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Headspace solid-phase microextraction-gas chromatography–mass spectrometry characterization of propolis volatile compounds
Accepted Manuscript Title: Headspace solid-phase microextraction-gas chromatography-mass spectrometry characterization of propolis volatile compounds Author: Federica Pellati Francesco Pio Prencipe Stefania Benvenuti PII: DOI: Reference:
S0731-7085(13)00246-X http://dx.doi.org/doi:10.1016/j.jpba.2013.05.045 PBA 9106
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
13-3-2013 24-5-2013 27-5-2013
Please cite this article as: F. Pellati, F.P. Prencipe, S. Benvenuti, Headspace solid-phase microextraction-gas chromatography-mass spectrometry characterization of propolis volatile compounds, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.05.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract
Raw propolis
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GC-MS
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HS-SPME
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*Highlights (for review)
A HS-SPME-GC-MS method was developed for the analysis of propolis volatile compounds. Ninety-nine constituents were identified in propolis from different Italian regions. Aromatic compounds and sesquiterpenes were the most abundant constituents.
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This technique is a new and reliable tool for the study of this apiary product.
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*Revised Manuscript
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Headspace solid-phase microextraction-gas chromatography-mass spectrometry
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characterization of propolis volatile compounds
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Federica Pellati*, Francesco Pio Prencipe, Stefania Benvenuti
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Department of Life Sciences, University of Modena and Reggio Emilia,
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Via G. Campi 183, 41125 Modena, Italy
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* Corresponding author:
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Dr. Federica Pellati
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Department of Life Sciences
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University of Modena and Reggio Emilia
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Via G. Campi 183
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41125 Modena, Italy
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Tel.: +39-059-205-5144
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Fax: +39-059-205-5131
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E-mail: [email protected]
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Abstract
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In this study, a novel and efficient method based on headspace solid-phase microextraction (HS-
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SPME), followed by gas chromatography-mass spectrometry (GC-MS), was developed for the
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analysis of propolis volatile compounds. The HS-SPME procedure, whose experimental parameters
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were properly optimized, was carried out using a 100 µm polydimethylsiloxane (PDMS) fiber. The
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GC-MS analyses were performed on a HP-5 MS cross-linked 5% diphenyl-95% dimethyl
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polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 m film thickness), under programmed-
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temperature elution.
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Ninety-nine constituents were identified using this technique in the samples of raw propolis
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collected from different Italian regions. The main compounds detected include benzoic acid (0.87-
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30.13%) and its esters, such as benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and
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benzyl cinnamate (0.34-3.20%). Vanillin was detected in most of the samples analyzed in this study
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(0.07-5.44%). Another relevant class of volatile constituents is represented by sesquiterpene
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hydrocarbons, such as -cadinene (1.29-13.31%), -cadinene (1.36-8.85%) and -muurolene (0.78-
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6.59%), and oxygenated sesquiterpenes, such as -eudesmol (2.33-12.83%), T-cadinol (2.73-
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9.95%) and -cadinol (4.84-9.74%). Regarding monoterpene hydrocarbons, they were found to be
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present at low level in the samples analyzed in this study, with the exception of one sample from
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Southern Italy, where -pinene was the most abundant constituent (13.19%). The results obtained
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by HS-SPME-GC-MS were also compared with those of hydrodistillation (HD) coupled with GC-
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MS.
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The HS-SPME-GC-MS method developed in this study allowed us to determine the chemical
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fingerprint of propolis volatile constituents, thus providing a new and reliable tool for the complete
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characterization of this biologically active apiary product.
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Keywords: Propolis; Volatile compounds; Essential oil; Solid-phase microextraction; Gas 2
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chromatography; Mass spectrometry.
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Abbreviations: HD = Hydrodistillation; HS = Headspace; PDMS = polydimethylsiloxane; DVB-
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CAR-PDMS = divinylbenzene-carboxen-polydimethylsiloxane; LRI = linear retention index.
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1. Introduction
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Propolis is a resinous material collected and processed by honeybees (Apis mellifera L.) from
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several tree species [1]. In regions with temperate climate, the resin is collected mainly from the
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buds and cracks in the bark of Populus species [2]. Once collected, this material is enriched with
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salivary and enzymatic secretions [1]. The resulting product is used by bees to seal holes in their
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hives, to exclude draught and to make the entrance of the hive weather tight or easier to defend [1].
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Another advantage for bees is the capacity of this material to reduce the incidence of bacteria and
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moulds within the hive [2].
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Propolis is a phytochemically complex mixture composed by 50% resin (containing flavonoids and
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phenolic acids), 30% wax, 10% essential oil, 5% pollen and 5% other organic compounds [1]. A
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series of biological properties have been described for propolis extracts, such as antibacterial,
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antifungal, antiviral, antioxidant, anti-inflammatory, antiproliferative, immunostimulating [3].
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Typical applications of propolis include herbal products for cold syndrome and dermatological
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preparations [4]. Propolis extracts are also used to prevent and treat oral inflammations [4].
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The detailed chemical composition of propolis is known to be very complex [2,4]. The most
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important classes of its biologically active compounds are characterized by polyphenols, including
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flavonoids, phenolic acids and their esters [1,5]. The content of polyphenols in poplar type propolis
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extracts may vary as a function of the origin of samples and these differences can affect the
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biological activity of preparations and therefore their pharmacological effects [1].
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In addition to phenolics, another important class of propolis constituents is represented by volatile
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compounds [4,6]. Previous studies performed on propolis volatile fraction have been focused on the
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gas chromatographic analysis of the essential oil extracted by hydrodistillation (HD) [7-11]. In this
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ambit, the composition of propolis essential oil from different countries has been described [7-13].
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In some of these studies, propolis essential oil has demonstrated antimicrobial activity mainly
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against Gram-positive bacteria, but it is also active on Gram-negative bacteria and fungi
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[4,7,10,12,14].
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However, it is well-known that the HD technique presents some shortcomings, such as loss of
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volatile compounds, low extraction efficiency and long extraction time [15]. In addition, high
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temperature and water can cause degradation or chemical modifications of volatile constituents
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[15]. In recent years, the most frequently used analytical techniques for the extraction and
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concentration of volatile compounds from aromatic and medicinal plants are those based on
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headspace (HS) analysis [16]. In this context, the characterization of propolis samples by HS
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coupled with gas chromatography-mass spectrometry (GC-MS) has been described [17-19]. In
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particular, Greenaway et al. [17] have trapped volatile compounds from propolis on Tenax GC,
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followed by desorption and GC-MS analysis. Yang et al. [18] have developed a dynamic HS
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sampling coupled with GC-olfactometry-MS to study the common aroma-active components in
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propolis samples collected in China. Nunes and Guerreiro [19] have characterized Brazilian green
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propolis by HS extraction coupled with GC-MS.
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In the ambit of HS methods, solid-phase microextraction (SPME) represents a reliable tool for the
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analysis of volatile organic compounds [20,21] and eliminates most drawbacks to extracting
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organics, including high cost and excessive preparation time. In particular, SPME is a simple and
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fast modern tool used to characterize the volatile fraction of medicinal plants [20] and foods [21]
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and offers a valid alternative to HD for gas chromatographic analysis of essential oils from different
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sources [20]. In the specific case of honey bee products, SPME has been successfully applied to the
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characterization and analysis of the volatile compounds from honey [22] and royal jelly [23].
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In this study, a HS-SPME technique combined with GC-MS was optimized and applied for the first
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time to study the volatile organic compounds of Italian raw propolis. No references have been found
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in the international scientific literature on the use of HS-SPME to describe propolis volatile
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fraction. Even if volatile compounds are present in low concentration in propolis, their aroma and
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biological activity make them of importance for the characterization of this product. In addition, the
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volatile composition can give valuable information about the origin of samples.
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In particular, after the optimization of the extraction conditions, comparative studies on the typical
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HS-SPME-GC-MS profiles of raw propolis samples collected from different Italian regions were
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performed with the aim of confirming the applicability of the method developed for the chemical
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characterization of their volatile compounds.
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2. Materials and methods
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2.1. Chemicals
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allo-Aromadendrene, α-bisabolol, camphene, β-caryophyllene, caryophyllene oxide, 1,8-cineole, α-
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copaene, dodecane, heneicosane, heptadecane, hexadecane, α-humulene, limonene, linalool, cis-
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linalool oxide, menthol, 2-methyl-butanoic acid, 2-methyl-propanoic acid, myrtenal, myrtenol,
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naphthalene, nonadecane, octadecane, phenylethyl alcohol, α-pinene, tricosane, tridecane, undecane
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and cis-verbenol were purchased from Sigma-Aldrich-Fluka (Milan, Italy). Citronellol was
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purchased from Curt Georgi Imes (Milan, Italy), thymol from Merck (Darmstadt, Germany) and α-
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terpineol from Roth (Karlsruhe, Germany).
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2.2. Propolis samples
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Nine samples of raw propolis (indicated in the text as RP-1/RP-9) were collected from Apis 5
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mellifera hives located in different Italian regions in Spring 2012 and stored at –20 °C until
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chemical analysis. In particular samples RP-1/RP-6 were from Northern Italy, while samples RP-
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7/RP-9 were from Southern Italy. The frozen samples were finely powdered using a mortar and a
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pestle before the extraction procedure. Sample labeled as RP-9 was used for the optimization of the
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extraction conditions, because it was available in higher amount.
2.3. HS-SPME procedure
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HS-SPME was performed using a manual holder and two different fibers: a 100 µm
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polydimethylsiloxane
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polydimethylsiloxane (DVB-CAR-PDMS) (Supelco, Bellefonte, PA, USA). The coating was 1 cm
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long for both fibers. Before GC-MS analysis, each fiber was conditioned in the injector of the GC
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system, according to the instructions provided by the manufacturer.
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A 2 g amount of finely powdered raw propolis was placed in a 10 ml flat-bottom headspace vial
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sealed with a magnetic crimp cap and PTFE/silicone septa (Supelco). Under the optimized
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conditions, the sample was heated for 30 min during the equilibrium time in a thermostatic bath at
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75 °C. The SPME device was then inserted into the sealed vial by manually penetrating the septum
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and the fiber was exposed to the headspace for 20 min during the extraction time.
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After sampling, the SPME fiber was immediately inserted into the GC injector and thermally
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desorbed. A desorption time of 1 min at 230 °C was used in the splitless mode. Before sampling,
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each fiber was reconditioned for 5 min in the GC injector port at 230 °C.
and
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divinylbenzene-carboxen-
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2.4. Hydrodistillation (HD) procedure
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The powdered propolis sample RP-9 (190 g) was placed into a 3 L round-bottom distillation flask
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and then 1 l of water was added. After that, the mixture was distilled for 3 h. The essential oil was 6
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collected from the condenser and dried over anhydrous sodium sulfate. The yield of the HD was
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0.13%. The obtained essential oil was stored at +4°C until analysis. Before GC-MS analysis, the
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sample was diluted 1:20 (v/v) with n-hexane.
