Food Chemistry 121 (2010) 724–731

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Food Chemistry

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Chemical composition, angiotensin I-converting enzyme (ACE) inhibitory,antioxidant and antimicrobial activities of the essential oil from Periplocalaevigata root barks

Mohamed Hajji, Ons Masmoudi, Nabil Souissi, Yosra Triki, Sadok Kammoun, Moncef Nasri *

Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, B.P. ‘‘W” 3038 Sfax, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 April 2009Received in revised form 18 November 2009Accepted 19 January 2010

Keywords:Periploca laevigataRoot barksEssential oilChemical compositionACE inhibitoryAntioxidantAntimicrobial

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.01.021

* Corresponding author. Tel.: +216 96 501 698; faxE-mail addresses: mon_nasri@yahoo.fr, moncef.na

The present study describes the chemical composition, and antimicrobial, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of essential oil from Periploca laevigata root barks (PLRB), anaromatic plant widely distributed in Tunisia and used as a traditional medicinal plant. Gas chromatogra-phy/mass spectrometry was used to determine the composition of the PLRB oil. Forty-three componentswere identified in the essential oil and the main compounds were benzaldehyde (56%), methyl 4-meth-oxysalicylate (6.55%) and carvacrol (4.75%). The PLRB essential oil exhibited a dose-dependent manner ofinhibitory activity toward ACE. The highest ACE inhibitory activity (54%) was observed at a concentrationof 30 lg/ml. The PLRB oil was also found to possess antioxidant activities, as evaluated by the 1,1-diphe-nyl-2-picrylhydrazyl (DPPH) radical method, b-carotene bleaching and reducing power assays. The anti-microbial activity of the essential oil was also investigated on several microorganisms. The inhibitionzones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 12–46 mm and 50–300 lg/ml, respectively. The inhibitory activity of the PLRB essential oil against Gram-positive bacteria was significantly higher than against Gram-negative. It also exhibited remarkable activ-ity against several fungal strains.

Our findings demonstrate that the essential oil from P. laevigata might be a good candidate for furtherinvestigations of new bioactive substances.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years there has been an increasing interest in applica-tions of natural substances, and some questions concerning thesafety of synthetic compounds have encouraged more detailedstudies of plant resources. Essential oils, and odorous and volatileproducts of plant secondary metabolism, have a wide applicationin folk medicine, food flavouring and preservation, as well as inthe fragrance industries. Recently, many essential oils and theirconstituents have been investigated for their multifunctionalproperties.

Angiotensin I-converting enzyme plays an important physiolog-ical role in the regulation of blood pressure (Skeggs, Kahn, & Shum-way, 1956). ACE can increase blood pressure by converting theinactive decapeptide angiotensin-I to the potent vasoconstrictorangiotensin-II (an octapeptide). ACE is a multifunctional enzymewhich also catalyses the degradation of bradykinin (a vasodilatingnonapeptide) (Erdös, 1975). Hypertension is related to the inci-dence of coronary heart disease and its treatment is effective in

ll rights reserved.

: +216 74 275 595.sri@enis.rnu.tn (M. Nasri).

reducing the risk of the disease (Collins et al., 1990). Therefore,inhibition of ACE activity is considered to be a useful therapeuticapproach in the treatment of high blood pressure, since it reducesthe activity of angiotensin-II and increases the level of bradykinin.Although synthetic ACE inhibitors are effective as antihypertensivedrugs, they cause adverse side effects, such as coughing, allergicreactions, taste disturbances and skin rashes. Therefore, researchand development, to find safer, innovative and economical ACEinhibitors, is necessary for the prevention and remedy ofhypertension.

Oxidation of polyunsaturated fatty acids, which occurs duringstorage, processing, and heat treatment of raw materials, and fur-ther storage of final products, is one of the major factors resultingin decrease of fatty food quality by formation of compounds withnegative effects on the aroma and nutritional value of foods (Shah-idi & Wanasundara, 1992). Lipid oxidation can be effectively pre-vented by using antioxidants. Moreover, it has been shown thatantioxidants and free radical-scavengers are crucial in the preven-tion of pathologies such as cancer, heart diseases, biological dam-age in living tissues, and neurodegenerative diseases, in whichreactive oxygen species (ROS) or free radicals are implicated (Mid-dleton, Kandaswamy, & Theoharides, 2000). Synthetic antioxidants

M. Hajji et al. / Food Chemistry 121 (2010) 724–731 725

have been used for stabilization of foods. The two most commonlyused are butylated hydroxyanisole (BHA) and butylated hydroxy-toluene (BHT) which are added to fatty and oily foods to preventoxidative deterioration (Löliger, 1991). However, use of thesechemical compounds has begun to be restricted because of theirinduction of DNA damage and their toxicity (Ito et al., 1986). Fromthis point of view, governmental authorities and consumers areconcerned about the safety of their food and about the potential ef-fect of synthetic additives on their health (Reische, Lillard, & Ein-tenmiller, 1998).

On the other hand, food-borne diseases caused by microorgan-isms are a major dilemma in the third world and developing coun-tries, and even in developed nations (Sokmen et al., 2004). Theconsumption of foods contaminated with some microorganismsrepresents a serious health risk to humans. The subsistence andgrowth of microorganisms in foods may lead to spoilage, formationof toxins and quality deterioration of food products (Celiktas et al.,2007).

In recent years, essential oils and plant extracts have attracted agreat deal of scientific interest due to their potential as a source ofnatural antioxidants and biologically active compounds, such asantibacterial, antifungal and insecticidal substances (Celiktaset al., 2007).

