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Advances in Environmental Biology, 5(2): 227-230, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Alternative Treatment of Infection by Compounds Isolated from Globularia Eriocephala
Leaves
1
2
Meddah Boumediene,
G. Krim
1,2
Tir touil Aicha, 2Leke André, 3Nahnouh Noreddine, 2J.P. Canarelli,
1
LRSBG, Faculté SNV – Université de Mascara, Algérie.
Service de Pédiatrie, CHU, Nord Amiens, France.
3
CH de MASCARA, Mascara, Algérie.
2
Meddah Boumediene, Tir touil Aicha, Leke André, Nahnouh Noreddine, J.P. Canarelli, G. Krim:
Alternative Treatment of Infection by Compounds Isolated from Globularia Eriocephala Leaves
ABSTRACT
Objective: The prevalence of antimicrobial resistance among clinical pathogens is reviewed and its clinical
impact on management is increased. Continued surveillance of resistance rates among clinical pathogens is
needed to ensure that appropriate recommendations can be made for treatment of infected patients. Since,
vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) are the most
important bacteria isolated in this case. Further studies addressing the clinical and bacteriological outcomes of
patients infected with a resistant pathogen are needed. The leaves of Globularia eriocephala Pomel (family
Globulariaceae) named “Tasselgha” are widely used in North region of Algeria as a folk medicine. Chloroform,
ethyl acetate and hydromethanol extracts of Globularia alypum and essential oil were tested against some
clinical pathogens bacteria. Methods used: Antibacterial activities were tested against bacteria isolated from
surgery in perioperative period using disc-diffusion method and MIC was determined. The compound of
essential oils was confirmed using chromatography techniques. Results: The hydromethanolic extract of
Globularia eriocephala leaves demonstrated antimicrobial activity against Staphylococcus aureus, Pseudomonas
aeruginosa, Klebsiella pneumoniae and inactivity against Proteus vulgaris and Escherichia coli. Antibacterial
activities were observed in ethyl acetate and hydromethanolic extracts of Globularia eriocephala leaves. The
MIC values of the compound against Staphylococcus aureus (75.0mg/ml), Pseudomonas aeruginosa
(63.5mg/ml) and Klebsiella pneumonia (62.0 mg/ml). The combination of Chloroform extract and essential oils
exerted wielded a synergistic effect for the inhibition against the growth of the Gram-negative and Grampositive bacteria when the MICs were applied. Conclusion: Ethyl acetate extract showed promising antibacterial
activity against bacterial responsible of nosocomial infection, and the essential oil compounds showed at all
a moderate activity against these bacteria. These results suggested Globularia eriocephala could be a potential
source of antibacterial agents. Further investigations are in progress to determine the active constituent(s) for
their application in medical research.
Key words: Antimicrobial, Medicinal plants, Globularia eriocephala, Polyphenols, Algeria.
Introduction
The plants of Globularia eriocephala Pomel
(family Globulariaceae) named in Arabic “Tasselgha”
are traditionally used in north Africa especially in
Tiaret region of Algeria as folk medicine in the
treatment of many illnesses. The leaves and stems of
Globularia eriocephala are used as decoction in the
treatment of hypoglycemia, rheumatic and infectious
diseases, its leaves are reported to be used in the
treatment of diabetes, in renal and cardiovascular
diseases [1,2,3]. Little is now about tis specie
Corresponding Author
Meddah Boumediene, LRSBG, Faculté SNV – Université de Mascara, Algérie.
Email: [email protected]
Adv. Environ. Biol., 5(2): 227-230, 2011
Globularia eriocephala but others species were
studied; for example, Çalis et al. [4,5] have isolated
the Iridoid glycosides from Globularia trichosantha
and G. davisiana, Sugar esters from G. orientalis [6].
Elbetieha et al. [7] showed the phytotoxic potentials
of G. arabica and G. alypum and Es-Safi et al. [8],
Saglam et al., [9], Djeridane et al., [10] have
determined the antioxidant constituents and the αtocopherol of G. alypum. In our research group,
many compounds with significant pharmacological
effects have been isolated from this plant. The aim
of this study was to evaluate the antibacterial effect
of plant extracts isolated from Globularia eriocephala
leaves against Clinical Pathogens Bacteria.
Materials and Methods
Plant material
Fresh Globularia eriocephala was collected
during the florescence phase in 2008 from the region
of Tiaret (Algeria). Taxonomic identification was
performed by Pr. K. Benabdeli (Ecobiological
Laboratory, S.N.V. Faculty, Sidi Belabes-Algeria).
Isolation of the Essential Oil (EO):
Oil extracts were obtained from 100 g of fresh
leaves after submition for 3 h, to hydrodistillation
water-distillation using a Clevenger-type apparatus.
The obtained essential oil (EO) was filtred and
stored at 4°C until antibacterial tested againts a
pathogens.
