Article pubs.acs.org/JAFC
Degradation of Caffeic Acid in Subcritical Water and Online HPLCDPPH Assay of Degradation Products Pramote Khuwijitjaru,*,† Boonyanuch Suaylam,† and Shuji Adachi‡ †
Department of Food Technology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailand ‡ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan S Supporting Information *
ABSTRACT: Caffeic acid was subjected to degradation under subcritical water conditions within 160−240 °C and at a constant pressure of 5 MPa in a continuous tubular reactor. Caffeic acid degraded quickly at these temperatures; the main products identified by liquid chromatography-diode array detection/mass spectrometry were hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol. The reaction rates for the degradation of caffeic acid and the formation of products were evaluated. Online highperformance liquid chromatography/2,2-diphenyl-1-picryhydrazyl assay was used to determine the antioxidant activity of each product in the solution. It was found that the overall antioxidant activity of the treated solution did not change during the degradation process. This study showed a potential of formation of antioxidants from natural phenolic compounds under these subcritical water conditions, and this may lead to a discovering of novel antioxidants compounds during the extraction by this technique. KEYWORDS: 3,4-dihydroxycinnamic acid, stability, free radical scavenging
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INTRODUCTION Subcritical water, which is defined as liquid water at 100−374 °C under pressurized conditions, is gaining much interest for use as an extraction solvent for components from several kinds of materials.1,2 The extraction of phenolic compounds has been studied particularly frequently by a number of researchers because these compounds exhibit a variety of bioactive activities, especially antioxidant and antimicrobial activities, and therefore can be used as food ingredients in place of synthetic compounds. However, the severe conditions required for subcritical water extraction tend to adversely affect thermally labile substances, including several phenolic compounds. Studies have shown that raising the temperature of the subcritical water above certain values resulted in decreased amounts of phenolic compounds.3,4 However, only a few quantitative studies on the degradation of phenolic compounds under subcritical water conditions have been reported.5,6 We recently reported a study on the kinetics of the decomposition of nine phenolic acids, caffeic, chlorogenic, p-coumaric, gallic, gentisic, p-hydroxybenzoic, protocatechuic, syringic, and vanillic acids, and catechin in subcritical water.7 Caffeic acid (3,4-dihydroxycinnamic acid) is found in several plants and exhibits relatively high antioxidant activity compared to those of other phenolic acids.8 In our previous study,7 we found that, despite degradation of caffeic acid, a solution of caffeic acid after treatment under subcritical water conditions still exhibited 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity; this implies that some products of the degradation of caffeic acid under subcritical water conditions might be active for DPPH radical scavenging and stable under these conditions. © 2014 American Chemical Society
The objective of this study was to evaluate the degradation kinetics of caffeic acid in subcritical water. In addition, the degradation products were identified, and their DPPH radical scavenging activities were estimated to demonstrate the potential of new antioxidants formation from this natural phenolic compound in subcritical water that can be considered as a green extraction technique.
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MATERIALS AND METHODS
Chemicals. Caffeic acid (≥95%), gallic acid (monohydrate, ≥98%), and 2,2-diphenyl-1-picryhydrazyl were purchased from Sigma−Aldrich (St. Louis, MO, USA). L-Ascorbic acid was purchased from Riedel-de Haën (Hanover, Germany). All other chemicals were of analytical grade. Degradation of Caffeic Acid in Subcritical Water. A continuous flow-type reactor was used to study the degradation of caffeic acid in the temperature range 160−240 °C (Figure 1). A caffeic acid solution (100 mg/L) was prepared in distilled water and degassed
Figure 1. Apparatus for subcritical water treatment. Received: Revised: Accepted: Published: 1945
October 29, 2013 January 27, 2014 February 1, 2014 February 1, 2014 dx.doi.org/10.1021/jf404850a | J. Agric. Food Chem. 2014, 62, 1945−1949
