Evidence-Based Complementary and Alternative Medicine

Volume 2013 (2013), Article ID 495703, 18 pages

http://dx.doi.org/10.1155/2013/495703

Review Article

Antiapoptotic Effects of EGb 761

Norma Serrano-García,¹ José Pedraza-Chaverri,² José Juan Mares-Sámano,³ Marisol Orozco-Ibarra,¹ Arturo Cruz-Salgado,⁴ Anabel Jiménez-Anguiano,⁵ Julio Sotelo,⁶ and Cristina Trejo-Solís⁶

¹Laboratorio de Neurobiología Molecular y Celular, INNN-UNAM, Instituto Nacional de Neurología y Neurocirugía “Manuel Velasco Suárez”, 14269 México, DF, Mexico

²Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, 04510 México, DF, Mexico

³Laboratorio Analítico de Compuestos del Tabaco, Instituto Nacional de Salud Pública, 014000 México, DF, Mexico

⁴Departamento de Salud Bucal, Centro Nacional de Programas Preventivo y Control de Enfermedades, 11800 México, DF, Mexico

⁵Área de Neurociencias, Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, 09340 México, DF, Mexico

⁶Departamento de Neuroinmunología, Instituto Nacional de Neurología y Neurocirugía “Manuel Velasco Suárez”, 14269 México, DF, Mexico

Received 19 February 2013; Revised 19 June 2013; Accepted 27 June 2013

Academic Editor: Alfredo Vannacci

Copyright © 2013 Norma Serrano-García et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Ginkgo biloba extracts have long been used in Chinese traditional medicine for hundreds of years. The most significant extract obtained from Ginkgo biloba leaves has been EGb 761, a widely used phytopharmaceutical product in Europe. EGb 761 is a well-defined mixture of active compounds, which contains two main active substances: flavonoid glycosides (24–26%) and terpene lactones (6–8%). These compounds have shown antiapoptotic effects through the protection of mitochondrial membrane integrity, inhibition of mitochondrial cytochrome c release, enhancement of antiapoptotic protein transcription, and reduction of caspase transcription and DNA fragmentation. Other effects include the reduction of oxidative stress (which has been related to the occurrence of vascular, degenerative, and proliferative diseases), coupled to strong induction of phase II-detoxifying and cellular defense enzymes by Nrf2/ARE activation, in addition to the modulation of transcription factors, such as CREB, HIF-1α, NF-κB, AP-1, and p53, involved in the apoptosis process. This work reviews experimental results about the antiapoptotic effects induced by the standardized extract of Ginkgo biloba leaves (EGb 761).

1. Introduction

Programmed cell death is a key cellular process in the development and maintenance of tissue homeostasis. Its function is to eliminate superfluous, damaged, infected, or transformed cells. This type of cell death, also known as apoptosis, was first described by Vogt in 1842, who discovered that some cells were morphologically different. In 1972, Kerr et al. [1] coined the word apoptosis from the Greek for “dropping off” of petals or leaves from plants or trees; in this way, they described the intrinsic mechanism of programmed cell suicide observed in normal hepatocyte replacement. These authors were the first to distinguish between the morphology of cell death caused by apoptosis and that produced by necrosis.

The processes of apoptosis and necrosis differ in that, in the latter, cell death is due to physical, thermal, or ischemic stimuli that produce swelling of the cell and mitochondria and rupture of the membrane and organelles, as well as damage to the cytoskeleton and nucleus, which eventually leads to cell death [2]. In contrast, apoptosis is characterized by persistence of membrane integrity until the final phases of the death process when the membrane starts to shrink as the cell volume diminishes. In the meantime, lysosomal content remains intact, cell fragmentation continues, and apoptotic bodies are formed; these are small membrane-associated vesicles that are phagocytized by neighboring cells.

The distinctive feature of apoptosis is DNA fragmentation. The morphologic and biochemical processes of apoptosis are mainly mediated by death effectors, such as proteases, which lead to nuclear and cellular fragmentation. Before these death effector pathways are activated, the life/death balance of the cell is modulated by a complex interaction between the different death effectors. When this balance tilts towards apoptosis, the cell death effectors start to act [2].

The present review discusses the regulatory effects of the proteins that participate in apoptosis. Firstly, the apoptotic pathways and the proteins that were involved in each will be described. Subsequently, the relevant characteristics of the Ginkgo biloba leaf extract (EGb 761) will be explained. Lastly, studies on the antiapoptotic function of EGb 761 will be analyzed.

2. Pathways Leading to Apoptosis Activation

The molecular mechanisms of apoptosis (cascade of events starting at the cell surface up to the final changes in the nucleus) have not been totally clarified; however, several key proteins are implicated in the regulation of programmed cell death [3, 4]. Two main pathways have been described that lead to apoptosis: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway [5–7]. Recently, evidence has been found that in certain types of cells the two pathways converge [6]. The mechanisms of apoptotic death, both intracellular and extracellular, combine to activate a group of proteases named caspases, specific for aspartates and cysteines. These are present as inactive proteins in live cells and are activated by proteolysis [8]. Initiator caspases are able to activate effector caspases or to amplify the signal, augmenting their own activation [9], leading to cell death [8, 10].

2.1. Extrinsic Pathway

The extrinsic pathway of programmed cell death requires the activation of membrane receptors [5]. These cell death receptors belong to the genic superfamily of receptors of the tumor necrosis factor (TNFR) and are characterized by a cysteine-rich extracellular domain and a homologous cytoplasmic sequence named death domain (DD).

2.1.1. Fas Receptor

The Fas receptor (also known as cytotoxicity-dependent protein, CD-95, or Apo-1) is a surface membrane protein of the death receptor superfamily, named tumor necrosis factor/nerve growth factor (TNF/NGF) [11–15]. It is abundantly expressed in several cells [12] including neurons [16, 17] and plays an important role in apoptosis [18]. Fas is a type I membrane protein with two N-glycosylation sites in the extracellular domain, a cysteine-rich region where the ligand binds and an intracellular death domain close to the carboxyl terminal [12, 15, 19].

2.1.2. FasL (FAS Receptor Ligand)

FasL belongs to the genic family of the tumor necrosis factor (TNF); it is a type II homotrimeric cell-surface glycoprotein with molecular weight of approximately 40 kDa, a cytoplasm-oriented N-terminal and an extracellularly oriented C-terminal [20]. After Fas receptor activation by its ligand, the protein forms microaggregates with the death domains of the Fas receptor. This involves recruitment of a cytoplasmic adaptor cell, which also contains a Fas-associated death domain (FADD).

2.1.3. FADD

The FADD protein presents a single serine phosphorylation site (Ser 194 for human and Ser 191 for mouse), which is essential for other cell functions: cell cycle regulation, survival, and proliferation in some cell types [21, 22]. The amino terminal of FADD is named death effector domain (DED) and recruits procaspase-8. The dimerization of this procaspase brings about its autoactivation, which leads to the mature form of the protein and the consequent onset of the apoptotic cascade. The transducer FADD protein is expressed in cytoplasm and in the nucleus [23]. After binding to the Fas receptor, the cytoplasmic protein is rapidly recruited to the cytoplasmic membrane where, together with the receptor and procaspase-8, it forms the death-inducing signaling complex (DISC) [24, 25]. Later, procaspase-8 autolytically splits producing caspase-8 (active forma), which, in turn, cleaves and activates a cascade of effector caspases (caspase-3). This provokes fragmentation of numerous substrates until cell death ensues [18]. Cell death induced by Fas in the nervous system [26] shares the same basic mechanisms as described for peripheral cells [4].

Once caspase-8 has been activated, it has two possibilities: to activate the previously described caspase cascade or to act on a member of the Bcl-2 protein family named Bid. The proteins of the Bcl-2 family are grouped into three subfamilies: the antiapoptotic proteins (Bcl-2, Bcl-XI, Mcl-1, and others); the multidomain-type proapoptotic protein family (Bax and Bak); and the BH3-type proapoptotic proteins (Bid, Bim, Bad, and others). The truncated form of Bid may translocate to the mitochondria where it activates the mitochondrial pathway by a mechanism yet to be clarified. At this point, the extrinsic and intrinsic pathways converge [27].

2.2. Intrinsic Pathway (Mitochondrial Pathway)

A pathway, independent of the death receptors, is the mitochondrial apoptosis (intrinsic pathway) [4, 28, 29]. This pathway is mediated by stimuli such as chemotherapeutic agents, UV radiation, stress molecules (reactive oxygen and nitrogen species), oncogenes, hypoxia, and survival factor deprivation, which induce the activity of the p53 protein [26–28]. This pathway can also be activated by death receptors to amplify the apoptosis-inducing signal [30].

Proteins of the Bcl-2 family are involved in control of apoptosis and participate as activators or inhibitors of cell death [27]. Bcl-2 is an oncoprotein of 26 kDa, mainly localized in the internal mitochondrial membrane. A neuroprotective role, opposite to apoptotic cell death, has also been attributed to it [31], probably by preventing the release of cytochrome c (induced by Bax) and the subsequent activation of effector caspases [4, 27, 32]. Once cytochrome c (cyt c) has been released from mitochondria, an apoptosome is formed by the assembly of the apoptotic protease activation factor (Apaf-1) with procaspase-9. The Apaf-1 factor is a cytoplasmic monomer of 130 kDa that contains a caspase recruiting domain [33]. Procaspase-9 recruitment by Apaf-1 through the so-called caspase recruitment domain (CARD) requires ATP. Later, procaspase-9 automatically cleaves producing active caspase-9, which, in turn, activates other caspases (caspase-3, -6, and -7) and provokes fragmentation of other substrates and cell death. The release of cyt c from mitochondria to the cytoplasm is an important regulating step in caspase activation; it activates Apaf-1 and interrupts the electron transference chain reducing energy production, thus increasing reactive oxygen species (ROS) [34].

3. Ginkgo biloba and EGb 761

Some herbal products have attracted interest in the so-called alternative treatments; among them, Ginkgo biloba is one of the most extensively studied. The tree has been present for some 250 million years; it is therefore considered a living fossil. The word ginkgo derives from the Chinese Yin-kuo for golden apricot; biloba refers to the bilobular morphology of the leaves [35]. In China, its leaves were considered medicinal as far back as 2800 B.C; infusions were prepared for the treatment of asthma and bronchitis [36]. During the past decade, evidence has been gathered suggesting that concentrates and purified extracts of Ginkgo biloba leaves promote protection from neuronal and vascular damage [36].

Various extracts from Ginkgo biloba leaves have been prepared; one of them is called EGb 761. This extract is a well-defined and standardized mixture and has been used for treatment of brain disorders (including dementia), neurosensory syndromes, peripheral blood flow disorders, and cerebral insufficiency [37]. The latter is defined as the set of hemodynamic disturbances in the arterial system, manifesting as impaired circulatory flow, causing a number of different clinical manifestations depending on the brain regions affected. Symptoms include localized pain in a brain artery, dizziness and balance disorder, optical disorders that can range from simple blurred vision to oculomotor paralysis transient bilateral, recurrent headaches, syncope, fainting, and waking disorders. This disease stands out as the most common cause of disability in adult people in Germany having a very high incidence (250 cases per 100,000 population per year) [38]. Therefore, in 1965, the German Dr. Willmar Schwabe characterized the pharmacological activity of Ginkgo biloba and registered it for therapeutic use as EGb 761. In 1978, the product was commercialized under the name Tebonin 761 as a 40 mg oral formulation, and in 1982, Schwabe himself commercialized Tebonin forte, an 80 mg oral formulation [39].

The EGb 761 consists of a mixture of active compounds obtained by multiple extractions. Its effects on vestibular disorders, sexual dysfunction induced by antidepressants, traumatic brain injury, and hypertension have been explored [35]. It has been found to show protective effects against hypoxia, to inhibit platelet-aggregation factor (PAF), to increase blood rheology, and reduce capillary permeability. The therapeutic effect of EGb 761 is attributed to the whole of its constituents [35]. However, select constituents have been identified as mediating a variety of more specific effects.

3.1. Chemical Composition of EGb 761

The Gingko biloba tree is composed of amino acids, sugars, polysaccharides, organic acids, inositols, and sterols among other constituents. The active principles are compounds with different chemical structure: flavonoids, present in approximately 26%, terpenoids, 6-7%, and organic acids in minor quantities (Figure 1) [35].

