Application of soybean extract in prevention and treatment of senescence-related cognitive impairment

Abstract

The invention discloses application of a soybean extracting solution to prevention and treatment of senescence-related cognitive impairment, and belongs to the technical field of application of plant extracts. A preparation method of the soybean extracting solution comprises the following steps that soybeans with the water content being 55% or above are flatly laid on a conveying belt, the soybeans are conveyed into extraction equipment disclosed by the patent ZL 201711472613.5 through the conveying belt, the soybeans sequentially pass through 11 plasma effect generator cracking reaction kettles, the reaction time of each plasma effect generator cracking reaction kettle is 40-60 seconds, and then the soybeans are extracted through the extraction equipment. The microwave power of the cracking reaction kettles of the first 10 plasma effect generators is 1-6 KW, and the microwave power of the cracking reaction kettles of the last plasma effect generator is 1-3 KW. The extracting solution is a natural plant liquid preparation and is high in safety. Experiments prove that the traditional Chinese medicine composition can significantly improve the working memory and spatial learning memory ability of mice with senescence-related cognitive impairment, regulate the neurotransmitter level, reduce oxidative stress and inflammatory response, and improve intestinal flora composition. The invention provides a brand-new, safe and effective natural medicine choice for preventing and treating senescence-related cognitive impairment, and can be used for preparing related medicines or foods.

Description

Technical Field

[0001] This invention belongs to the field of plant extract application technology, and in particular relates to the application of soybean extract in the prevention and treatment of age-related cognitive impairment. Background Technology

[0002] Age-related cognitive impairment primarily manifests as memory loss, decreased learning ability, impaired executive function, and slowed information processing speed, commonly seen in Alzheimer's disease (AD) and vascular dementia (VD). Its pathological features include neuronal loss, synaptic dysfunction, β-amyloid deposition, and neurofibrillary tangles, accompanied by oxidative stress, mitochondrial dysfunction, and exacerbated neuroinflammation. These impairments not only reduce patients' quality of life but also increase the burden of care on families and social healthcare costs; in severe cases, it can lead to complete loss of independent living ability. With the increasing global aging population, cognitive impairment has become a significant challenge in the field of public health.

[0003] Strategies for preventing and treating age-related cognitive impairment encompass lifestyle interventions (such as the Mediterranean diet, regular exercise, and cognitive training), pharmacological interventions (such as cholinesterase inhibitors and NMDA receptor antagonists), and the development of natural medicines. Natural medicines have attracted significant attention due to their multi-target nature and low toxicity; for example, curcumin protects neurons through anti-inflammatory and antioxidant effects, ginkgo biloba extract improves cerebral blood flow and neuroplasticity, and ginsenosides regulate neurotransmitter metabolism. These components offer new directions for preventing and treating cognitive decline.

[0004] Soybeans are rich in isoflavones (such as daidzein and genistein), lecithin, and polyphenols, possessing antioxidant, anti-inflammatory, and estrogen-like effects. Studies have shown that soy isoflavones can alleviate oxidative stress by activating the Nrf2 pathway and relieve neuroinflammation by inhibiting NF-κB signaling. Clinical studies also suggest a positive correlation between soy intake and cognitive function in older adults, making it a safe and accessible dietary supplement. However, the preparation of natural components faces multiple challenges: the extraction process requires the use of large amounts of organic solvents, potentially leaving residues that could harm the user's health. The extraction process is complex, requiring multiple separation and purification steps; therefore, there is an urgent need to find a safe and efficient natural extract that can be used to prevent and treat age-related cognitive impairment. Summary of the Invention

[0005] This invention provides an application of soybean extract in the preparation of products for preventing and treating age-related cognitive impairment. The preparation method of the soybean extract is as follows: Soybeans with a moisture content of more than 55% are spread flat on a conveyor belt and transported by the conveyor belt into the extraction equipment disclosed in patent ZL 201711472613.5. The soybean extract passes through 11 plasma effect generator pyrolysis reactors in sequence. The reaction time of each plasma effect generator pyrolysis reactor is 40-60 seconds. The microwave power of the first 10 plasma effect generator pyrolysis reactors is 1-6KW, and the microwave power of the last plasma effect generator pyrolysis reactor is 1-3KW. The soybean extract is obtained by condensation and recovery.