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for 15 min using an ultrasonic bath (Tru-Sweep 275D, Crest Ultrasonic, Malaysia). The solution was delivered from an amber glass bottle, which was connected via a helium gas balloon to prevent oxygen contamination, through a tubular reactor comprising stainless steel tubing (0.75 mm i.d., 1.58 mm o.d.) immersed in a temperaturecontrolled silicone fluid bath using a high-performance liquid chromatography (HPLC) pump (LC-20AD, Shimadzu, Kyoto, Japan). The length of the reactor coil and the flow rate were adjusted to obtain the desired treatment times (40−1080, 20−340, 20−240, 10−80, and 5−30 s for 160, 180, 200, 220, and 240 °C, respectively). A thermocouple (type K) was connected via a tee connector near the initial part of the reactor (∼10 cm under the fluid surface) to monitor the temperature of the solution inside the tube. After treatment, the solution flowed through a cooling coil immersed in an ice−water bath to rapidly stop the reaction. A miniature back-pressure regulator (P880, Upchurch Scientific, WA, USA) was connected to the end of the tube to maintain a constant pressure of 5 MPa. This pressure was high enough to ensure that the solution remained in the liquid state throughout the temperature range. HPLC-DAD Analysis of Caffeic Acid and Its Degradation Products. Caffeic acid and its degradation products in the treated solution were quantitatively analyzed using an HPLC system (Shimadzu, Kyoto, Japan) comprised of a solvent delivery unit (LC20AD), a diode array detector (DAD; SPD-M20A), and a system controller (CBM-20A). The sample was chromatographically separated using an Inertsil ODS-3 column (4.6 mm × 150 mm, GL Sciences, Tokyo, Japan). The mobile phase consisted of 0.5% v/v aqueous acetic acid (A) and methanol (B), and the following gradient was used at a flow rate of 1.5 mL/min: 0−5 min, 5% B; 5−9 min, linear increase to 50% B; and 9−12 min, 50% B. The treated sample was filtered through a 0.45 μm nylon syringe filter, and 20 μL of sample was injected into the HPLC instrument. Caffeic acid and its degradation products were monitored at 280 nm with authentic caffeic acid as a calibration standard. However, since one of the major degradation products (4-vinylcatechol) is not commercially available, the concentrations of degradation products were estimated, after identification by liquid chromatography-diode array detection/mass spectrometry (LC-DAD/MS), using a calibration curve of caffeic acid corrected by the ratio of the molar absorptivities of caffeic acid (ε = 18 200 M−1 cm−1) and the products, that is, 2951 M−1 cm−1 for hydroxytyrosol,9 10 233 M−1 cm−1 for protocatechuic aldehyde,10 and 2692 M−1 cm−1 for 4-vinylcatechol.11 Identification of Degradation Products Using LC-DAD/MS. Agilent 1100 LC/MSD Trap SL (Agilent Technologies, CA, USA) equipped with a solvent delivery unit (G113A), a diode array detector (G1315D), and an ion-trap mass spectrometer (G2445D) with an electrospray ionization ion source was used to identify the degradation products. Chromatographic separation was accomplished using the same column described above. The mobile phase consisted of 0.5% v/ v aqueous acetic acid and methanol (85:15 v/v) and flowed at a rate of 0.4 mL/min. The treated sample was filtered through a 0.45 μm nylon syringe filter, and 30 μL of sample was injected. Both positive and negative modes were performed over the m/z range 50−400. Online HPLC-DPPH Assay. Evaluation of the DPPH radical scavenging activity of caffeic acid and each degradation product in the treated samples was conducted using an online HPLC-DPPH assay.12 This method is based on the chromatographic separation of components in the sample and a postcolumn reaction with DPPH solution. In this study, only one DAD detector was used to monitor caffeic acid, the degradation products at 280 nm, and the negative peaks due to DPPH bleaching at 517 nm. A first pump was used to deliver the mobile phase that consisted of 0.5% v/v aqueous acetic acid and methanol (85:15 v/v) at a flow rate of 1 mL/min for separating the compound on the column described above, while another pump delivered 6 × 10−5 M DPPH in methanol or pure methanol at a flow rate of 0.8 mL/min through tubing that was connected via a tee connector to the column outlet. A reaction coil made with PEEK tubing (28.8 m) was connected to enable the compounds eluted from the column to react with DPPH for 47 s. Ascorbic acid (AA) was used
for calibration of the DPPH radical scavenging assay via the negative peak area.
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RESULTS AND DISCUSSION Degradation Products of Caffeic Acid in Subcritical Water. A typical HPLC chromatogram of the solution treated under subcritical water conditions is shown in Figure S1 (Supporting Information). There were many compounds in the treated solution, but only the following three main compounds, which could be accurately identified by the LC-DAD/MS, are proposed to be degradation products of caffeic acid: hydroxytyrosol (3,4-dihydroxyphenylethanol), protocatechuic aldehyde (3,4-dihydroxybenzaldehyde), and 4-vinylcatechol (2hydroxy-4-vinylphenol) (Figure 2). The mass/charge ratio (m/
Figure 2. Proposed pathway of caffeic acid degradation in subcritical water.
