Determination of Antioxidant Capacities, α-Dicarbonyls, and Phenolic

Article pubs.acs.org/JAFC

Determination of Antioxidant Capacities, α‑Dicarbonyls, and Phenolic Phytochemicals in Florida Varietal Honeys Using HPLCDAD-ESI-MSn Sara M. Marshall, Keith R. Schneider, Katherine V. Cisneros, and Liwei Gu* Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, United States ABSTRACT: Honeys contain phenolic compounds and α-dicarbonyls with antioxidant and antimicrobial capacities, respectively. The type and concentration of these compounds vary depending on the floral source and geographical location where the honey is produced. Seventeen varietal honeys, including 12 monofloral and 5 multifloral honeys, were sampled from different regions of Florida. The monofloral honeys included those from citrus, tupelo, palmetto, and gallberry. These honeys were evaluated for their antioxidant capacity, total phenolic content, and free radical scavenging capacity and compared with three New Zealand Manuka honeys. Phenolic phytochemicals and α-dicarbonyls were identified and quantified using HPLCDAD-MSn. Several honey varieties from gallberry, Manuka, and multifloral displayed a total phenolic content >1000 μg GAE/g. A citrus honey had the lowest total phenolic content of 286 μg GAE/g. The oxygen radical absorbance capacity of the honeys ranged from 1.48 to 18.2 μmol TE/g. All honeys contained 3-deoxyglucosone at a higher concentration than methylglyoxal or glyoxal. Manuka honeys had higher concentrations of methylglyoxal than other varieties. Plant hormones 2-cis,4-trans-abscisic acid and 2-trans,4-trans-abscisic acid were the most abundant phytochemicals in all honeys. Coumaric acid, rutin, chrysin, pinocembrin, quercetin, luteolin, and kaempferol were also found in samples but at lower concentrations. KEYWORDS: honey, Manuka, carbonyls, methylglyoxal, phytochemicals

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INTRODUCTION Honey is a natural product produced by bees from the nectar of various plants. The composition of honey is known to vary depending on the season, geographical location, and floral source. Honey is mostly composed of fructose and glucose, but it has also been shown to contain phenolic phytochemicals. These compounds are mostly phenolic acid derivatives and flavonoid aglycones, although some flavonoid glycosides have also been identified.1,2 Flavonoids are ubiquitous in plants, and they possess antioxidant, radical scavenging, antimuntagenic, anti-inflammatory, and anticarcinogenic functions.3 Phenolic compounds, specifically flavonoids, are known to contribute to the sensory characteristics of honey, such as color, flavor, and taste as well as potential health-promoting properties. The composition of these compounds was proposed as a quicker and easier way than pollen analysis to determine the floral source of honeys.1,4−8 Honeys have also been shown to contain α-dicarbonyls including glyoxal, methylglyoxal, and 3-deoxyglucosone. These compounds react with proteins to produce advanced glycation end products (AGEs) through the Maillard reaction.9,10 Manuka honey from New Zealand and Australia have welldocumented antimicrobial properties.11−13 The high concentration of methylglyoxal in Manuka honeys is considered the major factor contributing to their high antimicrobial capacity.11,12,14 Manuka honeys are classified and sold according to their methylglyoxal concentration or “Unique Manuka Factor”; the larger the Unique Manuka Factor value, the higher the antimicrobial capacity. In addition to αdicarbonyls, some phenolic compounds such as kaempferol, © XXXX American Chemical Society

quercetin, and myricetin also possess antimicrobial properties.13,15 There are several studies on the phenolic composition of honey from Europe, New Zealand, and Asia, but little exists on the phenolic and α-dicarbonyl composition of North American honey.16−18 The present work aims to identify and quantify the phenolic compounds from various Florida honeys while determining their antioxidant capacities. Furthermore, the identification and quantification of the main α-dicarbonyls in honey were also performed.

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MATERIALS AND METHODS

Chemicals. 2,2′-Azobis(2-amidinopropane) (AAPH) was a product of Wako Chemicals Inc. (Bellwood, RI, USA). Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), 2,2-diphenyl-1-picrylhydrazyl (DPPH), glyoxal (40% aqueous solution), pinocembrin, ellagic acid, luteolin, apigenin, morin, and galangin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 3-deoxyglucosone standard was a product of Toronto Research Chemicals Inc. (Toronto, Canada). Folin−Ciocalteu reagent, HPLC grade methanol, formic acid, acetic acid, 2-cis,4-trans-abscisic acid, coumaric acid, chlorogenic acid, syringic acid, naringenin, hesperetin, rutin, quercetin, chrysin, and kaempferol were purchased from Fisher Scientific (Pittsburgh, PA, USA). The C18 (3 mL) solid phase extraction cartridges were from Dionex (Sunnyvale, CA, USA). Methylgloxal (40% aqueous solution) was purchased from MP Biomedicals, LLC (Solon, OH, USA).