[0006] Preferably, the moisture content of the soybeans is 60%.

[0007] More preferably, the soybeans are laid out with a thickness of 2 cm.

[0008] More preferably, the reaction time of each plasma effect generator pyrolysis reactor is 50 seconds.

[0009] More preferably, the microwave power of the first 10 plasma effect generator pyrolysis reactors is 5KW.

[0010] More preferably, the microwave power of the last plasma effect generator is 2KW.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The soybean extract of this invention is a natural plant liquid preparation with high safety. It can be well applied to drugs and foods for the prevention and treatment of age-related cognitive impairment, providing a new, safe and effective natural medicine for the prevention and treatment of age-related cognitive impairment. Attached Figure Description

[0012] Figure 1 The percentage of mice alternating in each group during the Y-maze experiment in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0013] Figure 2 The values ​​represent the new object recognition indices of mice in each group during the new object recognition experiment in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0014] Figure 3 The values ​​represent the initial latency of mice in each group during the Morris water maze experiment in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0015] Figure 4The values ​​represent the cumulative percentage of time spent in the target area by mice in each group during the Morris water maze experiment in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0016] Figure 5 The values ​​represent the levels of various neurotransmitters in the brain tissue of mice in each group in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0017] Figure 6 The values ​​represent the levels of inflammatory factors and oxidative factors in the brain tissue of mice in each group in Example 3. All values ​​are expressed as mean ± SD. Different letters on the box plot indicate significant differences (p < 0.05).

[0018] Figure 7 The values ​​represent the levels of inflammatory and oxidative factors in the serum of mice in each group in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0019] Figure 8 The values ​​represent the levels of inflammatory and oxidative factors in the livers of mice in each group in Example 3. All values ​​are expressed as mean ± SD, and different letters on the box plot indicate significant differences (p < 0.05).

[0020] Figure 9 This refers to the α-diversity of the gut microbiota in Example 3.

[0021] Figure 10 This refers to the β-diversity of the gut microbiota in Example 3.

[0022] Figure 11 This refers to the differences in the composition of the gut microbiota at the phylum level in Example 3.

[0023] Figure 12 This refers to the differences in the composition of the gut microbiota at the genus level in Example 3. Detailed Implementation

[0024] Example 1: Preparation method of soybean extract This invention provides a method for preparing soybean extract, the specific extraction steps of which are as follows: Soybeans, soaked to obtain a moisture content of 60%, are conveyed via conveyor belt into an extraction device (disclosed in patent ZL201711472613.5), spread to a thickness of approximately 2 cm, and then fed into 11 plasma effect generator pyrolysis reactors. The reaction time in each plasma effect generator pyrolysis reactor is 50 seconds (conveyor belt speed 1.2 m / min, reactor working chamber). The total length is 11 meters (one chamber is 1 meter). The microwave power of the first 10 plasma effect generator cracking reaction kettles is 6 KW, and the microwave power of the last 1 plasma effect generator cracking is 3 KW. The extract is obtained by condensation and recovery.

[0025] Wuhan Maiwei Metabolic Biotechnology Co., Ltd. was commissioned to detect the amino acids in the extract, and the results are as follows: Table 1

[0026] Example 2 In this example, the acute poisoning reaction, target organs and death of rats after oral administration of the soybean extract prepared in Example 1 were observed. The specific process is as follows: 40 SPF-grade SD rats (20 males and 20 females) were randomly divided into a control group (given pure water) and a drug administration group (60 ml / kg body weight, intragastric administration three times a day), and the observation period was 14 days. The results showed that there were no abnormal poisoning symptoms in the drug administration group during the observation period, and there were no significant statistical differences in body weight and feed consumption compared with the control group; no abnormalities were found in the main organs such as the heart, liver, spleen, lungs, and kidneys after dissection. The conclusion pointed out that under the experimental conditions, the maximum tolerated dose of SD rats intragastrically administered with soybean extract was greater than 60 ml / kg, which was equivalent to 40 times the clinically proposed body weight dose for humans.

