A novel chemo-enzymatic synthesis of hydrophilic phytosterol derivatives

Accepted Manuscript A novel chemo-enzymatic synthesis of hydrophilic phytosterol derivatives Wen-Sen He, Di Hu, Yu Wang, Xue-Yan Chen, Cheng-Sheng Jia, Hai-Le Ma, Biao Feng PII: DOI: Reference:

S0308-8146(15)01057-2 http://dx.doi.org/10.1016/j.foodchem.2015.07.047 FOCH 17842

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

4 November 2014 8 July 2015 10 July 2015

Please cite this article as: He, W-S., Hu, D., Wang, Y., Chen, X-Y., Jia, C-S., Ma, H-L., Feng, B., A novel chemoenzymatic synthesis of hydrophilic phytosterol derivatives, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/ j.foodchem.2015.07.047

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1

A novel chemo-enzymatic synthesis of hydrophilic phytosterol

2

derivatives

3 4

Wen-Sen He a, *, Di Hu a, Yu Wang a, Xue-Yan Chen a, Cheng-Sheng Jia b, Hai-Le

5

Ma a, Biao Feng b

6 7

a

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Zhenjiang 212013, Jiangsu, China

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b

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School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,

State Key Laboratory of Food Science and Technology, School of Food Science and

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

11 12

*

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Tel.: +86-511-88780201; Fax: +86-511-88780201.

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E-mail: [email protected] (W. S. He)

Corresponding Author.

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1

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ABSTRACT

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In this study, a novel method was developed for chemo-enzymatic synthesis of

20

hydrophilic phytosterol derivatives, phytosteryl polyethylene glycol succinate (PPGS),

21

through an intermediate phytosteryl hemisuccinate (PSHS), which was first

22

chemically prepared and subsequently coupled with polyethylene glycol (PEG)

23

through lipase-catalyzed esterification. The chemical structure of intermediate and

24

goal product were finally confirmed to be PSHS and PPGS by FT-IR, MS and NMR,

25

suggesting that hydrophilic phytosterol derivatives were successfully synthesized. The

26

effects of various parameters on the conversion of PSHS to PPGS were investigated

27

and the highest conversion (>78%) was obtained under the selected conditions: 75

28

mmol/L PSHS, 1: 2 molar ratio of PSHS to PEG, 50 g/L Novozym 435, 120 g/L 3 Å

29

molecular sieves in tert-butanol, 55 oC, 96 h and 200 rpm. The solubility of

30

phytosterols in water was significantly improved by coupling with PEG, facilitating

31

the incorporation into a variety of foods containing water.

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Keywords:

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Phytosterols / Hydrophilic / Polyethylene glycol / Lipase / Esterification

34

2

35

1. Introduction

36

Phytosterols, mainly including -sitosterol, stigmasterol, campesterol and

37

brassicasterol, are essential triterpenoid molecules that stabilize phospholipid bilayers

38

of cell membranes in plants (Hamedi, Ghanbari, Saeidi, Razavipour, & Azari, 2014).

39

Phytosterols are generally extracted from the deodorizer distillates produced during

40

vegetable oil refining and from tall oil, a by-product of the paper pulping industry

41

(González-Larena, García-Llatas, Vidal, Sánchez-Siles, Barberá, & Lagarda, 2011;

42

Fernandes, & Cabral, 2007). Recently, phytosterols have been attracting much interest

43

because of its well-known cholesterol-lowering property (Tan, & Shahidi, 2012;

44

Sakamoto, Nakahara, & Shibata, 2013). In addition, phytosterols exhibit a variety of

45

other health benefits in vivo such as anti-oxidative (Gupta, Sharma, Dobhal, Sharma,

46

& Gupta, 2011; Tan, & Shahidi, 2013), anti-tumor, anti-inflammatory, anti-diabetic as

47

well as immunomodulatory functions (Hamedi, Ghanbari, Saeidi, Razavipour, &

48

Azari, 2014; Rudkowska, 2010; Bradford, & Awad, 2007).

49

However, the unique chemical structure of phytosterols determines that they are

50

insoluble in water and poorly soluble in oil and fat, which drastically limits their

51

widespread application in food, medical, cosmetic and other industries. To overcome

52

this problem, many studies have focused on the chemical modification of phytosterols

53

with fatty acids or its anhydride to improve the solubility in oil and fats (Miao, Liu,

54

Jiang, Yang, Xia, & Zhang, 2014; No, Zhao, Lee, Lee, & Kim, 2013; Valange,

55

Beauchaud, Barrault, Gabelica, Daturi, & Can, 2007; Deng et al., 2011). Previously, a

56

series of phytosteryl or phytostanyl fatty acid esters were synthesized in the presence 3

57

of lipase, ionic liquid or acid-surfactant-combined catalyst in our previous studies (He

58

et al., 2010; Yang, He, Jia, Ma, Zhang, & Feng, 2012; He et al., 2012a). Compared

59

with the free phytosterols, phytosteryl fatty acid esters had higher oil solubility and

60

lower melting temperature (He et al., 2012a; Miao, Liu, Jiang, Yang, Xia, & Zhang,

61

2014). Furthermore, it has been reported that phytosterol esters have similar

62

cholesterol-lowering effect to the free phytosterols. For example, equimolar

63

phytosterols and phytosteryl laurate could decrease serum TC in mice by 11.5% and

64

13.2%, respectively (He et al., 2011). Earlier studies reported that plant sterol esters

65

were rapidly hydrolyzed by intestinal enzymes, producing the physiologically active

66

free plant sterols (Moreau, Whitaker, & Hicks, 2002), indicating that phytosterol

67

derivatives linked by ester bond would retain the biological activity of free

68

phytosterols.

69

So far, little research has been reported to improve the solubility of phytosterols in

70

water. A route to make them more soluble in water is to utilize emulsification or

71

self-assembly method to increase their dispersity in micro-emulsion or molecular

72

solution. Leong et al. prepared water-soluble phytosterol nanodispersions using an

73

emulsification-evaporation method and obtained the smallest particle size about 50

74

nm (Leong, Lai, Long, Man, Misran, & Tan, 2011). It has been reported that some

75

natural sterol conjugates, such as steryl glycoside, showed hydrophilic properties

76

owing to the carbohydrate moiety of the conjugate (Nyström, Schär, & Lampi, 2012).

