Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol

Transcription

1 J. Ind. Eng. Chem., Vol. 13, No. 3, (2007) 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 , 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 adjusting 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 1 H-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-ethyl-5-cholestene-3-ol) has been acquiring greater importance 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]. However, 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 studies 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. ( dwchung@suwon.ac.kr) lyst such as sodium bisulfate [4]; many synthetic methods using fatty acid anhydride [5], acyl chloride [6], or a basic catalyst [2-(dimethylamino)pyridine] with an activator (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 fatty acid methyl ester derived from canola oil in the presence of sodium ethoxide at o C under vacuum, finally entered the United States market in 1999 [10,11]. On the other hand, approaches to increase the water-solublility of β-sitosterol for potential application in nonlipid-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 synthesis of hydrophilic derivatives of β-sitosterol have been reported. In the present studies, hydrophilic β-sitosterol derivatives (HPSs) were synthesized by two step reactions,

2 368 Dae-won Chung and Young Tai Choi TEA, the reaction mixture was heated for 2 h at 80 o C. 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 1 H-NMR spectroscopy in CDCl 3. 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 polymer (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-dimethylaminopyridine (DMAP) were purchased from Aldrich and used without further purification. β-sitosterol contains % of campersterol and % of dihydrobrassicasterol. Gas chromatography was performed using a capillary column [HP-1 (30 m 0.25 mm i.d µm)] and a M600D instrument (Young-Lin Instrument Co.). FT-IR spectroscopy was performed using a Nexus instrument (Nicolet Instrument Corp.). 1 H-NMR spectra were obtained using a Bruker FT-NMR spectrometer (500 MHz) and CDCl 3 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 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 dropwise to the reaction mixture at 30 o C. The reaction was continued at 30 o C until the CES peak disappeared (TLC). Dicyclohexylurea was removed by filtration, and then the filtrate was evaporated. Recrystallization in isopropyl alcohol afforded 20.6 g of HPS. The structure of HPS was confirmed by 1 H-NMR spectroscopy in CDCl 3. 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 o C to the reaction mixture. The reaction was continued at 30 o C until the CES peak disappeared in TLC. Dicyclohexylurea was removed by filtration, and then the filtrate was extracted with water four times to remove unreacted PEG. Recrystallization three times from isopropyl alcohol afforded 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 o C for 2 h. After the solution became clear, it was kept at 25 or 4 o C for 48 h. If HPS was not soluble at 35 o C or the precipitation appeared during the storage at 25 or 4 o C, 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 concentration where the solution was visually clear. 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 dichloroethane (DCE). After adding 16.7 ml (0.12 mol) of

3 Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol H-NMR spectra for cholesterol and cholesterol acetate [17]. The absence of succinic anhydride in CES was confirmed by GC. Figure 2. 1 H-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 carboxyethylcholesterol, with a minor modification. The chemical structure of CES was confirmed by 1 H-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 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 ester group in CES can be cleaved under acidic conditions. In fact, the reaction between CES and PEG in the presence 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 reliably 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 1 H-NMR spectrum of the product showed a remarkable peak at 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), regardless 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 various DS were synthesized through coupling reactions under various molar ratios of CES to PEG, whose molecular weight was fixed at The results are summarized 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 1 H-NMR spectrum, 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

4 370 Dae-won Chung and Young Tai Choi Table 1. Reaction Conditions and Results Feeding molar ratio (PEG : CES) Reaction time (hr) DS * Yield** (%) 1.00 : : : : : * Calculated from 1 H-NMR spectra. ** Yield was calculated according to the following equation: {weight of product/( *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 between 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 produce three different compounds (unreacted PEG, Mono, and Di) because PEG is a di-functional compound and CES is mono-functional. As described in the experimental section, pure Mono was synthesized by coupling CES with an excess (5 equivalents) of PEG to prevent the formation of Di, with subsequent purification to remove unreacted PEG. No significant difference in the 1 H-NMR spectra of Mono and HPS with DS=1.08 was observed because DS obtained from the 1 H-NMR spectral data means the average number of sterol moieties in HPS. The distribution of isomers in pure Mono was investigated 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 range of mass (m/e), is considered to be unreacted PEG. The slightly higher and narrower 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 compared to HPS with DS=1.08. On the other hand, in spectrum (E) of Di, no significant peaks assigned to PEG and Mono are observed.

5 Synthesis and Solubility of Hydrophilic Derivatives of β- Sitosterol 371 Table 2. Solubility of HPSs Critical Concentration * Critical Concentration * (g of HPS / ml of water) DS (mg of β-sitosterol moiety in HPS / ml of water) 4 o C 25 o C 35 o C 4 o C 25 o C 35 o C 1.00 (Mono) (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 g/ml. It is clear that the introduction of bulky hydrophobic groups (β-sitosterol) at both ends of PEG limit the water-solubility 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 calculated from the following equation: Weight ratio of β-sitosterol moiety in HPS = 515 DS / ( DS) where 515 is the molecular weight of CES and 1500 is the average molecular weight of PEG; thus, 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 water-solubility of Mono was higher than that of HPS with DS = 1.08, even though the contents of β-sitosterol moiety of both compounds were almost the same. This result strongly suggests that the absence of Di in HPS is desirable 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 accomplished 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 purification 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. References 1. H. T. Vanhanen and T. A. Miettinen, Clin. Chim. Acta, 205, 97 (1992). 2. H. Gylling and T. A. Miettinen, J. Lipid Res., 37, 1776 (1996). 3. P. J. H. Jones, D. E. MacDougall, F. Ntanios, and C. A. Vanstone, Can. J. Physiol. Pharmacol., 75, 217 (1997). 4. U. S. Patent 6,184,397 (2001). 5. F. H. Mattson, R. A. Volpenhein, and J. B. Martin, J. Lipid Res., 5, 374 (1964). 6. K. G. Pinter, J. G. Hamilton, and J. E. Muldrey, J. Lipid Res., 5, 273 (1964). 7. D.-W. Chung and Y. T. Cho, J. Korean Ind. Eng. Chem., 17, 375 (2006). 8. U. S. Patent 6,660,491 (2003). 9. P. J. H. Jones, D. E. MacDougall, F. Ntanios, and C. A. Vanstone, Can, J. Pharmacol., 75, 217 (1997). 10. U. S. Patent 5,502,045 (1996). 11. J. A. Weststrate and G. W. Meijer, Eur. J. Clin.

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