Natural Products as Novel Neuroprotective Agents; Computational Predictions of the Molecular Targets, ADME Properties, and Safety Profile

Neurodegenerative diseases (NDs) are one of the most challenging public health issues. Despite tremendous advances in our understanding of NDs, little progress has been made in establishing effective treatments. Natural products may have enormous potential in preventing and treating NDs by targeting microglia; yet, there have been several clinical concerns about their usage, primarily due to a lack of scientific evidence for their efficacy, molecular targets, physicochemical properties, and safety. To solve this problem, the secondary bioactive metabolites derived from neuroprotective medicinal plants were identified and selected for computational predictions for anti-inflammatory activity, possible molecular targets, physicochemical properties, and safety evaluation using PASS online, Molinspiration, SwissADME, and ProTox-II, respectively. Most of the phytochemicals were active as anti-inflammatory agents as predicted using the PASS online webserver. Moreover, the molecular target predictions for some phytochemicals were similar to the reported experimental targets. Moreover, the phytochemicals that did not violate important physicochemical properties, including blood-brain barrier penetration, GI absorption, molecular weight, and lipophilicity, were selected for further safety evaluation. After screening 54 neuroprotective phytochemicals, our findings suggest that Aromatic-turmerone, Apocynin, and Matrine are the most promising compounds that could be considered when designing novel neuroprotective agents to treat neurodegenerative diseases via modulating microglial polarization.


Introduction
Once the body is exposed to damage caused by external or internal harmful stimuli, the immune system will defend against these threats and initiate the repairing process [1,2]. After recognition of foreign agents, inflammatory processes will begin where many inflammatory mediators are released, such as tumor necrosis factor-α (TNF-α), interleukins (ILs), leukotrienes, nitric oxide (NO), and prostaglandin E2 (PGE2), besides the activation of inflammatory pathways such as nuclear factor-kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase signal transducer and activator of transcription (JAK/STAT) to minimize the impending of the damage [1]. After that, inflammation resolution is mediated by reducing mediators' production, which leads to diluting the chemokine gradients and reducing the white blood cells (WBC) sensation at the site of disease (AD), Parkinson's disease (PD), and Multiple sclerosis (MS) by investigating their pharmacokinetic properties, predicting their biological targets, assessing their safety/toxicity profiles, and cytochrome enzyme inhibition using computational techniques.

Study Design
Below is the study design that involves several steps, as shown in Figure 1.

AD
The prevalence of AD greatly rises with age [28], and in 1997, approximately 2.32 million people in the United States had Alzheimer's disease, and by 2047, it is expected that 8.64 million individuals will be diagnosed with AD, resulting in a massive societal and economic burden [29]. Although no treatments are available to stabilize or reverse the neurodegenerative process, several palliative disease-modifying medicines are now in development with early clinical investigations [30]. Natural products are a viable treatment option. A wide range of phytochemical compounds and secondary bioactive metabolites has been studied pre-clinically and clinically to prevent and attenuate the multifactorial pathologies of AD (chemical structures are summarized in Figure 2  The prevalence of AD greatly rises with age [28], and in 1997, approximately 2.32 million people in the United States had Alzheimer's disease, and by 2047, it is expected that 8.64 million individuals will be diagnosed with AD, resulting in a massive societal and economic burden [29]. Although no treatments are available to stabilize or reverse the neurodegenerative process, several palliative disease-modifying medicines are now in development with early clinical investigations [30]. Natural products are a viable treatment option. A wide range of phytochemical compounds and secondary bioactive metabolites has been studied pre-clinically and clinically to prevent and attenuate the multifactorial pathologies of AD (chemical structures are summarized in Figure 2) via microglial modulation.
In the case of physiological conditions, microglia's number and functions are tightly regulated. Nonetheless, if stimuli bind to the pattern-recognition receptors [PRRs] on the surface of microglia [31], microglia will be over-activated to respond to the insult through shifting into different functional states, modifying its proliferation, morphology, phagocytic activity, antigen presentation, and the production of inflammatory markers such as cytokines and chemokines [32]. The process involves a diverse set of signaling pathways, including but not limited to tumor necrosis factors (TNFs), interferons (IFNs), chemokines, colony-stimulating factors (CSFs), and interleukins (ILs) [33]. This sustained over-activation of microglia has been observed in various neurodegenerative diseases, and targeting these pathways is one of the proposed mechanisms of multiple phytochemical compounds, as discussed in detail below. In the case of physiological conditions, microglia's number and functions are tightly regulated. Nonetheless, if stimuli bind to the pattern-recognition receptors [PRRs] on the surface of microglia [31], microglia will be over-activated to respond to the insult through shifting into different functional states, modifying its proliferation, morphology, phagocytic activity, antigen presentation, and the production of inflammatory markers such as cytokines and chemokines [32]. The process involves a diverse set of signaling pathways, including but not limited to tumor necrosis factors (TNFs), interferons (IFNs), chemokines, colony-stimulating factors (CSFs), and interleukins (ILs) [33]. This sustained overactivation of microglia has been observed in various neurodegenerative diseases, and targeting these pathways is one of the proposed mechanisms of multiple phytochemical compounds, as discussed in detail below.

