Application of composition

Abstract

The invention discloses application of the composition. The composition comprises egg oil, semen raphani oil, perilla seed oil and the like, and can remarkably improve behavioral performance of an Alzheimer's disease model, hippocampal injury of the Alzheimer's disease model, inflammation and oxidative stress, hippocampal acetylcholinergic transfer of the Alzheimer's disease model and intestinal inflammation of the Alzheimer's disease model. According to the present invention, the egg yolk oil composition has the characteristics that the egg yolk oil composition has the characteristic that the egg yolk oil composition is close to the existing chemical drug donepezil for treating the Alzheimer's disease in a plurality of detection indexes, the egg yolk oil composition is significantly superior to the donepezil in the cognitive index index, and the egg yolk oil composition has the cognitive disorder treatment effect, and is superior to the existing drug donepezil to a certain extent.

Description

Technical Field

[0001] The present invention relates to biomedicine, and in particular to the use of a composition. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with an insidious onset and progressive development. Clinically, it is characterized by comprehensive dementia manifestations such as memory impairment, agnosia, apraxia, aphasia, reduced executive function, personality and behavioral changes. It was first diagnosed by neuropathologist Alois Alzheimer and was named after him. Alzheimer's disease (AD) is a common type of senile dementia. The clinical manifestations are mainly insidious onset and slowly progressive dementia. It is generally believed that the cause of Alzheimer's disease is β-amyloid protein (Aβ) and Tau amyloid protein. Deposition leads to neuropathology. Aβ positivity is the core basis for the diagnosis of Alzheimer's disease. The symptoms of the disease may first appear after the age of 60, and the risk increases with age, 5%-6% for those over 65 years old, 10% for those 70 years old, and 48% for those 90 years old. Although AD drugs have a wide audience, their research and development is difficult. Although there have been some chemical drug treatment options for the clinical treatment of AD in recent decades, none of them have achieved satisfactory results (Reardon, 2023). The development of safe and effective treatments for AD is still urgently needed.

[0003] In the prior art, it has been confirmed that drugs such as donepezil, rivastigmine, and galantamine hydrobromide have a certain improvement effect on learning and memory disorders caused by chemical substances, but these chemical substances have strong toxic side effects. There is an urgent need to develop a safe and effective drug for treating Alzheimer's disease in this field. Summary of the invention

[0004] The object of the present invention is to provide a use of a composition.

[0005] Another object of the present invention is to provide a method for preventing and / or treating Alzheimer's disease or its complications.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides a use of a composition, the composition comprising egg yolk oil, radish seed oil and perilla seed oil; the composition is used for:

[0007] (i) prevention and / or treatment of Alzheimer's disease or its complications;

[0008] (ii) preparing a medicament for preventing and / or treating Alzheimer's disease or its complications;

[0009] (iii) improving cognitive impairment or neuroinflammation induced by chemicals;

[0010] (iv) reducing excessive microglial activation;

[0011] (v) reducing the level of pro-inflammatory factors and / or reducing the level of oxidative stress in the hippocampus; and / or

[0012] (vi) increase the expression level of cholinergic receptors, increase the level of acetylcholine and / or increase the activity of key cholinergic enzymes.

[0013] In some preferred embodiments, the composition is a pharmaceutical composition.

[0014] In some preferred embodiments, the composition is a health care product composition.

[0015] In some preferred embodiments, the chemical substance is selected from at least one of scopolamine, trihexyphenidyl, and atropine.

[0016] In some preferred embodiments, the cognitive disorders include learning and memory disorders, working memory disorders, and spatial positioning memory disorders.

[0017] In some preferred embodiments, the complications of Alzheimer's disease include intestinal inflammation caused by Alzheimer's disease.

[0018] In some preferred embodiments, the uses described in (iv)-(vii) are non-therapeutic uses in vitro, such as use as scientific research reagents.

[0019] In some preferred embodiments, the composition further comprises Noni pulp and cinnamon oil.

[0020] In some preferred embodiments, the composition consists of egg yolk oil, perilla seed oil, radish seed oil, noni pulp and cinnamon oil.

[0021] In some preferred embodiments, in the composition, the mass of the egg yolk oil accounts for 55-70% of the total mass of the composition.

[0022] In some preferred embodiments, in the composition, the mass proportion of the egg yolk oil is more than 50%.

[0023] In some preferred embodiments, in the composition, the mass of the perilla seed oil accounts for 15-25% of the total mass of the composition.

[0024] In some preferred embodiments, in the composition, the mass of the radish seed oil accounts for 15-25% of the total mass of the composition.

[0025] In some preferred embodiments, in the composition, the mass of the Noni pulp accounts for 3-7% of the total mass of the composition.

[0026] In some preferred embodiments, in the composition, the mass of the cinnamon oil accounts for 0.1-5% of the total mass of the composition.

[0027] In some preferred embodiments, the composition comprises:

[0028] 55-70 parts by weight of egg yolk oil; and

[0029] Perilla seed oil 15-25 parts by weight.

[0030] In some preferred embodiments, the composition comprises the following:

[0031] 55-60 parts by weight of egg yolk oil; and

[0032] Perilla seed oil 15-20 parts by weight.

[0033] In some preferred embodiments, the composition comprises the following:

[0034] 55-70 parts by weight of egg yolk oil;

[0035] Perilla seed oil 15-25 parts by weight;

[0036] 15-25 parts by weight of radish seed oil;

[0037] 3-7 parts by weight of Noni pulp; and

[0038] 0.1-5 parts by weight of cinnamon oil.

