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 INTRODUCTION 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 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 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 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 The layout of the thesis The thesis has 149 pages including: Introduction pages, Chapter Overview of 33 pages, Chapter Materials and Methods 13 pages, Chapter Results 54 pages, Chapter Discussions 16 pages, Conclusions and Recommendations pages, List of publications 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 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), 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 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 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 MATERIALS AND METHODS 2.1 Materials and study media 2.1.1 Studied samples, tested strains and cell lines - Studied samples: samples of sponges, 11 samples of soft corals, samples of seaweeds and 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, samples of soft corals, samples of seaweeds and samples of marine sediments were collected from Nha Trang Bay (12o18’N; 109o31’E) at a depth of 5-10 m, February 2016; samples of sponges, 14 samples of soft corals, 16 samples of seaweeds and 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 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 - 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 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) 2.2.9 Processing research data The experiments were repeated times and the data expressed as a mean ± standard deviations were calculated using Microsoft Excel 2010 CHAPTER 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 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 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, 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 selected fungal strains The results showed that the crude extracts of 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 11 01NT.1.12 - 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 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 01NT.1.9.4 - Grey brown, round, 15-18 mm in diameter Filamentous surface, umbonate Produce dark grey soluble pigment Entire margin 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 168ST.35.2 - Mossy green, round, 20-24 mm in diameter Smooth surface Not produce soluble pigment Entire margin 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 selected strains of fungi belong to Ascomycota Of which, there are 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 01NT.1.1.5 - Conidia globose, size 2,5-3 µm - Vesicle globose, 35-45 µm wide - Conidiaphores with rough wall 01NT.1.5.4 - Conidia globose, size 3,5-4,5 µm, rough surface Vesicle globose, 30-75 µm wide Conidiaphores with smooth wall 01NT.1.12.3 - Conidia globose, size 2-2,5 µm - Vesicle globose, 25-35 µm wide - Conidiaphores with rough wall 168ST.16.1 - Conidia globose, size 2-2,5 µm - Vesicle globose, 25-35 µm wide - Conidiaphores with transparent wall 13 01NT.1.9.4 - Gray mycelium, 2,5–3,9 µm wide, sparse branching - Mycelium with smooth and transparent wall 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 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 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 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 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 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 and 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 has signals of methyl groups, methine groups, methoxy group (δH 3.89/H-12) and aromatic ring proton at δH 5.62 (H-4) Two olefinic carbons, ketone 15 carbons, carbons are directly linked to oxygen, and 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, 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 are similar to that of phomaligol A isolated from the fungus Paecilomyces lilacinus F-9, except for the OH group at C-9 Compound is a new compound and is named as phomaligol A2 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 methine group at δH 2.47 (H-7), methylene groups at H-16, H-10, H- 9, H-6, H-15, methyl groups, 10 quaternary carbon signals, carbonyl ketone at δC 218.0 (C-14), carbonyl ester at δC 165.7 (C-1), conjugated oxidized carbons at δC 97.6 (C-2), 107.5 (C-4), 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 closely resemble ochraceopone E, an α-pyrone merosesquiterpenoid from Aspergillus ochraceopetaliformis SCSIO 05702 isolated from Antarctica The difference between compound and ochraceopone E is compound without hydroxyl group at C-9 Therefore, the structure of compound 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 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 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 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 