Plant Sterols: Chemical and Enzymatic Structural ... - ACS Publications

Review Cite This: J. Agric. Food Chem. 2018, 66, 3047−3062

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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*,‡ †

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

‡

ABSTRACT: Plant sterols have attracted increasing attention due to their excellent cholesterol-lowering activity. However, free plant sterols have some characteristics of low oil solubility, water insolubility, high melting point, and low bioavailability, which greatly limit their application in foods. Numerous studies have been undertaken to modify their chemical structures to improve their chemical and physical properties in meeting the needs of various applications. The present review is to summarize the literature and update the progress on structural modifications of plant sterols in the following aspects: (i) synthesis of plant sterol esters by esterification and transesterification with hydrophobic fatty acids and triacylglycerols to improve their oil solubility, (ii) synthesis of plant sterol derivatives by coupling with various hydrophilic moieties to enhance their water solubility, and (iii) mechanisms by which plant sterols reduce plasma cholesterol and the effect of structural modifications on plasma cholesterollowering activity of plant sterols. KEYWORDS: plant sterols, phytosterol esters, chemical modification, cholesterol-lowering activity, cholesterol

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INTRODUCTION Sterols are essential components of eukaryotic cell membranes and play an important role in regulating the physicochemical properties of cell membranes.1 They are mainly classified into phytosterols (plant origin), zoosterols (animal origin), and mycosterols or ergosterol (fungal origin). All types of sterols share a structure of similar chemical backbone and differ in the number and position of double bonds or lengths of side carbon chains (Figure 1). So far, more than 250 plant sterol species have been identified in various plants and marine organisms. On the basis of the difference in the number of methyl groups on carbon-4, plant sterols are further divided into 4,4-dimethyl sterols, 4α-monomethyl sterols, and 4-desmethyl sterols. In nature, 4,4-dimethyl sterols and 4α-monomethyl sterols are quantitatively minor, whereas 4-desmethyl sterols are quantitatively abundant.2 In this regard, the most frequently referred plant sterols are 4-desmethyl sterols, mainly including βsitosterol, campesterol, stigmasterol, and brassicasterol (Figure 1). These plant sterols exist in the following five forms: free sterols, fatty acid esters, hydroxycinnamic acid esters, sterol glycosides, and acylated sterol glycosides.3 In the industrial process, plant sterols are primarily obtained from the deodorizer distillates produced during the refining process of vegetable oil production or from tall oil, a byproduct of the pulping of pine wood.4 Plant sterols have been shown to be effective in decreasing blood total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) by inhibiting intestinal cholesterol absorption in both humans and animals.5 In addition, they also exhibit various other health benefits including anticancer,6 anti-inflammation,7 antioxidation,8 neuroprotection,9 and cardiovascular protection.10,11 As a functional ingredient, plant sterols have been widely used in foods, medicines, cosmetics, nutraceuticals, and other applications. On one hand, plant sterols, commonly in the form of crystalline powder, have some © 2018 American Chemical Society

characteristics of low oil solubility and high melting point, which limit their application in foods of hydrophobic matrix. On the other hand, the water insolubility of plant sterols hinders their application in foods of hydrophilic matrix. For the needs of various applications to be met, numerous studies on chemical modifications have been undertaken to improve the oil or water solubility of plant sterols.12−15 More than 1400 scientific articles published in the last two decades can be retrieved from the Web of Science using the terms “plant sterols” and “phytosterols”. Among these publications, there are more than 150 reviews, most of which have addressed the health benefits of plant sterols regarding their plasma lipid reduction,16−19 antitumor,20,21 anti-inflammation,22,23 synergistic interaction with other active ingredients,24,25 and potential protection against cardiovascular diseases,10,26 dementia27 and central nervous system disorders.28 The other review articles have focused on other aspects of plant sterols including their production,29,30 diversity,31 analysis,32 oxidized phytosterols,33−35 conjugated sterols,1 and related derivatives.36−38 However, a thorough review on structural modification of plant sterols and effect of chemical structural modifications on the biological activity of plant sterols is lacking. The present review summarizes the process and the reaction mechanism of various chemical modifications of plant sterols and briefs the effect of chemical modifications of plant sterols on their cholesterol-lowering activity. Received: Revised: Accepted: Published: 3047

January 4, 2018 March 8, 2018 March 9, 2018 March 9, 2018 DOI: 10.1021/acs.jafc.8b00059 J. Agric. Food Chem. 2018, 66, 3047−3062

Review

Journal of Agricultural and Food Chemistry

the basic physical and chemical properties of original plant sterols. The C-3 hydroxyl group is the major functional group of plant sterols. The chemical modifications of plant sterols mainly occur at the C-3 position including (1) esterification with hydrophobic fatty acids to improve their oil solubility and (2) coupling with hydrophilic components to enhance their water solubility. Improving the Oil Solubility of Plant Sterols. Plant sterols have a relatively high melting point (usually at least 135 °C) and a poor solubility in vegetable oils, thus hindering their application into fat-based foods. In general, plant sterols are easy to crystallize when they are added directly into food, rendering the food an undesirable texture and seriously affecting the quality of the food. Given that plant sterol fatty acid esters have a higher solubility in fats and oils, numerous studies have concentrated on the synthesis of plant sterol fatty acid esters to improve the oil solubility of plant sterols by enzymatic or nonenzymatic esterification or transesterification (Figure 2). These common acyl donors include the saturated and unsaturated fatty acids, fatty acid methyl or ethyl esters, triacylglycerols originating from different vegetable oils, and fatty acid halogenide or anhydride. Synthesis of Plant Sterol Esters by Acid-Catalyzed Esterification and Transesterification. Plant sterol esters can be synthesized by direct esterification and transesterification in the presence of some acid catalysts. This reaction is a nucleophilic acyl substitution reaction based on the electrophilicity of carbonyl carbon and the nucleophilicity of an alcohol. The general mechanism of acid-catalyzed esterification and transesterification of plant sterols is given in Figure 3. The acid-catalyzed esterification mainly involves the following reactions: (1) a proton transfer from an acid catalyst to carbonyl oxygen of a fatty acid, (2) nucleophilic sterol (ROH) attacks the carbonyl carbon of a fatty acid, forming an oxonium ion, (3) deprotonation of oxonium ion and protonation of another hydroxyl group of activated complex produces a new oxonium ion, (4) the new oxonium ion loses a water molecule, and (5) deprotonation produces a sterol fatty acid ester. The acid-catalyzed transesterification mainly involves the following reactions: (i) protonation of carbonyl oxygen on a triacylglycerol by the acid catalyst, (ii) nucleophilic attack of sterol (ROH) on the carbonyl carbon, forming a tetrahedral intermediate, (iii) proton migration, (iv) breakdown of the intermediate, forming diacylglycerols, and (v) deprotonation and formation of a sterol fatty acid ester. The commonly used acid catalysts include homogeneous acid catalysts (sulfuric acid,

Figure 1. Chemical structures of different-origin sterols.

