Optimization of ultrasound-accelerated synthesis of enzymatic caffeic acid phenethyl ester by response surface methodology

Ultrasonics Sonochemistry 18 (2011) 455–459

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Optimization of ultrasound-accelerated synthesis of enzymatic caffeic acid phenethyl ester by response surface methodology Hsiao-Ching Chen a, Jiann-Hwa Chen b, Cheng Chang c, Chwen-Jen Shieh c,* a

Department of Bioindustry Technology, Dayeh University, Chang-Hua 515, Taiwan Graduate Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan c Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan b

a r t i c l e

i n f o

Article history: Received 2 November 2009 Received in revised form 20 July 2010 Accepted 26 July 2010 Available online 30 July 2010 Keywords: Caffeic acid phenethyl ester Lipase Optimization Response surface methodology (RSM) Ultrasound

a b s t r a c t The ultrasound-accelerated enzymatic synthesis of caffeic acid phenethyl ester (CAPE) from caffeic acid and phenethyl alcohol was investigated in this study. A commercial immobilized lipase from Candida antarctica, called NovozymÒ 435, was used as the catalyst. A 5-level-4-factor central-composite rotatable design (CCRD) and response surface methodology (RSM) were employed to evaluate the effects of reaction time, substrate molar ratio, enzyme amount, and ultrasonic power on percent molar conversion of CAPE. The results indicated that reaction time, substrate molar ratio, and ultrasonic power significantly affected percent molar conversion, whereas enzyme amount did not. A model for synthesis of CAPE was established. Based on ridge max analysis, the optimum condition for CAPE synthesis was predicted to be reaction time 9.6 h, substrate molar ratio 1:71, enzyme amount 2938 PLU, and ultrasonic power 2 W/cm2 with the molar conversion value of 96.03 ± 5.18%. An experiment was performed under this optimal condition and molar conversion of 93.08 ± 0.42% was obtained. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Plant materials contain various phenolic acids, such as caffeic acid, cinnamic acid, p-coumaric acid, and ferulic acid [1]. The phenolic nucleus of phenolic acids can readily form resonance-stabilized phenoxy radicals which are good radical scavengers [2]. Phenolic acids, therefore, carry strong anti-oxidation activities. Although phenolic acids extracted from plant materials have been approved for use as food anti-oxidants [3], their low solubility and low stability in various solvent systems have limited their uses in the food industry. One way to increase the solubility of phenolic acids in oil-based formulas and emulsions is to esterify the compounds with aliphatic alcohols. On the other hand, propolis is a resinous mixture of various plant materials collected by honeybees. It has several important biological properties including anti-inflammatory [4], anti-microbial [5], anti-oxidation, and anti-tumor activities [6]. Of the many chemical components identified so far in propolis, caffeic acid phenethyl ester (CAPE) is one major component and has been shown to carry the same biological activities as propolis including the anti-oxidation activity [7,8]. The structure of CAPE is shown in Fig. 1, indicating that it is a phenolic acid ester which should have good oil solubility. However, extraction of CAPE directly from prop* Corresponding author. Tel.: +886 4 2284 0452x5121; fax: +886 4 2286 1905. E-mail address: [email protected] (C.-J. Shieh). 1350-4177/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.07.018

olis is difficult and costly, precluding its general use as a food antioxidant. Many studies thus have focused on chemical synthesis of CAPE from another two plant compounds, caffeic acid and phenethyl alcohol. For instances, Chen et al. [9] used caffeic acid and phenethyl alcohol at molar ratio 1:5 to synthesize CAPE at room temperature in the presence of dicyclohexyl carbodiimide (DCC) and a 38% yield of CAPE was achieved. Lee et al. [10] synthesized CAPE by first heating a solution containing caffeic acid (5.6 mmol) and SOCl2, followed by adding phenethyl alcohol (8.4 mmol) and heating the resulting solution in vacuum. The crude product was then subjected to column chromatography purification and an 86% yield of CAPE was achieved. The disadvantages for chemical synthesis of CAPE is that, although the two substrates were natural materials, the reagents used for the synthesis were harmful to human and might caused environmental pollution. Recently, enzymatic synthesis of CAPE from caffeic acid and phenyl ethanol has been developed (Fig. 1). In contrast to the chemical synthesis, enzymatic synthesis of CAPE offers the advantages of specificity as well as milder reaction conditions and reagents. Lee et al. [11] used NovozymÒ 435 for the synthesis of ethyl ferulate (EF) and octyl methoxycinnamate (OMC) with a yield of 87% within 2 days at 75 °C for the synthesis of EF, while the OMC was 90% conversion at 80 °C within 1 day. High reaction temperature results were better for the conversion of cinnamic acid derivatives. Twu et al. [12] investigated NovozymÒ 435 to catalyze the direct esterification of hydroxyphenylpropionic acid and octanol

