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Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical composition and antimicrobial activity against food spoilage pathogens
Accepted Manuscript Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical composition and antimicrobial activity against food spoilage pathogens M.N. Boukhatem, A. Kameli, F. Saidi PII:
S0956-7135(13)00167-9
DOI:
10.1016/j.foodcont.2013.03.045
Reference:
JFCO 3212
To appear in:
Food Control
Received Date: 16 December 2012 Revised Date:
24 March 2013
Accepted Date: 30 March 2013
Please cite this article as: BoukhatemM.N., KameliA. & SaidiF., Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical composition and antimicrobial activity against food spoilage pathogens, Food Control (2013), doi: 10.1016/j.foodcont.2013.03.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Highlights
► Analysis of rose geranium (Pelargonium graveolens) essential oil (EO) from Algeria.
► Higher antimicrobial activity was observed in the vapor phase.
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► Geranium EO may be used as natural preservative for food.
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► EO exhibited good antibacterial effect against Gram positive bacteria and yeast.
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Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical
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composition and antimicrobial activity against food spoilage pathogens
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M.N. Boukhatem ab,*, A. Kameli a, F. Saidi b
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a
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Kouba, Alger, Algérie. (Email : [email protected] ; Tél : +213557283091)
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b
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Blida, Algérie.
Laboratoire éco-physiologie végétale, Département des sciences naturelles, Ecole Normale Supérieure, BP 92, 16050
Département de Biologie, Faculté des sciences agro-vétérinaire, Université Saad Dahleb, BP 270, Route Soumaa,
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Corresponding author: Boukhatem Mohamed Nadjib. Email : [email protected] ; Tél : +213557283091
11 Abstract
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The aim of this study was to analyze the chemical composition of essential oil of rose-scented geranium (Pelargonium
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graveolens L.) growing in Algeria and to test the efficacy of the oil against food spoilage and food-borne pathogens.
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The chemical composition of the oil was analysed by Gas Chromatography-Mass Spectrometry (GC-MS). A total of
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45 compounds representing 94.2% of the essential oil was identified. The main constituents were citronellol (30.2%),
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citronellyl formate (9.3%) and geraniol (7.6%).
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The antimicrobial activity of essential oil was evaluated against 23 food spoilage microorganisms in liquid and vapour
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phase at three different concentrations (10, 20 and 30 µl/disc).
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The oil exhibited promising antibacterial effect against Gram positive more than negative bacteria and provides a
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good inhibitory effect against Candida strains. Furthermore, the zone of inhibition increased with increasing oil
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concentration. Significantly higher antimicrobial activity was observed in the vapour phase. This is the first report on
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the antimicrobial properties of the essential oil of Algerian rose geranium.
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Our results suggest that rose-scenred geranium oil could be used for the development of novel types of antibacterial
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agents to control food spoilage and food-borne pathogens.
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Keywords: rose geranium ; Pelargonium graveolens ; essential oil ; GC-MS ; antimicrobial activity ; vapor phase ;
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geraniol.
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1. Introduction
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In recent years there has been a considerable pressure from consumers to reduce or eliminate chemically synthesized
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additives in their foods. Many naturally occurring compounds present in plants have been shown to possess
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antimicrobial effect against food-borne pathogens. In particular, the antimicrobial properties of plant essential oils
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ACCEPTED MANUSCRIPT (EOs) and their constituents have been widely demonstrated (Burt, 2004; Dorman & Deans, 2000). Use of EOs as
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antimicrobial agents in food systems may be considered as additional intrinsic determinant to increase the safety and
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shelf life of food.
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Rose-scented geranium (Pelargonium species) is a multi-harvest high value, aromatic plant cultivated for its EO
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which is widely used in cosmetic industry and as flavouring for foods (Lis-Balchin, 2005). Pelargonium graveolens L.
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is native of South Africa and was introduced into North Africa as an ornamental plant. It has adapted well to the
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Mediterranean climate (Lis-Balchin, 2002; Boukhatem et al., 2011).
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Geranium essential oil is obtained from the scented leaves of a number of Pelargonium cultivars grown mainly in
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Reunion, China and Algeria. The volatile oil of rose geranium species has been attributed a number of biological
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properties including: antibacterial (Rosato et al., 2007; Lalli, Van Zyl, Van Vuren, & Viljoen, 2008), antifungal
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(Naeini, Nazeri, & Shokri, 2011; Hassan et al., 2011) and also some other pharmacological properties such as anti-
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inflammatory (Maruyama, Ishibash, & Hu, 2006), spasmolytic (Lis-Balchin, Hart, & Roth, 1997) and hypoglycaemic
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effects (Boukhris et al., 2012).
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Rose geranium oil and their major components have gained acceptance in the food industry since they have been
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generally recognized as safe (GRAS) by FEMA (1965) and approved by the American Food and Drug Administration
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(FDA) for food use.
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Although several studies have focused on the chemical composition of Pelargonium EOs (Araya, Soundy, & Steyn,
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2006; Rajeswara Rao, Kaul, Syamasundar, & Ramesh, 2002; Juliani et al., 2006; Gomes, Mata, & Rodrigues, 2007),
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only a few reports evaluated their activity against pathogenic and food spoilage bacteria and fungi (Si et al., 2006).
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As far as our knowledge of the literature is concerned, this study could be considered as the first report on the
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antimicrobial activities of rose geranium growing in Algeria. Since the volatile oils of rose geranium have been used
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widely as pharmaceuticals and flavouring agents, it is necessary to assess their antimicrobial activity for the
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improvement of food safety. Further, guided by ethnobotanical literature and availability from natural resources, our
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main objective is to validate the use of selected Algerian aromatic plants for their antimicrobial activities and to
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emphasise the need to promote their natural botanical resources in North Africa.
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Moreover, no systematic studies comparing antimicrobial activity (in liquid and vapour phase) of the geranium oil are
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available. Several approaches have been proposed to minimize EO concentrations. One of them is use of EOs in
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vapour phase to reduce the required concentration. EO in vapour phase could be highly effective against foodborne
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pathogens at relatively lower concentrations than the liquid phase, thereby causing minimum effect on organoleptic
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properties (Tyagi & Malik, 2011).
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The main objectives of this study were (1) to determine the chemical composition of rose geranium EO and (2) to
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investigate the antimicrobial activity of the oil by disc diffusion and vapour phase methods against 23 selected
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spoiling and pathogenic microorganisms, in an attempt to contribute to the use of these as alternative products for
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microbial control and food preservation.
67 2. Material and methods
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2.1. Plant material
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Rose-scented geranium (Pelargonium graveolens) was grown in the field station of aromatic and medicinal plants of
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“Extral Bio” Company, Chiffa, Blida city (Algeria). The aerial parts of rose geranium were collected in May 2011
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during the flowering period. Identification of the species was confirmed by the Botanical Laboratory in the National
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Institute of Agronomy (Algiers, Algeria).
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74 2.2. Extraction of the essential oil
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Samples of 100 g of the air-dried aerial parts of plants were subjected to hydrodistillation for 2 h using a
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Clevenger apparatus. The obtained EO was dried over anhydrous sodium sulphate and, after filtration,
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stored at + 4 °C until tested. All experiments were conducted in triplicates and results were expressed on
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the basis of dry matter weight.
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2.3. Gas chromatography/mass spectrometry (GC/MS) analysis conditions
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The analysis of the EO was performed using a Hewlett Packard 5890 II GC, equipped with a HP-5 MS (Crosslinked
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5% PH ME Siloxane) capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness) and a mass spectrometer 5972 of
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the same company as detector. For GC-MS detection an electron ionization system was used with ionization energy of
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70 eV. Helium was the carrier gas, at a flow rate of 1 ml min-1 min. Injector and detector (MS transfer line)
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temperatures were set at 220 and 290 °C, respectively. Column temperature was initially at 50 °C, then gradually
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increased to 150 °C at a 3 °C min-1 rate, hold for 10 min and finally increased to 250 °C at 10 °C min-1. Diluted
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samples (1/100 in acetone) of 1.0 µl were injected manually and splitless.
