Structure Characterization and Anti-tyrosinase Mechanism of

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Structure Characterization and Antityrosinase Mechanism of Polymeric Proanthocyanidins Fractionated from Kiwifruit Pericarp Wei-Ming Chai, Yan Shi, Hui-Ling Feng, Lian Xu, Zhi-Hao Xiang, Yu-Sen Gao, and Qing-Xi Chen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jun 2014 Downloaded from http://pubs.acs.org on June 27, 2014

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Journal of Agricultural and Food Chemistry

Structure Characterization and Antityrosinase Mechanism of Polymeric Proanthocyanidins Fractionated from Kiwifruit Pericarp

Wei-Ming Chai,†,‡ Yan Shi,† Hui-Ling Feng,† Lian Xu,† Zhi-Hao Xiang,† Yu-Sen Gao,† and Qing-Xi Chen*,†,§

†

State Key Lab of Cellular Stress Biology, Key Lab of the Ministry of Education for Coastal

and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China ‡

Key Laboratory of Small Fuctional Organic Molecule, Ministry of Education and College of

Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China §

Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen

361005, China

* Corresponding authors. E-mail: [email protected]

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ABSTRACT:

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To provide information on structure, activity, and structure-activity relationship of

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Kiwifruit (Actinidia chinensis) pericarp proanthocyanidins (PAs), they were separated into

4

three fractions. These fractions were further identified by MALDI-TOF MS and

5

HPLC-ESI-MS methods. Spectra results revealed that they are complex mixtures of B-type

6

propelargonidins, procyanidins, procyanidins gallate, and prodelphinidins. Enzymatic activity

7

analysis showed that these compounds strongly inhibit the activity of tyrosinase, indicating

8

that they are reversible and mixed type inhibitors of the enzyme. The results obtained from

9

fluorescence quenching showed PAs inhibit the enzyme activity by interacting with substrate

10

and enzyme. This study confirmed that mean degree of polymerization (mDP) of PAs

11

produces positive effect on their antityrosinase activity. In addition, the antioxidant analysis

12

indicated that PAs possess potent antioxidant activity. Aboved conclusions mean kiwifruit

13

pericarp PAs may be explored as insecticide, food preservatives, and cosmetic additives.

14

KEYWORDS: kiwifruit pericrap PAs; antityrosinase; antioxidant; fractionation; degree of

15

polymerization; structure-activity

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Introduction

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Tyrosinase (EC 1.14.18.1) is a mixed-function enzyme widely distributed in

19

microorganisms, plants, and animals.1 It plays important roles in melanogenesis,2 insect

20

development,3 and fruit and vegetable browning4. Thus tyrosinase inhibitors have the possible

21

use as whitening agents, insect control agents, and food additives. Some reports have found

22

that proanthocyanidins (PAs) extracted from different plants show potent tyrosinase inhibitory

23

activity.5,6

24

PAs are polyphenol compounds universally distributed in the foods of plant origin. They

25

are major parts of human diets and play a positive role in health and nutrition.7 These

26

compounds have been reported to possess many health beneficial effects by acting as

27

antioxidant, anticarcinogen, cardiopreventive, antimicrobial, anti-viral, and neuro-protective

28

agents.8 However, the structure of plant PAs is generally recognized to decide their bioactivity

29

capacity.9 Therefore, there is necessary to study structure of PAs. Their structure vary

30

depending upon the nature (stereochemistry and hydroxylation pattern) of the flavan-3-ol

31

starter and extension units, the position and stereochemistry of the linkage, the degree of

32

polymerization, and the presence or absence of modifications such as esterification of the

33

3-hydroxyl group.10,11 Thus it remains very difficult to obtain qualitative and quantitative

34

imformation of PAs.

35

Kiwifruit, also known as Mi-hou-tau, is a fruit with a high commercial value for their

36

health benefits and taste.12 Phenolics have been thought as bioactive components responsible

37

part for the health benefits in the kiwifruit13 and their compositions were partially

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characterized by Dawes et al.14 However, antioxidant, antityrosinase activities and detailed

39

structure information about purified PAs’ profile of kiwifruit pericarp have not been reported.

40

The present study therefore aims to provide a more comprehensive picture of kiwifruit

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pericarp PAs structure. Gel chromatographic fractionation, thiolysis, reversed-phase HPLC–

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ESI-MS, normal-phase HPLC–ESI-MS, positive-ion reflectron mode and linear mode of

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MALDI-TOF-MS analytical techniques are used for the first time on kiwifruit pericarp PAs to

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elucidate the monomer units, nature of the interflavan linkage, and distribution of

45

polymerization degree. Their antityrosinase activity, mechanism, and antioxidant activity

46

information are also provided in this study.

47 48

Materials and Methods

49

Reagents and Standards

50

Mushroom

tyrosinase,

L-tyrosine,

3,4-dihydroxyphenylalanine,

DPPH,

ABTS,

51

2,4,6-Tripyridyl-S-triazine, ascorbic acid, trolox, Sephadex LH-20, HPLC standards,

52

trifluoroacetic acid, benzyl mercaptan, Amberlite IRP-64 cation-exchange resin, cesium

53

chloride, and 2,5-dihydroxybenzoic acid were purchased from Sigma Aldrich (St. Louis, MO,

54

USA). All analytical grade solvents, including acetone, petroleum ether, and ethyl acetate, and

55

methanol, and HPLC-grade acetonitrile, dichloromethane, methanol for analytical

56

HPLC-ESI-MS were obtained from Sinopharm (Sinopharm, Shanghai, China).