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Lipase

+

+

H 2O

Isooctane Caffeic acid

2-Phenyl ethanol

CAPE

Water

Fig. 1. Lipase-catalyzed synthesis of CAPE from caffeic acid and 2-phenyl ethanol.

in a solvent-free system, with a reaction time in 58.2 h and 95.9% molar conversion. In recent years, ultrasound has been used in extraction, emulsification, and chemical and/or enzymatic syntheses of chemical compounds [13,14]. High-energy ultrasonic waves would cause cavitation in the liquid solution. While subsequent collapses of the cavitation bubbles cause a thorough mixing and stirring of the liquid solution, the energy thus released could accelerate the chemical and/or enzymatic reactions that occurred in the solution [15,16]. For instance, Ribeiro et al. [17] demonstrated decrease in the reaction time for the rate of hydrolysis of ethyl 3-hydroxy-3phenylpropanoate by pig liver esterase (PLE), Pseudomonas cepacia lipase (PCL), Candida rugosa lipase (CRL) with the aid of ultrasound. Xiao et al. [18] investigated the effects of solvents, enzymes, chain lengths of the acyl donor, and ultrasonic power on synthesis of sugar esters in nonaqueous solvents, and found that higher power and continuous operation had a significantly positive effect. So far, there are few reports regarding the use of ultrasound in the chemical or enzymatic synthesis of phenolic acid ester. In this study, lipase-catalyzed synthesis of CAPE from caffeic acid and 2-phenyl ethanol by either sonication or mechanical mixing was investigated and the results were compared. The relationships between condition factors (reaction time, substrate molar ratio, enzyme amount, and ultrasonic power) and the response results (percent molar conversion) were revealed using a central-composite rotatable design (CCRD) and response surface methodology (RSM) analysis. Attempts were made to obtain optimal conditions for the enzymatic synthesis of CAPE from caffeic acid and 2-phenyl ethanol. 2. Materials and methods 2.1. Reagents Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3; NovozymÒ 435) from Candida antarctica supported on acrylic resin beads was purchased from Novo Nordisk Bioindustrials, Inc. (Bagsvaerd, Denmark). The catalytic activity of NovozymÒ 435 was 10,000 PLU (Propyl Laurate Units)/g containing 1–2% (w/w) water. Caffeic acid and 2-phenyl ethanol (99.5% pure) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Isooctane was purchased from Tedia Company Inc. (Fairfield, OH, USA). Methanol was purchased from Sigma Chemical Co (Steinheim, Germany). Molecular sieve (4 Å) was purchased from Davison Chemical (Baltimore, MD, USA). All other chemicals were analytical reagent grade. 2.2. Equipment The synthesis of enzymatic CAPE by ultrasound-acceleration was carried out in a 40 kHz ultrasonic bath. Ultrasound was input from a flat-plate ultrasonic transducer in the bath bottom (d = 4.5 cm). Ultrasonic power was adjusted using a scaled dial disc on the bath panel. The acoustic power was determined using the calorimetric method [19]. When the intensity of ultrasound is reported as W/cm2, the area of the transducer must be known to calculate the power delivered to a given volume of the bath.

