Phenolic Profile of Honeydew Honeys from theNorth-East Part of Romania
Mircea Oroian*, Sorina Ropciuc, Amelia Buculei, Sergiu Pădureț, Elena Todosi

Faculty of Food Engineering, Stefan cel Mare University of Suceava, Suecava, Romania*Corresponding author, e-mail: m.oroian@fia.usv.ro1

Bulletin UASVM Food Science and Technology 73(2)/2016
ISSN-L 2344-2344; Print ISSN 2344-2344; Electronic ISSN 2344-5300
DOI: 10.15835/buasvmcn-fst:12316

Abstract
The aim of this study was todetermine the physicochemical (moisture content, pH, free acidity, electricalconductivity, colour (L*, a*, b*, chroma, hue angle), ash content, fructose and glucose content) and to determine
the phenolic profile (quercetin, apigenin, myricetin, isorhamnetin, kaempherol, caffeic acid, chrysin, galangin,luteolin, p-coumaric acid, gallic acid and pinocembrin) of five samples of honeydew honeys from the North-Eastpart of Romania. Allanalysed honey samples had a moisture content below the maximum level of 20%establishedby the European Directive 110/2001 regarding honey. The acidic nature of the honeydew was confirmed by thelevel of the pH and free acidity of the samples, andis influenced mainly by the organic acids; all the samples had
a free acidity lower than 50 meq acid/kg. The honey colour was dark which is confirmed by the level of the CIE
L*a*b* parameters (lower values of L*, a* and b*). The inverted sugar level (was higher than 60 g/ 100g. The
myricetin ranged between 0 – 0.37 mg/100 g honey, p-coumaric acid ranged between 0-4.35 mg/100 g honey,
chrysin ranged between 0-0.16 mg/100 g honey, caffeic acid ranged between 0-1.92 mg/100 g honey, pinocembrin
ranged between 0.27-4.36 mg/100 g honey, quercetin ranged between 0.10 – 2.79 mg/100 g honey, apigenin
ranged between 0-1.10 mg/100 g honey, kaempherol 0–0.60 mg/100 g honey, isorhamentin 0-0.12 mg/100 ghoney, luteolin ranged between 0-0.11 mg/100 g honey, gallic acid ranged between 0.02-0.26 mg/100 g honey andgalangin 0.02-0.49 mg/100 g honey, respectively.
Keywords: honeydewhoney,phenolic profile, physicochemical parameters

INTRODUCTION
Honey is a natural product proved to provide
beneficial effects to the human health, due to a highconcentration of sugars, water, proteins, organicacids, minerals, phenolic acids, flavonoids andenzymes (Halouzka et al., 2016). According to thepresentEuropean Directive 110/2001, there arethree types of honeys withregard to their origin:(1) nectar honey – made fromplant nectar thatcan be monofloral or multifloral,(2) honeydew –made mostly from the secretion ofinsects feedingon plant juices or plant secretion, and(3) mixed
honey of honeydew and nectar honey.
Honeydew honey refers to honey produced
by bees that collect the excretion of plant-sucking
insects on the living parts of plants. Honeydew
itself has been historically considered as delightful

syrup used as food and medicine by people, to theextent that the famous term “manna’ often refers
to the crystallized honeydew produced by scale
insects feeding on the tree (de Miguel et al., 2014).
Phenolic compounds that can be found inhoney are free phenols (volatilecompounds),phenolic acids, polyphenols (usually in the formof flavonoids), anthocyanins, procyanidins andpigments.Lately, the phenolic compounds havebeen intensively used by researchers to determine
the botanical and geographical origin of honeys(Escricheet al. 2014, Halouzka et al., 2016, Gomeset al. 2010; Bertoncelj et al. 2011; Manzanares etal. 2011; Juan-Borras et al. 2014; Karabagias etal. 2014). The kaempferol has been establishedas marker for rosemary honeys, quercetin for

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OROIAN et al.

sunflower honey, while hesperitin for citrus honey(Thomas-Barberan et al., 2001)
The aim of this study was to analyse from melissopalynological point of view and to establish
the phenolic profile (quercetin, apigenin, myricetin, isorhamnetin, kaempherol, caffeic acid, chrysin, galangin, luteolin, p-coumaric acid, gallic acidand pinocembrin) of the honeydew honeys fromthe North-East part of Romania using an HPLC-UV
methods; to our knowledge, no other studies havebeen reported on the phenolic profile determination of the honeydew honeys from the North-Eastpart of Romania.

