Chemical Composition, Antimicrobial Activity and Potential Cytotoxic Effect of Mentha viridis (Spearmint) Extracts from Saudi Arabia

Many medicinal plants have been used to treat and prevent illnesses in Saudi Arabia. The present study aimed to investigate the chemical composition of Mentha viridis obtained from Albaha region of Saudi Arabia and evaluate its antimicrobial and antiproliferative potential. The extract was obtained from plant fresh material and identified by gas chromatography-mass spectrometry (GC-MS). The antimicrobial and antiproliferative potential of the plant extract was analysed by performing four subsequent extracts: ethanol, petroleum ether, chloroform, and methanol. The GC-MS analysis showed carvone as a main component, as it comprised 64.82% of the plant extract. In antimicrobial activity, methanol extract showed significant activity against Pseudomonas aeruginosa with zone of inhibition of 15 mm. The MTT assay showed that petroleum ether and chloroform extracts have moderate cytotoxic effect against MCF-7 breast cancer cell line with IC50 values of 193.23 μg/mL and 131.86 μg/mL, respectively. Chloroform extract also showed mild activity against HCT-116 colorectal cancer cell line with IC50 value of 189.2 μg/mL. This study highlights the potential of M. viridis extracts as powerful bioactive phytochemicals with possible role in diseases and cancer therapy.


INTRODUCTION
Presently, the effectiveness, low cost, and fewer side effects have increased the worldwide demand of medicinal plants. Many pharmaceutical companies are engaged in large-scale pharmacologic screening of medicinal plants for developing new drugs 1 . Medicinal plants are rich resources of traditional medicines and many modern medicines, including aspirin, digoxin, quinine and morphine are obtained from willow bark foxglove, cinchona bark, opium poppy, respectively 2 . The effectiveness of medicinal plants has been proved on individual body systems, for example, they have profound antioxidant, anti-inflammatory, antimicrobial and immunostimulatory properties 1 .
Mentha species is a member of the Lamiaceae (Labiatae) family and are mass distributed across all continents 3,4 . According to latest data, Lamiaceae family is considered one of the largest families of plants that produce flowers with around 4000 species that grow worldwide 5 and is considered to have the highest number of medicinal plants 6 . The use of Mentha plants in treating many diseases, including common cold, fever, throat infection, bronchitis, ulcerative colitis, and digestive issues has been known for a long time 7,8 . Moreover, its use as an antimicrobial, antioxidant, anti-motion sickness, anti-inflammatory and anticarcinogenic agent has also been reported . [9][10][11] Mentha viridis, commonly known as spearmint, has well known industrial importance. The leaves of the M. viridis are used as flavouring agent in culinary purposes including iced drinks and jellies 12 .
The medicinal uses of M. virids are also well documented. It is considered as a relaxant, antispasmodic, and soothing agent in nausea and vomiting 13 . Moreover, it is widely known as a strong stimulant and carminative 14 . M. viridis extract contains various terpenes, fatty acid esters and Vitamin E, which explains its antioxidant potential 15 . The essential oil from leaves of M. viridis has potent antimicrobial activity 16 .
Previously, no study has been reported on M. viridis cultivated in Albaha region. Its medicinal properties and safe usage have made it an ideal option for studying. In addition, plants growing in different geographical and weather conditions tend to have different phytochemical composition and different biological activities. This study aims to characterize the bioactive compounds of M. viridis from Albaha region and investigate its extracts for antimicrobial and antiproliferative potential.

Plant materials
The plant was obtained from local farmer market in Albaha region, Saudi Arabia in March 2019. Dr. Haider authenticated its botanical identification. An authenticated specimen was deposited at the Botany Laboratory, Department of Biology, Albaha University.

