MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY OF
SCIENCE AND TECHNOLOGY

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

Phan Thi Hoai Trinh

STUDY ON CHEMISTRY AND BIOLOGICAL ACTIVITY OF
SOME COMPOUNDS FROM MARINE FUNGI ISOLATED
IN CENTRAL VIETNAM

Major: Biotechnology
Code: 9.42.02.01

SUMMARY OF BIOLOGY DOCTORAL THESIS

Ha Noi – 2019


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

Advisor 1: Assoc. Prof. Dr. Phi Quyet Tien
Advisor 2: Assoc. Prof. Dr. Tran Thi Thanh Van

1st Reviewer:
2nd Reviewer:

marine fungi. Tran Hong Quang et al. (2018) detected five new
compounds such as modiolide D-G and 1-(2,5-dihydroxyphenyl)-3methoxy-butane-1-one, including three known compounds as modiolide
A-B and 1- (2,5-dihydroxyphenyl) -2-buten-1-one were obtained from
the culture fermentation of endophytic fungus Paraconiothyrium sp.
VK-13. Two out of five obtained compounds demonstrated antiinflammatory activity. Besides, the fungus strain Penicillium sp. KMM
4672, derived from Padina spp. collected in Van Phong Bay of Khanh
Hoa province, also produced 3 new compounds, including 4-hydroxy3,6-dimethyl-2-pyrone, 4-methoxy-isoquinolin-1 (2H) -one and N, Ndiethyl- 3-methyl-benzamide. With aim to obtain natural bioactive
compounds from marine fungi in order to find new sources of
pharmaceutical materials, we carry out the study: “Study on chemistry
and biological activity of some compounds from marine fungi
isolated in Central Vietnam”


2
2. The objectives of the thesis
Obtaining of secondary metabolites from marine fungi isolated in the
Central

Vietnam

and

identifying

some

biological

activities


14-trihydroxyconfertifolin (11), 6β, 9α, 14-trihydroxycinnamolide (12),
andrastin A (13), and citreohybridonol (14).
- Four new compounds were identified including phomaligol A2 (1),
ochraceopone F (7), 6β,7α,14-trihydroxyconfertifolin (11), and
6β,9α,14-trihydroxycinnamolide (12).
CHAPTER 1. OVERVIEW
Marine fungi are considered as a valuable marine microorganism
resource with the ability to synthesize many antibiotic, antiinflammatory, anti-cancer and antioxidant compounds being researched
and applied in medicine. It is the process of adapting to the harsh living
conditions of the marine environment such as high salinity, low
nutrition, high pressure, temperature variation and competition with
other organisms that have created conditions for marine microorganisms
biosynthesis of secondary compounds with new biological activities,
superior to terrestrial microorganisms.
According to published reports, marine microorganisms are diverse
and are present in almost all marine ecosystems including seawater,
marine sediments, wood substrates in the ocean, symbiotic association
with seaweeds, corals, seaweeds and other organisms. Scientists
estimate that as many as 1500 species of marine fungi exist, but so far,
less than 10% of marine fungus biodiversity has been discovered and is
in the process of research. Studies indicated that changing fermentation
conditions or culture with other microorganisms will activate the
metabolic pathways of microorganisms to produce new secondary
metabolic compounds. To meet today's new drug needs, scientists have
expanded their scope of research on marine fungi from temperate to


4
tropical waters, and also in areas with year-round low temperatures such
as Antarctica. Vietnam is a country located in a tropical region with a long

2.1.2. Study media
- Isolation medium: Sabouraud agar medium supplemented with
antibiotics including 10 g of peptone, 20 g of glucose, 18-20 g of agar, 1000
mL of natural seawater, 1.5 g of penicillin, 1.5 g of streptomycin, pH 6.0-7.0.
- Solid fermentation medium (RYE): medium prepared in a 500 mL
Erlenmeyer flask consisting of 20 g of rice, 20 mg of yeast extract, 10
mg KH2PO4 and 40 mL of seawater.
2.2. Methods
2.2.1. Isolation of marine fungi
Marine fungi were isolated on Sabouraud medium at 28°C.
2.2.2. Evaluation of antimicrobial activity of marine fungi
Determined by diffusion method on agar plates of Becerro et al. (1994).
2.2.3. Analysis of extraction residues of microorganisms with high
antimicrobial activity
Crude extracts of high antibiotic activity were analyzed on thin layer
chromatography (TLC Silica gel 60 F254) with solvent toluen:
isopropanol (6:1, v/v) and 1H NMR.
2.2.4.

