Aromatic plants have been extensively used in the past for culinary purposes and in traditional medicine. Nowadays, there is an increased interest in the pharmacological properties of aromatic plants that can be in part attributed to their essential oils. These are volatile mixtures of secondary metabolites, with a distinct odour, that can be extracted from all plant organs e.g., flowers, buds, stems, bark, leaves fruits, etc., and are soluble only in organic solvents. Traditionally, essential oils have been used for their biological activities, including analgesic, antiseptic, sedative, spasmolytic, anesthetic and anti-inflammatory effects. Because of their stimulant or sedative properties, they are also employed in aromatherapy [1
The plant kingdom has always represented an attractive source for novel therapeutics. Among phytochemicals, essential oils, although known since antiquity, have recently regained interest due to their wide variety of bioactivities. It is only during the last decades that systematic studies have been initiated to explore, in more detail, their bioactive potential and relate it with their phytochemical profile.
In particular, their antimicrobial, antioxidant and anticancer activities are of special interest as they are associated with health-promoting properties. Currently, due to the growing health concerns of the use of synthetic antimicrobials in preventing pathogenic microbes and food spoilage, civil authorities are increasing the pressure on food manufacturers to substitute harmful synthetic preservatives with alternative natural ones. In this context, the use of essential oils with antimicrobial activity represents an attractive alternative. In addition, some essential oils possess antioxidant properties which may have a positive impact on food production by preventing oxidation [3
]. On the other hand, oxidative stress is linked to many pathological conditions being the result of an imbalance between Reactive Oxygen Species (ROS) generation and their metabolism by cellular antioxidants. More specifically, oxidative stress can lead to DNA damage, mutagenicity, genotoxicity, etc., and ultimately contribute to disease development, including carcinogenesis [2
]. Thus, compounds with antioxidant properties exert beneficial effects by protecting cells against oxidative cellular damage and thus acting as “protective shields” against carcinogenesis. It is also true that essential oils have recently gained great interest in their use as anticancer agents, as they have been found to exert their anti-proliferative potential through different mechanisms of action [4
]. Conventional chemotherapy, on the other hand, is compromised by drug resistance and undesirable side-effects. Consequently, there is a need for novel agents with specific toxicity against cancer cells that will enhance the efficacy of standard treatment and may also alleviate any undesired cytotoxic side effects. Quite interestingly, studies have shown synergistic effects of conventional chemotherapeutic drugs when administered together with specific essential oils or some of their major components, responsible for exerting such effects [5
]. This further supports the notion that nutritional intervention with natural phytochemicals, such as essential oils, may be very advantageous in enhancing the therapeutic potential of any existing therapy (e.g., chemotherapy) and furthermore diminish any adverse side effects [9
In the present study, the essential oil volatiles of four widely used aromatic culinary Greek herbs, namely Ocimum basilicum (sweet basil), Mentha spicata (spearmint), Pimpinella anisum (anise) and Fortunella margarita (kumquat) were investigated for their chemical composition and their antimicrobial, antioxidant and antiproliferative properties in vitro. The selected plants are very popular in Greece and of high economic significance. Besides their utilization as spices, they are also used as food ingredients, and in beverages, and are well-known as home remedies in the treatment of different diseases or ailments. Thus, the purpose of this study was to further explore the potential benefits of these plants as a source of naturally occuring bioactive agents.
3. Materials and Methods
3.1. Essential Oil Extraction and GC/MS Analysis
Essential oils were obtained at the VIORYL facilities by hydrodistillation. Chopped leaves and stems of the plant material were used for the species Ocimum basilicum
(collected during the months of May and June), and Mentha spicata
(collected during spring and autumn) without further drying. Seeds of Pimpinella anisum
(collected during mid-summer) and the chopped peel of Fortunella margarita
fruits (collected on the island of Corfu between January and March) were directly treated and processed for hydrodistillation. Following decantation, essential oils were dried over anhydrous sodium sulfate. In all cases, hydrodistillation took place immediately after the harvesting period (respecting seasonality restrictions) so that the plants/seeds/fruit peels would provide the most of their essential oils. A Dean Stark apparatus was used for hydrodistillation [44
] where the studied material was placed along with 6 L of distilled water. After hydrodistillation (8 h, 90–120 °C), the essential oil was isolated. Subsequently samples were dried with Na2
and collected to sealed vials for further use. GC/MS analysis was carried out with a GC-MS (GC: 6890A and MSD: 5973, Agilent Technologies, Santa Clara, CA, USA) using a Factor Four VF 1 ms column (25 m, 0.2 mm i.d., 0.33 μm film thickness, Agilent Technologies). Essential oil (0.1 μL) was directly injected and a 1:100 split ratio was applied. The oven temperature was set at 50 °C for 1 min, followed by a temperature gradient of 2.5 °C/min to 160 °C for 20 min and then 50 °C/min to 250 °C for 15 min. Helium was used as carrier gas (flow rate 1 mL/min). Injector and transfer line temperatures were set to 200 °C and 250 °C, respectively. The mass spectrometer operated in the electron impact mode with the electron energy set to 70 eV. Identification of the compounds was carried out according to the standard method of Kováts Indices.
