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Pressurized Hot Water Extraction of anthocyanins from red onion: A study on extraction and degradation rates
Analytica Chimica Acta 663 (2010) 27–32
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Pressurized Hot Water Extraction of anthocyanins from red onion: A study on extraction and degradation rates Erik V. Petersson, Jiayin Liu, Per J.R. Sjöberg, Rolf Danielsson, Charlotta Turner ∗ Uppsala University, Department of Physical and Analytical Chemistry, P.O. Box 599, SE-751 24, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 10 November 2009 Received in revised form 7 January 2010 Accepted 11 January 2010 Available online 1 February 2010 Keywords: Anthocyanins Subcritical water extraction Degradation Rates Red onion
a b s t r a c t Pressurized Hot Water Extraction (PHWE) is a quick, efficient and environmentally friendly technique for extractions. However, when using PHWE to extract thermally unstable analytes, extraction and degradation effects occur at the same time, and thereby compete. At first, the extraction effect dominates, but degradation effects soon take over. In this paper, extraction and degradation rates of anthocyanins from red onion were studied with experiments in a static batch reactor at 110 ◦ C. A total extraction curve was calculated with data from the actual extraction and degradation curves, showing that more anthocyanins, 21–36% depending on the species, could be extracted if no degradation occurred, but then longer extraction times would be required than those needed to reach the peak level in the apparent extraction curves. The results give information about the different kinetic processes competing during an extraction procedure. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Anthocyanins (AC) form a class of natural pigments found in flowers, fruits and berries. They belong to the polyphenolic class of compounds and usually occur in the plant as glycosides and acylglycosides of the anthocyanidin aglycones. Anthocyanins can be useful as colorants (red and blue colors), or for human health, as they are antioxidants and free radical scavengers. Therapeutic effects include preventive action against cancer [1], improvement of visual functions [2] and cardiovascular health [3], and preventive action against Alzheimer’s and Parkinson’s diseases [4]. These factors have led to an increased interest in using AC in food and pharmaceutical industries, to substitute synthetic colorants and antioxidants. Anthocyanins are particularly unstable, being especially sensitive to light, alkaline pH, and heat [5,6]. Therefore, to avoid degradation, conventional extraction of AC is sometimes performed with large amounts of organic solvent, for example methanol, at low temperatures (down to −25 ◦ C) over a long time (up to four days), as described by Revilla et al., where several different procedures used for extraction of AC from red grapes were compared [7]. With non-automated methods, one sample at a time, the whole
∗ Corresponding author. Current address: Lund University, Department of Organic Chemistry, P.O. Box 124, SE-221 00 Lund, Sweden. Tel.: +46 2228125; fax: +46 2228209. E-mail addresses: [email protected], [email protected] (C. Turner). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.01.023
process is not only time consuming, but also has a negative effect on the environment because of the solvents used. The use of Pressurized Fluid Extraction (PFE) is an attractive alternative, since it allows fast extraction, small solvent consumption, and automated extraction procedures. Sometimes referred to as Pressurized Liquid Extraction (PLE® ), Pressurized Solvent Extraction (PSE® ) and Accelerated Solvent Extraction (ASE® ), PFE has been used for extraction of thermolabile AC from various plants [8–10]. Pressurized Hot Water Extraction (PHWE) is a variety of PFE, where the solvent is mainly water. A comprehensive review on the PHWE technique was written by Kronholm et al. [11]. PFE in general implies that the temperature of the extraction solvent is raised above the atmospheric boiling point, and pressure is applied to maintain the solvent in liquid state. Under such circumstances, in the case of PHWE, the fast movement of the water molecules disorders the intermolecular hydrogen bonding, and the dielectric constant (specific permittivity) is lowered, which gives water more non-polar solvent characteristics [11]. Moreover, the viscosity and surface tension of the solvent decrease, and the solubility and diffusion rate of the target compounds increase. The penetration of the solvent into the matrix and the transfer of the compounds out from the matrix are faster than in a similar extraction process performed at room temperature [11]. Hence, compared to conventional extraction methods, the PHWE technique attains more rapid and efficient extraction with minimal consumption of organic solvent [11]. There is a potential problem in using PHWE for extraction of AC; an increasing temperature will certainly achieve higher AC yield in a shorter period of time, but at the same time also lead to
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degradation of these thermolabile compounds. Commercially available PFE systems operate in batch mode, which means that the solvent, and the analytes, stay in the cell during pre-heating, extraction, and the cooling stage of the procedure. It usually takes 5–8 min to heat up the solvent and samples in the extraction cell to the target temperature. Some AC could already have started to degrade before being transferred to the collection vial. Degradation of AC has been shown to follow first-order reaction kinetics [12,13], although there is a lack of reliable data concerning degradation kinetics of AC at temperatures above 100 ◦ C [14]. Furthermore, most studies so far have been done using fruit juice or lab-produced food products that contain already extracted AC, therefore these studies are focused on the stability of AC in food products rather than the degradation during an extraction procedure. To make PHWE a more advantageous and applicable technique for AC extraction, the study of extraction and degradation rates from a natural vegetable matrix at high temperature is of vital importance. A positive side-effect of the high temperature used is that it accelerates extraction and degradation rates, which makes the experiment quicker to perform. It also gives an indication of potential extraction and degradation effects competing at lower temperatures. The aim of this paper was to investigate and separate extraction and degradation rates when extracting AC from red onion using PHWE in a static batch extraction. A methodology to construct a total extraction curve was used, showing the extraction curve with the previously degraded AC included. The overall goal was to develop a strategy, especially for thermolabile compounds, to distinguish between extraction efficiency from a sample and degradation/reaction in the extraction solvent.
