Physicochemical quality parameters, antibacterial properties and cellular antioxidant activity of Polish buckwheat honey

Journal Pre-proof Physicochemical quality parameters, antibacterial properties and cellular antioxidant activity of Polish buckwheat honey Dżugan Małgorzata, Grabek-Lejko Dorota, Swacha Sylwia, Tomczyk Monika, Bednarska Sabina, Kapusta Ireneusz PII:

S2212-4292(18)31050-2

DOI:

https://doi.org/10.1016/j.fbio.2020.100538

Reference:

FBIO 100538

To appear in:

Food Bioscience

Received Date: 31 October 2018 Revised Date:

27 January 2020

Accepted Date: 28 January 2020

Please cite this article as: Małgorzata Dż., Dorota G.-L., Sylwia S., Monika T., Sabina B. & Ireneusz K., Physicochemical quality parameters, antibacterial properties and cellular antioxidant activity of Polish buckwheat honey, Food Bioscience (2020), doi: https://doi.org/10.1016/j.fbio.2020.100538. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Małgorzata Dżugan: Conceptualization; Methodology; Project administration; Supervision; Writing – review & editing; Funding acquisition Dorota Grabek-Lejko: Investigation, Writing – original draft; Visualization; Resources Sylwia Swacha: Investigation, Resources Monika Tomczyk: Investigation, Writing – original draft, Data curation; Software Sabina Bednarska: Investigation Ireneusz Kapusta: Investigation; Formal analysis; Validation

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Physicochemical quality parameters, antibacterial properties and cellular antioxidant activity

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of Polish buckwheat honey

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Running title: Polish buckwheat honey bioactivity

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Dżugan Małgorzata1*, Grabek-Lejko Dorota2, Swacha Sylwia1, Tomczyk Monika1,, Bednarska

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Sabina3, Kapusta Ireneusz4

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University of Rzeszów, Rzeszów, Poland

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2

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Nutrition, University of Rzeszów, Rzeszów, Poland

Department of Chemistry and Food Toxicology, Institute of Food Technology and Nutrition,

Department of Bioenergetics, Food Analysis and Microbiology, Institute of Food Technology and

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3

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of Rzeszów, Rzeszów, Poland

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4

13

Nutrition, University of Rzeszów, Rzeszów, Poland

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*corresponding author: Małgorzata Dżugan, Department of Chemistry and Food Toxicology,

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Institute of Food Technology and Nutrition, University of Rzeszów, Ćwiklińskiej 1a St., 35-601

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Rzeszów, Poland, phone no. +48 17 8721730, fax + 48 17 872 12 65, e-mail: [email protected]

Department of Biochemistry and Cell Biology, Institute of Biology and Biotechnology, University

Department of Food Technology and Human Nutrition, Institute of Food Technology and

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1

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Abstract

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Buckwheat honey is the darkest Polish honey and has the strongest antibacterial and antioxidant

20

activity; however, the mechanism of this bioactivity remains unknown. To determine the factors

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responsible for the bioactivity of buckwheat honey, antioxidant power, radical scavenging activity,

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and total phenolic and flavonoid contents of 20 buckwheat honey samples from southeastern Poland

23

were measured. The antibacterial activity of the honey was studied using 4 bacterial strains. The

24

effect of catalase on the antibacterial action of the honey was determined. Five buckwheat honey

25

samples with different antioxidant and antibacterial activities were selected, and their phenolic

26

profiles were characterized in detail with UPLC-PDA-MS/MS. In vivo experiments showed that

27

these samples protected cells of the yeast Saccharomyces cerevisiae exposed to hydrogen peroxide,

28

which was used as a hydroxyl radical generator. The antibacterial activity was significantly

29

correlated with antioxidant activity and phenolic compounds (p<0.05). The removal of H2O2 by

30

catalase partially eliminated (30-50%) the bacteriostatic activity of the honeys. The results indicated

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that among the 13 phenolic compounds identified in buckwheat honeys, only quercetin, rutin,

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chlorogenic acid and caffeic acid were correlated with its antioxidative and antibacterial activity,

33

which was shown by using Principal Component Analysis (PCA). The protective effect of

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buckwheat honey resulting from its polyphenols content was confirmed (p<0.05) against in situ-

35

generated hydroxyl radicals using the S. cerevisiae yeast cells as a biological model.

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Keywords: buckwheat honey, polyphenols, Saccharomyces cerevisiae, Polish honey

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2

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1. Introduction

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Honey is one of the oldest traditional medicines, which is considered a remedy for microbial

40

infections. It is also recognized as an efficacious topical antimicrobial agent in the treatment of

41

burns and wounds (Brudzynski, 2006). The healing properties of honey could be due to various

42

physical and chemical properties. The floral source of honey has an important role in its biological

43

properties. In general, dark-colored honey contains increased concentrations of compounds showing

44

antibacterial and antioxidant properties (Gheldof et al., 2002).

45

Buckwheat honey, produced from buckwheat flowers, is characterized by a dark brown,

46

almost black, color and a strong aroma with a flavor similar to molasses (Pasini et al., 2013).

47

Among honeys, this variety is the richest source of antioxidants that can reduce the oxidative stress

48

induced by reactive oxygen species (ROS) (Gheldof et al., 2003). Buckwheat honey included in the

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human diet, supply the body with exogenous antioxidants that support the endogenous antioxidant

50

defense system. Moreover, buckwheat honey was found to help reduce cholesterol levels in the

51

blood, which can improve heart health and even reduce high blood pressure (Giménez-Bastida and

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Zieliński, 2015). Additionally, buckwheat honey is more effective for respiratory infections, such as

53

colds, than some over-the-counter cough medicines (Paul et al., 2007).

54

Although the basic composition and nutritional profile of all types of honey are similar,

55

buckwheat honey has higher concentrations of macronutrients, trace elements, and vitamins

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(Kędzierska-Matysek et al., 2018; Wilczyńska, 2010). It has been reported that buckwheat honey

57

stands out from other varieties of honey with exceptionally high antibacterial (Paul et al., 2007) and

58

antioxidant activity (Gheldof et al., 2002). The antibacterial activity of honey is due to both

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enzymatic (glucose oxidase, catalase) and nonenzymatic (phenolics acids, flavonoids, ascorbic

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acids, organic acids, methylglyoxal, bee defensin-1) components (García-Tenesaca et al., 2018;

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Kwakman and Zaat, 2012). Among them, there is strong evidence suggesting that the hydrogen

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peroxide produced by glucose oxidase and the phenolic compounds in honey, including buckwheat3

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specific flavonoids, i.e., hesperetin and rutin (Cheng et al., 2017; Gheldof et al., 2003) are the main

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contributors to this activity (Brudzynski et al., 2012; Crushnie and Lamb, 2005; Sowa et al., 2017;

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Xie et al., 2015). Brudzynski et al. (2012) provided the first evidence that honeys with high

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bacteriostatic activity has significantly higher levels of phenolic compounds showing stronger

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radical scavenging activities than honeys with low bacteriostatic activity. They also suggested that

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the same polyphenols could become powerful pro-oxidants and could be involved in the generation

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of substantial amounts of hydrogen peroxide, which in the presence of transition metals such as

70

Cu(I) or Fe(II) can be converted into hydroxyl radicals using the Fenton reaction (Brudzyński et al.,

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2012). Buckwheat honey has all the necessary substrates for the Fenton reaction in high

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concentrations, including hydrogen peroxide produced by glucose oxidase, polyphenols and

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transition metal ions (Bogdanov et al., 2007). This coupling reaction is associated with the

74

bacteriostatic activity of honey (Brudzynski et al. 2012).

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The aim of this study was to explore the factors influencing the antioxidant and antibacterial

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action of buckwheat honey and to determine the role of polyphenols in the antibacterial activity of

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this type of honey. Moreover, the protective effect of buckwheat honey against oxidative stress in in

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vivo experiments using yeast cells was tested.

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2. Materials and methods

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2.1.

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Twenty three samples of buckwheat honey collected by various beekeepers working in southeastern

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Poland in the Lublin region in the 2017 season were studied. The honey variety was declared by

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producers based on the availability of bee nectar flow. Freshly centrifuged honey samples were

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filtered using a honey stainless sieve (0.2 mm mesh diameter) (Łysoń, Klecza Dolna, Poland).

