Characterization of Secondary Metabolites, Biological Activity and GlandularTrichomes of Stachys tymphaea Hausskn. from the Monti Sibillini National

Park (Central Apennines, Italy)

by Alessandro Venditti*a), Armandodoriano Biancoa), Marcello Nicolettib), Luana Quassintic),Massimo Bramuccic), Giulio Lupidic), Luca Agostino Vitalic), Fabrizio Papac), Sauro Vittoric),

Dezemona Petrellid), Laura Maleci Binie), Claudia Giulianie), and Filippo Maggic)

a) Department of Chemistry, La Sapienza University, Piazzale Aldo Moro 5, IT-00185 Rome(phone: þ39-0649913229; fax: þ39-0649913841; e-mail: alessandro.venditti@uniroma1.it)

b) Department of Environmental Biology, La Sapienza University, Rome, Italyc) School of Pharmacy, University of Camerino, Camerino, Italy

d) School of Biosciences and Biotechnology, University of Camerino, Camerino, Italye) Department of Biology, University of Florence, Florence, Italy

Stachys tymphaea (Lamiaceae) is a perennial herb growing in forest openings and dry meadows ofcentral and southern Italy. It was investigated for the first time here, determining the content ofsecondary metabolites, the micromorphology of glandular trichomes, the histochemical localization ofsecretion, and the biological activity of the volatile oil, namely, the cytotoxic, antioxidant, andantimicrobial properties. The plant showed a peculiar molecular pattern, being rich of biophenoliccompounds as flavonoids, phenylethanoid glycosides, and caffeoylquinic acid derivatives, but poor ofiridoids, which are known as marker compounds of the genus Stachys. The essential oil was characterizedby GC-FID and GC/MS analyses, revealing a high percentage of sesquiterpene hydrocarbons (54.6%),with germacrene D (30.0%) and (E)-b-farnesene (12.4%) as the most abundant compounds, while othermain components were representatives of the diterpenes (19.2%), represented mainly by (E)-phytol(11.9%). This composition supported the taxonomic relationships in the genus Stachys, which comprisesoil-poor species producing essential oils rich in hydrocarbons, with germacrene D as one of thepredominant components. The micromorphological study revealed three types of glandular hairs, i.e.,Type A peltate trichomes, being the primary sites of essential oil biosynthesis, Type B short-stalkedtrichomes, typical mucopolysaccharide producers, and Type C long capitate trichomes, secreting acomplex mixture of both lipophilic and hydrophilic substances, with a major phenolic fraction. Moreover,the MTT assay revealed the potential of the volatile oil to inhibit A375, HCT116, and MDA-MB 231tumor cells lines (IC50 values of 23.9–34.4 mg/ml).

Introduction. – The genus Stachys L. is one of the largest genera of the Lamiaceaefamily, comprising ca. 450 taxa [1], mostly represented by annual or perennial herbs orsmall shrubs, distributed in warm and temperate regions of the Mediterranean Basinand southwestern Asia, with secondary centers in North America [2]. In Italy, 31species and subspecies are reported, five of which are considered as endemic taxa [3].

S. tymphaea Hausskn. is a perennial herb with erect flowering stems up to 90 cm,usually branched above, densely adpressed tomentose-villous, without persistentterminal rosettes of leaves. Basal and cauline leaves are paired, cordate at base,sericeous-tomentose above, and floccose-tomentose beneath, oblong-ovate or ovatethe former, ovate or ovate-lanceolate the latter, both with margins crenate to crenate-

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� 2014 Verlag Helvetica Chimica Acta AG, Z�rich

serrate. Inflorescences are pinkish-purple verticillasters, 12 –45 flowered, with the calyxdensely tomentose-villous, with glandular hairs on teeth, and corolla sericeous-tomentose on the outside [2]. The species grows in forest openings and dry meadows ofcentral and southern Italy (from Emilia-Romagna to Calabria, with the exception ofPuglia) [3].

Plants of the genus Stachys have been used for centuries as herbal remedies in thetreatment of several complaints [4 –6]. Some of them were proven to exhibit anti-inflammatory, antitoxic, antibacterial, antioxidant, and cytotoxic effects [7]. Phenyl-ethanoid glycosides, iridoids, triterpenoids, steroids, diterpenes, flavonoids, fatty acids,polysaccharides, and acidic metabolites have been identified as main secondarymetabolites [8 – 13].

In continuation of our studies of secondary metabolites produced by Lamiaceaespecies growing in central Italy [14 – 17], we investigated S. tymphaea, which grows inthe Monti Sibillini National Park (central Apennines, Italy) and which has not beenstudied in much detail so far. No information is available on the traditional uses of thisspecies in Italy.

The phytochemical analysis of the constituents of medium polarity obtained fromthe EtOH extracts of the flowering aerial parts of S. tymphaea together with the volatileconstituents contained in the hydrodistilled essential oil was reported. Furthermore, invitro assays to evaluate the cytotoxic, antioxidant, and antimicrobial activities of thevolatile oil were performed, namely, the MTT test on human tumor cell lines, theDPPH . and ABTS .þ radical-scavenging assays, the ferric-reducing antioxidant power(FRAP) assessment, and the agar disk-diffusion technique. To improve the value of oursurvey, a detailed micromorphological study of glandular and non-glandular trichomespresent on the vegetative and reproductive organs was carried out, with special focus onthe histochemical localization of secretion.

Results and Discussion. – Essential-Oil Analysis. Despite the high number ofStachys species, the essential oil compositions of many of them remain to beinvestigated. The present work represents the first study of the essential-oilcomposition of S. tymphaea, a taxon representative of mountain environments. Atotal of 62 compounds were identified in the essential oil of S. tymphaea, correspondingto 91.5% of the oil composition (Table 1). The very low oil yield (0.04%) was consistentwith those reported in the literature for other species of the genus Stachys [18] [19] andconfirmed that Lamioideae comprise oil-poor plants [20].

The essential oil of S. tymphaea was mostly constituted by sesquiterpene hydro-carbons (54.6%), with germacrene D (30%) and (E)-b-farnesene (12.4%) as the mostabundant components. The oil contained also high levels of diterpenes (19.2%),represented by (E)-phytol (11.9%) and two unknown compounds, one with amolecular weight of 272 (2.7%) and the other one with a molecular weight of 288(4.7%). The mass-fragmentation patterns of these two compounds are reported inFig. 1. The abundance of these compounds in S. tymphaea is consistent with thatreported for other representatives of the section Eriostomum, which comprises speciesaccumulating a significant amount of diterpenoids [19]. Furthermore, the large amountof germacrene D appears to be one of the main characters of members of this section aswell [18]. Finally, the oil presented moderate amounts of aliphatic n-alkanes (9.4%),

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Table 1. Composition of the Essential Oil Isolated from the Aerial Parts of Stachys tymphaea

Compound name and classa) RFb) RIexpc) RIlit

d) Content [%]e) Identificationf)

