MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY NGUYEN THI THU HIEN STUDY ON CHEMICAL COMPOSITIONS AND BIOLOGICAL ACTIVITIES OF Annona glabra L Major: Organic chemistry Major Code: 62.44.01.14 SUMMARY OF DOCTORAL THESIS HA NOI - 2016 This thesis was completed at: Institute of Marine Biochemistry Vietnam Academy of Science and Technology Advisors: Prof Dr Phan Van Kiem Institute of Marine Biochemistry - Vietnam Academy of Science and Technology Dr Hoang Le Tuan Anh Institute of Marine Biochemistry - Vietnam Academy of Science and Technology 1st Reviewer: Prof Dr Sci Phan Tong Son Hanoi University of Sciences 2nd Reviewer: Prof Dr Nguyen Thi Ngoc Dao Institute of Biotechnology, Vietnam Academy of Science and Technology 3rd Reviewer: Prof Dr Tran Viet Hung National Institute of Drug Quality Control The thesis will be defended at the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, No.18 Hoang Quoc Viet, Cau Giay District, Ha Noi City at hour date month 2016 Thesis can be found in the library of the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, No.18 Hoang Quoc Viet, Cau Giay District, Ha Noi City I INTRODUCTION Preface Annona glabra L., belonging Annonaceae family, is a tropical tree and wildly distributes in the America and Asia It is used in traditional medicine to treat several diseases such as insecticide, inflammation, and cancer Phytochemical investigation of A glabra led to the isolation of numberous acetogenins, entkauranes, peptides, and alkaloids In addition, a lot of isolated compounds exhibited potent anticancer, anti HIV-reserve transcriptase, and anti-malarial activities This research aims to clarify chemical compositions and biological activities of Annona glabra L So, the thesis’s title was proposed to be "Study on chemical compositions and biological activities of Annona glabra L." Subject and contents of the thesis - Subject is the leaves and fruits of Annona glabra - The aims of the thesis: Extraction and isolation of secondary metabolites from A glabra; Determination of chemical structure of the isolated compounds; Evaluation of cytotoxic activity of isolated compounds; Evaluation of anti-inflammatory activity of isolated compounds New contributions of the thesis 3.1 New contributions on chemical stuty of Annona glabra fruits as belowing: 3.1.1 Five new compounds including: 7β,16α,17-trihydroxy-ent-kauran-19-oic acid (1), 7β,17-dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-D-glucopyranoside ester (2), 7β,17-dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β-D-glucopyranoside ester (3), 16α-hydro-ent-kauran-17,19-dioic acid 17,19-di-O-β-D-glucopyranoside ester (4), (2E,4E,1′R,3′S,5′R,6′S)-dihydrophaseic acid 1,3′-di-O-β-Dglucopyranoside (13) were isolated; 3.1.2 Among isolated compounds, seven compounds, including paniculoside IV (5), (2E,4E,1′R,3′S,5′R,6′S)-dihydrophaseic acid 3′-O-β -D-glucopyranoside (14), cucumegastigmane I (15), icariside B1 (17), icariside D2 (18), icariside D2 6′-O-βD-xylopyranoside (19), 3,4-dimethoxyphenyl 1-O-β-D-glucopyranoside (20) were isolated from the Annona genus for the first time; 3.1.3 Two compounds, 16α,17-dihydroxy-ent-kaurane (6) and 3,4dihydroxybenzoic acid (21) were first isolated from the Annona glabra species 3.2 This is the first report on cytotoxic effects toward five human cancer cell lines (LU-1, MCF-7, SK-Mel2, HL-60 and KB) of isolated compounds (1-22) The results indicated that compounds 3, 4, 6, 14, and 15 potentially exhibited cytotoxic effects toward tested cancer cells except HL-60 Of those, three ent-kaurane type compounds 3, 4, and showed the highest cytotoxic activity with their IC 50 values ranging from 0.65 ữ7.39 àM Megastigmane type compounds 14 and 15 exhibited cytotoxic effects with the IC50 values ranging from 2.79 ữ11.17 àM on four cancer cell lines Phenolic 18 and acetogenin 22 (squamocin M) not only displayed cytotoxic activity toward all tested cancer cells (IC 50 6.30 ữ10.61 àM) but also nontoxified in case of normal cells (HEL-299) 3.3 This is the first time, the mechanism of cytotoxic activities on HL-60 cell line was studied The results showed that compounds 18 and 22 induced apoptosis via alteration of expression of apoptosis-related proteins in HL-60 cells 3.4 19 compounds (1-3, 5-18, 20, 22) were first evaluated their inhibitory activity on LPS-stimulated nitric oxide (NO) production in RAW 264.7 macrophages As the results, five compounds 1, 3, 8, 12, and 13 potentially inhibited NO production with the IC50 values ranging from 0.01 ữ 0.42 àM The layout of the thesis The thesis consists of 138 pages containing 26 tables, 62 pictures, 147 references The layout of the dissertation: Preface (2 pages), Chapter 1: Overview (35 pages), Chapter 2: Objects and methods (7 pages), Chapter 3: Experimental (9 pages), Chapter 4: Results and discussion (64 pages), Conclusions (3 pages), Recommendations (1 page), Publications (1 page), References (17 pages), and Appendix II CONTENTS OF THE THESIS PREFACE This part mentioned scientific