85

Samples were stored in the laboratory at 20°C until the time of analysis but no more than 3 months.

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The botanical origin of honey was verified by pollen analysis according to Panseri et al. (2013). The

Honey samples

4

87

microscopic analysis of honey (10 g) sediment (obtained by centrifugation at 1,250 x g (4000 rpm

88

in a 11459 rotor, model 351R, MPW Med. Instruments, Warsaw, Poland) for 15 min at 21ºC) which

89

allowed the establishment of the percentage of the buckwheat grains in the total honey pollen. Such

90

analysis was carried out personally by one of author (Swacha S.) based on her own collection of

91

floral

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(https://globalpollenproject.org/). The examination of 300 pollen grains was carried out under the

93

microscope (CX21 LED, Olympus, Tokyo, Japan) at a magnification of 400x. Twenty samples

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containing >45% buckwheat pollen grains were subjected to further analysis, the rest 3 were

95

rejected.

96

Physicochemical properties

97

Basic parameters, i.e., sugar and water content were measured directly using an Abbe refractometer

98

(Optica, Ponteranica, Italy) exactly according to the International Honey Commission (IHC) (2009).

99

Honey pH value was measured using a digital pH-meter in 10% (w/v) solution at 21°C whereas

100

total acidity was determined by titrating a 10% (w/v) solution of honey at 21ºC with 0.1 M NaOH

101

to pH 8.3 using a pH-meter (CP-401, Elmetron, Zabrze, Poland). Results were expressed as mval

102

kg-1.

103

A specific rotation was determined according to the procedure described by Bogdanov (2009).

104

Briefly, 12 g (p) of honey was dissolved in < 70ml distilled water, then 10 ml Carrez I solution

105

(10.6%

106

Zn(CH3COO)2⋅2H2O acidified using 3 g glacial acetic acid) were added and the solution brought to

107

100 ml. After 24 h storage at 21°C, the solution was filtered using medium filter paper of 65 g m-2

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(Poch S.A. Gliwice, Poland) and filtrate was transferred to a 1-dm polarimetric tube (l). Angular

109

rotation (α) was measured using a polarimeter (POL-1 Optika, Ponteranica, Italy), and specific

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rotation was calculated using the follow equation:

111

[α]20 D = (α × 100) / (l × p)

pollen

w/v

microscopic

aqueous

preparations

K4Fe(CN)6⋅3H2O)

and

and

10

an

ml

available

Carrez

II

pollen

solution

(24%

atlas

w/v

5

112

Results were expressed as + or - degree/g of honey (+ or - °).

113

Color intensity was determined spectrophotometrically according to Pontis et al. (2014). The

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absorbance (A) of a 50% aqueous honey solution centrifuged at 14,300 x g (14000 rpm in a 11204a

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rotor, model 55, MPW Med. Instruments) for 10 min at 21ºC was measured with a Biomate 3

116

spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

117

The level of 5-hydroxymethylfurfural (HMF) was measured using HPLC according to the Polish

118

standard for honey (IHC, 2009). The HPLC analyses were done in the Laboratory of Plant

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Biotechnology “Aeropolis" in Rzeszów (Poland) using a Gilson (Middleton, WI, USA)

120

chromatographic set equipped with a binary pump (Gilson 322), a diode array detector (Gilson

121

172), a column thermostat (Knauer, Berlin, Germany) and an autosampler with a fraction collector

122

(GX-271 Liquid Handler, Gilson). The separation was carried out using a Eurospher 100-5 C-18

123

column (250 x 4 mm and 5 µm grain diameter) (Knauer) at 35°C. The HPLC conditions were as

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follows: isocratic mobile phase, 90% water with 1% acetic acid, and 10% methanol; flow rate, 1 ml

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min-1; injection volume, 20 µl; time of analysis, 15 min. All the solvents were HPLC grade (Sigma

126

Aldrich Co., St. Louis, MO, USA). The detection was done at 285 nm. 5-hydroxymethylfurfural

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(HMF) content was calculated using an external calibration curve prepared for HMF standard

128

(Sigma Aldrich) in the range of 12.5 – 300 µg ml-1 (R2=0.999). The analyses were done in triplicate

129

and expressed as mg kg-1. Trilution software v.3.0 (Gilson) was used for data acquisition and

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processing.

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2.2.

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The antioxidant properties of the aqueous honey solutions (20% w/v) were determined using a ferric

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reducing/antioxidant power assay (FRAP assay) according to a modified procedure described by

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Wesołowska and Dżugan (2017). Aliquots of 0.2 ml 10% (w/v) honey solution were mixed with 1.8

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ml FRAP reagent (2.5 ml 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (Sigma Aldrich) in 40 mM

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HCl, 2.5 ml 20 mM FeCl3 (Sigma Aldrich) and 25 ml 0.3 M acetate buffer (pH 3.6)), and after 10

Antioxidant properties

6

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min incubation at 37°C absorbance was measured at 593 nm. A calibration curve was prepared

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using Trolox (Sigma Aldrich) solutions at 0-300 nmol ml-1. The results were expressed as the FRAP

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value (µmol Trolox equivalent (TE) 100 g-1 of honey).

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The antiradical activity of honey was investigated using the 2,2-diphenyl-picrylhydrazyl radical

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(DPPH) as described by Wilczyńska (2010) with some modifications. Exactly 1.5 ml 0.1 mM

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DPPH (Sigma Aldrich) methanolic solution was added to 0.2 ml aqueous honey solution (20%

143

w/v). After 60 min, the absorbance (A) was measured at 517 nm. The antioxidant activity (AA) was

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expressed as the percentage of DPPH discoloration using the following formula:

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AA [%]=([Acontrol-Asample]/Acontrol)×100.

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2.2.1. Total phenolic content

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The total phenolic content (TPC) was measured using a modified method described by Wilczyńska

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(2010). Aliquots of 0.2 ml 10% (w/v) honey solution were mixed with 1 ml Folin-Ciocalteu reagent

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(Merck KGaA, Darmstadt, Germany) previously diluted 1:10 with distilled water followed by the

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addition of 0.8 ml 7.5% (w/v) Na2CO3 (Poch). After incubation at room temperature (21º±2) for

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120 min, the absorbance of the reaction mixture was measured at 760 nm against a blank. Gallic

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acid (GA) (Sigma Aldrich) was used (0-250 µg ml-1) for calibration. The results were expressed as

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mg GAE 100 g-1 of honey.

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2.2.2. Flavonoid content

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The total flavonoid content (TFC) of the aqueous honey solutions (20% w/v) was measured using

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an aluminum chloride spectrophotometric assay modified by Pontis et al. (2014). A honey solution

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(0.5 ml) was mixed with 1.5 ml 5% (w/v) aluminum chloride (Sigma Aldrich) methanolic solution.

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After 30 min incubation at room temperature, the absorbance was measured at 437 nm against a

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methanol blank. A standard curve of quercetin (Q) (Sigma Aldrich) was prepared within a

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concentration range of 0-40 µg ml-1, and the results were expressed as mg QE 100 g-1 of honey. 7

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2.2.3. Polyphenolic profile

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Sample extraction

163

For UPLC-MS analysis, phenolic compounds were extracted from honey as reported by Gómez-

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Caravaca et al. (2006). Approximately 20 g of honey was dissolved in 5 parts (100 ml) of acidified

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water (pH 2 with HCl (Poch, Gliwice, Poland) and was stirred at room temperature until the mixture

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was homogenous. The solution was filtered through a 0.45 µm nylon filter disc (Sigma Aldrich) and

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passed through a Sep Pak C18 Cartridge (Waters, Milford, CT, USA) preconditioned with water

168

and subsequently rinsed with 10 ml distilled water. The phenolic compounds remained on the

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column, while sugars and other polar compounds were eluted with the aqueous solvent. The whole

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phenolic fraction was then eluted with 10 ml HPLC grade methanol (Poch), and the solvent was

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evaporated in a rotary evaporator Hei-VAP Advantage (Heidolph Instruments, Walpersdorfer,

172

Schwabach, Germany) at 50ºC under reduced pressure. The residue was redissolved in 1 ml of

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50:50 (v:v) HPLC grade acetonitrile (Poch) and water mixture. Finally, all extracts were filtered

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through 0.45 µm nylon filter discs and subjected to UPLC–MS analysis.

