Nghiên cứu phân lập, chuyển hóa và đánh giá tác dụng sinh học của steroid từ loài sao biển acanthaster planci tt tieng anh

MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCES AND TECHNOLOGY

-----------------------------

DINH THI HA

ISOLATION, CHEMICAL MODIFICATION AND BIOLOGICAL
EVALUATION OF STEROIDS FROM THE STARFISH
ACANTHASTER PLANCI

Speciality: Natural Products Chemistry
Code: 9 44 01 07

ABSTRACT DISSERTATION Dr. CHEMISTRY

HANOI-2020


This dissertation was completed in: Graduate university of Sciences and
Technology – Vietnam Academy of Science and Technology.

Supervisor 1: Assoc. Prof. Tran Thi Thu Thuy
Supervisor 2: Assoc. Prof. Ngo Dai Quang

Reviewer 1:

Reviewer 2:
Reviewer 3:

The thesis will be defended in front of the Academy Thesis
Evaluation Council at the: Graduate university of Sciences and Technology
– Vietnam Academy of Science and Technology.
At the time..… hour…., August…, 2020

Thesis can be found at the:
- Library of Graduate university of Sciences and Technology
- Vietnam National Library


INTRODUCTION
1. Reason to choose the topic
Vietnam has extremely diverse and abundant marine resources with
hundreds of thousands of different species of plants, animals and
microorganisms... However, the values provided for medicinal resources of
marine species is still very limited. Nowaday, there have been many steroid
subtances isolated from marine organisms and evaluated for biological activity. In
particular, many polar steroid compounds exhibit biological activity against many
cancer cell lines, and can be applied in medicine and pharmacy. These steroids are
important to organic synthesis. They have become a model to synthesize from
popular natural steroids by more efficient and economical methods.
The starfish class belong to the echinoderm phylum, which is one of the
rich sources provide of polarizing steroid compounds. This substance class has
diverse structure and they exhibit many biological activities such as: antibacterial,
antifungal, anti-inflammatory, anti-cancer, anti-viral, inhibiting fertilization.... So,
polar steroids from starfish have been attracting the attention of many scientists
around the world. In Vietnam, there is very little research on this substance class.

Studies are only in the direction of isolating natural compounds and evaluating
some of their biological activity. At present, there has not been any overall
research on isolation, chemical metabolism and evaluation of the bioactivity of
steroids isolated from marine organisms. Starfish Acanthaster planci is a common
starfish in Vietnam's waters, they are a threat to the survival of living coral reefs
because coral is their preferred food source. Preliminary studies also show that the
main chemical composition of starfish Acanthaster planci is steroids, especially
polar steroids. Follow this direction, the author to choose the research topic of the
thesis: "Isolation, chemical modification and biological evaluation of
steroids from the starfish Acanthaster planci ".
2. Research objectives of the thesis
1


Study on chemical composition of starfish Acanthaster planci of Vietnam,
synthesizing hydroxyl and oxime derivatives from a steroid isolated from this
starfish and assessing biological activity of isolated and synthesized compounds.
3. The main research content of the thesis
To achieve the above objectives, the thesis has implemented the following contents:
•
•
•

Isolation and determination of chemical structure of compounds from
starfish Acanthaster planci, especially steroids
Convert derivatives in the direction of hydroxylation and oximelation
from a high steroid in starfish Acanthaster planci.
Evaluation some biological activities of isolated and synthesized compounds.
CHAPTER 1. OVERVIEW DOCUMENT

The literature review is a collection of national and international research on:
1.1. General introduction to starfish class (Asteroidea)
1.2. Studies on polar steroids from the starfish
1.3. Biological activity of polar steroids from the starfish
1.4. Overview of research subjects
1.5. Situation of research on synthesis of polyhydroxysteroid and
hydroximinosteroid compounds from natural steroids
CHAPTER 2. SUBJECT AND METHODS OF THE STUDY
This section describes information of research subject; methods of isolation,
structure determination of compounds and methods of evaluation of biological activity
2.1. Research subject
Starfish Acanthaster planci was collected at a depth of 5-10 m, in Van
Phong Bay, Nha Trang, Khanh Hoa, Vietnam. Species name was determined by
Assoc. Do Cong Thung, Institute of Marine Resources and Environment Vietnam Academy of Science and Technology.
2


The upper side of the sample

The underneath of the sample

2.2. Research methods
2.2.1. Method of isolation of compounds
The chromatographic methods used to isolate compounds including: thin-layer
chromatography (TLC), normal or reverse-phase silica gel column chromatography
(RP-C18), Polychrome1, Sephadex LH-20 column chromatography, highperformance liquid chromatography (HPLC), method of crystallization also used.
2.2.2. Methods of determining structure
Common methods for determining the chemical structure of compounds
include: Electrospray Ionization Mass Spectrometry (ESI-MS), High-resolution
Electrospray Ionization Mass Spectrometry (HR ESI-MS), extreme rotation ([α]D),

