Accepted Manuscript

Characterisation of phenolic compounds in Algerian honeys by RP-HPLC coupled to

electrospray time-of-flight mass spectrometry

Salim Ouchemoukh, Nadia Amessis-Ouchemoukh, María Gómez-Romero, Farid

Aboud, Alonzo Guiseppe, Alberto Fernández-Gutiérrez, Antonio Segura-Carretero,
Hayette Louaileche
PII: S0023-6438(16)30774-5
DOI: 10.1016/j.lwt.2016.11.084
Reference: YFSTL 5893

To appear in: LWT - Food Science and Technology

Received Date: 30 June 2016

Revised Date: 29 November 2016
Accepted Date: 30 November 2016

Please cite this article as: Ouchemoukh, S., Amessis-Ouchemoukh, N., Gómez-Romero, M., Aboud,
F., Guiseppe, A., Fernández-Gutiérrez, A., Segura-Carretero, A., Louaileche, H., Characterisation
of phenolic compounds in Algerian honeys by RP-HPLC coupled to electrospray time-of-flight mass
spectrometry, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2016.11.084.

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 ACCEPTED MANUSCRIPT

1 Characterisation of phenolic compounds in Algerian honeys by RP-HPLC

2 coupled to electrospray time-of-flight mass spectrometry

3 Salim Ouchemoukh1,*, Nadia Amessis-Ouchemoukh2, María Gómez-Romero3, Farid

4 Aboud4, Alonzo Guiseppe4, Alberto Fernández-Gutiérrez3, Antonio Segura-Carretero3,5,

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5 Hayette Louaileche1
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6 Laboratoire de Biochimie appliquée, Faculté des Sciences de la Nature et de la Vie,

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7 Université de Bejaia, Route de Targa-Ouzemour 06000 Bejaia, Algeria.

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8 Laboratoire de Biomathématique, Biochimie, Biophysique et Scientométrie, Faculté des

9 Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria.

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10 Department of Analytical Chemistry, Faculty of Sciences, University of Granada,

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c/.Fuentenueva s/n, 18071, Granada, Spain.
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12 Department of Agriculture and Forestry, University of Palermo, Viale delle Scienze edifice
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13 4, 90128 Palermo, Italy.
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14 Functional Food Research and Development Center, PTS Granada, Avd. del Conocimiento,
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15 Edificio Bioregion, 18016 Granada, Spain.

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16 * Corresponding author : Salim OUCHEMOUKH

17 Laboratoire de Biochimie appliquée, Faculté des Sciences de la Nature et de la Vie,

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18 Université de Bejaia, Route de Targa-Ouzemour 06000 Bejaia, Algeria

19 email: salimouchemoukh@yahoo.fr
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20 Tel/Fax: 0021334214762
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26 Abstract

27 A total of 35 honey samples from different regions of Algeria were studied to determine their

28 phenolic profiles. Phenolic compounds, products of the secondary metabolism of plants, were

29 extracted with amberlite XAD-4 and analysed by liquid chromatography, with diode array

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30 detection and electrospray ionisation mass spectrometry in negative ion polarity. By using

31 colorometric assays, Erica honeys showed the highest content of phenolic compounds and

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32 flavonoids (245 ± 54 mg GAE/100 g and 29 ± 6 mg QE/100 g, respectively). More than 30

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33 compounds were identified in the honey samples studied including 14 phenolic acids and 16

34 flavonoids. In general, honey samples showed different chromatographic profiles. It has been

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35 shown that 4-hydroxybenzoic acid, apigenin, chrysin, galangin, kaempferol, isorhamnetin,

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luteolin and pinocembrin were present in all honey extracts. Moreover, caffeic, p-coumaric

and vanillic acids; abscisic and syringic acids; and benzoic acid were detected in 34, 33 and
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38 32 honey samples, respectively. The members of flavonol subclass were the most abundant of

39 the identified flavonoids. Gallic and homovanillic acids, daidzein and myricetin were less
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40 present: 7, 5, 7 and 6 in honey samples, respectively. Caffeic and p-coumaric acids were
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41 potential floral markers for Capparis spinosa and Trifoliumn honeys, respectively.

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43 Keywords: Honey extracts ; Flavonoids ; Phenolic profiles ; Separation ; Floral marker.

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51 1. Introduction

52 Honey is the natural sweet substance produced by honey bees from the nectar of plants or

53 from secretions of living parts of plants or excretions of plant sucking insects on the living

54 parts of plants, which the bees collect, transform by combining with specific substances of

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55 their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature (Council

56 Directive 2001/110/EC). The majority of its components are derived from the plants, some are

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57 added by the bees and others are due to the maturation of the honey (Anklam, 1998). Honey

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58 possesses valuable nourishing, healing and prophylactic properties, which result from its

59 chemical composition. It has potential therapeutic value in the treatment of heart disease,

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60 cancer, cataracts and several inflammatory diseases (Noor, Sarfraz, Ali, & Shahid, 2014).

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Although the major components of honey are sugar and water, it has a wide range of minor

constituents, such as phenolic compounds, which may be responsible for the biological
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63 activities of honey (Jaganathan, & Mandal, 2009). Phenolic compounds, or polyphenols, are

64 one of the important groups of phytochemicals that occur in plants, in which they are widely
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65 distributed. They are products of the secondary metabolism of plants, being fundamental for
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66 their normal development and an important key in their defence mechanism (Bravo, 1998).

67 Flavonoids and phenolic acids (both benzoic and cinnamic-acid derivatives) constitute the
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68 most important classes of polyphenols and the most abundant on the diet, accounting for the

69 60 and 30 % of the total, respectively, with more than 8000 phenolic structures known.
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70 Flavonoids can be subdivided into 13 classes, including flavonols, flavanones, flavones,

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71 anthocyanidins and isoflavones (Robards, Prenzler, Trucker, Swatsitang, & Glover, 1999).

72 Phenolic compounds contribute to the organoleptic properties, such as color, taste or flavour

73 of honey. They also have antioxidant activities, together with other honey substances (e.g.

74 enzymes (glucose oxidase, catalase), vitamins (ascorbic acid) or organic acids) (Gheldof,

75 Wang, & Engeseth, 2002). Honey can serve as a source of natural antioxidants and the

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76 variations in the antioxidant activities of honey are due to the qualitative and quantitative

77 nature of their phenolic constituents (Aljadi, & Kamaruddin, 2004).