Pattern Recognition Receptors (PRRs)
Pattern recognition receptors (PRRs) are present on the plasma membrane of microglia that are capable of detecting foreign bodies that stimulate microglia. PRR subfamilies that are predominantly expressed by microglia include toll-like receptors (TLR), inflammasome-forming nucleotide-binding oligomerization domain (nod)-like receptors (NLRs), triggering receptor expressed on myeloid cells (TREMs), and other receptors [34]. Inflammatory factors such as IL-1β, IL-6, TNF-α, ROS, and Cyclooxygenase-2 (COX-2) are produced due to the interaction between the ligand and PRR receptor, as well as boosting microglial phagocytic activity in the short term microglial activation. However, chronic activation will impair this protective mechanism and might exacerbate neurodegeneration [34]. TLR4 signaling pathways, for example, are activated in microglia during neuroinflammation, resulting in caspase-8 and caspase-3 activation, nuclear translocation of NF-κB, and expression of genes implicated in the inflammatory response; inhibiting TLR4 activation and signaling is thus a beneficial mechanism.
For instance, Eriodictyol, a natural flavonoid found in citrus fruits and peanuts, has been shown to alleviate neuroinflammation, amyloidogenesis, and memory impairment

Pattern Recognition Receptors (PRRs)
Pattern recognition receptors (PRRs) are present on the plasma membrane of microglia that are capable of detecting foreign bodies that stimulate microglia. PRR subfamilies that are predominantly expressed by microglia include toll-like receptors (TLR), inflammasomeforming nucleotide-binding oligomerization domain (nod)-like receptors (NLRs), triggering receptor expressed on myeloid cells (TREMs), and other receptors [34]. Inflammatory factors such as IL-1β, IL-6, TNF-α, ROS, and Cyclooxygenase-2 (COX-2) are produced due to the interaction between the ligand and PRR receptor, as well as boosting microglial phagocytic activity in the short term microglial activation. However, chronic activation will impair this protective mechanism and might exacerbate neurodegeneration [34]. TLR4 signaling pathways, for example, are activated in microglia during neuroinflammation, resulting in caspase-8 and caspase-3 activation, nuclear translocation of NF-κB, and expression of genes implicated in the inflammatory response; inhibiting TLR4 activation and signaling is thus a beneficial mechanism.
For instance, Eriodictyol, a natural flavonoid found in citrus fruits and peanuts, has been shown to alleviate neuroinflammation, amyloidogenesis, and memory impairment induced by Lipopolysaccharide (LPS) through many mechanisms, one of which is via inhibiting TLR4 activation [35]. Furthermore, NLRP3, which belongs to the NOD-like receptors (NLRs) family, is another target of Esculentoside A and Pterostilbene according to in-vitro models, where they inhibit the Aβ 1−42 induced NLRP3/caspase-1 inflammasome in BV-2 cells, as shown in Table 1 [36,37]. Transcription factors are proteins that are involved in the regulation of the expression of genes. NF-κB represents a family of transcription factors that control the expression of a variety of genes involved in cell death, inflammation, proliferation, and differentiation [78]. Multiple studies have revealed that NF-κB is activated in several NDs and engaged in microglia-mediated Aβ toxicity, making it one of the most important transcription factors for the expressions of pro-inflammatory cytokines [79]. The activation of NF-κB results in the phosphorylation of NF-κB inhibitor, IκB, via the IκB kinase (IKK) signalosome complex leading to transcription of pro-inflammatory mediators, such as iNOS, COX-2, TNF-α, and IL-1β [80,81] Therefore, inhibiting the NF-κB will suppress the release of these inflammatory markers, which is a mechanism of a variety of natural plants, such as Piperlongumine, Aromatic-turmerone, Oridonin, and Andrographolide, as demonstrated in pre-clinical studies that shown in Table 1. Epigallocatechin-3-gallate, a polyphenolic compound found in green tea, has been shown to suppress the expression of TNFα, Il-β, Il-6, and iNOS in Aβ-stimulated EOC 13.31 mouse immortalized microglial cells [49]. It is worth noting that a phase III clinical trial for Epigallocatechin-3-gallate is being conducted to treat the early stages of Alzheimer's disease; however, the results have not yet been published [82].
Moreover, signal transducer and activator of transcription (STATs), another family of the transcription factors that expressed and mediated various functions, including proliferation, apoptosis, and differentiation in response to cytokines [83]. STAT1 is assumed to be a key signaling regulator via IFNs involved in innate immune responses, including type I and type II IFNs [84]. STAT3, on the other hand, mediates the cells' survival and proliferation of the IL-6 through regulating the expression of genes involved in the cell cycle and suppression of apoptosis [84]. STAT proteins are phosphorylated by the Janus kinase family, which includes JAK1, JAK2, and TYK2, causing them to translocate to the nucleus and stimulate transcription of their target genes. The abnormal activation of JAK/STAT signaling in innate immune cells has been linked to AD and MS [84].
Resveratrol, a naturally occurring dietary polyphenolic compound found in abundance in the skin of grapes and blueberries, reduced pro-inflammatory IL-6 and TNF-α production via inhibiting STAT1 and STAT3, as well as NF-κB pathways. Additionally, oral administration of Resveratrol suppressed microglial activity associated with the production of cortical amyloid plaques in a mouse model of cerebral amyloid deposition [45]. It is worth mentioning that Resveratrol has undergone a phase II clinical trial to investigate its beneficial role in delaying or altering the deterioration of memory and daily functioning in AD [85].
Activator protein-1 (AP-1) is also another transcription factor that regulates proinflammatory genes, including COX-2 and iNOS, and this signaling is inhibited by Sulforaphane, leading to reducing the expression of many inflammatory mediators and proinflammatory cytokines [47]. Indeed, multiple transcription factors are potential targets of herbal medicines as the mutations of transcription factors are one of the causes of neurodegenerative diseases, including AD.

