Article pubs.acs.org/JAFC
Quantification of Individual Phenolic Compounds’ Contribution to Antioxidant Capacity in Apple: A Novel Analytical Tool Based on Liquid Chromatography with Diode Array, Electrochemical, and Charged Aerosol Detection Merichel Plaza,*,† James Kariuki,§ and Charlotta Turner† †
Department of Chemistry, Centre for Analysis and Synthesis, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Department of Science, Augustana Campus, University of Alberta, 4901-46 Avenue, Camrose, Alberta, Canada T4V 2R3
§
S Supporting Information *
ABSTRACT: Phenolics, particularly from apples, hold great interest because of their antioxidant properties. In the present study, the total antioxidant capacity of different apple extracts obtained by pressurized hot water extraction (PHWE) was determined by cyclic voltammetry (CV), which was compared with the conventional antioxidant assays. To measure the antioxidant capacity of individual antioxidants present in apple extracts, a novel method was developed based on highperformance liquid chromatography (HPLC) with photodiode array (DAD), electrochemical (ECD), and charged aerosol (CAD) detection. HPLC-DAD-ECD-CAD enabled rapid, qualitative, and quantitative determination of antioxidants in the apple extracts. The main advantage of using CAD was that this detector enabled quantification of a large number of phenolics using only a few standards. The results showed that phenolic acids and flavonols were mainly responsible for the total antioxidant capacity of apple extracts. In addition, protocatechuic acid, chlorogenic acid, hyperoside, an unidentified phenolic acid, and a quercetin derivative presented the highest antioxidant capacities. KEYWORDS: antioxidant capacity, charged aerosol detector, cyclic voltammetry, electrochemical detector, hyphenation, phenolic compounds
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INTRODUCTION Flavonoids represent a highly diverse class of secondary plant metabolites with about 9000 structures.1 Flavonoids are polyphenolic compounds derived from 2-phenylchromone, commonly found in many plants, vegetables, and flowers.2 They have received considerable attention in the literature, specifically due to their biological and physiological importance. They are also known to have benefits to human health, including antioxidant activity,3 metal chelation,4 and antiproliferative, anticarcinogenic, antibacterial, anti-inflammatory, antiallergenic, and antiviral effects.5−7 In this context, the apple is an interesting vegetable plant species due to its natural high content of polyphenols (500− 2700 mg/kg) and its widespread popularity all over the world.8 Thus, there is considerable research interest toward the production of polyphenols and their enriched extracts, for use as food supplements or active ingredients by the cosmetics and pharmaceutical industries. Due to this ongoing interest, in this work, various apple extracts were investigated as sources of natural antioxidants. Pressurized hot water extraction (PHWE) has been successfully used at an analytical level for the quantitative recovery of target analytes.9−13 For instance, in a previous study, PHWE was used to extract antioxidants from apple byproducts.14 This study showed the optimum conditions to extract antioxidants while minimizing the formation of undesirable compounds, such as Maillard and caramelization reaction products, during the extraction. The extraction © 2013 American Chemical Society
conditions used in the present work to extract phenolic compounds from different types of apples were based on that previous, study although slightly modified. There is an increased interest in assessing the total antioxidant capacity of complex samples, that is, the total number of moles of a given free radical that is scavenged by the sample. The antioxidant capacity should be distinguished from the antioxidant activity, of which the latter corresponds to the rate constant of a reaction between a specific antioxidant and a specific oxidant.15 Several methods to measure antioxidant properties have been proposed and were recently reviewed.16,17 Among these, trolox equivalent antioxidant capacity (TEAC), scavenging of stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), total radical-trapping antioxidant parameter (TRAP), ferric reducing antioxidant power (FRAP), and cupric ions (Cu2+) reducing power (Cuprac) were employed in foods.16 These methods are related to the capacity of a sample to compete for and scavenge a specific reactive oxygen species (ROS). Even though these methods provide useful information, they are not sufficient for the evaluation of the overall antioxidant profile of biological fluids, tissue homogenate, or plant extract. Besides, these procedures present some drawbacks because Received: Revised: Accepted: Published: 409
September 23, 2013 December 14, 2013 December 18, 2013 December 18, 2013 dx.doi.org/10.1021/jf404263k | J. Agric. Food Chem. 2014, 62, 409−418
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system shows great potential in all applications involving the analysis of antioxidants in complex samples.
