Ginkgo Biloba and Long COVID: In Vivo and In Vitro Models for the Evaluation of Nanotherapeutic Efficacy

Coronavirus infections are neuroinvasive and can provoke injury to the central nervous system (CNS) and long-term illness consequences. They may be associated with inflammatory processes due to cellular oxidative stress and an imbalanced antioxidant system. The ability of phytochemicals with antioxidant and anti-inflammatory activities, such as Ginkgo biloba, to alleviate neurological complications and brain tissue damage has attracted strong ongoing interest in the neurotherapeutic management of long COVID. Ginkgo biloba leaf extract (EGb) contains several bioactive ingredients, e.g., bilobalide, quercetin, ginkgolides A–C, kaempferol, isorhamnetin, and luteolin. They have various pharmacological and medicinal effects, including memory and cognitive improvement. Ginkgo biloba, through its anti-apoptotic, antioxidant, and anti-inflammatory activities, impacts cognitive function and other illness conditions like those in long COVID. While preclinical research on the antioxidant therapies for neuroprotection has shown promising results, clinical translation remains slow due to several challenges (e.g., low drug bioavailability, limited half-life, instability, restricted delivery to target tissues, and poor antioxidant capacity). This review emphasizes the advantages of nanotherapies using nanoparticle drug delivery approaches to overcome these challenges. Various experimental techniques shed light on the molecular mechanisms underlying the oxidative stress response in the nervous system and help comprehend the pathophysiology of the neurological sequelae of SARS-CoV-2 infection. To develop novel therapeutic agents and drug delivery systems, several methods for mimicking oxidative stress conditions have been used (e.g., lipid peroxidation products, mitochondrial respiratory chain inhibitors, and models of ischemic brain damage). We hypothesize the beneficial effects of EGb in the neurotherapeutic management of long-term COVID-19 symptoms, evaluated using either in vitro cellular or in vivo animal models of oxidative stress.


Introduction
Long COVID involves continuous, long-term manifestations of the residual damages and sequelae of coronavirus infection caused by human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a large positive-stranded enveloped RNA virus that generally provokes respiratory diseases [1,2]. SARS-CoV-2 predominantly affects the respiratory system but can also invade the nervous system and cause multiple neurological disorders [3][4][5]. Patients at risk following COVID-19 infection may still exhibit a range of long-term neurologic and psychiatric disorders ( Figure 1) and may not fully recover several months post-infection. These sequelae include mild symptoms, such as headaches; extreme tiredness (fatigue); loss of smell, taste, or tactile sensing functions; cognitive impairment; depression; delirium; and psychosis [5]. More severe documented outcomes include cases of encephalitis, Guillain-Barre syndrome, and stroke [5]. These Due to their neuroinvasive nature, coronaviruses may invade the central nervous system (CNS), causing inflammation and demyelination [12]. According to recent studies, SARS-CoV-2 can reach the CNS in many different ways, including (i) the hematopoietic pathway and subsequent blood-brain barrier (BBB) rupture, (ii) blood-cerebrospinal fluid (B-CSF) distribution, (iii) transsynaptic viral spreading from the peripheral nerve, (iv) via circumventricular organs (CVOs), and (v) olfactory bulb penetration due to the interaction between the virus spike 1 (S1) protein and the angiotensin-converting enzyme 2 (ACE2) receptor [3,13,14]. The latter is widely expressed in neurons, oligodendrocytes, and astrocytes throughout the brain. Additional evidence of these entry pathways has been obtained using genome sequencing and via viral detection in the cerebrospinal fluid (CSF) of several patients [15]. The most frequent symptoms persisted after 3 and 12 months in 216 respondents from a cohort of suspected and confirmed COVID-19 cases. Adapted with permission from [11]. Copyright {2022} Science-HHS Public Access (PubMedCentral).
Due to their neuroinvasive nature, coronaviruses may invade the central nervous system (CNS), causing inflammation and demyelination [12]. According to recent studies, SARS-CoV-2 can reach the CNS in many different ways, including (i) the hematopoietic pathway and subsequent blood-brain barrier (BBB) rupture, (ii) blood-cerebrospinal fluid (B-CSF) distribution, (iii) transsynaptic viral spreading from the peripheral nerve, (iv) via circumventricular organs (CVOs), and (v) olfactory bulb penetration due to the interaction between the virus spike 1 (S1) protein and the angiotensin-converting enzyme 2 (ACE2) receptor [3,13,14]. The latter is widely expressed in neurons, oligodendrocytes, and astrocytes throughout the brain. Additional evidence of these entry pathways has been obtained using genome sequencing and via viral detection in the cerebrospinal fluid (CSF) of several patients [15].
It has been shown that EGb 761, a standardized extract that contains bilobalide, 24% flavone glycosides (quercetin, kaempferol, and isorhamnetin), and 6% terpenes, increases the synthesis of INF-γ, while reducing the release of pro-inflammatory cytokines in Figure 2. Primary bioactive compounds in Ginkgo biloba and their pharmacological effects (created with BioRender using information from [21,22]). Up and down arrows indicate the upregulation and downregulation of relevant biomarkers, respectively.
It has been shown that EGb 761, a standardized extract that contains bilobalide, 24% flavone glycosides (quercetin, kaempferol, and isorhamnetin), and 6% terpenes, increases the synthesis of INF-γ, while reducing the release of pro-inflammatory cytokines in pe-Pharmaceutics 2023, 15, 1562 4 of 34 ripheral blood leukocytes [22]. Docking simulations and inhibition kinetic studies have demonstrated that ginkgolic acids and bioflavonoids derived from Ginkgo biloba exhibit comparatively potent SARS-CoV-2 3CLpro inhibitory capabilities. Thus, several promising leading compounds have been identified for the advancement of antiviral medication research by targeting the 3CLpro enzyme [19]. However, there is currently limited scientific literature on the possible use of EGb nanotherapy for managing long-term neurological problems related to SARS-CoV-2 or an overview of several models to evaluate the therapeutic outcome of EGb. Recent studies on Ginkgo biloba have examined the efficiency of various EGb extracts against age-related disorders, including dementia, Alzheimer's disease, and mild cognitive impairment (MCI). These studies include those by Barbalho et al., Singh et al., and Tomino et al. [23][24][25]. Al-Kuraishy et al., however, discussed the use of EGb in managing COVID-19 severity but did not thoroughly review the effectiveness of EGb nanotherapies [26].
This review gives a comprehensive overview of the potential uses of Ginkgo biloba as an antiviral, anti-inflammatory, and antioxidant agent capable of regulating the neurological complications of SARS-CoV-2 infection. We summarize the reported findings of in vivo and in vitro models mimicking oxidative stress conditions and aiming at the discovery of novel nanotherapeutics based on Ginkgo biloba ingredients. We hypothesize that the presented results can be of interest to the neurotherapeutic management of long COVID.

