Plant Sterols: Chemical and Enzymatic Structural Modifications and

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Plant Sterols: Chemical and Enzymatic Structural Modifications and Effects on Their Cholesterol-Lowering Activity Wen-Sen He, Hanyue Zhu, and Zhen-Yu Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00059 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 10, 2018

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Journal of Agricultural and Food Chemistry

Plant Sterols: Chemical and Enzymatic Structural Modifications and Effects on Their Cholesterol-Lowering Activity

Wen-Sen He,†,‡,§ Hanyue Zhu,‡,§ Zhen-Yu Chen‡,*

†

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, China

‡

Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China

_________________________________________________________ * Correspondence: Dr. Zhen-Yu Chen, Room 179, Science Center, School of Life Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China; email: [email protected]; tel: (852) 3943 6382; fax: (852) 2603-7246. §

W-S He and H Zhu contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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Plant sterols have attracted more and more attention due to their excellent

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cholesterol-lowering activity. However, free plant sterols have some characteristics

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of low oil solubility, water insolubility, high melting point and low bioavailability,

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which greatly limit their application in foods. Numerous studies have been

6

undertaken to modify their chemical structures in order to improve their chemical

7

and physical properties in meeting the needs of various applications. The present

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review is to summarize the literature and update the progress on structural

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modifications of plant sterols in the following aspects: (i) synthesis of plant sterol

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esters by esterification and transesterification with hydrophobic fatty acids and

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triacylglycerols to improve their oil solubility; (ii) synthesis of plant sterol derivatives

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by coupling with various hydrophilic moieties to enhance their water solubility; and

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(iii) mechanisms by which plant sterols reduce plasma cholesterol and the effect of

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structural modifications on plasma cholesterol-lowering activity of plant sterols.

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Keywords: plant sterols, phytosterol esters, chemical modification, cholesterollowering activity, cholesterol;

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INTRODUCTION

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Sterols are essential components of eukaryotic cell membranes and play an

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important role in regulating the physicochemical property of cell membranes.[1] They

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are mainly classified into phytosterols (plant origin), zoosterols (animal origin) and

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mycosterols or ergosterol (fungal origin). All types of sterols share a structure of

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similar chemical backbone and differ in a number and position of double bonds or a

25

length of side carbon chain (Figure 1). So far, more than 250 plant sterol species have

26

been identified in various plants and marine organisms. Based on the difference of

27

the number of methyl groups on carbon-4, plant sterols are further divided into 4,4-

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dimethyl sterols, 4α-monomethyl sterols, and 4-desmethyl sterols. In nature, 4,4-

29

dimethyl sterols and 4α-monomethyl sterols are quantitatively minor, while 4-

30

desmethyl sterols are quantitatively abundant.[2] In this regard, the most frequently

31

referred plant sterols are 4-desmethyl sterols, mainly including β-sitosterol,

32

campesterol, stigmasterol and brassicasterol (Figure 1). These plant sterols exist in

33

following five forms namely free sterols, fatty acid esters, hydroxycinnamic acid

34

esters, sterol glycosides and acylated sterol glycosides.[3] In the industrial process,

35

plant sterols are primarily obtained from the deodorizer distillates produced during

36

the refining process of vegetable oils production, or from tall oil, a by-product of the

37

pulping of pine wood.[4]

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Plant sterols have shown to be effective in decreasing blood total cholesterol (TC)

39

and low-density lipoprotein cholesterol (LDL-C) by inhibiting intestinal cholesterol

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absorption in both humans and animals.[5] In addition, they also exhibit various other

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health benefits including anti-cancer,[6] anti-inflammation,[7] anti-oxidation,[8]

42

neuroprotection,[9] and cardiovascular-protection.[10,11] As a functional ingredient,

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plant sterols have been widely used in foods, medicines, cosmetics, nutraceuticals

44

and other applications. On the one hand, plant sterols, commonly in a form of

45

crystalline powder, have some characteristics of low oil solubility and high melting

46

point, which limit their application in foods of hydrophobic matrix. On the other

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hand, the water insolubility of plant sterols hinders their application in foods of

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hydrophilic matrix. To meet the needs of various applications, numerous studies on 3



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chemical modifications have been undertaken to improve the oil or water solubility

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of plant sterols.[12-15]

51

More than 1400 scientific articles published in the last two decades can be

52

retrieved from the Web of Science using the terms “plant sterols” and “phytosterols”.

53

Among these publications, there are more than 150 reviews, most of which have

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addressed the health benefits of plant sterols regarding their plasma lipid-

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reduction,[16-19] anti-tumor,[20,21] anti-inflammation,[22,23] synergistic interaction with

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other active ingredients,[24,25] and potential protection on cardiovascular

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diseases,[10,26] dementia[27] and central nervous system disorders.[28] The other review

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articles have focused on other aspects of plant sterols including their production,[29,30]

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diversity,[31] analysis,[32] oxidized phytosterols,[33-35] conjugated sterols,[1] and related

60

derivatives.[36-38] However, a thorough review on structural modification of plant

61

sterols and effect of chemical structural modifications on the biological activity of

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plant sterols is lacking. The present review is to summarize the process and the

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reaction mechanism of various chemical modifications of plant sterols, and to brief

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the effect of chemical modifications of plant sterols on their cholesterol-lowering

65

activity.

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CHEMICAL AND ENZYMATIC STRUCTURAL MODIFICATIONS OF PLANT

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STEROLS

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Chemical or enzymatic structural modification is to change the chemical

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structure and molecular weight of plant sterols by introducing one or more

71

substituent groups, and thereby alter the basic physical and chemical properties of

72

original plant sterols. C-3 hydroxyl group is the major functional group of plant

73

sterols. The chemical modifications of plant sterols mainly occur at C-3 position

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including (1) esterification with hydrophobic fatty acids to improve their oil solubility,

75

and (2) coupling with hydrophilic components to enhance their water solubility.

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Improving the Oil Solubility of Plant Sterols

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Plant sterols have a relatively high melting point (usually at least 135oC) and a

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poor solubility in vegetable oils, thus hindering their application into fat-based foods.

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In general, plant sterols are easy to crystallize when they are added directly into food,

80

rendering the food undesirable texture and seriously affecting the quality of food. In

81

view that plant sterol fatty acid esters have a higher solubility in fats and oils,

82

numerous studies have concentrated on the synthesis of plant sterol fatty acid esters

83

to improve the oil solubility of plant sterols by enzymatic- or non-enzymatic

84

esterification or transesterification (Figure 2). These common acyl donors include the

85

saturated and unsaturated fatty acids, fatty acid methyl or ethyl esters,

86

triacylglycerols originating from different vegetable oils, fatty acid halogenide or

87

anhydride.

88

Synthesis of Plant Sterol Esters by Acid-catalyzed Esterification and

89

Transestrification. Plant sterol esters can be synthesized by direct esterification and

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transesterification in the presence of some acid catalysts. This reaction is a

91

nucleophilic acyl substitution reaction based on the electrophilicity of carbonyl

92

carbon and the nucleophilicity of an alcohol. The general mechanism of acid-

93

catalyzed esterification and transesterification of plant sterols is given in Figure 3.

94

The acid-catalyzed esterification mainly involves the following reactions: (1) a proton

95

transfer from an acid catalyst to carbonyl oxygen of a fatty acid; (2) nucleophilic

96

sterol (ROH) attacks the carbonyl carbon of a fatty acid, forming an oxonium ion; (3)

97

deprotonation of oxonium ion and protonation of another hydroxyl group of

98

activated complex produces a new oxonium ion; (4) the new oxonium ion loses a

99

water molecule; and (5) deprotonation produces a sterol fatty acid ester. The acid-

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catalyzed transesterification mainly involves the following reactions: (i) protonation

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of carbonyl oxygen on a triacylglycerol by the acid catalyst; (ii) nucleophilic attack of

102

sterol (ROH) on the carbonyl carbon, forming a tetrahedral intermediate; (iii) proton

103

migration; (iv) breakdown of the intermediate, forming diacylglycerols; (v)

104

deprotonation and formation of a sterol fatty acid ester. The commonly used acid

105

catalysts include homogeneous acid catalysts (sulfuric acid, toluene-p-sulfonic acid,

106

lewis acid) and heterogeneous acid catalysts (immobilized heteropolyacid).[39]

5



107

Homogeneous acid catalysts have advantages of a low cost and a high efficiency,

108

but they have some disadvantages of difficult separation and easy corrosion of

109

equipment. Concentrated sulfuric acid is a catalyst used earlier to synthesize plant

110

sterol esters. Using H2SO4 as a catalyst, conversion of plant sterols to their fatty acid

111

esters could achieve 98% by direct esterification with oleic acid.[40] However, the side

112

products such as dehydrated sterols sometimes could reach as high as 19%.[40,41] To

113

avoid the production of side products, other acid catalysts have been subsequently

114

used for the synthesis of plant sterol esters. Deng et al. optimized the reaction

115

parameters of sodium bisulfate-catalyzed synthesis of plant sterol polyunsaturated

116

fatty acid esters,[42] finding that the yield of esterification could reach 96%. Zhou et al.

