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Biosynthesis of phenolic antioxidants in carrot tissue increases with wounding intensity
Food Chemistry 134 (2012) 615–624
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Biosynthesis of phenolic antioxidants in carrot tissue increases with wounding intensity Bernadeth B. Surjadinata, Luis Cisneros-Zevallos ⇑ Department of Horticultural Sciences, Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX 77843-2133, USA
a r t i c l e
i n f o
Article history: Received 4 June 2011 Received in revised form 21 December 2011 Accepted 26 January 2012 Available online 4 February 2012 Keywords: Wounding intensity Phenolics Antioxidant capacity Fresh-cut Carrots
a b s t r a c t Biosynthesis of phenolic antioxidants in three carrots cultivars (Navajo, Legend and Choctaw) were studied under different wounding intensities (A/W) during storage. Generated A/W of 4.2, 6.0 and 23.5 cm2/g, corresponded to slices, pies, and shreds, respectively. Soluble phenolics, antioxidant capacity (AOX), and PAL activity increased with A/W for all cultivars. Intense wounding (23.5 cm2/g) induced an 2.5- and 12.4-fold increase in soluble phenolics and AOX, respectively, after 4 days compared to whole carrots. Wounding induced the synthesis of mainly chlorogenic acid (5-CQA) and 3,5-dicaffeoylquinic acid. A higher proportion of 5-CQA in the phenolic mixture was responsible for an increasing specific AOX (809 and 1619 lg Trolox/mg phenolics for whole carrots and shreds, respectively, for Choctaw cultivar). Wounded carrots can be promoted as an inexpensive rich source of phenolic antioxidants for the regular diet. By simply increasing wounding stress intensity it is possible to enhance the biosynthesis of phenolic antioxidants. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction One of the procedures involved in fresh-cut fruit and vegetable processing is cutting the produce into smaller pieces. This cutting action essentially is causing the tissue to endure wounding stress. Wounding will cause some physiological effects to the tissue and it is understood that a more severe cutting process will elicit a greater wounding response decreasing the quality of the produce. Wounding has been known to increase respiration rate (Surjadinata & Cisneros-Zevallos, 2003) and ethylene production of many different tissues (Rolle & Chism, 1987; Saltveit, 1997). Many responses occur because wounding stress is involved in the generation, translocation, perception, and transduction of signals in order to activate the expression of wound-inducible genes (Leon, Rojo, & Sanchez-Serrano, 2001), either locally by the injured cells or systemically by the adjacent cells. Plant cells are contained inside rigid walls, making each cell capable of triggering defence mechanisms. When wounding stress occurs, the cell can activate the specific transcriptional genes (Leon et al., 2001; Salveit, 2000) with the purpose of adjusting the metabolism to repair and heal the damage and to synthesise substances to prevent invasion by the predators. These processes could occur between a few minutes to several hours after wounding (Leon et al., 2001).
⇑ Corresponding author. Tel.: +1 979 8453244; fax: +1 979 8450627. E-mail address: [email protected] (L. Cisneros-Zevallos). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.01.097
In plants, many compounds have been proposed to play a role in wound signalling, such as phytohormones (e.g. ethylene, jasmonic acid, ABA), reactive oxygen species (ROS), salicylic acid, systemin, oligosaccharides, as well as electrical pulses and hydraulic waves (Leon et al., 2001; Rakwal & Agrawal, 2003; Salveit, 2000). Recently, Jacobo-Velazquez, Martinez-Hernandez, Rodriguez, Cao, and Cisneros-Zevallos (2011) showed that ROS plays a key role as secondary signal in the biosynthesis of phenolic antioxidants in carrots as a response to wounding. Furthermore, they proposed a model where ATPs would play a primary signal role. Improved knowledge of this signalling mechanism would be very important in order to understand the effect of a stress on the tissue and to develop strategies to avoid it or enhance it. A stress or an injury to a plant cell will trigger two types of responses in phenolic metabolism (Rhodes & Wooltorton, 1978). The first response is the oxidation of the existing phenolic compounds as a result of rupture of the cell membrane, causing the phenolics to combine with the oxidative enzyme systems. The other response involves the synthesis of monomeric or polymeric phenolics to repair the wounding damage. This second response are caused by changes in phenylalanine ammonia lyase activity (PAL, EC 4.3.1.5) since it is the key metabolic enzyme in the phenylpropanoid pathway (Babic, Amiot, Nguyen-The, & Aubert, 1993; Kang & Saltveit, 2002). Increase in phenolic content is one of the most studied phenomena in response to wounding (Toivonen & DeEll, 2002). For example, Babic et al. (1993) found that storing shredded carrots in air accumulated chlorogenic acid while Leja, Mareczek, Wojciechowska, and Rozek (1997) found that peels of carrot slices
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stored for 4 days at 20 °C produced more chlorogenic acid and isocoumarin. However, a first report showing increased antioxidant capacity in wounded tissue was presented by Heredia and Cisneros-Zevallos (2002) in a study using carrots as a model system. These findings of increased antioxidant activity with wounding stress were later confirmed in lettuce leaf (Kang & Saltveit, 2002), purple-flesh potato (Reyes & Cisneros-Zevallos, 2003) and in a variety of fresh produce (Reyes, Villarreal, & Cisneros-Zevallos, 2007). Furthermore, all of these studies were done with only one type of cuts (e.g. slices), therefore the need to study how wounding intensity affects the biosynthesis of phenolic antioxidants. It is well known the importance phenolic antioxidant have as promoters of human health through their antiradical scavenging activity by preventing chronic diseases associated to oxidative stress. In this study we characterised the effects of wounding stress under the hypothesis that the biosynthesis of phenolic antioxidants will increase with wounding intensity. In this paper we present information confirming how wounding intensity influences phenolic accumulation during storage, PAL activity, the phenolic profiles and their effects on antioxidant activity and the influence of carrot cultivars used. This work is based on a dissertation by Surjadinata (2005). The generated information may be used to promote wounded carrots as an inexpensive rich source of phenolic antioxidants for the regular diet. 2. Materials and methods 2.1. Chemical reagents All chemicals and standards used, Folin–Ciocalteu reagent, sodium carbonate (Na2CO3), Trolox and 2,2-diphenyl-1-picrylhydrazyl (DPPH), polyvinylpyrrolidine (PVPP), sodium hydroxide (NaOH), boric acid, 2-mercaptoethanol, chlorogenic acid, p-hydroxybenzoic acid, and ferulic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1,3-Dicaffeoylquinic acid (cynarin) was purchased from ChromaDex, Inc. (Santa Ana, CA, USA). 1-Aminocyclopropene-1-carboxylic acid (ACC) was purchased from MP Biomedicals (Aurora, OH, USA). Methanol, ethanol, hexane, and acetonitrile were reagents of HPLC grade. 2.2. Carrots and wounding stress studies Whole-topped commercial carrots cultivars Navajo, Legend and Choctaw used in this study were kindly provided by Grimmway Farms, Bakersfield, CA. The carrots were grown under similar conditions and were harvested within 2 weeks, shipped to Texas A&M and stored at 2–5 °C until used (within 2 weeks period). The carrots were conditioned overnight at 15 °C before the wounding studies. All carrots used were of similar size (2.5–3 cm in diameter and 22–25 cm in length) and free of visual damage to reduce experimental variability. Wounding intensity studies were performed cutting carrots into different shapes, including slices, pies and shreds. Wounding intensity (A/W) was defined by the ratio of the new surface area created by wounding in cm2 over the tissue weight in g (Surjadinata & Cisneros-Zevallos, 2003). The wounded surface areas of carrots slices, pies, and shreds were manually measured. The slices were 5 mm thick while the pies were obtained from the carrots slices and were cut further into quarters. For shreds, the A/W was estimated from 40 pieces of shredded carrots of about 5.1 g, which gave an area of 119.7 cm2. The calculated wounding intensities (A/W) were 4.2, 6.0, and 23.5 cm2/g for carrot slices, pies, and shreds, respectively. Whole carrots used as controls have an A/W of 0.0 cm2/g since they were not cut, thus no wounded surface area was present (Fig. 1).
Fig. 1. Wounding intensity (A/W) defined by the ratio of the new surface area created by wounding in cm2 over the tissue weight in g. The calculated A/W for whole carrots, slices, pies and shreds were 0, 4.2, 6.0 and 23.5 cm2/g, respectively.
On the day of processing, carrots were taken from the 15 °C storage, washed with 100 ppm chlorinated water and air-dried for 2–3 h at room temperature. Shreds (considered as extreme wounding) were obtained from carrot cylinders of 150 g using a food processor (High Performance model, West Band Co., West Band, WI). After cutting, wounded and non-wounded carrots were placed in 4 L closed clear glass jars. The jars were stored in the dark at 15 °C and were ventilated periodically (every 6–8 h) to maintain aerobic respiration and avoid accumulation of carbon dioxide (<0.5%). The carrot shreds were stored for 0, 2, 4, and 8 days while carrot slices, pies and non-wounded carrots (controls) were stored 4 days monitoring total phenolics, phenolic profiles antioxidant capacity, and phenylalanine ammonia lyase (PAL) activity. After 8 days of storage, the water loss of the shredded carrots was approximately 3% and showed development of off-odour, possibly due to a combined effect of increase respiration, ethylene production and membrane deterioration at 15 °C. This temperature was used in all experiments only to accelerate the wounding response and it is not recommended for maintaining quality. Lower temperature would be more appropriate. 2.3. Total soluble phenolics assay The phenolic content was analysed using the procedure of Swain and Hillis (1959) with some modifications. Five grams of fresh tissue samples were mixed with 25 ml of methanol and homogenised until reaching uniform consistency using an Ultra Turrax (T 25 basic, IKA Labortechnik, Staufen, Germany). Extracts were stored in covered plastic tubes overnight at 4 °C and then centrifuged at 30,000g for 20 min (Beckmann Coulter J2-21, Fullerton, CA). The centrifuge was equipped with a rotor (Beckman Coulter JA-17, Fullerton, CA). A 150 ll sample of the clear supernatant was collected and diluted with 2400 ll of nanopure water. At the same time, a blank (150 ll of nanopure water) was prepared by following the same procedure as the sample. To each diluted sample and blank, 150 ll of 0.25 N Folin–Ciocalteau reagent was added, vortexed and allowed to react at room temperature for 3 min, followed by the addition of 300 ll of 1 N Na2CO3. This mixture was allowed to react at room temperature in the dark for 2 h. Readings were done at 725 nm using a photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany), which was previously blanked with methanol. Total phenolics were expressed as mg chlorogenic acid equivalent/100 g fresh weight tissue (FW), based on a standard curve.
