Artikel Jurnal
Artikel Jurnal
Artikel Jurnal
Marwa CHRAIBI1, Abdellah FARAH2, Mounyr BALOUIRI1, Hassan BARKAI1, Moulay SADIKI1,
Kawtar FIKRI BENBRAHIM1*
1
Laboratory of Microbial Biotechnology, Faculty of Science and Technology Saïss.Sidi Mohamed Ben Abdellah University, P.O.Box 2202. Fez, Morocco.
2
Laboratory ofApplied Organic Chemistry, Faculty of Science and Technology Saïss.SidiMohamed Ben Abdellah University, P.O.Box 2202. Fez, Morocco.
Article history: In the aim of valorizing aromatic and medicinal plants from Morocco, this work focuses on the chemical
Received on: 03/10/2016 characterization and antimicrobial activity of Pelargonium asperum essential oil against 11 microbial strains
Revised on: 17/10/2016 causing problems in the medical and food domain. The chemical profile of the volatile oil was investigated by
Accepted on: 05/11/2016 GC/MS. The major compounds were citronellol (26.98%), geraniol (14.12%), isomenthone (8.80%), linalool
Available online: 28/12/2016 (4.97%), citronellylformate (3.1%), followed by geranylformate (4.07%) and guai-6,9-diene (4.24%). The
results of antimicrobial activity by using the broth microdilution method indicated that essential oil of
Key words: Pelargonium asperum exhibited significant antimicrobial activity against all tested microorganisms with the
Pelargonium asperum; strongest inhibitory effect against yeasts. The MICs values ranged from 0.003% to 0.25% (v/v) for all strains,
essential oil; antimicrobial except Pseudomonas aeruginosa, which was least susceptible and inhibited by 2% (v/v). These results suggest
activity; bacteria; fungi. that Pelargonium asperum oil could be used for the development of new antimicrobial agents.
© 2016 Marwa Chraibi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
Chraibi et al. / Journal of Applied Pharmaceutical Science 6 (12); 2016: 042-046 043
Indeed the use of antimicrobial properties of essential μL of bacterial inoculum, previously prepared and adjusted to 0.5
oils does not start today (Beylier-Maurel, 1976), while nowadays McFarland, were added to each well to reach the final
the need to their applications became urgent. Therefore, the concentration of 106CFU/mL. After incubation at 37°C for 24 h,
present study aims to determine the chemical composition of 10 μL of resazurin were added to each well as a bacterial growth
Pelargonium asperum essential oil and to investigate its indicator.
antimicrobial activity by micro-dilution methods against 11 After further incubation at 37°C for 2 h, the bacterial
selected spoiling and pathogenic microorganisms, in an attempt to growth was revealed by the change of coloration from purple to
contribute to the use of these as alternative products for microbial pink. Experiments were carried out in triplicate.
control and food preservation.
Determination of Minimum inhibitory concentration against
MATERIAL AND METHODS fungal strains
To investigate the antifungal activity of the studied
Plant material
essential oil against Aspergillus niger and Penicillium expansum a
Fresh aerial part of Pelargonium asperum was harvested
modified microdilution technique described by (Daouk et al.,
from the garden of National Institute of the Medicinal and
1995) was used. Firstly, 50 μl of malt extract broth were added
Aromatic Plants (NIMAP). The botanical identification was
from the second to the 12thwell. The essential oil was diluted in
performed, and then the voucher specimen was deposited at the
Tween 20 1% (v/v) at a final concentration of 40% (v/v), then 100
Herbarium of NIMAP (Morocco).
µL of this solution were deposed in the first well. Afterwards,
scalar dilution was made by transferring 50 µL from the 1 st to the
Essential oil extraction
11th well. The 12th well was considered as growth
The fresh aerial part of P. asperum (leaves and stems)
control. Thereafter 50 μL of the fungal spore suspension was
was hydrodistilled for 3 h using a Clevenger-type apparatus. The
added to each well to reach a final concentration of 10 6 spores/mL.
essential oil was then kept in dark at 4°C until further use.