sense, practicality, object, objectives and tasks of the dissertation research CHAPTER 1: OVERVIEW 1.1 Introduction to Annona genus 1.2 Introduction to Annona galabra species CHAPTER 2: OBJECTS AND METHODS 2.1 Plant materials The leaves and fruits of A glabra were collected in Ho Chi Minh City, Vietnam during May 2013, and taxonomically identified by Dr Bui Van Thanh, Institute of Ecology and Biological Resources, VAST A voucher specimen (AG1605) was deposited at the Herbarium of the Institute of Marine Biochemistry, VAST 2.2 Methods using for isolation of compounds Chromatographic methods include thin-layer chromatography (TLC), column chromatography (CC) 2.3 Methods using for determination of chemical structure of compounds General method using for determination of chemical structure of the compounds is a combination of physical parameters and modern spectroscopic methods such as melting point (Mp), the optical rotation ([]D), mass spectrometry and highresolution mass spectrometry (ESI-MS, HR-ESI-MS), magnetic resonance spectrum (1D, 2D-NMR), Circular dichroism (CD) and chemical method 2.4 Method using for evaluation biological activities 2.4.1 Cytotoxic activity Cytotoxic activity was determined by MTT assay 2.4.2 Anti-inflammatory activity Anti-inflammatory activity of the compounds is assessed on the basis of inhibiting NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages CHAPTER 3: EXPERIMENTALS 3.1 Extraction and Isolation This section presents the process of making methanol extracts, fractions and isolated compounds from A glabra Figure 3.1 Preparation of crude extract and fractions from A glabra Figure 3.2 Isolation of compounds from dichloromethane extract Figure 3.3 Isolation of compounds from water extract 3.2 Physical and spectroscopic data 3.2.1 Compound 1: 7β,16α,17-Trihydroxy-ent-kauran-19-oic compound) White powder acid (new  25D : –64,9 (c 0,1, MeOH) Mp: 298-299oC H-NMR (500 MHz, CD3OD): δH 0,93 (1H, m, Ha-1), 1,87 (1H, m, Hb-1), 1,44 (1H, m, Ha-2), 1,96 (1H, m, Hb-2), 1,08 (1H, dd, J = 4,5 Hz, 13,5 Hz, Ha-3), 2,17 (1H, d, J = 13,5 Hz, Hb-3), 1,17 (1H, d, J = 9,0 Hz, H-5), 1,98 (1H, m, Ha-6), 2,11 (1H, m, Hb-6), 3,63 (1H, br s, H-7), 1,43 (1H, d, J = 7,5 Hz, H-9), 1,57 (1H, m, Ha11), 1,64 (1H, m, Hb-11), 1,57 (1H, m, Ha-12), 1,68 (1H, m, Hb-12), 2,08 (1H, m, H-13), 1,70 (1H, dd, J = 4,0 Hz, 12,0 Hz, Ha-14), 1,83 (1H, d, J = 12,0 Hz, Hb-14), 1,56 (1H, d, J = 13,5 Hz, Ha-15), 1,74 (1H, d, J = 13,5 Hz, Hb-15), 3,62 (1H, d, J = 11,5 Hz, Ha-17), 3,72 (1H, d, J = 11,5 Hz, Hb-17), 1,18 (3H, s, H-18), 1,00 (3H, s, H-20) 13 C-NMR (125 MHz, CD3OD): δC 41,71 (C-1), 20,29 (C-2), 34,23 (C-3), 44,25 (C4), 48,09 (C-5), 30,48 (C-6), 78,05 (C-7), 49,00 (C-8), 51,08 (C-9), 40,44 (C-10), 19,11 (C-11), 27,64 (C-12), 46,09 (C-13), 37,50 (C-14), 50,13 (C-15), 82,86 (C16), 66,71 (C-17), 29,27 (C-18), 182,00 (C-19), 16,14 (C-20) HR-ESI-MS: m/z 375,2159 [M+Na]+ Calcd for [C20H32O5Na]+: 375,2142 Molecular formula C20H32O5, M = 352 3.2.2 Compound 2: 7β,17-Dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-Dglucopyranoside ester (new compound) White powder  25D : –40,7 (c 0,1, MeOH); Mp: 185-186oC H-NMR (500 MHz, CD3OD): δH 0,94 (1H, m, Ha-1), 1,88 (1H, m, Hb-1), 1,45 (1H, m, Ha-2), 1,94 (1H, m, Hb-2), 1,13 (1H, m, Ha-3), 2,22 (1H, m, Hb-3), 1,78 (1H, d, J = 13,0 Hz, H-5), 1,97 (1H, m, Ha-6), 2,18 (1H, dd, J = 13,0 Hz, 14,5 Hz, Hb-6), 3,50 (1H, br s, H-7), 1,43 (1H, m, H-9), 1,57 (1H, m, Ha-11), 1,63 (1H, m, Hb-11), 1,43 (1H, m, Ha-12), 1,63 (1H, m, Hb-12), 2,11 (1H, m, H-13), 1,08 (1H, m, Ha-14), 1,80 (1H, d, J = 11,5 Hz, Hb-14), 1,12 (1H, m, Ha-15), 1,71 (1H, dd, J = 3,5 Hz, 10,0 Hz, Hb-15), 1,94 (1H, m, H-16), 3,35 (2H, m, H-17), 1,22 (3H, s, H-18), 0,99 (3H, s, H-20), 5,42 (1H, d, J = 8,0 Hz, H-1′), 3,38 (1H, m, H-2′), 3,45 (1H, m, H-3′), 3,39 (1H, m, H-4′), 3,39 (1H, H-5′), 3,71 (1H, dd, J = 4,0 Hz, 11,5 Hz, Ha6′), 3,86 (1H, d, J = 11,5 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 41,77 (C-1), 20,20 (C-2), 39,09 (C-3), 44,69 (C4), 49,05 (C-5), 30,68 (C-6), 78,70 (C-7), 49,80 (C-8), 50,62 (C-9), 40,47 (C-10), 19,51 (C-11), 32,93 (C-12), 39,46 (C-13), 37,17 (C-14), 42,58 (C-15), 44,66 (C16), 67,66 (C-17), 28,82 (C-18), 178,67 (C-19), 16,28 (C-20), 95,66 (C-1′), 74,07 (C-2′), 78,67 (C-3′), 71,14 (C-4′), 78,56 (C-5′) and 62,42 (C-6′) HR-ESI-MS: m/z 521,2732 [M+Na]+ Calcd for [C26H42O9Na]+: 521,2721 Molecular formula C26H42O9, M = 498 3.2.3 Compound 3: 7β,17-Dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β-Dglucopyranoside ester (new compound) White powder  25D : –38,5 (c 0,1, MeOH) Mp: 280-281oC H-NMR (500 MHz, CD3OD): δH 1,03 (1H, dd, J = 3,5 Hz, 13,5 Hz, Ha-1), 1,87 (1H, d, J = 13,5 Hz, Hb-1), 1,44 (1H, dt, J = 5,0 Hz, 10,0 Hz, Ha-2), 1,96 (1H, m, Hb-2), 1,12 (1H, dd, J = 4,0 Hz, 13,5 Hz, Ha-3), 2,22 (1H, m, Hb-3), 1,78 (1H, m, H-5), 1,96 (1H, m, Ha-6), 2,23 (1H, m, Hb-6), 3,59 (1H, br s, H-7), 1,39 (1H, d, J = 7,5 Hz, H-9), 1,58 (1H, m, Ha-11), 1,64 (1H, m, Hb-11), 1,52 (2H, m, H-12), 2,57 (1H, m, H-13), 1,42 (1H, dd, J = 7,5 Hz, 10,5 