Article pubs.acs.org/JAFC
Efficient Synthesis and Characterization of Ergosterol Laurate in a Solvent-Free System Wen-Sen He,*,† Ji Yin,† Han-Shan Xu,† Qiu-Ying Qian,† Cheng-Sheng Jia,‡ Hai-Le Ma,† and Biao Feng*,‡ †
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, People’s Republic of China ‡ State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, People’s Republic of China ABSTRACT: Ergosterol and its derivatives have attracted much attention for a variety of health benefits, such as antiinflammatory and antioxidant activities. However, ergosterol esters are advantageous because this compound has better solubility than the free ergosterol. In this work, ergosterol laurate was efficiently synthesized for the first time by direct esterification in a solvent-free system. The desired product was purified, characterized by Fourier transform infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance, and finally confirmed to be ergosterol laurate. Meanwhile, the effect of various catalysts, catalyst dose, reaction temperature, substrate molar ratio, and reaction time were studied. Both the conversion of ergosterol and the selectivity of the desired product can reach above 89% under the selected conditions: sodium dodecyl sulfate + hydrochloric acid as the catalyst, 2:1 molar ratio of lauric acid/ergosterol, catalyst dose of 4% (w/w), 120 °C, and 2 h. The oil solubility of ergosterol and its laurate was also compared. The results showed that the solubility of ergosterol in oil was significantly improved by direct esterification with lauric acid, thus greatly facilitating the incorporation into a variety of oil-based systems. KEYWORDS: ergosterol, ergosterol laurate, oil solubility, solvent-free, esterification
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INTRODUCTION Ergosterol (ergosta-5,7,22-trien-3β-ol) is a type of natural steroid alcohol found in ergot and fungi, occurring in the vast majority of fungi.1 Its content is closely associated with structural and growing fungal characteristics,2 such as maturation, hyphal formation, and sporulation3 as well as the fungal species.4 Ergosterol is a characteristic component of the fungal cell membrane, which plays an essential role in membrane function, affecting membrane rigidity, fluidity, and permeability,5 while it is completely or nearly absent in animal, plant, and bacterial cells.1 In recent years, ergosterol has attracted much attention because of its important role. On the one hand, ergosterol is the precursor of vitamin D2, which can be converted into vitamin D2 after photolysis and thermal rearrangement.2,3 On the other hand, ergosterol and its derivatives exhibit a variety of health benefits, such as anti-inflammatory,6 antihyperlipidemic,7,8 antioxidant,9 and antiangiogenic activities.10 Hu et al. observed that ethyl acetate extract and methanol extract from Pleurotus citrinopileatus had a significant antihyperlipidemic effect in rats, in which ergosterol was one of the major components.7 Schneider et al. also reported that the beneficial effects of oyster mushroom on blood parameters (triglyceride, total cholesterol, and oxidized low-density lipoprotein) in humans may be attributed to the presence of linoleic acid, ergosterol, and ergosterol derivatives.8 Moreover, ergosterol and its derivatives have shown melanogenesis and epidermal cell proliferation inhibiting effects.10 Despite its potential attractiveness, the practical use of ergosterol has been greatly limited by its poor solubility in oils and fats. It has been reported that esterification of sterols with © 2014 American Chemical Society
lipo-soluble components, such as fatty acids, would improve their lipophilicity.11 A variety of strategies have been explored for the synthesis of sterol esters with fatty acids, such as chemical esterification and lipase-catalyzed esterification. In recent years, an enzymatic route has been gaining importance because of its remarkable properties, such as allowing for mild and environmentally friendly reaction conditions. A large number of studies have also been carried out on the lipasecatalyzed synthesis of phytosterol fatty acid esters.10,12,13 Nevertheless, these disadvantages, such as the high cost of enzymes and low productivity, greatly limit its industrial application. Thus far, the chemical route remains mainstream for commercial production. Numerous studies have been performed to synthesize fatty acid sterol esters by