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Wild Sicilian Rosemary: Phytochemical and Morphological Screening and Antioxidant Activity Evaluation of Extracts and Essential Oils by Edoardo M. Napoli a ), Laura Siracusa a ), Antonella Saija b ), Antonio Speciale b ), Domenico Trombetta b ), Teresa Tuttolomondo c ), Salvatore La Bella c ), Mario Licata c ), Giuseppe Virga c ), Raffaele Leone c ), Claudio Leto d ), Laura Rubino a ), and Giuseppe Ruberto* a ) a

) Istituto del C.N.R. di Chimica Biomolecolare, Via Paolo Gaifami 18, IT-95126 Catania (phone: þ 39-0957338347; fax: þ 39-0957338310; e-mail: [email protected]) b ) Dipartimento Scienze del Farmaco e dei Prodotti per la Salute, Universit— di Messina, Contrada Annunziata, IT-98168 Messina c ) Dipartimento di Scienze Agrarie e Forestali (SAF), Universit— di Palermo, Viale delle Scienze 13, IT-90128 Palermo d ) Co.Ri.S.S.I.A. Consorzio di Ricerca per lo Sviluppo di Sistemi Innovativi Agroambientali, Via Libert— 203, IT-90100 Palermo

To identify the best biotypes, an extensive survey of Sicilian wild rosemary was carried out by collecting 57 samples from various sites, followed by taxonomic characterization from an agronomic perspective. All the biotypes collected were classified as Rosmarinus officinalis L. A cluster analysis based on the morphological characteristics of the plants allowed the division of the biotypes into seven main groups, although the characteristics examined were found to be highly similar and not areadependent. Moreover, all samples were analyzed for their phytochemical content, applying an extraction protocol to obtain the nonvolatile components and hydrodistillation to collect the essential oils for the volatile components. The extracts were characterized by LC-UV-DAD/ESI-MS, and the essential oils by GC-FID and GC/MS analyses. In the nonvolatile fractions, 18 components were identified, namely, 13 flavones, two organic acids, and three diterpenes. In the volatile fractions, a total of 82 components were found, with as predominant components a-pinene and camphene among the monoterpene hydrocarbons and 1,8-cineole, camphor, borneol, and verbenone among the oxygenated monoterpenes. Cluster analyses were carried out on both phytochemical profiles, allowing the separation of the rosemary samples into different chemical groups. Finally, the total phenol content and the antioxidant activity of the essential oils and extracts were determined with the Folin–Ciocalteu (FC) colorimetric assay, the UV radiation-induced peroxidation in liposomal membranes (UV-IP test), and the scavenging activity of the superoxide radical (O .2¢ ). The present study confirmed that the essential oils and organic extracts of the Sicilian rosemary samples analyzed showed a considerable antioxidant/free radical-scavenging activity.

1. Introduction. – The Mediterranean area is a peculiar environment, in which Sicily Island plays a predominant role, due its position, its coastal extension, and its weather, characterized by mild winter and warm to hot summer seasons. This climate favors a large plant biodiversity, and numerous aromatic plants of the Lamiaceae family find their optimal habitat. For this reason, several studies have been carried out in Sicily, to preserve the plant genetic resources [1 – 4] and to improve the know-how on cultivation techniques. Particularly, rosemary is one of the most interesting aromatic species, because of the various properties and applications. Rosemary is a woody, perennial herb native to the Mediterranean region, commonly used in cookery as spice and well-known in the folk and traditional Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

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medicine as remedy for several diseases. The use of this herb as aromatic plant and as food flavoring in the Mediterranean cuisine is widely recognized. As medicinal plant, rosemary belongs to the pool of herbs whose efficacy is largely acknowledged and listed in the official Pharmacopoeia of several countries. Several biological activities have been ascribed to rosemary, and it is used to improve the memory [5] [6], in the treatment of neurodegenerative diseases [7 – 9] and hypertension [10], as antidepressant [11], and for its anticancer [12], anti-inflammatory [13] [14], antimicrobial [15 – 18], antithrombotic [19], and antioxidant properties [20 – 25]. Most of the aforesaid assets of rosemary are due to the large presence of polyphenol derivatives evidenced in many phytochemical studies of its extracts, i.e., flavonoids (genkwanin (1) and cirsimaritin (2)), aromatic terpenoids (carnosol (3) and carnosic acid (4)), and organic acids (rosmarinic acid (5) and caffeic acid (6)) [26 – 29]. Nowadays, one of the main industrial uses of rosemary is the extraction of its essential oils (EOs), which are widely used in cosmetics and the controversial aromatherapy [30]. The antimicrobial [31] [32], insecticidal and larvicidal [33] [34], and antioxidant [35 – 37] properties of rosemary EO are widely documented. Furthermore, the evidenced high and promising biological efficacy of this natural matrix prompted new and innovative applications of this product in several fields, e.g., as feed additive [38], in new packaging systems [39] [40], and other innovative applications [41 – 43]. Moreover, efforts are made to improve the extraction conditions [44] and methods, such as solvent-free microwave extraction [31]. From a chemical point of view, rosemary EO has been classified in three chemotypes, namely, cineoliferum (high content in 1,8-cineole), camphoriferum (high content in camphor), and verbenoniferum (high content in verbenone). However, many other chemotypes have been recognized, according to the relative abundance of other relevant compounds, and Sicilian wild rosemary populations show a certain compositional complexity, like other labiates collected from the wild [45 – 48]. The main aim of this study was to give a large ÐsnapshotÏ of the actual wild Sicilian rosemary populations, based on their morphoagronomic characteristics and phytochemical composition. Several cluster analyses (CA) were applied to select the best biotypes from an agronomic point of view and to distinguish homogenous chemotypes by their chemical differences. 2. Results and Discussion. – 2.1. Morphological and Agronomic Characteristics. In total, 57 sites where rosemary was growing wild were identified. For each of the sampling sites, the main geographical, topographical, and ecological characteristics were obtained. All the biotypes (samples) were classified as Rosmarinus officinalis L. (Table 1). Cluster analysis based on the morphological characteristics examined, led to the division of the 57 biotypes into seven main groups (Table 2). The results shown in Table 2 are descriptive only and seek to illustrate the morphological and biometric characteristics of the biotypes as grouped by CA. Only the biotypes gathered in Siracusa formed a separate group (Group 4); those from the other areas formed groups with biotypes from different geographical areas. The first group (Group 1, n ¼ 10) contained plants with the greatest number of leaves per cm of branch and the smallest leaf size. Group 4 (n ¼ 4) contained plants with

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Table 1. Location ( Province) and Altitude of the Collection Sites of the 57 Sicilian Rosemary Samples Sample

Province a )

Altitude [m.a.s.l.]

