Nghiên cứu phân lập, chuyển hóa và đánh giá tác dụng sinh học của steroid từ loài sao biển acanthaster planci tt tieng anh

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: 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 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 " Research objectives of the thesis 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 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 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 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 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 weeks The tumors formed were 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 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 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 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 new compounds and known compounds Table 4.21: Summary table of substances isolated from starfish A.planci AP1 Planciside A (new compound) AP2 Planciside B (new compound) 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 * 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 3+ 1187.4533 Intens x106 +MS, 0.8-1.4min #44-83 -MS, 1.3-1.8min #76-100 1+ 437.1941 2+ 605.2223 2 1+ 526.3922 11141.4735 2570.2335 1163.4545 1+ 1269.4566 497.2043 774.6342 977.4046 424.1754 1367.4546 849.3573 1127.4573 995.4151 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 methyl groups, characterized by steroid nucleus including methyl groups CH3-18 (s, 0.58 ppm) and CH3-19 (s, 0.94 ppm); signal in CH3-21 weaker field (s, 2.08 ppm) 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 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 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 aglycon is Fuc(1→2)-Qui3(1→4)-[Qui4(1→2)]-Qui2(1→3)-Qui1-Aglycon Based on the signals on the 1D-, 2D-NMR spectrum, we can determine the carbon and proton values of oligosaccharide moiety (see Table 4.9) Fig 4.26: Chemical structure of AP11 Fig 4.27: Key interactions in the HMBC and ROESY spectrum of AP11 The configuration of the sugar units in AP11 was demonstrated by the method of Leontein et al, the results of the GC spectral analysis showed that all of sugar units of AP11 was D-configuration On the basis of the above mentioned data, the structure of AP11 was established to be sodium 6α-O-{β-D-fucopyranosyl-(1→2)-β-D-quinovopyranosyl(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovo-pyranosyl-(1→3)-β-Dquinovopyranosyl}-6α-hydroxy-5α-pregn-9(11)-en-20-one-3β-yl sulfate, and named Acanthaglycoside G Acanthaglycoside G is rare asterosaponin, the carbohydrate moiety of which includes only β-D-fucopyranosyl and β-D-quinovopyranosyl units Thus, Acanthaglycoside G (AP11) is the first new substance isolated from nature 10 Table 4.8: Aglycon spectrum data of AP11 Cac Hab (JHz) 35,8 63,2 12,9 1,63 m 1,38 m 2,81 (brd, J 13,6 Hz) 1,89 (brq, J 12,5 Hz) 4,87 m 3,45 (brd, J 12,6Hz) 1,70 m 1,48 m 3,78 m 2,66 m 1,28 m 2,06 m ‒ ‒ 5,24 (brt, J 4,2 Hz) 2,14 brs ‒ 1,33 m 1,76 m 1,20 m 2,34 (brq, J 10,9 Hz) 1,61 m 2,51 (t, J 8,7 Hz) 0,58 s 19 19,0 0,94 s 20 21 208,0 30,8 ‒ 2,08 s C 1β 1α 2α 2β 4α 4β 7β 7α 10 11 12 13 14 15α 15β 16β 16α 17 18 a 29,3 77,4 30,6 49,1 80,1 41,3 35,4 146,0 35,8 115,8 40,4 42,3 53,5 25,3 23,0 ROESY (H→H) H-11, H3-19 H-3, H-11 HMBC (H→C) H3-19 H-1, H-5 H3-19 H-3, H-7 H3-19 H-5 H3-18, H3-19 H-1 H-14, H-17 H-12, H-17 H3-18 H3-18 H-12, H-14, H3-21 H-8, H-15, H-16 H-1, H-2, H-4, H-6, H-8 H-17 C-13, C-18 