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Molecules 2017, 22(11), 1909; doi:10.3390/molecules22111909

Communication
Headspace Solid-Phase Microextraction and Ultrasonic Extraction with the Solvent Sequences in Chemical Profiling of Allium ursinum L. Honey
Igor Jerković 1,*Orcid and Piotr M. Kuś 2
1
Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, Rudera Boskovica 35, 21000 Split, Croatia
2
Department of Pharmacognosy, Wroclaw Medical University, ul. Borowska 211a, 50-556 Wroclaw, Poland
*
Correspondence: Tel.: +385-21-329-422; Fax: +385-21-329-461
Received: 17 October 2017 / Accepted: 4 November 2017 / Published: 6 November 2017

Abstract

:
A volatile profile of ramson (wild garlic, Allium ursinum L.) honey was investigated by headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) followed by gas chromatography and mass spectrometry (GC-FID/GC-MS) analyses. The headspace was dominated by linalool derivatives: cis- and trans-linalool oxides (25.3%; 9.2%), hotrienol (12.7%), and linalool (5.8%). Besides direct extraction with dichloromethane and pentane/diethyl ether mixture (1:2, v/v), two solvent sequences (I: pentane → diethyl ether; II: pentane → pentane/diethyl ether (1:2, v/v) → dichloromethane) were applied. Striking differences were noted among the obtained chemical profiles. The extracts with diethyl ether contained hydroquinone (25.8–36.8%) and 4-hydroxybenzoic acid (11.6–16.6%) as the major compounds, while (E)-4-(r-1′,t-2′,c-4′-trihydroxy-2′,6′,6′-trimethylcyclohexyl)but-3-en-2-one predominated in dichloromethane extracts (18.3–49.1%). Therefore, combination of different solvents was crucial for the comprehensive investigation of volatile organic compounds in this honey type. This particular magastigmane was previously reported only in thymus honey and hydroquinone in vipers bugloss honey, while a combination of the mentioned predominant compounds is unique for A. ursinum honey.
Keywords:
Allium ursinum L. honey; headspace solid-phase microextraction (HS-SPME); ultrasonic solvent extraction (USE) with the solvent sequence; (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one; hydroquinone; methyl syringate; 4-hydroxybenzoic acid

1. Introduction

Ramson (wild garlic, Allium ursinum L.) is a perennial plant, widely distributed in Europe. Phytochemical investigations of this plant revealed the presence of S-alk(en)yl-l-cysteine-sulfoxides (methiin, alliin, isoalliin, propiin, and ethiin) and their degradation products ((poly)sulfides, dithiins, or ajoenes) [1,2]. Apart from the abovementioned, various sulphur compounds have also been detected as constituents of its essential oil, e.g., disulfides, trisulfides, and tetrasulfides [3,4]. A. ursinum has been also reported to be a rich source of phenolic compounds (up to 27.9 g GAE (gallic acid equivalent)/100 g) [5]. Similar to organosulfur compounds, it was found to contain steroidal saponins that are also commonly found in the Allium genus [1,6]. Other identified constituents of interest include lectins, polysaccharides, and fatty acids [1]. A great number of in vitro and in vivo experiments showed that A. ursinum is a plant with antimicrobial, cytotoxic, antioxidant, and cardio-protective effects [1,7].
A. ursinum provides excellent spring bee pasture with a good nectar flow [8,9]. Allium species tend to secrete highly concentrated nectar, and the daily nectar production of A. ursinum ranged from 0.1 to 3.8 µL per flower, with sugar concentrations of 25% to 50%. However, the floral nectar volume and concentration varies in different populations of A. ursinum which can be also strongly affected by the varying conditions in different natural habitats. Nevertheless, the honey cannot be produced on a regular basis and its production is limited [8].
In continuation of the chemical fingerprinting of different unifloral honey types in search of specific or nonspecific chemical markers of botanical origin, the focus of this work was on not yet investigated volatile organic compounds (VOCs) of Allium ursinum L. honey of Croatian origin (a very rare sample). Headspace solid-phase microextraction (HS-SPME) followed by gas chromatography and mass spectrometry (GC-FID/GC-MS) analysis was applied to investigate its headspace chemical profile. To complement the honey profiling with data on less volatile organic compounds, ultrasonic solvent extraction (USE) was applied with solvents of different polarities, and the obtained extracts were analysed by GC-FID/GC-MS.