Preparation of samples for GC-MS analysis
The leaves and stem (representing fresh aerial parts of the plant), with a weight of 25 g, was transferred to 15-mL screw test tube, mixed with 5 mL methanol, capped, vortexed for 5 min, sonicated for 30 min, mixed with about 2 g anhydrous sodium sulfate, filtered through a filtration disc of the PTFE syringe, 0.22 micron thickness. The produced filtrate is then concentrated to 1 mL using room temperature Nitrogen gas in the form of a gentle stream. The extract contains both polar and nonpolar components of the plant material. A portion from the clear extract was transferred to autosampler vial. GC-MS analysis sample was prepared by injecting 1.5 μl into the vial. In this assay, Helium (purity 99.9999%) was used as a carrier gas, with 0.90 mL/min flow rate. Source (EI+): source was set to 215˚C temperature, GC inlet line was set to 265˚C temperature, with 70 eV Electron energy, and 100V trap-emission. The oven programming went as follows: 50°C temperature initially (with a 5 min hold), then raised to 260˚C (at a rate of 10˚C/min, with 5 min hold), then raised again to 280˚C (at a rate of 10˚C/min, with 2 min hold). Temperature of the injector was set to 265°C, 1.0 μL was injected, and a 50:1 ratio was used for splitting. A total MS scan from 40 to 500 m/z (500 scan/sec) was applied to acquire the sample. The eluted compounds were characterized using NIST 2008, as reported in Mosbah et al.,(2018).

Extraction of crude extracts
The leaves and stem (representing fresh aerial parts of the plant) were air dried at room temperature (25 ± 2 o C) for about 7 days. The dried material was ground using an electric blender machine (Pulverizer HR-30B, USA). 200 g powder material was macerated in 600 mL methanol with shaking for 3 days. Then they were filtered through Whatman no1 filter paper. The residue was further extracted two times by using the same fresh solvent. All filtrates were compiled for further evaporating. The resulting residue was air dried and further extracted with solvents of increasing polarity namely petroleum ether, chloroform, and ethanol by using similar procedure carried out for the methanol extraction. Finally, rotary evaporator (IKA RV-10, Germany) was used under reduced pressure and low temperature to evaporate solvent from each filtrate extract until dryness was achieved. 19

Antimicrobial evaluation
The antimicrobial activity of the four plant extracts (methanol, ethanol, petroleum ether, and chloroform) was tested against standard strains of four bacteria and one fungus. King Abdulaziz University Hospital, Jeddah, KSA provided the organisms through their microbiology laboratory. These strains were: Staphylococcus aureus (reference: ATCC 29213) and Bacillus subtilis (Reference: ATCC 6633), both Gram-positive bacteria, as well as Escherichia coli (Reference: ATCC 35218) and Pseudomonas aeruginosa (Reference: ATCC 27853), both Gram-negative bacteria and finally fungus: Candida albicans (Reference: ATCC 76615).
Agar diffusion technique was used for the initial screening of the antibacterial and antifungal activities, as previously described (20). Briefly, Muller-Hinton agar (25 mL) containing 1 mL bacterial culture (1 × 10 6 CFU/mL) was used to fill Petri dishes with a capacity of 90 mm. The strains were inoculated separately. Seven holes (4 mm in diameter) were prepared in the seeded agar dishes, which were then filled with 50 μL of each extract (10 mg/mL), as well as a negative control agent (10% dimethyl sulfoxide (DMSO)). Dishes were then incubated at 37°C for 24 hours. Success of the Inhibitory activity was marked by the absence of bacterial growth in the area surrounding the holes. Triplicates were carried out against each of the tested microorganisms. The growth inhibition zones' diameters were measured using a calliper and averaged at the end of the incubation period. The mean values were tabulated.

Acquisition of cell lines and culture medium preparation
Human breast cancer MCF-7 and colorectal cancer HCT-116 cell lines were obtained from Dr. Thikryat Neamatallah, Pharmacology and Toxicology laboratory, Faculty of medicine, King Abdulaziz University, Jeddah, KSA. Dulbecco's Modified Eagles Medium (DMEM)/high glucose medium was used as a culture medium, augmented with 10% fetal bovine serum (FBS), as well as 10,000 units/mL penicillin/ streptomycin (Pen/Strep) and 1% glutamine). All reagents were purchased from Thermo Fisher (Thermo Fisher Scientific UK Ltd, Leicestershire, UK) except Pen/Strep, which was obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA). The cell lines were cultured in 75 cm 2 flasks were used to culture the cells, and were sustained at 37°C in a 5% CO 2 humid incubator. A Class II Safety Flow Hood was used to carry out the cell culture procedure, under aseptic conditions.

