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Effect of bioclimatic area on thecomposition and bioactivity of TunisianRosmarinus officinalis essential oilsMariem Ben Jemiaa, Rosa Tundisb, Alessandro Puglieseb, FrancescoMenichinib, Felice Senatorecd, Maurizio Brunoe, Mohamed ElyesKchouka & Monica Rosa Loizzob

a Laboratoire des Plantes Extremophiles – Biotechnologic CenterBorj-Cedria Technopark, B.P. 901, 2050 Hammam-Lif, Tunisiab Department of Pharmacy, Health Sciences and Nutrition,University of Calabria, I-87036 Rende (CS), Italyc Department of Chemistry of Natural Products, University ofNaples “Federico II”, Via D. Montesano, 49-80131 Naples, Italyd Department of Pharmacy, University of Naples “Federico II”, ViaD. Montesano, 49-80131, Naples, Italye Department STEBICEF, University of Palermo, Viale delleScienze, Parco d'Orleans II, 90128 Palermo, ItalyPublished online: 07 Aug 2014.

To cite this article: Mariem Ben Jemia, Rosa Tundis, Alessandro Pugliese, Francesco Menichini,Felice Senatore, Maurizio Bruno, Mohamed Elyes Kchouk & Monica Rosa Loizzo (2014): Effect ofbioclimatic area on the composition and bioactivity of Tunisian Rosmarinus officinalis essential oils,Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.942305

To link to this article: http://dx.doi.org/10.1080/14786419.2014.942305

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Effect of bioclimatic area on the composition and bioactivity of TunisianRosmarinus officinalis essential oils

Mariem Ben Jemiaa, Rosa Tundisb*, Alessandro Puglieseb, Francesco Menichinib,

Felice Senatorecd, Maurizio Brunoe, Mohamed Elyes Kchouka and Monica Rosa Loizzob

aLaboratoire des Plantes Extremophiles – Biotechnologic Center Borj-Cedria Technopark, B.P. 901, 2050Hammam-Lif, Tunisia; bDepartment of Pharmacy, Health Sciences and Nutrition, University of Calabria,I-87036 Rende (CS), Italy; cDepartment of Chemistry of Natural Products, University of Naples “FedericoII”, Via D. Montesano, 49-80131 Naples, Italy; dDepartment of Pharmacy, University of Naples “FedericoII”, Via D. Montesano, 49-80131 Naples, Italy; eDepartment STEBICEF, University of Palermo, Vialedelle Scienze, Parco d’Orleans II, 90128 Palermo, Italy

(Received 6 June 2014; final version received 3 July 2014)

The chemical composition of eight Tunisian Rosmarinus officinalis L. populations(A–H) from different bioclimatic areas has been examined by gas chromatography(GC) and GC-mass spectrometry. The essential oils are characterised by high amountsof oxygenated monoterpenes (58.2–71.7%) followed by monoterpene hydrocabons(15.1–26.7%). 1,8-Cineole, camphor, a-pinene and borneol are the mainrepresentative components. The antioxidant activity was investigated by 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), ferric reducing ability power assay andb-carotene bleaching test. Samples showed antiradical activity by inhibiting DPPHradical with IC50 values ranging from 375.3 to 592.8mgmL21 for samples F and A,respectively. Sample A also showed the most promising activity in b-carotenebleaching test (IC50 of 31.9mgmL21). The essential oils were also screened foracetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity.Sample G showed the highest activity against AChE (IC50 of 64.7mgmL21) whilesample D (IC50 of 29.5 mgmL21) exhibited the most potent activity against BChE.

Keywords: Rosmarinus officinalis; essential oil; GC-MS analysis; antioxidantproperties; cholinesterase inhibitory activity

1. Introduction

Rosmarinus officinalis L. (Lamiaceae) is an aromatic evergreen shrub widely distributed in the

Mediterranean area. Several factors, such as place of collection, time of harvest, environmental

and agronomic conditions and method of extraction, affect the chemical composition of

R. officinalis essential oil (Jordan et al. 2013). The Tunisian R. officinalis essential oil chemical

composition was previously investigated (Zaouali et al. 2010; Jordan et al. 2011, 2013; Yosr

et al. 2013).

