GNPL#1709193, VOL 0, ISS 0

Essential oil compositions of fruticans, T. scordium subsp. scordioides and T. siculum growing in Sicily and Malta

Rossella Gagliano Candela, Vincenzo Ilardi, Natale Badalamenti, Maurizio Bruno, Sergio Rosselli, and Filippo Maggi

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Q1 Please provide volume number for the references “Bruno et al. 2019.” Q2 Please update the year of publication and citation for the references “Euro Check List; World Checklist of Selected Families, Royal Botanic Gardens, Kew.” Q3 Please provide publisher location for the reference “Horvat et al. 1974.” Q4 Please provide publisher name for the reference “Tutin and Wood 1972.” Q5 Please note that the ORCID section has been created from information sup- plied with your manuscript submission/CATS. Please correct if this is inaccurate. 1 NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2019.1709193 2 3 4 5 Essential oil compositions of Teucrium fruticans, 6 T. scordium subsp. scordioides and T. siculum growing 7 8 in Sicily and Malta 9 Rossella Gagliano Candelaa, Vincenzo Ilardib, Natale Badalamentia, Maurizio 10 Q5 Brunoa, Sergio Rossellic and Filippo Maggid 11 12 aDepartment of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), b 13 University of Palermo, Palermo, Italy; Department of Earth and Marine Sciences (DISTeM), University of Palermo, Palermo, Italy; cDepartment of Agricultural and Forest Sciences (SAF), University of 14 Palermo, Palermo, Italy; dSchool of Pharmacy, University of Camerino, Camerino, Italy 15 16 ABSTRACT ARTICLE HISTORY 17 In the present study, the chemical composition of the essential Received 7 December 2019 18 oils from the aerial parts of Teucrium fruticans L. collected in Sicily Accepted 15 December 2019 19 and Malta, Teucrium scordium subsp. scordioides (Schreb.) Arcang. and Teucrium siculum (Raf.) Guss., collected in Sicily, were eval- KEYWORDS 20 Teucrium uated by GC-MS. The main volatile components of both T. fruti- ssp.; Volatile 21 components; Germacrene D; cans collections were germacrene D (29.4% and 50.0%), (E)- b b (E)- -Caryophyllene; 1- 22 -caryophyllene (19.6% and 21.9%), and 1-octen-3-ol (19.7% and Octen-3-ol; 23 7.4%); T. scordium subsp. scordioides essential oil was rich in car- Chemotaxonomy yophyllene oxide (25.8%), a-pinene (19.4%) and b-pinene (8.5%); 24 T. siculum essential oil was rich in (E)-b-caryophyllene (30.9%), 1- 25 octen-3-ol (9.0%), a-humulene (8.6%) and germacrene D (8.0%). 26 The chemotaxonomic relationship with other members of the 27 respective botanical sections was discussed. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 CONTACT Sergio Rosselli [email protected] ß 2019 Informa UK Limited, trading as Taylor & Francis Group 2 R. GAGLIANO CANDELA ET AL.

