Ginkgo biloba: A Leaf of Hope in the Fight Against Alzheimer’s Dementia: Clinical Trial Systematic Review

1. Introduction

Alzheimer's disease (AD) was first described in 1906 by the German psychiatrist Aloysius Alzheimer. This disease is an insidious and progressive neurodegenerative disorder that gradually deteriorates memory and cognitive functions [1]. Not only is it the leading cause of dementia in the elderly population worldwide, but it is also a major cause of disability. Typically, it displays the characteristic symptoms of being 75 years old, with around 4% of cases being early and severe, affecting language and visuospatial abilities associated with memory. Indeed, the manifestations and indications of AD extend beyond mere memory impairments, encompassing challenges in executing routine activities, disrupted sleep patterns, and even alterations in personality and mood [2,3,4,5,6,7]. Figure 1 illustrates various indicators and manifestations of AD, as well as lifestyle practices that can aid in the management of the condition.

Macroscopically, there is an irreversible loss of 15 to 35 percent of encephalic mass, with significant damage to the white matter, diffuse cortical atrophy, bilateral and symmetrical, and subsequent cerebral gyri narrowing and sulci enlargement affecting primarily the hippocampus. These abnormalities occur due to the accumulation of β-amyloid protein toxic plaques in the nervous tissue's extracellular matrix, hyperphosphorylated tau protein in neurofibrillary tangles, and apolipoprotein E, which is the main risk factor for the development of AD. These changes impair the number of neurons, dendritic branching, and synapse zones [3,8,9,10].

The aggregation of β-amyloid protein is considered to initiate all biochemical alterations in AD. It is a byproduct of the division (split) of amyloid precursor protein (APP), a membrane protein present in neurons. This reaction is catalyzed by three enzymes: alpha-secretase, beta-secretase, and gamma-secretase, each with a specific site of action on APP just above, slightly above, and within the phospholipid bilayer, respectively. The formation of B-amyloid protein toxic plaques is contingent upon the coordination of beta and gamma-secretases within the amyloidogenic pathway. Furthermore, tau protein, a microtubule-associated protein widely distributed inside axons that is responsible for retrograde and anterograde transport of substances, becomes hyperphosphorylated and undergoes a shift associated with the generation of malformed helical filaments, which can then be found inside neurons' soma and dendrites. These neurofibrillary tangles, as they are known, cause the nucleus of the parenchyma to be displaced, thereby preventing communication between parts of the cell and favoring their death and apoptosis [11,12,13,14,15,16] (Figure 2).

Figure 2. General mechanisms of damage in Alzheimer's Disease (AD). Many factors such as inflammation and free radicals can interfere with enzymes activation and the formation of βA plaques. This scenario leads to increase in inflammatory factors release and installation of oxidative stress leading to neurotoxicity and AD development. ↑ – increase; ↓ –decrease; APP – amyloid precursor protein; βA – β-amyloid.

Figure 2. General mechanisms of damage in Alzheimer's Disease (AD). Many factors such as inflammation and free radicals can interfere with enzymes activation and the formation of βA plaques. This scenario leads to increase in inflammatory factors release and installation of oxidative stress leading to neurotoxicity and AD development. ↑ – increase; ↓ –decrease; APP – amyloid precursor protein; βA – β-amyloid.

Traditionally, AD has been treated with drugs that relieve symptoms, such as cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) and N-methyl-D-aspartate receptor (NMDAR) antagonists (memantine). In AD, some neurons' acetylcholine secretion is significantly reduced, and NMDAR is greatly stimulated, promoting a deleterious calcium influx sufficient to cause cell death and synaptic dysfunction. Thus, it must be advantageous to prolong the action of the neurotransmitter's remaining molecules attached to cholinergic receptors by preventing recycling and reducing excessive NMDAR activation [17]. Despite this, a cure for AD remains elusive because current pharmacotherapy cannot prevent APP enzymatic cleavage, the first step in the pathogenesis [18]. Hence, alternative treatments may improve patients' quality of life with AD. Novel therapeutic opportunities for AD strategies include improving cognitive function and reducing amyloid deposition, opening up new treatment options, including monoclonal antibodies [6,19,20,21,22,23,24,25].