3.2. Microbial Strains
Salmonella enterica subsp. enterica ser. Enteritidis FMCC Β56 PT4 (kindly provided by G.J.E. Nychas, Agricultural University of Athens, Athens, Greece), Salmonella enterica subsp. enterica ser. Typhimurium DSMZ 554, Listeria monocytogenes NCTC 10527 serotype 4b, Escherichia coli ATCC 25922, Staphylocccus epidermidis FMCC B-202 C5M6 (kindly provided by Nisiotou A., Wine Institute of Athens, ELGO “DEMETER”, Lykovrysi, Greeceand Staphylococcus aureus ATCC 25923 were grown in Brain Heart Infusion (BHI) broth (LABM, Heywood, UK) at 37 °C for 24 h. Saccharomyces cerevisiae uvaferm NEM (Lallemand, Montreal, QC, Canada) was grown in YPD broth (yeast extract 10 g/L, glucose 20 g/L and peptone 20 g/L) at 28 °C for 3 days. Aspergillus niger 19111 (kindly provided by G.J.E Nychas) was grown on malt extract agar (LABM) for 7 days at 37 °C.
3.3. Antimicrobial Assays
The antimicrobial activity of the tested essential oil was monitored using the two following methods [45
3.3.1. Disk Diffusion Assay
For the antibacterial screening, the disk diffusion assay was performed. The bacterial suspensions were 10-fold diluted in ¼ Ringer’s solution (LABM). A 0.1 mL portion from the appropriate dilution was spread on Brain Heart Infusion (BHI) agar (LABM), in order to provide initial inoculums of 105 or 107 cfu/mL. Subsequently, sterile paper disks (Whatman No. 2) of 5 mm diameter were placed onto the inoculated agar surface containing 5 μL (4700 μg spearmint, sweet 4600 μg basil, 4200 μg kumquat, 4800 μg anise) of the essential oils. Petri dishes were incubated at 37 °C for 24 h. After incubation, the diameter of the inhibition zones were measured in mm. The same procedure was also followed for the screening of the activity against yeasts, using S. cerevisiae suspensions 10-fold diluted in ¼ Ringer’s solution (LABM) and spread on YPD agar, which were then incubated at 28 °C for 3 days and then the inhibition zones were measured in mm. For the antifungal activity, 100 fungal spores/plate from A. niger were spread on Malt Extract agar (LABM) and the above procedure was followed. The diameter of the inhibition zones were measured daily in petri dishes were incubated at 37 °C for 10 days. Ciproxin (5 μg) (Oxoid Ltd., Basingstoke, UK) was used as positive control for bacteria and amphotericin B (10 μg) (Mast Group Ltd., Merseyside, UK) for yeast and fungi. Sterile water was used as negative control. All experiments were carried out at least in triplicate, and the mean values are presented.