57

Sample Preparation

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Ripe kiwifruit fruits were purchased from fruit market nearby Xiamen University in

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September 2011. The fruits were selected for uniformity of shape and colour without physical

60

damage and injury because of insects or fungal infection. They were manually separated into

61

pulp and pericarp. The latter was immediately washed and freeze-dried for 72 h, and then they

62

were ground into fine powders. These fine powders were stored at −20 °C before analyses.

63

Extraction and Fractionation of PAs

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Freeze-dried powders (10.0 g) were ultrasonically extracted with 7:3 (v/v) acetone−water

65

solution containing 0.05 % ascorbic acid (3 × 250 mL) at room temperature. Each extract was

66

filtered and pooled, and the acetone was removed by evaporation under vacuum (40 °C). The

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remaining aqueous residue was subsequently extracted thrice with petroleum ether (3 × 150

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mL) and ethyl acetate (3 × 150 mL), yielding dried crude tannin extracts. The crude tannin

69

fraction was chromatographed on a 50 × 1.5 cm i.d. Sephadex LH-20 column (Pharmacia

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Biotech, Uppsala, Sweden), the column was washed with 500 mL methanol-water (50:50, v/v)

71

to remove sugars, glycosides and monomeric polyphenols, then eluted with methanol-H2O

72

(80:20, v/v),

73

obtain fraction A (FA), fraction B (FB), and fraction C (FC) (each 500 mL) respectively.

74

Organic solvents were removed and the remaining aqueous fractions were freeze-dried. Their

75

yields were calculated and listed in Table 1. Three fractions were freeze-dried after the

76

organic solvents were eliminated and stored at −20 °C before analyses. Fractionation

77

experiment was repeated three times. In addition, UV–vis spectra of three fractions showed a

78

pronounced and symmetrical peak near 280 nm with no band broadening beyond 300 nm.

79

This kind of spectra is typical of the PAs.

80

MALDI-TOF-MS Analysis

acetone-methanol-H2O (40:40:20, v/v/v), and acetone-water (70:30, v/v) to

81

MALDI-TOF-MS analysis was performed by using a Bruker Reflex III MALDI-TOF mass

82

spectrometer (Bruker, Bremen, Germany). The parameters for positive mode spectra in the

83

reflectron mode and linear mode were set according to our previous report.15 Parameters for

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positive pattern spectra in the reflectron pattern were as follows: accelerating voltage, 20 kV;

85

reflectron voltage, 23.0 kV; delayed extraction voltage, 16.32 kV; lens voltage, 9.45 kV.

86

Parameters for positive pattern spectra in the linear pattern were as follows: accelerating

87

voltage, 20.0 kV; delayed extraction voltage, 16.25 kV; lens voltage, 10.0 kV. Cs+ was used as

88

the cationization and 2, 5-Dihydroxybenzoic acid was selected as the matrix. Amberlite

89

IRP-64 cation-exchange resins (Sigma-Aldrich, USA) were used to deionize the analyte and

90

matrix solution. An aqueous solution of cesium chloride (1.5 µL, 1.52 mg/mL in aqueous) was

91

added to the sample solution (1.5 µL, 10 mg/mL in 30 % aqueous acetone) followed by

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addition of an equal volume of 2,5-dihydroxybenzoic acid (10 mg/mL in aqueous). The

93

mixture (1 µL) was spotted to a steel target and introduced into the mass spectrometer.

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Thiolysis of Polymeric PAs for Reversed-phase HPLC-ESI-MS Analysis

95

A thiolysis method based on that described by Guyot et al.16 was carried out with a minor

96

modification. Degradation products of thiolysis were analyzed by reversed-phase HPLC–

97

ESI-MS. PAs used for thiolysis were 5 mg/mL. The high performance liquid chromatograph

98

(HPLC) was performed on an Agilent 1200 system (USA). The thiolysis medium was further

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analyzed using LC/MS (QTRAP 3200, USA). Two solvents, 0.5 % trifluoroacetic acid (A)

100

and acetonitrile (B), were used. The linear gradient elution process was: 0−45 min, 12−80 %

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B; 45−50 min, 80−12 % B. The column temperature was 25 °C and the flow rate was 1

102

mL/min. The detection wavelength was set at 280 nm. Degradation products were identified

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on chromatograms according to their retention times and LC/MS. The calculation of the mean

104

degree of polymerization (mDP) was based on the following equation:

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mDP = (total area of the extender units)/(total area of the terminal units) + 1.

106

Normal-phase HPLC–ESI-MS Analysis

107

The HPLC equipment consisted of an Agilent 1200 liquid chromatograph system as

108

described above. Concentration values of the solutions used for analysis were 5 mg/mL. The

109

column temperature was 35 °C and the injection volume was 20 µL. The mobile phase

110

consisted of A, dichloromethane/methanol/water/acetic acid (41:7:1:1, v/v/v/v), and B,

111

dichloromethane/methanol/water/acetic acid (5:43:1:1, v/v/v/v). The 60 min linear gradient

112

was: 0−13.5 % B (0−20 min); 13.5−29.2 % B (20−50 min); 29.2−100 %B (50−55 min); 100 %

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B (55−60 min).17 And then the eluting stream (1 mL/min) was introduced into a QTRAP 3200

114

mass spectrometer. Peaks were identified on chromatograms according to their retention times

115

and LC/MS.

