Honey is a highly consumed commodity due to its potential health benefits upon certain consumption, resulting in a high market price. This fact indicates the need to protect honey from fraudulent acts by delivering comprehensive analytical methodologies. In this study, targeted, suspect and non-targeted metabolomic workflows were applied to identify botanical origin markers of Greek honey. Blossom honey samples (n = 62) and the unifloral fir (n = 10), oak (n = 24), pine (n = 39) and thyme (n = 34) honeys were analyzed using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) system. Several potential authenticity markers were revealed from the application of different metabolomic workflows. In detail, based on quantitative targeted analysis, three blossom honey markers were found, namely, galangin, pinocembrin and chrysin, while gallic acid concentration was found to be significantly higher in oak honey. Using suspect screening workflow, 12 additional bioactive compounds were identified and semi-quantified, achieving comprehensive metabolomic honey characterization. Lastly, by combining non-targeted screening with advanced chemometrics, it was possible to discriminate thyme from blossom honey and develop binary discriminatory models with high predictive power. In conclusion, a holistic approach to assessing the botanical origin of Greek honey is presented, highlighting the complementarity of the three applied metabolomic approaches.

Propolis samples from a geographical part of northwest Greece (Prespa National Park, PNP), which is characterized as a plant endemism center and biodiversity hotspot, were characterized through pollen analysis, chemically analyzed, and biologically evaluated. The majority of the studied propolis showed typical chemical constituents (phenolic acids, flavonoids, and chalcones) of European type, while a sample of Mediterranean-type propolis (rich in diterpenes) was also identified. The palynological characterization was implemented to determine the botanical origin and to explain the chemical composition. The total phenolic content and the DPPH assay showed that the European-type propolis samples possessed strong antioxidant activity (86–91% inhibition at 200 μg/mL). Moreover, promising antibacterial activity of the extracts (MIC values 0.56–1.95 mg/mL) and moderate antifungal activity (MIC values 1.13–2.40 mg/mL) were noticed, while the sample with the highest activity had a significant content in terpenes (Mediterranean type). Propolis samples from the PNP area represent a rich source of antibacterial and antioxidant compounds and confirm the fact that propolis is a significant natural product with potential use for improving human health and stimulating the body’s defense. Finally, it is noteworthy that a significant chemical diversity was demonstrated, even in samples from a limited geographical area as this of PNP.

Commercially available bee pollen from Northwestern Greece was subjected to headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry (HS-SPME/GC–MS) analysis for the identification of its aroma compounds. Two different procedures were followed: (a) volatile compounds analysis of ground bee pollen and (b) volatile compounds analysis of aqueous solution (1:1, w/v) of ground bee pollen. The results showed that the volatile pattern of bee pollen was mainly dominated by aldehydes, followed by ketones, terpenoids, and minor contributions of other classes of volatile compounds. The aqueous medium affected the total volatile composition by means of increasing mainly the contribution of aldehydes to the total volatile pattern. However, aroma compounds, such as (E)-2-hexenal, γ -valerolactone, and γ-butyrolactone were not identified in the aqueous bee pollen solution. Based on the volatile compounds that were identified, the aroma profile of bee pollen may be characterized as ‘’green’, ‘’floral’’, and ‘’pleasant’’. Given the limited available literature in the analysis of volatile compounds of bee pollen and the use of bee pollen as a natural odorant, Greek bee pollen at different physical state may be proposed as a flavoring agent in different foodstuffs, depending however, on the goods of interest.

In the present study, we aimed to examine the antioxidant, antiaging and photoprotective properties of Greek honey samples of various botanical and geographical origin. Ethyl-acetate extracts were used and the and the total phenolic/flavonoid content and antioxidant capacity were evaluated. Honey extracts were then studied for their cytoprotective properties against UVB-induced photodamage using human immortalized keratinocytes (HaCaT) and/or reconstituted human skin tissue models. Specifically, the cytotoxicity, oxidative status, DNA damage and gene expression levels of specific matrix metalloproteinases (MMPs) were examined. Overall, the treatment of HaCaT cells with honey extracts resulted in lower levels of DNA strand breaks and attenuated the decrease in cell viability following UVB exposure. Additionally, honey extracts significantly decreased the total protein carbonyl content of the irradiated cells, however, they had no significant effect on their total antioxidant status. Finally, the extracts alleviated the UVB-induced up-regulation of MMPs-3, -7 and -9 in a model of reconstituted skin tissue. In conclusion, honey extracts exhibited significant photoprotective and antiaging properties under UVB exposure conditions and thus could be further exploited as promising agents for developing novel and naturally-based, antiaging cosmeceutical products.