2. Results

A rare sample of A. ursinum honey from Croatia was confirmed to be unifloral according to performed mellisopalynological analysis. It contained 58% of Allium ursinum L. pollen grains accompanied by the pollen from Prunus spp. (19%), Acer spp. (14%), and a minor contribution from the grains of Salix spp. (2%), Fraxinus excelsior (1%), Tilia spp. (1%), Asteraceae (1%), Ericaceae (1%), and Brassicaceae (1%).
At the time of blooming, A. ursinum plants emit a strong garlic odour that can also be smelled in the nectar and in front of the beehives. However, it has been reported that the odour of the corresponding ripe honey is different, with a pleasant, particular aroma [8]. Therefore, significant differences among the chemical profiles obtained from A. ursinum honey VOCs and the corresponding plant VOCs were expected. To investigate in detail the headspace, volatile, and semi-volatile compounds from A. ursinum honey, up to-date complementary methodologies were applied: headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE) followed by GC-FID/GC-MS analyses. Striking differences were found among the chemical profiles obtained by those methods and the plant VOCs.

2.1. The Headspace Chemical Profile

The headspace of A. ursinum honey (Table 1) dominated with monoterpenes—linalool derivatives such as cis- and trans-linalool oxides (9.2%; 25.3%), hotrienol (12.7%), and linalool (5.8%).
Few benzene derivatives, often found in different honey types [10], were detected by HS-SPME with minor abundance, e.g., benzaldehyde (1.1%), phenylacetaldehyde (1.7%), 2-phenylethanol (3.0%), 4-methoxybenzaldehyde (1.1%), and phenylacetonitrile (1.9%). 4-Ketoisophorone (2.8%) was the only norisoprenoid detected in the headspace in distinction from the extracts. Dimethyl disulfide (1.2%) was the only headspace compound that could be connected to the plant VOCs (it was found in A. ursinum essential oil). The majority of the essential oil constituents, such as typical sulphides, disulfides, and trisulfides, were not present in the honey [3,4]. As was mentioned before, ripe ramson honey possesses a pleasant, particular aroma, and the probably typical sulfur volatile organic compounds were lost during the honey maturation in the hive. In addition, it is well known that honey VOCs usually significantly differ from the corresponding plant VOCs [11].