Figure 1: Chemical structures that represent some EGb 761 constituents (taken from [35]).

Flavonoids, also known as phenylbenzopyrones or phenylchromones, are a group of substances of low molecular weight broadly distributed in the plant kingdom. The flavonoids present in EGb 761 are flavones, flavonols, tannins, biflavones (amentoflavones, bilobentol, 5-metoxibilobetol, ginkgetin, isoquingetin and ciadopitysin), quercetin glucosides, and kaempferol [36]. These compounds act as antioxidants/free radical scavengers, enzyme inhibitors, and cation chelating agents [38]. In general, flavonoids show low bioavailability because they are poorly absorbed by the intestine in their glycosylated form and are rapidly eliminated. They are absorbed only when presented as aglycones [40]. Nonabsorbed flavonoids that reach the colon are subject to bacterial enzyme degradation; then, the metabolites may be absorbed. Once absorbed, flavonoids are metabolized in the liver into their conjugated derivatives [41]. EGb 761 also contains terpenoids, which are nonsaponifiable lipids of the cyclic ester type (lactones). Two types of terpenoids are present in the extract: ginkgolides and bilobalides [42]. The ginkgolides are classified into 5 types: A, B, C, J, and M. Types A, B, and C are present in 3.1% of the total extract of the Gingko biloba leaf. Bilobalide is a sesquiterpene trilactone present in 2.9% of the total extract [41].

The polyvalent action of EGb 761 is responsible for the efficacy in the treatment of clinical disorders of multifactorial origin [42]. The multiple effects of EGb 761 on different therapeutic targets are due to the synergistic activity of its constituents, their additive effect, and even their antagonistic interactions [41]. The ginkgolides are PAF antagonists and able to reduce platelet activation and aggregation, and therefore having the potential to improve blood circulation. Bilobalide can reduce cerebral edema and damage from cerebral ischemia. The antioxidant effect of the flavonoid fraction may be achieved by direct attenuation of ROS, chelation of prooxidant transitional metal ions, expression of antioxidant proteins like superoxide dismutase (SOD), or the increase in antioxidant metabolites such as glutathione. Therefore, the flavonoids react preferentially with hydroxyl radicals (OH•) and directly scavenge them [41].

4. Antiapoptotic Effects of EGb 761

EGb 761 has shown antiapoptotic effects in diverse tissues associated with various multifactorial and synergistic actions. Among these are reduction in the number of apoptotic cells, maintaining of the mitochondrial integrity, inhibition of cyt c release from the mitochondria, increase in antiapoptotic protein Bcl-2 transcription rate, and decrease in caspase transcription rate and DNA fragmentation.

4.1. Reduction in the Number of Apoptotic Cells

The protective and recuperative effects of EGb 761 on different cell types have been demonstrated in animal models. Ergun et al. [43] showed death inhibition by EGb 761 at a dose of 150 μg/mL in human lymphocytes exposed to gossypol, a toxin that causes cell death via apoptosis. Similar results have been observed in thymus cells pretreated with EGb 761 (100 μg/mL) and then exposed to ferrous sulfate in hydrogen peroxide (H₂O₂) [44]. In addition, in lymphocytes isolated from spleen of aged mice treated with 100 mg/Kg EGb 761 for 2 weeks, less susceptibility to ROS-induced apoptosis was found [45]. In the peripheral nervous system, posttreatment with EGb 761 (100 mg/kg) decreased the number of apoptotic cells in injured rat spinal cord [46]. In central nervous system, treatment with EGb 761 (40 mg/kg) reduced neuronal death in the substantia nigra pars compacta from an experimental model of Parkinson’s disease [47].

4.2. Mitochondrial Preservation

Mitochondria play a key role in apoptosis; changes in its structure and function develop when free radicals accumulate [48]. Reduction of the mitochondrial membrane potential triggers the intrinsic pathway of apoptosis. In a model of ageing in rats, treatment with 100 mg/kg EGb 761 prevented morphological changes and diminished oxidative stress in liver and brain mitochondria [49]. Flavonoids, which are components of EGb 761, prevent lipid peroxidation as they interact with molecules of the lipid bilayer, contributing to the stability and functionality of the mitochondrial membrane [50]. Pretreatment with EGb 761 (200 μg/mL) of PC12 cells preserves the mitochondrial membrane potential, allowing increased ATP production and preventing the damage caused by oxidative stress [51]. In the nervous system of adult animals, the administration of EGb 761 increases mitochondrial ATP content [52, 53].

4.3. Regulation of cyt c, Bax, Bcl-2, and Apaf-1 Expression

As described previously, the release of cyt c into the cytoplasm triggers apoptosis via the caspase-9 pathway. However, other alternatives have been recently described to explain apoptosis generated by mitochondria. For example, it has been proposed that cyt c is released instantly prior to the transitory opening of the permeability pore and to the loss of membrane potential. Moreover, Bax may promote this cyt c release without involving the pore. In a healthy cell, the external mitochondrial membrane expresses Bcl-2 on the surface, which binds to Apaf-1, which is kept inactivated. Any alteration at the internal equilibrium of the cell, for example the accumulation of ROS, causes mitochondrial cyt c release. In turn, Bcl-2 liberates Apaf-1 that then binds to cyt c [31, 34]. EGb 761 administration decreases the expression or activation of the proteins that participate in the apoptotic signaling cascade of the intrinsic pathway. For example, cell death is induced by release of mitochondrial cyt c in testicles and heart of rats when doxorubicin is administered. Pretreatment with EGb 761 prevents the release of cyt c and activation of p53 and Apaf-1, thus acting against doxorubicin [54, 55]. The effects of EGb 761 on Bax and Bcl-2 have been studied using various experimental models that generate neuronal toxicity. Pretreatment with EGb 761 (100 μg/mL) increased Bcl-2 mRNA expression in cerebellum cells incubated with OH• [56]. A neuroprotective effect obtained by an increase in Bcl-2 expression has also been reported in PC12 cells damaged by staurosporine [57]. EGb 761 administrations diminish Bax expression in the hippocampus of rats suffering from accelerated senescence [58]. Jiang et al. [59] observed a protective effect of EGb 761 in apoptotic spinal cord neurons exposed to oxidative stress; the antiapoptotic effect was caused by regulation of Bcl-2 and Bax expression. The protective effect of EGb 761 against paraquat-induced cell death in PC12 cells is partially due to an increase in Bcl-2 expression [60]. Studies on a focal ischemia model have shown that EGb 761 reduces the amount of Bax in cerebral cortex, increasing the amount of Bcl-2 [61]. Also, administration of EGb 761 during 5 days inhibits Bax activity in diverse brain regions when global ischemia is applied to mice with accelerated senescence [62]. As a whole, these studies show that EGb 761 increases the expression of Bcl-2 and inhibits the expression of Bax when there is a cellular damage.

4.4. Caspase Activity

EGb 761 treatment inhibits the activity of different caspases involved in both the intrinsic and the extrinsic pathway. Posttreatment with EGb 761 inhibited caspase-9 activity in six brain regions of mouse following global ischemia in senescence-accelerated mice [62]. In agreement, 1 mg/mL EGb 761 prevented the increase in the activity of caspases-3 and -9 in bone-marrow mesenchymal stem cells cultured under hypoxic conditions [63]. Also, in cultures of rat cortical neurons incubated with 200 nM staurosporine to induce apoptosis, EGb 761 inhibited caspase-3 activity [64]. The inhibitory action of EGb 761 on caspase-3 activity has been reported in different apoptosis models [51, 60, 65, 66]. These studies evidence the inhibiting effect of EGb 761 on caspases of both the intrinsic and the extrinsic pathways.

4.5. DNA Fragmentation

In the process of apoptosis, one phase involves the degradation of proteins and nucleic acids. DNA is fragmented by endonucleases [1]. In endothelial lung cells incubated with 100 μg/mL EGb 761 and exposed to cigarette smoke, the DNA fragmentation was diminished [66]. Similar effects were found in cells exposed to paraquat; DNA fragmentation was reduced by pretreatment with EGb 761 at doses of 10, 20, and 40 μg/mL [60]. Furthermore, in cerebellum granular cells, EGb 761 has shown a neuroprotective effect in the DNA fragmentation caused by OH• [56].

5. Antiapoptotic Mechanisms from EGb 761

Defects in the physiological pathways of apoptosis, leading to defective cell survival, are thought to be involved in several major diseases for which therapy is lacking [67]. Oxidative stress has been widely implicated in neuronal death associated with chronic neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s diseases or amyotrophic lateral sclerosis [68]. Oxidative stress is caused by an enhanced production of ROS and reactive nitrogen species (RNS), including superoxide anion (), OH•, peroxyl radical (ROO•), alkoxy radical (RO•), and peroxynitrite anion (ONOO⁻), as well as nonradical species such as singlet oxygen (¹O₂), ozone (O₃), and H₂O₂ [69, 70]. The main source of cellular ROS is the mitochondria electron transport chain, located in mitochondria, through which continuous aerobic respiration generates [70]. The moderately reactive is the substrate for SOD enzymes, yielding H₂O₂, which in turn is the precursor of the highly toxic HO•, which was generated in the presence of reduced iron (Fe²⁺) or copper (Cu²⁺) through Fenton reaction. In addition of mitochondrial electron transport chain, low levels of ROS are produced by membrane-localized NADPH oxidase enzymes, peroxisomes, and the cytochrome P450 system [71, 72]. At low levels, ROS participate in cellular signaling; however, at high levels, ROS can cause irreversible oxidative damage to lipids, proteins, and DNA, which induce apoptosis in a variety of cell types [73]. Other mechanisms have been proposed for ROS-induction apoptosis. As reported by Wang et al. [74], when there is an increase of H₂O₂, it can cause apoptosis by upregulating Fas-FasL system (extrinsic pathway of apoptosis). H₂O₂ could also disrupt the mitochondrial membrane potential thereby leading to the release of proapoptotic components present in the mitochondria. In addition, it activates several transcriptional factors such as NF-κB, AP-1, and p53 and causes them to translocate into the nucleus. They could drive the expression of proapoptotic genes and also the inhibitors of survival-related genes [75, 76]. Complex antioxidant defense mechanisms have evolved to protect cells from oxidative injury, including enzymatic and nonenzymatic systems. Antioxidant enzymes include catalase, SOD, and glutathione peroxidase (GPx) [77, 78].

5.1. Antioxidant Properties of EGb 761

The flavonoid fraction (quercetin, kaempferol, and isorhamnetin glycosides) and terpenoid fraction (ginkgolides and bilobalides) constituents of EGb 761 inhibit apoptosis by its effect as antioxidants [79–82]. These latter compounds may act directly “scavenging” free radicals such as superoxide anion radical (), OH•, peroxyl radical, and related ROS like H₂O₂ and oxoferryl species. Jiang et al. [59] reported that EGb 761 protects against H₂O₂-induced death through inhibition of intracellular ROS production and modulation of apoptotic regulating genes (increased Bcl-2 and decreased Bax), suggesting that oxidative stress directly induces apoptosis and that EGb 761, a powerful antioxidant agent, can effectively protect the cell from apoptosis induced by oxidative stress. H₂O₂ reduces Bcl-2 gene expression and tends to increase Bax expression. Bcl-2, as an antioxidant, prevents ROS accumulation and other subsequent events such as mitochondrial membrane depolarization, Bax relocalization, cyt c release, caspase activation, and nuclear fragmentation [83]. The antioxidant action of EGb 761 is due mainly to the flavonoid fraction effective against superoxide anion, , OH•, and peroxyl radicals [82, 84]. Flavonoids also prevent lipid peroxidation in membranes, particularly owing to their ability to interact with and penetrate the lipid bilayers [50]. The terpenoid fraction has been found to have antioxidant effects in a model of neurodegeneration [85]. The protective effects of terpenes appear to involve inhibition of free radical formation [86], increasing intracellular levels of enzymatic antioxidants, such as SOD and catalase [87].

