Journal of ApiProduct and ApiMedical Science 1 (2): 43 - 50 (2009)

DOI 10.3896/IBRA.4.01.2.04

IBRA 2009

ORIGINAL RESEARCH ARTICLE

Antioxidant properties and phenolic content of different floral origin honeys

J. Piljac-egarac1*, T. Stipevi2, and A. Belak1
1 2

Department of molecular biology, Institute Ruer Bokovi, Bijenika c. 54, P.O. Box 180, 10000 Zagreb, Croatia Department of molecular medicine, Institute Ruer Bokovi, Bijenika c. 54, P.O. Box 180, 10000 Zagreb, Croatia

Received 29 January 2009, accepted subject to revision 20 February 2009, accepted for publication 12 March 2009. *Corresponding author: Email: jpiljac@irb.hr

Summary
Twenty six honey preparations of different floral origin (11 monofloral, 7 heterofloral, 8 special) were tested for total phenol (TP) content and antioxidant/antiradical properties. Commonly employed analytical techniques used to quantify plant antioxidants were used for analysis of various floral origin honeys. The Folin-Ciocalteu test was used to determine the TP content, ferric reducing antioxidant power (FRAP) assay for reducing capacity and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays for radical scavenging capacity. The colour intensity measure, ABS450, was used to approximate the contribution of coloured phytochemicals (carotenoids, flavonoids) to the overall antioxidant capacity of honey. Phenolics were most concentrated in Velebit forest honey (116.97 mg ascorbic acid equivalents (AAE)/100 g) and honey with added sour cherry juice (109.81 mg AAE/100 g), which also exhibited the highest FRAP value (459.21 mM Fe(II)) as well as DPPH and ABTS+ radical scavenging capacities (43.04 and 65.24 mg AAE/100 g, respectively). Overall, heterofloral honeys exhibited the highest mean TP content (58.75 mg GAE/100 g), the highest mean reducing capacity (57.66 mM Fe(II)) and the best mean radical scavenging properties with respect to both DPPH (16.66 mg AAE/100 g) and ABTS+ radicals (35.27 mg AAE/100 g).

Keywords: honey analysis, heterofloral, monofloral, polyphenols, antioxidant capacity, FRAP, DPPH, ABTS, colour intensity, Croatia.

Introduction
It has been proven that oxidative stress plays a significant role in the onset of numerous pathological conditions including coronary heart disease (Soydin et al., 2007), strokes (Vibo et al., 2007), cancer (Bentz, 2007). There are also preliminary reports pointing to the role of oxidative stress in ageing (Gilca et al., 2007; Muller et al., 2007), thus, foods containing significant levels of antioxidants which can inhibit or delay oxidation of a substrate represent a healthy and logical diet choice. Honey, as well as wax, pollen, and propolis have been termed value-added products ever since the initial studies confirmed that antioxidant properties of polyphenols lie at the heart of their cosmetic (Jimnez et al., 1994), medical (Cooper and Molan, 1999) and alimentary applications (Nagai et al., 2006). The focus of the scientific community has been directed to various honey components such as amino acids, proteins, trace elements, sugars and pollen (Anklam, 1998; Latorre et al., 2000; Popek, 2002), as

well as antioxidants (Antony et al., 2000; Beretta et al., 2005; Blasa

et al., 2006). The lack of a widely accepted standardized method in

evaluation of antioxidant capacity of foods is the reason why most studies reporting the antioxidant capacity of honeys employ a battery of various antioxidant capacity assays. The choice of floral honey samples used in this study was based on the assumption that varying total phenol content and antioxidant capacity is expected for honeys produced from varying floral sources on different geographic locations in Croatia that include the mountains (Velebit), the coastal belt (Dalmatia and the islands), the hills of Zagorje, and the plains of Slavonia. Honey producers have throughout the past years successfully exploited the resulting diversity of plant species existing in Croatia, as a consequence, there is quite a selection of different floral honeys available on the market. However, virtually no data is available on the phenolic antioxidants of the resulting honey preparations. The aim of our study was to apply available analytical techniques and analyse a representative portion

