chemical profiling of polyfloral belgian honey: ellagic acid and … · 2019. 7. 30. ·...

Research ArticleChemical Profiling of Polyfloral Belgian Honey: Ellagic Acid andPinocembrin as Antioxidants and Chemical Markers

Izabela Jasicka-Misiak,1 Steven Gruyaert,1 Anna Poliwoda,1 and PaweBKafarski2

1Faculty of Chemistry, Opole University, Oleska 48, 45-052 Opole, Poland2Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland

Correspondence should be addressed to Izabela Jasicka-Misiak; [email protected]

Received 4 July 2017; Revised 6 November 2017; Accepted 13 November 2017; Published 31 December 2017

Academic Editor: Sevgi Kolaylı

Copyright © 2017 Izabela Jasicka-Misiak et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Chemical profiling of northern Belgian polyfloral honeys was performed to analyse their phenolic compound content (flavonoidsand phenolic acids). First, samples were subjected to a standard analysis of their physicochemical properties, and then, the phenolicfraction was isolated and analysed using a HPLC/PAD method. All of the tested honeys showed a common and specific phenolicprofile that could be the basis for the differentiation of polyfloral honeys of the Antwerp region from other polyfloral honeys.Chromatographic data indicated a high content of ellagic acid (9.13–13.66mg/100 g honey), as well as the flavonoid pinocembrin(1.60–1.85mg/100 g honey) in these honeys. Ellagic acid, a compound with well-defined prohealth activities, might be used as achemical marker for these honeys. With respect to total phenolic and flavonoid contents, 1,1-diphenyl-2-picrylhydrazyl (DPPH)assays were determined spectrophotometrically. The honey exhibited a moderate antioxidant activity, typical for light honeys.

1. Introduction

Because of their health-promoting activities, polyphenoliccompounds are always of interest among substances ofnatural origin. Until now, the biological activity of a varietyof phenolic acids and flavonoids of plant origin has beendescribed in some detail.Themost important feature of thesecompounds is their antibacterial and antioxidant activity [1–5]. Dietary studies have clearly shown a correlation betweenthe content of phenolic compounds in plant foods and adecrease in the incidence of lifestyle diseases [6, 7]. Froma wide variety of honeys, consumers frequently chooseunifloral honeys because their specific medicinal propertiesare well defined. On the other hand, polyfloral honey,called thousand flowers honey, is still the most popular andmost widely consumed variety. Polyphenolic compounds arepresent in honeys due to their direct transfer from the nectarof plants by bees.The chemical patterns of these compounds,namely, their composition and relative concentration, havealready been used for the authentication of honey bars [8,9]. However, most studies focusing on unifloral honey are

devoted to the identification of individual markers of theirbotanical origin [9–11].

In this study, we focused on the determination of thechemical profiles, namely, the identification of phenolic acidsand flavonoids, of Belgian polyfloral honeys from three api-aries located in the Antwerp province to determine the utilityof this approach for the determination of the geographicalorigin of honey. Furthermore, the antioxidant propertiesof these honeys were determined and correlated with thecontent of phenolic compounds.

2. Materials and Methods

2.1. Reagents. All chemicals were of analytical reagentgrade. Methanol, dibasic sodium phosphate heptahydrate(Na2HPO4× 7H2O), hydrochloric acid, and 85% phosphoric

acid were purchased from POCH S. A. (Gliwice, Poland).Ten flavonoids, galangin, kaempferol, chrysin, quercetin,myricetin, (+)-naringenin, pelargonidin chloride, pinocem-brin, and rutin, as well as 11 phenolic acids, 3-hydroxybenzoic

HindawiJournal of ChemistryVolume 2017, Article ID 5393158, 8 pageshttps://doi.org/10.1155/2017/5393158

https://doi.org/10.1155/2017/5393158

2 Journal of Chemistry

acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, caf-feic acid, vanillic acid, syringic acid, p-coumaric acid, chloro-genic acid, rosmarinic acid, ferulic acid, and ellagic acid,which were used as standards, were purchased from Sigma-Aldrich (Poznan, Poland). Furthermore a isoprenoid, (±)-abscisic acid (Sigma-Aldrich, Poznan, Poland), was used as astandard. Stock solutions of each standardwere preparedwithHPLC-grademethanol at a concentration of 0.01mmol/L andwere kept at 4∘C protected from light. Amberlite XAD-2 wasused as an adsorption resin for the extraction step, and it wasobtained from Supelco (Bellefonte, PA, USA).

