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Process Biochemistry xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

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hemical composition, angiotensin I-converting enzyme (ACE) inhibitory,ntioxidant and antimicrobial activities of the essential oil from south Tunisianjuga pseudoiva Rob. Lamiaceae

ohamed Ben Mansoura,b, Rafik Balti a,∗, Lotfi Rabaouia,d, Ali Bougatef c, Mokhtar Guerfel a

Higher Institute of Applied Biology of Medenine, University of Gabes, Km 22.5 Road El Djorf 4119, Medenine, TunisiaLaboratoire de Génétique, Biodiversité et valorisation des Bioressources (LR 11-ES 41), University of Monastir, TunisiaHigher Institute of Biotechnology of Sfax, University of Sfax, Km 4 Road Soukra 3038, Sfax, TunisiaUniversity of Tunis El Manar – Faculty of Science of Tunis, Research Unit of Integrative Biology and Evolutionary and Functional Ecology of Aquatic Systems, University Campus092 Tunis, Tunisia

r t i c l e i n f o

rticle history:eceived 9 November 2012eceived in revised form 24 January 2013ccepted 18 February 2013

eywords:juga pseudoiva

a b s t r a c t

The essential oil of Ajuga pseudoiva, collected from Tunisia, was analyzed using gas chromatography–massspectroscopy. Thirty-two compounds accounting for 95.76% of the total oil were identified. Sesquiter-penes were found to be the most abundant components of A. pseudoiva oil. And they were mainlyrepresented by viridiflorol (30.17%), germacrene B (9.26%) (�, � and �)-eudesmol (8.11%) and aromaden-drene (7.45%). The essential oil of A. pseudoiva showed radical scavengers activity (IC50 = 0.72 mg/mL)and displayed lipid peroxidation inhibitory activity (IC50 = 0.6 mg/mL). A. pseudoiva essential oil was also

CE inhibitory activityntibacterial activityntioxidant activityssential oil

found to exhibit a dose-dependent ACE inhibitory activity with an IC50 value of 65.5 �g/mL. Moreover, theantimicrobial activity of the essential oil was tested against 17 species of microorganisms, and the resultsobtained showed significant antibacterial activity against the Gram-positive and Gram-negative bacteria,with inhibition zones and minimal inhibitory concentration values of 14–32 mm and 84–137 �g/mL and6–21 mm and 105–336 �g/mL, respectively. Higher activity was also found against several fungal strains.

. Introduction

Oxidation processes are intrinsic to the energy management ofll living organisms and are therefore kept under strict control byeveral cellular mechanisms [1]. However, the excessive produc-ion of free radicals and the unbalanced mechanisms of antioxidantrotection result in the onset of numerous chronic disorders:ancer, cardiovascular disease, diabetes, and other ageing-relatediseases [2]. In the last decades, the use of synthetic antioxidantsuch as butylated hydroxyanisole (BHA) and butylated hydroxy-oluene (BHT) in stabilization of foods in agro-alimentary industryre suspected to have negative effects on consumer’s health [3].ccordingly, attention is focused on the use of naturally occur-ing safe antioxidants and biologically active compounds fromedicinal plants. Several plants were found to exhibit antioxidant

roperties, which are mainly attributed to a variety of active natural

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ntioxidants including flavonoids, polyphenols, alkaloids, antho-yanins, terpenoids, carotenoids and vitamins [4].

∗ Corresponding author. Tel.: +216 75 633 919; fax: +216 75 633 918.E-mail address: rafikbalti1981@gmail.com (R. Balti).

359-5113/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ltd. All rittp://dx.doi.org/10.1016/j.procbio.2013.02.022

Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.

Numerous studies have been published on the antiviral, antimi-crobial and cytotoxic activities of plant extracts. Some of thesestudies claim that the phenolic compounds present in plant extractsmight also play a major role in their biological effects [5,6]. How-ever, essential oils from traditional medicinal plants have beenfrequently used as a source of natural antioxidants, antifungal andantibacterial agents [7–10]. Additionally, some of these oils havebeen reported to reduce the risk of hypertension and coronary heartdisease. In fact, these essential oils have been identified to possessan in vitro Angiotensin I-Converting Enzyme (ACE) inhibitory activ-ity. ACE is a zinc metallopeptidase that can increase blood pressureby converting the inactive decapeptide angiotensin I to the potentvasoconstrictor angiotensin II (octapeptide) and by the degradationof a hypotensive peptide, bradykinin [7,10].

