Exploring marine resources against neurological disorders– the neuroprotective and anti-inflammatory potential of the brown seaweed Bifurcaria bifurcata

Oxidative stress is strongly involved in the pathogenesis of neurodegenerative diseases, like Parkinson´s disease (PD). Particularly, an excess of reactive oxygen species (ROS) released by the cells promotes an oxidative stress condition, which is a main cause of tissue injury leading to nervous system dysfunction. In this work, the antioxidant, neuroprotective and anti-inflammatory activities of different fractions from the brown seaweed Bifurcaria bifurcata are presented and related with their chemical profile. The antioxidant capacity was evaluated by the Folin-Ciocalteu method, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Neuroprotective capacity was evaluated to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells, and their anti-inflammatory effects on RAW 264.7 macrophages. The ethyl acetate fractions (100 µg mL−1) exhibited significant antioxidant and neuroprotective activities in the in vitro models assayed. Furthermore, two of the most polar fractions obtained with methanol and water also evidenced a significant neuroprotective potential. Bifurcaria bifurcata fractions treatment decreased ROS production, mitochondrial dysfunction, and Caspase-3 activity. Regarding the anti-inflammatory potential, five fractions (100 µg mL−1) inhibited nitric oxide (NO) production and reduced the interleukin – 6 (IL-6) and tumor necrosis factor (TNF-α) levels. Mannitol, identified as the major component of the most bioactive fraction, protected SH-SY5Y cells against the 6-OHDA neurotoxicity mediating ROS generation mitigation, mitochondrial dysfunction, and DNA damage, together with the Caspase-3 activity inhibition. Results suggest that B. bifurcata is a relevant source of neuroprotective agents, with particular interest for preventive therapeutics.