Nuclear Receptors (NRs)
Nuclear Receptors are responsible for regulating microglia phenotypes by activating transcription factors such as Peroxisome proliferator-activated receptors (PPARs) and nuclear factor erythroid 2-related factor 2 (Nrf2) [86]. PPARs are a nuclear receptor family composed of three subtypes, one of which is PPARγ, which suppresses the expression of pro-inflammatory mediators such as TNF-α, IL-6, IL-1β, and IL-12 while also promoting the production of anti-inflammatory cytokines such as TGF-β and IL-10 [87]. PPARγ agonists, such as β-caryophyllene and Curcumin, have been shown in pre-clinical trials to alter microglia polarization to the M2 phenotype, as shown in Table 1. Moreoever, it is worth mentioning that Curcumin has been clinically studied. Phase II clinical trials were carried out, one for treating patients with mild to moderate Alzheimer's disease [88] and the other for studying the combination of Curcumin and Ginkgo for treating mild to severe dementia [89]. The beneficial effects of PPARγ agonists are proposed to be due to the suppression of microglial pro-inflammatory activity as well as the promotion of their phagocytic activity [90,91].
In addition, Nrf2 is a nuclear receptor that governs antioxidant responses initiated in oxidative damage, which is a feature of many neurodegenerative disorders [92]. Nrf2 expression in macrophages directly suppresses inflammation by blocking RNA polymerase II to IL-6 and TNF, as well as modulating antioxidative defense proteins such as heme oxygenase-1 (HO-1) [93]. As a result, Nrf2 activation is hypothesized to be involved in neuroprotection for Alzheimer's disease patients. An in-vitro study conducted by Yeon Seo, Ji et al. [52] showed that Andrographolide activates the Nrf2/Keap1-mediated HO-1 signaling pathway, leading to a decrease in the expression of iNOS and COX-2 in BV-2 cells [52].

Protein Kinases (PKs)
MAPKs are one of the most important kinase groups in inflammatory cells. They include Extracellular signal-regulated kinase (ERK 1/2 ), also known as p44/42 MAPK, and c-Jun N-terminal kinase (JNK), as well as p38 MAPK pathways [94]. Activation of these MAPK pathways causes phosphorylation of nuclear transcription factors and other cytoplasmic protein kinases, which results in increased expression of target inflammatory genes. For example, p38 MAPK activation via multiple pathways is necessary for the productions of IL-1, IL-6, TNF-α, COX-2, and iNOS, implying that p38 MAPK activity is associated with the hallmark lesions of Alzheimer's disease [94]. Hence, targeting these activations through suppressing phosphorylation of the proteins is a proposed mechanism of many herbal medicines, such as Curcumin and Aromatic-turmerone [38,41]. Furthermore, Silibinin, Triptolide, Xanthoceraside, and Eriodictyol are natural plants that have been studied in-vitro and in-vivo to treat AD by inhibiting different MAPK pathways, as summarized in Table 1.
Similarly, the mammalian target of rapamycin (mTOR) kinase, a member of the phosphatidylinositol 3-kinase-related kinase (PIKKs) protein kinase family, is implicated in the neuroinflammation process. mTOR activation will eventually result in the activation of the NF-κB signaling pathway. As a result, blocking mTOR can reduce microglial cell activation and enhance M2 phenotypic conversion. Paeoniflorin, a traditional Chinese herb, has been proven in a rat model to suppress the mTOR/NF-κB pro-inflammatory pathway [56].

Cytokines
Cytokines are small proteins that have a role in controlling innate and adaptive immune responses. They are also involved in cell growth, survival, differentiation, and activities regulation [95]. Various types of CNS cells, including tissue infiltrating immune cells, neurons, and astrocytes, have been identified as CNS cytokine sources. However, microglia appears to be a major source of both pro-inflammatory and immune-regulatory cytokines. Several cytokines and their receptors have been discovered to exist and function in the CNS. TNF-α, IFNs, ILs including IL-1, -2, -3, -4, -6, -10, -12, -15, and -18, TGFβ, and CSFs are some of them [96]. During CNS inflammation, microglia produce two main pro-inflammatory cytokines, IL-1 and TNF-α, which are involved in BBB disruption [97]. Thereby, inhibiting activation of microglia and attenuating production of pro-inflammatory and anti-inflammatory cytokines are proposed mechanisms of many phytochemical compounds to treat AD, as shown in Table 1. For example, Oridonin extracted from Rabdosia rubescens has been shown to reduce NO production as well as the attenuation of iNOS, IL-1β, and IL-6 expressions that are involved in the development of neuroinflammation and neurodegeneration [59]. Moreover, Luo et al. (2018) found that the administration of Paeoniflorin, derived from Paeonia lactiflora, inhibits the productions of IL-1β, IL-6, TNF-α, and NO, while upregulating IL-10 and TGF-β1, which promote the transition of M1 to M2 phenotypes in microglia [56].