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Retention index (RI) of all the constituents were determined by the Kovats method by co-injection of the samples with
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a solution containing the homologous series of n-alkanes (C8-C24) (Fluka, Buchs/sg, Switzerland) on the HP-5 MS
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column. Identification of the components was made by visual interpretation, comparing their retention indices and
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mass spectra with data published in the literature (Adams, 2001) and by matching their recorded mass spectra with
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reference spectra in the computer library (NIST MS library, Version 2.0). Quantification was computed as the
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percentage contribution of each compound to the total amount present.
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2.4. Microbial strains and growth conditions
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The in vitro antimicrobial activity of the EO from rose geranium was tested against 23 food-related microorganisms
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including: (1) clinically isolated strains from the Food Microbiology Laboratory of Blida Health Institute and Pasteur institute of
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Algeria (IPA): Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Citrobacter
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freundii, Proteus vulgaris, Serratia marcescens, Candida albicans, Candida lipolytica, Candida tropicalis and
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Candida sake.
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(2) referenced strains: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6051, Enterococus feacalis ATCC
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2921, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 19430, Klebsiella pneumoniae ATCC 700603,
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Pseudomonas aeruginosa ATCC 2785, Enterobacter aerogenes ATCC 13043, Candida albicans ATCC 90028,
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Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 and Rhodotorula glutinis ATCC 16740.
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All the strains were grown on Mueller-Hinton agar (MHA) for the bacteria and Saboureaud Dextrose Agar (SDA)
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with Chloramphenicol for yeasts. The medium were obtained from IPA (Algiers, Algeria). Inocula were prepared in
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sterile saline from 24 h and 48 h cultures of bacteria and yeasts respectively, and standardized against 0.5 McFarland
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Standard to obtain suspensions containing approximately 108 cfu mL-1 of bacteria and 106 cfu mL-1 of yeasts.
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2.5. Antimicrobial activity assays
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Two techniques were used to test the antimicrobial activity of rose geranium EO: agar disc diffusion method
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(NCCLS, 1997) and the vapour diffusion method (Tyagi & Malik, 2011).
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2.5.1. Disk diffusion method
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Briefly, a suspension of the tested microorganism (0.1 ml of 108 cells ml-1) was spread on the solid media plates. Filter
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paper discs (6 mm in diameter) were impregnated with 3 different concentrations (10, 20 and 30 µl per disc) of the oil
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and placed on the inoculated plates (MHA/SDA). The plates were incubated at 37 °C for 24 h for bacteria and at 30
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°C for 48 h for yeasts. The zone of inhibition was measured with a calliper. Tests were performed in triplicate, and
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mean value was calculated. Antibiotic discs of Streptomycin (25 µg/disc) and Amphotericin B (20 µg/disc), purchased
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from Antibiotical company of Saidal (Medea, Algeria) were also used as positive controls for bacteria and yeasts
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respectively.
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2.5.2. Vapour diffusion method
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Antibacterial activity of EO in vapour phase was evaluated by disc volatilization method at three different volumes
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(10, 20 and 30 µl per disc). In brief, solidified medium was inoculated over the surface of MHA/SDA with 0.1 ml of
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microorganism suspension. A paper disc (diameter 9 mm, Sigma, Steinheim, Germany) was laid on the inside surface
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of the upper lid and 10 µl of EO was placed on each disc.
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The plate inoculated was immediately inverted on top of the lid and sealed with parafilm to prevent leakage of the EO
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vapour. After the incubation period, the effectiveness of the EO was calculated by measuring the diameter of the
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inhibition zone. All tests were carried out in triplicate.
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All data were reported as means ± standard deviation of three triplicates. One-way analysis of variance (ANOVA) was
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applied to the data to determine differences (P < 0.05). To ascertain significant differences between the levels of the
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main factor, Fisher LSD (least significant difference) test was applied between means. ANOVAs were made with the
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following factors: strains (23 levels) and volume of essential oil tested (10, 20 and 30 µl with disc diffusion and
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vapour diffusion methods). Statistical data analysis was undertaken using XLStat. Pros statistical software (XLStat,
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Paris, France). P values < 0.05 were considered as significant.
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141 3. Results and Discussion
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3.1. Yield and chemical composition of essential oil
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The EO was obtained by hydrodistillation from air-dried P. graveolens aerial parts with a yield of 0.15% (v/w).
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The rose geranium oil obtained is liquid and has yellowish-green colour. It has a strong rose scent with a less
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pronounced mint note. Therefore, this oil may be valuable for the flavouring of foods.
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The EO was analyzed by GC-MS. Qualitative and quantitative analyses of the oil volatile profiles are listed in Table 1
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according to their elution order. In total, 45 compounds constituting 94.21% of the essential oil have been identified.
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Results showed that citronellol (30.2%), citronellyl formate (9.3%), geraniol (7.6%) and guai-6,9-diene (5.4%) were
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found to be the major compounds in the oil with minor amounts of isomenthone (4.1%) and menthone (3.6%). Other
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components were found in less quantity (< 3%). Linalool which is present in 3.2% is an important constituent in
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fragrances because of its pleasing aroma.
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The rose geranium EO consisted mainly of monoterpenic alcohols (41.2%), monoterpenic esters (23%) and
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sesquiterpenic hydrocarbons (13.9%) (Table 2).
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The market value of the essential oil is determined by terpenoid composition: in particular, besides geraniol,
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citronellol, linalool, and their esters, isomenthone, the sesquiterpenoid hydrocarbon guaia-6,9-diene, and alcohol 10-
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epi-g-eudesmol play a key role, as they allow to distinguish between oils of different origin, and different varieties
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and quality (Lis-balchin, 2002).
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In our study, it appears that EO of rose geranium analyzed could be therefore classified as ‘‘citronellol chemotype”.
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composition of the oil was similar to Chinese oils, with the same content of guaia-6,9-diene and the absence of 10-epi-
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y-eudesmol. This oil is characterized by high levels of citronellyl formate (9.3%), 6,9 guaia-6,9-diene (5.4%) and
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trace amounts of 10-epi-y-eudesmol (0.88%) (Table 2) while the bourbon and Egyptian geranium oils contained high
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levels of 10-epi-y-eudesmol and traces of guaia-6,9- diene.
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In agreement with results obtained by other authors, the chromatographic analysis of rose geranium EO showed that
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citronellol was the component present in the greatest percentage (Jalali-Heravi, Zekavat, & Sereshti, 2006; Gomes,
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Mata, & Rodrigues, 2007; Araya, Soundy, & Steyn, 2006).
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Regarding other components, our results diverge from those published by other authors (Saxena, Banerjee, & Gupta,
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2004; Juliani et al., 2006).
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The studies of Lalli (2006) on the essential oils from South Africa showed a significant difference in their
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composition, consisting of isomenthone 65.8% and decanoid acid 12.9%. Very low levels of citronellol (0.4%),
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citronellyl formate (0.05%) were obtained. Furthermore, geraniol was not present.
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173 3.2. Antimicrobial activity
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3.2.1. Direct diffusion method
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The antimicrobial activity of rose geranium EO, both by direct contact or through vapour phase, was qualitatively and
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quantitatively assessed by the presence or absence of inhibition zone. The diameter of the inhibition zone (DIZ) is
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given in Table 3 and Table 4.
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To the best of our knowledge, the antimicrobial activity of rose geranium EO growing in Algeria has never been
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reported. This work is therefore the first report on the EOs from this aromatic plant.
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The results obtained from the disc diffusion method, indicated that the oil exhibited antimicrobial activity against all
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Gram-positive bacteria and 3 Gram-negative bacteria at the concentration of 10 µl per disc. As can be seen from the
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Table 2, the EO of rose geranium showed various degrees of antimicrobial activity depending on the tested bacterial
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strains.
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Among the Gram-negative bacteria, the EO was more effective against E. coli and Enterobacter aerogenes ATCC
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13043 with inhibition zones measured at 15 and 14 mm respectively. In contrast, P. aeruginosa and K. pneumoniae
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were the most resistant strains to the oils while Proteus vulgaris, an important food pathogen, shown a modest
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sensibility.
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Among the Gram-positive bacteria, S. aureus ATCC 6538 and E. faecalis ATCC 29212 (DIZ 21.17 mm) were the
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most sensitive strains to the EO, followed by B. subtilis ATCC 6051(20.5 mm) and S. epidermidis (16.17 mm).
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among all the tested microorganisms in comparison with the amphetorecin B (Table 4).
