Polyphenolic content and antioxidant capacity of honeydew honey: comparison between Romanian and other south European declared honeydew honeys.
The composition of honey depends on the place the honeybees collect their raw material. If the bees collect nectar from flowers, nectar honey will be generated, if they collect fluids that exude from plants, after plant-sucking insects visit the plants, honeydew honey will be generated by the bees. Naturally occurring antioxidants are important ingredients of many foods, and keenly sought in many 'healthy foods'. They are believed to help protect people from diseases like cancer, cardiovascular disorders, neurodegenerative diseases and aging. They operate by mopping up potentially damaging free radicals that are released in the body. The increasing interest in the antibacterial properties of various types of honey is being supported by their well known therapeutic potential, mostly accepted in the alternative medicine . Honey is a natural source of antioxidants and among all honey types, honeydew honey is the most important. Honeydew honeys differ from nectar honeys in respect of chemical composition , mineral content and thus in antioxidative properties also. Honeydew honey has proven to be a powerful antibacterial, antiviral, antifungal agent, killing bacteria such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Pseudomonas aeruginosa .
Components that were identified and quantified as honey antioxidants include phenolic compounds, ascorbic acid, enzymes (glucose oxidase, catalase, peroxidase and others). Additional research on single phenolic and other compounds in honey indicate that the antioxidant capacity is due to combination of a wide range of honey active compounds beyond phenolics . It is interesting to know if the geographical origin of different honey samples belonging to the same type, exhibit different antioxidant activity as an efect of possesing different amounts of antioxidative components. Different methods were proven to be appropriate for assessing the antioxidant activity: ferric reducing antioxidant power (FRAP) , 2.2diphenyl-1-picrylhydrazyl (DPPH assay) , Trolox equivalent antioxidant activity (TEAC) , oxygen radical absorbance capacity (ORAC)  and in most cases it is necessary to use several combined tests to obtain good reliability . The main objective of this study was to investigate honeydew honey samples from Romania (Transylvania region), Bulgaria, Croatia, Turkey and Greece in respect of their antioxidant activity and polyphenolic content (as the main responsabile class of compounds for antioxidant activity). All analyses were performed in Laboratory for Quality Control of Bee Products APHIS Cluj-Napoca.
MATERIAL AND METHODS
Honeydew honey samples from different counties of Transylvania were purchased from the open market, directly from beekeepers, retailers and from supermarket. 7 declared honeydew honey samples were investigated for their polyphenolic content and antioxidant activity. 15 honeydew honey samples (2 from Bulgaria, 4 from Greece, 2 from Turkey and 5 from Croatia) were kindly offered by LAVES-Institut fiir Bienenkunde Celle, Germany.
Reagents and equipments
Gallic acid, sodium carbonate and Folin reagent, Trolox (6-hydroxy- 2,5,7,8tetramethylchroman carboxylic acid), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid acid) diammonium salt (ABTS), 2,2-diphenyl-1-pycrylhydrazil were purchased from SigmaAldrich (St.Louis MO). Iron chloride, sulfate 7-hydrate (FeSO4-7H2O) were obtained from BDH (Poole, UK). 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) was purchased from Fluka Chemie AG (Bushs, Switzerland). Methanol was obtained from Merck (Darmstadt, Germany) and all other chemicals used were analytical grade purity.
Spectrophotometric measurements were made on Synergy HT Multi-Detection Microplate Reader 96 well plates (BioTek Instruments Inc, P).
The concentration of total phenolics in diluted honey samples (5%, w/v) was determined with a modified method developed by Singleton et al. (1999) (Folin-Ciocalteu method) . Each honey sample (1 g) was diluted to 20 ml with methanol and filtered through Whatman filter paper. The method used was adapted for the microplate reader. Shortly, 25[micro]l of honey extract was mixed with 125 [micro]l of Folin Ciocalteu reagent (0.2 N) in a 96 well microplate and mixed for 5 min. 100 [micro]l sodium carbonate (75 g/l) was added and incubated for 120 min. in the dark. The absorbance was read at 760 nm against a blank of methanol, Folin reagent and sodium carbonate. Gallic acid (Sigma Aldrich Germany)(0.01-0.25 mg/ml) was used as standard, to make the calibration curve (r2=0.9995). The mean of three readings was used and the total phenolic content was expressed as mg of Gallic acid equivalents (GAE)/100 g of honey.