[0039] In some preferred embodiments, the composition comprises the following:

[0040] 55-60 parts by weight of egg yolk oil;

[0041] Perilla seed oil 15-20 parts by weight;

[0042] 15-20 parts by weight of radish seed oil;

[0043] 3-5 parts by weight of Noni pulp; and

[0044] 0.1-3 parts by weight of cinnamon oil.

[0045] A second aspect of the present invention provides a method for preventing and / or treating Alzheimer's disease or its complications, the method comprising the steps of:

[0046] A therapeutically effective amount of the composition as described in the first aspect of the present invention is administered to the subject.

[0047] In some preferred embodiments, the composition is administered by intragastric administration or oral administration, preferably oral administration.

[0048] The third aspect of the present invention provides a use of a health care product composition, wherein the health care product composition comprises an active ingredient and an excipient acceptable to health care products, wherein the active ingredient comprises the composition as described in the first aspect of the present invention; the health care product composition is used for:

[0049] (iii) improving cognitive impairment induced by chemicals;

[0050] (iv) reducing excessive microglial activation;

[0051] (v) reducing the levels of pro-inflammatory factors and / or reducing the levels of oxidative stress in the hippocampus;

[0052] (vi) increase the expression level of cholinergic receptors, increase the level of acetylcholine and / or increase the activity of key cholinergic enzymes;

[0053] (vii) Improve neuroinflammation induced by chemicals.

[0054] The fourth aspect of the present invention provides a method for reducing the transcription level of inflammatory signal factors in nerve cells in vitro, the method comprising the steps of: culturing nerve cells in a culture system containing the composition described in the first aspect of the present invention.

[0055] In some preferred embodiments, the neural cells are microglia.

[0056] In some preferred embodiments, the microglia are derived from mammals, preferably humans or mice.

[0057] In some preferred embodiments, the nerve cells are induced to produce inflammatory signal factors by chemical substances.

[0058] Compared with the prior art, the present invention has at least the following advantages:

[0059] (1) The composition of the present invention can significantly improve the behavioral performance of the Alzheimer's model, improve the damage of the hippocampus of the Alzheimer's model, reduce inflammation and oxidative stress, improve the acetylcholine transmission in the hippocampus of the Alzheimer's model and improve the intestinal inflammation of the Alzheimer's model. In many detection indicators, it is close to the chemical drug donepezil currently used to treat Alzheimer's disease, and the egg yolk oil group is significantly better than donepezil in the cognitive index indicator. The present invention has the effect of treating cognitive impairment and is superior to the existing drug donepezil to a certain extent;

[0060] (2) The composition of the present invention is derived from food, has safe ingredients, and has almost no toxic side effects. It can be used as a health product for potential Alzheimer's patients or middle-aged and elderly people to take for a long time.

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0063] Figure 1 EPRCN was shown to prevent scopolamine-induced cognitive dysfunction in mice. (A) Experimental schedule. Oa: oral administration; Ip: intraperitoneal injection. NOR: novel object recognition; MWM: Morris water maze. (B) Escape latency during Morris water maze training. (C) Representative track images on days 1 and 4 of training (MWM test). (D) Stay time in the target quadrant, (E) platform crossing time, (F) average swimming speed of mice in the navigation test. Spontaneous alternation (G) and total arm entries (H) of the Y maze. Number of mouse explorations of novel objects (I) and recognition index of mice (J) in novel object recognition. (K) Representative track images of mice on day 3 (NOR test). Data are presented as mean ± standard error of the mean, and each open point represents one sample. *p<0.05, ns, no significant difference.

[0064] Figure 2 The results showed that EPRCN inhibited scopolamine-induced microglia-related neuroinflammation and oxidative stress in the hippocampus. (A) The number of microglia in the CA1 area of ​​the hippocampus per square millimeter ( / mm 2 ) Quantitative analysis of the number of Iba-1-positive microglia. (B) Representative images of Iba-1-positive microglia. Black arrows indicate activated microglia. (C) Levels of IL-6, (D) levels of MDA, (E) SOD activity, and (F) levels of GSH in the hippocampus. Data are presented as mean ± standard error of the mean, and each hollow dot represents one sample. *p < 0.05, ns, no significant difference.

[0065] Figure 3Transcriptome analysis revealed the molecular mechanism by which EPRCN improves the AD-like phenotype in mice. Volcano plot of differentially expressed genes (DEGs) identified from the comparisons of (A) Sco vs Vco, (B) EPRCN vs Sco, (C) Don vs Sco, and (D) EPRCN vs Don. The top 5 most enriched KEGG pathways enriched for up- and down-regulated DEGs identified from the comparisons of (E) Sco vs Vco, (F) EPRCN vs Sco, (G) Don vs Sco, and (H) EPRCN vs Sco.

[0066] Figure 4 Figure 3. EPRCN reshaped the metabolic profile of mice with AD-like phenotypes. Scatter plot of differentially abundant metabolites identified from the comparison of (A) Don vs Sco and (B) EPRCN vs Sco. (C) Heat map of the top 50 most abundant metabolites with annotations. KEGG topology map of differentially abundant metabolites identified from the comparison of (D) Don vs Sco and (E) EPRCN vs Sco.

