TOTAL PHENOL, ANTIOXIDANT ACTIVITY, FLAVONOIDS,ANTHOCYANINS AND COLOR OF HONEY AS AFFECTED BYFLORAL ORIGIN FOUND IN THE ARID AND SEMIARIDMEDITERRANEAN AREASTAHA M. RABABAH1,6, MOHAMAD AL-OMOUSH1, SUSAN BREWER2, MOHAMMAD ALHAMAD1,WADE YANG3, MOHAMMAD ALRABABAH1, ABD AL-MAJEED AL-GHZAWI1,MUHAMMAD AL- U’DATT1, KHALIL EREIFEJ1, FAWZI ALSHEYAB4, RANYA ESOH1 and ALI ALMAJWAL5

1Faculty of Agriculture, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan2Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, USA3Food Science and Human Nutrition Department, University of Florida, Gainesville, FL, USA4Faculty of Science and Arts, Jordan University of Science and Technology, Irbid, Jordan5Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia

6Corresponding author.TEL: 00962-2-7201000;FAX: 00962-2-7201087;EMAIL: trababah@just.edu.jo orrebabah@gmail.com

Accepted for Publication December 9, 2012

doi:10.1111/jfpp.12071

ABSTRACT

Honey samples representing 12 different Mediterranean floral sources in Jordanwere investigated for their phytochemical compounds and color. The floral com-position (% pollen), total phenolics, total flavonoids, antioxidant activity, antho-cyanins and color varied between locations. Total phenolics were highest (86.3 mgGAE/100 g) in the multifloral summer honey from Sarrout location and lowest(33.7 mg GAE/100 g) in the early spring harvest from the Jordan Valley. The fla-vonoid, anthocyanins and antioxidant activities were highest in summer honeyfrom Umm Alyanabea location (4.6 mg/100 g, 4.1 mg/100 g and IC50 = 24.5 mg/mL, respectively) and were lowest in the early spring honey harvest from theJordan Valley. The colors of Jordanian honey were visually noticeable, varyingfrom yellowish, yellowish green to darker colors. Significant differences existed inthe phytochemical compounds in different honey samples from different Mediter-ranean areas in Jordan. These differences mainly depended on floral sources.

PRACTICAL APPLICATIONS

Honey is a natural product produced primarily from the nectar secreted by flow-ering plants gathered and processed by the honeybee (Apis millifera). It is animportant and unique food product containing bioactive compounds derivedfrom plant nectar or added by bees, and these compounds are known to act asnatural antioxidants that play an important role in food preservation and humanhealth through combating damage caused by oxidizing agents. Honey collectedfrom different nectar of plant flower species contains different levels of phenolicsthat possess antioxidant activity, so different honey properties were expected sincethe composition of active compounds in honey from different locations should bedifferent. Therefore, this study was conducted to evaluate the bioactive com-pounds and color of honeys obtained from various plant vegetations in the aridand semiarid Mediterranean areas in Jordan.

INTRODUCTION

Honey is a natural product produced primarily from thenectar secreted by flowering plants gathered and processed

by the honeybee (Apis millifera). It usually contains manysubstances in addition to pollen grains that are gatheredfrom a wide spectrum of flowers from flowering plants,resulting in honey with different colors, aromas and flavors

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(Blasa et al. 2006). Honey is an important and unique foodproduct containing bioactive compounds derived fromplant nectar or added by bees (Al-Mamary et al., 2002).These compounds are known to act as natural antioxidantsthat play an important role in food preservation andhuman health through combating damage caused by oxi-dizing agents. Natural and synthetic antioxidants have along history of use as preservatives in food (The NationalHoney Board 2003). Antioxidants include vitamins Cand E, flavonoids and phenolic compounds. Phenoliccompounds are one of the most important groups ofcompounds occurring in plants, comprising about 8,000different known structures (Bravo 1998). Honey collectedfrom different nectar of plant flower species contains dif-ferent levels of phenolics that possess antioxidant activity(Bertoncelj et al. 2007). So different honey properties wereexpected since the composition of active compoundsin honey from different locations should be different(Estevinho et al. 2008).

The antioxidant capacity of honey results from the inter-action of many compounds, including phenolics, composi-tion, organic acids, enzymes, peptides and other minorcompounds. The composition and amount of honey phe-nolics depend on the floral source, seasonal and environ-mental factors, geographic origin and storage conditions(Singleton et al. 1999; Kesic et al. 2009; Dobre et al. 2010).Many studies indicated that the antioxidant activity ofhoney varies widely, depending on the floral source; whileprocessing, handling and storage affect honey antioxidantactivity only to a minor degree (Al-Mamary et al., 2002;Beretta et al. 2005).