2.2. Honey Samples. Twenty-seven Belgian honey samplesfrom the Antwerp province were studied.Theywere obtainedfrom three beekeepers operating in the vicinity of Antwerp,namely, in Mortsel (M, the beekeeper guild was establishedthere in 1695), Lichtaart (L), and Kasterlee (K). Honey sampleoriginates from 3 successive years (2007, 2008, and 2009). Allof the samples, after being acquired from the beekeepers, werestored in the dark at 4∘C. Before they were studied, they werecharacterized by the determination of their moisture content,electrical conductivity, specific rotation, and invertase activ-ity as well as by using classical visual and organoleptic testing.All samples of honeys were certified from their producers.The botanical origin of the analysed honeys was confirmedand certified by pollen analysis. The proper documents havebeen presented by beekeepers supplying the honey.

2.3. Isolation of Phenolic Compounds. Extraction was carriedout as described previously [9] with some modifications.Thestudied polyfloral honey samples (5 g)were thoroughlymixedwith five parts (25mL) of distilled water and sonicated for1 hour until completely homogenized. The aqueous samplewas then passed through filter paper to remove any solidparticles.The filtrate wasmixed with 40 g of Amberlite XAD-2 (pore size 9 nm, particle size 0.3–1.2mm) and stirred witha magnetic stirrer for 10min to absorb phenolic compounds.TheAmberlite particles were then packed into a glass column(3.5 × 65 cm), and the column was washed with a dilutedhydrochloric acid (0.05M, 120mL) and then with distilledwater (100mL). The phenolic compounds were retained onthe column, while all sugars and other polar compoundseluted. The phenolic fraction was then eluted from the col-umn with methanol (150mL) and evaporated under reducedpressure (40∘C). The obtained residue was redissolved in amixture of distilled water (1mL) and HPLC-grade methanol(1mL), and themixture was analysed with the use of a HPLC-PDA system. The applied extraction method enabled therecovery values for analysed compounds to be higher than85%.

2.4. Total Phenolic Content (TPC). To determine the TPC ofthe honey extracts, the Folin-Ciocalteu method was applied[9, 12, 13]. Briefly, 5mL of aqueous eluate of honey (0.5 ghoney/50mL of distillate water) was placed into a 10mLvolumetric flask. Folin-Ciocalteu (FC) reagent (0.5mL) and1.5mL of a 20% Na

2CO3solution were added to the eluate.

After filling the flaskwith distilledwater to themark and thor-oughly agitating the reaction mixture, it was left to stand for

120min at room temperature, and the TPC was determinedspectrophotometrically (Hitachi U-2810) at 760 nm againstthe blank (water). The TPC was expressed in milligrams ofgallic acid equivalents (GAE/100 g) and was an average valuefrom three parallel measurements.

2.5. Total Flavonoid Content (TFC). The total flavonoid con-tent was determined colometrically according to the methoddescribed by Lin and Tang [14] with minor modifications.Five millilitres of a honey solution (0.5 g/mL) was mixedwith 5mL of 2% aluminium chloride (AlCl

3). A flavonoid-

aluminium complex was formed after 30min of incubationtime at room temperature.The formation of the complex wasmeasured at 415 nm using a Hitachi U-2810 double beamspectrophotometer. Quercetin (0–100mg/L) was used as astandard for obtaining the calibration curve. The TFC wascalculated as themean value of triplicate assays and expressedas mg of quercetin equivalents (QE) in 100 g of honey.