2. Materials Standards of cyanidin-3,5-di-O-glucoside chloride and cyanidin-3-O-glucoside chloride (HPLC grade) were obtained from Extrasynthese (Geney, France). Ethanol (EtOH) and formic acid (FA) were purchased from Merck (Darmstadt, Germany). Ultrapure water (MQ) was obtained from a purification system (Milli-Q; Millipore, Bedford, MA). Nitrogen (99.996 vol.%) was bought from AGA (Stockholm, Sweden). Red onions (Allium cepa L.) were purchased from a local supermarket in Uppsala, Sweden. A batch of fresh red onions was cut into small pieces of approximately 5 mm × 5 mm × 2 mm in average, and then frozen to −18 ◦ C. Extraction and degradation experiments were performed in a pressurized static batch reactor with a cell volume of 100 mL (Mini Reactor, Autoclave Engineers, Erie, PA, USA). The reactor features heating; stirring; inlet of N2 gas; and filtered inlet and outlet of sample. Temperature was set to 110 ◦ C, and the over-pressure inside the reactor thereby reached 1–3 bar. Samples (1 mL) were taken out by turning the outlet valve, allowing the over-pressure to push out the extract into an HPLC vial. Before each sampling, the dead volume of the tubings (1 mL) was flushed out to waste. The inlet of the sampling tube was altered by a homemade fitting joint, to avoid blocking caused by small onion pieces. A round 4 mm micropore metal filter (10 m) was built into the fitting to filtrate extraction solvent before being collected. The sample extracts were then separated on a Synergi Max-RP 2 mm × 100 mm 2.5 m HPLC column (Phenomenex, Torrance, CA, USA) and analyzed with an Ultimate 3000 HPLC with Diode-Array detection (Dionex Softron GmbH, Germering, Germany). Elution was performed using mobile phase A (1% formic acid in acetonitrile, vol.%) and mobile phase B (1% formic acid in water, vol.%). The flow rate was 200 L min−1 and the main wavelength monitored was 520 nm. The optimal elution profile was a multistep linear solvent gradient: 0–2 min 0% A, 2–62 min 0–15% A, 62–70 min 15% A, 70–75 min 15–0% A, and 75–85 min 0%
A. The mass spectrometer was a Bruker Daltonics HCT ESI Ion Trap (Bruker Daltonik GmbH, Bremen, Germany) operated in positive mode and Auto MS2 . The MS-parameters were set to: capillary voltage 3500 V, End Plate Offset −500 V, Skimmer 40 V, Cap Ext 205.2 V, Oct 1 DC 12.00 V, Oct 2 DV 3.70 V, Trap Drive 106.3, Oct RF 200 Vpp, Lens 1 −5 V and Lens 2 −60 V. MATLAB software was supplied by MathWorks, Inc. (Natick, MA, USA) for statistical evaluation. 3. Methods 3.1. Combined extraction/degradation experiment In this experiment, 8.0 g red onion was put in the batch reactor together with 80.0 mL of the extraction liquid MQ:EtOH:FA (94:5:1, v/v/v). Temperature was set to 110 ◦ C, which resulted in a few bars pressure inside the closed reactor vessel. Stirring was set to 300 rpm, and 2–5 bar of N2 was applied to make sample outlet easier. The pre-heating time from room temperature to 110 ◦ C was 8 min. Regarding sampling, one sample (1 mL) was taken out halfway through pre-heating. When pre-heating was finished, another sample was taken out, and thereafter every 10 min, up to 100 min after target temperature was reached (that is 108 min total time). Before each sample, 1 mL was purged and discarded, in order to flush out remains of the former sample. At all times, calculations were made to compensate the volume loss due to sample collection. 3.2. Degradation experiment 3.2.1. Preparation of extract for degradation experiment In this experiment, 9.0 g red onion was put in the batch reactor together with 90.0 mL of the extraction liquid MQ:EtOH:FA (94:5:1, v/v/v). Extraction and pre-heating conditions were as in experiment (3.1). After 10 min of extraction time, the reactor vessel was cooled down and opened. The extract was taken away from the reactor and further cooled to room temperature, and quickly filtered, in order to remove solids and thereby stop extraction. All in order to get an extract where only degradation occurs—for the degradation study in Section 3.2.2. 3.2.2. Degradation of extract from Section 3.2.1 From the filtered extract prepared above, 80.0 mL was put in the batch reactor. A sample for the starting point was taken out, then extraction and pre-heating conditions were applied as in experiment (3.1). A sample (1 mL) was taken out halfway through pre-heating, followed by the same sampling procedure as in experiment (3.1). However, no calculations were made to compensate for volume loss due to sample collection, since the volume change of the extraction solvent had no effect on the concentration of the extract (due to solids had been removed and thereby extraction was stopped). 3.3. Data evaluation The aim of the data evaluation was to reconstruct a “total extraction” curve by adding the estimated amount of degraded AC to the observed extraction/degradation curve. A basic assumption for the approach taken is that the rate of degradation is solely dependent on the current AC concentration and therefore possible to obtain from the degradation curve. For pure degradation, the change in AC concentration during a small time interval t can be approximated by (y)degr =
dy dt
· t = f (y) · t
(1)
where y is the AC concentration taken at the end of the interval. For the combined extraction/degradation process the concentration
E.V. Petersson et al. / Analytica Chimica Acta 663 (2010) 27–32
29
Fig. 1. Typical chromatogram of an HPLC-UV/vis-DAD run of a red onion extract (at 520 nm). The identity of the labeled peaks is shown in Table 1.
change is the sum of the changes due to pure extraction and pure degradation, respectively. Hence, (y)extr = (y)extr/degr − (y)degr and at time point ti we arrive at yextr,i = yextr/degr,i − ˙f (yextr/degr,i ) · t
(2)
where the summation includes all previous time points tj , j ≤ i. The terms of the sum can be evaluated from the experimental degradation curve by a look-up procedure: f (yextr/degr,j ) · t = ydegr,m − ydegr,m−1
(3)
where the time index m is selected so that ydegr,m = yextr/degr,j . The four major peaks (labeled 1–4) from the HPLC chromatogram (Fig. 1) were chosen for evaluation. The calculations were performed with MATLAB software supplied by Math Works, Inc. (Natick, MA, USA). For each of the four peaks, the experimental points for the extraction and the degradation experiments were loaded into MATLAB and smoothed curves were fitted by non-linear least squares regression. By visual inspection it was found that for pure degradation (experiment 3.2.2) a single-term exponential function y = a·ebt and for the combined extraction/degradation (experiment 3.1) a twoterm exponential function y = a·ebt + c·edt gave adequate fits. These smoothing functions were evaluated with one point per minute, compared to 10 min between experimental points. The fitted degradation curves in this study correspond to firstorder reactions with the rate of change f(y) = −k·y where k is the kinetic rate constant. Then Eq. (2) can be simplified to yextr,i = yextr/degr,i + k · ˙yextr/degr,j (i0 < j ≤ i)
(4)
where k
is a dimensionless kinetic parameter, approximately equal to k·t. The onset of degradation, at point i0 , was chosen to correspond to end of the pre-heating period. The k value can be obtained from the fitted degradation curve simply as k =
ydegr,i − ydegr,i−1 ydegr,i
(5)
for any two consecutive points i − 1 and i. In addition to this method, k was also calculated from the last two points of the fitted extraction/degradation curve, assuming that no further extraction took place at that stage of the combined process.