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CHEMICAL AND ENZYMATIC STRUCTURAL MODIFICATIONS OF PLANT STEROLS Chemical or enzymatic structural modification is to change the chemical structure and molecular weight of plant sterols by introducing one or more substituent groups and thereby alter

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. 3048

DOI: 10.1021/acs.jafc.8b00059 J. Agric. Food Chem. 2018, 66, 3047−3062

Review


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

catalysts include metallic hydroxides and alkoxides as well as other oxides.39,46,47 The general mechanism of base-catalyzed transesterification of plant sterols with a fatty acid methyl ester is given in Figure 4. The base-catalyzed transesterification

toluene-p-sulfonic acid, lewis acid) and heterogeneous acid catalysts (immobilized heteropolyacid).39 Homogeneous acid catalysts have advantages of low cost and high efficiency, but they have some disadvantages of difficult separation and easy corrosion of equipment. Concentrated sulfuric acid is a catalyst used earlier to synthesize plant sterol esters. Using H2SO4 as a catalyst, conversion of plant sterols to their fatty acid esters could achieve 98% by direct esterification with oleic acid.40 However, the side products such as dehydrated sterols could sometimes reach as high as 19%.40,41 To avoid the production of side products, other acid catalysts have been subsequently used for the synthesis of plant sterol esters. Deng et al. optimized the reaction parameters of sodium bisulfate-catalyzed synthesis of plant sterol polyunsaturated fatty acid esters,42 finding that the yield of esterification could reach 96%. Zhou et al. investigated the catalytic activity of several Lewis acids and Lewis acid-surfactant-combined catalysts for the synthesis of phytostanol esters,43 demonstrating that cuprum dodecyl sulfate [Cu(DS)2] displayed the highest catalytic activity with a conversion of 98% when lauric acid was used as an acyl donor.43 When sodium dodecyl sulfate was chosen as a catalyst, the yield of phytostanol laurate could reach 92%.44 Heterogeneous catalysts such as immobilized heteropolyacid have a high catalytic activity and good reusability with minimum pollution. Meng et al. synthesized the food-grade phytosteryl esters using 0.2% tungstosilicic acid in silica gel as a catalyst in a solvent-free system with a good yield of 90%.45 The catalyst could be used at least six times without having a significant loss of catalytic activity. Recently, acidic ionic liquids (ILs) have gained increasing attention due to their excellent catalytic activity for esterification or transesterification. Yang et al. reported a highly efficient process to synthesize the plant sterol esters in the presence of Lewis acid ILs, demonstrating the esterification rate could reach 92% when ChCl·2SnCl2 was used as a catalyst with a 3:1 molar ratio of lauric acid to plant sterols at 150 °C for 4 h.41 Synthesis of Plant Sterol Esters by Base-Catalyzed Transesterification. Plant sterol esters could be synthesized by a chemical reaction of base-catalyzed transesterification of plant sterols with fatty acid esters or triacylglycerols. The base

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

mainly involves the following reactions: (1) reaction of a base (B) with a sterol (ROH) produces an alkoxide (RO) and a protonated catalyst (BH+), (2) nucleophilic attack of the alkoxide (RO) on the carbonyl group of fatty acid ester generates a tetrahedral intermediate, (3) the intermediate breaks down to produce a sterol fatty acid ester and a corresponding anion (R′O), and (4) R′O deprotonates the catalyst (BH+) to generate the active form of base (B) and the corresponding alcohol (R′OH). Base-catalyzed transesterification provides the following advantages including a fast reaction speed, a high conversion rate, a low cost, and a wide availability.48 At the same time, base-catalyzed transesterification suffers some disadvantages including a low selectivity and a high rate of byproducts derived from the formation of soap, 3049


Review


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

polymerization, and oxidation.47 Most importantly, these homogeneous catalysts are corrosive and difficult to separate from the final products and produce the excessive waste.47 Sodium ethylate was patented as a catalyst in 1996 to produce plant sterol esters by transesterification of β-sitostanol with rapeseed oil fatty acid methyl ester.49 Subsequently, numerous heterogeneous catalysts such as metallic oxide were successively used for the synthesis of plant sterol esters to overcome the shortcoming of homogeneous catalyst.39,47 Pouilloux et al. compared the catalytic performance of some solid base catalysts, including Na2CO3, MgO, LiMgO, and ZnO, for the synthesis of plant sterol esters from β-sitosterol and methyl dodecanoate in a solvent-free system, reporting a yield of 78% with 10% stigmasta-3,5-diene as a side product when using MgO as a catalyst.39 Valange et al. evaluated the catalytic ability of different La2O3 oxides and found that La2O3 oxide could inhibit the side reaction of dehydration while the yield of plant sterol esters could achieve 89% and the selectivity was 90−96%.47 Two alumina-supported magnesium oxide (MgO-Al2O3-SG) and alumina-supported zinc oxide (ZnO/ Al2O3) as catalysts were also tested and investigated by Meng et al. and Robles-Manuel et al.40,50 It was found that, compared with traditional homogeneous catalysts, immobilized ZnO had a maximum production of plant sterol esters (98%) after 8 h at lower temperature (170 °C) with a higher selectivity (>90%), better reusability, and lower corrosivity.40 To avoid the use of poisonous catalysts and the production of side products, He et al. developed a process to synthesize plant sterol esters in the absence of catalyst and solvent.46 Under the optimized autocatalyzed reaction conditions, the conversion and yield could reach 99 and 95%, respectively.46 Most of these reactions catalyzed by metallic oxides require a high temperature (>170 °C), and therefore, the side products are inevitably formed. The content of diene in products ranges from 2 to 6.3%, and the level of other side products, such as oxide sterols, are between 3 and 9% when the esterification or transesterification of plant sterols is performed at 230−250 °C.40 There is a indeed a need to develop some mild methods for the synthesis of plant sterol esters. In this regard, JulienDavid et al. utilized 4-dimethylaminopyridine (DMAP) as a catalyst to synthesize the plant sterol ester at room temperature

for 24 h in the presence of dichloromethane and N,N′dicylohexylcarbodiimide, and the yield of plant sterol oleate reached 78%.51 Synthesis of Plant Sterol Esters or Ethers by Other Chemical Reactions. Plant sterol esters could also be synthesized via other acylation reactions. Yang et al. presented an alternative method of synthesizing phytosterol ester from soybean sterol and acetic anhydride in the absence of catalyst and solvent.52 It was found that the esterification rate could reach 99% at 135 °C for 1.5 h. As an effective acyl donor, the fatty acid anhydrides are more reactive than the free acids or the corresponding esters. However, this method appears only suitable for esterification of phytoserols with short-chain fatty acids. Ishida proposed a method to synthesize plant sterol esters from plant sterols and acyl halide using DMAP as a catalyst in dry pyridine.53 In a similar way, Hang produced a series of plant sterol esters with a yield of 79−94%.54 Brown and Hang prepared a phytosterol-octadecyl ether from phytosterol and octadecyl iodide in the presence of sodium hydride.54,55 Similarly, Wang et al. prepared 3β-methoxy and 3β-ethoxy ethers of β-sitosterol from methyl or ethyl iodide and β-sitosterol in the presence of sodium hydride.56 Enzyme-Catalyzed Synthesis of Plant Sterol Esters. In recent years, the enzyme-catalyzed synthesis of plant sterol esters has attracted increasing attention as these methods have advantages of having mild and environmentally friendly reaction conditions, a higher selectivity, and fewer sideproducts. Lipases as a biocatalyst play a crucial role in enzyme-catalyzed esterification. The catalytic activity of lipase is structure-dependent. The X-ray crystallography on the threedimensional structures of lipases has shown that they have an α/β-hydrolase fold, an oxyanion hole, and, in most cases, a “lid” formed by an α-helix that covers the active site.57 Most importantly, almost all lipases have an active site formed by a Ser-His-Asp/Glu triad, which is essential in esterification reactions. The catalytic serine has been found to be located in the same place on the central β-sheet,57 suggesting that the mechanism of the lipase-catalyzed reaction is similar or identical.57 Taking Candida rugosa lipase as an example,57−60 the mechanism of lipase-catalyzed esterification mainly involves the following reactions (Figure 5): (1) The catalytic triad and 3050


