Food Chemistry 113 (2009) 568–574
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Antioxidant activity and phenolic composition of herbhoneys Robert Socha, Lesław Juszczak *, Sławomir Pietrzyk, Teresa Fortuna Department of Analysis and Evaluation of Food Quality, University of Agriculture, Balicka 122 Street, 30-149 Krakow, Poland
a r t i c l e
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Article history: Received 21 March 2008 Received in revised form 4 June 2008 Accepted 11 August 2008
Keywords: Herbhoneys Antioxidant activity Phenolic content Flavonoid composition
a b s t r a c t A study of 10 herbhoneys of various origin revealed differences in their antioxidant activity and profiles of phenolic acids and flavonoids. The total phenolic content of herbhoneys determined spectrophotometrically varied between 21.7 and 75.3 mg gallic acid equivalents per 100 g product, and the total flavonoid content ranged from 6.9 to 28.5 mg quercetin equivalents per 100 g. Dark herbhoneys, such as raspberry, thyme, hawthorn and black chokeberry, exhibited a high antioxidant activity and contained high total levels of polyphenols and flavonoids. There was a significant linear correlation between total phenolic and flavonoid contents and antioxidant activity in the reactions with DPPH and ABTS+ free radicals. The profiles of phenolic acids and flavonoids determined by HPLC depended on the variety of herbhoney. Among the products studied, raspberry and thyme herbhoneys were the richest in phenolic acids and flavonoids. The dominant phenolic acid in most samples was p-coumaric acid; the herbhoneys contained also a considerable amount of gentisic acid. The dominant flavonoids were hesperetin and naringenin. Thyme herbhoney had an especially high quercetin content. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Herbhoney is a honey-like product obtained from bees fed on a saccharose-based food containing herbal extracts or fruit juices. The great variety of potential sources of such plant-derived additives makes it possible to give specific sensory characteristics (colour, aroma, taste) to herbhoneys, and thus considerably expand the commercial range of bee products. Herbhoneys have the medicinal properties of herbs or other raw materials contained in the food fed to bees, which makes them usable in pharmacy and medicine, both as drug components, prophylactic agents and diet supplements. The basic chemical composition of herbhoneys is similar to that of natural honeys and most of them meet the quality criteria concerning acidity, water content, hydroxymethylfurfural content and _ diastase number, applied to the latter (Juszczak, Socha, Roznowski, Fortuna, & Nalepka, 2008). Due to a different method of production, and especially a high availability of such biologically active substances as antioxidants in the food fed to bees, herbhoneys may somewhat differ from natural honeys in the profile of phenolic acids and flavonoids. Being natural antioxidants, these compounds play a major role in human nutrition. Phenolic acids and flavonoids may also be used as biomarkers for the origin of a honey (TomásBarberán, Martos, Ferreras, Radovic, & Anklam, 2001). It should be taken into account, however, that the levels of individual phenolic compounds are also closely connected with the climatic conditions of an area (Kenjeric´, Manic´, Primorac, Bubalo, & Perl, 2007; Cˇeksteryte˙, Kazlauskas, & Racˇys, 2006). * Corresponding author. Fax: +48 12 6624746. E-mail address:
[email protected] (L. Juszczak). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.029
The characteristic polyphenols present in honeys that are able to perform the role of biomarkers are flavonoids such as hesperetin, kaempferol, quercetin and chrysin, and phenolic acids: abscisic, ellagic, p-coumaric, gallic and ferulic (Ferreres, Andrade, & TomásBarberán, 1994; Kenjeric´ et al., 2007; Soler, Gil, Garcia-Viguera, & Tomás-Barberán, 1995; Tomás-Barberán et al., 2001; Yao et al., 2003; Yaoa, Jiang, Singanusong, Datta, & Raymont, 2005). The present study focuses on herbhoneys of various origins with the aim of investigating their antioxidant properties and phenolic acid and flavonoid profiles.
