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:
Trang 1GRADUATE 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
Trang 2Advisor 1: Assoc Prof Dr Phi Quyet Tien
Advisor 2: Assoc Prof Dr Tran Thi Thanh Van
Thesis can be found in:
- The library of the Graduate University Science and Technology
- National Library of Vietnam
Trang 3INTRODUCTION
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 methoxy-butane-1-one, including three known compounds as modiolide A-B and 1- (2,5-dihydroxyphenyl) -2-buten-1-one were obtained from
1-(2,5-dihydroxyphenyl)-3-the culture fermentation of endophytic fungus Paraconiothyrium sp
VK-13 Two out of five obtained compounds demonstrated
anti-inflammatory 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 3,6-dimethyl-2-pyrone, 4-methoxy-isoquinolin-1 (2H) -one and N, N-diethyl- 3-methyl-benzamide With aim to obtain natural bioactive compounds from marine fungi in order to find new sources of
4-hydroxy-pharmaceutical materials, we carry out the study: “Study on chemistry and biological activity of some compounds from marine fungi isolated in Central Vietnam”
Trang 42 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),
Trang 5mactanamide (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, anti-inflammatory, 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
Trang 6tropical 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), PC-
3 (cancer prostate cancer) and MDA-MB-231 (mammary carcinoma) provided by Korea Institute of Oceance Science and Technology
Trang 7- 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
Trang 8environmental 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)
-2,5-diphenyltetrazolium bromide)
Trang 92.2.9 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)
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
Figure 3.1
Number of marine fungi were isolated from Ninh Thuan, Nha Trang and Da Nang
Trang 10aureus 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
Trang 11strains, 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, 045-357-2, 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
Trang 12Figure 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
Morphological characteristics
1 01NT.1.1.5 - Cream yellow, round, 22-25 mm in diameter
- Filamentous surface with many cream-yellow spores, curled
- Producing pale yellow soluble pigment
- Filamentous margin
2 01NT.1.5.4 - 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
168ST.16.1
01NT.1.5.4
045-357-2
Trang 133 01NT.1.12.
3
- Grey white, round,18-22 mm in diameter
- Smooth filamentous surface with many yellow spores in the middle of colonies
olive 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
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