117

investigated the catalytic activity of several Lewis acids and Lewis acid-surfactant-

118

combined catalysts for the synthesis of phytostanol esters,[43] demonstrating that

119

cuprum dodecyl sulfate [Cu(DS)2] displayed an highest catalytic activity with a

120

conversion of 98% when lauric acid was used as an acyl donor.[43] When sodium

121

dodecyl sulfate was chosen as a catalyst, the yield of phytostanol laurate could reach

122

92%.[44]

123

Heterogeneous catalysts such as immobilized heteropolyacid have a high catalytic

124

activity and a good reusability with minimum pollution. Meng et al. synthesized the

125

food grade phytosteryl esters using 0.2% tungstosilicic acid in silica gel as a catalyst

126

in solvent-free system with a good yield of 90%.[45] The catalyst could be used at

127

least six times without having a significant loss of catalytic activity. Recently, acidic

128

ionic liquids (ILs) have gained more and more attention due to their excellent

129

catalytic activity for esterification or transesterification. Yang et al. reported a highly

130

efficient process to synthesize the plant sterol esters in the presence of Lewis acid ILs,

131

demonstrating the esterification rate could reach 92% when ChCl·2SnCl2 was used as

132

a catalyst with 3:1 molar ratio of lauric acid to plant sterols at 150⁰C for 4 h.[41]

133 134

Synthesis of Plant Sterol Esters by Base-Catalyzed Transesterification. Plant

135

sterol esters could be synthesized by a chemical reaction of base-catalyzed

136

transesterification of plant sterols with fatty acid esters or triacylglycerols. The base

137

catalysts include metallic hydroxides and alkoxides as well as other oxides.[39,46,47] 6


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The general mechanism of base-catalyzed transesterification of plant sterols with a

139

fatty acid methyl ester is given in Figure 4. The base-catalyzed transesterification

140

mainly involves the following reactions: (1) reaction of a base (B) with a sterol (ROH)

141

produces an alkoxide (RO—) and a protonated catalyst (BH+); (2) nucleophilic attack

142

of the alkoxide (RO—) on the carbonyl group of fatty acid ester generates a

143

tetrahedral intermediate; (3) the intermediate breaks down to produce a sterol fatty

144

acid ester and a corresponding anion (R’O—); (4) R’O— deprotonates the catalyst (BH+)

145

to generate the active form of base (B) and the corresponding alcohol (R’OH). Base-

146

catalyzed transesterification provides the following advantages including a fast

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reaction speed, a high conversion rate, a low cost and a wide availability.[48] At the

148

same time, base-catalyzed transesterification suffers some disadvantages including a

149

low selectivity and a high rate of byproducts derived from formation of soap,

150

polymerization and oxidation.[47] Most importantly, these homogeneous catalysts are

151

corrosive and difficult to separate from the final products and produce the excessive

152

waste.[47]

153

Sodium ethylate was patented as a catalyst in 1996 to produce plant sterol esters

154

by transesterification of β-sitostanol with rapeseed oil fatty acid methyl ester.[49]

155

Subsequently, numerous heterogeneous catalysts such as metallic oxide were

156

successively used for the synthesis of plant sterol esters to overcome the

157

shortcoming of homogeneous catalyst.[39,47] Pouilloux et al. compared the catalytic

158

performance of some solid base catalysts, including Na2CO3, MgO, LiMgO and ZnO,

159

for the synthesis of plant sterol esters from β-sitosterol and methyl dodecanoate in a

160

solvent-free system, reporting a yield of 78% with 10% stigmasta-3, 5-diene as a side

161

product when using MgO as a catalyst.[39] Valange et al. evaluated the catalytic

162

ability of different La2O3 oxide and found that La2O3 oxide could inhibit the side

163

reaction of dehydration while the yield of plant sterol esters could achieve 89%, and

164

the selectivity was 90-96%.[47] Two alumina-supported magnesium oxide (MgO-Al2O3-

165

SG) and alumina-supported Zinc Oxide (ZnO/Al2O3) as catalysts were also tested and

166

investigated by Meng et al and Robles-Manuel et al.[40,50] It was found that compared

167

with traditional homogeneous catalysts, immobilized ZnO had a maximum

168

production of plant sterol esters (98%) after 8 h at lower temperature (170oC) with a 7



169

higher selectivity (>90%), a better reusability and a lesser corrosivity.[40] To avoid the

170

use of poisonous catalysts and the production of side products, He et al. developed a

171

process to synthesize plant sterol esters in the absence of catalyst and solvent.[46]

172

Under the optimized auto-catalyzed reaction conditions, the conversion and yield

173

could reach 99% and 95%, respectively.[46]

174

Most of these reactions catalyzed by metallic oxides require a high temperature

175

(>170oC) and therefore the side products are inevitably formed. The content of diene

176

in products ranges from 2% to 6.3%, and the level of other side products, such as

177

oxide sterols are between 3 to 9% when the esterification or transesterification of

178

plant sterols is performed at 230-250 oC.[40] There is a need in deed to develop some

179

mild methods for the synthesis of plant sterol esters. In this regards, Julien-David et

180

al. utilized 4-dimethylaminopyridine (DMAP) as a catalyst to synthesize the plant

181

sterol ester at room temperature for 24 h in the presence of dichloromethane and

182

N,N’-dicylohexylcarbodiimide and the yield of plant sterol oleate reached 78%.[51]

183 184

Synthesis of Plant Sterol Esters or Ethers by Other Chemical Reactions. Plant

185

sterol esters could be also synthesized via other acylation reaction. Yang et al.

186

presented an alternative method of synthesizing phytosterol ester from soybean

187

sterol and acetic anhydride in the absence of catalyst and solvent.[52] It was found

188

that the esterification rate could reach 99% at 135 °C for 1.5 h. As an effective acyl

189

donor, the fatty acid anhydrides are more reactive than the free acids or the

190

corresponding esters. However, this method appears only suitable for esterification

191

of phytoserols with short-chain fatty acids. Ishida proposed a method to synthesize

192

plant sterol esters from plant sterols and acyl halide using DMAP as a catalyst in dry

193

pyridine.[53] In a similar way, Hang produced a series of plant sterol esters with a

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yield of 79-94%.[54] Brown and Hang prepared a phytosterol-octadecyl ether from

195

phytosterol and octadecyl iodide in the presence of sodium hydride.[54,55] Similarly,

196

Wang et al prepared 3β-methoxy and 3β-ethoxy ethers of β-sitosterol from methyl

197

or ethyl iodide and β-sitosterol in the presence of sodium hydride.[56]

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Enzyme-Catalyzed Synthesis of Plant Sterol Esters. In recent years, enzyme-

201

catalyzed synthesis of plant sterol esters has attracted more and more attention as

202

these methods have advantages of having mild and environmental-friendly reaction

203

conditions, a higher selectivity and fewer side-products. Lipases as a biocatalyst play

204

a crucial role in enzyme-catalyzed esterification. The catalytic activity of lipase is

205

structures-dependent. The x-ray crystallography on the three-dimensional structures

206

of lipases has reviewed they have an α/β-hydrolase fold, an oxyanion hole, and, in

207

most cases, a “lid” formed by an α-helix that covers the active site.[57] Most

208

importantly, almost all lipases have an active site formed by a Ser-His-Asp/Glu triad,

209

which is essential in esterification reaction. The catalytic serine has been found to be

210

located in a same place on the central β-sheet,[57] suggesting that the mechanism of

211

lipase-catalyzed reaction is similar or identical.[57] Taking Candida rugosa lipase as an

212

example,[57-60] the mechanism of lipase-catalyzed esterification mainly involves the

213

following reactions (Figure 5): (1) The catalytic triad and serine residue form a