z) and maximum absorbance (λmax) values were used to identify these compounds, as shown in Table 1. Only the standard Table 1. Characteristics of Caffeic Acid and Its Degradation Products Obtained by LC-DAD/MS λmax (nm)
[M − H]− m/z
220, 279 228, 278, 311 324
153 137
3
hydroxytyrosol protocatechuic aldehyde caffeic acid
179
4
4-vinylcatechol
259
135
peak no. 1 2
compd
negative ions m/z 153 137 − 179 − 135
[M − H]− [M − H]−; 108 [M CHO]− [M − H]−; 135 [M COOH]− [M − H]−
caffeic acid was injected to confirm the accuracy of the mass spectrometer. Decarboxylated products are the main products of the degradation of phenolic acids under subcritical water conditions. For example, degradation of vanillic acid gave 2methoxy-phenol,5 whereas degradation of syringic acid gave syringol (1,3-dimethoxy-2-hydroxybenzene).6 In this study, 4vinylcatechol was detected as a decarboxylated product and was a main product in the treated solution (∼38−57 mol %). It is known that 4-vinylcatechol can be formed through thermal degradation13 and photodegradation14 and can also be prepared by heating caffeic acid at 110 °C using a sodium acetate catalyst.15 However, 4-ethylcatechol, which was also formed during pyrolysis, was not detected in this study. Hydroxytyrosol was found at moderate amounts in the treated solution (∼14− 43 mol %); this compound is an effective antioxidant that is naturally found in olive oil.16 However, the pathway for the 1946
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Figure 3. Caffeic acid degradation products at (a) 160, (b) 180, (c) 200, (d) 220, and (e) 240 °C. Caffeic acid (▲), hydroxytyrosol (◇), protocatechuic aldehyde (□), and 4-vinylcatechol (○).
formation of hydroxytyrosol from caffeic acid remains unclear. A small amount of protocatechuic aldehyde was also found in the treated solution. It has been reported that thermal degradation of chlorogenic acid, which is an ester of caffeic acid and quinic acid, can form a small amount of protocatechuic aldehyde.17 In addition, protocatechuic aldehyde has been reported to be a main oxidation product of caffeic acid in the Fenton oxidation system.18 Kinetics analysis was performed along the pathways shown in Figure 2. The reactions can be described using the following first-order rate equations: dCCA = −kHTCCA − kPACCA − k4VCCCA dt
dC HT = kHTCCA dt
(2)
dC PA = kPACCA dt
(3)
dC4VC = k4VCCCA dt
(4)
where CCA, CHT, CPA, and C4VC are the concentrations of caffeic acid, hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol, respectively, and the k terms are the rate constants for each reaction step. Equations 1−4 were solved by integration and substitution to generate the following equations:
(1) 1947
dx.doi.org/10.1021/jf404850a | J. Agric. Food Chem. 2014, 62, 1945−1949
Journal of Agricultural and Food Chemistry CCA = CCA0e−k totalt
Article
C HT =
kHT CCA0(1 − e−k totalt ) k total
(6)
C PA =
kPA CCA0(1 − e−k totalt ) k total
(7)
C4VC =
k4VC CCA0(1 − e−k totalt ) k total
in Figure 5. The total radical scavenging activity of the treated solution, which was calculated from the areas of all seven
(5)
(8)
where ktotal is the overall degradation rate constant of caffeic acid and therefore equal to kHT + kPA + k4VC, and CCA0 is the initial concentration of caffeic acid. First, the overall degradation rate constant (ktotal) was determined by nonlinear regression fitting of eq 5 to the changes in the caffeic acid concentration with time; then, eqs 6−8 were fitted to obtain the rate constants. All the reaction rate constants and their standard errors were estimated using the nls function in R statistical language;19 the results are shown in Figure 3. The standard errors of the estimated rate constants were less than 5% in all cases. It is evident that the experimental results were in good agreement with the proposed reaction pathway. The overall degradation rate constants (ktotal) increased with temperature and followed the Arrhenius relationship (Figure S2 in the Supporting Information) with an activation energy (Ea) of 93 kJ/mol and a pre-exponential factor (A) of 1.78 × 108 s−1. This value is significantly higher than that for vanillic acid degradation (57 kJ/mol) in the temperature range 280−375 °C.5 The formation rate constant for each product also obeyed the Arrhenius equation with Ea values of 103, 85, and 87 kJ/mol and A values of 8.24 × 108, 1.06 × 10 6 , and 2.46 × 10 7 s −1 for hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol, respectively. Online HPLC-DPPH Assay. The antioxidant activity of caffeic acid and its degradation products during subcritical water treatment at 240 °C were estimated using an online HPLC-DPPH assay. A typical chromatogram is shown in Figure 4. The seven peaks that were detected at 280 nm appeared as negative peaks at 517 nm, which indicates their ability to quench the DPPH radicals. Changes in the DPPH radical scavenging activity of each identified product are shown
Figure 5. DPPH radical scavenging activity of caffeic acid (▲), hydroxytyrosol (◇), protocatechuic aldehyde (□), and 4-vinylcatechol (○) in the solution treated by subcritical water at 240 °C. The overall activity (●) was calculated from the summation of the areas of the negative peaks in Figure 5.