Preparation of plant extracts:
Fresh aerial parts (leaves) were washed and airdried in shade at room temperature. They were then
mechanically powdered and sieved. The powdered
plant material (50 g) was extracted, for 3h under
occasional shaking with 250 ml of chloroform and
The obtained crude preparation was centrifuged at
5000x g for 30 min. After filtration, the supernatant
filtrates were concentrated under reduced pressure at
45°C and the crude extracts were filtered over
Whatman n° 1 filter paper. The combined extracts
were reduced and pooled, the w/w yield in terms of
dry starting material was 1.43%. The obtained
residue was dried and re-extracted with Ethyl acetat
and filtred for a yield of 2.82%. The final residue
was dried and re-extracted with methanol-water (1:1,
v/v) for 24h at room temperature in darkness, filtred
and concentrated for yielding 19.36%. The extracts
were sterilized by membrane filtration using 0.45 μm
pore size filters, and were kept at 4°C for further
investigation. All the extractions were repeated three
times.
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2.4. Microbial strains:
Five clinical microbial strains tested were
isolated from surgery patients in perioperative
period in Mascara Hospital. Four Gram negativebacteria, Pseudomonas aeroginosa, Klebsiella
pneumonia, Proteus vulgaris, Escherichia coli; one
Gram-positive bacteria, Staphylococcus aureus. All
microorganisms were identified by recommandation
of bergy’s manual Methods by culture in specific
media followed by Gram coloration and biochemical
test using automate microbiological system
identification (API system). Inoculm for the assays
were prepared by diluting scraped cell mass in 0.85%
NaCl solution, adjusted to McFarland scale 0.5 and
confirmed by spectrophotometric reading at 580 nm.
Cell suspensions were finally diluted to 106 CFU /ml.
The microbial isolates were maintained on agar slant
at 4 NC in the Laboratory LRSBG (Faculty of S.N.V,
University of Mascara) where the antimicrobial tests
were performed.
2.4.1. Determination of Minimum Inhibitory
Concentration (MIC) and Minimum Bacteriocidal
Concentration (MBC) by microdilution:
MIC test were carried out according to Ellof [11]
using Muller Hilton Broth on a tissue culture test
plate (96-well). Briefly, 100 ml of each concentration
from the substance (compounds or essential oils) was
added in the first well, and serial dilutions were
performed (1/2) and at the last 100 ml of cell
suspension was added to all wells. Hydro Methanol
extract was dissolved in DMSO (2%), it used as
negative control. The plates were agitated and
incubated at 37°C for 48 h.
The lowest concentration showing no culture was
considered as the MIC and its express as (μg/ml, or
ml/ml). For The MBC determination, 100 ml of
liquid from each well that showed no change culture
was plated on GNA and incubated at 37NC for 24 h.
The lowest concentration that yielded no growth after
this sub-culturing was taken as the MBC.
2.4.1. Sensitivity test: Agar Disc Diffusion Assay:
Antibacterial activity was carried out using a
disc-diffusion method. Petri plates were prepared with
20 ml of sterile Mueller Hinton Agar (MHA) (Sigma,
France) inoculate by suspension of cell (1ml)
adjusted by McFarland 0.5 method (106 CFU /ml).
The test cultures were swabbed on the top of the
solidified media and allowed to dry for 10min. The
tests were conducted at different concentrations of
the plant extract in Sterile filter paper discs (6 mm).
The loaded discs were placed on the surface of the
medium and left for 30 min at room temperature for
compound diffusion. The inhibition zones produced
Adv. Environ. Biol., 5(2): 227-230, 2011
by the plant extract were compared with the
inhibition zones produced by commercial standard
antibiotics: gentamycin and vancomycin (50mg/disc).
DMSO were used as the negative control. The zone
of inhibition (mm) was measured from the boundary
of the disc after cultivation at 37°C for 48 h.
2.5. Statistical analysis:
Results were expressed as the mean±SD of five
replicates. Data were analyzed using one-way
analysis of variance (ANOVA), P values <0.05 were
considered significant.
Statistical analysis:
Results and Discussion
The results of the experiments assessing the
bacteriostatic effects of essential oil and solvent
extracts compounds of the plants under study on
Gram-negative and Gram-positive bacteria
Pseudomonas aeroginosa, Klebsiella pneumonia,
Proteus vulgaris, Escherichia coli, Staphylococcus
aureus are presented in Table 1. Effectively The
ethyl acetate extract from Globularia eriocephala
leaves was the best compounds demonstrated
antibacterial activity against all clinical pathogens
bacteria with lower concentrations, the MIC were
including 55 and 75 mg/ml. Hydromethanolic extract
showed activities against only Klebsiella pneumonia,
Pseudomonas aeroginosa and Staphylococcus aureus,
with concentration between 62 and 75 mg/ml
respectivelly. Ethanolic extracts of the same species
have been mentioned in the littérature for their
antibacterial activity on S. aureus, P. aeruginosa and
E. coli [12]. The essential oil compounds showed at
all a moderate activity (150-500mg/ml) against these
bacteria. The most binteresting result from this study
was the combination of Chloroform extract and
essential oil wich exerted wielded a synergistic effect
for the inhibition against the growth of the Gramnegative and Gram-positive bacteria especially, S.
aureus (67mg/ml), P.aeruginosa (74,2mg/ml) andK.
