STUDY ON CHEMICAL COMPOSITIONS AND BIOLOGICAL ACTIVITIES OF annona glabra l

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 AN

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:

1 Prof Dr Phan Van Kiem

Institute of Marine Biochemistry - Vietnam Academy of Science and Technology

2 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

1 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,

ent-kauranes, 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."

2 Subject and contents of the thesis

- Subject is the leaves and fruits of Annona glabra

- The aims of the thesis:

1 Extraction and isolation of secondary metabolites from A glabra;

2 Determination of chemical structure of the isolated compounds;

3 Evaluation of cytotoxic activity of isolated compounds;

4 Evaluation of anti-inflammatory activity of isolated compounds

3 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-D-glucopyranoside (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 D-xylopyranoside (19), 3,4-dimethoxyphenyl 1-O-β-D-glucopyranoside (20) were isolated from the Annona genus for the first time;

6′-O-β-3.1.3 Two compounds, 16α,17-dihydroxy-ent-kaurane (6) and

3,4-dihydroxybenzoic 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 6 showed the highest cytotoxic activity with their IC50 values ranging from 0.65 ÷7.39 µM Megastigmane type compounds 14 and 15 exhibited cytotoxic effects with the IC50 values ranging from 2.79 ÷11.17 µM on four cancer cell lines Phenolic 18 and acetogenin 22 (squamocin M) not only displayed cytotoxic activity toward all tested cancer cells (IC50 6.30 ÷10.61 µM) but also non-toxified 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 ÷ 0.42 µM

4 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 high-resolution 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

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 acid (new compound)

(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, Ha-11), 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 1), 20,29 2), 34,23 3), 44,25 4), 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 (C-16), 66,71 (C-17), 29,27 (C-18), 182,00 (C-19), 16,14 (C-20)

6′), 3,86 (1H, d, J = 11,5 Hz, Hb-6′)

13 C-NMR (125 MHz, CD3OD): δC 41,77 1), 20,20 2), 39,09 3), 44,69 4), 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 (C-16), 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′)

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 1), 20,17 2), 39,10 3), 44,71 4), 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 (C-16), 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′)

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, Hb-11), 1,47 (1H, m, Ha-12), 1,71 (1H, m, Hb-12), 2,55 (1H, m, H-13), 1,17 (1H, m,

20,14 11), 28,05 12), 41,09 13), 41,87 14), 42,69 15), 46,62 16), 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′′).

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, Ha-14), 2,02 (1H, m, Hb-14), 1,42 (1H, m, Ha-15), 1,58 (1H, m, Hb-15), 3,62 (1H, m, Ha-17), 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, 1′), 3,40 (1H, m, 2′), 3,42 (1H, m, 3′), 3,90 (1H, m, 4′), 3,43 (1H,

H-5′), 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 1), 19,62 2), 39,05 3), 45,10 4), 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 (C-16), 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′)

13 C-NMR (125 MHz, CDCl3): δC 42,02 1), 18,28 2), 42,06 3), 33,26 4), 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 (C-16), 66,38 (C-17), 33,56 (C-18), 21,55 (C-19) and 17,72 (C-20).

-Hb-3), 0,77 (1H, m, H-5), 1,25 (1H, m, Ha-6), 1,51 (1H, m, Hb-6), 1,37 (2H, m, 7), 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

H-(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 1), 18,82 2), 42,10 3), 33,27 4), 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 (C-16), 69,90 (C-17), 33,60 (C-18), 21,60 (C-19) and 17,57 (C-20)

H-2,0 Hz; 1H-2,0 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)

13 C-NMR (125 MHz, CDCl3): δC 41,02 1), 19,47 2), 35,28 3), 49,84 4), 57,90 (C-5), 20,71 (C-6), 43,08 (C-7), 44,69 (C-8), 57,19 (C-9), 40,65 (C-10), 19,82 (C-11), 27,77 (C-12), 42,20 (C-13), 39,41 (C-14), 53,09 (C-15), 80,74 (C-16), 70,61 (C-17), 24,57 (C-18), 207,87 (C-19) and 16,85 (C-20)

-3), 1,34 (1H, t, J = 3,5 Hz, Hb-3), 0,99 (1H, dd, J = 5,0 Hz, 12,0 Hz, H-5), 1,62

(1H, m, Ha-6), 2,43 (1H, m, Hb-6), 1,52 (1H, m, Ha-7), 1,95 (1H, m, Hb-7), 0,99 (1H, m, H-9), 1,57 (1H, m, Ha-11), 1,65 (1H, m, Hb-11), 1,52 (1H, m, Ha-12), 1,62 (1H, m, Hb-12), 2,52 (1H, br s, H-13), 0,86 (1H, dd, J = 13,5, 3,5 Hz, Ha-14), 1,10

(1H, dd, J = 13,5, 3,5 Hz, Hb-14), 1,54 (1H, m, Ha-15), 1,91 (1H, m, Ha-15), 2,92

(1H, dt, J = 12,0 Hz, 6,0 Hz, H-16); 0,95 (3H, s, H-18), 3,34 (1H, d, J = 11,0 Hz, Ha

-19), 3,73 (1H, d, J = 11,0 Hz, Hb-19), 1,05 (3H, s, H-20)

13 C-NMR (125 MHz, CD3OD): δC 41,72 1), 19,06 2), 36,73 3), 39,78 4), 58,25 (C-5), 21,62 (C-6), 43,19 (C-7), 45,54 (C-8), 59,00 (C-9), 40,41 (C-10), 19,32 (C-11), 28,50 (C-12), 41,08 (C-13), 41,56 (C-14), 43,34 (C-15), 46,71 (C-16), 178,42 (C-17), 27,83 (C-18), 65,16 (C-19) and 18,79 (C-20)

-Hz, 13,0, Hb-15), 2,94 (1H, m, H-16); 0,94 (3H, s, H-18), 3,88 (1H, d, J = 11,0 Hz, Ha

-19), 4,20 (1H, d, J = 11,0 Hz, Hb-19), 1,01 (3H, s, H-20), 2,04 (3H, s, H-22)

13 C-NMR (125 MHz, CD3OD): δC 40,23 1), 18,20 2), 36,33 3), 37,04 4), 56,71 (C-5), 20,64 (C-6), 41,87 (C-7), 44,35 (C-8), 57,17 (C-9), 39,13 (C-10), 18,01 (C-11), 27,31 (C-12), 39,80 (C-13), 40,55 (C-14), 41,69 (C-15), 45,29 (C-16), 179,95 (C-17), 27,53 (C-18), 67,19 (C-19), 18,07 (C-20), 171,47 (C-21) and 21,02 (C-22)

m, Hb-11), 1,53 (1H, m, Ha-12), 2,05 (1H, m, Hb-12), 2,74 (1H, m, H-13), 1,52 (1H,

m, Ha-14), 2,27 (1H, m, Hb-14), 1,52 (1H, m, Ha-15), 1,92 (1H, m, Hb-15), 3,15 (1H, m, H-16), 1,27 (3H, s, H-18) and 0,95 (3H, s, H-20)

13 C-NMR (125 MHz, C5D5N): δC 39,98 1), 18,66 2), 43,68 3), 71,02 4), 58,10 (C-5), 19,79 (C-6), 41,03 (C-7), 44,63 (C-8), 57,41 (C-9), 40,05 (C-10),

20,16 11), 28,05 12), 40,17 13), 41,17 14), 43,15 15), 46,03 16), 175,91 (C-17), 23,43 (C-18) and 17,39 (C-20)

H-m, H-3′) 1,19 (1H, H-m, Ha-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

(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

3,48 (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