Composition for synergistically enhancing cognitive ability and memory

Abstract

The invention provides a composition for synergistically enhancing cognitive ability and memory and application thereof, specifically, the composition comprises phosphatidylserine and 3 '-sialic acid lactose or salt thereof, and the two components have a synergistic effect in enhancing cognitive ability and memory and / or improving cognitive function degeneration.

Description

Technical Field

[0001] This invention relates to the food industry, and more specifically to a composition that synergistically enhances cognitive abilities and memory, and its application. Background Technology

[0002] Cognitive impairment not only manifests as memory loss, decreased attention, and impaired executive function, but is also often accompanied by mood disorders, behavioral abnormalities, and loss of social function, severely impacting patients' quality of life and imposing a heavy burden on families and society. Furthermore, the cognitive development of children and adolescents is crucial to their future education, career, and social outcomes. With societal development, children and adolescents face increasingly diverse cognitive challenges. Sociodemographic characteristics, such as growing global socioeconomic inequality, also significantly influence the trajectory of children's cognitive development. These factors collectively lead to the need for effective cognitive enhancement strategies.

[0003] Currently, clinical strategies for improving cognitive decline mainly fall into two categories: pharmacological treatment and non-pharmacological interventions. Traditional drugs such as cholinesterase inhibitors (donepezil, galantamine) and NMDA receptor antagonists (memantine), while widely used, have limited efficacy and are accompanied by adverse reactions such as nausea, diarrhea, and dizziness, leading to poor long-term adherence. Furthermore, these drugs only temporarily relieve symptoms and cannot halt the fundamental progression of neurodegenerative diseases. Non-pharmacological approaches such as cognitive training, physical exercise, and dietary adjustments offer some protection, but their effectiveness varies greatly among individuals, and standardized implementation plans are lacking. In recent years, nutritional supplements and functional foods have gained widespread attention due to their high safety and ease of acceptance, particularly in the field of products targeting children and adolescents' cognitive development. Consumers generally view improved cognitive abilities as a major reason for changing dietary habits or using dietary supplements, presenting a significant opportunity for public health. However, current products on the market targeting cognitive function are mostly single-ingredient, lacking targeted and scientifically validated compound products. Furthermore, single-component supplements often fail to fully address the multifactorial pathological mechanisms of cognitive decline, including oxidative stress, neuroinflammation, mitochondrial dysfunction, and neurotransmitter imbalance, thus limiting their overall intervention efficacy. Against this backdrop, developing synergistic and comprehensive intervention programs is particularly necessary. Summary of the Invention

[0004] This invention aims to provide a composition that enhances cognitive ability and memory and / or improves cognitive function decline, specifically comprising phosphatidylserine and 3'-sialyl lactose or their salts. The functional properties of this composition are primarily verified through zebrafish experiments, with verification indicators including zebrafish color recognition, memory tests, acetylcholinesterase activity, and related genes. bdnf and gdnfaThe expression level of the components in this composition is high, and the components in the composition have a synergistic effect in enhancing cognitive ability and memory and improving cognitive function decline.

[0005] The present invention provides a composition for (a) enhancing cognitive ability and memory and / or (b) improving cognitive decline, comprising phosphatidylserine and 3'-sialyl lactose or a salt thereof.

[0006] In one or more embodiments, the ratio of PS to 3'-SL in the composition ranges from 1:0.05 to 1:60, preferably from 1:0.12 to 1:50, for example 1:0.1 to 1:10, 1:0.12 to 1:9.6, 0.12 to 1:0.6, 1:0.12 to 1:2.4, 1:0.12 to 1:12, 1:0.12 to 1:48, 1:0.48 to 1:0.6, 1:0.48 to 1:2.4, 1:0.48 to 1:12, 1:0.48 to 1:48, 1:0.6 to 1:2.4, 1:0.6 to 1:12, 1:0.6 to 1:48, 1:2.4 to 1:12, 1:2.4 to 1:48, 1:12 to 1:48.

[0007] In one or more embodiments, the salt of the 3'-sialyl lactose is a sodium salt.

[0008] In one or more embodiments, the cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0009] In one or more embodiments, the components in the composition synergistically enhance the cognitive abilities and memory of the subject and / or improve cognitive decline.

