Enhancement of Galloylation Efficacy of Stigmasterol and Î²-Sitosterol

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Bioactive Constituents, Metabolites, and Functions

Enhancement of Galloylation Efficacy of Stigmasterol and #Sitosterol Followed by Evaluation of Cholesterol Reducing Activity Shanshan Wang, Kai Ye, Tong Shu, Xiuwen Tang, Xiu Jun Wang, and Songbai Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06983 • Publication Date (Web): 02 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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Journal of Agricultural and Food Chemistry

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Enhancement of Galloylation Efficacy of Stigmasterol and β-Sitosterol Followed

2

by Evaluation of Cholesterol Reducing Activity

3 4

Shanshan Wang†, Kai Ye†, Tong Shu§, Xiuwen Tang‡, Xiu Jun Wang‡, Songbai

5

Liu*†,§

6 7

†Department

8

Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food

9

Technology and Equipment, Zhejiang University, 866 Yuhangtang Road, Hangzhou

of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang

10

310058, China

11

‡Department

12

University, 866 Yuhangtang Road, Hangzhou 310058, China

13

§Qinghai

of Biochemistry & Pharmacology, School of Medicine, Zhejiang

Food Inspection and Testing Institute, 12 Beidajie, Xining 810000, China

14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


23 24

ABSTRACT

25

In this study, incorporation of gallic acid into typical phytosterols (β-sitosterol and

26

stigmasterol) through Steglich esterification was optimized employing protection and

27

deprotection strategy. A novel mechanism leading to side esterification was discovered.

28

Complication of the phenolic hydroxyl groups and side reaction were successfully

29

reduced under the optimized condition. The structural identity and purity of galloyl

30

stigmasterol and galloyl β-sitosterol were confirmed by NMR, FT-IR, and HPLC-MS.

31

Evaluation of galloyl β-sitosterol and galloyl stigmasterol revealed their excellent

32

antioxidant and cholesterol reducing activities. Significant enhancement of cholesterol

33

reducing activity by galloylation was unveiled especially for β-sitosterol. Galloyl β-

34

sitosterol had slightly better antioxidant activity at ambient temperature and better

35

cholesterol reducing activity. Molecular modeling suggested that subtle difference of

36

galloyl β-sitosterol and galloyl stigmasterol in activities could be attributed to variation

37

of molecular rigidity and conformation. The excellent properties of galloyl β-sitosterol

38

and galloyl stigmasterol suggested their great potential application in food industry.

39 40

Keywords: galloylation; phytosterol; gallic acid; antioxidant activity; cholesterol

41

reducing activity; Steglich esterification

42 43 44 2


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INTRODUCTION

47

Phytosterols are important structural and functional components in plant cells,

48

stabilizing the cellular membranes.1 To date, more than 250 phytosterols and relating

49

compounds have been identified in nuts, legumes, and seeds.2,3 As naturally abundant

50

phytosterols, β-sitosterol and stigmasterol have been intensively studied.2 Various

51

beneficial effects of phytosterols have been unveiled including anti-inflammatory,4

52

anti-cancer5 and cardiovascular-protective capabilities6. Especially, the cholesterol

53

reducing activity of phytosterols has received great attention in food industry.1,7-9 As

54

a result, the European Commission approved application of phytosterols as novel food

55

ingredients.10 In 2007, the Ministry of Health of China also granted phytosterols as

56

new resource foods.10 However, application of phytosterols is largely limited by their

57

poor solubility in oil (3-4 % in oil) resulting from self-aggregation.11 Usually,

58

phytosterols are derivatized through esterification to enhance oil or water

59

solubility.12,13

60

Gallic acid (GA) is widely spread in plants14 including Anacardiaceae, Fabaceae,

61

Myrtaceae and the fungi of the genus Termitomyces15,16. GA exhibits various

62

important biological activities like antioxidation, anti-cancer, anti-HIV, and anti-

63

tyrosinase.17-19 Moreover, orally taken GA is non-toxic up to the level of 5000 mg per

64

kg body weight.20 As a result, GA and its derivatives have been generally applied in

65

food and medicinal industry as analgesics, astringents and antimicrobial agents.21

66

Hence, incorporation of gallic acid into phytosterols through esterification would 3



67

be useful to ameliorate their solubility and also deliver the excellent antioxidant

68

activities to phytosterols. Furthermore, the portion of gallic acid would modulate the

69

molecular conformation of phytosterols and possibly enhance the important

70

cholesterol reducing activity. In our previous study, a novel galloyl phytosterol

71

antioxidant was first developed via this strategy.22 However, the cholesterol reducing

72

activity had not been explored and the preparation performed by Steglich reaction

73

suffered complication of the phenolic hydroxyl groups. Moreover, the specific roles

74

of individual phytosterols had not been examined since a mixture of phytosterols was

75

applied in that study. Therefore, new strategy should be developed to optimize

76

preparation of galloyl phytosterols. In addition, galloylation effect on the typical

77

individual phytosterols regarding antioxidant activity and especially cholesterol

78

reducing activity should be explored to facilitate their application.

