Summary of Chemistry Doctoral thesis: Study on chemical constituents and biological activities from Culcita Novaeguineae Müller & Troschel, 1842 and Pentaceraster Gracilis (Lutken, 1871) in

The objective of the thesis is to study to clarify the main chemical composition of two species C.novaeguinea and P. gracilis in the Northeast sea of ​​Vietnam. Evaluate the cytotoxic activity of some isolated compounds to search for bioactive compounds.

1 MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY Bui Thi Ngoan STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM CULCITA NOVAEGUINEAE MÜLLER & TROSCHEL, 1842 AND PENTACERASTER GRACILIS (LUTKEN, 1871) IN VIETNAM Major: Organic chemistry Code: 62.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2018 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Prof Dr Chau Van Minh Adviser 2: Dr Nguyen Hoai Nam 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 2018 Thesis can be found in: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - National Library INTRODUCTION The urgency of the thesis The starfish are invertebrates belonging to the class Asteroidea, phylum Echinodermata Following the classification of Blacke (1987), the class Asteroidea including Brisingida, Forcipulatida, Notomyotida, Paxillosida, Spinulosida, Valvatida, and Velatida From the 1997 to 2007, the aproximately 98 starfish species were investigated on the chemical conponents as the report of Guang Dong et al The secondary metabolites from the starfish are characterized by a diversity of polar steroids, including polyhydroxylated steroids and steroid glycosides There are two main structural groups of steroid glycosides from the starfish, namely asterosaponins and glycosylated polyhydroxysteroids Those compounds showed several biological properties, such as cytotoxic, hemolytic, and anti-microbial effects In the Vietnam sea, the starfish belong to the phylum Echinodermata, which was known as the 350 species of 58 phylums, and are divided into five classes Holothuroidea (sea cucumbers), Asteroidea (starfishes), Echinoidea (sea urchins and sand dollars), Crinoidea (crinoids and sea lilies), and Ophiuroidea (brittle stars and basket stars) Up to date, the chemical constituents and biological acivities of starfish species abundant in the Vietnam sea were studied, including Archaster typicus, Asterina batheri, Asteropsis carinifera, Astropecten polyacanthus, Astropecten monacanthus, Protoreaster nodosus, Acanthaster planci, Linckia laevigata, and Anthenea aspera Their structures were determined as the steroid and steroid glycoside which displayed the cytotoxic activity and antiinlammatory In this study, the chemical components and biological activities of two starfish C novaeguineae and P gracilis in Vietnam were identified The results will be contributed in the medicinal and pharmaceutical industry as an important role for the discovery and development of new drugs Therefore, thesis title was chosen to be: “Study on chemical constituents and biological activities from Culcita novaeguineae Müller & Troschel, 1842 and Pentaceraster gracilis (Lutken, 1871) in Vietnam” The objectives of the thesis  Investigated on the chemical constituents of the two starfish C novaeguinea and P gracilis in the East-North sea of Vietnam  Studied on the cytotoxic activity of the isolated compounds to find the bioactive compounds The main contents of the thesis The extraction and isolation of compounds from the starfishes C novaeguineae and P gracilis in Viet Nam using the chromatography methods  The structure determination of the