MATERIALS AND METHODS

Materials. The honeydew honeys (5 samples,each sample of 500 g) were purchased in September 2015 from the local beekeepers from theSuceava county, Romania.The samples were preserved at 20 °C until they were analysed.
Melissopalynological analysis. The pollen
analysis was performed according to the methodof Louveaux et al. (1970), using a non-acetolytic
method. Ten grams of honey were mixed with
about 40 ml of distilled water; then, centrifugedat 4500 rpm (3383 × g) for 15 minutes and thesupernatant was carefully removed. The residue
was re-dissolved again and centrifuged for other15 minutes. The full sediment was used to prepare
the slide. The pollen spectrum of each honey sample was determined by a light microscopy (Motic ×40) by counting at least 800 pollen grains. Quantitative evaluation of each pollen type was determined and expressed as percentage according toDobreet al. 2012.
Physicochemical properties determination. The physicochemical parameters(moisture
content, pH, free acidity, electrical conductivity,
colour (L*, a*, b*, chroma, hue angle), fructose andglucose content) have been made based on themethod described by Bogdanov et al. (2002).
Phenolics extraction. The phenolics extraction was realized using the method describedby Baltrušaitytė et al. (2007) and Escriche et al.
(2014). Sixty grams of Amberlite XAD-2 resin,pore size 9 nm, and particle size 0.3–1.2 mm were
soaked in methanol for 10 minutes, then, the mostof methanol was decanted and replaced by distilled water. The mixture was stirred, allowed tostand for 5–10 min and packed into a glass column, 25×2 cm. The honey samples (25 g) wereBulletin UASVM Food Science and Technology 73(1) / 2016

thoroughly mixed with 250 mL of distilled waterand adjusted to pH 2 with concentrated HCl. The
solution was slowly filtered through the column
packed as previously described. The column waswashed with 250 mL of acidified water (pH 2with HCl) and subsequently rinsed with 300 mLof neutral distilled water to remove all sugars andother polar compounds of honey. The flavonoids
and phenolic compounds were eluted from thesorbentwith 250 mL of methanol.  The methanol
extracts were concentrated under vacuum at 40°C
in rotary evaporatorHS-2005S-N (HAHNVAPOR,China). The residuewas dissolved in 5 mL of distilled water and extracted three times with 5 mLof diethyl ether. The dried residue was then redissolved in 1 mL of methanol (HPLC grade) andfiltered through a membrane filter with a 0.45 μm
pore size. Three replicate extractions were performed for each sample.
HPLC analysis of phenolics. The phenolic
compounds were separated and quantified using
the method described by Coneac et al. (2008). A
High Performance Liquid Chromatography (HPLC)(Shimadzu, Kyoto, Japan) system equipped with a
LC-20 AD liquid chromatograph, SIL-20A auto samples, CTO-20AC auto sampler and a SPD-M-20A diode array detector was used. The separation wascarried out on a Zorbax SP-C18 column, with 150
mm length, 4.6 mm i.d., and 5 µm-diameterparticle
was used; the phenolics detection was set at 200nm and 210 nm. The mobile phase was acetonitrile: water ratio 48:52, temperature was of 25°C,
with a flow of 0.3 ml/min, the injected sample volume was of 20 µl. The diluted standard solutionsof quercetin, apigenin, myricetin, isorhamnetin,kaempherol, caffeic acid, chrysin, galangin, luteolin, p-coumaric acid, gallic acid and pinocembrin
were analyzed under the same HPLC conditions
and furthermore the calibration of the detector response was made. Data collection and subsequentprocessing were performed using the LC solution
software 1.21 version (Shimadzu, Kyoto, Japan).
The quantitative results were expressed as mg ofcompound per 100 g honey.