Received: April 17, 2014 Revised: July 9, 2014 Accepted: August 7, 2014

A

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Samples. A total of 20 honey samples, 15 monofloral and 5 multifloral honeys, were collected from Florida apiarists. The monofloral honeys included those from citrus, tupelo, palmetto, gallberry, and Manuka honeys. Their harvesting regions are listed in Table 1. Three Manuka honeys with certified antimicrobial capacity

(0.5 M, 6.5 pH) was mixed with 0.6 mL of 1% (w/v) ophenylenediamine in phosphate buffer. The honey solutions were kept in darkness at room temperature for at least 12 h for the reaction to complete. After derivatiztion, solutions were filtered through a membrane (0.45 μm), and 20 μL was injected for HPLC analysis. An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, and a diode array detector (Agilent Technologies, Palo Alto, CA, USA) were interfaced to a HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). A Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm particle size, Agilent Technologies) was used for the separation. Column temperature was set at 30 °C. The binary mobile phase consisted of (A) methanol/acetic acid (99.85:0.15 v/v) and (B) methanol/acetic acid (99.97:0.03 v/v). The 49 min gradient is described as follows: 0−2 min, 20% B isocratic; 2−22 min, 20−40% B linear; 22−37 min, 40−100% B linear; 37−42 min, 100% B isocratic; 42−49 min, 100−20% B linear, at a flow rate of 0.8 mL/min followed, by 5 min of re-equilibration of the column before the next sample run.12 The detection wavelength was 312 nm. The estimated limits of detection for glyoxal, methylglyoxal, and deoxyglucosone were 0.083, 0.063, and 0.130 μg/mL, respectively. Electrospray ionization in positive mode was performed using nebulizer, 50 psi; drying gas, 10 L/min; drying temperature, 290 °C; and capillary, 4000 V. The full scan mass spectra of the quinoxalines were recorded in a range of m/z 100−3000. Auto MS2 was conducted with 80% compound stability and 80% trap drive level. Pure compounds of methylglyoxal, glyoxal, and 3-deoxyglucosone were used as external standards to quantify the quinoxalines formed after derivatization. The concentration ranges of external standard for 3-deoxglucosone, glyoxal, and methylglyoxal were 3−520, 0.1−60, and 0.1−250 mg/L, respectively. The α-dicarbonyls were identified on the basis of full scan and product ion mass spectra and UV−vis spectra on a diode array detector and compared to authentic standards.10,12,24 HPLC Analyses of Phenolic Phytochemicals. The procedure for the extraction of phenolic phytochemicals from honeys was adapted from that of Hadjmohammadi et al.25 Honey (10 g) was mixed with 40 mL of deionized water. The solution was filtered to remove any solid residue. The SPE C18 cartridges (0.5 g packing) were preconditioned with 6 mL of methanol followed by 3 mL of deionized water. The honey solutions were loaded on the individual cartridges and rinsed with 15 mL of water followed by 3 mL of 10% (v/v) methanol to remove potential sugars. The phenolic fractions were recovered by eluting the cartridge with 6 mL of methanol. The eluent was dried in a SpeedVac concentrator (Thermo scientific ISS110, Waltham, MA, USA) overnight at room temperature. The dried extracts were then dissolved in 500 μL of methanol, sonicated for 10 min, and centrifuged at 17000g for 5 min. Twenty microliters of the supernatant was injected for analysis on HPLC. A Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm particle size, Agilent Technologies) was used for the separation. The binary mobile phase consisted of (A) water/formic acid (99.5:0.5, v/v) and (B) methanol at a constant flow rate of 0.8 mL. A 60 min gradient was adapted from a published paper.7,26 The gradient is described as follows: 0−5 min, 5% B isocratic; 5−20 min, 5−30% B linear; 20−32 min, 30−35% B linear; 32−45 min, 35−50% B linear; 45−50 min, 50−80% B linear; 50−60 min, 80−5% B linear, followed by 5 min of re-equilibration of the column before the next injection. The detection wavelengths were set at 290 and 340 nm, because a majority of the phenolics’ UV absorption maxima are at these wavelengths.5,27−29 Electrospray ionization in negative mode was performed using nebulizer, 50 psi; drying gas, 11 L/min; drying temperature, 350 °C; and capillary, 4000 V. The full scan mass spectra of the phytochemicals were recorded from m/z 100 to 1000. Auto MS2 was conducted with 80% compound stability and 50% trap drive level. Galangin, pinocembrin, quercetin, 2-cis,4-trans-abscisic acid, luteolin, rutin, coumaric acid, chyrsin, and kaempferol were used as external standards to quantify flavonoids and phenolic acids that were identified from mass analysis. Phenolic phytochemicals were identified using a combination of full scan and product ion mass spectra, UV−vis spectra on diode array detector with comparison to authentic standards, and comparison with published papers.3