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2.4. Determination of phenolic profiles The same extracts used for the total phenolics assay were used for determining the phenolic profiles using an HPLC method described by Hale (2003). The samples were run using Waters Millennium 3.2 software (Milford, MA, USA). The HPLC system was equipped with a binary pump system (Waters 515, Milford, MA, USA), an auto-injector (Waters 717 plus, Milford, MA, USA), a photodiode array (PDA) detector (Waters 996, Milford, MA, USA), and a column heater (SpectraPhysics SP8792, San Jose, CA, USA). The column used to separate the phenolic compounds was a 4.6 150 mm, 5 lm, C-18 reverse-phase column (Waters Atlantis, Milford, MA, USA), which was maintained at 40 °C. The injection volume was 10 ll. Two mobile phases were used: water/HCl adjusted to pH 2.3 as solvent A and acetonitrile as solvent B. The gradient system was 0/85, 5/85, 30/0, 35/0 (min/% solvent A). Both solvents were filtered and degassed before used. Prior to injection, the samples were filtered through a 0.2 lm nylon syringe filter. Identification and quantification was based on developed standard curves, retention times and UV–vis spectra. Commercial standards of chlorogenic acid, ferulic acid, p-hydroxybenzoic acid and 1,3-dicaffeoylquinic acid were used for peak identification when possible. Other chlorogenic acid derivatives and isocoumarin were determined as described bellow. 2.4.1. Determination of dicaffeoylquinic acid (diCQA and isocoumarin (ISO) by time of flight mass spectrometry (TOF-MS) Based on the HPLC method described above, the obtained peaks between 16 and 18 min (corresponding to diCQA) were collected using a fraction collector (Waters Fraction Collector II, Milford, MA, USA) connected after the PDA detector (Waters 996, Milford, MA, USA). The eluent in each collected fraction was evaporated using a Speed-Vac Concentrator (SC 100, Savant, NY, USA) and then freeze dried (PD-6-54A, FTS Systems, Inc., NY, USA). The isolated compounds were dissolved in water/methanol (50:50 v/v) acidified with 1% acetic acid and injected into a time of flight (TOF) mass spectrometer equipped with electron spray ionisation (ESI) in negative ion mode (M-H) (PE Sciex API QStar Pulsar, Concord, Ontario, Canada). Isocoumarin extraction was done using a method by Talcott and Horward (1999) with some modifications. Ten kilogram of whole carrots were sprayed with 100 ppm of 1-aminocyclopropane-1carboxylic acid (ACC), exposed to 1000 ppm of ethylene and sealed in 4 L glass jars during 4 days in the dark at room temperature. The jars were ventilated every 12 h to avoid CO2 accumulation (>0.5%). At the end of storage, carrots were peeled. About 600 g of peels were obtained and these peels were then extracted with 3 L of hexane for 20–24 h. The next day, the liquid phase was partitioned with 1 L of ethanol. The ethanol fraction was divided into 10 vials of 1 ml and injected to the HPLC using the protocol described before. A peak that appeared around 24 min was confirmed to be isocoumarin by monitoring the kmax of the PDA spectra (absorbance at 267 and 302 nm). In order to accumulate significant amount of isocoumarin, the injection volume used was 125 ll. Each vial was injected three times. The total fractions collected were around 30 ml and these were evaporated with Speed-Vac Concentrator (SC 100, Savant, NY, USA) and further freeze-dried (Kinetics, NY, USA). The resulting powder was then re-dissolved in 1 ml of ethanol. To quantify the amount of isocoumarin present in the dried powder, the absorbances at 267 and 302 nm of this ethanolic sample were measured with photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany). The ratio between the two absorbances was around 2.25–2.29, which is very similar to the pure isocoumarin (ratio = 2.47) (Sondheimer, 1957). Quantification of isocoumarin collected was performed using a molar extinction coefficient in ethanol (e = 14,800/M cm)
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(Sondheimer, 1957). In 1 ml of ethanolic samples there was 1.1031 mg of isocoumarin. Several dilutions of this ethanolic sample were injected back into the HPLC to create a standard curve. This time the injection volume was set to be 10 ll, which was the volume injection condition used for the wounded and nonwounded carrot extracts. Isocoumarin powder collected from the freeze-drier was also re-dissolved in ethanol and injected into a time of flight (TOF) mass spectrometer (PE Sciex API QStar Pulsar, Concord, Ontario, Canada) equipped with ESI in positive ion mode (M+H)+ combined with (M+Li)+ for further identification and confirmation. The isolated compounds were dissolved in water/methanol acidified with 1% acetic acid. The capillary voltage was 4.5 kV. The mass spectrometry procedure was done in collaboration with the Chemistry Department of Texas A&M University (College Station, TX, USA). 2.5. Antioxidant capacity (AOX) assay AOX was quantified using the procedure of Brand-Williams, Cuvelier, and Berset (1995). The same extracts prepared for the total phenolics assay were also used for the AOX assay. A diluted solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared from the mother stock solution using methanol to obtain an absorbance of 1.1 ± 0.02. After centrifugation of the extracts at 30,000g (Beckmann Coulter J2-21 equipped with a rotor JA-17, Fullerton, CA) at 4 °C for 20 min, 150 ll of the clear supernatant were mixed with 2850 ll of the diluted DPPH solution in a clean plastic vial, vortexed and then closed. At the same time, a blank of 150 ll of methanol mixed with the DPPH diluted solution was prepared. The sample extract and DPPH was allowed to react in the dark at room temperature for 24 h or until reaching steady state. AOX was measured at 515 nm using a photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany), that was previously blanked with methanol/DPPH solution. AOX was expressed as lg Trolox equivalents/g FW, based on a standard curve using Trolox. 2.6. Phenylalanine ammonia lyase (PAL) enzyme activity assay The PAL activity was measured using the procedure of Ke and Salveit (1986) with slight modifications. For each replicate, 1 g of tissue was mixed with 0.2 g polyvinylpyrrolidine (PVPP) and homogenised in 30 ml of 50 mM cold borate buffer (pH 8.5) at low speed to a uniform consistency. The borate buffer was a mixture of boric acid and sodium hydroxide in nanopure water containing 400 ll of 2-mercaptoethanol per 1 L buffer. The extracts were then filtered through 4 layers of cheesecloth and centrifuged at 30,000g (Beckmann Coulter J2-21 equipped with a rotor JA-17, Fullerton, CA) for 15 min at 4 °C. Throughout the analysis, sample extracts were kept in ice and in dark condition. After centrifugation, clear supernatant was collected and incubated for 5 min in a 40 °C water-bath. Two sets of 10 ml glass tubes were prepared. The first set is for the water control and the second set is for the samples to which 100 mM of L-phenylalanine was added as substrate. Measurements of the enzyme activity were done at 0 and 1 h of incubation in a 40 °C water-bath. PAL activity was quantified as lmoles of t-cinnamic acid/h g FW. It was calculated using a standard curve developed for t-cinnamic acid in the borate buffer. 2.7. Statistical analysis Statistics analysis was done using the ANOVA procedure from SAS Statistical Analysis System for Windows v8.1 software (SAS Institute Inc., Cary, NC, USA). The treatment means were compared with Tukey’s Studentized Range test at a = 0.05. Six replicates per treatment were used in this study (n = 6).
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storage period. The total phenolics of individual carrot replicates (n = 6) showed a linear relationship with their corresponding total AOX for each sampling day of storage with R2 ranging from 0.71 to 0.98. There was a distinct line for each sampling day (data not shown). This pattern was similar for all cultivars and the linear relationship showed a trend to pass through 0 for both X and Y axes. The slopes of these linear relationships increased as the storage time increased. The slopes correspond to the specific phenolic antioxidant capacity, which is the antioxidant capacity expressed on a phenolic basis (ratio between total AOX and total phenolics) (Jacobo-Velazquez & Cisneros-Zevallos, 2009). The specific AOX gives information of the effectiveness of the phenolic mixture present on neutralising the free radicals. Thus, a higher specific AOX would mean that the phenolics present stabilise a greater number of free radicals. Accordingly, Navajo, Legend and Choctaw carrots
3. Results and discussion 3.1. Effects of wounding stress on carrot tissue during storage Intense wounding stress or shredding induced the accumulation of phenolic compounds in carrot tissue through storage time (P < 0.05), however, there was no difference between carrot cultivars (P > 0.05). After 8 days storage there was a 5.7-fold increase in total phenolics compared to day 0 (45 mg/100 g for controls) (Fig. 2A). There was an increase in AOX with storage time for all wounded carrot cultivars (P < 0.05). Total AOX showed a 15.5-, 18.5-, and 17.0-fold increase after 8 days storage for Navajo, Legend, and Choctaw cultivar, respectively, compared to day 0 (150–235ug Trolox/g for controls) (Fig. 2B). This increase in AOX is related to the accumulation of phenolic compounds observed in that same 300
Phenolics (mg Chlorogenic Acid/100g)
A
Navajo Legend
250
Choctaw
200 150 100 50 0 0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
6000
B AOX (ug Trolox Eq./g)
5000 4000 3000 2000 1000 0
2
PAL
(umol t -Cinnamic Ac/hr g)
C
1.6
1.2
0.8
0.4
0 Day Fig. 2. Total soluble phenolics (A), antioxidant capacity (B), and PAL activity (C) of three cultivars of shredded carrots during 8 storage at 15 °C. Vertical bars represent SD (n = 6).