2. Materials and methods 2.1. Materials The study used 10 herbhoneys: aloe, black chokeberry, camomile, hawthorn, marigold, mint, nettle, pine, raspberry and thyme, produced in 2006 and supplied by P.P. Apipol (Krakow, Poland). The raspberry herbhoney sample came from an experimental apiary, the rest were commercial samples. All samples were stored in a refrigerator at 4° C. Immediately before analyses they were heated at a temperature below 40° C to dissolve crystals. 2.2. Determination of antioxidant activity in the reaction with DPPH (1,1-diphenyl-2-picrylhydrazyl) radical The antioxidant activity of herbhoneys was determined using a procedure described by Turkmen, Sari, and Velioglu (2006). Onegram samples of herbhoneys were dissolved in 5 ml of distilled
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water, centrifuged at 4350g, and filtered through paper filters. Then, 0.5 ml of the solutions were mixed with 1.5 ml of 0.1 mM methanol solution of DPPH (Sigma–Aldrich, Steinheim, Germany). The control test was made with distilled water in place of herbhoney solution. The reaction mixtures were vortex-mixed and left in the dark at room temperature for incubation during 60 min. Absorbance was measured at k = 517 nm against methanol, using a UV/Vis V-530 spectrophotometer (Jasco, Tokyo, Japan). The antioxidant activity of methanolic extracts of herbhoneys against DPPH was determined analogically. In this case 0.5 ml of the solution containing 100 ll of methanolic extract and 400 ll of methanol was applied instead of the water solution of herbhoney. Antioxidant activity was expressed as a percent inhibition of DPPH radical, and calculated from the equation:
AA½% ¼ ðAbscontrol Abssample Þ=Abscontrol 100 2.3. Determination of antioxidant activity in the reaction with ABTS+ cation radical The antioxidant activity of herbhoneys was determined in accordance with Baltrusaityte˙, Venskutonis, and Ceksteryte˙ (2007). ABTS+ was obtained in the reaction of a 2 mM stock solution of 2,20 -azino-di(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma–Aldrich, Steinheim, Germany) in phosphate-buffered with potassium persulfate (POCh, Gliwice, Poland). The mixture was left to stand for 24 h. Prior to analysis, the ABTS+ solution was diluted with phosphate buffer to produce a solution with an absorbance of 0.80 ± 0.03 at k = 734 nm. Onegram samples of herbhoneys were diluted with distilled water to 5 ml, centrifuged at 4350 g, and filtered through paper filters. Then, 100 ll of a herbhoney solution was mixed with 6 ml of the ABTS+ cation radical solution and after 15 min absorbance was measured at a wavelength of 734 nm by using a UV/Vis V-530 spectrophotometer (Jasco, Tokyo, Japan). The antioxidant activity of methanolic extracts of herbhoneys in the reaction with ABTS+ was determined analogically. In this case 50 ll of methanolic extracts was applied instead of the water solution of herbhoney. Antioxidant activity was expressed as a percent inhibition of ABTS+, calculated from the same equation as for DPPH. 2.4. Determination of total phenolic content To determine the total phenolic content of herbhoneys, the method described by Meda, Lamien, Romito, Millogo, and Nacoulma (2005) was employed. Herbhoney solutions with a concentration of 5 g/50 ml were centrifuged at 4350 g and filtered through paper filters. After that, 0.5 ml of the obtained solution was mixed with 2.5 ml of 0.2 N solution of Folin–Ciocalteau reagent (Sigma–Aldrich, Steinheim, Germany), and then 2 ml of sodium carbonate solution (POCh, Gliwice, Poland) was added. Following incubation for 2 h, absorbance of the reaction mixture was measured at k = 760 nm against a methanol blank using a UV/Vis V-530 spectrophotometer (Jasco, Tokyo, Japan). The standard curve was produced for gallic acid (Sigma–Aldrich, Steinheim, Germany) within the concentration range from 0 to 200 mg/100 ml. The total phenolic content was expressed as gallic acid equivalents in mg/100 g of herbhoney sample.