116

Enzyme Assay

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Inhibitory Effects Assay

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Enzyme reaction was performed by using 3 mL system established by our laboratory.18 In

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brief, L-tyrosine was selected as substrate for monophenolase reaction assay and

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3,4-dihydroxyphenylalanine was selected as substrate for diphenolase reaction assay. The

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reaction medium (3 mL) contained 12 mM L-tyrosine or 0.5 mM 3,4-dihydroxyphenylalanine

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in 50 mM sodium phosphate buffer (pH 6.8). The final concentrations of tyrosinase were

123

33.33 µg/mL for monophenolase reaction and 6.67 µg/mL for diphenolase reaction.

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Absorption was detected by using a Beckman UD-800 spectrophotometer (California, USA).

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The extent of inhibition by the addition of the sample was expressed as the percentage

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necessary for 50 % inhibition (IC50) for enzyme activity assay. A lower value of IC50 indicates

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better antityrosinase activity. Controls, without inhibitor but containing equal H2O, were

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routinely carried out. All measurements were carried out at 30 °C.

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Inhibitory Mechanism and Type Assay

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Inhibitory mechanism assay was carried out by changing enzyme concentration in reaction

131

medium. The plots of the remaining enzyme activity versus the concentrations of enzyme in

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the presence of inhibitors with diffierent concentration gave a family of straight lines. If the

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inhibition was reversible, all straight lines intersect at the origin; when the inhibition was

134

irreversible, these straight lines are parallel. Inhibition type assay was achieved by changing

135

substrate concentration in reaction medium. The inhibition type was assayed by the

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Lineweaver–Burk plot.

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Fluorescence Emission Spectra of Mushroom Tyrosinase in Solutions with Different

138

Concentration of PAs

139 140

The fluorescence assay was measured with a Varian Cary Eclipse fluorescence spectrophotometer. The reaction medium (3 mL) contained 600 µL mushroom tyrosinase

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solution, 2.1 mL of 50 mM sodium phosphate buffer (pH 6.8), and 300 µL sample solution

142

with different concentration. The experiment was performed with an excitation wavelength of

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290 nm at a constant temperature of 25 °C.

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Fluorescence Emission Spectra of L-tyrosine or 3,4-dihydroxyphenylalanine in Solutions

145

with Different Concentrations of PAs

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The fluorescence assay was carried out on a Varian Cary Eclipse fluorescence

147

spectrophotometer. In brief, L-tyrosine (12 mM) or 3,4-dihydroxyphenylalanine (0.5 mM)

148

solution (200 µL) was added into 50 mM sodium phosphate buffer (pH 6.8), and sample

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solution was then added at a constant temperature of 25 °C (the final medium is l mL),

150

preincubated for 30 s before fluorescence spectra measurements with an excitation

151

wavelength of 290 nm.

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DPPH Radical Scavenging Capacity

153

DPPH method was achieved in compliance with the report of Brand-Williams et al.19 with

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minor modification. Mainly, sample solution (0.1 mL) was mixed with 3 mL of DPPH

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solution (25 mg/L in methanol). Methanol (0.1 mL) and 3 mL DPPH served as the control.

156

After the mixture was shaken and kept at room temperature for 30 min, the absorbance at 517

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nm was measured. The calculated equation for scavenging percentage of DPPH was:

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DPPH % inhibition = [(A1–A2)/A1] ×100.

159

where A1 = the absorbance of the control reaction; A2 = the absorbance in the presence of the

160

sample. The quality of the antioxidants about the PAs was determined by the IC50 values (the

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concentration that led to 50 % decrease in absorbance). A lower value of IC50 indicates better

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antioxidant activity. In addition, Trolox and ascorbic acid were selected as reference. All

163

fractions were analyzed in three replicates.

164

ABTS Radical Scavenging Activity 8


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ABTS assay was conducted in accordance with the procedure described by Re et al.20

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ABTS+ was generated by reacting 7 mM ABTS and 2.45 mM potassium persulfate after

167

incubation at room temperature in dark for 16 h. The ABTS+ solution was then diluted with

168

80 % ethanol to an absorbance of 0.700 ± 0.050 at a wavelength of 734 nm. Tested sample

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solution (0.1 mL) was added to ABTS+ solution (3.9 mL) and the mixture was mixed well.

170

The absorbance of reactive mixture was recorded at 734 nm on a Beckman UD-800

171

spectrophotometer (California, USA) after keeping at room temperature for 6 min. The results

172

were expressed the same to DPPH assay described previously, with ABTS % inhibition and

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IC50 value.

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Ferric Reducing Antioxidant Power (FRAP)

175

FRAP assay was performed according to the method of Benzie and Strain21 with slight

176

modification. Mainly, 3 mL FRAP reagent (10 mM TPTZ, 20 mM ferric chloride and 300 mM

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sodium acetate buffer were mixed at a ratio of 1:1:10) was mixed with test sample solution

178

(0.1 mL) or methanol (blank). The absorbance of reaction mixture at 593 nm was measured

179

after incubation at room temperature for 10 min. The FRAP values, expressed in millimoles of

180

ascorbic acid equivalents (AAE)/g fraction, were derived from a standard curve. A higher

181

absorbance corresponds to a higher ferric reducing power.

182

Statistical Analysis

183

All datas were expressed as means ± standard deviation of three independent

184

determinations. One-way analysis of variance was used, and the differences were considered

185

to be significant at P < 0.05. All statistical analyses were performed with SPSS 13.0 for

186

Windows.

187 188

Results and Discussion

189

MALDI-TOF MS Analysis

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Reflectron MALDI-TOF MS spectra (figure 1) of three fractions were obtained when

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2,5-Dihydroxybenzoic acid was selected as the matrix and Cs+ was used as the cationization.

