Analytical grade chemicals were purchased from Fisher Scientific (UK) and Merck (Germany). Dimethyl sulfoxide (DMSO) and H2O2 were purchased from Univar (Australia). High performance liquid chromatography (HPLC) grade chemicals and standards, gallic acid, quercetin, rutin, colchicine and mitomycin C were obtained from Sigma Chemical Co. (UK). HPLC grade acetonitrile was purchased from F S Chemicals (India). Ultrapure water used was purified using the Milli-Q-plus filter system by Millipore (USA).
Fresh Coriandrum sativum roots, leaves and stems were purchased from the wet market in Selayang, Kuala Lumpur, Malaysia. The plant was identified by Dr. M. Sugumaran, Institute of Biological Sciences, University of Malaya. A voucher specimen (KLU47742) was deposited in the University of Malaya Herbarium. C. sativum roots were separated from the leaves and stems using a knife. The plant parts were washed under running tap water to remove dirt and soil and finally rinsed with distilled water. The plant parts were freeze-dried, weighed, ground into fine powder and stored at -20°C until extraction.
Preparation of plant extracts
Powdered roots, leaves and stems of C. sativum were extracted through sequential extraction using hexane, dichloromethane, ethyl acetate, methanol and water. Briefly, powdered roots (20 g) and powdered leaves and stems (120 g) were extracted in 100 and 600 ml of hexane (1:5 w/v), respectively, for 6 h at 40°C on a hotplate with stirring. Extracts were then filtered through filter paper Whatman no. 1 and the resulting plant residues were re-extracted twice with fresh hexane. The remaining plant residue was then extracted using dichloromethane, followed by ethyl acetate, methanol and water (three times in each solvent). Each filtrate (except for the aqueous extract) was concentrated to dryness under reduced pressure at 40°C using a rotary evaporator. The aqueous extract was concentrated to dryness in a freeze-dryer. The dried extracts were stored at -20°C.
For use in cell culture treatment, the dried extracts were dissolved in DMSO and diluted in ultrapure water to obtain stock solutions which were sterile filtered through 0.2 μm syringe filters. Stock solutions were diluted in ultrapure water to make appropriate extract concentrations for testing. The final concentration of DMSO in the cell culture reaction mixture was less than 1%. Extracts were kept at 4°C.
Determination of total phenolic content
Total phenolic content (TPC) of C. sativum extracts was determined using the Folin-Ciocalteau method  with some modifications. Briefly, 500 μl of 1:10 Folin-Ciocalteau phenol reagent was added to 10 μl of sample (dissolved in 10% DMSO), standard or positive control. The mixture was allowed to stand for 5 min before the addition of 350 μl of 10% sodium carbonate (Na2CO3). The resulting reaction mixture was incubated in the dark at room temperature (RT) for a further 2 h. Absorbance was then measured at 765 nm using a spectrophotometer. Gallic acid (50–500 mg/l in 10% DMSO) was used as the standard. Rutin and quercetin were used as positive controls. Results were expressed in milligrams of gallic acid equivalents (GAE) per gram dried extract. All experiments were carried out in triplicate.
Ferric reducing antioxidant power (FRAP) assay
The antioxidant activity based on the ferric reducing ability of C. sativum extracts was estimated based on the assay by Benzie & Strain  with some modifications. A working reagent was prepared fresh by mixing 10 ml of 300 mM acetate buffer with 1 ml of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM of hydrochloric acid (HCl) and 1 ml of 20 mM FeCl3.6H2O. The freshly prepared FRAP reagent was pre-warmed at 37°C for 5 min after which a blank reading was taken at 595 nm using a plate reader. Subsequently, 3 μl of sample, standard or positive control (each dissolved in 10% DMSO) and 9 μl of water was added to 90 μl of the FRAP reagent. Absorbance readings were measured instantly upon addition of the FRAP reagent and again at 4 min after the start of the reaction. The change in absorbance in the 4 min reaction was calculated by comparison to the absorbance changes of FeSO4.7H2O against a standard curve (100–1000 μM) tested in parallel. Rutin and quercetin were used as positive controls. Results were expressed as mmol ferric reducing activity of the extracts per gram of dried extract. All experiments were carried out in triplicate.