2.6. Determination of the Radical Scavenging Activity (DPPH).The scavenging activity of the honey was determined accord-ing to a slightly modified procedure described by Turkmenet al. [15] against the radical 1,1-diphenyl-2-picrylhydrazyl(DPPH). All honey samples were prepared by dissolving1 g of honey in 5mL of distilled water. The samples werehomogenized by shaking.Then, 1mL of the aqueous solutionfrom each sample was transferred into a centrifuge tube andcentrifuged for 10min at 20∘C (10,000 rpm).Then, 0.25mL ofthe solution was mixed with 0.75mL of 0.1mmol/L DPPH inmethanol. A control test was performed using distilled waterinstead of the honey solution. The reaction mixtures werevortex-mixed and incubated for 1 hour at room temperaturein darkness. After incubation, the mixtures were centrifugedfor 10min at 20∘C (10,000 rpm), and the absorbance wasmeasured at 517 nm against methanol using a Hitachi U-2810double beam spectrophotometer. The antioxidant activity isexpressed as a percentage of inhibition of the DPPH radicaland calculated from the equation:

AA% = [(Absc − Abss)Absc

] × 100, (1)

where AA (%) is the percentage of disappearing DPPHradical; Absc is the absorbance of the control sample.

2.7. HPLC Analysis. HPLC analyses of the honey extractswere performed using an Ultimate 3000 Dionex HPLCsystem with a photodiode array detector (PDA) and theChromeleon 6.8 software (Dionex, Sunnyvale, CA, USA).Separations were carried out on a reversed-phase Gemini5 𝜇m C-18 column (Merck, Darmstadt, Germany; 250 × 4.60146mm, particle size 5𝜇m). The mobile phase consistedof 0.01mol/dm3 phosphate buffer at pH 2.5 (solvent A)and methanol (solvent B). A constant solvent flow rateof 1mL/min was applied. The optimized gradient elutionscheme is reported in Table 1.The temperature of the columnoven was set at 30∘C. The eluate was monitored at 214,280 nm and 320, 340 nm because most natural phenoliccompounds show their UV absorption maxima around these

Journal of Chemistry 3

Table 1: Gradient elution profile for HPLC-PAD analysis.

Time[min]

Solvent A(phosphate buffer at pH 2.5) (%)

Solvent B(methanol) (%)

0 90 1013.5 60 4039.0 10 9042.0 0 10046.0 0 10055.0 90 10

Table 2: Calibration curves and detection limits of studied flavonoids and phenolic acids for HPLC-PAD analysis.

Analyte Retention time[min]

Correlation coefficient(𝑟2)

LOD[ug/ml]

LOQ[ug/ml]

Precision (RSD)%(𝑛 = 5)

Interday Intraday3,4-Dihydroxybenzoic acid 10.56 0.9993 0.03 0.07 1.9 3.4Chlorogenic acid 14.36 0.9992 0.16 0.47 2.6 3.24-Hydroxybenzoic acid 15.11 0.9912 0.02 0.07 2.1 2.93-Hydroxybenzoic acid 15.65 0.9998 0.04 0.13 2.5 3.1Vanillic acid 16.06 0.9983 0.10 0.28 1.9 2.8Caffeic acid 16.34 0.9992 0.13 0.41 2.5 3.2Syringic acid 17.06 0.9994 0.11 0.35 2.1 2.9Ferulic acid 19.44 0.9993 0.16 0.48 1.6 2.4p-Coumaric acid; 19.85 0.9968 0.08 0.25 2.2 2.7Rosmarinic acid 21.21 0.9987 0.14 0.43 2.4 3.1Ellagic acid, 24.05 0.9979 0.15 0.47 2.5 3.1Myricetin 25.46 0.9991 0.38 1.13 1.8 2.8(+)-naringenin 26.70 0.9982 0.11 0.34 2.2 3.2Quercetin 29.20 0.9967 0.26 0.80 1.6 2.4Kaempferol 32.05 0.9998 0.31 0.92 1.9 2.6Pinocembrin 32.75 0.9996 0.08 0.23 1.6 2.5Chrysin 35.57 0.9985 0.28 0.61 2.1 2.9Galangin 36.10 0.9990 0.15 0.48 2.3 3.1LOD, limit of detection at the signal three times at the baseline noise; LOQ, limit of quantification.

wavelengths.The comparison of theUV spectra and retentiontimes with standard compounds allowed the identificationof the phenolic acids and flavonoids presented in the anal-ysed honey extracts. They were quantified using externalstandards. Parameters of calibration equations and validationcharacteristics of phenolic compounds are listed in Table 2.