Neuroinvasive and Neurotoxic Potential of Coronavirus Linked to Neurodegeneration
The CNS has been proven to be susceptible to viral infections [3,4,27,28]. A series of recent studies have demonstrated the neuroinvasive potential of SARS-CoV-2, like the previously established neurovirulence of human coronaviruses, such as SARS-CoV, MERS-CoV, HcoV-229E, and HcoV-OC43 [28][29][30][31][32]. According to Boroujeni et al., COVID-19 impacts the cerebral cortex of patients. This effect is characterized by activated microglia that amplify the inflammatory activation of astrocytes and is accompanied by low glutathione levels and upregulation of inflammation [33]. Palpagama et al. asserted that the activation of glial cells is involved in the pathology of neurodegenerative disorders [34]. The elevated number of such cells can be associated with neuroinflammation and brain tissue damage.
Other researchers have detected the presence of SARS-CoV-2 RNA in the cerebrospinal fluid after the virus infects the patient's CNS and causes meningitis and encephalitis [10,32,35,36]. Alternatively, evidence has revealed that 50% of patients with COVID-19 develop intestinal inflammation [37][38][39]. These results support the gut-driven inflammation hypothesis of Parkinson's disease (PD) pathogenesis, which starts in the intestines and advances through inflammation toward the CNS. The associated increased levels of α-synuclein initiate aggregation in the gut and brain [40]. Magnetic resonance imaging (MRI) data have also shown brain alteration in the cortical region (posterior gyrus rectus) that is associated with olfaction. This fact suggests that SARS-CoV-2 can invade the brain through the olfactory pathway and cause olfactory dysfunction, among other neurological disorders [41]. Additionally, olfaction-related brain changes in the posterior gyrus rectus of the brain have been observed using magnetic resonance imaging (MRI) [41]. This finding suggests that SARS-CoV-2 can enter the brain via the olfactory pathway and induce neurological diseases, such as olfactory impairment and other neurological conditions.
It is hypothesized that the neuroinvasive potential of SARS-CoV-2 plays a subordinate role in the pathogenesis of long COVID [4,[42][43][44]. The virus enters the CNS and the PNS via the engagement of hematogenous or transsynaptic pathways (through the nasal cavity or the bloodstream) and triggers neuroinflammation [45]. As shown in Figure 3, SARS-CoV-2 invades the host cells by binding to the ACE2 receptor with its spike (S) protein and then priming with the S protein through the activities of the transmembrane protease, serine 2 (TMPRSS2) [46]. ACE2 is highly expressed in the brain of humans and animals, notably in some brain locations, such as the choroid plexus and paraventricular nuclei of the thalamus and in non-neuron cells (mainly astrocytes and oligodendrocytes) as well as various vessel calibers in the frontal cortex [47]. The presence of ACE2 in primary human brain microvascular endothelial cells (hBMVECs) alleviates the ability of SARS-CoV-2 to compromise the activity of the BBB [47,48]. Coronavirus infections are primarily associated with cytokine production, inflammation, and apoptosis in accordance with the pathophysiological process of oxidative stress [49]. protease, serine 2 (TMPRSS2) [46]. ACE2 is highly expressed in the brain of humans and animals, notably in some brain locations, such as the choroid plexus and paraventricular nuclei of the thalamus and in non-neuron cells (mainly astrocytes and oligodendrocytes) as well as various vessel calibers in the frontal cortex [47]. The presence of ACE2 in primary human brain microvascular endothelial cells (hBMVECs) alleviates the ability of SARS-CoV-2 to compromise the activity of the BBB [47,48]. Coronavirus infections are primarily associated with cytokine production, inflammation, and apoptosis in accordance with the pathophysiological process of oxidative stress [49]. The hypothesized mechanism of coronavirus-induced neuronal damage via oxidative stress and mitochondria dysfunction. SARS-CoV-2 infection occurs when TMPRSS2 primes the spike proteins for proteolysis, allowing the binding to ACE2. The affinity interaction triggers the binding of Ang II to the angiotensin type 1 receptor (AT1R), activating NADPH oxidase. This leads to mitochondrial electron transport chain (ETC) damage via the release of oxidative and nitrosative species, subsequently increasing the formation of mitochondrial reactive oxygen species (mtROS). The signaling pathways mediated by mtROS trigger the production of inflammatory cytokines, which can compromise the blood-brain barrier, thus resulting in neuronal damage. Additionally, mtROS cause nuclear and mitochondrial damage, which prolongs mitochondrial dysfunction and encourages inflammatory senescence (created with BioRender).

Oxidative Stress and Redox Signaling, Players in SARS-CoV-2 Neurological Damage
It has been shown that Parkinson's disease and SARS-CoV-2 infection cause similar oxidative stress and the activation of nuclear factor kappa B (NF-κB) [18,50,51]. In addition, activating pro-inflammatory mediators, such as IL-1 and IL-6, can lead to amyloid beta (Aβ) deposition and accumulation, thus establishing a link between neuroinflammation and Alzheimer's disease [14,52,53]. The hypothesized mechanism of coronavirus-induced neuronal damage via oxidative stress and mitochondria dysfunction. SARS-CoV-2 infection occurs when TMPRSS2 primes the spike proteins for proteolysis, allowing the binding to ACE2. The affinity interaction triggers the binding of Ang II to the angiotensin type 1 receptor (AT1R), activating NADPH oxidase. This leads to mitochondrial electron transport chain (ETC) damage via the release of oxidative and nitrosative species, subsequently increasing the formation of mitochondrial reactive oxygen species (mtROS). The signaling pathways mediated by mtROS trigger the production of inflammatory cytokines, which can compromise the blood-brain barrier, thus resulting in neuronal damage. Additionally, mtROS cause nuclear and mitochondrial damage, which prolongs mitochondrial dysfunction and encourages inflammatory senescence (created with BioRender).

