Cytotoxicity sceening of extracts from combretum quadrangulare on MCF-7 breast cancer cell line.pdf

NGUYEN TAT THANH UNIVERSITY NGUYEN TAT THANH True learning, true practice, true success, true future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS CYTOTOXICITY SCREENING OF EXTRACTS FROM Combretum quadrangulare ON MCF-7 BREAST CANCER CELL LINE Student’s name : Thu Thi My Tran Student ID :1411534235 Supervisor : Hung Huu Nguyen, PhD Phuong Thi Nguyen, MSc Ho Chi Minh City, 2019 NGUYEN TAT THANH UNIVERSITY NGUYEN TAT THANH True learning, true practice, true success, true future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS CYTOTOXICITY SCREENING OF EXTRACTS FROM Combretum quadrangulare ON MCF-7 BREAST CANCER CELL LINE Student’s name : Thu Thi My Tran Student ID : 1411534235 Major : Biotechnology Supervisors : Hung Huu Nguyen, PhD Phuong Thi Nguyen, MSc Ho Chi Minh City, 2019 NGUYEN TAT THANH UNIVERSITY SOCIALIST REPUBLIC OF VIETNAM FACULTY OF BIOTECHNOLOGY Independence - Liberty - Happiness -oOo ASSIGNED TASK OF GRADUATION THESIS Full name: Thi My Thu Tran Student ID: 1411534235 Major: Biotechnology Class: 14DSH02 Thesis’s title: Cytotoxicity screening of extracts from Contbretunt quadrangulare on MCF-7 breast cancer cell line Objectives - Screening for extracts or fractions from Combretum quadrangulare based on cytotoxic activity on MCF-7 breast cancer cells using MTT assay - Study on other bioactivity of the best cytotoxic fraction as antibacterial and antioxidant ability Contents - Determination of time culture for MTT assay - Screening extracts or fractions at each extraction phase of Combretum quadrangulare based on toxic effect on breast cancer cells MCF-7 through cell morphology and IC50 values - Determine the fraction with the best cytotoxicity on MCF-7 cells through IC50 values to isolate bioactive compounds - Determine antibacterial and antioxidant activity of the best cytotoxic fraction Execution time: from 12/2018 to 09/2019 Supervisor: Huu Hung Nguyen, PhD Co-supervisor: Phuong Thi Nguyen, MSc Contents and requirements of this thesis were adopted by subject HCM city, .2019 Subject Supervisor ACKNOWLEDGEMENTS With sincere feelings, I would like to express my gratitude to my supervisors Hung Huu Nguyen, PhD and Phuong Thi Nguyen, MSc Faculty of Biotechnology, Nguyen Tat Thanh University (NTTU) in particular and all of brothers, sisters, friends in the stem cell laboratory who have devotedly helped me to complete this graduation thesis I would also like to thank the teachers in NTTU who have taught me a lot of useful knowledge to carry out the thesis and have a solid luggage for my future career Because of the limitations of knowledge and reasoning ability of myself, there are many shortcomings and limitations, I hope to receive the instruction and contribution of teachers so that my thesis will be improved Thu Thi My Tran Faculty of Biotechnology Nguyen Tat Thanh University TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii ABSTRACT iv LIST OF FIGURES V LIST OF TABLES vi LIST OF ACRONYMS vii INTRODUCTION viii CHAPTER LITERATURE REVIEW 10 1.1 Cancer 10 1.1.1 Cancer overview 10 1.1.2 Breast cancer 11 1.2 Combretum quadrangulare overview .13 1.2.1 Characteristics 13 1.2.2 Bioactivity of Combretum quadrangulare extracts and compounds 14 CHAPTER CONTENTS AND METHODS 18 2.1 Place of administration 18 2.2 Contents 18 2.3 Methods 18 2.3.1 Cells preparation 18 2.3.2 MTT method 20 2.3.3 Agar Well Diffusion 23 CHAPTER RESULTS AND DISCUSSION 25 3.1 Determination of time culture for MTT assay 25 ii 3.2 Screening of the extracts based on cytotoxicity ability 26 3.2.1 Screening of c quadrangualre Crude extracts 26 3.2.2 Screening of Liquid-Liquid extracts 27 3.2.3 Screening of cytotoxicity of P1-P14 and PAX1 fractions on MCF-7 cells 29 3.2.4 Other Bio-activity of P12 fraction 33 CONCLUSIONS AND RECOMMENDATIONS 35 REFERENCES 36 APPENDICES 37 iii ABSTRACT Breast cancer is the most common type of cancer in women and is the second most common cancer in the world Worldwide estimates have more than 508,000 women died in 2011 due to breast cancer In Vietnam, the proportion of women with breast cancer has increased rapidly in recent years with more than 10,000 new patients each year and 4,500 deaths Therefore, the research on compounds, with activity to kill breast