PD
Parkinson's disease (PD) is a progressive age-related neurodegenerative condition characterized by resting tremors, muscle rigidity, bradykinesia, and postural reflex deficits [98]. There is scientific proof that oxidative stress, peptide misfolding, and the death of dopaminergic neurons in the substantia nigra pars compacta are the fundamental features of Parkinson's disease pathophysiology [99]. Although Levodopa is the gold standard for symptomatic management of Parkinson's disease, long-term usage has been linked to the development of dyskinesia. Besides that, there are no pharmacological options that provide neuroprotection or slow the onset of PD. As a result, more efforts are required to discover therapy methods that alter the course of PD progression as well as relieve symptoms [100]. Therefore, numerous studies on phytochemical compounds have been conducted to investigate secondary metabolites' efficacy and mechanisms in treating PD, some of which will be summarized in Figure 3 and addressed below.

Nuclear Receptors (NRs)
In PD patients, clinical trials with pioglitazone, a PPARγ agonist, have shown encouraging results [131]. Moreover, Macelignan is a plant-derived from Myristica fragrans that exhibits a PPARγ agonist activity and has been demonstrated to protect dopaminergic neurons [124]. Nrf2, a nuclear receptor that defends against oxidative stress and inflammatory process, is a target for Licochalcone E herb extracted from Glycyrrhiza inflata. Lico-E activates the Nrf2-antioxidant response element (ARE) system and up-regulates HO-1 [132].
Protein Kinases (PKs) Kim et al. (2019) [121] found that Galangin suppressed the phosphorylation of p38 MAPK and JNK pathways, which significantly reduced the production of NO, iNOS, and IL-1β [107]. Similarly, phytochemical compounds such as Biochanin A, Baicalein, Myricetin, Macelignan, and Ginsenoside Rg1, which are listed in Table 2, have also been shown in pre-clinical studies to treat PD via targeting MAPKs pathways. Further, suppressing the phosphorylation of ERK 1/2 is one of the mechanisms of Licochalcone A, according to invitro and in-vivo experiments in which the LPS-stimulated production of pro-inflammatory mediators and microglial activation was inhibited [121].

Cytokines
Growing evidence revealed that activation of microglia in the PD brain resulted in higher expression of pro-inflammatory cytokines, in which the productions of IL-1β, IL-6, and TNF-α were enhanced in activated microglia [133]. Several phytochemical compounds have been studied pre-clinically to treat PD, as shown in Table 2, and it has been noted that they exert their activity by inhibiting pro-inflammatory cytokines releases, such as Capsaicin and Icariin.

MS
Multiple Sclerosis (MS) is a chronic degenerative neuroinflammatory disease that affects the central nervous system (CNS) and manifests in a range of clinical presentations. It is characterized by immunological abnormalities that result in myelin degradation in grey and white matter plaques [134,135]. The neurological symptoms are associated with the visible inflammatory lesions made up of lesser amounts of microglia and other types of cells that are all involved in the demyelinating process.
Currently, there is no cure for MS; however, there are two available approaches for management. The first is known as disease-modifying drugs, which include recombinant interferon β-1a and β-1b (e.g., Avonex and Betaferon), in addition to glatiramer acetate [136]. These agents are used to prevent relapses and improve neuropsychological deficits by inhibiting gamma interferon and enhancing the production of anti-inflammatory cells [137,138]. The second approach involves utilizing γ-aminobutyric acid type B (GABA-B) receptor agonists (e.g., baclofen) and α2 adrenergic receptor agonists (e.g., tizanidine) to manage MS symptoms such as pain and spasticity, with moderate benefits [139,140]. Multiple research, on the other hand, has studied the role of bioactive metabolites ( Figure 4) as a therapeutic alternative for MS, which will be mentioned below.

Pattern Recognition Receptors (PRRs)
According to Peng H et al. (2016) [141], Dimethyl fumarate, the methyl ester of fumaric acid, is strongly suppressed NF-κB activation, besides other pathways, leading to a reduction of pro-inflammatory cytokines and chemokines production, which eventually improves the survival of oligodendrocytes and neurons [141]. It is worth mentioning that Dimethyl fumarate has been approved by the FDA to manage relapsing-remitting MS.

Nuclear Receptors (NRs)
Some natural plants have been studied to treat MS through activating Nrf2, which modulates the anti-oxidant stress response. As an example, Dimethyl fumarate, it has been reported that activation of Nrf2 receptor will lead to inhibit the phosphorylation of NF-κB signaling [142]. Moreover, Foresti et al. (2013) [143] identified Carnosol, a traditional medicine derived from Rosmarinus officinalis [Rosemary] and Salvia officinalis, to be a potent activator of the Nrf/Ho-1 pathway [143].