the one-dimensional magnetic resonance spectrum (1H, 13C, DEPT, TOCSY1D) and
two-dimensional (HSQC, HMBC, COSY, NOESY, ROESY) recorded on Bruker
Avance 500MHz or Bruker Avance III 700MHz using TMS is internal standards.
2.2.3. The methods of activity evaluation
2.2.3.1. Cytotoxic assay
The cytotoxic activity of compounds was assessed by MTS method
performed at: Institute of Natural Products Chemistry-Vietnam Academy of
Science and Technology (Hep G2, HeLa); Research Center for Natural
Compounds - Korea Institute of Science and Technology (T98G); Pacific Institute
of Organic Biochemistry - Russian Federal Academy of Sciences - Vladivostok
(HCT-116, HT-29, RPMI-7951, T-47D, MDA-MB-231).
2.2.3.2. Soft agar assay
The test substances added to the cell culture medium at non-toxic
concentrations 5, 10 and 15 μM, incubated for 4 weeks. The tumors formed were
3


measured using a microscope and imaging software using Colburn's method. The
test was done at the Pacific Institute of Organic Biochemistry - Russian Federal
Academy of Sciences - Vladivostok.
2.2.3.3. Migration-cell Wound-healing assay
The test substances (at concentration 10 μM) were added into the medium
that was prepared a wound on MDA-MB-231 mammary carcinoma cell and check
after 48 hours. The closure of the lesion area was measured and determined the
percentage of distance traveled by the cell. The test was done at the Pacific Institute
of Organic Biochemistry - Russian Federal Academy of Sciences – Vladivostok.
CHAPTER 3. EXPERIMENT
3.1. Isolation of compounds from starfish Acanthaster planci
This section details how to isolate compounds from A. planci starfish.
The separation of compounds was summarized in the diagram in Figure 3.1.

Figure 3.1. Diagram of isolated compounds from starfish A.planci
4


3.2. Physical constants and spectrum data of isolated compounds
3.3. Summary of cholesterol derivatives
AP7 compound was identified as cholesterol, selected as the start material
to convert into polyhydroxysteroid and hydroximinosteroid derivatives.
3.3.1. Synthesis of polyhydroxyl derivatives of cholesterol
3.3.2. Synthesis of hydroximinosteroid derivatives from cholesterol
3.4. Biological activity of isolated compounds and synthetic derivatives
3.4.1. Biological activity of polar steroid compounds isolated from starfish
Acanthaster planci
3.4.2. Cytotoxic activity of cholesterol derived derivatives
The hydroxysteroid compounds (15c-21c), hydroximinosteroid compounds
(23c, 25c, 29c, 31c) and intermediate compounds (22c, 24c, 26c-28c, 30c) were
evaluated for cytotoxic activity on three cancer cell lines as Hep G2, HeLa and T98G.
CHAPTER 4. RESULTS AND DISCUSSION
This chapter presents the results of research on isolating, metabolizing and
determining the structure of compounds, results of cytotoxic activity, tumor suppression
activity on soft agar and inhibitory activity metastases of the tested compounds.
4.1. Study on chemical composition of starfish Acanthaster planci
This section details the results of the structure determination of 14 compounds
isolated from A. planci, including 4 new compounds and 8 known compounds.
Table 4.21: Summary table of substances isolated from starfish A.planci

AP1. Planciside A (new compound)

AP2. Planciside B (new compound)

5


AP4. Planciside D

AP3. Planciside C (new compound)

AP5. 3-O-sulfothornasterol A

AP6. 5-ergost-7-en-3-ol

AP7. Cholesterol

AP8. Astaxanthin

AP9. Thymin

AP10. Uracil

AP11. Acanthasglycoside G
(new compound)

AP12. Pentareguloside G

AP13. Acanthasglycoside A

AP14. Maculoside
6

* The following shows the spectral analysis and chemical structure
determination of a new glycoside steroid called Acanthaglycoside G (AP11).
•

AP11 compound: Acanthaglycoside G (new compound)
AP11 compound was isolated as solid, amorphous. The molecular
formula of AP11 was determined to be C51H81O26SNa (M = 1164) from the
[M+Na]+ sodium adduct ion peak at m/z 1187,4533 (theoretical calculations for
molecular formula is C51H81Na2O26S = 1187,4532) in the (+) HR-ESI-MS
spectrum and the [M–Na]− ion peak at m/z 1141,4735 (theoretical calculations
for molecular formula is C51H81O26S = 1141,4737) in the (-) HR-ESI-MS, where
M is the molecular mass of the intact sodium salt. In addition, the (–) HR-ESIMS/MS spectrum of the [M–Na]− ion at m/z 1141 exhibited fragment ion peak
at m/z 995 [(M–Na)–C6H10O4]−, 849 [(M–Na)–2xC6H10O4]−, 703 [(M–Na)–
3xC6H10O4]−, 557 [(M–Na)–4xC6H10O4]−, 411 [(M–Na)–5xC6H10O4]−,
corresponding to the successive losses of one, two, three, four, and five 6deoxyhexose units, respectively, and 393 [(M–Na)–4 x C6H10O4 –C6H12O5]−,
corresponding to the loss of an oligosaccharide chain in AP11. The MS
spectroscopic data confirmed the presence of five 6-deoxyhexoses in the
carbohydrate moiety of AP11.
Intens.
x105
4

