Nghiên cứu hóa học và hoạt tính sinh học hai loài sao biển anthenea sibogae và anthenea aspera của việt nam tt tieng anh

MINISTRY OF EDUCATION AND TRANING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCES AND TECHNOLOGY - NGUYEN ANH HUNG CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF TWO STARFISH ANTHENEA SIBOGAE AND ANTHENEA ASPERA COLLECTED FROM VIETNAMESE COAST Major: Natural Products Chemistry Code: 44 01 17 SUMMARY OF CHEMICAL DOCTORAL THESIS HA NOI - 2020 This dissertation was completed in: Graduate university of Sciences and Technology – Vietnam Academy of Science and Technology Supervisor 1: Assoc Prof Dr Tran Thi Thu Thuy Supervisor 2: Dr.Sc Alla Anatolievna Kicha 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…., ………, 2020 Thesis can be found at the: - Library of Graduate university of Sciences and Technology - Vietnam National Library PREAMBLE The necessary of the thesis Vietnam is endowed with more than million km2 of sea area, tropical monsoon climate, densely estuarine which are ideal conditions for a diverse and rich marine organison Since the 1970s there have been a few studies on natural compounds from marine organison However, comparing to the potential source of marine life in our country, up to now, the research in this field is still too few and scattered, especially the studies in echinoderms Starfish are invertebrates, belonging to echinoderms and have long been known as a nutritious source About 1800 species of starfish occur in all the world’s oceans, from tropics to frigid polar water However, only about 80 species have been studied for their chemical composition and biological activity The Oreasterdae family includes 20 genera: Acheronaster, Anthaster, Anthenea, Astrosarkus, Bothriaster, Choriaster, Culcita, Goniodiscaster… Currently, there are only species of the Oreasterdae family which were studied in the world, of which species have been studied in Vietnam, namely Anthenea chinensis and Culcita novaeguineae The results of those studies showed that substances isolated from the starfish of the Oreasterdae family have anti-inflammatory, analgesic, hypotensive, cytotoxic, antibacterial and antifungal actioity In the aim to find active ingredients for use in medicine and pharmacy from Vietnamese medicinal remedies, we have selected starfish species of the genus Anthenea "Chemical constituents and biological activities of two starfish Anthenea sibogae and Anthenea aspera collected from Vietnamese coast” Research objectives of the thesis - Chemical investigation of two starfish Anthenea sibogae and Anthenea aspera - Biological evaluation of isolated compounds from starfish Anthenea sibogae and Anthenea aspera The main research content of the thesis To achieve the above objectives, the thesis has implemented the following contents: • Isolation of pure compounds from starfish Anthenea aspera and Anthenea sibogae collected from Vietnam’s coast • Structural elucidation of the chemical structure of isolated compounds • Biological evaluation of isolated compounds: anticancer activities CHAPTER INTRODUCTION The literature review is a collection of national and international researchs on: 1.1 Introduction about starfish 1.2 Chemical constituents of the Oreasteridae starfish 1.3 Biological activity of starfish CHAPTER MATERIALS AND METHODS 2.1 Materials Starfish samples were collected at Van Boi island, Quang Ninh province in June 2012 and identified by Assoc Do Cong Thung, Institute of Marine Resources and Environment, Vietnam Academy of Science and Technology The voucher specimens are deposited at Anthenea sibogae: DG02-BTL, Anthenea aspera: SBĐ 12 2.2 Methods for chemical and biological