C-12, C-13, C-14, C-17 C-1, C-5, C-9, C-10 C-17, C-20 C5D5N, b 500,13 MHz, c 125,75 MHz Table 4.9: Oligosaccharide NMR spectrum data of AP11 C Qui 1 δCac δHab (JHz) 104,9 4,82 (d, J 7,6 Hz) 73,8d 3,97 (t, J 8,3 Hz) ROESY H-6 of aglycon; H-3, H-5 Qui1 HMBC (H→C) C-6 of aglycon C-1, C-3 Qui1 11 Qui 91,0 74,4 71,7d 18,2 3,77 (t, J 8,6 Hz) 3,55 (t, J 9,0 Hz) 3,69 m 1,60 (d, J 6,0 Hz) H-1 Qui1, H-1 Qui2 H-6 Qui1 H-1 Qui1 H-4 Qui1 C-1 Qui C-3, C-6 Qui1 103.6 4,97 (d, J 6,8 Hz) C-3 Qui1 Qui 82.3 75.1 85.7 71.4 18,0 4,09 (t, J 7,6 Hz) 4,12 (t, J 8,7 Hz) 3,56 (t, J 8,7 Hz) 3,90 m 1,73 (d, J 6,1 Hz) H-3, H-5 Qui2; H-3 Qui1 H-1 Qui4 H-1 Qui2 H-6 Qui2, H-1 Qui3 H-1 Qui2 H-4 Qui2, H-1 Qui3 102,3 4,84 (d, J 7,9 Hz) C-4 Qui2 84,3 4,00 (t, J 8,3 Hz) H-3, H-5 Qui3; H-4, H6 Qui2 H-1 Fuc Fuc 77,5 75,7 72,8 17,7d 4,13 (t, J 9,3 Hz) 3,62 (t, J 8,9 Hz) 3,71 m 1,48 (d, J 6,0 Hz) H-1, H-5 Qui3 H-6 Qui3 H-1, H-3 Qui3 H-4 Qui3 106,9 73,7d H-3, H-5 Fuc; H-2 Qui3 74,9 Qui 72,3 71,8d 16,9 5,03 (d, J 7,7 Hz) 4,41 (dd, J 8,6; 9,5 Hz) 4,06 (dd, J 3,6; 9,5 Hz) 3,99 (d, J 4,0 Hz) 3,78 (q, J 6,5 Hz) 1,49 (d, J 6,2 Hz) H-5, H-6 Fuc H-3, H-4 Fuc H-4 Fuc C-3 Fuc C-1, C-4 Fuc C-4, C-5 Fuc 104,9 5,27 (d, J 7,7 Hz) H-3, H-5 Qui4; H-2 Qui2 C-2 Qui2 C-4, C-5 Qui1 C-2 Qui2 C-3 Qui2 C-4, C-5 Qui2 C-3 Qui3, C-1 Fuc C-2, C-4 Qui3 C-5, C-6 Qui3 C-3 Qui3 C-4, C-5 Qui3 C-2 Qui3 C-1, C-3 Fuc H-1, H-5 Fuc 76,2 4,04 (t, J 8,8 Hz) 77,5 4,12 (t, J 8,8 Hz) H-1 Qui4 75,5 4,01 (t, J 8,7 Hz) H-6 Qui4 73,4 3,70 m H-1 Qui4 17,8d 1,79 (d, J 6,1 Hz) H-4 Qui4 C-4, C-5 Qui a C5D5N, b 500,13 MHz, c 125,75 MHz, d Assignments may be interchanged 12 4.2 Chemical metabolism of cholesterol Polyhydroxysteroid, hydroximinosteroid compounds with cholesterollike branched blood are evaluated as potential compounds with toxic activity on many cancer cell lines Therefore, cholesterol has been chosen as the first raw material for metabolic reactions to produce hydroxyl and oxidized products 4.2.1 Metabolism of polyhydroxysteroid derivatives Cholesterol (AP7) is hydroxylated to form many -OH groups around the C5/C6 double connection The hydroxylation agents used are mainly oxidizing agents and are capable of producing diol-type products Seven hydroxysteroid compounds (15c-21c) were synthesized from cholesterol according to the diagram 4.1 below Although synthetic compounds are known substances, the content of this study provides effective synthesis methods for active substances, only through one reaction step These methods can also be used on other steroid compounds from starfish to synthesize polyhydroxysteroid derivatives Diagram 4.1 Synthesis of polyhydroxysteroid derivatives from cholesterol Reagents and reaction conditions: (i): BH3.THF, H2O2, NaOH, 0oC, 1h (15c: 71%, 16c: 9%); (ii): SeO2, dioxane, H2O, 80oC, 80h (17c: 50%, 18c: 2,0%, 19c: 3,5%); (iii): 4% OsO4/H2O, NMO, reflux, 48h (20c: 74%); (iv): HCOOH 88%, THF/H2O2, 12h, KOH 3% in MeOH (21c: 75%) 13 4.2.2 Metabolism of hydroximinosteroid derivatives Recent studies have shown that the position of the oxime groups and the branch circuit types at the C-17 position of the cholestane-like steroid framework is more active than other types of branch circuits Therefore, cholesterol was chosen as the first raw material for the synthesis of hydroximinosteroid derivatives Products with an oxime group at C-3 position; or C-3 and C-6; and epoxy ring at position C-4,5 Four hydroximinosteroid (23c, 25c, 29c, 31c) including new substances (29c, 30c) and intermediates (15c, 22c, 24c, 26c, 27c, 28c, 30c) including new substance (30c) has been summarized according to Figure 4.2 below Diagram 4.2 Synthesis of hydroximinosteroid derivatives from cholesterol Reagents and reaction conditions: (i): PCC/CH2Cl2, rt, 48h (22c: 80,0 %, 24c: 82,0 %); (ii): BH3.THF/H2O2, NaOH, 0oC, 1h (15c: 80,0 %); (iii): CeCl3.7H2O/NaBH4, CH2Cl2&MeOH (1:1), rt, 1h (26c: 89,0 %); (iv): mCPBA/CH2Cl2, Dess-Martin/CH2Cl2, 0oC (27c: 12,3 %, 28c: 14,5 %); (v): NH2OH.HCl/Pyridine, 24h (23c: 85,0 %, 25c: 81,0 %, 29c: 19,3 %, 30c: 13,4 %, 31c: 22,5 %) 14 From cholesterol, it is metabolized in different directions to create ketone-mediated derivatives (C = O) at C-3 and C-6 positions; in the C-4,5 position there is a double bond (22c), no double bonds (24c) or an epoxy ring (27c) Finally, these ketones are transformed into hydroximinosteroid products (> C = N-OH), respectively (23c, 25c, 29c, 31c) by hydroxylamine hydrochloride (NH2OH.HCl) in pyridine by tissue method described by Javier In particular, when performing chemical oxidation 27c (with epoxy ring at position C-4,5 and ketone group at C-3), two products were obtained, one of which was oxidized (29c) and one product that is not oxidized but opens the epoxy ring in position C-4,5 (30c) The products have been successfully oxidized due to the apparent change in the chemical shift of the carbonyl group within δC 195-210 ppm to the oxime group at δC 155-160 ppm Combining modern physico-chemical methods and nuclear magnetic resonance spectroscopy structures of all proven products 4.3 Results of bioactive test 4.3.1 The activity of steroidal glycoside compounds isolated from starfish Acanthaster planci a Biological activity of polyhydroxysteroid glycoside compounds AP1 was tested for cytotoxic activity on three HCT-116, T-47D and RPMI7951 cell lines The result of AP1 is likely to be cytotoxic on HCT-116 and RPMI7951 cell lines with IC50 values of 36 and 58 µM, compared to cisplatin-positive control Both AP1 and cisplatin were non-toxic on T-47D cell lines (see table 4.23) Table 4.23: In vitro cytotoxic activity of compound AP1 Cell lines (IC50 µM) AP1 Cisplatin HCT-116 36 75 T-47D >150 > 150 RPMI-7951 58 43 15 AP1 inhibited the proliferation of T-47D cell line after 72 hours was 35%, the RPMI-7951 cell line after 48 hours was 27% (Figure 4.54 B, C) While cisplatin almost completely inhibits the growth of T-47D and RPMI7951 cell lines (Figure 4.54 B, C) A B C AP1, 15 μM Control Cisplatin, 15 μM Fig 4.54: Effects of AP1 on the proliferation of HCT-116, T-47D and RPMI-7951 cell lines at a concentration of 15 μM b Biological activity of asterosaponin compounds AP13 and AP14 compounds have the potential to cause toxic effects on HT-29 rectal cancer cells; MDA-MB-231 mammary carcinoma cells, but not toxic RPMI-7951 malignant melanoma cells at concentrations above 150 µM The value of IC50 concentration for each cell line showed in Table 4.24 Table 4.24: Cytotoxic activity and affecting tumor formation on soft agar of asterosaponin compounds AP11-AP14 Com AP11 AP12 AP13 AP14 RPMI-7951 IC50, µM IF50, µM >150 >15 >150 >15 >150 15 >150 14 HT-29 IC50, µM IF50, µM >150 >15 >150 >15 109 11 90 MDA-MB-231 IC50, µM IF50, µM >150 >15 >150 >15 30 13 24 IC50: the concentration of compounds that caused a 50% reduction in cell viability of human cancer cells; IF50: the concentration of compounds that caused a 50% reduction in colonies