Hz, Ha-14), 2,06 (1H, d, J = 10,5 Hz, Hb-14), 5,81 (1H, s, H-15), 4,13 (2H, d, J = 1,0 Hz, H-17), 1,22 (3H, s, H-18), 1,02 (3H, s, H-20), 5,42 (1H, d, J = 7,5 Hz, H-1′), 3,38 (1H, m, H-2′), 3,42 (1H, m, H-3′), 3,40 (1H, m, H-4′), 3,39 (1H, H-5′), 3,71 (1H, dd, J = 4,0 Hz, 12,0 Hz, Ha-6′), 3,85 (1H, dd, J = 2,0 Hz, 12,0 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 41,72 (C-1), 20,17 (C-2), 39,10 (C-3), 44,71 (C4), 48,30 (C-5), 29,28 (C-6), 75,62 (C-7), 54,25 (C-8), 43,56 (C-9), 40,88 (C-10), 19,65 (C-11), 26,33 (C-12), 42,19 (C-13), 43,51 (C-14), 132,13 (C-15), 148,11 (C16), 61,21 (C-17), 28,79 (C-18), 178,58 (C-19), 16,10 (C-20), 96,64 (C-1′), 74,06 (C-2′), 78,70 (C-3′), 71,13 (C-4′), 78,69 (C-5′) and 62,41 (C-6′) HR-ESI-MS: m/z 519,2550 [M+Na]+ Calcd for [C26H40O9Na]+: 519,2565 Molecular formula C26H40O9, M = 496 3.2.4 Compound 4: 16α-Hydro-ent-kauran-17,19-dioic acid 17,19-di-O-β-Dglucopyranoside ester (new compound) White powder  25D : – 40 (c 0,1, MeOH) Mp: 191-192oC H-NMR (500 MHz, CD3OD): δH 0,87 (1H, m, Ha-1), 1,88 (1H, m, Hb-1), 1,52 (1H, m, Ha-2), 1,69 (1H, m, Hb-2), 1,11 (1H, m, Ha-3), 1,21 (1H, d, J = 14,0 Hz, Hb-3), 1,15 (1H, m, H-5), 1,88 (1H, m, Ha-6), 2,00 (1H, m, Hb-6), 1,57 (1H, m, Ha7), 1,96 (1H, m, Hb-7), 1,08 (1H, m, H-9), 1,43 (1H, m, Ha-11), 1,94 (1H, m, Hb11), 1,47 (1H, m, Ha-12), 1,71 (1H, m, Hb-12), 2,55 (1H, m, H-13), 1,17 (1H, m, Ha-14), 2,16 (1H, d, J = 12,0 Hz, Hb-14), 1,59 (1H, m, Ha-15), 1,97 (1H, m, Hb-15), 3,06 (1H, m, H-16), 1,24 (3H, s, H-18), 0,97 (3H, s, H-20), 5,53 (1H, d, J = 8,0 Hz, H1′), 3,35 (1H, m, H-2′), 3,48 (1H, m, H-3′), 3,42 (1H, m, H-4′), 3,40 (1H, H-5′), 3,71 (1H, dd, J = 2,0 Hz, 11,5 Hz, Ha-6′), 3,84 (1H, d, 11,5 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 41,44 (C-1), 19,15 (C-2), 39,04 (C-3), 45,11 (C4), 58,61 (C-5), 23,19 (C-6), 42,91 (C-7), 45,62 (C-8), 57,59 (C-9), 40,83 (C-10), 20,14 (C-11), 28,05 (C-12), 41,09 (C-13), 41,87 (C-14), 42,69 (C-15), 46,62 (C16), 175,32 (C-17), 29,04 (C-18), 178,43 (C-19), 16,36 (C-20), 95,61 (C-1′), 74,04 (C-2′), 78,68 (C-3′), 71,11 (C-4′), 78,68 (C-5′) and 62,40 (C-6′), 95,61 (C-1′′), 74,04 (C-2′′), 78,68 (C-3′′), 71,11 (C-4′′), 78,68 (C-5′′) and 62,34 (C-6′′) HR-ESI-MS: m/z 681,3095 Tính tốn lý thuyết cho công thức [C32H50O14Na]+: 681,3093 Molecular formula C32H50O14, M = 658 3.2.5 Compound 5: Paniculoside IV White powder  31D : +56 (c 0,1, MeOH) Mp: 192-193oC H-NMR (500 MHz, CD3OD): δH 0,91 (1H, m, Ha-1), 1,88 (1H, m, Hb-1), 1,62 (2H, m, H-2), 1,12 (1H, m, Ha-3), 2,21 (1H, m, Hb-3), 1,10 (1H, dd, J = 2,0 Hz, 12,0 Hz, H-5), 1,87 (1H, m, Ha-6), 2,01 (1H, m, Hb-6), 1,52 (1H, m, Ha-7), 1,68 (1H, m, Hb-7), 1,03 (1H, m, H-9), 1,42 (1H, m, Ha-11), 1,97 (1H, m, Hb-11), 1,50 (1H, m, Ha-12), 1,61 (1H, m, Hb-12), 2,01 (1H, br s, H-13), 1,72 (1H, m, H a-14), 2,02 (1H, m, Hb-14), 1,42 (1H, m, Ha-15), 1,58 (1H, m, H b-15), 3,62 (1H, m, Ha17), 3,71 (1H, m, Hb-17), 1,23 (3H, s, H-18), 0,99 (3H, s, H-20), 5,43 (1H, d, J = 8,0 Hz, H-1′), 3,40 (1H, m, H-2′), 3,42 (1H, m, H-3′), 3,90 (1H, m, H-4′), 3,43 (1H, H5′), 3,71 (1H, dd, J = 6,0 Hz, 11,5 Hz, Ha-6′), 3,85 (1H, dd, J = 2,5 Hz, 11,5 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 41,80 (C-1), 19,62 (C-2), 39,05 (C-3), 45,10 (C4), 58,53 (C-5), 23,16 (C-6), 43,32 (C-7), 45,77 (C-8), 57,32 (C-9), 40,88 (C-10), 20,10 (C-11), 27,16 (C-12), 46,20 (C-13), 38,07 (C-14), 53,69 (C-15), 82,99 (C16), 66,87 (C-17), 29,02 (C-18), 178,38 (C-19), 16,35 (C-20), 95,60 (C-1′), 74,03 (C-2′), 78,67 (C-3′), 71,11 (C-4′), 78,67 (C-5′) and 62,41 (C-6′) Molecular formula C26H42O9, M = 498 3.2.6 Compound 6: 16α,17-Dihydroxy-ent-kaurane Colorless needle  31D : -25 (c 0,1, CHCl3) Mp: 153-154oC H-NMR (500 MHz, CDCl3): δH 0,77 (1H, dd, J = 2,0 Hz, 12,0 Hz, H-5), 1,12 (1H, dt, J = 4,5 Hz, 14,0 Hz, , H-9), 1,59 (2H, br d, H-14), 1,43 (2H, m, H-15), 3,65 (1H, d, J = 11,0 Hz, Ha-17), 3,77 (1H, d, J = 11,0 Hz, Hb-17), 0,84 (3H, s, H18), 0,80 (3H, s, H-18) and 1,01 (3H, s, H-18) C-NMR (125 MHz, CDCl3): δC 42,02 (C-1), 18,28 (C-2), 42,06 (C-3), 33,26 (C4), 56,17 (C-5), 20,45 (C-6), 37,31 (C-7), 44,75 (C-8), 56,72 (C-9), 39,38 (C-10), 18,59 (C-11), 26,32 (C-12), 45,51 (C-13), 40,33 (C-14), 53,39 (C-15), 81,89 (C16), 66,38 (C-17), 33,56 (C-18), 21,55 (C-19) and 17,72 (C-20) 13 Molecular formula C20H34O2, M = 306 3.2.7 Compound 7: 16β,17-Dihydroxy-ent-kaurane Colorless needle  31D : -32 (c 0,1, CHCl3) Mp: 151-152oC H-NMR (500 MHz, CDCl3): δH 0,76 (1H, m, Ha-1), 1,82 (1H, d, J = 12,0 Hz, Hb1), 1,41 (1H, overlep, Ha-2), 1,63 (1H, m, Hb-2), 1,12 (1H, m, Ha-3), 1,46 (1H, m, Hb-3), 0,77 (1H, m, H-5), 1,25 (1H, m, Ha-6), 1,51 (1H, m, Hb-6), 1,37 (2H, m, H7), 1,12 (1H, H-9), 1,63 (1H, m, Ha-11), 1,87 (1H, m, Hb-11), 1,55 (1H, m, Ha-12), 1,75 (1H, m, Hb-12), 2,07 (1H, m, H-13), 0,99 (1H, br d, J = 12,0 Hz, Ha-14), 1,99 (1H, br d, J = 12,0 Hz, Hb-14), 1,38 (1H, m, Ha-15), 1,42 (1H, m, Hb-15), 3,38 (1H, d, J = 11,0 Hz, Ha-17), 3,46 (1H, d, J = 11,0 Hz, Ha-17), 0,84 (3H, s, H-18), 0,79 (3H, s, H-19) and 1,02 (3H, s, H-20) 13 C-NMR (125 MHz, CDCl3): δC 40,45 (C-1), 18,82 (C-2), 42,10 (C-3), 33,27 (C4), 56,21 (C-5), 20,05 (C-6), 41,93 (C-7), 43,58 (C-8), 57,06 (C-9), 39,43 (C-10), 18,64 (C-11), 26,76 (C-12), 40,89 (C-13), 38,32 (C-14), 52,84 (C-15), 79,82 (C16), 69,90 (C-17), 33,60 (C-18), 21,60 (C-19) and 17,57 (C-20) ESI-MS: m/z 329,2 [M+Na]+ Molecular formula C20H34O2, M = 306 3.2.8 Compound 8: 16β,17-Dihydroxy-ent-kauran-19-al Colorless needle  31D : – 45 (c 0,1, CHCl3) Melting point: 186-187oC H-NMR (500 MHz, CDCl3): δH 0,88 (1H, m, Ha-1), 1,90 (1H, m, Hb-1), 1,44 (1H, m, Ha-2), 2,07 (1H, m, Hb-2), 1,06 (1H, m, Ha-3), 2,12 (1H, m, Hb-3), 1,23 (1H, dd, J = 2,5 Hz; 12,5 Hz, H-5), 1,59 (H, m, Ha-6), 1,63 (1H, m, Hb-6), 1,52 (2H, m, H7), 1,16 (1H, m, H-9), 1,72 (1H, m, Ha-11), 1,89 (1H, m, H b-11), 1,49 (1H, m, Ha12), 1,84 (1H, m, Hb-12), 2,08 (1H, H-13), 1,13 (1H, m, H a-14), 2,00 (1H, dd, J = 2,0 Hz; 12,0 Hz, Hb-14), 1,45 (2H, m, H-15), 3,32 (1H, d, J = 11,5 Hz, Ha-17), 3,43 (1H, d, J = 11,5 Hz, Hb-17), 1,00 (3H, s, H-18), 9,75 (1H, d, J =1,5 Hz, H-19) and 0,93 (3H, s, H-20) 11 20,16 (C-11), 28,05 (C-12), 40,17 (C-13), 41,17 (C-14), 43,15 (C-15), 46,03 (C16), 175,91 (C-17), 23,43 (C-18) and 17,39 (C-20) Molecular formula C19H30O3, M = 306 3.2.13 Compound 13: (2E,4E,1′R,3′S,5′R,6′S)-Dihydrophaseic acid 1,3′-di-O-βD-glucopyranoside (new compound) White powder  25D : −25,0 (c 0,1, MeOH) Mp: 229-230oC CD (c = 1,5 ×10-5, MeOH), [θ] (λmax, nm) –52481 (237) H-NMR (500 MHz, CD3OD): δH 5,86 (1H, s, H-2), 8,05 (1H, d, J = 16,0 Hz, H4), 6,62 (1H, d, J = 16,0 Hz, H-5), 2,14 (3H, s, H-6), 1,82 (1H, m, Ha-2′), 2,02 (1H, m, Hb-2′), 4,28 (1H, m, H-3′), 1,82 (1H, m, Ha-4′), 2,21 (1H, m, Hb-4′), 3,78 (1H, d, J = 7,0 Hz, Ha-7′), 3,83 (1H, d, J = 7,0 Hz, Ha-7′), 0,96 (3H, s, H-8′), 1,19 (3H, s, H-9′), 5,54 (1H, d, J = 8,0 Hz, H-1′′), 3,17 (1H, t, J = 8,0 Hz, H-2′′), 3,30 (2H, m, H-3′′, H-4′′), 3,42 (1H, m, H-5′′), 3,70 (1H, m, Ha-6′′), 3,87 (1H, m, Hb-6′′), 4,38 (1H, d, J = 8,0 Hz, H-1′′′), 3,40 (1H, m, H-2′′′), 3,46 (1H, t, J = 8,0 Hz, H-3′′′), 3,39 (2H, m, H-4′′′, H-5′′′), 3,70 (1H, m, Ha-6′′) and 3,87 (1H, m, Hb-6′′) 13 C-NMR (125 MHz, CD3OD): δC 166,00 (C-1), 117,78 (C-2), 154,00 (C-3), 131,78 (C-4), 136,43 (C-5), 21,32 (C-6), 48,83 (C-1′), 42,86 (C-2′), 73,99 (C-3′), 42,83 (C-4′), 87,63 (C-5′), 83,25 (C-6′), 77,13 (C-7′), 16,34 (C-8′), 19,72 (C-9′), 95,44 (C-1′′), 73,86 (C-2′′), 78,04 (C-3′′), 71,66 (C-4′′), 78,79 (C-5′′), 62,76 (C-6′′), 103,04 (C-1′′′), 73,99 (C-2′′′), 77,96 (C-3′′′), 71,14 (C-4′′′), 78,08 (C-5′′′), 62,37 (C-6′′′) HR-ESI-MS: m/z 629,2431[M+Na]+ Tính tốn lý thuyết: [C27H42O15Na]+ 629,2416 Molecular formula C27H42O15, M = 606 3.2.14 Compound 14: (2E,4E,1′R,3′S,5′R,6′S)-Dihydrophaseic acid 3′-O-β-Dglucopyranoside White powder  31D : -110 (c 1,0, MeOH) Mp: 199-200oC H-NMR (500 MHz, CD3OD): δH 5,85 (1H, s, H-2), 7,78 (1H, d, J = 16,0 Hz, H4), 6,31 (1H, d, J = 16,0 Hz, H-5), 2,17 (3H, s, H-6), 1,96 (2H, m,H-2′), 4,28 (1H, m, H-3′) 1,19 (1H, m, H a-4′), 2,17 (1H, m, Hb-4′), 3,76 (1H, d, J = 7,5 Hz, Ha-7′), 3,82 (1H, dd, J = 2,0, 7,5 Hz, Ha-7′), 0,95 (3H, s, H-8′),1,18 (3H, s, H-9′), 4,37 (1H, d, J = 8,0 Hz, H-1′′), 3,16 (1H, dd, J = 8,0, 9,0 Hz, H-2′′), 3,30 (1H, m, H-3′′), 3,29 12 (1H, m, H-4′′), 3,39 (1H, m, H-5′′), 3,29 (1H, dd, J = 2,0, 12,0 Hz, Ha-6′′) and 3,90 (1H, dd, J = 5,0, 12,0 Hz, Hb-6′′) 13 C-NMR (125 MHz, CD3OD): δC 174,50 (C-1), 126,76 (C-2), 142,57 (C-3), 132,95 (C-4), 130,98 (C-5), 20,62 (C-6), 49,85 (C-1′), 42,89 (C-2′), 74,09 (C-3′), 42,83 (C-4′), 87,63 (C-5′), 83,16 (C-6′), 77,19 (C-7′), 16,34 (C-8′), 19,74 (C-9′), 103,16 (C-1′′), 75,13 (C-2′′), 78,09 (C-3′′), 71,69 (C-4′′), 77,94 (C-5′′) and 62,79 (C-6′′) Molecular formula C21H32O10, M = 444 3.2.15 Compound 15: Cucumegastigmane I Yellow wax  31D : + 35 (c 0,1, MeOH) H-NMR (500MHz, MeOD): δH 2,18 (1H, d, J = 17,0 Hz, Ha-2), 2,53 (1H, d, J = 17,0 Hz, Hb-2), 5,90 (1H, s, H-4), 5,92 (d, J = 16,0 Hz, H-7), 5,81 (1H, dd, J = 5,5 Hz; 16,0 Hz, H-8), 4,22 (1H, m, H-9), 3,53 (1H, dd, J = 5,5 Hz; 11,0 Hz, Ha-10), 3,48 (1H, dd, J = 7,0 Hz, 11,0 Hz, Hb-10), 1,06 (3H, s, H-11), 1,04 (3H, s, H-12) and 1,94 (3H, d, J = 1,0 Hz, H-13) 13 C-NMR (125 MHz, CD3OD): δC 42,38 (C-1), 50,72 (C-2), 201,25 (C-3), 127,16 (C-4), 167,30 (C-5), 80,13 (C-6), 132,54 (C-7), 132,43 (C-8), 73,62 (C-9), 67,28 (C-10), 23,43 (C-11), 24,50 (C-12) and 19,56 (C-13) Molecular formula C13H20O4, M = 240 3.2.16 Compound 16: Blumenol A White powder  31D : +25 (c 0,3, CHCl3) Mp: 112-113oC H-NMR (500 MHz, CDCl3): δH 2,22 (1H, d, J = 17,0 Hz, Ha-2), 2,43 (1H, d, J = 17,0 Hz, Hb-2), 5,91 (1H, br s, H-4), 5,77 (1H, d, J = 16,0 Hz, H-7), 5,86 (1H, dd, J = 5,0, 16,0 Hz, H-8), 4,41 (1H, m, H-9), 1,30 (1H, d, J = 6,5 Hz, H-10), 1,02 (3H, s, H-11), 1,08 (3H, s, H-12) and 1,90 (3H, s, H-13) 13 C-NMR (125 MHz, CDCl3): δC 41,16 (C-1), 49,70 (C-2), 198,06 (C-3), 126,88 (C-4), 162,60 (C-5), 79,04 (C-6), 135,74 (C-7), 129,02 (C-8), 68,02 (C-9), 23,74 (C-10), 22,90 (C-11), 24,03 (C-12) and 18,89 (C-13) Molecular formula C13H20O3, M = 224 3.2.17 Compound 17: Icariside B1 White powder  31D : -42 (c 