It could be noted that a kinetic model with two first-order consecutive reactions, A → B → C, results in a two-term exponential function for the concentration of B. However, then there are only three independent parameters kA → B , kB → C and cA (0), which implies a restriction for the four parameters of the fitting function a·ebt + c·edt . In this study the aim was to derive a “total extraction” curve from the experimental extraction/degradation curve, possibly using the experimental degradation curve. Since the computational work was substantially reduced assuming a first-order degradation, and since the fit of an exponential decay was quite good, a first-order model for the degradation step was applied. No further attempt on modeling the extraction step was made, the numerical procedure outlined in Eqs. (1)–(3) is rather based on the first principles of mass balance. 4. Results and discussion After HPLC-UV/vis-DAD analysis, the peak areas at 520 nm were integrated, and concentrations expressed as Area Units. A typical chromatogram is shown in Fig. 1. The identified peaks can be seen in Table 1. 4.1. Peak identification Anthocyanins were tentatively identified (see Table 1) by serially coupled UV/vis-DAD and MS detectors. Mass spectrometric data was compared with molecular masses known for AC in red onion from literature [15,16]. Only peaks with characteristic absorbance for AC (520 nm) were evaluated for molecular mass by MS. In Table 1, the columns labeled “Loss” show the mass of the neutral species leaving the molecule when it is fragmented by MS/MS. This information, in combination with molecular and fragment masses formed, helps solving the puzzle of the structure of the molecule. All losses in Table 1 correspond to neutral molecules minus water: 162 corresponds to a hexose sugar (i.e. glucose), 324 corresponds to two of the former or a disaccharide, 248 corresponds to a combination of hexose and malonoyl species, 204 corresponds to a combination of hexose and acetoyl species, 410 corresponds to a combination of a disaccharide and malonoyl species, and 290 corresponds to a combination of hexose, malonoyl and acetoyl species. Peaks b and d are isobaric, but could be separated by retention
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Table 1 Identification of the peaks from a red onion extract by HPLC-UV/vis-DAD–MS. In “Loss” columns molecular mass is shown; and in M+ columns m/z is shown. Rt (min)
M+
Loss [x]
M+ − [x]
36.18 40.93 45.89 47.54 48.99 51.14 53.13 56.01 60.37 62.40
449 611 535 627 491 627 535 697 465 577
162 324 248 162 204 162 248 410 162 290
287 287 287 465 287 465 287 287 303 287
a b
Loss [y]
M+ − [y]
324
303
324
303
Peak in Fig. 1
Tentative identification
1 2 a b c d 3 4 e f
Cyanidin 3-O-glucoside Cyanidin 3-laminaribioside Cyanidin 3-(3 -malonoyl)glucoside Delphinidin 3,5-digalactosidea Cyanidin 3-(3 -acetoyl)glucosideb Delphinidin 3,5-diglucosidea Cyanidin 3-(6 -malonoylglucoside) Cyanidin 3-(6 -malonoyl-laminaribioside) Delphinidin 3-glucosidea Cyanidin 3-(malonoyl)(acetoyl)glucosideb
Not reported before in this matrix. Weak MS signal.
time, and tentatively identified by the shorter retention time of the galactoside compared to the glucoside as reported in similar chromatographic setups in literature [15]. Only cyanidin 3-glucoside could be obtained as standard for AC common in red onion, the rest of the AC present are only tentatively identified in this paper. For higher certainty, the identity of all compounds would have to be confirmed with standards (though not all are available), and possibly utilizing MSn combined with the use of Nuclear Magnetic Resonance (NMR) spectroscopy. 4.2. Working hypothesis The total yield of AC (if no degradation occurs) will remain constant after a certain time of extraction, when all AC available for extraction have been extracted. In reality the actual curve will be a function of both extraction and degradation. The actual extraction/degradation and degradation curves can be measured separately from experiments, after which the degradation effect can be compensated for by calculations, and the total extraction curve can be constructed. 4.3. Combined extraction/degradation experiment The experimental extraction/degradation curves were obtained for the main occurring AC in chopped red onion (peaks 1–4 in Fig. 1). As an example, the curve for peak 3, cyanidin 3-(6 malonoylglucoside), is shown in Fig. 2, as a solid line. During the extraction of unstable compounds, degradation and extraction will occur at the same time. This is certainly the case for AC; after a certain time, a maximum level of AC will be reached, then degradation effects will overcome the extraction effects, and the level decreases. A peak level for different AC is reached after approximately 13–25 min extraction (out of which 8 min is preheating time) in the reactor, but then degradation effects takes over, as can be seen in Fig. 2.
with and without yellow onion matrix (blank matrix, free from AC). It was shown that degradation was quicker without matrix in solution—the time to reach 95% degradation of the standard (cyanidin-3,5-di-O-glucoside chloride) was shorter without matrix (60 min) than with matrix (90 min). As pointed out earlier, some AC are more stable than others. In some cases we found that the explanation for this could be that in the reactor, during the experiments, di-glucosylated species degrade to similar but mono-glucosylated AC. Thereby the apparent degradation of the mono-glucosylated will be slower, since the levels of these will get a net increase by the degradation of the di-glucosylated ones. In summary, the safe residence time at 110 ◦ C, without degradation, is very short—since degradation occurs already during pre-heating. 4.5. Total extraction curve The derived total extraction curve, i.e. the extraction curve compensated for the simultaneous degradation, was calculated as described in Section 3. Two methods were utilized to evaluate the degradation rate parameter; (A) any two points from the pure degradation curve, and (B) the last two points from the extraction/degradation curve. Fig. 3 shows that both methods, A and B, give quite similar results, although they have their advantages and disadvantages. Method A is based on the degradation experiment in which all the
4.4. Degradation experiment Fig. 2, dashed line, exemplifies the degradation of cyanidin 3(6 -malonoylglucoside) in a red onion extract obtained from the experiment above. Degradation starts immediately, even during the 8 min pre-heating stage. For some compounds, in the worst case, the level after pre-heating is just 87% of the starting value, and after 10 min of extraction at the set temperature, there is only 67% left. Experiments with a standard (cyanidin-3,5-di-Oglucoside chloride) showed that the corresponding values are 70% and 40% left, respectively. The vegetable matrix seems to have a “protective effect”, which may be due to the extraction of other antioxidant species that prevent the degradation of the AC. The protection effect was also seen in an experiment under similar conditions when the same AC standard was put in the batch reactor
Fig. 2. Extraction/degradation curve (solid) and degradation curve (dashed) of peak 3, cyanidin 3-(6 -malonoylglucoside), at 110 ◦ C. All data points are means from triplicate experiments.
