Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals

Food and Chemical Toxicology 50 (2012) 2766–2773

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Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals Juan Zhou a, Peng Li b, Ni Cheng a, Hui Gao a, Bini Wang a, Yahui Wei b,⇑, Wei Cao a,⇑ a b

Department of Food Science and Engineering, College of Chemical Engineering, Northwest University, Xi’an 710069, China College of Life Science, Northwest University, Xi’an 710069, China

a r t i c l e

i n f o

Article history: Received 3 March 2012 Accepted 27 May 2012 Available online 7 June 2012 Keywords: Buckwheat honey Antioxidant activity DNA damage Phenolic acids

a b s t r a c t To understand the antioxidant properties of buckwheat honeys, we investigated their antioxidant effects on hydroxyl radical-induced DNA breaks in the non-site-specific and site-specific systems, the physicochemical properties, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity, chelating, and reducing power assays), total phenolic content and individual phenolic acids were also determined. Total phenolic content of buckwheat honeys ranged from 774 to 1694 mg PA/kg, and p-hydroxybenzoic and p-coumaric acids proved to be the main components in buckwheat honeys. All the buckwheat honey samples possess stronger capability to protect DNA in the nonsite-specific systems than in the site-specific systems from being damaged by hydroxyl radicals. In the non-site-specific and site-specific system, buckwheat honeys samples prevented OH-induced DNA breaks by 21–78% and 5–31% over control value, respectively. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Buckwheat honey is produced by honeybees which collect nectar from the flowers of buckwheat (Fagopyrum esculentum Möehch). Buckwheat honey is also a dark-colored honey. Actually the dark purple color of buckwheat honey looks almost black. It is characterized by a strong malty, rich, molasses flavor, which is not oversweet. The previous studies founded that buckwheat honey had the highest antioxidant activity of honeys from 14 different floral sources (Gheldof et al., 2002) and had the highest oxygen radical absorbance capacity (ORAC) values from seven different floral sources (Gheldof and Engeseth, 2002). In addition, a recent study indicates that consumption of buckwheat honeys can increases antioxidant capacity of human serum (Gheldof et al., 2003). Many studies have reported that honey contains a variety of preservative substances such as phenolic acids and flavonoids (Alvarez-Suarez et al., 2010a, 2012a; Tenore et al., 2012), ascorbic acid, protein and carotenoid (Alvarez-Suarez et al., 2010a). The therapeutic role of honey in the treatment of various ailments has been receiving considerable attention recently. They include antibacterial (Estevinho et al., 2008; Gomes et al., 2010; Rodríguez et al., 2012), antifungal (Feás and Estevinho, 2011), antimutagenicity (Saxena et al., 2012), hypertensive (Erejuwa et al., 2012) and anti-inflammatory effects (Ahmad et al., 2012). Honey also displays a significant antioxidant activity; the antioxidant activities of var-

⇑ Corresponding authors. Tel./fax: +86 29 88302213. E-mail address: [email protected] (W. Cao). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.05.046

ious floral sources honeys have received much attention due to the increasing interest in human health (Alvarez-Suarez et al., 2010b, 2012a,b; Brudzynski et al., 2012). During the past decade, there has been a remarkable increment in scientific research dealing with natural antioxidants and their potential health benefits. Oxidative stress, the consequence of an imbalance between ROS (reactive oxygen species) generation and antioxidants in the organism, initiates a series of harmful biochemical events which are associated with diverse pathological processes which can lead to various cellular damages and diseases (Sastre et al., 2003). Antioxidants are considered as possible protection agents reducing oxidative damage to important biomolecules, including lipoprotein and DNA (deoxyribonucleic acid) from ROS (Gulcin et al., 2003). In a previous study, it was also demonstrated that honey has a similar antioxidant activity as many fruits and vegetables on a fresh weight basis (Gheldof and Engeseth, 2002), and its antioxidant substances might be responsible for its ability to protect against oxidative reactions (Gheldof et al., 2003). However, very little if any research has been done on the antioxidant properties of buckwheat honeys from China, especially undertaking on protective effects on DNA damage caused by hydroxyl radicals. The objectives of our study were: (1) to investigate the effects of buckwheat honeys on DNA oxidative damage induced by hydroxyl radicals; (2) to determine antioxidant activity of the eight buckwheat honeys by scavenging activity on DPPH and hydroxyl radical, chelating activity on Fe2+, and reducing power; (3) to determine the physicochemical properties, total phenolic contents and main phenolic compounds of buckwheat honeys.