Introduction
Degenerative brain disorders such as Parkinson´s disease (PD) are the consequence of pathological brain aging, which are characterized by the loss of neurons in the substantia nigra region (Hannan et al. 2020). The current therapeutic options only alleviate symptoms but do not halting the progression of PD. Therefore, there are continued efforts to discover the potential therapeutic agents that can target disease pathogenesis, but without causing adverse effects on patient's health (Hannan et al. 2020). The mechanisms responsible for neuronal degeneration in PD are complex and remain to be fully elucidated. However, several studies indicate that oxidative stress (ROS generation), nitric oxide (NO) production, mitochondrial dysfunction, inflammation and accumulation of misfolded proteins contribute to the cascade of events leading to degeneration of dopaminergic neurons (Hwang 2013;Blesa et al. 2015;Hemmati-Dinarvand et al. 2019).
Oxidative stress triggers several molecular pathways leading to the progressive loss of neuronal structures and functions, a process termed neurodegeneration, and is considered a major factor in PD development (Chen et al. 2017). The endogenous triggers of ROS generation include biological processes that release ROS as by-products, such as the mitochondrial electron transport chain. Example of this is the membrane-bound NADPH oxidase (NOX) family and nitric oxidase synthase, as responses to bacterial invasions and cytokines' release (Zorov et al. 2014;Di Meo et al. 2016). The most of free radicals in cells are generated by the mitochondria oxidative phosphorylation, through the electron leak from the electron transport chain (Zhao et al. 2019). These radicals, as superoxide radical, are formed at complexes of the electron transport chain that lead to ROS formation, through O 2 consumption by mitochondria (Zhao et al. 2019). Thus, several studies have been demonstrated that ROS production is strongly dependent from the membrane potential of the mitochondria, showing high values resulting in increased rates of ROS generation (Kausar et al. 2018;Warraich et al. 2020). Another radical that is produced in the mitochondria through the oxidation of l-arginine to l-citrulline is NO, that can mediate neurotoxicity causing neuronal cell death, being suspected to be directly related with PD pathogenesis (Abou-sleiman et al. 2006). Nitric oxide radical is synthesized by cytosolic NO synthases (NOSs) using NADPH and molecular oxygen. Three isoforms of NOS: nNOS (neuronal NOS), eNOS (endothelial NOS) and inducible NOS (iNOS) have been described (Kleinert et al. 2010). nNos and eNos are constitutively expressed with their activity depending on intracellular calcium levels both present in the central nervous system, while the iNOS is expressed in astrocytes and microglia and their activity is induced during cellular inflammatory response (Aquilano et al. 2008). Upregulation of iNOS generates high levels of nitric oxide that breaks down into hydroxyl radicals promoting further intensification of the inflammatory response, leading to the expression of cytokines. These cytokines play an important role in the regulation of the inflammatory response in neuronal cells, such as tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β), which are positively regulated in the brain tissue during the inflammatory response in PD (Wink et al. 2011). In addition, other interleukins, such as IL-6 and IL-10, are also secreted by microglia and astrocytes during inflammatory response (Burmeister and Marriott 2018). An excessive inflammatory response in PD leads to an increased release of pro-inflammatory cytokines and production of free radicals, causing neuronal damage (Guo et al. 2018). All of these consequences result in the activation of protein kinases cascades, which can lead to the activation of the transcription nuclear factor kappa B (NF-kB), which migrates to the nucleus intervening in the transcription of many proteins involved in the inflammatory process, such as COX-2, TNF-α, IL-1β, IL-6, iNOS and metalloproteinases (MMP) .
In recent years several researchers have studied the neuroprotective effects of exogenous antioxidant molecules, since they have been shown to be fundamental in preventing the damage induced by oxidative stress reducing the production of ROS and reactive nitrogen species (RNS) (Tan et al. 2018). As a consequence, the interest in antioxidant molecules for therapeutic applications has been increasing. For example, Man Anh et al. (2019) studied the neuroprotective effect of vitamin C in a Drosophila model with PDlike phenotype and demonstrated its potential to reduce PD symptoms. However, they observed that its administration in high doses, as well as long-term use, lead to significant side-effects. On the other hand, Ma and collaborators (2018) verified that pretreatment with proanthocyanidis, a class of natural flavonoids, reduced the rotenone-induced oxidation in human neuroblastoma cells SH-SY5Y. In addition, they also observed that flavonoids were able to considerably block rotenone-induced apoptosis through Caspase-9 and Caspase-3 activity inhibition, as well as the inhibition of the mitogen-activated protein kinases, p38, JNK and ERK (Man Anh et al. 2019).
Therefore, there is still a medical need to search for new PD therapeutic agents that not only treat symptoms, but also have the capacity to reduce disease the progression. In this way, new naturally occurring antioxidant molecules could be of utmost interest. For example, marine organisms such as seaweeds are a rich source of new bioactive compounds, and exhibit diverse biological activities, including strong antioxidant capacity (Salehi et al. 2019;Cotas et al. 2020). Among the various species of seaweeds, brown algae have shown the ability to produce a wide variety of secondary metabolites with unique structural features such as phlorotannins (Cotas et al. 2020). They belong to a group of phenolic compounds identified in several families of brown algae in the Alariaceae, Fucaceae and Sargassaceae and are the main group of phenolics detected in this group (Sathya et al. 2017).
Bifurcaria bifurcata is a brown seaweed of the Sargassaceae family. Most studies regarding B. bifurcata in recent years have been related to its content of specific diterpenoids such as eleganolone, eleganonal and others, which have shown antiprotozoal, antifouling and antioxidant activities (Culioli et al. 1999;Gallé et al. 2013;Culioli and Köck 2013;Silva et al. 2019). However, the place and season of collection, the maturation stage, and extraction procedures are factors that can influence the composition and bioactivities of the extracts.
In this study twenty-three fractions from B. bifurcata harvested on the Portuguese coastline were investigated regarding their antioxidant, neuroprotective and anti-inflammatory potential in different in vitro models. Additionally, fractions showing the greatest potential were analyzed by the means of chromatographic and spectroscopic techniques, aiming the identification of compounds responsible for the above described bioactivities.

Quantification of total phenolic content (TPC)
TPC of B. bifurcata polar fractions was determined using the Folin-Ciocalteu reagent as described by Singleton and Rossi (1965) with slight modifications. After 1 h of reaction in the dark, the absorbance was measured at 755 nm (Synergy H1 Multi-Mode Microplate Reader, BioTek Instruments, USA). Gallic acid was used as standard, and TPC was expressed in milligrams of phloroglucinol equivalents per gram of dry extract (mg PE g −1 of extract).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
DPPH free radical scavenging effect was determined according to the protocol described by Pinteus et al. (2017). The reaction mixtures were incubated in the dark for 30 min at room temperature. The absorbance was then measured at 517 nm. The samples were tested at a maximum concentration of 100 µg mL −1 . Dose-response analysis (10-100 µg mL −1 ) was performed for the samples with high activity (DPPH reduction > 50%) for EC 50 determination.