The color of honey is a useful parameter to characterizethe product. It has been used both to typify the honey andto set a market price. Color can vary from water white tonearly black. Some honeys are colorless and transparent,including Acacia honey (Robinia pseudoacacia L.). Othersare white after crystallization (Rosmarinus officinalis, Citrussp.). Most multifloral honeys are amber, but some are verydark, nearly black in color (Negueruela and Perez-Arquillue2000). Honey color depends on the potential alkalinity andash content, as well as on the antioxidatively active pig-ments, such as carotenoids and flavonoids (Frankel et al.1998).

The content and composition of the different types ofhoney vary with different floral sources as well as climaticand environmental conditions (Mohamed et al. 2010). Also,different types of honey type have a diverse content of phe-nolics and, consequently, different antioxidant activity. Dueto its potential and proven positive medicinal properties,honey is particularly recommended for children (over theage of 2 years) and sportsmen, and it can help improve thehealth of elderly and invalids. Because of that, it is quiteimportant to determine the antioxidative potential of honey

and how can this parameter be of any help in differentiationof various honey types (Krpan et al. 2009).

Many researchers have studied the correlations amongthe phenolic content, flavonoids and the antioxidant activityin honey samples worldwide (Cook and Sammon 1996;Chang et al. 2001; Al-Mamary et al. 2002; Meda et al. 2005;Bertoncelj et al. 2007; Ferreira et al. 2009; Makawi et al.2009). Flavonoids have a great effect on many biologicalactivities (e.g., antibacterial, anti-inflammatory and anti-allergic) (Cook and Sammon 1996; Makawi et al. 2009). Themost important class of antioxidant polyphenols are the fla-vonoids and phenolic acids (Ayoub et al. 2009). Theamount and type of these antioxidant compounds alsodepend largely upon the floral source/variety of the honey(Kesic et al. 2009, Khalil et al. 2010). Researchers havestudied the phenolic and flavonoid contents of honey todetermine if a correlation exists with floral origins. Newstudies evaluated the rheological aspects (Oroian et al.2013) and the physicochemical and rheological properties(Oroian 2012) of honey samples. Therefore, the presentstudy was conducted to assess the total phenol, antioxidantactivity, flavonoids, anthocyanins and color of honeysobtained from various plant vegetations in the arid andsemiarid Mediterranean areas in Jordan.

MATERIALS AND METHODS

Materials

All chemicals used in this investigation were of analyticalgrade. DPPH (2,2-diphenyl-1-picrylhydrazyl), (+)-Catechenhydrate, Folin–Ciocalteu’s phenol reagent, gallic acid andascorbic acid, aluminum chloride, butylated hydroxytoluene(BHT) and methanol (HPLC grade) were purchased fromlocal sources (Irbid, Jordan).

Samples Collection

Honey samples (n = 3) were collected from each of 12 dif-ferent locations in northern Jordan during the 2009 harvestseason. The beehives were maintained under similar man-agement conditions by one beekeeper. The honey sampleswere classified according to the vegetation that prevailed ineach location (Table 1). The samples were stored at 4°C inthe dark until analysis within 2 weeks. The pollen analysis(melissopalynology) was used to identify the source ofnectar of the investigated honey samples. The melissopa-lynological analysis was carried out in accordance with themethod set by the International Commission for Plant-BeeRelationships of the International Union of the BiologicalScience (Louveaux et al. 1978). Table 1 shows the botanicalorigin (% pollen) of the honey samples.

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Total Phenolic Determination

The total phenolics (TP) content was estimated by theFolin–Ciocalteu method as described by Singleton et al.(1999). Honey samples (5 g) were diluted in 50 mL of dis-tilled water then filtered through Whatman no. 1 filterpaper; 0.1 mL of the diluted aliquot was transferred to a testtube and mixed with 2.5 mL of 0.2 N Folin–Ciocalteu’s

reagent and allowed to stand for 5 min at room tempera-ture. Subsequently, 2 mL of 75 g/L sodium carbonate(Na2CO3) solution was added, and the mixture was allowedto stand for 2 h at room temperature in the dark. The absor-bance at 760 nm was determined against a methanol blankusing a spectrophotometer (CELL, model CE 1020, CecilInstruments, Cambridge, U.K.). Gallic acid (0–400 mg/L)was used as a standard to produce the calibration curve.