Total Antioxidant Activity (FRAP Assay)
The ferric reducing-antioxidant assay (FRAP) is based on the reduction at low pH of ferric 2, 4, 6-tris(2-pyridyl)-1,3,5-triazine [Fe(III)-TPTZ] to the ferrous complex followed by spectrophotometric analysis . The reagent was prepared by mixing 300 mmol/l acetate buffer (pH 3.6) with 10mmol/l TPTZ in 40 mmol/l hydrochloric acid and 20mmol/l ferric chloride in distilled water. Quantitative analysis was done using the external standard method (ferrous sulfate, 0.1-1 mmol), correlating the absorbance (k = 593 nm, Synergy HT MultiDetection Microplate Reader) with the concentration. The sample (honey solution) with FRSAP reagent was incubated at 370C for the monitoring period (4 min.).
The results were expressed as millimoles [Fe.sup.2+] per 100g of honey. Regression coefficient for calibration curve (0.1-1 mmol/l FeSO4) was [r.sup.2]=0.9976.
Antiradical Activity (DPPH Assay)
The antiradical capacity was determined by the 2,2 diphenyl-1-picrylhydrazyl (DPPH) assay . Honeydew honey samples were diluted first in ultra pure water (1:5, w/v). Spectrophotometric readings were carried out with a Synergy HT Multi-Detection Microplate Reader spectrophotometer at 517 nm. DPPH assay was carried out in triplicate for each sample.
The absorbance of DPPH radical without the sample, i.e. the control, was determined. Quantitative analysis was made using the external standard method (Trolox). A calibration curve in the range of 0.01-0.1 mmol/L was used for Trolox and data were expressed as Trolox equivalent antioxidant capacity (TEAC, mmol/100g). Each sample (40 [micro]L of previously prepared concentration 5 g/100ml) was mixed with 200 [micro]L of DPPH 0.02 mmol/ml in methanol. The mixtures was shaken and left for 15 min at room temperature in the dark.
The absorbance was measured against a control made of 40 [micro]l of methanol and 200 [micro]l of DPPH (the bank was read at t = 0 min and at t = 15 min).
Trolox equivalent antioxidant capacity
Procedure of Re et al. (1999)  was used for Trolox equivalent antioxidant capacity determination from honeydew honey samples. This method is based on the scavenging of 2,2azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS radical) depolarization and Monitoring at 734 nm. ABTS radical was induced by the reaction of 7 mM ABTS solution and potasium persulfate solution (2.45 mM), stored in the dark at room temperature for 16 hours. The absorbance at 300C after 6 min was read for sample (17 [micro]l sample and ABTS radical solution having absorbance of 0.700) against the bank. Calibration curve in the range of 0.040.5 mmol Trolox (r2=0.9969) was used for quantitative determination. Results were expressed as mmols Trolox/100 g honey.
RESULT AND DISCUSSION
Total phenolic content (mgGAE/100 g honey) investigated using a modified Folin-Ciocalteu assay, which is sensitive to phenol and polyphenol entities and other electronDonating antioxidants, presented the highest values in honeydew honeys origination from Romania (mean of 137.33 mgGAE/100 g honey), followed by Bulgarian honeys (125.79 mgGAE), Croatian honeys (116.37 mgGAE) and lower concentration in Greece (80.85 mgGAE) And Turkey samples (70.45 mgGAE/100 g) (figure 1).
The total polyphenolic content was significantly higher (p<0.05) in Romanian, Bulgarian and Croatian samples than in Turkish and Greece samples.
For all honey samples, origin was determined in order to know the plant or insect that was responsible for honeydew flow. According to results received from LAVES Institute, Celle, Germany, these 20 honey samples have 3 different honeydew origins: chestnut, oak and pine/fir. Using the standard curve of gallic acid ([r.sup.2] = 0.9995), the total phenolic content varied from 59.28 mgGAE/100 g in pine/fir honeydew honey, to 140.11 mgGAE/100 g honey in oak honeydew honey and 162.78 mgGAE/100 g honey in chestnut honey (figure 2).
Latest investigations show many differences between the results obtained between the test systems in the determination of antioxidant activity. It is obvious that for assessing correctly this property at least 2-3 tests must be performed .
The present research used different methods based on the capacity of reducing oxidants (ferric ions) and on the capacity to scavenge free radicals. DPPH method is one of the most rapid and easy methods to determine the scavenging activity of substrates possessing antioxidant components. Taking into consideration the location of the analyzed samples, DPPH values were not significantly different. Their values ranged between 0.78 mmol/100g in Romanian samples and 1.39 mmol Trolox/100 g honey in Croatian samples.
The ferric reducing antioxidant power differed significantly from one geographical origin to another. Samples originating from Bulgaria exhibit the lowest antioxidant power (1.45 mmol [Fe.sup.2+]/100 g honey), comparing to Croatian samples, which exhibit the highest value (9.67 mmol [Fe.sup.2]+/100 g honey). Significantly differences (p<0.05) were noticed for Bulgarian, Romanian and Turkish honey samples, towards Croatian and Greece honeys.
