Nghiên cứu hoá học và hoạt tính sinh học của một số hợp chất tử vi nấm biển phân lập tại miền trung việt nam tt tiếng anh

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:

3rd Reviewer:

The thesis will be defended at Graduate University Science and
Technology – Vietnam Academy of Science and Technology, at
hour

date

month

2019

Thesis can be found in:
- The library of the Graduate University Science and Technology
- National Library of Vietnam


1
INTRODUCTION
1. The rationale of the thesis
Nowadays, along with the socio-economic development, health
protection is always a great concern of each country. Although human
modern medicine has reached a certain level of progress, infectious and
incurable diseases are still big public health issues. Therefore,
approaching natural compounds with new biological activities,
especially compounds derived from marine fungi, is attracting the
research interest of scientists worldwide.
Vietnam is located in a tropical region, with a diverse marine
ecosystem which is potential for biodiversity research. This is the basis
source for studies on biodiversity as well as bioactive substances from

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

(antibiotimicrobial, cytotoxic, antioxidant, neuroprotective activities) of
obtained individual compounds.
3. The main contents of the thesis
- Isolation, evaluation of antibiotic activity and selection of marine
fungus strains for further research.
- Determine suitable solid fermentation conditions for the
biosynthesis of crude antibiotics of selected marine fungal strains.
- Separation, purification and determination of the structure of
secondary metabolic compounds from fermented extraction residues of
selected fungal strains.
- Determine the bioactivities of obtained secondary metabolites.
4. The layout of the thesis
The thesis has 149 pages including: Introduction 2 pages, Chapter 1.
Overview of 33 pages, Chapter 2. Materials and Methods 13 pages,
Chapter 3. Results 54 pages, Chapter 4. Discussions 16 pages,
Conclusions and Recommendations 2 pages, List of publications 2
pages, References 27 pages.
THE NEW FINDINGS OF THE THESIS
- The thesis is a new study on isolation and evaluation of antimicrobial
activity of marine fungal strains isolated in Central Vietnam.
- Purification and evaluation of antibiotic, cytotoxic, antioxidant, and
neuroprotective activities of 14 secondary metabolites obtained from 3
marine fungal strains of Aspergillus flocculosus 01NT.1.1.5, Aspergillus
sp. 01NT.1.12.3 and Penicillium chrysogenum 045-357-2, including:
phomaligol A2 (1), wasabidienone E (2), aspertetranone D (3),


3
mactanamide (4), cycloechinulin (5), asteltoxin (6), ochraceopone F (7),
asterriquinone C1 (8), dihydroaspyrone (9), aspilactonol F (10), 6β, 7α,

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

coastline and diverse ecosystems. However, according to the literature,
the number of research works is limited compared to the potential of
marine fungus exploitation in Vietnam. Therefore, the research direction
to discover natural compounds from Vietnamese marine fungi is
necessary, contributing to the exploitation of new biological substances
while protecting the national sovereignty over the sea.
CHAPTER 2. MATERIALS AND METHODS
2.1. Materials and study media
2.1.1. Studied samples, tested strains and cell lines
- Studied samples: 4 samples of sponges, 11 samples of soft corals,
9 samples of seaweeds and 8 samples of marine sediments collected
from Ninh Thuan sea area (11o70'N; 109o21'E) at a depth of 10-20 m,
June 2013; 17 samples of sponges, 3 samples of soft corals, 8 samples
of seaweeds and 9 samples of marine sediments were collected from
Nha Trang Bay (12o18’N; 109o31’E) at a depth of 5-10 m, February
2016; 8 samples of sponges, 14 samples of soft corals, 16 samples of
seaweeds and 4 samples of sediment were collected from Da Nang
waters (16o11'N; 108o31'E) at a depth of 8-15 m, August 2016.
- Tested microorganisms: B. cereus ATCC 11778, E. coli ATCC 25922,
S. aureus ATCC 25923, P. aeruginosa ATCC 27853, S. faecalis ATCC
19433, L. monocytogenes ATCC 19111 và C. albicans ATCC 10231
provided by Nha Trang Institute of Technology Research and Application.
- Human cancer cell lines: HCT-15 (colon cancer), NUGC-3
(stomach cancer), NCI-H23 (lung cancer), ACHN (kidney cancer), PC3 (cancer prostate cancer) and MDA-MB-231 (mammary carcinoma)
provided by Korea Institute of Oceance Science and Technology.


5
- Neuroblastoma cell line Neuro2a (ATCC® CCL-131™) provided by
Pacific Institute of Bioorganic Chemistry-Russian Academy of Sciences.

