Summary of chemistry dotoral thesis: Study on chemical constituents, cytotoxic and antiinflammatory activities of the sponges Rhabdastrella providentiae and Xestospongia muta living in the

MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
-----------------------------

STUDY ON CHEMICAL CONSTITUENTS, CYTOTOXIC
AND ANTI-INFLAMMATORY ACTIVITIES OF THE
SPONGES Rhabdastrella providentiae AND Xestospongia muta
LIVING IN THE SEA AREA OF CENTRAL VIETNAM
Major: Organic chemistry
Code: 9.44.01.14

SUMMARY OF CHEMISTRY DOTORAL THESIS

Ha Noi - 2019


1

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

Advisor 1:
Advisor 2:

Reviewer 1:
Reviewer 2:

Reviewer 3:

This thesis will be defended at Graduate University of Science and
Technology - Vietnam Academy of Science and Technology at hour
date month 2019.

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.


2

INTRODUCTION
1. The urgency of the thesis
In recent years, with the fast development of advanced techniques, a
large number of natural compounds have been actively isolated and
evaluated for biological activities. Many drugs from marine species have
been available on the market by major pharmaceutical companies in the
world such as: Cytarabine, Halaven, Ziconotide, Vidarabine, Trabectedin ...
Vietnam has the advantage of a long coastline with more than 3,260
km from North to South and many coastal islands, notably Truong Sa and
Hoang Sa archipelagoes located in the middle of East Sea. Such
geographical conditions have brought many advantages and potentials for
rich natural resources, creating an extreme diversity and abundant marine
ecosystem. The research showed that there were about 160 marine sponge
species distributed mainly in coastal and offshore islands. Among them,
there were only 20 species which have been studied on the chemical
composition and biological activity. Particularly, the sponge Rhabdastrella

providentiae has not been studied in Vietnam and over the world. The
sponge Xestospongia muta has not been studied in Vietnam.
The study and survey of chemical constituents and biological
activity of marine species are topic of interest for scientists in the world. In
Vietnam, there were some studies on chemical constituents and biological
activity of marine species published in prestigious international magazines
but not many. Therefore, the task of studying the chemical composition and
biological activities of marine life in our country is very important. From
that point, the topic "Study on chemical constituents, cytotoxic and antiinflammatory activities of the sponges Rhabdastrella providentiae and
Xestospongia muta living in the sea area of Central Vietnam" was
chosen.
Objectives of the thesis:
+ Determine the chemical constituents of two marine sponge species
Rhabdastrella providentiae and Xestospongia muta living in the sea area of
Central Vietnam.


3

+ Evaluate cytotoxic and anti-inflammatory activities of isolated
compounds to seek for active compounds for further studies to develop
healthy care products for the community.
The content of the thesis includes:
1. Isolate compounds from two species of marine sponges
Rhabdastrella providentiae and Xestospongia muta living in the sea area of
Central Vietnam by chromatographic methods:
2. Determine the chemical structure of compounds isolated by
physical and chemical methods
3. Evaluate in vitro cancer cell toxicity activity of isolated
compounds;

4. Evaluate in vitro anti-inflammatory activity of isolated
compounds.
2. The objectives of the thesis
Study on chemical constituents of two marine sponge species named
Rhabdastrella providentiae and Xestospongia muta.
Evaluation of cytotoxic and anti-imflammatory biological activities of
isolated compounds to seek for potential compounds for further researches.
3. The main contents of the thesis
Isolation of compounds from the sponges Rhabdastrella providentiae
and Xestospongia muta living in the sea area of Central Vietnam.
Determination of chemical structures of isolated compounds.
Evaluation of cytotoxic and anti-imflammatory biological activies of
isolated compounds to provide scientific evidences for applied researches.

CHAPTER 1: OVERVIEW
This chapter provides an overview on the sponges in all over the world
and in Vietnam.
1.1. Introduction to sponges
This section introduces generally about the characteristics,
distribution, studies of chemical constituents and biological activities of
marine sponges.


4

1.2. The study of sponges of the genus Rhabdastrella
1.3. The study of sponges of the genus Xestospongia

CHAPTER 2: EXPERIMENT AND RESULTS
2.1. Sponge materials

Sample of Rhabdastrella providentiae (Dendy, 1916) was collected at
Con Co, Quang Tri, Vietnam.
Sample of Xestospongia muta (Schmidt, 1870) was collected at Vinh
Moc, Quang Tri, Vietnam.
2.2. Methods
2.2.1. Methods for isolation of secondary metabolites
2.2.2. Methods for determination of chemical structure of compounds
2.2.3. Cytotoxic assay
2.3. Isolation of compounds
2.3.1. Isolation of compounds from R. providentiae

Figure 2.1. Isolation of compounds from R. providentiae


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2.3.2. Isolation of compounds from X. muta

Figure 2.2. Isolation of compounds from X. muta
2.4. Physical and spectroscopic data of compounds
2.5. Results on activity of compounds
2.5.1. Results on anti-inflammatory activity of compounds from R.
providentiae
Table 2.1. The results of evaluation inhibited NO production activity in BV2 cells
of compounds RP1-RP18
Compounds
RP2
RP5
RP7
RP8