[0067] Figure 5 EPRCN promotes AEA biosynthesis in SH-SY5Y cells (human neuroblastoma cells) and transcription of Trpv1 and Nrf2 in BV-2 cells. (A) Aβ (25-35) Schematic diagram of qPCR analysis of the induced neuronal (SH-SY5Y) neuroinflammation model. qPCR analysis showing (B) NAPE-PLD or (C) FAAH levels in EPRCN-treated SH-SY5Y cells, regardless of whether Aβ (25-35) Treatment (n=3 per group). (D) Aβ (25-35) Schematic diagram of qPCR analysis of induced astrocytes (SW-1783) and microglia (BV-2) in the neuroinflammation model. qPCR analysis showing (E) TRPV1 or (F) Trpv1 levels in EPRCN-treated SW-1783 and BV-2 cells, regardless of whether Aβ was used. (25-35) qPCR analysis shows (G) CNR1 or (H) Cnr1 levels in EPRCN-treated SW-1783 and BV-2 cells, regardless of whether Aβ was used or not. (25-35) Treatment (n = 3 per group). qPCR analysis shows the levels of (I) CNR2 (cannabinoid receptor 2) or (J) Cnr2 in SW-1783 and BV-2 cells treated with EPRCN, regardless of whether Aβ (25-35) qPCR analysis showing (K) NRF2 and (L) Nrf2 levels in EPRCN-treated SW-1783 and BV-2 cells, with or without Aβ(25-35) Treatment (n = 3 per group). Data are expressed as mean ± standard error of the mean. #p < 0.05 vs. (25-35) Compared with the cell group treated with 0 μg / mL EPRCN; *p<0.05 compared with Aβ (25-35) Comparison of the cell groups treated with 0 μg / mL EPRCN.

[0068] Figure 6 Demonstrated that EPRCN and its components inhibit Aβ (25-35) Induced neuroinflammation in BV-2 cells. (A) Schematic diagram of the cell experiment design. (B) EPRCN and its components affect Aβ (25-35) Effects of EPRCN and its components on the transcription of IL-6 induced by BV-2 cells. (C) Effects of EPRCN and its components on the transcription of IL-6 induced by Aβ (25-35) Effect of induced Tnf-α transcription in BV-2 cells. ^ There is a significant difference between the target group and the negative control group (dark gray column); * represents a significant difference between the target group and the model group (blank column); # represents a significant difference between the target group and the EPRCN group (light gray column). Data are expressed as mean ± standard error of the mean. Referring to the formula of EPRCN, the dosage of each group is as follows: EPRCN group: 150μg / ml; Egg yolk oil group: 87μg / ml; Perilla seed oil group: 28.2μg / ml; Raphani seed oil group: 28.2μg / ml; Noni Puree group: 6μg / ml; Cinnamon oil group: 0.6μg / ml.

[0069] Figure 7 A schematic diagram showing the possible mechanism of action of EPRCN in preventing cognitive deficits in the AD mouse model.

[0070] Figure 8 A schematic diagram showing the localization of hippocampal CA3 and DG is shown.

[0071] Fig. 9 Figure 2 OPLS-DA (orthogonal principal component analysis) score diagram between (A) EPRCN vs Sco group mice and (B) Don group and Sco group mice based on the metabolic profiles of positive and negative ion modes.

[0072] Fig.10 Metabolites that improve cognitive impairment were upregulated in the EPRCN group compared with the Sco and Don groups. (A) Dehydroascorbic Acid. (B) Citric Acid. (C) Plumbagin. (D) Cabergoline.

[0073] Fig.11 The integrated analysis of transcriptome and metabolome data showed that (A) Trpv1 expression and its activator (B) anandamide production were promoted. Data are presented as mean ± standard error of the mean. *p<0.05, ns indicates no significant difference. DETAILED DESCRIPTION

[0074] In the present invention, we developed a pharmaceutical composition EPRCN formulation consisting of five safe ingredients, which can prevent scopolamine-induced learning and memory impairment in mice. It also improved neuroinflammation and oxidative stress in the hippocampus of these mice. Multi-omics and in vitro studies have shown that the pharmaceutical composition of the present invention can inhibit neuroinflammation and oxidative stress by activating the AEA-Trpv1-Nrg2 pathway. In addition, the components in the pharmaceutical composition of the present invention work synergistically, and the therapeutic effect is significantly better than that of the individual components.

[0075] the term

[0076] As used in this article, the terms "egg yolk oil", "egg yolk oil", "chicken oil" and "egg butter" can be used interchangeably to refer to the oil extracted from the yolk of eggs. Egg yolk oil has the effects of clearing heat and detoxifying, astringing, stopping bleeding and relieving pain. It is mostly used for burns, eczema, ear pus, sores and the like.

[0077] As used herein, the term "radish seed oil" refers to the oil separated from radish seeds, which are the dried mature seeds of the plant radish (RaphanussativusL) of the genus Raphanus (RaphanusL) of the cruciferous family. The plants are harvested in summer when the fruits are ripe, dried, the seeds are rubbed out, and the impurities are removed and then dried again. Radish seeds belong to the lung, spleen, and stomach meridians, are flat in nature, and have a pungent and sweet taste. They have the functions of descending qi and resolving phlegm, digesting food and removing bloating. They are mainly used to treat food stagnation, constipation, abdominal distension and pain, phlegm-blocked cough and wheezing, and stagnation and diarrhea.

[0078] As used herein, the term "perilla seed oil" refers to a natural oil obtained from the mature seeds of the medicinal plant Perilla frutesccns of the Labiatae family, which is a highly unsaturated oil. Perilla is mainly used to treat colds, fever, cough, asthma, chest and abdominal distension, and fetal movement disorder.

[0079] As used herein, the terms "cinnamon oil" and "cassia bark oil" are used interchangeably and refer to a clear liquid that is yellow or yellow-brown; it has the distinctive aroma of cinnamon and tastes sweet and spicy. Cinnamon oil is an aromatic oil obtained by distilling the bark, branches and leaves of the cinnamon plant of the Lauraceae family. It is mainly used to treat spleen and stomach yang deficiency, cold limbs and weak pulse, cold accumulation in the abdomen, abdominal pain and diarrhea, cold hernia and running pigs; it is used externally to treat rheumatism and skin itching.

