Tóm tắt luận án Tiến sĩ Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.

1

MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND ECHNOLOGY
------------------------

Lai Hop Hieu

STUDY ON CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITIES FROM THE LEAVES OF
EXCOECARIA AGALLOCHA L. AND EXCOECARIA
COCHINCHINENSIS Lour.

Major: Organic chemistry
Code: 9.44.01.14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

Hanoi – 2021


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This thesis was completed at: Graduate University Science and
Technology - Vietnam Academy of Science and Technology

Adviser 1: Prof. Dr. Ngo Dai Quang
Adviser 2: Dr. Nguyen Van Thanh

1st Reviewer: ……………………………………………………....
2nd Reviewer: : …………………………………………………….
3rd Reviewer: : ……………………………………………………..

The thesis will be defended at Graduate University of Science and
Technology - Vietnam Academy of Science and Technology, at
hour

date

month

2021.

Thesis can be found in:
- The library of the Graduate University of Science and Technology,
Vietnam Academy of Science and Technology
- National Library


1

INTRODUCTION
1. The urgency of the thesis
Throughout human history, marine microorganisms and natural
plants have become potential sources in the discovery of novel drugs for
the treatment of human diseases. Nowadays, more than 70% of anticancer drugs in the market are derived from natural products or

synthesized based on the structure of natural compounds. Besides
cancer, which is a major issue of concern for scientists, the emergence
of antibiotic drug resistance is also a big threat to human health
worldwide. Antibiotic drug resistance occurs when microorganisms such
as viruses, fungi or parasites change their mechanism of action in
response to the existing antimicrobial treatments. Several factors
contribute to antibiotic resistance such as the overuse/misuse of
antibiotics and the self-medication with antibiotics.
The important role of natural bioactive compounds has been
investigated from traditional medicine to modern medicine. Their value
is not only for direct use as a medicine but also as a structure lead
compound for the discovery and development of new drugs. In an
attempt to investigate and research medicinal materials for public health
care programs, the study on natural compounds which exhibit several
biological activities such as cytotoxicity, anti-cancer, antimicroorganisms... for treatment of cancer and antibiotic multidrugresistance is one of the main goals of scientists around the world.
Marine organisms and mangrove plants raise much attention to the
scientists in the field of biomedicine and pharmacology. Several studies
have been carried out to investigate new bioactive compounds derived
from mangrove plants.
Therefore, the thesis namely “Study on chemical constituents
and biological activities from the leaves of Excoecaria agallocha L.
and Excoecaria cochinchinensis Lour.” was conducted to investigate
potential bioactive compounds from E. agallocha and E.
cochinchinensis in order to demonstrate more clearly the therapeutic
uses in traditional medicine and increase the scientific value of these
plants in Vietnam.
2. The objectives of the thesis
 Isolation and determination of chemical structures of the
isolated compounds from the leaves of Excoecaria agallocha L. and
Excoecaria cochinchinensis Lour.

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 Studied the cytotoxic, anti-inflammatory, and antimicrobial
activities of the isolated compounds to find the bioactive compounds.
3. The main contents of the thesis
 Isolation of compounds from the leaves of Excoecaria
agallocha and E. cochinchinensis using various chromatographic
separations. Determination of chemical structures of the isolated
compounds.
 Evaluation of the cytotoxic, anti-inflammatory, and
antimicrobial activities of the isolated metabolites to find out potential
compounds.
CHAPTER I. OVERVIEW
This chapter presents the overview of domestic and international
studies related to the chemical compositions and biological activities of
E. agallocha and E. cochinchinensis.

CHAPTER II. RESEARCH OBJECTIVE AND RESEARCH
METHODOLOGY
II.1. Research objective

Figure II.1. E. agallocha

Figure II.2. E. cochinchinensis

The leaves of E. agallocha were collected in Xuan Thuy, Nam
Dinh, Vietnam in July 2013. The leaves of E. cochinchinensis were
collected in Van Giang, Hung Yen, Vietnam in April 2016. Two

samples were identified by Dr. Nguyen The Cuong, Institute of Ecology
and Biological Resources, VAST. The voucher specimens were
deposited at the Institute of Ecology and Biological Resources and
Institute of Marine Biochemistry, VAST, Vietnam.


