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Screening of the essential oil composition of wild Sicilian thyme
Biochemical Systematics and Ecology 38 (2010) 816–822
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Screening of the essential oil composition of wild Sicilian thyme Edoardo M. Napoli, Giusy Curcuruto 1, Giuseppe Ruberto* Istituto del CNR di Chimica Biomolecolare, Via Paolo Gaifami, 18–95126 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2010 Accepted 8 August 2010
Thirty samples of wild thyme collected from as many Sicilian locations have been analysed for their qualitative and quantitative essential oil profiles. The oils, obtained by hydrodistillation, have been analysed by a combination of GC-FID-MS; in all, 46 components, representing more than 98% of the oils, were fully characterized. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented components: the former with a range of 8–61% and the latter with a range of 31–86%. Carvacrol was the main compound in 29 samples, ranging between 49 and 83%, suggesting that Thymus capitatus Hoff. et Link. [syn. Coridothymus capitatus (L.) Rchb.f., Satureja capitata L., Thymbra capitata (L.) Cav.] is the most widespread wild species in the Sicilian area. Only one sample, identified as Thymus longicaulis C. Presl., collected from the North-East of Sicily showed a different composition, p-cymene and thymol being the main compounds with 40 and 16%, respectively. Statistical analyses allowed establishing a single broad group, confirming the substantial compositional uniformity of the essential oil profiles of the wild Sicilian thyme. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Thymus capitatus Hoff. et Link Thymus longicaulis C. Presl. Lamiaceae Essential oil Carvacrol p-Cymene GC–MS Cluster analysis Sicily
1. Introduction It is well-known that most spices, especially those belonging to the Lamiaceae family, have a wide range of biological and pharmacological activities. The geomorphologic and climatic conditions on the island of Sicily in the heart of the Mediterranean, provide an optimal habitat for many aromatic plants of this family to grow in the wild; indeed, the cultivation of aromatic plants is an important vocation here. They have become the common raw materials for many traditional uses such as natural remedies for human and animal diseases, for combating pests and parasites and making homemade perfumes and spices for cooking. Pursuing our project aimed at the valorisation and exploitation of wild aromatic Sicilian plants, we report here the results of a study on the essential oil of wild Sicilian thyme (Thymus capitatus Hoff. et Link.) or Thymbra capitata (L.) Cav. Thyme is a woody, perennial herb native to the Mediterranean region. Its name probably derives from the Latin “thymus” which means “perfumed” or from Greek “thymos” which means “courage” or “strength”. The Thymus genus, with its estimated 250–350 taxa (species and varieties) of wild growing plants (Morales, 2002; Lawrence and Tucker, 2002), complicated further by the presence of several chemotypes regarding the essential oil profiles associated to several species (Senatore, 1996), is one of the most taxonomically complex genera in the Lamiaceae family. As an aromatic plant, thyme is an important spice in home cooking as well as in the food and aroma industries. At the same time it is a widely used medicinal plant, with an ancient tradition dating back to the Egyptians, who used it in unguents for embalming and then the Greeks and Romans who used it for therapeutic purposes (Barros et al., 2010). Infusions are used for treating ulcers, dermatitis and some types of rheumatic pains, while thyme is used to fight pathologies of the respiratory
* Corresponding author. Tel.:þ39 0957338347; fax: þ39 0957338310. E-mail address: [email protected] (G. Ruberto). 1 Present address: Dipartimento di Metodologie Fisiche e Chimiche per l’Ingegneria (DMFCI), Università di Catania, V.le A. Doria, 6 - 95125 Catania, Italy. 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.08.008
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apparatus owing to its expectorant, spasmolytic and antiseptic properties (Zarzuelo and Crespo, 2002; Mulas, 2006). All the aforesaid activities can be ascribed to the considerable presence of polyphenol derivatives (Vila, 2002) in thyme extracts and/ or infusions. However, it is the essential oil (EO) that has a broad spectrum of bioactivities. In fact, it serves as a preservative for food due to the antimicrobial properties of its volatile components (Bagamboula et al., 2004; Baydar et al., 2004; Rasooli et al., 2006; Omidbeygi et al., 2007; Solomakos et al., 2008; Nedorostova et al., 2009), as an antioxidant (Lee et al., 2005; Bozin et al., 2006), as an additive to enhance organoleptic characteristics (Viuda-Martos et al., 2009) and physical properties (Bensmira et al., 2007). The thyme EO is also used as a raw material in perfumery and cosmetics (Zarzuelo and Crespo, 2002; Inouye and Abe, 2006). From a chemical point of view thyme essential oils are characterized by a large amount of monoterpenes, which normally account for 90% of oil. The phenolic monoterpenes thymol and carvacrol occur more frequently, always accompanied by the couple p-cymene/g-terpinene, the four monoterpenes being biogenetically closely correlated. Linalool, borneol, 1,8-cineole are the other oxygenated monoterpenes, in order of importance, singled out in thyme essential oils (Stahl-Biskup, 2002, 2004). In particular, concerning the capitatum species, or T. capitata, carvacrol generally proves to be the characteristic constituent (Karousou et al., 2005; Bounatirou et al., 2007). However, as aforementioned, different chemotypes have been reported for many Thymus species, so this species also shows different chemical profiles: thymol, carvacrol and thymol/ carvacrol (Karousou et al., 2005; Miceli et al., 2006). 2. Materials and methods 2.1. Plant material Table 1 lists the thirty samples of thyme collected in different locations of Sicily, which are reported in Fig. 1. All the plant samples were harvested in the years 2006–2007 at bloom stage and were naturally dried at the collection point. Voucher specimens were deposited in the herbarium of the respective Regional Unit listed in Table 1, which also carried out the botanical identification. 2.2. Isolation of the essential oil The air-dried aerial part of each plant (50–100g) was subjected to hydrodistillation in accordance with current European Pharmacopoeia (European Pharmacopoeia, 2008) until there was no significant increase in the volume of oil collected (3h). Table 1 Collection data and essential oil yield of analysed samples of thyme (Thymus capitatus Hoff. et Link.). Sample
Site
Regional Unit
Date
Yield % (v/w)
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 D1 N1 N2 N3 N4 N5 N6 N7 N8 N9
Partanna (Trapani) Partanna (Trapani) Santa Elisabetta (Agrigento) Sant’Angelo Muxaro (Agrigento) Palazzo Adriano (Palermo) Mazara del Vallo (Trapani) Favignana (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Alcamo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Cambobello di Mazara (Trapani) Cambobello di Mazara (Trapani) Palazzo Adriano (Palermo) Galati Mamertino (Messina)a Noto (Siracusa) Noto (Siracusa) Calascibetta (Enna) Calascibetta (Enna) Calascibetta (Enna) Caltagirone (Catania) Caltagirone (Catania) Noto (Siracusa) Noto (Siracusa)
SOPAT Partanna SOPAT Partanna UOT Aragona UOT Aragona UOT Lercara Friddi UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Alcamo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Castelvetrano UOT Castelvetrano UOT Lercara Friddi UOT Sant’Agata di Militello UOT Acireale UOT Acireale UOT Enna UOT Enna UOT Enna UOT Acireale UOT Acireale UOT Palazzolo Acreide UOT Palazzolo Acreide
2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007 2006 2006 2006 2006 2006 2007 2007 2007 2007
2,80 3,30 4,48 2,50 2,70 4,50 3,20 2,20 2,80 3,20 2,60 1,90 3,90 4,30 4,30 3,20 4.50 3,00 3,00 4,60 1,40 5,30 4,80 3,50 3,40 4,20 5,60 4,60 5,00 5,00
a
Thymus longicaulis C. Presl.
818
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Fig. 1. Sicilian collection localities of investigated wild thyme samples.