0,05, MeOH) 13 Mp: 183-184oC H-NMR (500 MHz, CD3OD): δH 1,50 (1H, dd, J = 4,0 Hz, 12,0 Hz, Ha-2), 2,11 (d, J = 12,0 Hz, Hb-2), 4,37 (1H, tt, J = 4,0 Hz, 12,0 Hz, H-3), 1,47 (1H, dd, J = 4,0 Hz, 12,0 Hz, Ha-4), 2,39 (1H, d, J = 12,0 Hz, Hb-4), 5,86 (1H, s, H-8), 2,21 (3H, s, H-10), 1,18 (3H, s, H-11), 1,41 (3H, s, H-12), 1,42 (3H, s, H-13), 4,46 (1H, d, J = 7,5 Hz, H-1′), 3,18 (1H, dd, J = 7,5 Hz, 9,0 Hz, H-2′), 3,36 (2H, m, H-3′, H-4′), 3,40 (1H, m, H-5′), 3,71 (1H, dd, J = 5,0 Hz, 12,0 Hz, Ha-6′) and 3,90 (1H, d, J = 12,0 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 36,99 (C-1), 48,11 (C-2), 72,57 (C-3), 46,61 (C4), 72,37 (C-5), 120,08 (C-6), 211,48 (C-7), 101,15 (C-8), 200,86 (C-9), 26,53 (C10), 32,24 (C-11), 29,40 (C-12), 30,79 (C-13), 102,66 (C-1′), 75,07 (C-2′), 78,11 (C-3′), 71,63 (C-4′), 77,87 (C-5′) and 62,72 (C-6′) Molecular formula C19H30O8, M = 386 3.2.18 Compound 18: Icariside D2 White powder  31D : -52 (c 0,1, MeOH) Mp: 151-152oC H-NMR (500 MHz, CD3OD): δH 7,16 (2H, d, J = 8,0 Hz, H-2, H-6), 7,04 (2H, d, J = 8,0 Hz, H-3, H-5), 2,78 (2H, t, J = 7,5Hz, H-7), 3,73 (2H, m, H-8), 4,88 (1H, d, J = 7,5 Hz, H-1′), 3,46 (3H, m, H-2′, H-3′, H-5′), 3,43 (1H, m, H-4′), 3,72 (1H, dd, J = 5,0, 12,0 Hz, Ha-6′), 3,89 (1H, dd, J = 2,0, 12,0 Hz, Hb-6′), 13 C-NMR (125 MHz, CD3OD): δC 134,29 (C-1), 130,85 (C-2), 117,82 (C-3), 157,64 (C-4), 117,82 (C-5), 130,85 (C-6), 39,41 (C-7), 64,36 (C-8), 102,56 (C-1′), 74,95 (C-2′), 78,11 (C-3′), 71,42 (C-4′), 78,02 (C-5′) and 62,54 (C-6′) Molecular formula C14H20O7, M = 300 3.2.19 Compound 19: Icariside D2 6′-O-β-D-xylopyranoside White powder  31D : -32 (c 0,05, MeOH) Mp: 178-179oC H-NMR (500 MHz, CD3OD): δH 7,08 (2H, d, J = 8,0 Hz, H-2, H-6), 7,18 (2H, d, J = 8,0 Hz, H-3, H-5), 2,79 (2H, t, J = 7,0 Hz, H-7), 3,74 (2H, t, J = 7,0 Hz, H-8), 4,88 (1H, d, J = 7,5 Hz, H-1′), 4,36 (1H, d, J = 7,5 Hz, H-1′′) 13 C-NMR (125 MHz, CD3OD): δC 134,33 (C-1), 130,96 (C-2), 117,81 (C-3), 157,45 (C-4), 39,36 (C-7), 64,32 (C-8), 102,32 (C-1′), 74,93` (C-2′), 77,84 (C-3′), 71,44 (C-4′), 77,34 (C-5′), 69,67 (C-6′), 105,23 (C-1′′), 74,88 (C-2′′), 77,56 (C-3′′), 71,44 (C-4′′), 66,81 (C-5′′) 14 Molecular formula C18H26O11, M = 418 3.2.20 Compound 20: 3,4-Dimethoxyphenyl 1-O-β-D-glucopyranoside White powder  31D : -26 (c 0,4, MeOH) Mp: 195-196oC H-NMR (500 MHz, CD3OD): δH 6,84 (1H, d, J = 2,0 Hz, H-2), 6,87 (1H, d, J = 8,5 Hz, H-5), 6,68 (1H, dd, J = 2,0, 8,5 Hz, H-6), 3,80 (3H, s, 3-OCH3), 3,83 (3H, s, 4-OCH3), 4,80 (1H, d, J = 7,5 Hz, H-1′), 3,69 (1H, dd, J = 5,0, 12,5 Hz, Ha-6′), 3,92 (1H, dd, J = 2,5, 12,5 Hz, Hb-6′) 13 C-NMR (125 MHz, CD3OD): δC 153,96 (C-1), 104,13 (C-2), 146,06 (C-3), 151,16 (C-4), 113,99 (C-5), 109,36 (C-6), 57,16 (3-OCH3), 56,62 (4-OCH3), 103,47 (C-1′), 74,98 (C-2′), 78,05 (C-3′), 71,57 (C-4′), 78,22 (C-5′) and 62,64 (C-6′) Molecular formula C14H20O8, M = 316 3.2.21 Compound 21: 3,4-Dihydroxybenzoic acid White powder Mp: 190-191oC H-NMR (500 MHz, CD3OD): δH 7,45 (1H, s, H-2), 6,82 (1H, d, J = 8,0 Hz, H-5), 7,44 (1H, d, J = 8,0 Hz, H-6) 13 C-NMR (125 MHz, CD3OD): δC 123,84 (C-1), 117,76 (C-2), 145,99 (C-3), 151,33 (C-4), 115,73 (C-5), 123,84 (C-6), 170,51 (COOH) Molecular formula C7H6O4, M = 154 3.2.22 Compound 22: Squamocin M White wax H-NMR (500 MHz, CDCl3): δH 3,39 (2H, m, H-15, H-24), 3,86 (4H, m, H-16, H19, H-20, H-23), 0,87 (t, J = 6,5 Hz, H-34), 6,98 (br s, H-35), 4,99 (q, J = 6,5 Hz, H-36), 1,40 (d, J = 7,0 Hz, H-37) 13 C-NMR (125 MHz, CDCl3): δC 173,86 (C-1), 134,34 (C-2), 74,05 (C-15), 83,14 (C-16), 81,76 (C-19), 81,76 (C-20), 83,14 (C-23), 74,05 (C-24), 14,08 (C-34), 148,83 (C-35), 77,37 (C-36) and 19,20 (C-37) Molecular formula C37H66O6, M = 606 CHAPTER RESULTS AND DISCUSSIONS 4.1 Chemical structure of compounds This section presents the detailed results of spectral analysis and structure determination of 22 isolated compounds from A glabra including new compounds and 17 known compounds 15 * Summary isolation of 22 compounds from A glabra is shown in Figure 4.60 and table 4.23: 1: 7β,16α,17-Trihydroxy-entkauran-19-oic acid 2: 7β,17-Dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-D-glucopyranoside ester 3: 7β,17-Dihydroxy-ent-kaur-15-en19-oic acid 19-O-β-Dglucopyranoside ester 4: 16α-Hydro-ent-kauran-17,19-dioic acid 17,19-di-O-β-D-glucopyranoside ester 5: R = COOGlc Paniculoside IV 6: R = CH3 16α,17-Dihydroxy-ent-kaurane 7: R = CH3 16β,17-Dihydroxy-ent-kaurane 8: R = CHO 16β,17-Dihydroxy-ent-kauran-19-al 9: R = COOH 16β,17-Dihydroxy-ent-kauran-19-oic acid 10: R = H annoglabasin E 11: R = Ac annoglabasin B 12: 19-nor-ent-kauran-4α-ol-17-oic acid 13: (2E,4E,1′R,3′S,5′R,6′S)- 14: (2E,4E,1′R,3′S,5′R,6′S)-Dihydrophaseic 16 Dihydrophaseic acid 1,3′-di-O-β-Dglucopyranoside 15: Cucumegastigmane I 18: R = H Icariside D2 19: R = Xylopyranosyl Icariside D2 6′-O-β-Dxylopyranoside acid 3′-O-β-D -glucopyranoside 16: Blumenol A 20: 3,4-Dimethoxyphenyl 1O-β-D-glucopyranoside 17: Icariside B1 21: 3,4-Dihydroxybenzoic acid 22: Squamocin M * Below details the method for determining the structure of a new compound 