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Daucus carota ssp. hispanicus Gouan. essential oils:chemical variability and fungitoxic activityAmel Bendiabdellaha, Mohammed El Amine Diba, Nassim Djaboua, Fayçal Hassanic, JulienPaolinia, Boufeldja Tabtia, Jean Costab & Alain Muselliba Laboratoire des Substances Naturelles et Bioactives (LASNABIO), Département de Chimie,Faculté des Sciences, Université Aboubekr Belkaïd, Tlemcen, Algérieb Université de Corse, Laboratoire Chimie des Produits Naturels, Campus Grimaldi, Corte,Francec Laboratoire d’Ecologie et Gestion des Ecosystèmes Naturels, Département d’Ecologie &Environnement, Faculté SNV-STU, Université Aboubekr Belkaïd, Tlemcen, AlgériePublished online: 19 Sep 2014.
To cite this article: Amel Bendiabdellah, Mohammed El Amine Dib, Nassim Djabou, Fayçal Hassani, Julien Paolini, BoufeldjaTabti, Jean Costa & Alain Muselli (2014) Daucus carota ssp. hispanicus Gouan. essential oils: chemical variability andfungitoxic activity, Journal of Essential Oil Research, 26:6, 427-440, DOI: 10.1080/10412905.2014.956189
To link to this article: http://dx.doi.org/10.1080/10412905.2014.956189
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RESEARCH ARTICLE
Daucus carota ssp. hispanicus Gouan. essential oils: chemical variability and fungitoxicactivity
Amel Bendiabdellaha, Mohammed El Amine Diba*, Nassim Djaboua, Fayçal Hassanic, Julien Paolinia, BoufeldjaTabtia, Jean Costab and Alain Musellib
aLaboratoire des Substances Naturelles et Bioactives (LASNABIO), Département de Chimie, Faculté des Sciences, UniversitéAboubekr Belkaïd, Tlemcen, Algérie; bUniversité de Corse, Laboratoire Chimie des Produits Naturels, Campus Grimaldi, Corte,
France; cLaboratoire d’Ecologie et Gestion des Ecosystèmes Naturels, Département d’Ecologie & Environnement, FacultéSNV-STU, Université Aboubekr Belkaïd, Tlemcen, Algérie
(Received 21 October 2013; accepted 15 August 2014)
The chemical composition of Algerian Daucus carota ssp. hispanicus Gouan. essential oils has been investigatedusing gas chromatography/retention indices (GC/RIs) and GC–mass spectrometry (GC–MS), and their antibacterialand antifungal activities were tested for the first time. Chemical analysis allowed the identification of sixty-eight com-pounds amounting to 92.3–98.5% of aerial part essential oils and eight components representing 97.4–99.4% of rootessential oils. Intra-species variations of the chemical compositions of essential oils from ten Algerian sample loca-tions were investigated using statistical analysis (principal component analysis and cluster analysis). In addition, rootessential oils of D. carota ssp. hispanicus were found to be strongly fungicidal and inhibitory to aflatoxin production.
Keywords: Daucus carota ssp. hispanicus; essential oils; phenylpropanoids; chemical variability; antimicrobial andantifungal activities; aflatoxin inhibitors
Introduction
Beneficial plants are widely distributed all over theworld, and they are rich sources of useful secondarymetabolites, often as compounds with therapeutic rolesin defense against a wide array of pathogens includingviruses, bacteria and fungi (1). According to the litera-ture, Aspergillus flavus is common fungus that normallythrives as saprobes in soils and on a wide variety ofdecaying organic matter. Besides being an etiologicalagent of systemic aspergillosis and allergic reactions,A. flavus has received much attention due to its abilityto produce the carcinogenic aflatoxins (2). The searchfor natural sources of novel inhibitors of aflatoxin bio-synthesis has been the subject of intense study and avariety of bioactive aflatoxin inhibitory compoundshave been reported from medicinal plants (3). Thechemical composition of the essential oils of Daucuscarota has been widely studied; according to geograph-ical and botanical origins of samples, monoterpenes,sesquiterpenes and phenylpropanoids have beenreported as the main component classes (Table 1) (5,8–11, 14–17, 19, 22, 23). In the present study, D. carotassp. hispanicus essential oils from Algeria were investi-gated in order to determine its biological activity aswell as its inhibitor potential of aflatoxin. For this pur-pose, the chemical composition of the essential oilobtained by hydrodistillation was first investigated
using gas chromatography/retention indices (GC/RIs)and GC–mass spectrometry (GC–MS). The essentialoils of separated organs (roots, total aerial parts, leaves,stems and flowers) were also analyzed and the intra-species variations in root and aerial part essential oilsfrom ten sample locations were studied using statisticalanalysis. Correlations between the essential oil chemicalcompositions and environmental parameters of the sam-ple locations were discussed. Secondly, the antibacterialand antifungal effects of D. carota ssp. hispanicusessential oils were tested against eight bacteria and twofungi involved in food-borne illnesses or consideredclinically important pathogenic microorganisms.Biological experiments were performed by means of apaper disc diffusion method and minimum inhibitoryconcentration (MIC) assays.
Experimental Plant material and oil isolation
The plant material (roots and aerial parts) of D. carotassp. hispanicus was collected in ten locations of Wes-tern Algeria. Information concerning the locations ofharvest such as the location names, latitudes, longi-tudes, nature of soils and climates were tabulated inTable 2. Voucher specimens were deposited with theherbarium of the University of Tlemcen. To obtainessential oils, the fresh roots and aerial parts (leaves,
*Corresponding author. Email: [email protected]
© 2014 Taylor & Francis
Journal of Essential Oil Research, 2014Vol. 26, No. 6, 427–440, http://dx.doi.org/10.1080/10412905.2014.956189
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stems and flowers) were subjected to hydrodistillationfor 5 hours using a Clevenger-type apparatus accordingto the European Pharmacopoeia (25). The essential oilyields were expressed in percent (w/dw) through theweight of dried plant material. Fresh plant material wasdried for five days at room temperature; water contentwas close to 82.5% of the plant weight.
Sample locations
The locations of the harvest were distributed in twoareas differentiated according to the altitude, the natureof soils and the climates. Area 1 includes Kihal,Amieur, Besekrane and Saf-Saf (S1–S4), four middle-
mountain locations (263–599 m) with calcareous soilrich in organic matter and dry climates. Area 2 includesMansourah, Beni Boublene, Lalla Setti, Mafrouche,Hafir and Terny (S5–S10), six mountainous locations(907–1220 m) with mineral-rich soils and humid andcooler climates (26, 27) (Table 2).
Gas chromatography
GC analyses were carried out using a PerkinElmerClarus 600 GC apparatus equipped with a dual flameionization detection system and two fused-silica capil-lary columns (60 m × 0.22 mm i.d., film thickness0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax
Table 1. Main components of the essential oils of Daucus carota from different origins previously reported.