Oxidative Stress and Redox Signaling, Players in SARS-CoV-2 Neurological Damage
It has been shown that Parkinson's disease and SARS-CoV-2 infection cause similar oxidative stress and the activation of nuclear factor kappa B (NF-κB) [18,50,51]. In addition, activating pro-inflammatory mediators, such as IL-1 and IL-6, can lead to amyloid beta (Aβ) deposition and accumulation, thus establishing a link between neuroinflammation and Alzheimer's disease [14,52,53].
It has been demonstrated that the receptor-binding domain (RBD) of the SARS-CoV-2 spike 1 (S1) protein binds to Aβ and tau proteins and causes their aggregation [54]. Additionally, SARS-CoV-2 infection has been linked to hypoxia and decreased oxygen levels [53]. The mitochondria of brain cells may undergo a rise in anaerobic metabolism due to this process, which can increase the levels of lactic acid, lipid peroxides, and oxygen-free radi-cals and deplete the antioxidant system. Consequently, the BBB is compromised, which may result in CNS complications. [36,49].
In regard to the role of oxidative stress in chronic diseases, Aranda-Rivera et al. highlighted the importance of nuclear factor erythroid 2-related factor 2 (Nrf2), a ubiquitous protein (containing 605 amino acids) that can modulate cellular oxidative stress [55]. During oxidative stress, Keap1 undergoes conformational change due to the oxidation of residues Cys 226, Cys 613, and Cys 624 by electrophiles and oxidants [56]. This mechanism enables Nrf2 to escape ubiquitination, leading to its release into the nucleus and regulating the expression of a number of antioxidant and detoxifying enzymes, such as glutamate-cysteine ligase modifier (GCLM) subunits, heme oxygenase, NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione S-transferase, and glutathione peroxidase [57,58].
It should also be emphasized that viral infections, such as SARS-CoV-2, have adverse effects on antioxidant systems and have been linked to phenomena involving the inhibition of Nrf2 and activation of the NF-kB pathway in favor of inflammation and oxidative stress. Olagnier et al. showed that during SARS-CoV-2 infection, the Nrf2 pathway is repressed, leading to the downregulation of heme oxygenase 1 (HO-1) and NQO1 [59]. Therefore, several antioxidant enzymes that guard against oxidative stress, including glutathione peroxide, peroxiredoxin, thioredoxin reductase, and thioredoxin, are affected.
Numerous studies have shown that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway controls oxidative stress by activating the transcription factor FOXO3 and initiating the transcription of antioxidant proteins, such as SOD-2, peroxiredoxins (PRDXs) 3 and 5, which are found in the mitochondria, and catalase, which is found in the peroxisomes [60][61][62]. Peroxisome-proliferator-activated receptor coactivator 1 (PGC-1), the master biogenesis regulator that promotes the transcription of antioxidant enzymes, interacts with the FOXO3 transcription factor to control oxidative stress in the mitochondria [61]. However, research has revealed that the ability of PGC-1 to promote gluconeogenesis and fatty acid oxidation is inhibited by protein kinase Akt2/protein kinase B (PKB), which acts as an intermediary trigger of phosphorylation and inhibition [62].
NF-κB is another transcription factor that regulates stress responses. It is activated by the phosphorylation of I-κB, thanks to the I-κB kinase (IKK) complex. In vitro studies by Wu et al. have reported that sustained exposure of human lens epithelial cells (HLECs) to increasing doses of H 2 O 2 (50-100 µM) for 4 h attenuates the TNFα-induced degradation of I-κB, accompanied by the activation of NF-κB and proteasome activity by 50-80% [63]. The obtained data have also indicated that the activation of NF-κB is an essential phenomenon that enables cells to recover from oxidative stress.

In Vivo and In Vitro Models of Oxidative Stress
Over the years, various oxidative stress models have been developed to study the pathogenesis of neurodegenerative disorders and discover new strategies for developing therapeutic agents. Such experimental techniques include the use of lipid peroxidation products, endogenous antioxidant depletion, mitochondrial respiratory chain inhibitors, neurotoxic agents (e.g., rotenone and N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)), and ischemic brain damage models [51]. The proposed in vitro cellular models and in vivo animal models have shed light on the molecular mechanisms underlying oxidative stress responses in the nervous system, such as cell survival and cell death. Among neurotoxic chemical agents, 6-hydroxydopamine (6-OHDA) has been used to induce neurotoxicity in the dopaminergic nigrostriatal system by inhibiting the mitochondrial electron transport chain of complexes I and IV and accelerating neuronal degeneration [64][65][66].
6-OHDA has been regarded as an endogenous toxic factor in the pathogenesis of PD. The neurotoxin 6-OHDA induces excessive production and accumulation of ROS and, therefore, oxidative stress. In fact, 6-OHDA induces caspase-3 activation in the cells mediated by the Fas or mitochondrial pathways [67]. Indeed, it has been demonstrated that MTPT/6-OHDA-induced NF-κB activation in SH-SY5Y neuroblastoma cells triggers caspase-3 activation, which results in the death of DCNs via the NF-κB pathway [68][69][70].
The advantages of cellular systems for studying oxidative stress are their low cost, adaptability, modularity, reproducibility, compatibility with high-throughput screening, and interest in cell mechanical investigations without systemic interferences. Other studies have demonstrated that hypoxia can increase cells' susceptibility to oxidative stress. The hypoxia-reoxygenation model is a relevant in vitro model of oxidative stress, given that cellular hypoxia appears to be a crucial signal that activates transcriptional regulators, specifically hypoxia-inducible factor-1 (HIF-1) [74], nuclear factor kappa B (NF-κB) [75], activator protein 1 (AP-1) [76,77], and some mitogen-activated protein kinase (MAPK) signaling pathways, and induces cell death and necrosis [76,77].
Genetically derived models of neurodegenerative diseases are gaining considerable interest because they are excellent surrogates, providing intrinsic validity to genetically based models of degenerative disorders [78]. Scientific investigations have reported that knockout of genes, including PINK1, DJ-1, LRRK2, and LRRK1, in rats leads to agedependent neurodegeneration of the dopaminergic neurons of PD [78][79][80]. Moreover, mutation in the copper-zinc superoxide dismutase 1 (SOD1) gene has been associated with ALS, while the alteration of MAPT genes or progranulin is linked to frontotemporal dementia (FTD) [81]. Other studies have highlighted that mutations in amyloid precursor protein (APP), presenilin 1, and presenilin 2 (PSEN1/2) are the main causes of autosomal dominant early-onset AD [82,83].
It has been documented that invertebrates can also mimic endogenous-generated ROS. Such a model has been widely implemented in Drosophila melanogaster and Caenorhabditis elegans (C. elegans) [84,85]. The initial longevity mutant, known as age-1, which contains increased levels of both SOD1 and catalase, is arguably the best-studied mutant in the nematode C. elegans. The age-1 mutant exhibits more significant levels of both SOD1 and catalase enzymes because this gene, which encodes for phosphatidylinositol-3 kinase, confers a longer lifetime phenotype when silenced, along with improved resilience to several forms of stress [85][86][87].
The aforementioned findings indicate that oxidative-stress-based pharmaceutical therapies can slow aging and degeneration, as summarized in Table 1. Therefore, the developed in vivo and in vitro models of oxidative stress can be of interest for evaluating the efficacy of nanotherapeutic management of long COVID conditions.

