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Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

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Himalayan balsam (Impatiens glandulifera Royle) is an invasive garden ornamental plant species originating from Asia, which produces significant amounts of nectar. In Croatia, it is widely distributed along the banks of the Mura River. Although this plant species is widespread in Europe, there are still no available scientific data about this unifloral honey type. The results showed that Himalayan balsam honey is characterized by the high presence of pollen grains in the pollen spectrum (59−85%), natural higher diastase activity (39.1 ± 7.98 DN), negative specific rotation (−21.2° ± 6.89) and an extra light amber color (48.5 ± 12.69 mm Pfund). The carbohydrate profile is characterized by monosaccharides fructose (39.34 ± 0.65 g/100 g) and glucose (31.91 ± 1.42 g/100 g) with a ratio >1.23, while the most commonly represented disaccharide was maltose (3.04 ± 0.79 g/100 g). The average total phenolic content was 130.97 ± 11.17 mg gallic acid/kg honey, and the average antioxidant capacity value was 225.38 ± 29.58 µM Fe(II). The major mineral element was K, with an average of 533.92 ± 139.70 mg/kg. The sensory profile was characteristic with a light orange color and medium-intensity odor and aroma. The crystallization rate was moderate and characterized by the appearance of opalescence and gelatinous forms of crystals. The results of this study provide the first insight into the melissopalynological, physico-chemical and sensory profile of Himalayan balsam honey.

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Strawberry tree (Arbutus unedo L.) honey (STH), also known as "bitter honey", is a traditional medicine widely used in the Mediterranean area. Regardless of geographical origin, it usually has a very high content of phenolic compounds and strong antioxidant capacity. Yet, little is still known about the effects of STH, its phenolic extract (STHE), and its main bioactive compound - homogentisic acid (HGA) - at the cell level. The aim of this study was to estimate total phenolic content, DPPH radical scavenging activity, and ferric reducing antioxidant power of STH made in Croatia and investigate cytotoxic and pro-oxidative effects of STH, STHE and HGA on three human cell lines: tongue squamous cell carcinoma (CAL 27), hepatocellular carcinoma (HepG2), and epithelial colorectal adenocarcinoma cells (Caco-2) cells. These substances were tested at four concentrations (0.5-5× average human daily intake of STH) and over 30 min and 1 and 2 h. Croatian STH had a total phenolic content of 1.67 g gallic acid equivalents (GAE) per kg of honey, DPPH radical scavenging activity of 2.96 mmol Trolox equivalents (TE) per kg of honey, and ferric reducing antioxidant power (FRAP) of 13.5 mmol Fe2+ per kg of honey. Our results show no clear and consistent time- or concentration-dependent cytotoxicity in any of the cell lines. ROS levels in all the three cell types at almost all exposure times were not significantly higher than control. The most important observation is that the tested substances have low cytotoxicity and high biocompatibility, regardless of concentration, which is a good starting point for further research of their biological effects in other models.

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Quality and safety of food, including honey, is one of the leading priorities regarding residues of anthropogenic chemicals with proven adverse health effects. In total, 61 honey samples of known botanical origin were collected in period 2018-2019 from Croatian registered organic and conventional beekeepers (N = 16 organic and N = 45 conventional honey samples). Eleven trace metal(loid)s (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn; quantitated by ICP-MS), 24 antibiotics (LC-MS/MS, ELISA, microbiological inhibition test), six indicator PCBs (GC-MS/MS) and 121 pesticides (LC-MS/MS, GC-MS/MS) originating from environment and/or beekeeping practice were measured to assess possible differences in contaminant residues between organic and conventional honeys. All honey samples had contaminant residues below the legal maximum levels and are considered safe for consumers. However, 2/16 organic and 34/45 conventional honeys contained one or two synthetic acaricides (most frequently coumaphos), while other pesticides, antibiotics and PCBs were not quantified. Also, organic honey contained lower levels of coumaphos, amitraz and amitraz metabolite N-(2,4-dimethylphenyl) formamide than conventional honeys, on average. Higher levels of Cr (p = 0.006) were detected in organic compared to conventional chestnut honeys. This study pinpointed beehive disease control treatment as prominent pesticide residue source, which was to some extent reduced in organic honeys. Quantified pesticide and metal(loid) levels were within range or lower than reported in recent literature regarding honey of the same botanical origin.