The oils, whose yield is listed in Table 1, were dried over anhydrous sodium sulphate and stored under N2 in a sealed vial until required. 2.3. GC and GC–MS analyses A Shimadzu GC-17A Gas Chromatograph equipped with a fused silica capillary column (Supelco SPBTM-5 15 m 0.1 mm 0.1 mm) and FID was used. The operating conditions were the following: 60 C for 1 min, 60–280 C at 10 C/min then 280 C for 1 min; injector temperature 250 C; detector temperature 280 C; carrier gas helium (1 mL/min); split mode (1:200), volume of injection 1 mL (4% essential oil/CH2Cl2 v/v). Percentages of compounds were determined from their peak areas in the GC-FID profiles. GC–MS analyses were performed on a Shimadzu GCMS-QP5050A with the same column and the same operating conditions used for analytical GC. Ionization was performed at 70 eV. Ion source temperature 180 C; mass spectral data were acquired in the scan mode in m/z range 40–400. Oil solutions were injected with the split mode (1:96). 2.4. Identification procedure The identity of components was based on their retention index relative to C9–C22 n-alkanes (Alltech Italy) on the SPB-5 column, computer matching of spectral MS data those from NIST MS 107 and NIST 21 libraries (NIST 1998), the comparison of the fragmentation patterns with those reported in literature (Adams, 2001) and, whenever possible, co-injections with authentic samples. Pure standards were purchased from Aldrich Chemical Co., Extrasynthese, France, and Fluka Chemie AG, Switzerland. 3. Statistical analysis All data were statistically analysed using the STATWIN 6.0 software package in order to determine the relationship between the different samples of thyme using the percentage composition of their essential oils. Euclidean distance was selected as a measure of similarity and the unweighted pair-group method with arithmetic average (UPGMA) was used for cluster definition. 4. Results and discussion Tables 2 and 3 list the composition of the essential oils of the thirty samples of thyme collected during the biennium 2006– 2007. Samples have been pooled according to a historical territorial subdivision of Sicily island: Val Mazara (M), Val di Noto
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Table 2 Chemical composition of wild Sicilian thyme essential oils from Val Mazara (M).a KIb
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20
1 2 3 4 5 8 10 11 12 13 14 16 17 19
Monoterpene Hydrocarbons a-Thujene a-Pinenec Camphenec Sabinenec b-pinenec b-Myrcenec a-Phellandrene p-Menth-1-(7),8-diene a-Terpinenec p-Cymemec Limonenec trans–b-Ocimene g-Terpinenec Terpinolenec
929 936 951 975 979 990 1003 1010 1016 1024 1028 1046 1058 1083
17,2 1,5 0,8 0,5 t 0,4 1,7 0,2 0,1 1,2 4,7 0,4 0,1 5,3 0,1
18,5 1,6 0,8 0,3 t 0,4 1,8 0,3 0,1 1,4 4,3 0,4 0,1 6,7 0,1
14,6 1,4 0,6 0,1 – 0,6 1,7 0,3 0,1 1,1 3,8 0,4 0,1 4,0 0,1
14,3 1,1 0,5 0,1 – 0,4 1,4 0,2 0,1 1,2 3,5 0,3 0,1 4,9 0,1
20,4 1,0 0,6 0,1 – 0,4 1,6 0,2 0,1 1,6 5,7 0,4 0,1 8,5 0,1
17,0 1,6 0,7 0,2 – 0,6 1,9 0,3 0,1 1,5 4,0 0,4 – 5,5 0,1
15,3 1,0 0,8 0,4 – 0,5 1,5 0,2 0,1 1,0 6,2 0,4 – 3,2 0.4
7,7 0,2 0,4 0,1 – 0,5 0,7 0,1 0,1 0,4 4,1 0,3 – 0,7 0,1
14,4 0,2 0,7 0,1 – 0,3 1,2 0,2 0,1 1,0 7,9 0,4 – 2,1 0,2
10,8 0,4 0,4 0,1 – 0,5 1,0 0,1 0,1 0,7 5,2 0,4 – 1,9 0,1
10,8 0,5 0,5 0,1 – 0,4 1,1 0,1 0,1 0,7 4,7 0,3 – 2,2 0,1
33,6 0,9 0,8 1,1 – 1,9 0,9 0,1 0,1 1,0 19,1 0,5 – 7,1 0,2
11,3 1,3 0,6 0,1 t – 1,6 0,2 0,1 0,7 4,1 0,4 t 1,9 0,1
14,5 1,4 0,6 0,1 – – 1,7 0,2 0,1 1,2 4,4 0,4 0,1 4,0 0,1
12,2 1,4 0,5 0,1 – – 1,6 0,2 0,1 0,9 3,8 0,4 0,1 2,8 0,1
14,5 0,9 0,6 0,1 t – 1,5 0,2 0,1 1,2 4,7 0,4 – 4,6 0,1
12,2 1,2 0,7 0,2 – – 1,5 0,2 0,1 0,8 4,9 0,4 – 1,9 0,1
14,1 0,9 0,5 0,1 t – 1,4 0,2 0,1 1,2 4,7 0,4 t 4,3 0,1
16,1 0,9 0,6 0,2 t – 1,5 0,2 0,1 1,4 5,0 0,4 0,1 5,6 0,1
21,5 0,8 0,6 0,1 – – 1,6 0,2 0,1 1,8 5,7 0,4 0,1 10,0 0,3
15 18 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 36 38
Oxygenated Monoterpenes 1,8-Cineolec Sabinene hydratec Linalool Camphorc Borneol Terpinen-4-olc p-Cymen-8-ol trans-Dihydro Carvone Nerol Thymol methyl ether Carvacrol methyl ether Neral Carvone Geraniol Geranial Thymol Carvacrol Thymol acetate Carvacrol acetate
1039 1065 1092 1148 1162 1172 1185 1202 1234 1248 1249 1254 1262 1266 1281 1284 1304 1347 1373
79,9 t 0,3 1,0 – 1,3 0,6 0,1 t 0,1 – – – 0,1 – 0,2 0,5 75,8 0,1 –
78,6 t 0,3 1,0 – 0,7 0,6 – – 0,1 – – – – – 0,1 0,4 75,2 0,2 –
82,2 – 0,3 0,4 – 0,1 0,6 – – 0,1 – – 0,1 0,1 t 0,2 0,4 80,1 – –
83,4 – 0,2 0,4 – 0,3 0,6 – – 0,1 – – – 0,5 – – 0,6 81,1 0,1 –
77,2 – 0,2 0,7 – 0,3 0,6 – – 0,1 – – 0,1 – – – 0,5 74,5 0,1 –
80,6 – 0,3 1,0 – 0,4 0,7 – t 0,1 – – 0,2 0,1 t t 0,4 77,6 t –
81,3 – – 1.0 – 1,1 0,7 – – 0,1 – – – – – 0,1 0,3 77,7 0,1 0,1
86,0 0,1 0,1 0,7 – 0,4 0,8 t 0,1 t 0,1 – 0,1 0,2 – t 0,3 83,2 – –
82,1 0,2 t 2,6 – 0,3 0,7 0,1 t t – – 0,1 0,1 – – 0,3 77,5 – –
84,5 t 0,1 0,3 – 0,2 0,7 – – 0,1 – – 0,1 0,1 – – 0,2 82,7 – –
85,1 t 0,1 3,1 – 0,2 0,8 t t 0,1 – – 0,1 0,1 t – 0,2 80,3 – –
62,5 0,2 0,5 0,2 0,3 2,9 0,9 0,2 – – – – 0,2 – – – 8,1 48,9 – 0,1
84,9 – 0,2 0,9 – 0,2 0,7 – 0,1 – t t – – 0,2 0,1 t 82,6 0,1 –
82,0 – 0,3 1,2 – 0,3 0,7 – – – 0,1 – – – 0,1 0,1 0,2 79,2 t –
84,0 – 0,3 1,1 – 0,4 0,6 – – – 0,1 – – – 0,1 0,1 0,2 81,1 0,1 –
82,4 – 0,3 1,8 – 0,3 0,6 – – – – – – – 0,1 0,0 0,2 79,1 – 0,1
84,7 – 0,6 3,2 – 0,7 0,6 0,1 t – t – – – 0,1 0,1 0,1 79,1 t –
83,0 t 0,3 1,8 – 0,3 0,5 – 0,1 – – – – – 0,1 0,1 0,1 79,7 0,1 0,1
80,6 – 0,3 1,4 – 0,4 0,6 – 0,1 – – – – – 0,1 – 0,2 77,4 0,1 0,2
73,6 – 0,2 – – 0,2 0,6 – – – – 0,1 – – 0,1 – 2,9 69,4 0,1 –
2,6 2,0 t 0,2 0,1 – – t 0,3
2,8 2,3 t 0,1 0,1 – – 0,1 0,3
2,9 2,4 0,2 0,1 – – – – 0,2
1,9 1,4 – 0,1 0,1 – – t 0,3
1,9 1,4 – 0,1 0,1 – – t 0,3
2,0 1,7 – 0,1 0,1 – – 0,1 0,1
2,5 1,8 – 0,1 0,1 – – – 0,5
4,7 3,6 – 0,1 – – – t 0,9
2,4 1,9 – 0,1 – – – – 0,4
3,1 2,4 – 0,1 t – – – 0,6
3,2 2,5 – 0,1 t t – – 0,6
2,9 1,3 0,3 0,1 0,4 0,1 – 0,3 0,5
2,4 2,0 – 0,1 – – – – 0,2
2,1 1,7 – 0,1 0,2 – – – 0,2
2,7 2,4 – 0,1 0,1 – – – 0,1
1,8 1,4 – 0,1 0,1 – – – 0,3
1,7 1,4 – 0,1 0,1 – – – 0,2
1,8 1,3 – 0,1 0,1 – – – 0,3
2,3 1,8 – 0,1 0,1 – – – 0,3
2,9 2,0 – 0,1 0,3 – 0,3 – 0,3
0,0 – – t – –
0,1 – – t – 0,1
0,2 – – 0,1 – 0,1
0,1 – – – – 0,1
0,3 – – 0,1 – 0,2
0,2 – – 0,1 – 0,1
0,1 – – 0,1 – –
0,6 – – 0,3 0,2 0,1
0,1 – – 0,1 – –
0,3 – – 0,2 0,1 –
0,4 – – 0,2 0,1 0,1
0,0 – – – – –
0,7 0,6 – 0,1 – –
0,7 0,6 – 0,1 – –
0,5 0,4 – 0,1 – –
0,5 0,5 – – – –
0,8 0,7 – 0,1 – –
0,5 0,5 – t – –
0,5 0,5 – t – –
0,8 0,4 – t 0,4 –
#
Components
Sesquiterpenes
b–Caryophyllene
39 40 41 42 43 44 45 46
Aromadendrene a-Humulene b-Bisabolene a-Cadinene trans–g-Bisabolene Spathulenol Caryophyllene Oxide
1408 1428 1442 1501 1523 1544 1565 1582
6 7 9 25 37
Others 1-Octen-3-ol 3-Octanone 3-Octanol Estragolec Eugenol
980 985 995 1193 1348
Bold numbers indicate the sum of the columns. a The numbering refers to elution order, and values (relative peak area percent) represent averages of 3 determinations (t ¼ trace, < 0.05%). b Retention index (KI) relative to standard mixture of n-alkanes on SPB-5 column. c Co-elution with authentic sample.