214

tetrahedral intermediate with a fatty acid molecule; (2) Water molecule is released

215

from the intermediate to form an acyl-enzyme complex; (3) A second tetrahedral

216

intermediate of lipase-ester complex is formed with a nucleophilic sterol (ROH); (4) a

217

sterol fatty acid ester is released from the lipase-ester complex.[57,59] The

218

conformational changes and kinetic study on lipase have demonstrated that lipase-

219

catalyzed esterification often follows Ping-Pong Bi-Bi mechanism.[61,62]

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Effect of Different Lipases. Production of plant sterol esters are lipase-

221

dependent. Over the past two decades, there are more than forty publications

222

reporting the synthesis of plant sterol fatty acid esters via lipase-catalyzed

223

esterification of sterols with free fatty acids or enzyme-catalyzed transesterification

224

with fatty acid esters or vegetable oils (Table 1). Among these studies, numerous

225

lipases originated from various sources were employed to catalyze plant sterol esters

226

synthesis and their relative catalytic activity had been compared.[63-66] Villeneuve et

227

al. evaluated the catalytic ability of several plant and microbial lipases from Carica

228

papaya, Ricinus communis, Rhizomucor miehei, Candida antarctica B, Candida

229

rugosa, finding Candida rugosa lipase had a highest catalytic activity with a 9



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230

conversion rate of 85% when 5% lipase was used at 35oC after 72 h.[63] Similarly,

231

Weber et al. tested the catalytic efficiency of microbial lipases from Rhizopus

232

arrhizus, Candida rugosa, Chromobacterium viscosum as the potential biocatalysts,

233

demonstrating Candida rugosa lipase could achieve a 90% conversion rate for

234

esterification of sitostanol with oleic acid under a vacuum of 20-40 mbar.[64] In

235

contrast, He et al. investigated the effects of various sourced-lipases on the

236

conversion of plant stanols to phytostanol laurate, showing a microbial lipase

237

Novozym 435 was the best biocatalyst for esterification of plant stanols with fatty

238

acids.[65]

239

Among microbial lipases, lipase from Candida rugosa is the most frequently used

240

biocatalyst for the synthesis of plant sterol esters. Candida rugosa lipase has been

241

widely used for the synthesis of plant sterol esters by direct esterification with

242

caprylic acid,[66] lauric acid,[67-69] oleic acid,[63,64,70-73] linoleic acid,[74] linolenic acid,[75-

243

77]

244

Ahiflower seed oil,[81] and transesterification with ethyl linolenate,[12] methyl

245

oleate[64] and triolein.[64] Miao et al. synthesized plant sterol esters of lauric acid in

246

the presence of free Candida rugosa lipase with a yield of 75% at 55oC after 48 h.[68]

247

Villeneuve et al. explored the feasibility of lipase-catalyzed esterification of canola

248

phytosterols with oleic acid using Candida rugosa lipase as a catalyst, finding the

249

yield could reach 85% after 72 h.[63] Kim et al. optimized the reaction parameters of

250

Candida rugosa lipase-catalyzed synthesis of plant sterol oleic acid esters using

251

response surface methodology and had a yield of 97% at 51.3 oC for 17 h.[70] Apart

252

from Candida rugosa lipase, lipases from Candida antarctica and Candida sp. 99-125

253

have also been widely used to synthesize plant sterol esters. Panpipat et al. used

254

Candida antarctica lipase A as a catalyst to synthesize β-sitosterol myristic acid ester

255

with a yield up to 98% at 40–50⁰C and 5–10% enzyme load for 24 h.[82] Zheng et al.

256

produced plant sterol ester of conjugate linoleic acid and obtained a yield of 86%

257

using Candida sp. 99-125 as a biocatalyst for 8 h.[83]

conjugated linoleic acid,[78] pinolenic acid,[79] fatty acids from butter fat

[80]

and

258

Although these free lipases have shown a high catalytic activity, there are still

259

some concerns such as a poor stability and a low reusability, which make their

260

application relatively difficult and expensive. Recently, the immobilization technique 10


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has been developed with a great success. Compared with free lipase, the

262

immobilized lipases retain its higher catalytic activity and a stability in addition to

263

easier separation and good recovery.[84] The lipases have been successfully

264

immobilized on synthetic organic polymers, biopolymers hydrogels and inorganic

265

supports, for examples, Novozyme 435 was immobilized on a macroporous acrylic

266

resin; Lipozyme TL IM was immobilized on silica granulation; Candida rugosa lipase

267

was immobilized on magnetic microspheres,[76] silica particles,[75] hyper-cross-linked

268

polymer-coated silica,[77] mesostructured magnetic hollow mesoporous silica

269

microspheres,[74] macroporous resin[67,79] and ZnO nanowires/macroporous SiO2.[71,73]

270

Torrelo et al. produced phytosterol esters using both free and immobilized Candida

271

rugosa lipases as a catalyst in solvent-free system, finding the former having a yield

272

of 94% while the latter having a yield of 99%.[80] In another study, plant sterol esters

273

was produced by a process of ultrafast transesterification between sterols and

274

triacylglycerols with a yield of above 90% within 15 min at 55oC using Candida rugosa

275

lipase immobilized on magnetic hollow mesoporous silica microspheres.[74] Most

276

important was that the catalytic activity of immobilized lipase was largely retained

277

after more than 50 successive reactions.[74] Pan et al. examined the catalytic activity

278

of three immobilized lipases from Candida antarctica (Novozyme 435), Thermomyces

279

lanuginosus (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM), and Candida sp.

280

99-125 lipase, finding Candida sp. 99-125 lipase was the most effective biocatalyst in

281

producing phytosterol oleate with a yield of 93.4% at 45 oC for 24 h.[85] The

282

conversion of plant sterols to their corresponding sterol esters is closely related to

283

the type of enzymes. In addition, the conversion is affected by many factors. The

284

reaction temperature not only influences the substrate solubility in solvent, but also

285

affects the activity and stability of lipase. In general, the higher the temperature is,

286

the greater the substrate solubility is, and the poorer the stability and activity of

287

lipase are. In fact, the optimum temperature of immobilized lipases is higher than

288

that of free form. For the Candida rugosa lipase (Table 1), the optimum temperature

289

for free form ranges from 30 to 50 oC, while the optimum temperature for their

290

immobilized from ranges from 40 to 60 oC.

11



291

Solvent Effect. The solvent is another crucial factor in enzyme-catalyzed

292

production of plant sterol ester. Apart from lipase, the reaction solvent is also an

293

important parameter for lipase-catalyzed synthesis of plant sterol esters. In general,

294

organic solvents not only are the medium for mass transfer by affecting the solubility

295

of substrate, but also they have a great effect on the activity and stability of lipase by

296

changing the structure of enzyme and water activity.[12] The Log P value is used for

297

describing the solvent hydrophobicity. The higher the Log P is, the stronger the

298

hydrophobicity of solvents.[12] Numerous organic solvents with different Log P value,

299

such as n-hexane,[67,70,85-87] cyclohexane,[81] n-heptane,[88] isooctane[12,71,73,75-77,89] and

300

toluene[90] have been proved to be efficient solvent for the synthesis of plant sterol

301

esters. Jiang et al. investigated the effect of several solvents on the esterification of

302

phytosterol acetate and found n-hexane with Log P of 3.5 was the optimum

303

solvent.[67] Zhang et al. compared the effects of different solvents including toluene,

304

cyclohexane, n-hexane, n-heptane and octane on the conversion of plant sterol ester

305

catalyzed by Candida rugosa lipase, demonstrating that n-heptane with a log P value

306

of 4.0 was the optimum solvent and had the highest conversion rate of 90% at 44oC

307

after 12 h reaction. In a study by Choi et al.,[81,88] cyclohexane with a Log P value of

308

4.0 was deemed to be the suitable reaction medium for simultaneous synthesis of

309

phytosterol esters and enrichment of stearidonic acid using Candida rugosa lipase as

310

a biocatalyst. Pan et al. studied the relationship between the conversion of plant

311

sterols and Log P values of solvent from -1.3 to 4.7 when using Candida sp. 99-125

312

lipase to catalyze the esterification of plant sterols, finding Isooctane with a Log P

313

value of 4.7 showed a higher conversion over n-hexane with a Log P value of 3.5.[85]

314

A similar trend was observed by He et al.