175

HPLC analysis

176

Polyphenolic compounds were measured using the UPLC-PDA-MS/MS Waters ACQUITY system

177

(Waters), consisting of a binary pump manager, sample manager, column manager, PDA detector

178

and tandem quadrupole mass spectrometer (TQD) with electrospray ionization (ESI). The

179

separation was done using a BEH C18 column (100 x 2.1 mm i.d., 1.7 µm, Waters) kept at 50°C.

180

For the polyphenolics, the following solvent system was applied: mobile phase A (0.1% formic acid

181

in water v/v) and mobile phase B (0.1% formic acid in 40% ACN in water v/v). All solvents were

182

HPLC grade purchased from Sigma Aldrich. The gradient program was as follows: 0 min 5% B;

183

from 0 to 8 min linearly increase to 100% B; and from 8 to 9.5 min washing and return to initial

184

conditions. The injection volume of the samples was 5 µl, and the flow rate was 0.35 ml min-1. The

185

following parameters were used for the TQD: capillary voltage, 3.5 kV; con voltage, 30 V in 8

186

negative mode; source temperature, 250°C; desolvation temperature, 350°C; con gas flow, 100 l h-1;

187

and desolvation gas flow, 800 l h-1. Argon was used as the collision gas at a flow rate of 0.3 ml min-

188

1

189

ratio and fragment ions obtained after collision-induced dissociation (CID). Quantification was

190

determined by injection of solutions of known concentrations ranging from 0.05 to 5 mg ml-1

191

(R2≤0.999) of the following phenolic compounds as standards: protocatechuic acid, p-

192

hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, quercetin-3-

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rutinoside, quercetin-3-glucoside, quercetin, myricetin, kaempferol, apigenin, galangin (Sigma

194

Aldrich). All determinations were done in triplicate and expressed as µg 100 g-1. The intra- and

195

inter-day variations were determined using relative standard deviation (RSD) values were <3.5%

196

for all the analyzed compounds. Waters MassLynx software v.4.1 was used for data acquisition and

197

processing.

198

2.3. Antibacterial properties

199

2.3.1. Hydrogen peroxide determination

200

Hydrogen peroxide concentrations accumulated in diluted honey were determined according to

201

Sowa et al. (2017). Briefly, honey samples (40 µl) were mixed with 135 µl reagent (50 µg ml-1 o-

202

dianisidine and 20 µg ml-1 horseradish peroxidase (Sigma Aldrich) in 10 mM sodium phosphate

203

buffer at pH 6.5). Samples were incubated for 5 min at room temperature, and the reaction was

204

stopped by the addition of 120 µl 6 M H2SO4. The absorption was measured at 540 nm. For the

205

calibration curve, 30% H2O2 (perhydrol) (Sigma Aldrich) at 0–100 mM was used. Results were

206

expressed as mmol of hydrogen peroxide l-1 of 30% honey solution.

207

Antibacterial assay

208

The antibacterial potency of buckwheat honey was tested against 4 bacterial strains: the Gram-

209

positive Staphylococcus aureus ATCC 25923 and the Gram-negative Escherichia coli ATCC

. The polyphenol detection and identification were based on specific PDA spectra, mass to charge

9

210

25922, Salmonella eneterica and Klebsiella pneumoniae ATCC 700600 all of which originated

211

from the collection of the Department of Biotechnology and Microbiology, Faculty of Biology and

212

Agriculture, University of Rzeszow (Rzeszów, Poland). The overnight bacterial cultures grown on

213

Mueller-Hinton (Sigma Aldrich) agar plates were suspended in water to obtain an optical density

214

(OD) at 600 nm = 0.132 (corresponds to 0.5 McFarland turbidity standard (Kuś et al., 2016), which

215

was previously confirmed experimentally (Kuś et al., 2016, Grabek-Lejko et al., 2018). Then

216

bacteria were diluted in double concentrated Mueller-Hinton broth (MHB) medium to final cells

217

concentrations of 1-5 x 106 CFU ml-1 and used for the determination of minimum inhibitory

218

concentration. Fifty % (w/v) of each honey sample was prepared in water and then sterilized using

219

filtration through a polytetrafluoroethylene (PTFE) membrane filter (0.45 µm) (Sigma Aldrich)

220

(Sowa et al., 2017).

221

Minimum inhibitory concentration (MIC)

222

The broth microdilution method was used to determine the MIC of the honey samples (Kuś et al.,

223

2016). Briefly, 2-fold serial dilutions from a stock solution of each honey sample were prepared and

224

mixed with equal volume of previously prepared bacterial suspensions in 100-well honeycomb

225

plates (Growth Curve Oy, Helsinki, Finland). The final concentration of inoculated honey samples

226

ranged from 0.375 to 25% (w/v). The plates were incubated at 37°C for 24 h in a Bioscreen cell

227

analyzer (Growth Curve Oy), with continuous medium shaking. OD at 600 nm was measured every

228

h. As a positive control, bacterial growth without honey addition was measured. The MIC value was

229

defined as the lowest concentration of honey inhibiting bacterial growth by a minimum of 90%. The

230

influence of catalase on the inhibition of bacterial growth was tested with the addition of catalase

231

(Sigma Aldrich) to a final concentration of 250 U ml-1.

232

Minimum bactericidal concentration (MBC)

233

To determine the MBC, 5 µl of suspensions from wells (from the previous experiment (MIC))

234

showing no visible sign of growth/turbidity at the MIC were subcultured on sterile Mueller-Hinton 10

235

agar plates and incubated at 37°C for 24 h. The lowest concentration of honey that did not show any

236

growth of the tested organisms was considered to be the MBC (Kacaniova et al., 2011).

237

2.4. Protective effect of buckwheat honey during oxidative stress

238

A wild-type strain of the yeast Saccharomyces cerevisiae (SP4 MATα leu1 arg4) (Bilinski et al,

239

1978) was used for this study. The yeast obtained from the laboratory yeast strains collection of the

240

Department of Cell Biology and Biochemistry, University of Rzeszow was grown in a standard

241

liquid yeast extract–peptone–dextrose (YPD) medium (1% yeast extract, 1% yeast bacto-peptone

242

(BD Difco, Becton, Dickinson and Co., Franklin Lakes, NJ, USA), and 2% glucose (Poch)) on a

243

rotary shaker at 150 rpm at 28°C. Cultured cells from the exponential growth phase (at 16 h of

244

growth) were centrifuged (4000 x g for 5 min at 4°C), washed twice and suspended at a density of

245

108 cells ml-1 in 100 mM sodium phosphate buffer at pH 7.0, containing 1 mM EDTA (Sigma

246

Aldrich) and 0.1% glucose. Honey was added to the cell suspensions (0.5 ml of 50% w/v of honey

247

solution per 10 ml of cell suspension), and the mixture was incubated at 28°C with shaking for 1 h.

248

Then, the cell samples were removed, centrifuged (as previously), washed and suspended in

249

phosphate buffer. The cells suspensions were immediately placed in a microplate and directly

250

treated with hydrogen peroxide (Sigma Aldrich, 2 mM final concentration prepared directly before

251

use). Then, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA (Sigma Aldrich) 10.3 µM final

252

concentration, stock in 96% ethanol) was added to quantify ROS. The kinetics of the increase in

253

fluorescence, due to the oxidation of the fluorogenic probe, was measured immediately after

254

addition of the probe using a Tecan Infinite M-200 microplate reader (Tecan Group Ltd.,

255

Männedorf, Switzerland) with excitation/emission maxima at 495/525 nm for DCF-DA at 28°C

256

(Cathcart et al. 1983). The possible interaction between the probes and honey in a blank, the buffer

257

without the cells, was measured. The ROS content were measured in 3 independent experiments,

258

and each sample was measured in 3 technical replicates.

11

259

2.5. Statistical analysis

260

The results are shown as the mean values with standard deviations (SD). Significant differences

261

(p<0.05) between honey parameters were determined using one-way analysis of variance followed

262

by Fisher’s least significant differences (LSD) test. The correlation between some parameters was

263

calculated using Spearman’s correlation test. The Shapiro-Wilk test was used to test the normality

264

of catalase influence on the antibacterial activity of honey. Then, the Wilcoxon signed-rank test was

265

used to determine the statistical significance of adding catalase to the honey samples compared with

266

honey without catalase. Principal component analysis (PCA) was used to evaluate the relationship

267

between individual phenolic compounds, antioxidant and antibacterial activity. All calculations

268

were obtained using Statistica 10.0 software (StatSoft, Inc., Tulsa, OK, USA).

