WO2016076689A1 - Pharmacological composition of oregano (essential oil) exhibiting an antibacterial effect on the jp2 clone of aggregatibacter actinomycetemcomitans - Google Patents
Pharmacological composition of oregano (essential oil) exhibiting an antibacterial effect on the jp2 clone of aggregatibacter actinomycetemcomitans Download PDFInfo
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- WO2016076689A1 WO2016076689A1 PCT/MA2014/000058 MA2014000058W WO2016076689A1 WO 2016076689 A1 WO2016076689 A1 WO 2016076689A1 MA 2014000058 W MA2014000058 W MA 2014000058W WO 2016076689 A1 WO2016076689 A1 WO 2016076689A1
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- essential oil
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This new application encompasses both the methodology and the results obtained.
- the technique used is a simple, clear, evidence-based method with improved accuracy and reproducibility of results.
- Oregano essential oil This is Origanum compactum from the Lamiaceae family. It was extracted from plants that were grown in Morocco. The authenticated specimens were deposited in the photochemistry laboratory herbarium of the National Institute of Medicinal and Aromatic Plants - University of Sidi Mohamed Ben Abdellah, Fez, Morocco, according to code FA / RP / INPMA / 107.
- a solution of 10% Tween 80 (diluted in distilled water) is autoclaved beforehand. Then, 900 ⁇ of this solution is added to 100 ⁇ of essential oil and then the whole is homogenized: it is the mother solution of the emulsified essential oil.
- GC gas chromatographic
- HP-5 capillary column 5% phenyl methyl silicone
- the characteristic of this column was: 30 m in length, 0.25 mm in diameter and 0.25 mm in film thickness.
- the temperature is programmed from 50 ° C (5min initial wait) to 200 ° C at 4 ° C / min.
- Gas chromatography conditions were as follows: IM2 as a carrier gas (1.8 ml / min); shared mode (flow rate: 72.1 ml / min, ratio: 1/50) was used, the temperatures of the injector and the detector are 275 ° C and 250 ° C respectively.
- the diluted samples (1/20 in hexane) of 1 ml were injected manually. The machine was run by a type of "HP ChemStation" computer system.
- the chromatographic conditions are as follows: injector and detector temperatures at 220 and 300 ° C respectively; carrier gas, helium at a flow rate of 1.4 ml / min, ramp program temperature of 40 to 300 ° C with a gradient of 4 ° C / min (maintenance of the initial and final temperature for 4 min).
- the relative amount of the individual components of the total oil was expressed as percentage of peak area relative to the total peak area.
- a literature search was conducted using the combination of NIST MS Research and literature. The components of the oils have also been identified by their relative retention indices for n-alkanes (C8-C24)
- the protocol firstly uses the technique of direct diffusion contact in agar medium (well), which allows to predict the in vitro efficacy of the essential oil. Then, for the calculation of the MIC (Minimum Inhibitory Concentration) of the oil tested effective, the 96-well microdilution method is used. The CMB (Minimal Bactericidal Concentration) is also measured according to the protocol below.
- This test is carried out by deposition of the essential oil in wells dug in the agar according to a modification of the Dorman and Deans 2000 technique. It ensures a total diffusion of the essential oil from a well by giving a zone clear inhibition and easily measurable diameter on agar seeded by the bacterial suspension.
- 1 ml of the prepared inoculum is seeded on the surface of the agar medium by flooding. After 15 min, wells are dug using Pasteur pipettes (the thick end of 6 mm). It should be noted that the inoculum thus prepared should not be used beyond 30 minutes at the risk of an increase in the density of the inoculum because of bacterial growth. Then, 50 ⁇ l of the essential oil is poured into each well. The pure essential oil is tested and diluted in 1/10 Tween 80 (10%).
- the doxycycline 30ug disc is used as a positive control, Tween 80 pure and diluted to 10% are used as a negative control.
- a flood-seeded petri dish was taken as a control for bacterial growth. All tests are done in triplicate.
- Incubation The seeded petri dishes are incubated in an oven at 37 ° C. in jars under 5% CO 2 for 48 hours.
- MIC minimum inhibitory concentration
- Aa Y Aggregatibacter actinomycetemeomitans
- successive serial dilutions by geometric progression of reason 2 of the stock solution obtained for each essential oil tested are prepared in a sterile culture medium (BHI), so as to successively obtain the dilutions: 1/2, 1/4 , 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512.
- these dilutions correspond, therefore, to: 5%, 2.5%, 1.25%, 0.6%, 0.3% %, 0.15%, 0.07%, 0.03%, 0.01%.
- 100 ⁇ l aliquots of each prepared dilution of oil tested were added to each microplate well to 100 ⁇ l of prepared inoculum.
- Tween 80 to 10% is used as a negative control. Growth control (inoculum alone) for each strain is also included in the test. The tests are carried out in triplicate on the same microplate and the experiment is repeated twice. The diagram of the experimental distribution at the level of the microplates is illustrated below (FIG. 1).
- a bacterial suspension in BHI (Brain Heart Infusion) broth equivalent to the McFarland 0.5 standard is prepared and added to the antibiotic solutions at different concentrations (0.01 mg / ml, 0.1 mg / ml, 1 mg / ml, 10 mg / ml).
- a negative control sterile water + inoculum
- a solution containing sterile broth is used as a contamination control.
- concentration of 10 mg / ml, the solution of which remained clear (not turbid) is considered the MIC of Arnoxicillin on Aa and which will be used as a positive control in our test.
- microplates covered with their lids, were incubated in plastic bags at 5% CO 2 at 37 ° C. for 48 hours.
- TTC Triphenyl Tetrazolium Chloride
- CMB Minimal Bactericidal Concentration
- Origanum compactum, Doxycycline and Tween 80 the one-factor analysis (ANOVA) with Bonferroni correction was performed. The value of P ⁇ 0.05 was considered statistically significant.
- the statistical analysis was conducted using SPSS for Windows (SPSS, Inc., Chicago, IL, USA).
- the diameters of the zones of inhibition observed for the crude Origanum compactum essential oil showed values higher than 20 mm (Table 2), expressing a high sensitivity of the strain Aggregatibacter actinomycetemcomitans clone JP2 to the oil studied.