DPPH radical scavenging activity
Radical scavenging activities of C. sativum sequential extracts were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay  with some modifications. The extract (20 μl) was added to 120 μl of 0.04 mg/ml DPPH solution in methanol. The extracts tested ranged from 0–5000 μg/ml (dissolved in 10% DMSO). The mixtures were mixed well and incubated in the dark for 30 min. The reduction of DPPH absorption was measured at 515 nm using a plate reader. Rutin and quercetin were used as the positive controls. All determinations were performed in triplicate. The DPPH radical scavenging activity was calculated using the following equation:
The IC50 value is the concentration of the plant extract required to scavenge 50% of the total DPPH radicals available.
Cell lines and cell culture
The human breast adenocarcinoma cell line, MCF-7, and the human mammary epithelial cell line, 184B5, were used in the anti-proliferation study. Assays of antioxidant enzymes and caspase activities, cell cycle analysis, and inhibition of cell migration were performed using MCF-7 cells. Mouse fibroblast cells, 3 T3-L1, were used in the comet assay. All cells were purchased from the American Type Culture Collection (ATCC), USA. MCF-7 cells were routinely cultured in RPMI-1640 (Sigma, UK). 3 T3-L1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, USA). 184B5 cells were cultured in Mammary Epithelial Basal Medium (MEBM) and supplemented with bovine pituitary extract (BPE), hydrocortisone, human epidermal growth factor (hEGF) and insulin using Mammary Epithelial Cell Growth Medium (MEGM) SingleQuots from Lonza, USA. All cells were supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were grown at 37°C in a humidified incubator with 5% CO2.
The inhibition of MCF-7 cell proliferation by C. sativum extracts was estimated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide assay as described by Mosmann . The ethyl acetate extract of C. sativum root showed the best antiproliferative activity on MCF-7 and was assessed for its toxicity on the human mammary cell line, 184B5. Briefly, cells supplemented with 5% FBS were seeded (5 × 103 cells/well) in 96-well microtiter plates and cultured at 37°C in a humidified atmosphere of 5% CO2. After 24 h of incubation, the cells were treated with various concentrations of extract (0–500 μg/ml) for another 48 h. Vehicle-control wells with cells only and diluent-control wells with similar DMSO concentrations as in treatment were included. At the end of the incubation period, 10 μl of 5 mg/ml MTT bromide in phosphate-buffered saline (PBS) was added to each well. The plates were re-incubated for a further 4 h after which media and MTT were removed by aspiration. DMSO (100 μl) was added to each well to dissolve the formazan crystals. Absorbance was read using a microtiter plate reader at 595 nm. All measurements were performed in triplicate. The percentage inhibition of cell proliferation was calculated by the following formula:
Estimation of antioxidant enzymes
Preparation of cell lysate
MCF-7 cells were seeded in a 6-well plate (1.5 × 106 cells/well) in RPMI-1640 supplemented with 5% FBS. After 24 h, cells were treated with the ethyl acetate extract of C. sativum roots at a final extract concentration of 200 μg/ml (IC50 concentration as determined from the MTT assay) in the well. DMSO was used to replace extracts in the untreated control samples. Cells were treated for 0, 6, 9, 15, 24 and 48 h. Following incubation, cells were washed with PBS, harvested using a cell scraper and collected for centrifugation at 1,000 rpm for 5 min at 4°C. The supernatant was discarded and the cell pellet resuspended in cold PBS. The number of cells was determined by the trypan blue dye exclusion method. Cells were then lysed while on ice using a sonicator and centrifuged for 10 min at 6,000 × g at 4°C. The supernatant was used in antioxidant enzyme assays.
Superoxide dismutase (SOD) assay
The SOD activity in MCF-7 cells treated with test samples was assayed in triplicate using the superoxide dismutase assay kit by Cayman Chemical (USA). The assay uses a tetrazolium salt for detection of superoxide anion radicals generated by xanthine oxidase. The assay was performed according to the manufacturer’s protocol. SOD activity was calculated using the following equation:
One unit is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide anion radical. SOD activity was expressed in U/ml per 106 cells.
Glutathione peroxidase (GPx) assay
The GPx activity in MCF-7 cells treated with test samples was assayed in triplicate using the glutathione peroxidase assay kit by Cayman Chemical (USA). This assay measures GPx activity indirectly by a coupled reaction with glutathione reductase. The assay was performed according to the manufacturer’s protocol. GPx activity was calculated using the following equation:
One unit is defined as the amount of enzyme that causes the oxidation of 1 nmol of NADPH to NADP+ per min at 25°C. GPx activity was expressed in nmol/min/ml per 106 cells.
