A Mixture of Ginkgo biloba L. Leaf and Hericium erinaceus (Bull.) Pers. Fruit Extract Attenuates Scopolamine-Induced Memory Impairments in Mice

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by loss of memory and cognitive impairment via dysfunction of the cholinergic nervous system. In cholinergic dysfunction, it is well known that impaired cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) signaling are major pathological markers and are some of the strategies for the development of AD therapy. Therefore, this study is aimed at evaluating whether a mixture comprising Ginkgo biloba L. leaf (GL) and Hericium erinaceus (Bull.) Pers. (HE) fruit extract (GH mixture) alleviated cognitive impairment induced in a scopolamine-induced model. It was discovered that GH reduced neuronal apoptosis and promoted neuronal survival by activating BDNF signaling in an in vitro assay. In addition, the GH (p.o. 240 mg/kg) oral administration group significantly restored the cognitive deficits of the scopolamine-induced mouse group (i.p. 1.2 mg/kg) in the behavior tests such as Y-maze and novel object recognition task (NORT) tests. This mixture also considerably enhanced cholinergic system function in the mouse brain. Furthermore, GH markedly upregulated the expressed levels of extracellular signal-regulated kinase (ERK), CREB, and BDNF protein levels. These results demonstrated that GH strongly exerted a neuroprotective effect on the scopolamine-induced mouse model, suggesting that an optimized mixture of GL and HE could be used as a good material for developing functional foods to aid in the prevention of neurodegenerative diseases, including AD.


Introduction
Alzheimer's disease (AD), which involves continuous memory and cognitive dysfunction, is an age-related neurodegenerative disease [1]. As mentioned in the World Health Organization (WHO) report, this disease is one of the rapidly increasing chronic diseases that is globally affecting approximately 30 million people over the age of 60 years each year [2]. Acetylcholine (ACh) released from the hippocampus and cortex plays a vital role in attention, memory, and learning [3]. However, the brain of AD patients has critically elevated levels of acetylcholinesterase (AChE), which is involved in the breakdown of ACh, resulting in a loss of memory and cognitive functions [4]. It is clear that dysfunction of cholinergic neurons is mainly involved in AD pathogenesis [5]. Hence, the development of AD therapy has mainly focused on the protective effect on the cholinergic system. Several drugs, as acetylcholinesterase inhibitors (e.g., tacrine, rivatigmine, galantamine, and donepezil), have been approved for the treatment of AD, but they only help in keeping symptoms from worsening [6]. However, they have short half-lives and side effects such as hepatotoxicity and nausea [7]. Thus, it is needed to find alternative drugs to treat AD which show strong effects without any toxicity.
Many studies have shown that the scopolamine-induced AD model is useful for evaluating AD progression [8,9].
Scopolamine, an ACh antagonist that leads to cholinergic dysfunction, is a tropane alkaloid that has been used for studying cognitive deficits in preclinical studies [10]. Numerous studies have already demonstrated that memory impairment in the scopolamine-induced mouse model is correlated with the production of oxidative stresses, such as through reactive oxygen species (ROS) from the brain via the dysfunction of antioxidant enzymes [11]. Moreover, it has been reported that scopolamine interferes with the neuronal protection mechanisms, including extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) mouse studies [12][13][14].
Meanwhile, cognitive enhancers, also known as nootropics, have been studied with respect to alleviating the suffering of AD patients [15]. Nootropics improve learning and memory through several mechanisms (i.e., blockage of calcium channels, inhibition of AChE activities, enhancement of the levels of antioxidants, and synaptic and mitochondrial responses genes) [16]. Indeed, these agents also show neuroprotective effects via amyloid beta accumulation, synaptic dysfunctions, apoptosis, inflammation, and oxidative stress [15]. Many types of nootropics, which are drugs or herbal agents that improve memory function, have been reported [17]. The two kinds of nootropics are synthetic compounds (e.g., piracetam) and natural/herbal nootropics (e.g., Ginkgo biloba L. leaf (GL) and Hericium erinaceus (Bull.) Pers.