Ajuga pseudoiva (L.) Schreiber (Lamiaceae), locally known as“Chendgoura” in Tunisia, Algeria and Morocco is potentially used inphytomedicine. A. pseudoiva is used in traditional medicine in Alge-ria to treat diabetes, and it is known to have anti-inflammatory,antifungal, antimicrobial, antifebrile, and anthelmintic activity

composition, angiotensin I-converting enzyme (ACE) inhibitory,Tunisian Ajuga pseudoiva Rob. Lamiaceae. Process Biochem (2013),

[11]. More recently, several studies have been published on thehypoglycemic [12], vasorelaxant [13] and hypolipidemic [14,15]activities of plant extracts. Some of these studies highlighted thepresence, in A. pseudoiva, of high phenolic compounds, flavonoids

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nd tannins. The latter compounds were reported to be activengredients that protect against coronary heart disease [13]. Inddition, Chenni et al. [15] and Bouderbala et al. [16] proved thathe plant aqueous extract is effective in improving the antioxi-ant status by reducing lipid peroxidation in plasma and tissuesnd enhancing the antioxidant enzymes in rats fed with high-holesterol diet. Furthermore, A. pseudoiva may reduce intestinalholesterol absorption.

However, in spite of the advantages and benefits of the con-eneric species A. pseudoiva, there is still a knowledge gap about theffect of its essential oil on biological activities. Within this context,he present work was carried out aiming to (i) study angiotensin-converting enzyme (ACE) inhibitory, antioxidant, antifungalnd antibacterial activities of the essential oil of A. pseudoivaxtracted by Clevenger distillation apparatus and to (ii) determineheir chemical composition using gas chromatography–masspectroscopy (GC–MS).

. Materials and methods

.1. Chemicals

Angiotensin I-converting enzyme from rabbit lung, the ACE synthetic substrateippuryl-l-histidyl-l-leucine (HHL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), BHA, �-arotene, linoleic acid, Tween 40, potassium ferricyanide, trichloroacetic acid (TCA)nd ferric chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA).ll culture media and standard antibiotics were purchased from Bio-Rad (Bio-Rad

aboratories, France). All other chemicals and solvents were of analytical grade. Allolutions were freshly prepared in distilled water.

.2. Plant material

The aerial parts (stems and leaves) of A. pseudoiva (L.) were collected from theouth-East of Tunisia on March 2011 (Toujane, bioclimatic zone: lower arid, rainfullmm/year): 100–200, Latitude: 33◦27′34′′N, Longitude: 10◦8′17′′E, altitude 520 m).he plant material was dried at room temperature in the shadows, for 2 weeks untilonstant weight. The dried preparation was ground further to obtain a fine powder,nd then stored at ambient temperature in a dry and dark place until being used.

.3. Extraction of the essential oil

The dried powder from A. pseudoiva (100 g) was subjected to hydrodistillationor 4 h, using a Clevenger-type apparatus (ST15 OSA, Staffordshire, UK). The obtainedistillate (100 mL) was extracted twice successively with 100 mL of n-hexane andethanol. The mixture was dried with anhydrous sodium sulphate. For the determi-

ation of the procedure yield, the solvent was evaporated using a rotatory vacuumvaporator. The yield was determined by weighing the remaining oil on an analyticalalance. The resulting essential oil was stored at −20 ◦C until further analysis.

.4. Essential oil analysis

The isolated volatile compounds were analyzed by GC/MS, using an Agilentechnologies 6890 N gas chromatograph. The fused HP-5MS capillary column wasonnected to the ion source of a mass spectrometer (Agilent Technologies 5975B).he column temperature was programmed at 50 ◦C for 1 min, then 7 ◦C/min to50 ◦C, and then left at 250 ◦C for 5 min. The temperature of the injector port waseld at 250 ◦C, the temperature of the detector was set at 280 ◦C. The carrier gasas helium, with a flow rate of 1.2 mL/min. The mass-spectrometer was operat-

ng (full. scan-mode) in the EI-mode at 70 eV and an ionization energy of 1294 V. Aample of 1.0 �L was injected, using split mode (split ratio, 1:100). The compositionas reported as a relative percentage of the total peak area. The identification of

he essential oil constituents was based on a comparison of their retention times to-alkanes mixture (C6–C24). Compounds were further identified and authenticatedsing their mass spectra compared to the Wiley 275 and NIST mass spectra libraries.

.5. Antioxidant activity

.5.1. DPPH• assayThe DPPH radical-scavenging activity of A. pseudoiva essential oil was deter-

ined as described by Kirby and Schmidt [17] with some modifications. A volumef 500 �L of essential oil at different concentrations (0.2–1.0 mg/mL) was addedo 375 �L of 99% methanol and 125 �L of DPPH• solution (0.2 mM in methanol) as

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ree radical source. The mixtures were incubated for 60 min in the dark at roomemperature. BHA was used as positive control. Scavenging capacity was measuredpectrophotometrically by monitoring the decrease in absorbance at 517 nm. Ints radical form, DPPH• has an absorption band at 517 nm which disappears uponeduction by an antiradical compound. Lower absorbance of the reaction mixture

PRESShemistry xxx (2013) xxx– xxx

indicated higher free radical-scavenging activity. DPPH radical-scavenging activitywas calculated as:

DPPH radical-scavenging activity % = A control − A sampleA control

× 100

where A control is the absorbance of the control reaction (containing all reagentsexcept the sample), and A sample is the absorbance of A. pseudoiva essential oil.Tests were carried out in triplicate.

