Anticandidal and anti-carcinogenic activities of Mentha longifolia (Wild Mint) extracts in vitro

2.1 Preparation of plant extracts

Leaves of M. longifolia were obtained from a local market in Riyadh, Saudi Arabia. Identification of plant material was performed by the Saudi herbarium of the Botany Department, College of Science, King Saud University, where vouchers were deposited (KSU_14683). Extraction procedure was performed using four organic solvents (diethyl ether, ethyl acetate, methanol and n-hexane) with different polarities to allow extraction of all phytochemical active components. Leaves of M. longifolia were disinfected using 0.5% sodium hypochlorite (NaOCl), washed using sterile distilled water for three consecutive times, and allowed to dry. Leaves were then macerated using a mechanical mortar to obtain a homogenous powder. 50 g of plant powder were soaked into four 500 mL Erlenmeyer flasks, each containing 200 mL of the four different organic solvents. Each flask was stirred over a magnetic stirrer at 25 °C for 48 h and then centrifuged at 9000 rpm for 10 min to get rid of plant residues. Supernatant of each solvent was filtered using Whatman filter paper no. 1 to attain clear filtrates. Finally, the clear filtrates were concentrated using a rotatory evaporator and kept at 4 °C until use. Calculation of the extract yields was carried out according to the following equation: Extract yield = (R/S) × 100;Where R is the weight of the plant extract residue and S is the weight of raw plant sample.

2.2 Fungal strains

Candidal strains (C. albicans, C. tropicalis and C. glabrata) were obtained from the culture collection of Botany and Microbiology Department, College of Science, King Saud University. The fresh inoculum was prepared by subculturing each Candida species onto Sabouraud dextrose agar (SDA) medium at 35 °C for 48 h.

2.3 Preparation of candidal inocula

Previously prepared SDA slants were inoculated with different Candida species. The Candida specimens grown on the SDA slants were harvested using a sterile saline solution. The absorbance of microbial suspension was adjusted at 560 nm using a spectrophotometer to obtain a viable cell count of 10⁷ colony forming unit (CFU)/mL for each Candida species.

2.4 Screening of the antifungal activity of different M. longifolia extracts

The disk diffusion method was performed to evaluate the antifungal activity of different M. longifolia extracts against different Candida strains. M. longifolia extracts were screened for their anticandidal activity in which 15 mL of SDA medium was poured into sterile petri dishes (base layer) followed by 10 mL of SDA containing a Candida suspension (1 mL of Candida for each 100 mL of medium to obtain a viable cell count of 10⁵ CFU/mL). Sterile filter paper disks (8 mm in diameter) were loaded with 10 mg of M. longifolia extracts then placed over the seeded layer once it had solidified (Mansourian et al., 2014; Mostafa et al., 2018). Fluconazole antifungal drug was used as a positive control with a concentration of 25 µg/disk according to the Clinical & Laboratory Standards Institute (CLSI, 2007). Fungal susceptibility to fluconazole, as an antifungal drug, was determined based on the inhibition zone diameter as follows: for C. albicans and C. tropicalis, a diameter ≥19 mm was interpreted as sensitive, 15–18 mm as dose dependent, and ≤14 mm as resistant; while for C. glabrata, a diameter ≥15 mm was interpreted as dose dependent, and finally a diameter ≤14 mm was interpreted as resistant. Plates were incubated at 4 °C for 2 h to allow the M. longifolia extracts to diffuse throughout the medium. Finally, plates were incubated at 35 °C for 48 h and the inhibition zone diameter was measured using Vernier caliper. Inhibition zone diameters were recorded and considered as an indication of antifungal efficiency of the mint extracts against different candidal pathogens.