269

3.

270

3.1. Physicochemical quality parameters

Results

271

The quality of the honey samples was evaluated based on the results of the basic

272

physicochemical parameters (Table 1). All samples met the legal requirements for nectar honey

273

according to water (<20%) and sugar content (>65%) as well as acidity (<50 mval kg-1). HMF

274

content, the parameter indicating the proper handling of honey, was detected at a very low level and

275

exceeded the limit in only one sample (2). The level of F. esculentum pollen in all the tested

276

samples of buckwheat honey, excluding 2 and 8, complied with the Polish requirements (minimum

277

45% of predominant pollen). Moreover, the results were comparable with the findings of domestic

278

and foreign authors, although this type of honey is not frequently tested (Azonwade et al., 2018;

279

Panseri et al., 2013; Pasini et al., 2013).

280

Other tested parameters are not regulated; however, they are commonly used in the quality

281

evaluation of honey (Alvarez-Suarez et al., 2010; Gheldof et al., 2002; Ramanauskiene et al., 2012).

282

The specific rotation, a common criterion for differentiation of nectar and honeydew honeys, was 12

283

negative for all tested samples. However, this parameter was not stable and probably resulted from

284

differences in sugar composition but not in total sugar content (Spearman’s rank r=0.273). A high

285

variability in the color intensity of the tested honeys was observed (coefficient of variability CV

286

43%). Moreover, it was observed that mostly flavonoids influenced the color of honey, which was

287

confirmed using the correlation coefficient between color and flavonoids (r=0.888) as well as color

288

and total phenolic compounds (r=0.663). This results are consistent with other authors, who

289

observed a strong relationship between honey color and its ingredients, especially polyphenols

290

(Gheldof et al., 2002; Pasini et al., 2013).

291

3.2. Antioxidant activity

292

The antioxidant activities of the honey samples were assayed using the DPPH (as antiradical

293

scavenging activity) and FRAP (as reducing capacity) tests (Table 2). Moreover, TPC and TFC

294

were determined (Table 2).

295

A strong but variable antioxidant activity for the buckwheat honey samples, regardless of

296

the method used, was observed (Table 3). The weak relationship (p≥0.05) between the results of

297

DPPH and other methods is mainly due to the different mechanisms of the tests; the DPPH method

298

serves to measure only hydrophobic antioxidants (Amarowicz et al., 2004).

299

The results are consistent with the findings of other studies examining Polish (Wesołowska

300

and Dżugan, 2017; Wilczyńska, 2010) and foreign (Gheldof et al., 2002) buckwheat honeys. In

301

comparison to other light and dark honeys, this variety of honey was characterized by the highest

302

antioxidant activity regardless of the method used. Similarly, the highest antioxidant activity of

303

buckwheat honey measured using the DPPH test (in the range of 35 to 57%) as compared to other

304

honey varieties, i.e., tilia - 12%, rape - 14%, heather - 28%, and honeydew - 33% was observed by

305

Wesołowska and Dżugan (2017). Simultaneously, analogous trends in the FRAP and TPC results

306

were observed.

13

307

3.3. Antibacterial activity

308

Because of the growing resistance of pathogenic microorganisms to antibiotics and the lack

309

of therapeutic options, new treatment approaches are needed (Morroni et al., 2018). Honey may be

310

one such potential therapeutic alternative. The susceptibility of 4 representative species of Gram-

311

positive and Gram-negative bacteria (S. aureus, E. coli, S. enterica, K. pneumoniae) to 20

312

buckwheat honeys was investigated. According to the World Health Organization, these bacteria

313

species are the most commonly reported antibiotic-resistant bacteria (WHO, 2014).

314

The MIC and MBC values for each honey sample are shown in Table 4. All samples

315

inhibited bacterial growth to different extents, with the highest antibacterial properties against

316

Gram-positive bacteria (S. aureus, the lowest MIC - 6.25%), followed by the Gram-negative

317

bacteria: E.coli > S. enterica > K. pneumoniae. Similarly, Hammond et al. (2016) observed that

318

among a broad spectrum of antimicrobial properties of buckwheat honey from the USA, Gram-

319

positive bacteria were slightly more susceptible than Gram-negative bacteria. They found the MIC

320

values 15 and 20% for S. aureus and K. pneumonia, respectively. Additionally, for Ethiopian

321

buckwheat honeys, a higher MIC was observed for K. pneumoniae than for S. aureus and E. coli

322

(Wasihun and Kasa, 2016). However, some authors did not observe any antibacterial effects of

323

honeys on K. pneumoniae.

324

The variation in bacterial sensitivity among species could be attributed to differences in the

325

growth rate and reduced cell wall permeability of the pathogen. For Canadian buckwheat honey,

326

MIC values against S. aureus ranged from 3.13 to 12.5% (v/v), but for most samples, it was 6.25%

327

(Brudzynski et al., 2012), which corresponds to the current results. Similar MIC values, ranging

328

from 3.13 to 12.5%, were observed for different types of honey from Greece (Stagos et al., 2018).

329

The MIC values of Canadian buckwheat honey against E. coli for most samples were similar to

330

those of Polish honey (12.5%). The percentage of samples with stronger antibacterial potential

331

against E. coli was comparable in Polish and Canadian buckwheat honey (12.5 - 15%). Consistent 14

332

with this study, Brudzynski et al. (2012) observed the same MIC and MBC values for some

333

samples, whereas other samples had higher MBC values. Compared with the buckwheat honey

334

tested in the current study, other Polish unifloral honey samples showed rather high antibacterial

335

potential (Grabek-Lejko et al., 2018; Kuś et al., 2016; Sowa et al., 2017). Moreover, Deng et al.

336

(2018) concluded that the antibacterial potential of buckwheat honey is comparable to that of

337

Manuka honey, which has well-known antibacterial and antioxidant potential.

338

3.4. Hydrogen peroxide-dependent antibacterial activity of buckwheat honey

339

Hydrogen peroxide was detected in all the buckwheat honeys (Table 4). As the level of

340

hydrogen peroxide is a measure of glucose oxidase activity, honey can be divided into two groups:

341

1 - with high glucose oxidase activity and 2 - with weak glucose oxidase activity. It is consistent

342

with Brudzynski et al. (2012) who reported large variability of glucose oxidase activity among

343

Canadian buckwheat honey samples. Similar variability in the glucose oxidase activity was

344

observed in melilot honey by Sowa et al. (2017). Hydrogen peroxide is a heat- and light-sensitive

345

substance, and its level in honey is strongly influenced by the handling method of beekeepers as

346

well as storage conditions (Brudzynski and Kim, 2011).

347

To investigate the contribution of H2O2 to the antibacterial action of honey, the samples

348

were treated with catalase (an enzyme that catalyzes the decomposition of H2O2 into water and

349

oxygen) and incubated with bacterial strains. The removal of H2O2 reduced (by ∼40-50%) the

350

antibacterial effect of buckwheat honey in most samples against all the tested bacteria (p<0.05) but

351

did not inhibit it completely (Fig. 1). Despite the suppression of activity related to oxidase, the

352

highest bacterial growth inhibition was observed mainly in samples with the strongest total

353

antibacterial activity. The present study has confirmed that the antibacterial activity of Polish

354

buckwheat honey is the result of both hydrogen peroxide and the occurrence of some non-peroxide

355

components, which is consistent with findings for Canadian buckwheat honey (Brudzyński et al.,

356

2012) and other honey varieties (Brudzynski et al., 2011; Sowa et al., 2017). 15

357

3.5. The in vitro antioxidant activity of honey

358

The polyphenolic profiles of 5 buckwheat honey samples with the highest (11 and 12),

359

moderate (18) and lowest (1 and 17) antioxidant activities, were determined using UPLC-PDA-

360

MS/MS (Table 5). Thirteen polyphenolic compounds, 6 phenolic acids (protocatechuic, p-

361

hydroxybenzoic, chlorogenic, caffeic, p-coumaric and ferulic) and 7 flavonoids (quercetin-3-

362

rutinoside, quercetin-3-glucoside, quercetin, myricetin, kaempferol, apigenin and galangin), were

363

found in all the samples. The phenolic compounds have been previously observed in various honey

364

types (Alvarez-Suarez et al., 2010; Ramanauskiene et al., 2012), and some had been found in

365

buckwheat honey (Jasicka-Misiak et al., 2012; Pasini et al., 2013). The comparison of the phenolic

366

compound profiles of samples 11, 12 and 18 with high antioxidant activity with those of samples 1

367

and 17 with lower activity provided some evidence that quercetin, rutin (quercetin-3-rutinoside),

368

chlorogenic and caffeic acids may be mainly responsible for the bioactivity of buckwheat honey.

