Study on chemical constituents and biological activities from culcita novaeguineae muller & troschel, 1842 and pentaceraster gracilis (lutken, 1871) in Vietnam

1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY 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 1 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 1 st Reviewer:................................................................. 2 nd Reviewer:................................................................ 3 rd 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 2 INTRODUCTION 1. 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 9 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 anti- inlammatory. 3 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”. 2. 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. 3. 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 1. 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...) 4 were the main groups with the cytotoxic, antibacteria, and anti- inflammatory 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 2. 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). 5 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 High- resolution 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. C8.5 (560 mg) C8.5A,B C8.5C (46mg CC, Silicagel EMW 10/1/0,1 C8.5D (18mg) C8.5E (76mg) C8.5F (150mg) C8.5G (150mg) CN4 (7,5mg) CN3 (4,0mg) CN5 (9,5mg) Silica gel CC D/M/W 5/1/0,1 H8.5F1 H8.5F2 CN8 (3,5mg) Sephadex LH-20 M/W 2/1 CN7 (5,0mg) CN6 (2,5mg) YMC CC A/W 1/1 CH2CL2 (15,2 g) C1 C8 (2 g) C9 MPLC SNAP-Sil DM 100:1 – 1:1 C8.1 C8.2 (500 mg) C8.3 (387mg) C8.4 (172 mg) C8.6 (185 mg) CC, YMC RP-18 MW 1:1 – 5:1 C4 C7 CN9 (5,4 mg) Silica gel CC E/M/W 10/1/0,1 YMC CC M/W 1,5/1 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 6 W3 8.5 g W3E 900 mg DMW: 4/1/0.15 W3 A 2.7 g YMC CC, MW: 1/1 W3B 700 mg DMWa: 2.5/1/0.15/0.002 W3B2 80 mg W3B1 50 mg W3C 720 mg W3C1 220 mg DMWa: 2.7/1/0.15/0.002 PG2 12 mg Sephadex MW: 1/1 Sephadex MW: 1/1 PG1 9 mg W3D 1.3 g DMWa: 2.5/1/0.15/0.002 W3D2 700 mg W3D1 210 mg W3D2a 240 mg EMW: 1.8/1/0.2 EMW: 2.5/1/0.15 W3D2a2 40 mg W3D2a1 60 mg W3D2a2a 25 mg MW: 1.5/1 W3E4 100 mg Sephadex MW: 2/1 W3E3 60 mg EMW: 5/1/0.1 W3E3a 25 mg W3E3b 10 mg Sephadex MW: 1/1 PG4 10 mg PG6 8 mg Sephadex MW: 1/1 Sephadex MW: 1/1 W3E1 80 mg W3E1a 50 mg DAW: 1/2/0.1 PG7 18 mg W3E2 120 mg W3E2a 80 mg W3E5 65 mg PG5 25 mg PG3 10 mg DAW: 1/3/0.1 EMW: 3.5/1/0.1 W3E5a 23 mg MW: 2.5/1 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. 1 H-NMR (DMSO-d6, 500 MHz) and 13 C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.1). 2.4.2. Compound CN2: Natri 6α-[(O-β-D-fucopyranosyl-(l2)-O-β- D-galactopyranosyl-(l4)-O-[β-D-quinovopyranosyl-(l2)]-O-β-D- xylopyranosyl-(l3)-O-β-D-quinovopyranosyl)-oxy]-5α-pregn- 9(11)-ene-20-one-3β-yl-sulfate White powder; 1 H-NMR (DMSO-d6, 500 MHz) and 13 C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.2). 2.4.3. Compound CN3: Linckoside B White powder; Molecular formula C40H68O14. 1 H-NMR (pyridine- d5, 500 MHz) and 13 C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.3). 2.4.4. Compound CN4: Halityloside E 7 White powder; Molecular formula C39H68O13. 1 H-NMR (pyridine- d5, 500 MHz) and 13 C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.4). 2.4.5. Compound CN5: Halityloside D White powder; melting point: 243-248 0 C; Molecular formula C39H68O14. 