J. Zhou et al. / Food and Chemical Toxicology 50 (2012) 2766–2773 Table 1 The most predominant pollen in the eight buckwheat honeys analyzed. Sample Frequency classa

Pollen identification (frequency)

Geographical origin

S1

Fagopyrum esculentum (70.4%) Robinia pseudoacacia (19%) Melilotus spp. (5.3%) Fagopyrum esculentum (62%) Robinia pseudoacacia (30.5%) Glycine max (5.3%) Fagopyrum esculentum (66.4%) Robinia pseudoacacia (23.7%) Melilotus spp. (3.1%) Fagopyrum esculentum (72%)

Shaanxi (2009)

S4

P S I P S I P S I P

S5

S I P

Robinia pseudoacacia (18.7%) Glycine max (6.1%) Fagopyrum esculentum (69.2%) Shaanxi Jingbian (2009) Robinia pseudoacacia (25.4%) Melilotus spp. (3.3%) Fagopyrum esculentum (57%) Neimenggu (2009) Robinia pseudoacacia (32.2%) Glycine max (8.4%) Fagopyrum esculentum (56%) Jilin (2009) Robinia pseudoacacia (25.9%) Melilotus spp. (5.7%) Fagopyrum esculentum (68%) Shanxi (2008) Robinia pseudoacacia (25.7%) Glycine max (11.6%)

S2

S3

S6

S7

S8

S I P S I P S I P S I

Shaanxi (2009)

Shaanxi (2009)

Shaanxi Jingbian (2009)

a Frequency classes: P-predominant pollen (more than 45% of pollen grains counted); S-secondary pollen (16–45%); I-important minor pollen (3–15%).

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2.4.2. pH The pH was measured by a pH-meter Delta 320 (China), with a precision of ±0.01 pH units. The pH of the honey was measured in solution of 5 g honey in 20 ml of CO2-free distilled water (AOAC, 1990; Official Method 962.19). 2.4.3. Ash The ash content was determined by placing 2–3 g of honey samples in a crucible in a muffle furnace and heating at 640 °C for 6 h. Measurements of ash were done in triplicate and the mean was expressed in g% (AOAC, 1990; Official Method 920.181). 2.4.4. Electrical conductivity Electrical conductivity was determined by a Delta 326 conductimeter, from a solution containing 10 g of honey in 75 mL of distilled water (Sancho et al., 1992). 2.4.5. Diastase activity Diastase activity was determined using a buffered solution of soluble starch and honey incubated in a thermostatic bath at 40 °C (AOAC, 1990; Official Method 958.09). Thereafter, 1 mL aliquot of this mixture was removed at 5 min intervals and the absorption of the sample was followed at 660 nm in a UV751-GD Spectrophotometer (shanghai, China). The diastase value was calculated using the time taken for the absorbance to reach 0.235, and the results were expressed in Gothe degrees as the amount (mL) of 1% starch hydrolyzed by an enzyme in 1 g of honey in 1 h. 2.4.6. Hydroxymethylfurfural content (HMF) Hydroxymethylfurfural was determined by using the standard method AOAC (1990) Official Method 980.23. Five grams of honey were dissolved in 25 mL of distilled water, treated with a clarifying agent (0.5 mL of Carrez I and 0.5 mL of Carrez II solutions) and volume made up to 50 mL. The solution was filtered, and the first 10 mL discarded. The absorbance of the filtered solution was measured at 284 and 336 nm against an aliquot of the filtered solution treated with NaHSO3. HMF was determined as:

HMF=100 g of honey ¼ ðAbs284  Abs336 Þ  14:97  ð5=g of sampleÞ:

2. Materials and methods 2.1. Samples Eight buckwheat honey samples from six geographical regions were obtained from Shaanxi Beemaster Biotechnology Co. Ltd. and Yanliang (Xi’an, China) Honey Co. Ltd., respectively (Table 1). Samples were kept at +4 °C until assayed.

