Variation of the Chemical Composition and Antimicrobial Activity of the Essential Oils of Natural Populations of Tunisian Daucus carota L. (Apiaceae)

Variation of the Chemical Composition and Antimicrobial Activity of theEssential Oils of Natural Populations of Tunisian Daucus carota L.

(Apiaceae)

by Nesrine Rokbenia), Yassine M�rabeta), Salma Dziria), Hedia Chaabanea), Marwa Jemlia),Xavier Fernandezb), and Abdennacer Boulila*a)

a) Laboratory of Natural Substances, National Institute of Research and Physico-Chemical Analyses,Biotechpole of Sidi Thabet, Ariana, 2020, Tunisia (phone: þ216-71537666; fax: þ216-71537688;

email: [email protected])b) Institut de Chimie de Nice, UMR 7272, Universite de Nice – Sophia Antipolis, CNRS, Parc Valrose,

F-06108 Nice, Cedex 2

The essential oils of Daucus carota L. (Apiaceae) seeds sampled from ten wild populations spreadover northern Tunisia were characterized by GC-FID and GC/MS analyses. In total, 36 compounds wereidentified in the D. carota seed essential oils, with a predominance of sesquiterpene hydrocarbons in mostsamples (22.63–89.93% of the total oil composition). The main volatile compounds identified were b-bisabolene (mean content of 39.33%), sabinene (8.53%), geranyl acetate (7.12%), and elemicin(6.26%). The volatile composition varied significantly across the populations, even for oils of populationsharvested in similar areas. The chemometric principal component analysis and the hierarchical clusteringidentified four groups, each corresponding to a composition-specific chemotype. The in vitroantimicrobial activity of the isolated essential oils was preliminarily evaluated, using the disk-diffusionmethod, against one Gram-positive (Staphylococcus aureus) and two Gram-negative bacteria(Escherichia coli and Salmonella typhimurium), as well as against a pathogenic yeast (Candidaalbicans). All tested essential oils exhibited interesting antibacterial and antifungal activities against theassayed microorganisms.

Introduction. – In recent years, essential oils have been used in wide-ranging fields,especially for food curing and flavoring as well as in the fragrance and pharmaceuticalindustries [1]. At present, there is a growing interest in essential oils and theircomponents, particularly for their broad-spectrum antibacterial and antifungalactivities, which can provide, for instance, alternative functional ingredients to extendfood products shelf life and afford microbial safety for consumers [2].

The genus Daucus is considered as one of the richest reservoirs of volatiles in theApiaceae family [3]. Furthermore, the essential-oil composition of Daucus species hasbeen found highly species-specific, with the volatile constitutes being a reliablecriterion for chemotaxonomy [4]. In the current literature, several studies have beenpublished concerning the volatile-fraction composition of many Daucus species, such asD. glaber (Forssk.) Thell. [5], D. gingidium L. [6], D. crinitus [7], D. guttatus [8], D.muricatus L. [9], and, particularly, D. carota L. that was investigated in the largestnumber of studies [10– 12].

D. carota L. (wild carrot, Queen Anne�s lace), is the most prevalent species of thegenus Daucus, spreading over the Mediterranean region, which is known as its

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)2278

� 2013 Verlag Helvetica Chimica Acta AG, Z�rich

endemism center [3]. According to Alapetite [13], D. carota can be divided into threesubspecies, viz., carota, maximus, and maritimus. Moreover, it is valued in thetraditional Tunisian medicine for its appetizer, emmenagogue, or diuretic propertiesand as remedy for the treatment of cutaneous infections [14]. Previous studiesdescribing the essential-oil composition of D. carota revealed a high qualitative andquantitative degree of variation in the constituents. Generally, the most prevalentcomponents detected have been b-bisabolene, (E)-methylisoeugenol, sabinene, a-pinene, elemicin, myrcene, geranyl acetate, and carotol. Nevertheless, their oil contentswere highly variable according to several factors. This chemical polymorphism of D.carota oil was mostly dependent on the geographical origin [10] [15], the stage ofdevelopment [11] [16– 18], and the part of the plants [8] [19– 21].

Moreover, the essential oils of D. carota were found to exhibit antibacterial activity,namely against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudo-monas aeruginosa [19] [22], as well as antifungal activity against Candida albicans andAlternaria alternate [10] [18] [23]. However, the identification of the volatile constit-uents responsible for these antimicrobial activities remains a matter of active research[24].

To the best of our knowledge, there is a limited number of studies that have focusedon the chemical oil composition of D. carota growing wild in Tunisia [11] [20]. Indeed,none of them highlighted the species� variability in terms of volatile compounds withina large-scale sampling from different locations and bioclimates. Therefore, to enrich theknowledge in this area, the main aim of the present study was to provide a bettercomprehension of the chemical polymorphism of D. carota essential oils by perform-ing a multi-site sampling. Furthermore, the antimicrobial activity of the oils wasassessed.

