Chemicals and essential oils
The essential oil (EO) of basil was purchased from KATO Aromatic Co., Giza, Egypt. Thyme EO was purchased from Canada Essential Oils Co. LTD, Canada. Both oils were prepared by hydro-distillation. The major components, thymol, euganol, linalool and carvacrol, were delivered from Sigma–Aldrich Chemicals. α-Tocopherol (vit E) and ascorbic acid (vit C) were obtained from Fluka Chemical company. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Aldrich chemical company. Other chemicals were pure reagent grade.
GC–MS identification and quantification
Separation of essential oil components has been carried out in 1 µL of sample solution (10 μg/mL) in hexane:diethyl ether (ratio 1:1) by GC–MS (Shimadzu-QP-2010S plus) instrument equipped with [AOC−20i + s] autosampler autoinjector and a capillary column (Rtx-1 30 m × 0.25 mm I.D., 0.25 μm). The oven temperature program was adjusted for an initial temperature of 60 °C for 1 min followed by a 4 °C/min temperature ramp to 260 °C. The final temperature was maintained for 1 min. Injector and mass interface temperatures were adjusted at 260 °C. The initial head pressure of the helium carrier gas (He) was 51.4 kPa and injection mode was split (ratio 1:10); the column flow was 2.62 mL/min with linear velocity 58.7 cm/sec and purge flow 4.1 mL/min. The mass parameters were set as following: ion source temp. 210 °C, solvent cut time 5.00 min, MS detector (EI mode) with start time 5.0 min and end time 51.3 min; the compounds were acquired by scan mode ACQ start m/z 75 and end m/z 600. Auto injector was set to be pre-rinsed with washing solvent for 3 times and post rinsed 3 times then rinsed with samples two times with rinse volume 6 µL in high plunger washing speed. Injector port Dwell time was 0.3 s. The integration was performed by Lab Solution software 4.1. Samples were injected in triplets to test the stability and reproducibility of the column. Identification was based on both comparing spectra against NIST 11 s library and interpreting spectra.
Determination of DPPH Scavenging activity
DPPH radical scavenging activity was determined according to the method of Brand-Williams et al. [20] with some modifications. The reaction mixture contained 0.1 mL of DPPH solution (5 mM) and different concentrations of tested samples ranged from 1 to 200 ppm. The total volume of the reaction mixture was 3 mL (final DPPH concentration 65.85 µg/mL). Absorption of DPPH radical at 517 nm was measured at different intervals up to reaching a plateau/steady state for each reaction against a blank solution (contains no DPPH). Methanol was used instead of sample in control. The results were calculated as follows:
$${\text{\% DPPH radical scavenging activity}}\,{ = }\,\left( {\left( {{\text{Ac}} - {\text{As}}} \right){\text{/Ac}}} \right)\,\, \times \,\,{100}{\text{.}}$$
where As and Ac are absorbance of sample and control, respectively. Time required to reach the steady-state (TEC50) is determined for each compound by plotting time vs. % scavenged DPPH.
The efficient concentration of antioxidant necessary to decrease the initial DPPH radical concentration by 50% (EC50) was calculated from means of three determinations of each concentrations using nonlinear- sigmoidal regression for dose/response four-parameter correlation implemented in OriginPro 2019b. Finally, the EC50 and TEC50 values were used to calculate the Antiradical Efficiency (ARE), expressed as mg antioxidant/g DPPH at the steady-state, as follows [21]:
$${\text{ARE}}\,{ = }\,{{1} \mathord{\left/ {\vphantom {{1} {({\text{EC}}_{50} }}} \right. \kern-\nulldelimiterspace} {({\text{EC}}_{50} }}\, \times \,{\text{T}}_{{{\text{Ec50}}}} )$$
To standardize DPPH results, the antioxidant activity index (AAI), proposed by Scherer and Godoy [22] was calculated as follows:
$${\text{AAI}}\, = \,[{{{\text{DPPH concentration in reaction mixture}}\,\left( {{{\mu {\text{g}}} \mathord{\left/ {\vphantom {{\mu {\text{g}}} {{\text{mL}}}}} \right. \kern-\nulldelimiterspace} {{\text{mL}}}}} \right)} \mathord{\left/ {\vphantom {{{\text{DPPH concentration in reaction mixture}}\,\left( {{{\mu {\text{g}}} \mathord{\left/ {\vphantom {{\mu {\text{g}}} {{\text{mL}}}}} \right. \kern-\nulldelimiterspace} {{\text{mL}}}}} \right)} {EC_{50} \left( {{{\mu {\text{g}}} \mathord{\left/ {\vphantom {{\mu {\text{g}}} {{\text{mL}}}}} \right. \kern-\nulldelimiterspace} {{\text{mL}}}}} \right)}}} \right. \kern-\nulldelimiterspace} {EC_{50} \left( {{{\mu {\text{g}}} \mathord{\left/ {\vphantom {{\mu {\text{g}}} {{\text{mL}}}}} \right. \kern-\nulldelimiterspace} {{\text{mL}}}}} \right)}}].$$
Antioxidant potency is classified according to AAI as poor (AAI < 0.5), moderate (0.5 < AAI < 1.0), strong (1.0 < AAI < 2.0) and very strong (AAI > 2.0) antioxidant activity.
