|
|
|
|
Research Article
|
|
Antimicrobial and Cytotoxic Activity of Marrubium alysson and Retama raetam Grown in Tunisia |
|
Edziri Hayet,
Ammar Samia,
Groh Patrick,
Mahjoub Mohamed Ali,
Mastouri Maha,
Gutmann Laurent,
Zine Mighri
and
Aouni Mahjoub
|
|
|
ABSTRACT
|
Antibacterial and antifungal activities of extracts obtained from M. alysson, R. raetam were tested using a solid medium technique. We showed that the petroleum ether extract of M. alysson had a Minimum Inhibitory Concentration (MIC) varied from 128 to 2000 μg mL-1 against different Enterobacteriaceae and antifungal activity against Candida glabrata, Candida albicans, Candida parapsilosis and Candida kreusei with a MIC of 256 μg mL-1. The ethyl acetate extract of R. raetam showed the best activity against Gram positive organisms with MICs of 128 to 256 μg mL-1 against methicillin resistant Staphylococcus aureus but low activity against the different Candida species.
|
|
|
|
|
INTRODUCTION Medicinal plants which contain many active principles of therapeutic values have been used for centuries as treatments for many human diseases. M. alysson is a perinial herb of the Lamiaceae family which is commonly distributed in Europe, Mediterranean, Asia (Mabberly, 1997) and in Tuinisia. Its flowered aerial parts as well as their aqueous and hydroalcoholic extracts are used in traditional medicine for treating cough but also as laxative during digestive and biliary complaints (Wichtel and Anton, 1999). Retama raetam, locally named as ‘R’tm’, is a wild plant belonging to the Fabaceae family. It is common to North, East Mediterranean regions (Mittler et al., 2001; Elbahri et al., 1999) and in Tunisia. It is used for traditional treatment of some renal diseases. Since it was reported to show significant diuretic activity (Caceres et al., 1987) it could be also be useful for treatment of hypertension (Archer and Pyke, 1991; Izhaki and Neeman, 1997; Kassem et al., 2000; Taylor, 1981). The aim of this study was to investigate the antimicrobial and antifungal activity of different extracts of M. alysson and of R. raetam. MATERIALS AND METHODS Plant materials: Plants materials were collected in 2004 in kerker region (Tunisia). Plant extraction: The aerial part of the dried powdered of M. alysson and R. raetam (75 g) was extracted for 5 h with 1 L of petroleum ether, ethyl acetate and methanol successively by Soxhlet extraction. Extracts of each solvent were evaporated under reduced pressure and the final residues were used for the bioassays. Microorganisms: The micro-organism strains employed in the biological assays are listed in Table 1. Different American Type Cell Culture (ATCC) reference bacteria and fungi were used as well as clinical isolates including methicillin susceptible (MSSA) and methicillin resistant S. aureus (MRSA) strains.
Determination of antimicrobial and antifungal activity: Various concentrations
(1 μg mL-1 up to 10 mg mL-1) of M. alysson
and R. raetam extracts were used to determine the antimicrobial activity.
Overnight broth cultures were adjusted to yield approximately 1x106
CFU mL-1 of bacteria or yeast. The Minimal Inhibitory Concentrations
(MICs) were determined on Brain Heart Infusion (BHI) agar plates (Bio-Rad, Marne
la Coquette, France) by a standard method (NCCLS, 1997) with a Steers-type replicator
device that delivered ca. 104 bacteria per spot. One milliliter of
each of extracts previously dissolved in 10% Dimethyl sulfoxide (DMSO) were
mixed for each concentration with 19 mL of BHI agar at 40°C and poured over
Petri dishes. The resulting DMSO concentration was at most 0.5%. Plates containing
only medium or medium with 0.5% DMSO were used as controls to ensure that DMSO
did not affect growth, a standard antibiotic (oxacilin, amoxicillin, ticarcillin
and cefotaxim) used in order to control the sensitivity of the tested microorganism.
After 18 h of incubation at 37°C, the Minimum Inhibitory Concentration (MIC)
was defined as the lowest concentration of the extract inhibiting the visible
growth of each microorganism. Each test was carried out in triplicate. The microorganisms
tested in this study were provided from European Hospital of George Pompidou
(HEGP) (France).
