Neuroprotective Effect of the Mixture of Gastrodiae elata and Glycyrrhizae uralensis In Vitro

Abstract

Background: This study investigated the effect of a mixture of Gastrodiae elata and Glycyrrhizae uralensis (GGW) on β-amyloid-induced neuronal damage in vitro. Methods: For finding the appropriate GGW ratio, we performed MTT assays using the ratios of 5:5, 6:4, 7:3, 8:2 and 9:1 in SK-N-SH cell and SH-SY-5Y cell. Treatment with β-amyloid (10 μM) caused cell death and overexpression of acetylcholinesterase (ACHE) in SH-SY-5Y cells. β-amyloid treatment increased the expression of mitogen-activated protein kinases (MAPKs). In addition, we detected the antioxidant activity of GGW using DCF-DA staining of SK-N-SH cells. To determine the effect of GGW on oxidative stress, we used a hydrogen peroxidase-induced in vitro model. Results: We selected the ratios of 5:5 and 7:3 mixtures with the least cytotoxicity. The 7:3 ratio of GGW (GGW73) decreased the mRNA expression of ACHE in SH-SY-5Y cell more than the 5:5 ratio of GGW (GGW55). GGW73 suppressed p-ERK protein expression in β-amyloid-treated SH-SY-5Y cells. Additionally, GGW73 regulated reactive oxygen species (ROS) in vitro. GGW73 treatment regulated apoptotic factors in β-amyloid-stimulated neuronal cells. Conclusions: These results suggest that GGW73 exerts neuroprotective and anti-inflammatory effects in vitro. These results also showed that GGW73 had a protective effect against H₂O₂ induced cell damage in an in vitro model. These results indicate the potential of GGW as a therapeutic agent for cognitive disorders.

1. Introduction

Worldwide population aging is emerging as a major long-term problem [1]. As population aging progresses worldwide, the number of people above the age of 65 are expected to steadily increase to two billion by 2050 [2], and the patients with neurodegenerative disease including dementia and cognitive disorder are also expected to increase to over 130 million [3]. Among the numerous age-related diseases, degenerative brain diseases, such as Alzheimer’s disease (AD), cause many additional diseases and excessive medical expenses [4]. AD is characterized by cognitive impairment and memory loss [5]. These degenerative brain disorders reduce learning and memory, affecting not only patients, but also their families and social behavior [6]. AD with cognitive function disorders is etiologically characterized by neuroinflammation, causing neuronal loss, amyloid deposition, and nerve fiber entanglement [7]. It is well known that β-amyloid (Aβ) and oxidative stress are involved in the pathogenesis of cognitive function disorder [8]. The oligomeric form of Aβ peptide plays a major role in neuronal and glial activation of inflammation. After activation, neuronal cells produce inflammatory agents, including chemokines and cytokines [9]. It induces apoptosis and destabilizes calcium homeostasis by Aβ peptide, ultimately leading to neurotoxicity both in vitro and in vivo [10]. Aβ peptide is released from amyloid precursor protein (APP) by β-and γ-secretases, which form senile plaques [11]. Therapies that modulate the Aβ pathway are promising targets for drug development for cognitive function disorders. In connection with this method, approaches are being developed to regulate the Aβ pathway by inhibition of β- or γ-secretase or anti-Aβ monoclonal antibodies in cognitive function disorders [12].

Acetylcholine (Ach), a neurotransmitter in the nervous system and brain, plays a key role in memory and learning [13]. General medications might be used for degenerative brain disorders, such as AD and cognitive function disorders, by inhibiting acetylcholinesterase (AChE), nicotinic acetylcholine receptors, and muscarinic acetylcholine receptor (mAChR) [14]. The expression of AChE, which breaks down ACh, is activated in patients with cognitive disorders or AD [15]. Generally, in clinical treatment, donepezil, tacrine, and rivastigmine are used to inhibit AChE. However, these medicines have side effects, such as somnipathy and nausea, and short half-lives [16]. In the amyloid pathway, β-secretase (BACE-1) is localized in endosomes and cleaved APP [17]. Therapeutically, inhibition of BACE-1 is a better treatment for AD or cognitive function disorders, aimed at suppressing the production of Aβ [18]. Additionally, Aβ peptide has been known to inhibit mitochondrial activity and alter intercellular calcium levels and induce cell death through oxidative and inflammatory stimulation [19].