D. carotassp. Organs
Origins(country) Main components References
maritimus Flowers Tunisia Sabinene (51.6%), terpinen-4-ol (11.0%) (4)Roots Myristicin (29.7%), dillapiole (46.6%)
maximus Ripe and mature fruits Egypt Shyobunone (16.8–24.3%), β-cubebene (3.5–12.7%),preisocalamendiol (17.9–32.7%)
(5)
sativus Umbels Poland, α-Pinene (40.0–46.0%), sabin[4]ene (12.0–24.0%), β-caryophyllene(4.6–13.2%)
(6)
Leaves Iran trans-Anethole (23.5%), myrcene (14.5%) (7)Seeds Poland Carotol (10.7–48.0%), α-pinene (9.0–18.0%) (8)Fruits Sweden Myrcene (25.7–44.6%), (E)-β-ocimene (8.0–11.3%), methyl
isoeugenol (19.7–55.3%)(9)
gummifer Fruits Spain Geranyl acetate (51.74–76.95%) (10)halophilus Flowering umbels Portugal Sabinene (28.3–33.8%). Limonene (11.0–11.8%) (11)
Ripe umbels Elemicin (26.0–31.0%), sabinene (27.6–29.0%)sativa Seeds China β-bisabolene (80.49%), α-asarone (8.8%), and cis-α-bergamoten
(5.51%)(12)
Stems and leaves China Caryophyllene (17.24%), myrcene (14.06%), (+) epi-bicyclo-sesquiphellandrene (10.14%)
(13)
Roots α-farnesene (17.1%), caryophyllene (10.9%), 1, 2, 4-Methano-1H-cyclobuta[β] cyclo (32.3%)
carota Umbels Italy β-Bisabolene (17.6–51.0%), carotol (2.4–25.1%), 11α-(H)-himachal-4-en-1-β-ol (9.0–21.6%), E-methylisoeugenol (1.3–10.0%)
(14)
Portugal α-Pinene (13.0–37.9%), geranyl acetate (15.0–65.0%)Umbels Tunisia Elemicin (31.5–35.3%), carotol (48.0–55.7%), 11-α-(H)-himachal-4-
en-1-β-ol (12.7–17.4%), sabinene (12.0–14.5%), α-selinene (7.4–8.6%)
(15)
Ripe, flowers, roots,leaves, and stems
Serbia α-Pinene (7.1–51.2%), sabinene (2.7–36.7%) (16)
Fruits Muurolene (8.2–10.9%)Seeds Turkey Carotol (68.0%), daucene (8.7%) (17)Aerial parts Corsica α-Pinene (15.9–24.9%), elemicin (11.4–16.3%), E-methyl-isoeugenol
(21.8–33.0%)(18, 19)
Herbs, umbels Poland α-Pinene (30.0–42.0%), sabinene (19.5–40.5%), myrcene (2.5–7.0%) (8, 20)Seeds Lithuania Sabinene (28.2–37.5%), α-pinene (16.0–24.5%), terpinen-4-ol (4.6–
7.5%), γ-terpinene (2.9–6.0%)(21)
Roots Vienna α-Terpinolene (26–56%) (22)Leaves α-Pinene (20.9–44.8%), sabinene (14.2–19.5%)Fruits α-Pinene (23.5–30.4%), sabinene (21.5–46.6%), geranyl-acetate (3.9–
28.1)Fruits Northern
SerbiaSabinene (18.7%), carotol (20.3%) (23)
Herbs, umbels Poland α-Pinene (16.1–42.7%), sabinene (21.3–45.3%), myrcene (4.0–12.9),limonene (3.55–6.75%)
(24)
428 A. Bendiabdellah et al.
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(polyethylenglycol). The oven temperature was pro-grammed from 60° to 230°C at 2°C/minute and thenheld isothermally at 230°C for 35 minutes. The injectorand detector temperatures were maintained at 280°C.Samples were injected in split mode (1/50), usinghelium as the carrier gas (1 mL/minute); the injectionvolume was 0.2 μL. RIs of the compounds were deter-mined from PerkinElmer software.
Gas chromatography–mass spectrometry
Samples were analyzed using a PerkinElmer Turbo-Mass quadrupole analyzer, directly coupled to a Perkin-Elmer Autosystem XL, equipped with two fused-silicacapillary columns (60 m × 0.22 mm, film thickness0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax(polyethylene glycol). Other GC conditions were thesame as described above. Ion source temperature:150°C; energy ionization: 70 eV; the electron ionizationmass spectra were acquired with a mass range of35–350 Da; scan mass: 1 second. Oil injected volume:0.1 μL; fraction injected volume: 0.2 μL.
Component identification and quantification
Identification of the components was based (i) on thecomparison of their GC RIs on non-polar and polar col-umns, determined using the retention times of a series ofn-alkanes with linear interpolation, with those of authen-tic compounds or literature data (28, 29) and (ii) on com-puter matching with commercial mass spectral libraries(31) and comparison of the spectra with those of the in-house laboratory library. The quantification of the essen-tial oils components was performed using methodologyreported in the literature (33) and adapted by our labora-tory (34). Component quantification was carried outusing peak normalization, including response factors,with an internal standard (tridecane: 0.7 g/100 g), andexpressed as normalized percent abundances.
Antimicrobial assays
Micro-organisms and culture conditions
The essential oils of roots and aerial parts from D. carotassp. hispanicus were tested against eight microorgan-isms, including Gram-positive Staphylococcus aureusATCC 25923, Enterococcus faecalis ATCC 49452,Listeria monocytogenes ATCC 15313, Bacillus cereusATCC 10876, Bacillus subtilis ATCC 6633, Gram-negative Escherichia coli ATCC 25922, Klebsiellapneumoniae ATCC 70063 and two fungal microorgan-isms, Candida albicans ATCC 10231 and Aspergillusflavus MNHN 994294. The bacteria were cultivated inbrain–heart infusion broth (BHI) at 37 ± 1°C. Candidaalbicans was cultivated in Sabouraud dextrose agar(DAS) at 27 ± 1°C. The inoculi were prepared by thedirect inoculation of colonies in 1 mL of sterile salinesolution and adjusted to the 0.5 standard of the McFar-land scale, corresponding to 1.5 × 108 CFU/mL for thebacteria and 2–5 × 106 CFU/mL for fungal strains (35).
Agar disk diffusion method
The standard agar disk diffusion method (36) was usedto evaluate the inhibitory spectrum of the essential oilagainst the micro-organisms analyzed in the presentstudy. The bacterial inoculi were seeded on Müller–Hinton agar solidified in Petri dishes, in such a way asto produce uniform growth throughout the dish. Oncethe dishes were prepared, 6-mm-diameter discs of filterpaper containing 10 μL of the undiluted essential oilwere pressed lightly against the surface of the agar.After 30 minutes at room temperature, the dishes wereincubated in a bacteriological oven at 37 ± 1°C for24 hours. For the cultures of C. albicans, incubationtime was 48 hours at 27 ± 1°C and the substrate wasDAS. At the end of the test period, the diameter of theinhibition zone formed over the agar culture wasmeasured in millimeters. All tests were conducted in
Table 2. Information concerning the locations of harvest of Daucus carota ssp. hispanicus from Western Algeria.
