Due to the variable composition of honey, identification of origin and detection of adulterations is a challenging task for food experts and authorities. There are no standard criteria in the European Union (EU) and worldwide, therefore it is a difficult task for identification of botanical and geographical origin of honeys. As quality of honey depends on several factors, such as, the nectar consumed by bees, natural structure of the nectar, climatic conditions and geographical origin, therefore the prediction of composition of honey also remains a challenging task [2]. There are several studies dealing with identification of origin based on physicochemical properties [10, 20, 45], spectral measurements [13,23,34], data acquired by electronic sensor instruments [46, 79, 80, 81], and sophisticated techniques like nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), time- of-flight (TOF)-mass spectrometry (MS) [72], etc.

TPC according to techniques, such as: Folin-Ciocalteu (TPC), ferric reduction antioxidant power (FRAP), cupric ion reducing antioxidant capacity (CTJPRAC), ABTS (2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging. Total flavonoid content (TFC) are also studied for identification of origin of different monofloral honeys [1, 7, 11, 33, 47, 62, 67]. However, these methods cannot provide information on the type of the distinct molecules providing the antioxidant properties, therefore individual phytochemical components may serve as more appropriate marker molecules [16] for the identification of honeys from different botanical sources.

Sections 3.2, 3.3, and 3.4 give an account of different types of phenolic components and flavonoids that are present in monofloral honeys and can qualify as possible tracer molecules for authentication.


Studies report that the antioxidant capacity (AOC) and total phenolic and flavonoid contents in honeys are directly correlated.


Flavonoids and phenolic acids are considered as efficient plant-derived antioxidants. According to literature, there are many assays available to determine the TP and flavonoid contents in honey. The total AOC of honey can be measured through polyphenols content in honey. According to main mechanisms governing these reactions, the techniques can be classified into two main groups, such as: (1) reactions based on hydrogen atom transfer (HAT);

  • (2) single-electron transfer (SET); and (3) mixed mechanisms, involving both pathways.
  • Methods Based on Hydrogen Atom Transfer (HAT) Reactions

HAT based techniques are aimed to determine the ability of antioxidants to quench the free radicals by hydrogen donation. In these reactions, the bond dissociation enthalpy is an important parameter to evaluate the antioxidant action: lower enthalpies of the antioxidants facilitate the reaction. These methods are usually fast and need only seconds or minutes to get the results. The two main HAT-based methods are [69]: the oxygen radical absorbance capacity (ORAC) and the total-radical trapping antioxidant parameter (TRAJP).

In ORAC method, the AOC is measured by measuring the intensity of a fluorescent signal from a probe that is quenched in the presence of free radicals. The antioxidants absorb the generated free radicals, reactive oxygen species, allowing thus the fluorescent signal to persist. The free radicals are generated from 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH) that produces a free radical (peroxyl).

The TRAP method monitors the capacity of the antioxidants in the sample to scavenge luminol-derived radicals, generated from AAPH decomposition. The reaction is followed by chemi-luminescence [69].

Other methods under HAT group are photochemi-luminescence (PCL), chemi-luminescence (CL) and total antioxidant scavenging capacity (TOSC) [69]. Methods Based on Single Electron Transfer (SET)

In SET reaction, the antioxidant delivers an electron to the free radical and itself becomes a radical cation. In this reaction, the ionization potential of the antioxidant is the most important factor affecting the antioxidant action. Lower ionization potentials make the electron abstraction easier [69]. SET measurements are generally more popular than HAT measurements. This also applies to the analysis of honey. Assays like ferric reducing antioxidant power (FRAP) and cupric ion reducing antioxidant power (CUPRAC) belong to this group [69].

FRAP reaction is based on the measurement of reduction of Fe^ to Fe^, which forms a colored complex with 2,4,6-tris (2-tripyridyl)-s-triazine

(TPTZ) -phiS color change is followed by spectrophotometry at 593 nm. The method is not suitable for the detection of thiols and proteins [69].

CUPRAC method is similar to FRAP method. It is based on the reduction of Cu++ to Cu+, which latter forms a colored complex with neocuproine. The CUPRAC can be used for both hydrophilic and lipophilic antioxidants. It has the advantage because this method works at a neutral pH similar to the physiological pH [69].

