Anthemis maritima L.

24

Chapter 3

Anthemis maritima L.

Species (binomial name): maritima L.

25 During the PhD project I collaborated in a work on Anthemis maritima.

Below, I am going to report the results obtained on this study, as the full paper that we have published, of which I am one of the authors.

(D. Ciccarelli, S. Giovanelli, L. Pistelli. Essential oils from Anthemis maritima flowers: infraspecific variability along the Adriatic coast (Italy). Chem. Biodiver. (2016) 13(5): 561-570. doi: 10.1002/cbdv.201500184)

3.1 Introduction

In traditional medicine, some species belonging to the genus Anthemis have been used for their anti-inflammatory, antibacterial and antispasmodic activity [4-6]. In previous phytochemical studies, sesquiterpene lactones, flavonoids, and coumarins have been identified as the main constituents of the genus [7-14]. The essential oils from different Anthemis taxa are characterised by oxygenated monoterpenes, as α- and β-thujone, yomogi alcohol, borneol, terpinen 4-ol, α-terpineol, and by terpene esters, especially cis-chrisanthenyl acetate and trans-chrysanthenyl acetate, and other chrysanthenyl esters [15-24].

The subject of the present study is A. maritima, a perennial plant (12 – 70 cm high), with pinnatifid leaves and white ligulate and yellow tubulose flowers, blooming from May to August [2]. A. maritima is one of the typical dune species distributed throughout the Mediterranean area, which is able to colonise both coastal sand dunes and rocky cliff ecosystems, exhibiting a high degree of leaf trait plasticity [25], so it contributes to coastal sand dune edification and restoration [3]. Recently, different chemotypes from A. maritima populations growing in different localities along the Tyrrhenian coast of Italy have been

26 circumscribed. The most interesting population was the one living on sea cliffs, because it showed a different chemical profile compared to those of the other populations growing on sand dunes [26]. In order to improve the knowledge about the chemical composition of the Italian A. maritima, we carried out a comparative analysis of the essential oils from five populations growing along the Adriatic coast. In particular, we wanted to answer the following questions: 1) Are the chemical compositions of the essential oil extracted from the flowers of A. maritima populations of Adriatic coast linked to the geographic origins? 2) Are there any differences regarding the chemical profile between sea cliffs/shingle beaches and sand dunes populations, as seen for the Tyrrhenian coast?

3.2 Phytochemicals and volatiles

Several studies on Anthemis spp. evidenced the presence of sesquiterpene lactones, flavonoids, and coumarins [7–13]. In particular, two new cytotoxic saturated and unsaturated cyclohexanones from the leaves of Sardinian A. maritima were isolated and identified [14]. The essential oils from Anthemis spp. are characterized by oxygenated monoterpenes, as α- and β-thujone, yomogi alcohol, borneol, terpinen 4-ol, and α-terpine4-ol, and by terpene esters, especially cis-chrisanthenyl acetate and other chrysanthenyl esters [15-21]. In a previous work on the essential oil from Corsican and Sardinian samples of A. maritima, the presence of several chrysanthenyl esters was evidenced, and the authors distinguished two clusters of samples, discriminated by the amount of trans-chrysanthenyl acetate [22].

The same trend of compounds were identified in other oils isolated from samples collected in different peninsular and insular Italian localities (along the Tirrenian coasts) [23]: these are constituted mainly of terpene esters (35.1–62.4%), as trans-chrysanthenyl acetate (16.7 –55.6%) and other chrysanthenyl esters. These oils presented an high amount of sesquiterpene hydrocarbons, comprising mainly (Z,E)-α-farnesene, germacrene D and valencene (from 29.2 and 36.5%), while the monoterpene composition showed significant differences between the oils of the six localities, exhibiting in general a predominance of monoterpene hydrocarbons (0.5 –23.9%) compared to oxygenated monoterpenes (0.3 –15.0%).

3.3 Aim of the work

In the present study the flowers of Anthemis maritima L. were collected in May 2013 from 5 different Italian localities (along the Eastern Italian coasts): Marina di Lesina [LES], Lido di Casalbordino [CAS], Torino di Sangro [TDS], Istmo di Varano [VAR] e Portocorsini [PC].