Adams [21] NIST08

(E)-Hex-2-enal 1.7 854 855 0.1 RI, MSa-Pinene 1.1 932 939 932 0.1 Stdb-Pinene 1.1 974 979 974 0.2 StdOct-1-en-3-ol 1.5 979 979 979 4.5 StdOctan-3-ol 1.5 998 991 999 trg) RI, MSa-Phellandrene 1.1 1003 1002 1003 0.1 Stdb-Phellandrene 1.1 1030 1029 1030 0.5 RI, MS(Z)-b-Ocimene 1.1 1043 1037 1042 0.6 RI, MS(E)-b-Ocimene 1.1 1053 1050 1052 0.1 RI, MSLinalool 1.5 1100 1096 1100 1.9 Stdn-Nonanal 1.7 1106 1100 1106 tr RI, MSa-Terpineol 1.5 1189 1188 1189 0.2 StdMethyl salicylate 1.7 1192 1191 1192 tr RI, MSb-Cyclocitral 1.0 1218 1219 1218 tr RI, MSa-Cubebene 1.1 1346 1348 1346 0.5 RI, MSEugenol 1.7 1358 1359 1358 tr Stda-Copaene 1.1 1370 1376 1370 2.1 RI, MSb-Bourbonene 1.1 1378 1388 1378 0.4 RI, MS(E)-b-Damacenone 1.0 1382 1384 1383 tr RI, MSb-Cubebene 1.1 1385 1388 1385 0.3 RI, MSb-Elemene 1.1 1387 1390 1387 1.2 RI, MSb-Ylangene 1.1 1410 1420 1405 0.3 RI, MS(E)-Caryophyllene 1.1 1412 1418 1412 0.3 Stdb-Copaene 1.1 1431 1432 0.4 RI, MSAromadendrene 1.1 1436 1441 1437 0.1 RI, MS6,9-Guaiadiene 1.1 1443 1444 0.1 RI, MSAlloaromadendrene 1.1 1452 1460 1452 0.2 RI, MS(E)-b-Farnesene 1.1 1459 1456 1459 12.4 Stdg-Gurjunene 1.1 1465 1477 1465 0.4 RI, MSGermacrene D 1.1 1474 1485 1474 30.0 RI, MS(E)-b-Ionone 1.0 1483 1488 1483 0.2 StdBicyclogermacrene 1.1 1488 1500 1488 0.8 RI, MSLedene 1.1 1488 1489 0.8 RI, MStrans-Muurola-4(14),5-diene 1.1 1491 1493 0.2 RI, MSa-Muurolene 1.1 1494 1500 1494 1.1 RI, MSg-Cadinene 1.1 1508 1513 1508 0.5 RI, MSb-Bisabolene 1.1 1505 1505 1505 0.5 Std(E,E)-a-Farnesene 1.1 1508 1505 1508 0.5 Stdd-Cadinene 1.1 1518 1523 1518 1.4 RI, MS(E)-Nerolidol 1.3 1564 1563 1564 0.6 StdGermacrene D-4-ol 1.3 1569 1575 1569 0.7 RI, MSViridiflorol 1.3 1582 1592 1582 1.6 Stdepi-a-Cadinol 1.3 1635 1640 0.4 RI, MSepi-a-Muurolol 1.3 1635 1642 0.4 RI, MSa-Muurolol 1.3 1640 1646 0.1 RI, MSa-Cadinol 1.3 1648 1654 0.8 RI, MSValeranone 1.3 1662 1675 1662 0.5 RI, MSepi-b-Bisabolol 1.3 1667 1671 0.3 RI, MS

with nonacosane as the most abundant representative (4.2%). Apart from the majorvolatiles reported above, only oct-1-en-3-ol (4.5%) and a-copaene (2.1%) exceeded acontent of 2% of the total oil composition, whilst the remaining constituents (53) werepresent in scarce amounts, most of them occurring at contents lower than 1%.

Analysis of the Polar Fraction. The molecular pattern recognized in S. tymphaearesulted very different from those of other members of the genus. Generally, polarmarker compounds of the genus Stachys are represented by iridoid glycosides, with

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Table 1 (cont.)

Compound name and classa) RFb) RIexpc) RIlit

d) Content [%]e) Identificationf)

Adams [21] NIST08

a-Bisabolol 1.3 1682 1685 1682 0.3 RI, MSHeptadecane 1.2 1700 1700 1700 0.3 Std6,10,14-Trimethylpentadecan-2-one 1.7 1846 1846 0.3 RI, MSUnknown diterpene (MW 272) 1.0 1949 2.7 MSHexadecanoic acid 1.0 1972 1960 1972 0.4 StdUnknown diterpene (MW 288) 1.0 2067 4.7 MSHeneicosane 1.2 2101 2100 2100 0.2 Std(E)-Phytol 1.0 2117 2117 11.9 StdLinoleic acid 1.0 2143 2133 2144 0.4 StdTricosane 1.2 2301 2300 2300 0.8 StdTetracosane 1.2 2400 2400 2400 0.2 StdPentacosane 1.2 2501 2500 2500 1.0 StdHeptacosane 1.2 2702 2700 2700 1.8 StdOctacosane 1.2 2802 2800 2800 0.3 StdNonacosane 1.2 2902 2900 2900 4.2 StdUntriacontane 1.2 3102 3100 3100 0.8 Std

Total identified 91.5

Monoterpene hydrocarbons 1.6Oxygenated monoterpenes 2.1Sesquiterpene hydrocarbons 54.6Oxygenated sesquiterpenes 5.6Diterpenes 19.2Alcohols 4.5Alkanes 9.4Others 1.3

Oil yield [%] 0.04

a) Compounds are listed in order of their elution from a HP-5 MS column. Their nomenclature is inaccordance with Adams [21]. b) RF: Relative response factor of the FID for the main compound classespresent in the oil. c) RIexp: Linear retention index experimentally determined on a HP-5 MS columnrelative to a homologous series of n-alkanes (C8 –C30). d) RIlit : Literature RI published by Adams [21]and/or listed in the NIST08 mass-spectral library [22]. e) Contents are given as means of threedeterminations; the relative standard deviations for the main components were below 5% in all cases.f) Identification method: Std, based on the comparison with authentic compounds; MS, based on thecomparison of mass spectra with those listed in the Wiley, ADAMS, and NIST08 mass-spectral libraries[22]; RI, based on the comparison of RIs with those reported by Adams [21] and listed in NIST08 [22].g) tr: Trace (mean value below 0.1%).