192

The results indicated that the studied polymeric mixture exhibit mass spectra with a

193

dominating set of peaks with differences of 288 Da, corresponding to the mass difference of

194

one (epi)catechin monomer unit. Other repeated patterns within each main set of peaks were

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the signals separated by the subset of masses 16 Da difference. These masses were produced

196

by propelargonidin-type (which lack one hydroxyl group at the 3′-position of the B-ring

197

compared to procyanidin-type flavan-3-ol units) and prodelphinidin-type flavan-3-ol units

198

(which possess one hydroxyl group at the 5 ′ -position of the B-ring compared to

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procyanidin-type flavan-3-ol units). It was further suggested that the PAs from fruit pericarps

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of kiwifruit contain prodelphinidin, procyanidin, and prodelphinidin when the absolute

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masses corresponding to each peak were obtained. Procyanidins were the dominating

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constituent of kiwifruit pericarp PAs. The series of peaks with more 132 Da (which produced

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by synchronous attachment of two Cs+ and absence of a proton [M+2Cs+−H]+)22 and 152 Da

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(corresponding to the addition of one galloyl group at the heterocyclic C-ring)23 were also

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detected. Additionally, the A-type interflavan linkages with characteristic signals 2 Da smaller

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than the B-type linked PAs (due to the loss of two hydrogen atoms with the formation of the

207

ether bond) were not detected in the mass spectra.

208

The distribution of polymerization degree and predominant polymers was clearly different

209

among three fractions, the DP varying from 2-mers to 10-mers, 3-mers to 18-mers, and

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3-mers to 23-mers for FA, FB, and FC, respectively. MALDI-TOF mass spectra provided the

211

evidences that PAs from kiwifruit pericarp possessed structural heterogeneity and distinct DP

212

distribution in each fraction. In this study, as high as 23-mers was present in the spectra of FC,

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which might be attributed to the improvement of fractionation in the MALDI-TOF-MS

214

analysis as stated by previous report.24 Therefore, fractionation of lower oligomers (FA) on 10


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Sephadex LH-20 column would significantly enhance the sensitivity of the detection of larger

216

polymers (FB) under MALDI-TOF-MS analysis.

217

PAs polymers extracted from kiwifruit pericarp were then characterized by MALDI-TOF

218

MS in the positive-ion (Cs+) linear mode (Figure 2). In the mass spectra, an equispaced series

219

of signal groups corresponding to the polymeric distribution of the PAs was observed. The

220

distance between each degrees of polymerization was 288 Da. It agreed well with the results

221

obtained from reflectron mode. PAs with DP up to 29-mers could be detected working in the

222

linear mode. So, the linear mode provided better information about the DP distribution of the

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PAs present in the sample. However, high mass resolution power in the reflectron spectrum

224

allowed distinguishing the mass of the isotopic peaks with enough accuracy (Figure 1).

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Reversed-phase HPLC-ESI-MS Analysis followed Thiolysis Reaction

226

The chromatograms of thiolytic products of FA, FB, and FC were illustrated in Figure 3.

227

The results showed that the terminal units contained catechin (peak 2), epicatechin (peak 4),

228

gallocatechin (peak 1), epigallocatechin (peak 3), catechin gallate (peak 5), and epicatechin

229

gallate (peak 6) for the FA. However, gallocatechin and epigallocatechin were not detected in

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the FB and FC. The extension units of FA contained (epi)afzelechin (peak 10), epicatechin

231

(peak 9), gallocatechin (peak 7) and epigallocatechin (peak 8), with the epicatechin

232

dominating. However, gallocatechin benzylthioether and epigallocatechin benzylthioether

233

were not found in the FB and FC. A-type PAs could not be detected in the chromatogram. The

234

profile after thiolysis demonstrated that PAs extracted from kiwifruit pericarp are B-type

235

procyanidin (the most abundant type of PAs in plants), propelargonidin, and prodelphinidin.

236

B-type procyanidin dominates. These results confirmed the finding of that obtained from

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MALDI-TOF MS analysis. In addition, the mDP for the polymers was estimated by thiolysis

238

to be in a range of 4.3 ± 0.3 to 13.6 ± 0.7 (Table 1). The contents of terminal units decreased

239

in the order FA > FB > FC (Table 1).

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Normal-phase HPLC-ESI-MS Analysis

241

The normal-phase HPLC profile of kiwifruit pericarp PAs was illustrated in Figure 4. The

242

result indicated the presence of PAs with different degrees of polymerization. The good

243

resolution between each oligomeric class suggested that the PAs are quite homogeneous,

244

showing only minor differences in their constituent units and the linkages between them.

245

HPLC performance implied the precise separation of PAs from monomers to 6-mers and the

246

presence of isomers. However, the polymers could not be resolved by normal-phase HPLC.

247

They showed a profile with a single broad peak at the end of the run with a retention time of

248

approximately 55.7 min. No PAs with DP higher than 10-mers could be separated. Our results

249

agreed well with the finding of Gu et al.17 on the analysis of PAs from lowbush blueberry.