negative peaks, was not affected by the treatment time (t test, p ≥ 0.05) even though the caffeic acid content gradually decreased. We found that the radical scavenging activity changed linearly with the concentration of each compound; from these relationships, the DPPH radical scavenging activity of each compound was estimated to be ∼88, 212, 230, and 292 g AA/mol for hydroxytyrosol, protocatechuic aldehyde, caffeic acid, and 4-vinylcatechol, respectively. However, because the concentrations of the degradation products were estimated, we can only conclude that protocatechuic aldehyde, caffeic acid, and 4-vinylcatechol showed higher DPPH radical scavenging activities than hydroxytyrosol. Obied et al. reported that caffeic acid was more active for DPPH radical scavenging than hydroxytyrosol.20 It should be noted that the DPPH radical scavenging activities of these four compounds accounted for more than 90% of the overall activity. From these results, it can be expected that, during an extraction of plant materials containing phenolic compounds using the subcritical water technique, which is usually performed in a batch type extractor for longer periods, similar circumstances might occur for caffeic acid and other compounds as well. More complex reactions might also be possible because there are several compounds existing in sample matrices. This can be an advantage if degradation products possess some required bioactive activities or a disadvantage if undesirable or even toxic products are formed.
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ASSOCIATED CONTENT
S Supporting Information *
HPLC chromatogram of caffeic acid after subcritical water treatment and Arrhenius plot of the reaction rates. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Figure 4. HPLC chromatogram of caffeic acid after subcritical water treatment detected at 280 and 517 nm. The negative peaks indicate DPPH radical scavenging activity.
*Phone: +66-034-219361; fax: +66-034-272194; e-mail: [email protected]. 1948
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Funding
(17) Moon, J.-K.; Shibamoto, T. Formation of volatile chemicals from thermal degradation of less volatile coffee components: Quinic acid, caffeic acid, and chlorogenic acid. J. Agric. Food Chem. 2010, 58, 5465−5470. (18) Antolovich, M.; Bedgood, D. R.; Bishop, A. G.; Jardine, D.; Prenzler, P. D.; Robards, K. LC-MS investigation of oxidation products of phenolic antioxidants. J. Agric. Food Chem. 2004, 52, 962−971. (19) R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2013. (20) Obied, H. K.; Prenzler, P. D.; Robards, K. Potent antioxidant biophenols from olive mill waste. Food Chem. 2008, 111, 171−178.
This work was supported by a cofunded project between the Thailand Research Fund, the Office of the Higher Education Commission, and the Faculty of Engineering and Industrial Technology, Silpakorn University (MRG5380123). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS PK thanks Associate Professor Dr. Rungnaphar Pongsawatmanit, Kasetsart University for her valuable discussion from the beginning of the project.
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REFERENCES
(1) Herrero, M.; Cifuentes, A.; Ibañez, E. Sub- and supercritical fluid extraction of functional ingredients from different natural sources: Plants, food-by-products, algae and microalgae: A review. Food Chem. 2006, 98, 136−148. (2) Wiboonsirikul, J.; Adachi, S. Extraction of functional substances from agricultural products or by-products by subcritical water treatment. Food Sci. Technol. Res. 2008, 14, 319. (3) Singh, P. P.; Saldaña, M. D. A. Subcritical water extraction of phenolic compounds from potato peel. Food Res. Int. 2011, 44, 2452− 2458. (4) Vergara-Salinas, J. R.; Bulnes, P.; Zúñiga, M. C.; Pérez-Jiménez, J.; Torres, J. L.; Mateos-Martín, M. L.; Agosin, E.; Pérez-Correa, J. R. Effect of pressurized hot water extraction on antioxidants from grape pomace before and after enological fermentation. J. Agric. Food Chem. 2013, 61, 6929−6936. (5) González, G.; Salvadó, J.; Montané, D. Reactions of vanillic acid in sub- and supercritical water. J. Supercrit. Fluids 2004, 31, 57−66. (6) Lindquist, E.; Yang, Y. Degradation of benzoic acid and its derivatives in subcritical water. J. Chromatogr. A 2011, 1218, 2146− 2152. (7) Khuwijitjaru, P.; Plernjit, J.; Suaylam, B.; Samuhaseneetoo, S.; Pongsawatmanit, R.; Adachi, S. Degradation kinetics of some phenolic compounds in subcritical water and radical scavenging activity of their degradation products. Can. J. Chem. Eng. doi:10.1002/cjce.21898. (8) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm.−Wiss. Technol. (1968−2004) 1995, 28, 25−30. (9) Owen, R. W.; Haubner, R.; Mier, W.; Giacosa, A.; Hull, W. E.; Spiegelhalder, B.; Bartsch, H. Isolation, structure elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes. Food Chem. Toxicol. 