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Phenolics (mg Chlorogenic Ac/100g)
A
250
Navajo Legend
200
Choctaw 150 100 50 0
B
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
3500
AOX (ug Trolox Eq/g)
3000 2500 2000 1500 1000 500 0
PAL (umol t-Cinnamic Ac/hr g)
C
2.5
2
1.5
1
0.5
0
A/W (cm2/g) Fig. 3. Total soluble phenolics (A), antioxidant capacity (B), and PAL activity (C) of three carrot cultivars under different wounding intensities (A/W) after 4 days storage at 15 °C. Vertical bars represent SD (n = 6).
showed increases in specific AOX between days 0 and 8 ranging from 542 to 1543, 476 to 1382 and 664 to 1748 lg Trolx/mg phenolics, respectively, indicating that the mixture of phenolics biosynthesised through time had higher radical scavenging properties. Despite that the total phenolics were similar for all cultivars at day 8, there seems to be differences in the phenolic profiles as depicted by differences in the specific AOX (e.g. Choctaw > Lengend and Navajo). Wounding stress induced an increase in PAL activity for all three cultivars reaching a maximum peak after 2 days storage and a further decrease through time (Fig. 2C). At day 2, Navajo, Legend and Choctaw cultivars showed a 7.1-, 7.5- and 11.7-fold increase, respectively, compared to day 0 (0.133–0.169 lmol tcinnamic acid/h g for controls). The total phenolics in wounded carrots continued to accumulate after 4 and 8 days storage since
the PAL-synthesizing system remained present. Even though its activity decreased, the enzyme was not completely inactivated, thus maintaining the ability to produce phenolic compounds. Between days 4 and 8 of storage, there was a decline in the rate of biosynthesised phenolics (Fig. 2A). In general, Choctaw cultivar had higher PAL activity throughout the storage period, which could be associated to higher levels of specific phenolic compounds being synthesised and not observed in the total phenolics assay. 3.2. Wounding intensity effects on soluble phenolics, phenolic profiles, AOX and PAL activity The phenolic content increased with wounding intensity for all three carrot cultivars (Fig. 3A). This increase was a 97%, 76%, and 252% for carrots slices, pies and shreds, respectively, after 4 days of
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Table 1 Individual phenolics determined by HPLC at 280 nm of different carrot cultivars and wounding intensities. Cultivar
Peak #
Ret time (min)
kmax (nm)
Navajo
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
241.3, 325.5
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
241.3, 325.5
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
Legend
Choctaw
Compound
234.2, 321.9 326.7 240.1, 326.7 302.3
234.2, 321.9 326.7 240.1, 326.7 302.3 241.3, 325.5 234.2, 321.9 326.7 240.1, 326.7 302.3
Amount (mg/100 g) Whole
Slices
Pies
Shreds
5-CQA p-HBA FA 3,4-diCQA** 3,5-diCQA** HBA derivative*** Isocoumarin
5.499 nd* nd nd 4.593 nd nd
15.71 nd 2.099 nd 7.670 nd 0.089
22.34 nd 2.200 nd 8.250 nd 0.095
52.54 0.819 2.740 1.399 8.214 1.718 0.734
5-CQA p-HBA FA 3,4-diCQA 3,5-diCQA HBA derivative Isocoumarin
5.291 nd nd nd 4.472 nd nd
12.64 nd 2.073 nd 6.441 nd 0.079
13.04 nd 2.055 nd 6.158 nd 0.080
38.67 0.157 2.386 1.656 7.887 0.616 0.301
5-CQA p-HBA FA 3,4-diCQA 3,5-diCQA HBA derivative Isocoumarin
6.302 nd nd nd 4.840 nd nd
15.44 nd 2.080 nd 6.613 nd 0.053
23.29 nd 2.810 nd 7.372 nd 0.088
71.68 0.639 3.032 1.363 8.677 2.239 0.580
*
nd = Not detected. Quantified using chlorogenic acid (5-CQA) as standard. *** Quantified using p-hydroxybenzoic acid (p-HBA) as standard. **
storage at 15 °C compared to non-wounded carrots (45–52 mg/ 100 g). Cultivars Choctaw and Navajo showed higher accumulation of phenolic compounds compared to cultivar Legend at wounding intensities P6.0 cm2/g (pies). HPLC phenolic profiles for wounded and non-wounded carrot tissue showed these were similar for all three cultivars (Table 1). Typical chromatograms for different wounding intensities are shown for Choctaw cultivars in Fig. 4. HPLC phenolic analysis of non-wounded tissue showed the presence of chlorogenic acid (5CQA, peak 1) and a dicaffeoylquinic acid isomer (diCQA, peak 5). Phenolic profiles of carrot slices and pies, showed an additional presence of ferulic acid (FA, peak 3) and isocoumarin (ISO, peak 7), while phenolic profiles of shredded carrots showed, in addition, peaks for p-hydroxybenzoic acid (pHBA, peak 2), an unknown hydroxybenzoic acid derivative (peak 6), and one more diCQA (peak 4). Peak 6 had very similar PDA spectra to p-hydroxybenzoic acid, however, the retention times differed by about 18 min, indicating this compound is less polar compared to p-HBA. Peaks 4 and 5 were identified as isomers of chlorogenic acid since the PDA spectra were similar to that of chlorogenic acid. This was then confirmed by TOF-MS analysis showing that m/z of the parent ion of peaks 4 and 5 were 515.11. According to Clifford, Johnston, Knight, and Kuhnert (2003), the m/z for the parent ion of 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA in coffee bean are 515.7, 515.2, and 515.4, respectively. There are 4 different diCQA: 1,3diCQA (also known as cynarin), 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA (Clifford, 1986; Clifford et al., 2003). In this study, commercially available 1,3-diCQA (m/z, 515.12) was injected in the HPLC system and its retention time was about 14 min (data not shown), differing from peak 4 (17.47 min) and peak 5 (18.15 min). In carrot tissue, 3,5-diCQA has been shown to be in higher amounts compared to other diCQA isomers (Alasalvar, Grigor, Zhang, Quantick, & Shahidi, 2001; Zhang & Hamauzu, 2004). Accordingly, peak 5 would correspond to 3,5-diCQA, while peak 4 which appeared before peak 5, would correspond to 3,4-diCQA since this isomer is more polar than 3,5-diCQA. Results showed that chlorogenic acid was the major phenolic compound present in all three carrot cultivars of wounded and
non-wounded tissue (Table 1). When carrots were shredded, this compound showed a 9.5-, 7.3-, and 11.3-fold increase in content for cultivar Navajo, Legend, and Choctaw, respectively (Table 1). Peak 7 was confirmed to be isocoumarin by TOF-MS analysis. The positive ionisation and injection of internal standard, lithium, gave m/z of 215.08. Isocoumarin has a mass of 208.21 and lithium has a mass of 7, therefore when combined, the m/z would be 215. The maximum level of isocoumarin produced by the wounded carrot tissue was approximately 0.30–0.73 mg/100 g FW. The phytoalexin isocoumarin is synthesised by wounding in carrot tissue and plays a major role in the plant defence system (Fan, Mattheis, & Roberts, 2000). This compound gives bitter flavour, which could decrease the quality of the wounded carrots. According to Lafuente, Lopez-Galvez, Cantwell, and Yang (1996), bitterness flavour in carrot tissue, is associated to isocoumarin content P20 mg/100 g FW. The amount of isocoumarin observed in the present study (61 mg/ 100 FW) will not induce a bitter taste to the tissue. It has been shown previously that other stresses such as ethylene exposure may elicit more isocoumarin synthesis compared to wounding (Lafuente et al., 1996). The AOX increased with the wounding intensity for all three cultivars studied (Fig. 3B). The AOX increased by 2- to 3.5-fold for slices and pies and up to 12.4-fold for shreds compared to non-wounded carrot after 4 days of storage at 15 °C (250– 300 lg Trolox/g for controls). AOX was similar (P > 0.05) between slices and pies for each cultivar. Cultivars Choctaw and Navajo showed higher AOX compared to Legend for all wounding intensities (P < 0.05). Increased wounding intensity also induced an increased in PAL activity among the tree cultivars studied (Fig. 3C). PAL activity increased by 19.2-, 27.9- and 266.2-fold for carrot slices, pies, and shreds, respectively, compared to non-wounded carrots (P < 0.05). Navajo and Legend showed very similar PAL activity (P > 0.05), however for Choctaw, PAL was higher only for shreds (P < 0.05). This higher PAL activity is likely associated to a higher biosynthesis of 5-CQA in Choctaw carrots (Table 1). Ke and Salveit (1989) previously found that PAL activity increased with number of punctures on lettuce tissue. In the present
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0.08
AU
0.06 0.04
1
0.02
5
0.00 5.00
10.00
15.00
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25.00
Minutes 0.10
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0.08
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0.06 0.04
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0.02
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3
7
0.00 5.00
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0.06 0.04
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Fig. 4. HPLC profiles of carrot tissue at 280 nm for Choctaw carrots. Peaks 1, chlorogenic acid (5-CQA); 2, p-hydroxybenzoic acid (pHBA); 3, ferulic acid (FA); 4, 3,4dicaffeoylquinic acid (3,4-diCQA); 5, 3,5-dicaffeoylquinic acid (3,5-diCQA); 6, hydroxybenzoic acid derivative; 7, isocoumarin (ISO). Y-axis is absorbance unit (AU) and X-axis is retention time in min.
study wounding stress from 0 to 23.5 cm2/g seem to originate an exponential response in PAL activity, but more data would be needed to confirm this non-linear relationship. Furthermore, it would be important to define the linear or non-linear association between wounding area created (W/A) and the abiotic stress signal. In general, reported experimental data related to increase in PAL activity with the increase of wounding intensity is scarce in the literature. Thus, this study is one of the first attempts to quantify wounding intensity effects on phenolic biosynthesis.
3.3. Wounding intensity effects on specific AOX AOX of individual carrot replicates presented a linear relationship with their corresponding total phenolic contents for all wounding intensities and this trend was similar for all carrot cultivars (R 0.48–0.92) (Fig. 5). The slopes of the linear relationship increased with wounding intensity indicating that wounding stress synthesised phenolic compounds with increased AOX properties. The slope defines the specific AOX of the phenolic mixture present
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A
4000
Slices Pies
R2 = 0.869
Shreds
3000
Linear (Whole)
2000 R2 = 0.849
1000
R2 = 0.749 R2
= 0.929
0 0
AOX (ug Trolox Eq./g)
B
50
100
150
200
2500
2000 R2 = 0.485
1500
1000 R2 = 0.879 R2 = 0.819
500 R2 = 0.842
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50
100
150
4000 R2 = 0.623
3000
2000 R2 = 0.868
1000
R2 = 0.754 R2
= 0.905
0 0
50
100
150
200
Phenolics (mg Chlorogenic Acid/100g) Fig. 5. Linear correlations between antioxidant capacity and total soluble phenolics of carrot cultivars Navajo (A), Legend (B), and Choctaw (C) under different wounding intensities using individual carrot samples (n = 6). Measurements were taken after 4 days storage at 15 °C.
Table 2 Relative proportion (%) of the three major hydroxycinnamic acids present in carrot tissue under different wounding intensities and the associated specific AOX.