studies 2.2.1 Isolation methods The chromatographic methods are used to isolate pure from starfish compounds including: thin - layer chromatography (TLC), normal or reverse phase silica gel column chromagraphy (RP - C18), Polychrome1, High Performance Liquid Chromatography (HPLC) 2.2.2 Structural determination The structures of isolated compounds are determined by the combination of physical parameters with modern spectroscopic methods such as melting point (Mp), optical rotation ([α]D), Mass Gas Chromatography Spectroscopy (GC-MS), Electrospray Ionization Mass Spectrometry (ESI-MS), High Resolution Electrospray Ionization Mass spectrometry (HR-ESI-MS), Nuclear Magnetic Resonance Spectroscopy including one-dimensional spectrum (1H, 13C and DEPT, TOCSY 1D) and two-dimensional spectrum (COSY, HSQC, HMBC and NOESY) recorded on Bruker Avance III 500 MHz or Bruker Avance III 700 MHz using TMS as internal standard 2.2.3 Biological evaluation methods 2.2.3.1 Cytotoxicity assay The cytotoxic activity was tested by MTS method on human breast cancer cell line T-47D The tests were conducted at the Pacific Institute of Organic Biochemistry, Russian Federal Academy of Sciences, Vladivostok 2.2.3.2 Cell proliferation assay The tested substances were added to the cell culture medium at concentrations (50 μM) then incubated for additional 24, 48, and 72 h at 37 0C in a 5% CO2 incubator The tumors formed were measured in using absorbance at 490/630 nm using Fedorov method 2.2.3.3 Soft agar clonogenic assay The tested substances were added to the cell culture medium at a concentration of 50 μM The culture was maintained at 37 0C in 5% CO2 incubator for weeks and the cells colonies visualized scored microscope and the ImageJ computer software CHAPTER EXPERIMENTAL PART 3.1 Extraction and isolation procedure for starfish Anthenea sibogae This section details how to isolate compounds from A sibogae The separation of compounds was summarized in the diagram in Figure 3.1 3.1.1 Sample treatment of starfish A sibogae Fresh A sibogae (2 Kg) H/E 40/1, 5/1 Extraction four times MeOH (t = 45 0C) ASB1 (7 mg) ASB2 (5 mg) Crude MeOH Crude CH2Cl2 CH2Cl2 (3x500 ml) E/Me 30/1, 10/1 ASB3 (6 mg) Residue 80 g ASB4 (7 mg) Dissolved in H2O (1.0 L) Elution AgNO3; EtOH EtOH Fraction 10,5 g CH2Cl2/MeOH (9/11:5) F1… F8 MeOH/H2O (15/15:1) F6 F6.1, F6.2, F6.5 CHCl3/EtOH (3:1Ò2:1) F6.3 (87 mg) F6.4 (25 mg) HPLC (Diasorb-130-C16T) F6.3.1 (6,9 mg) 75% EtOH,; 25 ml/min F6.3.2 (2,3 mg) F6.3.3 (3,2 mg), F6.3.4 (5,8 mg), F6.4.1 (13,5 mg), HPLC (Diasfer-110-C18) 83%MeOH; 0,5 ml/min HPLC (Diasfer-110-C18) 80%MeOH; 0,5 ml/min HPLC (Diasfer-110-C18) 85% MeOH: 0,5 ml/min ASB7 (0,3 mg; tR = 31,7 min) ASB8 (1,2mg; tR = 30,4 min) ASB5 (2,4 mg; tR = 35 min) ASB6 (2,1 mg; tR = 39,8 min) ASB9 (0,6 mg; tR = 27,6 min) ASB10 (1,0 mg; tR = 27,5 min) ASB11 (1,9 mg; tR = 58,2 min) Figure 3.1 Diagram for the isolation procedure of starfish A sibogae 3.1.2 Physical properties and spectral data of isolated compounds 3.2 Extraction and isolation procedure for starfish A aspera This section details how to isolate compounds from A aspera The separation of compounds was summarized in the diagram in Figure 3.2 3.2.1 Sample treatment of starfish A aspera Fresh Anthenea aspera (10 Kg) Chopped Extracted by EtOH (3x8L), filtered Evaporated Crude EtOH 213 g EtOAc (1Lx3) n-hexane (1Lx3) MeOH (1Lx3) Crude hexane (SDH) 45 g Crude MeOH (SDM) 96 g Crude EtOAc (SDE) 68 g CHCl3/MeOH gradien hexane/CH2Cl2 100:0-0:100 SDH1 … CH2Cl2/ MeOH 10:0-8:2 CH2Cl2/ MeOH 100:0-0:100 SDH4 … SDH6 SDH9 SDE1 …… SDE3 SDE7 SDE9 SDM1 … SDM4 SDM7 … SDM11 Hexane/EtOAc gradien RP-C18 MeOH/H2O/NH4OH 70/29/1 AA1 0,17 g AA2 0,53 g CH2Cl2/ MeOH 05/1 AA4 15 mg AA3 1,02 g CHCl3/MeOH/H2O 4/1/0,1 CH2Cl2/ MeOH 3/1 CHCl3/MeOH/H2O 5/1/0,1 AA5 48 mg AA6 30 mg AA7 17 mg AA8 33 mg CHCl3/MeOH/H2O 2/1/0,1 AA10 10 mg AA11 mg AA9 mg Figure 3.2 Diagram for the isolation procedure of starfish A aspera 3.2.2 Physical properties and spectral data of isolated compounds CHAPTER - RESULTS AND DISCUSSION This chapter presents the results of isolation and structural elucidation of isolated compounds from starfish A aspera and A sibogae, cytotoxic activity, tumor suppression activity on soft agar and anti-proliferative activity of some isolated compounds 4.1 Structural elucidation of isolated compounds from the starfish A sibogae This section details the results of the structural determination of 11 compounds isolating from A sibogae, including new compounds and known compounds ASB2 Thymine ASB1 Cholesterol ASB3 L-tyrosine ASB4 Tryptophan ASB6 Anthenoside S2 (new compound) ASB5 Anthenoside S1 (new compound) ASB7 Anthenoside S3 (new compound) ASB8 Anthenoside S4 (new compound) ASB9 Anthenoside S5 (new compound) ASB10 Anthenoside S6 (newcompound) ASB11 Mixture of Anthenoside J Anthenoside K 4.1.5 ASB5 compound: (20R, 22E)-7-O- (6-O-methyl-β-D-galactofuranosyl) 16-O- (3-O-methyl- β -D-glucopyranosyl) -24-nor- 5α-cholesta-8(14), 22(23) diene-3α, 6β, 7β, 16α-tetraol (anthenoside S1, new compound) The molecular formula of ASB5 was determined to be of C40H66O14 from the [M + Na]+ sodium adduct ion peak at m/z 793,4346 in the (+)-HR-ESI-MS spectrum The 1H- and 13 C-NMR spectroscopic data reffered to the steroidal nucleus of ASB5 revealed the chemical shifts of H- and C-atoms of two angular Me groups H3–C(18) [δ(H) 0,91 (s), δ(C) 20,3] and H3–C(19) [δ(H) 0,84 (s); δ(C) 15,5], an 8(14) double bond (δ(C) 126,6; 147,6), two HC–O groups, including H‒ C(3) [δ(H) 4,07 ‒ 4,08 (m, W = 11,8); δ(C) 67,5] and H‒C(6) [δ(H) 3,64 (t, J = 2,8); δ(C) 75,2], as well as two HC–O groups bearing Omonosaccharide residues, including H‒C(7) [δ(H) 4,23 (d, J = 2,8); δ(C) 78,5] and H‒C(16) [δ(H) 4,63 (td, J = 9,0; 5,0); δ(C) 79,2] The width of the multiplet of H‒C(3) about 12 Hz corresponded well to the 3α-OH configuration while the width of the multiplet of H‒C(3) at the 3β-OH configuration is more than 30 Hz The H- and C-atom resonances of the H3‒C(18), H3‒C(19), H‒C(3), H‒C(6), H‒C(7), and H‒C(16) were similar to the same signals in the NMR spectra of anthenoside Q and testified about a Δ8(14)-3α,6β,7β,16α-tetrahydroxysteroidal nucleus glycosylated at the C(7) and C(16) positions in ASB5 The NMR spectra of the aglycon side chain showed the presence of three secondary Me groups H3–C(21) [δ(H) 1,10 (d, J = 7,0); δ(C) 23,8], H3–C(26) [δ(H) 0,98 (d, J = 6,7); δ(C) 23,2], and H3–C(27) [δ(H) 0,97 (d, J = 6,7); δ(C) 23,1], and a 22(23) double bond [δ(H) 5,74 (ddd, J = 15,3; 8,8; 1,2), 5,30 (dd, J = 15,3; 6,8); δ(C) 133,9; 137,2] Based on these data, a (22E)-Δ22-24-nor-cholestane side chain has been assumed in ASB5 A thorough analysis of the COSY, HSQC, HMBC, and ROESY spectra led to the assignment of all the H- and C-atom resonances of the steroidal moiety in ASB5 (Tables 4.6 and 4.7, Fig 4.1.27) The H- and C-atom sequences at H-C(1) to H-C(7), H-C(9) to H-C(12) through H-C(11), H-C(15) to H-C(17), H-C(17) to H-C(20), H-C(20) to H-C(22), H-C(23) to H3-C(27) were ascertained using the COSY and HSQC experiments The total structure of the steroidal aglycon of ASB5 was supported by the key HMBC correlations H-C(6)/C(8), C(10); H-C(15)/C(8), C(14), C(17); H3-C(18)/C(12), C(13), C(14), C(17); H3-C(19)/С(1), С(9), С(10); H3C(21)/C(17), C(20), C(22); H-C(22)/C(25); and H-C(23)/C(20), C(25), C(26), C(27) The key ROESY