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

The morphological characteristics and scientific name of fungi were
determined according to Raper and Thom (1949), Samson et al. (2011),
Crous and Groenewald (2015), Stolk and Samson (1972). In addition,
the fungi were classified based on sequence analysis of ITS/28S rDNA
and compared with corresponding gene sequences on the Gen Bank.
2.2.5. Determination of suitable solid fermentation conditions for
antibiotic biosynthesis of marine fungi
The fermentation factors including time, salt concentration and


6
environmental pH were investigated to assess the effect of fermentation
conditions on RYE medium on the ability of antibiotic biosynthesis of
selected fungal strains.
2.2.6. Separation of secondary metabolites from marine fungi
The fungal biomass and fermentation medium were extracted with ethyl
acetate at room temperature in a static state for 48 hours and carried out at
40°C to collect crude ethyl acetate residue. The residue is further separated
based on chromatographic methods including thin-layer chromatography
(TLC), column chromatography (CC) and high-performance liquid
chromatography (HPLC) to obtain individual compounds..
2.2.7. Determination of the chemical structure of secondary
metabolites from marine fungi
The chemical structure of compounds is determined based on a
combination of modern spectroscopic methods such as nuclear magnetic
resonance (NMR) and mass spectrometry (ESI-MS or HR-ESI-MS).
2.2.8. Determination of biological activity of secondary metabolites
from marine fungi
2.2.8.1. Determining antimicrobial activity
Evaluation by the method of determining the minimum inhibitory

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

marine fungi
were isolated
from
Ninh
Thuan, Nha
Trang and Da
Nang

Analysis of colony morphological characteristics of 273 strains of
fungi obtained showed that most strains had round colony shape (81.6%,
n=235),

smooth

surface

(54.2%,

n=148).

Flat

and

entire

contoured/border colonies were also recorded at high rates of 55.3%
(n=151) and 74.7% (n=204), respectively. Fungal strains have the
surface of colonies of different color groups, in which the green/mossy
green colonies occupies the highest rate (31.9%, n=87).

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.1.9.4,

01NT.1.12.3, 045-357-2, 168ST.16.1, 168ST.35.2 and 168ST.51.1
should be selected for further studies on the analysis of crude extraction
residues on TLC and NMR spectra and at the same time identify the
specific morphological characteristics and classification.
3.2. Analyze the crude extracts and determine the classification
characteristics of 8 selected fungal strains
The results showed that the crude extracts of 8 fungal strains showed
streaks with different colors and retardation factor on the TLC plate (Figure
3.9). Three strains of 01NT.1.1.5, 01NT.1.12.3 and 045-357-2 were predicted
to have a variety of layers of substances contained in the extraction residue,
followed by strains of 01NT.1.5.4 and 168ST.16.1. The remaining three


9
strains, 01NT.1.9.4, 168ST.35.2 and 168ST.51.1, showed unclear streaks on
the chromatograph. Therefore, five strains of 01NT.1.1.5, 01NT.1.12.3, 045357-2, 01NT.1.5.4 and 168ST.16.1 were selected for the analysis of crude
extraction residues on the 1H NMR spectrum.
The 1H NMR spectrum of the extracts from 3 fungal strains 01NT.1.1.5,
01NT.1.12.3 and 045-357-2 clearly shows proton signals at low field regions
(5-8 ppm), so it is expected containing aromatic ring structures in the residue
obtained. The residue from 2 strains 168ST.16.1 and 01NT.1.5.4 recorded very
few proton signals in low field area and 1H NMR spectrum showed simpler
signals than 3 strains 01NT.1.1.5, 01NT.1.12.3 and 045-357-2 (Figure 3.10).

Figure 3.9. Analysis of crude extracts of 8 selected fungal strains on
TLC (solvent system toluene : isopropanol, 6:1 v/v)
01NT.1.1.5

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

colonies

Morphological characteristics
- Cream yellow, round, 22-25 mm in diameter
- Filamentous surface with many cream-yellow
spores, curled
- Producing pale yellow soluble pigment
- Filamentous margin
-

Grey and yellow, round, 22-25 mm in diameter
Filamentous surface with many cream-yellow
spores then change to black
Producing brown soluble pigment
Filamentous margin


11
3

01NT.1.12.
3

-

Grey white, round,18-22 mm in diameter
Smooth filamentous surface with many oliveyellow spores in the middle of colonies
Producing yellow soluble pigment
Thick filamentous margin

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

- Exudate pigment on the surface
- Entire margin

7

168ST.35.2

-

Mossy green, round, 20-24 mm in diameter
Smooth surface
Not produce soluble pigment
Entire margin

8

168ST.51.1

-

White, round, 22-26 mm in diameter
Filamentous surface
Not exudate pigment on the surface, produce
brown soluble pigment
Entire margin

-

-

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

1

01NT.1.1.5

- Conidia globose, size 2,5-3 µm
- Vesicle globose, 35-45 µm wide
- Conidiaphores with rough wall

2

01NT.1.5.4

-

Conidia globose, size 3,5-4,5 µm, rough
surface
Vesicle globose, 30-75 µm wide
Conidiaphores with smooth wall

3

01NT.1.12.3

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

4

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

- Spore-shaped sporangia, with one to three
spores.
- Spores smooth, ellipse, size 2-3 x 1,5-2,5
µm.
- Conidiaphores with thick wall.