[0027] Example 3 Prevention and treatment effect of soybean extract on senescence-related cognitive impairment mice 1. Experimental animals SPF-grade male ICR mice were selected and provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. (production license number SCXK (Zhejiang) 2019-0001). Animal quality certificate number: 20200909Abzz0619000660. The test animals were housed in the barrier facilities of the experimental animal center at a temperature of 20-26 °C and a relative humidity of 40-70%. Irradiated sterilized feed and bedding were both provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., Su Feed Certificate (2014) 01008.

[0028] 2. Preparation of modeling agent Preparation of D-gal solution (500 mg / kg): Dissolve 10 g to a volume of 100 mL and inject at 5 mL / kg BW.

[0029] 3. Dose selection and administration method of test substance Based on the recommended human intake of the test sample, low, medium, and high dose groups were defined as 5, 10, and 20 times the recommended human intake. The experiment included a blank control group (NC), a model group (MC), and three dose groups of the test substance: low (LD, 5 mL / kg BW), medium (MD, 10 mL / kg BW), and high (HD, 20 mL / kg BW), administered via gavage.

[0030] 3. Animal grouping and treatment Fifty mice were housed in cages at a temperature of 25±1 ℃, humidity of 50-55%, and a photoperiod of 12 h / 12 ​​h, with free access to food and water. The experiment began after one week of acclimatization. Mice were randomly divided into 5 groups (n=10 per group) according to body weight. Except for the control group, all other groups received subcutaneous injections of D-gal solution (500 mg / kg) in the neck and back daily for 8 weeks. Thirty minutes after D-gal injection, the soybean extract-treated group was administered the corresponding test substance by gavage, while the blank control group and model group were administered physiological saline by gavage at 5 mL / kg BW. The mice's body weight and food intake were recorded at fixed times each week.

[0031] 4. Behavioral tests 4.1 Y-maze experiment The Y-maze test is a classic experiment for evaluating working memory in mice. After 8 weeks of modeling drug treatment, the test mice are placed at the end of one arm of a darkened Y-shaped maze device with a three-pronged passage (20cm×4cm×40cm) and are allowed to explore for 8 minutes. The mice's walking path and the number of times they enter the arm are recorded. After each mouse's test, the device is cleaned with 75% ethanol. The behavioral performance in the Y-maze is calculated using the percentage of alternation: percentage of alternation = total number of times the mice entered the three prongs without repetition / total number of times the mice entered the arm × 100%.

[0032] 4.2 New Object Recognition Experiment The new object recognition experiment was used to evaluate the short-term memory ability of mice. The experiment was divided into three phases: adaptation phase, training phase and testing phase. (1) Adaptation phase: On the first day, each mouse was placed in an open area without any objects and allowed to explore freely for 5 minutes. (2) Training phase: On the second day, two identical green cubes without odor were placed in a box, and each mouse was placed in the box for 10 minutes of exploration. (3) Testing phase: On the third day, the same procedure as the training phase was used, but one of the green cubes was replaced by a yellow cylinder. The equipment was cleaned with 75% ethanol during each experimental interval. The exploration behavior was defined as follows: when a mouse licked, smelled and touched an object with its paw or head within 2 cm of the object, the time and number of exploration behaviors were recorded. The new object recognition index was used to define the new object recognition ability of mice. The new object recognition index = (new object exploration time - old object exploration time) / total exploration time 4.3 Morris Water Maze Experiment The Morris water maze is a classic experiment for evaluating the spatial learning and memory abilities of mice. The main body of the water maze consists of a circular water tank. The water tank is divided into four quadrants by an animal behavior monitoring system. One of the quadrants is selected as the target quadrant, and a small platform is placed 1 cm underwater in the target quadrant. The experiment is divided into two stages: the orientation cruise stage and the spatial exploration stage. (1) Orientation cruise stage: For the first five days of the experiment, the mice freely explore the water maze with the escape platform for 1 minute. If the mouse finds the platform within 1 minute, it is placed on the platform to rest and orient itself for 10 seconds, and then it is returned to the cage. If the mouse does not find the platform within 1 minute, it is manually guided to the platform by the experimenter and rests and orients itself on the platform for 10 seconds. (2) Spatial exploration stage: On the sixth day of the experiment, the escape platform is removed, and the mice freely explore the water maze without the escape platform for 1 minute. The time required for each mouse to reach the platform (escape latency) is recorded using a video monitoring system. The time / distance the mouse stays in the target quadrant and the number of times it crosses the platform are also recorded. Platform crossing count refers to the number of times a mouse crosses the original platform location after platform removal within a specified time. Target quadrant percentage refers to the proportion of time a mouse spends exploring the original platform quadrant after platform removal within the specified time. After each mouse's test, its fur was dried with a towel to reduce stress.