TABLE 1. TOTAL PHENOL, TOTAL FLAVONOIDS, ANTHOCYANIN, AND ANTIOXIDANT ACTIVITY (IC50) OF HONEY FROM DIFFERENT FLORAL ORIGINGROWN IN DIFFERENT LOCATIONS IN MEDITERRANEAN AREAS IN JORDAN

No. Location Floral origin (% of pollen)Total phenlics(mg/100 g)

Flavonoids(mg/100 g)

Anthocyanins(mg/100 g)

IC50

(mg/mL)

1 Jdaitta Multiflora 49.1 � 0.7de 3.9 � 0.1b 2.19 � 0.08e 45.7 � 3.5f

Sinapis arvensis (21%)Balluta undulata (16%)Centaurea hyalolepis (43%)

2 Umm Alyanabea Multiflora 58.8 � 2.9c 4.6 � 0.3a 4.1 � 0.12a 24.5 � 1.4j

Sinapis arvensis (14%)Acacia spp. (14%)Centaurea hyalolepis (44%)

3 Jordan Valley (1) Multiflora 33.7 � 1.2h 0.9 � 0.2e 0.91 � 0.03j 111 � 3.8a

Citrus spp. (44%)Sylbum marianum (14%)Calendula arvensis (22%)

4 Sbairreh Centaurea hyalolepis (45%) 37.5 � 1.7gh 1.6 � 0.2cd 2.13 � 0.11f 47.1 � 1.7e

Acacia spp. (14%)Sylbum marianum (14%)Sinapis arvensis (26%)

5 Jordan valley (2) Ziziphus spinsa-christi (L) 53.2 � 0.7d 3.9 � 0.1b 2.44 � 0.1d 41.0 � 2.1g

Multiflora6 Al-Hashemiyah (1) Centaurea hyalolepis (43%) 48.6 � 0.2de 4 � 0.2ab 2.55 � 0.1c 39.2 � 1.4h

Sylbum marianum (42%)Sinapis arvensis (15%)

7 Al-Hashemiyah (2) Centaurea hyalolepis 51.9 � 0.5d 4.4 � 0.3ab 3.67 � 0.12b 27.0 � 1.5i

Multiflora8 Sarrout (1) Eucalyptus (43%) 79.7 � 2.4b 3.9 � 0.3b 2.02 � 0.11g 49.6 � 1.9d

Sinapis arvensis (35%)Portulaca oleracea (21%)Multiflora

9 Sarrout (2) Eucalyptus spp. (52%) 86.3 � 2.7a* 1.9 � 0.1cd 1.54 � 0.1i 65.0 � 2.8b

Centaurea hyalolepis (14%)Echinops polycerus (33%)Multiflora

10 Irbid Centaurea aryngioides (28%) 60.7 � 2.7c 4.4 � 0.2ab 2.56 � 0.12c 39.2 � 1.3h

Sylbum marianum (57%)Sinapis arvensis (14%)Multiflora

11 Irhaba Centaurea aryngioides (37%) 44.7 � 6.5ef 2.2 � 0.1c 1.81 � 0.11h 55.4 � 2.1c

Eucalyptus spp. (53%)Echinops polycerus (9%)Multiflora

12 Ras moneif Centaurea hyalolepis (8%) 40.2 � 1.2fg 1.5 � 0.1de 2.03 � 0.12g 49.3 � 1.3d

Sylbum marianum (21%)Sinapis arvensis (53%)Prunus amygdalus (17%)

* Values (mean � SD) followed by different letters within column are significantly different at P � 0.05.

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Estimation of TP was conducted in duplicate and concen-trations were calculated from the gallic acid calibrationcurve. Total phenolics were expressed as gallic acid equiva-lents (mg GAE/100 g of honey).

Determination of Total Flavonoids

The total flavonoid content was determined using a colori-metric method as described by Zhishen et al. (1999). Eachsample (0.5 mL) was mixed with 2 mL of distilled waterthen with 0.15 mL of a NaNO2 solution (15%). After 6 min,0.15 mL of an AlCl3 solution (10%) was added and allowedto stand for 6 min, then 2 mL of NaOH solution (4%) wasadded to the mixture. The volume was made up to 5 mL;the mixture was thoroughly mixed and allowed to stand foranother 15 min. Absorbance was determined at 510 nmversus a water blank using a spectrophotometer (CELL,model CE 1020, Cecil Instruments). Results were expressedas catechin equivalents (mg catechin/g of honey sample).Total flavonoids contents were calculated on the basis of thestandard curve for (+)-catechen hydrate solutions (10 mg/L–200 mg/L).