harpagide (1) as one of the most representatives [16] [17] [23 – 26]. However, it was verydifficult to isolate only a few mg of this compound from 5 g of crude S. tymphaea extractas starting material. On the other hand, phenolics were the major compounds. Amongthese, we isolated verbascoside (2) [27] and stachysoside A (¼2-(3,4-dihydroxyphe-nyl)ethyl O-a-l-arabinopyranosyl-(1!2)-O-6-deoxy-b-l-mannopyranosyl-(1!3)-4-O-[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-b-d-glucopyranoside, 3 ;Fig. 2), two phenylethanoid glycosides, the former almost ubiquitous, while the latter,containing an a-arabinose unit, seems to be more characteristic, since it was isolatedonly from S. sieboldii [28]. The IUPAC name for compound 3 was reported to avoid anyconfusion, because it has to be considered that the same name, i.e., stachysoside A, wasalso used for an iridoid glucoside by other authors [29]. Phenylethanoid glycosides areknown to be present in Asteridae, together with iridoid glycosides [30], and thischaracteristic is of chemotaxonomic importance. In a preliminary analysis of the crudeextract, iridoid glucosides seemed to be absent. After a careful separation work, a littlequantity of harpagide (1) was recognized [31], confirming the co-occurrence of thesecompounds also in S. tymphaea. However, the metabolic profile of this plant is strongly

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Fig. 1. Mass spectra fragmentation patterns of two unknown diterpenes with a MW of a) 272 and b) 288,respectively, detected in the essential oil isolated from the aerial parts of Stachys tymphaea

oriented toward the synthesis of biophenolics. In fact, a particularly high quantity ofchlorogenic acid (4) [32] was isolated, and 4 was revealed as the main phenoliccomponent of the species. Also three monoacetylated flavonoid diglycosides related toisoscutellarein were identified, i.e., 7-{[2-O-(6-O-acetyl-b-d-allopyranosyl)-b-d-gluco-pyranosyl]oxy}-5,8-dihydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (5), 7-{[2-O-(6-O-acetyl-b-d-allopyranosyl)-b-d-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-hy-droxyphenyl)-4H-1-benzopyran-4-one (6), and 7-[(6-O-acetyl-2-O-b-d-allopyranosyl-b-d-glucopyranosyl)oxy]-5,8-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzo-pyran-4-one (7). Among these, the presence of a new monoacetylated derivative, i.e., 7,was recognized, since the Ac residue seems to be connected to the OH group of theC(6’’) of glucose. On the contrary, the known monoacetylated derivatives have the Acsubstitution only on the allose moiety [33] [34]. The connection of the Ac group to theOH group of the C(6’’) of glucose was evident in the 1H-NMR spectrum. In fact, thesinglet relative to the Me group of the Ac moiety resulted more deshielded than2.0 ppm. Differently, the derivatives that are monoacetylated on the allose moietypresented the singlet of the Me group at ca. 1.98 ppm. Also the H�C(6’’) signals at 4.37–4.27 ppm could be assigned to glucose, since the H�C(6’’’) signals (allose) generally

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Fig. 2. Structures of the polar compounds isolated from the EtOH extract of Stachys tymphaea. 1,Harpagide; 2, verbascoside; 3, stachysoside A; 4, chlorogenic acid; 5, 7-{[2-O-(6-O-acetyl-b-d-allopyranosyl)-b-d-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one;6, 7-{[2-O-(6-O-acetyl-b-d-allopyranosyl)-b-d-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; 7, 7-[(6-O-acetyl-2-O-b-d-allopyranosyl-b-d-glucopyranosyl)oxy]-5,8-dihy-

droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one.

result less deshielded (ca. 4.0 ppm) [34]. Additional evidence for this kind ofsubstitution was recognized in the 13C-NMR spectra, where the signal at ca. 65 ppmcould be assigned only to the C(6’’) of glucose, due to the deshielding effect of the Acgroup (ca. 3 ppm), since deshielding due to acetylation of the C(6’’’) of allose caused ashift that reached ca. 63 ppm [35]. Acetylation at C(6’’) exerted also a shielding effectof 3 – 4 ppm on the adjacent H�C(5’’); in fact, by 2D-NMR experiments (HSQC), asignal was found at d(H) 4.12 that was directly connected with the C-atom thatresonated at 72.2, and since it had a H-atom multiplicity of three (triplet), it could beassigned to the H�C(5’’) of the glucose moiety. These data are in accordance with theproposed structure for the new isoscutellarein derivative 7, monoacetylated on theglucose moiety.

Acetylated isoscutellarein derivatives containing allose have taxonomic relevance,since they were isolated only from some correlated genera of Lamiaceae, such asPogostemon, Sideritis, Galeopsis, and Stachys [36]. The new compound (7) may beconsidered a molecular marker of S. tymphaea. Acetylated flavonoid glycosidescontaining allose were recently investigated for their structure�activity relationship byUriate-Pueyo and Calvo [37]. The authors found that methylation at OH�C(4’) madethe antioxidant reactivity of the investigated flavonoid glycosides kinetically faster.Moreover, the diacetylated derivatives showed a higher antioxidant activity withrespect to the monoacetylated ones. Finally, the contemporaneous presence ofmonoacetylation and methylation at OH�C(4’) resulted beneficial for the acetylcho-linesterase-inhibitory and neuroprotective activities.

Glandular Trichomes: Distribution, Morphology, and Histochemistry. For the genusStachys, various morphotypes of trichomes have already been described in previousarticles [38] [39]. A more detailed study of stems, leaves, and floral organs of S.tymphaea allowed the observation of the following types of glandular hairs (Fig. 3).

The Type A trichome, scattered on stems, leaves, bracts, calyces, and corollas(Fig. 3,n and o), is a peculiar peltate hair with an elongated basal epidermal cell (Fig. 3,a and b), so that a well-developed stalk is formed [40]. This kind of hair has also beenobserved in other Lamiaceae species lacking the typical sessile peltate trichomes [39].The secreting material proved positive to all the lipophilic stains, particularly to theNadi reagent (Fig. 3,c and d), whereas the hydrophilic dyes gave negative results(Fig. 3, e), indicating the exclusive secretion of terpenoids.

The Type B trichome, considered the most common glandular hair of Lamiaceae[40] [41] and occurring on the whole plant (Fig. 3,n and o), is a short-stalked capitatehair composed by a basal cell, a unicellular stalk, and by two to four glandular cellssurrounded by a thin subcuticular chamber (Fig. 3, f). The secretion of Type Btrichomes stained positive with Ruthenium Red and Alcian Blue, indicating onlyhydrophilic secretion of polysaccharides (Fig. 3, f – h).

The Type C trichome, abundant and present only on calyces and corollas (Fig. 3,oand p), is a long capitate trichome with a multicellular stalk and a head of eight totwelve glandular cells, each developing on the apex its own small subcuticular space(Fig. 3, i). The stalk shows a micropapillate surface and is composed of longer cells atthe base graduating into shorter ones above. The secretion showed a positive responseto all the histochemical dyes, indicating the production of polysaccharides, lipids, and

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phenolic compounds (Fig. 3, j – l). Copious amounts of secreting material was oftenobserved along the stalk and on the epidermis.