Obtained directly from beekeepers in Portugal and Greece, some rare honey samples were subjected to melissopalynological, physicochemical (°Brix, moisture, pH, electrical conductivity, free acidity, total dissolved solids, salinity, liquid resistivity, specific weight, CIELAB colour parameters, Pfund, and colour intensity), biochemical (diastase activity, vitamin C, antioxidant activity, and total phenolic content), and volatile compounds analyses. Results showed that honey samples from Portugal were dominated by pollen grains of Anarrhinum bellidifolium (L.), whereas those from Greece were honeydew of not specific type, possessing a diverse pollen spectrum and few honeydew elements. The implementation of one-way analysis of variance showed significant differences (p < 0.05) among the two honey types in the majority of physicochemical, biochemical, and to a lesser extent in the volatile compounds. These were 34 in total, belonging to alcohols, aldehydes, benzene derivatives, esters, hydrocarbons, ketones, sulfur compounds, and terpenoids. Application of correlation, regression, and stepwise linear discriminant analyses indicated some potential fitting among the investigated parameters, and classified perfectly honey samples according to botanical origin based on electrical conductivity, salinity, 2-methylbutanal, and alpha-pinene, providing a rapid tool for the classification of less common honey types.

A less common honeydew honey, "dryomelo", from different regions in Greece was characterized based on palynological, physico-chemical and bioactivity parameter analyses. Results showed that dryomelo had diverse proportions (%) of Castanea saliva, Polygonum aviculare, Quercus ilex, minor pollen grains and few to medium honeydew elements. Analysis of variance showed significant differences (p < 0.05) in hydroxymethylfurfural, diastase, colour parameters, and % in vitro antioxidant activity values according to region. Application of factor analysis to predominant pollen and the significant parameters, in combination with the Kaiser-Meyer-Olkin test, strengthened the effect of geographical origin and their potential use for quality control analysis of dryomelo.

Twenty-two honey samples, namely clover and citrus honeys, were collected from the greater Cairo area during the harvesting year 2014–2015. The main purpose of the present study was to characterize the aforementioned honey types and to investigate whether the use of easily assessable physicochemical parameters, including color attributes in combination with chemometrics, could differentiate honey floral origin. Parameters taken into account were: pH, electrical conductivity, ash, free acidity, lactonic acidity, total acidity, moisture content, total sugars (degrees Brix-°Bx), total dissolved solids and their ratio to total acidity, salinity, CIELAB color parameters, along with browning index values. Results showed that all honey samples analyzed met the European quality standards set for honey and had variations in the aforementioned physicochemical parameters depending on floral origin. Application of linear discriminant analysis showed that eight physicochemical parameters, including color, could classify Egyptian honeys according to floral origin (p < 0.05). Correct classification rate was 95.5% using the original method and 90.9% using the cross validation method. The discriminatory ability of the developed model was further validated using unknown honey samples. The overall correct classification rate was not affected. Specific physicochemical parameter analysis in combination with chemometrics has the potential to enhance the differences in floral honeys produced in a given geographical zone.

The purpose of the present study was to investigate and chart pollen characteristics of Egyptian clover honey in order to evaluate its purity with respect to authentication. For this purpose, 8 clover honey samples were randomly collected from local products shops in the greater Cairo area and subjected to melissopalynological analysis, the official method for the determination of honey botanical origin, by providing exhaustive details on specific pollen grains that contribute to the overall pollen grains profile (PGP). Results showed that, the dominant pollen grains (>45%) in 7 of the 8 honey samples analyzed were Trifolium alexandrinum L. followed by lower pollen grain percentages of Brassica sp., Melilotus sp., Umbeliferae, and Compositae, along with others. Given that, research on PGP involving Egyptian clover honey is limited, the present study contributes to the characterization of clover honey that enters the domestic or international market

The aim of the present study was to investigate the volatile fraction and discriminate 34 commercial thyme honeys from Morocco, Egypt, Spain, and Greece according to geographical origin, using key volatile compounds in combination with chemometrics. Sixty-two volatile compounds belonging to different classes were identified and semi-quantified using headspace solid phase micro-extraction coupled to gas chromatography/mass spectrometry (HS-SPME-GC/MS). Applying chemometric analyses to 17 volatiles (p < 0.05), honeys were successfully discriminated according to geographical origin. Correct classification rate was 88.2% using the cross-validation method. Volatile compounds proved to be a powerful tool for discriminating commercial thyme honeys from different countries.

