Chemopreventive and Antioxidant Activity of the Chamazulene-RichEssential Oil Obtained from Artemisia arborescens L. Growing on the Isle

of La Maddalena, Sardinia, Italy

by Luigi Ornanoa), Alessandro Vendittia), Mauro Balleroa), Cinzia Sannaa), Luana Quassintib),Massimo Bramuccib), Giulio Lupidib), Fabrizio Papab), Sauro Vittorib), Filippo Maggi*b), and

Armanodoriano Biancoa)

a) Consorzio Interuniversitario CoSMeSe, Dipartimento di Scienze Botaniche, Universita di Cagliari,Dipartimento di Chimica, Universita di Roma �La Sapienza�, IT-Rome

b) School of Pharmacy, University of Camerino, Via Pontoni 5, IT-62032 Camerino(phone: þ39-0737-404506; fax: þ39-0737-404508; e-mail: filippo.maggi@unicam.it)

The essential oils of Artemisia arborescens growing in Sardinia (Italy), collected during three plantgrowth stages, i.e., from the vegetative stage to post-blooming time, were characterized. Moreover, the invitro antiproliferative and antioxidant activities of the oil isolated from aerial parts collected in Februarywere evaluated. The essential oils belonged to the b-thujone/chamazulene chemotype, notably with thehighest amount of chamazulene (ca. 52%) ever detected up to now in the genus Artemisia and, in general,in essential oils. Quantitative variations in the oil composition were observed as the plant passes from thevegetative to the blooming stage. The oil was tested for its potential tumor cell growth-inhibitory effecton T98G, MDA-MB 435S, A375, and HCT116 human cell lines, using the MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The highest activity was observedon A375 and HCT116 cell lines, with IC50 values of 14 mg/ml. Moreover, the in vitro antioxidant and freeradical-scavenging assays revealed the oil to be an effective scavenger of the ABTS radical cation, with anactivity comparable to that of Trolox�. These results support the use of A. arborescens oil for thetreatment of inflamed skin conditions. Finally, the composition of the polar fraction of the A. arborescensaerial parts was also examined, and the main component detected was 5-O-caffeoylquinic acid, which wasidentified for the first time in this plant.

Introduction. – Artemisia arborescens L. (Asteraceae), well-known in theMediterranean area as �arboreus absinth� or �arborent mugwort�, is a bushy shrubcovered by a thick gray tomentum. It has leaves divided in profound and linear laciniasand flower heads assembled in terminal panicles. It grows in ruderal environments oncalcareous soils, in the southern part of the Mediterranean area, where it is consideredendemic.

Sparsely used in folk medicine, it is not included in the European Pharmacopoeia,probably due to the presence of thujones. Recently, the essential oil of A. arborescenshas been used as an ecological pesticide in both conventional and organic agricultureand in domestic and public use [1].

Some studies have been published on the essential-oil composition of A.arborescens growing in several countries, leading to the identification of severalchemotypes characterized by different amounts of b-thujone and chamazulene [2]. Thechemical composition of the oils seems to vary greatly according to geographic and

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� 2013 Verlag Helvetica Chimica Acta AG, Z�rich

genetic factors, so that such a variability could be considered a chemotaxonomicmarker of the genus. Nonetheless, the biological potential of the essential oil of thisplant has not been systematically investigated. As a matter of fact, only recently, the oilwas proven to inhibit the Herpes simplex virus type 1 (HSV-1) [3].

In the present study, the variation of the essential-oil composition during thephenological cycle in an A. arborescens population growing on La Maddalena Island(Sardinia, Italy) was investigated, and, for the first time, the radical-scavengingactivities in the DPPH (¼2,2-diphenyl-1-picrylhydrazyl) and ABTS (¼2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assays and the chemopreventive potential onsome human tumor cell lines, assessed by the MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, of the oil were evaluated. Finally, thecomposition of the polar fraction of A. arborescens (EtOH extract) was examined aswell.

