Journal of Ethnopharmacology 104 (2006) 104 107
Antimicrobial activities of the leaf extracts of two Moroccan
Cistus L. species
a,d," b b c d
H. Bouamama , T. Noël , J. Villard , A. Benharref , M. Jana
a
Laboratoire des Sciences Végétales, Département de Biologie, Université Cadi Ayyad, Faculté des Sciences et Techniques,
BP 549, Marrakech 40000, Morocco
b
Laboratoire des Sciences Végétales, Université Renée Descartes Paris V, Faculté de Pharmacie, 4 Avenue de l observatoire,
75270 Paris Cedex 06, France
c
Laboratoire de Chimie des Substances Naturelle, Département de Chimie, Université Cadi Ayyad, Faculté des
Sciences-Semlalia, BP S 15, Marrakech 40000, Morocco
d
Laboratoire des Plantes Médicinales, Département de Biologie, Université Cadi Ayyad, Faculté des Sciences-Semlalia,
BP S 15, Marrakech 40000, Morocco
Received 21 January 2004; received in revised form 12 August 2005; accepted 26 August 2005
Available online 6 October 2005
Abstract
We used the standard M27-T technique to study organic and aqueous leaf extracts of two Moroccan Cistus L. species: Cistus villosus L. and Cistus
monspeliensis L. (Cistaceae L.) used in traditional medicine, for their antimicrobial properties against microorganisms, Staphylococcus aureus,
Pseudomonas aeruginosa, Candida albicans, Candida krusei, Candida glabrata and Aspergillus fumigatus. The broth dilution method M27-T,
standardized by the National Committee for Clinical Laboratory Standards (NCCLS) allowed to determine the minimum inhibitory concentrations
(MICs) of different extracts. Results showed that the different extracts differed clearly in their antimicrobial activities. Cistus villosus extracts
exhibited more interesting activity than Cistus monspeliensis extracts when used on Staphylococcus aureus (MIC = 0.78 mg/ml) and Candida
glabrata (MIC = 0.19 mg/ml), which are the most susceptible microorganisms. On the other hand, Candida krusei and Aspergillus fumigatus
were the least susceptible microorganisms to all Cistus extracts. Comparison results were carried out using chloramphenicol, amoxicillin and
amphotericin B as standard antibiotics.
© 2005 Published by Elsevier Ireland Ltd.
Keywords: Cistus villosus L.; Cistus monspeliensis L.; Antimicrobial activity
1. Introduction All the Cistus species are frequently used in many traditional
medicines for their antimicrobial (Chinou et al., 1994; Demet-
The genus Cistus L. rock rose, holly rose (Hocking, 1997) zos et al., 1999), antitumor (Polunin, 1969; Dimas et al., 2000),
(Cistaceae L.) comprises several medicinal plants of perennial antiviral (Dimas et al., 2000) and anti-inflammatory (Yesilada et
shrubs. These shrubs grow wild in the Moroccan and Mediter- al., 1997; Singh et al., 1998; Demetzos et al., 2001) properties.
ranean edges with 16 Cistus species (Heywood et al., 1968). Previous pharmacological investigations showed that Cistus
The leaves of all species secrete essential oils and resins. The leaf extracts have antimicrobial properties (Chinou et al.,
resin [common names: ladanum, labdanum or ladano (Anonis, 1994; Bouamama et al., 1999) against many bacteria and fungi
1995; Demetzos et al., 1999)] is composed mainly of flavonoids responsible of human infections (Koch, 1981; Russell, 1991).
(as aglycones and glycosides), pro-anthocyanidins, terpenoids These previous results encouraged us to deepen the studies
and tannins (Demetzos et al., 1989; Vogt and Gülz, 1991; on antimicrobial properties of the Cistus genus by evaluating
Danne et al., 1993; Demetzos et al., 1999). the minimal concentration of inhibition (MIC) of Cistus extracts
using the standard M27-T broth microdilution technique of
the National Committee for Clinical Laboratory Standards
" (NCCLS) (Barchiesi et al., 1994; Espinel-Ingroff et al., 1995;
Corresponding author. Tel.: [phone]; fax: [phone].