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Piljac-egarac, Stipevi, Belak

of honey products available in Croatia, with special emphasis on three different groups of honey, natural honey (monofloral, heterofloral) and honey preparations with added juice or extracts (special). These additives were added by the producers with the aim of boosting the antioxidant potency and the potential health benefits of the final products. In attempting to analyse the antioxidant activity of a complex mixture of substances, such as honey, we made use of a combination of analytical methods that takes into account the sample's reducing capacity such as the FolinCiocalteu and ferric reducing antioxidant power (FRAP) tests as well as antiradical activity, namely, the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays. Honey colour intensity was also quantified (Frankel et al., 1998), and correlations were draw between the results of employed methods. To the best of our knowledge, there are no published studies focused on investigating the influence of floral origin and additives on antioxidant properties of such a large set of monofloral, heterofloral and special honeys.

check whether the main sugar components interfere in the assays. The analogue was prepared by dissolving 1.5 g of sucrose, 7.5 g of maltose, 40.5 g of fructose and 33.5 g of glucose in 17 ml of deionized water (Cooper et al., 2002). Each honey sample (5 g) was distributed into test tubes and diluted to 50 ml with distilled water, using a vortex mixer. The solution was then filtered through Whatman No.1 and analysed for TP content and antioxidant activity (Meda et al., 2005). Estimation of total phenol content (TP) The determination of the TP content of honey was performed according to the Folin-Ciocalteau method with slight modifications (Singleton et al., 1999). An 0.5 ml aliquot of the previously prepared honey solution was added to 2.5 ml of 0.2 N Folin-Ciocalteau reagent and mixed for 5 min, followed by the addition of 2 ml of 75 g/l sodium carbonate (Na2CO3). After incubation at room temperature for 2 h, the absorbance of the reaction mixture was measured at 760 nm against a sugar analogue blank. The TP content was expressed as mg of gallic acid equivalents GAE/100 g of honey and mg of ascorbic acid equivalents AAE/100 g of honey, using the calibration curves of gallic acid (0-200 mg/l) and ascorbic acid (0-125 mg/l) standards (Meda et al., 2005). All determinations were performed in

Materials and methods

Chemicals and instruments Except for the Folin-Ciocalteu reagent (Fluka, Switzerland) and FeSO47H2O (Kemika, Croatia) all the chemicals and reagents used in this study were of analytical grade and supplied by Sigma Chemical Co. (St. were Louis, MQ, USA). on a Spectrophotometric double-beam UV-VIS measurements performed

triplicate. Ferric reducing antioxidant power (FRAP) The reducing capacity of honey samples was assayed with the original method of Benzie and Strain (Benzie and Strain, 1996), adjusted to analysis of honey samples. Honey preparations were diluted as described above (5 g/50 ml) and all measurements were performed as follows: to 100 l of honey solution a volume of 900 l of freshly prepared FRAP reagent was added (up to completing 1 ml). The FRAP reagent was prepared by mixing 25 ml of 300 mM acetate buffer, with 2.5 ml of 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) solution and 2.5 ml of FeCl3 6H2O. The mixture was shaken and absorbance readings were taken after 4 min at 593 nm, against the reagent blank of sugar analogue. The results, obtained from triplicate analyses, were expressed as m of FeSO47H2O (Sigma) of the 10% honey solution, and derived from a calibration curve determined for this standard (0-500 m). Colour intensity: ABS450 Colour intensity was quantified as the net absorbance defined as the difference between spectrophotometric absorbance at 450 and 720 nm. For this purpose, the honey samples were prepared as follows: diluted to 50% (w/v) with warm water (4550C), sonicated for 5 min and filtered (0.45 m pore size) to eliminate large particles. The results were expressed as mAU (Beretta et al., 2005).

