Chemical Composition and in vitro Biological Activities of the Essential Oil of Vepris macrophylla ( Baker ) I.Verd. Endemic to Madagascar by Filippo Maggi* a ), Richard Fortune ´ Randriana b ), Philippe Rasoanaivo b ) c ), Marcello Nicoletti d ), Luana Quassinti a ), Massimo Bramucci a ), Giulio Lupidi a ), Dezemona Petrelli e ), Luca A. Vitali a ), Fabrizio Papa a ), and Sauro Vittori a ) a ) School of Pharmacy, University of Camerino, IT-62032 Camerino (e-mail: [email protected]) b ) De ´ partement de Ge ´nie Chimique, Ecole Supe ´rieure Polytechnique, Universite ´ d)Antananarivo, Antananarivo, Madagascar c ) Institut Malgache de Recherches Applique ´es, Antananarivo, Madagascar d )Department of Environmental Biology, La Sapienza University, IT-Rome e ) School of Biosciences and Biotechnology, University of Camerino, IT-62032 Camerino Vepris macrophylla is an evergreen tree occurring in sub-humid forest of Madagascar and traditionally used in the Island to treat several complaints as well as to prepare aromatic teas and alcoholic drinks. In the present work, the essential oil distilled from the leaves was analyzed for the first time by gas chromatography (GC-FID) and gas chromatography/mass spectrometry (GC/MS). The major compounds were citral (56.3%), i.e. , mixture of neral (23.1%) and geranial (33.2%), citronellol (14.5%), and myrcene (8.3%). The essential oil exhibited antimicrobial activity against S. aureus , P. aeruginosa, and C. albicans as determined by vapor-diffusion assay, supporting the traditional use of the plant for preparing steam bath for the treatment of infectious diseases. The essential oil was evaluated for cytotoxic activity on human tumor cell lines by MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide) assay, showing inhibitory effects comparable to those of cisplatin, notably on MDA-MB 231 (human breast adenocarcinoma) and HCT116 (human colon carcinoma) cell lines. Finally, the essential oil was also subjected to screening for its antioxidant activity and the free radical scavenging capacity. Introduction. – The genus Vepris Comm. ex A.Juss. (Rutaceae) comprises ca. 100 species according to different botanical databases, namely TROPICOS, International Plant Names Index, and Plant List. It consists of shrubs and trees, widespread mainly in tropical Africa, Zanzibar, Madagascar, and the Mascarene Islands [1]. Twenty-five species are found in Madagascar, of which 24 are endemic to the Island according to data published in the (Flore de Madagascar et des Comores) [2] . In the Catalogue of the Vascular Plants of Madagascar (http://www.tropicos.org/project/mada), 31 species are reported. Nearly half of them, known under the common name ampody/ampoly , are used for making alcoholic drinks (local name betsabetsa) commonly used during circumcision rituals as a euphoristic, aphrodisiac, and stimulant [3]. Seven species, i.e. , V. sclerophylla H.Perrier, V. lepidota Capuron, V. madagascarica ( Baill.) H.Per- rier, V. schmidelioides ( Baker ) I.Verd. , V. macrophylla ( Baker) I.Verd. , V. elliotii ( Radlk.) I.Verd. , and V. nitida ( Baker) I.Verd. , are reported to be aromatic [3]. Particularly, the essential oils of V. madagascarica and V. nitida, known under the vernacular names manitranzety (anise perfume) and anzety (derived from the CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 356 # 2013 Verlag Helvetica Chimica Acta AG, Zɒrich
Transcript
Chemical Composition and in vitro Biological Activities of the Essential Oil ofVepris macrophylla (Baker) I.Verd. Endemic to Madagascar
by Filippo Maggi*a), Richard Fortune Randrianab), Philippe Rasoanaivob)c), Marcello Nicolettid),Luana Quassintia), Massimo Bramuccia), Giulio Lupidia), Dezemona Petrellie), Luca A. Vitalia),
Fabrizio Papaa), and Sauro Vittoria)