  • Methods Based on Mixed Set and Hat Mechanisms
  • 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or trolox equivalent antioxidant capacity (TEAC) measurement belongs to this group, where the antioxidant ability of the test compound is determined based on the loss of color that is determined by their reaction with the ABTS‘+ radical cation, produced prior to the assay. The most appropriate wavelengths for following the radical scavenging reaction are 415 nm and 734 nm.

A similar assay is 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which is based on the reducing capacity of the antioxidants towards DPPH radical causing change of color that can be measured at 515 mn. The method has the drawback related with steric hindrance of DPPH radical, which is not easily accessible for the antioxidants in the solution. The method is suitable for hydrophilic and lipophilic antioxidants. Other Methods for Determination of Total Polyphenol

The formal content of total polyphenols (TPC) is determined by popular Folin-Ciocalteu method. This is based on the reduction of a mixture of phos- potungstate-phospomolibdate by the antioxidants in the sample, resulting in the appearance of a blue color measured usually at 750 mn or 765 nm by spectrophotometry. The basic mechanism is a redox reaction. During this reaction, the phenolic group is oxidized and the metal ion is reduced. The method has the drawback of low specificity. It can detect many other reducing compounds, such as reducing sugars [75].

The TFC assay is often used to measure AOC of honey. This technique is based on the complication of flavonoids with aluminum chloride, which results in a change of color, detectable by spectrophotometry at 430 nm [74].

The total antioxidant status (TAS) and total oxidant status (TOS) measurements are also available. Usually, commercially available kits are used for the determination of these properties. Using TAS and TOS values, an oxidative stress index can be calculated. The spectrophometrically detected change of color is dependent on the total amount of antioxidant molecules in the sample. The methods can be calibrated with H,02 or trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).

As mentioned above, several in vitro assays are available for the detection of AOC in foods, however, all of them have unique advantages and disadvantages. Although new assays are continuously being developed, yet there is no “universal” method. Therefore, usually several techniques have been used to characterize the samples [31, 66].


Although there has been no attempt to identify or quantify the individual compounds in these studies, yet the overall antioxidant properties of honeys are indicative in some cases for the botanical or geographical origins of honey.

Turkish researchers determined the TAS and TOS of honey samples from seven different regions in Turkey. They found significant differences between honeys from the different regions in terms of both parameters, due to the differences in the climatic conditions and the soil quality in the study zones [8].

Three different botanical types of honey (mainly: Acacia tortilis (Summer), Ziziphus spina-crhisti (Sidr) and multiflora honey) were investigated for their total polyphenol (TPC), total flavonoid (TFC) contents and their antioxidant capacity (DPPH) in Oman. The results revealed the differences between different types of honeys. Summer honey is richest in antioxidant compounds compared to other types. The researchers reported that their results were different from those reported from other countries, which can be the consequence of different climatic conditions [1].

Portuguese researcher investigated thyme, orange, strawberry, locust pod-shrub, rosemary, eucalyptus, and heather honeys. Honeys were classified based on their antioxidant capacities and concentration of minerals. Results showed that rosemary and orange honeys had lower TPC and AOC. AOC was increased in the following order: thyme honey>strawberry honey> locust pod-shrub honey> heather honey. Due to their low AOC, rosemary honey and orange honey were completely distinguished from the other types of honey. In addition, results showed that if AOC parameters are further combined with the mineral profile, this could represent a more efficient tool in the differentiation between honeys from various botanical types [7].

Bertoncelj and his co-workers [11] studied TPC, antioxidant activities of acacia, linden, chestnut, fir, spruce, forest, and multiflora honeys with FRAP and DPPH methods. For checking the significant differences among samples from different botanical origin, ANOVA test was applied, followed by Duncans’s post-hoc test. ANOVA test showed that there were significant differences between different floral groups. Acacia honeys had significantly lower AOC and TPC than other botanical types. Linden honeys showed significantly higher values in TPC and DPPH in comparison to acacia honey, but significantly lower than other types. Forest and fir honeys had the significantly highest TPC and FRAP values. These results show that the phenolic content of honey is responsible for its antioxidant power, and these parameters can be suitable to obtain a satisfactory differentiation among samples of different botanical origin [11]. In this study, a linear discriminant analysis (LDA) model was used among antioxidant parameters, water content, electrical conductivity, pH, acidic parameters, color (L*a*b*) and optical rotation. Based on this statistical analysis, acacia, and multiflora honeys were 100% correctly classified; and linden, chestnut, and fir honeys also showed good separation and classification scores (>80%). Therefore, if routine physicochemical parameters are combined with the antioxidant properties and the results are chemo metrically evaluated, the botanical differentiation of honeys can be significantly improved [12].