Results and Discussion. – Essential-Oil Yield and Composition. Prospection carriedout during autumn 2010 over the natural distribution area of D. carota led to thecollection of seed samples from ten populations (P1 –P10) located in differentgeographical regions of northern Tunisia. The sampled populations grow wild indifferent bioclimatic zones, i.e., in lower humid, sub-humid, upper semi-arid, and lowersemi-arid bioclimatic zones as classified according to Emberger�s Q2 pluviothermiccoefficient [25]. The main ecological traits and the location of the prospected sites arereported in Table 1 and Fig. 1.

The yields of the essential oils obtained by hydrodistillation of D. carota seeds,presented in Table 2, ranged between 0.5 and 2.6% (w/w, based on the dry weight of theseeds). These values are in accordance with previous results obtained for carrot seedessential-oil yields, which varied from 0.6 to 3.3% [10] [22]. D. carota essential oil fromplants at the stage of ripe umbels with mature seeds is commonly used as naturalproduct in fragrance, flavor, and pharmaceutical industries.

Qualitative and quantitative analysis of volatile compounds was mainly investigatedusing both GC/MS and GC-FID techniques. Altogether, 36 compounds were identifiedrepresenting 82.12 to 97.89% (depending on the population) of the total oil (Table 2).Sesquiterpenes were highly predominant (74.37 – 90.38%) in the essential oils ofpopulations P2– P5, and P8. On the other hand, the essential oils derived frompopulations P6 and P10 mainly consisted of monoterpenes (more than 50% of the total

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 2279

https://www.researchgate.net/publication/285632366_Angiospermes-Dicotyledones_Apetales-_Dialypetales?el=1_x_8&enrichId=rgreq-83563f3d-3146-419e-a1f2-3da7e64e7e72&enrichSource=Y292ZXJQYWdlOzI1OTI3MTQ5NTtBUzoxMTQ1MDQ4MDUzOTIzODRAMTQwNDMxMTQ4MzExMw==

oil), followed by sesquiterpenes (33.95 and 38.82%, resp.). Finally, the oils obtainedfrom populations P1, P7, and P9 were dominated by sesquiterpenes (50.70, 64.04, and48.88%) and, to a lesser extent, by monoterpenes (29.38, 22.81, and 30.49%, resp.).


Table 1. Location and Main Ecological Factors of the Ten Daucus carota Populations Studied

Popula-tioncode

Locality name Geographi-cal region

Latitude(N)

Longitude(E)

Bio-climaticzonea)

Annualrainfall[mm/year]

P1 Takelsa Cap Bon 36848’9.64’’ 10838’13.81’’ II 600–800P2 Menzel Salem Cap Bon 3780’38.08’’ 10855’59.88’’ II 600–800P3 Al Houaria Cap Bon 3783’7.24’’ 1181’9.93’’ II 600–800P4 Zaouit El Megaiez Cap Bon 36856’42.10’’ 10853’46.37’’ II 600–800P5 Laarima Medjerda 3780’42.19’’ 9837’42.31’’ II 600–800P6 Joumine Medjerda 36855’32.53’’ 9823’15.70“ I >800P7 Zahret Medyen Medjerda 36851’22.14’’ 9816’44.28’’ I >800P8 Wed Maleeg Medjerda 36821’27.32’’ 8842’28.15’’ III 400–600P9 Zaafrana Tell 3689’33.93’’ 8849’10.99’’ III 400–600P10 Monastir Sahel 35845’9.73’’ 10846’53.16’’ IV 300–400

a) Bioclimatic zones according to Emberger [25]: I, lower humid; II, sub-humid; III, upper semi-arid; IV,lower semi-arid.

Fig. 1. Bioclimatic map of northern Tunisia showing the geographical location of the studied Daucuscarota populations. For the population codes (P1–P10), cf. Table 1. I – IV: bioclimatic zones.


Tabl

e2.

Com

posi

tion

ofth

eSe

edE

ssen

tial

Oils

Obt

aine

dfr

omT

enW

ild-G

row

ing

Dau

cus

caro

taP

opul

atio

ns(P

1–

P10

)

Com

poun

dna

me

and

clas

sa )R

Ib)

Con

tent

[%]c )

F-T

estd

)

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

Mea

n

a-P

inen

e93

51.

94b

1.47

c0.

57e

2.38

a0.

36g

1.43

c–

0.45

f0.

86d

0.84

d1.

03**

*Sa

bine

ne97

218

.78

b0.

76g

0.37

h1.

85e

0.14

j42

.03

a15

.03

c0.

27i

4.9

d1.

19f

8.53

***

b-P

inen

e97

57.

06a

1.06

f0.

73g

1.45

e0.

42i

3.05

c2.

27d

0.68

h3.

