Neuroactive Responses In Vivo To Ginkgo Biloba Egb 761

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An important limitation of the in vitro assay of gene expression analysis of herbal extracts is that it does not account for the processes that affect bioavailability and biotransformation. These biological processes of the alimentary tract and the liver will modify herb components. Therefore, an in vivo study was undertaken in normal mice to define the potential in vivo transcriptional activity of EGb 761 (3). The hypothesis was that a centrally active herbal extract should change the gene expression profile in the brain. Survey of the literature identified several reports that indicated central effects of orally administered EGb 761 in rodents (23,24) and in humans (25,26).

Figure 7 Glutathione levels are elevated after EGb 761 treatment of cultured cells (HEK, human keratinocytes; T-24, human bladder cells; HepG2, liver cells; and RAW, murine macrophages) (19).

Cortex and hippocampus were chosen as target neuroa-natomical sites for gene expression analysis of ~ 12,000 mouse genes (Affymetrix, Murine Genome Mu74Av2). Mice were fed a diet supplemented with Ginkgo biloba extract as previously described (3) which was supplemented with 1mg of EGb 761 per day. Plasma was obtained from blood samples and analyzed for flavonoids as previously described (27). Dietary supplementation for 30 days increased the presence of flavonoids, quercetin, and kempherol, in plasma of mice supplemented with EGb 761 (3). These data support previous observations that orally administered extract is bioavailable in rodents (28,29) and in humans (30,31). The extract was well tolerated as indicated by absence of any effects on the dietary intake or the whole body weight of mice.

Analysis of the expression of 12,000 mouse genes in cortex and hippocampus showed that the orally administered

Phase 2 Enzymes







Y-glutamyl synthetase HO-1

Mitochondrial Genes Vesicular Proteins Glutathione Transferases NAD(P)H-Quinone Reductase UDP-glucoronosyltransferases

Figure 8 Antioxidant and electrophile response elements appear to be the common targets of transcription factors activated by Ginkgo biloba extract EGb 761 (18).

extract was centrally active (3). The expression of 43 genes and 13 genes was up regulated by at least twofold in cortex and hippocampus, respectively. A small number of genes such as those encoding growth hormone, prolactin, serum albumin LINE-1 repeat, and serine protease inhibitor were activated in both the brain regions. These observations are remarkable for three reasons. First, they provide molecular evidence for the action of a dietary supplement in the brain. Second, they show that EGb 761 has differential effects in the two brain regions that were selected for gene analysis. Third, the extract increased the abundance of the affected mRNAs suggesting increase in transcription although we cannot exclude the possibility of increased stability of mRNAs.

The heterogeneity in the responsiveness of the two neu-roanatomical regions raises an important issue of targeting specific brain regions by dietary supplementation for desired molecular outcome and its effects on physiological, pathophy-siological, and behavioral outcomes. Functional classification of the affected genes showed that Ginkgo biloba extract affected the expression ofgenes encoding transcription factors, ion-channels, growth factors and neuromodulators, synaptic vesicles and transport, cell surface, and protein kinases and phosphates.

The selective induction and transtheyretin transcript in hippocampus but not in cortex is noteworthy. Defects in hip-pocampal functions have been described in Alzheimer's disease (32,33). Transthyretin, a tetrameric protein important in the transport of vitamin A and an accessory protein for the transport of thyroxine, plays a role in the formation of amyloid products present in degenerating brain (34,35). The activation of transthyretin gene by EGb 761 suggests that the extract may regulate the metabolism of amyloid products in vivo. The mechanisms by which transthyretin may affect the formation of neuronal amyloid is poorly understood. However, a role forthyroxine may be implicated because it binds to trasnsthyretin and affects neuronal growth and development (36). A gene related to that of transthyretin, serum albumin gene with LINE-1 repeat, was induced (eightfold) in cortex and to a lesser magnitude (threefold) in hippocampus. These observations emphasize the differential effects of herbal extract in the brain.

EGb 761 also activated the transcription of genes that encode peptides important in cellular growth and function. These included growth hormone, prolactin, oxytosin neuro-physin 1, placental growth factor, brain derived neurothro-pic factor, platelet derived growth factor, and insulin like growth factor receptor 1. Also noteworthy was the simultaneous induction of the genes for oligodendrocyte basic protein and proteolipid protein, both of which are essential for the biogenesis and assembly of myelin (37-39). Collectively, these observations provide some of the potential molecular targets through which the extract of Ginkgo biloba confers neuroprotective effects observed in rodent models of human stroke (40,41) and may contribute to regeneration of neurons.

The complex processes of learning and memory recruit multiple cellular types and molecular pathways that involve ion-channels (42,43). The expression of several genes encoding proteins of ion-channels was induced in the brain cortex and hippocampus after 30 days of dietary supplementation (3). These included the genes for chloride protein 3, calcium activated potassium channel, alpha subunit of GABAa recep-

Table 1 Effect of Ginkgo Biloba Extract (EGb 761), Ginkgolide A, B, and Bilobalide on GSH Levels in (HaCaT) Human Kerati-nocytes (18)

Table 1 Effect of Ginkgo Biloba Extract (EGb 761), Ginkgolide A, B, and Bilobalide on GSH Levels in (HaCaT) Human Kerati-nocytes (18)


3.32 ± 0.20


EGb 761 (100 mg/mL)

6.40 ± 0.26


Ginkgolide A 4 mg/mL

3.17 ± 0.34


Ginkgolide B 4 mg/mL

2.97 ± 0.33


Bilobalide 4 mg/mL

2.92 ± 0.20


tor, alpha-2 subunit of glutamate receptor, and type VII alpha polypeptide of voltage gated sodium channel. The induction of the ion-channel genes was accompanied by the activation of several genes encoding proteins in the signal transduction pathways. These included calmodulin 3, neuronal tyrosine threonine phosphatase 1, the beta subunit of phosphatidylinositol 4-phosphate 5-kinase, cAMP dependent regulatory subunit of protein kinase, magnesium dependent protein phosphatase 1B, protein tyrosine phosphatese IF2p, protein tyrosine phosphatase-1, serine/arginine rich protein specific kinase 2, Janus N-terminal kinase 2, Gz subunit of GTP binding protein and epsilon subunit of protein kinase C.

In summary, gene expression profiling of hippocampus and cortex from mice whose diets were supplemented with EGb 761 for 4 weeks showed significant increases in the transcripts encoding brain proteins. Many of these proteins play a vital role in neuronal and synaptic plasticity. The data reviewed here identify a molecular phenotype of EGb 761 actions. The behavioral phenotype of EGb 761 in health and in neurological disease remains to be defined. The data may offer some molecular correlates of behavioral changes observed in some studies (24,44). Future studies that combine the tools of functional genomics and behavioral analysis to study the effects Ginkgo biloba leaves are essential for critical and objective analysis of the in vivo effects of the dietary anti-oxidant supplements.


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