2.5. Preparation of buckwheat honey samples Two grams of buckwheat honey were put into a 10 ml calibrated flask and dissolved with 5 ml water. The flask was covered and then placed in an ultrasound (US) water bath apparatus for 10 min. Then, the honey sample was homogenized and filtered through 0.45 lm membrane filter. And then, it was kept in the freezer for chromatographic analysis. 2.6. Determination of phenolic acids by HPLC analysis

2.2. Chemicals and reagents Fish DNA and plasmid pBR322 DNA were obtained from Takara Biomedicals (Japan). 1,1-Diphenyl-2-picrylhydrazyl(DPPH), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-40 ,400 -disulfonic acid monosodium salt (namely, ferrozine), 1,1,1-tris (hydroxymethyl) ethane (Tris), protocatechuic acid (PA), gallic acid, benzoic acid, p-hydroxybenzoic acid, caffeic acid, p-coumaric acid, and Folin–Ciocalteu reagent were purchased from Sigma–Aldrich (Steinheim, Germany). HPLC grade methanol was purchased from Merck (Darmstadt, Gemany). HPLC grade water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Trichloroacetic acid (TCA), thiobarbituric acid (TBA), ethylenediamine tetraacetic acid (EDTA), and ascorbic acid (Ac) were purchased from Beijing Chemical Co. (Beijing, China). All other reagents used were of analytical grade and were purchased from Xi’an Chemical Co. (Xi’an, China). 2.3. Pollen analysis The botanical origin of the samples was determined by the method of (Lutier and Vaissière, 1993). For floral identification, 5 g of diluted honey sample was centrifuged at 10,000 rpm for 15 min, to separate the pollens. Samples of separated pollen grains were spread with the help of a brush on a slide containing a drop of lactophenol. The slides were examined microscopically at 45, using a bright-field microscope (Olympus, Tokyo). The following terms were used for pollen frequency classes: predominant pollen (P, more than 45% of pollen grains counted), secondary pollen (S, 16–45%) and important minor pollen (I, 3–15%). 2.4. Physiochemical analysis 2.4.1. Moisture content The conventional-drying oven method was carried out as described in the Association of Official Analytical Chemists (AOAC) method number 925.45 (AOAC, 1990). The moisture content was determined by drying a weighed amount of the sample at 105 °C for 3 h (or until a constant weight was obtained). Samples were analyzed in triplicate, and the corresponding moisture content was calculated as a percentage.

HPLC was performed with an Agilent 1100 liquid chromatography system (Agilent Technologies Deutschland, Waldbronn, Germany), equipped with a vacuum degasser, a quaternary solvent delivery pump, a manual chromatographic valve, a thermostated column compartment, a diode-array detector (Agilent, Palo Alto, CA, USA) and a HP1049A programmable electrochemical detector (HP, USA) was used. The samples were dissolved in water and filtered through a 0.45 lm membrane filter. The injection volume was 10 ll and the separation temperature was 30 °C. The column was a Zorbax SB-C18 (150  4.6 mm, 5.0 lm) connected with a Zorbax SB-C18 (20  4.0 mm, 5 lm). The mobile phase consisted of 4% aqueous acetic acid (A) and methanol (B) (v/v) using a linear gradient elution of 5–20% B at 0–10 min, 20–40% B at 10–15 min, 40–60% B at 15–20 min, 60% B at 20–25 min. The flow-rate was 1.0 ml/min. The diode-array detector was performed at 288 and 320 nm and the electrochemical detector was set at 1.0 V in the oxidative mode (Liang et al., 2009). 2.7. Determination of total phenolic content (TPC) TPC was determined using a modified version of the Folin–Ciocalteu method (Socha et al., 2011). 0.4 ml of sample (0.2 g/ml) was added to 1.0 ml of Folin–Ciocalteu reagent (previously diluted to 10 folds with distilled water) and the mixture was kept at room temperature for 5 min. Five milliliters of sodium carbonate (1 M) was added to the mixture and the whole mixed gently. The total volume of the mixture was adjusted to 10 ml with distilled water. After the mixture was kept at room temperature for 1 h, the absorbance was read at 760 nm. The standard calibration (0.02–0.12 mg/ml) curve was plotted using protocatechuic acid. The total phenolic content was expressed as the protocatechuic acid equivalents. 2.8. Analysis of antioxidant activities 2.8.1. Reducing power assay The reducing power of the buckwheat honey was determined by the method of Estevinho et al. (2008) with a slight modification. Different volumes of samples (0.2 g/ml) were mixed with 2 ml of phosphate buffer (0.2 M, pH 6.6) and 1 ml of

