Raw Millefiori honey is packed full of antioxidants

Total polyphenols, flavonoids and antioxidant power of raw honey samples from two of the most common Italian varieties, i.e., Millefiori and Acacia, were evaluated. Phenolic content, expressed as caffeic acid equivalents, ranged from 12.5 to 17.5 mg/100 g and from 3 to 11 mg/100 g in Millefiori and Acacia honeys, respectively. All Millefiori samples exhibited the highest flavonoid concentration being between 1.23 and 2.93 mg catechin equivalents (CE)/100 g honey. Total flavonoids in 100 g Acacia honeys were in the range of 0.45–1.01 mg CE. Acacia honeys had lower total antioxidant power, as assessed by ferric reducing/antioxidant power assay, than Millefiori. The relationship between phenolic content and antioxidant power was discussed. Comparative experimental analysis was performed with an artificial honey and processed honeys. Raw Millefiori honey is rich in both amount and variety of antioxidant substances, and its inclusion in the diet may be recommended to complement other polyphenol sources.     

Flavonoids,phenolic acids and abscisic acid in Australian and New Zealand Leptospermum honeys

Flavonoids, phenolic acids and abscisic acid of Australian and New Zealand Leptospermum honeys were analyzed by HPLC. Fifteen flavonoids were isolated in Australian jelly bush honey (Leptospermum polygalifolium), with an average content of 2.22 mg/100 g honey. Myricetin (3,5,7,3′,4′,5′-hexahydroxyflavone), luteolin (5,7,3′,4′-tetrahydroxyflavone) and tricetin (5,7,3′,4′,5′-pentahydroxyflavone) were the main flavonoids identified. The mean content of total phenolic acids in jelly bush honey was 5.14 mg/100 g honey, with gallic and coumaric acids as the potential phenolic acids. Abscisic acid was quantified as twice the amount (11.6 mg/100 g honey) of the phenolic acids in this honey. The flavonoid profile mainly consisted of quercetin (3,5,7,3′,4′-pentahydroxyflavone), isorhamnetin (3,5,7,4′-tetrahydroxyflavone 3′-methyl ethyl), chrysin (5,7-dihydroxyflavone), luteolin and an unknown flavanone in New Zealand manuka (Leptospermum scoparium) honey with an average content of total flavonoids of 3.06 mg/100 g honey. The content of total phenolic acids was up to 14.0 mg/100 g honey, with gallic acid as the main component. A substantial quantity (32.8 mg/100 g honey) of abscisic acid was present in manuka honey. These results showed that flavonoids and phenolic acids could be used for authenticating honey floral origins, and abscisic acid may aid in this authentication.     

Flavonoid profile of Robinia honeys produced in Croatia

In this work specific pollen content, selected physicochemical parameters and flavonoid profile of 40 Croatian Robinia honeys from two production seasons were analysed. Results showed good compliance with national and international regulatory requirements, as well as with values typical for Robinia monofloral honey. All analysed samples showed same, typical flavonoid profile. Flavonoid content was different for two seasons, but rates of individual compounds remained unchanged. Higher concentrations of flavonoids were found in samples produced during dry season with high temperatures.     

Phenolic profile,antioxidant activity and palynological analysis of stingless bee honey from Amazonas,Northern Brazil

In this study honey samples produced by Melipona (Michmelia) seminigra merrillae, collected in seven counties distributed in the central and southern region of Amazonas state in Brazil, were analysed for their botanical origin, content and profile of phenolic compounds, and antioxidant and antimicrobial activities. Twenty-two pollen types were identified. The total phenolic content ranged from 17 to 66 mg GAE/g of extract; the highest contents were found in honeys produced from pollen types such as Clidemia and Myrcia. The antioxidant activity was higher in the samples that contained higher quantities of phenolic compounds. In relation to the antibacterial activity, samples CAD3, CAD4 and SAD3 presented the best results. Fourteen phenolic compounds were determined. Among them, we identified the flavonoid taxifolin, which has not previously been described in honeys from stingless bees, and we report the identification of catechol in Brazilian honey samples for the first time.     

Major flavonoids of Argentinean honeys. Optimisation of the extraction method and analysis of their content in relationship to the geographical source of honeys

In the present research we optimised an extraction procedure for the flavonoid aglycones: myricetin, quercetin and luteolin from honeys (as natural biological matrices), based on Amberlite XAD-4 resin followed by HPLC quantification. In addition, honeys from three geographical regions of Argentina were analysed with regard to the contents of these flavonoids. The extraction procedure was optimised for XAD-4 resin considering: resin/honey ratio, elution volume to desorb flavonoids and colour intensity of honeys. Differences in flavonoid recoveries were observed depending on the colour intensity. The flavonoid aglycones contents, in accordance with differences in geochemical characteristics and typical vegetation, varied with the geographical origin of honeys. The results obtained allowed us to consider these three flavonoids as chemical markers for the phytogeographical origin of honeys. In the case of monofloral honeys, the contribution of each one of the flavonoids was associated with the presence of a dominant pollen kind in these samples.     