However, these studies did not highlight significantly differences among varieties or

population or distribution. In Tunisia, R. officinalis grows mainly in sub-humid, semi-arid

superior and arid superior bioclimatic stages according to Emberger’s classification (Emberger

1996). Several works reported the ability of rosemary essential oil as a memory enhancer and to

treat cognitive disorders, including neurodegenerative diseases such as Alzheimer’s disease

(AD) (Adsersen et al. 2006; Posadas et al. 2009). The current treatment for AD is restricted to

q 2014 Taylor & Francis

*Corresponding author. Email: rosa.tundis@unical.it

Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.942305

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drugs whose principal action is to augment the availability of the neurotransmitter acetylcholine

via the inhibition of either the cholinesterase group of enzymes (acetylcholinesterase, AChE,

and butyrylcholinesterase, BChE). Many human diseases including AD are caused by oxidative

stress and several essential oils have been shown to be effective as natural antioxidants (Beal

1995; Loizzo et al. 2009a). In this study we screened the chemical composition, antioxidant

properties and cholinesterase inhibitory activity of eight essential oils obtained from

R. officinalis representative of the global distribution area of the species in Tunisia. The

knowledge of the Tunisian rosemary could help to promote additional resources to rural

populations according to their local natural resources.

2. Results and discussion

R. officinalis analysed in this work was collected in different bioclimatic stages according to

Emberger’s rainfall temperature coefficient, Q2 ¼ 2000P/M 2 2 m 2, in which P is the mean of

annual rainfall (mm),M is the mean of maximal temperatures for the hottest month and m is the

mean of minimal temperatures for the coldest month (Emberger 1996).

Qualitative and quantitative differences in the composition of R. officinalis essential oil were

observed (Table 1). Seventy-two compounds were identified which represent 94.9–97.7% of the

volatile components. The oxygenated monoterpenes represent the most abundant fraction in all

analysed samples with percentage in the range of 58.2–71.7% followed by monoterpene

hydrocarbons (15.1–26.7%). Among monoterpene hydrocarbons, a-pinene is the main

representative component. In the oxygenated fraction, 1,8-cineole is the most abundant

compound followed by camphor. 1,8-Cineole was identified as the main abundant compound in

all samples except sample F that was characterised by the presence of camphor as principal

constituent. In all samples the main components of the sesquiterpene fraction were trans-

caryophyllene and d-cadinene. Other identified compounds are a-humulene and g-muurolene.

According to previous studies, the yield of oil extraction and the total content of volatiles are not

affected by the different bioclimatic area in which plants are collected (Zaouali et al. 2010;

Jordan et al. 2011, 2013; Yosr et al. 2013). Zaouali et al. (2010) described the variation in

essential oil yielded from Tunisian R. officinalis var. typicus and R. officinalis var.

troglodytorum, growing in different bioclimatic areas. In disagreement with our results that

did not evidence modification, the oil yield for the variety typicus was higher in upper semi-arid

zones than that obtained from sub-humid regions. The same author evidenced that variations in

the chemical composition of the oil should be attributed almost exclusively to varieties rather

than bioclimatic conditions. On the contrary, Tigrine-Kordjani et al. (2012), who analysed the

chemical composition of 32 R. officinalis samples collected at the same time from different sites

in the north of Algeria, highlighted a strong correlation between the chemical composition and

the place of collection. Elamrani et al. (2000) studied the R. officinalis essential oils from

Morocco finding three different chemotypes: a-pinene-chemotype, camphor-chemotype and the

1,8-cineole-chemotype. Eight-seventy populations of rosemary collected in Spain were

investigated (Varela et al. 2009). Among these samples, 38 showed 1,8-cineole content

of . 24%, 6 showed high 1,8-cineole/linalool ratio and 3 samples had high linalool content. A

more recent study reported the chemical variability of R. officinalis samples collected in south-

eastern part of Spain, identifying three major chemotypes: 1,8-cineol-a-pinene-camphor;

camphor-1,8-cineole-a-pinene and 1,8-cineole-camphor-a-pinene (Jordan et al. 2011). The

same trend was observed in our samples. Papageorgiou et al. (2008) investigated the chemical

composition of Greece R. officinalis oil. 1,8-Cineole, borneol and a-terpineol were the principalconstituents.

