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GC, GC-MS and 13C NMR Spectroscopy IntegratedAnalyses and in vitro Antibacterial Activity of Ridolfiasegetum Essential Oils from TunisiaAymen Jabrane a , Hichem Ben Jannet a , Fethia Harzallah-Skhiri b , Joseph Casanova c &Zine Mighri aa Laboratoire de Chimie des Substances Naturelles et de Synthése Organique (99/UR/ 12–26),Faculté des Sciences de Monastir , Université de Monastir , 5000 , Monastir , Tunisiab Institut Supérieur de Biotechnologie de Monastir , Monastir , 5000 , Tunisiac Université de Corse-CNRS , UMR 6134 SPE, Equipe chimie et Biomassa, Route desSanguinaires , 20000 , Ajaccio , FrancePublished online: 12 Mar 2013.

To cite this article: Aymen Jabrane , Hichem Ben Jannet , Fethia Harzallah-Skhiri , Joseph Casanova & Zine Mighri (2009) GC,GC-MS and 13C NMR Spectroscopy Integrated Analyses and in vitro Antibacterial Activity of Ridolfia segetum Essential Oils fromTunisia, Journal of Essential Oil Bearing Plants, 12:5, 521-530, DOI: 10.1080/0972060X.2009.10643752

To link to this article: http://dx.doi.org/10.1080/0972060X.2009.10643752

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GC, GC-MS and 13C NMR Spectroscopy Integrated Analyses and in vitroAntibacterial Activity of Ridolfia segetum Essential Oils from Tunisia

Aymen Jabrane 1, Hichem Ben Jannet 1*, Fethia Harzallah-Skhiri 2Joseph Casanova 3 and Zine Mighri 1

1 Laboratoire de Chimie des Substances Naturelles et de Synthèse Organique(99/UR/ 12-26), Faculté des Sciences de Monastir, Université de Monastir,

5000, Monastir, Tunisia2 Institut Supérieur de Biotechnologie de Monastir, 5000, Monastir, Tunisia3 Université de Corse-CNRS, UMR 6134 SPE, Equipe chimie et Biomassa,

Route des Sanguinaires, 20000 Ajaccio, France

Abstract: The present work describes the chemical composition and evaluates theantibacterial properties of the leaf and the flower oils from Ridolfia segetum, a traditional medicinalplant widely distributed in Tunisia. The essential oils were analysed by combination of GC, GC-MS and 13C NMR and 17 components, accounting for 88.7 % and 88.6 % of the oil, respectively,were identified. The oils from fresh flowers and leaves were characterized by high contents of α-phellandrene (34.7 % and 47.8 %), terpinolene (23.7 and 9.0 %), p-cymene (4.8 and 8.7 %), β-phellandrene (4.8 and 7.2 %) and limonene (3.7 and 3.9 %). Five oxygenated components wereisolated from the flower oil. The essential oils and isolated compounds were evaluated for theirantibacterial activity using the microdilution assay resulting in the inhibition of a number of commonhuman pathogenic bacteria as well as of some clinical and environmental isolated strains. Theminimum inhibitory concentrations (MICs) varied between 1.25 and 5 mg/mL for essential oils andbetween 0.62 and 5 mg/mL for pure compounds and hydrocarbon fraction. The minimum bactericidalconcentrations (MBCs) were 5 mg/mL for most strains and they varied between 1.25 and more than10 mg/mL for pure compounds. The results may suggest that the essential oils of R. segetum containscompounds with antibacterial activity, and therefore can be explored as a natural preservativeingredient in food and/or pharmaceutical preparations.

Keywords: Ridolfia segetum (L.); essential oils; Antibacterial activity GC(RI),GC-MS, 13C NMR.