77

In a previous study by Sánchez-Ferrer et al. the glycosylated sterols were shown to

78

form various chiral nanostructures by self-assembly (Sánchez-Ferrer, Adamcik, & 4

79

Mezzenga, 2012). These structures were soluble in aqueous environments, and could

80

hence be applied to foods with higher water contents than the common sterol-enriched

81

functional foods (margarine, yoghurt). Alternatively, hydrophilic phytosterol

82

derivatives was synthesized by conjugating them to a polar group via ester bond to

83

enhance the solubility due to the presence of hydrophilic groups. Up to now, the

84

related research on the synthesis of hydrophilic phytosterol derivatives is rare in the

85

literature. Chung et al. reported that hydrophilic -sitosterol derivatives with various

86

degrees of substitution were synthesized by two step chemical modification in the

87

presence of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) (Chung, &

88

Choi, 2007). Subsequently, hydrophilic derivatives of -sitosterol (the main

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phytosterols) with polyethylene glycol (PEG) were proved to have comparable effects

90

to -sitosterol in lowering blood cholesterol level in rats (Chung, Kim, Noh, & Dong,

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2008). In a previous study, we tried to prepare hydrophilic phytostanol esters via

92

enzymatic method and finally established a chemo-enzymatic routes for the synthesis

93

of phytostanol esters by coupling phytostanols and D-sorbitol (He et al., 2012b).

94

Furthermore, phytostanyl sorbitol succinate have been confirmed to have similar

95

cholesterol-lowering effect to the free phytostanols in vivo (He et al., 2013). However,

96

no significant improvement for phytostanols in water solubility when coupled with

97

D-sorbitol.

98

In recent years, enzymatic synthesis has been attracted much attention due to its

99

potential advantages, such as mild reaction conditions and reagents, which has been

100

widely used for the synthesis of phytosteryl fatty acid esters and phenolates (Kim, & 5

101

Akoh, 2007; Villeneuve et al., 2005; Tan, & Shahidi, 2012, 2013). In a previous study

102

by Kim et al. (2007), phytosteryl oleic acid esters were synthesized catalyzed by

103

Candida rugosa lipase in hexane. Tan and Shahidi (2012) successfully produced

104

phytosteryl caffeate by chemo-enzymatic route and evaluated their antioxidant activity.

105

However, little research has been performed on the synthesis of hydrophilic

106

phytosterol derivatives by lipase-catalyzed synthesis.

107

The present study was aimed to establish a novel chemo-enzymatic preparation of

108

phytosteryl polyethylene glycol succinate (PPGS) by chemical acylation of

109

phytosterols with succinic anhydride followed by lipase-catalyzed esterification of

110

phytosteryl hemisuccinate (PSHS) with polyethylene glycol 1000 (PEG 1000). The

111

effects of various parameters on the lipase-catalyzed conversion of PSHS to PPGS

112

were investigated. And the chemical structure of intermediate product and hydrophilic

113

derivatives were confirmed by fourier transform infrared spectroscopy (FT-IR) and

114

mass spectra (MS). Meanwhile, the solubility of phytosterols, PSHS and PPGS in

115

water was also compared.

116

2. Materials and methods

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2.1. Materials

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Phytosterols (purity>95%) was a generous gifts from Jiangsu Spring Fruit

119

Biological Products Co., Ltd. (Taixing, China). Succinic anhydride was provided by

120

TCI Chemicals Co., Ltd. (Shanghai, China). Novozym 435 (lipase B from Candida

121

antarctica, immobilized on a macroporous acrylic resin, 10,000 PLU/g) and

122

Lipozyme RM IM (lipase from Rhizomucor miehei, immobilized on an anionic 6

123

exchange resin, 275 IUN/g) were obtained from Novo Nordisk Co., Ltd. (Shanghai,

124

China). Candida rugosa lipase (lyophilized powder, Type VII, 700 U/mg) was

125

supplied by Sigma-Aldrich Co., Ltd. (Shanghai, China). Methanol used for HPLC

126

analysis were of spectral grade and provided from Tedia Company Inc. (Shanghai,

127

China). PEG 1000, acetone, tert-pentanol, tert-butanol, n-hexane, methanol,

128

petroleum ether (60-90 oC), formic acid, ethyl acetate, trifluoroacetic acid (TFA),

129

toluene, pyridine, 3 Å molecular sieves and other common reagents used were of

130

analytical grades and purchased from Sinopharm Chemical Reagent Co., Ltd.

131

(Shanghai, China).

132

2.2. Preparation and separation of intermediate product

133

The intermediate product, PSHS, was prepared by esterification of phytosterols

134

with succinic anhydride using pyridine and toluene as catalyst and solvent,

135

respectively. At the end of the reaction, the solvent and catalyst of reaction mixtures

136

was removed by rotary evaporation under vacuum. The intermediate product, PSHS,

137

were purified by silica gel column chromatography and eluted with petroleum ether

138

(60~90

139

containing the intermediate products PSHS were collected by rotary evaporation. The

140

isolated PSHS was dried under vacuum at 50 ◦C for 24 h and used as substrate for the

141

following lipase-catalyzed reaction.

142

2.3. Lipase-catalyzed reaction of hydrophilic phytosterol derivatives

o

C)/ethyl acetate/formic acid (10/10/0.02, v/v/v). The fractions only

143

All reaction solvents used were dried with 4 Å molecular sieves at 0.1 g/mL for at

144

least 24 h prior to use. PSHS (0.125-0.75 mmol), PEG 1000 (0.5-2.5 mmol), lipase 7

145

(0.1-0.4 g), 3 Å molecular sieves (0.15-0.90 g) and solvent (5 mL) were added into a

146

15 mL screw-capped vial in sequence. The vial was placed in a water-bath shaker

147

(45-75 ◦C) and the reaction mixtures were shaken at 200 rpm. Over the time course of

148

the reactions, a portion of the reaction mixture (100 μL) was periodically taken out

149

from the reaction and used for quantitative analysis.

150

2.4. Purification of hydrophilic phytosterol derivatives

151

At the end of the lipase-catalyzed reaction, the reaction mixtures of PSHS with

152

PEG 1000 were filtered under vacuum to remove molecular sieves and lipase. The

153

solvent was removed by rotary evaporation and the solid reaction mixtures were

154

obtained. The preliminary separation was achieved by liquid-liquid extraction

155

between brine and ethyl acetate to remove excess PEG 1000. The reaction mixtures

156

mainly containing PSHS and PPGS were obtained and used for silica gel column

157

chromatography. The samples were eluted with ethyl acetate/methanol/formic acid

158

(9/1/0.1, v/v/v) at the flow rate of 0.3 mL/min. The eluent was collected and the purity

159

of product was detected by HPLC analysis. The fractions only containing PPGS were

160

collected by rotary evaporation under vacuum.

