VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ~~~~~***~~~~~ GRADUATION THESIS STUDY ON THE ANTI-OXIDATION AND ANTICANCER OF ARTHROSPIRA PLATENSIS’S EXTRACTION Student : TRAN NGOC DANG Student code : 610600 Class : K61CNSHE Supervisor : PHI THI CAM MIEN, Ph.D HANOI - 2021 STATUTORY DECLARATION I herewith formally declare that I have written the summited thesis independently under the guidance of Phi Thi Cam Mien, Ph.D Faculty of Biotechnology, Vietnam National University of Agriculture Data and results written in the essay completely truthful I did not use any outside support except for the quoted literature and other sources mentioned at the end of this thesis Hanoi, 5th February 2021 Student Tran Ngoc Dang i ACKNOWLEDGEMENT First and foremost, I would like to express my sincere thanks to my supervisor, Phi Thi Cam Mien, Ph.D, Faculty of Biotechnology, of the Vietnam National University of Agriculture who enthusiastically guided and helped me in the process of practicing and implementing the thesis I would like to thank the teachers of the Institute of Biotechnology and Institute for Research and Development of Microalgae of the Vietnam National University of Agriculture for creating favorable conditions to allow me to conduct experiments related to my graduation thesis I also would like thanks to Associate Professor Nguyen Duc Bach, Ms Hien, and all interns at the Institute for Research and Development of Microalgae for helping me in the research process here Last but no mean least, I would like to acknowledge my parents for their endless love, care and have most assistance and motivation for me for the whole of my life I also would like to explain my thanks to my siblings, brothers and sisters for their support and care for me all the time Hanoi, 5th February 2021 Student Tran Ngoc Dang ii TABLE OF CONTENTS STATUTORY DECLARATION i ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ABSTRACT ix CHAPTER I INTRODUCTION 1.1 Introduction 1.2 Objective 1.3 Requirements CHAPTER II LITERATURE REVIEW .2 2.1 Introduction in Arthrospira platensis 2.2 Taxonomy of Arthrospira platensis 2.3 Morphology of Arthrospira platensis 2.4 Distributions of Arthrospira platensis 2.5 Biochemical composition of Arthrospira platensis 2.6 Application of Arthrospira platensis 2.7 Methods of plant extraction 13 2.7.1 Soxhlet extraction method 13 2.7.2 Steam distillation method 14 2.8 Methods of assessing the antioxidant capacity of plant extracts 15 2.8.1 DPPH method 15 2.8.2 MDA method 16 2.8.3 ABTS˙+ method 17 iii 2.9 Some methods of assessing the ability of plant extracts to inhibit cancer cell growth 17 2.9.1.MTT method 17 2.9.2 Monks method 18 2.9.3 WST-8 method 18 2.10 The Arthrospira platensis studies in Vietnam and in the world 19 2.10.1 In Vietnam 19 2.10.2 In the world 20 CHAPTER III MATERIALS, CONTENT AND METHOD OF RESEARCH 22 3.1 Material, place and time of the study 22 3.1.1 Material 22 3.1.3 Location and time of study 23 3.2 The research contents 23 3.3 The Research Methods 23 3.3.1 Sample preparation method 23 3.3.2 The extraction method with Soxhlet to obtain extracts 23 3.3.3 Evaluate the antioxidant ability of microalgae extracts by the DPPH method 24 3.3.4 Evaluate the antioxidant ability of microalgae extracts by the MDA method 24 3.3.5 Evaluate the anti-cancer ability of microalgae extracts by MTT method 25 CHAPTER IV RESULTS AND DISCUSSION 28 4.1 Evaluate the effect of sample state on microalgae extraction efficiency 28 4.2 Evaluate the effect of solvent type on microalgae extraction efficiency 28 4.3 Evaluate the antioxidant ability of microalgae extracts by the DPPH method 31 iv 4.4 Evaluate the antioxidant ability of microalgae extracts by the MDA method 35 4.5 Evaluate the anti-cancer ability of microalgae extracts by MTT method 38 CHAPTER V CONCLUSION AND SUGGESTION .42 5.1 Conclusion 42 5.2 Request 42 REFERENCES .44 v LIST OF TABLES Table 2.1 Arthrospira platensis scientific classification .3 Table 2.2 Amino acid composition in Arthrospira platensis Table 2.3 Vitamin composition in Arthrospira platensis Table 2.4 The fatty acid composition in Arthrospira platensis Table 2.5 Mineral composition in Arthrospira platensis Table 2.6 Pigmentation composition in Arthrospira platensis .8 Table 4.1 The effect of sample state on microalgae extraction efficiency 28 Table 4.2 The effect of solvent type on microalgae extraction efficiency 29 Table 4.3 Antioxidant activity of extracts in the test by DPPH