Zvonimir Marijanović

Headspace, volatile and semi-volatile organic compounds diversity and radical scavenging activity of ultrasonic solvent extracts from Amorpha fruticosa honey samples.

Volatile organic compounds of Amorpha fruticosa honey samples were isolated by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry analyses (GC, GC-MS), in order to obtain complementary data for overall characterization of the honey aroma. The headspace of the honey was dominated by 2-phenylethanol (38.3–58.4%), while other major compounds were trans- and cis-linalool oxides, benzaldehyde and benzyl alcohol. 2‑Phenylethanol (10.5–16.8%) and methyl syringate (5.8–8.2%) were the major compounds of ultrasonic solvent extracts, with an array of small percentages of linalool, benzene and benzoic acid derivatives, aliphatic hydrocarbons and alcohols, furan derivatives and others. The scavenging ability of the series of concentrations of the honey ultrasonic solvent extracts and the corresponding honey samples was tested by a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Approximately 25 times lower concentration ranges (up to 2 g/L) of the extracts exhibited significantly higher free radical scavenging potential with respect to the honey samples.

Oak (Quercus frainetto Ten.) honeydew honey - approach to screening of volatile organic composition and antioxidant capacity (DPPH and FRAP assay).

Two samples of oak honeydew honey were investigated. Headspace solid-phase microextraction (HS-SPME) combined with GC and GC/MS enabled identification of the most volatile organic headspace compounds being dominated by terpenes (mainly cis- and trans-linalool oxides). The volatile and less-volatile organic composition of the samples was obtained by ultrasonic assisted extraction (USE) with two solvents (1:2 (v/v) pentane -diethyl ether mixture and dichloromethane) followed by GC and GC/MS analysis. Shikimic pathway derivatives are of particular interest with respect to the botanical origin of honey and the most abundant was phenylacetic acid (up to 16.4%). Antiradical activity (DPPH assay) of the honeydew samples was 4.5 and 5.1 mmol TEAC/kg. Ultrasonic solvent extracts showed several dozen times higher antiradical capacity in comparison to the honeydew. Antioxidant capacity (FRAP assay) of honeydew samples was 4.8 and 16.1 mmol Fe2+/kg, while the solvent mixture extracts showed antioxidant activity of 374.5 and 955.9 Fe2+/kg, respectively, and the dichloromethane extracts 127.3 and 101.5 mmol Fe2+/kg.

Organic Extractives from Mentha spp. Honey and the Bee-Stomach: Methyl Syringate, Vomifoliol, Terpenediol I, Hotrienol and Other Compounds

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.

Screening of natural organic volatiles from Prunus mahaleb L. honey: coumarin and vomifoliol as nonspecific biomarkers.

Headspace solid-phase microextraction (HS-SPME; PDMS/DVB fibre) and ultrasonic solvent extraction (USE; solvent A: pentane and diethyl ether (1:2 v/v), solvent B: dichloromethane) followed by gas chromatography and mass spectrometry (GC, GC-MS) were used for the analysis of Prunus mahaleb L. honey samples. Screening was focused toward chemical composition of natural organic volatiles to determine if it is useful as a method of determining honey-sourcing. A total of 34 compounds were identified in the headspace and 49 in the extracts that included terpenes, norisoprenoids and benzene derivatives, followed by minor percentages of aliphatic compounds and furan derivatives. High vomifoliol percentages (10.7%–24.2%) in both extracts (dominant in solvent B) and coumarin (0.3%–2.4%) from the extracts (more abundant in solvent A) and headspace (0.9%–1.8%) were considered characteristic for P. mahaleb honey and highlighted as potential nonspecific biomarkers of the honey’s botanical origin. In addition, comparison with P. mahaleb flowers, leaves, bark and wood volatiles from our previous research revealed common compounds among norisoprenoids and benzene derivatives.