79

In this study, incorporation of gallic acid into typical individual phytosterols (β-

80

sitosterol and stigmasterol) through esterification was optimized employing protection

81

and deprotection strategy. The complication of the phenolic hydroxyl groups and side

82

reaction was successfully reduced under the optimized condition. Specific antioxidant

83

activity and cholesterol reducing activity of galloyl β-sitosterol and galloyl

84

stigmasterol were further evaluated. Significant enhancement of cholesterol reducing

85

activity of β-sitosterol and stigmasterol after galloylation was unveiled. Herein the

86

details of the study were described as follows.

87 88

MATERIALS AND METHODS 4


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Chemicals. Stigmasterol and β-sitosterol were purchased from Shanghai Yuanye

90

Biotechnology (Shanghai, China). Sodium bicarbonate, sodium phosphate dibasic

91

dodecahydrate, sodium chloride, sodium phosphate monobasic dihydrate, anhydrous

92

magnesium sulphate, ethyl acetate, ethyl alcohol, n-hexane, petroleum ether, toluene,

93

hydrochloric acid and methanol were obtained from Sinopharm Chemical Reagent

94

(Shanghai, China). N,N-dicyclohexylarbodiimide (DCC), gallic acid, butylated

95

hydroxy

96

butylhydroquinone (TBHQ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), triethylamine,

97

N,N-Dimethylformamide (DMF), 4-dimethyaminopyridine (DMAP), hydrazinium

98

hydrate solution (80%), glycerol trioleate, sodium taurocholate, cholesterol (Ch), oleic

99

acid, isobutyric anhydride were obtained from Aladdin Reagent (Shanghai, China). All

100

anisole

(BHA),

2,6-di-tert-butyl-4-methylphenol

(BHT),

tert-

chemicals were of analytical grade.

101

Protection of Gallic Acid by Isobutyric Anhydride. To a solution of 1.020 g (6

102

mmol) gallic acid and 73.3 mg (0.6 mmol) DMAP in 3 mL DMF was added 4.477 mL

103

isobutyric anhydride (27 mmol) and 3.763 mL triethylamine (27 mmol). The resulted

104

mixture was stirred at ambient temperature for 2 h. Then, the reaction solution was

105

transferred to a larger container, 1 N hydrochloric acid was added to acidify the solution,

106

and the white precipitate was washed by water for three times. After dried on filter

107

paper, the protected product, tri-isobutyroyl gallic acid was afforded (yield: 85%) as an

108

analytically pure white solid.

109

Optimization of Galloylation of Phytosterols. Stigmasterol was employed to

110

optimize galloylation condition of phytosterols. Typically, to a solution of 5



111

stigmasterol (0.018 mmol), tri-isobutyroyl gallic acid (0.018 or 0.027 mmol), and

112

DMAP (0.0018 or 0.0009 mmol) in 0.4 mL solvent (n-hexane, toluene or CH2Cl2) was

113

added a solution of DCC (0.022 or 0.027 mmol) in 0.2 mL solvent (n-hexane, toluene

114

or CH2Cl2), then stirred and monitored by thin layer chromatography (TLC) at ambient

115

temperature.

116

Preparation of Galloyl Stigmasterol. To a solution of 285.2 mg (0.75 mmol) tri-

117

isobutyroyl gallic acid, 206.3 mg (0.5 mmol) stigmasterol and 3.05 mg of DMAP (0.025

118

mmol) in 12 mL toluene was added a solution of DCC (154.7 mg, 0.75 mmol) in 1 mL

119

toluene and then stirred at ambient temperature. After three hours, the reaction mixture

120

was added 13 mL 95 % ethanol, followed by addition of 3 mmol hydrazine hydrate (80

121

%), and stirred further for 1 h. The solvent was removed to separatory funnel. Then the

122

solution was acidified by 1 N hydrochloric acid, extracted with ethyl acetate and washed

123

by water for three times. Purification over a silica gel chromatography eluted with

124

petroleum ether/ ethyl acetate (2:1.5, V/V) gave the product (93%) as a white solid.