cross-peaks, such as H3-C(19)/Hβ-C(2), Hβ-C(4), HβC(11); H3-C(18)/Hβ-C(12), Hβ-C(15), H-C(16); H-C(5)/Hα-C(1), Hα-C(9); HαC(4)/Hα-C(6); Hβ-C(4)/H-C(19); and Hα‒C(7)/Hα-C(15), along with the values of the coupling constants of H-C(6), H-C(7), and H-C(16), confirmed the 3α,6β,7β,16α relative configurations of O-bearing substituents and 5α/9α/10β/13β steroidal nucleus in ASB5 The resonance of H3-C(21) at δ(H) 1,10 (δ(H) 1,10 for (20R)-Δ22- and δ(H) 1,00 for (20S)-Δ22-steroids) as well as the ROESY correlations of H3-C(18)/H-C(20), H3-C(21); H3-C(21)/Hβ-C(12); and H-C(22)/HC(16) allowed us to assume the (20R)-configuration As a result, we proposed the (20R,22E)-24-nor-5α-cholesta-8(14),22(23)-diene-3α,6β,7β,16α-tetraol structure as the aglycon moiety of ASB5 The 1H-NMR spectrum of ASB5 showed two resonances of anomeric Hatoms at δ(H) 4,33 and 5,02, correlated in the HSQC experiment with corresponding C-atom signals at δ(C) 102,9 and 108,4, resp The (+)-ESI-MS/MS spectrum of the [M + Na]+ ion peak at m/z 793 exhibited fragment ion peaks at m/z 599 ([(M + Na) – С7H14O6]+) and 217 ([С7H14O6 + Na]+) The (‒)-ESIMS/MS spectrum of the [M ‒H]- ion peak at m/z 769 displayed fragment ion peaks at m/z 575 ([(M – H) – С7H14O6]-) and 193 ([С7H13O6]-) All peaks corresponded to the loss of O-methyl-hexose residue The chemical shifts and coupling constants of H-C(1)-H-C(6) of two O-methyl-hexose units were determined by the irradiation of anomeric H-atoms in the 1D TOCSY 2,5) 6’ 3,88(dd, J=11,6; 2,5) 3,70(dd, J=11,6; 5,5) OMe 3,63 (s) 6-OMe-βD-Galf 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 2,2) 3,86(dd, J=11,6; 2,2) 3,71(dd, J=11,6; 5,1) 3,56 (s) 6-OMeβ-D-Galf 3,86(dd, J=11,6, 2,5) 3,65(dd,J=11,6, 5,7) 3,86(dd, J=11,6, 2,5) 3,63(dd, J=11,6, 5,6) 3,65(br d, J=6,1) 3,62 (s) 6-OMe-β-DGalf 3,62 (s) 6-OMe-βD-Galf 3,41 (s) 3,62 (dd, J = 11,2; 7,2) 3,59 (dd, J = 11,2, 4,5) 6-OMeβ-DGalf 4.99 (d, J = 2.3) 3.91 (dd, J = 3.8, 2.3) 3.95 (dd, J = 6.2, 3.8) 3.88 (dd, J = 6.2, 3.9) 3.82 ‒ 3.85 (m) 5,02 (d, J = 2,0) 3,90 (dd, J = 3,7; 2,0) 3,94 (dd, J = 6,2; 3,7) 3,903,91(m) 5,01 (d, J 5,05 (d, J = 2,0) = 2,0) 3,893,90 (dd, J = 3,91 (m) 4,3, 2,0) 5.04 (d, J = 2.0) 3.90 (dd, J =3.6, 2.0) 3,94 (dd, J = 6,1; 3,6) 3,893,91 (m) 3,90-3,96 (m) 3.93 (dd, J = 6.8, 3.6) 3,91(dd, J = 5,8, 3,4) 3.92 (dd, J = 6.8, 3.5) 3,82 (ddd, J = 7,0; 5.2; 3,4) 3,82 (ddd, J = 7,0; 5,2, 3,3) 3,53 (dd, J = 10,1; 5,2) 3,52 (dd, J = 10,1; 7,0) 3,38 (s) 3,82 (ddd, J = 7.2, 4.8, 3.4) 3.82 (ddd, J = 7.1, 4.8, 3.5) 3,53(dd, J = 10,1, 4,8) 3,52 (dd, J = 10,1; 7,2) 3,54 (dd, J = 10.1, 4.8) 3,52 (dd, J = 10.1, 7,1) 3,53 (d, J = 6,0) 3,38 (s) 3,38(s) 3,39(s) 3,53 (dd, J = 10,3; 5,2) 3,52 (dd, J = 10,3;7,0) OMe 3,38 (s) 13 Measured in CD3OD, 700 MHz 13 Table 4.7 C-NMR spectroscopic data of compounds ASB5-ASB10 Position ASB5 ASB6 ASB8 ASB9 ASB10 34.6 34.5 34.6 34.5 34.5 29.6 29.6 29.6 29.5 29.6 67.5 67.5 67.3 67.4 67.5 33.3 33.3 33.2 33.4 33.3 38.0 38.0 37.9 37.7 38.0 75.2 75.5 75.5 77.4 75.2 78.5 78.7 78.7 72.1 78.4 126.6 126.6 126.6 128.4 127.0 45.9 45.9 45.6 45.8 45.8 10 38.8 38.8 38.9 38.7 38.9 11 19.5 19.5 19.6 19.4 19.5 12 37.2 37.1 37.6 36.9 37.3 13 45.4 45.4 45.4 44.9 45.1 14 147.6 147.6 148.0 146.8 147.4 15 34.3 34.5 34.2 33.5 33.8 16 79.2 79.4 80.1 76.9 77.7 17 18 19 20 21 22 23 24 25 26 27 28 1’ 2’ 3’ 4’ 62.8 20.3 15.5 37.2 23.8 133.9 137.2 32.3 23.2 23.1 62.8 20.3 15.4 37.1 23.8 133.8 137.4 32.3 23.2 23.1 3-OMe-β-DGlcp 4-OMeβ-DGlcp 103.0 75.2 78.2 81.2 102.9 75.2 87.9 71.5 62.7 19.7 15.5 34.1 20.8 34.6 33.0 158.4 34.9 22.6 22.4 108.6 3-OMe-βD-Glcp 102.6 75.1 87.8 71.7 14 62.6 20.5 15.3 37.1 24.6 137.7 129.5 43.2 29.9 22.8 3-OMeβ-DGalf 108.2 80.9 88.9 84.3 62.7 20.1 15.4 32.9 21.4 