spectrophotometer Bio-Spec-1601 (Shimadzu Corporation, Kyoto, Japan). The pH of 10% honey solution was determined using the Metrohm 744 pH meter. Honey samples Eighteen honey samples of varying floral origin (11 monofloral, 7 heterofloral), and 8 special honey preparations with different additives (propolis, silver fir tincture, fruit juices, mushroom and broccoli extracts or lavender essential oil), were used for this study. Honey samples were obtained from a donation from the Croatian apiarists union or purchased at local stores. Monofloral honey was defined as honey deriving at least 45% of the pollen from a single floral source, heterofloral honeys had more than 55% pollen contributions from more than one floral source (Von der Ohe et al., 2004). All tested honey samples originated from geographically separate regions (Table 1) of Croatia. Honey samples were between 1-3 years old, with the majority deriving from 2007. Honey samples were stored at 4C in the dark until analysed. A sugar analogue (an artificial honey whose composition reflects the approximate sugar composition of honey) was used to

Antioxidant properties of honey preparations

45

DPPH radical scavenging DPPH radical scavenging of honeys was tested according to the method previously described by Zhang and Hamauzu (2004). Each honey sample was precisely diluted to 4Bx with distilled water using the Atago - 5000a digital refractometer. A 1.5 ml aliquot of 0.1 mM DPPH solution in methanol was mixed with 0.5 ml of honey solution. The reaction mixture was mixed on a vortex and left to stand at 25C in the dark for 60 min. Absorbance at 517 nm was measured against a methanol blank. Radical scavenging capacity was expressed as mg AAE/100 g of honey solution, using the appropriate calibration curve of ascorbic acid (0-25 mg/l). ABTS
+

Results
TP content, reducing capacity (FRAP) and colour intensity (ABS450) All tested honeys and honey preparations contained significant levels of polyphenols, as shown in Table 2. The TP content per 100 g of honey ranged from 12.64 mg GAE (green) to 90.57 mg GAE (Velebit forest), which represents a 7.2-fold difference. There was no significant difference (p > 0.05) between the mean TP content of monofloral (42.24 mg GAE/100 g) and heterofloral honeys (58.75 mg GAE/100 g). The sequence of honeys according to TP content, expressed in AAE, is the same as the sequence obtained for GAE. The ability of the 10% honey solution to reduce the Fe3+/Fe2+ couple was determined using the FRAP assay. We observed a 38.0fold difference between the sample with the greatest reducing capacity (459.21 m Fe(II), sour cherry juice) and the lowest ranking honey (12.06 m Fe(II), acacia). The mean reducing capacity of heterofloral honeys amounted to 157.66 m Fe(II), while the mean reducing capacity of monofloral honeys amounted to 82.31 m Fe (II). Special honey preparations came in between with a mean reducing capacity of 142.42 m Fe(II). The colours of analysed honeys and honey preparations evaluated by visual observation varied from practically transparent (acacia) to dark brown (e.g. Velebit forest, meadow, and fruit flower) and dark green (honey with added broccoli extract). Net absorbance of analysed honeys and honey preparations, the ABS450 parameter, varied from 25 mAU in amorpha honey to 1058 mAU in flower-mix honey with sour cherry juice.

radical scavenging

A modification of the original method of Re et al. (1999) was applied to assess the scavenging capacity of honey samples in a reaction with the ABTS
+

radical. ABTS

radical solution was generated by

oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt stock solution with potassium persulfate (K2S2O8). Stock solutions of ABTS (7 mM) and potassium persulfate (140 mM) in water were prepared, and ABTS+ radical solution was produced by reacting 10 ml of the ABTS stock solution with 175 l of potassium persulfate solution. The mixture was left to stand in the dark at room temperature for 1216 hours before use. For the evaluation of antioxidant capacity, the ABTS
+

solution was diluted

with ethanol (96%) to obtain the absorbance of 0.700 0.020 at 734 nm. 2 ml of ABTS+ solution were mixed with 100 l of honey solution in a cuvette and the decrease in the absorbance was measured after 6 min. The reagent blank was prepared by adding 100 ml of ethanol instead of the sample. Ascorbic acid was used as the standard. Different solutions (0-100 mg/l) of ascorbic acid were prepared in 96% ethanol, and assayed under the same procedure as the samples. The means of three values were obtained and expressed as mg AAE/100 g of honey. Statistical analysis All measurements were performed in triplicate and are presented as mean SD. Correlations were drawn between different methods employed, and quantified in terms of the correlation coefficient, r. The correlation coefficients calculated between each pair of antioxidant capacity measures indicate whether the interdependence exists between the variables, or simply, whether the results of one antioxidant capacity test are in accordance with the results of the remaining tests. One-way ANOVA (performed in SigmaStat 3.5) was used to determine whether the differences between measurements of honeys from different subsets (monofloral, heterofloral and special) are significant. Differences at p < 0.05 were considered to be significant.

