Sonochemical effects on free phenolic acids under ultrasound treatment in a model system

Ultrasonics Sonochemistry 20 (2013) 1017–1025

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Sonochemical effects on free phenolic acids under ultrasound treatment in a model system Liping Qiao, Xingqian Ye, Yujing Sun ⇑, Jieqi Ying, Yan Shen, Jianchu Chen Department of Food Science and Nutrition, School of Biosystems, Engineering and Food Science, Zhejiang University, Hangzhou 310058, PR China

a r t i c l e

i n f o

Article history: Received 3 June 2012 Received in revised form 16 December 2012 Accepted 22 December 2012 Available online 5 January 2013 Keywords: Ultrasound Phenolic acids Sonochemical effects Kinetics Degradation

a b s t r a c t Sonochemical effects on seven free phenolic acids under ultrasound treatment in a model system have been investigated. The degradation products have also been tentatively identified by FTIR and HPLC-UV-ESIMS. Five phenolic acids (protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, and ferulic acid) proved to be stable, while two others (caffeic acid and sinapic acid) were degraded under ultrasound treatment. The nature of the solvent and the temperature has been identified as important factors in determining the degradation reaction. Liquid height, ultrasonic intensity, and duty cycle of the ultrasound exposure affected only the degradation rate and did not change the nature of the degradation. The degradation rates of caffeic acid and sinapic acid decreased with increasing temperature. The degradation kinetics of these two acids under ultrasound conformed to zeroth-order reactions at 5 to 25 °C. Both decomposition and polymerization reactions occurred when caffeic acid and sinapic acid were subjected to ultrasound. Degradation products, such as the corresponding decarboxylation products and their dimers, have been tentatively identified. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction Phenolic acids constitute a subclass of a larger category of metabolites commonly referred to as ‘‘phenolics’’. They possess one carboxylic acid functional group. When describing plant metabolites, they comprise a distinct group of organic acids. These naturally occurring phenolic acids contain two distinguishing constituent carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures. Although the basic skeleton remains the same, the numbers and positions of the hydroxyl groups on the aromatic ring create the variety. Caffeic acid, p-coumaric acid, vanillic acid, ferulic acid, and protocatechuic acid are present in nearly all plants. Other acids are found in selected foods or plants (e.g., gentisic acid and syringic acid). Phenolic acids account for almost one-third of dietary phenols [1]. They possess antioxidant [2–4] and antibacterial activities [5,6], and can also reduce the incidence of tumors [7–9] and cardiovascular disease [10–12]. However, phenolic acids are sensitive to degradation due to their hydroxyl, methoxyl, carboxyl, and ethylene groups. Studies have been carried out to assess the impact of food processing on the stability of phenolic acids. For example, chlorogenic acid, caffeic acid, and cinnamic acid in apple juice were found to be degraded during ozonolysis of cloudy apple juice [13]. Roasting of ⇑ Corresponding author. Tel./fax: +86 57188982151. E-mail address: [email protected] (Y. Sun).

coffee resulted in the degradation of chlorogenic acid [14]. In roasted coffee, caffeic acid, quinic acid, and chlorogenic acid degraded to volatiles, and catechol derived from caffeic acid under anaerobic conditions was not oxidatively degraded [15,16]. Tanchev [17] studied the kinetics of the thermal degradation of gallic acid and protocatechuic acid, and found that the degradation of both acids depended on the pH and temperature. Degradation of phenolic acids may also occur in the course of their extraction from fruit and vegetables. For example, while gentisic acid, gallic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid were found to be stable at up to 100 °C during microwave-assisted extraction, at 125 °C they were significantly degraded [18]. Ultrasound-assisted extraction has been widely used for the extraction of phenolic acids due to the high extraction efficiency and extraction rate. However, the cavitation effect of ultrasound may accelerate or trigger chemical reactions in the extraction medium. For example, ultrasound treatment has been reported to cause an off-flavor in edible oils [19–22], the degradation of flavonoids [23] and carotenoids [24], and the aggregation and decomposition of polysaccharides [25,26]. The effects of high-power ultrasound on phenolic acids have seldom been reported. Ma et al. [27] found that the extraction yields of phenolic acids from citrus peel decreased with extended time at a relatively high temperature. Atanassova et al. [28] found that a combined ultrasonic/catalytic process could decompose 4-hydroxybenzoic acid in olive mill