315

sterols to plant sterol linolenate was positively correlated with the Log P values (-1.3

316

to 4.7) of solvents with isooctane (Log P 4.7) giving a highest conversion. In general,

317

Isooctane (Log P 4.7) and n-hexane (Log P 3.5) were the two most frequently used

318

solvents. Under the certain conditions, isooctane was superior to n-hexane for

319

lipase-catalyzed synthesis of plant sterol esters. He et al. also investigated the

320

relationship between the solvent hydrophobicity and the lipase stability after

321

exposure to different solvents

[12]

, who showed the conversion of plant

[12]

. The stability of Candida rugosa lipase gradually 12


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322

decreased as the Log P values of solvent decreased from 4.7 to -1.3. The lipase

323

treated with dimethyl sulfoxide (DMSO) showed no activity in isooctane. This was

324

because the solvent with a strong polarity (low Log P values) rapidly deprived the

325

necessary water from lipase molecules, thus making the enzyme completely

326

deactivated.

327

Recently, some new solvents were introduced into the esterification of plant

328

sterols and fatty acids. In a study conducted by Zeng et al., a water-in-ionic liquid

329

microemulsion ([Bmim]PF6/Tween 20/H2O) was applied as a reusable reaction

330

medium for Candida rugosa lipase-catalyzed esterification of phytosterols with fatty

331

acids and the conversion of 87.9% was achieved in 24 h under optimized

332

conditions.[69] Two bio-based solvents (limonene and p-cymene) were also used for

333

lipase B from Candida antarctica-catalyzed synthesis of β-sitosterol ester and

334

showed higher initial reaction rates than n-hexane, obtaining 75% conversion.[91]

335

King et al. explored the feasibility of lipase-catalyzed esterification between

336

sitostanol and fatty acids in supercritical carbon dioxide and achieved a yield of 92%

337

and 99% for caprylic and palmitic acid, respectively, when chirazyme L-1 lipase from

338

Burkholderia cepacia was used as a biocatalyst.[92]

339

Enzyme-catalyzed production of plant sterol esters can be operated in a solvent-

340

free system. In fact, lipase-catalyzed synthesis of plant sterol esters conducted in

341

solvent-free system has some advantages including the improvement of food safety,

342

easy operation and less environmental hazards.[74] Martínez et al. demonstrated the

343

feasibility of preparing wood sterol esters in a solvent-free system via lipase-

344

catalyzed transesterification of wood sterols with sunflower fatty acid methyl

345

esters.[93] In general, solvent-free synthesis of plant sterol esters often requires a

346

higher reaction temperature to make the substrate fully melted. Negishi et al.

347

prepared plant sterol esters by lipase-catalyzed transesterification at 100oC using

348

sunflower oil containing 10% plant sterols as substrates.[94] Lanctôt et al. reported

349

solvent-free lipase-catalyzed preparation of β-sitosterol esters at 90 oC.[91] Most of

350

the reported studies regarding the solvent-free synthesis of plant sterol esters were

351

carried out using an excess of fatty acids or their methyl or ethyl esters. Using

352

immobilized Candida rugosa lipase, plant sterol esters was successfully synthesized 13



Page 14 of 54

353

with 5:1 molar ratio of fatty acids or triacylglycerols to plant sterols in a solvent-free

354

medium.[74] To reduce the excess use of acyl donor, Torres et al.[78] and Torrelo et

355

al.[80] established an efficient route for solvent-free synthesis of plant sterol esters

356

with a 1:1 molar ratio of plant sterols to conjugated linoleic acid or fatty acids from

357

butterfat by stepwise addition of plant sterols.

358

Other Assisted Methods. Ultrasound and microwave have shown to accelerate

359

the enzyme-catalyzed production of plant sterol esters. Ultrasound mainly decreases

360

a particle size, maximize the surface area of substrate and enzyme, and facilitate the

361

access of substrate to the active site of enzymes.[83] Zheng et al. found that

362

ultrasound

363

transesterification of plant sterols with fatty acids or triacylglycerols, having an

364

overall conversion being more than 2-fold as that of stirring process for 16–24 h

365

without affecting the lipase activity.[83] Apart from ultrasound, microwave is also

366

used for plant sterol ester synthesis. Microwave irradiation can enhance the activity

367

and thermal stability of a lipase by a direct coupling of microwave energy with the

368

molecules (solvents, substrates, catalysts), resulting in a short reaction time as well

369

as a high yield.[71] Shang et al. used the microwave irradiation to catalyze

370

esterification of plant sterols and oleic acid in the presence of immobilized Candida

371

rugosa lipase, demonstrating a conversion rate of 95% could be achieved in 1 h,

372

while the conventional methods would require 6 h to obtain the same conversion.[71]

373

Water (or methanol, ethanol) is the side product of the reversible esterification (or

374

transesterification). The excessive production of water would further enhance the

375

reverse reaction and thereby decrease the conversion rate. In this regard, numerous

376

esterification or transesterification processes of plant sterols are developed under

377

vacuum in order to remove the excessive side products.[64,78-80,93,95,96] Weber et al.

378

established an enzymatic method for the preparation of plant sterol esters in

379

vacuum and achieved a yield of 95% at 20-40 hPa using Thermomyces lanuginosus

380

lipase.[95] No et al. synthesized plant sterol esters of pinolenic acid using the

381

immobilized Candida rugosa lipase under vacuum with a maximum conversion of

382

93% being achieved at 80 kPa.[79]

pretreatment

for

8-10

h

could

accelerate

14


esterification

or

Page 15 of 54


383

Physical Properties of Plant Sterol Esters. The melting point and the oil

384

solubility of free and esterified plant sterols have been investigated in numerous

385

studies (Table 2).[12,64,65,72,96,104] The melting point of plant sterols ranges from 137 to

386

145 oC.[41,68,105,106] Kobayashi et al. compared the melting points of free and esterified

387

sitosterol by the differential scanning calorimetric analysis, finding the melting point

388

of β-sitosterol decreased from 130-140 to 28oC after esterification with oleic acid.[72]

389

Vu et al. synthesized a series of plant sterol esters of medium chain fatty acids (C

390

6:0~C12:0), demonstrating all the plant sterol esters had a lower melting point than

391

the corresponding free sterols. It should be pointed out that the melting points

392

would increase from 58 oC to 85 oC when the carbon number of saturated fatty acids

393

increased from 6 to 12.[99] Vaikousi et al. also found that the melting points of soy

394

phytostanol esters increased with increasing chain length of the fatty acid moiety

395

(C8-C12).[105] Free plant sterols have a poor solubility in vegetable oils, for example,

396

the solubility of plant sterols in soybean oil was only 1.24 g/ 100 mL at 20 oC.[42] The

397

solubility of plant sterols was increased by 3 times via lipase-catalyzed esterification

398

with lauric acid and could reach 4.28 g/100 mL in sunflower oil at 30 oC.[68] The

399

solubility of a plant sterol would further improve by above 25 times if it was esterifed

400

with an unsaturated fatty acid, such as oleic acid, linoleic acid or linolenic acid.[12,68,77]

401

In this connection, the oil solubility of plant sterols has been greatly improved by

402

chemical or enzymatic esterification with fatty acids or transesterification with fatty

403

acid esters. At present, plant sterol or stanol fatty acid esters as functional

404

ingredients have been widely used in many foods, such as margarine, butter, dairy

405

products, mayonnaise, and salad dressings.

406 407

Enhancing the water solubility of plant sterols.

