Summary of Chemistry Doctoral thesis: Study on chemical constituents and biological activities from Culcita Novaeguineae Müller & Troschel, 1842 and Pentaceraster Gracilis (Lutken, 1871) in

The objective of the thesis is to study to clarify the main chemical composition of two species C.novaeguinea and P. gracilis in the Northeast sea of ​​Vietnam. Evaluate the cytotoxic activity of some isolated compounds to search for bioactive compounds.

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

Bui Thi Ngoan

STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM

CULCITA NOVAEGUINEAE MÜLLER & TROSCHEL, 1842 AND PENTACERASTER GRACILIS (LUTKEN, 1871) IN VIETNAM

Major: Organic chemistry

Code: 62.44.01.14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

Hanoi - 2018

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

Adviser 1: Prof Dr Chau Van Minh

Adviser 2: Dr Nguyen Hoai Nam

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 2018

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

The starfish are invertebrates belonging to the class Asteroidea, phylum Echinodermata Following the classification of Blacke (1987), the class Asteroidea including Brisingida, Forcipulatida, Notomyotida, Paxillosida, Spinulosida, Valvatida, and Velatida From the 1997 to 2007, the aproximately 98 starfish species were investigated on the chemical conponents as the report of Guang Dong et al The secondary metabolites from the starfish are characterized by a diversity of polar steroids, including polyhydroxylated steroids and steroid glycosides There are two main structural groups of steroid glycosides from the starfish, namely

compounds showed several biological properties, such as cytotoxic, hemolytic, and anti-microbial effects

In the Vietnam sea, the starfish belong to the phylum Echinodermata, which was known as the 350 species of 58 phylums, and are divided into five classes Holothuroidea (sea cucumbers), Asteroidea (starfishes), Echinoidea (sea urchins and sand dollars), Crinoidea (crinoids and sea lilies), and Ophiuroidea (brittle stars and basket stars) Up to date, the chemical constituents and biological acivities of 9 starfish species abundant in the Vietnam sea were

studied, including Archaster typicus, Asterina batheri, Asteropsis

carinifera, Astropecten polyacanthus, Astropecten monacanthus, Protoreaster nodosus, Acanthaster planci, Linckia laevigata, and Anthenea aspera Their structures were determined as the steroid and

steroid glycoside which displayed the cytotoxic activity and inlammatory

anti-In this study, the chemical components and biological

activities of two starfish C novaeguineae and P gracilis in Vietnam

were identified The results will be contributed in the medicinal and pharmaceutical industry as an important role for the discovery and development of new drugs Therefore, thesis title was chosen to be:

“Study on chemical constituents and biological activities from

Culcita novaeguineae Müller & Troschel, 1842 and Pentaceraster gracilis (Lutken, 1871) in Vietnam”

2 The objectives of the thesis

 Investigated on the chemical constituents of the two starfish C

novaeguinea and P gracilis in the East-North sea of Vietnam

 Studied on the cytotoxic activity of the isolated compounds to find the bioactive compounds

3 The main contents of the thesis

The extraction and isolation of compounds from the starfishes C

novaeguineae and P gracilis in Viet Nam using the chromatography

methods

 The structure determination of the isolated compounds based

on the physical and chemical methods

 Studied the cytotoxic activity of the isolated compounds

Numerous studies on the chemical components of the starfish

in the world were reported The steroid, glycoside (glycoside of polyhydroxysteroid, asterosaponin and cyclic steroid glycoside )

were the main groups with the cytotoxic, antibacteria, and inflammatory activities

anti-1.2 The previous studies about the chemical constituents and biologycal activity from the starfish in Vietnam

Up to date, the several starfishes were investigated the

chemical constituents from the Vietnam sea, including Archaster

typicus, Asterina batheri, Asteropsis carinifera, Astropecten polyacanthus, Astropecten monacanthus, Protoreaster nodosus, Acanthaster planci, Linckia laevigata, and Anthenea aspera The

polyhydroxysteroides, asterosaponin were evaluated with their cytotoxic and anti-inflammatory activities

