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Review

Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys

by
Marco Ciulu
,
Nadia Spano
,
Maria I. Pilo
and
Gavino Sanna
*
Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, via Vienna 2, 07100 Sassari, Italy
*
Author to whom correspondence should be addressed.
Molecules 2016, 21(4), 451; https://doi.org/10.3390/molecules21040451
Submission received: 28 January 2016 / Revised: 11 March 2016 / Accepted: 25 March 2016 / Published: 8 April 2016
(This article belongs to the Special Issue 20th Anniversary of Molecules—Recent Advances in Natural Products)
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Abstract

:
Honey is one of the most renowned natural foods. Its composition is extremely variable, depending on its botanical and geographical origins, and the abundant presence of functional compounds has contributed to the increased worldwide interest is this foodstuff. In particular, great attention has been paid by the scientific community towards classes of compounds like phenolic compounds, due to their capability to act as markers of unifloral honey origin. In this contribution the most recent progress in the assessment of new analytical procedures aimed at the definition of the qualitative and quantitative profile of phenolic compounds of honey have been highlighted. A special emphasis has been placed on the innovative aspects concerning the extraction procedures, along with the most recent strategies proposed for the analysis of phenolic compounds. Moreover, the centrality of validation procedures has been claimed and extensively discussed in order to ensure the fitness-for-purpose of the proposed analytical methods. In addition, the exploitation of the phenolic profile as a tool for the classification of the botanical and geographical origin has been described, pointing out the usefulness of chemometrics in the interpretation of data sets originating from the analysis of polyphenols. Finally, recent results in concerning the evaluation of the antioxidant properties of unifloral honeys and the development of new analytical approaches aimed at measuring this parameter have been reviewed.

Graphical Abstract

1. Introduction

Without any doubt honey is the most recognized and famous natural food produced by bees (Apis mellifera) from nectar and honeydew. Its historic, cultural and economic relevance make it the major beekeeping product [1]. It exhibits functional properties [2], and its significance in traditional medicine has been recognized in various cultures [3] and sacred texts like the Bible (“My son, eat honey, for it is good…”, The proverbs, 24:13) and Quran (honey is “... the healing for mankind”, 16:69). In principle, honey could be defined as an aqueous solution supersaturated in sugars (mainly fructose and glucose), but its chemical composition is much more complex and extremely variable, depending on a number of factors among which geographical and botanical origin are the most representative. Indeed, beyond glucose and fructose, it is possible to find many minor mono- and oligosaccharides [4,5], sometimes useful in order to gain information that helps identify the botanical origin of honey. Moreover honey is rich in hundreds of analytes other than sugars, usually present in a mass ratio between 10−3 and 10−6 (w/w), representing in principle a detailed “chemical fingerprint” that may be a very efficient tool for identifying both the botanical and geographical origin of honey as well as revealing adulterations or frauds [6]. In this context, it is worth remembering classes of compounds like nonaromatic organic acids [7,8,9,10], vitamins [11,12,13,14,15], free amino acids [16,17,18,19,20], inorganic elements [21] and—among others—phenolic compounds.
The most important sources of phenolic compounds in honey can be traced to the vegetal kingdom. These compounds are plant-derived secondary metabolites, biosynthesized mainly for protection against stress and oxidative damage and transferred via the nectar to the honey. The phenolic compounds of honey can be classified into two main families: phenolic acids with their related derivatives (Figure 1), and flavonoids (Figure 2 and Figure 3). While some of the most representative phenolic acids found in honey are shown in Figure 1, flavonoids, all characterized by the presence of an x-phenyl-1,4-benzopyrone backbone (where x = 2, 3), can be further classified in a number of subfamilies, reported in Figure 2, whereas a selection of the most important flavonoids identified in honey is available in Figure 3.
The qualitative and quantitative dissimilarities in the phenolic profile of honeys belonging to different floral sources are a direct consequence of the natural variability of these compounds in the plants from which they originate. This variability represents the scientific basis of the two main research themes regarding the study of honey phenolic fraction. The first approach is focused on the evaluation of the overall bioactive properties of honeys from different botanical (or—sometimes—geographical) origins, while the second one tries to attribute the floral and/or the geographical origin of honey on the basis of the presence and the abundance of one (or more) specific phenolic compounds, hence proposed as chemical marker(s) of origin. The outcomes of these studies are meaningful in both directions: honeys of different origins have shown a broad range of health-promoting properties like antibacterial, antioxidant and radical-scavenging activity [22,23,24,25,26,27]; on the other hand valuable results have been obtained in proposing a number of phenolic compounds as possible candidate markers of unifloral honeys [28,29,30,31,32,33,34,35,36,37,38]. In addition, first attempts of geographical attribution of honey according to differences in the phenolic profiles have been described in the literature [6,39].
The complexity of a food matrix like honey implies that the target analytes are usually present in low concentrations, and this demands the adoption of a multi-step analytical procedure able to provide a careful measurement of these quantities. In this context, the need to provide a proper validation protocol for the whole procedure of analysis in order to obtain reliable analytical data is nowadays felt much more than before [40].
Furthermore, the recent literature reports numerous attempts to provide a comprehensive view of the health-promoting properties of unifloral honeys and the attribution of their floral/geographical origin. In order to do this, different chemometric approaches have been used to obtain (or to process) analytical data of the phenolic profile in honey samples.
At the best of our knowledge, no recently published review provides a specific and updated state of the art on the analysis of the phenolic compounds in unifloral honey, related to the evaluation of its health-promoting properties and to the classification of its origin. Hence, the primary aim of this contribution is to fill this gap. Since the results of the less recent studies have been already reported in previous reviews [3,6,21,40,41,42], this article is primarily aimed at summarizing the pertinent studies carried out during the last decade. Within the chosen topics and this timespan, special attention has been devoted to studies where the quality of data produced is demonstrated by an adequate validation of the analytical method, and to those where a chemometric approach was used to manage analytical data and maximize the amount of information obtained.

2. Analytical Methods for the Determination of the Phenolic Profile of Unifloral Honey

2.1. General Remarks

The comprehensive characterization of the phenolic compounds of unifloral honeys generally begins with a proper sampling phase, aimed to obtain a large enough number of samples to provide representative results for a certain botanical and/or geographical origin. Obviously, the reliability of the information obtained from the phenolic pattern is strictly related to the authenticity and freshness of samples.
Usually floral source can be ascertained by means of melissopalynological analysis [43], while freshness can be proved quantifying the concentration of 5-hydroxymethyl-2-furaldehyde (HMF) [44,45], virtually absent in fresh samples, but whose concentration tends to increase after thermal treatments, improper storage or too long storage time.
For this reason, after sampling, honey should be stored in the dark and at low temperatures (typically 4 °C or less) until analysis, in order to preserve its chemical composition. Just before starting with the analysis, the analytical sample is allowed to reach the room temperature, and then it is homogenized. If sugar crystals are visible in the sample, they have to be dissolved by gentle heating, performed at temperatures never exceeding 40 °C.
According to the most of the published analytical procedures, and with only rare exceptions [38,46,47,48], phenolic compounds in honey need to be purified by means of both an extraction and clean-up phases followed by the separation and the identification steps, usually performed by chromatographic [28,29,30,31,32,34,35,36,37,38,39,40,41,42] or electrophoretic [33,41] approaches. The choice of the instrumental technique and the selection of the operative parameters strongly depend on the analytical goals and the type of characterization (qualitative and/or quantitative).

2.2. Extraction and Clean-Up

This represents a key step in the definition of the qualitative and quantitative profile of phenolic compounds in unifloral honeys. The aim of this phase is to guarantee an increased concentration of the target analytes and the simultaneous removal of any potential interfering compounds, such as sugars and other polar substances. The extraction and clean-up should represent the best compromise to maximize the recoveries for analytes, even when they belong to different chemical classes (flavonoids, phenolic acids, etc.).
In the last ten years, Amberlite XAD-2 resin has been one of the most popular adsorbent media for the extraction of phenolic compounds from honey. As described by Das and co-workers [49], the sample is dissolved in an aqueous solution of HCl (pH = 2), filtered and then passed through a column containing Amberlite XAD-2. Elution, accomplished first with aqueous HCl solution (pH = 2) and after with water, allows one to separate the phenolic fraction (retained on the column) from the polar interfering substances like sugars. Then, the analytes are eluted with methanol; the extracts are first evaporated to dryness at reduced pressure and then dissolved again in water. The clean-up phase can be performed by extraction with a proper organic solvent (diethyl ether [49] or ethyl acetate [50]). The organic extracts are finally evaporated and dissolved again in methanol for the HPLC analysis. The whole procedure is depicted in Figure 4.
Some authors have chosen to simplify this step by adopting SPE methods, where phenolic compounds are retained by means of hydrophobic interactions with a solid sorbent. In this way it is possible to combine the extraction and clean-up phases, maximizing the recoveries and achieving considerable savings of time and solvents. As observed for the extraction with Amberlite, the sample is generally dissolved in acidified water. Prior its use, the SPE cartridge is washed and activated with a proper solvent mixture, depending on the nature of the sorbent phase (e.g., C18 [51,52] and Strata-X-SPE [53]). It is advisable that the sorbents strongly interact with a wide range of phenolic compounds. After the complete removal of polar interfering substances, the elution of analytes is usually performed with methanol. In Figure 5, the extraction/clean-up protocol using SPE cartridges, as described by Truchado and co-workers [51], is reported.
Michalkiewicz’s research group compared the performances of four sorbents (Bond Elut octadecyl C18, Oasis HLB, Strata-X and Amberlite XAD-2) for the isolation and preconcentration of six phenolic acids (gallic, p-HBA, p-coumaric, vanillic, caffeic and syringic acid) and three flavonols (rutin, quercetin and kaempferol) from honey samples. Oasis HLB sorbent phase, washed with 50 mL of acidified water (pH = 2) and eluted with methanol provided the best results [54]. Recently, Liu and coworkers proposed a new sorbent material, a nano-Al2O3 coated mesoporous silica (Al2O3/SiO2), to be used for the SPE of flavonoids. Its extraction properties were evaluated by using myricetin, quercetin, luteolin and kaempferol as the test analytes, and the extraction efficiency was apparently better than those of commercial C18 sorbents [55].
More consolidated procedures, like liquid-liquid extraction (LLE), have also been reported in recent studies [56,57] for the analysis of phenolic compounds in honey. In both cases, repeated extractions with ethyl acetate were performed on a solution obtained dissolving honey in pure water [56] or in a 2% NaCl aqueous solution [57]. While Tuberoso’s research group [56] performed, on the ethyl acetate extracts, a TLC clean-up aimed to isolate a specific phenolic compound (i.e., the methyl syringate, proposed marker for the asphodel unifloral honey), the contribution of Kečkeš et al. [57] was focused on the definition of the phenolic profile of a number of Serbian unifloral honeys and, in this case, any additional clean-up phase was performed before the UHPLC-HESI-MS/MS analysis. More recently, Campone and co-workers reported an example of dispersive liquid–liquid microextraction (DLLME) of phenolic compounds from honey, obtaining recoveries normally higher than 70%. Subsequent analysis of these extracts were accomplished by means of a HPLC-UV method [58]. DLLME was also used in a similar way by Campillo et al. to determine flavonoid aglycones in honey using the HPLC-DAD-TOF-MS technique [59].
An appealing and recent improvement is represented by the use of multiwalled carbon nanotubes (MWCNTs) as sorbents for phenolic compounds [60,61]. MWCNTs are added to an acidified solution of honey, then the mixture is magnetically stirred in order to promote the adsorption of phenolic compounds onto the surface of the nanotubes. The sorbent material is first separated from the solution by vacuum filtration and then washed with water. Then, the treatment of the MWCNT with methanol causes the solubilisation of phenolic compounds. The methanolic solution is evaporated to dryness at 40 °C. The solid residue is dissolved with water and extracted three times with diethyl ether. The organic extract is evaporated to dryness, and the residue is finally dissolved in methanol for the HPLC analysis. The main advantages of this approach lie in the possibility to simultaneously extract a really wide number of phenolic species belonging to various classes (phenolic acids, flavonoids and the relevant derivatives) with high recoveries and reproducibility. Furthermore, the excellent regeneration properties of the MWCNTs let envisage their use for further extraction cycles of phenolic compounds in honey.
The literature also reports a number of contributions where no conventional extraction or clean-up procedure has been used. In these studies, the honey sample was analysed just after its solubilisation (in water [46,48], or in the HPLC mobile phase [47]) and the sonication for a few tens of minutes. This is the so-called “ultrasonic extraction” [62], that should not be confused with ultrasound assisted liquid–liquid extraction, extensively described in a review by Luque De Castro and Priego-Capote [63]). In this way Liang and coworkers [46] measured the amounts of four phenolic compounds (i.e., caffeic acid, p-coumaric acid, ferulic acid, and hesperetin) in Chinese citrus honey by HPLC-ECD. The low number of analytes measured, the selectivity and sensitivity of the detector and the absence of a complex pre-treatment of the matrix allowed the authors to achieve almost quantitative recoveries for the compounds considered. An ultrasonic extraction technique was also used by Zhang et al. [47] to assess a very interesting multivariate calibration technique (second-order calibration method based on a trilinear decomposition algorithm) in the development of a HPLC-DAD method aimed at the quantification of nine polyphenols in five unifloral honey samples. This calibration technique involves the mathematical decomposition of the overlapped chromatographic profile into the pure profiles of each chemical species even in the presence of unknown interferences or uncompleted chromatographic resolution of peaks, overcoming also the problem of the baseline drift. According to the authors, the procedure is characterized by rapidity (the whole chromatographic run is completed in less than 8 min), linearity and good recoveries (within the range 90%–110% for all analytes). In a similar way, in a paper concerning the HPLC-UV determination of thirteen phenolic compounds in Italian honeys [48], samples were simply dissolved with distilled water and placed in an ultrasound bath at 25 °C for 10 min. Despite of the absence of any control system for the column temperature (not reported in the experimental section, and confirmed by the inconsistency of the retention times displayed in the published chromatograms), and notwithstanding the unsatisfactory chromatographic resolution, the authors reported outstanding recovery performances (ranging—for all analytes—from 98.50% for p-coumaric acid to 100.80% for quercetin) and precision (RSD values never exceeding 3%).
In a recent study published by Biesaga and Pyrzynska [62], aimed at checking the stability of phenolic compounds in ultrasonic- or microwave-assisted extraction, the authors demonstrated that the ultrasound-assisted extraction normally provides better extraction yields than conventional extraction performed by shaking immiscible solvents. However, the ultrasonic extraction of aglycones of flavonols (like quercetin) shows, under these conditions, very low yields (recovery values always less than 2% for different honey samples), due to their instability.
Despite the growing interest inthe application of advanced liquid extraction techniques aimed to obtain phenolic extracts, only one example of accelerated solvent extraction (ASE) has been found in the literature during the last ten years [64]. The extraction is performed dissolving the sample in acidified water (pH = 2, HCl), at 25 °C by means of four different static cycles. Polyphenols are eluted with methanol, the solution is evaporated until dryness, and the residue is resuspended in distilled water and extracted three times with diethyl ether. Extracts are again dried and dissolved in a methanol/water solution before the HPLC analysis.