33.8 33.3 157.7 34.9 22.5 22.3 107.2 β-DGalf 107.6 83.7 78.3 84.4 5’ 6’ OMe 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ OMe 77.8 63.2 61.0 6-OMe -βD-Galf 77.2 62.7 60.8 6-OMeβ-DGalf 77.9 63.4 61.0 6-OMe-βD-Galf 108.4 83.4 78.7 85.0 70.8 75.5 59.4 108.4 83.4 78.7 85.0 70.8 75.5 59.4 108.5 83.4 78.7 85.0 70.8 75.5 59.4 73.2 65.1 58.1 72.4 65.4 6OMeβ-DGalf 108.4 83.5 78.7 85.0 70.8 75.5 59.4 Measured in CD3OD, 176 MHz 4.2 Structure elucidation of isolated compounds from the starfish A aspera sterols and other compounds were isolated for the first time from hexane, ethyl acetate and methanol extracts of starfish A aspera collected in Northeast Vietnam AA1 Cholesterol AA2 Lathosterol AA4.Cholestan-3β,5α,6β,15α,16β-26-hexol AA3 Cholest-4-ene-3β,6β-diol AA6 L-glycine-L-propyl AA5 Cyclo(L-glycine-L-proline) 15 AA8 L-Phenylalanine AA7.Cyclo(L-alanine-4-hydroxyl-L-prolyl AA9 Tyramine AA10 Thymine AA11 Uracil 4.2.1 AA1 compound: cholesterol Compound AA1 has melting point, Rf and NMR spectrum coincide with compound ASB1 4.2.2 AA2 compound: Lathosterol (Cholest-7,8-ene-3 -ol) Fig 4.2.8 Chemical structure of AA2 Table 4.14 NMR spectrum data of AA2 and reference substance Position 10 11 12 13 C Ca,c Ha,b(mult., J, Hz) 37.1 31.3 70.7 37.8 40.2 29.6 117.2 139.3 49.4 34.1 21.5 39.5 43.2 37.1 31.4 71.1 38.0 40.3 29.7 117.4 139.6 49.5 34.2 21.6 39.5 43.4 1.82 m/ 1.07 m 1.80 m/ 1.61 m 3.60 m 1.27 m/ 1.72 m 1.40 m 1.76 m 5.15 m 1.61 m 1.57 m, 1.45 m 1.20; 2.02 m - # 16 14 15 16 17 18 19 20 21 22 23 24 25 26 27 54.9 55.0 1.80 overlap 22.9 23.0 1.40 m; 1.52 m 27.9 27.9 1,88 m; 1.26 m 56.1 56.1 1.20 m 11.8 11.8 0.53 s 12.9 13.0 0.79 s 36.1 36.0 1.36 m 18.8 18.8 0.92 d (6.5) 36.1 36.2 0.99 m; 1.34 m 23.9 23.9 1.14 m, 1.34 m 39.4 39.6 1.13-1.10 m 27.9 28.0 1.52 m 22.5 22.6 0.86 d (7.0) 22.7 22.8 0.87 d (7.0) a CD3OD, b500 MHz, c125 MHz, #δC data of [58] 4.2.3 AA3 compound: cholest-4-ene-3 ,6 -diol Fig 4.2.13 Chemical structure of AA3 Table 4.15 NMR spectrum data of AA3 and reference substance Position 10 11 12 Ca,c Ha,b(mult., J, Hz) 37.4 29.6 69.2 121.4 149.5 68.6 43.7 35.8 55.9 38.9 22.1 41.1 1.76 (m); 1.32 (m) 1.91 (m); 1.49 (m) 4.11-4.16 (trùng H-6) 5.67 (d, J 1.5 Hz) 4.11-4.16 (trùng H-3) 0.87 (m) 1.58 (m) 0.75 (m) 1.39; 1.53 (m) 2.04-2.06 (m) 17 C 15 16 17 18 19 20 21 22 23 24 25 26 δCa,c 25.2 29.2 57.6 12.4 19.2 37.1 19.2 37.3 24.9 40.7 29.1 23.2 Ha,b(mult., J, Hz) 1.64 (m); 1.40 (m) 1.88 ( m), 1.32 (m) 1.14 (m) 0.75 (s) 1.08 (s) 1.43 (m) 0.95 (d, J 6.5) 1.39 (m); 1.05 (m) 1.14-1.22 (m) 1.10-1.21 (m) 1.55 (m) 0.89 (d, 6.5) 13 14 43.2 57.4 27 22.9 1.07 (m) a CD3OD, b500 MHz, c125 MHz, #δC data of [77] 4.2.4 AA4 compound: cholestane 3 ,5,6 ,15,16 ,26-hexol 0.86 (d, 6.5) Fig 4.2.21 Chemical structure of AA4 Table 4.16 NMR spectrum data of AA4 and reference substance # Position C Cac Hab, mult (J = Hz) HMBC (HC) NOESY 31.7 31.7 1.79 m; 1.51 m C-5, C-19 33.5 33.5 1.62 m; 1.35 m C-10 68.4 68.3 4.03 m (5.5) 41.6 41.5 2.08 dd (11.5; 13.0) C-3, C-5 76.6 76.6 76.6 76.4 3.49 dd (2.5; 3.0) C-4, C-5, C-8, H-4, H-7 C-10 35.4 35.2 1.89 m C-6, C-8, C-9, C-14 32.2 31.1 2.01 m C-7, C-9, C-14 46.7 46.6 1.41 m 10 39.5 39.3 11 22.0 21.9 1.38 m C-13 12 42.1 42.0 1.98 m; 1.20 m C-9, C-14 13 44.9 44.7 14 61.2 60.9 0.98 m C-13, C-16, C-18 15 85.0 85.1 3.76 dd (2.5; 10.0) C-8, C-14, C-16 H-18 16 83.2 83.0 3.99 dd (2.5; 7.5) C-13, C-15 H-17 17 60.1 59.9 1.27 m C-13, C-18, C-20 18 15.2 15.1 0.93 s C-12, C-13, C-14, C-17 18 19 17.2 17.3 20 21 22 23 24 25 26 31.0 18.6 37.5 24.8 35.0 37.0 68.6 31.0 18.6 37.4 24.8 34.9 37.0 68.4 1.20 s C-1, C-5, C-9, C-10 C-17 C-17, C-20, C-22 C-21 C-24 C-27 1.89 m 0.98 d (5.5) 1.08 m 1.46 m; 1.23 m 1.43 m; 1.06 m 1.58 m 3.45 dd (6.0; 10.5); C-24, C-25, C-27 3.34 overlapped 27 17.3 17.4 0,93 d (6,5) C-24, C-25, C-26 a b c # CD3OD, 500 MHz, 125 MHz, δC data of [78] 4.2.5 AA5 compound: cyclo(L-glycine-L-proline) Fig 4.2.27 Chemical structure of AA5 Table 4.17 NMR spectrum data of AA5 and reference substance δaH,c Position (mult, J, Hz) cyclo(LGly-LPro) [79] Gly NH Pro 1′ 2′ 4.10 3.87 (dd) 7.15 4.11 δaH,c (mult, J, δaC,b Hz) (AA5) HMBC (H→C) (AA5) 4.10 163.6 19 175.6 C-1, C-1′ 46.5 46.2 C-1, C-2′ 170.1 58.5 * δCd ,e L-Gly-LPro [80] (AA5) 4.10* 3.90 (dd, 4.5; 16.5) 7.35 (brs)  C-3’, C-4’ 169.3 59.6 3′ 4′ 5′ 2.38 (m)/2.06* C-1′, C-2′, 28.4 29.2 (2.34-2.41) C-4′, C-5′ * 1.75-2.55 1.92 (m)/2.06 C-2′, C-3′, 22.3 23.6 (1.86-2.11) C-5′ 3.58 (m) 3.64 (m)/3.56 C-1, C-2′, (m) 45.2 C-3′, C-4′ 43.0 (3.58, m) a CDCl3, , b125 MHz, c500 MHz, *signal overlap, dD2O, eCD3OD 1.75-2.55 4.2.6 AA6 compound: L-glycine-L-prolin Fig 4.2.31 Chemical structure of AA6 Table 4.18 NMR spectrum data of AA6 and reference substance δC [80] # Position L-Gly-L-Pro δ a ,b C (AA6) δaH, c (mult, J.,Hz) HMBC (AA6) (H→C) (AA6) Gly 175.6 172.0 4.12 (ddd, 17,0; 2,0; 46.2 47.0 1,0) C-1, C-1’ 3.76 (d, 17.0) NH Pro 1′ 169.3 166.5 2′ 59.6 59.9 3′ 29.2 29.3 4′ 23.6 23.3 5′ 46.3 4.25 (m) 2.35 (m) 1.99 (m) 2.04 (m) 1.96 (m) 46.3 a # C-3’ 3.52-3.60 (m) b c MeOD–d4, D2O, 125 MHz, 500 MHz 20 C-5’ C-4’ 4.2.7 AA7 compound: cyclo(L-alanyl-4-hydroxyl-L-prolyl) Fig 4.2.37 Chemical structure of AA7 Table 4.19 NMR spectrum data of AA7 and reference substance Position 1’ 2’ 3’ 4’ C Cac Hab, mult (J = Hz) 163.6 46.5 169.1 52.1 15.7 172.8 58.9 38.2 69.1 4.26 - # 170.1 58.5 28.4 22.3 4.54 2.30 (dd, dd, 6.5; 13.5) 4.49 2.11 (ddd, 4.0 ; 11.0; 13.5) 5’ 45.2 55.2 3.69 (dd,4.5; 13.0) 3.45 (d,13.0) N-H 4.63 a b c CD3OD, 500 MHz, 125 MHz, #δC data of [81] 4.2.8 AA8 compound: L-phenylalanine HMBC (HC) C-1 C-1 C-1’ C-1’ C-1 - Fig 4.2.43 Chemical structure of AA8 Table 4.20 NMR spectrum data of AA8 and reference substance # Position C Cac Hab, mult (J = Hz) HMBC (HC) 135.3 137.3 128.7 130.0 7.28-7.38 (m, 5H, H-Ar); 129.7 130.4 7.28-7.38 (m, 5H, H-Ar); 130.7 128.4 7.28-7.38 (m, 5H, H-Ar); 21 129.7 128.7 37.8 130.4 130.0 38.3 7.28-7.38 (m, 5H, H-Ar); 7.28-7.38 (m, 5H, H-Ar); 3.33 (dd, 1H, J = 4.5; 14.5 Hz, H-7), 3.02 (dd, 1H, J = 9.0; 14.5 Hz, H-7) 55.5 57.6 3.79 (dd, 1H, J = 4.5; 9.0 Hz, H-8); C-8, C-2, C-6, C-1, C-9 C-7, C-1, C-9 174.2 173.8 b c # CD3OD, 500 MHz, 125 MHz, δH data of [82] in CD3OD 4.2.9 AA9 compound: tyramine a Fig 4.2.46 Chemical structure of AA9 Table 4.21 NMR spectrum data of AA9 and reference substance Position C=O CH3 C # Cac Hab, mult (J = Hz) 128.3 128.5 130.5 130.8 7.11 d (8.5) 116.2 116.7 6.79 d (8.5) 156.6 157.8 116.2 116.7 6.79 d (8.5) 130.5 130.8 7.11 d (8.5) 35.1 34.0 2.88 dd (8.0; 7.0) 42.3 42.3 3.13 dd (8.0; 7.0) 177.0 10.5 a CD3OD, b500 MHz, c125 MHz, #δC data of [83] in CD3OD 4.2.10 AA10 compound: thymine Phổ 1H-NMR Rf điểm nóng chảy AA10 hoàn toàn đồng với liệu ASB2 4.2.11 AA11 compound: uracil 22 Fig 4.2.51 Chemical structure of AA11 Table 4.22 NMR spectrum data of AA11 and reference substance C Cac Hab, mult (J = Hz) 167.5 110.4 139.2 150.3 12.1 164.3 100.2 142.1 151.5 11.8 5.44 ppm (J =7,5Hz, H-2) 7.38 ppm (J =7,5Hz, H-3) 11.0 # Position N-H a CD3OD, b500 MHz, c125 MHz, #δc data of TLTK [84,85] 4.3 Anticancer activity of steroid glycosides from starfish species A sibogae 4.3.1 Cytotoxic activity ASB5-ASB11 compounds were tested for cytotoxicity on human breast cancer cell lines T-47D using MTS method Cisplatin was used as positive control The results showed that compounds ASB5, ASB6, ASB8 and the mixture ASB11 as well as cisplatin were not cytotoxic to T-47D cell line at concentrations up