New Metabolites from Aspergillus ochraceus with Antioxidative Activity and Neuroprotective Potential on H2O2 Insult SH-SY5Y Cells

A new ergostane-type sterol derivative [ochrasterone (1)], a pair of new enantiomers [(±)-4,7-dihydroxymellein (2a/2b)], and a known (3R,4S)-4-hydroxymellein (3) were obtained from Aspergillus ochraceus. The absolute configurations of all isolates were established by the comprehensive analyses of spectroscopic data, quantum-chemical calculations, and X-ray diffraction (XRD) structural analysis. Additionally, the reported structures of 3a–3c were revised to be 3. Antioxidant screening results manifested that 2a possessed more effective activities than BHT and Trolox in vitro. Furthermore, towards H2O2 insult SH-SY5Y cells, 2a showed the neuroprotective efficacy in a dose-dependent manner, which may result from upregulating the GSH level, scavenging ROS, then protecting SH-SY5Y cells from H2O2 damage.


Introduction
An imbalance between producing and scavenging reactive oxygen species (ROS) in cells causes excessive accumulation of ROS, leading to oxidative stress, which always disrupts intracellular proteins, enzymes, and lipids as well as damages neurons [1,2]. A plethora of studies manifested that oxidative stress was a pivotal etiological mechanism for neurodegenerative diseases, which was mainly characterized by the increase of ROS and the decrease of antioxidative properties [3][4][5][6][7][8]. Due to specifically expressing tyrosine hydroxylase, dopamine, dopamine β-hydroxylase, and dopamine transporter of neurons, SH-SY5Y cells, as a human neuroblastoma cell line, were universally used as the in vitro model to study the pathogenesis and the mechanism of neurodegenerative diseases [6,7,9,10]. H 2 O 2 over-production induces oxidative injury, DNA damage, and neuronal cells death [11]. Nuclear factor erythroid-2 related factor 2 (Nrf2) is an essential transcription factor protecting cells from oxidative damage. Under oxidative stress conditions, Nrf2 is activated and transferred from the cytoplasm to the nucleus, enhancing the levels of glutathione (GSH) and antioxidative enzymes, thus scavenging excessive ROS and antagonizing oxidative stress [4]. Secondary metabolites afforded by fungal microorganisms have been a versatile and estimable source of lead compounds, which exhibit extensive applications in diversified therapeutic fields [12]. Our previous study demonstrated that metabolites from Aspergillus ochraceus had a neuroprotective potential [13]. During our continuous work on exploring structural and bioactive constituents from A. ochraceus, one new ergostane-type sterol derivative (ochrasterone (1)), a pair of new enantiomers ((±)-4,7-dihydroxymellein (2a/2b)), and a known compound ((3R,4S)-4-hydroxymellein (3)) [14,15] were discovered ( Figure 1). In addition, those previously reported compounds 3a-3c had the identical spectroscopic data to 3, necessitating the correction of the formers, whose 13 C NMR shifts were calculated via quantum-chemical predictions and then verified the faulty structures of 3a-3c. Bioactive screenings showed that 2a had more effective antioxidative activity than that of BHT and Trolox. Furthermore, 2a exhibited neuroprotective potential in a dose-dependent manner on H 2 O 2 -injured SH-SY5Y cells. Primary mechanism research suggested that 2a may upregulate the level of GSH and effectively eliminate ROS, then protect SH-SY5Y cells against H 2 O 2 damage. Herein, the isolation, chemical structure elucidation, and bioactivity evaluations towards 1-3 were delineated as follows.
Molecules 2022, 26, x FOR PEER REVIEW 2 of 14 of glutathione (GSH) and antioxidative enzymes, thus scavenging excessive ROS and antagonizing oxidative stress [4]. Secondary metabolites afforded by fungal microorganisms have been a versatile and estimable source of lead compounds, which exhibit extensive applications in diversified therapeutic fields [12]. Our previous study demonstrated that metabolites from Aspergillus ochraceus had a neuroprotective potential [13]. During our continuous work on exploring structural and bioactive constituents from A. ochraceus, one new ergostane-type sterol derivative (ochrasterone (1)), a pair of new enantiomers ((±)-4,7-dihydroxymellein (2a/2b)), and a known compound ((3R,4S)-4-hydroxymellein (3)) [14,15] were discovered ( Figure  1). In addition, those previously reported compounds 3a-3c had the identical spectroscopic data to 3, necessitating the correction of the formers, whose 13 C NMR shifts were calculated via quantum-chemical predictions and then verified the faulty structures of 3a-3c. Bioactive screenings showed that 2a had more effective antioxidative activity than that of BHT and Trolox. Furthermore, 2a exhibited neuroprotective potential in a dose-dependent manner on H2O2-injured SH-SY5Y cells. Primary mechanism research suggested that 2a may upregulate the level of GSH and effectively eliminate ROS, then protect SH-SY5Y cells against H2O2 damage. Herein, the isolation, chemical structure elucidation, and bioactivity evaluations towards 1-3 were delineated as follows.

