Fitoterapia 90 (2013) 94–103

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Phytochemical analysis, biological evaluation andmicromorphological study of Stachys alopecuros (L.) Benth.subsp. divulsa (Ten.) Grande endemic to central Apennines, Italy

Alessandro Venditti a,⁎, Armandodoriano Bianco a, Marcello Nicoletti b, Luana Quassinti c,Massimo Bramucci c, Giulio Lupidi c, Luca Agostino Vitali c, Dezemona Petrelli d, Fabrizio Papa c,Sauro Vittori c, Domenico Lucarini e, Laura Maleci Bini f, Claudia Giuliani f, Filippo Maggi c

a Department of Chemistry, La Sapienza University, Rome, Italyb Department of Environmental Biology, La Sapienza University, Rome, Italyc School of Pharmacy, University of Camerino, Camerino, Italyd School of Biosciences and Biotechnology, Italye Museum-Botanical Garden, University of Camerino, Camerino, Italyf Department of Biology, University of Florence, Florence, Italy

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +39 0649913229; faxE-mail address: alessandro.venditti@uniroma1.it (

0367-326X/$ – see front matter © 2013 Elsevier B.V.http://dx.doi.org/10.1016/j.fitote.2013.06.015

a b s t r a c t

Article history:Received 15 April 2013Accepted in revised form 14 June 2013Available online 1 July 2013

Stachys alopecuros subsp. divulsa (Lamiaceae), a perennial herb endemic to central Italy growingon mountain pastures, was investigated for the first time for the content of secondarymetabolites, for the micromorphology and histochemistry of glandular trichomes, and for thebiological activity of the volatile oil, namely cytotoxic, antioxidant and antimicrobial. Theplant showed the molecular pattern of iridoids, among which a new iridoid diglycoside(4′-O-β-D-galactopyranosyl-teuhircoside) was detected, together with a sterol glucoside and aphenylethanoid glycoside. The essential oil from the flowering aerial parts was characterizedby a high proportion of sesquiterpene hydrocarbons (65.1%), with (E)-caryophyllene (33.2%)as the most abundant, while other main components were germacrene D (7.6%), α-humulene(6.4%) and the oxygenated cis-sesquisabinene hydrate (10.2%). Taken together, polar andapolar chemical profiles support the classification of the species within the section Betonica ofthe genus Stachys. Micromorphological study revealed three types of glandular hairs secretingdifferent classes of compounds, with type A peltate hairs producing the bulk of the essentialoil. MTT assay revealed the potential of the volatile oil in inhibiting A375, HCT116 andMDA-MB 231 tumor cells (IC50 values below 20 μg/ml).

© 2013 Elsevier B.V. All rights reserved.

Keywords:Stachys alopecuros subsp. divulsaLamiaceaeIridoidsVerbascosideEssential oilBiological activity

1. Introduction

With about 450 taxa, the genus Stachys L. (subfamilyLamioideae), known as woundwort, is one of the largestgenera of the Lamiaceae family [1]. It comprises annual orperennial herbs or small shrubs. Species of the genus aredistributed in warm temperate regions of the Mediterra-nean and south-western Asia, with secondary centers in

: +39 0649913841.A. Venditti).

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North America [2]. Thirty-one species and subspecies havebeen reported for Italy, of which five taxa are consideredendemic [3].

Many Stachys species are known as “mountain tea” andused in the preparations of yogurt or jelly or traditionally asflavorings and seasonings [4]. Furthermore, plants of this genushave been used for centuries as herbal remedies in thetreatment of several complaints [5–7]. Teas prepared from thewhole plant or leaves are used in phytotherapy for sedative,antispasmodic, diuretic and emmenagogue properties [7–9]. Inrecent years, pharmacological studies on different taxa of this

95A. Venditti et al. / Fitoterapia 90 (2013) 94–103

genus demonstrated some effects of extracts or isolatedcomponents such as anti-inflammatory, antitoxic, antibacterial,antioxidant and cytotoxic [4]. Phytochemical studies on Stachysspecies reported the presence of phenylethanoid glycosides,iridoids, triterpenoids, steroids, diterpenes, flavonoids, fattyacids, polysaccharides and acidic metabolites [10–15].

In continuation of our study of secondary metabolitesproduced by Lamiaceae species growing in central Italy [16,17],in the present work we investigated Stachys alopecuros(L.) Benth. subsp. divulsa (Ten.) Grande belonging to thesubgenus Betonica (L.) Bhattacharjee (=Stachys sectionBetonica Bentham) and endemic to central Apennines(Monti Sibillini National Park, Italy), which has not beenstudied in much detail so far. No information is available onits traditional uses.

S. alopecuros subsp. divulsa is a hirsute, perennial herb upto 60 cm, with persistent terminal rosettes of triangular orovate-cordate, coarsely crenate or crenate–dentate leaves. In-florescences are pale-yellow verticillasters held in a dens spike.The species is endemic to central Italy (Marche, Umbria, Lazio,Abruzzo and Molise) [3] where it grows on mountain pastures,meadows, scrub and screes with preference with calcareoussoils. Compared with the other S. alopecuros subspecies, this isgenerally larger and presentsmore teeth (18–28) on each side ofthe basal leaf margin [18].

In the present paper, we report on the phytochemicalanalysis of constituents of medium polarity obtained from theethanol extracts of flowering aerial parts of S. alopecuros subsp.divulsa together with the volatile constituents contained in thehydrodistilled essential oil. Furthermore, we performed somein vitro assays to evaluate the cytotoxic, antioxidant andantimicrobial activity of the volatile oil, namely MTT assay onhuman tumor cell lines, DPPH and ABTS radical scavengingmethod, determination of ferric reducing antioxidant power(FRAP), and agar disk diffusion test. To improve the value of oursurvey, we carried out a detailed micromorphological studyof glandular and non-glandular trichomes present on thevegetative and reproductive organs, with special focus on thehistochemical localization of secretion.