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Fig. 3. Micrographs obtained by SEM and LM showing the types, micromorphology, histochemistry, anddistribution of Stachys tymphaea trichomes. a) and b) Micromorphology of Type A peltate trichomes(bars, 25 mm (a) and 50 mm (b)). c) –e) Histochemistry of Type A trichomes. Staining was made withFluoral Yellow-088 (c), Nadi reagent (d), and Alcian Blue (e); bars, 25 mm. f) Micromorphology of TypeB capitate trichomes (bar, 20 mm). g)– i) Histochemistry of Type B trichomes. Staining was made withRuthenium Red (g), Alcian Blue (h), and Nile Red (i); bars, 10 mm. j) Micromorphology of Type Ccapitate trichomes (bar, 25 mm). k) –m) Histochemistry of Type C trichomes. Staining was made withRuthenium Red (k), Nadi reagent (l), and FeCl3 (m); bars, 25 mm. n) Leaf abaxial surface (bar, 100 mm).

o) Calyx adaxial surface (bar, 500 mm). p) Corolla abaxial surface (bar, 500 mm).

Within the Lamiaceae family, a great taxonomic significance is attributed to calyxtrichomes, in particular to Type C capitate hairs, for better distinguishing plants at thesubgeneric or subspecific levels [41]. Accordingly, the presence of this morphotype onthe inflorescences of S. tymphaea further enhances the taxonomic value of theindumentum in Stachys [39] [41].

Recently, there has been renewed interest in the use of histochemical techniques tostudy the nature of trichome contents [39]; in general, each type of trichome ischaracterized by a main or exclusive type of secretion. The present study of S. tymphaeatrichomes is consistent with this suggestion, since Type A hairs, as the sessile peltatetrichomes, are known to be the primary sites of essential-oil biosynthesis [40], whereasshort-stalked Type B capitate trichomes have been identified as typical mucopolysac-charide producers [41]. The secretion of Type C trichomes was characterized by acomplex mixture of both lipophilic and hydrophilic substances, with a major phenolicfraction. Their glandular cells are probably able to produce different classes ofcompounds, but generally, only one type is prevailing [42].

Cytotoxic Activity. To investigate the cytotoxic activity of S. tymphaea essential oil,its effect on a selection of human tumor cell lines was evaluated. The susceptibility tothe oil of the MDA-MB 231 human breast adenocarcinoma, A375 human malignantmelanoma, and HCT116 human colon carcinoma cell lines was tested using the MTTassay. All cell lines were submitted to increasing concentrations of S. tymphaeaessential oil for 72 h. The results, collected in Table 2, show that the essential oilexhibited a significant cytotoxic effect towards the three human tumor cell linesexamined. Indeed, it induced a concentration-dependent growth-inhibitory effect onthe tested cells at dilutions ranging from 0.78 to 200 mg/ml. The IC50 values of the oilwere 23.93, 25.40, and 34.35 mg/ml towards MDA-MB 231, A375, and HCT116 celllines, respectively.

Previously, the essential oils from different Stachys species were found to havecytotoxic activity towards C32 amelanotic melanoma and ACHN adenocarcinoma celllines [7]. In the present study, it was shown that also S. tymphaea essential oil resultedactive on a human melanoma cell line and, in addition, it also showed cytotoxic activitytowards human breast and colon carcinoma cell lines. The cytotoxic activity of S.tymphaea essential oil may be attributed to specific components of the oil. Somecompounds found in S. tymphaea essential oil have been tested for cytotoxic properties.Germacrene D was reported to be active on human breast adenocarcinoma (MDA-MB

Table 2. In vitro Cytotoxic Activity of the Stachys tymphaea Essential Oil in Comparison with that ofCisplatin

IC50 [mg/ml]a)

MDA-MB 231b) HCT116c) A375d)

S. tymphaea oil 23.9 (20.4–28.1) 34.4 (30.5–38.7) 25.4 (20.8–31.0)Cisplatin 3.0 (2.2 –3.2) 2.5 (2.1 –2.9) 0.4 (0.3–0.5)

a) IC50 : Concentration of sample that affords a 50% reduction in cell growth after 72 h of incubation;values are means with 95% confidence intervals in parentheses (n¼3). b) Human breast adenocarci-noma cell line. c) Human colon carcinoma cell line. d) Human malignant melanoma cell line.

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231 and MCF-7), human ductal carcinoma (Hs 578T), and human hepatocellularcarcinoma (Hep G2) cell lines [43]. (E)-Phytol also exerted cytotoxic activity againstseveral solid tumor cell lines as KB, MCF-7, A549, and CasKi cells, whereas it resultedless active on HCT116 cells [44]. To our knowledge, data reporting the cytotoxicactivity of (E)-b-farnesene are missing. The presence of germacrene D (30.0%) and(E)-phytol (11.9%) can partially explain the cytotoxicity of the essential oil. However,it should be considered that also the minor components could contribute to thecytotoxic activity and might also be involved in some type of synergism with the other,more abundant active compounds.

Antioxidant Activity. Antioxidant properties of essential oils are of great interest forthe food-processing industry, since a growing tendency to possibly use natural additivesto replace synthetic antioxidants with natural ones is emerging. Previous pharmaco-logical studies suggested that different polar extracts of Stachys species possess highradical-scavenging activity [45]. Like many other representatives of the familyLamiaceae, Stachys species produce essential oils that have potential antioxidantactivities that have to be evaluated, to obtain an insight into the beneficial effects of thisplant species related to the possible use against inflammation, to reduce the risk ofcardiovascular diseases, and as cancer prevention [7] [9].

The antioxidant potential of essential oils can be evaluated using numerous assays.The first step is the screening of the potential antioxidant activity by different in vitrotests. Each of those is based on one aspect of the antioxidant activity, such as the abilityto scavenge free radicals, the inhibition of lipid peroxidation, the chelation of transitionmetal ions, etc. Hence, to get relevant data, a single method for testing the antioxidantactivity of plant products is not recommended, due to their complex composition.Therefore, the antioxidant potential of the S. tymphaea essential oil was evaluated bythe DPPH . and ABTS .þ radical-scavenging and the ferric-reducing antioxidant power(FRAP) assays, and the results are summarized in Table 3 in comparison with Trolox�,used as control. The activity was expressed as antioxidant index, determined with eachof the three different methods used. The essential oil exhibited significant, but muchlower antioxidant activity than Trolox�, with IC50 values ca. 71- and 28-fold higher thanthose shown by Trolox � for the DPPH . and ABTS .þ radical-scavenging activity,respectively. Therefore, also the FRAP of the oil was determined. The FRAP isassociated with antioxidant activity [46] and may serve as a significant indicator ofpotential antioxidant activity. The FRAP value reported in Table 3 confirms that S.tymphaea oil has a significant capacity for iron binding, which is related to its possibleaction as peroxidation protector. From our data, the essential oil showed a meaningfulability as free-radical inhibitor and the antioxidant effectiveness of the oil observed isprobably due to the relatively high content of sesquiterpene hydrocarbons andditerpenes that together represent 73.8% of the total oil composition. It was shown thatthese terpene hydrocarbons, whose antioxidant activity is close to that of phenoliccompounds, break free-radical chain reactions, which could be accompanied by theirirreversible oxidation into inert compounds [47], and essential oils that contain thesekind of compounds have good antioxidant properties [48] [49].