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filters. To 1 ml of the solution, 4 ml of distilled water and 0.3 ml of sodium nitrate(III) (POCh, Gliwice, Poland) solution with 15 g/100 ml concentration were added. This was followed by adding 0.3 ml of aluminum chloride (POCh, Gliwice, Poland) solution (10 g/100 ml) and then 4 ml of sodium hydroxide (POCh, Poland) solution (4 g/100 ml). The whole was made up to 10 ml with distilled water, stirred and left to stand for 15 min. The total flavonoid content was calculated on the basis of the standard curve for quercetin solutions (Sigma–Aldrich, Germany) and expressed as quercetin equivalents in mg/100 g of herbhoney sample. 2.6. Extraction and analysis of phenolic compounds Phenolic compounds were extracted by using ethyl acetate as extracting agent (Wahdan, 1998). Herbhoney solutions with a concentration of 10 g/50 ml were acidified with HCl solution to reach pH 2, and then saturated with sodium chloride (POCh, Gliwice, Poland) in the amount of 3 g/10 ml. The resulting solutions were extracted with three portions of ethyl acetate (POCh, Gliwice, Poland): one of 50 ml and two of 25 ml each. The extracts were combined, and ethyl acetate was vacuum-evaporated at 40° C under argon atmosphere. The dry residue was dissolved in 5 ml of methanol (POCh, Gliwice, Poland) and stored at 18° C. Prior to the chromatographic analysis, the extracts were purified by using Millex-LCR syringe filters (PTFE). The recovery of phenolic compounds for such extraction was from 82% for chlorogenic acid to 106% for gallic acid. Chromatographic analyses of phenolic compounds were made by high-performance liquid chromatography (HPLC–LaChrom, Hitachi, Tokyo, Japan). Phenolic acids such as caffeic, chlorogenic, pcoumaric, ferulic, gallic, sinapic and syringic acid were detected at k = 280 nm, while gentisic acid and flavonoids (chrysin, galangin, hesperetin, kaempferol, naringenin and quercetin) were determined at k = 330 nm. Gradient elution was conducted at a flow rate of 1 ml/min using a solution of acetic acid (POCh, Gliwice, Poland), 2.5 g/100 ml, and acetonitrile (Merck, Darmstadt, Germany) as a mobile phase. The solvent system was a linear gradient from 3% of acetonitrile then increased to 8% for 10 min, this was increased to 15% at 20 min, 20% at 30 min, 30% at 40 min and 40% at 50 min. Finally, the column was eluted isocratically with acetonitrile before the next injection. The compounds studied were separated on a RP18 Lichrosorb column (250 4.5 lm) (Merck, Darmstadt, Germany) at a temperature of 25° C. Quantification of individual phenolic acids and flavonoids was based on comparison with standards from Sigma– Aldrich (Steinheim, Germany) and Fluka Chemie AG (Buchs, Switzerland). 2.7. Statistical analysis For the estimation of the significant of differences between the means, a one-way analysis of variance and the test of least significant differences (LSD) at p = 0.05 was applied. Calculations were performed with statistical software package Statistica 8.0 (StatSoft Inc. Tulsa, USA).
3. Results and discussion
2.5. Determination of total flavonoid content
3.1. Antioxidant activity
The total flavonoid content of herbhoneys was established in the reaction with aluminum chloride using the methods described by Zhishen, Mengcheng, and Jianming (1999), Ardestani and Yazdanparast (2007) Herbhoney solutions with a concentration of 0.2 g/ml were centrifuged at 4350g and filtered through paper
Figs. 1 and 2 show the antioxidant activity of herbhoney solutions in the reactions with DPPH and ABTS+ radicals, respectively. For the former, the values of this parameter varied between 27.2% (camomile herbhoney) and 86.8% (hawthorn herbhoney), whereas for the latter they ranged from 21.9% (camomile herbhoney) to
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80 60 40 20
e th ym
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Fig. 2. Antioxidant activity of herbhoney solutions in reaction with ABTS+ cation radical (LSD0.05 = 2.31).
79.2% (raspberry herbhoney). Dark red herbhoneys (raspberry, black chokeberry) and intensely green herbhoney (thyme) tended to be highly active in the reaction involving DPPH. The highest antioxidant activity in the reaction with ABTS+, more than twice that of most samples, was exhibited by raspberry herbhoney. The other samples with dark red (black chokeberry) or vividly green colours (pine and thyme) were much less active. In both reactions, pale yellow–green herbhoneys (camomile, mint, and aloe) showed the lowest antioxidant activity. A relationship between colour parameters and antioxidant activity in natural Slovenian honeys was observed by Bertoncelj, Doberšek, Jamnik, and Golob (2007), who found light honeys (e.g., acacia) to be least active and dark honeys to be most active against DPPH. The linear correlation between the results of the two assays (R = 0.61) was significant at p = 0.05. A somewhat stronger linear correlation between the antioxidant activities determined with DPPH and ABTS+ was found by Baltrusaityte˙ et al. (2007). The honeys examined by the latter authors displayed a greater antioxidant activity for ABTS+. The differences in the antioxidant activities between the DPPH and ABTS+ assays, observed in the present study, may be attributed to the different concentrations of the substrates (herbhoney samples) on the one hand, and to the different kinetics of the two reactions, on the other hand (Baltrusaityte˙ et al., 2007).