Determination of hydrogen peroxide scavenging activity
Hydrogen peroxide scavenging activity was determined according to the reported method [23] with some modification. The reaction mixture is composed of 1 mL hydrogen peroxide solution (35.4 mM) and different concentrations of samples ranged from 12.5 to 62.5 ppm with total volume of the reaction mixture 3 mL. Absorption of hydrogen peroxide at 230 nm was determined after 3 min against a blank solution without hydrogen peroxide while methanol substituted the sample in the control experiment.
The percentage of scavenging activity was calculated as follows:
$$\% \,\,{\text{of}}\,\,{\text{scavenging}}\,\,{\text{of}}\,{\text{H}}_{{2}} {\text{O}}_{2} = \,(1 - ({\text{As}}\,{\text{/Ac}}) \times 100$$
As and AC are absorbance of sample and control, respectively.
Reducing power method (RP)
The reducing power was determined according to the method of [24]. A volume of 0.4 mL of ethanolic solution of each sample with different concentrations, 12.5, 25, 37.5, 50 and 62.5 ppm was added to 1 mL potassium ferricyanide (1%) and 1 mL phosphate buffer (0.2 M, pH 6.6). The reaction mixture was incubated at 50 °C for 20 min then 1 mL trichloroacetic acid (10%) was added to the mixture and centrifuged at 650 ×g for 10 min. Two mL of the supernatant was mixed with 2 mL distilled water and 0.4 mL ferric chloride (0.1%) then absorbance was read at 700 nm. All experiments were performed in triplicates. Absorbance is used as reducing power indicator where higher absorbance of the reaction mixture indicates greater reducing power.
Evaluation of anticancer activity in tissue cells
Cytotoxicity of the thyme and basil oils was tested by sulforhodamine B (SRB) assay using the method of Skehan [25]. Tumor cell lines were U251 (Brain tumor cell line) and HEPG2 (liver carcinoma cell line) in addition to THLE2 normal liver cell line which is used as negative control. Doxorubicin (DOX) was used as positive control. Cells were plated in 96-multiwell plate (104 cell/well) for 24 h before treatment with the essential oils to allow attachment of cell to the plate wall. Different concentrations of the essential oils or DOX (0, 1, 2, 5, and 10 µg/mL) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the essential oils for 48 h at 37 °C under atmosphere of 5% CO2. After 48 h cells were fixed with 50% trichloroacetic acid (TCA) for one hour then washed five times with tap water; plates were air dried and then stored until use then stained with 20% SRB stain. Excess stain was washed with 1% acetic acid and attached stain was recovered with Tris–EDTA buffer. Color intensity was measured in an ELISA reader. The relationship between surviving fraction and drug concentration is plotted to get the surviving curve of each tumor cell line with essential oil.
Computational methods
All calculations were performed at the level of DFT/ B3LYP with a basis dataset 6-311-G (d,p) using the Gaussian 09 package. HOMO energy was generated by Huckel calculations. Calculations of parent compounds were executed at restricted closed shell level while calculations of radicals were accomplished at unrestricted open shell level. Harmonic vibrational frequencies were computed for thermochemical correction of electronic thermal enthalpies of all species. Bond dissociation energy (BDE), ionization potential (IP), proton dissociation energy (PDE), proton affinity (PA), electron transfer energy (ETE) were calculated by subtracting the electronic thermal enthalpy of the products from those of reactants according to Eqs. 1, 2, 3, 4, 5, respectively. Electron enthalpy was used as calculated by Fermi–Dirac statistics [26]. Correction factor is considered as suggested by the program manual to eliminate systematic error in thermal energies of frequency calculations. Spin density and charges were calculated by NBO calculations.
Statistical analysis
Analysis of variance one-way ANOVA using Duncan's multiple range test at significant level p ≤ 0.05 was computed by SPSS statistics package version 22.0.