Periploca laevigata (Asclepiadaceae family) is native to the Med-iterranean region and widely distributed in the Sahara area. InTunisia, it is predominantly found in the south of the country,especially in the mountains. It is used as a food ingredient (tea)and as a herbal preparation because of its reputed medicinal prop-erties, e.g. for the treatment of headaches and diabetes (Askri,Mighri, Bui, Das, & Hylands, 1989). The most studied Periploca spe-cies, Periploca sepium, Periploca graeca and Periploca nigrescens,were reported to have various biological activities, such as antipro-liferative (Spera, Siciliano, De Tommasi, Braca, & Vessières, 2007),antitumor (Itokawa, Xu, & Takeya, 1988) and hypotensive effects(Askri et al., 1989).

Previous studies, dealing with chemical composition, and anti-oxidant and radical-scavenging activities of different solvent ex-tracts from P. laevigata, have been reported (Hajji et al., 2009). Inaddition, Hichri et al. (2003) reported the antibacterial activity ofoleanolic acid derivatives isolated from P. laevigata fruits. To thebest of our knowledge, there are no available reports on the chem-ical composition and biological activities of the essential oil from P.laevigata root barks. Therefore, the aim of the present work was tostudy in vitro ACE inhibitory, antioxidant and antimicrobial activi-ties of the essential oil of P. laevigata. The chemical composition ofthe essential oil was also determined by GC–MS.

2. Materials and methods

2.1. Chemicals

Angiotensin I-converting enzyme from rabbit lung, the ACE syn-thetic substrate hippuryl-L-histidyl-L-leucine (HHL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), BHA, b-carotene, linoleic acid, Tween 40,potassium ferricyanide, trichloroacetic acid (TCA), ferric chlorideand p-iodonitrotetrazolium violet (INT) were purchased from Sig-ma Chemical Co. (St. Louis, MO, USA). All culture media and stan-dard antibiotics were purchased from Bio-Rad (Bio-Radlaboratories, France). All other chemicals and solvents were of ana-lytical grade. All solutions were freshly prepared in distilled water.

2.2. Plant material

Fresh roots of P. laevigata were collected from Matlegg Moun-tain (Regueb, Tunisia, on December 20, 2007). The raw material

was washed with distilled water and barks were separated,ground, and then dried at 80 �C for at least 5 h to obtain P. laevigataroot bark (PLRB) powder. The dried preparation was ground furtherto obtain a fine powder, and then stored in glass bottles at roomtemperature (Hajji et al., 2009).

2.3. Extraction of the essential oil

The dried powder from PLRB (100 g) was subjected to hydrodi-stillation for 4 h, using a Clevenger-type apparatus (ST15 OSA, Staf-fordshire, UK). The obtained distillate (100 ml) was extracted twicewith 100 ml of n-hexane and dried with anhydrous sodium sulfate.For the determination of the procedure yield, the solvent was evap-orated using a rotatory vacuum evaporator (EYELA N1000). Theyield was determined by weighing the remaining oil on an analyt-ical balance. The resulting essential oil was stored at �20 �C priorto further analyses.

2.4. GC–MS analysis

The essential oil was analysed using an Agilent-Technologies6890 N Network GC system equipped with a flame ionizationdetector and HP-5MS capillary column (30 m � 0.25 mm, filmthickness 0.25 lm; Agilent-Technologies, Little Falls, CA, USA).The injector and detector temperatures were set at 220 �C and290 �C, respectively. The column temperature was programmedfrom 80 �C to 220 �C at a rate of 4 �C/min, with the lower and uppertemperatures being held for 3 and 10 min, respectively. The flowrate of the carrier gaz (helium) was 1.0 ml/min. A sample of1.0 ll was injected, using split mode (split ratio, 1:100). All quan-tifications were carried out using a built-in data-handling pro-gramme provided by the manufacturer of the gaschromatograph. The composition was reported as a relative per-centage of the total peak area. The identification of the essentialoil constituents was based on a comparison of their retention timesto n-alkanes, compared to published data and spectra of authenticcompounds. Compounds were further identified and authenticatedusing their mass spectra compared to the Wiley version 7.0 library.

2.5. Determination of the angiotensin I-converting enzyme (ACE)inhibition activity

The ACE inhibition activity was assayed as reported by Nakam-ura et al. (1995). A volume of 80 ll, with different concentrations(10, 20 and 30 lg/ml) of PLRB essential oil, was added to 200 llof 5 mM hippuryl-L-histidyl-L-leucine (HHL), and then preincu-bated for 3 min at 37 �C. PLRB essential oil and HHL were preparedin 100 mM borate buffer, pH 8.3, containing 300 mM NaCl. Thereaction was then initiated by adding 20 ll of 0.1 U/ml ACE fromrabbit lung, prepared in the same buffer and incubated for30 min at 37 �C. The enzyme reaction was stopped by the additionof 250 ll of 0.1 M HCl. The released hippuric acid (HA) was ex-tracted by the addition of 1.7 ml of ethyl acetate. After mixing byvortex for 15 s, 1 ml of the upper layer was transferred into a glasstube and evaporated at 90 �C for 15 min. The released hippuric acidwas redissolved in 1 ml of distilled water, and the absorbance wasmeasured at 228 nm, using a spectrophotometer (T70, UV/VISspectrometer, PG Instruments Ltd, China).

The average value from three determinations at each concentra-tion was used to calculate the ACE inhibition rate as follows:

ACE inhibition ð%Þ ¼ B� AB� C

� �� 100

where A is the absorbance of HA generated in the presence of ACEinhibitor component, B is the absorbance of HA generated without

726 M. Hajji et al. / Food Chemistry 121 (2010) 724–731

ACE inhibitors and C is the absorbance of HA generated without ACE(corresponding to HHL autolysis in the course of enzymatic assay).

The IC50 value was defined as the concentration of inhibitor (lg/ml) required to inhibit 50% of the ACE inhibitory activity.