formation of human cancer cells 16 AP13 and AP14 compounds effectively inhibit tumor formation of HT-29 and MDA-MB-231 cell lines, and are less effective with RPMI-7951 cell lines IF50 values corresponding to cell lines showed in Table 4.24 AP13 and AP14 compounds at a concentration of 10 µM can prevent the movement of MDA-MB-231 cells at 26% and 45%, respectively, compared to the control after 48 hours of incubation (Figure 4.55) Meanwhile, compounds AP11 and AP12 cannot stop the movement of these cells Fig 4.55: Effects of asterosaponins AP11-AP14 on MDA-MB-231 breast adenocarcinoma migration in humans 4.3.2 Biological activity of cholesterol derivatives a Biological activity of polyhydroxysteroid derivatives Three substances 16c, 18c, 21c show Hep G2 cytotoxic activity with IC50 value of 11,69; 11,89 and 6,87 μM Only 21c exhibited T98G cytotoxic activity with IC50 = 2,28 μM, compared with the positive control Paclitaxel (see Table 4.25) Table 4.25: Biological activity of substances 15c-21c on Hep G2 and T98G cell lines IC50 µM IC50 µM Com 15c 16c 17c 18c Com Hep G2 T98G >100 11,59 >100 11,89 >100 >100 >100 >100 19c 20c 21c Paclitaxela a Hep G2 T98G >100 >100 6,87 0,040 >100 >100 2,28 0,023 positive control Two pairs of substances 15c and 16c; 20c and 21c differ in the OH group configuration at C-6 positions (6α-OH in substances 15c and 20c; 6β17 OH in substances 16c and 21c) While 16c has toxic activity on Hep G2 cell lines and 21c has toxic activity on both Hep G2 and T98G cell lines, substances 15c and 20c not show activity on these tests Thus, the configuration of the OH group at C-6 of this type of structure may be one of the factors determining their cytotoxic activity b Biological activity of hydroximinosteroid derivatives and intermediate compounds Three derivatives 3,6-dihydroximino (23c, 25c, 31c) have stronger cytotoxic activity than 3-hydroximino-6α-hydroxy (29c) on test cell lines The 3,6-dihydroximino 25c has no double bonds at position C4/5, which can cause toxic inactivation on HepG2 and HeLa cell lines compared with Δ43,6-dihydroximino 23c While 31c has two oxime groups at position C-3, C6 and epoxy ring at position C-4,5 with selective cytotoxic activity on T98G cell line (IC50 = 2,9 μM ) 29c has an oxime group at C-3 and epoxy rings at C-4/5 but does not have this activity Table 4.26: Cytotoxic activity of substances 22c- 31c on Hep G2, HeLa, T98G cell lines Com 22c 23c 24c 25c 26c Paclitaxel a IC50 (µM) HepG2 HeLa >100 >100 42,4 68,6 >100 >100 >100 >100 >100 >100 0,040 0,031 Com T98G >100 70,3 >100 69,8 >100 0,023 27c 28c 29c 30c 31c Paclitaxela IC50 (µM) HepG2 HeLa 41,8 72,4 >100 74,6 >100 >100 >100 >100 >100 >100 0,040 0,031 T98G >100 >100 >100 18,5 2,9 0,023 positive control Three intermediates (27c, 28c, 30c) with the presence of elemental oxygen at C-4 or C-5 have moderately toxic activity on at least one cell line while the other compounds not active As such, these steroids have double bonds at the C4/5 position or are linked to the oxygen element at the C-4 position, C-5 may have a positive effect on cytotoxic activity on cancer cell lines tested 18 CONCLUSIONS From the starfish Acanthaster planci was isolated 14 compounds The structure of these compounds was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and other physicochemical methods The isolated and identified compounds include: planciside A (AP1); planciside B (AP2); planciside C (AP3); planciside D (AP4); (3-Osulfothornasterol