Protein Kinases (PKs)
18β-Glycyrrhe acid derived from Glycyrrhiza glabra is demonstrated by Zhou J. et al. (2015) [144] in a mice model to block the release of neurotoxic pro-inflammatory mediators induced by IFN-γ through inhibiting the phosphorylation of the MAPK pathways, ERK 1/2 and p38 in microglia [144].

Cytokines
Most of the natural plants proposed to treat MS share the inhibition of IFN-γ cytokines, which function as effector cells damaging CNS cells by phagocytosis and the release of cytotoxic substances such as glutamate, nitric oxide, superoxide, and pro-inflammatory cytokines [145]. As shown in Table 3, Cannabidiol, 3H-1,2-dithiole-3-thione, Oleanolic Acid, Astragaloside IV, and Glycyrrhizin are all compounds that have been studied and found to suppress IFN-γ.  [136]. These agents are used to prevent relapses and improve neuropsychological deficits by inhibiting gamma interferon and enhancing the production of anti-inflammatory cells [137,138]. The second approach involves utilizing γ-aminobutyric acid type B (GABA-B) receptor agonists (e.g., baclofen) and α2 adrenergic receptor agonists (e.g., tizanidine) to manage MS symptoms such as pain and spasticity, with moderate benefits [139,140]. Multiple research, on the other hand, has studied the role of bioactive metabolites ( Figure 4) as a therapeutic alternative for MS, which will be mentioned below.  Glycyrrhizin, a compound extracted from licorice root, was studied by Sun Y. et al. (2018) [146] who showed that glycyrrhizin had an anti-inflammatory effect against MS through suppressing microglial M1 activation via reducing TGF-β1, IFN-γ, TNF-α, IL-17A, and IL-6 cytokines while increasing IL-4 [146]. On the other hand, Sativex®[Nabiximols®], a derived mixture of delta-9-tetrahydrocannabinol and Cannabidiol, is an investigational product in Phase III for the spasticity and pain associated with MS in the US [147].

Target Prediction
We have investigated the possible targets of the bioactive metabolites of 54 plants using a Molinspiration webserver that predict the probability of the compound's activity as G protein-coupled receptors ligand, ion channel modulator, a kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor.

GPCR Ligand
G protein-coupled receptors (GPCRs) expressed by microglia had already been exhibited to regulate various aspects of their activation process, such as cell proliferation, migration, and differentiation into M1 or M2 phenotypes [160]. GPCRs, among these numerous different receptor types, play an important role in the modulation of different components of microglial activation. As a direct consequence, the involvement of GPCRs and their subtypes in neurological diseases has been implicated in many studies. Furthermore, many other unstudied GPCR subtypes are highlighted in microglial activation and need to be investigated for their potential therapeutic and molecular activity in Alzheimer's disease [161,162]. Several types of research have concluded that GPCRs are novel targets for treating neuropsychiatric illnesses such as anxiety, depression, and cognition in Alzheimer's disease, Parkinson's disease, Huntington's disease, and schizophrenia.
Cannabinoid receptor 2 (CB2R) is a subfamily of GPCRs found on cell membranes. Although CB2R is abundant on peripheral immune cells, it is only found in very small amounts in the normal brain, primarily in microglia [163]. Interestingly, Cheng Z et al. (2014) [58] Founded that β-Caryophyllene intragastric administration (48 mg/kg, for 10 weeks) to APP/PS1 rats might prevent cognitive impairments and reverse neurodegeneration [58]. This was linked to a reduction in microglial M1 activation and inflammatory cytokines via the CB2R and PPAR-pathway [58]. However, in the Molinspiration biological predictions, our results showed that β-caryophyllene is not active as GPCR with a result of -0.34, as shown in Table 4.
In-silico predictions suggested compounds Andrographolide, Cannabidiol, and Carnosol are active as GPCR-targeting. However, the reported studies have not investigated these possible targets suggesting further mechanistic studies are warranted.

Ion Channel Modulators
Microglial functions, including the proliferation, morphological alterations, migration, cytokine release, and reactive oxygen species generation, are all regulated by ion channels and transporters, which regulate ionic flux [164]. In microglial cells, ion channel expression is carefully controlled, with most ion channel types expressing differently depending on the cells' functional state. Even though microglia are non-excitable cells, the abundance of voltage-gated ion channels shows that they play an important role in both normal and pathological conditions. Inflammation in the brain is a hallmark of Alzheimer's disease, and multiple studies have shown that microglia can directly interact with neurons to cause inflammation [165].
As illustrated in Table 4, the findings of Resveratrol, Epigallocatechin-3-gallate, Andrographolide, Paeoniflorin, β-caryophyllene, Oridonin, Dihydromyricetin, Triptolide, Isobavachalcone, Tripchlorolide, Triptolide, Carnosol, and Tanshinone IIA suggest that these bioactive metabolites could modulate ion channels; however, inadequate published data is investigating phytochemical compounds as ion channel modulators.    As microglia ion channels are key regulators of microglial function and morphology. New evidence on the presence of specific ion channel localization on microglia and the possibility of enhanced ion channel expression in neurodegeneration may open up a new method for selectively targeting microglia and reducing the ongoing inflammatory process [166]. Among the six potential transient receptors (TRP) subfamilies, only the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin) are expressed in microglia [167]. Capsaicin, a TRPV1 agonist, has been demonstrated by Young C et al. (2017) [105] to be useful in treating Parkinson's disease. Using the in-vivo model, Capsaicin (0.5 mg/kg, i.p.) was found to restore nigrostriatal dopaminergic neurons in MPTP-injected mice, resulting in improved motor function. This, however, did not match our in-silico predictions as shown in Table 4 that Capsaicin had activity as Ion Channel Modulator with a score of −0.15 [105].
Despite the lack of studies that evaluate these natural products, the in-silico prediction illustrated that β-caryophyllene, Oridonin, and Tripchlorolide are considered ion channel modulators with the activity of 0.28, 0.27, and 0.24, respectively.