2.5 Determination of the minimum inhibitory concentration (MIC)

The lowest concentration of M. longifolia extract exhibiting anticandidal activity was identified as the minimum inhibitory concentration (MIC). The MIC was evaluated for the n-hexane extract of M. longifolia as it showed the highest antimicrobial efficiency. For such, 15 mL of SDA medium was poured into sterile petri dishes (base layer) and 10 mL of seeded SDA was poured on top and left to solidify. Sterile filter paper disks (8 mm in diameter) with different concentration of n-hexane extracts of M. longifolia (62.5, 125, 250, 500, 1000, 2000 µg/disk) were placed over the seeded medium. Plates were stored at 5 °C for 2 h to allow the mint extracts to diffuse. Finally, plates were incubated at 35 °C for 48 h and the inhibition zone diameters were measured. MIC was recorded for the different Candida strains tested.

2.6 Determination of the minimum fungicidal concentration (MFC)

The lowest concentration of the M. longifolia n-hexane extract in which pathogens show no growth is defined as MFC. Freshly prepared SDA plates were inoculated with streaks taken from the inhibition zone area of the MIC and the two successive concentrations. Plates were then incubated at 35 °C for 48 h and examined for candidal growth. The lowest concentration of the n-hexane extract of M. longifolia showing no Candida growth was recorded as the minimum fungicidal concentration (MFC).

2.7 Cytotoxicity assay

Human breast cancer (MCF7) cell lines were obtained from the collection of the zoology department, college of science, King Saud University, Saudi Arabia. The anti-cancer activity of Mentha longifolia (diethyl ether, ethyl acetate, methanolic and n-hexane) extracts against MCF7 cell lines was evaluated using the 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay (Famuyide et al., 2019). The cells were cultured in minimal essential medium (MEM, Highveld Biological, South Africa) supplemented with 0.1% gentamicin (Virbac) and 5% foetal calf serum (Adcock-Ingram) in a 5% CO₂ incubator. Incubation of MCF7 cells in 96-well plate at 37 °C for 24 hr. in 5% CO₂ incubator to allow adherence of cells. The crude M. longifolia extracts were dissolved in methanol (10 mg/mL), and appropriate dilutions were prepared. MCF7 cells were treated with the M. longifolia extracts at concentration range of (0.0065–1 mg/ml). After a 48hr exposure, removal of supernatant was performed and MTT solution was added to the wells at a concentration of 5 mg/ml. Incubation of plates at 37 °C for 4 hr and then supernatants were removed. Finally, 50 μl of DMSO was added to the wells to allow the solubilization of the formed formazan crystals. Absorbance was measured at wavelength of 570 nm. The concentration causing 50% inhibition of cell viability (IC₅₀) was calculated.

2.8 Gas chromatography–mass spectrometry (GC–MS) of M. longifolia extracts

Phytochemical analysis of the extracts was carried out using (an Agilent 7890 gas chromatograph and an Agilent 5975C Mass Spectrometer, USA) to investigate the phytochemical active components of different M. longifolia extracts. The gas chromatograph was set as follows: a HP-5MS UI capillary column (30 m × 0.25 mm; 0.25 μm film thickness), helium carrier gas with a flow rate of 1 mL/min, an oven temperature of 40 °C and adjusted to 250 °C at 5 °C/min, an injection volume of 1 μl, injector and detector temperatures of 250 °C, and a split ratio of 1:50. The mass spectrometry was set as follows: an ionization potential of 70 eV, a mass range from 40 to 400 amu, and an electron multiplier energy of 2000 V. Identification of the phytochemical components of leaf extracts of M. longifolia was determined by comparing the results of the GC–MS analysis with the reference retention time and spectral mass data of the NIST database.

3.1 Extract yields

The highest yield of M. longifolia extracts was obtained using methanol (5.49%) as solvent, followed by diethyl ether (3.82%), ethyl acetate (1.27%), and n-hexane (0.29%).

3.2 Antifungal activity

In our current study, C. albicans strain expressed resistance to fluconazole and that was coincident with the results reported by Nasrollahi et al., (2015) in which 94% of C. albicans strains isolated from vaginitis patients were resistant. Moreover, the resistance of C. glabrata strain to fluconazole drug was detected and the previous result was similar to that of Hasanvand et al., (2017) who reported the fluconazole resistance of three C. glabrata strains isolated from vaginitis patients.