39b

0.66

h2.

07**

*b

-Myr

cene

988

0.73

b0.

10fg

0.24

def

0.45

c0.

05j

0.98

a0.

41cd

0.05

j0.

32cd

e0.

22ef

g0.

35**

*p-

Cym

ene

1025

0.73

b0.

22g

0.39

e0.

48d

0.14

h1.

6a

0.63

c0.

06i

0.32

f0.

14h

0.47

***

Lim

onen

e10

34tr

tr–

trtr

trtr

trtr

trtr

Bor

nyla

ceta

te12

820.

09c

0.04

etr

0.06

d0.

05e

1.34

b2.

87a

tr0.

05e

0.04

e0.

45**

*a

-Lon

gipi

nene

1348

0.13

c–

–tr

–0.

02d

tr0.

02d

3.09

b4.

38a

0.77

***

Ger

anyl

acet

ate

1379

0.04

e0.

02e

––

0.02

e0.

08d

1.59

ctr

20.6

3b

48.7

5a

7.12

***

b-C

ubeb

ene

1388

0.49

a0.

02e

0.02

e0.

05d

0.03

e0.

32b

0.30

b0.

06d

0.08

c0.

04d

0.14

***

a-G

urju

nene

1397

tr0.

04d

––

–0.

03d

0.10

c0.

43a

0.2

b0.

26b

0.11

***

b-C

aryo

phyl

lene

1417

2.68

a0.

85g

1.23

b1.

13c

0.11

i1.

01d

0.90

f0.

46h

0.46

h1.

00e

0.98

***

g-E

lem

ene

1431

0.68

d0.

85c

0.52

e1.

46a

0.98

b0.

34f

0.65

d0.

59e

0.39

f0.

19g

0.67

***

a-B

erga

mot

ene

1439

0.93

d1.

07c

0.66

f1.

94a

1.16

b0.

27i

0.38

h0.

74e

0.44

g0.

18j

0.78

***

a-H

imac

hale

ne17

470.

08c

tr–

––

0.02

d–

–0.

12b

0.16

a0.

04**

*a

-Hum

ulen

e14

524.

15a

0.94

e0.

85f

1.55

d0.

76g

3.93

b3.

51c

0.90

e0.

81f

0.57

h1.

80**

*b

-Far

nese

ne14

580.

31b

0.08

e–

0.11

dtr

0.38

a0.

26c

–0.

02f

0.02

f0.

12**

*g

-Muu

role

ne14

770.

54a

trtr

––

0.12

b0.

62a

––

0.03

b0.

13**

*G

erm

acre

neD

1479

10.5

8a

1.07

f0.

66h

1.28

e1.

51d

5.38

c6.

39b

0.83

g0.

40i

0.84

g2.

89**

*b

-Sel

inen

e14

864.

55b

0.28

g0.

48f

0.31

g0.

04i

0.08

h9.

29a

4.07

c3.

48d

0.64

e2.

32**

*a

-Sel

inen

e14

943.

02c

–0.

27ef

–0.

29e

0.24

f3.

85b

4.67

a–

2.39

d1.

47**

*(E

)-M

ethy

liso

euge

nol

1496

0.23

f1.

27c

–1.

83b

–0.

44e

0.65

d0.

41e

6.69

a–

1.15

***

b-H

imac

hale

ne14

990.

11e

0.09

e0.

19d

0.16

d0.

33c

––

–1.

27b

1.69

a0.

38**

*a

-Am

orph

ene

1505

3.79

a–

––

–3.

19b

2.33

c–

––

0.93

***

b-B

isab

olen

e15

093.

06i

70.6

0b

69.2

6c

61.9

0d

80.4

8a

3.63

h1.

67j

60.1

9e

27.8

6f

14.6

g39

.33

***

(Z)-

a-B

isab

olen

e15

140.

13b

0.13

b0.

33a

0.08

d0.

12c

–0.

07e

0.08

e0.

13b

0.12

c0.

12**

*d

-Cad

inen

e15

21–

––

0.42

b–

–1.

18a

0.33

c0.

25d

0.10

e0.

23**

*b

-Ses

quip

hella

ndre

ne15

231.

89b

0.33

e0.

30f

–0.

40d

2.88

a–

––

0.76

c0.

66**

*C

adin

e-1,

4-di

ene

1535

0.61

a–

––

–0.

47c

0.23

d–

–0.

53b

0.18

***

a-C

adin

ene

1538

0.31

j3.

32e

3.72

a3.

38d

3.71

b0.

33i

0.59

h3.

65c

1.85

f1.

34g

2.22

***


Tab

le2

(con

t.)

Com

poun

dna

me

and

clas

sa )R

Ib)

Con

tent

[%]c )

F-T

estd

)

P1

P2

P3

P4

P5

P6

P7

P8

P9

P10

Mea

n

Ele

mic

in15

541.

