Composition for preventing, alleviating or treating alzheimer's disease, comprising gongjindan composition as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating or treating Alzheimer's disease, comprising a Gongjindan composition as an active ingredient. The Gongjindan composition enhances memory or cognitive function, alleviates cholinergic dysfunction, strengthens neurotrophic factors, and has antioxidant and anti-inflammatory effects.

Description

Composition for preventing, improving or treating Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient

[0001] The present invention relates to a composition for preventing, improving or treating Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient.

[0002] Alzheimer's disease (AD) is the most common form of dementia and a significant global health problem, impacting both individuals and society. According to reports, an estimated 6.7 million people aged 65 and older in the United States are living with AD. Furthermore, the number of Americans with AD is projected to reach approximately 14 million by 2060 (Non-patent Document 1). Despite this serious problem, the exact pathogenesis remains unclear, and it is only known that AD is characterized by neurodegeneration accompanied by severe symptoms such as cognitive and memory impairment (Non-patent Document 2). A possible neuropathological mechanism is closely related to the abnormal accumulation of senile plaques and neurofibrillary tangles in brain tissue, particularly in the hippocampus region (Non-patent Document 3).

[0003] Studies have shown that these conditions are due to cholinergic system dysfunction in conditions of loss and deficit of cholinergic neurons, which is primarily caused by increased severe oxidative stress, accumulation of amyloid-β, and abnormal expression of neuroinflammatory factors (Non-patent Document 4). These abnormal events lead to the generation of excessive amounts of reactive oxygen species (ROS) and reactive nitrogen species, as well as the persistent induction of lipid damage in brain tissue, ultimately resulting in cholinergic neuron loss and deficits in behavioral and memory functions (Non-patent Document 5). However, no treatment has been developed to date for AD, and most available treatments rely on alleviating symptoms or delaying the progression of the pathological condition through the use of cholinesterase inhibitors or dietary supplements such as folic acid and vitamin E (Non-patent Document 6). Therefore, a clear strategy is needed to develop AD treatments that possess antioxidant and anti-inflammatory properties while improving cholinergic dysfunction (Non-patent Document 7).

[0004] According to traditional Korean medicine, Gongjindan is a popular prescription in East Asian countries to help people with frail constitutions, especially the elderly (Non-patent Document 8). Gongjindan's pharmacological effects range from chronic fatigue disorder to central nervous system disorders and short-term sleep disorders (Non-patent Document 9). Previous studies have shown that Gongjindan may be a potential drug candidate for treating AD by improving cognitive function through its potent antioxidant effects (Non-patent Documents 8, 10).

[0005] The traditional Gongjindan recipe consists of deer antler (Cervus nippon T., Cervus elaphus L.), angelica gigas Nakai, cornelian cherry (Cornus Officinalis), and musk (Moschus moschiferus). The Gongjindan used in Non-Patent Document 8 consists of deer antler (Cervus nippon Temminck), angelica gigas Nakai, cornelian cherry (Cornus officinalis Siebold et Zucc.), musk (Moschus moschiferus L.), ginseng (Panax ginseng C.A. Meyer), and royal jelly (A. indica Rodosz kowski). The resonance-inducing agent used in non-patent literature No. 10 is composed of glutinous rice (Rehmannia glutinosa), deer antler (Cervi pantotrichum), angelicae radix, Chinese yam (Dioscorea polystachya), cornelian cherry (Cornus officinalis), poria cocos (Wolfiporia extensa), aesculus (Alisma canaliculatum), moutan bark (Moutan Cortex Radicis), musk, and agarwood (Aquilaria agallocha Roxburgh).

[0006] Task 1 is to elucidate the neuropharmacological characteristics and underlying mechanisms of GJD by adopting a mouse model of scopolamine-induced cognitive impairment.

[0007] Task 2 is to provide a composition for preventing, improving or treating Alzheimer's disease, which comprises a resonance diagnosis composition according to the present invention as an active ingredient.

[0008] Solution 1 is,

[0009] A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient,

[0010] The above-mentioned resonance diagnostic composition is characterized by comprising musk (Moschus moschiferus Linne), asparagus cochinchinensis MERR, Chinese yam (Dioscorea polystachya Turcz.), angelica (Angelica acutiloba (Siebold & Zucc.) Kitag.), cornus (Cornus officinalis Siebold & Zucc.), deer antler (Cervus elaphus L.), bean beetle (larva) (Popillia flavosellata Fairmaire), cattail (Typha orientalis Presl), bovine milk colostrum (Bovine Milk Colostrum), chaga mushroom (Inonotus obliquus), and royal jelly (Apis indica Rodosz Kowski).

[0011] Solution 2 is, in Solution 1,

[0012] The above resonance diagnosis composition is a composition characterized in that it is a mixed extract obtained by mixing materials.

[0013] Solution 3 is, in Solution 1,

[0014] The weight ratio of the materials included in the above resonant diagnostic composition is:

[0015] A composition characterized by being 30:360:360:60:24:400:100:60:100:15:50.

[0016] Solution 4 is, in Solution 1,

[0017] The above resonance diagnosis composition is a composition characterized in that it improves memory or cognitive function, improves cholinergic dysfunction, enhances neurotrophic factors, and has antioxidant and anti-inflammatory effects.