Oxygen radical absorbance capacity (ORAC-fluorescein)
ORAC assay was performed according to the protocol described by Dávalos et al. (2004). The samples (20 µL) and fluorescein (120 µL; 70 nM, final concentration) were placed in the wells of a 96-wells microplate. The mixture was preincubated for 15 min, at 37 °C. Then, AAPH (2,2′-azobis(2methylpropionamidine) dihydrochloride solution) (60 µL; 12 mM, final concentration) was added rapidly. The microplate was immediately placed in the reader and the fluorescence (λ excitation: 458 nm; λ emission: 520 nm) recorded every minute for 240 min, and automatically shaken prior to each reading. Trolox was used as antioxidant standard. ORAC values were expressed as trolox equivalents by using the standard curve calculated for each assay. The results were expressed in micromol of trolox equivalents per gram of dry extract (µmol TE g −1 of extract).

Ferric reducing antioxidant power (FRAP)
The capacity of seaweed fractions to reduce Fe (III) to Fe (II) through electron donation was performed according to Benzie and Strain (1996) with slight modifications (Silva et al. 2019). The seaweed fractions (100 µg mL −1 ) were mixed with FRAP reagent (0.3 M acetate buffer (pH = 3.6), 10 mM of 2,4,6-tri(2-pyridyl)-s-triazine, (TPTZ) in 40 mM HCl and 20 mM ferric solution using FeCl 3 (10:1:1)) preheated at 37 ºC, in the dark, for 30 min. The absorbance was then read at 593 nm. The difference between the absorbance of test fractions and the blank reading was calculated and results expressed as micromolar of FeSO 4 per gram of extract (µM FeSO 4 g −1 of extract).

Cell culture maintenance
Biological activities of fractions were conducted on an in vitro cellular model of human neuroblastoma (SH-SY5Y cells, strain number ACC 209) previously acquired from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) biobank. SH-SY5Y cells grew in Dulbecco's Modified Eagle's Medium: F12 (DMEM:F12) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin/ streptomycin and kept at 37 °C in a humidified incubator with 5% CO 2 .

Evaluation of the neuroprotective potential and study of associated mechanisms
Cell viability The neuroprotective activity was evaluated on SH-SY5Y cells according to Silva et al. (2019). Cells were treated with seaweed fractions (100 µg mL −1 ) for 24 h, and cell viability estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Briefly, MTT (0.5 mg mL −1 ) was added to SH-SY5Y cells and the cells incubated for 1 h at 37 °C. After this time, DMSO was added and absorbance was read at 570 nm (Synergy H1 Multi-Mode Microplate Reader, BioTek Instruments, USA).

Mitochondrial membrane potential (MMP) assay
The MMP (ΔΨm) was assessed using JC -1 probe according to Silva et al. (2019). The cells were exposed to 6-OHDA (100 μM) in the presence/absence of seaweed fractions (100 µg mL −1 ) Fig. 1 Schematic representation of the extraction and fractionation process of Bifurcaria bifurcata. VLC-vacuum liquid chromatography; SPE-solid phase extraction; CC-column chromatography. MeOH -methanol; EA -Ethyl acetate for 6 h. Then SH-SY5Y cells were washed twice with phosphate-buffered saline (PBS) and incubated with JC-1 solution (3 µM) in the dark, for 15 min, at 37 ºC. FCCP (2.5 µM) plus oligomycin A (1 µg mL −1 ) conjugate solution was used as positive control. The fluorescence was then read at 530 nm (monomers) and at 590 nm (aggregates) and 490 nm emission/ excitation wavelengths, respectively. The results were calculated from the ratio between JC-1 monomers and aggregates and expressed in percentage of control.

Measurement of Caspase -3 activity
The induction of apoptosis via 6-OHDA has been shown to increase the activity of Caspase-3 involved in the executioner pathway. Thus, cells were exposed to 6-OHDA (100 μM) in the presence/ absence of seaweed fractions (100 µg mL −1 ) for 6 h. Then Caspase-3 activity was evaluated using the Caspase-3 fluorometric assay kit (Sigma, CASP3F-KIT, USA) according to the manufacturers' instructions. Cells were then washed with PBS, harvested, and enzyme activity determined by reading the fluorescence at 360 nm and 460 nm emission and excitation wavelengths, respectively. The results were expressed as percentage of control.