Kinase Inhibitors
Kinases have become attractive drug targets because they are involved in nearly all cellular activities, such as cell growth, survival, proliferation, differentiation, and metabolism, and dysregulation of their activity has been linked to a variety of diseases, including CNS disorders such as AD, PD, and MS [168].
Unfortunately, most of the compounds showed no activity as a kinase inhibitor. However,   [54] suggested that the Andrographolide suppressed NF-κB nuclear translocation by suppressing NF-κB phosphorylation in BV-2 cells, which were supported by our in-silico study [54]. Moreover, Leung et al. (2005) [169] studied the novel mechanism of inhibition of NF-κB DNA-binding activity by diterpenoids found in the compound Oridonin to treat inflammatory diseases [169]. However, the study did not find Oridonin to be active as a kinase inhibitor. Nevertheless, Oridonin works as a Nuclear Receptor Ligand and Enzyme Inhibitor based on Molinspiration biological predictions. Additionally, using the prediction analysis, only Epigallocatechin-3-gallate, Dihydromyricetin, Silibinin, Quercetin, Apigenin, Galangin, Baicalein, Myricetin, Myricitrin, and Nobiletin showed a good activity as kinase inhibitors. Moreover, Quercetin and Myricetin were the most active, with a score of 0.28 for both. Goldmann et al. demonstrate that 18β-Glycyrrhetinic Acid targeted the MAPK, but this did not represent our in-silico prediction [170].

Nuclear Receptor Ligand
Nuclear receptors have attracted a lot of attention in the last 10 years as prospective therapeutic targets for neurodegenerative diseases. Effective treatments for progressive neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, and ALS have eluded researchers for years, making non-traditional therapeutic targets like nuclear receptors an appealing alternative. The involvement of nuclear receptors in several neurodegenerative disorders, most notably Alzheimer's disease, has been studied extensively in mice models of disease and several therapeutic studies [86].
Zun-jing et al. (2016) [86] reported that Curcumin inhibited the NF-κB signaling pathway and reduced the production of pro-inflammatory mediators from M1 microglia by specifically targeting PPAR-γ which is a Nuclear Receptor, and this was obvious in the Molinspiration biological predictions with an activity of 0.12 [86]. Moreover, Cheng et al. (2014) [40] showed that β-caryophyllene intragastric treatment (48 mg/kg, for 10 weeks) to APP/PS1 mice could prevent cognitive decline and reverse neurodegeneration through the activation of the CB2R and PPAR-pathways. This correlates with the reduction in microglial M1 activation and inflammatory cytokines [40]. Interestingly, all these results were supported by the Molinspiration webserver. Moreover, as shown in Table 4, some of the data were favorable as a Nuclear Receptor ligand, especially for compound PD-4. The results of the Galangin matched those of Min-ji and his colleagues in their 2017 study in which authors suggest in LPS-stimulated BV-2 cells, Galangin is a well-known PPAR activator that inhibits M1 inflammatory responses and increases the Nrf2/CREB signaling pathway from 10 to 50 µM [58]. Additionally, Sativex®(Sativex-like combination of Phytocannabinoids) therapy alone exhibited potential results in TMEV-IDD (Theiler's murine encephalomyelitis virus-induced demyelinating disease) models as a modulatory drug for increasing microglia polarization to M2 phenotype to establish cytoprotective milieu. The therapeutic effects of Sativex may be due to (tetrahydrocannabinol-botanical drug substance) THC-induced upregulation of both CB1R and CB2R expression, as well as CBD-induced PPAR activation, and this matched the in-silico of Cannabidiol which showed a good activity (0.38) as nuclear receptor ligand [171]. Furthermore, compounds Andrographolide, Oridonin, Oleanolic Acid, 18β-Glycyrrhetinic Acid, and Carnosol demonstrated high scores of 0.94, 0.73, 0.77, 0.79, and 0.51 as nuclear receptor ligand, respectively.