Herbal medicine's therapeutic strategies are one of the available options for alleviating symptoms [26]. Gingko biloba (GB) is one of the plants whose extracts can be used as an adjuvant in the treatment of AD [27,28]. GB is a gymnosperm from Japan, China, and Korea that belongs to the Ginkgoaceae family and the Ginkgoopsida class of plants. For centuries, it has been used in popular medicine to treat a variety of health problems due to the abundance of bioactive substances such as terpenoids (Ginkgolides A, B, and C), polyphenols, organic acids, and flavonoids (quercetin, kaempferol, and isorhamnetin), which are associated with anti-inflammatory, antioxidant, and antiapoptotic effects (Figure 2) [28,29]. Indeed, the standardized extract of GB (EGB 761) is a popular dietary supplement among the elderly to improve memory and prevent cognitive decline. These effects can be applied to Alzheimer's treatment.

In the Ginkgo Evaluation of Memory Study (GEMS) (a longitudinal analysis), blood amyloid levels and dementia risk were assessed. This study examined the levels of beta-amyloid protein and other substances, such as vitamin B12, liver enzymes, and creatinine, in patients who had their Apolipoprotein E genotyped. For 8.5 years, the authors studied baseline β-amyloid (Aβ) levels in plasma and incident dementia in 2840 individuals aged 75 and up. From this total, 2381 were classified as cognitively normal, while 450 had mild cognitive impairment. The study found that higher plasma levels of Aβ1-40 and Aβ1-42 were associated with age, gender (women), low education, stroke history, hypertension, and creatinine levels. Normal subjects with dementia had lower levels of Aβ1-42 and Aβ1-42/Aβ1-40 compared to those without dementia. Aβ levels did not predict dementia in individuals with mild cognitive impairment, which is noteworthy [30].

This study aims to address the current issues in treating AD and dementia, where the efficacy of GB remains uncertain. Some studies found significant improvements in cognitive function, neuropsychiatric symptoms, and functional abilities, whereas others found no significant difference between the GB-treated and placebo groups. Given these inconsistencies, the study's goal is to provide a comprehensive synthesis of existing clinical data, identify gaps in current research, and guide future studies. Furthermore, we set the objective to explore the potential of GB as an alternative treatment for AD and dementia, particularly considering the limitations and side effects of current AD medications. Through a systematic review and synthesis of clinical trials on the effects of GB extract/EGb 761® on dementia patients, this study examines the effectiveness of GB's bioactive compounds.

2. Methods

This systematic review aimed to investigate the potential benefits of GB for AD or other types of dementia. Only studies published in English were considered, sourced from MEDLINE–PubMed, EMBASE, and Cochrane databases. The search utilized MeSH terms including ‘Ginkgo biloba’ and ‘Alzheimer´s disease’ or ‘dementia,’ or cognition. These terms guided the identification of studies examining GB's effects on AD or dementia. Furthermore, we applied filters to enhance the quality of our search, namely "Clinical Trial" and "Randomized Controlled Trial". Consequently, conferences, abstracts, letters to editors, and other emerging sources were evaluated but not included. Study identification and inclusion were carried out by S.M.B. and M.T., with conflicts resolved by a third author, M.T. Inclusion criteria were limited to human interventional studies, while exclusion criteria comprised studies not in English, reviews, editorials, case reports, poster presentations. and studies with animal models. The search for clinical trials had no temporal restriction. Data extraction followed the PICO (Population, Intervention, Comparison, and Outcomes) format, and study selection adhered to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [31,32]. The risk of bias in selected trials was evaluated using the Cochrane Handbook for systematic reviews of interventions [33].

3. Overview of the Included Studies and Result Findings

Initially, 1,702 studies were identified according to the search terms. After applying the inclusion/exclusion criteria, we identified 15 clinical trials involving the use of GB in AD and dementia that met these criteria. Figure 2 shows the selection of the studies in accordance with PRISMA guidelines. Fifteen studies compared the outcomes of GB or GB extract treatment to a placebo. The dosage ranged from 120 to 240 mg, with treatment periods ranging from four to 24 weeks. Four studies found no significant differences between groups treated with GB and placebo. Eleven studies found that administering GB extract/EGb 761® improved cognitive function, neuropsychiatric symptoms, and functional abilities in both types of dementia. Significant differences were found in the Mini-Mental State Examination (MMSE), Short Cognitive Performance Test (SKT), and Neuropsychiatric Inventory (NPI) scores.

Figure 2. Flow diagram showing the study selection (PRISMA Guidelines) [31,32].

Figure 2. Flow diagram showing the study selection (PRISMA Guidelines) [31,32].

Table 1 lists the articles synthesized in this systematic review, while Table 2 displays the risk of bias assessments for the studies included. This systematic review reveal that GB can benefit Alzheimer's conditions.