2. Results and discussion

2.1. Essential oil analysis

Despite the high number of Stachys species, the essentialoil compositions of many of them remain to be investigated.The present work represents the first investigation of theessential oil compositions of S. alopecuros subsp. divulsa, ataxon representative of mountain environments.

A total of eighty-two compounds were identified in theessential oil of S. alopecuros subsp. divulsa, corresponding to96.4% of the total composition. The calculated oil yield (0.06%)was consistent with those reported in literature for otherspecies of the genus [19,20] and confirmed that the subfamilyLamioideae comprise oil-poor plants [21].

The essential oil was characterized by its high proportion ofsesquiterpene hydrocarbons (65.1%), with (E)-caryophyllene(33.2%) as the major compound followed by germacreneD (7.6%) and α-humulene (6.4%). The second most abundantfraction was constituted by oxygenated sesquiterpenes(24.5%), with cis-sesquisabinene hydrate (10.2%) as the most

predominant. Diterpenes were not detected in the oil. Apartfrom the major constituents, only four compounds exceed 2%of the total whereas the remaining (74 components) gave alower contribution. These molecules were 1-octen-3-ol (2.8%),β-curcumene (2.0%), caryophyllene oxide (2.8%) and epi-β-bisabolol (2.7%). The detected composition was little differentwith respect to that reported by Skaltsa [19] and collaboratorsfor S. alopecuros growing in Greece (subspecies not specified byauthors), where caryophyllene oxide (8.9%), (E)-caryophyllene(7.6%) andα-calacorene (13.4%) resulted themajor constituents.Notably, 54 out of 82 componentswere in common between thetwo studies, whilst 28 volatiles were detected only in thesubspecies analyzed by us. Among them, cis-sesquisabinenehydrate (10.2%), α-acorenol (2.6%) and epi-β-bisabolol (2.7%)were the major. Anyway, the abundance of sesquiterpenehydrocarbons and oxygenated sesquiterpenes detected by uswas in accordance with that reported for the species belongingto the subgenus Betonica [7,19,20]. On the other hand, labdanediterpenes previously reported for S. alopecuros [19] have notbeen detected in the subsp. divulsa. The highlighted dif-ferences may be attributed to the different subspeciesanalyzed (divulsa in the present study vs. probably alopecurosin the study from Greece).

Although themain volatile constituents vary among differentspecies included in the section Betonica, for instance Stachysofficinalis and Stachys scardica [19,22,23], the abundance ofsesquiterpene hydrocarbons and oxygenated sesquiterpenesdetected by us seems to be a chemotaxonomic feature of thesection [7,19,20].

2.2. Analysis of the fraction of medium polarity

The investigation of the S. alopecuros led to the isolation ofseveral classes of secondary metabolites (Fig. 1). A sterolglucoside, 3-O-β-sitosterol-glucoside, 1 [24], widely distribut-ed in the plant kingdom, together with verbascoside, 4 [25], aphenylethanoid glycoside almost ubiquitous inmany genera ofAsteridae. Furthermore, the iridoids acetyl-harpagide, 3 andharpagide, 2 [26] were identified. From the chemotaxonomicpoint of view, the last isolation is perfectly on line with thosereported in previous studies on this genus [27–29]. Also theco-occurrence of iridoids and phenylethanoids in this genuswas previously reported [30]. In addition to these commoncompoundswe have also isolated a new iridoid diglycoside (5),related to teuhircoside (6) and allobetonicoside (7), whichwere isolated from Lamiaceae family. In particular (7) was alsoreported in other species related to Betonica section, as Stachysmacrantha [31], Stachys glutinosa [28] and Betonica officinalis[32]. On the other hand, teuhircoside (6) was found only innear genera as Teucrium and Ajuga [33,34] but never reportedfrom Stachys spp.

The structure of the new iridoid was elucidated by meansof spectroscopic methods, namely 1D and 2D-NMR experi-ments and comparing the obtained data with those reportedin literature for the known similar compounds. In fact thespectral data were much similar to those reported for 7, withthe difference that no anomeric signal, attributable to anallose moiety, was observed. In fact we observed in 1H NMRspectrum of 5 an anomeric proton signal at 4.91 ppm, with acoupling constant of 8.0 Hz, and in 13C NMR spectrum thecorresponding anomeric carbon signal at 98.3 ppm. On the

OO

OH

OH

OH

OH

O

OH OH

HOGlc

OR

O

O

O

OH

OH

O

O

OH

O

OH

OH

OH

OH

OH

OH

O

OOH

OH

O

OOH

OH

OHOH

OH

1

O

3

4

5

9

8

7

6

O

10

OH

H

β-sitosterol-3-O-β-D-glucoside, 1

R=H, harpagide, 2R=Ac, acetyl harpagide, 3

verbascoside, 4

4'-O-β-D-galactopyranosyl-teuhircoside, 5

1'

1''

4'

Fig. 1. Structures of isolated compounds.

96 A. Venditti et al. / Fitoterapia 90 (2013) 94–103

contrary the allose anomeric proton gives a signal at 5.23 ppmwith a coupling constant of 8.0 Hz and its anomeric carbon at~102 ppm.

Some heteronuclear correlations in HMBC experimentwere particularly diagnostic (Fig. 3) for the assignment ofthe structure. No correlation between H1″ and C5 wasobserved and this is an evidence of the absence of anylinkage of the second glycosidic moiety to C5 carbon ofiridoidic skeleton, as in the case of 7. On the other hand onthe spectra we observed correlation between the anomericproton at 4.91 and the carbon at 77.1 ppm which could beassigned to the C4′ of glucose moiety where the secondglycosidic unit should be linked. Also the reverse correla-tion between H4′ at 3.94 (assigned by HSQC experiment)and C1″ at 98.3 ppm was recognized. The HMBC experimentswere performed using different values of heteronuclearcoupling constants, to explore different coupling systemsand no correlation between C5 and H1″ was neverobserved.