Protease Inhibitors
Gene transcription, the initiation process of precursor forms, and interactions with endogenous protease inhibitors are all mechanisms that closely regulate protease activity. Once activated, proteases can cause irreversible breakage of peptide bonds in various proteins. Some substrates are inactivated after cleavage, while others are activated to gain new functionalities. As a result, microglial proteases are thought to have both positive and negative effects. According to Table 4, only compounds Epigallocatechin-3-gallate, Andrographolide, Paeoniflorin, Oridonin, Dihydromyricetin, Silibinin, Triptolide, Tenuigenin, Isobavachalcone, Tripchlorolide, Triptolide, Naringin, Matrine, Oleanolic Acid, and Glycyrrhizin appear to have good activity as protease inhibitors. Defects in proteostasis are thought to be associated with various neurodegenerative disorders, including Parkinson's disease. While the proteasome fails to destroy large protein aggregates, such as alphasynuclein (α-SYN) in PD, drug-induced autophagy can effectively remove clusters and prevent dopaminergic neuron degeneration. As a result, maintaining these pathways is critical for preserving all cellular functions that rely on a properly folded proteome [172]. The Molinspiration analysis indicated that Tenuigenin, Isobavachalcone, Tripchlorolide, Triptolide, and Naringin act as Protease Inhibitors.

Enzyme Inhibitors
The aggregation of misfolded amyloid-β and hyperphosphorylated tau and α-synuclein are linked to the pathogenesis of AD and PD, respectively. To cure the diseases, multiple small molecules have been developed to regulate the aggregation pathways of these amyloid proteins. In addition to controlling the aggregation of amyloidogenic proteins, maintaining the levels of the proteins in the brain by amyloid degrading enzymes (ADE); neprilysin (NEP), insulin-degrading enzyme (IDE), asparagine endopeptidase (AEP), and ADAM10 is also essential to cure AD and PD. Therefore, numerous biological molecules and chemical agents have been investigated as either inducers or inhibitors against the levels and activities of amyloid degrading enzymes [173]. All the AD and PD compounds showed enzyme inhibitor activity except Aromatic-turmerone, Xanthoceraside, Esculentoside A. α-asarone, Apocynin, Icariin, Tanshinone I, Salvianolic acid B, Licochalcone E, and Ginsenoside Rg1.
Moreover, reactive oxygen species (ROS) possess a physiological role in various cellular regulation processes. Antioxidant enzyme therapy may be advantageous for treating MS as ROS scavengers may interfere at numerous levels during the formation of MS lesions [174].  Table 4. 3.3. Absorption, Distribution, Metabolism, and Excretion (ADME) ADME properties were predicted using SwissADME, an online web server. Furthermore, the BBB can prevent chemicals from entering the brain and acts as a natural barrier against numerous poisons and infected cells in the bloodstream, but it also restricts the uptake of diagnostic and therapeutic substances in the brain, diminishing therapeutic efficiency and targeted delivery, therefore, small (often less than 500 Da) and lipophilic compounds can effectively penetrate the BBB and enter the brain. Thus, as disease-targeting strategies molecular weight (MW), blood-brain barrier penetration (BBB), high solubility (logS), and P-glycoprotein substrate, all are essential characteristics of the drug to be promising as a neuroprotective molecule [175].

Molecular Weight (MW)
Considering Lipinski's rule limit of MW of 500 g/mol, all compounds were within the recommended range, which improves their chances to be absorbed orally in the gastrointestinal tract except for Hesperidin, Xanthoceraside, Esculentoside A, Icariin, Tenuigenin, Salvianolic acid B, Ginsenoside Rg1, Naringin, Astragaloside IV, and Glycyrrhizin, which have molecular weights of 610. 56

Blood-Brain Barrier (BBB) Permeability
All the studied compounds could not cross the blood-brain barrier (BBB) except for Aromatic-turmerone, Resveratrol, Pterostilbene, 4-O-methylhonokiol, Piperlongumine, Capsaicin, α-asarone, Apocynin, Tanshinone I, Licochalcone E, Licochalcone A, Macelignan, Cannabidiol, Matrine, Carnosol, and Tanshinone IIA. Moreover, these sixteen compounds possess an advantage of blood-brain barrier penetration that allows them to be used in treating neurodegenerative diseases and targeting microglia [177]. Furthermore, α-asarone is one of the most studied compounds to cross the blood-brain barrier in more than one scientific study as an effective treatment for Parkinson's disease. For example, according to Chinese medicine, Xiao et al. (2015) [178] showed that α-asarone had been used to treat dementia, amnesia, and stroke as an orifice-opening medicinal because of the adequate and appropriate BBB permeability [178]. Similarly, Carnosol can cross through the BBB and subsequently produce an anti-inflammatory effect on M1 microglia in the CNS, according to Xing . [158]

Solubility (Log S)
The aqueous solubility of substances that have a direct impact on oral absorption is referred to as Log S. Within the specified range (−6.5 to 0.5), all compounds demonstrated soluble to moderate solubility except for Nobiletin, Tanshinone I, Astragaloside IV, and Tanshinone IIA with log S values of −6.82, −6.91, and −6.71 which were poorly soluble.

Inhibition of the Cytochromes P450
Herbs can accelerate or decrease the expected activity of prescribed medication, resulting in undesired side effects or therapeutic failure. Herbal active components can dramatically affect a drug's pharmacokinetic and pharmacodynamic properties, raising concerns regarding herb-drug interactions. The inhibition or induction of cytochrome P450 (CYP450) has been proposed as one of the key mechanisms for herb-drug interactions. Thus, to evaluate the potential interactions between the bioactive metabolites of natural herbs and cytochrome P450 enzymes SwissADME webserver was utilized [179].