Review


Table 1. Summary of Recent Publications on the Lipase-Catalyzed Synthesis and Production of Plant Sterol Fatty Acid Estersa enzyme C. rugosa lipase

substrate phytosterols and caprylic acid phytosterols and lauric acid phytosterols and lauric acid

immobilized C. rugosa lipase

C. antarctica lipase B (Novozyme 435)

C. antarctica lipase A

Candida sp. 99-125

immobilized Candida sp. 99−125 Lipozyme TL IM

phytosterols and oleic acid 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 sitostanol and oleic acid phytosterols and oleic, linoleic, linolenic acids phytosterols and linolenic acid phytosterols and pinolenic acid from pine nut oil phytosterols and algae, camellia, rapeseed, linseed, sunflower oils sitostanol and methyl oleate sitostanol and triolein canola phytosterols and oleic acid phytostanols and lauric acid 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 β-sitosterol and myristic acid phytosterols and mixed fatty acids from sunflower oil phytosterols and rapeseed oil

immobilized Ylip2

phytosterols and oleic acid β-sitosterol and linseed oil β-sitosterol and fish oil sitostanol and methyl oleate sitostanol and oleic acid phytosterols and fatty acids from linseed oil sitostanol and methyl oleate sitostanol and methyl oleate sitostanol and triolein β-sitosterol and conjugated linoleic acid β-sitosterol and conjugated linoleic acid sitostanol and caprylic acid/palmitic acid phytosterols and oleic acid

Pseudomonas lipase

sitosterol and PUFA

Lipozyme RM IM

AYS from C. rugosa immobilized chirazyme L-2 C2 chirazyme L-1

conversion (%)

reaction parameters

ref

n-hexane, 45 °C, 2.15:1 A/S, 7.9% lipase, 9.2 h n-hexane, 45 °C, 3:1 A/S, 10% lipase, 48 h, Na2SO4/ Na2SO4·10H2O water-in-[Bmim]PF6, 50 °C, 2:1 A/S, 10% lipase, 48 h, pH 7.4 n-hexane, 51.3 °C, 2.1:1 A/S, 7.2% lipase, 17 h n-hexane, 35 °C, 3:1 A/S, 5% lipase, 72 h 30 °C, 5:1 A/S, 24 h 40 °C, 1.2:1 A/S, 10% lipase, 120 h, 100 mbar, stepwise addition 50 °C, 1.1:1 A/S,10% lipase, 48 h, 100 mbar, stepwise addition n-heptane, 44.2 °C, 3:1 A/S, 0.84% lipase, 12 h cyclohexane, 30 °C, 1:1 A/S, 10% lipase, 6 h

98 74.9

66 68

95.1

69

97 85 80 85

70 63 72 78

94

80

90.8 81

88 81

isooctane, 40 °C, 1.75:1 A/S, 20 g/L lipase, 2 h n-hexane, 40 °C, 2:1 A/S, 7.5% lipase, 10 h 50 °C, 1.1:1 A/S,10% lipase, 9 h, 100 mbar, stepwise addition isooctane, 50 °C, 2:1 A/S, 2 g/L lipase, 1 h, microwave irradiation, aw 0.11 isooctane, 50 °C, 2:1 A/S, 50 mg lipase, 6 h, aw 0.11 40 °C, 3:1 A/S, 20% lipase, 2 h, 20−40 mbar 55 °C, 5:1 A/S, 10% lipase, 15 min

95.9 96.6 99

12 67 80

95.4

71

96.5 96.8 90.9−95.3

73 64 74

isooctane,55 °C, 1.5:1 A/S, 20 g/L lipase, 6−15 h 60 °C, 4:1 A/S, 10% lipase, 1.5 h, 80 kPa

92.1−95.3 93

75−77 79

55 °C, 5:1 A/S, 10% lipase, 15 min

92.1−96.3

74

74.3 95.1 16.1

64 64 63

79.3 34.6 88.7 96.8 92 75 88 93−98 90.1

65 85 96 96 97 91 91 82 83

93.5

83

93.4 70 57 95 63.8 79

85 86 86 95 96 98

83.4 93.2 95.7 26.8−28.3 72.6 92−99

95 96 96 99 100 92

91.1

87

85.3−92.7

101

40 °C, 3:1 A/S, 20% lipase, 8 h, 20−40 mbar 40 °C, 3:2 A/S, 20% lipase, 8 h, 20−40 mbar n-hexane, 55 °C, 1:1 A/S, 5% lipase, 24 h n-hexane, 55 °C, 4:1 A/S, 40 g/L lipase, 96 h n-hexane, 45 °C, 1:1 A/S,140% lipase, 24 h 80 °C, 3:1 A/S, 20−40 mbar, 50 mg, 48 h 80 °C, 3:1 A/S, 20−40 mbar, 50 mg, 48 h SC-CO2, 85 °C, 1% lipase, 1 h, 1 MPa Limonene, 90 °C, 24 h 90 °C, 24 h n-hexane, 40−50 °C, 1:1 A/S, 5−10% lipase, 24 h isooctane, 60 °C, 1.5:1 A/S, 20 g/L lipase, 8 h, ultrasonic pretreatment (35 kHz, 200 W, 1 h) isooctane, 60 °C, 1.5:1 A/S, 20g/L lipase, 10h, ultrasonic pretreatment (35 kHz, 200W, 1h), isooctane, 45 °C, 1:1 A/S, 140% lipase, 24 h n-hexane, 60 °C, 1:2 A/S, 10% lipase, 24 h n-hexane, 60 °C, 1:2 A/S, 10% lipase, 24 h 80 °C, 3:1 A/S, 38% lipase, 24 h, 20−40 hPa 80 °C, 3:1 A/S, 48 h, 20−40 mbar, 50 mg ethyl acetate, 50 °C, 1:1 A/S, 10% lipase, 24 h 80 °C, 3:1 A/S, 38% lipase, 48 h, 20−40 hPa 80 °C, 3:1 A/S, 24h, 20−40mbar, 50 mg 80 °C, 3:2 A/S, 48h, 20−40mbar, 50 mg n-hexane, 55 °C, 3:1 A/S, 15% lipase,48 h n-hexane,50 °C, 1:1 A/S, 20 g/L lipase, 72 h SC-CO2, 50 °C, 27.6 MPa n-hexane, 50 °C, 3:1 A/S, 10000 U lipase/g substrate, 78 h, aw 0.15 30% water, 40 °C, 3:1 A/S, 3000 U/g lipase, 24 h