4.1.4 Compound 4: 16α-Hydro-ent-kauran-17,19-dioic acid 17,19-di-O-β-Dglucopyranoside ester (new) Figure 4.24 The chemical structure of and the reference compound Compound was isolated as a white amorphous powder and its molecular was determined to be C32H50O14 by HR-ESI-MS at m/z 681.3095 (Calcd for C32H50O14Na: 681.3093) The 1H-NMR spectrum of showed the signals for two tertiary methyl groups at δH 0.97 (3H, s) and 1.24 (3H, s), assigned to entkauranes tructure; two anomeric protons at δ H 5.43 (d, J = 8.0 Hz) and 5.53 (d, J = 8.0 Hz), suggested the presence of two sugar moieties The 13C-NMR and DEPT spectra of revealed signals for 32 carbons including two carbonyl, three quaternary, fourteen methine, eleven methylene, and two methyl carbons The H- and 13C-NMR data of were very similar to those of 16α-hydro-ent-kauran17,19-dioic (4a) acidexcept for the addition of two sugar moieties at C-17 and C- 17 19 [71] The HMBC correlations between H-18(δH1.24) and C-3 (δC 39.0)/C-4 (δC 45.1)/C-5 (δC 58.6)/C-19 (δC 178.4) suggested the presence of both methyl and carboxyl groups at C-4 The HMBC correlations from H-13 (δH 2.55)/H-15 (δH 1.59 and 1.97)/H-16 (δH 3.06) to C-17 (δC175.3) confirmed the position of carboxyl group at C-16 The HMBC correlations between H-1′ (δH 5.53) and C17 (δC 175.3); H-1″ (δC 5.43) and C-19 (δC 178.4) confirmed the positions of two glucopyranosyl moieties at C-17 and C-19 The observation of NOESY correlation between H-18 (δH 1.24) and H-5 (δH 1.15) as well as no observation of NOESY correlation between H-18 (δH 1.24) and H-20 (δH 0.97) confirmed the β configuration of the methyl group at C-4 Moreover, the NOESY correlations of H-16 (δH 3.06) and H-13 (δH 2.55); H16 (δH 3.06) and Hα-15 (δH 1.59); H-9 (δH 1.08) and Hβ-15 (δH 1.97) were observed confirming the α configuration of H-16 Acid hydrolysis of provided the D-glucose (identified as TMS derivative) [17] In addition,the coupling constants of glcH-1′/glc H-2′; glc H-1″/glc H-2″,J = 8.0 Hz indicated these protons were allaxial orientation Consequently, compound was elucidated to be 16α-hydro-entkauran-17,19-dioic acid 17,19-di-O-β-D-glucopyranoside ester, a new compound named annoglabasin H Figure 4.25: The important HMBC, COSY, and NOESY correlations of Table 4.4 NMR data for compound and the reference compound C δC(#) DEPT δC(@) δH(@) 40,0 CH2 41,44 0,87(m)/1,88 (m) 20,3 CH2 19,15 1,52 (m)/1,69 (m) 41,1 CH2 39,04 1,11 (m) 1,21 (d, J = 14,0 Hz) 44,8 C 45,11 58,2 CH 58,61 1,15 (m) 23,5 CH2 23,19 1,88 (m)/2,00 (m) 41,6 CH2 42,91 1,57 (m)/1,96 (m) 45,6 C 45,62 57,8 CH 57,59 1,08 (m) 18 10 40,8 11 19,3 12 28,5 13 40,5 14 43,1 15 42,0 16 46,7 17 178,1 18 29,5 19 180,6 20 16,8 17-O-Glc 1' 2' 3' 4' 5' 6' 19-O-Glc 1'' 2'' 3'' 4'' 5'' 6'' #δ C C CH2 CH2 CH CH2 CH2 CH C CH3 C CH3 40,83 20,14 28,05 41,09 41,87 42,69 46,62 175,32 29,04 178,43 16,36 1,43 (m)/1,94 (m) 1,47 (m)/1,71 (m) 2,55 (m) 1,17 (m)/2,16 (d, J = 12,0 Hz) 1,59 (m)/1,97 (m) 3,06 (m) 1,24 (s) 0,97 (s) CH CH CH CH CH CH2 95,61 74,04 78,68 71,11 78,68 62,40 5,53 (d, J = 8,0 Hz) 3,35 (m) 3,48 (m) 3,42 (m) 3,40 (m) 3,71 (dd, J = 2,0 Hz, 11,5 Hz) 3,84 (d, J = 11,5 Hz) CH CH CH CH CH CH2 95,61 74,04 78,68 71,11 78,68 62,34 5,43 (d, J = 8,0 Hz) 3,38 (m) 3,48 (m) 3,42 (m) 3,40 (m) 3,71 (dd, J = 2,0 Hz, 11,5 Hz) 3,84 (d, J = 11,5 Hz) of 16α-hydro-ent-kauran-17,19-dioic acid (4a) in CD3OD [71], @ in CD3OD Figure 4.26 HR-ESI-MS spectrum of Figure 4.27 1H-NMR spectrum of 19 Figure 4.28 13 C-NMR spectrum of Figure 4.30 HSQC spectrum of Figure 4.32 COSY spectrum of 4.2 Evaluation of biological activities Figure 4.29 DEPT spectrum of Figure 4.31 HMBC spectrum of Figure 4.33 NOESY spectrum of t 4.2.1 Cytotoxic activity of crude fractions Cytotoxic screening results indicated that the methanolic extract of the leaves and fruits of A glabra showed cytotoxic effect toward both of LU-1 and KB cell lines with the IC50 values ranging from 12,57 3,62 ữ 38,19 1,23 àg/mL Of these, cytotoxic effects of extract from the fruits exhibited stronger than that of the leaves Moreover, dichloromethane and water fractions from A glabra fruits showed potent cytotoxic effects with the IC50 in the range 1,08 ± 0,14 ÷ 3,70 ± 0,98 µg/mL Therefore, they were selected for chemical studies to clarify active constituents 20 Table 4.24 Cytotoxicity of extracts/fractions from A glabra Extracts/fractions IC50 (µg/mL) LU-1 Part used Leaves Fruits 32,43 ± 2,12 14,59 ± 3,16 MeOH extract 25,15 ± 2,70 16,15 ± 1,04 n-Hexane fraction 9,02 ± 1,34 2,21 ± 0,15 Dichloromethane fraction > 100 > 100 Ethyl acetate fraction > 100 3,47 ± 0,29 Water fraction 0,34 ± 0,09 Ellipticine 4.2.2 Cytotoxic effects of isolated compounds KB Leaves Fruits 38,19 ± 1,23 12,57 ± 3,62 30,35 ± 1,08 13,10 ± 1,23 6,85 ± 0,11 1,08 ± 0,14 > 100 > 100 > 100 3,70 ± 0,98 0,36 ± 0,08 Among 12 ent-kaurane type compounds isolated from A glabra, compounds 3, and displayed strong cytotoxic activity towards four human cancer cell lines including LU-1, MCF-7, SK-Mel2 and KB Their IC50 values were in the range of 0,65 