Catalase (CAT) assay
The CAT activity in cells treated with test samples was assayed in triplicate using the catalase assay kit by Cayman Chemical (USA). The assay is based on the reaction of CAT with methanol in the presence of H2O2 which produces formaldehyde. Formaldehyde is measured colorimetrically using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. The assay was performed according to the manufacturer’s protocol. CAT activity was calculated using the following equation:
One unit is defined as the amount of enzyme that will cause the formation of 1 nmol of formaldehyde per min at 25°C. The CAT activity was expressed in nmol/min/ml per 106 cells.
Colorimetric assays of caspase-3, -8 and -9
A time- and dose-dependent study of caspase-3, -8, and -9 activities in MCF-7 cells was performed in triplicate using Caspase-3/CPP32, FLICE/Caspase-8 and Caspase-9 colorimetric assay kits by BioVision (USA). The test assays for the activities of caspase-3, -8 and -9 that recognise the amino acid sequence, DEVD, IETD, and LEHD, respectively. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (p NA), which is released from labeled substrates after cleavage by caspase. In a 6-well plate, MCF-7 cells (1.5 × 106 cells/well) were seeded with RPMI-1640 containing 5% FBS. After 24 h of incubation, cells were treated with C. sativum (root) ethyl acetate extract at a final concentration of 200 μg/ml (IC50) and 276 μg/ml (IC70) in the well. DMSO was used in place of the extract for control wells. Colchicine for caspase-3 and mitomycin C for caspases-8 and -9 at 1 μM were used as positive controls. Cells were treated for 6 and 24 h and then harvested. Caspase activities of cell lysates were assayed according to the manufacturer’s protocol. Briefly, in a 96-well plate, 50 μg of protein sample was diluted in 50 μl of cell lysis buffer and 50 μl of 2× reaction buffer (containing 10 mM DTT) was added into each well. For the caspase-3 assay, 5 μl of 4 mM DEVD-p NA substrate (200 μM final concentration) was added into wells and the mixture incubated at 37°C for 2 h. For analysis of caspase-8 and caspase-9, the substrates IETD-p NA and LEHD-p NA, respectively, were used. The absorbance of the wells was read at 405 nm. The data was presented as fold change.
Cell cycle analysis
To determine the distribution of extract-treated MCF-7 cells in different phases of the cell cycle, DNA content in cells was detected by propidium iodide (PI) staining and flow cytometry. MCF-7 cells were cultured in 75 cm2 flasks at a density of 4 × 106 cells in 5% FBS. After 24 h of incubation, cells were treated with the ethyl acetate extract of the roots, at a final concentration of 276 μg/ml (IC70) in the flask for another 24, 48 and 72 h. Untreated control samples were performed using DMSO to replace extracts. The cells were collected, washed and fixed in 70% ethanol at -20°C overnight. Cells were then washed in PBS, stained in 500 μl of PI/RNase staining buffer (Becton Dickinson, USA) and incubated in RT for 15 min in the dark. Cell cycle phase distribution was determined using BD FACSCanto II flow cytometer instrument and BD FACSDiva software (Becton Dickinson, USA). A total of 15,000 events per sample were collected for analysis. The fluorescence intensity of the sub-G1 cell fraction represents the apoptotic cell population.
Scratch motility assay
MCF-7 cells (3.5 × 105 cells/well) were seeded in a 24-well plate and grown for 24 h. The confluent cell monolayer was then scratched vertically with a pipette tip, washed twice with PBS and incubated with media containing C. sativum (root) ethyl acetate extract (0, 100, 150, 200, 250 and 300 μg/ml) with 5% FBS. H2O2 was added into each well at a final concentration of 1 μM in the cell suspension to stimulate the proliferation and migration of MCF-7 cells. The number of cells in the denuded area were photographed and counted at 0- and 24-h incubation. The experiment was performed in triplicate. The percent inhibition was calculated as described by Sato & Rifkin . Percent inhibition = 100 – [(cell no. in denuded area of sample / cell no. in denuded area of control) × 100].
