influence of heat treatment on antioxidant capacity and (poly)phenolic compounds of selected vegetables. Food Chemistry, 197, 466-473. https://doi.org/10.1016/j.foodchem. 2015.10.139. Kim, Y. J., Yamabe, N., Choi, P., Lee, J. W., Ham, J., & Kang, K. S. (2013). Efficient Thermal Deglycosylation of Ginsenoside Rd and Its Contribution to the Improved Anticancer Activity of Ginseng. Journal of Agricultural and Food Chemistry, 61(38), 9185-9191. https://doi: 10.1021/jf402774d. Kumar, C., & Karim, M. A. (2019). Microwave-convective drying of food materials: A critical review. Critical Reviews in Food Science and Nutrition, 59(3), 379-394. https://doi:10.1080/10408398. 2017.1373269. Kumar, C., Millar, G., & Karim, M. A. (2015). Effective Diffusivity and Evaporative Cooling in Convective Drying of Food Material. Drying Technology, 33(2), 227-237. https://doi:10. 1080/07373937.2014.947512. Li, Y. F., Ouyang, S. H., Chang, Y. Q., Wang, T. M., Li, W. X., Tian, H. Y., He, R. R. (2017). A comparative analysis of chemical compositions in Camellia sinensis var. puanensis Kurihara, a novel Chinese tea, by HPLC and UFLC-Q-TOF-MS/MS. Food Chemistry, 216, 282-288. https://doi.org/10.1016/j.foodchem.2016.08.017. Liu, B. M., Zhang, J., Bai, C. L., Wang, X., Qiu, X. Z., Wang, X. L., Liu, B. (2015). Spectroscopic study on flavonoid–drug interactions: Competitive binding for human serum albumin between three flavonoid compounds and ticagrelor, a new antiplatelet drug. Journal of Luminescence, 168, 69-76. https://doi:10.1016/j.jlumin.2015.07.037. Nan, Z., Hao, C., Ye, X., Feng, Y., & Sun, R. (2019). Interaction of graphene oxide with bovine serum albumin: A fluorescence quenching study. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 210, 348-354. https://doi.org/10.1016/j.saa.2018.11.028. Negi, J. S., Singh, P., Joshi, G. P., Rawat, M. S., & Bisht, V. K. (2010). Chemical constituents of Asparagus. Pharmacognosy reviews, 4(8), 215-220. https://doi:10.4103/0973-7847.70921. Peng, W., Ding, F., Jiang, Y. T., Sun, Y., & Peng, Y. K. (2014). Evaluation of the biointeraction of colorant flavazin with human serum albumin: insights from multiple spectroscopic studies, in silico docking and molecular dynamics simulation. Food & Function, 5(6), 1203-1217. https://doi:10.1039/C3FO60712G. Silva, D., Cortez, C. M., & Louro, S. R. W. (2004). Chlorpromazine interactions to sera albumins: A study by the quenching of fluorescence. Spectrochimica Acta Part A: Molecular & 18

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461

Biomolecular Spectroscopy, 60(5), 1215. https://doi:10.1016/j.saa.2003.08.003. Sun, L., Chen, W., Meng, Y., Yang, X., Yuan, L., & Guo, Y. (2016). Interactions between polyphenols in thinned young apples and porcine pancreatic α-amylase: Inhibition, detailed kinetics and fluorescence quenching. Food Chemistry, 208, 51-60. https://doi.org/10.1016/j.foodchem. 2016.03.093. Sun, L., Gidley, M. J., & Warren, F. J. (2018). Tea polyphenols enhance binding of porcine pancreatic α-amylase with starch granules but reduce catalytic activity. Food Chemistry, 258, 164-173. https://doi.org/10.1016/j.foodchem.2018.03.017. Talens, C., Arboleya, J. C., Castro-Giraldez, M., Fito, P. J. (2017). Effect of microwave power coupled with hot air drying on process efficiency and physico-chemical properties of a new dietary fibre ingredient obtained from orange peel. LWT - Food Science and Technology, 77, 110-118. https://doi.org/10.1016/j.lwt.2016.11.036. Vázquez-Castilla, S., Jaramillo-Carmona, S., Fuentes-Alventosa, J. M., Jiménez-Araujo, A., Rodriguez-Arcos, R., Cermeño-Sacristán, P., Guillén-Bejarano, R. (2013). Optimization of a Method for the Profiling and Quantification of Saponins in Different Green Asparagus Genotypes.