2003, 41, 703−717. (10) Achenbach, H.; Benirschke, G. Joannesialactone and other compounds from Joannesia princeps. Phytochemistry 1997, 45, 149− 157. (11) Albert, S.; Horbach, R.; Deising, H. B.; Siewert, B.; Csuk, R. Synthesis and antimicrobial activity of (E) stilbene derivatives. Bioorg. Med. Chem. 2011, 19, 5155−5166. (12) Niederländer, H. A. G.; van Beek, T. A.; Bartasiute, A.; Koleva, I. I. Antioxidant activity assays on-line with liquid chromatography. J. Chromatogr. A 2008, 1210, 121−134. (13) Stadler, R. H.; Welti, D. H.; Stämpfli, A. A.; Fay, L. B. Thermal decomposition of caffeic acid in model systems: Identification of novel tetraoxygenated phenylindan isomers and their stability in aqueous solution. J. Agric. Food Chem. 1996, 44, 898−905. (14) Le Person, A.; Lacoste, A.-S.; Cornard, J.-P. Photo-degradation of trans-caffeic acid in aqueous solution and influence of complexation by metal ions. J. Photochem. Photobiol., A 2013, 265, 10−19. (15) Terpinc, P.; Polak, T.; Šegatin, N.; Hanzlowsky, A.; Ulrih, N. P.; Abramovič, H. Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food Chem. 2011, 128, 62−69. (16) Cornwell, D. G.; Ma, J. Nutritional benefit of olive oil: The biological effects of hydroxytyrosol and its arylating quinone adducts. J. Agric. Food Chem. 2008, 56, 8774−8786. 1949
dx.doi.org/10.1021/jf404850a | J. Agric. Food Chem. 2014, 62, 1945−1949
Degradation of Caffeic Acid in Subcritical Water and Online HPLCDPPH Assay of Degradation Products Pramote Khuwijitjaru,*,† Boonyanuch Suaylam,† and Shuji Adachi‡ †
Department of Food Technology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailand ‡ Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan S Supporting Information *
ABSTRACT: Caffeic acid was subjected to degradation under subcritical water conditions within 160−240 °C and at a constant pressure of 5 MPa in a continuous tubular reactor. Caffeic acid degraded quickly at these temperatures; the main products identified by liquid chromatography-diode array detection/mass spectrometry were hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol. The reaction rates for the degradation of caffeic acid and the formation of products were evaluated. Online highperformance liquid chromatography/2,2-diphenyl-1-picryhydrazyl assay was used to determine the antioxidant activity of each product in the solution. It was found that the overall antioxidant activity of the treated solution did not change during the degradation process. This study showed a potential of formation of antioxidants from natural phenolic compounds under these subcritical water conditions, and this may lead to a discovering of novel antioxidants compounds during the extraction by this technique. KEYWORDS: 3,4-dihydroxycinnamic acid, stability, free radical scavenging
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INTRODUCTION Subcritical water, which is defined as liquid water at 100−374 °C under pressurized conditions, is gaining much interest for use as an extraction solvent for components from several kinds of materials.1,2 The extraction of phenolic compounds has been studied particularly frequently by a number of researchers because these compounds exhibit a variety of bioactive activities, especially antioxidant and antimicrobial activities, and therefore can be used as food ingredients in place of synthetic compounds. However, the severe conditions required for subcritical water extraction tend to adversely affect thermally labile substances, including several phenolic compounds. Studies have shown that raising the temperature of the subcritical water above certain values resulted in decreased amounts of phenolic compounds.3,4 However, only a few quantitative studies on the degradation of phenolic compounds under subcritical water conditions have been reported.5,6 We recently reported a study on the kinetics of the decomposition of nine phenolic acids, caffeic, chlorogenic, p-coumaric, gallic, gentisic, p-hydroxybenzoic, protocatechuic, syringic, and vanillic acids, and catechin in subcritical water.7 Caffeic acid (3,4-dihydroxycinnamic acid) is found in several plants and exhibits relatively high antioxidant activity compared to those of other phenolic acids.8 In our previous study,7 we found that, despite degradation of caffeic acid, a solution of caffeic acid after treatment under subcritical water conditions still exhibited 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity; this implies that some products of the degradation of caffeic acid under subcritical water conditions might be active for DPPH radical scavenging and stable under these conditions. © 2014 American Chemical Society
The objective of this study was to evaluate the degradation kinetics of caffeic acid in subcritical water. In addition, the degradation products were identified, and their DPPH radical scavenging activities were estimated to demonstrate the potential of new antioxidants formation from this natural phenolic compound in subcritical water that can be considered as a green extraction technique.