369

Moreover, the level of these phenolic compounds was strongly correlated with the antioxidant

370

activity measured using the DPPH assay (Spearman’s rank r > 0.432) as well as the FRAP (r >

371

0.551) and TPC (r > 0.500) tests.

372

Based on PCA analysis, the antioxidant activity measurements were negatively correlated

373

with the MIC values (Fig. 2). The lower the MIC the stronger the antibacterial activity. These

374

results were consistent with the antioxidant and antibacterial activities of buckwheat honey being

375

positively correlated. A similar calculation showed that some polyphenols (quercetin, rutin,

376

chlorogenic and caffeic acids) significantly affected the antibacterial activity of buckwheat honey.

377

Results are consistent with Pasini et al. (2013), who investigated polyphenols in Italian buckwheat

378

honey.

379

The protective effect of the same samples on the S. cerevisiae cells exposed to hydrogen

380

peroxide (very strong ROS generator) was determined (Fig. 3). Because the marker molecule

381

H2DCF-DA penetrates the plasma membrane and is oxidized only inside the cell by ROS, the 16

382

increase in fluorescence is a measure of the cellular free radical level. In the control yeast cells, the

383

protective effect of honey against endogenic ROS was observed in all samples (Fig. 3). Due to

384

differences in antioxidant activity, the concentration of ROS in the yeast cells incubated with honey

385

differed slightly between honey samples. Compared to the untreated cells, a significant increase in

386

peroxide production was observed after the addition of hydrogen peroxide. The highest ROS level

387

was found in the control yeast, and a significant inhibition of ROS was observed in all groups,

388

which were protected by the addition of honey (Fig. 3). The strongest protective effect was seen

389

with samples 11 and 12 (with the highest quercertin, rutin and chlorogenic acid contents), and the

390

weakest protective effect was observed with honey sample 1 (the lowest polyphenol content).

391

Moreover, antioxidant activity and H2O2 content in honey samples were negatively correlated

392

(p<0.05) with ROS level in yeasts cells (Spearman’s rank from 0.816 for H2O2 to 0.948 for FRAP).

393

Meanwhile, antibacterial activity (expressed using MIC) was positively correlated (p<0.05) with

394

ROS level in H2O2 treated yeasts (Spearman’s rank from 0.744 for S. enterica to 0.794 for other

395

bacterial strains). This suggested that antioxidant and antibacterial activities of buckwheat honey

396

were directly interlinked with its protection effect on yeasts cells during endogenous generated

397

stress. These results are consistent with Alugoju et al. (2017), who showed that quercetin protects

398

S. cerevisiae cells against oxidative stress. Additionally, several cinnamic acid derivatives (trans-

399

cinnamic, p-coumaric, caffeic and ferulic acids, as well as caffeic acid-methyl and caffeic acid-

400

propyl esters) were found to protect cells from oxidative stress-induced DNA damage (Kitsati et

401

al., 2012). Zhou et al. (2012) observed a protective effect of buckwheat honey on fish DNA

402

damage induced by H2O2. It was also shown that among different honeys, buckwheat honey,

403

which has a high antioxidant, protected mouse lymphocytes against hydrogen peroxide-induced

404

DNA damage (Cheng et al., 2017). The authors suggested that the phenolic acids in honey can

405

penetrate into lymphocytes and protect DNA from oxidative damage by scavenging hydrogen

406

peroxide and/or chelating ferrous ions. 17

407

According to the available data, the protective effect of buckwheat honey in the yeast model

408

has not previously been observed. However, the beneficial protective effect of pretreatment with

409

honey against oxidative stress was observed in another eukaryotic model organism, Drosophila

410

melanogaster (Cruz et al., 2014), following exposure to paraquat and iron. The protective effect of

411

Manuka honey against oxidative damage was observed in macrophages (inflammation mediatory

412

immune cells), which was related to the suppression of ROS and nitrite production as well as the

413

protection of lipid, protein and DNA damage (Gasparrini et al., 2018). Based on the similar

414

antibacterial power of buckwheat and Manuka honeys (Deng et al., 2018), their protective action is

415

hypothesized to be based on a similar mechanism; however, further studies are needed.

416

4.

Conclusions

417

The health benefits of Polish buckwheat honey as well as the factors influencing its

418

antioxidant activity in vitro and in vivo were studied. It was shown that the antibacterial activity of

419

buckwheat honey is due to both enzymatic and nonenzymatic factors. In the samples with the

420

highest antibacterial activity, a strong inhibition of antimicrobial activity was observed when

421

catalase was used to decompose hydrogen peroxide (enzyme-dependent factor). Moreover, it was

422

shown that phenolic compounds, especially quercetin, rutin, chlorogenic and caffeic acids, were

423

probably responsible for the antioxidant and antibacterial activity of buckwheat honey. The

424

protective effect of buckwheat honey against endogenous ROS was shown using S. cerevisiae as a

425

biological model.

426

Conflict of Interest

427

The authors confirm that they have no conflict of interest with respect to the work described in

428

the manuscript.

429

Acknowledgments

18

430

This study was financially supported by the Polish Ministry of Science and Higher Education

431

(Project no PB/KCHTZ/2017 University of Rzeszów in the year 2017) and the project financed

432

under the program of the Minister of Science and Higher Education entitled "Regional Initiative

433

of Excellence "in 2019-2022 project no. 026/RID/2018/19.

434

References

435

Alugoju, P., Janardhanshetty, S.S., Subaramanian, S., Periyasamy, L., & Dyavaiah, M. (2017).

436

Quercetin protects yeast Saccharomyces cerevisiae pep4 mutant from oxidative and apoptotic stress

437

and extends chronological lifespan. Current Microbiology, https://doiorg/101007/s00284-017-1412-

438

x

439

Alvarez-Suarez, J.M., González-Paramáz, A.M., Santos-Buelga, C., & Battino, M. (2010).

440

Antioxidant characterization of native monofloral Cuban honeys. Journal of Agricultural and Food

441

Chemistry, 58, 9817–9824.

442

Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., & Weil, J.A. (2004). Free-radical

443

scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies.

444

Food Chemistry, 84, 551–56.

445

Azonwade, F.E., Paraïso, A., Dossa, C.P.A., Dougnon, V.T., N’tcha, C., Mousse, W., & Baba-

446

Moussa, L. (2018). Physicochemical characteristics and microbiological quality of honey produced

447

in Benin. Journal of Food Quality, Article ID 1896057, 13 pages.

448

Bilinski, T., Lukaszkiewicz, J., & Sledziewski, A. (1978). Demonstration of anaerobic catalase

449

synthesis in the cz1 mutant of Saccharomyces cerevisiae. Biochemical and Biophysical Research

450

Communications, 83(3), 1225-1233.

451

Bogdanov, S. (2009). Honey composition, Chapter 5, In: The Honey Book. Bee Product Science

452

www.bee-hexagon.net, accessed: 25.09.2018.

19

453

Bogdanov, S., Haldimann, M., Luginbühl, W., & Gallmann, P. (2007). Minerals in honey:

454

Environmental, geographical and botanical aspects. Journal of Apicultural Research and Bee

455

World, 46(4), 269–275.

456

Brudzynski, K. (2006). Effect of hydrogen peroxide on antibacterial activities of Canadian honeys.

457

Canadian Journal of Microbiology, 52, 1228–1237.

458

Brudzynski, K., & Kim, L. (2011). Storage-induced chemical changes in active components of

459

honey deregulate its antibacterial activity. Food Chemistry, 126, 1155–1163.

460

Brudzynski, K., Abubaker, K., St Martin, L., & Castle, A. (2011). Re-examining the role of

461

hydrogen peroxide in bacteriostatic and bactericidal activities of honey. Frontiers in Microbiology,

462

2(213), 1-9.