a) School of Pharmacy, University of Camerino, IT-62032 Camerino (e-mail: [email protected])b) Departement de Genie Chimique, Ecole Superieure Polytechnique, Universite d�Antananarivo,
Antananarivo, Madagascarc) Institut Malgache de Recherches Appliquees, Antananarivo, Madagascard) Department of Environmental Biology, La Sapienza University, IT-Rome
e) School of Biosciences and Biotechnology, University of Camerino, IT-62032 Camerino
Vepris macrophylla is an evergreen tree occurring in sub-humid forest of Madagascar andtraditionally used in the Island to treat several complaints as well as to prepare aromatic teas andalcoholic drinks. In the present work, the essential oil distilled from the leaves was analyzed for the firsttime by gas chromatography (GC-FID) and gas chromatography/mass spectrometry (GC/MS). Themajor compounds were citral (56.3%), i.e., mixture of neral (23.1%) and geranial (33.2%), citronellol(14.5%), and myrcene (8.3%). The essential oil exhibited antimicrobial activity against S. aureus, P.aeruginosa, and C. albicans as determined by vapor-diffusion assay, supporting the traditional use of theplant for preparing steam bath for the treatment of infectious diseases. The essential oil was evaluated forcytotoxic activity on human tumor cell lines by MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, showing inhibitory effects comparable to those of cisplatin, notably onMDA-MB 231 (human breast adenocarcinoma) and HCT116 (human colon carcinoma) cell lines.Finally, the essential oil was also subjected to screening for its antioxidant activity and the free radicalscavenging capacity.
Introduction. – The genus Vepris Comm. ex A.Juss. (Rutaceae) comprises ca. 100species according to different botanical databases, namely TROPICOS, InternationalPlant Names Index, and Plant List. It consists of shrubs and trees, widespread mainly intropical Africa, Zanzibar, Madagascar, and the Mascarene Islands [1]. Twenty-fivespecies are found in Madagascar, of which 24 are endemic to the Island according todata published in the �Flore de Madagascar et des Comores� [2]. In the Catalogue of theVascular Plants of Madagascar (http://www.tropicos.org/project/mada), 31 species arereported. Nearly half of them, known under the common name ampody/ampoly, areused for making alcoholic drinks (local name betsabetsa) commonly used duringcircumcision rituals as a euphoristic, aphrodisiac, and stimulant [3]. Seven species, i.e.,V. sclerophylla H.Perrier, V. lepidota Capuron, V. madagascarica (Baill.) H.Per-
rier, V. schmidelioides (Baker) I.Verd., V. macrophylla (Baker) I.Verd., V. elliotii(Radlk.) I.Verd., and V. nitida (Baker) I.Verd. , are reported to be aromatic [3].Particularly, the essential oils of V. madagascarica and V. nitida, known under thevernacular names manitranzety (anise perfume) and anzety (derived from the
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� 2013 Verlag Helvetica Chimica Acta AG, Z�rich
malgachization of the French word anisette) in reference to the aniseed odor of theplants rich in anethole and/or estragole, were exported in the past to China and Japanfor making anise-flavored mouthwash or toothpastes [4].
V. macrophylla (vernacular names ampodiberavina, itampody, mampodifotsy), thesubject of the present study, is a 10– 15-m-high evergreen tree endemic to Madagascar,occurring in subhumid forest, from sea-level up to 600 m altitude. Besides its use aseuphoristic plant, a leaf infusion is taken as an astringent. A root infusion is drunk totreat fatigue, apathy, and depression. From the aromatic fruits, a steam bath is made forpatients recovering from infectious diseases [3]. In our ethnobotanical field work, welearned that leaves are also used to make a tea. Previously, some acridone alkaloidswere isolated from the leaves of this plant [5], whilst no previous investigations of itsessential oil (EO) have been reported.