Results and Discussion. – Essential-Oil Composition. The composition of theessential oils obtained from the aerial parts of A. arborescens collected during threedifferent growth periods is reported in Table 1. A total of 58 volatile components weredetected, corresponding to 87.4 –96.1% of the total oil compositions. The majorcompound classes were represented by sesquiterpene hydrocarbons (32.0 –55.3%) andoxygenated monoterpenes (37.7 –57.7%), with chamazulene (25.6 – 51.5%) and b-thujone (33.8– 53.20%) as the main compounds, respectively. Other components,occurring at lower amounts, but with contents >1%, were germacrene D (3.2 – 5.4%)and (E)-caryophyllene (0.4 – 2.7%) among the sesquiterpene hydrocarbons andterpinen-4-ol (0.6 – 1.4%) among the oxygenated monoterpenes. Despite the abun-dance of the cis isomer of the ketone thujone (b-thujone), the trans form (a-thujone)was only present in small amounts (0.6 – 1.0%). Noteworthy is the occurrence in theessential oils of an unknown diterpene (MW¼286, LRI¼2005) at contents rangingfrom 2.7 to 7.7%, whose MS fragmentation is shown in Fig. 1. This compound could bethe same unidentified diterpene previously reported in [4] [5], being responsible for thearoma impact of the oil.

The ketones a- and b-thujone, known to have psychotropic activity, are also typicalof other species of the genus Artemisia, notably wormwood (A. absinthium), which are

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Fig. 1. Mass spectrum of an unknown diterpene (LRI¼2005) occurring in the essential oil of Artemisia

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Table 1. Composition of the Essential Oils Isolated from the Aerial Parts of Artemisia arborescensCollected on La Maddalena Island (Sardinia, Italy) during July 2011, February 2012, and May 2012

Compound name and classa) LRIexpb) LRIlit

c) Content [%]d) Methode)

Adams NIST08 July2011

February2012

May2012

a-Pinene 932 939 932 0.2 StdSabinene 971 975 972 trf) 1.3 LRI, MSb-Pinene 974 979 974 0.1 Std6-Methylhept-5-en-2-one 991 985 0.1 0.1 LRI, MSMyrcene 991 990 991 0.1 Stda-Phellandrene 1003 1002 1003 0.1 0.3 StdIsobutyl 2-methylbutanoate 1004 1004 tr LRI, MSa-Terpinene 1017 1017 1017 tr 0.2 StdIsoamyl isobutyrate 1021 1021 0.1 LRI, MSp-Cymene 1026 1024 1026 0.1 0.2 Stdb-Phellandrene 1030 1029 1029 tr LRI, MS1,8-Cineole 1033 1031 1033 0.1 0.2 Stdg-Terpinene 1061 1059 1061 0.1 0.4 StdIsopentyl butanoate 1065 1058 1064 tr LRI, MScis-Sabinene hydrate 1069 1070 1070 0.1 tr 0.6 LRI, MSTerpinolene 1087 1088 1087 tr 0.1 Stdtrans-Sabinene hydrate 1097 1098 tr tr 0.1 LRI, MSa-Thujone 1102 1102 1102 0.6 1.0 1.3 Std2-Methylbutyl 2-methylbutanoate 1107 1100 1107 0.1 0.2 0.7 LRI, MSb-Thujone 1114 1114 1114 38.0 33.8 53.2 Stdiso-3-Thujanol 1136 1138 0.1 0.2 0.2 LRI, MStrans-Sabinol 1141 1142 tr 0.1 tr LRI, MSTerpinen-4-ol 1176 1177 1175 0.6 1.4 1.0 Stda-Terpineol 1190 1188 1190 0.2 0.6 0.6 StdMethyl salicylate 1192 1191 1192 tr LRI, MSMyrtenol 1194 1195 1194 tr Std(Z)-Hex-3-enyl 2-methylbutanoate 1237 1232 tr tr tr LRI, MSThymol, methyl ether 1237 1235 1237 tr LRI, MSCumin aldehyde 1239 1241 1239 tr tr LRI, MS(Z)-Hex-3-enyl 3-methylbutanoate 1241 1235 tr tr LRI, MS2,3-Dimethoxytoluene 1245 1245 0.2 LRI, MSCarvotanacetone 1247 1247 1247 0.1 LRI, MSPerilla aldehyde 1271 1271 1271 0.3 0.2 LRI, MSEugenol 1358 1359 1358 tr Stda-Copaene 1370 1376 1370 0.1 0.5 0.4 LRI, MSb-Bourbonene 1377 1388 1377 0.1 0.3 0.1 LRI, MSb-Cubebene 1384 1388 1384 tr tr LRI, MSb-Elemene 1387 1390 1387 0.3 LRI, MS(Z)-Jasmone 1397 1392 1397 tr LRI, MSMethyl eugenol 1409 1403 1409 0.2 0.2 0.2 LRI, MS(E)-Caryophyllene 1409 1419 1411 0.4 2.7 1.3 Stdb-Copaene 1421 1432 tr 0.1 tr LRI, MSa-Humulene 1445 1454 1444 0.2 0.1 StdSelina-4(14),7(11)-diene 1469 1469 0.1 LRI, MSGermacrene D 1473 1485 1473 3.2 5.4 4.3 LRI, MSb-Selinene 1477 1490 1477 0.7 tr LRI, MS