46 1. Introduction 47 Teucrium is a plant genus of the family, represented mostly by perennial, 48 bushy or herbaceous , living commonly in sunny habitats. The genus consists of 49 415 accepted taxa, including , subspecies, varieties, forms and hybrids (World 50 Checklist), that are divided in ten botanical sections (McClintock and Epling 1946; 51 Tutin and Wood 1972). 52 The species of the genus Teucrium grow in moderate climate zones, particularly in 53 the Mediterranean basin and Central Asia. As the species can be found in southern, 54 south-western and south-eastern part of Europe, the continent is regarded as one of 55 the main centre of differentiation of the genus. A significant number of species have 56 been also observed in south-western Asia, north-western Africa, southern North 57 America and south-western South America. As for Australia, the species of genus 58 Teucrium are distributed in both southern parts of the continent and certain nearby 59 islands (Mesuel et al. 1978, Hollis and Brummitt 1992) 60 In Sicily the genus Teucrium is represented by ten taxa: Teucrium fruticans L., 61 Teucrium campanulatum L., Teucrium siculum (Raf.) Guss., Teucrium scordium subsp. 62 scordioides (Schreb.) Arcang., Teucrium spinosum L., Teucrium chamaedrys L., Teucrium 63 flavum L., Teucrium montanum L., Teucrium luteum (Mill.) Degen, and Teucrium capita- 64 65 tum L. (Pignatti 2017) 66 Teucrium fruticans is a Mediterranean species occurring in Southern Europe and 67 North Africa growing mainly on calcareous rocks near the seaside. Due to its blue 68 flowers with its evergreen foliage, it is largely used as an ornamental plant. In Italy, it 69 grows along the Tyrrhenian coasts up to Tuscany, Sicily and Sardinia, and in almost all 70 the minor islands. It also grows in Maltase archipelago (Euro Check List). In Tuscany, “ ” 71 where it is known as Erba di Santa Lucia the infusion of the leaves is used as depura- 72 tive and diuretic (Maccioni et al. 1997), whereas on Mt Etna (Sicily), where it is known “ ” 73 as Erba ricottara , the leaves are used to treat hemorrhoids (Tuttolomondo et al. 74 2014). Phytochemical investigations on the extracts of this species showed that it is 75 quite rich of diterpenoids. Researches, performed on the aerial parts of a Sicilian acces- 76 sion, allowed to identify three new neo-clerodanes and a new seco-neo-clerodane 77 derivative (Savona et al. 1978a; 1978b; Bruno et al. 1992), whereas the analysis of its 78 roots showed the presence of three new rearranged abietane diterpenoids (Bruno 79 et al. 1990; Cuadrado et al. 1992). Other metabolites isolated from the aerials parts of 80 the same population were triterpenoids (Passannanti et al. 1983), an unusual fatty acid 81 ester (Fontana et al. 1999) and several flavonoids (Kisiel et al. 2014). The investigation 82 of plants collected in Barcellona (Spain), showed the occurrence, apart from the previ- 83 ously detected metabolites, of nine neo-clerodanes. All these compounds were tested 84 for their antifeedant activity against Spodoptera littoralis, showing for some of them a 85 moderate activity (Coll and Tandron 2004; 2005). Similar antifeedant studies on natural 86 and synthetic derivatives indicated a good potency of isofruticolone (Bruno et al. 87 1999). Recently, the investigation of the aerial parts of T. fruticans, cultivated in China, 88 showed the occurrence of other seven new neo-clerodane diterpenoids. All the diter- 89 penes were evaluated for their cytotoxic activities on three human cancer cell lines, 90 and for their ability to inhibit LPS-induced nitric oxide production in RAW 264.7 mac- rophages. None of the compounds displayed cytotoxic activities on the cancer cell NATURAL PRODUCT RESEARCH 3