Ginkgo Biloba Antioxidative and Anti-Inflammatory Effects
Ginkgo biloba (GB) is one of the medicinal plants that ameliorate capillary blood circulation, provide brain oxygenation, and thus improve age-related disorders. The ginkgo tree is monotypic and belongs to the class Ginkgoopsida, considered the oldest tree alive in the world (ginkgo species are from the Permian period, around 286-248 million years ago) [23]. The currently available herbal medicines based on Ginkgo biloba extract (EGb) are Tebonin ® and Tanakan ® , which are mostly standardized on ginkgo flavone glycosides and terpene lactones-EGb 761 ® [26]. The standardized extract of Ginkgo biloba leaves includes 6% terpenoids, of which 3.1% are ginkgolides A, B, C, and J and 2.9% are bilobalide. It contains 24% flavonoid glycosides, including quercetin, kaempferol, and isorhamnetin, and 5-10% organic acids ( Figure 2) [100]. Ginkgo biloba leaf extract is mentioned in the British Herbal Compendium as a treatment for mild-to-severe dementia, including Alzheimer's disease, and for the treatment of neurological symptoms attributed to loss of concentration and poor memory, confusion, depression, anxiety, vertigo, tinnitus, and headache [101].
The neuroprotective effect of EGb 761 extract has been examined in a rat model of cerebral injury following ischemia/reperfusion (I/R). The treatment resulted in a decrease in MDA levels, the downregulation of pro-inflammatory cytokines (TNF-α and IL-1β), and an increase in the expression of anti-inflammatory cytokines (IL-10) and enzymatic SOD and myeloperoxidase (MPO) activities, which can control neurological impairments [102]. It can be suggested that the beneficial effects of EGb on cerebral ischemia/reperfusion I/R injury result from the reduction in oxidative stress due to the inhibition of nitric oxide production and inflammation induced by I/R [102].
Kaempferol is one of the most important constituents of Ginkgo biloba, and its action accounts for the upregulation of the glutamate-cysteine ligase catalytic (GCLC) subunit, brainderived neurotrophic factor (BDNF), B-cell lymphoma protein 2 (Bcl-2), and GSH [103][104][105]. Kaempferol inhibits ROS generation by scavenging free radicals and efficiently protects neuronal cells from oxidative injury. Additionally, it inhibits the production of pro-apoptotic proteins, including Bax and caspase-3, and modulates the downregulation of the NF-κB pathway to exert anti-apoptotic effects [106]. A study by Zhou et al. showed that kaempferol can inhibit mitochondrial membrane transition (mPTP) opening and suppresses the release of cytochrome C via GSK-3β inhibition [107]. Kaempferol is also involved in the inhibition of serotonin breakdown by monoamine oxidase, reduces neurotoxicity induced by 3-nitropropionic acid (3-NP), and induces the upregulation of heme oxygenase 1 (HMOX-1) [108].
Quercetin, bilobalide, and isorhamnetin are other essential compounds in Ginkgo biloba extracts. Bilobalide decreases the expression of reactive species induced through H 2 O 2 , thus inhibiting ER stress [109]. It can also suppress pro-inflammatory activation, NF-κB, and COX-2 activities [110]. Studies have reported the beneficial effects of bilobalide in the upregulation of c-myc and p53 proteins, inhibition of the degradation of membrane phospholipids, and increased cellular proliferation of neurons in the hippocampus [109,111,112]. According to Wang et al., pretreatment with bilobalide substantially reduced COX-2, iNOS, and phosphorylated p65 in sepsis-induced CLP mouse models, while inducing I-kB activation in the lungs [111]. Additionally, bilobalide reduced oxidative stress by increasing HO-1 expression in lung tissues and antioxidative enzyme genes, including catalase, MnSOD, CuZnSOD, and GPx-1 [111]. Moreover, bilobalide and EGb50 can modulate the expression of TLR4, NF-B, and MyD88, preventing the onset of acute lung injury (ALI) [111,113,114]. Ginkgo biloba components may therefore prevent the onset of ALI and the cytokine storm syndrome in COVID-19 by inhibiting pro-inflammatory signaling via the NF-κB and TLR4 signaling pathways. A study showed that the administration of EGb50 substantially lowers TNF-and IL-1 levels and prevents the related signal transduction through the p38 MAPK and NF-B p65 pathways in LPS-stimulated microglial cells [115]. In a separate study, EGb50 demonstrated a potential anti-inflammatory action by suppressing NLRP3-inflammasome-induced microglial activation [116]. For comparison, isorhamnetin has been linked with the inhibition of apoptosis and the suppression of DNA fragmentation [117].
In a recent study, Liu et al. compared the antioxidant capacity of various Ginkgo biloba extracts by evaluating the mechanisms of ginkgolides A (GA), B (GB), K (GK), and bilobalide (BB) against oxidative stress caused by transient focal cerebral ischemia [119].
In vivo studies have been performed in a developed middle cerebral artery occlusion (MCAO) model of cerebral ischemic injury using male SD rats, followed by reperfusion and Ginkgo biloba treatments [119]. Neuroblastoma cells (SH-SY5Y) were subjected to oxygenglucose deprivation (OGD) for 4 h, followed by 6 h of reoxygenation using ginkgolides and bilobalide. The in vitro experimental findings revealed that GA, GB, GK, and BB significantly reduce ROS and increase SOD activities and protein levels, including HO-1 and Nqo1. Additionally, p-Akt and p-Nrf2 levels considerably increased following ginkgolide and BB treatments, with GB demonstrating greater efficacy than GA and GK. These upregulations could be reduced in a dose-dependent manner by LY294002, a PI3K inhibitor. The triphenyl tetrazolium chloride (TTC) staining performed demonstrated that GB significantly reduced the infarct volume ratios in MCAO rats in a dose-dependent manner ( Figure 4a). Additionally, GB markedly increased the amounts of the proteins HO-1, Nqo1, SOD, p-Akt, p-Nrf2, and Nrf2 through the modulation of the Akt/Nrf2 signaling pathway, shielding neurons from oxidative-stress-related damage (Figure 4b) [119].  Antioxidant-related proteins in the MCAO group drastically decreased after cerebral ischemic injury compared to the normal group, while rats treated with various concentrations of GB showed a marked increase in the expression of HO-1, Nqo1, and SOD1. Data represented as the mean ± SD from eight rats of each group. (# p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the sham group; * p < 0.05 and *** p < 0.001 vs. the I/R group). Adapted with permission from [119]. Copyright {2019} Science-HHS Public Access (PubMedCentral). Table 2 summarizes the chemical structures of the active constituents identified in Ginkgo biloba extracts and their reported biological activities of potential interest to recovery from neurological long COVID syndrome. MCAO rats were treated with various GB doses for 72 h, followed by triphenyl tetrazolium chloride (TTC) staining and statistical analysis of the cerebral infarct area. The results revealed a significant reduction in infarct volume ratios after GB treatments in a dose-dependent manner. (B) Results of Western blot analysis and semiquantitative measurements of the levels of HO-1, Nqo1, and SOD1 proteins in the ischemic penumbra region of MCAO rats treated with various doses of Ginkgo biloba for 24 h. Antioxidant-related proteins in the MCAO group drastically decreased after cerebral ischemic injury compared to the normal group, while rats treated with various concentrations of GB showed a marked increase in the expression of HO-1, Nqo1, and SOD1. Data represented as the mean ± SD from eight rats of each group. (# p < 0.05, ## p < 0.01, and ### p < 0.001 vs. the sham group; * p < 0.05 and *** p < 0.001 vs. the I/R group). Adapted with permission from [119]. Copyright {2019} Science-HHS Public Access (PubMedCentral). Table 2 summarizes the chemical structures of the active constituents identified in Ginkgo biloba extracts and their reported biological activities of potential interest to recovery from neurological long COVID syndrome.