100 80 60 40 20 0
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Fig. 1. Antioxidant activity of herbhoney solutions in reaction with DPPH radical (LSD0.05 = 2.02).
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The antioxidant activity of natural herbhoneys depends largely on their chemical composition, i.e., phenolic and flavonoid content, hence on their origin. Antioxidant activity is also affected by the processing (e.g., heating) and storage method of the material. As shown by Turkmen et al. (2006), the antioxidant activity of natural honeys rises when the temperature and time of heating are increased. In natural honeys, antioxidant activity is due to the presence of many various substances such as enzymes, organic acids, amino acids, Maillard reaction products, phenolic compounds, flavonoids, tocopherols, catechins, ascorbic acid, and carotenoids (Meda et al., 2005). To produce herbhoneys, bees receive food containing plant extracts or fruit juices. Since antioxidant substances or other bioactive components are present in higher concentrations in such a food, they may be more easily available to bees. In addition, the total antioxidant activity of herbhoneys may partly be due to substances that do not occur, or occur in tiny amounts in natural honeys. Baltrusaityte˙ et al. (2007) showed that honeys obtained using plant (birch, pine and nettle) extracts have a higher antioxidant activity and contain much more apigenin than natural honeys. The antioxidant activity of methanolic extracts of herbhoneys in the reaction with DPPH and ABTS+ is shown in Figs. 3 and 4. The highest antioxidant activity in the reaction with DPPH was exhibited by the extracts obtained from thyme, raspberry and hawthorn herbhoneys, analogically to the water solutions of herbhoneys. The significantly lower activity was exhibited by the extract of black chokeberry herbhoney, that may be attributed to the significant participation in antioxidant activity of compounds which were not extracted under the employed conditions. The lowest antioxidant activity in the reaction with DPPH was exhibited by the extract from aloe herbhoney, where water solution also exhibited low activity in this reaction. A significant linear correlation was observed (R = 0.91) between the antioxidant activity against DPPH, which was carried out using water solutions of herhoneys and their methanolic extracts. Analogically to the water solutions of herbhoneys, the highest antioxidant activity against ABTS+ was exhibited by extracts obtained from raspberry, hawthorn, and thyme herbhoneys, whereas the extract from aloe herbhoney characterized the lowest activity. In the reaction with ABTS+ the correlation between the results for the water solutions and methanolic extracts of herbhoneys was lower (R = 0.79), but statistically significant (p = 0.05). Whereas, considerably higher linear correlation (R = 0.96) was observed between the reactions of methanolic extracts of herbhoneys against DPPH and ABTS+, in comparison to the water solutions of herbhoneys.
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570
Fig. 3. Antioxidant activity of herbhoney extracts in reaction with DPPH radical (LSD0.05 = 3.22).
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30
Quercetin equivalents [mg/100g]
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Fig. 4. Antioxidant activity of herbhoney extracts in reaction with ABTS+ cation radical (LSD0.05 = 5.91).
Fig. 6. Total flavonoid content of herbhoneys expressed as quercetin equivalent (LSD0.05 = 1.58).