27 The collection sites, their soil features and their geo-climatic information are reported in Fig. 3.1 and Table 3.1. Based on literature [3], we analysed all the Adriatic regions were the presence of A. maritima was registered in Italy.

All the samples were dried and then hydrodistilled for 1 hours in a Clevenger apparatus. The amount of each essential oil were less than 0.01%.

Table 3.1 Localities of collection

Location (Sample name) Latitude Longitude Soil type Bioclimatic Region* Porto Corsini (PC) 44°29’57.3” 012°17’00.3” sand dunes Continental Torino di Sangro (TDS) 42°13’30.1” 014°33’38.0” rocky cliffs Continental Casalbordino Lido (CAS) 42°11’49.5” 014°37’33.5” sand dunes Continental Marina di Lesina (LES) 41°54’58.2” 015°19’39.4” sand dunes Mediterranean Istmo di Varano (VAR) 41°54’51.3” 015°42’02.8” sand dunes Mediterranean

*Sensu Directive 92/43/EEC

Figure 3.1 Map showing the localization of the different collection sites (indicated by an asterisk) of the Anthemis maritima

populations. 1 = Porto Corsini (PCO), 2 = Torino di Sangro (TDS), 3 = Casalbordino Lido (CAS), 4 = Marina di Lesina (LES), 5 = Istmo di Varano (VAR).

28

- Population Study Based on Chemical Composition of the Essential Oils.

The compositions of the essential oils obtained from the different populations of Anthemis maritima are reported in Table 3.2. Totally 162 compounds were identified, with percetages of identification of 94.4% for [TDS], 94.7% for [LES], 96.9% for [CB], 98.6% for [VAR] and 99.2% for [POR].

The composition of the EOs showed the presence of high amount of terpene esters in all samples (from 15.5 and 28.5%) (see Figure 3.2) comprising mainly trans-chrysanthenyl acetate (from 3.9 and 21.6%) and cis-chrysanthenyl isobutyrate (2.8 and 5.8%), and other chrysanthenyl esters, in agreement with previous studies reported in the literature.

Among the analyzed essential oils, the sample collected in PC differs from those of the other populations, showing the higher amount of oxygenated monoterpenes (28.5%): cis-chrysanthenol (16.0%) and 1,8-cineole (6.2%). Moreover the oils obtained from [PC] and [VAR] presented high amount of monoterpene hydrocarbons (17.9% and 15.9%, respectively), where α-pinene was the mainly compound (6.8 and 3.6%, respectively). The other samples ([VAR], [LES], [TDS]) are characterized by the presence of oxygenated sesquiterpenes (from 11.0 and 14.2%). The amount of sesquiterpene hydrocarbons (from 4.9 and 11.8%) and aliphatic compounds (ranges from 5.3 and 13.2%, less in TDS sample) are present in similar amount inside all EOs. This composition is different than the other samples studied before and collected in the Tirrenian coast, characterized by an high amount of sesquiterpene hydrocarbons. These discrepancies in EO composition could be due to the differences in geographical area origin, growing conditions, climate and soil type.

Figure 3.2. Composition and relative percentage concentrations of volatile compounds

29

- Cluster Analysis (CA) and Principal Coordinate Analysis (PCO).