Table 1. Floral Sources and Harvesting Region of Honeysa sample

floral type

1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c

citrus citrus citrus gallberry gallberry gallberry Manuka 16+ Manuka 15+ Manuka 12+ palmetto palmetto palmetto

5a 5b 5c 6 7 8 9 10

tupelo tupelo tupelo multifloral multifloral multifloral multifloral multifloral

region of Florida (FL, USA) and season central, West Bradenton, FL, spring 2011 north, Lake City, FL, spring 2011 south/central, Zolfo Springs, FL, spring 2011 central, Land O’ Lakes, FL, spring 2011 north, Wacissa, FL, spring 2011 central, Winter Park, FL, spring 2011 New Zealand New Zealand New Zealand north, Gulf Breeze, FL, spring 2010 central, Geneva, FL, summer 2010 south/central, Zolfo Springs, FL, spring/summer 2011 central, Land O’ Lakes, FL, spring 2011 north, Bristol, FL, spring 2011 north, Chipley, FL, spring 2011 north, Fleming Island, FL, early summer 2010 south, Fort Myers, FL, fall 2010 central, Geneva, FL, late summer 2010 south, Fort Myers, FL, fall 2010 north, Lake City, FL, spring 2011

a

Due to the year-round blooming of Leptospermum scoparium, the harvesting time for commercial Manuka honey was unknown. North region is defined above 29.28° N, the central region from 29.21° to 27.08° N, and the south region below 26.95° N. indices of 12+, 15+, and 16+ were purchased from New Zealand. A lower index number corresponds to a lower capacity, whereas a higher number corresponds to stronger antimicrobial capacity based on the Unique Manuka Factor index. The honeys were stored in darkness at 4 °C prior to analysis. On the day of analysis honeys were removed from refrigeration, and samples were run under warmed water to help create a homogeneous liquid sample. Total Phenolic and Antioxidant Capacity Assay. Total phenolic content was determined using the Folin−Ciocalteu assay.19,20 Contents were expressed as microgram gallic acid equivalents per gram of honey (μg GAE/g). The DPPH scavenging activities of honeys were measured according to a published method.21 DPPH scavenging activities were expressed as micromole Trolox equivalents per gram of honey (μmol TE/g). Oxygen radical absorbance capacity (ORAC) values for each honey were determined using a modified method from Huang et al.22 Samples (1 g) were diluted with 10 mL of the phosphate buffer solution and then brought to a final dilution of 1:300. No acetone/water extraction solution was used. Antioxidant capacities were expressed as micromole Trolox equivalents per gram of honey (μmol TE/g). Color Assay. The color of honeys was determined using a published method on a SPECTRAmax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA).23 Honey was diluted 50% (w/v) with warm water. The solutions were sonicated for 5 min and filtered through 0.45 μm filter units to remove large particles. The absorbance was measured at 450 nm using 720 nm as a reference wavelength to determine the color of honeys. Color was expressed as absorbance units (AU) at the 450 nm wavelength. Derivatization and HPLC Analysis of α-Dicarbonyls. αDicarbonyls were analyzed as their corresponding quinoxalines after derivatization with o-phenylenediamine following the method from Mavric et al.12 One milliliter of 15% (w/v) honeys in phosphate buffer B

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Table 2. Total Phenolic Content, Free Radical Scavenging, Antioxidant Capacities, and Color Analysis of Honeysa honey

total phenolics (μg/g)

DPPH (μmol TE/g honey)

ORAC (μmol TE/g honey)

ABS450 (AU, 50% w/v)

1a, citrus 1b, citrus 1c, citrus 2a, gallberry 2b, gallberry 2c, gallberry 3a, Manuka 16+ 3b, Manuka 15+ 3c, Manuka 12+ 4a, palmetto 4b, palmetto 4c, palmetto 5a, tupelo 5b, tupelo 5c, tupelo 6, multifloral 7, multifloral 8, multifloral 9, multifloral 10, multifloral range average