2.8. Statistical Analysis. All tests were repeated in triplicate,except for those applying the Folin-Ciocalteu method, whichwere repeated twice. The values obtained in experimentswere expressed as the mean ± standard deviation (or relativestandard deviation, RSD). The standard deviations and RSDwere calculated using spreadsheet software (Excel�) [9].

3. Results and Discussion

Nine Belgian honey samples were purchased from 3 dif-ferent beekeepers, which are indicated by the name of the

neighbouring city, namely, Mortsel (M), Lichtaart (L), andKasterlee (K). All sampleswere classified as polyfloral honeys.Honeys were preliminary characterized by determining theirwater content, electrical conductivity, specific rotation, andactivity of invertase as well as by visual and organoleptictesting (Table 3). Samples of 3 successive years, 2007, 2008,and 2009, were studied.

3.1. Total Phenolic Content (TPC). Phenolic compounds arean important group of compounds that influence the appear-ance and functional properties of honey. Thus, identifyingand quantifying the total phenolic content are of significance,considering the initial assessment of the properties of honey.These substances can also be used as indicators of botanicaland/or geographical origin and the source of honey [16]. Thetotal phenolic contents of the analysed honeys related to gallicacid (mg of GAE/100 g of honey; 𝑅2 = 0.9970) are presented


Table3:Th

estand

ardchem

icalparameterso

fhon

ey(Polish

QualityStandard

(PN-88/A–77626).

Generalsta

ndards

Mortsel

Lichtaart

Kaste

rlee

2007

2008

2009

2007

2008

2009

2007

2008

2009

Av.,𝑛=3

Moistu

recontent

≤20%,excepth

eather-,baker

honey:≤23

%Note≥

20%:sho

rtshelflives,risk

offerm

entatio

n

16.1%

18.9%

17.3%

17.4%

16.3%

16.4%

16.9%

16.2%

18.1%

Electricalcond

uctiv

ity

Flow

erho

neyandblended

honey:

≤800𝜇

S/cm

Leaveh

oney,chestn

utho

ney,and

blends:>8

00𝜇S/

cm

252𝜇

S/cm

461𝜇S

/cm

221𝜇S

/cm

178𝜇

S/cm

166𝜇

S/cm

696𝜇

S/cm

169𝜇

S/cm

206𝜇

S/cm

446𝜇

S/cm

Specificr

otation

−11.3

−9.5

−10.8

−12.5

−14.0

−2.0

−11.8

−12.3

−7.5

Invertase

General:≥5

0U/kg(Siegenthaler

scaleu

nits)

,hon

eywith

low

enzymea

ctivity≥20

U/kg

122U

/kg

152U

/kg

92U/kg

54U/kg

117U/kg

188U

/kg

51U/kg

78U/kg

173U

/kg


in Table 4. It varied from 26.98 to 48.54mg, with a mean of36.56 ± 1.78mg (Table 4).

The total content of phenolic compounds was found to berelatively different among the honey samples collected in theconsecutive years and were found to be year-dependent. Thelowest content of TPC was observed in all samples collectedduring the production season of 2008, while the highestcontent was found in the honeys harvested in 2009 (Table 4).It is worth mentioning that the variation in TPC was notobserved during honey storage. The quantified levels of thephenolic compounds in Belgian honeys were significantlyhigher than those reported by Socha et al. [13] andWieczoreket al. [17] in Polish polyfloral honeys. Similar values of TPCwere observed by Gasic et al. [18] in Serbian polyfloral honey,whereas significantly higher values were found in the case ofhoney from north western Spain [19] and in honey samplesfrom an Atlantic European area [20].