63g

10.0

4c

14.2

7b

9.08

d0.

06i

0.07

i0.

51h

15.8

9a

6.94

e4.

10f

6.26

***

Ger

mac

radi

en-5

-ol

1574

0.67

btr

0.04

g0.

11d

0.08

e1.

1a

0.25

c0.

05g

0.06

f0.

08e

0.24

***

Car

otol

1599

0.07

ef0.

11de

0.09

ef0.

12de

0.15

d1.

02c

27.0

8a

0.05

f1.

82b

0.04

f3.

06**

*a

-Cad

inol

1641

10.1

9a

0.14

f0.

06gh

0.08

g0.

03h

9.14

b4.

17e

0.09

g5.

68d

8.13

c3.

77**

*(E

)-A

saro

ne16

760.

18g

1.66

d2.

49c

4.29

b5.

41a

0.15

h0.

10i

2.41

c0.

49e

0.44

f1.

76**

*Se

lin-(

7)11

-en-

4a-o

l16

951.

71a

0.06

i0.

13h

0.28

e0.

19d

0.06

i0.

17g

0.22

f0.

48c

0.73

b0.

4**

*U

nkno

wn

1771

3.04

a–

––

––

––

––

0.3

***

Tota

lid

enti

fied

82.1

296

.66

97.8

996

.26

97.0

585

.13

88.1

197

.66

93.4

895

.23

Yie

ld[%

(w/w

)]e )

0.5

2.6

2.4

2.2

1.7

0.5

0.7

2.3

1.9

2.6

Tot

alm

onot

erpe

nes

29.5

03.

672.

316.

681.

1850

.53

22.8

11.

5533

.56

56.2

2M

onot

erpe

nehy

droc

arbo

ns29

.37

3.61

2.30

6.62

1.11

49.1

118

.35

1.53

12.8

87.

43O

xyge

nate

dm

onot

erpe

nes

0.13

0.06

0.01

0.06

0.07

1.42

4.46

0.02

20.6

848

.79

Tot

alse

squi

terp

enes

50.7

080

.02

78.8

374

.37

90.3

833

.95

64.0

477

.41

48.8

838

.82

Sesq

uite

rpen

ehy

droc

arbo

ns38

.06

79.7

078

.50

73.7

889

.93

22.6

332

.38

77.0

140

.84

29.8

4O

xyge

nate

dse

squi

terp

enes

12.6

40.

320.

330.

590.

4511

.32

31.6

60.

408.

048.

98P

heny

lpro

pano

ids

2.04

12.9

716

.76

15.2

05.

470.

661.

2718

.71

14.1

24.

55

a )Id

enti

fica

tion

ofth

eco

mpo

unds

base

don

the

com

pari

son

ofre

tent

ion

indi

ces

(RIs

)an

dm

ass

spec

tra

wit

hth

ose

ofG

C/M

Slib

rari

esan

dlit

erat

ure

data

(cf.

Exp

er.

Par

t).

b)

RI,

Ret

enti

onin

dex

dete

rmin

edre

lati

veto

ase

ries

ofn-

alka

nes

(C8–

C30

)on

the

apol

arH

P-5

MS

colu

mn.

c )P

1–

P10

,in

divi

dual

cont

ents

(mea

nva

lues

,n¼

3)in

the

oils

ofpo

pula

tion

sP

1–

P10

(for

popu

lati

onco

des

and

deta

ils,c

f.T

able

1);M

ean,

mea

nco

nten

tofa

llpo

pula

tion

s(P

1–

P10

);va

lues

,fol

low

edby

diff

eren

tit

alic

lett

ers

wit

hin

the

sam

eco

lum

n,di

ffer

sign

ific

antl

yby

Dun

can�

sm

ulti

ple

rang

ete

st(P<

0.05

,n¼

3);t

r,tr

aces

(�0.

01%

);–,

notd

etec

ted.

d)

F-T

esti

non

e-w

ayA

NO

VA

(F37

/30

degr

ees

offr

eedo

m);

***,

high

lysi

gnif

ican

tatP

<0.

001.

e )E

ssen

tial

-oil

yiel

dex

pres

sed

in%

(w/w

),i.e

.,g/

100

gof

seed

dry

mas

s.

Despite these similar contents of sesquiterpenes and monoterpenes, a difference in thephenylpropanoid content between the former two populations and P9 was noticed(2.04 and 1.27 vs. 14.12%, resp.).

