J. Ind. Eng. Chem., Vol. 13, No. 3, (2007) 367-372

Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol

Dae-won Chung† and Young Tai Choi

Department of Polymer Engineering, College of Engineering, Suwon University, Suwon 400-600, Korea

Received November 28, 2006; Accepted December 31, 2006

Abstract: In this study, hydrophilic derivatives of β-sitosterol, which is known to have potential to reduce blood cholesterol levels, were synthesized by the esterification of poly(ethylene glycol) (PEG) and β-sitosterol with carboxylic functionality in the presence of dehydrating agents and a basic catalyst. By adjust-ing the molar ratios of reactants in the esterification reaction, hydrophilic β-sitosterol derivatives (HPSs) with various degrees of substitution (DS, average molar number of β-sitosterol moieties per molecule of HPS) were synthesized. The chemical structure and the distribution of isomers in HPSs were characterized by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry, respectively. The solubility of HPS in water was significantly affected by the value of DS of HPS.

Keywords:β-sitosterol, hydrophilic derivatives, poly(ethylene glycol), esterification

Introduction

1)

Phytosterols refer to all of the alcohol compounds with steroid moietics found in higher plant life. They can be classified as stigmasterol, spinasterol, campesterol, and sitosterol; for example, sitosterol has α, β, and γ- types. Among all the phytosterols, β-sitosterol (24-eth-yl-5-cholestene-3-ol) has been acquiring greater im-portance for the production of steroidal drugs and as a food ingredient that functions as a cholesterol lowering agents. β-Sitosterol lowers the serum cholesterol level by reducing the absorption of cholesterol in the intestines through competition with LDL-cholesterol [1-3]. How- ever, the hydrophobic and lipophobic properties of β- sitosterol have prevented its widespread application; it can be formulated only as a tablet or capsule, making it difficult and inefficient as a food ingredient. To utilize β-sitosterol as a food ingredient, many stud-ies have focused on the chemical modification of β-sitosterol, mainly using its 3-hydroxy group. The first approaches to increase the solubility of β-sitosterol in fats were carried out by esterification of the 3-hydroxy group with fatty acids in the presence of an acidic cata-

† To whom all correspondence should be addressed.(e-mail: dwchung@suwon.ac.kr)

lyst such as sodium bisulfate [4]; many synthetic meth-ods using fatty acid anhydride [5], acyl chloride [6], or a basic catalyst [2-(dimethylamino)pyridine] with an acti-vator (dicy- clohexylcarbodiimide) [7] were investigated. Lipase-catalyzed syntheses of β-sitosterol esters were recently reported [8]. β-Sitosterol ester compounds are reported as having the same cholesterol lowering effect. For example, when β-sitosteryl oleate was given to an adult at 2 g per day for 5 days, the serum cholesterol level was reduced by ca 33 % [9]. A margarine containing a β-sitosterol ester, produced by transesterification of β-sitosterol with a fat-ty acid methyl ester derived from canola oil in the pres-ence of sodium ethoxide at 90∼120 oC under vacuum, finally entered the United States market in 1999 [10,11]. On the other hand, approaches to increase the water-solu-blility of β-sitosterol for potential application in non- lipid-based foods were carried out only by formulation with an emulsifier. Lecitine-emulsified micelles of β- sitosterol were reported as having the same cholesterol lowering effect [12]. However, no studies on the syn-thesis of hydrophilic derivatives of β-sitosterol have been reported. In the present studies, hydrophilic β-sitosterol de-rivatives (HPSs) were synthesized by two step reactions,

Dae-won Chung and Young Tai Choi368

Figure 1. Synthesis of HPS.

as described in Figure 1. The first step was the synthesis of an intermediate (carboxyethyl-β-sitosterol, CES), which afforded carboxylic functionality to β-sitosterol; the second step was the coupling of a hydrophilic poly-mer (PEG) to CES in the presence of a basic catalyst and dehydrating agents. HPSs with various values of DS were synthesized by adjusting the molar ratios of CES to PEG in the second reaction. For comparison, an HPS with one sterol moiety per molecule of PEG (Mono) was also synthesized. The solubility of HPS in water and the effect of the value of DS on the solubility were also investigated.