- MIC (0.03 ⁇ 0.01%) and CMB (0.07 ⁇ 0%) indicate effective antibacterial activity.
- Origanum compactum essential oil The CMB / MIC ratio obtained is less than 4, which gives information on the nature of the antibacterial effect of the essential oil tested which thus proves to be bactericidal.
- CM1 Minimal Inhibitory Concentration
- CMB Minimal Bactericidal Concentration
Abstract
The invention relates to the application of the antibacterial effect of the essential oil of oregano (Origanum compactum) of Moroccan origin on a virulent oral bacterial strain, the JP2 clone of Aggregatibacter actinomycetemcomitans. Said original strain has never been subjected to antimicrobial tests based on essential oils. This novel application relates to both the methodology and the results obtained. The technique used is a simple and clear evidence-based method which provides a higher level of precision and improved reproducibility of results.
Description
COMPOSITION PHARMACOLOGIQUE DE L'ORIGAN (HUILE ESSENTIELLE) A EFFET ANTIBACTERIEN SUR D'AAGGREGATIBACTER ACTINOMYCETEMCOMITANS PHARMACOLOGICAL COMPOSITION OF ORIGAN (ESSENTIAL OIL) WITH ANTIBACTERIAL EFFECT ON AAGGREGATIBACTER ACTINOMYCETEMCOMITANS
Description : Description:
Notre invention concerne la phytothérapie, en particulier l'effet antibactérien de l'huile essentielle d'Origan {Origanum compactum) d'origine Marocaine sur une souche bactérienne orale virulente; Aggregatibacter actinomycetemcomitans clone JP2. Cette souche originelle n'a jamais fait l'objet de tests antimicrobiens à base d'huiles essentielles. Our invention relates to herbal medicine, in particular the antibacterial effect of Oregano essential oil (Origanum compactum) of Moroccan origin on a virulent oral bacterial strain; Aggregatibacter actinomycetemcomitans clone JP2. This original strain has never been the subject of antimicrobial tests based on essential oils.
Cette nouvelle application englobe aussi bien la méthodologie que les résultats obtenus. La technique utilisée est une méthode simple, claire, basée sur la preuve et offrant une meilleure précision et une meilleure reproductibilité des résultats. This new application encompasses both the methodology and the results obtained. The technique used is a simple, clear, evidence-based method with improved accuracy and reproducibility of results.
Aggregatibacter actinomycetemcomitans {Aa) clone JP2 est une bactérie orale à Gram négatif anaérobie facultative, connue virulente, incriminée comme agent étiologique principal dans les parodontites agressives, réel problème de santé publique au Maroc. Il s'agit de maladies parodontaies destructrices entraînant des pertes osseuses sévères et aboutissant à la perte des dents chez de jeunes adolescents Marocains (Haubek et al. 2001, Haubek et al. 2008, Ennibi et al. 2012). Ces dernières décennies, en raison de l'incidence croissance de ces parodontites, de la résistance accrue des bactéries orales aux antibiotiques et des effets secondaires liés aux agents antibactériens fréquemment utilisés en dentisterie, la recherche d'un nouvel agent thérapeutique alternatif sans danger pour la santé s'impose. Ainsi, les huiles essentielles, utilisés en Médecine traditionnelle, peuvent être considérées comme une bonne alternative thérapeutique. Ceci d'autant plus que le Maroc figure parmi les principaux pays producteurs d'huiles essentielles. Aggregatibacter actinomycetemcomitans {Aa) clone JP2 is an facultative facultative anaerobic gram-negative oral bacterium, known to be virulent, implicated as the main etiological agent in aggressive periodontitis, a real public health problem in Morocco. These are destructive periodontal diseases that cause severe bone loss and lead to loss of teeth in young Moroccan adolescents (Haubek et al., 2001, Haubek et al., 2008, Ennibi et al., 2012). In recent decades, due to the increasing incidence of these periodontitis, increased resistance of oral bacteria to antibiotics and side effects related to antibacterial agents frequently used in dentistry, the search for a new alternative therapeutic agent safe for the health is necessary. Thus, essential oils, used in traditional medicine, can be considered as a good therapeutic alternative. This is especially true as Morocco is one of the leading producers of essential oils.
A la date d'aujourd'hui, il n'existe pas de procédures standardisées concernant la détermination du pouvoir antimicrobien des huiles essentielles telles qu'il en existe pour les antibiotiques selon les recommandations de CLSI par National Committee for Clinical Laboratory Standards, Wayne, PA, USA (NCCLS). Toutefois, l'évaluation de l'activité antibactérienne de ces agents a fait l'objet de plusieurs publications dont celles sur Aggregatibacter actinomycetemcomitans (clone non-JP2), et ce^ suivant différentes méthodes non standardisées pour la détermination de la CMI (Concentration Minimale Inhibitrice) des huiles sur cette bactérie. En effet, on retrouve une grande divergence entre les études concernant le protocole employé ainsi que le matériel utilisé. Certains auteurs utilisent la méthode de diffusion en milieu solide (Shih et al. 2013, Kçdzia et al. 2013, Gursoy et al. 2009), d'autres rapportent la méthode de diffusion en milieu liquide, en macrodilution pour certains (Cha et al. 2005, 2007, Kulik et al. 2000) et en microdilution pour d'autres (Cunha et al. 2013, Francesca et al. 2013, Park et al. 2012, Taweechaisupapong et al. 2010, Takarada et al 2004, Shapiro et al 1994).
