Antioxidant activity of Myxobacteria isolated from soil.pdf

MINISTRY OF EDUCATION AND TRAINING NGUYEN TAT THANH UNIVERSITY oOo SUMMARY REPORT ON STUDENT PROGRAM SCIENTIFIC RESEARCH 2020 Topic’s name: ANTIOXIDANT ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Topic’s code: Topic manager: NGUYEN THI KIM HONG Scientific instructor: M.Sc Pharm NGUYEN THI NGOC YEN Industry: Pharmacology The members participate: No First and last name Nguyen Thi Kim Hong Nguyen Phuong Cham Code 1511540916 1511540569 Ho Chi Minh city, 10/2020 Class 15DDS8A 15DDS8A CONTENTS CONTENTS i LIST OF ACRONYMS iv LIST OF TABLES V LIST OF FIGURES vi ABSTRACT vii INTRODUCTION CHAPTER LITERATURE REVIEW 1.1 The SOIL MICROBIOTA ANDPOTENTIAL SECONDARY METABOLITES 1.1.1 Role in organisms 1.1.2 Role in ecosystem 1.1.3 Role in medical use 1.2 THE MYXOBACTERIA 1.2.1 The ecology of the myxobacteria 1.2.2 Genome 1.2.3 Enzymatic activity 1.2.4 Phylogeny of Myxobacteria 1.2.5 Morphological facts seemto explain the special traits of the myxobacteria 10 1.3 THE DISCOVERY OF BIO ACTIVE METABOLITES 15 1.4 ISOLATION AND IDENTIFICATION 16 1,4.1 Isolation of myxobacteria 16 1.4.2 Purification of isolates 18 1.4.3 Identify myxobacteria strains 18 1.5 ANTIOXIDANTION ACTIVITY AND MICROBIAL SOURCE 19 1.5.1 Antioxidant agents and myxobacteria 19 1.5.2 Folin-Ciocalteu assay for polyphenols total 20 1.5.3 ABTS radical cation scavenging activity .20 CHAPTER MATERIALS AND RESEARCH METHODS .22 2.1 SUBJECTS AND MATERIALS 22 2,1.1 Subjects 22 2,1.2 Instruments and equipments 22 2.1.3 Media and chemicals 23 2.2 RESEARCH METHODS 24 2.2.1 Samples collection and storage conditions 24 2.2.2 Treatment of soil samples 24 2.2.3 Isolation of myxobacterial strains 25 2,2.4 Purification methods .26 2.2.5 Storage of isolates 27 2,2,6 Identify the Myxobacteria strains 27 2,2.7 Extraction of myxobacterial crude extract 29 2.2.8 Screening antioxidant activity 31 CHAPTER RESULTS AND DISCUSSION 34 3.1 Collection of samples 34 3.2 Isolation and identification 35 3.2.1 Isolation 35 3.2.2 Identification 36 3.2.3 Discussion 43 3.3 Screening antioxidant activity 45 3.3.1 Folin-Ciocalteu method for totalpolyphenols 45 3.3.2 ABTS radicalcation scavenging activity .48 3.3.3 Discussion 51 CHAPTER CONCLUSION AND SUGGESTION 53 4.1 CONCLUSION 53 ii 4.2 SUGGESTION 53 REFERENCES .54 iii LIST OF ACRONYMS CFU Central Processing Unit LB broth Luria Bertani Broth P-medium Peptone medium wcx Water agar with cycloheximide 100 pg/ml Con Concentration Abs Absorbance ABTS 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonate DPPH 2,2-diphenyl-1 -picrylhydrazyl FC Folin-Ciocalteu GAE Gallic acid equivalent TEAC Trolox equivalent antioxidant capacity ANOVA Analysis of variance iv LIST OF TABLES Table 1.1 Overview of myxobacterial strains found in soil Table 1.2 A list of published bioactive compounds 16 Table 2.1 List of laboratory instruments used in this research .22 Table 2.2 Some chemicals and reagents are used throughout the process 23 Table 3.1 Statistical table of soil samples collected in Vietnam 34 Table 3.2 The image of morphological Myxobacteria 36 Table 3.3 The number of identified strains from different provinces 38 Table 3.4 Myxobacterial strains isolated by different methods 39 Table 3.5 Identitification of new isolates based on 16S rDNA analysis 40 Table 3.6 Data for establishing gallic acid calibration curve 45 Table 3.7 Data of absorbance and concentration of each sample 46 Table 3.8 ANOVA analysis of polyphenol content data of samples 48 Table 3.9 Data for establishing Trolox calibration graph 48 Table 3.10 The Trolox equivalent concentration of extracts from p and VY3 media 49 Table 3.11 ANOVA analysis of polyphenol content data of samples 51 V LIST OF FIGURES Figure 1.1 Group and solitary-based predation by Myxococcus xanthus Figure 1.2 Stereophotomicrographs of Byssovorax cruenta on filter paper Figure 1.3 Taxonomy of Myxococcales Figure 1.4 The predatory life cycle of Myxococcus xanthus 10 Figure 1.5 Stereophotomicrographs of swarm colony surface Chondromyces 11 Figure 1.6 Morphology of two myxobacteria suborders 12 Figure 1.7 The variety of myxobacterial fruiting bodies 14 Figure 1.8 Wild rabbit dung 17 Figure 1.9 The reaction mechanism of 2,2’-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) with antioxidant 20 Figure 2.1 The study procedure 24 Figure 2.2 Illustration of three isolation methods 26 Figure 2.3 The myxobacterial purification 27 Figure 2.4 Gram staining procedure 28 Figure 2.5 Decomposing filter paper 28 Figure 2.6 Mega X and phylogenetic tree 29 Figure 2.7 The procedure of crude extract preparation 31 Figure 2.8 The mechanism of Folin-Ciocalteu method 32 Figure 3.1 Apperances of fruiting bodies from isolation 35 Figure 3.2 The gram-stained images of vegetative cells 37 Figure 3.3 The number of 43 myxobacterial strains isolated 39 Figure 3.4 Myxobacterial strains isolated by different methods 39 Figure 3.5 Gallic acid calibration curve in the Folin-Ciocalteu method 45 Figure 3.6 The sample concentration expressed as gallic acid equivalents 47 Figure 3.7 Trolox calibration curve in ABTS assay 49 Figure 3.8 The sample concentration expressed as Trolox equivalents 50 VI ABSTRACT DISSERTATION SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE PHARMACIST - ACADEMIC YEAR 20 - 20 ANTIOXIDANT ACTIVITY OF MYXOBACTERIA ISOLATED FROM SOIL Nguyen Thi Kim Hong Supervisor: M.Sc Pharm Nguyen Thi