Plant material
Based on ethnopharmacological data and using the help of traditional medical practitioners, fresh leaves of Melissa officinalis L. were collected, in January 2018, in Dhamna Village, located 10 km from the city of El Jadida, Middle Morocco. The plant was identified with botanist of the Laboratory of Quality control in Bioindustry and Bio-active Molecules, Faculty of Science, Chouaib Doukkali University, a voucher specimen (no. RAB76712) was deposited in the Herbarium of Botany Department of Scientific Institute of Rabat.
Leaves were dried at room temperature and ground to a fine homogeneous powder using an electric grinder prior to extraction.
Preparation of plant extracts
Soxhlet extractions of Melissa officinalis leaves (40 g) were conducted with n-hexane and dichloromethane (600 mL) for 8 h. The ethanol extract was obtained by maceration of 10 g of powdered material in 100 mL of ethanol for 7 days. The extracts were concentrated to dryness by rotary evaporation and the residues were stored at 4 °C for subsequent experiments.
Chemicals and reagents
Acetic acid, acetonitrile, n-hexane, lithospermic acid, and methanol were obtained from E. Merck (Darmstadt, Germany). Caffeic acid, 3,4-dihydroxyphenyl lactic acid, 3,4-dihydroxybenzoic acid, luteolin-7-O-glucoside, methyl caffeate, and rosmarinic acid from Extrasynthese (Lyon Nord, Genay, France). All solutions were made up in either doubly distilled water, or methanol, unless otherwise stated.
Gas chromatography-mass spectrometry (GC-MS)
This was conducted by the methods of Owen et al. (2000) on an Agilent 5973 mass quadruple spectrometer coupled to an Agilent 6890 gas chromatograph [19].
Analytical HPLC
Analytical HPLC was conducted as described by us previously [20].
HPLC-ESI-MS
HPLC-ESI-MS was conducted exactly as assayed by us previously [21] on an Agilent 1100 HPLC coupled to an Agilent single-quadrupole mass-selective detector (HP 1101; Agilent Technologies, Waldbronn, Germany). Chromatographic separations of extracts were dissolved in methanol and HPLC-MS separations were conducted using a column of the same type and dimensions as for analytical HPLC (Phenomenex, Aschaffenburg, Germany).
Phenolic compounds were detected by their UV absorbance (A) at 278 and 340 nm at 30 °C. Negative-ion mass spectra, were generated under the following conditions: fragmentor voltage, 100; capillary voltage, 2500 V; nebulizer pressure, 30 psi; drying gas temperature, 350 °C; m/z scan range, 100–1500 D. Positive-ion spectra, were generated under the following conditions: fragmentor voltage, 200; capillary voltage, 1500 V; nebulizer pressure, 30 psi; drying gas temperature, 350 °C; m/z scan range, 100–1500 D. For HPLC-ESI-MS-MS experiments, in negative-ion mode, the fragmentor voltage was increased to 300. Quantitation of the polyphenolic compounds was conducted against standard curves (optical absorbance vs. concentration) in the range 0.05–1.0 mM (50, 100, 250, 500, 750, and 1000 μM) prepared using the following authentic commercial samples, namely caffeic acid, 3,4-dihydroxyphenyl lactic acid, 3,4-dihydroxybenzoic acid, lithospermic acid, luteolin-7-O-glucoside, methyl caffeate and rosmarinic acid. Cis-rosmarinic acid, methyl rosmarinate, and rosmarinic acid glucoside were quantitated against the standard curve of rosmarinic acid, whereas isolithospermic acid, methyl isolithospermate, and methyl lithospermate were quantitated against the standard curve of lithospemic acid with relevant molecular weight corrections. Instrument control and data handling were performed with the same software as for analytical HPLC.
Cell culture
Human prostate adenocarcinoma PC3 and LNCAP and breast adenocarcinoma MCF7 cancer cell lines were used in this study. Cells were kindly provided by Dr. L’Houcine Ouafik (Laboratoire de transfert d’oncologie, Marseille). PC3, and MCF7 cells were maintained in DMEM medium and LNCAP cells were cultured in RPMI medium. The medium was supplemented with 10% (v/v) fetal calf serum and 1% penicillin/streptomycin mixture (10,000 IU/mL). All cell lines were kept under standard conditions of temperature (37 °C), humidity (95%) and carbon dioxide (5%), and subcultured at 80% confluency.
Evaluation of cell viability by MTT assay
The cytotoxic effect of ethanol and dichloromethane extracts from Melissa officinalis on three tumor cell lines was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay [22]. Briefly, cells in the exponential growth phase were plated in 96-multiwell plates, at a cellular density of 8000 cells/well in 0.1 mL medium. After 24 h, 100 μL of fresh medium, containing serial concentrations ranging from 12.5 to 400 μg/mL of each extract (dissolved in DMSO) was added to the cells and incubated for 72 h at 37 °C. At the end of treatment, 10 μL MTT (5 mg/mL) were added to each well, and plates were reincubated for an additional 4 h at 37 °C. The purple-blue MTT formazan precipitate was dissolved in 100 μL DMSO. The reduced MTT was spectrophotometrically analyzed at 570 nm using a microplate reader. Untreated cells were considered as a negative control, mitomycin C was used as a positive control. Experiments were conducted in duplicate. The percentage of cytotoxicity and cell viability were calculated using following equations:
$$ \%\mathrm{Viability}=100-\%\mathrm{Cytotoxicity} $$
$$ \%\mathrm{Cytotoxicity}=1-\left(\mathrm{mean}\ \mathrm{absorbance}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{cells}/\mathrm{mean}\ \mathrm{absorbance}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}\right). $$
Statistical analysis
The data represented in cytotoxic study are mean SEM of two identical experiments made in duplicates; the statistical differences between the treatments and the positive control were tested by One-way analysis of variance (ANOVA), followed by a multiple comparison to assess the difference of the IC50 values of the same extract on different cell lines. p < 0.05 was considered to be statistically significant. The IC50 calculations, statistical analysis and graphs plots were done using Graph Pad Prism Data Editor for Windows, Version 6.0 (Graph Pad software Inc., San Diego, CA).