(HE)) [17][18][19][20]. In particular, natural nootropics act through various mechanisms in the brain [15]: (1) modulating neurotransmitter release, (2) improving neuronal function, (3) protecting against oxidative damage, and (4) serving as an energy booster. In addition, the application of nootropics for chronic treatment shows improvement in memory function via the regulation of ERK/CREB/BDNF pathways on the hippocampus. In view of the aforementioned points, nootropics from natural resources are recognized as alternative materials used to improve the cognitive impairment in AD.
Clinical studies with GL in dementia patients have pointed out that it slows down the decline in mental function and neuropsychiatric symptoms. In addition, GL showed efficacy and safety for treatment of cognitive impairment and dementia in clinical studies [21,22]. HE revealed that it ameliorated BDNF levels and depressive behavior in chronically stressed animals, improved memory impairment, protected neurons from neurotoxicity (e.g., beta-amyloid, hydrogen peroxide, and lipopolysaccharide), and restored hippocampal damage after pilocarpine-induced status epilepticus [19,[23][24][25]. The well-known edible nutrient nootropics, such as GL and HE, are cognitive enhancers. Nevertheless, it is reported that each such agent may not have any effect on memory dysfunction. This study was thus carried out to investigate the synergic effects of the improvement in cognitive function, both in vitro and in vivo.

High-Performance Liquid Chromatography (HPLC)
Analysis of GH. GH, which was mixed in a ratio 1 : 5 of GL and HE, was analyzed using a Waters e2695 Separation Module (Waters Corp., Milford, MA, USA) with a 2998 Photodiode Array Detector. For the analysis of kaempferol, quercetin, isorhamnetin, and acacetin from GH, twenty milligrams of GH mixture and standard compounds (0.1, 1, and 10 μg/mL) were dissolved in 50% acetonitrile/water. The mobile phase consisted of 0.4% phosphoric acid/water (solvent A) and acetonitrile (solvent B) with the following gradient: 0-10 min, 10% B; 10-40 min, 10-70% B; 40-44 min, 70-100% B; and 44-47 min, 0% B, with a flow rate of 1 mL/min [26]. For the analysis of hericene A and hericene D from GH, three milligrams of GH mixture and standard compounds (0.1, 1, and 10 μg/mL) were dissolved in 30% ethyl acetate/methanol. For isocratic analyses, the mobile phase consisted of 97% methanol/3% water with a flow rate of 1 mL/min [27]. For the analysis of ginkgolide A and ginkgolide B, twenty milligrams of GH mixture and standard compounds (1, 10, and 100 μg/mL) were dissolved in 50% methanol/water. For isocratic analyses, the mobile phase consisted of 72.5% water/17.5% water/10% isopropanol with a flow rate of 1 mL/min [28]. All analyses used a Kromasil C 18 column (150 mm × 4:6 mm, 5 μm). The column temperature was set at 30°C, and the PDA detector was set at 280-340 nm to acquire chromatograms.

Measurement of Mitochondrial Membrane Potential.
The mitochondrial membrane potential assay was carried out using a JC-10 kit (Abcam, CA, USA) [31]. SH-SY5Y cells were seeded in 96-well plates and pretreated with various concentrations of GH (dissolved in PBS). After incubation for 1 h at 37°C, scopolamine was cotreated with GH for 24 h at 37°C. After treatment, the cell mitochondria were stained using 50 μL of JC-10 solution for 30 min at 37°C and kept away from light. After incubation, 50 μL of buffer B solution was added into the JC-10 loading plate before reading the fluorescence intensity, and this was analyzed using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The fluorescence intensities were measured at Ex/Em = 490/525 nm and 490/590 nm for ratio analysis.