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2.5. High performance liquid chromatography (HPLC) analysis

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The reaction samples periodically removed from the reaction mixtures were diluted

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in 2 mL absolute ethyl alcohol. The samples were analyzed by Agilent 1100 HPLC

164

using a symmetry-C18 column (5 μm, 4.6 mm × 150 mm, Waters, USA) eluted with

165

methanol/TFA (1000/1, v/v) at the flow rate of 0.8 mL/min. The eluate was monitored

166

with a Schambeck ZAM 4000 evaporative light scattering detector (ELSD) at 60 ◦C 8

167

and nitrogen as carrier gas at the pressure of 0.5 bar. The purified PPGS were used as

168

standards and the calibration curve was prepared for quantitative analysis. The

169

conversion was defined as the molar ratio of PPGS at the end of the reaction to that of

170

PSHS at the beginning of the reaction.

171

2.6. FT-IR analysis

172

The purified PSHS and PPGS was dried under vacuum and then analyzed. FT-IR

173

measurement was performed on a FT-IR spectrophotometer (Thermo Nicolet IS50

174

FT-IR, USA) using attenuated total reflectance method with the spectral scanning

175

scope for 600-4000 cm-1, number of scans: 32, resolution: 4 cm-1.

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2.7. MS analysis

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The substrate PEG 1000 and the purified PPGS were determined by MS analysis.

178

Mass spectra were obtained by a liquid chromatography ion trap mass spectrometry

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(Thermo LXQ, USA) with positive electron spray ionization (ESI) mode. The MS

180

parameters were as follows: sheath gas flow rate 35 arb, aux gas flow rate 5 arb, spray

181

voltage 4.5 kV, capillary temperature 300 oC, capillary voltage 30 V, tube lens 120 V,

182

and mass scan range 600-1700 amu.

183

2.8. NMR analysis

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The isolated PPGS was examined by nuclear magnetic resonance spectroscopy

185

(NMR). 1H NMR spectra of phytosterols, PSHS and PPGS were recorded with CDCl3

186

as solvent with a Bruker NMR spectrometer (Avance Ⅲ 400 MHz, Switzerland),

187

operating at 400 MHz.

188

2.9. Determination of substrate solubility 9

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To determine the effect of the substrate solubility on the conversion, the solubility

190

of PSHS and PEG 1000 in various solvents was investigated based on the previous

191

literature with some modification (Jia, Zhao, Feng, Zhang, & Xia, 2010). In brief, an

192

amount of 2.0 g of PSHS or PEG 1000 was added into screw-capped vial with 5 mL

193

tert-pentanol, tert-butanol, acetone or 10 mL n-hexane. These solutions were

194

incubated in a water-bath shaker (30 oC) at 200 rpm for 2 h. The solutions were

195

centrifuged at 5000 rpm at 30 oC for 10 min. Subsequently, 2 mL of the upper phase

196

was accurately taken out, weighed, recorded and then dried under vacuum to remove

197

the solvent. At the end of drying, the remaining solid sample was weighed and

198

recorded. The substrate solubility was calculated according to the following formula:

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Substrate solubility (mg/mL) = The sample weight at the end of drying under

200

vacuum (mg) / The solvent volume (mL)

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2.10. Determination of water solubility

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The solubility of phytosterols, PSHS and PPGS in water was investigated according

203

to previous literature with minor modification (He et al., 2012b). Briefly, 1.0 g of

204

phytosterols, PSHS and PPGS was added into screw-capped vial with 5 mL pure

205

water. These vials were incubated in a water-bath shaker (30 oC) at 200 rpm for 5 h.

206

100 L upper solution of each were withdrawn by pipette and then diluted in 5 mL of

207

methanol/TFA (1000/1, v/v). The sample was quantified by HPLC analysis. The

208

amount of phytosterols, PSHS or PPGS was determined by comparison with the peak

209

areas of the corresponding standard materials.

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2.11. Determination of the residual enzyme activity 10

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After decanting the solvent, the lipase and 3 Å molecular sieve were washed five

212

times with warm tert-butanol, and then dried under vacuum at room temperature for

213

24 h. After removing 3 Å molecular sieve, the lipase was stored and used for the

214

recycling test. The residual activity was determined under the same optimum

215

conditions. The residual enzyme activity was expressed as the product conversion of

216

the repeated lipase to that of the fresh lipase.

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3. Results and discussion

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3.1. Product analysis

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3.1.1. HPLC Analysis

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The conversion of PSHS to PPGS and the purity of hydrophilic phytosterol

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derivatives was determined by HPLC with ELSD. The peaks of PEG 1000 and PSHS

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were characterized on the basis of their retention time with reference to standards.

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PEG 1000 was firstly eluted with the retention time of 2.7 min. Phytosterols mainly

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contained four components, -sitosterol, stigmasterol, campesterol and brassicasterol,

225

so PSHS also included four constituents, -sitosteryl, stigmasteryl, campesteryl and

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brassicasteryl hemisuccinate. PSHS were eluted with the retention time of 9.4 min,

227

10.5 min, 10.9 min and 11.5 min, respectively. PEG 1000 was a mixture with different

228

degree of polymerization (DP) between 16 and 24, so the product PPGS was also a

229

mixture. From HPLC chromatogram of the reaction mixtures, a wide peak at 7.9 min

230

was new additional peak and corresponded to the product PPGS. Apparently, the

231

products can be clearly distinguished from the reaction substrates.

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3.1.2. FT-IR Analysis 11

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The FT-IR spectral data of phytosterols and the potential functional groups were

234

shown in Table 1(a). The medium peak at 3446 cm-1 corresponded to the stretching

235

vibration of hydroxyl group. The weak peak at 3026 cm-1 was the signal of C-H in

236

–C=C-H. The peaks at 2956 cm-1 and 2869 cm-1 were the asymmetrical and

237

symmetrical stretching vibration of C-H in -CH3 group, respectively. The medium

238

peak at 1376 cm-1 was the bending vibration of C-H in -CH3 group. The peaks at 2933

239

cm-1 and 1459 cm-1 were the asymmetrical and the bending stretching vibration of

240

C-H in -CH2 group, respectively. The medium peak at 1622 cm-1 was the absorption

241

signal of C=C.