Herein, we report for the first time the chemical composition of the essential oilobtained from the leaves of the plant, together with some biological properties, namelythe cytotoxic effects on human tumor cell lines by MTT assay, the antioxidant capacityby ABTS, DPPH, and FRAP assays1), and the antimicrobial activity by microdilutionand vapor-diffusion assay.
Results and Discussion. – 1. Chemical Analysis of the Essential Oil. A total of 44volatiles were identified in V. macrophylla, corresponding to 99% of the total oil(Table 1). About half of the constituents (24), corresponding to 90% of the essential oil(EO), were identified by comparison with commercially authentic compounds, whilstthe remaining ones were identified according to the International Organization of theFlavor Industry (IOFI, http://www.iofi.org/) statement (see Exper. Part). The EO wasalmost entirely constituted by monoterpenoids (97.7%), of which the major volatileswere citral (56.3%), represented by the isomeric aldehydes neral (23.1%) and geranial(33.2%), the alcohol citronellol (14.5%), and the hydrocarbon myrcene (8.3%).Together, they accounted for 79% of the total composition. The major fractionconsisted mainly of aldehydes (61.7%), while a lower contribution was furnished byalcohols (18.6%), hydrocarbons (10.6%), and acetates (5.4%).
To the best of our knowledge, the EO of V. macrophylla has so far never beenstudied, while other species of this genus (V. madagascarica, V. heterophylla, V.leandriana, V. elliotii, and V. nitida), of which three are endemic to Madagascar, werepreviously investigated [11 –15] for EO composition. Notably, the chemical profile of V.macrophylla oil, nevertheless qualitatively richer, resulted in major components similarto those of V. leandriana growing in the northeast of Madagascar. In the EO of thelatter, neral (19.5%), geranial (27.0%), and citronellol (33.6%) were detected as majorcomponents [13]. Furthermore, some minor less-known volatiles such as 6,7-epoxymyrcene (0.1%), exo-citral (0.7%), and (Z)- and (E)-isocitral (1.7 and 2.5%,resp.) were reported for the first time for the genus Vepris, and their structures aredepicted in the Figure. Based on the data published so far, the Malagasy Vepris spp. canbe classified into two chemotypes: the anethole/estragole chemotype (V. madagascar-
ica, and V. nitida) and the citral/citronellol chemotype (V. elliotii, V. leandriana, and V.macrophylla).
The major constituent of V. macrophylla oil is citral (¼ 3,7-dimethylocta-2,6-dienal), a natural mixture of the isomeric acyclic aldehydes neral and geranial. It isresponsible for the pleasant odor of the essential oils obtained from Citrus limon (L.)Burm. f. epicarp., as well as from the leaves of lemon balm (Melissa officinalis L.) andlemongrass (Cymbopogon citratus (DC.) Stapf. ). Citral is a generally recognized as asafe (GRAS) food additive, and it has been approved by the Food and DrugAdministration (FDA) for use in foods. Moreover, it has been registered by theEuropean Commission for use as flavoring in foodstuffs, because it does not pose healthrisk to the consumer [16]. Regarding the biological properties, citral possessesantifungal activity against plant and human pathogens [17], and bactericidal andinsecticidal effects [18] [19]. Recently, it has been reported as a promising agent to beused in the treatment of inflammation [20], as potent antimicrotubule [21] andchemopreventive agent [22] [23], and as capable to affect animals� spatial learning andmemory [24]. Citral, together with citronellol, was also found to exhibit anticonvulsanteffects [25]. The alcohol citronellol and the hydrocarbon myrcene, occur in significant
Figure. Chemical structures of some less-known volatiles detected in Vepris macrophylla
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Table 1 (cont.)
