Piotr Marek Kuś

Terpenes in honey: occurrence, origin and their role as chemical biomarkers

The major developments and concepts in the study of terpenes (mainly monoterpenes) as well as C9-, C10-, C13-, C14- and C15- norisoprenoids in honey are summarized. Their occurrence and biosynthetic correlations (terpene transformations, particularly the generation of linalool derivatives as well as carotenoid degradation and the corresponding variety of norisoprenoids) are discussed considering the plant/nectar/bee-stomach composition and hive conditions (e.g. warm and acidic conditions that can lead to oxidative degradation of compounds). Terpenes up to C15 have been found as major compounds of the essential oils, but honeys from the same plant sources exhibit only partial similarity to the oil composition (e.g. lavender, sage or citrus essential oils/honeys). The formation of heat-derived or prolonged storage artefacts in honey (mainly the products of Maillard reactions and Strecker degradation reactions) influence the honey composition, and hotrienol is particularly labile among the chemical groups discussed. Stimulated by the challenges of fingerprinting methods and structure identification, exploratory studies (including our published results) on terpenes and norisoprenoids are summarized with emphasis on identifying specific or nonspecific chemical markers of the botanical origin of honey. In general, nonspecific biomarkers dominate in different honey types (e.g. monoterpenes: linalool, linalool oxide isomers or lilac alcohol/aldehyde isomers; norisoprenoids: isophorone and vomifoliol derivatives) while only a few specific biomarkers are found (e.g. anhydrolinalool oxide isomers, 3,4-dihydro-3-oxoedulan isomers, 3-oxo-retro-α-ionol isomers, kamahine A-C or meliracemoic acid).

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.

Volatile Profile, Phytochemicals and Antioxidant Activity of Virgin Olive Oils from Croatian Autochthonous Varieties Mašnjača and Krvavica in Comparison with Italian Variety Leccino

Virgin olive oils (VOOs) obtained from the fruits of Croatian autochthonous varieties Mašnjača and Krvavica were extensively characterized for the first time. Investigated oils were compared with the oil obtained from Italian variety Leccino, grown and processed under the same conditions. Headspace volatile profile, tocopherols, chlorophylls, carotenoids and total phenolic content, peroxide value, % acidity, K232, K270 as well as antioxidant activity (DPPH) of the oils’ hydrophilic fractions (HFs) including their phenolic composition were assessed by means of HS-SPME/GC-MS, HPLC-FL, HPLC-DAD and spectrophotometric methods, respectively. Most of the studied quality parameters varied between the cultivars. The main volatile compounds detected in all tested olive oils were the C6 compounds derived from polyunsaturated fatty acids through the lipoxygenase pathway. Krvavica oil was characterized by hexanal (8.8%–9.4%). Leccino oil contained the highest percentage of (E)-hex-2-enal (73.4%–74.0%), whereas (Z)-hex-3-enal (21.9%–25.0%) and (E)-hex-2-enal (27.6%–28.9%) dominated in Mašnjača oil. Leccino oil contained the highest amount of tocopherols (312.4 mg/kg), chlorophylls (7.3 mg/kg), carotenoids (4.2 mg/kg) and total phenols (246.6 mg/kg). The HF of Leccino oil showed the highest antioxidant capacity (1.3 mmol TEAC/kg), while the HFs of Mašnjača and Krvavica oils exhibited the activity of 0.5 mmol TEAC/kg.

Phytochemical and physical-chemical analysis of Polish willow (Salix spp.) honey: identification of the marker compounds

The case study of Polish Salix spp. honey was compared with published data on willow honey from other regions. GC-FID/MS (after HS-SPME and ultrasonic solvent extraction) and targeted HPLC-DAD were applied. Phenolic content, FRAP/DPPH assays and the colour coordinates were determined spectrophotometrically. Beside ubiquitous linalool derivatives, borneol (up to 10.9%), bicyclic monoterpenes with pinane skeleton (pinocarvone up to 10.6%, myrtenal up to 4.8% and verbenone up to 3.4%) and trans-β-damascenone (up to 13.0%) dominated in the headspace. The main compounds of the extractives were vomifoliol (up to 39.6%) and methyl syringate (up to 16.5%) along with not common 4-hydroxy-3-(1-methylethyl)benzaldehyde (up to 11.1%). Abscisic acid (ABA) was found (up to 53.7 mg/kg) with the isomeric ratio (Z,E)-ABA:(E,E)-ABA=1:2. The honey exhibited low antioxidant potential with pale yellow colour. The composition of Polish willow honey is similar to Mediterranean willow honeys with several relevant differences.