3

3+
1187.4533

Intens.
x106
4

+MS, 0.8-1.4min #44-83

-MS, 1.3-1.8min #76-100

3

1+
437.1941

2+
605.2223

2

2

1+
526.3922

1

11141.4735

2570.2335

1

1163.4545

1+
1269.4566

497.2043
774.6342

977.4046

424.1754

1367.4546

0

849.3573

1127.4573

995.4151

0
400

500

600

700

800

900

1000

1100

1200

1300

400

m/z

Fig. 4.21: (+) HR-ESI-MS spectrum of AP11

500

600

700

800

900

1000

1100

1200

m/z

Fig. 4.22: (-) HR-ESI-MS spectrum of AP11

The 1H-NMR spectrum of AP11 showed signals of 3 methyl groups,
characterized by steroid nucleus including 2 methyl groups CH3-18 (s, 0.58
ppm) and CH3-19 (s, 0.94 ppm); 1 signal in CH3-21 weaker field (s, 2.08 ppm).
7


There is also the signal of a proton olefin at δH 5.24 (brt, J 4.2 Hz); an oxymetin
group linked to a sulfate group at δH 4.87 (m) ppm; an oxymetin group is
directly linked to a carbohydrate chain at the CH-6 position with δH 3.78 (m). In
the weak field region, five doublet signals of five proton anomers of 5
monosaccharide units at δH 4.82 (1H, d, J = 7.6 Hz), 4.84 (1H, d, J = 7.9 Hz),
4.97 (1H, d, J = 6.8 Hz), 5.03 (1H, d, J = 7.7 Hz), 5.27 (1H, d, J = 7.7 Hz).

Fig. 4.23: 1H-NMR spectrum of AP11

Fig. 4.24: 13C-NMR spectrum of AP11

The 13C-NMR spectrum of AP11 showed the presence of 51 carbon
atoms, including: eight CH3 groups, seven CH2 groups, thirty two CH groups
and four quater carbons. The presence of olefin carbon was triple substituted
in the molecule was determined at δC 116.4/145.8 ppm. Five carbon CH
signals at δC 102,3; 103,6; 104,9; 104,9; 106,9 ppm were identified as carbon
anomers of the five sugar units. In addition, in the region of 60 - 90 ppm,

there are resonance signals of 23 carbon atoms directly linked to the oxygen
atom, including 22 carbon oximetin with 2 carbon of aglycon fraction at δC
77,4 (C-3) bind to sulfate group; δC 80,1 (C-6) linked to carbohydrate chain;
and 20 carbons of sugar molecules at δC 73,8; 91,0; 74,4; 71,7; 82,3; 75,1;
85,7; 71,4; 84,3; 77,5; 75,7; 72,8; 73,7; 74,9; 72,3; 71,8; 76,2; 77,5; 75,5;
73,4 ppm; and a carbon of C=O group at δC 208,0 ppm.
The sequences of proton at C-1 to C-8, C-11 to C-12, and C-8 to C-17
were ascertained from the COSY and HSQC experiments. The HMBC
8


correlation H3-18/C-12, C-13, C-14, C-17; H3-19/C-1, C-5, C-9, C-10; and H321/C-17, C-20 supported the whole structure of the steroidal aglycon. The key
ROESY correlations, such as H-5/H-3α, H-7α; H-14/H-12α, H-17; H3-18/H-8,
H-15β, H-16β; and H3-19/H-2β, H-4β, H-6β, H-8 confirmed
5α/8β/10β/13β/14α/17α steroidal nucleus and 3β, 6α relative configurations of
O-bearing substitutions in AP11. In HMBC spectrum, there is interaction of
proton anomer H-1 of Qui1 unit at δH 4,82 ppm with C-6 at δC 80,1 ppm of
aglycon, and in ROESY spectrum there is interaction of proton anome H-1 of
Qui1 at δH 4,82 ppm with H-6 at δH 3,78 ppm of aglycon. This proves that the
position linked of oligosaccharide chain to aglycon is C-6 position.

Fig. 4.25: 1D TOCSY spectrum of AP11
1D TOCSY experiments with the irradiation of anomeric proton indicated
the resonaces H-1 – H-6 of four quinovose units and H-1 – H-4 of one fucose
units, whereas the irradiation of the resonance of the corresponding methyl
group resulted in a signal for H-5 of the fucose unit. The positions of the
interglycosidic linkages and the attachment of the oligosacchride moiety to the
steroidal aglycon at C-6 were elucidated from long-range correlations in the
ROESY abd HMBC spectra. The key cross-peaks between H-1 of Quip1 and H6 (C-6) of aglycon, H-1 of Quip2 and H-3 (C-3) of Quip1, H-1 of Quip3 and H-4
(C-4) of Quip2, H-1 of Fucp and H-2 (C-2) of Quip3, H-1 of Quip4 and H-2 (C-2)

of Quip2 were detected. Therefore, it is possible to infer the position of the link
between sugar units and between oligosaccharide moiety to the steroidal
9


aglycon is

Fuc(1→2)-Qui3(1→4)-[Qui4(1→2)]-Qui2(1→3)-Qui1-Aglycon.

Based on the signals on the 1D-, 2D-NMR spectrum, we can determine the
carbon and proton values of oligosaccharide moiety (see Table 4.9).

