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In vitro anticancer activity of Betanin, fresh juice and hydro-alcoholic root extract of Beta vulgaris on HL-60 cell lines M. Pharm Dissertation Protocol Submitted to Rajiv Gandhi University of Health Sciences, Karnataka Bangalore– 560 041 By Ms. K. Sirisha, B. Pharm. Under the Guidance of Dr. Kalyani Divakar, M. Pharm, Ph. D. Professor & Head 2010-2012 Department of Pharmacology, Acharya & B.M. Reddy College of Pharmacy, Soldevanahalli, Chikkabanavara (Post), Hesaraghatta Main Road, Bangalore – 560 090. 1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1. Name of the candidate & Address 2. Name of the Institution 3. Course of the study & subject 4. Date of admission 5. Title of the Topic 6. Brief resume of intended work Ms. K. Sirisha, D/o K. Nageswara Rao, D.No. 8-15-2, Sujana appartment -A, flat no B2, Gandhi nagar, Kakinada, Andhra Pradesh. Acharya & B.M. Reddy College of Pharmacy Soldevanahalli, Hesaraghatta Road, Chikkabanavara Post, Bangalore-560090. Phone No: 080 65650815 Fax No: 080 28393541 M.Pharmacy (Pharmacology) 02/09/2010 In vitro anticancer activity of Betanin, fresh juice and hydro-alcoholic root extract of Beta vulgaris on HL-60 cell lines 6.1 Introduction and need of the work Enclosure I 6.2 Review of Literature Enclosure II 6.3 Aim and Objective of the study Enclosure III 2 7. Materials & Methods 7.1 Source of data Enclosure IV 7.2 Methods of collection of data Enclosure V 7.3 Does the study require investigation on animals? No 7.4 Has ethical clearance been obtained from your institution in case of 7.3 Not applicable 8. List of references (About 1 – 19) 9. Signature of the candidate 10. Remarks & Signature of the guide 11. Name & Designation of Guide 12. Name & Signature of HOD Enclosure VII Dr. Kalyani Divakar, M. Pharm, Ph. D. Professor & Head, Dept. of Pharmacology, Acharya & B.M. Reddy College of Pharmacy Dr. Kalyani Divakar, M. Pharm, Ph. D. Professor & Head, Dept. of Pharmacology, Acharya & B.M. Reddy College of Pharmacy 13. Remarks of the Principal 14. Signature of Principal Dr. Divakar Goli, M. Pharm, Ph. D. Principal Acharya & B.M. Reddy College of Pharmacy 3 ENCLOSURE-I 6. BRIEF RESUME OF INTENDED WORK 6.1 Introduction and need of work: 6.2 Cancer is a leading cause of death worldwide. The disease accounted for 7.4 million deaths (or around 13% of all deaths worldwide) in 2008. The main types of cancer leading to overall cancer mortality each year are: Lung (1.4 million deaths/year) Stomach (740 000 deaths) Colorectal (610 000 deaths) Liver (700 000 deaths) Breast (460 000 deaths) Cancer is a generic term for a large group of diseases that can affect any part of the body. Other terms used are malignant tumours and neoplasms. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. This process is referred to as metastasis which is the major cause of death from cancer. [1] More than 70% of all cancer deaths occurred in low-and middle-income countries. Deaths from cancer worldwide are projected to continue rising, with an estimated 13 million deaths in 2030. [1] Several attempts have been made to improve existing treatments to increase the survival of patient. Hence, there is need of new therapeutic agents that are less toxic to normal cells and produce an enhanced anti tumor effect. [2, 3] 4 Most cancer patients in India reel under the pressure of expensive treatment. Most American pharma companies producing and selling these drugs in India have a patent over them. The patients have no choice but to pay the market price since none of the Indian companies are producing those medicines here except one drug, which is much less expensive and equally efficient manufactured by Dr Reddy. Although the Indian government does provide free/subsidized treatment at the cancer centers, a patient has to go for expensive advanced-level treatment to increase his chances of survival. But in India, it is beyond the reach of most needy patients, hence need to find the cost effective newer drugs. [4] A high intake of vegetables and fruits can reduce the risk of developing cancer and other diseases. Beetroot has unique chemicals (e.g. Betalains) and high levels of important micronutrients, which make it a valuable vegetable to include in the diet as a means of deterring the onset of cancer and other diseases. The Betalains also act as antioxidants. The therapeutic use of beetroot in cancer treatment came to prominence with the work of the Hungarian physician Alexander Ferenczi in the 1950s. He introduced a revolutionary new treatment for cancer using nothing but raw beetroot juice. In his papers from the late 1950s and early 1960s, he reported remarkable success in treating cancer patients. His patients suffered from a range of different cancers. His reputation grew and beetroot juice became a sought-after treatment for cancer. Ferenczi’s treatment was based on consuming a liter of beetroot juice daily, for at least two to three months. Rosenberg concluded that beetroot’s effect on cancer cells is probably due to the combined effects of betanine, allantoine, vitamin C and other compounds present, such as farnesol and rutine.