Determination of Antioxidant ActivityUsing DPPH

The antioxidant activity of honeys was determined accord-ing to Turkmen et al. (2006), with some modifications. Fivegrams of honey were dissolved in 25 mL of methanol, cen-trifuged at 4,350 ¥ g, and filtered with filter paper(Whatman no.1). Then 0.2 mL of methanol solution ofDPPH (50 mg/100 mL) was mixed with different levels ofmethanol extracts, and the mixture was made up to a totalvolume of 4.0 mL with the corresponding extractionsolvent. The mixture was mixed thoroughly and allowed tostand for 60 min in the dark. Absorbance was measuredat 517 nm using a spectrophotometer (general-purposeUV/Vis spectrophotometer, Beckman DU 520). The radicalscavenging activity of the tested samples was expressed asinhibition % of the free radicals according to the followingformula:

Inhibition absorb of control absorb of sampleabsorb o

% . ..( ) = −( )

ff control( ) ×100 (1)

IC50 is defined as the concentration of extract in mg/mLneeded to scavenge 50% of the DPPH radical. The IC50

for each honey sample extract was calculated from itsconcentration–response curve.

Determination of Anthocyanins

Anthocyanins were extracted as described by Rabino andMancinelli (1986). Ten grams of honey was diluted in

50 mL of 1% HCL methanol (w/v) and incubated in a waterbath at 60C for 1 h with occasional shaking, then filteredusing Whatman # 1 filter paper into a 50 mL volumetricflask and made up to volume with the same solvent. Absor-bance of the extract was measured at 657 and 530 nm usinga spectrophotometer (general-purpose UV/Vis spectropho-tometer, Beckman DU 520). The net absorbance was calcu-lated by the equation:

Net absorb Absorb at nm Absorb at nm. . . .= − ( )530 0 25 657

(2)

Anthocyanin content was calculated based on cyanidin-3-glycoside (Cyd-3-glu), which has a molecular weight of449.1 and an extinction coefficient of 29,600 (Giusti andWrolstad 2001).

Anthocyanin in mg g net absorbanceMW DF V Wt

( ) = ( )× × × ( )

29 600,(3)

where MW = 449.1, DF = dilution factor, V = total volume(mL) and Wt = sample weight (g).

Instrumental Color Measurements

Color of honey samples was measured by a reflectance spec-trocolorimeter (12MM Aperture U 59730 Inc., Pittsford,NY) and recorded in the L*, a*, b* color system. The spec-trolorimeter was calibrated throughout the study by using astandard white ceramic reference Commission Internation-alle De L’eclairage (L* = 97.91, a* = -0.68, and b* = +2.45).Honey samples were heated to 50°C to dissolve sugar crys-tals then placed in a plastic container 7 cm in diameter andcovered with a plastic plate. The measured layer was 1 cmthick. In addition, total color difference (DE) and chromawere calculated using the following equations:

Δ Δ Δ ΔE a b L= ( ) + ( ) + ( )⎡⎣ ⎤⎦2 2 2 1 2

(4)

Chroma = ( ) + ( )⎡⎣ ⎤⎦a b2 2 1 2. (5)

Statistical Analyses

Data were analyzed using the general linear model (GLM)procedure with JMP statistical package (JMP InstituteInc., Cary, NC). Means were separated by least significantdifference analysis (P < 0.05). The correlation coefficientsbetween all the parameters were also determined. Also,the relationships (R2) among the total phenolics, antioxi-dant activity, anthocyanins and total flavonoids wereinvestigated.

RESULTS AND DISCUSSION

Pollen analysis is a useful tool to indicate the plant sourcesof honey. Pollen analysis of 12 different locations in Medi-

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terranean areas in Jordan (Table 1) shows that the largerproportion of honeys were obtained from a variety ofwild flowers, including Sinapis arvensis, Sylbum marianumand Centaure aryngioides; while a lesser proportion wasobtained from Citrus spp. and Acacia spp. flowers. Pollenanalysis also shows that nine honey samples are considereda multifloral honey obtained from different floral origin.Multiflora honey is expected since Jordan is characterizedby diverse plant community and because distances betweendifferent geographical regions are short.