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2.5. GC-MS conditions
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Chromatographic analyses were carried out on a GC 6890 N (Agilent Technologies, Waldbronn,
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Germany), coupled with 5973 Network mass spectrometer (Agilent Technologies). Compounds
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were separated using a HP-5 MS cross-linked 5% diphenyl–95% dimethyl polysiloxane capillary
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column (30 m × 0.25 mm I.D., 1.00 µm film thickness, Agilent Technologies). The oven
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temperature was programmed from initial 40 °C to final 280 °C at 3 °C/min, which was maintained
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for 5 min. Splitless injection was used for HS-SPME-GC-MS. As for HD-GC-MS, the injection
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volume was 0.1 l , with a 1:100 split ratio. Helium was used as the carrier gas at a flow rate of 0.7
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ml/min. The injector, transfer line and ion-source temperatures were 230, 280 and 230 °C,
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respectively. MS detection was performed with electron ionization (EI) at 70 eV, operating in the
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full-scan acquisition mode in the m/z range 40-400.
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A mixture of aliphatic hydrocarbons (C7-C30) in n-hexane (Sigma-Aldrich) was loaded onto the
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PDMS fiber and injected under the same conditions to calculate the linear retention index (LRI) of
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each compound.
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2.6. Qualitative and semi-quantitative analysis
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Compounds were identified by comparing the retention times of the chromatographic peaks with
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those of authentic reference compounds run under the same conditions and by comparing the LRIs
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with the literature [24]. Peak enrichment on co-injection with authentic reference compounds was
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also carried out. The comparison of the MS fragmentation pattern of the target analytes with those 7
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of pure compounds was performed. Mass spectrum database search was carried out using the
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National Institute of Standards and Technology (NIST) mass spectral database (version 2.0d, 2005).
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The percentage relative amount of individual components was expressed as percent peak area
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relative to total peak area, in agreement with previous papers on the GC composition of propolis
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essential oil [7-13]. Semi-quantitative data were the mean of three analyses.
3. Results and discussion
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3.1. Optimization of HS-SPME conditions
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In this study, HS-SPME combined with GC-MS was applied for the first time to the extraction and
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analysis of volatile compounds in Italian samples of raw propolis. Bicchi et al. [20] have
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highlighted the importance of the effect of the fiber coating on HS-SPME of volatile compounds
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from various aromatic and medicinal plants. Therefore, the first step of the method optimization
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was the selection of the best fiber coating for HS-SPME.
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In this study, two fiber types, including a PDMS (100 m) and a DVB-CAR-PDMS (50/30 m),
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which have been frequently employed for the extraction of the volatile fraction from natural
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products [20], were tested for the analysis of propolis volatile compounds. Fig. 1 shows the results
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of the fiber selection carried out by HS-SPME-GC-MS. Each fiber was exposed to the HS for the
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same time at the same temperature.
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Fig. 1
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Of these two fibers, the DVB-CAR-PDMS showed a strong extraction affinity for carboxylic acids
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and, in particular, for benzoic acid [25]; in samples RP-1, RP-2 and RP-5, where benzoic acid was
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present at high level, this caused a significant peak broadening and tailing, with the consequent
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overlapping of adjacent compounds. The PDMS fiber allowed to obtain a better profile for all
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classes of propolis volatile compounds and, therefore, it was selected for this study. As previously 8
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described in the literature [20,26], although multi-component fibers have proved to be very
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effective, most of the routine applications in the aromatic and medicinal plant fields adopt a PDMS
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fiber.
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Other parameters are known to influence the extraction efficiency of the HS-SPME technique [26].
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Therefore, to obtain the optimal HS-SPME conditions, additional variables were chosen, including
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sample amount, extraction temperature, equilibrium time and extraction time. Other parameters,
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such as salt addition and sample agitation during the equilibrium time, were also studied. The sum
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of peak areas was adopted to optimize the experimental parameters.
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It is well-known that, for a given vial volume, sample amount has usually a positive effect on peak
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areas of the compounds extracted by SPME. Indeed, the results showed that the peak areas of the
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extracted analytes increased by increasing the propolis amount from 1 to 2 g in a 10 ml vial and
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then reached a plateau for larger amounts (e.g. 3 g). Therefore, 2 g of powdered sample in a 10 ml
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vial was used for further studies as the optimal sample amount.
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In relation to the effect of temperature, an increase in sampling temperature usually increases the
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headspace concentration of volatile compounds, favouring their extraction. However, if temperature
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is too high, analytes can be desorbed from the SPME fiber, thus reducing the overall sensitivity. In
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this study, the effect of temperature was studied over the range 70-100 °C and the results showed
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that a significant jump occurred between 70 and 75 °C; when the temperature was further increased
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to 80, 85 and 100 °C, no significant increase in the response was observed. Thus, 75 °C was
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selected as the optimal temperature.
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The extraction profile was also evaluated by changing the equilibrium time from 15 to 30 min and
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the extraction time from 5 to 20 min. The results showed that the best global response, within the
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range studied, was reached with an equilibrium time of 30 min and an extraction time of 20 min.
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Finally, the addition of a 25-30% (w/v) sodium chloride solution (from 0.5 to 1.5 ml) and the
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sample agitation during the equilibrium time were not found to increase the HS-SPME efficiency.
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3.2. Analysis of volatile compounds in raw propolis by HS-SPME-GC-MS
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Volatile compounds in propolis essential oil extracted by HD have been previously determined by
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GC techniques [7-11]; due to their aroma and biological activity, these compounds are very useful
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for the chemical characterization of this product [4,6]. Similar to reports on the phenolic
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composition of hydroalcoholic extracts [1], variations in the chemical profile of propolis volatile
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fraction between temperate and tropical regions have been described [6]. The volatile chemical
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composition of propolis is also known to be strongly dependent on the local flora at the site of
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collection [2,4]. Some authors have reported that the volatile fraction is also influenced by the bee
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species, because Apis mellifera and Melipona beechei in the same region of Yucatán (Mexico) have
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produced propolis with different volatile compounds [13]. The same has occurred in Turkey, where
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phenols and terpenes present in propolis samples were dependent on the race of honeybees [14].
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In the present study, the powdered raw propolis from different Italian regions was analyzed by HS-
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SPME-GC-MS. Ninety-nine constituents were identified as shown in Table 1. The typical total ion
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current (TIC) chromatogram of a representative sample (RP-9) is shown in Fig. 1.
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Table 1
The main volatile compounds identified in propolis samples are benzoic acid (0.87-30.13%) and its
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esters, including benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and benzyl
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cinnamate (0.34-3.20%). It must be pointed out that benzyl salicylate and benzyl cinnamate are two
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known allergens present in propolis and in other matrixes as well [27]. Vanillin, a further aromatic
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compound previously identified in propolis essential oil [7,8], was detected in most of the samples
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analyzed in this study (0.07-5.44%). Another relevant class of volatile compounds is represented by
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sesquiterpene hydrocarbons, such as -cadinene (1.29-13.31%), -cadinene (1.36-8.85%) and -
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(0.78-6.59%), and oxygenated sesquiterpenes, such as -eudesmol (2.33-12.83%), T-
257
cadinol (2.73-9.95%) and -cadinol (4.84-9.74%). Germacrene D-4-ol was detected only in sample
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RP-7 (6.32%). Regarding monoterpene hydrocarbons, they were present at low level in the samples
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analyzed in this study, with the exception of sample RP-7 from Southern Italy, where -pinene was
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the most abundant constituent (13.19%). As for oxygenated monoterpenes, menthol was determined
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mainly in sample RP-6 (2.54%) and thymol in samples RP-4 and RP-6 (2.08 and 3.27%,
262
respectively). Hemiterpenes were detected at low percentages in all the samples and were absent in
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sample RP-7. The presence of naphthalene (0.97%) in sample RP-5 is probably due to pollution
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attributable to unexpected material collected by bees [8] or to inappropriate environmental storage
265
conditions of propolis and confirms a previous observation that propolis could be used as a marker
266
of pollution [9].
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The analysis of variance (ANOVA) was used to evaluate the statistical significance of the measured
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differences between HS-SPME-GC-MS data of propolis samples of different geographic origin
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(Northern and Southern Italy), with a P level set at 0.05. The statistical analysis was performed
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using Statistica 6 for Windows (StatSoft® Italia, Vigonza, Italy). ANOVA was applied to the
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volatile compounds shared by all samples of at least one geographic origin. Sample RP-7 was
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excluded from the statistical analysis, due to its peculiar composition. Significant differences
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between propolis samples from Northern and Southern Italy were observed for 2-methyl-2-buten-1-
274
ol, 2-methyl-butanoic acid, linalool, -cadinol and heptadecane.
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The chemical composition of propolis volatile fraction determined in the present study by means of
276
HS-SPME-GC-MS was found to be in agreement with previous reports [7,8]. In general, propolis
277
constituents are known to be directly related to those of bud exudates collected by honeybees from
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various trees [2,4]. Indeed, several volatile compounds identified in propolis volatile fraction have
279
been previously detected from leaf buds of Populus nigra L., which represents one of the main
280
botanical sources of propolis constituents in temperate regions [2]. In particular, the essential oil
281
from leaf buds of P. nigra is composed mainly by oxygenated sesquitepenes (35.7-41.7%) and
282
sesquiterpene hydrocarbons (35.2-36.5%) [28]. The most abundant volatile compound is -
283
eudesmol (19.1-19.6%) [28]. Hemiterpenes have also been identified (2.2-7.6%) [28], while
284
monoterpenes have been determined in lower percentages (1.6-5.7%) [28]. Aliphatic and aromatic
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alcohols, carbonyl compounds and aliphatic acids have been characterized among non-terpene
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volatiles (9.8-13.5%) [28].
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According to Petri et al. [7], propolis from temperate zones can be separated in two types, based on
288
the presence of representative amounts of -eudesmol (40-60%) or benzyl benzoate (20-40%) in the
289
essential oil. In a subsequent study on the volatile compounds isolated from propolis collected in
290
two different regions of Croatia, benzoic acid (27.0%), benzyl alcohol (18.2%) and benzyl benzoate
291
(3.6%) were the predominant constituents of the essential oil from Slavonia [8]. Another study
292
carried out on the essential oil composition in propolis samples from various regions of Greece
293
indicated a uniformly elevated concentration of -pinene [10], except in a sample from one
294
location, in which junipene was determined as the main constituent.