[0018] Solution 5 is,

[0019] A health functional food composition for preventing or improving Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient.

[0020] The above-mentioned resonance diagnostic composition is characterized by comprising musk (Moschus moschiferus Linne), asparagus cochinchinensis MERR, Chinese yam (Dioscorea polystachya Turcz.), angelica (Angelica acutiloba (Siebold & Zucc.) Kitag.), cornus (Cornus officinalis Siebold & Zucc.), deer antler (Cervus elaphus L.), bean beetle (larva) (Popillia flavosellata Fairmaire), cattail (Typha orientalis Presl), bovine milk colostrum (Bovine Milk Colostrum), chaga mushroom (Inonotus obliquus), and royal jelly (Apis indica Rodosz Kowski).

[0021] The resonance diagnosis composition according to an embodiment of the present invention showed efficacy in preventing and improving amnesia in a scopolamine-induced dementia animal model.

[0022] The resonance diagnosis composition according to the present invention exhibited antioxidant prevention and improvement effects in a scopolamine-induced dementia animal model.

[0023] Figure 1 is a drawing showing the results of analyzing the main components of a resonance diagnosis unit according to an embodiment of the present invention using UPHLC.

[0024] Figure 2 is a graph showing the results of a Morris Water Maze Task experiment of a resonance diagnosis unit according to an embodiment of the present invention.

[0025] Figures 3 and 9 are graphs showing the results of an antioxidant experiment of a resonance diagnosis unit according to an embodiment of the present invention.

[0026] Figure 4 is a graph showing the results of an anti-inflammatory experiment of a resonance diagnosis device according to an embodiment of the present invention.

[0027] Figure 5 is a graph showing the results of an experiment to improve cholinergic dysfunction of a resonance diagnosis device according to an embodiment of the present invention.

[0028] Figure 6 is a graph showing the results of an experiment to strengthen neurotrophic factors of a resonance diagnosis unit according to an embodiment of the present invention.

[0029] Figure 7 is a drawing describing the experimental design details of the present invention.

[0030] Figure 8 is a drawing describing information about PCR primers used in the experiment of the present invention.

[0031] Figure 9 is a graph showing the antioxidant effect of a resonance diagnosis device according to an embodiment of the present invention.

[0032] Figure 10 shows the results of TUNEL analysis of the effect of the resonance diagnosis on the dentate gyrus according to an embodiment of the present invention.

[0033] Figure 11 shows the results of IHC analysis of the effect of the resonance diagnosis on the dentate gyrus according to an embodiment of the present invention.

[0034] We investigated the neuropharmacological properties of Gongjindan (GJD) on scopolamine-induced cognitive impairment using a mouse model. C57 / BL6J mice were orally administered various doses of GJD (0, 100, 200, or 400 mg / kg daily) or tacrine (THA, 10 mg / kg) for 14 days under scopolamine (2 mg / kg) or vehicle-injected conditions. Mice were trained and performed the Morris Water Maze task to acquire learning and memory behavioral assays. GJD administration enhanced learning and memory abilities, as evidenced by improvements in escape latency, path length, and time spent in the target quadrant. Furthermore, scopolamine induced severe hippocampal oxidative and inflammatory states, but these abnormalities were significantly improved by GJD treatment through enhanced antioxidant capacity. GJD treatment also restored hippocampal cholinergic dysfunction, including acetylcholinesterase (ACHE) and choline acetyltransferase, and overactivated ACHE activity, through enhancement of brain-derived neurotrophic factor and cAMP response element binding protein. Our findings strongly support the clinical use of GJD, which has a potentially potent anti-amnestic effect, and the underlying mechanism may involve regulation of cholinergic activity through the activation of neurotrophic factors.

[0035]

[0036] Experimental Example 1. Materials, Reagents, and Methods

[0037] 1.1 Preparation for GJD

[0038] In this study, GJD (sample name) was prepared by modifying the original prescription based on clinical practice. All ingredients were obtained from Song Gwang Oriental Medical Clinic (South Chungcheong Province, South Korea). The ingredients of GJD are listed in Table 1.

[0039] A total of 1,559 g of mixed materials were ground, and the powdered material was mixed with distilled water (DW), extracted overnight at 4°C, passed through a 45 μm filter, and then stored at -20°C.

[0040] Ingredients Content (g) Musk (Moschus moschiferus Linne) 30 Asparagus cochinchinensis MERR) 360 Dioscorea polystachya Turcz.) 360 Angelica acutiloba (Siebold & Zucc.) 60 Cornus officinalis (Cornus officinalis Siebold & Zucc.) 24 Deer antler (Cervus elaphus L.) 400 Bean beetle (larva) (Popillia flavosellata Fairmaire) 100 Cattail (Typha orientalis Presl) 60 Bovine milk colostrum (Bovine Milk Colostrum) 100 Chaga mushroom (Inonotus obliquus) 15 Royal jelly (Apis indica Rodosz Kowski) 50

[0041] 1.2 Component Analysis

[0042] To investigate the chemical composition of GJD, we performed analyses using an Acela ultra-high-performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a high-resolution LTQ-Orbitrap XL mass spectrometer (MS). The UHPLC system was equipped with an ACQUITY UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm; Waters Corporation, Milford, MA, USA), which separated chemical compounds based on polarity (flow rate 0.4 mL / min, injection volume 3 μL). The mobile phase was a gradient condition for a gradient of solvent composition. Solvent A for DW containing 0.1% (v / v) formic acid and solvent B for acetonitrile containing 0.1% (v / v) formic acid were introduced into the column as follows: 95% A / 5% B at 0–1 min, 30% A / 70% B at 20 min, 0% A / 100% B at 24 min, 95% A / 5% B at 27.1 min, and 95% A / 5% B at 30 min. Chemical components were detected at various wavelengths from 200 to 600 nm. MS conditions in positive and negative HESI modes were optimized as follows: capillary temperature of 300 °C, capillary voltage of 35 V, and spray voltage value of 4.0 kV.