Sample

Province a )

Altitude [m.a.s.l.]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

AG AG AG AG SR SR SR SR ME ME ME ME ME ME ME ME ME ME ME ME AG AG AG AG AG AG AG AG AG AG

390 420 450 410 108 209 155 169 660 710 680 590 400 435 415 437 157 168 210 170 68 97 74 56 115 120 107 103 186 174

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

AG AG AG AG AG AG ME ME ME ME ME ME ME ME AG AG AG AG AG AG AG AG TP TP TP TP TP

175 167 120 137 115 128 390 481 502 428 60 57 43 62 94 78 87 82 105 97 89 106 29 33 60 51 42

Average Range

215 29 – 710

a ) Abbreviation of the provinces of the sample origin: AG, Agrigento; ME, Messina; SR, Siracusa; TP, Trapani.

few leaves but of a larger size. The fact that the number of leaves was found to be inversely proportional to the leaf size was found for nearly all of the groups. Only the samples of Group 7 (n ¼ 6) had few and small leaves as well as the lowest values for all of the parameters examined. Intermediate values, compared to the other groups, for all the parameters examined were found for the samples of Group 6, which also were the most numerous (n ¼ 19). 2.2. Phytochemical Profiles. 2.2.1. Composition of EOs. The yield and number of identified components of the EOs (obtained by hydrodistillation) of 40 among the 57 rosemary samples investigated are given in Table 3. For 17 samples, insufficient plant material was available to obtain their EOs. The average essential-oil yield of the 40 samples was 1.2% (v/w). The general chemical composition, indicating the content range of the identified compounds in the 40 rosemary samples, can be found in Table 4 (the detailed chemical

1.1 (30.7%) 2.5 (5.9%) 9.4 (7.8%) 7.9 (25.3%) 18.0 (11.3%) 16.8 (11.9%) 9.6 (21.1%) 3.3 (2.9%) 29.6 (2.8%) 15.3 (16.8%)

1.4 (18.4%) 1.8 (3.8%) 14.4 (9.0%) 6.8 (23.4%) 20.0 (6.8%) 18.9 (9.8%) 15.0 (14.6%) 2.5 (13.1%) 21.4 (4.2%) 17.3 (8.0%)

2.5 (58.0%) 1.8 (14.2%) 13.2 (12.3%) 5.4 (44.3%) 11.5 (14.9%) 11.7 (20.0%) 12.8 (23.8%) 2.3 (12.1%) 22.2 (3.6%) 15.3 (9.2%)

0.8 (42.7%) 1.7 (14.4%) 8.0 (29.8%) 3.8 (47.7%) 8.3 (17.0%) 9.7 (14.2%) 9.8 (24.9%) 2.2 (3.6%) 18.8 (14.1%) 7.7 (28.0%)

a ) Composition of the Groups: Group 1, Samples 9, 11, 21 – 24, and 49 – 52; Group 2, Samples 26, and 29 – 32; Group 3, Samples 37 – 40; Group 4, Samples 5 – 8; Group 5, Samples 25, 33 – 36, and 45 – 48; Group 6, Samples 1 – 4, 10, 12 – 20, 27, 28, 53, 55, and 56; Group 7, Samples 41 – 44, 54 , and 57. b ) Results are given as average values with coefficients of variation in parentheses. c ) Leaf density: Number of leaves per cm of branch.

10.2 (18.0%) 2.4 (2.4%) 13.0 (7.1%) 6.6 (16.6%) 13.1 (2.3%) 13.4 (1.7%) 13.2 (18.5%) 2.4 (0.6%) 24.3 (0.0%) 23.9 (10.0%)

1.8 (47%) b ) 2.1 (15.2%) 13.3 (13.5%) 5.2 (35.1%) 12.6 (13.1%) 12.2 (17.4%) 26.6 (10.6%) 2.1 (12.7%) 18.2 (14.4%) 16.4 (20.1%)

Branch weight [g] Diameter [mm] Internode number Apical internode length [mm] Median internode length [mm] Basal internode length [mm] Leaf density c ) Width (30 leaves) [mm] Length (30 leaves) [mm] Stem length [cm]

3.4 (6.9%) 1.6 (16.7%) 15.3 (11.3%) 6.3 (13.5%) 16.1 (11.8%) 14.8 (14.9%) 23.8 (12.7%) 2.3 (12.3%) 19.9 (5.5%) 27.9 (6.6%)

Group 1 (n ¼ 10) a ) Group 2 (n ¼ 5) Group 3 (n ¼ 4) Group 4 (n ¼ 4) Group 5 (n ¼ 9) Group 6 (n ¼ 19) Group 7 (n ¼ 6)

Characteristics

Table 2. Morphological Characteristics of the Seven Groups of Sicilian Rosemary Samples Obtained by Cluster Analysis Based on these Characteristics

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Table 3. Yield and Number of Components of the Essential Oils and Yields of the AcOEt and EtOH Extracts of Rosemary Samples 1 – 57 Extract yield [%] a )

Sample Essential oil b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

c

Yield [%] ) N ) AcOEt

EtOH

1.3 1.5 1.5 1.5 1.6 1.1 1.3 0.8 0.8 0.7 0.8 0.8 0.9 1.1 0.9 0.9 0.8 0.8 0.7 0.7 2.0 2.2 1.9 n.a. n.a. n.a. n.a. n.a. n.a.

2.37 5.77 3.70 4.70 8.07 13.13 14.43 3.63 12.30 12.23 16.20 10.73 10.30 8.07 6.00 13.97 14.33 12.63 12.03 11.53 4.53 2.50 1.67 1.90 3.93 7.33 3.37 2.47 1.73

39 43 40 40 37 55 57 67 53 48 45 49 59 59 51 58 57 55 58 54 35 34 33 n.a. n.a. n.a. n.a. n.a. n.a.

11.80 17.20 14.67 12.30 16.90 21.93 16.30 10.87 16.50 17.70 20.73 25.00 11.10 12.10 9.33 18.17 22.90 11.90 25.90 19.27 20.07 12.63 19.23 9.53 24.60 12.07 13.40 18.70 16.27

Sample

b

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Average

Extract yield [%] a )

Essential oil c

Yield [%] ) N ) AcOEt

EtOH

2.8 2.3 n.a. d ) n.a. 2.3 2.3 2.2 0.9 0.9 0.8 0.8 0.9 0.7 0.9 1.0 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 0.7 n.a. 0.8 0.8 0.7 1.2

3.03 5.57 1.67 6.70 2.50 2.47 4.40 4.40 3.40 4.43 7.80 7.33 5.00 2.73 7.87 5.23 3.87 3.97 5.07 1.37 n.a. 5.77 5.27 3.63 10.93 19.03 3.23 15.30 6.63

30 34 n.a. n.a. 36 35 40 54 51 53 49 54 56 54 54 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 56 61 51 48

15.87 13.10 14.10 18.07 13.50 14.00 14.57 11.60 18.57 13.20 11.57 10.07 17.63 12.47 10.73 17.77 11.00 13.23 13.73 16.53 n.a. 11.13 12.27 22.70 10.37 12.07 20.80 11.80 15.29

a

) The extract yields are given as percentage (w/w) based on the dry weight. b ) The essential oil yields are given as percentage (v/w). c ) N: Number of essential oil components identified. d ) n.a.: Not available (insufficient plant material).

composition of each sample can be obtained as Supplementary Material 1)). In total, 82 compounds were identified and grouped in four chemical classes, viz., monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes, and others (non-terpenoid compounds). In all EOs, monoterpenes comprised the main compound classes, with the oxygenated fraction as the most represented, with the exception of Sample 8, in which monoterpene hydrocarbons clearly prevailed over the oxygenated ones. The contents of the other two compound classes, namely sesquiterpenes and others, were much lower. The main compounds, present in the EOs at higher amounts, were a-pinene and 1)

Tables A – C, listing the detailed chemical composition of each essential-oil sample, are available as Supplementary Material from the corresponding author.