3.2. Total Flavonoid Content (TFC). The flavonoid contentwas determined by using a method that used aluminiumchloride, which is based on the formation of a yellow complexbetween the aluminium ion, Al (III), and the carbonyl andhydroxyl groups of flavones and flavonols [21]. Using astandard curve generated for quercetin (𝑅2 = 0.989), thetotal flavonoid content in the honey samples (mg ofQE/100 g)varied from 3.11 to 5.12mg, with a mean value of 4.08 ±0.21mg (Table 4). The highest TFC values were recorded inall of the samples of honey harvested in 2009.

3.3. Antioxidant Activity. The standard DPPH method wasused for the determination of the antioxidant activity. It isa rapid, easy, and widely used screening method for thequantification of antioxidants in foods, including honey [22].The radical scavenging activity of Belgian polyfloral honeysvaried from 36.77% to 44.28% in the DPPH test (Table 4).Thus, their antioxidant activities are comparable to the valuesreported previously [13, 23].

Furthermore, a significant correlation among the TPCand TFC values and the antioxidant activity was observed(𝑅2 = 0.951 and 𝑅2 = 0.872, respectively). The phenoliccontent of the honey samples is partially responsible for theirantioxidant activity, which supports the relevance of this typeof honey as an important source of antioxidant compoundsand its possible use as a natural, therapeutically valuedproduct. In addition, the values obtained by theDPPHstudieshave shown that this activity does not undergo significantchanges between samples collected in subsequent years,which confirms that the activity depends on the compositionof the phenolic fraction. Overall, our study confirms that allof the investigated polyfloral honey samples are substantiallygood sources of phenolic acids and flavonoids, which isreflected in their good antioxidant potential.

3.4. Profiles of Phenolic Compounds. One of the emergingmethods of differentiating between honeys of various originsis chemical profiling. Thus, the presence and relative levelof a chosen set of phenolic acids in all of the samples havebeen determined by means of HPLC. Eighteen commerciallyavailable compounds were chosen, including 11 phenolic

M07 M08 M09 L07 L08 L09 K07 K08 K09Honey types

Abscisic acidEllagic acidRosmarinic acidp-Coumaric acidFerulic acidSyringic acid

Caffeic acidVanillic acid3-Hydroxybenzoic acid4-Hydroxybenzoic acidChlorogenic acid3,4-Dihydroxybenzoic acid

0

10

20

30

40

50

60

70

80

90

100

Con

tent

(%)

Figure 1: The chemical profiling of phenolic acids and abscisicacid in polyfloral honeys fromnorthern Belgium.M07–M09 (honeysamples from Mortsel apiary), L07–L09 (honey samples fromLichtaart apiary), and K07–K09 (honey samples from Kasterleeapiary).

acids (3-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid,p-coumaric acid, chlorogenic acid, rosmarinic acid, ferulicacid, and ellagic acid), six flavonoids (galangin, kaempferol,chrysin, quercetin, myricetin, and pinocembrin), and anisoprenoid, (±)-abscisic acid, which is a plant hormone thathas been previously recognised as a marker of heather honey[9]. The levels of the individual phenolic compounds in theanalysed honeys are shown in Table 4.

It is obvious that the composition of phenolic acids inhoney mainly results from the composition of plant nectarcollected by bees. The most abundant compounds found inall studied samples are five acids, including ellagic, syringic,caffeic, vanillic, and chlorogenic acids. A comparison of therelative quantities of phenolic acids in the honey samplesallowed the construction of the profiles of their phenoliccompounds. As observed from Figure 1, these profiles aresignificantly similar to each other. Thus, a HPLC fingerprintof phenolic acids could be considered as one of the ratio-nal means to determine the identity of polyfloral honeysproduced in the northern part of Belgium. Moreover, theexamination of Figure 1 indicates that the similarities betweenthese profiles are even more substantial when comparinghoneys from different sources, taking into consideration theyear of production. Thus, they also reflect the influence of


Table4:Th

elevelof

theind

ividually

identifi

edph

enoliccompo

unds

andabscisica

cid(m

g/100g

ofprod

uct),

thec

ontent

oftotalpheno

liccompo

unds

(GAE:

gallica

cidequivalent),andthe

contento

ftotalflavono

ids(QE:

quercetin

equivalent)inpo

lyflo

ralB

elgian

honeys.

