Neuroprotective Activity of Some Marine Fungal Metabolites in the 6-Hydroxydopamin- and Paraquat-Induced Parkinson’s Disease Models

A new melatonin analogue 6-hydroxy-N-acetyl-β-oxotryptamine (1) was isolated from the marine-derived fungus Penicillium sp. KMM 4672. It is the second case of melatonin-related compounds isolation from microfilamentous fungi. The neuroprotective activities of this metabolite, as well as 3-methylorsellinic acid (2) and 8-methoxy-3,5-dimethylisochroman-6-ol (3) from Penicillium sp. KMM 4672, candidusin A (4) and 4″-dehydroxycandidusin A (5) from Aspergillus sp. KMM 4676, and diketopiperazine mactanamide (6) from Aspergillus flocculosus, were investigated in the 6-hydroxydopamine (6-OHDA)- and paraquat (PQ)-induced Parkinson’s disease (PD) cell models. All of them protected Neuro2a cells against the damaging influence of 6-OHDA to varying degrees. This effect may be realized via a reactive oxygen species (ROS) scavenging pathway. The new melatonin analogue more effectively protected Neuro2A cells against the 6-OHDA-induced neuronal death, in comparison with melatonin, as well as against the PQ-induced neurotoxicity. Dehydroxylation at C-3″ and C-4″ significantly increased free radical scavenging and neuroprotective activity of candidusin-related p-terphenyl polyketides in both the 6-OHDA- and PQ-induced PD models.

The 2,5-diketopiperazine alkaloid mactanamide (6) was isolated from the Vietnamese sediment-derived fungus Aspergillus flocculosus. The NMR data (Figures S14 and S15) for this compound were identical with earlier published data [31]. It is only the third case of isolation this compound.
The 2,5-diketopiperazine alkaloid mactanamide (6) was isolated from the Vietnamese sediment-derived fungus Aspergillus flocculosus. The NMR data (Figures S14 and S15) for this compound were identical with earlier published data [31]. It is only the third case of isolation this compound.