4. Discussion

4.1. Ginkgo Biloba, General Aspects

Ginkgo, also known as GB, is a tree that is notable for its distinctive characteristics. The earliest documented records of the Ginkgo tree date back to the 11th century in the Yangtze River region of China. Despite this, it is now grown in a wide range of countries, including New Zealand, Brazil, the Netherlands, Chile, Japan, Belgium, Austria, England, Spain, Italy, Germany, France, Korea, Australia, and India, owing primarily to its ability to thrive in deep and sandy soils found in temperate and subtropical climate regions worldwide. In fact, Ginkgo is known for having a large genome and remarkable tolerance to various stress factors, whether abiotic or biotic; this species demonstrates remarkable resilience and adaptability in the face of adverse conditions, which contributes to its survival and longevity; additionally, it has the ability to cope with various forms of stress, whether caused by the physical environment or interactions with other organisms [49,50,51].

Its leaves, with their distinctive fan-shaped shape and green-yellow color, emit a strong odor. One notable feature of this tree is its impressive height, which ranges between 20 and 30 meters. Ginkgo is also well-known for its incredible longevity, which can last over 1,000 years. The leaves are divided into two distinct lobes, hence the botanical name "biloba". This unique feature makes the tree easily identifiable and distinct in its natural environment. Another interesting aspect is the Ginkgo's reproductive system. This species' trees have distinct male and female flowers, and instead of true seeds, the plant uses an outdated reproductive system that disperses "ovules."[49,52].

The first mention of the internal use of GB leaves for medicinal purposes appears in Wen-Tai's text "Ben Cao Pin Hue Jing Yaor," written in 1505 AD. This work provides information on the traditional uses of GB leaves. Over time, modern Chinese pharmacopeias recognized the therapeutic properties of these leaves, particularly in the treatment of heart and lung disorders [53,54].

GB contains several flavonoids, including quercetin, kaempferol, isorhamnetin, rutin, ginkgetina, and bilobetina. Terpenoids (ginkgolides) include ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J and ginkgolide M. Terpenoids (bilobalides) include bilobalide A and bilobalide B, as well as proanthocyanidins and organic acids like caffeic acid, ginkgolic acid, and quinic acid. The composition contains phytosterols, including β-sitosterol and stigmasterol. Figure 3 and Table 3 summarize the bio compounds composition of GB. The use of Ginkgo to improve symptoms in patients with oxidative inflammation-related diseases is thought to be associated with improved tissue perfusion and hypoxia tolerance. Ginkgo's flavonoid glycosides exhibit antioxidant and anti-inflammatory properties, which can help reduce endothelial cell damage caused by free radical oxidation [28,55,56,57,58]. Figure 4 shows the major bioactive components found in this plant, parts of the GB tree, and the chemical compounds classified by class and and Table 1 lists the chemical structure of bioactive compounds, parts of the GB tree, and their effects.

Tissue damage activates an inflammatory cascade, a natural response that includes complex molecular reactions and cellular responses. Lysosomal enzymes, such as phospholipase A2 (PLA2), are critical at the start of the cascade because they catalyze the hydrolysis of the sn2 position of membrane glycerophospholipids, resulting in 1-acylphospholipids and free fatty acids such as arachidonic acid. Cyclooxygenase (COX) and lipoxygenase (LOX) degrade arachidonic acid, producing prostaglandins, thromboxanes, leukotrienes, and lipoxins. These substances play a role in vasodilation, platelet aggregation, leukocyte chemotaxis, and monocyte adhesion. When activated excessively or persistently, inflammation can damage organs and systems, resulting in decompensation, organ dysfunction, and death [93,94,95,96].

Genetic links between inflammation-associated factors strengthen the link between inflammation and AD. The amyloid peptides of neuritic plaques and tau protein of neurofibrillary tangles are alterations resulting from post-translational modifications that affect multiple genes, making AD a polygenic neurodegenerative complex disease. β-amyloid accumulation and blood-brain barrier dysfunction contribute to neuroinflammation. Furthermore, neurodegenerative diseases have been linked to neuroinflammation and viral infections, with research indicating that exposure to common viral pathogens increases the risk of conditions such as AD [97,98,99].

The accumulation in the parenchyma and blood vessels induces microglial migration and promotes acute and chronic anti-aggregate inflammatory responses, resulting in the production of nitric oxide, reactive oxygen species (ROS), and proinflammatory cytokines such as Interleukin (IL)-1, IL-6, Tumor Necrosis Factor-α (TNF-α), and prostaglandins (PGE2). This can eventually promote neuronal death (Figure 2) [100,101,102,103,104].