The antioxidant activity of R. officinalis essential oils was analysed by: 2,2-diphenyl-1-

picrylhydrazyl radical (DPPH), ferric reducing ability power (FRAP) assay and b-carotene

2 M. Ben Jemia et al.

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Table

1.Chem

ical

constituentsoftheessential

oilsfrom

eightR.officinalispopulation.

RIa

RIb

Compound

AB

CD

EF

GH

I.m.c

928

1014

Tricyclene

0.1

trtr

trtr

0.5

0.4

0.4

1,2

931

1023

a-Thujene

trtr

trtr

trtr

tr1,2

938

1032

a-Pinene

7.1

9.5

8.3

8.0

9.1

10.4

10.1

9.4

1,2,3

953

1076

Cam

phene

2.1

3.2

3.0

2.9

3.0

11.1

10.4

9.1

1,2,3

980

1118

b-Pinene

3.0

2.5

3.2

1.7

2.1

0.8

0.9

0.8

1,2,3

993

1174

Myrcene

0.7

0.6

0.7

0.6

0.7

0.4

0.4

0.5

1,2,3

1005

1150

a-Phellandrene

trtr

trtr

tr0.2

tr1,2

1011

1159

d-3-Carene

t1,2

1012

1189

a-Terpinene

0.5

0.5

0.5

tr0.6

0.5

0.6

0.6

1,2,3

1020

1187

o-Cymene

0.2

1,2

1025

1278

p-Cymene

0.2

1.4

2.1

0.2

2.7

1,2,3

1038

1045

(Z)-b-ocimene

tr1,2

1049

1265

(E)-b-O

cimene

trtr

tr1,2

1057

1256

g-Terpinene

0.6

0.5

0.7

0.5

0.5

0.6

0.8

0.7

1,2,3

1086

1265

Terpinolene

1.0

0.3

0.3

t0.3

0.3

0.3

1,2,3

Monoterpenes

15.1

19.3

16.7

15.1

16.3

26.7

24.5

24.2

1034

1213

1,8-Cineole

52.6

44.9

51.4

39.1

50.3

23.2

25.9

26.0

1,2,3

1063

1555

cis-Sabinenehydrate

tr0.2

trtr

tr0.2

tr1,2

1086

1474

trans-Sabinenehydrate

trtr

trtr

tr1,2

1098

1553

Linalool

0.5

0.6

0.7

0.6

tr1,2,3

1120

1592

Fenchylalcohol

trtr

trtr

1,2

1128

1498

a-Cam

pholenal

tr1,2

1145

1532

Cam

phor

7.8

8.0

7.2

12.0

10.5

27.5

21.1

24.3

1,2,3

1167

1719

Borneol

4.1

6.2

5.9

10.0

5.9

3.3

6.0

4.4

1,2,3

1176

1611

Terpinen-4-ol

0.8

0.7

0.8

0.8

0.8

1.0

1.1

1.2

1,2,3

1189

1706

a-Terpineol

3.5

2.6

3.1

3.1

3.3

1.7

1.6

2.1

1,2,3

1286

1567

Bornylacetate

0.6

2.1

0.9

1.3

0.3

3.5

4.2

2.1

1,2,3

Oxygenated

monoterpenes

69.4

65.2

69.9

67.0

71.7

58.2

60.1

60.0

1352

1466

a-Cubebene

tr0.3

0.4

0.4

0.3

1,2

1373

1493

a-Y

langene

trtr

trtr

0.1

trtr

1,2

1377

1497

a-Copaene

0.4

0.5

0.6

tr0.6

1,2

1415

1612

trans-Caryophyllene

4.1

7.7

3.5

4.1

4.3

1.1

2.1

2.4

1,2,3

1422

1565

b-Y

langene

trtr

trtr

tr1,2

(Continued

)

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Table

1.(Continued

)

