Identification and Biological Evaluation of Secondary Metabolites from Marine Derived Fungi-Aspergillus sp. SCSIOW3, Cultivated in the Presence of Epigenetic Modifying Agents

Chemical epigenetic manipulation was applied to a deep marine-derived fungus, Aspergillus sp. SCSIOW3, resulting in significant changes of the secondary metabolites. One new diphenylether-O-glycoside (diorcinol 3-O-α-D-ribofuranoside), along with seven known compounds, were isolated from the culture treated with a combination of histone deacetylase inhibitor (suberohydroxamic acid) and DNA methyltransferase inhibitor (5-azacytidine). Compounds 2 and 4 exhibited significant biomembrane protective effect of erythrocytes. 2 also showed algicidal activity against Chattonella marina, a bloom forming alga responsible for large scale fish deaths.


Introduction
With the recent completion of fungal genomes, it has become clear that the number of gene clusters encoding secondary metabolites greatly outnumbers the characterized compounds from these organisms [1]. It has been demonstrated that chemical epigenetic method is an effective technique for promoting the transcription of silent biosynthetic pathways and is well suited for the generation of structurally unique secondary metabolites [2][3][4]. Several different types of commercially available chemical epigenetic agents have been explored by several laboratories and provided valuable outcomes, including Zn 2+ type histone deacetylase (HDAC) inhibitors: suberoylanilidehydroxamic acid (SAHA) and suberohydroxamic acid (SBHA); NAD type HDAC inhibitors: sirtinol, splitomycin, nicotinamide, and 2-anilino-benzamide; and DNA methyltransferase (DNMT) inhibitors: RG-108, procaine, and 5-azacytidine (5-AZA) [5]. Moreover, studies on the effect of the concomitant addition of these inhibitors have been conducted. A marked increase was observed in the secondary metabolite produced by Isariatenuipes and Gibellulaformosana cultivated in the presence of both SBHA and RG108 [6,7]. In order to maximize the opportunity for detecting novel secondary metabolites, we have begun using chemical epigenetic induction as a routine part of our screening program involving the we successfully obtained four new eremophilane-type sesquiterpenes from a deep marine derived fungi, Aspergillus sp. SCSIOW2, in the presence of a combination of SBHA and 5-AZA [8].
In this study, we applied a test bed of 72 marine derived fungi (strain numbers and properties shown in Table S1, Supporting Information) to assess changes in their biosynthetic products induced by adding a combination of 1 mM SBHA and 1 mM 5-AZA. The crude ethyl acetate extracts (from broths + or − SBHA and 5-AZA) were profiled by analytical RP C18 high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) to observe differences between the matched pairs. One member of this set, Aspergillus sp. SCSIOW3, showed noteworthy changes in the accumulation of some new peaks in HPLC (3, 5 and 8 were new emerging peaks; the amount of 6 was increased, 7 was decreased, and 1, 2 and 4 were not significantly changed. Figure 1A) and was chosen for further study. Therefore, the EtOAc extract of this culture was scaled-up and separated by using column chromatography and semi-preparative HPLC, resulting in the isolation of one new compound, diorcinol 3-O-α-D-ribofuranoside (1), along with seven known compounds, diorcinal (2), 3,3′-dihydroxy-5,5′-dimethyldibenzofuran (3), cordyol (4), gibellulin B (5), cyclo-(L-Trp-L-Phe) (6), sydonic acid (7), and sydowic acid (8) ( Figure 1B).
Diorcinal (2) [13,14], 3,3 -dihydroxy-5,5 -dimethyldibenzofuran (3) [15], cordyol (4) [12], gibellulin B (5) [16], cyclo-(L-Trp-L-Phe) (6) [17], sydonic acid (7) [18], and sydowic acid (8) [19] were known compounds, whose structures were elucidated by comparisons with the literature (MS, 1 H and 13 C-NMR data of known compounds are available from supporting information). Biomembrane protective activity of the representative compounds 1, 2, 4, 7 and 8 (which have enough amounts) was tested using the erythrocytes protective assay [20]. Among the five compounds, the diphenyl ethers 2 and 4 showed a relatively strong protective effect against free radicals, with EC 50 at 8.7 and 4.9 µM, respectively ( Figure 2). While the glycosylated compound 1 showed an