[0080] As used herein, the term "Noni pulp" refers to the pulp extracted from Noni fruit, which is the fruit of Morinda officinalis, and has the effects of dispelling wind evil, strengthening yang and kidney, and strengthening tendons and bones.

[0081] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0082] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0083] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0084] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0085] 1. Experimental Methods

[0086] 1.1 Drug preparation

[0087] The composition (EPRCN) of the present invention is a mixture of the following ingredients: 0.29 g of egg yolk oil (G. gallus domesticus) (58.0%), 0.094 g of perilla seed oil (P. frutescens) (18.8%), 0.094 g of radish seed oil (R. Sativus) (18.8%), 0.02 g of noni pulp (M. citrifolia) (4.0%) and 0.002 g of cinnamon oil (C. spp.) (0.4%).

[0088] The composition of the present invention is encapsulated in 0.5 gram capsules to prepare capsules.

[0089] According to the "3R" principle of animal experiment ethics, the dose of EPRCN was calculated using the equivalent dose ratio of 20 mg / kg, resulting in a daily intake of 0.36 to 0.52 mg for mice.

[0090] 1.2 Animal Model and Dosing Regimen

[0091] 36 SPF-grade eight-week-old male ICR mice, weighing 18-26 g, were housed at 22±2°C and maintained in a 12-h light-dark cycle. All mice were provided by SLAC Laboratory Animal Co., Ltd., Shanghai, China (license number SCXK(Hu)2022-0004). This study has been approved by the Experimental Animal Ethics Committee of Shanghai Institute of Biomedical Technology (protocol code 2023-30, approval date March 28, 2023). EPRCN and donepezil (product number D332795, Aladdin) were dissolved in 0.5% CMC-Na (product number HY-Y0703, MedChemExpress) and administered orally. Scopolamine (product number S302239, Aladdin) was dissolved in normal saline and administered by intraperitoneal injection. The control group mice were orally administered with an equal volume of 0.5% CMC-Na and intraperitoneally injected with an equal volume of normal saline. The doses of scopolamine (3 mg / kg / d) and donepezil (3 mg / kg / d) were based on previous studies (Yu-Min et al., 2019). All mice were randomly divided into the following four groups: control group (Vco group) (normal saline 5 ml / kg / d + 0.5% CMC-Na 5 ml / kg / d, n = 9), scopolamine (Sco) group (scopolamine 3 mg / kg / d + 0.5% CMC-Na 5 ml / kg / d, n = 9), donepezil (Don) group (scopolamine 3 mg / kg / d + donepezil 3 mg / kg / d, n = 9) and EPRCN (EPRCN) group (scopolamine 3 mg / kg / d + EPRCN 20 mg / kg / d, n = 9). Mice were orally administered 0.5% CMC-Na, donepezil, or EPRCN once daily for 24 days. The body weight of mice was measured every 7 days to adjust the dosage. Figure 1 A. Behavioral testing lasted for 10 days, and drug administration was performed simultaneously during the testing period.

[0092] 1.3Y maze spontaneous alternation test

[0093] The Y-maze (Shanghai Xinruan Information Technology Co., Ltd., China) spontaneous alternation test was used to measure working memory as previously reported (Luo et al., 2020). The Y-maze apparatus had three identical arms (35 × 5 × 15 cm) with a visual cue at the end of each arm at 120°. Mice were placed in one of the three arms and allowed to explore freely for 7 min. The trajectories were videotaped, and the spontaneous alternation rate was calculated as follows: spontaneous alternation rate = (number of alternations / total number of arm entries-2) x 100 (%). Spontaneous alternation was defined as three consecutive entries of a mouse into different arms. At the end of the experiment, the arms were cleaned with 75% alcohol to remove the odor of the previous mouse.

[0094] 1.4 New Object Recognition (NOR) Test

[0095] The NOR test was performed after the Y-maze spontaneous alternation test. The apparatus consisted of a 50 × 50 × 20 cm rectangular apparatus (Shanghai Xinruan Information Technology Co., Ltd., China), a cylinder, and a triangle of the same wood material. The NOR test was divided into three parts: an adaptation phase, a training phase for familiar object memory, and a recognition phase for novel objects. On the first day, each mouse was placed in an open field and allowed to explore freely for 5 min. At the end of this period, the mouse was removed, the box was wiped with 75% ethanol to remove the odor, and then the next trial was performed. On the second day, two identical objects were placed in a rectangular box and fixed with double-sided tape, and the mouse was placed in the apparatus to explore freely for 10 min, after which the mouse was removed and the box was wiped with 75% ethanol to remove the odor. On the third day, a familiar object was replaced by a novel object, and the mouse was allowed to explore the apparatus for 10 min. The time the mouse explored each object was recorded by a video camera mounted above and automatically monitored and recorded by VsiuTrack animal behavior analysis software (Shanghai Xinruan Information Technology Co., Ltd., China).

[0096] 1.5 Morris water maze (MWM) test

[0097] The MWM apparatus consisted of a circular water tank (diameter, 120 cm; depth, 50 cm) filled with water and painted black to hide the platform (diameter, 10 cm). Different visual cues were placed in four locations: north, south, east, and west. The hidden platform was placed in the southeast center. In the training part (days 1–4), mice were placed in the MWM apparatus and allowed to swim freely for 60 s until they found the hidden platform. If the mice did not find the platform within 60 s, they were carefully placed on the platform. Thirty seconds after the mice found the platform, they were returned to their cages. Training was performed three times a day for four days. In the detection part, mice were allowed to swim freely for 60 s without the hidden platform. The time and distance that the mice stayed in the target quadrant, the number of escape platform crossings, and the average swimming speed were analyzed using VsiuTrack Animal Behavior Analysis Software (Shanghai Xinruan Information Technology Co., Ltd., China).