The microplate was sealed and incubated for 72 h at 30 °C. The
lowest essential oil concentration that prevents visible fungal
Target strains
growth was defined as the MIC. Likewise, the MIC determination
Tested bacteria include seven isolates of Escherichia coli
against Candida albicans and Candida tropicalis, was performed
ATCC 25922, Pseudomonas aeruginosa ATCC27853,
in 96 well-microplate according to the protocol previously
Micrococcus luteus ATCC 14452, Staphylococcus aureus ATCC
described (Balouiri et al., 2016) with slight modifications. The
29213, Bacillus subtilis ATCC 6633, Salmonella enterica (serovar
essential oil was also serially diluted in YPG broth supplemented
typhimurium) and Bacillus cereus. Before use, strains were
with agar at 0.15% (w/v). The 12th well was also considered as
revivified by subcultures in Luria-Bertani (LB) plates at 37°C for
growth control. Then, 50 μL of fungal inoculum were added to
24 h. As regards the tested fungi, they include Candida albicans,
each well at a final concentration of 103 CFU/mL. Finally, the
Candida tropicalis, Aspergillus niger and Penicillium expansum.
microplate was sealed and incubated at 30°C for 48 h.
Revivification of molds was made by subcultures in malt extract-
Experiments were carried out in triplicate. Similarly, the lowest
agar plates (malt extract 30 g/L and agar 20 g/L) at 25°C for 7
essential oil concentration that prevents visible fungal growth was
days. After incubation, their spores were harvested by scraping the
defined as the MIC.
culture surface in sterile Tween 20 (1%) solution. Then the spore
suspension was concentrated by centrifugation at 10000 g for 15
RESULTS AND DISCUSSION
min at 4°C until a concentration of 106 spores/mL (counted with an
hemocytometer).While, yeast strains were inoculated in yeast- The studied essential oil was previously subjected to a
peptone-glucose agar (YPG) (yeast extract 10 g/L, peptone 20 g/L, gas chromatography-mass spectrometry analysis. This analysis
glucose 20 g/L and agar 20 g/L) and incubated at 30°C for 48 h. revealed 61 different compounds accounting for 99.96% of the
whole Pelargonium asperum essential oil, where the major
Determination of minimum inhibitory concentration against constituents were citronellol (26.98%), geraniol (14.12%),
bacteria isomenthone (8.80%), linalool (4.97%), geranylformiate (4.07%),
The minimum inhibitory concentration was determined guaï-6,9-diene (4.24%) and citronellyl formiate (3.1%). Likewise,
in 96 well-microplate using the microdilution assay according to several previous studies have found citronellol as the principal
the protocol previously described by (Balouiri et al., 2016) with major component of this essential oil (Boukhatem et al., 2013;
slight modifications. Bacteriological agar at 0.15 % (w/v) was Boukhris et al., 2013; Bouzenna and Krichen, 2013; Gomes et al.,
used as an emulsifier of the essential oil in the culture medium. 2007; Jalali-Heravi et al., 2006).
For bacteria, the essential oil was serially diluted in Muller Hinton
broth supplemented with agar to obtain the final concentrations Antibacterial effect of Pelargonium asperum essential oil
ranging between 8% and 0.007% (v/v). The 12th well was This study focused on the Pelargonium asperum
considered as growth control (free-essential oil control). Then, 50 essential oil bioactivity. Results found of its antimicrobial activity
044 Chraibi et al. / Journal of Applied Pharmaceutical Science 6 (12); 2016: 042-046
evaluated against 11 microbial strains are compiled in Tables (Tab. part responsible for the intrinsic resistance of Gram-negative
1, 2 and 3). compared to the Gram-positive bacteria to the essential oils
As regards to the antibacterial effect, it can be seen in the constituents (Gachkar et al., 2007; Trombetta et al., 2005).