2.3. Analysis

Separation, identification and quantification are the next steps towards the definition of phenolic profile of unifloral honeys. The most used analytical technique is very often a HPLC approach, almost always in its reverse phase configuration (RP-HPLC). Selected features of recent HPLC methods used for the analysis of honey phenolic extracts have been summarized in Table 1.
The separation of the analytes is usually achieved by the use of C18 (ODS) columns, except for the Discovery HS PEG [59], a polyethylene glycol reversed phase column. In the last years, the application of ultra-high performance liquid chromatography (UHPLC) systems has proved advantageous in the analysis of the phenolic constituents of honey, improving the resolution, sensitivity and accuracy of the methods and reducing time of analysis [56,58,61,67]. The reason for the better analytical performances lies in the smaller size of the stationary phase particles (usually smaller than 2 µm) and the possibility to deliver the liquid phase at very high pressures. The mobile phase is generally composed of a gradient of two solvents: A) an aqueous acid solution of formic acid [47,50,51,53,57,58,59,60,61,67] or, alternatively, acetic acid [46,64], trifluoroacetic acid [65,66], phosphoric acid or phosphate buffers [48,49,52,56] and B) methanol or acetonitrile.
The most used HPLC detection system for measuring the phenolic profile of unifloral honeys is still based on the measurement of the UV absorption, sometimes performed using diode-array devices (DAD) [47,50,51,56,60,61,65,67,68]. The choice of appropriate absorption wavelengths is fundamental to maximize the method sensitivity, especially when the target compounds do not belong to a specific class. Unfortunately, HPLC-UV identification of polyphenols is possible only by the comparison of retention times and by the peak spiking method, and these ways are practicable only when the analyte under quantification is effectively available as chromatographic standard. When the molecular detection is performed by a diode-array spectrophotometer, also the whole UV-Vis spectrum can contribute to the identification of peaks. The principal drawback of all spectrophotometric detectors is the inability to provide a direct structural information, and this strongly limits the characterization of compounds which were not previously identified. For this reason, in the last years many studies have been focused on the assessment of methods that involve different couplings between HPLC and mass spectrometry.
Among others, the most common instrumental combinations used are UHPLC-MSn (1 ≤ n ≤ 3) [57,61], HPLC-MSn (1 ≤ n ≤ 3) [56,69,70,71,72], HPLC-DAD-MSn (1 ≤ n ≤ 3) [51,59,73] and UHPLC-DAD-MSn (1 ≤ n ≤ 3) [67,74]. The growing interest in the exploitation of mass spectrometry and tandem-mass spectrometry as detection systems in HPLC led to the identification of new possible markers for honeys from specific botanical origins, like the methyl syringate for asphodel honey [56]. Moreover, the study of the fragmentation patterns allows performing the structural investigations on particular classes of compounds whose discrimination is made difficult by the strong similarities in their structures. In a recent study performed by Truchado and coworkers [71], the HPLC-ESI-MSn characterization of O-glycosyl flavones of honeys produced by Tetragonula carbonaria bees consented to determine the nature of the inter-glycosidic linkage and to identify several flavonoid mono-, di- and triglycosides. Both ion trap and triple quadrupole mass spectrometers, along with the more recent hybrid instruments, have been used for the characterization of the phenolic fraction of honey. The ESI source, which is the most frequently installed in this kind of spectrometers, is generally set on the negative ion mode but also the positive mode can be helpful for the identification of some of the analytes [53,73].
Heated electrospray ionization probes (HESI) can be used to enhance desolvation during the ionization phase improving sensitivity. For example, this expedient was adopted by Kečkeš et al. [57] for the identification of polyphenols and other phytochemicals in Serbian unifloral honeys. In addition, Petrus and coworkers performed the determination of seven flavonoids in unifloral honeys by HPLC coupled with coulometric electrode array detection (CEAD), but confirmed the previously made attributions with HPLC-ESI-MS evidences [72].
Only some rare examples of analytical methods assessed by the use of techniques different from the traditional RP-HPLC are reported in the scientific literature. In this context, it is possible to consider the study by Jandric’s research group [83], performed by means of a multimethodological analytical approach (i.e., an analysis of the elements, a stable isotope analysis, metabolomics findings, and NIR, FT-IR, and Raman spectroscopic fingerprinting) and chemometric instruments, the contribution of Sergiel et al. [84], aimed to explore the suitability of a right-angle geometry three-dimensional synchronous fluorescence spectroscopy for the differentiation and classification of unifloral honeys, and the recent results obtained by Lenhardt and coworkers [85], that coupled fluorescence measurements with parallel factor analysis and partial least squares discriminant analysis for the characterization and classification of honey of different botanical origin.

2.4. Validation

Validation of an analytical method represents an essential component of the measures that any laboratory should implement to produce reliable analytical data. According to the 2nd Edition (2014) of The Fitness for Purpose of Analytical Methods Eurachem Guide [86], validation “…is basically the process of defining an analytical requirement, and confirming that the method under consideration has capabilities consistent with what the application requires.” According to ISO/IEC 17025 [87], a method should be validated whenever it is: (a) a non-standard method; (b) a laboratory-designed (or developed method); (c) a standard method used outside its intended scope; (d) an amplification and/or modification of standard method. Even if outside of the field of application of ISO/IEC 17025, some sectors (including food analysis) are anyway subject to validation requirements prescribed by international organizations like EC [88] or FAO [89].
These introductory definitions and considerations should provide sufficient support of the mandatory need, for any analytical chemist involved in food analysis, to provide—whenever necessary, and certainly when a new analytical method is proposed—at least the key parameters of a validation protocol, i.e., limits of detection (LOD) and quantification (LOQ), the working range, the precision and the trueness.
Although nowadays the attention for the presence of basic validation data in scientific publications is surely higher than in the last years, unfortunately more than 30% of the methods reported in Table 1 are completely unvalidated and this fact must raise some doubts on the reliability on any of the analytical data there reported.
As regards methods including some validation parameters, it is evident that precision and—above all—trueness play a key role for the correct interpretation of the qualitative and quantitative profile of phenolic compounds of unifloral honeys. In fact, when the analytical bias is statistically outside the interval of values typical for a known analyte concentration [90] a systematic error can affect the meaning of the conclusions obtained on the basis of such data. In the case of honey, the absence of suitable certificated reference materials (CRMs) and the frequent impossibility of comparing the results coming from the proposed procedure to the ones deriving from independent analytical methods, imply that the only feasible option for evaluating accuracy is represented by recovery tests. Unfortunately, even this approach may be problematic when increasing the number of analytes. On the other hand, precision is also an important data quality parameter. It is crucial to remember that precision varies with the level of analyte concentration [91] and depending on its measuring mode (e.g., within the same analytical session or in different analytical sessions), hence the evaluation of a single precision data is in general scarcely informative. With only few exceptions [58,59], the validation data reported in studies here considered are too often inconsistent or unreliable. In addition, no fitness-for-purpose assessment for precision and accuracy data has been evaluated, neither on the basis of the guidelines that have long been available in the literature [90,91].

3. Phenolic Profile as a Powerful Tool for Origin Classification and Evaluation of Health-Promoting Properties of Unifloral Honeys

3.1. Phenolic Compounds for the Classification of Botanical (or Geographical) Origin of Unifloral Honeys

Beyond being strictly associated to a number of health-promoting properties, the qualitative and quantitative profile of the phenolic compounds of unifloral honeys represents a powerful instrument for the verification of their origin. In the last years, the phenolic compounds of unifloral honeys have been characterized in order to identify components that—alone or, more easily, in combination with other compounds (phenolic or not)—could be descriptive of a specific floral origin. One of the most remarkable examples of specificity of a single phenolic acid as chemical marker of floral origin is still homogentisic acid for the strawberry tree honey [34,38,69]. Now more than 15 years since its discovery, this marker has never been found in any other unifloral honey, whereas many other candidate chemical markers have been later found also in honeys of other origins, thus losing any aspect of strict specificity towards a single unifloral honey. It is clear that almost all the scientific efforts made in this direction have been successful when devoted towards the quantification of one (or more) molecule rather than the presence/absence of a specific chemical marker. This is the case of methyl syringate, proposed as a marker of asphodel honey [56], but afterwards found in a number of different unifloral honeys [53,76,77,78,79], even if not in the same concentration levels. The research group of Tuberoso proposed additional chemical markers for the strawberry tree honey [69], Jerković and coworkers found useful chemical markers for unifloral honey by Coffea spp. [80], whereas, in a recent study on sage (Sage officinalis L.) honey from Croatia [67], four chemical species (i.e., the flavonol kaempferol, present in quite high concentrations, boron and potassium among minerals, and turanose among sugars) were proposed as authentication markers for honeys of this botanical origin. Moreover, some compounds can be considerably useful to discriminate honeys which show similar properties and palinological features. This is the case of two typical honeys from New Zealand, like Manuka (Leptospermum scoparium) and Kanuka (Kunzea erikoides) honeys, which are indistinguishable by means of a melissopalynological analysis. In spite of the fact that Manuka and Kanuka honeys share most of the phenolic profiles, Stephens et al. [53] observed that 2-methoxybenzoic acid and trimethoxybenzoic acid are characteristic of Manuka honey while 4-methoxyphenylacetic acid is distinctive of the phenolic pattern of Kanuka honey.
Besides the characterization of honeys of a particular floral origin, the definition of phenolic profile has also been applied to investigate on honeys produced by subspecies of the common honeybee. For instance, some studies [73,92] have been devoted to the analytical characterization of honeys produced by the Sicilian black honeybee (Apis mellifera ssp. Sicula), with the identification and quantification of a number of phenolic acids and flavonoids. Furthermore, honeys produced by two different species of stingless honeybees have been investigated. More specifically, four phenolic acids, three flavonoids and the isomers of abscisic acid were identified and quantified in Jandaíra (Melipona subnitida) honey from Brazil [50], and the qualitative characterization of the O-glycosyl flavones of Tetragonula carbonaria honeys from Australia was performed in the already cited study by Truchado et al. [51].
Furthermore, attention has been focused on the use of the polyphenolic pattern to characterize honeys from particular geographical origins. In the study by Habib and coworkers [64] a significant difference in the content of phenolic compounds of honeys produced in non-arid and arid regions was found. The dissimilarity in the phenolic profile was explained on the basis of the different climate and sunlight exposure indicating the latter as the responsible for the higher content of polyphenols in honeys produced in arid regions. The phenolic profile of Serbian unifloral honeys was investigated by Kečkeš and coworkers [57], who suggested that quercetin and eriodictyol could be proposed as floral markers for local sunflower honeys. As a further example, phenolic profile of Sulla (Hedysarum spp.) honeys produced in Southern Italy resulted influenced by their geographical origin [93], whereas the concentrations of gallic, chlorogenic, caffeic, p-coumaric and ferulic acids showed the highest variation as a function of the production site of this honey.
As previously said, the definition of the qualitative and quantitative profile of phenolic compounds in unifloral honeys is surely suitable to give key information on their botanical and/or their geographical origin, but data obtained are reliable only if they are originated by an adequate number of samples. Since large data sets can be very difficult to be properly managed and correctly interpreted, a chemometric approach may be decisive to distinguish data from noise and to maximize analytical information [94]. Chemometrics is a powerful and interdisciplinary science finalized to extract and to maximize information from chemical systems by both descriptive and predictive viewpoints. Recently Gašić and coworkers [67] explored a number of classes of analytes (i.e., polyphenolic profiles, the total phenolic contents, the compositions of minerals, sugars and sugar alcohols, and the radical scavenging activities), creating a dataset that was interpreted using PCA and targeted to the authentication of unifloral Salvia officinalis L. honey. Again, Petretto and coworkers [95] performed this methodological approach in classifying, by means of PCA, the botanical origin of fifty one Sardinian samples belonging to ten different kind of unifloral honeys according to the phenolic content, antioxidant power and physico-chemical properties. Also Kečkeš and coworkers [57] used PCA in order to rationalize the phenolic profile of forty four unifloral honey from Serbia, whereas—in a very recent contribution—Zhao et al. [81] used PCA and discriminant analysis (DA) to correctly classify more than 85% of the honey samples, according to their botanical origin. Kuś and van Ruth attempted the discrimination of Polish unifloral honeys using proton transfer reaction mass spectrometry (PTR-MS) and HPLC-DAD fingerprints combined with PCA and k-nearest neighbor classification [82]. Whereas models based on HPLC fingerprints may be useful as universal methods of classification, the model based exclusively on PTR-MS findings is only exploitable for quick targeted on-line screenings and for specific unifloral honeys. Chemometric models have also been used for the rationalization of data by analytical techniques other than HPLC. The already cited study performed by Jandric and coworkers [83] allowed to determine which technique (or combination of techniques) is able to provide the best classification and prediction abilities towards a group of authentic unifloral honeys from New Zealand. This result was accomplished using chemometric tools such as orthogonal partial least square discriminant analysis. In addition, the contribution of Lenhardt et al. [85] proposed a new method for unifloral honey characterization and classification based on fluorescence data treated with parallel factor analysis and partial least squares discriminant analysis.
Moreover, chemometric techniques allowed providing reliable information concerning the geographical origin of unifloral honeys also on the basis of their phenolic composition. This is the case of the study of Karabagias et al. [96], who differentiated, according to the geographical origin, thirty five samples of Greek thyme honey from four different sites. Differentiation was accomplished on the basis of the phenolic content and the conventional physicochemical parameters by means of multivariate analysis of the variance (MANOVA) and Linear Discriminant Analysis. Furthermore Pasquini et al. [97] used principal component analysis, discrimination methods, like linear and quadratic discriminant analysis, and classification and regression trees in order to accomplish geographic differentiation of fifty honey samples among its mineral contents, the total phenolic concentrations and the radical scavenging capacity. Classification and regression trees were found to be the model with the best predictive ability, specificity and sensitivity (66.67%, 80% and 67%, respectively).
Finally, the chemometric treatment (cluster analysis and PCA) of data from HPLC-ECD determination of the phenolic profile allowed Wang and coworkers to identify an acacia honey adulteration with rape honey [98]. In particular, chlorogenic acid and ellagic acid were hypothesized as possible markers of acacia and rape honeys, respectively.