NCCLS, 1995).
E-mail address: [e-mail] (H. Bouamama).
0378-8741/$  see front matter © 2005 Published by Elsevier Ireland Ltd.
doi:10.1016/j.jep.2005.08.062
H. Bouamama et al. / Journal of Ethnopharmacology 104 (2006) 104 107 105
We report here the results of antimicrobial testing of Cistus 2.3. Fungal and bacterial strains
villosus and Cistus monspeliensis extracts against Escherichia
coli, Enterococcus hirea, Staphylococcus aureus, Pseudomonas Tests were performed on four fungi and four bacteria refer-
aeruginosa, Candida albicans, Candida glabrata, Candida kru- ence strains obtained from American Type Culture Collection
sei and Aspergillus fumigatus. (ATCC, Rockville, USA), Centraalbureau voor Schimmelcul-
tures (CBS, Baarn, The Netherlands) and Pasteur Institute (CIP,
Lyon France): Candida albicans (ATCC 2091), Candida krusei
2. Materials and methods
(ATCC 6258) and Candida glabrata (ATCC 90030), Aspergillus
fumigatus (CBS 14489), Escherichia coli (CIP 54.8), Staphylo-
2.1. Plant material
coccus aureus (CIP 53154), Enterococcus hirea (CIP 5855) and
Pseudomonas aeruginosa (CIP A22).
Leaves of Cistus villosus L. and Cistus monspeliensis L.
were collected in July 1996 from Ourika (Marrakesh, Morocco).
2.4. Standard microdilution method (SMM)
Specimens were botanically identified. Voucher specimens
Cv.O.96 for Cistus villosus and Cm.O.96 for Cistus monspelien-
2.4.1. Antifungal activity
sis, (coll. Bouamama) were deposited in the Department of
We used the method M27-T described by the NCCLS
Biology, Faculty of Sciences and Technology, Cadi Ayyad Uni-
as stated above. The culture medium is the RPMI-1640
versity of Marrakesh, Morocco.
(Sigma), buffered to pH 7.0 with morpholinopropanesulfonic
acid (MOPS, Sigma) and supplemented with 2% glucose.
Tests are performed in sterile U-bottom 96-well plates (Fal-
2.2. Extracts preparation
con 3077: Becton Dickinson Labware, NJ, USA). Amphotericin
B (Sigma) was used as standard antibiotic with concentrations
Dried and finely powdered leaves (200 g) of each plant were
ranging from 12.5 to 0.04 g/ml. All antifungal dilutions were
exhaustively extracted with methanol using a Soxhlet apparatus
made in RPMI-1640 and 100 l of RPMI were poured in each
for 48 h. Methanol extracts were filtered and evaporated to dry-
well. The first column of the plate was reserved for negative con-
ness using a rotary evaporator (Buchii Laboratory technique,
ć%
trol wells (without inocula) and the last column, for the positive
Switzerland) at 45 C, yielding 59 g (29%) from Cistus villo-
growth control wells (without antifungal agent). In each well,
sus and 50 g (25%) from Cistus monspeliensis. Both extracts
100 l of the antifungal dilution was added to 10 l of inocula.
were redissolved in distilled water and successively extracted
The antifungal concentrations ranged from 200 to 0.19 mg/ml.
with hexane, dichloromethane, ethyl acetate and butanol. Each
After inoculation, the plates were incubated for 24, 48 and 72 h
organic extract was then evaporated to dryness and labeled as
ć%
until day 7 at 35 C.
indicated in Table 1.
Stocks extracts solutions were prepared at 200 mg/ml in dis-
tilled water. The pH was adjusted between 5 and 7. Extracts 2.4.2. Antibacterial activity
were sterilized over a membrane filter unit of 0.2 m of We used the same method M27-T for the antibacterial test.
