Nghiên cứu hoạt tính gây độc một số dòng tế bào ung thư của các hợp chất phân lập từ ba loài san hô mềm sinularia nanolobata, sinularia leptoclados, sinularia conferta thu thập ở vùng biển trung bộ việt nam TT TA

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY --- Ninh Thi Ngoc CELL LINES OF ISOLATED COMPOUNDS FROM THREE SOFT CORALS SINULARIA NANOLOBATA, SINULARIA LEPTOCLADOS, SINULARIA CONFERTA

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

-

Ninh Thi Ngoc

CELL LINES OF ISOLATED COMPOUNDS FROM THREE

SOFT CORALS SINULARIA NANOLOBATA, SINULARIA LEPTOCLADOS, SINULARIA CONFERTA COLLECTED

FROM THE SEA AREA OF CENTRAL VIETNAM

Major: Biochemistry Code: 9.42.01.16

SUMMARY OF BIOLOGY DOTORAL THESIS

Ha Noi - 2021

This thesis was completed at: Graduate university of Science and

Technology - Vietnam Academy of Science and Technology

- Institute of Biotechnology

- Institute of Marine Biochemistry

This thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at hour date month 2021

The thesis can be found in:

- The Library of Graduate University of Science and Technology, Vietnam Academy of Science and Technology

- National Library of Vietnam

- Institute of Biotechnology

INTRODUCTION

1 The urgency of the thesis

In recent years, many countries have exploited bioactive substances from marine organisms to serve researches to find drugs to treat dangerous diseases such as cancer, hepatitis, and diseases infections and viruses Up to this point, a number of pharmaceuticals derived from marine organisms have reached the users' hands, such as Cytarabine, Vidarabine, Eribulin, Trabectedin To achieve this achievement, research institutes around the world has screened the biological activity of millions of compounds from marine species, and invested financial resources and time in pre-clinical and clinical research phases for potential compounds

With the advantage of owning a long coastline of over 3.260 km along with many islands and bays, Vietnam has great potential for exploiting a diverse marine resources, rich in both species composition and storage amount However, up to now, there are not many studies searching for valuable active ingredients from Vietnamese marine organisms and limited

in the in vivo activity test step and research on drug-cell interaction

mechanisms cancer The studies are currently in the early stages compared with other countries in the region and far behind the advanced countries The reason is that there are still some difficulties such as: surveying and collecting samples of marine organisms requires modern equipment, compounds isolated from marine organisms often have very small concentrations, complex structures impurities, some compounds that are easy to decompose right in the analysis process Therefore, an urgent requirement for our country is to develop research to step by step systemize the chemical composition and biological activity of marine species

Genus Sinularia is one of the genus of soft corals that is interested in

research by many scientists around the world Up to now, there have been many studies on the chemical composition and biological activities of many

compounds isolated from soft corals belonging to this genus However,

studies on many soft corals of the genus Sinularia such as S nanolobata, S

leptoclados, S conferta in Vietnam are few and almost no systematic and

methodical studies on these species mentioned above Stemming from the

above fact, I chose the thesis topic "Study on cytotoxic activity of some

cancer cell lines of isolated compounds from three soft corals Sinularia nanolobata, Sinularia leptoclados, Sinularia conferta collected from the

sea area of Central Vietnam" was chosen

2 The objectives of the thesis

- Determine the chemical composition of three soft coral species S

nanolobata, S conferta, S leptoclados collected in the central sea of

Vietnam

- Detecting active substances with cytotoxic activity in soft corals studied, orienting their application for biomedical and pharmacological studies

3 The main contents of the thesis

- Determine the scientific names of three soft corals collected from the sea area of Central Vietnam by molecular markers