No. Componenta) RFb) RIc) RI Lit.d) %e) Method ofidentificationf)Adams NIST08
a) Compounds are listed in order of their elution from a HP-5 column. Their nomenclature was inaccordance with Adams [6]. b) Relative response factor (RF) of FID detector for the main chemicalgroups occurring in essential oil [7]. c) Linear retention index on HP-5 column, experimentallydetermined using homologous series of C8 – C30 alkanes. d) Linear relative retention index taken fromAdams [6] and/or NIST08 [8] for DB-5 and HP-5 capillary column, respectively. e) Percentage values aremeans 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 oncomparison with Wiley, Adams, and NIST08 MS database; RI, based on comparison of RI with thosereported in Adams and NIST08. g) Tr, traces (mean value below 0.1%). h) Retention indices and MStaken from NIST08, Brophy et al. [9], and Silva et al. [10].
levels in citronella oils from the genus Cymbopogon, that are used in Asian and Africancuisine for sauces and soups with a strong lemon- and rose-like odor. The EO containsalso a little amount of monoterpenoid acetates (5.4%) that are very fragrantcompounds. Main representatives of this group are geranyl acetate (1.5%), citronellylacetate (1.7%), and and neryl acetate (1.8%). Thus, the composition of the oil supportssome of the ethnobotanical uses of the plant in the island.
2. Antimicrobial Activity. In the microdilution test, V. macrophylla EO was notactive against the two Gram-negative species E. coli and P. aeruginosa (MIC and MBC� 4096 mg/l). The same result was obtained with citral and citronellol (Table 2). Theywere used as reference because they accounted together for 71% of the EO. On theother hand, the Gram-positive species S. aureus and E. faecalis showed a moderatesusceptibility towards the EO. In the case of E. faecalis, the activity was small (4096 mg/l), while S. aureus was inhibited at a twofold lower dilution (1024 mg/l). The EO wasbactericidal, as the value of the MIC essentially matched that of the MBC (1024 –2048 mg/l). The reference compound citral showed the same activity.
The antimicrobial activity was higher against the yeast C. albicans. The MIC andMBC values were 256 and 512 mg/l, respectively. Also in this case, the activity could beconsidered as bactericidal (Table 2).
V. macrophylla EO was additionally subjected to the vapor-diffusion assay. Theresults are compiled in Table 3. Activities were detected against S. aureus, C. albicans,and P. aeruginosa. Contrary to what was observed in the microdilution test, the vapor ofthe oil was not active against E. faecalis, while it was inhibitory against the Gram-negative P. aeruginosa with a diameter of the inhibition zone of ca. 15 mm. Again, thehighest activity was recorded against Candida with a diameter of 38 mm. Under thesame conditions, also the vapor from the two reference compounds citral andcitronellol was assayed. Remarkably, vapor from citral was a strong inhibitor of growthof the three species susceptible to the EO, thus confirming reports in literature [17].Citronellol was only active against the fungal species. An attempt to normalize data wasconducted by comparing the activity of V. macrophylla EO with those of pure referencecompounds. The resulting relative activity R was calculated as the ratio between thediameters obtained with the vapor of V. macrophylla oil and the sum of the diametersmeasured for citral and citronellol corrected for their relative abundance in the EO, i.e.,56 and 15%, respectively. The values for R are collected in Table 3. A relative activity R
Table 2. Minimal Inhibitory Concentrations (MIC [mg/l]) and Minimal Bactericidal Concentrations(MBC, in mg/l) of V. macrophylla Essential Oil and Two Major Components against Representative
Microbial Species
Microorganism V. macrophylla EO Citral Citronellol
of ca. 1 was observed for both S. aureus and C. albicans. The simplest explanation is thatthe activity of the EO could be completely accounted for by the contribution of its twomain components, citral (mixture of neral and geranial) and citronellol. On thecontrary, the activity of the EO vapor against P. aeruginosa is not determined by thesum of the relative activity of citral and citronellol (R>1).
The results from the microdilution tests evidenced an inhibitory effect of V.macrophylla EO against S. aureus, E. faecalis, and C. albicans. More than 50% of theEO is constituted by citral that was most probably the main contributor to thedetermined antimicrobial activity, which was also microbicidal. In fact, citral has a well-known activity against bacteria as well as against fungi [26] [27].