2.8. Animal Study. Institute of Cancer Research (ICR, 6 weeks old, male) mice were obtained from Orient Bio Inc. (Gyeonggi-do, Korea). Each mouse was housed in ventilated cages under a specific pathogen-free environment (temperature, 23 ± 2°C; relative humidity, 55 ± 5%). Mice were fed an AIN-76A purified rodent diet (Central Laboratory Animal Inc., Seoul, Korea) and given water ad libitum. All mice (n = 60) were randomly divided into six groups (n = 10 per group): normal (10 mL/kg, per os (p.o.)), scopolamine (Sco, 1.2 mg/kg, intraperitoneal injection (i.p.)), donepezil (PC, 1 mg/kg, p.o.), G. biloba L. leaf (GL, 40 mg/kg, p.o.), H. erina-ceus (Bull.) Pers. fruit (HE, 200 mg/kg, p.o.), and a mixture of GL and HE (GH, 240 mg/kg, p.o.). All samples were dissolved in 0.9% saline and administered for 1 week. Afterward, each group was induced with 1.2 mg/kg scopolamine (dissolved in 0.9% saline) 30 min before Y-maze and NORT tests on the first day. Body weight was measured once weekly from day 0 and did not change among groups ( Figure 1S). The Y-maze (n = 5 per group) and novel objective recognition task (NORT (n = 5 per group)) were performed from day 7 to day 8 (see Figure 1(a)) [32,33]. After behavior tests, all mice (n = 10 per group) were sacrificed and collected whole brain and then analyzed for histology (n = 3 per group) and biochemical assessments (n = 7 per group). This in vivo study was carried out according to the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Gachon University, Korea (Approval No. GIACUC-R2019001-2). In addition, all behavior tests were analyzed by using SMART 3.0® video tracking software (Panlab, Barcelona, Spain).
2.9. Y-Maze Test. The Y-maze is a maze of three identical arms (60 × 15 × 12 cm 3 ). Each arm was marked as A, B, and C. Each mouse was placed in the center of this tool, and the sequence (e.g., ABC) and the numbers of arm entries were manually recorded through video analysis. In this behavior test, the actual alternation was defined as entries into arms on consecutive choices (i.e., ABC, BCA, or BAC but not ACA). This tool was cleaned using 70% ethanol. The percentage of spontaneous alternation performance (%) was calculated by using equation (1)  . Each mouse was placed in the box with two different objects for 3 min on the 1 st day of training. The next day, mice were placed in the box again in which one of the identical objects had been replaced with a novel object. The exploration time of familiar and novel objects was recorded.
2.11. Acetylcholine (ACh) and Acetylcholinesterase (AChE) Activity Assay. After the behavioral tests, each mouse group was sacrificed, and the brain was collected and stored at -80°C. Before the ACh and AChE activity assay, whole brain (n = 7 per group) with PBS (Ca. 20-fold volumes) buffer was homogenized using an MP FastPrep-24 Instrument (MP Biomedicals Inc., SA, USA). The homogenates in PBS were centrifuged at 12,000 × g for 30 min at 4°C. The upper solution was used for ACh and AChE activity. The amount of brain lysate solution was calculated using the Bio-Rad Bradford kit (Bio-Rad). ACh levels were analyzed using an EnzyChrom™ acetylcholine assay kit (BioAssay System, CA, USA). Briefly, 20 μL of brain lysate solution was mixed with 80 μL working 3 Oxidative Medicine and Cellular Longevity reagent in a 96-well plate. The color intensity was detected using a microplate reader (Bio-Rad) at 570 nm from 0 min to 10 min, compared with that of the standard (ACh) [34].