La méthode de diffusion en milieu solide est moins précise et présente certaines limites dû au cloisonnement des composants de l'huile dans la gélose en fonction de leur affinité avec l'eau (Budzynska et al. 2009). Concernant la méthode diffusion en milieu liquide en macrodilution, la CMI est généralement déterminée visuellement ce qui également manque de précision. Quant à la technique de dilution en milieu liquide en microdilution, elle reste la plus sensible, la plus précise et la plus reproductible pour le calcul de la CMI (Budzynska et al. 2009). Cependant, afin de mener à bien cette technique et obtenir des résultats probants, l'utilisation d'un indicateur de croissance est recommandée selon plusieurs auteurs (tel : Triphényl Tétrazolium Chloride (TTC), piodonitrotetrazolium violet (INT)...). Or, les études divergent concernant le choix de la dose appropriée de cet indicateur, sachant que ce dernier peut être toxique et peut avoir un effet inhibiteur sur la croissance bactérienne à haute concentration. Ainsi, dans notre méthodologie adoptée, nous avons utilisé une concentration appropriée de TTC (qui ne montre aucun effet inhibiteur sur la croissance bactérienne), en se référant à l'étude de Rahman et al. 2004 sur les bactéries anaérobies à Gram négatif. As of today, there are no standardized procedures for the determination of the antimicrobial potency of essential oils as exists for antibiotics as recommended by CLSI by National Committee for Clinical Laboratory Standards, Wayne, PA, USA (NCCLS). However, evaluation of antibacterial activity of these agents has been the subject of several publications including those on Aggregatibacter actinomycetemcomitans (non-JP2 clone), and following ^ various non standardized methods for determining the MIC (Minimum Concentration Inhibitory) oils on this bacteria. Indeed, there is a great divergence between the studies concerning the protocol used as well as the equipment used. Some authors use the solid medium diffusion method (Shih et al 2013, Kçdzia et al 2013, Gursoy et al., 2009), others report the liquid diffusion method, macrodilution for some (Cha et al. 2005, 2007, Kulik et al., 2000) and microdilution for others (Cunha et al 2013, Francesca et al 2013, Park et al 2012, Taweechaisupapong et al., 2010, Takarada et al 2004, Shapiro et al. al 1994). The solid medium diffusion method is less precise and has some limitations due to the partitioning of the oil components in the agar depending on their affinity for water (Budzynska et al., 2009). Regarding the diffusion method in liquid medium in macrodilution, the MIC is generally determined visually which also lacks precision. The microdilution dilution technique remains the most sensitive, the most accurate and the most reproducible for calculating the MIC (Budzynska et al., 2009). However, in order to carry out this technique and obtain convincing results, the use of a growth indicator is recommended according to several authors (such as: Triphenyl Tetrazolium Chloride (TTC), Piodonitrotetrazolium Violet (INT) ...). However, the studies differ on the choice of the appropriate dose of this indicator, knowing that the latter may be toxic and may have an inhibitory effect on the high concentration of bacterial growth. Thus, in our adopted methodology, we used an appropriate TTC concentration (which shows no inhibitory effect on bacterial growth), referring to the study by Rahman et al. 2004 on Gram-negative anaerobic bacteria.
Il est important de signaler que les protocoles rapportés sur l'étude de l'effet des huiles essentielles sur Aggregatibacter actinomycetemcomitans sont souvent ambiguës, non clairs et/ou utilisant des matériaux ou appareils coûteux souvent compliqués tels que les systèmes automatisés (plaques microscan avec le système WalkAway-96 (Saiman et al. 2003), système de microbiologie automatisé Vitek (bioMérieuxVitek, lnc.)(Barry et al. 2003)...) ou semiautomatisés (spectrophotomètres, traitement d'image assistée par ordinateur (Canton et al. 2000, Korgenski et al. 1998)...). It is important to point out that the reported protocols on the study of the effect of essential oils on Aggregatibacter actinomycetemcomitans are often ambiguous, unclear and / or using expensive materials or devices often complicated such as automated systems (microscan plates with the WalkAway-96 system (Saiman et al., 2003), automated microbiology system Vitek (bioMérieuxVitek, Inc.) (Barry et al., 2003) ...) or semi-automated (spectrophotometers, computer-assisted image processing (Canton et al. 2000, Korgenski et al., 1998) ...).
Par ailleurs, il est judicieux de noter qu'il n'existe pas de standards internationaux concernant les tests de susceptibilité antimicrobienne pour les bactéries orales. Par conséquent, notre présent travail se propose de présenter une méthodologie précise basée sur la preuve, aboutissant à des résultats reproductibles et fiables. In addition, it is worth noting that there are no international standards for antimicrobial susceptibility testing for oral bacteria. Therefore, our present work proposes to present a precise methodology based on the proof, resulting in reproducible and reliable results.
Matériel et méthodes : Material and methods :
• Matériel : • Material:
L'huile essentielle d'Origan: Il s'agit de Origanum compactum de la famille des Lamiaceae. Elle a été extraite de plantes qui ont été cultivées au Maroc. Les spécimens authentifiés ont été déposés dans l'herbier de laboratoire de photochimie de l'Institut national de plantes médicinales et aromatiques - Université de Sidi Mohamed Ben Abdellah, Fès, Maroc, selon le code FA/RP/INPMA/107. Oregano essential oil: This is Origanum compactum from the Lamiaceae family. It was extracted from plants that were grown in Morocco. The authenticated specimens were deposited in the photochemistry laboratory herbarium of the National Institute of Medicinal and Aromatic Plants - University of Sidi Mohamed Ben Abdellah, Fez, Morocco, according to code FA / RP / INPMA / 107.
Une solution mère de travail de cette huile est d'abord préparée selon la technique de Benjilali et al. 1986 comme suit: A working mother solution of this oil is first prepared according to the technique of Benjilali et al. 1986 as follows:
Une solution de Tween 80 à 10% (diluée dans l'eau distillée) est préalablement autoclavée. Ensuite, 900 μΙ de cette solution est ajoutée à 100 μΙ d'huile essentielle et puis l'ensemble est homogénéisé : c'est la solution mère de l'huile essentielle émulsifiée.
Souche bactérienne : Aggregatibacter actinomycetemcomitons, clone JP2, sérotype b. Il s'agit d'un isolât clinique issu de prélèvements de plaque sous-gingivale de patients atteints de parodontite agressive, au Service de Parodontologie du Centre de Consultation et de traitement dentaire de Rabat. A solution of 10% Tween 80 (diluted in distilled water) is autoclaved beforehand. Then, 900 μΙ of this solution is added to 100 μΙ of essential oil and then the whole is homogenized: it is the mother solution of the emulsified essential oil. Bacterial strain: Aggregatibacter actinomycetemcomitons, clone JP2, serotype b. This is a clinical isolate from subgingival plaque samples from patients with aggressive periodontitis, at the Periodontology Department of Rabat Dental Consultation and Treatment Center.