The second glycosidic unit appeared to be a galactoseunit, because the observed chemical shifts for glycosecarbons (in particular the signals between 72 and 69 ppm)are a characteristic of galactose [35]. The identity of galactosefor the second sugar unit is also supported by the value ofchemical shift of the anomeric proton (4.91 ppm in 1H NMRspectrum); the value of 8.0 Hz for the coupling constant

between H1 and H2 indicates a β configuration for theinter-glycosidic linkage.

Therefore the structure of 4-O-β-D-galactopyranosyl-β-D-glucopyranose appeared to be demonstrated for thedisaccharidic unit linked to C1 of iridoid moiety of 5. All ofthese evidences are therefore in agreement with the structureof 4′-β-D-galactopyranosyl-teuhircoside, for the new com-pound 5.

The presence of galactose derivatives is not unexpect-ed, in fact galactosyl glycosides were yet isolated fromStachys genus: linked to a flavonoid [36]; as a constituentof stachyose, an oligosaccharide [37]; and as methylα-D-galactopyranoside, an artifact arising from extractionprocedure [38].

From a phytochemical point of view the presence of thisteuhircoside derivative, since related substances were notpreviously recognized from this genus, seems to have achemotaxonomic relevance in S. alopecuros subsp. divulsa.

2.3. Morphology, distribution and histochemistry of trichomes

Glandular and non-glandular trichomes have alreadybeen described in several Stachys species [39,40]. A moredetailed study of the vegetative and reproductive organs ofS. alopecuros subsp. divulsa allowed observation of the followingtypes of trichomes (Fig. 2).

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2.3.1. Type APeltate hairs are composed of one basal cell, one very

short stalk cell and a round head (60–70 μm in diameter) ofeight secretory cells arranged in a single disk and surroundedby a large subcuticular space (Fig. 2a). They are tightly closeon the interveinal regions of the leaf abaxial side (Fig. 2e) andscattered all over the abaxial surfaces of calyx and corolla(Fig. 2e,g and i).

Peltate trichomes, generally regarded as typical essentialoil producers [41], present an abundant secretion that stainspositive both with all lipophilic dyes (Fig. 2j–k) and withRuthenium Red (Fig. 2l) and AlCl3 (Fig. 2m), indicating thepresence of terpenoids, non-cellulosic polysaccharides andpolyphenols. Such a complex composition of the secretionwas already reported in other members of the Betonicagroup [39].

2.3.2. Type BShort-stalked capitate trichomes, widespread in the whole

Lamiaceae family, are composed of one basal cell, one neck celland a head of four secretory cells (20–30 μm in diameter) witha thin subcuticular chamber (Fig. 2a). They occur along theveins of both leaf surfaces (Fig. 2f) and all over sepals and petals(Fig. 2g and i).

The exudates of type B trichomes stain positive with bothhydrophilic and lipophilic dyes (Fig. 2n–p), indicating theproduction of different classes of compounds, whereas theygenerally exhibit a main or exclusive polysaccharidic secretion[42].

2.3.3. Type C1Long-stalked capitate trichomes are composed of one

basal cell, a stalk of variable length (35–60 μm) and a uni- orbicellular ovoid head (25 μm in diameter) developing aspherical subcuticular space where the secretion accumulatestemporarily (Fig. 2b). Type C1 hairs occur only on leaves andare distributed in irregular rows at the edge of the lamina(Fig. 2d); their exudate is a typical terpenoidic secretion, wellstained with all the lipophilic stains (Fig. 2q–s).

These trichomes were already described in several Stachysspecies lacking peltate trichomes as primary sites of essentialoil biosynthesis [39], however they are here reported for thefirst time in a member of the subgenus Betonica.

2.3.4. Type DNon-glandular trichomes, present on the whole plant and

varying in length between 120 and 350 μm, are multicellularand uniseriate, pointed, erect or leaning towards the organapex (Fig. 2d–i). They consist of three to seven cells, and aresupported by a cellular pedestal formed by a group of six to tenepidermal cells arranged in a circle around the base (Fig. 2c).

S. alopecuros subsp. divulsa exhibits three types ofglandular hairs producing different classes of compounds,however they all are able to synthesize significant amounts ofterpenoids. Anyway based on the distribution pattern oftrichomes and on their different density on the whole plant,type A peltate glands do seem to produce the bulk of theessential oil.

The occurrence of both types A and C1 trichomes in thesame taxon, i.e. S. alopecuros subsp. divulsa, has never recorded

previously; accordingly, this report provides new lights on thecontroversial Betonica group.