Content [%] b ) 20.9 – 65.6 0.1 – 0.4 11.8 – 39.8 3.2 – 12.1 n.d.–2.0 n.d.–1.9 n.d.–5.1 0.1 – 1.9 0.1 – 1.3 n.d.–2.8 n.d.–1.1 n.d.–2.8 n.d.–7.9 n.d.–0.6 n.d.–0.1 n.d.–0.8 n.d.–3.6 27.7 – 74.3 0.1 – 62.7 n.d.–0.7 n.d.–5.0 n.d.–0.4 n.d.–0.4 n.d.–0.2 2.6 – 30.5 n.d.–0.4 n.d.–0.4 n.d.–2.7 n.d.–0.2 3.8 – 12.0 n.d.–1.5

Compound name and class Monoterpene hydrocarbons Tricyclene a-Pinene Camphene Thuja-2,4(10)-diene Verbenene b-Pinene b-Myrcene a-Phellandrene p-Mentha-7,8-diene a-Terpinene p-Cymene Limonene cis-b-Ocimene trans-b-Ocimene g-Terpinene Terpinolene Oxygenated monoterpenes 1,8-Cineole cis-Sabinene hydrate Linalool a-Pinene oxide endo-Fenchol exo-Fenchol Camphor Camphene hydrate cis-Verbenol trans-Pinan-3-one Pinocarvone Borneol cis-Pinan-3-one

2 3 4 5 6 7 10 12 13 14 15 16 18 20 21 23

17 22 24 25 26 27 28 29 30 31 32 33 34

40 7 39 12 27 14 40 39 26 27 26 40 26

40 40 40 22 24 17 40 40 23 36 38 18 11 10 35 26

Nsample c ) n.d.–0.3 n.d.–0.8 n.d.–4.1 n.d.–0.1 n.d.–0.3 n.d.–0.3 n.d.–0.2 0.6 – 7.2 n.d.–0.2 n.d.–0.4 n.d.–0.4 n.d.–2.9 n.d.–0.4 n.d.–0.5 n.d.–0.3 n.d.–0.1 n.d.–0.6 n.d.–0.2 n.d.–0.7 n.d.–0.6 n.d.–0.4 n.d.–0.3 n.d.–0.7 n.d.–0.4 n.d.–0.3 n.d.–0.7 n.d.–0.9 n.d.–0.1 n.d.–0.3 n.d.–0.8 n.d.–0.4

Sesquiterpenes a-Ylangene a-Copaene Isocaryophyllene b-Caryophyllene Neryl acetone a-Humulene g-Muurolene Curcumene Germacrene D g-Amorphene Germacrene A a-Muurolene a-Bisabolene b-Bisabolene d-Amorphene g-Cadinene g-Bisabolene d-Cadinene a-Cadinene b-Calocarene Caryophyllene alcohol Caryophyllene oxide Globulol 54 55 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

Content [%] b )

Geranial Thymol Bornyl Acetate Carvacrol Verbenyl Acetate Piperitenone Myrtanol acetate

Compound name and class

47 48 49 50 51 52 56

Elution order a )

Table 4. Components Identified in the 40 Samples of Rosemary Essential Oil

Elution order )

a

28 29 9 33 30 31 27 8 18 12 31 17 12 13 34 21 10 8 17 25 17 21 31

14 36 36 31 16 9 13

Nsample c )

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Terpinen-4-ol m-Cymen-8-ol p-Cymen-8-ol a-Terpineol Myrtenal Myrtenol Verbenone b-Citronellol trans-Carveol Nerol cis-Myrtanol trans-Myrtanol

35 36 37 38 39 40 41 42 43 44 45 46

0.6 – 1.2 0.1 – 0.4 n.d.–0.4 n.d.–4.6 n.d.–0.5 n.d.–1.0 n.d.–13.7 n.d.–0.2 n.d.–0.2 n.d.–1.0 n.d.–1.7 n.d.–2.0

Content [%] b ) 40 40 19 39 22 24 27 25 26 25 27 24

Nsample c )

n.d.–0.6 n.d.–0.3 n.d.–0.3 n.d.–0.3 0.2 – 2.2 n.d.–0.3 n.d.–0.7 n.d.–0.8 n.d.–0.3 n.d.–0.6 n.d.–0.3 n.d.–0.2

Others ( Z )-Hexen-3-ol Oct-1-en-3-ol Octan-3-one Octan-3-ol Benzene acetaldehyde Eugenol Methyl eugenol 1 8 9 11 19 53 57

Content [%] b )

epi-a-Muurolol Cubenol a-Cadinol a-Bisabolol

Compound name and class

79 80 81 82

Elution order a )

24 8 39 10 23 32 20

12 7 8 13

Nsample c )

a ) Elution order of the components on the SPB-5 GC column. b ) The contents (relative peak-area percentages) are given as range of three determinations; n.d., not detected. c ) Nsample : Number of oil samples in which the component was detected.

Compound name and class

Elution order a )

Table 4 (cont.) CHEMISTRY & BIODIVERSITY – Vol. 12 (2015) 1081

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camphene among the monoterpene hydrocarbons and 1,8-cineole, camphor, borneol, and verbenone among the oxygenated monoterpenes. A cluster analysis was applied to the EO compositional data, to distinguish possible groups among the samples and, mainly, to obtain a better comparison of all samples. Fig. 1 shows the resulting dendrogram obtained from this analysis. The clustering pattern allowed the division of the 40 samples in two large groups, a first one (Group A) comprising 24 samples and a second one (Group B) grouping 13 samples. The three remaining samples, namely Samples 6 – 8, belonged to none of the groups and were separated from each other. This division appeared to be mainly correlated with the content of monoterpenes. In fact, the EOs of Group A showed a homogeneous content of the principal oxygenated components, i.e., 1,8-cineole (median content of 11.7%), camphor (13.4%), borneol (10.9%), and verbenone (10.8%), and a somewhat lower content of linalool (4.2%). Among the monoterpene hydrocarbons present in the EOs of this group, a-pinene reached an average content of 16.4% and camphene of only 3.6%, being the main components of this class. On the other hand, the EOs of Group B were predominated by 1,8-cineole (median content of 48.8%). These high 1,8-cineole contents together with the total absence of verbenone and the very low contents of linalool were the most distinguishing characteristics of the EOs of this group. Camphor (8.4%), borneol (7.1%), and a-terpineol, with a comparatively high content of 4.0%, were the other significant oxygenated monoterpenes present in the EOs of Group B. Among the monoterpene hydrocarbons, no significant differences between the EOs of Group A and B were observed. The highest amounts of monoterpene hydrocarbons and the correspondingly lowest contents of oxygenated monoterpenes, with respect to the rosemary samples discussed

Fig. 1. Dendrogram obtained by cluster analysis of the essential-oil compositions of 40 samples of wild Sicilian rosemary