78 It has been found that phenolic compounds present in honey can come from flower nectar,

79 pollen, propolis and/or beeswax (Gil, Ferreres, Ortiz, Subra, & Tomás-Barberán, 1995) and,

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80 consequently, it can be reasonably expected that the composition of honey will be different

81 depending on its origin. As a matter of fact, the content of phenolic compounds in honey is

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82 strongly affected by the floral source and geographical origin, as well as by the climatic

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83 characteristics of the site of collection. Therefore, the analysis of phenolic compounds has

84 been regarded as a very promising technique for studying the floral and geographical origins

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85 of honeys. The extraction of phenolics from the honey matrix is the first step involved in their

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analysis, and different protocols, including both liquid-liquid and solid-phase extraction

strategies, have been employed for the isolation of this fraction. The use of non-ionic
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88 polymeric resins has the advantage of eliminating numerous polar and ionic interfering

89 substances such as sugars and acids. Amberlite XAD type resins are polymeric adsorbents
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90 which have proved to be the best materials for extracting honey flavonoids with yields higher
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91 than 80 %. Amongst them, we have chosen amberlite XAD-4 because it has a higher surface

92 area, and so a higher efficiency, compared with the XAD-2 type (Iurlina, Saiz, Fritz, &
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93 Manrique, 2009). Identification and quantification of polyphenols in honey have been carried

94 out using different techniques: colorometric reactions, thin-layer chromatography (TLC), gas
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95 chromatography (GC), high-performance liquid chromatography (HPLC) and capillary

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96 electrophoresis (CE), the three later coupled to diverse detection systems (Pyrzynska, &

97 Biesaga, 2009 ; Mattonai, Parri, Querci, Degano, & Ribechini, 2016). Moreover, UPLC-MS

98 was used to study the phenolic acids and flavonoids contained in several unifloral honey

99 samples; the developed method enabled the identification of 37 phenolic compounds within

100 14 min (Gasic et al., 2014 ; Trautvetter, Koelling-Speer, & Speer, 2009). Nevertheless, the

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101 method of choice for phenolic compounds analysis is reversed-phase (RP)-HPLC coupled to

102 UV detector and, more recently, to mass spectrometry (MS). In RP-HPLC, the stationary

103 phase consists of a non-polar octadecylsilane (C18) bonded phase and the mobile phase is a

104 polar solvent, so that the more polar the phenolic compounds, the earlier they will elute.

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105 Numerous mobile phases have been employed but binary systems comprising an aqueous

106 component and a less polar organic solvent (acetonitrile or methanol) remain as the most

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107 common (Pyrzynska, & Biesaga, 2009). In general, gradient elution is usually used in

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108 recognition of the complexity of the phenolic profile of honey samples.

109 Numerous flavonoids (such as apigenin, chrysin, galangin, kaempferol, luteolin, pinocembrin,

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110 pinobanksin, quercetin) and phenolic acids (caffeic, gallic, cinnamic, p-coumaric and

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protocatechuic acids) were identified in honey samples (Gasic et al., 2014). Caffeic and p-

coumaric acids, chrysin, galangin, hesperetin, kaempferol, luteolin, pinocembrin and

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113 quercetin were detected in lemon and orange Spanish honeys (Escriche, Kadar, juan-Boras, &

114 Domenech, 2011). In addition, a total of 43 polyphenols were identified in serbian unifloral
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115 honeys (Keckes, Gasic, Velickovic, Milojkovic-Opsenica, Natic, & Tesic, 2013).
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116 Several studies have revealed that the analysis of flavonoids and other phenolic compounds

117 constitute a very promising technique to study the geographical and floral origin of honey.
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118 Flavonoids originating from propolis are not helpflul for the determination of the botanical

119 origin of the honey, as the levels of such flavonoids depend on the presence and content of
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120 propolis in the honey. Pinocembrin, pinobanksin, galangin and chrysin are characteristic
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121 flavonoids of propolis and they are determined in european honey samples (Gasic et al., 2014;

122 Tomas-Barberan et al., 2001, Yao, Datta, Tomas-Barberan, Ferreres, Martos, & Singanusong,

123 2003). Specific flavonoids have been described as markers of the botanical origin of unifloral

124 honeys (Soler, Gil, García-Viguera, & Tomás-Barberán, 1995) as is the case of the flavanone

125 hesperetin for citrus honey (Ferreres, García-Viguera, Tomás-Lorente, & Tomás-Barberán,

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126 2008), the flavone kaempferol for rosemary honey (Gil et al., 1995) or the flavonol quercetin

127 for sunflower honey (Tomás-Barberán, Martos, Ferreres, Radovic, & Anklam, 2001).

128 Additionally, some phenolic acids have also been used as floral marker substances (Andrade,

129 Ferreres, & Amaral, 1997); some examples include ellagic acid in heather honey (Ferreres,

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130 Andrade, Gil, & Tomás-Barberán, 1996), homogentisic acid in strawberry honey (Cabras et

131 al., 1999) and gallic in the Eucalyptus one (Yao et al., 2004). Due to the significance of

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132 natural phenolic compounds as natural antioxidants and to their use as markers in plant

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133 chemotaxonomic studies, interest in their identification and quantification in honey samples

134 has significantly increased in recent years (Bertoncelj, Polak, Kropf, Korošec, & Golob, 2011;

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135 Pyrzynska, & Biesaga, 2009; Martos, Ferreres, Yao, D’arcy, Caffin, & Tomás-Barberán,

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137
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2000; Michalkiewicz, Biesaga, & Pyrzynska, 2008 ; Yao et al., 2003).

Honey production in Algeria has a very long tradition. However, its composition and
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138 bioactive properties have not been exhaustively analysed hitherto. There are publications

139 dealing with the study of the pollen analysis, physico-chemical and antioxidant properties of
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140 Algerian honeys (Makhloufi, Kerkvliet, D’albore, Choukri, & Samar, 2010; Makhloufi,
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141 Kerkvliet, & Schweitzer, 2015; Mouhoubi-Tafinine, Ouchemoukh, & Tamandjari, 2016;

142 Ouchemoukh, Louaileche, & Schweitzer, 2007; Ouchemoukh, Schweitzer, Bachir Bey,
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143 Djoudad-Kadji, & Louaileche, 2010). According to our knowledge, this is the first research of

144 phenolic profiles in Algerian honeys. The major purpose of this study was the identification of
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145 the phenolic compounds contained in honey samples by means of RP-HPLC-ESI-TOF-MS.

