MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY CAO DUC DANH STUDY ON ISOLATION AND INVESTIGATION OF ANTIBIOTIC AND CYTOTOXIC ACTIVITY OF SECONDARY COMPOUNDS FROM THREE ACTINOMYCETES STRAINS STREPTOMYCES G246, G261, G248 COLLECTED IN SOME CENTRAL EAST VIETNAM SEA Major: Organic Chemistry Code: 44 01 14 SUMMARY OF CHEMISTRY DOCTORAL THESIS HA NOI- 2019 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Advisor 1: Prof Dr Habil Pham Van Cuong Advisor 2: Dr Tran Dang Thach 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour … date … month … 2019 The thesis can be found in - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - The National Library of Vietnam INTRODUCTION The urgency of the thesis The ocean accounts for 70% of the earth's surface, where there is the largest biological diversity on earth It is home to 34 of the 36 animal and plant sectors on earth with more than 300,000 known species The marine environment has been known as a rich source of natural compounds, such as a huge pharmaceutical warehouse awaiting exploration and discovery Characteristics of harsh deep sea habitats are the conditions for forming organic compounds with unique chemical structure characteristics and precious biological activity The study of secondary active ingredients produced from marine microorganisms in the world has achieved many remarkable achievements Among them are many secondary compounds with interesting chemical structure and biological activity At the same time many of these compounds are being tested further for medical applications Vietnam is located in the Pacific region (with sovereignty with an area of about 1,000,000 km2) with a rich and diverse marine system, with great potential for marine resources The Government of Vietnam has oriented the development of marine economy, exploitation of natural resources and the study of natural products from the sea However, the study of secondary compounds from Vietnam's marine microbial source has only just begun, there are very few published studies, although the source of marine diversity of our country is very large Infectious diseases account for the majority of human and animal diseases Around the second half of the 19th century, it was discovered that microorganisms are the cause of infectious diseases Therefore, chemotherapy aims at pathogenic microorganisms that have been developed into the main therapy In 1928, Alexander Fleming discovered Penicillin - a powerful antibiotic compound and purified by Abraham, Chain, and Florey in a stable form that had a therapeutic effect in 1941 Penicillin antibiotics became famous because It saved many lives in World War II Since then, antibiotics have become an early miracle pharmaceutical that occupies the world's leading position in the pharmaceutical field, with increasingly new results, with increasing demand and increasing production volume Currently, there are more antibiotics that are extracted from fungi, bacteria, actinomycetes, which occupy most of them with marine actynomycetes But more and more microorganisms cause disease resistance to existing antibiotics Therefore, it is necessary to continue to research, find and discover new antibiotics, active ingredients with tuberculosis and anti-cancer resistance Therefore, we carried out this thesis with the title: “Study on isolation and investigation of antibiotic and cytotoxic activity of secondary compounds from three actynomycetes strains Streptomyces G246, G261, G248 collected in some central East Vietnam sea” 2 The aim of the thesis - Study to isolate and determine the structure of secondary compounds from cultivation of strains of microorganisms isolated from central East Viet Nam sea - Survey of cytotoxic activity and testing antimicrobial activity of isolated substances as a scientific basis for the research and application of these compounds The main contents of the thesis - Review of previous studies on secondary compounds as well as biological activity from marine microbial strains - Finding procedures for handling culture fluid to create extracts Purify these extracts on column chromatography to obtain fractions - Refining substances in the segments to obtain compounds - Determine the chemical structure of isolated compounds - Testing antimicrobial activity to test isolated substances - Testing anticancer activity in vitro for some cancer cell lines such as KB, MCF-7, Hep-G2, Lu-1 of compounds isolated CHAPTER OVERVIEW 1.1 The distribution and diversity of marine microorganisms 1.2 Bacterial radiation and the formation of secondary compounds from actinomycetes 1.3 Common marine bacteriophage families 1.4 Secondary compounds with biological activity from marine bacteriophages 1.4.1 Finding in the world 1.4.1.1 The compounds have antibiotic activity 1.4.1.2 The compounds are active against tuberculosis 1.4.1.3 The compounds with cytotoxic activity 1.4.2 Finding in our country CHAPTER MATERIALS AND METHODS 2.1 Materials and research equipment 2.1.1 Material 2.1.3 Device 2.2 Research Methods 2.2.1 Sample collection method 2.2.2 Marine bacteriophage isolation method 2.2.3 Methods for cleaning strains with loop implants 2.2.4 Method to keep the actinomycetes after isolation 2.2.5 Activation and culture methods 2.2.6 Method of extraction from cultured fluid for biological activity screening 2.2.7 Method of identifying bacteriophages 2.2.8 Mass birth method 2.2.9 Methods of isolation of secondary compounds 2.2.10 Methods for determining the chemical structure of clean compounds can be extracted 2.2.11 Method of testing antibiotic antimicrobial activity 2.2.12 Test method of cytotoxic activity 2.2.13 Test method for anti-tuberculosis activity CHAPTER EXPERIMENT AND RESULTS 3.1 Sample collection results 25 samples have been collected from central East Vietnam sea, namely: - In Thanh Hoa sea