used to make alcoholic beverages and vermouths. Owing to its toxicity, in Europe,maximum levels of thujone (the isomeric mixture) in alcoholic and nonalcoholicbeverages are set to 35 and 0.5 mg/kg, respectively [8]. It has to be noted that theserestrictions are due mainly to the a-isomer of thujone.

In the last few years, several studies reported new biological activities of thesecompounds, such as the potential to correct the lipid profile (cholesterol andtriglycerides) in diabetic rats [9], to inhibit lung metastasis induced by B16F-10melanoma cells in mice [10], and to enhance the immune response of mice [11].

Chamazulene, an azulenic compound, is a degradation product of matricin,occurring in the flowers of chamomile (Matricaria recutita). It is responsible forthe blue color of the flower distillate. Chamazulene is one of the most active

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Table 1 (cont.)

Compound name and classa) LRIexpb) LRIlit

c) Content [%]d) Methode)

Adams NIST08 July2011

February2012

May2012

g-Himachalene 1480 1482 1480 0.1 LRI, MSBicyclogermacrene 1487 1500 1487 tr 0.2 0.1 LRI, MSd-Cadinene 1517 1523 1518 tr 0.2 0.1 LRI, MSElemol 1544 1549 1544 0.4 0.3 0.4 LRI, MSCaryophyllene oxide 1572 1583 1572 0.5 0.1 StdNeryl isovalerate 1585 1583 tr 0.1 tr LRI, MSGeranyl 2-methylbutanoate 1603 1601 tr 0.3 0.1 LRI, MSGeranyl isovalerate 1610 1607 1613 0.4 1.8 1.0 LRI, MSJunenol 1615 1619 0.4 LRI, MS(Z)-Methyl jasmonate 1647 1649 1652 0.3 0.1 LRI, MSa-Bisabolol 1679 1685 1680 0.1 tr LRI, MSChamazulene 1718 1731 1718 51.5 34.2 25.6 LRI, MSUnknown diterpene 1 (MW 286)g) 1997 tr 0.7 0.2 MSUnknown diterpene 2 (MW 286)g) 2005 3.9 7.7 2.7 MSUnknown diterpene 3 (MW 300)g) 2082 0.2 MS

Total identified [%] 96.1 87.4 95.9

Monoterpene hydrocarbons 0.4 2.9Oxygenated monoterpenes 40.0 37.7 57.7Sesquiterpene hydrocarbons 55.3 45.2 32.0Oxygenated sesquiterpenes 0.4 3.1 0.5Diterpenes 3.9 8.6 2.9Others 0.3 1.0 2.3

a) Compounds are listed in order of their elution from a HP-5 MS column. b) LRIexp : Linear retentionindex experimentally determined on a HP-5 MS column, relative to a homologous series of alkanes(C8 –C30). c) LRIlit : Linear retention index according to Adams [6] and/or the NIST 08 library [7]. d) Thecontents are given as means� standard deviations (n¼3) and expressed as percentages of the total oilcomposition. e) Identification methods: Std, coinjection with authentic compounds; MS, comparison ofmass spectra with those registered in the following mass spectral libraries: WILEY275, NIST 08, Adams,and a home-made library; LRI, comparison of LRIs with those reported by Adams [6] and/or listed in theNIST 08 library [7]. f) tr: Trace (< 0.1%). g) Tentative identification based on MS fragmentationpattern.

