American Journal of Ethnomedicine, 2016, Vol. 3, No. 1 ISSN: 2348-9502 Available online at http://www.ajethno.com © American Journal of Ethnomedicine Assessment of the Cytotoxic Potential of Tecoma castanifolia (D. Don) Melch Flower Extract Against MCF-7 Cell Line R.Vidhya* and Dr. Albin T. Fleming P.G. & Research Department of Advanced Zoology & Biotechnology, Loyola College, Nungambakkam, Chennai-600082, Tamil Nadu, India *Corresponding author e-mail: [email protected] ABSTRACT Objective: Bignoniaceae is a family having 100 genera of flowering plants. This family has many species with effective cytotoxic activity. One of its genuses, Tecoma contains 14 species and they are referred as trumpet trees. Tecoma species has various uses recorded in folk medicine and are being explored scientifically. Flowers of one of the species T.castanifolia is assessed for its potential. Method: Fresh and healthy flowers were collected and were cold extracted with solvents viz., chloroform, ethyl acetate and methanol; filtered through Whatmann No.1 filter paper and evaporated to dryness. The extracts were subjected to MTT assay against MCF-7 cell line. Result: The extraction yielded a gummy extract which were stored in a glass vial. The preliminary data shows that of the extracts tested the ethyl acetate extract has the highest cytotoxic activity in vitro against MCF-7 cell line. The 50% growth inhibition concentration of the ethyl acetate extract was obtained at 180µg/ml. Concluison: The study shows that the ethyl acetate extract has fair cytotoxicity and can be tested with other cell lines. Further, studies pertaining to their composition of phytochemicals etc need to be done. Keywords- Plant extract, Cytotoxicity, MCF-7, MTT assay, Tecoma. INTRODUCTION International Union Against Cancer indicate that 12 million cases of cancer were Worldwide, one in seven deaths is diagnosed last year, with 7 million deaths due to cancer. Cancer is one of the leading worldwide; these numbers are expected to causes of deaths as it causes more deaths double by 2030 (27 million cases with 17 than AIDS, tuberculosis, and malaria 1 million deaths. combined. Current estimates from the Breast cancer is the most commonly American Cancer Society and from the diagnosed cancer among women in the vast Page 1 www.ajethno.com American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502 majority (140 of 184) of countries Melch was analysed in this study. The part worldwide, making it the only cancer that is of the plant chosen was the flowers. common among women in all regions of the world (IARC Cancer Base). The treatments MATERIALS AND METHODS that are available slow down tumor progression and/or eradicate cancer cells. Materials But this treatment does not always promise a The solvents viz., chloroform, complete recovery based on various factors ethylacetate and methanol were of analytical such as time of cancer diagnosis, patient grade and purchased from HiMedia age, pathological condition of the tumor. Laboratories Ltd., Mumbai. 3-(4,5–dimethyl This calls for the need for discovery of thiazol–2–yl)–5–diphenyl tetrazolium higher potent drug candidates. Majority of bromide (MTT), Fetal Bovine serum (FBS), such research has been on the traditional Phosphate Buffered Saline (PBS), Modified medicines given in literature and on the folk Eagle’s Medium (MEM) and Trypsin were medicines of various tribes. obtained from Sigma Aldrich Co, St Louis, Nature has been a source of USA. EDTA, Glucose and antibiotics from medicinal agents for thousands of years and Hi-Media Laboratories Ltd., Mumbai. an impressive number of modern drugs have Dimethyl Sulfoxide (DMSO) and Propanol been isolated from natural sources2. India is from E.Merck Ltd., Mumbai, India. usually referred as “Botanical garden of the world” due to its large diversity in flora. Collection and Extraction of Plant Material Many traditional medicines in use are The fresh flowers of the plant were derived from medicinal plants, minerals and collected in Chennai and the plant was organic matter3. Botanicals are less authenticated from Botanical Survey of expensive and easily available because of India, Coimbatore. The voucher number for their natural occurrence in abundance.4 the same is BSI/SRC/5/23/2014- Under the order Lamiales, the 15/Tech./842. The healthy flowers were cold Bignoniaceae constitutes the family of extracted with various solvents for a period flowering plant. This family contains more of 7 days with continuous shaking. The than 800 species spread throughout India. solvents were changed thrice during the Many species of Bignoniaceae have some period and filtered through Whatmann No.1 use,either commercially or ethnobotanically. filter paper. It was then rotary evaporated to Several species are known to be used as a obtain a gummy extract which were stored folk medicine for diabetes, skin disorders, in a glass vial. gastro and urinary disorders etc.5 Tecoma is a genus of 14 species of Cell lines and Culture medium shrubs or small