Statistical analysis was carried out using the most abundant compounds with an amount ≥ 1.5% in at least one population. Based on Bray-Curtis coefficient matrix, the CA classified the studied populations in two groups with a similarity > 70% (Fig. 3.3): cluster I, formed only by POC (population from North Italy); and cluster II, which was made of populations from Central and South Italy. Inside the second cluster, TDS and CAS (populations from Central Italy) showed the highest similarity (> 75%). This classification was supported by Principal Coordinate Analysis, that resumed 88.7% of the total variability. The first axis (PCO1) explained 60.6% of the chemical variation, while PCO2 accounted for 28.1% (Fig. 3.4). POC population was characterised by a high content of β-pinene (20), γ-terpinene (38), and β-caryophyllene (112). In the cluster II, VAR sample could be distinguished from the others by the highest values of cis-chrysanthenyl acetate (83), trans-cis-chrysanthenyl isobutyrate (99) and cubenol (158). Both samples from South Italy (VAR and LES) shared high values of cis-carveol propionate (128) and α-zingiberene (129). Lastly, TDS and CAS (populations from Central Italy) could be distinguished for the highest amount of trans-chrysanthenyl acetate (79). Similarly, trans-chrysanthenyl acetate distinguished the A. maritima essential oils of Sardinian samples from Corsican samples [22]. If we compare our data with those of [26], the populations analysed in the present work are comprised in a different cluster from the Tyrrhenian samples, as suggested by Cluster analysis and PCO results (Figures 3.4 and 3.5). The main compounds that played a crucial role for discriminating the Italian populations were cubenol (absent in all the Tyrrhenian samples), and (E)-β-farnesene (absent in all the Adriatic samples).

Figure 3.4. Dendrogram obtained by average-linkage cluster analysis based on the Bray-Curtis distances among the essential

30

Figure 3.4. PCO ordination (using the Bray-Curtis dissimilarity metric) of the Anthemis maritima populations based on the

major essential oil components (content ≥ 1.5%). For the abbreviations of the sampling sites, cf. Table 1 and Fig. 1. All the compounds have a Spearman correlation coefficient > 0.9 with the two axes. 20 = β-pinene, 38 = γ-terpinene, 79 = trans-chrysanthenyl acetate, 83 = trans-chrysanthenyl acetate, 99 = trans-trans-chrysanthenyl isobutyrate, 112 = β-caryophyllene, 128 =

cis-carveol propionate, 129 = α-zingiberene, 158 = cubenol

3.5 Conclusions

The present work highlighted the relationship between the chemical composition of essential oils extracted from the flowers of A. maritima populations and their geographical locations. Hence, we can answer the questions posed at the beginning of the paper.

1) The following three different chemotypes have been circumscribed: one group living in North Italy (POC population) characterised by the highest content of pinene, γ-terpinene, and β-caryophyllene; a second chemotype in Central Italy (CAS and TDS) with the highest amount of trans-chrysanthenyl acetate; and a third group living in South Italy (LES and VAR) with a more heterogeneous chemical profile characterised by the highest values of cis-chrysanthenyl acetate, trans-chrysanthenyl isobutyrate, cis-carveol propionate, α-zingiberene, and cubenol.

2) Since we did not find any appreciable differences between TDS and CAS populations, growing on shingle beaches and sand dunes respectively; the infra-specific variability of this plants can be explained mainly by the climatic factors along the Adriatic coast.

In conclusion, we can confirm the role of A. maritima as plant particularly interesting for the flavour and fragrance industry.

31

Table 3.2. Volatile composition of the EOs of the five samples of A..maritima

No. Class Compound LRI Area [%]