242

The FT-IR spectral data of the intermediate product and the potential functional

243

groups were shown in Table 1(b). The weak peak at 3030 cm-1 was the signal of C-H

244

in -C=C-H. The peaks at 2936 cm-1 and 2866 cm-1 were the asymmetrical and

245

symmetrical stretching vibration of C-H in -CH3 group. The peak at 1376 cm-1 was

246

the bending vibration of C-H in -CH3 group. The peaks at 2905 cm-1 and 1465 cm-1

247

were the asymmetrical stretching and bending vibration of C-H in -CH2 group. The

248

strong peak at 1177 cm-1 was the signal of the stretch vibration of C-O in ester or

249

carboxyl group. The wide and medium peak between 2400 cm-1 and 3500 cm-1

250

corresponded to the vibration of hydroxyl group in carboxyl group, indicating the

251

presence of the free carboxyl group. The strong peaks at 1727 cm-1 and 1709 cm-1

252

were the stretching vibration of C=O in ester and carboxyl group, respectively,

253

suggesting the existing of ester and carboxyl group. Compared with phytosterols, the

254

disappearance of the hydroxyl group signal and the presence of ester bond and 12

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carboxyl group were observed, indicating that PSHS were successfully synthesized.

256

The FT-IR spectral data of the hydrophilic derivatives and the potential functional

257

groups were shown in Table 1(c). The wide and medium peak at 3439 cm-1

258

corresponded to the vibration of hydroxyl group. The peaks at 2931 cm-1 and 2868

259

cm-1 were the signal of the asymmetrical and symmetrical stretching vibration of C-H

260

in -CH3 group. The weak peak at 1456 cm-1 was the bending vibration of C-H in -CH2

261

group. The strong peak at 1731 cm-1 was the signal of C=O in ester group. The strong

262

peak at 1093 cm-1 corresponded to the vibration of C-O in ester group. The absorption

263

signal of the free carboxyl group was observed in PSHS, but not in hydrophilic

264

derivatives. Only one absorption peak of carbonyl group in ester bond and no signal

265

of carbonyl group in carboxyl group were found in Table 1(c), suggesting that PPGS

266

was successfully synthesized.

267

3.1.3 MS analysis

268

The mass spectra of PEG 1000 and PPGS were acquired in the positive ESI mode

269

and their results were shown in Fig 1(a) and Fig 1(b), respectively. In general, the

270

protonated molecular ion [M+H]+ or [M+Na]+ of the compound was observed in the

271

positive ESI mode. The molecular weight of PEG 1000 and PPGS and their [M+Na]+

272

was displayed in Table S1. PEG 1000 was a mixture with various DP value and the

273

corresponding molecular weight was directly correlated with DP value. The

274

calculation formula of molecular weight of PEG was as follows: M=44*DP+18. For

275

example, the molecular weight of PEGDP=20 was 898, [M+H]+DP=20 and [M+Na]+DP=20

276

were 899 and 921, respectively. In Fig. 1(a), the m/z 921 can be observed and 13

277

corresponded to [M+Na]

278

contained PEGDP=20.

+

DP=20,

suggesting that PEG 1000 used in this study

279

The molecular weight of PEG was correlated with DP value, so the molecular

280

weight of their hydrophilic derivatives, PPGS, was also related to DP value. The

281

molecular weight of the major phytosterols, -sitosterol, was 414, so -sitosteryl

282

hemisuccinate corresponded to 514. The calculation formula of molecular weight of

283

-sitosteryl polyethylene glycol succinate was M=44*DP+514. As for PPGS, the

284

molecular weight of [M+H]+ and [M+Na]+ were 1395 and 1417 when DP value was

285

20. Similarly, the m/z 1417 can be observed from Fig. 1(b) and corresponded to

286

[M+Na]+DP=20, indicating that PPGS was successfully synthesized.

287

3.1.4 NMR analysis

288

The chemical structure of PPGS was confirmed by 1H-NMR analysis. As displayed

289

in Fig. S1, the proton of the 3-position at 3.5 ppm of free phytosterols (A) shifted to

290

4.6 ppm (B), as a result of the formation of ester bond from hydroxyl group. The same

291

phenomena were observed in the previous study by Lim et al (2012). Compared with

292

Fig. S1 (B), the resonances between 3.5 and 3.8 ppm in Fig. S1 (C) were mainly

293

ascribable to methylene units in PEG moieties, indicating that PPGS was successfully

294

synthesized.

295

3.2. Chemical preparation of PSHS

296

In recent years, enzymatic catalysis have been gaining importance because of its

297

remarkable properties such as regio, stereo, and substrate specificity, allowing mild

298

and environment friendly reaction conditions. The original object of the present study 14

299

was to develop a two-enzymatic route for the synthesis of hydrophilic phytosterol

300

derivatives. In preliminary experiment, enzymatic synthesis of PSHS employing

301

phytosterols and succinic acid or succnic anhydride as substrates and utilizing several

302

commercially available lipases as biocatalyst was attempted. However, none of them

303

led to successful coupling between phytosterols and succinic anhydride. Low

304

conversion (<5%) of phytosterol to PSHS was obtained in the presence of Candida

305

rugosa lipase at 48 h when using succinic anhydride as acyl donor. This may be

306

ascribed to that the lipase activity was inhibited by the two carboxyl groups, which

307

made direct esterification of succinic acid more difficult. The objective of the present

308

study was to synthesize hydrophilic phytosterol derivatives from PSHS with PEG. The

309

intermediate product PSHS, as the substrate of the second step reaction, had a great

310

amount of requirement. Consequently, chemical route was selected and used for the

311

synthesis of PSHS.

312

There were many known routes used to synthesize ester compounds by coupling

313

hydroxyl and carboxyl groups. The common method was using pyridine or DMAP as

314

catalyst. In a previous study by Chung et al. carboxyethyl-β-sitosterol was synthesized

315

in the presence of triethylamine and DMAP using dichloroethane as solvent and the

316

yield reached 92% (Chung, & Choi, 2007). DMAP was not easily removed from the

317

reaction mixtures, while pyridine could be quickly eliminated by rotary evaporation.