CHAPTER 2 EXPERIMENT AND RESULTS

2.1 Materials

2.1.1 The starfish Culcita novaeguineae

The starfish C novaeguineae was collected at Quang Ninh,

Vietnam, in October 2013, and identified by Prof Do Cong Thung

The starfish P gracilis was collected at Bac Van, Co To,

Quang Ninh, Vietnam, in March 2014, and identified by Prof Do Cong Thung

2.2 Methods

2.2.1 The extraction and isolation of compounds methods

The combination of the chromatography methods including thin

chromatography, and the column chromatography (CC)

2.2.2 The structure determination of isolated compound methods

The structure identification is the combination of the physical properties and the model spectroscopic methods, including the Electrospray Ionisation Mass Spectrometry (ESI-MS), the High-

and Nuclear magnetic resonance spectroscopy (NMR)

2.2.3 The cytotoxic activity assay

The cytotoxic activity of the isolated compounds was

2.3 Extraction and isolation of compounds

2.3.1 The extraction and isolation of compounds from Culcita novaeguineae

This part showed the extraction and isolation experiments of

the compounds isolated from the starfish C novaeguineae

C8.5 (560 mg)

C8.5A,B C8.5C (46mg

CC, Silicagel EMW 10/1/0,1 C8.5D

(18mg) C8.5E (76mg)

C8.5F (150mg)

C8.5G (150mg)

CN8

(3,5mg)

Sephadex LH-20 M/W 2/1

CH2CL2 (15,2 g)

C8.6 (185 mg)

Figure 2.4-6 The extraction and isolation of compounds isolated from the

water layer of C novaeguineae

2.3.2 Extraction and isolation of compounds from P gracilis

This part showed the extraction and isolation experiments of

compounds from the starfish P gracilis

Sephadex MW: 1/1

PG1

W 3 D 1.3 g

PG4

10 mg

PG6

Sephadex MW: 1/1 Sephadex MW: 1/1

2.4 Physical properties and spectroscopic data of the isolated

compounds from C novaeguineae and P gracilis

2.4.1 Compound CN1: Novaeguinoside E (New compound)

-quinovopyranosyl)-oxy]-5α-pregn-9(11)-ene-20-one-3β-yl-sulfate

White powder; Molecular formula C39H68O13

1H-NMR

IV.1.6)

2.4.7 Compound CN7: Halityloside B

1H-NMR

Table IV.1.7)

2.4.8 Compound CN8: Halityloside A

1H-NMR

IV.1.8)

2.4.9 Compound CN9: heptol

1H-NMR

Table IV.1.9)

2.4.10 Compound PG2: Protoreasteroside

Table IV.2.1)

2.4.11 Compound PG1: Maculatoside

Table IV.2.2)

2.4.12 Compound PG3: Pentaceroside A (New compound)

1

13

2.4.13 Compound PG4: Pentaceroside B (New compound)

IV.2.5)

2.4.15 Compound PG6:

1H-NMR

IV.2.6)

2.4.16 Compound PG7: heptol

1H-NMR

Table IV.2.7)

2.5 Results on cytotoxic activity of compounds

The cytotoxic activity of isolated compounds was investigated on five cancer cell lines, including LNCaP (human

prostate carcinoma), MCF7 (human breast cancer), KB (human epidermoid carcinoma), HepG2 (human hepatoma), and SK-Mel-2 (human melanoma) The all experiments were evaluated at the Biologycal activity laboratory in the Institute of Biotechnology

2.5.1 Cytotoxic effects of compounds isolated from C novaeguineae

Table 4.1 IC50 values of compounds isolated from C novaeguineae

on five human cell lines

a

Positive control

2.5.2 Cytotoxic effects of the compounds isolated from P gracilis

Table 4.2 The IC50 value of the compounds isolated from

Pentaceraster gracilis on the five line cells

-fucopyranosyl-(l 2)-O-β-D -galactop yranosyl-(l 4)-O-[β-D -quinovopyra nosyl-(l 2)]-O-β-D -xylopyranosyl- (l 3)-O-β-D -quinovopyranosyl)-oxy]