125

Preparation of Galloyl β-Sitosterol. The galloyl β-sitosterol was prepared in a

126

similar way to galloyl stigmasterol. Tri-isobutyroyl gallic acid, β-sitosterol and DMAP

127

was added in toluene at a molar ratio of 1.5:1:0.5 (tri-isobutyroyl gallic acid / β-

128

sitosterol / DMAP), and the molar ratio of DCC to β-sitosterol used in the reaction was

129

1.5:1. The solution was constantly stirred at ambient temperature. Then the same

130

volume of ethanol as toluene was added to the reaction solution before addition of

131

hydrazine hydrate (80 %). Purification over a silica gel chromatography eluted with

132

petroleum ether / ethyl acetate / 95 % ethyl alcohol (2.5:1:0.1, V/V/V) gave the product 6


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(92%) as a light yellow solid.

134

NMR, FT-IR and MS Analysis. The 1H and

13C

NMR of the prepared galloyl

135

stigmasterol and galloyl β-sitosterol were performed on a 400 MHz NMR spectrometer

136

(Bruker Corporation, Fällanden, Zürich, Switzerland) at room temperature, respectively,

137

employing acetone-d6 (1H = 2.05 ppm, 13C = 29.84 ppm) and DMSO-d6 (1H = 2.50 ppm,

138

13C

= 39.52 ppm) as solvents.23

139

FT-IR analysis was performed on AVA TAR370 spectrophotometer (Thermo

140

Nicolet Corporation, Madison, WI, USA) applying attenuated total reflectance method

141

with the spectral scanning scope of 400-4000 cm-1.

142

Mass spectra were obtained on a Thermo Finnigan LCQ Deca XP Max system

143

(Thermo Fisher Scientific, Waltham, MA, USA) employing positive and negative ion

144

electron spray ionization (ESI) mode with scan range of m/z 50-1500.

145

HPLC-MS Analysis. The HPLC-MS was applied to analyze GSt and GSi on

146

Agilent 1200 system (Agilent, Santa Clara, CA, USA). An Agilent SB-C18 (150*2.1

147

mm, 4.5 μm) column was employed. The column temperature was maintained at 35 ˚C

148

and the sample injection volume was 10 μL. The mobile phase comprised acetonitrile

149

containing 0.1 % formic acid aqueous solution (A) and acetonitrile (B). The gradient

150

elution profile started with 90 % B, after 10 min B was gradually increased to 100 %

151

within 5 min, then with a final hold of 35 min. The mobile phase was delivered at a

152

flow rate of 0.2 mL/min and signals were monitored at 277 nm with DAD detection.

153

Mass spectra were obtained on a Thermo Finnigan LCQ Deca XP Max system (Thermo

154

Fisher Scientific, Waltham, MA, USA) employing positive and negative ion electron 7



155

spray ionization (ESI) mode with scan range of m/z 50-1500.

156

DPPH Scavenging Activity Assay. The DPPH scavenging activity was estimated

157

according to the method described by Sánchez-Moreno et al. with slight modification.24

158

Briefly, 0.1 mL of methanol or ethanol absolute solution of different antioxidation

159

concentrations was added to 3.9 mL of 0.025 g/L DPPH methanol solution. The mixture

160

was placed in the dark for 30 min at ambient temperature. Then the absorbance was

161

measured at 515 nm on a UV-vis spectrophotometer (Model SP-756, Shanghai

162

Spectrum Corporation, Shanghai, China). The percentage of scavenged DPPH

163

(%DPPHSCA) was calculated according to the following equation. Appropriate solvent

164

and sample blank were run in each essay.

165 166

[

(%𝐷𝑃𝑃𝐻𝑆𝐶𝐴) = 1 ―

𝐴𝑠𝑎𝑚𝑝𝑙𝑒 - 𝐴𝑏𝑙𝑎𝑛𝑘 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙

]

∗ 100%

167

Where Asample is the absorbance of the mixture of methanol or ethanol absolute

168

solution of different antioxidation concentrations (0.1 mL) and 0.025 g/L DPPH

169

methanol solution (3.9 mL), Ablank is the absorbance of the mixture of methanol or

170

ethanol absolute solution of different antioxidation concentrations (0.1 mL) and

171

methanol or ethanol absolute solution (3.9 mL), Acontrol is the absorbance of the mixture

172

of methanol or ethanol absolute solution (0.1 mL) and 0.025 g/L DPPH methanol

173

solution (3.9 mL).25

174

Oxidation Stability Evaluated by Rancimat Method. Oxidation stability of

175

antioxidants was determined by the Rancimat method (Model 743, Metrohm AG, 8


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Herisau, Switzerland) in glycerol trioleate according to ISO 6886:2006.26 The

177

accelerated oxidation test was carried out by heating a sample to 120 °C in a sealed test

178

tube while passing an air flow (20 L/h) through it, and the volatile oxidation products

179

such as acetic acid and formic acid were dragged by the air flow into distilled water.