ữ7,39 àM; megastigmanes 14 and 15, phenolic 18 and acetogenin 22 IC50 values ranging from 2,79 ữ11,17 àM Compounds 1, 2, 5, 7, 19 and 17 showed moderate cytotoxic activity Remaining compounds were inactivity in our tested conditions In addition, our research program on finding anti-leukemia agents, compounds 7, 8, 18 and 22 were found potent anti-proliferation of HL-60 cells Their IC50 values were in range of 6,94 ÷ 9,38 µM in comparison with positive control mitoxantrone (IC50: 6,8 µM) Those of compounds were further evaluated their effects on the HEL-299 normal cell line Our results indicated that, only compounds 18 and 22 didn’t show toxicity on HEL-299 normal cells at their corresponding IC50 concentrations (cells viability were 93.25 and 94.69 %, respectively) Thus, both of compounds 18 and 22 were further investigated whether their cytotocix effects might come from induction of apoptosis Comp Table 4.25 Cytotoxic activity of compounds 1-22 % ss(*) IC50 (µM) LU-1$ MCF-7$ SK-Mel2$ KB$ HL-60# HEL-299# 55,68 62,36 53,66 58,38 55,76 88,47 94,10 61,85 80,80 >100 0,65 4,46 1,79 1,73 58,79 4,06 4,64 3,68 4,42 68,22 67,07 77,97 84,58 80,66 >100 1,70 7,39 6,44 3,69 35,64 25,59 55,46 93,17 23,27 6,94 >100 78,10 >100 >100 75,72 >100 9,38 >100 82,17 21 10 11 12 13 14 15 16 17 18 19 20 21 22 Ellipticine Mitoxantrone 74,58 >100 >100 >100 16,72 5,33 2,79 >100 92,67 6,30 80,50 >100 >100 6,75 3,50 >100 >100 >100 >100 37,94 7,28 11,17 >100 80,91 10,07 74,43 >100 >100 10,61 3,73 >100 >100 >100 >100 >100 >100 >100 >100 18,96 19,93 5,36 6,71 5,13 5,58 >100 >100 90,05 117,07 8,50 6,63 93,97 >100 >100 >100 >100 >100 9,57 7,93 3,35 4,04 56,54 66,87 32,61 30,17 25,82 >100 >100 >100 >100 9,02 49,09 32,23 64,61 8,72 >100 93,25 >100 94,69 6,8 *HEL-299 normal cell viability at IC50 concentration of tested compounds, #Experiments were performed at College of Pharmacy, Chungnam National University, Korea; $Experiments were performed at Biological Testing Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology Treatment of compounds 18 and 22 increased the sub-G1 hypodiploid cell population in a time-dependent manner The Bcl-2 family is separated into antiapoptotic proteins, such as Bcl-2, and pro-apoptotic proteins, such as Bax The Bax induces apoptosis by the releasing of cytochrome c from mitochondria In contrast, Bcl-2 inhibits the releasing of cytochrome c During apoptosis, released cytochrome c induces the cleavage of caspase-9, which is followed by the cleavage of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) Therefore, to investigate the possible mechanism underlying the induction of apoptosis, we examined the levels of apoptosis-related proteins When treated with compounds 18 (9.0 µM) and 22 (8.7 µM) for 24 and 48 h, we could observe the alteration of expression of apoptosis-related proteins such as up-regulation of Bax, down-regulation of Bcl-2, cleaves of caspase-3, and cleaves of PARP These results indicated that compounds 18 and 22 induced apoptosis via alteration of expression of apoptosis-related proteins in HL-60 cells The PI3K/AKT signaling pathways regulate cell survival, cell growth, and apoptosis Especially, activated AKT contributes the survival and the growth of cancer cell via c-myc The c-myc is frequently overexpressed in various types of 22 tumors In order to investigate intracellular signaling induced by compounds 18 and 22, we analyzed the phosphorylation of AKT and the level of c-myc by Western blotting As the results, the treatment of compounds 18 and 22 decreased phosphorylation of AKT in conditions that could induce apoptosis in HL-60 cells Furthermore, that was also accompanied by down-regulation of c-myc These findings provide evidence demonstrating that the apoptosis-inducing effects of compounds 18 and 22 are mediated by down-regulation of p-AKT and c-myc 4.2.3 Anti-inflammatory activity of isolated compounds At a concentration of 30 µM, compounds 1, 3, 8, 12, and 13 potentially inhibited NO production Their inhibitory effects were found better than that of positive control, dexamethasone Particularly, compound strongly inhitbited NO producion in LPS stimulated RAW 264.7 macrophages with a IC 50 value of 0.01 µM Compounds 1, 8, 12 and 13 inhibited NO production with their IC50 values of 0.39, 0.32, 0.10 and 0.42 µM, respectively Table 4.26 Inhibitory effects of isolated compounds on LPS stimulated NO production in RAW 264.7 macrophages Comp IC50 (µM) Comp IC50 (µM) 10 11 0,39 12 >30 13 0,01 14 3,1 15 17,06 16 6,20 17 0,32 18 12,1 20 >30 22 >30 Dexamethasone 0,10 0,42 14,7 >30 16,3 >30 1,21 1,84 3,21 0,80 CONCLUSIONS From A glabra species, 22 compounds were isolated and determined their chemical structures as listed belowing:  new compounds:  new diterpenoids belonging ent-kaurane-type: 7β,16α,17-trihydroxyent-kauran-19-oic acid (1), 7β,17-dihydroxy-16α-ent-kauran-19-oic acid 19-O-β-D-glucopyranoside ester (2), 7β,17-dihydroxy-ent-kaur- 23 15-en-19-oic acid 19-O-β-D-glucopyranoside ester (3), and 16α-hydroent-kauran-17,19-dioic acid 