(N) and Val Demona (D) as represented in Fig. 1. In total, 46 components were fully identified covering more than 96% of the total composition. For an easier comparison of the oils, the components were grouped into four classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes and others. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented classes: the former with a range of 8–61% and the latter with a range of 31–86%. The sesquiterpenes and other classes were in all cases the least represented. A first and general survey of compositional data shows that carvacrol is by far the main compound in almost all samples: indeed in twenty-eight samples it ranges between 70 and 83%, though in one sample (M12) it is the main component with 49%, and in another sample (D1) it reaches only 3%. This peculiar composition strongly influences the cluster analysis carried out on the compositional data. The resulting dendrogram reported in Fig. 2, reflects the compositional uniformity of wild Sicilian thyme essential oils, placing most of the samples in a single Group A, with the exclusion of the aforesaid samples: M12
820
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Table 3 Chemical composition of wild Sicilian thyme essential oils from Val Demona (D) and Val di Noto (N).a KIb
D1
N1
N2
N3
N4
N5
N6
N7
N8
N9
1 2 3 4 5 8 10 11 12 13 14 16 17 19
Monoterpene Hydrocarbons a-Thujene a-Pinenec Camphenec Sabinenec b-pinenec b-Myrcenec a-Phellandrene p-Menth-1-(7),8-diene a-Terpinenec p-Cymemec Limonenec trans–b-Ocimene g-Terpinenec Terpinolenec
929 936 951 975 979 990 1003 1010 1016 1024 1028 1046 1058 1083
61,0 2,3 1,6 2,0 0,2 – 0,3 0,2 0,1 2,6 40,4 0,5 t 10,4 0,4
13,5 1,4 0,7 0,3 t 0,4 1,7 0,2 0,1 0,9 3,8 0,4 0,1 3,3 0,1
18,7 1,8 0,7 0,2 t 0,4 1,9 0,3 0,1 1,5 4,4 0,4 0,1 6,6 0,1
17,6 0,9 0,6 0,1 – 0,6 1,7 0,2 0,1 1,5 5,9 0,4 0,1 5,2 0,2
16,9 0,8 0,6 0,1 – 0,6 1,7 0,2 0,1 1,4 5,9 0,4 0,1 4,8 0,2
13,9 0,7 0,6 0,2 – 0,4 1,7 0,2 0,1 1,2 4,4 0,4 0,1 3,7 0,2
16,4 1,3 0,6 0,2 – 0,4 1,7 0,2 0,1 1,3 5,0 t 0,4 5,0 0,1
16,1 1,2 0,6 0,1 – 0,4 1,7 0,2 0,1 1,2 4,8 0,4 4,3 0,3 0,7
16,7 0,9 0,7 0,2 – t 1,8 0,3 0,1 1,5 5,0 0,4 0,1 5,5 0,2
17,1 1,1 0,7 0,1 – t 1,9 0,3 0,1 1,6 4,5 0,5 0,1 6,0 0,2
15 18 20 21 22 23 24 27 28 29 30 31 32 33 34 35 36
Oxygenated Monoterpenes 1,8-Cineolec Sabinene hydratec Linalool Camphorc Borneol Terpinen-4-olc p-Cymen-8-ol Nerol Thymol methyl ether Carvacrol methyl ether Neral Carvone Geraniol Geranial Thymol Carvacrol Thymol acetate
1039 1065 1092 1148 1162 1172 1185 1234 1248 1249 1254 1262 1266 1281 1284 1304 1347
31,2 0,2 1,9 0,6 0,1 4,0 2,6 0,1 – 1,8 0,9 – – – – 16,1 2,7 0,1
84,3 t 0,3 0,8 – 0,8 0,6 – 0,1 – – 0,1 0,1 t – 0,4 81,1 –
78,7 – 0,3 0,9 – 0,5 0,7 – t – – 0,1 0,1 t 0,1 0,1 76,0 –
78,4 – 0,2 0,9 – 0,3 0,7 – – – – – 0,1 – – 0,2 76,0 –
79,2 – 0,2 0,8 – 0,3 0,7 – – – – 0,1 0,1 – – 0,2 76,8 –
82,8 – 0,2 0,9 – 0,4 0,8 – 0,1 – – 0,1 0,1 – – 0,3 79,9 –
80,7 – 0,3 0,7 – 0,4 0,6 – – – – – – – 0,1 0,3 78,3 –
81,5 – 0,1 0,3 – 0,6 0,1 – – – – – – – 0,1 0,3 79,5 –
80,1 t 0,2 0,7 – 0,3 0,6 – – – – – – 0,1 0,1 0,2 77,8 0,1
79,9 t 0,3 0,9 – 0,2 0,6 – – 0,1 – – – 0,1 0,1 0,1 77,7 t
39 40 41 42 43 45 46
Sesquiterpenes b–Caryophyllene Aromadendrene a-Humulene b-Bisabolene a-Cadinene Spathulenol Caryophyllene Oxide
1408 1428 1442 1501 1523 1565 1582
2,7 1,7 – 0,2 0,4 – – 0,5
2,2 1,4 0,1 0,1 0,3 – 0,2 0,2
2,4 2,1 0,1 0,1 t – t 0,2
3,4 2,8 – 0,1 0,1 – – 0,3
3,1 2,5 – 0,1 0,2 – – 0,3
2,8 2,3 – 0,1 0,2 – – 0,2
2,3 1,4 0,1 0,1 0,2 0,2 – 0,3
2,4 1,6 0,1 0,1 0,2 0,1 – 0,3
2,1 1,7 – 0,1 0,1 – – 0,3
1,8 1,3 – 0,1 0,2 – – 0,2
6 7 9 37
Others 1-Octen-3-ol 3-Octanone 3-Octanol Eugenol
980 985 995 1348
4,3 2,7 1,4 0,2 –
0,1 – – t 0,1
0,1 – – 0,1 t
t – – t –
0,1 – – 0,1 –
– – – – –
0,1 – – 0,1 –
t – – t –
0,6 0,4 – 0,2 –
0,5 0,5 – t –
#
Components
Bold numbers indicate the sum of the columns. a The numbering refers to elution order, and values (relative peak area percent) represent averages of 3 determinations (t ¼ trace, < 0.05%). b Retention index (KI) relative to standard mixture of n-alkanes on SPB-5 column. c Co-elution with authentic sample.
