Plant Extracts and Phytochemicals Targeting α-Synuclein Aggregation in Parkinson's Disease Models

α-Synuclein (α-syn) is a presynaptic protein that regulates the release of neurotransmitters from synaptic vesicles in the brain. α-Syn aggregates, including Lewy bodies, are features of both sporadic and familial forms of Parkinson's disease (PD). These aggregates undergo several key stages of fibrillation, oligomerization, and aggregation. Therapeutic benefits of drugs decline with disease progression and offer only symptomatic treatment. Novel therapeutic strategies are required which can either prevent or delay the progression of the disease. The link between α-syn and the etiopathogenesis and progression of PD are well-established in the literature. Studies indicate that α-syn is an important therapeutic target and inhibition of α-syn aggregation, oligomerization, and fibrillation are an important disease modification strategy. However, recent studies have shown that plant extracts and phytochemicals have neuroprotective effects on α-syn oligomerization and fibrillation by targeting different key stages of its formation. Although many reviews on the antioxidant-mediated, neuroprotective effect of plant extracts and phytochemicals on PD symptoms have been well-highlighted, the antioxidant mechanisms show limited success for translation to clinical studies. The identification of specific plant extracts and phytochemicals that target α-syn aggregation will provide selective molecules to develop new drugs for PD. The present review provides an overview of plant extracts and phytochemicals that target α-syn in PD and summarizes the observed effects and the underlying mechanisms. Furthermore, we provide a synopsis of current experimental models and techniques used to evaluate plant extracts and phytochemicals. Plant extracts and phytochemicals were found to inhibit the aggregation or fibril formation of oligomers. These also appear to direct α-syn oligomer formation into its unstructured form or promote non-toxic pathways and suggested to be valuable drug candidates for PD and related synucleinopathy. Current evidences from in vitro studies require confirmation in the in vivo studies. Further studies are needed to ascertain their potential effects and safety in preclinical studies for pharmaceutical/nutritional development of these phytochemicals or dietary inclusion of the plant extracts in PD treatment.

α-Synuclein (α-syn) is a presynaptic protein that regulates the release of neurotransmitters from synaptic vesicles in the brain. α-Syn aggregates, including Lewy bodies, are features of both sporadic and familial forms of Parkinson's disease (PD). These aggregates undergo several key stages of fibrillation, oligomerization, and aggregation. Therapeutic benefits of drugs decline with disease progression and offer only symptomatic treatment. Novel therapeutic strategies are required which can either prevent or delay the progression of the disease. The link between α-syn and the etiopathogenesis and progression of PD are well-established in the literature. Studies indicate that α-syn is an important therapeutic target and inhibition of α-syn aggregation, oligomerization, and fibrillation are an important disease modification strategy. However, recent studies have shown that plant extracts and phytochemicals have neuroprotective effects on α-syn oligomerization and fibrillation by targeting different key stages of its formation. Although many reviews on the antioxidant-mediated, neuroprotective effect of plant extracts and phytochemicals on PD symptoms have been well-highlighted, the antioxidant mechanisms show limited success for translation to clinical studies. The identification of specific plant extracts and phytochemicals that target α-syn aggregation will provide selective molecules to develop new drugs for PD. The present review provides an overview of plant extracts and phytochemicals that target α-syn in PD and summarizes the observed effects and the underlying mechanisms. Furthermore, we provide a synopsis of current experimental models and techniques used to evaluate plant extracts and phytochemicals. Plant extracts and phytochemicals were found to inhibit the aggregation or fibril formation of oligomers. These also appear to direct α-syn oligomer formation into its unstructured form or promote non-toxic pathways and suggested to be valuable drug candidates for PD and related synucleinopathy. Current evidences from in vitro studies require confirmation in the in vivo studies. Further studies are needed to ascertain their potential effects and safety in preclinical studies for pharmaceutical/nutritional development of these phytochemicals or dietary inclusion of the plant extracts in PD treatment.
Several recent reviews highlighted the neuroprotective potential of plant extracts and phytochemicals in PD through antioxidant and anti-inflammatory activities (Sarrafchi et al., 2016;da Costa et al., 2017;Mazo et al., 2017;Morgan and Grundmann, 2017;Wang et al., 2017;Zhang et al., 2017;Amro and Srijit, 2018). However, despite the enormous success of antioxidants (whether of synthetic or natural origin) in preclinical studies, coenzyme Q10 (Beal et al., 2014), creatine (Attia et al., 2017), and vitamin E (Ahlskog, 1994) either failed or showed marginal neuroprotection in patients. Recently, α-syn antibodies (PRX002) showed safety in phase 1 studies and were indicated for further phases of clinical studies (Schenk et al., 2017;Jankovic et al., 2018). Similarly, natural products (mainly plant extracts and phytochemicals) emerged to specifically target α-syn (Masuda et al., 2006;Meng et al., 2009Meng et al., , 2010Caruana et al., 2011;Marchiani et al., 2013). Yet, no comprehensive review is available on these plant extracts and phytochemicals, or on how they target the different steps leading to α-syn oligomerization or fibrillation.
This review, therefore, focuses on the neuroprotective properties and mechanism of action of plant extracts, extractbased formulations, and plant-derived phytochemicals that target α-syn oligomerization, fibrillation, aggregation, and toxicity in various experimental PD models. Furthermore, we also elaborate on the suitability of biochemical, biophysical, and neurochemical techniques to evaluate plant extracts and phytochemicals that ameliorate α-syn neurotoxicity. The source of phytochemicals, the models used, and the effect/mechanisms observed are presented in Tables 1-7. The chemical structures of these phytochemicals are presented in Figure 1. A scheme on the action of the plant extracts and phytochemicals targeting α-syn is presented in Figure 2.