POR CB LES VAR TDS

1 EST 2-methyl butanoic acid, ethyl ether 850 tr

2 EST Ethyl isovalerate 861 tr

3 ALD (E)-2-Hexanal 860 tr

4 ALC (E)-3-Hexen-1-ol 868 tr tr

5 EST Methyl tiglate 871 tr

6 ALC n-Hexanol 875 tr tr tr

7 EST Isopenthyl acetate 876 tr

8 ALCH 1-Nonene 895 tr tr tr 9 ALD Heptanal 903 tr tr tr tr 10 MH Santolina triene 910 tr 3.1 0.6 6.5 6.2 11 MH α-Thujene 930 tr tr tr 12 MH α-Thujene 932 0.7 0.2 0.1 0.2 13 MH tricyclene 934 tr tr 14 MH α-Pinene 940 7.5 2.1 1.1 4.9 1.9 15 MH Camphene 955 0.1 0.1 tr 0.1 tr 16 MH Thuja-2,4(10)-diene 959 tr tr tr tr 17 ALD Benzaldehyde 965 tr tr tr tr 18 ALC 1-Heptanol 973 tr 19 MH Sabinene 978 1.1 0.2 0.1 1.0 0.4 20 MH β-Pinene 981 3.5 0.6 1.1 2.0 1.0 21 KET 6-Methyl-5-hepten-2-one 990 7.9 3.4 6.5 5.2 3.7 22 MH Myrcene 993 1.9 1.5 0.8 1.8 1.3 23 ALC 3-Octanol 994 tr tr 24 OM Yomogi alcohol 998 tr tr tr 25 MH m-Mentha-1(7),8-diene 1001 tr 26 ALD n-Octanal 1005 tr tr tr tr 27 MH α-Phellandrene 1006 0.2 0.1 tr tr 0.4 28 MH δ-3-Carene 1012 1.5 0.8 tr tr 0.5 29 MH α-Terpinene 1019 0.3 0.1 tr 0.2 0.1 30 MH o-Cymene 1026 0.2 0.2 0.1 31 MH p-Cymene 1028 0.6 0.3 0.3 0.4 0.3 32 MH Limonene 1032 tr 0.2 0.2 0.3 0.2 33 OM 1,8-Cineole 1036 7.3 tr 1.3 4.0 1.7 34 OM Santolina alcohol 1037 tr 35 MH (Z)-β-Ocimene 1042 tr tr tr tr tr

36 ALD Benzene acetaldehyde 1047 tr tr

37 MH (E)-β-Ocimene 1053 0.2 tr 0.2 0.3 tr

38 MH γ-Terpinene 1062 2.0 0.3 0.6 0.7 0.3

39 unknown 0.1 0.2

32

41 ALC 1-Octanol 1075 tr tr tr tr

42 ALC 1-Nonen-3-ol 1083 0.1 tr 0.2

43 ALC Artemisia alcohol 1086 tr

44 MH para-Mentha-2,4(8)-diene 1086 tr 45 MH α-Terpinene 1090 tr 46 MH Terpinolene 1090 0.2 tr tr 0.1 tr 47 ALC 3-Nonanol 1097 tr 48 OM trans-Sabinene hydrate 1101 tr 49 OM Linalool 1102 1.0 0.4 50 ALD n-Nonanal 1104 0.1 0.4 0.3 0.3 0.3

51 EST Isopenthyl isovalerate 1106 0.1 tr

52 OM β-Thujone 1114 1.3 0.6 1.1 53 OM cis-para-Menth-2-en-1-ol 1125 tr tr tr tr 54 OM Chrysanthenone 1128 0.2 0.1 55 OM α-Campholenal 1130 0.1 0.2 0.1 56 OM trans-Chrysanthenol 1133 0.3 0.2 0.1 0.1 0.2 57 OM trans-Pinocarveol 1142 tr 58 OM trans-para-Menth-2-en-1-ol 1145 tr tr tr

59 ALD Lilac aldehyde C 1146 0.5

60 OM Camphor 1148 0.4 0.5

61 OM cis-Verbenol 1149 0.3 0.1 0.1

62 ALD Lilac aldehyde B 1150 0.6

63 unknown 0.2 64 OM cis-Chrysanthenol 1166 18.3 5.7 5.4 5.8 4.4 65 OM para-Mentha-1,5-dien-8-ol 1166 0.2 66 OM δ-Terpineol 1170 0.4 0.3 0.1 67 OM 3-Thujanol 1171 0.3 0.2 0.2 68 OM Terpinen-4-ol 1180 1.4 0.3 0.9 1.0 0.8 69 OM α-Terpineol 1193 0.8 tr 0.3 0.4 0.1