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146 2. Material and methods

147 2.1 Reagents and standards

148 All chemicals and reagents used were either analytical or HPLC reagent grade. Aluminum

149 chloride, Folin-Ciocalteu reagent, sodium nitrite, sodium carbonate and sodium hydroxide

150 were from Biochem chemopharm (Nievre, France).

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151 Doubly deionised water with a conductivity of 18.2 MΩ was obtained by using a Milli-Q

152 system (Millipore, Bedford, USA). The organic solvents acetic acid and methanol were from

153 Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain), respectively. All solutions

154 were filtered through a 0.2 µm Millipore (Bedford, USA) membrane filter before injection

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155 into the column.

156 Twenty four phenolic standards were bought from different sources: gallic, protocatechuic,

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157 vanillic, 3,4-dihydroxyphenylacetic, 4-hydroxyphenylacetic, caffeic, gentisic, p-coumaric,

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158 benzoic and cinnamic acids, myricetin, quercetin, hesperetin, chrysin and luteolin were from

159 Sigma-Aldrich (St. Louis, MO, USA); 4-hydroxybenzoic, ferulic, syringic and homovanillic

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160 acids were from Fluka (Buch, Switzerland); isosakuranetin, galangin, isorhamnetin,

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pinocembrin and kaempferid were purchased from Extrasynthese (Lyon, France).

2.2 Honey samples

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163 Thirty five honey samples were harvested from nine different regions of Algeria (Table 1).

164 The samples were stored in a refrigerator in airtight plastic containers until analysis.
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165 2.3 Pollen analysis

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166 The identity of honey samples was based on pollen analysis following the method suggested

167 by Louveaux, Maurizio, & Vorwhol (1978). The microscopic preparations were made without
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168 acetolysis to preserve all of the components in the sediments extracted. Samples were treated

169 with aqueous sulphuric acid to remove colloidal matter. Pollen identification was based on the
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170 collection of reference pollens. Monofloral honeys were considered as such whenever the
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171 dominant pollen was found at over 45 % of the total pollen.

172 2.4 Total phenolic compounds content

173 Total phenolic compounds of the honey samples were determined by the method of Naithani,

174 Nair, & Kakkar (2006). A volume of 0.1 mL of honey solution (0.1 g/mL) was mixed with 0.1

175 mL of Folin–Ciocalteu reagent. The mixture was shaken and 2.2 mL of Na2CO3 solution (2

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176 g/100 g) was added. After 30 min in the dark, the absorbance of the reaction mixture was

177 measured at 720 nm. The content of total polyphenols was expressed as mg of gallic acid

178 equivalents per 100 g of honey (mg GAE/100 g) through the calibration curve with gallic acid

179 (y = 1.6841x - 0.013 ; R2=0.9951).

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180 2.5 Total flavonoids content

181 The total flavonoid content was measured by a colorimetric assay described by Al, Daniel,

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182 Moise, Bobis, Laslo, & Bogdanov (2009). An aliquot of 1 mL of the honey solution (0.5 g /

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183 mL) was mixed with NaNO2 (5 g/ 100 mL). The mixture was shaken and after 5 min, 300 µL

184 of AlCl3 (10 g/100 mL) were added. After 6 min, 2 ml of NaOH (1 mole/L) were added to the

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185 mixture. Absorbance of the mixture was determined at 510 nm. The concentrations of

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flavonoids were expressed as mg of quercetin equivalents per 100 g of honey (mg QE/100 g)

(y = 2.4469 x ; R2 = 0.9968).
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188 2.6 Phenolic compounds extraction from honey

189 Phenolic compounds were extracted according to a previously developed method (Tomás-
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190 Barberán, Ferreres, Blazquez, García-Viguera, & Tomas-Lorente, 1993). Twenty five g of
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191 honey were thoroughly mixed with 5 parts of acidified water (pH 2 with HCl) until

192 completely fluid and filtered through cotton to remove solid particles. The filtrate was then
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193 passed through a glass column (50 x 5 cm) filled of amberlite XAD-4 resins (0.25-0.84 mm,

194 mean pore size 4 nm, Supelco, Bellefonte, USA). The various phenolic compounds remained
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195 in the column while sugars and other polar compounds were eluted with the aqueous solvent.
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196 The column was washed with acid water (100 mL) and subsequently with distilled water (300

197 mL). The whole phenolic fraction was then eluted with methanol (400 mL) and concentrated

198 under reduced pressure (40 °C). The residue was redissolved in 5 mL of water and extracted

199 with diethyl ether (5 mL x 3). The ether extracts were combined and concentrated under

200 reduced pressure in a rotavapory evaporator at 30 °C. The dried residue was then redissolved

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201 with 0.5 mL of methanol and filtered through a 0.45 µm membrane filter, ready for HPLC

202 analysis.

203 2.7 HPLC/MS analysis

204 The separation of the phenolic compounds from honey extracts was performed using an

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205 Agilent 1200 Series Rapid Resolution Liquid Chromatography system (RRLC, Agilent

206 Technologies, CA, USA) equipped with a vacuum degasser, an autosampler, a binary pump

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207 and a thermostated column department, and using a reversed-phase C18 analytical column (4.6

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208 x 150 mm, 1.8 µm particle size; ZORBAX Eclipse plus). The eluting mobile phase, consisting

209 of 0.5 mL/ 100 mL of acetic acid in deionised water (solvent A) and methanol (solvent B),

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210 was pumped at 1.2 mL/min. The following multi-step linear gradient was applied: 0 min, 5 %

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B; 30 min, 76.1 % B; 33 min, 100 % B; 34 min, 5 % B. The initial conditions were held for 6

min before each analysis. The column temperature was maintained at 35 °C and the injection
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213 volume was 5 µL.