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Fig. 3. Derived total extraction curves for the four major peaks calculated by two different methods (method A: upper right, dashed; method B: upper right, solid). The figure also shows the combined extraction/degradation data points as circles (triplicates) with the model values (calculated for each minute) as an adjacent solid curve. The degradation data points are shown as crosses (triplicate means) and the corresponding model as an adjacent dashed curve.
solids were removed when high levels of AC were present, and on the assumption that the degradation rate is independent of the solid content. However, removal of the solids, or dissolved matrix components, may possibly accelerate the degradation and lead to an overestimation of the degradation rate. This was at least seen by the quicker degradation rate of an AC standard solution without matrix as mentioned earlier. A “protection effect” from the plant matrix, solid or dissolved, could be the reason that the degradation rate in a real sample is lower. In method B, the kinetic rate constant was determined using the very last part of the extraction/degradation curve, assuming that the extraction was complete at this stage, and only degradation effects were present. The merit of method B is that it reflects a somewhat more real degradation rate during an actual extraction process, with sample matrix present during the degradation. In a separate experiment, it was tested whether the extraction was complete at this stage. A sample was prepared as in experiment 3.1 in Section 3, but taken out after 8 min pre-heating plus 60 min extraction, and the solids were collected and put back in the reactor. Then 10 mL fresh solvent was added, and the sample was extracted again at the same temperature during 10 min (plus 6.5 min pre-heating time). Results showed that no more AC Table 2 Degradation rate parameter k (corresponding to the first-order rate constant in min−1 ) and peak value of extraction yield for different anthocyanins.
k (method A) k (method B) Peak value (%)
Peak 1
Peak 2
Peak 3
Peak 4
0.0149 0.0146 64
0.0117 0.0116 66
0.0270 0.0264 74
0.0257 0.0246 79
could be extracted, and therefore method B was valid in that sense. Table 2 shows the degradation rate parameter k calculated by both methods and the relative peak value, i.e. the ratio between the peak concentration and the calculated final value corresponding to 100% yield. The two methods agree well, and it is clearly shown in Fig. 3 that more AC could be obtained if the observed degradation could be avoided or minimized to the utmost extent. Our results show that the extraction of AC is quite slow with this kind of static (batch) extraction. An alternative technique that should give less degradation at the same temperature (due to shorter holding time), and most likely also quicker extraction, is dynamic extraction. With that technique, the compounds can be continuously transported away from the hot extractor to cooled collection vials (e.g. on ice-bath), using an optimal flow rate of the extraction solvent.
5. Conclusions In this paper, extraction and degradation rates of AC were successfully separated by calculations based on experimental data obtained from a pressurized batch reactor at 110 ◦ C. It was observed that extraction and degradation effects compete already from the start of the extraction process. A total extraction curve, taking into account the degradation of AC during the extraction, was established using two different methods, giving similar results. Results showed that 20–35% higher yield of AC, compared to the observed peak value, could be obtained from red onions if degradation could be avoided. However, this would require longer extraction times
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than those needed to reach the peak level in the observed extraction curves. Acknowledgements Marcus Sjödin is acknowledged for technical assistance with MS-equipment. Charlotta Turner acknowledges the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS, 2006-1346), the Swedish Foundation for Strategic Research (SSF, 0073/13) and the Swedish Research Council the Swedish Research Council (VR, 2006-4084). References [1] D.X. Hou, Potential mechanisms of cancer chemoprevention by anthocyanins, Current Molecular Medicine 3 (2003) 149–159. [2] H. Matsumoto, Y. Nakamura, S. Tachibanaki, S. Kawamura, M. Hirayama, Journal of Agricultural and Food Chemistry 51 (2003) 3560–3563. [3] D.R. Bell, K. Gochenaur, Direct vasoactive and vasoprotective properties of anthocyanin rich extracts, Journal of Applied Physiology 100 (2006) 1164–1170. [4] J. Kong, L. Chia, N. Goh, T. Chia, R. Brouillard, Analysis and biological activities of anthocyanins, Phytochemistry 62 (2003) 923–933. [5] F.C. Stintzing, A.S. Stintzing, R. Carle, B. Frei, R.E. Wrolstad, Color and antioxidant properties of cyanidin-based anthocyanin pigments, Journal of Agricultural and Food Chemistry 50 (2002) 6172–6181. [6] M. Dyrby, N. Westergaard, H. Stapelfeldt, Light and heat sensitivity of red cabbage extract in soft drink model systems, Food Chemistry 72 (2001) 431–437.