2.6. Antioxidant activity

2.6.1. DPPH� assayThe DPPH radical-scavenging activity of PLRB essential oil was

determined by the method of Kirby and Schmidt (1997) with somemodifications. A volume of 500 ll of essential oil at different con-centrations (0.2–1.0 mg/ml) was added to 375 ll of 99% methanoland 125 ll of DPPH� solution (0.2 mM in methanol) as free radicalsource. The mixtures were incubated for 60 min in the dark atroom temperature. Scavenging capacity was measured spectro-photometrically (T70 UV–visible spectrometer, PG instrumentsLtd, Wibtoft, China) by monitoring the decrease in absorbance at517 nm. In its radical form, DPPH� has an absorption band at517 nm which disappears upon reduction by an antiradical com-pound. Lower absorbance of the reaction mixture indicated higherfree radical-scavenging activity. BHA was used as positive control.DPPH radical-scavenging activity was calculated as:

DPPH radical� scavenging activity ð%Þ

¼ A control� A sampleA control

� 100

where A control is the absorbance of the control reaction (contain-ing all reagents except the sample), and A sample is the absorbanceof PLRB essential oil. Tests were carried out in triplicate.

2.6.2. ß-Carotene bleaching by linoleic acid assayThe ability of PLRB essential oil to prevent bleaching of b-caro-

tene was assessed as described by Koleva, van Beek, Linssen, deGroot, and Evstatieva (2002). A stock solution of b-carotene/lino-leic acid was prepared by dissolving 0.5 mg of b-carotene, 25 llof linoleic acid and 200 ll of Tween 40 in 1 ml of chloroform.The chloroform was completely evaporated under vacuum in arotatory evaporator at 40 �C; then 100 ml of distilled water wereadded and the resulting mixture was vigorously stirred. The emul-sion obtained was freshly prepared before each experiment. Ali-quots (2.5 ml) of the b-carotene/linoleic acid emulsion weretransferred to test tubes containing different essential oil concen-trations. Following incubation for 2 h at 50 �C, the absorbance ofeach sample was measured at 470 nm. BHA was used as a positivestandard. The control tube contained no sample. Tests were carriedout in duplicate.

2.6.3. Ferric-reducing activityThe reducing power of the essential oils was determined by the

method of Yildirim, Mavi, and Kara (2001). Sample solutions(0.5 ml) with different concentrations of the essential oil weremixed with 1.25 ml of 0.2 M phosphate buffer (pH 6.6) and1.25 ml of (10 g/l) potassium ferricyanide solution. The mixtureswere incubated for 30 min at 50 �C. After incubation, 1.25 ml of(100 g/l) trichloroacetic acid were added and the reaction mixtureswere centrifuged for 10 min at 3000g. A 1.25 ml aliquot of thesupernatant from each sample mixture was mixed with 1.25 mlof distilled water and 0.25 ml of (1.0 g/l) ferric chloride solutionin a test tube. After a 10 min reaction time, the absorbance wasmeasured at 700 nm. Higher absorbance of the reaction mixtureindicated higher reducing power. Values presented are the meansof triplicate analyses.

2.7. Antimicrobial activity

2.7.1. Microbial strainsAntibacterial activities of PLRB oil were tested against seven

strains of bacteria: Staphylococcus aureus (ATCC 25923), Micrococ-cus luteus (ATCC 4698), Escherichia coli (ATCC 25922), Pseudomonasaeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 13883),Bacillus cereus (ATCC 11778) and Enterococcus faecalis (ATCC29212). Antifungal activities were tested using Aspergillus niger,Fusarium solani, Fusarium oxysporum and Aspergillus clavatus ES1.The first three strains were kindly provided from the microbial col-lection of Center of Biotechnology, Sfax-Tunisia. A. clavatus ES1 wasisolated in our laboratory from wastewater (Hajji, Kanoun, Nasri, &Gharsallah, 2007).

2.7.2. Agar diffusion methodAntimicrobial activity assays were performed according to the

method described by Berghe and Vlietinck (1991). The essentialoil (50 ll) was dissolved in 100% dimethylsulfoxide (DMSO)(950 ll) and sterilized by filtration through a 0.22 lm Nylon mem-brane filter. Culture suspension (200 ll) of the tested microorgan-isms (106 colony-forming units (cfu)/ml of bacteria cells (estimatedby absorbance at 600 nm) and 108 spores/ml of fungal strains(measured by Malassez blade) were spread on Muller–Hinton agarand PDA medium, respectively. Then, bores (3 mm depth, 4 mmdiameter) were made using a sterile borer and were loaded with50 ll of PRLB oil at 50 mg/ml. A well with only 50 ll of DMSO(without essential oil) was used as a negative control. Gentamycinand cycloheximide were used as positive references for bacteriaand fungi, respectively. The Petri dishes were kept, first for 1 h at4 �C, and then incubated for 24 h at 37 �C for bacteria and 72 h at30 �C for fungal strains. Antimicrobial activity was evaluated bymeasuring the diameter of the growth inhibition zones in millime-tres (including well diameter of 4 mm). The measurements of inhi-bition zones were carried out for three sample replications, andvalues are the averages of three replicates.

2.7.3. Determination of the minimum inhibitory concentration (MIC)MIC values, which represent the lowest essential oil concentra-

tion that completely inhibits the growth of microorganisms, weredetermined, based on a micro-well dilution method (Eloff, 1998).The essential oil was dissolved (at 50 mg/ml) in 100% DMSO andthen dilution series were prepared in a 96-well microtitre plate.The antibiotic gentamycin was used as reference in each assay.Oil-free solution, containing 50 ll distilled water and 950 llDMSO, was used as a negative control. Each well of the microplatesincluded 40 ll of the growth medium, 10 ll of the inoculum(106 cfu/ml) and 50 ll of the diluted essential oil. Then the micro-plates were incubated overnight at 37 �C. As an indicator of micro-organism growth, 40 ll of p-iodonitrotetrazolium violet (INT)dissolved in water were added to the wells and incubated at37 �C for 30 min. The colourless tetrazolium salt acts as an electronacceptor and is reduced to a red-coloured formazan product bybiologically active organisms (Eloff, 1998). Where microbialgrowth was inhibited, the solution in the well remained clear afterincubation with INT. The determinations of MIC values were donein triplicate.