Experimental

Materials β-Sitosterol, PEG (MW = 1500), succinic anhydride, 1,3-dicyclohexylcarbodiimide (DCC), and 4-dimethyla-minopyridine (DMAP) were purchased from Aldrich and used without further purification. β-Sitosterol contains 20∼30 % of campersterol and 10∼30 % of dihydro- brassicasterol. Gas chromatography was performed using a capillary column [HP-1 (30 m × 0.25 mm i.d. × 0.25 µm)] and a M600D instrument (Young-Lin Instrument Co.). FT-IR spectroscopy was performed using a Nexus instrument (Nicolet Instrument Corp.). 1H-NMR spectra were ob-tained using a Bruker FT-NMR spectrometer (500 MHz) and CDCl3 as solvent. A Voyager Biospectrometry Workstation (Applied Biosystem Inc.) was used for the measurement of Matrix-Assisted Laser Desorption Ionization (MALDI-TOF) Mass Spectra.

Preparation of CES 50.0 g (0.12 mol) of β-sitosterol and 24.1 g (0.24 mol) of succinic anhydride were dissolved in 250 mL of di-chloroethane (DCE). After adding 16.7 mL (0.12 mol) of

TEA, the reaction mixture was heated for 2 h at 80 oC. After confirming the completion of the reaction with thin layer chromatography (TLC), the solvent was evaporated from the reaction mixture. The remaining solid residue was dissolved in 500 mL of ethyl alcohol and then 11 mL of concentrated HCl was added dropwise. A white precipitate was collected by filtration after adding 500 mL of water. Recrystallization from hexane afforded 56.8 g of the intermediate compound (yield; 92 %). The structure of CES was confirmed by 1H-NMR spectro-scopy in CDCl3.

Preparation of HPS 15.0 g of PEG having a molecular weight of 1500, 2.7 g (13 mmol) of DCC, and 0.1 g (0.8 mmol) of DMAP were dissolved in 100 mL of methylene chloride (MC) at room temperature. 7.7 g (1.5 times the number of moles of PEG) of CES pre-dissolved in MC was then added drop-wise to the reaction mixture at 30 oC. The reaction was continued at 30 oC until the CES peak disappeared (TLC). Dicyclohexylurea was removed by filtration, and then the filtrate was evaporated. Recrystallization in iso-propyl alcohol afforded 20.6 g of HPS. The structure of HPS was confirmed by 1H-NMR spectroscopy in CDCl3.

Preparation of Mono (R=H in Figure 1) 15.0 g (10 mmol) of PEG having a molecular weight of 1500, 0.58 g (2.8 mmol) of DCC, and 0.24 g (2.0 mmol) of DMAP were dissolved in 100 mL of MC at room temperature. 2.1 g (4 moles) of CES pre-dissolved in MC was then added dropwise at 30 oC to the reaction mixture. The reaction was continued at 30 oC until the CES peak disappeared in TLC. Dicyclohexylurea was re-moved by filtration, and then the filtrate was extracted with water four times to remove unreacted PEG. Recrystallization three times from isopropyl alcohol af-forded 2.1 g of Mono.

Solubility Test of HPS The solubility was determined visually after 600 mg of HPS in 1 mL of water was stirred at 35 oC for 2 h. After the solution became clear, it was kept at 25 or 4 oC for 48 h. If HPS was not soluble at 35 oC or the precipitation ap-peared during the storage at 25 or 4 oC, 1 mL of water was added, and the same procedure was repeated until the solution appeared clear. The critical concentration at each temperature was defined as the maximum concen-tration where the solution was visually clear.

Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol 369

Figure 2. 1H-NMR spectra of β-sitosterol (A), CES (B), and HPS with DS=1.08 (C).

Results and Discussion

Preparation of CES Although the synthesis of CES has not been reported, previously the preparation of carboxyethyl-cholesterol (cholesteryl hemisuccinate), which acts as a stabilizer for phosphatidylcholine membranes [13] and an enhancer for the specific immunogenicity of tumor cell [14], is well known. In this paper, CES was synthesized according to the reported method [15,16] for preparing carboxyethyl- cholesterol, with a minor modification. The chemical structure of CES was confirmed by 1H-NMR spectroscopy studies. As described in (A) and (B) of Figure 2, the proton of the 3-position at 3.5 ppm [b in (A)] of free β-sitosterol shifted to 4.6 ppm [b in (B)], as a result of the vicinal hydroxy group having changed into an ester group. Similar phenomena were observed in

1H-NMR spectra for cholesterol and cholesterol acetate [17]. The absence of succinic anhydride in CES was con-firmed by GC.