Identification

of

morphological

characteristics

and

classification of marine fungi

concentration (MIC).
2.2.8.2. Determination of cytotoxic activity
Determined by SRB dyeing method (sulforhodamine B).
2.2.8.3. Determination of antioxidant activity
Determined according to its ability to eliminate free radicals DPPH
(2,2-diphenyl-1-picrylhydrazyl)

and

ABTS

(2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulphonic acid).
2.2.8.4. Determine neuroprotective activity
Determined by the method MTT (3- (4,5-dimethylthiazol-2-yl) -2,5diphenyltetrazolium bromide).


7
2.2.9. Processing research data
The experiments were repeated 3 times and the data expressed as a
mean ± standard deviations were calculated using Microsoft Excel 2010.
CHAPTER 3. RESULTS AND DISCUSSIONS
3.1. Isolation and screening of antimicrobial activity of marine fungi
From 29 samples of sponges, 28 samples of soft corals, 33 samples
of seaweeds and 21 samples of marine sediment collected from the
coastal areas of Da Nang, Nha Trang and Ninh Thuan, 273 strains of
marine fungi were isolated and purified (Figure 3.1).
Figure 3.1.
Number of

Results of antimicrobial activity screening showed that 54.2%
(n=148) strains exhibited antibiotic activity for at least one tested
pathogenic. The study also found that 43.9% (n=109) strains against B.
cereus, 34.4% (n=94) against S. faecalis, 42.1% (n=115) against S.


8
aureus and 29.7% (n=81) against L. monocytogenes. Resistance to
Gram-negative bacteria including E. coli, P. aeruginosa and yeast C.
albicans was recorded at a lower rate, 4.4% (n=12), 2.2% (n=6) and
4.8% (n=13), respectively. The number of fungal strains isolated from
Nha Trang Bay showing antibiotic activity accounts for a higher
proportion than Da Nang and Ninh Thuan. Specifically, the S. aureus
resistance activity of fungal strains collected from 3 coastal areas of Nha
Trang, Ninh Thuan and Da Nang accounted for 57, 42 and 21%,
respectively. Survey results were similarly recorded for B. cereus, S.
faecalis and L. monocytogenes. It is predicted that different ecosystems
in the seas have affected the biological characteristics of the studied
fungal strains. Zhou et al. (2016) demonstrated that location and source
of isolation not only relate to the diversity of fungal species but also the
ability to biosynthesize biologically active substances from them.
Among 273 strains of fungus studied, 8 strains exhibited high
antimicrobial activity and broad spectrum resistance for most of the tested
pathogenics

including

01NT.1.1.5,

01NT.1.5.4,


01NT.1.12.3


10
045-357-2

168ST.16.1

01NT.1.5.4

Figure 3.10. 1H NMR spectra of crude extracts of 5 fungal strains
01NT.1.1.5, 01NT.1.12.3, 045-357-2, 168ST.16.1 and 01NT.1.5.4
Morphological characteristics of 8 selected marine fungi were
determined after 5-10 days of incubation on Sabouraud agar medium at
28oC and described in Table 3.4.
Table 3.4. Morphological characteristics of 8 selected marine fungi
N
o
1

Fungal
strains
01NT.1.1.5

2

01NT.1.5.4

Photos of

4

168ST.16.1

-

Cream yellow, round, 18-25 mm in diameter
Hyphae mycelium on surface
Exudate pigment on the surface and produce
brown yellow soluble pigment
Entire margin

5

01NT.1.9.4

-

Grey brown, round, 15-18 mm in diameter
Filamentous surface, umbonate
Produce dark grey soluble pigment
Entire margin

6

045-357-2

- Grey and mossy green, round, 20-25 mm in
diameter
- Smooth surface, straight wall, curled

Based on the morphological characteristics observed under the
microscope, four strains of studied fungi including 01NT.1.1.5,
01NT.1.5.4, 01NT.1.12.3 and 168ST.16.1 were identified as Aspergillus
genus. Strains 045-357-2 were identified as genus Penicillium (Table 3.5).
From the combination of morphological characteristics and sequencing
analysis of ITS/28S rDNA region, the results of classification of 8 selected
strains of fungi belong to Ascomycota. Of which, there are 7 strains


