Separation and Identification of Anthocyanins Extracted from

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Separation and identification of anthocyanins extracted from blueberry wine lees and the pigment binding properties toward #-glucosidase Qian Wu, Yang Zhang, Hu Tang, Yashu Chen, Bijun Xie, Chao Wang, and Zhida Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04244 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

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Journal of Agricultural and Food Chemistry

Separation and identification of anthocyanins extracted from blueberry wine lees and the pigment binding properties toward β-glucosidase Qian Wu,† Yang Zhang,§ Hu Tang,ǁ Yashu Chen,§ Bijun Xie,§ Chao Wang, *,† Zhida Sun,*,§ †

Hubei Collaborative Innovation Center for Industrial Fermentation, Research Center

of Food Fermentation Engineering and Technology of Hubei, Hubei University of Technology, Wuhan, Hubei 430068, China. §

Natural Product Laboratory, Department of Food Science and Technology, Huazhong

AgriculturalUniversity, Wuhan, Hubei 430070, People’s Republic of China ǁ

Department of Product Processing and Nutriology, Oil Crops Research Institute, Chinese

Academy of Agricultural Sciences, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Ministry of Agriculture Key Laboratory of Oil Crops Biology, Wuhan 430062, China *Author to whom correspondence should be addressed; E-Mail:,[email protected], [email protected]; Tel.: +86-27-87-28-3201; Fax: +86-27-87-28-2966.

ACS Paragon Plus Environment


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1

ABSTRACT: Anthocyanins were isolated from blueberry wine lees using Sephadex

2

LH-20 column chromatography and semi-preparative high performance liquid

3

chromatography

4

HPLC-DAD-ESI-MS/MS. Our results show that malvidin-3-hexose (Mv-3-hex) and

5

malvidin-3-(6'acetyl)-hexose (Mv-3-ace-hex) are the major components in the

6

anthocyanins extracts of blueberry wine lees (>90%). The binding characteristics of

7

Mv-3-hex and Mv-3-ace-hex with β-glucosidase were investigated by fluorescence

8

spectroscopy, circular dichroism (CD) spectroscopy and molecular docking.

9

Spectroscopic analysis reveals that β-glucosidase fluorescence quenched by Mv-3-hex

10

and Mv-3-ace-hex follows a static mode. Binding of Mv-3-hex and Mv-3-ace-hex to

11

β-glucosidase mainly depends on electrostatic force. The result from CD spectra

12

shows that adaptive structure rearrangement and increase of β-sheet structure occur

13

only in the presence of Mv-3-ace-hex. A molecular docking study suggests that

14

Mv-3-ace-hex has stronger binding with β-glucosidase than Mv-3-hex.

(semi-preparative

HPLC)

and

then

identified

by

15

blueberry

wine

lee;

malvidin-3-hexose

16

KEYWORDS:

17

malvidin-3-(6'acetyl)-hexose (Mv-3-ace-hex); β-glucosidase; interaction

18 19 20 21 22 1


(Mv-3-hex);

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23

INTRODUCTION

24

Blueberries, which belongs to the family Ericaceae, the genus Vaccinium.1 The

25

genus Vaccinium has a largely circumpolar dispersion in America, Europe and Asia,

26

particularly in North America.2 Blueberries consumption has become important in

27

population health promotion, mainly due to their phenolic compounds, such as

28

proanthocyanidins and anthocyanins, which own anti-inflammatory, anti-proliferative,

29

gastroprotective, antimicrobial and other biological activities.3 Thus, it has been

30

associated with protection against different pathologies, e.g. several types of human

31

cancer.4 One of the most important groups of phenolic compounds in blueberries is

32

anthocyanins, a major cluster of water-soluble pigments from the flavonoid group.5

33

With the development of blueberry processed industry, more and more blueberry

34

wine is produced and entered the markets, however, the remaining wine lees, as an

35

abundant by-product which composed of fine particles of blueberry residue and yeasts

36

are often discarded as waste.6-9 They are possible sources of proanthocyanidins and

37

anthocyanins, since these compounds in the blueberry are only partially transferred to

38

the wine. Pérez-Serradilla, J. A. and Luque de Castro, M. D.10 had extracted some

39

kinds of phenolic compounds from blueberry wine lees such as malvidin-3-glucoside

