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 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.

1
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, 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."
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-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


2
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 IC 50 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 (IC 50 6.30 ÷10.61 µM) 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 ÷ 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.


3
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


4

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.

1
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 =


5
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.
1

3.2.3. Compound 3: 7β,17-Dihydroxy-ent-kaur-15-en-19-oic acid 19-O-β-Dglucopyranoside ester (new compound)
White powder.


6

 25D : –38,5 (c 0,1, MeOH).
Mp: 280-281oC.
1
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.
1
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),


7
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 toán 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.
1

3.2.6. Compound 6: 16α,17-Dihydroxy-ent-kaurane
Colorless needle.

 31D : -25 (c 0,1, CHCl3)
Mp: 153-154oC.
1
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).

8
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.
1
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).
1


9
C-NMR (125 MHz, CDCl3): δC 41,02 (C-1), 19,47 (C-2), 35,28 (C-3), 49,84 (C4), 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 (C16), 70,61 (C-17), 24,57 (C-18), 207,87 (C-19) and 16,85 (C-20).
ESI-MS: m/z 319,2 [M-H]-.
Molecular formula C20H32O3, M = 320.
13

3.2.9. Compound 9: 16β,17-Dihydroxy-ent-kauran-19-oic acid
Colorless needle.

 31D : – 57 (c 0,01, MeOH).
Mp: 296-297oC.
1
H-NMR (500 MHz, C5D5N): δH 3,77 (1H, d, J = 10,5 Hz, Ha-17), 3,84 (1H, d, J =
10,5 Hz, Ha-17), 1,34 (3H, s, H-18) and 1,21 (3H, s, H-20).
13
C-NMR (125 MHz, C5D5N): δC 41,16 (C-1), 19,85 (C-2), 38,73 (C-3), 43,91 (C4), 57,07 (C-5), 22,48 (C-6), 42,50 (C-7), 43,96 (C-8), 56,72 (C-9), 40,07 (C-10),
19,43 (C-11), 27,57 (C-12), 41,67 (C-13), 38,58 (C-14), 53,38 (C-15), 79,81 (C16), 70,49 (C-17), 29,37 (C-18), 180,18 (C-19) and 15,96 (C-20).
ESI-MS: m/z 335,2 [M-H]-.
Molecular formula C20H32O4, M = 336.
3.2.10. Compound 10: Annoglabasin E
White powder.

 31D : – 70 (c 0,07, MeOH).
Mp: 203-204oC.
H-NMR (500 MHz, CD3OD): δH 1,87 (1H, m, Ha-1), 2,11 (1H, d, J = 11,5 Hz, Hb1), 1,41 (1H, m, Ha-2), 1,68 (1H, m, Hb-2), 0,93 (1H, dd, J = 3,5 Hz, 13,5 Hz, Ha3), 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, H a-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, Ha19), 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 (C-1), 19,06 (C-2), 36,73 (C-3), 39,78 (C4), 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 (C16), 178,42 (C-17), 27,83 (C-18), 65,16 (C-19) and 18,79 (C-20).
1


10
ESI-MS: m/z 319 [M-H]−
Molecular formula C20H32O3, M = 320.
3.2.11. Compound 11: Annoglabasin B
White powder.

 31D : – 40 (c 0,1, CHCl3).
Mp: 166-167oC.
H-NMR (500 MHz, CD3OD): δH 0,78 (1H, dd, J = 3,5 Hz, 13,0 Hz, Ha-1), 1,81
(1H, br d, J = 13,0, Hb-1), 1,40 (1H, m, Ha-2), 1,52 (1H, m, Hb-2), 0,98 (1H, m, Ha3), 1,68 (1H, m, Hb-3), 0,95 (1H, m, H-5), 1,33 (1H, m, Ha-6), 1,67 (1H, m, Hb-6),
1,50 (2H, m, H-7), 1,08 (1H, m, H-9), 1,52 (1H, m, Ha-11), 1,63 (1H, m, Hb-11),
1,51 (1H, m, Ha-12),1,66 (1H, m, Hb-12), 2,57 (1H, m, H-13), 1,05 (1H, m, Ha-14),
2,04 (1H, m, Hb-14), 1,55 (1H, dd, J = 6,5 Hz, 13,0, Ha-15), 1,90 (1H, dd, J = 6,5
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, Ha19), 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 (C-1), 18,20 (C-2), 36,33 (C-3), 37,04 (C4), 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 (C16), 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).
ESI-MS: m/z 361 [M-H]-.
Molecular formula C22H34O4, M = 362.
1