Journal

of

Agricultural

and

Food

Chemistry,

61(26),

6250-6258.

https://doi:10.1021/jf401462w. Wang, B. S., Chang, L. W., Wu, H. C., Huang, S. L., Chu, H. L., & Huang, M. H. (2011). Antioxidant and antityrosinase activity of aqueous extracts of green asparagus. Food Chemistry, 127(1), 141-146. https://doi.org/10.1016/j.foodchem.2010.12.102. Wang, Y., Curtis-Long, M. J., Lee, B. W., Yuk, H. J., Kim, D. W., Tan, X. F., & Park, K. H. (2014). Inhibition of tyrosinase activity by polyphenol compounds from Flemingia philippinensis roots.

Bioorganic

&

Medicinal

Chemistry,

22(3),

1115-1120.

https://doi.org/10.1016/j.bmc.2013. 12.047. Wang, Y., Zhang, G., Yan, J., & Gong, D. (2014). Inhibitory effect of morin on tyrosinase: insights from spectroscopic and molecular docking studies. Food Chemistry, 163(3), 226-233. https://doi. org/10.1016/j.foodchem.2014.04.106. Yang, J., Wang, P., Wu, W., Zhao, Y., Idehen, E., & Sang, S. (2016). Steroidal Saponins in Oat Bran. Journal of Agricultural and Food Chemistry, 64(7), 1549-1556. https://doi:10.1021/acs. jafc.5b06071. Yu, Q., Li, J., & Fan, L. (2019). Effect of Drying Methods on the Microstructure, Bioactivity Substances, and Antityrosinase Activity of Asparagus Stems. Journal of Agricultural and Food Chemistry, 67(5), 1537-1545. https://doi:10.1021/acs.jafc.8b05993. Yu, Q., Fan, L., & Duan Z. (2019). Five individual polyphenols as tyrosinase inhibitors: Inhibitory activity, synergistic effect, action mechanism, and molecular docking. Food Chemistry, 297, 124910. https://doi.org/10.1016/j.foodchem.2019.05.184. Yu, X. P., Su, W. C., Wang, Q., Zhuang, J. X., Tong, R. Q., Chen, Q. X., & Chen, Q. H. (2016). Inhibitory mechanism of cardanols on tyrosinase. Process Biochemistry, 51(12), 2230-2237. https://doi.org/10.1016/j.procbio.2016.09.019. Zhang, L., Zhao, X., Tao, G. J., Chen, J., & Zheng, Z. P. (2017). Investigating the inhibitory activity and mechanism differences between norartocarpetin and luteolin for tyrosinase: A combinatory kinetic study and computational simulation analysis. Food Chemistry, 223, 40-48. https://doi.org/10.1016/j.foodchem.2016.12.017. Zhang, R., Qiu, W., Zhang, M., Ho Row, K., Cheng, Y., & Jin, Y. (2018). Effects of different heating 19

462 463 464 465 466 467 468 469 470 471 472 473 474

methods on the contents of nucleotides and related compounds in minced Pacific white shrimp and Antarctic krill. LWT - Food Science & Technology, 87, 142-150. https://doi:10.1016/j.lwt. 2017.08.078. Zhong, L., Wu, G., Fang, Z., Wahlqvist, M. L., Hodgson, J. M., Clarke, M. W., Johnson, S. K. (2019). Characterization of polyphenols in Australian sweet lupin (Lupinus angustifolius) seed coat by HPLC-DAD-ESI-MS/MS. Food Research International, 116, 1153-1162. https://doi.org/10. 1016/j.foodres.2018.09.061. Zhu, G. L., Hao, Q., Li, R. T., & Li, H. Z. (2014). Steroidal saponins from the roots of Asparagus cochinchinensis. Chinese Journal of Natural Medicines, 12(3), 213-217. https://doi.org/10. 1016/S1875-5364(14)60035-2. Zu, F., Yan, F., Bai, Z., Xu, J., Wang, Y., Huang, Y., & Zhou, X. (2017). The quenching of the fluorescence of carbon dots: A review on mechanisms and applications. Microchimica Acta, 184(7), 1899-1914. https://doi:10.1007/s00604-017-2318-9.