[5] 5 There are two distinct classes of betalains, the red/purple betacyanins and the yellow betaxanthins.[6] The present protocol is proposed to find out the in-vitro anticancer activity of Betanine, fresh juice and hydro-alcoholic extract of root of Beta vulgaris against HL-60 cell lines. Further this study is also aimed to find out the difference in the activity between Betanine and fresh juice and hydro-alcoholic extract of root of Beta vulgaris. Since Betanine is main pigment of beet root whether itself is sufficient to show the anticancer activity (or) any other phyto-constituents of root of Beta vulgaris is also responsible for its anticancer activity, is also a part of the evaluation. 6 ENCLOSURE-II 6.2 Review of Literature: Plant selected for study:[7] Name of the plant selected for the present study is “Beta vulgaris” Description: Botanical name: Beta vulgaris Family: Amaranthaceae Synonyms: Garden beet, Table beet, Red beet. Beetroots are an excellent source of red and yellow pigments, and this trait has fostered research and industrial interest in the context of natural colorants. Beet pigments, collectively known as betalains.[6] The betalains found in beetroot peel extract were vulgaxanthin I, vulgaxanthin II, indicaxanthin, betanin, prebetanin, isobetanin and neobetanin. Also cyclodopa glucoside, N-formylcyclodopa glucoside, glucoside of dihydroxyindolcarboxylic acid, betalamic acid, l-tryptophan, p-coumaric acid, ferulic acid and traces of unidentified flavonoids were detected. [8] Betanine content in beetroot varies from 100 mg/100 g fresh product to 16-38 mg/100 g dried vegetable product. Therefore, the plant species and harvesting conditions are important factors to be considered when selecting the suitable raw material. [9] About 0.5-1% of extracted beet juice solids are betalains. In addition to the pigments, the root contains about 700-800 mg oxalic acid and 5-6 mg ascorbic acid per 100 g beetroot .[10] The beet juice may inhibit or enhance carcinogenic MA (N-nitrosodimethylamine) formation in human gastric juice depending on the pH of gastric juice. In acid medium, there was a trend to inhibit MA synthesis, while in neutral and alkaline medium, MA synthesis is activated.[11] 7 Beetroot is also rich in ferulic acid derivatives. The specific betalains routinely identified in these fractions were vulgaxanthin I, vulgaxanthin II (glutamic acid replaces glutamine, and (iso) betanin, all of which have been reported as major betalains in beetroots.[6] In the 1990s, cell culture and animal studies, such as those conducted by Edenharder et al., and Kapadia et al., respectively, confirmed that beetroot juice had significant antimutagenic effects against Salmonella typhimurium TA98 and TA100 and tumorinhibiting againt prostate and breast cancer cell lines,[12] and in-vivo mice skin and lung cancer.[13] Beetroot crude extracts contained multiple antioxidant and phase II enzymeinducing activities. In some studies, antioxidant function has been attributed directly to betalains in terms of reducing power and inhibition of iron-induced lipid peroxidation.[6] Beetroot extract able to show anti inflammatory and immunosuppressive activity in peripheral blood mononuclear cells (PBMC), because inflammation is strongly involved in the development and progression of several clinical conditions including coronary heart disease and cancer, beneficial effect of beetroot extract may relate to this anti-inflammatory capacity.