-5α-pregn-9(11)-ene-20-one-3β-yl-sulfate

CN3: Linckoside B

CN7: Halityloside B CN8: Halityloside A CN9: 5α-cholestane-

3β, 6β,7α,8β,15α,16β,26-heptol

3.1.1 Compound CN1: Novaeguinoside E (New compound)

powder Its molecular formula was determined as C56H91NaO27S by high-resolution electrospray ionization mass spectrometry (HR-ESI-

MS) at m/z 1273.5257 [M + Na]+ The NMR features indicated an asterosaponin, one of the main constituents of starfish The 1

H and 13

C NMR data (in DMSO-d6) of CN1 were similar to those of

protoreasteroside, except for differences in the data of sugar moieties

274.2765

353.2659 413.2667

566.2132 648.2584

803.5400

1273.5257 +MS, 1.4min #83

Figure 3.1 The HR-ESI-MS spectra of CN1

Figure 3.2a 1H-NMR spectrum of CN1 in DMSO-d6

Figure 3.2 1H-NMR spectrum of CN1 in pyridine-d5

Figure 3.3 13C-NMR spectra of CN1

Analysis of 1D and 2D NMR spectra confirmed that the

aglycone of CN1 contained three oxymethine groups [δC 75.2 (C-3),

78.1 (C-6), and 76.2 (C-22)/δ 3.83–3.87 (H-3), 3.45–3.47 (H-6), and

3.04–3.06 (H-22), each 1H, m], one oxygenated quaternary carbon

atom [δC 75.1 (C-20)], two trisubstituted double bonds [δC145.2 (s,

C-9) and 115.7 (d, C-11)/δH 5.24 (1H, d, J = 5.0 Hz, H-11); δC 124.0

(d, C-24)/δH 5.20 (1H, t, J = 7.0 Hz, H-24) and δC 130.4 (s, C-25)],

and five tertiary methyl groups [δC 13.0 18), 19.0 19), 19.9

(C-21), 17.8 (C-26), and 25.6 (C-27)/δH 0.72 (H-18), 0.88 (H-19), 1.06 (H-21), 1.55 (H-26), and 1.65 (H-27), each 3H, s] Locations of the oxygenated quaternary carbon atom at C-20, one oxymethine group

at C-22, and one double bond at C-24/C-25 were identified by 1H–1H correlation spectroscopy (COSY) peaks of H-22/H-23/H-24 and combination with heteronuclear multiplebond correlation (HMBC)

cross-peaks of H-21 (δH 1.06) with C-17 (δC 75.1)/C-22 (δC

53.8)/C-20 (δC 76.2) and those of H-26 (δH 1.55)/H-27 (δC 124.0)/C-25 (δH

1.65) with C-24 (δC130.4) Detailed analysisof the other HMBC and COSY peaks unambiguously identified the planar structure of the aglycone

Figure 3.4 HSQC spectrum of CN1

In the rotating frame Overhause effect spectroscopy

observed between H-6 (H 3.45-3.47) and H-8 (H 1.97-1.99)/H-19

the borientation of H-6 (Figure 3.8) The13C NMR chemical shift of C-21 at  19.9 (in DMSO-d ) indicated the relative R* configuration

at C-20 [3] For determination of the stereochemistry at C-22, the1

chemical shift of H-21 at H 1.64 (pyridine-d5) clearly indicated the relative S* configuration at C-22

Figure 3.5 The COSY spectrum of CN1 Figure 3.6 The HMBC spectrum of CN1

Table 3.3 1H and 13C NMR spectroscopic data of CN1

mult (J = Hz)

HMBC (H  C)

Figure 3.7 ROESY spectrum of CN1 Figure 3.8 COSY, HMBC, and ROESY correlation of CN1

anomeric protons (each 1H, d, J = 7.5 Hz) at H 4.31 1), 4.53

compounds was only observed in the fourth sugar moiety The 13NMR chemical shifts of this sugar in DMSO-d6 at C 100.1 (C-1),

by hypothetic biosynthesis with the coexistence of compounds CN1

and novaeguinoside A, containing the same pentaglycoside chain in

the starfish C novaeguineae.