and D1. Under this aspect, then, Sicilian wild thyme shows a completely different behaviour with respect to the other previous investigated aromatic wild Sicilian plants, namely oregano, fennel and rosemary (Napoli et al., 2009, 2010a,b), which show a much more variegated composition. However, on examining the results of the statistical analysis in depth, it is possible to sub-divide the Group A in two subgroups: IA and IIA with sixteen and twelve samples, respectively Table 4. Their differentiating aspect is mainly due to the quantitative complementarity of the two main classes of components, namely monoterpene hydrocarbons and oxygenated monoterpenes. In fact, the sub-group IA shows a lower content of the hydrocarbons between 8 and 16% with respect to those of sub-group IIA comprised between 16 and 22%, whereas the contrary is true for the oxygenated monoterpenes. This is essentially due to the lower amount of g-terpinene in the first sub-group and the corresponding higher amount of carvacrol, and vice-versa in second sub-group, given the well-known biosynthetic relationship between the two monoterpenes. Furthermore, inside each sub-group some samples stand out from the others for some peculiar compositional characteristic. This is true for the N7, M5 and M20 samples: indeed, N7, belonging to IA sub-group, shows a relatively high content of transb-ocimene (4.3%) against an average content lower than 1%; M5 and M20 of the IIA sub-group, have the highest content of
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Fig. 2. Dendogram obtained by cluster analysis of 30 samples of wild thyme.
g-terpinene (8.5 and 10.0%), against an average of 5.6% for this component in the IIA sub-group. Furthermore, M20 stands out even more owing to the anomalous presence of 2.9% of thymol, which in all other samples is generally present below 1%. The Group B, constituted by the single sample M12, present a lower content of carvacrol (49%), followed by p-cymene (19%) and thymol (8%). Finally, the other one sample Group C, D1, is characterized by the presence of p-cymene as main component (40%) followed by thymol (16%) and g-terpinene (10%), and a very low amount of carvacrol (>3%). This sample has been classified as Thymus longicaulis C. Presl. (Chorianopoulos et al., 2004; De Martino et al., 2009). It is comprised in the botanic section Serpyllum, which contains the largest number of thymus species (Morales, 2002; Sáez and Stahl-Biskup, 2002), often mistaken for each others, but all characterized by a prostate and creeping growth, unlike the capitatus species which grows erect. This species is widespread all over the Italian peninsula, though it is more consistently present in the north-eastern area of Sicily (Pignatti, 1982), where our sample was collected. Therefore, the salient character of wild Sicilian thyme emerging form this study is the substantial compositional homogeneity of the essential oils of the different analysed populations. In practice, no essential oil polymorphism, which is fairly widespread among many species of the Lamiaceae family, with the presence of several and well differentiated chemotypes, has been observed for the local T. capitatus Hoff. et Link (T. capitata (L.) Cav.). On the basis of the data collected in this study, it is possible to establish that it is present as a single chemical phenotype, namely carvacrol. However, this feature, far being a handicap, reinforces the typicalness of this plant and represents a local resource which can be economically exploited.
Table 4 Grouping by main components percentages of thyme samples from the cluster analysis. GROUPS SUB-GROUPS Samples
A
B
C
IA
IIA
–
–
M3, M4, M7–M11, M13- M18, N1, N5, N7
M1, M2, M5, M6, M19, M20, N2-N4, N6, N9
M12
D1
% Monoterpene Hydrocarbons -a-Thujene -a-pinene -b-Myrcene -a-Terpinene -p-Cymene -g-Terpinene
8–16 t–1 <1 <1–2 <1–2 4–8 t–5
16–22 1–2 <1 <1–2 1–2 4–6 5–10
34 1 1 2 t 19 7
61 2 2 t 3 40 10
Oxygenated Monoterpenes -Sabinene hydrate -Linalool -Borneol -Terpinen-4-ol -Thymol -Carvacrol
81–86 <1 1–3 t–1 <1 <1 78–83
74–81 <1 0–1 t-1 <1 t–3 70–78
63 1 t 3 1 8 49
31 2 1 4 3 16 3
2–5 1–4
2–3 1–3
3 1
3 2
Sesquiterpenes -b–Caryophyllene
Bold numbers indicate the percentage range of the class into the samples.
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Acknowledgements We wish to thank Assessorato Agricoltura e Foreste – Regione Siciliana for financial support. The authors express their thanks to the staff of Acireale, Aragona, Catania, Cianciana, Lercara Friddi, Mazara del Vallo, Palazzolo Acreide, Partanna, S. Agata di Militello, Salemi and Spadafora Territorial Regional Units of Regione Siciliana for the collection and first treatment of plant material. Thanks are also due to Ms. Tonia Strano (ICB-CNR, Catania) for skilful technical assistance, and to Mr. Antonio Giuffrida (ISA-CRA, Acireale CT) for helpful assistance in statistical analyses. References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatographic/Quadrupole Mass Spectrometry. Allured Publ. Corp., Carol Stream, IL, USA. Bagamboula, C.F., Uyttendaele, M., Debevere, J., 2004. Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri. Food Microbiol. 21, 33–42. Barros, L., Heleno, S.A., Carvalho, A.M., Ferreira, I.C.F.R., 2010. Lamiaceae often used in Portuguese folk medicine as a source of powerful antioxidants: vitamins and phenolic. Food Sci. Technol. 43, 544–550. diç, O., Özcan, G., Karadogan, T., 2004. Antibacterial activity and composition of essential oils from Origanum, Thymbra, and Satureja, species Baydar, H., Sag with commercial importance in Turkey. Food Control 15, 169–172. Bensmira, M., Jiang, B., Nsabimana, C., Jian, T., 2007. Effect of lavender and thyme incorporation in sunflower seed oil on its resistance to frying temperatures. Food Res. Int. 40, 341–346. Bounatirou, S., Smiti, S., Miguel, M.G., Faleiro, L., Rejeb, M.N., Neffati, M., Costa, M.M., Figueiredo, A.C., Barroso, J.G., Pedro, L.G., 2007. Chemical composition, antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link. Food Chem. 105, 146–155. Bozin, B., Mimica-Dukic, N., Simin, N., Anackov, G., 2006. Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem. 54, 1822–1826. Chorianopoulos, N., Kalpoutzakis, E., Aligiannis, N., Mitaku, S., Nychas, G.-J., Haroutounian, S.A., 2004. Essential oils of Satureja, Origanum, and Thymus species: chemical composition and antibacterial activities against food-borne pathogens. J. Agric. Food Chem. 52, 8261–8267. De Martino, L., Bruno, M., Formisano, C., De Feo, V., Napolitano, F., Rosselli, S., Senatore, F., 2009. Chemical composition and antimicrobial activity of the essential oils from two species of Thymus growing wild in southern Italy. Molecules 14, 4614–4624. Inouye, S., Abe, S., 2006. Predominant adsorption of sesquiterpenes constituents of lavender, tea tree, lemongrass, and thyme thymol oils on hairless mouse, and human hairs in an aromatic bath. Int. J. Aromather. 16, 75–83. Karousou, R., Koureas, D.N., Kokkini, S., 2005. Essential oil composition is related to the natural habitats: Coridothymus capitatus and Satureja thymbra in NATURA 2000 sites of Crete. Phytochemistry 66, 2668–2673. Lawrence, B.M., Tucker, A.O., 2002. The genus Thymus as a source of commercial products. Thyme. The genus Thymus. In: Stahl-Bishup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 252–262. Lee, S.-J., Umano, K., Shibamoto, T., Lee, K.-G., 2005. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem. 91, 131–137. Miceli, A., Negro, C., Tommasi, L., 2006. Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy). Biochem. System. Ecol. 34, 528–535. Morales, R., 2002. The history, botany and taxonomy of the genus Thymus. Thyme. The genus Thymus. In: Stahl-Bishup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 1–43. Mulas, M., 2006. Traditional uses of Labiatae in the Mediterranean area. 2006. Proc. First Int. Symp. On the Labiatae: advances in production, biotechnology and utilisation. Acta Hort. 723, 25–32. Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., Pulkrabek, J., 2009. Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria. Food Control 20, 157–160. Napoli, E.M., Curcuruto, G., Ruberto, G., 2009. Screening the essential oil composition of wild Sicilian oregano. Biochem. System. Ecol. 37, 484–493. Napoli, E.M., Curcuruto, G., Ruberto, G., 2010a. Screening the essential oil composition of wild Sicilian fennel. Biochem. System. Ecol. 38, 213–223. Napoli, E.M., Curcuruto, G., Ruberto, G., 2010b. Screening the essential oil composition of wild Sicilian rosemary. Biochem. System. Ecol. doi:10.1016/j.bse. 2010.04.001. Omidbeygi, M., Barzegar, M., Hamidi, Z., Naghdibadi, H., 2007. Antifungal activity of thyme, summer savory and clove essential oils against Aspergillum flavus in liquid medium and tomato paste. Food Control 18, 1518–1523. Pignatti, S., 1982. Flora d’Italia, vol. II. Edagricole, Bologna, Italy, pp. 488–493. Rasooli, I., Rezaei, M.B., Allameh, A., 2006. Ultrastructural studies on antimicrobial of thyme essential oils on Listeria monocytogenes. Int. J. Infect. Dis. 10, 236–241. Sáez, F., Stahl-Biskup, E., 2002. Essential oil polymorphism in the genus Thymus. Thyme. The genus Thymus. In: Stahl-Biskup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 125–142. Senatore, F., 1996. Influence of harvesting time on yield and composition of the essential oil of thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food Chem. 44, 1327–1332. Solomakos, N., Govaris, A., Koidis, P., Botsoglou, N., 2008. The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in minced beef during refrigerated storage. Meat Sci. 80, 159–166. Stahl-Biskup, E., 2002. Essential oil chemistry of the genus Thymus – a global view. Thyme. The genus Thymus. In: Stahl-Bishup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 75–124. Stahl-Biskup, E., 2004. Thyme in Handbook of Herbs and Spices, vol. 2, ed. Peter K.V., Woodhead Publish. Lim., England and CRC Press LCC, USA, pp. 297– 320. Vila, R., 2002. Flavonoids and further polyphenols in the genus Thymus. Thyme. The genus Thymus. In: Stahl-Bishup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 144–176. Viuda-Martos, M., Ruiz-Navajas, Y., Fernandez-Lopez, J., Perez-Alvarez, J.A., 2009. Effect of adding citrus waste water, thyme and oregano essential oil, on the chemical, physical and sensory characteristic of a bologna sausage. Inn. Food Sci. Emerging Tech. 10, 655–660. Zarzuelo, A., Crespo, E., 2002. The medicinal and non –medicinal uses of Thyme. Thyme. The genus Thymus. In: Stahl-Bishup, E., Sáez, F. (Eds.), Medicinal and Aromatic Plants – Industrial Profiles, vol. 17. Taylor & Francis, London, pp. 263–292. ˇ