Samples Locations Latitude (N)Longitude(E)
Altitude(m) Nature of soils Climates
S1 Kihal 35° 12’ 16” 1° 11’46” 490 Calcareous soil, rich in organic matter (agriculturalsoil)
DryS2 Amieur 34° 02’ 05” 1° 14’17” 319S3 Bensekrane 34° 04’ 49” 1° 13’10” 263S4 Saf Saf 34° 53’ 58” 1° 16’49” 599S5 Mansourah 34° 51’ 27” 1° 20’ 50” 907 Red soil, fersiallitic to vertisol, high water content
(rich in clays)Humid
S6 BeniBoublene
34° 51’ 41” 1° 20’ 15” 908
S7 Lalla Setti 34° 51’ 45” 1° 18’ 56” 1030S8 Mafrouche 34° 51’ 00” 1° 17’ 48” 1140 Brown fersiallitic soil originating on limestone rock,
rich in Mg2+, Ca2+ and K+Humid andcoolerS9 Hafir 34°49’60" 1° 22’ 0” 1100
S10 Terny 34° 47’ 44” 1° 21’ 32” 1220
Note: Nature of soils and climate was reported by the Algerian Minister of Agriculture and Rural Development (26, 27).
Journal of Essential Oil Research 429
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triplicate and the inhibition zones formed in theexperimental dishes were compared with those of thecontrols.
Determination of the minimum inhibitory concentration(MIC)
The MIC assays were performed by standard dilutionmethods (37). The MIC was defined as the lowestproduct concentration that prevented visible growth ofbacteria. All tests were performed on Müller–Hintonagar. Briefly, 30 μL of two-fold serial dilutions in dim-ethylsulfoxyde (DMSO; Sigma-Aldrich) were added to15 mL of agar to obtain concentrations ranging from0.1 to 5 mg/mL of the tested product. The resultingagar solutions were mixed at high speed for 15 seconds,immediately poured into sterile Petri dishes andallowed to set for 30 minutes. The plates were thenspot inoculated by pipetting 105 CFU of the desiredstrain on the spot on the plates. A negative control wasprepared without essential oil, using only DMSO. Gen-tamycin and amphotericin B (Sigma-Aldrich) were usedas positive controls. Inoculated plates were incubated at37°C for 24 hours. After the incubation period, theplates were observed and recorded for the presence orabsence of growth. Each test was repeated at least threetimes.
Antifungal assay
Direct method
Antifungal assays were performed using the agar med-ium assay (38). Yeast extract sucrose (YES) mediumwith different concentrations of essential oil (1.0, 2.0and 4.0 μL/mL) were prepared by adding the appropri-ate quantity of the essential oils and Tween 80, tomelted medium, followed by manual rotation of theErlenmeyer flask to disperse the essential oil in themedium. About 20 mL of the medium were pouredinto glass Petri dishes (9 cm). Each Petri dish was inoc-ulated at the centre with a mycelial disc (6 mm diame-ter) taken at the periphery of A. flavus colonies grownon potato dextrose agar (PDA) for 48 h. Control plates(without essential oil) were inoculated following thesame procedure. Plates were incubated at 25°C forseven days and the colony diameter was recorded eachday. The MIC was defined as the lowest concentrationof essential oil in which no growth occurred. Theinhibited fungal discs of the oil treated sets were re-inoculated into fresh medium, and revival of theirgrowth was observed. Minimal fungicide concentration(MFC) is the lowest concentration at which no growthoccurred on the plates. The diameter of the fungal colo-nies of treatment and control sets was measured, and
the percentage inhibition (PI) of fungal growth wascalculated according to following formula (39):
PI ¼ 1� Dt
Dc� 100
where Dt is the diameter of growth zone in the testplate and Dc is the diameter of growth zone in thecontrol plate.
Statistical analysis
Chemical data analyses were performed using principalcomponent analysis (PCA) and cluster analysis (CA)(40). Both methods aim at reducing the multivariatespace in which objects (oil samples) are distributed butare complementary in their ability to present results(41). Indeed, PCA provides the data for diagrams inwhich both objects (oil samples) and variables (oilcomponents) are plotted while canonical analysisinforms a classification tree in which objects (sampleregions) are gathered. PCA was carried out using the‘PCA’ function from the statistical R software. Thevariables (volatile components) have been selectedusing functions from the statistical software. CA pro-duced a dendrogram (tree) using Ward’s method ofhierarchical clustering, based on the Euclidean distancebetween pairs of oil samples.
Results and discussion
Chemical compositions of D. carota ssp. hispanicusessential oils
The chemical compositions of essential oils from roots,total aerial parts, leaves, stems and flowers of D. carotassp. hispanicus harvested form Hafir (S9) were investi-gated by GC–RI and GC–MS analysis. Eight, sixty-eight, sixty-one, fifty-three and forty-seven componentsaccounting for 99.1%, 97.4%, 96.1%, 95.0% and98.9% were identified in the essential oils of roots, totalaerial parts, leaves, stems and flowers, respectively(Table 3). Their RIs and normalized percent abun-dances are shown in Table 3. All components wereidentified by comparison of their EI–MS and GC–RIswith those of our laboratory-produced ‘Arômes’ library,with the exception of five components that wereidentified by comparison with spectral data and RIsfrom the literature (Table 3). Among them, twenty-eightsesquiterpenes, twenty monoterpenes, fourteen aliphat-ics, five phenolics and one diterpene compounds wereidentified. Phenylpropanoid is the dominant class ofcompounds of essential oils; they accounted for67.2–96.9%. However, two types of essential oils wereproduced by D. carota ssp. hispanicus according to theaerial or subterranean part of the plant. Apiole (80.3%)
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Table 3. Composition of the essential oils of Daucus carota ssp. hispanicus (roots, leaves, stems and flowers).