[0043] After UPHLC was completed, data were acquired and processed using Xcalibur software (see Figure 1). Metabolite profile data were analyzed based on literature reviews, and major components were identified based on high-resolution mass and MS / MS spectral library searches. Table 2 below lists the major components.

[0044] RT(min)m / z([M+H])Formula([M+H])Error ppmIdentificationSource2.14166.0856C9H 11O2N-3.764PhenylalanineCervus elaphus L.3.96355.1015C 16 H 19 O9-2.559Chlorogenic acidCornus officinalis siebold & Zucc.5.38369.1169C 17 H 21 O9-3.0585-Feruloylquinic acidCornus officinalis siebold & Zucc.6.89409.1506C 20 H 25 O9-3.101NodakeninAngelica acutiloba (Siebold & Zucc.) Kitag.8.92187.1323C 10 H 18 O3-2.94410-HDAApis indica Rodosz Kowski90.11301.0361(-)C 15 H 10 O71.821QuercetinTypha orientalis Presl10.09271.0594(-)C 15 H 11 O5-0.740NaringeninTypha orientalis Presl13,73287.1998C 19 H 25 O2-2.565Androstemdionecolostrum14.64345.1324C 19 H 20 O6-2.651Diosbulbin BDioscorea polystachya Turcz.16.64329.1370C 19 H 21 O5-4.163DcursinAngelica acutiloba (Siebold & Zucc.) Kitag.16.79329.1370C 19 H 21 O5-4.163Decursinol angelateAngelica acutiloba (Siebold & Zucc.) Kitag.17.78869.4884C 45 H 72 O16 -1.095DioscinDioscorea polystachya Turcz.18.69520.3386C 26 H 51 O7NP-2.317LysoPC 18:2Cervus elaphus L.19.62496.3384C 24 H 51 O7NP-2.671LysoPC 16:0Cervus elaphus L.20.22522.3542C 26 H 55 O7NP-2.347LysoPC 18:1Cervus elaphus L.22.00524.3696C 26 H 55 O7NP-2.738LysoPC 18:0Cervus elaphus L.

[0045] 1.3. Preparation for animal testing

[0046] All animal experiments were approved by the Institutional Animal Care and Use Committee of Daejeon University (DJUARB2023-051). A total of 70 pathogen-free C57 / BL6J male mice (10 weeks old, weighing 24–26 g) were purchased from Daehan Biolink (Chungcheongbuk-do, South Korea). All mice had free access to water and food (Cargill Agripurina, Gyeonggi-do, South Korea) and were housed in cages maintained at 23±2°C on a 12-h:12-h light / dark cycle. The animals were acclimated for 1 week and randomly assigned to six experimental groups: control (n=10), scopolamine (2 mg / kg, n=12), GJD treatment (100, 200, and 400 mg / kg, n=12 each), and positive control (10 mg / kg, tacrine (THA), n=12). GJD and THA were dissolved in distilled water and administered orally via gavage to each group for 14 days. Scopolamine was dissolved in 0.9% neutral saline and administered intraperitoneally once. The dose of GJD was determined based on the results of the pre-screening.

[0047] Details of the experimental design are shown in Figure 7. After 7 days of acclimation, mice were orally administered with distilled water, various doses of Gongjin-Dan, or Tacrine for 7 days. On the 7 th Day of the experiment, mice were injected with a single injection of scopolamine intraperitoneally (2 mg / kg) and continuously administered with drugs. All mice were trained in the Morris Water Maze Task for a total of 7 days and analyzed the behaviors for 6days. (n=10 for vehicle group, n=12 for others).

[0048]

[0049] 1.4 Morris water maze task

[0050] To evaluate the pharmacological effects of GJD on locomotion, the Morris water maze task was performed according to a standard protocol. Briefly, a circular pool (diameter 100 cm × height 50 cm) with a circular acrylic platform (diameter 10 cm × height 35 cm) in the target quadrant, identifiable by visual cues, was filled with milk water (23 ± 1 °C) for each session. All behavioral and data from the experiment were recorded by a video camera connected to software.

[0051] Mice were placed on the platform for 10 seconds and then removed from the pool, and then acquired for 6 days. A video camera recorded both escape latency and cumulative path length throughout each test session. On the 7th day, all mice were tested for 120 seconds without platform conditions to assess spatial learning and memory activity and to analyze the time spent in the target quadrant.