to 150 μM after 24 hours and 48 hours 4.3.2 Anti-proliferative activity Compounds ASB5, ASB6 and ASB8 did not show significant proliferative inhibitory activity against T-47D cell lines at a concentration of 50 μM, while the mixture ASB11 inhibited T-47D cell proliferation after 24 h, 48 hours and 72 hours at the same concentration cisplatin After 24 hours of ASB11 mixture decreased T-47D cell proliferation by 10% while control cisplatin decreased T-47D cell proliferation by 20% After 48 hours of ASB11 mixture decreased T-47D cell proliferation by 20% while cisplatin decreased T-47D cell proliferation by 50% The ASB11 mixture (50 μM) reduced T-47D cell 23 proliferation after 72 hours by 47%, the cisplatin control (15 μM) reduced T-47D cell proliferation after 72 hours by 67% (Figure 4.3.1) Hình 4.3.1 The time dependent cell proliferation cells T-47D of ASB11 and cisplatin 4.3.3 Investigation of tumor cell formation on soft agar Human breast cancer cells T-47D (8x103) were treated with the steroid glycosides ASB5, ASB6, ASB8 and a mixture of ASB11 at a concentration of 50 μg/ml in a soft agar matrix and cells were incubated at 37 0C in an incubation chamber with 5% CO2 at atmospheric pressure for weeks Cells (colony) are counted under a microscope with the help of ImageJ software Hình 4.3.2 The inhibitory effects of ASB5, ASB6, ASB8 and the mixture of ASB11 on colonies number (B) and size (C, D) in cells T-47D comparing with untreated control cells Results showed that a concentration of 50 μM compounds ASB5, ASB6 and ASB8 showed no inhibition of the number or size of T-47D breast cancer cells when compared with controls Meanwhile, the mixture ASB11 did not inhibit the number of cell masses but reduced the size of T-47D cell masses by 24 more than half Cisplatin (1 μM) inhibited cancer cell mass formation by 48% (Figure 4.3.2.A) Previous biological experiments also showed that a mixture of anthenoside J + K has more potent cytotoxic and anti-proliferative activity than other anthenosides Therefore, it is necessary to continue to study the mechanism of action of this mixture, as well as the relationship between the structure and activity of anthenosides isolated from the genus Anthenea CHAPTER CONSLUSION Chemical consituents study Using a combination of chromatographic methods and modern spectroscopy methods, 22 compounds from the two starfish A aspera and A sibogae have been isolated and determined, including new compounds From starfish A sibogae 11 compounds (ASB1-ASB11), including new compounds (ASB5-ASB10) were isolated: anthenoside S1, anthenoside S2, anthenoside S3 , anthenoside S4, anthenoside S5, anthenoside S6 and known compounds are cholesterol (ASB1), thymine (ASB2), L-tyrosine (ASB3), tryptophan (ASB4) and mixture of anthenoside J and anthenoside K (ASB11) From starfish A aspera 11 compounds (AA1-AA11) were isolated: cholesterol (AA1), lathosterol (AA2), cholest -4-ene-3β, 6β-diol (AA3), cholestane 3β,5α,6β,15α,16β,26-hexol) (AA4), cyclo (L-glycine-L-proline) (AA5), L-glycine-L-prolin (AA6), cyclo (L-alanine-4-hydroxyl-L-proline) (AA7), L-phenyl alanine (AA8), tyramine (AA9), thymine (AA10) and uracil (AA11) All these compound were isolated from the genus Anthenea For the first time Biological activity evaluation For the first time, the new steroid glycoside compounds: anthenoside S1S6 isolated from starfish A sibogae have been studied for cytotoxic activity, antiproliferative activity cell and ability to inhibit colony formation on soft agar plates on human T-47D breast cancer cells The compounds ASB5, ASB6 and ASB8 did not show inhibitory activity against T-47D cell lines at a concentration of 50 μM, while the