3051


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Journal of Agricultural and Food Chemistry Table 1. continued enzyme cholesterol esterase from Trichoderma sp. AS59 LPL 311 Aspergillus oryzae NRRL 6270 sterol esterases from Aspergillus strains sterol esterase from Ophiostoma piceae lipase TL from Pseudomonas stutzeri PL-836 lipase QLM (Alcaligenes sp.) a

substrate

reaction parameters

stigmasterol and stearic acid

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

phytosterols and DHA phytosterols and sunflower oil, caprylic acid β-sitosterol and lauric acid

n-hexane, 45 °C, 2:1 A/S, 9% lipase, 24 h room temperature, 12:1 A/S, 18% lipase, 120 h

conversion (%)

ref

96

102

96 92.1

13 103

toluene, 35 °C, 2:1 A/S, 160U/g, 168 h

85

90

β-sitostanol and lauric acid/oleic acid

isooctane, 28 °C, 1:1 A/S, 3U/mL, 3 h

90

89

wood sterols and fatty acid methyl esters from sunflower oil phytosterols and sunflower oil

60 °C, 10% lipase, 8 h, 2 mbar

95

93

97.1

94

100 °C, 9:1 A/S, 1% lipase, 7 h

A/S, molar ratio of acyl donor to sterols; aw, water activity; PUFA, polyunsaturated fatty acids.

parameters of C. rugosa lipase-catalyzed synthesis of plant sterol oleic acid esters using response surface methodology and had a yield of 97% at 51.3 °C for 17 h.70 Apart from C. rugosa lipase, lipases from C. antarctica and Candida sp. 99−125 have also been widely used to synthesize plant sterol esters. Panpipat et al. used C. antarctica lipase A as a catalyst to synthesize βsitosterol myristic acid ester with a yield up to 98% at 40−50 °C and 5−10% enzyme load for 24 h.82 Zheng et al. produced plant sterol ester of conjugate linoleic acid and obtained a yield of 86% using Candida sp. 99−125 as a biocatalyst for 8 h.83 Although these free lipases have shown a high catalytic activity, there are still some concerns such as a poor stability and a low reusability, which make their application relatively difficult and expensive. Recently, an immobilization technique has been developed with a great success. Compared with free lipase, the immobilized lipases retain their higher catalytic activity and stability in addition to easier separation and good recovery.84 The lipases have been successfully immobilized on synthetic organic polymers, biopolymers, hydrogels, and inorganic supports; for example, Novozyme 435 was immobilized on a macroporous acrylic resin, Lipozyme TL IM was immobilized on silica granulation, C. rugosa lipase was immobilized on magnetic microspheres,76 silica particles,75 hyper-cross-linked polymer-coated silica,77 mesostructured magnetic hollow mesoporous silica microspheres,74 macroporous resin,67,79 and ZnO nanowires/macroporous SiO2.71,73 Torrelo et al. produced phytosterol esters using both free and immobilized C. rugosa lipases as a catalyst in a solvent-free system, finding the former having a yield of 94% and the latter having a yield of 99%.80 In another study, plant sterol esters were produced by a process of ultrafast transesterification between sterols and triacylglycerols with a yield above 90% within 15 min at 55 °C using C. rugosa lipase immobilized on magnetic hollow mesoporous silica microspheres.74 Most important was that the catalytic activity of immobilized lipase was largely retained after more than 50 successive reactions.74 Pan et al. examined the catalytic activity of three immobilized lipases from C. antarctica (Novozyme 435), Thermomyces lanuginosus (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM), and Candida sp. 99−125 lipase, finding Candida sp. 99−125 lipase was the most effective biocatalyst in producing phytosterol oleate with a yield of 93.4% at 45 °C for 24 h.85 The conversion of plant sterols to their corresponding sterol esters is closely related to the type of enzymes. In addition, the conversion is affected by many factors. The reaction temperature not only influences the substrate solubility in solvent but also affects the activity and stability of lipase. In general, higher

serine residue form a tetrahedral intermediate with a fatty acid molecule. (2) A water molecule is released from the intermediate to form an acyl-enzyme complex. (3) A second tetrahedral intermediate of lipase-ester complex is formed with a nucleophilic sterol (ROH). (4) A sterol fatty acid ester is released from the lipase-ester complex.57,59 The conformational changes and kinetic study on lipase have demonstrated that lipase-catalyzed esterification often follows a Ping-Pong Bi−Bi mechanism.61,62 Effect of Different Lipases. Production of plant sterol esters are lipase-dependent. Over the past two decades, there has been more than 40 publications reporting the synthesis of plant sterol fatty acid esters via lipase-catalyzed esterification of sterols with free fatty acids or enzyme-catalyzed transesterification with fatty acid esters or vegetable oils (Table 1). Among these studies, numerous lipases originating from various sources were employed to catalyze plant sterol ester synthesis, and their relative catalytic activities were compared.63−66 Villeneuve et al. evaluated the catalytic ability of several plant and microbial lipases from Carica papaya, Ricinus communis, Rhizomucor miehei, Candida antarctica B, and Candida rugosa, finding that C. rugosa lipase had the highest catalytic activity with a conversion rate of 85% when 5% lipase was used at 35 °C after 72 h.63 Similarly, Weber et al. tested the catalytic efficiency of microbial lipases from Rhizopus arrhizus, C. rugosa, and Chromobacterium viscosum as potential biocatalysts, demonstrating C. rugosa lipase could achieve a 90% conversion rate for esterification of sitostanol with oleic acid under a vacuum of 20−40 mbar.64 In contrast, He et al. investigated the effects of variously sourced lipases on the conversion of plant stanols to phytostanol laurate, showing that microbial lipase Novozym 435 was the best biocatalyst for esterification of plant stanols with fatty acids.65 Among microbial lipases, lipase from C. rugosa is the most frequently used biocatalyst for the synthesis of plant sterol esters. C. rugosa lipase has been widely used for the synthesis of plant sterol esters by direct esterification with caprylic acid,66 lauric acid,67−69 oleic acid,63,64,70−73 linoleic acid,74 linolenic acid,75−77 conjugated linoleic acid,78 pinolenic acid,79 fatty acids from butter fat80 and Ahiflower seed oil81 and transesterification with ethyl linolenate,12 methyl oleate,64 and triolein.64 Miao et al. synthesized plant sterol esters of lauric acid in the presence of free C. rugosa lipase with a yield of 75% at 55 °C after 48 h.68 Villeneuve et al. explored the feasibility of lipase-catalyzed esterification of canola phytosterols with oleic acid using C. rugosa lipase as a catalyst, finding the yield could reach 85% after 72 h.63 Kim et al. optimized the reaction 3052