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above, were observed in the EOs of Samples 6 – 8. This was the most striking for the EO of Sample 8, where the monoterpene hydrocarbons reached ca. 66% of the total composition, due to very high contents of a-pinene (39.8%), camphene (12.1%), and also limonene (7.9%). On the contrary, the oxygenated monoterpenes showed the lowest level (ca. 28%) in this EO, due to the very low content of 1,8-cineole and comparatively lower contents of camphor (8.8%), verbenone (5.5%), borneol (4.8%), and linalool (2.2%). It is to underline that this sample presented the lowest amount of 1,8-cineole (0.1%) of all Sicilian rosemary samples subjected to this study. A very low amount (0.4%) of this latter compound was also present in the EO of Sample 6, which, in turn, presented a high content of camphor (30.5%), a significant amount of borneol (10.8%) and verbenone (4.4%), and no trace of linalool. a-Pinene, camphene, and limonene were the main monoterpene hydrocarbons. Finally, the EO of Sample 7 showed a high content of 1,8-cineole (25.4%) and intermediate contents of verbenone (5.6%), camphor (5.5%), borneol (5.4%), a-terpineol (3.1%), and linalool (3.0%). The main monoterpene hydrocarbons present in this sample were also a-pinene (30.5%) and camphene (5.3%). 2.2.2. Composition of Extracts. In total, 18 compounds were identified in the rosemary extracts, i.e., 13 flavonoids, three terpenoids, and two common organic acids, namely caffeic acid (6) and rosmarinic acid (5; Table 5 and Fig. 2). One of the distinctive characteristics of rosemary, especially if compared with other herbs from the same family, is the dominance of flavones (2-phenylchromen-4-ones), which were found as the sole subclass of flavonoids present in detectable amounts in the extracts (Table 5 and Fig. 2). Other authors, e.g., Okamura et al. [49] and more recently Ozarowski et al. [50], found in addition two flavanones (hesperidin and eriocitrin) and the flavonol isorhamnetin. Okamura et al. also isolated and characterized two acetyl-glucuronide derivatives of the flavone luteolin (7; compounds with elution orders 7 and 8 in Table 5). Their presence is known to be substantially characteristic for this plant [49 – 51]. Conversely, the presence of flavone derivatives with several MeO groups is quite common among the Lamiaceae, as previously reported [4]; these particular compounds have been found of external occurrence (excretion flavonoids) and have therefore successfully been used in chemotaxonomic studies [52]. The detailed composition of each sample is listed in Table 6. Quantitatively speaking, terpenoids, with carnosic acid (4) as the dominant compound, were the main components in all samples analyzed, with the unique exception of Sample 44. Rosmarinic acid (5) was the sole compound found in both extracts (AcOEt and EtOH), despite the fact that its contents were quite different among the samples (Tables 5 and 6). Genkwanin (1), salvigenin (12), 6-hydroxyluteolin glucoside (8), and scutellarein 7-O-glucoside (9) were the most abundant compounds found among the flavones. A cluster analysis was also performed with the compositional data of the extracts, and Fig. 3 shows the resulting dendrogram obtained from this analysis. The clustering pattern allowed dividing the 56 samples in three groups, viz., Group a, comprising 30 samples, Group b, composed of 10 samples, and Group c, consisting of 14 samples. The two remaining samples, namely Samples 11 and 44, belonged to none of the groups and were separated from each other. This division appeared to be mainly correlated with

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Table 5. Content of the Chemical Markers in the AcOEt and EtOH Rosemary Extracts Elution order a ) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Marker Caffeic acid (6) Luteolin glucosideglucuronide ( L glc-glu) 6-Hydroxyluteolin glucoside (8) Scutellarein 7-Oglucoside (9) Rosmarinic acid (5) Luteolin glucuronide (L glu) Luteolin acetylglucuronide 1 (L Ac-glu 1) Luteolin acetylglucuronide 2 (L Ac-glu 2) Luteolin (7) Scutellarein (10) Hispidulin (11) Cirsimaritin (2) Cirsimaritin isomer (CI) Genkwanin (1) Salvigenin (12) Carnosol (3) Carnosic acid (4) Methyl carnosate (13)

MW

lmax [nm]

Content [mg/kg dry weight] AcOEt Extract

EtOH Extract

Both extracts b )

180.16 624.00

330 330

n.d. ¢ 3.52 n.d. ¢ 6.64

n.d. ¢ 3.52 n.d. ¢ 6.64

448.00

330

n.d. ¢ 31.40

n.d. ¢ 31.40

462.00

330

n.d. ¢ 22.90

n.d. ¢ 22.90

360.33 462.00

330 330

0.03 ¢ 172.00 0.01 – 13.80

0.03 – 188.00 0.01 – 13.8

504.00

330

n.d. ¢ 9.10

n.d. ¢ 9.10

504.00

330

n.d. ¢ 8.88

n.d. ¢ 8.88

286.24 286.24 300.00 314.30 314.00

330 330 330 330 330

0.10 – 1.43 0.13 – 2.71 0.14 – 9.28 0.32 – 5.11

n.d. ¢ 7.30

n.d. ¢ 7.30 0.10 . 1.43 0.13 – 2.71 0.14 – 9.28 0.32 – 5.11

284.00 328.00 330.00 318.00 332.00

330 330 280 280 280

0.05 – 21.80 0.77 – 16.10 7.72 – 85.60 29.60 – 408.00 n.d. ¢ 96.70

n.d. ¢ 23.20

0.05 – 21.80 0.77 – 16.10 7.72 – 85.60 29.60 – 408.00 n.d. ¢ 96.70

a ) Compounds (markers) are listed according to their HPLC order (see Exper. Part). b ) Summed-up content of both extracts.

the content of the terpene derivatives, which were the main components present in the extracts. Compared to the extracts of the two other groups (b and c), the extracts of the first and largest group (30 samples; Group a) were characterized by intermediate contents of terpenoid components (332 mg/kg), flavones (38.8 mg/kg), and organic acids (35.3 mg/kg). The extracts of Group b contained low amounts of terpenes (235 mg/kg) and the highest amounts of flavones and organic acids (54.9 and 129 mg/kg, resp.), whereas the extracts of Group c presented the opposite quantitative proportions, namely the highest contents of terpenes (452 mg/kg) and the lowest contents of the other two classes of compounds (29.4 and 17.4 mg/kg for flavones and organic acids, resp.). Sample 11 showed high contents of all three classes of compounds, whereas Sample 44 showed the lowest content of terpenes (37.3 mg/kg). These characteristics explain the clear separation of these two samples (more marked for Sample 44) from all the other ones (Fig. 3).

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Fig. 2. Flavonoids, terpenes, and organic acids characterized in the extracts of the Sicilian rosemary samples. 1, Genkwanin; 2, cirsimaritin; 3, carnosol; 4, carnosic acid; 5, rosemarinic acid; 6, caffeic acid; 7, luteolin; 8, 6-hydroxyluteolin glucoside; 9, scutellarein 7-O-glucoside; 10, scutellarein; 11, hispidulin; 12, salvigenin; 13, methyl carnosate.