Preparation of HPS There are many known methods used to prepare ester compounds by coupling OH and COOH groups. The most common method is using an acidic catalyst, such as P-toluenesulfonic acid (PTSA) [18,19]; however, the es-ter group in CES can be cleaved under acidic conditions. In fact, the reaction between CES and PEG in the pres-ence of PTSA in toluene afforded the target compound (HPS), but free β-sitosterol was also observed in TLC at the initial stage of reaction, suggesting that the ester group in CES was partially hydrolyzed under these conditions. DCC is a powerful dehydrating agent commonly used for the preparation of amides, esters, and anhydrides [20]. In general, ester formation reactions proceed reli-ably through O-acylurea or anhydride intermediates, but require the presence of an acylation catalyst [21]. When the combination of DCC and DMAP was applied for the esterification reactions between β-sitosterol and fatty acids, the yields were over 90 % and no side products were observed [7]. In this study, DMAP was also used as an acylation catalyst, and the reactions were completed during 6∼8 h. As described in (C) of Figure 2, the 1H-NMR spectrum of the product showed a remarkable peak at 3.5∼3.7 ppm (e), which is assigned to the methylene protons of PEG. The most characteristic peak appeared at 4.3 ppm (d). This triplet can be assigned to terminal methylene protons of PEG coupled with CES, and its appearance is considered to be direct evidence for the esterification of CES with PEG. The integration ratio of peaks “a”, “b”, “c”, and “d” corresponded to the ideal ratio (1:1:4:2), re-gardless of the DS value. Because one chain of PEG has -OH groups at both ends, two sterol moieties can be introduced into one PEG chain. However, HPS having two sterol moieties at both ends may not be soluble in water. Thus, HPSs with vari-ous DS were synthesized through coupling reactions un-der various molar ratios of CES to PEG, whose molec-ular weight was fixed at 1500. The results are summar-ized in Table 1. In Table 1, the reaction time is the time required for the CES peak to disappear completely in TLC, and the DS value of HPS was calculated, based on a 1H-NMR spec-trum, according to the following equation:

DS = n*c /e,

where n is the degree of polymerization of PEG (in this case, n=34), and c and e are the integrations of the peaks

Dae-won Chung and Young Tai Choi370

Table 1. Reaction Conditions and Results

Feeding molar ratio(PEG : CES) Reaction time (hr) DS* Yield**

(%)

1.00 : 1.00 6 1.08 941.00 : 1.25 7 1.31 931.00 : 1.50 7 1.59 891.00 : 1.75 7 1.72 90

1.00 : 2.00 8 1.96 86* Calculated from 1H-NMR spectra.** Yield was calculated according to the following equation:

{weight of product/(1500 + 515*DS)}/moles of PEG reacted.

assigned in (C) of Figure 2. As the ratio of CES increased, more time was required for the completion of the reaction. Good agreement be-tween the DS values and the feeding molar ratios of CES to PEG and the high yield (over 86 %) suggest that the coupling reaction occurred quantitatively without any side reactions.

Synthesis of Mono The coupling reaction between CES and PEG can pro-duce three different compounds (unreacted PEG, Mono, and Di) because PEG is a di-functional compound and CES is mono-functional. As described in the ex-perimental section, pure Mono was synthesized by cou-pling CES with an excess (5 equivalents) of PEG to pre-vent the formation of Di, with subsequent purification to remove unreacted PEG. No significant difference in the 1H-NMR spectra of Mono and HPS with DS=1.08 was observed because DS obtained from the 1H-NMR spec-tral data means the average number of sterol moieties in HPS. The distribution of isomers in pure Mono was in-vestigated using MALDI-TOF mass spectrometry.