Biological Activities of the Studied Compounds
2.2.1. 6-Hydroxy-N-acetyl-β-oxotryptamine (1) 6-Hydroxy-N-acetyl-β-oxotryptamine (1) was not cytotoxic against neuroblastoma Neuro2a cell up to 100 μM. This compound scavenged DPPH radicals by 48% at 100 μM ( Table 2). 83.5 ± 0.2 ->100 Melatonin-like compound 1 showed a statistically significant reduction of reactive oxygen species (ROS) level on 18% in the neuronal 6-OHDA-treated cells in the in vitro experiment ( Figure  2). Melatonin (1a), a well-known antioxidant and neuroprotective compound, was used to compare with 1. It decreased ROS formation in the 6-OHDA-treated neuronal cell stronger in comparison with 1. Figure 2. Influence of compounds 1-6 on reactive oxygen species (ROS) formation in Neuro2a cells treated with 6-hydroxydopamine (6-OHDA) for 30 min. * Difference between data for compounds and for 6-OHDA was statistically significant with p ≤ 0.05. The neuroprotective effect of 1 was shown both in the Neuro2a cells treated 1 h before, as well as 1 h after, adding of 6-OHDA by 23% and 28%, respectively, at a concentration of only 10 µM. Melatonin (1a) did not increase the viability of cells treated with neurotoxin in this experiment (Figure 3a). Our experiments showed that 6-hydroxy-N-acetyl-β-oxotryptamine (1) more effectively protected Neuro2a cells against 6-OHDA-induced neuronal death, in comparison with melatonin (1a). The neuroprotective effect of 1 was shown both in the Neuro2a cells treated 1 h before, as well as 1 h after, adding of 6-OHDA by 23% and 28%, respectively, at a concentration of only 10 μM. Melatonin (1a) did not increase the viability of cells treated with neurotoxin in this experiment ( Figure 3a). Our experiments showed that 6-hydroxy-N-acetyl-β-oxotryptamine (1) more effectively protected Neuro2a cells against 6-OHDA-induced neuronal death, in comparison with melatonin (1a). In the PQ-induced PD model, compounds 1 and 1a (at concentration of 10 μM) were more effective in comparison with their influence in the 6-OHDA-induced model, and decreased ROS formation in the PQ-treated cells by 35% and 22%, respectively ( Figure 4). As a result, increase of the PQ-treated cell viability, by 40% and 24%, was observed ( Figure 5).   All compounds were added to the cell suspension 1 h before treatment with PQ. * Difference between data for compounds and for PQ was statistically significant with p ≤ 0.05. (3) 3-O-Methylorsellinic acid (2) and 8-methoxy-3,5-dimethylisochroman-6-ol (3) were not cytotoxic against neuroblastoma Neuro2a cells up to 100 μM.
3-O-Methylorsellinic acid (2) statistically significantly increased 6-OHDA-treated cell viability by 26%, when the compound was added to cells 1 h before adding 6-OHDA. When compound 2 was added to cells 1 h after adding 6-OHDA, it had no neuroprotective effect.
3-O-Methylorsellinic acid (2) statistically significantly increased 6-OHDA-treated cell viability by 26%, when the compound was added to cells 1 h before adding 6-OHDA. When compound 2 was added to cells 1 h after adding 6-OHDA, it had no neuroprotective effect.
3-O-Methylorsellinic acid (2) statistically significantly increased 6-OHDA-treated cell viability by 26%, when the compound was added to cells 1 h before adding 6-OHDA. When compound 2 was added to cells 1 h after adding 6-OHDA, it had no neuroprotective effect.
On the other hand, compounds 2 and 3 did not exhibit any protective effect on based on the viability of cells treated with PQ, despite the fact that they reduced ROS formation in these cells (Figures 4 and 5).
Candidusin A (4) did not have any effect on ROS formation in the 6-OHDA-treated cells ( Figure 2). 4"-Dehydroxycandidusin A (5) decreased the ROS level in 6-OHDA-treated cells by 34%, being more effective as a radical scavenger. Candidusin A (4) had no effect on the viability of cells when it was added 1 h before treatment with 6-OHDA, and increased 6-OHDA-treated cell viability by 24% at a concentration of only 10 µM, when it was added 1 h after 6-OHDA (Figure 3c). 4"-Dehydroxycandidusin A (5) increased cell viability by more than 80% (when the compound was added 1 h before 6-OHDA) and 62% (when it was added 1 h after 6-OHDA). These effects were observed at a concentration of 10 µM only. When the concentration of compound 5 was reduced tenfold, its neuroprotective effect was not preserved.
In the PQ-induced model, compound 4 decreased ROS formation by 27% at a concentration of 10 µM. Compound 5 was more effective and statistically significantly decreased ROS formation by 19% and 40%, at concentrations of 1 and 10 µM, respectively ( Figure 4). Nevertheless, compound 4 did not have any effect on the viability of the PQ-treated cells, but its 4"-dehydroxylated derivative (5) statistically significantly increased the viability of these cells by 17% at concentration of 10 µM only ( Figure 5).
Thus, the presence of hydroxy groups at C-3" and C-4" in candidusins decreases the radical scavenging activity of these compounds in the cell-free assay. Moreover, the hydroxylation at C-4" results in the significant decreasing their neuroprotective effect.
In the PQ-treated cells, mactanamide (6) decreased ROS formation by 32% and 37%, at concentrations of 1 and 10 µM, respectively ( Figure 4). However, compound 6 did not show any neuroprotective activity on the viability of the PQ-treated cells ( Figure 5).
Earlier, mactanamide showed fungistatic activity against Candida albicans, and an influence on osteoclast differentiation without any cytotoxicity [31,37]. Antioxidant and neuroprotective properties of mactanamide were demonstrated, for the first time, in this investigation.
Thus, compounds 1, 2, 3, and 6 were non-cytotoxic for Neuro2a cells up to a concentration of 100 µM. Compounds 4 and 5 demonstrated low cytotoxicity with an IC 50 of 75.7 and 78.9 µM, respectively. This allowed investigating the neuroprotective activity of all compounds in non-toxic concentrations of 1 and 10 µM. Neuroprotective effects of the compounds were studied in two PD in vitro models using 6-OHDA and PQ as inducers of neuronal cell damage.