180

The conductivity of the distilled water was monitored and a significant change of

181

conductivity was detected at induction time due to accumulation of the oxidation

182

products in the water.27

183

Cholesterol Reducing Activity in Micelle. The cholesterol reducing activity of

184

samples in vitro was measured by the method of Zhang et al. with slight modification.28

185

Specifically, cholesterol micellar solution (1 mL) containing 10 mmol/L sodium

186

taurocholate, 0.5 mmol/L cholesterol, 1 mmol/L oleic acid, 132 mmol/L NaCl and 15

187

mmol/L sodium phosphate buffer (pH 7.4), was prepared by sonication, which was

188

placed at ambient temperature for 2 h to reach equilibrium. Then, 2.5 mmol/L of sample

189

was added to 1 mL of micellar solution, and the micellar solution without sample was

190

used as a blank. Then the mixtures were incubated in a 37 ˚C shaker bath, then

191

centrifuged at 15550 rpm for 20 min at 37 ˚C after 3 h. The UHPLC was used to

192

measure the amount of cholesterol in the supernatant on Agilent 1290 Infinity system

193

(Agilent, Santa Clara, CA, USA). An Agilent ZORBAX Eclipse XDB-C18 (150*2.1

194

mm, 3.5 μm) column was applied and signals were monitored at 205 nm with DAD

195

detection. The column temperature was maintained at 35 ˚C and the sample injection

196

volume was 5 μL. The mobile phase for UHPLC determinations was methanol, and the

197

time of isocratic elution was 10 min with a flow rate of 0.4 mL/min. The percentage of 9



198

cholesterol (%CHOs) dissolved in the micelle was calculated as the peak area of

199

cholesterol in the supernatant of the blank divided by the peak area in the supernatant

200

of the samples. The inhibition rate of cholesterol micelle (%CHOi) was calculated

201

according to the following equation.

202 203

(%𝐶𝐻𝑂𝑖) = (1 ― %𝐶𝐻𝑂𝑠) ∗ 100% Statistical Analysis. All the experiments were performed in triplicate, and the data

204

was expressed as mean ± standard deviation (SD). The least significant difference (LSD)

205

in one-way analysis of variance (ANOVA) was employed to analysis the differences

206

among the results applying the STATVIEW software. Differences with a p-value less

207

than 0.05 were considered significant.

208

Molecular Modeling. Molecular modeling was performed in Chimera (Version

209

1.13.1).29 The three-dimensional structures of the molecules were built with the default

210

protocols. The structures were energetically minimized applying conjugate gradient

211

method with consideration of H-bonds. Charges to standard and other residues were

212

assigned by AMBER ff14SB and AM1-BCC respectively. Superposition of the

213

structures was performed by employment of the match command.

214 215

RESULTS AND DISCUSSION

216

New Strategy for Preparation of Galloyl Stigmasterol and Galloyl β-Sitosterol.

217

Incorporation of gallic acid into phytosterols was initially attempted by Liu’s group22

218

through a mild Steglich esterification which was first described by Steglich30 in 1978.

219

However, the yield of the product was unsatisfactory resulting from the complication 10


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of phenolic hydroxyls. The phenolic hydroxyls of gallic acid were acidic and readily

221

participated in the esterification coupling reaction which resulted in the problematic

222

self aggregation of gallic acid. Although many approaches for esterification

223

phytosterols were developed in our and other groups,31,32 an easily scalable preparation

224

method was preferred in this study. To address this issue, the strategy of protection of

225

the phenolic hydroxyls was adopted in this study. The new synthetic route was

226

demonstrated in Figure 1. Initially, various protecting agents including propionic

227

anhydride, acetic anhydride, benzyl chloroformate and isobutyric anhydride were

228

attempted to shield the phenolic hydroxyls. It was revealed that protection with

229

isobutyric anhydride provided best performance owing to excellent reactivity, superior

230

stability and simple purification.

231

Then the protected gallic acid was employed in the Steglich esterification with

232

phytosterols. Unfortunately, application of the typical reaction condition for Steglich

233

esterification afforded significant amount of a by-product (Figure 1). As exemplified

234

by stigmasterol, the identity of the by-product was further determined as isobutyroyl

235

phytosterol by ESI-MS spectrum ([M+Na]+: 505.47). To minimize production of the

236

by-product, numerous experiments were then carried out to find out the origin of the

237

by-product. Controlled experiments exhibited that there was no by-product produced

238

without addition of either the coupling agent (DCC) or the catalyst (DMAP) in the

239

reaction mixture, which suggested that the by-product resulted not from direct acyl

240

transfer from the protected gallic acid but from the generated intermediate during

241

Steglich reaction. Therefore, the overall mechanism during this reaction was proposed 11



242

as in Figure 2A. Briefly, the protected gallic acid at first attacked DCC and formed the

243

O-acylisourea intermediate which was exposed to the action of DMAP. Owing to the

244

activated electrophilicity of the generated O-acylisourea intermediate, DMAP could

245

react with the O-acylisourea intermediate at either the galloyl or isobutyroyl group.