cancer cells, extracted from plants is promising The topic "Cytotoxicity screening of the extracts from Combretum quadrangulare on MCF-7 breast cancer cell line" is carried out from 12/2018 to 09/2019 in Cancer cell and Stem cell laboratory, Faculty of Biotechnology and Hi-tech Institute, Nguyen Tat Thanh University With the goal: To screening the cytotoxicity possibility of the extracts/fractions from Combretum quadrangulare MCF-7 breast cancer and other bioactivities of the fraction For the purpose: To determine the fraction from Combretum quadrangulare that have the best effect on MCF-7 cells for the use of bioactive compounds isolation The topic has two main contents: (1) Screening for extracts or fractions from Combretum quadrangulare based on cytotoxic activity on MCF-7 breast cancer cells using MTT assay (2) Study on other bioactivity of the best cytotoxic fraction as antibacterial and antioxidant ability With the obtained results: (1) EtOH extract from the leaf was determined as the best cytotoxicity on MCF-7 cells; H:Ea was determined as best solvent for EtOH leaf extract; from H: Ea extract, fraction P12 was capable of causing the highest MCF-7 cell toxicity (2) Fraction P12 had quite good antibacterial effect on Staphylococcus aureus, Bacillus cercus, Escheriachia coll, Pseudomonas aeruginosa and also good antioxidant ability with IC50 was 96.3 ± 1.8 pg/ml IV LIST OF FIGURES Figure 1.1 Map showing the number of cancer cases by country and region in the world in 2016 (https://ourworldindata.org/cancer) 11 Figure 1.2 Chart showing the number of cancers of each type of cancer in the world in 2016 (https://ourworldindata.org/cancer) 12 Figure 1.3 The gourd Combretum quadrangulare5 13 Figure 3.1 Graph of growth curve of MCF-7 breast cancer cell 24 Figure 3.2 Cytotoxic activity on MCF-7 of Doxorubicin, EtOH extract from leaf, root, seed The cell death rate is expressed as the mean of three repetitions and SD standard deviation 26 Figure 3.3 Breast cancer cytotoxic activity MCF-7 of the fraction Bu, Ea, H, H:Ea and Doxorubicin The cell death rate is expressed as the mean of three repetitions and SD standard deviation 28 Figure 3.4 MCF-7 cytotoxic activity of the fraction Pl- P14, PAX1 and Doxorubicin The cell death rate is expressed as the mean of three repetitions and SD standard deviation 30 Figure 3.5 The antibacterial activity of P12 fraction and 10 pg/mL Kanamycin (Kan) were implemented against two Gram positive bacteria (Ổ cereus-BC and aureusSA) and two Gram negative bacteria (P aeniginosa-PA and E coli-EC) 33 Figure 3.6 DPPH scavenging activity of (A) Ascorbic acid (Vitamin C) and (B) P12 fraction from c quadrangulare Inhibition (%) is expressed as the mean of three repetitions and SD standard deviation .34 V LIST OF TABLES Table 3.1 Changes in MCF-7 morphology after treating EtOH extracts from leaf, root, seed at a concentration of 50 pg/ml at 24, 48 and 72 hours of culture 26 Table 3.2 IC50 value of EtOH leaf, root, and seed extract of c quadrangulare on MCF7 27 Table 3.3 Changes in MCF-7 morphology after treating Bu, H: Ea, Ea and H extracts at a concentration of 50 pg/ml at 24, 48 and 72 hours of culture 28 Table 3.4 IC50 value of H, H:Ea, Ea, Bu extract of c quadrangulare on MCF-7 29 Table 3.5 Changes in MCF-7 morphology after treating P1-P14, PAX1 fractions at a concentration of 50 pg/ml at 24, 48 and 72 hours of culture 30 Table 3.6 IC50 value of Pl-P14 and PAX1 fractions of Cotnbretum quadrangulare on breast cancer cell MCF-7 32 vi LIST OF ACRONYMS WHO World Health Organization DMSO Dimethyl sunfoxide FBS Fetal Bovine Serum PBS Phosphate Buffered Saline EDTA Ethylendiamin Tetraacetic Acid MHA Mueller Hinton Agar DPPH 2,2-Diphenul-1 -picrylhydrazyl Vil ... fraction with a strong biological activity on cancer cell line For the purpose of screening of extracts from Combretum quadrangulare based on MCF-7 breast cancer cytotoxicity to find the fraction... the cytotoxicity possibility of the extracts/ fractions from Combretum quadrangulare MCF-7 breast cancer and other bioactivities of the fraction For the purpose: To determine the fraction from Combretum. .. research on compounds, with activity to kill breast cancer cells, extracted from plants is promising The topic "Cytotoxicity screening of the extracts from Combretum quadrangulare on MCF-7 breast cancer