Physico chemical and bioactive properties of honeys from Northwestern Argentina

Honey is used for its nutritional and functional properties. The Argentinean Northwest is a region with a growing potential for honey production, but up to now, few physicochemical and biological studies have been carried out. The aim of this study is to characterize monofloral (Prosopis sp and Citrus lemon) and multifloral honey samples from the Argentinean Northwest from a physicochemical and functional standpoint. The results showed that the honeys had good properties of stability and freshness. The highest content of flavonoid and phenolic compounds correspond to multifloral honeys. A positive correlation was observed between colour intensity and flavonoid or phenolic compounds content (R² = 0.98 and R² = 0.92, respectively). The flavonoids, chrysin and pinocembrin were present in all samples analyzed, while hesperidin and hesperetin were numerically more important in lemon honey (>1 mg/kg), providing a valuable marker of botanical origin.The highest antioxidant activity against ABTS radical cation was detected in the darkest honey samples. All tested honeys showed antibacterial activity with MIC values between 0.10 and 0.25 g/mL on Gram-positive and Gram-negative antibiotic resistant bacteria. Neither pH nor osmolarity affected bacterial growth. The phenolic compounds and hydrogen peroxide were responsible for antimicrobial activity by bioautographic assays.The antioxidant and antimicrobial properties found in honeys from the Argentinean Northwest make them products of high added value and excellent quality.     

Rhus verniciflua Stokes flavonoid extracts have anti-oxidant,anti-microbial and α-glucosidase inhibitory effect

We isolated four compounds, fustin, gallic acid, 3′,4′,7-trihydroxyflavone, and fisetin from Rhus verniciflua. These compounds showed electron donation ability (87–94%) that was stronger than butylated hydroxyanisole (52%). Gallic acid (OD₇₀₀ = 1.98) showed the highest reducing power, and the other isolated compounds (OD₇₀₀ = 0.66–1.31) showed stronger activity than α-tocopherol (OD₇₀₀ = 0.21). The ethyl-acetate fraction had the highest phenolic content (723 mg GAE/g), followed by the 80% ethanolic extract (597 mg GAE/g). For Gram-negative bacteria, fisetin had the most potent anti-bacterial activity (MIC = 8 μg/ml) against Escherichia coli. 3′,4′,7-Trihydroxyflavone (106%), the 80% ethanolic extract (101%), and ethyl-acetate (98%) had the most powerful α-glucosidase inhibitory effect at 50 μg/ml. R. verniciflua extracts have potential as functional food additives.     

Ecuadorian stingless bee (Meliponinae) honey: A chemical and functional profile of an ancient health product

Stingless bee honey samples from west Amazonian Ecuador were studied for their physiochemical, chemical and functional properties. Reducing sugars (44.9 ± 5.72 g/100 g), water (34.1 ± 4.34 g/100 g), free acidity (31.8 ± 4.05 meq/100 g), diastase activity (1.60 ± 0.20 u.d./g), hydroxymethylfurfural (15.0 ± 1.91 mg/kg), electrical conductivity (0.48 ± 0.06 mS/cm), ash (0.28 ± 0.04 g/100 g), colour (150 mm Pfund) were determined as physicochemical parameters. Melissopalynological analyses were processed evidencing pollen belonging to 14 plant families. Glucose (25.5 ± 3.41 g/100 g), fructose (25.2 ± 3.37 g/100 g) and sucrose (3.72 ± 0.49 g/100 g) contents were determined by HPLC, evidencing equal concentrations between fructose and glucose. Coumarins and flavonoids were determined by densitometric HPTLC: fraxin and bergamotin (0.065 ± 0.009; 0.035 ± 0.005 μg/g) among coumarins; luteolin (0.045 ± 0.006 μg/g), quercitrin (0.020 ± 0.003 μg/g), isoramnetin (0.015 ± 0.002 μg/g) among flavonoids. Among the vitamin E isomers, evaluated by HPLC, the occurrence of the only β-tocopherol (1.12 ± 0.15 μg/g) was noted. All these results were compared with those acquired for two multifloral Apis mellifera honeys. DPPH and β-carotene bleaching tests were performed, showing interesting values for Ecuadorian honey samples, higher than those shown by multifloral A. mellifera honeys (88.1 ± 11.1 DPPH inhibition%; 70.8 ± 8.90 β-carotene inhibition%). Antibacterial activity, against both Gram positive and Gram negative bacteria, revealed MIC values (10–50 μg/ml) always lower than those of A. mellifera honeys. Ecuadorian Meliponinae honey samples also showed anti-mutagenic activity assayed with Saccharomyces cerevisiae D7 strain, inhibiting back mutation over the entire range of concentrations.     

