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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCES AND TECHNOLOGY ----------------------------- DINH THI HA ISOLATION, CHEMICAL MODIFICATION AND BIOLOGICAL EVALUATION OF STEROIDS FROM THE STARFISH ACANTHASTER PLANCI Speciality: Natural Products Chemistry Code: 9 44 01 07 ABSTRACT DISSERTATION Dr. CHEMISTRY HANOI-2020 This dissertation was completed in: Graduate university of Sciences and Technology – Vietnam Academy of Science and Technology. Supervisor 1: Assoc. Prof. Tran Thi Thu Thuy Supervisor 2: Assoc. Prof. Ngo Dai Quang Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended in front of the Academy Thesis Evaluation Council at the: Graduate university of Sciences and Technology – Vietnam Academy of Science and Technology. At the time..… hour…., August…, 2020 Thesis can be found at the: - Library of Graduate university of Sciences and Technology - Vietnam National Library INTRODUCTION 1. Reason to choose the topic Vietnam has extremely diverse and abundant marine resources with hundreds of thousands of different species of plants, animals and microorganisms... However, the values provided for medicinal resources of marine species is still very limited. Nowaday, there have been many steroid subtances isolated from marine organisms and evaluated for biological activity. In particular, many polar steroid compounds exhibit biological activity against many cancer cell lines, and can be applied in medicine and pharmacy. These steroids are important to organic synthesis. They have become a model to synthesize from popular natural steroids by more efficient and economical methods. The starfish class belong to the echinoderm phylum, which is one of the rich sources provide of polarizing steroid compounds. This substance class has diverse structure and they exhibit many biological activities such as: antibacterial, antifungal, anti-inflammatory, anti-cancer, anti-viral, inhibiting fertilization.... So, polar steroids from starfish have been attracting the attention of many scientists around the world. In Vietnam, there is very little research on this substance class. Studies are only in the direction of isolating natural compounds and evaluating some of their biological activity. At present, there has not been any overall research on isolation, chemical metabolism and evaluation of the bioactivity of steroids isolated from marine organisms. Starfish Acanthaster planci is a common starfish in Vietnam's waters, they are a threat to the survival of living coral reefs because coral is their preferred food source. Preliminary studies also show that the main chemical composition of starfish Acanthaster planci is steroids, especially polar steroids. Follow this direction, the author to choose the research topic of the thesis: "Isolation, chemical modification and biological evaluation of steroids from the starfish Acanthaster planci ". 2. Research objectives of the thesis 1 Study on chemical composition of starfish Acanthaster planci of Vietnam, synthesizing hydroxyl and oxime derivatives from a steroid isolated from this starfish and assessing biological activity of isolated and synthesized compounds. 3. The main research content of the thesis To achieve the above objectives, the thesis has implemented the following contents: • • • Isolation and determination of chemical structure of compounds from starfish Acanthaster planci, especially steroids Convert derivatives in the direction of hydroxylation and oximelation from a high steroid in starfish Acanthaster planci. Evaluation some biological activities of isolated and synthesized compounds. CHAPTER 1. OVERVIEW DOCUMENT The literature review is a collection of national and international research on: 1.1. General introduction to starfish class (Asteroidea) 1.2. Studies on polar steroids from the starfish 1.3. Biological activity of polar steroids from the starfish 1.4. Overview of research subjects 1.5. Situation of research on synthesis of polyhydroxysteroid and hydroximinosteroid compounds from natural steroids CHAPTER 2. SUBJECT AND METHODS OF THE STUDY This section describes information of research subject; methods of isolation, structure determination of compounds and methods of evaluation of biological activity 2.1. Research subject Starfish Acanthaster planci was collected at a depth of 5-10 m, in Van Phong Bay, Nha Trang, Khanh Hoa, Vietnam. Species name was determined by Assoc. Do Cong Thung, Institute of Marine Resources and Environment Vietnam Academy of Science and Technology. 