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1% potassium ferricyanide in 10 ml test tubes. The mixtures were incubated in water bath for 20 min at 50 °C. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixtures. The upper layer (2 ml) was mixed with 2 ml of distilled water and 0.4 ml of 0.1% ferric chloride, and the absorbance was measured at 700 nm. The reducing power tests were performed in triplicate. Increased absorbance of the reaction mixture correlates with greater reducing power. 2.8.2. Ferrous ion-chelating activity The ferrous ion-chelating activity of buckwheat honey was investigated according to the method of Nandita and Rajini (2004). Ferrous ion-chelating ability of buckwheat honey was evaluated by measuring the absorbance of ferrozine–Fe2+ complex at 562 nm. Briefly, the reaction mixture, containing 0.2 ml of sample (0.2 g/ml), FeSO4 (1 mmol/L) 60 ll, and ferrozine (1 mM) 300 ll, was adjusted to a total volume of 3 ml with methanol, shaken well and incubated for 10 min at room temperature. The absorbance of the mixture was measured at 562 nm against the blank. The standard calibration (0.02–0.12 mg/ml) curve was plotted using Na2EDTA. Ferrous ion-chelating activity was expressed as the Na2EDTA equivalents (mg Na2EDTA/g honey). 2.8.3. DPPH radical scavenging activity The effects of buckwheat honeys on the DPPH radical inhibition was determined by the method of Wang et al. (2012). The samples (200 ll, 0.2 g/ml) were added to a methanolic solution (1 ml) of DPPH radical (the final concentration of DPPH was 0.2 mM). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min; the absorbance of the resulting solution was then measured spectrophotometrically at 517 nm. The percent inhibition of activity was calculated according to the following equation:

% inhibition ¼ ððA0  At Þ=A0  100Þ;

where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the sample. All the tests were performed in triplicate and the graph was plotted with the mean values. 2.8.4. Hydroxyl radical scavenging activity The OH scavenging ability of buckwheat honeys were examined by following the procedure previously described (Cheng et al., 2003), with some modifications. Briefly, the reaction mixtures contained 2-deoxyribose (8 mg/ml), EDTA–FeSO4 (1 mM)100 ll, Vc (1 mM) 50 ll, H2O2 (1 mM) 100 ll, and 50 ll samples (0.2 g/ ml). The total volume of mixture was adjusted to 1 ml with phosphate buffer (0.2 M, pH7.4). The mixture was vortexed and incubated in water bath at 37 °C for 10 min. Thereafter, 1 ml of 10% TCA and 1 ml of 1.0% TBA were added to each tube. Samples were heated in water bath at 80 °C for 30 min. The extent of oxidation was estimated from the absorbance of the solution at 532 nm. The hydroxyl radical-scavenging activity of the honey was reported as the percentage of inhibition of deoxyribose degradation and was calculated according to the following equation:

% inhibition ¼ ððA0  At Þ=A0  100Þ; where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the sample. All the tests were performed in triplicate and the graph was plotted with the mean values. 2.8.5. Assay for testing the effects of buckwheat honeys on hydroxyl radical-mediated DNA strand breaks To test the effects of buckwheat honey samples on the extent of damage to DNA induced by hydroxyl radical (OH) in vitro, the reaction was conducted in an Eppendorf tube. Briefly, 0.25 lg of DNA was incubated with 2 ll of 1.0 mM EDTA–FeSO4, 1 ll of 1% H2O2, and 2 ll of 0.2 g/ml honey samples, final volume was made up to 12 ll with 50 mM phosphate buffer (pH 7.0). The reaction system was involved in the production of site-specific radicals with the absence of EDTA and non-site-specific without EDTA. Then the mixture was incubated in water bath at 37 °C for 30 min according to the procedure described by Yeung et al. (2002) with some modifications. After the incubation, the mixture was subjected to 1% agarose gel electrophoresis. DNA bands (supercoiled, linear, and open circular) were stained with ethidium bromide and quantified by scanning the intensity of bands with quantity one programme (version 4.2.3, BioRad Co.). Evaluations of antioxidant effects on DNA were based on the increase or loss percentage of supercoiled monomer, compared with the control value. To avoid the effects of photoexcitation of samples, experiments were done in the dark. 2.9. Statistical analysis Data were expressed as means ± standard deviation determined from triplication analysis. Analysis of variance was performed using the ANOVA procedure. Significant differences among the means were determined using Duncan’s multiple-range test. p 6 0.05 was considered to be statistically significant.