12
belong to Eurotiales including A. flocculosus 01NT.1.1.5 (MG972941),
A. niger 01NT.1.5.4 (MH095994), Aspergillus sp. 01NT.1.12.3
(MH101466), Aspergillus sp. 168ST.16.1 (MG920345), P. chrysogenum
045-357-2 (MH753592), Talaromyces sp. 168ST.35.2 (MK080561) and
Talaromyces sp. 168ST.51.1 (MK072976). One strain belongs to the
order Dothideales is Biatriospora sp. 01NT.1.9.4 (MK072974). It is
showed that the fungi of the genus Aspergillus and Penicillium have the
ability to produce most of the bioactive natural compounds.
Table 3.5. Morphological characteristics of 08 marine fungal strains
observed under a microscope
No

Fungal
strains

Photos of
morphological
characteristics

Morphological characteristics

168ST.16.1

- Conidia globose, size 2-2,5 µm
- Vesicle globose, 25-35 µm wide
- Conidiaphores with transparent wall


13
5

01NT.1.9.4

- Gray mycelium, 2,5–3,9 µm wide, sparse
branching.
- Mycelium with smooth and transparent
wall.

6

045-357-2

- Conidia smooth, original ellipse, size 2-2,5
x 2,5-3 µm, then change spherical
- Conidiaphores smooth, with many
partitions, typical branching, size up to 100
µm

7

168ST.35.2

14
3.3. Determining suitable solid fermentation conditions for
antibiotic biosynthesis of 03 selected marine fungal strains
- Strain A. flocculosus 01NT.1.1.5 produces 363 mg of crude
extract/40 g of rice in an environment with salt concentration of 35 g/L,
initial environmental pH 6.0 and after 20 days of fermentation.
- Strain Aspergillus sp. 01NT.1.12.3 produces 564 mg of crude
extract/40 g of rice in an environment with a salt concentration of 25
g/L, initial environmental pH 6.0 and after 22 days of fermentation.
- Strain P. chrysogenum 045-357-2 produces 264 mg of crude
extract/40 g of rice in an environment with a salt concentration of 35
g/L, initial environmental pH 7.0 and after 14 days of fermentation.
3.4. Extraction, purification and identification of structures of
secondary metabolites from selected marine fungal strains
3.4.1. Extraction, purification and identification of structures of
compounds from A. flocculosus 01NT.1.1.5
The extract of A. flocculosus 01NT.1.1.5 was separated on C18
chromatography column and purified by HPLC to obtain 8 individual
compounds 1-8 (Figure 3.15). Based on ESI-MS spectrum analysis, HRESI-MS combined with NMR spectroscopy data and publications have
identified the names of 8 compounds as phomaligol A2 (1),
wasabidienone E (2), aspertetranone D (3), mactanamide (4),
cycloechinulin (5), asteltoxin (6), ochraceopone F (7) and asterriquinone
C1 (8). In which, compounds 1 and 7 are identified as new compounds.
Compound 1: Phomaligol A2 (New compound)
Yellow oil, ESI-MS (m/z 300,88 [M+H]+), molecular formula
C14H20O7. 1H NMR spectrum of compound 1 has signals of 4 methyl
groups, 2 methine groups, 1 methoxy group (δH 3.89/H-12) and 1
aromatic ring proton at δH 5.62 (H-4). Two olefinic carbons, 3 ketone



Brown oil, HR-ESI-MS (m/z 397,1987 [M+Na]+), molecular formula
C22H30O5. 1H and 13C-NMR spectra data together with COSY, HSQC
spectra showed the appearance of 1 methine group at δH 2.47 (H-7), 5
methylene groups at H-16, H-10, H- 9, H-6, H-15, 6 methyl groups, 10
quaternary carbon signals, 1 carbonyl ketone at δC 218.0 (C-14), 1
carbonyl ester at δC 165.7 (C-1), 2 conjugated oxidized carbons at δC
97.6 (C-2), 107.5 (C-4), 2 quaternary carbons with oxygen at δC 80.4 (C-