• Méthode expérimentale : • Experimental method:
Extraction de l'huile essentielle Extraction of the essential oil
Une portion (100 g) des parties aériennes des plantes a été hydrodistillée, pendant au moins trois heures, en utilisant un appareil de type Clevenger. Pour éliminer toute trace d'eau, l'huile extraite a été traitée avec du sulfate de sodium anhydre (Na2S04), filtrée et ensuite stockée à l'obscurité à 4 ° C. Le rendement en huiles essentielles est calculé selon la formule : Yield = x WQ A portion (100 g) of the aerial parts of the plants was hydrodistilled for at least three hours using a Clevenger type apparatus. To remove all traces of water, the extracted oil was treated with anhydrous sodium sulphate (Na2SO4), filtered and then stored in the dark at 4 ° C. The yield of essential oils is calculated according to the formula: Yield = x WQ
Ce dernier est exprimé en ml/100 g de matière sèche. The latter is expressed in ml / 100 g of dry matter.
Analyse chromatographique et caractérisation de l'huile essentielle Chromatographic analysis and characterization of the essential oil
CG : analyse par la chromatographie en phase gazeuse (CG) est effectuée sur un chromatographe Hewlett -Packard ( HP 6890 ) gazeuse ( FID) , équipé d'une colonne capillaire HP- 5 (5% de phényl méthyl silicone ) . La caractéristique de cette colonne était de: 30 m de longueur, 0,25 mm de diamètre et de 0,25 mm d'épaisseur de film. La température est programmée de 50 ° C (5min d'attente initiale) à 200 ° C à 4 ° C / min . Des conditions de chromatographie en phase gazeuse ont été les suivantes : IM2 comme gaz porteur (1,8 ml / min) ; mode partagé (débit: 72,1 ml / min , le rapport : 1/50 ) a été utilisée , les températures de l'injecteur et le détecteur sont de 275 ° C et 250 ° C respectivement . Les échantillons dilués (1/20 dans l'hexane) de 1 ml ont été injectés manuellement. La machine a été dirigée par un type de système informatique « HP ChemStation ». GC: gas chromatographic (GC) analysis is performed on a Hewlett-Packard (HP 6890) gas chromatograph (FID) equipped with a HP-5 capillary column (5% phenyl methyl silicone). The characteristic of this column was: 30 m in length, 0.25 mm in diameter and 0.25 mm in film thickness. The temperature is programmed from 50 ° C (5min initial wait) to 200 ° C at 4 ° C / min. Gas chromatography conditions were as follows: IM2 as a carrier gas (1.8 ml / min); shared mode (flow rate: 72.1 ml / min, ratio: 1/50) was used, the temperatures of the injector and the detector are 275 ° C and 250 ° C respectively. The diluted samples (1/20 in hexane) of 1 ml were injected manually. The machine was run by a type of "HP ChemStation" computer system.
GC / MS : chromatographie en phase gazeuse /spectrométrie de masse : La composition chimique des huiles essentielles ont été analysées en utilisant un chromatographe en phase gazeuse (GC Ultra TRACE ) monté sur un spectromètre de masse (Polaris Q-lon Trap MS). Fonctionnement en mode impact électronique d'assurance-emploi (70 eV). VB- 5 ( méthylpolysiloxane 5% phényle ) et une colonne ( 30m x 0,25 mm x épaisseur de 0,25 pm ) ont été utilisés (Centre national de la recherche scientifique et technique - ( CNRST ) , Rabat , Maroc ) . Les conditions chromatographiques sont les suivantes: températures de l'injecteur et du détecteur à 220 et 300 ° C respectivement; gaz porteur, de l'hélium à un débit de 1,4 ml / min, température de programme rampe de 40 à 300 ° C avec un gradient de 4 ° C / min (maintien de la température initiale et finale pendant 4min). La quantité relative des composants individuels de l'huile totale a été exprimée en pourcentage d'aire du pic par rapport à la zone de pic totale. Une recherche bibliographique a été réalisée en utilisant la combinaison de NIST MS Recherche et de la littérature. Les composants des huiles ont également été identifiés par leurs indices de rétention relatifs à n-alcanes (C8 - C24) GC / MS: Gas Chromatography / Mass Spectrometry: The chemical composition of the essential oils were analyzed using a gas chromatograph (GC Ultra TRACE) mounted on a mass spectrometer (Polaris Q-lon Trap MS). Operation in electronic impact mode of employment insurance (70 eV). VB-5 (methylpolysiloxane 5% phenyl) and a column (30m x 0.25mm x 0.25μm thickness) were used (National Center for Scientific and Technical Research - (CNRST), Rabat, Morocco). The chromatographic conditions are as follows: injector and detector temperatures at 220 and 300 ° C respectively; carrier gas, helium at a flow rate of 1.4 ml / min, ramp program temperature of 40 to 300 ° C with a gradient of 4 ° C / min (maintenance of the initial and final temperature for 4 min). The relative amount of the individual components of the total oil was expressed as percentage of peak area relative to the total peak area. A literature search was conducted using the combination of NIST MS Research and literature. The components of the oils have also been identified by their relative retention indices for n-alkanes (C8-C24)
• Etude de l'activité antibactérienne in vitro de Origanum compactum • Study of the in vitro antibacterial activity of Origanum compactum
Le protocole fait appel dans un 1er temps à la technique de contact direct par diffusion en milieu gélosé (en puits), qui permet de prévoir l'efficacité in vitro de l'huile essentielle. Ensuite, pour le calcul de la CMI (Concentration Minimale Inhibitrice) de L'huile testée
efficace, la méthode de microdilution sur microplaque de 96 puits est utilisée. La CMB (Concentration Minimale Bactéricide) est également mesurée suivant le protocole ci-après. The protocol firstly uses the technique of direct diffusion contact in agar medium (well), which allows to predict the in vitro efficacy of the essential oil. Then, for the calculation of the MIC (Minimum Inhibitory Concentration) of the oil tested effective, the 96-well microdilution method is used. The CMB (Minimal Bactericidal Concentration) is also measured according to the protocol below.
