Glutamate is considered to be an important excitatory neurotransmitter that mediates it effects by binding to and activating ionotropic and metabotropic glutamate receptors in the brain [1
]. Both in vitro and in vivo studies have demonstrated that over activation of ionotropic glutamate receptors (such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N
-aspartate (NMDA), and kainate receptors) through exposure to high levels of glutamate can lead to excitotoxicity. It has been hypothesized that chronic excitotoxicity contributes to the onset of a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease [1
]. The NMDA and the two “non-NMDA” kainate and AMPA receptors have subtly different functions within the brain. The AMPA receptors are predominantly responsible for mediating the influx of sodium while the kainate receptors regulate the influx of both sodium and potassium ions. The NMDA receptors have the greatest conductivity by allowing the influx of positive ions. While both NMDA and kainic acid are physiological ligands for these receptors, over-activation can result in excitotoxicity due to an increased influx into the cell of calcium ions (Ca2+
). Increase in the cellular concentration of Ca2+
activates enzymatic signaling cascades that are integral to the regulation of numerous cellular functions including the oxidative stress response, inflammation, mitochondrial impairment, and caspase activation. The dysregulation of these pathways ultimately leads to the death of neuronal cells. In recent decades, the interest in herbal medicines as a therapeutic resource in disease prevention, health promotion, and recovery has grown in many countries. There is some evidence demonstrating the use of botanicals as useful modulators of Brain-derived neurotrophic factor (BDNF) in central nervous system (CNS) diseases [2
]. Further preclinical and clinical studies are needed to support the safe use of herbal medicines as prophylactic and/or therapeutic strategies for the treatment of neurodegenerative diseases [2
Over the last decade there has been increasing evidence to support the therapeutic benefits of Panax ginseng
and Ginkgo biloba
and their components in neurodegenerative brain disease [3
]. The beneficial effects observed have been attributed predominantly, but not exclusively, to the immunomodulatory and antioxidative properties of the herbal medicines. The pharmacological effects of P. ginseng
are due largely to the action of ginsenosides, which are considered to be the major active components. However, other bioactive ingredients of P. ginseng,
such as the phytosterols, sesquiterpene, flavonoids, polyacetylese, alkaloids, and phenolic compounds, are involved in the important role of eliciting the beneficial effects of the ginsenosides [5
]. Water extract of P. ginseng
has been demonstrated to have a protecting effect against 1-methyl-4-phenylpyridinium-iodide (MPP+)-induced apoptosis in in vitro models of Parkinson’s disease [9
]. Other studies have demonstrated that ginsenoside Rb1 can protect dopaminergic neurons, SH-SY5Y cells, and PC12 cells from 6-OHDA- or MPP+-induced toxicity [10
]. Ginsenoside Rd has been demonstrated in male ischemic rat models to increase extracellular glutamate clearance by the upregulation of GLT-1 expression, mediated by the activation of PI3K/AKT and ERK1/2 signaling pathways [13
]. Further to this, Ginsenoside Rd has been shown to decrease levels of apoptotic proteins such as PARP1 and Bax, via adenylate cyclase-associated protein 1 (CAP1) regulation in an in vitro model of Alzheimer’s disease [14
]. Ginsenoside Rg1 reduced the amyloid β-stimulated expression of Toll-like receptors and TNF-α in a NG108-15 neuroglia cell line. P. ginseng
extracts showed neuroprotective effects by ameliorating the advanced glycation end-product-induced memory impairment and reducing the pathophysiological changes through down regulation of the RAGE/NF-kB pathway [15
]. Furthermore, in Alzheimer-like rat models, ginsenoside reduced the d
-galactose- and aluminum chloride (AlCl3)-induced spatial memory impairment through restoration of neurotransmitter levels, tau phosphorylation, and amyloid β formation [16
]. In an in vitro model of Huntington’s disease, ginsenosides protected striatal neurons in an Huntington’s disease (HD) mouse model from glutamate toxicity [17
Research conducted with the G. biloba
extract (containing 49% total flavones; 28.7% glycosides; 11.6% gingkolides (sum of A, B, C, and bilobalide); and 3.3% gingkolide A) in human astrocytes demonstrated reduced neuroinflammation by blocking the generation of pro-inflammatory cytokines and oxygen-glucose deprivation (OGD)-induced signal transducer and activator of transcription (STAT3) activation [18
]. The same authors observed that G. biloba
was able to attenuate cerebral infarction and neuronal apoptosis and reduce neurological deficiencies in cerebral ischemic rats [18
]. The extract inhibited the Aβ induced activation of NF-κB and MAPK pathways in the neuroblastoma cell line N2a, thereby protecting the neuronal cells from Aβ toxicity [19
]. Kim and colleagues observed that pretreatment with daily administration of Egb761®
extract induced a neuroprotective effect on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the rat brain [20
]. The neuroprotective effects of G. biloba