1350-4177/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.12.007

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wastewater and was thus very promising for environmental applications. However, the mechanism of degradation of free phenolic acids under ultrasound treatment remains unclear. The objective of this study has been to determine the effects of different factors of ultrasound treatment on the stability of free phenolic acids in a model system, as well as the kinetics and products of their degradation. The results should further our understanding of the degradation mechanisms of free phenolic acids during ultrasound treatment. 2. Materials and methods 2.1. Chemicals HPLC-grade methanol was purchased from Tedia Company, Inc. (USA). Ethanol, acetone, and acetic acid (analytical grade) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Standard samples of protocatechuic acid (purity P97.0%), hydroxybenzoic acid (purity P99.5%), vanillic acid (purity P97.0%), caffeic acid (purity P98.0%), p-coumaric acid (purity P98.0%), ferulic acid (purity P99.0%), and sinapic acid (purity P98.0%) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Ultrasound treatment Ultrasound treatments (US) were carried out with a probe ultrasonic processor (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ningbo, China). This probe ultrasonic processor has a maximum ultrasound power output of 900 W, a frequency of 20–25 kHz, and a horn microtip of diameter 6 mm. An 8.0 lg/mL solution of standard phenolic acid was prepared in a volumetric flask. Aliquots of this solution were placed in brown glass tubes (3 cm diameter  10–20 cm height), which were in turn immersed in low-temperature thermostatic ethanol (T 6 0 °C) or water (T P 5 °C) bath (DC-1006, Safe Corporation, Ningbo, China) to maintain a constant temperature. The solutions were then treated with ultrasound. Apart from specific ultrasound conditions indicated in the results section, the general ultrasound conditions were as follows. The probe was placed 1 cm from the top surface of the extraction cell; the liquid height, measured as the distance from the horn microtip to the bottom of the tube, was 4 cm; the temperature was 5 °C (ethanol) or 5 °C (water); pulsed mode (2 s on and 2 s off) was applied; the treatment time was 60 min; and the ultrasound intensity was 2.3 W/cm2, as determined by a calorimetric method. A sample subjected to maceration under the same conditions was used as a control check (CK). The US and CK solutions were filtered through 0.45 lm polyvinylidene fluoride microfiltration membranes (Shanghai Xingya Purification Material Co., Shanghai, China) and then stored at 18 °C for subsequent HPLC analysis. 2.3. Calculation of ultrasonic intensity The ultrasonic intensity dissipated from the probe microtip was calculated according to the following formula [24]:

I¼

P

pr 2

2.4. Analytical method for phenolic acids HPLC analysis of phenolic acids was carried out on an LC-20 (Shimadzu) linked to an SPD-M20A. Treated phenolic acid solution (1 lL) was injected onto a reversed-phase 2.0  100 mm  2.9 lm CAPCELL PAK C18 MG S3 column (Shiseido Co., Japan). The column thermostat was set at 40 °C. Solvent A was 4% acetic acid/water, solvent B was methanol (A:B = 80:20), and the flow rate was 0.2 mL/ min. These conditions were in accordance with those of Xu et al. [29], with some revision. Hydroxycinnamic acid and hydroxybenzoic acid were monitored at 320 and 260 nm, respectively. All identified phenolic acids were quantified with external standards by UFLC analysis. The concentration of phenolic acids is expressed as micrograms per milliliter solution volume (lg/ mL). Seven different standard stock solutions with varying phenolic acid concentrations were prepared. Within the range 1–12 lg/ mL, the equation of linear regression was good with R2 > 0.997 for all measured phenolic acids. The repeatability of intra-day analysis ranged from RSD 0.13% to 1.23% (n = 3). For protocatechuic acid, hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid, the detection limits were 0.024, 0.028, 0.053, 0.035, 0.033, 0.047, and 0.056 lg/mL, respectively, and the quantification limits were 0.081, 0.094, 0.176, 0.117, 0.111, 0.155, and 0.188 lg/mL, respectively. 2.5. Modeling of degradation kinetics The reaction order of the degradation of phenolic acids under ultrasound treatment was determined using the integral method [30]. This method uses a trial-and-error procedure to identify the reaction order. If the order assumed is correct, the appropriate plot of the concentration (c)–time (t) data [c against t (zeroth-order), ln c against t (first-order), c1 against t (second-order), and c2 against t (third-order)] should be linear. The result showing the best correlation coefficient (R) was selected. The zeroth-order, first-order, second-order, and third-order models were as follows:

c  c0 ¼ kt

ð2Þ

ln c=c0 ¼ kt

ð3Þ

1=c  1=c0 ¼ kt

ð4Þ

1=c2  1=c20 ¼ kt

ð5Þ

where c is the concentration of the reactant at a given time, c0 is the initial concentration of the reactant, k is the rate constant, and t is the treatment time. 2.6. Determination of degradation products by FTIR spectroscopy The functional groups of the degradation products obtained at 5 to 25 °C were analyzed by FTIR spectroscopy using a Nicolet 5700 spectrometer (Thermo Fisher Scientific, USA). The wavenumber range covered was 400–4000 cm1. The spectral resolution was 1 cm1 and the collection time was about 1 min. The FTIR peaks were analyzed with Origin-lab 7.5.