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The volatile composition of the Italian samples of raw propolis investigated in this work indicates a
296
close relationship with bud exudates of Populus species, which is also the well-known source of
297
typical poplar bud phenolics (including phenolic acids and flavonoids), identified in the same
298
samples by HPLC [29]. In the case of sample RP-7, originated from a region of Southern Italy
299
(Adriatic coast) where poplars are not present, the high percentage of -pinene suggests that the
300
botanical source of bee glue may be attributed to Pinus species [30,31], in agreement with what has
301
been previously observed in a propolis sample from Portugal (Mogadouro region) [32].
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3.3. Repeatability
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The repeatability was determined by performing five replicate HS-SPME-GC-MS experiments on
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the same propolis sample (RP-9), under the optimized extraction conditions. The relative standard
307
deviation (RSD) values of % relative peak area of volatile constituents were ≤ 11%, indicating a
308
satisfactory repeatability of the developed method. As previously indicated in the literature, the
309
widespread use of the PDMS fiber is not only attributable to its good recovery for the HS
310
components of medicinal and aromatic plants, because, in general, its polarity is from medium to 12
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low, but also to both the consistency of its performance and repeatability, especially when a large
312
number of samples are involved [20].
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3.4. Comparison of HS-SPME-GC-MS and HD-GC-MS
315
In this study, the propolis volatile profile obtained by HS-SPME-GC-MS was compared with that of
317
traditional HD-GC-MS. As shown in Fig. 2, the TIC chromatograms of these techniques share the
318
same components and comparable values of % relative peak area were observed in most cases.
319
However, some differences between HS-SPME-GC-MS and HD-GC-MS in terms of the relative
320
amount of some compounds, such as hemiterpenes and sesquiterpenes, were found. In particular,
321
compared with the HD-GC-MS method, the proposed HS-SPME-GC-MS technique allowed to
322
obtain higher % relative peak area values for -cadinene, T-cadinol and -cadinol, but lower for 3-
323
methyl-3-buten-1-ol and 2-methyl-2-buten-1-ol. These differences might result from the higher
324
extraction efficiency of the PDMS fiber for less polar compounds. Benzoic acid and vanillin were
325
detected by HS-SPME-GC-MS only.
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Fig. 2
Although the composition of samples extracted by HD and HS-SPME is sometimes similar, the %
328
relative peak areas of an analyte obtained with the two techniques are not easily interchangeable,
329
because they are obtained from entirely different approaches [20]. It is well-known that these
330
intrinsic methodological differences greatly influence the resulting quantitative composition and, to
331
a lesser extent, also the qualitative profile [20]. An additional factor of discrimination in HS-SPME
332
is the nature of the polymeric coatings of the fiber, which conditions the composition of the volatile
333
fraction recovered [20].
Ac
327
334 335
4. Conclusions
336 13
Page 15 of 25
The present study describes a new, rapid and simple HS-SPME-GC-MS method for the analysis of
338
propolis volatile compounds. The parameters affecting the extraction efficiency were optimized.
339
Compared with HD-GC-MS, the HS-SPME-GC-MS procedure developed in this study can greatly
340
simplify and shorten the extraction and analysis of volatile compounds from propolis. In addition,
341
this technique needs much less sample amount and is environmentally friendly.
342
The application of the developed technique to samples of raw propolis collected from different
343
Italian regions allowed to obtain the volatile profile of this biologically active apiary product.
344
In conclusion, HS-SPME-GC-MS represents an effective technique for the analysis of volatile
345
compounds in bee glue. The developed method could also be applied to the evaluation of the
346
antimicrobial activity in the vapour phase of propolis constituents against different bacterial and
347
fungal strains.
an
us
cr
ip t
337
349
M
348
Acknowledgements
351
ed
350
The authors are grateful to Kontak (Pozzo d’Adda, Milan, Italy) for the financial support.
ce pt
352
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354
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volatile and extractable compounds of crude royal jelly, J. Chromatogr. B 885-886 (2012) 109-116.
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433 434
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Ac
432
ip t
414
435 436 437 438 439 17
Page 19 of 25
440
Figure captions
441
Figure 1: Total ion current (TIC) chromatograms obtained by HS-SPME-GC-MS analysis of
443
volatile compounds from propolis (sample RP-9) using (A) a 100 µm PDMS fiber and (B) a
444
stableflex 50/30 µm DVB-CAR-PDMS fiber. For peak identification, see Table 1. Experimental
445
conditions as in sections 2.3 and 2.5.
ip t
442
cr
446
Figure 2: Total ion current (TIC) chromatograms of volatile compounds from propolis (sample RP-
448
9) by (A) HS-SPME-GC-MS and (B) HD-GC-MS. For peak identification, see Table 1.
449
Experimental conditions as in sections 2.3, 2.4 and 2.5.
Ac
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ed
M
an
us
447
18
Page 20 of 25
1.77 ± 0.11 0.35 ± 0.01 0.10e 0.24 ± 0.02 0.13 ± 0.01 0.54 ± 0.03 0.16 ± 0.01 0.06e 1.16 ± 0.12 1.07 ± 0.06 0.07e 3.91 ± 0.41 0.06e 0.18 ± 0.02 0.26 ± 0.02 1.05 ± 0.11 8.29 ± 0.45 0.56 ± 0.06 0.12 ± 0.01 -
1.19 ± 0.13 0.32 ± 0.01 0.38 ± 0.04 0.37 ± 0.03 0.12e 0.22 ± 0.02 0.46 ± 0.05 0.14e 0.07e 0.23 ± 0.01 0.76 ± 0.05 0.09e 0.06e 0.61 ± 0.05 12.08 ± 1.29 0.39 ± 0.03 -
an
1.94 ± 0.19 0.28 ± 0.02 0.48 ± 0.03 0.41 ± 0.02 0.37 ± 0.01 0.24 ± 0.02 0.07 ± 0.01 0.30 ± 0.02 0.19e 0.38 ± 0.01 0.20e 0.07e 1.54 ± 0.09 0.17e 2.07 ± 0.20 0.17 ± 0.01 0.36 ± 0.01 1.40 ± 0.11 20.78 ± 0.09 0.22e -
M
1.82 ± 0.09 0.30 ± 0.01 0.27e 0.36e 0.07e 0.20e 0.39e 0.23 ± 0.01 0.46 ± 0.02 0.04e 1.59 ± 0.07 0.06e 1.02 ± 0.03 21.79 ± 0.19 -
d
606 625 722 736 769 782 800 829 839 890 911 922 931 941 956 959 966 987 1003 1019 1037 1037 1039 1039 1078 1099 1101 1105 1119 1137 1154 1157 1165 1168 1178 1181 1187 1194
ep te
Acetic acid 2-Methyl-3-buten-2-ol 3-Methyl-3-buten-1-ol 2-Methyl-propanoic acid 2-Methyl-2-buten-1-ol 3-Methyl-2-butenal Hexanal 3-Methyl-butanoic acid 2-Methyl-butanoic acid Styrene 2-Methyl-2-butenoic acid Prenyl acetate Tricyclene α-Pinene α-Fenchene Camphene Benzaldehyde β-Pinene Octanal Δ-3-Carene Benzyl alcohol Limonene 1,8-Cineole β-Phellandrene cis-Linalool oxide Undecane Linalool Nonanal Phenylethyl alcohol -Campholenal cis-Verbenol trans-Verbenol Benzoic acid Benzyl acetate Pinocarvone Menthol Terpinen-4-ol p-Cymen-8-ol
Ac c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
cr
RP-5c
us
Table 1 Volatile components identified in raw propolis samples (RP-1/RP-9) by HS-SPME-GC-MSa Peak Compoundb LRI RP-1c RP-2c RP-3c RP-4c number
ip t
Table 1
0.67 ± 0.03 0.06e 0.06e 0.10e 0.13 ± 0.01 0.16 ± 0.01 0.22 ± 0.02 0.88 ± 0.02 0.08e 0.05e 0.09e 0.08e 0.39 ± 0.01 30.13 ± 2.60 -
RP-6c
RP-7c
RP-8c
RP-9c
Identification methodd
1.33 ± 0.10 0.39 ± 0.03 0.20e 0.56 ± 0.05 0.32 ± 0.02 0.38 ± 0.03 0.56 ± 0.06 0.09e 0.10e 0.75 ± 0.08 0.07e 0.14 ± 0.02 0.73 ± 0.08 3.12 ± 0.31 0.43 ± 0.05 2.54 ± 0.28 -