DPPH and ABTS+ radical scavenging The radical scavenging capacity of tested honeys was evaluated in the DPPH and ABTS+ radical reaction systems. All honeys and honey preparations exhibited scavenging potential towards both radicals. A simple glance at Figure 1 shows that the mean AAE value determined in the ABTS assay (29.89 mg AAE/100 g) is significantly higher (46.7%) than the mean AAE determined in the DPPH assay (13.95 mg AAE/100 g). The highest scavenging potential with respect to both DPPH and ABTS+ radicals was observed for flower-mix honey with added sour cherry juice (43.04 and 65.24 mg AAE/100 g, respectively), while the green (2.67 mg AAE/100 g) and amorpha (6.83 mg AAE/100 g) honeys exhibited the lowest ABTS+ and DPPH radical scavenging capacities, respectively. In addition to flower-mix honey with added sour cherry juice, Velebit forest (DPPH assay: 32.14 mg AAE/100 g; ABTS assay: 57.76 mg AAE/100 g) and queen of honey (DPPH assay: 21.74 mg AAE/100 g; ABTS assay: 57.15 mg AAE/100 g) stand out from the rest of the samples with respect to their radical scavenging potential.

46

Table 1. Descriptive data for 18 Croatian honeys and 8 honey preparations.

Sample

no.

Common name Floral origin

Year

Geographical origin in Croatia

Appearance

pH

Monofloral Paliurus spina-christi Mill. Helianthus annuus L. Salvia officinalis L. Satureja montana L Satureja montana L. Amorpha fruticosa L. Castanea sativa L. Tilia cordata Mill. Robinia pseudoacacia L. Brassica napus oleifera Solidago virga aurea L. unknown Leaf forest Apple, cherry, plum, pear flowers Lime, acacia, thyme unknown Acacia, chestnut, sage unknown Composition Sage honey with propolis Sage honey with 0.75% eucalyptus and silver fir tincture Flower honey with 8% sour cherry concentrate Flower honey with 5% lemon concentrate Meadow honey with 12.5% blackberry juice Cream-honey with 2.5% powdered shiitake mushroom Acacia honey with 4% broccoli Meadow honey with lavender essential oil Zadar, Dalmatia, coastal Croatia Zaprei, continental Croatia Zagreb, continental Croatia Zagreb, continental Croatia Karlovac, continental Croatia Karlovac, continental Croatia Dalmatia hinterland, coastal Croatia Zagreb, continental Croatia beige (opaque) light brown (clear) ruby red (clear) golden-yellow (clear) medium brown (opaque) dark brown (opaque) dark green (opaque) dark golden-brown (clear) 4.98 4.96 3.22 3.13 4.38 4.88 4.90 4.39 Slavonia, continental Croatia Velebit mountain, continental Croatia Posavina, continental Croatia Zaprei, continentalCroatia Lika, continental Croatia Velebit mountain, continental Croatia Zaprei, continental Croatia Zagorje, continental Croatia Slavonia, continental Croatia Podravina, continental Croatia unknown unknown Posavina, continental Croatia unknown Velebit mountain, continental Croatia Velebit mountain, continental Croatia Slavonia, continental Croatia golden-brown (opaque) medium brown (clear) golden-brown (clear) light brown (opaque) light orange (opaque) medium brown (opaque) light yellow (clear) transparent yellow light brown (opaque) golden-brown (opaque) dark brown (clear) medium brown (clear) dark brown (clear) golden-yellow (clear) dark brown (clear) light brown (opaque) golden-brown (opaque) Dalmatia, coastal Croatia golden-brown (clear) 4.49 4.00 4.52 3.98 4.09 4.27 5.05 4.56 4.89 4.56 4.21 5.06 4.95 4.81 5.05 4.29 4.48 5.10

1.

Jerusalem thorn honey

2007.

2.

Sunflower honey

2006.

3.