Neuroprotective, Anti-Apoptotic, and Anxiolytic Drug Effects of EGb
Results from microarray experiments have established the neuroprotective effect of Egb 761 against ischemic-induced neuronal injury [139]. The data revealed that the upregulation of Bcl-2 protein may be mediated by the activation of cAMP-responseelement-binding protein (CREB) [139]. EGb 761 increases CREB phosphorylation via the activation of PI3K/Akt and extracellular-signal-regulated kinase (ERK) signaling pathways [140]. The consequently released BNDF protects the neurons against ischemia. Moreover, Tchantchou et al. affirmed that EGb 761 can reduce Aβ oligomerization and promote neurogenesis by the phosphorylation of CREB. This was evidenced by enhanced cell proliferation in the hippocampus of TgAPP/PS1 mice [141]. Overall, these studies have demonstrated that flavonoids, the primary active constituents of EGb 761, may upregulate the CREB-BDNF pathway and therefore exert neuroprotection.
Combination therapy of EGb 761 with bone-marrow-derived mesenchymal stem cells (BMSCs) has shown a synergistic effect in animals with autoimmune encephalomyelitis. The therapeutic mechanism involves the inhibition of proinflammatory cytokines, demyelination, and protection axons and neurons [142]. Other studies have documented that GA prevents p-Tau deposition and thus protects cells from toxicity associated with Tau hyperphosphorylation. Interestingly, the degree of dementia is closely correlated with the production of hyperphosphorylated Tau aggregates, making EGb 761 crucial to counteract the neurodegenerative process [143].
Ginkgo biloba extracts, mainly flavonoids and ginkgolides, exert inhibitory effects on acetylcholinesterase activity. In fact, cholinergic agonists can reduce inflammation by blocking inflammatory signals, particularly the ubiquitous nuclear protein HMGB1 that is released by dying cells or activated innate immunity cells to promote inflammation [144]. It has been suggested that nicotinic receptors nAChRs may control the expression of ACE2 and serve as a binding receptor for S1 protein, leading to an inflammatory response. However, EGb may counteract the central inhibitory and anti-inflammatory effects of GABAergic neurons, leading to increased cortical neuronal activity and an increased risk of convulsion [145,146]. However, meta-analysis research has proven that there is no convulsion risk associated with the anxiolytic action of EGb, which is mediated through the regulation of GABAergic neurons [147]. In patients with dementia, EGb 761 ® has shown promise in alleviating comorbid neurosensory symptoms and improving memory deficits [147].
In another study, EGb 761 showed neuroprotective effects against oxidative-stressinduced apoptotic cell death by inhibiting apoptosis in a p53-dependent pathway, preventing mitochondrial membrane damage, reducing the release of cytochrome C from

Neuroprotective, Anti-Apoptotic, and Anxiolytic Drug Effects of EGb
Results from microarray experiments have established the neuroprotective effect of Egb 761 against ischemic-induced neuronal injury [139]. The data revealed that the upregulation of Bcl-2 protein may be mediated by the activation of cAMP-responseelement-binding protein (CREB) [139]. EGb 761 increases CREB phosphorylation via the activation of PI3K/Akt and extracellular-signal-regulated kinase (ERK) signaling pathways [140]. The consequently released BNDF protects the neurons against ischemia. Moreover, Tchantchou et al. affirmed that EGb 761 can reduce Aβ oligomerization and promote neurogenesis by the phosphorylation of CREB. This was evidenced by enhanced cell proliferation in the hippocampus of TgAPP/PS1 mice [141]. Overall, these studies have demonstrated that flavonoids, the primary active constituents of EGb 761, may upregulate the CREB-BDNF pathway and therefore exert neuroprotection.
Combination therapy of EGb 761 with bone-marrow-derived mesenchymal stem cells (BMSCs) has shown a synergistic effect in animals with autoimmune encephalomyelitis. The therapeutic mechanism involves the inhibition of proinflammatory cytokines, demyelination, and protection axons and neurons [142]. Other studies have documented that GA prevents p-Tau deposition and thus protects cells from toxicity associated with Tau hyperphosphorylation. Interestingly, the degree of dementia is closely correlated with the production of hyperphosphorylated Tau aggregates, making EGb 761 crucial to counteract the neurodegenerative process [143].
Ginkgo biloba extracts, mainly flavonoids and ginkgolides, exert inhibitory effects on acetylcholinesterase activity. In fact, cholinergic agonists can reduce inflammation by blocking inflammatory signals, particularly the ubiquitous nuclear protein HMGB1 that is released by dying cells or activated innate immunity cells to promote inflammation [144]. It has been suggested that nicotinic receptors nAChRs may control the expression of ACE2 and serve as a binding receptor for S1 protein, leading to an inflammatory response. However, EGb may counteract the central inhibitory and anti-inflammatory effects of GABAergic neurons, leading to increased cortical neuronal activity and an increased risk of convulsion [145,146]. However, meta-analysis research has proven that there is no convulsion risk associated with the anxiolytic action of EGb, which is mediated through the regulation of GABAergic neurons [147]. In patients with dementia, EGb 761 ® has shown promise in alleviating comorbid neurosensory symptoms and improving memory deficits [147].
In another study, EGb 761 showed neuroprotective effects against oxidative-stressinduced apoptotic cell death by inhibiting apoptosis in a p53-dependent pathway, preventing mitochondrial membrane damage, reducing the release of cytochrome C from