91 lines, and only 11-hydroxyfruticolone showed weak NO inhibitory activity (Lv et al. 92 2015). The methanol extract of the leaves of T. fruticans collected in Northern Morocco 93 was tested for its antioxidant and antibacterial activities. The results indicated moder- 94 ate antioxidant properties and poor activity against Staphylococcus spp. and B. subtilis 95 (Boudkhili et al. 2012). Also, the ethanol extract of the inflorescences of plants col- 96 lected on Mt. Etna (Sicily) inhibited the growth of several bacterial strains, with better 97 activity against Gram þ bacteria (Acquaviva et al. 2018). To the best of our knowledge, 98 only one paper on the analysis of the essential oil of T. fruticans has been published 99 and regards plants collected in Tuscany (Italy) (Flamini et al. 2001). 100 Teucrium scordium subsp. scordiodes [Bas.: Teucrium scordioides Schreb.; sinonymus: T. 101 scordium L. subsp. scordioides (Schreb.) Maire & Petitm.; Syn.: T. scordioides var. lanugino- 102 sum (Hoffmanns. & Link) Lojac.] is a perennial species distributed in southern and south- 103 eastern Europe, Asia Minor and Morocco. It grows mainly on wet soil and swamps 104 (Horvat et al. 1974). Chemical investigation on a Serbian accession of this taxa showed 105 the presence of several flavonoids in the methanol extract (Kundakovicetal.2011). In 106 the ethanol extract of T. scordium ssp. scordioides collected in Spain many free flavone 107 aglycones and glycosides were identified (Harborne et al. 1986). The cyclohexane and 108 dichloromethane extracts of T. scordium subsp. scordiodes have shown to possess high 109 cytotoxicity against MDA-MB-361 and MB-453 cell lines (Kundakovicetal.2011). Further 110 studies indicated for this taxa antioxidant, proapoptotic and cytotoxic activity against 111 HeLa and K562 cancer cell lines (Stankovicetal.2011; 2012; 2015). With regard to the 112 analysis of the essential oil only one report has been published and concerns a Serbian 113 population of T. scordium subsp. scordiodes (Radulovicetal.2012). 114 Teucrium siculum [Bas.: Scorodonia sicula Raf., T. scorodonia subsp. siculum (Raf.) 115 Nyman, T. scorodonia subsp. crenatifolium (Guss.) Arcang., T. scorodonia L. var. crenati- 116 folium Guss., T. pseudo-scorodonia Ces., Passer. & Gibelli non Desf.] is an herbaceous 117 perennial . It is widely spread in Middle and Southern peninsular Italy, as well as 118 in Sicily. Moreover, small disjointed areas of the latter species are known on the 119 Euganean hills and in Vicenza and Trento outskirts (Servettaz et al. 1994). In Europe it 120 121 is present only in Greece and Malta. (Euro Check List). The essential oils of some 122 Italian accessions have been studied (Servettaz et al. 1994) and recently an excellent 123 antimicrobial activity of the ethanolic extract of T. siculum against Staphylococcus aur- 124 eus, S. epidermidis and Enterococcus faecalis has been reported (Acquaviva et al. 2018). 125 Up to now, of the ten Teucrium taxa growing in Sicily only two species have been inves- 126 tigated (Servettaz et al. 1994;Prestietal.2010).Consequently, in the frame of our ongoing 127 researches on Sicilian plants and Teucrium species (Bruno et al. 2019;Zitoetal.2013; 128 Senatore et al. 2008; Bruno et al. 2003) we decided to investigate the phytochemical profile 129 of the essential oils of the Sicilian accession of the three above mentioned Teucrium taxa in 130 order to increase the knowledge on species of this genus growing in Sicily. 131 132 2. Results and discussion 133 134 A total of ten volatile components were identified by GC-MS in the essential oil from a 135 Sicilian accession of T. fruticans (TFS), accounting for 96.9% of the total composition (Table 1), expressed in terms of peak area percentages. The oil was dominated by 4 R. GAGLIANO CANDELA ET AL.