[0052]

[0053] 1.5 Sample Preparation

[0054] After completion of the maze test, mice were sacrificed with Avertin (250 mg / kg, ip) and perfused transcardially with 10% neutral formalin solution for immunohistochemical (IHC) analysis (n=2 in each group, four hippocampal regions from both hemispheres). For biochemical analysis, whole brain tissue was isolated from the mice and further dissected from fresh whole brains to obtain hippocampal tissue. It was then temporarily stored in liquid nitrogen and stored in a -80°C freezer (n=8–10 for whole brain tissue samples in each group). For hippocampal samples (n=6–9 for each group), we homogenized them using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher SCIENTIC, Cat No. 89900, Waltham, MA, USA) and obtained protein lysates or TRIZOL reagent (Sigma-Aldrich, T-9424-200ML, St. Louis, MO, USA) to obtain (mRNA) samples, respectively.

[0055]

[0056] 1.6 Biochemical analysis of oxidative stress and antioxidant components

[0057] We measured hippocampal protein levels of oxidative stress and antioxidant components using commercially available kits according to the manufacturer's protocol (n=8–10 in each group). Nitric oxide (NO) levels were determined using a colorimetric nitric oxide assay kit (Abcam, Cambridge, UK, #ab65328), and reactive oxygen species (ROS) levels were measured using homogenized hippocampal lysates in 10 mM PBS (2 μM, Thermo Fisher Scientific, Waltham, MA, USA #C6827) by reading the fluorescence (488 / 520 nm) of CM-H2DCFDA. Lipid peroxidation, the end product of oxidative stress, was measured as malondialdehyde (MDA) levels using a lipid peroxidation / fluorometric kit (Catalog #K739, BioVision Inc., Milpitas, CA, USA). Hippocampal protein levels of total glutathione (GSH) content were measured using a GSH assay kit (Catalog # CS0260, Sigma-Millipore, St. Louis, MO, USA). Superoxide dismutase (SOD) activity was determined using a SOD assay kit (Sigma-Aldrich, #19160) at concentrations ranging from 0.01 to 50 U / mL by calculating a standard curve with bovine erythrocyte SOD (Sigma-Aldrich, #57494). Catalase activity was assessed using the Amplex™ Red Catalase assay kit (Catalog # A22180, Thermo Fishers). Absorbance was measured at a wavelength of 450 nm using a plate reader. All assays were read with a plate reader at specific wavelengths according to the manufacturer's instructions.

[0058]

[0059] 1.7 IHC analysis

[0060] IHC was applied to determine the dentate gyrus using 4-hydroxyneonyl (HNE) staining of the hippocampus, specifically formalin-fixed, paraffin-embedded (FFPE) samples. Briefly, FFPE sections were sectioned into 4-μm thick sections, deparaffinized in xylene, gradient ethanol solutions, and rehydrated in deionized water (DW). Slides were gently rinsed under running tap water and microwaved for 5 min in sodium citrate buffer (pH 6.0, 10 mM) for antigen retrieval. After cooling to room temperature (RT), the slides were rinsed under running tap water. To remove red blood cells, the slides were soaked in 3% hydrogen peroxide for 10 min and rinsed again. Slides were blocked in blocking buffer containing 10% normal donkey serum for 40 min at RT, followed by incubation overnight at 4°C with anti-mouse 4-HNE antibody (1:200, ABCam, #ab48506). The primary antibody was washed, and the slides were treated with secondary antibodies using a commercially available kit (VECTASTAIN® Elite®ABC Universal Kit, Peroxidase, RTU (Horse Anti-Mouse / Rabbit IgG, #PK-7200, VEOR LABORATORY, CA). The visualized signals were detected using 3,3'-diaminobenzidine (DAB) substrate (ImPACT® DAB Substrate Kit, Peroxidase, #SK-4105 VEOR LABORATORY). Images were captured using optimal microscope conditions (Leica Microsystems, Wetzlar, Germany, 100x). Positive signals were quantified using Image J 1.64 software (NIH, Bethesda, MD, USA).

[0061]

[0062] 1.8 Western blot analysis

[0063] Hippocampal protein expression levels of ChAT (CHAT Rabbit anti-Human Polyclonal antibody, LSBIo, (aa 517-732) LS-B15027, Shirley, MA. LSBIo), AChE (anti-goat polyclonal antibody, NB100-1519, Centennial, CO, Novus Bio), BDNF (anti-rabbit antibody [EPR1292], ab108319 Abcam, UK), p-Creb (anti-rabbit monoclonal antibody, #9198 CST, Beverly, MA, USA), and Creb (anti-rabbit monoclonal antibody, #4820, CST) were assessed by Western blot. Protein lysates were separated by 10% polyacrylamide gel electrophoresis. After running, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane and immediately blocked in 5% skim milk. All membranes were incubated overnight at 4°C with primary antibodies. The membranes were washed with 0.1% TBST and incubated for 2 hours with HRP-conjugated anti-mouse, rabbit, or goat antibodies. Western blots were detected using an enhanced chemiluminescence (ECL) advanced kit (Thermo Fishers).