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II.2. Research methodology
II.2.1. Methods for extraction
The samples were cut into pieces and extracted three times with
MeOH at room temperature (for 3 days) or in an ultrasonic bath (three
times, each time 45 min). Evaporation of the solvent in vacuo obtained a
residue, which was suspended in distilled water and partitioned in turn
with n-hexane, CH2Cl2, and EtOAc.
2.2.2. Methods for metabolites isolation
Combining a number of chromatographic methods including thinlayer chromatography (TLC), column chromatography (CC), silica gel,
RP-18, and Sephadex LH-20...
II.2.2. Methods for determination of the chemical structure of compounds
The general method used to determine the chemical structure of
compounds is the combination between physical parameters and modern
spectroscopic including optical rotation ([α]D), electrospray ionization mass
spectrometry (ESI-MS), and high-resolution ESI-MS (HR-ESI-MS), one/twodimension nuclear magnetic resonance (NMR) spectra.
II.2.3. Methods for evaluation of biological activities
 Cytotoxic activity was evaluated against three human cancer
cell lines, MCF-7 (human breast cancer cells), LU-1 (human lung
adenocarcinoma), and KB (human epidermoid carcinoma) by the MTT
and SRB assays.
 Anti-inflammatory activity of isolated compounds was assessed
based on inhibiting NO production in lipopolysaccharide (LPS)

activated RAW264.7 cells.
 The antimicrobial activity of the isolated metabolites against a
selected panel of the Gram-positive (Bacillus subtillis ATCC11774 and
Staphylococcus aureus ATCC11632) and Gram-negative (Escherichia
coli ATCC25922, and Pseudomonas aeruginosa ATCC27853) bacteria,
as well as a set of yeast molds (Aspergillus niger 439, Fusarium
oxysporum M42, Candida albicans ATCC7754, and Saccharomyces
cerevisiae SH 20), were also determined.


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CHAPTER III. EXPERIMENT AND EMPIRICAL RESULTS
III.1. Isolation of compounds
III.1.1. Isolation of compounds from E. agallocha
This part showed the extraction and isolation experiments of the
compounds isolated from the leaves of E. agallocha.
The leaves of
Excoecaria agallocha
(2.5 kg dried)

A: Acetone
CC: Chromatography column
D: Dichloromethane

Extraction with ultrasonic bath
MeOH, 3 times, 45min, 45-60oC.

M: Methanol
H: n-Hexane

MeOH extract
(A, 200 g)

W: water

Add water (1L)
Add CHCl3 (1L×3 times)

CHCl3/H2O 1:1

CHCl3 fraction
(94 g)

H2O layer
Add EtOAc (1L×3 times)

n-Hexane/60% Aq. MeOH

n-Hexane fraction
(H, 80 g)

MeOH fraction
(C, 14 g)

H2O layer
(W)

EtOAc fraction
(E, 7 g)

n-Hexane, n-Hexane-acetone
gradien
100:1, 70:1,...0:100

C-1

C-2

(2.6 g)

( 1.1 g)

C-3
CC, Silica gel
HA 3:1, DA 4:1

C-3A

(10.1 g)

C-3B
RP-18, CC,
MW 1:1

C-3C
RP-18, CC,
MW 1:2

EA-5

EA-3

(5,8 mg)

(8 mg)

Figure III.1. Isolation of compounds from the CHCl3 fraction of E. agallocha
Cặn
ethylfraction
acetate
EtOAc

(E, 7 g)
RP-C18, CC,
H2O/MeOH :2/1

E-1

E-2

E-3

E-4

(0,45 g)

(1,5 g)

(2,7g)

(2,24 mg)

Sephadex
H2O/MeOH:2/1

E-2B

E-2B
CC, CH2Cl2/MeOH:
2/1 v/v

EA-9
(8,2 mg)

E-1

E-2

E-3

(0,45 g)

(1,5 g)

(2,7g)

Sephadex
H2O/MeOH:1/1

Sephadex
H2O/MeOH:1/1

EA-4
EA-6

EA-7

(12 mg)

(7 mg)

(10,2 mg)

Figure III.2. Isolation of compounds from the EtOAc fraction of E. agallocha


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Water layer
Diaion HP-20
MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v)

W-1

W-2A

W-2B

Silica gel CC,
CHCl3-MeOH (30:1, 20:1, v/v)

W-3A

W-3C

RP-18 CC
MeOH-H2O (3:3, v/v)

W-3A1
Silica gel CC,
n-Hexane-acetone (5:2)

W-4

W-3

W-2

Silica gel CC,
CHCl3-MeOH (50:1, 25:1, v/v)

W-3A2

W-3B

W-3D

RP-18 CC
Acetone-H2O (1:3,5, v/v)