Activity of Polish unifloral honeys against pathogenic bacteria and its correlation with colour, phenolic content, antioxidant capacity and other parameters

The use of honey as an antimicrobial agent gains importance due to often ineffectiveness of conventional treatment. However, activity of honey depends mainly on its botanical and geographical origin. To date, antimicrobial potential of Polish honeys has not yet been entirely investigated. In this study, 37 unifloral samples of 14 honey types (including rare varieties) from Poland were analysed and compared with manuka honey. The most active were cornflower, thyme and buckwheat honeys. Their MICs ranged from 3·12 to 25·00%, (depending on tested micro‐organism) and often were lower than for manuka honey. Additionally, colour, antioxidant activity, total phenols, pH and conductivity were assessed and significant correlations (P < 0·05) of MICs with several parameters were found. The most active were darker honeys, with strong yellow colour component, rich in phenolics, with high conductivity and water content. The honey antibacterial properties depended mainly on peroxide mechanism and were vulnerable to excessive heating, but quite stable during storage in cold. A number of honey samples showed potential as effective antimicrobial agents. The observed correlations of MICs and physical–chemical parameters help to understand better the factors impacting the antibacterial activity.

The volatile profiles of a rare apple (Malus domestica Borkh.) honey: shikimic acid-pathway derivatives, terpenes, and others.

The volatile profiles of rare Malus domestica Borkh. honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44–45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid‐pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H‐indole‐3‐acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car‐2‐en‐4‐one (10.2%). CH2Cl2 and pentane/Et2O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9–3.9%), benzoic acid (2.0–11.2%), terpendiol I (0.3–7.4%), coumaran (0.0–2.8%), 2‐phenylacetic acid (2.0–26.4%), methyl syringate (3.9–13.1%), vomifoliol (5.0–31.8%), and methyl 1H‐indole‐3‐acetate (1.9–10.2%). Apple honey contained also benzyl alcohol, 2‐phenylethanol, (E)‐cinnamaldehyde, (E)‐cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles.

Screening of Coffea spp. honey by different methodologies: theobromine and caffeine as chemical markers

Coffea spp. honey was screened by UV/VIS, HS-SPME/GC-MS/FID, USE/GC-MS/FID and HPLC-DAD. The direct HPLC-DAD methodology overcame the major limitations of the other methods used. The obtained results constitute a breakthrough since dominant xanthine derivatives were found (theobromine and caffeine with HPLC-DAD and caffeine by USE/GC-MS/FID). Phenylacetaldehyde was the major headspace compound.

Bioorganic diversity of rare Coriandrum sativum L. honey: unusual chromatographic profiles containing derivatives of linalool/oxygenated methoxybenzene.

The compounds responsible for highly individual aroma profile of Coriandrum sativum L. honey were isolated by headspace solid‐phase microextraction (HS‐SPME; used fibers: A: polydimethylsiloxane (PDMS)/divinylbenzene (DVB) and B: divinylbenzene/carboxen/polydimethylsiloxane), as well as ultrasonic solvent extraction (USE; used solvents: A: pentane/Et2O 1 : 2 (v/v) and B: CH2Cl2) and analyzed by gas chromatography (GC) and mass spectrometry (MS). Unusual chromatographic profiles were obtained containing derivatives of linalool/oxygenated methoxybenzene. trans‐Linalool oxide (11.1%; 14.6%) dominated in the headspace, followed by other linalool derivatives (such as cis/trans‐anhydrolinalool oxide (5.0%; 5.9%), isomers of lilac aldehyde/alcohol (14.9%; 13.8%) or p‐menth‐1‐en‐9‐al (15.6%; 18.5%)), octanal, and several low‐molecular‐weight esters. The major compounds in the solvent extracts were oxygenated methoxybenzene derivatives such as 3,4,5‐trimethoxybenzyl alcohol (26.3%; 24.7%), methyl syringate (23.8%; 11.7%), and 3,4‐dimethoxybenzyl alcohol (5.6%; 13.9%). Another group of abundant compounds in the extracts were derivatives of linalool (e.g., (E)/(Z)‐2,6‐dimethylocta‐2,7‐diene‐1,6‐diol (17.8%; 16.1%)). Among the compounds identified, cis/trans‐anhydrolinalool oxides and 3,4,5‐trimethoxybenzyl alcohol can be useful as chemical markers of coriander honey.