AChE activity was analyzed using the QuantiChrom™ Acetylcholinesterase Assay Kit (BioAssay System). The AChE assay was conducted in a 96-well plate, and 10 μL of brain lysate solution was mixed with a 190 μL working reagent in a 96-well plate. The intensities of samples were detected using a microplate reader (Bio-Rad) at 412 nm from 0 min to 10 min [34]. AChE activity of each group was calculated as the percentage, compared to the control group. The exploration times of the familiar and novel objects were recorded. Each group was orally administered with GH (240 mg/kg), GL (40 mg/kg), HE (200 mg/kg), PC (1 mg/kg donepezil), or water. Before behavior tests (30 min), Sco was intraperitoneally injected for constructing the memory deficit model. All behavior tests were analyzed by using video tracking software. Each behavior represents the mean ± SEM (n = 4-5). # P < 0:05 and ## P < 0:01 vs. control group. * * P < 0:01 and * * * P < 0:001 vs. Sco-treated group. 4 Oxidative Medicine and Cellular Longevity (iNtRON Biotechnology, Seongnam, Korea) to obtain proteins. The protein amount from each sample was calculated using the Bio-Rad Bradford kit (Bio-Rad). An equal amount (30 μg) of protein was loaded and separated using 10-12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Millipore Corp., MA, USA). Blocking was performed with 5% skimmed milk for 2 h, and the blots were incubated for 1 d with primary antibodies against Bax, Bcl-2, cleavedcaspase-3, BDNF, CREB, pCREB, ERK, pERK, AKT, pAKT, GSK3β, pGSK3β, and GAPDH (dilution 1 : 1000). The blots were then incubated with secondary antibodies (dilution, 1 : 2000). Protein bands were analyzed using the Chemi Doc-XRS + imaging system (Bio-Rad).
2.13. Histopathological Analysis. We obtained whole mouse brain by carrying out perfusion for blood removal according to a previous study [27]. Brain tissues (n = 3 per group) were collected from all groups and then fixed in 10% formalin. Fixed tissues were processed to obtain 4 μm paraffinembedded sections. Each section was stained with hematoxylin and eosin (H&E) and analyzed at 4-40x magnification using a microscope (Olympus, Tokyo, Japan). The optical density of H&E-stained tissue sections in the dentate gyrus (DG), cornu ammonis 1 (CA1), and cornu ammonis 2/3 (CA2/3) regions was analyzed using the ImageJ software (Bethesda, MD, USA).
2.14. Immunohistochemistry Analysis. The obtained 4 μm paraffin-embedded sections (n = 3 per group) were deparaffinized using xylene and rehydrated with EtOH (100%, 90%, 80%, and 70%), followed by treatment with an endogenous peroxidase blocker, and finally washed with PBS. These sections were incubated with the primary antibody, BDNF (dilution 1 : 500), at 4°C. After washing in PBS, each section was incubated with a biotinylated anti-goat and anti-rabbit IgG (dilution 1 : 200) for 1 h and then with avidin-biotin horseradish peroxidase complex (Vector Laboratories, CA, USA). The optical density of BDNF immunoreactivity in the CA1 and CA2/3 regions was analyzed using ImageJ software. The images were photographed at 4-40x magnification using a microscope (Olympus).

Statistical Analysis.
In this study, all data were analyzed using GraphPad Prism 5 software (CA, USA) and represented as means ± standard error of the mean (SEM). All results were analyzed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer's t-test for post hoc analysis. Differences with a P value less than 0.05 were considered statistically significant.

GH Improves Neuroprotective Effects on Scopolamine-
Induced Cytotoxicity in SH-SY5Y Cells. Scopolamine showed cytotoxicity in neuronal cells with cholinergic dysfunction [10]. For studying the cytotoxicity effect of scopolamine in SH-SY5Y cells, the SH-SY5Y cells were treated with 1, 3, or 5 mM scopolamine for 1 day. As shown in Figure 2(a), treatment with 5 mM scopolamine significantly decreased cell viability (63:33 ± 3:96%, ### P < 0:001) compared to that of the control group. Therefore, further tests were performed using 5 mM scopolamine to investigate the neuroprotective effects of GH on scopolamine-induced cytotoxicity. In addition, GH was mixed with GL and HE in a 1 : 5 ratio, since the cytotoxicity of GL and HE on scopolamine-induced SH-SY5Y cells was significantly suppressed at 50 μg/mL (GL, 22:02 ± 5:14%, * P < 0:05) and 250 μg/mL (HE, 24:82 ± 2:28 %, * * P < 0:001), respectively. SH-SY5Y cells were pretreated with 50, 100, and 250 μg/mL GH for 1 h and then cotreated with 5 mM scopolamine for 24 h. In the MTT assay, GH significantly increased cell viability compared with that in the negative (scopolamine only) group (Figure 2(b)). Moreover, the MTT assay showed that GH did not exert any cytotoxicity. As shown in Figure 2(b), the treatment of scopolamine progressively changed the cellular morphology such as shrinkage of cells and decreased density of cells. Meanwhile, pretreatment with 250 μg/mL GH prevented the observed situation. These results suggest that GH prevented scopolamine-induced cytotoxicity.