RIa

RIb

Compound

AB

CD

EF

GH

I.m.c

1437

1628

Aromadendrene

tr0.1

tr1,2

1455

1689

a-H

umulene

0.6

1.1

1.0

0.7

0.7

tr0.2

0.3

1,2

1466

1656

9-epi-(E)-Caryophyllene

trtr

1,2

1475

1715

b-Selinene

0.1

tr1,2

1476

1669

g-G

urjunene

trtr

tr1,2

1478

1704

g-M

uurolene

0.4

0.4

0.6

0.6

0.5

0.5

0.2

1,2

1487

1679

a-A

morphene

trtr

tr1,2

1490

1612

b-G

uaiene

trtr

1,2

1493

1698

d-Selinene

trtr

tr0.2

1,2

1494

1740

Valencene

0.3

trtr

tr1,2

1503

1740

a-M

uurolene

trtr

trtr

trtr

1,2

1509

1746

cis-(Z)-a-Bisabolene

tr1,2

1515

1776

g-Cadinene

0.3

0.2

0.6

1.0

0.4

1,2

1526

1773

d-Cadinene

1.1

1.0

1.5

1.8

1.3

1.2

0.8

1.7

1,2

1533

1802

Cadina-1,4-diene(Cubenene)

0.1

trtr

trtr

tr1,2

1535

2093

a-Cadinene

0.1

trtr

tr1,2

1542

1918

a-Calacorene

trtr

trtr

0.1

0.2

0.1

0.3

1,2

1629

1611

Calarene

trtr

0.6

1,2

Sesquiterpenes

7.5

10.8

7.7

8.8

7.3

4.1

3.9

5.8

1578

2150

Spathulenol

0.3

tr1,2,3

1580

2008

Caryophylleneoxide

1.4

1.2

1.7

0.8

0.5

0.4

1,2,3

1632

2371

Caryophylla-3,8(13)-dien-5a-ol

0.3

1.0

tr0.7

0.4

1,2

1638

2185

g-Eudesmol

0.9

0.8

1,2

1640

2316

Caryophylla-4(12),8(13)-dien-5b-ol;

CaryophylladienolI

0.4

0.2

tr0.6

0.4

t1,2

1640

2185

t-Cadinol

0.7

0.5

1.2

tr0.8

0.6

1,2

1642

2209

t-Muurolol

trtr

trtr

0.3

0.3

1,2

1645

2145

Torreyol

tr0.1

t1,2

1648

2258

b-Eudesmol

0.2

1.1

1.3

1.0

1,2

1649

2255

a-Cadinol

trtr

tr0.1

1,2

1653

2252

a-Eudesmol

0.3

0.2

1.7

3.0

1.5

1,2

Oxygenated

sesquiterpenes

3.3

2.5

0.7

4.2

1.2

4.4

5.8

4.6

1293

2198

Thymol

trtr

trtr

tr0.2

0.3

1,2,3

1299

2239

Carvacrol

0.2

trtr

trtr

0.3

0.1

1,2,3

4 M. Ben Jemia et al.

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1353

2186

Eugenol

tr0.2

trtr

0.2

0.2

tr1,2,3

1396

2296

Isoeugenol

tr1,2

1405

2031

Methyleugenol

0.4

0.3

0.5

tr0.5

1.1

0.8

1.0

1,2

Phenols

0.6

0.5

0.5

t0.5

1.3

1.5

1.4

1287

1593

2-U

ndecanone

tr0.1

trtr

0.1

tr1,2

1302

1797

40 -M

ethoxyacetophenone

trtr

trtr

tr0.2

tr1,2,3

1908

7-Ethenyl-1,2,3,4,4a,5,6,7,

8,9,10,10a-dodecahydro-1,1,4a,7-

tetram

ethylphenanthrene

tr0.2

1,2

1943

2185

Cem

brene

0.1

tr0.4

1,2

1989

2393

Manoyloxide

0.3

1,2

2054

2524

Abietatriene

0.3

0.2

0.4

0.3

0.1

tr1,2

2329

trans-Ferruginol

0.3

0.4

0.4

0.4

0.4

0.2

0.3

0.2

1,2

Others

1.0

0.9

1.2

0.4

0.7

0.2

0.7

0.2

Total

96.9

97.3

96.7

95.7

97.7

94.9

96.5

96.2

aRetentionindices

relativeto

C8–C24n-alkanes

ontheHP5MScolumn.

bRetentionindices

relativeto

C8–C24n-alkanes

ontheHPInnowax

column.

c1,Comparisonofretentiontimes;2,ComparisonofmassspectrawithMSlibraries;3,Comparisonwithauthenticcompounds;tr,trace(,

0.05%).