Comprehensive Study of Mediterranean (Croatian) Propolis Peculiarity: Headspace, Volatiles, Anti-Varroa-Treatment Residue, Phenolics, and Antioxidant Properties

Eight propolis samples from Croatia were analyzed in detail, to study the headspace, volatiles, anti‐Varroa‐treatment residue, phenolics, and antioxidant properties. The samples exhibited high qualitative/quantitative variability of the chemical profiles, total phenolic content (1,589.3–14,398.3 mg GAE (gallic acid equivalent)/l EtOH extract), and antioxidant activity (11.1–133.5 mmol Fe2+/l extract and 6.2–65.3 mmol TEAC (Trolox® equivalent antioxidant capacity)/l extract). The main phenolics quantified by HPLC‐DAD at 280 and 360 nm were vanillin, p‐coumaric acid, ferulic acid, chrysin, galangin, and caffeic acid phenethyl ester. The major compounds identified by headspace solid‐phase microextraction (HS‐SPME), simultaneous distillation extraction (SDE), and subsequent GC‐FID and GC/MS analyses were α‐eudesmol (up to 19.9%), β‐eudesmol (up to 12.6%), γ‐eudesmol (up to 10.5%), benzyl benzoate (up to 28.5%), and 4‐vinyl‐2‐methoxyphenol (up to 18.1%). Vanillin was determined as minor constituent by SDE/GC‐FID/MS and HPLC‐DAD. The identified acaricide residue thymol was ca. three times more abundant by HS‐SPME/GC‐FID/MS than by SDE/GC‐FID/MS and was not detected by HPLC‐DAD.

Screening of Polish fir honeydew honey using GC-MS, HPLC-DAD and physical-chemical parameters: benzene derivatives and terpenes as chemical markers

GC/MS of headspace solid phase micro extraction (HS‐SPME) and solvent extractives along with targeted HPLC‐DAD of Polish fir (Abies alba Mill.) honeydew honey (FHH), were used to determine the chemical profiles and potential markers of botanical origin. Additionally, typical physical‐chemical parameters were also assigned. The values determined for FHH were: conductivity (1.2 mS/cm), water content (16.7 g/100 g), pH (4.5), and CIE chromaticity coordinates (L* = 48.4, a* = 20.6, b* = 69.7, C* = 72.9, and h° = 73.5). FHH contained moderate‐high total phenolic content (533.2 mg GAE/kg) and antioxidant activity (1.1 mmol TEAC/kg) and (3.2 mmol Fe2+/kg) in DPPH and FRAP assays. The chemical profiles were dominated by source plant‐originated benzene derivatives: 3,4‐dihydroxybenzoic acid (up to 8.7 mg/kg, HPLC/honey solution), methyl syringate (up to 14.5%, GC/solvent extracts) or benzaldehyde (up to 43.7%, GC/headspace). Other markers were terpenes including norisoprenoid (4‐hydroxy‐3,5,6‐trimethyl‐4‐(3‐oxobut‐1‐enyl)cyclohex‐2‐en‐1‐one, up to 20.3%, GC/solvent extracts) and monoterpenes, mainly linalool derivatives (up to 49%, GC/headspace) as well as borneol (up to 5.9%, GC/headspace). The application of various techniques allowed comprehensive characterisation of FHH. 4‐Hydroxy‐3,5,6‐trimethyl‐4‐(3‐oxobut‐1‐enyl)cyclohex‐2‐en‐1‐one, coniferyl alcohol, borneol, and benzaldehyde were first time proposed for FHH screening. Protocatechuic acid may be a potential marker of FFH regardless of the geographical origin.