3. Results and discussion 3.1. Pollen analysis Table 1 shows the floral origin of buckwheat honeys was determined by microscopy pollen analyses. The data indicate that all the honey samples were monofloral. Fagopyrum esculentum pollen was detected in 56–72% of all analyzed samples. 3.2. Physiochemical analysis 3.2.1. Ash content and electrical conductivity The percentage ash content is an indicator of the mineral content. It is considered as a quality criterion indicating the possible botanical origin of honey. The results found (0.16–0.37%) (Table 3) are within the limit allowed for floral honeys (0.6%). The variability in the ash content of honeys could be due to harvesting processes, beekeeping techniques and the material collected by the bees during the foraging on the flora (Finola et al., 2007). The electrical conductivity (mS/cm) values in honey samples varied in the range of 0.35–0.63 (Table 3), were within the allowed parameters (lower than 0.8 mS/cm) and corresponded to those previously reported by Alvarez-Suarez et al. (2010a). It is closely related to the concentration of mineral salts, organic acids and proteins. The coefficient of correlation between electrical conductivity and ash was found to be 0.92, which indicated a strong positive correlation between the two parameters. Similarly, a correlation value of 0.92 has been found to exist between the electrical conductivity and ash content for some Algerian honeys (Ouchemoukh et al., 2007). 3.2.2. pH All the buckwheat honeys analyzed were found to have an acidic character. Their pH values ranged from 3.92 to 4.70 (Table 3). These results are comparable to 3.9–4.9 for Cuban (Alvarez-Suarez et al., 2010a) and 3.87–5.12 for Southern Africa (Serem and Bester, 2012) honeys. 3.2.3. Moisture content Moisture content is a good criterion to establish honey quality; high moisture content can produce honey fermentation during storage, resulting in the formation of ethyl alcohol and carbon dioxide. The alcohol can further be oxidized into acetic acid and water with the ensuing sour taste (Chirife et al., 2006). The moisture content (%) in the investigated samples ranged from 14.3% to 16.3%, which are well below to the imposed limit of 620% (Codex Alimentarius, 2001). In our samples, the values were similar to those previously reported for this organic honeys from TrásOs-Montes region whose corresponding values ranged from 14.5% to 16.3% (Estevinho et al., 2012). 3.2.4. Diastase activity Diastase is a natural enzyme of honey. Its level depends upon geographic and floral origins of the product, as well as on its freshness. As with HMF, diastase activity can be used as indicative of aging and temperature abuse, but with precaution, since its variability has been higher, confirmed in several honeys (Fallico et al., 2006). Diastase activity (°Gothe) varied between 9.6 and 17.1 for the buckwheat honeys. All honeys under analysis in the present study fall within imposed limits. 3.2.5. HMF The HMF content is widely recognized as a parameter of honey samples freshness, because it is absent in fresh honeys and tends to increase during processing and/or aging of the product. Several factors influence the levels of HMF, such as temperature and time

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J. Zhou et al. / Food and Chemical Toxicology 50 (2012) 2766–2773 Table 2 Penolic acids and TPC (mg/kg) of buckwheat honeys. Sample S1 S2 S3 S4 S5 S6 S7 S8

Gallic acid

Protocatechuic acid d

0.250 ± 0.009 0.278 ± 0.011cd 0.413 ± 0.012a 0.291 ± 0.007bc 0.316 ± 0.023b 0.276 ± 0.033cd 0.265 ± 0.011d 0.283 ± 0.019cd

b

0.565 ± 0.019 0.609 ± 0.021a 0.628 ± 0.029a 0.213 ± 0.017d 0.345 ± 0.007c 0.116 ± 0.005f 0.155 ± 0.015 0.314 ± 0.033c

p-Hydroxybenzoic acid

Caffeic acid

g

p-Coumaric acid b

12.267 ± 0.179 29.128 ± 0.297c 34.061 ± 0.134b 18.600 ± 0.125f 22.347 ± 0.120e 5.826 ± 0.198h 40.682 ± 0.231a 25.178 ± 0.067d

Total phenolic

c

0.319 ± 0.032 0.241 ± 0.012b 0.256 ± 0.032b 0.260 ± 0.011b 0.240 ± 0.098b 0.483 ± 0.044a 0.319 ± 0.022b 0.234 ± 0.099b

1102 ± 55c 1104 ± 52c 1350 ± 75b 780 ± 35e 774 ± 31e 1694 ± 90a 1461 ± 83b 978 ± 73d

1.708 ± 0.102 4.031 ± 0.110b 1.435 ± 0.083d 0.793 ± 0.043e 0.851 ± 0.011e 3.853 ± 0.132b 4.933 ± 0.230a 1.233 ± 0.092d

Results are displayed with mean ± SE (n = 3). Values with different superscripts in each row are significantly different (p 6 0.05).