MTT assay
Both types of cells were seeded at (1 × 10 5 cells/mL) into a plate containing 96 wells, together with 3 duplicates. The whole assay was incubated at 37°C through the night for attachment in a humid atmosphere containing 5% CO 2 as described by Mansour et al., (2016), but slightly modified. In this assay, plant extracts (methanol, ethanol, petroleum ether, and chloroform) at 7 serial dilutions (1000-10 μg/mL) were introduced in 3 identical settings (triplicates) and incubated for 72 h, at a temperature of 37°C, and 5% CO 2 . 0.1% DMSO was used as a vehicle to dissolve the drugs in. Untreated cells were used as control. Afterwards, 100 μL of full medium containing 10% of 3-(4,5-dimethylthiaxolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (10 mg/ mL) was used as replacements for each well at each recorded time point. Cells were incubated again at a temperature of 37°C, and 5% CO 2 for 4 hours. 100 μL of DMSO was added after removing the media, and incubation at a temperature of 37 o C, and 5% CO 2 was done for an additional 5 minutes. Spectra Max M3 plate reader at 570 nm was used to quantify the plates.
The following for mula was used to determine the viability percentage: Cell viability (%) = (A of treated cells/A of control cells) ×100.

Statistical analysis
Triplicates of three independent experiments were carried out and data are expressed as the mean ± SD. IC 50 value was calculated by ED50 GraphPad Prism software (GraphPad Prism 5.0, GraphPad Software, Inc., CA, USA).

GC-MS Analysis
Th analysis showed that aerial parts (stem and leaves) of M. viridis methanolic extract had a variety of phytochemicals, which are shown in the GC-MS chromatograms (Fig. 1). Thirty-two distinct phytochemical compounds were present in M. viridis methanolic extract in different ratio and accounted for 98.6% of the total components. It was found that carvone was the most abundant phytochemical, as it was present in 64.82% followed by eucalyptol (10.44%), oleamide (6.34%) and phytol (5.40%). Table 1 shows the identified components in M. viridis methanolic extract. Fig. 2 illustrates the chemical structure of some active components detected in M. viridis methanolic extract.

Antimicrobial Activities
The In vitro antifungal and antibacterial bioassays were carried out using the extracts from M. viridis. A variety of bacteria (both Grampositive and Gram-negative) as well as fungal strains were used as test strains and agar diffusion assays were carried out to analyse the antimicrobial properties of M. viridis. The results were determined on the basis of formation of zones of inhibition (measured in mm) around the extract on the agar plates which were seeded with test microbial strains. The methanolic extract showed significant antibacterial activity against P. aeruginosa with zone of inhibition of 15 mm while ethanol, chloroform and petroleum ether extracts did not show any antimicrobial activity against the tested microorganisms. The results are given in Table 2.
The diameters of the inhibition zones. Values are the means ± SD of three cultures.

MTT (IC 50 ) Assay
The antiproliferative effect of the extract of M. viridis on human cells' viability was analysed using MTT assay. Two cancer cell lines, human breast cancer (MCF-7) and colorectal cancer (HCT-116) cell lines were selected and tested against M. viridis extracts. Readings were taken using spectrophotometer Spectra Max M3 and are given in Table 3. Petroleum ether and chloroform extracts showed moderate cytotoxic effects against MCF-7 with IC 50 values 193.23 μg/mL and 131.86 μg/mL, respectively. Chloroform extract also showed mild activity against HCT-116 with IC 50 value of 189.2 μg/mL. Ethanol and methanol extracts showed IC 50 higher than 200 μg/mL against the tested cell lines, which is indicative of no cytotoxicity.  GC-MS profiling of our study also indicated the presence of fatty acid methyl esters (tridecanoic acid, linolenic acid) terpenoids and terpenoid alcohol (eucalyptol, phytol). Most of the plant terpenoids and their derivatives are biologically active and are used extensively as traditional herbal remedies for many diseases 24 . They are also used worldwide in food, cosmetics and pharmaceutical industries 25 .