2 The upper side of the sample The underneath of the sample 2.2. Research methods 2.2.1. Method of isolation of compounds The chromatographic methods used to isolate compounds including: thin-layer chromatography (TLC), normal or reverse-phase silica gel column chromatography (RP-C18), Polychrome1, Sephadex LH-20 column chromatography, highperformance liquid chromatography (HPLC), method of crystallization also used. 2.2.2. Methods of determining structure Common methods for determining the chemical structure of compounds include: Electrospray Ionization Mass Spectrometry (ESI-MS), High-resolution Electrospray Ionization Mass Spectrometry (HR ESI-MS), extreme rotation ([α]D), the one-dimensional magnetic resonance spectrum (1H, 13C, DEPT, TOCSY1D) and two-dimensional (HSQC, HMBC, COSY, NOESY, ROESY) recorded on Bruker Avance 500MHz or Bruker Avance III 700MHz using TMS is internal standards. 2.2.3. The methods of activity evaluation 2.2.3.1. Cytotoxic assay The cytotoxic activity of compounds was assessed by MTS method performed at: Institute of Natural Products Chemistry-Vietnam Academy of Science and Technology (Hep G2, HeLa); Research Center for Natural Compounds - Korea Institute of Science and Technology (T98G); Pacific Institute of Organic Biochemistry - Russian Federal Academy of Sciences - Vladivostok (HCT-116, HT-29, RPMI-7951, T-47D, MDA-MB-231). 2.2.3.2. Soft agar assay The test substances added to the cell culture medium at non-toxic concentrations 5, 10 and 15 μM, incubated for 4 weeks. The tumors formed were 3 measured using a microscope and imaging software using Colburn's method. The test was done at the Pacific Institute of Organic Biochemistry - Russian Federal Academy of Sciences - Vladivostok. 2.2.3.3. Migration-cell Wound-healing assay The test substances (at concentration 10 μM) were added into the medium that was prepared a wound on MDA-MB-231 mammary carcinoma cell and check after 48 hours. The closure of the lesion area was measured and determined the percentage of distance traveled by the cell. The test was done at the Pacific Institute of Organic Biochemistry - Russian Federal Academy of Sciences – Vladivostok. CHAPTER 3. EXPERIMENT 3.1. Isolation of compounds from starfish Acanthaster planci This section details how to isolate compounds from A. planci starfish. The separation of compounds was summarized in the diagram in Figure 3.1. Figure 3.1. Diagram of isolated compounds from starfish A.planci 4 3.2. Physical constants and spectrum data of isolated compounds 3.3. Summary of cholesterol derivatives AP7 compound was identified as cholesterol, selected as the start material to convert into polyhydroxysteroid and hydroximinosteroid derivatives. 3.3.1. Synthesis of polyhydroxyl derivatives of cholesterol 3.3.2. Synthesis of hydroximinosteroid derivatives from cholesterol 3.4. Biological activity of isolated compounds and synthetic derivatives 3.4.1. Biological activity of polar steroid compounds isolated from starfish Acanthaster planci 3.4.2. Cytotoxic activity of cholesterol derived derivatives The hydroxysteroid compounds (15c-21c), hydroximinosteroid compounds (23c, 25c, 29c, 31c) and intermediate compounds (22c, 24c, 26c-28c, 30c) were evaluated for cytotoxic activity on three cancer cell lines as Hep G2, HeLa and T98G. CHAPTER 4. RESULTS AND DISCUSSION This chapter presents the results of research on isolating, metabolizing and determining the structure of compounds, results of cytotoxic activity, tumor suppression activity on soft agar and inhibitory activity metastases of the tested compounds. 