Volatile Compounds of Asphodelus microcarpusSalzm. et Viv. Honey Obtained by HS-SPME and USE Analyzed by GC/MS

Chemical analysis of Asphodelus microcarpus Salzm. et Viv. honey is of great importance, since melissopalynology does not allow the unambiguous determination of its botanical origin. Therefore, the volatile compounds of eight unifloral asphodel honeys have been investigated for the first time. The honey extracts were obtained by headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE) and analyzed by GC and GC/MS. In the honey headspace, 31 volatile compounds were identified with high percentages of 2‐phenylacetaldehyde (2; 14.8–34.7%), followed by somewhat lower percentages of methyl syringate (1; 10.5–11.5%). Compound 2 is not a specific marker of the botanical origin of the honey, but its high percentage can be emphasized as headspace characteristic of asphodel honey. The extraction solvent for all the samples was selected after extracting a representative sample with pentane, Et2O, pentane/Et2O 1 : 2 (v/v), and CH2Cl2. Compound 1 was the major constituent of all the USE extracts (46.8–87.0%). According to these preliminary results, all the honey samples were extracted by USE with the solvent pentane/Et2O 1 : 2. A total of 60 volatile compounds were identified with 1 as predominant compound (69.4–87.0%), pointing out 1 as Asphodelus honey volatile marker.

Contribution of the Bees and Combs to Honey Volatiles: Blank‐Trial Probe for Chemical Profiling of Honey Biodiversity

This research is focused on the immediate contribution of the bees and combs to honey volatiles in order to exclude these compounds as botanical‐origin biomarkers for honey authentification. Therefore, the bees were closed in a hive containing empty combs under controlled food‐flow conditions (saccharose solution). The obtained ‘saccharose honey’ probe samples were subjected to ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry analyses (GC and GC/MS). A total of 66 compounds were identified. Higher alcohols made up ca. 50% of the total volatiles, mainly (Z)‐octadec‐9‐en‐1‐ol, hexadecan‐1‐ol, and octadecan‐1‐ol, with minor percentages of undecan‐1‐ol, dodecan‐1‐ol, tetradecan‐1‐ol, pentadecan‐1‐ol, and heptadecan‐1‐ol. Other abundant compounds were saturated long‐chain linear hydrocarbons, C10–C25, C27, and C28, particularly C23, C25, and C27). Identified chemical structures were related to the composition of combs and cuticular waxes, and less to the bee pheromones. In addition, the impact of two‐hour heat treatment at 80° and one‐year storage at room temperature on the same probe was investigated in order to identify thermal and storage artefacts. These findings can be considered as blank‐trial probe (no plant source) for honey chemical profiling and identification of reliable botanical origin biomarkers.

Screening of volatile composition of Lavandula hybrida Reverchon II honey using headspace solid-phase microextraction and ultrasonic solvent extraction.

The volatiles of unifloral Lavandula hybrida Reverchon II honey were isolated by means of headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) and analyzed by gas chromatography and mass spectrometry (GC, GC/MS). A total of 23 compounds were identified in the headspace with hexan-1-ol, hexanal, acetic acid, hotrienol, and 2-phenylacetaldehyde as the principal components. Three solvents of different polarity were used for USE, and a total of 53 compounds were identified. The extracts with pentane/Et(2)O 1 : 2 (v/v) were the most representative for USE method containing the majority of the honey floral origin compounds and potential biomarkers (hexanol, acetic acid, butane-1,3-diol, butane-2,3-diol, benzoic acid, coumarin, and 2-phenylacetic acid). The total number of identified compounds (USE and HS-SPME) was 59. In general, the comparison with volatiles of other lavandin honeys of different geographic origins indicated several similarities, while acetic and formic acids were identified with high percentages in L. hybrida Reverchon II honey.