ð1Þ

where r is the radius of the probe microtip, and P is the ultrasound power absorbed by the liquid as determined by a calorimetric method. In the present study, the levels of ultrasound power output were adjusted to 5%, 15%, 25%, 35%, and 45% of the total ultrasound power output (900 W), equating to 45, 135, 225, 315, and 405 W, respectively. The corresponding ultrasonic intensities were 2.1, 2.5, 2.3, 1.9, and 1.8 W/cm2, respectively.

2.7. Determination of degradation products by HPLC-UV-ESIMS HPLC-UV-ESIMS analyses were carried out on an Agilent 6460 Triple Quad LC/MS fitted with an ESI source. The HPLC conditions were in accordance with the above description with some revision. Solvent A was changed to water containing 0.1% formic acid. Data acquisition and processing were performed using Mass Hunter software. Positive- and negative-ion mass spectra of the column

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eluate were recorded in the range m/z 50–1000. The fragmentor was set at 135 V, the gas temperature was set at 325 °C, and the gas flow was 5 L/min. The sheath gas temperature was 250 °C and the sheath gas flow was 11 L/min. The nozzle voltage was 500 V. The capillary voltage was 4000 V in positive-ion mode and 3500 V in negative-ion mode. 2.8. Statistical analysis Each treatment was performed in triplicate. The results are expressed as mean ± SD. All the data were subjected to statistical analysis using SPSS16.0. The main effect of each factor of ultrasound treatment was subjected to analysis of variance and Duncan’s multiple range tests using the one-way ANOVA procedure. Mean values were considered significantly different when p < 0.05. 3. Results and discussion 3.1. Effect of solvent on the stability of free phenolic acids The effects of different solvents on the concentrations of seven phenolic acids treated by ultrasound are shown in Table 1 and Fig. 1(a) and (b). Protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, and ferulic acid were fairly stable in all of the solutions. Only caffeic acid and sinapic acid degraded significantly (p < 0.05) in the solutions, and the degradation rates in seven solvents were significantly different. Compared with the initial concentration, the concentrations of sinapic acid in water and 80% ethanol under ultrasound treatment were reduced by 16.34% and 11.88%, respectively, while that in methanol decreased by just 2.38%. The concentration of caffeic acid in 80% ethanol under ultrasound treatment was reduced by 8.90%, while that in water was decreased by just 1.02%. It could be concluded that ultrasound had much stronger chemical effects on sinapic acid in water and caffeic acid in 80% ethanol than on these acids in acetone and ethanol. The results indicated that the stability of phenolic acids under ultrasound depends not only on the nature of the phenolic acid but also on the solvent. Some phenolic acids, such as ferulic acid, proved to be stable in all of the solvents, but caffeic acid and sinapic acid degraded significantly in the respective media and their degradation rates in seven solvents were significantly different. Other researchers have found that polyphenols do not degrade under ultrasound treatment in polar or apolar solvents. For example, Pingret et al. [31] found that the main polyphenols (epicatechin, phloridzin, and chlorogenic acid) did not degrade during ultrasoundassisted water extraction of polyphenols from apple pomace, and that the total phenolics content and antioxidant activity of this ex-