correlated to the regulation of the content of copper in the brain, as observed in animal models of Parkinson’s disease [21
]. In vitro studies with PC12 neuronal cells investigating Aβ (1–42) treatment (aggregated and soluble form) showed that G. biloba
extracts have the potential to prevent Aβ-induced reactive oxygen species (ROS) production, cytotoxicity, glucose uptake, and apoptosis as well as the development of Aβ-derived diffusible neurotoxic ligands. These neurotoxic ligands have been implicated in mediating the neurotoxic effect of Aβ [22
]. In C. elegans, G. biloba
alleviates Aβ-induced pathological behavior, inhibits Aβ oligomerization and deposits (not by reducing oxidative stress), and attenuates both the basal and Aβ-induced levels of H2
-related reactive oxygen species in Alzheimer’s disease models of neurodegeneration [23
]. A study conducted by Liu et al. using a transgenic mouse model investigated the anti-inflammatory activity and underlying molecular pathways impacted by treatment with G. biloba
extract in the context of Alzheimer’s disease. The results showed inhibition of neuroinflammation, reduction of cognitive deficit and synaptic impairment, and enhanced autophagy, as well as the prevention of Aβ-induced microglial inflammatory activation [25
]. In a recent systematic review, Reay and colleagues investigated both the psychological and physiological impact of the combination of G. biloba
and P. ginseng
) in human subjects. In particular, the most interesting results were the observed benefits of treatment on both the circulatory and cardiovascular systems as well as the improvement in the “secondary memory” performance in the total patient population. The conclusion of that review was that the Gincosan®
treatment can improve the physiological and cognitive functions in humans [26
In the present study, in vitro models of excitotoxicity were used to examine the neuroprotective effects of P. ginseng G115® and G. biloba GK501® extracts. The extracts were tested alone and in combination (mix), maintaining the same ratio as the commercial product Gincosan®.
Excitotoxicity-induced neurodegeneration is a determinant in various neurodegenerative diseases such as ischemia-induced brain damage, traumatic injury, disease, Parkinson’s disease, and Huntington’s disease [1
]. Ginseng and ginkgo have been two of the most extensively used herbal medicines in Eastern Asian countries for more 2000 years [3
]. Various studies have reported that ginseng and ginkgo protect against injury in numerous neurological diseases, including Parkinson’s disease [9
], Alzheimer’s disease [14
] and Huntington’s disease [17
]. The neuroprotective effects were also observed for ginkgo in ischemia [18
], against Aβ toxicity [19
], and in Parkinson’s models [20
]. Additionally, the activation of the PI3K/Akt pathways attenuates injury and alleviates damage to the brain in many neurological diseases [29
], and ginseng and ginkgo have been shown to independently activate these pathways [13
]. Neuroprotection of ginseng and ginkgo extracts is also related to their anti-inflammatory properties [31
In this study, we utilized in vitro experimental models of excitotoxicity to explore the neuroprotective effect of native P. ginseng and G. biloba GK501®. We demonstrated in our models that both extracts reduced excitotoxic insult-induced neuronal damage in the hippocampus and cortex, and that the protective effects of the extracts were improved when used in combination.
Brain function is dependent on good nutrition and a healthy lifestyle. Plant extracts such as P. ginseng
and G. biloba
can also contribute to a healthy, cognitive function. In our study, we demonstrated that organotypic hippocampal slices and mixed cortical cells exposed to the extracts resulted in a marked attenuation of neuronal damage following excitotoxic injury. Slices exposed to the combination had reduced CA1 (NMDA model) cell death compared to slices treated with single extracts alone. When slices were treated with ginseng and ginkgo alone, a significant attenuation of neuronal damage induced by kainic acid in the CA3 area of organotypic slices was observed. Again, the combination of two extracts showed a more enhanced neuroprotective effect. The doses of extracts were selected in succession to achieve a probable clinical application, and they were extrapolated from other antecedent in vitro and in vivo preclinical studies and clinical use [26
]. In the mixed cortical cells model of exitotoxicity, the neuroprotective effect of both ginseng and ginkgo was dose-dependent showing the greatest impact at the highest concentrations. As with the organotypic hippocampal slices model, treatment of the mixed cortical cells with the combination of extracts exhibited a higher degree of neuroprotection as compared to single extracts alone. When used at different time points after the NMDA insult, only the combination preserved significant neuroprotective effects. For the first time, our results provide evidence of the additive neuroprotective effects of the combination of P. ginseng
and G. biloba
and their safety in neuron samples. Differentiating properties of the combination from the single extracts, our results suggest a role of the mix as a new potential approach in clinical settings to counteract those mechanisms that were found to be the cause of some neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.