D. carota ssp. hispanicusfIdentificationg
No. Componentsa 1RIab RIac RIpd RFe Roots Aerial parts Stems Leaves Flowers
1 Pentan-3-one 654 652 954 1.40 – tr – tr – RI, MS2 Heptane 700 700 700 1.01 – 0.1 – 0.1 – RI, MS3 2-Methyl pentan-3-one 750 747 991 1.40 – 0.1 0.1 tr – RI, MS4 Hexanal 770 771 1049 1.40 – tr 0.1 tr – RI, MS5 (E)-2-Hexenal 832 830 1204 1.40 – 0.1 0.1 tr tr RI, MS6 α-Thujene 932 925 1014 1.01 – 0.3 4.3 0.5 0.1 RI, MS7 α-Pinene 936 931 1016 1.01 – tr 0.1 0.2 – RI, MS8 Thuja-2,4(10)-diene 946 943 1115 1.01 – 0.1 – tr – RI, MS9 Camphene 950 947 1062 1.01 – 0.1 tr – – RI, MS10 6-Methylhept-5-en-2-one 966 961 1325 1.40 – tr tr – – RI, MS11 Sabinene 973 968 1111 1.01 – tr tr – 0.1 RI, MS12 β-Pinene 978 974 1102 1.01 – 0.2 1.2 0.4 0.2 RI, MS13 2-Pentylfuran 981 978 1353 1.59 – 0.1 tr tr – RI, MS, Ref.14 Myrcene 987 980 1152 1.01 – 0.3 0.2 – tr RI, MS15 Isobutyl-2-methyl butyrate 994 991 1102 1.55 – 0.2 tr tr – RI, MS16 α-Phellandrene 1002 997 1155 1.01 – 0.1 0.2 0.2 – RI, MS17 α-Terpinene 1008 1011 1267 1.01 – 0.1 1.9 2 0.1 RI, MS18 p-Cymene 1011 1015 1256 1.01 – 0.1 0.9 tr tr RI, MS19 Limonene 1025 1022 1195 1.01 – 0.8 0.4 0.3 tr RI, MS20 (Z)-β-Ocimene 1024 1027 1221 1.01 – 1.1 2.6 tr 1.1 RI, MS21 (E)-β-Ocimene 1034 1037 1237 1.01 – tr 0.4 tr 0.1 RI, MS22 γ-Terpinene 1051 1051 1233 1.01 – 0.2 0.2 tr – RI, MS23 m-Tolualdehyde 1053 1055 1575 1.40 – 0.1 tr – 0.1 RI, MS24 4-Methyl benzaldehyde 1060 1064 1591 1.40 – 0.2 tr – 0.1 RI, MS25 Terpinolene 1082 1079 1274 1.01 – 0.3 0.1 – – RI, MS26 Nonanal 1076 1081 1387 1.40 – tr tr 0.1 tr RI, MS27 3-Methyl butyl isovalerate 1098 1090 1267 1.55 – tr 0.2 tr tr RI, MS28 2-Methyl butyl isovalerate 1102 1097 1284 1.55 – 0.3 0.2 tr tr RI, MS29 (Z)-Ocimene oxide 1115 1115 1365 1.59 – tr tr tr tr RI, MS, Ref.30 allo-Ocimene 1120 1121 1359 1.01 – tr tr tr – RI, MS31 (E)-Ocimene oxide 1125 1127 1377 1.59 – 0.1 tr tr – RI, MS, Ref.32 (E)-2-Nonenal 1136 1134 1522 1.40 – 0.1 tr tr – RI, MS33 Lyratol 1150 1148 1769 1.34 – 0.1 – 0.1 – RI, MS34 (E)-2-Nonen-1-ol 1153 1152 1672 1.34 – 0.2 tr – tr RI, MS35 (E)-2-Decenal 1240 1237 1646 1.40 – tr 0.5 tr – RI, MS36 Bornyl acetate 1270 1265 1571 1.55 – 0.1 0.3 0.4 – RI, MS37 α-Longipinene 1360 1357 1465 1.0 – tr tr tr tr RI, MS38 α-Ylangene 1376 1371 1470 1.0 – 0.3 0.2 – tr RI, MS39 α-Copaene 1379 1382 1457 1.0 – 0.1 0.2 tr 0.1 RI, MS40 β-Bourbonene 1386 1385 1510 1.0 – 0.2 – tr tr RI, MS41 β-Ylangene 1420 1413 1560 1.0 – 0.5 1.1 0.4 0.3 RI, MS42 (E)-β-Caryophyllene 1421 1416 1579 1.0 – 0.2 tr – tr RI, MS43 (E)-α-Bergamotene 1434 1430 1572 1.0 – 0.2 0.2 0.1 tr RI, MS44 α-Himachalene 1450 1447 1630 1.0 – 0.1 0.2 0.1 0.1 RI, MS45 α-Humulene 1455 1460 1660 1.0 – 0.4 tr 0.2 0.1 RI, MS46 α-Curcumene 1473 1468 1763 1.0 tr 0.1 1.0 0.9 0.1 RI, MS47 Germacrene D 1479 1477 1704 1.0 0.3 3.1 3.0 2.9 6.4 RI, MS48 Zingiberene 1489 1489 1717 1.0 – 0.3 0.3 0.4 0.2 RI, MS49 Bicyclogermacrene 1494 1495 1720 1.0 – 0.3 0.2 – 0.7 RI, MS50 Myristicin 1489 1499 2186 1.25 16.6 73.2 66.9 80.2 83.8 RI, MS51 δ-cadinene 1507 1503 1742 1.0 – 0.2 1.6 0.2 0.2 RI, MS52 Elemicin 1518 1520 2232 1.25 – 0.3 0.1 tr 0.6 RI, MS53 (E)-α-Bisabolene 1531 1526 1776 1.0 0.1 0.1 0.1 tr 0.1 RI, MS54 Elemol 1541 1539 2070 1.34 – 0.4 0.1 0.2 tr RI, MS55 Epiglobulol 1558 1550 2013 1.34 1.4 5.1 2.1 3.1 1.2 RI, MS56 Epoxy salvial-1,5,4(14)-ene 1560 1562 1902 1.55 – 0.1 1.4 0.1 0.2 RI, MS57 Spathulenol 1572 1568 2110 1.34 – 0.2 0.3 0.3 tr RI, MS58 cis-Sesquisabinene hydrate 1586 1566 2099 1.34 0.1 0.3 0.1 0.1 tr RI, MS
(Continued)
Journal of Essential Oil Research 431
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was the ultra-major component, followed by myristicin(16.6%) in the root essential oils, while myristicin(66.9–83.8%) was alone the main component in aerialpart oils (Figure 1, sample of S4). On moving from thebottom to the top of the plant, it is noteworthy that thenormalized abundances of myristicin increased as fol-lows: 16.6% in the roots, 66.9% in the stems, 80.2% inthe leaves and 83.8% in the flowers. Concentrations ofterpenic compounds were very much lower in rootessential oils (2.2%) and higher (12.6–28.6%) in theaerial part essential oils. This compound class wasmainly represented by germacrene D (2.9–6.4%) andepiglobulol (1.2–5.1%). Only a few quantitative differ-ences occurred between the chemical compositions ofessential oils from separate organs (leaves, stems andflowers) and those of aerial parts. Relative to the stud-ies reported previously in the literature (Table 1), thechemical compositions of the essential oils from Alge-rian D. carota ssp. hispanicus exhibit singular original-ity. Aerial part essential oils were clearly different fromthose of other origins in which myristicin has neverbeen reported. As myristicin (29.7%) and dillapiole(46.6%) were identified as main components in the rootoil of D. carota ssp. maritimus from Tunisia, the rootessential oil of Algerian D. carota ssp. hispanicus wasdifferent, shown by the occurrence of apiole, as
reported here for the first time, as a D. carota essentialoil component.
Chemical variability of D. carota ssp. hispanicusessential oils
GC–RI and GC–MS analysis of D. carota ssp. hispani-cus essential oils obtained from the aerial parts androots from ten Algerian locations allowed the character-ization of 92.3–98.5% and 97.4–99.4% of the oils,respectively (Table 4). Although the ten essential oilsof aerial parts were qualitatively similar, there were afew differences in the normalized percent abundancesof their main components: myristicin (62.9–86.2%),epiglobulol (1.1–6.6%), germacrene D (1.2–5.3%), api-ole (1.1–4.1%) and viridiflorol (0.5–2.3%). Consideringthe essential oils from roots, there are significant differ-ences in the normalized percent abundances of theirmain components. For instance, apiole ranged from13.2% to 81.3% and myristicin ranged from 15.6% to83.4% (Table 4). PCA and CA (dendrograms) wereapplied to identify possible relationships between theessential oil compositions and geographical origins ofsamples. The data presented in Figures 2 and 3 wereobtained from the correlation matrix and the standard-ized matrix linking the essential oil compositions to
Table 3. (Continued).