GB has exceptional anti-inflammatory properties due to its unique composition of bioactive compounds. GbE (standardized GB extract) inhibits the secretion of proinflammatory mediators and cytokines, such as nitric oxide, PGE2, TNF-α, IL-6, and IL-1β (Figure 2). Ginkgolide A is the primary compound of GbE that can inhibit cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), preventing the production of inflammatory substances [56,105,106,107]. According to studies, GbE treatment significantly altered proteins involved in inflammation (mast cell protease-1, complement C3, T-kininogen 1) and oxidative stress (peroxiredoxin 1) (Figure 4). These proteins are related to the oxidation-reduction process, which is closely linked to inflammation [108,109]. GbE upregulates the expression of lactoylglutathione lyase (glyoxalase I), an enzyme that has anti-inflammatory properties. This enzyme is responsible for neutralizing methylglyoxal, a highly toxic compound produced during glycolysis and lipid peroxidation that can trigger inflammation [58,110].

GB inhibits the activation of nuclear factor kappa B (NF-κB), a transcription factor that activates genes that produce proinflammatory cytokines (Figure 2). This reduces the expression of these cytokines and inflammation mediators. By inhibiting NF-kB, GB also decreases the expression of adhesion molecules, which are proteins found on the surface of cells involved in the immune response and are crucial for guiding immune cells to inflamed areas. There are various types of adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and VCAM-1 (Vascular Cellular Adhesion Molecule 1), that are required for leukocyte adhesion to the surface of blood vessels and subsequent migration to inflammation. By decreasing the expression of these molecules, GB can reduce leukocyte adhesion and migration, resulting in a more controlled and less intense inflammatory response (Figure 2) [111,112,113,114,115,116].

GB has the ability to enhance the generation of nitric oxide by endothelial cells. This is attributed to its active compounds, such as flavonoids, which have the capacity to facilitate vasodilation. As a result, GB causes the relaxation of blood vessel walls, leading to an augmentation in blood flow. Furthermore, this plant also exhibits antiplatelet aggregation properties. These combined factors decrease the likelihood of inflammation caused by damage to the endothelium and the development of atherosclerotic plaques, thus safeguarding multiple systems in the human body. It is important to note that following an injury, GB strengthens blood vessel walls, reducing blood vessel permeability. This inhibits the movement of inflammatory cells into the surrounding tissues [117,118]. Another mechanism proposes that GB may enhance the expression of proteins such as Bcl-xL and Bcl-2. Promoting the production of these proteins increases cell survival because fewer undergo apoptosis, lowering the likelihood of inflammation being triggered (Figure 2). On the other hand, it can inhibit the activity of pro-apoptotic proteins like Bax, effectively blocking the pro-apoptotic signaling pathway [119,120].

4.2. Ginkgo Biloba and Antioxidant Effects

Oxidative stress is a complex pathological process that occurs in the body as a result of aging, infections, psychological stress, and exposure to radiation and chemical agents such as drugs and pollutants over a lifetime. Increased production of ROS and free radicals, which are reactive and attack various cellular components indiscriminately, is one of the damage mechanisms. They can interact with normal structures via electron donation/acceptance, hydrogen removal, addition reactions, self-annihilation, or disproportionation, resulting in irreversible cumulative injury if the endogenous antioxidant system is compromised or there is insufficient intake of exogenous plant-derived antioxidant substances (Figure 2). Therefore, oxidative stress has been linked to the onset of a variety of medical conditions, as well as the worsening of pre-existing pathologies [28,121,122,123,124].

It is important to note that the human body is constantly producing ROS, which are unavoidable byproducts of aerobic metabolism. In aerobic eukaryotic cells, cytochrome oxidase in the electron transport chain reduces more than 90% of oxygen to water via four-electron mechanisms that do not release ROS. The remainder produces superoxide (•O2^-) via the hydroperoxyl radical, while mitochondrial respiratory byproducts at 7.4 pH and NADPH oxidase catalyze the synthesis of hydrogen peroxide (H₂O₂). Superoxide can undergo a transformation into hydrogen peroxide. This hydrogen peroxide can then be converted into different reactive oxygen species (ROS) such as hydroxyl radicals (•OH) and hydroxyl anions (OH^-). Finally, catalase facilitates the conversion of these ROS into water [125,126,127,128].