2.8. Statistical analysis

Values were expressed as means ± standard deviation. Analysisof variance was conducted and differences between variables weretested for significance by one-way ANOVA with a SPSS 11 (Statis-tical Package for the Social Sciences) programme. Differences atP < 0.05 were considered statistically significant. Correlation andregression analysis were carried out using the EXCEL programme.

M. Hajji et al. / Food Chemistry 121 (2010) 724–731 727

3. Results and discussion

3.1. Chemical composition of the essential oil

The yield of PLRB essential oil obtained by hydrodistillation ofdry material was 0.51% (w/w). The oil was colourless, with a strongperfumery odour. Physical analysis of PLRB essential oil showed arefractive index of 1.531 and a density of 0.965 at 25 �C.

The essential oil of P. laevigata was analysed by GC–MS and theprofile obtained is depicted in Fig. 1. The individual identified com-ponents, with their relative percentages, are given in Table 1.Forty-three different components, representing about 97% of thetotal oil, were identified. From the data obtained, the essential oilcontained a complex mixture of several components, predominatlyaldehydes and oxygenated monoterpenes. Benzaldehyde (56.3%),methyl 4-methoxysalicylate (6.55%), carvacrol (4.75%), 2,4-decadi-enal (2.55%), salicylaldehyde (1.34%), and 1,8-cineole (1.15%) werefound to be the major components. Benzaldehyde, the major com-pound of PLRB oil, was not detected in the essential oil of Peripolocasepium roots (Miyazawa et al., 2004), which contained the 4-meth-oxy-salicyl aldehyde as the most abundant substance (87.8%). Asreported in the literature, many factors, such as the geographicalorigin, the genetic factors, the plant material and the season inwhich the plants were collected, may be responsible for the varia-tion of the chemical composition of the essential oils (Sivropoulouet al., 1997).

To the best of our knowledge, there are few reports on thechemical composition of the essential oil isolated from the plantbelonging to the genus Periploca (Miyazawa et al., 2004) and no re-ports describe the chemical composition of essential oil of P. laevig-ata species.

3.2. ACE inhibitory activity of PLRB essential oil

It has been well demonstrated that the inhibition of ACE mightdecrease blood pressure in animal models and in humans with var-ious types of hypertension (Skeggs et al., 1956). The essential oilwas then assayed for ACE inhibition activity. As shown in Fig. 2,PLRB essential oil exhibited dose-dependent ACE inhibitory activi-

Retention

Abu

ndan

ce

5.00 10.00 15.00 20.00

20004000600080001000012000140001600018000200002200024000260002800030000320003400036000380004000042000440004600048000

->

Fig. 1. GC–MS profile of

ties of 40.5%, 47.8% and 57.4%, respectively, with 10, 20 and 30 lg/ml of oil, respectively. The IC50 value, defined as the concentrationof inhibitor required to inhibit 50% of the ACE inhibitory activity,was calculated to be 22 lg/ml.

To the best of our knowledge, there are no reports on the ACEinhibitory activity of plant essential oils. However, several classesof ACE inhibitory compounds derived from plant solvent extractshave been identified, e.g. hydrolysable tannins (Oh, Kang, Lee, &Lee, 2002), flavonoids (Wagner, Elbl, Lotter, & Guinea, 1991), andterpenoids (Hansen et al., 1995). The flavonoids vitexin and isovit-exin, at 0.33 mg/ml, isolated from the plants, inhibited the ACEactivity by 20% and 45%, respectively (Lacaille-Dubois, Franck, &Wagner, 2001).

3.3. Antioxidant activity

3.3.1. GeneralIn this study, various antioxidant assays, including 1,1-diphe-

nyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducingpower and b-carotene assay were employed to evaluate the antiox-idant activity of the essential oil from PLRB.

3.3.2. 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 and theabsorbance is reduced (Shimada, Fujikawa, Yahara, & Nakamura,1992). The decrease in absorbance is taken as a measure of radi-cal-scavenging activity. This is a widely used method to investigatethe scavenging activity of some natural compounds.

As can be seen in Fig. 3, the scavenging activity of PLRB oil isconcentration-dependent. The IC50 value of the essential oil is0.76 mg/ml, whereas the IC50 of BHA is 11 lg/ml. The free radi-cal-scavenging activity of PLRB essential oil was higher than thatof Marrubium globosum essential oil which had an IC50 value of1.20 mg/ml (Sarikurkcu, Tepe, Daferera, Polissiou, & Harmandar,2008). Previous study on the antioxidant activity of the polarsub-fraction of PLRB methanol extract showed an IC50 of 4.0 lg/ml, more effective than the PLRB essential oil (Hajji et al., 2009).

time (min)0 25.00 30.00 35.00

PLRB essential oil.

Table 1Essential oil components of PLRB essential oila.

No. Rt (min)b Compoundc (%)d

1 4.74 Cyclohexane 0.102 5.03 a-Pinene 0.603 5.24 Cyclohexene 0.224 5.33 a-Terpinene 0.425 5.64 1,8-Cineole 1.156 6.16 n-Dodecanol 0.557 6.45 Loxanol 0.208 6.78 Myristic alcohol 0.209 6.96 n-Pyridinone 1.0510 7.29 1-Octyne 0.8511 7.68 1-Nonyne 0.5012 8.13 cis-p-Menth-8-ene 0.1813 8.51 Methyl-8-hexadecyn-1-ol 0.6214 9.62 Eugenol acetate 0.7015 10.32 Eugenol 0.5416 13.08 Carvacrol 4.7517 13.11 Camphor 0.9218 13.15 p-Cymen-2-ol 1.8419 13.23 Isothymol 1.5320 13.29 2-Hydroxy-p-cymene 1.1421 13.55 2,4-Decadienal 2.5522 14.05 Benzaldehyde 56.3423 14.34 2-Hydroxy-4-methoxybenzaldehyde 2.3524 14.57 p-Anisaldehyde 1.0525 14.66 Vanillic aldehyde 0.3526 14.74 Orcylaldehyde 0.6827 14.84 Salicylaldehyde 1.3428 15.34 Pinocarvone 0.4029 16.44 Cinnamyl acetate 0.230 18.13 Methyl 4-methoxysalicylate 6.5531 19.53 2,6-bis-(1,1-dimethylethyl) phenol 2.0132 21.36 Vianol 0.8133 22.05 Ionole 1.1534 22.79 Lioxin 0.3035 23.46 Resorcylaldehyde 0.1036 24.96 4-Dihydroxy-6-methylbenzene 0.2537 26.31 Methylorsellinate 0.1038 27.56 Stavox 0.1539 36.78 a-Caryophyllene 0.6040 37.66 a-Calacorene 0.4541 38.79 Caryophyllene oxide 0.8042 39.22 a-Cadinol 0.4043 39.72 Hexenyl benzoate 0.10