2.4. Cytotoxic activity

The essential oil of S. alopecuros subsp. divulsawas testedin vitro for the potential tumor cell growth-inhibitory effecton MDA-MB 231 human breast adenocarcinoma cell line,A375 human malignant melanoma cell line, and HCT116human colon carcinoma cell line, using MTT assay. Theresults, collected in Table 2, show that essential oil exhibiteda strong inhibitory effect against the human cancer cellsexamined. The highest activity was observed on A375 cellline, with IC50 value of 10.60 μg/ml. Lower activity was ob-tained on HCT116, with IC50 value of 19.03 μg/ml. Essential oilsfrom different Stachys species were found to be cytotoxicagainst C32 amelanotic melanoma and ACHN adenocarcinomacell lines [4]. In the present study we also showed that theessential oil from S. alopecuros subsp. divulsa resulted active onbreast adenocarcinoma cell line with IC50 value of 17.67 μg/ml.The (E)-caryophyllene antiproliferative activity was testedon our tumor cell lines. IC50 values were 43.27 μg/ml(65.47 μM) for MDA-MB 231, 58.57 μg/ml (286.61 μM) forHCT116, and 67.14 μg/ml (328.55 μM) for A375. α-Humulenealso exerts cytotoxic activity against several solid tumor cell linesas MCF-7, PC-3, A-549, DLD-1, M4BEU, and CT-26 [43].Germacrene Dwas reported active against human breast adeno-carcinoma (MDA-MB 231 andMCF-7), human ductal carcinoma(Hs 578T), and human hepatocellular carcinoma (Hep G2)[44]. However, the concentrations of (E)-caryophyllene(33.2%), germacrene D (7.6%), and α-humulene (6.4%) cannotfully explain the cytotoxic activity of S. alopecuros essential oil,whichmeans that some other compounds, probably sesquiter-penes, contributed to the activity of the essential oil [45].

In theUSNCI plant screening program, a crude plant extractis generally considered promising if the IC50 value followingincubation between 48 and 72 h, is less than 20 μg/ml [46]. Thesignificant inhibition values detected make the essential oil ofS. alopecuros subsp. divulsaworthy of further investigation as apromising chemopreventive agent.

2.5. Antioxidant activity

The antioxidant capacity of the essential oil obtainedfrom S. alopecuros subsp. alopecuros was evaluated and theresults are summarized in Table 3 compared with Troloxused as control. The activity is indicated by the antioxidantindex (IC50) determined for each of the two differentmethods used: DPPH, ABTS. From our data the essential oilexhibited significant antioxidant activities with IC50 valuesof about 65 and 50 folds lower than that shown by Troloxagainst DPPH and ABTS radicals, respectively. The reducingcapacity power (FRAP assay) may serve also as a significantindicator of potential antioxidant activity. The FRAP valuereported for the essential oil of S. alopecuros subsp. divulsa(Table 3) reveals that the oil has a significant capacityfor iron binding that is related to its possible action asperoxidation protector. Among the identified compoundsin the essential oil the antioxidant effectiveness, observed,probably, can be in part, attributed to the high content ofsesquiterpene hydrocarbons (Table 1). In particular it has

98 A. Venditti et al. / Fitoterapia 90 (2013) 94–103

been reported [47] that (E)-caryophyllene, a sesquiterpenecommon constituent of the essential oil of numerous plantvegetables, fruits, medicinal herbs and foods (main constituentof S. alopecuros subsp. divulsa essential oil, for the 33.2%, isa natural antioxidant [47]) that showed higher inhibitorycapacity on lipid peroxidation than probucol, α-humulene andα-tocopherol and has also high scavenging activities againsthydroxyl radical and superoxide anion [47]. Probably also otherphenolic and alcoholic components (which constituted 2% ofthe total oil) can exert either the synergistic or additive actionstowards the total antioxidant activity but to study thescavenging mechanism of these major compounds alone or insynergy with other compounds found in the essential oilfurther investigations are needed.

2.6. Antimicrobial activity

The essential oil from the aerial parts of S. alopecuros subsp.divulsa did not show any valuable activity against the twobacterial species tested and in the experimental conditionsused (inhibition zonediameter = 6–7 mm). The activity of thepure major compound (E)-caryophyllene gave an inhibitionzone diameter of 8 mm, while the control antibiotic ciproflox-acin showed a diameter of N20 mm. Therefore, the activity ofthe essential oil was not further investigated nor by othermethods neither against other microbial species.

3. Materials and methods

3.1. Plant material

Flowering aerial parts of S. alopecuros subsp. divulsa werecollected at the end of June 2012 from a natural populationgrowing on stony slopes of Pizzo Tre Vescovi (Bolognola,Marche, Italy) at an altitude of 1520 m (GPS coordinates: N42°58′07″, E 13°14′10″). The voucher specimen was identi-fied by D. Lucarini using available literature [18], anddeposited in the Herbarium Camerinensis (included in theonline edition of Index Herbariorum: http://sweetgum.nybg.org/ih/) [49] of School of Environmental Sciences(University of Camerino, Italy) under the accession CAME24825; it is also available at the following website: htpp://erbariitaliani.unipg.it.

3.2. Essential oil isolation

Air-dried aerial parts of S. alopecuros subsp. divulsa (170 g)were cut into small pieces and submitted to hydro-distillationfor 3 h in a Clevenger type apparatus using n-hexane (3 ml) ascollector solvent. The yellow-colored essential oils were driedover anhydrous sodium sulfate, then solvent was evaporatedunder a N2 flow; they were stored in sealed vials protectedfrom the light at −20 °C before GC-FID and GC–MS analyses.The oil yield (0.06%) was estimated on a dry weight basis.

3.3. Chemicals

α-Pinene, β-pinene, 1-octen-3-ol, myrcene, limonene,linalool, α-terpineol, (E)-caryophyllene, (Z)-β-farnesene,α-humulene, (E)-β-farnesene, (E)-β-ionone, β-bisabolene,(E)-nerolidol, caryophyllene oxide,n-heptadecane, hexadecanoic

acid, n-heneicosane, linoleic acid, n-docosane, n-tricosane,n-tetracosane, n-pentacosane, n-heptacosane, n-nonacosane,and n-untriacontane were purchased from Sigma–Aldrich(Milan, Italy). For the retention index determination, a mix ofhydrocarbons ranging from n-octane (C8) to n-dotriacontane(C30) (Supelco, USA) was used and run at the experimentalconditions reported below. All compounds were of analyticalstandard grade. Analytical grade n-hexane solvent was pur-chased from Carlo Erba (Milan, Italy); it was successivelydistilled by a Vigreux column before use.