246

When DMAP reacted at the isobutyroyl group, the isobutyroyl phytosterol by-product

247

was produced.

248

Once the origin of the by-product was unraveled, it was relatively simple to optimize

249

the reaction condition. To suppress the undesired attack at the isobutyroyl group, less

250

polar solvents including n-hexane and toluene were employed because the isobutyroyl

251

group was less electrophilic in nonpolar solvents (Figure 2B). It was disclosed that

252

application of n-hexane did completely eliminate production of the by-product but the

253

reaction rate was too slow. To our delight, reaction in toluene successfully suppressed

254

the by-product and proceeded in a reasonable reaction rate. As a result, toluene was

255

applied as the ideal solvent and the ratio of reaction agents was further optimized. The

256

results of the optimizing process including solvents and ratio of reagents monitored by

257

TLC were demonstrated in Figure 2B. Higher ratio of the protected gallic acid and

258

DCC afforded better conversion of phytosterol. Less loading of the DMAP catalyst

259

lowered the side reaction product. Eventually, the optimal condition for the

260

esterification reaction was set as 1.5 mole equivalent of the protected gallic acid, 1.5

261

mole equivalent of DCC and 0.05 mole equivalent of DMAP to 1.0 mol equivalent of

262

phytosterol. Deprotection of the protected galloyl phytosterol went smoothly by the

263

action of aqueous hydrazine. Under the optimized condition, the naturally abundant 12


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phytosterols, β-sitosterol and stigmasterol were galloylated respectively in good yield

265

(>85%) for further investigation. Excellent lipase preparation works have been done

266

previously.33,34 The advantage of the enzymatic methods was that they would be

267

potentially greener and more acceptable in the market. On the other hand, the synthetic

268

approaches were more cost efficient.

269

Structural Analysis of Galloyl Stigmasterol and Galloyl β-Sitosterol. The

270

structural identity of galloyl stigmasterol (GSt) and galloyl β-sitosterol (GSi) was

271

confirmed by NMR, FT-IR, and further analyzed by HPLC-MS.

272

1H-NMR

of GSt (400 MHz, acetone-d6) δ 8.13 (br, 3 H; phenolic proton), 7.12 (s, 2

273

H; aromatic proton of the galloyl group), 5.42 (d, J=4.2 Hz, 1 H; olefin proton of the

274

cyclic skeleton of stigmasterol), 5.22 (dd, J=15.2, 8.7 Hz, 1 H; olefin proton of the side

275

chain of stigmasterol), 5.08 (dd, J=15.2, 8.7 Hz, 1 H; olefin proton of the side chain of

276

stigmasterol), 4.70 (m, 1 H; oxygen adjacent proton of stigmasterol), 2.52-0.66 (m, 43

277

H; aliphatic protons of stigmasterol).

278

13C-NMR

of GSt (100 MHz, acetone-d6) δ 166.09, 146.03, 140.86, 139.38, 138.63,

279

130.14, 123.16, 122.40, 109.84, 74.60, 57.71, 56.83, 52.18, 51.12, 43.00, 41.39, 40.51,

280

39.04, 37.93, 37.45, 32.75, 32.72, 32.63, 29.75, 28.65, 26.11, 24.99, 21.77, 21.71, 21.45,

281

19.71, 19.38, 12.61, 12.45. The 13C chemical shifts of 166.09 ppm corresponded to the

282

signal of the galloyl carbon, 146.03 to 109.84 ppm corresponding to the signals of

283

aromatic and olefinic carbons, 74.60 to 12.45 ppm corresponding to the signals of

284

aliphatic carbons.

285

1H-NMR

of GSi (400 MHz, DMSO-d6) δ 9.20 (s, 2 H; phenolic protons), 8.90 (s, 1 13



286

H; phenolic proton), 6.94 (s, 2 H; aromatic proton of the galloyl group), 5.37 (br, 1 H;

287

olefin proton of the cyclic skeleton of β-sitosterol), 4.61 (m, 1 H; oxygen adjacent

288

proton of β-sitosterol), 2.42-0.61 (m, 47 H; aliphatic protons of β-sitosterol).

289

13C-NMR

of GSi (100 MHz, DMSO-d6) δ 165.18, 145.52, 139.54, 138.35, 122.17,

290

119.70, 108.50, 73.28, 56.16, 55.47, 49.51, 45.16, 41.88, 37.85, 36.61, 36.14, 35.55,

291

31.40, 28.71, 27.84, 27.53, 25.47, 23.89, 22.62, 20.59, 19.72, 19.02, 18.94, 18.63, 11.79,

292

11.67. The 13C chemical shifts of 165.18 ppm corresponded to the signal of the galloyl

293

carbon, 145.52 to 108.50 ppm corresponding to the signals of aromatic and olefinic

294

carbons, 73.28 to 11.67 ppm corresponding to the signals of aliphatic carbons.