W-3B1

W-3B2

W-3B3

Sephadex LH-20,
MeOH-H2O (1:2)

Sephadex LH-20,
MeOH-H2O (1:2)

EA-8

EA-1

EA-2

(4 mg)

(3.5 mg)

(5 mg)

Figure III.3. Isolation of compounds from the water layer of E. agallocha

III.1.2. Isolation of compounds from E. cochinchinensis
This section presents the process of isolating 13 compounds from
the leaves of E. cochinchinensis.
The leaves of Excoecaria

cochinchinensis (3 kg dried)
Extraction with ultrasonic bath
MeOH, 3 time, 45min, 45-55oC

MeOH extract
(M, 450 g)
n-Hexane/H2O 1:1

Add water (1L)
Add n-hexane (1.5L × 3 time)

H2O layer

n-Hexane fraction
(H, 120 g)

add EtOAc (1.5L
× 3 time)
EtOAc/H2O 1:1

EtOAc fraction
(E, 100 g)

H2O layer
(W)

Figure III.4. The partitioned MeOH extract of E. cochinchinensis


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Water layer
Diaion HP-20
MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v)

W-1

W-3

W-2

Silica gel CC,
CHCl3-MeOH (50:1, 25:1, v/v)

W-2A

W-2B

W-4

(85 g)
Silica gel CC,
CHCl3-MeOH (30:1, 20:1, v/v)

W-3B

W-3A

W-3C

W-3D

RP-C18 CC
Acetone-H2O (1:3,5, v/v)

W-3B1

W-3B3

W-3B2

Sephadex LH-20
(MeOH-H2O, 1:2)

EC-6

EC-2

(3 mg)

(8 mg)

Silica gel CC, n-Hexane-acetone (5:2); Sephadex LH-20, MeOH-H2O (1:1)

EC-11

EC-10

EC-9

EC-8

(2,7 mg)

(6,6 mg)

(3 mg)

(3 mg)

Silica gel CC, CH2Cl2-MeOH-H2O (5:1:0,1); RP-C18, MeOH-H2O (1:1)

EC-5

EC-4

EC-3

EC-1

(5,5 mg)

(2 mg)

(2 mg)

(2,2 mg)

Silica gel CC, CH2Cl2-MeOH-H2O (6:1:0,05); Sephadex LH-20, MeOH-H2O (1:1)

EC-13

EC-12

EC-7

(20 mg)

(21 mg)

(3 mg)

Figure III.7. Isolation of compounds from the water layer of E. cochinchinensis

III.1.3. Physical properties and spectroscopic data of the isolated
compounds
III.1.3.1. Physical properties and spectroscopic data of the isolated
compounds from E. agallocha
This section presents physical properties and spectroscopic data
of 09 compounds from E. agallocha.
III.1.3.2. Physical properties and spectroscopic data of the isolated
compounds from E. cochinchinensis
This section presents physical properties and spectroscopic data
of 13 compounds from E. cochinchinensis.


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III.2. Results on cytotoxic activities of isolated compounds
III.2.1. Results on cytotoxic activity of extract from E. agallocha
Table III.1. The effects of the MeOH extract from E. agallocha

Cell line
LU-1

%
inhibition

IC50
(µg/mL)

%
inhibition

IC50
(µg/mL)

MCF7
%
IC50
inhibition (µg/mL)

MeOH
extract

81.90

19.77

85.03

15.23

65.38

57.57

Ellipticine
10 µg/mL

97.18

0.39

96.35

0.50

95.73

0.48

Sample

KB

III.2.2. Results on antimicrobial activity of compounds from E.
agallocha
Table III.3. The effects of isolated compounds from E. agallocha
Minimum inhibitory concentration (MIC, g/mL)
Samples

Gram (-)
Ec

Streptomycin
(57,5 g/mL)
Nystatin
(92,5 g/mL)
Tetracyclin
(44 g/mL)

*

Gram (+)

Pa

*

Bc

*

Sa

Fungus
*

*

An

Fo

*

Sc*

Ca*

-

-

7.188

14.375

-

-

-

-

-

-

-

-

23.125

11.563

5.781

11.563

5.5

11

-

-

-

-

-

-

EA-1

>50

-

-

-

-

-

-

>50

EA-2

-

-

>50

-

>50

-

-

-

EA-3

-

-

-

-

>50

-

-

-

EA-4

-

-

>50

-

-

-

-

-

EA-5

-

>50

-

-

-

50

-

-

EA-6

-

-

-

-

-

-

-

-

EA-7

-

-

-

-

-

-

-

-

EA-8

-

-

-

-

-

-

-

-

EA-9

>50

-

-

-

>50

-

>50

-

MeOH
extract

-

-

200

-

-

-

-

-

Streptomycin, nystatin, and tetracyclin were used as the positive control. Ec (Escherichia coli),
Pa (Pseudomonas aeruginosa), Bc (Bacillus subtillis), Sa (Staphylococcus aureus), An

(Aspergillus niger), Fo (Fusarium oxysporum), Sc (Saccharomyces cerevisiae), and Ca
(Candida albicans). (-) No detection.