214 2.8 ESI-TOF-MS

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215 The RRLC system was coupled to a TOF mass spectrometer (Bruker Daltonics, Bremen,
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216 Germany) equipped with an ESI (interface operating in the negative ion mode, with spectra

217 acquired over a mass range of 50-1100 m/z and using a capillary voltage of + 3.7 kV. The
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218 other optimum values of the ESI-MS parameters were as follows: drying gas temperature, 190

219 °C; drying gas flow, 9.0 L/min; and nebulising gas pressure, 2 * 105 Pa.
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220 External instrument calibration was performed using sodium formate clusters by switching the
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221 effluent from the RRLC system to a solution containing 0.005 mole/L of sodium hydroxide

222 and 0.2 g/100 mL of formic acid in water/isopropanol (1:1; mL/mL). This calibration solution

223 was also injected at the beginning of each run and all the spectra were calibrated prior to the

224 compound identification. The accurate mass data of the molecular ions were processed

225 through the SmartFormula Editor within the DataAnalysisTM 4.0 software (Bruker Daltonics,

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226 Bremen, Germany). This tool provides a list of potential elemental molecular formulae by

227 combining the accurate mass and isotopic distribution, reflected in their error and sigma

228 values, respectively. An error of 5 ppm and a Sigma value of 0.05 are generally considered

229 appropriate.

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230 Retention times and chromatographic comparisons with authentic standards were the basic

231 criteria considered for the polyphenols identification.

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232 3. Statistical analysis

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233 All the experiments were done in triplicates and the results were given as mean ± standard

234 deviation (SD). One-way analysis of variance (ANOVA) with a post hoc least significant

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235 difference test was used to determine significance difference (p < 0.05) with STATISTICA

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5.5 software for the groups of honey samples.

4. Results and discussion

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238 4.1 Melissopalynological analysis

239 The pollen analysis indicated that 37 % of the honey samples were polyfloral and 63 % were
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240 monofloral, belonging to seven different botanical families (Table 1). Eucalyptus sp. was a
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241 predominant source used by honeybees in Algeria and this genus is important melliferous

242 plants in the mediterrean area. Multifloral honeys contained several pollen types (Eucalyptus,
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243 Erica, Rubus, Castanea sativa, Prunus/pyrus, Brassicaceæ, Taraxacum, Mahinot, Achillea

244 type, Lavandula stœchas, Rubus, Malva spp., Myrtus communis, Lamiaceæ, Carduus, Acacia
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245 spp., Hæmatoxylon...).

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246 4.2 Contents of antioxidant components

247 The amounts of total phenolic compounds and flavonoids of honey samples varied from 90 to

248 318 mg GAE/100 g and 0.30 to 36 mg QE/100 g, respectively (Table 2). The results obtained

249 were different from those found by Al et al. (2009) on the Romanian honeys (2 to 125 mg/

250 100 g) and Kishore, Sukari, Nurul, and Sirajudeen (2011) for Malaysian honeys (25 to 50 mg

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251 EQ/ 100 g), respectively. Moreover, Erica honeys had higher total phenolic compounds and

252 flavonoids contents than other honeys (Table 2).

253 4.3 Identification of phenolic compounds

254 Several phenolic compounds were identified in Algerian honeys by HPLC-ESI-TOF-MS

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255 (Table 3). These compounds were characterised by their retention times (3.70 to 28.59 min)

256 and absorption in the UV. Phenolic acids were eluted first (before 16.50 min), with exception

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257 of cinnamic acid (20.74 min). Myricetin was the first flavonoid eluted. The extraction of

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258 phenolics from source materials is the first step involved in their analysis. The small particles

259 of XAD-4 allowed the separation of all compounds in less than 30 min. Retention time, in

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260 general, followed the expected reversed-phase pattern of benzoic acids<cinnamic

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acids<flavonoid glycosides<flavonoid aglycones. Phenolic acids are mostly derivatives of

benzoic and cinnamic acids, namely hydroxybenzoic and hydroxycinnamic acids. The
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263 configurations of the substituents also affect the strengths of the interactions and therefore the

264 order of elution (for instance, isomers of coumaric acid). Among flavonoids, hydroxylation
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265 decreases retention owing to increasing polarity (Biesaga, & Pyrzynska, 2009). For this
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266 reason, myricetin is eluted at first because it has three OH-groups in B-ring.

267 The comparaison with the standards and the EIC (Extracted Ion Chromatogram) allowed the
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268 identification of more than 30 phenolic compounds. All honey extracts had on common

269 hydroxybenzoic acid and seven flavonoid aglycones (isorahmnetin, luteolin, chrysin,
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270 galangin, apigenin, kaempferol and pinocembrin) (Table 3). Caffeic, p-coumaric and vanillic
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271 acids were detected in 34 samples, exception for M09, syringic acid in 33, benzoic acid in 32,

272 quercetin in 30, ferulic and 4-hydroxyphenylacetic acids in 28 and isosakuranetin in 27 honey

273 samples. Cinnamic and 3, 4 dihydroxyphenylacetic acids genistein were present in 21 honey

274 samples; hesperitin in 18 and kaempferid in 17 samples.

275 4.3.1 Phenolic acids

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276 Fourteen phenolic acids were detected in Algerian honey samples: 4-hydroxybenzoic,

277 cinnamic, 4-hydroxyphenylacetic, protocatechuic, p-coumaric, gallic, caffeic, homovanillic,

278 vanillic, syringic and ferulic acids. These identified phenolic compounds were already

279 reported in honeys (Andrade et al., 1997; Keckes et al., 2013; Yao et al., 2003). Moreover, it

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280 has been found by Can, Yildiz, Sahin, Turumtay, Silici, and Kolayli (2015) that benzoic,

281 caffeic and p-coumaric acids were present in differing amounts in all unifloral Turkish

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282 honeys. A common fragmentation pathway based on the loss of the CO2 group was observed

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283 for the phenolic acids. For polyfloral honeys, gallic and gentisic acids were present in two

284 samples, H10 and H12; H03 and H10, respectively. In Eucalyptus honeys, homovanillic acid

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285 was present only in H15 and H16. Gallic, gentisic and homovanillic acids were not present in

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Apiaceae honeys.

