Abstract: Mentha longifolia (L.) Huds. is a perennial rhizomatous herb distributed from Southern Africa, Europe, the Mediterranean region and eastwards into Asia. Mentha species are also generally known under name of Puneh in Iran where they have been used for centuries as tonics, carminative, digestive, stomachic, antispasmodic and anti-inflammatory agent in folk medicine. In the present work, we study antimicrobial and cytotoxic activity of the plant extracts. The antibacterial and antifungal activity of the plant extracts were evaluated using disk diffusion method. We evaluated cytotoxicity of M. longifolia with MTT assay, as well as. Present finding revealed that the methanol extract of the plant leaves were active against all tested bacteria and fungi. The highest inhibitory effect was observed against Erwinia carotovora, a common plant pathogen bacteria, with MIC value of 128 μg mL-1 and inhibition zone of 41 mm. The extract also exhibited high antibacterial activity with MIC value rang of 192-512 μg mL-1 and inhibition zone of 34-40 mm against S. aureus, Enterococcus faecalis, Streptococcus agalactiae and Escherichia coli. The methanol extract of the plant displayed modest to strong antifungal activity against Candida kefyr, Candida albicans, Sclerotinia sclerotiorum and Aspergillus niger with MIC value range of 576-800 μg mL-1 and inhibition zone of 26-33 mm. Our finding showed that M. longifolia methanol extract has cytotoxic activity. The extract reduced the viability of McCoy cells with RC50 value of 1.92 mg mL-1. It was be concluded that M. longifolia extract cab be used as an antiseptic agent and may be also a good candidate to construction of a new plant biopesticide.
INTRODUCTION
The Labiatae family comprised of 220 genera of aromatic plants which are widely used for various purposes world wide. The genus Mentha is one of the important members of the family represented by species in the Flora of Iran (Mozaffarian, 1996). Mentha species are generally known under name of Puneh in Iran where they have been used for centuries as tonics, carminative, digestive, stomachic, antispasmodic and anti-inflammatory agent in folk medicine (Amin, 2005).
Mentha longifolia (L.) Huds. is the most widespread species of the genus in Iran. It is a perennial rhizomatous herb with erects to straggling stems reach 120 cm in height. The plant is an extremely variable species with a widespread distribution in Southern Africa, Europe, the Mediterranean region and eastwards into Asia (Rechinger, 1982).
There is many reports on bioactivity of M. longifolia in the literature. It was previously shown that the plant posses antioxidant (Nickavar et al., 2008), antimicrobial (Al-Bayati, 2009) and hepatoprotective activity (Nimica-Dukic et al., 1999).
Phytochemical studies revealed the presence of different flavonoides (Ghoulami et al., 2001), monoterpene ketones (Heganauer, 1953), tannins and saponins (Do Nascimento et al., 2009) in the plants of the genus Mentha. These chemicals are responsible for different pharmacological and biological activity of these plants. In the recent work, we focus on cytotoxic activity and antimicrobial properties of M. longifolia extracts on some human and plant pathogen microorganisms.
MATERIALS AND METHODS
Plant materials: The leaves of M. longifolia were collected from around of Ardabil, Iran during June 2009. A voucher specimen was deposited at the herbarium of faculty of sciences, University of Mohaghegh Ardabili, Ardabil, Iran (No. 1389-3).
Preparation of the extracts: Air-dried plant leaves were Soxhlet extracted with n-hexane, dichloromethane and methanol, respectively. The extracts were dried in vacuum (Razavi et al., 2009).