Fig. 4.26: Chemical structure of AP11

Fig. 4.27: Key interactions in the HMBC and ROESY spectrum of AP11
The configuration of the sugar units in AP11 was demonstrated by the
method of Leontein et al, the results of the GC spectral analysis showed that
all of sugar units of AP11 was D-configuration.
On the basis of the above mentioned data, the structure of AP11 was
established to be sodium 6α-O-{β-D-fucopyranosyl-(1→2)-β-D-quinovopyranosyl(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovo-pyranosyl-(1→3)-β-Dquinovopyranosyl}-6α-hydroxy-5α-pregn-9(11)-en-20-one-3β-yl sulfate, and named
Acanthaglycoside G. Acanthaglycoside G is rare asterosaponin, the carbohydrate
moiety of which includes only β-D-fucopyranosyl and β-D-quinovopyranosyl units.
Thus, Acanthaglycoside G (AP11) is the first new substance isolated from nature.
10


Table 4.8: Aglycon spectrum data of AP11
Cac

Hab (JHz)

35,8

63,2
12,9

1,63 m
1,38 m
2,81 (brd, J 13,6 Hz)
1,89 (brq, J 12,5 Hz)
4,87 m
3,45 (brd, J 12,6Hz)
1,70 m
1,48 m
3,78 m
2,66 m
1,28 m
2,06 m
‒
‒
5,24 (brt, J 4,2 Hz)
2,14 brs
‒
1,33 m
1,76 m
1,20 m
2,34 (brq, J 10,9 Hz)
1,61 m
2,51 (t, J 8,7 Hz)
0,58 s

19

19,0

0,94 s

20
21

208,0
30,8

‒
2,08 s

C
1β
1α
2α
2β
3
4α
4β
5
6
7β
7α
8
9

10
11
12
13
14
15α
15β
16β
16α
17
18

a

29,3
77,4
30,6
49,1
80,1
41,3
35,4
146,0
35,8
115,8
40,4
42,3
53,5
25,3
23,0

ROESY (H→H)
H-11, H3-19
H-3, H-11

HMBC (H→C)

H3-19
H-1, H-5
H3-19
H-3, H-7
H3-19
H-5
H3-18, H3-19

H-1
H-14, H-17
H-12, H-17
H3-18
H3-18
H-12, H-14, H3-21
H-8, H-15, H-16
H-1, H-2, H-4, H-6,
H-8
H-17

C-13, C-18
C-12, C-13, C-14,
C-17
C-1, C-5, C-9, C-10

C-17, C-20

C5D5N, b 500,13 MHz, c 125,75 MHz

Table 4.9: Oligosaccharide NMR spectrum data of AP11
C
Qui 1
1

δCac

δHab (JHz)

104,9

4,82 (d, J 7,6 Hz)

2

73,8d

3,97 (t, J 8,3 Hz)

ROESY
H-6 of aglycon;
H-3, H-5 Qui1

HMBC (H→C)
C-6 of aglycon
C-1, C-3 Qui1

11


3
4
5
6
Qui 2
1

91,0
74,4
71,7d
18,2

3,77 (t, J 8,6 Hz)
3,55 (t, J 9,0 Hz)
3,69 m
1,60 (d, J 6,0 Hz)

H-1 Qui1, H-1 Qui2
H-6 Qui1
H-1 Qui1
H-4 Qui1

C-1 Qui 2
C-3, C-6 Qui1

103.6

4,97 (d, J 6,8 Hz)

C-3 Qui1

2
3
4
5
6
Qui 3
1

82.3
75.1
85.7
71.4
18,0

4,09 (t, J 7,6 Hz)
4,12 (t, J 8,7 Hz)
3,56 (t, J 8,7 Hz)
3,90 m
1,73 (d, J 6,1 Hz)

H-3, H-5 Qui2; H-3
Qui1
H-1 Qui4
H-1 Qui2
H-6 Qui2, H-1 Qui3

H-1 Qui2
H-4 Qui2, H-1 Qui3

102,3

4,84 (d, J 7,9 Hz)

C-4 Qui2

2

84,3

4,00 (t, J 8,3 Hz)

H-3, H-5 Qui3; H-4, H6 Qui2
H-1 Fuc

3
4
5
6
Fuc
1
2

77,5
75,7
72,8
17,7d

4,13 (t, J 9,3 Hz)
3,62 (t, J 8,9 Hz)
3,71 m
1,48 (d, J 6,0 Hz)

H-1, H-5 Qui3
H-6 Qui3
H-1, H-3 Qui3
H-4 Qui3

106,9
73,7d

H-3, H-5 Fuc; H-2 Qui3

3

74,9

4
5
6
Qui 4
1

72,3
71,8d
16,9

5,03 (d, J 7,7 Hz)
4,41 (dd, J 8,6;
9,5 Hz)
4,06 (dd, J 3,6;
9,5 Hz)
3,99 (d, J 4,0 Hz)
3,78 (q, J 6,5 Hz)
1,49 (d, J 6,2 Hz)

H-5, H-6 Fuc
H-3, H-4 Fuc
H-4 Fuc

C-3 Fuc
C-1, C-4 Fuc
C-4, C-5 Fuc

104,9

5,27 (d, J 7,7 Hz)

H-3, H-5 Qui4;
H-2 Qui2

C-2 Qui2

C-4, C-5 Qui1

C-2 Qui2
C-3 Qui2

C-4, C-5 Qui2

C-3 Qui3, C-1
Fuc
C-2, C-4 Qui3
C-5, C-6 Qui3
C-3 Qui3
C-4, C-5 Qui3
C-2 Qui3
C-1, C-3 Fuc

H-1, H-5 Fuc

2
76,2
4,04 (t, J 8,8 Hz)
3
77,5
4,12 (t, J 8,8 Hz) H-1 Qui4
4
75,5
4,01 (t, J 8,7 Hz) H-6 Qui4
5
73,4
3,70 m
H-1 Qui4
6
17,8d 1,79 (d, J 6,1 Hz) H-4 Qui4
C-4, C-5 Qui 4
a