[0098] 1.6 Sample Collection

[0099] Mice were anesthetized and decapitated immediately after the behavioral experiment. Mouse brains were divided into left and right hemispheres. The right hemispheres of five mice were placed in 4% paraformaldehyde for immunohistochemistry, and the left hemispheres were used for metabolome and biochemical marker analysis. The right hemispheres of the remaining mice (n=3) were subjected to RNA sequencing, and the left hemispheres were prepared for qRT-PCR.

[0100] 1.7 Immunohistochemistry

[0101] After dehydration with 30% sucrose, the brain was cut into 40 μm coronal sections using a freezing microtome (CM3050S, Leica, USA), and then the brain sections were placed in 1× PBS. For long-term storage, PBS was replaced with antifreeze solution (5mM Na2HPO4, 30% sucrose, 20mM NaH2PO4, 30% ethylene glycol and 1% polyvinyl pyrrolidone) and stored at -20°C. After three PBS washes, the cells were incubated with 3% hydrogen peroxide for 15 minutes to inhibit endogenous peroxidase. Then, the sections were washed three times with PBS and blocked with blocking buffer (PBS containing 0.3% Triton X-100, 5% goat serum and 2% BSA) at room temperature for 1.5 hours, followed by incubation with primary antibodies at 4°C overnight. Rabbit polyclonal anti-Iba-1 antibody (Product No. 019-19741, Wako) was used. Images were acquired using a Nikon confocal microscope (Nikon, USA).

[0102] 1.8 RNA sequencing

[0103] Total RNA from mouse hippocampal tissue was purified by RNApure Fast Tissue&Cell Kit (Product No. CW0599S, CWBIO), and the quality of RNA was checked by ND-1000 (NanoDrop), Bioanalyzer 2100 (Agilent), and agarose gel electrophoresis. mRNA with polyadenylation was captured by magnetic beads (Product No. 88802, ThermoFisher) and fragmented by the magnesium RNA fragmentation module. A sequencing library with a fragment size of 300±50bp was constructed and sequenced in PE150 sequencing mode using NovaSeq X Plus. The raw data was processed using Cutadapt-1.9 software to remove low-quality spliced ​​sequences and repeat sequences. The clean data was compared with the genome using HISAT2-2.0.4 (https: / / daehwankimlab.github.io / hisat2 / ). FPKM was calculated as FPKM = total_exon_fragments / mapped_reads (million) × exon_length (kB) using the ballgown package. The DESeq2 R package (http: / / www.bioconductor.org / packages / release / bioc / vignettes / DESeq2 / inst / doc / DESeq2.html) (version 1.16.1) based on negative binomial distribution was used to analyze the differential expression of the four groups, and genes with log2|fold change|≥1 and p<0.05 were defined as differentially expressed genes (DEGs), and KOBAS (http: / / bioinfo.org / kobas) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, with a threshold of p value <0.05.

[0104] 1.9 Metabolomics

[0105] Untargeted metabolomics experiments were performed as described previously (Li et al., 2022). The extraction simplified steps and conditions were as follows: 1 ml of 70% v / v methanol precooled to 4 °C was added to a 2.0 ml microcentrifuge tube containing 100 mg of powdered sample stored at 4 °C, extracted overnight, centrifuged at 10,000 g for 10 min, and then the supernatant was collected immediately. A CNWBOND Carbon-GCB SPE column (250 mg, 3 ml) was used; it was subsequently absorbed with ANPEL (Shanghai, China). The collected extracts were then further analyzed in the following section. The sample extracts were analyzed using an LC-ESI-MS / MS system. The identified metabolites were annotated using the HMDB (https: / / hmdb.ca / ) and KEGG compound databases (http: / / www.kegg.jp / kegg / compound / ), and the annotated metabolites were subsequently mapped to the KEGG pathway database (http: / / www.kegg.jp / kegg / pathway.html). Significantly differentially expressed metabolites between groups were determined by VIP (variable projection importance) ≥ 1 and |log2FC (fold change)| ≥ 1. VIP values ​​were extracted from orthogonal partial least squares discriminant analysis (OPLS-DA) results, which also included score plots and arrangement plots, and were generated using the R package MetaboAnalystR (version 1.0.1). Significantly enriched pathways were determined using the hypergeometric test (p < 0.05). Heatmaps were produced using TBtools-II (version 2.130) (Chen et al., 2023).

[0106] 1.10 Cell culture and drug treatment

[0107] Immortalized mouse microglia BV-2 cells (Cat. No. CL-0493) and human neuroblastoma SH-SY5Y cells (Cat. No. CL-0208) purchased from Pronocell Life Sciences were cultured in Dulbecco's modified Eagle medium (DMEM, Cat. No. 11965092, Thermofisher) containing 10% heat-inactivated fetal bovine serum (FBS, Cat. No. SH30406.02, HyClone), 1% penicillin / streptomycin (P / S, Cat. No. G4003, Servicebio), and DMEM / F12 (Cat. No. 11320033, ThermoFisher) supplemented with 10% FBS, 1% non-essential amino acids (NEAA, Cat. No. 11140050, ThermoFisher) and 1% P / S at 37°C, 5% CO2. Human astrocyte SW-1783 cell line (Cat. No. STCC11003P) was purchased from Servicebio Cell Cultures and cultured in RPMI-1640 (Cat. No. 11875093, Servicebio) containing 10% hot FBS and 1% PS at 37°C and 5% CO2.