70 EST Methyl salicylate 1193 tr

71 OM Myrtenol 1195 tr tr tr

72 OM Safranal 1200 0.1 tr 0.1 tr tr

73 unknown tr 0.3

74 ALD n-Decanal 1206 tr

75 OM cis-Sabinene hydrate acetate 1221 0.2 0.2 0.1 0.1 0.1

76 OM trans-(3)10-Caren-2-ol 1226 2.1 0.44 0.3 0.3 0.2

77 OM neo-iso-Dihydro carveol 1229 tr tr

78 EST 3-methyl-3-Hexen-1-yl butanoate 1236 tr tr

79 TE trans-Chrysanthenyl acetate 1237 28.1 5.1 tr 20.1

80 OM Cuminaldehyde 1244 tr

81 OM Carvone 1248 tr tr tr tr

82 OM Geraniol 1259 tr

33

84 OM Perilla aldehyde 1271 tr

85 ACID Nonanoic acid 1276 tr tr tr

86 TE cis-Verbenil acetate 1283 0.4 0.2 0.5

87 EST Isobornyl acetate 1287 tr 0.1 tr

88 OM Thymol 1293 tr tr tr 0.1 tr

89 OM Carvacrol 1301 tr tr tr tr

90 OM trans-2-Caren-4-ol 1304 0.2 0.2

91 ALD Undecanal 1305 0.2

92 EST n-Nonanol acetate 1312 tr

93 ALD 2E,4E-Decadienal 1318 tr tr tr tr 94 SH Silphiperfol-5-ene 1327 tr tr tr tr 95 TE cis-Chrysanthenyl propanoate 1340 0.3 tr 0.3 96 SH 7-epi-Silphiperfol-5-ene 1345 0.1 0.3 tr tr 97 SH α-Cubebene 1351 tr tr 98 TE trans-Chrysanthenyl isobutyrate 1360 2.0 1.7 3.3 4.5 2.3 99 SH Cyclosativene 1371 tr tr tr tr 100 SH Longicyclene 1374 0.1 101 unknown 0.2 0.3 102 SH α-Copaene 1376 0.4 0.7 0.6 0.4 0.4 103 SH β-Maaliene 1379 0.2 0.4 0.2 tr 104 SH β-Bourbonene 1383 0.3 0.4 0.2 0.2 0.2 105 SH β-Cubebene 1390 tr 0.2 0.2 tr tr 106 TE cis-Chrysanthenyl isobutyrate 1400 4.5 3.8 5.8 7.8 3.7 107 unknown 0.3 0.3 0.1 108 PP Methyl eugenol 1407 tr 0.2 tr tr 109 SH α-Gurjunene 1410 0.5 1.0 tr 110 unknown 1.0 111 SH β-Caryophyllene 1418 5.4 3.8 3.0 1.6 3.0 112 SH γ-Elemene 1431 tr 113 SH β-Copaene 1432 tr tr 114 SH β-Gurjunene 1434 0.2 0.6 tr 115 SH trans-α-Bergamotene 1438 tr tr 0.2 0.1

116 EST 2-methyl buthyl benzoate 1437 tr

117 TE cis-Chrysanthenyl butyrate 1440 1.3 0.6 1.4 1.4 1.0 118 TE trans-Chrysanthenyl isovalerate 1452 0.6 0.5 0.9 0.6 0.4 119 SH α-Humulene 1456 0.9 0.7 0.6 0.4 0.5 120 SH α-Patchoulene 1456 tr 121 SH allo-Aromadendrene 1461 0.4 1.0 0.2 122 SH 9-epi-(E)-Caryophyllene 1467 1.4 2.5 3.1 1.3 123 unknown 0.3 124 SH trans-Cadina-1(6),4-diene 1470 tr tr tr 125 SH γ-Muurolene 1477 tr tr tr tr 126 SH Germacrene D 1481 3.6 3.4 4.5 2.1 2.0

34 127 TE cis-Carveol propionate 1489 4.2 4.6 11.3 11.8 6.1 128 SH Bicyclogermacrene 1495 0.5 0.6 1.1 0.2 0.6 129 SH α-Zingiberene 1496 5.2 5.4 9.0 13.0 6.8 130 SH α-Muurolene 1499 tr 131 SH trans-β-Guaiene 1500 tr tr tr tr 132 SH (E.E)-α-Farnesene 1507 0.6 0.2 tr 0.2 133 SH (Z)-β-Bisabolene 1508 tr 134 SH trans-γ-Cadinene 1513 0.2 0.9 1.1 0.6 0.9 135 SH epi-α-Selinene 1519 0.2 136 SH δ-Cadinene 1523 0.4 0.9 0.8 0.9 1.1 137 SH Selina-3,7(11)-diene 1542 tr 138 unknown 1.1 139 TE Limonen-6-ol pivalate 1550 0.6 140 unknown 0.1 tr tr tr 141 OS epi-Longipinanol 1564 tr 142 OS (E)-Nerolidol 1566 0.3 1.4 0.4 0.2 0.4