3.2.12. Compound 12: 19-nor-ent-kauran-4α-ol-17-oic acid
White powder.

 31D : -61 (c 0,1, MeOH).
Mp: 278-279oC.
1
H-NMR (500 MHz, C5D5N): δH 1,76 (1H, m, Ha-1), 2,28 (1H, m, Hb-1), 1,52 (1H,
m, Ha-2), 2,02 (1H, m, Hb-2), 1,62 (1H, m, Ha-3), 1,93 (1H, m, Hb-3), 1,38 (1H, m,
H-5), 1,52 (1H, m, Ha-6), 2,20 (1H, m, Hb-6), 1,12 (1H, dd, J = 4,0; 6,5 Hz, Ha-7),
2,07 (1H, m, Hb-7), 1,18 (1H, d, J = 7,5 Hz, H-9), 1,51 (1H, m, Ha-11), 2,25 (1H,
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 (C-1), 18,66 (C-2), 43,68 (C-3), 71,02 (C4), 58,10 (C-5), 19,79 (C-6), 41,03 (C-7), 44,63 (C-8), 57,41 (C-9), 40,05 (C-10),


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).
1
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 toán 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.
1
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.
1

3.2.16. Compound 16: Blumenol A
White powder.

 31D : +25 (c 0,3, CHCl3)
Mp: 112-113oC.
1
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.
1

 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.
1

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′′).
1


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.
1
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.
1
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.
1
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 4. 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 5 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 4 and the reference compound
Compound 4 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 4 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 4 revealed signals for 32 carbons including two carbonyl, three
quaternary, fourteen methine, eleven methylene, and two methyl carbons. The
1
H- and 13C-NMR data of 4 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 4 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 1 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 4
Table 4.4. NMR data for compound 4 and the reference compound
C
δC(#)
DEPT
δC(@)

δH(@)
1
40,0
CH2
41,44
0,87(m)/1,88 (m)
2
20,3
CH2
19,15
1,52 (m)/1,69 (m)
3
41,1
CH2
39,04
1,11 (m)
1,21 (d, J = 14,0 Hz)
4
44,8
C
45,11
5
58,2
CH
58,61
1,15 (m)
6
23,5
CH2
23,19

1,88 (m)/2,00 (m)
7
41,6
CH2
42,91
1,57 (m)/1,96 (m)
8
45,6
C
45,62
9
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 4

Figure 4.27. 1H-NMR spectrum of 4


19

Figure 4.28.

13

C-NMR spectrum of 4

Figure 4.30. HSQC spectrum of 4

Figure 4.32. COSY spectrum of 4

4.2. Evaluation of biological activities

Figure 4.29. DEPT spectrum of 4

Figure 4.31. HMBC spectrum of 4

Figure 4.33. NOESY spectrum of t 4

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 ÷ 38,19 ± 1,23 µg/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,
4 and 6 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 ÷7,39 µM; megastigmanes 14 and 15, phenolic 18 and acetogenin 22 IC50
values ranging from 2,79 ÷11,17 µM. 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.
1
2
3
4
5
6
7
8

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
9
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 3 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)
1
2

3
5
6
7
8
9
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
1. From A. glabra species, 22 compounds were isolated and determined their
chemical structures as listed belowing:


5 new compounds:



4 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:



8 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);



4 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);



4 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).



7 compounds (5, 14, 15, 17-20) were first isolated from Annona genus
and 2 compounds (6, 21) were first isolated from A. glabra species.

2. 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.
3. 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, 4 and 6 exhibited strong cytotoxic effects with
the IC50 values ranging of 0,65 ÷7,39 µM; 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.
4. 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