[0108] SH-SY5Y cells were cultured at 1×10 per well. 5 Cells were seeded in 12-well culture plates at a density of 10 cells / mL. After 24 h of incubation, cells were pretreated with 0, 50, 100, 150, or 200 μg / ml for 0.5 h and then treated with or without 20 μg / mL Aβ (25-35) (Cat. No. HY-P0128, MedChemExpress) for 24 hours. After treatment, the medium was removed, the cells were washed three times with PBS, and collected for RNA extraction and biochemical index determination. BV-2 and SW-1783 cells were plated at 1×10 5 Cells were seeded at a density of 10 cells / mL in 12-well culture plates. After 24 h of incubation, cells were pretreated with 0, 50, 100, 150, or 200 μg / ml EPRCN for 0.5 h and then treated with or without 20 μg / ml Aβ (25-35)Stimulate for 24 hours. For BV-2 cells, antagonists were added to the culture medium 15 minutes before EPRCN incubation. AM404 (Cat. No. HY-101388, MedChemExpress), AMG9810 (Cat. No. HY-101736, MedChemExpress) and brusatol (BT, Cat. No. HY-19543, MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO, Cat. No. HY-Y0320, MedChemExpress) to obtain a stock solution. The DMSO concentration in the culture medium was kept at 0.1% and below. The antagonist concentrations used were as follows: AM404 (50 μM), AMG9810 (1 μM) and BT (200 nM). After treatment, the culture medium was removed, the cells were washed three times with PBS, and collected for RNA extraction and biochemical index determination.

[0109] 1.11RT-qPCR

[0110] RNA extraction and cDNA reverse transcription were performed as described above. All qRT-PCR primers were designed according to the corresponding sequences using Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi) and are listed in Supplementary Table 1. All primers used were required to span an exon-exon junction. The 20 μL reaction volume contained the following components: 10 μL SYBR Green qPCR Master Mix (Cat. No. G3328, Servicebio), 0.4 μL forward and reverse primers, 2 μL cDNA, and 7.2 μL nuclease-free water. qRT-PCR analysis was performed in a two-step method with the following cycle parameters: 94°C for 30 seconds, 40 cycles of denaturation (94°C, 5 seconds) and annealing (60°C, 30 seconds). All reactions were repeated two or three times with β-actin or GAPDH as the internal reference, and the relative mRNA expression data used 2 ▲▲ The results were calculated by Ct method and expressed as mean ± standard error of the mean (SEM).

[0111] 1.12 Biochemical analysis

[0112] The activities of SOD (superoxide dismutase), GSH-px (glutathione peroxidase) and the contents of MDA (malondialdehyde) and reduced GSH (glutathione) were detected using kits (Cat. No. A001-2, A005-1, A003-1, and A006-2, Nanjing Jiancheng Biotechnology Institute). The cortex and hippocampus of each group were pretreated with 1× PBS (Cat. No. G4202, Servicebio). The homogenate was centrifuged at 5000×g for 10 minutes at 4°C. The supernatant was stored in an Eppendorf tube, and the levels and activities of SOD, GSH-px, MDA and reduced GSH were detected using a universal microplate spectrophotometer (Bio-Rad, Hercules, USA) according to the manufacturer's instructions. IL-6 detection was performed using a microplate reader (Bio-Rad) at 450nm according to the manufacturer's instructions (Cat. No. E-EL-M0044, Elabscience).

[0113] 1.13 Statistical analysis

[0114] Statistical analysis was performed using GraphPad Prism version 9.5 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± standard error of the mean (SEM), and each open point on the bar graph represents the measurement of one sample. One-way or two-way ANOVA was used for comparison between groups, with Tukey's multiple comparison correction when necessary. # and * indicate p < 0.05, which was considered statistically significant.

[0115] 2. Results

[0116] 2.1EPRCN rescues scopolamine-induced cognitive impairment in mice

[0117] To evaluate the protective effect of EPRCN against AD, 9-week-old scopolamine-induced mice were orally treated with EPRCN (20 mg / kg / d) or donepezil (5 mg / kg / d, used as a positive control) for 24 days ( Figure 1 A). The mice were then tested in Morris water maze, Y maze and novel object recognition (NOR).

[0118] Morris water maze test showed that during the training day, the average escape latency of the scopolamine model group (Sco) was significantly longer than that of the control group (Vco) ( Figure 1 B). In contrast, the mean escape latency of the EPRCN (EPRCN) and donepezil (Don) groups was significantly shorter than that of the Sco group. Notably, the mean escape latency of the EPRCN group was significantly lower than that of the Don group on day 4. Mice in the Sco group showed disorganized tracks and spent more time searching for the platform, which was improved after EPRCN or donepezil administration ( Figure 1 C). Mice in the EPRCN or Don group spent relatively more time in the target quadrant ( Figure 1 D), and crossed the platform more times than the Sco group ( Figure 1 E), and EPRCN was close to donepezil in effect at selected concentrations. There was no significant difference in swimming speed between the Sco, Don, and EPRCN groups, demonstrating that swimming speed was not improved ( Figure 1 F).

[0119] In the Y-maze spontaneous alternation test, the EPRCN and Don groups showed more spontaneous alternation behaviors than the Sco group ( Figure 1 G). Scopolamine-induced mice treated with EPRCN and donepezil entered the arms more often than scopolamine-induced mice, indicating that both drugs improved the exploratory activity of scopolamine-induced mice ( Figure 1 H). In addition, a novel object recognition test was performed to examine the cognitive ability of mice. The scopolamine-induced decrease in the time spent on the novel object was reversed by EPRCN and donepezil administration ( Figure 1 I). Interestingly, EPRCN administration was more effective than donepezil in improving the recognition index in scopolamine-induced mice ( Figure 1 J). In the representative trace, it was found that the Sco group mice tended to explore familiar objects rather than novel objects, which was reversed in the Don and EPRCN groups ( Figure 1 K).