D. carota ssp. hispanicusfIdentificationg
No. Componentsa 1RIab RIac RIpd RFe Roots Aerial parts Stems Leaves Flowers
59 Caryophyllene oxide 1578 1574 1937 1.59 – 0.4 0.5 tr – RI, MS60 4(14)-Salvialene-1-one 1592 1585 2109 1.31 – 0.5 0.1 0.1 0.1 RI, MS, Ref.61 Viridiflorol 1592 1594 2083 1.34 0.3 2.3 0.5 0.7 0.3 RI, MS62 Guaiol 1593 1589 2090 1.34 – 0.6 0.1 0.1 tr RI, MS63 Aromadendrene oxide II 1623 1620 1996 1.59 – 0.1 0.3 0.1 0.1 RI, MS64 s-Muurolol 1633 1626 2138 1.34 – 0.1 0.1 0.1 0.3 RI, MS65 s-Cadinol 1633 1632 2160 1.34 – 0.8 0.4 0.1 0.3 RI, MS66 α-Cadinol 1643 1641 2223 1.34 – 0.2 0.8 0.1 0.1 RI, MS67 Apiole 1649 1646 2402 1.25 80.3 1.1 0.2 0.1 1.7 RI, MS, Ref.68 (E)-Phytol 2114 2015 2568 1.34 – 0.1 tr 0.1 tr RI, MSTotal identification % 99.1 97.4 96.1 95.0 98.9Essential oil yields % (w/w) 0.4 2.2 0.1 1.6 2.1
% Hydrocarbon compounds 0.4 9.9 20.6 8.8 10.0% Monoterpene hydrocarbons – 3.7 12.5 3.6 1.7% Sesquiterpene hydrocarbons 0.4 6.1 8.1 5.2 8.3% Non-terpenic hydrocarbon compounds – 0.1 – – –% Oxygenated compounds 98.7 87.5 75.5 86.2 88.9% Oxygenated monoterpenes – 0.6 1.2 0.5 –% Oxygenated sesquiterpenes 1.8 11.1 6.8 5.1 2.6% Non-terpenic oxygenated compounds – 0.8 0.3 0.2 –% Oxygenated diterpenes – 0.1 – 0.1 –% Phenylpropanoids 96.9 74.9 67.2 80.3 86.3
Notes: aOrder of elution is given on apolar column (Rtx-1). bRetention indices of literature on the apolar column (lRIa) reported from the literature(40). cRetention indices on the apolar Rtx-1 column (RIa). dRetention indices on the polar Rtx-Wax column (RIp). eResponse factors (RF). fSam-ple: S9, Hafir. Percentages (means of three analyses). gRI, retention indices; MS, mass spectrometry in electronic impact mode; Ref., compoundsidentified from literature data (40).
432 A. Bendiabdellah et al.
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sample locations. The distribution of the six discrimi-nated compounds (Z)-β-ocimene, myristicin, germac-rene D, epiglobulol, viridiflorol and apiol is illustratedin Figure 2. As shown in Figures 2 and 3, the principalfactorial plane accounts for 83.32% of the chemicalvariability of the essential oil variance. The dendrogramand plot established using the first two axes suggeststhat there are two main clusters of D. carota ssp. hispa-nicus oils (Figures 2 and 3). One cluster included allsample oils from aerial parts and the other clusterincluded all sample oils from roots. Sample oils fromaerial parts represented a homogeneous group (S1–10)characterized by high amounts of myristicin (66.7–86.2%). Sample oils from roots were divided into twosub-groups according to the normalized percent abun-dances of myristicin and apiole (Table 5). The first sub-group (S1–S4) was myristicin rich (58.0–83.4%) withapiole (13.2–39.7%) and the second sub-group (S5-S10) contained higher amounts of apiole (54.1–81.3%)and lower amounts of myristicin (15.6–39.2%).
Correlation between essential oils chemical variabilityand environmental parameters of sample locations
PCA and CA analysis revealed the chemical variabilityof the root essential oils of D. carota ssp. hispanicusfrom ten locations (Figures 2 and 3). It is noteworthythat the normalized percent abundances of both majoroil components differed greatly according to environ-mental parameters of sample locations. Specimens fromKihal, Amieur, Besekrane and Saf-Saf locations (Area1: S1–S4) growing on calcareous soils with a dry cli-mate produced myristicin-dominant essential oils, whilespecimens from Mansourah, Beni Boublene Lalla Setti,Mafrouche, Hafir and Terny (Area 2: S5–S10) growingon mineral-rich soils with humid and cooler climates
produced apiole-dominant essential oils. By contrast, itseems that essential oils of aerial parts from D. carotassp. hispanicus were not affected by the nature of soilsand the climate. In addition, it is interesting to note thedirect correlation between the essential oil yields andthe area of harvest. This correlation occurs for D. carotassp. hispanicus roots and aerial parts. Specimensoriginating from calcareous soils with a dry climate(Area 1: S1–S4) exhibited lower yields (0.13–0.22%for the root oils and 0.4–0.85% for the aerial part oils),and specimens from mineral-rich soils and humid andcooler climates (Area 2: S5–S10) exhibited higheressential oil yields (1.0–1.6% for the root oils and1.2–3.1% for the aerial part oils).
Generally, the observed differences in chemicalcomposition of the various oils can be the consequenceof many factors. Such factors may include differencesin climatic conditions, geographical locations and soiltypes (42, 43). Biotic and abiotic stresses exert a con-siderable influence on the production of several second-ary metabolites in plants (44). Drought is one of themost important abiotic stress factors (45), affectingplant growth and leaf photosynthesis (46) and alteringthe biochemical properties of plants (47). In the sameway, changes in the chemical compositions of theessential oils have been reported according to soil type(48, 49). To advance in the study of the chemical vari-ability of D. carota ssp hispanicus from Algeria, itcould be interesting to determine the genetic diversityof the populations studied here.
Antibacterial activity
The essential oil was evaluated for antibacterial activityagainst pathogenic strains of Gram-positive (S. aureus,E. faecalis, L. monocytogenes, B. cereus and B. subtilis)
Figure 1. Chromatogram of aerial part and root of essential oil of Daucus carota ssp. hispanicus from Chelaida (S4).
Journal of Essential Oil Research 433
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Table
4.Chemical
compo
sitio
nsof
Dau
cuscarota
ssp.
hispanicus
essentialoilsfrom
Algeria.
D.carota
ssp.
hispan
icus
samples
e
Roo
tsAerialparts
No.
