Optimizing Nucleophilic Depolymerization of Proanthocyanidins in

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Optimizing nucleophilic depolymerization of proanthocyanidins in grape seeds to dimeric proanthocyanidin B1 or B2 kui-shan wen, xiao ruan, jing wang, li yang, Feng Wei, ying-xian zhao, and qiang wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01188 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Agricultural and Food Chemistry

Optimizing nucleophilic depolymerization of proanthocyanidins in grape seeds to dimeric proanthocyanidin B1 or B2

Kui-Shan WEN

a,b,

Xiao RUAN

a,b,

Jing WANG c, Li YANG

a,b,

Feng WEI

a,b,

Ying-Xian ZHAO

a,b,

and

Qiang WANG a,b *

a Ningbo

Institute of Technology, Zhejiang University, Ningbo 315100, China;

b Ningbo

Research Institute, Zhejiang University, Ningbo 315100, China;

c Ningbo

Osaki Biotech Co., Ltd, Ningbo 315800, China;

Corresponding author: [email protected];

Tel.: +86-13777135491

1

ACS Paragon Plus Environment


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1

Abstract: Depolymerization of polymeric proanthocyanidins (PPC) in grape seeds into oligomeric

2

proanthocyanidins (OPC), especially the dimers, has important academic significance and practical

3

value. Reaction conditions including nucleophilicreagent/PPC mass ratio, HCl concentration,

4

reaction time and temperature were systematically optimized by central composite design to

5

maximize the yield of the dimeric product B2 or B1. The yield of B2 reached 3.35 mg mL-1 under

6

the conditions of (-)-epicatechin/PPC mass ratio = 2.8, HCl concentration= 0.06 mol, reaction time

7

= 16 min and temperature = 36 °C, and that of B1 reached 3.64 mg mL-1 under the conditions of

8

(+)-catechin /PPC mass ratio = 2.8, HCl concentration= 0.07 mol, reaction time = 17 min and

9

temperature = 34 °C. Overall, this study has provided a theoretical guidance and practical approach

10

to improve reaction process and economic value of proanthocyanidins in GSPE.

11

Keywords: grape seed proanthocyanidins extract (GSPE), polymeric proanthocyanidins (PPC),

12

depolymerization, mechanism, optimization

13 14 15 16 17 18 19 20 21 2


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22


Introduction

23

As a group of very important polyphenolic compounds with vital functions to resist microbial

24

pathogens [1], insect pests[2] and larger herbivores[3], proanthocyanidins are widely present in natural

25

plants of grape, cranberry, cocoa, etc., particularly in skin and seed of these plants [4]. Up to now, a

26

great deal of research interests have been generated for proanthocyanidins due to their important

27

roles in stabilizing color and enhancing mouth-feel of red wines

28

sources of proanthocyanidins in nature, grape seed contains much richer polyphenolic compounds

29

than its skin and stem. In fact, most products of proanthocyanidin in market originally come from

30

grape seed

31

and polymers (PPC) with flavan-3-ol monomeric units[9], such as (+)-catechin, (−)-epicatechin and

32

(−)-epicatechin-3 -O-gallate (CATs), linked mainly through C4→C8 or C4→C6 (B-type)[10,11]. The

33

difference in constitutional units, bonding position and configuration gives structural diversity of

34

the polymerized proanthocyanidins, and the number of isomers increases markedly with increasing

35

the degree of polymerization (DP) [12].

[7,8].

[5, 6].

As one of the most abundant

Grape seed proanthocyanidins extracts (GSPE) are composed of oligomers (OPC)

36

Substantial investigations have demonstrated that proanthocyanidins could broadly provide

37

pharmacological and therapeutic functions against oxidative stress and degenerative diseases such

38

as acute and chronic stress, gastrointestinal distress, neurological disorders, pancreatitis,

39

cardiovascular dysfunctions, various stages of neoplastic processes and carcinogenesis including

40

detoxification of carcinogenic metabolites[13,14], and the curative efficacy. However, it would largely

41

depend on their structures and DPs contents. Jiang et al[15] evaluated the effect of GSPE fractions

42

with different DPs on blood glucose, lipids and hepatic oxidative stress in diabetic rats, and

43

confirmed that GSPE gave a positive effect on diabetes in rats, and its oligomeric form might be 3



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44

more effective than other forms. Moreover, GSPE exhibited potent abilities to scavenge free

45

radicals in both vitro and vivo models. It has been lately verified that PPCs possessed the highest

46

antioxidant activities followed by OPCs, whereas CATs gave a lower antioxidant activity,

47

indicating that the vitro antioxidant activities of proanthocyanidins were positively related to their

48

DP [16]. In fact, the dimers, trimers and tetramers of proanthocyanidin could be preferably absorbed

49

and presented in blood circulation, whereas its polymers with higher molecular weight could not [17].

50

Besides, Tomás-Barberan et al [18] proved that the increase of monomers in proanthocyanidins could

51

enhance the bio-availability. Among various proanthocyanidins, dimeric proanthocyanidins B1 and

52

B2 have attracted more research interests, their physiological mechanism has been generally

53

recognized and application value been highly affirmed so far [19-21].

54

Extraction and separation of proanthocyanidins from plants still raise a major challenge to

55

scientists due to the structural diversity and complexity. There have been a large number of studies

56

on the fractionation of proanthocyanidins during the last two decades. A variety of separation

57

methods such as size-exclusion chromatography

58

extraction

59

of proanthocyanidins corresponding to DP. Zhang et al

60

GSPEs by high-speed counter-current chromatography (HSCCC), and under the optimal conditions

61

of HSCCC, GSPEs could be separated into seven distinct fractions (F1-F7) with increasing the

62

mean degree of polymerization from 1.44 to 6.95.

63

[24]

and normal phase chromatography

[25]

[22],

solid-phase extraction

[23],

enzymatic

were tentatively adopted for the fractionation [26]

conducted the preparative separation of

Several methods of chemical reaction have been also developed to depolymerize [27-30].

64

proanthocyanidin polymers into oligomers for the enhancement of bio-availability

The

65

acid-catalyzed hydrolysis is to cleave the interflavanic C-C bonds in proanthocyanidin molecules 4


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66

into the terminal units (CATs) and the upper intermediate units or fragments as carbocations which

67

react with nucleophiles to form stable conjugates. Foo and Porter [27] heated palm proanthocyanidin

68

polymers with epicatechin in ethanol-acetic acid at 95℃ for 22 h and then identified some dimers.

69

Moreover, polymeric proanthocyanidins from grape seeds or hazelnut skins were reacted with

70

flavan-3-ols under acidic conditions for the semi-synthesis of dimmers

71

experiments, the HCl-catalyzed depolymerization with catechin or epicatechin was convenient to

72

operate with simple apparatus and the yield of oligomers was relatively high

73

understanding of knowledge, however, the depolymerization process of PPC from GSPE will

74

generate a complex mixture of various intermediate products (see in Fig.1). With a proper design

75

based on the reaction mechanism and by optimizing reaction conditions, the use of HCl and the

76

addition of (+)-catechin and (-) –epicatechin as nucleophile agents, maybe be profitable to enhance

77

the proportion of dimers proanthocyanidins B1 or B2 in the final mixture of products.

[30].

Among these reaction

[28].