Evaluation of antioxidant ability of ethanolic extract from dill (Anethum graveolens L.) flower

Antioxidant activities of ethanolic extract from dill flower and its various fractions were evaluated with 2,2-diphenyl-1-picrylhydrazyl radical scavenging, Trolox equivalent antioxidant capacity, reducing power, chelating power, and β-carotene bleaching assays. The flower extract was successively separated into n-hexane, ethyl acetate and ethanol soluble fractions by liquid–liquid partition. Dill leaf and seed extracts were used for comparison. In all assays, the flower extract showed higher antioxidant activity than the leaf and seed extracts. With regard to various fractions of the flower extract, the sequence for antioxidant activity was ethyl acetate fraction > ethanol fraction > original flower extract > n-hexane fraction. Phenols including flavonoids and proanthocyanidins should be responsible for antioxidant abilities of the flower extract. Chlorogenic acid, myricetin, and 3,3’,4′,5,7-pentahydoxyflavan (4 → 8)-3,3′,4′,5,7-pentahydoxyflavan were the major phenolic acid, flavonoid, and proanthocyanidin, respectively, in the dill flower extract.     

Analysis of the flavonoid component of bioactive New Zealand mānuka (Leptospermum scoparium) honey and the isolation,characterisation and synthesis of an unusual pyrrole

The flavonoid components of New Zealand mānuka (Leptospermum scoparium) honey have been quantified in a series of 31 honeys of varying non-peroxide antibacterial activity to clarify discrepancies between previous studies reported in the literature. Total flavonoid content was 1.16 mg/100 g honey. The principal flavonoids present were pinobanksin, pinocembrin, luteolin and chrysin and together these represented 61% of the total flavonoid content. 1, 2-formyl-5-(2-methoxyphenyl)-pyrrole, which was weakly correlated with the non-peroxide antibacterial activity, was isolated from the flavonoid fraction and separately synthesised. 1 did not display inhibitory activity against Staphylococcus aureus in vitro and thus the origin of the correlation, which is still unknown, is not a direct contribution.     

Distribution of quercetin-3,4′-O-diglucoside,quercetin-4′-O-monoglucoside,and quercetin in different parts of the onion bulb (Allium cepa L.) influenced by genotype

Flavonoids have gained much attention because of their proposed positive effects for human health. Onions are a rich source of flavonoids, consisting mainly of the major flavonols quercetin-3,4′-O-diglucoside (QDG) and quercetin-4′-O-monoglucoside (QMG) in the bulb and the aglycone quercetin in the outer scales. In this study, distribution of these three flavonoids was determined in 16 onion cultivars (Allium cepa) using HPLC–DAD. Three different parts of the onion bulb, the inner layers, the middle layers and the outer scales were analysed. The analysis showed varietal differences in the selected onion cultivars. The cultivars with the highest total flavonoid content were the red skinned ‘Red Baron’ and the yellow skinned cultivars ‘Ailsa Craig’ and ‘Prilep’. The distribution of the total flavonoid content in the different parts of the onion bulb showed the following order: middle layers > outer scales > inner layers. In the inner layers QDG was the major flavonoid, while in the middle layers QDG and QMG were in equal amounts. In the outer scales quercetin was the major flavonoid prior to QMG.     

蜂蜜中原蜜黄酮类化合物HPLC图谱比较分析

利用高效液相色谱比较分析不同蜂种、不同产地、加工与否以及不同蜜源蜂蜜中原蜜黄酮类化合物的种类及含量差异。结果表明,意大利蜜蜂油菜原蜜和中华蜜蜂油菜原蜜总峰数和峰形都比较相似,意大利蜜蜂油菜原蜜的总黄酮含量比中华蜜蜂油菜原蜜总黄酮含量高。不同产地的意大利蜜蜂油菜原蜜的图谱峰形整体相似,总黄酮含量和黄酮类化合物种类大致相同。加工后的商品蜜总黄酮含量减少,黄酮类化合物的种类也减少,商品蜜的高效液相色谱图显示其黄酮类化合物的出峰时间靠前,主要密集分布在水溶性的分离相中。3个不同蜜源蜂蜜原蜜黄酮类化合物高效液相色谱图的总峰数和峰形都有较大差异,油菜原蜜、洋槐原蜜及柑橘原蜜的总峰数分别为65、58、70个,柑橘原蜜的总黄酮含量最高,达161.62μg/100 g。单花蜂蜜原蜜黄酮类化合物的高效液相色谱图峰形具有一定的特异性,可以作为指纹图谱用于蜂蜜蜜源的鉴定。     