Gastrodia elata (GE), a traditional herbal medicine in South Korea, and its constituents have been used to treat various diseases, such as cardiovascular system disorders [20], convulsions [21], neurotransmitter disorders [22], and cognitive function disorders by regulating APP cleavage [23]. Gastrodin, one of the major constituents of Gastrodia elata, has been reported to have beneficial effects on the nervous system, including suppression of microglial activation, anti-inflammation, regulation of oxidative stress, and modulation of neurotransmitters [24]. Furthermore, gastrodin showed a protective effect on the midbrain by increasing the levels of GSH, HO-1, and SOD in MPTP intoxicated mice [25]. These results suggest that GE has the potential to treat cognitive function disorders. Glycytthiza ularesis (GU), belonging to the Leguminosae family [26], has been used as an herbal medicine and a natural sweetener [27]. In addition, GU has traditionally been used to treat skin inflammation, digestive system diseases, and hepatitis [28]. GU has been reported to contain bioactive compounds such as triterpenoids (glycyrrhizic acid, glycyrrhetinic acid) and flavonoids (liquilitin, liquilitigenin, and licochalcone A) [29,30], which possess a variety of biological activities [28]. A previous study showed that GU decreases Aβ-induced neurotoxicity by regulating oxidative stress in cultured cortical neurons [31]. In addition, GU has been reported to ameliorate cognitive dysfunction in an AD mice model [32]. The constituents of GU have been studied for their effects on neuronal diseases by attenuating kainic acid-induced neuronal cell death [33] and anti-apoptotic activity in dopaminergic neurons [34]. Various effects of GE and GU on neurological diseases have been reported; however, the efficacy of a mixture of GE and GU has not yet been evaluated. The mixture of GE and GU as herbal medicines, each with effects on neuronal diseases, is expected to have a better improvement effect on cognitive function disorders.

This study aimed to determine whether a mixture of GE and GU could ameliorate neuronal damage in vitro. In addition, we investigated the mechanism of AChE and apoptosis in Aβ-stimulated cell damage by applying a mixture of GE and GU. We evaluated the protection effect against Aβ-induced cell death and AChe and BDNF expression using SH-SY-5Y cells, and regulatory effect against Aβ-induced apoptotic factors such as BCl-2 and Bax using SK-N-SY neuroblastoma cells.

2. Materials and Methods

2.1. Reagents

Minimum Essential Medium (MEM) used in cell culture was purchased from Welgene (Gyoungsan, Gyoungsangbuk-do, Republic of Korea). Dulbecco’s modified Eagle’s medium (DEME), fetal bovine serum (FBS), and penicillin streptomycin were purchased from Gibco (Grand Island, NY, USA). The Amyloid β-Protein Fragment 25–35 (Aβ25–35) (purity 95%) and lipopolysaccharide (LPS) from Escherichia coli 0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 1–42 (Human) HFIP-treated Aβ 1–42 peptide (purity 95%) was purchased from GenicBio Synthetic Peptide (Shanghai, China). All primary antibodies used for Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

2.2. Preparation of GE and GU Extracts

The rhizome of gastrodia elata (GER) was provided by MJ Health Food Co., Ltd. (Muju-gun, Jeollabuk-do, Republic of Korea). Then, 100 g of GER was decocted into 10 times volume of distilled water (1 L) at 90 °C for 6 h. The extract was filtered through 25 μm polyethersulfone (PES) syringe filter, then vacuum evaporated at 65 °C. Subsequently, the concentrate was frozen at −75 °C for 24 h, and then frozen-dried at −85 °C and 5 mTorr condition for 60 h. The GER yield was 13.8%. G. ularensis radix of glycyrrhiza ularensis (GUR) was provided by Mj Health Food Co., Ltd. (Muju-gun, Jeollabuk-do, Republic of Korea). Next, 100 g of GUR was decocted with 10 times volume of distilled water (1 L) at 100 °C for 3 h. The extract was filtered through 25 μm polyethersulfone (PES) syringe filter, then vacuum evaporated at 65 °C. Subsequently, the concentrate was frozen at −75 °C for 24 h, and then frozen at −85 °C and 5 mTorr condition for 60 h. The GUR yield was 17.4%. Various ratios (GER:GUR = 5:5, 6:4, 7:3, 8:2, 9:1) of a mixture of GER and GUR were prepared by mixing GER and GUR.