[7] E. Revilla, J. Ryan, G. Martn-Ortega, Comparison of several procedures used for the extraction of anthocyanins from red grapes, Journal of Agricultural and Food Chemistry 46 (1998) 4592–4597. [8] P. Arapitsas, C. Turner, Pressurized solvent extraction and monolithic columnHPLC/DAD analysis of anthocyanins in red cabbage, Talanta 74 (2008) 1218–1223. [9] A.M. Gizir, N. Turker, E. Artuvan, Pressurized acidified water extraction of black carrot [Daucus carota ssp sativus var. atrorubens Alef.] anthocyanins, European Food Research and Technology 226 (2008) 363–370. [10] Z.Y. Ju, L.R. Howard, Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin, Journal of Agricultural and Food Chemistry 51 (2003) 5207–5213. [11] J. Kronholm, K. Hartonen, M.L. Riekkola, Analytical extractions with water at elevated temperatures and pressures, Trends in Analytical Chemistry 26 (2007) 396–412. [12] E. Sadilova, F.C. Stintzing, R. Carle, Thermal degradation of acylated and nonacylated anthocyanins, Journal of Food Science 71 (2006) 504–512. [13] E. Sadilova, R. Carle, F.C. Stintzing, Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity, Molecular Nutrition & Food Research 51 (2007) 1461–1471. [14] N. Harbourne, J.C. Jacquier, D.J. Morgan, J.G. Lyng, Determination of the degradation kinetics of anthocyanins in a model juice system using isothermal and non-isothermal methods, Food Chemistry 111 (2008) 204–208. [15] X. Wu, R.L. Prior, Identification and characterization of anthocyanins by highperformance liquid chromatography–electrospray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains, Journal of Agricultural and Food Chemistry 53 (2005) 3101– 3113. [16] E.V. Petersson, A. Puerta, J. Bergquist, C. Turner, Analysis of anthocyanins in red onion using capillary electrophoresis–time of flight-mass Spectrometry, Electrophoresis 29 (2008) 2723–2730.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Pressurized Hot Water Extraction of anthocyanins from red onion: A study on extraction and degradation rates Erik V. Petersson, Jiayin Liu, Per J.R. Sjöberg, Rolf Danielsson, Charlotta Turner ∗ Uppsala University, Department of Physical and Analytical Chemistry, P.O. Box 599, SE-751 24, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 10 November 2009 Received in revised form 7 January 2010 Accepted 11 January 2010 Available online 1 February 2010 Keywords: Anthocyanins Subcritical water extraction Degradation Rates Red onion
a b s t r a c t Pressurized Hot Water Extraction (PHWE) is a quick, efficient and environmentally friendly technique for extractions. However, when using PHWE to extract thermally unstable analytes, extraction and degradation effects occur at the same time, and thereby compete. At first, the extraction effect dominates, but degradation effects soon take over. In this paper, extraction and degradation rates of anthocyanins from red onion were studied with experiments in a static batch reactor at 110 ◦ C. A total extraction curve was calculated with data from the actual extraction and degradation curves, showing that more anthocyanins, 21–36% depending on the species, could be extracted if no degradation occurred, but then longer extraction times would be required than those needed to reach the peak level in the apparent extraction curves. The results give information about the different kinetic processes competing during an extraction procedure. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Anthocyanins (AC) form a class of natural pigments found in flowers, fruits and berries. They belong to the polyphenolic class of compounds and usually occur in the plant as glycosides and acylglycosides of the anthocyanidin aglycones. Anthocyanins can be useful as colorants (red and blue colors), or for human health, as they are antioxidants and free radical scavengers. Therapeutic effects include preventive action against cancer [1], improvement of visual functions [2] and cardiovascular health [3], and preventive action against Alzheimer’s and Parkinson’s diseases [4]. These factors have led to an increased interest in using AC in food and pharmaceutical industries, to substitute synthetic colorants and antioxidants. Anthocyanins are particularly unstable, being especially sensitive to light, alkaline pH, and heat [5,6]. Therefore, to avoid degradation, conventional extraction of AC is sometimes performed with large amounts of organic solvent, for example methanol, at low temperatures (down to −25 ◦ C) over a long time (up to four days), as described by Revilla et al., where several different procedures used for extraction of AC from red grapes were compared [7]. With non-automated methods, one sample at a time, the whole
∗ Corresponding author. Current address: Lund University, Department of Organic Chemistry, P.O. Box 124, SE-221 00 Lund, Sweden. Tel.: +46 2228125; fax: +46 2228209. E-mail addresses: [email protected], [email protected] (C. Turner). 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.01.023
process is not only time consuming, but also has a negative effect on the environment because of the solvents used. The use of Pressurized Fluid Extraction (PFE) is an attractive alternative, since it allows fast extraction, small solvent consumption, and automated extraction procedures. Sometimes referred to as Pressurized Liquid Extraction (PLE® ), Pressurized Solvent Extraction (PSE® ) and Accelerated Solvent Extraction (ASE® ), PFE has been used for extraction of thermolabile AC from various plants [8–10]. Pressurized Hot Water Extraction (PHWE) is a variety of PFE, where the solvent is mainly water. A comprehensive review on the PHWE technique was written by Kronholm et al. [11]. PFE in general implies that the temperature of the extraction solvent is raised above the atmospheric boiling point, and pressure is applied to maintain the solvent in liquid state. Under such circumstances, in the case of PHWE, the fast movement of the water molecules disorders the intermolecular hydrogen bonding, and the dielectric constant (specific permittivity) is lowered, which gives water more non-polar solvent characteristics [11]. Moreover, the viscosity and surface tension of the solvent decrease, and the solubility and diffusion rate of the target compounds increase. The penetration of the solvent into the matrix and the transfer of the compounds out from the matrix are faster than in a similar extraction process performed at room temperature [11]. Hence, compared to conventional extraction methods, the PHWE technique attains more rapid and efficient extraction with minimal consumption of organic solvent [11]. There is a potential problem in using PHWE for extraction of AC; an increasing temperature will certainly achieve higher AC yield in a shorter period of time, but at the same time also lead to
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degradation of these thermolabile compounds. Commercially available PFE systems operate in batch mode, which means that the solvent, and the analytes, stay in the cell during pre-heating, extraction, and the cooling stage of the procedure. It usually takes 5–8 min to heat up the solvent and samples in the extraction cell to the target temperature. Some AC could already have started to degrade before being transferred to the collection vial. Degradation of AC has been shown to follow first-order reaction kinetics [12,13], although there is a lack of reliable data concerning degradation kinetics of AC at temperatures above 100 ◦ C [14]. Furthermore, most studies so far have been done using fruit juice or lab-produced food products that contain already extracted AC, therefore these studies are focused on the stability of AC in food products rather than the degradation during an extraction procedure. To make PHWE a more advantageous and applicable technique for AC extraction, the study of extraction and degradation rates from a natural vegetable matrix at high temperature is of vital importance. A positive side-effect of the high temperature used is that it accelerates extraction and degradation rates, which makes the experiment quicker to perform. It also gives an indication of potential extraction and degradation effects competing at lower temperatures. The aim of this paper was to investigate and separate extraction and degradation rates when extracting AC from red onion using PHWE in a static batch extraction. A methodology to construct a total extraction curve was used, showing the extraction curve with the previously degraded AC included. The overall goal was to develop a strategy, especially for thermolabile compounds, to distinguish between extraction efficiency from a sample and degradation/reaction in the extraction solvent.