Evaluation of biological activities of Bifurcaria bifurcata on an in vitro cellular model of inflammation
Cell culture maintenance RAW 264.7 macrophages were obtained from American Type Culture Collection (ATCC) (TIB -71). Cells grown in Dulbecco's Modified Eagle's Medium without phenol red and supplemented with 10% (v/v) FBS, 1% penicillin/ streptomycin, 1% of sodium pyruvate and kept at 37 °C in a humidified incubator with 5% CO 2 .

Evaluation of the anti-inflammatory potential Cytotoxicity
The cytotoxicity of seaweed fractions was evaluated on RAW 264.7 macrophages (5 × 10 4 cells mL −1 ) after seeding in 96-well plates and incubated overnight. Cells were treated with fractions (100 µg mL −1 ) for 24 h. Cellular viability was then estimated through the MTT method and absorbance was measured at 570 nm using a microplate reader (Sinergy H1 Multi-Mode Microplate Reader, Biotek Instruments, USA). The results were expressed as percentage of control.

Determination of nitric oxide (NO) production
The inflammatory and anti-inflammatory effects of B. bifurcata fractions were estimated through the nitric oxide (NO) production, according to Freitas et al. (2020). RAW 264.7 cells (5 × 10 4 cells mL −1 ) were pretreated (1 h) with seaweed fractions (100 µg mL −1 ) and stimulated with LPS (1 µg mL −1 ) for 24 h. NO levels were determined by measuring nitrite levels in the culture media using the Griess reagent (1% sulphanilamide in phosphoric acid (2.5%) + 0.1% naphtylethylenediamine dihydrochloride). The results are expressed in percentage of control.
Measurement of IL-6 and TNF-α RAW 264.7 cells (1 × 10 5 cells mL −1 ) were pre-treated for 1 h with seaweed fractions and stimulated with LPS (1 µg mL −1 ) for 18 h. The concentrations of IL-6 and TNF-α were determined using ELISA kits according to the manufacturer's instructions.

Chemical characterization
The chemical profiles of the B. bifurcata most bioactive fractions were evaluated by NMR, HPLC-DAD and GC-MS techniques.
NMR spectra were acquired on a Bruker Avance 400 spectrometer with a frequency of 400 MHz for 1 H, and 100 MHz for 13 C. Samples were dissolved in 500 µL of CDCl 3 or D 2 O (Sigma-Aldrich, USA). Chemical shifts were expressed in ppm and reported to the residual solvent signals. Coupling constants (J) were expressed in Hertz (Hz).
GC-MS qualitative analysis was performed in a Shimadzu QP2010-Plus GC/MS system equipped with a TRB5MS (30 m × 0.25 mm i.d. × 0.25 µm film thickness) capillary column (Teknokroma, Spain) operating in the linear velocity mode. The carrier gas was helium 5.0 (Linde, Portugal), at a constant flow of 1 mL min −1 . Samples were dissolved in dichloromethane and automatically injected. Injections were performed in split mode, with a ratio of 1/9. The injector port was heated to 280 °C. The initial column temperature of 60 °C was held for 2 min, followed by a temperature ramp of 30 °C min −1 to 300 °C held for 15 min. All mass spectra were acquired in electron impact (EI) mode at 70 eV. The operating temperatures were 200 °C for MS ion source, and 250 °C for the liner interface. The analysis was performed in full scan mode with mass ranging from 10 to 800 m/z Compounds were identified by matching the mass fragmentation patterns with those stored in the GC-MS mass spectral databases (Wiley 229 and NIST-National Institute of Standards and Technology libraries).

Data and statistical analysis
When applicable, results are presented as mean ± standard error of the mean (SEM). The determination of EC 50 was attained from sigmoidal dose-response variable-slope curves using the GraphPad Prism V.8 software (Graph-Pad Software Inc., USA). One-way analysis of variance (ANOVA) with Dunnett's multiple comparison of group means was employed to determine significant differences (p < 0.05) relatively to the control treatment. All other posthoc analyses were conducted using Tukey's test. All data were checked for normality and homoscedasticity. Comparisons concerning variables, which did not meet variance or distributional assumptions, were carried out with Kruskal-Wallis non-parametric tests. At least three independent experiments were carried out in triplicate.