Table 3 Distribution data for physicochemical parameters in buckwheat honey samples. Sample

Moisture (%)

pH

EC (mS/cm)

Ash (%)

Diastase activity (°Gothe)

HMF (mg/kg)

S1 S2 S3 S4 S5 S6 S7 S8

14.3 ± 0.0a 14.7 ± 0.2a 16.3 ± 0.1b 16.0 ± 0.2b 16.1 ± 0.1b 15.5 ± 0.0c 15.7 ± 0.3bc 16.1 ± 0.2b

4.25 ± 0.03c 4.09 ± 0.07cd 4.58 ± 0.05ab 4.46 ± 0.01b 4.70 ± 0.04a 4.07 ± 0.04d 4.60 ± 0.13ab 3.92 ± 0.22d

0.55 ± 0.09ab 0.46 ± 0.07cd 0.63 ± 0.04a 0.59 ± 0.01ab 0.37 ± 0.07d 0.49 ± 0.04bc 0.47 ± 0.06cd 0.35 ± 0.07d

0.31 ± 0.04ab 0.23 ± 0.02cd 0.37 ± 0.09a 0.34 ± 0.07ab 0.18 ± 0.02d 0.27 ± 0.04bc 0.19 ± 0.01cd 0.16 ± 0.01d

13.4 ± 0.2d 15.7 ± 0.4b 14.2 ± 0.1c 9.6 ± 0.8e 13.2 ± 0.3d 12.0 ± 0.4e 15.1 ± 0.5b 17.1 ± 0.3a

17.8 ± 1.1b 14.2 ± 1.2d 18.7 ± 0.9ab 19.9 ± 0.3a 18.3 ± 0.5b 17.4 ± 0.4b 15.9 ± 0.4c 11.3 ± 0.7e

Results are displayed with mean ± SE (n = 3). Values with different superscripts in each row are significantly different (p 6 0.05).

of heating, storage conditions, pH and floral source, thus it provides an indication of overheating and storage in poor conditions (Fallico et al., 2006; Khalil et al., 2010). In all samples the HMF content was found to be lower than the values recommended by the Codex Alimentarius (2001) (40 mg/kg). The HMF values were between 11.3 and 19.9 mg/kg (Table 3).

and p-hydroxybenzoic acids quantities in honey were associated with its botanical origin (Gómez-Caravaca et al., 2006). Therefore,

A

250

150

100

50

0 0

5

10

15

20

25

time/min

B

350 300 250

response/nA

Since phenolic substances have been shown to be responsible for the antioxidant activity of honey, the total phenol content of the honey samples were investigated. The obtained values of TPC and five phenolic acids are shown in Table 2. As can be seen, TPC of buckwheat honey samples ranging from 774 to 1694 mg PA / kg of honey. For complete honey, sample S6 (1694 mg PA /kg) had the highest value of phenolics, while sample S5 (774 mg PA / kg) had the lowest value. It was observed that the TPC showed significant differences between the different samples. Apart from the region term, the analyses of variance also considered a nested term of sampling locations within a region. A similar level of phenolic content was also observed for Algerian and Slovenian honeys for which the phenolic content varied from 64 to 1304 and 448 to 2414 mg GAE/kg, respectively (Bertoncelj et al., 2007; Ouchemoukh et al., 2007). The highest obtained amount was 1210 mg GAE/kg for Polish buckwheat honeys (Jasicka-Misiak et al., 2012). HPLC analysis of phenolic acids in honey samples showed that gallic, protocatechuic, caffeic, p-coumaric and p-hydroxybenzoic acids are presented in all honey samples analyzed. Fig. 1 shows the typical HPLC chromatograms obtained for all honey samples. The main phenolic acids are p-hydroxybenzoic acid and p-coumaric acid in buckwheat honeys. The content of p-hydroxybenzoic and p-coumaric acids ranging from 5.826 to 40.682 mg/kg and 0.793 to 4.933 mg/kg, respectively, are much higher than gallic, protocatechuic and caffeic acids which present in small amounts in buckwheat honeys. Available data on contents of p-coumaric and p-hydroxybenzoic acids in buckwheat honey have indicated relatively high quantities (Gheldof et al., 2002; Ramanauskiene et al., 2012; Jasicka-Misiak et al., 2012). Dependence of p-coumaric