α-SYNUCLEIN AS A THERAPEUTIC TARGET FOR PD
α-syn, a 140-residue presynaptic protein in the brain, plays a key role in the trafficking and fusion of synaptic vesicles and it regulates dopamine release at presynaptic terminals (Burre et al., 2010;Bendor et al., 2013). The physiological concentration of α-syn is 1 µM in the normal human brain and 70 pM in cerebrospinal fluid (Borghi et al., 2000). It exists natively as an unfolded monomer and attains an α-helical secondary structure after binding lipid vesicles. Upon destabilization, this leads to the misfolding and aggregation of α-syn in neurons (Ruipérez et al., 2010;Bartels et al., 2011;Broersen et al., 2018). Monomeric α-syn is an intrinsically disordered protein found in different conformational states. It plays a significant role in many key biochemical processes (Tompa, 2005), as well as in a rising number of diseases involving misfolding, notably PD (Uversky and Dunker, 2010). In dopaminergic neurons, the intracytoplasmic inclusions of α-syn (Spillantini et al., 1998), synphilin-1 (Wakabayashi et al., 2000) and ubiquitin (Kuzuhara et al., 1988) form Lewy bodies, a pathological characteristic of PD. The cascade of α-syn aggregation begins with dimer formation, then tiny oligomers/protofibrils that lead to the development of β-sheet-rich α-syn fibrils. These eventually lead to end-stage fibrils and aggregated α-syn that are the major component of Lewy bodies (Ghiglieri et al., 2018). Thus, in the multistep process of α-syn-mediated neuronal toxicity, oligomerization of α-syn monomers is the primary phase that facilitates the development of intracytoplasmic inclusions and fibrils (Spillantini et al., 1997).
Numerous theories have been proposed on the role of α-syn in initiating dopaminergic neurodegeneration in PD (Herrera et al., 2017;Ghiglieri et al., 2018). These include the interaction of α-syn aggregates with biomolecules, impaired fusion, and trafficking of vesicles, excessive free radical generation, mitochondrial dysfunction, endoplasmic reticulum stress, and synaptic dysfunction (Herrera et al., 2017;Longhena et al., 2017;Ghiglieri et al., 2018). The α-syn protein consists of three distinct domains, where the central region is critical for α-syn fibril aggregation, a key component of Lewy bodies. α-syn aggregation assay and α-syn induced cytotoxicity in SH-SY5Y cells and induced A53T α-syn PC12 in cells Increases α-syn solubility and prevents oligomerization Attenuates apoptosis, ROS, and mitochondrial depolarization Reduces formation, aggregation, and accumulation of α-syn Downregulates mTOR/p70S6K signaling and recovers suppressed macroautophagy Binds to preformed oligomers/fibrils, alter the hydrophobic surface Binds specifically to oligomer intermediates and reduces numbers Ono and Yamada, 2006;Pandey et al., 2008;Wang et al., 2010;Liu et al., 2011;Gadad et al., 2012;Jiang et al., 2013;Singh et al., 2013 Curcumin-glucoside α-syn aggregation biochemical assay Prevents oligomer and fibrilformation Improves binding with oligomers and enhances α-syn solubility and prevents fibrillation of α-syn Solubilizes oligomers by disintegrating preformed fibrils Gadad et al., 2012 Curcumin derivatives: Dehydrozingerone, O-methyl, zingerone, biphenyl analogs α-syn aggregation biochemical assay and PC12 cells model of PD Biphenyl analogs of dehydrozingerone and O-methyl-dehydrozingerone inhibit α-syn aggregation process Displays the best antioxidant properties Marchiani et al., 2013 Curcumin pyrazole and curcumin isoxazole α-syn aggregation biochemical, biophysical and cell based assays Curcumin pyrazole and derivative exhibit better potency Arrests fibrillization and disrupting preformed fibrils Prevents A11 conformation in protein that imparts toxicity Decreases fast aggregating A53T mutant form of α-syn

Ahsan et al., 2015
Curcumin α-syn in genetic synucleinopathy mouse line overexpresses wild-type α-syn Improves gait impairments Increases phosphorylated α-syn in presynaptic terminals without affecting α-syn aggregation Spinelli et al., 2015 Curcumin with β-cyclodextrin α-syn aggregation assay Inhibits aggregation and Brakes up preformed aggregates, exhibit synergy in their action at low concentrations Gautam et al., 2014 Curcumin, myricetin, rosmarinic acid, nordihydroguaiaretic acid, and ferulic acid Biophysical assays for α-syn and electrophysiological assays for long-term potentiation in mouse hippocampal slices Inhibits α-syn oligomerization and structure conversion Directly bound to the N-terminal region of α-syn Ameliorates α-syn aggregation and α-syn synaptic toxicity Prevents process, reducing the neurotoxicity of αS oligomers Ameliorates α-syn synaptic toxicity in long-term potentiation Takahashi et al., 2015 (Continued) Frontiers in Pharmacology | www.frontiersin.org  α-syn-induced synaptic toxicity in rat hippocampal cells and human induced pluripotent stem cell (iPSC)-derived neurons Reduces the α-syn-induced loss of synaptophysin Enhances synaptic vesicles recycling in the presence of α-syn Protects iPSC-derived neurons and maintain firing activity Protects against mutated β-SynP123H-induced synaptic activity Alhebshi et al., 2014 α-syn can adopt a wide range of conformational structures ranging from compact to fully extended (Winner et al., 2011). The interactions between the N-and C-termini of α-syn play a role in its stabilization into a compact, monomeric conformation that is non-toxic (Bertoncini et al., 2005). The agents that bind to α-syn and form a loop structure between the N-and Cterminus are believed to confer neuroprotection. In contrast, the agents which induce more compressed structures are considered neurotoxic in nature (Karpinar et al., 2009;Lashuel et al., 2013). Mutations in α-syn can contribute to multiple forms of PD including genetic and rare forms of PD with early onset (Singleton et al., 2003;Simon-Sanchez et al., 2009). Monomeric α-syn is a potential therapeutic target as it is an upstream form of the protein during the aggregation process and the etiopathogenesis of PD (Winner et al., 2011;Lashuel et al., 2013;Brundin et al., 2017;Ghiglieri et al., 2018). The agents stabilizing, promoting clearance, degrading misfolded proteins, solubilizing oligomers, or inhibiting the propagation of α-syn aggregates are pharmacologically appropriate and a clinically relevant therapeutic strategy for PD. Interaction of curcumin and α-syn in genetic synucleinopathy of α-syn-GFP mouse line overexpresses α-syn Chronic and acute curcumin treatment improves gait impairments and increases phosphorylated forms of α-syn at cortical presynaptic terminals in α-syn-GFP line Increases phosphorylated α-syn in terminals without affecting α-syn aggregation Spinelli et al., 2015 Alginate-curcumin nanocomposite