3.4. GC-FID and GC–MS analyses

For gas chromatographic separations, an Agilent 4890Dinstrument coupled to an ionization flame detector (FID) wasused. Volatile components were separated on a HP-5 capillarycolumn (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.;0.17 μm film thickness) (J and W Scientific, Folsom, CA), withthe following temperature program: 5 min at 60 °C, subse-quently 4 °C/min up to 220 °C, then 11 °C/min up to 280 °C,held for 15 min, for a total run of 65 min. Injector and transferline temperatures were 280 °C. Heliumwas used as the carriergas, at a flow rate of 1.4 ml/min; injection volume: 1 μl; splitratio, 1:34. A mixture of aliphatic hydrocarbons (C8–C30)(Sigma, Milan, Italy) in n-hexane, was directly injected intothe GC injector under the above temperature program, in orderto calculate the retention index (as Kovats index) of eachcompound. Oil samples were diluted 1:100 in n-hexane andinjected at a volume of 1 μl. Analysis was repeated three times.Data were collected by using HP3398A GC Chemstationsoftware (Hewlett Packard, Rev. A.01.01). The relative amountsof essential oil components, expressed as percentages, wereobtained by FID peak-area normalization by calculating theresponse factor (RF) of the FID for eight different classes ofvolatiles (Table 1). Standard compounds, each representing thechemical classes determined, were selected among thoseavailable in the authors' laboratory i.e. β-pinene, limoneneand γ-terpinene formonoterpene hydrocarbons; p-cymene foraromatic monoterpenes; 1,8-cineole and linalool for oxygen-ated monoterpenes; (E)-caryophyllene and α-humulene forsesquiterpene hydrocarbons; caryophyllene oxide for oxygen-ated sesquiterpenes; n-octanal for aldehydes; n-octane andn-octadecane for alkanes; benzaldehyde and p-vinylanisole foraromatics. Each standard, diluted in n-hexane, was injected atfour different concentrations (0.04, 0.08, 0.16 and 0.40 mg/ml)using n-octane and n-octadecane as internal standards at aconcentration of 0.04 mg/ml. For each standard a regressionline was obtained by plotting the ratio of internal standard(mean value of n-octane and n-octadecane) to referencestandard peak area versus the ratio of internal standard toreference standard concentration. Each dilution was analyzedfor five times. The correlation coefficients (R2) obtained forcalibration curves of representative standards were all higherthan 0.992. The correction factor of each reference standardcompound was calculated as the slope of the linear regressionequation. Wherever more than one reference analyte for eachclass were considered, the correction factor was calculated bythe mean of the slopes of all representative compounds. Usingthe generalized response factor for compounds within theeight classes, the derived quantitative data may be consideredas an approximation of the absolute quantification.

Table 1Composition of the essential oil from the aerial parts of S. alopecuros subsp. divulsa.

N. Componenta RFb RIc RI lit.d %e IDf

ADAMS NIST08

1 (2E)-Hexenal 1.7 854 855 Trg RI,MS2 α-Thujene 1.1 926 930 926 Tr RI,MS3 α-Pinene 1.1 932 939 932 0.7 Std4 Sabinene 1.1 971 975 971 Tr RI,MS5 β-Pinene 1.1 974 979 974 Tr Std6 1-Octen-3-ol 1.5 979 979 979 2.8 Std7 Myrcene 1.1 991 990 991 0.2 Std8 3-Octanol 1.5 998 991 999 Tr RI,MS9 Limonene 1.1 1030 1029 1028 Tr Std10 (Z)-β-ocimene 1.1 1043 1037 1042 0.1 RI,MS11 Benzene acetaldehyde 1.7 1050 1042 1050 Tr RI,MS12 (E)-β-ocimene 1.1 1053 1050 1052 Tr RI,MS13 Linalool 1.5 1100 1096 1100 0.7 Std14 n-Nonanal 1.7 1106 1100 1106 0.1 RI,MS15 Benzofuran, 2-methyl- 1.7 1169 1168 0.1 RI,MS16 α-Terpineol 1.5 1189 1188 1189 0.1 Std17 Methyl salicylate 1.7 1192 1191 1192 Tr RI,MS18 α-Cubebene 1.1 1346 1348 1346 0.1 RI,MS19 α-Copaene 1.1 1370 1376 1370 0.1 RI,MS20 epi-α-Funebrene 1.1 1373 1382 Tr RI,MS21 β-Bourbonene 1.1 1378 1388 1378 1.5 RI,MS22 (E)-β-damacenone 1.0 1382 1384 1383 Tr RI,MS23 β-Cubebene 1.1 1385 1388 1385 0.1 RI,MS24 β-elemene 1.1 1387 1390 1387 0.3 RI,MS25 7-epi-sesquithujene 1.1 1387 1391 0.3 RI,MS26 α-Cedrene 1.1 1400 1411 1400 0.1 RI,MS27 Sesquithujene 1.1 1404 1405 0.6 RI,MS28 (E)-caryophyllene 1.1 1412 1418 1412 33.2 Std29 β-Copaene 1.1 1431 1432 0.3 RI,MS30 α-Trans-bergamotene 1.1 1432 1434 1433 0.6 RI,MS31 Aromadendrene 1.1 1436 1441 1437 Tr RI,MS32 (Z)-β-farnesene 1.1 1440 1442 1.4 Std33 Myltayl-4(12)-ene 1.1 1445 1447 0.5 RI,MS34 α-Humulene 1.1 1446 1454 1446 6.4 Std35 (E)-β-farnesene 1.1 1459 1456 1459 2.0 Std36 α-Acoradiene 1.1 1467 1466 1464 Tr RI,MS37 Germacrene D 1.1 1474 1485 1474 7.6 RI,MS38 γ-Curcumene 1.1 1477 1482 2.0 RI,MS39 γ-Selinene 1.1 1477 1477 1.8 RI,MS40 ar-curcumene 1.1 1480 1480 1480 0.5 RI,MS41 (E)-β-Ionone 1.0 1483 1488 1483 0.1 Std42 δ-Selinene 1.1 1483 1492 1484 0.1 RI,MS43 Bicyclogermacrene 1.1 1488 1500 1488 0.6 RI,MS44 α-Zingiberene 1.1 1493 1493 1493 0.2 RI,MS45 α-Muurolene 1.1 1494 1500 1494 0.1 RI,MS46 γ-Cadinene 1.1 1508 1513 1508 0.2 RI,MS47 β-Bisabolene 1.1 1505 1505 1505 0.2 Std48 β-Curcumene 1.1 1509 1515 3.1 RI,MS49 δ-Amorphene 1.1 1510 1512 Tr RI,MS50 (Z)-γ-bisabolene 1.1 1511 1515 0.2 RI,MS51 δ-Cadinene 1.1 1518 1523 1518 0.8 RI,MS52 α-Alaskene 1.1 1520 1521 0.2 RI,MS53 (E)-γ-bisabolene 1.1 1529 1531 0.1 RI,MS54 cis-sesquisabinene hydrate 1.3 1542 1544 10.2 RI,MS55 (E)-nerolidol 1.3 1564 1563 1564 0.2 Std56 Germacrene D-4-ol 1.3 1569 1575 1569 0.2 RI,MS57 Spathulenol 1.3 1569 1578 1570 0.2 RI,MS58 Caryophyllene oxide 1.3 1573 1583 1573 2.8 Std59 trans-sesquisabinene hydrate 1.3 1576 1579 1.2 RI,MS60 Salvial-4(14)-en-1-one 1.3 1594 1594 Tr RI,MS61 Cedrol 1.3 1599 1600 1599 0.4 RI,MS62 Humulene epoxide II 1.3 1608 1608 0.5 RI,MS63 α-Acorenol 1.3 1630 1632 2.6 RI,MS64 epi-α-muurolol 1.3 1635 1642 0.5 RI,MS65 β-Acorenol 1.3 1638 1637 0.9 RI,MS66 epi-β-bisabolol 1.3 1667 1671 2.7 RI,MS