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bleaching test (Table 2). All the oils showed antiradical activity by inhibiting DPPH radicals

with IC50 values ranging from 375.3 to 592.8mgmL21 for samples F and A, respectively.

Previously, Beretta et al. (2011) analysed the DPPH radical scavenging activity of Italian

R. officinalis at the flowering, post-flowering and vegetative stages finding IC50 values of 36.78,

79.69 and 111.94mgmL21, respectively.

In b-carotene bleaching test, overall results were better than those provided by the radical

scavenging activity. Sample A exhibited the most promising activity with an IC50 value of

31.9mgmL21. Wang et al. (2008) investigated the antioxidant activity of the main constituent

present in R. officinalis essential oil. In the DPPH test, free radical scavenging activity of

R. officinalis essential oil, 1,8-cineole, a-pinene and b-pinene, was determined to be 62.45, 42.7,

45.61 and 46.21% (v/v), respectively. In the b-carotene bleaching test system, IC50 values were

determined as 2.04, 4.05, 2.28 and 2.56% (v/v) for R. officinalis essential oil, 1,8-cineole,

a-pinene and b-pinene, respectively. A concentration–response relationship was observed for

all samples in the FRAP assay. Values ranging from 1.0 to 23.0mM Fe(II) g21 for E and F

samples, respectively, were found.

A variety of plants have been reported to show cholinesterase inhibitory activity and so may

be relevant to the treatment of neurodegenerative disorders such as AD (Loizzo et al. 2008).

The inhibition of the two key enzymes in AD treatment, AChE and BChE, was herein tested

by Ellman’s colorimetric assay. Rosemary oils showed a concentration– response

relationship. As reported in Table 2, sample G exhibited the most promising activity against

AChE with an IC50 of 64.7mgmL21 followed by sample B (IC50 of 98.2mgmL21). The

investigation on the activity on BChE is of certain interest since in late stages of AD, levels of

AChE decline by up to 85% and BChE represents the predominant cholinesterase in the brain so

is the isoform that will be inhibited to obtain a pro-cholinergic effect. In our study, oils exhibited

lower inhibitory activity against BChE than against AChE, except for sample D (IC50 of

29.5mgmL21). Moss et al. (2003) reported that rosemary essential oil produced a significant

enhancement of memory performance and overall quality of memory in healthy adult volunteers.

These pieces of evidence moved several researchers to investigate the cholinesterase inhibitory

activity of R. officinalis from different geographical areas. Mata et al. (2007) investigated the

AChE inhibitory activity of R. officinalis oil from Portugal finding an IC50 of 69.8mgmL21

while Orhan et al. (2008) investigated the cholinesterase inhibitory effect of Turkish

R. officinalis oil finding a higher inhibition towards BChE than towards AChE with a percentage

of inhibition of 63.7 and 74%, respectively, at 1mgmL21. A moderate cholinesterase activity

was observed by Perry et al. (1996) who reported an AChE percentage of inhibition of 16.8% at

0.1ml mL21. A recent study demonstrated that rosemary powder at the dose nearest normal

culinary consumption had positive effects on speed of memory (Pengelly et al. 2012). Although

many of the major identified compounds were found to be active against AChE and BChE

(Satomi et al. 2009; Bonesi et al. 2010), these compounds are in low concentration and are

affected by synergistic or antagonistic interaction (Savelev et al. 2003). The findings revealed

that the bioactivity is a result of a complex interaction between oil constituents, which produce

both synergistic and antagonistic responses between the main components. Understanding such

interactions is important in comparing species on the basis of chemical composition.