C5D5N, b 500,13 MHz, c 125,75 MHz, d Assignments may be interchanged

12


4.2. Chemical metabolism of cholesterol
Polyhydroxysteroid, hydroximinosteroid compounds with cholesterollike branched blood are evaluated as potential compounds with toxic activity on
many cancer cell lines. Therefore, cholesterol has been chosen as the first raw
material for metabolic reactions to produce hydroxyl and oxidized products.
4.2.1. Metabolism of polyhydroxysteroid derivatives
Cholesterol (AP7) is hydroxylated to form many -OH groups around the
C5/C6 double connection. The hydroxylation agents used are mainly oxidizing agents
and are capable of producing diol-type products. Seven hydroxysteroid compounds
(15c-21c) were synthesized from cholesterol according to the diagram 4.1 below.
Although synthetic compounds are known substances, the content of
this study provides effective synthesis methods for active substances, only
through one reaction step. These methods can also be used on other steroid
compounds from starfish to synthesize polyhydroxysteroid derivatives.

Diagram 4.1. Synthesis of polyhydroxysteroid derivatives from cholesterol
Reagents and reaction conditions: (i): BH3.THF, H2O2, NaOH, 0oC, 1h (15c: 71%,
16c: 9%); (ii): SeO2, dioxane, H2O, 80oC, 80h (17c: 50%, 18c: 2,0%, 19c: 3,5%); (iii):
4% OsO4/H2O, NMO, reflux, 48h (20c: 74%); (iv): 1. HCOOH 88%, THF/H2O2, 12h,
2. KOH 3% in MeOH (21c: 75%).
13


4.2.2. Metabolism of hydroximinosteroid derivatives
Recent studies have shown that the position of the oxime groups and
the branch circuit types at the C-17 position of the cholestane-like steroid

framework is more active than other types of branch circuits. Therefore,
cholesterol was chosen as the first raw material for the synthesis of
hydroximinosteroid derivatives. Products with an oxime group at C-3
position; or C-3 and C-6; and epoxy ring at position C-4,5. Four
hydroximinosteroid (23c, 25c, 29c, 31c) including 2 new substances (29c,
30c) and 7 intermediates (15c, 22c, 24c, 26c, 27c, 28c, 30c) including 1 new
substance (30c) has been summarized according to Figure 4.2 below.

Diagram 4.2. Synthesis of hydroximinosteroid derivatives from cholesterol
Reagents and reaction conditions: (i): PCC/CH2Cl2, rt, 48h (22c: 80,0 %, 24c:
82,0 %); (ii): BH3.THF/H2O2, NaOH, 0oC, 1h (15c: 80,0 %); (iii):
CeCl3.7H2O/NaBH4, CH2Cl2&MeOH (1:1), rt, 1h (26c: 89,0 %); (iv): 1. mCPBA/CH2Cl2, 2. Dess-Martin/CH2Cl2, 0oC (27c: 12,3 %, 28c: 14,5 %); (v):
NH2OH.HCl/Pyridine, 24h (23c: 85,0 %, 25c: 81,0 %, 29c: 19,3 %, 30c: 13,4 %,
31c: 22,5 %).
14


From cholesterol, it is metabolized in different directions to create
ketone-mediated derivatives (C = O) at C-3 and C-6 positions; in the C-4,5
position there is a double bond (22c), no double bonds (24c) or an epoxy
ring (27c). Finally, these ketones are transformed into hydroximinosteroid
products (> C = N-OH), respectively (23c, 25c, 29c, 31c) by hydroxylamine
hydrochloride (NH2OH.HCl) in pyridine by tissue method. described by
Javier. In particular, when performing chemical oxidation 27c (with epoxy
ring at position C-4,5 and ketone group at C-3), two products were obtained,
one of which was oxidized (29c) and one product that is not oxidized but
opens the epoxy ring in position C-4,5 (30c).
The products have been successfully oxidized due to the apparent change
in the chemical shift of the carbonyl group within δC 195-210 ppm to the oxime
group at δC 155-160 ppm. Combining modern physico-chemical methods and

nuclear magnetic resonance spectroscopy structures of all proven products.
4.3. Results of bioactive test
4.3.1. The activity of steroidal glycoside compounds isolated from starfish
Acanthaster planci
a. Biological activity of polyhydroxysteroid glycoside compounds
AP1 was tested for cytotoxic activity on three HCT-116, T-47D and RPMI7951 cell lines. The result of AP1 is likely to be cytotoxic on HCT-116 and RPMI7951 cell lines with IC50 values of 36 and 58 µM, compared to cisplatin-positive
control. Both AP1 and cisplatin were non-toxic on T-47D cell lines (see table 4.23).
Table 4.23: In vitro cytotoxic activity of compound AP1
Cell lines
(IC50 µM)
AP1
Cisplatin
HCT-116
36
75
T-47D
>150
> 150
RPMI-7951
58
43

15


AP1 inhibited the proliferation of T-47D cell line after 72 hours was
35%, the RPMI-7951 cell line after 48 hours was 27% (Figure 4.54 B, C).
While cisplatin almost completely inhibits the growth of T-47D and RPMI7951 cell lines (Figure 4.54 B, C).