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Screening of the essential oil composition of wild Sicilian thyme Edoardo M. Napoli, Giusy Curcuruto 1, Giuseppe Ruberto* Istituto del CNR di Chimica Biomolecolare, Via Paolo Gaifami, 18–95126 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 May 2010 Accepted 8 August 2010
Thirty samples of wild thyme collected from as many Sicilian locations have been analysed for their qualitative and quantitative essential oil profiles. The oils, obtained by hydrodistillation, have been analysed by a combination of GC-FID-MS; in all, 46 components, representing more than 98% of the oils, were fully characterized. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented components: the former with a range of 8–61% and the latter with a range of 31–86%. Carvacrol was the main compound in 29 samples, ranging between 49 and 83%, suggesting that Thymus capitatus Hoff. et Link. [syn. Coridothymus capitatus (L.) Rchb.f., Satureja capitata L., Thymbra capitata (L.) Cav.] is the most widespread wild species in the Sicilian area. Only one sample, identified as Thymus longicaulis C. Presl., collected from the North-East of Sicily showed a different composition, p-cymene and thymol being the main compounds with 40 and 16%, respectively. Statistical analyses allowed establishing a single broad group, confirming the substantial compositional uniformity of the essential oil profiles of the wild Sicilian thyme. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Thymus capitatus Hoff. et Link Thymus longicaulis C. Presl. Lamiaceae Essential oil Carvacrol p-Cymene GC–MS Cluster analysis Sicily
1. Introduction It is well-known that most spices, especially those belonging to the Lamiaceae family, have a wide range of biological and pharmacological activities. The geomorphologic and climatic conditions on the island of Sicily in the heart of the Mediterranean, provide an optimal habitat for many aromatic plants of this family to grow in the wild; indeed, the cultivation of aromatic plants is an important vocation here. They have become the common raw materials for many traditional uses such as natural remedies for human and animal diseases, for combating pests and parasites and making homemade perfumes and spices for cooking. Pursuing our project aimed at the valorisation and exploitation of wild aromatic Sicilian plants, we report here the results of a study on the essential oil of wild Sicilian thyme (Thymus capitatus Hoff. et Link.) or Thymbra capitata (L.) Cav. Thyme is a woody, perennial herb native to the Mediterranean region. Its name probably derives from the Latin “thymus” which means “perfumed” or from Greek “thymos” which means “courage” or “strength”. The Thymus genus, with its estimated 250–350 taxa (species and varieties) of wild growing plants (Morales, 2002; Lawrence and Tucker, 2002), complicated further by the presence of several chemotypes regarding the essential oil profiles associated to several species (Senatore, 1996), is one of the most taxonomically complex genera in the Lamiaceae family. As an aromatic plant, thyme is an important spice in home cooking as well as in the food and aroma industries. At the same time it is a widely used medicinal plant, with an ancient tradition dating back to the Egyptians, who used it in unguents for embalming and then the Greeks and Romans who used it for therapeutic purposes (Barros et al., 2010). Infusions are used for treating ulcers, dermatitis and some types of rheumatic pains, while thyme is used to fight pathologies of the respiratory
* Corresponding author. Tel.:þ39 0957338347; fax: þ39 0957338310. E-mail address: [email protected] (G. Ruberto). 1 Present address: Dipartimento di Metodologie Fisiche e Chimiche per l’Ingegneria (DMFCI), Università di Catania, V.le A. Doria, 6 - 95125 Catania, Italy. 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.08.008
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apparatus owing to its expectorant, spasmolytic and antiseptic properties (Zarzuelo and Crespo, 2002; Mulas, 2006). All the aforesaid activities can be ascribed to the considerable presence of polyphenol derivatives (Vila, 2002) in thyme extracts and/ or infusions. However, it is the essential oil (EO) that has a broad spectrum of bioactivities. In fact, it serves as a preservative for food due to the antimicrobial properties of its volatile components (Bagamboula et al., 2004; Baydar et al., 2004; Rasooli et al., 2006; Omidbeygi et al., 2007; Solomakos et al., 2008; Nedorostova et al., 2009), as an antioxidant (Lee et al., 2005; Bozin et al., 2006), as an additive to enhance organoleptic characteristics (Viuda-Martos et al., 2009) and physical properties (Bensmira et al., 2007). The thyme EO is also used as a raw material in perfumery and cosmetics (Zarzuelo and Crespo, 2002; Inouye and Abe, 2006). From a chemical point of view thyme essential oils are characterized by a large amount of monoterpenes, which normally account for 90% of oil. The phenolic monoterpenes thymol and carvacrol occur more frequently, always accompanied by the couple p-cymene/g-terpinene, the four monoterpenes being biogenetically closely correlated. Linalool, borneol, 1,8-cineole are the other oxygenated monoterpenes, in order of importance, singled out in thyme essential oils (Stahl-Biskup, 2002, 2004). In particular, concerning the capitatum species, or T. capitata, carvacrol generally proves to be the characteristic constituent (Karousou et al., 2005; Bounatirou et al., 2007). However, as aforementioned, different chemotypes have been reported for many Thymus species, so this species also shows different chemical profiles: thymol, carvacrol and thymol/ carvacrol (Karousou et al., 2005; Miceli et al., 2006). 2. Materials and methods 2.1. Plant material Table 1 lists the thirty samples of thyme collected in different locations of Sicily, which are reported in Fig. 1. All the plant samples were harvested in the years 2006–2007 at bloom stage and were naturally dried at the collection point. Voucher specimens were deposited in the herbarium of the respective Regional Unit listed in Table 1, which also carried out the botanical identification. 2.2. Isolation of the essential oil The air-dried aerial part of each plant (50–100g) was subjected to hydrodistillation in accordance with current European Pharmacopoeia (European Pharmacopoeia, 2008) until there was no significant increase in the volume of oil collected (3h). Table 1 Collection data and essential oil yield of analysed samples of thyme (Thymus capitatus Hoff. et Link.). Sample
Site
Regional Unit
Date
Yield % (v/w)
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 D1 N1 N2 N3 N4 N5 N6 N7 N8 N9
Partanna (Trapani) Partanna (Trapani) Santa Elisabetta (Agrigento) Sant’Angelo Muxaro (Agrigento) Palazzo Adriano (Palermo) Mazara del Vallo (Trapani) Favignana (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Pantelleria (Trapani) Alcamo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Mazara del Vallo (Trapani) Cambobello di Mazara (Trapani) Cambobello di Mazara (Trapani) Palazzo Adriano (Palermo) Galati Mamertino (Messina)a Noto (Siracusa) Noto (Siracusa) Calascibetta (Enna) Calascibetta (Enna) Calascibetta (Enna) Caltagirone (Catania) Caltagirone (Catania) Noto (Siracusa) Noto (Siracusa)
SOPAT Partanna SOPAT Partanna UOT Aragona UOT Aragona UOT Lercara Friddi UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Alcamo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Mazara del Vallo UOT Castelvetrano UOT Castelvetrano UOT Lercara Friddi UOT Sant’Agata di Militello UOT Acireale UOT Acireale UOT Enna UOT Enna UOT Enna UOT Acireale UOT Acireale UOT Palazzolo Acreide UOT Palazzolo Acreide
2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2007 2007 2007 2007 2006 2006 2006 2006 2006 2007 2007 2007 2007
2,80 3,30 4,48 2,50 2,70 4,50 3,20 2,20 2,80 3,20 2,60 1,90 3,90 4,30 4,30 3,20 4.50 3,00 3,00 4,60 1,40 5,30 4,80 3,50 3,40 4,20 5,60 4,60 5,00 5,00
a
Thymus longicaulis C. Presl.