829 ± 17.8 b 593 ± 28.4 d 286 ± 3.30 d 1004 ± 13.7 a 694 ± 47.6 c 736 ± 11.9 c 1080 ± 14.2 a 774 ± 27.0 b 1030 ± 46.1 a 767 ± 27.1 c 851 ± 12.3 b 530 ± 7.76 d 820 ± 23.0 b 773 ± 15.7 c 691 ± 15.8 c 936 ± 3.84 b 1040 ± 12.6 a 981 ± 6.82 a 662 ± 12.1 d 478 ± 10.2 d 286−1080 778

1.40 ± 0.00500 a 0.282 ± 0.115 d 0.384 ± 0.251 d 0.826 ± 0.326 d 0.970 ± 0.0770 c 0.870 ± 0.128 d 1.37 ± 0.665 b 1.35 ± 0.218 b 1.38 ± 0.244 a 0.679 ± 0.231 d 1.22 ± 0.250 c 1.22 ± 0.130 c 1.36 ± 0.324 b 1.13 ± 0.182 c 1.78 ± 0.0990 a 1.08 ± 0.0140 c 1.49 ± 0.233 a 1.28 ± 0.132 b 2.07 ± 0.124 a 1.27 ± 0.113 b 0.282−2.07 1.17

6.16 ± 3.35 c 1.48 ± 1.36 d 4.87 ± 2.08 d 16.5 ± 1.78 a 3.18 ± 0.575 d 11.6 ± 2.91 b 15.4 ± 9.31 a 6.92 ± 0.233 c 11.3 ± 3.22 b 5.09 ± 0.420 c 12.8 ± 0.961 a 4.77 ± 0.632 d 7.69 ± 0.639 b 6.86 ± 1.14 c 12.7 ± 4.56 b 18.2 ± 5.10 a 15.9 ± 1.68 a 4.67 ± 3.16 d 6.70 ± 0.0400 c 9.34 ± 6.24 b 1.48−18.2 9.11

0.348 ± 0.0180 a 0.0770 ± 0.00500 d 0.0840 ± 0.00300 d 0.272 ± 0.0100 b 0.115 ± 0.000 d 0.172 ± 0.115 c 0.298 ± 0.0180 b 0.310 ± 0.0140 a 0.224 ± 0.0100 c 0.163 ± 0.00800 d 0.248 ± 0.0650 b 0.284 ± 0.0650 b 0.241 ± 0.0170 c 0.145 ± 0.00300 d 0.192 ± 0.0100 c 0.220 ± 0.0170 c 0.341 ± 0.0190 a 0.480 ± 0.0350 a 0.380 ± 0.0560 a 0.299 ± 0.0350 b 0.077−0.508 0.245

Results are the mean ± SD of three determinations on a fresh weight basis. ORAC values are the mean of two determinations. Values in each column are grouped into four quartiles (a > 75th, b = 50th−75th, c = 25th−50th, and d < 25th percentile). a

Statistical Analysis. Data was expressed as the mean ± standard deviation of three independent observations per each honey sample unless otherwise stated. Total phenolic content, antioxidant capacities, and color of honeys were grouped into four quartiles. The lowest and highest values were in quartiles 1st−25th and 75th−100th, respectively. Pearson correlation for the different comparable assays was determined using SigmaPlot software (version 12, Systat software Inc., Chicago, IL, USA).

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RESULTS AND DISCUSSION Total Phenolic Content, Antioxidant Capacity, and Color Analysis of Honeys. Table 2 shows the total phenolic content, antioxidant capacity, and color analysis results of the 20 honeys analyzed, including the 3 New Zealand Manuka honeys. The total phenolic content in the monofloral and multifloral honeys ranged from 286 to 1080 μg GAE/g on a fresh weight basis. This range of values was in agreement with previously published values of honeys from other regions of the world.3,17,23,30,31 Manuka honeys had the highest total phenolic content (averaging 961 μg GAE/g) among all of the honeys. This value is similar to those reported in honeydew honeys (1150 μg GAE/g) from Burkina Faso.31 The highest total phenolic content in the multifloral honeys was 1040 μg GAE/g in sample 6b. This honey was collected from southern Florida during the fall season. There was no apparent correlation between total phenolic content and harvesting time or location for the multifloral honeys. This may be due to the fact that Florida’s subtropical climate allows for the same plants to be grown in multiple seasons and multiple areas of the state. A previous study from Cuba and Portugal showed that the highest total phenolic content for honeys produced in these countries was 700 μg GAE/g.32,33 These contents were comparable to Florida monofloral honeys of tupelo and palmetto, which had average total phenolic contents of 761 and 716 μg GAE/g, respectively. The multifloral varieties had a higher average total