a Data are the means of two replicates.b Retention time.c Identification of components based on GC–MS Wiely 7.0 version library.d Percentages are the means of two runs and were obtained from electronic

integration measurements using a selective mass detector.

0

10

20

30

40

50

60

10 20 30Concentration (µg/ml)

AC

E in

hibi

tion

(%

)

Fig. 2. Angiotensin-converting enzyme inhibitory effect of PLRB essential oil.

0

20

40

60

80

100

120

0.2 0.4 0.6 0.8 1

Scav

engi

ng a

ctiv

ity

(%)

Concentration (mg/ml)

PLRB EO

BHA

Fig. 3. Free radical-scavenging capacities of PLRB essential oil and BHA as positivecontrol measured by DPPH� assay.

728 M. Hajji et al. / Food Chemistry 121 (2010) 724–731

Ruberto and Baratta (2000) studied the antioxidant activity of 98pure essential oils chemical components and showed that mono-terpene hydrocarbons had a significant protective effect, with sev-eral variants due to the different functional groups. Furthermore,some researchers have shown that some essential oils rich innon-phenolic compounds also have antioxidant potential (El-Massry, El-Ghorab, & Farouk, 2002). Table 1 shows that essentialoil of P. laevigata is notably rich in non-phenolic components.

3.3.3. Antioxidant activity measured by the b-carotene bleaching assayThe antioxidant assay using the discolouration of b-carotene is

widely used to measure the antioxidant activity of bioactive com-pounds. In this assay, oxidation of linoleic acid produces hydroper-oxyl radicals evolving toward lipid hydroperoxides, conjugateddienes, and volatile by-products, which simultaneously attack thechromophore of b-carotene, resulting in bleaching of the reactionemulsion (Frankel, 1998). In this test, b-carotene undergoes rapiddiscolouration in the absence of antioxidant, which results in areduction in absorbance of the test solution with increasing reac-tion time. The presence of antioxidant hinders the extent of bleach-ing by neutralizing the linoleic hydroperoxyl radicals formed(Kulisic, Radonic, Katalinic, & Milos, 2004).

As can be seen in Fig. 4, antioxidant activity increased withincreasing essential oil concentration. The IC50 was found to be0.5 mg/ml, similar to the IC50 exhibited by fresh leaves essentialoils of Cymbopogon schoenanthus L. Spreng (IC50 = 0.47 mg/ml)measured by the same test (Khadri et al., 2008), less effective than

0

20

40

60

80

100

120

0.2 0.4 0.6 0.8 1

An

tiox

idan

t ac

tivi

ty (

%)

Concentration (mg/ml)

PLRB EO

BHA

Fig. 4. Antioxidant activities of PLRB essential oil and BHA as positive controlmeasured by b-carotene bleaching method.

Table 2Antibacterial activity of the essential oil of P. laevigata root barksa.

Strains of bacteria IZDb (mm) MIC (lg/ml)

PLRB EOc Gentamycind PLRB EO

E. coli 30.0 ± 1.0 29.0 ± 1.0 125 ± 12.5P. aeruginosa 12.0 ± 1.0 35.0 ± 2.0 250 ± 12.5K. pneumonia 24.0 ± 2.0 31.0 ± 2.0 300 ± 25.0S. aureus 18.0 ± 1.0 25.0 ± 1.0 100 ± 12.5B. cereus 46.0 ± 2.0 30.0 ± 2.0 75.0 ± 6.25E. faecalis 20.0 ± 2.0 30.0 ± 2.0 50.0 ± 0.00M. luteus 46.0 ± 2.0 24.0 ± 1.0 60.0 ± 6.25

a Values represent averages ± standard deviations for triplicate experiments(p 6 0.05).

b Inhibition zone diameter.c P. laevigata root barks essential oil.d The concentration of gentamycin used was 10 lg/well.

M. Hajji et al. / Food Chemistry 121 (2010) 724–731 729

essential oils from Bidens pilosa (IC50 = 0.0497 mg/ml) (Deba, Xuan,Yasuda, & Tawata, 2008) and more effective than essential oilsfrom Mosla chinensis Maxim (IC50 = 0.588 mg/ml) (Cao et al., 2009).

3.3.4. Reducing powerThe reducing power assay is often used to evaluate the ability of

natural antioxidant to donate an electron or hydrogen (Shimadaet al., 1992). Different studies have reported that there is a directcorrelation between antioxidant activities and reducing power ofcertain bioactive compounds. It has been widely accepted thatthe higher the absorbance at 700 nm, the greater is the reducingpower.

In this study, the ability of the essential oil, and BHA as a posi-tive control, to reduce Fe3+ to Fe2+ was determined (Shimada et al.,1992). As can be seen in Fig. 5, the reducing capacity of the essen-tial oil and BHA increased with increasing concentration. Thereducing power activity of the essential oil from PLRB was similarto that of Thymus algeriensis essential oil (Hazzit, Baaliouamer,Veríssimo, Faleiro, & Miguel, 2009). However, the essential oil ofPLRB showed lower reducing power activity than did BHA and sol-vent extracts from the same material. The reducing powers ofmethanol, water, ethyl acetate and chloroform extracts, at a con-centration of 200 lg/ml, were 1.84, 1.32, 0.96 and 0.36, respec-tively (Hajji et al., 2009), whilst those of essential oil and BHAwere 0.15 and 2.02, respectively.

