Estrogenic Isoflavones in Clover Plants, Flower Nectar, Unripe Honeys and Mature Honeys: A Natural Biochemical Transformation of Isoflavones by Honeybees.

This study is the first to report on the presence of oestrogenic compounds in different clover flower nectar samples, in bee-deposited nectars collected from hive combs (unripe honey) and in mature honeys harvested from the same hives. The clover species investigated were two red clover (Trifolium pratense) cultivars, bred specifically for high isoflavone content, alongside a sainfoin (Onobrychis viciifolia) and a purple clover (T. purpureum) cultivar. A total of eight isoflavones, four of them non-glycosidic (biochanin A, formononetin, genistein and daidzein) the others glycosidic (sissotrin, ononin, genistin and daidzin), were targeted for identification and quantification in this study using high-performance thin-layer chromatography (HPTLC). Leaves and flower bracts of the clover samples were also investigated. Different isoflavone profiles were found across the four clover species and also in the different samples collected from each species indicating that, most likely due to the activity of honeybee (Apis mellifera) salivary enzymes, biochemical conversions take place when these bioactive compounds transition from flower nectar into ripe honey. Among the four investigated clover species, the two red clover cultivars, including their honeys, were found to contain higher levels of estrogenic compounds compared to other two cultivars.

Antioxidant Activity and Phenolic Compound Identification and Quantification in Western Australian Honeys.

This study reports on the total phenolic content and antioxidant activity as well as the phenolic compounds that are present in Calothamnus spp. (Red Bell), Agonis flexuosa (Coastal Peppermint), Corymbia calophylla (Marri) and Eucalyptus marginata (Jarrah) honeys from Western Australia. The honey's total phenolic content (TPC) was determined using a modified Folin-Ciocalteu assay, while their total antioxidant activity was determined using FRAP and DPPH assays. Phenolic constituents were identified using a High Performance Thin-Layer Chromatography (HTPLC)-derived phenolic database, and the identified phenolic compounds were quantified using HPTLC. Finally, constituents that contribute to the honeys' antioxidant activity were identified using a DPPH-HPTLC bioautography assay. Based on the results, Calothamnus spp. honey (n = 8) was found to contain the highest (59.4 ± 7.91 mg GAE/100 g) TPC, followed by Eucalyptus marginata honey (50.58 ± 3.76 mg GAE/100 g), Agonis flexuosa honey (36.08 ± 4.2 mg GAE/100 g) and Corymbia calophylla honey (29.15 ± 5.46 mg GAE/100 g). In the FRAP assay, Calothamnus spp. honey also had the highest activity (9.24 ± 1.68 mmol Fe2+/kg), followed by Eucalyptus marginata honey (mmol Fe2+/kg), whereas Agonis flexuosa (5.45 ± 1.64 mmol Fe2+/kg) and Corymbia calophylla honeys (4.48 ± 0.82 mmol Fe2+/kg) had comparable FRAP activity. In the DPPH assay, when the mean values were compared, it was found that Calothamnus spp. honey again had the highest activity (3.88 ± 0.96 mmol TE/kg) while the mean DPPH antioxidant activity of Eucalyptus marginata, Agonis flexuosa, and Corymbia calophylla honeys were comparable. Kojic acid and epigallocatechin gallate were found in all honeys, whilst other constituents (e.g., m-coumaric acid, lumichrome, gallic acid, taxifolin, luteolin, epicatechin, hesperitin, eudesmic acid, syringic acid, protocatechuic acid, t-cinnamic acid, o-anisic acid) were only identified in some of the honeys. DPPH-HPTLC bioautography demonstrated that most of the identified compounds possess antioxidant activity, except for t-cinnamic acid, eudesmic acid, o-anisic acid, and lumichrome.

The Development and Application of a HPTLC-Derived Database for the Identification of Phenolics in Honey.

This study reports on the development and validation of a HPTLC-derived database to identify phenolic compounds in honey. Two database sets are developed to contain the profiles of 107 standard compounds. Rich data in the form of Rf values, colour hues (H°) at 254 nm and 366 nm, at 366 nm after derivatising with natural product PEG reagent, and at 366 nm and white light after derivatising with vanillin-sulfuric acid reagent, λ max and λ min values in their fluorescence and λ max values in their UV-Vis spectra as well as λ max values in their fluorescence and UV-Vis spectra after derivatisation are used as filtering parameters to identify potential matches in a honey sample. A spectral overlay system is also developed to confirm these matches. The adopted filtering approach is used to validate the database application using positive and negative controls and also by comparing matches with those identified via HPLC-DAD. Manuka honey is used as the test honey and leptosperine, mandelic acid, kojic acid, lepteridine, gallic acid, epigallocatechin gallate, 2,3,4-trihydroxybenzoic acid, o-anisic acid and methyl syringate are identified in the honey using the HPTLC-derived database.