5.2. Influence of EGb 761 on Gene Expression

Functional and genomic studies reveal that EGb 761 upregulates and downregulates various signaling pathways as well as the transcription of some genes. These events may increase stress resistance and stabilize the cellular redox state in living organisms [40, 88, 89]. Bilobalide upregulates two mitochondrial DNA-encoded genes: subunit III of cytochrome c oxidase (complex IV) and subunit ND1 of NADH dehydrogenase (complex I) of the respiratory chain, indicating a fundamental mechanism that may underline EGb 761 induced neuroprotection [90, 91]. Enhancement of the respiratory control ratio, improving ATP synthesis, and preserving the mitochondrial outer membrane integrity prevent cyt c release, thereby blocking the formation of the apoptosome and the apoptotic caspase cascade [89]. Tendi et al. [91] suggest that EGb 761 and bilobalide might indirectly increase ND1 transcription by modulating the expression and/or the binding of transcription factors in the promoters of genes involved in mitochondrial transcription. In addition, both flavonoid and terpenoid constituents of EGb 761 decrease the expression of iNOS, which might oppose the deleterious effects of excessive production of NO• [92], which inhibit the complex I of respiratory chain by S-nitrosation, tyrosine nitration, and damage to FeS centers [93] and could increase Bax expression and decrease Bcl-2 expression subsequently leading to neural death [94]. Mak et al. [58] have suggested that EGb 761 protects the hippocampus from neural death by reducing the NO• levels through changes in Bax to Bcl-2 expression ratio. EGb 761 also increases the transcription of antiapoptotic Bcl-2-like protein inhibiting the proapoptotic factor Bax [59] and attenuates the transcription of proapoptotic genes, such as Fas, Bax and Bcl-xs, and caspase-7, -8, and -12 [57, 95]. Thus, the regulation of gene expression of proteins of the Bcl-2 family during brain injury could be relevant to explain the beneficial effects of EGb 761 extract in treating AD, as well as its possible benefits for treating other neurodegenerative and ischemic disorders of the nervous system whose pathogenesis involves oxidative stress and apoptosis.

6. Molecular Targets of EGb 761

The ability of EGb 761 to induce expression of genes affects apoptosis through NRF2 and also modulates transcription factors such as CREB, HIF-1α, NF-κB, AP-1, and p53.

6.1. Nrf2

Nuclear transcription factor E₂-related factor-2 (Nrf2) plays an important role in detoxification from carcinogenic agents and in the modulation of the antioxidant cell defense system. In normal conditions, Nrf2 is localized in the cell cytoplasm, forming a complex with the inhibitory protein Keap1 (Kelch-like ECH-associated protein 1) [96]. However, oxidative stress leads to dissociation of Nrf2 from Keap1, thereby rescuing Nrf2 from proteasomal degradation and inducing its translocation into the nucleus where, together with other transcription factors, it binds to ARE to regulate the expression of target genes [97–100]. For example, NAD(P)H:quinone oxidoreductase-1 (NQO1), SOD, GPx, heme oxygenase-1 (HO-1), catalase, thioredoxin (Tx1), γ-glutamyl-cysteinyl-synthase (γ-GCS), glutamate cysteine ligase catalytic subunit (GCLC), and glutathione-S-transferase subunit-P1 (GST-P1). Besides, Nrf2 also shows antiinflammatory effects [96, 97].

EGb 761 upregulates the expression of genes (and their respective protein products) that encodes antioxidant enzymes such as mitochondrial SOD, GPx, GCLC, and HO-1 [101–105]. HO-1, a heat shock protein of 32 kDa, degrades prooxidant free heme to iron, carbon monoxide (CO), and biliverdin which is reduced to bilirubin (BR); both are direct antioxidants [106]. Also, the induction of HO-1 increase levels of the antiapoptotic phosphorylates Akt and Bcl-xl [107]. Hsu et al. [66] have shown that EGb 761 confers protection from oxidative stress-related apoptosis induced by cigarette smoke extracts in human lung endothelial cells. This effect depends on transcriptional upregulation of HO-1 by EGb 761 via activation of MAPKs such as JNK, ERK, and p38 as well as nuclear translocation of Nrf2. Polyphenols including epigallocatechin-3-gallate, quercetin, and resveratrol, upregulate HO-1 via MAPKs/Nrf2 pathway [108–110]. Additionally, EGb 761 increases mRNA and protein levels of GST-P1 and NQO1 in vitro through the Nrf2-Keap1-ARE signaling pathway [111]. Liu et al. suggest that EGb 761 may have dual effects on Keap1; first, EGb 761 interacts with keap1 (or Nrf2) to dissociate the Nrf2-Keap1 complex, facilitating the release and nuclear translocation of Nrf2; second, EGb 761 reduced expression or enhanced degradation of Keap1 enables Nrf2 nuclear translocation.

Phase 2 gene inducers react much more avidly with Keap1 than with Nrf2; both contain multiple cysteine residues, which react with phase 2 inducers [98]. Wakabayashi et al. [112] have suggested that the cysteines C273 and C288 in the intervening region of keap1 may sequester one molecule of Nrf2 between two DGR domains in the cytosol and ensure its rapid turnover by targeting it for proteasomal degradation. Upon exposure to inducers, the reactive C273 and C288 residues form an intermolecular disulfide bond, covalently linking two monomers of Keap1 and changing its conformation accordingly. The DGR domains are then separated to release Nrf2, which translocates to the nucleus, activating the expression of phase 2 genes.

6.2. CREB

cAMP responsive element binding protein (CREB) is a transcription factor with multiple functions [113] believed to play a key role in cell survival. Reportedly, CREB mediates the expression of several neuroprotective proteins, including Bcl-2 and BNDF [114]. CREB is phosphorylated by several different proteins, including Akt, (PKA protein kinase A), (PKC protein kinase C), (CSNK casein kinase), and (CaMKs calmodulin kinases) [115]. Phosphorylation by these kinases can either increase or decrease the activity of CREB. Smith et al. [57] has reported, by microarray assay, an upregulation of Bcl-2 in the neuronal cells treated with EGb 761, suggesting that it would be mediated via CREB activation. EGb 761 increases CREB phosphorylation via the activation of Akt, releasing BNDF and consequently protecting the neurons injured by ischemia [116]. Similarly, EGb 761 restored impaired phosphorylation of CREB and brain-derived neurotrophic factor (BDNF) expression in β-amyloid-expressing neuroblastoma cells enhancing hippocampal neurogenesis and phosphorylation of CREB in a transgenic mouse model of Alzheimer’s disease (AD) [117, 118]. These studies suggest that flavonoids, the major active constitutes of EGb 761, may upregulate the CREB-BDNF pathway.

Quercetin and kaempferol, flavonols extracted from leaves of ginkgo, improve impaired neuroplasticity in transgenic cortical neurons and hippocampus of transgenic mice upregulating the CREB-BDNF signaling pathway and might represent a potential treatment for AD [119]. Bilobalide and quercetin increased significantly cell proliferation in hippocampal neurons in a dose-dependent manner and enhanced phosphorylation of CREB, elevating the contents of pCREB and BNDF in brain of mice [120]. Furthermore, inhibitors of upstream signaling pathways of CREB, including protein kinase C, ERK, ribosomal S6 kinase (RSK) 90, and nitric oxide pathway differentially blocked the effects of the individual components of EGb 761, ginkgolide C, quercetin, and bilobalide. These results suggest diverse effects of the EGb 761 individual components and provide direct insights about the mechanisms underlying the effect of EGb 761 in the enhancement of the cognitive performance of AD patients [118].

6.3. HIF-1

The hypoxia-inducible factor-1 (HIF-1) transcription factor is a heterodimer composed by the oxygen-regulated HIF-α subunit and the constitutively expressed HIF-1β subunits. Three isoforms of the α-subunits (HIF1α, HIF2α, and HIF3α) and two isoforms of the β-subunits (HIF-1β and HIF-2β) are known to be involved in the in vivo response to hypoxia [121–123]. Under aerobic conditions, HIF-1α is hydroxylated by specific PHDs (prolyl hydroxylases) in its oxygen-dependent degradation domain (ODD); it binds subsequently and is degraded by the von Hippel-Lindau protein (pVHL) leading to HIF-1α ubiquitination and degradation by the 26S proteasome [121, 124]. Under hypoxic conditions, PHDs lose their activity, which prevents hydroxylation and subsequent pVHL binding. This results in the stability of HIF-α, nuclear translocation, and heterodimerization with HIF-1β and binds at specific DNA sequences known as hypoxia-response elements (HREs) of target genes [125] involved in crucial physiological functions including angiogenesis (VEGF and transforming growth factor-β3: TGF-β3), cell survival (insulin-like growth factor 2: IGF2), transforming growth factor α: TGFα), and glucose metabolism (hexokinase 1: HK1; glucose transporter 1: GLUT1; and enolase 1: ENO1) [125, 126]. The proteins encoded by these genes mediate adaptive physiological responses such as angiogenesis and glycolysis that either serve to increase O₂ delivery or allow metabolic adaptation to reduced O₂ availability [125].

When HIF-1 is activated by hypoxia, it could play an antiapoptotic role by its ability to prompt anaerobic glycolytic metabolism, or expression of antiapoptotic proteins, such as Bcl-2, Bcl-xl, Mcl-1, BNIP-3, VEGF, enolase, and erythropoietin, or downregulation of proapoptotic proteins such as Bid, Bax, Bak, TRAIL-R1, PAF, and caspases-3, -9, and -8 [127–129].

Under hypoxic conditions, EGb 761 has shown neuroprotective effects in different experiments in vivo and in vitro [130–135]. The nonflavone fraction is responsible for the antihypoxic activity of EGb 761 [134, 135]. Zhu et al. [136] reported that ginkgolides, the main constituent of the nonflavone fraction of EGb 761, have a significant protective role against chemical and physical hypoxia-induced injury in neurons and PC12 cells; ginkgolides could significantly increase the expression of HIF-1α mRNA and protein, increasing the expression of erythropoietin protein and the cell viability and simultaneously decreasing the release of LDH in hypoxic neurons. Furthermore, they demonstrated that ginkgolides could induce a significant increase in the activation of the p42/p44 (ERK) MAPK pathway, which play a key role in the upregulation of the HIF-1α expression and enhance HIF-1 DNA binding activity, which might also be associated with a neuroprotective role of ginkgolides under hypoxic condition promoting the expression of the target genes of HIF-1 [137].

6.4. NF-κB

The nuclear factor-kappa B (NF-κB) transcription factor has emerged as a major regulator of programmed cell death via apoptosis and necrosis, activated by oxidative stress and inhibited by a variety of antioxidants [138]; NF-κB resides inactive in the cytoplasm; the most abundant form is a heterodimer p50–p65, in which the p65 subunits contain the transcriptional activation domain [139]. In unstimulated cells, NF-κB is retained in the cytoplasm through the binding of a family of inhibitors IκB (IκBα, IκBβ, and IκBγ) [140]. Activation of NF-κB requires sequential phosphorylation of IκB by IκB kinase (IKKα or IKKβ), ubiquitination, and degradation by the proteasome, followed by the translocation de p50–p65 to the nucleus and binding to the specific κB DNA consensus sequence in the enhancer region of a variety of κB-responsive proapoptotic-genes, such as the death receptor Fas (CD95), TRAIL receptors DR4, DR5, and DR6, the death-inducing ligands FasL, TNFα and TRAIL, tumor suppressor p53, the proapoptotic protein Bax and Bcl-xs as well as caspase-11, iNOS, and COX-2 [141–147]. NF-κB signaling is also modulated by posttranslational modifications including reversible acetylation of the p65 subunits [148]. For example, transcriptional activity of p65 requires acetylation of Lys 310, which can be deacetylated by SIRT1, a class III histone deacetyltransferase [149]. It has been shown that EGb 761 reduces NO• and PGE2 production in a model of inflammation [150], an effect possibly related with the inhibition of NF-κB activation. EGb 761 also activates SIRT1 in N2a cells, which in turn suppresses the β-amyloid peptide mediated NF-κB activation by promoting the deacetylation of Lys 310 of subunits p65, inducing protection from the neurotoxicity of β-amyloid [151]. Furthermore, EGb 761 inhibits ERK1/2, an upstream signaling pathway of NF-κB. The authors suggested that as the flavonoids present in EGb 761 are well-known antioxidants, they could scavenge H₂O₂ and consequently attenuate NF-κB activity, since H₂O₂ is a well-known activator of NF-κB [151]. On the other hand, antioxidants inhibit the activity of NF-κB [138]. For instance, quercetin decreases the phosphorylation of IκBβ, which decreased the activation of NF-κB [152]. However, it has been reported that bilobalide blocks apoptosis of dopaminergic neurons through the suppression of the expression of p65 protein decreasing its nuclear translocation in rat substantia nigra [153] and that administration of ginkgolide B significantly suppresses gene expression of TLR-4 and NF-κB, lessens concentrations of TNFα, IL-1β, and IL-6, and reduces the number of apoptotic neuronal cells in hemorrhagic rat brain [154]. TLR4 stimulates the phosphorylation and degradation of IκB, resulting in nuclear translocation of NF-κB, which initiates transcription of genes associated with apoptosis and inflammation [155, 156]. Production of proinflammatory cytokines like IL-1β, TNFα, and chemokines and the expression of iNOS involve NF-κB activation, which may contribute to neuronal degeneration [156]. In addition, Wang et al. [157] have reported that ginkgolides (A + B) inhibit the nuclear translocation of p65 and downregulates the expression of NF-κB target genes such as c-myc through the inhibition of the phosphorylation of NIK-IKKα-mediated NF-κB activation signaling pathway (NIK/IKK/IκBα/NF-κB).