[0033] 4.4 Experimental Results The Y-maze spontaneous alternation is a behavioral test based on animals' natural curiosity about exploration. These animals typically tend to explore new branches of the maze rather than return to previously visited branches. The lower the alternation percentage, the deeper the memory impairment. Figure 1Based on the results of the Y-maze experiment, the percentage of alternation in the model group mice was significantly lower than that in the control group, while the percentage of alternation in the soybean extract treatment group was significantly increased. The low-dose and medium-dose groups were significantly higher than the control group, showing a strong level of improvement in working memory.

[0034] The novel object recognition experiment uses the fact that animals are willing to spend more time smelling new objects than familiar ones to determine the ability of mice to distinguish novelty in complex environments with different objects. The lower the novel object recognition index, the deeper the memory impairment. Figure 2 The results of the novel object recognition experiment in mice showed that although there were no significant differences between the groups, the novel object recognition ability of the model group mice decreased compared with the control group. The mean and median values ​​of the soybean extract groups were greater than those of the control group, indicating that it has the potential to improve the short-term memory of mice.

[0035] In the water maze test, mice had to learn complex behavioral strategies to escape from the water to the platform, and the severity of memory impairment was positively correlated with the escape latency. Figure 3-4 These are the results of the Morris water maze experiment in mice. The initial latency time (ITT) is the time it takes for a mouse to reach the platform position for the first time after being placed in the maze. A shorter ITT indicates better spatial learning and memory ability. Compared to the control group, the initial latency time in the model group was significantly longer, with an average of >15 seconds. The soybean extract groups significantly reduced the initial latency time. Statistical analysis showed no significant difference between the soybean extract groups and the control group. The cumulative time to the target area is the ratio of the total time spent by the mouse in the quadrant where it was initially located to the total experimental time. A higher value indicates a longer search time in the target area, indirectly reflecting the mouse's spatial learning and memory ability. The results show that although there were no significant differences between the soybean extract groups and the model / control group, the mean and median values ​​were significantly higher. Furthermore, there were no significant differences between the low, medium, and high dose soybean extract groups and the control group. Therefore, Figure 3 and Figure 4 The results indicate that soybean extract can significantly improve spatial learning and memory abilities in mice. In conclusion, behavioral experiments demonstrate that soybean extract can significantly improve working memory and spatial learning and memory abilities in mice, and also has a certain effect on enhancing short-term memory.

[0036] 5. Effects of soybean extract on neurotransmitters in the brain tissue of aging mice 5.1 Brain tissue sample collection After the behavioral experiments, the mice were fasted overnight but allowed free access to water. Blood was collected from the orbital fossa, and the mice were then euthanized by cervical dislocation to collect brain tissue. Some of the collected brain tissue was stored at -80°C for later use.

[0037] 5.2 Determination of neurotransmitter biochemical indicators The total protein content, 5-hydroxytryptamine (5-HT), dopamine (DA), glutamate (Glu), γ-aminobutyric acid (GABA), acetylcholine (Ach), acetylcholinesterase (AchE), and cyclic adenosine monophosphate (cAMP) were measured according to the kit instructions.