250

Inhibition Effect, Mechanism and Type of the Kiwifruit Pericarp PAs on the

251

Monophenolase Activity and Diphenolase Activity of Mushroom Tyrosinase

252

The monophenolase and diphenolase activity may be both negatively affected by adding of

253

kiwifruit pericarp PAs as shown in Table 2. The IC50 values for the monophenolase activity

254

were estimated to be 180.2 ± 6.5, 80.1 ± 4.1, and 48.9 ± 4.6 µg/mL for FA, FB, and FC,

255

respectively. As for diphenolase activity, the inhibitor concentrations leading to 50 % (IC50)

256

enzyme activity decrease were estimated to be 390.2 ± 12.6, 192.6 ± 10.3, and 64.9 ± 3.2

257

µg/mL for FA, FB and FC, respectively. In previous reports, the methanol extracts from

258

sorghum distillery residue25 and red koji extracts26 (containing a high percentage of PAs)

259

showed inhibitory effect with the IC50 value of 580 µg/mL and 5570 µg/mL on mushroom

260

tyrosinase activity, respectively. Therefore PAs from kiwifruit pericarp in this study were

261

potent inhibitors on tyrosinase. In present study, we further found that DP, i.e., molecular

262

weight had an obvious effect on the inhibition of mushroom tyrosinase activity. The PAs with

263

higher DP possessed a better inhibition on the activity of tyrosinase. This revealed the

264

possible use of these compounds (especially FB and FC) as whitening agents, insect control

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agents, and food additives.

266

Inhibitory mechanism and type of three fractions on the monophenolase and diphenolase

267

enzyme reaction were then investigated. The results obtained from inhibitory mechanism

268

analysis showed that the inhibition was reversible. It could be concluded that the presence of

269

inhibitor did not bring down the amount of enzyme, but just led to a decrease in activity of the

270

enzyme for oxidation of L-tyrosine and 3,4-dihydroxyphenylalanine. This further indicated

271

that DP had no effect on the inhibitory mechanism. Inhibition type analysis indicated that they

272

were mixed type inhibitors of the enzyme. The results revealed that these fractions combined

273

with free enzymes as well as enzyme-substrate complexes. The results also displayed that

274

three fractions had the same inhibition type on the enzyme.

275

Fluorescence Quenching Analysis

276

Fluorescence of tryptophan which existed in tyrosinase molecules has been frequently

277

examined to obtain information about conformational changes.27 The interaction of tyrosinase

278

with PAs and its conformational alteration were evaluated by measuring the intrinsic

279

fluorescence intensity of the protein in the presence or absence of PAs. The fluorescence

280

emission spectra were collected from a range of 300-500 nm with excitation wavelength set at

281

290 nm. The result showed that the addition of fractions caused a dramatic decrease in the

282

fluorescence emission spectra. The fluorescence intensity decreased by 51.3 %, 80.0 %, and

283

85.6 % (Figure 5A) for FA, FB, and FC at 100 µg/mL. Undoubtedly, FC had better

284

interaction with tryptophan residue in the enzyme than those of FA and FB. This indicated that

285

PAs with high DP combined tryptophan residue more effectively. Therefore these findings

286

may be one reason for the strong inhibition of PAs on the tyrosinase. In addition, an obvious

287

blue shift was also present in the spectra of FB and FC. These results indicated that PAs

288

induced the change of enzyme conformation and they also led to the inactivation of the

289

enzyme after binding to the enzyme molecule. Moreover, the fact that the interaction of PAs

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290

with the tryptophan of tyrosinase implied that the tyrosinase became disagglomerated and its

291

structure got loosened. It was concluded that the tryptophan residue is one of the essential

292

groups of the enzyme activity and it is situated on the surface of the enzyme and easily

293

influenced by PAs. However, the results showed that PAs of FA did not affect the

294

conformation of tyrosinase. Further study should be carried out to illustrate it. Tyrosinase

295

owns two copper ions in the active center and they are of great importance for tyrosinase

296

activity.28 The inhibitory effects of PAs on the tyrosinase might be due to binding with copper

297

ions (which is the metal at the centre of the active site of tyrosinase) with their –OH group.

298

Because the –OH group of PAs presents anionic form at pH 6.8, they might interact

299

electrostatically with copper ions in the mushroom tyrosinase. The copper-chelating

300

properties of the PAs were thought to be another reason for their strong inhibitory activity on

301

the tyrosinase.29

302

Fluorescence Emission Spectra of L-tyrosine or 3,4-dihydroxyphenylalanine in Solutions

303

with Different Concentration of PAs

304

The interaction of three fractions with L-tyrosine which used as substrate of

305

monophenolase was first investigated by the fluorescence quenching method. The fluorescence

306

emission spectra were recorded from a range of 300-400 nm with excitation wavelength set at

307

290 nm. The results showed that fluorescence intensity decreased distinctly with the

308

increasement of PAs concentration. The fluorescence intensity decreased by 63.7 %, 83.7 %

309

and 89.1 % (Figure 5B) for FA, FB, and FC at 100 µg/mL.

310

Then fluorescence emission spectra of 3,4-dihydroxyphenylalanine which used as substrate

311

of diphenolase were then recorded in the presence of PAs with different concentration. The

312

excitation was the same as L-tyrosine assay. The results showed that fluorescence intensity

313

decreased distinctly with the increasement of PAs concentration. The fluorescence intensity

314

decreased by 47.9 %, 75.5 % and 80.7 % (Figure 5) for FA, FB and FC at 100 µg/mL.

14


Page 15 of 31


315

These results provided the first evidence that PAs are the effective binding agent of

316

L-tyrosine and 3,4-dihydroxyphenylalanine. Obviously, PAs could effectively stop the

317

reaction of substrate and tyrosinase by this way. It could be another reason for the strong

318

inhibition of PAs on the tyrosinase catalytic reaction. These findings were significant in both

319

designing and screening of potent novel tyrosinase inhibitiors. And the results also indicated

320

that combining capacities with L-tyrosine and 3,4-dihydroxyphenylalanine decrased in the

321

order: FC > FB > FA. These were also the proof that DP is an important factor for the

322

inhibition of PAs on the enzyme.