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The paper examines the antiproliferative, antimicrobial and antioxidative effects of fir (Abies alba Mill.) honeydew honey from mountain region of Croatia (Gorski kotar) as a potential replacement for standard antibiotics and chemotherapeutic agents. Cell viability, annexin V assay and flow cytometry analysis served to analyse the antiproliferative effect on, apoptosis induction in and cell death of cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1, MIA PaCa-2 and normal diploid human fibroblasts (BJ). Antimicrobial activity was tested against Staphylococcus and Acinetobacter strains by agar well diffusion and microdilution assays. The DPPH˙ assay determined the radical scavenging activity, while mathematical models helped to evaluate the kinetic data of DPPH˙ inhibition. Antiproliferative effect on all tested cell lines and the prominent effect on normal diploid human fibroblasts (BJ), colorectal adenocarcinoma (SW620, metastatic) and breast epithelial adenocarcinoma (MCF-7, metastatic) was observable. The mechanisms of antiproliferative effect included accumulation of cells in the sub-G1 phase in all tested cells and induction of apoptosis in SW620 and MCF-7 cells predominantly. The antibacterial assays showed that antibiotic-resistant strains of both bacteria, including multi-resistant strain A. baumannii ATCC® BAA-1605™, were sensitive to all tested honey samples. Radical scavenging assay suggests that antioxidants present in the honey possess different radical suppressing abilities and that they react at different rates with radicals, thereby causing two steps of reaction. The results of the study indicate that Croatian fir honeydew honey has a therapeutic potential due to the strong biological activity and can serve to protect human health.

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Samples of Satsuma mandarin (Citrus unshiu Marc.) nectar, honey sac content and honey were analyzed by FTIR-ATR spectroscopy and reference methods. The spectral analysis allowed detection of the major chemical constituents in C. unshiu nectar-to-honey transformation pathway thus providing information on the intensity and location of the compositional changes occurring during this process. The preliminary results showed that in average more than one-third of sugar-related nectar-to-honey conversion takes place directly in the honey sac; the average sugar content (w/w) was 17.93% (nectar), 47.03% (honey sac) and 79.63% (honey). FTIR-ATR results showed great spectral similarity of analyzed honey samples and small degree variations in both sugar and water content in nectar samples. The spectral data revealed distinctive differences in the chemical composition of individual honey sac contents with the most intensive and complex absorption envelope in the spectral region between 1175 and 950cm-1 (glucose, fructose and sucrose absorption bands).

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Headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by GC-MS/FID, were applied for monitoring the nectar (NE)/honey-sac (HoS)/honey (HO) pathways of the headspace, volatiles, and semi-volatiles. The major NE (4 varieties of Citrus unshiu) headspace compounds were linalool, α-terpineol, 1H-indole, methyl anthranilate, and phenylacetonitrile. Corresponding extracts contained, among others, 1H-indole, methyl anthranilate, 1,3-dihydro-2H-indol-2-one and caffeine. The major HoS headspace compounds were linalool, α-terpineol, 1,8-cineole, 1H-indole, methyl anthranilate, and cis-jasmone. Characteristic compounds from HoS extract were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl anthranilate, and phenylacetonitrile. However, HO headspace composition was significantly different in comparison to NE and HoS with respect to phenylacetaldehyde and linalool derivatives abundance that appeared as the consequence of the hive conditions and the bee enzyme activity. C. unshiu honey traceability is determined by chemical markers: phenylacetaldehyde, phenylacetonitrile, linalool and its derivatives, as well as 1H-indole, 1,3-dihydro-2H-indol-2-one, and caffeine.