2.5.2. Ferric-reducing activityThe reducing power of the essential oils was determined as described by Yildirim

et al. [18]. Sample solutions (0.5 mL) with different concentrations of the essentialoil were mixed with 1.25 mL of 0.2 M phosphate buffer pH 6.6 and 1.25 mL of 10 g/Lpotassium ferricyanide solution. The mixtures were incubated for 30 min at 50 ◦C.After incubation, 1.25 mL of 100 g/L trichloroacetic acid was added and the reac-tion mixtures were centrifuged for 10 min at 3000 × g. An aliquot of 1.25 mL of thesupernatant from each sample mixture was mixed with 1.25 mL of distilled waterand 0.25 mL of 1.0 g/L ferric chloride solution in a test tube. After a 10 min reactiontime, the absorbance was measured at 700 nm. Higher absorbance of the reactionmixture indicated higher reducing power. Tests were carried out in triplicate.

2.5.3. ˇ-Carotene bleaching by linoleic acid assayThe ability of A. pseudoiva essential oil to prevent bleaching of �-carotene was

assessed as described by Koleva et al. [19]. A stock solution of �-carotene/linoleicacid was prepared by dissolving 0.5 mg of �-carotene, 25 �L of linoleic acid and200 �L of Tween 40 in 1 mL of chloroform. The chloroform was completely evap-orated under vacuum in a rotatory evaporator at 40 ◦C; then 100 mL of distilledwater were added and the resulting mixture was vigorously stirred, the emulsionwas prepared before each experiment. Aliquots (2.5 mL) of the �-carotene/linoleicacid emulsion were transferred to test tubes containing different essential oil con-centrations, followed by incubation for 2 h at 50 ◦C, the absorbance of each sampleand BHA used as reference were measured at 470 nm and the relative antioxidantactivity was calculated according to the following formula:

Antioxidant activity (%) =(

1 − A0 − At

A00 − A0t

)× 100

where A0 is the absorbance at the beginning of the incubation with the essential oil;At is the absorbance after incubation with the essential oil; A00 is the absorbance atthe beginning of the incubation without the essential oil and A0t is the absorbanceafter incubation without the essential oil. Tests were carried out in triplicate.

2.6. Antimicrobial activity

2.6.1. Microbial strainsAntimicrobial activities of A. pseudoiva essential oil were tested against ten

strains of bacteria: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 13883),Bacillus subtilis (ATCC 26633), Bacillus cereus (ATCC 11778), Listeria monocytogenes(ATCC 15313), Enterococcus faecium (ATCC 12755), Salmonella typhimurium (ATCC19430) and Enterococcus faecalis (ATCC 29212). Antifungal activities were testedusing Aspergillus niger (ATCC 6275), Aspergillus fumigatus (ATCC 9142), Saccha-romyces cerevisiae (ATCC 561), Candida albicans (ATCC 10231), Candida rugosa (ATCC10571), Fusarium solani, and Fusarium oxysporium. The Fusarium species were kindlyprovided by the Centre of Biotechnology of Sfax, Tunisia.

2.6.2. Agar diffusion methodAntimicrobial activity was performed according to the method described by

Berghe and Vlietinck [20]. A. pseudoiva essential oil (50 �L) was dissolved in100% HPLC grade dimethylsulfoxide (DMSO) (950 �L) and sterilized by filtrationthrough 0.22 �m Nylon membrane filter. Culture suspension (200 �L) of the testedmicroorganisms 106 colony-forming units (cfu)/mL of bacteria cells (estimated byabsorbance at 600 nm) and 108 spores/mL of fungal strains (measured by Malassezblade) were spread on Muller-Hinton agar and PDA medium, respectively. Then,bores (3 mm depth, 4 mm diameter) were made using a sterile borer and were loadedwith 50 �L of A. pseudoiva essential oil at 30 mg/mL. A well with only 50 �L of DMSO(without essential oil) was used as a negative control. Tetracycline, vancomycin andnystatin were used as positive references for bacteria and fungi. The Petri disheswere kept, first, for 1 h at 4 ◦C, and then incubated for 24 h at 37 ◦C for bacteria andfor 72 h at 30 ◦C for fungal strains. Antimicrobial activity was evaluated by mea-suring the diameter of the growth inhibition zones in millimetres (including welldiameter of 4 mm). The measurements of inhibition zones were carried out for threesample replications, and values are the means of three replicates.