Antimicrobial Activity. The S. tymphaea essential oil did not show any valuableactivity against the two bacterial species Staphylococcus aurens and Escherichia colitested under the experimental conditions used (inhibition-zone diameter of 6– 7 mm).

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In comparison, the control antibiotic ciprofloxacin showed a diameter of >20 mm.Therefore, the activity of the essential oil was not further investigated, neither by othermethods, nor against other microbial species.

Conclusions. – The analysis of the volatile constituents of S. tymphaea supported thetaxonomic relationship within Stachys species as well as that within the Lamiaceaefamily, for which the oil-poor species belong to the subfamily Lamioideae and produceessential oils rich in hydrocarbons, with germacrene D often being one of thepredominant components [50].

The composition of the polar fraction of this plant resulted very different from whatwas expected for a species of the genus Stachys. As a matter of facts, characteristiciridoid glucosides as harpagide (1) and its acetylated derivatives, considered as markercompounds for this genus, were poorly represented, since only a very low amount ofcompound 1 was detected in the polar fraction and no evidence of related derivativeswas found. The majority of the polar compounds were represented by biophenolics,viz., the caffeolylquinic acid 4 (chlorogenic acid), three acetylated flavonoids, and twophenylethanoid glycosides. Compound 4 was found to be the most abundantcomponent of the polar fraction, followed by the three isoscutellarein derivatives 5 –7. Among these latter, compound 7 was identified as a new monoacetylated flavonoiddiglycoside containing allose. These compounds (5– 7) are of taxonomic relevance,since they were isolated only from some related genera of the Lamiaceae family, such asPogostemon, Sideritis, and Galeopsis, and the monoacetylated flavonoid diglycoside 7may be a molecular marker for S. tymphaea. Also the phenylethanoid glycosides have ataxonomic relevance, when recognized together with iridoid glucosides.

The biological assays revealed the potential of the volatile oil to inhibit tumor-cellgrowth and/or act as antioxidants, activities that might be dependent on the highcontent of germacrene D. In contrast, no antibacterial activity was detectable using thepure essential oil of the plant.

Finally, the micromorphological study of the glandular trichomes enhanced the highmorphological and functional diversity of trichomes in Stachys species, confirming thatthe variability of these structures has not yet been fully understood and properlyclassified [39]. In S. tymphaea, each type of trichome was characterized by a main orexclusive type of secretion, and their histochemical profiles and distribution patterns

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Table 3. In vitro Antioxidant Activity of the Stachys tymphaea Essential Oil in Comparison to that ofTrolox�

DPPHa) ABTSb) FRAPc)

TEACd) IC50 [mg/ml]e) TEAC IC50 [mg/ml] TEAC

S. tymphaea oil 55.9�0.6 764.0�23.9 142.0�6.4 101.0�9.6 194.1�12.6Trolox� – 10.7�0.2 – 3.6�0.2 –

a) DPPH .-Scavenging activity. b) ABTS .þ-Scavenging activity. c) Ferric ion reducing antioxidant power.d) Trolox � equivalent (TE) antioxidant concentration expressed in mmol TE/g; values are means�standard deviations. e) Concentration of sample that reduces the presence of radicals by 50%; values aremeans� standard deviations.

explained the poorness of essential oil and the abundance of the phenolic fraction in thespecies.

The authors thank the persons in charge of the Sibillini Mountains National Park for allowingcollection of the species investigated in this study.

Experimental Part

Plant Material. Flowering aerial parts of S. tymphaea were collected at the end of June 2012 fromnatural populations on calcareous rocks and stony slopes of the Monte Prata (Castelsantangelo sul Nera,Marche, Italy) at an altitude of 1650 m (GPS coordinates: N 42852’08’’; E 13811’44’’). The plant materialwas identified by F. M., based on available literature [51], and a voucher specimen has been depositedwith the Herbarium Camerinensis, School of Environmental Sciences (University of Camerino, Italy),included in the online edition of the Index Herbariorum [52], under the accession code CAME 24826. Itis also available on the web site in the anArchive system [53].

Essential-Oil Isolation. The air-dried aerial parts of S. tymphaea (250 g) were cut into small piecesand submitted to hydrodistillation for 3 h in a Clevenger-type apparatus using hexane (3 ml) as collectorsolvent. The yellow-colored essential oil was dried (Na2SO4), then the solvent was evaporated under aflow of N2, and the oil was stored in sealed vials, protected from light, at �208 before analyses. The oilyield (0.04%) was estimated on the dry-weight basis.

Chemicals. a-Pinene, b-pinene, oct-1-en-3-ol, a-phellandrene, linalool, a-terpineol, eugenol, (E)-caryophyllene, (E)-b-farnesene, (E)-b-ionone, b-bisabolene, (E,E)-a-farnesene, (E)-nerolidol, viridi-florol, heptadecane, hexadecanoic acid, heneicosane, linoleic acid, tricosane, tetracosane, pentacosane,heptacosane, octacosane, nonacosane, and untriacontane were purchased from Sigma-Aldrich (Milan,Italy). (E)-Phytol was previously isolated from Onosma echioides [54]. Viridiflorol was kindly providedby Michael Russell, Department of Primary Industries, Industry and Investment NSW, Wollongbar, NSW,Australia. For the determination of retention indices (RIs), a series of n-alkanes (Supelco, USA),ranging from octane (C8) to dotriacontane (C30), was used and analyzed at the experimental conditionsreported below. All compounds were of analytical standard grade. The solvent analytical grade hexanewas purchased from Carlo Erba (Milan, Italy) and was successively distilled on a Vigreux column beforeuse.

GC-FID Analysis. For the GC-FID analyses, an Agilent 4890D apparatus coupled to an ionizationflame detector (FID) was used. The volatile components were separated on a HP-5 cap. column (5%phenylmethylpolysiloxane, 25 m�0.32 mm i.d., film thickness 0.17 mm; J&W Scientific, Folsom, CA).The oven temp. was programmed isothermal at 608 for 5 min, then rising from 60 to 2208 at 48/min andfrom 220 to 2808 at 118/min, and finally held isothermal at 2808 for 15 min (total run time of 65 min);injector temp., 2808 ; transfer-line temp., 2808 ; carrier gas, He (1.4 ml/min); injection volume, 1 ml (oilsamples dil. 1 : 100 in hexane); split ratio, 1 : 34. A mixture of aliphatic hydrocarbons (C8 –C30; Sigma,Milan, Italy) in hexane was directly injected into the GC injector under the above temp. program, tocalculate the RI (as Kovats index) of each compound. The analysis was repeated three times, and datawere collected by using HP3398A GC Chemstation software (Hewlett-Packard, Rev. A.01.01). Therelative contents of the essential oil components, expressed as percentages, were obtained by FID peak-area normalization by calculating the response factor (RF) of the FID for eight different classes ofvolatiles (Table 1). Standard compounds, each representing the chemical classes determined, wereselected among those available in the authors� laboratory, i.e., b-pinene, limonene, and g-terpinene formonoterpene hydrocarbons, p-cymene for aromatic monoterpenes, 1,8-cineole and linalool for oxy-genated monoterpenes, (E)-caryophyllene and a-humulene for sesquiterpene hydrocarbons, caryophyl-lene oxide for oxygenated sesquiterpenes, octanal for aldehydes, octane and octadecane for alkanes, andbenzaldehyde and p-vinylanisole for aromatics. Each standard, diluted in hexane, was injected at fourdifferent concentrations (0.04, 0.08, 0.16, and 0.40 mg/ml) using octane and octadecane as internalstandards at a concentration of 0.04 mg/ml. For each standard, a regression line was obtained by plottingthe ratio of the peak area of the internal standard (mean value of octane and octadecane) to that of thereference standard vs. the ratio of the concentration of the internal standard to that of the reference