[0064]

[0065] 1.9 Quantitative real-time PCR analysis

[0066] mRNA expression of the target gene in the hippocampus was determined by real-time PCR. Hippocampal mRNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Ambion, Austin, TX, USA). Real-time PCR was performed using SYBRGreen PCR Master Mix (Applied Biosystems; Foster City, CA, USA), followed by PCR amplification using the Eppendorf Realplex polymerase chain reaction (PCR) system (Eppendorf North America, Hauppauge, NY, USA). Briefly, PCR reactions were performed in a total volume of 10 μL (5 μL of SYBR Green Supermix, 1 μL of each 10-pM primer pair, 1 μL of RNase-free water, and 2 μL of cDNA sample). The entire PCR reaction was performed according to the manufacturer's instructions as follows: 1) 2 min uracil-DNA glycosylase (UDG) activation at 50°C, 2) 2 min DNA polymerase amplification at 95°C, and 3) 40 cycles of amplification (95°C for 15 s, 60°C for 15 s, and 72°C for 15 s). After completion of all steps, a melting curve for each PCR primer was determined using an internal program. The final gene expression level was analyzed using the expression level of β-actin as an internal control. The values ​​of the final product at the mRNA level were evaluated using a standard curve. Information about the PCR primers is described in Figure 8.

[0067]

[0068] 1.10 Measurement of choline acetyltransferase (ChAT) activity, acetylcholine (Ach) levels, and acetylcholine esterase (AchE) activity

[0069] Hippocampal ChAT activity was determined using a ChAT activity assay kit (Elabscience, Huston, TX, USA) according to the manufacturer's protocol. ACh levels and AChE activity were measured using the Amplex Red ACh / AChE assay kit (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. The final products of each reaction were read using a microplate.

[0070]

[0071] 1.11 Statistical Analysis

[0072] We expressed all data as mean ± standard error of the mean (SEM). Statistical significance of differences between groups was determined using one-way or two-way analysis of variance (ANOVA) with Bonferroni t-test as a post hoc multiple comparison using Prism ver. 10.01 (GraphPad, CA, USA). P < 0.05 was used to determine statistically significant differences. (Reference: Data were expressed mean ± SEM ## P< 0.01 and ### P<0.001 for vs. Vehicle group; *P< 0.05, **P< 0.01, or ***P< 0.001 for vs. Scopolamine group (n=4 for each group))

[0073]

[0074] Experimental Example 2. Experimental Results

[0075] 2.1 Chemical composition analysis of GJD

[0076] UHPLC-MS / MS analysis was performed to characterize the chemical composition of GJD. In GJD, we identified a total of 16 compounds. For Cornus officinalis Siebold & Zucc. (Cornus officinalis), two compounds, chlorogenic acid and 5-Feruloylquinic acid, were detected with retention times of 3.96 and 5.38 min. For Angelica acutiloba (Siebold & Zucc.) Kitag (Danggui), Nodakenin, Decursin, and Decursinol Angelate were detected with retention times of 6.89, 16.64, and 16.79 min, respectively. For Typha orientalis Presl (Budle), Quercetin and Naringenin were detected with retention times of 9.11 and 10.09 min, respectively. For Dioscorea polystachya Turcz (Diosbulbin B and Dioscin) with retention times of 14.64 and 17.78 min, respectively. were detected with retention time. In Apis indica Rodosz Kowski (royal jelly), 10-HDA was detected with a retention time of 8.92 minutes. In Cervus elaphus L. (deer antler), phenylalanine, LysoPC 18:2, LysoPC 16:0, LysoPC 18:1, and LysoPC 18:0 were detected with retention times of 2.14, 18.69, 19.62, 20.22, and 22.00 minutes. In Clolstrum (bovine colostrum), androstendione was detected at a retention time of 13.73 minutes.

[0077]

[0078] 2.2 Effect of GJD on anti-amnesia in the Morris Water Maze Task

[0079] To verify the amnesia-inhibiting effect of GJD, the Moris Water Maze Task was performed over a 6-day experimental period. We confirmed that scopolamine injection significantly prolonged escape latency compared to the control (Vehicle) group (Figure 2-A, P < 0.001). Furthermore, the cumulative path length on day 6 was also significantly increased by scopolamine injection compared to the control group (Figure 2-B, P < 0.001). On the final day of the experiment, we measured the time spent in the target quadrant, and the scopolamine group showed a significant decrease compared to the control group (Figure 2-C, P < 0.01).

[0080] However, when administered together with GJD, these cognitive impairment abnormalities were significantly restored compared to the scopolamine group (Fig. 2-A~C, P< 0.05, 0.01, or 0.001). Tacrine, used as a positive drug, showed effects similar to those of GJD treatment (Fig. 2-A~C, P< 0.05, 0.01, or 0.001).

[0081]

[0082] 2.3 Antioxidant effect of GJD on scopolamine-induced hippocampal oxidation

[0083] IHC for 4-HNE showed that scopolamine injection significantly increased 4-HNE positive signals compared to the control group (P<0.01), whereas GJD administration significantly decreased 4-HNE signals throughout the hippocampus in the dentate gyrus region compared to the scopolamine group (Fig. 3-A and B, P<0.01 for 200 and 400 mg / kg).

[0084] Hippocampal protein levels of NO and ROS were significantly increased by scopolamine injection compared to the control group (Fig. 3-C and D, P < 0.01 and 0.05, respectively). The end product of oxidative stress, measured by MDA content in the hippocampal region, was significantly increased in the scopolamine group compared to the control group (Fig. 3-E, P < 0.001). In contrast, GJD administration significantly reduced these oxidative stress markers at the hippocampal protein level compared to the control group (Fig. 3-C to E, P < 0.05 or 0.01, respectively).