Tab. 1 that all tested bacterial strains were susceptible to the Furthermore, concerning the antifungal activity against molds and
studied essential oil. The antibacterial effect unveiled seems to be yeasts (Tab. 3 and 4), the screening test revealed that all tested
strain- dependent. In fact, strong inhibitory effect has been shown fungal strains were susceptible to the Pelargonium asperum oil.
against all Gram-positive bacteria, especially Bacillus cereus and Indeed, the strongest inhibitory effect was exerted against yeasts
Micrococcus luteus, since they were inhibited by very low (both Candida species) with MIC values of 0.003% and 0.007%
concentrations 0.007% and 0.015% (v/v) respectively. In addition, (v/v) against Candida albicans and Candida tropicalis
the concentration of 0.031% (v/v) was sufficient to inhibit the respectively. These results are in agreement with those of previous
growth of S. aureus and B. subtilis. Against Gram-negative works (Boukhatem et al., 2013; Hassane et al., 2011). As regards
bacteria the tested essential oil was more active against E. coli and to the molds they were inhibited with MIC values of 0.312% and
S. enterica serovar typhimurium with a minimum inhibitory 0.15% (v/v) against Penicillium expansum and Aspergillus niger
concentration of 0.125% (v/v). In contrast, P. aeruginosa was the respectively. The broad spectrum and the significant antimicrobial
most resistant strain with MIC value of 2% (v/v). Previous studies activity of the tested essential oil may be attributed to its richness
confirmed that this bacterial strain was the most resistant in terpenic alcohols (citronellol, geraniol, linalool), which
(Boukhatem et al., 2013; Ghannadi et al., 2012; Haloui et al., represent 46.07% of its total composition. In fact, these molecules
2015). Usually, the Gram-positive strains were more susceptible to are well-known for their greater efficiency as antimicrobials
the essential oils than the Gram-negative bacteria. This difference (Hammer et al., 2003; Inouye et al., 2001; Satrani et al., 2006). In
is closely related to their cell wall compositions, since the addition, among the identified compounds in this oil, some
antibacterial activity of the essential oils has been explained by molecules were previously reported to exhibit antimicrobial
molecular interactions of the functional groups of their activity such as limonene (Mazzanti et al., 1998), geraniol (Araújo
components and the bacterial wall, which inflict several damages et al., 2003), carvacrol and citronellol (Sacchetti et al., 2005).
to the cell (Calo et al., 2015). In addition, this antimicrobial outcome could also be
In fact, the outer membrane of Gram-negative bacteria is attributed to the synergistic interaction between the various
characterized by the presence of lipopolysaccharides (75%), which components of this oil. In fact, it has been reported in previous
have hydrophilic character that makes the outer membrane of these studies that the inhibitory activity of an essential oil results from a
bacteria invulnerable to the most hydrophobic molecules (i.e. complex interaction between its different constituents (Burt, 2004;
Hydrocarbons terpenes). Thus, this structural particularity is in Viuda-Martos et al., 2008; Xianfei et al., 2007).
Table 3: Antifungal activity of Pelargonium asperum essential oil against Candida albicans and Candida tropicalis.
Concentrations % (v/v)
Strains 4% 2% 1% 0.5% 0.25% 0.125% 0.062% 0.031% 0.015% 0.007% 0.003%
Control
(v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v)
C. albicans - - - - - - - - - - - +
C. tropicalis - - - - - - - - - - + +
C. albicans: Candida albicans and C. tropicalis: Candida tropicalis.
Table 4: Antifungal activity of Pelargonium asperum essential oil against Aspergillus niger and Penicillium expansum.
Concentrations v/v
Strains 20% 10% 5% 2.5% 1.2% 0.625% 0.312% 0.15% 0.075% 0.037% 0.018%
control
(v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v) (v/v)
A. niger - - - - - - - - + + + +
P. expansum - - - - - - - + + + + +
A. niger : aspergillus niger ; P. expansum : Penicillium expansum.
Ghannadi A, Bagherinejad MR, Abedi D, Jalali M, Absalan B, Sadiki M, Balouiri M, Barkai H, Maataoui H, Ibnsoud S, Elabed
Sadeghi N. Antibacterial activity and composition of essential oils from S. Synergistic antibacterial effect of Myrtus communis and Thymus
Pelargonium graveolens L’Her and Vitex agnus-castus L. Iran J vulgaris essential oils fractional inhibitory concentration index. Int J
Microbiol, 2012; 4:171–176. Pharm Pharm Sci, 2014; 6:121–124.