3.2. Total phenolic and flavonoid content
Meda et al. (2005) observed a significant relationship between proline content and antioxidant activity. Many authors report that total phenolic content is strongly linearly correlated with antioxidant activity (Buratti, Benedetti, & Cosio, 2007; Meda et al., 2005). In the present study, a significant (p = 0.05) linear correlation was observed between total phenolic content and antioxidant activity of herbhoney solutions and extracts against DPPH (R = 0.73, R = 0.70) and ABTS+ (R = 0.96, R = 0.81), respectively. The total flavonoid content of herbhoneys, expressed as quercetin equivalent, was the highest for raspberry herbhoney (28.5 mg/100 g; Fig. 6). Hawthorn, thyme and black chokeberry herbhoneys also contained a considerable amount of flavonoids. This confirms the results indicating a high antioxidant activity of those products in the reactions with DPPH and ABTS+ (Figs. 1–4). The flavonoid content was lowest for camomile herbhoney (6.9 mg/100 g). Again, flavonoid content appeared to be closely connected with colour; herbhoneys with strong red or green colours (raspberry, black chokeberry, thyme) had a high flavonoid content, while those with pale yellow or pale green colours (camomile, mint) contained a small amount of these compounds. The total flavonoid content determined in this study is significantly higher than the values reported for natural honeys (Blasa et al., 2006; Meda et al., 2005). This may be due to the fact that the food fed to bees in the production of herbhoneys constitutes a richer source of flavonoids; therefore, the products obtained in such a way have a much higher flavonoid content than natural honeys. In addition, intensely red herbhoneys, such as raspberry and black chokeberry, may also contain anthocyanin pigments. Although the determination of phenolics by using the Folin– Ciocalteu reagent and the determination of flavonoids by using aluminum chloride are based on different mechanisms of the reaction, and the reactants exhibit different affinities to individual substrates, a significant (p = 0.05) linear correlation (R = 0.83) between total phenolic content and total flavonoid content was observed in the present study. There was also a significant (p = 0.05) linear correlation between total flavonoid content and antioxidant activity of herbhoney solutions and extracts: R = 0.70, R = 0.90 for ABTS+, and R = 0.95, R = 0.91 for DPPH, respectively. Blasa et al. (2006) report that total flavonoid content is strongly linearly correlated with antioxidant activity.
The total phenolic content of herbhoneys, expressed as gallic acid equivalent, ranged from 21.7 mg/100 g for the camomile sample to 75.3 mg/100 g for the raspberry product (Fig. 5). Being in general within the broad range reported in the literature, these values often were even higher than those of natural honeys (Al-Mamary, Al-Meeri, & Al-Habori, 2002; Aljadi & Kamaruddin, 2004; Baltrusaityte˙ et al., 2007; Bertoncelj et al., 2007; Blasa et al., 2006; Küçük et al., 2007; Meda et al., 2005). As with antioxidant activity, the total levels of phenolic compounds were highest in dark red herbhoneys (raspberry, black chokeberry) and intensely yellow–green ones (hawthorn, thyme), and lowest in light-coloured products (camomile, mint). This supports the observations made by others on the correlation between the phenolic content and antioxidant activity of natural honeys and their colour (Bertoncelj et al., 2007). In particular, Meda et al. (2005) found that dark honeys, such as honeydew ones, have a higher phenolic content. It should be taken into account, however, that herbhoneys may somewhat differ from natural honeys in the profile of compounds reducing the Folin–Ciocalteu reagent, among them phenolics. Besides, they may contain other compounds, e.g., amino acids, that contribute to the total antioxidant activity of the product. A significant correlation between the levels of individual amino acids and antioxidant activity was found by Pérez, Iglesias, Pueyo, González, and de Lorenzo (2007) for natural Spanish honeys. Also
Gallic acid equivalents [mg/100g]
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3.3. Profile of phenolic acids and flavonoids
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Fig. 5. Total phenolic content of herbhoneys expressed as gallic acid equivalent (LSD0.05 = 2.50).
The levels of individual phenolic acids in herbhoneys are specified in Table 1. Among the phenolic acids, p-coumaric acid was present in the largest amounts, followed by gentisic acid; the
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Table 1 Phenolic acid content of herbhoneys Herbhoney variety
Caffeic acid (lg/100 g)
Aloe Black chokeberry Hawthorn Raspberry Mint Marigold Nettle Camomile Pine Thyme
10.1a 118.9b 517.4 35.8c 25.7c 106.7b 20.4a 35.5c 27.7c 52.0
Chlorogenic acid (lg/100 g) 13.7a 336.4 24.9a
p-Coumaric acid (lg/100 g)
Ferulic acid (lg/100 g)
Gallic acid (lg/100 g)
Gentisic acid (lg/100 g)
Sinapic acid (lg/100 g)
794.6a 446.4b 361.0b 3676.7 240.8c 316.7b 350.8b 199.6c 629.7a 1141.9
112.8a 41.4b 113.2a 55.6b 61.3b 13.2c 18.3c 46.3b 22.4c 178.6
39.3a,b 111.3b,c 16.3a 1126.0 92.7c 74.3b,c 61.0a 12.1a 153.3c 23.1a
189.2a 229.2a,b 1012.8 136.8a 1645.8 576.9 123.3a 337.8b 204.6a 142.3a
44.6a 274.9 24.9b 24.3b 104.6 23.6b 43.4a 48.6a
Syringic acid (lg/100 g) 7.8a 58.8
14.4a 1.4 9.7a 119.0
Means in the same columns with the same superscript letters are not significantly different at p = 0.05.