Figure 1. HPLC chromatogram of α-dicarbonyls in Manuka honey 3b (A) and multifloral honey 6 (B) after derivatization. Peaks 1, 2, 3, 4, and 5 are o-phenylenediamine, glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, respectively.

phenolic content of 819 μg GAE/g than most of the monofloral varieties examined. It has been shown that color can also be an indicator of total phenolic content and honey’s antioxidant capacity. Darker honeys often have a higher phenolic content and antioxidant capacity.17,23 The color of honeys ranged from 77 mAU in a C

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Table 3. α-Dicarbonyl Content of Honeysa sample

3-deoxyglucosone (μg/g)

glyoxal (μg/g)

methylglyoxal (μg/g)

total carbonyl (μg/g)

1a, citrus 1b, citrus 1c, citrus 2a, gallberry 2b, gallberry 2c, gallberry 3a, Manuka 16+ 3b, Manuka 15+ 3c, Manuka 12+ 4a, palmetto 4b, palmetto 4c, palmetto 5a, tupelo 5b, tupelo 5c, tupelo 6, multifloral 7, multifloral 8, multifloral 9, multifloral 10, multifloral range average

684 ± 34.5 b 288 ± 11.9 d 206 ± 21.4 d 517 ± 3.15 c 466 ± 24.9 d 701 ± 39.9 b 636 ± 48.8 b 558 ± 13.2 c 646 ± 40.2 b 474 ± 35.1 c 831 ± 3.46 a 730 ± 28.7 a 614 ± 23.4 c 502 ± 23.5 c 259 ± 12.7 d 372 ± 17.2 d 754 ± 17.6 a 678 ± 109 b 884 ± 21.8 a 727 ± 8.95 a 206−884 576

4.53 ± 1.01 a 5.70 ± 0.462 a 2.19 ± 0.345 d 3.75 ± 1.39 b 2.76 ± 0.694 c 7.35 ± 0.935 a 4.39 ± 0.660 b 5.13 ± 0.0740 a 3.09 ± 0.182 c 2.95 ± 0.222 c 6.10 ± 0.315 a 2.23 ± 0.109 d 2.90 ± 1.19 c 3.93 ± 1.22 b 2.68 ± 0.257 d 2.44 ± 0.778 d 4.11 ± 1.56 b 3.96 ± 0.915 b 2.49 ± 0.321 d 3.27 ± 0.245 c 2.19−7.35 3.80

6.09 ± 1.27 b 6.19 ± 1.46 b 3.68 ± 1.49 d 7.09 ± 2.52 b 4.88 ± 1.05 d 13.0 ± 4.30 a 86.9 ± 0.967 a 483 ± 10.7 a 92.1 ± 0.343 a 5.80 ± 0.255 c 10.8 ± 1.77 a 4.53 ± 1.01 d 4.85 ± 0.619 d 5.90 ± 1.35 c 4.97 ± 0.582 c 5.23 ± 1.15 c 6.80 ± 1.98 b 5.31 ± 0.948 c 3.63 ± 0.284 d 6.38 ± 0.187 b 3.63−483 38.4

695 ± 33.2 b 300 ± 12.7 d 212 ± 21.6 d 528 ± 6.55 c 474 ± 26.6 d 722 ± 35.3 b 727 ± 49.8 b 1047 ± 23.6 a 741 ± 39.8 b 483 ± 34.8 c 848 ± 23.2 a 737 ± 29.1 b 622 ± 24.8 c 512 ± 24.5 c 266 ± 13.3 d 380 ± 19.0 d 765 ± 18.0 a 687 ± 111 c 890 ± 22.3 a 753 ± 37.7 a 212−890 619

Results are the mean ± standard deviation of three determinations on a fresh weight basis. Values in each column are grouped into four quartiles (a > 75th, b = 50th−75th, c = 25th−50th, and d < 25th percentile).

a

Figure 2. HPLC chromatogram of phenolic phytochemicals in palmetto honey 4a (A), palmetto honey 4b (B), Manuka honey 3b (C), and citrus honey 1a (D). Peak number and identification are listed in Table 4.