tract were higher than those of a conventional extract. Achat et al. [32] found that ultrasound-assisted maceration of olive leaves greatly facilitated the enrichment of phenolic compounds in the extract and improved its antioxidant activity compared to that obtained by conventional maceration; tyrosol and hydroxytyrosol were not significantly degraded by sonication. Japón-Luján et al. [33] found that a dynamic ultrasound-assisted approach enhanced the contents of biophenols (oleuropein, verbascoside, apigenin7-glucoside, and luteolin-7-glucoside) in edible oils from olive leaves. The difference between the above literature reports and the present study may be due to the different polyphenols, as the main phenolic acid of apple pomace is chlorogenic acid, and phenolic acids are not among the main polyphenols of olive leaves. In other studies, we also found chlorogenic acid to be stable in the seven solvents, and that most flavonoids, with the exception of quercetin, are stable under ultrasound (unpublished). The seven polar solvents (methanol, ethanol, acetone, 80% ethanol, 80% acetone, 80% methanol, water) examined in the present study are commonly used for the extraction of phenolic acids by traditional methods [34–37]. Considering the extraction yields of phenolic acids, mixtures of alcohol (methanol or ethanol)/water or acetone/water are the best extraction solvents [34], but a pure organic solvent (methanol) may be the best extraction solvent under conditions of ultrasound treatment considering the stabilities of the seven studied phenolic acids. A mixture of ethanol and water is the most popular medium for the extraction of phenolics because of its green character and high extraction yields. However, in the present study, we found that caffeic acid and sinapic acid in 80% ethanol showed poor stability under ultrasound treatment. To elucidate this observation, further experiments were carried out using 80% ethanol. 3.2. Effect of temperature on the stability of free phenolic acids Seven phenolic acids in 80% ethanol were treated with ultrasound at 5, 5, 25, 45, and 65 °C. The range of temperatures studied was kept below the thermal degradation temperatures of the seven phenolic acids. The degrees of degradation of caffeic acid and sinapic acid in 80% ethanol decreased with increasing temperature (p < 0.05) (Fig. 2), and the other five phenolic acids were stable under the studied conditions (data not listed), either the present or the following conditions. The concentrations of caffeic acid and sinapic acid under ultrasound treatment were 83% and 78% of those in untreated samples at 5 °C, but were 98% and 98% of those in the untreated samples at 65 °C. The results may be explained in terms of decreasing cavitation intensity with increasing temperature. The physical properties (surface tension, viscosity, vapor pressure) of a solvent are the main factors affecting cavitation intensity, and the most important

Table 1 Effect of solvent on the stability of seven phenolic acids under ultrasound treatment (US) and maceration (CK). Solvent

Protocatechuic

p-Hydroxybenzoic

Vanillic

Caffeic

p-Coumaric

Ferulic

Sinapic

Methanol-CK Methanol-US Ethanol-CK Ethanol-US Acetone-CK Acetone-US 80% Ethanol-CK 80% Ethanol-US 80% Acetone-CK 80% Acetone-US 80% Methanol-CK 80% Methanol-US Water-CK Water-US

8.09 ± 0.07 8.13 ± 0.07 8.01 ± 0.03 7.94 ± 0.05 8.08 ± 0.30 7.87 ± 0.46 8.06 ± 0.07 7.84 ± 0.18 8.00 ± 0.17 7.82 ± 0.15 7.95 ± 0.03 7.82 ± 0.06 8.27 ± 0.12 8.13 ± 0.13

8.07 ± 0.06 8.15 ± 0.04 8.02 ± 0.02 8.10 ± 0.04 8.10 ± 0.29 7.93 ± 0.29 7.91 ± 0.05 7.99 ± 0.02 8.06 ± 0.10 8.08 ± 0.09 7.94 ± 0.04 7.96 ± 0.07 8.25 ± 0.10 8.25 ± 0.11

8.03 ± 0.09 8.09 ± 0.05 8.13 ± 0.08 8.04 ± 0.04 8.12 ± 0.28 7.99 ± 0.29 8.07 ± 0.09 8.05 ± 0.04 8.01 ± 0.26 8.08 ± 0.09 7.99 ± 0.09 8.00 ± 0.14 8.24 ± 0.26 8.05 ± 0.08

8.08 ± 0.10 7.85 ± 0.08 8.16 ± 0.03 7.57 ± 0.03 8.06 ± 0.30 7.37 ± 0.28 8.01 ± 0.09 7.30 ± 0.04 8.12 ± 0.17 7.88 ± 0.10 7.98 ± 0.06 7.41 ± 0.07 8.23 ± 0.14 8.14 ± 0.09

8.09 ± 0.06 8.11 ± 0.05 8.06 ± 0.04 8.15 ± 0.05 8.09 ± 0.37 8.10 ± 0.37 8.02 ± 0.10 8.19 ± 0.02 8.02 ± 0.18 8.02 ± 0.19 8.02 ± 0.06 8.07 ± 0.06 8.26 ± 0.21 8.19 ± 0.15

8.12 ± 0.03 8.17 ± 0.03 8.06 ± 0.03 8.13 ± 0.02 7.97 ± 0.14 7.84 ± 0.15 8.05 ± 0.05 7.92 ± 0.26 8.04 ± 0.10 8.26 ± 0.18 7.98 ± 0.07 7.83 ± 0.14 8.27 ± 0.08 8.14 ± 0.10

8.18 ± 0.05 7.99 ± 0.06 8.10 ± 0.02 7.59 ± 0.05 8.04 ± 0.44 7.57 ± 0.33 8.01 ± 0.19 7.06 ± 0.07 8.09 ± 0.20 7.41 ± 0.09 8.01 ± 0.06 7.79 ± 0.13 8.19 ± 0.10 6.85 ± 0.13