Sample

name

3- HBA

3,4-

DHBA

4- HBA

CHA

VACA

SAFA

p- CARA

EAMC

QC

KCH

GP

ABA

Totalpheno

liccontent

[mgGAE/100g±

SD]

(Av.,𝑛=3)

Totalfl

avon

oids

content

[mgQE/100g±

SD]

(Av.,𝑛=3)

Radical

scavenging

activ

ityDPP

H(%

)

Levelsof

individu

alidentifi

edcompo

unds

ofteste

dho

neys[m

g/100g

ofprod

uct](

Av.,𝑛=3)

M07

0.11

0.04

0.11

1.58

0.41

0.20

0.41

-0.12

-12.37

0.10

0.31

0.10

0.60

0.00

31.8

5-

41.20±3

.114.28±0

.23

42.37

±SD0.03

0.01

0.04

0.23

0.14

0.04

0.11

0.02

1.12

0.03

0.07

0.02

0.08

0.001

0.46

M08

--

0.28

1.50

0.20

1.70

1.73

-0.10

-9.27

1.13

0.27

0.32

0.54

0.00

21.7

70.66

35.01±

1.17

4.01±0

.21

39.11

±SD0.08

1.14

0.001

0.37

0.17

M09

--

0.06

0.47

0.04

0.04

0.42

-0.02

-13.33

0.01

0.22

0.15

0.53

-1.7

0-

41.95±2

.20

4.72±0

.35

40.92

±SD0.02

2.10

0.21

L07

-0.07

0.10

0.67

0.17

1.43

1.69

--

-10.96

0.00

40.26

0.14

0.55

0.00

41.6

90.02

31.81±

1.32

3.47±0

.1638.87

±SD0.02

0.04

1.65

0.002

0.32

0.00

9L0

8-

0.05

0.07

0.41

0.20

0.66

1.91

-0.56

-11.03

0.09

0.29

0.12

0.58

0.00

61.7

30.02

28.05±1

.41

3.16±0

.1437.15

±SD0.02

0.04

2.07

0.003

0.44

0.00

4L0

9-

0.04

0.22

0.26

0.48

0.52

1.45

-0.60

-12.30

0.08

0.51

0.07

1.12

0.00

51.7

1-

48.54±2

.135.35±0

.23

44.28

±SD0.01

0.06

2.23

0.003

0.18

K07

-0.10

0.27

0.54

1.50

1.40

0.68

-0.57

-12.32

0.17

0.27

0.15

0.85

0.00

81.8

3-

30.27±1

.44

3.52±0

.1 738.93

±SD0.05

0.08

2.11

0.00

0.25

K08

--

0.09

0.41

1.60

1.40

1.74

-1.4

30.14

9.13

0.99

0.30

0.05

0.45

0.00

71.8

10.04

26.98±0

.98

3.11±0

.1536.77

±SD0.03

1.72

0.003

0.31

0.01

K09

--

0.11

0.30

0.04

0.13

0.43

-0.12

0.10

13.66

0.59

0.31

0.02

0.77

0.00

31.6

00.11

45.22±2

.06

5.12±0

.27

42.13

±SD0.03

2.31

0.001

0.19

0.05

Average

36.56±1

.78

4.08±0

.21

40.06

Av.:average;3-HBA

:3-hydroxybenzoica

cid;3,4-DHBA

:3,4-hydroxybenzoica

cid;CH

A:chlorogenicacid;4-H

BA:4-hydroxybenzoica

cid;VA

:vanillicacid;C

A:caffeica

cid;SA

:syringica

cid;FA

:ferulicacid;p-

CA:p-cou

maricacid;R

A:rosmarinicacid;E

A:ellagica

cid;MC:

myricetin;Q

C:qu

ercetin

;K:kaempferol;C

H:chrysin;G

:galangin;

P:pino

cembrin,A

BA:abscisic

acid;–:not

detected.