[14] 8 ENCLOSURE-III 6.3 Objectives of the study a) To find out the invitro anticancer activity of Betanine, fresh juice and hydroalcoholic extract of root of Beta vulgaris. b) To compare the activity between Betanine and juice and hydro-alcoholic extract of root of Beta vulgaris. c) To explore the mechanism involved in the anticancer activity of Betanine, fresh juice and hydro-alcoholic extract of root of Beta vulgaris through the following parameters. d) In-vitro estimations using HL-60 cell lines by conducting the following tests: a. Clonogenic assay [15] b. MTT assay [16] c. Trypan Blue exclusion technique [16] d. DNA fragmentation analysis [17] e. Determination of cell viability in presence of caspase-3 inhibitor [16] f. Glutathione estimation [18] g. Lactate dehydrogenase estimation [kit] h. Nitric oxide estimation [19] 9 ENCLOSURE-IV 7. MATERIALS AND METHODS 7.1 Source of Data: The data will be obtained from experiments which involve: A) Laboratory based studies B) Literature survey, abstracts C) National & International Journals D) Text books E) Internet 10 ENCLOSURE-V 7.2 Methodology: Collection and Extraction of Root of Beta vulgaris: a. Clonogenic Assay:[14] Tumor colony forming units will be cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 0.3% agar and 20% fetal calf serum. The cultures will be incubated at 37°C in a fully humidified atmosphere containing 10% CO2 in air. Colonies (greater than 40 cells) will be scored after 10-20 days using a dissecting microscope at 45 X. A linear relation of the number of cells plated and colonies could be established. The same procedure will be followed using various concentrations of drug(s). b. MTT Assay:[15] The 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a common method used to assess cell proliferation and cytotoxicity. Briefly, 1x104 exponentially growing cells will be seeded per well in 96 well plates and exposed to various concentrations of drug(s) and incubated to certain period. MTT will be added and incubated at 37°C for 4 h. The precipitated formazan salt will be dissolved in DMSO and the samples will be read at 570 nm. The 50% inhibitory concentration (IC50) of drug will be calculated. c. Trypan Blue exclusion technique:[15] The effect of drug on the HL-60 Cancer cell will be determined by means of Trypan Blue exclusion technique. Briefly, 3x104 cells will be seeded per well in 24 well plates and then treated with various concentrations of drug(s). The plate will be incubated at 37°C and the number of cultured cells in the different wells will be counted using a hemocytometer after staining with 0.4% Trypan Blue every 24 h to calculate the doubling time. d. Estimation of percent DNA fragmentation: Induction of apoptotic mode of cell death in cells was also confirmed by quantitative determination of DNA fragmentation following a method given by sellins & cohen with 11 slight modification.Treated or untreated tumor cells were lysed in 0.5 ml of Tris-EDTA buffer, pH 7.4, containing 0.2% (v/v) triton x-100 and the fragmented DNA was separated from intact chromatin in a microfuge tube (labeled as B) by centrifugation at 13,000× g at 4°C for 10 min. supernatant containing the fragmented DNA was transferred to another microfuge tube (labeled as T). A volume of 0.5 ml of 25% TCA was added to each T and B tube and vortexed vigorously. DNA was precipitated overnight at 4°C and collected at 13,000× g at 4°C for 10 min supernatant was discarded and 80 μl of 5% TCA was added to each pellet. DNA was hydrolyzed by heating at 90°C for 15 min .At this stage, a blank was included containing 80 µl of 5% TCA. Then 160 µl of freshly prepared diphenylamine (DPA) reagent (150 mg diphenyleamine in 10 ml glacial acetic acid, 150 µl concentrated H2SO4 and 50 µl of acetaldehyde solution) was added and the tubes were allowed to stand overnight at room temperature to develop color. 100 µl of this colored solution was transferred to the wells of a 96-well flat-bottomed ELISA plate and observance was measured at 600 nm in a microtitre ELISA plate reader percent DNA fragmentation was calculated as: DNA fragmentation (%) = [T/(T+B)]×100 Where T= absorbance of fragmented DNA and T+B= absorbance of total DNA. e. Determination of cell viability in presence of caspase-3 inhibitor:[15] The viability of HL-60 cells treated with caspase-3 inhibitor and drug will be evaluated using MTT assay. After incubation with caspase-3 inhibitor for 1 h, cells will be treated with various concentrations of drug(s) for 48 h after which viability will be determined. f. Glutathione estimation:[16] Cultured HL 60 cell lines homogenate will be prepared in 0.02 M ethylenediamine tetra acetic acid (EDTA). Aliquots of 0.5 ml of the tissue homogenates will be mixed in test tubes with 1-5 ml of 0.2 M Tris buffer, pH 8.2, and 0.1 ml of 0.01 M DTNB. The mixture will be brought to 10.0 ml with 7.9 ml of 0.5% of dodecyl sulphate. The reaction mixture will be 12 centrifuged at approximately 3000 g at