2.3. Experimental design A 5-level-4-factor CCRD including 27 experiments was employed in this study. To avoid bias, the 27 runs were performed in a random order. The variables and their levels selected for the study of CAPE synthesis were reaction time 4–12 h, caffeic acid and 2-phenyl ethanol molar ratio 1:20–1:100, enzyme amount 1000–5000 PLU, and ultrasonic power 1.46–1.98 W/cm2. All experiments were performed in an isooctane (2 mL) system. Table 1 shows the independent factors (xi), levels, and experimental design in terms of codes and uncodes. 2.4. Lipase-catalyzed syntheses of CAPE Caffeic acid, 2-phenyl ethanol, and isooctane were dehydrated by 4 Å molecular sieve for 24 h before use. Initially, lipase-catalyzed synthesis of CAPE was carried out at 70 °C for 3, 6, 9 and 12 h either in an ultrasonic water bath at an ultrasonic power of 2 W/cm2 or in a shaking water bath at an agitation speed of 180 rpm. The reaction mixture contained 1 mM caffeic acid, 100 mM 2-phenyl ethanol, and 1000 PLU NovozymÒ 435 in isooctane. In another experiment, synthesis of CAPE was carried out in an ultrasonic bath with various ultrasonic powers for various reaction times. The reaction mixture contained 1 mM caffeic acid and different amounts of 2-phenyl ethanol and NovozymÒ 435 in isooctane (Table 1). 2.5. Confirmation and analysis of CAPE Methanol (10 mL; 5-fold v/v) was added in the synthesis reaction tube and filtrate, a 20 lL aliquot of the reaction mixture was loaded onto a Thermo C18 capillary column (5 lM, 250  4.6 mm, Agilent, USA) and HPLC (Hewlett–Packard 1100 series, USA) was performed in splitless mode. Elution was carried out using 0.1% trifluoroacetic acid (TFA) in acetonitrile at a flow rate of 0.7 mL/min, and the eluents were examined by UV light at a wavelength of 345 nm. 2.6. Statistical analysis The experimental data (Table 1) were analyzed by response surface regression (RSREG) procedures using SAS software to fit the following second-order polynomial Eq. (1):

Y ¼ b0 þ

4 X i¼1

bi xi þ

4 X

bii x2i þ

i¼1

3 4 X X

bij xi xj

ð1Þ

i¼1 j¼iþ1

where Y is the response (percent of molar conversion); b0 is a constant, bi, bii and bij are coefficients; xi and xj are the uncoded independent variables. The options of RSREG SAS and RIDGE MAX were employed to compute the estimated ridge of maximum response for increasing radii from the center of the original design. 3. Results and discussion 3.1. Prime experiment Lipase-catalyzed synthesis of CAPE from caffeic acid and 2-phenyl ethanol was carried out with either ultrasonic mixing

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H.-C. Chen et al. / Ultrasonics Sonochemistry 18 (2011) 455–459 Table 1 The 5-level-4-factor central-composite rotatable design for lipase-catalyzed synthesis of CAPE and response surface analysis of the experimental data. Treatment no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 a b

Factors Reaction time (h) X1

Substrate molar ratio (CA/PE)b X2

Enzyme amount (PLU) X3

Ultrasonic power (W/cm2) X4

Molar conversion (%) Y ± SD

1(6)a 1(6) 1(6) 1(6) 1(10) 1(10) 1(10) 1(10) 0(8) 1(6) 1(6) 1(6) 1(6) 1(10) 1(10) 1(10) 1(10) 0(8) 2(4) 2(12) 0(8) 0(8) 0(8) 0(8) 0(8) 0(8) 0(8)

1(1:40) 1(1:40) 1(1:80) 1(1:80) 1(1:40) 1(1:40) 1(1:80) 1(1:80) 0(1:60) 1(1:40) 1(1:40) 1(1:80) 1(1:80) 1(1:40) 1(1:40) 1(1:80) 1(1:80) 0(1:60) 0(1:60) 0(1:50) 2(1:20) 2(1:100) 0(1:60) 0(1:60) 0(1:60) 0(1:50) 0(1:50)

1(2000) 1(4000) 1(2000) 1(4000) 1(2000) 1(4000) 1(2000) 1(4000) 0(3000) 1(2000) 1(4000) 1(2000) 1(4000) 1(2000) 1(4000) 1(2000) 1(4000) 0(3000) 0(3000) 0(3000) 0(3000) 0(3000) 2(1000) 2(5000) 0(3000) 0(3000) 0(3000)