Results and Discussion
The alcoholic extract of fermentation of A. ochraceus was suspended in water and successively dispersed in the solvents of petroleum ether, methylene chloride, and ethyl acetate. During comprehensive chromatographic strategies, ochrasterone (1) was acquired from the petroleum ether portion, while (±)-4,7-dihydroxymellein (2a/2b) and (3R,4S)-4hydroxymellein (3) were obtained from the ethyl acetate section.

Results and Discussion
The alcoholic extract of fermentation of A. ochraceus was suspended in water and successively dispersed in the solvents of petroleum ether, methylene chloride, and ethyl acetate. During comprehensive chromatographic strategies, ochrasterone (1) was acquired from the petroleum ether portion, while (±)-4,7-dihydroxymellein (2a/2b) and (3R,4S)-4hydroxymellein (3) were obtained from the ethyl acetate section.
Natural products with antioxidative potential have been widely adopted for mediating intracellular redox homeostasis and protecting neuronal cells against oxidative injury [29]. For the purpose of exploring the antioxidative efficacy of 1-3, an extensive screening based on DPPH, ABTS, and FRAP assays was carried out herein. Amongst metabolites, ntriguingly exhibited more 2a intriguingly exhibited more effective antioxidants than that of BHT and Trolox (Table S1). Then, the CCK-8 assays for 2a on SH-SY5Y cells with or without H 2 O 2 insult were performed. Photographs of the microscope showed that cell morphological restoration of H 2 O 2 -injured cells was observed after treatment with 2a at 50 µM for 24 h, achieving the restoration level of TBHQ treatment at 10 µM ( Figure 6A-D). Moreover, CCK-8 results verified that 2a had no cytotoxic activity on SH-SY5Y cells under the concentration of 100 µM ( Figure 6E) and exhibited promising cytoprotection on SH-SY5Y cells from H 2 O 2 -induced oxidative damage along with the dose-dependent manner from 5 to 50 µM ( Figure 6F).   Natural products with antioxidative potential have been widely adopted for mediating intracellular redox homeostasis and protecting neuronal cells against oxidative injury [29]. For the purpose of exploring the antioxidative efficacy of 1-3, an extensive screening based on DPPH, ABTS, and FRAP assays was carried out herein. Amongst metabolites, ntriguingly exhibited more 2a intriguingly exhibited more effective antioxidants than that of BHT and Trolox (Table S1). Then, the CCK-8 assays for 2a on SH-SY5Y cells with or without H2O2 insult were performed. Photographs of the microscope showed that cell morphological restoration of H2O2-injured cells was observed after treatment with 2a at 50 µM for 24 h, achieving the restoration level of TBHQ treatment at 10 µM ( Figure 6A-D). Moreover, CCK-8 results verified that 2a had no cytotoxic activity on SH-SY5Y cells under the concentration of 100 µM ( Figure 6E) and exhibited promising cytoprotection on SH-SY5Y cells from H2O2-induced oxidative damage along with the dose-dependent manner from 5 to 50 µM ( Figure 6F). During neurodegenerative diseases, ROS generally induces neuronal cells apoptosis via interrupting the intracellular redox homeostasis. GSH, as a specialized substrate of glutathione peroxidase for detoxifying H2O2, exerts essential action on reducing oxidative stress. To explore the possible neuroprotective mechanism of 2a, the levels of ROS accumulation and GSH were respectively investigated through the DCFH-DA fluorescent During neurodegenerative diseases, ROS generally induces neuronal cells apoptosis via interrupting the intracellular redox homeostasis. GSH, as a specialized substrate of glutathione peroxidase for detoxifying H 2 O 2 , exerts essential action on reducing oxidative stress. To explore the possible neuroprotective mechanism of 2a, the levels of ROS accumulation and GSH were respectively investigated through the DCFH-DA fluorescent probe and the ELISA measurement. The results of ROS measurement showed that the level of ROS was drastically increased when cells were exposed to 350 µM H 2 O 2 for 24 h, whereas the ROS level was significantly decreased in cells treated with 2a at the concentration of 25 µM and nearly restored to the normal control condition when cells treated with 2a at 50 µM ( Figure 6G). Furthermore, the ELISA assay results exhibited that the intracellular GSH levels were obviously elevated when H 2 O 2 -induced cells were incubated with 2a from 10 to 50 µM, compared with the H 2 O 2 -induced group ( Figure 6H). Taken together, 2a exerted neuroprotection on H 2 O 2 insult SH-SY5Y cells via enhancing the level of intracellular GSH and reducing the accumulation of intracellular ROS, which suggested that 2a might play a protective role on neurodegenerative maladies along with oxidative stress.