2.3. Cell Culture

Human neuroblastoma cells (SH-SY5Y and SK-N-SH) and a macrophage cell line (RAW 264.7) were purchased from the Korea Cell Line Bank (Jongno-gu, Seoul, Republic of Korea). SH-SY5Y and SK-N-SH were cultured in MEM containing 10% FBS and 1% penicillin-streptomycin, and the RAW 264.7 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin. All cell lines were incubated at 37 °C with 5% CO₂.

2.4. Cell Viability

The cytotoxicity of the complex mixture was measured using the MTT assay. SH-SY-5Y cells (3 × 10⁴ cells per well) were added to a 96-well plate and incubated at 37 °C and 5% CO₂ for 24 h. After that, the mixtures were added for 4 h, and β-amyloid (25–35) was added at a concentration of 10 μM, incubated for 24 h, and 5 mg/mL of 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT; Sigma-Aldrich Co.) solution was added to each well and incubated for an additional 5 h, after which the supernatant from each well was removed. To completely dissolve the remaining formazan crystals, 200μL of dimethyl sulfoxide (DMSO) was added to the wells, and the absorbance was measured at 540 nm using a microplate reader (Synergy HTX Multi-Mode Reader, BioTek, Santa Clara, CA, USA).

2.5. Western Blot Analysis

Cells were seeded at a density of 3 × 10⁶ in 6 cm dishes. After 24 h, it was replaced with 0% medium, and various concentrations of GGW were added. After 2 h, 2 μM of Aβ was added. After 45 min, the cells were washed with 1 × PBS (ELPIS BIOTECH, Seo-gu, Daejeon, Republic of Korea), collected, and centrifuged for 5 min at 3515× g. The supernatant was removed, and the cells were lysed with PRO-PREP (iNtRON Biotech, Seongnam, Gyeonggi-do, Republic of Korea). Centrifugation was performed at 18,894× g for 10 min at 4 °C.

The supernatant was collected and transferred into a sterile tube. Proteins were quantified using the DC Protein Assay Reagent (BIO-RAD Laboratories, Hercules, Califonia, USA).

Quantified samples (5 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After blocking with 5% BSA for 2 h, the membranes were washed with 0.05% TBST at 15 min intervals for 1 h 30 min. Primary antibodies were incubated overnight with the transferred membrane. The membranes were washed with 0.05% TBST at intervals of 15 min for 1 h 30 min. After stabilization with 2.5% BSA, the secondary antibody diluted 1/10 with sterile distilled water and shaken for 2 h. The membranes were washed with 0.05% TBST at 10-min intervals for 1 h 30 min. After using Western blotting luminol reagent (Santa Cruz, CA, USA), it was visualized using LuminoGraph III Lite (ATTO, Tokyo, Japan).

2.6. Reactive Oxygen Species (ROS) Analysis

ROS activities in RAW 264.7, and SK-N-SH cells were determined using a DCFDA/H₂DCFDA-cellular ROS assay kit (Abcam, London, UK). The cells (3 × 10⁵) were seeded in a 24-well plate. After 24 h, various concentrations of GGW were added, and 4 h later, 200 μM hydrogen peroxide (H₂O₂) or 1 μM Aβ was added. The cells were washed with sterile 1 × PBS and observed under a fluorescence microscope (Carl Zeiss, Berlin, Germany).

2.7. Quantitative Real Time Polymerase Chain Reaction (qPCR)

The cells were seeded into a 6 cm dish at a density of 3 × 10⁶ and incubated at 37 °C, 5% CO₂ for 20 h. After that, various concentrations of GGWs were treated for 8 h, and then β-amyloid (25–35) was added at a concentration of 2 μM and incubated for 24 h.

Total RNA was isolated using an easy-BLUE™ Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Gyeonggi, Republic of Korea). cDNA was synthesized using the HelixCript™ Easy cDNA Synthesis Kit (NanoHelix, Republic of Korea).