RP9
RP10
RP11
RP12
RP13
RP14
RP15
RP16

Butein

IC50 (µM)
7.4 ± 0.4
75.3 ± 3.8
17.5 ± 0.9
46.8 ± 2.3
22.9 ±1.1
39.2 ± 2.0
26.3 ± 1.3
19.4 ± 1.0
29.4 ± 1.5
17.1 ± 0.9
19.5 ± 1.0
43.8 ± 2.2
4.5 ± 0.5


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2.5.2. Results on anti-inflammatory activity of compounds from X. muta

Table 2.2. Inhibited NO production activity in BV2 cells of compounds XM1XM11
Compounds
XM1
XM6
XM8

IC50 (µM)
4.9±0.24
11.5± 0.57
8.2 ± 0.41
4.4 ± 0.5

Butein

2.5.3. Results on cytotocic activity of compounds from R. providentiae
Table 2.3. Cytotocic activity of compounds RP1-RP18
Compounds

RP1
RP2
RP3
RP7
Pos.

IC50 (µM)
Hep-G2

LU-1

MCF-7

HL-60

SK-Mel 2

84.70±3.45
13.99±2.13
71.26±3.99
56.03±2.25
2.03 ± 0.3

84.82±6.67
14.75±1.30
63.38±2.45
62.20±3.41
1.96 ± 0.2

77.92±4.54
16.04±2.04
63.76±3.91
55.85±2.96
1.71 ± 0.2

56.14± 4.08
14.76± 1.31
46.33± 2.58
48.30± 3.73
2.16 ± 0.1

75.39± 4.83

11.16± 1.40
75.52± 6.69
37.96± 0.07
2.12 ± 0.3

Positive control: Ellipticine
2.5.4. Results on cytotocic activity of compounds from X. muta
Table 2.4 .Cytotocic activity of compounds XM1-XM11
Compounds
XM1
XM2
XM3
XM4
XM5
XM6
XM7
XM8
XM11
Pos.

Hep-G2
0.43±0.03
0.75±0.11
6.58±0.94
5.06±0.39
5.55±0.98
6.85±0.76
30.35±3.04
19.52±1.45
34.31±3.43

1.54 ± 0.37

LU-1
0.76±0.09
0.96±0.09
9.20±1.21
5.63±0.19
5.84±0.45
9.88±0.98
32.59±2.56
22.25±1.26
34.83±0.54
1.38 ± 0.16

Positive control: Ellipticine

IC50 (µM)
MCF-7
0.44±0.05
0.79±0.05
7.36±1.16
5.32±0.67
5.68±0.89
7.82±0.53
24.85±1.21
24.85±0.91
37.96±1.01
1.18 ± 0.12

HL-60

0.62±0.08
0.88±0.17
7.84±0.85
5.65±0.42
6.58±0.94
9.19±0.72
22.95±0.95
16.79±0.74
19.14±1.58
1.34 ± 0.24

SK-Mel 2
0.77±0.13
1.02±0.11
11.23±0.33
5.45±0.91
6.24±0.96
7.51±0.69
35.92±4.87
23.04±2.47
36.63±1.40
1.91 ± 0.28


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Evaluation of anti-cancer mechanism of XM1 against MCF-7 - human
breast cancer cell line: XM1 triggers cell apoptosis by altering the
expression level of related proteins in human breast cancer cells MCF-7. At
the same time, this compound also affects MCF-7 human breast cancer cell

cycle in G2/M phase

CHAPTER 3: DISCUSSIONS
3.1. Determination of chemical structure of compounds from R.
providentiae
3.1.12. Compound RP12: rhabdaprovidine G (new compound)

Figure 3.30. Chemical structure of compound RP12 and RP10
Compound RP12 is a light yellow amorphous powder. The
molecular formula of compound RP12 was determined to be C30H46O4 by
the exhibition of a quasi-molecular ion peak at m/z 493.3289 [M+Na]+
(calcd for C30H46O4Na: 493.3294) in the HR-ESI-MS. The 1H-NMR
spectrum of RP12 contained signals corresponding to seven methyl groups
at δH 0.95, 1.05, 1.09, 1.49, 1.61, 1.68, 1.71, two olefinic protons at δH 5.10,
5.81, and three oxygenated methines at δH 4.15, 4.24, 4.70 ppm. The 13CNMR of RP12 contained signals corresponding to 30 carbon atoms that
were preliminary assigned, according to HSQC data, as one carbonyl
carbon at δC 219.9; four olefinic carbons at δC 121.8, 124.1, 131.6, and
141.9; and three oxygenated methines at δC 68.1, 76.3, 77.0. The NMR
spectral data of RP12 were compared with the corresponding data of
compound RP10 (its structure was elucidated) and found to match from
carbons C-1 to C-8, and different from carbon C-9, suggesting that the ring
C (5C) of RP12 was changed. This suggestion was further confirmed by
HMBC and COSY correlations at rings A and B (Figure 3.32).