143 EST (Z)-3-Hexenyl benzoate 1570 tr

144 OS Caryophyllene oxide 1582 0.6 0.7 0.8 0.4 1.0 145 OS di-epi-α-Cedrene 1586 0.4 146 OS Viridiflor 1591 1.1 147 OS Longiborneol 1596 0.4 0.5 tr 148 OS Humulene oxide II 1607 tr 0.2 tr 0.5 1.0 149 unknown 0.8 tr 150 OS 1,10-di-epi-Cubenol 1614 tr 0.3 tr tr 151 OS 1-epi-Cubenol 1630 0.1 0.2 0.2 tr 0.4 152 OS Eremoligenol 1632 tr 153 unknown 0.3 1.2 0.6 154 unknown 1.4 4.3 155 OS Caryophylla-4(14),8(15)-dien-5-ol 1636 tr 156 OS epi-α-Cadinol 1642 0.5 4.8 5.9 4.0 6.2 157 OS Cubenol 1642 1.3 0.2 0.9 1.5 0.4 158 OS α-Cadinol 1655 0.6 1.3 1.8 1.1 1.5 159 OS epi-β-Bisabolool 1672 0.4 0.3 tr 0.2 160 OS Valeranone 1675 0.2 161 unknown 0.1 162 unknown 0.2 0.3 0.2 163 TE 8-hydroxy-isobornyl isobutyrate 1675 0.2 0.3 0.4 0.5 0.3 164 OS Khusinol 1680 0.6 0.4 tr 0.2 165 OS epi-α-Bisabolool 1685 0.1 166 unknown 0.8 0.3 0.6 0.4 0.2 167 unknown 0.2 168 ALD β-Sinensal 1701 0.2 169 OS Curcumen-15-al 1704 tr 0.1 0.2 tr 0.1

35 170 ALD Pentadecanal 1717 0.1 171 OS (E)-Nerolidol acetate 1719 0.3 172 OS (Z)-Nuciferol 1725 tr 0.2 0.3 tr 173 unknown 0.2 tr 174 OS (E)-Nuciferol 1758 tr 0.4 0.5 0.3 0.2 175 OS α-Sinensal 1759 tr tr 176 unknown 0.4 0.6 0.4 0.3

177 EST Benzyl benzoate 1766 0.2 0.2

178 OS (Z)-Lanceol 1769 0.3 tr 0.8 179 TE (Z)-α-Santalol acetate 1779 tr tr 180 OS 14-hydroxy-δ-Cadinene 1800 tr MH Monoterpene hydrocarbons 20.1 9.9 4.4 18.5 12.9 OM Oxygenated monoterpenes 32.6 9.0 10.8 12.5 9.2 SH Sesquiterpene hydrocarbons 19.0 21.3 26.5 20.7 17.7 OS Oxygenated sesquiterpenes 3.8 11.6 12.3 9.1 13.8 TE Terpene esters 14.5 41.0 32.5 32.3 36.4 PP Phenyl propanoids 0.2 AE Acid/Esters 0.3 0.3 ALD/KET Aldehydes/Ketons 9.2 3.8 7.3 5.5 4.0 ALK/ALC Alkanes/Alcohols 0.1 0.2 UNK Unknowns 1.8 3.7 5.0 2.4 5.6 Total identified [%] 99.2 96.9 94.7 98.6 94.4

LRI, Linear retention index. Compounds with relative percentages smaller or equal to 0.1% are considered as traces (tr.). The components are listed in order of their elution on the DB-5 column. POR, Portocorsini; CB, Casalbordino; LES, Lesina; VAR,

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