As highlighted in boldface in Table 2, the main compounds for the analyzed sampleswere b-bisabolene (mean content of 39.33%), sabinene (8.53%), geranyl acetate(7.12%), elemicin (6.26%), and a-cadinol (3.77%) and for one sample (P7) carotol.Statistical analysis showed that the amounts of all identified compounds differedsignificantly from one population to another (one-way ANOVA and Duncan�s tests,P<0.001). The b-bisabolene contents ranged from 1.67 to 80.48%, and this compoundwas the major component in both sub-humid (P2 – P5) and upper semi-arid populations(P8 and P9). In the oils of two populations, it was dethroned by sabinene, whichdominated the oils of P1 (sub-humid zone; 18.78%) and P6 (lower humid zone;42.03%). Moreover, sabinene occurred in the second position in the oil of populationP7 (lower humid zone; 15.03%). In most samples, geranyl acetate (an oxygenatedmonoterpene) was either not detected or found in low amounts (traces – 1.59%), butcomposed 20.63 and 48.75%, respectively, of the oil of populations P9 and P10, whichgrow in semi-arid climatic zones. Except in the oils of samples P1and P5 –P7, thepresence of considerable amounts (4.10 to 15.89%) of the phenylpropanoid elemicinwas also noted. The sesquiterpene alcohol carotol was found as the major oilcomponent in population P7 (27.08%), while it was detected only at low contents(0.04 –1.82%) in the oils of the other populations.

There are many factors, such as the plant part, the stage of development, theenvironment, and the geographical origin, that can considerably influence thecomposition pattern of D. carota oils, as exemplified by previous studies (Table 3).In term of occurrence frequency, the oils obtained from seeds or ripe umbels(containing seeds) were mostly dominated by monoterpenes, i.e., sabinene and geranylacetate. However, there are a few exceptions where sesquiterpenes prevailed the oilcompositions, with b-bisabolene [10] [26] or carotol as the main compounds; although,the latter is more expected in cultivated D. carota [22] [28].

Chemical Population Structure. Principal component analysis (PCA) based on theessential-oil composition was used to facilitate chemotype distinction. Indeed, PCAallowed determining the combination of essential-oil components that accounted formost of the variation observed between samples. As shown in Fig. 2, the PCA plotrepresents graphically the relationship between the populations, using as input thecontents of all constituents (Table 2). The first three principal components (PC1, PC2,and PC3) had the greatest eigenvalues and accounted for 73.39% of the total variation(41.59, 18.21, and 13.62%, resp.). The PCA plot (Fig. 2) allowed distinguishing fourmajor population groups, each corresponding to a particular chemotype, characterizedby the dominant compounds. Chemotype I, characterized by high oil contents ofsabinene, was represented by populations P1 and P6. Chemotype II, the b-bisabolenechemotype, comprised populations P2– P5 and P8. Chemotype III, the b-bisabolene/geranyl acetate or geranyl acetate/b-bisabolene chemotype, consisted of populationsP9 and P10, whereas Chemotype IV, characterized by oils with carotol as the maincompound, was represented only by P7.

In addition to the PCA and with the aim to better characterize the populationgrouping, a hierarchical cluster analysis (HCA) was applied by calculating the mean



Table 3. Origin, Plant Part, and Main Components of the Essential Oils of Wild-Growing and Cultivated Daucuscarota Samples Previously Studied (between 2004–2013)

Samples Origin Wild or culti-vated

Plant organs Main components (content [%]) Refer-ence

D. carota Corsica Cultivated Aerial parts (E)-Methylisoeugenol (33.0), a-pinene(24.9), elemicin (11.4), b-bisabolene(4.4), sabinene (3.7)

[17]

D. carotassp. sativus

Poland Cultivated Floweringumbels

a-Pinene (42.4–46.2), myrcene(19.5–23.7), b-caryophyllene (4.6�6.2),carotol (1.2–5.6)

[26]

D. carotasativa

Uzbekistan Cultivated Seeds b-Bisabolene (80.49), a-asarone (8.82),cis-a-bergamoten (5.51)

[25]

D. carota cv.Chanteney

Serbia Cultivated Seeds Carotol (20.3), sabinene (18.7), a-pinene(7.95), b-caryophyllene (5.04), geranylacetate (4.40)

[22]

D. carota Turkey Cultivated Seeds Carotol (66.78), daucene (8.74), (Z,Z)-a-farnesene (5.86), germacrene D (2.34)

[27]

D. carotassp. halo-philus

Portugal Wild Floweringumbels

Sabinene (28.3–33.8), a-pinene (12.6–16.0), limonene (11.0 –11.8), elemicin(5.9–6.2), b-pinene (5.1 –2.3)

[18]

Ripe umbelswith seeds

Sabinene (29–27.6), elemicin (26.0–31.0),a-pinene (12.2–10.1), limonene (5.5 –6.5)

D. carota. ssp.maritimus

Tunisia Wild Flowers Sabinene (51.6), terpinen-4-ol (11.0), p-cymene (4.2)

[20]

Roots Dillapiole (46.6), myristicine (29.7), li-monene (3.6)