A Comprehensive Survey of Phenolic Constituents Reported in Monofloral Honeys around the Globe.

The aim of this review is to provide a comprehensive overview of the large variety of phenolic compounds that have to date been identified in a wide range of monofloral honeys found globally. The collated information is structured along several themes, including the botanical family and genus of the monofloral honeys for which phenolic constituents have been reported, the chemical classes the phenolic compounds can be attributed to, and the analytical method employed in compound determination as well as countries with a particular research focus on phenolic honey constituents. This review covers 130 research papers that detail the phenolic constituents of a total of 556 monofloral honeys. Based on the findings of this review, it can be concluded that most of these honeys belong to the Myrtaceae and Fabaceae families and that Robinia (Robinia pseudoacacia, Fabaceae), Manuka (Leptospermum scoparium, Myrtaceae), and Chestnut (Castanea sp., Fagaceae) honeys are to date the most studied honeys for phenolic compound determination. China, Italy, and Turkey are the major honey phenolic research hubs. To date, 161 individual phenolic compounds belonging to five major compound groups have been reported, with caffeic acid, gallic acid, ferulic acid and quercetin being the most widely reported among them. HPLC with photodiode array detection appears to be the most popular method for chemical structure identification.

Australian Honeypot Ant (Camponotus inflatus) Honey-A Comprehensive Analysis of the Physiochemical Characteristics, Bioactivity, and HPTLC Profile of a Traditional Indigenous Australian Food.

Despite its cultural and nutritional importance for local Aboriginal people, the unusual insect honey produced by Western Australian honeypot ant (Camponotus inflatus) has to date been rarely investigated. This study reports on the honey's physicochemical properties, its total phenolic, major sugars and 5-hydroxymethylfurfural contents, and its antioxidant activities. The honey's color value is 467.63 mAU/63.39 mm Pfund, it has a pH of 3.85, and its electric conductivity is 449.71 µSiemens/cm. Its Brix value is 67.00, corresponding to a 33% moisture content. The total phenolics content is 19.62 mg gallic acid equivalent/100 g honey. Its antioxidant activity measured using the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing-antioxidant power) assays is 1367.67 µmol Trolox/kg and 3.52 mmol Fe+2/kg honey, respectively. Major sugars in the honey are glucose and fructose, with a fructose-to-glucose ratio of 0.85. Additionally, unidentified sugar was found in minor quantities.

An investigation of the suitability of melissopalynology to authenticate Jarrah honey.

This study reports on the analysis of eleven Jarrah (Eucalyptus marginata) honeys, of which nearly half (n = 5) were re-classified as Blackbutt (E. patens) honey on the grounds of the predominant flower pollen identified by melissopalynology. Based on a comprehensive analysis of the honeys' physico- and phytochemical characteristics and antioxidant activity data, taking into account pH, electrical conductivity, refractive index and Brix values as well as moisture content, individual fructose and glucose content and derived fructose to glucose ratio alongside total phenolic content and antioxidant activity determined by the DPPH assay, no statistically significant difference was found amongst the eleven honeys classified by pollen analysis into two honey groups, 'Jarrah' or 'Blackbutt'. This study therefore draws into question the value of melissopalynology as an analysis tool to authenticate Jarrah honey.

Optimisation of Bee Pollen Extraction to Maximise Extractable Antioxidant Constituents.

This paper presents the findings of a comprehensive review on common bee pollen processing methods which can impact extraction efficiency and lead to differences in measured total phenolic content (TPC) and radical scavenging activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) data. This hampers the comparative analysis of bee pollen from different floral sources and geographical locations. Based on the review, an in-depth investigation was carried out to identify the most efficient process to maximise the extraction of components for measurement of TPC, DPPH and FRAP antioxidant activity for two bee pollen samples from western Australia (Jarrah and Marri pollen). Optimisation by Design of Experiment with Multilevel Factorial Analysis (Categorical) modelling was performed. The independent variables included pollen pulverisation, the extraction solvent (70% aqueous ethanol, ethanol, methanol and water) and the extraction process (agitation, maceration, reflux and sonication). The data demonstrate that non-pulverised bee pollen extracted with 70% aqueous ethanol using the agitation extraction method constitute the optimal conditions to maximise the extraction of phenolics and antioxidant principles in these bee pollen samples.