1(1.85) 1(1.59) 1(1.59) 1(1.85) 1(1.59) 1(1.85) 1(1.85) 1(1.59) 0(1.72) 1(1.59) 1(1.85) 1(1.85) 1(1.59) 1(1.85) 1(1.59) 1(1.59) 1(1.85) 0(1.72) 0(1.72) 0(1.72) 0(1.72) 0(1.72) 0(1.72) 0(1.72) 2(1.46) 2(1.98) 0(1.72)

50.37 ± 2.68 42.91 ± 0.03 51.70 ± 2.27 56.96 ± 3.19 41.56 ± 1.34 55.78 ± 1.46 78.73 ± 2.39 71.73 ± 2.51 50.04 ± 2.06 26.27 ± 0.07 49.27 ± 3.19 53.28 ± 2.28 39.34 ± 1.37 56.38 ± 0.11 41.07 ± 0.02 67.05 ± 0.91 79.48 ± 0.51 52.60 ± 1.97 37.80 ± 1.35 61.79 ± 1.23 28.76 ± 1.46 60.95 ± 1.16 52.08 ± 0.20 60.97 ± 3.70 63.40 ± 2.28 79.14 ± 4.73 49.76 ± 1.16

Numbers in parenthesis represent actual experimental amounts. CA: caffeic acid; PE: 2-phenyl ethanol.

100

180 rpm with ultrasonic power

80

Molar conversion (%)

Molar conversion (%)

100

60 40 20 0

0

3

6

9

12

15

Reaction time (h)

80 60 40 20 0

1.46

1.59

1.72

1.85

Ultrasonic power (W/cm 2 )

1.98

Fig. 2. Comparison between the ultrasonic mixing (2 W/cm2) and the mechanical agitation (180 rpm) on lipase-catalyzed synthesis of CAPE. The reaction conditions were set such that the substrate molar ratio was 1:100, the enzyme amount was 1000 PLU, and the reaction temperature was set at 70 °C.

Fig. 3. Effect of ultrasonic power on the synthesis of CAPE. The synthesis conditions include a reaction time of 12 h, a substrate molar ratio of 1:100, an enzyme amount of 5000 PLU, and a reaction temperature of 70 °C.

(2 W/cm2) or mechanical agitation (180 rpm). The reaction conditions were set with a substrate molar ratio (caffeic acid: 2-phenyl ethanol) of 1:100, an enzyme amount of 1000 PLU, a reaction temperature of 70 °C, and reaction times of 3, 6, 9 and 12 h. The results are shown in Fig. 2, which indicate that a higher molar conversion was obtained with ultrasonic mixing than with mechanistic mixing. This result is similar to that obtained by Ramachandran et al. [14], who reported that hydrolysis rates measured with ultrasonic mixing were better than mechanical agitation. In a subsequent experiment, lipase-catalyzed syntheses of CAPE in an ultrasonic bath at different ultrasonic powers (1.46–1.98 W/cm2) were carried out. The reaction conditions were set such that the substrate molar ratio was 1:100, the enzyme amount was 5000 PLU, the reaction temperature was set at 70 °C, and reaction time was 12 h. The results are shown in Fig. 3. It appears that higher molar

conversion was obtained with higher ultrasonic power. Xiao et al. [18] also demonstrated the ultrasound-accelerated enzymatic synthesis of sugar esters. They suggested that the acceleration was likely due to an increase in collisions between the two substrates and the enzyme by ultrasound. In order to systemically understand the relationships between reaction time, substrate molar ratio, enzyme amount, and ultrasonic power for the synthesis of CAPE, a 5-level-4-factor CCRD was applied and a total of 27 treatments (experiments) were carried out. RSM was then applied to analyze the experimental data. The results are shown in Table 1. Of the total of 27 treatments, treatment 17 (reaction time 10 h, substrate molar ratio 1:80, enzyme amount 4000 PLU, and ultrasonic power 1.85 W/cm2) resulted in the greatest molar conversion (79.48 ± 0.51%), whereas treatment 10 (reaction time 6 h, substrate molar ratio 1:40,

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enzyme amount 2000 PLU, and ultrasonic power 1.59 W/cm2) resulted in the smallest molar conversion (26.27 ± 0.07%) (Table 1).

cm2, respectively, affected the response molar conversion significantly (p < 0.01), but enzyme amount (x3) in the range of 1000– 5000 PLU did not exert significant effect on the response molar conversion (p = 0.5855).