A

B

C

AP1, 15 μM

Control

Cisplatin, 15 μM

Fig. 4.54: Effects of AP1 on the proliferation of HCT-116, T-47D and RPMI-7951
cell lines at a concentration of 15 μM

b. Biological activity of asterosaponin compounds
AP13 and AP14 compounds have the potential to cause toxic effects on
HT-29 rectal cancer cells; MDA-MB-231 mammary carcinoma cells, but not
toxic RPMI-7951 malignant melanoma cells at concentrations above 150 µM.
The value of IC50 concentration for each cell line showed in Table 4.24.
Table 4.24: Cytotoxic activity and affecting tumor formation on soft agar of
asterosaponin compounds AP11-AP14
Com.
AP11
AP12
AP13
AP14

RPMI-7951
IC50, µM IF50, µM
>150

>15
>150
>15
>150
15
>150
14

HT-29
IC50, µM IF50, µM
>150
>15
>150
>15
109
11
90
7

MDA-MB-231
IC50, µM IF50, µM
>150
>15
>150
>15
30
13
24
8

IC50: the concentration of compounds that caused a 50% reduction in cell viability of human
cancer cells; IF50: the concentration of compounds that caused a 50% reduction in colonies
formation of human cancer cells

16


AP13 and AP14 compounds effectively inhibit tumor formation of
HT-29 and MDA-MB-231 cell lines, and are less effective with RPMI-7951
cell lines. IF50 values corresponding to cell lines showed in Table 4.24.
AP13 and AP14 compounds at a concentration of 10 µM can prevent the
movement of MDA-MB-231 cells at 26% and 45%, respectively, compared to the
control after 48 hours of incubation (Figure 4.55). Meanwhile, compounds AP11
and AP12 cannot stop the movement of these cells.

Fig. 4.55: Effects of asterosaponins AP11-AP14 on MDA-MB-231 breast
adenocarcinoma migration in humans
4.3.2. Biological activity of cholesterol derivatives
a. Biological activity of polyhydroxysteroid derivatives
Three substances 16c, 18c, 21c show Hep G2 cytotoxic activity with IC50
value of 11,69; 11,89 and 6,87 μM. Only 21c exhibited T98G cytotoxic activity with
IC50 = 2,28 μM, compared with the positive control Paclitaxel (see Table 4.25).
Table 4.25: Biological activity of substances 15c-21c on Hep G2 and T98G cell lines
IC50 µM

IC50 µM

Com.
15c
16c

17c
18c

Com.
Hep G2

T98G

>100
11,59
>100
11,89

>100
>100
>100
>100

19c
20c
21c
Paclitaxela

a

Hep G2

T98G

>100

>100
6,87
0,040

>100
>100
2,28
0,023

positive control
Two pairs of substances 15c and 16c; 20c and 21c differ in the OH
group configuration at C-6 positions (6α-OH in substances 15c and 20c; 6β17


OH in substances 16c and 21c). While 16c has toxic activity on Hep G2 cell
lines and 21c has toxic activity on both Hep G2 and T98G cell lines,
substances 15c and 20c do not show activity on these tests. Thus, the
configuration of the OH group at C-6 of this type of structure may be one of
the factors determining their cytotoxic activity.
b. Biological activity of hydroximinosteroid derivatives and intermediate compounds
Three derivatives 3,6-dihydroximino (23c, 25c, 31c) have stronger
cytotoxic activity than 3-hydroximino-6α-hydroxy (29c) on 3 test cell lines.
The 3,6-dihydroximino 25c has no double bonds at position C4/5, which can
cause toxic inactivation on HepG2 and HeLa cell lines compared with Δ43,6-dihydroximino 23c. While 31c has two oxime groups at position C-3, C6 and epoxy ring at position C-4,5 with selective cytotoxic activity on T98G
cell line (IC50 = 2,9 μM ) 29c has an oxime group at C-3 and epoxy rings at
C-4/5 but does not have this activity.
Table 4.26: Cytotoxic activity of substances 22c- 31c on Hep G2, HeLa, T98G
cell lines
Com.
22c

23c
24c
25c
26c
Paclitaxel
a

IC50 (µM)
HepG2 HeLa
>100
>100
42,4
68,6
>100
>100
>100
>100
>100
>100
0,040 0,031

Com.
T98G
>100
70,3
>100
69,8
>100
0,023

27c
28c
29c
30c
31c
Paclitaxela

IC50 (µM)
HepG2 HeLa
41,8
72,4
>100
74,6
>100
>100
>100
>100
>100
>100
0,040 0,031

T98G
>100
>100
>100
18,5
2,9
0,023

positive control

Three intermediates (27c, 28c, 30c) with the presence of elemental
oxygen at C-4 or C-5 have moderately toxic activity on at least one cell line
while the other compounds not active.
As such, these steroids have double bonds at the C4/5 position or are
linked to the oxygen element at the C-4 position, C-5 may have a positive
effect on cytotoxic activity on cancer cell lines tested.
18