Neuroprotective, Anti-Apoptotic, and Anxiolytic Drug Effects of EGb
Results from microarray experiments have established the neuroprotective effect of Egb 761 against ischemic-induced neuronal injury [139]. The data revealed that the upregulation of Bcl-2 protein may be mediated by the activation of cAMP-responseelement-binding protein (CREB) [139]. EGb 761 increases CREB phosphorylation via the activation of PI3K/Akt and extracellular-signal-regulated kinase (ERK) signaling pathways [140]. The consequently released BNDF protects the neurons against ischemia. Moreover, Tchantchou et al. affirmed that EGb 761 can reduce Aβ oligomerization and promote neurogenesis by the phosphorylation of CREB. This was evidenced by enhanced cell proliferation in the hippocampus of TgAPP/PS1 mice [141]. Overall, these studies have demonstrated that flavonoids, the primary active constituents of EGb 761, may upregulate the CREB-BDNF pathway and therefore exert neuroprotection.
Combination therapy of EGb 761 with bone-marrow-derived mesenchymal stem cells (BMSCs) has shown a synergistic effect in animals with autoimmune encephalomyelitis. The therapeutic mechanism involves the inhibition of pro-inflammatory cytokines, demyelination, and protection axons and neurons [142]. Other studies have documented that GA prevents p-Tau deposition and thus protects cells from toxicity associated with Tau hyperphosphorylation. Interestingly, the degree of dementia is closely correlated with the production of hyperphosphorylated Tau aggregates, making EGb 761 crucial to counteract the neurodegenerative process [143].
Ginkgo biloba extracts, mainly flavonoids and ginkgolides, exert inhibitory effects on acetylcholinesterase activity. In fact, cholinergic agonists can reduce inflammation by blocking inflammatory signals, particularly the ubiquitous nuclear protein HMGB1 that is released by dying cells or activated innate immunity cells to promote inflammation [144]. It has been suggested that nicotinic receptors nAChRs may control the expression of ACE2 and serve as a binding receptor for S1 protein, leading to an inflammatory response. However, EGb may counteract the central inhibitory and anti-inflammatory effects of GABAergic neurons, leading to increased cortical neuronal activity and an increased risk of convulsion [145,146]. However, meta-analysis research has proven that there is no convulsion risk associated with the anxiolytic action of EGb, which is mediated through the regulation of GABAergic neurons [147]. In patients with dementia, EGb 761 ® has shown promise in alleviating comorbid neurosensory symptoms and improving memory deficits [147].
In another study, EGb 761 showed neuroprotective effects against oxidative-stressinduced apoptotic cell death by inhibiting apoptosis in a p53-dependent pathway, preventing mitochondrial membrane damage, reducing the release of cytochrome C from the mitochondria, upregulating the anti-apoptotic protein Bcl-2, and inhibiting PARP cleavage [148]. The anti-apoptotic effects are schematically presented in Figure 5. the mitochondria, upregulating the anti-apoptotic protein Bcl-2, and inhibiting PARP cleavage [148]. The anti-apoptotic effects are schematically presented in Figure 5. The work of Wang et al. showed that EGb may be used to increase cerebral blood flow and cognitive function by co-administration with 75 mg of aspirin toward the treatment of vascular cognitive impairment of non-dementia [149]. In a randomized, double-blind exploratory study, the authors demonstrated that administration of EGb (Symfona ® forte) at a dose of 120 mg/twice daily for at least 6 months may improve dualtask-related gait performance in patients with MCI [150]. Moreover, extensive work by Kuo et al. found that GA exhibits a strong therapeutic promise, like memantine, for treating AD by blocking NMDA and AMPA receptors. GA also suppresses c-Jun Nterminal kinase (JNK) activation at different doses (1-200 μM) in Aβ-induced neuronal depolarization in mice ( Figure 5) [151].
In parallel, Yu et al. demonstrated the neuroprotective effect of Ginkgo biloba dropping pills (GBDP) in the amelioration of PD [152]. In that study, the pharmacological effects of GBDP and EGb 761 were exploited in both in vivo and in vitro models of PD. The work of Wang et al. showed that EGb may be used to increase cerebral blood flow and cognitive function by co-administration with 75 mg of aspirin toward the treatment of vascular cognitive impairment of non-dementia [149]. In a randomized, double-blind exploratory study, the authors demonstrated that administration of EGb (Symfona ® forte) at a dose of 120 mg/twice daily for at least 6 months may improve dual-task-related gait performance in patients with MCI [150]. Moreover, extensive work by Kuo et al. found that GA exhibits a strong therapeutic promise, like memantine, for treating AD by blocking NMDA and AMPA receptors. GA also suppresses c-Jun N-terminal kinase (JNK) activation at different doses (1-200 µM) in Aβ-induced neuronal depolarization in mice ( Figure 5) [151].
In parallel, Yu et al. demonstrated the neuroprotective effect of Ginkgo biloba dropping pills (GBDP) in the amelioration of PD [152]. In that study, the pharmacological effects of GBDP and EGb 761 were exploited in both in vivo and in vitro models of PD. Following GBDP and EGb 761 treatments, the viability of DA neurons in zebrafish was assessed via tyrosine hydroxylase immunostaining. Dopaminergic neurons in zebrafish were significantly lost after exposure to 400 mM 6-OHDA for 48 h. Nevertheless, administration of 250 or 500 mg/mL of GBDP or 250 mg/mL of EGb 761 rescued the death of dopaminergic neurons induced with 6-OHDA. No protection was observed with further GBDP and EGb 761 dosages ( Figure 6A,B). Moreover, GBDP reduced cognitive impairment and neuronal damage in MPTP-induced PD mice and reversed the effect of 6-OHDAinduced dopaminergic neuronal loss in zebrafish ( Figure 6C). In vitro findings revealed that the neuroprotective effects of GBDP can be mediated through the Akt/GSK3β pathway ( Figure 6D,E) [152].  Figure 6A,B). Moreover, GBDP reduced cognitive impairment and neuronal damage in MPTP-induced PD mice and reversed the effect of 6-OHDA-induced dopaminergic neuronal loss in zebrafish ( Figure 6C). In vitro findings revealed that the neuroprotective effects of GBDP can be mediated through the Akt/GSK3β pathway ( Figure 6D,E) [152].   Adapted with permission from [152]. Copyright {2021} Science-HHS Public Access (PubMedCentral). Table 3 summarizes the reported neuroprotective effects of EGb. EGb-associated numerical non-significant trends found after 6 months for dual-task-related gait velocity and stride time variability [150]

Bioavailability and Safety of EGb
Acute toxicity studies have shown that EGb has a lethal dose (LD50) of 1.1 g/kg, 1.9 g/kg, and 7.73 g/kg in mice and 1.1 g/kg, 2.1 g/kg, and >10 g/kg in rats when administered intravenously, intraperitoneally, and orally, respectively [168,169]. In the older population, EGb is frequently used in the management of type 2 diabetes (T2DM), hypertension, or rheumatism. However, the interaction of EGb with other drug candidates can lead to bleeding. In a retrospective case series study, a 73-year-old man who had been taking 75 mg of a ginkgo supplement for 6 months as an aid to improve his memory deficits experienced episodes of spontaneous bleeding. The supplement included 27% ginkgo flavone glycosides and 10% terpene lactones [170]. A case report revealed that a 38-year-old lady who had been taking thiamine and Ginkgo biloba extract (240 mg/day) for 4 years experienced a brain hemorrhage [171]. Another study documented the case of a 70-year-old man with spontaneous iris bleeding (hyphema). This clinical occurrence took place 1 week after the patient started taking Ginkoba, a different natural Ginkgo biloba supplement (40 mg twice a day), in combination with 325 mg of aspirin every day. Ginkoba was withdrawn from therapy after monitoring the case for 3 months, and no further bleeding incidents were observed. Thus, it was proposed that the bleeding was caused by interactions between aspirin and gingko biloba extracts [172].
From the examples cited, research connecting Ginkgo biloba therapy with bleeding risk includes case reports. In addition, comprehensive analyses of randomized controlled studies have not identified any elevated risk of bleeding in patients using Ginkgo biloba extracts [173,174]. For example, in a prospective, double-blind, randomized, placebocontrolled trial, the administration of EGb 761 to young, healthy male volunteers at three different doses (120, 240, and 480 mg/day) had no effect on platelet function or coagulation [175]. Moreover, it has been reported that EGb 761 at a dosage of 120 mg/day inhibits platelet aggregation and thromboxane B2 synthesis [176]. In general, there is inconclusive evidence that Ginkgo biloba extracts are associated with bleeding. However, further research is necessary to resolve these controversies.