area, sediment samples were collected - In Quang Binh sea area, sediment samples, sea form, seaweed samples and coral sample were collected In the Quang Tri sea area, there were sediment samples, sea pattern, seaweed sample - In the water of Son Tra peninsula, Da Nang obtained sample of sediment, samples of seaweed, sample of seaweed, sample of thorny skin, sample of bag - In Cu Lao Cham coastal area, Quang Nam obtained sediment samples, model of thorny skin, soft body sample, sea rabbit model, solid tail pattern 3.2 Result of isolation of actinomycete strains - From 25 samples collected in the central waters of Vietnam, 32 bacteria strains with different morphology and colors were isolated - From 12 samples collected in the waters of Thanh Hoa, Quang Binh and Quang Tri, 12 bacteria strains were isolated - From 13 samples collected in Da Nang and Quang Nam waters, 20 bacteria strains were isolated 3.3 Results of bioactivity test of actinomycete strains Antiretroviral resistance results: 29/32 active strains (see details in table 3.1) Results of anti-tuberculosis screening were performed at UIC University for Mycobacterium tuberculosis H37Rv strain The results showed that 21/32 strains showed antituberculosis activity, in which strain showed very good activity with> 90% inhibitory value, strains showed good activity with inhibitory%> 50% (see details in table 3.2) Coarse residues of cultured extracts of 32 studied strains were tested for cytotoxic activity against human cancer cell line (KB - carcinoma (CCL-17) Toxic cancer of KB epithelial cells (see details in Table 3.3) 3.4 Results of identification of bioactive antibiotic strains 3.4.1 Observe morphological characteristics of studied strains 3.4.2 Multiplication of 16S RNA riboxom gene 3.4.3 Genome sequencing The nucleotide sequence of the 16S ARN riboxom gene section of strains G246, G261, G248 showed that the studied strains belong to genus Streptomyces sp (see PLIII.1.4) 3.5 The results of high biomass of strains have good biological activity The strains of G246, G261, G248 have good biological activity, they are fermented in large quantities of 50 liters in suitable conditions to conduct further studies 3.6 Extraction and chemical structure of secondary compounds from Streptomyces sp G246 3.6.1 Sample processing, creating a residue 3.6.2 Isolate substances from the extract Figure Diagram of isolation of Streptomyces sp G246 3.6.3 Physical parameters and spectral data of compounds isolated from Streptomyces sp G246 3.7 Extraction and chemical structure of secondary compounds from Streptomyces sp G261 3.7.1 Sample processing, creating a residue 3.7.2 Isolate from Streptomyces sp G261 Figure Diagram of isolation of Streptomyces sp G261 3.7.3 Physical parameters and spectral data of secondary compounds from the Streptomyces sp G261 3.8 Extraction and chemical structure of secondary compounds from Streptomyces sp G248 3.8.1 Sample processing, creating a residue 3.8.2 Isolate substances from the extract 3.8.3 Physical parameters and spectral data of secondary compounds from the Streptomyces sp G248 Figure 3 Diagram of isolation of Streptomyces sp G248 3.9 Results of bioactivity test of compounds isolated from actinomycete strains 3.9.1 Performance test results for test microorganisms of some isolated compounds Of the 26 substances isolated, we have tested antimicrobial activity As a result, there are 13 substances with antimicrobial antibiotic activity, many of which have very strong activity such as G246-2, G248-11, G248-12, G248-9 and many substances with good antifungal activity as G246-6, G261-11, G248-11 (see details in table 3.5) 3.9.2 The results of anti-tuberculosis activity test of some isolated compounds The results of tuberculosis resistance testing were performed at UIC University Some substances tested for anti-tuberculosis activity with Mycobacterium tuberculosis H37Rv, in which G246-1 exhibited the best activity with MIC value of 6.00 µg / ml, G248-10 showed good activity with MIC value = 11.17 µg / ml, while G248-9 showed weak activity with MIC value of 48.02 µg/ ml The remaining substances did not show activity at the study concentration (50 µg/ ml) (see details table 3.6) 3.9.3 Test results of cytotoxic activity of some isolated compounds 14 compounds with tested antimicrobial activity, tuberculosis resistance from three research strains (G246, G261 and G248) were tested for cytotoxic activity with four cancer cell lines in: KB - Schedule cancer tissue (CCL - 17TM); Hep G2 - liver cancer (HB - 8065TM); MCF-7 - breast cancer (HTB - 22TM) and LU-1 - lung cancer (HTB-57TM) The results are detailed in Table 3.5 The result has compounds with cytotoxic activity, including active substances with KB cell line, active compounds with liver cancer cell line Hep-G2, active compounds calculated with Lu-1 cell lines, active compounds with MCF7 cell lines (see details in table 3.7) CHAPTER DISCUSSION OF RESULTS 4.1 Sample collection results A total of 25 samples included: 12 samples in the waters of Thanh Hoa, Quang Binh and Quang Tri; 13 samples in Da Nang and Quang Nam waters Specifically, there are 10 samples of sediment, samples of seaweed, samples of seaweed, sample of soft corals, samples of echinoderms and other samples 4.2 Result of isolation of actinomycete strains From 25 samples, 32 isolates with different morphology and colony color were isolated from the collected sample (details are described in Appendix PL III.1.2) 4.3 Results of bioactivity test of strains obtained - The results of the test of antimicrobial activity with crude scale showed that 29/32 isolated strains are resistant to the strains of test microorganisms - The results showed that 21/32 strains exhibited anti-tuberculosis activity - The results showed that 20 strains had toxic activity of KB epithelial cancer cells (IC50