2.6.3. Determination of the minimal inhibitory concentration (MIC)MIC values, which represent the lowest essential oil concentration that com-

composition, angiotensin I-converting enzyme (ACE) inhibitory,Tunisian Ajuga pseudoiva Rob. Lamiaceae. Process Biochem (2013),

pletely inhibits the growth of microorganisms, were determined, based on amicro-well dilution method [21]. The essential oil was dissolved (at 30 mg/mL) in100% DMSO and then dilution series were prepared in a 96-well microtitre plate.Oil-free solution, containing 50 �L distilled water and 950 �L DMSO, was usedas a negative control. Each well of the microplates included 40 �L of the growth

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edium, 10 �L of the inoculum (106 cfu/mL), and 50 �L of the diluted essential oil.he microplates were thereafter incubated overnight at 37 ◦C. As an indicator oficroorganism growth, 40 �L of p-iodonitrotetrazolium violet (INT) dissolved inater were added to the wells and incubated at 37 ◦C for 30 min. The colourless

etrazolium salt acts as an electron acceptor and is reduced to a red-coloured form-zan product by biologically active organisms [21]. And hence, when the microbialrowth was inhibited, the solution in the corresponding well remains clear, afterncubation with INT. The determinations of MIC values were carried out by replicate.

.7. Determination of the angiotensin I-converting enzyme (ACE) inhibitionctivity

The ACE inhibition activity was measured as reported by Nakamura et al. [22]. Aample solution (80 �L), of different concentrations (25, 50, 75, 100 and 150 �g/mL)f A. pseudoiva essential oil, was added to 200 �L containing 5 mM hippuryl-l-istidyl-l-leucine (HHL), and then preincubated for 3 min at 37 ◦C. A. pseudoivassential oil and HHL were prepared in 100 mM borate buffer, pH 8.3, containing00 mM NaCl. The reactions were then initiated by adding 20 �L of 0.1 U/mL ACErom rabbit lung prepared in the same buffer and incubated for 30 min at 37 ◦C.he enzymatic reaction was terminated by the addition of 250 �L of 1.0 M HCl.he released hippuric acid (HA) was quantified by RP-HPLC on a Vydac C18 col-mn connected to a system composed of a Waters TM 600 automated gradientontroller pump module, a WaterWisp 717 automatic sampling device and a Waters96 photodiode array detector. The sample was thereafter eluted using an acetoni-rile gradient from 0 to 28% and from 28 to 47% in 0.1% trifluoroacetic acid (TFA)v/v) for 50 and 20 min, respectively. The eluate was followed at 228 nm and thepectral and chromatographic data were stored on a NEC Image 446 computer. Mil-ennium software was used to acquire, analyze and plot chromatographic data. Theverage value estimated from three determinations at each concentration was usedo calculate the ACE inhibition rate as follows:

CE inhibition (%) =[

B − A

B − C

]× 100

here A is the absorbance of HA generated in the presence of ACE inhibitor com-onent, B the absorbance of HA generated without ACE inhibitors and C is thebsorbance of HA generated without ACE (corresponding to HHL autolysis in theourse of enzymatic assay).

.8. Statistical analysis

Values were expressed as means ± standard deviation. Analysis of variance wasonducted and differences between variables were tested for significance by one-ay ANOVA with a SPSS statistic program (SPSS 10.0 for Windows, SPSS Inc., Chicago,

L). Differences at p < 0.05 were considered statistically significant.

. Results and discussion

.1. Chemical composition of A. pseudoiva essential oil

The yield of essential oil obtained by hydrodistillation from theerial part of A. pseudoiva was 0.35% (w/w). The percentages andhe retention indices of the identified components are listed inable 1 according to their elution on the HP-5MS column. GC–MSnalysis of A. pseudoiva essential oil led to the identification ofhirty-two compounds, accounting for 95.76% of the total oil. Theseompounds were divided into five classes which are monoterpeneydrocarbons, oxygenated monoterpenes, sesquiterpene hydro-arbons, oxygenated sesquiterpenes and oxygenated diterpenesTable 1). The most abundant compounds were viridiflorol (30.17%),ermacrene B (9.26%), aromadendrene (7.45%) and phytol (6.42%).n addition, the oil was mainly constituted of sesquiterpenes84.1%), of which oxygen-containing sesquiterpenes (55.32%) pre-ailed over sesquiterpene hydrocarbons (28.86%). Moreover, 11xygen-containing sesquiterpenes were particularly identified inhe oil extracted and the results obtained highlighted the preva-ence of viridiflorol (30.17%), �-copaen-4-�-ol (5.54%) and (�, � and)-eudesmol (8.11%). As for the 14 sesquiterpene hydrocarbons,

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heir analysis showed that the most abundant were found to beromadendrene (7.45%) and germacrene B (9.26%). Regarding thexygenated diterpenes, it is worth nothing that they were mainlyresented by phytol (6.42%).

positive control measured by DPPH• assay. Values presented are the means of trip-licate analysis.