295

IR of GSt (cm-1) 3327 (s, νO-H), 2954 (s, νC-H), 2867 (s, νC-H), 1709 (m, νC=O), 1676

296

(s, νC=C), 1625 (s, νC=C, benzene skeleton vibration), 1534 (m, νC=C, benzene skeleton

297

vibration), 1514 (m, νC=C, benzene skeleton vibration), 1455 (s, δO-H), 1375 (s, δO-H),

298

1247 (s, νC-O), 1099 (m, δC-H), 1030 (s, δC-H), 926 (m, δC-H), 769 (m, δC-H), 752 (m, δC-

299

H).

300

the hydroxyl groups of gallic acid. The absorption bands at 1709 and 1247 cm−1 were

301

attributed to C=O and C–O–C stretching vibration, which confirmed the formation of

302

ester bonds between gallic acid and stigmasterol.

The absorption bands at 3327 cm-1 corresponded to -OH stretching vibration from

303

IR of GSi (cm-1) 3327 (s, νO-H), 2931 (s, νC-H), 2851 (s, νC-H), 1709 (w, νC=O), 1661

304

(m, νC=C), 1627 (s, νC=C, benzene skeleton vibration), 1576 (m, νC=C, benzene skeleton

305

vibration), 1536 (m, νC=C, benzene skeleton vibration), 1510 (w, νC=C, benzene skeleton

306

vibration), 1464 (m, δO-H), 1375 (s, δO-H), 1312 (s, δC-H), 1245 (s, νC-O), 1088(w, δC-H),

307

1030 (m, δC-H), 769 (w, δC-H), 752 (w, δC-H) , 641 (w, δC-H). The absorption bands at 14


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3327 cm-1 corresponded to -OH stretching vibration from the hydroxyl groups of gallic

309

acid. The absorption bands at 1709 and 1245 cm−1 were attributed to C=O and C–O–C

310

stretching vibration, which confirmed the formation of ester bonds between gallic acid

311

and β-sitosterol.

312

Then the identity and purity of GSt and GSi were further investigated by HPLC-MS.

313

As shown in Figure 3, the individual components were fully separated and the

314

corresponding ESI-MS spectrum (negative ion mode) of each component demonstrated

315

their structural identities (GSt: [M-H]- 563.50 , [M+Cl]- 598.96; GSi: [M-H]- 565.57,

316

[M+Cl]- 601.12). The results of HPLC-MS analysis revealed that the purities of GSt

317

and GSi were 93% and 86% respectively. The good purity of the prepared products

318

assured further studies of their properties.

319

Antioxidant Activity of Galloyl Stigmasterol and Galloyl β-Sitosterol.

320

Antioxidant activities of the prepared galloyl stigmasterol (GSt) and galloyl β-sitosterol

321

(GSi) were evaluated employing the representative approaches including DPPH assay

322

and Rancimat method. DPPH assay is based on measurement of free radical scavenging

323

activity of antioxidants, which assesses the scavenging capacity of hydrogen donating

324

antioxidants toward the DPPH free radical.35 The odd electron of nitrogen atom in

325

DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding

326

hydrazine.36

327

DPPH radical scavenging efficiency of GSt, GSi and the corresponding starting

328

materials gallic acid (GA), stigmasterol (St) and sitosterol (Si) were determined under

329

different concentrations. The typical lipophilic antioxidants including BHA, BHT and 15



330

TBHQ were employed in the study for comparison. As shown in Figure 4, GSt and

331

GSi inherited extraordinary DPPH radical scavenging activity from GA. The starting

332

materials, St and Si, exhibited no antioxidant activity. Under the percentages of

333

scavenged DPPH radicals between 20 % to 80 % with variation of the concentrations

334

of antioxidants, the corresponding EC50 (the concentration of the antioxidant needed to

335

decrease the initial radical concentration by 50 %) of each antioxidant was derived to

336

evaluate the antioxidant activity. The obtained EC50 values were shown as follows in

337

ascending order: GSi (0.366 mM), GSt (0.41 mM), TBHQ (0.622 mM), BHA (0.655

338

mM), BHT (1.37 mM).

339

As a result, the EC50 values indicated that the antioxidant activities of GSt and GSi

340

were significantly superior to the commonly used BHT, BHA and even TBHQ.

341

Regarding specific galloyl phytosterol, the antioxidant activity of GSi was slightly

342

better than that of GSt. Presumably, the double bond of the side chain portion of GSt

343

the molecular structure of GSt was more rigid than GSi and accordingly the GSt

344

molecules would more easily stack together and was more sluggish in radical

345

scavenging.