According to our

78

In this work, the nucleophilic catalytic depolymerization of polymeric proanthocyanidins was

79

conducted, the effects of various reaction variables on the yields of dimeric proanthocyanidins B1

80

or B2 were comprehensively investigated by using response surface methodology (RSM) to

81

experiments of a central composite design (CCD) with four factors (reaction time, temperature, pH,

82

and ratio of reactant PPC / nucleophile catechin or epicatechin) and five levels, and the maximum

83

yield of dimer B1 or B2 under optimal reaction conditions was evaluated.

84

Materials and Methods

85

Materials and reagents

86

The powder of freeze-dried grape seed proanthocyanidin extract (GSPE) was supplied by

87

Tianding, Inc. Ningbo, China. Standard samples of (+)-catechin (≥98%), (-)-epicatechin (≥98%), 5



acid

(≥98%),

Epicatechin-3-O-gallate

(≥98%),

proanthocyanidin

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88

gallic

B1

(≥96%),

89

proanthocyanidin B2 (≥96%), proanthocyanidin B3 (≥96%) and proanthocyanidin B4 (≥96%) were

90

provided by the National Institute for the Control of Pharmaceutical and Biological Products,

91

Beijing, China. Vanillin, DPPH, acetonitrile and methanol in spectral grade were purchased from

92

Sigma Chemical, Louis, MO, USA, and all the other chemicals and reagents in analytical grade

93

purchased from commercial sources.

94

Isolation of polymeric proanthocyanidins

95

250 mL aqueous solution containing 2.5 g GSPE powder was mixed with 750 mL ethyl acetate,

96

and then the mixture refrigerated at 4 °C for 12 h. Next, ethyl acetate supernatants and aqueous

97

phase were separated, and the two fractions of three repeated operations were combined

98

respectively. Afterward, the solvents were removed from ethyl acetate fraction and water fraction

99

by using rotary evaporator to give the product of OPC and PPC respectively. Of which, the PPC

100

was used as a raw material for depolymerization reaction.

101

Depolymerization reaction

102

Depolymerization reaction was conducted according to the procedure adopted in previous [11,28]

103

study

with minor modification. In a typical run, 100 mg of (-)-epicatechin (or (+)-catechin)

104

and 100 mg of PPC were dissolved in 8.0 mL methanol, resulting in the 1:1 mass ratio of

105

epicatechin (or catechin) to PPC. Next, the solution was immediately mixed with 2.0 mL

106

methanolic HCl at 0.5 mol to give an H+ ion concentration of 0.1 mol, and then incubated in a water

107

bath at 40 °C for 40 min. The reaction was terminated by adding a certain amount of NaHCO3

108

solution to adjust pH of reaction mixture into 5-6. By the end of reaction, the products were filtered

109

through a 0.22 m nylon filter and sampled for UPLC analysis. 6


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110


UPLC analysis

111

Chromatographic analyses of GSPE, extraction fractions, and deploymerization products were

112

performed on an Ultra High Performance Liquid chromatography (UPLC) system (Agilent 1290,

113

Santa Clara, CA, USA). This system contains a binary pump, an auto-sampler, a column thermostat

114

and DAD detector. Chromatographic conditions were set as follows: injection volume of 5 uL, flow

115

rate at 1 mL min-1, column temperature in 30 °C, detected wavelength at 280 nm; column with 1.8

116

m, 4.6 mm×100 mm i.d. (Agilent Technologies, Beijing, China); 0.2% aqueous formic acid (v/v,

117

solvent A) or acetonitrile (solvent B) as mobile phase. Gradient elution was performed linearly by

118

10 - 15% A at 0-2 min, 15 - 16% A at 2 - 5 min, 16 - 20% A at 5 - 8 min, 20 - 45% A at 8 - 9 min

119

and 45 - 10% A at 9 - 10 min, and followed by 5 min for column re-equilibration before the next

120

injection. Gallic acid, catechin and epicatechin, dimeric proanthocyanidins B1, B2, B3 and B4, and

121

epicatechin-3-O-gallate as reference materials were assayed by UPLC.

122

Based on the retention time eluted on the chromatogram, the contents of standard materials

123

were calculated by referring to the calibration curve plotted with six concentration points of each

124

material. Relative Standard Deviation (RSD) was calculated to evaluate the precision of the assay

125

by the standard solution with six replicates. For catechin as an example, the RSD was 0.71%, and

126

the accuracy was determined by recovery experiment in a half and double concentration levels of

127

the same solution, giving the determined recoveries to be 99.4 and 101.2%, respectively.

128

Determination of polymerization degree

129

The mass content of proanthocyanidin was determined by vanillin-methanol method and the [31]

130

previous procedure

with minor modification. In detail, 1.0 mL sample was mixed with 5.0 mL

131

methanolic vanillin reagent containing 0.75% (M/V) vanillin and 4% (V/V) HCl in dark water bath 7



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132

for 15 min at 25 °C, and then the absorbance was measured at 500 nm wavelength on a UV-Vis

133

microplate reader. Calibration curve was drawn using catechin as the standard material. Butler et al

134

[32]

135

flavan-3-ol unit could react with vanillin, and therefore the molar number of proanthocyanidin

136

would be determined. When the vanillin-acetic acid method was used, analogously, 1.0 mL sample

137

was mixed with 5.0 mL glacial acetic acid -vanillin solution containing vanillin 1.5% (M/V)

138

vanillin and 4% (V/V) HCl in dark water bath for 10 min at 20 °C, and then the absorbance was

139

measured at 500 nm wavelength on a UV-Vis microplate reader. Besides, the proanthocyanidin

140

sample should be dissolved in a minimum volume of methanol and diluted in glacial acetic acid.

141

Based on the mass weight and molecular weight of proanthocyanidin, the mean degree of

142

polymerization (mDP) could be calculated by the following equation:

143 144

revealed that when the reaction carried out in the solvent of glacial acetic acid, only the terminal

mDP 

m Mr  n

(1)

Where m is mass content (μg·mL-1), n was molecular content (μmol mL-1), Mr was relative

145

molecular mass of catechin with a value of 290.

146

Quantitative analysis of broad peak in chromatogram

147

Proanthocyanidins with DP over 3 would be eluted as a broad peak in the chromatogram[33]. In

148

order to measure the mass content of the substance in this broad peak, the product obtained from

149

water fraction (section 2.2) was taken as the reference to establish the calibration curve using UPLC.

150

The water fraction was dried out by rotary evaporator to get brownness solid product. Next, 10 mg

151

mL-1 of methanolic product solution was prepared and diluted into six sample solutions at

152

concentrations of 0.25, 0.5, 1.0, 2.5, 5 and 10 mg mL-1, respectively. Each of the sample solutions

153

would be measured using the UPLC method in section 2.4, and the calibration curve of peak area 8


Page 9 of 32


154

versus mass concentration could be drawn. The regression equation of calibration curve was given

155

as Y=2010.2X- 485.7 with linear range of 0.25-10.0 mg mL-1, where Y was peak area and X was

156

concentration (mg mL-1).