[0085] Nonenzymatic antioxidant components and total GSH content in hippocampal protein levels were significantly reduced by scopolamine injection compared to the control group (Fig. 3-F, P < 0.01). Scopolamine injection also reduced hippocampal protein levels of enzymatic antioxidants, such as SOD and catalase activities, compared to the control group (Fig. 3-G and H, P < 0.01 and 0.05, respectively). GJD significantly restored both nonenzymatic and enzymatic antioxidant components compared to the scopolamine group (Fig. 3-F to H, P < 0.05 or 0.01, respectively).

[0086] The mRNA expression levels in the hippocampus also showed a reduced oxidative stress status of approximately 0.41-fold, 0.34-fold, 0.59-fold, and 0.37-fold in Gss, Gpx3, Cat, and Sod2 compared to the control group, indicating the antioxidant capacity of GJD against scopolamine injection (Fig. 9, P< 0.05 or 0.01) (Reference: Gss: glutathione synthetase, Grs; glutathione reductase, Gpx; glutathione peroxidase, Cat: catalase, Sod; superoxide dismutase)

[0087] The positive control drug, THA 10, showed similar effects of GJD on antioxidant properties against scopolamine-induced hippocampal oxidation compared to the control (Fig. 3-A to H, P < 0.05 or 0.01).

[0088]

[0089] 2.4 Anti-inflammatory effect of GJD on scopolamine-induced hippocampal inflammation

[0090] Scopolamine administration significantly upregulated the hippocampal mRNA expression levels of inflammatory cytokines, including NF-α, IL-1β, and IL-6, by approximately 2.78-fold, 2.45-fold, and 4.20-fold compared to the control group (Fig. 4-A~C, P< 0.05 or 0.01), whereas the mRNA expression level of IL-100 was downregulated by 0.37-fold compared to the control group (Fig. 4-D, P< 0.001).

[0091] Hippocampal protein levels of TNF-α, IL-1β, and IL-6 were significantly increased in the scopolamine group compared to the control group (Fig. 4-E~G, P< 0.01 or 0.001).

[0092] However, GJD significantly reduced the elevated cytokine levels compared to the scopolamine group (Fig. 4-A~C, Fig. 4-E~G, P< 0.05, P< 0.01, or P< 0.001).

[0093]

[0094] 2.5 Improvement of cholinergic dysfunction in GJD

[0095] This study is about the ameliorating effect of GJD on scopolamine-induced cholinergic dysfunction in the hippocampus.

[0096] Western blot analysis of ChAT and AChE showed that they were significantly altered by scopolamine injection compared with the control group (P<0.001 and P<0.01, respectively), whereas GJD administration significantly normalized these alterations (Fig. 5-A and B, P<0.05 or P<0.01, respectively).

[0097] Hippocampal protein levels ChAT and AChE activities were significantly altered (decreased in ChAT and increased in AChE) in the scopolamine group (Figure 5-A and B, P< 0.001), whereas GJD administration resulted in a significant increase in ChAT as well as a decrease in AChE compared to the scopolamine group (Figure 5-A and B, P< 0.05, P< 0.01, or P< 0.001).

[0098] These alterations and effects of GJD are supported by quantitative protein level analyses of ACh content and ChAT activity in the hippocampal region (Fig. 5-C and D, P<0.05, P<0.01, or P<0.001). Hippocampal protein levels of AChE activity were significantly increased by scopolamine injection, whereas GJD reduced these abnormalities compared to the scopolamine group (Fig. 5-E, P<0.01 and P<0.001, respectively).

[0099] The positive control drug, THA, had pharmacological properties similar to those of GJD on cholinergic molecules (Fig. 5-A-E, P<0.05, P<0.01, or P<0.001).

[0100]

[0101] 2.6 Enhanced neurotrophic factor effect of GJD

[0102] This study is about the enhanced effect of neurotrophic factors in the hippocampal region induced by scopolamine.

[0103] Hippocampal mRNA expression levels of Machr1, Trka, and Trkab were significantly reduced by 0.43-fold, 0.59-fold, and 0.38-fold, respectively, compared to the control group (Fig. 6A–C, P<0.05 or P<0.01). The mRNA expression level of synaptophysin was downregulated by scopolamine injection by approximately 0.74-fold compared to the control group without statistical significance (Fig. 6D, P>0.05). These changes in mRNA expression levels were significantly upregulated by GJD administration compared to the control group (Fig. 6A–D, P<0.05, 0.01, or 0.001).

[0104] Scopolamine significantly induced a decrease in neurotrophic factors such as BDNF and p-Creb / Creb ratio as evidenced by Western blot analysis compared with the control group (P<0.001), whereas GJD significantly restored these abnormal changes in hippocampal proteins (Fig. 6-E and F, P<0.05, P<0.01, or P<0.001).

[0105] THA 10 showed neurotrophic factor-like effects on both hippocampal mRNA expression and protein levels (Fig. 6-A-F, P< 0.05, P< 0.01, or P< 0.001).