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Caffeic acid concentration(µg/ml)

8

CK US a

a b

(b)

ab

a

a cd

c

a

b

7 6 5 4 3 2 1 0 l l l e e ol ano ano ton ton ethano than Eth Ace 0%Eth Ace Me M % 8 80 80%

CK US

a a c

d

a

Sinapic acid concentration(µg/ml)

(a)

8

cd

cd

ab

ab

a c

a b

d

7

e

6 5 4 3 2 1 0 l ano Eth

nol tha Me

ter Wa

ab

a

ab

l l ne ne ano ano eto eto eth Eth Ac Ac M % % 80 80 80%

ter Wa

Solvent

Solvent

Fig. 1. Effect of solvent on the stability of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US) and maceration (CK). Different letters on bars show significant differences (p < 0.05).

(a)

CK US a

a

a

c

a

b

e

f

7

a d

8

Sinapic acid concentration (µg/ml)

Caffeic acid concentration (µg/ml)

8

CK US

(b)

6 5 4 3 2 1

a

a

a

a

c

a ab

d

7

e

f

6 5 4 3 2 1 0

0 -5

5

25

45

65

Temperature(°C)

-5

5

25

45

65

Temperature(°C)

Fig. 2. Effect of temperature on the stability of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US) and maceration (CK). Different letters on bars show significant differences (p < 0.05).

of these properties is the vapor pressure [38]. The vapor pressure of a solvent is negatively correlated with cavitation intensity and increases with increasing temperature. Therefore, cavitation intensity decreases with increasing temperature. These results are in agreement with the previous finding that the cavitation intensity decreased with an increase in temperature [24]. Romdhane et al. [39] found that the oxidation rate of aqueous potassium iodide decreased with increasing temperature. It was inferred that sonochemical reactions do not conform to Arrhenius theory.

3.3. Effect of liquid height on the stability of free phenolic acids Fig. 3 shows the effect of liquid height (the distance from the horn microtip to the bottom of the tube) on the two phenolic acids in 80% ethanol. As can be seen from the figure, the caffeic acid and sinapic acid concentrations increased significantly as the height was increased from 2 to 12 cm (p < 0.05). This may have been due to the fact that the cavitation intensity decreases with increasing

height because of attenuation of the waves caused by absorption and scattering. Other authors have also found that the maximum ultrasound power was observed in the vicinity of the radiating surface of the ultrasonic horn, and that ultrasonic intensity decreased rather abruptly with increasing distance from the radiating surface [40,41]. Somewhat different results were obtained by Sun et al. [24], who found that b-carotene concentration under ultrasound decreased markedly for heights ranging from 2 to 6 cm, then increased slightly for heights ranging from 6 to 12 cm. The difference may be ascribed to differences in the attenuation coefficients of the respective solutions.

3.4. Effect of ultrasonic intensity on the stability of free phenolic acids Fig. 4 shows the effects of ultrasonic intensity on the concentrations of caffeic acid and sinapic acid in 80% ethanol. The concentrations of the two phenolic acids did not change significantly on increasing the ultrasonic intensity from 1.8 to 2.5 W/cm2. Although the ultrasound power

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L. Qiao et al. / Ultrasonics Sonochemistry 20 (2013) 1017–1025

8

(b)

a

7 e

a

8

b

b

c

c

d

Sinapic acid concentration(µg/ml)

Caffeic acid concentration(µg/ml)

(a)

6 5 4 3 2 1 0

7

cd

bc

b

8

10

12

de

e f

6 5 4 3 2 1 0

CK

2

4

6

8

10

12

CK

2

4

6

Liquid height (cm)

Liquid height (cm)

Fig. 3. Effect of liquid height on the stability of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US). Different letters above the plotted points show significant differences (p < 0.05).

8

Caffeic acid concentration(µg/ml)

(b)

a b

b

b

b

b

7 6 5 4 3 2 1 0

Sinapic acid concentration(µg/ml)

(a)

8

a

7

b

b

b

1.8

1.9

2.1

b

b

2.3

2.5

6 5 4 3 2 1 0

CK

1.8

1.9

2.1

2.3

2.5

2

Ultrasonic intensity(W/cm )

CK

2

Ultrasonic intensity(W/cm )

Fig. 4. Effect of ultrasonic intensity on the stability of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US). Different letters above the plotted points show significant differences (p < 0.05).

output ranged from 45 to 405 W, the corresponding ultrasonic intensity ranged only from 1.8 to 2.5 W/cm2. It may be that it is easier to form cavitation bubbles and the bubbles collapse more violently with increasing ultrasonic power output in the range 45–225 W [42]. In the ultrasonic power output range 225–415 W, the cavitation bubbles may grow too large to collapse or they might collapse only weakly, thereby reducing the cavitation effect. Moreover, too many bubbles may hamper propagation of the ultrasonic wave [43].