compounds of chamomile. Notably, it was proven to possess anti-inflammatory activityin vivo, by inhibiting the leukotriene synthesis, and to inhibit lipid peroxidation[12] [13]. It has also been reported as one of the main constituents of the essential oilsof other species of the genus Artemisia, such as A. absinthium, A. gallica ssp. densiflora,A. rehan, A. santonicum, A. copa, and A. feddei [14 – 17]. However, to the best of ourknowledge, similar contents to those detected in the present samples (51.5%) havenever been reported.

When comparing the compositions of the essential oils obtained from the aerialparts of A. arborescens collected at three different phenological stages, i.e., in July,February, and May, mostly quantitative differences were observed (Fig. 2). Notably,the highest content of chamazulene (51.5%) was reached in summer (July), while thatof b-thujone (53.2%) was detected in spring (May). The essential oil obtained from theaerial parts collected in July showed the highest amount of sesquiterpene hydrocarbons(55.3%), while that from plant material collected in May exhibited the highest contentof oxygenated monoterpenes. In both cases, chamazulene and b-thujone, respectively,were the principal compounds responsible for this difference. With respect to theessential oil obtained from plants harvested in winter, the May and July samplesshowed a lower content of oxygenated sesquiterpenes (0.5 and 0.4 vs. 3.1%) andditerpenes (2.9 and 3.9 vs. 8.6%). From a qualitative point of view, the oil from plantscollected in July was poorer in constituents than those obtained from the aerial partsharvested in February and May, since only 23 vs. 50 and 47 components were detectedin the oil of this growth stage. These differences can be explained by the fact that thisMediterranean species increases the volatile production during the early months of theyear up to flowering (May), while after this period, the plant undergoes dehydrationwith a consequent loss in volatiles. In conclusion, the phenological stage influencedsignificantly the quantitative composition of the essential oils, leading to an increase ofoxygenated monoterpenes and a decrease of sesquiterpenes as the plant passes from

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Fig. 2. Content of the main classes of volatiles in Artemisia arborescens during different phenologicalstages. MH, Monoterpene hydrocarbons; MO, oxygenated monoterpenes; SH, sesquiterpene hydro-

carbons; SO, oxygenated sesquiterpenes; D, diterpenes.

the vegetative to the blooming stage. These results were consistent with those reportedfor Sicilian populations [2].

To date, different chemotypes have been identified for the essential oils of A.arborescens: a b-thujone/camphor type occurring in Sardina (Italy, around Usellus) andMorocco [3] [4], a chamazulene/camphor type existing in the north-west of the U.S.A.and in southern parts of Italy, namely Calabria, Sicily, and the Eolian Islands [4] [5],and a b-thujone/chamazulene type detected in Liguria (Sacco) [18], Sicily [2], Sardinia[19], and Algeria [20].

The present results revealed that the essential oil from the A. arborescenspopulation growing on La Maddalena Island belongs to the latter chemotype.However, its peculiarity was the absence of camphor (like in the oil from Algeria[20]), which, instead, was detected in significant levels in the essential oils of plantsfrom Sicily and Sardinia itself [2] [19]. Another characteristic of the investigatedpopulation worthy of mention was the high amount of chamazulene (ca. 52%) detectedin the essential oils, the highest ever found up to now.

The above-mentioned chemical differences confirm that the genus Artemisia showshigh variability, either at interspecific or intraspecific levels, with different chemotypesdepending on the geographic origin and environmental conditions. Moreover, thechemical variability reflects the morphological variation of this species [2].

