VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ~~~~~***~~~~~ GRADUATION THESIS STUDY ON THE ANTI-OXIDATION AND ANTI- CANCER 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. ii TABLE OF CONTENTS. iii LIST OF TABLES .v LIST OF FIGURES.
vii LIST OF ABBREVIATIONS. Introduction in Arthrospira platensis. Taxonomy of Arthrospira platensis. Morphology of Arthrospira platensis.
Distributions of Arthrospira platensis. Biochemical composition of Arthrospira platensis. Application of Arthrospira platensis. Methods of plant extraction.
Soxhlet extraction method. Steam distillation method. Methods of assessing the antioxidant capacity of plant extracts. Some methods of assessing the ability of plant extracts to inhibit cancer cell growth.
The Arthrospira platensis studies in Vietnam and in the world. In the world. MATERIALS, CONTENT AND METHOD OF RESEARCH. Material, place and time of the study.
Location and time of study. The research contents. The Research Methods. Sample preparation method.
The extraction method with Soxhlet to obtain extracts. Evaluate the antioxidant ability of microalgae extracts by the DPPH method. Evaluate the antioxidant ability of microalgae extracts by the MDA method. Evaluate the anti-cancer ability of microalgae extracts by MTT method.
RESULTS AND DISCUSSION. Evaluate the effect of sample state on microalgae extraction efficiency. Evaluate the effect of solvent type on microalgae extraction efficiency. Evaluate the antioxidant ability of microalgae extracts by the DPPH method.
Evaluate the antioxidant ability of microalgae extracts by the MDA method. Evaluate the anti-cancer ability of microalgae extracts by MTT method. CONCLUSION AND SUGGESTION .44 v LIST OF TABLES Table 2. Arthrospira platensis scientific classification.
Amino acid composition in Arthrospira platensis. Vitamin composition in Arthrospira platensis. The fatty acid composition in Arthrospira platensis. Mineral composition in Arthrospira platensis.
Pigmentation composition in Arthrospira platensis. The effect of sample state on microalgae extraction efficiency. The effect of solvent type on microalgae extraction efficiency. Antioxidant activity of extracts in the test by DPPH method.
The EC50 value of types of extracts in the DPPH test. The MDA antioxidant activity of studied samples. The cytotoxicity of the sample on cell lines .39 vi LIST OF FIGURES Figure 2. Arthrospira platensis powder.
Steam distillation apparatus. Human hepatocellular carcinom cells (HepG2 cells). Human breast carcinoma cells (MCF-7 cells). Extraction of Arthrospira platensis by Soxhlet extractor.
Extracts of Arthrospira platensis. Arthrospira platensis extract. The correlation of concentration and% of antioxidant activity of extracts. Image of color change of sample in DPPH test.
Experimental plates of three samples extract and control in MDA inhibitory reaction. The correlation between concentration and percentage inhibition of samples on HepG2 cell line. 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 3 different solvents was different, using the 96% ethanol solvent resulted in the highest extracting efficiency (1.6%) and the total extract is 43.
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. 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 CHAPTER I. Introduction Research looking for natural sources of compounds with antioxidant and cancer-preventing abilities is a growing trend.
Obtained from a variety of sources such as plants, microorganisms, fungi, bacteria, and microalgae. In these objects, microalgae have many advantages such as being able to synthesize many different compounds including fats, proteins, vitamins, pigments and secondary metabolic compounds including antioxidants, anticancer agents, antibiotics, antifungal and antibacterial. In addition, microalgae are able to grow quickly in a simple environment and easily replicate on a large scale. Therefore, the investigation of the antioxidant and cancer-preventing capabilities of natural compounds from microalgae opens up prospects in the exploitation and application of these compounds.
I conducted the project “Study on the anti-oxidation and anti-cancer of Arthrospira platensis’s extraction” to evaluate the antioxidant and cancer-preventing ability of the microalgae 1. Objective Study on the anti-oxidation and anti-cancer of Arthrospira platensis’s extraction 1. Requirements Evaluate the effect of sample state on microalgae extraction efficiency Evaluate the effect of solvent type on microalgae extraction efficiency Evaluate the antioxidant ability of microalgae extracts by DPPH method Evaluate the antioxidant ability of microalgae extracts by MDA method Evaluate the anticancer ability of microalgae extracts by MTT method 1 CHAPTER II. Introduction in Arthrospira platensis Arthrospira platensis has been harvested for food purposes since ancient times, it appeared about 3.5 billion years ago.