17,19-di-O-β-D-glucopyranoside ester (4);  a new megastigmane: (2E,4E,1′R,3′S,5′R,6′S)-dihydrophaseic acid 1,3′di-O-β-D-glucopyranoside (13)  17 known compounds:  ent-kaurane-type diterpenes: paniculoside IV (5), 16α,17-dihydroxyent-kaurane (6), 16β,17-dihydroxy-ent-kaurane (7), 16β,17-dihydroxyent-kauran-19-al (8), 16,17-dihydroxy-ent-kauran-19-oic acid (9), annoglabasin E (10), annoglabasin B (11), and 19-nor-ent-kauran-4ol-17-oic acid (12);  megastigmanes: (2E,4E,1′R,3′S,5′R,6′S)-dihydrophaseic acid 3′-O-βD-glucopyranoside (14), cucumegastigmane I (15), blumenol A (16), and icariside B1 (17);  phenolic compounds: icariside D2 (18), icariside D2 6′-O-β-Dxylopyranoside (19), 3,4-dimethoxyphenyl 1-O-β-D-glucopyranoside (20), and 3,4-dihydroxybenzoic acid (21);  an acetogenin compound: squamocin M (22)  compounds (5, 14, 15, 17-20) were first isolated from Annona genus and compounds (6, 21) were first isolated from A glabra species The cytotoxic activity of methanol extracts from leaves and fruits of A glabra and their fractions (n-hexane, dichoromethane, ethyl acetate and water fractions) toward KB and LU-1 human cancer cell lines were screened Result showed that methanol extract of the leaves and fruits of A glabra exhibited cytotoxic effects on tested cancer cells with the IC50 values in the range of 12.57 ± 3.62 ÷ 38.19 ± 1.23 µg/mL In addition, both of dichloromethane fraction and water layer from A glabra fruits displayed strong cytotoxic effects with the IC50 values from 1.08 ± 0.14 to 3.70 ± 0.98 µg/mL Those are important evidences for chemical study of A glabra All of isolated compounds were evaluated their cytotoxic effects towards four human cancer cell lines including LU-1, MCF-7, SK-Mel2, and KB Among them, three ent-kaurane-type diterpenes 3, and exhibited strong cytotoxic effects with the IC50 values ranging of 0,65 ữ7,39 àM; two megastigmanes 14 and 15, a phenolic 18 and an acetogenin 22 were also exhibited potent cytotoxic activity with the IC50 values from 2.79 to 11.17 µM Compounds 18 and 22 not only displayed cytotoxic activity toward HL-60 cancer cells (IC50 9,02 and 8,72 µM, respectively) but also non-toxified with 24 normal cells (HEL-299) Further studies indicated that both of those compounds induced apoptosis via alteration of expression of apoptosis-related proteins in HL60 cells The anti-inflamatory activity of isolated compounds 1-22 were also evaluated via inhibiting NO production in LPS stimulated RAW 264.7 macrophages Compounds 1, 3, 8, 12, and 13 were found to inhibit NO production better than that of positive control dexamethasone (IC50 values in range of 0.01 ữ 0.42 àM) RECOMENDATIONS Our results suggested that: Two new ent-kaurane-type diterpenes 3, showed potent anti-proliferation on all of four human cancer cell lines (LU-1, MCF-7, SK-Mel2 and KB) Further studies to clarify activity mechanism and pharmacoglogical study of those compounds should be carried Compounds 1, 3, 8, and 12 strongly inhibited NO production in LPS-stimulated RAW 264.7 macrophages (better than positive control in our tested experiments) Their IC50 values were found in range of 0.42 – 0.01 µM The results suggested that ent-kaurane-type diterpenes might be played important roles in the antiinflammatoty activity of A glabra Further experiments should be required to aprove anti-inflammatory activity of compounds 1, 3, 8, and 12 before in vivo studies 25 PUBLICATIONS WITHIN THE SCOPE OF THESIS Nguyen Xuan Nhiem, Nguyen Thi Thu Hien, Bui Huu Tai, Hoang Le Tuan Anh, Dan Thi Thuy Hang, Tran Hong Quang, Phan Van Kiem, Chau Van Minh, Wonmin Ko, Seungjun Lee, Hyuncheol Oh, Seung Hyun Kim, and Young Ho Kim New ent-kauranes from the fruits of Annona glabra and their inhibitory nitric oxide production in LPS-stimulated RAW264.7 macrophages Bioorganic and Medicinal Chemistry Letters, 2015, 25, 254-258 Hoang Le Tuan Anh, Nguyen Thi Thu Hien, Dan Thi Thuy Hang, Tran Minh Ha, Nguyen Xuan Nhiem, Truong Thi Thu Hien, Vu Kim Thu, Do Thi Thao, Chau Van Minh and Phan Van Kiem ent-Kaurane Diterpenes from Annona glabra and Their Cytotoxic Activities Natural Products Communications, 2014, 9, 16811682 Nguyen Thi Thu Hien, Nguyen Xuan Nhiem, Duong Thi Hai Yen, Dan Thi Thuy Hang, Bui Huu Tai, Tran Hong Quang, Hoang Le Tuan Anh, Phan Van Kiem, Chau Van Minh, Eun-Ji Kim, Seung Hyun Kim, Hee Kyoung Kang, and Young Ho Kim Chemical constituents of the Annona glabra fruit and their cytotoxic activity Pharmaceutical Biology, 2015, 53 (11), 1602-1607 Nguyễn Thị Thu Hiền, Phạm Hải Yến, Dương Thị Dung, Đan Thị Thúy Hằng, Nguyễn Xuân Nhiệm, Trần Minh Đức, Ninh Khắc Bản, Hoàng Lê Tuấn Anh, Châu Văn Minh, Phan Văn Kiệm Các hợp chất đitecpenoit phân lập từ Na biển Annona glabra (Phần 1).Tạp chí Hóa học, 2015, 53 (3), 392-395 Nguyễn Thị Thu Hiền, Đan Thị Thúy Hằng, Dương Thị Dung, Nguyễn Thị Cúc, Dương Thị Hải Yến, Hoàng Lê Tuấn Anh, Nguyễn Xuân Nhiệm, Châu Văn Minh, Phan Văn Kiệm Các hợp chất đitecpenoit phân lập từ loài Na biển Annona glabra (Phần 2) Tạp chí Hóa học, 2015, 53 (4), 526-530