3.3.2. Determination of Minimum Inhibitory Concentration (MIC) and Non-Inhibitory Concentration (NIC)
Determination of MIC and NIC values was carried out as recently described [45
]. In brief, bacterial growth in BHI broth (LABM) was monitored through changes in optical density of bacterial suspensions in the presence of multiple concentrations of essential oils. Stock solutions (ranging 43–9300 mg/L) of the essential oils were prepared by mixing them directly with BHI broth. Aliquots (0.180 mL) of growth medium mixed with the essential oils were transferred to the wells of a 96-well microplate. The bacterial suspensions were diluted tenfold in ¼ Ringer’s solution and a 0.070 mL portion from the appropriate dilution was added to the wells containing the growth medium (final volume 0.250 mL), in order to result in a population of approximately 103
cfu/mL. Microplates were incubated in Microplate Reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA, Softmaxpro v. 5.0 software) at 37 °C for 24 h. Optical density measurements were carried out every 10 min at 610 nm. Ciproxin (positive control) stock solutions (0.5–4 mg/L) were prepared by mixing the antibiotic directly with BHI broth. BHI broths with no inoculum and inoculated BHI broths with no essential oils were used as negative controls. The calculation of MIC and NIC values was based on the Lambert-Pearson model (LPM) [14
]. In brief, the effect on the growth, measured by the optical density method, is manifested by a reduction in the area under the OD/time or curve relative to control well at any specified time. By calculating the area using the trapezoidal rule, the relative amount of growth were obtained using the ratio of the test area to that of the control, termed the fractional area, fa. Data were fitted to the LPM using non-linear least squares regression analysis assuming equal variance.
3.4. Cell Lines and Cell Cultures
The human hepatocellular carcinoma HepG2, the human breast adenocarcinoma MCF-7, the human colon adenocarcinoma Caco2 and the human leukemic monocytic THP-1 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). HepG2 and MCF-7 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MD, USA) while Caco2 and THP-1 cells were cultured and maintained in RPMI-1640 medium (Gibco), both supplemented with 10% fetal bovine serum (FBS), and penicillin (100 U/mL) (Biosera, Boussens, France) and were incubated at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. Stock cultures were passaged at 2- to 3-day intervals. Cells were seeded at a density of 3.0–5.0 × 103 cells/well in 96-well plates for the sulforhodamine B (SRB) assay. THP-1 cells were seeded at a density of 2.0 × 103 cells/well in round bottom 96-well plates for the XTT assay.
3.5. Antioxidant Activity
3.5.1. DPPH Assay
The radical scavenging activity of the essential oils was estimated using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), as described previously with few modifications [46
]. Different concentrations of the essential oils (basil oil (0.0049–49 mg/mL), anise oil (0.00485–48.5 mg/mL), kumquat oil (0.0043–43 mg/mL) and spearmint oil (0.0048–4.8 mg/mL)) were prepared using dimethyl sulfoxide (DMSO, Biotium, Fremont, CA, USA) as the solvent. Ten microliters of each concentration were placed in a 96-well plate, and 190 μL of 300 μM methanolic solution of DPPH (Calbiochem, Darmstadt, Germany) was added. Ten microliters of DMSO with 190 μL DPPH was used as the control. Ascorbic acid was used as a positive control (Sigma-Aldrich, St. Louis, MO, USA). The plate was left in darkness for 30 min, and then the absorbance was measured at 517 nm using an Elisa plate reader (EnSpire Multimode Plate Reader, Perkin Elmer, Waltham, MA, USA). The % inhibition of the DPPH radical for each concentration was determined by making use of the following formula: % DPPH radical scavenging activity = [(ODcontrol
)] × 100.
3.5.2. ABTS Assay
The ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] decoloration assay was performed as described previously with few modifications [47
]. Seven mmoles of ABTS (Sigma-Aldrich) dissolved in water were mixed with 2.45 mM potassium persulfate (final concentration, Sigma-Aldrich), and were let to stand in the dark for 16 h in order to allow the formation of the ABTS radical cation (ABTS•+
). The cation was further diluted in ethanol (Scharlau, Barcelona, Spain) in order to obtain absorbance of 0.8 at 734 nm. Different concentrations of the essential oils were prepared in DMSO. Ten microliters of each concentration were placed in a 96-well plate, and 190 μL of ABTS•+
was added. Ten microliters of DMSO with 190 μL ABTS•+
was used as the control. The plate was left in darkness for 15 min, and then the absorbance was measured at 734 nm using an Elisa plate reader (EnSpire Multimode Plate Reader, Perkin Elmer) against a standard curve with ascorbic acid. The % inhibition of the ABTS•+
cation for each concentration was determined by making use of the following formula: % ABTS•+
radical scavenging activity = [(ODcontrol
)] × 100. Furthermore, the results are expressed as micromoles ascorbic acid equivalent per gram of essential oil (mmolEA/g).