323

Determination of Antioxidant Capacity

324

DPPH, ABTS and FRAP assay were simultaneously used for measuring the antioxidant

325

properties of three fractions to better reflect their potential protective effects. Table 3 showed

326

the scavenging effect on the DPPH radical decreased in the order: FC ≈ FB > FA ≈

327

ascorbic acid >Trolox. And the order for the ABTS radical scavenging activity was: FC > FB >

328

FA > ascorbic acid > Trolox. The antioxidant activity of kiwifruit pericarp PAs was also

329

measured by FRAP assay. The FRAP values for the FA, FB, and FC were 7.4 ± 0.2, 9.6 ± 0.3,

330

and 9.6 ± 0.9 mmol AAE/g (Table 3), respectively. At each concentration, FB and FC

331

exhibited distinctly higher reducing power than that of Trolox. In addition, with increasing the

332

concentration of the compounds, the antioxidant activity of PAs was observably increased in a

333

concentration-dependent pattern.

334

Yokozawa et al.30 confirmed that an increase of galloyl groups, molecular weight (DP), and

335

ortho-hydroxyl structure enhanced the scavenging activity of tannins. In this study, the results

336

obtained from three antioxidant analyses revealed that PAs were potent antioxidant. This can

337

be explained that PAs possessed abundant hydroxyl groups. FB and FC possessed obviously

338

higher antioxidant properties than that of FA. The result revealed that DP was a key factor for

339

the antioxidant capacity of PAs. Hagerman et al.31 provided insights into the mechanism of

15



Page 16 of 31

340

procyanidin as the potential antioxidants, which showed that high molecular weight and

341

hydroxyl groups were important factors for free radical scavenging by tannins. Our results

342

agreed well with the findings of Hagerman’s. In conclusion, our results indicated for the first

343

time that PAs from kiwifruit pericarp might be a good resource for further development as an

344

antioxidant. This indicated the possible use of these compounds (especially for FB and FC) in

345

medical, cosmetic, food and agricultural industry.

346

Structure-Activity of PAs from Kiwifruit Pericarp

347

Fractions FA, FB, and FC, with distinguishing DP ranges, were selected for discussing the

348

structure-activity relation of PAs. In previous reports, many researchers had focused on the

349

relationship between DP and antioxidant activity. Ariga et al.32 found that the ability to

350

scavenge peroxyl radicals was proportional to DP after testing several oligomeric flavonoids.

351

Zhou et al.33 thought that 9–10 mDP PAs should be considered as a dividing point for

352

predicting the structure-activity of mangosteen pericarp PAs. In the present study, antioxidant

353

activity of kiwifruit pericarp PAs increased from mDP = 4.3 (fraction F1) to 9.1 (fraction F2),

354

but kept stable after that when using DPPH and FRAP assay. However, an increasing

355

antioxidant activity of PAs was displayed following the increase of mDP when using ABTS

356

assay. Our results partly agreed with the finding of Zhou et al33. As for antityrosinase activity,

357

our results revealed that activities (the monophenolase activity and the diphenolase activity)

358

increased following the increase of PAs mDP. However, Shoji et al.34 fractionated the

359

procyanidins according to the DP using normal-phase chromatography, and no correlation

360

between the DP and tyrosinase inhibitory activity was observed. It was suggested that

361

methods are used to separate PAs from other polyphenols and to fractionate them according to

362

the DP important. In a word, appropriate fractionation might be a feasible way to screen better

363

antioxidant and antityrosinase agent.

364

In conclusion, the structures of proanthocyanidin fractions (FA, FB, and FC) from kiwifruit

16


Page 17 of 31


365

pericarp were successfully characterized and elucidated. They possessed structural

366

heterogeneity in monomer units, polymer length. Furthermore, the PAs exhibited excellent

367

efficiency in inhibiting the activities of the mushroom tyrosinase, including the

368

monophenolase activity and the diphenolase activity. The antityrosinase activity of kiwifruit

369

PAs increased with mDP. They were found to be reversible and mixed type inhibitors of the

370

enzyme. Moreover, the inhibition was carried out mainly through the interaction of the

371

hydroxyl groups of the condensed tannins with the active center of the enzyme and the

372

substrate of the enzymatic reaction. Additionally, these proanthocyanidin fractions, especially

373

FB and FC, could effectively scavenge DPPH, ABTS radical and Fe3+-TPTZ. The antioxidant

374

activity of kiwifruit pericarp PAs increased from mDP = 4.3 (fraction F1) to 9.1 (fraction F2),

375

but kept stable after that when using DPPH and FRAP assay. However, the antioxidant

376

activity of these compounds increased with mDP when using ABTS assay. The elucidation of

377

the antityrosinase mechanisms and antioxidant activity of the PAs are significant in both

378

designing and screening of novel tyrosinase inhibitiors and potent antioxidant.