2. Materials Standards of cyanidin-3,5-di-O-glucoside chloride and cyanidin-3-O-glucoside chloride (HPLC grade) were obtained from Extrasynthese (Geney, France). Ethanol (EtOH) and formic acid (FA) were purchased from Merck (Darmstadt, Germany). Ultrapure water (MQ) was obtained from a purification system (Milli-Q; Millipore, Bedford, MA). Nitrogen (99.996 vol.%) was bought from AGA (Stockholm, Sweden). Red onions (Allium cepa L.) were purchased from a local supermarket in Uppsala, Sweden. A batch of fresh red onions was cut into small pieces of approximately 5 mm × 5 mm × 2 mm in average, and then frozen to −18 ◦ C. Extraction and degradation experiments were performed in a pressurized static batch reactor with a cell volume of 100 mL (Mini Reactor, Autoclave Engineers, Erie, PA, USA). The reactor features heating; stirring; inlet of N2 gas; and filtered inlet and outlet of sample. Temperature was set to 110 ◦ C, and the over-pressure inside the reactor thereby reached 1–3 bar. Samples (1 mL) were taken out by turning the outlet valve, allowing the over-pressure to push out the extract into an HPLC vial. Before each sampling, the dead volume of the tubings (1 mL) was flushed out to waste. The inlet of the sampling tube was altered by a homemade fitting joint, to avoid blocking caused by small onion pieces. A round 4 mm micropore metal filter (10 m) was built into the fitting to filtrate extraction solvent before being collected. The sample extracts were then separated on a Synergi Max-RP 2 mm × 100 mm 2.5 m HPLC column (Phenomenex, Torrance, CA, USA) and analyzed with an Ultimate 3000 HPLC with Diode-Array detection (Dionex Softron GmbH, Germering, Germany). Elution was performed using mobile phase A (1% formic acid in acetonitrile, vol.%) and mobile phase B (1% formic acid in water, vol.%). The flow rate was 200 L min−1 and the main wavelength monitored was 520 nm. The optimal elution profile was a multistep linear solvent gradient: 0–2 min 0% A, 2–62 min 0–15% A, 62–70 min 15% A, 70–75 min 15–0% A, and 75–85 min 0%
A. The mass spectrometer was a Bruker Daltonics HCT ESI Ion Trap (Bruker Daltonik GmbH, Bremen, Germany) operated in positive mode and Auto MS2 . The MS-parameters were set to: capillary voltage 3500 V, End Plate Offset −500 V, Skimmer 40 V, Cap Ext 205.2 V, Oct 1 DC 12.00 V, Oct 2 DV 3.70 V, Trap Drive 106.3, Oct RF 200 Vpp, Lens 1 −5 V and Lens 2 −60 V. MATLAB software was supplied by MathWorks, Inc. (Natick, MA, USA) for statistical evaluation. 3. Methods 3.1. Combined extraction/degradation experiment In this experiment, 8.0 g red onion was put in the batch reactor together with 80.0 mL of the extraction liquid MQ:EtOH:FA (94:5:1, v/v/v). Temperature was set to 110 ◦ C, which resulted in a few bars pressure inside the closed reactor vessel. Stirring was set to 300 rpm, and 2–5 bar of N2 was applied to make sample outlet easier. The pre-heating time from room temperature to 110 ◦ C was 8 min. Regarding sampling, one sample (1 mL) was taken out halfway through pre-heating. When pre-heating was finished, another sample was taken out, and thereafter every 10 min, up to 100 min after target temperature was reached (that is 108 min total time). Before each sample, 1 mL was purged and discarded, in order to flush out remains of the former sample. At all times, calculations were made to compensate the volume loss due to sample collection. 3.2. Degradation experiment 3.2.1. Preparation of extract for degradation experiment In this experiment, 9.0 g red onion was put in the batch reactor together with 90.0 mL of the extraction liquid MQ:EtOH:FA (94:5:1, v/v/v). Extraction and pre-heating conditions were as in experiment (3.1). After 10 min of extraction time, the reactor vessel was cooled down and opened. The extract was taken away from the reactor and further cooled to room temperature, and quickly filtered, in order to remove solids and thereby stop extraction. All in order to get an extract where only degradation occurs—for the degradation study in Section 3.2.2. 3.2.2. Degradation of extract from Section 3.2.1 From the filtered extract prepared above, 80.0 mL was put in the batch reactor. A sample for the starting point was taken out, then extraction and pre-heating conditions were applied as in experiment (3.1). A sample (1 mL) was taken out halfway through pre-heating, followed by the same sampling procedure as in experiment (3.1). However, no calculations were made to compensate for volume loss due to sample collection, since the volume change of the extraction solvent had no effect on the concentration of the extract (due to solids had been removed and thereby extraction was stopped). 3.3. Data evaluation The aim of the data evaluation was to reconstruct a “total extraction” curve by adding the estimated amount of degraded AC to the observed extraction/degradation curve. A basic assumption for the approach taken is that the rate of degradation is solely dependent on the current AC concentration and therefore possible to obtain from the degradation curve. For pure degradation, the change in AC concentration during a small time interval t can be approximated by (y)degr =
dy dt
· t = f (y) · t
(1)
where y is the AC concentration taken at the end of the interval. For the combined extraction/degradation process the concentration
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Fig. 1. Typical chromatogram of an HPLC-UV/vis-DAD run of a red onion extract (at 520 nm). The identity of the labeled peaks is shown in Table 1.
change is the sum of the changes due to pure extraction and pure degradation, respectively. Hence, (y)extr = (y)extr/degr − (y)degr and at time point ti we arrive at yextr,i = yextr/degr,i − ˙f (yextr/degr,i ) · t
(2)
where the summation includes all previous time points tj , j ≤ i. The terms of the sum can be evaluated from the experimental degradation curve by a look-up procedure: f (yextr/degr,j ) · t = ydegr,m − ydegr,m−1
(3)
where the time index m is selected so that ydegr,m = yextr/degr,j . The four major peaks (labeled 1–4) from the HPLC chromatogram (Fig. 1) were chosen for evaluation. The calculations were performed with MATLAB software supplied by Math Works, Inc. (Natick, MA, USA). For each of the four peaks, the experimental points for the extraction and the degradation experiments were loaded into MATLAB and smoothed curves were fitted by non-linear least squares regression. By visual inspection it was found that for pure degradation (experiment 3.2.2) a single-term exponential function y = a·ebt and for the combined extraction/degradation (experiment 3.1) a twoterm exponential function y = a·ebt + c·edt gave adequate fits. These smoothing functions were evaluated with one point per minute, compared to 10 min between experimental points. The fitted degradation curves in this study correspond to firstorder reactions with the rate of change f(y) = −k·y where k is the kinetic rate constant. Then Eq. (2) can be simplified to yextr,i = yextr/degr,i + k · ˙yextr/degr,j (i0 < j ≤ i)
(4)
where k
is a dimensionless kinetic parameter, approximately equal to k·t. The onset of degradation, at point i0 , was chosen to correspond to end of the pre-heating period. The k value can be obtained from the fitted degradation curve simply as k =
ydegr,i − ydegr,i−1 ydegr,i
(5)
for any two consecutive points i − 1 and i. In addition to this method, k was also calculated from the last two points of the fitted extraction/degradation curve, assuming that no further extraction took place at that stage of the combined process.