[0106]

[0107] 2.7 Analysis of the effect of GJD on the dentate gyrus

[0108] 도 10은 TUNEL assay of thedentate gyrusin hippocampus regions에 대한 것이다.(TUNEL:Terminal deoxynucleotidyl transferase dUTP nick-end labeling)(A) Histopathological analysis of TUENL assay. (B) Quantitative analysis of positive cell counts. Representative photomicrographs were taken at magnifications of 100 ×. Data were expressed mean ㅁ S.E.M. ### P< 0.001for vs. Vehicle group; ** P< 0.01 and *** P< 0.001 for vs. Scopolamine group (n=4 for each group). 도 11은 IHC analysis against DCX in thedentate gyrusof hippocampus regions에 대한 것이다. (DCX: doublecortin protein) DCX-positive staining in immature neurons is shown in the subgranular zone of the dentate gyrus. Representative photomicrographs were taken at magnifications of 100 and 400×. (B) Quantitative analysis of positive signals. Representative photomicrographs were taken at magnifications of 100 ×. Data were expressed mean ㅁ S.E.M. ### P< 0.001 for vs. Vehicle group; ** P< 0.01 and ***P<0.001 for vs. Scopolamine group (n=4 for each group).

[0109]

[0110] Experimental Example 3. Overall Analysis

[0111] Previous studies have shown that inflammation, as well as impaired cholinergic activity and unpredictable oxidation in brain tissue, particularly in the hippocampus, are important pathological mechanisms of AD. Therefore, maintaining a balance between ACh levels and AChE activity is crucial.

[0112]

[0113] In this study, we investigated the effects of GJD on behavioral analysis using the Morris Water Maze Task. As expected, GJD improved cognitive impairment by reducing escape latency, path length, and time spent in the target quadrant compared to scopolamine-injected mice (Figure 2, A–B). Our data on cholinergic system parameters supported the observation that GJD not only increased depleted ChAT content and AChE at the hippocampal protein level (Figure 4, A–B), but also restored both ACh levels and ChAT activity (Figure 4, C–D). These characteristics ultimately contributed to reducing AChE activity, a key pathological factor in the development and progression of AD (Figure 4, E).

[0114]

[0115] Next, we confirmed the antioxidant and anti-inflammatory effects of GJD, with strong evidence of decreased oxidative stress activity, as evidenced by increased total GSH content and increased antioxidant enzyme activity, such as SOD and catalase, at the hippocampal protein and gene expression levels (Figure 3, A–H). Previous studies have shown that hippocampal oxidation is a common phenotype in the scopolamine-induced amnesia model, characterized by increased 4-hydroxymethylcellulose (4-HNE) production (a marker of lipid peroxidation) due to severe ROS and NO production, and by depletion of endogenous antioxidants. Therefore, we believe that the neuropharmacological effects of GJD are well-established as antioxidant effects by inducing a balance between redox biology. Oxidative stress inevitably accompanies an inflammatory response. In brain tissue, microglia are the most common inducers of inflammatory responses, mediated by their activation and secretion of proinflammatory cytokines and oxidative stress mediators. As a result of these cellular events, neurons undergo damage and cell death. We also found that the neuropharmacological effects of GJD, with a focus on anti-inflammation, decreased the release of pro-inflammatory cytokines and increased anti-inflammatory cytokines at both the mRNA and protein levels in the hippocampus (Fig. 4, A–H).

[0116]

[0117] Our IHC data showed that GJD administration improved apoptosis and impaired cell proliferation in hippocampal neurons (Fig. 9, A and B; Fig. 4, A and B). Hippocampal neurons were damaged by a complex combination of oxidation, inflammation, and cholinergic dysfunction. Cholinergic activity is regulated by CREB phosphorylation. Once activated, CREB translocates to the nucleus for transcription of target genes that play crucial roles in neuronal development, growth, proliferation, differentiation, and survival, respectively. Therefore, there is an interaction between CREB activity and hippocampal-dependent memory capacity. BDNF, a diagnostic biomarker for patients with early-stage Alzheimer's disease and cognitive impairment, is known as an important neurotrophic factor that regulates memory and also participates in CREB activation. Our results showed that the pharmacological properties of GJD reached the deterioration of CREB and BDNF at the hippocampal protein level, and also restored the mRNA expression level of the muscarinic cholinergic receptor, Chrm1 (Fig. 6, A, E, and F). Furthermore, the BDNF receptors TrkA and TrkB in the hippocampus normalized the abnormal downregulation of mRNA expression levels (Fig. 6, B and C). As a key synaptic vesicle molecule, synaptophysin is closely related to memory-related behaviors and is regulated by BDNF in animal studies.

[0118] In this study, we applied positive pharmacodynamic control using the AD drug THA, which improved cognitive abilities with reduced AChE activity, and GJD exhibited similar characteristics. Therefore, GJD may be a potential therapeutic candidate for the treatment and management of AD. Furthermore, we were able to elucidate the neuropharmacological effects of GJD, which were originally prescribed as GJD, through enhanced antioxidant capacity and anti-inflammatory effects. Furthermore, long-term pathological events such as cholinergic dysfunction, neurogenesis, proliferation, and differentiation are likely to be the most targeted signaling pathways for the pharmacological actions of GJD. The underlying mechanism of GJD is activation of the BDNF / CREB signaling pathway.