Sage honey

2007.

4.

Velebit winter savory honey

2007.

5.

Winter savory honey

2007.

6.

Amorpha honey

2007.

7.

Chestnut honey

2007.

8.

Linden honey

2007.

9.

Acacia honey

2007.

10.

Oilseed rape honey

2007.

11.

Goldenrod honey

2006.

Heterofloral

12.

Velebit forest honey

2007.

13.

Honeydew honey

2007.

14.

Fruit flower honey

2005.

15.

Mountain meadow honey

2007.

16.

Velebit meadow honey

2007.

17.

Queen of honey

2007.

18.

Continental meadow honey

2005.

Special

19.

Sage + propolis honey

2007.

20.

Bronchial honey

2007.

21.

Flower-mix honey + sour cherry juice

2007.

22.

Flower-mix honey + lemon juice

2007.

23.

Meadow honey + blackberry juice

2006.

24.

Meadow honey + shiitake mushroom

2007.

25.

Green honey

2007.

Piljac-egarac, Stipevi, Belak

26.

Lavender honey

2007.

Antioxidant properties of honey preparations

47

70 DPPH 60 50 ABTS

mg AAE / 100 g

40 30 20 10 0

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

Monofloral honeys

Heterofloral honeys

Special honey preparations

Fig. 1. Ascorbic acid equivalent (AAE) antioxidant capacity determined for 18 honeys and 8 honey preparations of various floral origin using DPPH and ABTS+ radical scavenging assays. Correlation between employed methods Correlations between the antioxidant capacity measures obtained by various techniques are shown in Table 3. The correlation matrix shows that significant linear correlation exists between the results of all five analytical methods employed. The colour intensity measure whose correlation factor r ranged from 0.795-0.820 between methods exhibited the lowest linear correlation, but still highly significant, while the r determined between all other methods exceeded 0.865. Slovenian honey samples, 71.0 478.5 m Fe(II) (Bertoncelj et al., 2007). However, the mean FRAP value (120.0 m Fe(II)) for our analysed set of 18 monofloral and heterofloral honeys is exceeded 3.8-fold by the mean FRAP value (458.24 m Fe(II)) obtained for a set of honeys analysed by Beretta et al. (2005). When the mean reducing capacities were compared among the two honey subsets, heterofloral honeys had a significantly higher (p < 0.05) mean reducing capacity (157.66 m Fe(II)), in comparison to the mean reducing capacity of monofloral honeys (82.31 m Fe(II)). None of the individual honey categories could be identified as exhibiting an overall darker or more intense coloration. The brightest, almost colourless, were acacia and amorpha honeys, while the darkest included flower-mix honey with added sour cherry juice and meadow honey with added shiitake mushrooms. The wide range of observed honey colours was directly reflected in the values of the ABS450 parameter, which was used to define the colour intensity of a 50% honey solution (w/v). The observed range of net absorbance (25 1058 mAU) for our honeys and honey preparations is comparable to the colour intensity of various honeys reported by other authors (Beretta et al., 2005, Mendiola et al., 2008). The ABS450 parameter may be interpreted as a reliable index of the presence of pigments with antioxidant activity such as carotenoids and certain flavonoids (Antony et al., 2000). It has been previously determined by other authors that a class of flavonoids present in functional drinks, the anthocyanins (Mendiola et al., 2008), as well as carotenoids (Furr, 2004) have absorption maxima at 450 nm. DPPH and ABTS+ radical scavenging The observation that the mean AAE value determined in the ABTS assay (29.89 mg AAE/100 g) is significantly higher (46.7%) than the mean AAE determined in the DPPH assay is justified by the fact that DPPH radical reacts only with lipophilic antioxidants while ABTS+