1. Méthode de diffusion en puits 1. Well diffusion method
Ce test est réalisé par dépôt de l'huile essentielle dans des puits creusés dans la gélose suivant une modification de la technique de Dorman et Deans 2000. Elle assure une diffusion totale de l'huile essentielle à partir d'un puits en donnant une zone d'inhibition claire et de diamètre facilement mesurable sur gélose ensemencée par la suspension bactérienne. This test is carried out by deposition of the essential oil in wells dug in the agar according to a modification of the Dorman and Deans 2000 technique. It ensures a total diffusion of the essential oil from a well by giving a zone clear inhibition and easily measurable diameter on agar seeded by the bacterial suspension.
> Préparation de l'inoculum > Preparation of the inoculum
A partir de souches conservées à -20°C (sous formes de billes), une mise en culture est réalisée sur gélose au sang cuit en condition d'anaérobiose sous 5% de C02 à 37°C pendant 24h. Une suspension bactérienne d'une densité de 0.5 Me Farland est ensuite préparée à partir de cette culture pure et jeune (âgée de 24 heures) dans une solution de 0,85% Nacl, en utilisant un étalon. La suspension ajustée devra ainsi contenir approximativement 108 CFU/ml (colony forming units /ml). From strains stored at -20 ° C. (in the form of beads), culturing is carried out on blood-agar plates cooked under anaerobic conditions under 5% CO 2 at 37 ° C. for 24 hours. A bacterial suspension of a density of 0.5 Me Farland is then prepared from this pure and young culture (aged 24 hours) in a solution of 0.85% Nacl, using a standard. The adjusted suspension should thus contain approximately 10 8 CFU / ml (colony forming units / ml).
> Ensemencement des boîtes de pétri : > Seeding petri dishes:
lml de l'inoculum préparé est ensemencé en surface du milieu gélosé par inondation. Après 15 min, des puits sont creusés à l'aide de pipettes Pasteur (l'extrémité épaisse de 6 mm). Il est à signaler que l'inoculum ainsi préparé ne doit pas être utilisé au delà de 30 minutes au risque d'une augmentation de la densité de l'inoculum à cause de la croissance bactérienne. Ensuite, 50 ul de l'huile essentielle est versée dans chaque puits. On teste l'huile essentielle pure et diluée dans du Tween 80 à 1/10 (10%). 1 ml of the prepared inoculum is seeded on the surface of the agar medium by flooding. After 15 min, wells are dug using Pasteur pipettes (the thick end of 6 mm). It should be noted that the inoculum thus prepared should not be used beyond 30 minutes at the risk of an increase in the density of the inoculum because of bacterial growth. Then, 50 μl of the essential oil is poured into each well. The pure essential oil is tested and diluted in 1/10 Tween 80 (10%).
La doxycycline en disque de 30ug est utilisée comme contrôle positif, Tween 80 pur et dilué à 10% sont utilisés comme contrôle négatif. Une boîte de pétri ensemencée par inondation a été prise comme témoin de croissance bactérienne. Tous les tests sont effectués en triplicata. The doxycycline 30ug disc is used as a positive control, Tween 80 pure and diluted to 10% are used as a negative control. A flood-seeded petri dish was taken as a control for bacterial growth. All tests are done in triplicate.
Incubation : Les boîtes de pétri ensemencées sont incubées à l'étuve à 37°C, dans des jarres, sous 5% de C02, pendant 48h. Incubation: The seeded petri dishes are incubated in an oven at 37 ° C. in jars under 5% CO 2 for 48 hours.
2. Méthode de microdilution 2. Microdilution method
La détermination de la concentration minimale inhibitrice (CMI) de l'huile testée sur Y Aggregatibacter actinomycetemeomitans (Aa) est réalisée sur microplaque à 96 puits de culture cellulaire selon une modification de la méthode décrite par Shapiro et al. 1994 et Carson et al. 1995. A partir de colonies de Aa datant de 48h, on inocule 2 tubes contenant 10 ml de BHI (Brain Heart Infusion) stérile. Après 24h d'incubation à 37°C dans une jarre sous 5% de C02, on ajuste la densité à 0.5 Me Farland. Parallèlement, des dilutions en série successives par progression géométrique de raison 2 de la solution mère obtenue pour chaque huile essentielle testée sont préparées dans un milieu de culture stérile (BHI), de façon à obtenir successivement les dilutions : 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512. En prenant en compte la solution mère de base préparée de l'huile essentielle (10%), ces dilutions correspondent, par conséquent, à : 5%, 2,5%, 1,25%, 0,6%, 0,3%, 0,15%, 0,07%, 0,03%, 0,01%. Ensuite, des aliquotes de 100 μΙ de chaque dilution préparée d'huile testée ont été ajoutées, dans chaque puits de microplaque, à 100 μΙ d'inoculum préparé. Le Tween
80 à 10% est utilisé comme contrôle négatif. Un contrôle de croissance (inoculum seul) pour chaque souche est inclus également dans l'essai. Les tests sont effectués en triplicata sur la même microplaque et l'expérimentation est répétée 2 fois. Le schéma de la distribution expérimentale au niveau des microplaques est illustré ci-après (Fig 1). The determination of the minimum inhibitory concentration (MIC) of the oil tested on Y Aggregatibacter actinomycetemeomitans (Aa) is carried out on a 96-well cell culture microplate according to a modification of the method described by Shapiro et al. 1994 and Carson et al. 1995. From Aa colonies dating from 48h, 2 tubes containing 10 ml of sterile Brain Heart Infusion (BHI) were inoculated. After 24h incubation at 37 ° C in a jar under 5% CO 2, the density is adjusted to 0.5 Me Farland. In parallel, successive serial dilutions by geometric progression of reason 2 of the stock solution obtained for each essential oil tested are prepared in a sterile culture medium (BHI), so as to successively obtain the dilutions: 1/2, 1/4 , 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512. Taking into account the prepared basic stock solution of the essential oil (10%), these dilutions correspond, therefore, to: 5%, 2.5%, 1.25%, 0.6%, 0.3% %, 0.15%, 0.07%, 0.03%, 0.01%. Then, 100 μl aliquots of each prepared dilution of oil tested were added to each microplate well to 100 μl of prepared inoculum. Tween 80 to 10% is used as a negative control. Growth control (inoculum alone) for each strain is also included in the test. The tests are carried out in triplicate on the same microplate and the experiment is repeated twice. The diagram of the experimental distribution at the level of the microplates is illustrated below (FIG. 1).