D. carotassp. carota

Italy Wild Floweringumbels

Carotol (25.1), 11a-H-himachal-4-en-1b-ol (21.6), b-bisabolene (17.6), elemicin(6.4)

[10]


b-Bisabolene (51.0), (E)-methylisoeuge-nol (10.0), 11a-H-himachal-4-en-1b-ol(9.0), elemicin (5.2)

Portugal Wild Floweringumbels

a-Pinene (37.9), geranyl acetate (15.0),(E)-caryophyllene (4.9)


Geranyl acetate (65.0), a-pinene (13.0),b-pinene (2.3)


Tunisia Wild Ripe umbelswith seedsfrom Tunis

Sabinene (14.5), 11a-H-himachal-4-en-1b-ol (12.7), eudesm-7(11)-en-4-ol (8.2),a-selinene (7.4), b-bisabolene (5.5), b-farnesene (4.5), carotol (3.5)

[11]

Ripe umbelsfrom Sajnan

Carotol (48.0), elemicin (31.5), b-farne-sene (2.6), b-caryophyllene (2.2).

D. carota Serbia Wild Seeds Geranyl acetate (53.2), geraniol (5.2),sabinene (5.1), linalool (4.3)

[8]

Aerial parts a-Pinene (29.3), sabinene (16.0), terpi-nen-4-ol (7.9), limonene (6.3), g-myrcene(4.0), germacrene D (3.4)


Algeria Wild Aerial parts a-Pinene (26.0), sabinene (14.0), limo-nene (13.0), b-pinene (11.2), myrcene(10.0)

[12]

Euclidian distances between the samples, followed by a dendrogram construction basedon the Ward�s algorithm. As can be observed in Fig. 3, the HCA allowed separating thesamples mainly into three major population groups (A, B, and C).

The first group (A) was represented by five populations (P2 –P5 and P8) growing insub-humid and upper semi-arid climatic zones. Their oil composition was stronglydominated by b-bisabolene, which was present in decreasing amounts (80.48 – 60.19%)in the oils of P5, P2, P3, P4, and P8 (from left to right in the dendrogram in Fig. 3). Theoils of Group A also showed high contents of elemicin (9.08– 15.89%), with theexception of that of P5 (0.06%). The second group (B) included the two populationsP10 and P9, growing in the lower semi-arid and the upper semi-arid zone, respectively,which afforded oils with geranyl acetate and b-bisabolene as the major compounds. Thethird group (C) was composed of the remaining three populations, i.e., P1, P6, and P7(growing in the sub-humid and lower humid zones). Their oils were not onlycharacterized by the highest contents of sabinene (15.03– 42.02%), germacrene D(5.38 –10.58%), and a-humulene (3.51 –4.15%), but also by the exclusive presence ofa-amorphene (2.33 – 3.79%).


Fig. 2. Plot of the principal component analysis performed on all populations according to the first threeaxes. Axes (PC1, PC2, and PC3) refer to the ordination scores obtained from the samples. The length ofeach stick indicates the value of the PCA score of each population compared to the first axis PC1. Theposition of each population against its relative stick indicates a positive (population below stick) or

negative (population above stick) value of the PCA score compared to PC1.

As illustrated in the dendrogram in Fig. 3, the oils of Groups A and B wererepresented by a single chemotype (II and III, resp.). Conversely, the oils of twochemotypes (I and IV) were clustered in the same group (C). Nevertheless, asdiscussed below, the projections of the population PCA coordinates (Fig. 2) haveclearly shown the emergence of four groups with, notably, P7 clearly separeted from P1and P6. Indeed, the essential oil of P7 exhibited some specific characteristics comparedto its neighbors, mainly the absence of a-pinene as well as the presence at high amountsof carotol, b-selinene, bornyl acetate, and d-cadinene. Hence, the combination of thesefactors displaced P7 into a particular location of the three-dimensional PCA space,which lies on the negative side of the PC3 axis. Moreover, Ward�s method reportedlytends to produce compact clusters with roughly the same size of observations, whichwould explain the underestimation of the chemotype number revealed by the HCA[29].

Based on the seed essential-oil composition, sabinene (Chemotype I), b-bisabolene(Chemotype II), and carotol (Chemotype IV) chemotypes have already previously beenreported (Table 3). However, Chemotype III, the b-bisabolene/geranyl acetate chemo-type, being rather unusual, showed two forms. Either the content of geranyl acetate washigher than that of b-bisabolene, a chemotype previously detected in the seed oils ofwild-growing D. carota in Serbia [8], or the b-bisabolene content was higher than thatof geranyl acetate, which was not reported previously in the literature.