8

Table 3.12. NMR spectral data of RP12 and reference compound
C
1

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

*

δCa,b

31.2
33.4
218.8
46.9
45.2
18.9
35.8
41,2
50.6
34.5
34.4
207.1
123.6
172.2
19.3
23.5
-

δCa,b
32.9
33.8
219.9
47.2
45.7
19.4
34.4

42,6
54.1
36,0
77.0
76.3
54.9
54.0
36.4
68.1
121.8
49.8
24.1
141.9
16.6
39.8
26.6
124.1
131.6
25.7
17.8
29.3
19.4
36.5

δHa,c (mult., J = Hz)
1.73 (m)/ 2.57 (m)
2.32 (m)/ 2.70 (m)
2.48 (dd, 2.5, 12.5)
1.41 (m)/ 1.58 (m)
1.40 (m)/ 2.04 (m)

1.90 (d, 3.0)
4.24 (br s)
4.15 (d, 5.5)
1.72 (d, 5.5)
1.13 (d, 13.5)/ 2.26 (dd, 6.0, 13.5)
4.70 (br dd, 6.5, 8.5)
5.81 (d, 8.5)
2.65 (d, 4.5)/ 2.76 (d, 4.5)
0.95 (s)
1.71 (s)
2.06 (m)/ 2.11 (m)
2.08 (m)/ 2.14 (m)
5.10 (t, 6.5)
1.68 (s)
1.61 (s)
1.09 (s)
1.05 (s)
1.49 (s)

CDCl3, b125MHz, c500MHz. *δC of RP10
The COSY cross peaks H-9 (δH 1.90)/ H-11 (δH 4.24)/ H-12 (δH
4.15)/ H-13 (δH 1.71) and HMBC correlation between H-30 (δH1.49) and C13 (δC 54.9) established five-membered ring C in RP12. The chemical
shifts of C-11 (δC 77.0), C-12 (δC 76.3), and C-13 (δC 54.9) characterized for
a


9

two oxygenated methines (C-11 and C-12) and one aliphatic methine (C13). Other connections of carbon backbone were established by consecutive
analysis of HMBC and COSY spectra, including HMBC correlations

between terminal methyl protons H-26 (δH 1.68)/ H-27 (δH 1.61) and C-25
(δC 131.6)/ C-24 (δC 124.1), COSY cross peaks H-24 (δH 5.10)/ H-23 (δH
2.08, 2.14)/ H-22 (δH 2.06, 2.11), HMBC correlations between H-21
(δH1.71) and C-22 (δC 39.8)/ C-20 (δC 141.9)/ C-17 (δC 121.8), COSY cross
peaks H-17 (δH 5.81)/ H-16 (δH 4.70)/ H-15 (δH 1.13, 2.26), HMBC
correlations between H-18 (δH 2.65, 2.76) and C-15 (δC 36.4)/ C-14 (δC
54.0)/ C-13 (δC 54.9). In addition, an HMBC correlation between H-16 (δH
4.70) and C-12 (δC 76.3) indicated an ether bridge between C-16 and C-12.
Also, the presence of an epoxide ring at C-14/C-18 was conclusively
confirmed by the molecular formula of RP12 (C30H46O4), chemical shift of
the methylene carbon C-18 (δC 49.8), the presence of a non-protonated
carbon C-14 (δC 54.0), the J-coupling constant of geminal protons H-18
(4.5 Hz), and by comparison with previously published data. The
stereochemistry of compound RP12 was then proposed by analysis of
NOESY and CD spectra. An E-configuration of double bond C-17/C-20
was confirmed by a NOESY correlation between H-16 (δH 4.70) and H-21
(δH 1.71). NOESY correlations between H-19 (δH 0.95) and H-9 (δH 1.90)/
H-29 (δH 1.05), H-5 (δH 2.48) and H-28 (δH 1.09)/ H-30 (δH 1.49) indicated
a trans-syn-trans junction between rings A-C (Figure 3.32). This means that
C-29, C-19, and H-9 are located on the same side, assuming a βconfiguration. Therefore, the NOESY correlations H-19 (δH 0.95)/ H-11
(δH 4.24), H-9 (δH 1.90)/ H-15ax (δH 2.26), H-15ax/ H-16 (δH 4.70)
indicated β-configurations for H-11 and H-16. Additional NOESY
correlations H-17 (δH 5.81)/ H-12 (δH 4.15), and H-12/H-13 (δH 1.71)
suggested an α-configuration of H-12 and H-13. The epoxide ring at C14/C-18 adopted an α-configuration based on the NOESY correlation
between H-30 (δH 1.49) and H-18 (δH 2.76). Finally, an absolute structure of
RP12 was deduced by the similarity of experimental CD spectrum of RP12
in comparison with that of calculated CD spectrum for enantiomer RP12a
(5R,8S,9S,10S,11S,12S,13S,14S,16S, Figure 3.33). Consequently, structure
of compound RP12 was established and named as rhabdaprovidine G.