408

Insolubility of plant sterols in water limits their application in foods of aqueous

409

medium. Despite a large number of studies have focused on improving the oil

410

solubility of plant sterols, research on chemical modification is needed to improve

411

the water solubility or hydrophilic property of plant sterols. The chemical structures

412

of some hydrophilic plant sterol derivatives are shown in Figure 6. Ramaswamy et al. 15



413

synthesized a novel hydrophilic phytostanol analogue (disodium ascorbyl

414

phytostanol phosphates) by chemical modification with ascorbic acid.[107] Hossen et

415

al. synthesized a new phosphatidyl derivative of plant sterols (phosphatidyl-

416

sitosterols) using phosphatidyl choline as a modifier via an enzymatic process

417

catalyzed by phospholipase D.[108] Pang et al. investigated the feasibility of

418

esterification of plant sterols with L-glutamic acid, finding the esterification could

419

reach 92% under optimum conditions.[109] Yuan et al. synthesized ten β-sitosterol

420

esters using N-phosphoryl amino acids as an acyl donor and the yield reached 60-87%

421

by employing dcyclohexylcarbodiimide(DCC)/DMAP as a catalyst system under

422

microwave irradiation.[110] He et al. established an efficient two-step chemo-

423

enzymatic method to synthesize plant stanol hydrophilic derivatives (plant stanol

424

sorbitol succinate) using D-sorbitol as a hydrophilic modifier.[111] Some hydrophilic

425

compounds with a high molecular weight were also used as a hydrophilic modifier,

426

for example, Chung et al. synthesized hydrophilic β-sitosterol derivatives with

427

various degrees of substitution using polyethylene glycol (PEG) with higher molecular

428

weight via a two-step chemical modification in the presence of triethylamine and 4-

429

dimethylaminopyridine.[112] He et al. further synthesized hydrophilic phytosterol

430

derivatives using PEG as a hydrophilic modifier by a chemo-enzymatic route and two-

431

step ionic liquid-catalyzed method. [15, 113] The solubility of plant sterols could be

432

significantly improved by conjunction with PEG 1000. The solubility of hydrophilic

433

phytosterol derivatives in water could reach 7-25 g/100 mL at 30oC.[14,15,112] The

434

water solubility of plant sterols can be improved to some extent by chemical

435

modification, but at present these hydrophilic plant sterols derivatives still cannot be

436

directly applied into food systems due to the lack of extensive safety evaluation.

437

Apart from their application in foods, plant sterols can also be used in

438

pharmaceutical and chemical industry. Klumphu et al. synthesized a β-sitosterol

439

derivative as a surfactant using monomethylated polyethylene glycol by a two-step

440

synthesis.[114] This surfactant can provide a desired micellar condition for transition-

441

metal-catalyzed reactions. Sánchez-Ferrer et al. synthesized a food-grade glucose-β-

442

sitosterol conjugate with intention to use it for constructing edible supramolecular

443

chiral nanostructure by its amphiphilic behavior. [115] Wang et al. synthesized a folate 16


Page 16 of 54

Page 17 of 54


444

mediated self-assembled phytosterol-alginate nanoparticle (FPA NPs) using plant

445

sterols as a hydrophobic moiety. The self-assembled FPA NPs could efficiently

446

encapsulate a highly hydrophobic anticancer drug (DOX) with a high drug-loading

447

capacity and show strong potential as a promising carrier for drugs to target cancer

448

cells. [116]

449

Chemical Modification with Other Compounds

450

Plant sterols naturally occur in plants as free and conjugated forms. The latter

451

are the esters of fatty acids and phenolic acids, glycosides and acylated glycosides.

452

Most research regarding plant sterols has focused on free sterols and their fatty acid

453

esters. Recently some plant sterol esters of phenolic acids have been identified, such

454

as sterol ferulates in rice, wheat, rye and corn, caffeates in canary seeds, and p-

455

coumaric acid esters of plant sterol in corn. [117] Natural sterol phenolates are present

456

in many plants but in a very small amount. Over the past decade, emerging studies

457

have concentrated on the synthesis plant sterol esters of phenolic acids. The

458

chemical structure of the major sterol phenolates are shown in Figure 7. Plant sterol

459

ferulates, as a major component of γ-oryzanol, have been successfully synthesized.

460

[118, 119]

461

step reactions, (a) preparation of trans-4-O-acetylferulic acid, (b) preparation of 3-O-

462

(trans-4-O-acetylferuloyl)-β-sitostanol by esterification of β-sitostanol with trans-4-

463

O-acetylferulic acid in the presence of DMAP and DCC, and (c) preparation of β-

464

sitostanol ferulate by selective deacetylation. The final yield of β-sitostanol ferulate

465

could reach 60%.[118] Tan et al. synthesized phytosterol ferulate by two-step

466

chemoenzymatic reactions: (a) chemical synthesis of vinyl ferulate and (b)

467

preparation of phytosterol ferulates by lipase-catalyzed transesterification of

468

phytosterols with vinyl ferulate. [119] In a similar way, plant sterol caffeates, sinapates

469

and vanillates were also synthesized via a chemo-enzymatic route by Tan et al. [120,121]

470

The synthetic plant sterol esters (ferulate, caffeate, sinapate and vanillate) of

471

phenolic acid exhibited a higher antioxidant activity than their corresponding free

472

plant sterols and phenolic acid tested in different models. Fu et al. prepared

473

phytosterol gallate through a mild chemical Steglich esterification reaction and the

474

product showed excellent antioxidant activity.[122] Wang et al. synthesized a series of

Condo et al. developed a synthetic process of β-sitostanol ferulate by three-

17



Page 18 of 54

475

plant sterol esters of phenolic acids including 4-hydroxybenzoic acid, vanillic acid, 4-

476

chlorophenylacetic acid, hydrocinnamic acid, 4-phenylbutyric acid, 5-phenylvaleric

477

acid, cinnamic acid, m-coumaric acid, ferulic acid and 3,4-dimethoxycinnamic acid

478

and tested their antioxidant activity, demonstrating these esters with 4-

479

hydroxybenzoate, vanillate and ferulate of plant sterols showed the potential for use

480

as food antioxidants. [123] Schär et al. recently developed a fully enzymatic procedure

481

for the synthesis of sterol phenolates[117,124] and evaluated their anti-oxidant capacity

482

in different systems. [117] Candida rugosa lipase was used to synthesize β-sitosterol

483

ferulates via direct esterification with ferulic acid as well as transesterification with

484

yields of 35 and 55%, respectively. [124] Lipase-catalyzed transestrification has also

485

been used for the synthesis of other sterol esters of hydroxycinnamic acid (sinapic

486

acid, m-coumaric acid, o-coumaric acid, p-coumaric acid, caffeic acid and phloretic

487

acid) using Candida rugosa lipase. [117] In addition to phenolic acids, lipoic acid and

488

dihydrogen lipoic acid also showed a good anti-oxidant capacity. To provide better

489

physiological functions, plant sterol esters of lipoic acid and dihydrogen lipoic acid

490

were also synthesized. [125,126] Madawala et al. synthesized phytosterol lipoate and

491

phytosterol dihydrolipoate using a chemical method in the presence of DMAP and 1-

492

ethyl-3-(3-dimethylaminopropyl)-carbodiimide

493

phytosterol dihydrolipoate displayed a better scavenging capacity of 1,1-diphenyl-2-

494

picrylhydrazyl (DPPH) free radical. Wang et al. developed an enzymatic route to

495

synthesize phytosterol lipoate with a conversion of 71% using Candida rugosa lipase

496

as a catalyst under optimal conditions for 96 h.

497

phytosterol have been greatly improved by conjugating with lipoic acid. Wang et al.

498

also investigated the oil solubility of phytosterols, their lipoate and ferulate

499

derivatives,

500

increase from 9.1 g/L of free sterol to 20.4 g/L and 22.5 g/L by esterification with

501

lipoic acid and ferulic acid, respectively.

[126]

hydrochloride.[125]

[126]

By

contrast,

The antioxidant ability of

finding that the solubility of phytosterol in rapeseed oil could

502 503 504

CHOLESTEROL-LOWERING ACTIVITY OF PLANT STEROLS/STANOLS, THEIR ESTERS AND OTHER CHOLESTEROL ANALOGS

505 18


Page 19 of 54


506

Hypercholesterolemia is one of the major risk factors for coronary heart

507

disease (CHD). Management of blood TC and LDL-C levels by cholesterol-lowering

508

nutraceuticals could slow the progression of atherosclerosis and reduce the risk of

509

CHD.[127] Phytosterols, comprising plant sterols and plant stanols as a healthy

510

supplement, have been widely used to treat hypercholesterolemia. The role of

511

phytosterols in lowering TC and LDL-C has been well recognized by various health

512

authorities worldwide, including EU, the US, Canada, and Australia/New Zealand.[128]

513

For instance, the European Foods Safety Authority (EFSA) recommends to consume

514

1.5-2.4 g/day of phytosterols in order to decrease blood cholesterol.[129] US Food and

515

Drug Administration (FDA) has approved the use of phytosterol esters into a low

516

saturated fat and cholesterol diet in reducing the risk of coronary heart disease.[130] It

517

has been suggested that the daily consumption of 2 g of phytosterols can effectively

518

lower plasma cholesterol by 9-14% in humans with little or no effect on high density

519

lipoprotein cholesterol and triacylglycerol levels. [131]