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The zone of inhibition increased with the increasing concentration of EO (Table 3). The zone of inhibition in the case
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of bacteria was less than that for the yeasts strains at 30 µl dose of EO. Hence, the zone of inhibition due to the same
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concentration of essential oil was bigger for yeasts than for bacteria.
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Overall, the EO showed very promising antibacterial activity. EO was more selective for the Gram-positive test
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pathogens than for the Gram-negative bacteria which is in accordance with studies reporting that Gram-positive
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bacteria are generally more sensitive than Gram-negative bacteria to EO (Gilles, Zhao, An, & Agboola, 2010;
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Oussalah, Caillet, & Lacroix, 2006).
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Our conclusions were in agreement with the results of an earlier study (Lis-Balchin, Deans & Eaglesham, 1998)
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wherein 24 cultivars were tested for antimicrobial activity of rose geranium EO against 25 test bacteria. Of these test
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bacteria, the most sensitive bacteria were Bacillus subtilis and Staphylococcus aureus, a group of predominantly
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Gram-positive organisms, whilst the least sensitive organisms were Escherichia coli, Klebsiella pneumonia and
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Pseudomonas aeruginosa, a group of predominantly Gram-negative organisms.
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Not all studies on essential oils have concluded that Gram-positives are more susceptible. (Zaika, 1988; Dorman &
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Deans, 2000)
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Pattnaik et al. (1996) tested geranium oil for antibacterial activity against 22 bacteria by disc diffusion. Only 12
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bacterial strains were inhibited by the Geranium oil, but all the fungi were inhibited.
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Lis-Balchin et al. (1998) found that Pelargonium EOs showed substantial in vitro activity against S. aureus, Proteus
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vulgaris, B. cereus and S. epidermidis.The latter two studies suggest that Pelargonium EO can potentially be used as
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novel antibacterial agents in the food and cosmetic industries.
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More recently, attempts have been made to identify the component(s) responsible for such bioactivities (Dorman &
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Deans, 2000). The antimicrobial properties of volatile oils and their constituents from a wide variety of plants have
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been assessed and reviewed.
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The antimicrobial activity of geranium EO could be due to citronellol and geraniol. Other geranium oils rich in
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citronellol and geraniol were previously demonstrated to have potent antimicrobial activities (Prashar, Hilli, Veness,
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& Evans, 2003; Si et al., 2006). Pelargonium also contains the oxygenated monoterpene linalool (3.2%) known to
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possess good antimicrobial activity (D’auria et al., 2005).
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More recently, a study carried out by Jirovetz et al. (2006) showed that essential oils with floral-rosy scent, such as
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geranium, possess high antimicrobial activities against various microorganisms and these effects are maintly the result
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of a combination of some biologically active principal aroma compounds (geraniol, nerol, citronellol and many of
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their derivatives).
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also contribute to the antimicrobial activity of the oils. It has been reported in the literature that the inhibitory activity
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of an essential oil results from a complex interaction between its different constituents (Xianfei, Xiaoqiang, Shunying,
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& Guolin, 2007).
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Few studied have examined the antifungal activity of geranium essential oils. In our study, EO exhibited the strongest
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inhibition effect against yeasts. These results are in agreement with those reported in the literature for other rose
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geranium oil rich in geraniol that showed a very strong action versus C. tropicalis and C. albicans (Hassan et al.,
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2011). Hammer et al. (1999) tested various essential oils and plant extracts for their antimicrobial effects. Rose
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geranium EO showed greater activity against Candida albicans than against S. aureus. These findings correlate with
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our results obtained in this study. A study carried out by Mahboobi et al. (2008) proved that rose geranium oil was the
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second most effective of the essential oils on Candida albicans and exhibited strong activity on isolates.
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Mechanism of anti-Candida activity of geraniol, geranyl acetate and citronellol appears to be associated with damage
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in the membrane integrity, inhibited germ tube induction at very low concentrations and inhibited Candida albicans
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cell cycle (Zore, Archana, Thakre, Rathod, & Karuppayil, 2010). These previously mentioned components are present
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in our essential oil sample.
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Dalleau et al. (2008) compared the anti-biofilm activity of molecules which are often prevalent in EOs. Thymol,
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carvacrol and geraniol exhibited the most significant antibiofilm activity against all three tested strains, including
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Candida albicans, C. parapsilosis and C. glabrata.
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3.2.2. Vapour diffusion method
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Comparative studies of antibacterial potential of the EO were also conducted in the vapour phase (Table 3 and Table
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4). As observed in the earlier assays using EO in solid agar, the zone of inhibition due to the oil vapours also
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increased with increasing concentration of the oil. Further, the zone of inhibition due to the EO vapours was bigger for
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Gram positive bacteria (S. aureus) and yeasts than for Gram negative bacteria (E. coli).
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The zone of inhibition resulting from the exposure to EO vapours was significantly larger than that from the same
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concentration of EO in the liquid phase. This effect was visible at both lower (10 µl) and more prominently at higher
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(30 µl) concentrations.
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EO showed higher activity in the vapour phase. P. aeruginosa, K. pneumoniae and S. marcescens were more resistant
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against the rose geranium oil vapours too.
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The yeasts (Candida albicans and C. parapsilosis) were inhibited completely by the oil vapours at 30 µl exposure.
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Our study clearly demonstrates the higher antimicrobial efficacy of rose gernium oil vapours. This could be attributed
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to the variation in the relative composition of the oil and vapours as the latter must be enriched in terms of its volatile
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the antimicrobial potential of rose geranium EO, very few studies exist on the activity of EO vapours.
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Hence, smaller doses of essential oil in the vapour phase can be inhibitory to spoilage bacteria. Our results are in
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agreement with previous reports (Tyagi & Malik, 2011). Pibiri (2006) also demonstrated that the EOs of Satureia
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Montana and Cinnamon in vapour phase have a lethal effect on S. aureus and P. aeruginosa, even in small doses.
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This demonstrates that the antimicrobial activity of different EO vapours can be achieved with a smaller amount than
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the EO in liquid phase. A study carried out by Goni et al. (2009) showed that antimicrobial activity of combination of
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cinnamon and clove EOs in vapour phase showed better antimicrobial with less active concentration in the vapour
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phase compared to liquid phase. Hence the evaluation of the antimicrobial properties of rose geranium EO in the
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vapour phase would open up a newer dimension with immense potential applications.
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265 4. Conclusions
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Our results demonstrated that citronellol, geraniol and their esters were the main constituents of EO samples of rose
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geranium growing in Algeria. EO exhibited significant antimicrobial activity against a range of food spoiling
269
microorganisms and the strongest inhibitory effect against yeasts. Our findings suggest that rose geranium EO is
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highly effective in vapour phase. Rose geranium EO could be considered suitable alternatives for use in the food
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industry as a natural antimicrobial agent. However, issues of safety and toxicity will always need to be addressed.
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272 References
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Adams, R.P. (2001). Identification of essential oil components by gas chromatography/quadrupole mass
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Pelargonium (Geraniaceae) species. South African Journal of Botany, 74, 153-157.
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ACCEPTED MANUSCRIPT Saxena, G., Banerjee, S., & Gupta, R. (2004). Composition of the essential oil of a new isomenthone-rich variant of
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357
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361
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362
inhibition of growth, dimorphism and sensitization. Mycoses, 54, 99-109.