6.5. AP-1

The activator protein-1 (AP-1) is a series of related dimeric protein complexes that is composed of members of Jun and Fos families, which responds to low levels of oxidants resulting in AP-1/DNA binding and gene expression that mediate diverse signaling events implicated in apoptosis and inflammation [158–160]. AP-1 activation is due to the induction of JNK activity by oxidants resulting in the phosphorylation of serine 63 and serine 73 in the c-jun transactivation domain [161]. JNK exhibits redox-sensitive cysteine residues and could bind to glutathione S-transferase. In the absence of oxidative stress, JNK is associated with glutathione S-transferase, resulting in the inhibition of JNK activity [162]. Under oxidative stress conditions characterized by H₂O₂ overproduction, glutathione S-transferase is dissociated from JNK, resulting in its activation [163]. Numerous studies show that the JNK-AP-1 pathway can stimulate expression of proapoptotic genes such as TNF and FasL [164, 165]. JNK also decreases the expression of prosurvival genes such as Bcl-2 and Bcl-xl [165]. It has been reported that EGb 761 prevents the activation of JNK in the presence of β-amyloid; the EGb 761 ability to decrease H₂O₂ could prevent the dissociation of glutathione S-transferase from JNK resulting in its activation, and this mechanism could also be implicated in the antiapoptotic effect of EGb 761 against oxidative stress induced by β-amyloid [151]. EGb 761 (50–100 μg/mL) inhibits H₂O₂-induced cell apoptosis in SH-SY5Y cells via inactivation of AKT, JNK, and caspase-3 [166]. Similarly, EGb 761 protects against β-amyloid-induced cell apoptosis and abrogates β-amyloid-induced cytotoxic events. Accumulation of ROS and mitochondrial dysfunction and activation of JNK, ERK1/2, and Akt signaling pathways suggest that quercetin and ginkgolide B may be involved in the inhibitory effects of EGb 761 on JNK and ERK1/2 and Akt signaling pathways [85]. EGb 761 also inhibits production of several cytokines, including TNFα, IL-2, IL-4, and IFN-γ, through the down-regulation of JNK/AP-1 signaling pathway [167]. Quercetin, one of the major phenolic constituents of EGb 761, is unique in its ability to inhibit TNF-α transcription by inhibiting the phosphorylation and activation of JNK/SAPK and, therefore, suppressing AP-1-DNA binding [168]. Furthermore, quercetin inhibits iNOS transcription suppressing the phosphorylation of JNK/SAPK as well as its downstream substrates, c-jun and ATF-2, dramatically decreasing the binding of the transcription factor AP-1 to DNA [169]. Shi et al. [85] reported that pretreatment of the SH-SY5Y cells with ginkgolide B totally inhibited PAF-generated increases in c-fos and c-jun mRNA expression.

6.6. P53

The tumor suppressor p53 protein has been proposed as a key mediator of stress response (i.e., oxidative stress) to play an essential role in cell death [170, 171]. P53 induces cell death by a multitude of molecular pathways involving activation of target genes (i.e., Bax. Fas, and DR5) and direct signaling at the mitochondria, leading to cyt c release and caspase activation [172–175]. In vivo and in vitro studies have demonstrated that EGb 761 inhibits the mitochondrion-dependent cell apoptosis, mediated by p53, whereas EGb 761 ameliorates the p53 mRNA expression, disturbs the Bcl-2 family protein balance, disrupts mitochondrial membrane potential, inhibits the release of cyt c to cytosol, and activates caspases [55]. EGb 761 reduced significantly the effects of oxidative stress induced by 6-hydroxydopamine in PC12 cells and inhibits cell apoptosis suppressing p53 and caspase-3 activation and decreasing the ration Bax and Bcl-2. It has been suggested that p53 is a direct transcriptional activator of the Bax gene [176]. Similarly, it was reported that bilobalide, the main constituent of the nonflavone fraction of EGb 761, protects neurons against oxidative stress; bilobalide may block apoptosis in the early stage and attenuate the elevation of c-myc, p53, and Bax and the activation of caspase-3 [177].

7. Conclusions

Apoptosis or programmed cell death may be activated by two signaling pathways: the extrinsic (mediated by death receptors) and the intrinsic (triggered by mitochondria) [32]. Caspases, a family of intracellular endopeptidases, are responsible for this type of cell death [178]. EGb 761 inhibits apoptosis through its antioxidant effects and free-radical trapping (Figure 2) and by the activation of Nrf2/ARE, CREB, and HIF-1 and by decreasing the activities of NF-κB, AP-1, and p53 transcription factors, which modulate antioxidant activity and anti- and proapoptotic genes expression (Figure 3).

Figure 2: Inhibition of apoptosis by EGb 761. This figure details some of the mechanisms by which apoptosis may be inhibited by EGb 761, as described in the text. Apoptosis pathways include those initiated by death-receptor ligand or mitochondrial stress (i.e., ROS). These pathways may be downregulated at several levels by EGb 761: blocking the cascade of activation of caspases (-3, -7, and -9), inhibition of Bax and Apaf-1 proapoptotic protein expression, inhibition of cyt c release from the mitochondrial inner membrane to cytosol, and increments of Bcl-2 expression. EGb 761 also inhibits apoptosis acting as antioxidant and increasing intracellular levels of some enzymatic antioxidants (SOD, Cat, and GPx).

Figure 3: Antiapoptotic mechanisms from EGb 761. EGb 761 inhibits apoptosis through its antioxidant effect and by induction of gene expression, activating NRF2, CREB, HIF1α, and also modulating transcription factors such as NF-κB, AP-1, and p53.

Further studies are needed to corroborate the antiapoptotic activity and mechanisms of action of this phytochemical, since it could constitute an alternative for the treatment of vascular and degenerative diseases among others in which cell death by apoptosis plays a major role (for details see Tables 1 and 2).

Table 1: Specific effects and mechanisms associated with EGb 761 administration in vivo in several models and the equivalent dosage for human.

Dosage of EGb 761
Experimental treatment
Tissue studied
Equivalent dose for human*
Effect
References

Traumatic spinal cord injury

100 mg/Kg i.g. SD rats
Daily, for 1, 7, 14, and 21 days after surgery
Spinal cord
112 mg
The percentage of iNOS-positive cells and apoptotic index of nerve cells in the EGb group was significantly lower than that in the control group, which suggested that EGb 761 suppressed iNOS expression and then prevented nerve cell death
[46]

Aging

100 mg/Kg in SD rats
Daily, for 4 and 12 months
Cochlea
112 mg
EGb 761 treatment significantly prevented aging-related caspase-3/7-induced activity. This result correlates with significant improvement of auditory steady state
[53]

2 mg/Kg in SAMP8 mice
Daily, for 3 and 9 months
Hippocampus and motor cortex
1.12 mg
The Bax/Bcl-2 expression ratio was significantly decreased in the 9-month-old hippocampus and in the 3-month-old motor cortex as compared with the control group
[58]

100 mg/kg in NMRI mice
Daily, for 14 days
Spleen
56 mg
The number of ROS-induced apoptotic cells was significantly reduced in the EGb 761 group
[45]

Ischemia

45 mg/kg, i.v. in SD rats
Just before reperfusion
Hippocampus
50 mg
EGb 761 treatment improved behavior score and enhanced the phosphorylations of Akt and CREB and the expression of BDNF
[116]

100 mg/Kg oral in SAMP8 mice
Daily, 3 days prior to surgery and further 4 days after
Frontal, parietal and temporal lobes, corpus striatum, cerebellum, and brainstem
56 mg
EGb 761 treatment significantly decreased Bax/Bcl-2 ratios as well as caspase-9 levels in all brain regions compared with control animals in both young and aged mice
[62]

100 mg/Kg in SD rats
One hour before the onset of MCAO
Cerebral cortex
112 mg
EGb 761 administration significantly decreased the number of TUNEL-positive cells and the ratio of Bcl-2 and Bax expression
[61]

100 mg/Kg in C57BLK/6 mice, HO- knockout
After 1.5 and 4 h of permanent distal MCAO
Cerebral cortex
56 mg
Treatment with EGb 761 decreased infarct volume and improved neurologic deficit scores. This protective effect was lost in HO-1 knockout
[105]

100 mg/Kg p.o. in C57BLK/6 mice, HO- knockout
Daily, for 7 days before induction of MCAO
Brain
56 mg
EGb 761 improved neurobehavioral function and decreased the infarct size. This effect was abolished in HO-1 knockout
[104]

Cardiovascular diseases

5 mg/Kg i.p. in SD rats
Three doses: One dose every 2 days for 6 days
Heart
5.6 mg
EGb 761 alleviated doxorubicin-induced cardiomyocyte apoptosis through stabilizing a cascade of mitochondrial-signalling effectors from p53, Bcl-2 proteins, cytochrome c, and mitochondrial potential to caspase-3 implicating the additional counteracting action of EGb 761 against doxorubicin apoptotic cardiotoxicity at multiple cellular levels
[55]

50 and 100 mg/Kg in Wistar rats
Daily, for 10 and 15 days
Aortic blood
56 and 112 mg
EGb 761 significantly increased the levels of polyunsaturated fatty acids in erythrocyte membranes, especially the eicosapentaenoic acid, and decreased the saturation index
[179]

Alzheimer’s disease

100 mg/Kg p.o. APPswe/PS1dE9 mice
Daily, for 30 days
Brain
56 mg
EGb 761 treatment had no effect on the size of existing senile plaques, but it had a straightening effect on curved neurites, indicating that neuronal plasticity is fast and still active in adult animals
[180]

100 mg/Kg in C57BL/6J and double transgenic TgAPP/PS1 mice
In diet for 30 days
Hippocampus
56 mg
EGb 761 reduced β-amyloid oligomers and restored CREB phosphorylation
[117]

100 mg/kg in SAMP8 mice
Daily, for 12 weeks
Hippocampus and platelets
56 mg
In platelets, EGb 761 protected against mitochondrial dysfunction, evaluated as cytochrome c oxidase activity, mitochondrial ATP and GSH content. In hippocampi, the protective effect of EGb 761 was observed only in old mice
[52]

Parkinson’s disease

40 mg/Kg i.p. in C57BL/6J mice
Daily, for 18 days
Striatum and midbrain
22.4 mg
EGb 761 administration upregulates the genes for tyrosine hydroxylase, vesicular monoamine transporter 2, dopamine transporter, dopamine D2 receptor, and transcription factors Pitx3 and Nurr1
[181]

40 mg/Kg i.p. in C57BL/6J mice
Daily, for 18 days
Striatum and substantia nigra pars compacta
22.4 mg
EGb 761 significantly attenuated MPTP-induced loss of striatal dopamine levels and tyrosine hydroxylase, blockade of lipid peroxidation, and downregulation of Mn-superoxide dismutase activity. Also, EGb761 improved MPTP-induced impairment of locomotion
[47]

Diabetes

25, 50, and 100 mg/kg i.p. in Wistar rats
Daily, for 14 days
Blood and liver
112, 28, and 56 mg
EGb 761 attenuated the increase of lipoperoxidation and urinary nitrite levels in comparison with control
[182]

The calculations of equivalent dose for human were according to Hernandez-Lopez [183]. The standard weight taken was of 70 kg. SD: Sprague-Dawley, MCAO: middle cerebral artery occlusion.