- Isolation and Determination of chemical structures of isolated

compounds from three soft corals S nanolobata, S conferta, S leptoclados

- Evaluation of cytotoxic activity in vitro of isolated compounds

- Evaluation of cytotoxicity mechanism of some typical compounds

CHAPTER 1: OVERVIEW

This chapter provides an overview domestic and international studies on the extraction of natural compounds from marine organisms in cancer treatment, research on cytotoxic activity, general characteristics of the soft

corals and genus Sinularia, about the chemical composition and biological activity of soft corals genus Sinularia

1.1 Test to evaluate cytotoxic activity

1.2 Natural compounds from marine organisms in cancer treatment 1.3 Introduction to soft corals

1.3.1 Characteristics of soft corals

1.3.2 Overview of soft corals of the genus Sinularia

1.3.3 Application of molecular indicators in the classification of soft corals

1.3.4 Study on the biological activity of isolated compounds from soft corals of the genus Sinularia

Statistics of published studies show that compounds isolated from soft

corals genus Sinularia mainly include sesquiterpen, diterpen and steroid

compounds Many of these compounds exhibit interesting biological activity such as cytotoxic, anti-inflammatory, antibacterial, antiviral, neuroprotective and antioxidant activities

CHAPTER 2: SUBJECTS AND METHODS

2.1 Subjects

Sample of S nanolobata Verseveldt, 1977 was collected at Lang Co, Hue, Vietnam (4/2015)

Sample of S leptoclados Ehrenberg, 1834 was collected at Con Co,

Quang Tri, Vietnam (5/2016)

Sample of S conferta Dana, 1846 was collected at Con Co, Quang Tri,

Vietnam (5/2015)

2.2 Methods

2.2.1 Determination of scientific name of soft coral samples by molecular markers (msh1 and 28S RNA)

2.2.1.2 Amplification and sequencing of marker DNA fragments

Hình 2.6 Electrophoresis of PCR-colony products from some colonies after

transformation of vector pTZ57R/T at 28S rRNA of soft coral samples SN (well

No 1-7), SLE (well No 8-14), SCO (well No 15-21) M: GeneRulerTM 1kb

DNA ladder

Hình 2.7 Electrophoresis of PCR-colony products from some colonies after

transformation of vector pTZ57R/T with msh1 gene attached to soft coral samples

SN (well No 22-28), SLE (well No 29-35), SCO (well No 36-42) M:

GeneRulerTM 1kb DNA ladder

28S rRNA gene segment using

28SF and 28SR primers (B)

msh1 gene segment using

MSHF and MSHR primers M: GeneRulerTM 1kb DNA ladder

M 22 23 24 25 26 27 28 29 30 31 32 33 34 M 35 36 37 38 39 40 41 42

M 1 2 3 4 5 6 7 M 8 9 10 11 12 13 14 M 15 16 17 18 19 20 21

2.2.2 Methods for isolation of secondary metabolites

2.2.2.1 Isolation of compounds from S nanolobata

Hình 2.8 Isolation of compounds from S nanolobata

2.2.2.2 Isolation of compounds from S leptoclados

Hình 2.9 Isolation of compounds from S leptoclados

2.2.2.3 Isolation of compounds from S conferta

Hình 2.10 Isolation of compounds from S conferta

2.2.3 Methods for determination of chemical structure of compounds 2.2.4 Methods of assessment of activity and mechanism of cytotoxicity

CHAPTER 3: RESULTS

3.1 Determination of species by molecular makers

3.1.1 Sequencing 28S rARN and msh1 gene fragments of 3 soft corals

- Nucleotide sequence msh1 gene segment of SN sample with length 639

- Nucleotide sequence of 28S rRNA gene segment of SN sample with

3.1.2 Sequence comparison of 3 soft corals by BLAST program

The results of analyzing the msh1 and 28S rRNA gene sequences of the

study samples with the reference sequences on the NCBI international gene bank showed that the sequences of the indicator genes of the study samples have similarities high with the corresponding sequences on the gene bank, namely:

- Sequence of msh1 gene fragment of SN sample had 100% similarity

with the corresponding sequence (code FJ621451.1) of the sample S

nanolobata vourcher RMNH coel 38441 on the NCBI gene bank

- Sequence of msh1 gene fragment of SLE sample has 100% similarity

with the corresponding sequence (code KC542857.1) of the sample S

leptoclados vourcher ZMTAU: CO35308 on the NCBI gene bank

- Sequence of msh1 gene fragment of SCO sample has 100% similarity

with corresponding sequence (code FJ621389.1) of sample S conferta

vourcher NTM C13972 on NCBI gene bank

- Sequence of 28S rRNA gene fragment of SN sample has 99.8%

similarity with corresponding sequence (code KF915519.1) of Sinularia sp

RMNH voucher: Coel.41326 on the NCBI gene bank

- Sequence of 28S rRNA gene fragment of SLE sample has 100%

similarity with the corresponding sequence (code KC542837.1) of the

sample S leptoclados voucher ZMTAU: CO34095 on the NCBI gene bank

- Sequence of 28S rRNA gene fragment of SCO sample has 99.6%

similarity with corresponding sequence (code MF817932.1) of Sinularia sp

on the NCBI gene bank

Table 3.1 The results of species identification of the soft coral samples

studied based on analysis of the similarity (%) of the sequence of the marker DNA segments (genes 28S rARN and msh1) of the study samples with the reference sequences on gene bank

Name

msh1 maker 28S rRNA maker The general

conclusion is based

on 2 DNA markers Species Ident Species Ident

Sinularia leptoclados

SCO Sinularia

conferta 100% Sinularia sp ≥ 99.6% Sinularia conferta

3.2 Determination of chemical structure of the compounds

3.2.1 Determination of chemical structure of compounds from S nanolobata

HO

1

3 5 6 9 10

11 13 15 17 18

19 20

21 22

23 25 28

Figure 3.3 Chemical structure of isolated compounds from S leptoclados

3.2.3 Determination of chemical structure of compounds from S conferta

HO

1

3 5 6 9 10

11 13 16 18

19 20

21 22 23 24 25 26

27 28

OH

Figure 3.4 Chemical structure of isolated compounds from S conferta

3.3 Evaluation of cytotoxic activity of compounds isolated from soft corals

3.3.1 Evaluation of cytotoxic activity of compounds isolated from S nanolobata

Table 3.2 Results of cytotoxic activity of SN compounds

Elipticine* 1.26± 0.20 2.07± 0.33 1.95± 0.28 2.36± 0.24 2.07± 0.28 1.46± 0.20 1.91± 0.37 2.24± 0.16

*: Positive control; "-": No activity

The results of evaluation of 10 SN compounds showed that: The new compound SN 6 showed an average cytotoxic activity on the line of 3 cells

HL-60, HepG2 and SW480 (IC50 from 33.53-71.02 µM) Compound SN 4

exhibits toxic activity on the HL-60 cell line The remaining compounds did not show activity

3.3.2 Evaluation of cytotoxic activity of compounds isolated from S leptoclados

Table 3.3 Results of cytotoxic activity of SLE compounds

*: Positive control; "-": No activity

The results of 15 SLE compounds showed that: 3 compounds SLE 10, SLE 27 and SLE 28 (IC50 in the range of 1.78 to 78.33 µM) showed

cytotoxic activity on 8 cancer cell lines test letter Compounds SLE 20 and

SLE 30 showed activity in 2-3 tested cancer cell lines The remaining

compounds did not show activity

3.3.3 Evaluation of cytotoxic activity of compounds isolated from S conferta

Table 3.4 Results of cytotoxic activity of SCO compounds

cancer cell line A549

a Evaluation of the effect of SCO 27 on morphological change in cancer

cells

Figure 3.5 Cell morphology A549 under the influence of SCO 27 at

different concentrations and positive control (camptothecin 5 µM) Arrows

indicate cells in a state of apoptosis

b Evaluate the ability of SCO 27 to stimulate production of the enzyme caspase 3

Figure 3.6 The ability to stimulate production of caspase 3 of SCO 27;