4.1. Study on chemical composition of starfish Acanthaster planci This section details the results of the structure determination of 14 compounds isolated from A. planci, including 4 new compounds and 8 known compounds. Table 4.21: Summary table of substances isolated from starfish A.planci AP1. Planciside A (new compound) AP2. Planciside B (new compound) 5 AP4. Planciside D AP3. Planciside C (new compound) AP5. 3-O-sulfothornasterol A AP6. 5-ergost-7-en-3-ol AP7. Cholesterol AP8. Astaxanthin AP9. Thymin AP10. Uracil AP11. Acanthasglycoside G (new compound) AP12. Pentareguloside G AP13. Acanthasglycoside A AP14. Maculoside 6 * The following shows the spectral analysis and chemical structure determination of a new glycoside steroid called Acanthaglycoside G (AP11). • AP11 compound: Acanthaglycoside G (new compound) AP11 compound was isolated as solid, amorphous. The molecular formula of AP11 was determined to be C51H81O26SNa (M = 1164) from the [M+Na]+ sodium adduct ion peak at m/z 1187,4533 (theoretical calculations for molecular formula is C51H81Na2O26S = 1187,4532) in the (+) HR-ESI-MS spectrum and the [M–Na]− ion peak at m/z 1141,4735 (theoretical calculations for molecular formula is C51H81O26S = 1141,4737) in the (-) HR-ESI-MS, where M is the molecular mass of the intact sodium salt. In addition, the (–) HR-ESIMS/MS spectrum of the [M–Na]− ion at m/z 1141 exhibited fragment ion peak at m/z 995 [(M–Na)–C6H10O4]−, 849 [(M–Na)–2xC6H10O4]−, 703 [(M–Na)– 3xC6H10O4]−, 557 [(M–Na)–4xC6H10O4]−, 411 [(M–Na)–5xC6H10O4]−, corresponding to the successive losses of one, two, three, four, and five 6deoxyhexose units, respectively, and 393 [(M–Na)–4 x C6H10O4 –C6H12O5]−, corresponding to the loss of an oligosaccharide chain in AP11. The MS spectroscopic data confirmed the presence of five 6-deoxyhexoses in the carbohydrate moiety of AP11. Intens. x105 4 3 3+ 1187.4533 Intens. x106 4 +MS, 0.8-1.4min #44-83 -MS, 1.3-1.8min #76-100 3 1+ 437.1941 2+ 605.2223 2 2 1+ 526.3922 1 11141.4735 2570.2335 1 1163.4545 1+ 1269.4566 497.2043 774.6342 977.4046 424.1754 1367.4546 0 849.3573 1127.4573 995.4151 0 400 500 600 700 800 900 1000 1100 1200 1300 400 m/z Fig. 4.21: (+) HR-ESI-MS spectrum of AP11 500 600 700 800 900 1000 1100 1200 m/z Fig. 4.22: (-) HR-ESI-MS spectrum of AP11 The 1H-NMR spectrum of AP11 showed signals of 3 methyl groups, characterized by steroid nucleus including 2 methyl groups CH3-18 (s, 0.58 ppm) and CH3-19 (s, 0.94 ppm); 1 signal in CH3-21 weaker field (s, 2.08 ppm). 7 There is also the signal of a proton olefin at δH 5.24 (brt, J 4.2 Hz); an oxymetin group linked to a sulfate group at δH 4.87 (m) ppm; an oxymetin group is directly linked to a carbohydrate chain at the CH-6 position with δH 3.78 (m). In the weak field region, five doublet signals of five proton anomers of 5 monosaccharide units at δH 4.82 (1H, d, J = 7.6 Hz), 4.84 (1H, d, J = 7.9 Hz), 4.97 (1H, d, J = 6.8 Hz), 5.03 (1H, d, J = 7.7 Hz), 5.27 (1H, d, J = 7.7 Hz). Fig. 4.23: 1H-NMR spectrum of AP11 Fig. 4.24: 13C-NMR spectrum of AP11 The 13C-NMR spectrum of AP11 showed the presence of 51 carbon atoms, including: eight CH3 groups, seven CH2 groups, thirty two CH groups and four quater carbons. The presence of olefin carbon was triple substituted in the molecule was determined at δC 116.4/145.8 ppm. Five carbon CH signals at δC 102,3; 103,6; 104,9; 104,9; 106,9 ppm were identified as carbon anomers of the five sugar units. In addition, in the region of 60 - 90 ppm, there are resonance signals of 23 carbon atoms directly linked to the oxygen atom, including 22 carbon oximetin with 2 carbon of aglycon fraction at δC 77,4 (C-3) bind to sulfate group; δC 80,1 (C-6) linked to carbohydrate chain; and 20 carbons of sugar molecules at δC 73,8; 91,0; 74,4; 71,7; 82,3; 75,1; 85,7; 71,4; 84,3; 77,5; 75,7; 72,8; 73,7; 74,9; 72,3; 71,8; 76,2; 77,5; 75,5; 73,4 ppm; and a carbon of C=O group at δC 208,0 ppm. The sequences of proton at C-1 to C-8, C-11 to C-12, and C-8 to C-17 were ascertained from the COSY and HSQC experiments. The HMBC 8