16
8), 78.2 (C-11), two aliphatic quaternary carbons at δC 53.1 (C-13), 40.4
(C-12). Spectral data also showed that tetracyclic rings of compound 7
closely resemble ochraceopone E, an α-pyrone merosesquiterpenoid
from Aspergillus ochraceopetaliformis SCSIO 05702 isolated from
Antarctica. The difference between compound 7 and ochraceopone E is
compound 7 without hydroxyl group at C-9. Therefore, the structure of
compound 7 was identified as 9-deoxy ochraceopone E and was named
as ochraceopone F.
3.4.2. Extraction, purification and identification of structures of
compounds from Aspergillus sp. 01NT.1.12.3
From the extract of marine fungi Aspergillus sp. 01NT.1.12.3,
separating on silica gel chromatography column and purifying by HPLC
collected 4 compounds 9-12 (Figure 3.26). Based on the analysis of HRESI-MS spectra in combination with NMR spectroscopy data and the
publications have identified the names of four compounds including
dihydroaspyrone

(9),

aspilactonol


obtained from A. versicolor CNC 327 isolated from seaweed Penicillus
capitatus in Bahamas island. In 2018, the compound 6β, 14-dihydroxy7α-methoxyconfertifoline continued to be obtained by Tan et al. from
A. ochraceus Jcma1F17 derived from Coelarthrum sp. collected in
southern China. The difference between compound 11 and 6β, 14dihydroxy-7α-methoxyconfertifoline is compound 11 with hydroxyl
group at C-7 instead of methoxy group. Therefore, compound 11 was
identified as a new compound and was named 6β, 7α, 14trihydroxyconfertifolin.
Compound 12: 6β,9α,14-trihydroxycinnamolide (New compound)
White powder, HR-ESI-MS (m/z 281,1390 [M-H]-), molecular
formula C15H22O5. Spectra data showed that the structure of compound
12 is similar to pereniporin B isolated from the fungus Perenniporia
medullaepanis Aj 8345. The difference between compound 12 and
pereniporin B is compound 12 with hydroxyl group attached to group
methyl at C-14. Therefore, compound 12 was identified as a new
compound and was named 6β, 9α, 14-trihydroxycinnamolide.
3.4.3. Extraction, purification and identification of structures of
compounds from P. chrysogenum 045-357-2
The extract of P. chrysogenum 045-357-2 was separated on C18
chromatography column and purified by HPLC to get 2 compounds 13
and 14 (Figure 3.31). Based on ESI-MS spectrum analysis combined
with NMR spectroscopy data and publications, the compounds were
identified as andrastin A (13) and citreohybridonol (14).
Andrastin A is a compound with the meroterpenoid frame structure
described first by Omura et al. (1996) obtained from Penicillium sp. FO-


18
3929. The study also showed that this compound is mainly produced
from fungal strains of the genus Penicillium when fermented in solid
medium and extracted with ethyl acetate, namely P. roqueforti CECT
2905, Penicillium sp. FO-3929, P. albocorenium IBT 16884, and P.

MIC values of 16-128 μg/mL. Although the structure of compound 1 has a
more hydroxyl group than that of phomaligol A, the ability against S. aureus
is reduced compared to phomaligol A (MIC, 31.2 µg/mL).
Table 3.7. Antimicrobial activity of compounds 1-14
Antimicrobial activity (MIC, µg/mL)
Gram (+) bacteria
Gram (-) bacteria
Yeast
Compounds
B. cereus S. faecalis S. aureus E. coli P. aeruginosa C. albicans
ATCC
ATCC
ATCC ATCC
ATCC
ATCC
11778
19433
25923
25922
27853
10231
Compounds from A. flocculosus 01NT.1.1.5
Phomaligol A2 (1)
128
32
128
64
16
16
Wasabidienone E (2)


16

64

128

16

32

Cycloechinulin (5)

64

64

> 256

128

64

64

Asteltoxin (6)
Ochraceopone F (7)

64


> 256

32

64

Compounds from Aspergillus sp. 01NT.1.12.3
Dihydroaspirone (9)
16
32
Aspilactonol F (10)

32

32

32

32

16

32

32

32

32



6β,7α,14-trihydroxyconfertifolin (11)
6β,9α,14-trihydroxycinnamolide (12)