method 32 Table 4.4 The EC50 value of types of extracts in the DPPH test 33 Table The MDA antioxidant activity of studied samples 35 Table 4.6 The cytotoxicity of the sample on cell lines 39 vi LIST OF FIGURES Figure 2.1 Arthrospira platensis powder Figure 2.2 Soxhlet extractor 14 Figure 2.3 Steam distillation apparatus 15 Figure 2.4 DPPH chemical 16 Figure 2.5 A spectrometer 17 Figure 3.1 Human hepatocellular carcinom cells (HepG2 cells) 27 Figure 3.2 Human breast carcinoma cells (MCF-7 cells) 27 Figure 4.1 Extraction of Arthrospira platensis by Soxhlet extractor 30 Figure 4.2 Extracts of Arthrospira platensis 30 Figure 4.3 Arthrospira platensis extract 31 Figure 4.4 The correlation of concentration and% of antioxidant activity of extracts 33 Figure 4.5 Image of color change of sample in DPPH test 34 Figure 4.6 Experimental plates of three samples extract and control in MDA inhibitory reaction 37 Figure 4.7 The correlation between concentration and percentage inhibition of samples on HepG2 cell line 40 Figure 4.8 The correlation between concentration and percentage inhibition of samples on MCF-7 cell line 40 vii LIST OF ABBREVIATIONS Abbreviations Signification LSD Least Significant Difference-LSD- Test CV % Coefficient of variation SC% Scavenging effect IC 50 Half maximal inhibitory concentration EC 50 Effective concentration DPPH 1,1-diphenyl-2-picrylhydrazyl MDA Malonyl dialdehyd viii ABSTRACT Arthrospira platensis is also known as Spirulina platensis, it is one of the photoautotrophic, planktonic, filamentous green-blue algae Arthrospira platensis is a widely known microalga with nutritional and therapeutic applications due to its richness in nutrients and bioactive elements The uses of Arthrospira platensis contain: anti-oxidant, anti-cancer and anti-hyperalgesic effects This study was conducted to evaluate the effect of Arthrospira platensis microalgae extraction The results showed that the extraction efficiency of different solvents was different, using the 96% ethanol solvent resulted in the highest extracting efficiency (1.6%) and the total extract is 43.55 ml In the method DPPH, the antioxidant activity of extract ethyl acetate samples was the highest at a concentration of 500 (µg/ ml) with a percentage of antioxidant capacity of 61.57% and in the MDA method is shown extracts with ethyl acetate showed the strongest antioxidant capacity (53.06%) with 500 (µg/ ml) In addition, the Arthrospira platensis extraction using ethanol (96% ethanol) give significantly higher levels of inhibitory effect on two human cancer cell lines than the Arthrospira platensis extraction using ethyl acetate At a concentration of 100 μg/ml, the Arthrospira platensis extracts with ethanol (96% ethanol) can inhibit and kill 94.21% of breast cancer cells and 96.57% of liver cancer cells ix results to plot the correlation of concentration and % of antioxidant activity of extracts (Figure 4.4) The relationship between concentration and SC% of the extracts 90 80 SC (%) 70 60 50 40 30 20 10 0 50 100 150 200 250 300 350 400 450 Concentration (µg/ml) SC% Etyl acetate SC% H2O SC% Ethanol SC% Trolox Figure 4.4 The correlation of concentration and% of antioxidant activity of extracts From the results in table 4.3 calculate the value of EC 50 The active samples (SC ≥ 50%) will be tested to find EC 50 value, the results are in Table 4.4 Table 4.4 The EC 50 value of types of extracts in the DPPH test EC 50 (µg/ml) DPPH SC% SC% SC% SC% Extract of ethyl Extract of H O Extract of Ethanol Trolox acetate 178.65 96% >400 (µg/ml) 285.72 33 67.34 Table 4.4 shows that the extract of ethyl acetate has the lowest EC 50 value among the three tested samples This proves that the DPPH activity of this extract is the highest Besides, the extract of water with a test concentration of 400 (µg/ml) has not reached the rate of 50% antioxidant activity, so EC 50 concentration has not been calculated so it shows the lowest resistance to oxidation The DPPH free radical capture activity among extracts has statistically significant differences with P-value = 0.05 After adding DPPH to samples, incubate in the dark for 30 minutes The reaction color is shown in Figure 4.5 The color of the sample changes from purple to red to orange and fades until the reaction is completed The lighter the color of the test specimen after the end of the reaction, the higher the antioxidant activity Trolox as a reference material Ethanol 96% H2O Ethyl acetate Trolox DPPH