Antimicrobial and Cytotoxic Activity of Marrubium alysson and Retama raetam Grown in Tunisia

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.