Supplemented with diet to Drosophila melangoster
Delays climbing disability in flies Reduces oxidative stress and apoptosis in the brain of PD flies Siddique et al., 2013b α-Linolenic acid Caenorhabditis elegans wild type N2 and transgenic (UA44) exposed to 6-OHDA Improves locomotion, pharyngeal pumping, and lifespan Shows a visibly significant reduction in neuronal degeneration Increases GFP expression within in neurons Shashikumar et al., 2015 Squamosamide 6-OHDA-induced PD in rats Improves motor dysfunction and behavior Enhances dopamine level and TH activity Decreases α-syn expression mediated by the Akt/mTOR pathway Reduces RTP801 expression, a protein in the pathogenesis of PD Bao et al., 2012 Geraniol (Monoterpene from rose oil, palmarosa oil, and citronella oil) MPTP-induced PD in C57BL/6 mice Reduces α-syn aggregation in dose dependent manner Improves nigral dopamine, TH and dopamineric terminals in striatum Improves neuromuscular disability and Lewy body aggregation Rekha et al., 2013 (Continued) Frontiers in Pharmacology | www.frontiersin.org

MEDICINAL PLANTS TARGETING α-SYNUCLEIN CASCADE AND TOXICITY
Recently, many plant extracts appear to inhibit oligomerization and fibrillization of α-syn, an emerging therapeutic target in PD (Lobbens et al., 2016;Ren et al., 2016;Briffa et al., 2017;Cheon et al., 2017). The plant extracts, which were shown to be neuroprotective in PD, target various pathogenic stages of αsyn conformations ranging from fibrillation to oligomerization in experimental models and are listed in Table 1 Figure 2 to In vitro and in vivo models Effects and observed mechanisms References Ellagic acid Cell-based and cell-independent in vitro showing nitrosative stress mediated S-nitrosylation (SNO), the SNO-PDI formation is linked to the aggregation of α-syn and α-syn:synphilin-1 deposits in the PD brain Scavenges NOx radicals and protect cells from SNO-PDI formation via rotenone insult both, cell-based and cell-independent in vitro Mitigates nitrosative-stress-induced aggregation of synphilin-1 but also α-syn and α-syn: synphilin-1 composites (Lewy-like neurites) in PC12 cells Lowers rotenone-instigated reactive oxygen species and reactive nitrogen species in PC12 cells Inhibits apoptosis and interferes with SNO-PDI formation
From the perspective of traditional medicine, targeting αsyn with plant extracts containing phytochemicals could be

Biochemical/biophysical techniques Events monitored in the system References
Fluorescence polarization technique α-syn aggregation Luk et al., 2007 Scanning for intensely fluorescent targets and atomic force microscopy α-syn oligomers Kostka et al., 2008 High performance liquid chromatography (HPLC), Circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), Size exclusion HPLC, small-angle X-ray scattering, and atomic force microscopy ( considered beneficial using dietary intervention. This could be due to the synergy in action and superior therapeutic effects, along with polypharmacological properties (Wagner and Ulrich-Merzenich, 2009;Wu et al., 2013). The fraction that termed active from Radix Polygalae was found more potent than the constituent, where onjisaponin B increased mutant huntingtin removal and reduced α-syn aggregation. This plant could be a good source of phytochemicals and a template for novel small molecule inhibitors of α-syn (Wu et al., 2013). Plantbased formulations, such as S/B which contain extracts of Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium and a traditional Chinese medicine decoction known as Tianma Gouteng Yin, were also found to diminish α-syn accumulation and aggregation in experimental PD models (Lin et al., 2011). The majority of plant extracts used in traditional medicines are based on long-established knowledge of their health benefits, time tested safety due to ancient use, and potential therapeutic effects. However, some plants are not as beneficial as documented or are detrimental; the essential oil from Myrtus communis, which is popular in the Zoroastrian community for aroma (Morshedi and Nasouti, 2016), increases α-syn fibrillation and enhances α-syn toxicity in human neuroblastoma cells (Morshedi and Nasouti, 2016). This study suggests that essential oils used in aromatherapy should be investigated for their potential neurotoxicity or neurodegenerative ability.
The attenuation of α-syn toxicity by plant extracts validates traditional claims of medicinal plants. It may also provide the basis for dietary or nutritional inclusion of these plants in foods to achieve neuroprotective effects. This is not only based on antioxidant approaches but also inhibition of α-syn aggregation. However, in-depth studies are needed for a dietary or therapeutic recommendation on the use of plant extracts in humans.

PLANT EXTRACTS AND PHYTOCHEMICALS AS PHARMACOLOGICAL CHAPERONES FOR PD
Pharmacological chaperoning is emerging as a potential therapeutic approach for the treatment of numerous diseases associated with single gene mutations (Srinivasan et al., 2014). These chaperones are small molecules that bind proteins and stabilize them against proteolytic degradation or protect  them from thermal denaturation. Furthermore, they assist in or prevent certain protein-protein assemblies similar to the molecular chaperones (Ringe and Petsko, 2009). Chaperoning is beneficial in cystic fibrosis (Chanoux and Rubenstein, 2012), Gaucher's disease (Sawkar et al., 2002), nephrogenic diabetes insipidus (Tamarappoo and Verkman, 1998), and retinitis pigmentosa (Noorwez et al., 2003). Mechanistically, ligand-mediated chaperoning is believed to correct receptor mislocalization and inhibit mutant proteins from forming toxic intracellular aggregates (Loo and Clarke, 2007). This has been shown to be successful with the pharmacological chaperone, tafamadis, in a clinical trial for the treatment of transthyretin familial amyloid polyneuropathy . Several of the molecular chaperones, such as Hsp70, Hsp40, and torsin A either prevent the misfolding of proteins or promote the degradation and elimination of misfolded proteins; they provide a novel therapeutic approach in PD (Dimant et al., 2012).
Although molecular chaperoning is therapeutically significant in α-syn-associated neurodegeneration, the structural heterogeneity and deficiency of persistent structural components for α-syn creates a major issue in the discovery, design, and development of small molecules targeting α-syn (Lester et al., 2009). Plant-derived phytochaperones are a good source of molecules that target protein misfolding in neurotherapeutics (Bernd, 2008). In a chaperone-based approach, Ginkgo biloba is being utilized to search for lead molecules in drug discovery and in the development of protein-misfolding diseases leading to neurodegeneration (Kastenholz and Garfin, 2009). Thus, plant extracts and phytochemicals are a novel source of pharmacological chaperones for a disease-modifying approach that could be promising against neurodegenerative diseases. Following the reductionist approach of drug discovery from plant extracts, it is also important to characterize the bioactive constituents contributing to these pharmacological effects.