Com
ponentsa
lRIa
bRIa
cRIp
dS1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
Id.f
1Penta-3-one
654
652
954
––
––
––
––
––
–tr
tr0.1
0.1
0.1
trtr
trtr
RI,MS
2Heptane
700
700
700
––
––
––
––
––
–tr
–0.1
0.1
trtr
tr0.1
0.1
RI,MS
32-Methy
lpentan-3-on
e75
074
799
1–
––
––
––
––
––
–tr
0.2
tr0.1
0.1
0.1
0.1
trRI,MS
4Hexanal
770
771
1049
––
––
––
––
––
–tr
tr0.1
tr0.2
0.1
0.1
tr0.1
RI,MS
5(E)-2-Hexenal
832
830
1204
––
––
––
––
––
–tr
–0.1
0.1
0.1
0.1
0.1
0.1
trRI,MS
6α-Thu
jene
932
925
1014
––
––
––
––
––
0.1
–tr
0.1
0.1
tr0.1
0.1
0.3
0.1
RI,MS
7α-Pinene
936
931
1016
––
––
––
––
––
tr0.3
tr0.5
0.8
0.2
0.9
0.9
tr0.1
RI,MS
8Thu
ja-2,4(10)-diene
946
943
1115
––
––
––
––
––
–tr
trtr
0.2
0.1
0.1
0.1
0.1
trRI,MS
9Cam
phene
950
947
1062
––
––
––
––
––
–tr
–0.1
0.3
trtr
tr0.1
trRI,MS
106-Methy
lhept-5-en-2-
one
966
961
1325
––
––
––
––
––
tr–
tr0.1
0.1
0.1
0.1
0.1
trtr
RI,MS
11Sabinene
973
968
1111
––
––
––
––
––
trtr
tr0.2
tr0.2
0.1
0.1
tr0.1
RI,MS
12β-Pinene
978
974
1102
––
––
––
––
––
0.1
0.1
0.2
0.4
0.9
0.8
1.1
0.8
0.2
1.8
RI,MS
132-Pentylfuran
981
978
1353
––
––
––
––
––
trtr
–0.1
0.1
0.2
trtr
0.1
0.2
RI,MS,
Ref.
14Myrcene
987
980
1152
––
––
––
––
––
0.2
0.1
0.1
0.3
tr0.1
0.2
0.2
0.3
0.4
RI,MS
15Isobutyl-2-m
ethyl
butyrate
994
991
1102
––
––
––
––
––
tr0.1
0.1
0.1
tr0.1
0.1
0.1
0.2
0.4
RI,MS
16α-Phelland
rene
1002
997
1155
––
––
––
––
––
tr0.1
–tr
0.1
0.1
0.2
0.2
0.1
0.3
RI,MS
17α-Terpinene
1008
1011
1267
––
––
––
––
––
tr0.1
–tr
0.1
0.1
1.9
0.9
0.1
0.2
RI,MS
18p-Cym
ene
1011
1015
1256
––
––
––
––
––
0.1
0.1
trtr
tr0.1
0.1
0.1
0.1
0.1
RI,MS
19Lim
onene
1025
1022
1195
––
––
––
––
––
tr0.3
0.5
0.4
0.6
0.1
0.9
0.7
0.8
0.9
RI,MS
20(Z)-β-Ocimene
1024
1027
1221
––
––
––
––
––
1.3
1.7
1.8
1.9
2.6
1.6
2.6
2.6
1.1
2.2
RI,MS
21(E)-β-Ocimene
1034
1037
1237
––
––
––
––
––
0.1
0.1
0.1
0.1
0.1
0.1
trtr
tr0.1
RI,MS
22γ-Terpinene
1051
1051
1233
––
––
––
––
––
0.1
0.1
tr0.2
0.1
tr0.2
0.2
0.2
0.3
RI,MS
23m-Tolualdehyd
e10
5310
5515
75–
––
––
––
––
–0.1
0.1
–0.1
0.1
tr0.1
0.1
0.1
trRI.MS
244-methyl-Benzaldehyde
1060
1064
1591
––
––
––
––
––
0.1
0.1
tr0.1
0.1
tr0.1
0.1
0.2
0.2
RI,MS
25Terpinolene
1082
1079
1274
––
––
––
––
––
0.1
0.1
0.1
0.1
0.1
tr0.2
0.2
0.3
0.4
RI,MS
26Non
anal
1076
1081
1387
––
––
––
––
––
0.1
0.1
tr0.2
0.3
tr0.1
0.1
trtr
RI,MS
273-Methy
lbu
tyl
isov
alerate
1098
1090
1267
––
––
––
––
––
0.1
tr0.1
tr0.2
0.1
0.1
0.1
tr0.1
RI,MS
282-Methy
lbu
tyl
isov
alerate
1102
1097
1284
––
––
––
––
––
0.1
–0.1
0.1
0.1
0.1
0.2
0.2
0.3
0.2
RI,MS
29(Z)-Ocimeneox
ide
1115
1115
1365
––
––
––
––
––
0.1
–0.1
tr0.2
0.1
0.3
0.3
trtr
RI,MS,
Ref.
30allo-O
cimene
1120
1121
1359
––
––
––
––
––
0.1
tr0.1
tr0.2
tr0.1
0.1
tr0.1
RI,MS
31(E)-Ocimeneox
ide
1125
1127
1377
––
––
––
––
––
0.1
tr0.1
0.1
0.1
tr0.2
0.2
0.1
0.2
RI,MS,
Ref.