3.2. Antioxidant activity

The antioxidant activity of the essential oil of A. pseudoiva wasevaluated by various antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing powerand �-carotene bleaching assay.

3.2.1. DPPH free radical-scavenging activityDPPH• is a stable free radical that shows maximum absorbance

at 517 nm. When DPPH radicals encounter a proton-donating sub-strate, such as an antioxidant, the radicals are scavenged andthe absorbance is reduced [23]. The decrease in absorbance istaken as a measure of radical-scavenging activity. Fig. 1 showsthe free radical scavenging activities of A. pseudoiva essential oiland BHA used as positive control, furthermore IC50 values weredetermined (0.72 mg/mL and 0.01 mg/mL, respectively). The freeradical-scavenging activity of A. pseudoiva essential oil was lowercompared to other Lamiaceae species such as Marrubium vulgare [8]and Teucrium marum [24] which showed IC50 values correspondingrespectively to 0.074 and 0.013 mg/mL, respectively. The obtainedherein were found to be in agreement with the findings of severalauthors who reported that the efficiency of an antioxidant compo-nent to reduce DPPH essentially depends on its hydrogen donatingability, which is directly related to the presence of phenolic com-pounds [25] and the abundance of monoterpenes hydrocarbons[26] and oxygenated monoterpenes [27].

3.2.2. Reducing powerThe reducing power assay is often used to evaluate the abil-

ity of natural antioxidant to donate an electron or hydrogen [23].The determination of the ferric reducing/antioxidant was based onthe reduction of Fe3+/ferricyanide complex to the ferrous form inpresence of reductants (antioxidants) in the tested samples. TheFe2+ was then monitored by measuring the formation of Perl’sPrussian blue at 700 nm. In fact, it is widely accepted that higherabsorbance at 700 nm is correlated to power reducing. As shownin Fig. 2, the reducing capacity of the essential oil from A. pseu-doiva increased in a concentration-dependant manner. However,the reducing power of essential oil from A. pseudoiva remained sig-nificantly lower than that of BHA. This difference may be explainedby lower content of electron donor compounds in the chemicalcomposition of this oil which was characterized by the abundanceof sesquiterpenes (84.1%). In accordance of our data, Shimada et al.[23] reported that the reductive potential may be related to the

composition, angiotensin I-converting enzyme (ACE) inhibitory,Tunisian Ajuga pseudoiva Rob. Lamiaceae. Process Biochem (2013),

presence of phenolic compounds, such as isothymol and carvacrol,due to hydroxyl substitutions in the aromatic ring, which possessespotent hydrogen-bonding abilities.

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Table 1Chemical composition, retention indices (RI) and percentage composition of A. pseudoiva essential oil.a

No. Compounds % RI Identification

1 �-Pinene 0.49 930 MS,IR2 �-Pinene 2.19 973 MS,IR3 1-Octen-3-ol 0.4 976 MS,IR4 �-Terpinene 0.23 1054 MS,IR5 �-Terpinolene 0.68 1095 MS,IR6 4-Vinyl-2-methoxy-phenol 0.79 1309 MS,IR7 �-Copaene 0.77 1371 MS,IR8 Aromadendrene 7.45 1436 MS,IR9 �-Gurjunene 0.34 1443 MS,IR10 �-Gurjunene 0.46 1449 MS,IR11 Alloaromadendrene 0.83 1458 MS,IR12 �-Selinene 0.7 1484 MS,IR13 Bicyclogermacrene 0.76 1492 MS,IR14 �-Cadinene 0.85 1512 MS,IR15 �-Cadinene 1.77 1517 MS,IR16 Germacrene B 9.26 1559 MS,IR17 �-Selinene 2.33 1566 MS,IR18 Spathulenol 0.87 1577 MS,IR19 Viridiflorol 30.17 1586 MS,IR20 �-Copaen-4-�-ol 5.54 1592 MS,IR21 Caryophyllene oxide 2.91 1601 MS,IR22 Eremophilene 1.36 1613 MS,IR23 Epiglobulol 2.94 1621 MS,IR24 2-Isopropyl-5-methyl-9-

methylene licycle[4.4.0]dec-1-ene

0.85 1625 MS,IR

25 �-Eudesmol 1.12 1639 MS,IR26 �-Eudesmol 2.98 1651 MS,IR27 �-Eudesmol 4.01 1653 MS,IR28 Farnesol 0.6 1711 MS,IR29 Hexahydrofarnesyl acetone 2.51 1827 MS,IR30 Dibutyl phthalate 2.78 1936 MS,IR31 Dihydrophytol 0.38 2049 MS,IR32 Phytol 6.42 2100 MS,IR