20

Figure Captions Fig. 1 Separation of (a) cinnamic acid, (b) ferulic acid, (c) quercetin, (d) protodioscin, and (e) dioscin by HPLC and UPLC-MS. Fig. 2 Comparison in the antityrosinase activity of (A) Group 11, (B) Group 11 and protodioscin, and (C) Group 11 and dioscin. The combined heat-processing treatment represents microwave 700 W for 20 min and 120

for 30 min.

Fig. 3 (A) (B) (C) Fluorescence spectra of tyrosinase in Group 11, Group 11 + protodioscin, and Group 11 + dioscin. (D) (E) (F) Fluorescence spectra of tyrosinase in Group 11, Group 11 + protodioscin, and Group 11 + dioscin after combined heating. Curve m, n, p, q, s, and t denote the emission spectrum of samples only. c (samples) = 0, 2, 5, 7, 10, 12, 15, 17, 20 uM for curves a→i, respectively. (a) The Stern-Volmer plots of F0/F vs [Samples]. (b) The corrected Stern-Volmer plots of ln(F0/F) vs [Samples]. Fig. 4 Comparison in the UPLC-MS chromatograms of polyphenols and saponins under different conditions. (A) UPLC-MS chromatograms of Group 11, Protodioscin, and Dioscin before heating treatment. (B) UPLC-MS chromatograms of Group 11 after heating treatment. (C) UPLC-MS chromatograms of Group 11 and protodioscin after heating treatment. (D) UPLC-MS chromatograms of Group 11 and dioscin after heating treatment. Fig. 5 Mass spectrum and proposed fragmentation structures of Est1 (a) and Est2 (b).

21

Table 1 Combinations and conditions of the polyphenols and saponins in 200 µL DMSO. Polyphenols/ Saponins

Sample 1 (mg)

Sample 2 (mg)

Sample 3 (mg)

Sample 4 (mg)

Sample 5 (mg)

Sample 6 (mg)

Quercetin Cinnamic acid Ferulic acid Protodioscin Dioscin

3 1.6 1.9 / /

3 1.6 1.9 1 /

3 1.6 1.9 / 1

3 1.6 1.9 / /

3 1.6 1.9 1 /

3 1.6 1.9 / 1

Conditions

/

/

/

Heating

Heating

Heating

Table 2 Fluorescence quenching, binding, and thermodynamics parameters Parameters

Sample 1

KSV (×105 L/mol） Fluorescence quenching parameters

R12

0.983

KFQ (×104 L/mol） R22

Ka (×10 L/mol) 5

Binding parameters

n △H （KJ/mol） Thermodyna mics parameters

△G （KJ/mol） △S (J mol-1 K-1)