Total Phenolic Contents

Table 1 showed the concentration of total phenolic com-pounds in 12 honey samples collected from different florallocations in Jordan. Phenolic content varied significantlyand ranged from 33.7 mg/100 g (Jordan Valley) to 86.3 mg/100 g (Sarrout). The highest values were obtained frommultifloral samples (Sarrout, Sarrout and Irbid: 86.3, 79.7and 60.7 mg/100 g, respectively). The lowest value of33.7 mg/100 g was obtained from the Jordan Valley (1)flora, which has a high proportion of citrus trees (44%).Comparable values were found by Kaškoniene et al. (2009)who studied the effect of botanical source on honey andfound the lowest phenolic content in citrus. Multifloralhoney that came from Sbeirreh and Ras Moneif also hadlow total phenolic compound values (37.5 and 40.2 mg/100 g, respectively), while the other values ranged between44 and 58 mg/100 g. Lachman et al. (2010a) reported thatthe total phenolic content varied from 32.59 mg/100 g inmultifloral honey to 93.66 mg/100 g in honeydew honey.This range is consistent with our results. Khalil et al. (2010)reported that there are considerable differences in bothcomposition and content of phenolic compounds in differ-ent unifloral honeys coming from different regions, rangingbetween 46 GAE mg/kg (acacia) and 796 GAE mg/kg (Illi-nois buckwheat), a value that is equivalent to one of oursamples (Sarrout [1]). The total phenolics of Indian honeyranged from 24 to 98 mg Gallic acid/100 g of honey (Saxenaet al. 2010), which also falls within the range of our results.However, the results obtained by Lachman et al. (2010b) fortotal phenolics, which ranged from 3.92 mg GAE/100 g(multifloral honey) to 16.71 mg GAE/100 g (honeydewhoney), by Mohamed et al. (2010) for total phenolics ofTualang honey (251.7 mg GAE /kg honey) and byBertoncelj et al. (2007), were all lower than what is reportedin this investigation.

Estevinho et al. (2008) reported that honey type andsource have a significant effect on phenolic contents. Com-paring our honey type in this study with other reports fromRomania clearly shows that total phenolics found in thisstudy were higher than Romanian honey as reported by Alaet al. (2009). The variation is mainly attributed to the

diverse floral sources that contain different phenolic acidsand other substances that affect the phenolic content. Totalphenolic contents are significantly dependent on the floralsources (Zheng and Wang 2001; Gheldof and Engeseth 2002;Piljac-Žegarac et al. 2009). Location of the collected honeysamples plays a role in total phenolics content as is reportedfor rhododendron honey where phenolics ranged between20 and 121 mg gallic acid/100 g, as reported by Silici et al.(2010).

Total Flavonoid Contents

The concentrations of total flavonoid compounds in 12honey samples collected from different floral locations inJordan are shown in Table 1. Flavonoids contents (Table 1)varied with the origin of honey samples and ranged from0.93 mg/100 g (Jordan Valley) to 4.6 mg/100 g (Umm Aly-anabea), which were moderate values compared with otherinvestigated samples. The lowest value was observed in mul-tifloral honey sample no. 3 (0.93 mg/100 g), which wasabout 44% citrus honey. The highest value was observed inhoney sample nos. 2, 7, 10 and 6 (4.6, 4.4, 4.4 and 4.0 mg/100 g, respectively) where all samples are multifloral exceptfor sample no. 7, which represents Boiss. Centaurea hyalol-epis honey. These results agree with those of Lachman et al.(2010a) who reported that the total flavonoid content ofhoney samples (mg QE/100 g) varied from 0.17 to 8.35 mg,with the highest observed in multifloral types of honey.Higher flavonoids contents were found by Kaškoniene, et al.(2009). In general, the flavonoid concentration in honey isapproximately 20 mg/kg (Ayoub et al. 2009). This value is inagreement with the samples nos. 9, 4 and 12 in the presentwork. The flavonoids present in honey samples from differ-ent geographical areas (Europe, North America, Equatorialregions, South America, China and Australia) have beenanalyzed by Tomás-Barberán et al. (1993). The preliminaryresults of their work show that flavonoid analysis could beused as an adjunct to geographical origin studies of honey.