Additionally, our study showed a high chemical polymorphism among the D. carotapopulations belonging to the same geographic area and thus growing under similarbioclimatic conditions. Notably, the northern populations P6 and P7 (growing both inthe lower humid zone) clustered separately into different chemotypes (I and IV, resp.),despite the fact that they are geographically very close. Nevertheless, the northeasternpopulations P2, P3, and P4, growing in the same region (sub-humid zone), affordedoils, as expected, with the same chemotype (II). Maxia et al. [10] reported aconsiderable variability of the composition of D. carota seed oils, depending on thegeographical origin (Italy and Portugal). Furthermore, Marzouki et al. [11] described


Fig. 3. Dendrogram generated by cluster analysis based on Euclidean distances. The scale represents thedissimilarity index calculated within Euclidean distances using Ward�s aggregation algorithm. Letters (A,

B, and C) indicate the groups (clusters) discussed in the text.

the existence of two distinct chemotypes in northern Tunisia, among two localities(Tunis and Sajnan, Bizerte governorate) distanced by 68 km (Table 3).

In the current study, the sampling locations P1 to P9 are nearly disposed along atransect of up to 200 km, covering northern Tunisia from east to west (Fig. 1). Asdescribed above, the co-occurrence of four chemotypes within this area was reportedhere. The results showed that the variability of the composition of the seed essential oilswas not always concordant with the bioclimatic zones and seemed rather to be linkedwith local selective forces acting on the chemotype diversity. Indeed, local abiotic(topography, moisture, temperature, and edaphic factors) and/or biotic selective factors(associated fauna and flora) might act on the loci of the terpene-biosynthesis pathwaysand contribute to the emergence of different chemical patterns [30].

Antimicrobial Activity of the Essential Oils. Preliminary screening of the in vitroantimicrobial activity of the essential oils isolated from D. carota seeds against fourpathogenic microorganisms was assessed using the filter-paper disk diffusion technique.The results showed that all tested essential oils exhibited similar degrees of inhibitionagainst both Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive (Staphylococcus aureus) bacteria as well as against the yeast Candida albicans(Table 4). These results were supported by previous studies of the antimicrobialactivity of D. carota oils isolated from seeds [19], aerial parts [24], and flowers [20].

Conclusions. – This study offered a comprehensive overview of the chemicalpolymorphism of the essential oils of wild-growing D. carota. Such variability washighlighted in the occurrence of four distinct chemotypes within northern Tunisia, as aresult of environmental and/or genetic factors. This assumption should be verified byphylogenetic experiments under controlled conditions. Based on the remarkableantimicrobial activities, confirmed here, it might be useful to consider D. carota oil ascandidate for therapeutic purposes.


Table 4. Antimicrobial Activity of Daucus carota Essential-Oil Samples P1 to P10 Determined with theDisk Diffusion Method

Microorganism Inhibition zone [mm]a)

Ampicillinb) P1 P2 P3 P4 P5

Escherichia coli 13.3�1.4 14.3�0.3 14.3�1.3 13.7�1.9 14.7�0.7 13.0�1.5Salmonella typhimurium 15.0�3.1 12.0�1.0 12.0�3.0 13.5�5.5 12.0�2.0 12.0�4.0Staphylococcus aureus 30.0�5.0 18.0�4.5 16.0�8.0 16.0�8.0 8.0�1.0 20.0�0.0Candida albicans 13.3�0.7 16.7�2.8 15.0�1.9 14.0�3.6 14.3�3.2 13.3�3.5

P6 P7 P8 P9 P10

Escherichia coli 12.7�0.3 9.0�1.7 10.7�0.9 16.3�0.7 13.7�0.9Salmonella typhimurium 13.0�2.1 8.7�0.7 10.0�1.0 11.7�1.7 9.0�1.0Staphylococcus aureus 26.7�3.3 14.0�5.6 11.5�2.5 18.3�5.9 12.7�2.7Candida albicans 15.7�3.0 11.0�2.3 16.3�1.4 16.3�1.3 15.7�0.7

a) Diameter of the growth-inhibition zone in mm (including disk diameter of 6 mm); values are means�SD (n¼3). b) Positive control (12 mg/disk).

The authors express their gratitude to the Ministry of Higher Education and Scientific Research andthe National Institute of Research and Physico-Chemical Analysis (Research grant LR10/INRAP02).

Experimental Part

Plant Material. Mature seeds of Daucus carota L. were collected during September 2010 from tennatural, Tunisian populations distributed over four different bioclimatic zones (Table 1). The plantmaterial was botanically authenticated by Prof. Nadia Ben Brahim (Laboratory of Botany andOrnamental Plants, National Institute of Agronomical Research of Tunisia) and voucher specimens(DCn84-sep2010) have been deposited with the above-mentioned laboratory to serve as a futurereference.

Essential-Oil Extraction. The D. carota essential oils were extracted by hydrodistillation (100 g of air-dried seeds per sample) for 3h using a standard distillation quick-fit apparatus. For each sample, threereplications of each extraction were performed. The obtained essential oils were dried (anh. Na2SO4) andstored at �208 until analysis.