2.2. The Extracts Chemical Profile

Ultrasonic extraction (USE) of the honey was first performed separately with two solvents: (a) the mixture of pentane and diethyl ether (1:2, v/v) (A), and (b) dichloromethane (B), as in our previous research [12,13]. Significant differences were found among chemical profiles of the extracts (Table 2). The extract A contained 1,4-benzenediol (25.8%) as the major compound followed by a variety of benzene derivatives, particularly benzoic acid and its p-substituted derivatives: 4-hydroxybenzoic acid (16.4%), benzoic acid (4.4%), and 4-methoxybenzoic acid (3.7%). 4-Hydroxybenzaldehyde (10.3%) and methyl syringate (9.8%) were also quite abundant. In contrast, the extract B contained as the major compound C13-norisoprenoid (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one (18.3%), which was present only with 3.1% in the extract A. Aromatic compounds were present among the major compounds (similar as in the extract A: methyl syringate (12.2%), 4-hydroxybenzaldehyde (9.9%), 4-methoxybenzoic acid (4.2%), and benzoic acid (3.1%)). However, the major difference in 4-hydroxybenzoic acid and hydroquinone abundance was noted among two extracts (dominated in A). Both of them also contained other C13-norisoprenoids (solvent A; solvent B): 3-oxo-α-ionone (1.8%; 1.5%), vomifoliol (1.1%; 2.6%), and 3-oxo-7,8-dihydro-α-ionone (0.5%; 0.1%). Higher aliphatic compounds were present among minor constituents in both extracts as well as trans- or cis-linalool oxides (furan type).
Since significant differences were found in the obtained chemical profiles, previously applied USE was modified and two solvent sequences (sequence I: pentane (C) → diethyl ether (D); sequence II: pentane (C) → pentane:diethyl ether (1:2, v/v) (E) → dichloromethane (F)) were applied for the honey extraction and more complete profiling by the fractionation of compounds according to their distribution among the solvents of different polarities. Pentane extract (C) contained methyl syringate (26.2%) and docosane (23.0%) as the major compounds. cis- and trans-Linalool oxides (furan type) were the most abundant among all extracts (3.3%; 1.2%). 3-Hydroxy-4-phenylbutan-2-one was only present in this extract (2.8%). Higher aliphatic compounds were also present (Table 2). However typical compounds found in direct extracts with solvents A and B were not present. Diethyl ether extract (sequence I, D) contained hydroquinone (36.8%) and 4-hydroxybenzoic acid (16.6%) as the major constituents. (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one was present with 6.2%, methyl syringate with 3.0%, and 4-hydroxybenzaldehyde with 5.3%. Other C13-norisoprenoids were found with minor abundance, such as 3-oxo-α-ionone, 3-oxo-7,8-dihydro-α-ionone, and vomifoliol. Only trans-linalool oxide (furan type) was found. It can be seen that this extract was purified from less polar compounds by previous extraction with pentane (sequence I). The extract with pentane:diethyl ether (1:2, v/v) applied in sequence II (E) contained as major compounds 1,4-benzenediol (27.7%), docosane (14.0%), 5-hydroxymethylfurfural (13.3%), 4-hydroxybenzoic acid (11.6%), methyl syringate (6.6%), and (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one (3.3%). Other C13-norisoprenoids (3-oxo-α-ionone, 3-oxo-7,8-dihydro-α-ionone, and vomifoliol) were present. Lot of similarities were noted among diethyl ether extract in sequence I (D) and the extract with the mixture of pentane:diethyl ether (1:2, v/v) in sequence II (E) regarding the distribution of hydroquinone, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, and C13-norisoprenoids. The major difference was the abundance of docosane in the extract E (sequence II). Since dichloromethane extract in sequence II (F) was applied after pentane extraction and after the extraction with pentane:diethyl ether (1:2, v/v), it was expected to contain the least compounds of all the extracts (Table 2). However, this extract was dominated by (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one (49.1%), which could be useful for its isolation from the honey matrix. Such a result was also expected and it is in accordance with the data obtained from the direct extraction with dichloromethane. It is interesting to note that other C13-norisoprenoids were extracted with pentane:diethyl ether (1:2, v/v) previously applied in sequence II (E), and they were not present in dichloromethane extract (F). Docosane was the second major compound (25.9%) in this extract, followed by (Z)-octadec-9-en-1-ol (8.9%) and octadecan-1-ol (2.8%).
In comparison with HS-SPME (Table 1 and Table 2), only a few compounds were similar, while linalool and its derivatives were found with significantly lower abundance in the extracts than in the headspace. Epoxidation of linalool gives 6,7-epoxylinalool, which undergoes further reactions to form linalool oxides, while hotrienol is derived from hydroxylated linalool derivatives [11]. Higher abundance of linalool, cis-, and trans-linalool oxide were found in the headspace of Coriandrum sativum L. [14] and Citrus spp. [13,15,16] honey types. Regarding the extract chemical profiles, no major similarity was found among the profiles of other honey types. A combination of predominant compounds (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one, hydroquinone, methyl syringate, and 4-hydroxybenzoic acid is unique to A. ursinum honey. 1,4-Dihydroxybenzene was proposed as a floral marker compound for vipers bugloss (Echium vulgare L.) honey [17]. High proportions of benzoic acid and its derivatives were found in Salix spp. honeydew extractives [18], but with a minor percentage of 4-hydroxybenzoic acid. The latter was found abundant by HPLC in buckwheat (Fagopyrum esculentum L.) honey [19]. (E)-4-(r-1′,t-2′,c-4′-trihydroxy-2′,6′,6′-trimethylcyclohexyl)but-3-en-2-one contains a megastigmane structure. Structurally, megastigmanes are C13-carbon skeleton compounds, which are commonly classified as C13-norisoprenoids, also assumed to be apocarotenoides. Megastigmanes possess a unique basic skeleton with a six-membered ring with a double bond within the ring system, followed by methyl and dimethyl substitutions and an attached four membered chain with a double bond in the trans-mode [20]. The biosynthesis of this compound can be envisaged as proceeding via the alkene with a double bond within the ring system and via one or both of the epoxides [20]. Although a wide variety of degraded carotenoid-like substances have been identified from different honey types [13], this appears to be a rare situation where a trihydroxy ketone has been found. In fact, it was previously isolated and characterized by X-ray crystallographic analysis as a dominant substance from the ether extracts of New Zealand thyme honey [21]. Its recorded MS spectra were m/z 224 (6%), 141 (9), 140 (8), 125 (55), 124 (l2), 123 (18), 109 (8), 99 (7), 97 (23), 95 (6), 83 (9), 71 (13), 69 (8), 55 (17), 43 (96) and the reported data [21] on MS of (E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one were m/z 224 (6%), 141 (9), 140 (8), 125 (43), 124 (l0), 123 (17), 109 (8), 99 (7), 97 (23), 95 (6), 83 (9), 71 (13), 69 (8), 55 (17), 43 (100). This compound exerted significant apoptotic activity in PC-3 prostate cancer cells at 100 μM, while it inhibited NF-κB phosphorylation and IL-6 secretion at a concentration range of 10−6–10−4 M [22].