[0120] 2.2EPRCN inhibits scopolamine-induced neuroinflammation and oxidative stress

[0121] We then investigated whether EPRCN could prevent scopolamine-induced abnormal activation of microglia in mice. As expected, abnormal activation of microglia was observed in the dentate gyrus (DG) of the hippocampus in the Sco group mice compared with the Vco group ( Figure 2 A, Figure 8 ). However, in scopolamine-induced mice treated with EPRCN or donepezil, microglial activation was significantly suppressed ( Figure 2 B). In addition, the levels of IL-6 and MDA were significantly decreased in the EPRCN group ( Figure 2 C, D), SOD activity and GSH (reduced) levels were higher ( Figure 2 E, F), indicating that EPRCN can inhibit scopolamine-induced neuroinflammation and oxidative stress in the mouse hippocampus.

[0122] 2.3EPRCN regulates multiple signaling pathways in the hippocampus of mice with AD-like phenotypes

[0123] To identify biological factors and signaling pathways associated with the efficacy of EPRCN, multiple comparisons were performed between the Vco, Sco, Don, and EPRCN groups by RNA sequencing. Comparison between the Sco and Vco groups revealed 151 differentially expressed genes (DEGs), including 90 upregulated and 61 downregulated genes ( Figure 3 A). When comparing the EPRCN and Sco groups, 457 DEGs were identified, of which 347 were upregulated and 110 were downregulated ( Figure 3 B). When comparing the Don and Sco groups, 384 DEGs were identified, of which 222 were upregulated and 162 were downregulated ( Figure 3 C). A total of 239 DEGs were identified between the EPRCN and Don groups, of which 182 were upregulated and 57 were downregulated ( Figure 3 D).

[0124] From the comparison between Sco and Vco groups, it can be observed that the five pathways with the most upregulated DEGs were related to neuroinflammation, while the five pathways with the most downregulated DEGs were related to disease and biosynthesis ( Figure 3 E). Neuroactive ligand-receptor interaction and PI3K-Akt signaling pathway were significantly enriched in upregulated DEGs in the comparison between Don and Sco and between EPRCN and Sco ( Figure 3 F, G). Interestingly, ECM-receptor interactions were enriched with up-regulated DEGs in the comparison between Sco and EPRCN. Xenobiotic metabolism via cytochrome P450 and tight junctions were enriched with up-regulated DEGs in the comparison between EPRCN and Don ( Figure 3 H). Compared with donepezil treatment, EPRCN significantly promoted antioxidant activity but reduced paracellular permeability, which played an important role in improving cognitive impairment.

[0125] 2.4EPRCN reshapes the metabolic profile of the mouse hippocampus with AD-like phenotypes

[0126] To further explore the potential mechanism of EPRCN's protective effect against AD, we performed an untargeted metabolomics study in the hippocampus. The OPLS-DA score plot clearly distinguished scopolamine-induced mice treated with EPRCN or donepezil from Sco model mice, with higher R 2 X, R 2 Y and Q 2 Y value( Fig. 9 In the comparison between Don and Sco and between EPRCN and Sco, 599 and 483 differential metabolites were identified, respectively ( Figure 4 A, B). Compared with donepezil, EPRCN increased the levels of metabolites that contribute to the improvement of cognitive impairment, including dehydroascorbic acid, cabergoline, plumbagin, and citric acid ( Figure 4 C, Fig.10 ).

[0127] KEGG pathway enrichment revealed significant changes in glycerophospholipid and amino acid metabolic pathways after treatment with EPRCN or donepezil ( Figure 4 D, E). Interestingly, the neuroactive ligand-receptor interaction pathway was significantly enriched in EPRCN but not in donepezil. Integrated analysis of transcriptomic and metabolomic data showed that after EPRCN administration, scopolamine-induced mice simultaneously promoted the expression of transient receptor potential cation channel subfamily V member 1 (Trpv1) and its activator anandamide (AEA) ( Fig.11 ).

[0128] 2.5EPRCN promotes Aβ (25-35) Induced biosynthesis of AEA in SH-SY5Y cells, and Aβ (25-35) Induced transcription of Trpv1 and Nrf2 in BV-2 cells

[0129] AEA is synthesized by N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and converted to arachidonic acid by fatty acid amide hydrolase (FAAH) in central neurons. (25-35) An in vitro neuroinflammatory neuronal model was constructed by co-culture of EPRCN and SH-SY5Y cells to explore the effect of EPRCN on AEA synthesis. Figure 5 A). The results showed that incubation with EPRCN significantly affected the expression of NAPE-PLD in SH-SY5Y cells only at a concentration of 150-200 μg / mL, but significantly increased Aβ at all concentrations. (25-35) NAPE-PLD expression in SH-SY5Y cells induced by Figure 5 B). In contrast, incubation with EPRCN at 100-200 μg / mL significantly reduced the expression of SH-SY5Y cells and Aβ under steady-state conditions. (25-35) Induced expression of FAAH in SH-SY5Y cells ( Figure 5 C).