Introduction: In recent years, scientists have focused on improving the food quality

*Corresponding author (Hichem Ben Jannet)E- mail: < hichem.benjannet@yahoo.fr >

ISSN 0972-060X

Received 26 June 2008; accepted in revised form 15 February 2009

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which becomes more and more urgent. Microorganisms have unfavourable effects on thequality, safety and shelf life of food. Therefore, the postharvest diseases caused bymicroorganisms are still an important problem. Synthetic chemicals are widely used in thecontrol of plant diseases. However, these chemicals are associated with undesirable effectsand some toxic residues in the products 1,2. Microorganisms causing infectious diseases inhumans may develop resistance to many antibiotics due to the indiscriminate use ofcommercial antibiotics 3,4.

Therefore, there has been a growing interest in research concerning alternativepesticides and antibacterial agents, including the plant extracts and essential oils that arerelatively more safe to the health and environment. Previous studies showed that the essentialoils constitute a class of very potent natural antibacterial and antioxidant agents 5-8. Theirbactericidal and antioxidant effects have established that their use in food system may beconsidered an additional intrinsic determinant to increase safety and to extend the shelf lifeof foods 5.

Ridolfia segetum (L.) Moris belongs to the Apiaceae family 9. It is widely distributedin the Mediterranean basin such as in Tunisia, Morocco, Sardinia, and in the central andSouthern parts of Spain (Castilla la Mancha and Andalucía provinces) as well as in theCanaries 9-11. It grows abundantly as a weed in the fields of cereals 9,10. It has been reportedthat this plant is used in Sardinian folk remedies for pathologies of slight entity. The driedfruits are used as eupeptic digestive in atony and digestive difficulty; the infusion of thefruits of R. segetum and Foeniculum vulgare Mill. is applied like carminative in the flatulence.It is indicated that the plant is believed, in folk medicine, to be an anti-haemorrhoid 10.

The essential oil of this plant collected from several areas of Morocco 12, Sardinia(Italy) 10, Central Spain, Castilla la Mancha Province 13, Andalucía province (South Spain)13 and Holy Land and Sinai 14 has been the subject of previous studies showing similaritiesand sometimes significant differences. Two types of oils may be distinguished, those largelydominated by monoterpene hydrocarbons and oxygenated monoterpenes such as α-phellandrene, terpinolene, (Z)-β-ocimene, p-cymene and piperitone oxide and those whichcontain also phenylpropanoids myristicine and dillapiole as major components or atappreciabe contents.

In previous works on this plant, we examined the kinetic of hydrodistillation and thecomposition of the flower oil all along the hydrodistillation process 15. Our findings revealedmyristicine and dillapiole as the main components of the oil, the contents of which varieddepending upon the duration of hydrodistillation (13.1 - 31.5 and 29.4 - 85.4 %, respectively).Dillapiole (47.4 %) and myristicine (19.2 %) were also the major components of a root oilsample from R. segetum 16, which possessed moderate antioxidant activity (evaluated usingthe 2,2’-diphenyl-1-picrylhydrazyl free radical scavenging method DPPH, IC50 = 38 mg/mL) and fair antibacterial activity (using the microdilution assay resulting in the inhibitionof a number of common human pathogenic bacteria as well as of some clinical andenvironmental isolated strains; MICs varied between 1.25 and 5 mg/mL).

In continuation of our efforts to study the phytochemistry and biological activity ofthis aromatic plant, we report here on the in vitro study of the antibacterial properties of theessential oils isolated from their leaves and flowers, in relation with their chemical

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composition investigated by mean of complementary techniques (GC, GC-MS and 13CNMR).

Materials and methods Plant material: Plant material was collected in the region of Kroussia (Tunisia), inMay 2006. Identification was performed at the Laboratoire de Biologie Végétale etBotanique, Institut Supérieur Agronomique de Chott Meriem, Ministère de l’Agricultureand Université de Sousse, Sousse, Tunisia. Voucher specimen has been deposited in theabove laboratory (N° RS-2).