This study was carried out to evaluate the antioxidant properties of Greek endemic Cistus creticus L. (rock rose) bee pollen and define its phenolic compounds. In the framework of our scientific studies on Greek bee keeping products, we report herein our research on three Greek bee pollen samples from Cistus. Their pollinic spectra were obtained by Louveaux's quantitative microscopical analysis and it showed that one of them had Cistus sp. (Cistaceae) as abundant pollen (together with low percentage of Brassica sp. (Cruciferae). Throughout the chemical analysis of the extracts, several secondary metabolites of flavonoid structure have been identified as major components. Specifically, quercetin-7-rhamnoside (1), quercetin-3-neohesperidoside (2), kaempferol-3-neohesperidoside (3), myricetin-3-neohesperidoside (4), kaempferol-3-glucoside (5) and quercetin-3-glucoside (6) have been isolated and their structures were elucidated on the basis of spectral evidence. Moreover, the total phenolic and flavonoid content was estimated and the free radical scavenging activity was determined by DPPH and ABTS assays. The antimicrobial activity of the extracts was tested against six Gram-positive and -negative bacteria and three pathogenic fungi, and the butanol extract showed a very interesting broad antimicrobial profile (MIC 1.98•10 −3 - 2.98•10 −3 mg/ml) against all the assayed microorganisms.

In the present study, the effect of different parameters on the correct geographical differentiation of Greek fir honey was investigated. Forty-three honey samples were collected from four regions and subjected to physicochemical and melissopalynological analyses, using official and instrumental methods. Results showed that fir honeys met the European regulatory quality standards, whereas significant differences (p<0.05) were recorded for all the determined parameters according to geographical origin. The highest differentiation rate (81.4%) was recorded using the combination of six physicochemical parameters and five phenolic compounds, as assessed by multivariate analyses.

The aim of the present study was to provide information on physicochemical (pH, free, lactonic, and total acidity, electrical conductivity, moisture, ash, lactonic/free acidity), colour parameters (L*, a*, b*), and mineral content (Ag, Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Ni, Pb, Sb, Se, Si, Ti, Tl, V, Zn) of 34 commercial thyme honeys produced in Egypt, Morocco, Greece, and Spain, in terms of characterization and geographical discrimination using MANOVA/LDA, chemometric analyses. Conventional physicochemical and colour parameters were determined using official methods of analysis. Minerals were quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that all honey samples analysed met the standard quality criteria set by the EU. In order to classify honeys according to geographical origin, MANOVA and LDA were applied in sequence. Respective classification rates based on cross-validation method were: 91.2, 76.5, and 88.2%, using 10 physicochemical and colour parameters, 12 minerals, and their combination, respectively. Testing whether the determined physicochemical parameters of commercial Mediterranean thyme honeys could be applied to routine quality control serving as honey origin predictors showed positive results. Finally, such a combination of data enhances honey quality control and authentication efforts, at an international level.

The objective of the present study was: i) to characterize Mediterranean citrus honeys based on conventional physicochemical parameter values, volatile compounds, and mineral content ii) to investigate the potential of above parameters to differentiate citrus honeys according to geographical origin using chemometrics. Thus, 37 citrus honey samples were collected during harvesting periods 2013 and 2014 from Greece, Egypt, Morocco, and Spain. Conventional physicochemical and CIELAB colour parameters were determined using official methods of analysis and the Commission Internationale de l' Eclairage recommendations, respectively. Minerals were determined using ICP-OES and volatiles using SPME-GC/MS. Results showed that honey samples analyzed, met the standard quality criteria set by the EU and were successfully classified according to geographical origin. Correct classification rates were 97.3% using 8 physicochemical parameter values, 86.5% using 15 volatile compound data and 83.8% using 13 minerals.