(continued on next page)

99A. Venditti et al. / Fitoterapia 90 (2013) 94–103

Table 1 (continued)

N. Componenta RFb RIc RI lit.d %e IDf

ADAMS NIST08

67 β-Bisabolol 1.3 1667 1675 1668 1.3 RI,MS68 epi-α-bisabolol 1.3 1679 1684 0.2 RI,MS69 α-Bisabolol 1.3 1682 1685 1682 0.3 RI,MS70 (Z)-α-trans-bergamotol 1.3 1690 1690 0.1 RI,MS71 n-Heptadecane 1.2 1700 1700 1700 Tr Std72 2-Pentadecanone, 6,10,14-trimethyl- 1.7 1846 1846 0.5 RI,MS73 Hexadecanoic acid 1.0 1972 1960 1972 Tr Std74 n-Heneicosane 1.2 2101 2100 2100 0.1 Std75 Linoleic acid 1.0 2143 2133 2144 Tr Std76 n-Docosane 1.2 2201 2200 2200 0.1 Std77 n-Tricosane 1.2 2301 2300 2300 0.5 Std78 n-Tetracosane 1.2 2400 2400 2400 Tr Std79 n-Pentacosane 1.2 2501 2500 2500 0.3 Std80 n-Heptacosane 1.2 2702 2700 2700 0.2 Std81 n-Nonacosane 1.2 2902 2900 2900 0.3 Std82 n-Untriacontane 1.2 3102 3100 3100 0.1 Std

Oil yield (%) 0.06Total identified (%) 96.4Grouped compounds (%)

Monoterpene hydrocarbons 1.0Oxygenated monoterpenes 0.8Sesquiterpene hydrocarbons 65.1Oxygenated sesquiterpenes 24.5Diterpenes 0.0Alcohols 2.8Alkanes 1.6Others 0.8

a Compounds are listed in order of their elution from a HP-5MS column. Their nomenclature was in accordance with Adams (2007).b Relative response factor (RF) of FID detector for the main chemical groups occurring in EO.c Linear retention index on HP-5MS column. experimentally determined using homologous series of C8–C32 alkanes.d Linear relative retention index taken from Adams (2007) and/or NIST08 (2008).e Percentage values are means of three determinations, with a RSD% for the main components below 5% in all cases.f Identification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, ADAMS and NIST08 MS database; RI,

based on comparison of RI with those reported in ADAMS and NIST08.g Tr, traces (mean value below 0.1%).

100 A. Venditti et al. / Fitoterapia 90 (2013) 94–103

GC–MS analysis was performed on an Agilent 6890N gaschromatograph coupled to a 5973Nmass spectrometer using aHP-5MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d.,0.1 μm film thickness) (J&W Scientific, Folsom) capillarycolumn. The temperature program was the same as above.Injector and transfer line temperatures were 280 °C. Heliumwas used as the carrier gas, at a flow rate of 1 ml/min. Split

Table 2In vitro cytotoxic activity of the essential oil from Stachys alopecuros subspdivulsa.