The vapor phase of the oil showed a different specific activity. It was ineffectiveagainst E. faecalis but had an appreciable inhibitory activity against P. aeruginosa.Interestingly, the resistance of P. aeruginosa to many essential oils as well as to citraland/or citronellol is paradigmatic and known since a long time [28]. Under ourexperimental conditions, the vapors of V. macrophylla EO showed a new andinteresting activity against this important human pathogen that is widespread in theenvironment. Moreover, the main components citral and citronellol do not seem toaccount for all the measured inhibitory activity. It might be hypothesized that at leastone minor volatile component other than citral and citronellol was responsible for theantipseudomonal activity. Further studies are needed to identify and characterize theseactive compounds.
3. Antiproliferative Activity. V. macrophylla EO was tested in vitro for the potentialtumor cell growth-inhibitory effect on T98G (human glioblastoma), MDA-MB 231(human breast adenocarcinoma), A375 (human malignant melanoma), and HCT116(human colon carcinoma cell lines), using MTT assay1).
The results, collected in Table 4, show that the EO exhibited strong inhibitoryeffects against the human cancer cells examined, with inhibition values comparable tothose of the anticancer drug cisplatin. The highest activity was observed on MDA-MB231 and HCT116 cell lines, with IC50 values of 3.14�0.21 and 3.21�0.22 mg/ml,respectively. Lowest activity was obtained on T98G, with an IC50 value of 28.4�2.2 mg/ml. The cytotoxic activity of the EO may be attributed to specific components such ascitral (56.3%), citronellol (14.5%), and myrcene (8.3%). It was reported that citral
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Table 3. Inhibitory Effects of V. macrophylla Essential Oil and Two Major Constituent�s Vapors againstRepresentative Microbial Species Assessed by Vapor-Diffusion Test.
Microorganism Inhibition zone diameter [mm] Ra)
V. macrophylla Citral Citronellol
S. aureus 13�3 23�4 0 1.01�0.4E. faecalis 0 0 0 N. D.E. coli 0 0 0 N. D.P. aeruginosa 15�1 18�1 7�1 1.36�0.2C. albicans 38�4 63�4 30�1 0.96�0.2
a) R¼ (diameter)V. macrophylla oil /(0.56 (diameter)Citralþ0.15 (diameter)Citronellol), given that the percentage ofthe citral (AþB, geranialþneral) in the essential oil is ca. 56% and that of citronellol is 15%. N. D., notdetermined.
exerted an antitumor effect on several hematopoietic cell lines [29] and human breastcancer cell line MCF-7 [22] via the induction of apoptosis and activation of caspase-3,and resulted cytotoxic in human cervix epithelioid carcinoma cells HeLa [30]. Thecitral antiproliferative activity was tested on our tumor cell lines. IC50 Values were5.73�0.34 mg/ml (37.6�2.2 mm) for MDA-MB 231, 8.1�0.4 mg/ml (53.1�2.6 mm) forHCT116, 11.3�0.6 mg/ml (74.1�3.9 mm) for A375, and 28.6�4.9 mg/ml (188.6�32.2 mm) for T98G cell lines. Citronellol was also active, whereas myrcene was notactive in our cell lines as reported in [31]. Therefore, these compounds, withdemonstrated cytotoxic activities, and the compounds present at high concentrations inthe oil were used to reconstitute 80.5% of the main composition of natural EO usingcommercially available compounds. Citral, citronellol, myrcene, citronellyl acetate, andgeraniol were, therefore, mixed at the same percentage reported in Table 1, and themixture was tested on the cell lines. As given in Table 4, the cytotoxic activities resultedsignificantly less (p<0.0001) than the original EO, indicating that the activities of themajor components are modulated by other minor molecules occurring in the EO.