The reductive potential may be related to the presence of phe-nolic compounds, such as isothymol and carvacrol, due to hydroxylsubstitutions in the aromatic ring, which possess potent hydrogen-donating abilities, as described by Shimada et al. (1992). Antioxi-dant properties of carvacrol and terpinene were reported previ-ously (Ruberto & Baratta, 2000). However, other compounds mayalso play an important role in the reducing power of the PLRBessential oil, e.g. 2-hydroxy-4-methoxybenzaldehyde and 2-hydro-xy-p-cymene.

3.4. Antibacterial activity

The antibacterial activity of PLRB essential oil was evaluatedagainst Gram-positive (B. cereus, E. faecalis, S. aureus and M. luteus)and Gram-negative (P. aeruginosa, E. coli and K. pneumoniae) bacte-ria. The antibacterial activity was assessed by evaluating the inhi-bition zone (IZ), and the determination of MIC values.

As can be seen in Table 2, essential oil of PLRB showed varyingdegrees of antibacterial activity against all strains tested. The inhi-bition zones were in the range of 12–46 mm. The essential oil wasfound to have a significant antibacterial activity against the four

0

0.5

1

1.5

2

2.5

0.2 0.4 0.6 0.8 1

Ab

sorb

ance

at

700

nm

Concentration (mg/ml)

PLRB EO

BHA

Fig. 5. Antioxidant capacities of PLRB essential oil, using ferric reducing powermethod.

Gram-positive bacteria tested compared to Gram-negative bacte-ria, with MIC values of 50–100 lg/ml and 125–300 lg/ml, respec-tively. These results are in line with several works reporting thatessential oils are slightly more active against Gram-positive thanGram-negative bacteria (Deba et al., 2008; Lambert, Skandamis,Coote, & Nychas, 2001). The antimicrobial activity is more potentagainst E. faecalis ATCC 29212, strain with a MIC value of 50 lg/ml. However, the highest MIC value occurred with K. pneumoniaeATCC 13883 (300 lg/ml).

The Gram-negative P. aeruginosa is known to have a high levelof intrinsic resistance to virtually all known antimicrobials andantibiotics, due to a very restrictive outer membrane barrier,highly resistant, even to synthetic drugs (Mann, Cox, & Markham,2000). Moreover, the results obtained are of a great importance,particularly in the case of B. cereus and S. aureus, which are well-known for being resistant to a number of phytochemical com-pounds and for the production of several types of enterotoxins thatcause gastroenteritis (Halpin-Dohnalek & Marth, 1989). On theother hand, Hazzit et al. (2009) reported that S. aureus was resis-tant to the essential oils from Thymus species.

It has been demonstrated that aldheydes, monoterpene hydro-carbons and oxygenated monoterpenes in essential oils are ableto destroy cellular integrity, and thereby inhibit respiration andion transport processes. The antibacterial activity of the PLRBessential oil can be attributed to its aldheyde components, suchas benzaldehyde, salicylaldehyde and anisaldehyde, which consti-tute about 60% of PLRB essential oil. In this respect, Skaltsa, Demet-zos, Lazari, and Sokovic (2003) reported that aldehydes andalcohols from essential oils are known to be active, with differentspecificities and levels of activity, which is related not only to thefunctional groups present but also to hydrogen bonding parame-ters. In addition, the antimicrobial effects of carvacrol, 1,8-cineoleand caryophyllene oxide were also reported (Lambert et al., 2001).

From these results, PLRB essential oil may be considered to be anatural preservative against food-borne pathogens for the foodproduction industry.

3.5. Antifungal activity

The antifungal activity was evaluated against A. niger, F. solani,F. oxysporum and A. clavatus ES1. The results showed that theessential oil of PLRB had a strong inhibitory effect on the growthof all studied fungi (Table 3). Carvacrol, found in appreciableamounts in the oil of PLRB, has been reported to exhibit antifungalactivity (Lambert et al., 2001). In addition, antifungal activity ofcaryophyllene oxide, present in PLRB essential oils of Fusarium spe-cies, was previously reported (Cakir, Kordali, Zengin, Izumi, & Hira-ta, 2004). Benzaldeyde, detected in PLRB essential oil (56%), may

Table 3Antifungual activity of the essential oil of P. laevigata root barksa.

Fungal strains IZDb (mm)

PLRB EOc Cycloheximidec

A. niger 44.0 ± 1.0 40.0 ± 2.0A. clavatus ES1d 50.0 ± 3.0 43.0 ± 1.0F. oxysporum 48.0 ± 2.0 45.0 ± 1.0F. solani 55.0 ± 2.0 48.0 ± 2.0

aValues represent averages ± standard deviations for triplicate experiments

(p 6 0.05).b Inhibition zone diameter.c The concentration of cycloheximide used was 10 lg/well.d The isolate strain is from the ‘‘Laboratoire de Genie Enzymatique et de Micro-

biolgie, ENIS, Sfax”.

730 M. Hajji et al. / Food Chemistry 121 (2010) 724–731

play an important role in both antibacterial and antifungal activi-ties. These chemical components exert their toxic effects againststudied microorganisms through the disruption of bacteria or fun-gal membrane integrity. The permeabilization of the membranes isassociated with loss of ions and reduction of membrane potential,collapse of the proton pump and depletion of the ATP pool (Lam-bert et al., 2001).