chemical esterification.14−16 For example, phytosterol esters can be efficiently prepared from natural sterols and methyl esters in the presence of magnesium oxide or zinc oxide.14 Currently, green synthesis has been attracting increasing attention. To avoid organic solvents for its environmentally unfriendly, solvent-free system is undoubtedly an important choice for chemical synthesis. In recent years, an acid− surfactant-combined catalyst has been widely used for catalyzing esterification and transesterification reactions.11,15,17 Ghesti et al. observed that cerium(III) trisdodecylsulfate trihydrate as a Lewis acid−surfactant-combined catalyst could be used for the efficient synthesis of alkyl esters by solvent-free Received: Revised: Accepted: Published: 11748
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transesterification and esterification reactions.17 Moreover, Lewis acid has also been proven to be active for the esterification reaction.18 Thus far, numerous studies concerning the synthesis of phytosterol esters have been reported. However, the related research on the synthesis of ergosterol esters is still rare in the literature, except for a recent report concerning the synthesis of ergosteryl oleate,10 in which the highest conversion (72.4%) was obtained using Lipozyme TL IM as the biocatalyst. The present study was aimed at the efficient synthesis of ergosterol ester by esterification using lauric acid as an acyl donor in the presence of a Lewis acid or an acid−surfactant-combined catalyst in a solvent-free system. The synthesized product was separated by silica gel column chromatography, and its chemical structure was characterized by Fourier transform infrared spectroscopy (FTIR), mass spectroscopy (MS), and nuclear magnetic resonance (NMR). The influence of different catalysts, catalyst dose, substrate molar ratio, reaction temperature, and reaction time was also considered. Furthermore, the solubility of ergosterol and its laurate in vegetable oil was studied and compared.
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as follows: 0.05−0.09 (lauric acid), 0.16−0.21 (ergosterol), and 0.85− 0.91 (ergosterol laurate). The sample (1.0 g) at the end of the reaction was dissolved in 20 mL of petroleum ether (60−90 °C)/ethyl acetate (9:1, v/v) and then applied to a silica gel column (12 × 1000 mm), eluted with petroleum ether (60−90 °C)/ethyl acetate (9:1, v/v) at the flow rate of 0.8 mL/ min. The eluent was collected, and the purity of the product was preliminarily detected by TLC analysis and further analyzed by HPLC. The fractions only containing ergosterol laurate were collected by rotary evaporation under vacuum. FTIR Analysis. The above purified ergosterol laurate was dried under vacuum and then characterized by FTIR analysis. FTIR measurement was performed on a FTIR spectrophotometer (Thermo Nicolet IS50 FT-IR, Waltham, MA) using an attenuated total reflectance method with the spectral scanning scope for 600−4000 cm−1, number of scans of 32, and resolution of 4 cm−1. MS Analysis. The mass spectrum was obtained by liquid chromatography ion trap mass spectrometry (Thermo LXQ, Waltham, MA) with positive-ion atmospheric pressure chemical ionization (APCI) mode. The MS operating parameters were as follows: vaporizer temperature, 350 °C; sheath gas flow rate, 25 arb; auxiliary gas flow rate, 5 arb; discharge current, 4.0 μA; capillary temperature, 275 °C; capillary voltage, 30 V; tube lens, 120 V; and mass scan range, 100−800 amu. NMR Analysis. The chemical structure of the purified product was further identified by NMR. In detail, 1H and 13C NMR and distortionless enhancement by polarization transfer 135 (DEPT 135) spectra of the product were recorded using CDCl3 as the solvent with a Bruker NMR spectrometer (Avance II 400 MHz, Switzerland). The 1 H NMR spectrum was acquired using the following settings: temperature, 294.5 K; frequency, 400.13 MHz; acquisition time, 4 s; and number of scans, 16. 13C NMR and DEPT 135 spectra were acquired using the same settings as follows: temperature, 295.2 K; frequency, 100.61 MHz; acquisition time, 1.4 s; and number of scans, 549. HPLC Analysis. The reaction sample (0.05 g) periodically removed from the reaction tube was diluted in 20 mL of absolute ethyl alcohol/ n-hexane (1:1, v/v). The sample (10 μL) was analyzed by Agilent 1100 HPLC using a symmetry-C18 column (5 μm, 4.6 × 150 mm, Waters, Milford, MA) controlled at 35 °C. The sample was eluted with methanol/n-hexane (9:1, v/v) as the mobile phase at the flow rate of 1.0 mL/min. The eluate was monitored with a Schambeck ZAM 4000 evaporative light scattering detector (ELSD) at 60 °C and nitrogen as the carrier gas at a pressure of 0.5 bar. The purified ergosterol laurate and ergosterol were used as standards, and the corresponding calibration curves were prepared for quantitative analysis. The conversion and selectivity were defined as follows:
MATERIALS AND METHODS
Materials. Ergosterol (purity of >95%) was provided by SigmaAldrich Co., Ltd. (Shanghai, China). Methanol and n-hexane used for high-performance liquid chromatography (HPLC) analysis were of spectral grade and provided from Tedia Company, Inc. (Shanghai, China). Petroleum ether (60−90 °C), ethyl acetate, formic acid, nhexane, ethanol, hydrochloric acid (HCl), sodium dodecyl sulfate (SDS), stannous chloride (SnCl2), cuprous chloride (CuCl), and other common reagents used were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silica gel (100−200 mesh) was obtained from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China). Preparation of the Catalyst. In this study, SDS, SDS + HCl, SnCl2, and CuCl were selected as the potential catalyst. Among these catalysts, SDS + HCl was prepared according to a previous report with minor modification.11 Briefly, 0.3 g of SDS was weighed and put into a mortar in a fume hood, and 0.2 mL of HCl was added to the mortar. The mixture was ground for 10 min and stood for 20 min in a fume hood to remove the excess HCl by evaporation. The catalyst SDS + HCl was finally obtained. This catalyst was newly prepared every time prior to use. The other catalysts were directly used as catalyst for the esterification. Esterification Reaction. The esterification reaction for ergosterol laurate synthesis was performed on a 20 mL special reaction tube. First, the reaction tube was placed and fixed in a digital oil bath heating device equipped with a magnetic stirring apparatus, and a magnetic stirrer was added. Then, the mixture of lauric acid (1.0−4.0 g) and catalyst (0.02−0.08 g) were added to the reaction tube and, subsequently, heated to the desired temperature under a constant nitrogen flow. A total of 2.0 g of ergosterol was added to the reaction system until lauric acid and catalyst melted completely, starting the reaction and lasting for some time (0.5−4.0 h). Over the time course of the reaction, a portion of the sample was periodically taken out for thin-layer chromatography (TLC) and HPLC analyses. At the end of the reaction, nitrogen flow was stopped until all samples were removed. TLC Analysis and Product Purification. The sample (0.05 g) removed periodically from the reaction system was dissolved in 10 mL of n-hexane/ethanol (1:1, v/v) and then used for TLC analysis. A small quantity of sample solution (10 μL) was evenly pointed on a activated TLC plate. Development was then carried out using petroleum ether (60−90 °C)/ethyl acetate (9:1, v/v) as the developing agent, and the TLC plate was located by iodine vapor staining for 20 min. Rf values of different substrates and products were
conversion of ergosterol (%) = (EB − EE)/EB × 100 selectivity of ergosterol laurate (%) = ELE/(EB − EE) × 100 where EB is the molar amount of ergosterol at the beginning of the reaction, EE is the molar amount of ergosterol at the end of the reaction, and ELE is the molar amount of ergosterol laurate at the end of the reaction. Determination of the Oil Solubility of Ergosterol and Its Laurate. The solubility of ergosterol and ergosterol laurate in vegetable oil was investigated as follows: 0.2 g of ergosterol or 0.5 g of ergosterol laurate was added to a 100 mL flask, successively. The flask was heated with a oil bath equipped with a magnetic stirring apparatus to 30 °C. Subsequently, the soybean oil was added dropwise until the sample was completely dissolved. Thereafter, the flask stood for 6 h with the help of magnetic stirring. The oil volume were promptly adjusted and recorded on the basis of the dissolution. The oil solubility can be calculated by the amount of sunflower oil to be added and expressed as grams per 100 mL at 30 °C. 11749