4.3.2 Flavonoids
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288 Five subclasses of flavonoids were detected in the samples analysed: flavones (galangin,

289 apigenin, chrysin, luteolin), flavanones (pinocembrin), flavanonols (pinobanksin), flavonols

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290 (myricetin, kaempferol, quercetin, isorhamnetin, myricetin, kaempferid) and isoflavones

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291 (genistein and daidzein). It is clear that in our case, flavonoids do not lose fragments because

292 there was not MS/MS fragmentation. In the negative mode, the loss of groups' such as H2O,
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293 CO, CO2 and C2H2 are common for all flavonoids (Gasic et al., 2014). Myricetin was observed

294 exclusively in one polyfloral honey (H03). Apiaceae and Erica honeys did not have myricetin.
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295 Quercetrin was present in these two types of honey. This compound was also identified in
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296 Capparis spinosa honey. Concerning multifloral and Eucalyptus honeys, it was detected in 4

297 and 3 honeys, respectively. Less than 20 compounds were identified in H09, H11, H33, H34

298 and H35. Some Honey samples from Bejaia (H02, H03, H12 and H14) had 24 phenolic

299 compounds.

300 4.4 Phenolic profiles of Erica and Eucalyptus honeys

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301 The base peak chromatogram (BPC) of honey extract 16 (H16), using the negative ionization

302 mode, was displayed in Fig. 1. The peaks identified were enumerated according to their

303 elution order and phytochemicals were summarised in table 4, including retention times,

304 experimental and calculated m/z, molecular formula, error and sigma values (comparison of

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305 theoretical with measured isotope patterns). Fourty substances were identified in this honey

306 samples: 16 phenols with standards (6 phenol acids and 10 flavonoids) and others were

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307 tentatively identified with SCIFINDER data base. Methoxychrysin and prenylcaffeate were

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308 reported by Gasic et al. (2014) and Keckes et al. (2013).

309 Peak 1 gave a molecular ion at m/z 211 and showed a molecular formula C10H11O5. The

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310 Compound 1 was tentatively identified as gallic acid propyl ester. This later was present also

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in 11 other samples especially in Erica honeys (Fig. 3) where the area of the peak was high.

In general, the chromatograms of Erica honeys, collected in the same region, some
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313 Eucalyptus honeys (H14, H15 and H16) and multifloral honeys from Blida showed similar

314 profiles, but they had differences in the area of the peaks. Trautvetter et al. (2009) showed that
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315 honeys of the same biological origin showed similar profiles in regard to their determined
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316 phenolic compounds.

317 With respect to fragments, 2 compounds were identified in honey samples: methyl syringate
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318 and abscisic acid. For the first compound, retention time was 17.71 min and gave the

319 fragments 181 and 196. Tuberoso, Bifulco, Jerkovic, Caboni, Cabras, and Floris (2009)
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320 identified this compound in Italian monofloral honeys with the same fragments. Also, these
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321 authors reported that methyl syringate was detected in honeys obtained from plants of

322 different botanical families but only asphodel honey reached the highest level.

323 Two compounds with the same m/z 263 and different retention time (18.55 ; 20.20 min) were

324 also identified. Comparing to results of Tuberoso, Bifulco, Caboni, Cottiglia, Cabras, and

325 Floris (2010), these are isomers of abscisic acid (cis-trans and trans-trans abscisic acid) with

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326 fragments 219 and 153. Stephens et al. (2010) obtained the same fragments in New Zealand

327 honeys. With respect to the literature, the first compound was trans-trans abscisic acid; the

328 second was cis-trans abscisic acid (Gasic et al., 2013; Yao et al., 2003).

329 4.5 Floral markers

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330 As can be seen from fig. 3a, Capparis spinosa honey (H28) was marked with caffeic acid

331 with respect to other honey samples. Nevertheless, this phenolic acid was the main compound

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332 in Spanish orange honeys and Malaysian honey extracts (Escriche et al., 2011). Trifolium and

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333 Annarhinum honeys (H33 and H34, respectively) had p-coumaric acid as floral marker (Fig.

334 3b, c). This phytochemical was present in significant amount in oak honey (Can et al., 2015).

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335 Phenolic profiles are related to the botanical origin of honey. Really, we did not detect

336

337
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chlorogenic and ellagic acids, acacetin, naringin, pinostrobin, rutin and taxifolin in analysed

honeys. These compounds are reported in other honeys (Gasic et al., 2014, Michalkiewicz,
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338 Biesaga, & Pyrzynska, 2008). Also, other phenols were reported to be the marker of some

339 honeys, quercetin and eriodictyol in Serbian honeys (Keckes et al., 2013).
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340 5. Conclusion
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341 In this study, the results of polyphenols screening in unifloral and multifloral Algerian honeys

342 were reported for the first time. In general, phenolic profiles varied within samples and the
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343 distribution of polyphenols was affected by the floral origins of honeys. The most abundant of

344 the identified flavonoids were the members of the flavonol subclass and all honey samples
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345 shared 4-hydroxybenzoic acid and seven flavonoids aglycones. In this paper, other tentatively
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346 phenolic compounds (gallic acid propyl ester, secologanin acetal, hexyl salicylate, anchoic

347 and camphoric acids...) were identified in Eucalyptus honey and some of these compounds

348 were not reported in the literature. Caffeic and p-coumaric acids are markers for Capparis and

349 Trifolium honey, respectively.

350 Acknowledgments

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351 The authors thank the Algerian ministry of high education and scientific research for

352 sponsoring this work.

353 References

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447 al. (2010). Phenolic compounds and methylglyoxal in some New Zealand manuka and kanuka

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461 markers of strawberry tree (Arbutus unedo L.) honey. Journal of Agricultural and Food

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463 Yao, L., Datta, N., Tomas-Barberan, F.A., Ferreres, F., Martos, I., & Singanusong, R. (2003).
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464 Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum

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466 Yao, L., Jiang, Y., D'Arcy, B., Singanusong R., Datta, N., Caffin, N. et al., (2004).

467 Quantitative high-performance liquid chromatography analyses of flavonoids in Australian

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468 Eucalyptus honeys. Journal of Agricultural and Food Chemistry, 52, 210-214.
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Figure captions:

Fig. 1 : Base peak chromatogram (BPC) of Eucalyptus honey (H16) by HPLC-MS.

Peak labeling represents the compound identified.

Reversed-phase C18 analytical column (4.6 x 150 mm, 1.8 µm particle size). Solvent A : acetic acid in

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deionised water ; solvent B : methanol. Solvent flow-rate 1.2 mL/min. Volume injection 5 µL. Linear

gradient was applied: 0 min, 5 % B; 30 min, 76.1 % B; 33 min, 100 % B; 34 min, 5 % B.