response/nA

200

3.3. Total phenolic content and phenolic acids in buckwheat honey samples

200 150 100

1

50 0

2

0

5

10

5

3 4 15

20

25

time/min Fig. 1. Chromatograms of standards (A) and buckwheat honey. (B) Peaks: (1) gallic acid, (2) protocatechuic acid, (3) p-hydroxybenzoic acid, (4) caffeic acid, (5) p-coumaric acid.

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p-coumaric and p-hydroxybenzoic acids can be considered as botanical markers of buckwheat honeys, in the course of preliminary estimation of the quality of this unifloral honey. 3.4. Antioxidant activity 3.4.1. Reducing power assay Fig. 2(a) shows the reducing power of buckwheat honeys. The presence of reducers (i.e. antioxidants) causes the reduction of the Fe3+/ferricyanide complex to the ferrous (II) form, which can be measured by the formation of Perl’s Prussian blue at 700 nm. Ferric ion-reduction is often used as an indicator for electrodonating activity of antioxidant, which is an important mechanism of phenolic antioxidant action (Yildirim et al., 2001). As shown in Fig. 2(a), the reducing activity of all samples increases steadily with their concentrations increasing. S4 and S5 buckwheat honeys possess lower reducing power than other samples, and S6 buckwheat honey have the highest reducing power at the same concentration. The correlation between the reducing power and TPC is 0.87. The results illustrate that the reducing power of buckwheat honeys might be related to their TPC. 3.4.2. Ferrous ion-chelating activity The ability of buckwheat honeys to chelate Fe2+ ion was evaluated and expressed as Na2EDTA equivalents (mg Na2EDTA/g honey). Bivalent transition metal ions play an important catalysitic role in the oxidative processes, whereby leading to the formation of hydroxyl radicals and hydroperoxide decomposition reactions via Fenton reaction (Halliwell, 1997). These processes can be delayed by iron chelation. The results are presented in Fig. 2(b). As seen from Fig. 2(b), all buckwheat honeys possess weak ionchelating activity, though S4 processes the highest ion-chelating activity among them, its chelating activity of 1 g buckwheat honeys is only less than 0.22 mg Na2EDTA. The results indicated that buckwheat honeys had weak chelating on iron ions. Generally, Sun et al. (2008) showed that both rutin and quercetin (with o-diphenolic groups in the 3,4-dihydroxy position in ring B and the ketol struc-ture, 4-oxo, 3-OH or 4-oxo, 5-OH in the C ring of the flavonols) could chelate irons effectively and might exert their inhibitory effects by chelating metal ions in the course of the Fenton reaction upon lipid peroxidation. However, only phenolic acids were identified in buckwheat honeys in our study, their iron-chelating activities are very low. The results demonstrate that ion-chelating activity contributes weakly to antioxidant activity of buckwheat honey samples. 3.4.3. DPPH scavenging activity DPPH is a stable nitrogen centered radical and has been widely used to test the free radical scavenging ability of various samples. The reduction capability of DPPH was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. Positive DPPH test suggests that the samples were free radical scavengers. The DPPH radical scavenging effect of buckwheat honeys is presented in Fig. 2c. The percentage DPPH scavenging activity ranged from 37.2% to 56.4%. The coefficient of correlation between DPPH scavenging activity and TPC was found to be 0.83, supporting the former statement on the contribution to antiradical of phenolic compounds (Yildirim et al. 2001). 3.4.4. Hydroxyl radical scavenging activity The hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells (Hochestein and Atallah, 1988). This radical has the capacity to join nucleotides in DNA and cause strand breakage which contributes to carcinogenesis, mutagenesis and

Fig. 2. Antioxidant activities of Chinese buckwheat honeys in iron reduction (a), in iron chelation (b), DPPH scavenging activity (c) and OH radical scavenging activity (d).