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1366 cm−1 was the bending vibration of C−H in the CH3 group. The peaks at 2932 and 1456 cm−1 were the asymmetrical and bending stretching vibrations of C−H in the CH2 group, respectively. The medium peak at 1658 cm−1 was the vibration signal of CC. The strong peak at 1031 cm−1 corresponded to the vibration of C−O. The FTIR spectral data of the product and the potential functional groups were displayed in Figure 1c. The weak peak at 3040 cm−1 was the signal of C−H in −CC−H. The peaks at 2952 and 2869 cm−1 were the asymmetrical and symmetrical stretching vibration of C−H in the CH3 group, respectively. The peak at 1380 cm−1 was the bending vibration of C−H in the CH3 group. The peaks at 2915 and 1460 cm−1 were the asymmetrical and bending stretching vibrations of C−H in the CH2 group, respectively. The weak peak at 1651 cm−1 was the vibration signal of CC. The strong peaks at 1740 and 1173 cm−1 were the characteristic absorption signals of CO and C−O in the ester group, respectively. In comparison to the FTIR spectrum of lauric acid, the characteristic absorption signal of the carbonyl group in the product moved to a higher wavenumber, indicating that presence of the ester group. The band at 724 cm−1 implied the presence of four or more CH2 groups in the carbon chain. The absorption signal of the free carboxyl group was observed in lauric acid but not in the product. The characteristic signal of the free hydroxyl group was observed in ergosterol. Nevertheless, the appearance of the ester group and the disappearance of the free hydroxyl group in the product suggesting that the new product may be ergosterol laurate. MS Analysis. A direct sample infusion APCI−MS with positive-ion mode was used to identify the product. A major advantage of this approach was no need of separating the targeted compound from other compounds prior to analysis. The positive APCI−MS spectrum of the purified product was displayed in Figure 2. Theoretically, the relative molecular weight for ergosterol laurate was 578. In Figure 2, the protonated molecular ion [M + H]+ at m/z 579 was observed, indicating that the product was ergosterol laurate. Similarly, the molecular ion [M + H]+ at m/z 661 for oleyl ergosterol was observed in a previous report of the analysis of ergosterol esters isolated from the fruiting bodies of Mycena chlorinella using positive APCI−MS.19 The base peak at m/z 379 [M + H − lauric acid]+ indicated the loss of lauric acid, providing strong evidence for the presence of ergosterol laurate. A similar result was also observed in a previous study, in which dehydrated molecular ions ([M + H − H2O]+) at m/z 379 were found in ergosterol with strong intensity using positive APCI−MS.20,21 NMR Analysis. Following the purification procedure, the purified product showed only a single peak in the HPLC chromatograms, indicating that the purity of the product was very high (>98%). The microstructure of the product was further analyzed by 1H and 13C NMR and DEPT 135 spectroscopy (Figure 3). The 1H and 13C chemical shifts for the product were assigned by the interpretation of their 1H and 13 C NMR and DEPT 135 spectra and further comparison to those of ergosterol in the literature.21 As displayed in Figure 3a, the 1H NMR spectrum was complex, owing to the overlapping of the absorption signal of methylene and/or methylidyne between 1.2 and 2.1 ppm. The resonances below 1.1 ppm were mainly ascribable to methyl units, and those above 2.1 ppm were due to methylidyne units. The 3-H signal at 4.7 ppm in the product had a downfield shift of 1.0 ppm in comparison to that of ergosterol (3.7 ppm),21
RESULTS AND DISCUSSION Product Analysis. The product was quantified by HPLC. After the purification procedure, the purity of the product was also determined by HPLC. The purified product was characterized by FTIR, MS, and NMR analyses. HPLC Analysis. The conversion of ergosterol to ergosterol laurate was determined by HPLC. ELSD was a kind of universal detector and selected for this study because of lauric acid without ultraviolet (UV) absorption. The peaks of ergosterol and lauric acid were characterized on the basis of their retention times with reference to standards. Lauric acid was first eluted with the retention time of 3.4 min, and then ergosterol was eluted with the retention time of 5.8 min. From the HPLC chromatogram of the reaction mixtures, a new peak at 22.4 min was observed and corresponded to the product ergosterol laurate. Apparently, the products can be clearly distinguished from the reaction substrates. FTIR Analysis. The FTIR spectral data of lauric acid and the potential functional groups were displayed in Figure 1a.