3.2. Model fitting In order to obtain a model for CAPE synthesis, the results from the 5-level-4-factor CCRD experiment (Table 1) were used and the RSREG procedure from SAS was employed to fit the second-order polynomial Eq. (1). Eq. (2) was thus generated and is given below:

3.3. Mutual effect of parameters The mutual effects between reaction time and substrate molar ratio on the molar conversion of CAPE were investigated by the response surface plot. The results from the 5-level-4-factor CCRD experiment (Table 1) were used for constructing the plot. With the enzyme amount set at 3000 PLU and ultrasonic power set at 1.72 W/cm2, Fig. 4A shows the effects of reaction time from 4 to 12 h and substrate molar ratio from 1:20 to 1:100 on percent molar conversion. With the lowest reaction time (4 h) and the lowest substrate molar ratio (1:20), the yield was only 25% molar conversion. With the highest reaction time (12 h) and the highest substrate molar ratio (1:100), the yield of CAPE reached 90% molar conversion. The mutual effects between substrate molar ratio and ultrasonic power on the molar conversion of CAPE were also investigated by the same strategy. With the enzyme amount set at 3000 PLU and reaction time set at 8 h, Fig. 4B shows the effects of substrate molar ratio from 1:20 to 1:100 and ultrasonic power from 1.46 to 1.98 W/cm2 on molar conversion of CAPE. It appears that increases in substrate molar ratio and ultrasonic power led to higher yields of CAPE.

Yð%Þ ¼ 701:7017  3:7510x1 þ 1:3936x2 þ 0:0065x3  876:1618x4  0:1604x21 þ 0:1194x1 x2 þ 1:4306x1 x4  0:0047x22  0:6732x2 x4  0:0045x3 x4 þ 279:7329x24

ð2Þ

Analysis of variance (Table 2) indicates that this second-order polynomial model was highly significant and adequate to represent the actual relationship between the response (percent molar conversion) and the variables. The p-value was <0.0001 and the coefficient of determination (R2) was 0.945. The overall effect of the four synthesis variables on the percent molar conversion was further analyzed by a joint test. As shown in Table 3, reaction time (x1), substrate molar ratio (x2), and ultrasonic power (x4) in the ranges of 4–12 h, 1:20–1:100, and 1.46–1.98 W/ Table 2 Analysis of variances for the variables in the CAPE synthesis experiment pertaining to response percent molar conversion. Degree of freedom

Sum of squares

Prob. > Fa

Model Linear Quadratic Cross product Lack of fit Pure error Total error R2

14 4 4 6 9 3 12 0.9459

4774.12 3567.88 823.279 382.962 268.502 4.54 273.042

<0.0001 <0.0001 0.0013 0.0606 0.0161

3.4. Obtaining optimum synthesis condition The optimum condition for the synthesis of CAPE was determined by ridge max analysis [20], which computes the estimated ridge of maximum response for increasing radii from the center of the original design. Table 4 shows the determination. Maximum molar conversion of 96.03 ± 5.18% could be expected with reaction time 9.6 h, substrate molar ratio 1:71, enzyme amount 2938 PLU, and ultrasonic power of 2 W/cm2. Since both of the substrates for CAPE synthesis, caffeic acid and 2-phenyl ethanol, contain a benzene ring (Fig. 1), it is suggested that these two benzene rings need to be properly positioned for the esterification reaction to occur. This reaction time (9.6 h) was much shorter and high molar ratio (1:71) were required for maximum molar conversion (96.03 ± 5.18%). Yet, this reaction time (9.6 h) was much shorter than those required to obtain similar yields from other lipase-catalyzed esterification reactions in which no ultrasound was used. Twu et al. [12] studied NovozymÒ 435-catalyzed esterification of hydroxyphenyl propionic acid and octanol and obtained yield molar conversion 95.9% with a reaction time of 58.2 h. López Giraldo et al. [21] studied the lipase-catalyzed synthesis of chlorogenate fatty esters using different lipases and obtained yield molar conversions of 61–93% with reaction time of 96 h.