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lipase B from C. antarctica-catalyzed synthesis of β-sitosterol ester and showed higher initial reaction rates than n-hexane, obtaining 75% conversion.91 King et al. explored the feasibility of lipase-catalyzed esterification between sitostanol and fatty acids in supercritical carbon dioxide and achieved yields of 92 and 99% for caprylic and palmitic acid, respectively, when chirazyme L-1 lipase from Burkholderia cepacia was used as a biocatalyst.92 Enzyme-catalyzed production of plant sterol esters can be operated in a solvent-free system. In fact, lipase-catalyzed synthesis of plant sterol esters conducted in solvent-free system has some advantages including the improvement of food safety, ́ easy operation, and less environmental hazards.74 Martinez et al. demonstrated the feasibility of preparing wood sterol esters in a solvent-free system via lipase-catalyzed transesterification of wood sterols with sunflower fatty acid methyl esters.93 In general, solvent-free synthesis of plant sterol esters often requires a higher reaction temperature to make the substrate fully melted. Negishi et al. prepared plant sterol esters by lipasecatalyzed transesterification at 100 °C using sunflower oil containing 10% plant sterols as substrates.94 Lanctôt et al. reported a solvent-free lipase-catalyzed preparation of βsitosterol esters at 90 °C.91 Most of the reported studies regarding the solvent-free synthesis of plant sterol esters were carried out using an excess of fatty acids or their methyl or ethyl esters. Using immobilized C. rugosa lipase, plant sterol esters was successfully synthesized with a 5:1 molar ratio of fatty acids or triacylglycerols to plant sterols in a solvent-free medium.74 To reduce the excess use of acyl donor, Torres et al.78 and Torrelo et al.80 established an efficient route for solvent-free synthesis of plant sterol esters with a 1:1 molar ratio of plant sterols to conjugated linoleic acid or fatty acids from butter fat by stepwise addition of plant sterols. Other Assisted Methods. Ultrasound and microwave have shown to accelerate the enzyme-catalyzed production of plant sterol esters. Ultrasound mainly decreases a particle size, maximizes the surface area of substrate and enzyme, and facilitates the access of substrate to the active site of enzymes.83 Zheng et al. found that ultrasound pretreatment for 8−10 h could accelerate esterification or transesterification of plant sterols with fatty acids or triacylglycerols, having an overall conversion being more than 2-fold that of the stirring process for 16−24 h without affecting the lipase activity.83 Apart from ultrasound, microwave is also used for plant sterol ester synthesis. Microwave irradiation can enhance the activity and thermal stability of a lipase by a direct coupling of microwave energy with the molecules (solvents, substrates, catalysts), resulting in a short reaction time as well as a high yield.71 Shang et al. used microwave irradiation to catalyze esterification of plant sterols and oleic acid in the presence of immobilized C. rugosa lipase, demonstrating that a conversion rate of 95% could be achieved in 1 h, whereas the conventional methods would require 6 h to obtain the same conversion.71 Water (or methanol, ethanol) is the side product of the reversible esterification (or transesterification). The excessive production of water would further enhance the reverse reaction and thereby decrease the conversion rate. In this regard, numerous esterification or transesterification processes of plant sterols have been developed under vacuum to remove excessive side products.64,78−80,93,95,96 Weber et al. established an enzymatic method for the preparation of plant sterol esters in vacuum and achieved a yield of 95% at 20−40 hPa using Thermomyces lanuginosus lipase.95 No et al. synthesized plant sterol esters of

temperature results in greater substrate solubility and poorer stability and activity of lipase. In fact, the optimum temperature of immobilized lipases is higher than that of the free form. For the C. rugosa lipase (Table 1), the optimum temperature for the free form ranges from 30 to 50 °C, and the optimum temperature for their immobilized form ranges from 40 to 60 °C. Solvent Effect. The solvent is another crucial factor in enzyme-catalyzed production of plant sterol ester. Apart from lipase, the reaction solvent is also an important parameter for lipase-catalyzed synthesis of plant sterol esters. In general, organic solvents are not only the medium for mass transfer by affecting the solubility of substrate but also have a great effect on the activity and stability of lipase by changing the structure of enzyme and water activity.12 The log P value is used for describing the solvent hydrophobicity. Higher log P results in stronger hydrophobicity of solvents.12 Numerous organic solvents with different log P values, such as n-hexane,67,70,85−87 cyclohexane,81 n-heptane,88 isooctane,12,71,73,75−77,89 and toluene90 have been proven to be efficient solvents for the synthesis of plant sterol esters. Jiang et al. investigated the effect of several solvents on the esterification of phytosterol acetate and found n-hexane with that a log P of 3.5 was the optimum solvent.67 Zhang et al. compared the effects of different solvents including toluene, cyclohexane, n-hexane, n-heptane, and octane on the conversion of plant sterol ester catalyzed by C. rugosa lipase, demonstrating that n-heptane with a log P value of 4.0 was the optimum solvent and had the highest conversion rate of 90% at 44 °C after 12 h reaction. In a study by Choi et al.,81,88 cyclohexane with a log P value of 4.0 was deemed to be the suitable reaction medium for simultaneous synthesis of phytosterol esters and enrichment of stearidonic acid using C. rugosa lipase as a biocatalyst. Pan et al. studied the relationship between the conversion of plant sterols and log P values of solvent from −1.3 to 4.7 when using Candida sp. 99−125 lipase to catalyze the esterification of plant sterols, finding isooctane with a log P value of 4.7 showed a higher conversion over nhexane with a log P value of 3.5.85 A similar trend was observed by He et al.,12 who showed the conversion of plant sterols to plant sterol linolenate was positively correlated with the log P values (−1.3 to 4.7) of solvents with isooctane (log P 4.7) giving the highest conversion. In general, isooctane (log P 4.7) and n-hexane (log P 3.5) were the two most frequently used solvents. Under certain conditions, isooctane was superior to n-hexane for lipase-catalyzed synthesis of plant sterol esters. He et al. also investigated the relationship between the solvent hydrophobicity and the lipase stability after exposure to different solvents.12 The stability of C. rugosa lipase gradually decreased as the log P values of solvent decreased from 4.7 to −1.3. The lipase treated with dimethyl sulfoxide (DMSO) showed no activity in isooctane. This was because the solvent with a strong polarity (low log P values) rapidly deprived the necessary water from lipase molecules, thus making the enzyme completely deactivated. Recently, some new solvents were introduced into the esterification of plant sterols and fatty acids. In a study conducted by Zeng et al., a water-in-ionic liquid microemulsion ([Bmim]PF6/Tween 20/H2O) was applied as a reusable reaction medium for C. rugosa lipase-catalyzed esterification of phytosterols with fatty acids, and the conversion of 87.9% was achieved in 24 h under optimized conditions.69 Two biobased solvents (limonene and p-cymene) were also used for 3053