and antioxidant capacity (Table 2). This observation helps to support the conclusion that color and total phenolic content in honey are directly correlated. The multifloral variety had an average absorbance of 344 mAU, which was higher than the average absorbance of monofloral honeys (212 mAU). The correlation coefficient (r) between total phenolic concentration and color was 0.47. This r value is lower than those reported

light colored citrus honey to 480 mAU in a multifloral honey. The range of absorbance is consistent with previously reported values.23,34 Citrus honeys 1b and 1c had the lowest absorbance and were visually fairer than the other varieties. Two multifloral honeys, 6c and 6d, had the highest absorbances at 480 and 380 mAU, respectively, among all of the honeys analyzed. Citrus honey 1c showed lower values for color, total phenolic content, D

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(r) between total phenolic concentration of the honeys and ORAC values was 0.60 with p < 0.05. The ORAC assay does not measure antioxidant capacity due to phenolics alone; amino acids and other nucleophiles may also quench the free radicals in this method. The r value between the ORAC and DPPH assays was 0.29. This low r value was likely caused by the differences in the chemistry of these two methods. The ORAC assay is based on a hydrogen atom transfer reaction, whereas the DPPH assay uses an electron transfer mechanism.39 Identification and Quantification of α-Dicarbonyls in Honey. Carbonyl compounds were detected using UV−vis detection at 312 nm after a derivatization step with ophenylenediamine to form quinoxalines (Figure 1). Peak 1 was excessive o-phenylenediamine. Peak 2 was tentatively identified as glucosone according to [M + H]+ 251 and a published study.24,40,41 Peak 3 produced m/z 235 [M + H]+, which fragmented into m/z 217 and 199. These spectra were consistent with 3-deoxyglucosone.24,41 Peak 4 was identified as glyoxal by comparing UV−vis and retention time with standard. Peak 5 yielded m/z 145 [M + H]+ and was identified as the quinoxaline formed after the reaction between o-phenylenediamine and methylglyoxal.12,24 The α-dicarbonyl profiles in honeys differed according to floral sources and harvesting locations. For example, Manuka honey (Figure 1A) showed a predominant peak for methylglyoxal, whereas the methylglyoxal concentration in all of the other varieties was much lower (Figure 1B). Table 3 provides the concentrations of glyoxal, methylglyoxal, and 3-deoxyglucosone in monofloral and multifloral honeys. Glucosone was not quantified in this experiment due to its artificial formation in select buffers and sample matrices. The contents of methylglyoxal in Manuka honeys 3a, 3b, and 3c were 86.9, 483, and 92.1 μg/g, respectively. Methylglyoxal concentration in other honeys ranged from 3.63 to 13.0 μg/g. Methylglyoxal in Manuka was 7−37 times more concentrated than the next highest (gallberry honey, 2c). Other studies have shown methylglyoxal concentrations ranging from undetectable to >1500 μg/g.11,12,14,18,42 Stephen et al. showed that the longer a Manuka honey was aged, the higher the methylglyoxal concentration became.18 The high levels of methylglyoxal in the Manuka honeys were not seen in any other foods.12 A number of researchers suggested that the high levels of methylglyoxal in Manuka honeys are the major contributing factor to high antimicrobial capacity.12 Glyoxal concentrations ranged from 2.19 to 7.35 μg/g, which is similar to the concentrations reported in honeys collected from other regions of the world.11,12,15,34 Among three α-dicarbonyls, 3-deoxyglucosone had the highest concentration in all honeys. The higher concentration of 3-deoxyglucosone relative to methylglyoxal and glyoxal in honeys is expected because 3-deoxyglucosone is the initial product of glucose degradation and honey has a high sugar content.10 Retro-aldolization of 3-deoxyglucosone produces methylglyoxal.10 Thus, when 3-deoxyglucosone decreases, methylglyoxal should increase. This was not confirmed as a processing study was not performed on honeys to examine this reaction. Identification and Quantification of Phenolic Phytochemicals. Figure 2 depicts the HPLC chromatograms of phenolic phytochemicals in different honeys after samples were purified using solid phase extraction. Chromatograms were recorded at 290, 340, and 520 nm, although only the chromatograms at 290 nm are shown. This is because most phenolic compounds can be detected at this wavelength.20

Table 4. Identification of Phytochemicals in Honeys by HPLC-DAD-ESI-MSna peak 1 2 3

compound

mol wt

retention time (min)

[M− H]− (m/z)

164 610 264

23.3 32.6 35.1

163 609 263

119 301 219, 204, 201

MS2 (m/z)

264

38.4

263

219, 153

5 6 7 8

coumaric acid rutin 2-trans,4-transabscisic acidb 2-cis,4-transabscisic acid unidentified quercetin unidentified luteolin