In ABTS radical decolorisation method, Turkish honeys posses the lowest TEAC Value (1.87 mmol Trolox/100g honey). Higher activity was noticed for Romanian samples (2.3 mmol Trolox/100g), Greece samples (3.51 mmols Trolox/100g), and highest value for Croatian (5.21 mmols/100g) and Bulgarian samples (5.37 mmols Trolox/100g) (table 1).
Antioxidant activity determined by the three different methods show significant differences when using FRAP and TEAC methods, but for DPPH assay, the radical scavenging capacity was similar for the three declared honeydew origin samples (oak, chestnut and pine/fir) (table 2).
Honeydew honey has higher levels of antioxidants than most other flower honeys. Chestnut honeydew honey has the highest level of polyphenols, followed by oak honeydew honey. The same classification was obtained when determining the antioxidant activity (DPPH, FRAP and TEAC). The highest levels were registered in chestnut honeydew honey followed closely by oak honey. Previous studies on oak honeydew honey from Croatia (7) show high radical scavenging activity and antioxidant power determined by DPPH and FRAPS method. The third source of honeydew (pine/fir), exhibits the lowest polyphenolic content and also the lowest antioxidant activity, measured by the three described methods.
Geographical origin of honey did not interfere in the content of antioxidant substances from the class of polyphenols. All oak honeydew honey samples from Romania, Bulgaria and Croatia, exhibit very high content of polyphenols, excepting one sample from Croatia (94.58mgGAE/100 g), all other samples ranging between 111.65 and 196.0 mgGAE/100 g. Taking into consideration that we have analyzed only one sample of chestnut honeydew, further studies have to be made on a larger number of samples. Also pine/fir honeydew honey samples originating from Greece and Turkey exhibit the same small quantities of polyphenols ranging between 43.71 and 80.09 mg GAE/100 g honey.
The authors are grateful to Dr. Werner von der Ohe from LAVES Institut fur Bienenkunde Celle, Germany and Prof. dr. Stefan Bogdanov from Swiss Bee Research Center Liebefeld, Switzerland for providing the honey samples and confirming the origin. The research was partly supported by the Grant POS CCE Nr. 206/20.07.2010 COD SMIS--618/12460.
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LIVIU ALEXANDRU MARGHITAC, DANIEL DEZMIREAN,
OLTICA GIORGIANA STANCIU, OTILIA BOBIC
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
Department of Beekeeping and Sericiculture, Manactir st. 3-5, Cluj-Napoca,
Table 1. Antioxidant activity of tested honeydew honey samples according to geographical location DPPH value FRAP value TEAC value Location of (mmol Trolox/ (mmol [Fe.sup.2+]/ (mmol Trolox/ samples 100 g honey) 100 g honey) 100 g honey) Romania (n=7) 0.78 3.13 2.30 Bulgaria (n=2) 1.17 1.45 5.37 Croatia (n=5) 1.39 9.67 5.21 Greece (n=4) 1.15 5.24 3.51 Turkey (n=2) 0.82 2.13 1.87 Table 2. Antioxidant activity of analyzed honey samples according to origin DPPH value FRAP value TEAC value Origin of (mmol Trolox/ (mmol [Fe.sup.2+]/ (mmol Trolox/ samples 100 g honey) 100 g honey) 100 g honey) Oak (n=12) 1.05 7.40 4.09 Chestnut (n=1) 1.27 9.61 6.08 Pine/Fir (n=7) 1.02 2.85 2.21 Figure 1. Polyphenolic content (mgGAE/100 g honey) in different honeydew honeys, according to geographical location Romania (n=7) 137,330 Bulgaria (n=2) 125,790 Croatia (n=5) 116,370 Greece (n=4) 80,850 Turkey (n=2) 70,450 Note: Table made from bar graph. Figure 2. Polyphenolic content (mgGAE/100 g Honey) according to honeydew origin Oak (n=12) 140,11 Chestnut (n=1) 162,78 Pine/Fir (n=7) 59,28 Note: Table made from bar graph.
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|Title Annotation:||PROCEEDINGS OF THE 1ST INTERNATIONAL ANIMAL HEALTH SCIENCE CONFERENCE: THE BEEKEEPING CONFERENCE|
|Author:||Marghitas, Liviu Alexandru; Dezmirean, Daniel; Stanciu, Oltica Giorgiana; Bobis, Otilia|
|Publication:||Economics, Management, and Financial Markets|
|Date:||Mar 1, 2011|
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