463

Brudzynski, K., Abubaker, K., & Wang, T. (2012). Powerful bacterial killing by buckwheat honeys

464

is conentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

465

Frontiers in Microbiology, 3(242), 1-9.

466

Cathcart, R., Schwiers, E., & Ames, B.N. (1983). Detection of picomole levels of hydroperoxides

467

using a fluorescent dichlorofluorescein assay. Analytical Biochemistry, 134 (1), 111-116.

468

Cheng, N., Wang, Y., & Cao, W. (2017). The protective effect of whole honey and phenolic extract

469

on oxidative DNA damage in mice lymphocytes using comet assay. Plant Foods for Human

470

Nutrition, 72, 388-395.

471

Cruz, L.C., Batista, J.E.S., Zemolin, A.P.P., Nunes, M.E.M., Lippert, D.B., Royes, L.F.F., Soares,

472

F.A., Pereira, A.B., Posser, T., & Franco, J.L. (2014). A study on the quality and identity of

473

Brazilian pampa biome honey: Evidences for its beneficial effects against oxidative stress and

474

hyperglycemia. International Journal of Food Science, 2014, Article ID 470214, 11 pages,

475

http://dxdoiorg/101155/2014/470214

476

Cushnie, T., & Lamb, A. (2005). Antimicrobial activity of flavonoids. International Journal of

477

Antimicrobial Agents, 26, 343-356. 20

478

Deng, J., Liu, R., Lu, Q., Hao, P., Xu, A, Zhang, J., & Tan, J. (2018). Biochemical properties,

479

antibacterial and cellular antioxidant activities of buckwheat honey in comparison to Manuka

480

honey. Food Chemistry, 252, 243-249.

481

García-Tenesaca, M., Navarrete, E.S., Iturralde, G.A.,Villacrés Granda, I.M., Tejera, E., Beltrán-

482

Ayala, P., Giampieri, F., Battino, M., & Alvarez-Suarez, J.M. (2018). Influence of botanical origin

483

and chemical composition on the protective effect against oxidative damage and the capacity to

484

reduce in vitro bacterial biofilms of monofloral honeys from the Andean region of Ecuador.

485

International Journal of Molecular Sciences, 19(1): 45. doi: 10.3390/ijms19010045.

486

Gasparrini, M., Afrin, S., Forbes-Hernández, T.Y., Cianciosi, D., Reboredo-Rodriguez, P., Amici,

487

A., Battino, M., & Giampieri, F. (2018). Protective effects of Manuka honey on LPS-treated RAW

488

264.7 macrophages. Part 2: Control of oxidative stress induced damage, increase of antioxidant

489

enzyme activities and attenuation of inflammation. Food and Chemical Toxicology, 120, 578-587.

490

Gheldof, N., Wang, X.H., & Engeseth, N.J. (2002). Identification and quantification of antioxidant

491

components of honeys from various floral sources. Journal of Agricultural and Food Chemistry, 50,

492

5870–5877.

493

Gheldof, N., Wang, X.H., & Engeseth, N.J. (2003). Buckwheat honey increases serum antioxidant

494

capacity in humans. Journal of Agricultural and Food Chemistry, 51, 1500–1505.

495

Giménez-Bastida, J.A., & Zieliński, H. (2015). Buckwheat as a functional food and its effects on

496

health. Journal of Agricultural and Food Chemistry, 63, 7896–7913.

497

Gómez-Caravaca, A.M., Segura-Carretero, A., & Fernández-Gutiérrez, A. (2006). Problems of

498

quantitative and qualitative estimation of polyphenols in honey by capillary electrophoresis with

499

UV–vis detection. Agro Food Industry Hi-Tech, 17, 68–71.

500

Grabek-Lejko, D., Słowik, J., & Kasprzyk, I. (2018). Activity of selected honey types against

501

Staphylococcus aureus methicillin susceptible (MSSA) and methicillin resistant (MRSA) bacteria

21

502

and its correlation with hydrogen peroxide, phenolic content and antioxidant capacity. Farmacia,

503

66(1), 37-43.

504

Hammond, E.N., Duster, M., Musuuza, J.S., & Safdar, N. (2016). Effect of United States

505

buckwheat honey on antibiotic-resistant hospital acquired pathogens. Pan African Medical Journal,

506

25, 212. doi:1011604/pamj20162521210414

507

IHC, 2009. Harmonised Methods of the International Honey Commission (Available on:

508

http://www.bee-hexagon.net/en/network.htm, accessed: 15.05.2019)

509

Jasicka-Misiak, I., Poliwoda, A., Dereń, M., & Kafarski, P. (2012). Phenolic compounds and

510

abscisic acid as potential markers for the floral origin of two Polish unifloral honeys. Food

511

Chemistry, 131, 1149–1156.

512

Kacaniova, M., Vukovic, N., Bobkova, A., Fikselova, M., Rovna, K., Hascik, P., Čuboň, J., Hleba,

513

L., & Bobko, M. (2011). Antimicrobial and antiradical activity of Slovakian honeydew honey

514

samples. Journal of Microbiology Biotechnology and Food Sciences, 1, 354–360.

515

Kędzierska-Matysek, M., Florek, M., Wolanciuk, A., Barłowska, J., & Litwińczuk, Z. (2018).

516

Concentration of minerals in nectar honeys from direct sale and retail in Poland. Biological Trace

517

Element Research, https://doi.org/10.1007/s12011-018-1315-0.

518

Kitsati, N., Fokas, D., Ouzouni, M.D., Mantzaris, M.D., Barbouti, A., & Galaris, D. (2012).

519

Lipophilic caffeic acid derivatives protect cells against H2O2-induced DNA damage by chelating

520

intracellular labile iron. Journal of Agricultural and Food Chemistry, 15, 7873-7879.

521

Kuś, P.M., Szweda, P., Jerković, I., & Tuberoso, C.I. (2016). Activity of Polish unifloral honeys

522

against pathogenic bacteria and its correlation with colour, phenolic content, antioxidant capacity

523

and other parameters. Letters in Applied Microbiology, 62, 269-276.

524

Kwakman, P.H., & Zaat, S.A. (2012). Antibacterial components of honey. IUBMB Life, 64(1), 448-

525

455.

22

526

Morroni, G., Alvarez-Suarez, J.M., Brenciani, A., Simoni, S., Fioriti, S., Pugnaloni, A., Giampieri,

527

F., Mazzoni, L., Gasparrini, M., Marini, E., Mingoia, M., Battino, M., & Giovanetti, E. (2018).

528

Comparison of the antimicrobial activities of four honeys from three countries (New Zealand, Cuba,

529

and Kenya). Frontiers in Microbiology, 25, 9:1378. doi: 10.3389/fmicb.2018.01378.

530

Panseri, S., Manzo, A., Chiesa, L. M., & Giorgi, A. (2013). Melissopalynological and volatile

531

compounds analysis of buckwheat honey from different geographical origins and their role in

532

botanical

533

http://dxdoiorg/101155/2013/904202

534

Pasini, F., Gardini, S., Marcazzan, G.L., & Caboni, M.F. (2013). Buckwheat honeys: Screening of

535

composition and properties. Food Chemistry, 141, 2802-2811.

536

Paul, I.M., Beiler, J., McMonagle, A., Shaffer, M.L., Duda, L., & Berlin, C.M.Jr. (2007). Effect of

537

honey, dextromethorphan, and no treatment on nocturnal cough and sleep quality for coughing

538

children and their parents. Archives of Pediatrics and Adolescent Medicine, 161, 1140-1146.

539

Pontis, J.A., da Costa, L.A.M.A., da Silva, S.J.R., & Flach A. (2014). Color, phenolic and flavonoid

540

content, and antioxidant activity of honey from Roraima, Brazil. Food Science and Technology, 34,

541

69-73.

542

Ramanauskiene, K., Stelmakiene, A., Briedis, V., Ivanauskas, L., & Jakštas, V. (2012). The

543

quantitative analysis of biologically active compounds in Lithuanian honey. Food Chemistry, 132,

544

1544–1548.

545

Sowa, P., Grabek-Lejko, D., Wesołowska, M., Swacha, S., & Dżugan, M. (2017). Hydrogen

546

peroxide-dependent antibacterial action of Melilotus albus honey. Letters in Applied Microbiology,

547

65, 82-89.