318

In this study, PSHS was synthesized in the presence of pyridine using toluene as

319

solvent. The major influencing factors, such as reaction temperature, catalyst load,

320

substrate molar ratio and time, were also considered (data not shown). Finally, the 15

321

conversion of phytosterols to PSHS can achieve above 89% by HPLC analysis under

322

the selected conditions: toluene as solvent, reaction temperature 110 oC, 1.5% (v/v) of

323

the catalyst, 1: 1.5 molar ratio of phytosterols to succinic anhydride, reaction time 20

324

h.

325

3.3. Lipase-catalyzed synthesis of hydrophilic phytosterol derivatives

326

On the basis of the preparation and purification of the intermediate PSHS,

327

hydrophilic phytosterol derivatives PPGS was successfully synthesized and

328

characterized. Meanwhile, the influence of reaction parameters on the lipase-catalyzed

329

conversion of PSHS to PPGS was investigated.

330

3.3.1. Effect of solvent

331

Reaction solvent is one of the most important parameters for lipase-catalyzed

332

esterification reaction due to its effect on the enzyme activity and stability and the

333

solubility of substrate. The Log P value was defined as the logarithm of the partition

334

coefficient of a given compound in the standard two-phase system of octanol/water

335

and mainly used for describing the solvent hydrophobicity (Jia, Zhao, Feng, Zhang, &

336

Xia, 2010). The higher the Log P was, the stronger the hydrophobicity of solvents.

337

Four organic solvents with Log P from -0.26 to 3.50 were selected based on the

338

previous report concerning the biosynthesis of phytosterol esters (He et al., 2012b).

339

The effect of reaction solvent on the conversion and the substrate solubility in

340

different solvents were shown in Fig. 2 and Table S2, respectively. n-Hexane, with log

341

P value of 3.5, had the strongest hydrophobicity, but the conversion at 72 h of PSHS

342

to PPGS was very low (<5%). This was mainly ascribed to the lowest solubility of 16

343

both PSHS and PPGS in n-hexane. With the decrease of Log P value and the

344

hydrophobicity, the polarity of solvents gradually increased. Meanwhile, the solubility

345

of both PSHS and PPGS in solvent gradually increased and the conversion of PSHS to

346

PPGS was regularly improved. The conversion in tert-butanol reached above 24% and

347

32% for 48 and 72 h, respectively. Although the solvent with a log P value of -0.26,

348

acetone, had lower hydrophobicity than tert-butanol, it displayed a remarkable

349

reduction in the conversion. This trend can be explained by the following two reasons.

350

On the one hand, the solubility of PEG 1000 in acetone improved when compared

351

with tert-butanol, while the solubility of PSHS significantly reduced, which probably

352

affected the esterification. On the other hand, acetone had more stronger polarity and

353

weaker hydrophobicity than tert-butanol, which partially reduced the enzyme activity

354

and then affected the esterification. Base on the above analyses, tert-butanol was

355

selected as the optimal solvent and used for subsequent experiments.

356

As reported in our previous study, tert-butanol was found to be the most suitable

357

solvent for the synthesis of phytostanyl sorbitol succinate (He et al., 2012b). This may

358

be explained by higher substrate solubility and lipase activity in tert-butanol when

359

compared with the other solvents. In a previous study by Degn et al. the highest

360

glucose solubility, the enzyme activity and the residual activity was observed in

361

tert-butanol for carbohydrate fatty acid ester synthesis in organic media by a lipase

362

from Candida antarctica (Degn, & Zimmermann, 2001). Similarly, the high

363

enzymatic activity was maintained and the stability of the lipase could be improved

364

significantly using tert-butanol as solvent for lipase-catalyzed esterification for 17

365

1,3-DAG preparation (Duan, Du, & Liu, 2010).

366

3.3.2. Effect of lipase

367

In the present study, several lipases in either immobilized or powdered forms were

368

investigated and used for the synthesis of hydrophilic phytosterol derivatives.

369

Novozym 435, Lipozyme RM IM and Lipozyme TL IM were immobilized lipases

370

from Candida antarctica, Rhizomucor miehei and Thermomyces lanuginosus,

371

respectively, while Candida rugosa lipase was free and powdered lipase. The effect of

372

different lipases on the conversion of PSHS in the lipase-catalyzed esterification were

373

displayed in Table S3. A remarkable difference in the conversion was observed among

374

different lipases for the same esterification reaction. The immobilized lipases,

375

Novozym 435, Lipozyme RM IM and Lipozyme TL IM showed different catalytic

376

efficiency under the same reaction condition. The conversion achieved 33% and 22%

377

employing Novozym 435 and Lipozyme RM IM as biocatalyst for 72 h, while only

378

3% of conversion was obtained using Lipozyme TL IM, suggesting that Novozym 435

379

was superior to the other two immobilized lipases. In our previous study, the effect of

380

Novozym 435, Lipozyme RM IM and Lipozyme TL IM on the conversion of

381

phytostanyl hemisuccinate were investigated and Lipozyme RM IM was found to be

382

the most suitable biocatalyst for phytostanyl sorbitol succinate synthesis (He et al.,

383

2013). This may be ascribed to be that the enzyme catalytic performance was highly

384

dependent on the substrates used. As reported in a recent study by Martins et al. the

385

immobilized lipases Novozym 435, Lipozyme RM IM and Lipozyme TL IM

386

displayed different catalytic activity for specific flavor esters synthesis. Novozym 435 18

387

was the most efficient enzyme in most cases, and only Lipozyme RM IM offered

388

better results than Novozym 435 in the production of ethyl butyrate (Martins et al.,

389

2014).

390

Furthermore, the free Candida rugosa lipase did not show any catalytic activity for

391

this reaction and no hydrophilic phytosterol derivatives were synthesized for 48 h and

392

72 h, respectively. This result was in disagreement with the previous report that

393

Candida rugosa lipase was effective for the synthesis of phytosterol fatty acid esters

394

in n-hexane (Kim, & Akoh, 2007). The discrepancy between catalytic activity and

395

conversion was mainly attributed to the difference of reaction solvent and substrate.