CONCLUSIONS
1. From the starfish Acanthaster planci was isolated 14 compounds.
The structure of these compounds was determined by mass spectrometry,
nuclear magnetic resonance spectroscopy and other physicochemical
methods. The isolated and identified compounds include: planciside A
(AP1); planciside B (AP2); planciside C (AP3); planciside D (AP4); (3-Osulfothornasterol A (AP5); 5-ergost-7-en-3-ol (AP6); cholesterol (AP7);
astaxanthin (AP8); thymin (AP9); uracil (AP10); acanthaglycoside G
(AP11); pentareguloside G (AP12); acanthaglycoside A (AP13); and
maculoside (AP14).
Among them, four steroidal glycoside are discovered for the first time
from nature, including three polyhydroxysteroidal glycosides, namely
planciside A (AP1); planciside B (AP2); planciside C (AP3); and an
asterosaponin, acanthaglycoside G (AP11). In addition, the compound
pentareguloside G (AP12) found the first time in Acanthaster planci starfish
in Vietnam.
2. The chemical modification of cholesterol, staring material isolated
from this starfish, leads to obtain seventeen derivatives including 7
polyhydroxysteroid derivatives (15c-21c), 4 hydroximinosteroid derivatives
(23c, 25c, 29c, 31c) and 6 intermediate derivatives (22c, 24c, 26c, 27c, 28c,
30c), namely: cholestane-3β,6α-diol (15c); cholestane-3β,6β-diol (16c);

cholestan-5-ene-3β,4β-diol
(17c);
cholestan-5-ene-3β,7β-diol
(18c);
cholestan-5-ene-3β,4β,7β-triol (19c); cholestane-3β,5α,6α-triol (20c);
cholestane-3β,5α,6β-triol (21c); cholest-4-ene-3,6-dione (22c); (3E,6E)dihydroximinocholest-4-ene (23c); cholestane-3,6-dione (24c); (3E,6E)dihydroximinocholestane (25c); cholest-4-ene-3β,6α-diol (26c); 6-hydroxy4,5-epoxycholestane-3-one (27c); 4α,5α-epoxycholestane-3,6-dione (28c);
4α,5α-epoxy-6-hydroxycholestane-3-oxime (29c); 4α,5α,6α-trihydroxycholestane-3-one (30c); and 4α,5α-epoxycholestane-3,6-dioxime (31c).
19


In particular, 4α,5α-epoxy-6-hydroxy cholestan-3-oxime (29c); 4α,
5α,6α-trihydroxy-cholestane-3-one
dioxime (31c) are new substances.

(30c);

4α,5α-epoxycholestane-3,6-

3. Have investigated cytotoxic activity and evaluated the effects of
compounds AP1, AP11, AP12, AP13, AP14 to colony formation on soft
agar of human cancer cell lines. The results showed that:
-

Compound AP1 exhibited a moderate cytotoxicity against human

colon cancer (HCT-116) and human melanoma (RPMI-7951) cell lines with
IC50 = 36 µM and 58 µM, respectively. AP1 compound inhibited cell
proliferation of HCT-116, T-47D, and RPMI-7951 cancer cell lines, but had
no effect on colony formation of these cells.
- Compounds AP13 and AP14 at the same doses moderate inhibited

cell viability of human colorectal carcinoma HT-29 and human breast
adenocarcinoma MDA-MB-231 cell lines, but not RPMI-7951 cells. IC50 of
AP13 and AP14 were 109 and 90 µM in HT-29 cells; and 30 and 24 µM in
MDA-MB-231 cells, respectively. Compounds AP13 and AP14 effectively
inhibited colony formation of colorectal HT-29 and breast cancer MDA-MB231 cells and less melanoma RPMI-7951 cells. The concentrations which
caused 50% inhibition of colonies number of AP13 and AP14 were 11 and 7
µM in HT-29 cells, 13 and 8 µM in MDA-MB-231 cells, and 15 and 14 µM
in RPMI-7951 cells, respectively.
4. The compounds AP11-AP14 were determined effectively on
migration of MDA-MB-231 cells which are human breast adenocarcinoma
cells with high metastatic potential by method of in vitro wound healing
assay. The results showed that: AP13 and AP14 at concentration 10 were
able to prevent migration of MDA-MB-231 cells by 26% and 45%,
respectively, compared to control after 48 h of cells incubation.
5. Cytotoxicity of 07 prepared polyhydroxysteroid derivatives (15c21c), 04 hydroximinosteroid derivatives (23c, 25c, 29c, 31c) and 06 their
20


intermediate derivatives (22c, 24c, 26c, 27c, 28c, 30c) against three human
cancer cell lines including hepatocellular carcinoma (Hep-G2), cervical
cancer (HeLa) and glioblastoma (T98G) were studied. The results showed:
- Five compounds (16c, 18c, 21c, 23c, 27c) exhibited cytotoxicity
activity against Hep-G2 cell lines with values of IC50 = 11,59; 11,89; 6,87;
42,40; and 41,80 µM, respectively.
- Three compounds (23c, 27c, 28c) inhibited cytotoxicity activity against
HeLa cell lines with values of IC50 = 68,6; 72,4 and 74,6 µM, respectively.
- Five compounds (21c, 23c, 25c, 30c, 31c) exhibited cytotoxicity
activity against T98G cell lines with IC50 = 2,28; 70,3; 69,8; 18,5 and 2,9
µM, respectively. In particular, 21c and 31c are two potential substances for
further research and evaluation of cytotoxic on glioblastoma cell line.