In a 12-well culture plate, 3 T3-L1 mouse fibroblasts (1 × 105 cells/well) were cultured in DMEM with 10% FBS for 24 h. The cells were pre-treated with the root ethyl acetate extract at concentrations of 100–400 μg/ml in the well for another 24 h. The control was performed using DMSO to replace extracts. After pre-treatment, cells were exposed to 100 μM of H2O2 (final concentration in the cell suspension) for 60 min on ice to induce DNA damage. Following H2O2 exposure, cells were harvested using a cell scraper, centrifuged and resuspended in 1 ml of PBS for use in comet assay . Briefly, 25 μl of the cell suspension was mixed with 75 μl of 0.6% low melting agarose. The suspension was spread on a frosted microscopic slide pre-coated with 250 μL of 0.8% normal melting agarose, covered with a cover slip, and then allowed to solidify on ice for 10 min. The cover slips were removed and the slides were immersed in cold lysis solution containing 1% sodium dodecyl sulfate, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100 and 10% DMSO (with the DMSO added just before use) for one hour at 4 °C in the dark. Then, slides were arranged in an electrophoresis tank filled with pre-chilled electrophoretic buffer (1 mM Na2EDTA and 300 mM NaOH) and incubated for 20 min. Electrophoresis was conducted in the same buffer, in a horizontal chamber, at 25 V (300 mA) for 20 min using a power supply (CBS Scientific company, USA). The slides were washed with 0.4 M Tris–HCl (pH 7.5) and stained with 20 μg/ml ethidium bromide for viewing under a BX50 fluorescence microscope (Olympus, Japan). Electrophoresis of the samples separates intact DNA from damaged fragments. The comet tail length is associated with DNA damage. Greater tail length signifies greater DNA damage . A total of 50 individual cells were screened per slide . The assay was carried out in triplicate. The comet tail length was measured using an ocular micrometer.
Results were expressed in% DNA protection calculated by the following formula:
Identification of compounds
High performance liquid chromatography (HPLC) analysis
The C. sativum (root) ethyl acetate extract was subjected to acid hydrolysis to release free polyphenols from their glycosides according to the method of Nuutila, Kammiovirta, & Oksman-Caldentey , with slight modifications. Briefly, 20 mg of dried extract in 0.4 ml of 6 N HCl and 1.6 ml of HPLC grade methanol with 20 mM butylated hydroxytoluene (BHT) as antioxidant was heated at 90°C for 2 h. The mixture was centrifuged at 10,000 rpm for 5 min and the supernatant was filtered through a 0.2 μm syringe filter and stored at 4°C for HPLC analysis. HPLC analysis was performed using a SPD-20A HPLC system (Shimadzu, Japan). Reverse phase separation was performed at 40°C using a Purospher STAR RP-18 endcapped column (5 μm) (Merck, Germany). The mobile phase consisted of trifluoroacetic acid in ultrapure water at pH 2.6 (solvent A) and acetonitrile (solvent B). The gradient program consisted of: 0% to 12.5% B for 2.5 min, 12.5% to 100% B for 17.5 min and 100% B for 10 min. The flow rate was kept at 1 ml/min and injection volume was 10 μl. The chromatogram peaks were detected at 254 nm. Data acquisition and processing was performed using LCsolution software (Shimadzu, Japan). The compounds were identified by comparing the retention times of peaks with standards. The extract was then spiked with the standards to confirm their presence. Unidentified peaks were collected manually and the mobile phase was air dried. The dried fraction (referred to as fraction S1) was stored at 4°C for analysis with GC-MS.
Gas chromatography–mass spectrometry (GC-MS) analysis
Prior to GC-MS analysis, chemical derivatisation was performed to reduce the polarity of functional groups by reconstituting 400 μg of the dried fraction S1 (unidentified peaks collected from HPLC) with 500 μl of HPLC grade ethyl acetate and 20 μl of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and heated at 70°C for 40 min. The GC-MS analyses were carried out in a GCMS-QP2010 system (Shimadzu, Japan) fitted with a ZB-5 (30 m × 0.25 mm i.d.) capillary column (Phenomenex, USA). The carrier gas was helium with a flow rate of 1.08 ml/min. The column temperature was set at 100°C for 5 min, 100–275°C at 10°C/min, and finally held for 20 min in 275°C. Sample volume injected was 1 μl with a split ratio of 2:1. The injector temperature was 250°C and the detector temperature was 290°C. The MS operating parameters were: ionisation potential, 70 eV, ion source temperature, 200°C, solvent delay, 3.0 min, scan speed, 2500 amu/s, scan range, 40–500 amu and detector voltage, 1.5 kV. Compound identification was verified based on mass spectral data by computer matching with Wiley 229, NIST 107, NIST 21 and PMW_tox2 libraries.
Data are presented as mean ± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons and the Student’s t-test. A P-value of < 0.05 was considered statistically significant. Pearson correlation coefficient was used to assess the correlation between TPC, FRAP and DPPH radical scavenging activity. SPSS, version 18.0 (Chicago, Ill, USA) and Microsoft Excel 2007 (Roselle, Ill, USA) statistical software were used for the statistical and graphical evaluations.