3.5. Effect of duty cycle of ultrasonic exposure on the stability of free phenolic acids The effect of duty cycle on the concentrations of sinapic acid and caffeic acid in 80% ethanol was investigated at a pulse width of 2 s. Fig. 5 shows that both pulsed and continuous ultrasound decreased the concentrations of caffeic acid and sinapic acid. The degradation rate of caffeic acid under pulsed ultrasound was higher than that under continuous ultrasound, but the degradation rate of sinapic acid under pulsed ultrasound showed no significant difference from that under continuous ultrasound. As yet, we can offer no clear explanation for this. Indeed, the effects of duty cycle on cavitation phenomena in different literature reports have been

inconsistent. Luque-Garc´la et al. [44] found that duty cycle was not a major factor in relation to the extraction of total fat from oleaginous seeds. Sun et al. [24] found that b-carotene was degraded most rapidly when the duty cycle was 66.7%.

3.6. Degradation kinetics of free phenolic acids under ultrasound treatment In the present study, we only investigated the degradation kinetics of the two phenolic acids under ultrasound between 5 and 25 °C since only minor degradation occurred between 25 and 65 °C under ultrasound, and the acids were stable to maceration under the studied conditions. The corresponding determination coefficients (R2) of c, ln c, 1/c, and 1/c2 versus time at 5, 5, 15, and 25 °C are summarized in Table 2. According to the trial-and-error procedure, the degradation kinetics of caffeic acid and sinapic acid in 80% ethanol under ultrasound conformed to a zeroth-order reaction at 5 to 25 °C. The kinetic curves obtained at 5 to 25 °C are presented in Fig. 6. The concentrations of caffeic acid and sinapic acid were proportional to the treatment time at 5 to 25 °C. The kinetic parameters k, R2, and t1/2 were calculated according to the models at each temperature (Table 3). It can also be seen

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L. Qiao et al. / Ultrasonics Sonochemistry 20 (2013) 1017–1025

(a)

(b)

a c

c

a

8 b

Sinapic acid concentration (µg/ml)

bc

8

Caffeic acid concentration (µg/ml)

b c

7 6 5 4 3 2 1

b

b

40

50

7

b

b

66.7

100

6 5 4 3 2 1 0

0 CK

33.3

40

50

66.7

CK

100

33.3

Duty cycle(%)

Duty cycle(%)

Fig. 5. Effect of duty cycle on the stability of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US).

Table 2 Correlation coefficients (R) of c, ln c, 1/c, 1/c2 of caffeic acid (a) and sinapic acid (b) versus time at 5, 5, 15, and 25 °C under ultrasound treatment. T (°C)

R Zero (c)

R First (ln c)

R Second (1/c)

Table 3 Degradation kinetic parameters k (rate constant), R2 (determination coefficients), and t1/2 (half-lives) of caffeic acid (a) and sinapic acid (b) under ultrasound treatment.

R Third (1/c2)

T (°C)

k (lg ml1 min1) (p < 0.05)

R2 (p < 0.05)

t1/2 (min)

0.0331 0.0239 0.0104 0.00475

0.949 0.869 0.837 0.914

166.163 230.126 528.846 1157.895

0.0288 0.0250 0.0237 0.0179

0.774 0.901 0.972 0.964

154.514 178.000 187.764 248.603

(a) 5 5 15 25

0.974 0.932 0.915 0.956

0.98 0.929 0.916 0.957

0.983 0.924 0.917 0.957

0.983 0.918 0.917 0.957

(a) 5 5 15 25

(b) 5 5 15 25

0.88 0.949 0.986 0.982

0.878 0.943 0.985 0.982

0.869 0.936 0.982 0.982

0.852 0.925 0.979 0.982

(b) 5 5 15 25

that the degradation rates of both caffeic acid and sinapic acid were much higher at low temperature than at high temperature by comparing the values of k and t1/2. For example, the degradation rate of caffeic acid at 5 °C was seven times faster than that at 25 °C. 3.7. Analysis of the degradation products The HPLC-DAD chromatographic peaks and FTIR spectra of caffeic acid treated by ultrasound between 5 and 25 °C were similar,

(a)