Distribution of Isomers Figure 3 shows MALDI-TOF mass spectra of PEG and HPSs with various DS values. Each peak has intervals of 44, which is equivalent to the mass of a monomeric unit of PEG. As shown in (C) of Figure 3, HPS with DS=1.08 contained three groups in the peak pattern. The first group, with a 1600∼1800 range of mass (m/e), is con-sidered to be unreacted PEG. The slightly higher and nar-rower range of the first group, compared to the spectrum (A in Figure 3) of PEG, suggests that PEG with a lower molecular weight reacted preferentially with CES so that the PEG with higher molecular weight remained unreacted. The second and third groups are equivalent to the spectra of pure Mono (B) and Di (E), as well as to the expected masses of Mono and Di (molecular weight of one sterol moiety is 515). HPS (D) with DS=1.59 shows a similar pattern, but with a significant decrease in the

Figure 3. MALDI-TOF mass spectra of PEG (A), Mono (B), HPS with DS = 1.08 (C), HPS with DS = 1.59 (D), and Di (E).

amount of unreacted PEG and an increase in Di com- pared to HPS with DS=1.08. On the other hand, in spec-trum (E) of Di, no significant peaks assigned to PEG and Mono are observed.

Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol 371

Table 2. Solubility of HPSs

DSCritical Concentration* (g of HPS / mL of water) Critical Concentration *

(mg of β-sitosterol moiety in HPS / mL of water)4 oC 25 oC 35 oC 4 oC 25 oC 35 oC

1.00 (Mono) 0.175 0.200 0.200 44.7 51.1 51.11.08 0.100 0.150 0.150 27.1 40.6 40.61.31 0.075 0.100 0.100 23.3 31.0 31.01.61 0.050 0.075 0.075 17.8 26.7 26.71.72 0.025 0.025 0.050 9.3 9.3 18.6

1.96 (Di) insoluble insoluble insoluble insoluble insoluble insoluble* Maximum concentration where the solution becomes visually clear.

Solubility Test As more sterol moieties are introduced into PEG, the hydrophilicities of the HPSs decreased. Therefore, it is important to figure out relationships between the value of DS and the solubility of the products. As summarized in Table 2, the water-solubility of the HPSs decreased as the DS values increased and the temperature decreased; Di was not soluble in water, even at 0.001 g/mL. It is clear that the introduction of bulky hydrophobic groups (β-sitosterol) at both ends of PEG limit the water-sol-ubility of HPS. However, considering the application of HPS, it is also important to evaluate the solubility based on the β-sitosterol moiety in HPS, which can be calcu-lated from the following equation:

Weight ratio of β-sitosterol moiety in HPS = 515 × DS / (1500 + 515 × DS)

where 515 is the molecular weight of CES and 1500 is the average molecular weight of PEG; thus, 1500 + 515 × DS corresponds to the average molecular weight of HPS. The water-solubility based on the β-sitosterol moiety in HPSs also decreased as the DS values increased and the temperature decreased. It is remarkable that the wa-ter-solubility of Mono was higher than that of HPS with DS = 1.08, even though the contents of β-sitosterol moi-ety of both compounds were almost the same. This result strongly suggests that the absence of Di in HPS is desir-able for potential application of HPS in non-lipid-based foods.

Conclusion

Hydrophilic β-sitosterol derivatives were synthesized through two-step reactions and their solubilities in water investigated. The following results are drawn: 1) In the presence of a basic catalyst and dehydrating agents, the coupling reactions of CES to PEG were ac-

complished without side reactions. HPSs with various DS values were obtained by adjusting the feeding molar ratios of CES to PEG in the coupling reaction. 2) Mono, with one sterol moiety per molecule of PEG, was synthesized by coupling CES with an excess of PEG, to prevent the formation of Di, and subsequent pu-rification to remove unreacted PEG. 3) The distribution of isomers of HPSs, including Mono and Di, could be determined clearly through MALDI-TOF mass spectrometries studies. 4) The solubilities of HPSs decreased as the DS values increased and the temperature decreased. The solubility of Mono was higher than that of HPS with DS = 1.08, even though the content of β-sitosterol moieties of both compounds was almost identical. This result strongly suggests that absence of Di in HPS is desirable for the development of water-soluble β-sitosterol derivatives.

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