0.17 ± 0.01 0.15 ± 0.01 13.19 ± 1.56 0.09e 0.22 ± 0.02 0.23 ± 0.02 0.07e 0.27 ± 0.03 0.27 ± 0.03 0.09 ± 0.01 0.23 ± 0.02 1.31 ± 0.15 1.01 ± 0.12 4.55 ± 0.29 0.87 ± 0.10 0.35 ± 0.04 0.28 ± 0.03
1.30 ± 0.15 0.29 ± 0.03 0.14 ± 0.01 0.66 ± 0.07 0.10 ± 0.01 0.94 ± 0.09 0.24 ± 0.01 0.27 ± 0.03 0.58 ± 0.05 0.12 ± 0.01 0.07e 0.20 ± 0.02 0.17 ± 0.01 0.05e 0.28 ± 0.02 18.62 ± 1.78 -
1.66 ± 0.14 1.71 ± 0.04 1.69 ± 0.04 0.56 ± 0.01 0.50 ± 0.01 0.13 ± 0.01 0.27 ± 0.01 1.44 ± 0.01 0.21 ± 0.02 0.86 ± 0.09 0.29 ± 0.01 1.39 ± 0.07 1.17 ± 0.05 0.12e 0.52e 0.77 ± 0.01 1.84 ± 0.01 4.69 ± 0.44 0.50 ± 0.02 -
b, d b, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d b, d a, b, c, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d a, b, c, d b, d a, b, c, d b, d a, b, c, d b, d bd b, d b, d a, b, c, d b, d b, d
Page 21 of 25
ip t
0.97 ± 0.07 0.13e 0.10 ± 0.01 0.13e 0.12e 0.23e 0.18 ± 0.01 0.12e 5.44 ± 0.24 0.36 ± 0.02 0.29 ± 0.02 0.78 ± 0.06 1.36 ± 0.13 1.29 ± 0.12 0.96 ± 0.05 0.25e -
0.16 ± 0.01 0.09 ± 0.01 0.29 ± 0.03 3.27 ± 0.19 0.17 ± 0.01 0.11e 0.30 ± 0.01 1.93 ± 0.09 0.07e 0.11 ± 0.01 0.09e 0.04e 2.60 ± 0.13 4.17 ± 0.36 6.59 ± 0.59 8.85 ± 0.48 13.31 ± 0.84 4.05 ± 0.15 2.06 ± 0.03 0.47 ± 0.05
cr
0.14 ± 0.01 0.13 ± 0.01 0.11 ± 0.01 2.08 ± 0.23 0.10e 0.31 ± 0.01 0.16 ± 0.01 0.06e 0.69e 0.50 ± 0.02 0.20 ± 0.02 0.23 ± 0.02 1.01 ± 0.02 2.39 ± 0.22 2.97 ± 0.20 3.88 ± 0.29 7.87 ± 0.43 1.36 ± 0.11 0.63 ± 0.02 0.71 ± 0.07 0.21 ± 0.02 0.54 ± 0.01
us
0.21 ± 0.02 0.12 ± 0.01 0.16 ± 0.02 0.50 ± 0.05 0.38 ± 0.04 0.20 ± 0.02 0.44 ± 0.03 0.62 ± 0.07 0.90 ± 0.05 0.49 ± 0.02 2.65 ± 0.22 0.32 ± 0.01 0.23 ± 0.02 0.15 ± 0.01 0.51 ± 0.02 0.43 ± 0.05 0.77 ± 0.08 1.34 ± 0.04 1.53 ± 0.02 2.18 ± 0.18 0.77 ± 0.08 1.34 ± 0.05 0.44 ± 0.01 0.40 ± 0.04 1.26 ± 0.01
an
0.23 ± 0.01 0.57 ± 0.03 0.18 ± 0.01 0.51 ± 0.02 0.90 ± 0.07 0.14 ± 0.01 0.70 ± 0.03 0.33 ± 0.01 0.37e 0.40 ± 0.03 2.67 ± 0.19 0.61 ± 0.05 0.25 ± 0.01 0.34 ± 0.03 1.04 ± 0.05 2.02 ± 0.11 1.95 ± 0.07 0.98 ± 0.08 0.65 ± 0.04 1.06 ± 0.05
M
0.20 ± 0.01 0.50 ± 0.02 0.36e 0.20e 0.48 ± 0.02 0.20 ± 0.01 0.28 ± 0.03 0.45 ± 0.03 3.21 ± 0.05 0.81 ± 0.07 0.26 ± 0.02 0.35 ± 0.02 1.14 ± 0.11 1.73 ± 0.09 1.75 ± 0.05 2.68 ± 0.24 1.76 ± 0.11 2.04 ± 0.24 1.22 ± 0.08 0.54 ± 0.05
d
1199 1200 1202 1206 1209 1210 1218 1229 1232 1257 1262 1282 1291 1298 1313 1355 1358 1362 1368 1370 1385 1388 1400 1405 1436 1438 1452 1472 1479 1484 1487 1510 1529 1534 1534 1537 1545 1548 1560 1598 1605 1609
ep te
Dodecane Naphthalene α-Terpineol Decanal Myrtenol Myrtenal Verbenone Citronellol β-Cyclocitral Nonanoic acid β-Phenylethyl acetate Cinnamaldehyde Thymol Tridecane Cinnamyl alcohol Decanoic acid α-Terpinyl acetate α-Cubebene Eugenol Acetocinnamone -Ylangene α-Copaene -Bourbonene Vanillin Cinnamic acid β-Caryophyllene Cinnamyl acetate α-Humulene allo-Aromadendrene ar-Curcumene γ-Muurolene α-Muurolene γ-Cadinene Germacrene D-4-ol δ-Cadinene Calamenene Dodecanoic acid Cadina-1,4-diene α-Calacorene Hexadecane Caryophyllene oxide Guaiol
Ac c
39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
0.79 ± 0.07 0.57 ± 0.01 0.24 ± 0.02 0.78 ± 0.03 1.11 ± 0.12 4.50 ± 0.15 0.34 ± 0.03 1.00 ± 0.12 0.73 ± 0.07 6.32 ± 0.04 2.90 ± 0.33 0.37 ± 0.03 3.30 ± 0.38 -
0.10e 0.52 ± 0.05 0.64 ± 0.06 0.12 ± 0.01 0.41 ± 0.04 0.29 ± 0.03 0.84 ± 0.09 1.51 ± 0.17 2.41 ± 0.26 3.10 ± 0.34 6.77 ± 0.74 1.29 ± 0.15 0.57 ± 0.05 0.47 ± 0.05 0.45 ± 0.05 -
0.88 ± 0.05 0.19 ± 0.01 0.35 ± 0.01 0.49e 0.12 ± 0.01 0.15 ± 0.01 0.21 ± 0.02 1.00 ± 0.03 0.45 ± 0.04 1.04 ± 0.07 2.27 ± 0.07 2.38 ± 0.10 3.58 ± 0.12 6.16 ± 0.08 1.99 ± 0.16 0.88 ± 0.04 0.46 ± 0.05 -
a, b, c, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d b, d b, d b, d a, b, c, d b, d a, b, c, d a, b, c, d b, d b, d b, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d
Page 22 of 25
an
M
ip t
4.96 ± 0.06 1.65 ± 0.15 2.33 ± 0.38 1.74 ± 0.20 0.99 ± 0.08 0.71 ± 0.06 1.98 ± 0.22 0.91 ± 0.10
3.42 ± 0.39 3.68 ± 0.24 8.90 ± 0.40 2.55 ± 0.07 9.74 ± 0.37 6.78 ± 0.54 0.62 ± 0.02 6.91 ± 0.15 1.25 ± 0.15 0.74 ± 0.03 0.79 ± 0.09 1.49 ± 0.01 1.81 ± 0.21
3.94 ± 0.10 1.36 ± 0.10 9.95 ± 0.06 2.37 ± 0.04 8.36 ± 0.31 3.08 ± 0.28 0.66 ± 0.01 0.40 ± 0.04 0.59 ± 0.06 3.37 ± 0.34
b, d b, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d b, d b, d a, b, c, d b, d a, b, c, d
Ac c
ep te
d
0.22 ± 0.02 3.78 ± 0.10 1.40 ± 0.07 7.96 ± 0.31 1.70 ± 0.18 0.48 ± 0.03 6.53 ± 0.10 2.88 ± 0.29 1.26 ± 0.13 0.16 ± 0.01 0.28 ± 0.03 0.31 ± 0.03 0.94 ± 0.09
cr
1.35 ± 0.12 1.93 ± 0.11 3.50 ± 0.29 0.92 ± 0.09 0.52 ± 0.02 4.84 ± 0.49 3.99 ± 0.10 0.51 ± 0.03 0.54 ± 0.02 13.05 ± 0.57 0.95 ± 0.04 1.90 ± 0.03 0.76 ± 0.01 3.20 ± 0.20 1.46 ± 0.11
us
81 Viridiflorol 1615 0.18 ± 0.02 82 Cedrol 1620 3.26 ± 0.31 83 Cubenol 1650 1.94 ± 0.18 1.10 ± 0.03 0.53 ± 0.03 3.64 ± 0.26 84 γ-Eudesmol 1655 2.05 ± 0.13 3.40e 2.47 ± 0.12 2.65 ± 0.30 85 1657 4.70 ± 0.02 3.44 ± 0.35 2.73 ± 0.17 9.03 ± 0.77 -Cadinol 86 δ-Cadinol 1662 1.31 ± 0.05 1.12 ± 0.02 0.72 ± 0.07 2.04 ± 0.03 87 α-Copaen-11-ol 1668 0.44 ± 0.04 0.72 ± 0.04 1.39 ± 0.14 88 α-Cadinol 1676 5.23 ± 0.30 7.94 ± 0.30 89 β-Eudesmol 1677 4.86 ± 0.51 12.83 ± 0.45 9.85 ± 0.17 7.49 ± 0.22 90 Heptadecane 1699 91 α-Bisabolol 1700 5.34 ± 0.55 92 Benzyl benzoate 1783 9.59 ± 0.47 5.30 ± 0.63 3.22 ± 0.29 1.92 ± 0.20 93 Octadecane 1801 94 Nonadecane 1899 0.44 ± 0.03 0.49 ± 0.01 0.53 ± 0.04 0.91 ± 0.09 95 Benzyl salicylate 1903 1.46e 0.74 ± 0.08 0.63 ± 0.07 0.34 ± 0.03 96 Manoyl oxide 2010 0.39 ± 0.04 97 Heneicosane 2100 0.47 ± 0.02 0.57e 0.57 ± 0.06 0.66e 98 Benzyl cinnamate 2109 2.29 ± 0.08 1.27 ± 0.10 1.99 ± 0.20 0.34 ± 0.01 99 Tricosane 2300 1.27 ± 0.08 1.88 ± 0.03 1.94 ± 0.14 1.54 ± 0.06 a Experimental conditions as in sections 2.3 and 2.5. b Compounds are listed in order of elution time. c Data are expressed as mean (n = 3) of % relative peak area values ± SD. For sample RP-9, n = 5. d a: retention time; b: LRI; c: peak enrichment; d: mass spectrum. e SD < 0.005.
Page 23 of 25
Figure 1
Abundance
A
85
240000 220000
88
200000
73
180000 160000 99
ip t
140000 71
120000
86
100000
89 33
3
70 69
29
5
84
23
40000
1 6 9
27
11
19
14
10
42
28
17
20000
60
48 50 49 53
34
54
47
25
67
77
30.00
40.00
Abundance
70.00
ce pt
700000 600000
21
500000
80.00
B
ed
800000
89 88 73
29
85 71
34
400000
11
84
42
12
300000
Ac
3
1
17
70
50 53
27 28
5
74
69
86
49 23
62 54 60
6
100000
60.00
33
900000
200000
50.00
94
an
20.00
97
M
10.00
90
78
62
0 Time-->
83
21
us
60000
12
cr
80000
74
9
14
77 78
47
19 10
83 67
25
90 94
97
99
0 Time-->
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Figure 1
Page 24 of 25
Figure 2
Abundance
A 85
240000 e 220000
88
0 200000
73
0 180000 0 160000 99
0 120000
71 86
0 100000
89
80000
33
3
12
0
5
23 6
48
27
11
9
19
14
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Figure 2
Page 25 of 25
S0731-7085(13)00246-X http://dx.doi.org/doi:10.1016/j.jpba.2013.05.045 PBA 9106
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
13-3-2013 24-5-2013 27-5-2013
Please cite this article as: F. Pellati, F.P. Prencipe, S. Benvenuti, Headspace solid-phase microextraction-gas chromatography-mass spectrometry characterization of propolis volatile compounds, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.05.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract
Raw propolis
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GC-MS
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HS-SPME
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*Highlights (for review)
A HS-SPME-GC-MS method was developed for the analysis of propolis volatile compounds. Ninety-nine constituents were identified in propolis from different Italian regions. Aromatic compounds and sesquiterpenes were the most abundant constituents.