*
Cultivar
Cut
A/W (cm2/g)
5-CQA
FA
3,5-diCQA
Specific AOX** (lg Trolox/mg phenolic*)
Navajo
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
54.5 61.7 68.1 82.7
0.0 8.2 6.7 4.3
45.5 30.1 25.2 12.9
456 ± 71.5 1052 ± 131.8 1180 ± 47.6 1604 ± 83.5
Legend
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
54.2 59.8 61.4 79.0
0.0 9.8 9.7 4.9
45.8 30.4 29.0 16.1
482 ± 84.2 936 ± 97.2 902 ± 43.4 1221 ± 211.1
Choctaw
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
56.6 64.0 69.6 86.0
0.0 8.6 8.4 3.6
43.4 27.4 22.0 10.4
809 ± 53.6 1198 ± 213.8 1275 ± 49.9 1619 ± 210.0
Phenolic content was quantified based on 5-CQA spectrophotometric standard curve. Average specific AOX ± SD, obtained by diving AOX and phenolic content for each carrot replicate (n = 6). Specific AOX also corresponds to the slopes of the regression line in Fig. 5.
**
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in the carrots and this phenolic mixture clearly showed different HPLC phenolic profiles for individual wounding intensities (Table 1). For example, Table 2 presents the relative proportions (phenolic profiles) of the three major hydroxycinnamic acids present in wounded and non-wounded carrot tissue: chlorogenic acid (5-CQA), ferulic acid (FA), and one of the chlorogenic acid isomers (3,5-diCQA). The relative proportions (%) of phenolic compounds will depend on wounding intensity (A/W). For non-wounded carrot tissue, there was similar proportion between 5-CQA and 3,5-diCQA, corresponding to a lower specific AOX capacity. For slices and pies, the phenolic profiles were similar, showing a higher amount of 5-CQA compared to 3,5-diCQA. These phenolic profiles also give similar specific AOX. This would explain why the slopes of the linear relationship between total AOX and total phenolics were similar for both wounding intensities (Fig. 5). For shreds, which showed the highest specific AOX, the relative proportions of phenolic compound indicate a much higher contribution of 5CQA compared to 3,5-diCQA suggesting that 5-CQA content has a large effect on the specific AOX of carrot tissue. 3.4. Wounded carrots as an inexpensive source of phenolic antioxidants In the present study we have shown that phenolic antioxidant accumulation in carrot tissue is dependent on wounding intensity. For example, the antioxidant levels may reach up to 3000 lg Trolox/g (11.98 lmol/g) in shredded carrots which is 6 times higher than whole carrots in just only 4 days of storage. Blueberries, which is considered a rich source of phenolic antioxidants provides 5500 lg Trolox/g (21.97 lmol/g) (Cevallos-Casals & Cisneros-Zevallos, 2003), however they are expensive and seasonal. The annual consumption per capita of blueberries in the US is 0.18 kg while consumption of carrots is 3.6 kg (ERS-USDA, 2003, 2004) making wounded carrots a potential higher source of phenolic antioxidants compared to blueberries (an estimated 10 times higher antioxidant source per capita). The recommended daily intake of antioxidants for humans is estimated to be in the range of 3000–3600 lmol Trolox/day, from which 1200 to 1640 lmol Trolox/day comes from the consumption of fruit and vegetables (representing <50%) (Prior & Cao, 2000). Accordingly, 100 g of shredded carrots may provide 33% to 40% of the required recommended intake of antioxidants and could be considered an inexpensive alternative source of phenolic antioxidants compared to more expensive fruits and vegetables. However, to obtain full advantage of the use of this stress it is important to understand the mechanism behind the response. It is thought that the induction of wound responses requires a simultaneous action of different signals and regulators and participation of any of these signals will depend on the plant species (Leon et al., 2001), therefore further studies are needed to identify and to understand the primary and secondary signals that trigger phenolic metabolism. Recently, our group proposed a model where ATPs and ROS play key roles as primary and secondary signals in the biosynthesis of phenolic antioxidants (Jacobo-Velazquez et al., 2011). Thus, according to that model, we hypothesise that increased wounding in carrots intensifies these signals, enhancing the biosynthesis of phenolic compounds as observed in the present study. In addition, Heredia and Cisneros-Zevallos (2009a, 2009b) found synergistic effects on the biosynthesis of phenolic antioxidants by combining wounding and hormones such as ethylene and methyl jasmonate in carrot tissue and other fresh produce. Similar synergistic responses have been reported for wounding and hyperoxia in carrot tissue as well (Jacobo-Velazquez et al., 2011). In general, understanding how wounding stress alone or in combination with other abiotic stresses (e.g. UV light, altered gas composition, hormones, chemical elicitors among others) elicits the biosynthesis
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of phenolic antioxidants would enable the design of simple strategies and technologies to enhance the health promoting properties of fresh produce for its use in the fresh and processing markets (Cisneros-Zevallos, 2003). 4. Conclusions In general, the HPLC data showed that chlorogenic acid and its related isomers were the major phenolics present and synthesised by wounding stress in carrot tissue and the phenolic profiles were dependent on wounding intensity. The relative higher proportions of synthesised 5-CQA with increased wounding intensity caused a higher capacity of these phenolic mixtures to scavenge free radicals (per phenolic basis) compared to whole carrots (Table 2). The latter observation is important since it indicates that controlling wounding intensity can be used as a simple tool to provide an inexpensive source of phenolic antioxidants for the fresh market such as fresh cut products, but also for alternative high value health markets including functional foods, dietary supplements and cosmetics, where bioactive phenolics are extracted and incorporated into different processed products. Acknowledgements Authors would like to thank Grimmway Farms for kindly supplying carrot samples used in this study. This project was funded through the Vegetable and Fruit Improvement centre with the Grant ‘‘Designing Foods for Health, USDA-CSREES 2001-3440213647’’. References Alasalvar, C., Grigor, J. M., Zhang, D., Quantick, P. C., & Shahidi, F. (2001). Comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties. Journal of Agricultural and Food Chemistry, 49, 1410–1416. Babic, I., Amiot, M. J., Nguyen-The, C., & Aubert, S. (1993). Changes in phenolic contents in fresh ready-to-use shredded carrots during storage. Journal of Food Science, 58, 351–356. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol., 28, 25–30. Clifford, M. N., Johnston, K. L., Knight, S., & Kuhnert, N. (2003). Hierarchical scheme for LC-MSn identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51, 2900–2911. Clifford, M. N. (1986). Coffee bean dicaffeoylquinic acids. Phytochemistry, 25, 1767–1769. Cevallos-Casals, B. A., & Cisneros-Zevallos, L. (2003). Stoichiometric and kinetic studies of phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato. Journal of Agricultural and Food Chemistry, 51, 3313–3319. Cisneros-Zevallos, L. (2003). The use of controlled postharvest abiotic stresses as a tool for enhancing the nutraceutical content and adding value of fresh fruits and vegetables. Journal of Food Science, 68, 1560–1565. ERS-USDA (2003). Trends in the US blueberry industry. Available from: (accessed 10.10.05). ERS-USDA (2004). US fresh-market carrots: Supply and disappearance. Available from: (accessed 10.10.05). Fan, X., Mattheis, J. P., & Roberts, R. G. (2000). Biosynthesis of phytoalexin in carrot root requires ethylene action. Physiologia Plantarum, 110, 450–454. Hale, A. L. (2003). Screening potato genotypes for antioxidant activity, identification of the responsible compounds and differentiating Russet Narkotah strains using AFLP and microsatellite marker analysis. PhD Dissertation, Texas A&M University. Heredia, J. & Cisneros-Zevallos, L. (2002). Wounding stress on carrots increases the antioxidant capacity and the phenolics content [abstract]. In: IFT annual meeting book of abstracts; June 15–19, 2002; Anaheim, Calif. Chicago, Ill.: Institute of Food Technologists (p. 180), Abstract No. 76C–14. Heredia, J., & Cisneros-Zevallos, L. (2009a). The effect of exogenous ethylene and methyl jasmonate on PAL activity, phenolic profiles and antioxidant capacity of carrots (Daucus carota) under different wounding intensities. Postharvest Biology and Technology, 51, 242–249. Heredia, J., & Cisneros-Zevallos, L. (2009b). The effect of exogenous ethylene and methyl jasmonate on the accumulation of phenolic antioxidants in selected whole and wounded fresh produce. Food Chemistry, 115, 1500–1508.
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Jacobo-Velazquez, D. A., & Cisneros-Zevallos, L. (2009). Correlations of antioxidant activity against phenolic content revisited: A new approach in data analysis for food and medicinal plants. Journal of Food Science, 74, R107–R113. Jacobo-Velazquez, D. A., Martinez-Hernandez, G. B., Rodriguez, S., Cao, C. M., & Cisneros-Zevallos, L. (2011). Plants as biofactories: Physiological role of reactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress. Journal of Agricultural and Food Chemistry, 59, 6583–6593. Kang, H.-M., & Saltveit, M. E. (2002). Antioxidant capacity of lettuce leaf tissue increases after wounding. Journal of Agricultural and Food Chemistry, 50, 7536–7541. Ke, D., & Salveit, M. E. (1986). Effects of calcium and auxin on russet spotting and phenylalanine ammonia-lyase activity in iceberg lettuce. HortScience, 21, 1169–1171. Ke, D., & Salveit, M. E. (1989). Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce. Physiologia Plantarum, 76, 412–418. Lafuente, M. T., Lopez-Galvez, G., Cantwell, M., & Yang, S. F. (1996). Factors influencing ethylene-induced isocoumarin formation and increased respiration in carrots. Journal of the American Society for Horticultural Science, 121, 537–542. Leja, M., Mareczek, A., Wojciechowska, R., & Rozek, S. (1997). Phenolic metabolism in root slices of selected carrot cultivars. Acta Physiologiae Plantarum, 19, 319–325. Leon, J., Rojo, E., & Sanchez-Serrano, J. J. (2001). Wound signalling in plants. Journal of Experimental Botany, 52, 1–9. Prior, R. L., & Cao, G. (2000). Analysis of botanicals and dietary supplements for antioxidant capacity: A review. Journal of AOAC International, 83, 950–956. Rakwal, R., & Agrawal, G. K. (2003). Wound signaling-coordination of the octadecanoid and MAPK pathways. Plant Physiology and Biochemistry, 41, 855–861. Reyes, L. F., & Cisneros-Zevallos, L. (2003). Wounding stress increases the phenolic content and antioxidant capacity of purple-flesh potatoes (Solanum tuberosum L.). Journal of Agricultural and Food Chemistry, 51, 5296–5300. Reyes, L. F., Villarreal, J. E., & Cisneros-Zevallos, L. (2007). The increase in antioxidant capacity after wounding depends on the type of fruit and vegetable tissue. Food Chemistry, 101, 1254–1262.