396

3.3.3. Effect of lipase load

397

The influence of lipase load on the conversion was evaluated varying the amount of

398

Novozym 435 from 20 g/L to 80 g/L and the results were shown in Fig. 3. It was

399

firstly observed that almost no formation of the desired product PPGS occurred in the

400

absence of lipase (data not shown). Moreover, it can be obviously found that the

401

conversion of PSHS to PPGS was linearly increased with the rise of lipase load from

402

20 g/L to 50 g/L. As shown in Fig. 3, the conversion of PSHS to PPGS can achieve

403

above 43% for 72 h at 50 g/L, while the conversion only reached 16% for 72 h at 20

404

g/L. However, the conversion was slightly improved from 43% to 47% with a further

405

rise in lipase load from 50 g/L to 80 g/L for 72 h, indicating that the lipase load (50

406

g/L) was enough to make substrate activated. These results were similar to a previous

407

report (Yang, Mu, Chen, Xiu, & Yang, 2013), in which no further improvement in the

408

conversion of feruloylated lysophospholipids with further rise of lipase concentration 19

409

when the lipase Novozym 435 load reached 60 g/L. When more than 60 g/L enzyme

410

load was used, the conversion did not change appreciably. Based on these results,

411

Novozym 435 was used for this esterification in all further experiments at an enzyme

412

load of 50 g/L.

413

3.3.4. Effect of temperature

414

Reaction temperature was crucial to enzymatic synthesis in non-aqueous media. On

415

the one hand, the substrate solubility in solvent was affected by reaction temperature.

416

In general, the higher the temperature, the greater the solubility. On the other hand,

417

the activity, stability and reusability of the lipase was strongly associated with reaction

418

temperature. Too high temperature was unfavorable for the stability and reusability of

419

the lipase. Furthermore, organic solvent was easily volatilized at high temperature.

420

The effect of reaction temperature on the conversion was investigated ranging from

421

35 oC to 75 oC. As shown in Fig. 4. the conversion of PSHS was gradually improved

422

as the temperature increased from 35 oC to 55 oC. The maximum conversion was

423

nearly reached at 55 oC and the conversion of 35% and 47% was observed in

424

lipase-catalyzed reaction for 48 h and 72 h, respectively. It was observed that there

425

was no significant variation in the conversion when the temperature was beyond 55 oC.

426

The conversion only reached 46.8% and 45.7% in the lipase-catalyzed reaction at 65

427

o

428

had optimal activity at 55 oC when applied to the synthesis of phytostanyl esters from

429

phytostanols with lauric acid, which was in close agreement with our results. Lue,

430

Karboune, Yeboah, & Kermasha (2005) also reported the lipase activity for Novozym

C and 75 oC for 72 h. Based on a study by He et al. (2010), the lipase Novozym 435

20

431

435 approached the maximum at 55 oC for the esterification of cinnamic acid with

432

oleyl alcohol.

433

3.3.5. Effect of substrate molar ratio and concentration

434

The influence of substrate molar ratio and substrate concentration on the conversion

435

was evaluated (Fig. 5). As expected, although equimolar ratio of both substrates can

436

appear as ideal in terms of economical cost and further separation for the final

437

products, it was found that such ratio was not advantageous for PPGS synthesis.

438

Actually, no displacement of the equilibrium occurred with equivalent molar of PSHS

439

to PEG 1000. Under the equimolar of PSHS to PEG 1000, the conversion of PSHS to

440

PPGS only reached 36% and 43% after 48 h and 72 h, respectively (Fig. 5a). In

441

general, a molar excess of one of the substrates was considered to be favorable

442

(Villeneuve et al., 2005). The conversion was improved from 43.2% to 50.1% for 72 h

443

as the rise of the molar ratio of PSHS to PEG 1000 from 1: 1 to 1: 2, and then slightly

444

varied from 50.1% to 49.4% for 72 h with a further increase of PSHS to PEG 1000

445

from 1: 2 to 1: 4, indicating that excessive PEG 1000 could not promote the

446

conversion when the molar ratio exceeded 1: 2. This may be attributed to that the

447

esterification have reached its equilibrium at 1: 2 molar ratio of PSHS to PEG 1000.

448

The effect of PSHS concentration from 25 mmol/L to 150 mmol/L on the

449

conversion of PSHS in lipase-catalyzed reaction was investigated under 1: 2 molar

450

ratio of PSHS to PEG 1000 (Fig. 5b). At fixing substrate molar ratio, the

451

concentration of another substrate, PEG 1000, also increased with the rise of PSHS

452

concentration. As shown in Fig. 5b, the product PPGS concentration gradually 21

453

increased from 7.7 mmol/L to 48.7 mmol/L with the increase of PSHS concentration

454

from 25 mmol/L to 150 mmol/L. However, the conversion of PSHS to PPGS firstly

455

enhanced from 30.9% to 46.9%, then decreased to 32.5% with further rise of PSHS

456

concentration and reached the maximum at 75 mmol/L PSHS. This trend was similar

457

to the results from the enzymatic synthesis of phytostanyl esters by He et al.( 2010).

458

The total solubility of substrate in reaction solvent was limited, and the undissolved

459

substrate in reaction solvent also increased with the excess rise of substrate

460

concentration, which may account for lower conversion at higher concentration (He et

461

al., 2012b). Excess substrates were strongly absorbed on the enzyme active site and

462

inhibited the lipase activity, which may also account for this phenomenon (Yadav, &

463

Dhoot, 2009).

464

3.3.6. Effect of molecular sieve concentration

465

Water played a critical role in lipase-catalyzed esterification performed in

466

non-aqueous media. As known to all, a minimal amount of water was essential for the

467

enzyme to ensure its optimal conformation and catalytic activity. However, excess

468

water was unfavorable for esterification and would affect the equilibrium conversion

469

as well as the distribution of products in solvent. Therefore, the removal of excess

470

water could shift the reaction equilibrium towards esterification and improve the

471

conversion of substrate to product. Molecular sieves have been widely and effectively

472

used for the removal of water from esterification reaction due to its low cost, easy

473

separation and regeneration.

474

According to our previous studies (He et al., 2010; He et al., 2012b), 3 Å molecular 22

475

sieve was found to have a superior water removal capability to 4 Å molecular sieve.

476

Therefore, 3 Å molecular sieve was selected and used for the removal of water in this

477

study. The effect of molecular sieve concentration on the conversion was studied. As

478

shown in Fig. S2, the conversion did not exceed 20% with addition of 30 g/L 3 Å

479

molecular sieve. The conversion of PSHS gradually increased with the rise of 3 Å

480

molecular sieve, and reached the maximum at 120 g/L for 72 h. The conversion varied

481

little from 64.2% to 62.2% with further increase of 3 Å molecular sieve when 3 Å

482

molecular sieve concentration surpassed 120 g/L. The molar conversion lowered at

483

higher concentrations of molecular sieves, which may be ascribed to be excess loss of

484

water absorbed by molecular sieves so that the lipase activity was attenuated (Gumel,

485

Annuar, Heidelberg, & Chisti, 2011). Based on these results, 120 g/L 3 Å molecular

486

sieve was used for the next experiment.