Cytotoxicity assay: The cytotoxicity were evaluated on VERO cells (a
line of green monkey kidney cells from ICN-Flow), grown on Dulbecco’s modified
MEM with 2% foetal calf serum (both from Gibco). The assays were performed in
duplicate in 24- well plates with about 5 104 cell per well. Cytotoxicity
was read after 48 h of incubation in an atmosphere of 5% CO2 at 37°C,
as the inhibition of cell multiplication in the presence of decreasing amounts
of the compounds under study, with the use of a light microscope. The Minimal
Toxic Dose (MTD) was considered to be the dose of the compounds, which reduced
cell multiplication by at least 50%, as compared to control (Fioravantin et
al., 1997).
Phytochemical screening: The screening of the chemical composition was carried out with the methanol extract using chemical methods (Allen, 1974; Harbone, 1976) RESULTS
Antibacterial activities were tested with the petroleum ether, ethyl acetate
and methanol extracts of M. alysson and R. raetam. Only active
plant extracts are given in Table 1 witch summarized the in
vitro inhibitory activity of the aerial part extracts of M. alysson and
R. raetam. The aerial part extracts of M. alysson and R. raetam
were active against Gram positive and Gram negative bacteria. Among the different
extracts, the petroleum ether extract of M. alysson showed the most significant
antimicrobial activity against E. cloacae, K. pneumoniae and S.
marcescens with MICs of 0.128-0.256 mg mL-1. It also showed the
best activity against the different Candida species.
Table 1: |
Antibacterial activity of Marrubium alyson and Retama
raetam against bacteria |
 |
aThe most appropriate reference antibiotic was
chosen. OXA, oxacillin; AMX, amoxicillin; TIC, ticarcillin; CTX, cefotaxim,
AB; amphotericin B; bMarrubium alyson petroleum ether
extract; cMarrubium alyson ethyl acetate extract; dRetama
raetam ethyl acetate extract; eNot done; fStrain,
sensitive to methicillin; gStrain resistant to methicillin |
Table 2: |
Antifungal activity of Marrubium alyson and Retama
raetam |
 |
AB: amphotericin B;
aMarrubium alyson petroleum ether extract; bMarrubium
alyson ethyl acetate extract; cRetama raetam ethyl
acetate extract |
Table 3: |
Cytotoxic activity of plants extracts |
 |
MTD5O: Minimal Toxic dose which inhibit 50 cellular growth;
aMarrubium alyson petroleum ether extract; bMarrubium
alyson ethyl acetate extract; cRetama raetam ethyl
acetate extract |
Table 4: |
Chemical screening of methanolique plants extracts |
 |
The ethyl acetate extract of R. raetam was the most active against
Gram positive bacteria and in particular S. pyogenes, S. agalactiae,
MSSA and MRSA with MICs being generally less than 1 mg mL-1. We observed
that the ethyl acetate extract of M. alysson had a moderate activity
in candida species with MIC ranges from 0.512 to 1 mg mL-1. The Candida
species were resistant to the ethyl acetate extract of R. raetam with
MIC ranges from 2 to 4 mg mL-1 (Table 2). These
extracts have a good antibacterial activity because they aren’t cytotoxic
(Table 3). The chemical screening show that M. alysson
contained alkaloids, diterpenoids and saponosids, but R. raeatm is rich
with flavonoids, tannins and alcaloids (Table 4).
DISCUSSION
The results obtained could indicate the existence of antibacterial compounds
in the aerial part extracts of M. alysson and R. raetam. Curiously,
only a low activity was recorded against E. coli the most prevalent Enterobacteriaceae.
These results give evidence to the ethnotherapeutic claims for treating skin
diseases (Venkatesan et al., 2005) with such plants since S. aureus
and S. pyogenes are the primary causative agents of skin and soft tissues
infections. The activities observed for some of the different extracts studied
in this study is in support for further isolation of the compounds responsible
for the observed antimicrobial activity which includes that against S. aureus,
streptococci and Candida sp. In particular the activity of the petroleum
ether extract of M. alysson against some Enterobacteriacae could
provide future active principles for the eradication of some pathogens responsible
for urinary tract and nosocomial infections (Prescot et al., 1999; Stanier
et al., 1986). It is interesting to note that these extracts were able
to inhibit some of the bacteria and yeast at concentration much lower than the
toxic dose. The screening of the chemical groups in the extracts of M. alysson
revealed the presence of tannins, alkaloids and flavonoids. Tsuchia et
al. (1999) linked the antimicrobial effects of flavonoids to their capacity
to form complexes with extracellular and soluble proteins and with the cell
wall; we suggest that presence of such compounds in Marrubium alysson extracts
and Retama raetam extract may play a role in their observed antifungal
and antibacterial activities. However, Cown (1999) showed that tannins are able
to inhibit the growth of the hypes of many fungi; these molecules are used as
antimicrobial agents (Scalbert, 1991).