10

Figure 3.31. Chemical structure and important HMBC, COSY and NOESY
correlations of compound RP12
3.1.19. Total compounds isolated from R. providentiae RP1-RP18
18 compounds (RP1-RP18) were isolated from the marine sponge
Rhabdastrella providentiae. These include: 12 new compounds (RP1RP12) named: rhabdastrellins G-K (RP1-RP5), rhabdaprovidines A-G
(RP6-RP12) and 6 known compounds: jaspolide C (RP13), globostelletin
C (RP14), globostelletin D (RP15), jaspiferin A (RP16), mollisolactone A
(RP17), gibepyrone F (RP18) (Figure 3.50). Besides, some of these
isolated compounds demonstrate some specically structural characteristics
such as:
- The new compound RP12 has a complex structure with 8 chiral carbons
and an epoxy ring closure forming a 5-rings system was first found in
isomalabaricane frame compounds. In this study, we have determined the
absolute configuration of RP12 through NOESY and CD spectrum
analyses.
- The new compound RP11 with a special three-ring stereotype is arranged
trans-syn-cis instead of trans-syn-trans often found in marine sponge
Rhabdastrella species. The absolute configuration of -RP11 is also
determined through NOESY and CD spectra.
- The new compound RP3 has a γ-lactone ring closure in the vascular
fraction is rarely found in isomalabaricane compounds. The absolute
configuaration of two chiral carbons at the side chain of this compound are
also determined through analysis of δC values, NOESY and CD spectral
interactions.
- In previous publications on the isomalabaricane skeleton, the 13E/13Z
isomers are often reported as a mixed form (as globostelletin C and
globostelletin D). In this study, new compounds RP4/ RP5, RP6/ RP7 and

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two known compounds RP14/RP15 are 13E/13Z isomer pairs, however,
they were separated one by one with high purity.

Figure 3.50. Chemical structure of compounds RP1-RP18
- The isomalabaricane skeleton compounds are specialized secondary
substances synthesized by marine species like Stelletta, Jaspis, Geodia and
Rhabdastrella (of the family Astrophorida). Therefore, the detection of new
isomalabaricane compounds (RP1-RP12) can be considered as marker
compounds to identify marine species of Rhabdastrella. The special
structure of compounds RP11 and RP12 can be used as an indicator for
identification of R. providentiae species.


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3.2. Determination of chemical structure of compounds from X. muta
3.2.1. Compound XM1: araguspongine C

Figure 3.51. Chemical structure of compound XM1
The compound XM1 was obtained in the form of a white,
amorphous powder. TLC analysis showed that XM1 reacted positively with
Dragendorff's solution suggesting for an alkaloid compound. Compound
XM1 was shown to have a chemical formula similar to that of
araguspongine C, C28H50O4N2, which was deduced from a cluster of quasi
molecular ion peaks in the HR-ESI-MS at m/z 479.3845 [M+H]+ (Calcd for
[C28H51O4N2]+, 479.3843), 501.3656 [M+Na]+ (Calcd for [C28H50O4N2Na]+,

501.3663), and 477.3701 [M−H]ˉ (Calcd for [C28H49O4N2]−, 477.3692). The
chemical structure of araguspongine C was presented to form by symmetric
cycloaddition of a pair of 9-hydroxy-1-oxaquinolizidine moieties through
C6 linear chains. The 1H NMR and 13C- NMR spectral data of XM1
indicated that two these compounds share the same macrocyclic bis-1oxaquinolizidine skeleton. Interestingly, only 14 carbon signals were
observed in the 13C NMR spectrum of XM1, which also suggested the
symmetricity of its chemical structure. Hence, detailed chemical structure
of XM1 was elucidated on a half of its molecule. HMBC correlations from
H-10 (δH 4.10) to C-4 (δC 53.2) and C-6 (δC 45.5), and their chemical shifts,
demonstrated connections of C-10, C-4, and C-6 via nitrogen atom to form
a quinolizidine backbone. Moreover, replacement by an oxygen atom at C-1
of the quinolizidine structure of XM1 was confirmed by the presence of
oxygenated carbon signals of C-10 (δC 91.0) and C-2 (δC 77.6) and by
HMBC correlation between H-10 and C-2. In addition, a singlet signal of


13

H-10 and HMBC correlations from H-10 to the non-protonated C-9 (δC
72.0), and methylene carbons C-8 (δC 33.8)/C-11 (δC 41.3) indicated the
presence of a hydroxy group and linear carbon chain at C-9. It was
hypothesized that C6-linear chain arises from the C-9. Thus, HMBC
correlation from H-2 to C-16′ and COSY cross peak of H-2/H-16′ indicated
head-to-tail cyclization of two half molecules of XM1 by connections C2/C-16′: C-16/C-2′. Due to the presence of a system with two fused sixmembered rings and three chiral carbons, stereochemistry of XM1 was
studied by NOESY and conformational analyses. It was assumed that both
six-membered rings were in a stable chair forms. Thus, as in decalin, cis- or
trans-fused conformations should be acquired for a 1-oxaquinolizidine
structure (Figure 3.53).
Table 3.19. NMR spectral data of XM1 and reference compound
δCd

δCb,d

δCa,b

δHa,c (mult., J = Hz)

2, 2′

76.8

76.4

77.6

3.60 (br dd, 10.5, 10.5)

3, 3′

26.4

25.9

26.9

4, 4′

52.9

52.4

53.2

6, 6′

44.6

44.2

45.5

7, 7′

21.3

20.8

21.8

1,79 (dddd, 4.0, 10.5, 13.0, 13.0)
1,13 (br d, 13.0)
3.15 (ddd, 2.5, 13.5, 13.5)
2,99 (br dd, 3.5, 13.5)
3.09 (br dd, 11.5, 11.5)
2.40 (br d, 11,5)
1.94 (m)/ 1.49 (m)