157

Assay of DPPH radical scavenging

158

The assay of DPPH radical scavenging activity was performed in a Biotek Synergy 2

159

Multi-Detection Microplate Reader (Biotek, Winooski, VT, USA) according to the procedure

160

described by Ruan et al.[34] with minor modification. A set of samples was prepared with various

161

concentrations in methanol. Fresh DPPH in methanol (3.9 mL) was added into 0.1 mL of each

162

sample solution. In all the experiments, methanol and l-ascorbic acid were selected as a negative or

163

positive control, respectively. The absorbance was measured at 516 nm wavelength after incubation

164

in dark for 30 min at room temperature. The radical scavenging activity of DPPH was calculated as

165

follows

166

167

 A negative control  A sample  Radical scavenging(%)    100 A negative control  

(2)

Experimental design and evaluation

168

A central composite design (CCD) with four variables at five levels, including epicatechin or

169

catechin to PPC mass ratio (X1= 1, 1.5, 2, 2.5, 3), HCl concentration (X2= 0.02, 0.04, 0.06, 0.08, 0.1

170

mol), reaction time (X3= 10, 15, 20, 25, 50 min) and temperature (X4=30, 35, 40, 45, 50 °C), was

171

applied to examine the impact of variables on the yield of proanthocyanidin B2 or B1 using

172

Design-Expert Software (Stat-Ease Inc., Minneapolis, MN, USA). To predict the targeted response,

173

total 30 experimental trials including 16 factorial, 8 axial and 6 central runs were conducted to

174

determine the parameters in a second-order polynomial regression equation as follows:

9



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175

Y  b0  i 1 bi X i  i 1 bii X i  i 1  j i 1 bi j X i X j

176

Where Y is response, Xi and Xj are independent variables, b0, is the offset term, bi and bii are the

177

linear effect and the quadratic effect of Xi, and bij is the interaction effect between Xi and Xj. The

178

fitted polynomial equation was expressed in 3D response surface. Parameters R2, adjust-R2,

179

variance analysis, and residuals analysis were employed to evaluate the model. The software was

180

used to fit the response surface to optimize the reaction conditions. The predicted optimal yield of

181

dimeric proanthocyanidin was examined by experiments with the selective optimal values of

182

variables.

183

Statistical analysis

4

4

2

3

4

(3)

184

Data were expressed as mean ± standard deviation. The standard curves were plotted using

185

Origin Pro 8.1 (Origin Lab, Northampton, MA). The significant difference among parameters of the

186

reaction were first examined by ANOVA (p 0.999) in a wide

195

concentration range of 0.01-1.0 mg mL-1.

196

Chromatogram of GSPE was demonstrated in Fig.2B. All the references mentioned in above 10


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197

and some unidentified substances eluted in time range of 0-9 min gave insignificant contents less

198

than 50 mg g-1, as presented in Table 1. A notable single broad peak appeared at 9.63 min and

199

accounted for 881.0 mg g-1 of GSPE, attributed to the mixture of proanthocyanidins with mDP over

200

3[28]. After the GSPE was separated into two fractions by extraction of ethyl acetate/water at 3/1

201

volume ratio, the supernatant fraction of ethyl acetate predominantly contained oligomeric

202

proanthocyanidin (OPC) with the yield of 19% GSPE (g/g) gave the mDP of 2.21, and the

203

polymeric proanthocyanidins (PPC) with the yield of 81% GSPE (g/g) remained in water phase and

204

gave the mDP of 5.65. In the extracts of ethyl acetate, the contents of monomers and dimers were

205

remarkably enhanced, such that the contents of catechin and epicatechin significantly increased

206

from 35 and 25 mg g-1 to 178 and 143 mg g-1 respectively (Fig.2C and Table 1). On the other hands,

207

the residues in water phase only contained PPC at the estimated content of 991.0 mg g-1, and almost

208

no signals of monomers or hardly visible signals of dimers appeared in the chromatogram (Fig.2D).

209

Depolymerization of polymeric proanthocyanidins (PPC)

210

In the depolymerization of PPC catalyzed by an acidic catalyst such as HCl, positive hydrogen

211

ions H+ initiated the cleavage of interflavan bonds in polymeric proanthocyanidins, so that the

212

terminal unit was released as a catechin (or epicatechin) while the rest body successively turned into

213

several carbocations with positively charged on C4 (pathwayⅠin Fig.1)

214

additional nucleophilic agents, these liberated carbocations might react backwards with the released

215

catechin (or epicatechin) and eventually reach a dynamic equilibrium with the forward reaction,

216

thus giving a low apparent conversion of the original reactant (PPC) and a small reduction of mDP

217

as indicated by the data on the bottom line of Table 1 or by the chromatogram G in Fig. 2. Once

218

nucleophiles such as flavan-3-ol (epicatechin or catechin) were added into the reaction system,

[27].

In the absence of

11



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219

however, these carbocations would be immediately captured by the nucleophile as bottom units to

220

form dimeric proanthocyanidins (path Ⅱin Fig.1)

221

PPC material contained no monomers or dimers. After depolymerized with adding epicatechin or

222

catechin as nucleophilic promoter, however, the dimeric products increased from zero (mg/g) to a

223

visible level (Table 1, and Fig. 2). Moreover, in the case of epicatechin as reaction promoter, the

224

predominant dimeric product was B2 with the yield of 98.2 mg g-1 (Table 1), while the released

225

terminal unit of catechin and other dimeric products B4, B1, B3 and ECG as well as some

226

unidentified compounds were also detected (Fig.2E). Similarly, in the case of catechin as promoter,

227

the primary dimeric product was B1 with the yield of 109 mg g-1 (Table 1), and several other

228

products were simultaneously found in the chromatogram (Fig. 2F).

[35].

As seen in Fig.2D and Table 1, the original

229

As showed in Fig.1, those newly-formed dimers as nucleophiles would continuously interact

230

with carbocation of epicatechin or catechin to produce trimeric proanthocyanidin (path III), so that

231

the broad peak with elution time near to 10 min was still found in the chromatogram (Fig.2).

232

Theoretically, such similar reaction would continue to synthesize higher polymer of

233

proanthocyanidin (Path IV), and form a closed circle of reaction pathway in the end. Based on the

234

suggested reaction mechanism and pathways of PPC depolymerization, it can be concluded that

235

various variables such as flavan-3-ol to PPC ratio, HCl concentration, reaction temperature and time

236

would influence the formation yields of the intermediate products in different ways and degrees. On

237

the other side, the positive hydrogen ion H+ could also attack and protonate the oxygen on the ring

238

of flavan-3-ol, causing the ring to open and form the positively charged ion of chalcane during the

239

reaction process. These chalcane carbocations would compete nucleophiles with the flavan-3-ol

240

carbocation of the cleaved PPC to generate chalcan- falvan-3-ol derivatives, so called gambiriins [28]. 12


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241


The formation pathways of by-products were shown as V and VI in Fig.1.

242

The structures of B-type dimeric proanthocyanidins were shown in Fig.1, and their

243

conformations and yields from the PPC deploymerization in HCL/ nucleophile system depended on

244

the carbocation configuration, nucleophile type and interflavan bonding patterns

245

interflavan bonded through C4→C8, dimeric proanthocyanidins B1-B4 were formed, and if bonded

246

through C4→C6, the formed dimers were B5-B8. By comparison, the dimeric proanthocyanidins

247

B1-B4 occurred more widely in nature due to the preferable conformation of C4→C8 bonding. The

248

extensional units of grape seed proanthocyanidins were predominated epicatechin, and thus the

249

cleaved carbocations were epicatechin positively charged ions too [37]. When epicatechin added, the

250

yields of proanthocyanidin B2 and B4 were 98.2 mg g-1 and 11.5 mg g-1 respectively, giving the

251

ratio about 8.5:1. When catechin added, the yields of proanthocyanidin B1 and B3 were 109.0 mg

252

g-1 and 12.6 mg g-1 respectively, giving the ratio about 8.6:1. In other words, the dimeric products

253

were abundant in B2 (or B1) and proportionally accompanied with fewer B4 (or B3). Therefore, we

254

have further targeted the maximum yield of proanthocyanidin B2 or B1 as the objective to optimize

255

the reaction conditions of PCC depolymerization.