Composition of Polar Fraction. The composition of the polar fraction, obtained byextraction of the aerial parts of A. arborescens with EtOH (96%) at r.t. for 2 d,appeared to be characterized by the presence of one ester of caffeic acid. Indeed, it wasconstituted mainly by 5-O-caffeoylquinic acid (Fig. 3), also known as neochlorogenicacid. The presence of this compound was constant during the examined phenologicalperiod, being quite similar in all examined samples. Hence, its content in A. arborescensappears to be unrelated with the time of collection, contrary to the behavior of theessential-oil components. Neochlorogenic acid as well as other caffeoyl derivatives ofquinic acid were reported for the first time in A. arborescens.

Antiproliferative Activity. The essential oil isolated from the aerial parts of A.arborescens collected in February (winter sample) was tested for the first time for itspotential in vitro tumor cell growth-inhibitory effect on the T98G human glioblastomacell line, the MDA-MB 435S human breast adenocarcinoma cell line, the A375 humanmalignant melanoma cell line, and the HCT116 human colon carcinoma cell line, usingthe MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay.The results, summarized in Table 2, show that the essential oil exhibited a variablegrowth-inhibitory effect on the human cancer cells examined. The highest activity was

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Fig. 3. Chemical structure of 5-O-caffeoylquinic acid (neochlorogenic acid)

observed against the A375 and HCT116 cell lines, with IC50 values of 14.28�2.53 and14.62�1.79 mg/ml, respectively. The lowest activity was obtained against the T98G cellline (IC50 value of 59.82�1.76 mg/ml).

The cytotoxic activity of A. arborescens essential oil might be attributed to specificoil components. The main compounds present in the essential oil were found to be b-thujone (33.8%), chamazulene (34.2%), and germacrene D (5.4%). There are fewdata in the literature related to the cytotoxic activity of these compounds. It has beenreported that a thujone-rich extract from Thuja occidentalis exerted cytotoxic,antiproliferative, and apoptotic activity on the malignant melanoma cell line A375[21]. In addition, thujone can inhibit lung metastasis induced by B16F-10 melanomacells in mice, through the inhibition of tumor-cell proliferation, adhesion, and invasion[10]. The presence of thujone in the essential oil of A. arborescens might partiallyexplain the observed cytotoxic activity on the human melanoma cell line A375.Antiproliferative activity of germacrene D against the MDA-MB 231, MCF7, Hs578T,PC-3, and Hep-G2 cell lines has been reported by Setzer et al. [22]. No information onthe cytotoxicity of chamazulene was found in the literature.

Antioxidant Activity. As shown in Table 3, the essential oil of A. arborescens (wintersample) was an effective scavenger of the ABTS radical cation (ABTS .þ ), with an IC50

value comparable to that of Trolox�. Indeed, the IC50 value was 21.9 mg/ml for theessential oil compared to 15.1 mg/ml for Trolox�. The radical-scavenging activity is animportant attribute of antioxidants. ABTS .þ , a protonated radical, has a characteristic

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Table 2. In vitro Cytotoxic Activity of the Essential Oil Isolated from the Aerial Parts of Artemisiaarborescens Harvested in February (winter sample)

IC50 [mg/ml]a)

HCT 116b) MDA-MB 435Sc) T98Gd) A375e)

Essential oil 14.62�1.79 27.79�3.17 59.82�1.76 14.28�2.53Cisplatinf) 2.29�0.1 7.37�0.63 2.34�0.13 0.18�0.02

a) IC50 : Concentration of essential oil or cisplatin that affords a 50% reduction in cell growth after 72 h ofincubation; values are means�SD (n¼4). b) Human colon carcinoma cell line. c) Human breastadenocarcinoma cell line. d) Human glioblastoma multiforme cell line. e) Human malignant melanomacell line. f) Cisplatin was used as positive control and reference compound.