A (AP5); 5-ergost-7-en-3-ol (AP6); cholesterol (AP7); astaxanthin (AP8); thymin (AP9); uracil (AP10); acanthaglycoside G (AP11); pentareguloside G (AP12); acanthaglycoside A (AP13); and maculoside (AP14) Among them, four steroidal glycoside are discovered for the first time from nature, including three polyhydroxysteroidal glycosides, namely planciside A (AP1); planciside B (AP2); planciside C (AP3); and an asterosaponin, acanthaglycoside G (AP11) In addition, the compound pentareguloside G (AP12) found the first time in Acanthaster planci starfish in Vietnam The chemical modification of cholesterol, staring material isolated from this starfish, leads to obtain seventeen derivatives including polyhydroxysteroid derivatives (15c-21c), hydroximinosteroid derivatives (23c, 25c, 29c, 31c) and intermediate derivatives (22c, 24c, 26c, 27c, 28c, 30c), namely: cholestane-3β,6α-diol (15c); cholestane-3β,6β-diol (16c); cholestan-5-ene-3β,4β-diol (17c); cholestan-5-ene-3β,7β-diol (18c); cholestan-5-ene-3β,4β,7β-triol (19c); cholestane-3β,5α,6α-triol (20c); cholestane-3β,5α,6β-triol (21c); cholest-4-ene-3,6-dione (22c); (3E,6E)dihydroximinocholest-4-ene (23c); cholestane-3,6-dione (24c); (3E,6E)dihydroximinocholestane (25c); cholest-4-ene-3β,6α-diol (26c); 6-hydroxy4,5-epoxycholestane-3-one (27c); 4α,5α-epoxycholestane-3,6-dione (28c); 4α,5α-epoxy-6-hydroxycholestane-3-oxime (29c); 4α,5α,6α-trihydroxycholestane-3-one (30c); and 4α,5α-epoxycholestane-3,6-dioxime (31c) 19 In particular, 4α,5α-epoxy-6-hydroxy cholestan-3-oxime (29c); 4α, 5α,6α-trihydroxy-cholestane-3-one dioxime (31c) are new substances (30c); 4α,5α-epoxycholestane-3,6- Have investigated cytotoxic activity and evaluated the effects of compounds AP1, AP11, AP12, AP13, AP14 to colony formation on soft agar of human cancer cell lines The results showed that: - Compound AP1 exhibited a moderate cytotoxicity against human colon cancer (HCT-116) and human melanoma (RPMI-7951) cell lines with IC50 = 36 µM and 58 µM, respectively AP1 compound inhibited cell proliferation of HCT-116, T-47D, and RPMI-7951 cancer cell lines, but had no effect on colony formation of these cells - Compounds AP13 and AP14 at the same doses moderate inhibited cell viability of human colorectal carcinoma HT-29 and human breast adenocarcinoma MDA-MB-231 cell lines, but not RPMI-7951 cells IC50 of AP13 and AP14 were 109 and 90 µM in HT-29 cells; and 30 and 24 µM in MDA-MB-231 cells, respectively Compounds AP13 and AP14 effectively inhibited colony formation of colorectal HT-29 and breast cancer MDA-MB231 cells and less melanoma RPMI-7951 cells The concentrations which caused 50% inhibition of colonies number of AP13 and AP14 were 11 and µM in HT-29 cells, 13 and µM in MDA-MB-231 cells, and 15 and 14 µM in RPMI-7951 cells, respectively The compounds AP11-AP14 were determined effectively on migration of MDA-MB-231 cells which are human breast adenocarcinoma cells with high metastatic potential by method of in vitro wound healing assay The results showed that: AP13 and AP14 at concentration 10 were able to prevent migration of MDA-MB-231 cells by 26% and 45%, respectively, compared to control after 48 h of cells incubation Cytotoxicity of 07 prepared polyhydroxysteroid derivatives (15c21c), 04 hydroximinosteroid derivatives (23c, 25c, 29c, 31c) and 06 their 20 intermediate derivatives (22c, 24c, 26c, 27c, 28c, 30c) against three human cancer cell lines including hepatocellular