[0119]

[0120] Example 1. Composition for preventing, improving or treating Alzheimer's disease containing the Gongjindan (GJD) composition of Experimental Example 1 as an active ingredient

[0121]

[0122] This Example 1 includes the GJD (Gongjindan) composition of Experimental Example 1 as an effective ingredient of the composition based on the above Experimental Example. That is, the Gongjindan composition includes a total of 11 kinds of materials: musk (Moschus moschiferus Linne), angelica (Asparagus cochinchinensis ME R), Chinese yam (Dioscorea polystachya Turcz.), angelica (Angelica acutiloba (Siebold & Zucc.) Kitag.), cornus (Cornus officinalis Siebold & Zucc.), deer antler (Cervus elaphus L.), bean beetle (larva) (Popillia flavosellata Fairmaire), cattail (Typha orientalis Presl), bovine colostrum (Bovine Milk Colostrum), chaga mushroom (Inonotus obliquus), and royal jelly (Apis indica Rodosz Kowski). Additionally, each material can be mixed in a weight ratio of 30:360:360:60:24:400:100:60:100:15:50. Additionally, after mixing, the materials can be extracted and used.

[0123]

[0124] The present invention can provide a preparation formulated in a pharmaceutical unit dosage form by including the above-mentioned resonance diagnosis composition as an active ingredient and adding a pharmaceutically acceptable carrier, excipient or diluent. Here, the carrier, excipient or diluent may include tozole, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0125]

[0126] In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination. In addition, when formulating the active ingredient, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. In addition, the pharmaceutical dosage form may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, each according to a conventional method.

[0127] The above solid preparation for oral administration may be prepared by mixing the extract with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

[0128]

[0129] The above-mentioned parenteral administration preparations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. The non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0130] Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0131]

[0132] The preferred dosage of the resonance diagnostic composition of the present invention varies depending on the patient's condition and weight, severity of the disease, age, sex, drug form, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. However, for desirable effects, the composition of the present invention is preferably administered at a dose of 0.001 to 300 mg / kg, and may be administered once daily or in several divided doses. The above dosage does not limit the scope of the present invention in any way.

[0133] The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. Any route of administration is conceivable, including oral, rectal, intravenous, intramuscular, and subcutaneous injection.

[0134]

[0135] The resonance diagnosis composition of the present invention may be provided as a health functional food composition or may be provided by adding a food auxiliary additive.

[0136] Food products include, for example, various foods, beverages, gum, tea, vitamin complexes, and health functional foods.

[0137] The amount of the above-mentioned effective ingredient in the food or beverage may be added in an amount of 0.01 to 20 wt% of the total weight of the food or beverage, and the health beverage composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml.

[0138]

[0139] As described above, the present invention is a composition using natural materials and has no side effects.

[0140]

[0141] The health functional beverage composition of the present invention has no particular limitations on other components other than the resonance diagnosis composition, and may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. Examples of the above-mentioned natural carbohydrates include conventional sugars such as monosaccharides, for example, glucose, fructose; disaccharides, for example, maltose, sucrose, etc.; and polysaccharides, for example, dextrin, cyclodextrin, etc.; and sugar alcohols, for example, xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned, natural flavoring agents (thaumatin, stevia extracts (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.)) can be advantageously used as flavoring agents. The proportion of the above-mentioned natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

[0142]

[0143] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0144]

[0145] Additionally, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

[0146]

[0147] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0148] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0149] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

Claims

A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient, The composition is characterized in that the resonance diagnostic composition comprises musk (Moschus moschiferus Linne), asparagus cochinchinensis MERR, Chinese yam (Dioscorea polystachya Turcz.), angelica (Angelica acutiloba (Siebold & Zucc.) Kitag.), cornus (Cornus officinalis Siebold & Zucc.), deer antler (Cervus elaphus L.), bean beetle (larva) (Popillia flavosellata Fairmaire), cattail (Typha orientalis Presl), bovine milk colostrum (Bovine Milk Colostrum), chaga mushroom (Inonotus obliquus), and royal jelly (Apis indica Rodosz Kowski). In claim 1, A composition characterized in that the above resonance diagnosis composition is a mixed extract obtained by mixing materials. In claim 1, The weight ratio of the materials included in the above resonant diagnostic composition is: A composition characterized by being 30:360:360:60:24:400:100:60:100:15:

50. In claim 1, A composition characterized in that the above resonant diagnostic composition promotes memory or cognitive function, improves cholinergic dysfunction, enhances neurotrophic factors, and has antioxidant and anti-inflammatory effects. A health functional food composition for preventing or improving Alzheimer's disease, comprising a resonance diagnosis composition as an active ingredient. The composition is characterized in that the resonance diagnostic composition comprises musk (Moschus moschiferus Linne), asparagus cochinchinensis MERR, Chinese yam (Dioscorea polystachya Turcz.), angelica (Angelica acutiloba (Siebold & Zucc.) Kitag.), cornus (Cornus officinalis Siebold & Zucc.), deer antler (Cervus elaphus L.), bean beetle (larva) (Popillia flavosellata Fairmaire), cattail (Typha orientalis Presl), bovine milk colostrum (Bovine Milk Colostrum), chaga mushroom (Inonotus obliquus), and royal jelly (Apis indica Rodosz Kowski).

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