Figure 4.5 Image of color change of sample in DPPH test The results of Table 4.4, Figure 4.4 and the conclusion in the test samples, the antioxidant activity of extract ethyl acetate samples was the highest at a 34 concentration of 400 (µg/ ml) with a percentage of antioxidant capacity of 61.57% 4.4 Evaluate the antioxidant ability of microalgae extracts by the MDA method We weight, the mixed samples into concentrations (µg / ml): 10,000, 2,000, 400, 80, 16 Then, we add 0.1 ml of the test sample at the tested concentrations to be reacted with ml of cerebral homogeneous fluid and add 0.8 ml of phosphate buffer, and 0.1 ml of Fenton system will make enough ml Incubate at 100°C for 15 minutes, cool, and conduct measurements at λ = 532 nm Trolox is used as reference material The results of the determination of antioxidant activity through inhibition of membrane lipid peroxidation (MDA test) are presented in Table 4.5 Table The MDA antioxidant activity of studied samples % MDA inhibitors Concentration Extract of Extract of Extract of ethyl acetate H2O Ethanol 96% 500 53.06 ± 0.34 31.04 ± 0.21 35.53 ± 0.46 81.25±0.42 100 18.2 ± 0.41 11.2 ± 0.134 12.92 ± 0.24 77.53 ± 0.39 20 9.12 ± 0.42 6.45 ± 0.34 7.75 ± 0.36 62.33 ± 0.38 3.85 ± 0.34 2.97 ± 0.54 3.65 ± 0.45 33.16 ± 0.35 0,8 1.42 ± 0.10 1.17 ± 0.45 1.35 ± 0.10 15.66 ± 0.57 CV% 1.68 3.30 2.90 1.00 5%LSD 0.52 0.69 0.70 0.956 IC 50 268.5 (µg/ml) >500 >500 10.04(µg/ml) (µg/ml) 35 Trolox MDA antioxidant activity of the research samples shows that the concentration of substances in the sample increases gradually, the ability to inhibit MDA increases This demonstrates that the oxidation resistance of the extractants increases proportionally with the increasing concentration of the test sample in the test tube, with a sample concentration of 0.8 (µg/ ml) a very low % inhibition of MDA, a sample extracted with ethyl acetate, the% inhibition rate of MDA was (1.42%), and the sample with water was% inhibited MDA is (1.17%), for samples extracted with ethanol the inhibitory% of MDA is (1.35%) The concentration of the test sample increased to 20 (µg / ml), all three samples showed the ability to inhibit MDA of samples extracted with ethyl acetate, samples extracted with water, samples extracted with ethanol, the % inhibition rates of MDA respectively is 9.12; 6.45; 7.75 The highest test sample concentration of 500 (µg / ml), the% MDA inhibition rates of the three test samples, respectively, were samples extracted with ethyl acetate (53.06%), water-extracted samples (31.04%), and samples extracted with ethanol (35.53%) The samples extracted with ethyl acetate showed the highest inhibition of lipid peroxidation of the three samples with IC50 = 268.5 (µg / ml) The two samples extracted with water and ethanol have not reached 50% inhibition of MDA Therefore, the IC50 value has not been determined The reference is Trolox which was stable in the experiment Observing Figure 4.6, a picture depicting the coloration reaction of the samples at different concentrations 36 Concentration (μg/ml) Lab disk image of samples in MDA test Extract of ethyl acetate Extract of Ethanol Extract of H O 96% 500 100 20 0.8 The negative control Concentration (μg/ml) Trolox 500 100 20 0.8 The negative control Figure 4.6 Experimental plates of three samples extract and control in MDA inhibitory reaction 37 Observing Figure 4.6 shows that the lighter the pink pattern, the higher the ability to inhibit MDA generation, the higher the resistance to oxidation The darker the pink pattern, the more MDA-generating lipid peroxidation process, the lower the antioxidant capacity, the higher the concentration of samples, the lighter the reaction color Arthrospira platensis samples extracted with water have the darkest color reaction, algae samples extracted with Ethyl acetate and Ethanol 96% are lighter in color 4.5 Evaluate the anti-cancer ability of microalgae extracts by MTT method The reagent (10 μl) mix in 10% DMSO was fed into the wells of the 96well tray to have a final study concentration range in the well of 100 μg / ml; 20 μg / ml; μg / ml; 0.8 μg / ml (out of 200 μl total) We will add to the test wells (already with 10 μl of reagent above) 190 μl of suitable cell count On the same test plate, arrange a number of wells as control without test sample, only the sample phase solvent is DMSO10% We place the culture plate in the incubator of CO2 at 37°C, 5% CO2, incubate for 72 hours After 72 hours, 10 µl of