Rhodes, M. J., & Wooltorton, L. S. C. (1978). The biosynthesis of phenolic compounds in wounded plant storage tissue. In G. Kahl (Ed.), Biochemistry of wounded tissues (pp. 243–286). Berlin, German: Walter de Gruyter & Co. Rolle, R. S., & Chism, G. W. (1987). Physiological consequences of minimally processed fruits and vegetables. Journal of Food Quality, 10, 157–177. Saltveit, M. E. (1997). Physical and physiological changes in minimally processed fruits and vegetables. In F. A. Tomas-Barberan & R. J. Robins (Eds.), Phytochemistry of fruit and vegetables (pp. 205–220). New York, NY: Oxford University Press Inc. Salveit, M. E. (2000). Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Biology and Technology, 21, 61–69. Sondheimer, E. (1957). The isolation and identification of 3-methyl-6-methoxy-8hydroxy-3,4-dihydroisocoumarin from carrots. Journal of the American Chemical Society, 79, 5036–5039. Surjadinata, B. B., & Cisneros-Zevallos, L. (2003). Modeling wound-induced respiration of fresh-cut carrots (Daucus carota L.). Journal of Food Science, 68, 2735–2740. Surjadinata, B. (2005). Wounding and ultraviolet light radiation stresses affect the phenolic profile and antioxidant capacity of carrot tissue. PhD Dissertation, Texas A&M University. Swain, T., & Hillis, W. E. (1959). The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. Journal of the Science of Food and Agriculture, 10, 63–68. Talcott, S. T., & Horward, L. R. (1999). Chemical and sensory quality of processed carrot puree as influenced by stress-induced phenolic compounds. Journal of Agricultural and Food Chemistry, 47, 1362–1366. Toivonen, P. M. A., & DeEll, J. R. (2002). Physiology of fresh-cut fruits and vegetables. In O. Lamikanra (Ed.), Fresh-cut fruits and vegetables science, technology, and market (pp. 91–124). Boca Raton: CRC Press. Zhang, D., & Hamauzu, Y. (2004). Phenolic compounds and their antioxidant properties in different tissues of carrots (Daucus carota L.). Food, Agriculture & Environment, 2, 95–100.
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Biosynthesis of phenolic antioxidants in carrot tissue increases with wounding intensity Bernadeth B. Surjadinata, Luis Cisneros-Zevallos ⇑ Department of Horticultural Sciences, Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX 77843-2133, USA
a r t i c l e
i n f o
Article history: Received 4 June 2011 Received in revised form 21 December 2011 Accepted 26 January 2012 Available online 4 February 2012 Keywords: Wounding intensity Phenolics Antioxidant capacity Fresh-cut Carrots
a b s t r a c t Biosynthesis of phenolic antioxidants in three carrots cultivars (Navajo, Legend and Choctaw) were studied under different wounding intensities (A/W) during storage. Generated A/W of 4.2, 6.0 and 23.5 cm2/g, corresponded to slices, pies, and shreds, respectively. Soluble phenolics, antioxidant capacity (AOX), and PAL activity increased with A/W for all cultivars. Intense wounding (23.5 cm2/g) induced an 2.5- and 12.4-fold increase in soluble phenolics and AOX, respectively, after 4 days compared to whole carrots. Wounding induced the synthesis of mainly chlorogenic acid (5-CQA) and 3,5-dicaffeoylquinic acid. A higher proportion of 5-CQA in the phenolic mixture was responsible for an increasing specific AOX (809 and 1619 lg Trolox/mg phenolics for whole carrots and shreds, respectively, for Choctaw cultivar). Wounded carrots can be promoted as an inexpensive rich source of phenolic antioxidants for the regular diet. By simply increasing wounding stress intensity it is possible to enhance the biosynthesis of phenolic antioxidants. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction One of the procedures involved in fresh-cut fruit and vegetable processing is cutting the produce into smaller pieces. This cutting action essentially is causing the tissue to endure wounding stress. Wounding will cause some physiological effects to the tissue and it is understood that a more severe cutting process will elicit a greater wounding response decreasing the quality of the produce. Wounding has been known to increase respiration rate (Surjadinata & Cisneros-Zevallos, 2003) and ethylene production of many different tissues (Rolle & Chism, 1987; Saltveit, 1997). Many responses occur because wounding stress is involved in the generation, translocation, perception, and transduction of signals in order to activate the expression of wound-inducible genes (Leon, Rojo, & Sanchez-Serrano, 2001), either locally by the injured cells or systemically by the adjacent cells. Plant cells are contained inside rigid walls, making each cell capable of triggering defence mechanisms. When wounding stress occurs, the cell can activate the specific transcriptional genes (Leon et al., 2001; Salveit, 2000) with the purpose of adjusting the metabolism to repair and heal the damage and to synthesise substances to prevent invasion by the predators. These processes could occur between a few minutes to several hours after wounding (Leon et al., 2001).
⇑ Corresponding author. Tel.: +1 979 8453244; fax: +1 979 8450627. E-mail address: [email protected] (L. Cisneros-Zevallos). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.01.097
In plants, many compounds have been proposed to play a role in wound signalling, such as phytohormones (e.g. ethylene, jasmonic acid, ABA), reactive oxygen species (ROS), salicylic acid, systemin, oligosaccharides, as well as electrical pulses and hydraulic waves (Leon et al., 2001; Rakwal & Agrawal, 2003; Salveit, 2000). Recently, Jacobo-Velazquez, Martinez-Hernandez, Rodriguez, Cao, and Cisneros-Zevallos (2011) showed that ROS plays a key role as secondary signal in the biosynthesis of phenolic antioxidants in carrots as a response to wounding. Furthermore, they proposed a model where ATPs would play a primary signal role. Improved knowledge of this signalling mechanism would be very important in order to understand the effect of a stress on the tissue and to develop strategies to avoid it or enhance it. A stress or an injury to a plant cell will trigger two types of responses in phenolic metabolism (Rhodes & Wooltorton, 1978). The first response is the oxidation of the existing phenolic compounds as a result of rupture of the cell membrane, causing the phenolics to combine with the oxidative enzyme systems. The other response involves the synthesis of monomeric or polymeric phenolics to repair the wounding damage. This second response are caused by changes in phenylalanine ammonia lyase activity (PAL, EC 4.3.1.5) since it is the key metabolic enzyme in the phenylpropanoid pathway (Babic, Amiot, Nguyen-The, & Aubert, 1993; Kang & Saltveit, 2002). Increase in phenolic content is one of the most studied phenomena in response to wounding (Toivonen & DeEll, 2002). For example, Babic et al. (1993) found that storing shredded carrots in air accumulated chlorogenic acid while Leja, Mareczek, Wojciechowska, and Rozek (1997) found that peels of carrot slices
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stored for 4 days at 20 °C produced more chlorogenic acid and isocoumarin. However, a first report showing increased antioxidant capacity in wounded tissue was presented by Heredia and Cisneros-Zevallos (2002) in a study using carrots as a model system. These findings of increased antioxidant activity with wounding stress were later confirmed in lettuce leaf (Kang & Saltveit, 2002), purple-flesh potato (Reyes & Cisneros-Zevallos, 2003) and in a variety of fresh produce (Reyes, Villarreal, & Cisneros-Zevallos, 2007). Furthermore, all of these studies were done with only one type of cuts (e.g. slices), therefore the need to study how wounding intensity affects the biosynthesis of phenolic antioxidants. It is well known the importance phenolic antioxidant have as promoters of human health through their antiradical scavenging activity by preventing chronic diseases associated to oxidative stress. In this study we characterised the effects of wounding stress under the hypothesis that the biosynthesis of phenolic antioxidants will increase with wounding intensity. In this paper we present information confirming how wounding intensity influences phenolic accumulation during storage, PAL activity, the phenolic profiles and their effects on antioxidant activity and the influence of carrot cultivars used. This work is based on a dissertation by Surjadinata (2005). The generated information may be used to promote wounded carrots as an inexpensive rich source of phenolic antioxidants for the regular diet. 2. Materials and methods 2.1. Chemical reagents All chemicals and standards used, Folin–Ciocalteu reagent, sodium carbonate (Na2CO3), Trolox and 2,2-diphenyl-1-picrylhydrazyl (DPPH), polyvinylpyrrolidine (PVPP), sodium hydroxide (NaOH), boric acid, 2-mercaptoethanol, chlorogenic acid, p-hydroxybenzoic acid, and ferulic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1,3-Dicaffeoylquinic acid (cynarin) was purchased from ChromaDex, Inc. (Santa Ana, CA, USA). 1-Aminocyclopropene-1-carboxylic acid (ACC) was purchased from MP Biomedicals (Aurora, OH, USA). Methanol, ethanol, hexane, and acetonitrile were reagents of HPLC grade. 2.2. Carrots and wounding stress studies Whole-topped commercial carrots cultivars Navajo, Legend and Choctaw used in this study were kindly provided by Grimmway Farms, Bakersfield, CA. The carrots were grown under similar conditions and were harvested within 2 weeks, shipped to Texas A&M and stored at 2–5 °C until used (within 2 weeks period). The carrots were conditioned overnight at 15 °C before the wounding studies. All carrots used were of similar size (2.5–3 cm in diameter and 22–25 cm in length) and free of visual damage to reduce experimental variability. Wounding intensity studies were performed cutting carrots into different shapes, including slices, pies and shreds. Wounding intensity (A/W) was defined by the ratio of the new surface area created by wounding in cm2 over the tissue weight in g (Surjadinata & Cisneros-Zevallos, 2003). The wounded surface areas of carrots slices, pies, and shreds were manually measured. The slices were 5 mm thick while the pies were obtained from the carrots slices and were cut further into quarters. For shreds, the A/W was estimated from 40 pieces of shredded carrots of about 5.1 g, which gave an area of 119.7 cm2. The calculated wounding intensities (A/W) were 4.2, 6.0, and 23.5 cm2/g for carrot slices, pies, and shreds, respectively. Whole carrots used as controls have an A/W of 0.0 cm2/g since they were not cut, thus no wounded surface area was present (Fig. 1).