[0038] 5.3 Experimental Results Brain function depends on signal transmission between different types of neurons and glial cells, primarily through neurotransmitters. Neurotransmitters in the brain include monoamines, cholinesterase, and amino acids. Imbalances in these neurotransmitters can lead to neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, autism, and depression. Figure 5 The levels of neurotransmitters in the brain tissue of mice in each group were measured. In this study, after D-galactose modeling, the levels of all six neurotransmitters in the brain tissue of the model group were significantly lower than those in the control group. Specifically, the soybean extract intervention group significantly increased the levels of γ-aminobutyric acid (GABA), acetylcholine, dopamine, and cyclic adenosine monophosphate (cAMP) compared to the model group. The low- and medium-dose soybean extract groups significantly increased the level of serotonin (5-HT). Regarding glutamate content, although there was no significant difference between the soybean extract groups and the model group, the average and median values ​​of the low-, medium-, and high-dose soybean extract groups were all higher than those of the model group. In conclusion, soybean extract has a certain ameliorative effect on the reduction of neurotransmitters in brain tissue caused by aging.

[0039] 6. Effects of soybean extract on antioxidant and anti-inflammatory biochemical indicators in serum, liver, and brain tissue of aging mice. 6.1 Sample Collection After the behavioral experiments, mice were fasted but allowed free access to water overnight. Blood was collected from the orbital fossa, incubated at room temperature for 2 hours, and then centrifuged at 3000 r / min for 10 min. The collected serum was then flash-frozen at -80℃ for subsequent experimental measurements. The mice were then euthanized by cervical dislocation, and liver and brain tissues were collected. A portion of the liver and brain tissue was stored at -80℃ for later use.

[0040] 6.2 Determination of antioxidant and anti-inflammatory biochemical indicators Serum: Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were detected according to the kit instructions.

[0041] Liver and brain tissue: Accurately weigh 80 mg of liver and brain tissue from each group of mice, add 0.72 ml of physiological saline, and prepare a 10% homogenate in an ice-water bath. Centrifuge at 3000 rpm / min for 10 min and collect the supernatant. Detect total protein content, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) according to the kit instructions. 6.3 Experimental Results Continuous injection of high doses of D-galactose leads to excessive accumulation of reactive oxygen species (ROS), resulting in oxidative stress and subsequently inducing cellular and tissue aging. Compared to other tissues, the brain is particularly sensitive to ROS-induced oxidative stress due to its high oxygen consumption, abundant lipids (especially polyunsaturated fatty acids in neuronal membranes), and relatively weak antioxidant defense system. Excessive ROS induces neuronal damage and cognitive impairment through lipid peroxidation, enzyme inactivation, and DNA modification. Furthermore, some studies suggest that oxidative stress and inflammatory responses are always interrelated, with oxidative stress potentially promoting the progression of inflammation. For example, D-galactose effectively increases oxidative damage and inflammation, as well as brain cell apoptosis, in the mouse brain, further exacerbating cognitive impairment. Therefore, this study detected inflammatory factors and oxidative markers in mouse serum, liver, and brain tissue. The results are as follows: Figure 6-8 .

[0042] Figure 6 The study investigated inflammatory factors and oxidative indicators in mouse brain tissue. The results showed that, compared to the control group, the model group mice exhibited significantly increased levels of inflammatory factors and MDA, while significantly decreased levels of SOD and GSH-Px, indicating that D-galactose can cause oxidative damage and promote inflammation in brain tissue. Intervention with soybean extract effectively improved this situation; compared to the model group, all soybean extract groups significantly reduced the levels of inflammatory factors and MDA, and increased the levels of SOD and GSH-Px.

[0043] Figure 7 The results show the inflammatory factors and oxidative indicators in mouse serum. Consistent with the trends observed in brain tissue, the significant increase in serum inflammatory factors induced by D-galactose injection was suppressed after intervention with soybean extract. Regarding the improvement of IL-6 and MDA, there were no significant differences between the medium- and high-dose soybean extract groups and the control group.

[0044] Figure 8 Results of inflammatory factors and oxidative markers in mouse liver. Figure 6 and Figure 7The results differed. Soybean extract did not significantly improve the significant increases in IL-1β and TNF-α and the significant decreases in SOD and GSH-Px in liver tissue after modeling. However, it showed a more significant improvement in IL-6 and MDA, with no significant difference between the soybean extract intervention group and the control group. This indicates that soybean extract has a certain ameliorative effect on inflammatory factors and oxidative indicators in liver tissue.