Real-time PCR (QuantGene 9600 real-time system, Bioer Tech, Hangzhou, China) analysis was performed using the RealHelix™ Premier qPCR Kit (NanoHelix, Republic Korea) and gene-specific primers (

Table 1. Primer sequence for qPCR.

Gene	Forward	Reverse
h GAPDH	5′-TCTAGACGGCAGGTCAGGTCCACC-3′	5′-CCACCCATGGCAAATTCCATGGCA-3′
h AChE	5′-ACAGGTCTGAGCAGCGATCCTGCTTGCT-3′	5′-TACGCCTACGTCTTGAACACCGTGCTTC-3
h iNOS	5′-GCTCTACACCTCCAATGTGACC-3′	5′-CTGCCGAGATTTGAGCCTCATG-3′
h Beclin-1	5′-CCATGCAGGTGAGCTTCGT-3′	5′-GAATCTGCGAGAGACACCATC-3′
h Bcl-2	5′-ATGTGTGTGGAGAGCGTCAA-3′	5′-GCCGGTTCAGGTACTCAGTC-3′
h BACE-1	5′- TCTGTCGGAGGGAGCATGAT-3′	5′- GCAAACGAAGGTTGGTGGT-3′

). Primers were purchased from Bioneer (Daejeon, Republic of Korea). The samples were denatured at 95 °C for 2 min, followed by 40 cycles of 20 s denaturation at 95 °C and 15 min of annealing/extension at 63 °C. All comparative data were normalized to those for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

2.8. Statistical Analysis

GraphPad Prism (version 5.0) was used for all analyses. Results are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical evaluation was performed using one-way analysis of variance (ANOVA) and Bonferroni’s post hoc analysis. p-values of < 0.05 was considered to be statistically significant.

3. Results

3.1. The Cytotoxicity of GGWs on Neuronal Cells

To choose the mix ratio of GEW and GUW, the authors evaluated cytotoxicity in neuronal cells using an MTT assay. The various ratio of GGWs (5:5, 6:4, 7:3, 8:2, 9:1) were treated on SH-SY-5Y cell. GGW64, GGW82 and GGW91 had cytotoxicity on SH-SY-5Y cell at concentrations of 100 and 1000 μg/mL. However, GGW55 and GGW73 had non-cytotoxicity on SH-SY-5Y cell. Moreover, cell viability increased following GGW55 and GGW73 treatment (Figure 1A). In SK-N-SH cells, GGW64 and GGW91 were cytotoxic. The cell viability was not affected by GGW55 or GGW73 treatment (Figure 1B). Therefore, the ratios of GGW55 and GGW73 were used for the in vitro study.

3.2. The Protective Effect of GGW on Aβ-Treated Neuronal Cells

Aβ 10 μM treatment significantly reduced the cell viability of both SH-SY-5Y cell and SK-N-SH cell. Cytotoxicity caused by Aβ treatment was increased by treatment with GGW55 and GGW73. Especially, GGW55 100 μg/mL and GGW73 100 μg/mL increased the cell viability significantly. In SK-N-SH cells, Aβ treatment reduced the cell viability, and GGW55 and GGW73 treatment increased thr cell viability, respectively (Figure 2). Compared to the protective effects of GGW55 and GGW73, GGW73 had better efficacy.

3.3. The Regulatory Effect of GGW on Aβ-Stimulated ACHE Pathway on SH-SY-5Y Cells

In order to confirm the mechanism of the neuroprotective effects of GGW55 and GGW73, the expression levels of Aβ-stimulated ACHE, BACE-1, and Brain-derived neurotrophic factor (BDNF) in the SH-SY-5Y cell were determined by real-time qPCR and ELISA. The mRNA expression of ACHE and BACE-1 was stimulated by Aβ 2 μM. Both GGW55 and GGW73 treatment downregulated the mRNA expression of ACHE and BACE-1 at a concentration of 100 μg/mL (Figure 3A,B). BDNF expression was decreased by Aβ stimulation. However, decreased expression of BDNF was recovered by dose-dependent GGW55 and GGW73 treatments (Figure 3C). These results suggest that GGW73 had a better neuroprotective effect than GGW55.