818
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Fig. 1. Sicilian collection localities of investigated wild thyme samples.
The oils, whose yield is listed in Table 1, were dried over anhydrous sodium sulphate and stored under N2 in a sealed vial until required. 2.3. GC and GC–MS analyses A Shimadzu GC-17A Gas Chromatograph equipped with a fused silica capillary column (Supelco SPBTM-5 15 m 0.1 mm 0.1 mm) and FID was used. The operating conditions were the following: 60 C for 1 min, 60–280 C at 10 C/min then 280 C for 1 min; injector temperature 250 C; detector temperature 280 C; carrier gas helium (1 mL/min); split mode (1:200), volume of injection 1 mL (4% essential oil/CH2Cl2 v/v). Percentages of compounds were determined from their peak areas in the GC-FID profiles. GC–MS analyses were performed on a Shimadzu GCMS-QP5050A with the same column and the same operating conditions used for analytical GC. Ionization was performed at 70 eV. Ion source temperature 180 C; mass spectral data were acquired in the scan mode in m/z range 40–400. Oil solutions were injected with the split mode (1:96). 2.4. Identification procedure The identity of components was based on their retention index relative to C9–C22 n-alkanes (Alltech Italy) on the SPB-5 column, computer matching of spectral MS data those from NIST MS 107 and NIST 21 libraries (NIST 1998), the comparison of the fragmentation patterns with those reported in literature (Adams, 2001) and, whenever possible, co-injections with authentic samples. Pure standards were purchased from Aldrich Chemical Co., Extrasynthese, France, and Fluka Chemie AG, Switzerland. 3. Statistical analysis All data were statistically analysed using the STATWIN 6.0 software package in order to determine the relationship between the different samples of thyme using the percentage composition of their essential oils. Euclidean distance was selected as a measure of similarity and the unweighted pair-group method with arithmetic average (UPGMA) was used for cluster definition. 4. Results and discussion Tables 2 and 3 list the composition of the essential oils of the thirty samples of thyme collected during the biennium 2006– 2007. Samples have been pooled according to a historical territorial subdivision of Sicily island: Val Mazara (M), Val di Noto
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Table 2 Chemical composition of wild Sicilian thyme essential oils from Val Mazara (M).a KIb
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20
1 2 3 4 5 8 10 11 12 13 14 16 17 19
Monoterpene Hydrocarbons a-Thujene a-Pinenec Camphenec Sabinenec b-pinenec b-Myrcenec a-Phellandrene p-Menth-1-(7),8-diene a-Terpinenec p-Cymemec Limonenec trans–b-Ocimene g-Terpinenec Terpinolenec
929 936 951 975 979 990 1003 1010 1016 1024 1028 1046 1058 1083
17,2 1,5 0,8 0,5 t 0,4 1,7 0,2 0,1 1,2 4,7 0,4 0,1 5,3 0,1
18,5 1,6 0,8 0,3 t 0,4 1,8 0,3 0,1 1,4 4,3 0,4 0,1 6,7 0,1
14,6 1,4 0,6 0,1 – 0,6 1,7 0,3 0,1 1,1 3,8 0,4 0,1 4,0 0,1
14,3 1,1 0,5 0,1 – 0,4 1,4 0,2 0,1 1,2 3,5 0,3 0,1 4,9 0,1
20,4 1,0 0,6 0,1 – 0,4 1,6 0,2 0,1 1,6 5,7 0,4 0,1 8,5 0,1
17,0 1,6 0,7 0,2 – 0,6 1,9 0,3 0,1 1,5 4,0 0,4 – 5,5 0,1
15,3 1,0 0,8 0,4 – 0,5 1,5 0,2 0,1 1,0 6,2 0,4 – 3,2 0.4
7,7 0,2 0,4 0,1 – 0,5 0,7 0,1 0,1 0,4 4,1 0,3 – 0,7 0,1
14,4 0,2 0,7 0,1 – 0,3 1,2 0,2 0,1 1,0 7,9 0,4 – 2,1 0,2
10,8 0,4 0,4 0,1 – 0,5 1,0 0,1 0,1 0,7 5,2 0,4 – 1,9 0,1
10,8 0,5 0,5 0,1 – 0,4 1,1 0,1 0,1 0,7 4,7 0,3 – 2,2 0,1
33,6 0,9 0,8 1,1 – 1,9 0,9 0,1 0,1 1,0 19,1 0,5 – 7,1 0,2
11,3 1,3 0,6 0,1 t – 1,6 0,2 0,1 0,7 4,1 0,4 t 1,9 0,1
14,5 1,4 0,6 0,1 – – 1,7 0,2 0,1 1,2 4,4 0,4 0,1 4,0 0,1
12,2 1,4 0,5 0,1 – – 1,6 0,2 0,1 0,9 3,8 0,4 0,1 2,8 0,1
14,5 0,9 0,6 0,1 t – 1,5 0,2 0,1 1,2 4,7 0,4 – 4,6 0,1
12,2 1,2 0,7 0,2 – – 1,5 0,2 0,1 0,8 4,9 0,4 – 1,9 0,1
14,1 0,9 0,5 0,1 t – 1,4 0,2 0,1 1,2 4,7 0,4 t 4,3 0,1
16,1 0,9 0,6 0,2 t – 1,5 0,2 0,1 1,4 5,0 0,4 0,1 5,6 0,1
21,5 0,8 0,6 0,1 – – 1,6 0,2 0,1 1,8 5,7 0,4 0,1 10,0 0,3
15 18 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 36 38
Oxygenated Monoterpenes 1,8-Cineolec Sabinene hydratec Linalool Camphorc Borneol Terpinen-4-olc p-Cymen-8-ol trans-Dihydro Carvone Nerol Thymol methyl ether Carvacrol methyl ether Neral Carvone Geraniol Geranial Thymol Carvacrol Thymol acetate Carvacrol acetate
1039 1065 1092 1148 1162 1172 1185 1202 1234 1248 1249 1254 1262 1266 1281 1284 1304 1347 1373
79,9 t 0,3 1,0 – 1,3 0,6 0,1 t 0,1 – – – 0,1 – 0,2 0,5 75,8 0,1 –
78,6 t 0,3 1,0 – 0,7 0,6 – – 0,1 – – – – – 0,1 0,4 75,2 0,2 –
82,2 – 0,3 0,4 – 0,1 0,6 – – 0,1 – – 0,1 0,1 t 0,2 0,4 80,1 – –
83,4 – 0,2 0,4 – 0,3 0,6 – – 0,1 – – – 0,5 – – 0,6 81,1 0,1 –
77,2 – 0,2 0,7 – 0,3 0,6 – – 0,1 – – 0,1 – – – 0,5 74,5 0,1 –
80,6 – 0,3 1,0 – 0,4 0,7 – t 0,1 – – 0,2 0,1 t t 0,4 77,6 t –
81,3 – – 1.0 – 1,1 0,7 – – 0,1 – – – – – 0,1 0,3 77,7 0,1 0,1
86,0 0,1 0,1 0,7 – 0,4 0,8 t 0,1 t 0,1 – 0,1 0,2 – t 0,3 83,2 – –
82,1 0,2 t 2,6 – 0,3 0,7 0,1 t t – – 0,1 0,1 – – 0,3 77,5 – –
84,5 t 0,1 0,3 – 0,2 0,7 – – 0,1 – – 0,1 0,1 – – 0,2 82,7 – –
85,1 t 0,1 3,1 – 0,2 0,8 t t 0,1 – – 0,1 0,1 t – 0,2 80,3 – –
62,5 0,2 0,5 0,2 0,3 2,9 0,9 0,2 – – – – 0,2 – – – 8,1 48,9 – 0,1
84,9 – 0,2 0,9 – 0,2 0,7 – 0,1 – t t – – 0,2 0,1 t 82,6 0,1 –
82,0 – 0,3 1,2 – 0,3 0,7 – – – 0,1 – – – 0,1 0,1 0,2 79,2 t –
84,0 – 0,3 1,1 – 0,4 0,6 – – – 0,1 – – – 0,1 0,1 0,2 81,1 0,1 –
82,4 – 0,3 1,8 – 0,3 0,6 – – – – – – – 0,1 0,0 0,2 79,1 – 0,1
84,7 – 0,6 3,2 – 0,7 0,6 0,1 t – t – – – 0,1 0,1 0,1 79,1 t –
83,0 t 0,3 1,8 – 0,3 0,5 – 0,1 – – – – – 0,1 0,1 0,1 79,7 0,1 0,1