PETITION
For Acanthaster planci starfish: further studies on chemical
composition, especially polyhydroxysteroid glycoside compounds, are
needed to develop health, prevention and support products treatment of
diseases such as cancer, anti-inflammation ...
Expand the semi-synthesis other derivatives of cholesterol and
isolated steroid compounds, and test the cytotoxic of these derivatives on a
number of other cancer cell lines.
NEW CONTRIBUTIONS OF THE THESIS
1. Fourteen compounds were isolated and identified from the extract of
starfish Acanthaster planci collected off from Vietnam coast. Among them,
four steroidal glycosides are discovered for the first time from nature,
including three polyhydroxysteroidal glycosides, namely planciside A
(AP1), planciside B (AP2), and planciside C (AP3); and an asterosaponin,
acanthaglycoside G (AP11).
21


2. The chemical modification of cholesterol, starting material isolated from
this starfish, leads to obtain seventeen derivatives including 07
polyhydroxysteroids, 04 hydroxyminosteroids and 06 intermediate
derivatives. The polyhydroxysteroid derivatives were prepared by short and
efficient synthetic pathways with 1-2 steps. Four hydroxyminosteroid
derivatives with oxime groups at C-3, C-6 and 4,5 double bond or oxygen
at C-4, C-5 were synthesized via 6 intermediates . Among these synthetic
derivatives, 3 compounds: 4α,5α-epoxy-6-hydroxycholestane-3-oxime (29c);
4α,5α,6α-trihydroxy-cholestane-3-one (30c); 4α,5α-epoxycholestane-3,6-dioxime
(31c) were described for the first time.
3. The cytotoxicity of isolated steroidal glycosides against 05 human cancer
cell lines (HCT-116, HT-29, RPMI-7951, T-47D and MDA-MB-231), the

inhibition of cancer cell colony formation on soft agar were investigated.
Compound AP1 exhibited moderate cytotoxic effect against HCT-116 and
RPMI-7951 cell lines with IC50 36 and 58 µM, respectively. Compound
AP13 and AP14 exhibited moderate cytotoxic effect against RPMI-7951,
HT-29, MDA-MB-231 cell lines with IC50 ranging from 24 to 109 µM. Two
asterosaponin AP13 and AP14 were found to prevent effectively the
migration of MDA-MB-231 cells by 26% and 45%, respectively.
The results related to isolated asterosaponins were found to be concised with
the hypothesis that a shoter side chain of steroidal moiety may have a
negative effect to the cytotoxicity of these asterosaponins.
4. All synthetic cholesterol derivatives were evaluated for the cytotoxicity
agaisnt three human cancer cell lines (HepG2, HeLa, T98G). Compounds
16c, 18c, 21c, 23c, 25c, 27c, 28c, 30c and 31c were showed to be cytotoxic
against at least one tested cancer cell lines. Especially, compounds 21c and
31c exhibited strongly and selectively cytotoxicity against human
glioblastoma cell line (T98G) with IC50 2,28 and 2,9 µM, respectively.
22


LIST OF PUBLISHED WORKS RELATED TO THE THESIS
1. Asterosaponins from the tropical starfish Acanthaster planci and their cytotoxic
and anticancer activities in vitro. Dinh T. Ha, Alla A. Kicha, Anatony I.
Kalinovsky, Timofey V. Malyarenko, Roman S. Popov, Olesya S. Malyarenko,
Svetlana P. Ermakova, Tran T. T. Thuy, Pham Q. Long, and Natalia V. Ivanchina.
Natural Product Research, 2019, 1-8. DOI: 10.1080/14786419.2019.1585845 (SCIE).
2. Three new steroid biglycosides, Plancisides A, B, and C, from the starfish
Acanthaster planci. Alla A. Kicha, Thi H. Dinh, Natalia V. Ivanchina, Timofey
V. Malyarenko, Anatony I. Kalinovsky, Roman S. Popov, Svetlana P. Ermakova,
Thi T. T. Tran, and Lan P. Doan. Natural Product Communications, 2014, Vol. 9,
No. 9, 1269-1274. (SCI-E)

3. Patent: (24S)-28-O-[BETA-D-GALACTOFURANOSYL-(1-5)-ALPHA-LARABINOFURANOSYL] -24-METHYL-5ALPHA-CHOLESTANE- 3BETA,
4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28-HEPTOL COMPOUND AND
ISOLATION METHOD OF THIS COMPOUND FROM STARFISH
ACANTHASTER PLANCI. Doan Lan Phuong, Tran Thi Thu Thuy, Dinh Thi Ha,
Alla A. Kicha, Natalia V. Ivanchina, Timofey V. Malyarenko, Anatoly I.
Kalinovsky, Roman S. Popov, Svetlana P. Ermakova, Pham Minh Quan, No.
18377, Decision No. 6820/QĐ-SHTT, 05/02/2018.
4. Useful solution: [(24S)-28-O-[ALPHA-L-FUCOPYRANOSYL-(1→2)-3-OMETHYL-BETA-D-XYLOPYRANOSYL]-24-METHYL-5ALPHACHOLESTANE-3BETA, 4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28HEPTOL; [(24S)-28-O-[2,4-DI-O-METHYL-BETA-D-XYLOPYRANOSYL(1→2)-ALPHA-L-ARABINOFURANOSYL]-24-METHYL-5ALPHACHOLESTANE-3BETA,4BETA, 6ALPHA, 8, 15BETA, 16BETA, 28HEPTOL] 6-O-SULFATE COMPOUNDS AND ISOLATION METHOD OF
THEM FROM STARFISH ACANTHASTER PLANCI. Doan Lan Phuong, Tran
Thi Thu Thuy, Dinh Thi Ha, Alla A. Kicha, Natalia V. Ivanchina, Timofey V.
23