Concernant le contrôle positif : nous avons utilisé l'Amoxicilline que nous avons préparé et dont nous avons déterminé la CMI sur Aa en utilisant la méthode de microdilution dans un bouillon de culture adapté. En l'absence de méthode standardisée d'antibiogramme de Clinical and Laboratory Standards Institute (CLSI) pour ce type de souche, nous avons adopté la procédure suivante en s'inspirant de celle des « entérobactéries » dans les recommandations de CLSI (2006): Regarding the positive control: we used the Amoxicillin that we prepared and whose MIC was determined on Aa using the microdilution method in a suitable culture broth. In the absence of a standardized method of Clinical and Laboratory Standards Institute (CLSI) antibiogram for this strain type, we have adopted the following procedure based on that of "enterobacteria" in the recommendations of CLSI (2006):
Une suspension bactérienne en bouillon BHI (Brain Heart Infusion) équivalente au standard McFarland 0,5 est préparée et ajoutée aux solutions antibiotiques à différentes concentrations (0,01mg/ml, 0,lmg/ml, lmg/ml, 10mg/ml). Un contrôle négatif (eau stérile + inoculum) est utilisé comme témoin de croissance bactérienne et une solution contenant du bouillon stérile est utilisée comme contrôle de contamination. Ainsi, la concentration de 10 mg/ml, dont la solution est restée claire (non trouble) est considérée comme la CMI de l'Arnoxicilline sur Aa et qui sera utilisée comme contrôle positif dans notre essai. A bacterial suspension in BHI (Brain Heart Infusion) broth equivalent to the McFarland 0.5 standard is prepared and added to the antibiotic solutions at different concentrations (0.01 mg / ml, 0.1 mg / ml, 1 mg / ml, 10 mg / ml). A negative control (sterile water + inoculum) is used as a bacterial growth control and a solution containing sterile broth is used as a contamination control. Thus, the concentration of 10 mg / ml, the solution of which remained clear (not turbid) is considered the MIC of Arnoxicillin on Aa and which will be used as a positive control in our test.
Les microplaques, recouvertes de leurs couvercles, ont été mises à l'étuve pour incubation dans des sachets en plastique sous 5% de C02 à 37°C pendant 48h. The microplates, covered with their lids, were incubated in plastic bags at 5% CO 2 at 37 ° C. for 48 hours.
Après la période d'incubation, 40 ul d'une solution à 2 mg / mL de Triphényl Tétrazolium Chloride (TTC) (indicateur de croissance bactérienne) (Shafiei et al. 2012) est ajouté dans chaque puits et la plaque est incubée à 37 ° C pendant 1 heure à 2 heures environ. La solution d'indicateur TTC change du clair au pourpre en présence d'activité bactérienne, tandis qu'elle reste claire lorsque la croissance microbienne est inhibée. CMI (Concentration Minimale Inhibitrice) est définie comme la plus faible concentration de l'huile essentielle qui ne montre aucune croissance bactérienne visible après la période d'incubation (pas de changement de couleur (claire) de TTC). Pour déterminer la Concentration Minimale Bactéricide (CMB), une aliquote de 10 ul est prélevée à partir de cultures au niveau des puits ne présentant pas de turbidité visible, et ensemencée sur des milieux de gélose de sang cuit et incubée pendant 48h à 37 ° C sous 5% de C02 (Hammer et al. 2003). La détermination des valeurs de CMB a été réalisée en triplicata. Le rapport CMB / CMI a également été calculé pour mettre en évidence la nature de l'effet antibactérien de l'huiles essentielle testée. Lorsque le rapport est inférieur à 4, l'huile essentielle est considérée comme une huile essentielle bactéricide et lorsque le ratio est supérieur à 4, elle est considérée comme une huile essentielle bactériostatique (Levison et al. 2004).
* Analyse statistique After the incubation period, 40 μl of a 2 mg / mL solution of Triphenyl Tetrazolium Chloride (TTC) (bacterial growth indicator) (Shafiei et al 2012) is added to each well and the plate is incubated at 37 μg. ° C for 1 hour to 2 hours. The TTC indicator solution changes from clear to purple in the presence of bacterial activity, while remaining clear when microbial growth is inhibited. MIC (Minimum Inhibitory Concentration) is defined as the lowest concentration of the essential oil that shows no visible bacterial growth after the incubation period (no color change (clear) TTC). To determine Minimal Bactericidal Concentration (CMB), a 10 μl aliquot is taken from cultures at wells with no visible turbidity, and seeded on cooked blood agar media and incubated for 48 hours at 37 ° C. under 5% CO2 (Hammer et al., 2003). The determination of the CMB values was done in triplicate. The CMB / MIC ratio was also calculated to highlight the nature of the antibacterial effect of the essential oil tested. When the ratio is less than 4, the essential oil is considered a bactericidal essential oil and when the ratio is greater than 4, it is considered a bacteriostatic essential oil (Levison et al., 2004). * Statistical analysis
Les diamètres des zones d'inhibition, la CMl et CMB, variables continues avec distribution normale, ont été présentés en moyenne ± écart type. Pour les différences statistiques entre Origanum compactum, Doxycycline et Tween 80, l'analyse à un facteur (ANOVA) avec correction Bonferroni a été effectuée. La valeur du P< 0.05 a été considérée comme statistiquement significative. L'analyse statistique a été menée en utilisant SPSS pour Windows (SPSS, Inc., Chicago, IL, USA). The diameters of the inhibition zones, CM1 and CMB, continuous variables with normal distribution, were presented on average ± standard deviation. For statistical differences between Origanum compactum, Doxycycline and Tween 80, the one-factor analysis (ANOVA) with Bonferroni correction was performed. The value of P <0.05 was considered statistically significant. The statistical analysis was conducted using SPSS for Windows (SPSS, Inc., Chicago, IL, USA).