Known commonly as Spirulina, blue-green cyanobacteria, Spirulina is a photosynthesizing cyanophyte (blue- green algae) that grows vigorously in strong sunshine under high temperatures and highly alkaline conditions. It was first described in 1940, by Dangeard, a French phycologist, who received a sample from a French colleague, in the French Army in equatorial Africa, near present-day Lake Chad. After that, Dangeard published a report it. In 1964, a botanist on a Belgian Trans-Saharan expedition, Jean Léonard, reported finding a curious greenish, edible cake being sold in native markets of Fort- Lamy (now N’Djamena) in Chad (Léonard, 1967).
These cakes are Arthrospira platensis algae from Lake Chad and then it dried. platensis are filamentous blue-green algae that have gained considerable popularity in the health food industry, medical and increasingly as a protein and vitamin supplement to aquaculture diets. It grows stability in water, can be harvested and processed easily and has very high macro- and micro-nutrient contents. In 1973, The Food and Agriculture Organization (FAO) and World Health Organization (WHO) officially recognized A.
platensis as a source of nutrients and rare medicinal herbs in anti-malnutrition and anti-aging. platensis has been studied since the 1970s. After the 1970s, there has been a lot of research on it, in the Institute of Biotẹchnology under the research of scientist Dang Dinh Kim, Bui Thi Kim Anh and partner used CO 2 from the brick factory in Ha Noi to experimentally produce rich-nutrient Spirulina platensis. Currently, our country has many establishments growing Arthrospira platensis algae such as Vinh Hao (Binh Thuan province) since 1979, Suoi Nghe (Dong Nai province).
Taxonomy of Arthrospira platensis In 1827, P. Turpin isolated Arthrospira platensis from a freshwater sample (Ciferri, 1983). In 1962, Arthrospira platensis was officially classified as prokaryotes, because it has many bacteria-like characteristics such as no phospholipid membranes, no mitochondria and chloroplasts (Henrikson, 1980). Arthrospira platensis scientific classification Taxonomy of Arthrospira Platensis Kingdom Prokaryota Phylum Cyanobacteria Class Chroobacteria Order Oscillatoriales Family Phormidiaceae Genus Arthrospira Species Arthrospira platensis In 1852 that the first taxonomic report written by Stizenberger, this new genus the name Arthrospira based on the septa presence, helical form and multicellular structure.
Ever since 1852, many species of this genus of helically coiled cyanobacteria have been described and isolated. In 1892, Gomont arranged the aseptate form to the Spirulina genus and the septal form to the Arthrospira genus. Nowadays, two genera reunified the designation Spirulina without considering the septum presence (Ciferri,1983). Morphology of Arthrospira platensis The Arthrospira genus comprises helical trichomes of varying size and with various degrees of coiling, including tightly coiled morphology to even a straight form.
Arthrospira platensis has a prokaryotic organization, with several thread- like structures (fibrils) of DNA, along with a photosynthetic system, a cell wall, ribosomes among others (Ali,2012). Arthrospira platensis has flank hairs (fimbria) - fibers 5 to 7 nm in diameter and 1-2 µm in length around the body. These hairs act as paddles to help the cyanobacteria work. Arthrospira platensis is capable of generating small gas vesicles (70 nm in diameter) and is structured from coiled protein fibers.
Arthrospira platensis powder 2. Distributions of Arthrospira platensis Arthrospira platensis is widely distributed in soil, freshwater, brackish water, saltwater and hot springs. It is plankton living freely in bicarbonate rich environments (HCO3-), highly alkaline (pH 8. Arthrospira platensis is distributed in many parts of the world.
The wide range of distribution: Africa, Asia, Europe. This distribution is due to natural selection, possibly for other reasons as well, such as due to migrations following some migratory birds, 4 typically the flamingo using blue algae as a food source in the Americas. Biochemical composition of Arthrospira platensis Arthrospira platensis is considered to be a perfect nutritional source in the food, announced by FAO and WHO. Besides, according to the NASA nutrition program for astronauts, it has all kinds of essential nutrients for the body such as protein, lipids, carbohydrates, about 30 micronutrients and essential vitamins.
Arthrospira platensis has a high nutritional content, especially a very high protein content that accounts for 50-70% of the dry weight. The protein content of algae is higher than that of traditional foods such as eggs 47%, cow's milk 37 %, 36% soybean, 45% beer yeast, 26% wheat germ (Ciferri, 1983). Besides, Arthrospira platensis contains 18 different amino acids with 8 essential amino acids that the body cannot itself. Among them, the highest value is Leucine (10.9% of total amino acids), Valine 7.
Amino acid composition in Arthrospira platensis Content Content Amino acid composition Amino acid composition (g/100g) (g/100g) Alanine 4.5 Source: The role of Parry Organic Spirulina in Health Management October 2010 5 Besides, in Arthrospira platensis found many water-soluble vitamins like B1, B12, B6, A, C, K.