Prob. > F: level of significance.

Table 3 Analysis of variances in the CAPE synthesis experiment by joint test. Degree of freedom

Sum of squares

Prob. > Fa

Reaction time (X1) Substrate molar ratio (X2) Enzyme amount (X3) Ultrasonic power (X4)

5 5 5 5

1481.74 2001.41 88.247 1259.23

0.0001 <0.0001 0.5855b 0.0004

Prob. > F: level of significance. Not significant at p  0.01.

B

A

100

0

rr at io

)

(h

e

tio

20 4

n

6

tim

8 80 60 Subs 40 trate mola r ratio

ac

0

100 80 60

20 1.9 1.8

Ultras

onic p

40

1.7

1.6

ower (W

/cm 2)

1.5

20

ola

12 10

20

40

m

40

60

e

60

80

bs tra t

80

Re

Molar conversion

(%)

100

Su

a b

Factor

sion (%) Molar conver

a

Source

Fig. 4. Response surface plot showing effects of (A) reaction time and substrate molar ratio on the synthesis of CAPE; (B) substrate molar ratio and ultrasonic power on the synthesis of CAPE.

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H.-C. Chen et al. / Ultrasonics Sonochemistry 18 (2011) 455–459 Table 4 Estimated ridge of maximum response for variable percent molar conversion. Coded radius

0 0.2 0.4 0.6 0.8 1 1.2 a

Estimated response (% conversion) 50.92 56.1 61.88 68.54 76.34 85.47 96.03

Standard error

2.75 2.67 2.49 2.39 2.72 3.66 5.18

Uncoded factor values X1 (h)

X2 (CA/PE)a

X3 (PLU)

X4 (W/cm2)

8 8.45 8.87 9.18 9.37 9.49 9.57

60 64.89 68.43 70.45 71.26 71.34 71.03

3000 3028.73 3030.25 3013.41 2989.09 2963.24 2937.69

1.72 1.74 1.78 1.83 1.89 1.94 2

(CA/PE): CA: caffeic acid; PE: 2-phenyl ethanol.

It appears that ultrasound provide very effective mixing and stirring in the reaction solution and increases the contacts between the substrates and the enzyme. As the result, the reaction time may be considerably shortened. 3.5. Model verification The adequacy of the predicted model (Eq. (2)) was examined by carrying out the CAPE synthesis experiment under the optimal condition afore-mentioned. The results showed molar conversion of 93.08 ± 0.42% under the optimal condition. This observed molar conversion value did not significantly differ from the predicted molar conversion value of 96.03 ± 5.18%. Thus, the model developed, as shown in Eq. (2), adequately predicts the percent molar conversion in the CAPE synthesis reaction. 4. Conclusions The use of ultrasound in the NovozymÒ 435-catalyzed synthesis of CAPE in isooctane from caffeic acid and 2-phenyl ethanol was investigated. A 5-level-4-factor CCRD and RSM were employed for the experimental design and data analysis. Four parameters, i.e., reaction time, enzyme amount, substrate molar ratio, and ultrasonic power, were evaluated. Reaction time, substrate molar ratio, and ultrasonic power significantly affected the molar conversion, but enzyme amount did not. The experimental data was used to establish a CAPE synthesis model, and optimal synthesis conditions could be deduced from the model. The optimal synthesis conditions include a reaction time of 9.6 h, a substrate molar ratio of 1:71, an enzyme amount of 2938 PLU, and an ultrasonic power of 2 W/cm2, with a molar conversion of 96.03 ± 5.18%. A CAPE synthesis was performed under these optimal conditions and molar conversion of 93.08 ± 0.42% was obtained. Therefore, optimization of this ultrasound-accelerated synthesis of CAPE by NovozymÒ 435 was successful. References [1] S. Karboune, S.-L. Richard, S. Kermasha, Enzymatic synthesis of structured lipids by acidolysis of flaxseed oil with selected phenolic acid, J. Mol. Catal. B: Enzym. 52–53 (2008) 96–105.