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Journal of Agricultural and Food Chemistry Table 2. Melting Points and Oil Solubility of Free and Esterified Plant Sterols compounda free sterols/stanols

esterified sterols

a

plant sterols β-sitosterol stigmasterol caproate caprylate caprate laurate oleate linoleate CLA ester linolenate PUFA esters

melting point (°C)

ref

137−145 136−140 164−168 58.7 66.8 70.2 81.3−85.8 25.6−28 12.5 15.3 2.3 6.1

41, 68, 105, 106 72 104 99 99 99 41, 99 72, 77 77 99 77 42

oil solubility (g/100 mL)

ref

1−3

12, 41, 42, 105, 68

4.28 32.1 36.2

68 77 77

35.8 32.5−34.6

12, 77 42

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

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

pinolenic acid using the immobilized C. rugosa lipase under vacuum with a maximum conversion of 93% being achieved at 80 kPa.79 Physical Properties of Plant Sterol Esters. The melting point and oil solubility of free and esterified plant sterols have been investigated in numerous studies (Table 2).12,64,65,72,96,104 The melting point of plant sterols ranges from 137 to 145 °C.41,68,105,106 Kobayashi et al. compared the melting points of free and esterified sitosterol by differential scanning calorimetric analysis, finding the melting point of β-sitosterol decreased from 130−140 to 28 °C after esterification with oleic acid.72 Vu et al. synthesized a series of plant sterol esters of medium chain fatty acids (C 6:0 ∼ C 12:0), demonstrating all the plant sterol esters had a lower melting point than the corresponding free sterols. It should be pointed out that the melting points would increase from 58 to 85 °C when the carbon number of saturated fatty acids increased from 6 to 12.99 Vaikousi et al. also found that the melting points of soy phytostanol esters increased with increasing chain length of the fatty acid moiety (C8−C12).105 Free plant sterols have a poor solubility in vegetable oils; for example, the solubility of plant sterols in soybean oil was only 1.24 g/100 mL at 20 °C.42 The solubility of plant sterols was increased by 3-times via lipase-catalyzed esterification with lauric acid and could reach 4.28 g/100 mL in sunflower oil at 30 °C.68 The solubility of a plant sterol would further improve by above 25-times if it was esterified with an

unsaturated fatty acids, such as oleic, linoleic, or linolenic acid.12,68,77 In this connection, the oil solubility of plant sterols has been greatly improved by chemical or enzymatic esterification with fatty acids or transesterification with fatty acid esters. At present, plant sterol or stanol fatty acid esters as functional ingredients have been widely used in many foods, such as margarine, butter, dairy products, mayonnaise, and salad dressings. Enhancing the Water Solubility of Plant Sterols. Insolubility of plant sterols in water limits their application in foods of aqueous medium. Despite a large number of studies having focused on improving the oil solubility of plant sterols, research on chemical modification is needed to improve the water solubility or hydrophilic property of plant sterols. The chemical structures of some hydrophilic plant sterol derivatives are shown in Figure 6. Ramaswamy et al. synthesized a novel hydrophilic phytostanol analogue (disodium ascorbyl phytostanol phosphates) by chemical modification with ascorbic acid.107 Hossen et al. synthesized a new phosphatidyl derivative of plant sterols (phosphatidyl sitosterols) using phosphatidyl choline as a modifier via an enzymatic process catalyzed by phospholipase D.108 Pang et al. investigated the feasibility of esterification of plant sterols with L-glutamic acid, finding the esterification could reach 92% under optimum conditions.109 Yuan et al. synthesized ten β-sitosterol esters using Nphosphoryl amino acids as an acyl donor, and the yield reached 3054


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Figure 7. Chemical structures of β-sitosterol phenolates.

show strong potential as a promising carrier for drugs to target cancer cells.116 Chemical Modification with Other Compounds. Plant sterols naturally occur in plants as free and conjugated forms. The latter are the esters of fatty acids and phenolic acids, glycosides, and acylated glycosides. Most research regarding plant sterols has focused on free sterols and their fatty acid esters. Recently, some plant sterol esters of phenolic acids have been identified, such as sterol ferulates in rice, wheat, rye, and corn, caffeates in canary seeds, and p-coumaric acid esters of plant sterol in corn.117 Natural sterol phenolates are present in many plants but in a very small amount. Over the past decade, emerging studies have concentrated on the synthesis of plant sterol esters of phenolic acids. The chemical structure of the major sterol phenolates are shown in Figure 7. Plant sterol ferulates, as a major component of γ-oryzanol, have been successfully synthesized.118,119 Condo et al. developed a synthetic process of β-sitostanol ferulate by three-step reactions: (a) preparation of trans-4-O-acetylferulic acid, (b) preparation of 3-O-(trans-4-O-acetylferuloyl)-β-sitostanol by esterification of β-sitostanol with trans-4-O-acetylferulic acid in the presence of DMAP and DCC, and (c) preparation of βsitostanol ferulate by selective deacetylation. The final yield of β-sitostanol ferulate could reach 60%.118 Tan et al. synthesized phytosterol ferulate by two-step chemoenzymatic reactions: (a) chemical synthesis of vinyl ferulate and (b) preparation of phytosterol ferulates by lipase-catalyzed transesterification of phytosterols with vinyl ferulate.119 In a similar way, plant sterol caffeates, sinapates, and vanillates were also synthesized via a chemoenzymatic route by Tan et al.120,121 The synthetic plant sterol esters (ferulate, caffeate, sinapate, and vanillate) of phenolic acid exhibited a higher antioxidant activity than their corresponding free plant sterols and phenolic acid tested in different models. Fu et al. prepared phytosterol gallate through a mild chemical Steglich esterification reaction, and the product showed excellent antioxidant activity.122 Wang et al. synthe-

60−87% by employing dicyclohexylcarbodiimide (DCC)/ DMAP as a catalyst system under microwave irradiation.110 He et al. established an efficient two-step chemoenzymatic method to synthesize plant stanol hydrophilic derivatives (plant stanol sorbitol succinate) using D-sorbitol as a hydrophilic modifier.111 Some hydrophilic compounds with a high molecular weight were also used as a hydrophilic modifier; for example, Chung et al. synthesized hydrophilic β-sitosterol derivatives with various degrees of substitution using polyethylene glycol (PEG) with higher molecular weight via a twostep chemical modification in the presence of triethylamine and 4-dimethylaminopyridine.112 He et al. further synthesized hydrophilic phytosterol derivatives using PEG as a hydrophilic modifier by a chemoenzymatic route and two-step ionic liquidcatalyzed method.15,113 The solubility of plant sterols could be significantly improved by conjunction with PEG 1000. The solubility of hydrophilic phytosterol derivatives in water could reach 7−25 g/100 mL at 30 °C.14,15,112 The water solubility of plant sterols can be improved to some extent by chemical modification, but at present, these hydrophilic plant sterol derivatives still cannot be directly applied into food systems due to the lack of extensive safety evaluation. Apart from their application in foods, plant sterols can also be used in the pharmaceutical and chemical industries. Klumphu et al. synthesized a β-sitosterol derivative as a surfactant using monomethylated polyethylene glycol by a twostep synthesis.114 This surfactant can provide a desired micellar condition for transition-metal-catalyzed reactions. SánchezFerrer et al. synthesized a food-grade glucose-β-sitosterol conjugate with the intention to use it for constructing an edible supramolecular chiral nanostructure by its amphiphilic behavior.115 Wang et al. synthesized a folate-mediated selfassembled phytosterol-alginate nanoparticle (FPA NP) using plant sterols as a hydrophobic moiety. The self-assembled FPA NPs could efficiently encapsulate a highly hydrophobic anticancer drug (DOX) with a high drug-loading capacity and 3055