sco-10 µM; sco-5 µM and sco-2,5 µM: The analyzed samples were

supplemented with SCO 27 at the respective concentrations Control: Analytical sample with no added SCO 27 compound Camptothecin:

analytical sample supplemented with camptothecin

c Determination of the ability of SCO 27 to induce apoptosis in lung

cancer cell line A549

concentrations of 5

µM (C), 10 µM (D), negative control (A) and positive control

Method of dyeing Annexin V/PI

Table 3.4 Apoptosis rate under the influence of SCO 27 on lung cancer

cell line A549

Compounds

The rate of survival cells (%)

The rate of early apoptosis cells (%)

The rate of late apoptosis cells (%)

The rate

of necrotic cells (%)

3.3.4.2 Study on cytotoxicity mechanism of compound SLE 27 in breast

cancer cell line MCF-7

a Evaluation of the effect of SLE 27 on morphological change in cancer

cells

Figure 3.8 Effects of compound SLE 27 at concentrations of 10, 30 and

100 µM on morphology of breast cancer cell line MCF-7 Arrows indicate cells in a state of apoptosis 10X and 20X magnification Control: negative

control - MCF-7 cells not supplemented with SLE 27 compound

b Determination of induction apoptosis of breast cancer cell line MCF-7 of

compound SLE 27

Table 3.5 Apoptosis rate under the influence of SLE 27 on the breast

cancer cell line MCF-7

Compounds

The rate of survival cells (%)

The rate of early apoptosis cells (%)

The rate of late apoptosis cells (%)

The rate of necrotic cells (%)

Table 3.6 The rate (%) of MCF-7 cells in G0/G1, S, G2/M phases and

apoptosis (sub-G1) after 48 hours of treatment with SLE 27 compound

Compounds

The rate of cells in all phases of the cell division

cycle (%) sub-G1

phase

G0/G1 phase

S phase

G2/M phase

at 48 h, using

cytometer Novocyte system

CHAPTER 4: DISCUSSIONS

4.1 Determination of species of soft coral based on DNA makers

After cloning and sequencing of marker gene segments from 3 soft coral

samples, the DNA sequences of Sinularia leptoclados (MW077896, MW077906); Sinularia conferta (MW077897, MW077907) and Sinularia

nanolobata (MW077898, MW077908) are registered on the NCBI

Sinularia-abruptaMF817864 Sinularia-slieringsiMH516803 Sinularia-penghuensisJX991181 Sinularia-molestaJX991172 Sinularia-abrubtaFJ621374 Sinularia-abruptaJX991168 Sinularia-leptocladosKC542857 SLE

Sinularia-compactaFJ621384 Sinularia-bisulcaFJ621378 Sinularia-acutaFJ621375 Sinularia-verseveldtiKC542859 Sinularia-robustaFJ621473 Sinularia-diffusaFJ621399

Sinularia-sp.KF915757 Sinularia-abhishiktaeFJ621373 Sinularia-tumulosaFJ621482 Sinularia-siaesensisFJ621478 Sinularia-polydactylaKU230374 Sinularia-confertaFJ621389 SCO

Sinularia-peculiarisJX023274 Sinularia-ornataJX991173 Sinularia-nanolobataFJ621451 SN

64 50

99

66

46

30 37

99 46

48 47

0.01

Figure 4.1 The results of classification analysis by the NJ

(Neighbor-Joining) method on MEGA6 of soft coral samples based on msh1 gene

sequence polymorphism of the studied samples and related soft corals samples on NCBI The tributary numbers are the Bootdtrap values that represent the reliability of the genetic branching of the sequences and

groupings

Analysis of genotypes of the msh1 gene segment based on the nucleotide

sequence was performed on 3 study sequences and 23 reference sequences

on NCBI The results in Figure 4.1 show that the SLE sample has a high

genetic similarity with the species that has been sequenced in the world

gene bank, S leptoclados KC542857 (bootstrap 66%) The SCO sample