syringic and chlorogenic acid contents were lowest. Raspberry herbhoney proved the richest in phenolic acids (5082 lg/100 g in total), followed by hawthorn herbhoney, while nettle and camomile products were poorest in this respect. This corresponds with the high antioxidant activity of raspberry and hawthorn herbhoneys in the DPPH (Fig. 1) and ABTS+ (Fig. 2) reactions and their high phenolic content (Fig. 3), on the one hand, and the low values of these parameters for nettle and camomile products, on the other. Chlorogenic acid, which was found only in three herbhoneys, reached its highest level in hawthorn herbhoney. It was also present in black chokeberry herbhoney. The presence of small amounts of chlorogenic acid in the latter has been mentioned in the literature (Oszmian´ski & Wojdyło, 2005). The chlorogenic acid was also determined in Australian and New Zealand honeys (Yao et al., 2003; Yaoa et al., 2005). Mint and raspberry herbhoneys proved rich in gentisic acid, and thyme herbhoney in ferulic acid. The gallic acid level was highest for raspberry herbhoney which contained 10 times more acid than most of the other products. Gallic acid has already been identified in natural honeys. Aljadi and Yusoff (2003) found its levels to range from 82 to 330 lg/100 g in Malaysian honeys, whereas Yaoa et al. (2005) determined much higher values in Australian honeys. The latter authors established that gallic acid is the main component of the phenolic acid profile and a potential marker of the origin of a honey. Yao et al. (2003) found gallic acid to be the dominant acid in some Australian and New Zealand honeys and observed a high level of abscisic acid in the natural honeys they examined. In the present study, significant (p = 0.05) linear correlations were observed between gallic acid content and total phenolic content (R = 0.88), and between the former and the antioxidant activity of herbhoney solutions against ABTS+ (R = 0.93). The amount of ferulic acid in most herbhoneys was at the level reported by Tomás-Barberán et al. (2001) for natural European honeys (20–100 lg/100 g), but in thyme herbhoney it was higher. Hawthorn herbhoney exhibited the highest caffeic acid content that was more than five times higher than the level found in most of the other products which were similar to European honeys in this respect. To compare, Tomás-Barberán et al. (2001) report the levels of caffeic acid in European honeys of different origin to vary between 20 and 160 lg/100 g, with the highest value being found for sunflower honey. The p-coumaric acid content was largest in raspberry herbhoney (more than 10 times larger than in most of the samples). Also thyme herbhoney had a high p-coumaric acid content which, however, was more than three times lower than in raspberry herbhoney. The fact that among the acids under study p-coumaric acid showed highest levels in 9 of the 10 products suggests that this acid is characteristic of the herbhoneys studied. A high level of pcoumaric acid was also observed by Baltrusaityte˙ et al. (2007) in natural honeys and in honeys with plant extracts; the level was