3.2. Phenolic Compounds in the Health-Promoting Properties of Unifloral Honeys

The many health-promoting effects of honey have been well known for millennia. Beyond being the only form of sweetener available at that time, honey has been used by ancient cultures as medicine, but also as ointment. The traditional experience of our fathers is now supported by a solid scientific background that has been summarized in a number of authoritative and recent reviews [2,3,21,25,99,100]. Below we reported the principal health properties of honey directly attributed to the phenolic profile.
First, it has been ascertained that honey inhibits the growth of micro-organisms and fungi, and the botanical origin of honey is one of the most important factors influencing its antimicrobial activity. These properties have been attributed both to enzymes hydrogen peroxide-producers, like glucose oxidase and catalase, and non-peroxide substances, like the phenolic compounds.
On the other hand, also the antioxidant action of the honey is well-known from ancient times. It has been associated to a number of different substances present in fresh honey, first of all enzymes and phenolic compounds, but also carotenoids derivatives, amino acids, proteins and—usually—low amounts of vitamins, all active against the so called “oxidative stress”. With this term the lack of equilibrium between the antioxidant protective activity in a given organism and the production of free radicals has been defined. There are several ways to measure in vitro the antioxidant activity of honey, and to compare it with the total amount of phenolic compounds [100]. Among the most effective methods there are the Ferric Reducing Antioxidant Power (FRAP) spectrophotometric assay, and the 2,2-DiPhenyl-1-PicrylHydrazyl (DPPH) radical scavenging method. In a recent contribution Moniruzzaman et al. exhaustively reviewed the most important analytical methods devoted to determine the antioxidant properties of honey [101].
There is an abundant literature concerning the evaluation of antimicrobial effects and antioxidant capabilities of unifloral honeys worldwide. These contributions describe the use of reference (or published) analytical methods to accomplish this task and they are often accompanied by the measure of some spectrophotometric parameters like the total polyphenolic and flavonoidic amounts and/or the colour. Less frequently, in these studies additional characterizations like a chromatographic phenolic profile, a melissopalynological analysis and a mineral composition of major and trace elements are present. In the last ten years, many unifloral honeys from New Zealand [23], Burkina Faso [26], Morocco [102], Italy [48,66,92,93,95,103,104,105,106,107], India [49,64], Brazil [50], United Arabian Emirates, Oman, Yemen, Pakistan, Australia [64], Japan [65], Croatia [67,79], Poland [76,78], Turkey [108,109], Portugal [110], Romania [111], Slovenia [112], Cuba [113,114,115,116], Greece [117] and Serbia [117] have been evaluated for their antimicrobial, antioxidants and radical scavenging properties evidencing their dependence on the presence of specific phenolic compounds like homogentisic acid, as well as on the botanical and geographical origin [93] and the climatic conditions [64]. Table 2 reports a selection of antioxidant and antiradical properties of the unifloral honeys described in last ten years.
In particular, Rosa et al. demonstrated that, among a group of seven honeys (i.e., acacia, asphodel, Citrus spp, eucalyptus, heather, honeydew and strawberry tree) from Italy, the strawberry tree one showed the highest concentration in total phenols and the major activity in the DPPH and FRAP tests. The amount of homogentisic acid (i.e., the chemical marker for strawberry tree honey) was more than 60% of the total phenols of such honey and this phenolic acid showed interesting antioxidant and antiradical activities as well as protective effect against thermal cholesterol degradation, comparable to those of a number of well-known antioxidants [105]. However researches performed by Tuberoso and coworkers [104], aimed to evaluate the antioxidant capacity and vasodilatory properties of three Mediterranean foods rich in phenolic compounds like the Cannonau red wine, myrtle berries liqueur and strawberry-tree honey, demonstrated that such honey—unlike to what shown by red wine and the liqueur—did not induce any vasodilation. This confirms the fact that the abundance of phenolic compounds in foods does not represent an assurance about their functional properties that have to be tested by suitable methods. Also unifloral honeys from Cuba have attracted a great interest in this last decade. For the first time the phytochemical composition of five important monofloral Cuban honeys and their possible relationships with their biological activities were thoroughly studied by Alvarez-Suarez et al. In particular, their antioxidant [113,114] and antimicrobial [114] properties were examined and discussed also in terms of correlation with colour, total amount of phenolic compounds and concentrations of flavonoid species, amino acids, proteins and carotenoids. The samples analysed possessed good antioxidant and antibacterial properties and meaningful concentrations of phenolic species, flavonoids and carotenoids. Later, the same research group successfully studied the protective effect of such unifloral honeys against lipid peroxidation in an in vitro model of rat liver homogenates [115] and the ability of the phenolic extracts of two unifloral honeys (i.e., the Christmas vine, Turbina corymbosa L., and the Linen vine, Gouania polygama) to inhibit the oxidative damage induced by the 2,2’-azobis(2-methylpropionamidine) dihydrochloride in erythrocytes [116].
Recently, attention has been paid to alternative analytical approaches for assessing the antioxidant capacity of honey and relating it to the presence of polyphenolic species. In particular, Avila and coworkers proposed a novel, simple and fast electrochemical method devoted to estimate the antioxidant capacity in honey samples using flow injection analysis. The method is based on a selective electrooxidation of polyphenolic compounds using two different anodic potentials at two different pH values. The significance of the proposed protocol vs. the traditional spectrophotometric method was enhanced by a chemometric evaluation of data obtained. The procedure is inherently versatile, because it allows the evaluation of the antioxidant activity under predesigned oxidation conditions. Finally, an electrochemical antioxidant index is proposed for the evaluation of the antioxidant capabilities of honey [117]. This work demonstrates that electrochemistry is an emerging and valuable tool in the direct evaluation of antioxidant capacity of natural complex extracts from foods. Indeed, one year later, Buratti proposed a similar method to evaluate the antioxidant power of honeybee products (i.e., honey, propolis and royal jelly) by an amperometric flow injection method [106]. Also this method is easy and rapid (one measure/minute). Finally, in 2013, Gorjanovića et al. published a contribution concerning the assessment of an electroanalytical method aimed to check the applicability of DC polarography in determination of hydrogen peroxide scavenging activity of honey [118]. The reported results, compared with selected antioxidant assays like DPPH, FRAP, TEAC and ORAC, demonstrated that the hydrogen peroxide scavenging activity is discriminant towards the floral origin of honeys.
Still few contributions in assessing methods and interpreting results on antioxidant activity of unifloral honeys comes from the application of chemometric tools. In 2005, for the first time, Beretta and coworkers successfully attempted to standardize the antioxidant properties of honey by spectrophotometric and by fluorimetric data treated with multivariate techniques (correlation matrix calculation and PCA) [107]. In particular, Authors investigated the antioxidant power and the radical scavenging capacity of fourteen commercial honeys of different floral and geographic origin, using many of the known spectrophotometric tests like Folin-Ciocalteu assay for phenol content, FRAP assay, DPPH assay for antiradical activity, ORAC for the antilipoperoxidant activity, whereas color intensity was evaluated measuring absorbance at 450 nm. Multivariate techniques allowed to find significant correlations for all the antioxidant markers: antioxidant properties are strictly correlated to the phenolic content and honey colour intensity. Hence, it is evident that only with a chemometric approach for data obtained from the combination of different antioxidant assays it is possible to achieve reliable guidelines for the characterization of the antioxidant activity of honey. Finally, the study of Escuredo et al. [119] has applied for the first time the near infrared spectroscopy to the selection of antioxidant variables in honey. Calibration models for phenols, flavonoids, vitamin C, antioxidant capacity (DPPH), oxidation index and copper in unifloral honeys were obtained using the modified partial least squares regression method. Such models were optimised by cross-validation, and the best model was evaluated according to multiple correlation coefficient, standard error of cross-validation, ratio performance deviation and root mean standard error in the prediction set. Hence, near infrared spectroscopy can be considered as a rapid and reliable tool for the non-destructive evaluation of chemical and health-promoting parameters in honey.

4. Conclusions

Honey is an important source of phenolic compounds, and the amount and the nature of phenolic acids and flavonoids is of great interest because they are responsible for a number of functional and nutraceutical properties typical of this natural food. Moreover, several and recent studies have also confirmed that the phenolic profile is strictly related to the botanical and—sometimes—the geographical origin of unifloral honeys, so it can be used as invaluable tool for classification and authentication. Taking this into account, many groups have intensified their efforts in order to develop new and reliable analytical protocols able to improve the qualitative and quantitative characterization of phenolic compounds in honey, but also to evaluate the bioactive properties of new unifloral honeys worldwide and to correlate them to a number of health-promoting features of this foodstuff. While recent studies have frequently been accompanied by at least a minimal validation, the application of chemometric instruments for the optimization of procedures of obtaining and managing the analytical data still appears insufficient, although the trend is in sharp increasing in the last years.

Author Contributions

Marco Ciulu and Gavino Sanna wrote the manuscript; Nadia Spano and Maria I. Pilo revised the manuscript; all authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
HMF5-Hydroxymethyl-2-furaldehyde
HPLCHigh Pressure Liquid Chromatography
SPESolid Phase Extraction
p-HBApara-HydroxyBenzoic Acid
LLELiquid-Liquid Extraction
TLCThin Layer Chromatography
UHPLCUltra High Pressure Liquid Chromatography
HESIHeated ElectroSpray Interface
MSMass Spectrometry
DLLMEDispersive Liquid-Liquid MicroExtraction
UVUltraViolet spectrophotometry detector
DADDiode Array Detector
TOFTime Of Flight detector
MWCNTMultiWalled Carbon NanoTubes
ECDElectroChemical Detector
RSDRelative Standard Deviation
ASEAccelerated Solvent Extraction
RPReverse Phase
MeOHMethanol
MeCNAcetonitrile
TFATriFluoroAcetic acid
NIRNear InfraRed spectroscopy
FT-IRFourier Transform InfraRed spectroscopy
ISOInternational Organization for Standardization
IECInternational Electrotechnical Commission
ECEuropean Community
FAOFood and Agriculture Organization
LODLimit Of Detection
LOQlimit Of Quantification
CRMCertificated Reference Material
PCAPrincipal Components Analysis
DADiscriminant Analysis
PTRProton Transfer Reaction
MANOVAMultivariate Analysis Of the Variance
FRAPFerric (ion) Reducing Antioxidant Power
TPTZ2,4,6-tripyridyl-s-triazine
DPPH2,2-diphenyl-1-picrylhydrazyl radical
TEACTrolox equivalent antioxidant capacity
ORACOxygen Radical Absorbance Capacity
QEACQuercetin Equivalent Antioxidant Content
AEACAscorbic acid Equivalent Antioxidant Content
TBARSThioBArbituric Reactive Substances
IC5050% Inhibitory Concentration