Table 3. Antioxidant Potential of Artemisia arborescens Essential Oil (winter sample) and the ReferenceCompound Trolox�

DPPH assay ABTS assay FRAP assay

IC50 [mg/ml]a) TEACb) IC50 [mg/ml] TEAC

Essential oil >200.0 2700�3.0 21.9�0.3 147.7�2.8Trolox� 4.36�0.3 15.1�0.4

a) IC50 : Concentration of essential oil or Trolox� reducing the free radical concentration by 50%.b) TEAC: Trolox� equivalent antioxidant concentration expressed in mm Trolox� equivalents (TE)/g ofessential oil.

absorption maximum at 734 nm. Scavenging of the long-life ABTS .þ by antioxidantsproduces a decrease in the absorbance at 734 nm [23]. The ABTS .þ-scavenging activityof the A. arborescens essential oil was found to be much higher than its capacity toscavenge the long-life DPPH radical (DPPH .).

Factors like the stereoselectivity of radical reactions or the solubility of the samplesin the different test systems have been reported to affect the capacity of samples toreact with and quench different radicals [24]. Wang et al. [25] showed that somecompounds with ABTS .þ-scavenging activity did not quench DPPH . [25]. Theantioxidant potential of the A. arborescens oil was further estimated by its ability toreduce the ferric tripyridyl triazine (Fe3þ-TPTZ) complex to ferrous tripyridyl triazine(Fe2þ-TPTZ). The ferric-reducing antioxidant power (FRAP) reported for the A.arborescens oil was 147.7 mmol TE/g of essential oil, hence relatively higher comparedto that of Trolox� (399.53 mmol/g). Based on these data, we can conclude that the A.arborescens essential oil exhibits a good ABTS .þ-scavenging activity, which may limitfree radical damage occurring in the human body. The detected activity may be due tothe high content of chamazulene, which was previously reported to be a potent .OHscavenger capable to effectively inhibit lipid peroxidation [11]. Moreover, the radical-scavenging ability of chamazulene is what is thought to be responsible for theeffectiveness of A. arborescens essential oil in treating inflamed skin conditions [26].

Conclusions. – The essential oils of the Sardinian population of A. arborescensinvestigated here belonged to the b-thujone/chamazulene chemotype. They might be ofinterest for the treatment of inflammatory skin and bowel diseases, due to their highcontent of chamazulene, the highest ever detected in an essential oil to date, or as apotential anticancer and antioxidant agent. Further pharmacological experiments areneeded in any case. Finally, neochlorogenic acid was reported for the first time in thisspecies.

Experimental Part

Plant Material. The aerial parts of A. arborescens were collected in North Sardinia (Isle of LaMaddalena, La Maddalena Archipelago, Sardinia, Italy) at three different growth stages, i.e., at the endof the flowering time (July 2011), during the vegetative stage (February 2012), and at the beginning ofthe flowering time (May 2012). The plant was identified by Prof. Mauro Ballero at the Dipartimento diScienze Botaniche, Universita di Cagliari, and voucher specimens (No 734/Herbarium CAG) have beendeposited with the herbarium of this department.

Extraction of Essential Oils. Aerial parts (450 g) of A. arborescens were subjected to hydrodistillationin a Clevenger-type apparatus for 4 h using 1000 ml of dist. H2O. At the end of each distillation, the oilswere collected in a Clevenger trap, dried (anh. Na2SO4), stored in hermetically sealed glass containerswith rubber lids, covered with aluminum foil to protect the content from light, and kept at 48 untilanalysis. The oil samples from plants harvested at the three different growth periods were obtained withthe same protocol. The oil yield, calculated on the dry-weight basis of the plant material, ranged from0.35 to 0.50% (w/w).

GC/MS Analysis. The GC/MS analyses were performed with an Agilent 6890N gas chromatographcoupled to a 5973N mass spectrometer and equipped with an HP-5 MS fused-silica cap. column (5%phenylmethylpolysiloxane, 30 m�0.25 mm i.d., film thickness 0.1 mm; J & W Scientific, Folsom). Theoven temp. was programmed isothermal at 608 for 5 min, then rising from 60 to 2208 at 48/min and from220 to 2808 at 118/min, and finally isothermal at 2808 for 15 min; injector temp., 2808 ; transfer line temp.,

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2808 ; carrier gas, He (1 ml/min); split ratio, 1 : 50. All mass spectra were acquired in electron-impact (EI)mode with an ionization voltage of 70 eV over an m/z range of 29–400. The oil samples were diluted1 : 100 in hexane, and the volume injected was 2 ml. The analyses were repeated three times. Data wereanalyzed by using the MSD ChemStation software version G1701DA D.01.00 (Agilent).