40

myricetin, quercetin, quercetin-3-β-glucoside etc., which suggested that wine lees

41

antioxidant extracts could be an economical and low-cost alternative to those obtained

42

from grape seeds.11-12 However, most researchers pay attention to the wine itself, or

43

that of berry skins and seeds,13 there are few reports on the wine lees composition

44

such as phenolic compounds, inorganic matter or tartaric acid.10,14 Thus, the 2



45

exploitation of wine by-products is of great importance, not only because of their

46

health benefits, but also from an environmental point of view as several million tons

47

of industry wastes are generated every year by the wine manufacturing industry.15 Our

48

research found that there were abundant anthocyanins in blueberry wine lees,

49

especially acylated anthocyanins, which didn’t appear in Wuhan blueberries. So

50

taking advantage of the blueberry wine lees as a source of anthocyanins could play an

51

important role in utilization of waste products.

52

Anthocyanins have the ability to interact with proteins that make them worthy of

53

attention by diverse areas such as agriculture, chemistry, medicine and food science.

54

In recent years, much attention has been paid to the interaction between

55

anthocyanidins and protein.16–18 However, to our knowledge, an accurate and full

56

basic data for clarifying the binding mechanisms of anthocyanins from blueberry wine

57

lees

58

glucohydrolase; E.C. 3.2.1.21) belongs to glycosyl hydrolase family 1 (GH 1),

59

involved in glucose metabolism and playing a key role in prevention of postprandial

60

hyperglycemia in diabetic patients.19 Anthocyanins as natural β-glucosidase inhibitors,

61

which delay digestion of carbohydrates, may be a therapeutic approach for type 2

62

diabetes (T2D).20 While, their inhibitory mechanism is not completely clear. Thus, the

63

in vitro data of interaction between anthocyanins and β-glucosidase may provide

64

appropriate explaination for inhibitory mechanism in biological system.

to

β-glucosidase

remain

unclear.

β-glucosidase

(β-D-glucopyranoside

65

In this study, anthocyanins were separated and purified from blueberry wine lees

66

with Sephadex LH-20 column chromatography and semi-preparative high 3


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performance liquid chromatography (semi-preparative HPLC) and further identified

68

by ESI-MS/MS, and the main component (Mv-3-hex/Mv-3-ace-hex) was selected to

69

study its binding properties with β-glucosidase in simulated physical conditions using

70

fluorescence, circular dichroism (CD) spectroscopy and molecular docking approach.

71

MATERIALS AND METHODS

72

Materials. β-glucosidase was purchased from Sigma, Germany. Blueberry wine

73

lees were provided by College of Life Science and Technology, Huazhong

74

Agricultural University, Wuhan, China. Sephadex LH-20 was bought from GE, USA,

75

organic solvents were chromatographic grade and bought from Fisher Scientific,

76

USA.

77

Isolation of crude anthocyanins from blueberry wine lees. Blueberry wine lees

78

(100 g) were added to 2 L 0.1% HCl acidified 70% (v/v) ethanol. Then the mixture

79

was stirred constantly with 50 °C water bath for 2 hours and the supernatant was then

80

concentrated on a rotavapor (40 °C) to about 50 mL. The resulting aqueous solution

81

(50 mL) was loaded onto a column (3.0 cm×80 cm) of cation-exchange resin

82

(Amberlite XAD-7HP; particle size: 20–60 mesh, wet, Sigma-Aldrich). The column

83

was washed with 2 L of deionized water at a flow rate of 1 mL/min to remove the

84

majority of proteins, sugars, organic acids and ions, and then elution of anthocyanins

85

was done using 1 L 0.1% HCl acidified 80% (v/v) ethanol at 1.5 mL/min. The eluate

86

(around 500 mL) was collected based on the color band and UV–vis detector at 520

87

nm. Finally, the eluate was concentrated by a rotary evaporator (30 rpm, 500 Pa, and

88

40 °C), and the resulting solution was freeze-dried. The content of anthocyanins was 4