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conducted, and most results have proven that plant sterols are effective in reducing plasma total cholesterol and LDL-C.133 Plant stanols with no double bond are the saturated forms of plant sterols. Saturation of β-sitosterol and campesterol gives rise to β-sitostanol and campestanol, respectively (Figure 1).134 β-Sitostanol and campestanol are found in nature in much smaller amounts than plant sterols. Since the first description of the use of plant stanols to lower plasma cholesterol by Heinemann et al. in 1986, plant stanols have been widely used in clinical treatment for reducing coronary heart disease incidence.135 The minor structural differences make plant sterols and stanols different from each other functionally and metabolically. Plant sterols are poorly absorbed in the intestine (0.4−3.5%), and absorption of plant stanols (0.02−0.3%) is even lower.136 The saturation of double bond increases hydrophobicity and decreases micellar solubility of stanols. This explains why plant stanols are less absorbed than plant sterols. It has been shown that consumption of stanol-containing mixtures is more effective in reducing circulating cholesterol concentrations than that of sterols.137,138 However, recent reports have demonstrated that plant sterols and stanols equally effectively reduce serum LDL-C and atherosclerotic risk.139,140 Interestingly, phytostanol consumption not only decreases the LDL-C concentration but also reduces serum plant sterols dosedependently.141 A great number of extensive safety evaluation studies have been conducted for plant stanols/sterols. To date, there has been no evidence showing a moderate consumption of plant sterols/stanols in the general population is associated with any increase in the risk of cardiovascular diseases, except for individuals with phytosterolemia, an inherited lipid disorder.142,143 A recent meta-analyses has demonstrated that plant sterol/ stanol can decrease LDL-C by up to 12% if their intake is 3 g/ day.18 However, the exact mechanisms by which plant sterols/ stanols reduce plasma LDL-C are still under investigation. It is well-established that phytosterols act as a cholesterolabsorption inhibitor via displacing cholesterol from bile emulsion in the intestine, thus leading to reduction in cholesterol absorption and plasma TC.144,145 HMG-CoA reductase is a rate-limiting enzyme in the cholesterol synthesis cascade. β-Sitosterol has been shown to inhibit cholesterol synthesis by inhibiting HMG-CoA reductase gene expression in Caco-2 cells.146 Cholesterol 7 alpha-hydroxylase (CYP7A1), which converts cholesterol to bile acids, has its gene expression upregulated by the plant sterol mixture in WKY and Wistar rats, resulting in more fecal sterol excretion and less cholesterol accumulation in blood vessels.147 The research conducted by Yang et al. also found that stigmasterol could inhibit sterol regulatory element-binding protein 2 (SREBP-2) processing and reduce cholesterol synthesis in cultured adrenal cells from ABCG 5/8 knockout mice.148 Cholesterol-Lowering Activity of Plant Sterol/Stanol Esters. The low solubility of free plant sterols and plant stanols limits their usage in functional foods. Plant sterol and stanol esters have a better solubility or incorporation into various foods without changing the taste and texture. Margarines, yogurt, cream cheese spreads, and cereal bars containing either plant stanol or sterol esters have been marketed in many countries. In these products, plant sterols and stanols are usually esterified with long chain fatty acids to increase their fat solubility from 2 to 20%.149

sized a series of plant sterol esters of phenolic acids including 4hydroxybenzoic acid, vanillic acid, 4-chlorophenylacetic acid, hydrocinnamic acid, 4-phenylbutyric acid, 5-phenylvaleric acid, cinnamic acid, m-coumaric acid, ferulic acid, and 3,4dimethoxycinnamic acid and tested their antioxidant activity, demonstrating these esters with 4-hydroxybenzoate, vanillate, and ferulate of plant sterols showed the potential for use as food antioxidants.123 Schär et al. recently developed a fully enzymatic procedure for the synthesis of sterol phenolates117,124 and evaluated their antioxidant capacity in different systems.117 C. rugosa lipase was used to synthesize β-sitosterol ferulates via direct esterification with ferulic acid as well as transesterification with yields of 35 and 55%, respectively.124 Lipase-catalyzed transestrification has also been used for the synthesis of other sterol esters of hydroxycinnamic acid (sinapic acid, m-coumaric acid, o-coumaric acid, p-coumaric acid, caffeic acid, and phloretic acid) using C. rugosa lipase.117 In addition to phenolic acids, lipoic acid and dihydrogen lipoic acid also showed a good antioxidant capacity. To provide better physiological functions, plant sterol esters of lipoic acid and dihydrogen lipoic acid were also synthesized.125,126 Madawala et al. synthesized phytosterol lipoate and phytosterol dihydrolipoate using a chemical method in the presence of DMAP and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride.125 By contrast, phytosterol dihydrolipoate displayed a better scavenging capacity of 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical. Wang et al. developed an enzymatic route to synthesize phytosterol lipoate with a conversion of 71% using C. rugosa lipase as a catalyst under optimal conditions for 96 h.126 The antioxidant ability of phytosterol has been greatly improved by conjugating with lipoic acid. Wang et al. also investigated the oil solubility of phytosterols and their lipoate and ferulate derivatives,126 finding that the solubility of phytosterol in rapeseed oil could increase from 9.1 g/L of free sterol to 20.4 and 22.5 g/L by esterification with lipoic and ferulic acids, respectively.

■

CHOLESTEROL-LOWERING ACTIVITY OF PLANT STEROLS/STANOLS, THEIR ESTERS, AND OTHER CHOLESTEROL ANALOGUES Hypercholesterolemia is one of the major risk factors for coronary heart disease (CHD). Management of blood TC and LDL-C levels by cholesterol-lowering nutraceuticals could slow the progression of atherosclerosis and reduce the risk of CHD.127 Phytosterols, comprising plant sterols and plant stanols as a healthy supplement, have been widely used to treat hypercholesterolemia. The role of phytosterols in lowering TC and LDL-C has been well recognized by various health authorities worldwide, including the EU, the US, Canada, and Australia/New Zealand.128 For instance, the European Foods Safety Authority (EFSA) recommends consuming 1.5−2.4 g/ day of phytosterols to decrease blood cholesterol.129 The US Food and Drug Administration (FDA) has approved the use of phytosterol esters into a low saturated fat and cholesterol diet in reducing the risk of coronary heart disease.130 It has been suggested that the daily consumption of 2 g of phytosterols can effectively lower plasma cholesterol by 9−14% in humans with little or no effect on high density lipoprotein cholesterol and triacylglycerol levels.131 Cholesterol-Lowering Activity of Plant Sterols/Stanols. Plant sterols were first demonstrated as a therapeutic agent to treat hypercholesterolemia in humans in 1953.132 Since that time, more than 200 clinical studies have been 3056