2.3. Antioxidant Properties. The EOs and extracts of aromatic plants are known to have antioxidant properties. This offers the possibility of using these products as preservatives for food and cosmetics [53]. In the present study, the protective capacity of 28 EOs and 56 AcOEt and EtOH extracts of Sicilian rosemary against oxidative

Group a

7 3 40 37 15 45 13 33 52 51 31 29 30 32 38 21 54 39 2 28 23 49 41 14 55 53 42 56 25 35

54.1 39.6 68.5 85.6 73.6 35.3 58.8 41.4 59.6 51.6 51.2 48.3 45.4 60.1 53.6 29.4 53.4 57.0 34.8 29.1 41.5 38.7 56.0 45.9 61.4 46.4 47.0 58.3 26.0 60.0

3 )

d

264 272 280 252 252 229 236 255 231 275 248 240 224 276 170 182 209 192 180 222 232 195 199 191 215 134 138 144 141 88.9

4 16.3 3.33 32.4 34.3 20.5 22.7 27.8 37.4 58.2 79.2 81.8 71.7 84.8 96.7 33.1 33.6 22.9 31.9 2.86 22.6 29.3 72.6 10.2 19.6 23.4 24.8 14.5 28.2 16.2 25.0

13

AcOEt (280 nm) c )

1.82 7.97 4.33 2.34 2.01 2.36 2.23 6.89 4.99 4.82 1.30 3.73 – 5.76 1.94 2.64 2.26 3.92 1.81 2.68 – 6.98 2.54 2.39 1.35 3.15 3.43 1.45 2.87 2.08

5 0.62 0.46 0.62 1.29 0.62 0.61 0.58 0.63 0.35 0.54 0.35 0.37 0.36 0.36 1.02 0.47 0.54 0.92 0.48 0.47 1.10 0.38 0.55 0.78 0.66 0.89 0.68 1.43 0.46 0.54

10 1.16 1.17 0.97 1.29 0.98 0.70 0.78 2.00 1.13 1.42 1.18 1.17 1.16 1.58 1.31 0.90 0.81 1.24 0.87 0.61 1.29 1.07 0.69 0.98 0.97 1.20 0.96 2.25 0.90 0.80

11 9.28 4.26 1.76 2.11 1.49 5.93 1.25 3.04 4.76 5.27 3.19 3.40 3.48 3.36 2.23 2.46 1.42 2.24 3.59 4.21 2.55 4.98 1.15 1.30 1.88 2.50 1.94 2.09 3.41 5.28

2

AcOEt (330 nm) c )

Group a ) Sample Content [mg/kg dry weight] b )

3.14 3.74 3.61 4.58 3.54 3.14 3.14 3.12 2.82 2.99 1.64 1.95 1.80 1.91 4.39 1.83 3.17 4.15 3.49 3.96 2.58 2.97 2.38 3.60 3.82 4.76 3.27 4.42 2.54 2.75

CI 4.91 4.37 0.87 0.79 0.43 10.8 0.28 33.1 6.69 6.69 10.8 9.88 9.86 13.2 0.66 10.0 0.42 0.94 4.45 5.17 4.87 7.66 0.52 0.35 0.60 0.79 0.66 0.60 10.7 5.26

1 3.11 2.68 11.5 12.4 8.67 1.61 8.46 1.58 2.66 2.71 1.09 1.04 1.14 1.14 11.40 1.35 6.68 11.8 2.51 2.27 1.94 2.63 5.86 9.17 9.73 10.9 8.18 10.90 1.15 10.3

12 1.72 1.44 1.01 0.78 1.14 1.55 2.14 1.15 2.94 3.52 0.53 0.20 0.49 0.14 1.36 0.65 0.93 0.93 1.31 0.47 0.15 0.19 1.65 1.83 2.88 0.86 1.04 0.43 0.75 0.27

6 – – 1.95 1.13 1.98 – 2.47 – – – 0.59 0.24 – – 2.10 – 1.33 1.33 – – – – 2.25 3.17 6.64 1.44 1.50 1.39 – 0.50

9

9.57 3.94 1.54 0.64 8.20 3.21 4.37 1.78 7.82 2.86 3.43 1.63 10.0 4.11 5.05 2.63 – – – – 1.98 1.78 1.17 0.93 1.46 1.32 1.10 0.83 7.21 2.78 2.85 1.29 5.75 1.96 5.27 2.04 2.45 1.13 0.41 0.32 0.53 0.22 – – 11.4 4.39 21.2 6.71 31.4 11.60 7.03 2.49 7.29 3.06 6.80 1.84 0.63 0.58 1.40 0.59

L glc- 8 glu

EtOH (330 nm) c ) L glu

43.9 13.80 56.2 1.31 48.5 3.50 17.4 1.89 30.2 3.43 49.6 5.46 52.6 4.07 48.9 9.27 66.2 3.73 79.1 4.53 15.8 4.25 7.38 2.22 9.68 3.49 6.95 2.00 24.6 4.05 18.5 5.07 18.4 3.18 25.3 3.09 26.9 3.14 10.8 1.61 0.72 1.92 15.3 1.24 45.8 5.07 67.9 9.43 73.2 13.70 18.2 3.22 25.4 3.22 4.79 3.68 18.6 3.40 5.31 1.83

5

3.80 0.88 1.66 1.32 2.38 1.60 3.03 1.88 0.84 1.03 0.54 0.38 0.53 0.33 2.51 1.88 1.70 1.83 0.73 0.42 0.54 0.28 2.29 4.43 9.10 1.94 1.78 1.52 0.42 0.34

8.88 2.23 1.38 1.20 1.61 4.14 1.61 6.53 3.99 4.38 3.86 2.16 2.77 2.03 2.69 4.88 1.06 1.79 2.50 1.20 0.66 1.22 1.15 2.12 6.47 1.09 0.74 1.29 2.31 1.34

L Ac- L Acglu 1 glu 2

3.80 0.55 1.84 0.99 1.99 3.26 2.17 4.91 1.99 1.96 2.20 1.09 1.79 0.97 1.96 2.49 1.27 1.46 1.35 0.86 0.70 0.71 2.56 5.30 7.30 1.41 1.65 1.21 1.20 1.04

7

Table 6. Composition of the Extracts ( AcOEt and EtOH) of 56 Rosemary Samples, Grouped According to the Cluster Analysis of Their Compositional Data (cf. Fig. 3)

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57.4 47.4 44.4 48.0 47.3 59.0 59.9 46.2 85.9 69.7 62.5 41.4 39.8 70.0 47.0 7.72