379 380

Abbreviations Used

381

PAs, proanthocyanidins; MALDI-TOF MS, matrix-assisted laser desorption/ionization

382

time-of-flight mass spectrometry; HPLC-ESI-MS, high performance liquid chromatography

383

electrospray ionization mass spectrometry; DP, degree of polymerization; ABTS,

384

2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic

385

2,2-diphenyl-1-picrylhydrazyl; FRAP, Ferric reducing antioxidant power.

acid)

diammonium

salt;

DPPH,

386 387

References

388

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13. Park, Y.-S.; Leontowicz, H.; Leontowicz, M.; Namiesnik, J.; Suhaj, M.; Cvikrová, M.;

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compounds and the level of antioxidant activity in different kiwifruit cultivars. J. Food

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14. Dawes, H. M.; Keene, J. B., Phenolic composition of kiwifruit juice. J. Agric. Food Chem. 1999, 47, 2398-2403. 15. Chai, W. M.; Chen, C. M.; Gao, Y. S.; Feng, H. L.; Ding, Y. M.; Shi, Y.; Zhou, H. T.;

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Chen, Q. X., Structural analysis of proanthocyanidins isolated from fruit stone of Chinese

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hawthorn with potent antityrosinase and antioxidant activity. J. Agric. Food Chem. 2014,

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classes of phenolic compounds in different tissue zones of a French cider apple variety

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(Malus domestica var. Kermerrien). J. Agric. Food Chem. 1998, 46, 1698-1705.

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17. Gu, L.; Kelm, M.; Hammerstone, J. F.; Beecher, G.; Cunningham, D.; Vannozzi, S.; Prior,

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R. L., Fractionation of polymeric procyanidins from lowbush blueberry and

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quantification of procyanidins in selected foods with an optimized normal-phase

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HPLC-MS fluorescent detection method. J. Agric. Food Chem. 2002, 50, 4852-4860.

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18. Li, Z. C.; Chen, L. H.; Yu, X. J.; Hu, Y. H.; Song, K. K.; Zhou, X. W.; Chen, Q. X.,

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Inhibition kinetics of chlorobenzaldehyde thiosemicarbazones on mushroom tyrosinase. J.

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Antioxidant activity applying an improved ABTS radical cation decolorization assay.

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21. Benzie, I. F. F.; Strain, J., The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal. Biochem. 1996, 239, 70-76.

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22. Xiang, P.; Lin, Y.; Lin, P.; Xiang, C.; Yang, Z.; Lu, Z., Effect of cationization reagents on

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the matrix-assisted laser desorption/ionization time-of-flight mass spectrum of Chinese

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gallotannins. J. Appl. Polym. Sci. 2007, 105, 859-864.

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23. Li, C.; Leverence, R.; Trombley, J. D.; Xu, S.; Yang, J.; Tian, Y.; Reed, J. D.; Hagerman,

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A. E., High molecular weight persimmon (Diospyros kaki L.) proanthocyanidin: a highly

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galloylated, A-linked tannin with an unusual flavonol terminal unit, myricetin. J. Agric.

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Food Chem. 2010, 58, 9033-9042.

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Monagas, M.; Quintanilla-López, J. E.; Gómez-Cordovés, C.; Bartolomé, B.;

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Lebrón-Aguilar, R., MALDI-TOF MS analysis of plant proanthocyanidins. J.

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Pharmaceut. Biomed. 2010, 51, 358-372.

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25. Wang, C. Y.; Ng, C. C.; Lin, H. T.; Shyu, Y. T., Free radical-scavenging and

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tyrosinase-inhibiting activities of extracts from sorghum distillery residue. J. Biosci.

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Bioeng. 2011, 111, 554-556.

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26. Wu, L.; Chen, Y.; Ho, J. A.; Yang, C., Inhibitory effect of red koji extracts on mushroom tyrosinase. J. Agric. Food Chem. 2003, 51, 4240-4246.

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Kim, D.; Park, J.; Kim, J.; Han, C.; Yoon, J.; Kim, N.; Seo, J.; Lee, C., Flavonoids as

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mushroom tyrosinase inhibitors: a fluorescence quenching study. J. Agric. Food Chem.

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2006, 54, 935-941.

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28. Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M., Crystallographic

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evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. J. Biol.

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Chem. 2006, 281, 8981-8990.

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29. Maisuthisakul, P.; Gordon, M. H., Antioxidant and tyrosinase inhibitory activity of mango seed kernel by product. Food Chem. 2009, 117, 332-341.

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30. Yokozawa, T.; Chen, C. P.; Dong, E.; Tanaka, T.; Nonaka, G. I.; Nishioka, I., Study on the

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inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2-picrylhydrazyl

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radical. Biochem. Pharmacol. 1998, 56, 213-222.

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31. Hagerman, A. E.; Riedl, K. M.; Jones, G. A.; Sovik, K. N.; Ritchard, N. T.; Hartzfeld, P.

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W.; Riechel, T. L., High molecular weight plant polyphenolics (tannins) as biological

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antioxidants. J. Agric. Food Chem. 1998, 46, 1887-1892.

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32. Ariga, T.; Hamano, M., Radical scavenging action and its mode in procyanidins B-1 and

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B-3 from Azuki beans to peroxyl radicals (food & nutrition). Agric. and Biol. Chem. 1990,

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54, 2499-2504.

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33. Zhou, H. C.; Lin, Y. M.; Wei, S. D.; Tam, N. F. y., Structural diversity and antioxidant

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activity of condensed tannins fractionated from mangosteen pericarp. Food Chem. 2011,

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129, 1710-1720.

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34. Shoji, T.; Masumoto, S.; Moriichi, N.; Kobori, M.; Kanda, T.; Shinmoto, H.; Tsushida, T.,

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Procyanidin trimers to pentamers fractionated from apple inhibit melanogenesis in B16

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mouse melanoma cells. J. Agric. Food Chem. 2005, 53, 6105-6111.