8

168ST.51.1

- Conidiaphores with tubular shape, smooth
wall, tapering at the top.
- Conidiaphores are produced directly from
mycelium, size 12-20 x 1,5-2,0 µm.
- Mycelium branching

Five studied fungal strains include A. flocculosus 01NT.1.1.5, A.
niger 01NT.1.5.4, Aspergillus sp. 01NT.1.12.3, P. chrysogenum 045357-2, Aspergillus sp. 168ST.16.1 showed quite diverse streaks on the
TLC plate. However, there are only 3 strains A. flocculosus 01NT.1.1.5,
Aspergillus sp. 01NT.1.12.3 and P. chrysogenum 045-357-2 clearly
show proton signals in the low field region on the 1H NMR spectrum,
predicting the presence of aromatic ring structures in the extracted
residue. Therefore, three fungal strains were selected for further studies
on investigating suitable fermentation conditions and separating natural
compounds. This is a new study of bioactive compounds from the fungi
A. flocculosus and P. chrysogenum isolated in the central coast of
Vietnam.

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

15
carbons, 2 carbons are directly linked to oxygen, and 1 methoxy carbon
was observed at 13C NMR spectrum. The other six carbon signals are
thought to be one sec-butyl (δC 20.2/C-10, 12.1/C-11, 68.4/C-9, và
46.5/C-8) and two methyl groups (δC 22.7/C-14 , 20.7/C-13). The 1H and
13

C NMR spectra data of compound 1 are similar to that of phomaligol

A isolated from the fungus Paecilomyces lilacinus F-9, except for the OH group at C-9. Compound 1 is a new compound and is named as
phomaligol A2.

2

1

4

3

5

8
6

7

Figure 3.15. Chemical structures of the compounds 1-8 isolated
from A. flocculosus 01NT.1.1.5
Compound 7: Ochraceopone F (New compound)

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

F

(10),

6β,

9α,

14-

trihydroxycinnamolide (11) and 6β, 7α, 14-trihydroxyconfertifoline (12). In
particular, compounds 11 and 12 are identified as new compounds.

10

9

11

12

Figure 3.26. Chemical strcutures of the compounds 9-12
from A. flocculosus 01NT.1.12.3
Compound 11: 6β,7α,14-trihydroxyconfertifolin (New compound)
White powder, HR-ESI-MS (m/z 305,1361 [M+Na]+), molecular


17
formula C15H22O5. Spectral data showed that the structure of compound
11 is similar to 6β, 14-dihydroxy-7α-methoxyconfertifoline first

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.

crustosum 1088.

13

14

Figure 3.31. Chemical structures of 13-14 from
P. chrysogenum 045-357-2
Similar to andrastin A, citreohybridonol is also produced by many
fungal strains of the genus Penicillium. In 2018, the citreohybridonol was
also discovered by the Özkaya et al. from the fungus Penicillium
atrovenetum originating from the sponge. However, Özkaya et al. Did not
identify the structure based on NMR spectra but performed on the basis of
analysis of single crystal X-ray diffraction. From the results, all 14
compounds collected from 3 selected strains of fungi contained aromatic
ring structures and matched the initial substance screening results based on
TLC and 1H NMR analysis.
3.5. Determination of bioactivities of 14 compounds isolated from selected
marine fungi
3.5.1. Determination of antimicrobial activity
The results showed that 14 isolated compounds exhibited antimicrobial
activity against most pathogens tested with MIC values of 8-128 µg/mL
(Table 3.7). In particular, the new compound ochraceopone F (7) from A.
flocculosus 01NT.1.1.5 and 4 compounds 9-12 from Aspergillus sp.
01NT.1.12.3 shows the effective inhibitory activity of the growth of 6 tested


19
pathogens (MIC, 8-32 µg/mL). The antimicrobial activity of the new
phomaligol A2 (1) from A. flocculosus 01NT.1.1.5 was also recorded with

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)

128

32

64

64

16

16

Aspertetranone D (3)

64

32

Mactanamide (4)

64

32

64

64

16

16

64

128

16

32

Cycloechinulin (5)