References

  1. Krell, R. Value-Added Products from Beekeeping; FAO Agricultural Service Bulletin No 124; Food and Agriculture Organization of the United Nations: Rome, Italy, 1996; Available online: http://www.fao.org/docrep/w0076E/w0076E00.htm (accessed on 28 January 2016).
  2. Viuda-Martos, M.; Ruiz-Navajas, Y.; Fernández-López, J.; Pérez-Alvarez, J.A. Functional properties of honey, propolis, and royal jelly. J. Food Sci. 2008, 73, 117–124. [Google Scholar] [CrossRef] [PubMed]
  3. Gómez-Caravaca, A.M.; Gómez-Romero, M.; Arráez-Roman, D.; Segura-Carretero, A.; Fernández-Gutiérrez, A. Advances in the analysis of phenolic compounds in products derived from bees. J. Pharm. Biomed. Anal. 2006, 41, 1220–1234. [Google Scholar] [CrossRef] [PubMed]
  4. Doner, L.W. The sugars of honey—A review. J. Sci. Food Agric. 1977, 28, 443–456. [Google Scholar] [CrossRef] [PubMed]
  5. Siddiqui, I.R. The sugars of honey. Adv. Carbohydr. Chem. 1970, 25, 285–309. [Google Scholar]
  6. Anklam, E. A review of the analytical methods to determine the geographical and botanical origin of honey. Food Chem. 1998, 63, 549–562. [Google Scholar] [CrossRef]
  7. White, J.W., Jr. Honey. In Advances in Food Research; Academic Press: New York, NY, USA, 1978; Volume 24. [Google Scholar]
  8. Mato, I.; Huidobro, J.F.; Simal-Lozano, J.; Sancho, M.T. Rapid Determination of Nonaromatic Organic Acids in Honey by Capillary Zone Electrophoresis with Direct Ultraviolet Detection. J. Agric. Food Chem. 2006, 54, 1541–1550. [Google Scholar] [CrossRef] [PubMed]
  9. Suarez-Luque, S.; Mato, I.; Huidobro, J.F.; Simal-Lozano, J.; Sancho, M.T. Rapid determination of minority organic acids in honey by High-Performance Liquid Chromatography. J. Chromatogr. A. 2002, 955, 207–214. [Google Scholar] [CrossRef]
  10. Spano, N.; Ciulu, M.; Floris, I.; Panzanelli, A.; Pilo, M.I.; Piu, P.C.; Salis, S.; Sanna, G. A direct RP-HPLC method for the determination of furanic aldehydes and acids in honey. Talanta 2009, 78, 310–314. [Google Scholar] [CrossRef] [PubMed]
  11. Viňas, P.; Balsalobre, N.; Lòpez-Erroz, C.; Hernández-Còrdoba, M. Liquid chromatographic analysis of riboflavin vitamers in foods using fluorescence detection. J. Agric. Food Chem. 2004, 52, 1789–1794. [Google Scholar] [CrossRef] [PubMed]
  12. Viňas, P.; Balsalobre, N.; Lopez-Erroz, C.; Hernandez-Cordoba, M. Determination of vitamin B6 compounds in foods using liquid chromatography with post-column derivatization fluorescence detection. Chromatographia 2004, 59, 381–386. [Google Scholar]
  13. Leon-Ruiz, V.; Vera, S.; Gonzalez-Porto, A.V.; San Andres, M.P. Vitamin C and sugar levels as simple markers for discriminating Spanish honey sources. J. Food Sci. 2011, C356–C361. [Google Scholar] [CrossRef] [PubMed]
  14. Ciulu, M.; Solinas, S.; Floris, I.; Panzanelli, A.; Pilo, M.I.; Piu, P.C.; Spano, N.; Sanna, G. RP-HPLC determination of water-soluble vitamins in honey. Talanta 2011, 83, 924–929. [Google Scholar] [CrossRef] [PubMed]
  15. Ciulu, M.; Spano, N.; Salis, S.; Pilo, M.; Floris, I.; Pireddu, L.; Sanna, G. Assay of B vitamins and other water soluble vitamins in honey. In Food and Nutritional Components in Focus” No. 4, “B Vitamins and Folate: Chemistry, Analysis, Function and Effects; Preedy, V.R., Ed.; The Royal Society of Chemistry: Cambridge, UK, 2013; Chapter 13; pp. 173–194. [Google Scholar]
  16. Conte, L.S.; Miorini, M.; Giomo, A.; Bertacco, G.; Zironi, R. Evaluation of Some Fixed Components for Unifloral Honey Characterization. J. Agric. Food Chem. 1998, 46, 1844–1849. [Google Scholar] [CrossRef]
  17. Hermosin, I.; Chicón, R.M.; Cabezudo, M.D. Free amino acid composition and botanical origin of honey. Food Chem. 2003, 83, 263–268. [Google Scholar] [CrossRef]
  18. Cotte, J.F.; Casabianca, H.; Giroud, B.; Albert, M.; Lheritier, J.; Grenier-Loustalot, M.F. Characterization of honey amino acid profiles using high-pressure liquid chromatography to control authenticity. Anal. Bioanal. Chem. 2004, 378, 1342–1350. [Google Scholar] [CrossRef] [PubMed]
  19. Nozal, M.J.; Bernal, J.L.; Toribio, M.L.; Diego, J.C.; Ruiz, A. Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection. J. Chromatogr. A 2004, 1047, 137–146. [Google Scholar] [CrossRef] [PubMed]
  20. Spano, N.; Piras, I.; Ciulu, M.; Floris, I.; Panzanelli, A.; Pilo, M.I.; Piu, P.C.; Sanna, G. Reversed-Phase liquid chromatographic profile of free amino acids in strawberry-tree (Arbutus unedo L.) honey. J. AOAC Int. 2009, 92, S1145–S1156. [Google Scholar]
  21. Bogdanov, S.; Jurendic, T.; Sieber, S.; Gallmann, P. Honey for Nutrition and Health: A Review. J. Am. Coll. Nutr. 2008, 27, 677–689. [Google Scholar] [CrossRef] [PubMed]
  22. Bogdanov, S. Nature and origin of the antibacterial substances in honey. Lebensm-Wiss. Technol. 1997, 30, 748–753. [Google Scholar] [CrossRef]
  23. Weston, R.J.; Mitchell, K.R.; Allen, K.L. Antibacterial phenolic components of New Zealand manuka honey. Food Chem. 1999, 64, 295–301. [Google Scholar] [CrossRef]
  24. Gheldof, N.; Wang, X.H.; Engeseth, N.J. Identification and quantification of antioxidant components of honeys from various floral sources. J. Agric. Food Chem. 2002, 50, 5870–5877. [Google Scholar] [CrossRef] [PubMed]
  25. Havsteen, B.H. The biochemistry and medical significance of the flavonoids. Pharm. Therap. 2002, 96, 67–202. [Google Scholar] [CrossRef]
  26. Meda, A.; Lamien, C.E.; Romito, M.; Millogo, J.; Nacoulma, O.G. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 2005, 91, 571–577. [Google Scholar] [CrossRef]
  27. Jerković, I.; Marijanović, Z.; Kezić, J.; Gigić, M. Headspace, volatile and semi-volatile organic compounds diversity and radical scavenging activity of ultrasonic solvent extracts from Amorpha fruticosa honey samples. Molecules 2009, 14, 2717–2728. [Google Scholar] [CrossRef] [PubMed]
  28. Ferreres, F.; García-Viguera, C.; Tomás-Lorente, F.; Tomás-Barberán, F.A. Hesperetin: A marker of the floral origin of citrus honey. J. Sci. Food Agric. 1993, 61, 121–123. [Google Scholar] [CrossRef]
  29. Ferreres, F.; Tomás-Barberán, F.A.; Soler, C.; García-Viguera, C.; Ortiz, A.; Tomás-Lorente, F. A simple extractive technique for honey flavonoid HPLC analysis. Apidologie 1994, 25, 21–30. [Google Scholar] [CrossRef]
  30. Ferreres, F.; Giner, J.M.; Tomás-Barberán, F.A. A comparative study of hesperetin and methyl anthranilate as markers of the floral origin of citrus honey. J. Sci. Food Agric. 1994, 65, 371–372. [Google Scholar] [CrossRef]
  31. Ferreres, F.; Andrade, P.; Gil, M.I.; Tomás-Barberán, F.A. Floral nectar phenolics as biochemical markers for the botanical origin of heather honey. Z. Lebensm. Unters. For. 1996, 202, 40–44. [Google Scholar] [CrossRef]
  32. Andrade, P.; Ferreres, F.; Amaral, M.T. Analysis of honey phenolic acids by HPLC, its application to honey botanical characterization. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 2281–2288. [Google Scholar] [CrossRef]
  33. Andrade, P.; Ferreres, F.; Gil, M.I.; Tomás-Barberán, F.A. Determination of phenolic compounds in honeys with different floral origin by capillary zone electrophoresis. Food Chem. 1997, 60, 79–84. [Google Scholar] [CrossRef]
  34. Cabras, P.; Angioni, A.; Tuberoso, C.; Floris, I.; Reniero, F.; Guillou, C.; Ghelli, S. Homogentisic acid: A phenolic acid as a marker of strawberry-tree (Arbutus unedo) honey. J. Agric. Food Chem. 1999, 47, 4064–4067. [Google Scholar] [CrossRef] [PubMed]
  35. Martos, I.; Ferreres, F.; Tomás-Barberán, F.A. Identification of flavonoid markers for the botanical origin of Eucalyptus honey. J. Agric. Food Chem. 2000, 48, 1498–1502. [Google Scholar] [CrossRef] [PubMed]
  36. Tomás-Barberán, F.A.; Martos, I.; Ferreres, F.; Radovic, B.S.; Anklam, E. HPLC flavonoid profiles as markers for the botanical origin of European unifloral honeys. J. Sci. Food Agric. 2001, 81, 485–496. [Google Scholar] [CrossRef]
  37. Yao, L.; Datta, N.; Tomás-Barberán, F.A.; Ferreres, F.; Martos, I.; Singanusong, R. Flavonoids, phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys. Food Chem. 2003, 81, 159–168. [Google Scholar] [CrossRef]
  38. Scanu, R.; Spano, N.; Panzanelli, A.; Pilo, M.I.; Piu, P.C.; Sanna, G.; Tapparo, A. Direct chromatographic methods for the rapid determination of homogentisic acid in strawberry tree (Arbutus unedo L.) honey. J. Chromatogr. A 2005, 1090, 76–80. [Google Scholar] [CrossRef] [PubMed]
  39. Yao, L.; Jiang, Y.; D’Arcy, B.; Singanusong, R.; Datta, N.; Caffin, N.; Raymont, K. Quantitative high-performance liquid chromatography analyses of flavonoids in Australian Eucalyptus honeys. J. Agric. Food Chem. 2004, 52, 210–214. [Google Scholar] [CrossRef] [PubMed]
  40. Pyrzynska, K.; Biesaga, M. Analysis of phenolic acids and flavonoids in honey. Trends Anal. Chem. 2009, 28, 893–902. [Google Scholar] [CrossRef]
  41. Merken, H.M.; Beecher, G.R. Measurement of food flavonoids by high-performance liquid chromatography: A review. J. Agric. Food Chem. 2000, 48, 577–599. [Google Scholar] [CrossRef] [PubMed]
  42. Kaškonienė, V.; Venskutonis, P.R. Floral Markers in Honey of Various Botanical and Geographic Origins: A Review. Compr. Rev. Food Sci. Food Saf. 2010, 9, 620–634. [Google Scholar] [CrossRef]
  43. Louveaux, J.; Maurizio, A.; Vorwohl, G. Methods of melissopalynology. Bee World 1970, 51, 125–138. [Google Scholar] [CrossRef]
  44. Jeuring, H.J.; Kuppers, F.J.E.M. High performance liquid chromatography of furfural and hydroxymethylfurfural in spirits and honey. J. Assoc. Off. Agric. Chem. 1980, 63, 1215–1218. [Google Scholar]
  45. Spano, N.; Casula, L.; Panzanelli, A.; Pilo, M.I.; Piu, P.C.; Scanu, R.; Tapparo, A.; Sanna, G. An RP-HPLC determination of 5-hydroxymethylfurfural in honey: The case of strawberry tree honey. Talanta 2006, 68, 1390–1395. [Google Scholar] [CrossRef] [PubMed]
  46. Liang, Y.; Cao, W.; Chen, W.-Y.; Xiao, X.-H.; Zheng, J.-B. Simultaneous determination of four phenolic components in citrus honey by high performance liquid chromatography using electrochemical detection. Food Chem. 2009, 114, 1537–1541. [Google Scholar] [CrossRef]
  47. Zhang, X.-H.; Wu, H.-L.; Wang, J.-Y.; Tu, D.-Z.; Kang, C.; Zhao, J.; Chen, Y.; Miu, X.-X.; Yu, R.-Q. Fast HPLC-DAD quantification of nine polyphenols in honey by using second-order calibration method based on trilinear decomposition algorithm. Food Chem. 2013, 138, 62–69. [Google Scholar] [CrossRef] [PubMed]
  48. Perna, A.; Intaglietta, I.; Simonetti, A.; Gambacorta, E. A comparative study on phenolic profile, vitamin C content and antioxidant activity of Italian honeys of different botanical origin. Int. J. Food Sci. Technol. 2013, 48, 1899–1908. [Google Scholar] [CrossRef]
  49. Das, A.; Datta, S.; Mukherjee, S.; Bose, S.; Ghosh, S.; Dhar., P. Evaluation of antioxidative, antibacterial and probiotic growth stimulatory activities of Sesamum indicum honey containing phenolic compounds and lignans. Food Sci. Technol. 2015, 61, 244–250. [Google Scholar] [CrossRef]