520 521

Cholesterol-Lowering Activity of Plant Sterols/Stanols

522

Plant sterols were first demonstrated as a therapeutic agent to treat the

523

hypercholesterolemia in humans in 1953. [132] Since that time, more than 200 clinical

524

studies have been conducted and most results have proven that plant sterols are

525

effective in reducing plasma total cholesterol and LDL-C. [133] Plant stanols with no

526

double bond are the saturated forms of plant sterols. Saturation of β-sitosterol and

527

campesterol gives rise to β-sitostanol and campestanol, respectively (Figure 1).[134]

528

β-Sitostanol and campestanol are found in nature in much smaller amounts than

529

plant sterols. Since the first description of the use of plant stanols to lower plasma

530

cholesterol by Heinemann et al. in 1986, plant stanols have been widely used in

531

clinical treatment for reducing coronary heart disease incidence.[135]

532

The minor structural differences make plant sterols and stanols different from

533

each other functionally and metabolically. Plant sterols are poorly absorbed in the

534

intestine (0.4-3.5%), while absorption of plant stanols (0.02-0.3%) is even lower.[136]

535

The saturation of double bond increases hydrophobicity and decreases micellar

536

solubility of stanols. This explains why plant stanols are less absorbed than plant 19



Page 20 of 54

537

sterols. It has been shown that consumption of stanols-containing mixtures is more

538

effective in reducing circulating cholesterol concentrations than that of sterols.[137, 138]

539

However, recent reports have demonstrated that plant sterols and stanols equally

540

effectively reduce serum LDL-C and atherosclerotic risk.[139,

541

phytostanol consumption not only decreases the LDL-C concentration, but also

542

reduces serum plant sterols dose-dependently. [141] A great number of extensive

543

safety evaluation studies have been conducted for plant stanols/sterols. Up to now,

544

there has been no evidence showing a moderate consumption of plant

545

sterols/stanols in the general population is associated with any increase in the risk of

546

cardiovascular diseases, except for individuals with phytosterolemia, an inherited

547

lipid disorder. [142,143]

140]

Interestingly,

548

A recent meta-analyses has demonstrated that plant sterol/stanol can decrease

549

LDL-C by up to 12% if their intake is 3 g/day.[18] However, the exact mechanisms by

550

which plant sterols/stanols reduce plasma LDL-C are still being under investigation. It

551

is well established that phytosterols act as a cholesterol absorption inhibitor via

552

displacing cholesterol from bile emulsion in the intestine, thus leading to reduction

553

in cholesterol absorption and plasma TC.[144,145] HMG-CoA reductase is a rate-limiting

554

enzyme in cholesterol synthesis cascade. β-Sitosterol has been shown to inhibit the

555

cholesterol synthesis by inhibiting HMG-CoA reductase gene expression in CaCo-2

556

cells.[146] Cholesterol 7 alpha-hydroxylase (CYP7A1), which convers cholesterol to bile

557

acids, its gene expression is up-regulated by plant sterol mixture in WKY and Wistar

558

rats, resulting in more fecal sterol excretion and less cholesterol accumulation in

559

blood vessels. [147] The research conducted by Yang et al. also found that stigmasterol

560

could inhibit sterol regulatory element-binding protein 2 (SREBP-2) processing and

561

reduce the cholesterol synthesis in cultured adrenal cells from ABCG 5/8 knockout

562

mice. [148]

563 564

Cholesterol-Lowering Activity of Plant Sterol/Stanol Esters

565

The low solubility of free plant sterols and plant stanols limits their usage in

566

functional foods. Plant sterol and stanol esters have a better solubility or

567

incorporation into various foods without changing the taste and texture. Margarines, 20


Page 21 of 54


568

yogurt, cream cheese spreads and cereal bars containing either plant stanol or sterol

569

esters have been marketed in many countries. In these products, plant sterols and

570

stanols are usually esterified with long chain fatty acids in order to increase their fat

571

solubility from 2% to 20%.[149]

572

The active forms of plant sterol/stanol esters are the free sterols/ stanols as they

573

are hydrolyzed to their corresponding free sterols/stanols and fatty acids in the

574

intestine.[150] It has been proven that plant stanol esters at a level of 2–3 g/d can

575

reduce LDL-C by 10–15% without any side effects.[151] The LDL-lowering efficacy of

576

stanol esters might be influenced by doses, frequency of administration, food vehicle

577

in which the stanol esters are incorporated, and background diet.[152] In addition, a

578

daily intake of 1.6 g sterol esters induces an additional reduction in LDL-C

579

concentrations in children with familial hypercholesterolemia consuming a

580

recommended diet [153] Hallikainen et al. compared the relative cholesterol-lowering

581

activity of plant sterol esters and plant stanol ester-enriched margarines in

582

hypercholesterolemic subjects on a low-fat diet, finding that margarines contain

583

plant sterol esters and plant stanol esters are equally effective in lowering plasma TC

584

and LDL-C.[154] It appears that the cholesterol absorption is similarly inhibited by

585

plant sterol esters and plant stanol esters.[155]

586

It is inconclusive if the fatty acid moieties affect the cholesterol-lowering activity

587

of the esterified phytosterols. He et al. compared the cholesterol-lowering activity of

588

phytosterol and phytosterol laurate in mice and demonstrated that the lauric acid -

589

esterified phytosterols retained the similar cholesterol-lowering activity as the free

590

phytosterols.[156] Nestel et al. found that phytosterol esters prepared by

591

esterification with fatty acids from soybean oil possessed a slightly but not

592

significantly greater LDL cholesterol reducing activity than the plant stanols.[157] A

593

study by Kobayashi et al. demonstrated that unesterified plant sterol were

594

potentially more effective in inhibiting the cholesterol absorption than plant sterol

595

oleates in rats, but the difference was substantially small.[158]

596

It is also inconclusive whether fatty acid moieties of plant sterol esters affect the

597

cholesterol metabolism. Liu et al. compared the cholesterol-lowering activity of 21



598

sterol esters of sunflower oil and the sterol esters of canola oil, showing that they

599

were equally effective in reducing plasma cholesterol in a dose-dependent manner

600

regardless the significant differences in their fatty acid compositions. [159] However,

601

the research conducted by Rasmussen et al. demonstrated that plant sterol esters

602

made with beef tallow and pure stearic acid were more effective than plant sterol

603

esters made with soybean oil in reducing the cholesterol absorption, liver cholesterol,

604

and plasma non-HDL cholesterol concentration.[160] It was also shown that the sterol

605

esters with fish oil had an greater influence on LDL-cholesterol concentrations

606

compared with the esters with vegetable oil. [161, 162] Additionally, the sterol ester

607

enriched with n-3 fatty acids from fish oil not only retained plasma cholesterol-

608

lowering activity of plant sterol, but also retained plasma triacylglycerol-lowering

609

and eicosanoid-modifying properties of the fish oil.[163]

610

Apart from fat-soluble phytosterol esters, some researchers have recently

611

studied the cholesterol-lowering activity of water-soluble or hydrophilic phytosterol

612

derivatives. A study by Wasan et al. found that hydrophilic phytostanol analog, FM-

613

VP4, decreased total and LDL cholesterol concentrations in gerbils.[164] A water-

614

soluble phytostanol analog, disodium ascorbyl phytostanyl phosphates (DAPP), had

615

been demonstrated to reduce plasma cholesterol more efficiently than free stanols

616

in hamsters.[165] He et al. successfully synthesized a hydrophilic phytostanol

617

derivative, phytostanol sorbitol succinate (PSS), via a chemical-enzymatic route, and

618

investigated their cholesterol-lowering activity in mice, demonstrating that PSS

619

possessed a hypocholesterolemic activity.[166] Chung et al. compared the effect of β-

620

sitosterol, its hydrophilic derivative (HPSS) and lipophilic derivative (LPSS) on blood

621

cholesterol concentration in rats and demonstrated that LPSS and HPSS had a

622

comparable activity with β-sitosterol in lowering blood cholesterol. [167]

623

Cholesterol-Lowering Activity of Other Cholesterol Analogs

624

Compounds with a similar structure generally have a similar biological activity.