32(E)-2-Non
enal
1136
1134
1522
––
––
––
––
––
0.1
–0.1
0.2
0.1
tr0.1
0.1
0.1
trRI,MS
33Lyratol
1150
1148
1769
––
––
––
––
––
0.1
tr0.1
0.1
0.1
0.1
trtr
0.1
trRI,MS
34(E)-2-Non
en-1-ol
1153
1152
1672
––
––
––
––
––
0.1
–0.1
0.1
0.2
0.1
trtr
0.2
trRI,MS
434 A. Bendiabdellah et al.
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35(E)-2-Decenal
1240
1237
1646
––
––
––
––
––
0.1
tr0.1
tr0.5
0.1
0.3
0.3
tr0.1
RI,MS
36Borny
lacetate
1270
1265
1571
––
––
––
––
––
0.1
tr0.1
0.1
0.1
tr0.2
0.2
0.1
trRI,MS
37α-Lon
gipinene
1360
1357
1465
––
––
––
––
––
0.1
tr0.1
tr0.2
0.1
0.1
0.1
tr0.1
RI,MS
38α-Ylang
ene
1376
1371
1470
––
––
––
––
––
0.1
0.1
0.1
0.2
0.3
0.2
0.1
0.2
0.3
0.3
RI,MS
39α-Cop
aene
1379
1382
1457
––
––
––
––
––
0.1
0.1
tr0.3
0.1
0.2
0.1
0.1
0.1
0.2
RI,MS
40β-Bou
rbon
ene
1386
1385
1510
––
––
––
––
––
0.1
0.1
tr0.1
0.1
0.2
0.2
0.2
0.2
0.1
RI,MS
41β-Ylang
ene
1420
1413
1560
––
––
––
––
––
0.1
0.1
0.3
0.2
0.9
0.3
0.5
0.5
0.5
0.5
RI,MS
42(E)-β-Caryo
phyllene
1421
1416
1579
––
––
––
––
––
0.1
0.1
tr0.1
0.1
0.1
0.2
0.2
0.2
0.1
RI,MS
43(E)-α-Bergamotene
1434
1430
1572
––
––
––
––
––
0.1
0.1
0.1
0.1
0.1
tr0.1
0.1
0.2
0.1
RI,MS
44α-Him
achalene
1450
1447
1630
––
––
––
––
––
0.1
0.1
tr0.2
0.2
0.1
0.1
0.1
0.1
0.2
RI,MS
45α-Hum
ulene
1455
1460
1660
––
––
––
––
––
0.1
0.1
0.4
0.4
0.9
0.3
0.6
0.6
0.4
0.8
RI,MS
46α-Curcumene
1473
1468
1763
––
––
––
––
––
0.1
0.1
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.2
RI,MS
47Germacrene
D14
7914
7717
04tr
0.3
0.1
0.1
1.2
1.2
0.4
0.4
0.3
0.2
1.2
2.2
2.3
1.9
3.2
2.1
3.1
3.1
3.1
5.3
RI,MS
48Zingiberene
1489
1489
1717
––
––
––
––
––
0.4
0.1
0.4
0.4
0.4
0.2
0.3
0.1
0.3
0.4
RI,MS
49Bicyclogerm
acrene
1494
1495
1720
––
––
––
––
––
0.1
0.1
–0.1
0.1
0.1
0.4
0.5
0.3
0.5
RI,MS
50Myristicin
1489
1499
2186
83.4
83.3
57.4
58.3
39.2
39.5
25.9
25.5
16.6
15.6
86.2
84.1
80.1
78.6
70.3
76.5
66.7
69.2
73.2
62.9
RI,MS
51δ-cadinene
1507
1503
1742
––
––
––
––
––
0.2
0.1
0.2
0.1
0.3
0.1
0.1
0.1
0.2
0.3
RI,MS
52Elemicin
1518
1520
2232
––
––
––
––
––
0.1
0.1
0.2
0.2
0.5
0.3
0.3
0.3
0.3
0.4
RI,MS
53(E)-α-Bisabolene
1531
1526
1776
0.3
0.3
0.1
0.1
0.5
0.5
0.1
0.1
0.1
0.1
0.2
0.1
0.1
0.1
0.1
tr0.2
0.2
0.1
0.2
RI,MS
54Elemol
1541
1539
2070
––
––
––
––
––
0.1
0.1
tr0.1
0.1
tr0.3
0.3
0.4
0.6
RI,MS
55Epiglob
ulol
1558
1550
2013
2.1
2.0
1.2
1.3
1.9
1.8
2.7
2.6
1.4
1.3
1.1
1.7
1.9
3.1
1.4
6.6
2.1
3.1
5.1
4.8
RI,MS
56Epo
xysalvial-1,5-4
(14)-ene
1560
1562
1902
––
––
––
––
––
0.2
0.1
0.1
0.3
0.3
0.1
0.3
0.3
0.1
0.1
RI,MS
57Spathulenol
1572
1568
2110
––
––
––
––
––
0.1
0.1
0.2
0.4
0.4
0.2
0.1
0.1
0.2
0.1
RI,MS
58cis-Sesqu
isabinene
hydrate
1586
1566
2099
0.1
0.1
0.1
0.2
0.4
trtr
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.3
0.2
RI,MS
59Caryophyllene
oxide
1578
1574
1937
––
––
––
––
––
0.1
0.1
0.1
0.4
0.5
0.5
0.2
0.2
0.4
0.2
RI,MS
604(14
)-Salvialene-1-on
e15
9215
8521
09–
––
––
––
––
–0.1
0.1
0.1
0.1
0.1
0.1
0.4
0.3
0.5
0.3
RI,MS,
Ref.
61Viridiflorol
1592
1594
2083
0.1
0.2
0.2
0.1
0.1
0.3
0.2
0.3
0.3
0.2
1.1
1.2
1.1
1.2
1.5
1.2
1.6
2.2
2.3
0.5
RI,MS
62Guaiol
1593
1589
2090
––
––
––
––
––
0.3
0.1
0.3
0.7
0.4
0.3
0.2
0.2
0.6
0.3
RI,MS
63Aromadendreneoxide
II1623
1620
1996
––
––
––
––
––
0.1
0.1
0.1
0.2
0.4
0.1
0.2
0.2
0.1
0.5
RI,MS
64s-Muu
rolol
1633
1626
2138
––
––
––
––
––
0.1
0.1
0.3
0.3
0.2
0.2
0.1
0.1
0.1
0.2
RI,MS
65s-Cadinol
1633
1632
2160
––
––
––
––
––
0.4
0.1
0.1
0.5
0.7
0.8
0.4
0.4
0.8
0.7
RI,MS
66α-Cadinol
1643
1641
2223
––
––
––
––
––
tr0.1
0.1
0.1
0.2
0.1
0.3
0.3
0.2
0.4
RI,MS
67Apiole
1649
1646
2402
13.3
13.2
39.7
38.8
55.4
54.1
69.1
69.9
80.3
81.3
1.1
1.2
1.3
1.4
1.5
1.2
1.7
1.8
1.1
4.1
RI,MS,
Ref.
68(E)-Phy
tol
2114
2015
2568
––
––
––
––
––
tr0.1
tr0.2
0.1
0.1
0.2
0.2
0.1
trRI,MS
Total
identification
%99
.399
.498
.898
.998
.797
.498
.498
.999
.198
.897
.796
.594
.198
.594
.697
.692
.394
.997
.494
.3%
Essentialoilyields
0.22
0.13
0.15
0.19
1.1
1.2
1.3
1.1
1.6
1.0
0.40
0.49
0.50
0.85
1.2
1.5
1.8
2.1
3.1
2.2
%Hyd
rocarbon
compou
nds
0.3
0.6
0.2
0.2
1.7
1.7
0.5
0.5
0.4
0.3
5.3
6.6
7.0
8.7
13.4
7.7
14.9
13.4
9.9
16.5
%Monoterpene
hydrocarbons
––
––
––
––
––
2.2
3.1
2.9
4.3
6.2
3.5
8.7
7.2
3.7
7.1
%Sesqu
iterpenehy
drocarbo
ns0.3
0.6
0.2
0.2
1.7
1.7
0.5
0.5
0.4
0.3
3.1
3.5
4.1
4.3
7.1
4.2
6.2
6.2
6.1
9.3
%Non
-terpenichy
drocarbo
ncompo
unds
––
––
––
––
––
––
–0.1
0.1
––
–0.1
0.1
%Oxy
genated
compou
nds
9998
.898
.698
.797
95.7
97.9
98.4
98.7
98.5
92.4
90.4
87.1
89.8
81.2
89.9
77.4
81.5
87.5
77.8
%Oxy
genatedmon
oterpenes
--
--
––
––
––
0.6
–0.6
0.4
0.8
0.4
91
0.6
0.5
(Con
tinued)
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Table
4.(Con
tinued).
D.carota
ssp.
hispan
icus
samples
e
Roo
tsAerialparts
No.
Com
ponentsa
lRIa
bRIa
cRIp
dS1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
Id.f
%Oxy
genatedsesquiterpenes
2.3
2.3
1.5
1.6
2.4
2.1
2.9
31.8
1.6
3.8
44.5
7.5
6.3
10.3
6.3
7.8
11.1
8.9
%Non
-terpenicox
ygenated
compo
unds
––
––
––
––
––
0.4
0.2
0.4
1.3
1.5
1.1
11
0.8
0.8
%Oxy
genatedditerpene
––
––
––
––
––
–0.1
–0.2
0.1
0.1
0.2
0.2
0.1
–%
Pheny
lpropano
ids
96.7
96.5
97.1
97.1
94.6
93.6
95.0
95.4
96.9
96.9
87.6
85.6
81.6
80.4
72.5
7868
.971
.574
.967
.6
Notes:a O
rder
ofelutionis
givenon
apolar
column(Rtx-1).
bRetentio
nindicesof
literatureon
theapolar
column(lRIa)reported
from
literature(40).c Retentio
nindiceson
theapolar
Rtx-1
column
(RIa).
dRetentio
nindiceson
thepo
larRtx-W
axcolumn(RIp).
e Algeriansamples:S1.