PHYTOCHEMICALS TARGETING α-SYNUCLEIN ASSEMBLY AND TOXICITY
The phytochemicals are non-nutritive secondary metabolites that are heavily utilized for drug discovery and development; they remain an important source of drugs (Beutler, 2009;Henrich and Beutler, 2013). The phytochemicals that target α-syn at different stages of pathogenicity are represented in Table 2 (in vitro studies), Table 3 (in vivo studies), and Table 4 (in vitro and in vivo, both studies), respectively. A benefit of the phytochemicals is their huge structural diversity that offers lead structures for drug discovery and development. They belong to many classes, such as alkaloids, saponins, carotenoids, lignans, glycosides, etc. Briefly, the alkaloids are a nitrogen-containing, structurally-diverse group of secondary metabolites that are protective against neurodegenerative diseases (Hussain et al., 2018). To name a few, galantamine is used in the pharmacotherapy of mild to moderate Alzheimer's disease. Many of the alkaloids, such as acetylcorynoline, 3α-acetoxyeudesma-1,4 (15),11 (13)-trien-12, 6α-olide, corynoxine B, dl-3-n-butylphthalide, isorhynchophylline, and squamosamide attenuate neurotoxicity in experimental models by directly inhibiting α-syn aggregation or fibril formation.
Dietary intake of polyphenolic compounds is protective against neurodegeneration as evidenced from many epidemiologic and experimental studies (Ho and Pasinetti, 2010;Caruana et al., 2016;da Costa et al., 2017). Popular polyphenols in food are curcumin (present in turmeric), oleuropein (present in olive oil), resveratrol (present in grapes), catechins (present in black and green tea), astaxanthin (a carotenoid present in vegetables and fruits) and lycopene (present in tomato) (da Costa et al., 2017). Polyphenols inhibit α-syn aggregation and fibrillation (Masuda et al., 2006;Caruana et al., 2011Caruana et al., , 2012Sivanesam and Andersen, 2016) and formation of amyloid protofilaments and fibrils (Kumar et al., 2012;Velander et al., 2017) and confer protective effects in neurodegenerative diseases. Masuda et al. (2006) tested 79 compounds from different chemical classes of compounds including polyphenols, benzothiazoles, terpenoids, steroids, porphyrins, lignans, phenothiazines, polyene macrolides, and Congo red and its derivatives for their potential to inhibit α-syn assembly. Out of 39 polyphenolic compounds tested, 26 were found to inhibit α-syn assembly. These findings establish that polyphenols constitute a major class of compounds that can inhibit the assembly of α-syn. Several of them inhibited α-syn filament assembly with IC50 values in the low micromolar range. Caruana et al. (2011) investigated 14 polyphenolic compounds and black tea extract containing theaflavins and found that baicalein, scutellarein, myricetin, (-)-epigallocatechin-3-gallate (EGCG), nordihydroguaiaretic acid and black tea extract are the ideal candidates to investigate in experimental models for their direct effect on the inhibition of α-syn oligomer formation. The polyphenolic compounds are believed to interact with receptors or plasma membrane transporters and activate intracellular signaling pathways. Among several polyphenols, EGCG associates with the laminin receptor on vascular cells (Tachibana et al., 2004). Currently, numerous polyphenolic compounds have been studied for their effect on α-syn aggregation, fibrillation, elongation, nitration, and oligomerization using biophysical and biochemical techniques (Meng et al., 2010;Caruana et al., 2011Caruana et al., , 2012Takahashi et al., 2015). The list of these compounds is presented in Table 5. A scheme is presented in Figure 2 to depict the mechanism of action of the plant extracts and phytochemicals on α-syn oligomerization, fibrillation, and aggregation. An overview of some important phytochemicals which target α-syn aggregation and fibrillation and appear ideal candidates for further development is presented below.

Baicalein
Baicalein is a flavone isolated from the roots of Scutellaria baicalensis Georgi ("Huang Qin" in Chinese), a reputed plant in traditional Chinese medicine (Gasiorowski et al., 2011) and Scutellaria pinnatifida grown in Iran (Sashourpour et al., 2017). In many studies, baicalein was shown to prevent α-syn oligomerization and fibrillation (Bomhoff et al., 2006;Meng et al., 2009;Caruana et al., 2011;Gasiorowski et al., 2011;Sashourpour et al., 2017). Baicalein interacts with α-syn through a tyrosine residue. Following oxidation, it generates quinone metabolites that bind covalently with a lysine side chain in α-syn. It prevents fibril formation and degrades preformed fibrils at low micromolar concentrations (Zhu et al., 2004). In another study, its non-covalent binding with α-syn and covalent modification by the oxidized form restricts the conformational changes in the unfolded protein that results in α-syn monomer and oligomer stabilization (Meng et al., 2009). The oligomers cause impairment of neuronal membrane integrity that results in disruption or permeabilization of the membrane, impairment of calcium homeostasis, and cell death (Caruana et al., 2012).
Baicalein prevents α-syn fibrillation and protects against neurotoxicity by preventing α-syn oligomer formation in SH-SY5Y and HeLa cells (Lu et al., 2011). It also stabilizes the oligomers, prevents further fibrillation (Hong et al., 2008) and tandem repeats of α-syn in the aggregation process (Bae et al., 2010). Further, it prevents the formation of annular protofibrils of α-syn induced by copper and reduces the β-sheet contents . In another study, using JC-1, a probe that binds the α-syn C-terminal region, baicalein differentiated the α-syn fibrillation states (monomeric, oligomeric intermediate, and fibrillar forms) and reconfirmed the defibrillation action of baicalein on α-syn (Lee et al., 2009). In PC12 cells, it ameliorates cytotoxicity, mitochondrial depolarization, and inhibits proteasome inhibition induced by E46K, an α-syn point mutation that mimics familial PD (Jiang et al., 2010). In a recent study, baicalein induces autophagy, increases cell viability and reduces α-syn in the media of dopaminergic cell lines (SN4741) overexpressing A53T-syn . Baicalein diminished the transmission of α-syn and prompted the polymerization of α-syn to a big complex rather than promoting clearance . A recent study in rotenone-induced PD in rats showed reduced α-syn oligomer formation along with behavioral improvement and neurotransmitters in the striatum. However, it failed to reduce α-syn mRNA expression but prevented the transition from α-syn monomers to oligomers . Furthermore, baicalein attenuated α-syn aggregate formation, induced autophagy, inhibited apoptosis, reduced inflammation, and restored dopamine in PD induced by MPP + infusion in the SNc of mice (Hung et al., 2016).
The baicalein derivative N ′ -benzylidene-benzohydrazide also attenuated oligomer formation (Kostka et al., 2008). Baicalein in combination with β-cyclodextrin (β-CD) synergistically inhibited α-syn aggregation and disaggregated preformed fibrils even at very low concentrations (Gautam et al., 2017). A combination of baicalein with specific proteolytic peptide sequences of α-syn was developed for targeted drug delivery and found to prevent α-syn fibrillation . Integrating evidence from in vitro and in vivo studies, baicalein appears to be a potential drug to inhibit α-syn aggregation, fibrillation, and propagation among the neurons.