Pneumonae (83,6mg/ml) (Table 1). Also, the
bacteriocidal activity (MBC) its extent varied, and
depending on the extract compounds as shouwn in
Table 1. The literature indictes that the antibcterial
activity is due to different chemical agents in the
extract, including essential oils, flavonoids and other
phenolic compounds or free hydroxyl groups [13].
As follows from the Table 2, ethyl acetate
extracts tested by means of agar disc diffusion
assay showed activies againts all pathoges bacteria
with an inhibition zone more than 20mm (Fig. 1).
An inhibition zone of 10 or greater was considred as
g o o d a n t i mi c r o b i a l a c t i v i t y [ 1 4 ] . T h e
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hydomethanolicextracts showed an activity more
important with an inhibition zone from 25 to 30 mm,
against Staphyloccocus aureus, Pseudomonas
aeruginosa and Klebsiella pneumoniae respectively
(Photo 1). Thus, no growth of E.coli and Proteus
vulgaris tested was observed in the presence of this
compound (Table 2). Clinical pathogens strains were
less susceptible to Essentials oil with an inhibition
zone from 10 to 16 mm (Fig. 1). The choroform
exrtracts provided a moderate zone of inhibition from
8 to 10 mm diameter on Staphylococcus aureus,
Pseudomonas aeruginosa and Klebsiella pneumonia
(Table 2). The solvent used as control DMSO,
exerted no effect against the bacteria. Thus, both
extracts plants by solvents and essential oils exhibit
pronounced bacteriostatic effects against Grampositive and Gram-negative pathogens bacteria.
Solvent extracts and essential oils differed
qualitatively in the relative content of polar
compounds. Ethyl actetate extracts contained
compounds with greater polarity than that of their
counterparts present in others solvents or aqueous
extracts. This may be the likely explanation for
significant differences in the bacteriostatic activity
between differents compound extracts of Globularia
eriocephala leaves
The mechanism of bactericidal activity of plant
extracts has not been fully understood; it is believed
that the active compounds of G. eriocephala leaves
exert direct effects against Clinical pathogens
Bacteria. Generally, the mechanism is probably due
to their ability to complex with extracellular and
soluble proteins and then to complex with bacterial
cell walls [15]. The availability of efflux pumps
inhibitors [17]. Inhibition and interferences with some
virulence factors Enzymes, coagulase, Dnase, lipase,
thermonuclease [16]. Inhibition of conjugative R_
plasmid transfer in enteric bacteria [17,18]
Conclusion:
The plant extracts from Globularia eriocephala
leaves may be a promising alternative treatment of
localized infections even with severe hospitalacquired strains (Nosocomial Infection). Further
investigations are in progress to determine the active
constituent(s) for their application in medical
research. It would be advantageous to standardize
methods of extraction, activity-guided fractionation,
and in vitro testing so the search could be more
systematic and reproductible, and the interpretation of
results would be facilitated. In vitro activity should
be to determine their efficacy, stability, and
bioavailability and their effects on beneficial normal
microbiota.
Adv. Environ. Biol., 5(2): 227-230, 2011
Table 1: MIC and MBC Results (mg/ml)
Sample
Staphylococcus aureus
----------------------------MIC
MBC
Hydromethanolic 75
150
Ethyl acetate
73
146
Chloroform
200
400
Essential oil
150
300
Chloroform +
Essential oils
67
134
DMSO -water (1:1) -
230
from G. eriocephala extract leaves against differents clinical pathogens bacteria
Pseudomonas aeruginosa Klebsiella pneumoniae E. coli
Proteus vulgaris
--------------------------------- ----------------------------- ---------------------- ----------------------MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
63.5
127
62
124
60
120
55.5
111
65
130
55
110
250
500
250
500
150
300
200
400
200
400
250
500
74.2
-
148.4
-
83.6
-
Table 2: Growth Inhibition zones of clinical pathogens bacteria (mm)
Sample
Staphylococcus aureus Pseudomonas aeruginosa
Hydromethanolic
25 ± 0.20
25±0.36
Ethyl acetate
30 ±0.41
30 ± 0.21
Chloroform
08 ±0.02
10 ±0.21
Essential oils
16 ±0.25
14 ±0.77
Gentamicin
21 ±0.20
25 ±0.21
Vencomycin
22 ±0.09
18 ±0.60
DMSO –water (1:1)
-
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