Cell line (IC50 μg/ml)

MDA-MB 231a HCT116b A375c

S. alopecuros subsp.divulsa oil

17.67 19.03 10.60

95% C.I. 14.35–21.86 15.96–22.85 8.14–13.81(E)-caryophyllene 43.27 58.57 67.1495% C.I. 36.29–51.60 52.23–65.68 56.12–80.33Cisplatin 2.96 2.45 0.4295% C.I. 2.23–3.15 2.05–2.89 0.29–0.49

IC50 = The concentration of compound that affords a 50% reduction in celgrowth (after 72 h of incubation). C.I. = confidence interval.

a Human breast adenocarcinoma cell line.b Human colon carcinoma cell line.c Human malignant melanoma cell line.

.

l

ratio: 1:50; acquisition mass range: 29–400 m/z. All massspectra were acquired in electron-impact (EI) mode with anionization voltage of 70 eV. Oil samples were diluted 1:100 inn-hexane and the volume injected was 2 μl. Analysis wasrepeated three times. Data were analyzed by using MSDChemStation software (Agilent, Version G1701DA D.01.00).Whenever possible volatile components were identified byco-injection with authentic standards. Otherwise, the peakassignment was carried out on the basis of the standard of theInternational Organization of the Flavor Industry (IOFI, http://www.iofi.org/) statement, i.e., by the interactive combinationof chromatographic linear retention indices thatwere consistentwith those reported in literature [50,51] for apolar stationaryphases, and MS data consisting in the computer matchingwith the WILEY275, NIST 08 ADAMS, and a home-made(based on the analyses of reference oils and commerciallyavailable standards) libraries.

3.5. Analysis of polar compounds

3.5.1. PurificationThe dried plant materials (113.0 g) were extracted

three times with hydro alcoholic solutions (the first 80%ethanol/20% water, to rehydrate the plant materials, then95% ethanol/5% water (500 ml each)). The obtained

Table 3In vitro radical scavenging activities of the essential oil from S. alopecuros subsp. divulsa.

DPPH ABTS FRAP

TEACμmol TE/g

IC50

μg/mlTEACμmol TE/g

IC50

μg/mlTEACμmol TE/g

S. alopecuros subsp. divulsa oil 61.11 (±1.7) 700 (±20) 78.43 (±2.1) 183 (±4.2) 66.90 (±0.8)Trolox – 10.7 (±0.2) – 3.59 (±0.2) –

IC50 = The concentration of compound that affords a 50% reduction in the assay.TEAC = Trolox equivalent (TE) antioxidant concentration.

101A. Venditti et al. / Fitoterapia 90 (2013) 94–103

extracts were concentrated under reduced pressure toeliminate the ethanol and the resulting water suspensionwas first freeze to −20 °C and then lyophilized to recover28.1 g of crude extract. A portion of 4.34 g was partitionedon silica gel column using chloroform/methanol 8:2 andrising the polarity during the chromatographic run. Inorder of elution were directly collected the fractions: F21–26where the presence of a triterpene glycoside was evidencedby MR spectroscopy; F53–73 containing acetyl harpagide(69.9 mg); F76–81 containing harpagide (145.8 mg) [26];F86–101, containing a mixture of iridoids with positivereaction to vanillin/HCl reagent. The fraction F21–26 wasfurther purified on silica gel column, eluting with chloroform/methanol 9:1, obtaining β-sitosterol-3-O-glucoside (18.0 mg)[24]. 1H NMR analysis on F86-101 revealed the presenceof harpagide and additional peaks attributable to iridoidiccompounds together with signals of a phenylethanoidcompound. To separate these components the fraction F86-101 was re-chromatographed on silica gel using butanol/methanol/water mixture (70:10:30). From this separation itwas possible to recognize verbascoside [25] (9.4 mg),harpagide(19.6 mg) and the new iridoid diglycoside inmixture with sugars (19.9 mg). This last fraction waspurified by absorption on a mixture of charcoal/polyamide10:1 until no reaction to vanilline/HCl reagent (3 g. ofvanilline dissolved in 100 ml of methanol and 3 ml of HClconc.). The obtained suspension was stratified on a sinteredglass funnel and washed with water to eliminate the freecarbohydrates. Then the iridoid was desorbed from charcoalby repeated washing with methanol. After evaporation of thesolvent was recovered 8.1 mg of the compound 5 wasrecovered and identified as 4′-galactopyranosyl-teuhircoside.

3.5.2. Spectroscopic identificationNMR spectra were recorded on Varian Mercury 300 MHz

and/or on Bruker Avance 400 MHz instruments using CDCl3,CD3OD or D2O as deuterated solvents, the chemical shift wasexpressed in ppm from TMS (the signal of HDO at 4.78 ppmis used as reference for spectra in D2O).

MS spectrawere performedonaQ-TOFMICRO spectrometer(Micromass, now Waters, Manchester, UK) equipped with anESI source, that was operated in the negative and/orpositive ion mode. The flow rate of sample infusion was10 μl/min. with 100 acquisitions per spectrum. Data wereanalyzed using the MassLynx software developed byWaters.

4′-O-β-D-galactopyranosyl-teuhircoside (5): 1H NMR,CD3OD, 400 MHz, δ: 6.51 (1H, d J = 6.4 Hz, H3), 6.17 (1H,bs, H7), 5.90 (1H, d J = 1.2 Hz,H1), 4.98 (1H, dd J = 6.4; 1.1 Hz,H4), 4.91 (1H, d J = 8.0 Hz, H1″), 4.65 (1H, d J = 7.9 Hz, H1′),

2.33 (3H, s, H10), other signal attributable to carbohydratesmoieties between 3.45 and 4.13. 13C NMR, CD3OD, 100 MHz, δ:205.1 (C6), 178.6 (C8), 144.2 (C3), 127.8 (C-7), 102.7 (C4), 98.7(C1′), 98.3 (C1″), 92.1 (C1), 77.4 (C5), 77.1 (C4′), 76.8 (C3′), 76.4(C5′), 73.9 (C2′), 71.9 (C5″), 71.6 (C3″), 71.3 (C4″), 69.4 (C2″),62.9 (C6′), 62.7 (C6″), 54.5 (C9), 17.7 (C10). ESI-MS:m/z 529.08[M + Na]+.