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This technique is a new and reliable tool for the study of this apiary product.
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*Revised Manuscript
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Headspace solid-phase microextraction-gas chromatography-mass spectrometry
2
characterization of propolis volatile compounds
3 4
Federica Pellati*, Francesco Pio Prencipe, Stefania Benvenuti
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Department of Life Sciences, University of Modena and Reggio Emilia,
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Via G. Campi 183, 41125 Modena, Italy
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* Corresponding author:
18
Dr. Federica Pellati
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Department of Life Sciences
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University of Modena and Reggio Emilia
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Via G. Campi 183
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41125 Modena, Italy
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Tel.: +39-059-205-5144
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Fax: +39-059-205-5131
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E-mail: [email protected]
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Abstract
27
In this study, a novel and efficient method based on headspace solid-phase microextraction (HS-
29
SPME), followed by gas chromatography-mass spectrometry (GC-MS), was developed for the
30
analysis of propolis volatile compounds. The HS-SPME procedure, whose experimental parameters
31
were properly optimized, was carried out using a 100 µm polydimethylsiloxane (PDMS) fiber. The
32
GC-MS analyses were performed on a HP-5 MS cross-linked 5% diphenyl-95% dimethyl
33
polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 m film thickness), under programmed-
34
temperature elution.
35
Ninety-nine constituents were identified using this technique in the samples of raw propolis
36
collected from different Italian regions. The main compounds detected include benzoic acid (0.87-
37
30.13%) and its esters, such as benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and
38
benzyl cinnamate (0.34-3.20%). Vanillin was detected in most of the samples analyzed in this study
39
(0.07-5.44%). Another relevant class of volatile constituents is represented by sesquiterpene
40
hydrocarbons, such as -cadinene (1.29-13.31%), -cadinene (1.36-8.85%) and -muurolene (0.78-
41
6.59%), and oxygenated sesquiterpenes, such as -eudesmol (2.33-12.83%), T-cadinol (2.73-
42
9.95%) and -cadinol (4.84-9.74%). Regarding monoterpene hydrocarbons, they were found to be
43
present at low level in the samples analyzed in this study, with the exception of one sample from
44
Southern Italy, where -pinene was the most abundant constituent (13.19%). The results obtained
45
by HS-SPME-GC-MS were also compared with those of hydrodistillation (HD) coupled with GC-
46
MS.
47
The HS-SPME-GC-MS method developed in this study allowed us to determine the chemical
48
fingerprint of propolis volatile constituents, thus providing a new and reliable tool for the complete
49
characterization of this biologically active apiary product.
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Keywords: Propolis; Volatile compounds; Essential oil; Solid-phase microextraction; Gas 2
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chromatography; Mass spectrometry.
53 54
Abbreviations: HD = Hydrodistillation; HS = Headspace; PDMS = polydimethylsiloxane; DVB-
55
CAR-PDMS = divinylbenzene-carboxen-polydimethylsiloxane; LRI = linear retention index.
56
1. Introduction
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Propolis is a resinous material collected and processed by honeybees (Apis mellifera L.) from
60
several tree species [1]. In regions with temperate climate, the resin is collected mainly from the
61
buds and cracks in the bark of Populus species [2]. Once collected, this material is enriched with
62
salivary and enzymatic secretions [1]. The resulting product is used by bees to seal holes in their
63
hives, to exclude draught and to make the entrance of the hive weather tight or easier to defend [1].
64
Another advantage for bees is the capacity of this material to reduce the incidence of bacteria and
65
moulds within the hive [2].
66
Propolis is a phytochemically complex mixture composed by 50% resin (containing flavonoids and
67
phenolic acids), 30% wax, 10% essential oil, 5% pollen and 5% other organic compounds [1]. A
68
series of biological properties have been described for propolis extracts, such as antibacterial,
69
antifungal, antiviral, antioxidant, anti-inflammatory, antiproliferative, immunostimulating [3].
70
Typical applications of propolis include herbal products for cold syndrome and dermatological
71
preparations [4]. Propolis extracts are also used to prevent and treat oral inflammations [4].
72
The detailed chemical composition of propolis is known to be very complex [2,4]. The most
73
important classes of its biologically active compounds are characterized by polyphenols, including
74
flavonoids, phenolic acids and their esters [1,5]. The content of polyphenols in poplar type propolis
75
extracts may vary as a function of the origin of samples and these differences can affect the
76
biological activity of preparations and therefore their pharmacological effects [1].
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In addition to phenolics, another important class of propolis constituents is represented by volatile
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compounds [4,6]. Previous studies performed on propolis volatile fraction have been focused on the
79
gas chromatographic analysis of the essential oil extracted by hydrodistillation (HD) [7-11]. In this
80
ambit, the composition of propolis essential oil from different countries has been described [7-13].
81
In some of these studies, propolis essential oil has demonstrated antimicrobial activity mainly
82
against Gram-positive bacteria, but it is also active on Gram-negative bacteria and fungi
83
[4,7,10,12,14].
84
However, it is well-known that the HD technique presents some shortcomings, such as loss of
85
volatile compounds, low extraction efficiency and long extraction time [15]. In addition, high
86
temperature and water can cause degradation or chemical modifications of volatile constituents
87
[15]. In recent years, the most frequently used analytical techniques for the extraction and
88
concentration of volatile compounds from aromatic and medicinal plants are those based on
89
headspace (HS) analysis [16]. In this context, the characterization of propolis samples by HS
90
coupled with gas chromatography-mass spectrometry (GC-MS) has been described [17-19]. In
91
particular, Greenaway et al. [17] have trapped volatile compounds from propolis on Tenax GC,
92
followed by desorption and GC-MS analysis. Yang et al. [18] have developed a dynamic HS
93
sampling coupled with GC-olfactometry-MS to study the common aroma-active components in
94
propolis samples collected in China. Nunes and Guerreiro [19] have characterized Brazilian green
95
propolis by HS extraction coupled with GC-MS.
96
In the ambit of HS methods, solid-phase microextraction (SPME) represents a reliable tool for the
97
analysis of volatile organic compounds [20,21] and eliminates most drawbacks to extracting
98
organics, including high cost and excessive preparation time. In particular, SPME is a simple and
99
fast modern tool used to characterize the volatile fraction of medicinal plants [20] and foods [21]
100
and offers a valid alternative to HD for gas chromatographic analysis of essential oils from different
101
sources [20]. In the specific case of honey bee products, SPME has been successfully applied to the
102
characterization and analysis of the volatile compounds from honey [22] and royal jelly [23].
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In this study, a HS-SPME technique combined with GC-MS was optimized and applied for the first
104
time to study the volatile organic compounds of Italian raw propolis. No references have been found
105
in the international scientific literature on the use of HS-SPME to describe propolis volatile
106
fraction. Even if volatile compounds are present in low concentration in propolis, their aroma and
107
biological activity make them of importance for the characterization of this product. In addition, the
108
volatile composition can give valuable information about the origin of samples.
109
In particular, after the optimization of the extraction conditions, comparative studies on the typical
110
HS-SPME-GC-MS profiles of raw propolis samples collected from different Italian regions were
111
performed with the aim of confirming the applicability of the method developed for the chemical
112
characterization of their volatile compounds.
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2. Materials and methods
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2.1. Chemicals
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allo-Aromadendrene, α-bisabolol, camphene, β-caryophyllene, caryophyllene oxide, 1,8-cineole, α-
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copaene, dodecane, heneicosane, heptadecane, hexadecane, α-humulene, limonene, linalool, cis-
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linalool oxide, menthol, 2-methyl-butanoic acid, 2-methyl-propanoic acid, myrtenal, myrtenol,
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naphthalene, nonadecane, octadecane, phenylethyl alcohol, α-pinene, tricosane, tridecane, undecane
122
and cis-verbenol were purchased from Sigma-Aldrich-Fluka (Milan, Italy). Citronellol was
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purchased from Curt Georgi Imes (Milan, Italy), thymol from Merck (Darmstadt, Germany) and α-
124
terpineol from Roth (Karlsruhe, Germany).
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2.2. Propolis samples
127 128
Nine samples of raw propolis (indicated in the text as RP-1/RP-9) were collected from Apis 5
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mellifera hives located in different Italian regions in Spring 2012 and stored at –20 °C until
130
chemical analysis. In particular samples RP-1/RP-6 were from Northern Italy, while samples RP-
131
7/RP-9 were from Southern Italy. The frozen samples were finely powdered using a mortar and a
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pestle before the extraction procedure. Sample labeled as RP-9 was used for the optimization of the
133
extraction conditions, because it was available in higher amount.
2.3. HS-SPME procedure
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HS-SPME was performed using a manual holder and two different fibers: a 100 µm
138
polydimethylsiloxane
139
polydimethylsiloxane (DVB-CAR-PDMS) (Supelco, Bellefonte, PA, USA). The coating was 1 cm
140
long for both fibers. Before GC-MS analysis, each fiber was conditioned in the injector of the GC
141
system, according to the instructions provided by the manufacturer.
142
A 2 g amount of finely powdered raw propolis was placed in a 10 ml flat-bottom headspace vial
143
sealed with a magnetic crimp cap and PTFE/silicone septa (Supelco). Under the optimized
144
conditions, the sample was heated for 30 min during the equilibrium time in a thermostatic bath at
145
75 °C. The SPME device was then inserted into the sealed vial by manually penetrating the septum
146
and the fiber was exposed to the headspace for 20 min during the extraction time.
147
After sampling, the SPME fiber was immediately inserted into the GC injector and thermally
148
desorbed. A desorption time of 1 min at 230 °C was used in the splitless mode. Before sampling,
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each fiber was reconditioned for 5 min in the GC injector port at 230 °C.
and
a
stableflex
50/30
µm
divinylbenzene-carboxen-
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(PDMS)
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150 151
2.4. Hydrodistillation (HD) procedure
152 153
The powdered propolis sample RP-9 (190 g) was placed into a 3 L round-bottom distillation flask
154
and then 1 l of water was added. After that, the mixture was distilled for 3 h. The essential oil was 6
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155
collected from the condenser and dried over anhydrous sodium sulfate. The yield of the HD was
156
0.13%. The obtained essential oil was stored at +4°C until analysis. Before GC-MS analysis, the
157
sample was diluted 1:20 (v/v) with n-hexane.