487

3.3.7. Effect of reaction time

488

Fig. S3 displayed the time course of PPGS yield for the esterification of PSHS with

489

PEG 1000 catalyzed by Novozym 435 in tert-butanol. As shown in Fig. S3, the

490

conversion of PSHS to PPGS rapidly increased to 55% with the first 48 h, and then

491

tended to gradually raise to 78% from 48 h to 96 h. After 96 h, the conversion varied

492

little, which meant that further extending reaction time beyond 96 h would not result

493

in a significant improvement in the conversion, suggesting that the esterification

494

nearly reached equilibrium at 96 h. He et al. (2010) reported that the lipase-catalyzed

495

synthesis of phytostanyl esters tended to gradually rise until 96 h in the presence of

496

Novozym 435, which was in agreement with our results. On the basis of the above 23

497

results, a high yield of PPGS (>78%) was obtained under the previously selected

498

conditions: 75 mmol/L PSHS, 150 mmol/L PEG 1000, 50 g/L Novozym 435, 120 g/L

499

3 Å molecular sieves in tert-butanol, 55 oC, 96 h and 200 rpm.

500

3.4. Recycling of the lipase

501

The recyclable property of the lipase under the optimum conditions was considered

502

(data not shown). Under the same time, slight decrease in the residual enzyme activity

503

was observed. The residual activity was still 89.2% after six recycles, suggesting that

504

the lipase Novozym 435 was an efficient biocatalyst for the synthesis of hydrophilic

505

phytosterol derivatives and can be used at least six times.

506

3.5. Comparison of synthetic route

507

In a previous study by Chung & Choi hydrophilic derivatives of β-sitosterol with

508

various DP values have been successfully synthesized in the presence of a basic

509

catalyst and dehydrating agents through two-step chemical routes (Chung, & Choi,

510

2007). The highest yield (94%) of hydrophilic derivatives of β-sitosterol with DP

511

value of 1.08 was obtained with equimolar PEG and PSHS for 6 h. The solubility of

512

hydrophilic β-sitosterol derivatives decreased as the DP values increased. Hydrophilic

513

derivatives of β-sitosterol plus mono-sterol exhibited the highest solubility, while

514

hydrophilic derivatives of β-sitosterol plus di-sterol were insoluble in water (Chung,

515

& Choi, 2007). In the present study, only hydrophilic derivatives of β-sitosterol plus

516

mono-sterol was synthesized in the presence of lipase. This may be related to high

517

selectivity and steric effect for lipase-catalyzed reaction. The highest conversion

518

(>78%) was achieved in the presence of Novozym 435, but this method offered a 24

519

good alternative for hydrophilic phytosterol derivatives production allowing mild and

520

environment friendly reaction conditions.

521

3.5. The comparison of solubility

522

The solubility of phytosterols, PSHS and PPGS in water at 30 oC was investigated

523

and compared. The solubility of phytosterols and PSHS in water were below 0.01

524

g/100 mL. As known to all, PEG was a kind of polymer of ethylene oxide with

525

various DP value, having good solubility in water. As the PEG 1000 were introduced

526

into PSHS to form PPGS, the hydrophilic property of phytosterols increased. The

527

solubility of PPGS could reach above 28.7 g/100 mL, indicating that 28.7 g PPGS

528

could be dissolved in 100 mL water at 30 oC by coupling with PEG 1000.

529

Theoretically, the water solubility of PPGS was directly correlated to the molecular

530

weight or DP value of PEG. The higher the molecular weight of PEG, the better of the

531

water solubility of PPGS. However, the mass ratio of the bioactive ingredient

532

(phytosterols) in PPGS decreased as the increase of the molecular weight or DP value

533

of PEG. Therefore, it’s more reasonable to evaluate the water solubility of the product

534

on the basis of the number of phytosterols molecules per PPGS. The solubility in

535

water of phytosterols should be calculated as follows:

536

The water solubility of phytosterols = the solubility of PPGS × 414/ (514+1000-18)

537

where 414 was the average molecular weight of phytosterols, 514 was the the average

538

molecular weight of PSHS, 1000 was the average molecular weight of PEG and 18 was the

539

average molecular weight of water.

540

By calculation, the actual solubility of phytosterols was 7.9 g / 100 mL water, 25

541

indicating that 7.9 g phytosterols could be dissolved in 100 mL water at 30 oC by

542

coupling with PEG 1000. In a previous study by Lim et al. (2012), the solubility of

543

the hydrophilic derivatives of β-sitosterol with PEG 1000 at 35 oC was 31.3 g /100

544

mL water, and the solubility of phytosterols was 8.3 g / 100 mL water. The

545

discrepancy may be ascribed to be the difference of the test method and test

546

temperature. The results showed that this route was effective to improve the water

547

solubility of phytosterols by coupling with PEG 1000, which greatly facilitated the

548

incorporation into a variety of foods containing water.

549

Phytosterols naturally occurred in five common forms: the free alcohol, fatty acid

550

esters, hydroxycinnamic acid esters, steryl glycosides, acylated steryl glycosides

551

(Moreau, Whitaker, & Hicks, 2002). So far, health claims for phytosterols (sterol

552

esters and free sterols) were accepted by both the European Food Safety Authority and

553

the Food and Drug Administration in the United States (Nyström, Schär, & Lampi,

554

2012). These glycosylated sterol conjugates showed hydrophilic properties owing to

555

the carbohydrate moiety of the conjugate. The recent studies also demonstrated that

556

glycosylated sterols were effective dietary components in cholesterol-lowering

557

(Nyström, Schär, & Lampi, 2012; Lin, Ma, Moreau, & Ostlund, 2011). However,

558

there were no direct report concerning the water solubility of the steryl glycosides and

559

acylated steryl glycosides.