Antioxidant capacity of Bifurcaria bifurcata
In order to evaluate the antioxidant potential of B. bifurcata fractions (MF1-MF7; MC18F1-MC18F5; AEF1 -AEF11) different approaches have been outlined, including the determination of total phenolic content (TPC) by the Folin-Ciocalteu method, DPPH radical scavenging activity, oxygen radical absorbance capacity (ORAC), and ferric reducing antioxidant power (FRAP). The results are shown in Table 1.
The main components analysis (PCA) carried out in this study allowed us to to correlate the antioxidant capacity with the total phenolic content. It is possible to observe a clear arrangement of two groups that differ according to their antioxidant capacity (Fig. 3). Figure 3 shows the ordination of the different antioxidant methods using PCA analysis of the fractions obtained from B. bifurcata. PC1 accounted for 80.7% and PC2 13.4% of the total variance. In PC1, the horizontal axis expresses an opposition between DPPH (right), and antioxidant methods FRAP and TPC (Left). Moreover, FRAP and TPC present a negative correlation with DPPH (Fig. 3). Since DPPH radical scavenging activity is expressed by EC 50 , it is possible to verify that fractions which presented high phenolic content and potential to reduce iron ions also exhibited high DPPH radical scavenging activity (Group I). On the other hand, the fractions present in group I, mainly MF7 and MF1, showed low levels of TPC, and exhibited low DPPH radical scavenging activity. Moreover, fractions MC18F2, MC18F4, AEF8, AEF9 and AEF10 showed high FRAP and TPC values when compared with the samples present in group II.

Cytotoxicity and neuroprotective activities of Bifurcaria bifurcata fractions on SH-SY5Y cells
The cytotoxic and neuroprotective effects of B. bifurcata fractions on SH-SY5Y cells were evaluated. Firstly, SH-SY5Y cells were treated with different B. bifurcata fractions (100 µg mL −1 ) for 24 h. Fractions with no cytotoxicity were further tested for their neuroprotective potential. The neuroprotective effects were evaluated on SH-SY5Y cells treated with 6-OHDA in the presence/ absence of B. bifurcata fractions (100 µg mL −1 ) after 24 h. The results are presented in Fig. 4A and 4B.

Study of the effects on Parkinson's Disease biomarkers
In order to better understand which cellular mechanisms are associated with the neuroprotective effects mediated by B. bifurcata fractions on SH-SY5Y cells, different hallmarks linked to PD development were studied, namely MMP, ROS production and Caspase-3 activity. The results are presented as percentage of control and depicted in Fig. 5. The treatment with seaweed fractions did not induce effects on the mitochondrial membrane potential, ROS production and Caspase-3 activity when compared with the vehicle (data not shown). SH-SY5Y cells were treated with 6-OHDA (100 µM) in the presence of B. bifurcata fractions for 6 h. Cells exposed to 6-OHDA increase ROS levels by about 65.6 ± 4.19% when compared with the vehicle situation (Fig. 5A). On the other hand, MC18F2, AEF9 and AEF10 fractions reduced ROS production in 52.90 ± 15.05%, 40.7 ± 1.78%, and 41.7 ± 4.19%, respectively. The effects on mitochondrial membrane potential (Δψm) were evaluated using the JC-1 probe. SH-SY5Y cells exposed to 6-OHDA induced a depolarization of Δψm (192.70 ± 16.86%) when compared to the vehicle. By the other side, fractions AEF9, AEF10 and AEF11 exhibited ability to restore the mitochondrial membrane potential loss promoted by 6-OHDA treatment (156.00 ± 12.53%, 108.80% ± 24.29% and 145.10 ± 18.55%, respectively) (Fig. 4B). Regarding Caspase-3 activity, the treatment of SH-SY5Y cells with 6-OHDA (239.90 ± 11.30%) led to a significant increased of the Caspase-3 activity when compared with vehicle (100.00 ± 33.69%). On the other hand, fractions MC18F2 (179.1% ± 5.23%) and AEFF9 (22.24% ± 1.54%) exhibited a significant decrease of Caspase-3 activity promoted by 6-OHDA (Fig. 5C).

Anti -inflammatory activity of Bifurcaria bifurcata fractions on RAW 264.7 cells
For the B. bifurcata fractions that displayed neuroprotective activities, their anti-inflammatory effects were studied on LPS-induced RAW264.7 cells. Firstly, the cytotoxicity of seaweed fractions (100 µg mL −1 ; 24 h) was evaluated on RAW264.7 cells and the effects were estimatedby the MTT method. Fractions without cytotoxicity were further tested for their inflammatory and anti-inflammatory potential on RAW264.7 cells. The inflammatory and anti-inflammatory effects of seaweed fractions were estimated through the NO production. The results are presented in Figs. 6A-C.