Organ Toxicity
During the development of new medicine, the most important consideration is always safety, which includes a variety of toxicities and adverse drug effects that should be assessed during the preclinical and clinical trial phases. Herein, we investigated the direct organ toxicity of bioactive metabolites using computational approaches [180].

Literature Search
A systematic search was conducted in databases such as PubMed, Google Scholar, and Science Direct to identify relevant studies using key-words such as Microglia, Neurodegenerative diseases, Alzheimer disease, Parkinson disease, Multiple sclerosis, M1, and M2, Neuroprotective, ADME, in-vitro, in-vivo, in-silico, clinical trial. The reported phytochemicals in the studies that demonstrated neuroprotective effects via microglia modulation in neurodegenerative diseases (AD, PD, and MS) were selected.

Computational Analysis
The 2D chemical structure of each bioactive constituent was drawn using Chemdraw, and the simplified molecular-input line-entry system (SMILES), was utilized to conduct the computational analysis. The following computational tools were used: PASS online, Molinspiration, SwissADME, and ProTox-II webservers.

PASS Online
The activity is predicted by finding similarities between the new compound chemical structure and a well-known biological active substrate in the database. The activity spectrum estimation algorithm uses a Bayesian method. The PASS prediction tool will predict the probability of active [Pa] to probability of inactive [Pi] ratio. According to leaveone-out cross-validation [LOO CV] estimation, the average prediction accuracy is around 95%. PASS prediction accuracy depends on detailed information on the biological activity spectrum for each molecule in the PASS training set, so the biological activity estimate is more accurate. The website ( www.way2drug.com, accessed on 25 May 2021) [181] can be accessed directly with the search term "PASS prediction" in multiple web browsers.

Molinspiration
Molinspiration (www.molinspiration.com, accessed on 26 December 2021) [182] is a free online tool that aids the internet chemistry community by calculating essential chemical characteristics and predicting bioactivity scores for the most important drug targets [GPCR ligands, kinase inhibitors, ion channel modulators, nuclear receptors]. A molecule with a bioactivity score greater than 0.00 is most likely to have significant biological activities, whereas values and scores less than −0.50 are considered inactive.

SwissADME
To enhance drug discovery, this webserver (www.swissadme.ch, accessed on 8 November 2021) [183] allows for computing physicochemical descriptors and estimating absorption, distribution, metabolism, and excretion [ADME] parameters, pharmacokinetic properties, druglike nature, and medicinal chemistry properties of one or more small molecules.

ProTox-II
ProTox-II (http://tox.charite.de/protox_II, accessed on 8 November 2021) [184] uses a total of 33 models based on molecular similarity, fragment propensities, most frequent features, and [fragment similarity-based CLUSTER cross-validation] machine learning to predict various toxicity endpoints like acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity. Toxicity classifications are determined using the globally harmonized system of classification of labeling of chemicals (GHS); toxic doses are frequently expressed as LD 50 values in milligrams per kilogram of body weight. The median lethal dose (LD 50 ) is the dose at which 50% of test subjects die after being exposed to a substance. The following are the classification and the (mg/kg) LD 50 values.

Conclusions and Future Directions
The reported biological activity of neuroprotective medicinal plants could result from the overall effects of several bioactive molecules on multiple targets that make it difficult to identify the specific biological activity of a phytochemical. Thus, in this study, we screened 54 phytochemicals that have been reported in-vitro and in-vivo to be neuroprotective against NDs, and several parameters important for drug design and development were evaluated.
One of the most crucial factors that limit the therapeutic applications of these phytochemicals for the treatment of NDs is the physicochemical properties. Thus, we have selected phytochemicals that exhibited a good pharmaceutical profile with 0 violation of the rule of five [ROF], and only 34 phytochemicals were selected. The second important criteria that were considered is the safety and toxicity profile; thus, phytochemicals classified as class 4 and above were chosen, and the selection included 27 phytochemicals that passed this criterion. Furthermore, since herb-drug interactions are as important as toxicity, we selected phytochemicals that exhibited no CYP enzymes inhibition, and phytochemicals are Aromatic-turmerone, Sulforaphane, Andrographolide, Piperlongumine, Apocynin, and 3H-1,2-dithiole-3-thione.
To conclude, natural products hold considerable promise for treating various NDs, even though numerous questions concerning their efficacy and safety remain unevaluated. After the screening of 54 phytochemicals with neuroprotective effects in microglia, we can draw a solid conclusion that Aromatic-turmerone, Sulforaphane, Andrographolide, Piperlongumine, Apocynin, and 3H-1,2-dithiole-3-thione are the most promising compounds that could be considered when designing novel biologically active anti-inflammatory agents to treat neurodegenerative diseases via targeting microglial polarization. These six compounds demonstrated excellent ADME properties, safety profile, and promising anti-inflammatory activity that could be utilized as lead compounds for further drug optimization and development.

Acknowledgments:
The authors want to express their sincerest gratitude to the College of Pharmacy (COP) at King Saud bin Abdulaziz University for Health Sciences (KSAU-HS) for their continued support.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.