  50. Sarmento Silva, T.M.; Pereira dos Santos, F.; Evangelista-Rodrigues, A.; Sarmento da Silva, E.M.; Sarmento da Silva, G.; Santos de Novais, J.; Ribeiro dos Santos, F.A.; Amorim Camara, C. Phenolic compounds, melissopalynological, physicochemical analysis and antioxidant activity of jandaíra (Melipona subnitida) honey. J. Food Compos. Anal. 2013, 29, 10–18. [Google Scholar] [CrossRef]
  51. Truchado, P.; Vit, P.; Heard, T.A.; Tomàs-Barberàn, F.A.; Ferreres, F. Determination of interglycosidic linkages in O-glycosyl flavones by high-performance liquid chromatography/photodiode-array detection coupled to electrospray ionization ion trap mass spectrometry. Its application to Tetragonula carbonaria honey from Australia. Rapid Commun. Mass Spectrom. 2015, 29, 948–954. [Google Scholar] [PubMed]
  52. Dimitrova, B.; Gevrenova, R.; Anklam, E. Analysis of Phenolic Acids in Honeys of Different Floral Origin by Solid-phase Extraction and High-performance Liquid Chromatography. Phytochem. Anal. 2007, 18, 24–32. [Google Scholar] [CrossRef] [PubMed]
  53. Stephens, J.M.; Schlothauer, R.C.; Morris, B.D.; Yang, D.; Fearnley, L.; Greenwood, D.R.; Loomes, K.M. Phenolic compounds and methylglyoxal in some New Zealand manuka and kanuka honeys. Food Chem. 2010, 120, 78–86. [Google Scholar] [CrossRef]
  54. Michalkiewicz, A.; Biesaga, M.; Pyrzynska, K. Solid-phase extraction procedure for determination of phenolic acids and some flavonols in honey. J. Chrom. A 2008, 1187, 18–24. [Google Scholar] [CrossRef] [PubMed]
  55. Liu, H.; Guo, Y.; Wang, X.; Liang, X.; Liu, X. Preparation of an Al2O3/SiO2 core–shell composite material for solid phase extraction of flavonoids. Anal. Methods 2015, 7, 3486–3492. [Google Scholar] [CrossRef]
  56. Tuberoso, C.I.G.; Bifulco, E.; Jerkovic, I.; Caboni, P.; Cabras, P.; Floris, I. Methyl Syringate: A Chemical Marker of Asphodel (Asphodelus microcarpus Salzm. et Viv.) Monofloral Honey. J. Agric. Food Chem. 2009, 57, 3895–3900. [Google Scholar] [CrossRef] [PubMed]
  57. Kečkeš, S.; Gašić, U.; Ćirković Veličković, T.; Milojković-Opsenica, D.; Natić, M.; Tešic, Z. The determination of phenolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry. Food Chem. 2013, 138, 32–40. [Google Scholar] [CrossRef] [PubMed]
  58. Campone, L.; Piccinelli, A.L.; Pagano, I.; Carabetta, S.; Di Sanzo, R.; Russo, M.; Rastrelli, L. Determination of phenolic compounds in honey using dispersive liquid-liquid microextraction. J. Chrom. A 2014, 1334, 9–15. [Google Scholar] [CrossRef] [PubMed]
  59. Campillo, N.; Viñas, P.; Férez-Melgarejo, G.; Hernández-Córdoba, M. Dispersive liquid-liquid microextraction for the determination of flavonoid aglycone compounds in honey using liquid chromatography with diode array detection and time-of-flight mass spectrometry. Talanta 2015, 131, 185–191. [Google Scholar] [CrossRef] [PubMed]
  60. Badjah Hadj Ahmed, A.Y.; Obbed, M.S.; Wabaidur, S.M.; Al Othman, Z.A.; Al-Shaalan, N.H. High-Performance Liquid Chromatography Analysis of Phenolic Acid, Flavonoid, and Phenol Contents in Various Natural Yemeni Honeys Using Multi-walled Carbon Nanotubes as a Solid-Phase Extraction Adsorbent. J. Agric. Food Chem. 2014, 62, 5443–5450. [Google Scholar] [CrossRef] [PubMed]
  61. Wabaidur, S.M.; Ahmed, Y.B.H.; Alothman, Z.A.; Obbed, M.S.; Al-Harbi, N.M.; Al-Turki, T.M. Ultra high performance liquid-chromatography with mass spectrometry method for the simultaneous determination of phenolic constituents in honey from various floral sources using multiwalled carbon nanotubes as extraction sorbents. J. Separ. Sci. 2015, 38, 2597–2606. [Google Scholar] [CrossRef] [PubMed]
  62. Biesaga, M.; Pyrzyńska, K. Stability of bioactive polyphenols from honey during different extraction methods. Food Chem. 2013, 136, 46–54. [Google Scholar] [CrossRef] [PubMed]
  63. Luque de Castro, M.D.; Priego-Capote, F. Ultrasound assistance to liquid-liquid extraction: A debatable analytical tool. Anal. Chim. Acta 2007, 583, 2–9. [Google Scholar] [CrossRef] [PubMed]
  64. Habib, H.M.; Al Meqbali, F.T.; Kamal, H.; Souka, U.D.; Ibrahim, W.H. Bioactive components, antioxidant and DNA damage inhibitory activities of honeys from arid regions. Food Chem. 2014, 153, 28–34. [Google Scholar] [CrossRef] [PubMed]
  65. Kumazawa, S.; Okuyama, Y.; Murase, M.; Ahn, M-R.; Nakamura, J.; Tatefuji, T. Antioxidant Activity in Honeys of Various Floral Origins: Isolation and Identification of Antioxidants in Peppermint Honey. Food Sci. Technol. Res. 2012, 18, 679–685. [Google Scholar] [CrossRef]
  66. Pichichero, E.; Canuti, L.; Canini, A. Characterisation of the phenolic and flavonoid fractions and antioxidant power of Italian honeys of different botanical origin. J. Sci. Food Agric. 2009, 89, 609–616. [Google Scholar] [CrossRef]
  67. Gašić, U.; Natić, M.; Mišić, D.; Lušić, D.; Milojković-Opsenica, D.; Tešic, Z.; Lušić, D. Chemical markers for the authentication of unifloral Salvia officinalis L. honey. J. Food Compos. Anal. 2015, 44, 128–138. [Google Scholar] [CrossRef]
  68. Tuberoso, C.I.G.; Bifulco, E.; Caboni, P.; Sarais, G.; Cottiglia, F.; Floris, I. Lumichrome and Phenyllactic Acid as Chemical Markers of Thistle (Galactites tomentosa Moench) Honey. J. Agric. Food Chem. 2011, 59, 364–369. [Google Scholar] [CrossRef] [PubMed]
  69. Tuberoso, C.I.G.; Bifulco, E.; Caboni, P.; Cottiglia, F.; Cabras, P.; Floris, I. Floral Markers of Strawberry Tree (Arbutus unedo L.) Honey. J. Agric. Food Chem. 2010, 58, 384–389. [Google Scholar] [CrossRef] [PubMed]
  70. Biesaga, M.; Pyrzynska, K. Liquid chromatography/tandem mass spectrometry studies of the phenolic compounds in honey. J. Chrom. A 2009, 1216, 6620–6626. [Google Scholar] [CrossRef] [PubMed]
  71. Truchado, P.; Ferreres, F.; Tomas-Barberan, F.A. Liquid chromatography-tandem mass spectrometry reveals the widespread occurrence of flavonoid glycosides in honey, and their potential as floral origin markers. J. Chrom. A 2009, 1216, 7241–7248. [Google Scholar] [CrossRef] [PubMed]
  72. Petrus, K.; Schwartz, H.; Sontag, G. Analysis of flavonoids in honey by HPLC coupled with coulometric electrode array detection and electrospray ionization mass spectrometry. Anal. Bioanal. Chem. 2011, 400, 2555–2563. [Google Scholar] [CrossRef] [PubMed]
  73. Mannina, L.; Sobolev, A.P.; Di Lorenzo, A.; Vista, S.; Tenore, G.C.; Daglia, M. Chemical Composition of Different Botanical Origin Honeys Produced by Sicilian Black Honeybees (Apis mellifera ssp. sicula). J. Agric. Food Chem. 2015, 63, 5864–5874. [Google Scholar] [CrossRef] [PubMed]
  74. Oelschlaegel, S.; Gruner, M.; Wang, P.-N.; Boettcher, A.; Koelling-Speer, I.; Speer, K. Classification and Characterization of Manuka Honeys Based on Phenolic Compounds and Methylglyoxal. J. Agric. Food Chem. 2012, 60, 7229–7237. [Google Scholar] [CrossRef] [PubMed]
  75. Reza Hadjmohammadi, M.; Saman, S.; Nazari, S.J. Separation optimization of quercetin, hesperetin and chrysin in honey by micellar liquid chromatography and experimental design. J. Sep. Sci. 2010, 33, 3144–3151. [Google Scholar] [CrossRef] [PubMed]
  76. Kuś, P.M.; Jerković, I.; Tuberoso, C.I.G.; Marijanović, Z.; Congiu, F. Cornflower (Centaurea cyanus L.) honey quality parameters: Chromatographic fingerprints, chemical biomarkers, antioxidant capacity and others. Food Chem. 2014, 142, 12–18. [Google Scholar] [PubMed]
  77. Jerković, I.; Kuś, P.M.; Tuberoso, C.I.G.; Šarolić, M. Phytochemical and physical-chemical analysis of Polish willow (Salix spp.) honey: Identification of the marker compounds. Food Chem. 2014, 145, 8–14. [Google Scholar] [CrossRef] [PubMed]
  78. Kuś, P.M.; Congiu, F.; Teper, D.; Sroka, Z.; Jerković, I.; Tuberoso, C.I.G. Antioxidant activity, color characteristics, total phenol content and general HPLC fingerprints of six Polish unifloral honey types. Food Sci. Technol. 2014, 55, 124–130. [Google Scholar] [CrossRef]
  79. Jerković, I.; Tuberoso, C.I.G.; Baranović, G.; Marijanović, Z.; Kranjac, M.; Svečnjak, L.; Kuś, P.M. Characterization of Summer Savory (Satureja hortensis L.) Honey by Physico-Chemical Parameters and Chromatographic/Spectroscopic Techniques (GC-FID/MS, HPLC-DAD, UV/VIS and FTIR-ATR). Croat. Chem. Acta 2015, 88, 15–22. [Google Scholar] [CrossRef] [Green Version]
  80. Jerković, I.; Tuberoso, C.I.G.; Kuś, P.M.; Marijanović, Z.; Kranjac, M. Screening of Coffea spp. honey by different methodologies: Theobromine and caffeine as chemical markers. RSC Adv. 2014, 4, 60557–60562. [Google Scholar] [CrossRef]
  81. Zhao, J.; Du, X.; Cheng, N.; Chen, L.; Xue, X.; Zhao, J.; Wu, L.; Cao, W. Identification of monofloral honeys using HPLC-ECD and chemometrics. Food Chem. 2016, 194, 167–174. [Google Scholar] [CrossRef] [PubMed]
  82. Kuś, P.M.; van Ruth, S. Discrimination of Polish unifloral honeys using overall PTR-MS and HPLC fingerprints combined with chemometrics. LWT—Food Sci. Technol. 2015, 62, 69–75. [Google Scholar] [CrossRef]
  83. Jandric, Z.; Haughey, S.A.; Frew, R.D.; McComb, K.; Galvin-King, P.; Elliott, C.T.; Cannavan, A. Discrimination of honey of different floral origins by a combination of various chemical parameters. Food Chem. 2015, 189, 52–59. [Google Scholar] [CrossRef] [PubMed]
  84. Sergiel, I.; Pohl, P.; Biesaga, M.; Mironczyk, A. Suitability of three-dimensional synchronous fluorescence spectroscopy for fingerprint analysis of honey samples with reference to their phenolic profiles. Food Chem. 2014, 145, 319–326. [Google Scholar] [CrossRef] [PubMed]
  85. Lenhardt, L.; Bro, R.; Zeković, I.; Dramićanin, T.; Dramićanin, M.D. Fluorescence spectroscopy coupled with PARAFAC and PLS DA for characterization and classification of honey. Food Chem. 2015, 175, 284–291. [Google Scholar] [CrossRef] [PubMed]
  86. Magnusson, B.; Örnemark, U. (Eds.) Eurachem Guide: The Fitness for Purpose of Analytical Methods—A Laboratory Guide to Method Validation and Related Topics, 2nd ed.; 2014; Available online: http://www.eurachem.org (accessed on 28 January 2016).
  87. ISO/IEC 17025:2005. General Requirements for the Competence of Testing and Calibration Laboratories; ISO: Geneva, Switzerland, 2005. [Google Scholar]
  88. Council Directive 93/99/EEC on the Subject of Additional Measures Concerning the Official Control of Foodstuffs, O.J.; 1993, L290, 14–17, Brussels, Belgium. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:31993L0099&from=EN (accessed on 28 January 2016).
  89. Procedural Manual of the Codex Alimentarius Commission, 10th ed.; FAO: Rome, Italy, 1997.
  90. AOAC International. AOAC Peer Verified Methods Program, Manual on Policies and Procedures; AOAC International: Arlington, VA, USA, 1998. [Google Scholar]
  91. Horwitz, W. Evaluation of analytical methods used for regulation of foods and drugs. Anal. Chem. 1982, 54, 67A–76A. [Google Scholar] [CrossRef]