80,6 – 0,3 1,4 – 0,4 0,6 – 0,1 – – – – – 0,1 – 0,2 77,4 0,1 0,2
73,6 – 0,2 – – 0,2 0,6 – – – – 0,1 – – 0,1 – 2,9 69,4 0,1 –
2,6 2,0 t 0,2 0,1 – – t 0,3
2,8 2,3 t 0,1 0,1 – – 0,1 0,3
2,9 2,4 0,2 0,1 – – – – 0,2
1,9 1,4 – 0,1 0,1 – – t 0,3
1,9 1,4 – 0,1 0,1 – – t 0,3
2,0 1,7 – 0,1 0,1 – – 0,1 0,1
2,5 1,8 – 0,1 0,1 – – – 0,5
4,7 3,6 – 0,1 – – – t 0,9
2,4 1,9 – 0,1 – – – – 0,4
3,1 2,4 – 0,1 t – – – 0,6
3,2 2,5 – 0,1 t t – – 0,6
2,9 1,3 0,3 0,1 0,4 0,1 – 0,3 0,5
2,4 2,0 – 0,1 – – – – 0,2
2,1 1,7 – 0,1 0,2 – – – 0,2
2,7 2,4 – 0,1 0,1 – – – 0,1
1,8 1,4 – 0,1 0,1 – – – 0,3
1,7 1,4 – 0,1 0,1 – – – 0,2
1,8 1,3 – 0,1 0,1 – – – 0,3
2,3 1,8 – 0,1 0,1 – – – 0,3
2,9 2,0 – 0,1 0,3 – 0,3 – 0,3
0,0 – – t – –
0,1 – – t – 0,1
0,2 – – 0,1 – 0,1
0,1 – – – – 0,1
0,3 – – 0,1 – 0,2
0,2 – – 0,1 – 0,1
0,1 – – 0,1 – –
0,6 – – 0,3 0,2 0,1
0,1 – – 0,1 – –
0,3 – – 0,2 0,1 –
0,4 – – 0,2 0,1 0,1
0,0 – – – – –
0,7 0,6 – 0,1 – –
0,7 0,6 – 0,1 – –
0,5 0,4 – 0,1 – –
0,5 0,5 – – – –
0,8 0,7 – 0,1 – –
0,5 0,5 – t – –
0,5 0,5 – t – –
0,8 0,4 – t 0,4 –
#
Components
Sesquiterpenes
b–Caryophyllene
39 40 41 42 43 44 45 46
Aromadendrene a-Humulene b-Bisabolene a-Cadinene trans–g-Bisabolene Spathulenol Caryophyllene Oxide
1408 1428 1442 1501 1523 1544 1565 1582
6 7 9 25 37
Others 1-Octen-3-ol 3-Octanone 3-Octanol Estragolec Eugenol
980 985 995 1193 1348
Bold numbers indicate the sum of the columns. a The numbering refers to elution order, and values (relative peak area percent) represent averages of 3 determinations (t ¼ trace, < 0.05%). b Retention index (KI) relative to standard mixture of n-alkanes on SPB-5 column. c Co-elution with authentic sample.
(N) and Val Demona (D) as represented in Fig. 1. In total, 46 components were fully identified covering more than 96% of the total composition. For an easier comparison of the oils, the components were grouped into four classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes and others. Monoterpenes, both hydrocarbons and oxygenated, were the most highly represented classes: the former with a range of 8–61% and the latter with a range of 31–86%. The sesquiterpenes and other classes were in all cases the least represented. A first and general survey of compositional data shows that carvacrol is by far the main compound in almost all samples: indeed in twenty-eight samples it ranges between 70 and 83%, though in one sample (M12) it is the main component with 49%, and in another sample (D1) it reaches only 3%. This peculiar composition strongly influences the cluster analysis carried out on the compositional data. The resulting dendrogram reported in Fig. 2, reflects the compositional uniformity of wild Sicilian thyme essential oils, placing most of the samples in a single Group A, with the exclusion of the aforesaid samples: M12
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E.M. Napoli et al. / Biochemical Systematics and Ecology 38 (2010) 816–822
Table 3 Chemical composition of wild Sicilian thyme essential oils from Val Demona (D) and Val di Noto (N).a KIb
D1
N1
N2
N3
N4
N5
N6
N7
N8
N9
1 2 3 4 5 8 10 11 12 13 14 16 17 19
Monoterpene Hydrocarbons a-Thujene a-Pinenec Camphenec Sabinenec b-pinenec b-Myrcenec a-Phellandrene p-Menth-1-(7),8-diene a-Terpinenec p-Cymemec Limonenec trans–b-Ocimene g-Terpinenec Terpinolenec
929 936 951 975 979 990 1003 1010 1016 1024 1028 1046 1058 1083
61,0 2,3 1,6 2,0 0,2 – 0,3 0,2 0,1 2,6 40,4 0,5 t 10,4 0,4
13,5 1,4 0,7 0,3 t 0,4 1,7 0,2 0,1 0,9 3,8 0,4 0,1 3,3 0,1
18,7 1,8 0,7 0,2 t 0,4 1,9 0,3 0,1 1,5 4,4 0,4 0,1 6,6 0,1
17,6 0,9 0,6 0,1 – 0,6 1,7 0,2 0,1 1,5 5,9 0,4 0,1 5,2 0,2
16,9 0,8 0,6 0,1 – 0,6 1,7 0,2 0,1 1,4 5,9 0,4 0,1 4,8 0,2
13,9 0,7 0,6 0,2 – 0,4 1,7 0,2 0,1 1,2 4,4 0,4 0,1 3,7 0,2
16,4 1,3 0,6 0,2 – 0,4 1,7 0,2 0,1 1,3 5,0 t 0,4 5,0 0,1
16,1 1,2 0,6 0,1 – 0,4 1,7 0,2 0,1 1,2 4,8 0,4 4,3 0,3 0,7
16,7 0,9 0,7 0,2 – t 1,8 0,3 0,1 1,5 5,0 0,4 0,1 5,5 0,2
17,1 1,1 0,7 0,1 – t 1,9 0,3 0,1 1,6 4,5 0,5 0,1 6,0 0,2
15 18 20 21 22 23 24 27 28 29 30 31 32 33 34 35 36
Oxygenated Monoterpenes 1,8-Cineolec Sabinene hydratec Linalool Camphorc Borneol Terpinen-4-olc p-Cymen-8-ol Nerol Thymol methyl ether Carvacrol methyl ether Neral Carvone Geraniol Geranial Thymol Carvacrol Thymol acetate
1039 1065 1092 1148 1162 1172 1185 1234 1248 1249 1254 1262 1266 1281 1284 1304 1347
31,2 0,2 1,9 0,6 0,1 4,0 2,6 0,1 – 1,8 0,9 – – – – 16,1 2,7 0,1
84,3 t 0,3 0,8 – 0,8 0,6 – 0,1 – – 0,1 0,1 t – 0,4 81,1 –
78,7 – 0,3 0,9 – 0,5 0,7 – t – – 0,1 0,1 t 0,1 0,1 76,0 –
78,4 – 0,2 0,9 – 0,3 0,7 – – – – – 0,1 – – 0,2 76,0 –
79,2 – 0,2 0,8 – 0,3 0,7 – – – – 0,1 0,1 – – 0,2 76,8 –
82,8 – 0,2 0,9 – 0,4 0,8 – 0,1 – – 0,1 0,1 – – 0,3 79,9 –
80,7 – 0,3 0,7 – 0,4 0,6 – – – – – – – 0,1 0,3 78,3 –
81,5 – 0,1 0,3 – 0,6 0,1 – – – – – – – 0,1 0,3 79,5 –
80,1 t 0,2 0,7 – 0,3 0,6 – – – – – – 0,1 0,1 0,2 77,8 0,1
79,9 t 0,3 0,9 – 0,2 0,6 – – 0,1 – – – 0,1 0,1 0,1 77,7 t
39 40 41 42 43 45 46
Sesquiterpenes b–Caryophyllene Aromadendrene a-Humulene b-Bisabolene a-Cadinene Spathulenol Caryophyllene Oxide
1408 1428 1442 1501 1523 1565 1582
2,7 1,7 – 0,2 0,4 – – 0,5
2,2 1,4 0,1 0,1 0,3 – 0,2 0,2
2,4 2,1 0,1 0,1 t – t 0,2
3,4 2,8 – 0,1 0,1 – – 0,3
3,1 2,5 – 0,1 0,2 – – 0,3
2,8 2,3 – 0,1 0,2 – – 0,2
2,3 1,4 0,1 0,1 0,2 0,2 – 0,3
2,4 1,6 0,1 0,1 0,2 0,1 – 0,3
2,1 1,7 – 0,1 0,1 – – 0,3
1,8 1,3 – 0,1 0,2 – – 0,2
6 7 9 37
Others 1-Octen-3-ol 3-Octanone 3-Octanol Eugenol
980 985 995 1348
4,3 2,7 1,4 0,2 –
0,1 – – t 0,1
0,1 – – 0,1 t
t – – t –
0,1 – – 0,1 –
– – – – –
0,1 – – 0,1 –
t – – t –
0,6 0,4 – 0,2 –
0,5 0,5 – t –
#
Components