isolated compounds based on the physical and chemical methods  Studied the cytotoxic activity of the isolated compounds CHAPTER OVERVIEW Overview of internal and international researches related to our study 1.1 The previous studies in the world about the chemical constituents and biologycal activities from the starfishes Numerous studies on the chemical components of the starfish in the world were reported The steroid, glycoside (glycoside of polyhydroxysteroid, asterosaponin and cyclic steroid glycoside ) were the main groups with the cytotoxic, antibacteria, and antiinflammatory activities 1.2 The previous studies about the chemical constituents and biologycal activity from the starfish in Vietnam Up to date, the several starfishes were investigated the chemical constituents from the Vietnam sea, including Archaster typicus, Asterina batheri, Asteropsis carinifera, Astropecten polyacanthus, Astropecten monacanthus, Protoreaster nodosus, Acanthaster planci, Linckia laevigata, and Anthenea aspera The steroides, glycosides, including the glycoside of polyhydroxysteroides, asterosaponin were evaluated with their cytotoxic and anti-inflammatory activities CHAPTER EXPERIMENT AND RESULTS 2.1 Materials 2.1.1 The starfish Culcita novaeguineae The starfish C novaeguineae was collected at Quang Ninh, Vietnam, in October 2013, and identified by Prof Do Cong Thung 2.1.2 The starfish Pentaceraster gracilis The starfish P gracilis was collected at Bac Van, Co To, Quang Ninh, Vietnam, in March 2014, and identified by Prof Do Cong Thung 2.2 Methods 2.2.1 The extraction and isolation of compounds methods The combination of the chromatography methods including thin layer chromatography (TLC), the preparative of thin lay chromatography, and the column chromatography (CC) 2.2.2 The structure determination of isolated compound methods The structure identification is the combination of the physical properties and the model spectroscopic methods, including the Electrospray Ionisation Mass Spectrometry (ESI-MS), the Highresolution electrospray ionisation mass spectrometry (ESI-MS), []D, and Nuclear magnetic resonance spectroscopy (NMR) 2.2.3 The cytotoxic activity assay The cytotoxic activity of the isolated compounds was evaluated by SRB method 2.3 Extraction and isolation of compounds 2.3.1 The extraction and isolation of compounds from Culcita novaeguineae This part showed the extraction and isolation experiments of the compounds isolated from the starfish C novaeguineae CH2CL2 (15,2 g) MPLC SNAP-Sil C1 DM 100:1 – 1:1 C4 C8 (2 g) C7 C9 CC, YMC RP-18 MW 1:1 – 5:1 C8.1 C8.2 (500 mg) C8.3 (387mg) C8.4 (172 mg) CC, Silicagel EMW 10/1/0,1 C8.5A,B C8.5C (46mg C8.5D (18mg) C8.5E (76mg) C8.5F (150mg) Silica gel CC D/M/W 5/1/0,1 YMC CC M/W 1,5/1 CN9 (5,4 mg) C8.6 (185 mg) C8.5 (560 mg) Silica gel CC E/M/W 10/1/0,1 Sephadex LH-20 M/W 2/1 H8.5F1 CN3 (4,0mg) CN4 (7,5mg) C8.5G (150mg) H8.5F2 YMC CC A/W 1/1 CN5 (9,5mg) CN8 (3,5mg) CN6 (2,5mg) CN7 (5,0mg) Figure 2.4-6 The extraction and isolation of compounds isolated from the water layer of C novaeguineae 2.3.2 Extraction and isolation of compounds from P gracilis This part showed the extraction and isolation experiments of compounds from the starfish P gracilis W3 8.5 g YMC CC, MW: 1/1 W3 A 2.7 g W3B 700 mg W3B1 50 mg W3B2 80 mg W3D 1.3 g W3C 720 mg DMWa: 2.5/1/0.15/0.002 DMWa: 2.7/1/0.15/0.002 W3C1 220 mg W3E 900 mg DMW: 4/1/0.15 DMWa: 2.5/1/0.15/0.002 W3D1 210 mg W3D2 700 mg Sephadex MW: 1/1 Sephadex MW: 1/1 W3D2a 240 mg PG2 12 mg EMW: 1.8/1/0.2 W3D2a1 60 mg W3D2a2 40 mg W3E1 80 mg Sephadex MW: 1/1 W3E1a 50 mg DAW: 