CONCLUSIONS In conclusion, our biological study on M. alysson and R. raetam plants showed that the ethyl acetate extract of R. raetam showed the best activity against Gram positive organism especially against methicillin resistant Staphylococcus aureus but it showed low antifungal activity, compared with the petroleum ether extract of M. alysson witch had an interesting antibacterial activity with MIC varied from 128 to 2000 μg mL-1 against different enterobacteriaceae and had good antifungal activity with MIC between 128 and 256 μg mL-1.
|
REFERENCES |
1: Allen, S.T., 1974. Chemical Analysis of Ecological Material. Blackwell Scientific Publication, New York, pp: 313.
2: Archer, S. and D.A. Pyke, 1991. Plant animal interactions affecting establishment and persistence on revegetated rangelands. J. Range Manage., 44: 558-565. Direct Link |
3: Caceres, A., L.M. Giron and A.M. Martinez, 1987. Diuretic activity of plants used for the treatment of urinary aliment sin Guatemela. J. Ethnopharmacol., 43: 197-201.
4: Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev., 12: 564-582. CrossRef | PubMed | Direct Link |
5: El-Bahri, L., M. Djegham, H. Bellil, 1999. Retama W: A poisonous plant of North Africa. Vet. Human Toxicol., 41: 33-35. Direct Link |
6: Fioravantin, R., M. Biava, G.C. Poretta, M. Artico, G. Lampis, D. Deidda and R. Pompei, 1997. N-substituted 1-aryl-2-(1H-imidazol-1-yl)-1-ethanamines with broad spectrum in vitro zntimycobacterial and antifungal activities. Med. Chem. Res., 7: 87-97.
7: Harbone, J.R., 1976. Phytochemical Methods a Guide to Modern Techniques of Plant Analysis. Charpan and Hall, London, pp: 78.
8: Izhaki, I. and G. Neeman, 1997. Hares (Lepus sp.) as seed dispersers of Retama raetam (Fabaceae) in a Sandy landscape. J. Arid Environ., 37: 343-354. Direct Link |
9: Kassem, M., S.A. Mosharrafa, N.AM. Saleh and S.M. Abdel-Wahab, 2000. Two new flavonoids from Retama raetam. Fitoterapia, 71: 649-654. Direct Link |
10: Mabberly, D.J., 1997. The Plant Book. 2nd Edn., Cambridje University Press, Cambridge, pp: 440.
11: Mittler, R., E. Merquiol, E. Hallak-Herr, A. Rachmilevitch, A. Kaplan and M. Cohen, 2001. Living under a dormant canopy: A molecular acclimation mechanisme of the desert plant Retama raetam. Plant J., 25: 407-416. Direct Link |
12: National Committee for Clinical Laboratory Standards, 1997. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically. 4th Edn., National Committee for Clinical Laboratory Standards, Wayne, PA.
13: Prescot, L.M., J.P. Harly and D.A. Klein, 1999. Microbiology. 4th Edn., McGraw-Hill, Boston, pp: 1-685.
14: Scalbert, A., 1991. Antimicrobial properties of tannins. Phytochemistry, 30: 3875-3883. CrossRef | Direct Link |
15: Stanier, R.Y., J.L. Ingraham, M.L. Wheelis and P.R. Painter, 1986. General Microbiology. 5th Edn., Macmillan Publishing Company, Lnodon, pp: 1-685.
16: Taylor, G.B., 1981. Effect of constant temperature treatments followed by fluctuating temperatures on the softening of hard seeds of Trifolium subterraneum L. Aust. J. Plant Physiol., 8: 547-558. CrossRef | Direct Link |
17: Tsuchiya, H., M. Sato, T. Miyazaki, S. Fujiwara and S. Tanigaki et al., 1996. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin resistant Staphylococcus aureus. J. Ethnopharmacol., 50: 27-34. CrossRef | Direct Link |
18: Venkatesan, M., M.B. Viswanathan and N. Ramesh, 2005. Lakshmanaperumalsamy, antibacterial potential from Indian S. angustifolia. J. Ethnopharmacol., 99: 349-352. Direct Link |
19: Wichtel, M. and R. Anton, 1999. Plantes Therapeutiques. Tec and Doc, Paris, pp: 341.
|
|
|
 |