Total identification 95.76%Yield (g/100 g dry weight) 0.35Monoterpene hydrocarbons 3.59Oxygenated monoterpenes 1.19Sesquiterpene hydrocarbons 28.86Oxygenated sesquiterpenes 55.32

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trol. This antioxidant activity was more important than Thymuscaramanicus of which the essential oil showed 79% inhibition at2 mg/mL [28].

Oxygenated diterpenes

a Values presented are the means of triplicate analysis.

.2.3. Antioxidant activity measured by the ˇ-carotene bleachingssay

The potential of A. pseudoiva oil to inhibit lipid peroxidation wasvaluated using the �-carotene/linoleic acid bleaching test. The lat-er test measures the essential oil potential to inhibit conjugatediene hydroperoxides formation from linoleic acid oxidation [27].he results of antioxidant activities of A. pseudoiva essential oil and

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tandard (BHA) are presented in Fig. 3. At the same concentration0.4 mg/mL), the antioxidant activity of the essential oil was two-old lower than the activity achieved by BHA (36.50 ± 3.1% versus

ig. 2. Antioxidant capacities of A. pseudoiva essential oil, using ferric reducingower method. Values presented are the means of triplicate analysis.

6.8

83 ± 2%). Moreover, at higher concentration used (1.0 mg/mL), thepotential of essential oil sample to inhibit lipid peroxidation wasimproved to 68.50 ± 4% versus 93 ± 1.50% for the positive con-

composition, angiotensin I-converting enzyme (ACE) inhibitory,Tunisian Ajuga pseudoiva Rob. Lamiaceae. Process Biochem (2013),

Fig. 3. Antioxidant activity of A. pseudoiva essential oil and BHA as positive con-trol, measured by �-carotene bleaching essay. Values presented are the means oftriplicate analysis.

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Table 2Antibacterial activity of A. pseudoiva essential oil.

Strains of bacteria Zone inhibition (mm)a MIC (�g/mL)a

Essential oil Vancomycinb Tetracyclineb Essential oil

Gram positiveB. subtilis 19 ± 0.5 16 ± 0.5 24 ± 1.0 128 ± 3.51B. cereus 19 ± 1.0 11 ± 0.5 29 ± 1.0 124 ± 5.25E. faecalis 26 ± 1.5 29 ± 2.5 – 90 ± 2.34S. aureus 32 ± 1.5 15 ± 1.5 26 ± 1.5 84 ± 1.14L. monocytogenes 14 ± 1.0 23 ± 0.5 28 ± 1.5 137 ± 5.00E. faecium 22 ± 1.5 22 ± 1.0 – 95 ± 1.50

Gram negativeP. aeruginosa 6 ± 0.5 9 ± 0.0 11 ± 0.0 336 ± 10.87E. coli 21 ± 1.5 22 ± 1.5 26 ± 1.0 105 ± 3.37K. pneumoniae 19 ± 1.0 10 ± 0.5 25 ± 1.0 246 ± 6.50S. typhimurium 15 ± 0.5 18 ± 1.0 21 ± 0.5 212 ± 3.50

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The IC50 of A. pseudoiva essential oil was estimated to be.6 mg/mL and it was found to be similar to the IC50 exhibited byresh leaves essential oils of Cymbopogon schoenanthus L. SprengIC50 = 0.47 mg/mL) [29], Thymus algeriensis (IC50 = 0.5 mg/mL) [10]nd Mosla chinensis oil (IC50 = 0.59 mg/mL) [30]. In contrast, it wasound to be less effective compared to the essential oils fromidens pilosa (IC50 = 0.0497 mg/mL) [31] and from M. vulgare L.IC50 = 0.036 mg/mL) [8].

In addition, the data obtained herein from the evaluation ofntioxidant activity of A. pseudoiva as well by DPPH as by �-arotene/linoleic acid bleaching tests lead to deduce the presencef high antioxidant effect, highlighting the considerable potentialf the plant essential oil as an antioxidant food additive.

.3. ACE inhibitory activity of A. pseudoiva essential oil

The inhibition of angiotensin I-converting enzyme (ACE) byietary anti-hypertensive agents is potentially an important strat-gy to manage hypertension. With this regard, it was demonstratedhat the ACE inhibition is considered as a useful therapeuticpproach in the treatment of high blood pressure. Since syntheticCE inhibitors may cause adverse side-effects, plants essential oilsould be used as natural and economical ACE inhibitors for hyper-ension prevention and treatment. The A. pseudoiva essential oilas then tested for ACE inhibition activity. As shown in Fig. 4, A.