560

In this study, a hydrophilic phytosterol derivatives that didn’t occur in nature were

561

successfully synthesized by chemo-enzymatic route and the solubility in water was

562

greatly improved. Chung et al. reported hydrophilic derivatives of β-sitosterol with 26

563

PEG had comparable effects to -sitosterol in lowering blood cholesterol levels but

564

they differed from -sitosterol in having a solubility advantage (Chung, Kim, Noh, &

565

Dong, 2008). In our previous study, phytostanyl sorbitol succinate was synthesized by

566

chemo-enzymatic route and confirmed to retain similar cholesterol-lowering effect to

567

the free phytostanols in vivo (He et al., 2013). These results indicated that the new

568

synthesized hydrophilic phytosterol derivatives linked by ester bond may retain the

569

biological activity of the free phytosterols.

570

The water solubility of the synthesized phytosterols derivatives (PPGS) was

571

directly correlated with the molecular weight or DP values of PEG, which may be

572

higher than that of natural steryl glycosides and acylated steryl glycosides. However,

573

the synthesized hydrophilic phytosterol derivatives can not be directly applied to

574

foods for safety consideration. The detailed metabolism pathway, toxicity and safety

575

of the synthesized products (PPGS) is unknown and still need further evaluation in the

576

next works.

577

4. Conclusion

578

In recent years, phytosterols and its derivatives have been attracting much attention

579

due to its strong biological activities as high value added products originated from

580

plants. In this work, a novel hydrophilic phytosterol derivative PPGS was successfully

581

synthesized by a two-step sequence of chemical acylation of phytosterols with

582

succinic anhydride followed by lipase-catalyzed esterification of PEG 1000 with

583

PSHS. The chemical structure of intermediate product and hydrophilic derivatives

584

were characterized by FT-IR and MS and finally confirmed to be PSHS and PPGS, 27

585

respectively. Meanwhile, the effects of various parameters on the conversion of PSHS

586

to PPGS in lipase-catalyzed esterification were investigated and a high yield of PPGS

587

(>78%) was obtained at the selected conditions. However, a series of hydrophilic

588

phytosterol derivatives with different DS need to be prepared, and its properties,

589

safety and application still need to be studied and compared in the future study.

590

Acknowledgements

591

This study was financially supported by the National Natural Science Foundation of

592

China (31401664), the China Postdoctoral Science Foundation Funded Project

593

(2014M560406), the Research Fund for the Doctoral Program of Higher Education of

594

China (20130093110010), the Research Fund for Advanced Talents of Jiangsu

595

University (13JDG070) and a project funded by the Priority Academic Program

596

Development of Jiangsu Higher Education Institutions (PAPD).

597

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Yang, H., Mu, Y., Chen, H., Xiu, Z., & Yang, T. (2013). Enzymatic synthesis of

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feruloylated lysophospholipid in a selected organic solvent medium. Food

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Chemistry, 141, 3317–3322.

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Yang, Y., He, W., Jia, C., Ma, Y., Zhang, X., & Feng, B. (2012). Efficient synthesis of

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phytosteryl esters using the lewis acidic ionic liquid. Journal of Molecular.

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Catalysis A: Chemical, 357, 39–43.

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Figure Captions

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Fig. 1 ESI mass spectra of PEG 1000 (a) and PPGS (b)

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Fig. 2 Effect of reaction solvent on the conversion of PSHS to PPGS (5 mL solvent,

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50 mmol/L PSHS, 50 mmol/L PEG 1000, 40 g/L Novozym 435, 60 g/L 3 Å molecular

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sieves, 55 oC and 200 rpm)

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Fig. 3 Effect of lipase load on the conversion of PSHS to PPGS (5 mL tert-butanol, 50

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mmol/L PSHS, 50 mmol/L PEG 1000, Novozym 435, 60 g/L 3 Å molecular sieves, 55

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o

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Fig. 4 Effect of reaction temperature on the conversion of PSHS to PPGS (5 mL

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tert-butanol, 50 mmol/L PSHS, 50 mmol/L PEG 1000, 50 g/L Novozym 435, 60 g/L 3

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Å molecular sieves and 200 rpm)

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Fig. 5 (a) Effect of molar ratio of PSHS to PEG 1000 on the conversion of PSHS to

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PPGS (5 mL tert-butanol, 50 mmol/L PSHS, 50 g/L Novozym 435, 60 g/L 3 Å

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molecular sieves, 55 oC and 200 rpm); (b) Effect of the concentration of PSHS on the

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conversion of PSHS to PPGS (5 mL tert-butanol, 1: 2 molar ratio of PSHS to PEG

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1000, 50 g/L Novozym 435, 60 g/L 3 Å molecular sieves, 55 oC, 48 h and 200 rpm)

C and 200 rpm)

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34

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Table 1 FT-IR spectra of phytostreols (a), phytosteryl hemisuccinate (b) and

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phytosteryl polyethylene glycol succinate (c)

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(a) Phytosterols Wavenumbers (cm-1) Intensity Adscription a Potential functional groups 3446 medium vOH -OH 3026 weak vCH -C=C-H2956 strong vCH -CH3 2869 strong vCH -CH3 2933 strong vCH -CH21622 medium vC=C -C=C1459 medium δCH -CH21376 medium δCH -CH3 a v, stretching vibration; δ, bending vibration.

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(b) Phytosteryl hemisuccinate Wavenumbers(cm-1) 2400-3500 3030 2936 2905 2866 1727 1709 1465 1376 1177

Intensity Adscription a Potential functional groups medium vOH -COOH weak vCH -C=C-H strong vCH -CH3 strong vCH -CH2strong vCH -CH3 strong vC=O R-CO-OR’ strong vC=O -COOH weak δCH -CH2weak δCH -CH3 medium vC-O R-CO-OR’, -COOH a v, stretching vibration; δ, bending vibration.

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(c) Phytosteryl polyethylene glycol succinate Wavenumbers(cm-1) 3439 2931 2868 1731 1456 1093

Intensity Adscription a Potential functional groups medium vOH -OH medium vCH -CH3 medium vCH -CH3 strong vC=O R-CO-OR’ weak δCH -CH2medium vC-O R-CO-OR’ a v, stretching vibration; δ, bending vibration.

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731 732

Fig. 1. (a) PEG 1000

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(b) PPGS

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36

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Fig. 2.

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37

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Fig. 3.

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38

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Fig. 4.

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39

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Fig. 5. (a)

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(b)

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Highlights

► A novel chemo-enzymatic route was developed. ► Hydrophilic phytosterol derivatives were successfully synthesized. ► The water solubility of phytosterols was greatly improved .