3.4. The Regulatory Effect of GGW on Aβ-Stimulatedmitogen Activated Protein Kinases MAPKs) Expression on Neuronal Cells

To determine the mechanism underlying the neuroprotective effect of GGW, the regulation of Aβ-induced phosphorylation of MAPKs was determined by Western blotting. In SH-Sy-5Y cells, GGW55 treatment regulated only phosphorylation of extracellular signal regulated kinase (ERK). However, GGW73 treatment attenuated the phosphorylation of ERK and p38 (Figure 4A,B). In SK-N-SH cells, GGW55 treatment only regulated phosphorylation of c-Jun N-terminal kinase JNK). However, GGW73 treatment attenuated the phosphorylation of ERK and JNK (Figure 4C,D).

3.5. The Attenuation Effect of GGW on Aβ-Stimulated Apoptotic Factors on SK-N-SH Cells

To clarify the neuroprotective effect of GGW, apoptotic factors were investigated using real-time qPCR and Western blotting. The mRNA expression of Bcl-2 was reduced by Aβ stimulation. GGW55 and GGW73 treatment increased Bcl-2 mRNA expression (Figure 5A). In addition, increased beclin-1 mRNA levels were decreased by GGW55 and GGW73 treatments (Figure 5B). Moreover, the protein expression of Bax and Bcl-2 was regulated by GGW55 and GGW73 treatments (Figure 5C,D). Therefore, from these results, GGW73 was more effective than GGW55.

3.6. The Regulatory Effect of GGW on H₂O₂-Stimulated ROS on SK-N-SH Cell and RAW264.7 Cell

DCF-DA staining was performed to determine the antioxidant effects of GGW on the neuronal cells. ROS were released by H₂O₂ or Aβ (data not shown) treatment of SK-N-SH cells. However, ROS production induced by H₂O₂ was decreased by treatment with GGW55 and GGW73 (Figure 6). RAW264.7 cells were stained for comparison with normal cells. These results indicated that GGW regulates oxidative stress in neuronal cells.

4. Discussion

The present study demonstrated that GGW inhibited cell death by Aβ-treatment in the neuronal cell lines of SH-SY-5Y cell and SK-N-SH cell. Additionally, GGW55 and GGW73 modulated the AChE pathway and MAPKs to prove the neuroprotective effect in SH-SY-5Y cell. Moreover, GGW55 and GGW73 regulated apoptotic factors such as BCl-2 and Bax.

GU has been shown to prevent oxidative damage and cognitive impairment in Aβ-treated mice [35]. In addition, memory deficiencies in mice, induced by diazepam and scopolamine, were improved by the administration of GU water extract [36]. GE has been reported to exert neuroprotective effects by inhibiting Aβ-induced PC12 cell death and ameliorating acute hypomnesia [19]. In addition, GE water extract recovered memory impairment in the Morris water maze test and passive avoidance test in a chronic restraint stress-stimulated mice model [37]. Gastrodin, a major compound of GE, decreased protein kinase/eukaryotic initiation factor-2alpha and BACE-1 expression in an AD mouse model [38]. These studies suggest that GE and GU are effective in improving cognitive function disorders, degenerative neurological disease, and that better effects could be expected using a combination of GE and GU. Among GGWs, GE, and GU treatments, GGW55, GGW73, GEW, and GRW did not show cytotoxicity in the neuronal cell line SH-SY-5Y cell (Figure 1A). Therefore, we chose GGW55 and GGW73 to explore the mechanisms underlying their neuroprotective effects. In addition, GGW55 and GGW73 significantly reduced cell death following Aβ treatment in SH-SY-5Y cell (normal group; 100.00 ± 6.67%, Aβ group; 67.52 ± 9.19%; GGW55 100 group; 79.57 ± 5.96%, GGW73 100 group; 87.04 ± 9.19%) and SK-N-SH cell (normal group: 100 ± 4.06%, GGW55 100 group; 87.72 ± 4.81%, GGW73 100 group; 88.26 ± 5.46%) (Figure 2). In particular, these results suggested that GGW73 treatment had more protective effect on neuronal damage than GGW55.

Impairment of cholinergic function, reduced ACh levels, and a role in the pathogenesis of dementia have been mainly reported in degenerative diseases such as AD and cognitive function disorder [39]. A previous study suggested that AChE, which downregulates ACh, is a biomarker of neurotoxicity [40]. In addition, AChE is associated with neuronal mineralization and differentiation [41]. Attempts to treat cognitive function disorders have been made using AChE inhibitors such as donepezil, tacrine, and rivastigmine [42]. Both GGW55 and GGW73 attenuated AChE mRNA expression, and GGW73 at 100 μg/mL significantly reduced AChE mRNA expression (Figure 3A).