[2] E. Graf, Antioxidant potential of ferulic acid, Free Radical Biol. Med. 13 (1992) 435–488. [3] E.M. Marinova, N.V. Yanishlieva, Effect of lipid unsaturation on the antioxidative activity of some phenolic acids, J. Am. Oil Chem. Soc. 71 (1994) 427–434. [4] S.L. De Castro, Propolis: biological and pharmacological activities. Therapeutic uses of this bee-product, Annu. Rev. Boimed. Sci. 3 (2001) 49–83. [5] G.A. Burdock, Review of the biological properties and toxicity of bee propolis (propolis), Food Chem. Toxicol. 36 (1998) 347–363. [6] H.B. Arjun, Y. Tezuka, S. Kadota, Review article recent progress in pharmacological research of propolis, Phytother. Res. 15 (2001) 561–571. [7] D. Grunberger, R. Banerjee, K. Eisinger, E.M. Oltz, L. Efros, M. Caldwell, V. Estevez, K. Nakanishi, Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis, Experientia 44 (1988) 230–232. [8] L. Liu, W.R. Hudgins, S. Shack, M.Q. Yin, D. Samid, Cinnamic acid: a natural product with potential use in cancer intervention, Int. J. Cancer 62 (1995) 345– 350. [9] J.H. Chen, Y. Shao, M.T. Huang, C.K. Chin, C.T. Ho, Inhibitory effect of caffeic acid phenethyl ester on human leukemia HL-60 cells, Cancer Lett. 108 (1996) 211– 214. [10] Y.J. Lee, P.H. Liao, W.K. Chen, C.C. Yang, Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells, Cancer Lett. 153 (2000) 51–56. [11] G.S. Lee, A. Widjaja, Y.H. Ju, Enzymatic synthesis of cinnamic acid derivatives, Biotechnol. Lett. 28 (2006) 581–585. [12] Y.K. Twu, I.L. Shin, Y.H. Yen, Y.F. Ling, C.J. Shieh, Optimization of lipasecatalyzed of octyl hydroxyphenylpropionate by response surface methodology, J. Agric. Food Chem. 53 (2005) 1012–1016. [13] Q.H. Chen, M.L. Fu, L. Jin, H.F. Zhang, G.Q. He, H. Ruan, Optimization of ultrasonic-assisted extraction (UAE) of betulin from white birch bark using response surface methodology, Ultrason. Sonochem. 16 (2009) 599–604. [14] K.B. Ramachandran, A.-Z. Sulaiman, C.S. Fong, C.W. Gak, Kinetic study on hydrolysis of oils by lipase with ultrasonic emulsification, Biochem. Eng. J. 32 (2006) 19–24. [15] V.G. Yachmenev, E.J. Blanchard, A.H. Lambert, Use of ultrasonic energy for intensification of the bio-preparation of greige cotton, Ultrasonics 42 (2004) 87–91. [16] C. Li, M. Yoshimoto, H. Ogata, N. Tsukuda, K. Fukunaga, K. Nakao, Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks, Ultrason. Sonochem. 12 (2005) 373–384. [17] C.M.R. Ribeiro, E.N. Passaroto, E.C.S. Brenelli, Ultrasound in enzymatic resolution of ethyl 3-hydroxy-3-phenylpropanoate, Tetrahedron Lett. 42 (2001) 6477–6479. [18] Y.M. Xiao, Q. Wu, Y. Cai, X.F. Lina, Ultrasound-accelerated enzymatic synthesis of sugar esters in nonaqueous solvents, Carbohyd. Res. 340 (2005) 2097–2103. [19] M.H. Entezari, P. Kruus, Effect of frequency on sonochemical reactions I: oxidation of iodide, Ultrason. Sonochem. 1 (1994) S75–S79. [20] SAS, SAS User’s Guide, SAS Institute, Cary, USA, 1990. [21] L.J. López Giraldo, M. Laguerre, J. LecomteM.-C.F. Espinoza, N. Barouh, B. Baréa, P. Villeneuve, Lipase-catalyzed synthesis of chlorogenate fatty esters in solvent-free medium, Enzyme Microb. Technol. 41 (2007) 721–726.