-5 5 15 25

0.0

as were those of sinapic acid. We list only the degradation products of caffeic acid and sinapic acid at 5 °C. Several new chromatographic peaks appeared after caffeic acid and sinapic acid were treated by ultrasound (Fig. 7). The corresponding ESI mass spectral data of the degradation compounds from caffeic acid and sinapic acid are shown in Table 4. For caffeic acid, the ESI mass spectra of peak 1 showed the molecular ion [MH] at m/z 137.1 and [M+H]+ at m/z 139.0. Therefore, this compound was identified as having a molecular -5 5 15 25

(b) 0.0

•

-0.5 -0.5

g/ml)

-1.5

C-C0(

C-C0(

g/ml)

-1.0

-2.0 -2.5

-1.0 -1.5 -2.0

-3.0

-2.5

-3.5 0

10

20

30

40

50

Time (min)

60

70

80

0

10

20

30

40

50

60

70

Time (min)

Fig. 6. Degradation kinetic curves of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US) at 5, 5, 15, and 25 °C.

80

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L. Qiao et al. / Ultrasonics Sonochemistry 20 (2013) 1017–1025

Fig. 7. HPLC traces of caffeic acid (a) and sinapic acid (b) treated by ultrasound at 5 °C.

weight of 138, in accordance with the formula C8H10O2. For peak 2, the HPLC retention time (5.28 min) and mass spectra ([MH] at m/z 179.1, [M+H]+ at m/z 181.0, [MH2O+H]+ at m/z 163.0) are identical to those of the caffeic acid standard, hence this peak was unambiguously identified as being due to caffeic acid. The molecular weight of the species responsible for peak 3 was 492 according to [MH] at m/z 491.1 and [M+Na]+ at m/z 514.9. The molecular weight of the species responsible for peak 4 was 220 based on [MH] at m/z 219.1, [M+H]+ at m/z 220.8, and [M+Na]+ at m/z 243.0. Peak 5 was tentatively identified as being due to caffeic acid dimer, based on four molecular ions of [MH]

at m/z 357.1, [M+Cl] at m/z 393.1, [M+H]+ at m/z 359.0, and [M+Na]+ at m/z 381.0. For peaks 6, 8, 9, 10, and 11, no response was found in ESI mass spectra, and so further identification was needed. The molecular weight corresponding to peak 7 was 160 according to the molecular ions [M+H]+ at m/z 161.0 and [M+Na]+ at m/z 183.0. For sinapic acid, the ESI mass spectrum of peak 1 featured the molecular ion [M+H]+ at m/z 169.0, and so this compound was identified as having molecular weight 168, in accordance with the formula C9H12O3. Peak 2 corresponded to the molecular ion [M+H]+ at m/z 259.0, indicating a molecular weight of 258. For

Table 4 Assignments of new chromatographic peaks obtained by HPLC-DAD after ultrasound treatment of caffeic acid (a) and sinapic acid (b). Peak no.

HPLC tR (min)

Molecular weight

HPLC – ESIMS (m/z)

HPLC + ESIMS (m/z)

Tentative identification

(a) 1 2 3 4 5 7

3.97 5.36 8.94 9.66 10.23 13.20

138 180 492 220 358 160

137.1 179.1 491.1; 219.1 357.1; 393.1 –

139.0 163.0;181.0 514.9 220.8; 243.0 359.0; 381.0 161.0; 183.0

C8H10O2 Caffeic acidstd Undentified Undentified Caffeic dimer Undentified

(b) 1 2 3 4 5 8 12

4.26 5.62 7.04 7.88 10.34 21.17 51.41

168 258 182 222 224 225 446

– – – 220.9 223.1 224.1 445.1

169.0 259.0 183.0 223.0 207.0;225.0;247.0 226.0;248.0 447.1;469.0;485.0

C9O3H12 Undentified C10H14O3 C11H10O5 Sinapic acidstd C11H13O5 Sinapic dimer

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L. Qiao et al. / Ultrasonics Sonochemistry 20 (2013) 1017–1025

CK US

(a) 1.4

(b)

1.4

FTIR Absorbance

1.2

FTIR Absorbance

CK US

1.6

1.0 0.8 0.6

2360

1.2 1.0

3315

2920 2830

2510

0.8 0.6

0.4

0.4

0.2

0.2 0.0

0.0 4000 3600 3200 2800 2400 2000 1600 1200

800

400

4000 3600 3200 2800 2400 2000 1600 1200

Wavenumbers (cm-1)

800

400

Wavenumbers (cm-1)

Fig. 8. FTIR spectra of caffeic acid (a) and sinapic acid (b) after ultrasound treatment (US) and maceration (CK) at 5 °C. OH