Figure 1. FTIR spectra of (a) lauric acid, (b) ergosterol, and (c) ergosterol laurate.
Obviously, the broad peak between 2450 and 3300 cm−1 was the characteristic absorption signal of −OH in −COOH, and the strong peak at 1694 cm−1 was the characteristic absorption signal of CO in −COOH, suggesting the presence of a free carboxyl group. The peak at 2953 cm−1 was the asymmetrical stretching vibration of C−H in the CH3 group. The peaks at 2913 and 2847 cm−1 were the asymmetrical and symmetrical stretching vibrations of C−H in the CH2 group, respectively. The peak at 1471 cm−1 was the bending vibration of C−H in the CH2 group. The band at 718 cm−1 indicated the presence of four or more CH2 groups in the carbon chain. The FTIR spectral data of ergosterol and the potential functional groups were displayed in Figure 1b. The broad and weak peak at 3430 cm−1 corresponded to the stretching vibration of the hydroxyl group. The weak peak at 3056 cm−1 was the signal of C−H in −CC−H. The peaks at 2953 and 2869 cm−1 were the asymmetrical and symmetrical stretching vibrations of C−H in the CH3 group, respectively. The peak at 11750
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Figure 2. MS spectrum of ergosterol laurate.
dienes.15,23 In addition, ergosterol in mushrooms has also been found to be converted into vitamin D2 by UV irradiation or sunlight exposure.24 In this study, the side reaction was partially inhibited under constant nitrogen flow. Therefore, the conversion of ergosterol and the selectivity of the desired product (ergosterol laurate) were synchronously considered. The yield of the desired product (ergosterol laurate) was directly correlated to the selectivity. Under the same conversion, the higher the selectivity, the higher the yield of ergosterol laurate. On the contrary, the lower the selectivity, the higher the yield of side products. Selection of the Catalyst. The effect of different catalysts on the conversion of ergosterol and the selectivity of ergosterol laurate was investigated, and the result is shown in Figure 4. These catalysts were used for the synthesis of ergosterol ester on the basis of the previous reports concerning the synthesis of phytostanyl esters.11,15 Furthermore, CuCl was also selected to explore the catalytic efficiency for ergosterol ester synthesis because of its potential catalytic activity.25 As shown in Figure 4, the catalysts displayed different catalytic activities for the esterification of ergosterol with lauric acid. Among these catalysts, although SnCl2 and CuCl showed excellent selectivity, lower conversion was observed when using SnCl2 or CuCl as the catalyst. In a previous study, SDS + HCl has been found to be effective for the synthesis of phytostanyl esters in a solvent-free system.11 In this study, the effect of SDS, HCl, or SDS + HCl on the conversion and selectivity was considered and compared for the synthesis of ergosterol ester. Almost no formation of the desired product occurred using HCl alone as the catalyst (data not shown), which was ascribed to be the rapid evaporation of hydrochloride at a high temperature. SDS had a comparable effect to SDS + HCl in the selectivity for this esterification, while the latter was better in the conversion. Therefore, SDS + HCl was used on the basis of the conversion and selectivity for the following experiments. Effect of the Reaction Temperature. The influence of the reaction temperature on the conversion of ergosterol and the selectivity of ergosterol laurate was evaluated varying the reaction temperature from 80 to 120 °C. As shown in Figure 5, the conversion of ergosterol gradually improved as the temperature increased from 80 to 120 °C and then slightly
indicating the presence of an ester bond in the ergosteryl moiety. The signals at 5.2 ppm were attributed to 22-H and 23H. Furthermore, The resonances at 5.38 or 5.56 ppm were owing to 6-H or 7-H. DEPT was a very useful method for determining the presence of primary, secondary, and tertiary carbon atoms. The DEPT experiment distinguished between CH, CH2, and CH3 groups by variation of the selection angle parameter. In DEPT 135, all CH2 signals faced downward (negative), CH or CH3 signals faced upward (positive), and quaternary carbon did not appear. The 13C NMR spectrum in Figure 3b illustrated the distribution of all carbon atoms in the product, while the DEPT 135 spectrum in Figure 3c only contained primary, secondary, and tertiary