■
MATERIALS AND METHODS
Chemicals. Caffeic acid (≥95%), gallic acid (monohydrate, ≥98%), and 2,2-diphenyl-1-picryhydrazyl were purchased from Sigma−Aldrich (St. Louis, MO, USA). L-Ascorbic acid was purchased from Riedel-de Haën (Hanover, Germany). All other chemicals were of analytical grade. Degradation of Caffeic Acid in Subcritical Water. A continuous flow-type reactor was used to study the degradation of caffeic acid in the temperature range 160−240 °C (Figure 1). A caffeic acid solution (100 mg/L) was prepared in distilled water and degassed
Figure 1. Apparatus for subcritical water treatment. Received: Revised: Accepted: Published: 1945
October 29, 2013 January 27, 2014 February 1, 2014 February 1, 2014 dx.doi.org/10.1021/jf404850a | J. Agric. Food Chem. 2014, 62, 1945−1949
Journal of Agricultural and Food Chemistry
Article
for 15 min using an ultrasonic bath (Tru-Sweep 275D, Crest Ultrasonic, Malaysia). The solution was delivered from an amber glass bottle, which was connected via a helium gas balloon to prevent oxygen contamination, through a tubular reactor comprising stainless steel tubing (0.75 mm i.d., 1.58 mm o.d.) immersed in a temperaturecontrolled silicone fluid bath using a high-performance liquid chromatography (HPLC) pump (LC-20AD, Shimadzu, Kyoto, Japan). The length of the reactor coil and the flow rate were adjusted to obtain the desired treatment times (40−1080, 20−340, 20−240, 10−80, and 5−30 s for 160, 180, 200, 220, and 240 °C, respectively). A thermocouple (type K) was connected via a tee connector near the initial part of the reactor (∼10 cm under the fluid surface) to monitor the temperature of the solution inside the tube. After treatment, the solution flowed through a cooling coil immersed in an ice−water bath to rapidly stop the reaction. A miniature back-pressure regulator (P880, Upchurch Scientific, WA, USA) was connected to the end of the tube to maintain a constant pressure of 5 MPa. This pressure was high enough to ensure that the solution remained in the liquid state throughout the temperature range. HPLC-DAD Analysis of Caffeic Acid and Its Degradation Products. Caffeic acid and its degradation products in the treated solution were quantitatively analyzed using an HPLC system (Shimadzu, Kyoto, Japan) comprised of a solvent delivery unit (LC20AD), a diode array detector (DAD; SPD-M20A), and a system controller (CBM-20A). The sample was chromatographically separated using an Inertsil ODS-3 column (4.6 mm × 150 mm, GL Sciences, Tokyo, Japan). The mobile phase consisted of 0.5% v/v aqueous acetic acid (A) and methanol (B), and the following gradient was used at a flow rate of 1.5 mL/min: 0−5 min, 5% B; 5−9 min, linear increase to 50% B; and 9−12 min, 50% B. The treated sample was filtered through a 0.45 μm nylon syringe filter, and 20 μL of sample was injected into the HPLC instrument. Caffeic acid and its degradation products were monitored at 280 nm with authentic caffeic acid as a calibration standard. However, since one of the major degradation products (4-vinylcatechol) is not commercially available, the concentrations of degradation products were estimated, after identification by liquid chromatography-diode array detection/mass spectrometry (LC-DAD/MS), using a calibration curve of caffeic acid corrected by the ratio of the molar absorptivities of caffeic acid (ε = 18 200 M−1 cm−1) and the products, that is, 2951 M−1 cm−1 for hydroxytyrosol,9 10 233 M−1 cm−1 for protocatechuic aldehyde,10 and 2692 M−1 cm−1 for 4-vinylcatechol.11 Identification of Degradation Products Using LC-DAD/MS. Agilent 1100 LC/MSD Trap SL (Agilent Technologies, CA, USA) equipped with a solvent delivery unit (G113A), a diode array detector (G1315D), and an ion-trap mass spectrometer (G2445D) with an electrospray ionization ion source was used to identify the degradation products. Chromatographic separation was accomplished using the same column described above. The mobile phase consisted of 0.5% v/ v aqueous acetic acid and methanol (85:15 v/v) and flowed at a rate of 0.4 mL/min. The treated sample was filtered through a 0.45 μm nylon syringe filter, and 30 μL of sample was injected. Both positive and negative modes were performed over the m/z range 50−400. Online HPLC-DPPH Assay. Evaluation of the DPPH radical scavenging activity of caffeic acid and each degradation product in the treated samples was conducted using an online HPLC-DPPH assay.12 This method is based on the chromatographic separation of components in the sample and a postcolumn reaction with DPPH solution. In this study, only one DAD detector was used to monitor caffeic acid, the degradation products at 280 nm, and the negative peaks due to DPPH bleaching at 517 nm. A first pump was used to deliver the mobile phase that consisted of 0.5% v/v aqueous acetic acid and methanol (85:15 v/v) at a flow rate of 1 mL/min for separating the compound on the column described above, while another pump delivered 6 × 10−5 M DPPH in methanol or pure methanol at a flow rate of 0.8 mL/min through tubing that was connected via a tee connector to the column outlet. A reaction coil made with PEEK tubing (28.8 m) was connected to enable the compounds eluted from the column to react with DPPH for 47 s. Ascorbic acid (AA) was used
for calibration of the DPPH radical scavenging assay via the negative peak area.