Fig. 1. Wounding intensity (A/W) defined by the ratio of the new surface area created by wounding in cm2 over the tissue weight in g. The calculated A/W for whole carrots, slices, pies and shreds were 0, 4.2, 6.0 and 23.5 cm2/g, respectively.
On the day of processing, carrots were taken from the 15 °C storage, washed with 100 ppm chlorinated water and air-dried for 2–3 h at room temperature. Shreds (considered as extreme wounding) were obtained from carrot cylinders of 150 g using a food processor (High Performance model, West Band Co., West Band, WI). After cutting, wounded and non-wounded carrots were placed in 4 L closed clear glass jars. The jars were stored in the dark at 15 °C and were ventilated periodically (every 6–8 h) to maintain aerobic respiration and avoid accumulation of carbon dioxide (<0.5%). The carrot shreds were stored for 0, 2, 4, and 8 days while carrot slices, pies and non-wounded carrots (controls) were stored 4 days monitoring total phenolics, phenolic profiles antioxidant capacity, and phenylalanine ammonia lyase (PAL) activity. After 8 days of storage, the water loss of the shredded carrots was approximately 3% and showed development of off-odour, possibly due to a combined effect of increase respiration, ethylene production and membrane deterioration at 15 °C. This temperature was used in all experiments only to accelerate the wounding response and it is not recommended for maintaining quality. Lower temperature would be more appropriate. 2.3. Total soluble phenolics assay The phenolic content was analysed using the procedure of Swain and Hillis (1959) with some modifications. Five grams of fresh tissue samples were mixed with 25 ml of methanol and homogenised until reaching uniform consistency using an Ultra Turrax (T 25 basic, IKA Labortechnik, Staufen, Germany). Extracts were stored in covered plastic tubes overnight at 4 °C and then centrifuged at 30,000g for 20 min (Beckmann Coulter J2-21, Fullerton, CA). The centrifuge was equipped with a rotor (Beckman Coulter JA-17, Fullerton, CA). A 150 ll sample of the clear supernatant was collected and diluted with 2400 ll of nanopure water. At the same time, a blank (150 ll of nanopure water) was prepared by following the same procedure as the sample. To each diluted sample and blank, 150 ll of 0.25 N Folin–Ciocalteau reagent was added, vortexed and allowed to react at room temperature for 3 min, followed by the addition of 300 ll of 1 N Na2CO3. This mixture was allowed to react at room temperature in the dark for 2 h. Readings were done at 725 nm using a photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany), which was previously blanked with methanol. Total phenolics were expressed as mg chlorogenic acid equivalent/100 g fresh weight tissue (FW), based on a standard curve.
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2.4. Determination of phenolic profiles The same extracts used for the total phenolics assay were used for determining the phenolic profiles using an HPLC method described by Hale (2003). The samples were run using Waters Millennium 3.2 software (Milford, MA, USA). The HPLC system was equipped with a binary pump system (Waters 515, Milford, MA, USA), an auto-injector (Waters 717 plus, Milford, MA, USA), a photodiode array (PDA) detector (Waters 996, Milford, MA, USA), and a column heater (SpectraPhysics SP8792, San Jose, CA, USA). The column used to separate the phenolic compounds was a 4.6 150 mm, 5 lm, C-18 reverse-phase column (Waters Atlantis, Milford, MA, USA), which was maintained at 40 °C. The injection volume was 10 ll. Two mobile phases were used: water/HCl adjusted to pH 2.3 as solvent A and acetonitrile as solvent B. The gradient system was 0/85, 5/85, 30/0, 35/0 (min/% solvent A). Both solvents were filtered and degassed before used. Prior to injection, the samples were filtered through a 0.2 lm nylon syringe filter. Identification and quantification was based on developed standard curves, retention times and UV–vis spectra. Commercial standards of chlorogenic acid, ferulic acid, p-hydroxybenzoic acid and 1,3-dicaffeoylquinic acid were used for peak identification when possible. Other chlorogenic acid derivatives and isocoumarin were determined as described bellow. 2.4.1. Determination of dicaffeoylquinic acid (diCQA and isocoumarin (ISO) by time of flight mass spectrometry (TOF-MS) Based on the HPLC method described above, the obtained peaks between 16 and 18 min (corresponding to diCQA) were collected using a fraction collector (Waters Fraction Collector II, Milford, MA, USA) connected after the PDA detector (Waters 996, Milford, MA, USA). The eluent in each collected fraction was evaporated using a Speed-Vac Concentrator (SC 100, Savant, NY, USA) and then freeze dried (PD-6-54A, FTS Systems, Inc., NY, USA). The isolated compounds were dissolved in water/methanol (50:50 v/v) acidified with 1% acetic acid and injected into a time of flight (TOF) mass spectrometer equipped with electron spray ionisation (ESI) in negative ion mode (M-H) (PE Sciex API QStar Pulsar, Concord, Ontario, Canada). Isocoumarin extraction was done using a method by Talcott and Horward (1999) with some modifications. Ten kilogram of whole carrots were sprayed with 100 ppm of 1-aminocyclopropane-1carboxylic acid (ACC), exposed to 1000 ppm of ethylene and sealed in 4 L glass jars during 4 days in the dark at room temperature. The jars were ventilated every 12 h to avoid CO2 accumulation (>0.5%). At the end of storage, carrots were peeled. About 600 g of peels were obtained and these peels were then extracted with 3 L of hexane for 20–24 h. The next day, the liquid phase was partitioned with 1 L of ethanol. The ethanol fraction was divided into 10 vials of 1 ml and injected to the HPLC using the protocol described before. A peak that appeared around 24 min was confirmed to be isocoumarin by monitoring the kmax of the PDA spectra (absorbance at 267 and 302 nm). In order to accumulate significant amount of isocoumarin, the injection volume used was 125 ll. Each vial was injected three times. The total fractions collected were around 30 ml and these were evaporated with Speed-Vac Concentrator (SC 100, Savant, NY, USA) and further freeze-dried (Kinetics, NY, USA). The resulting powder was then re-dissolved in 1 ml of ethanol. To quantify the amount of isocoumarin present in the dried powder, the absorbances at 267 and 302 nm of this ethanolic sample were measured with photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany). The ratio between the two absorbances was around 2.25–2.29, which is very similar to the pure isocoumarin (ratio = 2.47) (Sondheimer, 1957). Quantification of isocoumarin collected was performed using a molar extinction coefficient in ethanol (e = 14,800/M cm)
617
(Sondheimer, 1957). In 1 ml of ethanolic samples there was 1.1031 mg of isocoumarin. Several dilutions of this ethanolic sample were injected back into the HPLC to create a standard curve. This time the injection volume was set to be 10 ll, which was the volume injection condition used for the wounded and nonwounded carrot extracts. Isocoumarin powder collected from the freeze-drier was also re-dissolved in ethanol and injected into a time of flight (TOF) mass spectrometer (PE Sciex API QStar Pulsar, Concord, Ontario, Canada) equipped with ESI in positive ion mode (M+H)+ combined with (M+Li)+ for further identification and confirmation. The isolated compounds were dissolved in water/methanol acidified with 1% acetic acid. The capillary voltage was 4.5 kV. The mass spectrometry procedure was done in collaboration with the Chemistry Department of Texas A&M University (College Station, TX, USA). 2.5. Antioxidant capacity (AOX) assay AOX was quantified using the procedure of Brand-Williams, Cuvelier, and Berset (1995). The same extracts prepared for the total phenolics assay were also used for the AOX assay. A diluted solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared from the mother stock solution using methanol to obtain an absorbance of 1.1 ± 0.02. After centrifugation of the extracts at 30,000g (Beckmann Coulter J2-21 equipped with a rotor JA-17, Fullerton, CA) at 4 °C for 20 min, 150 ll of the clear supernatant were mixed with 2850 ll of the diluted DPPH solution in a clean plastic vial, vortexed and then closed. At the same time, a blank of 150 ll of methanol mixed with the DPPH diluted solution was prepared. The sample extract and DPPH was allowed to react in the dark at room temperature for 24 h or until reaching steady state. AOX was measured at 515 nm using a photodiode array spectrophotometer (Hewlett–Packard 8425A, Waldbronn, Germany), that was previously blanked with methanol/DPPH solution. AOX was expressed as lg Trolox equivalents/g FW, based on a standard curve using Trolox. 2.6. Phenylalanine ammonia lyase (PAL) enzyme activity assay The PAL activity was measured using the procedure of Ke and Salveit (1986) with slight modifications. For each replicate, 1 g of tissue was mixed with 0.2 g polyvinylpyrrolidine (PVPP) and homogenised in 30 ml of 50 mM cold borate buffer (pH 8.5) at low speed to a uniform consistency. The borate buffer was a mixture of boric acid and sodium hydroxide in nanopure water containing 400 ll of 2-mercaptoethanol per 1 L buffer. The extracts were then filtered through 4 layers of cheesecloth and centrifuged at 30,000g (Beckmann Coulter J2-21 equipped with a rotor JA-17, Fullerton, CA) for 15 min at 4 °C. Throughout the analysis, sample extracts were kept in ice and in dark condition. After centrifugation, clear supernatant was collected and incubated for 5 min in a 40 °C water-bath. Two sets of 10 ml glass tubes were prepared. The first set is for the water control and the second set is for the samples to which 100 mM of L-phenylalanine was added as substrate. Measurements of the enzyme activity were done at 0 and 1 h of incubation in a 40 °C water-bath. PAL activity was quantified as lmoles of t-cinnamic acid/h g FW. It was calculated using a standard curve developed for t-cinnamic acid in the borate buffer. 2.7. Statistical analysis Statistics analysis was done using the ANOVA procedure from SAS Statistical Analysis System for Windows v8.1 software (SAS Institute Inc., Cary, NC, USA). The treatment means were compared with Tukey’s Studentized Range test at a = 0.05. Six replicates per treatment were used in this study (n = 6).