8, 8′

30.0

29.6

33.8

9, 9′

71.1

70.7

72.0

1.54 (ddd, 4.0, 11.0, 11.0)
1,39 (br d, 11,0)
-

10, 10′

90.7

90.3

91.0

4.10 (s)

11, 11′

39.0

38.5

41.3

1.70 (ddd, 4.0, 13.0, 13.0)/1.23 (m)

12, 12′

23.0

22.5

23.7

1.47 (m)/ 1.35 (m)

C

#

13, 13′
32.7
32.2
33.0 1.30 (m)/ 1.28 (m)
14, 14′
31.9
31.5
31.0 1.45 (m)/ 1.28 (m)
15, 15′

25.3
24.9
26.3 1.60 (m)/ 1.33 (m)
16, 16′
36.7
36.3
37.6 1.58 (m)/1.38 (m)
a
CD3OD, b125MHz, c500MHz, dCDCl3, #δC of araguspongine C


14

In the 1H-NMR spectrum, the splitting pattern of signals of H-2 (δH
3.60), H-3a (δH 1.79), H-4a (δH 3.15), H-6a (δH 3.09), and H-8a (δH 1.54)
showing a large J axial-axial coupling constant (10.5 ~ 13.5 Hz) indicated
that they are axial orientations. The NOE correlations between H-2 (δH
3.60) and H-10 (δH 4.10), H-10 and H-4a (δH 3.15) were observered
confirming the axial orientation of H-10 in ring B of compound XM1. The
NOE correlation between H-8a (δH 1.54) and H-11 (δH 1.70) suggested the
equatorial orientation of C-11 and hydroxy group at C-9 should be in an
axial orientation. Finally, the occurrence of a fusion in the (b)-cis form
between the two six-membered rings was deduced by NOE correlation
between H-3a (δH 1.79) and H-6a (δH 3.09) as shown in Figure 3.54.
Therefore, the chemical structure of XM1 has been determined. This
compound has been isolated from a number of species such as X. exigua,
Haloclona exigua and is often called araguspongine C. 13C-NMR data of
XM1 was reanalyzed in CDCl3 and found to completely similar with the
reported 13C-NMR data of araguspongine C in the same NMR measurement
solvent.

Figure 3.52. Sustainable configuration of 1-oxa-quinolizidine flame

Figure 3.53. The main NOE interactions in the 1-oxa-quinolizidine skeleton
of compound XM1


15

3.2.12. Total of compounds isolated from X. muta XM1-XM11
From the marine sponge of Xestospongia muta, 11 compounds
(XM1-XM11) were isolated and determined the chemical structures (Figure
3.78): all of 11 compounds are macrocyclic bis-quinolizidine alkaloids
skeleton, including 1 new compound (XM2) named meso - araguspongine
C and 10 known compounds: araguspongine C (XM1), araguspongines N-P
(XM3-XM5), araguspongine A (XM6), araguspongine E (XM7),
araguspongine L (XM8), petrosin (XM9), petrosin A (XM10),
aragupetrosine A (XM11). The structures of these compounds contains 1oxa-quinolizidine unit, two quinolizidine units, or a combination of 1-oxaquinolizidine and quinolizidine units. Some compounds consist of
symmetrically chemical structure such as XM1, XM2, XM3, XM9, XM10.
The highlights in structural analyses of compounds isolated from X. muta
species are mentioned below:

Figure 3.78. Chemical structure of XM1-XM11 compounds


16

- The stereochemistry of new compound XM2 was determined based on a
detailed analysis of the J coupling constant, structural analyses and spatial
interaction on NOESY.

- Although XM3-XM4 are known compounds, however they have been
structurally and chemically analyzed herein for the first time.
- Because up to now, only more than 20 compounds of macrocyclic bis-1oxa-quinolizidine skeleton have been reported in Xestospongia species, so
that, compounds XM1-XM11 can also be used as marker compounds to
indentify Xestospongia species.
3.3. Evaluation of the biological activities of isolated compounds
3.3.1. Anti-inflammatory activity of compounds isolated from R.
providentiae
After a preliminary assessment of the effect of inhibiting NO
production in BV2 cell was stimulated by LPS of 18 compounds (RP1RP18) at concentration of 80 µM, the compounds RP2, RP5, RP7-RP16
are not cytotoxic and capable of inhibiting> 50% of NO production in BV2
cell. Therefore, these substances are further tested at different
concentrations to determine the IC50 value. The results (Table 2.1) showed
that: RP2 compounds exhibited the strongest inhibitory effect on NO
production in BV2 cell with IC50 values of 7.4 ± 0.4 µM. Compounds RP7,
RP9, RP11-RP15 exhibit remarkable inhibitory effects on NO production
with IC50 values of 17.1-29.4 µM. The compounds RP8, RP10, RP16 show
inhibitory effects on NO production with IC50 values of 39.2-46.8 µM. The
remaining compound RP5 shows the weakest inhibitory effect on NO
production in BV2 cell with the value of IC50 75.3 ± 3.8 µM.
According to the literature review, there have been no studies of
anti-inflammatory activity of isomalabaricane analog skeleton compounds.
This study can be used as a premise for further studies on the antiinflammatory activity of compounds RP2, RP7, RP9, RP11-RP15.