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Riboflavin (vitamin B(2)) and its metabolite lumichrome were quantified in 117 samples from 11 unifloral honeys types (Arbutus unedo L., Asphodelus microcarpus Salzm. et Viv., Citrus spp., Eucalyptus spp., Hedysarum coronarium L., Castanea sativa L. honeydew, Mentha spp., Paliurus spina-christi., Salix spp., Salvia officinalis L., Satureja spp.). The quantification of these two compounds was performed by LC-DAD method which does not require sample purification. The proposed method in our study has low limits of detection and quantification, very good linearity in a large concentration range and very good precision. It allows simultaneous determination of 5-hydroxymethylfurfural (HMF) and known chemical biomarkers of unifloral honeys such as abscisic acid diastereomers, homogentisic acid, methyl syringate and kynurenic acid. No statistical correlation was observed between riboflavin and lumichrome content. Although, the concentration of vitamin B(2) in honey may be too low (<6.1mg/kg) to generate interest in the field of nutrition, the presence of its main metabolite lumichrome may be useful to determine the botanical origin of certain unifloral honeys. In fact, the analysis of 11 unifloral honey types showed that Dalmatian sage (S. officinalis L.) honey is characterised by unusual high levels of lumichrome (20.2±2.6mg/kg). The botanical origin of lumichrome from sage flower was assessed by analysing bee-stomach extracts. Other analytical parameters, such as total phenols, antioxidant and antiradical activities, HMF and diastase activity were studied in Dalmatian sage honey.

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Samples of unifloral sulla (Hedysarum coronarum L.) honey from Sardinia (Italy) were analysed. To investigate the chemical composition of the honey volatiles two solvent systems were used for ultrasonic solvent extraction (USE): 1) a 1:2 (v/v) pentane and diethyl ether mixture and 2) dichloromethane. All the extracts were analysed by GC and GC/MS. These procedures have permitted the identification of 56 compounds that include norisoprenoids, benzene derivatives, aliphatic compounds and Maillard reaction products. Norisoprenoids were the major compounds in both extracts, dominated by vomifoliol (5.3-11.2%; 9.6-14.0%) followed by minor percentages of other norisoprenoids such as α-isophorone, 4-ketoisophorone, 3-oxo-α-ionol or 3-oxo-α-ionone. Other abundant single compounds in the extracts were 3-hydroxy-4-phenylbutan-2-one (0.8-5.4%; 0.6-5.7%) and methyl syringate (3.0-5.7%; 2.2-4.1%). The composition of the volatiles and semi-volatiles in the obtained extracts suggests that sulla honey is quite distinctive relative to the other honeys that have been chemically studied by GC/MS, but no specific markers of the honey botanical origin were found.

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The GC and GC/MS analyses of the solvent organic extractive from the stomach of the bees, having collected Mentha spp. nectar, revealed the presence of methyl syringate (6.6%), terpendiol I (5.0%) and vomifoliol (3.0%) that can be attributed to the plant origin. Other major compounds from the bee-stomach were related to the composition of cuticular waxes and less to pheromones. Organic extractives from Mentha spp. honey were obtained by solvent-free headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) and analyzed by GC and GC/MS. The major honey headspace compounds were hotrienol (31.1%-38.5%), 2-methoxy-4-methylphenol (0.5-6.0%), cis- and trans-linalool oxides (0.9-2.8%), linalool (1.0-3.1%) and neroloxide (0.9-1.9%). Methyl syringate was the most abundant compound (38.3-56.2%) in the honey solvent extractives followed by vomifoliol (7.0-26.6%). Comparison of the honey organic extractives with the corresponding bee-stomach extractive indicated that methyl syringate and vomofoliol were transferred to the honey while terpendiol I was partially transformed to hotrienol in ripened honey.

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Oxidant/antioxidant status, estrogenic/anti-estrogenic activity and gene expression profile were studied in mice fed with Cystus incanus L. (Cistaceae) reach bee pollen from location in Central Croatia's Dalmatia coast and offshore islands. Seven phenolic compounds (out of 13 tested) in bee pollen sample were detected by high performance liquid chromatography (HPLC) analysis. Phenolics detected in C. incanus L. bee pollen belong to flavonol (pinocembrin), flavanols (quercetin, kaempferol, galangin, and isorhamnetin), flavones (chrysin) and phenylpropanoids (caffeic acid). Bee pollen as a food supplement (100mg/kgbw mixed with commercial food pellets) compared to control (commercial food pellets) modulated antioxidant enzymes (AOE) in the mice liver, brain and lysate of erythrocytes and reduced hepatic lipid peroxidation (LPO). Bee pollen induced 25% of anti-estrogenic properties while no estrogenic activity was found. Differential gene expression profile analyses after bee pollen enriched diet identify underexpressed gene Hspa9a, Tnfsf6 (liver) and down-regulated gene expression of Casp 1 and Cc121c (brain) which are important in the apoptosis pathway and chemotaxis. These results indicate that used bee pollen possess a noticable source of compounds with health protective potential and antioxidant activity.

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