3. Materials and Methods

A rare and representative Allium ursinum L. honey sample was collected from a professional beekeeper in Croatia (more unifloral samples were not available). The sample was stored in a hermetically closed glass bottle at 4 °C until the volatiles were isolated. Melissopalynological analysis was performed according to the International Commission for Bee Botany [23]. Microscopical examination was carried out on a Hund H 500 light microscope (Helmut Hund GmbH, Wetzlar, Germany) attached to a digital camera (Motic m 1000, Motic Deutschland GmbH, Wetzlar, Germany) and coupled to an image analysis system (Motic Images Plus software, Motic Deutschland GmbH) for the morphometry of pollen grains.

3.1. Headspace Solid-Phase Microextraction (HS-SPME)

The headspace solid-phase extraction (HS-SPME) was performed using a manual SPME holder using polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber that was conditioned prior to the usage according to Supelco (Bellefonte, PA, USA) instructions. The honey/saturated water solution (5 mL, 1:1 (v/v); saturated with NaCl) was placed in a 15-mL glass vial and hermetically sealed with polytetrafluorethylene (PTFE)/silicone septa. The vial was maintained in a water bath at 60 °C during equilibration (15 min) and HS-SPME (45 min) under constant stirring (1000 rpm) with a magnetic stirrer, and the sample was kept below the water level of the water bath. After sampling, the SPME fiber was withdrawn into the needle, removed from the vial, and inserted into the injector (250 °C) of the GC-FID and GC-MS for 6 min, where the extracted volatiles were thermally desorbed directly to the GC column. The experiment was performed in triplicate.

3.2. Ultrasonic Solvent Extraction (USE)

Ultrasound-assisted solvent microextraction (USE) was performed in an ultrasound cleaning bath (Clean 01, MRC Scientific Instruments, London, UK) by the indirect sonication mode at a frequency of 37 kHz at 25 ± 3 °C. The advantage of using USE is the isolation of volatile and semi-volatile as well as water-soluble organic compounds without the application of heat. Different solvents were used for USE: a mixture of pentane/diethyl ether, 1:2 (v/v), dichloromethane, pentane, and diethyl ether. The mixture and dichloromethane were separately used for the extractions. A previously developed USE method was modified with the solvent sequences that were applied for the honey extraction. Sequence I consisted of the extraction with pentane followed by the extraction with diethyl ether (pentane → diethyl ether). Sequence II consisted of pentane extraction followed by the extraction with pentane:diethyl ether 1:2 (v/v) and afterwards with dichloromethane (pentane → pentane:diethyl ether 1:2 (v/v) → dichloromethane). For each extraction, 40 grams of the honey was dissolved in distilled water (22 mL) in a 100-mL flask. Magnesium sulfate (1.5 g) was added and vortexed (10 min). The solvent volume was 20 mL and the sonication was applied for 30 min. After the sonication, the organic layer was separated by centrifugation and filtered over anhydrous MgSO4. The aqueous layer was returned to the flask and another batch of the same extraction solvent was added and extracted for 30 min. The organic layer was separated in the same way as the previous layer and filtered over anhydrous MgSO4, and the aqueous layer was sonicated a third time for 30 min with another batch of the extraction solvent. Combined organic extracts were concentrated to 0.2 mL by distillation with a Vigreaux column, and 1 µL was used for GC-FID/GC-MS analyses. The experiments were performed in triplicate.