3. Experimental

3.1. Chemicals and reagents

Solvents of analytical grade were purchased from VWR International s.r.l. (Milan, Italy). DPPH,

ascorbic acid, ABTS solution, Trolox, b-caroten, linoleic acid, Tween 20, propyl gallate,

tripyridyltriazine (TPTZ), FeCl3, FeSO4, butylated hydroxytoluene (BHT), 5,50-dithiobis (2-

nitrobenzoic-acid), butyrylthiocholine iodide, acetylthiocholine iodide, physostigmine, AChE

6 M. Ben Jemia et al.

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Table

2.AntioxidantactivityofR.officinalisessential

oils.

DPPH

assay

(IC50mgmL21)

b-Carotene

bleachingtest

(IC50mgmL21)

FRAP

assay

(mM

Fe(II)g21)

AChE

BChE

SI

(BChE/AChE)

Essential

oil

A592.8^

2.2

a31.9^

1.3a

16.1^

2.4

a498.9^

2.8

a924.2^

4.8

a1.9

B582.7^

2.4

a98.4^

2.2a

3.4^

2.7

a98.2^

1.9

a346.7^

3.8

a3.5

C435.5^

3.1

a76.7^

1.2a

10.5^

1.0

a200.5^

2.1

a697.8^

4.9

a3.5

D343.1^

2.0

a.

100

7.8^

0.8

a122.8^

1.4

a29.5^

1.5

a0.2

E520.1^

3.3

a97.0^

1.8a

1.0^

0.2

a478.0^

2.3

a918.2^

5.3

a1.9

F375.7^

1.9

a90.7^

2.2a

23.0^

2.3

a108.8^

1.5

a122.7^

1.7

a1.1

G471.4^

3.5

a98.7^

2.0a

15.9^

1.0

a64.7^

1.2

a353.1^

2.9

a5.4

H375.3^

2.0

a86.2^

2.5a

14.9^

0.9

a227.2^

2.6

a647.4^

3.8

a0.3

Positivecontrol

Ascorbic

acid

5.0^

0.07

Propylgallate

1.0^

0.04

BHT

63.2^

2.3

Physostigmine

0.2^

0.02

2.4^

0.04

12

Notes:Dataareexpressed

asmeans^S.D.(n¼

3).DPPH:one-way

ANOVA***p,

0.0001(F

¼76,260,R

2¼

1.0)followed

byamulti-comparisonDunnett’stest:ap,

0.01compared

withascorbicacid;b-Carotenebleachingtest:one-way

ANOVA***p,

0.0001(F

¼1084,R

2¼

0.998)followed

byamulti-comparisonDunnett’stest:ap,

0.01compared

withpropyl

gallate;FRAP:One-way

ANOVA***p,

0.0001(F

¼535.5,R

2¼

0.996)followed

byamulti-comparisonDunnett’stest:ap,

0.01compared

withBHT.AChE,acetylcholinesterase

Assay;BChE,butyrylcholinesterase

assay.AChE:one-way

ANOVA***p,

0.0001(F

¼41,600,R

2¼

0.999)followed

byamulti-comparisonDunnett’stest:ap,

0.01compared

with

physostigmine,BChE:one-way

ANOVA

***p,

0.0001(F

¼189,100,R

2¼

1.0)followed

byamulti-comparisonDunnett’stest:ap,

0.01compared

withphysostigmine.

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from Electrophorus electricus (EC 3.1.1.7, Type VI-S) and BChE from equine serum

(EC 3.1.1.8) were purchased from Sigma-Aldrich S.p.A. (Milan, Italy).

3.2. Plant material and extraction procedure

R. officinalis populations were collected in different bioclimatic areas and are representative of

the global distribution of the species in Tunisia. Plant samples were identified by Prof. M.E.

Kchouk (Centre of Biotechnologie of Borj Cedria Technopark. Tunisia) (Table 3). The essential

oils were obtained by hydrodistillation for 3 h, using a Clevenger-type apparatus (Clevenger

1928). The oils were dried and stored under N2 at þ48C in brown bottles until they were

analysed and tested.