room temperature for 15 minutes. The absorbance of the supernatants will be read using spectrophotometer at 412 nm in 1 cm quartz cells. g. Lactate dehydrogenase estimation: [kit] This is carried out using commercial kit. h. Nitric oxide estimation:[17] The cultured cells will be weighed and used for preparation of homogenates in 0.05 M phosphate buffer. The tubes will centrifuged at 1500 x g for 20 min and 40 ml supernatant will be used for nitric oxide (NO) estimation. Nitric oxide (NO) production will be estimated by Griess reaction (Giuseppina et al., 1999) which will be expressed in the form of nitrite accumulation. In brief, 100 µl of tissue extract will be mixed with 100 µl of Griess reagent [equal volumes of 1% (w/v) sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) napthylethylenediamine hydrochloride (NEDH)] and will be incubated at room temperature for 10 min, and then the absorbance at 550 nm will be measured in a UV-double beam spectrophotometer. The amount of nitrite in the sample (µM unit) will be calculated from a sodium nitrite standard curve. The results will be expressed as µM nitrite/mg of protein. 13 ENCLOSURE-VI 7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. Not applicable 7.4 Has ethical clearance been obtained from your institution in case of 7.3? Not applicable 14 ENCLOSURE-VII 8. References: 1. World Health Organization [online]. February 2008 [cited 2011 may 8]; URL: http://www.who.int/mediacentre/factsheets/fs297/en/ 2. Manish S, Began G, Suraag P, Dora B, Sunil C, Rajagopal R. Vitamin E succinate in combination with mda-7 results in enhanced human ovarian tumour cell killing through modulation of extrinsic and intrinsic apoptotic pathways. Cancer Lett 2007;254:217-26. 3. Faivre S, Djelloul S, Raymond E. New paradigms in anticancer therapy: targeting multiple signaling pathways with kinase inhibitors, semin. Oncol 2006;33:407-20. 4. Cost of cancer treatment. Citizen journalist [online]. 2007 june 20 [cited 2011 may 9]; [1 screen]. Available from: URL: http://www.ibnlive.in.com/news/money-killscancer-patients/40047-20.html. 5. Stephen Nottingham: Beetroot: Health and Nutrition. Chapter 6. 2004. p. 1-17. 6. Lee CH, Wettasinghe M, Bolling BW, Ji L, Parkin KL. Betalains, Phase II enzymeinducing components from red beetroot (Beta vulgaris L.) Extracts. Nutr cancer 2005; 53(1):91-103. 7. Beta vulgaris. The plant database [online]. 2006 March 6 [Cited 2011 may 11]: URL: http://www.scientificweb.com/en/Biology/Plants/Magnoliophyta/BetaVulgaris01.html 8. Kujala T, Loponen J, Pihlaja K. Betalains and phenolics in red beetroot (Beta vulgaris) peel extracts: extraction and characterisation 2001; 56c:343-8. 9. Sturzoiu A, Stroescu M, Stoica A, Dobre T. Betanine extraction from beta vulgaris experimental research and statistical modelling. U.P.B. Sci Bull 2011;73(1):145-56. 10. Winkler C, wirleitner B, Schroecksnadel K, Schennach H, Fuchs D. In vitro effects of beet root juice on stimulated and unstimulated peripheral blood mononuclear cells. Am J Biochem Biotech 2005;1(4):180-85. 15 11. Rajesh K. The wealth of India: A dictionary of Indian raw materials and industrial products. Newdelhi: NISCAIR; 2007. vol-1(A-Ci). p. 139. 12. Edenharder R, Kurz P, John k, Burgard S, Seeger K. In vitro effect of vegetable and fruit juices on the mutagenicity of 2-amino-3-methylimidazole[4,5-f]quinoline, 2,amino,3,8-dimethylimide-3-ol[4,5f]quinoxaline. Food Chem Toxicol 1994;32:44359. 13. Kapadia GJ, Tokudab H, Konoshimac T, Nishinod H. Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract. Cancer Lett 1996;100:211-4. 14. Eastwood MA, Nyhlin H. Beeturia and colonic oxalic acid. QJM 1995;88(10):711-7. 15. Nicol BM, Prasad SB. The effect of Cyclophosphamide alone and in combination with ascorbic acid against murine ascites Dalton’s lymphoma. Indian J Pharmacol 2006;38(4):260-5. 16. Sharma M, Sharma PD, Bansal MP, Singh J. Lantadene A-induced apoptosis in human leukemia HL-60 cells. Ind J Pharmacol 2007;39(3):140-4. 17. Vishvakarma NK, Singh SM. Mechanisms of tumor growth retardation by modulation of pH regulation in the tumor-microenvironment of a murine T cell lymphoma. BIOPHA 2010;2957. 18. Ellwart JW, Kremer JP, Dormer P. Drug Testing in Established Cell Lines by Flow Cytometric Vitality Measurements versus Clonogenic Assay. Cancer Res 1988;48:5722-5. 19. Dolan D, Sandip M, Asankur SD, Maitrayee M, Chandan M. Aqueous extract of black tea (Camellia sinensis) prevents ethanol + cholecystokinin-induced pancreatitis in a rat model. Life Sci 2006;78:2194-203. 16