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seudoiva essential oil exhibited dose-dependent ACE inhibitoryctivities of 28.3%, 53.8% and 74.5%, at 25, 75 and 150 �g/mL ofil, respectively. The IC50 value, defined as the concentration of

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50

75

100

25 50 75 10 0 15 0

Concentration (μg/ml)

AC

E i

nh

ibit

ion

(%

)

ig. 4. Angiotensin I-converting enzyme inhibitory effect of A. pseudoiva essentialil. Values presented are the means of triplicate analysis.

inhibitor required to inhibit 50% of the ACE inhibitory activity, wascalculated to be 65.5 �g/mL.

To the best of our knowledge, there are few reports on the ACEinhibitory activity of plant essential oils. Recently, Sami et al. [32]reported that 100 �g/mL of A. herba-alba essential oil exhibitedin vitro ACE inhibitory activity of 25.4%. On the other hand, sev-eral chemical classes of ACE inhibitors compounds derived fromplant extracts have been described such as tannins [33], flavonoids[34] and peptides [35]. Furthermore, Tundis et al. [36] reported a72.56% and 52.56% ACE inhibition by the methanol and ethyl acetateextracts of Senecio samnitum at 330 �g/mL.

3.4. Antibacterial activity

The in vitro antimicrobial activity of A. pseudoiva essential oilagainst the microorganisms employed and its activity potentialswere qualitatively and quantitatively assessed by the presence orabsence of inhibition zones, zone diameters and MIC values.

According to the results given in Table 2, the essential oil ofthe investigated species showed an important in vitro potential ofantimicrobial activities against the ten bacteria tested. The dataobtained from the disc diffusion method indicated that the essen-tial oil displayed a variable degree of antimicrobial activity on thedifferent tested strains. Our data indicated that Gram-positive S.aureus was the most sensitive tested strain to the oil of A. pseudoivawith a strongest inhibition zone of 32 mm. The Enterococcus groupwas found to be more sensitive among Gram-positive bacteria, withE. faecalis and E. faecium as the most sensitive showing inhibitionzones of 26 mm and 22 mm respectively. The oil also exhibited highantimicrobial activity against B. subtilis and B. cereus.

Modest activities were observed against important foodpathogens such as L. monocytogenes, with inhibition zones of14 mm. Among these, Gram-negative strains also displayed vari-able degrees of susceptibility against the investigated oil. Maximumactivity was observed against E. coli (21 mm), followed by K.pneumoniae (19 mm) and S. typhimurium (15 mm). Gram-negativebacteria, P. aeruginosa exhibited weak inhibition zones (6 mm),since it is known to have high level of intrinsic resistance to virtu-ally all known antimicrobials and antibiotics due to a combinationof a very restrictive outer membrane barrier, highly resistant evento synthetic drugs [37]. Moreover, the results obtained are of greatimportance, particularly in the case of B. cereus and S. aureus, whichare well-known for being resistant to a number of phytochemical

composition, angiotensin I-converting enzyme (ACE) inhibitory,Tunisian Ajuga pseudoiva Rob. Lamiaceae. Process Biochem (2013),

compounds and for the production of several types of enterotoxinsthat cause gastroenteritis [38].

In vitro activity of A. pseudoiva essential oil was also evalu-ated by a broth microdilution method. Antimicrobial activity was

ARTICLE ING Model

PRBI-9776; No. of Pages 7

6 M. Ben Mansour et al. / Process Bioc

Table 3Antifungal activity of A. pseudoiva essential oil.

Fungal strains Zone inhibition (mm)a MIC (�g/mL)a

Essential oil Nystatinb Essential oil

Aspergillus niger (ATCC 6275) 27 ± 1.5 14 ± 0.5 3.62 ± 0.4Aspergillus fumigatus (ATCC 9142) 30 ± 2.5 9 ± 0.5 3.74 ± 0.2Saccharomyces cerevisiae (ATCC 561) 20 ± 1.5 20 ± 0.5 5.79 ± 0.1Candida albicans (ATCC 10231) 38 ± 2.0 27 ± 1.0 2.14 ± 0.5Candida rugosa (ATCC 10571) 36 ± 1.0 17 ± 1.0 2.28 ± 0.0Fusarium solani 19 ± 0.5 14 ± 0.5 4.57 ± 0.7Fusarium oxysporium 21 ± 0.5 10 ± 0.5 5.22 ± 0.1

(

ertiwtbvrab

3

nTiTC(emdn

psaooattbrtERaf(cersbga

r

[

a Values represent means ± standard deviations for triplicate experimentsp < 0.05).

b The concentration of Nystatin used was 30 �g/well.

xpressed as minimum inhibitory concentration (MIC) and theesults of the MIC are shown in Table 2. The data showed thathe oil exhibited various levels of antimicrobial activity against thenvestigated food pathogens. In liquid medium, the essential oil

as active against all the tested strains. The essential oil was foundo have a significant antibacterial activity against Gram-positiveacteria tested compared to Gram-negative bacteria, with MICalues of 84–137 �g/mL and 105–336 �g/mL, respectively. Theseesults are consistent to several works reporting that essential oilsre slightly more active against Gram-positive than Gram-negativeacteria [7,39].