Compound Identification. For 15 out of the 58 compounds, i.e., a-phellandrene, a-terpinene, p-cymene, 1,8-cineole, g-terpinene, terpinolene, a-thujone, b-thujone, terpinen-4-ol, a-terpineol, myrtenol,eugenol, (E)-caryophyllene, a-humulene, and caryophyllene oxide, corresponding to 39.2–40.6% of thetotal oil composition, the identification was carried out by coinjection with authentic standards purchasedfrom Sigma-Aldrich (Milan, Italy). Otherwise, the peak assignment was in accordance with the standardof the International Organization of the Flavor Industry [27]. Briefly, the oil components were identifiedby comparison of their linear retention indices (LRIs; determined rel. to the tR of a series of n-alkanes(C8�C30; Sigma, Milan, Italy) with those reported by Adams [6] and listed in the NIST 08 library [7] andby computer matching of the mass spectra with those registered in the following mass spectral libraries:WILEY275, NIST 08, ADAMS, and a home-made library (based on the analyses of reference oils andcommercially available standards).

The contents of the essential oil components were obtained by internal peak-area normalizationwithout using response factors.

MTT Cytotoxicity Assay. The human glioblastoma multiforme cell line T98G was cultured in Eagle�sminimum essential medium (EMEM) with 2 mm l-glutamine, 0.1 mm non-essential amino acids, 1 mm

sodium pyruvate, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; PAA Laboratories GmbH, Austria). The human breastadenocarcinoma cell line MDA-MB 435S and the human malignant melanoma cell line A375 werecultured in Dulbecco�s modified Eagle�s medium (DMEM) with 2 mm l-glutamine, 100 IU/ml penicillin,100 mg/ml streptomycin, and supplemented with 10% HI-FBS. The human colon carcinoma cell lineHCT116 was cultured in RPMI1640 medium with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/mlstreptomycin, and supplemented with 10% HI-FBS. Cells were cultured in a humidified atmosphere at378 in presence of 5% CO2. The MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide) assay was used as a relative measure of cell viability. Cell viability assays were carried out asdescribed in [28] [29]. Briefly, cells were seeded at the density of 2�104 cells/ml. Quadruple cell sampleswere grown in 96-well micro-titer plates (Iwaki, Tokyo, Japan). After 24 h, samples were exposed todifferent concentrations (1.56 –200 mg/ml) of essential oil isolated from the aerial parts of A. arborescenscollected in February (winter sample) in a final volume of 100 ml of culture medium. Cells were incubatedfor 72 h in a humidified atmosphere of 5% CO2 at 378. At the end of incubation, each well received 10 mlof MTT (5 mg/ml in phosphate-buffered saline (PBS)), and the plates were incubated for 4 h at 378. Theformazan crystals formed were solubilized in 100 ml of DMSO after aspirating the medium. The extent ofMTT reduction was measured spectrophotometrically at 540 nm using a Titertek Multiscan microElisa(Labsystems, FI-Helsinki). Cytotoxicity was expressed as the concentration of compound inhibiting cellgrowth by 50% (IC50). The IC50 values were determined with the GraphPad Prism 4 computer software(GraphPad Software, S. Diego, CA, USA).