Table 2: Modulation of molecular signals in apoptosis by EGb 761 and its constituents.

Compounds
Target/mechanisms
References

EGb 761
Reduction of the apoptotic cell death
[44–46]

Reduction of Fas mRNA
[57, 95]

Stabilization of mitochondrial transmembrane potential
[49–51]

Enhancement of energy metabolism
[61]

Increase of the antiapoptotic Bcl-2 protein
[57–59, 62, 95]

Increase of mRNA of proapoptotic genes such as Bax and Bcl-xs and caspases-7, -8, and -12
[57, 62, 95]

Inhibition of cyt c release and activation of Apaf-1
[54, 55]

Reduction of caspase-9 and -3 activities
[51, 60, 62, 63, 65, 66]

Prevention of nuclear DNA fragmentation
[56, 60, 66, 83]

Inhibition of the production of cytokines, including TNFα, IL-2, IL-4, IFN-, and PGE2
[167]

Free radical scavenging activity
[50, 79–84]

Increase of Mn-SOD, GPx, GCLC, GST-P1, and NQO1 mRNA and protein
[101–105, 111]

Induction of Nrf2 nuclear translocation by the increase of the degradation of Keap1
[111, 112]

Induction of CREB phosphorylation via activation of Akt, releasing BDNF
[107, 116]

Activation of SIRT1
[151]

Downregulation of JNK/AP-1 signaling pathway
[164, 165]

Suppression of the activation of p53
[55]

Flavone fraction

Quercetin
Increase of cell proliferation and inhibition of apoptosis
[79–82]

Free radical scavenging activity
[82, 84]

Inhibition of TNF transcription
[167]

Upregulation of HO-1 via the MAPKs/Nrf-2 pathway
[110]

Induction of nuclear translocation of Nrf 2
[110]

Up-regulation of the CREB-BDNF pathway
[120]

Decrease in IkBβ phosphorylation
[152]

Inhibition of phosphorylation and activation of JNK and suppression of AP-1-DNA binding
[169]

Inhibition of ERK activation
[85]

Kaempferol
Inhibition of apoptosis
[79–82, 184–188]

Increase of Bcl-2 protein and decrease of Bax protein.
[184–188]

Inhibition of cyt c release
[187, 188]

Inhibition of caspase-3 activity and internucleosomal DNA fragmentation
[184, 187, 188]

Free radical scavenging activity
[82, 84, 185, 186]

Inhibition of the ascorbate-dependent NADH oxidase
[185]

Induction of GCLC expression and increase of glutathione level
[187]

Up-regulation of HO-1 via the JNK/Nrf-2 pathway
[187]

Suppression of p53 activation
[188]

Inhibition of NF-kB p65 protein
[189]

Isorhamnetin
Prevention of cell death and suppression of apoptosis
[190]

Up-regulation of Bcl-2-related genes
[190]

Down-regulation of the BH3 only and Bax-like-related genes
[190]

Inhibition of cyt c release from the mitochondria into the cytosol
[190]

Inactivation of caspases-3 and -9 and cleavage of PARP
[190]

Attenuation of DNA fragmentation
[190]

ROS-scavenging effect
[82, 84, 190]

Inactivation of the ERK pathway
[190]

Suppression of p53 activation
[190]

Terpenoid fraction

Ginkgolides
Increase of cell proliferation and inhibition of apoptosis
[90, 91]

Free radical scavenging activity
[85–87]

Decrease of the release of LDH, TNFα, IL-1β, and IL-6
[90, 91, 154]

Increase of the activation of the p42/p44 (ERK) MAPK pathway
[137]

Increase of mRNA and protein levels of HIF-1α
[137]

Inhibition of the NIK/IKKα/IkB/NF-kB signaling pathway
[157]

Decreases of c-fos and c-jun mRNA expression
[85]

Bilobalides
Inhibition of apoptosis
[177]

Up-regulation of the subunit III of cytochrome C-oxidase and subunit ND1 of NADH dehydrogenase
[90, 91]

Decrease of c-myc, p53, and Bax proteins
[177]

Inhibition of caspase-3 activity
[177]

Suppression of p65 NF-kB protein
[153]

Up-regulation of the CREB-BDNF pathway
[120]

Conflict of Interests

The authors do not have any conflict of interests with the content of the paper. In addition, they do not have direct financial relation with the commercial identity mentioned in the review.

Acknowledgment

This work was submitted in partial fulfillment of the requirements for the doctoral degree in Experimental Biology for NSG at the Universidad Autónoma Metropolitana, Mexico City, Mexico.

References

J. F. Kerr, A. H. Wyllie, and A. R. Currie, “Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics,” British Journal of Cancer, vol. 26, no. 4, pp. 239–257, 1972.

M. Ebadi and R. F. Pfeiffer, Parkinson’s disease, Cap. 43, CRC, Washington, DC, USA, 2005.

R. A. Kinloch, J. M. Treherne, L. M. Furness, and I. Hajimohamadreza, “The pharmacology of apoptosis,” Trends in Pharmacological Sciences, vol. 20, no. 1, pp. 35–42, 1999.

P. S. Sastry and K. S. Rao, “Apoptosis and the nervous system,” Journal of Neurochemistry, vol. 74, no. 1, pp. 1–20, 2000.

A. Ashkenazi and V. M. Dixit, “Death receptors: signaling and modulation,” Science, vol. 281, no. 5381, pp. 1305–1308, 1998.

S. Gupta, “Molecular steps of cell suicide: an insight into immune senescence,” Journal of Clinical Immunology, vol. 20, no. 4, pp. 229–239, 2000.

D. Nijhawan, N. Honarpour, and X. Wang, “Apoptosis in neural development and disease,” Annual Review of Neuroscience, vol. 23, pp. 73–87, 2000.

N. A. Thornberry and Y. Lazebnik, “Caspases: enemies within,” Science, vol. 281, no. 5381, pp. 1312–1316, 1998.

Z.-B. Wang, Y.-Q. Liu, and Y.-F. Cui, “Pathways to caspase activation,” Cell Biology International, vol. 29, no. 7, pp. 489–496, 2005.

G. M. Cohen, “Caspases: the executioners of apoptosis,” Biochemical Journal, vol. 326, no. 1, pp. 1–16, 1997.

N. Itoh, S. Yonehara, A. Ishii et al., “The polypeptide encoded by the cDNA for human cell surface antigen fas can mediate apoptosis,” Cell, vol. 66, no. 2, pp. 233–243, 1991.

R. Watanabe-Fukunaga, C. I. Brannan, N. Itoh et al., “The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen,” Journal of Immunology, vol. 148, no. 4, pp. 1274–1279, 1992.

S. Nagata and P. Golstein, “The Fas death factor,” Science, vol. 267, no. 5203, pp. 1449–1456, 1995.

S. Nagata, “Fas ligand-induced apoptosis,” Annual Review of Genetics, vol. 33, pp. 29–55, 1999.

J. R. Orlinick, A. K. Vaishnaw, and K. B. Elkon, “Structure and function of Fas/Fas ligand,” International Reviews of Immunology, vol. 18, no. 4, pp. 293–308, 1999.

I. Bechmann, G. Mor, J. Nilsen, et al., “FasL (CD95L, Apo1L) is expressed in the normal rat and human brain,” Glia, vol. 27, no. 1, pp. 62–74, 1999.

C. Choi, J. Y. Park, J. Lee et al., “Fas ligand and Fas are expressed constitutively in human astrocytes and the expression increases with IL-1, IL-6, TNF-α, or IFN-γ,” Journal of Immunology, vol. 162, no. 4, pp. 1889–1895, 1999.

D. J. MacEwan, “TNF ligands and receptors—a matter of life and death,” British Journal of Pharmacology, vol. 135, no. 4, pp. 855–875, 2002.

C. Lambert, A. M. Landau, and J. Desbarats, “Fas-beyond death: a regenerative role for Fas in the nervous system,” Apoptosis, vol. 8, no. 6, pp. 551–562, 2003.

T. Suda, T. Okazaki, Y. Naito et al., “Expression of the Fas ligand in cells of T cell lineage,” Journal of Immunology, vol. 154, no. 8, pp. 3806–3813, 1995.

C. Scaffidi, J. Volkland, I. Blomberg, I. Hoffmann, P. H. Krammer, and M. E. Peter, “Phosphorylation of FADD/MORT1 at serine 194 and association with a 70- kDa cell cycle-regulated protein kinase,” Journal of Immunology, vol. 164, no. 3, pp. 1236–1242, 2000.

J. Zhang, D. Zhang, and Z. Hua, “FADD and its phosphorylation,” IUBMB Life, vol. 56, no. 7, pp. 395–401, 2004.

M. Gómez-Angelats and J. A. Cidlowski, “Molecular evidence for the nuclear localization of FADD,” Cell Death and Differentiation, vol. 10, no. 7, pp. 791–797, 2003.

F. C. Kischkel, S. Hellbardt, I. Behrmann et al., “Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor,” EMBO Journal, vol. 14, no. 22, pp. 5579–5588, 1995.

A. Algeciras-Schimnich, L. Shen, B. C. Barnhart, A. E. Murmann, J. K. Burkhardt, and M. E. Peter, “Molecular ordering of the initial signaling events of CD95,” Molecular and Cellular Biology, vol. 22, no. 1, pp. 207–220, 2002.

U. Felderhoff-Mueser, D. L. Taylor, K. Greenwood et al., “Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain,” Brain Pathology, vol. 10, no. 1, pp. 17–29, 2000.

J. M. Adams and S. Cory, “The Bcl-2 protein family: arbiters of cell survival,” Science, vol. 281, no. 5381, pp. 1322–1326, 1998.

K. C. Zimmermann, C. Bonzon, and D. R. Green, “The machinery of programmed cell death,” Pharmacology and Therapeutics, vol. 92, no. 1, pp. 57–70, 2001.

P. C. Ashe and M. D. Berry, “Apoptotic signaling cascades,” Progress in Neuro-Psychopharmacology and Biological Psychiatry, vol. 27, no. 2, pp. 199–214, 2003.

S. D. Mundle and A. Raza, “Defining the dynamics of self-assembled Fas-receptor activation,” Trends in Immunology, vol. 23, no. 4, pp. 187–194, 2002.

D. Hockenbery, G. Nunez, C. Milliman, R. D. Schreiber, and S. J. Korsmeyer, “Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death,” Nature, vol. 348, no. 6299, pp. 334–336, 1990.

J. Yuan and B. A. Yankner, “Apoptosis in the nervous system,” Nature, vol. 407, no. 6805, pp. 802–809, 2000.

H. Zou, W. J. Henzel, X. Liu, A. Lutschg, and X. Wang, “Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3,” Cell, vol. 90, no. 3, pp. 405–413, 1997.

J. C. Reed, “Cytochrome c: can't live with it—can't live without it,” Cell, vol. 91, no. 5, pp. 559–562, 1997.

F. V. DeFeudis, , Ginkgo Biloba Extract (EGb761): From Chemistry To the Clinic, Ullstein Medical, Wiesbaden, Germany, 1998.

K. M. Maclennan, C. L. Darlington, and P. F. Smith, “The CNS effects of Ginkgo biloba extracts and ginkgolide B,” Progress in Neurobiology, vol. 67, no. 3, pp. 235–257, 2002.

J. Kleijnen and P. Knipschild, “Ginkgo biloba for cerebral insufficiency,” British Journal of Clinical Pharmacology, vol. 34, no. 4, pp. 352–358, 1992.

B. Hochmann, J. Coelho, J. Segura, M. Galli, C. Ketzoian, and M. Pebet, “The incidence of cerebrovascular accidents in the town of Rivera, Uruguay,” Revista de Neurologia, vol. 43, no. 2, pp. 78–83, 2006.

F. V. DeFeudis, “A brief history of EGb 761 and its therapeutic uses,” Pharmacopsychiatry, vol. 36, supplement 1, pp. S2–S7, 2003.