Some of our results agreed with those obtained by Medaet al. (2005). However, most of total flavonoid contents arein agreement with the results obtained by Yao et al. (2004)and Kaškoniene, et al. (2009) who used monofloral honeyaccording to the apiarists claim. Chang et al. (2001) alsoreported that each type (origin) of honey, wild flower andsunflower, alfalfa and prairie flower, have different totalflavonoid contents (18, 15 and 2 mg/g, respectively). So thevariation in the flavonoids content appears to be attribut-able to the variation in the flora source from which thehoney originated.

Antioxidant Activity

The antioxidant activity in the honey samples is shown inTable 1. The antioxidant activities varied with the location

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from which the honey originated; IC50 values rangedbetween 24.5 and 110.5 mg/mL. The multifloral honeysample from Umm Alyanabea and the monofloral honey(C. hyalolepis) from Al-Hashemiah (2) had the highest anti-oxidant capacity (24.5 and 27 mg/mL, respectively), whileother samples representing multifloral honeys from differ-ent locations showed a reasonable amount of antioxidantactivity (Table 1). The lowest IC50 values honey from theJordan Valley (1) followed by that from Sarrout (2).

Estevinho et al. (2008) determined the radical scavengingactivity by measuring IC50 and reported values of68.17 mg/mL in clear honey and 27.24 mg/mL in darkhoney. Ferreira et al. (2009) studied the antioxidant proper-ties of Portuguese honeys and reported values for the lighthoney (90.78 mg/mL) similar to our white honey samplefrom the Jordan Valley (1). The antioxidant activity of thesamples investigated in this study were lower than thoseobtained by Bertoncelj et al. (2007) and higher than thoseobtained by Krpan et al. (2009). The antioxidant activity ofour investigated honey samples were higher than thoseobtained for samples collected from northeast Portugal asreported by Estevinho et al. (2008). Kesic et al. (2009)showed that the total antioxidant activity of forest honeysamples was higher by 82.67 % than for acacia honeysamples and 40.19% for meadow samples. He too con-cluded that antioxidant activity of honey depends on thebotanical origin. Khalil et al. (2010) showed that antioxi-dant content varied more than 20-fold, from a high value of4.32 ¥ 10-3 eq for Illinois buckwheat honey to a low value of21.3 ¥ 10-5 eq for California button sage honey. Theseresults demonstrate that the variations in antioxidant con-tents of honey samples and their ability to scavenge the freeradicals are a function of different locations so differentfloral sources of nectar (Dobre et al. 2010; Mohamed et al.2010).

Total Anthocyanin

Table 1 shows the concentration of total anthocyanin com-pounds in honey samples collected from different florallocations in Jordan. Anthocyanin contents among rangedfrom 0.91 to 4.1 mg/100 g of honey sample. The highestvalues were observed in honey sample nos. 2, 10, 6 and 5(4.1, 2.56, 2.55 and 2.44 mg/100 g, respectively); while thelowest values were obtained from sample nos. 3, 9 and 11(0.91, 1.54 and 1.81 mg/100 g, respectively). There are noreports in the literature regarding the effect of floral sourceson anthocyanin contents of honey; however, our resultscould be due to different floral sources of nectar fromdifferent location.

Color Measurements

The lightness (L*), redness (a*), yellowness (b*), DE andchroma values of honeys are shown in Table 2. These resultsshow that the lightness, redness, yellowness, DE and chromavaried among different floral areas. The colors of Jordanianhoneys were visually apparent, varying from yellowish,yellowish green to darker colors. Honey color could beclassified to be light in color if the L* > 50. Significancedifferences were observed between the citrus sample, multi-floral sample no. 12 and multifloral sample no. 2, which hadthe highest lightness values (76.4, 73.5 and 69.4, respec-tively). These observations have been reported in otherstudies. Raezke (2006) reported a value differences due tobotanical source of polyflora, wild blossom and foresthoneys (67.4, 52.8 and 86.0, respectively). In addition,Khalil et al. (2010) reported that the color of the honey col-lected by the bees varies according to the floral source,which usually ranges from water white to dark amber incolor, and bears no relationship to total phenolics or anti-

TABLE 2. LIGHTNESS (L*), REDNESS (A*),YELLOWNESS (B*), DE AND CHROMA VALUESOF HONEY COLLECTED FROM DIFFERENTFLORAL ORIGIN GROWN IN DIFFERENTLOCATIONS IN MEDITERRANEAN AREASIN JORDAN