3.3. GC and GC-MS analyses

Analytical gas chromatography (GC) was carried out on a Perkin-Elmer Sigma 115 gas

chromatograph fitted with a HP-5 MS capillary column as previously described (Zito et al.

2013). GC-MS analysis was performed on an Agilent 6850 Ser. II apparatus (Agilent

Technologies, Inc., Santa Clara, CA, USA), as described elsewhere (Zito et al. 2013).

3.4. DPPH radical scavenging activity assay

Radical scavenging capacity was determined as previously reported (Loizzo et al. 2009b).

Decolourisation of DPPH was determined at 517 nm. Ascorbic acid was used as positive control.

3.5. b-Carotene bleaching test

Antioxidant activity was determined as previously described (Loizzo et al. 2009a). Propyl

gallate was used as positive control. The measurement was carried out at initial time (t ¼ 0) and

successively at 30 and 60min.

3.6. Ferric reducing ability power assay

The FRAP method measures the reduction of (2,4,6-tripyridyl-s-triazine (TPTZ))-Fe3þ to the

TPTZ-Fe2þ in the presence of antioxidants. Briefly, FRAP reagent containing 2.5mL of 10mM

Table 3. Place of collection and essential oil % yield of Tunisian R. officinalis populations selected for thestudy.

Samples Place of collectionBioclimaticzone Q2

a Latitude LongitudeAltitude(m)

Essentialoil % yield

A Fadj Atfal Sh 70–110 358210N 98150E 763 1.32B Dj. Zaghouan Sh 70–110 368260N 108190E 1295 1.71C Dj. Fragha Sh 70–110 358520N 98010E 742 1.33D Dj. Khamess Sh 70–110 358990N 98460E 143 1.56E Dj. Chaambi Usa 45–70 358160N 98 060E 935 1.36F El Aamra Usa 45–70 338520N 108120E 291 1.38G Matmata (Bni Zoltan) Ua 10–45 338550N 108100E 202 1.01H Toujane Ua 10–45 338460N 108140E 720 0.71

Note: Bioclimatic zones were defined according to Emberger’s classification [12].a Pluviothermic coefficient Q2 ¼ 2000P/M2 2 m 2 where P is the average of annual rainfall (mm), M is the mean ofmaximal temperature (K) for the warmest month (July) and m is the average of minimal temperature (K) for the coldestmonth (February). Sh, sub-humid; Usa, upper semi-arid; Ua, upper arid.

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TPTZ solution in 40m mol L21 of HCl plus 2.5mL of 20mM of FeCl3 and 25mL of 0.3M

acetate buffer was freshly prepared. BHT was used as positive control.

3.7. Cholinesterase inhibition assay

Cholinesterase (AChE and BChE) inhibiting activities were measured by slightly modifying

Ellman’s method (Loizzo et al. 2009a). Physostigmine was used as positive control. Results are

reported in Table 2.

3.8. Statistical analysis

The concentration giving 50% inhibition (IC50) was calculated by nonlinear regression with the

use of Prism GraphPad version 4.0 for Windows (GraphPad Software, San Diego, CA, USA).

The concentration–response curve was obtained by plotting the percentage inhibition versus

concentration. Differences within and between groups were evaluated by one-way analysis of

variance (ANOVA) test followed by a multi-comparison Dunnett’s test compared with the

positive controls.

4. Conclusions

Eight R. officinalis essential oils were investigated for their chemical composition and

bioactivity. Samples were obtained from three different bioclimatic zones of Tunisia. Seventy-

two compounds were identified. Oxygenated monoterpenes represent the most abundant

fraction. Our results are in agreement with several works reported in the literature that evidenced

how the yield of oil extraction and the total content of volatiles are not affected by the different

bioclimatic areas in which plants are collected. Essential oils showed in vitro cholinesterase

inhibitory activities and antioxidant effects. Our results confirm that food plant-derived natural

compounds are an important source for the development of cholinesterase inhibitors useful in the

treatment of neurodegenerative diseases.

Acknowledgements

The GC and GC-MS analyses were performed at the ‘C.S.I.A.S.’ of the University ‘Federico II’, Napoli.The assistance of the staff is gratefully appreciated.

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