548

Stagos, D., Soulitsiotis, N., Tsadila, C., Papaeconomou, S., Arvanitis, C., Ntontos, A., Karkanta, F.,

549

Adamou-Androulaki, S., Petrotos, K., Spandidos, D.A., Kouretas, D., & Mossialos, D. (2018).

determination.

Journal

of

Chemistry,

Article

ID

904202,

11

pages,

23

550

Antibacterial and antioxidant activity of different types of honey derived from Mount Olympus in

551

Greece. International Journal of Molecular Medicine, 42(2), 726-734.

552

Wasihun, A.G., & Kasa, B.G. (2016). Evaluation of antibacterial activity of honey against

553

multidrug resistant bacteria in Ayder Referral and Teaching Hospital, Northern Ethiopia.

554

SpringerPlus, 5:842, doi: 10.1186/s40064-016-2493-x

555

Wesołowska, M., & Dżugan, M. (2017). The use of Photochem device in evaluation of antioxidant

556

activity of Polish honey. Food Analytical Methods, 10, 1568–1574.

557

Wilczyńska, A. (2010). Phenolic content and antioxidant activity of different types of Polish honey:

558

A short report. Polish Journal of Food and Nutritional Science, 60(4), 309-313.

559

World Health Organization (2014). WHO’s first global report on antibiotic resistance reveals

560

serious,

561

http://www.who.int/mediacentre/news/releases/2014/amr-report/en/, accessed: 09.10.2018

562

Xie, Y., Yang, W., Tang, F., Chen, X., & Ren, L. (2015). Antibacterial activities of flavonoids:

563

Structure-activity relationship and mechanism. Current medicinal chemistry, 22, 132–149.

564

Zhou, J., Li, P., Cheng, N., Gao, H., Wang, B., Wei, Y., & Cao, W. (2012). Protective effects of

565

buckwheat honey on DNA damage induced by hydroxyl radicals. Food and Chemical Toxicology,

566

50, 2766–2773.

worldwide

threat

to

public

health.

567

24

568

Table 1 Physicochemical parameters of the buckwheat honey samples

82.1±0.2a,c 80.5±0.0a,b 81.3±0.0a,b 80.3±0.0 80.5±0.0a,b 77.6±0.2b 79.5±0.0 79.0±0.0 76.6±0.5b,c 79.3±0.0 80.3±0.0 80.3±0.0 78.5±0.0 81.3±0.0a,b 79.8±0.4 79.8±0.0 78.8±0.0 78.8±0.0 80.3±0.0 79.3±0.0 76.6 82.1 79.7 1.5 1.8% 137.1 0.000

Water content (%) 16.2±0.1a 17.8±0.1 17.0±0.1a,c 18.0±0.1 17.8±0.1 20.8±0.1b,c 18.8±0.1 19.4±0.1 21.5±0.1b 19.2±0.1 18.0±0.1 17.6±0.1a,c 18.8±0.1 20.1±0.1b,c 18.8±0.1 19.0±0.1 19.6±0.1 19.8±0.1 18.0±0.1 19.2±0.1 16.2 21.5 18.7 1.2 7.6% 167.2 0.000

Titratable acidity (mval kg-1) 12±1 9.0±1.4a 13.0±0.0 10.0±0.0a,b 15.0±0.0 12.0±0.0 14.0±0.0 7.5±0.7a 20±1b 15.0±0.0 14±1 14.0±0.0 15.0±0.0 12±1 12.0±0.0 21.0±0.0b 9.0±0.0a 14±1 14.0±0.0 13.0±0.0 7.5 21.0 13.3 3.7 27.5% 93.6 0.000

3.801

3.772

10.42

Parameter/ Sample no.

Sugar extract (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Min Max Mean SD Variability F-value p-value LSD0.05 value

3.47±0.01a,c 3.44±0.01a 3.49±0.01a,c 3.51±0.01a,c 3.50±0.00a,c 3.45±0.00a 3.57±0.01 3.51±0.01a,c 3.44±0.01a 3.60±0.00c 3.55±0.00a,c 3.50±0.00a,c 3.40±0.01a 3.6±0.1a,c 3.50±0.00a,c 3.52±0.02a,c 3.47±0.01a,c 3.7±0.1b,c 3.8±0.1b 3.60±0.00c 3.40 3.7 3.53 0.09 2.68% 21.27 0.000

Specific rotation [α]20/D -7.9±0.0 -8.3±0.0 -11.3±0.3 -12±1a -11.1±0.0 -11.3±0.0 -8.7±0.3 -9.4±0.1 -11±1 -7.9±0.0 -12±1 -9.7±0.0 -10±1 -8.0±0.1 -8.5±0.3 -7.5±0.5b -8.7±0.0 -8.9±0.3 -8.7±0.0 -10.3±0.0 -12 -7.5 -10.1 1.7 16.3% 38.0 0.000

Color intensity (mAU) 1.43±0.01a 1.31±0.01a 1.90±0.05a 1.89±0.05a 2.80±0.02 2.75±0.01 1.84±0.02a 1.94±0.01a 1.59±0.02a 2.29±0.01a 2.41±0.01 2.51±0.00 1.98±0.01a 1.61±0.01a 2.13±0.01 4.97±0.03b 1.51±0.00a 3.9±0.1 3.9±0.2 1.95±0.01a 1.31 4.97 2.36 1.02 43.3% 837.0 0.000

HMF (mg 100 g1 ) 0.84±0.01a 7.9±0.2b 1.4±0.3a 0.3±0.1a 2.13±0.05a 0.36±0.01a 0.8±0.2a 3.2±0.1 0.8±0.1a 0.9±0.1a 0.96±0.05a 1.6±0.1a 0.7±0.2a 0.75±0.05a 1.4±0.2a 2.1±0.1a 1.39±0.04a 0.4±0.1a 0.9±0.2a 1.0±0.1a 0.3 7.9 1.74 2.07 119% 333.3 0.000

0.182

4.364

2.634

4.941

pH

569

The results for each sample are reported as the mean value of 3 repetitions

570

a, b, c - different letters indicate significant differences (p<0.05) within the column

571

25

572

Table 2 Antioxidant activity and phenolic compounds of the studied buckwheat honey samples Parameter/ Sample no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Min Max Mean SD Variability F-value p-value LSD0.05 value

DPPH (% inhibition) 87±3a 51±2b 84.5±0.5a 93±1a 92±1a 94.3±0.3a 92±2a 83±2a 90.7±0.4a 91.2±3.5a 92±3a 91.3±3.4a 93±2a 94±1a 94.1±0.4a 89.2±0.3a 79.6±0.2a 94.0±0.1a 93.3±0.4a 95.2±0.4a 51 95.2 87 12 14.0% 72.20 0.000 29.04

FRAP (µmol TE 100 g-1) 220±30a 195±5a 313±1 410±40 357±1 351±10 260±10a 200±10a 414±1 360±50 420±30 320±50 440±50 490±30 335±5 680±50b 200±3a 340±10 640±50b 350±10 195 680 370 143 38.5% 38.66 0.000 385.1

TPC (mg GAE 100 g-1) 181±5a 240±30 240±20 300±30 259±4 230±10 204±1a 205±10a 320±50 262±3 281±2 230±10 282±3 230±10 238±5 355±30b 182±3a 250±10 340±10b 218±3 181 355 254 52 20.6% 14.96 0.000 143.8

573

The results for each sample are reported as the mean value of 3 repetitions

574

a, b – different letters indicate significant differences (p<0.05) within the column

TFC (mg QE 100 g-1) 9.2±0.1a 8.0±0.1a 12.7±0.04a 15.3±0.3 15.6±0.2 12.8±0.5a 11.2±0.4a 9.7±0.7a 13.5±0.1a 14.2±0.1a 17.1±0.1 10.7±0.5a 14±1a 10.6±0.3a 11.5±0.5a 30.4±0.1b 8.4±0.3a 13.7±0.3a 20.0±0.2 11.9±0.1a 8.0 30.4 14.0 5.9 42.4% 363.7 0.000 14.69

575

26

576

Table 3 Correlation between antioxidant activity (tested with DPPH, FRAP, TPC and TFC

577

methods), MIC value (determined for S. aureus E. coli S. enterica K. pneumoniae bacteria strains)

578

and H2O2 concentration calculated based on Spearman's rank order correlation coefficients.