No. Location L* a* b DE Chroma

1 Jdaitta 64.9 � 3.2fg -3.4 � 0.2g 6.4 � 0.2bc 65.3 � 3.2ef 7.2 � 0.3c

2 Umm Alyanabea 69.4 � 4.1c -1.7 � 0.1de 6.7 � 0.2ab 69.8 � 2.5c 6.9 � 0.3c

3 Jordan valley (1) 76.4 � 2.3a** -4.0 � 0.2h 3.7 � 0.2e 76.8 � 2.8a 7.0 � 0.3c

4 Sbairreh 61.9 � 2.5ij -1.2 � 0.1d 5.3 � 0.1d 62.1 � 2.9h 5.4 � 0.2e

5 Jordan valley (2) 59.6 � 3.3k 6.2 � 0.2a 6.7 � 0.2ab 60.2 � 2.4i 9.1 � 0.4a

6 Al-Hashemiyah (1) 66.1 � 2.4e -1.8 � 0.1de 7.1 � 0.2a 66.5 � 2.6d 7.3 � 0.4c

7 Al-Hashemiyah (2) 62.9 � 2.8hi 4.1 � 0.3b 6.9 � 0.2a 63.4 � 3.1g 8.1 � 0.3b

8 Sarrout (1) 67.3 � 3.8d -1.6 � 0.1de 6.1 � 0.2c 67.6 � 3.2d 6.3 � 0.3d

9 Sarrout (2) 65.9 � 2.4ef -2.6 � 0.1f 2.0 � 0.1f 65.9 � 2.7de 3.3 � 0.1g

10 Irbid 63.8 � 2.4gh 0.7 � 0.0c 6.8 � 0.2ab 64.1 � 2.6g 6.8 � 0.3cd

11 Irhaba 61.7 � 2.8j -2.3 � 0.1ef 4.2 � 0.1e 61.9 � 3.1h 4.8 � 0.2f

12 Raas moneif 73.6 � 2.3b -6.3 � 0.3i 5.3 � 0.2d 74.0 � 3.5b 8.2 � 0.4b

** Values (mean � SD) followed by different letters within column are significantly different atP � 0.05.

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oxidants. Another researcher has reported that dark honeyhas better antioxidant activity (lower IC50 values) than otherhoneys (Dobre et al. 2010; Lachman et al. 2010b). Ourresults were in contrast; the dark color honeys did not havehigher antioxidant activities or total phenolics. For example,sample no. 5 had a darker color but does not have thehighest antioxidant activities or phytochemical contents.This variation in phytochemicals in our areas could be duemore to floral sources than to honey color. This is also truein fruits and vegetables; the deeper color, in general, doesnot mean the fruit or vegetable has more phytochemicalsthan others with lighter colors (Karlidag et al. 2009).

Pearson Correlation Coefficients (r)among Different Color Components andBioactive Compounds

The correlation results among the investigated honeybeeparameters are presented in Table 3. Lightness is signifi-cantly and negatively correlated with redness (r = -0.650)and positively correlated with DE (r = 0.999). While theredness was positively correlated with yellowness (r =0.593), flavonoids (r = 0.596), anthocyanins (r = 0.594) andIC50 (r = 0.581), it was negatively correlated with DE (r =-0.650). The yellowness was more highly correlated withflavonoids (r = 0.832) and anthocyanins (r = 0.895), but

negatively correlated with IC50 (r = -0.903). IC50 was nega-tively correlated with flavonoids (r = -0.868) and anthocya-nins (r = -0.998).

Relationship between Phenolic Contentsand Other Bioactive Compounds of Honey

The relationship (R2) between total phenolics and antioxi-dant value, between total phenolics and total anthocyaninsand between total phenolics and total flavonoids in thehoney samples investigated were 0.55, 0.55 and 0.8 respec-tively. These results indicate that phenolics are the compo-nent responsible for the scavenging activity and can beattributed mainly to flavonoid compounds available inJordanian honey. There was a slight positive relationship(R2 = 0.55) between the concentration of total phenolic andtotal anthocyanin of the honey samples (Fig 1). That rela-tion was nearly as strong as that reported by Teh et al.(2005), who reported an R2 of 0.86 between anthocyaninand phenolics content.