  92. Tenore, G.C.; Ritieni, A.; Campiglia, A.; Novellino, E. Nutraceutical potential of monofloral honeys produced by the Sicilian black honeybees (Apis mellifera ssp. sicula). Food Chem. Toxicol. 2012, 50, 1955–1961. [Google Scholar] [CrossRef] [PubMed]
  93. Gambacorta, E.; Simonetti, A.; Garrisi, N.; Intaglietta, I.; Perna, A. Antioxidant properties and phenolic content of sulla (Hedysarum spp.) honeys from Southern Italy. Int. J. Food Sci. Technol. 2014, 49, 2260–2268. [Google Scholar] [CrossRef]
  94. Ruoff, K.; Karoui, R.; Dufour, E.; Luginbuhl, W.; Bosset, J.O.; Bogdanov, S.; Amado, R. Authentication of the botanical origin of honey by front-face fluorescence spectroscopy. A preliminary study. J. Agric. Food Chem. 2005, 53, 1343–1347. [Google Scholar] [CrossRef] [PubMed]
  95. Petretto, G.L.; Cossu, M.; Alamanni, M.C. Phenolic content, antioxidant and physico-chemical properties of Sardinian monofloral honeys. Int. J. Food Sci Technol. 2015, 50, 482–491. [Google Scholar] [CrossRef]
  96. Karabagias, I.K.; Vavoura, M.V.; Badeka, A.; Kontakos, S.; Kontominas, M.G. Differentiation of Greek Thyme Honeys According to Geographical Origin Based on the Combination of Phenolic Compounds and Conventional Quality Parameters Using Chemometrics. Food Anal. Methods 2014, 7, 2113–2121. [Google Scholar] [CrossRef]
  97. Pasquini, B.; Goodarzi, M.; Orlandini, S.; Beretta, G.; Furlanetto, S.; Dejaegher, B. Geographical characterisation of honeys according to their mineral content and antioxidant activity using a chemometric approach. Int. J. Food Sci. Technol. 2014, 49, 1351–1359. [Google Scholar] [CrossRef]
  98. Wang, J.; Xue, X.; Du, X.; Cheng, N.; Chen, L.; Zhao, J.; Zhen, J.; Cao, W. Identification of Acacia Honey Adulteration with Rape Honey Using Liquid Chromatography-Electrochemical Detection and Chemometrics. Food Anal. Methods 2014, 7, 2003–2012. [Google Scholar] [CrossRef]
  99. Alvarez-Suarez, J.M.; Tulipani, S.; Romandini, S.; Vidal, A.; Battino, M. Contribution Methodological Aspects about Determination of Phenolic Compounds and In Vitro Evaluation of Antioxidant Capacity in the Honey: A Review. Curr. Anal. Chem. 2009, 5, 293–302. [Google Scholar] [CrossRef]
  100. Alvarez-Suarez, J.M.; Tulipani, S.; Romandini, S.; Bertoli, E.; Battino, M. Contribution of honey in nutrition and human health: A review. Mediterr. J. Nutr. Metab. 2010, 3, 15–23. [Google Scholar] [CrossRef]
  101. Moniruzzaman, M.; Khalil, M.I.; Sulaiman, S.A.; Gan, S.H. Advances in the analytical methods for determining the antioxidant properties of honey: A review. Afr. J. Tradit. Complement. Altern. Med. 2012, 9, 36–42. [Google Scholar] [CrossRef] [PubMed]
  102. Petretto, G.L.; Tuberoso, C.I.G.; Vlachopoulou, G.; Atzei, A.; Mannu, A.; Zrirad, S.; Pintore, G. Volatiles, Color Characteristics and Other Physico-chemical Parameters of Commercial Moroccan Honeys. Nat. Prod. Res. 2016, 30, 286–292. [Google Scholar] [CrossRef] [PubMed]
  103. Pasini, F.; Gardini, S.; Marcazzan, G.L.; Caboni, M.F. Buckwheat honeys: Screening of composition and properties. Food Chem. 2013, 141, 2802–2811. [Google Scholar] [CrossRef] [PubMed]
  104. Tuberoso, C.I.G.; Boban, M.; Bifulco, E.; Budimir, D.; Pirisi, F.M. Antioxidant capacity and vasodilatory properties of Mediterranean food: The case of Cannonau wine, myrtle berries liqueur and strawberry-tree honey. Food Chem. 2013, 140, 686–691. [Google Scholar] [CrossRef] [PubMed]
  105. Rosa, A.; Tuberoso, C.I.G.; Atzeri, A.; Melis, M.P.; Bifulco, E.; Dessì, M.A. Antioxidant profile of strawberry tree honey and its marker homogentisic acid in several models of oxidative stress. Food Chem. 2011, 129, 1045–1053. [Google Scholar] [CrossRef] [PubMed]
  106. Buratti, S.; Benedetti, S.; Cosio, M.S. Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis. Talanta 2007, 71, 1387–1392. [Google Scholar] [CrossRef] [PubMed]
  107. Beretta, G.; Granata, P.; Ferrero, M.; Orioli, M.; Facino, R.M. Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics. Anal. Chim. Acta 2005, 533, 185–191. [Google Scholar] [CrossRef]
  108. Silici, S.; Sagdic, O.; Ekici, L. Total phenolic content, antiradical, antioxidant and antimicrobial activities of Rhododendron honeys. Food Chem. 2010, 121, 238–243. [Google Scholar] [CrossRef]
  109. Kucuk, M.; Kolayli, S.; Karaoglu, S. Biological activities and chemical composition of three honeys of different types from Anatolia. Food Chem. 2007, 100, 526–534. [Google Scholar] [CrossRef]
  110. Ferreira, I.C.F.R.; Aires, E.; Barreira, J.C.M.; Estevinho, L.M. Antioxidant activity of Portuguese honey samples: Different contributions of the entire honey and phenolic extract. Food Chem. 2009, 114, 1438–1443. [Google Scholar] [CrossRef]
  111. Marghitas, L.A.; Dezmirean, D.; Moise, A.; Bobisa, O.; Lasloa, L.; Bogdanov, S. Physico-chemical and bioactive properties of different floral origin honeys from Romania. Food Chem. 2009, 112, 863–867. [Google Scholar]
  112. Bertoncelj, J.; Dobersek, U.; Jamnik, M.; Golob, T. Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chem. 2007, 105, 822–828. [Google Scholar] [CrossRef]
  113. Alvarez-Suarez, J.M.; González-Paramás, A.M.; Santos-Buelga, C.; Battino, M. Antioxidant Characterization of Native Monofloral Cuban Honeys. J. Agric. Food Chem. 2010, 58, 9817–9824. [Google Scholar] [CrossRef] [PubMed]
  114. Alvarez-Suarez, J.M.; Tulipani, S.; Díaz, D.; Estevez, Y.; Romandini, S.; Giampieri, F.; Damiani, E.; Astolfi, P.; Bonpadre, S.; Battino, M. Antioxidant and antimicrobial capacity of several monofloral Cuban honeys and their correlation with color, polyphenol content and other chemical compounds. Food Chem. Toxicol. 2010, 48, 2490–2499. [Google Scholar] [CrossRef] [PubMed]
  115. Alvarez-Suarez, J.M.; Giampieri, F.; Damiani, E.; Astolfi, P.; Fattorini, D.; Regoli, F.; Quiles, J.L.; Battino, M. Radical-scavenging Activity, Protective Effect against Lipid Peroxidation and Mineral Contents of Monofloral Cuban Honeys. Plant Foods Hum. Nutr. 2012, 67, 31–38. [Google Scholar] [CrossRef] [PubMed]
  116. Alvarez-Suarez, J.M.; Giampieri, F.; González-Paramás, A.M.; Damiani, E.; Astolfi, P.; Martinez-Sanchez, G.; Bonpadre, S.; Quiles, J.L.; Santos-Buelga, C.; Battino, M. Phenolics from monofloral honeys protect human erythrocyte membranes against oxidative damage. Food Chem. Toxicol. 2012, 50, 1508–1516. [Google Scholar] [CrossRef] [PubMed]
  117. Ávila, M.; Crevillén, A.G.; González, M.C.; Escarpa, A.; Vela Hortigüela, L.; de Lorenzo Carretero, C.; Pérez Martín, R.A. Electroanalytical Approach to Evaluate Antioxidant Capacity in Honeys: Proposal of an Antioxidant Index. Electroanalysis 2006, 18, 1821–1826. [Google Scholar] [CrossRef]
  118. Gorjanovića, S.Z.; Alvarez-Suarez, J.M.; Novaković, M.M.; Pastor, F.M.; Pezo, L.; Battino, M.; Sužnjevića, D.Ž. Comparative analysis of antioxidant activity of honey of different floral sources using recently developed polarographic and various spectrophotometric assays. J. Food Compos. Anal. 2013, 30, 13–18. [Google Scholar] [CrossRef]
  119. Escuredo, O.; Seijo, M.C.; Salvador, J.; González-Martín, M.I. Near infrared spectroscopy for prediction of antioxidant compounds in the honey. Food Chem. 2013, 141, 3409–3414. [Google Scholar] [CrossRef] [PubMed]
Molecules 21 00451 g001 550
Figure 1. Phenolic acids and their derivatives in honey.
Figure 1. Phenolic acids and their derivatives in honey.
Molecules 21 00451 g001
Molecules 21 00451 g002 550
Figure 2. General structures of flavonoid subfamilies.
Figure 2. General structures of flavonoid subfamilies.
Molecules 21 00451 g002
Molecules 21 00451 g003a 550Molecules 21 00451 g003b 550
Figure 3. Most significant flavonoids in honey.
Figure 3. Most significant flavonoids in honey.
Molecules 21 00451 g003aMolecules 21 00451 g003b
Molecules 21 00451 g004 550
Figure 4. Extraction/clean-up of polyphenols from unifloral honey on Amberlite XAD-2, according [49].
Figure 4. Extraction/clean-up of polyphenols from unifloral honey on Amberlite XAD-2, according [49].
Molecules 21 00451 g004
Molecules 21 00451 g005 550
Figure 5. Extraction/clean-up of phenolic compounds from honey on C18 cartridge, according [51].
Figure 5. Extraction/clean-up of phenolic compounds from honey on C18 cartridge, according [51].
Molecules 21 00451 g005
Table
Table 1. Features of recent chromatographic methods for the analysis of phenolic compounds in unifloral honey.
Table 1. Features of recent chromatographic methods for the analysis of phenolic compounds in unifloral honey.
TechniqueStationary Phase (Length, mm × id, mm × Particle Size, µm)Mobile Phase aValidationChemometricsFloral/Geographical Origin of HoneyAnalytes bRef.
HPLC-ECDZorbax SB-C18 (150 × 4.6 × 5)A: 4% CH3COOH (aq)
B: MeOH		y Citrus honey from ChinaCaffeic acid, p-coumaric acid, ferulic acid, hesperetin[46]
HPLC-DADWonda-Sil C18 (150 × 4.6 × 5)MeOH 43% (v/v) and HCOOH (aq), pH 2.54 (57%, v/v)yMultivariate calibrationMilk vetch, wild chrysanthemum, jujube flower and acacia honeys from ChinaGallic acid, chlorogenic acid, protocatechuic acid, caffeic acid, p-hydroxybenzoic acid syringic acid, p-coumaric acid, ferulic acid, rutin[47]
HPLC-UVHypersil gold C18 (250 × 4.6 × 5)A: KH2PO4 (aq), pH 2.92
B: MeOH		y Chestnut, eucalyptus, citrus and sulla honeys from ItalyGallic acid; gallocatechin; epicatechin; catechin; chlorogenic acid; caffeic acid;, benzoic acid; p-coumaric acid; ferulic acid; rutin; myricetin; quercetin[48]
HPLC-UVNova-Pak C18, (150 × 3.9)A: H3PO4 (aq) pH 2.5;
B: MeCN		nSesamum indicum honey from Hooghly district, West BengalRutin, quercetin, apigenin and myricetin, ferulic acid[49]
HPLC-DADShimpack CLC-ODS, (250 × 4.6 × 5)A: 5% HCOOH (aq)
B: MeOH		n Jandaira honey from state of Paraiba, BrazilNaringenin, quercetin, isorhamnetin, gallic acid, vanillic acid, 3,4-dihydroxybenzoic acid, cumaric acids, trans–trans abscisic acid, cis–trans abscisic acid[50]
HPLC-DAD-MSnLiChroCART RP-18 (250 × 4 × 5)A: 1% HCOOH (aq)
B: MeOH		n Tetragonula carbonaria honey from AustraliaO-glycosyl flavones[51]
HPLC-UVSpherisorb ODS-2A: phosphate buffer (pH 2.92)
B: MeOH		y Chestnut, acacia, lime, eucalyptus, lavender, rapeseed, sunflower, rosemary, orange, lemon, fior di sulla, Echium plantagineum, heather, bell heather and ling heather honey from Germany, Denmark, Italy, Spain, France, Netherlands, UK and PortugalBenzoic acid, salicylic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, protocatechuic acid, gallic acid, syringic acid; vanillic acid; trans-cinnamic acid, o-; m- and p-coumaric acids, caffeic acid, ferulic acid, phenylacetic acid, l-β-phenyllactic acid, dl-p-hydroxy-phenyllactic acid[52]
HPLC-MS/MSPhenomenex Luna C-18 (150 × 2 × 3)A: 0.2% HCOOH (aq)