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III.2.3. Results on anti-inflammatory activity of isolated compounds
from E. cochinchinensis
Table III.4. Effects of compounds on the LPS-induced NO production
on RAW264.7 cells from E. cochinchinensis
Compounds

Concentration
(µM)

Inhibition (%)

Growth of cell (%)

EC-1

38.72 ± 0.56

76.12 ± 1.36

EC-2

46.78 ± 0.35

78.89 ± 1.32

EC-3

75.83 ± 0.77

86.65 ± 1.54

EC-4

31.09 ± 1.60

56.49 ± 0.97

EC-5

42.02 ± 1.01

75.51 ± 1.52

EC-6

68.18 ± 0.67

73.59 ± 0.67

39.22 ± 1.07

78.60 ± 2.18

EC-8

94.96 ± 0.26

80.41 ± 1.66

EC-9

82.91 ± 1.03

83.19 ± 2.37

EC-10

27.87 ± 0.81

84.55 ± 0.98

EC-11

30.47 ± 0.69

82.91 ± 1.43

EC-12

38.38 ± 0.19

70.16 ± 1.77

EC-13

35.01 ± 0.76

55.50 ± 2.54

0.3

33.89 ± 0.51

95.35 ± 0.75

3

88.80 ± 0.51

86.00 ± 1.55

100

EC-7

Cardamonin
Cardamonin was used as a positive control. Data are presented as the mean ± standard
deviation (SD) of at least three independent experiments performed in triplicate.

Table III.5. The IC50 values of selected compounds
Compounds

IC50 values (µM)

EC-3

13.80 ± 1.23

EC-6

58.10 ± 2.04

EC-8

6.17 ± 0.25

EC-9

12.02 ± 0.73

Cardamonin

1.57 ± 0.24

Cardamonin was used as a positive control. Data are presented as the mean ± standard
deviation (SD) of at least three independent experiments performed in triplicate.


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CHAPTER IV. DISCUSSIONS
IV.1. Determination of the chemical structure of compounds from
E. agallocha
This section presents the detailed results of spectral analysis and

structure determination of 09 isolated compounds from E. agallocha.
The detailed methods for the determination of the chemical structure of
a new compound are introduced in the following section.
IV.1.1. Excoecarin L (EA-1, new compound)

Figure IV.1. Structure of EA-1 and keys COSY, HMBC correlations
and reference compounnd
Intens.
x105

+MS, 1.5min #90
355.1761

3.0

343.1897

2.5

2.0

1.5

1.0

373.1984

0.5

397.2241

325.1961

311.1806

0.0
300

310

320

382.1945 388.3920

339.2025

330

340

350

360

370

380

390

m/z

Figure IV.2. HR-ESI-MS spectrum of EA-1
Compound EA-1 was obtained as an amorphous white powder.
Its molecular formula was determined by HR-ESI-MS as C19H28O4 on
the basis of the [M + Na]+ sodiated-molecular ion peak observed at m/z
343.1897 (calcd. for C19H28O4Na+, 343.1880). The 13C NMR and HSQC
spectra revealed the presence of 19 carbon atoms corresponding to four
quaternary carbons, six methines, eight methylenes, and one methyl.
Among them, two olefinic methines (δC 135.2 and 135.5), four
oxygenated carbons (two methylenes, one methine, and one quaternary
carbon resonating at δC 68.66, 69.4, 71.1, and 98.6, respectively) were
evident. With six degrees of unsaturation established from the molecular
formula, compound EA-1 was suggested to contain five rings and one
double-bond. The 1H NMR spectrum confirmed the presence of one secmethyl group [δH 1.12 (3H, d, J = 7.0 Hz, H-18)], one oxymethine group