RESULTS AND DISCUSSION

The honey samples were submitted to the melissopalynological analysis for confirming the authenticity of it. The range of pollen grains number
was between 1650 to 2450The honeydew honeywas poor inpollen, recording an average of 2083

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Phenolic Profile of Honeydew Honeys from the North-East Part of Romania

Tab. 1. The main pollen types in honeydew honeys samples
Honey typeSamples

Principal pollen type

Secondary pollen types

Honeydew

5Brassica napus (18.1%)

Quercus (14.3%)
Trifolium repens (11.8%)
Castanea sativa (8.6%)

Tab.2. Physicochemical parameters of the honeydew honeys
Variable

Other significant
pollen types
Helianthus annuus

Minimum

Maximum

Mean

Std. deviation

Moisture content (%)

14.44

17.04

16.13

1.04

Free acidity(meq acid/kg)

11.80

20.00

15.50

3.49

Ash (%)

0.45

pHElectrical conductivity (µS/cm)

4.19

5.16

4.75

0.48

923.3

1276.81077.3

149.6

L*

19.59

21.52

20.41

0.82

b*

4.15

6.90

5.45

a*

ChromaHue-angle

Fructose (%)
Glucose (%)

4.16
6.53
0.15

35.96
32.98

pollen grains,similar to other honeydew honyes
reported into the literature (Dobre et al. 2013).
The principal pollen types presented into the honeydew honeys are shown in Tab. 1. The palynological analysis provides useful information regarding the vegetation where the honey was collected.
The presence of Brassica napus, Trifolium repens,
Castanea sativa, Quercus and Helianthus annuus
are giving information regarding the time of yearof the honey production and it can be considereda spring honey.
In the case of honeydew samples, they canbe authenticated using the electrical conductivity
parameter (EC 110/2001) If the electricalconductivity is higher than 800 µS/cm, then thehoney is a honeydew one (EC 110/2001). The
analyzed honeys had electrical conductivityhigher
than 800 µS/cm (Tab. 2).

0.62
7.68
9.47
1.15

40.98
36.54

0.52
5.55
7.86
0.67

38.73
35.06

0.07
1.38
1.06
1.26
0.45
2.03
1.50

In the Tab. 2 are presented the physicochemicalparameters (moisture content, pH, free acidity,
electrical conductivity, colour (L*, a*, b*, chroma,hue angle), fructose and glucose content) of thefive samples of honeys analysed.
One of the most important parameter for thehoney quality is the moisture content. Regarding
this parameters, the UE (2001) has established athreshold of 20% in the case of honeys. A higher
content of water in honey will promote thefermentation processes (Escriche et al., 2014). In
the case of the honeys analysed all the samples
respected the regulation established by the UE (EC110/2001).
Honey contains less than 0.5% of organic
acids. They are a group of constituents that
contribute to several properties of this food,such as its color, aroma, taste, pH, acidity, and to
Bulletin UASVM Food Science and Technology 73(1) / 2016

108OROIAN et al.

a lesser extent, electrical conductivity (Sanchoet al., 2013). Small variations in the range of pH
in relation to the large swings in the free acidvalues were attributed to the buffer properties ofhoney, due to mineral salts such as phosphates,carbonates and other (Bogdanov 2009, Sancho etal., 2013). In the case of the present study, the pH
ranged between 4.19 to 5.16  being in agreementwith those reported in the case of Spanish honeys(Oroian et al., 2013, Escriche et al., 2014) andRomanian honeys (Oroian 2012).
The honey acidity is influenced in principal
by the organic acids (White 1979). Lactones
are internal esters of organic acids and do notcontribute to honey’s active acidity (Bogdanov2009). Lactones hydrolyze over time, therefore
increasing honey free acid. Total acidity is the sumof free acid and lactones. In terms of free acidity theCodex Alimentarius (2001) established a level of
50 meq acid/kg for honeydew honeys respectively.
All the samples analysed respected the regulation
established by Codex Alimentarius (2001).
Electrical conductivity is a physical propertyof honey mainly related to the content of mineralsalts, and to a lesser extent to the content of organic