Curcumin
Curcumin, chemically known as diferuloylmethane, is one of the most popular natural leads to drug discovery and development from turmeric (Molino et al., 2016). It is reputed for its dietary importance and health benefits and is the most studied phytochemical in experimental and clinical studies (Molino et al., 2016). It is a beneficial treatment in neurodegenerative diseases, including PD, and has antioxidant, anti-inflammatory, and antiapoptotic properties (Kim et al., 2012;Singh et al., 2013;Ji and Shen, 2014). Ono and Yamada (2006) found that curcumin possesses anti-fibrillogenic activity by inhibiting α-syn fibril formation and destabilizing preformed fibrils (Ono and Yamada, 2006). It was found to inhibit oligomerization of mutant α-syn into higher molecular weight aggregates (Pandey et al., 2008) and induce the dissociation of α-syn fibrils (Shoval et al., 2008). Curcumin treatment on mesencephalic cells did not affect α-syn fibril formation but enhanced LRRK2 mRNA and protein expression in rats (Ortiz-Ortiz et al., 2010). In neuroblastoma cells, curcumin attenuates cytotoxicity from aggregated α-syn, ROS generation, and diminished caspase-3 activation (Wang et al., 2010). In PC12 cells, curcumin ameliorates A53T mutant α-syn-induced PD . Further, curcumin reduces mutant α-syn accumulation by restoring macroautophagy, a process in the degradation pathway that clears proteins in cells by activating the mTOR/p70S6K signaling pathway (Jiang et al., 2013). Mechanistically, curcumin preferentially binds oligomeric intermediates rather than monomeric α-syn . Also, it binds strongly to the hydrophobic nonamyloid-β component of α-syn (Ahmad and Lapidus, 2012). The ordered structure is vital for effective binding and affects the extent of binding and potential in inhibiting oligomers or fibrils . The conformational and reconfiguration changes appear to govern the binding of curcumin to α-syn (Ahmad and Lapidus, 2012;Tavassoly et al., 2014). Curcumin, in combination with β-cyclodextrin, showed a synergistic inhibition of α-syn aggregation and degraded the preformed aggregates into monomers at very low concentrations (Gautam et al., 2014(Gautam et al., , 2017. Gautam et al. (2017) further demonstrated that a balanced arrangement of the phenolic groups, benzene rings, and flexibility attributes to the ability of curcumin. The phenolic groups enhance curcumin interactions with α-syn monomers as well as oligomers. In PC12 cells transfected with recombinant plasmids, α-syn-pEGFP-A53T downregulated αsyn expression or oligomer formation by regulating apoptosismediated mitochondrial membrane potential (Chen et al., 2015a). The effect of curcumin on α-syn observed in vitro was reconfirmed in vivo in genetic mouse models of synucleinopathy (Spinelli et al., 2015). Curcumin increased phosphorylated forms of α-syn at cortical presynaptic terminals but had no direct effect on α-syn aggregation. However, curcumin improved motor and behavioral performance (Spinelli et al., 2015).
Curcumin is less stable and soluble and has limited oral bioavailability. To improve its stability, solubility, and oral bioavailability, many nanoformulations or structural analogs have been developed (Gadad et al., 2012;Kundu et al., 2016;Taebnia et al., 2016). Curc-gluc, a modified curcumin preparation, inhibits α-syn oligomerization and fibrillation (Gadad et al., 2012). In another study, dehydrozingerone, zingerone; an O-methyl derivative of dehydrozingerone and their biphenyl analogs were investigated for their cytoprotective effects in PC12 cells challenged with H 2 O 2 , MPP + , and MnCl 2 (Marchiani et al., 2013). The biphenyl analogs of dehydrozingerone and O-methyl-dehydrozingerone prevent αsyn aggregation; the biphenyl zingerone analog is the most potent inhibitor and has the most potent antioxidant activity. This activity was attributed to the hydroxylated biphenyl scaffold in the pharmacophore (Marchiani et al., 2013). In another study, stable curcumin analogs, such as curcumin pyrazole, curcumin isoxazole, and their derivatives, were evaluated against α-syn aggregation, fibrillation, and toxicity. Curcumin pyrazole and its derivative N-(3-Nitrophenyl pyrazole) curcumin reduces A53T-α-syn-induced neurotoxicity by preventing fibrillation and disrupting preformed fibrils (Ahsan et al., 2015). Taebnia et al. (2016) developed amine-functionalized mesoporous silica nanoparticles of curcumin to enhance its bioavailability and evaluated its effect against cytotoxicity and α-syn fibrillation (Taebnia et al., 2016). This nanoformulation showed interaction with α-syn species and prevented fibrillation with negligible effect on cytotoxicity (Taebnia et al., 2016). A nanoformulation containing curcumin and piperine with glyceryl monooleate nanoparticles coated with various surfactants was developed for targeted delivery to enhance its bioavailability in the brain (Kundu et al., 2016). The nanoformulation has been shown to attenuate oxidative stress, apoptosis, prevent α-syn oligomerization and fibrillation, and induce autophagy. Another nanoformulation prepared with lactoferrin by sol-oil chemistry ameliorates rotenone-induced neurotoxicity in dopaminergic SK-N-SH cells (Bollimpelli et al., 2016). This nanoformulation exhibited better availability, improved cell viability, attenuated oxidative stress, and reduced tyrosine hydroxylase and αsyn expression. Nine curcumin analogs were synthesized by substitution of groups on the aromatic ring which alters the hydrophobicity, promotes stability, and facilitates binding with the fibrils as well as oligomers (Jha et al., 2016). Some of the analogs showed improved stability and appeared to interact with oligomers and preformed fibrils. The analogs exhibited differential binding patterns and augmented α-syn aggregation, generating different kinds of amyloid fibrils. The liposomal nanohybrid of curcumin with polysorbate 80-modified cerasome was developed for targeted drug delivery in the striatum and showed better half-life and bioavailability (Zhang et al., 2018). This nanoformulation ameliorated motor deficits and improved dopamine and tyrosine hydroxylase expression by promoting α-syn clearance in a mouse model of MPTPinduced PD (Zhang et al., 2018). Curcumin inhibited α-syn aggregates in dopaminergic neurons and attenuated oxidative stress, inflammation, apoptosis, and motor deficits in a rat model of lipopolysaccharide-induced PD (Sharma and Nehru, 2018). These reports demonstrate the effect of curcumin on α-syn aggregation-and fibrillation-induced neurotoxicity but further studies are still needed to demonstrate therapeutic success.