the highest in those with birch extracts. Compared to our results, the p-coumaric acid contents of various natural European honeys, established by Tomás-Barberán et al. (2001), are much lower (10–180 lg/100 g). The level of p-coumaric acid was found to influence the antioxidant activity and total phenolic content of herbhoneys. There were significant (p = 0.05) linear correlations between p-coumaric acid content and total phenolic content (R = 0.91), and between acid content and antioxidant activity of herbhoneys solutions against ABTS+ (R = 0.92), confirming the findings on the high antioxidant activity of raspberry and thyme herbhoneys in the reactions with DPPH and ABTS+. The high p-coumaric acid content of thyme herbhoney is supported by the literature data on thyme. Namely, Wojdyło, Oszmian´ski, and Czemerys (2007), investigating the antioxidant activity of various herb species observed a high level of this acid in thyme. Sinapic acid reached its highest level in hawthorn herbhoney, and was not observed in two herbhoneys. Syringic acid content was largest in thyme herbhoney, while the acid did not occur in four products. Studies by other authors have also found that syringic acid occurs only in some varieties of honey (Yao et al., 2003; Yaoa et al., 2005). The levels of individual flavonoids determined in herbhoneys are shown in Table 2. The totals were largest for thyme herbhoney, followed by raspberry herbhoney. This corresponds with the high antioxidant activity of both products in the reactions with DPPH and ABTS+ (Figs. 1–4). The two herbhoneys had also a high total flavonoid content (Fig. 6). Aloe herbhoney contained the smallest amount of the flavonoids determined, which corresponds with its relatively low antioxidant activity (Figs. 1–4) and average total flavonoid content (Fig. 4). By contrast, black chokeberry herbhoney, containing a low amount of the determined flavonoids, had a relatively high antioxidant activity (Figs. 1 and 2) and total phenolic content (Fig. 6). In this case, the high antioxidant activity may be connected with the presence of anthocyanin pigments responsible for the dark red colour of the product. The same is true for dark red raspberry herbhoney whose high antioxidant activity and total phenolic content are due to anthocyanin pigments. Of all samples, the latter product contained the largest amount of chrysin (Table 2), while six herbhoneys showed its absence. The presence of chrysin in Australian and New Zealand honeys is rather limited (Yao et al., 2003), but it appears to be the main flavonoid (along with kaempferol and apigenin) of Lithuanian honeys (Baltrusaityte˙ et al., 2007). Kenjeric´ et al. (2007) reported a much larger range of chrysin contents than that found in the present study and noted that chrysin made more than 50% of the total flavonoid content of Robinia honeys from Croatia. They also observed significant differences in flavonoid profiles due to climatic conditions; honeys produced in a year with a sunny and warm (high UV–B radiation) summer season with low rainfalls contained much more flavonoids. The range of chrysin contents determined by Tomás-
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R. Socha et al. / Food Chemistry 113 (2009) 568–574 Table 2 Flavonoid content of herbhoneys Herbhoney variety Aloe Black chokeberry Hawthorn Raspberry Mint Marigold Nettle Camomile Pine Thyme
Chrysin (lg/100 g)
Galangin (lg/100 g)
3.5a 23.3b 104.0
5.8a,b 7.2b
4.7a
2.6a
6.7a 27.9b
7.7b 48.5
Hesperetin (lg/100 g)
Kaempferol (lg/100 g)
Naringenin (lg/100 g)
Quercetin (lg/100 g)
25.7a 10.5 28.5a 212.1 130.9 37.2a,b 172.8 257.8 50.0b 279.9
14.3a 14.5a 102.4b 98.3b 3.94a 95.4b 244.7 61.6c 68.4c 101.9b
2.9a 31.3b 122.7c 252.5 110.5c 24.4b 189.7d 195.3d 2.8a 14.6a,b
21.7a,b 11.6a 32.1a,c 87.5d 43.9b,c 46.7b,c 72.5d 64.3d 20.7a 1199.9
Means in the same columns with the same superscript letters are not significantly different at p = 0.05.