89

determined using the AOAC official Method. 21

90

Separation and purification of Mv-3-hex and Mv-3-ace-hex. To obtain the major

91

monomeric anthocyanins with high purity from the column fractions, Sephadex

92

LH-20 column chromatography and semi-preparative high performance liquid

93

chromatography (HPLC) were performed by Yang’s method.22

94

On the other hand, semi-preparative scale HPLC (LC-6AD, Shimadzu, Japan) was

95

performed using a semi-preparative column [Shim-pack PREP-ODS (H) kit, 20.0 mm

96

× 250 mm, 5 m] to obtain other monomers. 10 mg of crude extract was dissolved in

97

100 µL ultrapure water and then injected into the column using a Rheodyne manual

98

sample injector. Mobile phases were composed of water-formic acid (98.5:1.5, v/v)

99

(mobile phase A) and water-acetonitrile-formic acid (48.5:50:1.5, v/v/v) (mobile

100

phase B). The linear gradient for elution was: 0-5 min, 12-30% B; 5-14 min, 30-100%

101

B; 14-20 min, 100-12% B, followed by washing and re-equilibration of the column

102

for 5 min, the total running time was 25 min, and chromatograms were recorded at

103

520 nm.

104

LC-MS Analysis. A TSK gel ODS-100Z column (4.6× 150 mm, 5 µm, TOSOH,

105

Japan) was used on an Agilent 1100 HPLC-MS system, which was equipped with an

106

ESI interface (Agilent Technologies Co. Ltd., Santa Clara, CA, USA). Mobile phases

107

were water-formic acid (98.5:1.5, v/v) (mobile phase A) and water-acetonitrile-formic

108

acid (48.5:50:1.5, v/v/v) (mobile phase B). The linear gradient for elution was: 0-25

109

min, 12-30% B; 25-34 min, 30-100% B; 34-40 min, 100-12% B; the column was then

110

allowed to re-equilibrate back to the starting mobile phase of 12% B for 5 min before 5


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111

the next injection. The total running time was 45 min, and chromatograms were

112

recorded at 520 nm using a diode array detector (DAD). An injection volume of 20 µL

113

was selected with a flow rate of 1 mL/min. Meanwhile, the eluent was also detected

114

by mass spectrometer. The mass fragmentation experiments were performed on an

115

electrospray ionization (ESI) mass spectrometer with a positive ion mode. Capillary

116

voltage, 3500 V; fragmentor voltage, 100 V; nebulizing pressure, 35 psi; dry gas

117

temperature, 325 °C; and mass range, m/z 100–1000.

118

Fluorescence spectroscopy. The stock solution of β-glucosidase was prepared with

119

Tris-HCl buffer (0.05 M, pH 7.4) containing NaCl (0.1 M) to control the pH and ionic

120

strength. Mv-3-hex and Mv-3-ace-hex were prepared by Sephadex LH-20 column

121

chromatography and semi-preparative HPLC, respectively. Fluorescence spectra of

122

β-glucosidase (1.0 mg·mL-1) in presence of Mv-3-hex/Mv-3-ace-hex (0-90 µM) were

123

recorded at 298, 303 and 308 K. The excitation wavelength was 280 nm and the

124

emission wavelength was ranged from 290 to 430 nm. The slid width of excitation

125

and emission were both set at 5.0 nm. Scanning speed was 1200 nm·min-1 and the

126

voltage was 400 V. In the synchronous fluorescence experiment, the excitation

127

wavelength was set at 250-330 nm (∆λ= 15 nm) and 200-350 nm (∆λ= 60 nm) at 303

128

K, pH 7.4.

129

Circular dichroism spectroscopy. In order to analyze the structural change of

130

β-glucosidase (1.0 mg·mL-1) by the addition of Mv-3-hex/Mv-3-ace-hex, the CD

131

spectra was recorded from 190 to 250 nm. The concentrations of anthocyanins were

132

varied at 2, 10 and 20 µM. 6



133

Molecular docking. The crystal structure of β-glucosidase used in the docking

134

study was extracted from the structure (PDB entry: 1E1E) available in Protein Data

135

Bank (PDB). Polar hydrogen atoms and Gasteiger charges were added to prepare

136

β-glucosidase molecule for docking analysis. For docking of Mv-3-hex and

137

Mv-3-ace-hex with β-glucosidase, the ground state geometry of Mv-3-hex and

138

Mv-3-ace-hex were drawn using Chemsketch 3.5 and energy minimized. The final

139

energy assessment was carried out with the free energy change (∆G0b) and a better

140

orientation was taken as the output for each ligands. The output from Autodock was

141

rendered with PyMol (http://www.pymol.org).