136 Table 1. Chemical composition of the essential oil of the aerial parts of T. fruticans from Sicily 137 (TFS) and Malta (TFM), and T. scordium subsp. scordiodes (TSS) and T. siculum (TS) from Sicily. a b 138 Ri Ri Compound TFS TFM TSS TS 139 842 846 (2E)-Hexenal 3.6 RI,MS 859 863 n-Hexenol 0.1 RI,MS 140 842 846 Heptanal 0.1 RI,MS 141 926 932 a-Pinene ––19.4 0.1 RI,MS,STD 932 931 Allyl isovalerate ––0.3 RI,MS 142 940 946 Camphene 0.2 RI,MS,STD 143 950 952 Benzaldehyde –––0.1 RI,MS,STD 966 974 b-Pinene – 3.0 8.5 RI,MS 144 973 974 1-Octen-3-ol 19.7 7.4 0.1 9.0 RI,MS,STD 145 986 984 2-Pentyl-furan 0.1 RI,MS – 146 987 988 Myrcene 2.4 0.4 0.1 RI,MS,STD 999 988 3-Octanol ––0.2 0.1 RI,MS 147 1000 1002 a-Phellandrene 0.1 RI,MS,STD 148 1019 1020 p-Cymene 0.1 RI,MS,STD 1025 1024 Limonene ––2.2 0.2 RI,MS,STD 149 1037 1032 (Z)-b-Ocimene ––1.3 0.1 RI,MS,STD 150 1038 1036 Benzene acetaldehyde 0.4 RI,MS 1047 1044 (E)-b-Ocimene ––0.1 0.1 RI,MS,STD 151 1099 1095 Linalool 6.0 3.2 7.6 RI,MS,STD 152 1102 1100 n-Nonanal 0.4 RI,MS 1123 1122 a-Campholenal 0.2 RI,MS 153 1133 1135 trans-Pinocarveol 0.4 RI,MS,STD 154 1141 1140 trans-Verbenol 0.4 RI,MS p 155 1147 1150 -Vinyl-anisole 0.1 RI,MS 1158 1160 Pinocarvone 0.3 RI,MS,STD 156 1161 1165 Borneol 0.1 RI,MS,STD 157 1184 1186 a-Terpineol –– 0.1 RI,MS,STD 1188 1190 Methyl salicylate 0.1 RI,MS 158 1191 1195 Myrtenal 0.2 RI,MS,STD 159 1192 1194 Myrtenol 0.1 RI,MS,STD 1237 1239 Carvone 0.3 RI,MS,STD 160 1258 1260 (2E)-Decenal 0.1 RI,MS 161 1281 1287 Bornyl acetate 0.4 RI,MS,STD 1282 1296 Dihydroedulan II 0.2 RI,MS 162 1287 1288 Theaspirane A 0.1 RI,MS 163 1304 1305 Theaspirane B 0.1 RI,MS a –– 164 1364 1374 -Copaene 0.1 RI,MS 1372 1387 b-Bourbonene –– 1.1 RI,MS 165 1376 1383 (E)-b-Damascenone 0.2 RI,MS,STD 166 1393 1408 (Z)-b-Caryophyllene 0.2 RI,MS 1402 1403 Methyl eugenol 0.1 RI,MS 167 1404 1417 (E)-b-Caryophyllene 19.6 21.9 4.4 30.9 RI,MS,STD 168 1415 1430 b-Copaene 0.3 RI,MS 1428 1432 a-trans-Bergamotene 2.4 RI,MS 169 1429 1431 b-Gurjunene 0.1 RI,MS 170 1435 1440 (Z)-b-Farnesene 1.4 1.1 RI,MS,STD 1437 14522 a-Humulene 5.6 3.3 0.5 8.6 RI,MS,STD 171 1444 1458 allo-Aromadendrene –– 0.3 RI,MS 172 1449 1457 Sesquisabinene 3.4 0.5 RI,MS b 173 1452 1454 (E)- -Farnesene 2.2 2.3 RI,MS,STD 1465 1484 Germacrene D 29.4 50.0 8.0 RI,MS 174 1468 1481 c-Curcumene 0.2 RI,MS 175 1471 1475 b-trans-Bergamotene 2.2 RI,MS 1474 1487 (E)-b-Ionone 0.1 RI,MS,STD 176 1479 1500 Bicyclogermacrene 0.2 RI,MS 177 1485 1483 a-Amorphene 0.1 RI,MS 1487 1493 a-Zingiberene 0.2 RI,MS 178 1498 1505 b-Bisabolene ––3.8 1.1 RI,MS 179 1499 1514 b-Curcumene –– 0.3 RI,MS trans 180 1507 1528 -Calamenene 7.3 2.0 RI,MS (continued) NATURAL PRODUCT RESEARCH 5