346

Then Rancimat method was applied to evaluate the antioxidant protective

347

performance of the antioxidants in edible oils which is very important for food safety.

348

The main composition of edible oils included glycerol tripalmitate (GTP), glycerol

349

tristearate (GTS) and glycerol trioleate (GTO).37 GTO was susceptible to oxidization

350

owing to the polyunsaturated bonds and was a good model to test oxidation. To exclude

351

interference of the added antioxidant in commercially available edible oils, 16


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352

analytically pure synthetic GTO was employed as a model of edible oils in Rancimat

353

assay.

354

The induction time of the antioxidants in GTO was investigated at a typical

355

concentration of 0.6 mM. As shown in Figure 4, induction times of GSt and GSi were

356

significantly higher than those of BHT, BHA and TBHQ. Notably, the performance of

357

GSt and GSi in Rancimat assay was much better than GA which demonstrated excellent

358

activity in DPPH assay. These results revealed significant improved physicochemical

359

property and antioxidant performance of the modified GSt and GSi in lipophilic

360

circumstance. Regarding specific galloyl phytosterol, there was no significant

361

difference between the antioxidant activities of GSi and GSt. Possibly, the molecules

362

of either GSi or GSt freely diffused at the high temperature (120 °C) and eliminated the

363

difference from molecular stacking property.

364

Investigation by the two typical approaches disclosed that GSt and GSi were

365

excellent antioxidants in lipophilic circumstance. GSt molecules would more easily

366

stack together and exhibited slightly lower antioxidant activity than GSi at the relatively

367

lower ambient temperature. Generally, GSt and GSi were superb fat-soluble

368

antioxidants and had great potential application.

369

Cholesterol Reducing Activity of Galloyl Stigmasterol and Galloyl β-Sitosterol.

370

As shown above, GSt and GSi preserved the exceptional antioxidant activity from the

371

portion of GA. Therefore, it was essential to investigate whether GSt and GSi inherited

372

the important cholesterol reducing activity from the other portion of the phytosterol.

373

The cholesterol reducing effect of phytosterols was recognized in the early 1950s.38,39 17



374

An average daily dose of 2 g phytosterols lowered plasma LDL-C by approximately

375

0.31-0.34 mmol/L or 8-10 % within 3-4 weeks.40,41 Usually, inhibition rate of

376

cholesterol in the bile salt micelles was employed as a model to evaluate the cholesterol

377

reducing effect of phytosterols because cholesterol was absorbed through incorporation

378

into the bile salt micelles.42

379

The micelle of cholesterol was prepared by sonication and then the test compound

380

was introduced. After incubation for 3 h, cholesterol in the micelle would be substituted

381

by the test compound. The inhibition rates of cholesterol in micelle by GSt, GSi, St, Si

382

and GA were shown in Figure 5. As expected, GA barely exhibited cholesterol

383

reducing activity. Si and especially St demonstrated significant cholesterol reducing

384

activities. To our delight, GSt and GSi indicated extraordinary cholesterol reducing

385

activities, suggesting successful integration of the activity from the phytosterol.

386

Surprisingly, galloylation generally improved cholesterol reducing activity of the

387

phytosterol presumably owing to enhanced aggregation capacity in micelle by the

388

increased molecular rigidity through introduction of the stiff aromatic galloyl group. In

389

particular, galloylation of Si greatly improved its cholesterol reducing activity. The

390

degree of improvement of cholesterol reducing activity through galloylation for St was

391

much smaller than Si. Probably, the molecular structure of St was already rigid enough

392

and further increment of rigidity only led to limited enhancement of the cholesterol

393

reducing activity. The outstanding cholesterol reducing activities of GSt and GSi were

394

intriguing for their further application in functional foods.

395

Rationalization of Antioxidant Activity and Cholesterol Reducing Capability 18


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Page 19 of 34


396

by Molecular Modeling. Exceptional antioxidant activity and cholesterol reducing

397

capability of GSt and GSi have been observed in this study. To understand the origin

398

of their remarkable properties, molecular modeling was conducted for GSt, GSi, St, Si

399

and cholesterol (Ch) applying the widely used Chimera program.29 The optimal

400

molecular conformation was afforded by minimization of the built molecular structure

401

with the reliable Amber force field.43 To readily compare the optimal conformations of

402

different molecules, the molecular structures were superposed together. As shown in

403

Figure 6, the molecular conformation of Si was notably more similar to Ch than St.

