Collection and pretreatment of plant material
Aerial fragments of wild M. spicata L. (Lamiaceae) at full flowering phase were collected in May 2017, from the Laghouat region (Algeria). The samples were identified at the Department of Agronomy, Faculty of Science, University of Laghouat (Algeria), and the voucher specimens (LGP Ms1–3/05/17) were deposited at the laboratory of Process Engineering, University of Laghouat. The dried plant material was stored in the laboratory at room temperature (25 °C), protected from light, until extraction.
Isolation of the essential oil
Essential oil was extracted from the dried aerial parts of M. spicata by hydro-distillation using an apparatus of Clevenger type. The extraction was carried out for 4 h to mix 200 g of plants in 1500 mL of distilled water. The extracts were dried with anhydrous sulphate and concentrated under reduced pressure by rotatory evaporator to evaporate water. The pure oil was stored at − 4 °C until further analyzed. The essential oils yield is demonstrated by the oil quality (in mL) obtained for 100 g of dried material.
Physicochemical properties
The physicochemical properties, namely refractive index, specific gravity, color, solubility and acid number, were determined following Association of Official Analytical Chemists (AOAC) standard methods [22]. The refractive index and specific gravity were measured at 20 °C.
Analysis of the essential oil
The essential oil chemical composition assessments and the identification of the main constituents were conducted by GC and GC-MS analyses. GC/MS analyses were performed with a Varian CP-3800 gas-chromatograph equipped with a DB-5 capillary column (30 m × 0.25 mm, coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were as follows: injector and transfer line temperatures 220 °C and 240 °C respectively; oven temperature programmed from 60 °C to 240 °C at 3 °C/min; carrier gas helium at 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. The identification of the constituents was based on the comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial [23] and home-made library mass spectra built up from pure substances and components of known oils and MS literature data [23, 24].
Antioxidant activity
DPPH radical scavenging activity
The antioxidant activity of the essential oil based on the scavenging activity of the stable 1,1-diphenyl-2- picrylhydrazyl free radical was determined by the method described by [25].A volume of 0.1 mL of essential oil from M. spicata prepared at different concentrations was mixed with 1.9 mL of 60 μM DPPH methanol solution. The disappearance of the DPPH was measured at 517 nm after 30 min of incubation at room temperature. The inhibition percentage of the DPPH radical by the essential oil was estimated using the following equation: Scavenging effect (%) = [100*(AC -AS/Ac)], where Ac is the absorbance of the control reaction (containing all reagents except the test sample) and AS the absorbance of the tested sample. The concentration of oil that could scavenge 50% of the DPPH radicals (IC50) was calculated. Ascorbic acid and BHT were used as standards for comparison.
ABTS radical scavenging activity
ABTS radical scavenging activity of M. spicata essential oil was measured by the ABTS cation decolorization assay as described by [26] with some modifications. The ABTS radical cation (ABTS•+) was produced by reaction of 7 mM stock solution of ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in dark at room temperature (25 °C) for 12 h before use. The ABTS•+ solution was diluted with methanol to give an absorbance of 0.7 ± 0.01 at 734 nm.20 μL of essential oil from M. spicata prepared at different concentrations were allowed to react with 1980 μL of the ABTS•+ solution and the absorbance was measured at 734 nm after 6 min. The scavenging rate and IC50 value were calculated using the equation described for DPPH assay. Ascorbic acid and BHT were used as standards for comparison.
Ferric-reducing power assay (FRAP assay)
The reducing power of the essential oil was measured by making use of the method described by [27] with some modifications. 0.1 mL of various concentrations of essential oil in methanol was taken separately and mixed with 2 mL of 0.2 M sodium phosphate buffer (pH 6.6). The diluted sample was then mixed with 2 mL of 1% potassium ferricyanide[K3Fe(CN)6] and the mixture was incubated at 50 °C for 20 min. 2 mL of 10% trichloroacetic acid (TCA) was added to the mixture and centrifuged at 3000 rpm for 10 min. 2 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL of 1% ferric chloride (FeCl3), and the absorbance was measured at 700 nm. The IC50 value is the effective concentration at which the absorbance is 0.5 and is obtained by the equation described for DPPH assay. Ascorbic acid and BHT were used as standards for comparison.
Antimicrobial activity
Microbial strains and growth conditions
Staphylococcus epidermidis ATCC 12228; a representative of Gram positive, Escherichia coli ATCC 29425; a representative of Gram negative and Candida glabrata ATCC 22553; fungi, were obtained from the Microbial Culture Collection Center of Medicine School at The University of Jordan, Jordan. Microorganisms were grown in nutrient broth medium and incubated, with shaking, at 37 °C and at 33 °C, for bacteria and candida respectively.
MIC determination
The minimum inhibitory concentration (MIC) determination, designated as the least concentration at which more than 80% of the microbial growth is inhibited, was carried out in 96 flat bottom microtiter plates (TPP, Switzerland) according to the microdilution method [28, 29]. Each microorganism was inoculated in each microtiter plate well at an inoculum size of 1 × 105 CFU mL− 1. Positive controls, Ampicillin for bacteria and Amphotericin B for fungi, and a negative control of the vehicle (DMSO), were prepared under the same experimental conditions. Bacterial testing plates were incubated for 48 h at 37 °C, whereas Candida plates were incubated for 48 h at 33 °C, with shaking. To evaluate microbial growth, optical densities were measured at 600 nm (OD600) using a Microplate Reader (Palo Alto, CA, USA).
Antiproliferative activity
Cells and cell culture conditions
The human colon cancer HCT-116, the human breast adenocarcinoma MCF-7, and the human ductal carcinoma T47D cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MD, USA) supplemented with 10% fetal bovine serum (FBS), and penicillin (100 U/mL) and were incubated at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. Cell cultures were passaged every 2–3-days or whenever reaching 80% confluent. Cells were seeded at a density of 6-8 × 103 cells/well in 96-well plates and incubated for 24 h for adhesion.
Cell proliferation assay (MTT)
The MTT colorimetric assay was employed to evaluate cell proliferation as previously described [5]. In brief, test samples were prepared by dissolving the essential oil in DMSO followed by further dilution with DMEM medium to reach the desired final concentration. The final DMSO concentration in the assay was kept as low as 0.1%. Test samples containing the desired concentration of the essential oil were added to the wells. After exposure period of 48 h, MTT solution was applied into each well and incubated for 4 h at 37 °C. Afterward, DMSO was added to each well to solubilize the purple formazan crystals formed. Then, absorbance was read using a micro-plate reader at 570 nm. Doxorubicin was used as the positive control and 0.1% DMSO in DMEM media as solvent control.
Statistical analysis
All experiments were conducted in triplicates and results are expressed as mean ± standard deviation (SD), being analysed using a Student’s t-test, with α = 0.05. The analyses were carried out using IBM SPSS Statistics for Windows, Version 22.0. (IBM Corp., Armonk, New York, USA).