181 Table 1. Continued. a b 182 Ri Ri Compound TFS TFM TSS TS 183 1508 15222 d-Cadinene – 1.7 0.8 RI,MS 1513 1521 b-Sesquiphellandrene ––5.9 0.3 RI,MS 184 1516 1533 trans-Cadina-1,4-diene 4.0 3.0 RI,MS 185 1531 1542 cis-Sesquisabinene hydrate 0.5 RI,MS 1554 1561 (E)-Nerolidol 0.8 RI,MS,STD 186 1558 1565 (3Z)-Hexenyl benzoate 0.2 RI,MS 187 1563 1582 Caryophyllene oxide 2.2 – 25.8 3.6 RI,MS,STD 188 1592 1608 Humulene epoxide II 2.0 0.7 RI,MS 1611 1627 1-epi-Cubenol 0.1 RI,MS 189 1615 1639 Caryophylla-4(12),8(13)-dien-5-a-ol 0.2 RI,MS 190 1621 1639 Caryophylla-4(12),8(13)-dien-5-b-ol 0.3 0.2 RI,MS 1626 1640 epi-a-Muurolol 1.9 0.3 RI,MS 191 1645 1652 a-Cadinol 1.2 0.4 RI,MS,STD 192 1658 1666 14-Hydroxy-(Z)-caryophyllene 1.7 RI,MS 1659 1674 b-Bisabolol –– 0.3 RI,MS 193 1674 1683 epi-a-Bisabolol 0.3 RI,MS 194 1678 1697 4-(1,5-Dimethylhex-4-enyl)cyclohex-2-enone 6.4 RI,MS 1702 1722 ar-Curcumen-15-al 0.3 RI,MS 195 1713 1740 Mint sulfide 0.2 RI,MS 196 1749 1759 Benzoyl benzoate 0.1 RI,MS 197 1833 1838 Neophytadiene 0.2 RI,MS 1843 1845 Phytone 0.6 0.4 RI,MS 198 1964 1959 Hexadecanoic acid 0.9 RI,MS,STD 199 2100 2104 Phytol 0.3 RI,MS,STD 2300 2300 n-Tricocosane 0.1 RI,MS,STD 200 2500 2500 n-Pentacosane 0.5 RI,MS,STD 201 2700 2700 n-Heptacosane 0.4 0.7 RI,MS,STD 2900 2900 n-Nonacosane 0.4 0.2 RI,MS,STD 202 Monoterpene hydrocarbons - 5.4 32.1 0.8 203 Oxygenated monoterpenes 6.0 3.2 2.1 8.0 Sesquiterpene hydrocarbons 65.9 81.9 24.0 58.9 204 Oxygenated sesquiterpenes 5.3 - 30.4 7.1 205 Others 19.7 7.4 8.4 18.8 206 TOTAL 96.9 97.9 97.0 93.6 a b 207 Ri : Retention index on a HP-5MS column; Ri : Retention index of literature. 208 209 sesquiterpene hydrocarbons (5 components, 65.9%), whereas oxygenated monoter- 210 penes (1 component, 6.0%) and oxygenated sesquiterpenes (3 components, 5.3%) 211 gave a minor contribution. The oil was devoid of monoterpene hydrocarbons. The 212 major constituents representing more than half oil were germacrene D (29.4%), 1- b 213 octen-3-ol (19.7%) and (E)- -caryophyllene (19.6%). An extremely similar chemical pro- 214 file was shown by the oil from plants collected in Malta (TFM) (Table 1). Also, in this 215 case the sesquiterpene hydrocarbons were the main class (6 compounds, 81.9%) with b 216 germacrene D (50.0%), and (E)- -caryophyllene (21.9%) as the main products of the oil 217 and 1-octen-3-ol was present in good quantity (7.4%). Consequently, T. fruticans could 218 be classified among Teucrium species producing germacrene D and (E)-b-caryophyl- 219 lene, as the main constituents, such as T. creticum L. (Valentini et al. 1997), T. orientale € 220 L. (Kucukbay et al. 2011; Ozek et al. 2012), T. orientale var. puberulens (T. Ekim) 221 (Kucukbay et al. 2011), T. parviflorum Schreb. (Bagci et al. 2011) and T. pestalozzae 222 Boiss. (Bas¸er et al. 1997) all belonging to the same section (genus Teucrium, section 223 Teucrium). Comparison of our data with the only analysis on this species, referring to 224 plants collected in Tuscany (Flamini et al. 2001), showed that although germacrene D 225 and (E)-b-caryophyllene occurred in high percentages (17.7% and 12.2%, respectively), the main class was represented by monoterpene hydrocarbons (50.8%), with b-pinene 6 R. GAGLIANO CANDELA ET AL.