[0130] We further evaluated the effects of EPRCN on the AEA downstream receptors Trpv1, CNR1 (cannabinoid receptor 1), and CNR2 (cannabinoid receptor 2) in two Aβ-induced (25-35) Effects on transcriptional levels in the induced glial neuroinflammation model (microglial cell line: BV-2; astrocyte cell line: SW-1783) Figure 5 D). EPRCN significantly increased the mRNA level of TRPV1 in SW-1783 cells under steady-state conditions at concentrations of 150 and 200 μg / mL, whereas EPRCN did not significantly affect Aβ(25-35) Induced transcription of TRPV1 in SW-1783 cells ( Figure 5 E). In contrast, EPRCN significantly increased steady-state or Aβ at concentrations of 50-200 μg / mL. (25-35) The mRNA level of Trpv1 in BV-2 cells under induced state ( Figure 5 F). Further data showed that EPRCN did not affect the transcription of CNR1 (or Cnr1) or CNR2 (or Cnr2) in microglial or astrocyte neuroinflammation models ( Figure 5 GJ).

[0131] TRPV1 can inhibit M1 macrophage polarization by activating Nrf2-dependent pathways. Therefore, we determined the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in EPRCN-treated SW-1783 and BV-2 cells and found that Nrf2 was only activated in the steady state or Aβ-induced by EPRCN. (25-35) The expression of β-catenin in BV-2 cells was significantly increased under induced conditions, but not in SW-1783 cells ( Figure 5 KL).

[0132] 2.6EPRCN and its components inhibit Aβ (25-35) Induced neuroinflammation in BV-2 cells

[0133] We further explored the effects of EPRCN and its components on Aβ through cell experiments. (25-35) Effects of induced neuroinflammation in BV-2 cells ( Figure 6 A) Cell experiments first showed that Aβ (25-35) After 24 h of induction, the transcription levels of IL-6 and Tnf-α in BV-2 cells increased significantly, and this trend was reversed after EPRCN treatment ( Figure 6 B and C). This phenomenon was also found when the single component of EPRCN acted alone; egg yolk oil, perilla seed oil, radish seed oil and noni pulp pretreatment significantly prevented Aβ (25-35) The transcriptional level of IL-6 in BV-2 cells induced by Aβ was significantly increased, while pretreatment with egg yolk oil, perilla seed oil and noni pulp significantly prevented the Aβ (25-35) The induced Tnf-α transcription level in BV-2 cells increased significantly. Compared with the single component effect, the inhibitory effect of EPRCN on IL-6 and Tnf-α transcription was significantly higher than that achieved by all single components at the same composition concentration, proving that the components of the EPRCN composition of the present invention have a synergistic effect.

[0134] 2.7. Conclusion

[0135] We demonstrated that EPRCN can prevent cognitive deficits in a mouse model of AD ( Figure 7 ). EPRCN treatment reversed the increased number of activated microglia, proinflammatory factors, and oxidative stress levels exhibited by AD-like phenotype mice, suggesting that EPRCN may be an effective treatment for improving AD-related symptoms. The possible mechanism of action of EPRCN in suppressing neuroinflammation and oxidative stress may involve activation of the AEA-Trpv1-Nrf2 pathway in scopolamine-induced mice.

[0136] Table 1 Primer list

[0137]

[0138] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A use of a composition, characterized in that: The composition comprises egg yolk oil, perilla seed oil and radish seed oil; the composition is used for: (i) prevention and / or treatment of Alzheimer's disease or its complications; (ii) preparing a medicament for preventing and / or treating Alzheimer's disease or its complications; (iii) improving cognitive impairment or neuroinflammation induced by chemicals; (iv) reducing excessive microglial activation; (v) reducing the level of pro-inflammatory factors and / or reducing the level of oxidative stress in the hippocampus; and / or (vi) increase the expression level of cholinergic receptors, increase the level of acetylcholine and / or increase the activity of key cholinergic enzymes.

2. The use according to claim 1, characterized in that The composition consists of egg yolk oil, perilla seed oil, radish seed oil, noni pulp and cinnamon oil.

3. The use according to claim 1, characterized in that The composition comprises: 55-70 parts by weight of egg yolk oil; 15-25 parts by weight of perilla seed oil; and 15-25 parts by weight of radish seed oil.

4. The use according to claim 1, characterized in that The composition of the composition is as follows: 55-70 parts by weight of egg yolk oil; Perilla seed oil 15-25 parts by weight; 15-25 parts by weight of radish seed oil; 3-7 parts by weight of Noni pulp; and 1-5 parts by weight of cinnamon oil.

5. The use according to any one of claims 1 to 4, characterized in that The composition is a pharmaceutical composition or a health care product composition.

6. The use according to any one of claims 1 to 4, characterized in that The chemical substance is selected from at least one of scopolamine, trihexyphenidyl and atropine.

7. The use according to any one of claims 1 to 4, wherein the cognitive impairment comprises learning and memory impairment, working memory impairment, and spatial positioning memory impairment.

8. A method for preventing and / or treating Alzheimer's disease or its complications, characterized in that: The method comprises the steps of: A therapeutically effective amount of a composition comprising egg yolk oil and perilla seed oil is administered to a subject.

9. A use of a health care product composition, characterized in that: The health care product composition comprises an active ingredient and an excipient acceptable to health care products, wherein the active ingredient comprises egg yolk oil, radish seed oil and perilla seed oil; the health care product composition is used for: (iii) improving cognitive impairment or neuroinflammation induced by chemicals; (iv) reducing excessive microglial activation; (v) reducing the level of pro-inflammatory factors and / or reducing the level of oxidative stress in the hippocampus; and / or (vi) increase the expression level of cholinergic receptors, increase the level of acetylcholine and / or increase the activity of key cholinergic enzymes.

10. A method for reducing the transcription level of inflammatory signal factors in nerve cells in vitro, characterized in that: The method comprises the steps of: culturing neural cells in a culture system containing the composition of claims 1-4.

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