3.3. GH Prevents the Scopolamine-Induced ROS, Mitochondrial Dysfunction, and Apoptosis Pathways in SH-SY5Y Cells. To evaluate whether GH-treated neuroprotective effects were regulated via the suppression of apoptosis pathways, we first measured ROS production in SH-SY5Y cells. The scopolamine-treated group (445:60 ± 6:29%, ### P < 0:001) showed a significant increase in ROS production compared with that in the control group, as shown in Figure 3(a). In addition, we measured the protection of mitochondrial dysfunction as a major hallmark of apoptosis in SH-SY5Y cells ( Figure  3(b)). The scopolamine-treated group (60:20 ± 1:18%, ## P < 0:01) demonstrated a significant loss in mitochondrial membrane potential, whereas GH-treated groups significantly recovered the loss of mitochondrial membrane potential in a dose-dependent manner. In particular, the scopolamine-treated group ( ### P < 0:001) showed increased levels of apoptosis-related proteins as the Bax/ Bcl-2 ratio and cleaved-caspase-3, while GH-treated groups showed a significant decrease in the protein levels (Figures 4(c) and 4(d)). These results indicated that GH has a neuroprotective effect by reducing ROS production and mitochondrial dysfunction and suppressing the apoptosis pathways.
3.4. GH Increases the Levels of BDNF and Activates GSK3β, ERK, and CREB Signaling Pathways in Scopolamine-Induced SH-SY5Y Cells. To investigate the neuronal 5 Oxidative Medicine and Cellular Longevity protection pathways in scopolamine-induced SH-SY5Y cells, we confirmed the signal transduction pathways (e.g., cell survival, growth, and proliferation), including BDNF, GSK3β, CREB, and ERK signaling ( Figure 4). Our data revealed that scopolamine significantly decreased BDNF, pGSK3β, pCREB, and pERK, whereas GH treatment inhibited the effects of scopolamine on the levels of pGSK3β, pCREB, and pERK. It was reported that BDNF, GSK3β, CREB, and ERK signaling play vital roles in the process of memory, learning, and modulation of cholinergic function, suggesting that GH has a neuroprotective effect via regulation of these signaling pathways [12].
3.5. GH Improves Scopolamine-Induced Memory Impairment in Behavior Tests. For elucidating the neuroprotective effects of GH on cognitive memory function, we carried out two behavior tests on the scopolamine-induced mouse model: Y-maze and NORT tests (Figure 1(a)). The mouse model used was six-week-old ICR males and was orally administered GL (40 mg/kg, p.o.), HE (200 mg/kg, p.o.), or GL+HE (GH, 240 mg/kg, p.o.) for 7 days before scopolamine injection (1.2 mg/kg, i.p.). In particular, the administration dose of GH was also mixed with GL and HE in a 1 : 5 ratio as shown in vitro data, and the administration dose of GL was referred from previous findings [35,36]. In addition, the positive control was used donepezil (1 mg/kg, p.o.), which is used for treating AD.