[0045] 7. Microbiome research based on high-throughput sequencing 7.1 Library preparation and sequencing Before library construction, DNA samples undergo quality control, checking their concentration (≥ 12.5 ug / ul), integrity (main peak on electrophoresis gel > 20 kb), and purity (free from protein, RNA / salt ion contamination). Samples meeting these criteria proceed to the library construction process. The specific library construction process is as follows: 1) Sample fragmentation. Take a certain amount of metagenomic DNA (extracted from intestinal contents) and fragment it using a Covaris ultrasonic disruptor.

[0046] 2) Fragment size selection. After the sample is broken down, the magnetic beads are used to select fragments so that the sample bands are concentrated in the range of 200-400bp.

[0047] 3) End repair, addition of an "A" base, and adapter ligation. Prepare the reaction system, react at an appropriate temperature for a certain time, repair the ends of the double-stranded cDNA, and add an "A" base to the 3' end. Prepare the adapter ligation reaction system, react at an appropriate temperature for a certain time, and ligate the adapter to the DNA.

[0048] 4) PCR reaction and product recovery. Prepare the PCR reaction system and set the reaction program to amplify the ligation product. Purify and recover the amplified product using magnetic beads.

[0049] 5) Product cyclization. After denaturing the PCR product into single strands, a cyclization reaction system is prepared, thoroughly mixed, and reacted at an appropriate temperature for a certain period of time to obtain a single-stranded circular product. After digesting the uncyclized linear DNA molecules, the final library is obtained.

[0050] 6) Library analysis. The concentration of cyclized products is determined before processing.

[0051] 7.2 Sequencing Libraries that pass the initial screening will be sequenced (DNBSEQ): Single-stranded circular DNA molecules replicate via rolling circle to form DNA nanospheres (DNBs) containing multiple copies. The resulting DNBs are then added to the mesh wells of a high-density DNA nanoarray using high-density DNA nanochip technology and sequenced using combined probe-anchored polymerization (cPAS) technology. Metagenomic sequencing is performed using the MGISEQ 2000 platform (or DNBSEQ T7) with PE150 sequencing.

[0052] 7.3 Bioinformatics Analysis 1) Read quality control and host removal: SOAPnuke software is used to filter and control the raw data, and Bowtie2 is used to align the host sequence and remove the sequence in the alignment to generate clean data.

[0053] 2) Functional Annotation: First, MEGAHIT was used to assemble the quality-controlled and host-removed sequences using k-mer-based assembly to generate contigs. Then, MetaGeneMark software was used to predict the gene sequences in the contigs. CD-HIT software was used to remove redundancy from the obtained genes. Salmon software was used to calculate the relative abundance table of each gene. Finally, DIAMOND or RGI was used to align the non-redundant genes to databases such as eggNOG, KEGG, BacMet, CARD, COG, CAZy, and Swiss-prot to complete gene functional annotation.

[0054] 3) Species annotation: Kraken2 and a self-built database (selecting NCBI NT database or UHGG database) are used to calculate the number of sequences of species contained in the sample, and then Kraken2 is used to estimate the actual abundance of species in the sample to complete the species annotation.

[0055] 4) Based on gene abundance tables, species abundance tables, and functional abundance tables, the distribution of genes, species, and functions can be visualized, their alpha diversity and beta diversity can be calculated, species or functional abundance cluster analysis can be performed, PCA, PLSDA, PCoA and NMDS dimensionality reduction analysis can be performed, sample cluster analysis can be performed, and Wilcoxon / Kruscal, T test / ANOVA, LEfSe analysis and KEGG pathway enrichment analysis (Reportscore method) can be used to explore the differences in species composition and functional composition among samples.

[0056] 5) In-depth correlation studies combining environmental factors, pathological indicators, or special phenotypes can provide a theoretical basis for further in-depth research and utilization of the species and functions of samples.