Under normal conditions, various mechanisms exist to combat free radicals. The endogenous antioxidant system consists of uncoupling protein enzymes (superoxide dismutase, glutathione peroxidase, coenzyme Q, and catalase) as well as non-enzymatic molecules that play an important role in the dissipation of free radicals. On the other hand, there is an exogenous antioxidant system that is derived from diet and plant sources such as vitamins A, C, and E, beta-carotene, lycopene, resveratrol, and flavonoids [128,129,130,131,132,133,134,135,136,137].

Regardless of how they work, their anti-aging, anti-cancer, anti-cataract, and antidiabetic properties have been demonstrated, as oxidative stress is largely responsible for disease pathophysiology. GB contains numerous compounds that are beneficial to brain health. Among them, Ginkgolide A stands out for its intriguing effects on cardiovascular and cerebrovascular diseases. Its mechanism of action involves inhibiting a biomarker of oxidative stress, 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is abundant in the brain following inflammation caused by, say, trauma [138]. GB's effects on oxidative stress and neuronal protection are linked to a reduction in ROS formation and action, such as inhibiting NADPH oxidase activation, downregulating the Mitogen-Activated Protein Kinases (MAPK) and activator protein-1 (AP-1) complex, inactivating Signal Transducer and Activator of Transcription 5 (STAT5), and several other molecules (Figure 2) [139,140,141].

Ginkgo biloba (GB) contains bioactive compounds, including flavonoids (approximately 28%) and terpenic lactones (2.8-3.4% of Ginkgolides A, B, and C, and 2.6-3.2% of bilobalide). These compounds have the ability to enhance brain circulation by reducing peroxide levels in cerebellar neurons and protecting cortical neurons from injuries caused by iron. Superoxide dismutase, catalase, and glutathione peroxidase are examples of antioxidant enzymes whose mRNA expression is positively regulated by this plant, which reduces the generation of ROS and free radicals [142,143,144,145]. Ginkgolides A, B, and C can reduce ROS production and levels (Figure 2). For example, ginkgolide A has been shown to reduce malonaldehyde production while increasing glutathione peroxidase and superoxide dismutase expression [146,147,148,149].

Kaempferol can also be found in GB, which is associated with an increase in the expression of brain-derived neurotrophic factor (BDNF), glutamate-cysteine ligase catalytic subunit, B-cell lymphoma protein 2 (BCL-2), and glutathione peroxidases. It can inhibit serotonin degradation, cytochrome C release, caspase-3 activity, and apoptosis (Figure 2). Kaempferol can reduce the neurotoxicity associated with 3-nitropropionic acid [145,150,151]. Quercetin, bilobalide, and isorhamnetin are also found in GB [71]. These phytocounphenols are related to the reduction of inflammatory processes and the production of ROS (Figure 2). Quercetin and bilobalide can work as free radical scavengers. Bilobalide is associated with the stimulation of the expression of cytochrome c oxidase subunit III and BCL-2. Isorhamnetin inhibits DNA fragmentation and apoptosis (Figure 2) [71,152,153,154].

EGB 761® inhibits the formation of hydrogen peroxide radicals, anion superoxide radicals, ROS, reactive nitrogen species, peroxyl radicals, and hydroxyl radicals. Nonetheless, EGB 761® plays a role in increasing the expression of glutathione peroxidase and superoxide dismutase [155,156,157]. Similarly, GB extract (GBE) has a broad pharmacological spectrum for preventing oxidative stress, which protects the central nervous system from depressive processes, improves aspects of impaired neuroplasticity, and increases cellular mitochondrial function (Figure 2). Secondarily, GBE's antioxidant and anti-inflammatory properties improve the prognosis of cardiovascular diseases [158,159,160,161].

4.3. Ginkgo Biloba and Alzheimer's Disease and Dementia: Evidence from Cellular and In Vivo Studies

In vitro, ginkgolide A treatment reduced the expression of pro-inflammatory mediators like COX2 and nitric oxide, as well as cytokines like TNF-α, IL-1, and IL-1β in mouse macrophages and differentiated human monocytes [162]. Furthermore, it has been shown to significantly reduce neurological deficit scores and brain infarct volume in rats suffering from cerebral ischemia/reperfusion damage [163], as well as repair mitochondrial dysfunction in cell culture [164]. Ginkgolide B has been shown to inhibit glutamate-induced apoptosis in astrocytes in vitro when glutamate metabolism is abnormal in the pathological environment of AD (Figure 2) [165]. According to studies, it improves neurological function by promoting the proliferation and differentiation of neural stem cells in rats with cerebral ischemia/reperfusion injury [166]. Electrophysiological recordings from the brain slices of rats exposed to hypoxia in vivo showed increases in the frequency of spontaneous discharge, the frequency of action potentials, and the magnitude of calcium currents. Pre-treatment with ginkgolide B, which may regulate Ca2+ influx in hippocampal neurons, suppressed all of these effects of hypoxia [167]. Figure 4 shows the effects of GB on AD.