Ngoc Yen Introduction: The objectives of thesis is to explore new strains capable of producing secondary metabolites with antioxidant activity that opened promising treatment direction on cancer disease in the future Materials and methods: Myxobacteria were isolated using three conventional methods (WCX with E coli bait, filter paper, and rabbit dung) Myxobacterial with morphological characteristics and 16S rRNA gene sequence analysis were identified Antioxidant activities were evaluated by (i) The Folin-Ciocalteu method for total phenol content and (ii) The ABTS radical cation scavenging activity Results: From 110 soil samples collected at 19 cities and provinces, 43 identified strains /68 pure isolates with high similarity (from 91.1-100%) were assigned in six genera, including Archangium, Angiococcus, Chondromyces, Corallococcus, Cystobacter, and Myxococcus The total contents of polyphenols are ranged from 7.522 to 67.864 pg GAE equivalents/ml and 0.250 - 89.776 pg GAE/ml for extracts from P-medium and VY3, respectively Extract of VL 2.5 gave the highest polyphenol total content in both culture media In ABTS method, the Trolox concentration ranges from 2.508 ±0.199 to 13.428 ±0.160 pg TEAC/ml, in which, the HG 2.25 extract exhibits the highest activity (equivalent 13.428 ± 0.160 pgTEAC/ml) InVY3 medium, the Trolox values ranges from 2.031 ±0.138 to 14.010 ± 0.142 pg TEAC/ml with the highest activity is GL 4.3 (equivalent 14.010 ± 0.142 pg TEAC/ml) Conclusion: Most of the myxobacterial strains exhibited antioxidant activity that has different concentrations and depends on the culture media The results are statistically significant differences in the Folin-Ciocalteu method and ABTS assay So, each strain must choose a suitable medium for fermentation The HG 2.25, HCM 8.1, VL 2.5, GL 4.3, TH 8.2 were the potential strains with the best antioxidant activity to proceed to the next active screening steps, e.g., antitumor activity vii Pharmaceutical Dissertation Introduction INTRODUCTION In recent years, there has been a great deal of research toward the free radical fields Various endogenous systems generate free radicals in our body, and a close relationship was demonstrated between an increase in free radicals and the risk of exposure to pathological states The high concentrations of free radicals promote oxidation, damage cellular structures, or biologically relevant molecules such as DNA, proteins, carbohydrates, and lipids Free radicals attack essential macromolecules leading to cell damage and degenerative diseases, cardiovascular disease, atherosclerosis, hypertension, diabetes, arthritis, weakened immune system, and the phenomenon of premature aging Therefore, a balance between free radicals and antioxidants is necessary for proper physiological function If free radicals overwhelm the body's ability of regulation, the supplement of antioxidants is essential to control them The research on antioxidants is increasingly interested in reducing free radicals and preventing diseases in the most effective way The use of a source of antioxidants from microorganisms is dominant because of their broad distribution in nature, easy cultivation on a large scale, low-cost cultural media, and rapid growth [2], [14] Myxobacteria is also one of the most promising strains since they were functionally characterized to assess their ability to produce antioxidant, antimicrobial, and anticancer metabolites Most compounds from myxobacteria were not found in other organisms and are unique to the bacterium [5], [20] According to our research, up to now, research on the biological activities of the myxobacterial extracts is still limited in Vietnam Therefore, this study is conducted to evaluate the antioxidant activity of crude extract from myxobacteria with the aim of finding more sources of the natural microbial system to protect human health The screening of active substances with antioxidant activity from myxobacteria strains is a beginning step for the detection of strains capable of producing anticancer substances So the thesis named "Antioxidant activity of myxobacteria isolated from the soil" was performed to satisfy four objectives: - Isolation of myxobacteria strains with three methods: (1) Isolation using wild rabbit dung, (2) Isolation using E coll baiting on wcx medium, and (3) Isolation of cellulolytic myxobacteria with ST21 medium (using filter paper); Nguyen Thi Kim Hong Pharmaceutical Dissertation Introduction - Purification and storage of myxobacteria strains; - Identification using morphological characteristics, gram staining, or 16S rRNA analysis; - Preparation of crude extracts from purified strains and evaluation of the antioxidant activity using the ABTS radical cation scavenging activity and Folin - Ciocalteu method Nguyen Thi Kim Hong ... capable of producing anticancer substances So the thesis named "Antioxidant activity of myxobacteria isolated from the soil" was performed to satisfy four objectives: - Isolation of myxobacteria. .. activities of the myxobacterial extracts is still limited in Vietnam Therefore, this study is conducted to evaluate the antioxidant activity of crude extract from myxobacteria with the aim of finding... sources of the natural microbial system to protect human health The screening of active substances with antioxidant activity from myxobacteria strains is a beginning step for the detection of strains