they require the use of specific reagents and they are tedious and time-consuming. Furthermore, the DPPH• and ABTS•+ radicals are not representative of biomolecules found in any biological system. In this context, cyclic voltammetry (CV) methodology allows a rapid production of the electrochemical profile of samples and is especially suitable for screening studies. It is based on the analysis of the anodic current (AC) waveform, which is a function of the reactive potential of a given compound in the sample or a mixture of compounds. The CV tracing indicates the ability of a compound to donate electrons at the potential of the anodic wave. A CV also provides information describing the integrated antioxidant capacity without the specific determination of the contribution of each individual component. Therefore, in the past couple of years, CV has been suggested as an instrumental methodology for the evaluation of the total antioxidant capacity of foods.18−21 Therefore, we have used CV methodology to evaluate the antioxidant capacity of apple extracts and then compared it with the traditional methods such as DPPH and TEAC. The main purpose of the current study was, however, to assess the contribution of individual components of apple extracts to the overall antioxidant capacity of the extracts. Recently, online HPLC methods for analyzing radicalscavenging activity have been developed.22−24 Such methods require a stable model free radical system, such as DPPH• and ABTS•+, with radical-scavenging activity assessed in comparison to the water-soluble synthetic vitamin E derivative 6-hydroxy2,5,7,8-tetramethylchroma-2-carboxylic acid (trolox). Online assessment of antioxidant activity allows complex mixtures to be separated by HPLC and the antioxidant contribution of individual components to be evaluated. Instead, likewise to CV, electrochemical properties of the individual compounds can be used for quantifying their antioxidant capacity. In our previous work, we have developed a separation system useful for characterizing antioxidants in complex samples, which was based on the hyphenation of high-performance liquid chromatography with diode array, electrochemical, and mass spectrometry detection (HPLC-DAD-ECD-MS/MS).25,26 However, a large drawback is that none of these detectors can give any information about the quantity of the analyzed compounds without their respective standards. Because chemical standards are rarely available for most flavonoids found in plants and herbs, and the available flavonoid standards are generally very expensive, the quantification of these compounds is a true challenge. Recently, a novel detector has been suggested to overcome this drawback; the charged aerosol detector (CAD), which is based on a technique that adds charges to each particle containing analytes eluting from HPLC column. The size of the charge is not dependent on chemical properties of the analytes, which makes the CAD relatively generic, thereby requiring only a limited number of chemical standards to quantify a large number of analytes in complex samples. The aim of this work was to develop a method to measure the total antioxidant capacity of apple extracts obtained by PHWE from different apple varieties and, additionally, to evaluate the contribution of individual compounds to the total antioxidant capacity of the extracts, as well as to quantify the compounds present. Hence, this paper presents for the first time the use of a hyphenated system, HPLC-DAD-ECD-CAD, to directly screen and characterize antioxidants in apples. This
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MATERIALS AND METHODS
Chemicals and Reagents. All of the chemicals were of analytical grade. 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Fluka (Buchs, Switzerland). Trolox, ammonium formate, potassium persulfate, chlorogenic acid, caffeic acid, protocatechuic acid, homovanillic acid, rutin, and phloridzin were supplied by Sigma-Aldrich (Steinheim, Germany). Formic acid was from Merck (Darmstadt, Germany). Methanol, LC-MS grade, was provided by Scharlau (Barcelona, Spain). The flavonoid standards were purchased from Extrasynthese (Lyon, France). Deagglomerated alumina (αAl2O3) suspensions with grain size = 0.1 μm was provided by Struers (Ballerup, Denmark). The ultrapure water used was obtained from a Milli-Q (Millipore, Billerica, MA, USA) instrument. Samples. The apple samples (Fuji, Golden Delicious, Granny Smith, Jonagold, Royal Gala, organic Golden Delicious, and organic Royal Gala, one of each) were obtained from a local market in Lund, Sweden. The apple