Melatonin-like compound 1 demonstrated an effect in increasing cell viability in both models, but the effect on PQ-treated cells was more pronounced. In both cases, neuroprotective effects were accompanied with a decrease of ROS formation in the 6-OHDA-and PQ-treated cells. Melatonin (1a) decreased ROS formation in both PD models, but it increased cell viability in the PQ-induced model only.
Polyketides 2 and 3 demonstrated ROS-decreasing effects in both PD models. Nevertheless, these compounds increased cell viability in the 6-OHDA-induced model only.
Candidusin A (4) and 4"-dehydrocandidusin A (5) have minimal differences between their chemical structures but this has a significant effect on their neuroprotective activity. In the 6-OHDA and PQ models, compound 5 produced a significant increase of cell viability, whereas compound 4 did not demonstrate any effect in the PQ model, and low increased cell viability on the 6-OHDA-treated cells. A similar influence of both compounds on ROS formation in the 6-OHDA-and PQ-treated cells was observed. Compound 4 had no significant effect on ROS formation in the 6-OHDA-treated cells, and decreased ROS formation in the PQ-treated cells, at a concentration of only 10 µM. By contrast, compound 5 was very effective in the 6-OHDA-induced PD model, and decreased ROS formation in the PQ-treated cells at concentrations 1 and 10 µM.
Mactanamide (6) demonstrated a significant decrease of ROS formation in both the 6-OHDAand PQ-induced PD models. However, this 2,5-diketopiperasine alkaloid increased viability of the 6-OHDA-treated cells only, and did not have any statistically significant effects on viability of the PQ-treated cells.
It should be noted that DPPH radical scavenging activity was shown for all compounds in varying degrees, and decreasing of ROS formation in 6-OHDA-and PQ-treated cells could be the result of radical scavenging by these compounds. However, differences between the effects of these compounds, on ROS formation and cell viability in different PD models, were observed.
In our investigation, two PD-like cell models, induced by neurotoxin 6-OHDA and pesticide paraquat, were used. Neurotoxins and pesticides share a common mechanism to induce damage to dopaminergic neurons that is correlated with an increased oxidative status caused by high levels of ROS, anions, and free radicals [6]. However, the effect of each of the inducers, on neurons, has the same differences.
6-OHDA has a specific neurotoxic effect on neurons containing dopamine, serotonin, and norepinephrine receptors. The structure of 6-OHDA is similar to dopamine and norepinephrine, and, therefore, this neurotoxin uses the same catecholaminergic transport system (the dopamine and norepinephrine transporters), and causes specific degeneration of dopaminergic and noradrenergic neurons [6]. Inside neurons, 6-OHDA is rapidly autooxidized to hydrogen peroxide and paraquinone, which are both highly toxic to mitochondria, by specifically affecting complex I. This process results in an increase of ROS generation and cell death [38]. Moreover, it was reported that 6-OHDA induces oxidative stress both during its autoxidation to p-quinone and, also, during one-electron reduction of p-quinone to p-semiquinone, catalyzed by flavoenzymes that transfer one electron [39]. In addition to these effects, 6-OHDA-induced cell death is dependent of such intracellular processes as neuroinflammation, mitochondria dysfunction, endoplasmic reticulum stress, and autophagy [40].
Paraquat causes oxidative stress in neuronal cells by another pathway. Divalent paraquat ion (PQ2+) is reduced to monovalent paraquat ion (PQ+) by NADPH-oxidase of mitochondrial complex I. Subsequently, PQ+ accumulates in dopaminergic neurons and reestablishes a new redox reaction intracellularly, leading to the generation of intracellular free radicals, such as superoxide and dopamine-reactive substances. This will eventually lead to dopaminergic neuron cell death [41].
Metabolic investigations of the molecular mechanisms associated with 6-OHDA and PQ toxicity were carried out by NMR spectroscopy and mass spectrometry. It was shown that PQ selectively upregulated the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents, and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increasing in glucose uptake, the translocation of glucose transporters to the plasma membrane, and adenosine monophosphate-activated protein kinase activation. In the contract, 6-OHDA did not demonstrate an influence on PPP. In addition, while paraquat induced a reduction in glucose-dependent glutamate-derived glutathione synthesis, 6-OHDA treatment increased this process [43,44].
In this study, we observed time differences between 6-OHDA and PQ effects on ROS formation in Neuro2a cells ( Figure S16). 6-OHDA caused an increase of ROS level of 30% for 30 min after addition to the cell suspension. The effect of PQ on ROS formation was insignificant after 30 min, and an increase of ROS levels in cells, by 39%, was observed 1 h after adding of PQ to the cell suspension.
Compounds 2, 3, and 6 demonstrated neuroprotective effects in the 6-OHDA-induced PD model only. For this reason, they could protect Neuro2a cells against the damaging influence of products of 6-OHDA autooxidation, due to their antioxidant properties. Compound 5 increased the viability of 6-OHDA-treated cells by 80%, but it increased viability of PQ-treated cells by 17% only. This suggests the same mechanism of action.
On the other hand, compounds 1 and 1a were more effective in the PQ-induced model, and increased cell viability by 40% and 24%, respectively, whereas in the 6-OHDA-induced model, compound 1 increased cell viability by 23% only, and melatonin (1a) was ineffective.
It was earlier published that melatonin and some related compounds demonstrated antioxidant activity in cell-free assays [45], and different neuroprotective effects in the in vitro experiments [46][47][48]. Pre-treating of PC12 cells with melatonin for 3 h increased viability of the cells, and prevented apoptosis in the 6-OHDA-induced PD model [49,50]. In addition, it was reported that melatonin diminished caspase-3 enzyme activity, cleavage of DNA fragmentation factor 45, and DNA fragmentation observed in the MPTP-treated neuroblastoma cells [46]. For this reason, melatonin-related compound 1 could influence on viability of the PQ-treated cells in a similar manner.