Perspectives for Use of Ginkgo Biloba in Nanotherapies of Neurological Disorders
Although preclinical studies of antioxidants to improve neuronal dysfunction have shown encouraging results, the outcomes of clinical trials have not always been conclusive. In general, antioxidant compounds have mainly suppressed clinical symptoms but are unable to halt or reverse disease progression [177]. In the absence of delivery systems, Ginkgo biloba (120 mg daily dosage) did not improve patient performance in neuropsychological tests of memory, attention, or speech during a 6-week, placebo-controlled, double-blind clinical study including 219 individuals [178]. Similarly, a feasibility investigation by Dodge et al. reported no difference in episodic memory deterioration in patients who received 240 mg/day of Ginkgo biloba or a placebo during an average follow-up period of 3.5 years [179]. The clinical translation of free antioxidant drugs may be hampered by several issues related to drug delivery efficacy. These problems include low drug bioavailability, low permeability through the CNS, limited half-life, and toxicity [180] (Figure 7). Significant efforts have been made to increase the clinical efficacy of both natural and synthetic antioxidants by using drug delivery systems to overcome their drawbacks. For example, investigations of drug conjugates, complexes, and nanocarriers of various polymeric materials have been performed [181][182][183].
for 4 years experienced a brain hemorrhage [171]. Another study documented the case of a 70-year-old man with spontaneous iris bleeding (hyphema). This clinical occurrence took place 1 week after the patient started taking Ginkoba, a different natural Ginkgo biloba supplement (40 mg twice a day), in combination with 325 mg of aspirin every day. Ginkoba was withdrawn from therapy after monitoring the case for 3 months, and no further bleeding incidents were observed. Thus, it was proposed that the bleeding was caused by interactions between aspirin and gingko biloba extracts [172].
From the examples cited, research connecting Ginkgo biloba therapy with bleeding risk includes case reports. In addition, comprehensive analyses of randomized controlled studies have not identified any elevated risk of bleeding in patients using Ginkgo biloba extracts [173,174]. For example, in a prospective, double-blind, randomized, placebocontrolled trial, the administration of EGb 761 to young, healthy male volunteers at three different doses (120,240, and 480 mg/day) had no effect on platelet function or coagulation [175]. Moreover, it has been reported that EGb 761 at a dosage of 120 mg/day inhibits platelet aggregation and thromboxane B2 synthesis [176]. In general, there is inconclusive evidence that Ginkgo biloba extracts are associated with bleeding. However, further research is necessary to resolve these controversies.

Perspectives for Use of Ginkgo Biloba in Nanotherapies of Neurological Disorders
Although preclinical studies of antioxidants to improve neuronal dysfunction have shown encouraging results, the outcomes of clinical trials have not always been conclusive. In general, antioxidant compounds have mainly suppressed clinical symptoms but are unable to halt or reverse disease progression [177]. In the absence of delivery systems, Ginkgo biloba (120 mg daily dosage) did not improve patient performance in neuropsychological tests of memory, attention, or speech during a 6-week, placebo-controlled, double-blind clinical study including 219 individuals [178]. Similarly, a feasibility investigation by Dodge et al. reported no difference in episodic memory deterioration in patients who received 240 mg/day of Ginkgo biloba or a placebo during an average follow-up period of 3.5 years [179]. The clinical translation of free antioxidant drugs may be hampered by several issues related to drug delivery efficacy. These problems include low drug bioavailability, low permeability through the CNS, limited half-life, and toxicity [180] (Figure 7). Significant efforts have been made to increase the clinical efficacy of both natural and synthetic antioxidants by using drug delivery systems to overcome their drawbacks. For example, investigations of drug conjugates, complexes, and nanocarriers of various polymeric materials have been performed [181][182][183].

Intranasal Administration and Biodistribution of Nanoparticulate Carriers
Delivering nanomedicines to the brain to treat CNS disorders is a major advantage of circumventing the BBB and reducing systemic exposure. Intranasal administration has attracted attention as a potential delivery mechanism to the brain for neuroprotection. The intranasal delivery method has been proposed for the treatment of CNS disorders (e.g., migraine, sleep disorders, brain tumors, multiple sclerosis, PD, and AD), thanks to its non-invasiveness and high patient compliance [184,[186][187][188]. Because the olfactory mucosa is in direct contact with the CNS, intranasal delivery of small and large molecules can successfully target the brain. This prevents the accumulation of drug molecules in vital organs, such as the liver, spleen, and kidney, which would lessen systemic adverse effects. Alternative delivery routes are via the lymphatic and vascular systems [189,190].
Nanotechnology has considerable potential for delivering therapeutic compounds to the brain through different mechanisms that allow blood-brain barrier passage. Nanoparticles (NPs) can improve drug solubility, increase the residence time of the active compounds at the target site, enhance their mucosal permeation and cellular internalization, regulate the release of encapsulated drugs, and lessen systemic side effects by limiting the distribution to non-targeted areas [189].
Wen et al. proposed nasal nanotherapy targeted to the brain with decreased immunogenicity with a drug delivery system. PEG-PLGA nanoparticles were modified with conventional lectin by combining a synthetic OL-conjugate PEG-PLGA (OL-PEG-PLGA) with PEG-PLGA [191]. For nanotherapy, odorranalectin (OL)-modified NPs were fabricated via the double-emulsion technique. A hemagglutination test was conducted to validate the biorecognitive activity of OL on the surface of the NPs. The results in Figure 8 show that OL-conjugated NPs possess hemagglutinating activity and demonstrate improved bioactivity. The nose-to-brain transport properties of the OL-conjugated NPs were examined using an in vivo fluorescent imaging approach with DiR dye as a tracer. The nanocarriers were co-administered with urocortin peptide via the intranasal route. The therapeutic efficacy of hemiparkinsonian rats was assessed using a rotation behavior test, a tyrosine hydroxylase test, and a neurotransmitter determination test. The obtained data suggested that the intranasal delivery of functionalized NPs to the brain enhances the therapeutic efficacy of nanodrugs in PD models [191].