10

Hình III.3. Phổ 1H NMR của hợp chất EA-1

Hình III.4. Phổ 13C NMR của hợp chất EA-1

Figure VI.5. HSQC spectrum of EA-1
[δH 3.75 (1H, ddd, J = 4.0, 11.0, 11.5 Hz, H-6)], two oxymethylene
groups [δH 3.40 (1H, d, J = 11.0 Hz, Ha-17)/3.45 (1H, d, J = 11.0 Hz,
Hb-17) and 3.80 (1H, d, J = 9.5 Hz, Ha-20)/3.89 (1H, dd, J = 3.5, 9.5
Hz, Hb-20)], and two olefinic protons of a disubstituted double bond [δH
5.73 (1H, d, J = 6.0 Hz, H-15) and 5.66 (1H, d, J = 6.0 Hz, H-16)]
(Table IV.1).

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Figure VI.6. HMBC spectrum of EA-1

Figure VI.7. COSY spectrum of EA-1
Detailed analysis of correlations provided by COSY and HMBC
experiments (Fig. IV.1) revealed that the planar structure of EA-1 was
similar to that of agallochin I, previously isolated from the same species,
except for the presence of an additional hydroxy group at C-17. In fact, the
HMBC cross-peaks from H-17 to C-12, C-13, C-14, and C-16 placed the
hydroxy group at C-17, whereas the other hydroxy group and the methyl
group were placed at C-6 and C-4, respectively, due to the COSY
correlations of H-18/H-4/H-5/H6/H-7. The downfield chemical shift of the
quaternary carbon at δC 98.6 (C-3) in conjunction with the HMBC
correlations from H-20 to C-1, C-3, C-5, and C-10 indicated that the ether
bridge was positioned between C-20 and C-3, and the last hydroxy group
was located at C-3.


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Figure VI.8. Keys NOESY correlations of EA-1

Figure IV.9. NOESY spectrum of EA-1
The relative stereochemistry of EA-1 was obtained through
analysis of 1H NMR coupling constants and NOESY experiment.
Specifically, the large J-values (J = 11.0 - 12.5 Hz) of H-5, H-6, Ha-7,
and H-9 indicated the axial orientation of these protons. The NOE
correlations between H-5/H-9, Ha-1, Ha-7; Ha-7/Ha-14, H-9; Hb-20/H15; H-15/H-16 and Ha-20/Ha-11, Hb-1, Ha-2 confirmed the structure of

beyer-15-ene diterpenoid skeleton. Finally, the configurations at C-4 and
C-6 were determined on the basis of the NOE correlations between H6/Hb-20, H-15, H-4 and between H3-18/Hb-2 (Fig. IV.8-IV.9).
Therefore, compound EA-1 was elucidated as 3β,20-epoxy-3,6α,17trihydroxy-19-nor-beyer-15-ene (excoecarin L).


13

Table IV.1. The NMR data of EA-1 and reference compound
δCa,b [35]

δCc,d

δCc,e (mult., J in Hz)

1

31.2

32.7

2

26.9

28.2

1.27 (1H, m)
2.09 (1H, ddd, 3.5, 12.5, 12.5)
1.71 (1H, m)
2.02 (1H, ddd, 3.5, 12.0, 13.5)

3

98.3

98.6

-

4

41.5

42.7

1.96 (1H, m)

5

56.9

57.9

1.03 (1H, dd, 5.0, 11.0)

6
7

69.5
44.7

71.1
46.2

3.75 (1H, ddd, 4.0, 11.0, 11.5)
1.43 (1H, dd, 11.5, 13.0)
1.85 (1H, dd, 4.0, 13.0)

8

49.4

50.2

-

9

44.5

46.4

1.20 (1H, dd, 4.5, 12.5)

10

36.3

37.5

-

11

20.7

21.4

1.08 (1H, m)/1.72 (1H, m)

12

31.9

28.0

1.28 (1H, m)/1.36 (1H, m)

13
14

43.7
60.4

51.1
56.6

1.09 (1H, m)
1.67 (1H, dd, 2.5, 9.5)

15

133.3

135.5

5.73 (1H, d, 6.0)

16
17

137.9
24.4

135.2
68.6

5.66 (1H, d, 6.0)
3.40 (1H, d, 11.0)
3.45 (1H, d, 11.0)

18
20

19.3
68.5

19.6
69.4

1.12 (3H, d, 7.0)
3.80 (1H, dd, 5.0, 9.5)
3.89 (1H, dd, 3.5, 9.5)

No.