Discussion
Polyphenol content, reducing capacity and colour intensity The sources of analysed honey samples as well as floral origin differed to a large extent, as shown in Table 1. Of 18 representative honey samples, 11 were monofloral and 7 were heterofloral. The 8 additional honey preparations were special preparations with different additives intended to increase the health benefits of honey consumption. The mean content of TP obtained for our samples is in good agreement with the TP content of honeys from various floral sources reported in the literature (Beretta et al., 2005; Gheldof et

al., 2002). In these three studies the greatest TP content was

reported for strawberry tree honey (78.96 mg GAE/100 g) and honeydew honey (114.75 mg GAE/100 g), which is by 12.8% lower and 26.6% higher, respectively, than our highest ranking Velebit forest honey (90.57 mg GAE/100 g). The FRAP results showed a greater difference in the antioxidant capacity of various honeys in comparison to the difference in the TP content evaluated by the Folin-Ciocalteu assay. The observed range of FRAP values (459.21 m Fe(II) to 12.06 m Fe(II)) is comparable to the reducing capacity range of raw Millefiori honey, 61.75 124.5 m Fe(II) (Blasa et al., 2006) and seventy

48

Piljac-egarac, Stipevi, Belak

Table 2. Colour intensity (ABS450), total phenol (TP) content with respect to gallic acid equivalents (GAE) and ascorbic acid equivalents (AAE), and ferric reducing antioxidant power (FRAP) of analysed honeys and honey preparations.

Sample no. Monofloral 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. MEAN Heterofloral 12. 13. 14. 15. 16. 17. 18. MEAN Special 19. 20. 21. 22. 23. 24. 25. 26. MEAN

ABS450 (mAU) 274 381 397 236 236 25 268 182 51 211 287 603 359 203 178 440 259 480 187 169 1058 85 585 780 68 381 -

TP (mg GAE/100 g SD) 48.58 0.95 54.63 1.20 55.40 1.14 44.43 2.83 44.17 2.24 25.66 0.99 43.09 2.68 40.88 1.05 21.61 0.63 36.92 2.53 49.24 2.02 42.24 90.57 2.73 56.22 2.05 20.20 2.08 45.85 1.77 65.76 0.77 81.11 1.98 51.55 1.19 58.75 36.71 0.55 19.18 1.37 85.02 1.25 21.05 0.67 64.61 1.01 66.54 1.69 12.64 0.65 44.09 2.11 43.73

TP (mg AAE/100 g SD) 62.77 1.23 70.57 1.59 71.55 1.47 57.35 3.65 57.08 2.88 33.14 1.32 55.57 3.39 52.80 1.37 27.92 0.84 47.65 3.28 63.60 2.60 54.55 116.97 3.56 72.61 2.62 26.02 2.60 59.20 2.28 84.94 1.04 104.73 2.59 66.55 1.53 75.86 47.42 0.69 24.81 1.78 109.81 1.62 27.16 0.86 83.45 1.33 85.95 2.23 16.33 0.85 56.93 2.69 56.48

FRAP (M Fe(II) SD) 113.49 2.91 113.81 9.72 121.27 4.70 118.57 4.54 99.68 3.99 23.02 2.79 84.60 2.62 73.81 6.75 12.06 1.98 52.22 6.14 92.86 1.65 82.31* 329.68 11.80 134.44 8.94 22.86 5.37 73.02 8.96 144.60 10.88 262.54 6.69 136.51 5.72 157.66* 109.52 6.62 44.76 6.08 459.21 20.55 68.25 6.21 161.11 8.94 141.43 13.74 18.25 5.72 136.83 2.15 142.42

A 95% confidence interval was applied in testing for significant differences between the mean values obtained for subsets. An asterisk denotes a significant difference between the two compared values (p < 0.05).

radical reacts with both hydrophilic and lipophilic antioxidants (Prior

honeys whose AAE values for 27 analysed samples ranged from 10.20 mg AAE/100 g (multifloral) to 65.86 AAE/100 g (Vitellaria) with a mean of 27.04 mg AAE/100 g. When mean ABTS+ and DPPH radical scavenging capacities are compared between honey subsets, heterofloral honeys once again performed the best exhibiting a mean scavenging potential of 35.27 mg AAE/100 g and 16.66 mg AAE/ 100 g, respectively. Significant difference was observed between the mean ABTS+ radical scavenging potential of monofloral and heterofloral honey substets (p < 0.05). The reasons behind the markedly higher radical scavenging

et al., 2005).
In evaluating the radical scavenging potential of honey solutions, the DPPH radical reaction (Meda et al., 2005; Beretta et

al., 2005; Baltrusaityt et al., 2007; Turkem et al., 2006) is used

more often than the ABTS+ radical reaction (Baltrusaityt et al., 2007). Considering the varying reaction conditions employed by different authors, it is difficult to make direct comparisons of radical scavenging capacities of honeys with available literature data. Our reaction conditions in the DPPH assay were the closest to those