286 302 272 286

39.5 40.2 41.0 42.0

285 301 271 285

9 10 11c 11c

kaempferol pinocembrin chrysin galangin

286 256 254 270

43.3 46.7 48.0 48.0

285 255 253 269

267, 252, 179, 151 253, 215, 243, 223, 151 267, 257, 213, 151 209 227, 213, 153

4

239 151 199, 151

197,

a Nine compounds were identified by comparison with authentic standards. Peaks 5 and 7 were unidentified. b2-trans,4-trans-Abscisic acid was tentatively identified using tandem mass spectra. cChrysin and galangin coeluted in peak 11.

between total phenolics and color in other foods.23,31 However, the correlation was significant at p < 0.05. The color of honeys can be attributed to flavonoids and Maillard reaction products, which can differ according to floral source and age of honey. Researchers have shown that proline content correlates with honey color.35 Also, vitamins such as ascorbic acid may have an influence on honey’s unique color. Carotenoids, minerals, and pollen are other pigment compounds that can be read at 450 nm and may be contributing to the low correlation between total phenolics and color.36 The DPPH scavenging activity of honeys ranged from 0.282 to 2.07 μmol TE/g honey. Multifloral honey 6d had the highest free radical scavenging capability among all honeys. The orange blossom honeys 1b and 1c had the lowest free radical scavenging values at 0.282 and 0.384 μmol TE/g honey, respectively. Honeys with paler colors, such as the citrus honeys, had lower absorbance and scavenging capacities compared to other varieties. It has been suggested that darker honeys generally have higher radical scavenging potential.23 The correlation between total phenolic concentration and DPPH concentration was close to significant with a p value of 0.058. Whereas correlation between total phenolic content and DPPH assay was shown for honeys, lack of correlation was also observed.37 The DPPH assay measures the presence of compounds able to donate an electron to scavenge the DPPH radical. Phenolic compounds are not the only compounds capable of donating an electron. Islam et al. showed that amino acids and other nonphenolic compounds were also able to scavenge DDPH.38 Honeys are complex matrices of compounds with different scavenging capabilities; therefore, it is possible that total phenolics and DPPH scavenging do not significantly correlate. The antioxidant capacity of honeys, expressed as ORAC values, ranged from 1.48 to 18.2 μmol TE/g honey, which are comparable with those reported previously.23 The ORAC value of multifloral honey 6a was higher than those of all other honeys tested. The multifloral varieties had an average ORAC value of 11 μmol TE/g, which is comparable to that of the gallberry honeys (10 μmol TE/g). The correlation coefficient E

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a

F

± 0.020 ± 0.080

± 0.020

± 0.050 ± 0.12 ± 0.14

± 0.030

± 0.070 ± 0.020 ± 0.010

rutin

0.232 0.555 0.597 ND ND 0.218 ND ND ND 0.337 0.534 0.360 ND ND ND 0.175 ND ND 0.441 0.301

6.36 ± 1.2 5.80 ± 0.080 2.03 ± 0.11 7.53 ± 0.49 26.1 ± 11 13.1 ± 1.0 0.710 ± 0.11 ND 3.08 ± 0.78 14.4 ± 0.40 12.7 ± 1.9 2.08 ± 0.73 19.1 ± 5.1 7.50 ± 0.36 4.53 ± 1.3 25.3 ± 4.3 6.46 ± 0.13 2.29 ± 0.060 0.723 ± 0.23 9.35 ± 1.1

2-trans,4-trans-abscisic acid 35.8 ± 4.0 15.1 ± 0.11 5.12 ± 0.27 24.0 ± 1.8 27.6 ± 12 24.3 ± 2.6 1.55 ± 0.19 0.525 ± 0.22 5.54 ± 1.4 25.5 ± 1.0 21.1 ± 2.5 8.00 ± 2.7 27.0 ± 1.9 9.70 ± 0.38 5.95 ± 0.78 72.9 ± 5.1 18.8 ± 0.27 8.95 ± 0.21 2.41 ± 0.49 17.5 ± 1.3