Effects of Bifurcaria bifurcata fractions on the pro-inflammatory cytokine's levels
The effects of pre-treatment with B. bifurcata polar fractions (100 µg mL −1 ; 1 h) on LPS-induced RAW264.7 cells was evaluated in the levels of TNF-α and IL-6 cytokines after 18 h (Fig. 7).
A general overview of the antioxidant, neuroprotective and anti-inflammatory activities of B. bifurcaria fractions is presented in Table 2. It possible to observe that AEF8 fraction showed high antioxidant capacity in all the evaluated methods when compared with other fractions. On the other hand, this fraction did not exhibit neuroprotective potential. Fig. 4 Neurotoxicity (100 µg mL -1 ) (A) and neuroprotective effects of non-toxic Bifurcaria bifurcata fractions (100 µg mL −1 , 24 h) on SH-SY5Y cells exposed to 6-OHDA (100 µM). (-) 6-OHDA (B). Values in each column represent the mean ± SEM of 3 or 4 independent experiments. Symbols represent significant differences (ANOVA, Dunnett's test, p < 0.05) when compared to: * vehicle; # to; 6-OHDA However, fractions AEF9, AEF10, AF11, MC18F1 and MC18F2 displayed neuroprotective activity and capacity to decrease ROS production, mitochondrial disfunction and Caspase-3 activity against 6-OHDA damage. Regarding the anti-inflammatory activity, all fractions, excepting fraction AEF9 decrease NO production induced by LPS treatment. Furthermore, seaweed fractions also revealed capacity to decreased IL-6 and TNF-α cytokines release. Accordingly, these five fractions that exhibited the best bioactive potential were studied for their chemical profile.