Cornflower (Centaurea cyanus L.) honey quality parameters: Chromatographic fingerprints, chemical biomarkers, antioxidant capacity and others

The samples of cornflower (Centaurea cyanus L.) honey from Poland were subjected to ultrasonic solvent extraction applying the mixture of pentane and diethyl ether 1:2v/v (solvent A) as well as dichloromethane (solvent B). The major compounds of the extracts (analysed by GC-MS/GC-FID) were C13 and C9 norisoprenoids. Among them, (E)-3-oxo-retro-α-ionol (2.4-23.9% (solvent A); 3.9-14.4% (solvent B)) and (Z)-3-oxo-retro-α-ionol (3.7-29.9% (solvent A); 8.4-20.4% (solvent B)) were found to be useful as chemical biomarkers of this honey. Other abundant compounds were: methyl syringate (0.0-31.4% (solvent A); 0.0-25.4% (solvent B)) and 3-hydroxy-4-phenylbutan-2-one (1.6-15.8% (solvent A); 5.1-15.1% (solvent B)). HPLC-DAD analysis of the samples revealed lumichrome (4.7-10.0mg/kg), riboflavin (1.9-2.7mg/kg) and phenyllactic acid (112.1-250.5mg/kg) as typical compounds for this honey type. Antioxidant and antiradical properties as well as total phenolic content of the samples were found to be rather moderate by FRAP (ferric reducing antioxidant power), DPPH (1,1-diphenyl-2-picrylhydrazyl radical) and Folin-Ciocalteu assays, respectively. Additionally, CIE L(∗)a(∗)b(∗)C(∗)h chromatic coordinates were evaluated. Colour attributes of cornflower honey were characterised by elevated values of L(∗) and particularly high values of b(∗) and h coordinates, which correspond to medium bright honey with intense yellow colour.

Volatiles from a Rare Acer spp. Honey Sample from Croatia

A rare sample of maple (Acer spp.) honey from Croatia was analysed. Ultrasonic solvent extraction (USE) using: 1) pentane, 2) diethyl ether, 3) a mixture of pentane and diethyl ether (1:2 v/v) and 4) dichloromethane as solvents was applied. All the extracts were analysed by GC and GC/MS. The most representative extracts were 3) and 4). Syringaldehyde was the most striking compound, being dominant in the extracts 2), 3) and 4) with percentages 34.5%, 33.1% and 35.9%, respectively. In comparison to USE results of other single Croatian tree honey samples (Robinia pseudoacacia L. nectar honey, Salix spp. nectar and honeydew honeys, Quercus frainetto Ten. honeydew as well as Abies alba Mill. and Picea abies L. honeydew) and literature data the presence of syringaldehyde, previously identified in maple sap and syrup, can be pointed out as a distinct characteristic of the Acer spp. honey sample. Headspace solid-phase microextraction (HS-SPME) combined with GC and GC/MS identified benzaldehyde (16.5%), trans-linalool oxide (20.5%) and 2-phenylethanol (14.9%) as the major compounds that are common in different honey headspace compositions.

Volatile Composition Screening of Salix spp. Nectar Honey: Benzenecarboxylic Acids, Norisoprenoids, Terpenes, and Others

Salix spp. nectar honey volatiles of Croatian origin were analyzed by headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE), followed by gas chromatography and mass spectrometry (GC, GC/MS). Isolated volatiles were found in the honey headspace and extracts with almost exclusive distribution of several abundant compounds (e.g., phenylacetic acid, pinocembrin, 8‐hydroxy‐4,7‐dimethylcoumarin, and 3‐hydroxy‐trans‐β‐damascone in the extracts, or safranal and lilac alcohols in the headspace). Comparison with Croatian Salix spp. honeydew honey revealed similarities regarding distribution of important shikimate pathway derivatives (e.g., high percentage of phenylacetic acid) and several norisoprenoids (α‐isophorone and 4‐oxoisophorone). On the other hand, distinct features of this honey were occurrence of compounds such as pinocembrin, 8‐hydroxy‐4,7‐dimethylcoumarin, phenylacetonitrile, norisoprenoids (major ones: 3‐hydroxy‐trans‐β‐damascenone and trans‐β‐damascone), more pronounced variability of linalool‐derived compounds, as well as the abundance of 3‐methylpropanoic acid, 3‐methylbutanoic acid, 2‐methylpentanoic acid, and 3‐methylpentan‐1‐ol.