activity reduction, with EC 50 at 20.8 µM. Compound 7, a benzoic acid with a C 8 aliphatic side chain, showed moderate activity (EC 50 at 17.1 µM). In contrast, compound 8, which had a six-membered ring side chain, showed a significant activity reduction (13.9% membrane protective rate at 100 µM, data not shown in Figure 2). Structures of 7 and 8 suggested the loss of activity might possibly come from the formation of six-membered ring which had a larger steric interference. Meanwhile, the same compounds did not exhibit any radical scavenging activity even at much higher concentrations (200, 400 µM) on DPPH assay, which was carried out at a non-cell condition [20] ( Table 2, five concentrations of each compound were examined, data of the highest conctration were shown). Several antioxidative effects of diphenyl ethers were reported before [21,22], whereas the biomembrane protective effects were only illustrated in this research.
The algicidal activity of diorcinal (2) and sydonic acid (7) (which we have enough amounts) against Chattonella marina, a bloom forming alga responsible for large scale fish deaths, at different concentrations (6.25, 12.5, 25, 50 and 100 µM) over time (15 and 120 min) were also examined. Greater algicidal activity was observed with higher concentrations and longer treatment times by 2 ( Figure 3). 7 did not show any activity even at the highest concentration over the above time period. 15 sulphonyl derivatives of diphenyl ethers were reported as potential algaecides against Chlorella fusca and Anabaena variabilis [23]. This is the first report about diphenyl esters against Chattonella marina. six-membered ring side chain, showed a significant activity reduction (13.9% membrane protective rate at 100 μM, data not shown in Figure 2). Structures of 7 and 8 suggested the loss of activity might possibly come from the formation of six-membered ring which had a larger steric interference. Meanwhile, the same compounds did not exhibit any radical scavenging activity even at much higher concentrations (200, 400 μM) on DPPH assay, which was carried out at a non-cell condition [20] ( Table 2, five concentrations of each compound were examined, data of the highest conctration were shown). Several antioxidative effects of diphenyl ethers were reported before [21,22], whereas the biomembrane protective effects were only illustrated in this research. The algicidal activity of diorcinal (2) and sydonic acid (7) (which we have enough amounts) against Chattonella marina, a bloom forming alga responsible for large scale fish deaths, at different concentrations (6.25, 12.5, 25, 50 and 100 μM) over time (15 and 120 min) were also examined. Greater algicidal activity was observed with higher concentrations and longer treatment times by 2 ( Figure 3). 7 did not show any activity even at the highest concentration over the above time period. 15 sulphonyl derivatives of diphenyl ethers were reported as potential algaecides against Chlorella fusca and Anabaena variabilis [23]. This is the first report about diphenyl esters against Chattonella marina.  The data were expressed as the means ± SD from four individual experiments and were analyzed using a t-test to determine any significant differences. *** p ≤ 0.001 compared to control group.   The data were expressed as the means ± SD from four individual experiments and were analyzed using a t-test to determine any significant differences. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to control group.

Strain
Fungus SCSIOW3 was isolated from a deep marine sediment sample collected in the South China Sea (111°36.160 E, 17°59.928 N) at a depth of 2134 m. This fungus was characterized as Aspergillus The data were expressed as the means ± SD from four individual experiments and were analyzed using a t-test to determine any significant differences. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to control group.

Strain
Fungus SCSIOW3 was isolated from a deep marine sediment sample collected in the South China Sea (111 • 36.160 E, 17 • 59.928 N) at a depth of 2134 m. This fungus was characterized as Aspergillus sp. based on the analysis of the ITS region sequence with GenebankS1. This fungus was deposited in the Marine Microbial Lab. College of Life Science, Shenzhen University (Shenzhen, China).