3.3. GC-FID and GC-MS Analyses

The GC-FID analyses were conducted with an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A equipped with a flame ionization detector (FID) and a HP-5MS capillary column (5% phenyl-methylpolysiloxane, Agilent J and W, Santa Clara, CA, USA). The GC conditions were described previously [13,18]. In brief, the oven temperature was programmed isothermal at 70 °C for 2 min, increasing from 70–200 °C at 3 °C·min−1, and held isothermally at 200 °C for 15 min; the carrier gas was He (1.0 mL·min−1); and the total run time was 65 min.
The GC-MS analyses were conducted with an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7820A equipped with a mass selective detector (MSD) model 5977E (Agilent Technologies) and a HP-5MS capillary column, under the same conditions as those described for the GC-FID analysis. The MSD (EI mode) was operated at 70 eV, and the mass range was 30–300 amu, as previously reported [13].
The identification was based on the comparison of VOC retention indices (RI), determined relative to the retention times of a homologous series of n-alkanes (C9–C25), with those reported in the literature and their mass spectra with authentic compounds available in our laboratories or those listed in Wiley 9 (Wiley, New York, NY, USA) and NIST 14 (D-Gaithersburg) mass spectral libraries. The percentage composition of the samples was computed from the GC peak areas using the normalization method (without correction factors). The average component percentages in Table 1 and Table 2 were calculated from duplicate GC-FID and GC-MS analyses.

4. Conclusions

The unusual chemical profile of A. ursinum honey was investigated and described for the first time. The headspace was dominated by linalool and its derivatives, which is not specific. The extracts showed remarkable variabilities according to the solvents applied, which is important to point out since the use of only one solvent could lead to incomplete results for A. ursinum honey. Namely, the extracts obtained with diethyl ether as the solvent contained 1,4-benzenediol and 4-hydroxybenzoic acid as the major compounds, while (E)-4-(r-1′,t-2′,c-4′-trihydroxy-2′,6′,6′-trimethylcyclohexyl)but-3-en-2-one predominated in the dichloromethane extracts. The applied sequence of solvents enabled the fractionation of the compounds according to polarity, and sequence II was useful for the concentration and possible isolation of (E)-4-(r-1′,t-2′,c-4′-trihydroxy-2′,6′,6′-trimethylcyclohexyl)but-3-en-2-one. More samples should be investigated to confirm these compounds as characteristic of this honey type.

Acknowledgments

This work has been supported by the Croatian Science Foundation under the project HRZZ-IP-11-2013-8547 and the Polish National Science Centre funding granted under the decision DEC-2014/15/N/NZ9/04058.