samples were cut in small cubes (3 mm) composed of skin and flesh. PHWE. Extractions were performed on a Dionex ASE 200 system (Thermo Fisher, Germering, Germany). At the beginning of the day, the water was sonicated for 40 min. Extractions were performed at 112 °C for 10 min on the basis of an experimental method used in a previous study.14 Prior to each experiment, heating of an extraction cell was carried out for 6 min. Likewise, all extractions were performed in 11 mL extraction cells, containing 5 g of fresh sample. Samples were prepared in duplicate. The dry weight of apple was calculated by subtracting water content from the total weight. The fresh apple was led to dryness in an oven at 125 °C for 24 h.27 This process was carried out in triplicate. The water contents in the apples, Fuji, Golden Delicious, Granny Smith, Jonagold, Royal Gala, organic Golden Delicious, and organic Royal Gala, were 84.5 ± 0.3, 85.1 ± 0.2, 85.3 ± 0.1, 86.1 ± 0.1, 83.4 ± 0.2, 88.1 ± 0.1, and 84.5 ± 0.4%, respectively. The extraction procedure was as follows: (i) the extraction cell was loaded into the oven; (ii) the cell was filled with solvent up to a pressure of 1500 psi; (iii) heating time was applied; (iv) a static extraction with all system valves closed was performed; (v) the cell was rinsed (with 60% cell volume using extraction solvent); (vi) solvent was purged from the cell with nitrogen gas; and (vii) depressurization took place. Between extractions, a rinse of the complete system was made to prevent any extract carry-over. The extracts obtained were protected from light, freeze-dried, and stored at −20 °C until analysis. To prepare the sample solutions, the freeze-dried extracts were redisolved in water at a concentration of 20 mg/mL. Evaluation of Antioxidant Capacity. TEAC Assay. The TEAC assay described by Re et al.28 with some modifications was used to measure the antioxidant capacity of the extracts. The ABTS radical cation (ABTS•+) was produced by reacting 7 mM ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12−16 h before use. The aqueous ABTS•+ solution was diluted with 5 mM phosphate buffer (pH 7.4) to an absorbance of 0.70 (±0.02) at 734 nm. Ten microliters of sample solution (four different concentrations) was added to 1 mL of diluted ABTS•+ radical solution. After 50 min at 30 °C, 300 μL of the mixture was transferred into a well of the microplate, and the absorbance was measured at 734 nm in a microplate spectrophotometer reader (Multiskan GO, Thermo Fisher). Trolox was used as a reference standard, and results were expressed as TEAC values (mmol trolox/g extract). These values were obtained from at least four different concentrations of each extract tested in the assay giving a linear response between 20 and 80% of the initial absorbance. All analyses were done in triplicate for each extract. DPPH Radical-Scavenging Assay. The antioxidant capacity of all the obtained extracts was measured using the DPPH radicalscavenging assay based on the protocol by Brand-Williams et al.29 Briefly, a solution was prepared by dissolving 23.5 mg of DPPH in 100 410
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mL of methanol. This stock solution was further diluted 1:10 with methanol. Both solutions were stored at 4 °C until use. Four different concentrations of extracts were tested. Twenty-five microliters of these solutions was added to 975 μL of DPPH diluted solution to complete the final reaction medium (1 mL). After 4 h at room temperature, 300 μL of the mixture was transferred into a well of the microplate, and the absorbance was measured at 516 nm in a microplate spectrophotometer reader (Multiskan GO, Thermo Fisher). A DPPH−methanol solution was used as a reference sample. The DPPH concentration remaining in the reaction medium was calculated from a calibration curve. The percentage of remaining DPPH against the extract concentration was then plotted to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%, or EC50. Therefore, the lower the EC50, the higher the antioxidant capacity. To enhance the clarity of the results, the antioxidant capacity was determined as the inverse value of the efficient concentration EC50 (μg/mL), representing a comparable term for the effectiveness of antioxidant and radical-scavenging capacity (1/EC50). The larger the antioxidant capacity, the more efficient the antioxidant. Measurements were done in triplicate for each extract. Cyclic Voltammetry. Cyclic voltammetric experiments were performed in 50:50 (methanol/water, v/v) extracts mixed with sodium formate buffer (pH 3.0) at a 1:1 (v/v) ratio and 