Cuminaldehyde
Cuminaldehyde is isolated from many edible plants including Artemisia salsoloides, Aegle marmelos, and spices cumin (Cuminum cyminum L.) and is used as a food additive and flavoring agent in many cuisines in the Middle East, South Asia, and Mediterranean countries. Cuminaldehyde isolated from Iranian cumin showed to inhibit α-syn fibrillation (Morshedi et al., 2015). It prevented α-syn fibrillation even in the presence of seeds with negligible disaggregating effect on the preformed fibrils of α-syn. Interestingly, it was found to be superior to baicalein, a known inhibitor of α-syn fibrillation and blocked protein assembly into β-structural fibrils that were attributed to interaction with amine and aldehyde groups in the chemical structure (Morshedi and Nasouti, 2016).

Catechins, Theaflavins, and (-)-Epigallocatechin-3-Gallate (EGCG)
Catechins, the polyphenolic compounds present in black and green tea, are protective in neurodegenerative diseases (Caruana and Vassallo, 2015;Jha et al., 2017;Xu et al., 2017;Pervin et al., 2018). Theaflavins present in fermented black tea inhibits fibrillogenesis of α-syn and amyloid-β formation (Grelle et al., 2011). These compounds facilitate the assembly of amyloidβ and α-syn into non-toxic, spherical aggregates which are unable to undergo seeding to form amyloid plaques. They were also found to remodel the formed amyloid-β fibrils into nontoxic aggregates and these effects were comparable to EGCG. Theaflavins also appeared less vulnerable to oxidation in air and exhibited better activity in oxidizing environments in comparison with EGCG (Grelle et al., 2011).
One of the most popular catechins, (-)-Epigallocatechin 3gallate (EGCG), is a flavonol compound predominantly present in green tea, a popular beverage across the world. EGCG inhibited α-syn aggregation and fibrillation in a concentrationdependent manner (Šneideris et al., 2015;Xu et al., 2017), and by disaggregating mature and large α-syn fibrils into smaller, non-toxic, amorphous aggregates (Ehrnhoefer et al., 2008). EGCG binds directly to the natively unfolded polypeptides and inhibits their conversion into toxic intermediates (Ehrnhoefer et al., 2008). It induces a conformational change without their disassembly into monomers or small diffusible oligomers (Bieschke et al., 2010). It appears to bind directly with βsheet-rich aggregates and reduces its concentration required to induce conformational changes (Liu et al., 2018). Furthermore, it showed neuroprotection against free radicals and α-syn toxicity by chelating Fe (III) in PC12 cells transfected with α-syn and exposed to β-sheet-enriched α-syn fibrils (Zhao et al., 2017). EGCG appears to disaggregate α-syn fibrils by preventing the amyloid formation of α-syn tandem repeat and destabilizing αsyn fibrils into soluble amorphous aggregates (Bae et al., 2010). This study also revealed that the tandem repeat of α-syn may be used as a molecular model to study the mechanism of αsyn aggregation (Bae et al., 2010). Further, EGCG also prevents α-syn aggregation and accumulation by activating the hypoxiainducible factor (HIF)-1 signaling mechanism that controls αsyn aggregation by regulating antioxidant and iron homeostasis (Weinreb et al., 2013). Lorenzen et al. (2014) showed that EGCG has potential to prevent α-syn oligomer formation and attenuate the oligomer cytotoxicity by preventing vesicle permeabilization and blocking the membrane affinity of syn to bind and immobilize in the C-terminal region. Though, it failed to affect the oligomer size distribution or secondary structure. Recently, in primary cortical neuron cultures challenged with oxidative injury, quercetin, EGCG, and cyanidin-3-glucoside inhibited fibrillation of α-syn and apoptosis (Pogacnik et al., 2016). Further, it decreased amyloid fibril formation on the surface of liposomal membranes and generates compact oligomers following off-pathway, as well as facilitating the conversion of active oligomers into amyloid fibrils . A combination of EGCG with specific α-syn proteolytic peptide sequences was developed for targeted drug delivery and found to prevent the α-syn fibrillation . In combination, this evidence suggests EGCG could be a promising treatment in neurodegenerative diseases and a good candidate for pharmaceutical development and dietary inclusion.

Gallic Acid
Gallic acid, a type of phenolic acid chemically known as 3,4,5trihydroxybenzoic acid, is found in free form or as part of the hydrolyzable tannins in many plants, such as gallnuts, sumac, witch hazel, tea leaves, and oak bark (Kosuru et al., 2018). Gallic acid and esters are well-known food additives, nutritional supplements, and a common reagent in the pharmaceutical analysis (Kosuru et al., 2018). Over the last few decades, many investigators showed the antioxidative, antiapoptotic, cardioprotective, neuroprotective and anticancer properties of gallic acid and gallates (Blainski et al., 2013;Choubey et al., 2018;Kosuru et al., 2018). It is used as a reference compound for the quantification of the phenolic contents in biochemical assays; Folin-Ciocalteau assay or Folin's phenol reagent or Folin-Denis reagent which determines the antioxidant power in gallic acid equivalents (Blainski et al., 2013). The polyphenolic compound gallic acid and its structurally similar benzoic acid derivatives elicit anti-aggregating effects (Ardah et al., 2014). Gallic acid impedes α-syn fibrillation and disaggregates the preformed fibrils of α-syn in a battery of biophysical, biochemical, and cell viability assays. In addition to inhibiting aggregation and disaggregation, it also binds to soluble and non-toxic oligomers devoid of βsheet content and confers structural stability. Numerous benzoic acid derivatives have been developed using structure-activity relationship and all inhibit α-syn fibrillation (Ardah et al., 2014). The number of hydroxyl groups and their presence on the phenyl ring in these structural derivatives of gallic acid are believed to attribute to the potential mechanism in binding and inhibiting αsyn fibrillation. Furthermore, gallic acid prevents α-syn amyloid fibril formation, stabilizes the extended intrinsic structure of α-syn, and reacts rapidly in biochemical assays .