4. Antioxidant Activity. In Table 5, the free radical-scavenging potentials of V.macrophylla EO and of the standard main components are collected. They weredetermined by the DPPH and ABTS assay1). In general, the EO was able to reduce thestable radical DPPH with an IC50 value of 340�8 mg/ml and was a significantly stronger
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Table 4. In vitro Cytotoxic Activities (IC50a) [mg/ml�SD]b)) of Essential Oil from V. macrophylla against
a) IC50 , Concentration of compound that affords a 50% reduction in cell growth (after 72 h ofincubation). b) SD, Standard deviation of the three determinations. c) Human glioblastoma multiformecell line. d) Human breast adenocarcinoma cell line. e) Human malignant melanoma cell line. f) Humancolon carcinoma cell line.g) *: p < 0.0001 vs. essential oil; **: p< 0.0004 vs. essential oil.
Table 5. Total Antioxidant Capacity of the Essential Oil from V. macrophylla and Some Major VolatileComponents
a) TEAC, Trolox-equivalent (TE) antioxidant concentration [mmol TE/g]. b) IC50 in mg/ml.
scavenger against the ABTSþ radical with an IC50 value of 210�3 mg/ml. To illustratethe relation between activity of EO and some main components, the radical scavengingcapacity of the two major components, citral and citronellol, was also studied.
Combining the results reported in Table 5, i.e., the free radical-scavenging activityof the EO, compared to the isolated components, may result from the synergistic actionof volatile constituents occurring in EO. The ferric-reducing power (determined by theFRAP assay) of V. macrophylla EO was evaluated as well. The activity of EO washigher than that of the two major constituents (Table 5). The FRAP assay evidencedthat EO has the reducing potential to react with ferric tripyridyl triazine (Fe3þ-TPTZ)complex producing blue-colored ferrous tripyridyl triazine (Fe2þ-TPTZ). FeIII
Reduction is often used as an indicator of electron-donating activity, which is animportant mechanism of antioxidant action [32] [33], and, generally, the reducingproperties are associated by breaking the free-radical chain by donating a H-atom [34].The results obtained for V. macrophylla EO indicate the possibility of using this plantproduct also for its antioxidant properties, and this would greatly reduce the need toobtain pure compounds via expensive industrial purification techniques.
Conclusions. – The obtained results highlight the connection between the chemistryand ethnobotany of V. macrophylla in Madagascar. The examined EO belongs to thecitral/citronellol chemotype of the genus Vepris, thus justifying its use for makingaromatic tea and alcoholic drinks. The antimicrobial activity, revealed especially by thevapour diffusion assay, supports the traditional use of the plant in preparing steam bathfor the treatment of infectious diseases. The cytotoxic effects of the EO against MDA-MB 231 (human breast adenocarcinoma) and HCT116 (human colon carcinoma) celllines are strongly related to the major compounds and comparable to those of cisplatin,thus encouraging further investigations on the mechanisms of action and in vivo studiesfor applications as natural anticancer drug.
Experimental Part
Plant Material. Leaves of V. macrophylla were collected in May 2011 in Sahamamy/Analalava(District of Mahavelona Toamasina II), eastern coastal forest of Madagascar. The plant was identified bybotanists at the Parc Botanique et Zoologique de Tsimbazaza, Antananarivo. A voucher specimen wasdeposited with the Herbarium of the Institut Malgache de Recherches Appliquees (IMRA), under theaccession code MAD-0724.
Extraction of the Essential Oil (EO) . Fresh leaves (5.5 kg) were extracted by steam distillation for4 h using a field alembic. Twenty-three ml (ca. 20 g) of EO were obtained (yield: 0.004%) afterdecantation. Before analysis, the oil was dried (Na2SO4), transferred into an amber glass flask, and keptat �208 before GC and biological experiments.