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3.3.2. Anti-inflammatory activity of compounds isolated from X. muta
The results of assessing the effect of 11 compounds (XM1-XM11) on
BV2 cell growth at the test concentrations (20, 40 and 80 µM) showed that

the XM2-XM5 and XM7 compounds are dead cells (> 30%) at
concentrations of 40 and 80 µM. However, these compounds do not affect
the normal development of BV2 cells at concentrations <20 µM.
Compounds XM9-XM11 inhibited <50% of NO production in BV2 cells at
the highest test concentration of 80 µM. Therefore, these compounds were
not allowed to further test to determine IC50. Three remaining compounds
XM1, XM6, XM8 were tested at different concentrations to determine the
IC50 values. The results (Table 2.2) showed: compounds XM1, XM6, XM8
exhibit strong anti-inflammatory activity with IC50 values in the range of
4.9-11.5 µM. In particular, the XM1 compound inhibits NO production in
BV2 cells with an IC50 value of 4.9 µM, roughly equivalent to buteinpositive control (IC50 4.4 µM). According to the literature review, there
have been no studies of anti-inflammatory activity of macrocyclic bisquinolizidine alkaloid skeleton compounds. This study can be used as a
premise for further studies on the anti-inflammatory activity of XM1, XM6,
XM8 compounds.
3.3.3. Cytotoxic activity of compounds isolated from R. providentiae
Results of in vitro cytotoxic activity evaluation of 18 compounds
isolated from marine sponge R. providentiae (RP1-RP18) on 5 cancer cell
lines: Hep-G2, LU-1, MCF- 7, HL-6, SK-Mel-2 (Table 2.3) shows: The
RP2 compound exhibits the most potent cytotoxic activity for all 5 cancer
cell lines tested with an IC50 value of about 11.16-16.04 (µM). Compounds
RP1, RP3, RP7 exhibited toxic effects on all cancer cell lines tested at IC50
prices in the range of 37.96-84.82 (µM). The remaining compounds do not
show cytotoxic activity (IC50> 100 µM) for the above 5 cell lines.
From the above results, it was found that the compounds RP1-RP3
with isomalabaricane analog 29C structure showed cytotoxic activity on
experimental cancer cell lines, while the remaining compounds having
isomalabaricane analog 19C until 25C structures did not show activity.
Compound RP7 with 13E isomer was active while 13Z isomer (RP6) did

18

not show activity. This is also quite consistent with the previous study that
analog isomalabaricane compounds with longer side chain showed stronger
cytotoxic activity than those with shorter side chain, 13E isomers exhibited
stronger activity than 13Z isomers. On the other hand, compound RP2 with
20(22)Z configuration of the double bond has significantly stronger
cytotoxicity than 20(22)E isomer (RP1) and the configuration of double
bond C-20/C-22 may also affect to the cytotoxic activity of isomalabaricane
analog compounds.
3.3.4. Cytotoxic activity of compounds isolated from X. muta
Results of in vitro cytotoxic activity evaluation of 11 compounds
isolated from marine sponge X. muta (XM1-XM11) against 5 cancer cell
lines in human: Hep-G2, LU-1, MCF -7, HL-6, SK-Mel-2 (Table 2.4)
show: compounds XM1, XM2 (IC50 0.43-1.02 µM) exhibit stronger
cytotoxic activity on 5 cancer cell lines than positive control (Ellipticine)
(IC50 1.18-1.91 µM). Compounds XM3-XM6 exhibit remarkable activity
with IC50 values of 4.71 to 11.23 µM. Compounds XM7, XM8, XM11
show potentially cytotoxic with an IC50 value of about 16.79-37.96 µM.
The other compounds did not show activity at the tested concentrations on 5
tested cancer cell lines.
Evaluation of anti-cancer mechanism of compound XM1:
Compound XM1 (araguspongine C) has an inhibitory activity on the
strongest test cancer cell lines that should be selected to further evaluate the
anti-cancer mechanism for breast cancer cell line MCF- 7.
a. Evaluate the impact of compound XM1 on human breast cancer cell
cycle MCF-7 after 48 hours of treatment with compound XM1 at
concentrations of 2 and 10µM:

Figure 3.79. Impact of XM1 compound on MCF-7 cell cycle

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Table 3.30. The percentage (%) of MCF-7 cells in phase G0/G1, S, G2/M
and apoptosis (sub-G1) after 48 hours of treatment with compound XM1 at
concentrations of 2 and 10µM
% Cells in phases of the cell division cycle
Sample

Control
XM1_2µM
XM1_10µM

% sub-G1
0.01
0.05
0.03

%G0/G1
54.92
53.10
40.00

%S
38.39
34.79
19.11

%G2/M

5.93
9.19
24.86

Through flow cytometry analysis, the ratio of MCF-7 cell at G0/G1
phase and S phase decreased with increasing concentration of compound
XM1. The percentage of cells at G2/M stage increased to 24.86% after
being treated with compound XM1 at concentration of 10µM after 48 hours
(Figure 3.79, Table 3.30). This showed that the compound XM1 acts on the
cell cycle at G2/M phase.
b. Evaluate the impact of compound XM1 on the change of cell morphology
MCF-7 at concentrations of 2 and 10µM.