Development of an HS-SPME-GC method to determine the methyl anthranilate in Citrus honeys

An efficient and simple method for determination of methyl anthranilate (MA) in Citrus spp. honeys by headspace-solid-phase microextraction-gas chromatography (HS-SPME-GC) was developed and validated. Experimental design was used to investigate the effects of the principal extraction parameters. The central composite design (CCD) and the desirability function were used to find the experimental conditions providing the optimal HS-SPME result. Validation was carried out in terms of specificity, linearity, limit of detection (LOD) and quantitation (LOQ) (0.149 and 0.324 μg/g, respectively), method precision (RSD 7%), LOQ precision (RSD 6.5%), and was resulted accurate and robust. Finally, the applicability of the method to the determination of MA in a number of commercial Citrus spp. honey samples was demonstrated, the content ranged from 0.63 to 3.26 μg/g.     

Evaluation of the phenolic content,antioxidant activity and colour of Slovenian honey

Honey samples from the seven most common honey types in Slovenia were screened for total phenolic content by the modified Folin–Ciocalteu method, for potential antioxidant activity using the ferric reducing antioxidant power (FRAP) assay and by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method for antiradical activity. In addition the colour characteristics of honey samples were analysed. The results of the study showed that total phenolic content, antioxidant activity and colour parameters differ widely among different honey types. Phenolic content expressed as gallic acid equivalent ranged from 44.8 mg/kg in acacia honey to 241.4 mg/kg in fir honey. Antioxidant activity was the lowest in the brightest acacia and lime honeys and the highest in darker honeys, namely fir, spruce and forest. The colour of the Slovenian honeys, analysed in this study was very variable and ranged from pale yellow to dark brown. Correlations between the parameters analysed were found to be statistically significant (p < 0.05).     

Storage-induced chemical changes in active components of honey de-regulate its antibacterial activity

To elucidate reasons for the observed variability in the antibacterial activity of honeys, we analysed a causal relationship between (a) honey floral sources and the activity and (b) the effect of honey storage on stability of compounds conferring this activity. Honeys from diverse floral sources were screened against Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633) using the broth microdilution method. Among “active” honeys, 37% originated from buckwheat, 18% from clover and 12% from blueberry, indicating that these floral sources produced phytochemical(s) that inhibited bacterial growth. The stability of the putative phytochemical(s) was analysed in “active” honeys (MIC₉₀ 6.25% v/v) by measuring the activity every 3–6 months for a period of 1–3 years. A sharp decline in activity against both bacteria was observed in the first 3–6 months of storage. The decline coincided with major changes in chemical composition of honeys which included a significant change in colour (p < 0.0025), extremely significant change in concentration of UV-absorbing compounds (p < 0. 0001) and appearance of melanoidins. While these changes reduced E. coli sensitivity to honey, it rendered B. subtilis completely insensitive. Thus, the data indicates that the presence of phytochemical(s) conferring the antibacterial activity is sensitive to storage. The de-regulation of the antibacterial activity with the concomitant appearance of melanoidins suggests that the active phytochemical components might be sequestered into melanoidin aggregates, losing their function.     

HPLC-fluorimetric method for analysis of amino acids in products of the hive (honey and bee-pollen)

An optimization of the OPA method has made feasible separation and quantification of 23 amino acids, which include 5 infrequently searched for. Detection limits ranged from 0.24 to 10.1 pmol in honey and from 29.1 to 0.42 pmol in bee-pollen; reproducibility (C.V.) ranged from 5.3% to 20.4%; recoveries were above 78.8%. Forty monovarietal honey samples from ilex, oak, heather and chestnut-tree were analyzed for their free amino acid profiles. α-Aminoadipic acid and homoserine are reported for the first time in honeys. Thirty-two samples of Spanish bee-pollen, made of a majority of pellets from Cistus Ladanifer (67.1%) and Echium plantagineum (8.9%), were analyzed for their free and total amino acid profiles. Free γ-aminobutyric acid was extensively found with an average of 0.53 mg/g, while Hser and Orn were infrequent. Manually separated monofloral pellets from Cistus ladanifer and Echium plantagineum were analyzed for their free amino acid contents (including proline): 32.46 and 21.87 mg/g for the former and 22.18 and 12.23 mg/g for the latter. In contrast, the total amino acid percentage (on a dry weight basis) was 13.95% for Cistus ladanifer and 32.22% for Echium plantagineum.     