Screening of the antifungal potency of ethyl acetate, diethyl ether and n-hexane extracts of M. longifolia exhibited a high efficiency at concentration of 10 mg/disc against different candidal strains with different susceptibility patterns. The M. longifolia extract using n-hexane as a solvent showed the highest antifungal activity against C. albicans, C. glabrata, and C. tropicalis, with inhibition zone diameters of 27.43, 16.33, and 22.63 mm, respectively (Table 1). These results were in accordance with that of Carretto et al. (2010) who proved the antimicrobial activity of Mentha sp. against Candida strains at concentration range of 7.8–250 mg/ml with inhibition zone diameters ranging from 5 to 10 mm.

On the other hand, the methanolic extracts of M. longifolia exhibited no antifungal activity against the three Candida strains which was ascertained by Gulluce et al., (2007) who found that methanolic extracts of M. longifolia leaves have no antimicrobial activity against C. albicans. In contrast, the diethyl ether extract of M. longifolia showed high antifungal activity against all three Candida strains with inhibition zone diameters of 14.93, 12.17, and 21.67 mm for C. albicans, C. glabrata, and C. tropicalis, while a moderate efficacy was detected for the ethyl acetate extract against the tested strains with inhibition zone diameters of 13.43, 13.20, and 16.57 mm respectively. Furthermore, the antifungal activity of the n-hexane extract of M. longifolia against non-albicans strains (C. glabrata and C. tropicalis) was significantly higher (p < 0.05) than that of fluconazole (control).

3.3 Determination of the minimum inhibitory concentration (MIC) of the M. longifolia n-hexane extract

The n-hexane extract of M. longifolia was the most effective extract against the concerned Candida strains and its MIC against C. albicans was 500 µg/disk, a value lower than that previously ascertained by Bakht et al., (2014) who reported MIC value by 2 mg/disk. MIC of n-hexane extract against non-albicans strains (C. tropicalis and C. glabrata) was 250 and 500 µg/disk respectively (Table 2). MIC results confirmed that C. tropicalis exhibited the highest sensitivity to the n-hexane extract of M. longifolia (Fig. 1).

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Fig. 1

Minimum inhibitory concentration (MIC) of the Mentha longifolia n-hexane extract against the three Candida pathogens tested.

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3.4 Determination of the minimum fungicidal concentration (MFC) of the M. longifolia n-hexane extract

The n-hexane extract of M. longifolia was tested for MFC against the three Candida strains used in order to detect the lowest concentration of the extract that shows no fungal growth. The minimum fungicidal concentration was achieved at 0.5 mg/disk against C. albicans and C. tropicalis while it was 1 mg/disk against C. glabrata. These results were contrasted with the previous study which reported the fungicidal activity of Mentha sp. extract at concentration range of 0.5–8 μl/mL against Candida pathogens (Saharkhiz et al., 2012).

3.5 Cytotoxicity assay

Methanolic extract exhibited the highest cytotoxic activity against MCF7 breast cancer cell line with IC₅₀ of 91.67 µg/ml (Fig. 2). Our results were in accordance with that of Al-Ali et al., (2013) who reported that the methanolic extract of M. longifolia possess potential anti-cancer activity against MCF7 cell line at concentration range of 20–320 µg/ml. Diethyl ether extract exhibited the lowest activity against MCF7 cell line with IC₅₀ of 244.7 µg/ml. Moderate cytotoxic activities of ethyl acetate and n-hexane extracts were detected with IC₅₀ of 136.3 and 183.2 µg/ml respectively. Our results were in accordance with that of Sharma et al. (2014) who reported the anticancer activity of Mentha sp. extracts against eight human (breast, colon, glioblastoma, lung, leukemia and prostate) cancer cell lines. Stringaro et al. (2018) ascertained that Mentha spp. extracts were a potential source of novel anticancer agents due to their high activity against different cancer cell lines.

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Fig. 2

IC₅₀ of different M. longifolia extracts against MCF7 cancer cell line.