acids, proteins, sugars, and polyols (Crane 1990).
It was found that the electrical conductivity was
directly proportional to ash content and acidityof honey (Sancho et al., 2013). The electricalconductivity ranged between 923.3 to 1276.8 µS/cm, being higher than 800 µS/cm recommended
by the literature in the case of honeydew honeys(EC 110/2001).
Color is an optical property of honey,
described as the result of different degrees ofabsorption of light at different wavelengths byhoney compounds (Sancho et al., 2013). Thehoneydew honeys are dark honeys (L* values
are lower) due to the high mineral contentand pigments (Oroian 2012). The lower the L*valuesare, the honey is darker.Due to the opacityof the honeythe a* and b* values are close to 0.The
taste of honeydew honeys is stronger and tend tohave relatively more maltose, minerals, acids andantioxidant flavonoids (Stanway 2012).
The sugar composition of honeys is influenced
by the honey origin. The main components of honeyare D-glucose and D-fructose which are originatedfrom the honeydey and from the enzymatichydrolysis of sucrose and other sugars from theFig.1.Relative quantities of analysed phenolics in honeydew honey samples
Bulletin UASVM Food Science and Technology 73(1) / 2016

Phenolic Profile of Honeydew Honeys from the North-East Part of Romania

nectar (Tomasik 2003). The concentration ofglucose and fructose in honey must be higher than
60 g/100 g honey (EC 110/2001). All the analysedhoneys met this requirements.
Polyphenols are another important group ofcompounds with respect to the appearance andthe functional properties of honey. Polyphenols inhoney are mainly flavonoids (e.g. quercetin, luteolin, kaempferol, apigenin, chrysin and galangin),phenolic acids, and phenolic acid derivates (Saad& Said 2011). In the case of the analysed honeydew honeys the following polyphenols were identified and quantified: myricetin (0 – 0.37 mg/100g honey), p-coumaric acid (0-4.35 mg/100 g honey), chrysin (0-0.16 mg/100 g honey), caffeic acid(0-1.92 mg/100 g honey), pinocembrin (0.27-4.36
mg/100 g honey), quercetin (0.10 – 2.79 mg/100 ghoney), apigenin (0-1.10 mg/100 g honey), kaempherol (0 – 0.60 mg/100 g honey), isorhamentin(0-0.12 mg/100 g honey), luteolin (0-0.11mg/100g honey), gallic acid (0.02-0.26 mg/100 g honey)
and galangin (0.02-0.49 mg/100 g honey).
The comparison of relative quantities of phenolic acids in different honeydew honey samples
demonstrated significant (P < 0.05) variations of
their quantitative composition (Fig. 1). From thefigure 1 it can be observed that the major polyphenols presented into the honeydew honeys arequercetin and pinocembrin.

CONCLUSION

The honey samples analysed respected theregulation of the European Union and Codex Alimentarius in terms of moisture content and glucose and fructose content. The honeydew colour
parameters were closed to the origin of the threeaxes (L*, a* and b*) due to their chemical composition. The phenolic profile of the honeydew samples do not presented one compound that can beconsidered a chemical marker. From the 12 individual phenolics studie, we observed thatquercetin and pinocembrin were in the highest concentration. The honeydew honeys analysed had a highcontent of phenolics and it can be considered asource of antioxidants.

Acknowledgments.This work was supported
by a grant of the Romanian National Authority for
Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-II-RU-TE-2014-4-0110

109

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