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Fig. 2 : HPLC phenolic profiles of Erica arborea honeys.

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Reversed-phase C18 analytical column (4.6 x 150 mm, 1.8 µm particle size). Solvent A : acetic acid in

deionised water ; solvent B : methanol. Flow-rate 1.2 mL/min. Volume injection 5 µL. Linear gradient

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was applied: 0 min, 5 % B; 30 min, 76.1 % B; 33 min, 100 % B; 34 min, 5 % B.

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Fig. 3 : Typical HPLC chromatograms of phenolic compounds of Capparis (a), Trifolium (b)
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and Annarhinum (c) honeys.

Reversed-phase C18 analytical column (4.6 x 150 mm, 1.8 µm particle size). Solvent A : acetic acid in
D

deionised water ; solvent B : methanol. Solvent flow-rate 1.2 mL/min. Volume injection 5 µL. Linear
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gradient was applied: 0 min, 5 % B; 30 min, 76.1 % B; 33 min, 100 % B; 34 min, 5 % B.

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Table 1 : Geographical and botanical origins of honey samples.

Sample Region Botanical origin

H01 Bejaia (Ait sidi elmohob), 2006

H02 Bejaia (Seddouk), 2006
H03 Bejaia (Melbou), 2006

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H04 Tizi-Ouzou, 2006
H05 Alger (Sidi moussa), 2006
H06 Blida, 2006 Multifloral

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H07 Blida, 2005
H08 Blida, 2005
H09 Mila, 2006

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H10 Bejaia, 2006
H11 Bejaia, 2006
H12 Bejaia (Akbou), 2004
H13 Bejaia (Akbou), 2004

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H14
H15
H16
Bejaia (Amizour), 2006
Tizi-Ouzou, 2006
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Bejaia (Oued-ghir), 2006

H17 Boumerdes, 2006 Eucalyptus

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H18 Blida, 2006
H19 Tlemcen, 2006
H20 Bejaia (Timezrit), 2004
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H21 Bejaia (El-Kseur), 2006

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H22 Bejaia (El-Kseur), 2006 Erica arborea

H23 Bejaia (Oued-des), 2006
H24 Bejaia (Adekar), 2006
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H25 Tizi-Ouzou, 2006 Apiaceae

H26 Bouira, 2006
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H27 Setif, 2006

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H28 Bejaia (Kherrata), 2006 Capparis

H29 Blida, 2006 Citrus
H30 Blida, 2006 Myrtaceae
H31 Jijel, 2006 Hedysarum
H32 Setif, 2006 Lotus
H33 Setif, 2006 Trifolium
H34 Batna, 2006 Annarrhinum
H35 Bejaia (Tazmalt), 2004 Melilotus
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Table 2 : Contents of total phenolic compounds and flavonoids in polyfloral, Eucalyptus, Erica and Apiaceae honey samples.

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Polyfloral Eucalyptus Erica arborea Apiaceae

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Total phenolic 164 ± 54b 166 ± 14b 245 ± 54a 194 ± 20b

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Mean ± SD
compounds
(mg GAE/100 g)

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Values 90 - 289 147 - 185 196 - 318 169 - 208

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Flavonoid Mean ± SD 16 ± 10b 19 ± 4b 29 ± 6a 21 ± 5b
(mg QE/100 g)

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Values 0,30 - 36,00 12 - 25 23 - 36 15 - 24

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Means with different letter are significantly different.
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Averages ± Standard deviation were obtained from three separate assays.
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EAG : Gallic Acid Equivalents.

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QE : Quercetin Equivalents.
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Table 3 : Phenolic compounds identified in all honey samples.

GA Prt HBA HPA Van Cou Caf Fer Syr Cin BA GeA HVA DPA PC IRM Chr PB Api Gal Gen Iso Myr Que Lut Kae Hes Qu Ka Dia

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Multifloral (polyfloral) honeys
H01 - + + + + + + + + + + - - + + + + + + + - + - + + + + + - -
H02 - + + + + + + + + + + - - + + + + + + + + + - + + + + + - +
H03 - + + + + + + + + - + + + + + + + + + + + - + + + + - + - +

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H04 - + + - + + + + + + + - - + + + + + + + + - - + + + + + - -
H05 - - + + + + + + - + + - - - + + + + + + + + - + + + + - + -
H06 - + + + + + + + + + - - - + + + + + + + + + - + + + + - + -

SC
H07 - + + + + + + + + + + - - - + + + + + + - + - + + + + - + -
H08 - + + + + + + + + - + - - - + + + + + + - + - - + + + - + -
H09 - - + - - - - - + - - - - - + + + + + + - + - + + + + - - -

U
H10 + + + - + + + + + - + + - + + + + + + + - + - + + + - - + -
H11 - + + - + + + - + - - - - - + + + + + + - + - - + + - - - -

AN
H12 + + + + + + + + + - + - + + + + + + + + + + - + + + - - + -
H13 - + + + + + + - + + + - - + + + + + + + + + - + + + - - + -
Eucalyptus honeys
H14 + + + + + + + + + + + + - + + + + + + + - - + + + + - + - -

M
H15 + + + + + + + + + + + + + + + + + + + + + + + + + + + + - +
H16 - - - + + - + + - + - - - + + + - + + - + - + + + - - + + -
H17 + + + + + + + + + + + + - + + + + + + + + + + + + + + - + -

D
H18 - - + + + + + + + + + - - + + + + + + + - + - + + + - - + -
H19 + + + + + + + + + - + + - + + + + + + + + + - + + + - - - -
H20 - - + + + + + + + - +
TE - - - +
Erica arborea honeys
+ + + + + + + - - + + + - - -

H21 - + + + + + + + + + + - - + + + + + + + - + - + + + + + - -
EP
H22 - + + + + + + + + + + + - + + + + + + + + + - + + + + + + -
H23 + + + + + + + + + + + - + + + + + + + + + - - + + + - + - +
H24 - + + + + + + - + + + - - - + + + + + + + + - - + + - + + -
Apiaceae honeys
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H25 - + + + + + + + + + + - - + + + + + + + + - - + + + - + - -
H26 - + + + + + + + + + + - - + + + + + + + + + - + + + + + + -
AC

H27 - + + + + + + + + - + - - - + + + + + + + + - + + + + + + -
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Table 3 : (Continued)