Kihal;S2.
Amieur;S3.
Bensekrane;
S4.
Chelaida;
S5.
Mansourah;S6.
BeniBoublene;
S7.
Mafrouche;S8.
Lalla
Setti;
S9.
Hafir;S10.Terny.
Percentages
(means
ofthreeanalyses).
f Id.,identifi
catio
n;RI,retentionindices;MS,massspectrom
etry
inelectronic
impact
mode;
Ref.,compounds
identified
from
literature
data
(40).
436 A. Bendiabdellah et al.
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and Gram-negative (E. coli and K. pneumoniae) bacte-ria. According to the results given in Table 6, bothessential oils exhibited strong antimicrobial activity
against C. albicans. The average zone of inhibition ofD. carota ssp. hispanicus essential oil against C. albicansis situated at 26 and 30 mm for the aerial parts and roots,
Figure 2. Principal component analysis (PCA) of chemical compositions of Daucus carota ssp. hispanicus oils. Distribution ofvariables (component codes corresponding to those of Table 4) and distribution of samples (coding numbers of locations).R, roots; Ap, aerial parts.
Figure 3. Cluster analysis (CA) of chemical compositions of Daucus carota ssp. hispanicus from Algeria. R, roots; Ap, aerial parts.
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respectively. However, B. subtilis was also prone togrowth moderate inhibition, with diameter zones ofinhibition ranging from 14 to 16 mm. The rest of thebacterial strains (L. monocytogenes, B. cereus, S. aur-eus, E. faecalis, K. pneumoniae and E. coli) showed noinhibition, with the diameter of zones of inhibitionranging from 6 to 10 mm (Table 6). The antimicrobialactivity of root and aerial part essential oils was con-firmed by the microdilution broth assay. As shown inTable 6, the most promising results were obtained fromthe aerial part oil, which had the lowest MIC value(0.078 mg/mL) against C. albicans. The aerial part oilalso showed an antimicrobial effect against B. subtilisand S. aureus, with an MIC of 1.2 and 4.8 mg/mL,respectively. It should be noted that the highest testedconcentration (5 mg/mL) had no effect on othermicroorganism growth (L. monocytogenes, B. cereus,S. aureus, E. faecalis, K. pneumoniae and E. coli). Theessential oil of D. carota ssp. hispanicus is mainlycomposed of two kind of phenolic compounds(myristicin and apiole). However, many phenolic com-pounds exhibit a wide range of biological effects (50,51), especially antimicrobial activity.
Fungicidal activity
The results of in vitro assays showed that the rootessential oil of D. carota ssp. hipanicus had a strongfungicidal effect against the growth of A. flavus. Theresults are given in Table 7. The MIC of root essentialoil of D. carota ssp. hispanicus was found to be4.0 μL/mL against a toxigenic strain of A. flavus. Theresults of mycelial percentage growth inhibition (PI)are given in Table 7 and indicated that the radialgrowth of strains was totally inhibited by the essentialoil. The PI was significantly (p < 0.05) influenced byincubation time and essential oil concentration. Myceliagrowth was considerably reduced with increasingconcentration of essential oil. The root essential oil wasmore active than the essential oil obtained from the aer-ial parts. The percentage of the inhibition zone and theMIC value of the root essential oil were recorded as100% and 4 μg/mL after eight days, respectively. Also,the concentration 4 μg/mL of the aerial part essentialoil exhibited a low inhibition, with a percentage reduc-tion of 42.22 after eight days (Table 7). The root essen-tial oil was found to be effective against A. flavus. Thebioactivity of the essential oil may be due to the pres-
Table 5. Clustering of Daucus carota ssp. hispanicus samples from the statistical analysis.
No.a Components
Roots
Group I (S1–4) Group II (S5–10)
Rangeb Averageb Rangeb Averageb
Phenylpropanoid compounds 96.8 99.850 Myristicin 58.3–83.4 70.6 15.6–39.5 31.471 Apiole 13.2–38.8 26.2 54.1–81.3 68.4
Notes: aThe numbering refers to those of Table 4. bNormalized percent abundances.
Table 6. Antibacterial activity of Daucus carota ssp. hispanicus essential oils (EO) using agar disc diffusion and minimal inhibi-tion concentration (MIC).
DD (mm) MIC (mg/mL)
Bacterial strains EO aerial parts EO roots EO aerial parts EO roots Gen (mg/mL) Am B (mg/mL)
Gram-positive bacteriumListeria monocytogenes 6.0 ± 0.00 6.0 ± 0.00 >5 >5 nt –Bacillus cereus 6.0 ± 0.00 6.0 ± 0.00 >5 >5 nt –Staphylococcus aureus 8.0 ± 0.1 10.0 ± 0.2 4.8 ± 0.6 4.2 ± 0.6 0.128 ± 0.02 –Bacillus subtilis 16.0 ± 0.6 14.0 ± 0.4 1.2 ± 0.4 1.5 ± 0.5 0.156 ± 0.09 –Enterococcus faecalis 6.0 ± 0.00 6.0 ± 0.00 >5 >5 nt –
Gram-negative bacteriumKlebsiella pneumoniae 6.0 ± 0.00 6.0 ± 0.00 >5 >5 0.625 ± 0.08 –Escherichia coli 6.0 ± 0.00 6.0 ± 0.00 >5 >5 0.256 ± 0.08 –
YeastsCandida albicans 26 ± 0.7 30 ± 0.9 0.078 ± 0.02 0.125 ± 0.04 – 0.312 ± 0.02
Notes: Am B, amphotericin B (10 µg/mL); Gen, gentamycin (10 µg/disc); DD, diameter of disc diffusion (mm); MIC, minimal inhibitory concen-trations (mg/mL); nt, not tested.
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ence of some highly fungitoxic components as phenyl-propanoids (52). For instance, apiole has been previ-ously reported as a specific inhibitor of aflatoxin (53).In the search for bioactive aflatoxin inhibitory com-pounds, D. carota ssp. hispanicus essential oils fromAlgeria were revealed to be interesting. The plants pro-duce essential oils dominated by myristicin and apiole,two phenylpropanoids compounds that accounted for93.6–97.1% and 67.2–86.3% in the roots and aerialparts, respectively. Root essential oils of D. carota ssp.hispanicus were found to be strongly fungicidal andinhibitory to aflatoxin production. Our study suggeststhat Algerian D. carota ssp. hispanicus essential oilshave the potential to be used as food preservatives.
AcknowledgementsThe authors are indebted to the Ministère des Affaires Etran-gères et Européennes throughout the ‘Partenariat HubertCurien Tassili’ research program.
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TestA. flavus colony diameters recorded (mm) Percentage myceliazone inhibition
EO (μg/mL) 3.0 3.5 4.0 3.0 3.5 4.0
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