Figure 3.80. Impact of compound XM1 at concentration 2 and 10µM on
human breast cancer cell morphology MCF-7
Results of MCF-7 cell morphological examination when treated with
compound XM1 after 48 hours showed that the cells had more contraction
and brighterness of some cell nuclei (due to the concentration of
chromosomes) and these characteristic bodies appear more at 10µM
concentration. MCF-7 cell density decreased significantly after treatment
with MCF-7 at 10µM concentration (Figure 3.80). These morphological
characteristics show that cells die on their own (apoptosis) after being treated
with compound XM1.


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c. Evaluate the impact of compound XM1 on the death process of human
breast cancer cell MCF-7 at concentrations of 2 and 10µM.

Figure 3.81. Impact of compound XM1 on the apoptosis of MCF-7 cells
The result (Figure 3.81) showed that compound XM1 causes the process
of self-death (apoptosis) on MCF-7 cell. The ratio of apoptotic cells
increased from 2.79% in control sample to 50.74% in sample treated with
compound XM1 at a concentration of 10µM.
d. Evaluate the effect of compound XM1 on expression of proteins on
MCF-7 cell line at concentrations of 1, 3 and 10µM

Figure 3.82. Effect of XM1 on expression of Caspase 3, Bcl-2, Bax
proteins on MCF-7 cell line at concentrations of 1, 3 and 10µM
Effect of compound XM1 on expression of Caspase 3, Bcl-2, Bax
proteins on MCF-7 cell line at concentrations of 1, 3 and 10µM was
assessed by Western blot method. The results show that the compound
XM1 reduced the expression of Bcl-2 protein and increased the expression
of Bax and Caspase -3 at concentrations of 3-10 µM (Figure 3.82).


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CONCLUSIONS
This is the first study on the chemical constituents and biological
activities of two marine sponge species Rhabdastrella providentiae and
Xestospongia muta living in the sea area of Central Vietnam.
1. Research on chemical composition
Using chromatographic methods, 29 compounds including 13 new
compounds were isolated from two marine sponge species Rhabdastrella
providentiae and Xestospongia muta. Their chemical structures were
determined by modern spectroscopic methods.
- From R. providentiae, 18 compounds (RP1-RP18) were isolated and
identified: including 12 new compounds (RP1-RP12) named:

rhabdastrellins G-K (RP1-RP5), rhabdaprovidines A-G (RP6-RP12) and 4
known compounds: jaspolide C (RP13), globostelletin C (RP14),
globostelletin D (RP15), jaspiferin A (RP16), mollisolactone A (RP17),
gibepyrone F (RP18).
- From X. muta, 11 compounds (XM1-XM11) were isolated and
determined: all 11 compounds belong to macrocyclic bis-quinolizidine
alkaloid skeleton, including 1 new compound (XM2) named meso araguspongine C and 10 known compounds: araguspongine C (XM1),
araguspongine N (XM3), araguspongine O (XM4), araguspongine P
(XM5), araguspongine A (XM6), araguspongine E (XM7), araguspongine
L (XM8), petrosin (XM9), petrosin A (XM10), aragupetrosine A (XM11).
2. Research on biological activity
The cytotoxic activity againsted 5 cancer cell lines in human: liver
cancer (Hep-G2), lung cancer (LU-1), breast cancer (MCF-7), acute
leukemia (HL-60), skin cancer (SK-Mel-2) of 18 compounds (RP1-RP18)
isolated from marine species R. providentiae and 11 compounds (XM1XM11) isolated from X. muta were evaluated and the results showed that:
Compound RP2 exhibited the strongest cytotoxic activity for all 5
cancer cell lines tested with IC50 values in the range of 11.16-16.04 µM.
Compounds RP1, RP3, RP7 showed toxic activity on all cancer cell lines
tested at IC50 in the range of 37.96-84.82 (µM).


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Compounds XM1 and XM2 (IC50 0.43-1.02 µM) exhibited cytotoxic
activity on 5 tested cancer cell lines stronger than ellipticine positive control
(IC50 1.18-1.91 µM). Compounds XM3-XM6 exhibited remarkable activity
with IC50 values of 4.71 to 11.23 µM. Compounds XM7, XM8, XM11 were
capable of cytotoxicity with value IC50 is in the range of 16.79-37.96 µM.
Research on mechanism of causing cancer cell MCF-7 of the cell
morphology compound XM1 at the molecular level has shown that

compound XM1 affects the cycle of human breast cancer cell MCF-7 in
G2/M. At the same time, this compound also causes cell apoptosis through
changes in expression levels of related proteins (increased expression of
Bax, Caspase-3 and reduced expression of Bcl-2) in human breast cancer
cell MCF-7.
The anti-inflammatory activity through inhibiting NO production against
BV2 cells of 18 compounds (RP1- RP18) isolated from R. providentiae and
11 compounds (XM1-XM11) isolated from X. muta were evaluated and the
results showed that:
Among compounds RP1-RP18, RP2 inhibited NO production with the
strongest IC50 value (7.4 ± 0.4 µM). Compounds RP7, RP9, RP11-RP15
inhibited NO production with a remarkable IC50 value of 17.1-29.4 µM,
compounds RP8, RP10, RP16 show anti-inflammatory activity with IC50
values of 39,2-46,8 µM.
Compounds XM1, XM6, XM8 exhibited remarkable anti-inflammatory
activity with IC50 values in the range of 4.98-11.58 µM. Especially,
compound XM1 inhibiting the production of NO in the cell BV2 cells with
IC50 values of 4.9 µM are roughly equivalent to butein positive controls
(IC50 4.4 µM).