In model rats with middle cerebral artery occlusion/reperfusion, ginkgolide C suppresses the CD40/NF-κB pathway to ameliorate cerebral ischemia or reperfusion-induced inflammatory impairments [162]. This compound can inhibit adipogenic factors and enzymes, increase lipolysis in a culture of differentiated adipocytes [168], and exhibit an intriguing anti-neoplastic effect in hepatocellular carcinoma cells [69], suggesting that other tissues may respond better to its application. It has been demonstrated that bilobalide increases neurogenesis and synaptogenesis in the cells of rat fetuses by stimulating the proliferation of hippocampal progenitor cells in a dose-dependent manner [169]. It supports the use of APP processing promoted by alpha-secretase in delaying the onset of AD by reducing the production of beta-amyloid in the human neuroblastoma cell line. Furthermore, studies have shown that bilobalide improves cognitive functions in AD mice [170].

In an in vitro model of AD, resveratrol can reduce oxidative damage to neurons caused by beta-amyloid via the mitophagy pathway [171]. It also had a significant effect on the integrity of the blood-brain barrier in AD-induced rats [172]. This substance was found to have good anticholinergic effects in mice with dementia when combined with other antioxidants such as vitamin E (Figure 2) [173].

In a culture of human umbilical endothelial cells, kaempferol was discovered to bind to vascular endothelial growth factor, improving some angiogenic functions [112]. Kaempferol increased dopaminergic and cholinergic neurotransmission in lab rats' prefrontal cortex, improving cognitive function [174]. It has a synergic effect on the learning and memory capabilities of model rats with AD [175]. Isorhamnetin has been shown to stimulate neurofilament production, which enhances neurite outgrowth and NGF-induced neurofilament expression. In addition,

4.4. Ginkgo Biloba and Neurotransmitters Related to Alzheimer's Disease

4.4.1. Acetylcholine

Several studies indicate that GB may have a direct and indirect impact on cholinergic function [176]. Scopolamine, a muscarinic receptor antagonist, has been shown to cause memory dysfunction in models, supporting this theory. Memory and cognitive function have been demonstrated to be negatively impacted by its administration's transient blockade of cholinergic muscarinic receptors [177]. In rodents, GB treatment reduces scopolamine-induced amnesia, indicating improved cognition. This condition was associated with GB's direct action on cholinergic receptors. This plant appears to directly affect presynaptic cholinergic nerve terminals, inhibiting choline absorption, which is a precursor to acetylcholine synthesis [178]. Other learning and memory models applied to rodents demonstrated the effectiveness of GB in memory acquisition and retention in the face of prolonged treatment [179], as well as an improvement in the animals' memory while using a maze [180]. The study found that using GB in healthy young and elderly humans improved both short-term [181] and long-term [182] memory. In Alzheimer's patients, a 3- to 6-month treatment with 120 to 240 mg of G. biloba extract has a small but significant effect on GB's clinical efficacy [183].

4.4.2. Glutamate and Dopamine

Ginkgolides, biologically active terpene lactones in GB, regulate glutamate transmission in the cortex and hippocampus. The binding of glutamate to receptors activates a short-term modulation pathway that allows ions to enter and exit the cell. The flow of calcium ions triggers the release of glutamate [184]. Ginkgolides influence phospholipase A2 and prevent kinase C activation. In turn, kinase C has a significant effect on calcium circulation by influencing endoplasmic reticulum calcium release. When this pathway is inhibited, calcium flow is blocked, and glutamate cannot be released into the synapse. Thus, ginkgolides' inhibitory function is demonstrated by their ability to reduce glutamate-induced damage in hippocampal neuronal cells in the face of cerebral ischemia [185]. Normal excitatory neurotransmission requires glutamate to be removed from the synapse by its transporters [186]. Failure to withdraw causes toxicity that can cause acute neurodegenerative diseases like epilepsy and hypoxia, as well as chronic neurodegenerative diseases like Alzheimer's and Huntington's Syndrome [187].