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ACCEPTED MANUSCRIPT Table 1 Chemical composition of essential oil from rose-scented geranium growing in Algeria N°
Rt b
Compounds a
RI c
Content (%)
29.210
α-Pinene
934
0.1
2
30.018
p-Cymene
1020
0.2
3
30.113
Linalool
1089
4
32.105
trans-Rose oxide
1100
5
32.110
cis-Rose oxide
1115
6
43.240
Menthone
1136
7
46.819
Isomenthone
8
49.211
α-Terpineol
9
58.689
Citronellol
10
58.871
Neral
11
59.500
Piperitone
12
59.834
Geraniol
13
60.402
Geranial
14
60.726
15
61.740
16
63.159
17
63.514
18
63.745
RI PT
1
3.2 2.2 1.3
SC
3.6 4.1
1165
0.2
M AN U
1149
30.2
1233
0.3
1247
0.3
1250
7.6
1262
2.6
Citronellyl formate
1266
9.3
Geranyl formate
1294
1.5
α-Cubebene
1348
0.2
Citronellyl acetate
1358
0.4
Geranyl acetate
1375
0.2
63.960
α-Copaene
1378
0.5
64.224
β-Bourbonene
1383
1.3
64.518
α-Ylangene
1385
0.3
22
65.227
γ-Caryophyllene
1400
0.6
23
65.521
α-Guaiene
1440
0.1
24
65.825
β-Caryophyllene
1453
0.6
25
65.967
Guaia-6,9-diene
1465
5.4
26
66.819
Citronellyl propionate
1476
1.0
20 21
EP
AC C
19
TE D
1220
ACCEPTED MANUSCRIPT 66.920
α-Caryophyllene
1478
0.9
28
67.042
Alloaromadendrene
1481
0.5
29
67.285
Germacrene D
1483
1.5
30
67.417
α-Salinene
1487
0.3
31
67.974
Citronellyl butyrate
1517
1.5
32
68.076
δ-Cadinene
1522
33
68.370
Calamenene
1526
34
68.806
Geranyl butyrate
1559
35
69.221
α-muurolene
1565
36
69.424
β-Phenylethyl tiglate
37
69.718
Geranyl isovalerate
38
70.042
39
RI PT
27
c
1.1
SC
0.1
1593
0.5
Citronellyl valerate
1601
1.4
70.144
10-epi-γ-eudesmol
1612
0.8
40
70.316
Geranyl valerate
1630
0.2
41
70.610
γ-Eudesmol
1650
0.3
42
71.745
Caryophyllene oxide
1686
1.4
43
72.161
Citronellyl tiglate
1692
0.5
44
77.797
Geranyl tiglate
1723
2.0
45
81.649
Geranyl propionate
1792
0.7
TE D
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2.3
EP b
0.5
1580
AC C
a
0.5
Total identified components
94.3
No identified
5.7
Compounds listed in order of elution from an HP-5 MS column.
Rt, Retention time (in minutes). RI, retention index calculated on the HP-5 MS column relative to C8-C28 n-alkanes.
ACCEPTED MANUSCRIPT Table 2 Chemical classes of essential oil extracted from rose-scented geranium growing in Algeria
Monoterpenic esters Sesquiterpenic hydrocarbons Monoterpenic ketones Monoterpenic oxides Monoterpenic aldehydes Sesquiterpenic alcohol
23.0 13.9 8.0 4.9 1.8 1.3 0.2
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Monoterpenic hydrocarbons
41.2
RI PT
Monoterpenic alcohols
Content (%)
SC
Chemical classes
AC C
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Total identified components
94.3
ACCEPTED MANUSCRIPT
Table 3 Zones of growth inhibition (mm) showing antibacterial activity of rose geranium essential oil Di sc di f f usi on me t h o d Antibiotic c
20 µl
30 µl
Staphylococcus aureus ATCC 6538
21.17±2.47fA
25.17±2.75iAB
30.83±5.06gB
Staphylococcus aureus
12.83±0.76bcA
20.83±1.76ghB
25.17±0.29efB
Staphylococcus epidermidis
16.17±1.04deA
20.50±0.50ghB
25.83±1.44fC
Bacillus subtilis ATCC 6051
20.50±0.87fA
22.17±0.29hB
23.17±0.29eB
Bacillus cereus
14.17±0.29cdB
17.83±0.29efC
Enterococcus feacalis ATCC 29212
21.17±0.29fA
Escherichia coli ATCC 25922 Escherichia coli
Di a met er of Inh i bit i on Zo ne
10 µl
20 µl
30µl
30
20.50±4.27fgA
32.83±3.33fB
61.17±8.81hC
18
33.50±7.70iC
41.17±1.89hD
46.17±3.62fD
20
15.50±0.50defA
20.83±0.76dB
31.83±1.76dD
10
22.83±0.29gB
36.50±0.87gC
50.83±1.44fgD
29.17±0.29gE
15
10.50±0.87bcdA
14.67±0.58cB
18.83±0.29cD
29.50±0.87kC
39.17±1.44iD
28
25.33±2.60ghB
48.50±0.87jE
54.50±2.60gF
11.33±4.04bAB
13.67±2.89cAB
16.00±1.73cB
32
10.00±1.73bcA
15.33±4.73cAB
23.33±3.06cC
15.50±0.87deC
16.83±0.29deD
19.67±0.58dE
22
7.50±0.87bA
10.33±0.58bB
11.33±0.58bB
Salmonella typhimurium ATCC 19430
-b
-
26
-
-
-
Citrobacter freundii
-
Klebsiella pneumoniae ATCC 700603
-
Pseudomonas aeruginosa ATCC 27853
-
Serratia marcescens Enterobacter aerogenes ATCC 13043
M AN U
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-
14
14,83±0,29deB
25.33±0.58eC
29.50±0.87dD
-
-
12
-
-
-
8.17±0.29bA
10.67±0.58bB
25
-
-
-
EP
Proteus vulgaris
SC
10 µl
11.33±0.58bB
14.33±1.15cC
15.33±1.15cC
20
-
-
-
-
-
-
-
-
-
-
15.67±0.58cdC
15.67±0.58cC
18
-
-
7.00±6.56bB
AC C
Mi cr o bi a l st r a in s
a
RI PT
Di a me t e r of I nhi bit i on Zo n e
Va p o ur d if f usi on me th o d
14.17±1.44cdC
a
Diameter of Inhibition Zone (mm) including disc diameter of 6 mm; Zone of growth inhibition values are presented as mean (mm) ± standard deviation of three replicates.
b
(-) no activity.
c
Streptomycin disc (25 µg) was used a positive reference standard for bacteria.
Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to LSD test. Means within the same line followed by the same capital letter are not significantly different (P > 0.05) according to LSD test.
ACCEPTED MANUSCRIPT
Table 4 Zones of growth inhibition (mm) showing antifungal activity of rose geranium essential oil against selected Candida strains Di sc di f f usi on me t h o d Antibiotic b
20 µl
30 µl
Candida albicans ATCC 90028
42.17±1.44iA
40.83±1.44mA
45.67±1.15jB
Candida albicans
38.33±2.89hA
41.33±1.15mA
47.83±2.02jA
Candida parapsilosis ATCC 22019
38.50±2.60hA
48.33±2.89nB
Candida krusei ATCC 6258
24.67±0.58gA
Candida lipolytica
Di a met er of Inh i bi t i o n Zo ne
10 µl
20 µl
30µl
10
42.17±0.29jA
49.00±1.73jC
54.00±1.73gD
15
78.33±11.55lB
81.67±5.77lB
85.00±0.00kB
53.83±2.02kC
13
59.33±1.15kD
64.17±1.44kE
78.50±2.60jF
31.83±0.29lB
35.50±0.87hC
19
24.00±1.73gA
30.83±0.29fB
38.17±0.29eD
11.67±0.58bA
19.83±0.29fgB
23.33±0.58eB
20
10.67±2.31bcdA
19.00±3.46dB
22.33±6.35cB
Candida tropicalis
17.67±1.15eB
18.33±0.58rfB
18.83±1.44dB
12
14.33±1.15cdeA
31.50±1.32fC
50.83±0.76fgD
Candida sake
21.50±0.87fB
29.17±0.29jkB
31.17±2.02gC
35
29.83±0.29hiBC
44.83±0.29iD
67.50±0.87iE
Rhodotorula glutinis ATCC 16740
22.50±0.87fgA
27.17±0.29ijC
29.83±0.29gD
12
17.17±1.44efA
48.50±2.60jE
54.50±0.87gF
M AN U
SC
10 µl
TE D
Mi cr o bi a l st r a in s
a
RI PT
Di a me t e r of I nhi bit i on Zo n e
Va p o ur d if f usi on me th o d
Diameter of inhibition zone (mm) including disc diameter of 6 mm; Zone of growth inhibition values are presented as mean (mm) ± standard deviation of three replicates.
b
Amphotericin B (20 µg) was used a positive reference standard for yeast.
EP
a
Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to LSD test.
AC C
Means within the same line followed by the same capital letter are not significantly different (P > 0.05) according to LSD test.