RESULTATS RESULTS
1. Composition chimique : 1. Chemical composition:
L'analyse chimique de l'huile essentielle étudiée a mis en évidence les constituants majeurs suivants -. y-terpinene (25.1 1%), Carvacrol (22.29%), Thymol (19.21 %), p-cymene (18.68 %) (Tableau 1). Chemical analysis of the essential oil studied revealed the following major constituents -. y-terpinene (25.1%), Carvacrol (22.29%), Thymol (19.21%), p-cymene (18.68%) (Table 1).
2. Activité antibactérienne : 2. Antibacterial activity:
> Essai de diffusion en puits : > Well diffusion test:
Après la période d'incubation (48h), des zones d'inhibition de croissance bactérienne sur gélose sont obtenues et mesurées par un pied à coulisse (Tableau 2). Cependant, aucun halo d'inhibition n'est obtenu pour les contrôles négatifs (Tween 80 pur et dilué à 10%). Concernant, le témoin de croissance utilisé, une croissance bactérienne sous forme d'un film recouvrant la gélose est obtenue. Le diamètre moyen des zones d'inhibition induits par la Doxycycline (22,67 ± 1,15 mm) est significativement plus petit que celui produit par l'huile essentielle testée à l'état pur (51,67 ± 5,77 mm). Les diamètres des zones d'inhibition observés pour l'huile essentielle de Origanum compactum à l'état brut, ont montré des valeurs supérieures à 20 mm (Tableau 2), traduisant une forte sensibilité de la souche Aggregatibacter actinomycetemcomitans clone JP2 à l'huile étudiée. After the incubation period (48h), zones of bacterial growth inhibition on agar are obtained and measured by a vernier caliper (Table 2). However, no halo inhibition is obtained for the negative controls (Tween 80 pure and diluted to 10%). Regarding the growth control used, a bacterial growth in the form of a film covering the agar is obtained. The average diameter of the inhibition zones induced by Doxycycline (22.67 ± 1.15 mm) is significantly smaller than that produced by the essential oil tested in the pure state (51.67 ± 5.77 mm) . The diameters of the zones of inhibition observed for the crude Origanum compactum essential oil, showed values higher than 20 mm (Table 2), expressing a high sensitivity of the strain Aggregatibacter actinomycetemcomitans clone JP2 to the oil studied.
> Essai de microdilution : > Microdilution test:
Les valeurs enregistrées de CMI, CMB et rapport CMB/CMI sont représentés dans le Tableau 3. La CMI (0,03 ±0,01 %) et la CMB (0,07±0 %) obtenues témoignent d'une activité antibactérienne effective de l'huile essentielle de Origanum compactum. Le rapport CMB/CMI obtenu est inférieur à 4, ce qui renseigne sur la nature de l'effet antibactérien de l'huile essentielle testée qui s'avère ainsi bactéricide. The recorded MIC, CMB and CMB / MIC values are shown in Table 3. MIC (0.03 ± 0.01%) and CMB (0.07 ± 0%) indicate effective antibacterial activity. Origanum compactum essential oil. The CMB / MIC ratio obtained is less than 4, which gives information on the nature of the antibacterial effect of the essential oil tested which thus proves to be bactericidal.
Liste des tableaux : List of paintings :
Fig 1: Distribution expérimentale de la microplaque pour l'huile testée pour la détermination de la CMl : Cl Fig 1: Experimental distribution of the microplate for the oil tested for the determination of CMl: Cl
— *-C : différentes concentrations de l'huile testée + inoculum, Ce : contrôle de croissance (inoculum seul), C- : contrôle négatif (tween 80 à 10% + inoculum), C+ : contrôle positif (Amoxicilline + inoculum) - * -C: different concentrations of the tested oil + inoculum, Ce: growth control (inoculum alone), C-: negative control (tween 80 at 10% + inoculum), C +: positive control (Amoxicillin + inoculum)
Tableau 1 : Composition chimique de Origanum compactum huile essentielle Table 1: Chemical composition of Origanum compactum essential oil
Tableau 2 : Diamètres d'inhibition (mm) obtenues par la méthode de diffusion en puits Table 2: Inhibition Diameters (mm) Obtained by the Well Diffusion Method
Tableau 3: Concentration Minimale Inhibitrice (CMl) et Concentration Minimale Bactéricide (CMB) Table 3: Minimal Inhibitory Concentration (CM1) and Minimal Bactericidal Concentration (CMB)
(%) (v/v) de l'huile essentielle testée sur la souche A. actinomycetemcomitans clone JP2
(%) (v / v) of the essential oil tested on the strain A. actinomycetemcomitans clone JP2
Claims
Revendications claims
1- Composition pharmacologique de l'huile essentielle d'Origan pour le traitement des infections à Aggregatibacter actinomycetemcomitans clone JP2 Caractérisée en ce qu'elle contient: 1- Pharmacological composition of the Oregano essential oil for the treatment of Aggregatibacter actinomycetemcomitans infections clone JP2 Characterized in that it contains:
γ-terpinene (25.1 1%), Carvacrol (22.29%), Thymol (19.21 %), p-cymene (18.68 %), a-thujene 0.22, a-pinene 0.54, Camphene 0.17, Sabinene 0.23, Sabinene 0.83, β- pinene 0.16, 2-octanol 0.86, Myrcene 2.21 , δ-2-carene 0.09, a-Phellandrene 0.26, δ-3- carene 0.08, a-terpinene 2.79, O-cymene 0.48, Camphor 0.05, β-Ε-ocimene 0.09, Cis- hydrate sabinene 0.15, m-cymenene 0.14, Terpinolene 0.07, linalool 1.24, borneol 0.20, terpinen-4-ol 0.34, p -cymen-8-ol 0.09, Ct-terpineol 0.99, E-caryophyllene 1.03, γ-cadinene 0.05, Caryophyllene oxide 0.06. γ-terpinene (25.1%), Carvacrol (22.29%), Thymol (19.21%), p-cymene (18.68%), α-thujene 0.22, α-pinene 0.54, Camphene 0.17, Sabinene 0.23, Sabinene 0.83, β- pinene 0.16, 2-octanol 0.86, Myrcene 2.21, δ-2-carene 0.09, α-Phellandrene 0.26, δ-3 carene 0.08, α-terpinene 2.79, O-cymene 0.48, Camphor 0.05, β-Ε-ocimene 0.09, Cis-hydrate sabinene 0.15, m-cymenene 0.14, Terpinolene 0.07, linalool 1.24, terminalol 0.20, terpinen-4-ol 0.34, p -cymen-8-ol 0.09, Ct-terpineol 0.99, E-caryophyllene 1.03, γ-cadinene 0.05 , Caryophyllene oxide 0.06.