Group c 36 34 47 26 46 48 1 22 8 43 5 27 24 4 11 44

29.6

373

381 346 317 338 363 353 350 342 353 299 315 286 408 419

72.4 89.6 121 105 113 112 216 218 181 168

4

–

69.8

48.1 53.1 20.2 30.5 23.9 18.0 – 27.0 6.66 27.4 – 21.2 28.5 –

29.2 20.7 37.6 21.5 26.9 33.3 22.0 32.6 31.0 32.4

13

0.60 0.53 0.58 0.47 0.55 0.66 0.45 0.47 0.65 1.02 0.53 0.52 0.43 0.78

0.73 0.71 0.36 0.47 0.60 1.16 0.73 0.96 0.57 1.07

10

2.19 1.93 0.71 0.78 0.59 0.85 0.51 0.76 0.98 1.51 1.60 1.27 0.80 1.20

1.48 1.01 0.66 0.81 0.91 1.29 1.03 1.08 0.89 1.60

11

4.63 3.08 6.57 4.09 6.01 7.61 3.99 2.59 4.59 2.03 6.98 3.47 1.59 4.24

3.34 6.17 4.33 4.70 4.45 7.02 5.70 1.99 2.61 7.05

2

3.72 2.98 3.67 3.81 3.53 4.10 3.60 2.58 4.66 4.64 0.93 3.87 2.00 3.07

3.28 3.53 1.73 2.48 2.77 3.81 3.38 5.11 2.97 4.65

CI 2.52 4.86 8.43 3.42 4.54 6.09 4.78 0.47 1.84 7.18

0.28 0.10 0.13 0.14 0.32

10.9 11.1 3.88 9.01 10.1 13.6 11.7 13.0 8.49 16.1

12

0.05

1.08 0.12 1.11 0.79 0.96 0.43 0.46 0.34 0.69 0.47 0.59 0.57 0.60 0.16

– 1.02 1.43 – – 1.20 1.08 2.72 1.71 1.30

6

0.77 1.66 3.71

–

– 0.18 0.81 – 0.94 0.49 – – – 0.90 1.16 – – –

2.13 – 5.67 – – – – – 4.05 –

17.7

7.83

4.31 0.45 2.85 1.07 2.82 1.27 0.89 0.82 1.80 4.08 3.48 0.38 0.52 0.35

13.2 9.45 11.5 9.31 8.67 11.0 7.56 14.3 17.5 10.1

L glc- 8 glu

EtOH (330 nm) c )

2.36 –

17.7 1.98 12.7 1.79 10.1 1.38 7.98 2.04 8.06 1.22 13.8 1.59 4.92 2.93 6.89 1.80 3.25 6.89 0.60 11.1 5.22 1.43 14.2 2.05 1.95 1.18 3.22 2.31

1

1.69 0.72 2.71 5.46 4.84 21.8

3.18 1.05 2.43 1.07 4.06 2.13 2.92 0.88 4.42 1.45 – 2.05 0.97 –

5.66 7.69 4.24 8.57 12.3 15.8 14.5 7.27 7.90 23.2

5

AcOEt (330 nm) c )

7.01

3.64

1.68 0.25 1.38 0.89 1.44 0.55 0.37 0.42 1.50 1.46 1.68 0.28 0.25 0.16

5.49 3.92 6.63 4.12 3.65 4.51 3.11 5.73 6.88 4.70

9

46.0

95.40

22.9 2.04 24.5 36.20 47.40 9.36 9.89 6.24 14.90 7.81 6.19 11.5 8.90 0.91

95.6 96.9 83.5 134 121 172 114 105 121 133

5

8.38

2.76

6.02 0.64 5.45 6.12 3.21 2.02 0.94 2.65 1.79 2.11 1.66 2.19 1.84 0.51

5.24 3.21 4.98 4.01 3.47 4.04 3.06 5.08 5.81 3.98

L glu

5.80

2.79

1.16 0.22 0.91 1.04 1.12 0.50 0.25 0.61 0.61 1.11 0.28 0.30 0.54 0.24

2.89 3.20 2.90 3.43 2.81 1.99 1.66 2.98 4.92 2.54

3.05

2.41

4.11 0.51 3.62 3.05 2.98 1.57 0.98 1.79 1.02 0.76 0.54 1.56 0.54 0.25

3.43 3.37 7.77 3.83 3.29 2.64 1.48 – 4.37 2.32

L Ac- L Acglu 1 glu 2

5.31

1.24

3.14 0.37 2.91 2.66 2.16 1.15 0.47 1.20 0.79 0.97 0.21 0.83 1.14 0.17

2.33 1.99 1.68 1.66 1.35 1.54 1.13 3.03 3.14 1.40

7

b

) Groups obtained by cluster analysis of the total content of the chemical markers in the extracts of the 56 samples of wild Sicilian rosemary (cf. Fig. 3). ) Values represent the average of three determinations; –, not detected. c ) Compounds were detected either at l 280 or 330 nm, cf. Exper. Part; d ) For the numbers and abbreviations of the compounds (chemical markers), cf. Table 5.

a

32.1 43.6 41.4 41.1 40.5 39.3 78.2 48.0 66.3 56.9

Group b 19 12 6 10 9 17 18 16 57 20

3 )

d

AcOEt (280 nm) c )

Group a ) Sample Content [mg/kg dry weight] b )

Table 6 (cont.)

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Fig. 3. Dendrogram obtained by cluster analysis of the total content (sum of both extracts, cf. Table 5) of the chemical markers in the extracts of 56 samples of wild Sicilian rosemary

damage was evaluated with i) two chemical assays, i.e., the Folin – Ciocalteu (FC) colorimetric assay and the superoxide dismutase (SOD)-mimetic activity assay, which measures the scavenging activity of superoxide radicals (O .2¢ ), the latter only for the rosemary extracts, and ii) a biomimetic model of phospholipidic membranes, i.e., with UV radiation-induced peroxidation in liposomal membranes (UV-IP assay). For the evaluation of the EOs, the data obtained with these antioxidant assays were analyzed and presented in Table 7, according to the results of the CA carried out on the corresponding compositional profiles of the rosemary EOs (Fig. 1). For the AcOEt and EtOH extracts, a CA based on the total polyphenol content determined with the FC assay was performed (Fig. 4) in addition to the analysis of their antioxidant capacity. The results of the antioxidant assays for the extracts is presented in Table 7, according to this latter CA. The analyzed rosemary EOs showed a discrete antioxidant/radical-scavenging activity both in the FC assay (range of 30 to 238 mg GAE (gallic-acid equivalents)/ml) and the UV-IP test (IC50 range of 0.93 to 2.37 mg/ml; Table 7). Their antioxidant activity was in accordance with the findings reported by several authors [35] [54] [55], which showed that rosemary oils possessed a good antioxidant power that depended on their widely variable chemical composition. Concerning a possible correlation between the compositional and antioxidant data of the EOs, the EOs of Group A possessed an only slightly higher antioxidant power than those of Group B in both assays employed, suggesting that the different amounts of the most abundant oxygenated monoterpenes present in the EOs did not play a main role in the antioxidant activity. In fact, the antioxidant effects of the EOs might be the result of a synergism between all oil constituents, as the activity of the main components might be modulated by other minor compounds [54] [55].