Ginsenosides
Ginseng, also known as red ginseng (Panax ginseng, Araliaceae), is a popular source of saponins and is reputed in the folk medicine of the Far East countries. It has shown neuroprotective effects in numerous neurodegenerative diseases including PD (Van Kampen et al., 2003;Chen et al., 2005;Luo et al., 2011). The ginseng extract, abbreviated as G115, confers neuroprotection against MPTP and its neurotoxic metabolite, MPP + in murine models of PD (Van Kampen et al., 2003). Van Kampen et al. (2014) reported that G115 treatment reduces dopaminergic cell loss, microgliosis, the buildup of α-syn aggregates, and improves locomotor activity and coordination in rats chronically exposed to the dietary phytosterol glucoside, β-sitosterol β-d-glucoside, which recapitulates features of PD.
Several studies identified the active constituents of ginseng known as ginsenosides, a group of 60 compounds that possess a wide range of pharmacological and physiological actions (Mohanan et al., 2018;Zheng et al., 2018). Several ginsenosides, Rg1, Rg3, and Rb1, were investigated for their effect on αsyn aggregation using biophysical and biochemical techniques (Ardah et al., 2015;Heng et al., 2016). Upon oral treatment, Rg1 attenuated neurodegeneration in a mouse model of MPTP-induced PD by inhibiting pro-inflammatory cytokines; reducing mortality, behavioral defects, and dopamine neuron loss; and correcting ultrastructure changes in the SNc (Heng et al., 2016). Rg1 also reduced oligomeric, phosphorylated, and disease-related α-syn in the SNc. In contrast, a separate study identified only Rb1 as a strong inhibitor of α-syn fibrillation; it also disaggregated preformed fibrils and inhibited the seeded polymerization of α-syn (Ardah et al., 2015). Further, Rb1 binds to the oligomers and causes stabilization of soluble, non-toxic oligomers with negligible involvement of β-sheets that depicts a novel mechanism of action (Ardah et al., 2015). Although the authors did not find a significant effect of Rg1 and Rg3 on αsyn aggregation in cellular models (Ardah et al., 2015). In one of the study, Rg3, another ginsenoside present in Panax ginseng, reduced α-syn expression in stress models . The evidence of Rg3-mediated changes in α-syn in stress models needs to be investigated in PD . A detailed investigation is required to understand the observed differences in the in vitro and in vivo studies.

Resveratrol
Resveratrol, a natural phytoestrogen found in grapes and red wine, is reputed for its neuroprotective properties by attenuating oxidative stress, mitochondrial impairment, inducing apoptotic cell death and promoting autophagy (Caruana et al., 2016;Ur Rasheed et al., 2016). Wu et al. (2011) showed that resveratrol enhanced α-syn degradation in PC12 cells expressing α-syn by activating autophagy and mediating the induction of AMP-activated protein kinase (AMPK) mammalian silent information regulator 2 (SIRT1) signaling mechanism. This reduces protein levels of microtubule-associated protein 1 light chain 3 (LC3-II) and preserves neuronal cells. AMPK is a serine/threonine kinase which acts as a metabolic energy sensor to maintain energy balance; upon activation, it induces neuronal cell apoptosis and decreases SIRT1 leading to activation of the ubiquitin-proteasome pathway by enhancing ubiquitination and promoting SUMOylation that may be important in reducing the progression of neurodegeneration (Wu et al., 2011). The induction of autophagy and apoptotic pathways represents an important approach in the therapeutic targeting of α-syn (Ghavami et al., 2014). Further, in MPTP-induced PD in mice, resveratrol corrected the behavioral and motor deficits and attenuated neurodegeneration by inducing autophagy of αsyn via activation of SIRT1 and subsequent deacetylation of LC3 . Recently, in an effort to enhance the bioavailability to attain therapeutic benefits, resveratrol was prepared with β-CD; this combination was found synergistic in showing activity at very low concentrations to prevent α-syn aggregation as well as disaggregate preformed fibrils (Gautam et al., 2017). Resveratrol treatment reduced α-syn oligomers in S1/S2 transfected human H4 neuroglioma cells by activating peroxisome proliferator-activated receptor γ (PPARγ), which regulates energy metabolism and mitochondrial biogenesis, and plays a role in the pathogenesis of PD (Eschbach et al., 2015). At the molecular level, resveratrol downregulates α-syn expression mediating miR-214 in the MPTP-induced mouse model of PD and MPP + induced neurotoxicity in neuroblastoma cells (Wang et al., 2015a).
The phytochemicals that inhibit fibrils and oligomer formation along with the ability to stabilize the α-syn oligomers or disaggregate α-syn oligomers can be potential compounds for pharmaceutical development. The in vitro data reveals success with many phytochemicals in ameliorating the fibrils and oligomer formation of α-syn as well as inducing degradation of α-syn and promoting autophagy. However, many polyphenolic compounds showed difficulty in crossing the blood brain barrier due to their non-lipophilic nature. Therefore, they may not attain the required concentration to exert effects in the brain (Pandareesh et al., 2015;Pogacnik et al., 2016). Several factors, such as stability, solubility in an acidic environment at gastric pH, absorption pattern, gut microflora, enterohepatic circulation, first pass metabolism, and metabolic pattern either phase I or phase II play a key role in achieving the ideal bioavailability of the phytochemicals in the brain (Scholz and Williamson, 2007). Additionally, the inconsistency between the in vitro concentration and in vivo dose in certain models encourages systematic pharmacokinetic evaluations to understand the variation between the in vitro and in vivo data.