256

[36].

When the

The activity and effect of natural antioxidants in foods has been the subject of extensive [13,38].

257

studies for a long time

As a class of prominent compounds in foods, for example,

258

proanthocyanidins have been attracting more and more attentions due to their antioxidant activity.

259

The activity of antioxidants is usually measured by several techniques such as DPPH-RSA

260

ABTS-RSA, FRAP assay [40] and CUPRAC (cupric reducing antioxidant capacity) assay [41]. In this

261

study, the scavenging free radicals capacity of antioxidant was quantified by the index IC50 of

262

eliminating 50% DPPH. For a reference, the conventional antioxidant molecules ascorbic acid gave

[39],

13



Page 14 of 32

263

the IC50 value of 71.2 mg mL-1. The GSPE, proanthocyanidins monomer, oligomer OPC and

264

polymer PPC gave the IC50 values of 51.2, 54.3, 53.2 and 50.6 mg mL-1 respectively. It indicated

265

that the capacity of various proanthocyanidins to scavenge free radicals was stronger than ascorbic

266

acid. Also, the IC50 of these proanthocyanidins decreased with increasing polymerization degree

267

and varied in the range of 50.6-54.3 mg mL-1, suggesting that the capacity of a proanthocyanidin to

268

scavenge free radicals in vitro was related to its concentration, dosage, size or DP.

269

Modelling based on the trials with central composite design

270

Following a preliminary investigation on the effects of various conditions on the

271

depolymeization of PPC by single factor experiment with changing one variable at a time, the

272

response surface methodology of experiment with central composite design has been applied to

273

maximize the production of dimeric proanthocyanidin B2 or B1 by combinatorial optimization of

274

four variables including epicatechin or catechin / PPC mass ratio (X1), HCl concentration (X2),

275

reaction time (X3) and temperature (X4). As showed by Table 2 in detail, the yield of either B2 or

276

B1 was set as response, the value of each factor X1 or X2 or X3 or X4 varied with five levels,

277

respectively. A total of 30 trials with adding epicatechin as nucleophile generated 30 response data

278

of Y2 in the range of 2.16 to 3.26 mg mL-1, and a total of additional 30 trials with adding catechin

279

as nucleophile gave other 30 response data of Y1 in the range of 2.34 to 3.58 mg mL-1.

280

The second-order polynomial equations for the yield of dimeric proanthocyanidin B2 or B1 as

281

function of variables X1, X2, X3 and X4 were obtained by the multiple regression fitting of data in

282

Table 2, as follows

283 284

Y2 =－5.08＋3.19 X1＋17.73 X2＋0.11 X3＋0.15 X4＋0.63 X1X2－0.03 X1X3－0.02 X1X4＋ 0.05 X1X3＋0.12 X2X4＋0.0004 X3X4－0.38 X12－195.8 X22－0.0018 X32－0.0017 X42 14


Page 15 of 32

285 286


Y1 = － 5.1 ＋ 3.59 X1 ＋ 20.94 X2 ＋ 0.09 X3 ＋ 0.13 X4 ＋ 0.07 X1X2 － 0.03 X1X3 － 0.02 X1X4-0.13 X1X3＋0.19 X2X4＋0.0007 X3X4－0.38 X12－205.3 X22－0.0014 X32－0.0015 X42

287

Analysis of variance (ANOVA) was performed to examine the reliability of the above

288

regression equations. The results of ANOVA in Table 2 confirmed that both models fitted the

289

experimental data adequately, as evidenced by the low P-value less than 0.001, higher R-square

290

values of 0.98 and 0.97, insignificant Lack of Fit 0.79 and 0.48, etc. Also, all the linear terms (X1,

291

X2, X3, X4), all the square items (X12 , X22 , X32 , X42) and two interaction items (X1X3, X1X4) had

292

p-value lower than 0.05, indicating they were significant factors with the confident level of more

293

than 95%. The other interaction terms (X1X2, X2X3, X2X4, X3X4) were not significant factors.

294

Overall, the models can be confidently applied for the correlation of real experimental data, and

295

thus the optimization of reaction conditions and the simulation of new experiments can be extended.

296

Optimization analysis of the response surface

297

The independent and interaction effects of four variables on the yields of dimeric

298

proanthocyanidin B2 and B1 in PPC depolymerization reaction were visually depicted by two set of

299

three-dimensional response curved surface graphs, as shown in Fig. 3 and Fig. 4 respectively.

300

Fig.3A or Fig.4A showed the effect of epicatechin or catechin to PPC mass ratio (X1) and HCl

301

concentration (X2) on the response values of proanthocyanidin B2 or B1 yield (Y2 or Y1), with

302

constant reaction time of 20 min (X3) and temperature of 40 °C (X4). One can see that higher yields

303

of B2 >3.11 mg mL-1 and B1 >3.25 mg mL-1 were obtained at HCl concentration between 0.04-0.07

304

mol and the material ratio over 2.5, respectively. In general, both reactant ratio and HCl

305

concentration gave significant effects on the yields. For dimeric proanthocyanidin B2, the yield

306

increased dramatically as the ratio increased from 1.0 to 2.5 at a certain HCl concentration and then 15



Page 16 of 32

307

this response curve surface became flatted at the ratio over 2.5. Such behavior phenomenon could

308

be interpreted by the mechanism and pathway of the depolymerization reaction. As previously

309

showed in Fig. 1, there were plenty of carbocations after the initial cracking of PPC catalyzied by

310

HCl (step I). At the lower ratio of nucleophilic additive to reactant, the addition of more

311

nucleophiles would promote their reaction with “free” carbocations to enhance the yield of dimeric

312

products (step II). When the addition of nucleophile approached a critical value corresponding to no

313

more carbocations to be available, adding more nucleophiles could hardly find free carbocation to

314

synthesize the dimers any more. On the other hand, the increase of dimers would potentially

315

compete with nucleophile for carbocation to form trimers (step 3), in turn, higher polymers (step

316

IV). So and so, a closed circle of reaction pathway was formed through step I to step IV, and each

317

step of reaction might take place reversibly in the presence of H+ ion. In terms of micro kinetic

318

mechanism, various parallel or reversible reactions would compete with each other to eventually

319

reach stable kinetic rates under certain conditions. From apparent thermodynamic behavior point of

320

view, the reaction system would gradually approach to a chemical equilibrium such as the yield of

321

dimers to stay on a steady level in the range of higher reactant ratio. Also as shown in Fig.3A or Fig.