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RESULTS AND DISCUSSION Degradation Products of Caffeic Acid in Subcritical Water. A typical HPLC chromatogram of the solution treated under subcritical water conditions is shown in Figure S1 (Supporting Information). There were many compounds in the treated solution, but only the following three main compounds, which could be accurately identified by the LC-DAD/MS, are proposed to be degradation products of caffeic acid: hydroxytyrosol (3,4-dihydroxyphenylethanol), protocatechuic aldehyde (3,4-dihydroxybenzaldehyde), and 4-vinylcatechol (2hydroxy-4-vinylphenol) (Figure 2). The mass/charge ratio (m/
Figure 2. Proposed pathway of caffeic acid degradation in subcritical water.
z) and maximum absorbance (λmax) values were used to identify these compounds, as shown in Table 1. Only the standard Table 1. Characteristics of Caffeic Acid and Its Degradation Products Obtained by LC-DAD/MS λmax (nm)
[M − H]− m/z
220, 279 228, 278, 311 324
153 137
3
hydroxytyrosol protocatechuic aldehyde caffeic acid
179
4
4-vinylcatechol
259
135
peak no. 1 2
compd
negative ions m/z 153 137 − 179 − 135
[M − H]− [M − H]−; 108 [M CHO]− [M − H]−; 135 [M COOH]− [M − H]−
caffeic acid was injected to confirm the accuracy of the mass spectrometer. Decarboxylated products are the main products of the degradation of phenolic acids under subcritical water conditions. For example, degradation of vanillic acid gave 2methoxy-phenol,5 whereas degradation of syringic acid gave syringol (1,3-dimethoxy-2-hydroxybenzene).6 In this study, 4vinylcatechol was detected as a decarboxylated product and was a main product in the treated solution (∼38−57 mol %). It is known that 4-vinylcatechol can be formed through thermal degradation13 and photodegradation14 and can also be prepared by heating caffeic acid at 110 °C using a sodium acetate catalyst.15 However, 4-ethylcatechol, which was also formed during pyrolysis, was not detected in this study. Hydroxytyrosol was found at moderate amounts in the treated solution (∼14− 43 mol %); this compound is an effective antioxidant that is naturally found in olive oil.16 However, the pathway for the 1946
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Figure 3. Caffeic acid degradation products at (a) 160, (b) 180, (c) 200, (d) 220, and (e) 240 °C. Caffeic acid (▲), hydroxytyrosol (◇), protocatechuic aldehyde (□), and 4-vinylcatechol (○).