L. M. L. Goh and P. J. Barlow, “Flavonoid recovery and stability from Ginkgo biloba subjected to a simulated digestion process,” Food Chemistry, vol. 86, no. 2, pp. 195–202, 2004.

F. V. DeFeudis and K. Drieu, “Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications,” Current Drug Targets, vol. 1, no. 1, pp. 25–58, 2000.

J. V. Smith and Y. Luo, “Studies on molecular mechanisms of Ginkgo biloba extract,” Applied Microbiology and Biotechnology, vol. 64, no. 4, pp. 465–472, 2004.

U. Ergun, E. Yurtcu, and M. A. Ergun, “Protective effect of Ginkgo biloba against gossypol-induced apoptosis in human lymphocytes,” Cell Biology International, vol. 29, no. 8, pp. 717–720, 2005.

Y.-M. Tian, H.-J. Tian, G.-Y. Zhang, and Y.-R. Dai, “Effects of Ginkgo biloba extract (EGb 761) on hydroxyl radical-induced thymocyte apoptosis and on age-related thymic atrophy and peripheral immune dysfunctions in mice,” Mechanisms of Ageing and Development, vol. 124, no. 8-9, pp. 977–983, 2003.

K. Schindowski, S. Leutner, S. Kressmann, A. Eckert, and W. E. Müller, “Age-related increase of oxidative stress-induced apoptosis in mice prevention by Ginkgo biloba extract (EGb761),” Journal of neural transmission, vol. 108, no. 8-9, pp. 969–978, 2001.

Q. Ao, X.-H. Sun, A.-J. Wang et al., “Protective effects of extract of Ginkgo biloba (EGb 761) on nerve cells after spinal cord injury in rats,” Spinal Cord, vol. 44, no. 11, pp. 662–667, 2006.

P. Rojas, N. Serrano-García, J. J. Mares-Sámano, O. N. Medina-Campos, J. Pedraza-Chaverri, and S. O. Ögren, “EGb761 protects against nigrostriatal dopaminergic neurotoxicity in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-induced Parkinsonism in mice: role of oxidative stress,” European Journal of Neuroscience, vol. 28, no. 1, pp. 41–50, 2008.

S. Desagher and J.-C. Martinou, “Mitochondria as the central control point of apoptosis,” Trends in Cell Biology, vol. 10, no. 9, pp. 369–377, 2000.

J. Sastre, A. Millán, J. García de la Asunción, et al., “A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress,” Free Radical Biology and Medicine, vol. 24, no. 2, pp. 298–304, 1998.

A. Saija, M. Scalese, M. Lanza, D. Marzullo, F. Bonina, and F. Castelli, “Flavonoids as antioxidant agents: importance of their interaction with biomembranes,” Free Radical Biology and Medicine, vol. 19, no. 4, pp. 481–486, 1995.

C. Shi, Z. Yao, J. Xu, and D. T. Yew, “Effects of Gingko extract (EGb761) on oxidative damage under different conditions of serum supply,” Journal of Bioenergetics and Biomembranes, vol. 41, no. 1, pp. 61–69, 2009.

C. Shi, S. Xiao, J. Liu et al., “Ginkgo biloba extract EGb761 protects against aging-associated mitochondrial dysfunction in platelets and hippocampi of SAMP8 mice,” Platelets, vol. 21, no. 5, pp. 373–379, 2010.

J. Nevado, R. Sanz, C. Sánchez-Rodríguez et al., “Ginkgo biloba extract (EGb761) protects against aging-related caspase-mediated apoptosis in rat cochlea,” Acta Oto-Laryngologica, vol. 130, no. 10, pp. 1101–1112, 2010.

Y.-C. Yeh, T.-J. Liu, L.-C. Wang et al., “A standardized extract of Ginkgo biloba suppresses doxorubicin-induced oxidative stress and p53-mediated mitochondrial apoptosis in rat testes,” British Journal of Pharmacology, vol. 156, no. 1, pp. 48–61, 2009.

T.-J. Liu, Y.-C. Yeh, C.-T. Ting et al., “Ginkgo biloba extract 761 reduces doxorubicin-induced apoptotic damage in rat hearts and neonatal cardiomyocytes,” Cardiovascular Research, vol. 80, no. 2, pp. 227–235, 2008.

W. Xin, T. Wei, C. Chen, Y. Ni, B. Zhao, and J. Hou, “Mechanisms of apoptosis in rat cerebellar granule cells induced by hydroxyl radicals and the effects of EGb761 and its constituents,” Toxicology, vol. 148, no. 2-3, pp. 103–110, 2000.

J. V. Smith, A. J. Burdick, P. Golik, I. Khan, D. Wallace, and Y. Luo, “Anti-apoptotic properties of Ginkgo biloba extract EGb 761 in differentiated PC12 cells,” Cellular and Molecular Biology, vol. 48, no. 6, pp. 699–707, 2002.

Y. T. Mak, W. Y. Chan, W. P. Lam, and D. T. Yew, “Immunohistological evidences of Ginkgo biloba extract altering Bax to Bcl-2 expression ratio in the hippocampus and motor cortex of senescence accelerated mice,” Microscopy Research and Technique, vol. 69, no. 8, pp. 601–605, 2006.

X. Jiang, B. Nie, S. Fu et al., “EGb761 protects hydrogen peroxide-induced death of spinal cord neurons through inhibition of intracellular ROS production and modulation of apoptotic regulating genes,” Journal of Molecular Neuroscience, vol. 38, no. 2, pp. 103–113, 2009.

X. Kang, J. Chen, Z. Xu, H. Li, and B. Wang, “Protective effects of Ginkgo biloba extract on paraquat-induced apoptosis of PC12 cells,” Toxicology in Vitro, vol. 21, no. 6, pp. 1003–1009, 2007.

P.-O. Koh, “Gingko biloba extract (EGb 761) prevents increase of Bad-Bcl-XL interaction following cerebral ischemia,” American Journal of Chinese Medicine, vol. 37, no. 5, pp. 867–876, 2009.

G. Lu, Y. Wu, Y. T. Mak et al., “Molecular evidence of the neuroprotective effect of Ginkgo biloba (EGb761) using bax/bcl-2 ratio after brain ischemia in senescence-accelerated mice, strain prone-8,” Brain Research, vol. 1090, no. 1, pp. 23–28, 2006.

S. Li, D. Tang, Z. Xue et al., “Biphasic effect of EGb761 on simulated ischemia-induced rat BMSC survival in vitro and in vivo,” Life Sciences, vol. 88, no. 19-20, pp. 853–863, 2011.

L. Massieu, J. Morán, and Y. Christen, “Effect of Ginkgo biloba (EGb 761) on staurosporine-induced neuronal death and caspase activity in cortical cultured neurons,” Brain Research, vol. 1002, no. 1-2, pp. 76–85, 2004.

D. C. Ren, G.-H. Du, and J. T. Zhang, “Protective effect of Ginkgo biloba extract on endothelial cell against damage induced by oxidative stress,” Journal of Cardiovascular Pharmacology, vol. 40, no. 6, pp. 809–814, 2002.

C.-L. Hsu, Y.-L. Wu, G.-J. Tang, T.-S. Lee, and Y. R. Kou, “Ginkgo biloba extract confers protection from cigarette smoke extract-induced apoptosis in human lung endothelial cells: role of heme oxygenase-1,” Pulmonary Pharmacology and Therapeutics, vol. 22, no. 4, pp. 286–296, 2009.

J. C. Reed, “Apoptosis-based therapies,” Nature Reviews Drug Discovery, vol. 1, no. 2, pp. 111–121, 2002.

M. B. Graeber and L. B. Moran, “Mechanisms of cell death in neurodegenerative diseases: fashion, fiction, and facts,” Brain Pathology, vol. 12, no. 3, pp. 385–390, 2002.

N. Shibata and M. Kobayashi, “The role for oxidative stress in neurodegenerative diseases,” Brain and Nerve, vol. 60, no. 2, pp. 157–170, 2008.

Y. Chen, E. McMillan-Ward, J. Kong, S. J. Israels, and S. B. Gibson, “Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells,” Cell Death and Differentiation, vol. 15, no. 1, pp. 171–182, 2008.

S. J. Chanock, J. El Benna, R. M. Smith, and B. M. Babior, “The respiratory burst oxidase,” Journal of Biological Chemistry, vol. 269, no. 40, pp. 24519–24522, 1994.

J. F. Turrens, “Mitochondrial formation of reactive oxygen species,” Journal of Physiology, vol. 552, part 2, pp. 335–344, 2003.

C. W. Olanow, “A radical hypothesis for neurodegeneration,” Trends in Neurosciences, vol. 16, no. 11, pp. 439–444, 1993.

L. Wang, N. Azad, L. Kongkaneramit et al., “The Fas death signaling pathway connecting reactive oxygen species generation and FLICE inhibitory protein down-regulation,” Journal of Immunology, vol. 180, no. 5, pp. 3072–3080, 2008.

Y. Qian, T. Guan, M. Huang, et al., “Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model,” Neurochemistry International, vol. 60, no. 8, pp. 759–767, 2012.

K. T. Turpaev and D. Y. Litvinov, “Redox-dependent regulation of the expression of nitric oxide-inducible genes,” Molecular Biology, vol. 38, no. 1, pp. 47–57, 2004.

J. B. Schulz, J. Lindenau, J. Seyfried, and J. Dichgans, “Glutathione, oxidative stress and neurodegeneration,” European Journal of Biochemistry, vol. 267, no. 16, pp. 4904–4911, 2000.

J. Lindenau, H. Noack, H. Possel, K. Asayama, and G. Wolf, “Cellular distribution of superoxide dismutases in the rat CNS,” Glia, vol. 29, no. 1, pp. 25–34, 2000.

L. Marcocci, J. J. Maguire, M. T. Droy-Lefaix, and L. Packer, “The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761,” Biochemical and Biophysical Research Communications, vol. 201, no. 2, pp. 748–755, 1994.

J. Pincemail, M. Dupuis, C. Nasr et al., “Superoxide anion scavenging effect and superoxide dismutase activity of Ginkgo biloba extract,” Experientia, vol. 45, no. 8, pp. 708–712, 1989.

I. Maitra, L. Marcocci, M. T. Droy-Lefaix, and L. Packer, “Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761,” Biochemical Pharmacology, vol. 49, no. 11, pp. 1649–1655, 1995.

C. Ferradini, M. T. Droy Lefaix, and Y. Christen, Cap 1, Advances in Gingko Biloba Extract Research, Elsevier, Paris, France, 1993.

D. J. Kane, T. A. Sarafian, R. Anton et al., “Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species,” Science, vol. 262, no. 5137, pp. 1274–1277, 1993.

L. Marcocci, L. Packer, M.-T. Droy-Lefaix, A. Sekaki, and M. Gardes-Albert, “Antioxidant action of Ginkgo biloba extract EGb 761,” Methods in Enzymology, vol. 234, pp. 462–475, 1994.

C. Shi, L. Zhao, B. Zhu et al., “Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells,” Chemico-Biological Interactions, vol. 181, no. 1, pp. 115–123, 2009.

S. Pietri, E. Maurelli, K. Drieu, and M. Culcasi, “Cardioprotective and anti-oxidant effects of the terpenoid constituents of Ginkgo biloba extract (EGb 761),” Journal of Molecular and Cellular Cardiology, vol. 29, no. 2, pp. 733–742, 1997.

W. Song, H.-J. Guan, X.-Z. Zhu, Z.-L. Chen, M.-L. Yin, and X.-F. Cheng, “Protective effect of bilobalide against nitric oxide-induced neurotoxicity in PC12 cells,” Acta Pharmacologica Sinica, vol. 21, no. 5, pp. 415–420, 2000.

K. Gohil, R. K. Moy, S. Farzin, J. J. Maguire, and L. Packer, “MRNA expression profile of a human cancer cell line in response to Ginkgo biloba extract: induction of antioxidant response and the golgi system,” Free Radical Research, vol. 33, no. 6, pp. 831–849, 2000.

F. V. DeFeudis, “Effects of Ginkgo biloba extract (EGb 761) on gene expression: possible relevance to neurological disorders and age-associated cognitive impairment,” Drug Development Research, vol. 57, no. 4, pp. 214–235, 2002.