1 H-NMR (pyridine-d5, 500 MHz) and 13 C-NMR (pyridine- d5, 125 MHz) (see Table IV.1.5). 2.4.6. Compound CN6: Culcitoside C5 Colorless powder; Molecular formula C38H66O14. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.1.6). 2.4.7. Compound CN7: Halityloside B White powder; Molecular formula C40H70O14. 1 H-NMR (pyridine-d5, 500 MHz) and 13 C-NMR (pyridine-d5, 125 MHz) (see Table IV.1.7). 2.4.8. Compound CN8: Halityloside A White powder; Molecular formula C40H70O15. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.1.8). 2.4.9. Compound CN9: 5α-cholestane-3β,6β,7α,8β,15α,16β,26- heptol White powder; Molecular formula: C27H48O7. 1 H-NMR (DMSO-d6, 500 MHz) and 13 C-NMR (DMSO-d6, 125 MHz) (see Table IV.1.9). 2.4.10. Compound PG2: Protoreasteroside White powder; Molecular formula: C56H92O27S. 1 H-NMR (pyridine-d5, 500 MHz) and 13 C-NMR (pyridine-d5, 125 MHz) (see Table IV.2.1). 8 2.4.11. Compound PG1: Maculatoside White powder; Molecular formular: C56H92O27S. 1 H-NMR (pyridine-d5, 500 MHz) and 13 C-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. 1 H-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. 1 H-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. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.2.5). 2.4.15. Compound PG6: (5α,25S)-Cholestane-3β,6α,8,15β,16β,26- hexol 3-O-[(2-O-methyl)-β-D-xylopyranoside] White powder; Molecular formula: C33H58O10. 1 H-NMR (CD3OD, 500 MHz) and 13 C-NMR (CD3OD, 125 MHz) (see Table IV.2.6) 2.4.16. Compound PG7: 5α-cholestane-3β,6α,7α,8β,15α,16β,26- heptol White powder; Molecular formula: C27H48O7. 1 H-NMR (DMSO-d6, 500 MHz) and 13 C-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 9 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 Compounds IC50 (µM) 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 a Positive control 10 2.5.2. Cytotoxic effects of the compounds isolated from P. gracilis Table 4.2. The IC50 value of the compounds isolated from Pentaceraster gracilis on the five line cells Comp. IC50 (µM) LNCaP MCF7 KB Hep-G2 SK-Mel2 PG1 39,753,34 47,347,01 36,530,78 16,750,69 19,441,45 PG2 >100 >100 >100 >100 >100 PG3 >100 >100 >100 >100 >100 PG4 >100 >100 >100 >100 >100 PG5 >100 >100 >100 >100 >100 PG6 >100 >100 >100 >100 >100 PG7 86,572,19 >100 >100 79,693,14 96,774,07 Elipticinea 1,990,16 1,950,12 2,070,12 1,710,16 2,150,24 a Positive control CHAPTER 3. DISCUSSIONS 3.1. The structure elucidation of compounds isolated from the starfish C. novaeguineae This part demonstrated the structure of 9 compounds from the starfish C. novaeguineae. CN1: Novaeguinoside E CN2: natri 6α-[(O-β-D- fucopyranosyl-(l2)-O-β-D-galactop yranosyl-(l4)-O-[β-D-quinovopyra nosyl-(l2)]-O-β-D-xylopyranosyl- (l3)-O-β-D-quinovopyranosyl)-oxy] -5α-pregn-9(11)-ene-20-one-3β-yl- sulfate 11 CN3: Linckoside B CN4: Halityloside E CN5: Halityloside D CN6: Culcitoside C5 CN7: Halityloside B CN8: Halityloside A CN9: 5α-cholestane- 3β, 6β,7α,8β,15α,16β,26-heptol 3.1.1. Compound CN1: Novaeguinoside E (New compound) Novaeguinoside E was isolated as a white amorphous powder. Its molecular formula was determined as C56H91NaO27S by high-resolution electrospray ionization mass spectrometry (HR-ESI- MS) at m/z 1273.5257 [M + Na] + . The NMR features indicated an asterosaponin, one of the main constituents of starfish. The 1H and 13 C NMR data (in DMSO-d6) of CN1 were similar to those of protoreasteroside, except for differences in the data of sugar moieties. 