1/2/0.1 PG7 18 mg MW: 1.5/1 W3D2a2a 25 mg W3E2 120 mg W3E3 60 mg Sephadex MW: 1/1 W3E2a 80 mg W3E3a 25 mg Sephadex MW: 1/1 PG4 10 mg W3E4 100 mg EMW: 5/1/0.1 W3E5 65 mg DAW: 1/3/0.1 W3E3b 10 mg Sephadex MW: 2/1 PG6 mg EMW: 3.5/1/0.1 PG5 25 mg W3E5a 23 mg MW: 2.5/1 PG3 10 mg EMW: 2.5/1/0.15 PG1 mg Figure 2.7-9 Extraction and isolation of compounds isolated from P gracilis 2.4 Physical properties and spectroscopic data of the isolated compounds from C novaeguineae and P gracilis 2.4.1 Compound CN1: Novaeguinoside E (New compound) White powder, HR-ESI-MS m/z 1273,5257 [M + Na]+, Molecular formula C56H91NaO27S 1H-NMR (DMSO-d6, 500 MHz) and 13C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.1) 2.4.2 Compound CN2: Natri 6α-[(O-β-D-fucopyranosyl-(l2)-O-βD-galactopyranosyl-(l4)-O-[β-D-quinovopyranosyl-(l2)]-O-β-Dxylopyranosyl-(l3)-O-β-D-quinovopyranosyl)-oxy]-5α-pregn9(11)-ene-20-one-3β-yl-sulfate White powder; 1H-NMR (DMSO-d6, 500 MHz) and 13C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.2) 2.4.3 Compound CN3: Linckoside B White powder; Molecular formula C40H68O14 1H-NMR (pyridined5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.3) 2.4.4 Compound CN4: Halityloside E White powder; Molecular formula C39H68O13 1H-NMR (pyridined5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.4) 2.4.5 Compound CN5: Halityloside D White powder; melting point: 243-2480C; Molecular formula C39H68O14 1H-NMR (pyridine-d5, 500 MHz) and 13C-NMR (pyridined5, 125 MHz) (see Table IV.1.5) 2.4.6 Compound CN6: Culcitoside C5 Colorless powder; Molecular formula C38H66O14 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz) (see Table IV.1.6) 2.4.7 Compound CN7: Halityloside B White powder; Molecular formula C40H70O14 1H-NMR (pyridine-d5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.7) 2.4.8 Compound CN8: Halityloside A White powder; Molecular formula C40H70O15 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz) (see Table IV.1.8) 2.4.9 Compound CN9: 5α-cholestane-3β,6β,7α,8β,15α,16β,26heptol White powder; Molecular formula: C27H48O7 1H-NMR (DMSO-d6, 500 MHz) and 13C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.9) 2.4.10 Compound PG2: Protoreasteroside White powder; Molecular formula: C56H92O27S 1H-NMR (pyridine-d5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz) (see Table IV.2.1) 2.4.11 Compound PG1: Maculatoside White powder; Molecular formular: C56H92O27S 1H-NMR (pyridine-d5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz) (see Table IV.2.2) 2.4.12 Compound PG3: Pentaceroside A (New compound) White powder; FT-ICR-MS: m/z 755.41935 [M + Na]+, Molecular formula: C37H64O14 1H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.2.3) 2.4.13 Compound PG4: Pentaceroside B (New compound) White powder; FT-ICR-MS: m/z 623.3771 [M + Na]+, Molercular formula: C32H56O10 1H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.2.4) 2.4.14 Compound PG5: Nodososide White powder; Molecular formula: C38H66O14 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz) (see Table IV.2.5) 2.4.15 Compound PG6: (5α,25S)-Cholestane-3β,6α,8,15β,16β,26hexol 3-O-[(2-O-methyl)-β-D-xylopyranoside] White powder; Molecular formula: C33H58O10 1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz) (see Table IV.2.6) 2.4.16 Compound PG7: 5α-cholestane-3β,6α,7α,8β,15α,16β,26heptol White powder; Molecular formula: C27H48O7 1H-NMR (DMSO-d6, 500 MHz) and 13C-NMR (DMSO-d6, 125 MHz) (see Table IV.2.7) 2.5 Results on cytotoxic activity of compounds The cytotoxic activity of isolated compounds was investigated on five cancer cell lines, including LNCaP (human prostate carcinoma), MCF7 (human breast cancer), KB (human epidermoid carcinoma), HepG2 (human hepatoma), and SK-Mel-2 (human melanoma) The all experiments were evaluated at the Biologycal activity laboratory in the Institute of Biotechnology 2.5.1 Cytotoxic effects of compounds isolated from C novaeguineae Table 4.1 IC50 values of compounds isolated from C novaeguineae on five human cell lines IC50 (µM) Compounds a LNCaP MCF7 KB HepG2 SK-Mel2 CN1 >100 >100 >100 >100 >100 CN2 >100 >100 >100 >100 >100 CN3 >100 >100 >100 >100 >100 CN4 >100 >100 >100 >100 >100 CN5 31,801,59 33,961,57 32,661,47 75,014,11 32,993,05 CN6 57,081,81 62,952,96 92,042,84 >100 89,762,47 CN7 39,682,65 39,992,65 44,373,00 80,223,67 50,094,06 CN8 48,592,30 51,612,70 70,703,56 >100 73,993,10 CN9 >100 >100 >100 >100 >100 Elipticinea 1,990,16 1,950,12 2,070,12 1,710,16 2,150,24 Positive control 13 3.04–3.06 (H-22), each 1H, m], one oxygenated quaternary carbon atom [δC 75.1 (C-20)], two trisubstituted double bonds [δC145.2 (s, C-9) and 115.7 (d, C-11)/δH 5.24 (1H, d, J = 5.0 Hz, H-11); δC 124.0 (d, C-24)/δH 5.20 (1H, t, J = 7.0 Hz, H-24) and δC 130.4 (s, C-25)], and five tertiary methyl groups [δC 13.0 (C-18), 19.0 (C-19), 19.9 (C21), 17.8 (C-26), and 25.6 (C-27)/δH 0.72 (H-18), 0.88 (H-19), 1.06 (H-21), 1.55 (H-26), and 1.65 (H-27), each 3H, s] Locations of the oxygenated quaternary carbon atom at C-20, one oxymethine group at C-22, and one double bond at C-24/C-25 were identified by 1H– 1H correlation spectroscopy (COSY) peaks of H-22/H-23/H-24 and combination with heteronuclear multiplebond correlation (HMBC) cross-peaks of H-21 (δH 1.06) with C-17 (δC 75.1)/C-22 (δC 53.8)/C20 (δC 76.2) and those of H-26 (δH 1.55)/H-27 (δC 124.0)/C-25 (δH 1.65) with C-24 (δC 130.4) Detailed analysis of the other HMBC and COSY peaks unambiguously identified the planar structure of the aglycone Figure 3.4 HSQC spectrum of CN1 In the rotating frame Overhause effect spectroscopy (ROESY), the correlation of H-3 (H 3.83-3.87) with H-5 (H 1.051.07) suggested an α-orientation of H-3 Spatial proximities were observed between H-6 (H 3.45-3.47) and H-8 (H 1.97-1.99)/H-19 (H 0.88) as well as H-8 (H 1.97-1.99) and H-18 (H 0.72), indicating the borientation of H-6 (Figure 3.8) The 13C NMR chemical shift of C-21 at C 19.9 (in DMSO-d6) indicated the relative R* configuration 14 at C-20 [3] For determination of the stereochemistry at C-22, the 1HNMR of CN1 was recorded again in pyridine-d5 The 1H-NMR chemical shift of H-21 at H 1.64 (pyridine-d5) clearly indicated the relative S* configuration at C-22 Figure 3.5 The COSY spectrum of CN1 Figure 3.6 The HMBC spectrum of CN1 Table 3.3 1H and 13C NMR spectroscopic data of CN1 Cc,d Aglycon 10 11 12 13 14 15 16 17 18 35.9 29.2 78.3 30.6 49.2 80.0 41.2 35.4 145.6 38.2 116.6 42.6 41.6 54.0 22.6 25.3 55.1 13.4 35.15 28.35 75.18 29.49 48.43 78.11 40.63 34.67 145.18 37.70 115.67 41.99 40.51 53.36 24.55 21.15 53.80 13.00 1.26 m/1.62 m 1.36 m/2.12 m 3.85 m 1.04 m/2.35 m 1.06 m 3.46 m 0.83 m/2.27 m 1.98 m 5.24 d (5.0) 1.96 m/2.18 m 1.15 m 1.10 m/1.63 m 1.62 m/1.75 m 1.83 m 0.72 s 19 19.2 19.04 0.88 s a C Hc,e C C b mult (J = Hz) HMBC (H  C) 10, 13 12, 13, 14,17 1, 5, 9, 10 15 20 21 22 23 24 25 26 27 Qui I 1 2 3 4 5 6 Xyl 1 2 3 4 5 Qui II 1 2 3 4 5 6 Fuc I 1 2 3 4 5 6 Fuc II 1 2 3 4 5 6 a e 76.4 21.5 77.8 29.6 124.2 131.8 17.4 25.6 75.14 19.91 76.24 28.99 124.04 130.40 17.85 25.64 1.06 s 3.05 m 1.75 m/2.35 m 5.20 t (7.0) 1.55 s 1.65 s 105.1 74.1 90.5 74.5 72.0 18.4 104.4 74.1 89.8 74.3 72.3 17.8 102.75 73.00 87.90 72.96 70.74 17.81 4.31 d (7.5) 3.18 f 