It could be noted that a kinetic model with two first-order consecutive reactions, A → B → C, results in a two-term exponential function for the concentration of B. However, then there are only three independent parameters kA → B , kB → C and cA (0), which implies a restriction for the four parameters of the fitting function a·ebt + c·edt . In this study the aim was to derive a “total extraction” curve from the experimental extraction/degradation curve, possibly using the experimental degradation curve. Since the computational work was substantially reduced assuming a first-order degradation, and since the fit of an exponential decay was quite good, a first-order model for the degradation step was applied. No further attempt on modeling the extraction step was made, the numerical procedure outlined in Eqs. (1)–(3) is rather based on the first principles of mass balance. 4. Results and discussion After HPLC-UV/vis-DAD analysis, the peak areas at 520 nm were integrated, and concentrations expressed as Area Units. A typical chromatogram is shown in Fig. 1. The identified peaks can be seen in Table 1. 4.1. Peak identification Anthocyanins were tentatively identified (see Table 1) by serially coupled UV/vis-DAD and MS detectors. Mass spectrometric data was compared with molecular masses known for AC in red onion from literature [15,16]. Only peaks with characteristic absorbance for AC (520 nm) were evaluated for molecular mass by MS. In Table 1, the columns labeled “Loss” show the mass of the neutral species leaving the molecule when it is fragmented by MS/MS. This information, in combination with molecular and fragment masses formed, helps solving the puzzle of the structure of the molecule. All losses in Table 1 correspond to neutral molecules minus water: 162 corresponds to a hexose sugar (i.e. glucose), 324 corresponds to two of the former or a disaccharide, 248 corresponds to a combination of hexose and malonoyl species, 204 corresponds to a combination of hexose and acetoyl species, 410 corresponds to a combination of a disaccharide and malonoyl species, and 290 corresponds to a combination of hexose, malonoyl and acetoyl species. Peaks b and d are isobaric, but could be separated by retention
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Table 1 Identification of the peaks from a red onion extract by HPLC-UV/vis-DAD–MS. In “Loss” columns molecular mass is shown; and in M+ columns m/z is shown. Rt (min)
M+
Loss [x]
M+ − [x]
36.18 40.93 45.89 47.54 48.99 51.14 53.13 56.01 60.37 62.40
449 611 535 627 491 627 535 697 465 577
162 324 248 162 204 162 248 410 162 290
287 287 287 465 287 465 287 287 303 287
a b
Loss [y]
M+ − [y]
324
303
324
303
Peak in Fig. 1
Tentative identification
1 2 a b c d 3 4 e f
Cyanidin 3-O-glucoside Cyanidin 3-laminaribioside Cyanidin 3-(3 -malonoyl)glucoside Delphinidin 3,5-digalactosidea Cyanidin 3-(3 -acetoyl)glucosideb Delphinidin 3,5-diglucosidea Cyanidin 3-(6 -malonoylglucoside) Cyanidin 3-(6 -malonoyl-laminaribioside) Delphinidin 3-glucosidea Cyanidin 3-(malonoyl)(acetoyl)glucosideb
Not reported before in this matrix. Weak MS signal.
time, and tentatively identified by the shorter retention time of the galactoside compared to the glucoside as reported in similar chromatographic setups in literature [15]. Only cyanidin 3-glucoside could be obtained as standard for AC common in red onion, the rest of the AC present are only tentatively identified in this paper. For higher certainty, the identity of all compounds would have to be confirmed with standards (though not all are available), and possibly utilizing MSn combined with the use of Nuclear Magnetic Resonance (NMR) spectroscopy. 4.2. Working hypothesis The total yield of AC (if no degradation occurs) will remain constant after a certain time of extraction, when all AC available for extraction have been extracted. In reality the actual curve will be a function of both extraction and degradation. The actual extraction/degradation and degradation curves can be measured separately from experiments, after which the degradation effect can be compensated for by calculations, and the total extraction curve can be constructed. 4.3. Combined extraction/degradation experiment The experimental extraction/degradation curves were obtained for the main occurring AC in chopped red onion (peaks 1–4 in Fig. 1). As an example, the curve for peak 3, cyanidin 3-(6 malonoylglucoside), is shown in Fig. 2, as a solid line. During the extraction of unstable compounds, degradation and extraction will occur at the same time. This is certainly the case for AC; after a certain time, a maximum level of AC will be reached, then degradation effects will overcome the extraction effects, and the level decreases. A peak level for different AC is reached after approximately 13–25 min extraction (out of which 8 min is preheating time) in the reactor, but then degradation effects takes over, as can be seen in Fig. 2.
with and without yellow onion matrix (blank matrix, free from AC). It was shown that degradation was quicker without matrix in solution—the time to reach 95% degradation of the standard (cyanidin-3,5-di-O-glucoside chloride) was shorter without matrix (60 min) than with matrix (90 min). As pointed out earlier, some AC are more stable than others. In some cases we found that the explanation for this could be that in the reactor, during the experiments, di-glucosylated species degrade to similar but mono-glucosylated AC. Thereby the apparent degradation of the mono-glucosylated will be slower, since the levels of these will get a net increase by the degradation of the di-glucosylated ones. In summary, the safe residence time at 110 ◦ C, without degradation, is very short—since degradation occurs already during pre-heating. 4.5. Total extraction curve The derived total extraction curve, i.e. the extraction curve compensated for the simultaneous degradation, was calculated as described in Section 3. Two methods were utilized to evaluate the degradation rate parameter; (A) any two points from the pure degradation curve, and (B) the last two points from the extraction/degradation curve. Fig. 3 shows that both methods, A and B, give quite similar results, although they have their advantages and disadvantages. Method A is based on the degradation experiment in which all the
4.4. Degradation experiment Fig. 2, dashed line, exemplifies the degradation of cyanidin 3(6 -malonoylglucoside) in a red onion extract obtained from the experiment above. Degradation starts immediately, even during the 8 min pre-heating stage. For some compounds, in the worst case, the level after pre-heating is just 87% of the starting value, and after 10 min of extraction at the set temperature, there is only 67% left. Experiments with a standard (cyanidin-3,5-di-Oglucoside chloride) showed that the corresponding values are 70% and 40% left, respectively. The vegetable matrix seems to have a “protective effect”, which may be due to the extraction of other antioxidant species that prevent the degradation of the AC. The protection effect was also seen in an experiment under similar conditions when the same AC standard was put in the batch reactor
Fig. 2. Extraction/degradation curve (solid) and degradation curve (dashed) of peak 3, cyanidin 3-(6 -malonoylglucoside), at 110 ◦ C. All data points are means from triplicate experiments.