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Table 7. Antioxidant Activity of the Rosemary Essential Oils and Extracts ( AcOEt and EtOH): Total Phenols (FC assay), O .2¢ -Scavenging Activity ( IC50-SOD ), and Radical-Scavenging Activity in the UV Radiation-Induced Peroxidation in Liposomal Membranes ( IC50-UV-IP ) Groups a )

Total phenols b )

IC50-SOD c )

IC50-UV-IP d )

Essential oils Group A (n ¼ 16) Group B (n ¼ 9) Sample 6 Sample 7 Sample 8

[mg GAE/ml] 138 œ 47.2 (80.6 – 238 ) 117 œ 51.7 (30.4 – 181) 168 131 119

– – – – – –

[mg/ml] 1.28 œ 0.37 (1.01 – 2.37) 1.39 œ 0.29 (1.03 – 1.81) 1.25 1.05 0.93

AcOEt Extracts Group AAcOEt (n ¼ 11) Group BAcOEt (n ¼ 31) Group CAcOEt (n ¼ 13) Sample 44

[mg GAE/mg] 38.8 œ 12.5 (24.3 – 58.2) 37.6 œ 10.2 (28.1 – 99.8) 44.0 œ 9.09 (25.7 – 58.2) 38.8

[mg/ml] 945 œ 890 (134 – 2672) 287 œ 115 (72.2 – 188) 224 œ 173 (114 – 742) 257

[mg/ml] 51.9 œ 12.1 (40.0 – 75.7) 50.1 œ 18.5 (39.1 – 294) 44.2 œ 3.52 (39.4 – 48.3) 44.6

EtOH extracts Group AEtOH (n ¼ 13) Group BEtOH (n ¼ 42 ) Sample 17

[mg GAE/mg] 67.1 œ 22.8 (37.5 – 92.6) 54.3 œ 21.3 (28.1 – 99.8) 72.9

[mg/ml] 102 œ 29.5 (76.8 – 176) 109 œ 50.8 (72.2 ¢ 188) 75.0

[mg/ml] 62.1 œ 21.2 (41.1 – 105) 72.2 œ 57.3 (39.1 – 295) 88.6

a

) Groups obtained by cluster analysis of the chemical profiles of the EOs and organic extracts: for the EOs, cf. Fig. 1, for the AcOEt extracts, cf. Fig. 4, a, and for the EtOH extracts, cf. Fig. 4, b; results are given as mean of n samples œ SD with the range in parentheses. b ) Total phenols determined with the Folin – Ciocalteu ( FC ) assay and expressed as gallic acid equivalents (GAE). c ) IC50-SOD : Concentration that scavenges 50% of the O .2¢ produced (SOD assay). d ) IC50-UV-IP : Concentration that inhibits 50% of the UV radiation-induced peroxidation in liposomal membranes ( UV-IP assay).

Also the rosemary extracts tested demonstrated varying degrees of efficiency in each screening assay used. Generally, if compared with the AcOEt extracts, the EtOH extracts showed a better antioxidant/radical scavenging power in the FC assay and the SOD-mimetic assay, but were less efficient in the UV-IP assay. In fact, the EtOH extracts were rich in antioxidant polyphenolic compounds, with compound 5 being the most abundant, while the AcOEt extracts contained mainly terpenoid compounds, including carnosol (3), carnosic acid (4), and methyl carnosate (11) [56]. Regarding the EtOH extracts, those belonging to Group AEtOH (n ¼ 13; Fig. 4, b) had a slightly better antioxidant capacity in all assays employed than those included in Group BEtOH (n ¼ 42), in accordance with their higher rosmarinic acid content. 3. Conclusions. – A broad, coast-to-coast sampling throughout Sicily of wild rosemary was carried out, leading to the collection of 57 samples. All the biotypes were identified as Rosmarinus officinalis L. A series of CAs was applied to study differences and similarities among the samples. The CA based on the morphological characteristics of the collected plants led to the division of the 57 biotypes into seven main groups, although the features examined were found to be highly similar and not areadependent. Only the samples collected around the Siracusa area formed a separate group. On the other hand, the phytochemical investigation of the EOs and extracts obtained from wild Sicilian rosemary allowed to differentiate biotypes according to

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Fig. 4. Dendrograms obtained by cluster analyses of the polyphenol contents (Folin – Ciocalteu assay) of a) the AcOEt extracts and b) the EtOH extracts of 56 samples of wild rosemary

their content of volatile and nonvolatile secondary metabolites, thus confirming the importance of chemotaxonomic markers as classification tool. The present study also showed that the EOs and organic extracts from the Sicilian rosemary samples analyzed had a considerable antioxidant and free radical-scavenging activity, as demonstrated with several in vitro chemical assays. Hence, they might be good candidates for the improvement of the dietary intake, such as in the food production and as nutraceuticals. Although clearly dependent on their widely variable chemical composition, the antioxidant properties of these EOs and extracts appeared to be the result of a synergism between various constituents, including those present in minor amounts. The authors wish to thank the Assessorato Agricoltura e Foreste – Regione Siciliana for financial support. Thanks are also due to Ms. Tonia Strano (ICB-CNR, Catania) and Dr. Broni Hornby (CoRiSSIA, Palermo) for their skillful technical assistance.

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Experimental Part Reagents and Standards. MeCN and H2O were of HPLC grade (Carlo Erba, IT-Milan), and all other solvents used were of high purity, meeting the American Chemical Society (ACS) requirements (Carlo Erba). The pure reference standards apigenin, luteolin, naringenin, eriodictyol, rosmarinic acid (5), and caffeic acid (6) were obtained from Fluka (IT-Milan), and the volatile standards were purchased from Aldrich Chemical Co., Extrasynthese (France), and Fluka Chemie AG (Switzerland). Plant Material. Sicilian sites where rosemary grows in the wild were found using available literature and indications provided by the Regional Operation Units of the Sicilian Regional Ministry of Food and Agriculture. In 2004 and 2005, following a field study of the sites, plants displaying homogeneous vegetative growth were identified. At each of the sites, samples of 30 branches/plant from flowering plants were collected (Table 1). The following biomorphological and production parameters were gathered from the plant material: average dry weight of branch [g], branch diameter [mm], number of branch internodes (n), internode length [mm] of apical, median, and basal internodes, number of leaves per cm/branch (n), leaf length, leaf width, and branch length ([cm]; Table 2). Isolation of Essential Oils. The air-dried aerial parts (50 – 100 g) of 40 among the 57 samples (for 17 samples insufficient plant material was available) were subjected to hydrodistillation, according to the method recommended by the current European Pharmacopoeia [57], until no significant increase in the volume of the collected oil was observed (3 h). The oils were dried (anh. Na2SO4 ) and stored under N2 in sealed vials until further analysis. GC-FID and GC/MS Analyses of Essential Oils. The GC-FID analyses were carried out with a Shimadzu GC-17A apparatus equipped with a flame ionization detector (FID), Class-VP Chromatography Data System version 4.3 operating software (Shimadzu), and a SPB-5 cap. column (15 m   0.10 mm i.d., film thickness 0.15 mm). The oven temp. was programmed isothermal at 608 for 1 min, then rising from 60 to 2808 at 108/min, and finally held isothermal at 2808 for 1 min; injector and detector temp., 250 and 2808, resp.; carrier gas, He (1 ml/min); injection in split mode (1 : 200); injection volume, 1 ml (4% (v/v) EO in CH2Cl2 ); linear velocity in column, 19 cm/s. The contents of the compounds, expressed in percentages, were determined from their peak areas in the GC-FID profiles. The GC/MS analyses were performed in the fast mode with a Shimadzu GCMS-QP5050A apparatus equipped with the same cap. column and using the same operative GC conditions as described above for the GC-FID analyses; operating software, GC/MS solution version 1.02 (Shimadzu); ionization voltage, 70 eV; electron multiplier, 900 V, ion-source temp., 1808. Mass spectral data were acquired in the scan mode over the m/z range 40 – 400. The same EO solns. (1 ml) as for the GC-FID analyses were injected in split mode (1 : 96). Identification of Essential-Oil Components. The identification of the components was based on the comparison of their retention indices (RIs), determined rel. to the tR of a series of n-alkanes (C9 – C22 ) on the SPB-5 column, with those reported in the literature [58], on the computer matching of their mass spectral data with those of the NIST MS library [59], on the comparison of their MS fragmentation patterns with those reported in literature [58], and, whenever possible, on the coinjection with authentic standards. Preparation of Extracts. Aliquots (30 g) of the dried aerial parts of each rosemary sample were finely grounded and defatted with hexane (3   200 ml), to eliminate the majority of fats, including sterols and EO components, which might interfere with the antioxidant assays. Cascade extractions with AcOEt (3   200 ml) and EtOH (3   200 ml) gave, after evaporation under reduced pressure, the corresponding extracts (their yields are reported in Table 3). EtOH was chosen over MeOH as industrial, polar, nontoxic solvent, while AcOEt was a good choice to extract flavonoid derivatives. The extractions were carried out overnight, in the dark, at r.t., and under vigorous mechanical stirring. The extracts were kept at 48 under N2 and unsealed immediately prior to use. Extract Characterization. Qualitative HPLC-PDA/ESI-MS Analyses. The HPLC analyses coupled with PDA and ESI-MS detection of the rosemary extracts were carried out using Waters instruments consisting of a 1525 binary HPLC pump, a PDA 996 photodiode array detector, and a Micromass ZQ mass analyzer equipped with a ESI Z-spray source (Waters Italia S.p.A., IT-Milan) operating in positive-