Antibacterial assay: The antibacterial activities of the plant extracts were determined against E. coli (PTCC 1047), S. aureus (PTCC 1112), E. faecalis (PTCC 1394), S. agalactiae (PTCC 1321), Erwinia carotovora (PTCC, 1675) and S. aureus (E38) by the disc diffusion method (Razavi and Nejad-Ebrahimi, 2009). Muller-Hinton Agar (MHA) (oxoid) was used for preparation of the media for bacteria. The filter paper discs (6 mm in diameter) were individually impregnated with 15 μL of stock solution of the plant extracts (100 mg mL-1) and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. The plates were inoculated at 37°C for 24 h. The diameters of inhibition zones were measured in millimeters. All the tests were performed in duplicate. Gentamicin (10 μg) and Erythromycin (15 μg) served as positive control. The MICs of the extracts against the test microorganisms were determined by the Agar dilution method (Razavi and Zarrini, 2010).
Antifungal assay: The antifungal activities of the plant extracts were determined against C. kefyr (ATCC 38296), C. albicans (ATCC 14053), A. niger (PLM 1140), Penicillium sp. and S. sclerotiorum by the disc diffusion method. Sabouraud Dextrose Agar (SDA) was used for preparation of the media for the fungal strains. The filter paper discs (6 mm in diameter) were individually impregnated with 15 μL of stock solution of the extracts (100 mg mL-1) and then placed onto the agar plates which had previously been inoculated with the tested microorganisms. Amphotericin B 10 μg disc was applied as positive control. The plates were inoculated with the fungi incubated at 30°C for 48 h. The diameters of inhibition zones were measured in millimeters. All the tests were performed in duplicate. The MICs of the extracts against the test microorganisms were determined by the Agar dilution method (Razavi and Zarrini, 2010).
Cytotoxic assay: MacCoy cell lines (Pasteur, C123) were grown in RPMI 1640 (Gibco, No. 51800-019) medium. Each 500 mL of the medium consisted of 5.2 g RPMI powder, 1 g of sodium bicarbonate, 1% w/v of penicillin/streptomycin and supplemented with 10% heat-inactivated Fetal Calf Serum (FCS) in demonized water (Razavi et al., 2010a). Completed medium was sterilized by filtering through 0.22 μm microbiological filters (Art No. 11107-25). Cell line was maintained in a humidified atmosphere of 5% CO2 at 37°C in incubator. The stock solutions of methanol extracts of Malva sylvestris flowers and leaves were prepared by dissolving the compound in DMSO (100 μL). The final concentration of the extract was 0.70, 0.50, 0.30, 0.3, 0.10 and 0.05 mg mL-1. Cells were plated in the appropriate media on 24-well microplates in a 500 μL total volume at a density of 6x105 cell mL-1. Triplicate wells were treated with media containing different concentration of the extract. The plates were incubated at 37°C in 5% CO2 for time course of 16 h. For evaluating of cell viability, each well was supplemented with 50 μL of a 5 mg mL-1) solution of MTT in uncompleted media and treated for 3 h at 37°C in 5% CO2. The media was carefully removed from each well and 1 mL of DMSO and placed in room temperature for 20 min. The plates were gently agitated until the color reaction was uniform and the OD570 was determined using a spectrophotometer. The amount of MTT converted to formazan is a sign of the number of viable cells. Media-only treated cells served as the indicator of 100% cell viability. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the control in the MTT assay. Viability percentage was evaluated as ODtreatment/ODcontrol (Razavi et al., 2010b).
RESULTS AND DISCUSSION
Table 1 presents the results of antibacterial and antifungal assays. This finding revealed that M. longifolia extracts indicated considerable antimicrobial activity. The methanolic extract of the plant leaves were active against all tested bacteria and fungi. The highest inhibitory effect was observed against E. carotovora, a common plant pathogen bacterium, with MIC value of 128 μg mL-1 and inhibition zone of 41 mm. The extract also exhibited high antibacterial activity with MIC value rang of 192-512 μg mL-1 and inhibition zone of 34-40 mm against S. aureus, E. faecalis, S. agalactiae and E. coli. The methanol extract of M. longifolia was found to have inhibitory effect against meticillin resistant strain of S. aureus (E38), as well as.