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Fig. 3. Derived total extraction curves for the four major peaks calculated by two different methods (method A: upper right, dashed; method B: upper right, solid). The figure also shows the combined extraction/degradation data points as circles (triplicates) with the model values (calculated for each minute) as an adjacent solid curve. The degradation data points are shown as crosses (triplicate means) and the corresponding model as an adjacent dashed curve.
solids were removed when high levels of AC were present, and on the assumption that the degradation rate is independent of the solid content. However, removal of the solids, or dissolved matrix components, may possibly accelerate the degradation and lead to an overestimation of the degradation rate. This was at least seen by the quicker degradation rate of an AC standard solution without matrix as mentioned earlier. A “protection effect” from the plant matrix, solid or dissolved, could be the reason that the degradation rate in a real sample is lower. In method B, the kinetic rate constant was determined using the very last part of the extraction/degradation curve, assuming that the extraction was complete at this stage, and only degradation effects were present. The merit of method B is that it reflects a somewhat more real degradation rate during an actual extraction process, with sample matrix present during the degradation. In a separate experiment, it was tested whether the extraction was complete at this stage. A sample was prepared as in experiment 3.1 in Section 3, but taken out after 8 min pre-heating plus 60 min extraction, and the solids were collected and put back in the reactor. Then 10 mL fresh solvent was added, and the sample was extracted again at the same temperature during 10 min (plus 6.5 min pre-heating time). Results showed that no more AC Table 2 Degradation rate parameter k (corresponding to the first-order rate constant in min−1 ) and peak value of extraction yield for different anthocyanins.
k (method A) k (method B) Peak value (%)
Peak 1
Peak 2
Peak 3
Peak 4
0.0149 0.0146 64
0.0117 0.0116 66
0.0270 0.0264 74
0.0257 0.0246 79
could be extracted, and therefore method B was valid in that sense. Table 2 shows the degradation rate parameter k calculated by both methods and the relative peak value, i.e. the ratio between the peak concentration and the calculated final value corresponding to 100% yield. The two methods agree well, and it is clearly shown in Fig. 3 that more AC could be obtained if the observed degradation could be avoided or minimized to the utmost extent. Our results show that the extraction of AC is quite slow with this kind of static (batch) extraction. An alternative technique that should give less degradation at the same temperature (due to shorter holding time), and most likely also quicker extraction, is dynamic extraction. With that technique, the compounds can be continuously transported away from the hot extractor to cooled collection vials (e.g. on ice-bath), using an optimal flow rate of the extraction solvent.
5. Conclusions In this paper, extraction and degradation rates of AC were successfully separated by calculations based on experimental data obtained from a pressurized batch reactor at 110 ◦ C. It was observed that extraction and degradation effects compete already from the start of the extraction process. A total extraction curve, taking into account the degradation of AC during the extraction, was established using two different methods, giving similar results. Results showed that 20–35% higher yield of AC, compared to the observed peak value, could be obtained from red onions if degradation could be avoided. However, this would require longer extraction times
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than those needed to reach the peak level in the observed extraction curves. Acknowledgements Marcus Sjödin is acknowledged for technical assistance with MS-equipment. Charlotta Turner acknowledges the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS, 2006-1346), the Swedish Foundation for Strategic Research (SSF, 0073/13) and the Swedish Research Council the Swedish Research Council (VR, 2006-4084). References [1] D.X. Hou, Potential mechanisms of cancer chemoprevention by anthocyanins, Current Molecular Medicine 3 (2003) 149–159. [2] H. Matsumoto, Y. Nakamura, S. Tachibanaki, S. Kawamura, M. Hirayama, Journal of Agricultural and Food Chemistry 51 (2003) 3560–3563. [3] D.R. Bell, K. Gochenaur, Direct vasoactive and vasoprotective properties of anthocyanin rich extracts, Journal of Applied Physiology 100 (2006) 1164–1170. [4] J. Kong, L. Chia, N. Goh, T. Chia, R. Brouillard, Analysis and biological activities of anthocyanins, Phytochemistry 62 (2003) 923–933. [5] F.C. Stintzing, A.S. Stintzing, R. Carle, B. Frei, R.E. Wrolstad, Color and antioxidant properties of cyanidin-based anthocyanin pigments, Journal of Agricultural and Food Chemistry 50 (2002) 6172–6181. [6] M. Dyrby, N. Westergaard, H. Stapelfeldt, Light and heat sensitivity of red cabbage extract in soft drink model systems, Food Chemistry 72 (2001) 431–437.
[7] E. Revilla, J. Ryan, G. Martn-Ortega, Comparison of several procedures used for the extraction of anthocyanins from red grapes, Journal of Agricultural and Food Chemistry 46 (1998) 4592–4597. [8] P. Arapitsas, C. Turner, Pressurized solvent extraction and monolithic columnHPLC/DAD analysis of anthocyanins in red cabbage, Talanta 74 (2008) 1218–1223. [9] A.M. Gizir, N. Turker, E. Artuvan, Pressurized acidified water extraction of black carrot [Daucus carota ssp sativus var. atrorubens Alef.] anthocyanins, European Food Research and Technology 226 (2008) 363–370. [10] Z.Y. Ju, L.R. Howard, Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin, Journal of Agricultural and Food Chemistry 51 (2003) 5207–5213. [11] J. Kronholm, K. Hartonen, M.L. Riekkola, Analytical extractions with water at elevated temperatures and pressures, Trends in Analytical Chemistry 26 (2007) 396–412. [12] E. Sadilova, F.C. Stintzing, R. Carle, Thermal degradation of acylated and nonacylated anthocyanins, Journal of Food Science 71 (2006) 504–512. [13] E. Sadilova, R. Carle, F.C. Stintzing, Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity, Molecular Nutrition & Food Research 51 (2007) 1461–1471. [14] N. Harbourne, J.C. Jacquier, D.J. Morgan, J.G. Lyng, Determination of the degradation kinetics of anthocyanins in a model juice system using isothermal and non-isothermal methods, Food Chemistry 111 (2008) 204–208. [15] X. Wu, R.L. Prior, Identification and characterization of anthocyanins by highperformance liquid chromatography–electrospray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains, Journal of Agricultural and Food Chemistry 53 (2005) 3101– 3113. [16] E.V. Petersson, A. Puerta, J. Bergquist, C. Turner, Analysis of anthocyanins in red onion using capillary electrophoresis–time of flight-mass Spectrometry, Electrophoresis 29 (2008) 2723–2730.