carcinoma (Hep-G2), cervical cancer (HeLa) and glioblastoma (T98G) were studied The results showed: - Five compounds (16c, 18c, 21c, 23c, 27c) exhibited cytotoxicity activity against Hep-G2 cell lines with values of IC50 = 11,59; 11,89; 6,87; 42,40; and 41,80 µM, respectively - Three compounds (23c, 27c, 28c) inhibited cytotoxicity activity against HeLa cell lines with values of IC50 = 68,6; 72,4 and 74,6 µM, respectively - Five compounds (21c, 23c, 25c, 30c, 31c) exhibited cytotoxicity activity against T98G cell lines with IC50 = 2,28; 70,3; 69,8; 18,5 and 2,9 µM, respectively In particular, 21c and 31c are two potential substances for further research and evaluation of cytotoxic on glioblastoma cell line PETITION For Acanthaster planci starfish: further studies on chemical composition, especially polyhydroxysteroid glycoside compounds, are needed to develop health, prevention and support products treatment of diseases such as cancer, anti-inflammation Expand the semi-synthesis other derivatives of cholesterol and isolated steroid compounds, and test the cytotoxic of these derivatives on a number of other cancer cell lines NEW CONTRIBUTIONS OF THE THESIS Fourteen compounds were isolated and identified from the extract of starfish Acanthaster planci collected off from Vietnam coast Among them, four steroidal glycosides are discovered for the first time from nature, including three polyhydroxysteroidal glycosides, namely planciside A (AP1), planciside B (AP2), and planciside C (AP3); and an asterosaponin, acanthaglycoside G (AP11) 21 The chemical modification of cholesterol, starting material isolated from this starfish, leads to obtain seventeen derivatives including 07 polyhydroxysteroids, 04 hydroxyminosteroids and 06 intermediate derivatives The polyhydroxysteroid derivatives were prepared by short and efficient synthetic pathways with 1-2 steps Four hydroxyminosteroid derivatives with oxime groups at C-3, C-6 and 4,5 double bond or oxygen at C-4, C-5 were synthesized via intermediates Among these synthetic derivatives, compounds: 4α,5α-epoxy-6-hydroxycholestane-3-oxime (29c); 4α,5α,6α-trihydroxy-cholestane-3-one (30c); 4α,5α-epoxycholestane-3,6-dioxime (31c) were described for the first time The cytotoxicity of isolated steroidal glycosides against 05 human cancer cell lines (HCT-116, HT-29, RPMI-7951, T-47D and MDA-MB-231), the inhibition of cancer cell colony formation on soft agar were investigated Compound AP1 exhibited moderate cytotoxic effect against HCT-116 and RPMI-7951 cell lines with IC50 36 and 58 µM, respectively Compound AP13 and AP14 exhibited moderate cytotoxic effect against RPMI-7951, HT-29, MDA-MB-231 cell lines with IC50 ranging from 24 to 109 µM Two asterosaponin AP13 and AP14 were found to prevent effectively the migration of MDA-MB-231 cells by 26% and 45%, respectively The results related to isolated asterosaponins were found to be concised with the hypothesis that a shoter side chain of steroidal moiety may have a negative effect to the cytotoxicity of these asterosaponins All synthetic cholesterol derivatives were evaluated for the cytotoxicity agaisnt three human cancer cell lines (HepG2, HeLa, T98G) Compounds 16c, 18c, 21c, 23c, 25c, 27c, 28c, 30c and 31c were showed to be cytotoxic against at least one tested cancer cell lines Especially, compounds 21c and 31c exhibited strongly and selectively cytotoxicity against human glioblastoma cell line (T98G) with IC50 2,28 