Tóm tắt Luận án Study on chemistry and biological activity of some compounds from marine fungi isolated in central Vietnam

inoma) 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) -2,5- diphenyltetrazolium 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). 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 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, 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 10 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 Fungal strains Photos of colonies 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 11 3 01NT.1.12. 3 - Grey white, round,18-22 mm in diameter - Smooth filamentous surface with many olive- yellow 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 045- 357-2, 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, HR- ESI-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 13C 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. 1 2 3 4 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 HR- ESI-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. 9 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-dihydroxy- 7α-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, 14- dihydroxy-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, 14- trihydroxyconfertifolin. 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 Compounds Antimicrobial activity (MIC, µg/mL) Gram (+) bacteria Gram (-) bacteria Yeast B. cereus ATCC 11778 S. faecalis ATCC 19433 S. aureus ATCC 25923 E. coli ATCC 25922 P. aeruginosa ATCC 27853 C. albicans ATCC 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 64 64 16 16 Mactanamide (4) 64 32 64 128 16 32 Cycloechinulin (5) 64 64 > 256 128 64 64 Asteltoxin (6) 64 64 > 256 128 64 64 Ochraceopone F (7) 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 32 32 32 32 Aspilactonol F (10) 16 32 32 32 32 32 6β,7α,14-trihydroxy- confertifolin (11) 32 32 32 32 32 32 6β,9α,14-trihydroxy- cinnamolide (12) 32 32 32 32 32 32 Compounds from P. chrysogenum 045-357-2 Andrastin A (13) 128 128 64 32 16 32 Citreohybridonol (14) 32 64 16 32 16 32 Positive control Amoxicillin 256 256 0.25 8 64 > 256 Cefotaxime 128 16 2 0.125 8 > 256 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 Compounds from A. flocculosus 01NT.1.1.5 Phomaligol A2 (1) 26.45 ± 2.14 19.77 ± 7.70 28.55 ± 5.74 19.62 ± 1.92 26.94 ± 3.78 19.74 ± 4.26 Wasabidienone E (2) 21.71 ± 3.44 17.91 ± 8.65 22.02 ± 2.12 21.05 ± 6.85 23.22 ± 0.45 23.68 ± 1.77 Aspertetranone D (3) 19.17 ± 2.64 25.82 ± 9.86 23.03 ± 6.92 20.20 ± 6.76 25.89 ± 5.18 37.16 ± 1.37 Mactanamide (4) 22.76 ± 2.09 19.14 ± 7.19 20.78 ± 3.51 22.22 ± 2.62 19.51 ± 0.28 16.51 ± 2.71 Cycloechinulin (5) 28.47 ± 2.49 24.60 ± 7.79 29.12 ± 5.27 25.33 ± 3.31 22.97 ± 3.72 26.66 ± 6.74 Asteltoxin (6) 16.19 ± 1.58 14.57 ± 6.32 19.45 ± 3.78 17.02 ± 2.15 18.59 ± 3.02 15.33 ± 2.19 Ochraceopone F (7) 19.32 ± 3.59 16.97 ± 6.92 21.03 ± 4.25 19.78 ± 5.21 20.67 ± 2.45 17.56 ± 3.22 Asterriquinone C1 (8) 81.58 ± 2.49 77.36 ± 5.52 83.58 ± 2.35 80.30 ± 3.32 88.07 ± 3.24 92.51 ± 2.08 Compounds from Asp