Physicochemical attributes and pollen spectrum of Portuguese heather honeys

The qualities of selected honey samples of “Serra da Lousã” (Portugal) from three consecutive harvests (20 samples from each harvest) were evaluated by determing the pollen spectrum and physicochemical attributes. The following determinations were carried out: moisture, electrical conductivity, hydroxymethylfurfural, diastase activity, pH, acidity (free, lactone and total), formol number, reducing sugars, apparent sucrose, insoluble material and ash. The samples were found to meet all major national and international honey specifications. Honeys were considered to be monofloral whenever the dominant pollen was found to be over 45% of total pollen. From the 60 studied samples, 70% were monofloral honeys from Erica sp., 17% monofloral honeys from Ericaceae (Erica sp. and Calluna vulgaris (L.) Hull) and 13% multifloral honeys with a high percentage of Erica sp.     

Evaluation of antioxidant activity and total phenolics of selected Czech honeys

Honey serves as a good source of natural antioxidants, which are effective in reducing the risk occurrence of heart disease, cancer, cataracts, different inflammatory processes and immune-system decline. In the fresh selected Czech honey samples originated mainly from the region North Moravia antioxidant activity and total polyphenol content were determined. A total of 40 honey samples (multifloral, lime, rape, raspberry, mixture and honeydew honeys) native to different stations gained in the period from May by August year 2006 were analysed. Total phenolics (TP) content was determined by the modified Folin-Ciocalteau method [TP was expressed as mg of gallic acid equivalent (GA eq.) per kg of honey]. For evaluation of the antioxidant activity (AOA) three different methods were used, the ferric reducing antioxidant power (FRAP) assay, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and the 2,2′-azinobis(3-ethylbenzothiazolin)-6-sulfphonate (ABTS) assay. AOA was expressed in mg ascorbic acid equivalent (AA eq.) per kg of honey. The results indicated that TP and AOA in Czech honey varied greatly between the honey kinds, location and time of the harvest. Average TP ranged from 89.9 mg GA eq. kg^-1 in lime honey to 215.2 mg GA eq. kg⁻¹ in honeydew honey. Antioxidant activity determined by the DPPH, ABTS and FRAP methods was lowest in floral honeys. The highest values were obtained for honeydew and mixture honeys. ABTS and FRAP assays have been shown to be the optimal methods for AOA determination in honey. A positive linear correlation between AOA and TP was observed (in FRAP assay R² = 0.852). It indicates that phenolics are one of the main components responsible for antioxidant behaviour of honey. The obtained results support and extend complete knowledge about the content of bioactive phenolics and antioxidant activity in the Czech honeys.     

Structure-dependent membrane interaction of flavonoids associated with their bioactivity

Plant foods contain various flavonoids with nutraceutical and health benefits. Structurally different flavonoids were compared by the potency to interact with liposomal membranes in the context of their mode of action. A series of fluorescence polarisation measurements showed that flavonoids (1–10 μM) structure-dependently acted on the deeper regions of lipid bilayers to decrease membrane fluidity. Their comparative effects on cell-mimetic membranes, consisting of unsaturated phospholipids and cholesterol, characterised the structure–membrane interactivity relationship: 3-hydroxylation of the C ring, non-modification of the B ring and 5,7-dihydroxylation of the A ring led to the greatest membrane interactivity, followed by 3′,4′-dihydroxylation of the B ring. Galangin and quercetin, meeting such a structural requirement, inhibited the proliferation of tumour cells at 10–100 μM, together with rigidifying cell membranes, but not membrane-inactive flavonoids. The structure-dependent membrane interaction, which modifies the fluidity, is mechanistically associated with flavonoid bioactivity in a membranous lipid phase.     

Flavonoids in Meliponinae honeys from Venezuela related to their botanical, geographical and entomological origin to assess their putative anticataract activity

 Honey was collected from 24 stinglessbee nests in Venezuela. The flavonoid compounds in the phenolic extracts were analysed and related to the botanical, geographical and entomological origin of the honey. Honeys produced in savannas were richer in flavonoids compared with honeys from the forests. It was found that phenolic extracts of honey of the same geographical origin had similar flavonoid profiles, whereas the same could not be said of honeys of the same entomological origin, although the botanical origin of the samples was variable. It is proposed that analysis of stinglessbee honey eyedrops in terms of their flavonoid content can be used as a basis of authenticating and controlling for their geographical origin. Received: 28 July 1997