322

4A, at a fixed ratio of reactants, the yield of dimeric product B2 or B1 increased as HCl

323

concentration increased from 0.02 to 0.06 mol, and then gradually decreased with the HCl

324

concentration over 0.06 mol. As seen from Fig.1, the increase of HCl concentration could generate

325

more flavan-3-ol carbocations (step I) to react with nucleophiles for synthesizing more dimmers

326

(step II). When extra H+ ions existed in system, however, the side reaction to produce a chalcane

327

positive ion by cleavage of the interflavan bond in a nucleophile (path V) could quickly increase,

328

and then these produced chalcane ions could subsequently react with nucleophiles to form 16


Page 17 of 32


329

gambiriins (path VI), resulting in the decrease of dimer yield. Besides, the interaction effect of the

330

material ratio and HCl concentration on the yield of diemric proanthocyanidin B2 and B1 seemed to

331

be insignificant, indicated by the p-value of X1X2 lager than 0.05 in Table 2 and also seen visually

332

from Fig. 3A and 4A. Overall, the optimal yield of B2 or B1 could be obtained at HCl concentration

333

of 0.04-0.07 mol and higher reactant ratio over 2.5.

334

Fig.3B and Fig.4B showed the change of Y1 and Y2 as a function of reactant ratio (X1) and

335

reaction time (X3) with the constant X2 of 0.06 mol and X4 of 40 °C. One can see that higher ratio

336

over 2.5 and shorter reaction time less than 20 min were favorable to the production of dimeric

337

proanthocyanidins. The yield of dimers decreased as reaction time prolonged over 20 min at a

338

higher reactant ratio, probably due to the competition of side reactions such as those by path Ⅲ and

339

pathⅤin Fig.1. Distinguished from the interaction effect of X1 and X2 in Fig.3A or Fig.4A, however,

340

the interaction between reactant ratio and reaction time (X1X3) gave very low p-values of 0.0002

341

and 0.0001 (Table 2), indicating a significant influence on the yields of B2 and B1. Overall, the

342

optimal yield of B2 or B1 could be obtained at a higher ratio and in a shorter time of reaction.

343

The response surface of B2 and B1 yield as a function of the reactant ratio (X1) and

344

temperature(X4) at constant X2 of 0.06 mol and X3 of 20 min were showed in Fig.3C and Fig.4C.

345

ANOVA in Table 2 showed that the change of temperature in the range of 30-50 °C gave no

346

significant effect on the yields of B2 and B1 respectively. In theory, an increase of temperature

347

could enhance the rates of various reactions, but high temperature could also cause breaking of

348

interfluves bonds and other covalent bonds such as those on C ring in the flavan-3-ol. As a result,

349

the yield of B1 or B2 slightly decreased at temperature over 45 °C due to the competitive increase

350

of byproducts from various side reactions. The individual effect of X1 on the yield of B1 or B2 was 17



Page 18 of 32

351

similar to those depicted in the above diagrams. Also approximatively to the interaction of X1X3,

352

the yields of B2 and B1 were significantly affected by the interaction of X1X4, with the p-values of

353

0.015 and 0.005, respectively. Overall, the optimal yield of B2 or B1 could be obtained in

354

temperature range of 30-40 °C at the ratio over 2.5.

355

Fig.3D and Fig.4D showed the response surface and the contour plot of B2 and B1 yield as

356

function of HCl concentration (X2) and reaction time (X3) with setting X1 and X4 to their central

357

values. High yield of B2 or B1 was obtained in the ranges of X2 between 0.04-0.08 mol and X3

358

between 14-22 min. Moreover, the interaction effect of HCl concentration and reaction time on the

359

yield was statistically insignificant with giving the p-values of 0.718 and 0.420, respectively.

360

The effects of HCl concentration (X2) and reaction temperature (X4) on the yields of B2 and

361

B1 were illustrated by the curved surfaces and the contour lines in Fig.3E and Fig.4E, while X1 and

362

X3 were set at constant central levels, respectively. The optimal yield of B2 or B1 was obtained in

363

temperature interval between 35 and 42C and HCl concentration range from 0.04 to 0.08 mol. It

364

should be noted that the lower yield was given in higher temperature and HCl concentration due to

365

the increased impact of competitive side reactions. The interaction effect by HCl concentration and

366

temperature was also insignificant for the yield of B2 or B1, evidenced by the p-values of 0.423 and

367

0.224 for X2X4 in Table 2.

368

Fig.3F and Fig.4F presented the profile of the yields of B2 and B1 as a function of reaction

369

time (X3) and temperature (X4), with fixing X1 and X2 to their respective central levels. The optimal

370

yield of B2 or B1 was gained in the temperature range of 35-40C and reaction period of 14-20 min.

371

The interaction effect of X3X4 gave the p-values of 0.490 and 0.273, ie p-value > 0.05, and thus was

372

not statistically significant to the yields. 18


Page 19 of 32


373

As a summary, the maximum yield of dimeric proanthocyanidin B1 or B2 from PPC

374

depolymerization in the system of HCl plus nucleophilic monomer could be reached under the

375

combinatorial conditions including reactant ratio of 2.8± 0.1, HCl concentration of 0.07± 0.01 mol

376

mL-1, reaction time of 17± 1 min, and reaction temperature of 35 ± 1C. Besides, it should be not

377

surprised that there may be slight difference between the optimal conditions to maximize B1 yield

378

and those to maximize B2 yield. From the stereochemistry point of view, catechin would present a

379

smaller steric hindrance than epicatechin when bonded to flavan-3-ol, so that the yield of B1 would

380

be slightly higher than B2 under the same reaction conditions.

381

Verification of optimal reaction conditions

382

More precise values of optimal conditions for maximization of B2 or B1 yield were

383

quantitatively determined by the simulation of the second-order polynomial equations. The ultimate

384

results revealed the maximum value of 3.35 mg mL-1 for B2 yield under the optimal conditions

385

including reactant ratio of 2.8, HCl concentration of 0.06 mol, reaction time of 16 min and

386

temperature of 36 °C, and also the maximum value of 3.64 mg mL-1 for B1 yield under the optimal

387

conditions including reactant ratio of 2.8, HCl concentration of 0.07 mol, reaction time of 17 min

388

and temperature of 34 °C, respectively. Under the same two groups of optimal conditions used in

389

the above model simulations, the supplementary experiments of PPC depolymerization verified the

390

maximum yield of 3.32 mg mL-1 for B2 and 3.75 mg mL-1 for B1, respectively. According to error

391

analysis, the relative errors between model simulation and experimental verification were 1.19%

392

and 3.2% respectively, indicating the model prediction matched with experiment investigation well.

393

Therefore, the established model was reliable, and the results were credible.

394

The proanthocyanidins in grape seed were extracted with ethyl acetate solution and separated 19



Page 20 of 32

395

into the supernatant phase of oligoproanthocyanidins (OPC) with 19% yield and 2.21 mDP, and the

396

residual phase of polyproanthocyanidins (PPC) with 81% yield and 5.65 mDP. In the presence of

397

HCL and (+)-catechin or (-)-epicatechin as nucleophile, the network of reaction pathways was

398

developed by analyzing the mechanism of various elementary reactions in PPC depolymerization.

399

Optimization of reaction conditions could promote the main reactions and inhibit the side reactions.

400

To maximize the yield of key dimeric procyanidine B2 or B1, the optimization of PPC

401

depolymerization process was carried out by the experiments of central composite design with the

402

four conditional variables at five levels, resulting in two second-order polynomial equation models

403

and two set of three-dimensional response surface diagrams. The response surface analyses, model

404

equation evaluations and additional experiment verifications unanimously confirmed that the

405

maximum yield of 3.35 mg mL-1 for B2 could be obtained under optimal conditions of

406

(-)-epicatechin/PPC mass ratio = 2.8, HCl concentration= 0.06 mol, reaction time = 16 min and

407

temperature = 36 °C, and the maximum yield of 3.64 mg mL-1 for B1 obtained under optimal

408

conditions of (+)-catechin /PPC mass ratio = 2.8, HCl concentration= 0.07 mol, reaction time = 17

409

min and temperature = 34 °C. Overall, this study has provided the theoretical guidance for

410

developing a new technology to improve the reaction process and practical value of

411

proanthocyanidins in grape seeds.