226 (21.1%) and myrcene (12.8%) as the main compounds. Furthermore, 1-octen-3-ol was 227 totally absent in this oil. 228 The oil of T. scordium subsp. scordiodes (TSS) was characterized by similar amounts of 229 oxygenated sesquiterpenes (30.4%), monoterpene hydrocarbons (32.1%) and sesquiter- 230 pene hydrocarbons (24.0%) (Table 1). The most abundant compound of the oil was 231 caryophyllene oxide (25.8%), followed by a-pinene (19.4%) and b-pinene (8.5%), whereas 232 among the sesquiterpene hydrocarbons the most representative products were b-ses- 233 quiphellandrene (5.9%) and (E)-b-caryophyllene (4.4%). Comparison of our data with the 234 only analysis of the essential oil of this taxa, i.e., from plants collected in Serbia 235 (Radulovicetal.2012), showed a complete different composition of the oil. In fact, the 236 latter one was characterized by the presence of menthofuran (11.9%), as the main com- 237 ponent, and by a complex mixture of fatty acids and fatty acid-derived compounds 238 (39.7%), being both totally absent in the Sicilian accession. Furthermore, the oil from 239 Serbia was almost devoid of caryophyllene oxide (0.9%), a-pinene (0.3%) and b-pinene 240 (0.5%), the principal compound of TSS. On the other hand, the oil of T. scordium col- 241 lected in Serbia (Kovacevic et al. 2001), and of T. melissoides Boiss. & Hausskn. from Iran 242 (Ahmadi et al. 2002), both belonging to same section of T. scordium subsp. scordiodes 243 (sect. Scordium), showed, similarly to TSS, high contents of a-pinene (17.7% and 27.7%, 244 respectively) and b-pinene (10.0% and 16.4%, respectively), although caryophyllene 245 oxide was present in lesser amounts with respect to TSS (3.2% and 0%, respectively). 246 The oil obtained from the aerial parts of T. siculum (TS) was quite rich in sesquiter- 247 pene hydrocarbons (58.9%) with (E)-b-caryophyllene (30.9%), a-humulene (8.6%) and 248 germacrene D (8.0%) as the main constituents. Among the oxygenated monoterpenes 249 (8.0%), linalool (7.6%) represented the principal one and, furthermore, it is noteworthy 250 the good occurrence of 1-octen-3-ol (9.0%). Previous investigations on the leaves of 251 Tuscanian and Sicilian accessions of this species (Servettaz et al. 1994) also indicated 252 (E)-b-caryophyllene as the main compound of the oils (32.9% and 25.6%, respectively), 253 although the Sicilian population contained high amounts of isoeugenol (23.8%) and 254 d-cadinene (18.6%) that are almost absent in TS (0% and 0.8%, respectively). On the 255 other hand, the oil from the leaves of a population of T. siculum collected in Northern 256 Italy (Servettaz et al. 1994), showed a very different profile, devoid of (E)-b-caryophyl- 257 lene, with a-pinene (28.6%), a-bergamotene (13.7%) and phenylacetaldehyde (9.8%) as 258 the main components. A review of literature showed that the oil of some other taxa, 259 belonging to the same section of T. siculum (sect. Scorodonia), namely T. canadensis L. 260 (Lawrence et al. 1972), T. kotschyanum Poech (Arnold et al. 1991), T. royleanum Wall. 261 ex Benth. (Mohan et al. 2010), T. salviastrum Schreb. (Velasco-Negueruela and Perez- 262 Alonso 1990), T. scorodonia ssp. baeticum (syn: T. pseudoscorodonia Desf.) (Djabou 263 et al. 2012) and T. scorodonia L. (Djabou et al. 2012; Maccioni et al. 2007) contained, 264 as TS, high quantity of (E)-b-caryophyllene, a-humulene and germacrene D. 265 266 267 3. Experimental 268 3.1. Plant material 269 270 Flowering aerial parts of T. fruticans (TFS) were collected at Capo Zafferano, 30 Km east of Palermo, Italy, on rocky cliffs near the sea (3806’53” N; 1330’29” E; 47 m a.s.l.), at NATURAL PRODUCT RESEARCH 7