K. Chandrasekaran, Z. Mehrabian, B. Spinnewyn, K. Drieu, and G. Fiskum, “Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia,” Brain Research, vol. 922, no. 2, pp. 282–292, 2001.

E. A. Tendi, F. Bosetti, S. F. Dasgupta, A. M. Giuffrida Stella, K. Drieu, and S. I. Rapoport, “Ginkgo biloba extracts EGB 761 and bilobalide increase NADH dehydrogenase mRNA level and mitochondrial respiratory control ratio in PC12 cells,” Neurochemical Research, vol. 27, no. 4, pp. 319–323, 2002.

H. S. Sharma, K. Drieu, P. Alm, and J. Westman, “Role of nitric oxide in blood-brain barrier permeability, brain edema and cell damage following hyperthermic brain injury. An experimental study using EGB-761 and Gingkolide B pretreatment in the rat,” Acta Neurochirurgica Supplement, vol. 76, pp. 81–86, 2000.

G. C. Brown and V. Borutaite, “Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols,” Biochimica et Biophysica Acta, vol. 1658, no. 1-2, pp. 44–49, 2004.

H. Matsuzaki, M. Tamatani, N. Mitsuda et al., “Activation of Akt kinase inhibits apoptosis and changes in Bcl-2 and Bax expression induced by nitric oxide in primary hippocampal neurons,” Journal of Neurochemistry, vol. 73, no. 5, pp. 2037–2046, 1999.

C. M. H. Watanabe, S. Wolffram, P. Ader et al., “The in vivo neuromodulatory effects of the herbal medicine Ginkgo biloba,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 12, pp. 6577–6580, 2001.

P. Talalay, “Chemoprotection against cancer by induction of Phase 2 enzymes,” BioFactors, vol. 12, no. 1–4, pp. 5–11, 2000.

Y. Zhang and G. B. Gordon, “A strategy for cancer prevention: stimulation of the Nrf2-ARE signaling pathway,” Molecular Cancer Therapeutics, vol. 3, no. 7, pp. 885–893, 2004.

A. T. Dinkova-Kostova, W. D. Holtzclaw, R. N. Cole et al., “Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 18, pp. 11908–11913, 2002.

L. M. Zipper and R. T. P. Mulcahy, “The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm,” Journal of Biological Chemistry, vol. 277, no. 39, pp. 36544–36552, 2002.

T. Satoh, S.-I. Okamoto, J. Cui et al., “Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophillic phase II inducers,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 3, pp. 768–773, 2006.

K. Li, P. Yao, S.-L. Zhou, and F.-F. Song, “Protective effects of extract of Ginkgo biloba against ethanol-induced oxidative injury in rat testes,” Journal of Hygiene Research, vol. 34, no. 5, pp. 559–562, 2005.

O. Coskun, F. Armutcu, M. Kanter, and G. M. Kuzey, “Protection of endotoxin-induced oxidative renal tissue damage of rats by vitamin E or/and EGb 761 treatment,” Journal of Applied Toxicology, vol. 25, no. 1, pp. 8–12, 2005.

X.-P. Liu, C. E. P. Goldring, H.-Y. Wang et al., “Extract of Ginkgo biloba induces glutamate cysteine ligase catalytic subunit (GCLC),” Phytotherapy Research, vol. 22, no. 3, pp. 367–371, 2008.

S. Saleem, H. Zhuang, S. Biswal, Y. Christen, and S. Doré, “Ginkgo biloba extract neuroprotective action is dependent on heme oxygenase 1 in ischemic reperfusion brain injury,” Stroke, vol. 39, no. 12, pp. 3389–3396, 2008.

Z. A. Shah, S. E. Nada, and S. Doré, “Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection,” Neuroscience, vol. 180, pp. 248–255, 2011.

R. Stocker, A. F. McDonagh, A. N. Glazer, and B. N. Ames, “Antioxidant activities of bile pigments: biliverdin and bilirubin,” Methods in Enzymology, vol. 186, pp. 301–309, 1990.

A. I. Goodman, P. N. Chander, R. Rezzani et al., “Heme oxygenase-2 deficiency contributes to diabetes-mediated increase in superoxide anion and renal dysfunction,” Journal of the American Society of Nephrology, vol. 17, no. 4, pp. 1073–1081, 2006.

C. K. Andreadi, L. M. Howells, P. A. Atherfold, and M. M. Manson, “Involvement of Nrf2, p38, B-Raf, and nuclear factor-κB, but not phosphatidylinositol 3-kinase, in induction of hemeoxygenase-1 by dietary polyphenols,” Molecular Pharmacology, vol. 69, no. 3, pp. 1033–1040, 2006.

S. Das, C. G. Fraga, and D. K. Das, “Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NFκB,” Free Radical Research, vol. 40, no. 10, pp. 1066–1075, 2006.

P. Yao, A. Nussler, L. Liu et al., “Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways,” Journal of Hepatology, vol. 47, no. 2, pp. 253–261, 2007.

X.-P. Liu, C. E. P. Goldring, I. M. Copple et al., “Extract of Ginkgo biloba induces phase 2 genes through Keap1-Nrf2-ARE signaling pathway,” Life Sciences, vol. 80, no. 17, pp. 1586–1591, 2007.

N. Wakabayashi, A. T. Dinkova-Kostova, W. D. Holtzclaw et al., “Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 7, pp. 2040–2045, 2004.

B. Mayr and M. Montminy, “Transcriptional regulation by the phosphorylation-dependent factor creb,” Nature Reviews Molecular Cell Biology, vol. 2, no. 8, pp. 599–609, 2001.

X. Tao, S. Finkbeiner, D. B. Arnold, A. J. Shaywitz, and M. E. Greenberg, “Ca²⁺ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism,” Neuron, vol. 20, no. 4, pp. 709–726, 1998.

K. Du and M. Montminy, “CREB is a regulatory target for the protein kinase Akt/PKB,” Journal of Biological Chemistry, vol. 273, no. 49, pp. 32377–32379, 1998.

Z. Zhang, D. Peng, H. Zhu, and X. Wang, “Experimental evidence of Ginkgo biloba extract EGB as a neuroprotective agent in ischemia stroke rats,” Brain Research Bulletin, vol. 87, no. 2-3, pp. 193–198, 2012.

F. Tchantchou, Y. Xu, Y. Wu, Y. Christen, and Y. Luo, “EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimer's disease,” FASEB Journal, vol. 21, no. 10, pp. 2400–2408, 2007.

Y. Xu, C. Cui, C. Pang, Y. Christen, and Y. Luo, “Restoration of impaired phosphorylation of cyclic AMP response element-binding protein (CREB) by EGb 761 and its constituents in Aβ-expressing neuroblastoma cells,” European Journal of Neuroscience, vol. 26, no. 10, pp. 2931–2939, 2007.

Y. Hou, M. A. Aboukhatwa, D.-L. Lei, K. Manaye, I. Khan, and Y. Luo, “Anti-depressant natural flavonols modulate BDNF and beta amyloid in neurons and hippocampus of double TgAD mice,” Neuropharmacology, vol. 58, no. 6, pp. 911–920, 2010.

F. Tchantchou, P. N. Lacor, Z. Cao et al., “Stimulation of neurogenesis and synaptogenesis by bilobalide and quercetin via common final pathway in hippocampal neurons,” Journal of Alzheimer's Disease, vol. 18, no. 4, pp. 787–798, 2009.

G. L. Wang and G. L. Semenza, “Purification and characterization of hypoxia-inducible factor 1,” Journal of Biological Chemistry, vol. 270, no. 3, pp. 1230–1237, 1995.

G. L. Wang, B.-H. Jiang, E. A. Rue, and G. L. Semenza, “Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 12, pp. 5510–5514, 1995.

B.-H. Jiang, G. L. Semenza, C. Bauer, and H. H. Marti, “Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension,” American Journal of Physiology, vol. 271, part 1, no. 4, pp. C1172–C1180, 1996.

S. Lahiri, A. Roy, S. M. Baby, T. Hoshi, G. L. Semenza, and N. R. Prabhakar, “Oxygen sensing in the body,” Progress in Biophysics and Molecular Biology, vol. 91, no. 3, pp. 249–286, 2006.

U. R. Jewell, I. Kvietikova, A. Scheid, C. Bauer, R. H. Wenger, and M. Gassmann, “Induction of HIF-1alpha in response to hypoxia is instantaneous,” The FASEB Journal, vol. 15, no. 7, pp. 1312–1314, 2001.

G. L. Semenza, “Targeting HIF-1 for cancer therapy,” Nature Reviews Cancer, vol. 3, no. 10, pp. 721–732, 2003.

K. Zaman, H. Ryu, D. Hall et al., “Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic enzymes, p21(waf1/cip1), and erythropoietin,” Journal of Neuroscience, vol. 19, no. 22, pp. 9821–9830, 1999.

N. Akakura, M. Kobayashi, I. Horiuchi et al., “Constitutive expression of hypoxia-inducible factor-lα renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient deprivation,” Cancer Research, vol. 61, no. 17, pp. 6548–6554, 2001.

L. Flamant, A. Notte, N. Ninane, M. Raes, and C. Michiels, “Anti-apoptotic role of HIF-1 and AP-1 in paclitaxel exposed breast cancer cells under hypoxia,” Molecular Cancer, vol. 9, article no. 191, 2010.

P. F. Smith, K. Maclennan, and C. L. Darlington, “The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF),” Journal of Ethnopharmacology, vol. 50, no. 3, pp. 131–139, 1996.

L. Zhu, J. Wu, H. Liao, J. Gao, X.-N. Zhao, and Z.-X. Zhang, “Antagonistic effects of extract from leaves of Ginkgo biloba on glutamate neurotoxicity,” Acta Pharmacologica Sinica, vol. 18, no. 4, pp. 344–347, 1997.

L. Zhu, J. Gao, Y. Wang, X. N. Zhao, and Z. X. Zhang, “Neuron degeneration induced by verapamil and attenuated by EGb761,” Journal of Basic and Clinical Physiology and Pharmacology, vol. 8, no. 4, pp. 301–314, 1997.

S. Bastianetto and R. Quirion, “Natural extracts as possible protective agents of brain aging,” Neurobiology of Aging, vol. 23, no. 5, pp. 891–897, 2002.

B. Ahlemeyer and J. Krieglstein, “Neuroprotective effects of Ginkgo biloba extract,” Cellular and Molecular Life Sciences, vol. 60, no. 9, pp. 1779–1792, 2003.

B. Ahlemeyer and J. Krieglstein, “Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease,” Pharmacopsychiatry, vol. 36, supplement 1, pp. S8–S14, 2003.

L. Zhu, X.-M. Wu, L. Yang, F. Du, and Z. M. Qian, “Up-regulation of HIF-1α expression induced by ginkgolides in hypoxic neurons,” Brain Research, vol. 1166, no. 1, pp. 1–8, 2007.

Z. Li, K. Ya, W. Xiao-Mei, Y. Lei, L. Yang, and Z. M. Qian, “Ginkgolides protect PC12 cells against hypoxia-induced injury by p42/p44 MAPK pathway-dependent upregulation of HIF-1α expression and HIF-1 DNA-binding activity,” Journal of Cellular Biochemistry, vol. 103, no. 2, pp. 564–575, 2008.

G. Gloire and J. Piette, “Redox regulation of nuclear post-translational modifications during NF-κB activation,” Antioxidants and Redox Signaling, vol. 11, no. 9, pp. 2209–2222, 2009.

H. L. Pahl, “Activators and target genes of Rel/NF-κB transcription factors,” Oncogene, vol. 18, no. 49, pp. 6853–6866, 1999.

K. G. Waddick and F. M. Uckun, “Innovative treatment programs against cancer: II. Nuclear factor-κB (NF-κB) as a molecular target,” Biochemical Pharmacology, vol. 57, no. 1, pp. 9–17, 1999.

M. A. Collart, P. Baeuerle, and P. Vassalli, “Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four κB-like motifs and of constitutive and inducible forms of NF-κB,” Molecular and Cellular Biology, vol. 10, no. 4, pp. 1498–1506, 1990.