mixture ASB11 25 inhibited the proliferation of the following T-47D cells 24, 48 and 72 hours at a concentration of 50 μM compared to the control cisplatin A mixture of ASB11 at a concentration of 50 μM reduced T-47D cell proliferation by 47% after 72 hours, cisplatin control at a concentration of 15 μM decreased by 67% of T-47D cell proliferation after 72 hours - At a concentration of 50 μg / ml of the steroid glycosides ASB5, ASB6, ASB8, ASB11 inhibits the tumor size of T-47D breast cancer cells 23%, 19%, 30%, 52% respectively PERSPECTIVE This is the initial research on biological activity of some steroid glycosides isolated from the starfish species A sibogae and A aspera These biological experiments just at the level of exploration, and need to be further investigation in mechanism of action It is necessary to test the anti-cancer activity of these compounds on other human cancer cell lines and some other biological activities such as antiinflammatory, antibacterial, and neuritogenic activity NEW CONTRIBUTIONS OF THE THESIS This is the first study on the chemical composition and biological activity of the starfish A sibogae in the world and the first study in Vietnam on the chemical composition of the starfish A aspera From the starfish A sibogae new compounds were isolated and identified: anthenoside S1 - anthenoside S6 From the starfish A aspera 11 compounds were isolated and identified of which 11 are isolated from the genus Anthenea for the first time Invitro anticancer activities of isolated glycoside steroides were tested on T-47D celine LIST OF PUBLICATIONS 26 A A Kicha, D T Ha, N V Ivanchina, T V Malyarenko, A I Kalinovsky, P S Dmitrenok, S P Ermakova, O S Malyarenko, N A Hung, T TT Thuy, P Q Long, Six new polyhydroxysteroidal glycoside Anthenosides S1-S6, from the starfish Anthenea sibogae, Chemistry & Biodiversity, 2018, 15 (3), e1700553 DOI: 10.1002/cbdv.201700553 (SCIE) Nguyen Anh Hung, Dinh Thi Ha, Tran Thi Thu Thuy, Pham Quoc Long, Alla Anatolievna Kicha Steroidal diglycoside from the starfish Anthenea sibogae, Vietnam Journal of Sciences and Technology, 2018, 56 (4A), 121-126 DOI: 10.15625/2525-2518/56/4A/13137 (ACI) Nguyen Van Tuyen Anh, Doan Lan Phuong, Nguyen Anh Hung, Pham Minh Quan, Tran Thi Thu Thuy, Biochemical constituents of some Vietnames starfish, Vietnam Journal of Science and Technology, 2016, 54 (2B), 263-269 (ACI) Đoàn Lan Phương, Phạm Văn Công, Đinh Thị Hà, Nguyễn Anh Hưng, Nguyễn Văn Tuyến Anh, Phạm Quốc Long, Trần Thị Thu Thủy, Một số hợp chất chứa nitơ phân lập từ loài biển Anthenea aspera Việt Nam, Tạp chí khoa học cơng nghệ, 2014, 52 (5A), 89-95 Phuong, D L., Thuy, T T T., Nguyet, N T., Huong, D T., Hung, N A., Bordoloi, M., & Long, P Q First study on chemical constituents of starfish Anthenea aspera from Vietnamese northeast sea Успехи наук о жизни, 2013, 7, 66-67 Nguyễn Anh Hưng, Nguyễn Thị Hải Yến, Đặng Thị Thúy Hồng, Trần Thị Thu Thủy, Phạm Quốc Long Phân lập hợp chất từ cặn dichloromethane lồi biển Anthenea sibogae, Tạp chí Khoa học trường Đại học Sư phạm Hà Nội 2, 2020 (Giấy chấp nhận đăng) 27 ... Đinh Thị Hà, Nguyễn Anh Hưng, Nguyễn Văn Tuyến Anh, Phạm Quốc Long, Trần Thị Thu Thủy, Một số hợp chất chứa nitơ phân lập từ loài biển Anthenea aspera Việt Nam, Tạp chí khoa học cơng nghệ, 2014,... Vietnamese coast” Research objectives of the thesis - Chemical investigation of two starfish Anthenea sibogae and Anthenea aspera - Biological evaluation of isolated compounds from starfish Anthenea. .. from Vietnamese medicinal remedies, we have selected starfish species of the genus Anthenea "Chemical constituents and biological activities of two starfish Anthenea sibogae and Anthenea aspera