158 159
2.5. GC-MS conditions
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Chromatographic analyses were carried out on a GC 6890 N (Agilent Technologies, Waldbronn,
162
Germany), coupled with 5973 Network mass spectrometer (Agilent Technologies). Compounds
163
were separated using a HP-5 MS cross-linked 5% diphenyl–95% dimethyl polysiloxane capillary
164
column (30 m × 0.25 mm I.D., 1.00 µm film thickness, Agilent Technologies). The oven
165
temperature was programmed from initial 40 °C to final 280 °C at 3 °C/min, which was maintained
166
for 5 min. Splitless injection was used for HS-SPME-GC-MS. As for HD-GC-MS, the injection
167
volume was 0.1 l , with a 1:100 split ratio. Helium was used as the carrier gas at a flow rate of 0.7
168
ml/min. The injector, transfer line and ion-source temperatures were 230, 280 and 230 °C,
169
respectively. MS detection was performed with electron ionization (EI) at 70 eV, operating in the
170
full-scan acquisition mode in the m/z range 40-400.
171
A mixture of aliphatic hydrocarbons (C7-C30) in n-hexane (Sigma-Aldrich) was loaded onto the
172
PDMS fiber and injected under the same conditions to calculate the linear retention index (LRI) of
173
each compound.
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2.6. Qualitative and semi-quantitative analysis
176 177
Compounds were identified by comparing the retention times of the chromatographic peaks with
178
those of authentic reference compounds run under the same conditions and by comparing the LRIs
179
with the literature [24]. Peak enrichment on co-injection with authentic reference compounds was
180
also carried out. The comparison of the MS fragmentation pattern of the target analytes with those 7
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181
of pure compounds was performed. Mass spectrum database search was carried out using the
182
National Institute of Standards and Technology (NIST) mass spectral database (version 2.0d, 2005).
183
The percentage relative amount of individual components was expressed as percent peak area
184
relative to total peak area, in agreement with previous papers on the GC composition of propolis
185
essential oil [7-13]. Semi-quantitative data were the mean of three analyses.
3. Results and discussion
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3.1. Optimization of HS-SPME conditions
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In this study, HS-SPME combined with GC-MS was applied for the first time to the extraction and
192
analysis of volatile compounds in Italian samples of raw propolis. Bicchi et al. [20] have
193
highlighted the importance of the effect of the fiber coating on HS-SPME of volatile compounds
194
from various aromatic and medicinal plants. Therefore, the first step of the method optimization
195
was the selection of the best fiber coating for HS-SPME.
196
In this study, two fiber types, including a PDMS (100 m) and a DVB-CAR-PDMS (50/30 m),
197
which have been frequently employed for the extraction of the volatile fraction from natural
198
products [20], were tested for the analysis of propolis volatile compounds. Fig. 1 shows the results
199
of the fiber selection carried out by HS-SPME-GC-MS. Each fiber was exposed to the HS for the
200
same time at the same temperature.
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Fig. 1
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Of these two fibers, the DVB-CAR-PDMS showed a strong extraction affinity for carboxylic acids
203
and, in particular, for benzoic acid [25]; in samples RP-1, RP-2 and RP-5, where benzoic acid was
204
present at high level, this caused a significant peak broadening and tailing, with the consequent
205
overlapping of adjacent compounds. The PDMS fiber allowed to obtain a better profile for all
206
classes of propolis volatile compounds and, therefore, it was selected for this study. As previously 8
Page 10 of 25
described in the literature [20,26], although multi-component fibers have proved to be very
208
effective, most of the routine applications in the aromatic and medicinal plant fields adopt a PDMS
209
fiber.
210
Other parameters are known to influence the extraction efficiency of the HS-SPME technique [26].
211
Therefore, to obtain the optimal HS-SPME conditions, additional variables were chosen, including
212
sample amount, extraction temperature, equilibrium time and extraction time. Other parameters,
213
such as salt addition and sample agitation during the equilibrium time, were also studied. The sum
214
of peak areas was adopted to optimize the experimental parameters.
215
It is well-known that, for a given vial volume, sample amount has usually a positive effect on peak
216
areas of the compounds extracted by SPME. Indeed, the results showed that the peak areas of the
217
extracted analytes increased by increasing the propolis amount from 1 to 2 g in a 10 ml vial and
218
then reached a plateau for larger amounts (e.g. 3 g). Therefore, 2 g of powdered sample in a 10 ml
219
vial was used for further studies as the optimal sample amount.
220
In relation to the effect of temperature, an increase in sampling temperature usually increases the
221
headspace concentration of volatile compounds, favouring their extraction. However, if temperature
222
is too high, analytes can be desorbed from the SPME fiber, thus reducing the overall sensitivity. In
223
this study, the effect of temperature was studied over the range 70-100 °C and the results showed
224
that a significant jump occurred between 70 and 75 °C; when the temperature was further increased
225
to 80, 85 and 100 °C, no significant increase in the response was observed. Thus, 75 °C was
226
selected as the optimal temperature.
227
The extraction profile was also evaluated by changing the equilibrium time from 15 to 30 min and
228
the extraction time from 5 to 20 min. The results showed that the best global response, within the
229
range studied, was reached with an equilibrium time of 30 min and an extraction time of 20 min.
230
Finally, the addition of a 25-30% (w/v) sodium chloride solution (from 0.5 to 1.5 ml) and the
231
sample agitation during the equilibrium time were not found to increase the HS-SPME efficiency.
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3.2. Analysis of volatile compounds in raw propolis by HS-SPME-GC-MS
234
Volatile compounds in propolis essential oil extracted by HD have been previously determined by
236
GC techniques [7-11]; due to their aroma and biological activity, these compounds are very useful
237
for the chemical characterization of this product [4,6]. Similar to reports on the phenolic
238
composition of hydroalcoholic extracts [1], variations in the chemical profile of propolis volatile
239
fraction between temperate and tropical regions have been described [6]. The volatile chemical
240
composition of propolis is also known to be strongly dependent on the local flora at the site of
241
collection [2,4]. Some authors have reported that the volatile fraction is also influenced by the bee
242
species, because Apis mellifera and Melipona beechei in the same region of Yucatán (Mexico) have
243
produced propolis with different volatile compounds [13]. The same has occurred in Turkey, where
244
phenols and terpenes present in propolis samples were dependent on the race of honeybees [14].
245
In the present study, the powdered raw propolis from different Italian regions was analyzed by HS-
246
SPME-GC-MS. Ninety-nine constituents were identified as shown in Table 1. The typical total ion
247
current (TIC) chromatogram of a representative sample (RP-9) is shown in Fig. 1.
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248
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Table 1
The main volatile compounds identified in propolis samples are benzoic acid (0.87-30.13%) and its
250
esters, including benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and benzyl
251
cinnamate (0.34-3.20%). It must be pointed out that benzyl salicylate and benzyl cinnamate are two
252
known allergens present in propolis and in other matrixes as well [27]. Vanillin, a further aromatic
253
compound previously identified in propolis essential oil [7,8], was detected in most of the samples
254
analyzed in this study (0.07-5.44%). Another relevant class of volatile compounds is represented by
255
sesquiterpene hydrocarbons, such as -cadinene (1.29-13.31%), -cadinene (1.36-8.85%) and -
256
(0.78-6.59%), and oxygenated sesquiterpenes, such as -eudesmol (2.33-12.83%), T-
257
cadinol (2.73-9.95%) and -cadinol (4.84-9.74%). Germacrene D-4-ol was detected only in sample
258
RP-7 (6.32%). Regarding monoterpene hydrocarbons, they were present at low level in the samples
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Page 12 of 25
analyzed in this study, with the exception of sample RP-7 from Southern Italy, where -pinene was
260
the most abundant constituent (13.19%). As for oxygenated monoterpenes, menthol was determined
261
mainly in sample RP-6 (2.54%) and thymol in samples RP-4 and RP-6 (2.08 and 3.27%,
262
respectively). Hemiterpenes were detected at low percentages in all the samples and were absent in
263
sample RP-7. The presence of naphthalene (0.97%) in sample RP-5 is probably due to pollution
264
attributable to unexpected material collected by bees [8] or to inappropriate environmental storage
265
conditions of propolis and confirms a previous observation that propolis could be used as a marker
266
of pollution [9].
267
The analysis of variance (ANOVA) was used to evaluate the statistical significance of the measured
268
differences between HS-SPME-GC-MS data of propolis samples of different geographic origin
269
(Northern and Southern Italy), with a P level set at 0.05. The statistical analysis was performed
270
using Statistica 6 for Windows (StatSoft® Italia, Vigonza, Italy). ANOVA was applied to the
271
volatile compounds shared by all samples of at least one geographic origin. Sample RP-7 was
272
excluded from the statistical analysis, due to its peculiar composition. Significant differences
273
between propolis samples from Northern and Southern Italy were observed for 2-methyl-2-buten-1-
274
ol, 2-methyl-butanoic acid, linalool, -cadinol and heptadecane.
275
The chemical composition of propolis volatile fraction determined in the present study by means of
276
HS-SPME-GC-MS was found to be in agreement with previous reports [7,8]. In general, propolis
277
constituents are known to be directly related to those of bud exudates collected by honeybees from
278
various trees [2,4]. Indeed, several volatile compounds identified in propolis volatile fraction have
279
been previously detected from leaf buds of Populus nigra L., which represents one of the main
280
botanical sources of propolis constituents in temperate regions [2]. In particular, the essential oil
281
from leaf buds of P. nigra is composed mainly by oxygenated sesquitepenes (35.7-41.7%) and
282
sesquiterpene hydrocarbons (35.2-36.5%) [28]. The most abundant volatile compound is -
283
eudesmol (19.1-19.6%) [28]. Hemiterpenes have also been identified (2.2-7.6%) [28], while
284
monoterpenes have been determined in lower percentages (1.6-5.7%) [28]. Aliphatic and aromatic
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Page 13 of 25
alcohols, carbonyl compounds and aliphatic acids have been characterized among non-terpene
286
volatiles (9.8-13.5%) [28].
287
According to Petri et al. [7], propolis from temperate zones can be separated in two types, based on
288
the presence of representative amounts of -eudesmol (40-60%) or benzyl benzoate (20-40%) in the
289
essential oil. In a subsequent study on the volatile compounds isolated from propolis collected in
290
two different regions of Croatia, benzoic acid (27.0%), benzyl alcohol (18.2%) and benzyl benzoate
291
(3.6%) were the predominant constituents of the essential oil from Slavonia [8]. Another study
292
carried out on the essential oil composition in propolis samples from various regions of Greece
293
indicated a uniformly elevated concentration of -pinene [10], except in a sample from one
294
location, in which junipene was determined as the main constituent.
295
The volatile composition of the Italian samples of raw propolis investigated in this work indicates a
296
close relationship with bud exudates of Populus species, which is also the well-known source of
297
typical poplar bud phenolics (including phenolic acids and flavonoids), identified in the same
298
samples by HPLC [29]. In the case of sample RP-7, originated from a region of Southern Italy
299
(Adriatic coast) where poplars are not present, the high percentage of -pinene suggests that the
300
botanical source of bee glue may be attributed to Pinus species [30,31], in agreement with what has
301
been previously observed in a propolis sample from Portugal (Mogadouro region) [32].