RECOMMENDATION
The study results of chemical composition and biological activity of R.
providentiae and X. muta demonstrate that:
Compound XM1 acts on G2/M phase of human breast cancer cell line
MCF-7 and causes cell apoptosis through altering the expression level of
related proteins in MCF-7, suggested that this is a potential compound for
research and development of drugs to treat breast cancer. Therefore, further


23

studies on cytotoxic mechanisms of different breast cancer cell lines need to
be conducted at the in vivo level.
Isomalabaricane analogs RP1-RP3, RP7 are new compounds isolated
from R. providentiae with potential to develop anti-cancer drugs. Therefore,
further research is needed to clarify the mechanism of these compounds,
especially rhabdastrellin H (RP2) with an IC50 value of 11.16-16.04 µM.
Compound XM1, RP2 exhibit inhibitory activity on NO production in
BV2 stimulated by LPS are interesting with IC50 values of 4.9 µM and 7.4
µM respectively. Therefore, further anti-inflammatory research of this
compound should be studied at in vivo level.

NEW FINDINGS OF THE THESIS
- This is the first study on the chemical constituents and biological activities
of marine sponge species Rhabdastrella providentiae in the world and is the
first study on the chemical constituents and biological activities of
Xestospongia muta in Vietnam.
- From the sponges Rhabdastrella providentiae and Xestospongia muta, 13
new compounds (rhabdastrellins G-K (RP1-RP5), rhabdaprovidines A-G
(RP6-RP12); meso - araguspongine C (XM2) were isolated and elucidated
the chemcial structure. Especially, the stereochemistry of two compounds
RP11, RP12 is determined by NOESY and CD spectra.
- This is the first time isomalabaricane and macrocyclic quinolizidine
skeletons were tested anti-inflammatory activity by inhibiting NO
production in BV2 cells.
- This is the first time 13 new compounds were tested cytotoxic activity
against 5 human cancer cell lines.


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LIST OF PUBLISHED ARTICLES
1. Phan Van Kiem, Duong Thi Dung, Pham Hai Yen, Nguyen Xuan Nhiem,

Tran Hong Quang, Bui Huu Tai, and Chau Van Minh. New isomalabaricane
analogues from the sponge Rhabdastrella providentiae and their cytotoxic
activity. Phytochemistry letters, 2018 (26), 199-204.
2. Duong Thi Dung, Pham Hai Yen, Nguyen Xuan Nhiem, Tran Hong Quang,
Bui Huu Tai, Chau Van Minh, Dong Cheol Kim, Hyuncheol Oh, Youn Chul
Kim, and Phan Van Kiem. New acetylated terpenoids from sponge
Rhabdastrella providentiae inhibit NO production in LPS stimulated BV2
cells. Natural Product Communications, 2018, 13 (6), 661-664.
3. Duong Thi Dung, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Tran Hong
Quang, Bui Huu Tai, Pham Hai Yen, Nguyen Thi Hoai, Do Cong Thung, and
Chau Van Minh, Phan Van Kiem. Rhabdaprovidines D-G, four new 6,6,5tricyclic terpenoids from the Vietnamese sponge Rhabdastrella providentiae.
Natural Product Communications, 2018, 13(10), 1251-1254.
4. Duong Thi Dung, Dan Thi Thuy Hang, Pham Hai Yen, Tran Hong Quang,
Nguyen Xuan Nhiem, Bui Huu Tai, Chau Van Minh, Youn-Chul Kim, Dong
Cheol Kim, Hyuncheol Oh, and Phan Van Kiem. Macrocyclic bisquinolizidine alkaloids from Xestospongia muta. Natural Products Research,
2018.1455043.
5. Dương Thị Dung, Đan Thị Thúy Hằng, Phạm Hải Yến, Nguyễn Xuân
Nhiệm, Trần Hồng Quang, Bùi Hữu Tài, Phan Văn Kiệm. Nghiên cứu thành
phần hóa học loài hải miên Rhabdastrella providentiae. Tạp chí Hóa học,
2018, 56(1), 81-85.
6. Duong Thi Dung, Nguyen Xuan Nhiem, Do Thi Trang, Pham Hai Yen,
Tran Hong Quang, Hoang Le Tuan Anh, Do Cong Thung, Bui Huu Tai,
Chau Van Minh, Phan Van Kiem. Isolation of isomalabaricane analog from
the sponge Rhabdastrella providentiae. Vietnam journal of chemistry, 2017,
55(6e), 38-41.
7. Duong Thi Dung, Nguyen Thi Cuc, Pham Hai Yen, Nguyen Xuan Nhiem,

Tran Hong Quang, Hoang Le Tuan Anh, Do Cong Thung, Do Thi Thao, Bui
Huu Tai, Chau Van Minh, Phan Van Kiem. Petrosin and petrosin A from the
Vietnamese sponge Xestospongia muta. Vietnam journal of chemistry, 2017,
55(6e), 72-75.