carbon atoms. Obviously, the peaks at 37.11, 37.11, 138.64, 141.47, and 173.33 ppm appearing in the 13 C NMR spectrum but disappearing in the DEPT 135 spectrum were ascribed to 10-, 13-, 5-, 8-, and 1′-quaternary carbon atoms, in agreement with proposed chemical structure for ergosterol laurate. In Figure 3c, the downward peaks between 21.0 and 39.1 ppm were mainly attributed to secondary carbon atoms. The resonances below 21.0 ppm were mainly ascribed to primary carbon atoms, and those above 39.1 ppm were mainly due to tertiary carbon atoms. By comparison, the number of primary, secondary, tertiary, and quaternary carbon atoms was in close agreement with the proposed chemical structure for ergosterol laurate. Furthermore, the 3-carbon atom signal at 72.5 ppm in the product exhibited a downfield shift of 2.0 ppm in comparison to that of ergosterol (70.5 ppm),21 indicating the presence of an ester bond on the 3-carbon atom of the ergosteryl moiety. These NMR results together with FTIR and MS results proved that ergosterol laurate was successfully synthesized. Determination of Reaction Parameters. For this esterification reaction, the main product was ergosterol laurate produced by direct esterfication of ergosterol with lauric acid, while there were also some unknown side products generated during the reaction. It has been reported that ergosterol was a rather unstable molecule and could be easily oxidized and photoxidized.22 Meng et al. considered that the high temperature may be in favor of the dehydration of sterols to 11751
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Figure 3. (a) 1H and (b) 13C NMR and (c) DEPT 135 spectra of ergosterol laurate.
decreased with a further increase of the reaction temperature from 120 to 140 °C. Moreover, the selectivity of ergosterol laurate varied slightly with the increase of the reaction temperature from 80 to 120 °C and then decreased when the temperature surpassed 120 °C. Although there was a high selectivity, the conversion of ergosterol was below 40% at 80 °C. The conversion was significantly improved at 140 °C, while the selectivity of ergosterol laurate was markedly decreased. In general, the esterification reaction would be enhanced as the substrates gradually melted and fully mixed with the gradual increase of the reaction temperature. Nevertheless, side
reactions, such as oxidation, dehydration, and degradation, may also be accelerated as the reaction temperature increased, thus resulting in the lower selectivity. Effect of the Catalyst Dose. The influence of the catalyst load on the conversion of ergosterol and the selectivity of ergosterol laurate was considered varying the amount of SDS + HCl from 1 to 5% (w/w), and the results were displayed in Figure 6. Although some ergosterol was consumed and the conversion below 8% was observed in the absence of SDS + HCl, almost no ergosterol laurate was formed at 120 °C (data not shown). As shown in Figure 6, the conversion of ergosterol 11752
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significantly promoted with the addition of 1% SDS + HCl and then gradually increased with a further increase of SDS + HCl and the highest conversion of ergosterol was achieved with 4% SDS + HCl. Meanwhile, the selectivity of ergosterol laurate also improved with the rise of SDS + HCl and then remained steady when the catalyst dose exceeded 4%. At a low catalyst dose, low conversion of ergosterol was still observed, while no ergosterol laurate was formed, which should be the occurrence of a side reaction under a high temperature. With the increase of the catalyst dose, the esterification of ergosterol with lauric acid was gradually activated and the conversion and selectivity also synchronously increased. However, the conversion and selectivity slightly varied with a further rise in the catalyst dose from 4 to 5%, indicating that 4% SDS + HCl was enough to make the substrate fully esterified. Effect of the Substrate Molar Ratio. The influence of the molar ratio of lauric acid/ergosterol on the conversion of ergosterol and the selectivity of ergosterol laurate was evaluated using a molar ratio of lauric acid/ergosterol from 1:1 to 4:1 (Figure 7). In general, equimolar lauric acid and ergosterol
Figure 4. Effect of different catalysts on the conversion of ergosterol and the selectivity of ergosterol laurate (2:1 molar ratio of lauric acid/ ergosterol, 100 °C, catalyst dose of 4%, and 2 h).