Extraction of the essential oils, fractionation and isolation of pure compounds:The fresh leaves and flowers (5 x 150g each) were submitted separately to hydrodistillationon a Clevenger-type apparatus for 4h. The essential oils were collected by decantation,dried over sodium sulphate, weighed and stored in sealed glass vials in a refrigerator at 4-5°C prior to analysis. Preparative TLC (3x PE:EtOAc = 95:5) of the flower oil afforded sixfractions. Five fractions contained each one a pure compound as evidenced by 1H and13C NMR spectra: R1 (p-cymen-8-ol, 5 mg), R2 (piperitenone oxide, 15 mg), R3 (piperitenone,13 mg), R4 (dillapiole, 14 mg), R5 (myristicine, 8 mg). The sixth fraction, R6 (58 mg) wasrich in hydrocarbons.

Analytical GC: GC analysis was carried out using a Perkin-Elmer Autosystemapparatus equipped with FID and two fused-silica capillary columns (50 m x 0.22 mm i.d,film thickness 0.25 μm), BP-1 (polydimethyl siloxane) and BP-20 (polyethylene glycol).The oven temperature was programmed from 60°C to 220 C at 2°C/min. and then heldisothermally at 220°C for 20 min; injector temperature, 250°C; detector temperature, 250°C;carrier gas, helium (1 mL/ min); split, 1/60. The relative proportions of the essential oilconstituents were expressed as percentages, obtained by peak area normalization. Retentionindices (RI) were determined relative to the retention times of a series of n-alkanes withlinear interpolation, using the Target Compounds software from Perkin-Elmer.

Analytical GC-MS: The analyses of the volatiles were run on an Autospec GC-HRMS system (GC: Agilent 6890N). The fused-silica HP5-MS 5% phenyl methylpoly-siloxane capillary column (30 m x 0.32 mm I.D, film thickness of 0.25 μm) was directlycoupled to the MS. The carrier gas was helium, with a flow rate of 1.2 mL/min. Oventemperature was programmed (50°C for 1 min., then 50 - 300°C at 2°C/min.) andsubsequently, held isothermally for 4 min. Injector port: 250°C, detector: 280°C, split ratio1:5. Volume injected: 1.0 μL of 1 % solution (diluted in hexane). HRMS Autospec-Ultimarecording at 70 eV; scan time 0.75 s; mass Range 40-300 amu. Software adopted to handlemass spectra and chromatograms was a ChemStation.

13C NMR Analysis: The 13C NMR spectrum of the essential oil was recorded on aBruker AVANCE 400 Fourier Transform spectrometer operating at 100.13 MHz for 13C-NMR, equipped with a 5 mm probe, in deuterochloroform, with all shifts referred to internal

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tetramethylsilane (TMS). The following parameters were used: pulse width, 4 μs (flip angle45°); acquisition time, 2.7 s for 128 K data table with a spectral width of 25 000 Hz (250ppm.); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulatedscans was 5000 (about 40 mg of the oil in 0.5 ml of CDCl3).

1H and 13C NMR spectra ofpure compounds R1-R5 were recorded in CDCl3 with a Bruker NMR-300 spectrometer,operating at 300 MHz for 1H and 75 MHz for 13C.

Identification of the compounds: Identification of compounds by GC and GC-MSwas based on comparison of their retention indices with those of authentic samples and bycomparison of their mass spectra with those of pure compounds compiled in computerizedlibraries. The identification of the individual components by 13C-NMR is based on thecomparison of the signals in the oil spectrum with those of reference spectra complied inthe laboratory spectral library, with the help of laboratory-made software, following amethodology developed in our laboratory 17-19.

Antibacterial activity: The antibacterial activity of essential oils and pure com-pounds was tested against a panel of microorganisms, including reference strains: P.aeruginosa ATCC 27853, E. coli ATCC 25922, S. aureus ATCC 25923 and E. faecalisATCC 29212, clinical strains: S. aureus (Muticilin-resisitant S. aureus: MRSA), K.pneumoniae (Extended-Spectrum Beta Lactamase: ESLb), S. marcescens, S. pneumonia,S. Typhimurium Shigella. Spp, E. coli (ESLb) and E. faecalis and environmental strains: P.aeruginosa and S. aureus. The bacterial strains were cultured overnight at 37°C in MullerHinton agar (MHA), with the exception of S. pneumoniae (MHA containing 50 mL citrateblood/L).