Chemicals. Standard compounds used for identification of volatiles were: a-pinene, b-pinene,myrcene, a-terpinene, p-cymene, 1,8-cineole, g-terpinene, terpinolene, linalool, trans-pinocarveol,citronellal, terpinen-4-ol, a-terpineol, myrtenal, citronellol, citral (mixture of neral and geranial),geraniol, myrtenyl acetate, citronellyl acetate, neryl acetate, geranyl acetate, (E)-caryophyllene,caryophyllene oxide; they were purchased from Sigma�Aldrich (I-Milan). For retention-index (RI)determination, a mixture of hydrocarbons (C8 –C30; Supelco, USA) was used and run at the exper.conditions reported below. All compounds were of anal.grade. Anal.-grade hexane was purchased fromCarlo Erba (I-Milan); it was successively distilled by a Vigreux column before use.
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GC-FID and GC/MS Analysis. For GC separations, an Agilent 4890D instrument coupled to anionization flame detector (FID) was used. Volatile components were separated on a HP-5 cap. column(5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17 mm film thickness; J and W Scientific, Folsom,CA), with the following temp. program: 5 min at 608, subsequently 48/min up to 2208, then 118/min up to2808, held for 15 min, for a total run of 65 min. Injector and transfer line temp. were 2808. He was used asthe carrier gas, flow rate, 1.4 ml/min; injection volume, 1 ml ; split ratio, 1 : 34. A mixture of aliphatichydrocarbons (C8 –C30; Sigma, I-Milan) in hexane, was directly injected into the GC injector according tothe above temp. program, in order to determine RI (as Kovats index) of each compound. Oil sampleswere diluted 1 :100 in hexane and injected at a volume of 1 ml. Analysis was repeated three times. Datawere collected with HP3398A GC Chemstation software (Hewlett–Packard, Rev. A.01.01). The GC-FIDanalyses were run in triplicate. Quantification of EO components was obtained by FID peak-areainternal normalization by calculating the response factor of the main chemical groups occurring in EO[7]. GC/MS Analysis was performed on an Agilent 6890N gas chromatograph coupled to a 5973N massspectrometer using a HP-5 MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 mm film thickness;J and W Scientific, Folsom), using the same temp. program described above. Injector and transfer-linetemps. were 2808. He was used as the carrier gas, at a flow rate of 1 ml/min. Injection volume, 2 ml ; splitratio, 1 : 50; acquisition mass range, m/z 29 –400. All mass spectra were acquired in electron-impact (EI)mode with an ionization voltage of 70 eV. EO Samples were diluted 1 : 100 in hexane, and the volumeinjected was 2 ml. Analysis was repeated three times. Data were analyzed with MSD ChemStationsoftware (Agilent, Version G1701DA D.01.00). For 24 out of 44 compounds, corresponding to 90.0% ofthe total oil, the identification was carried out by co-injection with authentic standards (see Table 1).Otherwise, the peak assignment was in accordance with the standard of the International Organization ofthe Flavor Industry (IOFI, http://www.iofi.org/) statement, i.e., by combining the computer matching withthe WILEY275, NIST08, and Adams libraries, with the correspondence of the calculated retentionindices with respect to those reported by Adams [6] and NIST08 [8].
Antimicrobial Activity. EO was tested against five microbial species including two Gram-positivebacteria, Staphylococcus aureus ATCC 25923 (American Type Culture Collection, Rockville, MD, USA)and Enterococcus faecalis ATCC 29212, two Gram-negative bacteria, Escherichia coli ATCC 25922 andPseudomonas aeruginosa ATCC 27853, and the yeast Candida albicans ATCC 24433. Bacterial strainswere cultured overnight at 35–368 in blood agar plates. C. albicans was grown in Sabouraud dextroseagar. Antibiotic susceptibility testing was performed by the microdilution method following theInternational Guidelines of the CLSI [35]. Prior to dilution in growth medium, the EO was prepared inDMSO at a final concentration of 0.1 mg/ml. The plates were incubated 18–20 h at 358 (48 h for C.albicans). The antimicrobials ciprofloxacin and nystatin were used as internal controls against bacteriaand fungi, respectively. Each test was repeated three times. The activity of the tested oil was consideredbactericidal if the value of the minimal bactericidal concentation (MBC) was equal to 1, or 2 times that ofthe minimal inhibitory concentration (MIC). Vapor-diffusion assay was performed according to theprocedure of Lopez et al. [36]. Briefly, a suspension of 108 cells per ml prepared in saline (106 per ml forC. albicans) was spread on the solid-media plates using a sterile cotton swab. Sterile filter paper discs(6 mm in diameter) were fixed on the medium-free cover of the Petri dish and spotted with 20 ml of a EOEt2O 1 : 1. The plates were sealed with Parafilm and incubated for 18 h at 358. The diameters of zoneinhibition were measured with a calliper. No zone inhibition was observed using Et2O alone.