Figure 5. Effect of the reaction temperature on the conversion of ergosterol and the selectivity of ergosterol laurate (SDS + HCl as the catalyst, 2:1 molar ratio of lauric acid/ergosterol, catalyst dose of 4%, and 2 h).
Figure 7. Effect of the molar ratio of lauric acid/ergosterol on the conversion of ergosterol and the selectivity of ergosterol laurate (SDS + HCl as the catalyst, 120 °C, catalyst dose of 4%, and 2 h).
Figure 6. Effect of the catalyst dose on the conversion of ergosterol and the selectivity of ergosterol laurate (SDS + HCl as the catalyst, 2:1 molar ratio of lauric acid/ergosterol, 120 °C, and 2 h).
should be ideal for direct esterification in view of the economic perspective and further purification. However, such a ratio was not favorable for the desirable product synthesis, and the conversion was below 80%. The conversion were improved with the rise of the molar ratio of lauric acid/ergosterol from 1:1 to 2:1, which was ascribed to a chemical equilibrium shift toward esterification. However, the conversion of ergosterol slightly varied with a further increase of lauric acid/ergosterol from 2:1 to 4:1, indicating that excessive lauric acid could not promote the conversion when the molar ratio exceeded 2:1. Low selectivity of ergosterol laurate was observed at a 1:1 molar ratio of lauric acid/ergosterol, and the selectivity was enhanced with the increase of the molar amount of lauric acid. For this reaction system, both the esterification reaction and side reaction were carried out at the same time, while the selectivity of ergosterol laurate was improved as the esterification shifted with the increase of the molar amount of lauric acid. Therefore, a 2:1 molar ratio of lauric acid/ergosterol was considered to be optimal. Effect of the Reaction Time. The effect of the reaction time on the conversion of ergosterol and the selectivity of ergosterol 11753
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Funding
laurate was investigated under the above parameters, and the results are shown in Figure 8. In this experiment, all parameters
This study was financially supported by the National Natural Science Foundation of China (31401664), the China Postdoctoral Science Foundation Funded Project (2014M560406), the Research Fund for the Doctoral Program of Higher Education of China (20130093110010), and the Research Fund for Advanced Talents of Jiangsu University (13JDG070). Notes
The authors declare no competing financial interest.
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Figure 8. Effect of the reaction time on the conversion of ergosterol and the selectivity of ergosterol laurate (SDS + HCl as the catalyst, 2:1 molar ratio of lauric acid/ergosterol, catalyst dose of 4%, and 120 °C).
used have been selected by the above works. Obviously, the conversion of ergosterol rapidly increased with the extension of the reaction time, and the conversion reached 89% at 2 h. Furthermore, the highest selectivity was observed at 0.5 h and then slightly decreased with the rise of the reaction time. The conversion tended to gradually increase as the reaction time further extended from 2 to 4 h, indicating that the esterification of ergosterol with lauric acid had nearly reached equilibrium at 2 h. However, the selectivity of ergosterol laurate still decreased with a further increase of the reaction time from 2 to 4 h, suggesting that a small quantity of ergosterol and its laurate may be converted into side products. Therefore, the conversion and selectivity should be excellent when the esterification of ergosterol with lauric acid was performed for 2 h. Comparison of Solubility. The solubility of ergosterol in vegetable oil was below 0.9 g/100 mL, while the solubility of ergosterol laurate could reach above 5.7 g/100 mL under the same conditions, indicating that the coupling of ergosterol and lauric acid by direct esterification can significantly improve the solubility of ergosterol in oil, thus greatly facilitating the incorporation into a variety of oil-based systems. In recent years, ergosterol and its derivatives have attracted much attention for a variety of health benefits, while the latter showed excellent advantage in solubility. In the present study, ergosterol laurate was successfully and efficiently synthesized for the first time by direct esterification using SDS + HCl as the catalyst in a solvent-free system. Nevertheless, the side reaction should be prohibited further by exploring some moderate routes. Although the desired product (ergosterol laurate) was purified and characterized, its properties and application still need to be studied.
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