Moreover, the methanolic extract of the plant displayed modest to strong antifungal activity against C. kefyr, C. albicans, S. sclerotiorum and A. niger with MIC value range of 576-800 μg mL-1 and inhibition zone of 26-33 mm (Table 2).
| Table 1: Antibacterial effects of M. longifolia extracts | |
| Table 2: Antifungal effects of M. longifolia extracts | |
The finding of present study has also showed that dichloromethanic and hexanic extract have no considerable antimicrobial effect against tested microorganisms.
On the other hand, our finding showed that M. longifolia methanol extract has modest cytotoxic activity. The extract reduced the viability of McCoy cells with IC50 value of 1.92 mg mL-1 (Fig. 1).
It was previously documented that methanol, ethanol and water extracts of M. longifolia have a good antimicrobial activity (Ikrani and Inam-ul-haq, 1980; Jawad et al., 1988; Akroum et al., 2009). However, no activity was reported against E. coli, S. aureus and C. albicans in these studies. Our finding depicted that the methanolic extract of the plant is very active against the mentioned human pathogen microorganisms. Therefore, it can be concluded that Iranian sample of M. longifolia has a great antimicrobial potential than other samples and could be regarded as a specific chemotype of the species.
Phytochemical studies revealed the presence of flavonoides in great quantity in M. longifolia (Ghoulami et al., 2001; Akroum et al., 2009). Many flavonoides like quercetin, luteolin, apigenin and kaempferol glycosylated derivatives were found in the plant leaves. It was realized that this compound are responsible for high antimicrobial activity of the plant methanol, ethanol and water extracts. It can be also concluded that high antimicrobial activity of the methanolic extract than dichloromethanic or hexanic ones should be attributed to prescience of flavonoides as polar compounds. It has also been shown that different glycosylated flavonoides exert a synergism effect in antimicrobial activity. Moreover, nonpolar volatile compounds like menthol isolated from M. longifolia essential oil were found to have strong antimicrobial activity (Al-Bayati, 2009).
| Fig. 1: | Cytotoxic activity of methanolic extract of M.
longifolia on Mc-Coy cell line using MTT test, Bars indicate standard
error |
|
The results herein reported showed that the plant methanolic extract has a great potential to suppress a meticillin resistant strain of S. aureus (E38). In the last decades, continuous using of antibiotics has caused some pathogen microorganism species ultimately evolved resistance to some antibiotics. The utilize of medicinal plant extracts or plant derived chemicals with antimicrobial activity could dissolve the problem.
The results of present study showed that M. longifolia methanol extract possess modest cytotoxic and antiproliferative activity. It is assumed that this biological effect of the plant extract is dependent to presence of phenolic compounds like flavonoides. A previous paper also demonstrated the M. longifolia extracts indicated high antioxidant potential (Nickavar et al., 2008). Antioxidants may act as free radicle scavengers which suppress the free radicle damages in biological systems and then may be associated with a reduced risk of cancer. Therefore, M. longifolia can be regarded as chemopreventive agent against cancer.
On the hand, Present findings showed that the M. longifolia extract exhibited a good inhibitory effect against plant pathogen microorganisms, E. carotovora and S. sclerotiorum. The former microorganism is a pathogen bacterium causes soft rot in a wide range of fruits and vegetable species and is considered as a serious problem in horticulture (Strange, 2003). The later organism causes stem rot in many plants and it is one of the most prevalent plant pathogen fungi. Therefore, the extract can be used as a biopesticide. Recently, the use of synthetic pesticides is claimed to negatively affect the environment and actually it dose not represent an appropriate tool for the control of plant pathogens developing resistance. So, natural derived materials can be exploited as an alternative for synthetic pesticides (Razavi and Nejad-Ebrahimi, 2009).
CONCLUSION
It was concluded that M. longifolia can posses antiseptic activity. The utilizing of this plant in treatment of sore throat and throat irritation in the Iranian folk medicine is validated scientifically by the results obtained in the present study.