Author Contributions

I.J. and P.M.K. planned the research, conceived and designed the experiments. P.M.K. and I.J. performed the experiments. I.J. and P.M.K. analyzed the data. I.J. and P.M.K. wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sobolewska, D.; Podolak, I.; Makowska-Wąs, M. Allium ursinum: Botanical, phytochemical and pharmacological overview. Phytochem. Rev. 2015, 14, 81–97. [Google Scholar] [CrossRef] [PubMed]
  2. Kubec, R.; Svobodova, M.; Velisek, J. Distribution of S-alk(en)ylcysteine sulfoxides in some Allium species. Identification of a new flavour precursor: S-ethylcysteine sulfoxide (ethiin). J. Agric. Food Chem. 2000, 48, 428–433. [Google Scholar] [CrossRef] [PubMed]
  3. Gođevac, D.; Vujisić, L.; Mojović, M.; Ignjatović, A.; Spasojević, I.; Vajs, V. Evaluation of antioxidant capacity of Allium ursinum L. volatile oil and its effect on membrane fluidity. Food Chem. 2008, 107, 1692–1700. [Google Scholar] [CrossRef]
  4. Błazewicz-Woźniak, M.; Michowska, A. The growth, flowering and chemical composition of leaves of three ecotypes of Allium ursinum L. Acta Agrobot. 2011, 64, 171–180. [Google Scholar] [CrossRef]
  5. Gîtin, L.; Dinicã, R.; Parnavel, R. The influence of extraction method on the apparent content of bioactive compounds in Romanian Allium spp. leaves. Not. Bot. Horti Agrobot. Cluj Napoca 2012, 40, 93–97. [Google Scholar]
  6. Sobolewska, D.; Janeczko, Z.; Kisiel, W.; Podolak, I.; Galanty, A.; Trojanowska, D. Steroidal glycosides from the underground parts of Allium ursinum L. and their cytostatic and antimicrobial activity. Acta Pol. Pharm. Drug Res. 2006, 63, 219–223. [Google Scholar]
  7. Sendl, A. Allium sativum and Allium ursinum: Part 1. Chemistry, analysis, history, botany. Phytomedicine 1995, 1, 323–329. [Google Scholar] [CrossRef]
  8. Farkas, Á.; Zajácz, E. Nectar production for the Hungarian honey industry. Eur. J. Plant Sci. Biotechnol. 2007, 1, 121–151. [Google Scholar]
  9. Farkas, Á.; Molnár, R.; Morschhauser, T.; Hahn, I. Variation in nectar volume and sugar concentration of Allium ursinum L. ssp. ucrainicum in three habitats. Sci. World J. 2012, 2012, 138579. [Google Scholar] [CrossRef]
  10. Jerković, I. Volatile benezene derivatives as honey biomarkers. Synllet 2013, 24, 2331–2334. [Google Scholar] [CrossRef]
  11. Jerković, I.; Kuś, P.M. Terpenes in honey: Occurrence, origin and their role as chemical biomarkers. RSC Adv. 2014, 4, 31710–31728. [Google Scholar] [CrossRef]
  12. Jerković, I.; Kranjac, M.; Marijanović, Z.; Zekić, M.; Radonić, A.; Tuberoso, C.I.G. Screening of Satureja subspicata Vis. honey by HPLC-DAD, GC-FID/MS and UV/VIS: Prephenate derivatives as biomarkers. Molecules 2016, 21, 377. [Google Scholar] [CrossRef] [PubMed]
  13. Jerković, I.; Prđun, S.; Marijanović, Z.; Zekić, M.; Bubalo, D.; Svečnjak, L.; Tuberoso, C.I.G. Traceability of Satsuma mandarin (Citrus unshiu Marc.) honey through nectar/honey-sac/honey pathways of the headspace, volatiles, and semi-volatiles: Chemical markers. Molecules 2016, 21, 1302. [Google Scholar] [CrossRef] [PubMed]
  14. Jerković, I.; Obradović, M.; Kuś, P.M.; Šarolić, M. Bioorganic diversity of rare Coriandrum sativum L. honey: Unusual chromatographic profiles containing derivatives of linalool/oxygenated methoxybenzene. Chem. Biodivers. 2013, 10, 1549–1558. [Google Scholar] [CrossRef] [PubMed]
  15. Alissandrakis, E.; Tarantilis, P.A.; Harizanis, P.C.; Polissiou, M. Evaluation of four isolation techniques for honey aroma compounds. J. Sci. Food Agric. 2005, 85, 91–97. [Google Scholar] [CrossRef]