3.28 f 2.91 t (9.0) 3.27 f 1.16 d (6.5) 104.5 81.9 75.6 78.8 64.5 104.0 82.7 75.1 78.3 64.2 102.49 82.68 73.99 76.54 63.07 4.53 d (7.5) 3.35 f 3.58 f 3.60 f 3.31/3.94 f 3 104.8 76.2 76.8 75.5 73.6 17.8 104.54 74.84 75.58 74.63 72.44 17.31 4.44 d (7.5) 3.06dd (7.5, 9.0) 3.12t (9.0) 2.86 t (9.0) 3.22 dd (6.0, 9.0) 1.19 d (6.0) 2 102.0 82.8 74.9 71.7 71.6 16.9 105.2 75.6 77.0 76.0 73.3 17.8 Qui 101.2 84.0 75.9 77.3 73.4 18.2 100.08 81.47 72.44 70.22 70.10 16.51 4.42 d (7.5) 3.44 f 3.50 f 3.45 f 3.58 f 1.14 d (6.0) 4 106.9 73.8 75.0 72.5 71.9 17.1 106.2 71.8 74.7 72.8 71.8 16.9 105.60 72.05 73.23 70.99 70.47 16.65 4.21 d (7.0) 3.32 f 3.51 f 3.38 br s 3.54 f 1.15 d (6.0) 2 17, 20, 22 24, 25, 27 24, 25, 26 4, 5 4, 5 4, 5 C of novaeguinoside A [4], C of protoreasteroside [3], measure in DMSO-d6, d125 MHz, 500 MHz, eoverlapped b c 16 Figure 3.7 ROESY spectrum of CN1 In addition, the 13 Figure 3.8 COSY, HMBC, and ROESY correlation of CN1 C-NMR of CN1 contained anomeric cacbon signal at C 102.7 (C-1), 102.5 (C-1), 104.5 (C-1), 100.1 (C-1) and 105.6 (C-1); which correlated with the corresponding anomeric protons (each 1H, d, J = 7.5 Hz) at H 4.31 (H-1), 4.53 (H1), 4.44 (H-1), 4.42 (H-1) and 4.21 (H-1) in the heteronuclear single quantum coherence (HSQC) spectrum, confirming the presence of five sugar moieties A detailed comparison of the 1H and oligosaccharide chain of compound CN1 13 C-NMR for the with those of protoreasteroside and a combination of the 1D total correlation spectroscopy (TOCSY), COSY, HMBC, HSQC, and ROESY data (Figure 3.9), indicated that the difference between these two compounds was only observed in the fourth sugar moiety The 13CNMR chemical shifts of this sugar in DMSO-d6 at C 100.1 (C-1), 81.5 (C-2), 72.4 (C-3), 70.2 (C-4), 70.1 (C-5) and 16.5 (C-6) were similar to those of novaeguinoside A in pyridine-d5 at C 102,0 (C-1), 82.8 (C-2), 74.9 (C-3), 71.7 (C-4), 71.6 (C-5) and 16.9 (C-6) and quite different from those of protoreasteroside in pyridine-d5 at C 101.2 (C-1), 84.0 (C-2), 75.9 (C-3), 77.3 (C-4), 73.4 (C-5), and 18.2 (C-6), indicated the fourth sugar is fucose This was also supported 17 by hypothetic biosynthesis with the coexistence of compounds CN1 and novaeguinoside A, containing the same pentaglycoside chain in the starfish C novaeguineae (A) (B) (C) (D) (E) Figure 3.9 1D TOCSY spectrum of CN1 In the 1D TOCSY of CN1, the magnetization transfer of anomeric proton: H-1 at H 4,53 (A), H-1 at H 4,44 (B), H-1 at H 4,42 (C), H-1 at H 4,31 (D), and H-1 at H 4,21 (E) were determined the protons of each sugars The HMBC correlation of the 18 anomeric proton H-1 (H 4.21) with C-2 (C 81.5), H-1 (H 4.42) with C-4 (C 76.5), H-1 (H 4.44) with C-2 (C 82.7) and H1 (H 4.53) with C-3 (C 87.9), indicated the attachment position of the fucose II at C-2, fucose I at C-4, quinovose II at C-2 and xylose C-3 Moreover, the anomeric proton H-1 (H 4.31) of quinovose I had an HMBC cross-peak with C-6 (C 78,1) demonstrated the common attachment position of the pentaglycoside chain at C-6 of the steroidal aglycone The large coupling constant (J = 7.5 Hz) of the anomeric protons indicated all β-glycosidic linkage Configuration of all five sugars was assigned as D by analogy with novaeguinoside A and all the reported asterosaponins Consequently, the structure of CN1 was elucidated as sodium (20R*,22S*)-6α-O-{-D-fucopyra nosyl-(12)--D-fucopyranosyl-(14)-[-D-quinovopyranosyl(12)]--D-xylopyranosyl-(13)--D-quinovopyranosyl}-3β,6α,20, 22- tetrahydroxy-5α-cholesta-9(11),24-dien-3β-yl sulfate This is the new compounds and named novaeguinoside E 3.2 The