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3.6 GC–MS analysis of M. longifolia extracts

Phytochemical analysis of the M. longifolia n-hexane extract exhibiting the highest anticandidal activity showed that menthone (18.37%) was the main phytochemical active component followed by butyloctanol (16.13%), 1,5-(1-bromo-1-methylethyl)-2-methyl-2-cyclohexen-1-one (14.89%), α-tocopherol (13.13%), eugenol (12.21%), α-resorcylamide (11.56%), citronellal (3.71%), butylated hydroxytoulene (3.21%), isocaryophyllene (2.81%), 2-hexyldecanol (2.12%), and tau-cadinol (1.87%) (Table 3). These components were similar to that reported by Okut et al. (2017) who reported that the chemical composition of M. longifolia extract was compromised of Menthone as a predominant chemical constituent (19.31%) followed by Pulegone (12.42%), Piperitone (11.05%), Dihydrocarvon (8.32%), Limonene (6.1%), 3-Terpinolenone (5.66%), 1,8-Cineole (4.37%), Germacrene D (3.38%) and Caryopyllene (3.19%), respectively. Other study reported that wild mint oil was mainly composed of menthone (34.13%) (Salman et al., 2015). Variation in the chemical composition of wild mint between our findings and previous studies may be attributed to a variety of factors, such as geographic variation, harvest time, environmental and agronomic conditions, the botanical parts of plants, and extraction methods (Kurihara et al., 2003; Fang et al., 2011; Xi et al., 2014; Bessada et al.,2016; Liu et al., 2016).

The reported potential antimicrobial activity of M. longifolia can be attributed to the presence of oxygenated monoterpenes in their phytochemical compounds (Mimica-Dukić et al., 2003; Şahin et al., 2003). However, Samber et al. (2015) reported that the main phytochemical components of Mentha sp. essential oil are menthone and carvone, and both of them act as antifungal agents due to their effect on ergosterol of cell membrane biosynthesis leading to microbial cell death.

Moreover, the diethyl ether extract of M. longifolia that exhibited a moderate anticandidal activity against the tested strains was composed of 3,5-dihydroxybenzamide (42.89%) as a main phytoactive component, followed by 5-(7a-isopropenyl-4,5-dimethyl-octahydroinden-4-yl)-3-methyl-pent-2-en-1-ol (11.03%), isoledene (10.91%), butylated hydroxytoluene (9.20%), epizonarene (4.92%), lycopersen (5.27%), epizonarene (4.92%), pyrrolo [3,2-d]pyrimidin-2,4(1H,3H)-dione (3.69%), naphthalene,1,1/-(1,10-decanediyl)bis*decahydro (3.37%), 1,1-naphthyl isocyanide (2.84%), benzenemethanol,3-hydroxy-5-methoxy- (2.09%), phosphinic acid, diisopropyl-,menthyl ester (1.89%), and phytol acetate (1.89%) (Table 4).

The M. longifolia ethyl acetate extract that exhibited a moderate antifungal activity against the concerned Candida strains whose GC–MS analysis showed that piperitone (15.75%), menthone (15.28%), and citronellal (14.87%) were the main active components (Table 5). Other study attributed the potent anticandidal activity of M. longifolia to their constituents of citronellal which could be used as a chemotherapeutic agent for the treatment of VVC or RVVC (Medeiros et al., 2017). The n-hexane and ethyl acetate extracts of M. longifolia were rich in menthone with percentage of 18.37% and 15.28% respectively. Menthone is a phenolic monoterpene that was reported to possess hydrophobic characteristics causing disruption of microbial selective permeability through its incorporation into microbial cell membranes (Saharkhiz et al., 2012).

The methanolic extract of M. longifolia exhibited the highest cytotoxic activity against MCF7 breast cancer cell line so can be used as a potential source of novel anticancer therapeutic agents. The main phytoactive compounds of methanolic extract were caryophyllene, t-cadinol, menthone, eugenol and menthol crotonate (Table 6). The potential anti-cancer activity of M. longifolia extracts may be attributed to their constituents of eugenol which possess the ability to induce apoptosis against the cancer cells Jaganathan and Supriyanto (2012).