625

Based on the structures of cholesterol and phytosterols, different cholesterol

626

analogs were designed to test their cholesterol-lowering activity. Wang et al. blocked

627

the hydroxyl group on β-sitosterol by methylating and ethylating to form β-sitostery 22


Page 22 of 54

Page 23 of 54


628

3 β-methoxy (SM) and β-sitostery 3β-ethoxy (SE) derivatives (Figure 8) and

629

investigated their effect on plasma lipoprotein profile in hypercholesterolemia

630

hamsters.[56] Results showed that β-sitosterol remarkably inhibited the cholesterol

631

absorption, while 3β-methoxy and β-sitostery 3β-ethoxy had no or little effect on

632

apparent cholesterol absorption, suggesting that the hydroxyl group was essential

633

for β-sitosterol to retain its cholesterol-lowering activity.[56] Similar with above

634

research, Lei et al. synthesized other two cholesterol analogs by blocking the

635

hydroxyl group on cholesterol with methyl and ethyl group, respectively, by

636

producing cholesteryl 3β-methoxy (CM) and cholesteryl 3β-ethoxy (CE) (Figure 8),

637

and then compared their effects on plasma cholesterol with that β-sitosterol.[168]

638

Results proved that β-sitosterol was effective in reducing plasma cholesterol, while

639

both cholesteryl 3β-methoxy and cholesteryl 3β-ethoxy had no cholesterol-lowering

640

activity.[168]

641

hypocholesterolemic sterols shall focus on the analogs having a different side chain

642

rather than these analogs having derivations on the ring. Dihydrocholesterol, also

643

called 5α-cholesterol, is a cholesterol analog. It has a same side chain as cholesterol,

644

but it has no double bond at the ∆5 position in B-ring. In 1953, Siperstein et al.[169]

645

had demonstrated that dihydrocholesterol could prevent the rise in plasma

646

cholesterol and atherosclerosis in cholesterol-fed chicken, while Nichols et al.[170] had

647

shown that dihydrocholesterol could decrease blood cholesterol in rabbits. The

648

recent research conducted by Wang et al. demonstrated that dihydrocholesterol was

649

effective in reducing plasma total cholesterol in hypocholesterolemia hamsters

650

comparable to that of β-sitosterol at a dose of 0.2%,[171] however, its application and

651

safety in management of hypercholesterolemia in humans remain largely unknown.

652

FUTURE PERSPECTIVES

[151]

Therefore, it is concluded that screening the potential

653

Plant sterol and stanol fatty acid esters have been produced on a large scale

654

by esterification or transesterification in food industries. Plant sterol and stanol fatty

655

acid esters can be easily incorporated into various fat- or oil-based foods. However,

656

production of plant sterol and stanol derivatives with a better water solubility or

657

hydrophilic property on a large scale is lacking in food industry. Future investigation 23



658

in a pilot or an industrial scale is in need to develop a highly-efficient process of

659

producing the hydrophilic plant sterol derivatives of food grade without

660

compromising their cholesterol-lowering activity. More efforts are also in need to

661

develop a process of synthesizing plant sterol and stanol derivatives conjugated with

662

phenolic acids or the other biological compounds so that these derivatives possess

663

not only plasma cholesterol-lowering activity but also other biological functions.

664 665

Acknowledgement

666

This study was financially supported by a grant from the Health and Medical

667

Research Fund, the Government of the Hong Kong Special Administrative Region,

668

China (13140111), the National Natural Science Foundation of China (31401664),

669

Hong Kong Scholars Program (XJ2017019), the China Postdoctoral Science

670

Foundation Funded Project (2014M560406), the Research Fund for Advanced

671

Talents of Jiangsu University (13JDG070) and a project funded by the Priority Academic

672

Program Development of Jiangsu Higher Education Institutions (PAPD).

673

Notes

674

The authors declare no competing financial interest.

24


Page 24 of 54

Page 25 of 54


675

REFERENCES

676

1

sterols in plants. Prog. Lipid Res. 2017, 67, 27–37.

677 678

Ferrer, A.; Altabella, T.; Arró, M.; Boronat, A. Emerging roles for conjugated

2

Brufau, G.; Canela, M. A.; Rafecas, M. Phytosterols: physiologic and metabolic

679

aspects related to cholesterol-lowering properties. Nutr. Res. 2008, 28(4), 217–

680

225.

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improve the lipid profile of dyslipidemic subjects more than do fish-oil or

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sunflower oil esters of plant sterols. Amer. J. Clin. Nutr. 2006, 84(6), 1534-1542.

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ventricle generation of thromboxane A2 in adult guinea pigs. J. Nutr. 2002,

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1167

cholesterol-lowering activity. Food Funct. 2015, 6(2), 630–634.

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169 Siperstein, M.; Nichols, C.; Chaikoff, I. Prevention of plasma cholesterol

1169

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1170

dihydrocholesterol. Circulation 1953, 7(1), 37–41.

1171

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1172

administration on plasma cholesterol and atherosclerosis in the rabbit. Exp. Biol.

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1176

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1177 1178

41



1179

Figure Captions

1180

Figure 1

1181

Chemical structure of different origin sterols.

1182 1183

Figure 2.

1184

Synthesis of plant sterol esters by esterification with free fatty acids, or

1185

transesterification with fatty acid esters, or triacylglycerols in the presence of acid

1186

catalyst, base catalyst or lipase.

1187 1188

Figure 3.

1189

General reaction mechanisms of acid-catalyzed synthesis of plant sterol esters by

1190

esterification and transesterification.

1191 1192

Figure 4

1193

General reaction mechanism of base-catalyzed synthesis of plant sterol esters by

1194

transesterification.

1195 1196

Figure 5

1197

Action mechanism of Candida rugosa lipase-catalyzed synthesis of plant sterol esters

1198

by esterification.

1199 1200

Figure 6

1201

Chemical structures of hydrophilic β-sitosterol derivatives.

1202 1203

Figure 7

1204

Chemical structures of β-sitosterol phenolates.

1205 1206

Figure 8

1207

Chemical structures of dihydrocholesterol, cholesteryl 3β-methoxy, cholesteryl 3β-

1208

ethoxy, β-sitostery 3β-methoxy and β-sitostery 3β-ethoxy.

1209 42


Page 42 of 54

Page 43 of 54


Table 1. A summary of recent publications on the lipase-catalyzed synthesis and production of plant sterol fatty acid esters Enzyme

Substrate Phytosterols and caprylic acid Phytosterols and lauric acid Phytosterols and lauric acid Phytosterols and oleic acid

Candida rugosa lipase

Canola phytosterols and oleic acid

Phytosterols and oleic acid Phytosterols from SODD and conjugated linoleic acid Phytosterols and fatty acids from butter fat Cotton seed oil deodorizer distillate

Phytosterols and fatty acids from Ahiflower™ seed oils Phytosterols and ethyl linolenate

Phytosterols and Lauric acid

Phytosterols and fatty acids from butter fat Phytosterols and oleic acid Phytosterols and oleic acid Immobilized Candida rugosa lipase

Sitostanol and oleic acid

Phytosterols and oleic acid, linoleic acid, linolenic acid Phytosterols and linolenic acid Phytosterols and pinolenic acid from pine nut oil Phytosterols and algae oil, camellia oil, rapeseed oil, linseed oil, sunflower oil Sitostanol and methyl oleate Sitostanol and triolein

Canola phytosterols and oleic acid Phytostanols and lauric acid Candida antarctica lipase B (Novozyme 435)

Phytosterols and oleic acid Sitostanol and oleic acid Sitostanol and methyl oleate Phytosterols and soybean oil