Gomes PB, Mata VG, Rodrigues AE. Production of rose Salah-Fatnassi KBH, Slim-Bannour A, Harzallah-Skhiri F,
geranium oil using supercritical fluid extraction. J Supercrit Fluids, 2007; Mahjoub MA, Mighri Z, Chaumont JP, Aouni M. Activités antivirale et
41:50–60. antioxydante in vitro d’huiles essentielles de Thymus capitatus (L.)
Guiraud JP. 2003. Microbiologie alimentaire. Paris, France: Hoffmans. & Link de Tunisie. Acta Bot Gall, 2010; 157:433–444.
Dunod: RIA, DL. Satrani B, Farah A, Talbi M. Effet de la distillation fractionnée
Haloui T, Farah A, Balouiri M, Chraibi M, Fadil M, Benbrahim sur la composition chimique et l’activité antimicrobienne des huiles
K, Alaoui A. Bacteriostatic and Bactericidal Profile of Leaves and Twigs essentielles du Myrte (Myrtus communis L.) du Maroc. Acta Bot Gall,
Essential oils of Moroccan Pistacia lentiscus L. J Appl Pharm Sci, 2015; 2006; 153:235–242.
5:50–53. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M,
Hammer KA, Carson CF, Riley T V. Antifungal activity of the Daniele C, Saija A, Mazzanti G, Bisignano G, Grazia M. Mechanisms of
components of Melaleuca alternifolia (tea tree) oil. J Appl Microbiol, Antibacterial Action of Three Monoterpenes Mechanisms of Antibacterial
2003; 95:853–860. Action of Three Monoterpenes. J Antimicrob Agents Chemother, 2005;
Hassane SOS, Ghanmi M, Satrani B, Mansouri N, Mohamed H, 49:2474–2478.
El Hajaji H, Chaouch A. Composition chimique et activités Viuda-Martos M, Ruiz-Navajas Y, Fernández-López J, Pérez-
antibactériennes, antifongiques et antioxydante de l’huile essentielle de Álvarez JA. Antibacterial activity of different essential oils obtained from
Pelargonium asperum Ehrh. ex Wilde des Comores. Acta Bot Gall, 2011; spices widely used in Mediterranean diet. Int J Food Sci Technol, 2008;
158:225–237. 43:526–531.
Inouye S, Takizawa T, Yamaguchi H. JAC Antibacterial Xianfei X, Xiaoqiang C, Shunying Z, Guolin Z. Chemical
activity of essential oils and their major constituents. J Antimicrob composition and antimicrobial activity of essential oils of Chaenomeles
Chemother, 2001; 47:565–573. speciosa from China. Food Chem, 2007; 100:1312–1315.
Jalali-Heravi M, Zekavat B, Sereshti H. Characterization of
essential oil components of Iranian geranium oil using gas
chromatography-mass spectrometry combined with chemometric
resolution techniques. J Chromatogr A, 2006 ;1114:154–163.
Mazzanti G, Battinelli L, Salvatore G. Antimicrobial properties
of the linalol-rich essential oil of Hyssopus officinalis L . var decumbens
( Lamiaceae ). Flavour Frag J, 1998; 13:289–294.
Nsambu M, Muhigwa B, Rubabura K, Bagalwa M, Bashwira S.
In vitro evaluation of the insecticidal activity of alkaloids, saponins,
terpenoids and steroids extracts Capscicum frutescens L. (Solanaceae) How to cite this article:
against Antestiopsis orbitalis ghesquierei, pest insects of cafeirs. Int J
Innov Appl Stud, 2014; 8:1231–1243.
Chraibi M, Farah A, Balouiri M, Barkai H, Sadiki M, Benbrahim
Sacchetti G, Maietti S, Muzzoli M, Scaglianti M, Manfredini S, KF. Chemical characterization and antimicrobial activity of
Radice M, Bruni R, Comparative evaluation of 11 essential oils of Moroccan Pelargonium asperum essential oil. J App Pharm Sci,
different origin as functional antioxidants, antiradicals and antimicrobials 2016; 6 (12): 042-046.
in foods. Food Chem, 2005; 91:621–632.