Figure 3.9 1D TOCSY spectrum of CN1

In the 1D TOCSY of CN1, the magnetization transfer of

determined the protons of each sugars The HMBC correlation of the

anomeric proton H-1 (H 4.21) with C-2 (C 81.5), H-1 (H

the fucose II at C-2, fucose I at C-4, quinovose II at C-2 and

the common attachment position of the pentaglycoside chain at C-6 of

anomeric protons indicated all β-glycosidic linkage Configuration of

all five sugars was assigned as D by analogy with novaeguinoside A and all the reported asterosaponins Consequently, the structure of

22- tetrahydroxy-5α-cholesta-9(11),24-dien-3β-yl sulfate This is the

new compounds and named novaeguinoside E.

3.2 The struture elucidation of compounds isolated from the starfish P gracilis

This part exhibited the structure determination of seven

compounds from the starfish P gracilis

PG3: Pentaceroside A (new compound) PG4: Pentaceroside B (new compound)

Pentaceraster gracilis as the white powder

The 1D and 2D NMR data of PG1 was displayed that PG1 is

a glycosid with the common signal of asterosaponin skeleton

contained 5 sugar units The aglycon of PG1 is a steroid with the

presence of double bond at 9(11) and one keton group

Figure 4.1 1H-NMR spectrum of PG1 Figure 4.2 13C-NMR spectrum of PG1

Figure 4.3 HSQC spectrum of PG1 Figure 4.4 COSY spectrum of PG1

Figure 4.5 HMBC spectrum of PG1 Figure 4.6 ROESY spectrum of PG1

Comparision of the NMR spectra data of PG1 with those of

the protoreasteroside showed that the main differrent of two compounds is the structure of the branched The NMR spectra of this

part of PG1 contained the chemical signal of quarternary carbon linked

oxygene, one tert-metyl group, one keton and two sec-metyl groups

Comparision of the NMR data of PG1 and the previous study,

combination with the correlation in the HSQC, HMBC, COSY and

ROESY spectra was determined the structure of PG1 was

maculatoside

Table 4.4 NMR spectroscopic data of PG1

C of protoreasteroside [3], b measured in pyridine-d 5 , c 125 MHz, d 500 MHz, *overlapped

4.4 Cytotoxic activity of the isolated compounds

4.4.1 Cytotoxic effects of the compounds isolated from the starfish

C novaeguineae

The results on cytotoxic activity of isolated compounds from

C novaeguineae showed that compounds CN5-CN8 exhibited

cytotoxic effects with IC50 value ranging from 31,80  1,59 to 92,04

Additionally, compound CN5 (halityloside D displayed the

4,11 µM) Halityloside B (CN7) exhibited moderate activity against

A (CN8) showed the moderate or weak activities on LNCaP, MCF7,

4.1.2 The cytotoxic activity of the isolated compounds from the starfish Pentaceraster gracilis

The results on cytotoxic activity of the isolated compound

from P gracilis was showed that the maculatoside (PG1) exhibited

CONCLUSIONS

This is the first study of chemical constituents and biological

activities from Culcita novaeguineae and Pentaceraster gracilis

which were collected in the Vietnam sea

on the model methods, including:

linckoside B (CN3), halityloside E (CN4), halityloside D (CN5),

2 Biological activity

The cytotoxic effects of the isolated compounds were

investigated in vitro in five human cancer cell lines: LNCaP, MCF7,

KB, Hep-G2, and SK-Mel2 The results showed that:

Halityloside D (CN5) exhibited significant cytotoxic activity

against LNCaP , MCF7 , KB, and SK-Mel2 cells and the weak effect on

Hep-G2 cell Halityloside B (CN7) showed significant cytotoxic effect

against LNCaP, MCF7, and KB cells; the moderate acitivity in

SK-Mel-2 cell and the weak activity in HepGSK-Mel-2 cell Two compounds, culcitoside

effect against LNCaP, MCF7, KB, and SK-Mel-2 cell , and inactive

Maculatoside (PG1) exhibited significant activity on LNCaP, KB ,

HepG2, and SK-Mel2 cells, and the moderate activity on MCF7

Compound PG7 showed the weak efffect on LNCaP, HepG2, and