GalanginChrysinPinocembrin

KaempferolQuercetinMyricetin

0

10

20

30

40

50

60

70

80

90

100

Flav

onoi

ds co

nten

t (%

)

Figure 2: The chemical profiling of flavonoids in polyfloral honeysfrom northern Belgium. M07–M09 (honey samples from Mortselapiary), L07–L09 (honey samples from Lichtaart apiary), andK07–K09 (honey samples from Kasterlee apiary).

weather conditions on honey composition. Specifically, it isworth emphasizing that a high content of ellagic acid is foundin all of the analysed honey samples. The relative amountof this component is between 47% and 88% among the 12studied phenolic compounds (Figure 1). Therefore, ellagicacid might be considered to be a geographical origin markerof Belgian polyfloral honeys. Ellagic acid, a structural unit ofellagitannins, has been found in several types of honey. Morespecifically, it has only been proposed as a floral marker ofheather honey to date [24, 25]. On the other hand, ellagic acidis a characteristic component in green tea and is also foundin other natural sources, such as pomegranate, blueberries,blackberries, raspberries, strawberries, and walnuts. Thiscompound has potential antimutagenic, antiviral, and antiox-idative properties. Because it exhibitswell-acknowledged bio-logical activities, including antioxidant, anti-inflammatory,and anticancer properties, northern Belgian honeys, as theyare a rich source of this acid, may be utilized as a prohealthfood.

Less indicative are the profiles of flavonoids (Figure 2).However, it is worth mentioning that they are characterizedby high levels of chrysin and pinocembrin, compounds thatmay be derived from propolis [26]. We also found that thechemical profiles of the unidentified compounds observed inthe HPLC chromatograms of the analysed Belgian polyfloralhoney extracts are also very similar to each other andalso might be useful as a part of their “HPLC fingerprint”(Figure 3).

4. Conclusions

Polyfloral honey samples from the Antwerp vicinity werestudied to determine if their chemical profiling could be used

0

10

20

30

40

50

60

70

80

90

100


U7U6U5

U4U3U2

U1

Uni

dent

ified

com

poun

ds co

nten

t (%

)

Figure 3: The chemical profiling of unidentified compounds inpolyfloral honeys from northern Belgium. M07–M09 (honey sam-ples from Mortsel apiary), L07–L09 (honey samples from Lichtaartapiary), and K07–K09 (honey samples from Kasterlee apiary).

for the identification of their geographical origin. Studiesperformed using the HPLC/PAD method indicated that theprofiles of the chosen phenolic compounds do not varyconsiderably within samples from year to year. All of thetested honeys were characterized by a very high content ofellagic acid and a significant level of syringic, caffeic, andchlorogenic acids. The chemical profiling of flavonoids andunidentified components of the phenolic fraction of honeys,although less indicative, might also be useful as indicatorsof their geographical origin. Flavonoids and phenolic acidsmight be considered to significantly contribute to the totalantioxidant activity in honey samples. Samples collected fromthe Antwerp province showed relatively high TPC and TFCvalues and are characterized by a good radical scavengingactivity (DPPH). These activities correlate well with the totalcontent of phenolic compounds. The obtained data are ofparticular interest in defining the effect of botanical origin inthe biological activity of honey and to confirm its importanceon the availability of phytochemistry compounds. Moreover,the high content of ellagic acid present in the northernBelgian polyfloral honeysmay support its use as a beneficiary,prohealth food.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This research was partially supported by the National ScienceCentre, Poland, Project 2014/2015/15/B/NZ9/02182.


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