(a)

O

O C

OH

HO

Caffeic dimer 1

C O

O HO

COOH

COOH COOH O

HO

HO

OH

Caffeic acid

Caffeic dimer 2

HO

HO

COOH

HO

COOH OH

CH2-CH3

HO

Caffeic dimer 3 OH

HO OH

Compound (Peak 1) OCH3 O H3CO

O C

OH

HO

(b)

OCH3

C O

O

Sinapic dimer1

H3CO H3CO COOH

H3CO

H3CO

COOH

COOH O

HO

HO

OCH3 H3CO

Sinapic acid

Sinapic dimer 2

H3CO

H3CO

COOH

HO

H3CO

peak 3, the molecular weight was 182 based on [M+H]+ at m/z 183.0. The molecular weight of the species responsible for peak 4 was 222, as indicated by [M+H]+ at m/z 223.0 and [MH] at m/z 220.9, in accordance with the formula C11H10O5. Peak 5 was unambiguously identified as being due to sinapic acid based on comparison of the HPLC retention time (10.26 min) and mass spectra ([MH] at m/z 223.1, [M+H]+ at m/z 225.0, [M+Na]+ at m/z 247.0, [MH2O+H]+ at m/z 207.0) with those of an authentic standard. The molecular weight of the species responsible for peak 8 was 225, in accordance with the formula C11H13O5, identified by [M+H]+ at m/z 226.0 and [M+Na]+ at m/z 248.0 along with [MH] at m/z 224.1. For peaks 6, 7, 9, 10, and 11, no response was found in ESI mass spectra. Peak 12 was tentatively identified as being due to sinapic acid dimer according to four molecular ions [MH] at m/z 445.1, [M+H]+ at m/z 447.1, [M+Na]+ at m/z 469.0, and [M+K]+ at m/ z 485.0. The FTIR spectra of caffeic acid before and after treatment by ultrasound at 5 to 25 °C were different, as were those of sinapic acid (Fig. 8). A new vibration peak at 2360 cm1 corresponding to m(CO2) was observed in the FTIR spectrum of caffeic acid treated by ultrasound (Fig. 8(a)). It indicated decarboxylation of caffeic acid under ultrasound treatment in this temperature range. The m(O–H) band in the range 2500–3500 cm1 of sinapic acid disappeared after treatment by ultrasound (Fig. 8(b)), indicating that the reaction of sinapic acid occurred at the site of hydroxylation. The ESI mass spectral data and the FTIR spectra of caffeic acid and sinapic acid treated by ultrasound indicated the occurrence of decomposition (decarboxylation) and polymerization reactions. The proposed degradation mechanisms of caffeic acid and sinapic acid under ultrasound are outlined in Fig. 9. In the present study, we only analyzed the molecular weights and functional groups of the degradation products. The structures of the degradation products will be further investigated in detail.

COOH

4. Conclusions

OCH3 HO

CH2 CH3

H3CO H3CO

H3CO

HO

CH3

OCH3 OH

Sinapic dimer 3

Compound (Peak 3) H3CO

Compound (Peak 1)

Fig. 9. The proposed degradation mechanisms of caffeic acid (a) and sinapic acid (b) under ultrasound treatment (US).

To obtain greater knowledge on the stability of phenolic acids during ultrasound-assisted extraction, we have analyzed the factors affecting degradation, the degradation kinetics, and degradation products in a model system. The results indicated that the seven studied phenolic acids were stable to maceration under the conditions examined. Five of these phenolic acids (protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, and ferulic acid) were stable, while two others (caffeic acid and sinapic acid) degraded under ultrasound treatment. The nature

L. Qiao et al. / Ultrasonics Sonochemistry 20 (2013) 1017–1025

of the solvent and the temperature proved to be important factors in determining the degradation reaction. Liquid height, ultrasonic intensity, and duty cycle of ultrasound exposure affected only the rate of degradation and did not change the nature of the degradation. The degradation rates of caffeic acid and sinapic acid decreased with increasing temperature. The degradation kinetics of caffeic acid and sinapic acid under ultrasound conformed to zeroth-order reactions at 5 to 25 °C. Decomposition and polymerization reactions occurred in caffeic acid and sinapic acid under ultrasound. The data collected in this study may prove very useful for application of the ultrasound techique for the extraction of phenolic acids.

Acknowledgments This project was supported by the National Natural Science Foundation of China (31071635; 31171784), the National Key Technology R&D Program (2012BAD31B06), and the Postdoctoral Foundation of China (316000-X91102; 316000-X91204).

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