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storage period. The total phenolics of individual carrot replicates (n = 6) showed a linear relationship with their corresponding total AOX for each sampling day of storage with R2 ranging from 0.71 to 0.98. There was a distinct line for each sampling day (data not shown). This pattern was similar for all cultivars and the linear relationship showed a trend to pass through 0 for both X and Y axes. The slopes of these linear relationships increased as the storage time increased. The slopes correspond to the specific phenolic antioxidant capacity, which is the antioxidant capacity expressed on a phenolic basis (ratio between total AOX and total phenolics) (Jacobo-Velazquez & Cisneros-Zevallos, 2009). The specific AOX gives information of the effectiveness of the phenolic mixture present on neutralising the free radicals. Thus, a higher specific AOX would mean that the phenolics present stabilise a greater number of free radicals. Accordingly, Navajo, Legend and Choctaw carrots
3. Results and discussion 3.1. Effects of wounding stress on carrot tissue during storage Intense wounding stress or shredding induced the accumulation of phenolic compounds in carrot tissue through storage time (P < 0.05), however, there was no difference between carrot cultivars (P > 0.05). After 8 days storage there was a 5.7-fold increase in total phenolics compared to day 0 (45 mg/100 g for controls) (Fig. 2A). There was an increase in AOX with storage time for all wounded carrot cultivars (P < 0.05). Total AOX showed a 15.5-, 18.5-, and 17.0-fold increase after 8 days storage for Navajo, Legend, and Choctaw cultivar, respectively, compared to day 0 (150–235ug Trolox/g for controls) (Fig. 2B). This increase in AOX is related to the accumulation of phenolic compounds observed in that same 300
Phenolics (mg Chlorogenic Acid/100g)
A
Navajo Legend
250
Choctaw
200 150 100 50 0 0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
6000
B AOX (ug Trolox Eq./g)
5000 4000 3000 2000 1000 0
2
PAL
(umol t -Cinnamic Ac/hr g)
C
1.6
1.2
0.8
0.4
0 Day Fig. 2. Total soluble phenolics (A), antioxidant capacity (B), and PAL activity (C) of three cultivars of shredded carrots during 8 storage at 15 °C. Vertical bars represent SD (n = 6).
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Phenolics (mg Chlorogenic Ac/100g)
A
250
Navajo Legend
200
Choctaw 150 100 50 0
B
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
3500
AOX (ug Trolox Eq/g)
3000 2500 2000 1500 1000 500 0
PAL (umol t-Cinnamic Ac/hr g)
C
2.5
2
1.5
1
0.5
0
A/W (cm2/g) Fig. 3. Total soluble phenolics (A), antioxidant capacity (B), and PAL activity (C) of three carrot cultivars under different wounding intensities (A/W) after 4 days storage at 15 °C. Vertical bars represent SD (n = 6).
showed increases in specific AOX between days 0 and 8 ranging from 542 to 1543, 476 to 1382 and 664 to 1748 lg Trolx/mg phenolics, respectively, indicating that the mixture of phenolics biosynthesised through time had higher radical scavenging properties. Despite that the total phenolics were similar for all cultivars at day 8, there seems to be differences in the phenolic profiles as depicted by differences in the specific AOX (e.g. Choctaw > Lengend and Navajo). Wounding stress induced an increase in PAL activity for all three cultivars reaching a maximum peak after 2 days storage and a further decrease through time (Fig. 2C). At day 2, Navajo, Legend and Choctaw cultivars showed a 7.1-, 7.5- and 11.7-fold increase, respectively, compared to day 0 (0.133–0.169 lmol tcinnamic acid/h g for controls). The total phenolics in wounded carrots continued to accumulate after 4 and 8 days storage since
the PAL-synthesizing system remained present. Even though its activity decreased, the enzyme was not completely inactivated, thus maintaining the ability to produce phenolic compounds. Between days 4 and 8 of storage, there was a decline in the rate of biosynthesised phenolics (Fig. 2A). In general, Choctaw cultivar had higher PAL activity throughout the storage period, which could be associated to higher levels of specific phenolic compounds being synthesised and not observed in the total phenolics assay. 3.2. Wounding intensity effects on soluble phenolics, phenolic profiles, AOX and PAL activity The phenolic content increased with wounding intensity for all three carrot cultivars (Fig. 3A). This increase was a 97%, 76%, and 252% for carrots slices, pies and shreds, respectively, after 4 days of
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Table 1 Individual phenolics determined by HPLC at 280 nm of different carrot cultivars and wounding intensities. Cultivar
Peak #
Ret time (min)
kmax (nm)
Navajo
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
241.3, 325.5
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
241.3, 325.5
1 2 3 4 5 6 7
6.85 7.92 15.57 17.47 18.15 23.19 24.19
193.1, 196.6, 195.4, 196.6, 197.8, 198.9, 214.2,
216.5, 254.2 216.5, 241.3, 271.7, 263.7 267.1,
Legend
Choctaw
Compound
234.2, 321.9 326.7 240.1, 326.7 302.3
234.2, 321.9 326.7 240.1, 326.7 302.3 241.3, 325.5 234.2, 321.9 326.7 240.1, 326.7 302.3
Amount (mg/100 g) Whole
Slices
Pies
Shreds
5-CQA p-HBA FA 3,4-diCQA** 3,5-diCQA** HBA derivative*** Isocoumarin
5.499 nd* nd nd 4.593 nd nd
15.71 nd 2.099 nd 7.670 nd 0.089
22.34 nd 2.200 nd 8.250 nd 0.095
52.54 0.819 2.740 1.399 8.214 1.718 0.734
5-CQA p-HBA FA 3,4-diCQA 3,5-diCQA HBA derivative Isocoumarin
5.291 nd nd nd 4.472 nd nd
12.64 nd 2.073 nd 6.441 nd 0.079
13.04 nd 2.055 nd 6.158 nd 0.080
38.67 0.157 2.386 1.656 7.887 0.616 0.301
5-CQA p-HBA FA 3,4-diCQA 3,5-diCQA HBA derivative Isocoumarin
6.302 nd nd nd 4.840 nd nd
15.44 nd 2.080 nd 6.613 nd 0.053
23.29 nd 2.810 nd 7.372 nd 0.088
71.68 0.639 3.032 1.363 8.677 2.239 0.580
*
nd = Not detected. Quantified using chlorogenic acid (5-CQA) as standard. *** Quantified using p-hydroxybenzoic acid (p-HBA) as standard. **
storage at 15 °C compared to non-wounded carrots (45–52 mg/ 100 g). Cultivars Choctaw and Navajo showed higher accumulation of phenolic compounds compared to cultivar Legend at wounding intensities P6.0 cm2/g (pies). HPLC phenolic profiles for wounded and non-wounded carrot tissue showed these were similar for all three cultivars (Table 1). Typical chromatograms for different wounding intensities are shown for Choctaw cultivars in Fig. 4. HPLC phenolic analysis of non-wounded tissue showed the presence of chlorogenic acid (5CQA, peak 1) and a dicaffeoylquinic acid isomer (diCQA, peak 5). Phenolic profiles of carrot slices and pies, showed an additional presence of ferulic acid (FA, peak 3) and isocoumarin (ISO, peak 7), while phenolic profiles of shredded carrots showed, in addition, peaks for p-hydroxybenzoic acid (pHBA, peak 2), an unknown hydroxybenzoic acid derivative (peak 6), and one more diCQA (peak 4). Peak 6 had very similar PDA spectra to p-hydroxybenzoic acid, however, the retention times differed by about 18 min, indicating this compound is less polar compared to p-HBA. Peaks 4 and 5 were identified as isomers of chlorogenic acid since the PDA spectra were similar to that of chlorogenic acid. This was then confirmed by TOF-MS analysis showing that m/z of the parent ion of peaks 4 and 5 were 515.11. According to Clifford, Johnston, Knight, and Kuhnert (2003), the m/z for the parent ion of 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA in coffee bean are 515.7, 515.2, and 515.4, respectively. There are 4 different diCQA: 1,3diCQA (also known as cynarin), 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA (Clifford, 1986; Clifford et al., 2003). In this study, commercially available 1,3-diCQA (m/z, 515.12) was injected in the HPLC system and its retention time was about 14 min (data not shown), differing from peak 4 (17.47 min) and peak 5 (18.15 min). In carrot tissue, 3,5-diCQA has been shown to be in higher amounts compared to other diCQA isomers (Alasalvar, Grigor, Zhang, Quantick, & Shahidi, 2001; Zhang & Hamauzu, 2004). Accordingly, peak 5 would correspond to 3,5-diCQA, while peak 4 which appeared before peak 5, would correspond to 3,4-diCQA since this isomer is more polar than 3,5-diCQA. Results showed that chlorogenic acid was the major phenolic compound present in all three carrot cultivars of wounded and
non-wounded tissue (Table 1). When carrots were shredded, this compound showed a 9.5-, 7.3-, and 11.3-fold increase in content for cultivar Navajo, Legend, and Choctaw, respectively (Table 1). Peak 7 was confirmed to be isocoumarin by TOF-MS analysis. The positive ionisation and injection of internal standard, lithium, gave m/z of 215.08. Isocoumarin has a mass of 208.21 and lithium has a mass of 7, therefore when combined, the m/z would be 215. The maximum level of isocoumarin produced by the wounded carrot tissue was approximately 0.30–0.73 mg/100 g FW. The phytoalexin isocoumarin is synthesised by wounding in carrot tissue and plays a major role in the plant defence system (Fan, Mattheis, & Roberts, 2000). This compound gives bitter flavour, which could decrease the quality of the wounded carrots. According to Lafuente, Lopez-Galvez, Cantwell, and Yang (1996), bitterness flavour in carrot tissue, is associated to isocoumarin content P20 mg/100 g FW. The amount of isocoumarin observed in the present study (61 mg/ 100 FW) will not induce a bitter taste to the tissue. It has been shown previously that other stresses such as ethylene exposure may elicit more isocoumarin synthesis compared to wounding (Lafuente et al., 1996). The AOX increased with the wounding intensity for all three cultivars studied (Fig. 3B). The AOX increased by 2- to 3.5-fold for slices and pies and up to 12.4-fold for shreds compared to non-wounded carrot after 4 days of storage at 15 °C (250– 300 lg Trolox/g for controls). AOX was similar (P > 0.05) between slices and pies for each cultivar. Cultivars Choctaw and Navajo showed higher AOX compared to Legend for all wounding intensities (P < 0.05). Increased wounding intensity also induced an increased in PAL activity among the tree cultivars studied (Fig. 3C). PAL activity increased by 19.2-, 27.9- and 266.2-fold for carrot slices, pies, and shreds, respectively, compared to non-wounded carrots (P < 0.05). Navajo and Legend showed very similar PAL activity (P > 0.05), however for Choctaw, PAL was higher only for shreds (P < 0.05). This higher PAL activity is likely associated to a higher biosynthesis of 5-CQA in Choctaw carrots (Table 1). Ke and Salveit (1989) previously found that PAL activity increased with number of punctures on lettuce tissue. In the present
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Whole
0.08
AU
0.06 0.04
1
0.02
5
0.00 5.00
10.00
15.00
20.00
25.00
Minutes 0.10
Slice
0.08
AU
0.06 0.04
1
0.02
5
3
7
0.00 5.00
10.00
15.00
20.00
25.00
Minutes 0.10
Pie
0.08
1
AU
0.06 0.04
5
3
0.02
7
0.00 5.00
10.00
15.00
20.00
25.00
Minutes 0.10
Shred
1
0.08
5
AU
0.06 0.04
7
3
0.02
6
4
2 0.00 5.00
10.00
15.00
20.00
25.00
Minutes
Fig. 4. HPLC profiles of carrot tissue at 280 nm for Choctaw carrots. Peaks 1, chlorogenic acid (5-CQA); 2, p-hydroxybenzoic acid (pHBA); 3, ferulic acid (FA); 4, 3,4dicaffeoylquinic acid (3,4-diCQA); 5, 3,5-dicaffeoylquinic acid (3,5-diCQA); 6, hydroxybenzoic acid derivative; 7, isocoumarin (ISO). Y-axis is absorbance unit (AU) and X-axis is retention time in min.