cytotoxicity. Hydroxyl radical scavenging capacity of an extract is directly related to its antioxidant activity (Babu et al., 2001). The

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compared with plasmid DNA control (Lane 0). Results in Fig. 3B show that buckwheat honeys of S1–S8 (Lanes 2–Lanes 9) prevented OH-induced DNA breaks by 5–31% over control value (Lane 1 in Fig. 3B, DNA treated with Fe2+ and H2O2). In the nonsite-specific system, EDTA forms a complex with iron ion, and hydroxyl radicals are generated in solution. In the site-specific system, EDTA is omitted. Iron ion can bind to DNA and produce hydroxyl radicals at the non-site-specific site (Aruoma et al., 1989). So iron chelating compounds can reduce the extent of DNA damage even if they are not effective hydroxyl radical scavengers. In the non-site-specific system, this only influences the results if the compounds form a more complex with iron ion than EDTA. Buckwheat honeys which protect more effectively in the non-site-specific system than in the site-specific system are due to its stronger hydroxyl radical scavenging activity than iron chelators in the present assay. In the Fe2+/EDTA/H2O2/Vc mediated DNA damage system, EDTA chelates iron ions from DNA, but iron-EDTA chelate is very effective in generating hydroxyl radicals, so that DNA is still degraded by hydroxyl radicals in ‘‘free’’ solution, rather than by hydroxyl radicals formed on DNA (Aruoma, 2003). Therefore, hydroxyl radicals generated attack DNA in a non-site-specific manner. In the ‘‘non-site-specific’’ system, one important mechanism of antioxidant action may be the chelation of Fe2+, which serves as catalysts in Fenton reaction. In addition, the chelating activities of most phenolic acids of buckwheat honeys are smaller than EDTA (Matsufuji and Shibamoto, 2004). Therefore, another important mechanism of antioxidant action is the scavenging hydroxyl radical activity, which is thought to be the main mechanism to inhibit non-site-specific hydroxyl radical-mediated DNA damage.

highly reactive hydroxyl radicals can cause oxidative damage to DNA, lipids and proteins (Trease and Evans, 1983). As shown in Fig. 2d, the buckwheat honeys displayed potential inhibitory effect of hydroxyl radical-scavenging activity. The percentage of OH scavenging activity ranged from 23.2% to 44.1% (Fig. 2d). Strong correlations are found between OH scavenging activity with TPC (r2 = 0.90). It is known that phenolics possess relevant radical scavenging activity, which would support their putative important role in the radical scavenging properties of honey (Henriques et al., 2006). 3.4.5. Effects of buckwheat honeys on hydroxyl radical-mediated DNA strand breaks Antioxidant of buckwheat honeys were investigated using a free radical-induced plasmid pBR322 DNA breaks system in vitro. With the attack of OH generated from the Fenton reaction, supercoiled plasmid DNA was broken into three forms, including supercoiled (SC), open circular (OC) and linear form (Linear). Effects of buckwheat honeys on the hydroxyl radical-induced DNA damage were investigated as shown in Fig. 3. In the non-site-specific system (Fig. 3A), the percentage of SC form in plasmid DNA decreased by 68.6% under the treatment of OH generated from the Fenton reaction (Lane 1), compared with plasmid DNA control (Lane 0). However, H2O2 or Fe2+ treatment alone resulted in no significant damage on DNA (Tian and Hua, 2005). Results in Fig. 3A show that buckwheat honeys of S1-S8 (Lanes 2–Lanes 9) prevented OHinduced DNA breaks by 21–78% over control value (Lane 1 in Fig. 3A, DNA treated with Fe2+ and H2O2), indicating an antioxidant effect. However, in the site-specific system (Fig. 3B), the percentage of SC form in plasmid DNA decreased by 95.2% under the treatment of OH generated from the Fenton reaction (Lane 1),

A

0

1

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8

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Open circular/ linear Supercoiled

B

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3

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5

6

7

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9

Open circular/linear Supercoiled

Fig. 3. Protective activity of buckwheat honey samples on hydroxyl radical–mediated DNA damage, ‘‘non-site-specific’’ (A); ‘‘site-specific’’ (B). Lane 0, no addition (only pBR322 DNA); Lane 1, FeSO4 and H2O2 (DNA damage control); Lanes 2–9, FeSO4 and H2O2 in presence of S1–S8 of buckwheat honeys with concentration of 0.2 mg/ml, respectively.

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