The PLRB essential oil contains relatively high proportions ofaldehydes and oxygenated monoterpenes (Table 1), and in generaloils containing high proportions of oxygenated monoterpenes havestronger antifungal activities than do essential oils relatively richin monoterpene hydrocarbons or sesquiterpenes (Kordali, Kotan,& Cakir, 2007). Thus, the potent antifungal activity of PLRB essen-tial oil could be attributed, amongst other components, to its rela-tively high proportions of oxygenated monoterpenes. The volatileoils consist of complex mixtures of numerous components. Eithermajor or trace compound(s) might give rise to the antifungal activ-ity. Possible synergistic and antagonistic effects of compounds alsoplay an important role in fungi inhibition.

4. Conclusions

Many plant species are currently used as sources of naturaladditives because of their beneficial properties. The results pre-sented in this study are the first information on the antioxidantand biological activities of essential oil from of P. laevigata. ThePLRB oil exerted strong antimicrobial and moderate antioxidantactivities. The results of this study also demonstrate the potentialof PLRB essential oil as a new antihypertensive agent. In fact,according to literature data, this the first study of in vitro ACEinhibitory activity of an essential oil.

The essential oil of PLRB may be an alternative additive forfoods, pharmaceuticals and cosmetic preparations (instead ofmany toxic synthetic antioxidants).

Acknowledgements

This work was funded by the ‘‘Ministry of Higher Education, Sci-entific Research and Technology-Tunisia”. The authors wish tothank Professor Hafedh Belghith of the ‘‘Centre of Biotechnologyof Sfax – Tunisia” for the GC/MS analysis.

References

Askri, M., Mighri, Z., Bui, A. M., Das, B. C., & Hylands, P. J. (1989). Medicinal plants ofTunisia. The structure of Periplocadiol, a new elemane-type sesquiterpeneisolated from the roots of Periploca laevigata. Journal of Natural Products, 52,792–796.

Berghe, V. A., & Vlietinck, A. J. (1991). Screening methods for antibacterial andantiviral agents from higher plants. Method for Plant Biochemistry, 6, 47–68.

Cakir, A., Kordali, S., Zengin, H., Izumi, S., & Hirata, T. (2004). Composition andantifungal activity of essential oils isolated from Hypericum hyssopifolium andHypericum heterophyllum. Flavor and Fragrance Journal, 19, 62–68.

Cao, L., Si, J. Y., Liu, Y., Sun, H., Jin, W., Li, Z., et al. (2009). Essential oil composition,antimicrobial and antioxidant properties of Mosla chinensis Maxim. FoodChemistry, 115, 801–805.

Celiktas, O. Y., Kocabas, E. E. H., Bedir, E., Sukan, F. V., Ozek, T., & Baser, K. H. C.(2007). Antimicrobial activities of methanol extracts and essential oils ofRosmarinus oficinalis, depending on location and seasonal variations. FoodChemistry, 100, 553–559.

Collins, R., Peto, R., MacMahon, S., Hebert, P., Fiebach, N. H., Eberlein, K. A., et al.(1990). Blood pressure, stroke, and coronary heart disease. Part II. The Lancet,335, 827–838.

Deba, F., Xuan, T. D., Yasuda, M., & Tawata, S. (2008). Chemical composition andantioxidant, antibacterial and antifungal activities of the essential oils fromBidens pilosa Linn. Var. Radiata. Food Control, 19, 346–352.

El-Massry, K. F., El-Ghorab, A. H., & Farouk, A. (2002). Antioxidant activity andvolatile components of Egyptian Artemisia judaica L.. Food Chemistry, 79,331–336.

Eloff, J. N. (1998). A sensitive and quick microplate method to determine theminimal inhibitory concentration of plant extracts for bacteria. Planta Medica,64, 711–713.

Erdös, E. G. (1975). Angiotensin I-converting enzyme. Circulation Research, 36,247–255.

Frankel, E. N. (1998). Hydroperoxide formation. In lipid oxidation. Dundee: The OilyPress. pp. 23–41.

Hajji, M., Kanoun, S., Nasri, M., & Gharsallah, N. (2007). Purification andcharacterization of an alkaline serine protease produced by a newly isolatedAspergillus clavatus ES1. Process Biochemistry, 42, 791–797.

Hajji, M., Masmoudi, O., Ellouz-Triki, Y., Siala, R., Gharsallah, N., & Nasri, M. (2009).Chemical composition of volatiles and antioxidant and radical-scavengingactivities of Periploca laevigata root bark extracts. Journal of the Science of Foodand Agriculture, 89, 897–905.

Halpin-Dohnalek, M. I., & Marth, E. H. (1989). Growth and production of enterotoxinA by Staphylococcus aureus in cream. Journal of Dairy Science, 72, 2266–2275.

Hansen, K., Nymann, U., Smitt, U. W., Adsersen, A., Gudiksen, L., Rajasekharan, S.,et al. (1995). In vitro screening of traditional medicines for antihypertensiveeffect based on inhibition of the angiotensin converting enzyme. Journal ofEthnopharmacology, 48, 43–51.

Hazzit, M., Baaliouamer, A., Veríssimo, A. R., Faleiro, M. L., & Miguel, M. G. (2009).Chemical composition and biological activities of Algerian Thymus oils. FoodChemistry, 116, 714–721.

Hichri, F., Ben jannet, H., Cheriaa, J., Jegham, S., & Mighri, Z. (2003). Antibacterialactivities of a few prepared derivatives of oleanolic acid and other naturaltriterpenic compounds. Comptes rendus de chimie, 6, 473–483.

Ito, N., Hirose, M., Fukushima, G., Tauda, H., Shira, T., & Tatematsu, M. (1986).Studies on antioxidant; their carcinogenic and modifying effects on chemicalcarcinogenesis. Food Chemistry and Toxicology, 24, 1071–1081.

Itokawa, H., Xu, J. P., & Takeya, K. (1988). Studies on chemical constituents ofantitumor fraction from Periploca sepium. IV. Structures of new pregnaneglycosides, periplocosides D, E, L, and M. Chemical and Pharmacology Bulletin, 36,2084–2089.