struture elucidation of compounds isolated from the starfish P gracilis This part exhibited the structure determination of seven compounds from the starfish P gracilis PG2: Protoreasteroside PG1: Maculatoside 19 PG3: Pentaceroside A (new compound) PG4: Pentaceroside B (new compound) PG6: (5α,25S)-cholestane-3β,6α,8,15β,16β, 26-hexol 3-O-[(2-O-methyl)-β-D-xylopyrano side] PG5: Nodososide PG7: 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol 3.2.1 Compound PG1: Maculatoside Compound PG1 was obtained from the starfish Pentaceraster gracilis as the white powder The 1D and 2D NMR data of PG1 was displayed that PG1 is a glycosid with the common signal of asterosaponin skeleton contained sugar units The aglycon of PG1 is a steroid with the presence of double bond at 9(11) and one keton group 20 Figure 4.1 1H-NMR spectrum of PG1 Figure 4.2 13C-NMR spectrum of PG1 Figure 4.3 HSQC spectrum of PG1 Figure 4.5 HMBC spectrum of PG1 Figure 4.4 COSY spectrum of PG1 Figure 4.6 ROESY spectrum of PG1 Comparision of the NMR spectra data of PG1 with those of the protoreasteroside showed that the main differrent of two compounds is the structure of the branched The NMR spectra of this part of PG1 contained the chemical signal of quarternary carbon linked oxygene, one tert-metyl group, one keton and two sec-metyl groups Comparision of the NMR data of PG1 and the previous study, combination with the correlation in the HSQC, HMBC, COSY and ROESY spectra was determined the structure of PG1 was maculatoside 21 Table 4.4 NMR spectroscopic data of PG1 Hb,d HMBC mult (J = Hz) (H  C) C Cb,c 35.9 29.2 78.3 30.6 49.2 80.0 41.2 35.4 145.6 38.2 116.6 42.6 41.6 54.0 22.6 25.3 55.1 13.4 19.2 76.4 21.5 77.8 29.6 35.97 29.46 77.64 30.74 49.37 80.33 41.67 35.39 145.55 38.29 116.84 42.79 41.61 54.07 22.58 25.38 55.06 13.63 19.29 76.34 21.38 78.24 30.62 24 25 26 27 Qui I 124.2 131.8 17.4 25.6 124.67 131.79 18.02 25.92 1.38 m/1.65 m 1.89 m/2.76 m 4.86 m 1.69 m/3.45 m 1.46 m 3.79 m 1.26 m/2.70 m 2.12 m 5.24 d (5.5) 2.14 m/2.35 m 1.33 m 2.08 m/2.48 m 1.30 m/1.84 m 2.40 m 1.13 s 0.96 s 1.66 s 3.88 m 2.45 m 2.92 br dd (6.5, 13.5) 5.66 t (6.5) 1.67 s 1.68 s 1 104.4 105.01 4.80 d (7.5) 2 74.1 74.16 3.98* 3 89.8 89.98 3.85* 4 74.3 74.53 3.57 t (9.0) 5 72.3 71.99 3.67* C Aglycon 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a 8, 10, 13 12, 13, 14,17 1, 5, 9, 10 17, 20, 22 24, 25, 27 24, 25, 26 22 17.8 18.47 1.55 d (6.0) 4, 5 1 104.0 104.35 5.07 d (7.5) 3 2 82.7 82.20 4.10* 3 75.1 75.55 4.23* 4 78.3 78.62 4.18* 5 64.2 64.41 3.81*/4.49 dd (5.0, 12.0) 1 105.2 105.12 5.28 d (7.5) 2 75.6 75.61 4.08* 3 77.0 77.47 4.14* 4 76.0 75.96 3.64* 5 73.3 73.71 3.70* 6 17.8 17.98 1.77 d (6.0) 4, 5 1 101.2 101.51 4.92 d (7.5) 4 2 84.0 84.96 3.98* 3 75.9 76.27 4.14* 4 77.3 76.93 4.08* 5 73.4 73.03 3.75* 6 18.2 18.17 1.50 d (6.0) 4, 5 1 106.2 107.01 4.97 d (7.5) 2 2 71.8 73.88 4.42 dd (7.5, 9.0) 3 74.7 74.96 4.06* 4 72.8 72.55 3.98 br s 5 71.8 71.99 3.73* 6 16.9 17.23 1.48 d (6.0) 6 Xyl Qui II 2 Qui III Fuc a 4, 5 C of protoreasteroside [3], measured in pyridine-d5, 125 MHz, 500 MHz, *overlapped b c d 4.4 Cytotoxic activity of the isolated compounds 4.4.1 Cytotoxic effects of the compounds isolated from the starfish C novaeguineae The results on cytotoxic activity of isolated compounds from C novaeguineae showed that compounds CN5-CN8 exhibited 23 cytotoxic effects with IC50 value ranging from 31,80  1,59 to 92,04  2,84 μM Other compounds were inactive with the IC50 > 100 µM Additionally, compound CN5 (halityloside D displayed the well activity against cancer cell lines, including LNCaP (IC50 = 31,80  1,59 µM), MCF7 (IC50 = 33,96  1,57 µM), KB (IC50 = 32,66  1,47 µM), and SK-Mel2 (IC50 = 32,99  3,05 µM) However, this compound showed weak effect against HepG2 cell (IC50 = 75,01  4,11 µM) Halityloside B (CN7) exhibited moderate activity against LNCaP (IC50 = 39,68  2,65 µM), MCF7 (IC50 = 39,99  2,65 µM), and KB cells (IC50 = 44,37  3,00 µM); the moderate activity on SKMel-2 cell (IC50 = 50,09  4,06 µM) and the weak activity on HepG2 cell (IC50 = 80,22  3,67 µM) Culcitoside C5 (CN6) and halityloside A (CN8) showed the moderate or weak activities on LNCaP, MCF7, KB, and SK-Mel2 cells with IC50 value ranging from 48,59  2,30 to 92,04  2,84 µM, and inactive on HepG2 cell (IC50> 100 µM) 4.1.2 The cytotoxic activity of the isolated compounds from the starfish Pentaceraster gracilis The results on cytotoxic activity of the isolated compound from P gracilis was showed that the maculatoside (PG1) exhibited significant activity against LNCaP (IC50 = 39,75  3,34 µM), KB (IC50 = 36,53  0,78 µM), HepG2 (IC50 = 16,75  0,69 µM), and SKMel-2 cells (IC50 = 19,44  1,45 µM), the moderate effect against MCF7 cell (IC50 = 47,34  7,01 µM) Compound PG7 displayed weak cytotoxicity effect on LNCaP (IC50 = 86,57  2,19 µM), HepG2 (IC50 = 79,69  3,14 µM), SK-Mel2 cells (IC50 = 96,77  4,07 µM), and inactive against other cell line (IC50 > 100 µM) Remaining compounds PG2PG6 was inactivity against all tested cells 24 CONCLUSIONS This is the first study of chemical constituents and biological activities from Culcita novaeguineae and Pentaceraster gracilis which were collected in the Vietnam sea Chemical investigations 16 compounds were isolated and identified from the two starfishes, includingone new compound and known compounds from C novaeguineae, two new compounds and known compounds from P gracilis Their structures were determined based on the model methods, including: Novaeguinoside E (CN1, new compound), natri 6α-[(O-β-Dfucopyranosyl-(l2)-O-β-D-galactopyranosyl-(l4)-O-[β-D-quino vopyranosyl-(l2)]-O-β-D-xylopyranosyl-(l3)-O-β-D-quinovopy ranosyl)-oxy]-5α-pregn-9(11)-ene-20-one-3β-yl-sulfate (CN2), linckoside B (CN3), halityloside E (CN4), halityloside D (CN5), culcitoside C5 (CN6), halityloside B (CN7), halityloside A (CN8), 5α-cholestane-3β,6β,7α,8β,15α,16β,26-heptol (CN9), maculatoside (PG1), protoreasteroside (PG2), pentaceroside A (PG3, new compound), pentaceroside B (PG4, new compound), nodososide (PG5), (5α,25S)-cholestane-3β,6α,8,15β,16β,26-hexol 3-O-[(2-Omethyl)-β-D-xylopyranoside] (PG6), and 5α-cholestane-3β,6α, 7α,8β,15α,16β,26-heptol (PG7) All compounds are the steroid derivatives, including two saponins (one new compound), polyhydroxy steroid glycosides and polyhydroxy steroid from C novaeguineae Among the compounds from P gracilis, saponins, polyhydroxy steroid glycosides (2 new compounds) and polyhydroxy steroid were found 25 Biological activity The cytotoxic effects of the isolated compounds were investigated in vitro in five human cancer cell lines: LNCaP, MCF7, KB, Hep-G2, and SK-Mel2 The results showed that: Halityloside D (CN5) exhibited significant cytotoxic activity against LNCaP , MCF7 , KB, and SK-Mel2 cells and the weak effect on Hep-G2 cell Halityloside B (CN7) showed significant cytotoxic effect against LNCaP, MCF7, and KB cells; the moderate acitivity in SK-Mel2 cell and the weak activity in HepG2 cell Two compounds, culcitoside C5 (CN6) and halityloside A (CN8), displayed the moderate or weak effect against LNCaP, MCF7, KB, and SK-Mel-2 cell , and inactive (IC50> 100 µM) on HepG2 cell Maculatoside (PG1) exhibited