General Experimental Procedures
NMR spectra were recorded in DMSO-d 6 and acetone-d 6 on a Bruker DPX-500 and DRX-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometers, using TMS as an internal standard. HRESIMS spectra were measured on a Maxis impact mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany).

Fungal Strain
The strain Penicillium sp. KMM 4672 was isolated from brown alga Padina sp. (Van Phong Bay, South China Sea, Vietnam) on malt extract agar, and identified on the basis of morphological and molecular features, as described earlier [20].
The strain Aspergillus sp. KMM 4676 was isolated from an unidentified colonial ascidian (Shikotan Island, Pacific Ocean) on malt extract agar, and identified on the basis of morphological and molecular features as described earlier [30].
The strain Aspergillus flocculosus was isolated from a sediment sample (Nha Trang Bay, South China Sea, Vietnam) by inoculating on modified Sabouraud medium (peptone 10 g, glucose 20 g, agar 18 g, natural sea water 1000 mL, penicillin 1.5 g, streptomycin 1.5 g, pH 6.0-7.0). The fungus was identified according to a molecular biological protocol by DNA amplification and sequencing of the ITS region (GenBank accession number MH101466.1). BLAST search results indicated that the sequence was 100% identical (796/796 bp) with the sequence of Aspergillus flocculosus strain NRRL 5224 (GenBank accession number EU021616.1).

Cultivation of Fungus
All the fungal strains were cultured at room temperature for three weeks in 500 mL Erlenmeyer flasks, each containing rice (20.0 g), yeast extract (20.0 mg), KH 2 PO 4 (10 mg), and natural sea water (40 mL).

Radical Scavenger Assay
DPPH radical scavenging activity of compounds was tested as described [51]. Compounds were dissolved in MeOH, and the solutions (160 µL) were dispensed into wells of a 96-well microplate. In all, 40 µL of the DPPH (Sigma-Aldrich, Steinheim, Germany) solution in MeOH (1.5 × 10 −4 M) was added to each well. Concentrations of compounds in mixture were 10 and 100 µM. The mixture was shaken and left to stand for 30 min, and the absorbance of the resulting solution was measured at 520 nm with a microplate reader MultiscanFC (ThermoScientific, Waltham, MA, USA).
Radical scavenging activity of all compounds at 100 µM were presented as percent of MeOH data, and the concentration of DPPH radical scavenging at 50% (EC 50 ) was calculated for some compounds.