GC-FID Analysis. The GC-FID analyses were performed using an Agilent 6890N gas chromatographequipped with a flame-ionization detector (FID), an electronic pressure control (EPC) injector (AgilentTechnologies, J&W Scientific Products, Palo Alto, CA, USA), and an apolar DB-5 cap. column (5%diphenyl 95% dimethylpolysiloxane; 30 m�0.25 mm i.d., film thickness 0.25 mm). The oven temp. wasprogrammed isothermal at 508 for 1 min, then rising from 50 to 2508 at 88/min, and, finally, heldisothermal at 2508 for 10 min; injector temp., 2208 ; detector temp., 2808 ; carrier gas, He (1 ml/min); splitratio, 1 :50; injection volume, 1 ml.

GC/MS Analysis. The GC/MS analyses were carried out with a gas chromatograph model HP 6890(Agilent, Palo Alto, CA) equipped with a mass selective detector MSD5975B (Agilent) and a HP-5 MScap. column (5% diphenyl 95% dimethylpolysiloxane; 30 m�0.25 mm i.d., film thickness 0.25mm). Theoven temp. was programmed rising from 50 to 3008 at 38/min; carrier gas, He (1 ml/min); ionizationvoltage, 70 eV; scan time, 1 s; mass range, 40–300 amu.

Volatile-Compound Identification. The identification of the volatile compounds was based on thecomparison of i) their retention indices (RIs), determined rel. to the retention times (tR) of a series of n-alkanes (C8 –C30), with those of the literature [31] and ii) their recorded mass spectra with those listed inthe mass-spectral libraries Wiley 07 (7th edn.) and NIST/EPA/NIH (NIST 05) and those published [31].The contents of all constituents expressed in percentages were determined from their GC-FID peak areaswithout using correction factors.

Microbial Strains. The essential oils of D. carota were individually tested against a set of pathogenicmicroorganisms, including the Gram-positive bacterium Staphylococcus aureus (ATCC 6538), the twoGram-negative bacteria Escherichia coli (ATCC 8739) and Salmonella typhimurium (ATCC 14028), aswell as the yeast Candida albicans (ATCC 10231). The bacterial and fungal reference strains wereobtained from the collections of the Pasteur Institute (Paris) and the Laboratory of Microbial Ecologyand Technology (National Institute of Applied Science and Technology, Tunisia).

Antimicrobial Activity. The antimicrobial activity of the essential oils was assessed by the paper-diskdiffusion method, according to the recommendations of the National Committee for Clinical LaboratoryStandards [32]. Briefly, microbial strains preserved in nutrient agar at 48 were revitalized inMueller�Hinton (MH) soln. and incubated at 378 for 18–24 h. A suspension of the tested micro-organisms (inoculum of 1�108 CFU/ml) was spread on solid MH media plates. Aliquots of 15 ml of theessential oils, dissolved beforehand in DMSO (1 : 1 (v/v)), were applied on filter-paper disks and placedon the inoculated plates. Then, the plates were incubated at 378 during 24 h for bacteria and at 308 during48 h for the yeast. Disks with DMSO alone were used as a negative control, and 12 mg of ampicillin/disk(USP grade, Biomatik, Wilmington, USA) was used as the positive reference. The antimicrobial activitywas evaluated by measuring the diameter of the growth-inhibition zones in mm (including disk diameterof 6 mm) of the test organisms.

Statistical Analysis. All experiments and analytical determinations were carried out in triplicate andthe results were expressed as mean values� standard deviations. One-way analysis of variance


https://www.researchgate.net/publication/200156675_Performance_Standards_for_Antimicrobial_Disk_Susceptibility_Test_Approved_Standard_In_M2-A9?el=1_x_8&enrichId=rgreq-83563f3d-3146-419e-a1f2-3da7e64e7e72&enrichSource=Y292ZXJQYWdlOzI1OTI3MTQ5NTtBUzoxMTQ1MDQ4MDUzOTIzODRAMTQwNDMxMTQ4MzExMw==

(ANOVA) followed by a Duncan�s multiple range test was performed to detect statistically significantdifferences between the relative contents of the volatile compounds among the samples (SPSS StatisticalSoftware version 15.0, Chicago, USA). These differences were considered statistically significant at P<0.05. The chemometric statistical analysis characterizing the population structure and the relationshipamong populations was performed by principal component analysis (PCA) based on the percentages ofall identified constituents. The divergence between populations was also estimated by the Euclideandistances calculated among population pairs, followed by hierarchical clustering using Ward�s linkagemethod as an agglomerative algorithm (RStudio version 0.96, Boston, USA).

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Received April 24, 2013




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Variation of the Chemical Composition and Antimicrobial Activity of the Essential Oils of Natural Populations of Tunisian Daucus carota L. (Apiaceae)