2- Composition pharmacologique de l'extrait d'Origan selon la revendication 1 caractérisée en ce que l'effet antibactérien se produit avec l'utilisation de l'extrait de l'huile essentielle de l'Origan entre 0,02 à 0,1 %; 2- Pharmacological composition of Oregano extract according to claim 1 characterized in that the antibacterial effect occurs with the use of Oregano essential oil extract between 0.02 to 0.1. %;
3- Composition pharmacologique de l'huile essentielle d'Origan selon la revendication 1 et 2 caractérisée en ce que l'effet est bactéricide sur espèce Aggregatibacter actinomycetemcomitans clone JP2 ; 3- Pharmacological composition of the Oregano essential oil according to claim 1 and 2, characterized in that the effect is bactericidal on Aggregatibacter actinomycetemcomitans species clone JP2;
4- Composition pharmacologique de l'extrait d'Origan selon la revendication 1,2 et 3 caractérisée en ce que l'effet antibactérien s'observe avec l'application locale de la composition sur les muqueuses ou sous les formes galéniques liquide et solide.
4- pharmacological composition of Oregano extract according to claim 1,2 and 3 characterized in that the antibacterial effect is observed with the local application of the composition on the mucous membranes or in the liquid and solid dosage forms.
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|---|---|---|---|---|
| US20060140884A1 (en) * | 2004-12-28 | 2006-06-29 | Colgate-Palmolive Company | Oregano oral care compositions and methods of use thereof |
| US20120183588A1 (en) * | 2011-01-13 | 2012-07-19 | Austin Research Labs Corp. | High load dispersions |
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| US20060140884A1 (en) * | 2004-12-28 | 2006-06-29 | Colgate-Palmolive Company | Oregano oral care compositions and methods of use thereof |
| US20120183588A1 (en) * | 2011-01-13 | 2012-07-19 | Austin Research Labs Corp. | High load dispersions |
Non-Patent Citations (8)
| Title |
|---|
| "OregaDENT -1 fl oz -North American Herb & Spice", 15 July 2014 (2014-07-15), XP055179740, Retrieved from the Internet <URL:https://web.archive.org/web/20140715042340/http://www.northamericanherbandspice.com/product/oregadent-1-fl-oz/> [retrieved on 20150327] * |
| "Oregano Oil & Periodontal Disease | LIVESTRONG", 16 August 2013 (2013-08-16), XP055179744, Retrieved from the Internet <URL:http://www.livestrong.com/article/521840-oregano-oil-periodontal-disease/> [retrieved on 20150327] * |
| "Oreganol P73 oil", 9 November 2013 (2013-11-09), XP055179739, Retrieved from the Internet <URL:https://web.archive.org/web/20131109050942/http://www.northamericanherbandspice.com/product/oreganol-p73-oil-1-fl-oz/> [retrieved on 20150327] * |
| ADEPAM, IAV HASSAN II, B.P. 6202, RABAT-INSTITUTS (MAROC): "Fiche technique de l'huile essentielle d'Origanum compactum Benth(Origan) du Maroc", 23 September 2007 (2007-09-23), XP002738117, Retrieved from the Internet <URL:http://corpolaseve.uqac.ca/manuel/chapitres/Annex1.pdf> [retrieved on 20150326] * |
| DORTE HAUBEK ET AL: "Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis", JOURNAL OF ORAL MICROBIOLOGY, vol. 108, no. 0, 14 August 2014 (2014-08-14), pages 136, XP055177870, DOI: 10.3402/jom.v6.23980 * |
| L LAKHDAR1 ET AL: "Antibacterial activity of essential oils against periodontal pathogens : a qualitative systematic review", 1 December 2012 (2012-12-01), XP055178063, Retrieved from the Internet <URL:http://www.researchgate.net/publication/236065647_Antibacterial_activity_of_essential_oils_against_periodontal_pathogens_a_qualitative_systematic_review> [retrieved on 20150320] * |
| LEILA LAKHDAR ET AL: "THESE N° : 28/14 CSVS THESE DE DOCTORAT EVALUATION DE L'ACTIVITE ANTIBACTERIENNE D'HUILES ESSENTIELLES MAROCAINES SUR AGGREGATIBACTER ACTINOMYCETEMCOMITANS : ETUDE IN VITRO Equipe de Recherche en Ecosystème buccal Présentée et soutenue par", 24 February 2015 (2015-02-24), XP055177857, Retrieved from the Internet <URL:http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCIQFjAA&url=http%3A%2F%2Fao.um5s.ac.ma%2Fjspui%2Fbitstream%2F123456789%2F14825%2F1%2FTHESE.pdf&ei=wCAMVfrsEsy8PbmjgdAB&usg=AFQjCNEQ7c8GNdc2GD57d0gdP1A47Mc2vA&sig2=pLPBGhd3wGOvR7ArVqddAg&bvm=bv.88528373,d.ZWU> [retrieved on 20150319] * |
| S BOUHDID ET AL: "Antibacterial and antioxidant activities of Origanum compactum essential oil", AFRICAN JOURNAL OF BIOTECHNOLOGY, 16 May 2008 (2008-05-16), pages 1563 - 1570, XP055007547, Retrieved from the Internet <URL:http://www.academicjournals.org/AJB/PDF/pdf2008/16May/Bouhdid et al.pdf> [retrieved on 20110920] * |
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