ć%
pore size (Minisart Sartorius) and preserved at +4 C until The culture medium was the Mueller Hinton broth (Difco Lab-
used. oratories). Amoxicillin and chloramphenicol (Sigma) were used
Table 1
Antibacterial and antifungal activities of Cistus L. extracts from Morocco
Microorganisms MIC (mg/ml)a MIC (mg/ml)a
Cistus villosus Cistus monspeliensis
P1 P2 P3 P4 P5 P6 P7 P8 AMX CHL APH B
Gram-positive bacteria
Staphylococcus aureus 0.78 1.56 0.78 1.56 25 1.56 1.56 3.125 0.0002 0.0012
Enterococcus hirea 3.125 1.56 1.56 6.25 25 3.125 1.56 6.25 0.0005 0.0005
Gram-negative bacteria
Escherichia coli 3.125 1.56 1.56 3.125 25 3.125 3.125 6.25 0.0031 0.0012
Pseudomonas aeruginosa 3.125 3.125 3.125 3.125 50 6.25 1.56 6.25 0.0002 0.0012
Yeast
Candida albicans 6.25 50 3.125 1.56 200 100 50 25 0.0015
Candida krusei 12.5 >200 12.5 6.25 100 100 12.5 25 0.0015
Candida glabrata 0.19 0.39 0.19 0.19 3.125 3.125 1.56 1.56 0.0007
Mold
Aspergillus fumigatus 100 100 100 200 100 25 200 200 0.025
P1 and P5, crude methanol extract; P2 and P6, ethyl acetate extract; P3 and P7, butanol extract; P4 and P6, remaining aqueous layer.
a
Replicate of three assays: AMX, amoxicilline; CHL, chloramphenicol and APH B, amphotericin B.
106 H. Bouamama et al. / Journal of Ethnopharmacology 104 (2006) 104 107
as standards. Concentrations ranged from 356 to 0.25 g/ml for inhibit the bacteria Escherichia coli, Enterococcus hirea and
amoxicillin and from 320 to 0.31 g/ml for chloramphenicol. Staphylococcus aureus (Table 1). P2 and P3 are the most active
ć%
After inoculation, the plates were incubated at 37 C for 24 and extracts (MIC = 1.56 mg/ml for Escherichia coli and Enterococ-
48 h. cus hirea). Staphylococcus aureus is the most susceptible species
to Cistus villosus with MIC values between 0.78 and 1.56 mg/ml.
2.5. Inocula preparation Regarding Cistus monspeliensis, Table 1 shows that ethyl
acetate (P6), butanol (P7) and aqueous (P8) extracts present the
Yeast colonies were obtained from 24 h cultures on lowest and therefore the most interesting MIC values.
ć%
Sabouraud broth at 35 C under agitation. The first suspen- Among them, ethyl acetate and butanol extracts show to
sions were used for the inocula preparation in RPMI-1640. The be the most active extracts inhibiting Staphylococcus aureus,
concentrations of yeast cells were determined with a counting Enterococcus hirea and Pseudomonas aeruginosa with MIC val-
chamber. Budding organisms were counted as two. The origi- ues = 1.56 mg/ml.
nal concentrations were adjusted to a concentration of 104 105
colony forming units (CFU)/ml with broth used in the suscepti- 3.2. Antifungal activity
bility test.