S0956-7135(13)00167-9
DOI:
10.1016/j.foodcont.2013.03.045
Reference:
JFCO 3212
To appear in:
Food Control
Received Date: 16 December 2012 Revised Date:
24 March 2013
Accepted Date: 30 March 2013
Please cite this article as: BoukhatemM.N., KameliA. & SaidiF., Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical composition and antimicrobial activity against food spoilage pathogens, Food Control (2013), doi: 10.1016/j.foodcont.2013.03.045. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Highlights
► Analysis of rose geranium (Pelargonium graveolens) essential oil (EO) from Algeria.
► Higher antimicrobial activity was observed in the vapor phase.
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► Geranium EO may be used as natural preservative for food.
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► EO exhibited good antibacterial effect against Gram positive bacteria and yeast.
ACCEPTED MANUSCRIPT 1
Essential oil of Algerian rose-scented geranium (Pelargonium graveolens): Chemical
2
composition and antimicrobial activity against food spoilage pathogens
3 4
M.N. Boukhatem ab,*, A. Kameli a, F. Saidi b
6
a
7
Kouba, Alger, Algérie. (Email : [email protected] ; Tél : +213557283091)
8
b
9
Blida, Algérie.
Laboratoire éco-physiologie végétale, Département des sciences naturelles, Ecole Normale Supérieure, BP 92, 16050
Département de Biologie, Faculté des sciences agro-vétérinaire, Université Saad Dahleb, BP 270, Route Soumaa,
SC
10
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Corresponding author: Boukhatem Mohamed Nadjib. Email : [email protected] ; Tél : +213557283091
11 Abstract
13
The aim of this study was to analyze the chemical composition of essential oil of rose-scented geranium (Pelargonium
14
graveolens L.) growing in Algeria and to test the efficacy of the oil against food spoilage and food-borne pathogens.
15
The chemical composition of the oil was analysed by Gas Chromatography-Mass Spectrometry (GC-MS). A total of
16
45 compounds representing 94.2% of the essential oil was identified. The main constituents were citronellol (30.2%),
17
citronellyl formate (9.3%) and geraniol (7.6%).
18
The antimicrobial activity of essential oil was evaluated against 23 food spoilage microorganisms in liquid and vapour
19
phase at three different concentrations (10, 20 and 30 µl/disc).
20
The oil exhibited promising antibacterial effect against Gram positive more than negative bacteria and provides a
21
good inhibitory effect against Candida strains. Furthermore, the zone of inhibition increased with increasing oil
22
concentration. Significantly higher antimicrobial activity was observed in the vapour phase. This is the first report on
23
the antimicrobial properties of the essential oil of Algerian rose geranium.
24
Our results suggest that rose-scenred geranium oil could be used for the development of novel types of antibacterial
25
agents to control food spoilage and food-borne pathogens.
26
Keywords: rose geranium ; Pelargonium graveolens ; essential oil ; GC-MS ; antimicrobial activity ; vapor phase ;
27
geraniol.
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28 29
1. Introduction
30
In recent years there has been a considerable pressure from consumers to reduce or eliminate chemically synthesized
31
additives in their foods. Many naturally occurring compounds present in plants have been shown to possess
32
antimicrobial effect against food-borne pathogens. In particular, the antimicrobial properties of plant essential oils
1
ACCEPTED MANUSCRIPT (EOs) and their constituents have been widely demonstrated (Burt, 2004; Dorman & Deans, 2000). Use of EOs as
34
antimicrobial agents in food systems may be considered as additional intrinsic determinant to increase the safety and
35
shelf life of food.
36
Rose-scented geranium (Pelargonium species) is a multi-harvest high value, aromatic plant cultivated for its EO
37
which is widely used in cosmetic industry and as flavouring for foods (Lis-Balchin, 2005). Pelargonium graveolens L.
38
is native of South Africa and was introduced into North Africa as an ornamental plant. It has adapted well to the
39
Mediterranean climate (Lis-Balchin, 2002; Boukhatem et al., 2011).
40
Geranium essential oil is obtained from the scented leaves of a number of Pelargonium cultivars grown mainly in
41
Reunion, China and Algeria. The volatile oil of rose geranium species has been attributed a number of biological
42
properties including: antibacterial (Rosato et al., 2007; Lalli, Van Zyl, Van Vuren, & Viljoen, 2008), antifungal
43
(Naeini, Nazeri, & Shokri, 2011; Hassan et al., 2011) and also some other pharmacological properties such as anti-
44
inflammatory (Maruyama, Ishibash, & Hu, 2006), spasmolytic (Lis-Balchin, Hart, & Roth, 1997) and hypoglycaemic
45
effects (Boukhris et al., 2012).
46
Rose geranium oil and their major components have gained acceptance in the food industry since they have been
47
generally recognized as safe (GRAS) by FEMA (1965) and approved by the American Food and Drug Administration
48
(FDA) for food use.
49
Although several studies have focused on the chemical composition of Pelargonium EOs (Araya, Soundy, & Steyn,
50
2006; Rajeswara Rao, Kaul, Syamasundar, & Ramesh, 2002; Juliani et al., 2006; Gomes, Mata, & Rodrigues, 2007),
51
only a few reports evaluated their activity against pathogenic and food spoilage bacteria and fungi (Si et al., 2006).
52
As far as our knowledge of the literature is concerned, this study could be considered as the first report on the
53
antimicrobial activities of rose geranium growing in Algeria. Since the volatile oils of rose geranium have been used
54
widely as pharmaceuticals and flavouring agents, it is necessary to assess their antimicrobial activity for the
55
improvement of food safety. Further, guided by ethnobotanical literature and availability from natural resources, our
56
main objective is to validate the use of selected Algerian aromatic plants for their antimicrobial activities and to
57
emphasise the need to promote their natural botanical resources in North Africa.
58
Moreover, no systematic studies comparing antimicrobial activity (in liquid and vapour phase) of the geranium oil are
59
available. Several approaches have been proposed to minimize EO concentrations. One of them is use of EOs in
60
vapour phase to reduce the required concentration. EO in vapour phase could be highly effective against foodborne
61
pathogens at relatively lower concentrations than the liquid phase, thereby causing minimum effect on organoleptic
62
properties (Tyagi & Malik, 2011).
63
The main objectives of this study were (1) to determine the chemical composition of rose geranium EO and (2) to
64
investigate the antimicrobial activity of the oil by disc diffusion and vapour phase methods against 23 selected
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2
ACCEPTED MANUSCRIPT 65
spoiling and pathogenic microorganisms, in an attempt to contribute to the use of these as alternative products for
66
microbial control and food preservation.
67 2. Material and methods
69
2.1. Plant material
70
Rose-scented geranium (Pelargonium graveolens) was grown in the field station of aromatic and medicinal plants of
71
“Extral Bio” Company, Chiffa, Blida city (Algeria). The aerial parts of rose geranium were collected in May 2011
72
during the flowering period. Identification of the species was confirmed by the Botanical Laboratory in the National
73
Institute of Agronomy (Algiers, Algeria).
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68
SC
74 2.2. Extraction of the essential oil
76
Samples of 100 g of the air-dried aerial parts of plants were subjected to hydrodistillation for 2 h using a
77
Clevenger apparatus. The obtained EO was dried over anhydrous sodium sulphate and, after filtration,
78
stored at + 4 °C until tested. All experiments were conducted in triplicates and results were expressed on
79
the basis of dry matter weight.
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2.3. Gas chromatography/mass spectrometry (GC/MS) analysis conditions
82
The analysis of the EO was performed using a Hewlett Packard 5890 II GC, equipped with a HP-5 MS (Crosslinked
83
5% PH ME Siloxane) capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness) and a mass spectrometer 5972 of
84
the same company as detector. For GC-MS detection an electron ionization system was used with ionization energy of
85
70 eV. Helium was the carrier gas, at a flow rate of 1 ml min-1 min. Injector and detector (MS transfer line)
86
temperatures were set at 220 and 290 °C, respectively. Column temperature was initially at 50 °C, then gradually
87
increased to 150 °C at a 3 °C min-1 rate, hold for 10 min and finally increased to 250 °C at 10 °C min-1. Diluted
88
samples (1/100 in acetone) of 1.0 µl were injected manually and splitless.