142

RESULTS

143

Identification of anthocyanins from blueberry wine lees. Total contents of

144

phenolics and anthocyanin in blueberry, blueberry wine lees and wine were shown in

145

Table S1. Both of blueberry fruit and blueberry wine lees were rich in polyphenols

146

and anthocyanins. Furthermore, acylated anthocyanins were not presented in

147

blueberries, but in wine and wine lees due to fermentation (Table S1). Four types of

148

anthocyanin fragmentation ions [delphinidin (De) at m/z 303, cyanidin (Cy) at m/z

149

287, petunidin (Pet) at m/z 317, and malvidin (Mal) at m/z 331] and three types of

150

glycosides [galactoside (Gal), glucoside (Glu), and arabinoside (Ara)] were detected

151

in blueberry wine lees. The diagrammatic structures were presented in Figure S1. In

152

order to obtain the purification grade of anthocyanins monomers, we employed

153

Sephadex LH-20 to separate individual anthocyanins from the crude anthocyanins

154

mixture.23 In our study, systematic investigations of the Sephadex LH-20 column with 7


Page 8 of 32

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155

different reagents showed that elution with 10% methanol containing 2% formic acid

156

is a very simple and effective method of fractionating anthocyanins. In this stage,

157

three major fractions were collected (Figure S2). The anthocyanin profiles in these

158

three fractions were determined by HPLC-DAD–MS/MS (Table 1). Fraction 1 mainly

159

consisted of Malvidin-3-galactoside (Mv-3-gal) and malvidin-3-glucoside (Mv-3-glu),

160

representing 90% of the total peak area (Figure 1a), fraction 2 included

161

malvidin-3-arabinoside

162

(Mv-3-ace-glu) (14%) (Figure 1b), and fraction 3 consisted of delphinidin-3-glucoside

163

(De-3-glu) (87%) and cyanidin-3-acetyl-glucoside (Cy-3-ace-glu) (12%) (Figure 1c).

164

The Mv-3-glu and Mv-3-gal in fraction 1 were pure isomers, so fraction 1 will be

165

used to investigate the interaction between β-glucosidase in the following experiment.

166

In the present study, six major fractions were collected by semi-preparative HPLC24

167

and further determined by HPLC-DAD–MS/MS (Figure S3, Figure 2, Table 2).

168

De-3-glu was the main component in fraction 1 (Figure 2a), petunidin-3-glucoside

169

(Pe-3-glu) in fraction 2 was the primary component (Figure 2b), fraction 3 consisted

170

of a mix of different anthocyanins, including Mv-3-glu, Mv-3-gal and Mv-3-ara

171

(Figure 2c), Mv-3-ara was the main component in fraction 4 (Figure 2d),

172

Mv-3-ace-glu and malvidin-3-acetyl-galactoside (Mv-3-ace-gal) appear to be the

173

major anthocyanins monomers in fraction 5 and 6 (Figure 2e, f), and the purity of

174

fraction 6 was higher than fraction 5, so fraction 6 was used for the interaction

175

experiments.

176

(Mv-3-ara)

(65%)

and

malvidin-3-acetyl-glucoside

Fluorescence quenching of β-glucosidase by Mv-3-hex and Mv-3-ace-hex. The 8



177

fluorescence intensity quenching of β-glucosidase upon stepwise addition of

178

Mv-3-hex and Mv-3-ace-hex were shown in Figure 3. With the increasing of

179

Mv-3-hex and Mv-3-ace-hex, the intrinsic fluorescence intensity of β-glucosidase

180

continued to decrease, which indicated that Mv-3-hex and Mv-3-ace-hex interacted

181

with β-glucosidase. The fluorescence maxima showed slightly red shift from 337.2 to

182

339.6 nm in the addition of Mv-3-hex, which indicated the hydrophobicity of the

183

microenvironment of amino acid residues increased due to the change of fluorescence

184

chromophore and conformation of β-glucosidase. While the addition of Mv-3-ace-hex

185

made the peak a slight blue shift from 341.0 to 338.8 nm, which suggested the

186

hydrophobicity of the microenvironment of aromatic amino acid residues reduced. It

187

illustrated that the compounds of Mv-3-hex and Mv-3-ace-hex quenched the

188

fluorescence intensity of β-glucosidase.