3.6. Micromorphological analysis

3.6.1. Scanning electron microscopy (SEM)Samples of leaves and flowers at different developmental

stages were fixed in 2.5% glutaraldehyde in 0.1 M phosphatebuffer, pH 7.2, for 7 days at 4 °C. Following rising in the samebuffer, the material was dehydrated in a graded acetoneseries, critical point dried and sputter coated with gold.Observations were carried out on a Philips XL 20 SEMscanning electron microscope at an accelerating voltage of15 kV.

3.6.2. HistochemistryThe main classes of compounds present in the trichome

secretions were investigated in transverse hand-cut sectionsof fresh material. Fluoral Yellow 088 for total lipids [52], NileRed for neutral lipids [53], Nadi reagent for terpenes [54],Ruthenium Red for pectines [55], Alcian Blue for acid poly-saccharides [56], Ferric Trichloride for polyphenols [57], andAluminium Trichloride for flavonoids [58]. Control procedureswere carried out at the same time. Observations were madewith a Leitz DM-RB Fluo optic microscope.

3.7. Cytotoxicity assay

HCT116 cells (human colon carcinoma cells) were culturedin RPMI1640 medium with 2 mM L-glutamine, 100 IU/mlpenicillin, 100 μg/ml streptomycin, and supplemented with10% heat-inactivated fetal bovine serum (HI-FBS) (PAALaboratories GmbH, Austria). MDA-MB 231 (human breastadenocarcinoma cells) and A375 cells (human malignantmelanoma cells) were cultured in Dulbecco's Modified Eagle'sMedium (DMEM)with 2 mM L-glutamine, 100 IU/ml penicillin,100 μg/ml streptomycin, and supplemented with 10% HI-FBS.Cells were cultured in a humidified atmosphere at 37 °C in thepresence of 5% CO2. The MTT assay was used as a relativemeasure of cell viability. Cell-viability assays were carried out asdescribed [59]. Briefly, cells were seeded at a density of2 × 104 cells/ml. After 24 h, samples were exposed to differentconcentrations of essential oil (0.78–200 μg/ml). Cells wereincubated for 72 h in a humidified atmosphere of 5% CO2 at37 °C. At the end of incubation, each well received 10 μl of

102 A. Venditti et al. / Fitoterapia 90 (2013) 94–103

3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl-tetrazolium bromide(MTT) (5 mg/ml in phosphate-buffered saline, PBS) and theplates were incubated for 4 h at 37 °C. The extent of MTTreduction was measured spectrophotometrically at 540 nmusing a Titertek Multiscan microElisa (Labsystems, FI-Helsinki).Experiments were conducted in triplicate. Cytotoxicity isexpressed as the concentration of compound inhibiting cellgrowth by 50% (IC50). The IC50 values were determinedwith GraphPad Prism 4 computer program (GraphPad Software,S. Diego, CA, USA).

3.8. Evaluation of Antioxidant activity

DPPH free radical scavenging activity of S. alopecurosessential oil was evaluated on a microplate analytical assayaccording to the previously described procedures [60], whilethe measurement of radical scavenging capacity with ABTSassaywas evaluated by amodifiedmethod originally describedby Re et al. [61] for application to a 96-well microplate assay[62]. Determination of antioxidant activity by ferric reducingantioxidant power (FRAP) assay was carried out according toMüller et al. [63]. The ability of the essential oil to scavenge thedifferent radicals in the assays was compared to that ofTrolox®, used as positive control, and the activity of the oilwas expressed as tocopherol equivalent antioxidant capacity(μmol TE/g of product).

3.9. Antimicrobial activity

Pure essential oil was tested directly against the Gram-positive Staphylococcus aureus (ATCC 25923) and the Gram-negative Escherichia coli (ATCC 25922). Growth conditions andantimicrobial activity tests were as previously described [64].10 μl per paper disk of the most abundant compound found inthe essential oil, (E)-caryophyllene, was used in the controlexperiments, while ciprofloxacin (5 μg disk) was used as thereference known antibiotic.

4. Conclusions

The essential oil composition investigated in this worksupported the taxonomic relationship in Stachys as well asthosewithin the familywhere the oil-poor species traditionallyincluded in the subfamily Lamioideae produce essential oil richin hydrocarbons, with germacrene D often being one of thepredominant components [48].

The investigation of polar fraction was focused in particularon iridoid glycosides. S. alopecuros showing the molecularpattern of iridoid characteristic of Stachys genus, as harpagideand its acetyl derivative, but it is noteworthy the presence of anew iridoid diglycoside (5), with the monoterpenic skeletonof 6, which was not detected in this plant, containing agalactose unit linked to the glucose at C1. Compound 5 couldbe considered as an additional taxonomic marker for thisspecies since teuhircoside derivatives were never reportedin Stachys genus.

The present survey also showed the high diversity of theindumentum in Stachys species and enhanced the putativeimportance of microcharacters, namely the glandular trichometypes, in terms of their taxonomic significance within thegenus. These micromorphological features together with the

composition of the essential oils may account for taxadelimitation, but further studies are needed to estimate thevalue of these parameters.

Finally, biological assays revealed the potential of the volatileoil in inhibiting tumor cells and antioxidant activity, probablydepending from the high content of (E)-caryophyllene, a com-mon constituent of many essential oils, occurring in numerousplants and foods forwhich antioxidant properties and activity onreduce the risk of developing chronic diseases have beenpreviously indicated.

Acknowledgments

The authors thank Sibillini Mountains National Park forallowing the collection of the species investigated in this study.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.fitote.2013.06.015.

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