Antioxidant properties of honey preparations

49

ABS450 ABS450 TP FRAP DPPH ABTS 0.795 0.820 0.820 0.795

TP

FRAP

DPPH

ABTS

To summarise, the employed analytical techniques have been validated on a large set of various floral origin honeys and indicate that the TP content may be considered a significant determinant of the antioxidant capacity of studied honeys, both their reducing ability as well as radical scavenging potential.

0.873 0.880 0.899 0.958 0.886 0.865

Conclusions
This is the first study reporting the results of a representative screening of Croatian honeys for antioxidant capacity, conducted on a sample set of 18 honeys and 8 special honey preparations. On the basis of employed analytical methods, the tested honeys may be considered easily accessible natural sources of antioxidants and valuable additions to everyday diet. Taken with fruit tea, they get transformed to functional fruit drinks. Although special honeys with the addition of various fruit/vegetable extracts and propolis were initially presumed to have the best antioxidant properties, the results have shown that heterofloral honeys are characterized by the highest mean TP content (58.75 mg GAE/100 g), the highest mean reducing capacity (57.66 mM Fe(II)) and the best mean radical scavenging properties with respect to both DPPH (16.66 mg AAE/100 g) and ABTS+ radicals (35.27 mg AAE/100 g) in the analyzed set of honey samples. From the consumers point of view, caution should be exercised in choosing honeys, since those often advertised as containing potent antioxidants may in fact contain additives that do not improve their antioxidant properties. In continuation of this work, a study aimed at direct comparison of the antioxidant properties of monofloral honeys with and without additives is planned.

Table 3. Correlation matrix between the results of employed assays for 18 honeys and 8 honey preparations. capacity, as well as ferric reducing antioxidant power, exhibited by heterofloral honeys probably lie in their diverse botanical origin. Antioxidant potential of honey is directly related to its plant source, and in our case, honeys derived from several floral sources performed better in all the antioxidant potency assays. However, a sample set of 18 honeys is too small the make the generalisation that heterofloral honeys exhibit overall better antioxidant properties. Among the special honey preparations, flower-mix honey with added sour cherry juice performed the best in the FRAP, DPPH and ABTS assays, which may be attributed to the high antioxidant capacity of the added sour cherry juice. It has been found that sour cherries contain two to three times more anthocyanins than sweet cherries (Kim et al., 2005) and that they rank 14th among the top 50 foods with the highest antioxidant content per serving size, surpassing red wine, prunes, dark chocolate, and orange juice (Halvorsen et al., 2006). Correlations Similar to our findings, Bertoncelj et al. (2007) and Beretta et al. (2005) also reported strong correlation between the antioxidant capacity according to FRAP and net absorbance (r = 0.853 and 0.918, respectively) indicating that honey colour intensity may be treated as a good initial indicator of its antioxidant capacity. Our results point to the highest linear correlation between the DPPH and FRAP assay results with r = 0.958, which is close to the correlation coefficient (r = 0.889) between these two assays reported by Beretta et al. (2005). Other authors also reported highly significant linear correlation between TP and FRAP measures, for example r = 0.869 (Aljadi and Kamaruddin, 2004) and r = 0.938 (Blasa et al., 2006), which is in agreement with our findings, that r = 0.873. This suggests that phenolics are the major components responsible for the antioxidant effects of honey. In fact, the positive correlation found between the two applied radical scavenging assays (r = 0.865) indicates that polyphenols are responsible for the radical scavenging properties of honey as well. Baltrusaityt et al. (2007) also reported strong correlation between the ABTS and DPPH assay results (r = 0.716).
2

Acknowledgments
This research has been financially supported by the TEST program technological research and implementation project no. E38/2005 financed by the Croatian Ministry of Science, Education and Sports. We are grateful to Antun Karlovi for valuable help in sample collection.

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