404

Therefore, the aggregation ability of Si would be similar to Ch and resulted in less

405

efficiency to replace the cholesterol in micelle. Then molecular solvent-excluded

406

surface (SES) was calculated to display the overall space occupation of the molecules.44

407

As depicted by SES, there was no significant difference between the areas of St, Si and

408

Ch. Nevertheless, the rigid side chain of St could be clearly observed which provided

409

its better aggregation ability in micelle.

410

Superposition of the minimized molecular structures of GSt and GSi revealed that

411

the optimal conformation varied dramatically after galloylation. The molecule of GSi

412

took a more extended conformation, suggesting increased molecular rigidity and

413

aggregation ability in micelle which was consistent with its notable enhanced

414

cholesterol reducing activity. Investigation of SES further demonstrated that the overall

415

shape of GSt took a curled conformation that would compromise increase of

416

aggregation ability in micelle, which was consistent with the limited enhancement of

417

cholesterol reducing activity after galloylation. As a result, although GSt was more rigid 19



418

and readily aggregated, its optimal conformation did not match well with that of

419

cholesterol. In addition, the stronger aggregation ability resulted in sluggish antioxidant

420

activity compared with GSi. Therefore, the optimal conformation of GSi matched better

421

with that of cholesterol leading to its good performance in reduction of cholesterol in

422

micelle. Furthermore, the less stacking effect owing to weaker aggregation ability

423

contributed to its stronger antioxidant activity at ambient temperature. Molecular

424

modeling provided a very good visualization to rationalize the molecular properties.

425

In conclusion, optimization of the preparation of galloyl phytosterols was achieved

426

through protection and deprotection strategy in this study. A novel mechanism leading

427

to side esterification was discovered and successfully suppressed under the optimized

428

condition. Both galloyl β-sitosterol and galloyl stigmasterol had excellent antioxidant

429

activity in lipophilic settings. Galloylation greatly improved cholesterol reducing

430

activity of β-sitosterol. Molecular modeling suggested that subtle difference of galloyl

431

β-sitosterol and galloyl stigmasterol in antioxidant and cholesterol reducing activities

432

could be attributed to variation of molecular rigidity and conformation. The excellent

433

properties of both galloyl β-sitosterol and galloyl stigmasterol suggested their great

434

potential application in food industry.

435 436

AUTHOR INFORMATION

437

Corresponding Author

438

*Songbai Liu. Phone: +86-15168319078. E-mail: [email protected].

439

Funding 20


Page 20 of 34

Page 21 of 34


440

This work was supported by the National Key Research and Development Program

441

(2016YFD0400805, 2017YFF0207800), Zhejiang Public Welfare Technology

442

Research Program (LGN18C200009), Qinghai Science and Technology Program

443

(2017-ZJ-Y06, 2016-NK-C22, 2015-NK-502), Foundation of Fuli Institute of Food

444

Science at Zhejiang University, Zhejiang Science and Technology Program

445

(2017C26004).

446

Molecular graphics and analyses performed with UCSF Chimera, developed by the

447

Resource for Biocomputing, Visualization, and Informatics at the University of

448

California, San Francisco, with support from NIH P41-GM103311.

449

Notes

450

The authors declare no conflict of interest.

451 452

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Cholesterol Atherosclerosis in the Rabbit. Circulation 1953, 7, 696-701.

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1157-1174. 26


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572 573


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574 575 576

Figure 1. New route for optimization of preparation of galloyl phytosterols applying

577

protection and deprotection strategy (A) and side reaction during Steglich esterification

578

with protected gallic acid (B). (Rxn = reaction mixture)

579

Figure 2. Plausible mechanism for the side reaction (A) and further optimization of the

580

reaction conditions (B). (TB-GA = tri-isobutyroyl gallic acid, St = stigmasterol, DCC

581

= N,N-dicyclohexylarbodiimide, DMAP = 4-dimethyaminopyridine)

582

Figure 3. HPLC-MS of galloyl stigmasterol (GSt) and galloyl β-sitosterol (GSi).

583

Figure 4. DPPH radical scavenging activity (A) and Rancimat assay (B).

584

Figure 5. Illustration of evaluation model (A) and cholesterol reducing activity (B).

585

Figure 6. Molecular modeling of galloyl stigmasterol (GSt), galloyl β-sitosterol (GSi),

586

stigmasterol (St), β-sitosterol (Si) and cholesterol (Ch). (SES = solvent-excluded

587

surface).

588 589

27



(A)

(B)

Figure 1


Page 28 of 34

Page 29 of 34


(A)

(B)

Figure 2



Figure 3


Page 30 of 34

Page 31 of 34


(A)

(B)

Figure 4



(A)

(B)

Figure 5


Page 32 of 34

Page 33 of 34


Figure 6



Graphic Abstract


Page 34 of 34

Enhancement of Galloylation Efficacy of Stigmasterol and Î²-Sitosterol

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