12 155.0736 274.2765 353.2659 413.2667 566.2132 648.2584 803.5400 1273.5257 +MS, 1.4min #83 0 2 4 6 8 4x10 Intens. 200 400 600 800 1000 1200 1400 m/z Figure 3.1. The HR-ESI-MS spectra of CN1 Figure 3.2a. 1 H-NMR spectrum of CN1 in DMSO-d6 Figure 3.2. 1 H-NMR spectrum of CN1 in pyridine-d5 Figure 3.3. 13 C-NMR spectra of CN1 Analysis of 1D and 2D NMR spectra confirmed that the aglycone of CN1 contained three oxymethine groups [δC 75.2 (C-3), 78.1 (C-6), and 76.2 (C-22)/δH 3.83–3.87 (H-3), 3.45–3.47 (H-6), and 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 (C- 21), 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)/C- 20 (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 1.05- 1.07) 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 13 C 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 1 H- NMR of CN1 was recorded again in pyridine-d5. The 1 H-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. 1 H and 13 C NMR spectroscopic data of CN1 C aC bC C c,d H c,e mult. (J = Hz) HMBC (H  C) Aglycon 1 35.9 35.15 1.26 m/1.62 m 2 29.2 28.35 1.36 m/2.12 m 3 78.3 75.18 3.85 m 4 30.6 29.49 1.04 m/2.35 m 5 49.2 48.43 1.06 m 6 80.0 78.11 3.46 m 7 41.2 40.63 0.83 m/2.27 m 8 35.4 34.67 1.98 m 9 145.6 145.18 - 10 38.2 37.70 - 11 116.6 115.67 5.24 d (5.0) 10, 13 12 42.6 41.99 1.96 m/2.18 m 13 41.6 40.51 - 14 54.0 53.36 1.15 m 15 22.6 24.55 1.10 m/1.63 m 16 25.3 21.15 1.62 m/1.75 m 17 55.1 53.80 1.83 m 18 13.4 13.00 0.72 s 12, 13, 14,17 19 19.2 19.04 0.88 s 1, 5, 9, 10 15 20 76.4 75.14 - 21 21.5 19.91 1.06 s 17, 20, 22 22 77.8 76.24 3.05 m 23 29.6 28.99 1.75 m/2.35 m 24 124.2 124.04 5.20 t (7.0) 25 131.8 130.40 - 26 17.4 17.85 1.55 s 24, 25, 27 27 25.6 25.64 1.65 s 24, 25, 26 Qui I 1 105.1 104.4 102.75 4.31 d (7.5) 6 2 74.1 74.1 73.00 3.18 f 3 90.5 89.8 87.90 3.28 f 4 74.5 74.3 72.96 2.91 t (9.0) 5 72.0 72.3 70.74 3.27 f 6 18.4 17.8 17.81 1.16 d (6.5) 4, 5 Xyl 1 104.5 104.0 102.49 4.53 d (7.5) 3 2 81.9 82.7 82.68 3.35 f 3 75.6 75.1 73.99 3.58 f 4 78.8 78.3 76.54 3.60 f 5 64.5 64.2 63.07 3.31/3.94 f Qui II 1 104.8 105.2 104.54 4.44 d (7.5) 2 2 76.2 75.6 74.84 3.06dd (7.5, 9.0) 3 76.8 77.0 75.58 3.12t (9.0) 4 75.5 76.0 74.63 2.86 t (9.0) 5 73.6 73.3 72.44 3.22 dd (6.0, 9.0) 6 17.8 17.8 17.31 1.19 d (6.0) 4, 5 Fuc I Qui 1 102.0 101.2 100.08 4.42 d (7.5) 4 2 82.8 84.0 81.47 3.44 f 3 74.9 75.9 72.44 3.50 f 4 71.7 77.3 70.22 3.45 f 5 71.6 73.4 70.10 3.58 f 6 16.9 18.2 16.51 1.14 d (6.0) Fuc II 1 106.9 106.2 105.60 4.21 d (7.0) 2 2 73.8 71.8 72.05 3.32 f 3 75.0 74.7 73.23 3.51 f 4 72.5 72.8 70.99 3.38 br s 5 71.9 71.8 70.47 3.54 f 6 17.1 16.9 16.65 1.15 d (6.0) 4, 5 a C of novaeguinoside A [4], bC of protoreasteroside [3], cmeasure in DMSO-d6, d125 MHz, e500 MHz, eoverlapped. 16 Figure 3.7. ROESY spectrum of CN1 Figure 3.8. COSY, HMBC, and ROESY correlation of CN1 In addition, the 13 C-NMR of CN1 contained 5 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 (H- 1), 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 1 H and 13 C-NMR for the oligosaccharide chain of compound CN1 with those of protoreasteroside and a combination of the 1D total correlation spectroscopy (TOCSY), COSY, HMBC, HSQC, and ROESY data (Figure 3.9), indicated tha