  16. Alissandrakis, E.; Tarantilis, P.A.; Harizanis, P.C.; Polissiou, M. Aroma investigation of unifloral Greek citrus honey using solid-phase microextraction coupled to gas chromatographic-mass spectrometric analysis. Food Chem. 2007, 100, 396–404. [Google Scholar] [CrossRef]
  17. Wilkins, A.L.; Tan, S.-T.; Molan, P.C. Extractable organic substances from New Zealand unifloral vipers bugloss (Echium vulgare) honey. J. Apicult. Res. 1995, 34, 73–78. [Google Scholar] [CrossRef]
  18. Jerković, I.; Marijanović, Z.; Tuberoso, C.I.G.; Bubalo, D.; Kezić, N. Molecular diversity of volatile compounds in rare willow (Salix spp.) honeydew honey: Identification of chemical biomarkers. Mol. Divers 2010, 14, 237–248. [Google Scholar] [CrossRef] [PubMed]
  19. Jasicka-Misiak, I.; Poliwoda, A.; Dereń, M.; Kafarski, P. Phenolic compounds and abscisic acid as potential markers for the floral origin of two Polish unifloral honeys. Food Chem. 2012, 131, 1149–1156. [Google Scholar] [CrossRef]
  20. Rao, A.S. Isolation, absolute configuration and bioactivities of megastigmanes or C13 isonorterpinoides. Chem. Int. 2017, 3, 69–91. [Google Scholar]
  21. Tan, S.T.; Wilkins, A.L.; Holland, P.T. Isolation and X-ray crystal structure of (E)-4-(r-l′,t-2′,c-4′-trihydroxy-2′,6′,6′-trimethylcyclohexy1)but-3-en-2-one, a constituent of New Zealand thyme honey. Aust. J. Chem. 1989, 42, 1799–1804. [Google Scholar] [CrossRef]
  22. Kassi, E.; Chinou, I.; Spilioti, E.; Tsiapara, A.; Graikou, K.; Karabournioti, S.; Manoussakis, M.; Moutsatsou, P. A monoterpene, unique component of thyme honeys, inducesapoptosis in prostate cancer cells via inhibition of NF-κB activityand IL-6 secretion. Phytomedicine 2014, 21, 1483–1489. [Google Scholar] [CrossRef] [PubMed]
  23. Louveaux, J.; Maurizio, A.; Vorwohl, G. Methods of melissopalynology. Bee World 1978, 59, 139–153. [Google Scholar] [CrossRef]
  • Sample Availability: The honey sample is available from the authors for limited time.
Table 1. The Headspace volatiles of the sample determined by HS-SPME/GC-MS.
Table 1. The Headspace volatiles of the sample determined by HS-SPME/GC-MS.
No.CompoundRI 1RI 2% 3NoCompoundRI 1RI 2% 3
1Dimethyl disulfide<9007471.214Hotrienol1106111012.7
2Butanoic acid<9007630.7152-Phenylethanol 4111611163.0
33-Methylbut-2-enal *<9007811.016Phenylacetonitrile114311411.9
4Octane<9008000.1174-Ketoisophorone114711472.8
53-Methylbutanoic acid<9008881.718Octanoic acid 4117611791.7
6Benzaldehyde 49659661.119Nonan-1-ol 4117811711.4
7Hexanoic acid 49809820.920trans-Linalool oxide (pyran type)118311831.5
8(E)-Hex-3-enoic 4 acid991/0.721α-Terpineol 4119411910.8
9(Z)-Hex-3-enoic acid101310130.8225-Hydroxymethylfurfural 4123012264.0
10Phenylacetaldehyde 4104810491.7234-Methoxybenzaldehyde 4125612581.1
11cis-Linalool oxide (furan type)1076107525.324Nonanoic acid 4127312764.0
12trans-Linalool oxide (furan type)109110919.2253,4,5-Trimethylphenol **1336-3.2
13Linalool 4110111015.826Hexadecanoic acid 4197019771.7
1 RI—retention indices on HP-5MS column relative to C9–C25 alkanes; 2 RI from the literature (National Institute of Standards and Technology (NIST) Chemistry WebBook, NIST Standard Reference Database Number 69, http://webbook.nist.gov/chemistry/); 3 Area percentages (%), 4 identification confirmed with standard compound; *—tentatively identified; **—correct isomer is not identified.
Table 2. The volatile organic compounds composition of the sample determined by ultrasonic solvent extraction (USE)/GC-FID; GC-MS.
Table 2. The volatile organic compounds composition of the sample determined by ultrasonic solvent extraction (USE)/GC-FID; GC-MS.
No.CompoundRI 1RI 2ABCDEF
% 3% 3% 2% 3% 3% 3
12-Furancarboxaldehyde<900835----0.60.1