The mold Aspergillus fumigatus was grown on malt agar at Cistus villosus extracts show the most interesting inhibi-
ć%
35 C for 7 days, to induce conidia formations. Then, the cul- tion activities against Candida, except the ethyl acetate extract
ture was washed with 2 ml of saline solution and the suspension which does not show any noticeable action on Candida kru-
was transferred to a sterile tube, where the heavy particles were sei. The species Candida glabrata is strongly inhibited by P1,
allowed to settle for 5 min. The upper homogeneous suspen- P3 and P4 (MIC = 0.19 mg/ml) and P2 (MIC = 0.39 mg/ml).
sions were transferred with a pipette Pasteur to a new sterile The order of susceptibility for the rest of the fungi tested
tube and filled with RPMI-1640, then vortexed for few seconds. was Candida albicans (MIC = 1.56 mg/ml for P4), Candida
This suspension is considered as the initial inoculum. After dilu- krusei (MIC = 6.25 mg/ml for P4) and Aspergillus fumigatus
tions in RPMI-1640, the inoculum was adjusted microscopically (MIC = 100 mg/ml for P1, P2 and P3).
to about 104 CFU/ml. Candida glabrata is the most sensitive strain to the Cistus
Stock bacterial inocula suspensions were obtained from 18 h monspeliensis extracts, but the MICs are not as significant as
ć%
culture on Mueller Hinton broth at 37 C. Those final suspen- for Cistus villosus extracts (Table 1). P7 and P8 show a MIC of
sions served for the inocula preparation. The cell density of each 1.56 mg/ml.
suspension was determined by using a counting chamber, and
then adjusted to a concentration of 105 106 CFU/ml by dilution 4. Conclusion
with Mueller Hinton broth.
Compared with other studies, our data show better antimi-
2.6. Readings and control crobial activities for Cistus species. Gnan and Demello (1999)
reported the antimicrobial activity of Goiaba leaf extract at
The experiments were repeated three times and the results a concentration of 6.5 mg/ml against Staphylococcus aureus.
were determined as an average value. The result readings were Chandrasekaran and Venkatesalu (2004) reported MIC values
made visually. The MIC endpoint was considered as the lowest of Syzygium jambolanum seed extracts ranged between 0.0031
drug concentration of antifungal or antibacterial agent inhibit- and 0.5 mg/ml against Escherichia coli, Staphylococcus aureus,
ing the total growth of microorganisms. MIC was detected by Pseudomonas aeruginosa and Candida albicans.
lack of visual turbidity (matching the negative growth con- The fact that the leaf extracts of Cistus species exhib-
trol). Subcultures were made from the clear wells which did ited inhibitory activities against some of the microorganisms
not show any growth after incubation during the MIC assays on involved in many infections and skin diseases. Yeasts such
Mueller Hinton agar for bacteria and Sabouraud agar for fungi. as Candida glabrata and Candida albicans, bacteria such as
Staphylococcus aureus, Escherichia coli, Enterococcus hirea
3. Results and discussion and Pseudomonas aeruginosa provide some scientific basis for
the utilization of this plant in folk medicine (Peyron and Alessan-
Using the M27-T technique, leaf extracts of Cistus villosus L. dri, 1986).
and Cistus monspeliensis L. show antimicrobial properties with Although fungi have been the main source of antibiotics up to
MIC values ranging from 0.78 to 50 mg/ml for bacteria and 0.19 now, the discovery of new plant products with potential antimi-
to 200 mg/ml for fungi. Cistus villosus extracts show the lowest crobial application is of considerable interest in view of the
MIC values (Table 1) and thus they could be considered as a increasing antibiotic resistance to many microorganisms (Irobi,
source of interesting antimicrobial compounds. 1997).
3.1. Antibacterial activity Acknowledgements
Methanol P1, ethyl acetate P2, butanol P3 and the aque- The authors wish to thank all the members of the Lab-
ous P4 extracts of Cistus villosus show the lowest MIC and oratory of Vegetal Sciences, Faculty of Pharmacy, Renée
H. Bouamama et al. / Journal of Ethnopharmacology 104 (2006) 104 107 107
Descartes University, Paris V (France) for their partial technical Committee for Clinical Laboratory Standards reference macrodilution
method M27-P for in vitro testing of fluconazole-resistant and -susceptible
assistance.
isolates of Candida albicans. Journal of Clinical Microbiology 33,
3154 3158.
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