GH Ameliorates the Pathological Changes of
Hippocampus in Mouse Brain Tissue. Histopathological changes in the mouse hippocampus were observed by the H&E staining assay (Figure 7). Neuronal damage was investigated in the dentate gyrus (DG), cornu ammonis (CA) 1, and CA2/3 region of the hippocampus in terms of the number of viable neurons, by using a microscope (40x magnification). The brain sections of normal mice displayed the normal structure of the hippocampus, such as the DG, CA1, and CA2/3 areas. In the scopolamine group, pyramidal cells of the stratum pyramidalis in the DG, CA1, and CA2/3 regions showed that scopolamine ( ### P < 0:001) caused significant damage in each region of the hippocampus, compared with those of the normal group. However, GH administration revealed a marked reversal of scopolamineinduced cell damage, similar to that by donepezil administration, indicating its neuroprotective potential.

GH Promotes the BDNF Expression in the Mouse
Hippocampus. BDNF, as a biomarker for synaptogenesis and synaptic plasticity, engages memory function by promoting neuronal cell survival and differentiation in hippocampal neurons [37]. In the present study, we examined whether GH mediates synapse formation in the scopolamine-induced hippocampus in mice by detecting the number of immunoreactive neurons in the CA1 and CA2/3 regions. The relative number of BDNF-reactive neurons in the CA1 and CA2/3 regions was significantly higher in both donepezil ( * P < 0:05) and GH-treated ( * * * P < 0:001) groups in comparison to that in the normal group ( Figure 8). Our data suggest that the administration of GH may be able to induce the expression of BDNF, which is attributed to memory enhancement.

Phytochemicals from GH Improves Neuroprotective
Effects on Scopolamine-Induced Cytotoxicity in SH-SY5Y Cells. As shown in Figure 9, the neuroprotective effects of phytochemicals as hericene A, hericene D, ginkgolide A, ginkgolide B, isorhamnetin, acacetin, quercetin, and kaempferol identified from GH were measured on scopolamineinduced cytotoxicity in SH-SY5Y cells. All phytochemicals prevented the cytotoxicity induced by scopolamine in a dose-dependent manner, and no cytotoxicity was detected.

Discussion
In this study, GH showed the effect of neuron protection on scopolamine-induced SH-SY5Y cells and improved scopolamine-induced cognitive impairments in a mouse model via behavioral tests including Y-maze and NORT. Moreover, we measured the neuroprotective mechanism of GH via in vitro and in vivo models. Interestingly, GH increased BDNF expression by activating CREB/ERK phosphorylation and downregulating ROS and AChE activities. Previous studies on the pharmacological effects of GL and HE extracts in GH mixture may partly explain our data [17,29]. GL ameliorates learning and memory impairments in animal studies and affects neurotransmitter levels, neuroplasticity, and suppression of the brain edema in clinical studies [15,18]. HE extract showed great potential in  Oxidative Medicine and Cellular Longevity improving the intellectual function of patients with cognitive impairment or against neurodegenerative diseases such as dementia and AD in the previous studies [29][30][31]. However, there is no report on a mixture of involved GL and HE showing a synergic effect on the scopolamine-induced mouse model. Thus, GL and HE were extracted from raw plant materials and arbitrarily mixed with a ratio of 1 : 5, since GL and HE significantly prevented scopolamineinduced cytotoxicity at 50 and 250 μg/mL concentrations, in scopolamine-induced cytotoxicity, respectively (see Figure 2S). Furthermore, the mixture showed a synergic effect in comparison with each extract of GL or HE extracts on the scopolamine-induced mouse model (Figures 1, 5-8). (HE) fruit extracts on the expressed levels of BDNF, pAKT, pERK, and pCREB in the brain. Whole brain from randomly selected mice in each group was analyzed for western blotting assay. (a-d) Western blotting assay of BDNF (a), pAKT/AKT (b), pERK/ERK (c), and pCREB/CREB (d) was carried out. GAPDH was used as a loading control. Quantification was performed using densitometric analysis with Bio-Rad Quantity software. Data represent the mean ± SEM ðn = 7Þ: # P < 0:05 and ## P < 0:01 vs. control group. * * P < 0:01 and * * * P < 0:001 vs. Sco-treated group. 10 Oxidative Medicine and Cellular Longevity

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Oxidative Medicine and Cellular Longevity Scopolamine has been widely used to investigate a model of amnesia in a preclinical study that contributes to increased oxidative stress with impairment of memory and cognitive functions [14]. GH significantly downregulated scopolamine-induced cytotoxicity (Figures 2(b) and 2(c)) and ROS (Figure 3(a) and Figure 6S) and showed greater protection against neuronal cell death ( Figure 3). Furthermore, GH suppressed the scopolamine-induced dephosphorylation of GSK3β, ERK, and CREB, which play vital roles in cell proliferation and survival signaling pathways (Figure 4). It was revealed that the inhibition of GSK3β by regulating pAKT reduces neuronal loss by suppressing apoptotic agents [38]. The activation of BDNF, ERK, and CREB signaling is related to neuronal cell survival and proliferation [12,14]. It seems that the neuronal protection effect of GH on the scopolamineinduced model may be mainly modulated by BDNF and then activated by CREB and ERK phosphorylation.
