Authentication of phacelia honeys (Phacelia tanacetifolia) based on a combination of HPLC and HPTLC analyses as well as spectrophotometric measurements
Introduction
Honey is a very popular food product in all parts of the world. A sweet taste, a pleasant aroma, good nutritional properties and antibacterial and antioxidant activity are its characteristic features. It allows for its inclusion into the “minimally processed” food category (Kuś et al., 2014). The composition of honey depends on the type and species of the plant from which bees collect the nectar. The main ingredients of honey are sugars (primarily glucose and fructose) and water. Honey also contains a cocktail of low-molecular-weight compounds (organic acids, amino acids, proteins, polyphenolic compounds and minerals), which significantly contribute to the honey biological activity being also responsible for its aroma, taste and colour. Honeys are offered as multifloral (produced by bees using nectar from many flower sources) or monofloral (from the nectar of one flower spices) ones, and their botanical origin affect the quality and price.
In this paper we are presenting the chemical composition of non-sugar factions of monofloral phacelia honeys. It is produced by honeybees by collection of nectar from Phacelia tanacetifolia alongside with these of other plants. This honey has a light beige or white colour, mild aroma and soft taste. Phacelia tanacetifolia is an herbaceous, flowering annual of the Hydrophyllaceae family. This plant gives high quality nectar and pollen that are very attractive to bees. It is on the list of the world's top twenty honey plants. Furthermore, many of the benefits of Phacelia tanacetifolia (for example, quick growth, rapid flowering, good growth in dry soil and climate adaptability) have made beekeepers more likely to produce phacelia honey (Adgaba et al., 2016; Farkas & Zajacz, 2007).
The available literature clearly indicates that honey is the product most susceptible to fraud in the food industry. Therefore, the control of the quality of honeys is of great importance (Jasicka-Misiak, Poliwoda, Dereń, & Kafarski, 2012; Soares, Amaral, Oliveira, & Mafra, 2017; Trifković, Andrić, Ristivojević, Guzelmeric, & Yesilada, 2017). Honey authentication began with melissopalynological analysis. In recent decades, new methods based on the identification of natural products, such as phenolic compounds, have been implemented (Karabagias et al., 2014). Polyphenols were found in individual varieties of honey in different contents and compositions. Unfortunately, the determination and identification of such compounds in honey is challenging because these occur there at low concentrations. Therefore, the described results are strongly dependent on the isolation and kind of analytical method applied for their identification (Pyrzyńska & Biesaga, 2009; Ciulu, Spano, Pilo, & Sanna, 2016; Kečkeš et al., 2013). High-performance liquid chromatography (HPLC) is the most commonly technique used for quantitative and qualitative method analysis of this class of compounds (Badjah Hadj Ahmed et al., 2016; Pascual-Maté, Osés, Fernández-Muiño, & Sancho, 2017). The technique used equally as frequently for studying low-molecular-weight compounds is high-performance thin layer chromatography (HPTLC) (Locher, Neumann, & Sostaric, 2017). These methods allow the construction of phenolics profiles, which indicate the differences and similarities between honeys. In recent decades, accumulation of the large amount of data available by various analytical methods requires the use of statistical methods to classify the botanical origin of honey. The most common statistical techniques that have been used to authenticate honey are hierarchical cluster analysis, principal component analysis and discriminant analysis (Kečkeš et al., 2013; Karabagias et al., 2014; Kuś et al., 2014; Zieliński, Deja, Jasicka-Misiak, & Kafarski, 2014).
Therefore, the aim of this study was to determine the characteristic phenolic HPLC and HPTLC profiles obtained for phacelia honey samples and to investigate their correlation with pollen analysis data and thus their authentication. In addition, selected parameters are presented, such as: the total phenolic and flavonoid contents (TPC and TFC), antioxidant activity (DPPH and ABTS test) and colour characteristics. These parameters are known to correlate with pro-health properties of honey (Parker, Byrne, & Hemsworth, 2016; Piljac-Žegarac, Stipčević, & Belščak, 2009).
Section snippets
Chemicals and materials
All chemicals used for the analyses were of analytical purity grade. Methanol, dibasic sodium phosphate heptahydrate (Na2HPO4 x 7H2O), 85% phosphoric acid, chloroform, ethyl acetate and formic acid were purchased from POCH S.A. (Gliwice, Poland). The standards: chrysin (5,7-dihydroxy-2-phenyl-4H-chromen-4-one), galangin (3,5,7-trihydroxy-2-phenylchromen-4-one), apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-chromen-4-one), pinocembrin (5,7-dihydroxy-2-phenyl-2,3-dihydro-4H-chromen-4-one),
Total phenolic and flavonoid contents, antioxidant activity and colour intensity
Table 1 presents the predominant plant sources of all tested honeys. As seen from the Table nearly all of them could be classified as phacelia brand. Samples ascribed as Ph1 and Ph9 (declared by producers as phacelia honeys) contained significant amounts of pollen from other plants (Ph1: Phacelia 38.9%, Salix 32.3%, Fagopyrum 10.1%, Brassicaceae 9.3%; Ph9: Brassica napus 40.3%, Phacelia 39.5%). Thus, these are typical examples of honeys obtained when bees feed on more than one source. This set
Conclusion
This research suggests that the exact classification of honey could be made either by spectrophotometric measurements but as well by HPLC and HPTLC analysis. The best solution, however, is to use several complimentary methods. The use of two different chromatographic methods provided complimentary information that was useful for the classification and differentiation of phacelia honey. These methods distinguished pure honeys containing the highest phacelia pollen content from honeys containing
Declarations of interest
The authors declare there is no conflict of interest.
Funding
This study was funded by No. 2014/2015/15/B/NZ9/02182.
Acknowledgements
This work was supported by The National Science Centre of Poland in the frame of Grant NO: 2014/15/B/N29/02182.
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