#

CDCl3, b75MHz; cCD3OD, d125MHz, e500MHz. #δC of agallochin I [35].

a


14

Figure IV.26. The structures of 9 compounds isolated from E. agallocha
IV.2. Determination of chemical structure of isolated compounds from
E. cochinchinensis
VI.2.1. 6α,7α-Epoxy-4β,5β,9α,13α-tetrahydroxy-rhamnofola-1,15-dien3-one 20-O-β-D-glucopyranoside (EC-1, new compound)

Figure IV.27. Structure of EC-1 and reference compound
Compound EC-1 was isolated as a white, amorphous powder.
Its molecular formula was determined to be C26H38O12 by the negative
HR-QTOF-MS ion peaks at m/z 541.2297 [M - H]– (calcd for
C26H37O12–, 541.2291), 577.2063 [M + Cl]– (calcd for C26H38ClO–,
577.2057), and 587.2346 [M + HCOO]– (calcd for C27H39O–,
587.2345), indicating eight degrees of unsaturation (Fig. IV.28).


15

Figure IV.28. HR-ESI-MS spectrum of EC-1

Figure IV.28. 1H NMR spectrum of EC-1

Figure IV.29. 13C NMR spectrum of EC-1


16

Figure IV.30. HSQC spectrum of EC-1
13

The C NMR and HSQC spectra revealed the presence of 26
carbon atoms including 6 non-protonated carbons, 13 methines, 4
methylenes, and 3 methyls. Among them, a typical α,β-unsaturated
carbonyl moiety [δC 209.9 (C-3), 134.8 (C-2), 163.1 (C-1)], two other
olefinic carbons [δC 145.9 (C-15), and 117.3 (C-16)], three oxygenated
tertiary carbons [δC 74.8 (C-4), 64.6 (C-6), and 77.3 (C-9)], three
oxymethines [δC 71.4 (C-13), 68.1 (C-5), and 62.3 (C-7)], and an
oxymethylene [δC 74.2 (C-20)], along with a glucopyranosyl unit [δC
104.8 (C-1′), 75.2 (C-2′), 78.0 (C-3′), 71.7 (C-4′), 78.0 (C-5′), and 62.8
(C-6′)] were observed (Table IV.9). Since one carbonyl group and two
double bonds accounted for three degrees of unsaturation, EC-1 was
determined to be a pentacyclic compound. Accordingly, the 1H NMR
spectrum showed the existence of three methyls [δH 1.70 (3H, s, H-17),
0.96 (3H, d, J = 7.0 Hz, H-18), and 1.76 (3H, d, J = 2.0 Hz, H-19)], one
terminal double bond [δH 4.98 (1H, d, J = 2.0 Hz, H-16a)/5.04 (1H, br s,
H-16b)], and one trisubstituted double bond [δH 7.66 (1H, br s, H-1)]
(Fig. IV.28-IV.29). The large coupling constant of the anomeric proton

[δH 4.33 (1H, d, J = 7.5 Hz, H-1′) confirmed the β-glucosidic linkage
(Fig. IV.26). Careful comparison of the 1H and 13C NMR spectroscopic
data for diterpenoidal nucleus of 1 (Table IV.9) with those of venenatin,
a daphnane-type diterpenoid, revealed that they were very similar and
these compounds had the same structure of A and B rings.


17

Figure IV.30. Keys COSY, HMBC, and NOESY correlations of EC-1

Figure IV.32. HMBC spectrum of EC-1
This deduction was also confirmed by COSY and HMBC
correlations as shown in Fig. IV.30. Besides, the COSY cross-peaks of
H-7/H-8/H-14/H-13/H-12/H-11/H-18 in combination with the HMBC
correlations from H3-18 to C-9, C-11, C-12, from H-7 to C-9 and C-14,
from H-8 to C-9, C-11, C-13, C-14, C-15, from H3-17 to C-14, C-15, C16, and from H2-16 to C-14, C-17 established structure of C ring, which
was fused to the B ring at the C-8 and C-9, and substituted with two
hydroxy groups at C-9 and C-13, a methyl group at C-11, and an


18

Figure III.33. COSY spectrum of EC-1

Figure IV.34. NOESY spectrum of EC-1
isopropenyl moiety at C-14. The downfield chemical shift of the
oxymethylene carbon at δC 74.2 (C-20) along with the HMBC
correlation from H-1′ to C-20 indicated the position of glucosyl moiety.
Thus, EC-1 was established as a rhamnofolane diterpene glucoside.