B: MeOH		n Manuka and kanuka honeys from New ZealandGallic acid; syringic acid; 2-methoxybenzoic acid; phenyllactic acid; methyl syringate; abscisic acid; 4-methoxybenzoic acid; 4-methoxyphenyllactic acid (tentative identification); trimethoxybenzoic acid (tentative identification); structural isomer of syringic acid (tentative identification); 4-methoxyphenyllactic acid (tentative identification)[53]
(1) HPLC-DAD
(2) HPLC-MS/MS		Lichrocart Purosher Star RP-18e (250 × 4 × 5)A: 0.2 M H3PO4 (aq)
B: MeCN 2 Water:MeCN 60:40 (v/v)		n Asphodel honey from Sardinia, ItalyMethyl syringate[56]
UHPLC-HESI-MSnHypersil gold C18 (50 × 2.1 × 1.9)A: 0.1% HCOOH (aq)
B: 0.1% HCOOH (MeCN)		yAnalytical data are interpreted in terms of principal component analysisAcacia, sunflower, linden, basil, buckwheat, oilseed rape and goldenrod honeys from SerbiaGallic acid; protocatechuic acid; 3-o-caffeoylquinic acid; caffeic acid; quercetin-3-o-rutinoside; p-coumaric acid; luteolin; quercetin; 2-cis,4-trans-abscisic acid; apigenin; kaempferol; chrysin; pinocembrin; galangin [57]
UHPLC–UVChromolith FastGradient RP-18e (2 × 50 × 2)A: 0.1% HCOOH (aq)
B: 0.1% HCOOH (MeOH)		y Acacia, sulla, thistle and citrus honeys from Calabria, Italy(±)-cis,trans-abscisic acid, apigenin, caffeic acid, chrysin, ferulic acid, hesperetin, pinobanksin, p-coumaric acid, quercetin, syringic acid, vanillic acid, galangin, kaempferol, luteolin, myricetin, pinocembrin[58]
HPLC-DAD-TOF-MSDiscovery HS PEG (150 × 4.6 × 5)A: 0.1% HCOOH (aq)
B: MeCN		y Lavender, orange blossom, rosemary, heather, eucalyptus, chestnut and thyme honeys. No declaration of geographical origin of honey was provided.Baicalein, hesperetin, fisetin, naringenin, chrysin, myricetin, quercetin, kaempferol[59]
HPLC-DADBetasil C18 (150 × 4.6 × 3)A: 1% HCOOH (aq)
B: MeOH		y Ziziphus Spina-Christi honey from YemenGallic acid; clorogenic acid; 4-hydroxybenzoic acid; 4-hydroxyphenyl acetic acid; caffeic acid; vanillic acid; syringic acid; p-coumaric acid; phenol; ferulic acid; sinapic acid; naringin; myricetin; quercetin; naringenin; cinnamic acid; kaempferol; apigenin; chrysin; galangin; thymol; carvacrol[60]
UHPLC-MSAcquity UHPLC BEH C18 (150 × 2.1 × 1.7)A: 0.1% HCOOH (aq)
B: Methanol		y Ziziphus Spina-Christi honey from YemenGallic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid, caffeic acid, chlorogenic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, phenol, myricetin, naringin, cinnamic acid, quercetin, naringenin, kaempferol, luteolin, apigenin, galangin, chrysin[61]
HPLC-UVWaters Xterra RP 18 (150 × 4.6 × 5)A: 1% CH3COOH (aq)
B: MeCN		n Prosopis juliflora, Ziziphus Spina-Christi, Acacia tortilis and Leptospermum scoparium honeys from UAE, Oman, Yemen and New ZealandGallic acid 4-hydroxy-3-methoxybenzoic acid; syringic acid p-coumaric acid; ferulic acid cinnamic acid; catechin, epicatechin, rutin[64]
HPLC-DADShiseido Capcell Pak C18 UG120, (250 × 4.6 × 5)A: TFA 0.1% (aq)
B: TFA 0.1% (MeOH)		n Peppermint honey from USA.p-coumaric acid and kaempferol[65]
HPLC-UVWhatman ODS-2 column (250 × 4.6 × 5)A: 87/3/10 (v/v/v) water/TFA/MeCN
B: 40/50/10 (v/v/v) water/TFA/MeCN		n Acacia, chestnut, savory, sulla, ailanthus, thymus and orange honeys from ItalyGallic acid; chlorogenic acid; p-coumaric acid; caffeic acid; myricetin; quercetin; genistein; kaempferol; apigenin; chrysin; galangin[66]
UHPLC-DAD MS/MSSyncronis C18 column (100 × 1 x 1.7)A: 0.1% HCOOH (aq)
B: MeCN		nAnalytical data are interpreted in terms of principal component analysis.Sage honey from CroatiaGallic acid; gallocatechin; protocatechuic acid; epigallocatechin; gentisic acid; p-hydroxybenzoic acid; chlorogenic acid; catechin; caffeic acid; gallocatechin gallate; epicatechin; p-coumaric acid; ferulic acid; rosmarinic acid; epigallocatechin gallate; cis,trans-abscisic acid; resveratrol; kaempferol; pinobanksin; quercetin; chrysin; pinostrobin; pinocembrin; hesperetin; galangin[67]
HPLC-DADPhenomenex Gemini C18 110° (150 × 4.60 × 3)A: 0.2 M H3PO4 (aq)
B: MeCN		n Thistle honey from Sardinia, Italy [68]Phenyllactic acid [68][68]
HPLC-DAD HPLC-MS/MSPhenomenex SynergiHydro-RP 80AC18 (150 × 4.60 × 4) Licrocart Purosher Star RP-18eA: 0.2 M H3PO4 (aq)
B: MeCN Water/MeCN 60:40 (v/v)		n Strawberry tree honey from Sardinia, Italy2-cis,4-trans-abscisic acid; 2-trans,4-trans-abscisic acid[69]
HPLC-ESI-MS/MSAtlantis C-18 (50 × 2.1 × 3)A: 2 mM HCOOH (aq)
B: MeOH		y Buckwheat honey. No declaration of geographical origin of honey was provided.Gallic acid, p-hydroxyphenylacetic, acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, myricetin, naringin, naringenin, quercetin, apigenin[70]
HPLC-DAD-ESI-MS/MSC18 LiChroCART (250 × 4 × 5)A: 1% CH3COOH (aq)
B: MeOH		n Canola, cherry blossom, eucalyptus, linden, lucerne, lavender, orange blossom, rapeseed, rhododendron, rosemary, sunflower, taraxacum and tilia honeys from Italy, Spain and SlovakiaFlavonoid glycosides[71]
HPLC-CEADHPLC-ESI-MSNucleodur Sphinx RP (150 × 4.6 × 5)c
d		y Acacia, buckwheat, maple, phacelia, pumpkin, raspberry, orange, cherry blossom, dandelion, melon, rhododendron, rosemary, citrus blossom, orange blossom, lavender, sage, thyme, pine tree and rape seed honeys from Italy, Austria, Croatia, Greece and Germany.Quercetin, naringenin, hesperetin, luteolin, kaempferol, isorhamnetin, galangin[72]
HPLC-DAD-MSnGemini C18 110 Å (150 × 2 × 5)A: 0.1% HCOOH (aq)
B: MeOH		n Sulla, dill, lemon, orange, and medlar honeys from Sicily, Italy4-methoxyphenyllactic acid; citric acid; phenylalanine; phenyllactic acid; dehydrovomifoliol; 3-hydroxy-1-(2-methoxyphenyl)penta-1,4-dione; p-hydroxybenzoic acid; riboflavin; kynurenic acid; methyl syringate; quercetin hexosyl rutinoside; quercetin rhamnosyl-hexosyl-rhamnoside; lumichrome; quercetin rutinoside; abscisic acid; pinobanksin methyl ether; kaempferol rutinoside; pinobanksin; pinocembrin; caffeic acid isoprenyl ester; pinobanksin acetate; pinobanksin butyrate.[73]
UPLC-DAD-MS/MSNucleodur C18 Pyramid (100 × 2.1 × 1.8)A: 0.1% HCOOH (aq)
B: 0.05% HCOOH (MeCN)		n Manuka honeys from New ZealandGallic acid, caffeic acid, phenyllactic acid, 4-methoxyphenyllactic acid, kojic acid, 4-hydroxybenzoic acid, syringic acid, 2-methoxybenzoic acid, phenylacetic acid, benzoic acid, methyl syringate, 2-trans,4-trans-abscisic acid, 2-cis,4-trans-abscisic acid, luteolin[74]
MLC-UVSpherisorb C18 column (250 × 4.6 × 10)7.8%v/v EtOH and 5.0%v/v CH3COOH in water, [SDS] = 0.124 mol/LyExperimental design (CCD) was used to optimize the chromatographic separationCitrus honey from IranQuercetin, hesperetin, chrysin[75]
HPLC-DADPhenomenex Gemini C18 110° (150 × 4.60 × 3)A: 0.2 M H3PO4 (aq)
B: MeCN		n Cornflower honey from Poland [76] Willow honey from Poland [77] Black locust, rapeseed, lime, goldenrod, heather and buckwheat honeys from Poland [78] Summer Savory honey from Poland [79]Methyl syringate: phenyllactic acid [76] Gallic acid, benzoic acid, p-coumaric acid, 4-hydroxybenzoic acid, kynurenic acid, methylbenzaldehyde, methyl syringate, vanillic acid, (±)-2-trans,4-trans-abscisic acid [77] p-hydroxybenzoic acid, methyl syringate, cis,trans-abscisic acid, gallic acid[78] Methyl syringate [79][76,77,78,79]
HPLC-DADPhenomenex Kinetex C18 (150 × 4.60 × 5)A: 0.2 M H3PO4 (aq)
B: MeCN		y Two samples of Coffea spp. honey of different geographical originKojic acid[80]
HPLC-DADRP-LiChrosorb Hibar C18 (250 × 2.4 × 5)A: 5% HCOOH (aq)
B: MeOH		nAnalytical data are interpreted in terms of principal component analysis.Jujube, longan and chaste honeys from ChinaCatechin, chlorogenic acid, syringic acid, p-hydroxycinnamic acid, ferulic acid, rutin, trans-cinnamic acid, quercetin, kaempferol, apigenin, galangin, pinocembrin, pinobanksin[81]
HPLC–ECD-DADZorbax SB-C18 (250 × 4.6 × 5)A: 1% CH3COOH (aq)
B: MeOH		yHoney classification has been accomplished by means of principal component analysis and discriminant analysisRapeseed, lime, heather, cornflower, buckwheat and black locust honeys from PolandGallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, ellagic acid, rosmarinic acid, cinnamic acid[82]
a MeOH = methanol, MeCN = acetonitrile, TFA = trifluoroacetic acid; b only quantified analytes are indicated; c Phase A: MeOH/0.02 M phosphate buffer (20:80, v:v) pH 3.2, Phase B: MeOH/0.02 M phosphate buffer (80:20, v:v) pH 3.2; d Phase A: 0.5% CH3COOH in MeOH/water (20:80, v:v); Phase B: 0.5% CH3COOH in MeOH/water (80:20, v:v); y = yes; n = no; methods in italic provided only qualitative findings of analytes.
Table
Table 2. Antioxidant and antiradical properties of unifloral honeys.
Table 2. Antioxidant and antiradical properties of unifloral honeys.
Floral and Geographical Origin of Unifloral HoneyAntioxidant and Antiradical PropertiesRef.
Combretaceae Honeys from Burkina FasoDPPH, IC50 (mg/mL ± SD): between 10.40 ± 0.50 and 17.97 ± 1.44[26]
AEAC, (mg/100 g ± SD): between 16.34 ± 0.25 and 23.40 ± 0.74
QEAC, (mg/100 g ± SD): between 6.89 ± 2.02 and 11.31 ± 0.28
Acacia Honey from Burkina FasoDPPH, IC50 (mg/mL ± SD): 10.40 ± 0.50
AEAC, (mg/100 g ± SD): 23.40 ± 0.74
QEAC, (mg/100 g ± SD): 11.31 ± 0.28
Vitellaria Honeys from Burkina FasoDPPH, IC50 (mg/mL ± SD): between 1.37 ± 0.03 and 2.43 ± 0.08
AEAC, (mg/100 g ± SD): between 57.72 ± 0.00 and 65.86 ± 0.10
QEAC, (mg/100 g ± SD): between 31.01 ± 0.03 and 33.34 ± 0.21
Lannea Honey from Burkina FasoDPPH, IC50 (mg/mL ± SD): 23.53 ± 0.40
AEAC, (mg/100 g ± SD): 11.27 ± 0.02
QEAC, (mg/100 g ± SD): 5.35 ± 0.01
Amorpha fruticosa honey from unknown geographical origin aDPPH, IC50 (mg/mL): 0.6 (data measured on pentane–diethyl ether (1:2, v/v) ultrasonic extracts from a 40% (w/w) aqueous solution of honey)[27]
Chestnut honey from ItalyDPPH, I% (% ± SD): 75.37 ± 7.87 [48]
Eucalyptus honey from ItalyDPPH, I% (% ± SD): 73.04 ± 7.52
Citrus honey from ItalyDPPH, I% (% ± SD): 55.06 ± 7.04
Sulla honey from ItalyDPPH, I% (% ± SD): 66.60 ± 12.71
Sesamum indicum honey from Hooghly district, West BengalDPPH, IC50 (mg/mL): 39.5 ± 0.4[49]
FRAP, (μmol Fe(II)/L): 2.75 × 106
Jandaíra honey from BrazilDPPH, IC50 (mg/mL ± SD): between 10.6 ± 0.6 and 12.9 ± 0.3[50]
ABTS, IC50 (mg/mL ± SD): between 6.1 ± 0.0 and 9.7 ± 0.1
Prosopis juliflora honeys from UAE bDPPH, I%: ca. 6[64]
ABTS, I%: ca. 74
FRAP, (μmol Fe(II)/100 g honey): ca. 800
Ziziphus Spina-Christi honeys from UAE, Oman, Yemen and PakistanbDPPH, I%: between ca. 3 and ca.14
ABTS, I%: between ca. 75 and ca. 80
FRAP, (μmol Fe(II)/100 g honey): between ca. 600 and ca. 900
Acacia tortilis honeys from UAE, Oman and Yemen bDPPH, I%: between ca.4 and ca.19
ABTS, I%: between ca. 72 and ca. 80
FRAP, (μmol Fe(II)/100 g honey): between ca. 500 and ca. 700
Leptospermum scoparium honeys from New Zealand bDPPH, I%: ca. 11
ABTS, I%: ca. 79
FRAP, (μmol Fe(II)/100 g honey): ca. 600
23 unifloral honeys from worldwideDPPH, I%, (% ± SD): between 4.7 ± 2.3 (Horse chestnut honey, Akita, Japan) and 51.9 ± 2.0 (Peppermint honey, USA)[65]
Acacia honey from ItalyDPPH, IC50 (mg/mL ± SD): 21.56 ± 1.08 [66]