Figure 2 shows the relationship between the total phe-nolic content with the antiradical scavenging activity; anti-oxidant capacity (1/IC50) of the honey samples. As observedin Fig. 2, there was also a slight positive relationship(R2 = 0.55) between the concentration of total phenolicsand total anthocyanin. This is consistent with results

TABLE 3. PEARSON CORRELATION COEFFICIENTS (R) AMONG INSTRUMENTAL COLOR MEASURES AND BIOACTIVE COMPOUNDS

Parameters Lightness Redness Yellowness DE Chroma Total phenolics Flavonoids Anthocyanins IC50

Lightness 1 -0.650* -0.193 0.999** 0.077 -0.091 -0.194 -0.21 0.2Redness 1 0.593* -0.650* 0.105 0.392 0.596* 0.594* -0.581*Yellowness 1 -0.193 0.547 0.218 0.832* 0.895** -0.903**DE 1 0.077 -0.091 -0.194 -0.21 0.2Chroma 1 -0.238 0.166 0.434 -0.448Total phenlics 1 0.568* 0.259 -0.242Flavonoids 1 0.871** -0.868**Anthocyanins 1 -0.998**

* significant (P < 0.05)** highly significant (P < 0.01)

y = 0.0735x - 1.0805R² = 0.55

0

1

2

3

4

5

25 35 45 55 65Total phenolics (mg/100g)

An

tho

cy

an

ins

(m

g/1

00

g)

FIG. 1. RELATIONSHIP BETWEEN TOTAL PHENOLIC CONTENTS MGGAE/100 G AND ANTHOCYANINS CONTENT MG Cyd-3-GLU/100 G

y = 0.0007x - 0.0108R² = 0.55

0

0.01

0.02

0.03

0.04

0.05

25 35 45 55 65

Total phenolics (mg/100g)

An

tira

dic

al (1

/Ic50)

FIG. 2. RELATIONSHIP BETWEEN TOTAL PHENOLIC CONTENTS MGGAE/100 G OF HONEY SAMPLES AND SCAVENGING ACTIVITY(1/IC 50) OF HONEY SAMPLES

T.M. RABABAH ET AL. TOTAL PHENOL, ANTIOXIDANT ACTIVITY, FLAVONOIDS

7Journal of Food Processing and Preservation •• (2013) ••–•• © 2013 Wiley Periodicals, Inc.

reported by others. Lachman et al. (2010b) showed higherrelationships than found in the present study for character-istic antioxidant activities (R2 = 0.96 for propolis and 0.90for honey). The same was obtained by Krpan et al. (2009)who reported a positive relationship between total phenoliccontent and antioxidant activity (R2 = 0.87), indicating thatphenolic compounds are mainly responsible for the antioxi-dant power of acacia honey. Also, four types of A. melliferahoney collected in the eastern region of Romania werescreened for their total phenolic content and for anti-radicalpower. A high relationship was found between total antioxi-dant activities and total phenol contents, indicating thatphenolics are the components responsible for the antioxi-dant effects of lime honey; but, obviously, other factors areinvolved. These might be different phenolic compositions orthe presence of nonphenol antioxidants such as ascorbate,a-tocopherol and b-carotene. Inoue et al. (2005) reportedthat there is a high relationship between 1/IC50 value andtotal phenolic content observed (R2 = 0.97) of studiedsamples from different floral sources (manuka, NewZealand; buckwheat, France; acacia, Japan, Hungary andChina). The same conclusion has been drawn by Bogdanov(2011).

As observed in Fig. 3, there was a good relationship(R2 = 0.80) between total phenolics and total anthocyanins.Marghitas et al. (2010) also showed a relationship betweentotal phenolic content and total flavonoid content of thesupplements used in honeybee. Meda et al. (2005) analyzed14 species of wild edible fruits from Burkina Faso for theirphenolic and flavonoid contents. He concluded that therewas a strong relationship between total phenolic andflavonoid levels, which is consistent with our findings.

CONCLUSIONS

This study evaluated 12 honey samples from different floralsources (origin) in the Mediterranean areas in Jordan forphytochemicals and color. The results indicated that honeysamples from each location had different floral makeup

(% pollen). The study also showed that the Jordanian honeysamples contain different amounts of total phenolics, anti-oxidant activity, flavonoids and anthocyanins; and thatthese depend mainly on the floral source. The increase inthe yellow color intensity seems to be related to mainly tofloral type and moderately to an increase in the antioxidantproperties and in the phenolic contents. The correlationresults indicate that phenolics are the component respon-sible for the scavenging activity and mainly attributed toflavonoids compounds in Jordanian honey.

ACKNOWLEDGMENTS

The authors extend their appreciation to the Deanshipof Scientific Research at King Saud University for fundingthe work through the research group project numberRGP-VPP-193.

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