β-sitosterol and stearic acid β-sitosterol and stearic acid

Candida antarctica lipase A

β-sitosterol and myristic acid

Candida sp. 99-

Phytosterols and mixed fatty acids from

Reaction parameters n-hexane, 45 oC, 2.15:1 A/S, 7.9% lipase, 9.2 h n-hexane, 45 oC, 3:1 A/S, 10% lipase, 48 h, Na2SO4/Na2SO4·10H2O water-in-[Bmim]PF6, 50 oC, 2:1 A/S, 10% lipase, 48 h, pH 7.4 n-hexane, 51.3oC, 2.1:1 A/S, 7.2% lipase, 17 h n-hexane, 35oC, 3:1 A/S, 5% lipase, 72 h 30 oC, 5:1 A/S, 24 h 40 oC, 1.2:1 A/S,10% lipase, 120 h, 100 mbar, stepwise addition 50 oC, 1.1:1 A/S,10% lipase, 48 h, 100 mbar, stepwise addition n-heptane, 44.2oC, 3:1 A/S, 0.84% lipase, 12 h Cyclohexane, 30oC, 1:1 A/S, 10% lipase, 6 h Isooctane, 40 oC, 1.75:1 A/S, 20 g/L lipase, 2 h n-hexane, 40 oC, 2:1 A/S, 7.5% lipase, 10 h 50 oC, 1.1:1 A/S,10% lipase, 9 h, 100 mbar, stepwise addition Isooctane, 50 oC, 2:1 A/S, 2 g/L lipase, 1 h, microwave irradiation, aw 0.11 Isooctane, 50oC, 2:1 A/S, 50 mg lipase, 6 h, aw 0.11 40 oC, 3:1 A/S, 20% lipase, 2 h, 20-40 mbar 55 oC, 5:1 A/S, 10% lipase, 15 min Isooctane,55 oC, 1.5:1 A/S, 20 g/L lipase, 6-15 h 60 oC, 4:1 A/S, 10% lipase, 1.5h, 80kPa 55 oC, 5:1 A/S, 10% lipase, 15 min 40 oC, 3:1 A/S, 20% lipase, 8 h, 20-40 mbar 40 oC, 3:2 A/S, 20% lipase, 8 h, 20-40 mbar n-hexane, 55oC, 1:1 A/S, 5% lipase, 24 h n-hexane, 55oC, 4:1 A/S, 40g/L lipase, 96h n-hexane, 45 oC, 1:1 A/S,140% lipase, 24 h 80oC, 3:1 A/S, 20-40mbar, 50 mg, 48h 80oC, 3:1 A/S, 20-40mbar, 50 mg, 48h SC-CO2, 85 oC, 1% lipase, 1h, 1MPa

Limonene, 90oC, 24h 90oC, 24h n-hexane, 40-50 oC, 1:1 A/S, 510% lipase, 24h Isooctane, 60 oC, 1.5:1 A/S,

43


Conversion (%)

References

98

[66]

74.9

[68]

95.1

[69]

97

[70]

85

[63]

80

[72]

85

[78]

94

[80]

90.8

[88]

81

[81]

95.9

[12]

96.6

[67]

99

[80]

95.4

[71]

96.5

[73]

96.8

[64]

90.9-95.3

[74]

92.1-95.3

[75-77]

93

[79]

92.1-96.3

[74]

74.3

[64]

95.1

[64]

16.1

[63]

79.3

[65]

34.6

[85]

88.7

[96]

96.8

[96]

92

[97]

75 88

[91] [91]

93-98

[82]

90.1

[83]


125

sunflower oil

Phytosterols and rapeseed oil

Immobilized Candida sp. 99– 125

Phytosterols and oleic acid

β-Sitosterol and linseed oil Lipozyme TL IM

β-Sitosterol and fish oil Sitostanol and methyl oleate

Sitostanol and oleic acid

Phytosterols and fatty acids from linseed oil Lipozyme RM IM

Sitostanol and methyl oleate Sitostanol and methyl oleate Sitostanol and triolein

20g/L lipase, 8h, ultrasonic pretreatment (35kHz, 200W, 1h), Isooctane, 60 oC, 1.5:1 A/S, 20g/L lipase, 10h, ultrasonic pretreatment (35kHz, 200W, 1h), Isooctane, 45 oC, 1:1 A/S, 140% lipase, 24 h n-hexane, 60 oC, 1:2 A/S, 10% lipase, 24 h n-hexane, 60 oC, 1:2 A/S, 10% lipase, 24h 80 oC, 3:1 A/S, 38% lipase, 24 h, 20-40 hPa

80 oC, 3:1 A/S, 48h, 20-40mbar, 50mg Ethyl acetate, 50 oC,1:1 A/S, 10% lipase, 24 h 80 oC, 3:1 A/S, 38% lipase, 48 h, 20-40 hPa 80 oC, 3:1 A/S, 24h, 20-40mbar, 50mg 80 oC, 3:2 A/S, 48h, 20-40mbar, 50mg

Page 44 of 54

93.5

[83]

93.4

[85]

70

[86]

57

[86]

95

[95]

63.8

[96]

79

[98]

83.4

[95]

93.2

[96]

95.7

[96]

AYS from Candida rugosa

β-Sitosterol and conjugated linoleic acid

n-hexane, 55oC, 3:1 A/S, 15% lipase,48 h

26.8-28.3

[99]

Immobilized Chirazyme L-2 C2

β-sitostero and conjugated linoleic acid

n-hexane,50oC, 1:1 A/S, 20 g/L lipase, 72 h

72.6

[100]

Chirazyme L-1

Sitostanol and caprylic acid/palmitic acid,

SC-CO2, 50oC, 27.6 Mpa,

92-99

[92]

Immobilized Ylip2

Phytosterols and oleic acid

n-hexane,50oC, 3:1 A/S, 10000 U lipase/g substrate, 78h, aw 0.15

91.1

[87]

Pseudomonas lipase

Sitosterol and PUFA

30% water, 40oC, 3:1 A/S, 3000 U/g lipase,24 h

85.3-92.7

[101]

Cholesterol esterase from Trichoderma sp. AS59

Stigmasterol and stearic acid

n-hexane, 20oC, 2:1 A/S, 200 U lipase, 19 h

96

[102]

LPL 311

Phytosterols and DHA

n-hexane, 45oC, 2:1 A/S, 9% lipase, 24h

96

[13]

Phytosterols and sunflower oil, caprylic acid

Room temperature, 12:1 A/S, 18% lipase, 120h

92.1

[103]

β-sitostero and lauric acid

Toluene, 35oC, 2:1 A/S, 160U/g, 168h

85

[90]

Sterol esterase from Ophiostoma piceae

β-Sitostanol and lauric acid/oleic acid

Isooctane, 28oC, 1:1 A/S, 3U/mL,3 h

90

[89]

Lipase TL from Pseudomonas stutzeri PL-836

Wood sterols and fatty acid methyl esters from sunflower oil

60oC, 10% lipase, 8 h, 2mbar

95

[93]

Lipase QLM (Alcaligenes sp.)

Phytosterols and sunflower oil

100oC, 9:1 A/S, 1% lipase, 7 h

97.1

[94]

Aspergillus oryzae NRRL 6270 Sterol Esterases from Aspergillus Strains

A/S, molar ratio of acyl donor to sterols; aw, water activity; PUFA, Polyunsaturated Fatty Acids. 44


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Table 2. Melting points and oil solubility of free and esterified plant sterols

Oil

Melting Compounds

point

Ref

solubility (g/100

o

( C)

Free sterols/stanols

Esterified sterols

mL)

[41,68,105,

Plant sterols

137-145

β-sitosterol

136-140

[72]

Stigmasterol

164-168

[104]

Caproate

58.7

[99]

Caprylate

66.8

[99]

Caprate

70.2

[99]

1-3

106]

81.3-

Laurate

Ref

85.8

[12,41,42,105, 68]

[41,99]

4.28

[68]

Oleate

25.6-28

[72,77]

32.1

[77]

Linoleate

12.5

[77]

36.2

[77]

CLA ester

15.3

[99]

Linolenate

2.3

[77]

35.8

[12,77]

PUFA esters

6.1

[42]

32.5-34.6

[42]

CLA, conjugated linoleic acids; PUFA, polyunsaturated fatty acids.

45



(a) Animal origin

(b) Plant origin

(c) Fungi origin

Figure 1. Chemical structure of different origin sterols.

46


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Figure 2

Figure 2. Synthesis of plant sterol esters by esterification with free fatty acids, or transesterification with fatty acid esters, or triacylglycerols in the presence of acid catalyst, base catalyst or lipase.

47



Figure 3. General reaction mechanisms of acid-catalyzed synthesis of plant sterol esters by esterification and transesterification.

48


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Figure 4

Figure 4 . General reaction mechanism of base-catalyzed synthesis of plant sterol esters by transesterification.

49



Figure 5. Action mechanism of Candida rugosa lipase-catalyzed synthesis of plant sterol esters by esterification.

50


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Figure 6. Chemical structures of hydrophilic β-sitosterol derivatives.

51



Figure 7 . Chemical structures of β-sitosterol phenolates.

52


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Figure 8. Chemical structures of dihydrocholesterol, cholesteryl 3β-methoxy, cholesteryl 3β-ethoxy, β-sitostery 3β-methoxy and βsitostery 3β-ethoxy.

53



TOC graphic

1210 54


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Plant Sterols: Chemical and Enzymatic Structural Modifications and

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