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and negative-ion mode under the following conditions: capillary voltage, 2.75 kV; cone voltage, 25 V; source temp., 1508; desolvation temp., 2508; carrier gas, N2 (400 l/h for the desolvation, 210 l/h for the cone). Chromatographic runs were performed on a Luna C18 reversed-phase column (250   4.6 mm, 5 mm particle size; Phenomenex Italia), with a mobile phase composed of 2.5% HCOOH in H2O (solvent A) and MeCN (solvent B), using the following elution program: 0 min, 5% B; 10 min, 15% B; 30 min, 25% B; 35 min, 30% B; 50 min, 90% B; 57 min, 100% B; 64 min, 100% B. The flow rate was 1 ml/min, the temp. was kept at 258, and the injection volume was 20 ml. The software used for the data analyses and elaboration was Mass Lynx v. 4.0 (Waters). Aliquots (10 – 20 mg) of each sample were dissolved in MeOH (1.5 ml), filtered with PTFE filters (15 mm diameter, 0.45 mm pore size, Chemtek Analytica, Milano) and put in 2-ml amber vials immediately prior to analysis. Quantitative HPLC-PDA Analyses. The quantitative HPLC analyses were carried out with a Dionex apparatus equipped with a P580 binary high pressure pump, a PDA-100 photodiode array detector, a TCC-100 thermostatted column compartment, and an ASI-100 automated sample injector. The collected data were processed using the Chromeleon chromatography information management system v. 6.70. The rosemary metabolites were eluted using the same chromatographic conditions (column, elution program, variables set) as described in the previous paragraph. Quantification was carried out at 330 nm for flavones, using apigenin (r2 ¼ 0.9995 ) or luteolin (r2 ¼ 0.9999) as external standards, based on the wavelengths of their absorption maxima. Compounds 5 and 6 were also quantified at 330 nm, using the corresponding commercially available standards to establish the calibration curves (r2 ¼ 0.9997 and 0.9999 for 5 and 6, resp.). Finally, 280 nm was the reference wavelength for the quantification of terpenoids, with carnosic acid (4) used as the external standard (r2 ¼ 0.9999). All analyses were carried out in triplicate. Antioxidant Activity. Folin – Ciocalteu (FC) Colorimetric Assay. The amount of total phenols was determined with the FC colorimetric method, according to Tomaino et al. [60], and expressed as gallicacid equivalents (GAE). Each determination was performed in triplicate and the results are expressed as mean ( œ SD) of three experiments. UV Radiation-Induced Peroxidation in Liposomal Membranes (UV-IP Assay). The protective effect of the samples against UVC-induced peroxidation was evaluated in phosphatidylcholine (PC) multilamellar liposome vesicles (MLVs), by monitoring the production of malondialdehyde (MDA), the final product of fatty-acid degradation [61]. Briefly, 100 mg of PC, dissolved in CHCl3 , were transferred to a small, stoppered tube. The lipid was thoroughly dried under a stream of N2 and then dissolved in warm EtOH (80 mg). An aliquot of 200 mg of a 25 mm Tris/HCl soln. of pH 7.4 was added, to yield a lipid/EtOH/H2O mixture of 100 : 80 : 200 (w/w/w). This mixture was heated to 608 for a few min and then allowed to cool to r.t., yielding a proliposome mixture. The proliposome mixture was finally converted to a liposome suspension by the drop-wise addition of 25 mm Tris/HCl soln. of pH 7.4 to a final volume of 10 ml. The suspension was vortex-mixed throughout this last stage. The liposome dispersion (1 ml in a glass flask with a 3 cm2 exposure surface area) was maintained at r.t. and exposed for 1 h to UVradiation generated by a 15 W Philips germicidal lamp (254 nm) at a distance of 10 cm; the dose rate of UV-radiation was 105 erg/mm2/s. Different amounts of the samples to be tested were added to the system, and an equal volume (50 ml) of the vehicle alone (MeOH) was added to control tubes. The MDA concentration in the mixture was measured as previously described [61]. All experiments were carried out in triplicate and repeated at least three times. The results were expressed as percentage MDA decrease with respect to controls, and mean inhibitory concentrations (IC50-UV-IP values) were calculated by using the Litchfield and Wilcoxon method. O .2¢ -Scavenging Activity. The superoxide dismutase (SOD) mimetic activity of the extracts under study was measured by the reduction of nitro blue tetrazolium (NBT), according to Tomaino et al. [60]. Briefly, the nonenzymatic system PMS/NADH generates O .2¢, which reduces NBT to a purple formazan derivative. Aliquots of 20 ml of the sample solns. to be tested dissolved in MeOH at different concentrations or of the vehicle alone were added to 1.5 ml of a mixture containing 500 ml of NADH (73 mm), 500 ml of NBT (50 mm), and 500 ml of PMS (15 mm). All reagents were prepared in Tris/HCl (16 mm, pH 8). After incubation for 2 min at r.t., the absorbance was measured at 560 nm, to determine the concentration of formazan formed. All experiments were carried out in duplicate and repeated at

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least three times. The results were expressed as percent decrease with respect to controls, and mean inhibitory concentrations (IC50-SOD values) were calculated by using the Litchfield and Wilcoxon method. Statistical Analysis. Hierarchical cluster analysis was applied to the morphological and production characteristics of the 57 biotypes of rosemary using the SPSS Statistics 17.0 software, based on the between-groups linkage method, while the distance selected was calculated by the squared Euclidean distance method. The chemical data (EO and extract compositions) were statistically analyzed using the statistiXL 1.8 software package, to determine the relationships between the different samples of rosemary. The Euclidean distance was selected as a measure of similarity, and single-linkage clustering (also known as nearest-neighbor clustering) was the method used for the cluster definition.

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