2.1 Samples
The Sorghum bicolor (L.), red sorghum bran was collected from the village area (Coimbatore district, Tamil Nadu, India). The plant was identified and authenticated (No.BSI/SRC/5/23/2011-12/Tech.1486) by Botanical survey of India (BSI), Tamil Nadu Agricultural University (TNAU), Coimbatore, India. The bran was collected and was stored at -20⁰C.
2.2 Sample extraction
The bran of red sorghum (200g) was extracted by incubation with 1% Hydrochloric acid in methanol. Overnight at room temperature, followed by a filtration through whatman filter paper no.4. Methanol was removed by a rotary evaporation under 35⁰C and the pigmented fraction extracts were stored for a further study.
2.3 Isolation of anthocyanin
The concentrated filtrates were then applied onto an amperlite XAD -7 resin column (Sigma Aldrich, Missouri, USA) (1.5 x 40 cm), and washed with distilled water, followed by an elution with 1% Trifluoroacetic acid in methanol (4,5). The elution was evaporated again after being collected by a fraction collector. Isolated anthocyanins were analysed on a UV-Visible spectrophotometer (Genesys 5 Technical trade links pvt.ltd). The fractions with the highest absorbance at 480nm were pooled and evaporated to remove methanol and dried under vaccum at 35⁰C.
2.4 Purification of Anthocyanin
5 ml of concentrated isolated methanolic pigments were fractioned by Sephadex LH-20 column (100 cm x 2.6 cm, sigma, USA) using water(0.1% TFA)- Methanol(40:60, v/v) as eluent (4,5). The flow rate was 0.3 mL/min. Fractions were collected by a fraction collector. The fractions were evaporated on a rotary evaporator to remove methanol to facilitate the removal of water remaining in the sample. The evaporation temperature was less than 38⁰C. The homogeneity of individual fractions was checked by HPLC analysis. The lyophilized sample was used for expression studies.
2.5 High – Performance Liquid Chromatography
The anthocyanin samples was diluted accordingly with methanol and HPLC analysis was carried out using a liquid chromatography system (Shimadzu, Kyoto, Japan) with a Photodidode array detector. The Detection was carried at 480 nm. The flow rate was maintained at 1.0ml/min. A Reverse Phase C18 column having a particle size of 5.0 μm was used for HPLC analysis of anthocyanins. The sample injection was 20 μl. A gradient mobile phase was used for elution : elution A was 10% Formic acid in water and B was Acetonitrile/water/formic acid ( 5:4:1). Apigenin-5-β-D-glucopyranoside was used as a standard.
2.6 Cell lines and Culture medium
MCF-7 (Human, Breast cancer cell line) was cultured in DMEM supplemented with 10% heat inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B (5 mg/ml) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 microtitre plates (Tarsons India Pvt. Ltd., Kolkata, India).
preparation of Test Solutions
For cytotoxicity studies, each weighed test drugs were separately dissolved in distilled DMSO and volume was made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies.
Determination of cell viability by MTT Assay
Principle: The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the number of cells present and on the mitochondrial activity per cell. The principle involved is the cleavage of tetrazolium salt 3-(4, 5 dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) into a blue coloured product (formazan) by mitochondrial enzyme succinate dehydrogenase. The number of cells was found to be proportional to the extent of formazan production by the cells used Francis and Rita, (1986).
Procedure:The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 ml of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37o C for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 ml of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37o C in 5% CO2 atmosphere. The supernatant was removed and 100 ml of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line.
2.7 RT-PCR for p53 and Bcl-2 gene expression.
1 X 106 MCF-7 cells were plated per well of 6 well tissue culture plate and incubated at 37oC/5% CO2 overnight. These cells were treated with 20 mg/ ml of the test sample dissolved in DMEM containing 10% FBS. As a negative control DMEM containing 10% FBS was used and as a positive control cells were treated with 5µg/ml Doxorubicin hydrochloride. Plates were incubated for further 48 hours. Total RNA was isolated from these cells and mRNA expression levels of p53 and BCl-2 were carried out using semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR).
2.2. Reverse Transcription (RT)
Reverse transcription of total RNA from MCF-7 control, MCF-7 cells treated with 20 mg/ml samples A (Luteolin-5-β-D-glucopyranoside), B (apigenin-5-β-D-glucopyranoside), and C (Control with out samples, Negative control) and MCF-7 cells treated with 5µg/ml Doxorubicin (Positive control) were performed separately using 200 units of RevertAid™ M-MuLV Reverse Transcriptase (Fermentas Life Sciences).
Polymerase Chain reaction (PCR)
PCR For β-actin:
The sequences of the primers used for β-actin are as follows
Forward: 5'-GTGGGGCGCCCCCAGGCACCA -3'
Reverse: 5'- CTCCTTAATGTCACGCACGATTTC-3'
The amplification was performed in a Master cycler® Thermocycler (Eppendorf, Germany) using the following program
Initial denaturation of 94oC for 2 minutes followed by 30 cycles of denaturation at 94oC for 1 min, annealing at 64oC for 1 min and extension at 72oC for 1.2 min and terminated after final extension of 72 oC for 8 min.
Expected size of product: 540 bp
PCR for p53
The sequences of the primers used for p53 are as follows
Forward: 5'-CTGAGGTTGGCTCTGACTGTACCACCATCC -3'
Reverse: 5'-CTCATTCAGCTCGGAACATCTCGAAGCG -3'
The amplification was performed in a Master cycler® Thermocycler (Eppendorf, Germany) using the following program
Initial denaturation of 94oC for 2 minutes followed by 30 cycles of denaturation at 94oC for 1 min, annealing at 69oC for 1 min and extension at 72oC for 1.2 min and terminated after final extension of 72 oC for 8 min
Expected size of product: 371 bp
PCR for Bcl-2
The sequences of the primers used for Bcl-2 are as follows
Forward: 5'- GTTCGGTGGGGTCATGTGTGTGGAGA-3'
Reverse: 5'- GCTGATTCGACGTTTTGCCTGAAGAC-3'
The amplification was performed in a Master cycler® Thermocycler (Eppendorf, Germany) using the following program
Initial denaturation of 94oC for 2 minutes followed by 30 cycles of denaturation at 94oC for 1 min, annealing at 64oC for 1 min and extension at 72oC for 1.2 min and terminated after final extension of 72 oC for 8 min.
Expected size of product: 462 bp