579

K. pneumoniae -0.199

Variable

DPPH

FRAP

TPC

TFC

S. aureus

E. coli

S. enterica

DPPH

1.000

0.446*

0.117

0.334

-0.161

-0.293

-0.310

FRAP

0.446*

1.000

0.758*

0.797*

-0.565*

-0.487*

-0.466*

-0.547*

-0.248

TPC

0.117

0.758*

1.000

0.835*

-0.509*

-0.472*

-0.461*

-0.500*

-0.044

TFC

0.334

0.797*

0.835*

1.000

-0.548*

-0.537*

-0.501*

-0.580*

-0.126

S. aureus

-0.161

-0.565*

-0.509*

-0.548*

1.000

0.805*

0.787*

0.762*

-0.285

E. coli

-0.293

-0.487*

-0.472*

-0.537*

0.805*

1.000

0.970*

0.715*

-0.376

S. enterica

-0.310

-0.466*

-0.461*

-0.501*

0.787*

0.970*

1.000

0.682*

-0.305

K. pneumoniae

-0.199

-0.547*

-0.500*

-0.580*

0.762*

0.715*

0.682*

1.000

-0.316

H 2O 2

-0.186

-0.248

-0.044

-0.126

-0.285

-0.376

-0.305

-0.316

1.000

*correlations statistically significant (p<0.05)

580

27

H 2O 2 -0.186

581

Table 4 Minimum inhibitory concentration (MIC 90), minimum bactericidal concentration (MBC)

582

and H2O2 level of tested buckwheat honey samples Sam ple 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

S.aureus MIC MBC

E.coli S. enterica MIC MBC MIC MBC % (w/w)

K. pneumoniae MIC MBC

25 25 12.5 6.25 6.25 12.5 12.5 12.5 6.25 25 6.25 6.25 6.25 12.5 12.5 6.25 25 25 12.5 12.5

25 25 12.5 12.5 12.5 12.5 25 25 12.5 25 6.25 6.25 6.25 25 12.5 12.5 25 25 12.5 12.5

25 >25 25 12.5 12.5 25 25 25 12.5 25 12.5 12.5 12.5 25 25 25 25 25 12.5 25

>25 25 12.5 12.5 12.5 12.5 25 25 12.5 25 6.25 6.25 6.25 25 25 12.5 25 25 12.5 12.5

>25 25 12.5 12.5 12.5 12.5 >25 25 12.5 >25 12.5 12.5 12.5 25 25 12.5 25 25 12.5 12.5

25 25 12.5 12.5 12.5 12.5 25 25 12.5 25 12.5 6.25 6.25 25 12.5 12.5 25 25 12.5 12.5

>25 >25 12.5 12.5 12.5 25 >25 >25 12.5 >25 12.5 12.5 12.5 >25 >25 25 >25 >25 25 12.5

>25 >25 25 12.5 12.5 25 >25 >25 12.5 >25 12.5 12.5 12.5 >25 25 25 >25 >25 25 25

H2O2 content* (mmol l-1) 0.12±0.01a 0.59±0.03 0.35±0.01 0.23±0.01a 0.55±0.02 0.24±0.01a 0.08±0.002a 0.25±0.01a 0.22±0.004a 0.05±0.002a 0.74±0.02 1.1±0.1b 0.62±0.03 0.10±0.001a 0.07±0.002a 0.04±0.001a 0.11±0.003a 0.31±0.02 0.06±0.001a 0.09±0.003a

583

*Statistical calculations for H2O2 content: mean value ± SD = 0.30 ± 0.30; and statistical differences LSD0.05

584

value = 0.88 (F 1065, p 0.000)

585

a, b - different letters indicate significant differences (p<0.05) within the last column

28

586

Table 5 Polyphenolic compounds content (µg 100 g-1) in chosen buckwheat honey samples determined by UPLC-PDA-MS/MS

No.

1 2 3 4 5 6 7 8

Component Protocatechuic acid pHydroxybenzoic acid Chlorogenic acid Caffeic acid p-Coumaric acid Ferulic acid Quercetin-3rutinoside Quercetin-3glucoside

Fragment Absorbance ions maxima (m/z) (nm)

Rt* (min)

[M-H] (m/z)

2.14

153

109

2.31

137

2.39 2.59 2.61

1

11

12

17

18

260, 294

83±2a

50±1b

55±2b

48±1b

39±1c

-0.090

-

121

254

360±10a

196±3b

410±10c

271±3d

189±5b

0.069

-

353

191

320, 238

51±2a

90±2b

82±2c

59±1d

60±2d

0.590

SA, EC, S

179

135

323, 240

26±1a

82±1b

70±1c

52±1d

64±2e

0.532

SA, EC, S

163

119

308

a

440±10 a

b

380±10 a

c

d

e

590±10

780±10

250±10

-0.649

-

b

b

b

SA, EC, S, KP

2.95

193

134

323, 293

73±2

66±2

166±4

164±3

154±4

-0.131

4.29

609

463, 301

254, 350

59±2a

85±2b

129±3c

58±1a

94±5b

0.822

4.48

463

301

255, 350

46±2a

22±1b

54±1c

49±1a

48±1a

-0.167

-

9

Quercetin

5.06

301

179, 151

255, 355

57±2a

339±4b

312±4c

22±1d

450±10e

0.823

10

Myricetin

5.16

317

179, 151

253, 372

42±1a

33±1b

13±1c

24.5±1d

37±3a,b

-0.140

SA, EC, S, KP -

d

b

146±3

-0.559

-

c

d

11

Kaempferol

5.35

285

121, 165

a

267, 360

119±3

a

b

77±2

a

c

140±10 b

179±4

12

Apigenin

5.39

269

151, 117

267, 340

60±1

57±1

70±1

33.5±1

80±2

0.851

S, KP

13

Galangin

5.85

269

195, 141

261, 351

20±1a

16±1b

21±1a

26±1c

17±1a,b

-0.756

-

1442±27

1489±26

2113±42

1766±26

1630±43

0.196

KP

Total (µg 100 g-1) 587 588 589 590

Correlation coefficient MIC DPPH value**

Honey sample No.

*Rt – retention time a, b, c, d – different letters indicate significant differences (p<0.05) in the columns **significant (p<0.05) correlations between particular phenolics compounds and MIC values for tested bacteria strains (SA – S. aureus, EC – E. coli, S – S. enterica, KP – K. pneumoniae) calculated by Spearman’s rank

29

591

Figure legends

592

Figure 1. Catalase influences on antibacterial properties of buckwheat honey (honey concentration

593

– 12.5%) against: a) S. aureus, b) E. coli, c) S. enterica, d) K. pneumoniae; white bars –

594

antibacterial properties of honey without catalase, black bars – antibacterial properties of honey

595

with addition of catalase. *- significant differences between samples with and without catalase

596

addition (p<0.05).

597

Figure 2. Principal component analysis (PCA) biplot of the antioxidant activity tested using

598

spectrophotometric methods (DPPH, FRAP, TPC and TFC), antibacterial activity against S. aureus,

599

E. coli, S. enterica and K. pneumoniae (expressed as MIC value) and particular phenolics

600

compounds content identified by UPLCPDA-MS/MS (marked as 1-13 in accordance with Table 5).

601

Figure 3. The effect of buckwheat honey samples on ROS generation in the yeast cells treated with

602

hydrogen peroxide after 1h of yeast cell incubation with honey; white bars - control yeast cells and

603

control yeast cells with addition of honey (samples 1, 11, 12, 17, 18), black bars – yeast cells

604

exposed to hydrogen peroxide.

605

a, b – significant differences between control cells and cells treated with honey (p<0.05); A, B – significant

606

differences between control cells and experimental cells treated with hydrogen peroxide (p<0.05).

607

30

608

609

610 31

611 612

Figure 1.

613

32

1,0

Total

5 13 11

0,5

2

6

8

7 H2O2

PC 2 : 26,8%

3 4

0,0 1

9

FRAP

12

K. pneumoniae S.aureus E.coli

DPPH

-0,5

TPC

TFC S. enterica

-1,0

10 -1,0

-0,5

0,0

0,5

1,0

PC 1 : 49,2%

614

Figure 2.

615

33

60

A ROS content [a.u.]

50 40

B

B

20

B

B

B

30

a a,b

a,b

b

a,b

a,b

10 0 control cells

1

11

12

17

18

sample number

616 617

Figure 3.

34