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.377 ± 0.068
Chestnut honey from ItalyDPPH, IC50 (mg/mL ± SD): 13.76 ± 0.82
FRAP, (mmol Fe(II)/Kg honey ± SD): 2.056 ± 0.103
Sulla honey from ItalyDPPH, IC50 (mg/mL ± SD): 54.74 ± 3.28
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.299 ± 0.080
Ailanthus honey from ItalyDPPH, IC50 (mg/mL ± SD): 64.09 ± 2.56
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.268 ± 0.063
Thymus honey from ItalyDPPH, IC50 (mg/mL ± SD): 31.4 ± 1.57
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.834 ± 0.092
Orange honey from ItalyDPPH, IC50 (mg/mL ± SD): 25.87 ± 1.29
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.265 ± 0.063
Savory honey from ItalyDPPH, IC50 (mg/mL ± SD): 10.85 ± 0.43
FRAP, (mmol Fe(II)/Kg honey ± SD): 3.702 ± 0.185
Cornflower honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.5 ± 0.2 [76]
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.5 ± 0.7
Willow honey from PolandDPPH, (mmol TEAC/kg ± SD): 2.1 ± 0.3 [77]
FRAP, (mmol Fe(II)/Kg honey ± SD): 0.5 ± 0.1
Black locust honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.3 ± 0.0 [78]
FRAP, (mmol Fe(II)/Kg honey ± SD): 0.6 ± 0.1
Rapeseed honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.4 ± 0.1
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.3 ± 0.3
Lime honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.4 ± 0.1
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.4 ± 0.4
Goldenrod honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.2 ± 0.1
FRAP, (mmol Fe(II)/Kg honey ± SD): 1.0 ± 0.1
Heather honey from PolandDPPH, (mmol TEAC/kg ± SD): 0.6 ± 0.1
FRAP, (mmol Fe(II)/Kg honey ± SD): 2.1 ± 0.5
Buckwheat honey from PolandDPPH, (mmol TEAC/kg ± SD): 1.2 ± 0.2
FRAP, (mmol Fe(II)/Kg honey ± SD): 5.7 ± 0.9
Summer Savory honey from PolandDPPH, (mmol TEAC/kg ± SD): 1.7 ± 0.2 [79]
FRAP, (mmol Fe(II)/Kg honey ± SD): 4.3 ± 0.6
Sulla honeys from Southern ItalyDPPH, (I% ± SD): between 47.06 ± 8.60 and 88.25 ± 9.85 [93]
FRAP, (μmol Fe(II)/100 g honey): between 98.26 ± 28.61 and 786.53 ± 91.28
Strawberry tree honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 51[95]
FRAP, (mg TE/100 g honey): ca. 89
Asphodel honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 4.5
FRAP, (mg TE/100 g honey): ca. 4
Cardoon honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 6
FRAP, (mg TE/100 g honey): ca. 6
Eucalyptus honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 8
FRAP, (mg TE/100 g honey): ca. 7
Thymus honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 4
FRAP, (mg TE/100 g honey): ca. 3
Chestnut honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 6.5
FRAP, (mg TE/100 g honey): ca. 8
Cistus honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 5.5
FRAP, (mg TE/100 g honey): ca. 7
Lavender honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 5
FRAP, (mg TE/100 g honey): ca. 4
Rosemary honey from Sardinia bDPPH, (mg TE/100 g honey): ca. 7
FRAP, (mg TE/100 g honey): ca. 5.5
Acacia honey from MoroccoDPPH, (mmol TE/Kg honey ± SD): 0.52 ± 0.01[102]
FRAP, (mmol Fe(II)/Kg honey ± SD): 2.15 ± 0.21
Eucalyptus honey from MoroccoDPPH, (mmol TE/Kg honey ± SD): 0.68 ± 0.01
FRAP, (mmol Fe(II)/Kg honey ± SD): 2.99 ± 0.09
Strawberry tree honey from ItalyDPPH, (mmol TE/Kg honey ± SD): 4.5 ± 1.1[104]
ABTS, (mmol TE/Kg honey ± SD): 5.9 ± 1.5
FRAP, (mmol Fe(II)/kg honey ± SD): 12.0 ± 2.2
Strawberry tree honey from Italy bDPPH, (mmol TE/Kg honey): ca. 4.7[105]
FRAP, (mmol Fe(II)/kg honey): ca. 11.7
Heather honey from Italy bDPPH, (mmol TE/Kg honey): ca. 1.45
FRAP, (mmol Fe(II)/kg honey): ca. 4.9
Eucalyptus honey from Italy bDPPH, (mmol TE/Kg honey ): ca. 0.45
FRAP, (mmol Fe(II)/kg honey): ca. 3.0
Asphodel honey from Italy bDPPH, (mmol TE/Kg honey): ca. 0.45
FRAP, (mmol Fe(II)/kg honey): ca. 4.3
Citrus honey from Italy bDPPH, (mmol TE/Kg honey): ca. 0.3
FRAP, (mmol Fe(II)/kg honey): ca. 1.65
Acacia honey from Italy bDPPH, (mmol TE/Kg honey): ca. 0.1
FRAP, (mmol Fe(II)/kg honey): ca. 0.55
Citrus honey from ItalyDPPH, IC50 (mg/mL ± SD): between 5.0 ± 0.3 and 15.1 ± 0.4[106]
Rhododendron honey from ItalyDPPH, IC50 (mg/mL ± SD): between 5.7 ± 0.3 and 15.5 ± 0.8
Acacia honey from ItalyDPPH, IC50 (mg/mL ± SD): between 8± 1 and 12.0 ± 0.6
Strawberry tree honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 1501.4 ± 60.2 [107]
DPPH, IC50 (mmol TE/Kg honey ± SD): 1.63 ± 0.17
ORAC, (mmol TE/Kg honey ± SD): 21.07 ± 0.34
Buckwheat honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 800.7 ± 23.8
DPPH, IC50 (mmol TE/Kg honey ± SD): 4.00 ± 0.44
ORAC, (mmol TE/Kg honey ± SD): 11.60 ± 0.027
Chestnut honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 388.6 ± 8.2
DPPH, IC50 (mmol TE/Kg honey ± SD): 7.93 ± 0.04
ORAC, (mmol TE/Kg honey ± SD): 8.90 ± 0.45
Sulla honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 155.2 ± 6.6
DPPH, IC50 (mmol TE/Kg honey ± SD): 16.90 ± 0.11
ORAC, (mmol TE/Kg honey ± SD): 5.66 ± 0.13
Clover honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 72.8±3.0
DPPH, IC50 (mmol TE/Kg honey ± SD): 25.00 ± 0.01
ORAC, (mmol TE/Kg honey ± SD): 2.15 ± 0.02
Dandelion honeys from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): from 212.2±2.2 to 224.4±6.0
DPPH, IC50 (mmol TE/Kg honey ± SD): from 24.39 ± 0.07 to 47.62 ± 0.39
ORAC, (mmol TE/Kg honey ± SD): from 2.00 ± 0.02 to 7.59 ± 0.60
Chicory honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 209.5±2.8
DPPH, IC50 (mmol TE/Kg honey ± SD): 5.81 ± 0.04
ORAC, (mmol TE/Kg honey ± SD): 6.72 ± 0.33
Acacia honey from ItalyFRAP, (μmol Fe(II)/Kg honey ± SD): 79.5±3.7
DPPH, IC50 (mmol TE/Kg honey ± SD): 45.45 ± 0.04
ORAC, (mmol TE/Kg honey ± SD): 2.12 ± 0.01
Rosemary honey from PortugalDPPH, IC50 (mg/mL ± SD): 168.94 ± 19.20[110]
Viper’s bugloss honey from PortugalDPPH, IC50 (mg/mL ± SD): 130.49 ± 1.38
Heather honey from PortugalDPPH, IC50 (mg/mL ± SD): 106.67 ± 2.48
Acacia honey from RomaniaDPPH, I%: between 35.80 and 45.27[111]
Sunflower honey from RomaniaDPPH, I%: between 36.60 and 40.91
Lime honey from RomaniaDPPH, I%: between 40.65 and 49.19
Acacia honey from SloveniaDPPH, IC50 (mg/mL): between 33.9 and 63.9[112]
FRAP, (μmol Fe(II)/100 g honey): between 56.8 and 86.0
Lime honey from SloveniaDPPH, IC50 (mg/mL): between 20.6 and 36.1
FRAP, (μmol Fe(II)/100 g honey): between 94.6 and 155.1
Chestnut honey from SloveniaDPPH, IC50 (mg/mL): between 7.8 and 14.0
FRAP, (μmol Fe(II)/100 g honey): between 238.3 and 469.5
Fir honey from SloveniaDPPH, IC50 (mg/mL): between 6.4 and 11.7
FRAP, (μmol Fe(II)/100 g honey): between 320.8 and 582.2
Spruce honey from SloveniaDPPH, IC50 (mg/mL): between 5.4 and 9.7
FRAP, (μmol Fe(II)/100 g honey): between 277.5 and 495.4
Linen vine honey from CubaORAC, (μmol of TE/g honey ± SD): 12.89 ± 0.28[113]
ABTS, (μmol of TE/g honey ± SD): 2.94 ± 0.23
Morning glory honey from CubaORAC, (μmol of TE/g honey ± SD): 9.26 ± 0.46
ABTS, (μmol of TE/g honey ± SD): 2.01 ± 0.21
Singing bean honey from CubaORAC, (μmol of TE/g honey ± SD): 8.12 ± 0.23
ABTS, (μmol of TE/g honey ± SD): 1.95 ± 0.14
Black mangrove honey from CubaORAC, (μmol of TE/g honey ± SD): 7.45 ± 0.37
ABTS, (μmol of TE/g honey ± SD): 1.22 ± 0.24
Christmas vine honey from CubaORAC, (μmol of TE/g honey ± SD): 4.59 ± 0.51
ABTS, (μmol of TE/g honey ± SD): 1.03 ± 0.28
Linen vine honey from CubaAEAC, (mg/100 g honey ± SD): 29.54 ± 1.62[114]
QEAC, (mg/100 g honey ± SD): 13.73 ± 1.32
DPPH, IC50 (mg/mL ± SD): 7.23 ± 1.17
TBARS, IC50 (mg/mL ± SD): 3.76 ± 0.42
Lipid hydroperoxides, (mmol ± SD): 32 ± 2.35
Morning glory honey from CubaAEAC, (mg/100 g honey ± SD): 16.14 ± 1.21
QEAC, (mg/100 g honey ± SD): 7.34 ± 1.40
DPPH, IC50 (mg/mL ± SD): 15.88 ± 1.57
TBARS, IC50 (mg/mL ± SD): 6.47 ± 0.72
Lipid hydroperoxides, (mmol ± SD): 39 ± 3.26
Singing bean honey from CubaAEAC, (mg/100 g honey ± SD): 19.7 ± 0.86
QEAC, (mg/100 g honey ± SD): 8.95 ± 0.49
DPPH, IC50 (mg/mL ± SD): 12.44 ± 1.56
TBARS, IC50 (mg/mL ± SD): 7.17 ± 0.52
Lipid hydroperoxides, (mmol ± SD): 46 ± 3.82
Black mangrove honey from CubaAEAC, (mg/100 g honey ± SD): 14.65 ± 1.03
QEAC, (mg/100 g honey ± SD): 6.65 ± 0.52
DPPH, IC50 (mg/mL ± SD): 15.53 ± 1.11
TBARS, IC50 (mg/mL ± SD): 7.28 ± 1.03
Lipid hydroperoxides, (mmol ± SD): 43 ± 2.48
Christmas vine honey from CubaAEAC, (mg/100 g honey ± SD): 10.85 ± 1.47
QEAC, (mg/100 g honey ± SD): 4.93 ± 0.74
DPPH, IC50 (mg/mL ± SD): 18.53 ± 1.92
TBARS, IC50 (mg/mL ± SD): 9.94 ± 1.31
Lipid hydroperoxides, (mmol ± SD): 51 ± 3.26
Pine honey from GreeceFRAP, (mmol TE/Kg honey ± SD): 4.05 ± 0.03[117]
ORAC, (mmol TE/Kg honey ± SD): 11.6 ± 0.2
TEAC, (mmol TE/Kg honey ± SD): 5.06 ± 0.02
DPPH, IC50 (mmol TE/Kg honey ± SD): 1.18 ± 0.03
Dead nettle honey from SerbiaFRAP, (mmol TE/Kg honey ± SD): 2.03 ± 0.03
ORAC, (mmol TE/Kg honey ± SD): 10.2 ± 0.3
TEAC, (mmol TE/Kg honey ± SD): 3.70 ± 0.04
DPPH, IC50 (mmol TE/Kg honey ± SD): 0.49 ± 0.01
Linden honey from SerbiaFRAP, (mmol TE/Kg honey ± SD): 0.61 ± 0.02
ORAC, (mmol TE/Kg honey ± SD): 9.5 ± 0.1
TEAC, (mmol TE/Kg honey ± SD): 2.04 ± 0.06
DPPH, IC50 (mmol TE/Kg honey ± SD): 0.25 ± 0.01
Acacia honey from SerbiaFRAP, (mmol TE/Kg honey ± SD): from 0.20 ± 0.00 to 0.26 ± 0.01
ORAC, (mmol TE/Kg honey ± SD): from 5.9 ± 0.1 to 6.5 ± 0.3
TEAC, (mmol TE/Kg honey ± SD): from 1.00 ± 0.02 to 1.02 ± 0.03
DPPH, IC50 (mmol TE/Kg honey ± SD): 0.00 ± 0.00
Acronyms meaning: FRAP: ferric reducing antioxidant power; DPPH: 2,2-diphenyl-1-picrylhydrazyl radical; TEAC: Trolox equivalent antioxidant capacity; ORAC: oxygen radical absorbance capacity; AEAC, ascorbic acid equivalent antioxidant content; QEAC, quercetin equivalent antioxidant content; TBARS, thiobarbituric reactive substances; TEAC, Trolox equivalent antioxidant capacity; IC50, 50% inhibitory concentration; TE: Trolox equivalents. a IC50 for the honey samples was undeterminable (at the maximum concentration of honey in water (45 g/L), I% it was measured only a 25% DPPH inhibition); b Values roughly inferred by figures reported by authors.

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Ciulu, M.; Spano, N.; Pilo, M.I.; Sanna, G. Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys. Molecules 2016, 21, 451. https://doi.org/10.3390/molecules21040451

AMA Style

Ciulu M, Spano N, Pilo MI, Sanna G. Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys. Molecules. 2016; 21(4):451. https://doi.org/10.3390/molecules21040451

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Ciulu, Marco, Nadia Spano, Maria I. Pilo, and Gavino Sanna. 2016. "Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys" Molecules 21, no. 4: 451. https://doi.org/10.3390/molecules21040451

APA Style

Ciulu, M., Spano, N., Pilo, M. I., & Sanna, G. (2016). Recent Advances in the Analysis of Phenolic Compounds in Unifloral Honeys. Molecules, 21(4), 451. https://doi.org/10.3390/molecules21040451

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