3.6. Cell Viability Assays
3.6.1. Sulforhodamine B Assay
The viability of the cancer cell HepG2, Caco2, and MCF-7 after treatment with the essential oils was determined using the SRB assay. SRB is a dye that binds to basic amino acids of cellular proteins and, then, the number of viable cells is estimated with colorimetric evaluation [48
]. Cells were plated in 96-well plates and treated with different concentrations of the essential oils [basil oil (0.00068–0.98 mg/mL), anise oil (0.00068–0.97 mg/mL), kumquat oil (0.0006–0.86 mg/mL) and spearmint oil (0.00067–0.96 mg/mL)] (dissolved in DMSO, 1:1 v
) for 72 h. The anticancer drug etoposide (Sigma-Aldrich) was used a positive control. Then, the cells were fixed with the addition of 25 μL of 50% (w
) cold trichloroacetic acid (TCA) (MP Biomedicals, Santa Ana, CA, USA) to the growth medium and incubation of the plates at 4 °C for 1 h. The cells were washed five times with tap water and then stained with 50 μL of 0.4% (w
) SRB (Sigma-Aldrich) in 1% (v
) acetic acid (Scharlau) for 30 min at room temperature. Then, the cells were rinsed five times with 1% (v
) acetic acid to remove the unbound dye. The fixed, stained plates were allowed to air-dry followed by solubilization of the bound dye by adding 100 μL of 10 mM Trizma base (Sigma-Aldrich) for at least 5 min. Absorbance was measured at 570 nm using an Elisa plate reader (EnSpire Multimode Plate Reader, Perkin Elmer), and the percent cellular survival was calculated using the formula: [(sample OD570
− media blank OD570
)/(mean control OD570
− media blank OD570
)] × 100.
3.6.2. XTT Cell Viability Assay
The viability of THP-1 cells was determined by the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S
-(phenylamino)carbonyl-2-tetrazolium hydroxide) assay [49
]. In all experiments, the XTT Cell Viability kit (Cell Signaling, Danvers, MA, USA) was used according to the manufacturer’s protocol. Briefly, cells were seeded in a 96-well-plate. After overnight incubation, cells were treated with increasing concentrations of essential oils (dissolved in DMSO, 1:1 v
) for 72 h. Control cells were treated with DMSO-containing medium at concentration <0.1% v
. The anticancer drug etoposide (Sigma-Aldrich) was used a positive control. At the end of the incubation, the XTT solution was added, and plates were placed in the incubator for 4 h and then absorbance was measured at 450 nm with a microplate reader (EnSpire Multimode Plate Reader, Perkin Elmer).
3.7. Data Analysis
All experiments were performed at least in triplicate. For MIC and NIC determination, each experiment was performed at least 4 times and standard deviation was calculated by Fig. P software (Fig.P Software Incorporated, Hamilton, ON, Canada). Significance was established at p < 0.05 and the results were analyzed for statistical significance with analysis of variance (ANOVA). Duncan’s multiple range test was used to determine significant differences among results (coefficients, ANOVA tables and significance (p < 0.05) were computed using Statistica v.5.0). The EC50 values (Effective Concentration; the concentration of test samples required to cause decrease of cancer cell viability by 50%) were calculated from the respective dose-response curves by regression analysis using a four-parameter logistic curve through the Sigma Plot Software v.10 (Systat Software Inc., San Jose, CA, USA).
The present work reports a comparative study of the chemical composition and the biological potential of essential oil volatiles from four widely used aromatic plants: Mentha spicata, Ocimum basilicum, Pimpinella anisum and Fortunella margarita, all grown in Greece. Chemical analysis by GC/MS showed that carvone, methyl chavicol, trans-anethole and limonene were the major components of Mentha spicata, Ocimum basilicum, Pimpinella anisum and Fortunella margarita, respectively. All essential oil preparations showed activity against the fungi Saccharomyces cerevisiae and Aspergillus niger, but only Mentha spicata and Ocimum basilicum were cytotoxic against common foodborne bacteria. Antioxidant evaluation by DPPH and ABTS radical scavenging activity assays revealed a variable degree of antioxidant potency. All essential oil preparations exhibited antiproliferative activity that also varied depending on the cancer model used, with the most potent one being Ocimum basilicum against an in vitro human colon carcinoma model. Further studies are required to correlate specific biological properties with active chemical components and/or possible compound synergy effects. In conclusion, it is of great interest to screen commonly used plants from the local flora for potent biological activities. Besides being safe (widely used for generations) and easily available, they could represent a new alternative source of bioactive substances for various applications in the food, pharmaceutical, neutraceutical and other industries.