Furthermore, in this study we have demonstrated that native P. ginseng
and G. biloba
extracts alone and in combination are able to activate pro-survival hippocampal signaling cascades including the Akt and the Erk1/2 pathways in organotypic hippocampal slices but not in mixed cortical cells. Activation of these pathways may, in part, increase the ability of neuronal cells to survive toxic insults such as the excitotoxic treatment that was mimicked by exposing hippocampal slices to NMDA and kainic acid. In this study, NMDA and kainic acid consistently induced death of neurons of the CA1 and CA3 regions of organotypic hippocampal slices, which correlated to the reduced hippocampal levels of p-Akt that we have previously observed [32
] but also of p-Erk1/2. In the presence of the both extracts, NMDA- and kainate-induced cell death was reduced and, in parallel, the levels of p-Akt and p-Erk1/2 remained high in the hippocampus. In cortical cells, we did not observe a decrease of the protein levels after NMDA treatment, but in the presence of both extracts, the phosphorylation increased. This supports the idea that there is a causal relationship between the neuroprotection observed with P. ginseng
and G. biloba
treatment and the activation of the PI3K/Akt and ERK1/2 pathways in the hippocampus. These findings are in agreement with a number of studies [3
], suggesting that these extracts may be used as prevention or to improve the pharmacological treatment of neurological diseases.
4. Materials and Methods
Male and female Wistar rat pups (7–9 days old), mouse CD1 pups (1–2 days old), and pregnant CD1 mice were obtained from Charles River (MI, Italy). Animals were housed at 23 ± 1 °C under a 12 h light–dark cycle (lights on at 07:00) and were fed a standard laboratory diet with ad libitum access to water. Experiments and animal use procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). The experimental protocols were approved by the Animal Care Committee of the Department of Health Sciences, University of Florence, in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123) project n 273/2016-PR (11/03/2016) and the European Communities Council Directive of 24 November 1986 (86/609/EEC). The authors further attest that all efforts were made to minimize the number of animals used and their suffering.
Native extract of P. ginseng and G. biloba GK501® extracts were a kind gift of Soho Flordis International Pty Ltd. Tissue culture reagents were obtained from Gibco-BRL (San Giuliano Milanese, Milano, Italy) and Sigma (St Louis, MO, USA).
4.3. Solubility Tests
The solubility tests were performed to determine the best solvent to use for each extract.
The G. biloba GK501® and P. ginseng G115® are contained in a commercially available product (Gincosan® capsules) in the amounts of 60 mg and 100 mg per capsule.
According to Pharmacopoeia European, the standardized P. ginseng G115® is obtained by ethanol extraction (40% v/ v) of the dried roots of P. ginseng and standardized on the total content of eight major ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg2) at 4.0% with a drug extract ratio (DER) of 3–7:1.
The extract is in line with the monographs and Pharmacopoeias world-wide, including the European Pharmacopoeia, World Health Organization (WHO) Ginseng Monograph, and ESCOP (Scientific Foundation for Herbal Medicinal Products) Monographs.
The standardized G. biloba GK501® is obtained by acetone/water extraction from the dried leaves of the G. biloba. The extract is standardized on the total content of ginkgo-flavone glycosides (22–27%) and of terpene lactones (5–7%). The extract conforms to the German Commission E Monograph and follows the indication of the WHO and ESCOP Monographs.
In our experiments, the native extracts of P. ginseng, which is the main constituent of the standardized P. ginseng G115®, and G. biloba GK501® were appropriately solubilized to prepare standard solutions that reflected the composition of Gincosan® and the posology of two capsules per day.
According to the specifications, 37.73 mg of this extract are equivalent to 100 mg of P. ginseng G115®. This aspect was considered for the solubility study and for the preparation of the samples for the pharmacological experiments.