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their cholesterol-lowering activity in mice, demonstrating that PSS possessed hypocholesterolemic activity.166 Chung et al. compared the effect of β-sitosterol, its hydrophilic derivative (HPSS), and lipophilic derivative (LPSS) on blood cholesterol concentration in rats and demonstrated that LPSS and HPSS had a comparable activity with β-sitosterol in lowering blood cholesterol.167 Cholesterol-Lowering Activity of Other Cholesterol Analogues. Compounds with a similar structure generally have a similar biological activity. On the basis of the structures of cholesterol and phytosterols, different cholesterol analogues were designed to test their cholesterol-lowering activity. Wang et al. blocked the hydroxyl group on β-sitosterol by methylating and ethylating to form β-sitostery 3β-methoxy (SM) and βsitostery 3β-ethoxy (SE) derivatives (Figure 8) and investigated

The active forms of plant sterol/stanol esters are the free sterols/stanols as they are hydrolyzed to their corresponding free sterols/stanols and fatty acids in the intestine.150 It has been proven that plant stanol esters at a level of 2−3 g/d can reduce LDL-C by 10−15% without any side effects.151 The LDL-lowering efficacy of stanol esters might be influenced by doses, frequency of administration, food vehicle in which the stanol esters are incorporated, and background diet.152 In addition, a daily intake of 1.6 g sterol esters induces an additional reduction in LDL-C concentrations in children with familial hypercholesterolemia consuming a recommended diet.153 Hallikainen et al. compared the relative cholesterollowering activity of plant sterol esters and plant stanol esterenriched margarines in hypercholesterolemic subjects on a lowfat diet, finding that margarines containing plant sterol esters and plant stanol esters are equally effective at lowering plasma TC and LDL-C.154 It appears that the cholesterol absorption is similarly inhibited by plant sterol esters and plant stanol esters.155 It is inconclusive if the fatty acid moieties affect the cholesterol-lowering activity of the esterified phytosterols. He et al. compared the cholesterol-lowering activity of phytosterol and phytosterol laurate in mice and demonstrated that the lauric acid-esterified phytosterols retained similar cholesterollowering activity as that of the free phytosterols.156 Nestel et al. found that phytosterol esters prepared by esterification with fatty acids from soybean oil possessed a slightly but not significantly greater LDL cholesterol-reducing activity than that of the plant stanols.157 A study by Kobayashi et al. demonstrated that unesterified plant sterol was potentially more effective in inhibiting the cholesterol absorption than plant sterol oleates in rats, but the difference was substantially small.158 It is also inconclusive as to whether fatty acid moieties of plant sterol esters affect cholesterol metabolism. Liu et al. compared the cholesterol-lowering activity of sterol esters of sunflower oil and the sterol esters of canola oil, showing that they were equally effective in reducing plasma cholesterol in a dose-dependent manner regardless of the significant differences in their fatty acid compositions.159 However, the research conducted by Rasmussen et al. demonstrated that plant sterol esters made with beef tallow and pure stearic acid were more effective than plant sterol esters made with soybean oil in reducing the cholesterol absorption, liver cholesterol, and plasma non-HDL cholesterol concentration.160 It was also shown that the sterol esters with fish oil had an greater influence on LDL cholesterol concentrations compared with the esters with vegetable oil.161,162 Additionally, the sterol ester enriched with n-3 fatty acids from fish oil not only retained plasma cholesterol-lowering activity of plant sterol but also retained plasma triacylglycerol-lowering and eicosanoid-modifying properties of the fish oil.163 Apart from fat-soluble phytosterol esters, some researchers have recently studied the cholesterol-lowering activity of watersoluble or hydrophilic phytosterol derivatives. A study by Wasan et al. found that hydrophilic phytostanol analogue FMVP4 decreased total and LDL cholesterol concentrations in gerbils.164 A water-soluble phytostanol analogue, disodium ascorbyl phytostanyl phosphate (DAPP), had been demonstrated to reduce plasma cholesterol more efficiently than free stanols in hamsters.165 He et al. successfully synthesized a hydrophilic phytostanol derivative, phytostanol sorbitol succinate (PSS), via a chemical-enzymatic route, and investigated

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

their effect on plasma lipoprotein profile in hypercholesterolemia hamsters.56 Results showed that β-sitosterol remarkably inhibited the cholesterol absorption, whereas 3β-methoxy and β-sitostery 3β-ethoxy had no or little effect on apparent cholesterol absorption, suggesting that the hydroxyl group was essential for β-sitosterol to retain its cholesterol-lowering activity.56 Similar with above research, Lei et al. synthesized other two cholesterol analogues by blocking the hydroxyl group on cholesterol with methyl and ethyl groups, respectively, by producing cholesteryl 3β-methoxy (CM) and cholesteryl 3βethoxy (CE) (Figure 8) and then comparing their effects on plasma cholesterol with that β-sitosterol.168 Results proved that β-sitosterol was effective at reducing plasma cholesterol, whereas both cholesteryl 3β-methoxy and cholesteryl 3β-ethoxy had no cholesterol-lowering activity.151,168 Therefore, it is concluded that screening the potential hypocholesterolemic sterols shall focus on the analogues having a different side chain rather than these analogues having derivations on the ring. Dihydrocholesterol, also called 5α-cholesterol, is a cholesterol analogue. It has the same side chain as cholesterol but has no double bond at the Δ5 position in the B-ring. In 1953, Siperstein et al.169 had demonstrated that dihydrocholesterol could prevent the rise in plasma cholesterol and atherosclerosis 3057


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Journal of Agricultural and Food Chemistry in cholesterol-fed chickens, and Nichols et al.170 showed that dihydrocholesterol could decrease blood cholesterol in rabbits. The recent research conducted by Wang et al. demonstrated that dihydrocholesterol was effective at reducing plasma total cholesterol in hypocholesterolemia hamsters comparable to that of β-sitosterol at a dose of 0.2%;171 however, its application and safety in the management of hypercholesterolemia in humans remain largely unknown.

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FUTURE PERSPECTIVES Plant sterol and stanol fatty acid esters have been produced on a large scale by esterification or transesterification in food industries. Plant sterol and stanol fatty acid esters can be easily incorporated into various fat- or oil-based foods. However, production of plant sterol and stanol derivatives with a better water solubility or hydrophilic property on a large scale is lacking in the food industry. Future investigation in a pilot or an industrial scale is needed for developing a highly efficient process of producing the hydrophilic plant sterol derivatives of food grade without compromising their cholesterol-lowering activity. More effort is also needed for developing a process of synthesizing plant sterol and stanol derivatives conjugated with phenolic acids or the other biological compounds so that these derivatives possess not only plasma cholesterol-lowering activity but also other biological functions.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; tel: (852) 3943 6382; fax: (852) 2603-7246. ORCID

Zhen-Yu Chen: 0000-0001-5615-1682 Author Contributions §

Wen-Sen He and Hanyue Zhu contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by a grant from the Health and Medical Research Fund, the Government of the Hong Kong Special Administrative Region, China (13140111), the National Natural Science Foundation of China (31401664), Hong Kong Scholars Program (XJ2017019), the China Postdoctoral Science Foundation Funded Project (2014M560406), the Research Fund for Advanced Talents of Jiangsu University (13JDG070), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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