Microwell dilution assay: MIC values were determined by a micro-titre plate dilutionmethod 20 dissolving the sample in 10 % DMSO solution. Sterile 10 % DMSO solution(100 μL) was pipetted into all wells of the micro-titre plate before transferring 100 μL ofstock solution to the microplate. Serial dilutions were made (starting from the first well) toobtain concentration ranging from 10 to 0.0775 mg/mL. Finally, 50 μL of 106 colony formingunits (cfu/mL) (according to Mc Farland turbidity standards) of standards microorganismsuspensions were inoculated onto microplates and incubated at 37°C for 24 h. At the end ofincubation period, the plates were evaluated for the presence or absence of growth. MICvalues were determined as the lowest concentration of the sample at which the absence ofgrowth was recorded. Each test was repeated twice. Vancomycin and Ceftazidim wereemployed as the positive control against Gram-positive and Gram-negative bacteria,respectively. The final concentration of DMSO in the well had no effect on bacterial growth.

Determination of minimum bactericidal concentration (MBC): Referring to theresults of the MIC assay, the wells showing complete absence of growth were identifiedand 5 μL of each well transferred to agar plates and incubation at previously-mentionedtimes and temperatures. 99.99% absence of growth was considered as the minimumbactericidal concentration 21.

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Results and discussionChemical composition: The yield of the essential oil from leaves and flowers of R.

segetum was 0.086 and 0.101 % (w/w), respectively. The leaf oil was light yellow whileflower oil was translucent, both oils were liquids at room temperature and their odours areagreeable. The composition of the oils was determined by GC, GC-MS and 13C NMR. Thepercentage composition, together with Retention Indices (RIs) calculated for eachcompound, and the identification methods are reported in table 1. A total of 17 constituents,accounting for 88.7 % and 88.6 % of the oil, respectively, were identified.

The studied leaf oil was rich in monoterpenes. Indeed, α-phellandrene (47.8 %) was,by far, the major component. Terpinolene, p-cymene and β-phellandrene (9.0 %; 8.7 % and7.2 %, respectively) were also important compounds of this oil. Limonene (3.9 %), α-pinene (3.1 %) and β-pinene (3.1 %) were present in lesser amount. Among the oxygenatedcompounds, piperitenone and its oxide represented 1.5 % and 0.4 % of the oil, respectively.p-Mentha-1(7),2-dien-6-ol (0.9 %) was the only identified monoterpenol. The phenolicfraction was represented only by dillapiole present at a low concentration (0.1 %).

The flower oil also contained a high proportion of monoterpene hydrocarbons, themajor components being α-phellandrene (34.7 %), terpinolene (23.7 %), p-cymene (4.8%), β-phellandrene (4.8 %) and limonene (3.7 %). Piperitenone and piperitenone oxide(2.4 % and 2.7 %, respectively), the only monoterpene ketones identified in the oil. Twophenylpropanoids, dillapiole and myristicine, have been also detected in the flower oil butthey constituted only 2.5 % and 1.5 % of the oil. The two identified monoterpenols, p-cymen-8-ol (1.0 %) and p-mentha-1(7),2-dien-6-ol (0.4 %) represented the minor oxygenatedmonoterpene fraction of this essential oil.

It is noticeable that α-phellandrene (47.8 and 34.7 % in our leaf and flower oils) wasthe main volatile compound of both flowers and leaves of R. segetum collected in othercountries 10-14: South Spain, 61.8 - 69.5 % leaves, 44.5 - 54.7 % flowers; Central Spain, 32.0- 33.8 % flowers; Egypt (Sinai), 44.1 - 48.9 % flowers; Italy (Sardinia), 24.7 %, flowers.Terpinolene (9.0 and 23.7 % in our samples) was present at appreciable content in theflower oil from various countries: Spain, 18.0 - 27.6 %; Sardinia, 24.7 % and Sinai 11.8 -16.3 %.