271 the beginning of May 2019, from plants at the full flowering stage. Typical specimens 272 (PAL 109702), identified by Prof. Vincenzo Ilardi, have been deposited in the 273 Herbarium Mediterraneum Panormitanum of the “Orto Botanico”, University of 274 Palermo, Italy. 275 Flowering aerial parts of T. fruticans (TFM) were collected at Paradise Bay, Malta 276 (3558’53” N, 1419’58” E; 50 m a.s.l.) on rocky cliffs near the sea at the end of April 277 2019, from plants at the full flowering stage. Typical specimens (PAL 109703), identi- 278 fied by Prof. Vincenzo Ilardi, have been deposited in the Herbarium Mediterraneum 279 Panormitanum of the “Orto Botanico”, University of Palermo, Italy. 280 Flowering aerial parts of T. scordium subsp. scordioides (TSS) were collected near 281 Piana degli Albanesi Lake (Contrada Maganoce), 40 km south of Palermo, Italy, in a 282 wet plateau, locally called “Margio” (37 57’ 35.20” N; 13 18’ 35.43” E; 775 m a.s.l.), at 283 the beginning of June 2019, from plants at the full flowering stage. Typical specimens 284 (PAL 109699), identified by Prof. Vincenzo Ilardi, have been deposited in the 285 Herbarium Mediterraneum Panormitanum of the “Orto Botanico”, University of 286 Palermo, Italy. 287 Flowering aerial parts of T. siculum (TS) were collected on a large area between 288 Scaletta d’Alfano, Cozzo Raimonda and Pizzo Argentiera (Madonie Mounts, Sicily, Italy) 289 (central point of collection 37 51’45.67” N; 14 07’05.68” E; 1368 m a.s.l.), at the begin- 290 ning of June 2019, from plants at the full flowering stage. Typical specimens (PAL 291 109700), identified by Prof. Vincenzo Ilardi, have been deposited in the Herbarium 292 Mediterraneum Panormitanum of the “Orto Botanico”, University of Palermo, Italy. 293 294 295 3.2. Isolation of the essential oil 296 The fresh materials were cut in small pieces and then subjected to hydrodistillation for 297 3 h using n-hexane as collector solvent, according to the standard procedure previ- 298 ously described (Ben Jemia et al. 2013). The oils were dried over anhydrous sodium 299 sulphate and then stored in sealed vials, at -20 C, ready for the GC-MS analyses. The 300 samples yielded 0.22% of oil (w/w), 0.18% of oil (w/w), 0.15% of oil (w/w), and 0.10% 301 of oil (w/w) for TFS, TFM, TSS and TS, respectively. 302 303 304 3.3. GC-MS analysis of the essential oil 305 Analysis of essential oil was performed according to the procedure reported by 306 Casiglia et al. (2017). Briefly, a gas chromatograph Agilent Technologies 6850 N 307 coupled with a mass spectrometer Agilent 5973 was used. Separation was achieved 308 309 using an apolar column (HP-5 MS, 5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., l 310 0.25 m film thickness; J & W Scientific, Folsom) using a temperature programmed gra- 311 dient (Casiglia et al. 2017). Temperatures of injector and detector were set up to 280 312 and 300 C, respectively. Helium was the carrier gas at a flow rate of 1 ml/min. Oil was 313 diluted in hexane (1:100) and 2 ml of the solution injected in split mode (1:50 split – 314 ratio). Electron impact (EI) spectra were acquired in the range m/z 29 400. For identifi- 315 cation of the volatile components, analytical standards available in the authors’ labora- tory were used (see Table 1) plus the combination of linear retention indices and mass 8 R. GAGLIANO CANDELA ET AL.