1.453 ± 0.07

6.538 ± 0.22

Sample 2 1.389 ± 0.01 0.939 6.154 ± 0.25

Sample 3 1.228 ± 0.09 0.979 5.866 ± 0.19

Sample 4 1.474 ± 0.07 0.984 6.458 ± 0.23 0.991

Sample 5

Sample 6

2.090 ± 0.02

1.906 ± 0.02

0.950

0.948

7.690 ± 0.30

7.790 ± 0.41

0.999

0.991

1.63 ± 0.037

1.20 ± 0.039

1.74 ± 0.034

1.38 ± 0.027

2.11 ± 0.052

1.43 ± 0.015

1.03 ± 0.06 1.09 ± 0.05 1.19 ± 0.04

1.09 ± 0.05 1.06 ± 0.08 1.06 ± 0.06

0.992

0.988

0.993

298 K

1.20 ± 0.039

1.21 ± 0.032

1.09 ± 0.024

303 K

1.21 ± 0.021

1.22 ± 0.027

1.12 ± 0.042

310 K

1.25 ± 0.024

1.33 ± 0.021

1.17 ± 0.031

298 K 303 K 310 K

1.09 ± 0.05 1.15 ± 0.08 1.11 ± 0.08

1.11 ± 0.07 1.08 ± 0.07 1.07 ±0.10

1.15 ± 0.08 1.04 ± 0.05 1.06 ± 0.03

1.30 ± 0.024 1.33 ± 0.040 1.37 ± 0.042 1.13 ± 0.08 1.09 ± 0.04 1.13 ± 0.05

2.31 ± 0.21

6.78 ± 0.43

5.90 ± 0.21

2.54 ± 0.27

21.06 ± 0.23

15.41 ± 0.65

-26.40 -26.88 -27.55

-28.98 -29.58 -30.42

-28.74 -29.32 -30.13

-29.20 -29.73 -30.48

-29.69 -30.54 -31.73

-29.01 -29.75 -30.80

170.2 ± 1.42

149.0 ± 0.69

298 K 303 K 310 K 298 K 303 K 310 K 298 K 303 K 310 K

96.3 ± 0.76

120.0 ± 0.90

116.2 ± 0.22

106.5 0.68

±

Sample 1 denotes the mixture of quercetin, ferulic acid, and cinnamic acid. Sample 2 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and protodioscin. Sample 3 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and dioscin. Sample 4 denotes the mixture of quercetin, ferulic acid, and cinnamic acid after heating treatments. Sample 5 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and protodioscin after heating treatments; Sample 6 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and dioscin after heating treatments.

Table 3 List of chemical compounds by UPLC-MS Peak name

Control (mg/mL)

Sample 4 (mg/mL)

Sample 5 (mg/mL)

Sample 6 (mg/mL)

tR (min)

UV (nm)

Parent ion (m/z)

Molecular formula

Product ions (m/z)

Proposed compound

Ca Fa Que Pd Di Est1 Est2

0.80 0.90 1.50 0.50 0.50 -

0.50 1.05 1.52 -

1.00 0.79 1.51 0.51 -

0.97 0.63 1.51 0.35 -

4.84 5.49 7.71 7.75 11.57 9.39 10.10

310 323 372 nd nd 249, 307, 373 254, 373

163[M-H]193[M-H]301[M-H]1047[M-H]867[M-H]601[M-H]361[M-H]-

C9H8O3 C10H10O4 C15H10O7 C51H84O22 C45H72O16 C30H18O14 C17H14O9

119 161, 134 178, 151 905, 603 867 299 300

4-Hydroxycinnamic acid trans-ferulic acid quercetin protodioscin dioscin ester 1 ester 2

Sample 4 denotes the mixture of quercetin, ferulic acid, and cinnamic acid after heating treatments. Sample 5 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and protodioscin after heating treatments; Sample 6 denotes the mixture of quercetin, ferulic acid, cinnamic acid, and dioscin after heating treatments.

Fig. 1 Separation of (a) cinnamic acid, (b) ferulic acid, (c) quercetin, (d) protodioscin, and (e) dioscin by HPLC and UPLC-MS.

Fig. 2 Comparison in the antityrosinase activity of (A) Group 11, (B) Group 11 and protodioscin, and (C) Group 11 and dioscin. The combined heat-processing treatment represents microwave 700 W for 20 min and 120

for 30 min.

A

B

C

D

E

F

a

b

Fig. 3 (A) (B) (C) Fluorescence spectra of tyrosinase in Group 11, Group 11 + protodioscin, and Group 11 + dioscin. (D) (E) (F) Fluorescence spectra of tyrosinase in Group 11, Group 11 + protodioscin, and Group 11 + dioscin after combined heating. Curve m, n, p, q, s, and t denote the emission spectrum of samples only. c (samples) = 0, 2, 5, 7, 10, 12, 15, 17, 20 uM for curves a→i, respectively. (a) The Stern-Volmer plots of F0/F vs [Samples]. (b) The corrected Stern-Volmer plots of ln(F0/F) vs [Samples].

Fig. 4 Comparison in the UPLC-MS chromatograms of polyphenols and saponins under different conditions. (A) UPLC-MS chromatograms of Group 11, Protodioscin, and Dioscin before heating treatment. (B) UPLC-MS chromatograms of Group 11 after heating treatment. (C) UPLC-MS chromatograms of Group 11 and protodioscin after heating treatment. (D) UPLC-MS chromatograms of Group 11 and dioscin after heating treatment.

(a)

(b)

Fig. 5 Mass spectrum and proposed fragmentation structures of Est 1 (a) and Est 2 (b).

Highlights 1. Combined heating enhance the antityrosinase ability of polyphenols and saponin. 2. Combined heating increases the affinity between enzyme and inhibitors. 3. The deglycosylation of saponins contribute to enhance antityrosinase ability. 4. The compounds Est1 and Est2 contribute to the antityrosinase ability.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The corresponding authors Liuping Fan