EXPERIMENTAL TECHNIQUES TO ASSESS THE α-SYN INHIBITORY ACTIVITY OF PHYTOCHEMICALS
Several biophysical and biochemical techniques used to assess the ability of phytochemicals and plant extracts in preventing α-syn oligomerization and fibrillation are represented in Table 6. These experimental techniques include surface plasmon resonance imaging (SPRi), Thioflavin-T (ThT) fluorescence, transmission electron microscopy (TEM), small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy, fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and absorption spectroscopy of Congo red (CR) binding assay (Luk et al., 2007;Kostka et al., 2008;Celej et al., 2009;Yamaguchi et al., 2010;Giehm et al., 2011;da Silva et al., 2013;Aelvoet et al., 2014;Coelho-Cerqueira et al., 2014;Cheng et al., 2015;Fazili and Naeem, 2015;Takahashi et al., 2015;Pujols et al., 2017;Das et al., 2018). The biochemical and biophysical assays employed to measure the α-syn aggregation are efficient in providing a high-resolution structure of α-syn oligomers, but not free from the restrictions and misconceptions on their efficacy (Coelho-Cerqueira et al., 2014). The polyphenolic nature of phytochemicals may cause some variations in interferences in spectrophotometric and fluorescent assays used to measure αsyn formation (Coelho-Cerqueira et al., 2014). Coelho-Cerqueira et al. (2014) show the drawbacks related to the application of ThT assays to examine α-syn fibrillation as ThT reacts with disordered α-syn monomer and augments protein fibrillation in vitro. As a result, phytochemicals may also bias the ThT assay and ambiguous results may be interpreted from the application of ThT based real-time assays in the screening of anti-fibrillogenic compounds. Therefore, a battery of techniques is recommended to support or confirm the anti-aggregatory and anti-fibrillogenic activity in side-stepping the possible artifacts associated with the measure of ThT fluorescence (Coelho-Cerqueira et al., 2014).
The knowledge of the stage, form, nature, and propagation of α-syn that play a key role in enhanced toxicity in PD highlights the multitude of techniques needed to gauge α-syn disposition in experimental situations (Giehm et al., 2011;Pujols et al., 2017). Recently, Moree et al. (2015) developed a novel assay to recognize compounds that control the conformation of monomeric αsyn in a direct manner to reduce the encounters associated with conventional small molecule screening of α-syn. This novel assay may aid in understanding the role of α-syn oligomers in PD and opens new avenues to evaluate α-syn-based potential neuroprotective agents.

EXPERIMENTAL MODELS FOR SCREENING AGENTS TARGETING α-SYN TOXICITY
Apart from biophysical and biochemical assays, numerous models including cell lines (in vitro) and animal models (in vivo) have been developed to study the role of α-syn in the etiology and disease modification of PD and evaluate test compounds against α-syn (Skibinski and Finkbeiner, 2011;Javed et al., 2016;Visanji et al., 2016;Ko and Bezard, 2017;Lázaro et al., 2017). The experimental models showing α-syn fibrillation, oligomerization, and neurotoxicity are summarized in Table 7. The toxicant models and mutation-based in vivo models are popularly used to mimic sporadic and familial PD, respectively (Javed et al., 2016). The experimental models often present issues and challenges in separating the complexities of cellular and molecular mechanisms and are infeasible for highthroughput screening and other drug development stages, such as dose-response toxicology studies. Cell-based models including stem cells and primary neurons with features of dopaminergic neurons give important insights into the cellular mechanisms of PD for drug discovery (Lázaro et al., 2017). This enables the recognition of agents targeting α-syn and their molecular mechanisms. Some of the prominent human cell models for PD drug screening are SK-N-SH, SHSY5Y, and SK-N-MC (Skibinski and Finkbeiner, 2011;Lázaro et al., 2017). The development of α-syn-based experimental models of sporadic or familial PD that show progressive forms of the disease will elucidate the mechanisms of neurodegeneration and aid in the identification of phytochemicals modulating α-syn. The uptake of recombinant α-syn from the culture medium has been reported in many cellular models (Reyes et al., 2015). Recently, Reyes et al. (2015) established a culture system that is a physiologically appropriate assay for the characterization of genetic modifiers or small molecules which prevent cell-to-cell transfer or propagation of α-syn.

CONCLUDING REMARKS AND FUTURE PROSPECTS
This comprehensive review presents an overview of the plant extracts and phytochemicals specifically targeting α-syn oligomerization, fibrillation, and aggregation in different models of PD and their underlying mechanisms. It also discusses the experimental techniques and models used to evaluate the plant extracts and phytochemicals. The literature review suggests that many phytochemicals are promising in targeting α-syn in the in vitro studies; however, the actions observed in vitro need to be reconfirmed in vivo. Indeed, the screening of phytochemicals or plant extracts in cell lines often lacks clinical applicability due to physiological, biochemical, and pharmacological relevancy. The available literature from a convincing number of in vitro studies and few in vivo studies demonstrates that phytochemicals, such as baicalein, curcumin, resveratrol, and epigallocatechin gallate have promising therapeutic potential in inhibiting α-syn oligomerization, fibrillation, aggregation, and accumulation. All these promising compounds should be studied in the in vivo studies to proceed further for clinical studies and thereon.
The process of α-syn oligomerization and fibrillation were well-recognized but the triggers that induce α-syn aggregation are not yet well-established. Thus, for a fair translational disease modifying approach, evaluation of phytochemicals in animal models involving α-syn aggregation and mimicking the progressive nature of PD pathogenesis is desired as a proof of concept. Although the available preclinical studies are encouraging, they are markedly speculative for clinical success. The issues, such as bioavailability, stability, metabolism, as well as long-term safety and toxicity, should be resolved before pharmaceutical development and further testing in humans. Based on the available preclinical studies, it can be concluded that these phytochemicals could possibly be novel drug candidates for neurodegenerative diseases, such as PD.

AUTHOR CONTRIBUTIONS
All the authors provided important intellectual content, reviewed the content and approved the final version of this manuscript. AA and SO conceptualized the idea for this review. MFNM, SA, HJ, and SO performed the literature search. HJ and SO wrote the first draft of the manuscript. SO and AA thoroughly revised and edited the manuscript. BS drew the chemical structures.