GA Prt HBA HPA Van Cou Caf Fer Syr Cin BA GeA HVA DPA PC IRM Chr PB Api Gal Gen Iso Myr Que Lut Kae Hes Qu Ka Dia
Other monofloral honeys

PT
H28 - + + + + + + + + + + - + + + + + + + + + - + + + + - + - +
H29 - - + - + + + + - + + - - + + + + + + + + + - + + + + - + -
H30 - + + + + + + + + + + + - + + + + + + + - + + + + + - - - -

RI
H31 - + + + + + + + + + + + - - + + + + + + - + - + + + + - + -
H32 - - + + + + + + + - + - - - + + + + + + - + - + + + + - - -
H33 - + + + + + + + + - + - - - + + + + + + - + - + + + - - - -

SC
H34 - + + - + + + - + - + - - - + + + + + + + - - + + + - - + +
H35 - - + + + + + - + - + - - - + + + + + + + - - - + + - - - +
TCH 7 27 35 21 34 34 34 28 34 21 32 9 5 21 35 35 35 34 35 35 22 27 7 30 35 35 16 16 17 7

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(+) : presence ; (-) : absence.

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GA = Gallic acid; Prt = Protocatechuic acid; HBA = 4-Hydroxybenzoic acid; HPA= 4-hydroxyphenylacetic acid; Van = Vanillic acid; Api = Apiginin; Cou = p-coumaric acid;

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Gal = Galangin; Chr = Chrysin; PB = Pinobanksin; IRM= Isorhamnetin ; Caf = Caffeic acid, Gen = Genistein; Fer = Ferulic acid; Iso = Isosakuranetin; Syr= Syringic acid;

Myr = Myricetin; Cin = Cinnamic acid; BA = Benzoic acid; Que = Quercetin; Lut = Luteolin; GeA = Gentisic acid; Kae = Kaempferol; HVA= Homovanillic acid; Hes =

D
Hesperetin; DPA = 3,4 dihydroxyphenylacetic acid; Qu = Quercetrin; PC = Pinocembrin; Ka = Kaempferid ; Dia = Diadzein; TCH: Total of each Compound in Honey samples.

.
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Table 4 : Phenolic compounds identified in Eucalyptus honey sample.

a
Peak tR (min) m/z experimental Molecular formula m/z calculated Error (ppm) mSigma value Compounds

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1 6.50 211.0622 C10H11O5 211.0612 -4.6 3.5 Gallic acid propyl ester

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2 8.59 137.0272 C7H5O3 137.0244 -20.0 13.1 4-Hydroxybenzoic acid b

3 10.11 121.0323 C7H5O2 121.0295 -23.3 6.6 Benzoic acid isomer

SC
6-Methylesculetin
4 10.38 191.0352 C10H7O4 191.0350 -1.4 12.4

U
5 10.54 179.0363 C9H7O4 179.0350 -7.5 16.4 Caffeic acid b

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6 11.61 197.0465 C9H9O5 197.0455 -4.7 10.2 Syringic acid b

M
7 12.15 433.1767 C19H29O11 433.1715 -12.0 30.1 Secologanin acetal

8 13.02 167.0372 C8H7O4 167.0350 -13.4 5.2 Vanillic acid b

D
9 13.35 163.0418 C9H7O3 TE 163.0401 -10.8 15.0 p-Coumaric acid b

10 13.59 165.0576 C9H9O3 165.0557 -11.3 6.8 Methylvanillin

EP

Hexyl salicylate
11 14.26 221.1182 C13H17O3 221.1183 0.6 11.5

12 15.34 195.0635 C10H11O4 195.0663 14.4 19.1 Trimethoxybenzaldehyde

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Benzoic acid b
AC

13 18.08 121.0323 C7H5O2 121.0295 -23.1 13.8

14 16.26 161.0627 C10H9O2 161.0608 -11.7 6.3 2-Acetylacetophenone

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15 16.93 301.0719 C16H13O6 301.0718 -0.3 22.5 Hesperetin isomer

PT
16 17.44 151.0414 C8H7O3 151.0401 -8.7 12.4 Vanillin

17 17.74 181.0151 C8H5O5 181.0142 -5.0 4.1 Methyl syringate

RI
18 18.56 263.1288 C15H19O4 263.1289 0.3 14.2 (trans,trans)-Abscisic acid

SC
19 18.99 251.1265 C14H19O4 251.1289 9.6 7.1 Ubiquinol 1 (isomer 1)

U
20 19.12 187.0989 C9H15O4 187.0976 -7.3 1.4 Anchoic acid

AN
21 19.41 219.1394 C14H19O2 219.1391 -1.0 2.1 Di-tert-butyl-1,4-benzoquinone

22 20.25 263.1286 C15H19O4 263.1289 1.2 8.6 (cis,trans)-Abscisic acid

M
23 21.17 199.0995 C10H15O4 199.0976 -9.7 5.4 Camphoric acid

D
24 21.50 301.0359 C15H9O7 301.0354 -1.9 19.0 Ubiquinol 1 (isomer 2)

25 21.99 271.0636 C15H11O5

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271.0612 -9.0 25.3 Naringenin b
EP
26 22.49 285.0407 C15H9O6 285.0405 -0.9 5.2 Luteolin b

27 23.02 315.0527 C16H11O7 315.0510 -5.2 34.6 Isorhamnetin b

C

28 23.51 165.0937 C10H13O2 165.0921 -9.6 10.3 2-t-Butylhydroquinone

AC

29 23.78 285.0398 C15H9O6 285.0405 2.3 15.7 Kaempferol b

30 24.26 269.0457 C15H9O5 269.0455 -0.7 12.4 Apigenin b

 ACCEPTED MANUSCRIPT

31 25.00 299.0556 C16H11O6 299.0561 1.8 12.1 Kaempferid b

32 26.87 255.0654 C15H11O4 255.0663 3.5 13.7 Pinocembrin b

PT
33 27.00 285.0765 C16H13O5 285.0768 1.1 16.7 Isosakuranetin b

RI
34 27.09 247.0986 C14H15O4 247.0976 -4.3 11.3 Prenyl caffeate

SC
35 27.41 247.0972 C14H15O4 247.0976 1.5 11.2 Prenyl caffeate isomer

36 28.13 253.0513 C15H9O4 253.0506 -2.4 13.3 Chrysin b

U
37 28.58 239.0682 C15H11O3 239.0714 13.2 30.9 2-Benzoylbenzoic acid methyl ester