BDNF is primarily responsible for maintaining the central nervous system and is widely expressed in the brain [43]. However, neurological inflammation significantly reduces BDNF expression in the brain, leading to cognitive function disorder [44]. β-secretase (BACE-1), together with γ-secretase, sequentially cleave APP to produce Aβ. Inhibiting the expression of BACE-1 to prevent degenerative disease is considered a strategy to remedy cognitive function disorders by lowering cerebral Aβ levels [45]. GGW55 and GGW73 increased BDNF expression upregulation by Aβ treatment (GGW55: normal group; 1855.60 ± 494.11 pg/mL, Aβ group; 1219.65 ± 119.52 pg/mL, GGW55 100 group; 1422.26 ± 450.03 pg/mL, GGW73: normal group; 2041.90 ± 213.48 pg/mL, Aβ group; 1019.04 ± 152.24 pg/mL, GGW73 100 group; 1608.13 ± 239.95 pg/mL) (Figure 3C) and decreased BACE-1 mRNA expression (Figure 3B). These results indicate that GGWs have a protective effect against the Aβ-induced neuronal damage. In addition, from the view point of central nervous system disorder, GGW is expected to have a positive effect on hydrocephalus, which occurs in the adult population with a ratio of 0.011% [46]. The previous study discovered the course of the hydrocephalus patients after shunting by observing brain volume and pressure change [47]. It requires more research into hydrocephalus using GGW as a natural origin-medicine treatment.

MAPKs signaling, especially ERK1/2, is necessary to activate transcription during long and short-term memory formation [48]. MAPKs, including ERK, (JNK), and p38, play important roles in attenuating neuronal inflammation and plasticity. Moreover, these separate pathways are related to memory and learning functions [49]. In this study, GGW55 and GGW73 did not significantly downregulate the phosphorylation of MAPKs signaling, but slightly decreased phosphorylation (Figure 4). These results suggest that the mixtures of GE and GU (GGW55 and GGW73) regulate Aβ-induced neuronal inflammation.

Increased expression of Bax and decreased expression of Bcl-2 have been observed in a memory and learning impairment mouse model [50]. In addition, dopamine-mediated neuronal cell death is associated with neurodegenerative disorders. In particular, the Bcl-2 signaling pathway affects dopaminergic synaptic apoptosis [51]. A previous study reported that regulation of the Bcl-2 signaling pathway is a way to treat neuronal damage [52]. Both GGW55 and GGW73 increased mRNA expression and protein expression of Bcl-2 that had been decreased by Aβ. It was also confirmed that GGW73 ameliorates the Bcl-2 signaling pathway at the protein level (Figure 5). These results indicate that GGW73 exerts a protective effect against neuronal cell apoptosis.

Oxidative stress is observed in early stages of neurodegenerative diseases [53]. Aβ induces ROS [54] and affects ROS activation, thus inducing NADPH oxidase (NOX) and neuronal cell death [55]. The previous study demonstrated that the antioxidant such as edaravone improved cognitive impairment in hypoxia-stimulated rat model by regulation oxidative stress [56]. In this study, DCFDA staining confirmed ROS expression in RAW264.7 cells and SK-N-SH cells. GGW55 and GGW73 inhibited ROS overexpression induced by Aβ and H₂O₂ stimulation (Figure 6). These results suggest that GGW55 and GGW73 exert protective effects against oxidative stress in the nervous system.

5. Conclusions

We investigated the protective effects of GGW55 and GGW73 against Aβ-induced neuronal cell damage. Both GGW55 and GGW73 helped repair Aβ-stimulated cell death and the Bcl-2 pathway. GGW55 and GGW73 treatment attenuated mechanisms in cognitive function disorder by regulating AChE and BACE-1 expression. In addition, GGW55 and GGW73 protected against apoptosis through the Bcl-2 signaling pathway and ROS regulation. Considering these results, GGW55 and GGW73 could be used in the medical treatment of cognitive function disorders, such as neurodegenerative diseases.