302
304
3.3. Repeatability
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305
The repeatability was determined by performing five replicate HS-SPME-GC-MS experiments on
306
the same propolis sample (RP-9), under the optimized extraction conditions. The relative standard
307
deviation (RSD) values of % relative peak area of volatile constituents were ≤ 11%, indicating a
308
satisfactory repeatability of the developed method. As previously indicated in the literature, the
309
widespread use of the PDMS fiber is not only attributable to its good recovery for the HS
310
components of medicinal and aromatic plants, because, in general, its polarity is from medium to 12
Page 14 of 25
311
low, but also to both the consistency of its performance and repeatability, especially when a large
312
number of samples are involved [20].
313 314
3.4. Comparison of HS-SPME-GC-MS and HD-GC-MS
315
In this study, the propolis volatile profile obtained by HS-SPME-GC-MS was compared with that of
317
traditional HD-GC-MS. As shown in Fig. 2, the TIC chromatograms of these techniques share the
318
same components and comparable values of % relative peak area were observed in most cases.
319
However, some differences between HS-SPME-GC-MS and HD-GC-MS in terms of the relative
320
amount of some compounds, such as hemiterpenes and sesquiterpenes, were found. In particular,
321
compared with the HD-GC-MS method, the proposed HS-SPME-GC-MS technique allowed to
322
obtain higher % relative peak area values for -cadinene, T-cadinol and -cadinol, but lower for 3-
323
methyl-3-buten-1-ol and 2-methyl-2-buten-1-ol. These differences might result from the higher
324
extraction efficiency of the PDMS fiber for less polar compounds. Benzoic acid and vanillin were
325
detected by HS-SPME-GC-MS only.
ed
M
an
us
cr
ip t
316
326
ce pt
Fig. 2
Although the composition of samples extracted by HD and HS-SPME is sometimes similar, the %
328
relative peak areas of an analyte obtained with the two techniques are not easily interchangeable,
329
because they are obtained from entirely different approaches [20]. It is well-known that these
330
intrinsic methodological differences greatly influence the resulting quantitative composition and, to
331
a lesser extent, also the qualitative profile [20]. An additional factor of discrimination in HS-SPME
332
is the nature of the polymeric coatings of the fiber, which conditions the composition of the volatile
333
fraction recovered [20].
Ac
327
334 335
4. Conclusions
336 13
Page 15 of 25
The present study describes a new, rapid and simple HS-SPME-GC-MS method for the analysis of
338
propolis volatile compounds. The parameters affecting the extraction efficiency were optimized.
339
Compared with HD-GC-MS, the HS-SPME-GC-MS procedure developed in this study can greatly
340
simplify and shorten the extraction and analysis of volatile compounds from propolis. In addition,
341
this technique needs much less sample amount and is environmentally friendly.
342
The application of the developed technique to samples of raw propolis collected from different
343
Italian regions allowed to obtain the volatile profile of this biologically active apiary product.
344
In conclusion, HS-SPME-GC-MS represents an effective technique for the analysis of volatile
345
compounds in bee glue. The developed method could also be applied to the evaluation of the
346
antimicrobial activity in the vapour phase of propolis constituents against different bacterial and
347
fungal strains.
an
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337
349
M
348
Acknowledgements
351
ed
350
The authors are grateful to Kontak (Pozzo d’Adda, Milan, Italy) for the financial support.
ce pt
352
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354
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volatile and extractable compounds of crude royal jelly, J. Chromatogr. B 885-886 (2012) 109-116.
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433 434
cr
us
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Ac
432
ip t
414
435 436 437 438 439 17
Page 19 of 25
440
Figure captions
441
Figure 1: Total ion current (TIC) chromatograms obtained by HS-SPME-GC-MS analysis of
443
volatile compounds from propolis (sample RP-9) using (A) a 100 µm PDMS fiber and (B) a
444
stableflex 50/30 µm DVB-CAR-PDMS fiber. For peak identification, see Table 1. Experimental
445
conditions as in sections 2.3 and 2.5.
ip t
442
cr
446
Figure 2: Total ion current (TIC) chromatograms of volatile compounds from propolis (sample RP-
448
9) by (A) HS-SPME-GC-MS and (B) HD-GC-MS. For peak identification, see Table 1.
449
Experimental conditions as in sections 2.3, 2.4 and 2.5.
Ac
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M
an
us
447
18
Page 20 of 25
1.77 ± 0.11 0.35 ± 0.01 0.10e 0.24 ± 0.02 0.13 ± 0.01 0.54 ± 0.03 0.16 ± 0.01 0.06e 1.16 ± 0.12 1.07 ± 0.06 0.07e 3.91 ± 0.41 0.06e 0.18 ± 0.02 0.26 ± 0.02 1.05 ± 0.11 8.29 ± 0.45 0.56 ± 0.06 0.12 ± 0.01 -
1.19 ± 0.13 0.32 ± 0.01 0.38 ± 0.04 0.37 ± 0.03 0.12e 0.22 ± 0.02 0.46 ± 0.05 0.14e 0.07e 0.23 ± 0.01 0.76 ± 0.05 0.09e 0.06e 0.61 ± 0.05 12.08 ± 1.29 0.39 ± 0.03 -
an
1.94 ± 0.19 0.28 ± 0.02 0.48 ± 0.03 0.41 ± 0.02 0.37 ± 0.01 0.24 ± 0.02 0.07 ± 0.01 0.30 ± 0.02 0.19e 0.38 ± 0.01 0.20e 0.07e 1.54 ± 0.09 0.17e 2.07 ± 0.20 0.17 ± 0.01 0.36 ± 0.01 1.40 ± 0.11 20.78 ± 0.09 0.22e -
M
1.82 ± 0.09 0.30 ± 0.01 0.27e 0.36e 0.07e 0.20e 0.39e 0.23 ± 0.01 0.46 ± 0.02 0.04e 1.59 ± 0.07 0.06e 1.02 ± 0.03 21.79 ± 0.19 -
d
606 625 722 736 769 782 800 829 839 890 911 922 931 941 956 959 966 987 1003 1019 1037 1037 1039 1039 1078 1099 1101 1105 1119 1137 1154 1157 1165 1168 1178 1181 1187 1194
ep te
Acetic acid 2-Methyl-3-buten-2-ol 3-Methyl-3-buten-1-ol 2-Methyl-propanoic acid 2-Methyl-2-buten-1-ol 3-Methyl-2-butenal Hexanal 3-Methyl-butanoic acid 2-Methyl-butanoic acid Styrene 2-Methyl-2-butenoic acid Prenyl acetate Tricyclene α-Pinene α-Fenchene Camphene Benzaldehyde β-Pinene Octanal Δ-3-Carene Benzyl alcohol Limonene 1,8-Cineole β-Phellandrene cis-Linalool oxide Undecane Linalool Nonanal Phenylethyl alcohol -Campholenal cis-Verbenol trans-Verbenol Benzoic acid Benzyl acetate Pinocarvone Menthol Terpinen-4-ol p-Cymen-8-ol
Ac c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
cr
RP-5c
us
Table 1 Volatile components identified in raw propolis samples (RP-1/RP-9) by HS-SPME-GC-MSa Peak Compoundb LRI RP-1c RP-2c RP-3c RP-4c number
ip t
Table 1
0.67 ± 0.03 0.06e 0.06e 0.10e 0.13 ± 0.01 0.16 ± 0.01 0.22 ± 0.02 0.88 ± 0.02 0.08e 0.05e 0.09e 0.08e 0.39 ± 0.01 30.13 ± 2.60 -
RP-6c
RP-7c
RP-8c
RP-9c
Identification methodd
1.33 ± 0.10 0.39 ± 0.03 0.20e 0.56 ± 0.05 0.32 ± 0.02 0.38 ± 0.03 0.56 ± 0.06 0.09e 0.10e 0.75 ± 0.08 0.07e 0.14 ± 0.02 0.73 ± 0.08 3.12 ± 0.31 0.43 ± 0.05 2.54 ± 0.28 -
0.17 ± 0.01 0.15 ± 0.01 13.19 ± 1.56 0.09e 0.22 ± 0.02 0.23 ± 0.02 0.07e 0.27 ± 0.03 0.27 ± 0.03 0.09 ± 0.01 0.23 ± 0.02 1.31 ± 0.15 1.01 ± 0.12 4.55 ± 0.29 0.87 ± 0.10 0.35 ± 0.04 0.28 ± 0.03