[0057] 7.4 Results Analysis 7.4.1 Species Diversity Alpha diversity refers to the diversity within a specific region or ecosystem, reflecting the species richness and evenness within the habitat. Common indices used to measure alpha diversity include Chao1, Shannon, and Simpson. A higher Chao1 index indicates a greater number of species; a higher Shannon index indicates greater species richness and evenness in the sample; and a higher Simpson index indicates higher community diversity. This study uses species data for alpha diversity analysis. Alpha diversity is analyzed at the genus level. Figure 9 It can be seen that there is no significant difference among the groups in Chao1, indicating no significant difference in the number of species among the groups. However, there are significant differences among the groups in Shannon and Simpson indices. The model group is significantly higher than the other groups, indicating that the diversity of gut microbiota increases after modeling. Beta diversity refers to the difference between samples. The distance matrix calculated by different distance methods is a statistical index of beta diversity. The distance matrix is ​​used to quantify the differences between different samples and is a quantitative indicator of beta diversity. In the distance matrix, each sample represents one dimension. When the number of samples exceeds three, the results cannot be displayed in a plane or three-dimensional space, and dimensionality reduction is required. PCoA (Principal Coordinates Analysis) is a non-constrained data dimensionality reduction analysis method that studies the similarity or dissimilarity of sample community composition based on the distance matrix (commonly Bray-curtis, JSD). In this study, the Bray-curtis method of PCoA was used to analyze the beta diversity of each group, and the results are as follows. Figure 10 As shown in the figure, the MC group was clearly separated from the other groups, indicating a significant difference between the MC group and the other groups. However, there was no significant difference between the low, medium, and high dose soybean extract groups and the NC group.

[0058] 7.4.2 Differences in species Species differences in gut microbiota were analyzed at both the phylum and genus levels.

[0059] Figure 11 The differences among the groups were at the phylum level. The results showed that the abundance of Bacillota (formerly known as Firmicutes) in the model group was significantly higher than in other groups, while the proportion of Bacteroidetes was significantly lower. There were no significant differences between the low, medium, and high dose groups of soybean extract and the control group.

[0060] Among the components of the gut microbiota, Firmicutes and Bacteroidetes are the two most abundant phyla, and their ratio (F / B ratio) is considered an important indicator of gut microbiota health. These two phyla typically account for over 90% of the total gut microbiota in humans and mice, and their dynamic balance is crucial for maintaining gut homeostasis and overall health. Firmicutes mainly includes various anaerobic and facultative anaerobic bacteria, such as *Lactobacillus*, *Bifidobacterium*, and *Clostridium*. These bacteria play important roles in energy metabolism, nutrient absorption, and immune regulation. In particular, some Firmicutes bacteria can produce short-chain fatty acids, such as butyrate, which is significant for maintaining intestinal barrier function and regulating immune responses. Bacteroidetes mainly includes *Bacteroides* and *Prevotella*, which play key roles in polysaccharide degradation, bile acid metabolism, and vitamin synthesis. Bacteroidetes bacteria can also produce various bioactive substances that participate in regulating host metabolism and immune function. Changes in the gut microbiota ratio (F / B ratio) are associated with various disease states. In the field of neurological diseases, increasing evidence suggests that abnormal F / B ratios are closely related to cognitive impairment, providing a new perspective for understanding and treating cognitive disorders. In Alzheimer's disease (AD) patients, the composition of the gut microbiota undergoes significant changes, with alterations in the F / B ratio being one of the most noteworthy features. Studies suggest that AD patients exhibit a significantly increased proportion of Firmicutes and a decreased proportion of Bacteroidetes in their gut, leading to an elevated F / B ratio. This change is believed to be related to multiple pathological mechanisms. First, an increased proportion of Firmicutes leads to an increase in harmful metabolites such as lipopolysaccharides (LPS). These substances can disrupt the integrity of the intestinal barrier, promote the entry of inflammatory factors into the bloodstream, and thus trigger neuroinflammation, accelerating the progression of AD. Second, certain Firmicutes species can produce neurotoxic substances such as hydrogen sulfide, which can damage neurons at high concentrations.