Cell Line and Culture Condition
The neuroblastoma cell line Neuro2a was purchased from ATCC. Cells were cultured in DMEM medium containing 10% fetal bovine serum (Biolot, St. Petersburg, Russia) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were incubated at 37 • C in a humidified atmosphere containing 5% (v/v) CO 2 .

Cell Viability Assay
Cell suspension (1 × 10 3 cells/well) was incubated with different concentration of compounds for 24 h. After that, cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) method, according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). The results were presented as percent of control data, and concentration required for 50% inhibition of cell viability (IC 50 ) was calculated.

6-Hydroxydopamine-Induced In Vitro Model of Parkinson's Disease
The neuroprotective activities of compounds in 6-hydroxydopamine-induced cell model of Parkinson's disease were examined, as described previously [52].
Neuroblastoma Neuro2a line cells (1 × 10 3 cells/well) were treated with 50 µM of 6-hydroxydopamine (Sigma-Aldrich, St. Louis, MO, USA) for 1 h and, after that, the investigated compounds were added to the neuroblastoma cell suspension at a concentration of 1 and 10 µM. In the other case, the substances were added to the cells 1 h before the addition of the neurotoxin. Cells incubated without 6-OHDA and compounds, and with 6-OHDA only, were used as positive and negative controls, respectively. After 24 h, viability of cells was measured using the MTT method. The results were presented as a percent of positive control data.

Paraquat-Induced In Vitro Model of Parkinson's Disease
Neuroblastoma Neuro2a line cells (1 × 10 3 cells/well) were treated with compounds at concentrations of 1 and 10 µM for 1 h, and then 500 µM of paraquat (Sigma-Aldrich, St. Louis, MO, USA) was added to the neuroblastoma cell suspension. Cells incubated without paraquat and compounds, and with paraquat only, were used as positive and negative controls, respectively. The viability of cells was measured after 24 h using the MTT method. The results were presented as percent of positive control data.

Reactive Oxygen Species (ROS) Level Analysis in 6-OHDA-and PQ-Treated Cells
Cell suspensions (1 × 10 3 cells/well) were incubated with compound solutions (10 µM) for 1 h. Then, 6-OHDA at a concentration of 50 µM was added in each well, and cells were incubated for 30 min. In other experiments, cells were incubated with PQ at a concentration of 500 µM for 30 min and 1 h. Cells incubated without 6-OHDA/PQ and compounds, and with 6-OHDA/PQ only, were used as positive and negative controls, respectively. To study ROS formation, 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (Molecular Probes, Eugene, OR, USA) was added to each well, such that the final concentration was 10 mM, and the microplate was incubated for an additional 10 min at 37 • C. The intensity of dichlorofluorescein fluorescence was measured with plate reader PHERAstar FS (BMG Labtech, Ortenberg, Germany) at λ ex = 485 nm, and λ em = 518 nm. The data were processed by MARS Data Analysis v. 3.01R2 (BMG Labtech, Ortenberg, Germany). The results were presented as a percentage of positive control data.

Conclusions
This study is the second case of isolation of melatonin-related compound from microfilamentous fungi. The neuroprotective activity in the 6-OHDA-and PQ-induced PD cell models of this and some other polyketides and alkaloids from marine-derived fungi were investigated. All of them protected Neuro2a cells against the damaging influence of 6-OHDA to varying degrees. We suppose that this effect is realized via a ROS scavenging pathway as one of the possibilities. The new melatonin analogue, 6-hydroxy-N-acetyl-β-oxotryptamine, protected Neuro2A cells more effectively against the 6-OHDA-induced neuronal death in comparison with melatonin. Moreover, 6-hydroxy-N-acetyl-β-oxotryptamine and melatonin protected Neuro2a cells against the damaging influence of PQ in a similar manner. It was shown that dehydroxylation at C-3" and C-4"significantly increases free radical scavenging and neuroprotective activity of candidusin-related p-terphenyl polyketides in both the 6-OHDA-and PQ-induced PD models.

Conflicts of Interest:
The authors declare no conflict of interest.