Khadri, A., Serralheiro, M. L. M., Nogueira, J. M. F., Neffati, M., Smiti, S., & Araujo, M.E. M. (2008). Antioxidant and antiacetylcholinesterase activities of essential oilsfrom Cymbopogon schoenanthus L. Spreng. Determination of chemicalcomposition by GC–mass spectrometry and 13C NMR. Food Chemistry, 109,630–637.

Kirby, A. J., & Schmidt, R. J. (1997). The antioxidant activity of Chinese herbs foreczema and of placebo herbs. Journal of Ethnopharmacology, 56, 103–108.

Koleva, I. I., van Beek, T. A., Linssen, J. P. H., de Groot, A., & Evstatieva, L. N. (2002).Screening of plant extracts for antioxidant activity: A comparative study onthree testing methods. Phytochemical Analysis, 13, 8–17.

Kordali, S., Kotan, R., & Cakir, A. (2007). Screening of antifungal activities of 21oxygenated monoterpenes in vitro as plant disease control agents. AllelopathyJournal, 19, 373–391.

Kulisic, T., Radonic, A., Katalinic, V., & Milos, M. (2004). Use of different methods fortesting antioxidative activity of oregano essential oil. Food Chemistry, 85,633–640.

Lacaille-Dubois, M. A., Franck, U., & Wagner, H. (2001). Search for potencialangiotensin converting enzyme (ACE)-inhibitors from plants. Phytomedicine, 8,47–52.

Lambert, R. J. W., Skandamis, P. N., Coote, P. J., & Nychas, G. J. E. (2001). A study ofthe minimum inhibitory concentration and mode of action of oregano essentialoil, thymol and carvacrol. Journal of Applied Microbiology, 91, 453–462.

Löliger, J. (1991). The use of antioxidants in foods. In free radicals and food additives.London: Taylor and Francis. pp. 121–150.

Mann, C. M., Cox, S. D., & Markham, J. L. (2000). The outer membrane ofPseudomonas aeruginosa NCTC6749 contributes to its tolerance to theessential oil of Melaleuca alternifolia (tea tree oil). Letters in AppliedMicrobiology, 30, 294–297.

Middleton, E., Kandaswamy, C., & Theoharides, T. C. (2000). The effects of plantflavonoids on mammalian cells: Implications for inflammation, heart disease,and cancer. Pharmacological Reviews, 52, 673–751.

Miyazawa, M., Fujita, T., Yamafuji, C., Matsui, M., Kasahara, N., Takaji, Y., & Ishikawa,Y. (2004). Chemical composition of volatile oil from the roots of Periplocasepium. Journal of Oleo Science, 53, 511–513.

M. Hajji et al. / Food Chemistry 121 (2010) 724–731 731

Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995).Purification and characterization of angiotensin I-converting-enzyme inhibitorsfrom Sour milk. Journal of Dairy Science, 78, 777–783.

Oh, H., Kang, D. G., Lee, S., & Lee, H. S. (2002). Angiotensin converting enzymeinhibitors from Cuscuta japonica Choisy. Journal of Ethnopharmacology, 83,105–108.

Reische, D. W., Lillard, D. A., & Eintenmiller, R. R. (1998). Antioxidants in food lipids.In C. C. Ahoh & D. B. Min (Eds.), Chemistry, nutrition and biotechnology(pp. 423–448). New York: Marcel Dekker.

Ruberto, G., & Baratta, M. T. (2000). Antioxidant activity of selected essentialoils components in two lipid model systems. Food Chemistry, 69, 167–174.

Sarikurkcu, C., Tepe, B., Daferera, D., Polissiou, M., & Harmandar, M. (2008). Studieson the antioxidant activity of the essential oil and methanol extract ofMarrubium globosum subsp. Globosum (lamiaceae) by three different chemicalassays. Bioresource Technology, 99, 4239–4246.

Shahidi, F., & Wanasundara, P. K. J. P. D. (1992). Phenolic antioxidant. Critical Reviewin Food Science and Nutrition, 32, 67–103.

Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidativeproperties of xanthum on the autoxidation of soybean oil in cyclodextrinemulsion. Journal of Agricultural and Food Chemistry, 40, 945–948.

Sivropoulou, A., Nikobu, C., Papanikolaou, E., Kokkini, S., Lanaras, T., & Arsenakis, M.(1997). Antimicrobial, cytotoxic and antiviral activities of Salvia fruticosaessential oil. Journal of Agricultural and Food Chemistry, 45, 3197–3201.

Skaltsa, H. D., Demetzos, C., Lazari, D., & Sokovic, M. (2003). Essential oil analysisand antimicrobial activity of eight Stachys from Greece. Phytochemistry, 64,743–752.

Skeggs, L. T., Kahn, J. E., & Shumway, N. P. (1956). The preparation and function of theangiotensin I-converting enzyme. Journal of Experimental Medicine, 103, 295–299.

Sokmen, M., Serkedjieva, J., Daferera, D., Gulluce, M., Polissiou, M., Tepe, B., et al.(2004). In vitro antioxidant, antimicrobial, and antiviral activities of theessential oil and various extracts from herbalparts and callus cultures ofOriganum acutidens. Journal of Agriculture and Food Chemistry, 52, 3309–3312.

Spera, D., Siciliano, T., De Tommasi, N., Braca, A., & Vessières, A. (2007).Antiproliferative cardenolides from Periploca graeca. Planta Medicine, 73,384–387.

Wagner, H., Elbl, G., Lotter, H., & Guinea, M. (1991). Evaluation of natural productsas inhibitors of angiotensin I-converting enzyme (ACE). Pharmaceutical andPharmacological Letter, 1, 15–18.

Yildirim, A., Mavi, A., & Kara, A. A. (2001). Determination of antioxidant andantimicrobial activities of Rumex crispus L. extracts. Journal of Agricultural andFood Chemistry, 49, 4083–4089.