[0061] Figure 12 This represents the differences between groups at the genus level. As shown in the graph, the model group... Ducaniella and Alistipes The relative abundance of these two genera was significantly lower than that of the other groups. And... Blautia , Acutalibacter , Mucispirillum The relative abundance of the three genera was higher than that of the other groups. Ducaniella Studies on this genus have focused solely on the association between its abundance variations and diet and metabolism, without clarifying its specific physiological functions. Existing research has found that a high-fat diet leads to changes in the gut microbiota of mice. Ducaniella The abundance of this genus was significantly reduced, suggesting that it may be involved in lipid metabolism in the body. However, its specific metabolic mechanisms and its regulatory effects on the gut microbiota have not been thoroughly studied and require further experimental verification. AlistipesThe genus *Alternaria* (also known as *Bacteroides*) is a group of Gram-negative obligate anaerobic bacteria belonging to the phylum Bacteroidetes. As commensal bacteria in the human gut, their role is dual: they provide protection against certain diseases and are also associated with the development and progression of others. Studies have shown that... Alistipes It is negatively correlated with cognitive ability, and increased abundance of it can impair hippocampal plasticity, thereby affecting cognitive function, but its specific mechanism of action is not yet unified. Although it contradicts the conclusions of this study, due to the dual nature of its effects, a more in-depth analysis combining model construction and mechanism of action is needed. Blautia (Broutella) Acutalibacter , Mucispirillum (Myxobolus) are all components of the intestinal flora, among which Blautia The functions of this genus have been extensively studied, and it has a clear association with cognitive impairment; while Acutalibacter and Mucispirillum Research on this genera is relatively scarce, and no direct evidence of a link between it and cognitive impairment has been found. Blautia It is significantly associated with cognitive impairment; in the gut of Alzheimer's patients Blautia The abundance was high, and its abundance was positively correlated with AD biomarkers. In this study, the model group Blautia High abundance may be one of the causes of cognitive impairment.

[0062] In recent years, with the deepening of research on the microbiome, scientists have gradually realized that there is a complex and sophisticated bidirectional communication network between the gut microbiota and brain function, a system known as the "microbiota-gut-brain axis." This concept has fundamentally changed our understanding of the pathogenesis of neurological diseases, particularly demonstrating immense research value in the field of cognitive impairment. Studies have shown that the gut microbiota can influence the integrity of the blood-brain barrier, regulate neuroinflammatory responses, participate in neurotransmitter synthesis, and affect neuroplasticity. When the gut microbiota is imbalanced, these normal physiological functions are disrupted, potentially leading to the occurrence and development of cognitive impairment. In this study, the model group showed significant differences in microbiota diversity and composition compared to the blank control group, while the low, medium, and high dose groups of soybean extract showed no significant differences in microbiota diversity and composition compared to the blank control group, indicating that soybean extract may improve cognitive impairment by improving gut microbiota composition.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a soybean extract in the preparation of a product for the prevention and treatment of cognitive impairment associated with aging, characterized in that, The preparation method of the soybean extract is as follows: soybeans with a water content of more than 55% are laid flat on a conveyor belt and transported into the extraction equipment disclosed in patent ZL 201711472613.5, and sequentially pass through 11 plasma effect generator cracking reaction kettles, the reaction time of each plasma effect generator cracking reaction kettle is 40-60 seconds, the microwave power of the first 10 plasma effect generator cracking reaction kettles is 1-6 KW, and the microwave power of the last plasma effect generator cracking is 1-3 KW, and the soybean extract is obtained by condensation recovery.

2. Use according to claim 1, characterized in that, The water content of the soybeans is 60%.

3. Use according to claim 2, characterized in that, The flat laying thickness of the soybeans is 2 cm.

4. Use according to claim 3, characterized in that, The reaction time of each plasma effect generator cracking reaction kettle is 50 seconds.

5. Use according to claim 4, characterized in that, The microwave power of the first 10 plasma effect generator cracking reaction kettles is 5 KW.

6. Use according to claim 5, characterized in that, The microwave power of the last plasma effect generator cracking is 2 KW.

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