We have previously demonstrated that Greek thyme honey inhibits significantly the cell viability of human prostate cancer cells. Herein, 15 thyme honey samples from several regions of Greece were submitted to phytochemical analysis for the isolation, identification and determination (through modern spectral means) of the unique thyme honey monoterpene, the compound trihydroxy ketone E-4-(1,2,4-trihydroxy-2,6,6-trimethylcyclohexyl)-but-3-en-2-one. We investigated the anti-growth and apoptotic effects of the trihydroxy ketone on PC-3 human androgen independent prostate cancer cells using MTT assay and Annexin V-FITC respectively. The molecular pathways involved to such effects were further examined by evaluating its ability to inhibit (a) the NF-κB phosphorylation (S536), (b) JNK and Akt phosphorylation (Thr183/Tyr185 and S473 respectively) and (c) IL-6 production, using ELISA method. The anti-microbial effects of the trihydroxy ketone against a panel of nine pathogenic bacteria and three fungi were also assessed. The trihydroxy ketone exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range 10−6–10−4 M. Akt and JNK signaling were not found to participate in this process. The trihydroxy ketone exerted significant anti-microbial profile against many human pathogenic bacteria and fungi (MIC values ranged from 0.04 to 0.57 mg/ml). Conclusively, the Greek thyme honey-derived monoterpene exerted significant apoptotic activity in PC-3 cells, mediated, at least in part, through reduction of NF-κB activity and IL-6 secretion and may play a key role in the anti-growth effect of thyme honey on prostate cancer cells.

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.

The aim of the present study was to investigate the possibility of characterisation and classification of Greek unifloral honeys (pine, thyme, fir and orange blossom) according to botanical origin using volatile compounds, conventional physico-chemical parameters and chemometric analyses (MANOVA and Linear Discriminant Analysis). For this purpose, 119 honey samples were collected during the harvesting period 2011 from 14 different regions in Greece known to produce unifloral honey of good quality. Physico-chemical analysis included the identification and semi quantification of fifty five volatile compounds performed by Headspace Solid Phase Microextraction coupled to gas chromatography/mass spectroscopy and the determination of conventional quality parameters such as pH, free, lactonic, total acidity, electrical conductivity, moisture, ash, lactonic/free acidity ratio and colour parameters L, a, b. Results showed that using 40 diverse variables (30 volatile compounds of different classes and 10 physico-chemical parameters) the honey samples were satisfactorily classified according to botanical origin using volatile compounds (84.0% correct prediction), physicochemical parameters (97.5% correct prediction), and the combination of both (95.8% correct prediction) indicating that multi element analysis comprises a powerful tool for honey discrimination purposes.

Greek thyme honeys (Thymus capitatus L.) were characterized and classified according to geographical origin based on the determination of volatile compounds and physicochemical parameters using MANOVA and Linear Discriminant Analysis. Forty two thyme honey samples were collected during the harvesting period in 2011 from 5 different regions in Greece known to produce thyme honey of good quality. The analysis of volatile compounds was performed by Headspace Solid Phase Microextraction coupled to Gas chromatography/Mass spectroscopy. Forty seven volatile compounds were identified and semi-quantified. Physicochemical analysis included the determination of pH, free, lactonic and total acidity, electrical conductivity, moisture, ash, lactonic/free acidity ratio and color parameters: L*, a*, b*. Using 9 volatile compounds and 11 physicochemical parameters the honey samples were satisfactorily classified according to geographical origin using volatile compounds (64.3% correct prediction), physicochemical parameters (92.7% correct prediction), and the combination of both (92.9% correct prediction).

The aim of the present study was to characterise and classify Greek pine honeys according to geographical origin, based on the determination of volatile compounds and physicochemical parameters using MANOVA and Linear Discriminant Analysis. Thirty-nine pine honey samples were collected during the harvesting period 2011 from 4 different regions in Greece known to produce good quality pine honey. The analysis of volatile compounds was performed by Headspace Solid Phase Microextraction-Gas Chromatography/Mass Spectroscopy. Fifty-five volatile compounds were tentatively identified and semi quantified. Physicochemical parameter analysis included the determination of pH, free, lactonic and total acidity, electrical conductivity, moisture, ash, lactonic/free acidity ratio and colour parameters L(∗), a(∗), b(∗). Using 8 selected volatile compounds and 11 physicochemical parameters, the honey samples were satisfactorily classified according to geographical origin using volatile compounds (84.6% correct prediction), physicochemical parameters (79.5% correct prediction) and the combination of both (74.4% correct prediction).