316 spectra with respect to those reported in commercial libraries such as ADAMS, FFNSC2 317 and NIST 17. Relative abundances of essential oil components were calculated in terms 318 of peak area percentage without calculating response factors. 319 320 4. Conclusion 321 322 Three taxa of Teucrium species collected in Sicily and Malta have been analyzed for 323 the composition of their essential oils: T. fruticans (TFS and TFM), T. scordium subsp. 324 scordioides (TSS) and T. siculum (TS). The essential oils showed a similar chemical pro- 325 file as generally shown by the other Teucrium taxa belonging to the same sections, 326 showing predominance of the sesquiterpene fraction. 327 328 Disclosure statement 329 330 No potential conflict of interest was reported by the authors. 331 332 ORCID 333 334 Filippo Maggi http://orcid.org/0000-0003-1375-4744 335 336 References 337 338 Acquaviva R, Genovese C, Amodeo A, Tomasello B, Malfa G, Sorrenti V, Tempera G, Addamo AP, Ragusa S, Rosa T, et al. 2018. Biological activities of Teucrium flavum L., Teucrium fruticans L., 339 and Teucrium siculum Rafin crude extracts. Plant Biosyst. 152(4):720–727. 340 Ahmadi L, Mirza M, Shahmir F. 2002. Essential oil of Teucrium melissoides Boiss. et Hausskn. ex 341 Boiss. J Ess Oil Res. 14(5):355–356. 342 Arnold N, Bellomaria B, Valentini G, Rafaiani SM. 1991. Comparative study on essential oil of 343 some Teucrium species from Cyprus. J Ethnopharmacol. 35(2):105–113. 344 Bagci E, Hayta S, Yazgin A, Dogan G. 2011. Composition of the essential oil of Teucrium parviflo- 345 rum L. (Lamiaceae) from Turkey. J Med Plant Res. 5:3457–3460. 346 Bas¸er KHC, Demirc¸akmak B, Duman H. 1997. Composition of the essential oils of three Teucrium species from Turkey. J Ess Oil Res. 9:545–549. 347 Ben Jemia M, Rouis Z, Maggio A, Venditti A, Bruno M, Senatore F. 2013. Chemical composition 348 and free radical scavenging activity of the essential oil of Achillea ligustica All. wild growing 349 in Lipari (Aeolian Islands, Sicily). Nat Prod Commun. 8:1629–1632. 350 Boudkhili M, Greche H, Bouhdid S, Zerargui F, Aarab L. 2012. In vitro antioxidant and antibacter- 351 ial properties of some Moroccan medicinal plants. Int J PharmTech Res. 4:637–642. 352 Bruno M, de la Torre MC, Savona G, Piozzi F, Rodrıguez B. 1990. A rearranged abietane diterpen- 353 oid from the root of Teucrium fruticans. Phytochemistry. 29(8):2710–2712. ı 354 Bruno M, Alcazar R, de la Torre M C, Piozzi F, Rodr guez B, Savona G, Perales A, Arnold N A. 1992. Neo- and seco-neo-clerodane diterpenoids from Teucrium gracile and T. fruticans. 355 Phtyochemistry. 31(10):3531–3534. 356 Bruno M, Ciriminna R, Piozzi F, Rosselli S, Simmonds M. 1999. Antifeedant activity of neo-clero- 357 dane diterpenoids from Teucrium fruticans and derivatives of fruticolone. Phytochemistry. 358 52(6):1055–1058. 359 Bruno M, Maggio AM, Piozzi F, Puech S, Rosselli S, Simmonds M. 2003. Neoclerodane diterpe- 360 noids from Teucrium polium subsp. polium and their antifeedant activity. Biochem Sys Ecol. 31(9):1051–1056. NATURAL PRODUCT RESEARCH 9

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