Conversely, myristicine which accounted for 13.6 % in a flower oil from Sardiniaand 33.0 % in a fruit oil from Morocco 10,12 reached only 1.5 % in our samples. Dillapiole(0.1 and 2.5 % in our samples) was mentioned as an important component (5.1 - 39.6 %) ofthe fruit oil from Central Spain 13. Dillapiole and myristicin were also the major componentsof a root oil sample from Tunisian R. segetum investigated in a previous study 16. Finally,piperitone oxide, one of the major components (8.3 - 18.0 %) of the flower oil from Sinai 14,was not detected in our samples.

Our previous work 15 on the flower oil of this plant collected in the region of M’saken(June 2004), not very far from the region from Kroussia (ca. 30 Km) reported on aphenylpropanoid-rich oil (dillapiole and myristicine) This surprising difference could beexplained by climatic factors which could change from a year to another, by the time ofharvest, as well as by genetic parameters which induce a chemical variability.

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Antibacterial activity: The antibacterial activity of leaf and flower oils of R. segetumwere assayed against various microorganisms: four reference strains, two environmentalstrains and eight clinical strains by the evaluation of MIC and MBC values (Tables 2). Thehydrocarbon monoterpenic fraction (R6) of the flower oil and the five isolated compounds(R1-R5, p-cymen-8-ol, piperitenone oxide, piperitenone, dillapiole and myristicine) weretested individually against four reference strains, two environmental strains and two clinicalstrains (Table 3).

The essential oils from leaves and flowers of R. segetum have a significant and broadspectrum of activity (Table 2). The MIC and MBC values for all bacterial strains were inmost cases in the range of 1.25 - 5 mg/mL and 2.5 - 5 mg/mL, respectively. Another resultfrom this study was that Gram-positive and Gram-negative bacteria exhibit practically thesame sensitivity to tested essential oils. The antibacterial activity of R. segetum leaf andflower oils may be attributed to the presence of the major monoterpenes: α-phellandrene,terpinolene, β-phellandrene, p-cymene and limonene. This result is in a good concordancewith previous data 22,23.

The hydrocarbon monoterpenic fraction (R6) exhibited a great activity against all thetested bacteria, the MIC values (0.62 - 1.25) being lower than that measured for the leaf andflower oils (2.5 - 5) (Table 3). On the other hand, p-cymen-8-ol (R1), piperitenone oxide(R2), piperitenone (R3), dillapiole (R4) and myristicine (R5) showed individually significanteffects towards the used strains. They may contribute to the antibacterial capacity of theleaf and flower oils of R. segetum.

Results reported here can be considered as the first detail document on the in vitroantibacterial features of R. segetum. It has shown that essential oils of this species may bepotentially useful source of especially natural antibacterial principles.

Acknowledgments: Thanks to Prof. Maha Mastouri for her contribution to theantibacterial assays, to Mrs Amna Benzarti and Mrs Cathy Lugrezi for their contribution toGC and NMR analysis.

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Table 1. Chemical composition of the leaf, flower and root oils of R. segetum

No Compoundsa Retention Index composition % Mode ofBP-1 BP-20 leaf flower Identification

1 α-Thujene 922 1025 0.4 0.2 RI, MS, 13C-NMR2 α-Pinene 930 1025 3.1 1.6 RI, MS, 13C-NMR3 Sabinene 965 1123 0.5 0.4 RI, MS, 13C-NMR4 β-Pinene 970 1112 3.1 1.4 RI, MS, 13C-NMR5 Myrcene 980 1163 1.1 1.0 RI, MS, 13C-NMR6 α-Phellandrene 999 1169 47.8 34.7 RI, MS, 13C-NMR7 p-Cymene 1012 1272 8.7 4.8 RI, MS, 13C-NMR8 Limonene 1021 1202 3.9 3.7 RI, MS, 13C-NMR9 β-Phellandrene 1021 1212 7.2 4.8 RI, MS, 13C-NMR

10 (Z)-β-Ocimene 1025 1233 1.0 1.8 RI, MS, 13C-NMR11 Terpinolene 1080 1286 9.0 23.7 RI, MS, 13C-NMR12 p-Cymen-8-ol 1159 1847 - 1.0 RI, MS, 13C-NMR13 p-Mentha-1(7), 1181 1805 0.9 0.4 RI, MS, 13C-NMR