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standard. Each dilution was analyzed five times. The correlation coefficients (r2) obtained for thecalibration curves of the representative standards were all higher than 0.992. The correction factor ofeach reference-standard compound was calculated as the slope of the linear-regression equation.Whenever more than one reference analyte per compound class was considered, the correction factorwas calculated as the mean of the slopes of all representative compounds. Using the generalized RF forcompounds within the eight classes, the derived quantitative data may be considered as an approximationof the absolute quantification.

GC/MS Analysis. The GC/MS analyses were performed on an Agilent 6890N gas chromatographequipped with a 5973N mass selective detector and a HP-5 MS cap. column (5% phenylmethylpoly-siloxane, 30 m�0.25 mm i.d., film thickness 0.1 mm; J&W Scientific, Folsom). The temp. program and theinjector and transfer-line temps. were the same as described above (cf. GC-FID Analysis). The carrier gaswas He (1 ml/min), the split ratio1 : 50, and the acquisition mass range 29 –40 amu. All mass spectra wereacquired in electron-impact (EI) mode with an ionization voltage of 70 eV. The oil samples were diluted1 : 100 in hexane and the volume injected was 2 ml. The analysis was repeated three times, and the datawere analyzed by using MSD ChemStation software (Agilent, Version G1701DA D.01.00). Wheneverpossible, the volatile components were identified by coinjection with authentic standards. Otherwise, thepeak assignment was carried out according to the standard statement of the International Organization ofthe Flavor Industry (IOFI) [55], i.e., based on the comparison of chromatographic RIs with thosereported in the literature [21] [22] for apolar stationary phases and on the computer matching of massspectral data with the commercial libraries WILEY275, NIST08, and ADAMS, and a home-made librarybased on the analyses of reference oils and commercially available standards.

Analysis of the Polar Fraction. Dried plant material (93.0 g) was extracted three times with aq. EtOHsolns. (first 450 ml of EtOH/H2O 80 : 20 to rehydrate the plant material, then 450 ml of EtOH/H2O 95 : 5).The obtained extracts were concentrated under reduced pressure to eliminate the EtOH, and theresulting H2O suspension was first frozen at �208 and then lyophilized to recover 21.8 g of crude extract.A portion of 5.0 g of crude extract was partitioned on a silica gel (SiO2) column using BuOH saturatedwith H2O as eluent. The chromatographic separation resulted directly in the isolation of 28.8 mg ofverbascoside (2), besides two big fractions (Frs. A and B) containing a mixture of phenolic compounds.Fr. A (699.4 mg) was mainly constituted by a mixture of flavonoids, and in Fr. B (560 mg), chlorogenicacid (4) was the principal compound. Moreover, Fr. B resulted positive to the vanillin/HCl spray-reagenttest, an evidence of the copresence of an iridoidic compound. Fr. A was repartitioned on SiO2 previouslytreated with gaseous CO2, using a mixture of CHCl3/MeOH 9 : 1, also insufflated with CO2, as startingeluent. The polarity was raised during the chromatographic run to CHCl3/MeOH 8 : 2 and then to CHCl3/MeOH 7 : 3. From this second fractionation, three acetylated flavonoids related to isoscutellarein, i.e., 5(11.1 mg), 6 (9.7 mg), and 7 (1.2 mg), as well as stachysoside A (3 ; 40.3 mg) were separated andidentified. Fr. B was partitioned on a SiO2 column using BuOH saturated with H2O as eluent. Thefractions positive to the vanillin/HCl reagent were collected (40 mg) and analyzed by 1H-NMR, resultingin a mixture composed by ca. 80% of chlorogenic acid. After evaporation of the deuterated solvent, thisfraction was suspended in H2O and treated with 10 mg of a mixture of charcoal/celite/polyamide(10 : 1 : 1) until no reaction to the vanilline/HCl reagent was observable. The resulting suspension wasfiltered on a glass-sintered funnel and washed first with H2O, then with a 70% MeOH soln. From theMeOH desorption, after evaporation of the solvent at reduced pressure, 5.9 mg of harpagide (1) wasrecovered.

Harpagide (1). 1H-NMR (300 MHz, D2O): 6.35 (d, J¼6.3, H�C(3)); 5.71 (br. s, H�C(1)); 5.02 (br. d,J¼6.3, H�C(4)); 4.71 (d, J¼7.8, H�C(1’)); 3.90 (d, J¼12.0, H�C(6)); 3.82–3.25 (overlapped glucosesignals); 2.53 (s, H�C(9)); 1.98 (dd, J¼14.5, 4.9, Ha�C(7)); 1.80 (dd, J¼14.0, 3.6, Hb�C(7)); 1.23 (s,Me(10)). 13C-NMR (75 MHz, D2O): 142.4 (C(3)); 108.1 (C(4)); 99.2 (C(1’)); 93.6 (C(1)); 83.8 (C(8));77.8 (C(6)); 77.5 (C(3’)); 77.0 (C(5’)); 74.4 (C(2’)); 71.2 (C(4’)); 62.3 (C(6)); 58.9 (C(9)); 46.9 (C(7)); 24.9(C(10)). ESI-MS: 387.14 ([MþNa]þ ).