Discussion
The prevalence of neurodegenerative disorders is growing worldwide. In parallel, there is an urgent need to find new compounds for the treatment of such impairments, in which oxidative stress is a common hallmark, being suggested to play a causative role in the etiology and progression of Parkinson's disease (PD) (Uttara et al. 2009). Several lines of evidence have shown that brains of PD patients have low levels of endogenous antioxidants, increased dopamine oxidation, and high levels of iron, suggesting that oxidative stress is a significant target to counteract the progression of the disease. Natural and endogenous antioxidants such as polyphenols, Coenzyme Q10 and vitamins A, C and E, have been proposed as therapeutic agents for preventing and delaying the development of PD (Tan et al. 2018). However, more efficient approaches to counteract this disease are necessary. Although some current drugs are effective in the early stages of the disease, long-term therapy has been associated with serious adverse effects. Over the last decade, in an attempt to search new alternative therapies for PD, basic science has focused on the discovery of marine natural products as a source of potent and effective agents in the treatment of this devastating pathology. In the present study, the antioxidant, neuroprotective and anti-inflammatory properties of several fractions from the brown seaweed B. bifurcata were evaluated. Regarding their antioxidant capacity, three complementary methods, DPPH, FRAP and ORAC, were assayed and the results correlated with their total phenolic content (TPC). Amongst the twenty-three evaluated fractions, the most polar ones revealed to have the best antioxidant potential. This capacity can be mediated by a great variety of compounds including phlorotannins, linear diterpenes and mannitol, commonly found in brown seaweeds and also identified in B. bifurcata. Molecules with great antioxidant capacity due to their redox properties, acting as reducing agents (Costa et al. 2011;Farasat et al. 2014) and hydrogen (H + ) donors, contribute to the production of less reactive radicals and for the removal of metal ions (Kasote et al. 2015).
Effectively, oxidative stress is related with several diseases, including neurodegenerative disorders like PD. Several studies have reported that marine-derived compounds exhibit neuroprotective effects against 6-OHDA neurotoxicity (Chen et al. 2012;Souza et al. 2018;Ye et al. 2019). So, in this work the 6-OHDA-induced neurotoxicity model in SH-SY5Y cell line was used, and it was verified that 6-OHDA per se reduced cells' viability by about 40%. Regarding the neuroprotection assay, B. bifurcata fractions exhibited capacity to promote cells' recovery (20 -40%) from the 6-OHDA-induced neurotoxicity. Tancheva et al. (2020) evaluated the neuroprotective action of three natural antioxidants, ellagic acid, α-lipoic acid and myrtenal, in an experimental model of PD that was induced in male Wistar rats through an intrastriatal injection of 6-OHDA. These authors verified that the three compounds improved learning and memory performances as well as neuromuscular coordination. Additionally, in the biochemical assays, all the three compounds substantially decreased lipid peroxidation (LPO) levels and restored Catalase (CAT) activity and DA levels that were impaired by the challenge with 6-OHDA. In this context, F8 fraction demonstrated high antioxidant activity, revealing capacity to inhibit peroxyl radicals, DPPH and iron ions, however it did not reveal neuroprotective effects on SH-SY5Y cells treated with 6-OHDA. These facts can be related with the methodologies accomplished. The antioxidant activity was evaluated using chemical/ cell-free systems, in which the antioxidants can interact chemically. However, in the assays accomplished with cellular models there are other facts, such as bioavailability and metabolic factors that can compromise their cellular and physiological activities, as previously observed in chemical/ cell free systems (Lü et al. 2010). On the other hand, the decrease of neurotoxicity associated with 6-OHDA by marine-derived compounds has already been reported in previous scientific works. Our results are consistent with other studies in which methanolic extracts from the brown seaweeds Sargassum muticum and Saccorhiza polyschides increased neuronal cell viability in about 35-40%, blocking the toxic effects of 6-OHDA . Chen et al (2012) also evaluated the protective effect of the mac-rolide11-dehydrosinulariolide, isolated from the soft coral Sinularia flaxibilis, against 6-OHDA -induced neurotoxicity in SH-SY5Y cell lines.
The mechanisms involved in the neuroprotective effects exhibited by seaweed fractions on SH-SY5Y cell viability were studied on different hallmarks associated to PD development. For this purpose different in vitro assays were performed (mitochondrial membrane potential, production of ROS, and Caspase-3 activity) on cells treated with the neurotoxin 6-OHDA, in the presence/absence of fractions. Oxidative stress has been intimately linked to mitochondrial dysfunction and mitochondria are responsible for more than 90% of cellular ROS production (Zorov et al. 2014), being completely dependent of an efficient antioxidant machinery to prevent oxidative stress conditions. In fact, mitochondrial dysfunctions play a central role in the neurodegeneration, especially in PD, leading to a disruption of the respiratory chain and, consequently, to an increase of ROS production, a decrease of mitochondrial Complex I enzyme activity and Caspase-3 activation (Zorov et al. 2014;Redza-Dutordoir and Averill-Bates 2016). Based on the results obtained in the present work, it was observed that B. bifurcata ethyl acetate fractions (AEF9, AEF10 and AEF11) were able to prevent ROS production and mitochondrial dysfunction promoted by 6-OHDA treatment. These properties can be related with the presence of linear diterpenes in fractions AEF9 and AEF10, which biological properties were recently reported by Pais et al. (2019). Di-n-octyl phthalate was the major component identified in fraction AEF11 and, although phthalate-derived compounds are normally regarded as synthetic contaminants, they can also be biosynthesized by several organisms. These group of compounds were recently reported to have a broad range of bioactivities, including antioxidant and antiinflammatory properties (Roy 2020).