1.30 ± 0.15 0.29 ± 0.03 0.14 ± 0.01 0.66 ± 0.07 0.10 ± 0.01 0.94 ± 0.09 0.24 ± 0.01 0.27 ± 0.03 0.58 ± 0.05 0.12 ± 0.01 0.07e 0.20 ± 0.02 0.17 ± 0.01 0.05e 0.28 ± 0.02 18.62 ± 1.78 -
1.66 ± 0.14 1.71 ± 0.04 1.69 ± 0.04 0.56 ± 0.01 0.50 ± 0.01 0.13 ± 0.01 0.27 ± 0.01 1.44 ± 0.01 0.21 ± 0.02 0.86 ± 0.09 0.29 ± 0.01 1.39 ± 0.07 1.17 ± 0.05 0.12e 0.52e 0.77 ± 0.01 1.84 ± 0.01 4.69 ± 0.44 0.50 ± 0.02 -
b, d b, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d b, d a, b, c, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d a, b, c, d b, d a, b, c, d b, d a, b, c, d b, d bd b, d b, d a, b, c, d b, d b, d
Page 21 of 25
ip t
0.97 ± 0.07 0.13e 0.10 ± 0.01 0.13e 0.12e 0.23e 0.18 ± 0.01 0.12e 5.44 ± 0.24 0.36 ± 0.02 0.29 ± 0.02 0.78 ± 0.06 1.36 ± 0.13 1.29 ± 0.12 0.96 ± 0.05 0.25e -
0.16 ± 0.01 0.09 ± 0.01 0.29 ± 0.03 3.27 ± 0.19 0.17 ± 0.01 0.11e 0.30 ± 0.01 1.93 ± 0.09 0.07e 0.11 ± 0.01 0.09e 0.04e 2.60 ± 0.13 4.17 ± 0.36 6.59 ± 0.59 8.85 ± 0.48 13.31 ± 0.84 4.05 ± 0.15 2.06 ± 0.03 0.47 ± 0.05
cr
0.14 ± 0.01 0.13 ± 0.01 0.11 ± 0.01 2.08 ± 0.23 0.10e 0.31 ± 0.01 0.16 ± 0.01 0.06e 0.69e 0.50 ± 0.02 0.20 ± 0.02 0.23 ± 0.02 1.01 ± 0.02 2.39 ± 0.22 2.97 ± 0.20 3.88 ± 0.29 7.87 ± 0.43 1.36 ± 0.11 0.63 ± 0.02 0.71 ± 0.07 0.21 ± 0.02 0.54 ± 0.01
us
0.21 ± 0.02 0.12 ± 0.01 0.16 ± 0.02 0.50 ± 0.05 0.38 ± 0.04 0.20 ± 0.02 0.44 ± 0.03 0.62 ± 0.07 0.90 ± 0.05 0.49 ± 0.02 2.65 ± 0.22 0.32 ± 0.01 0.23 ± 0.02 0.15 ± 0.01 0.51 ± 0.02 0.43 ± 0.05 0.77 ± 0.08 1.34 ± 0.04 1.53 ± 0.02 2.18 ± 0.18 0.77 ± 0.08 1.34 ± 0.05 0.44 ± 0.01 0.40 ± 0.04 1.26 ± 0.01
an
0.23 ± 0.01 0.57 ± 0.03 0.18 ± 0.01 0.51 ± 0.02 0.90 ± 0.07 0.14 ± 0.01 0.70 ± 0.03 0.33 ± 0.01 0.37e 0.40 ± 0.03 2.67 ± 0.19 0.61 ± 0.05 0.25 ± 0.01 0.34 ± 0.03 1.04 ± 0.05 2.02 ± 0.11 1.95 ± 0.07 0.98 ± 0.08 0.65 ± 0.04 1.06 ± 0.05
M
0.20 ± 0.01 0.50 ± 0.02 0.36e 0.20e 0.48 ± 0.02 0.20 ± 0.01 0.28 ± 0.03 0.45 ± 0.03 3.21 ± 0.05 0.81 ± 0.07 0.26 ± 0.02 0.35 ± 0.02 1.14 ± 0.11 1.73 ± 0.09 1.75 ± 0.05 2.68 ± 0.24 1.76 ± 0.11 2.04 ± 0.24 1.22 ± 0.08 0.54 ± 0.05
d
1199 1200 1202 1206 1209 1210 1218 1229 1232 1257 1262 1282 1291 1298 1313 1355 1358 1362 1368 1370 1385 1388 1400 1405 1436 1438 1452 1472 1479 1484 1487 1510 1529 1534 1534 1537 1545 1548 1560 1598 1605 1609
ep te
Dodecane Naphthalene α-Terpineol Decanal Myrtenol Myrtenal Verbenone Citronellol β-Cyclocitral Nonanoic acid β-Phenylethyl acetate Cinnamaldehyde Thymol Tridecane Cinnamyl alcohol Decanoic acid α-Terpinyl acetate α-Cubebene Eugenol Acetocinnamone -Ylangene α-Copaene -Bourbonene Vanillin Cinnamic acid β-Caryophyllene Cinnamyl acetate α-Humulene allo-Aromadendrene ar-Curcumene γ-Muurolene α-Muurolene γ-Cadinene Germacrene D-4-ol δ-Cadinene Calamenene Dodecanoic acid Cadina-1,4-diene α-Calacorene Hexadecane Caryophyllene oxide Guaiol
Ac c
39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
0.79 ± 0.07 0.57 ± 0.01 0.24 ± 0.02 0.78 ± 0.03 1.11 ± 0.12 4.50 ± 0.15 0.34 ± 0.03 1.00 ± 0.12 0.73 ± 0.07 6.32 ± 0.04 2.90 ± 0.33 0.37 ± 0.03 3.30 ± 0.38 -
0.10e 0.52 ± 0.05 0.64 ± 0.06 0.12 ± 0.01 0.41 ± 0.04 0.29 ± 0.03 0.84 ± 0.09 1.51 ± 0.17 2.41 ± 0.26 3.10 ± 0.34 6.77 ± 0.74 1.29 ± 0.15 0.57 ± 0.05 0.47 ± 0.05 0.45 ± 0.05 -
0.88 ± 0.05 0.19 ± 0.01 0.35 ± 0.01 0.49e 0.12 ± 0.01 0.15 ± 0.01 0.21 ± 0.02 1.00 ± 0.03 0.45 ± 0.04 1.04 ± 0.07 2.27 ± 0.07 2.38 ± 0.10 3.58 ± 0.12 6.16 ± 0.08 1.99 ± 0.16 0.88 ± 0.04 0.46 ± 0.05 -
a, b, c, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d b, d a, b, c, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d b, d b, d b, d a, b, c, d b, d a, b, c, d a, b, c, d b, d b, d b, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d
Page 22 of 25
an
M
ip t
4.96 ± 0.06 1.65 ± 0.15 2.33 ± 0.38 1.74 ± 0.20 0.99 ± 0.08 0.71 ± 0.06 1.98 ± 0.22 0.91 ± 0.10
3.42 ± 0.39 3.68 ± 0.24 8.90 ± 0.40 2.55 ± 0.07 9.74 ± 0.37 6.78 ± 0.54 0.62 ± 0.02 6.91 ± 0.15 1.25 ± 0.15 0.74 ± 0.03 0.79 ± 0.09 1.49 ± 0.01 1.81 ± 0.21
3.94 ± 0.10 1.36 ± 0.10 9.95 ± 0.06 2.37 ± 0.04 8.36 ± 0.31 3.08 ± 0.28 0.66 ± 0.01 0.40 ± 0.04 0.59 ± 0.06 3.37 ± 0.34
b, d b, d b, d b, d b, d b, d b, d b, d b, d a, b, c, d a, b, c, d b, d a, b, c, d a, b, c, d b, d b, d a, b, c, d b, d a, b, c, d
Ac c
ep te
d
0.22 ± 0.02 3.78 ± 0.10 1.40 ± 0.07 7.96 ± 0.31 1.70 ± 0.18 0.48 ± 0.03 6.53 ± 0.10 2.88 ± 0.29 1.26 ± 0.13 0.16 ± 0.01 0.28 ± 0.03 0.31 ± 0.03 0.94 ± 0.09
cr
1.35 ± 0.12 1.93 ± 0.11 3.50 ± 0.29 0.92 ± 0.09 0.52 ± 0.02 4.84 ± 0.49 3.99 ± 0.10 0.51 ± 0.03 0.54 ± 0.02 13.05 ± 0.57 0.95 ± 0.04 1.90 ± 0.03 0.76 ± 0.01 3.20 ± 0.20 1.46 ± 0.11
us
81 Viridiflorol 1615 0.18 ± 0.02 82 Cedrol 1620 3.26 ± 0.31 83 Cubenol 1650 1.94 ± 0.18 1.10 ± 0.03 0.53 ± 0.03 3.64 ± 0.26 84 γ-Eudesmol 1655 2.05 ± 0.13 3.40e 2.47 ± 0.12 2.65 ± 0.30 85 1657 4.70 ± 0.02 3.44 ± 0.35 2.73 ± 0.17 9.03 ± 0.77 -Cadinol 86 δ-Cadinol 1662 1.31 ± 0.05 1.12 ± 0.02 0.72 ± 0.07 2.04 ± 0.03 87 α-Copaen-11-ol 1668 0.44 ± 0.04 0.72 ± 0.04 1.39 ± 0.14 88 α-Cadinol 1676 5.23 ± 0.30 7.94 ± 0.30 89 β-Eudesmol 1677 4.86 ± 0.51 12.83 ± 0.45 9.85 ± 0.17 7.49 ± 0.22 90 Heptadecane 1699 91 α-Bisabolol 1700 5.34 ± 0.55 92 Benzyl benzoate 1783 9.59 ± 0.47 5.30 ± 0.63 3.22 ± 0.29 1.92 ± 0.20 93 Octadecane 1801 94 Nonadecane 1899 0.44 ± 0.03 0.49 ± 0.01 0.53 ± 0.04 0.91 ± 0.09 95 Benzyl salicylate 1903 1.46e 0.74 ± 0.08 0.63 ± 0.07 0.34 ± 0.03 96 Manoyl oxide 2010 0.39 ± 0.04 97 Heneicosane 2100 0.47 ± 0.02 0.57e 0.57 ± 0.06 0.66e 98 Benzyl cinnamate 2109 2.29 ± 0.08 1.27 ± 0.10 1.99 ± 0.20 0.34 ± 0.01 99 Tricosane 2300 1.27 ± 0.08 1.88 ± 0.03 1.94 ± 0.14 1.54 ± 0.06 a Experimental conditions as in sections 2.3 and 2.5. b Compounds are listed in order of elution time. c Data are expressed as mean (n = 3) of % relative peak area values ± SD. For sample RP-9, n = 5. d a: retention time; b: LRI; c: peak enrichment; d: mass spectrum. e SD < 0.005.
Page 23 of 25
Figure 1
Abundance
A
85
240000 220000
88
200000
73
180000 160000 99
ip t
140000 71
120000
86
100000
89 33
3
70 69
29
5
84
23
40000
1 6 9
27
11
19
14
10
42
28
17
20000
60
48 50 49 53
34
54
47
25
67
77
30.00
40.00
Abundance
70.00
ce pt
700000 600000
21
500000
80.00
B
ed
800000
89 88 73
29
85 71
34
400000
11
84
42
12
300000
Ac
3
1
17
70
50 53
27 28
5
74
69
86
49 23
62 54 60
6
100000
60.00
33
900000
200000
50.00
94
an
20.00
97
M
10.00
90
78
62
0 Time-->
83
21
us
60000
12
cr
80000
74
9
14
77 78
47
19 10
83 67
25
90 94
97
99
0 Time-->
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Figure 1
Page 24 of 25
Figure 2
Abundance
A 85
240000 e 220000
88
0 200000
73
0 180000 0 160000 99
0 120000
71 86
0 100000
89
80000
33
3
12
0
5
23 6
48
27
11
9
19
14
42
28
17
10
34
60
50 49
53 54
47
25
0 10.00
20.00
30.00
40.00
>
0
0
0
0
67
77
78
62
Abundance
3
60.00
70.00
80.00
0
0
0
0
B
88 85
ce pt
700000
89
42
500000 400000
94
50.00
ed
5
600000
90
97
M
Time--> 0
800000
83 84
21
0 40000 0 20000
70 69
29
us
0 60000
cr
74
an
0
ip t
0 140000
73
27
71
28
300000
Ac
200000
17
12
6
100000
9
99 84
29
21
70 74 69 83
25 19
23
34
49 48
54 60
47 50
11
86
67
77 78
97 90
53
10 14
94
0 Time-->
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Figure 2
Page 25 of 25