Fermentation, Extraction, and Isolation
Both seed and production media have the same constituents (2.0% glucose, 1.0% peptone, 0.5% yeast extract, with the pH adjusted to 7.5). Aspergillus sp. SCSIOW3 was cultured in 250 mL Erlenmeyer flasks containing 75 mL of seed medium. After growing at 28 • C, 220 rpm for two days, the cellular material was placed in a sterile Falcon tube and mixed by vortexing for several minutes to create a uniform fungal cell/spore suspension. Aliquots (5 mL) of seed cultures were inoculated into 250 mL of production medium in 1000 mL Erlenmeyer flasks. At the time of inoculation, 0.5 mL aliquots of DMSO-dissolved SBHA and water-dissolved 5-AZA was added in triplicate, resulting in final concentrations in the liquid media of 1 mM SBHA and 1 mM 5-AZA. The same amount of DMSO and water were added to the control group. The resulting cultures were fermented at 28

Antioxidant Assays
The direct antioxidative activity of the isolated compounds were evaluated by the modified 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) radical scavenging assay [13], in which compounds solutions (0.4 M HOAc/NaOAc buffer at 3:1 ratio) were mixed with 20% (m/v) DPPH ethanol solution, followed with a 30 min incubation in dark and detection at 517 nm. The biomembrane protective assay was tested in erythrocytes which were obtained by centrifuging whole rabbit heart vein blood with 3.2% citrate at 700 g for 10 min at 4 • C. 6 × 10 8 /mL of the washed erythrocytes were treated with the compounds for 30 min at 37 • C, followed with the addition of 100 mM, 2 -Azobis (2-amidinopropane) dihydrochloride (AAPH) to induce hemolysis for 60 min at 37 • C. The supernatant was measured at 545 nm after a centrifugation at 700 g, 2 min. The biomembrane protective activity was expressed as percentage of a negative control, in which intact erythrocytes in 0.9% saline were measured. Curcumin was used as a positive control.

Algicidal Activity
Chattonella marina was cultured in YMNL GXZ Constant temperature illuminate incubator (Nanjing Yimaneli Instrument Equipment Co., Ltd., Nanjing, China).The proliferation of algal was detected through OLYMPUS IX51 inverted microscope (OLYMPUS, Tokyo, Japan). C. marina was donated by the Algal Culture Collection of the Institute of Hydrobiology at Jinan University in Guangzhou, China. It was cultivated under a 12:12 h light-dark cycle with 50 µmol photons m −2 ·s −1 at 22 • C in a sterilized f/2 medium prepared with natural seawater. Exponential growth phase algal cultures were divided into aliquots for further study. C. marina in the exponential growth phase were divided into aliquots and plated in a 12-well plate at a density of 2.9 × 10 4 cells/well. The tested samples were dissolved in dimethyl sulfoxide (DMSO), and then two-fold serial dilutions were performed. 2 µL of each sample solution was then added in the culture medium to make the final concentrations of 100, 50, 25, 12.5 and 6.5 µM, respectively. 2 µL of DMSO was used as a solvent control. Four parallel repetition was set in each group. The alga was cultured under the conditions described above. Living alga cells in 100 µL culture were counted by a DengXun DSJ-01 counting chamber for zooplankton (Xiamen Den Instrument Co., Ltd., Fujian, China) to detect the proliferation of the algal cells over 15 min and 120 min.

Statistical Analysis
All results were expressed as mean ± SD. Statistical significance (p values) of the results was calculated by Student's t-test. The results were considered to be significant when p < 0.05 (*), and to be highly significant when p < 0.01 (**).
Supplementary Materials: The following are available online: Table S1: Fungal strains used in epigenetic experiments. MS, 1 H-and 13 C-NMR data of known compounds are also available from supporting information.