2-cis,4-trans-abscisic acid

quercetin 0.353 ± 0.12 0.650 ± 0.040 1.15 ± 0.060 0.317 ± 0.24 0.341 ± 0.16 0.344 ± 0.070 0.552 ± 0.060 0.495 ± 0.24 0.792 ± 0.21 0.689 ± 0.24 ND 0.711 ± 0.25 1.22 ± 0.070 0.436 ± 0.040 ND 0.614 ± 0.19 0.473 ± 0.010 0.617 ± 0.020 0.578 ± 0.12 0.557 ± 0.16

luteolin 0.401 ± 0.13 ND ND ND ND ND 1.05 ± 0.11 0.907 ± 0.16 1.26 ± 0.30 0.277 ± 0.060 ND 0.386 ± 0.16 ND ND ND 0.525 ± 0.17 0.797 ± 0.070 0.135 ± 0.00 0.196 ± 0.020 0.226 ± 0.080

kaempferol 0.497 ± 0.16 1.55 ± 0.11 3.12 ± 0.16 0.392 ± 0.030 0.322 ± 0.17 0.425 ± 0.040 0.693 ± 0.090 0.260 ± 0.11 0.804 ± 0.34 1.49 ± 0.13 0.583 ± 0.14 1.52 ± 0.52 2.11 ± 0.050 0.610 ± 0.32 1.18 ± 0.30 0.625 ± 0.30 1.08 ± 0.31 ND 0.636 ± 0.12 1.75 ± 0.50

pinocembrin 1.65 ± 0.27 1.07 ± 0.010 0.146 ± 0.010 0.357 ± 0.040 0.084 ± 0.020 1.04 ± 0.16 0.742 ± 0.090 2.75 ± 0.69 1.03 ± 0.30 0.064 ± 0.020 1.17 ± 0.19 2.25 ± 0.76 2.65 ± 0.080 0.152 ± 0.050 1.63 ± 0.14 1.24 ± 0.17 0.107 ± 0.00 1.50 ± 0.00 0.258 ± 0.050 1.57 ± 0.19

2.24 ± 0.14 1.16 ± 0.050 ND 0.348 ± 0.010 0.164 ± 0.040 1.03 ± 0.10 2.04 ± 0.19 4.56 ± 0.65 1.04 ± 0.080 0.140 ± 0.020 1.40 ± 0.090 2.94 ± 1.0 3.17 ± 0.12 0.393 ± 0.050 1.46 ± 0.19 0.686 ± 0.12 0.144 ± 0.020 2.42 ± 0.14 0.408 ± 0.050 1.73 ± 0.39

chrysin + galangin

total 47.6 ± 11 26.4 ± 4.7 12.2 ± 1.7 33.1 ± 7.7 55.0 ± 12 41.0 ± 8.2 7.34 ± 0.65 9.49 ± 1.5 13.5 ± 1.8 43.7 ± 8.7 37.5 ± 7.3 19.5 ± 2.4 55.8 ± 9.6 19.0 ± 3.6 15.1 ± 2.1 102 ± 24.0 28.0 ± 6.0 18.3 ± 2.7 6.27 ± 0.66 33.3 ± 5.7

Results are the mean ± standard deviation of three determinations on a fresh weight basis. ND, not detected. 2-trans,4-trans-abscisic acid was quantified using the 2-cis,4-trans-abscisic acid as a standard.

coumaric acid

ND 0.437 ± 0.26 ND 0.185 ± 0.050 0.295 ± 0.14 0.508 ± 0.16 ND ND ND 0.745 ± 0.030 ND 1.21 ± 0.49 0.604 ± 0.29 0.219 ± 0.040 0.355 ± 0.18 0.278 ± 0.10 0.166 ± 0.010 2.39 ± 0.33 0.616 ± 0.10 0.272 ± 0.21 c

sample

1a, citrus 1b, citrus 1c, citrus 2a, gallberry 2b, gallberry 2c, gallberry 3a, Manuka 16+ 3b, Manuka 15+ 3c, Manuka 12+ 4a, palmetto 4b, palmetto 4c, palmetto 5a, tupelo 5b, tupelo 5c, tupelo 6, multifloral 7, multifloral 8, multifloral 9, multifloral 10, multifloral

Table 5. Content of Phenolic Compounds in Honeys (μg/g)a

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf501329y | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

The concentrations of the 10 phenolic compounds quantified in the honeys are listed in Table 5. Figure 2 shows the phenolic phytochemical profiles were similar in honeys of the same variety. These profiles in palmetto honey show the phenolic “fingerprints” of honeys and may help to identify the floral source of unknown honeys. It was speculated that the multifloral honeys could be classified according to composition of phenolic phytochemicals. The Manuka honey (sample 3a) in Figure 2C had two large peaks that were unidentifiable from mass data. Honey is a complex mixture that may include other compounds such as pesticides, antibiotics, peptides, amino acids, dicarboxylic acids, and other residues that may not be easily identifiable with the current method.35,36,55,56 The concentrations and profiles of phenolics in the multifloral honeys varied according to seasons, locations, and apiarists. There was a higher concentration of abscisic acids in multifloral honey 6 than in all other honeys. A majority of the phenolic compounds identified and quantified were in small amounts of