89
Retention index (RI) of all the constituents were determined by the Kovats method by co-injection of the samples with
90
a solution containing the homologous series of n-alkanes (C8-C24) (Fluka, Buchs/sg, Switzerland) on the HP-5 MS
91
column. Identification of the components was made by visual interpretation, comparing their retention indices and
92
mass spectra with data published in the literature (Adams, 2001) and by matching their recorded mass spectra with
93
reference spectra in the computer library (NIST MS library, Version 2.0). Quantification was computed as the
94
percentage contribution of each compound to the total amount present.
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2.4. Microbial strains and growth conditions
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The in vitro antimicrobial activity of the EO from rose geranium was tested against 23 food-related microorganisms
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including: (1) clinically isolated strains from the Food Microbiology Laboratory of Blida Health Institute and Pasteur institute of
101
Algeria (IPA): Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Citrobacter
102
freundii, Proteus vulgaris, Serratia marcescens, Candida albicans, Candida lipolytica, Candida tropicalis and
103
Candida sake.
104
(2) referenced strains: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6051, Enterococus feacalis ATCC
105
2921, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 19430, Klebsiella pneumoniae ATCC 700603,
106
Pseudomonas aeruginosa ATCC 2785, Enterobacter aerogenes ATCC 13043, Candida albicans ATCC 90028,
107
Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258 and Rhodotorula glutinis ATCC 16740.
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All the strains were grown on Mueller-Hinton agar (MHA) for the bacteria and Saboureaud Dextrose Agar (SDA)
109
with Chloramphenicol for yeasts. The medium were obtained from IPA (Algiers, Algeria). Inocula were prepared in
110
sterile saline from 24 h and 48 h cultures of bacteria and yeasts respectively, and standardized against 0.5 McFarland
111
Standard to obtain suspensions containing approximately 108 cfu mL-1 of bacteria and 106 cfu mL-1 of yeasts.
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2.5. Antimicrobial activity assays
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Two techniques were used to test the antimicrobial activity of rose geranium EO: agar disc diffusion method
115
(NCCLS, 1997) and the vapour diffusion method (Tyagi & Malik, 2011).
116
2.5.1. Disk diffusion method
117
Briefly, a suspension of the tested microorganism (0.1 ml of 108 cells ml-1) was spread on the solid media plates. Filter
118
paper discs (6 mm in diameter) were impregnated with 3 different concentrations (10, 20 and 30 µl per disc) of the oil
119
and placed on the inoculated plates (MHA/SDA). The plates were incubated at 37 °C for 24 h for bacteria and at 30
120
°C for 48 h for yeasts. The zone of inhibition was measured with a calliper. Tests were performed in triplicate, and
121
mean value was calculated. Antibiotic discs of Streptomycin (25 µg/disc) and Amphotericin B (20 µg/disc), purchased
122
from Antibiotical company of Saidal (Medea, Algeria) were also used as positive controls for bacteria and yeasts
123
respectively.
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2.5.2. Vapour diffusion method
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Antibacterial activity of EO in vapour phase was evaluated by disc volatilization method at three different volumes
127
(10, 20 and 30 µl per disc). In brief, solidified medium was inoculated over the surface of MHA/SDA with 0.1 ml of
4
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microorganism suspension. A paper disc (diameter 9 mm, Sigma, Steinheim, Germany) was laid on the inside surface
129
of the upper lid and 10 µl of EO was placed on each disc.
130
The plate inoculated was immediately inverted on top of the lid and sealed with parafilm to prevent leakage of the EO
131
vapour. After the incubation period, the effectiveness of the EO was calculated by measuring the diameter of the
132
inhibition zone. All tests were carried out in triplicate.
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135
All data were reported as means ± standard deviation of three triplicates. One-way analysis of variance (ANOVA) was
136
applied to the data to determine differences (P < 0.05). To ascertain significant differences between the levels of the
137
main factor, Fisher LSD (least significant difference) test was applied between means. ANOVAs were made with the
138
following factors: strains (23 levels) and volume of essential oil tested (10, 20 and 30 µl with disc diffusion and
139
vapour diffusion methods). Statistical data analysis was undertaken using XLStat. Pros statistical software (XLStat,
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Paris, France). P values < 0.05 were considered as significant.
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141 3. Results and Discussion
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3.1. Yield and chemical composition of essential oil
144
The EO was obtained by hydrodistillation from air-dried P. graveolens aerial parts with a yield of 0.15% (v/w).
145
The rose geranium oil obtained is liquid and has yellowish-green colour. It has a strong rose scent with a less
146
pronounced mint note. Therefore, this oil may be valuable for the flavouring of foods.
147
The EO was analyzed by GC-MS. Qualitative and quantitative analyses of the oil volatile profiles are listed in Table 1
148
according to their elution order. In total, 45 compounds constituting 94.21% of the essential oil have been identified.
149
Results showed that citronellol (30.2%), citronellyl formate (9.3%), geraniol (7.6%) and guai-6,9-diene (5.4%) were
150
found to be the major compounds in the oil with minor amounts of isomenthone (4.1%) and menthone (3.6%). Other
151
components were found in less quantity (< 3%). Linalool which is present in 3.2% is an important constituent in
152
fragrances because of its pleasing aroma.
153
The rose geranium EO consisted mainly of monoterpenic alcohols (41.2%), monoterpenic esters (23%) and
154
sesquiterpenic hydrocarbons (13.9%) (Table 2).
155
The market value of the essential oil is determined by terpenoid composition: in particular, besides geraniol,
156
citronellol, linalool, and their esters, isomenthone, the sesquiterpenoid hydrocarbon guaia-6,9-diene, and alcohol 10-
157
epi-g-eudesmol play a key role, as they allow to distinguish between oils of different origin, and different varieties
158
and quality (Lis-balchin, 2002).
159
In our study, it appears that EO of rose geranium analyzed could be therefore classified as ‘‘citronellol chemotype”.
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161
composition of the oil was similar to Chinese oils, with the same content of guaia-6,9-diene and the absence of 10-epi-
162
y-eudesmol. This oil is characterized by high levels of citronellyl formate (9.3%), 6,9 guaia-6,9-diene (5.4%) and
163
trace amounts of 10-epi-y-eudesmol (0.88%) (Table 2) while the bourbon and Egyptian geranium oils contained high
164
levels of 10-epi-y-eudesmol and traces of guaia-6,9- diene.
165
In agreement with results obtained by other authors, the chromatographic analysis of rose geranium EO showed that
166
citronellol was the component present in the greatest percentage (Jalali-Heravi, Zekavat, & Sereshti, 2006; Gomes,
167
Mata, & Rodrigues, 2007; Araya, Soundy, & Steyn, 2006).
168
Regarding other components, our results diverge from those published by other authors (Saxena, Banerjee, & Gupta,
169
2004; Juliani et al., 2006).
170
The studies of Lalli (2006) on the essential oils from South Africa showed a significant difference in their
171
composition, consisting of isomenthone 65.8% and decanoid acid 12.9%. Very low levels of citronellol (0.4%),
172
citronellyl formate (0.05%) were obtained. Furthermore, geraniol was not present.
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173 3.2. Antimicrobial activity
175
3.2.1. Direct diffusion method
176
The antimicrobial activity of rose geranium EO, both by direct contact or through vapour phase, was qualitatively and
177
quantitatively assessed by the presence or absence of inhibition zone. The diameter of the inhibition zone (DIZ) is
178
given in Table 3 and Table 4.
179
To the best of our knowledge, the antimicrobial activity of rose geranium EO growing in Algeria has never been
180
reported. This work is therefore the first report on the EOs from this aromatic plant.
181
The results obtained from the disc diffusion method, indicated that the oil exhibited antimicrobial activity against all
182
Gram-positive bacteria and 3 Gram-negative bacteria at the concentration of 10 µl per disc. As can be seen from the
183
Table 2, the EO of rose geranium showed various degrees of antimicrobial activity depending on the tested bacterial
184
strains.
185
Among the Gram-negative bacteria, the EO was more effective against E. coli and Enterobacter aerogenes ATCC
186
13043 with inhibition zones measured at 15 and 14 mm respectively. In contrast, P. aeruginosa and K. pneumoniae
187
were the most resistant strains to the oils while Proteus vulgaris, an important food pathogen, shown a modest
188
sensibility.
189
Among the Gram-positive bacteria, S. aureus ATCC 6538 and E. faecalis ATCC 29212 (DIZ 21.17 mm) were the
190
most sensitive strains to the EO, followed by B. subtilis ATCC 6051(20.5 mm) and S. epidermidis (16.17 mm).