In our in vivo results by using behavioral tests (Y-maze test, NORT test), it was found that GH improved scopolamine-induced memory impairments. These scopolamine-induced mouse models are widely used to analyze short-term memory [32]. Donepezil, an AChE inhibitor, is commonly used for treating AD, and it has also been shown to positively affect episodic memory function [39]. Thus, donepezil was used as a positive control in our designed in vivo model. In this study, GH displayed a similar or superior activity to that of donepezil (Figure 1), indicating that GH may be involved in cognitive enhancement.
BDNF affects synaptogenesis and synaptic plasticity in the central nervous system (CNS). Neurotrophic signaling involving ERK, CERB, and AKT is stimulated by the activation of BDNF, which contributes to memory and thinking. Indeed, GH considerably increased the expression levels of pERK, pCREB, and BDNF in the mouse brain ( Figure 6), similar to that in in vitro data. Moreover, GL or HE could modulate BDNF/CREB/ERK in the brain and attenuate cognitive deficits in an amnesia mouse model. This indicates that the activation of the BDNF-induced signaling cascade by treating GL and/or HE could have neuroprotective effects on a scopolamine-induced mouse model ( Figure 6). Furthermore, GH showed highly immunoreactive neurons by BDNF in the CA1 and CA2/3 hippocampal regions, indicating that GH treatment can increase synaptogenesis that is associated with boosting memory function ( Figure 8).
Furthermore, the number of neurons observed by staining the doublecortin (DCX) was measured in the DG region of the hippocampus. DCX, a cytoskeletal protein, is expressed through neuronal cells and is widely used as a biomarker for neurogenesis [40]. Scopolamine refers to a decrease in DCX-immunoreactive cells in the DG and damage of dendritic development of the new immature neurons [41]. The decreased levels of stained DCX in the scopolamine-induced group were clearly observed. In the

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Oxidative Medicine and Cellular Longevity GH group, the number of immunoreactive neurons in the DG region was also improved in the scopolamine-induced mouse model, suggesting that GH engages in immature neuron differentiation in the hippocampus ( Figure 7S). Previous studies revealed that anti-AChE agents enhance the levels of ACh in the synaptic cleft and restore memory deficits [37]. Therefore, we also evaluated the effect of GH on the levels of ACh and AChE in a scopolamine-induced mouse model. Scopolamine increased AChE activity (0:61 ± 0:09 units/mg protein) and reduced ACh levels (12:15 ± 3:32 nmol/mg protein). Meanwhile, the administration of GH significantly reversed each level (AChE activity, 0:26 ± 0:03 units/mg protein; ACh level, 29:96 ± 8:62 nmol/ mg protein) in the mouse brain ( Figure 5). GH might be an efficient potential therapeutic source for preventing cholinergic dysfunction associated with AD. Also, we found that HE can strongly increase the content of ACh, while the activity of AChE became lower than that by GL treatment. This finding was in agreement with a few previous studies, which demonstrated that the AChE inhibitory activity of HE extracts involving hericenone and hericene components showed a mild effect [39,40].