Barberán et al. (2001) for a number of natural European honey varieties (9–415 lg/100 g) was also much broader than that established in herbhoneys in the present study, except for chestnut and citrus honeys whose chrysin levels were similar to our values. A significant (p = 0.05) linear correlation occurred only between chrysin content and antioxidant activity in the ABTS+ reaction of herbhoneys solutions (R = 0.96). Herbhoneys contained also a relatively small amount of galangin that was identified and determined in five samples among which thyme herbhoney was richest. The other herbhoneys had a lower galangin content than Robinia honeys from Croatia (Kenjeric´ et al., 2007). Considerable levels of chrysin and galangin were also observed in Portuguese heather honeys (Ferreres et al., 1994). The amount of hesperetin in herbhoneys was quite large, the largest in thyme herbhoney, followed by camomile nad raspberry. In the thyme and raspberry products, the high level of this flavonoid correlates well with their high antioxidant activity and total flavonoid content. Soler et al. (1995) found hesperetin to be a characteristic flavonoid of French citrus honeys but not of other varieties of French honeys. Kaempferol constitutes one of the main flavonoids occurring in natural honeys and has already been determined by many authors ˇ eksteryte˙ et al., 2006; Ferreres et al., (Baltrusaityte˙ et al., 2007; C 1998; Kenjeric´ et al., 2007; Martos et al., 2000; Soler et al., 1995; Yao et al., 2003). Among the herbhoneys studied, nettle herbhoney contained the highest amount of kaempferol. The second richest products were herbhoneys with a high antioxidant activity, i.e., hawthorn, raspberry and thyme. The levels of this flavonoid were close to those reported by Ferreres et al. (1998) for Spanish rosemary honey. Besides apigenin and chrysin, kaempferol is one of the main flavonoids found in Lithuanian honeys (Baltrusaityte˙ et al., 2007). Our values are similar to those obtained by Yao et al. (2003) for natural Australian and New Zealand honeys, but slightly higher than the levels observed by Kenjeric´ et al. (2007) ˇ eksteryte˙ et al. (2006) for Lithuanian for Croatian honeys and C honeys. Investigating the flavonoid profile of natural European honeys, Tomás-Barberán et al. (2001) noted significant differences in kaempferol contents. Among these honeys, heather and rapeseed ones were especially abundant in kaempferol, much more than our herbhoneys, while accacia honey was poorest in this respect. Another important flavonoid occurring in honeys is quercetin. Soler et al. (1995) found it to be a characteristic flavonoid of sunflower honey. In our studies, the richest herbhoney was the thyme product with its over 20 times higher quercetin content compared to the other herbhoneys studied (Table 2.). This is closely connected with the origin of the herbal extract used in its production. The quercetin content of the other herbhoneys fell within the broad ranges reported by Tomás-Barberán et al. (2001) for natural
European honeys and by Yao et al. (2003) for Australian and New Zealand honeys, and was significantly higher that the quercetin levels determined by Cˇeksteryte˙ et al. (2006) in Lithuanian honeys. All the herbhoneys contained naringenin (Table 2). Its amount was largest in raspberry herbhoney, and smallest in pine and aloe products. In three products (raspberry, hawthorn, and black chokeberry), naringenin was the most abundant flavonoid determined. Whether naringenin occurs in natural honeys or not, has not yet been mentioned in the literature. 4. Conclusion Herbhoneys differed in antioxidant activity and the profiles of phenolic acids and flavonoids. Dark-coloured products, such as raspberry, thyme, hawthorn, and black chokeberry herbhoneys, showed high antioxidant activities (for both free radicals) and total phenolic and flavonoid contents. There were significant linear correlations between these parameters. The phenolic acid and flavonoid profiles depended on the kind of product, with raspberry and thyme herbhoneys being richest in phenolic acids and flavonoids. Among the phenolic acids, p-coumaric acid prevailed in most herbhoneys. The products contained also a considerable amount of gentisic acid. Among the flavonoids determined in these studies, hesperetin and naringenin were dominant. Thyme herbhoney contained an especially large amount of quercetin. To determine the whole profile of polyphenols in herbhoneys, it would be necessary to undertake further research considering anthocyanin pigments and other biologically active compounds characteristic of the herbs or fruit juices used in herbhoney production. In herbhoneys, such compounds may have a significant effect on antioxidant activity and total phenolic and flavonoid contents References Aljadi, A. M., & Yusoff, K. M. (2003). Isolation and identification of phenolic acids in Malaysian honey with antibacterial properties. Turkish Journal of Medical Sciences, 33, 229–236. Aljadi, A. M., & Kamaruddin, M. Y. (2004). Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry, 85, 513–518. Al-Mamary, M., Al-Meeri, A., & Al-Habori, M. (2002). Antioxidant activities and total phenolics of different types of honey. Nutrition Research, 22, 1041–1047. Ardestani, A., & Yazdanparast, R. (2007). Antioxidant and free radical scavenging potential of Achillea santolina extracts. Food Chemistry, 104, 21–29. Baltrusaityte˙, V., Venskutonis, P. R., & Ceksteryte˙, V. (2007). Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chemistry, 101, 502–514. Bertoncelj, J., Doberšek, U., Jamnik, M., & Golob, T. (2007). Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chemistry, 105, 822–828. Blasa, M., Candiracci, M., Accorsi, A., Piacentini, M. P., Albertini, M. C., & Piatti, E. (2006). Raw Millefiori honey is packed full of antioxidants. Food Chemistry, 97, 217–222.
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