189

The quenching mechanism. Fluorescence quenching can be classified as dynamic

190

and static quenching by analyzing the equation of Stern–Volmer:

191

F0/F=1+Kqτ0[Q]=1+Ksv[Q]

192

Where F0 and F are the fluorescence intensities in the absence and presence of

193

Mv-3-hex and Mv-3-ace-hex, respectively. Kq is the bimolecular quenching constant,

194

and τ0 is the lifetime of fluorescence in the inexistence of quencher (usually 10-8 s). [Q]

195

is the concentration of the quencher, and Ksv is the Stern-Volmer quenching constant.

196

Usually, the Ksv along with the change of temperature could be used to illustrate the

197

mechanisms of quenching. Static quenching results from the formation of a ground

198

state complex between the quencher and the fluorophore, whereas dynamic quenching

（1）

9


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199

refers to a process in which the quencher and the fluorophore come into contact

200

during the transient existence of the excited state, dynamic and static quenching can

201

be distinguished by their binding constants on viscosity and temperature, or preferably

202

by their lifetimes.25-26

203

In order to elucidate the quenching type of the quenchers (Mv-3-hex and

204

Mv-3-ace-hex) and β-glucosidase, the Stern-Volmer curve of β-glucosidase

205

fluorescence quenching at various temperatures were constructed (Figure S4).

206

Obviously, there was a good linear regression of a plot of F0/F against [Q]. The value

207

of Ksv decreased with the temperature and the value of Kq was much greater than the

208

maximum scatter collision quenching constant value 2×1010 L/mol/s (Table S2), this

209

confirms that static quenching was the single type between β-glucosidase and the

210

quenchers ( Mv-3-hex and Mv-3-ace-hex).

211

Calculation of binding parameters and determination of the interaction force

212

between β-glucosidase and Mv-3-hex/Mv-3-ace-hex. The following equation was

213

used to calculate the binding constants (Ka) and the number of binding sites (n) of

214

small molecules interaction with protein.26

215

log[(F0-F)/F]=logKa+nlog[Q]

216

Ka and n are the binding constant and the number of binding sites, respectively. The

217

binding constant Ka and binding points n were obtained at different temperature

218

according to equation (2). The calculated results were listed in Table 5, the number of

219

binding sites was close to 1, which indicated that the binding between quenchers

220

(Mv-3-hex and Mv-3-ace-hex) and β-glucosidase were at 1:1 ratio, and the binding

(2)

10



Page 12 of 32

221

constants were close, while the value of n of Mv-3-hex-β-glucosidase was much

222

higher than that of Mv-3-ace-hex-β-glucosidase, which indicated that the interaction

223

between

224

Mv-3-ace-hex-β-glucosidase. On the other hand, the value of binding constants Ka

225

were not great enough and just close to 104 mol/L, with the increasing of temperatures,

226

the Ka didn’t show regular changes. It may be indicated that the complexes of

227

Mv-3-hex-β-glucosidase and Mv-3-ace-hex-β-glucosidase didn’t have good stabilities.

228

The following equations were used to determine the thermodynamic parameters in

Mv-3-hex

and

β-glucosidase

was

much

more

forceful

than

229

the drug-protein binding process:

230

lnKa=-∆H/RT+∆S/R

(3)

231

∆G=∆H-T∆S

(4)

232

Where Ka is the binding constant at each temperature T (absolute temperature) and R

233

is the gas constant (8.314 J/mol/K). Ploting lnK versus 1/T, the Van’t Hoff plots gave

234

relatively good straight lines and the ∆S and ∆H values calculated from the slope and

235

intercept of the plot were shown in Table S3. The free energy change (∆G) was

236

obtained from Eq. (4) at each temperature T.

237

Generally, the interactions of parameters with biological macromolecules belong to

238

non-covalent binding, the type of binding force includes Van der Waals, hydrogen

239

bonding, electrostatic and hydrophobic interactions. The thermodynamic parameters

240

(∆G, ∆H, ∆S) can be used to confirm the type of the binding force by using the Van’t

241

Hoff equation (Eq.5). When ∆H>0, ∆S>0, the hydrophobic effect was the prime force

242

of interaction; if ∆H

Separation and Identification of Anthocyanins Extracted from

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