Verbascoside (2). 1H-NMR (300 MHz, MeOD): 7.59 (d, J¼15.9, H�C(b’’); 7.05 (d, J¼1.8,H�C(2’’)); 6.96 (dd, J¼8.1, 1.8, H�C(2)); 6.77 (1 H, d, J¼8.0, H�C(5’’)); 6.69 (d, J¼2.0, H�C(2));6.67 (d, J¼8.0, H�C(5)); 6.56 (dd, J¼8.0, 2.0, H�C(6)); 6.27 (d, J¼15.9, H�C(a’’)); 5.18 (d, J¼1.3,H�C(1’’’)); 4.37 (d, J¼7.8, H�C(1’)); 2.79 (br. t, J¼7.4, CH2(b)); 1.09 (d, J¼6.1, Me�C(6’’’)). 13C-NMR

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(75 MHz, MeOD): 168.3 (COO); 149.8 (C(4’’)); 148.0 (C(b’’)); 146.8 (C(3’’)); 146.1 (C(3)); 144.6 (C(4));131.4 (C(1)); 127.6 (C(1’’)); 123.2 (C(6’’)); 121.2 (C(6)); 117.1 (C(5)); 116.5 (C(5’’)); 116.3 (C(2)); 115.2(C(2’’)); 114.7 (C(a’’)); 104.2 (C(1’)); 103.0 (C(1’’’)); 81.6 (C(3’)); 76.2 (C(2’)); 76.0 (C(5’)); 73.8 (C(4’’’));72.4 (C(2’’’)); 72.3 (C(3’’’)); 72.0 (C(a)); 70.6 (C(4’)); 70.4 (C(5’’’)); 62.3 (C(6’)); 36.6 (C(b)); 18.5(C(6’’’)). ESI-MS: 623.14 ([M�H]� ), 647.28 ([MþNa]þ ), 663.26 ([MþK]þ ).

Stachysoside A (¼2-(3,4-Dihydroxyphenyl)ethyl O-a-l-arabinopyranosyl-(1!2)-O-6-deoxy-b-l-mannopyranosyl-(1!3)-4-O-[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-b-d-glucopyrano-side ; 3). 1H NMR (300 MHz, D2O): 7.59 (d, J¼15.7, H�C(b’’)); 7.05 (br. s, H�C(2’’)); 6.96 (br. d, J¼8.5,H�C(6’’)); 6.77 (d, J¼8.0, H�C(5’’)); 6.69 (br. s, H�C(2)); 6.68 (d, J¼8.8, H�C(5)); 6.56 (br. d, J¼8.6,H�C(6)); 6.27 (d, J¼15.7, H�C(a’’)); 5.49 (br. s, H�C(1’’’)); 4.38 (d, J¼7.9,H�C(1’)); 4.30 (d, J¼7.1,H�C1’’’’)); 4.02–3.45 (overlapped sugar signals); 3.26 (t, part. overlapped with solvent signal, CH2(a));2.79 (t, J¼7.2, CH2(b)); 1.06 (d, J¼6.3, Me�C(6’’’)). 13C-NMR (75 MHz, D2O): 168.3 (COO); 147.7(C(3’’)); 147.6 (C(b’’)); 144.5 (C(4’’)); 144.0 (C(4)); 142.4 (C(3)); 131.4 (C(1)); 126.7 (C(1’’)); 123.1(C(6’’)); 121.3 (C(6)); 116.8 (C(5)); 116.3 (C(5’’)); 116.2 (C(2)); 115.3 (C(a’’)); 113.6 (C(2’’)); 105.7(C(1’’’’)); 102.3 (C(1’)); 100.7 (C(1’’’)); 81.3 (C(2’’’)); 80.9 (C(3’)); 74.2 (C(2’)); 74.0 (C(3’’’’)); 73.9 (C(5’));73.7 (C(4’’’)); 72.3 (part. overlapped C(2’’’’) and C(a)); 72.2 (C(3’’’)); 69.9 (C(4’)); 69.4 (C(4’’’’)); 68.5(C(5’’’); 66.4 (C(5’’’’)); 62.6 (C(6’)); )), 34.6 (C(b)), 17.3 (C(6’’’)). ESI-MS: 779.29 ([MþNa]þ ), 755.06([M�H]� ).

Chlorogenic Acid (4). 1H-NMR (400 MHz, D2O): 7.63 (d, J¼16.0, H�C(b)); 7.17 (d, J¼1.8,H�C(2’)); 7.11 (dd, J¼8.2, 1.8, H�C(6’)); 6.94 (d, J¼8.2, H�C(5’)); 6.38 (d, J¼15.9, H�C(a)); 5.38–5.30(m, H�C(3)); 4.18–4.14 (m, H�C(4)); 3.72 (dd, J¼8.5, 3.1, H�C(5)); 2.27–2.12 (m, 1 H each, Ha�C(2),Ha�C(6)); 2.05 (dd, J¼13.4, 11.4, 1 H each, Hb�C(2), Hb�C(6)). 13C-NMR (100 MHz, D2O): 180.0(COOH�C(1)); 168.9 (COO�C(a)); 147.1 (C(b)); 146.1 (C(3’)); 144.2 (C(4’)); 126.0 (C(1’)); 122.7(C(6’)); 116.0 (C(5’)); 115.0 (C(a)); 114.2 (C(2’)); 75.9 (C(1)); 74.4 (C(3)); 72.8 (C(4)); 71.7 (C(5)); 38.2(C(2)); 37.1 (C(6)). ESI-MS: 377.01 ([MþNa]þ ), 353.02 ([M�H]� ).

7-{[2-O-(6-O-Acetyl-b-d-allopyranosyl)-b-d-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-methoxyphen-yl)-4H-1-benzopyran-4-one (5). 1H-NMR (300 MHz, MeOD): 8.02 (d, J¼8.6, 1 H each, H�C(2’),H�C(6’)); 7.10 (d, J¼8.6, 1 H each, H�C(3’), H�C(5’)); 6.79 (s, H�C(3)); 6.68 (s, H�C(6)); 5.07 (d, J¼8.4, H�C(1’’’)); 4.96 (overlapped with solvent signal, H�C(1’’)); 4.04 (dd, J¼11.5, 5.3, CH2(6’’’)); 3.90–3.46 (overlapped sugar signals); 1.98 (s, MeCO). 13C-NMR (75 MHz, MeOD): 183.1 (C(4); 173.7(MeCO); 164.9 (C(2)); 163.2 (C(4’)); 152.7 (C(7)); 150.7 (C(5)); 143.0 (C(9)); 128.2 (C(2’),C(6’)); 126. 5(C(8)); 123.1 (C(1’)); 114.2 (C(3’),C(5’)); 102.9 (C(1’’’)); 101.5 (C(6)); 100.4 (C(1’’)); 82.6 (C(2’’)); 76.7(C(5’’)); 76.1 (C(3’’)); 72.0 (C(3’’’)); 71.7 (C(5’’’)); 70.9 (C(2’’’)); 69.8 (C(4’’)); 68.0 (C(4’’’)); 63.1 (C(6’’’));61.3 (C(6’’)); 54.8 (MeO); 19.4 (MeCO). ESI-MS: 667.24 ([MþH]þ ), 689.26 ([MþNa]þ ), 665.03 ([M�H]� ).

