CHAPTER 3 IN VITRO ANTICANCER ACTIVITY OF FLOWER EXTRACTS OF COUROUPITA GUIANENSIS 3. INTRODUCTION Plants are the basic source of knowledge of modern medicine. Almost all the parts of the plant, namely leaves, flowers, fruits, roots, stem and seeds are known to have various medicinal properties. The trend of using natural products has increased and the active plant extracts are frequently screened for new drug discoveries (Manimegalai and Rakkimuthu, 202).In particular traditional medicinal plants have recently attracted the attention of the pharmaceutical and scientific communities. This has involved the isolation and identification of secondary metabolites produced by plants and their use as active principles in medicinal preparations (Taylor et al., 200).One of the important plant that is used in traditional medicine is Couroupita guianensis which belongs to the family Lecythidaceae. In Ayurveda, it is called as ayahuma; it is used extensively as an ingredient in many preparations which cure gastritis, scabies, bleeding piles, dysentery, scorpion poison and many various part of this medicinal plant have been reported to contain oils, keto steroids, glycosides, couroupitine, indirubin, isatin and phenolic substances (Kavitha et al.,20). Juice made from the leaves of this plant is used to cure skin diseases, and shamans of South America have even used various parts of this plant for treating malaria (Kumar et al., 20). The leaves of C guianesis exhibited potential antioxidant, anti-arthritic and anti platelet activities (Elumalai et al., 202). Native people from Amazonian region and other states of North of Brazil use infusions obtained from the leaves and flowers to treat hypertension, tumors, pain, and inflammatory processes (Revilla, 2002). The ethanol extract of Couroupita guianensis Aubl. Leaves as 29
well as its fractions have antinociceptive action in the acetic acid-induced writhing, tail flick, and hot plate test mediated, inpart, by opioid and cholinergic systems and nitric oxide pathway (Pinheiro et al., 200). In the present investigation invitro cytotoxic potential of the medicinal plant Couroupita guianensis was evaluated against various cancer cell lines viz., Caco2 (Human epithelial colorectal adenocarcinoma), MCF- 7(Human Breast carcinoma), A-43(Human skin epidermoid carcinoma), HeLa (Cervix adenocarcinoma). 3.2 MATERIALS AND METHODS 3.2.Chemicals 3-(4,5 dimethyl thiazol 2 yl) 5 diphenyl tetrazolium bromide (MTT), Fetal Bovine serum (FBS), Phosphate Buffered Saline (PBS), Dulbecco s Modified Eagle s Medium (DMEM) and Trypsin were obtained from Sigma Aldrich Co, St Louis, USA. EDTA, Glucose and antibiotics from Hi-Media Laboratories Ltd., Mumbai. Dimethyl Sulfoxide (DMSO) and Propanol from E.Merck Ltd., Mumbai, India. 3.2.2 Cell lines and Culture medium Caco-2 (Human epithelial colorectal adenocarcinoma), MCF-7(Human Breast carcinoma), A-43(Human skin epidermoid carcinoma) and HeLa (Cervix adenocarcinoma), cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Stock cells were cultured in DMEM supplemented with 0% inactivated Fetal Bovine Serum (FBS), penicillin (00 IU/ml), streptomycin (00 g/ml) and amphotericin B (5 g/ml) in an humidified atmosphere of 5% CO 2 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 cm 2 culture flasks and all 30
experiments were carried out in 96 well microtitre plates (Tarsons India Pvt. Ltd., Kolkata, India). 3.2.3 Preparation of Test Solutions For cytotoxicity studies, each weighed test drugs were separately dissolved in distilled DMSO and the volume was made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of mg/ml concentration and sterilized by filtration. Serial two fold dilutions were made for carrying out cytotoxic studies. 3.2.4 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, 986). Procedure: The monolayer cell culture was trypsinized and the cell count was adjusted to.0 x 0 5 cells/ml using DMEM containing 0% FBS. To each well of the 96 well microtitre plate, 0. ml of the diluted cell suspension (approximately 0,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 00 l of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37 o C for 3 days in 5% CO 2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug 3