MTT1) Cytotoxicity Assay. Human glioblastoma multiforme cell line T98G was cultured in Eagle�sminimum essential medium (EMEM) with 2 mm l-glutamine, 0.1 mm non-essential amino acids, 1 mm
sodium pyruvate, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (PAA Laboratories GmbH, Austria). Human breastadenocarcinoma cell line MDA-MB 231 and human malignant melanoma cell line A375 were culturedin Dulbecco�s modified Eagle�s Medium (DMEM) with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with 10% HI-FBS. Human colon carcinoma cell line HCT116 wascultured in RPMI1640 medium with 2 mml-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, andsupplemented with 10% HI-FBS. Cells were cultured in a humidified atmosphere at 378 in presence of5% CO2. The MTTassay was used as a relative measure of cell viability. Cell-viability assays were carriedout as described in [37] [38]. Briefly, cells were seeded at the density of 2�104 cells/ml. Quadruple cell
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samples were grown in 96-well microtiter plates (Iwaki, Tokyo, Japan). After 24 h, samples were exposedto different concentrations of EO or standard compounds (0.39–200 mg/ml) in a final volume of 100 ml ofculture medium. Cells were incubated for 72 h in a humidified atmosphere of 5% CO2 at 378. At the endof incubation, each well received 10 ml of MTT (5 mg/ml in phosphate-buffered saline (PBS)), and theplates were incubated for 4 h at 378. The formazan crystals formed were solubilized in 100 ml DMSO afteraspirating the medium. The extent of MTT reduction was measured spectrophotometrically at 540 nmusing a Titertek Multiscan microElisa (Labsystems, FI-Helsinki), and the cell survival was expressed aspercentage over the vehicle. Experiments were conducted in triplicate. Cytotoxicity was expressed as theconcentration of compound inhibiting cell growth by 50% (IC50). The IC50 values were determined withGraphPad Prism 4 computer program (GraphPad Software, S. Diego, CA, USA).
Evaluation of Antioxidant Activity. DPPH1) Free radical scavenging activity was assessed on amicroplate anal. assay according to the procedures described by Srinivasan et al. [39], while the totalradical-scavenging capacity of EO and its main constituents (citral and citronellol) was measured byABTS1) assay modified by Re et al. [40] for application to a 96-well microplate assay. Determination ofantioxidant activity by FRAP1) assay was carried out according to the procedure described in [32] bymonitoring the reduction of Fe3þ-tripyridyl triazine (TPTZ) to blue-colored Fe2þ-TPTZ. The ability ofthe samples tested to scavenge the different radicals in the assays was compared to trolox used asstandard. The activities of EO and of its main constituents were expressed as trolox-equivalentantioxidant capacity mmol TE/g of product.
Statistical Analysis. Each experiment was repeated at least three times. The unpaired t test was usedfor comparisons between two groups. Statistical significance was indicated by p values<0.001. Data arereported as the mean�SD.
The authors are grateful to Professor Suzanne Ratsimamanga, President of the Institut Malgache deRecherches Appliquees, for her precious support during this study. We thank the local communities ofAnalalava for helping us to collect V. macrophylla.
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