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192
among all the tested microorganisms in comparison with the amphetorecin B (Table 4).
193
The zone of inhibition increased with the increasing concentration of EO (Table 3). The zone of inhibition in the case
194
of bacteria was less than that for the yeasts strains at 30 µl dose of EO. Hence, the zone of inhibition due to the same
195
concentration of essential oil was bigger for yeasts than for bacteria.
196
Overall, the EO showed very promising antibacterial activity. EO was more selective for the Gram-positive test
197
pathogens than for the Gram-negative bacteria which is in accordance with studies reporting that Gram-positive
198
bacteria are generally more sensitive than Gram-negative bacteria to EO (Gilles, Zhao, An, & Agboola, 2010;
199
Oussalah, Caillet, & Lacroix, 2006).
200
Our conclusions were in agreement with the results of an earlier study (Lis-Balchin, Deans & Eaglesham, 1998)
201
wherein 24 cultivars were tested for antimicrobial activity of rose geranium EO against 25 test bacteria. Of these test
202
bacteria, the most sensitive bacteria were Bacillus subtilis and Staphylococcus aureus, a group of predominantly
203
Gram-positive organisms, whilst the least sensitive organisms were Escherichia coli, Klebsiella pneumonia and
204
Pseudomonas aeruginosa, a group of predominantly Gram-negative organisms.
205
Not all studies on essential oils have concluded that Gram-positives are more susceptible. (Zaika, 1988; Dorman &
206
Deans, 2000)
207
Pattnaik et al. (1996) tested geranium oil for antibacterial activity against 22 bacteria by disc diffusion. Only 12
208
bacterial strains were inhibited by the Geranium oil, but all the fungi were inhibited.
209
Lis-Balchin et al. (1998) found that Pelargonium EOs showed substantial in vitro activity against S. aureus, Proteus
210
vulgaris, B. cereus and S. epidermidis.The latter two studies suggest that Pelargonium EO can potentially be used as
211
novel antibacterial agents in the food and cosmetic industries.
212
More recently, attempts have been made to identify the component(s) responsible for such bioactivities (Dorman &
213
Deans, 2000). The antimicrobial properties of volatile oils and their constituents from a wide variety of plants have
214
been assessed and reviewed.
215
The antimicrobial activity of geranium EO could be due to citronellol and geraniol. Other geranium oils rich in
216
citronellol and geraniol were previously demonstrated to have potent antimicrobial activities (Prashar, Hilli, Veness,
217
& Evans, 2003; Si et al., 2006). Pelargonium also contains the oxygenated monoterpene linalool (3.2%) known to
218
possess good antimicrobial activity (D’auria et al., 2005).
219
More recently, a study carried out by Jirovetz et al. (2006) showed that essential oils with floral-rosy scent, such as
220
geranium, possess high antimicrobial activities against various microorganisms and these effects are maintly the result
221
of a combination of some biologically active principal aroma compounds (geraniol, nerol, citronellol and many of
222
their derivatives).
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ACCEPTED MANUSCRIPT In addition, the components in lower amount such as, cymene, linalool, rose oxyde, geranial and caryophyllene could
224
also contribute to the antimicrobial activity of the oils. It has been reported in the literature that the inhibitory activity
225
of an essential oil results from a complex interaction between its different constituents (Xianfei, Xiaoqiang, Shunying,
226
& Guolin, 2007).
227
Few studied have examined the antifungal activity of geranium essential oils. In our study, EO exhibited the strongest
228
inhibition effect against yeasts. These results are in agreement with those reported in the literature for other rose
229
geranium oil rich in geraniol that showed a very strong action versus C. tropicalis and C. albicans (Hassan et al.,
230
2011). Hammer et al. (1999) tested various essential oils and plant extracts for their antimicrobial effects. Rose
231
geranium EO showed greater activity against Candida albicans than against S. aureus. These findings correlate with
232
our results obtained in this study. A study carried out by Mahboobi et al. (2008) proved that rose geranium oil was the
233
second most effective of the essential oils on Candida albicans and exhibited strong activity on isolates.
234
Mechanism of anti-Candida activity of geraniol, geranyl acetate and citronellol appears to be associated with damage
235
in the membrane integrity, inhibited germ tube induction at very low concentrations and inhibited Candida albicans
236
cell cycle (Zore, Archana, Thakre, Rathod, & Karuppayil, 2010). These previously mentioned components are present
237
in our essential oil sample.
238
Dalleau et al. (2008) compared the anti-biofilm activity of molecules which are often prevalent in EOs. Thymol,
239
carvacrol and geraniol exhibited the most significant antibiofilm activity against all three tested strains, including
240
Candida albicans, C. parapsilosis and C. glabrata.
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3.2.2. Vapour diffusion method
243
Comparative studies of antibacterial potential of the EO were also conducted in the vapour phase (Table 3 and Table
244
4). As observed in the earlier assays using EO in solid agar, the zone of inhibition due to the oil vapours also
245
increased with increasing concentration of the oil. Further, the zone of inhibition due to the EO vapours was bigger for
246
Gram positive bacteria (S. aureus) and yeasts than for Gram negative bacteria (E. coli).
247
The zone of inhibition resulting from the exposure to EO vapours was significantly larger than that from the same
248
concentration of EO in the liquid phase. This effect was visible at both lower (10 µl) and more prominently at higher
249
(30 µl) concentrations.
250
EO showed higher activity in the vapour phase. P. aeruginosa, K. pneumoniae and S. marcescens were more resistant
251
against the rose geranium oil vapours too.
252
The yeasts (Candida albicans and C. parapsilosis) were inhibited completely by the oil vapours at 30 µl exposure.
253
Our study clearly demonstrates the higher antimicrobial efficacy of rose gernium oil vapours. This could be attributed
254
to the variation in the relative composition of the oil and vapours as the latter must be enriched in terms of its volatile
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256
the antimicrobial potential of rose geranium EO, very few studies exist on the activity of EO vapours.
257
Hence, smaller doses of essential oil in the vapour phase can be inhibitory to spoilage bacteria. Our results are in
258
agreement with previous reports (Tyagi & Malik, 2011). Pibiri (2006) also demonstrated that the EOs of Satureia
259
Montana and Cinnamon in vapour phase have a lethal effect on S. aureus and P. aeruginosa, even in small doses.
260
This demonstrates that the antimicrobial activity of different EO vapours can be achieved with a smaller amount than
261
the EO in liquid phase. A study carried out by Goni et al. (2009) showed that antimicrobial activity of combination of
262
cinnamon and clove EOs in vapour phase showed better antimicrobial with less active concentration in the vapour
263
phase compared to liquid phase. Hence the evaluation of the antimicrobial properties of rose geranium EO in the
264
vapour phase would open up a newer dimension with immense potential applications.
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265 4. Conclusions
267
Our results demonstrated that citronellol, geraniol and their esters were the main constituents of EO samples of rose
268
geranium growing in Algeria. EO exhibited significant antimicrobial activity against a range of food spoiling
269
microorganisms and the strongest inhibitory effect against yeasts. Our findings suggest that rose geranium EO is
270
highly effective in vapour phase. Rose geranium EO could be considered suitable alternatives for use in the food
271
industry as a natural antimicrobial agent. However, issues of safety and toxicity will always need to be addressed.