Similarly, the presence of ginkgolides appears to modulate the neurotransmitter dopamine, which regulates cognition, voluntary movements, and the activation of the punishment and reward system [188]. Chronic ginkgolide administration increases dopaminergic and noradrenergic transmission in the frontal cortex of the brain [186]. Furthermore, ginkgolides boost dopaminergic activity in rats' paraventricular nuclei [189]. This effect can be explained by the inhibitory effect of ginkgolides on MAO (monoamine oxidase). MAO is responsible for the elimination of norepinephrine at synapses. Yoshaitatake et al. demonstrated that a single oral dose did not affect monoamine concentration levels [186]. Dysregulation of dopamine homeostasis is associated with the pathogenesis of neurodegenerative diseases. For example, growth hormone receptor 1α (GHSR1α)-induced disruption of dopamine D1 receptor (DRD1) function exacerbates the pathophysiology of AD [190].

4.4.3. Serotonin - 5HT

Changes in behavior, as in the case of depression, can be identified in individuals with AD [191], suggesting functional changes in the monoaminergic system, and not only in the cholinergic system [192]. Some studies have shown that serotonin receptors, in addition to increasing cholinergic neurotransmission, also improve neurogenesis processes, neuronal plasticity and reduce amyloid load in the brain [193,194].

In relation to serotonin 5-HT1A receptors, a decrease in their expression is observed in the aging phase [195]. However, this condition can be reversed with treatment with GB [196]. Although the mechanisms of action of GB in neuroprotective events are not well understood, the modulation of serotonin levels was observed, as well as the increase in dopamine levels due to the reduction of mannoamine oxidase (MAO) activity in the prefrontal cortex in the presence of this substance [197,198,199,200].

In rats that received GB treatment for 3 weeks with an average consumption of 50-300 mg of GB, a slight increase in serotonin levels was observed in brain regions such as the prefrontal cortex and hippocampus [201].

4.5. Ginkgo Biloba and miAlzheimer’s Disease and Dementia: The Results of Clinical Trials

Patients treated with MEMO (a combination of 750 mg of lyophilized royal jelly and 120 mg of standardized extracts of GB) had higher MMSE (Mini-Mental State Examination) scores than the control group (+2,07 and +0,13, respectively). The trial was randomized, double-blind, and placebo-controlled, which was a plus; however, the follow-up period was brief [34]. The protective effect of GB extract on the incidence of Alzheimer's disease was not supported by conclusive research. The study's sample size included over 2,500 randomly assigned patients who were evaluated on a five-year cycle. Despite this, the trial failed to demonstrate a protective effect because the number of dementia events was significantly lower than expected, resulting in a lack of statistical power to detect effects. Furthermore, more than 700 patients dropped out, which was a negative point [35].

The efficacy of GB was evaluated, as well as his tolerability in AD. The GB-treated group's MMSE scores increased from 16.52+-4.124 to 16.76+-4.116, with no significant difference (p>0.05) [34]. GB enhanced cognitive functioning, neuropsychiatric symptoms, and functional abilities in dementia patients. The SKT total score (drug-placebo differences: 1.7 for AD, p<0.001, and 1.4 for VaD, p<0.05) and the NPI total score (drug-placebo differences: 3.1 for AD, p<0.001, and 3.2 for VaD, p<0.05) demonstrate a significant drug-placebo difference. The results could not be properly interpreted due to the high prevalence of mixed pathologies and the challenges associated with accurate diagnostic classification. Nevertheless, the sample selected was representative of the patients with dementia encountered in daily practice and was appropriately allocated to each type of dementia diagnosed [39].

A once-daily dosage of EGb 761 significantly improves patients' neuropsychiatric symptoms and cognitive function, making it an effective treatment for dementia. On the SKT total score, patients' improvements ranged from 2.2 to 3.5 points. There was adequate follow-up and randomization; however, the patients' low average age and enrollment as outpatients may have limited the generalizability of the results [202].

Patients who received GB extract improved by -1.4 points on the SKT and -3.2 points on the NPI total score, whereas those who received placebo deteriorated by +0.3 on the SKT and did not change on the NPI total score. Ginkgo outperformed placebo on all secondary outcome measures. Positive aspects of the study include the requirement for a computer tomography or magnetic resonance imaging that is no more than one year old to confirm the inclusion diagnosis and to show no evidence of other brain lesions that could account for the cognitive deficit, which increases the research's reliability, as well as small and balanced losses between the groups at the end of the trial. The fact that there are more women than men included in the study could be a source of bias. According to the authors, a once-daily Ginkgo dosing regimen is both safe and effective for dementia [203]. One newly developed GB leaf extract is a safe, effective, and, at the very least, adjuvant treatment option for patients with mild cognitive impairment, improving symptoms of forgetfulness, impaired concentration, and impaired memory. The short duration of treatment may be a bias in this study, and the use of medications may result in an improvement in mental capacity during the study [40].