24-Methyloctane<900/0.1-0.10.1--
31,3-Dimethylbenzene **<9008641.5-0.60.70.6-
42-Furanmethanol<900866----0.1-
5Ethylbenzene<9008680.2-0.60.20.1-
63-Methylbutanoic acid (Isovaleric acid) <900888----0.1-
73-Methylbut-2-enoic acid *<900/0.10.2-0.10.1-
8Ethenylbenzene<9008920.1--0.1--
91,2-Dimethylbenzene **<9008970.3-0.80.1--
10Methoxybenzene912/0.1--0.2--
112-Acetylfuran918914----0.1-
12Benzaldehyde 49659660.10.20.70.1--
135-Methylfurfural 4970966----0.1-
14(E)-Hex-3-enoic acid 4991/0.50.3-0.2--
15(Z)-Hex-3-enoic acid101310130.10.2-0.10.1-
16Pantolactone1044/0.10.2-0.10.10.2
17Phenylacetaldehyde 4104810490.10.20.60.1--
18Acetophenone 410651065---0.1--
19cis-Linalool oxide (furan type)107610750.70.33.30.21.30.1
20trans-Linalool oxide (furan type)109110910.20.21.2-0.4-
21Linalool 4110211010.1-----
22Hotrienol110611100.10.20.6---
232-Phenylethanol 4111611160.70.71.60.30.4-
242,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one 114311490.20.3-0.3--
25Benzoic acid 4118111784.43.12.02.21.20.3
26Terpendiol I119111910.70.50.70.3--
275-Hydrohymethylfurfural 1123012260.82.6-1.113.30.8
284-Methoxybenzaldehyde 4125912580.10.20.7-0.3-
29Phenylacetic acid 4126912700.80.8-0.60.1-
30Nonanoic acid 4127312760.10.20.7---
311,4-Benzenediol 4 (Hydroquinone)1328/25.82.4-36.827.70.7
323,4,5-Trimethylphenol **133613310.30.51.60.3-0.2
333-Hydroxy-4-phenylbutan-2-one13541348--2.8---
34Phenylpropanoic acid 4135913611.81.6-0.4--
351-Hydroxylinalool **1365/0.30.3--0.1-
364-Hydroxybenzaldehyde 41393/10.39.9-5.32.51.2
374-Hydroxy-3-methoxy-benzaldehyde (Vanillin) 4141213940.30.7----
384-Methoxybenzoic acid (p-Anisic acid) 4145214513.74.2-2.50.70.4
39(E)-3-Phenylprop-2-enoic acid (trans-Cinnamic acid) 4145514570.90.7-0.40.10.1
40Methyl 4-hydroxybenzoate 41482/0.20.3----
414-Hydroxy-phenylacetonitrile *1502/1.01.3-0.80.30.3
42Methyl 4-hydroxy-3-methoxybenzoate153015270.20.2----
434-Hydroxybenzoic acid 41558155816.40.2-16.611.6-
443,5,5-Trimethyl-4-(3-oxo-1-butenyl)cyclohex-2-en-1-one (3-Oxo-α-ionone)166516611.81.52.4-0.3-
45Syringaldehyde 416681667-0.7--0.1-
463,5,5-Trimethyl-4-(3-oxobutyl)cyclohex-2-en-1-one (3-Oxo-7,8-dihydro-α-ionone)168216810.50.10.70.30.3-
47Heptadecane 4170017000.2-0.90.1--
48Methyl syringate 4174417449.812.226.23.06.61.0
494-Hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)cyclohex-2-en-1-one (Vomifoliol)180217961.12.6-1.10.9-
50Hexadecan-1-ol 4188218830.10.21.20.20.41.5
51(E)-4-(r-1′,t-2′,c-4′-trihydroxy-3′,6′,6′-trimethylcyclohexyl)-but-3-en-2-one1960/3.118.3-6.23.349.1
52Hexadecanoic acid 4197019771.14.11.80.70.10.1
53(Z)-Octadec-9-en-1-ol 4206020600.86.23.12.00.18.9
54Octadecan-1-ol 4208420810.11.50.80.22.22.8
55(Z)-Octadec-9-enoic acid 4214221401.52.42.81.70.10.1
56Docosane 4220022000.11.023.00.214.025.9
57Tricosane 4230023000.71.04.30.70.10.1
1 RI—retention indices on HP-5MS column relative to C9–C25 alkanes; 2 RI from the literature (NIST Chemistry WebBook, NIST Standard Reference Database Number 69, http://webbook.nist.gov/chemistry/); 3 Area percentages (%); 4 identification confirmed with standard compound; *—tentatively identified; **—correct isomer is not identified; - indicates that compound is not identified. A—USE with pentane:diethyl ether (1:2, v/v); B—USE with dichloromethane; C—sequence I/II: USE with pentane; D—sequence I: USE with diethyl ether after C (pentane extraction), E—sequence II: USE with the mixture pentane:diethyl ether (1:2, v/v) after C (pentane extraction); F—sequence II: USE with dichloromethane after E (the extraction with the mixture pentane:diethyl ether (1:2, v/v)) and C (pentane extraction).
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