412

Abbreviations Used

413

PPC: polymeric proanthocyanidins, OPC: oligomeric proanthocyanidins, GSPE: grape seed

414

proanthocyanidins extract, DP: degree of polymerization, TNFα: tumor necrosis factor α, PMA:

415

phorbol 12-myristate 13-acetate, LPS: lipopolysaccharide, ROS: reactive oxygen species, ERK1/2:

416

extracellular signal-regulated kinase, IKKb: IkB kinase beta, HSCCC: high-speed counter-current 20


Page 21 of 32


417

chromatography, RSM: response surface methodology, CCD: central composite design, UPLC:

418

ultra high performance liquid chromatography system, RSD: relative standard deviation, ANOVA:

419

analysis of variance.

420

Reference

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

[1] Riihinen, K.R.; Ou, Z.M.; Goedecke, T.; Lankin, D.C.; Pauli, G.F.; Wu, C.D. The antibiofilm activity of lingonberry flavonoids against oral pathogens is a case connected to residual complexity. Fitoterapia 2014, 96, 78-86. [2] Haukioja, E. Putting the insect into the birch-insect interaction. Oecologia 2003, 136 (2),161-168. [3] McMahon, L.R.; McAllister, T.A.; Berg, B.P.; Majak, W.; Acharya, S.N.; Popp, J.D.; Coulman, B.E.; Wang, Y.; Cheng, K.J. A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Can. J. Plant Sci. 2000, 80 (3), 469-485. [4] Peters, D.J.; Constabel, C.P. Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonolreductase from trembling aspen (Populustremuloides). Plant J. 2002, 32 (5), 701-712. [5] Lambert, S.G.; Asenstorfer, R.E.; Williamson, N.M.; Iland, P.G.; Jones, G.P. Copigmentation between malvidin-3-glucoside and some wine constituents and its importance to colour expression in red wine. Food Chem. 2011, 125, 106–115. [6] Ma, W.; Waffo-téguo, P.; Jourdes, M.; Li, H.; Teissedre, P.L. First evidence of epicatechin vanillate in grape seed and red wine. Food Chem. 2018, 259, 302-310. [7] Santos-Buelga, C.; Francia-Aricha, E.M.; Escribano-Bailon, M.T. Comparative flavan-3-ol composition of seeds from differentgrape varieties. Food Chem. 1995, 53 (2), 197-201. [8] Fuleki, T.; Silva, J.M.R.D. Catechin and procyanidin composition of seeds from grape cultivars grown in Ontario. Journal of Agricultural and Food Chem. 1997, 45 (4), 1156–1160. [9] Awika, J.M.; Dykes, L.; Gu, L.; Rooney, L.W.; Prior, R.L. Processing of sorghum (Sorghum bicolor) and sorghum products alters procyanidin oligomer and polymer distribution and content. Journal of Agricultural and Food Chem. 2003, 51, 5516–5521. [10] Dixon, R.A.; Xie, D.Y.; Sharma, S.B. Proanthocyanidins - a final frontier in flavonoid research?. New Phytol. 2005, 165 (1), 9-28. [11] Esatbeyoglu, T.; Winterhalter, P. Preparation of dimeric procyanidins B1,B2, B5, and B7 from a polymeric procyanidin fraction of black chokeberry (Aroniamelanocarpa). J. Agr. Food Chem. 2010, 58(8), 5147–5153. [12] Nakashima, S.; Oda, C.; Masuda, S.; Tagashira, M.; Kanda, T. Isolation and structure elucidation of tetramericprocyanidins from unripe apples (Maluspumila cv. Fuji) by NMR spectroscopy. Phytochemistry 2012, 83(11), 144-152. [13] Bagchi, D.; Bagchi, M.; Stohs, S.J.; Das, D.K.; Ray, S.D.; Kuszynski, C.A.; Joshi, S.S.; Pruess, H.G. Free radicals and grape seed proanthocyanidin extract:importance in human health and disease prevention. Toxicolog 2000, 148 (2), 187-197. [14] Ma, Z.F.; Zhang, H.X. Phytochemical constituents, health benefits, and industrial application of grape seeds: A mini-review. Antioxidants.2017, 6, 71-82. [15] Jiang, M.; Wu, Y.L.; Li, X.; Zhang, Y.; Xia, K.L.; Cui, B.W.; Lian, L.H.; Nan, J.X. Oligomeric proanthocyanidin derived from grape seeds inhibited NFkB signaling in activated HSC: Involvement of 21



458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501

Page 22 of 32

JNK/ERK MAPK and PI3K/Akt pathways. Biomed. Pharmacother. 2017, 93, 674-680. [16] Spranger, I.; Sun, B.S.; Mateus, A.M.; de Freitas, V.; Ricardo-da-Silva, J.M. Chemical characterization and antioxidant activities of oligomeric and polymeric procyanidin fractions from grape seeds. Food Chem. 2008, 108, 519-532. [17] Ou, K.Q.; Gu, L.W. Absorption and metabolism of proanthocyanidins. J. Funct. Foods 2014, 7, 43-53. [18]Tomás-Barberan, F.A.; Cienfuegos-Jovellanos, E.; Marin, A.; Muguerza, B.; Gil-Izquierdo, A.; Cerda, B.; Zafrilla, P.; Morillas, J.; Mulero, J.; Ibarra, A.; Pasamar, M.A.; Ramón, D.; Espín, J.C. A new process to develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy humans. J. Agr. Food Chem. 2007, 55 (10), 3926-3935. [19] Mackenzie, G.G.; Delfino, J.M.; Keen, C.L. Dimeric procyanidins are inhibitors of NF-kappa B-DNA binding. Biochem. Pharmacol.. 2009, 79(9), 1252-1262. [20] Yin, M.; Zhang, P.; Yu, F.; Zhang, Z.; Cai, Q.; Lu, W.; Li, B.; Qin, W.; Cheng, M.; Wang, H.; Gao, H. Grape seed procyanidin B2 ameliorates hepatic lipid metabolism disorders in db/db mice. Mol. Med. Rep. 2017, 16 (3), 2844-2850. [21] Terra, X.; Palozza, P.; Fernandez-Larrea, J.; Ardevol, A.; Blade, C.; Pujadas, G.; Salvado, J.; Arola, L.; Blay, M.T. Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes. Free Radical Res. 2011, 45 (5), 611-619. [22] Yanagida, A.; Shoji, T.; Shibusawa, Y. Separation of proanthocyanidins by degree of polymerization by means of size-exclusionchromatography and related techniques. J. Biochem. Bioph. Meth. 2003, 56 (1-3), 311-322. [23] Mezadri, T.; Villano, D.; Fernandez-Pachon, M.S.; García-Parrilla, M.C.; Troncoso, A.M. Antioxidant compounds and antioxidant activity in acerola (Malpighiaemarginata DC.) fruits and derivatives.J. Food Compos. Anal. 2008, 21 (4), 282-290. [24] Fernandez, K.; Vega, M.; Aspe, E. An enzymatic extraction of proanthocyanidins from Pais grape seeds and skins. Food Chem. 2015, 168, 7-13. [25] Chen, J.; Xu, Z.; Zhu, W.; Nie, R.; Li, C.M. Novel proanthocyanidin dimer analogues with the C-ring-opened diaryl-propan-2-gallate structural unit and enhanced antioxidant activities. J. Func. Foods 2016, 21, 290-300. [26] Zhang, S.; Li, L.; Cui, Y.; Luo, L.; Li, Y.; Zhou, P.; Sun, B. Preparative high-speed counter-current chromatography separation of grape seed proanthocyanidins according to degree of polymerization. Food Chem. 2017, 219, 399-407. [27] Foo, L.Y.; Porter, L.J. Synthesis and Conformation of Procyanidin Diastereoisomers. J. Chem. Soc. Perk. T. 1983, 41(7), 1535-1543. [28] Köhler, N.; Wray, V.; Winterhalter, P. New approach for the synthesis and isolation of dimeric procyanidins. J. Agr. Food Chem. 2008, 56(13), 5374–5385. [29] Gu, L.; House, S.E.; Rooney, L.W.; Prior, R.L. Sorghum extrusion increases bioavailability of catechins in weanling pigs. J. Agr. Food Chem. 2008, 56(4), 1283-1288. [30] Esatbeyoglu, T.; Juadjur, A.; Wray, V.; Winterhalter, P. Semisynthetic preparation and isolation of dimeric procyanidins B1-B8 from roasted hazelnut skins (Corylusavellana L.) on a large scale using countercurrent chromatography. J. Agr. Food Chem. 2014, 62, 7101-7110. [31] Price, M.L.; Vanscoyoc, S.; Butler, L.G. Critical evaluation of vanillin reaction as an assay for tannin in sorghum grain. J. Agr. Food Chem. 1978, 26 (5), 1214-1218. [32] Butler, L. G.; Price, M. L.; Brotherto, J. E. Vanillin assay for proanthocyanidins (Condensed Tannins): Modification of the solvent for estimation of the degree of polymerization. J. Agr. Food Chem. 1982, 30 22