formation of hydroxytyrosol from caffeic acid remains unclear. A small amount of protocatechuic aldehyde was also found in the treated solution. It has been reported that thermal degradation of chlorogenic acid, which is an ester of caffeic acid and quinic acid, can form a small amount of protocatechuic aldehyde.17 In addition, protocatechuic aldehyde has been reported to be a main oxidation product of caffeic acid in the Fenton oxidation system.18 Kinetics analysis was performed along the pathways shown in Figure 2. The reactions can be described using the following first-order rate equations: dCCA = −kHTCCA − kPACCA − k4VCCCA dt
dC HT = kHTCCA dt
(2)
dC PA = kPACCA dt
(3)
dC4VC = k4VCCCA dt
(4)
where CCA, CHT, CPA, and C4VC are the concentrations of caffeic acid, hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol, respectively, and the k terms are the rate constants for each reaction step. Equations 1−4 were solved by integration and substitution to generate the following equations:
(1) 1947
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C HT =
kHT CCA0(1 − e−k totalt ) k total
(6)
C PA =
kPA CCA0(1 − e−k totalt ) k total
(7)
C4VC =
k4VC CCA0(1 − e−k totalt ) k total
in Figure 5. The total radical scavenging activity of the treated solution, which was calculated from the areas of all seven
(5)
(8)
where ktotal is the overall degradation rate constant of caffeic acid and therefore equal to kHT + kPA + k4VC, and CCA0 is the initial concentration of caffeic acid. First, the overall degradation rate constant (ktotal) was determined by nonlinear regression fitting of eq 5 to the changes in the caffeic acid concentration with time; then, eqs 6−8 were fitted to obtain the rate constants. All the reaction rate constants and their standard errors were estimated using the nls function in R statistical language;19 the results are shown in Figure 3. The standard errors of the estimated rate constants were less than 5% in all cases. It is evident that the experimental results were in good agreement with the proposed reaction pathway. The overall degradation rate constants (ktotal) increased with temperature and followed the Arrhenius relationship (Figure S2 in the Supporting Information) with an activation energy (Ea) of 93 kJ/mol and a pre-exponential factor (A) of 1.78 × 108 s−1. This value is significantly higher than that for vanillic acid degradation (57 kJ/mol) in the temperature range 280−375 °C.5 The formation rate constant for each product also obeyed the Arrhenius equation with Ea values of 103, 85, and 87 kJ/mol and A values of 8.24 × 108, 1.06 × 10 6 , and 2.46 × 10 7 s −1 for hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol, respectively. Online HPLC-DPPH Assay. The antioxidant activity of caffeic acid and its degradation products during subcritical water treatment at 240 °C were estimated using an online HPLC-DPPH assay. A typical chromatogram is shown in Figure 4. The seven peaks that were detected at 280 nm appeared as negative peaks at 517 nm, which indicates their ability to quench the DPPH radicals. Changes in the DPPH radical scavenging activity of each identified product are shown
Figure 5. DPPH radical scavenging activity of caffeic acid (▲), hydroxytyrosol (◇), protocatechuic aldehyde (□), and 4-vinylcatechol (○) in the solution treated by subcritical water at 240 °C. The overall activity (●) was calculated from the summation of the areas of the negative peaks in Figure 5.
negative peaks, was not affected by the treatment time (t test, p ≥ 0.05) even though the caffeic acid content gradually decreased. We found that the radical scavenging activity changed linearly with the concentration of each compound; from these relationships, the DPPH radical scavenging activity of each compound was estimated to be ∼88, 212, 230, and 292 g AA/mol for hydroxytyrosol, protocatechuic aldehyde, caffeic acid, and 4-vinylcatechol, respectively. However, because the concentrations of the degradation products were estimated, we can only conclude that protocatechuic aldehyde, caffeic acid, and 4-vinylcatechol showed higher DPPH radical scavenging activities than hydroxytyrosol. Obied et al. reported that caffeic acid was more active for DPPH radical scavenging than hydroxytyrosol.20 It should be noted that the DPPH radical scavenging activities of these four compounds accounted for more than 90% of the overall activity. From these results, it can be expected that, during an extraction of plant materials containing phenolic compounds using the subcritical water technique, which is usually performed in a batch type extractor for longer periods, similar circumstances might occur for caffeic acid and other compounds as well. More complex reactions might also be possible because there are several compounds existing in sample matrices. This can be an advantage if degradation products possess some required bioactive activities or a disadvantage if undesirable or even toxic products are formed.
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ASSOCIATED CONTENT
S Supporting Information *
HPLC chromatogram of caffeic acid after subcritical water treatment and Arrhenius plot of the reaction rates. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Figure 4. HPLC chromatogram of caffeic acid after subcritical water treatment detected at 280 and 517 nm. The negative peaks indicate DPPH radical scavenging activity.
*Phone: +66-034-219361; fax: +66-034-272194; e-mail: [email protected]. 1948
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Funding
(17) Moon, J.-K.; Shibamoto, T. Formation of volatile chemicals from thermal degradation of less volatile coffee components: Quinic acid, caffeic acid, and chlorogenic acid. J. Agric. Food Chem. 2010, 58, 5465−5470. (18) Antolovich, M.; Bedgood, D. R.; Bishop, A. G.; Jardine, D.; Prenzler, P. D.; Robards, K. LC-MS investigation of oxidation products of phenolic antioxidants. J. Agric. Food Chem. 2004, 52, 962−971. (19) R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2013. (20) Obied, H. K.; Prenzler, P. D.; Robards, K. Potent antioxidant biophenols from olive mill waste. Food Chem. 2008, 111, 171−178.
This work was supported by a cofunded project between the Thailand Research Fund, the Office of the Higher Education Commission, and the Faculty of Engineering and Industrial Technology, Silpakorn University (MRG5380123). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS PK thanks Associate Professor Dr. Rungnaphar Pongsawatmanit, Kasetsart University for her valuable discussion from the beginning of the project.
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REFERENCES
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