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

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 A

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

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

Thesis can be found in:

- The library of the Graduate University of Science and Technology,

Vietnam Academy of Science and Technology

- National Library

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 anti-cancer 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, anti-microorganisms for treatment of cancer and antibiotic multidrug-resistance 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

 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

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 layer chromatography (TLC), column chromatography (CC), silica gel,

thin-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/two-dimension 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

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

Add water (1L) Add CHCl3 (1L×3 times)

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

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

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

MeOH-H2O (3:3, v/v)

Diaion HP-20 MeOH-H 2 O (gradient 0:100, 25:75, 50:50, v/v)

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

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

Silica gel CC, CHCl 3 -MeOH (30:1, 20:1, v/v)

RP-C18 CC Acetone-H 2 O (1:3,5, v/v)

Diaion HP-20 MeOH-H 2 O (gradient 0:100, 25:75, 50:50, v/v)

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

Sample

Cell line

% inhibition

IC 50

(µg/mL)

% inhibition

IC 50

(µg/mL)

% inhibition

Minimum inhibitory concentration (MIC, g/mL)

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

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

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

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 sec-methyl group [δ 1.12 (3H, d, J = 7.0 Hz, H-18)], one oxymethine group

311.1806 325.1961 339.2025

343.1897

355.1761

373.1984 382.1945 388.3920

397.2241 +MS, 1.5min #90

Hình III.3 Phổ 1

H NMR của hợp chất 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)

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

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/H-15; 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 H-6/Hb-20, H-15, H-4 and between H3-18/Hb-2 (Fig IV.8-IV.9)

Therefore, compound EA-1 was elucidated as trihydroxy-19-nor-beyer-15-ene (excoecarin L)

3β,20-epoxy-3,6α,17-Table IV.1 The NMR data of EA-1 and reference compound

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 3-one 20-O- β- D -glucopyranoside (EC-1, new compound)

6α,7α-Epoxy-4β,5β,9α,13α-tetrahydroxy-rhamnofola-1,15-dien-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

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

Figure IV.30 HSQC spectrum of EC-1

The 13C 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 [δC104.8 (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

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, C-

16, 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