The relative configuration of EC-1 was assigned by analysing
proton-proton coupling constants and NOESY data. The H-14 signal
(dd, J = 10.0, 12.5 Hz) exhibited two large coupling with the H-8 (br d,
J =12.5 Hz) and H-13 (ddd, J = 4.5, 10.0, 10.5 Hz) revealed the transdiaxial orientation of these protons. The equatorial orientation of Ha-12
(ddd, J = 4.5, 4.5, 12.5 Hz) was deduced by the small coupling constants
with its vicinal protons. In the NOESY spectrum, the correlations from


19

Table IV.9. The NMR data of EC-1 and reference compound
No.

Venenatin [106]

δCa,b

δHa,c (mult., J in Hz)

1
2
3
4
5
6
7
8
9
10
11

12
13
14
15
16
17
18
19
20

162.2
135.5
209.9
74.6
70.5
64.0
65.4
39.1
79.8
51.0
39.5
39.1
75.4
79.4
147.2
114.7
19.3
18.3
9.9
65.0

163.0
134.7
209.9
74.7
68.1
64.6
62.3
40.0
77.3
51.5
39.4
41.1
71.4
54.2
145.9
117.2
18.9
18.9
9.9
74.2

1'
2'
3'
4'
5'
6'

-

104.8
75.2
78.0
71.7
78.0
62.8

7.66 (1H, br s)
4.30 (1H, br s)
3.19 (1H, br s)
3.10 (1H, br d, 12.5)
4.15 (1H, dd, 2.5, 3.0)
2.10 (1H, m)
1.60 (1H, m)/1.73 (1H, m)
3.52 (1H, m)
2.80 (1H, dd, 10.0, 12.5)
4.98 (1H, d, 2.0)/5.04 (1H, br s)
1.70 (3H, s)
0.96 (3H, d, 7.0)
1.76 (3H, d, 2.0)
3.45 (1H, d, 11.0)
4.27 (1H, d, 11.0)
4.33 (1H, d, 7.5)
3.20 (1H, *)
3.36 (1H, *)
3.27 (1H, *)
3.26 (1H, m)
3.64 (1H, dd, 5.5, 12.0)
3.86 (1H, dd, 1.0, 12.0)

a

CD3OD, b125 MHz, c500 MHz. *Overlapped signals. 13C NMR of venenatin [106].

H-8 to H-11, H-13, H3-17 and H-7, and from H-7 to Ha-20 and H3-17,
and from H3-17 to H-13 and H-16b, from H3-18 to H-1 established the
β-configuration for H-8, H-11, H-13, H-7, and C-6-C-20 bond. On the
other hand, the NOESY cross-peaks from H-14 to Hb-12 and Ha-16,
and from H-5 to H-10 suggested that these protons were α-oriented. The
configuration of OH-4 and OH-9 was assigned as β and α-orientation,
respectively, by comparing the 13C NMR data with those of venenatin as
well as the coexistence of two compounds in this plant. Furthermore,


20

most related [5-7-6]tricyclic diterpenoids (daphnanes and tiglianes) of
Euphorbiaceae family have been found to possess trans-A/B ring and
trans-B/C ring. Therefore, compound EC-1 was determined as 6α,7αepoxy-4β,9α,13α,20β-tetrahydroxy-rhamnofola-1,15-dien-3-one 20-Oβ-D-glucopyranoside.

Figure IV.53. The structures of 13 compounds isolated from E.
cochinchinensis


21

IV.3. Biological activities of isolated compounds
IV.3.1. Cytotoxic activity of extracts and compounds isolated from
Excoecaria agallocha

Cytotoxicity testing method is carried out to evaluate the ratio of
living cells and dead cells after treatment cells with tested samples. This
is a basic method to screening new compounds for the development of
anti-cancer agents. All isolated compounds were examined in three
cancer cells line: human breast cancer (MCF-7), human lung cancer
(LU) and human epithelial cancer.
The results showed that MeOH extract inhibited more than 50%
of the growth of all three cancer cell lines. The positive compound
Ellipticine was used to determine the stability of the experiment. The
values had high accuracy with r2 ≥ 0,99 (Table II.1). However, all
isolated compounds exhibited weak or no cytotoxic activity against
tested cell lines (IC50 > 100 µM).
III.3.2. Anti-microorganism activity of extracts and compounds
isolated from Excoecaria agallocha
Screening results showed that MeOH extract inhibited the growth
of only one Gram-positive bacteria B. subtillis strain (MIC = 200
µg/mL). Isolated compounds were tested the anti-microorganism in 8
bacterial strains including 2 Gram-negative bacteria strains: E. coli, P.
aeruginosa, 2 Gram-positive bacteria strains: B. subtillis, S. aureus, 2
fungal strains: A. niger, F. oxysporum, 2 yeast strains: S. cerevisiae và
C. albicans. Results demonstrated that compound blumenol A (EA-6)
exhibited good inhibition activity against the growth of F. oxysporum
strain (MIC = 50 µg/mL).
III.3.3. Anti-inflammatory activity of compounds isolated from
Excoecaria cochinchinensis
NO production process is one of the body's self-protective
responses, but NO overproduction leads to cell and tissue damage,
promotes inflammation, and causes acute and chronic inflammatory
diseases. Therefore, the level of NO production is considered as one of
the indicators for the inflammatory process. Compounds that have the