2-dien-6-ol14 Piperitenone 1309 1924 1.5 2.4 RI, MS, 13C-NMR15 Piperitenone oxide 1332 1959 0.4 2.7 RI, MS, 13C-NMR16 Myristicine 1487 2267 - 1.5 RI, MS, 13C-NMR17 Dillapiole 1591 2361 0.1 2.5 RI, MS, 13C-NMR

Monoterpene hydrocarbons 85.8 78.1Oxygenated monoterpenes 2.8 6.5Phenolic derivatives 0.1 4.0Total 88.7 88.6

a The constituents are arranged according to their elution on the apolar BP-1 capillarycolumn.

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Table 2. Antibacterial activities of the leaf and floweressential oils from R. segetum

Microorganisms leaf flowerMICa MBCb MIC MBC

Reference strainsP. aeruginosa ATCC 27853 2.5 5 2.5 5E. coli ATCC 25922 2.5 5 2.5 >5S. aureus ATCC 25923 2.5 >5 5 5E. faecalis ATCC 29212 5 >5 2.5 5Environmentally isolated strainsP. aeruginosa 5 >5 5 5S. aureus 2.5 5 5 5Clinically isolated strainsS. aureus (MRSA) 5 >5 5 5K. pneumoniae (ESLb) 5 >5 5 5S. marcescens 2.5 5 2.5 5S. pneumonia 2.5 5 2.5 5S. Typhimurium 2.5 5 2.5 >5Shigella. Spp 2.5 5 2.5 2.5E. coli (ESLb) 1.25 5 1.25 5E. faecalis 2.5 5 5 >5

a MIC, Minimum inhibitory concentration (as mg/mL).b MBC, Minimum bactericidal concentration (as mg/mL).

Hichem Ben Jannet et al. / Jeobp 12 (5) 2009 pp 521 - 530 529

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Tabl

e 3.

Ant

ibac

teri

al a

ctiv

ities

of t

he fr

actio

ns R

1-R6 i

sola

ted

from

R. s

eget

um fl

ower

oil

Mic

roor

gani

sms

R1

R2

R3

R4

R5

R6

MIC

aM

BC

aM

ICM

BC

MIC

MB

CM

ICM

BC

MIC

MB

CM

ICM

BC

P. a

erug

inos

a AT

CC

278

532.

55

2.5

>10

2.5

52.

55

2.5

51.

252.

5E.

col

i ATC

C 2

5922

2.5

52.

55

1.25

51.

255

2.5

51.

252.

5S.

aur

eus A

TCC

259

232.

55

1.25

51.

255

2.5

>10

5>1

01.

252.

5E.

faec

alis

ATC

C29

212

1.25

52.

5>1

02.

5>1

05

55

>10

1.25

5

Envi

ronm

enta

lly is

olat

ed st

rain

s

P. a

erug

inos

a2.

55

2.5

51.

255

2.5

> 10

1.25

50.

622.

5S.

aur

eus

2.5

52.

55

1.25

51.

252.

50.

622.

50.

622.

5

Clin

ical

ly is

olat

ed s

train

s

S. T

yphi

mur

ium

1.25

52.

55

2.5

51.

252.

50.

622.

506

21.

25Sh

igel

la S

pp.

1.25

51.

255

2.5

51.

252.

51.

252.

50.

622.

5

a M

IC, M

inim

um in

hibi

tory

con

cent

ratio

n (a

s mg/

mL)

b M

BC

, Min

imum

bac

teric

idal

con

cent

ratio

n (a

s mg/

mL)

R 1 = p

-cym

en-8

-ol

R 2 = p

iper

iteno

ne o

xide

R 3 = p

iper

iteno

neR 4

= dilla

piol

eR 5

= m

yris

ticin

e Fr

actio

nR 6 c

onta

ined

mon

oter

pene

hyd

roca

rbon

s

Hichem Ben Jannet et al. / Jeobp 12 (5) 2009 pp 521 - 530 530

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