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 compounds 9-12 from Aspergillus sp 01NT.1.12.3 shows the effective inhibitory activity of the growth of 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 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 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 64 > 256 Cefotaxime 128 16 0.125 > 256 Positive control 20 Mactanamide (4) is able to inhibit the growth of 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 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 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 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 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 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, 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 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%) 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, 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 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 Isolation, purification and determination of the chemical 24 structures of 14 compounds from selected fungal strains: - Strain A flocculosus 01NT.1.1.5: Isolation of 08 compounds, 02 new compounds are phomaligol A2 (1) and ochraceopone F (7), and 06 known compounds including wasabidienone E (2), aspertetranone D (3), mactanamide (4), cycloechinulin (5), asteltoxin (6) and asterriquinone C1 (8) - Strain Aspergillus sp 01NT.1.12.3: Isolation of 04 compounds, 02 new compounds are 6β,7α,14-trihydroxyconfertifolin (11), 6β,9α,14-trihydroxycinnamolide (12) and 02 known compounds including dihydroaspyrone (9) and aspilactonol F (10) - Strain P chrysogenum 045-357-2: Isolation of 02 compounds including andrastin A (13) and citreohybridonol (14) Obtained compounds exhibited antimicrobial activity with MIC values of 8-128 µg/mL Asterriquinone C1 (8) has the ability to inhibit the growth of cancer cell lines including HCT-15, NUGC-3, NCI-H23, ACHN, PC-3 and MDA-MB-231 with IC50 values 30-40 µM Mactanamide (4) exhibits ABTS free radical scavenging activity with EC50 1.72 µg/mL and increases 42% the viability of Neuro2a treated by 6-OHDA RECOMENDATIONS Continuing to study gene clusters for biosynthesis biologically active substances from marine fungi strains isolated Testing the effect of bioactive compounds on test animal models LIST OF PUBLICATIONS AN Yurchenko, PTH Trinh, EV Girich, OF Smetanina, AB Rasin, RS Popov, SA Dyshlovoy, GV Amsberg, EM Menchinskaya, TTT Van, SS Afiyatullov Biologically active metabolites from the marine sediment-derived fungus Aspergillus flocculosus Marine Drugs, 2019, 17(10), 579 HJ Shin, BK Choi, PTH Trinh, HS Lee, JS Kang, TTT Van, HS Lee, JS Lee, YJ Lee, JH Lee Suppression of RANKL-induced 25 osteoclastogenesis by the metabolites from the marine fungus Aspergillus flocculosus isolated from a sponge Stylissa sp Marine Drugs, 2018, 16(1), 14 EA Yurchenko, ES Menchinskaya, EA Pislyagin, PTH Trinh, EV Ivanets, OF Smetanina, AN Yurchenko Neuroprotective activity of some marine fungal metabolites in the 6-hydroxydopamin- and paraquat-induced Parkinson’s disease models Marine Drugs, 2018, 16(11), 457 PTH Trinh, TTT Van, BM Ly, BK Choi, HJ Shin, JS Lee, HS Lee, PQ Tien Antimicrobial activity of natural compounds from sponge – derived fungus Aspergillus flocculosus 01NT.1.1.5 Vietnam Journal of Biotechnology, 2018, 16(4), 729-735 PTH Trinh, PQ Tien, NTD Ngoc, BM Ly, TTT Van Isolation and screening marine fungi with antimicrobial activity from samples collected in Nha Trang Bay, Vietnam Vietnam Journal of Biotechnology, 2018, 16(1), 181-187 PTH Trinh, NTK Chanh, NTD Ngoc, PQ Tien, BM Ly, TTT Van Secondary metabolites from a marine-derived fungus Penicillium chrysogenum 045-357-2 Vietnam Journal of Science and Technology, 2017, 55 (1A), 65-72 PTH Trinh, NTD Ngoc, VTD Trang, LQ Phong, PQ Tien, BM Ly, TTT Van Antimicrobial activity of marine fungi isolated from the Son Tra peninsula, Da Nang, Vietnam Vietnam Journal of Biology, 2017, 39(4), 457-462 PTH Trinh, NTD Ngoc, VTD Trang, PQ Tien, BM Ly, TTT Van, PD Thinh, PT San Effect of culture conditions for antimicrobial activity of marine - derived fungus Aspergillus flocculosus 01NT.1.1.5 Vietnam Journal of Biotechnology, 2017, 15(4), 721-728 PTH Trinh, NTD Ngoc, PQ Tien, BM Ly, TTT Van Effect of cultural conditions on antimicrobial activity of marine – derived fungus Penicillium chrysogenum Vietnam Journal of Biotechnology, 2016, 14(4), 727-733 ... 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... antimicrobial activity from samples collected in Nha Trang Bay, Vietnam Vietnam Journal of Biotechnology, 2018, 16(1), 181-187 PTH Trinh, NTK Chanh, NTD Ngoc, PQ Tien, BM Ly, TTT Van Secondary... Nang, Vietnam Vietnam Journal of Biology, 2017, 39(4), 457-462 PTH Trinh, NTD Ngoc, VTD Trang, PQ Tien, BM Ly, TTT Van, PD Thinh, PT San Effect of culture conditions for antimicrobial activity