ability to inhibit the production of NO are anti-inflammatory agents. 13
compounds (EC-1 – EC-13) isolated from E. cochinchinensis were
tested the anti-inflammatory effect in LPS-induced RAW264.7
macrophages cells. Results showed that compounds EC-3, EC-8, and
EC-9 inhibited the NO production significantly (Table II.2-II.3) at the
concentration of 100 µM. The IC50 of three compounds EC-3, EC-8 and


22

EC-9 were 13.80 ± 1.23, 6.17 ± 0.25, and 12.02 ± 0.73 µM, respectively
compared with the IC50 of positive control Cardamonin (1.57 ± 0.24
µM). At the concentration of 100 µM, compound EC-6 showed medium
inhibitory activity, other compounds exhibited weak activity (IC50 > 50
µM.
These findings indicated high similarity with the previously
published results of active compounds from the genus Excoecaria. This
contributes to demonstrate more clearly the therapeutic uses in
traditional medicine and increase the scientific value of these plants in
Vietnam.


23

CONCLUSIONS
Chemical composition investigations:
By using various chromatographic methods, 22 compounds were
isolated from Excoecaria agallocha L. and E. cochinchinensis Lour.
Their chemical structures were determined by NMR, electrospray
ionization (ESI)-MS, and as well as by comparison with those reported

in the literature.
- 09 compounds were isolated and identified from E. agallocha,
including two new compounds, named excoecarin L (EA-1) and
excoecarin O (EA-2), as well as seven known compounds: aquillochin
(EA-3), (+)-isolariciresinol (EA-4), (+)-epipinoresinol (EA-5),
blumenol A (EA-6), blumenol B (EA-7), kaempferol (EA-8), and
methyl gallate (EA-9).
- 13 compounds were isolated and identified E. cochinchinensis,
including two new compounds, named 6α,7α-epoxy-4β,5β,9α,13αtetrahydroxy-rhamnofola-1,15-dien-3-one
20-O-β-D-glucopyranoside
(EC-1)
and
acid
3-(2-O-β-D-glucopyranosyl-3-hydroxyphenyl)
propanoic (EC-9), as well as 11 known compounds: venenatin (EC-2),
glochionionol A (EC-3), (6R,9S)-roseoside (EC-4), isofraxoside (EC5), pinoresinol-4'-O-β-D-glucoside (EC-6), liriodendrin (EC-7),
rhamnocitrin 3-glucoside (EC-8), sinapyl alcohol 4-O-β-Dglucopyranoside (EC-10), 2,3-dihydroxypropyl-benzoate 3-O-α-(4''methoxyglucuronide) (EC-11), phenethyl rutinoside (EC-12, and
benzyl-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (EC-13).
Investigation of biological activity
- The cytotoxic effects of MeOH extract were investigated in
vitro in three human cancer cell lines: KB, LU-1, and MCF7. The results
showed that MeOH extract showed the strong cytotoxic effect in three
tested cell lines KB, LU-1, and MCF-7.
- Anti-microorganism activity testing showed that MeOH extract
from E. agallocha inhibited the development of only one Gram-positive
bacteria B. subtilli strain with the MIC of 200 µg/mL. Compound
blumenol A (EA-6) revealed an inhibitory effect against the growth of
F. oxysporum strains (MIC = 50 µg/mL).
- 13 compounds from E. cochinchinensis were used to measure
the anti-inflammatory activity in LPS-induced RAW264.7 macrophage

cells. Three compounds EC-3, EC-8, and EC-9 inhibited the NO
production significantly with the IC50 of 13.80 ± 1.23, 6.17 ± 0.25, and
12.02 ± 0.73 µM, respectively.