Bold numbers indicate the sum of the columns. a The numbering refers to elution order, and values (relative peak area percent) represent averages of 3 determinations (t ¼ trace, < 0.05%). b Retention index (KI) relative to standard mixture of n-alkanes on SPB-5 column. c Co-elution with authentic sample.
and D1. Under this aspect, then, Sicilian wild thyme shows a completely different behaviour with respect to the other previous investigated aromatic wild Sicilian plants, namely oregano, fennel and rosemary (Napoli et al., 2009, 2010a,b), which show a much more variegated composition. However, on examining the results of the statistical analysis in depth, it is possible to sub-divide the Group A in two subgroups: IA and IIA with sixteen and twelve samples, respectively Table 4. Their differentiating aspect is mainly due to the quantitative complementarity of the two main classes of components, namely monoterpene hydrocarbons and oxygenated monoterpenes. In fact, the sub-group IA shows a lower content of the hydrocarbons between 8 and 16% with respect to those of sub-group IIA comprised between 16 and 22%, whereas the contrary is true for the oxygenated monoterpenes. This is essentially due to the lower amount of g-terpinene in the first sub-group and the corresponding higher amount of carvacrol, and vice-versa in second sub-group, given the well-known biosynthetic relationship between the two monoterpenes. Furthermore, inside each sub-group some samples stand out from the others for some peculiar compositional characteristic. This is true for the N7, M5 and M20 samples: indeed, N7, belonging to IA sub-group, shows a relatively high content of transb-ocimene (4.3%) against an average content lower than 1%; M5 and M20 of the IIA sub-group, have the highest content of
E.M. Napoli et al. / Biochemical Systematics and Ecology 38 (2010) 816–822
821
Fig. 2. Dendogram obtained by cluster analysis of 30 samples of wild thyme.
g-terpinene (8.5 and 10.0%), against an average of 5.6% for this component in the IIA sub-group. Furthermore, M20 stands out even more owing to the anomalous presence of 2.9% of thymol, which in all other samples is generally present below 1%. The Group B, constituted by the single sample M12, present a lower content of carvacrol (49%), followed by p-cymene (19%) and thymol (8%). Finally, the other one sample Group C, D1, is characterized by the presence of p-cymene as main component (40%) followed by thymol (16%) and g-terpinene (10%), and a very low amount of carvacrol (>3%). This sample has been classified as Thymus longicaulis C. Presl. (Chorianopoulos et al., 2004; De Martino et al., 2009). It is comprised in the botanic section Serpyllum, which contains the largest number of thymus species (Morales, 2002; Sáez and Stahl-Biskup, 2002), often mistaken for each others, but all characterized by a prostate and creeping growth, unlike the capitatus species which grows erect. This species is widespread all over the Italian peninsula, though it is more consistently present in the north-eastern area of Sicily (Pignatti, 1982), where our sample was collected. Therefore, the salient character of wild Sicilian thyme emerging form this study is the substantial compositional homogeneity of the essential oils of the different analysed populations. In practice, no essential oil polymorphism, which is fairly widespread among many species of the Lamiaceae family, with the presence of several and well differentiated chemotypes, has been observed for the local T. capitatus Hoff. et Link (T. capitata (L.) Cav.). On the basis of the data collected in this study, it is possible to establish that it is present as a single chemical phenotype, namely carvacrol. However, this feature, far being a handicap, reinforces the typicalness of this plant and represents a local resource which can be economically exploited.
Table 4 Grouping by main components percentages of thyme samples from the cluster analysis. GROUPS SUB-GROUPS Samples
A
B
C
IA
IIA
–
–
M3, M4, M7–M11, M13- M18, N1, N5, N7
M1, M2, M5, M6, M19, M20, N2-N4, N6, N9
M12
D1
% Monoterpene Hydrocarbons -a-Thujene -a-pinene -b-Myrcene -a-Terpinene -p-Cymene -g-Terpinene
8–16 t–1 <1 <1–2 <1–2 4–8 t–5
16–22 1–2 <1 <1–2 1–2 4–6 5–10
34 1 1 2 t 19 7
61 2 2 t 3 40 10
Oxygenated Monoterpenes -Sabinene hydrate -Linalool -Borneol -Terpinen-4-ol -Thymol -Carvacrol
81–86 <1 1–3 t–1 <1 <1 78–83
74–81 <1 0–1 t-1 <1 t–3 70–78
63 1 t 3 1 8 49
31 2 1 4 3 16 3
2–5 1–4
2–3 1–3
3 1
3 2
Sesquiterpenes -b–Caryophyllene
Bold numbers indicate the percentage range of the class into the samples.
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Acknowledgements We wish to thank Assessorato Agricoltura e Foreste – Regione Siciliana for financial support. The authors express their thanks to the staff of Acireale, Aragona, Catania, Cianciana, Lercara Friddi, Mazara del Vallo, Palazzolo Acreide, Partanna, S. Agata di Militello, Salemi and Spadafora Territorial Regional Units of Regione Siciliana for the collection and first treatment of plant material. Thanks are also due to Ms. Tonia Strano (ICB-CNR, Catania) for skilful technical assistance, and to Mr. Antonio Giuffrida (ISA-CRA, Acireale CT) for helpful assistance in statistical analyses. References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatographic/Quadrupole Mass Spectrometry. Allured Publ. Corp., Carol Stream, IL, USA. Bagamboula, C.F., Uyttendaele, M., Debevere, J., 2004. Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri. 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