485

Note: The present investigation was supported by the Natural Science Foundation of China

486

(Grant Nos. 31071611 and 31070522), the National Science Foundation for Fostering Talents

487

in Basic Research of the National Natural Science Foundation of China (Grant No.

488

J1310027/J0106) and by the Science and Technology Foundation of Fujian Province (Grant

489

2010N5013)

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Figure Captions Figure 1 MALDI-TOF positive-ion (Cs+) mode mass spectra of polymeric kiwifruit pericarp PAs in the reflectron mode. Figure 2 MALDI-TOF positive-ion (Cs+) mode mass spectra of polymeric PAs extracted from kiwifruit pericarp in the linear mode. Figure 3 Reversed-phase HPLC chromatograms of polymeric PAs extracted from kiwifruit pericarp after thiolytic degradation. Peaks are: 1, gallocatechin (305 Da, [M−H]−); 2, catechin (289 Da, [M−H]−); 3, epigallocatechin (305 Da, [M−H]−); 4, epicatechin (289 Da, [M−H]−); 5, catechin gallate (441 Da, [M−H]−); 6, epicatechin gallate (441 Da, [M−H]−); 7, gallocatechin benzylthioether (427 Da, [M−H]−); 8, epigallocatechin benzylthioether (427 Da, [M−H]−); 9, epicatechin benzylthioether (411 Da, [M−H]−); 10, (epi)afzelechin benzylthioether (395 Da, [M−H]−); and 11, benzyl mercaptan. Figure 4 Normal-phase HPLC chromatograms of PAs from kiwifruit pericarp. The degree of polymerization of the peaks is labeled in expanded inserts. Figure 5 A, Relative fluorescence intensity of tyrosinase in solution with different concentration of PAs, the concentrations of PAs were 0, 12.5, 25, 50, and 100 µg/mL, respectively. B, Relative fluorescence intensity of L-tyrosine in solution with different concentration of PAs, the concentrations of PAs were 0, 25, 50, and 100 µg/mL. C, Relative fluorescence intensity of 3,4-dihydroxyphenylalanine in solution with different concentration of PAs, the concentrations of PAs were 0, 25, 50, and 100 µg/mL, respectively. The excitation wavelength for fluorescence analyses was set at 290 nm. Different letters (a, b, c) in the same column show significant differences from each other at P < 0.05 level. 490

22


Page 23 of 31


Table 1. The Yield and Thiolysis Results of Different Kiwifruit Pericarp PAs Fractions Eluted from Sephadex LH-20 Column. Contents (mg/g PAs) Fractions

FA

Yield (%)

0.93 ± 0.10

mDP

Catechin c

4.30 ± 0.30

b

Epicatechin a

16.5 ± 0.80

b

a

8.78 ± 0.30

b

Gallo-

Epigallo-

Eatechin

catechin

catechin

gallate

1.74 ± 0.80

0.66 ± 0.01

Epicatechin gallate a

6.20 ± 0.60a

b

1.99 ± 0.02

FB

1.43± 0.20

9.10 ± 0.60

5.70 ± 0.60

1.98 ± 0.05

---

---

0.31 ± 0.01

2.90 ± 0.30b

FC

0.28 ± 0.05

13.60 ± 0.70a

4.50 ± 0.40c

0.88 ± 0.01c

---

---

0.21 ± 0.01c

1.90 ± 0.10c

Values are expressed as mean of triplicate determinations ± standard deviation; Different letters (a, b, c) in the same column show significant differences from each other at P < 0.05 level.

23



Page 24 of 31

Table 2. Effects of PAs Fractions from Kiwifruit Pericarp on the Activity of Mushroom Tyrosinase for the Oxidation of L-tyrosine and 3,4-Dihydroxyphenylalanine. Monophenolase Samples IC50 (µg/mL) Inhibition Inhibition type FA FB FC

180 .2 ± 6.5c 80.1 ± 4.1b 48.9 ± 4.6a

reversible reversible reversible

mixed mixed mixed

Diphenolase IC50 (µg/mL)

Inhibition

Inhibition type

390.2 ± 12.6c 192.6 ± 10.3b 64.9 ± 3.2a

reversible reversible reversible

mixed mixed mixed

Values are expressed as mean of triplicate determinations ± standard deviation; Different letters (a, b, c) in the same column show significant differences from each other at P < 0.05 level.

24


Page 25 of 31


Table 3. Antioxidant Activities of the PAs Fractionated from Kiwifruit Pericarp Using the DPPH Free Radical Scavenging Assay, ABTS Free Radical Scavenging Assay, and the FRAP Assay. Samples FA FB FC Ascorbic acid Trolox

Antioxidant activity IC50/DPPH (µg/mL) b

105.3±1.8 67.7±1.5a 69.3±0.9a 102.6±3.9b 111.9±2.9c

IC50/ABTS(µg/mL) c

74.7±2.5 60.1±1.8b 39.5±1.2a 82.5±0.6d 89.1±2.7e

FRAP (mmol AAE/g) 7.4±0.2c 9.5±0.3a 9.6±0.9a ---8.3±0.4b

Values are expressed as mean of duplicate determinations ± standard deviation; Different letters (a, b, c, d, e) in the same column show significant differences from each other at P < 0.05 level.

25



Page 26 of 31

Figure 1

26


Page 27 of 31


Figure 2

27



Page 28 of 31

Figure 3

28


Page 29 of 31


Figure 4

29



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Figure 5

30


Page 31 of 31


Graphical Abstract

31


Structure Characterization and Anti-tyrosinase Mechanism of

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