Since the Caspase-3 enzyme is a key player in cell death by apoptosis, its activity was also determined to understand if the neuroprotective effects of seaweed fractions were mediated by apoptotic pathways. The exposure of SH-SY5Y cells to 6-OHDA increased its activity, and the treatment with B. bifurcata fractions MC18F2 and AEF9 led to a marked decrease of Caspase-3 activity.
Microglial activation in the SNpc is another hallmark of PD that leads to a progression of inflammation, being the main cause of this disease condition, triggering the overproduction of pro-inflammatory mediators (Kany et al. 2019;Baek et al. 2020). The macrophages are known to cross the leaky blood-brain barrier in PD to interact with microglia and stimulate the secretion of inflammatory cytokines causing brain damage via neuroinflammation. These are important immune regulatory cells that play essential roles in inflammatory responses by producing various inflammatory mediators, such as nitric oxide (NO), as well as different pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 ( Kany et al. 2019). LPS is the main component of endotoxins from Gram-negative bacteria, inducing a host inflammatory response that results in increased production of chemokines, cytokines, and pro-inflammatory mediators by the immune system (Dickson and Lehmann 2019). This way, macrophages exposed to LPS during microorganisms' infections stimulate the immune system to produce cytokines and chemokines, followed by inflammatory events (Monguió-Tortajada et al. 2018;Kany et al. 2019). Thus, inhibiting macrophage activation by LPS is an important focus for therapeutic strategies aiming the treatment of diseases where inflammation is involved, including in PD (Ji et al. 2020). In this study macrophages were exposed to LPS to induce inflammation in the presence of the most promising B. bifurcata fractions in previous trials. Based on the results presented in Figs. 5 and 6, it is possible to observe that all fractions, excepting AEF9, prevented inflammatory conditions by decreasing NO production. On the other hand, all fractions prevented inflammation by reducing TNF-α and IL-6 levels. Furthermore, to deeply characterize the inhibition of apoptosis and inflammation events by seaweed fractions treatment, further studies should be conducted targeting other biomarkers (e.g. Caspase-9, cytochrome c release, Caspase-7, Bcl-2 family proteins expression; COX-2 and iNOS, respectively), using different techniques such as ELISA and/or Western blot, to reinforce the data here attained.
The anti-inflammatory properties of linear diterpenes (Pais et al. 2019) and phthalate derivatives (Roy 2020) can explain, at least in part, the positive effect of those fractions. Neverthless, fraction MC18F2 seems to be the most prominent in a great part of the in vitro assays here performed and, as described above, mannitol was the major metabolite present in this fraction. Mannitol, a sugar alcohol that is used as a sweetener in diabetic food, is present in many seaweeds such as Sargassum mangarevense, Turbinaria ornata, Ascophyllum nodosum and Laminaria hyperborea and is reported to have a broad range of applications namely in the pharmaceutical sector (Zubia et al. 2008;Gomez-Zavaglia et al. 2019). Clinically, mannitol can act as a diuretic, promoting the urinary excretion of toxic substances. Additionally, it elevates blood plasma osmolality, resulting in enhanced flow of water from tissues, including the brain and cerebrospinal fluid, into interstitial fluid and plasma. As a result, cerebral edema, elevated intracranial pressure, and cerebrospinal fluid volume and pressure are reduced.
Mannitol was identified as a major component the promising fraction, suggesting that the evidenced bioactivities can be mediated by this compound. Therefore, in this work, the neuroprotective effect of mannitol was evaluated for the first time in an in vitro model of PD induced by 6-OHDA, and it showed capacity to increase cellular viability, to decrease ROS production and Caspase-3 activity. There are reports referring that the neuroprotective effects of mannitol can be related to its antioxidant properties (Conrozier et al. 2014;Andr 2017), improvement of the blood brain barrier permeability (Cerri et al. 2014;Choi et al. 2018) and also to its capacity to interact with key proteins involved in PD (Shaltiel-Karyo et al. 2013). Additionally, the ability of mannitol to interfere with the aggregation process of α-synuclein on in vitro and in vivo models of PD, and its blood-brain barrier-disrupting properties were also previously verified (Shaltiel-Karyo et al. 2013). Specifically, high concentrations of mannitol significantly decreased the formation of α-synuclein fibril tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein, suggesting potential alternative pathways for aggregation. When administered to a transgenic mice model, mannitol evidenced a general neuroprotective effect, which included the dopaminergic system, whereas no adverse effects were observed in control animals. Based on these findings, mannitol (Shaltiel-Karyo et al. 2013) and mannitol-based molecules (Paul et al. 2019) can be regarded as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson's disease.
In conclusion, the current lack of studies on seaweedderived neuroprotective agents can open new research lines in the near future. This work has shown that B. bifurcata bioactive fractions markedly inhibited 6-OHDA neurotoxicity induced in human neuroblastoma SH-SY5Y cells, suggesting that the neuroprotective effects are mediated by the mitigation of ROS generation and mitochondrial dysfunctions, together with the reduction of Caspase-3 activity. In addition, this work also showed that fractions inhibited inflammation processes promoted by LPS on macrophages, through a decrease of NO production and a reduction of proinflammatory interleukins, such as TNF-α and IL-6. Mannitol was identified as a major component of the most promising fraction, suggesting that the evidenced bioactivities can be mediated by this compound. However, the hypothesis that other minor components and their synergistic effects may also be involved in those properties cannot be discarded. Studies are ongoing aiming a more detailed chemical characterization of bioactive samples as well as the isolation and structural characterization of the compounds responsible for the neuroprotective and anti-inflammatory properties.