study wounding stress from 0 to 23.5 cm2/g seem to originate an exponential response in PAL activity, but more data would be needed to confirm this non-linear relationship. Furthermore, it would be important to define the linear or non-linear association between wounding area created (W/A) and the abiotic stress signal. In general, reported experimental data related to increase in PAL activity with the increase of wounding intensity is scarce in the literature. Thus, this study is one of the first attempts to quantify wounding intensity effects on phenolic biosynthesis.
3.3. Wounding intensity effects on specific AOX AOX of individual carrot replicates presented a linear relationship with their corresponding total phenolic contents for all wounding intensities and this trend was similar for all carrot cultivars (R 0.48–0.92) (Fig. 5). The slopes of the linear relationship increased with wounding intensity indicating that wounding stress synthesised phenolic compounds with increased AOX properties. The slope defines the specific AOX of the phenolic mixture present
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A
4000
Slices Pies
R2 = 0.869
Shreds
3000
Linear (Whole)
2000 R2 = 0.849
1000
R2 = 0.749 R2
= 0.929
0 0
AOX (ug Trolox Eq./g)
B
50
100
150
200
2500
2000 R2 = 0.485
1500
1000 R2 = 0.879 R2 = 0.819
500 R2 = 0.842
0 0
C
50
100
150
4000 R2 = 0.623
3000
2000 R2 = 0.868
1000
R2 = 0.754 R2
= 0.905
0 0
50
100
150
200
Phenolics (mg Chlorogenic Acid/100g) Fig. 5. Linear correlations between antioxidant capacity and total soluble phenolics of carrot cultivars Navajo (A), Legend (B), and Choctaw (C) under different wounding intensities using individual carrot samples (n = 6). Measurements were taken after 4 days storage at 15 °C.
Table 2 Relative proportion (%) of the three major hydroxycinnamic acids present in carrot tissue under different wounding intensities and the associated specific AOX.
*
Cultivar
Cut
A/W (cm2/g)
5-CQA
FA
3,5-diCQA
Specific AOX** (lg Trolox/mg phenolic*)
Navajo
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
54.5 61.7 68.1 82.7
0.0 8.2 6.7 4.3
45.5 30.1 25.2 12.9
456 ± 71.5 1052 ± 131.8 1180 ± 47.6 1604 ± 83.5
Legend
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
54.2 59.8 61.4 79.0
0.0 9.8 9.7 4.9
45.8 30.4 29.0 16.1
482 ± 84.2 936 ± 97.2 902 ± 43.4 1221 ± 211.1
Choctaw
Whole Slices Pies Shreds
0.0 4.2 6.0 23.5
56.6 64.0 69.6 86.0
0.0 8.6 8.4 3.6
43.4 27.4 22.0 10.4
809 ± 53.6 1198 ± 213.8 1275 ± 49.9 1619 ± 210.0
Phenolic content was quantified based on 5-CQA spectrophotometric standard curve. Average specific AOX ± SD, obtained by diving AOX and phenolic content for each carrot replicate (n = 6). Specific AOX also corresponds to the slopes of the regression line in Fig. 5.
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in the carrots and this phenolic mixture clearly showed different HPLC phenolic profiles for individual wounding intensities (Table 1). For example, Table 2 presents the relative proportions (phenolic profiles) of the three major hydroxycinnamic acids present in wounded and non-wounded carrot tissue: chlorogenic acid (5-CQA), ferulic acid (FA), and one of the chlorogenic acid isomers (3,5-diCQA). The relative proportions (%) of phenolic compounds will depend on wounding intensity (A/W). For non-wounded carrot tissue, there was similar proportion between 5-CQA and 3,5-diCQA, corresponding to a lower specific AOX capacity. For slices and pies, the phenolic profiles were similar, showing a higher amount of 5-CQA compared to 3,5-diCQA. These phenolic profiles also give similar specific AOX. This would explain why the slopes of the linear relationship between total AOX and total phenolics were similar for both wounding intensities (Fig. 5). For shreds, which showed the highest specific AOX, the relative proportions of phenolic compound indicate a much higher contribution of 5CQA compared to 3,5-diCQA suggesting that 5-CQA content has a large effect on the specific AOX of carrot tissue. 3.4. Wounded carrots as an inexpensive source of phenolic antioxidants In the present study we have shown that phenolic antioxidant accumulation in carrot tissue is dependent on wounding intensity. For example, the antioxidant levels may reach up to 3000 lg Trolox/g (11.98 lmol/g) in shredded carrots which is 6 times higher than whole carrots in just only 4 days of storage. Blueberries, which is considered a rich source of phenolic antioxidants provides 5500 lg Trolox/g (21.97 lmol/g) (Cevallos-Casals & Cisneros-Zevallos, 2003), however they are expensive and seasonal. The annual consumption per capita of blueberries in the US is 0.18 kg while consumption of carrots is 3.6 kg (ERS-USDA, 2003, 2004) making wounded carrots a potential higher source of phenolic antioxidants compared to blueberries (an estimated 10 times higher antioxidant source per capita). The recommended daily intake of antioxidants for humans is estimated to be in the range of 3000–3600 lmol Trolox/day, from which 1200 to 1640 lmol Trolox/day comes from the consumption of fruit and vegetables (representing <50%) (Prior & Cao, 2000). Accordingly, 100 g of shredded carrots may provide 33% to 40% of the required recommended intake of antioxidants and could be considered an inexpensive alternative source of phenolic antioxidants compared to more expensive fruits and vegetables. However, to obtain full advantage of the use of this stress it is important to understand the mechanism behind the response. It is thought that the induction of wound responses requires a simultaneous action of different signals and regulators and participation of any of these signals will depend on the plant species (Leon et al., 2001), therefore further studies are needed to identify and to understand the primary and secondary signals that trigger phenolic metabolism. Recently, our group proposed a model where ATPs and ROS play key roles as primary and secondary signals in the biosynthesis of phenolic antioxidants (Jacobo-Velazquez et al., 2011). Thus, according to that model, we hypothesise that increased wounding in carrots intensifies these signals, enhancing the biosynthesis of phenolic compounds as observed in the present study. In addition, Heredia and Cisneros-Zevallos (2009a, 2009b) found synergistic effects on the biosynthesis of phenolic antioxidants by combining wounding and hormones such as ethylene and methyl jasmonate in carrot tissue and other fresh produce. Similar synergistic responses have been reported for wounding and hyperoxia in carrot tissue as well (Jacobo-Velazquez et al., 2011). In general, understanding how wounding stress alone or in combination with other abiotic stresses (e.g. UV light, altered gas composition, hormones, chemical elicitors among others) elicits the biosynthesis
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of phenolic antioxidants would enable the design of simple strategies and technologies to enhance the health promoting properties of fresh produce for its use in the fresh and processing markets (Cisneros-Zevallos, 2003). 4. Conclusions In general, the HPLC data showed that chlorogenic acid and its related isomers were the major phenolics present and synthesised by wounding stress in carrot tissue and the phenolic profiles were dependent on wounding intensity. The relative higher proportions of synthesised 5-CQA with increased wounding intensity caused a higher capacity of these phenolic mixtures to scavenge free radicals (per phenolic basis) compared to whole carrots (Table 2). The latter observation is important since it indicates that controlling wounding intensity can be used as a simple tool to provide an inexpensive source of phenolic antioxidants for the fresh market such as fresh cut products, but also for alternative high value health markets including functional foods, dietary supplements and cosmetics, where bioactive phenolics are extracted and incorporated into different processed products. Acknowledgements Authors would like to thank Grimmway Farms for kindly supplying carrot samples used in this study. This project was funded through the Vegetable and Fruit Improvement centre with the Grant ‘‘Designing Foods for Health, USDA-CSREES 2001-3440213647’’. References Alasalvar, C., Grigor, J. M., Zhang, D., Quantick, P. C., & Shahidi, F. (2001). Comparison of volatiles, phenolics, sugars, antioxidant vitamins, and sensory quality of different colored carrot varieties. Journal of Agricultural and Food Chemistry, 49, 1410–1416. Babic, I., Amiot, M. J., Nguyen-The, C., & Aubert, S. (1993). Changes in phenolic contents in fresh ready-to-use shredded carrots during storage. Journal of Food Science, 58, 351–356. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol., 28, 25–30. Clifford, M. N., Johnston, K. L., Knight, S., & Kuhnert, N. (2003). Hierarchical scheme for LC-MSn identification of chlorogenic acids. Journal of Agricultural and Food Chemistry, 51, 2900–2911. Clifford, M. N. (1986). Coffee bean dicaffeoylquinic acids. Phytochemistry, 25, 1767–1769. Cevallos-Casals, B. A., & Cisneros-Zevallos, L. (2003). Stoichiometric and kinetic studies of phenolic antioxidants from Andean purple corn and red-fleshed sweetpotato. Journal of Agricultural and Food Chemistry, 51, 3313–3319. Cisneros-Zevallos, L. (2003). The use of controlled postharvest abiotic stresses as a tool for enhancing the nutraceutical content and adding value of fresh fruits and vegetables. Journal of Food Science, 68, 1560–1565. ERS-USDA (2003). Trends in the US blueberry industry. Available from:
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