Ginkgo-Biloba-Based Nanotherapy for Neuroprotection and Regeneration from SARS-CoV-2 Neurological Damage
In a recent study, Ginkgo-biloba-extract-loaded chitosan nanoparticles (Gb-CsNPs) were synthesized via an ionic gelation method. The outcomes revealed that the average size of the Gb-CsNPs was 104.4 nm, with a zeta potential of 29.3 mV and a polydispersity index (PDI) of 0.09. The encapsulation efficacy and drug-loading capacity were 40% and

Ginkgo-Biloba-Based Nanotherapy for Neuroprotection and Regeneration from SARS-CoV-2 Neurological Damage
In a recent study, Ginkgo-biloba-extract-loaded chitosan nanoparticles (Gb-CsNPs) were synthesized via an ionic gelation method. The outcomes revealed that the average size of the Gb-CsNPs was 104.4 nm, with a zeta potential of 29.3 mV and a polydispersity index (PDI) of 0.09. The encapsulation efficacy and drug-loading capacity were 40% and 97.4%, respectively. The neuroprotective efficacy of the Gb-CsNPs was examined in an oxidativestress-induced cellular model (SH-SY5Y). The results showed increased cell survival from 60% to 92.3%, proving the NPs' efficacy and biocompatibility [192]. Additionally, the encapsulation of EGb in chitosan NPs improved its neuroprotective properties [192].
Wang et al. developed Ginkgo biloba extract nanoparticles to enhance the oral bioavailability of GBE in Sprague-Dawley rats at a dosage of 40 mg kg −1 . The Cmax value of the flavonoids in raw GBE and GBE nanoparticles was reported to be 2.949 lg mL −1 at 0.5 h and 4.302 lg mL −1 in 0.333 h, respectively [193]. In a separate study, Zhao et al. demonstrated the ability of GBE NPs to transport across barriers, including the chorion, the GI barrier, the BRB, and the BBB. GBE was encapsulated by using poly(ethylene glycol)co-poly(ε-caprolactone) (PEG-PCL) nanoparticles. The developed nanoparticles facilitated the sustained release and enhanced brain uptake of GBE in the plasma of treated animals to treat PD [194].
Additionally, the delivery of quercetin across the BBB was achieved by using SLNs via intravenous administration to improve the therapeutic efficacy of this molecule [195]. The high-pressure homogenization procedure was used to successfully formulate SLNs loaded with Ginkgo biloba extract. The SLNs exhibited an appropriate particle size and shape, sustained the release profile, and improved the loading efficiency of the active substance [196].
In contrast, Xu et al. used an aqueous extract of Ginkgo biloba leaves to synthesize AgNPs with a mean particle size of 40.2 ± 1.2 nm, a polydispersity index of 0.091 ± 0.011, and a zeta potential of −34.56 mV [197]. In vitro results showed that EGb-AgNP treatment significantly increases intracellular ROS levels, facilitates cytochrome C release from the mitochondria into the cytosol, and facilitates caspase-9 and caspase-3 cleavage. This indicated that EGb-AgNPs can induce the activation of caspase-dependent mitochondrial apoptotic pathways, which are significant for various therapeutic applications [197].
As a potential nanotherapy for Parkinson's disease (PD), Wang et al. synthesized biodegradable poly(ethylene glycol)-b-poly(trimethylene carbonate) nanoparticles (PPNPs) to deliver ginkgolide B. This enhanced the accumulation of bioactive molecules in the blood and brain [198]. The fabricated GB-PPNPs effectively promoted the sustained release of ginkgolide B for 48 h. Moreover, the GB-PPNPs at various concentrations (50,100,200, and 400 µg/mL) prevented the neurotoxicity induced by MPP+ and protected zebrafish embryos or larvae, while decreasing the level of MDA protein expression in GB-PPNPtreated mice compared to MPTP-treated mice (Figure 9). Further research revealed that mice treated with GB-PPNPs had higher levels of SOD and GSH-Px than mice treated with MPTP. Additionally, GB-PPNPs elevated the concentration of DOPAC (10.66 ± 1.12, 1.65 ± 0.18 µg/g) and HVA (5.17 ± 0.60 µg/g). These values were significantly higher than those observed in the disease group [198].

Green Synthesis of Ginkgo Biloba nanoEGb
Ginkgo biloba is synthesized as nanoEGb using a variety of nanocarriers, such as liposomes, polymers, cyclodextrins, micelles, and carbon-based nanoconjugates, to address its limited water solubility and poor bioavailability and improve its half-life and retention time (Table 4) [199]. The poor bioavailability of EGb has been associated with the presence of diterpenoid molecules, particularly in ginkgolides A, B, and C [199]. The biosynthesis of Ginkgo biloba leaves has been realized using gold nanoparticles (AuNPs), Fe 3 O 4 magnetic nanoparticles (MNPs), and silver nanoparticles (AgNPs) [197,200,201]. This technique was demonstrated by Elshazly et al., who developed AgNPs using Ginkgo biloba extract. The nanoparticles exhibited a mean particle size ranging from 5.46 to 19.40 nm and an average diameter of 11.81 nm. In vitro experiments demonstrated that AgNPs have a moderate inhibition against MERS-CoV and HCoV-229E, which share similar sequence homology with SARS-CoV-2 [202]. Figure 10 demonstrates the potential uptake mechanism of an EGb nano-conjugate via transcytosis.  biosynthesis of Ginkgo biloba leaves has been realized using gold nanoparticles (AuNPs), Fe3O4 magnetic nanoparticles (MNPs), and silver nanoparticles (AgNPs) [197,200,201]. This technique was demonstrated by Elshazly et al., who developed AgNPs using Ginkgo biloba extract. The nanoparticles exhibited a mean particle size ranging from 5.46 to 19.40 nm and an average diameter of 11.81 nm. In vitro experiments demonstrated that AgNPs have a moderate inhibition against MERS-CoV and HCoV-229E, which share similar sequence homology with SARS-CoV-2 [202]. Figure 10 demonstrates the potential uptake mechanism of an EGb nano-conjugate via transcytosis.

Conclusions
It is indispensable to continue studying the mechanisms that underlie the pathophysiological process of SARS-CoV-2 infection. This will enable researchers to uncover the therapeutic targets that may be used for their management. According to this review, it may be suggested that Ginkgo biloba has potential positive effects, including anxiolytic, antineurotoxic, anti-inflammatory and anti-apoptotic functions, and has been explored in treating neurological disorders, particularly AD, PD, and dementia. Nevertheless, further studies are needed to corroborate the activity and mechanisms of action of this phytochemical since it could constitute an alternative for the treatment of vascular and degenerative diseases. Nanotechnology-based drug delivery systems could be an approach to address the limitations of antioxidant compounds, which include insufficient dosing, limited bioavailability, restricted transport to the CNS, transient retention, and low antioxidant capacity to completely scavenge the effect of ROS. The development of experimental techniques to mimic ROS has made it possible to study oxidative stress in the CNS. These methods will be fundamental for future discoveries related to the role of oxidative stress in neurological diseases.

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