MTT (final concentration mg/ ml) was added to each well After hours, removing the medium, formazan crystals were dissolved with 50 µl (DMSO) 100% The OD value measured at a wavelength of 540 nm using a spectrophotometer The experimental results are presented in table 4.6 38 Table 4.6 The cytotoxicity of the sample on cell lines % Inhibitors Concentration (µg/ml) Extract of Ethyl acetate Extract of Ethanol 96% HepG2 MCF-7 HepG2 MCF-7 100 57.35 51.06 96.57 94.21 20 10.19 15.12 87.56 72.80 3.51 -1.33 51.40 44.56 0.8 -6.54 -4.27 11.05 16.36 IC 50 84.98 ±4.53 94.96 ±1.39 4.75±0.59 5.63±0.61 Ellipticine Concentration (µg/ml) HepG2 MCF-7 100 105.40 93.71 20 74.50 79.89 53.29 46.52 0.8 27.40 25.97 IC 50 0.39±0.02 0.43±0.05 Note: Ellipticine has been tested at concentrations of 10-2-0.4-0.08 µg / ml 39 110 %Inhibitors 90 70 50 30 10 Ethyl acetate -10 0.8µg/ml Ethanol 96% 4µg/ml 20µg/ml Ellipticine 100µg/ml Figure 4.7 The correlation between concentration and percentage inhibition of samples on HepG2 cell line 110 90 %Inhibitors 70 50 30 10 -10 Ethyl acetate 0.8µg/ml Ethanol 96% 4µg/ml Ellipticine 20µg/ml 100µg/ml Figure 4.8 The correlation between concentration and percentage inhibition of samples on MCF-7 cell line 40 The above results showed that extract of ethyl acetate samples showed activity on cancer cell lines HepG2 and MCF-7 with IC50 values of 84.98 ± 4.53µg/ ml and 94.96 ± 1.39, respectively The extract of the ethanol sample showed activity on HepG2 and MCF-7 cancer cell lines with IC50 values of 4.75 ± 0.59 µg/ ml and 5.63 ± 0.61, respectively The samples all showed a higher percentage of inhibition in HepG2 liver cancer cells compared with MCF-7 breast cancer cells In HepG2 liver cancer cells, the extract of ethanol sample showing the inhibitory percentage at the highest concentration of 100 μg/ ml was 96.57% higher than that of the extract of ethyl acetate sample of 57.35% On breast cancer cells MCF-7 sample extract of ethanol exhibited the inhibitory percentage at the highest concentration of 100 μg/ ml which was 94.21% higher than the extract of ethyl acetate sample of 51.06% The Ellipticine positive control was stable in the assay 41 CHAPTER V CONCLUSION AND SUGGESTION 5.1 Conclusion On the basis of obtained experimental results, we can conclude: The results showed that, samples of Arthrospira platensis harvested at Vietnam National University of Agriculture when extracting the dried Arthrospira platensis (1.71%) is always higher than when extracting the fresh Arthrospira platensis (0.05%) The extract with an ethanol 96% solvent has the highest efficiency (1.6%), if extract with the solvent ethyl acetate the efficiency is the lowest in the three solvents (1.55%) In the method DPPH, at the highest concentration of 400 (µg/ml), the extract of ethyl acetate sample has the highest inhibition of oxidation (61.57%) higher than the extract of water sample (43.66%) and Ethanol 96% sample (53.16%) Algae samples extracted with water have the darkest color reaction demonstrated antioxidant capacity is the weakest (31.04%), two other samples extracted have lighter in color, ethyl acetate sample showed the strongest antioxidant capacity (53.06%) The samples showed the percentage of inhibition in HepG2 liver cancer cells higher compared with MCF-7 breast cancer cells On the HepG2, at the highest concentration of 100μg/ ml, the extract of ethanol sample showing the inhibitory percentage (96.57%) higher than the extract of ethyl acetate sample (57.35%) On the MCF-7, at the highest concentration of 100μg/ ml, the extract of ethanol sample showing the inhibitory percentage (94.21%) higher than the extract of ethyl acetate sample (51.06%) 5.2 Request Arthrospira platensis algae have many different groups of compounds with high therapeutic ability From the results obtained, it is necessary to 42 analyze to determine other compound components in the extracts by the soxhlet method, suggesting further studies on chemical composition as well as biological activity of Arthrospira platensis algae 43 REFERENCES Ali, S.K (2012) Spirulina, an overview School of Bio Scienses and Technology, VIT University, Vellore India Ahsan M., Habib B and Parvin M.2008 A review on Culture, prodcution and use of Spirulina as food for human and feeds for domestic animals and fish FAO fisheries and aquaculture circular; 1034: 33 Briot, R, Jouvencel, D de, Raginel, L., Rouille, G (2012) 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