0.5 M NaNO3 following the method of Zettersten et al.26 The sodium formate buffer consisted of 200 mM NaHCOO/HCOOH in water. The sodium formate buffer and NaNO3 served as supporting electrolytes for the voltammetric measurement. Cyclic voltammetry measurements were performed in a sealedbottom three-electrode cell in which the electrode area was defined by an elastomeric O-ring (area ∼ 0.5 cm2) using a glassy carbon electrode. A platinum auxiliary electrode and a silver/silver chloride reference electrode were used. The glassy carbon electrode was slightly polished on a polishing cloth with Al2O3 before each voltammetric experiment. All voltammograms were obtained at a scan rate of 20 mV/s. The homemade three-electrode cell used had angled openings allowing the reference and the working electrodes to be placed as close as possible to each other. The cell was connected to a battery-powered potentiostat (PalmSens, Palm Instruments BV, Houten, The Netherlands). The potentiostat was controlled by an HP iPAQ Pocket PC (HP 2200 series, Hewlett-Packard Co.) using PalmScan software (vs 1.4.2, Palm Instruments BV). All apple extract solutions (concentration = 10 mg/mL) were purged for 5 min with nitrogen gas prior to use. Measurements were done in triplicate. For the purpose of testing, the total anodic peak wave area (S) of the voltammogram was calculated.30 This method is based on the correlation between the total anodic peak wave area of cyclic voltammograms and the antioxidant capacity of the sample and reference substance. For the reference, an 80% methanol solution of trolox within the concentration range of 0.05−2.50 mM was used, and the results were expressed as micromoles of Trolox per gram of dry extract. HPLC with Hyphenated Diode Array, Electrochemical, and Charged Aerosol Detection. Instrumentation. The HPLC-DADECD-CAD setup is described schematically in Figure 1. It consisted of an UltiMate-3000 HPLC system from Dionex (Thermo Fisher) with an online degasser, a quarternary solvent pump, and an autosampler with cooler, column oven, and photodiode array detector (DAD) with scanning capabilities, all controlled by Chromeleon 6.80 (Thermo
Fisher) software. The detection wavelengths used were 200, 280, 350, 370, and 520 nm. An ECD instrument was attached just after the DAD. The online amperometric detection setup included a thin layer flow cell (Bioanalytical System Inc., West Lafayette, IN, USA) with a series dual glassy carbon disk electrodes (3 mm diameter) working electrode imbedded in a PEEK block (Bioanalytical System Inc.). A spacer gasket with a thickness of 16 μm (Bioanalytical System Inc.) was placed between the PEEK block comprising the working electrode and the stainless steel block serving as counter electrode. This resulted in a total cell volume of 1.0−1.2 μL. The reference electrode was fixed in a compartment in the stainless steel block. The ECD redox potential measured at +0.6 V vs Ag/AgCl was found to yield antioxidant capacity results comparable with those of the DPPH and ABTS assays. A battery-powered potentiostat (Palmsens, Palm Instrument BV) was connected to the flow cell and was controlled by an HP iPAQ Pocket PC (HP 2200 series, Hewlett-Packard Co.). PalmTime software (vs 2.3.0.0, Palmsens) was used to collect data. A Corona CAD instrument from ESA Biosciences Inc. (part of Thermo Fisher) was placed in series after the ECD detector. Data processing was carried out with Chromeleon 6.8 software (Thermo Fisher). Nitrogen gas flow rate was regulated automatically and monitored by the CAD device regulated at 35 psi. Response range was set to 20 pA full scale. Medium filter was applied. Chromatographic Separation. A porous-shell fused core Ascentis Express C18 (150 mm × 2.1 mm, 2.7 μm) from Supelco (Bellefonte, PA, USA) was used as an analytical column for LC separation. The mobile phases consisted of (A) ammonium formate buffer (pH 3.0), 60 mM (NH4HCOO/HCOOH) in water; and (B) methanol with 0.2% of formic acid in a gradient elution analysis programmed as follows: 0 min, 5% (B); 0−5 min, 5% (B); 5−35 min, 40% (B); 35−40 min, 40% (B), with 10 min of post-time at a flow rate of 300 μL/min. The mobile phases were purged with nitrogen to remove oxygen. The column temperature was set at 50 °C, the injection volume was 2 μL, and the vial tray was held at 4 °C. Statistical Analysis. The KaleidaGraph 4.03 (Synergy Software, Reading, PA, USA) program was employed for statistical analysis of the data with the level of significance set at 95%. Student’s t test was used to assess significant differences between antioxidant assays.