H. Wu and G. Lozano, “NF-κB activation of p53. A potential mechanism for suppressing cell growth in response to stress,” Journal of Biological Chemistry, vol. 269, no. 31, pp. 20067–20074, 1994.

S. Kasibhatla, T. Brunner, L. Genestier, F. Echeverri, A. Mahboubi, and D. R. Green, “DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1,” Molecular Cell, vol. 1, no. 4, pp. 543–551, 1998.

T. M. Baetu, H. Kwon, S. Sharma, N. Grandvaux, and J. Hiscott, “Disruption of NF-κB signaling reveals a novel role for NF-κB in the regulation of TNF-related apoptosis-inducing ligand expression,” Journal of Immunology, vol. 167, no. 6, pp. 3164–3173, 2001.

S.-J. Kim, S.-G. Hwang, D. Y. Shin, S.-S. Kang, and J.-S. Chun, “p38 kinase regulates nitric oxide-induced apoptosis of articular chondrocytes by accumulating p53 via NF κB-dependent transcription and stabilization by serine 15 phosphorylation,” Journal of Biological Chemistry, vol. 277, no. 36, pp. 33501–33508, 2002.

J. Kucharczak, M. J. Simmons, Y. Fan, and C. Gélinas, “To be, or not to be: NF-κB is the answer—role of Rel/NF-κB in the regulation of apoptosis,” Oncogene, vol. 22, no. 56, pp. 8961–8982, 2003.

Y. Tao, D. Yan, Q. Yang, R. Zeng, and Y. Wang, “Low K+ promotes NF-κB/DNA binding in neuronal apoptosis induced by K+ loss,” Molecular and Cellular Biology, vol. 26, no. 3, pp. 1038–1050, 2006.

L.-F. Chen, Y. Mu, and W. C. Greene, “Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB,” EMBO Journal, vol. 21, no. 23, pp. 6539–6548, 2002.

F. Yeung, J. E. Hoberg, C. S. Ramsey et al., “Modulation of NF-κB-dependent transcription and cell survival by the SIRT1 deacetylase,” EMBO Journal, vol. 23, no. 12, pp. 2369–2380, 2004.

Y.-M. Park, J.-H. Won, K.-J. Yun et al., “Preventive effect of Ginkgo biloba extract (GBB) on the lipopolysaccharide-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 via suppression of nuclear factor-κB in RAW 264.7 cells,” Biological and Pharmaceutical Bulletin, vol. 29, no. 5, pp. 985–990, 2006.

F. Longpré, P. Garneau, Y. christen, and C. Ramassamy, “Protection by EGb 761 against β-amyloid-induced neurotoxicity: involvement of NF-κB, SIRT1, and MAPKs pathways and inhibition of amyloid fibril formation,” Free Radical Biology and Medicine, vol. 41, no. 12, pp. 1781–1794, 2006.

M. P. Nair, S. Mahajan, J. L. Reynolds et al., “The flavonoid quercetin inhibits proinflammatory cytokine (tumor necrosis factor alpha) gene expression in normal peripheral blood mononuclear cells via modulation of the NF-κ β system,” Clinical and Vaccine Immunology, vol. 13, no. 3, pp. 319–328, 2006.

L.-Y. Li, X.-L. Zhao, X.-F. Fei, Z.-L. Gu, Z.-H. Qin, and Z.-Q. Liang, “Bilobalide inhibits 6-OHDA-induced activation of NF-κB and loss of dopaminergic neurons in rat substantia nigra,” Acta Pharmacologica Sinica, vol. 29, no. 5, pp. 539–547, 2008.

Y.-Y. Hu, M. Huang, X.-Q. Dong, Q.-P. Xu, W.-H. Yu, and Z.-Y. Zhang, “Ginkgolide B reduces neuronal cell apoptosis in the hemorrhagic rat brain: possible involvement of Toll-like receptor 4/nuclear factor-kappa B pathway,” Journal of Ethnopharmacology, vol. 137, no. 3, pp. 1462–1468, 2011.

K. V. Anderson, “Toll signaling pathways in the innate immune response,” Current Opinion in Immunology, vol. 12, no. 1, pp. 13–19, 2000.

T. Ueno, Y. Sawa, S. Kitagawa-Sakakida et al., “Nuclear factor-κB decoy attenuates neuronal damage after global brain ischemia: a future strategy for brain protection during circulatory arrest,” Journal of Thoracic and Cardiovascular Surgery, vol. 122, no. 4, pp. 720–727, 2001.

X. Wang, Z. H. Qin, H. Shi, et al., “Protective effect of Ginkgolids (A+B) is associated with inhibition of NIK/IKK/IkappaB/NF-kappaB signaling pathway in a rat model of permanent focal cerebral ischemia,” Research, vol. 1234, pp. 8–15, 2008.

R. Wisdom, “Ap-1: one switch for many signals,” Experimental Cell Research, vol. 253, no. 1, pp. 180–185, 1999.

E. Shaulian and M. Karin, “AP-1 as a regulator of cell life and death,” Nature Cell Biology, vol. 4, no. 5, pp. E131–E136, 2002.

M. Karin and E. Shaulian, “AP-1: linking hydrogen peroxide and oxidative stress to the control of cell proliferation and death,” IUBMB Life, vol. 52, no. 1-2, pp. 17–24, 2001.

H. Kamata, S.-I. Honda, S. Maeda, L. Chang, H. Hirata, and M. Karin, “Reactive oxygen species promote TNFα-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases,” Cell, vol. 120, no. 5, pp. 649–661, 2005.

T. Wang, P. Arifoglu, Z. Ronai, and K. D. Tew, “Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus,” Journal of Biological Chemistry, vol. 276, no. 24, pp. 20999–21003, 2001.

V. Adler, Z. Yin, S. Y. Fuchs et al., “Regulation of JNK signaling by GSTp,” EMBO Journal, vol. 18, no. 5, pp. 1321–1334, 1999.

N. Matsumoto, R. Imamura, and T. Suda, “Caspase-8- and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 production,” FEBS Journal, vol. 274, no. 9, pp. 2376–2384, 2007.

C. Trejo, D. Jimenez, S. Rodriguez et al., “Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation,” BMC Cancer, vol. 12, article 156, 2012.

C. Shi, L. Zhao, B. Zhu et al., “Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells,” Chemico-Biological Interactions, vol. 180, no. 3, pp. 389–397, 2009.

S.-M. Cheng, S.-P. Yang, L.-J. Ho et al., “Down-regulation of c-jun N-terminal kinase-activator protein-1 signaling pathway by Ginkgo biloba extract in human peripheral blood T cells,” Biochemical Pharmacology, vol. 66, no. 4, pp. 679–689, 2003.

T. L. Wadsworth, T. L. McDonald, and D. R. Koop, “Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factor-α,” Biochemical Pharmacology, vol. 62, no. 7, pp. 963–974, 2001.

T. L. Wadsworth and D. R. Koop, “Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced release of nitric oxide,” Chemico-Biological Interactions, vol. 137, no. 1, pp. 43–58, 2001.

B. Vogelstein, D. Lane, and A. J. Levine, “Surfing the p53 network,” Nature, vol. 408, no. 6810, pp. 307–310, 2000.

D. A. Liebermann, B. Hoffman, and D. Vesely, “p53 induced growth arrest versus apoptosis and its modulation by survival cytokines,” Cell Cycle, vol. 6, no. 2, pp. 166–170, 2007.

T. Miyashita and J. C. Reed, “Tumor suppressor p53 is a direct transcriptional activator of the human bax gene,” Cell, vol. 80, no. 2, pp. 293–299, 1995.

R. S. Morrison, Y. Kinoshita, M. D. Johnson, W. Guo, and G. A. Garden, “p53-dependent cell death signaling in neurons,” Neurochemical Research, vol. 28, no. 1, pp. 15–27, 2003.

S. Matsuyama and J. C. Reed, “Mitochondria-dependent apoptosis and cellular pH regulation,” Cell Death and Differentiation, vol. 7, no. 12, pp. 1155–1165, 2000.

P. Bonini, S. Cicconi, A. Cardinale et al., “Oxidative stress induces p53-mediated apoptosis in Glia: p53 transcription-independent way to die,” Journal of Neuroscience Research, vol. 75, no. 1, pp. 83–95, 2004.

J. Wang, Y. Cheng, J. Yin, Q. Lu, X. Xu, and X. Yin, “Protective effects of Ginkgo biloba extract on 6-hydroxydopamine-induced apoptosis in PC12 cells,” Neural Regeneration Research, vol. 6, no. 33, pp. 2565–2572, 2011.

L.-J. Zhou and X.-Z. Zhu, “Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide,” Journal of Pharmacology and Experimental Therapeutics, vol. 293, no. 3, pp. 982–988, 2000.

M. Chen and J. Wang, “Initiator caspases in apoptosis signaling pathways,” Apoptosis, vol. 7, no. 4, pp. 313–319, 2002.

K. Drieu, R. Vranckx, C. Benassayad et al., “Effect of the extract of Ginkgo biloba (EGb 761) on the circulating and cellular profiles of polyunsaturated fatty acids: correlation with the anti-oxidant properties of the extract,” Prostaglandins Leukotrienes and Essential Fatty Acids, vol. 63, no. 5, pp. 293–300, 2000.

M. Garcia-Alloza, L. A. Borrelli, B. T. Hyman, and B. J. Bacskai, “Antioxidants have a rapid and long-lasting effect on neuritic abnormalities in APP:PS1 mice,” Neurobiology of Aging, vol. 31, no. 12, pp. 2058–2068, 2010.

P. Rojas, E. Ruiz-Sánchez, C. Rojas, and S. O. Ögren, “Ginkgo biloba extract (EGb 761) modulates the expression of dopamine-related genes in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced Parkinsonism in mice,” Neuroscience, vol. 223, pp. 246–257, 2012.

R. Taliyan and P. L. Sharma, “Protective effect and potential mechanism of Ginkgo biloba extract EGb 761 on STZ-induced neuropathic pain in rats,” Phytotherapy Research, vol. 26, no. 12, pp. 1823–1829, 2012.

C. Hernández-López, “Nuevo marco regulatorio de los ensayos clínicos de primera administración en humanos,” Investigación Clínica y Bioética Digital, no. 50, pp. 1–10, 2007.

Y. Zhang and D. Liu, “Flavonol kaempferol improves chronic hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function,” European Journal of Pharmacology, vol. 670, no. 1, pp. 325–332, 2011.

A. K. Samhan-Arias, F. J. Martín-Romero, and C. Gutiérrez-Merino, “Kaempferol blocks oxidative stress in cerebellar granule cells and reveals a key role for reactive oxygen species production at the plasma membrane in the commitment to apoptosis,” Free Radical Biology and Medicine, vol. 37, no. 1, pp. 48–61, 2004.

K. Murota, S. Shimizu, S. Miyamoto et al., “Unique uptake and transport of isoflavone aglycones by human intestinal Caco-2 cells: comparison of isoflavonoids and flavonoids,” Journal of Nutrition, vol. 132, no. 7, pp. 1956–1961, 2002.

S. S. Gao, B.-M. Choi, X. Y. Chen et al., “Kaempferol suppresses cisplatin-induced apoptosis via inductions of heme oxygenase-1 and glutamate-cysteine ligase catalytic subunit in HEI-OC1 cells,” Pharmaceutical Research, vol. 27, no. 2, pp. 235–245, 2010.

J. Xiao, G.-B. Sun, B. Sun et al., “Kaempferol protects against doxorubicin-induced cardiotoxicity in vivo and in vitro,” Toxicology, vol. 292, no. 1, pp. 53–62, 2012.

Y. K. Byung, S. Kim, K.-H. Lee et al., “Transcriptional profiling in human HaCaT keratinocytes in response to kaempferol and identification of potential transcription factors for regulating differential gene expression,” Experimental and Molecular Medicine, vol. 40, no. 2, pp. 208–219, 2008.

B. Sun, G.-B. Sun, J. Xiao et al., “Isorhamnetin inhibits H₂O₂-induced activation of the intrinsic apoptotic pathway in H9c2 cardiomyocytes through scavenging reactive oxygen species and ERK inactivation,” Journal of Cellular Biochemistry, vol. 113, no. 2, pp. 473–485, 2012.