Dimethyl sulfoxide (DMSO) and EtOH:H2O at different ratios (50:50, 60:40, and 70:30) were evaluated for suitability as solvents for both extracts. EtOH:H2O 50:50 was selected as the most appropriate solvent mixture, and was also compatible with the cell culture. The concentrations of standard solutions were: 1.08 mg/mL of native extract of P. ginseng, 1.78 mg/mL of G. biloba GK501®, and 2.79 mg/mL of mix.
4.4. Organotypic Rat Hippocampal Slice Models of Excitotoxicity
Organotypic hippocampal slice cultures were prepared as previously reported [34
]. Briefly, hippocampi were removed from the brains of 7–9 days old Wistar rats (Charles River Laboratories, Milano, Italy), and transverse slices (420 µm) were prepared using a McIlwain tissue chopper and transferred onto semiporous membrane inserts and maintained in culture for 14 days in vitro. The slices were incubated with 5 µM kainic acid or 10 µM NMDA for 24 h [35
]. Native P. ginseng
and G. biloba
extracts alone or in combination (mix) were incubated during the 24 h treatment. Cell death was evaluated by using the fluorescent dye propidium iodide (5 µg/mL), and fluorescence was viewed using an inverted fluorescence microscope. Images were analyzed using morphometric analysis software. For cellular death, the CA3 and CA1 hippocampal subfields were identified, respectively, for kainic acid and NMDA toxicity, and were quantified using the image software v. 1.40g (ImageJ; NIH, Bethesda, MD, USA) that detected the optical density of PI fluorescence.
4.5. Cortical Mice Cell Cultures Model of Excitotoxicity
Cortical mice cell cultures were prepared as previously reported [37
]. Cerebral cortices were dissected from embryonic days (ED) 17–18 CD1 mice (Charles River, MI, Italy), cells were suspended and plated on a layer of confluent astrocytes. After 4–5 days in vitro (DIV), non-neuronal cell division was halted with cytosine arabinoside. After two weeks of maturation, the cells were exposed to 300 µM NMDA for 10 min as previously described [38
]. NMDA neurotoxicity was obtained by exposing cultures to 300 µM NMDA for 10 min in HEPES medium after the original treatment medium was restored, and cells were stored in normal condition for 24 h, followed by assessment of the extent of neuronal death. To obtain the maximal neuronal injury, the cultures were exposed for 24 h to 1 mM glutamate. Native P. ginseng
and G. biloba
extracts alone or as a mix were present in the incubation medium during NMDA treatment and during the 24 h of recovery period. The quantity of lactate dehydrogenase (LDH) released was measured 24 h after exposure to NMDA to evaluate the cell damage. Background LDH release was determined in control cultures. The resulting values correlated linearly with the degree of cell loss estimated by observation of cultures under phase-contrast microscopy.
4.6. Western Blotting
Western blotting was conducted as previously described [27
]. Four slices for sample and mixed cortical cells (3 wells/sample) were dissolved in 1% SDS. BCA (bicinchoninic acid) Protein Assays were used to quantify the total protein levels. Lysates (20 µg/lane of protein) were resolved by electrophoresis on a 4–20% SDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto nitrocellulose membranes. After blocking, the blots were incubated overnight at 4 °C with polyclonal-rabbit antibody against phospho-ERK1/2 (Thr202/Thr204) and phospho-Akt (Ser473) (all from Cell Signaling Technology, Beverly, MA, USA) diluted 1:1000 in TBS-T containing 5% bovine serum albumin. GAPDH was used as a loading control (monoclonal antibody purchased from Sigma (St Louis, MO, USA)). Immunodetection was performed with HRP-conjugated secondary antibodies (1:2000 anti-mouse, anti-rabbit, or anti-goat IgG from donkey, Amersham Biosciences, UK) in TBS-T containing 5% non-fat dry milk. After washing, membranes and reactive bands were detected using chemiluminescence (ECLplus; Euroclone, Padova, Italy). Quantity One analysis software was used for quantitative analysis (Bio-Rad, Hercules, CA, USA). Results are presented as the mean minus the standard error of the mean (SEM) of different gels and expressed as arbitrary unit (AU), which depicts the ratio between levels of target protein expression (for pERK1/2 both the bands were quantitated) and GAPDH normalized to basal levels [27
4.7. Statistical Analysis
Data are presented as means ± SEM of n experiments. The statistical significance of differences between PI fluorescence intensities, LDH release, or Western blot optical densities was analyzed using one-way ANOVA with a post hoc Tukey’s w-test for multiple comparisons. All statistical calculations were performed using GRAPH-PAD PRISM v. 5 for Windows (GraphPad Software, San Diego, CA, USA). A probability value p < 0.05 was considered significant.