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:

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 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.

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




+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



900

1000

1100

1200

1300

400

m/z

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

500

600

700

800

900

1000

1100

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)


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

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


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


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


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


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

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


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

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

>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

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

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

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

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