solutions in the wells were discarded and 50 l of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37 o C in 5% CO 2 atmosphere. The supernatant was removed and 00 l 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 of growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC 50 ) is generated from the dose-response curves for each cell line. Mean ODof individual test group % Growth Inhibition = 00- X 00 Mean ODof controlgroup 3.3 RESULTS AND DISCUSSION In the present investigation invitro anticancer activity of the medicinal plant Couroupita guianensis was evaluated against the cancer cell lines viz, Caco2 (Human epithelial colorectal adenocarcinoma), MCF-7(Human Breast carcinoma), A-43(Human skin epidermoid carcinoma), and HeLa (Cervix adenocarcinoma). The anticancer activity displayed by extract of this plant was found 80.63±0.6% at µg concentration against the Caco 2 human epithelial colorectal adenocarcinoma and the CTC 50 value was recorded as 367.93 µg/ml (Table 3.). 32
Table 3. Cytotoxic potential of flower extracts of the medicinal plant Couroupita guianensis against Caco-2 cell line % Cytotoxicity CTC 50 ( µg/ml) 80.63±0.6 79.70±.0 6.29±2.9 367.93±. 25 3.6±0.6 0.24±0.2 Values are means of three independent analyses ± standard deviation (n = 3). The anticancer activity was recorded 79.5±0.9 % against MCF-7(Human Breast carcinoma) and the CTC 50 value was found to be 56.67 µg/ml (Table 3.2). Table 3.2 Cytotoxic potential flower extracts of the medicinal plant Couroupita guianensis against MCF-7 cell line % Cytotoxicity CTC 50 ( µg/ml) 79.5±0.9 68.9±0.3 56.46±0.3 56.67±0.4 25 49.07±0.2 38.0±0.5 Values are means of three independent analyses ± standard deviation (n = 3). Against A-43(Human skin epidermoid carcinoma) the extract of this plant showed 77.24±.9% of activity at the concentration of µg/ml and in the mean time the CTC 50 was found 66.67 µg/ml(table 3.3). 33
Table 3.3 Cytotoxic potential flower extracts of the medicinal plant Couroupita guianensis against A-43 cell line % Cytotoxicity CTC 50 (µg/ml) 77.24±.9 60.3±0.7 57.32±. 66.67±.0 25 48.05±0.2 44.7±.2 Values are means of three independent analyses ± standard deviation (n = 3). Moderate anticancer activity 59.39±0.3% was recorded against HeLa Cervix cancer cell line at the concentration of µg/ml and the CTC 50 value was found 426.67 µg/ml (Table 3.4) Table 3.4 Cytotoxic potential of flower extracts of the medicinal plant Couroupita guianensis against HeLa cell line % Cytotoxicity CTC 50 ( µg/ml) 59.39±0.3 52.77±0.2 43.86±0.9 426.67±0.4 25 23.50±0. 7.97±0.5 Values are means of three independent analyses ± standard deviation (n = 3) The anticancer activity was recorded in a dose dependent manner in all types of cell lines treated with extracts of this medicinal plant. The cytotoxic potential of flower extract of Couroupita guianensis may be due to the presence of phenolic and flavonoid compounds. Prior reports also suggested that phenolic compounds and flavonoids 34
exhibited antineoplastic activity (Kim et al., 993) and antioxidant activity (Pietta, 2000). Madan Ranjit et al.,(204) reported that ethyl acetate and aqueous extract of Couroupita guianensis displayed potential anticancer activity against MCF-7 Breast cancer cell line with the IC 50 value of 685.6 µg/ml. In our findings the methanol extract of Couroupita guianensis inhibited the growth of the MCF-7 Breast cancer cell line with the IC 50 value of 56.67±0.4 µg/ml. This indicates that the active fraction for this antiproliferative activity was present in the methanol extract of Couroupita guianensis flowers. Hence, it s suggested from the present investigation that targeting the active compounds present in the methanol extract of the medicinal plant Couroupita guianensis may lead to the development of a novel potential drug to treat breast cancer. 3.4 CONCLUSION In the present investigation the methanol extract of Couroupita guianensis was evaluated for it s potential anticancer activity against various human cancer cell lines viz., Caco2 (Human epithelial colorectal adenocarcinoma), MCF-7(Human Breast carcinoma), A-43(Human skin epidermoid carcinoma) and HeLa (Cervix adenocarcinoma), cell lines. Except the HeLa cervix adenocarcinoma line high potential activity was recorded against all the rest of the cancer cell lines. Further invivo experiments in animal models and isolation of a responsible compound from the extract of this plant will be an interesting task in the development of novel anticancer compound 35