64

64

> 256

128

64

64

Asteltoxin (6)
Ochraceopone F (7)

64

64

> 256

128

64

64

8

8

8

32

16

16

Asterriquinone C1 (8)

32

32

> 256

> 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

32

32

32

32

32

32

32

32
32
Compounds from P. chrysogenum 045-357-2
Andrastin A (13)
128
128

32

32

32

32

Citreohybridonol (14)

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

64

32

16

32

32

64

16

32

16

32

Amoxicillin

256

256

0.25

8

64

> 256

Cefotaxime

128

16

2

0.125

8

> 256

Positive control


20
Mactanamide (4) is able to inhibit the growth of 6 tested pathogens, of
which the resistance to C. albicans has been confirmed by a previous
report of Lorenz et al. (1998). The results are also consistent with the
author Wang et al. (2015) on asteltoxin (6) which did not show

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

Inhibition of the growth of cancer cells at the concentration of 30 µg/mL (%)
HCT-15

NUGC-3

NCI-H23

ACHN

PC-3

MDA-MB-231

19.77 ± 7.70
17.91 ± 8.65
25.82 ± 9.86
19.14 ± 7.19
24.60 ± 7.79
14.57 ± 6.32
16.97 ± 6.92
77.36 ± 5.52

28.55 ± 5.74
22.02 ± 2.12
23.03 ± 6.92
20.78 ± 3.51
29.12 ± 5.27
19.45 ± 3.78
21.03 ± 4.25
83.58 ± 2.35

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

-

-

12.25 ± 2.56
-

-

9.41 ± 3.42
-

8.62 ± 2.37
-

33.25 ± 2.67
36.72 ± 3.05

29.86 ± 3.92
35.19 ± 4.06

34.55 ± 4.01
32.15 ± 3.78

26.74 ± 2.15 39.54 ± 3.08
34.22 ± 3.03 40.25 ± 2.97

22.36 ± 1.78
38.65 ± 2.31

80.07 ± 6.96

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)

6β,7α,14-trihydroxyconfertifolin (11)
6β,9α,14-trihydroxycinnamolide (12)
Compounds from P. chrysogenum 045-357-2
Andrastin A (13)
Citreohybridonol (14)
Positive control
Adriamycin (0.3 µg/mL)
“-”: Inactive


22
Phomaligol A2 (1) exhibited weak inhibitory activity towards 6 tested
cancer cell lines. However, according to the announcement of Elbandy et
al. (2009), phomaligol A is not capable of toxicing cancer cell lines
including A-549 (lung cancer), SK-OV-3 (ovarian carcinoma), SKMEL-2 (skin cancer) and HCT-15 (colon cancer) when tested at a
concentration of 30 µg/mL. From the results it is possible to predict the
cytotoxic activity of phomaligol A2 related to hydroxyl group at C-9. The
remaining compounds have no inhibitory or weak inhibition to the growth
of test cell lines.
3.5.3. Determination of antioxidant activity
The study showed that out of 8 compounds obtained from A.
flocculosus 01NT.1.1.5, mactanamide (4) and wasabidienone E (2) were
able to catch ABTS free radicals most effectively with EC50 values of
1.72 and 12.03 µg/mL, respectively. Four compounds including
dihydroaspyrone

(9),

aspilactonol

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

that

the

structural

characteristics

of

D-2,6-

dihydroxyphenylalanine amino acids in the molecule together with oxygen
functional groups are related to the biological activity of the compound.
CONCLUSIONS
1. Isolation and description morphology characteristics of 273 fungal
strains from 29 samples of sponges, 28 samples of soft corals, 33 samples
of seaweeds and 21 samples of sediment collected from the waters of
Ninh Thuan, Nha Trang and Da Nang. Most of the fungal strains collected
had green/mossy green colonies (31.9%), round shapes (86.1%), smooth
surface (54.2%) and entire borders (74.7%).
2. Screening of 148/273 strains showing antimicrobial activity for
at least one tested pathogen. Morphological and sequence analysis of
ITS/28S rDNA region showed subspecific results of 08 broadspectrum antibiotic fungi including A. flocculosus 01NT.1.1.5, A.
niger 01NT.1.5.4, Aspergillus sp. 01NT.1.12.3, P. chrysogenum 045357-2, Aspergillus sp. 168ST.16.1, Biatriospora sp. 01NT.1.9.4,
Talaromyces sp. 168ST.35.2, and Talaromyces sp. 168ST.51.1. Of
which, 3 strains A. flocculosus 01NT.1.1.5, Aspergillus sp.
01NT.1.12.3 and P. chrysogenum 045-357-2 have crude extracts that
show substance diversity on TLC and have aromatic ring signals on

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