Antimicrobial Activities
The significant antimicrobial activity was only shown by methanol extract whose concentration was 10 mg/ mL, forming an inhibition zone as of 15 mm when interacting with P. aeruginosa. However, no antimicrobial activity was noted by other extracts (10 mg/mL) obtained from M. viridis against the tested microorganisms. The results are similar to those by Mkaddem et al., (2009) who stated that no antimicrobial activity of essential oil extract (15 μl/ mL) from Tunisian M. viridis was observed against E. coli and S. aureus. Referring to Silva et al., (2015), the essential oil from M. viridis showed antimicrobial activity against E. coli (a Gram-ve bacterium) and S.aureus (a Gram+ve bacterium) forming zones of inhibition of 6 mm and 8 mm, respectively, when the minimal inhibitory concentration (MIC) was 62.5 μl/ mL. Other study showed that MIC of 12.5 mg/mL is the lowest concentration at which all the tested microorganisms are inhibited 16 . Our study found different results, which may be associated with differences in the concentration of essential oil and crude extracts of M. viridis used against the tested microorganisms. In addition, the disagreement was probably due to the difference in amounts and the nature of the components that presented in the essential oil and crude extracts of M. viridis, because Silva et al.,(2015) found linalool (40.70%), carvone (13.52%) and α-terpinene (8.56%) as the chief components.

MTT (IC 50 ) Assay
The antiproliferative effects of M. viridis extracts (10 μg/mL) on human breast cancer (MCF-7) and colorectal cancer (HCT-116) cell lines, were analysed using the MTT (IC 50 ) assay. Chloroform extract showed moderate antiproliferative activity and had IC 50 (μg/mL) values less than 200 against both the MCF-7 and HCT-116 cell lines. Petroleum ether extract showed inhibition effect only against MCF-7 with IC 50 values 193.23 μg/mL. However, no cytotoxic activity was observed in methanol and ethanol extracts against the tested cell lines. Sharma et al.,(2014), evaluated the anticancer potential of methanolic and aqueous extracts of whole plant of M. viridis against MCF-7 and HCT-116 In vitro, using a 100 μg/mL concentration, and sulforhodamine Blue (SRB) assay. Methanolic extract exhibited cytotoxicity against MCF-7 while aqueous extract was found active against HCT-116 27 . Our study found different results, which can be attributed to the difference in doses concentration applied against the tested cancer cell lines.
In this study, it is noticed that the nonpolar solvents (petroleum ether and chloroform) used for the plant extraction exhibited more cytotoxic effects against tested cancer cells. These effects are most likely due to the presence of more bioactive terpenoids and sesquiterpenes in these solvents. It has been reported that carvone and its derivatives do not exhibit cytotoxic effects against cancer cells 28 while many of the plant terpenoids inhibit different human cancer cells and are used as anticancer drugs 25,29,30 .
From the current findings we can safely conclude that extracts from M. viridis can be ideal candidates for novel therapeutic research. The difference observed in the antibacterial and antiproliferative properties of M.viridis between our results and other reported findings is certainly due to the chemical composition, methods used, strains tested, dose concentrations applied, growing conditions and regions. The emergence of novel infectious diseases and development of bacteria resistance against the available antibiotics have made it inevitable that medicinal plants, especially from novel environments, should be explored for their therapeutic potentials. The present study is among the very first studies that have investigated and explored the M. viridis species of Albaha Region, KSA, and it highlights the need and importance of similar studies.

ACKNOwLEDGEMENT
The principal investigator of this study gratefully acknowledges the Deanship of Scientific Research, Albaha University, Albaha, KSA for supporting by grant #17-1439.