and 2,9 µM, respectively 22 LIST OF PUBLISHED WORKS RELATED TO THE THESIS Asterosaponins from the tropical starfish Acanthaster planci and their cytotoxic and anticancer activities in vitro Dinh T Ha, Alla A Kicha, Anatony I Kalinovsky, Timofey V Malyarenko, Roman S Popov, Olesya S Malyarenko, Svetlana P Ermakova, Tran T T Thuy, Pham Q Long, and Natalia V Ivanchina Natural Product Research, 2019, 1-8 DOI: 10.1080/14786419.2019.1585845 (SCIE) Three new steroid biglycosides, Plancisides A, B, and C, from the starfish Acanthaster planci Alla A Kicha, Thi H Dinh, Natalia V Ivanchina, Timofey V Malyarenko, Anatony I Kalinovsky, Roman S Popov, Svetlana P Ermakova, Thi T T Tran, and Lan P Doan Natural Product Communications, 2014, Vol 9, No 9, 1269-1274 (SCI-E) Patent: (24S)-28-O-[BETA-D-GALACTOFURANOSYL-(1-5)-ALPHA-LARABINOFURANOSYL] -24-METHYL-5ALPHA-CHOLESTANE- 3BETA, 4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28-HEPTOL COMPOUND AND ISOLATION METHOD OF THIS COMPOUND FROM STARFISH ACANTHASTER PLANCI Doan Lan Phuong, Tran Thi Thu Thuy, Dinh Thi Ha, Alla A Kicha, Natalia V Ivanchina, Timofey V Malyarenko, Anatoly I Kalinovsky, Roman S Popov, Svetlana P Ermakova, Pham Minh Quan, No 18377, Decision No 6820/QĐ-SHTT, 05/02/2018 Useful solution: [(24S)-28-O-[ALPHA-L-FUCOPYRANOSYL-(1→2)-3-OMETHYL-BETA-D-XYLOPYRANOSYL]-24-METHYL-5ALPHACHOLESTANE-3BETA, 4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28HEPTOL; [(24S)-28-O-[2,4-DI-O-METHYL-BETA-D-XYLOPYRANOSYL(1→2)-ALPHA-L-ARABINOFURANOSYL]-24-METHYL-5ALPHACHOLESTANE-3BETA,4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28HEPTOL] 6-O-SULFATE COMPOUNDS AND ISOLATION METHOD OF THEM FROM STARFISH ACANTHASTER PLANCI Doan Lan Phuong, Tran Thi Thu Thuy, Dinh Thi Ha, Alla A Kicha, Natalia V Ivanchina, Timofey V 23 Malyarenko, Anatoly I Kalinovsky, Roman S Popov, Svetlana P Ermakova, Pham Minh Quan, No 1637, Decision No 4539/QĐ-SHTT, 31/01/2018 Chemical composition of the starfish Acanthaster planci from Vietnam’s sea Doan Lan Phuong, Pham Quoc Long, Dinh Thi Ha, Doan Thi Huong, Nguyen Tien Dung, Nguyen Van Tuyen Anh, Tran Thi Thu Thuy Chemistry journal, 2013, Vol.51, No 6ABC, 131-134 Synthesis and cytotoxicity of polyhydroxylated cholesterol derivatives Dinh Thi Ha, Doan Lan Phuong, Pham Quoc Long, Ngo Dai Quang, Tran Thi Thu Thuy Vietnam Journal of Science and Technology, 2018, 56 (4), 467-473 Synthesis of two new hydroximinosteroids from cholesterol and their biological evaluation Dinh Thi Ha, Baskar Salvaraja, Pham Quoc Long, Ngo Dai Quang, Do Huu Nghi, Lee Jae Wook, Tran Thi Thu Thuy Vietnam Journal of Science and Technology, 2019, 57 (5), 527-538 New steroid glycoside compounds from two Vietnamese starfish species Acanthaster planci and Echinaster luzonicus Pham Quoc Long, Nguyen Anh Hung, Alla A.Kicha, Natalia V Ivanchina, Dinh Thi Ha, Anatoly I Kalinovsky, Timofey V Malyarenko, Tran Thi Thu Thuy, Doan Lan Phuong, Trinh Thi Thu Huong, Valentin A Stonik, Proceedings of Scientific Conference on Biodiversity and Biologically active substances 40th anniversary of the establishment of Vietnam Academy of Science and Technology, 2015, 265-269 24 ... 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... and biological evaluation of steroids from the starfish Acanthaster planci " Research objectives of the thesis Study on chemical composition of starfish Acanthaster planci of Vietnam, synthesizing... 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