trees in the trumpet MCF-7 (Human breast carcinoma) vine family. Tecoma sambucifolia H.B.K. cell line was procured from National Centre alcoholic extract of flowers also for Cell Sciences (NCCS), Pune, India. demonstrated cytotoxicity against human Stock cells were cultured in MEM hepatoma cell line6. One species T.stans supplemented with 10% inactivated Fetal have been known to posses anti diabetic Bovine Serum (FBS), penicillin (100 activity and the compounds like tecomine IU/ml), streptomycin (100 g/ml) and and tecostanin have been isolated from it7-11. amphotericin B (5 g/ml) in an humidified Based on such literatures, the cytotoxic atmosphere of 5% CO2 at 37C until potential of one of the members of confluent. The cells were dissociated with Bignoniaceae, Tecoma castanifolia (D. Don) TPVG solution (0.2% trypsin, 0.02% Page 2 American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502 EDTA, 0.05% glucose in PBS). The stock inhibition was calculated using the cultures were grown in 25 cm2 culture flasks following formula and all experiments were carried out in 96 microtitre plates (Tarsons India Pvt. Ltd., % Growth Inhibition = 100 – Kolkata, India). Mean OD of individual test group X 100 Preparation of Test Solutions Mean OD of control group For Cytotoxicity studies, each weighed test drugs were separately RESULT dissolved in distilled DMSO and volume was made up with MEM supplemented with MTT Assay 2% inactivated FBS to obtain a stock The concentration required to inhibit solution of 1 mg/ml concentration and cell growth by 50% was assessed using sterilized by filtration. Serial two fold MTT assay. The effectiveness of inhibition dilutions were prepared from this for of the three extracts against MCF-7 cell line carrying out cytotoxic studies. is graphically represented in Graph 1. MCF- 7 is a hormone dependant breast cancer cell Determination of cell viability by MTT line frequently used to test cytotoxic activity Assay of various compounds and/or extracts. The The monolayer cell culture was GI50 values of chloroform, ethyl acetate and trypsinized and the cell count was adjusted methanol extracts are 378.3µg/ml, to 1.0 x 105 cells/ml using MEM containing 180.0µg/ml and >1000µg/ml respectively. 10% FBS. To each well of the 96 well Of the three extracts the ethyl acetate extract microtitre plate, 0.1 ml of the diluted cell at 180µg/ml shows the most effective suspension (approximately 10,000 cells) was cytotoxicity at lowest concentration. The added. After 24 h, when a partial monolayer growth inhibitory concentration gives the was formed, the supernatant was flicked off, preliminary support to further analyse the the monolayer was washed once with extract for cytotoxic activity. medium and 100 l of different test concentrations of test drugs were added on DISCUSSION to the partial monolayer in microtitre plates. o The genus of Tecoma has been The plates were then incubated at 37 C for 3 traditionally used in various parts of the days in 5% CO2 atmosphere, and world to treat a variety of conditions and microscopic examination was carried out cancer is recorded to be one of it. The leaves and observations were noted every 24 h of the plant is known to contain phenolic interval. After 72 h, the drug solutions in the acid, beta-setosterol, triterpenoids etc., both wells were discarded and 50 l of MTT in phenol and flavonoids possessing PBS was added to each well. The plates antioxidant activity. Tecoma fabrisi and were gently shaken and incubated for 3 h at o Tecoma impetignosa are used to treat some 37 C in 5% CO2 atmosphere. The forms of cancer14. supernatant was removed and 100 l of The three solvent extracts showed propanol was added and the plates were potent cell growth inhibitory activity. This gently shaken to solubilize the formed may be due to their action on any one of the formazan. The absorbance was measured following- mitosis, biochemical processes using a microplate reader at a wavelength of 12,13 etc. The biological activity shown by plants 540 nm . The percentage growth are mainly due to their secondary metabolites like alkaloids, phenolics, Page 3 American Journal of Ethnomedicine ________________________________________ ISSN: 2348-9502 flavonoids, terpenoids, saponins etc. These ACKNOWLEDGEMENT are referred to as bioactive phyto compounds. Several phytochemical studies I would like to thank my Supervisor revealed that the extracts from many species and colleagues for their continuing support. of Bignoniaceae contained secondary metabolites such as saponins, tannins, REFERENCES flavonoids, quinones, alkaloids, anthralene 1. American Cancer Society. Global Cancer derivatives, reducing sugars, glycosides, Facts & Figures 3rd Edition,2015. carbohydrates, querletin, kaempferol, â- 2. Dyamavvanahalli Lakshmanachar Shrisha et sitosterol, terpenes, steroids, coumarins etc., al., Bioprospecting of selected medicinal and their derivatives15. plants for antibacterial activity against some Out of the three crude flower leaf pathogenic bacteria; J.