significant activity on LNCaP, KB , HepG2, and SK-Mel2 cells, and the moderate activity on MCF7 Compound PG7 showed the weak efffect on LNCaP, HepG2, and SKMel2 cells, and inactive (IC50> 100 µM) in the other cell lines Compounds PG2PG6 showed the no significant effect at the all of cancer cell lines tested with IC50> 100 µM RECOMMENDATIONS Halityloside D (CN5), halityloside B (CN7), and maculatoside (PG1) displayed the good activity in the cancer cell line Therefore, further studies to clarify activity mechanism and pharmacoglogical study of these compounds should be carried Continuously, the biologycal activities of the isolated compounds should be widely to find the active compounds for the futher study 26 NEW FINDINGS OF THE THESIS Chemical investigations - 03 new compounds: Novaeguinoside E (CN1); pentaceroside A (PG3), and pentaceroside B (PG4) - Compounds CN2-CN9 were isolated as the first time from the starfish C novaeguineae in Vietnam - Compounds PG1-PG2, PG5-PG7 were isolated and identified as the first time from the starfish P gracilis - This is the first time, the chemical constituents of C novaeguineae and P gracilis were collected and reported in the EastNorth sea of Vietnam Biological activity This is the first time to evaluate cytotoxic effects of isolated compounds from the C novaeguineae and P gracilis in the Vietnam sea toward cancer cell lines The effects of 16 compounds against five cancer cell lines (LNCaP, MCF7, KB, HepG2, and SK-Mel-2) were evaluated The results showed that 03 compounds (CN5, CN7, and PG1) exhibited good significant cytotoxicity against LNCaP, MCF7, KB, and SK-Mel-2 cells; 02 compounds (PG1 and PG7) showed significant effects toward HepG2 cell 27 PUBLICATIONS WITHIN THE SCOPE OF THESIS Bui Thi Ngoan, Tran Thi Hong Hanh, Le Thi Vien, Chau Ngoc Diep, Nguyen Phuong Thao, Do Thi Thao, Nguyen Van Thanh, Nguyen Xuan Cuong, Nguyen Hoai Nam, Do Cong Thung, Phan Van Kiem, Young Ho Kim, and Chau Van Minh Asterosaponins and glycosylated polyhydroxysteroids from the starfish Culcita novaeguineae and their cytotoxic activities J Asian Nat Prod Res., 2015, 17(10), 1010–1017 Le Thi Vien, Bui Thi Ngoan, Tran Thi Hong Hanh, Le Ba Vinh, Do Cong Thung, Do Thi Thao, Nguyen Van Thanh, Nguyen Xuan Cuong, Nguyen Hoai Nam, Phan Van Kiem, and Chau Van Minh Steroid glycosides from the starfsh Pentaceraster gracilis J Asian Nat Prod Res., 2017, 19(5), 474–480 Bui Thi Ngoan, Tran Thi Hong Hanh, Le Thi Vien, Chau Ngoc Diep, Nguyen Phuong Thao, Nguyen Van Thanh, Nguyen Xuan Cuong, Nguyen Hoai Nam, Do Cong Thung, Phan Van Kiem, and Chau Van Minh A polyhydroxylated sterol and a saponin isolated from the starfish Culcita novaeguineae Vietnamese J Chem., 2015, 53(2e), 23-27 Bùi Thị Ngoan, Trần Thị Hồng Hạnh, Lê Thị Viên, Nguyễn Văn Thanh, Nguyễn Xuân Cường, Nguyễn Hồi Nam, Đỗ Cơng Thung, Châu Văn Minh Các hợp chất steroit diglycosit phân lập từ loài biển Culcita novaeguineae Tạp chí Hóa học, 2016, 54(2e), 44-48 ... discovery and development of new drugs Therefore, thesis title was chosen to be: ? ?Study on chemical constituents and biological activities from Culcita novaeguineae Müller & Troschel, 1842 and Pentaceraster. .. Extraction and isolation of compounds 2.3.1 The extraction and isolation of compounds from Culcita novaeguineae This part showed the extraction and isolation experiments of the compounds isolated from. .. extraction and isolation of compounds isolated from the water layer of C novaeguineae 2.3.2 Extraction and isolation of compounds from P gracilis This part showed the extraction and isolation experiments