Evaluation of the Antioxidant Activity. The ABTS (¼2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) assay was performed following the procedure described by Re et al. [30], adapted to a 96-wellmicroplate assay by Esparza Rivera et al. [31]. Stock solns. of 7.4 mm ABTSþ and 2.6 mm K2S2O8 wereprepared. The working soln. was obtained by mixing the two stock solns. in equal quantities and allowingthem to react for 12 h at r.t. in the dark. This soln. was then diluted with MeOH (1 : 60) to obtain anabsorbance of ca. 1 unit at 734 nm. Fresh ABTS .þ soln. was prepared for each assay, and Trolox� wasused as reference compound. The standard curve was linear between Trolox� concentrations of 25 and600 mm. The results were expressed in mm Trolox� equivalents (TE)/g of essential oil. Additional dilutionwas needed, if the value measured was outside the linear range of the standard curve.

The DPPH (¼2,2-diphenyl-1-picrylhydrazyl) free radical-scavenging activity was evaluated using amicroplate analytical assay according to the procedure previously described by Srinivasan et al. [32]. Thestock soln. was prepared by dissolving DPPH in MeOH and then stored at �208 until needed. Theworking soln. was obtained by diluting the stock soln. with MeOH to obtain an absorbance of ca. 1 unit at515 nm. The standard curve set up with Trolox� was linear between 25 and 800 mm. The results were

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expressed in mm TE/g essential oil. Additional dilution was needed, if the value measured was outside thelinear range of the standard curve.

The FRAP (ferric-reducing antioxidant power) assay was carried out according to Firuzi et al. [33],with minor modifications. Acetate buffer (0.3 m, pH 3.6) and stock solns. of 10 mm TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mm HCl and 20 mm FeCl3 · 6 H2O were prepared. The fresh working soln.(FRAP soln.) was obtained by mixing 25 ml of acetate buffer, 2.5 ml of TPTZ soln., and 2.5 ml of FeCl3 ·6 H2O soln. The FRAP soln. was then warmed to 378 before use. Aliquots of dil. essential oil soln. inMeOH were allowed to react with 500 ml of the FRAP soln. for 30 min in the dark. Samples werecentrifuged at 10000 rpm, and aliquots (280 ml) of the supernatant were transferred to a 96-wellmicroplate. The absorption of the colored product (Fe2þ-TPTZ) was then measured at 593 nm. Thestandard curve was linear between 25 and 800 mm of Trolox�. The results were expressed in mm TE/gessential oil. Additional dilution was needed, if the value measured was outside the linear range of thestandard curve.

Polar Fraction. Aerial parts of A. arborescens collected during the vegetative stage (1 kg) wereexhaustively extracted with EtOH (96%) at r.t. for 2 d. Each extract was evaporated in vacuo, and acrude fraction was obtained, which was suspended in H2O and finally lyophilized. The crude extract(4.98 g) was chromatographed on silica gel (SiO2; 1 : 20) eluting with H2O sat. BuOH; 80 mg of 5-O-caffeoylquinic acid were obtained. Its identification was made by comparison with authentic samplepresent in our laboratory. The same procedure was performed on samples obtained at the vegetative andflowering stages, affording comparable results.

1H-NMR (300 MHz, MeOD): 7.57 (d, J ¼ 15.9, H�C(b)); 7.05 (d, J¼1.8, H�C(2’)); 6.94 (dd, J ¼ 8.2,1.8, H�C(6’)); 6.29 (d, J ¼ 15.9, H�C(a)); 6.77 (d, J¼8.2, H�C(5’)); 5.35 (br. d, H�C(3)); 4.30 (s,H�C(5)); 3.86 (br. d, H�C(4)); 2.0–2.2 (m, CH2(6)); 2.0–2.2 (m, CH2(2)). 13C-NMR (75 MHz, MeOD):181.1 (COOH�C(1)); 169.2 (C¼O); 149.6 (C(4’)); 147.2 (C(b)); 146.8 (C(3’)); 127.8 (C(1’)); 123.0 (C(6’));116.6 (C(5’)); 115.4 (C(a)); 115.3 (C(2’)); 77.7 (C(1)); 73.8 (C(4)); 71.9 (C(3)); 71.8 (C(5)); 38.0 (C(6));40.3 (C(2)). ESI-MS (pos.): 377.1 ([MþNa]þ ). ESI-MS (neg.): 353.1 ([M�H]� ).

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1721, 174.

Received December 20, 2012

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