.5. Antifungal activity

The antifungal activity was evaluated against A. fumigatus, A.iger, F. solani, F. oxysporium, S. cerevisiae, C. albicans and C. rugosa.he results showed that A. pseudoiva essential oil had a strongnhibitory effect on the growth of all studied fungi (Table 3).he highest antifungal activity of this oil was observed against. albicans (38 mm), C. rugosa (36 mm), followed by A. fumigatus30 mm) and A. niger (27 mm). Besides, A. pseudoiva essential oilxhibited a very strong activity against the fungus C. albicans, whichay be significant since C. albicans was reported to be able to invade

ifferent parts of the human body causing cutaneous, mucocuta-eous, and opportunistic infections.

The remarkable antimicrobial and antifungal activities of A.seudoiva essential oil might be related to its predominantlyesquiterpenes components which constituted 84.18% of the totalnalyzed oil (Table 1). Particularly, the relatively high proportionf sesquiterpenoids (55.32%) detected in A. pseudoiva essentialil, may play an important role in both activities. This was ingreement with the study of Costa et al. [40] who reported thathe sesquiterpenes compounds in essential oils are responsible ofhe antibacterial activity. However, the evaluation of antimicro-ial activity of the essential oil of Amazon Guatteriopsis friesianaevealed significant antibacterial activity for all microorganismsested, displaying strong activity for B. subtilis, S. epidermides,nterococcus hirae, C. albicans, Micrococcus luteus, S. aureus andhodococcus equi with MIC values of 60, 100, 100, 125, 125, 125nd 50 �g/mL, respectively. The potent activity of the oil of G.riesiana might be attributed to its high sesquiterpene content97.13%). In addition, the antimicrobial activity of G. friesianaould result from the sesquiterpenoids �-eudesmol (51.60%), �-udesmol (23.70%), and �-eudesmol (14.56%), which was found toepresent 89.86% of the total of the oil analyzed [39]. Furthermore,esquiterpenoids and their derivatives are credited with variousiological actions, including antiasthmatic, antibacterial, antifun-

Please cite this article in press as: Ben Mansour M, et al. Chemicalantioxidant and antimicrobial activities of the essential oil from south

http://dx.doi.org/10.1016/j.procbio.2013.02.022

al, hypotensive activity, anti-inflammatory, and antineoplasticctivities [41,42].

On the other hand, several studies focusing on the antibacte-ial activities of different plants essential oils tried to attribute

[

PRESShemistry xxx (2013) xxx– xxx

these activities to the abundance of oxygenated monoterpenes andmonoterpenes hydrocarbons compounds which are able to affectcellular integrity resulting in inhibition of respiration and alterationin permeability [7,10]. In addition, Kordali et al. [43] proved that theessential oils containing high proportions of oxygenated monoter-penes have stronger antifungal activities than the essential oilsrelatively rich in monoterpene hydrocarbons or sesquiterpenes.

Thus, it is difficult to attribute the antifungal and antibacte-rial activities to a single or particular component of essential oilstaking into consideration the possible synergistic and antagonisticeffects of compounds which also play an important role in fungiand bacteria inhibition.

4. Conclusion

To the best of our knowledge, the essential oil composition,antioxidant, antimicrobial and ACE inhibitory activities of A. pseu-doiva has not been reported before and therefore our resultscan be considered as the first report about its biological proper-ties correlated to the chemical composition. The A. pseudoiva oilexerted strong antimicrobial and moderate antioxidant activities.The results of this study suggest the potential of A. pseudoiva essen-tial oil as a new antihypertensive agent. Moreover, the results of thisstudy suggested the possibility of using the essential oil or someof their components as natural food preservatives, because the oilpossesses strong antibacterial activity. Further research is neededin order to obtain information regarding the practical effectivenessof essential oil to prevent the growth of food borne and spoilingmicrobes under specific application conditions.

Acknowledgements

This work was funded by the Ministry of Higher Education andScientific Research, Tunisia. The authors are grateful to two anony-mous reviewers who helped, through their constructive commentsand suggestions, to improve the quality of the manuscript.

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