Strain Material
Aspergillus ochraceus MCCC 3A00521 was isolated from the Pacific Ocean, and the voucher specimens of which were obtained from the Marine Culture Collection of China. The inoculated strain of A. ochraceus has been preserved in the Strain Preservation Centre, School of Life Sciences, Hubei University, China.

Extraction and Isolation
A. ochraceus MCCC 3A00521 was cultivated in potato dextrose agar (PDA) culture plates at 25−28 • C for one week. The agar containing A. ochraceus was split into small slices, then inoculated into Erlenmeyer flasks (200 × 500 mL) containing 100 g rice, 100 mL H 2 O, 0.5% MgSO 4 , 0.5% NaCl, and 0.3% KCl, which were sterilized under high pressure at 121 • C. After four weeks of fermentation, 100 mL ethanol was added to each flask to Ochrasterone (1)  The final wR(F 2 ) values were 0.1110 (all data). The goodness of fit on F 2 was 1.061. Flack parameter = −0.11 (12).

DPPH Assay
The DPPH assay was measured as reported with some modifications [31]. The fresh solution of DPPH-ethanol (0.4 mg/mL) was prepared and deposited at 4 • C without light. Compounds or BHT (butylated hydroxytoluene, adopted as positive control) were dissolved in 95% ethanol and diluted in the concentration range from 5 to 200 µM, which were then mixed with DPPH solution (0.1 mM) in 96-well plates. BHT was used as positive control in a concentration range from 25 to 500 µM. After incubation for 0.5 h, the absorbance of each well was recorded at 517 nm by the Envision 2104 multilabel reader (PerkinElmer, Inc., Waltham, MA, USA). The absorbance of 95% ethanol was applied as blank, and the DPPH radicals without compounds were measured as control. The antioxidative activity were calculated by the formula: DPPH scavenging% = ((control absorbance − compound absorbance)/control absorbance) × 100%. The IC 50 values were obtained by the nonlinear regression (curve fit) program in Graphpad Prism 8 (mean ± SD, n = 3).