Page 23 of 32

502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526


(6), 1087-1089. [33] Gu, L.; Kelm, M.; Hammerstone, J.F.; Beecher, G.; Cunningham, D.; Vannozzi, S.; Prior, R.L. Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normalphase HPLC–MS fluorescent detection method. J. Agr. Food Chem. 2002, 50(17), 4852–4860. [34] Ruan, X.; Yang, L.; Cui, W.X.; Zhang, M.X.; Li, Z.H.; Liu, B.; Wang, Q. Optimization of supercritical fluid extraction of total alkaloids, peimisine, peimine and peiminine from the bulb of fritillaria thunbergii miq, and evaluation of antioxidant activities of the extracts. Materials 2016, 9(7), 524. [35] Liu, H.; Zou, T.; Gao, J.; Gu, L. Depolymerization of cranberry procyanidins using (+)-catechin, (-)-epicatechin, and (-)-epigallocatechingallate as chain breakers. Food Chem. 2010, 141 (1), 488-494. [36] Karonen, M.; Leikas, A.; Loponen, J.; Sinkkonen, J.; Ossipov, V.; Pihlaja, K. Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles. Phytochem. Analysis 2007, 18, 378-386 [37] Bosso, A.; Guaita, M.; Petrozziello, M. Influence of solvents on the composition of condensed tannins in grape pomace seed extracts. Food Chem. 2016, 207, 162-169. [38] Chen, M.; Yu, S. Characterization of lipophilized monomeric and oligomeric grape seed flavan-3-ol derivatives. J. Agr. Food Chem. 2017, 65 (40), 8875-8883. [39] Fan, J.; Ding, X.; Gu, W. Radical-scavenging proanthocyanidins from sea buckthorn seed. Food Chem. 2007, 102 (1), 168-177. [40] Zhou, H.; Lin, Y.; Li, Y.; Li, M.; Wei, S.; Chai, W.; Tam, N. Antioxidant properties of polymeric proanthocyanidins from fruit stones and pericarps of Litchi chinensis Sonn. Food Res. Int. 2011, 44 (2), 613-620. [41] González-Centeno, M.R.; Jourdes, M.; Femenia, A.; Simal, S.; Rosselló, C.; Teissedre, P.L. Proanthocyanidin Composition and Antioxidant Potential of the Stem Winemaking Byproducts from 10 Different Grape Varieties (Vitisvinifera L.). J. Agr. Food Chem. 2012, 60 (48), 11850-11858.

527 528

Funding sources

529

The authors are grateful to the Natural Science Foundation of China (NSFC, Project No.

530

31670631), and Department of Science and Technology of Ningbo (DSTNB, Project No.

531

2017C110004, 2017C10017, 2017C10070) for the financial support of the work.

532

23



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Page 24 of 32

FIGURE CAPTIONS Fig.1 Reaction mechanism and pathways in PPC depolymerization Ⅰ: the acid-catalyzed cleavage of interflavan bonds; Ⅱ: formation of dimeric proanthocyanidin by bonding C4-C8 or C4-C6; Ⅲ: formation of trimeric proanthocyanidin; Ⅳ: formation of polymeric proanthocyanidin; Ⅴ: formation of the positive charged chalcane ion; Ⅵ: formation of gambiriins. Fig.2 UPLC (280 nm) chromatograms of various samples （A, B, C, D, E, F and G） A: standard materials with peak identification of 1: gallic acid (1.52 min), 2: proanthocyanidin B1 (2.46 min), 3: proanthocyanidin B3 (2.93 min), 4: catechin (3.11 min), 5: proanthocyanidin B4 (3.32 min), 6: proanthocyanidin B2 (3.49 min), 7: epicatechin (4.02 min), 8: epicatechin-3-O-gallate (ECG, 7.74 min); B: GSPE; C: ethyl acetate extract of GSPE; D: water raffinate (PPC) of GSPE; E: the product from PPC depolymerization in epicatechin/HCl system under conditions ： PPC/epicatechin ratio of 1:1, HCl concentration of 0.1 mol/L, 40°C and 20 min; F: the product from PPC depolymerization in catechin/HCl system under conditions：PPC/epicatechin ratio of 1:1, HCl concentration of 0.1 mol/L, 40°C and 20 min; G: the product from PPC depolymerization catalyzed by 0.1 mol/L HCl at 40°C for 20min. Fig.3. Response surface plots of B2 yield as a fuction of conditional variables . A: epicatechin/PPC ratio and HCl concentration at constant time and temperature; B: epicatechin/PPC ratio and time at constant HCl concentration and temperature; C: epicatechin/PPC ratio and temperature at constant HCl concentration and time; D: HCl concentration and time at constant epicatechin/PPC ratio and temperature; E: HCl concentration and temperature at constant epicatechin/PPC ratio and time; F: time and temperature at constant epicatechin/PPC ratio and HCl concentration. Constant values of parameters as follow: epicatechin/PPC ratio=2, HCl concentration= 0.06 mol, time= 20 min, temperature=40°C. Fig.4. Response surface plots of B1 yield as a function of conditional variables A: catechin/PPC ratio and HCl concentration at constant time and temperature; B: catechin/PPC ratio and time at constant HCl concentration and temperature; C: catechin/PPC ratio and temperature at constant HCl concentration and time; D: HCl concentration and time at constant catechin/PPC ratio and temperature; E: HCl concentration and temperature at constant catechin/PPC ratio and time; F: time and temperature at constant catechin/PPC ratio and HCl concentration. Constant values of parameters same as those in Fig.3.