64

32

16

32

32

64

16

32

16

32

Amoxicillin

256

256


antibacterial activity against S. aureus and E. coli when tested with
concentration of 100 μg/mL. Dihydroaspyrone (9) was isolated from
Aspergillus sp. 01NT.1.12.3 has strong resistance against tested strains.
However, Liu et al. (2015) indicated that this compound did not show
resistance to aquatic pathogens including Aeromonas hydrophila, Vibrio
anguillarum and V. harveyi. Two compounds of andrastin A (13) and
citreohybridonol (14) from P. chrysogenum 045-357-2 are resistant to 2
Gram (-) bacteria including E. coli and P. aeruginosa and yeast C.
albicans with MIC values of 16-32 μg/mL. This is the first study to
evaluate the antimicrobial activity of wasabidienone E (2), aspertetranone
D (3), cycloechinulin (5), asterriquinone C1 (8), aspilactonol F (10) and
citreohybridonol (14) isolated from marine fungi.
3.5.2. Determination of cytotoxic activity
Among the studied compounds, only asterriquinone C1 (8) showed
the ability to effectively inhibit all 6 cancer cell lines tested with IC50
values of 30-40 µM. In addition, asterriquinone C1 has been reported to
inhibit other human cancer cell lines including NCI-H460 lung cancer,
MCF-7 breast cancer and glial tumor cell with IC50 values of 24.2; 4.1
and 25.7 μM, respectively. The new compound, ochraceopone F (7),
although the structure has a more hydroxyl group compared to the
ochraceopone E, the cytotoxic activity has almost no change. Wang et
al. (2015) noted that ochraceopone E does not exhibit toxic activity for
all 7 cancer cell lines tested including K-562, MCF-7, A-549, HeLa,
DU-145, HL-60 and HT-29 (Table 3.8).


21
Table 3.8. Cytotoxic activity of compounds 1-14
Compounds



19.62 ± 1.92
21.05 ± 6.85
20.20 ± 6.76
22.22 ± 2.62
25.33 ± 3.31
17.02 ± 2.15
19.78 ± 5.21
80.30 ± 3.32

26.94 ± 3.78
23.22 ± 0.45
25.89 ± 5.18
19.51 ± 0.28
22.97 ± 3.72
18.59 ± 3.02
20.67 ± 2.45
88.07 ± 3.24

19.74 ± 4.26
23.68 ± 1.77
37.16 ± 1.37
16.51 ± 2.71
26.66 ± 6.74
15.33 ± 2.19
17.56 ± 3.22
92.51 ± 2.08

-


78.01 ± 6.00

80.52 ± 2.58

79.58 ± 6.60 84.22 ± 1.03

81.79 ± 4.86

Compounds from A. flocculosus 01NT.1.1.5
Phomaligol A2 (1)
Wasabidienone E (2)
Aspertetranone D (3)
Mactanamide (4)
Cycloechinulin (5)
Asteltoxin (6)
Ochraceopone F (7)
Asterriquinone C1 (8)

26.45 ± 2.14
21.71 ± 3.44
19.17 ± 2.64
22.76 ± 2.09
28.47 ± 2.49
16.19 ± 1.58
19.32 ± 3.59
81.58 ± 2.49

Compounds from Aspergillus sp. 01NT.1.12.3
Dihydroaspirone (9)
Aspilactonol F (10)

F

(10),

6β,

7α,

14-

trihydroxyconfertifolin (11) and 6β, 9α, 14-trihydroxycinnamolide (12)
were obtained from Aspergillus sp. 01NT.1.12.3 did not show
antioxidant activity. Meanwhile, two compounds andrastin A (13) and
citreohybridonol (14) isolated from P. chrysogenum 045-357-2 were
able to catch ABTS free radicals effectively with EC50 values of 6.86
and 11.06 µg/mL, respectively.
3.5.4. Determination of neuroprotective activity
Aspertetranone D (3), mactanamide (4), dihydroaspyrone (9),
aspilactonol F (10), 6β, 7α, 14-trihydroxyconfertifolin (11) were noncytotoxic against Neuro-2a cells at concentrations up to 100 μM.
Compounds 3, 4 and 9-11 were further determined to protect neuronal
activity Neuro2a treated with 6-OHDA. However, only mactanamide (4)
showed a 42% increase in the viability of Neuro2a cells treated with 6-


23
OHDA at 50 µM. This is the first study to evaluate the neuroprotective
activity of mactanamide in a 6-OHDA neurotoxic supplement model. The
scientists

predict

the 1H NMR spectrum should be selected for further studies.
3. Three strains of A. flocculosus 01NT.1.1.5, Aspergillus sp.
01NT.1.12.3 and P. chrysogenum 045-357-2 biosynthesis of crude
antibiotics reach the highest in RYE medium with salt concentration
of 25-35 g/L, initial pH 6.0-7.0 and incubation period of 14-22 days.
4. Isolation, purification and determination of the chemical