The combined treatment of GB plus donepezil was found to be consistently and slightly more effective than administering these medications separately. Positive aspects of the study include the recruitment of patients from a large proportion of disabled Alzheimer's patients with behavioral and psychological symptoms, as well as the requirement of computer tomography or magnetic resonance imaging to confirm the diagnosis of probable AD. The study's limitations include a small sample size [41].

Ginkgo is ineffective in preventing or delaying the onset of all-cause dementia in participants older than 75 years of age. This was demonstrated in the largest clinical trial to date evaluating the impact of GB on the incidence of dementia, involving over three thousand patients. The study's strengths include the large number of patients under investigation and the balanced proportion of men and women (46% at baseline). Furthermore, the researchers included measures of overall cognitive decline and disability as secondary outcomes of the study but did not provide data in the paper, which could help us understand whether Ginkgo has a short-term effect on memory [42]. More evidence on the efficacy and safety of Ginkgo in treating cognitive and non-cognitive symptoms of dementia has been added. The treatment group improved by -3.2 points on the SKT, while the placebo group deteriorated by +1.3 points on the same test. One of the study's major strengths was that the patients were recruited outside of the norm of daily practice, with less stringent eligibility criteria than are typically used in other dementia studies. The study's drawback is that the small age average of 64 years may account for the patients' high responsiveness to drug treatment, as well as the absence of death and, thus, the low rate of premature discontinuation, both of which situations may distort the analysis's results [43].

The outcomes of patients who received daily doses of GB extract, donepezil, or a placebo were compared. The study found statistical differences in MMSE and SKT scores between the groups, demonstrating GB's effectiveness. There were no differences between the donepezil and EGb groups in this study. The study's limitation was the small number of participants, which was insufficient for the baseline assessment [44]. Some studies on the use of GB extract in mild to moderate Alzheimer's dementia proved inconclusive. There were no significant differences found among the groups in the entire sample. Nonetheless, patients who received the placebo experienced a minor decline in cognitive and functional aspects [204]. One study found no statistically significant differences in mean score changes between the Ginkgo-treated and placebo groups. There were minimal or no differences between the groups, and none of the main outcome measures were statistically significant (SKT:0.4; CGI-2:0.0; NAI-NAA:0.0). The ineffective multi-purpose design, unequal treatment distribution, and inconsistent findings with other studies are the main weaknesses of the study. The significant number of participants who left the study early is another disadvantage [48].

After EGb 761 was administered for six months, the patients in that group exhibited a small improvement. The findings from both the ITT and evaluable data show that EGb is effective in two assessment domains, specifically cognitive performance (ADAS-Cog). In terms of ADAS-Cog scores, the placebo group experienced a significant decline from their initial baseline score, whereas the EGb group showed a trend of improvement. One of the strengths of this study is its duration, which contributes to the study's reasonable efficacy. The study suggests that more research is needed to determine the role of GB in dementia treatment and its potential as an alternative or adjunct to cholinesterase inhibitors [47].

One study found that Ginkgo had no effect on each of the main outcome measures for participants compared to a placebo over the course of the 24-week treatment period. Furthermore, no benefits from higher doses or longer periods of Ginkgo treatment were discovered. The trial results indicate that this plant is ineffective for treating older people with age-related memory impairment or mild to moderate dementia. The study's strengths include a rigorous comparison, a comprehensive review of the existing literature, and methodological consistency. Nevertheless, the primary limitation of the study lies in its failure to establish the effectiveness of GB in enhancing cognitive and functional outcomes, thereby contradicting the prevailing body of research in this domain [48].

5. Conclusion

This systematic review of clinical trials on the efficacy of GB in AD and cognitive impairment provides a nuanced perspective. While some studies showed improvements in cognitive functioning and neuropsychiatric symptoms after GB treatment, others found no significant differences when compared to placebo or donepezil. Notably, the combination of GB and donepezil provided marginal benefits, especially in patients with AD behavioral symptoms. However, large-scale trials revealed that GB did not effectively prevent or delay dementia onset in older people. The limitations of these studies, such as small sample sizes, inconsistent findings, and diagnostic challenges, had a significant impact on the interpretation of results. Furthermore, some trials did not establish a protective effect of GB extract against AD. In light of these conflicting findings, more research with robust methodologies and larger sample sizes is needed to elucidate the role of GB extract in AD and cognitive impairment. Future investigations should explore optimal dosages, long-term effects, and potential synergies with existing treatments to enhance our understanding of GB's therapeutic potential in dementia management.