567 568

24


Page 25 of 32


569 570 571 572

Table 1 Basic data of GSPE, extract and raffinate from ethyl acetate extraction of GSPE, and products of PPC depolymerization X/GS

Gallic 1mDP

PE

(mg/g)

2C

EC

B1

B2

B3

B4

ECG

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

(mg/g)

Broad

DPPH

Peak

IC50

(mg/g)

mg/mL

GSPE

1

3.56

2.0

35.0

25.0

4.8

10.0

1.8

2.3

1.4

881.0

51.2

extract of GSPE

0.19

2.21

9.0

178.1

143.0

25.4

45.3

11.0

13.4

6.7

330.0

53.3

0.81

5.65

0

0

0

0

0

0

0

0

991.0

50.6

—

1.74

0.5

9.0

351.5

0.9

98.2

1.9

11.5

18

251.5

53.6

—

1.68

0.6

341.5

11.5

109.0

3.5

12.6

0.8

3.5

254

51.7

—

4.46

0.8

24.2

10.5

4.1

10.1

1.3

1.5

1.07

878.4

52.3

raffinate of GSPE (PPC) 3PPC

depolymerization

with epicatechin and HCl 4PPC

depolymerization

with catechin and HCl 5PPCdepolymerization

with HCl only

573 574 575 576

acid

1

Mean degree of polymerization; 2 Standard material same as those in Fig.2; 3 10 mg mL-1 epicatechin reacted with 10 mg mL-1 PPC

in 0.1 mol HCl methanolic solution; 4 10 mg mL-1 catechin reacted with 10 mg mL-1 PPC in 0.1 mol HCl methanolic solution; 5 10 mg mL-1 PPC reacted with 0.1 mol HCl methanol solution.

25



Page 26 of 32

Tab.2 Central Composited Design (CCD) of Experiment and Analyses of Variance (ANOVA) for the Yield of Dimeric Proanthocyanidin B2 or B1 Variable Trial No.

X1

X2 mol/mL

Yield

X3

X4

min

。C

B2 mg/mL

ANOVA B1 mg/mL

B2 Sum of Squares

Source

Meam Square

F-value

P-value

B1 Sum of Squares

Meam Square

F-value

P-value

1

1.5

0.08

15

35

2.46

2.72

Model

2.365

0.169

53.36

< 0.001

2.443

0.175

41.50

< 0.001

2

2.0

0.06

20

40

3.03

3.32

X1

1.803

1.803

569.5

< 0.001

1.820

1.820

432.8

< 0.001

3

1.5

0.08

25

45

2.56

2.83

X2

0.013

0.013

4.03

0.043

0.025

0.025

6.01

0.027

4

2.5

0.08

25

35

3.00

3.19

X3

0.052

0.052

16.38

0.001

0.026

0.026

6.14

0.026

5

2.0

0.06

20

40

3.12

3.35

X4

0.003

0.003

0.89

0.360

0.006

0.006

1.51

0.238

6

2.0

0.06

20

40

2.94

3.19

X1 X2

0.001

6×10-4

0.20

0.660

9×10-6

9×10-6

0.001

0.964

7

2.5

0.04

15

35

3.22

3.54

X1 X3

0.077

0.077

24.26

< 0.001

0.114

0.114

27.14

< 0.001

8

1.0

0.06

20

40

2.16

2.34

X1 X4

0.024

0.024

7.49

0.015

0.045

0.045

10.58

0.005

9

1.5

0.04

25

45

2.51

2.77

X2 X3

0.000

4×10-4

0.13

0.718

0.003

0.003

0.69

0.420

10

1.5

0.08

25

35

2.51

2.79

X2 X4

0.002

0.002

0.68

0.423

0.006

0.006

1.47

0.244

11

2.5

0.08

15

35

3.26

3.58

X3 X4

0.002

0.002

0.50

0.490

0.005

0.005

1.29

0.273

12

2.5

0.08

15

45

3.15

3.42

X12

0.245

0.245

77.27

< 0.001

0.249

0.249

59.17

< 0.001

13

2.0

0.06

20

40

3.09

3.22

X22

0.168

0.168

53.15

< 0.001

0.185

0.185

44.02

< 0.001

14

2.5

0.08

25

45

2.90

3.15

X32

0.062

0.062

19.50

< 0.001

0.037

0.037

8.82

< 0.001

15

2.5

0.04

15

45

3.09

3.22

X42

0.047

0.047

14.71

0.002

0.040

0.040

9.55

0.007

16

1.5

0.04

15

35

2.51

2.67

Residual

0.048

0.003

0.063

0.004

17

2.0

0.06

20

30

2.85

3.07

18

1.5

0.08

15

45

2.51

2.77

Lack of Fit

0.025

0.003

0.044

0.004

1.12

0.48

19

2.0

0.02

20

40

2.65

2.86

20

2.5

0.04

25

45

2.84

3.09

Pure Error

0.022

0.005

0.019

0.004

21

1.5

0.04

15

45

2.46

2.70

22

3.0

0.06

20

40

3.16

3.41

Cor-Total

2.413

2.510

23

2.0

0.06

10

40

2.91

3.11

24

2.0

0.06

20

50

2.90

3.14

R2

0.981

0.974

25

2.0

0.10

20

40

2.80

3.00

26

2.0

0.06

20

40

2.98

3.31

Adj-R2

0.962

0.951

27

2.0

0.06

20

40

3.04

3.25

28

2.5

0.04

25

35

2.99

3.24

cPred-R2

0.927

0.889

29

1.5

0.04

25

35

2.44

2.73

30

2.0

0.06

30

40

2.78

3.12

Adeq Precision

28.31

25.87

0.56

0.79

26


Page 27 of 32


X1 EC (or C) to PCC mass ratio; X2 HCl concentration; X3 reaction time;

X4 reaction temperature

27



Page 28 of 32

Fig.1 OH OH

HO

OH

OH

OH

OH HO

HO OH

O

8 6

OH

OH

4

O

HO

OH OHHO

O

OH

OH

OH

8

OH

OH

HO

+

O

6

OH

OH

OH

OH

OH

4

OH HO

O

8

OH

6

+

[H] OH

OH

OH

OH

4

carbocation

OH

OH

OH

OH

OH

OH

HO

O

HO

OH

OH HO

O

HO

OH

+

OH

O

HO

4 HO

OH OH

OH

O

8

4

OH HO

8 6

OH

OH

O

HO

6

O

HO OH

OH

OH

O 4

OH HO

OH HO

HO

OH

OH

nucleophile(EC/C)

4

OH

4

HO OH

O

8

O 4

OH HO

8

R1

4 OH

OH

R2 O

OH HO

+

OH

OH

R3

6

OH R1 R2 OH

O

R4

R4 R3

HO OH

28


OH

O

OH

OH

carbocation

OH

[H]+

OH

+

OH HO

OH

carbocation OH

OH

OH OH

OH

OH

6

OH

O

HO

OH

4 OH HO

flavonoid-3-ol OH

8 6

OH

HO

O

OH OH OH

Page 29 of 32


Fig.2

29



Page 30 of 32

Fig.3

30


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Fig.4

31



Page 32 of 32

Graphic for table of contents

32


Optimizing Nucleophilic Depolymerization of Proanthocyanidins in

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