Can and his co-workers [24] examined AOC of honeys from different floral sources (such as: chestnut, astralagus, heather, clover, lavender, lime, Jerusalem tea, common eryngo, chaste tree, rhododendron, oak, pine, acacia, and multiflora) by TPC, DPPH, FRAP, and TFC methods. Their results showed significant differences based on all parameters in relation to the botanical source. They concluded that honeydew, pine, and oak honeys were completely different from other types of honey in terms of each measured parameter. Heather honey was also outstanding due to high amount of phenolic compounds and elevated antioxidant activity [24].

In an analogous Romanian study, acacia, sunflower, forest, multiflora, linden, and sea buckthorn honeys were evaluated based on their pH, ash content, color, protein, free amino acid, TPC, and antioxidant capacities by ABTS and DPPH methods. Results indicated that TPC content varied significantly according to the botanical origin. Highest TPC level was obtained for forest honeys, while acacia honeys showed lowest values [12,24]. DPPH and ABTS results showed similar trends, revealing similar differences related to the floral origin. Principal component analysis (PCA) model was built for measurement parameters revealing the different botanical groups [20].

Irish researchers reported concomitant measurements of TPC and physicochemical parameters of honeys (electrical conductivity, pH, and color). Simultaneous evaluation of these properties proved to play an important role in differentiating among botanical types of honey [47].

Group of Romanian researchers evaluated the TPC and TFC of acacia, linden, sunflower, and honeydew honeys. The results showed differences according to the botanical origin. In agreement with other reports, acacia honeys had lowest TPC and TFC, followed by linden and sunflower honeys, while honeydew honey had the highest values [3].

Also, Spanish researchers examined total concentration of polyphenols and flavonoids in chestnut, blackberry, heather, eucalyptus, multiflora, and honeydew honeys. Significant differences were found with ANOVA test followed with pair wise-comparison. Honeydew, heather, and chestnut honeys showed significantly higher TPC and TFC content than other types of honeys [29].

Brazilian researchers determined the antioxidant capacities by DPPH and FRAP methods and total phenolic content in nectar and honeydew honeys. Results confirmed the superior antioxidant qualities of honeydew honeys compared to blossom honeys. Therefore, these methods can be eligible for the differentiation between these two main types of honey [19].

Gul and Pehlivan [33] studied 23 different types of honeys for their antioxidant activities and total phenolic compounds by FRAP and DPPH methods. TPC was highest for chestnut, parsley, carob, and rhododendron honeys, while acacia and mint honeys showed lower values. All results revealed significant differences among the samples [33].

Maurya and co-workers [58] reviewed the antioxidant properties of honeys from different botanical and geographical origins. They reported that total phenolic content and antioxidant activities by FRAP, ORAC, DPPH, and ABTS methods were powerful tools to differentiate honeys from different botanical sources. The authors also concluded that there were differences in honeys from the same floral sources but different geographical origins. These differences might be attributed to climate and environmental factors, soil, and the surrounding flora [58].

Results of research carried out in Hungary were in agreement with these abovementioned studies regarding the relationships between antioxidant qualities and the botanical and geographical origins. In an investigation, antioxidant activities and TPC measured by CUPRAC, FRAP methods in 79

honeys were determined from various botanical and geographical sources. Acacia, canola, silkgrass, and sunflower honeys reached the lowest total polyphenol concentration and antioxidant activities, measured by FRAP method. With the same method, chestnut, pine, and honeydew honeys showed highest scores [13, 46].

In another study, classification models were built based on pH, electrical conductivity, color, ash content, and TPC of acacia, chestnut, linden, and multiflora honeys. Evaluation of the set of data by LDA provided 89.3% and 90.5% recognition and prediction abilities, respectively for the classification of honeys according to their botanical origin. Models were also built for samples from the same floral source but different geographical zones. Results showed that 100% correct classification was obtained in case of chestnut honey for different topographical sources; while the results for acacia honey were less conclusive [45]. Other researchers obtained lowest antioxidant levels for acacia and significantly higher values for chestnut honeys [3, 11, 29,33].

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