AN
38 28.70 269.0462 C15H9O5 269.0455 -2.4 8.6 Galangin b

M
39 29.37 283.0615 C16H11O5 283.0612 -1.0 16.5 Methoxychrysin

D
40 26.90 255.0665 C15H11O4 255.0663 -0.8 7.9 Pinocembrin isomer
TE
a
Peak numbers assigned according to the overall elution order.
EP
b
Compounds confirmed by standard.
C
AC
 ACCEPTED MANUSCRIPT

Intens.
5
x10
26

PT
25
6 23

RI
SC
4

U
7 8 32,33
1 22
18
10

AN
2 24
27 28 34 36
14 20 21 30 31 37
12 29 35 38 40

M
9 16 19
2 5 11 13 39
34 6
15 17
0

D
5 10 15 20 25 30
TE Time [min]

Identified compounds : 1: Gallic acid propyl ester, 2: 4-Hydroxybenzoic acid, 3: Benzoic acid isomer, 4: 6-Methylesculetin, 5: Caffeic acid, 6: Syringic
acid, 7: Secologanin acetal, 8: Vanillic acid, 9: p-Coumaric acid, 10: Methylvanillin, 11: Hexyl salicylate, 12: Trimethoxybenzaldehyde, 13: Benzoic acid,
EP

14: 2-Acetylacetophenone, 15: Hesperetin isomer, 16: Vanillin, 17: Methyl syringate, 18: (trans,trans)-Abscisic acid, 19: Ubiquinol 1 (isomer 1), 20:
Anchoic acid, 21: Di-tert-butyl-1,4-benzoquinone, 22: (cis,trans)-Abscisic acid, 23: Camphoric acid, 24: Ubiquinol 1 (isomer 2), 25: Naringenin, 26:
C

Luteolin, 27: Isorhamnetin, 28: 2-t-Butylhydroquinone, 29: Kaempferol, 30: Apigenin, 31: Kaempferide, 32: Pinocembrin, 33: Isosakuranetin, 34: Prenyl
caffeate, 35: Prenyl caffeate isomer, 36: Chrysin, 37: 2-Benzoylbenzoic acid methyl ester, 38: Galangin, 39: Methoxychrysin, 40: Pinocembrin isomer.
AC

Fig. 1
 ACCEPTED MANUSCRIPT
Intens.
x105 H21
23
4
28
1
3
26
27
2

18 22 25
1 7
8 14 20-21 36
2 5 13 16 31
0 34
5 10 15 20 25 30 Time [min]

PT
Intens.
x105 H22
8
1

RI
6
23
4
26

SC
7 18 22
2 8 25 31
14 24 27 36
2 35 28
0
5 10 15 20 25 30 Time [min]

U
Intens.
x105
8
1 H23

7
AN
4
23
M
2 8 10 31
5 18 22 25 26 27
2 3 12 14 28
0
5 10 15 20 25 30 Time [min]
D

Intens.
x106
H24
1
TE

0.8

0.6
23
7
EP

0.4
26
8 10 18 22
0.2 14 21 25 31
5 24
2 3 13 16 20 28 32 36 37
C

0.0
5 10 15 20 25 30 Time [min]
AC

Identified compounds : 1: Gallic acid propyl ester, 2: 4-Hydroxybenzoic acid, 3: Benzoic acid
isomer, 4: 6-Methylesculetin, 5: Caffeic acid, 7: Secologanin acetal, 8: Vanillic acid, 10: Methylvanillin,
12: Trimethoxybenzaldehyde, 13: Benzoic acid, 14: 2-Acetylacetophenone, 16: Vanillin, 18:
(trans,trans)-Abscisic acid, 20: Anchoic acid, 21: Di-tert-butyl-1,4-benzoquinone, 22: (cis,trans)-
Abscisic acid, 23: Camphoric acid, 24: Ubiquinol 1 (isomer 2), 25: Naringenin, 26: Luteolin, 27:
Isorhamnetin, 28: 2-t-Butylhydroquinone, 31: Kaempferide, 32: Pinocembrin, 36: Chrysin, 37: 2-
Benzoylbenzoic acid methyl ester.
Fig. 2
 ACCEPTED MANUSCRIPT
Intens. a
5
x10
23

4 24
25
3 26

2 5
19 27
28
1
2 3 10 12 18 20 22 30 31 32

PT
0 5 10 15 20 25 30
Time [min]

Intens.
5 9 b

RI
x10

3
23 24

SC
2
25
32,33
1 26 36
20 37

U
5 10 15 20 25 30
Time [min]

Intens.
x105
AN c
2.0 23
M
1.5
9 24
25 32,33
1.0
26
D

17 36
0.5
10 35 37
TE

20 22 28 31
0.0 3 30
5 10 15 20 25 30
Time [min]
EP

Identified compounds : 2: 4-Hydroxybenzoic acid, 3: Benzoic acid isomer, 5: Caffeic acid, 9: p-

Coumaric acid, 10: Methylvanillin, 12: Trimethoxybenzaldehyde, 17: Methyl syringate, 18: (trans, trans)-
C

Abscisic acid, 19: Ubiquinol 1 (isomer 1), 20: Anchoic acid, 22: (cis,trans)-Abscisic acid, 23: Camphoric
acid, 24: Ubiquinol 1 (isomer 2), 25: Naringenin, 26: Luteolin, 27: Isorhamnetin, 28: 2-t-Butylhydroquinone,
AC

30: Apigenin, 31: Kaempferide, 32: Pinocembrin, 33: Isosakuranetin, 35: Prenyl caffeate isomer, 36:
Chrysin, 37: 2-Benzoylbenzoic acid methyl ester.

Fig. 3
 ACCEPTED MANUSCRIPT

Highlights :

Several phenolic compounds have been identified in honey extracts.

Caffeic and p-coumaric acids are markers for Capparis and Trifolium honey, respectively.

PT
All honey samples shared hydroxybenzoic acid and seven flavonoids aglycones.

Chromatograms of Erica honeys and some of Eucalyptus honeys showed similar profiles.

RI
Algerian honeys contain antioxidants that may have biological activities.

U SC
AN
M
D
TE
C EP
AC