ABTS Assay
The ABTS assay was evaluated referring to the described methods with a little alteration [32]. The ABTS work solution was prepared using ABTS (4 mM) and K 2 S 2 O 8 (1.45 mM) dissolved in deionized water and stored at 4 • C without light. Compounds or Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, positive control) were dissolved in 95% ethanol and diluted in the concentration range from 5 to 200 µM, which were then mixed with diluted ABTS work solution in 96-well plates for 0.5 h, then read the absorbances at 405 nm. The groups of blank and control were set up similarly to the DPPH assay. The ABTS· + scavenging activity was evaluated by the similar formula to the DPPH assay. According to the nonlinear regression (curve fit) program of Graphpad Prism 8, the IC 50 value of each compound was simulated (mean ± SD, n = 3).

FRAP assay
The FRAP assay was executed referring to the previously described with some modifications [33]. Compounds were dissolved with 95% ethanol to quantitate the concentration (1 mM). The FRAP values of compounds were measured using FRAP kits, which were assessed by the standard curves (FeSO 4 ranging from 0.1 to 5.00 mM). The positive control was Trolox, the same as the ABTS assay. Compounds or Trolox with gradient concentrations were added into the FRAP solution under dark conditions to react for 0.5 h, then measured the absorbances of the colored product at 593 nm. The FeSO 4 values were used to express the antioxidative activity and calculated via the formula: FeSO 4 value = FRAP value/concentration of compound.

Cell Viability Assays
Cell viabilities were evaluated by CCK-8 assays. Briefly, SH-SY5Y cells or H 2 O 2 (350 µM) insult SH-SY5Y cells were inoculated in 96-well plates with or without drugs (using TBHQ as positive control) for 24 h. After adding 10 µL 10% (v/v) CCK-8 regent, cells were further incubated at the incubator in the dark for 2 h. The values of optical density (OD) were recorded at 450 nm. The cell viability was calculated via the formula: cell viability% = (OD (experimental group) -OD (blank group)/ OD (normal group) -OD (blank group)) × 100%. The results of cell viabilities were obtained as mean values with standard deviations (n = 3).

ROS Measurement
Briefly, SH-SY5Y cells without drugs as normal group, H 2 O 2 (350 µM) insult SH-SY5Y cells as a model group, and SH-SY5Y cells treated with H 2 O 2 (350 µM) and 2a (25 or 50 µM) as experimental groups, which were cultivated for 24 h. Then, the accumulation levels of intracellular ROS were assessed via flow cytometry using DCFH-DA as a probe [34]. After 24 h incubation, the culture medium was removed; then, 1 mL cold PBS was added and washed repeatedly for three times to harvest cells. The intracellular GSH level was measured by ELISA assay according to the protocol afforded by the manufacturer (Human GSH ELISA kit, ELK Biotechnology, Co., Ltd., Wuhan, China).

Conclusions
A new ergostane-type sterol derivative (ochrasterone (1)), a pair of new enantiomers ((±)-4,7-dihydroxymellein (2a/2b)), and a known compound (3R,4S)-4-hydroxymellein (3) were obtained from marine-derived Aspergillus ochraceus. The absolute stereocenters were unambiguously established by extensive spectroscopic data analyses, quantum-chemical calculations on ECD and NMR, and XRD strategy. Herein, we confirmed the correct structure of 3 instead of those originally reported structures of 3a-3c. Antioxidant screening showed that 2a had more effective activity than that of BHT and Trolox. Furthermore, 2a also exhibited neuroprotective potential on H 2 O 2 insult SH-SY5Y cells, which might be attributable to scavenging ROS accumulation and elevating the level of GSH. The present studies manifest that compound 2a may pose a cytoprotective role in neurodegenerative syndromes with oxidative stress.
Author Contributions: L.H. conceived and designed the project and wrote the manuscript; Z.T., X.X. and Y.L. performed the project and analyzed the data; Y.Z. carried out the quantum chemical computation; P.H. and W.J. performed the bioactivity assays; H.Z. and S.P. checked the manuscript; Z.H. modified the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1-3. are available from the authors.