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RESULTS AND DISCUSSION As mentioned in the Introduction, the aim of this work was mainly to develop a method that can be used to characterize and quantify antioxidants in complex samples. Hence, the focus was not to identify the different types of antioxidants found in apples. Antioxidant Capacity of Apple Extracts Produced by PHWE. PHWE is an environmentally friendly technique that presents important advantages over traditional solvent extraction techniques, mainly achieving safe, green, and rapid extractions. Increasing the temperature decreases the dielectric constant of water, resulting in the possibility of tuning its solvent properties, as has been shown for aromatic plants.11 In this work, PHWE was used to obtain a variety of extracts from seven different apples: Fuji, Golden Delicious, Granny Smith, Jonagold, Royal Gala, organic Golden Delicious, and organic Royal Gala. The extraction yield (percent dry weight) obtained from apple PHWE extraction was around 63% (Table 1), being maximum in Jonagold (67%) and minimum in organic Golden Delicious (60%). In this study, we conducted a critical evaluation of a cyclic voltammetry method for the determination and rapid screening of antioxidant capacity of different apple type extracts compared with ABTS and DPPH assays. The representative cyclic voltammograms of the apple extracts (10 mg/mL concentration) were recorded from 0 to 700 mV at a scan rate of 20 mV/s (Figure 2). The obtained voltammograms
Figure 1. Employed online HPLC-DAD-ECD-CAD system. 411
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Table 1. Extraction Yield and DPPH, ABTS, and Cyclic Voltammetry Antioxidant Capacity Values Obtained for the Different Varieties of Apple Extracts after PHWE at 112 °C and 10 mina extraction yield
ABTS
DPPH
apple variety
% (dry wt)
RSD (%)
TEAC (mmol/g dry extract)
RSD (%)
Golden Delicious Royal Gala Fuji Granny Smith Jonagold organic Golden Delicious organic Royal Gala
60 64 64 62 67 59 63
1.7 4.9 5.5 1.7 4.8 8.0 2.4
0.12 0.082 0.050 0.13 0.18 0.16 0.10
4.0 7.8 8.0 1.9 3.3 6.0 4.0
CV
1/EC50 (μg/mL) RSD (%) 3.4 2.7 1.5 3.5 4.9 4.2 2.9
1.7 2.2 2.0 1.0 2.2 4.9 2.3
mmol trolox/g dry extract
RSD (%)
7.9 7.4 5.1 7.4 12 9.3 6.7
3.0 1.1 6.1 4.7 4.3 7.1 8.7
a
All measurements were done in triplicate for each extraction. The data are presented as the average of six measurements from two extracts (mean and relative standard deviation (RSD)).
ences in their antioxidant capacity, although the extraction yields are similar. To correlate the CV data for different apple varieties with the ABTS and DPPH, the results in Figure 3 have been normalized so that the values range from 0 to 1, 1 being the maximum value of total antioxidant capacity in each method. The results from CV strongly correlate with those determined using wellknown spectrophotometric methodologies such as DPPH and ABTS assays. There was no statistically significant difference between the different assays (p > 0.05). From the above results it follows that a good correlation exists between antioxidant capacity (obtained from the oxidation peak areas in CV) and antiradical power, indicating that the voltammetric method can be used for the determination of antioxidant capacity, in the same way as the DPPH and ABTS assays. At least this is true for water-soluble antioxidants in apples. This means that the values of the oxidation potentials can be interpreted in the same way as the antiradical power values obtained from DPPH and ABTS, and the quality of the information obtained is the same in all cases. This result is in agreement with other studies, in which CV was shown to be an efficient instrumental tool for evaluating the antioxidant capacity of plant, food, and biological samples.21,30,33,34 An advantage of the electrochemical measurement compared to the DPPH and ABTS methods is that it is fast. The CV measurement is carried out in