7-{[2-O-(6-O-Acetyl-b-d-allopyranosyl)-b-d-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-hydroxyphen-yl)-4H-1-benzopyran-4-one (6). 1H-NMR (400 MHz, MeOD): 7.86 (d, J¼8.9, 1 H each, H�C(2’),H�C(6’)); 6.91 (d, J¼8.9, 1 H each, H�C(3’), H�C(5’)); 6.74 (s, H�C(3)); 6.57 (s, H�C(6)); 5.06 (d, J¼7.9, H�C(1’’’)); 4.91 (d, J¼7.5, H�C(1’’)); 4.04 (dd, J¼10.9, 5.9, Ha�C(6’’’)); 3.96 (d, J¼12.0, Hb�C(6’’’));3.87–3.44 (overlapped sugars signals); 1.98 (s, MeCO). 13C-NMR (101 MHz, MeOD): 184.39 (C(4));173.00 (MeCO)); 166.5 (C(2)); 162.8 (C(4’)); 154.0 (C(7)); 151.8 (C(5)); 145.2 (C(9)); 129.8 (C(2’),C(6’)); 127.6 (C(8)); 123.1 (C(1’)); 116.9 (C(3’), C(5’)); 107.7 (C(10)); 104.3 (C(1’’’)); 103.7 (C(6)); 102.8(C(3)); 101.7 (C(1’’)); 84.0 (C(2’’)); 78.4 (C(5’’)); 77.5 (C(3’’)); 73.4 (C(2’’’)); 73.0 (C(5’’’)); 72.6 (C(3’’’));70.8 (C(4’’)); 68.4 (C(4’’’)); 65.0 (C(6’’’)); 62.3 (C(6’’)); 20.9 (MeCO)). ESI-MS: 675.19 ([MþNa]þ ),650.94 ([M�H]� ).

7-[(6-O-Acetyl-2-O-b-d-allopyranosyl-b-d-glucopyranosyl)oxy]-5,8-dihydroxy-2-(3-hydroxy-4-me-thoxyphenyl)-4H-1-benzopyran-4-one (7). 1H-NMR (400 MHz, MeOD): 7.60 (dd, J¼8.7, 2.2, H�C(6’));7.49 (d, J¼2.3, H�C(2’)); 7.11 (d, J¼8.7, H�C(5’)); 6.80 (s, H�C(3)); 6.65 (s, H�C(6)); 5.08 (d, J¼8.3,H�C(1’’’)); 4.95 (d, J¼7.4, H�C(1’’)); 4.33 (dd, J¼11.9, 2.5, Ha�C(6’’)); 4.24 (dd, J¼11.8, 5.2, Hb�C(6’’));4.12 (t, J¼3.0, H�C(5’’)); 3.96 (s, MeO); 3.82–3.40 (overlapped sugar signals); 2.00 (s, MeCO).13C-NMR (101 MHz, MeOD): 184.3 (C(4)); 173.3 (MeCO); 166.1 (C(2)); 154.13 (C(7)); 152.89 (C(4’));152.13 (C(5)); 148.27 (C(3’)); 143.8 (C(9)); 129.8 (C(8)); 125.0 (C(1’)); 120.5 (C(6’)); 114.1 (C(5’)); 112.7(C(2’)); 104.4 (C(6)); 104.2 (C(10)); 104.0 (C(1’’’)); 103.0 (C(3)); 101.8 (C(1’’)); 83.7 (C(2’’)); 73.4

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(C(5’’)); 73.1 (C(3’’)); 72.3 (C(2’’’)); 71.6 (C(5’’’)); 71.1 (C(3’’’)); 70.2 (C(4’’)); 68.5 (C(4’’’)); 65.0 (C(6’’));62.3 (C(6’’’)); 56.5 (MeO); 20.8 (MeCO). ESI-MS: 680.99 ([M�H]� ), 705.35 ([MþNa]þ ).

Micromorphological Analysis by Scanning Electron Microscopy. Stems, leaves, floral buds, andmature flowers were fixed for 7 d at 48 in 2.5% glutaraldehyde in 0.1m phosphate buffer pH 7.2.Following rising with the same buffer, the plant material was dehydrated in a graded acetone series,critical-point dried, and sputter coated with gold. The observations were carried out with a Philips XL 20scanning electron microscope at an accelerating voltage of 15 kV.

Histochemistry. The main classes of compounds present in the trichome secretions were investigatedin transverse, hand-cut sections of fresh plant material. The following chemicals were used to stain thesamples: Fluoral Yellow 088 for total lipids [56], Nile Red for neutral lipids [57], Nadi reagent forterpenes [58], Ruthenium Red for pectines [59], Alcian Blue for acid polysaccharides [60], FeCl3 forpolyphenols [61], and AlCl3 for flavonoids [62]. Simultaneous control procedures were carried out, andthe observations were made with a Leitz DM-RB Fluo optic microscope.

Cytotoxicity Assay. HCT116 cells (human colon carcinoma cells) were cultured in RPMI 1640medium with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with10% heat-inactivated fetal bovine serum (HI-FBS). MDA-MB 231 (human breast adenocarcinoma) andA375 (human malignant melanoma) cells were cultured in Dulbecco�s Modified Eagle�s Medium(DMEM) with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with10% HI-FBS. The cells were cultured in a humidified atmosphere at 378 in presence of 5% CO2. TheMTT assay was used as a relative measure of cell viability and was carried out as described by Quassintiet al. [63]. Briefly, the cells were seeded at a density of 2�104 cells/ml. After 24 h, the cells were exposedto different concentrations of essential oil (0.78–200 mg/ml) and incubated for 72 h in a humidifiedatmosphere at 378 in presence of 5% CO2. At the end of incubation, each well received 10 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; 5 mg/ml in phosphate-bufferedsaline), and the plates were incubated for 4 h at 378. The extent of MTT reduction was measuredspectrophotometrically at 540 nm using a Titertek Multiscan microElisa apparatus (Labsystems, FI-Helsinki). The experiments were conducted in triplicate, and the cytotoxicity was expressed as IC50 value,i.e., as the concentration of essential oil (mg/ml) inhibiting the cell growth by 50%. The IC50 values weredetermined with the GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA).

Antioxidant Activity. The DPPH .-scavenging activity of the S. tymphaea essential oil was evaluatedby using a microplate analytical assay according to the procedure described by Srinivasan et al. [64],while the ABTS .þ-scavenging capacity was determined with the method originally described by Re et al.[65] and adapted to the application in 96-well microplates by Esparza Rivera et al. [66]. Thedetermination of the ferric ion reducing antioxidant power (FRAP) was carried out according to M�lleret al. [67]. The ability of the essential oil to scavenge the different radicals was compared to that ofTrolox�, used as positive control, and the activity of the oil was expressed as tocopherol equivalent (TE)antioxidant capacity (mmol TE/g of sample).

Antimicrobial Activity. The essential oil was tested directly against the Gram-positive strain S. aureus(ATCC 25923, American Type Culture Collection, Rockville, MD, USA) and the Gram-negative strain E.coli (ATCC 25922). The growth conditions and the antimicrobial activity tests performed were aspreviously described by Nicoletti et al. [68]. Ciprofloxacin (5 mg/disc) was used as reference antibiotic(positive control).

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Received July 30, 2013

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