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272 References
274
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ACCEPTED MANUSCRIPT Table 1 Chemical composition of essential oil from rose-scented geranium growing in Algeria N°
Rt b
Compounds a
RI c
Content (%)
29.210
α-Pinene
934
0.1
2
30.018
p-Cymene
1020
0.2
3
30.113
Linalool
1089
4
32.105
trans-Rose oxide
1100
5
32.110
cis-Rose oxide
1115
6
43.240
Menthone
1136
7
46.819
Isomenthone
8
49.211
α-Terpineol
9
58.689
Citronellol
10
58.871
Neral
11
59.500
Piperitone
12
59.834
Geraniol
13
60.402
Geranial
14
60.726
15
61.740
16
63.159
17
63.514
18
63.745
RI PT
1
3.2 2.2 1.3
SC
3.6 4.1
1165
0.2
M AN U
1149
30.2
1233
0.3
1247
0.3
1250
7.6
1262
2.6
Citronellyl formate
1266
9.3
Geranyl formate
1294
1.5
α-Cubebene
1348
0.2
Citronellyl acetate
1358
0.4
Geranyl acetate
1375
0.2
63.960
α-Copaene
1378
0.5
64.224
β-Bourbonene
1383
1.3
64.518
α-Ylangene
1385
0.3
22
65.227
γ-Caryophyllene
1400
0.6
23
65.521
α-Guaiene
1440
0.1
24
65.825
β-Caryophyllene
1453
0.6
25
65.967
Guaia-6,9-diene
1465
5.4
26
66.819
Citronellyl propionate
1476
1.0
20 21
EP
AC C
19
TE D
1220
ACCEPTED MANUSCRIPT 66.920
α-Caryophyllene
1478
0.9
28
67.042
Alloaromadendrene
1481
0.5
29
67.285
Germacrene D
1483
1.5
30
67.417
α-Salinene
1487
0.3
31
67.974
Citronellyl butyrate
1517
1.5
32
68.076
δ-Cadinene
1522
33
68.370
Calamenene
1526
34
68.806
Geranyl butyrate
1559
35
69.221
α-muurolene
1565
36
69.424
β-Phenylethyl tiglate
37
69.718
Geranyl isovalerate
38
70.042
39
RI PT
27
c
1.1
SC
0.1
1593
0.5
Citronellyl valerate
1601
1.4
70.144
10-epi-γ-eudesmol
1612
0.8
40
70.316
Geranyl valerate
1630
0.2
41
70.610
γ-Eudesmol
1650
0.3
42
71.745
Caryophyllene oxide
1686
1.4
43
72.161
Citronellyl tiglate
1692
0.5
44
77.797
Geranyl tiglate
1723
2.0
45
81.649
Geranyl propionate
1792
0.7
TE D
M AN U
2.3
EP b
0.5
1580
AC C
a
0.5
Total identified components
94.3
No identified
5.7
Compounds listed in order of elution from an HP-5 MS column.
Rt, Retention time (in minutes). RI, retention index calculated on the HP-5 MS column relative to C8-C28 n-alkanes.
ACCEPTED MANUSCRIPT Table 2 Chemical classes of essential oil extracted from rose-scented geranium growing in Algeria
Monoterpenic esters Sesquiterpenic hydrocarbons Monoterpenic ketones Monoterpenic oxides Monoterpenic aldehydes Sesquiterpenic alcohol
23.0 13.9 8.0 4.9 1.8 1.3 0.2
M AN U
Monoterpenic hydrocarbons
41.2
RI PT
Monoterpenic alcohols
Content (%)
SC
Chemical classes
AC C
EP
TE D
Total identified components
94.3
ACCEPTED MANUSCRIPT
Table 3 Zones of growth inhibition (mm) showing antibacterial activity of rose geranium essential oil Di sc di f f usi on me t h o d Antibiotic c
20 µl
30 µl
Staphylococcus aureus ATCC 6538
21.17±2.47fA
25.17±2.75iAB
30.83±5.06gB
Staphylococcus aureus
12.83±0.76bcA
20.83±1.76ghB
25.17±0.29efB
Staphylococcus epidermidis
16.17±1.04deA
20.50±0.50ghB
25.83±1.44fC
Bacillus subtilis ATCC 6051
20.50±0.87fA
22.17±0.29hB
23.17±0.29eB
Bacillus cereus
14.17±0.29cdB
17.83±0.29efC
Enterococcus feacalis ATCC 29212
21.17±0.29fA
Escherichia coli ATCC 25922 Escherichia coli
Di a met er of Inh i bit i on Zo ne
10 µl
20 µl
30µl
30
20.50±4.27fgA
32.83±3.33fB
61.17±8.81hC
18
33.50±7.70iC
41.17±1.89hD
46.17±3.62fD
20
15.50±0.50defA
20.83±0.76dB
31.83±1.76dD
10
22.83±0.29gB
36.50±0.87gC
50.83±1.44fgD
29.17±0.29gE
15
10.50±0.87bcdA
14.67±0.58cB
18.83±0.29cD
29.50±0.87kC
39.17±1.44iD
28
25.33±2.60ghB
48.50±0.87jE
54.50±2.60gF
11.33±4.04bAB
13.67±2.89cAB
16.00±1.73cB
32
10.00±1.73bcA
15.33±4.73cAB
23.33±3.06cC
15.50±0.87deC
16.83±0.29deD
19.67±0.58dE
22
7.50±0.87bA
10.33±0.58bB
11.33±0.58bB
Salmonella typhimurium ATCC 19430
-b
-
26
-
-
-
Citrobacter freundii
-
Klebsiella pneumoniae ATCC 700603
-
Pseudomonas aeruginosa ATCC 27853
-
Serratia marcescens Enterobacter aerogenes ATCC 13043
M AN U
TE D -
-
14
14,83±0,29deB
25.33±0.58eC
29.50±0.87dD
-
-
12
-
-
-
8.17±0.29bA
10.67±0.58bB
25
-
-
-
EP
Proteus vulgaris
SC
10 µl
11.33±0.58bB
14.33±1.15cC
15.33±1.15cC
20
-
-
-
-
-
-
-
-
-
-
15.67±0.58cdC
15.67±0.58cC
18
-
-
7.00±6.56bB
AC C
Mi cr o bi a l st r a in s
a
RI PT
Di a me t e r of I nhi bit i on Zo n e
Va p o ur d if f usi on me th o d
14.17±1.44cdC
a
Diameter of Inhibition Zone (mm) including disc diameter of 6 mm; Zone of growth inhibition values are presented as mean (mm) ± standard deviation of three replicates.
b
(-) no activity.
c
Streptomycin disc (25 µg) was used a positive reference standard for bacteria.
Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to LSD test. Means within the same line followed by the same capital letter are not significantly different (P > 0.05) according to LSD test.
ACCEPTED MANUSCRIPT
Table 4 Zones of growth inhibition (mm) showing antifungal activity of rose geranium essential oil against selected Candida strains Di sc di f f usi on me t h o d Antibiotic b
20 µl
30 µl
Candida albicans ATCC 90028
42.17±1.44iA
40.83±1.44mA
45.67±1.15jB
Candida albicans
38.33±2.89hA
41.33±1.15mA
47.83±2.02jA
Candida parapsilosis ATCC 22019
38.50±2.60hA
48.33±2.89nB
Candida krusei ATCC 6258
24.67±0.58gA
Candida lipolytica
Di a met er of Inh i bi t i o n Zo ne
10 µl
20 µl
30µl
10
42.17±0.29jA
49.00±1.73jC
54.00±1.73gD
15
78.33±11.55lB
81.67±5.77lB
85.00±0.00kB
53.83±2.02kC
13
59.33±1.15kD
64.17±1.44kE
78.50±2.60jF
31.83±0.29lB
35.50±0.87hC
19
24.00±1.73gA
30.83±0.29fB
38.17±0.29eD
11.67±0.58bA
19.83±0.29fgB
23.33±0.58eB
20
10.67±2.31bcdA
19.00±3.46dB
22.33±6.35cB
Candida tropicalis
17.67±1.15eB
18.33±0.58rfB
18.83±1.44dB
12
14.33±1.15cdeA
31.50±1.32fC
50.83±0.76fgD
Candida sake
21.50±0.87fB
29.17±0.29jkB
31.17±2.02gC
35
29.83±0.29hiBC
44.83±0.29iD
67.50±0.87iE
Rhodotorula glutinis ATCC 16740
22.50±0.87fgA
27.17±0.29ijC
29.83±0.29gD
12
17.17±1.44efA
48.50±2.60jE
54.50±0.87gF
M AN U
SC
10 µl
TE D
Mi cr o bi a l st r a in s
a
RI PT
Di a me t e r of I nhi bit i on Zo n e
Va p o ur d if f usi on me th o d
Diameter of inhibition zone (mm) including disc diameter of 6 mm; Zone of growth inhibition values are presented as mean (mm) ± standard deviation of three replicates.
b
Amphotericin B (20 µg) was used a positive reference standard for yeast.
EP
a
Means within the same column followed by the same small letter are not significantly different (P > 0.05) according to LSD test.
AC C
Means within the same line followed by the same capital letter are not significantly different (P > 0.05) according to LSD test.