[0010] In one or more embodiments, the enhanced cognitive ability and memory include: enhancing memory, promoting brain development, and promoting the expression of cognitive function-related genes.

[0011] In one or more embodiments, the cognitive decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes.

[0012] In one or more embodiments, the cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0013] In one or more embodiments, the subjects of the enhancement of cognitive abilities and memory and / or improvement of cognitive decline are animals. In one or more embodiments, the subjects may or may not have experienced cognitive decline. In one or more embodiments, the subjects are infants, children, or adolescents. In one or more embodiments, the subjects are individuals aged 0-18 years.

[0014] In one or more embodiments, the animal includes livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, rats, rabbits, and humans.

[0015] In one or more embodiments, the animal is a cypriniformes species belonging to the Cyprinidae family, including carp, crucian carp, grass carp, snakehead, and minnow, preferably zebrafish.

[0016] The present invention also provides a formulation comprising the composition described in any of the embodiments herein.

[0017] In one or more embodiments, the formulation is a powder, pill, capsule, granule, tablet, liquid formulation, or gel.

[0018] The present invention also provides a product comprising the composition described in any embodiment herein and / or the formulation described in any embodiment herein.

[0019] In one or more embodiments, the product is a food, health product, or pharmaceutical composition.

[0020] In one or more embodiments, the types of food include: plant-based foods, animal-based foods, microbially fermented foods, and processed foods.

[0021] In one or more embodiments, the food also includes food additives and / or nutritional fortifiers.

[0022] In one or more embodiments, the additive includes one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifier includes one or more of the following: vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0023] In one or more embodiments, the food includes one or more of the following: dairy products, soy products, probiotic powder, probiotic oil droplets, dietary fiber supplements, nutrition bars, rice cereal, fruit puree, fruit and vegetable juice, solid food beverages, fruit juice, ice cream, candy, biscuits, infant formula, infant food for special medical purposes.

[0024] In one or more embodiments, the food is infant food, children's food, adolescent food, youth food, middle-aged food, or elderly food.

[0025] In one or more embodiments, the infants and young children include infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

[0026] In one or more embodiments, the dosage form of the health product includes, but is not limited to, powder, tablet, granule, capsule, solution, emulsion, and suspension.

[0027] In one or more embodiments, the health product further includes additives and / or nutritional fortifiers.

[0028] In one or more embodiments, the additive includes one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifier includes one or more of the following: vitamins, minerals, amino acids, fatty acids, dietary fiber.

[0029] In one or more embodiments, the health product is a powder, pill, capsule, granule, tablet, oil drop, liquid preparation or gel.

[0030] In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0031] In one or more embodiments, the product has the use of (a) enhancing cognitive abilities and memory and / or (b) improving cognitive decline.

[0032] In one or more embodiments, the improvement of cognition and enhancement of cognitive ability and memory includes: repair of cognitive dysfunction, enhancement of memory, promotion of brain development, and promotion of the expression of cognitive function-related genes.

[0033] In one or more embodiments, the cognitive decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes.

[0034] In one or more embodiments, the subjects of the enhancement of cognitive abilities and memory and / or improvement of cognitive decline are animals. In one or more embodiments, the subjects may or may not have experienced cognitive decline. In one or more embodiments, the subjects are infants, children, or adolescents. In one or more embodiments, the subjects are individuals aged 0-18 years.

[0035] In one or more embodiments, the animal includes livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, rats, rabbits, and humans.

[0036] In one or more embodiments, the animal is a cypriniformes species belonging to the Cyprinidae family, including carp, crucian carp, grass carp, snakehead, and minnow, preferably zebrafish.

[0037] The present invention also provides the use of the compositions and formulations described in any embodiment herein in the preparation of products that enhance cognitive abilities and memory and / or improve cognitive decline.

[0038] In one or more embodiments, the enhanced cognitive ability and memory include: enhancing memory, promoting brain development, and promoting the expression of cognitive function-related genes.

[0039] In one or more embodiments, the cognitive decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes.

[0040] In one or more embodiments, the cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0041] In one or more embodiments, the product is a food, health product, or pharmaceutical composition.

[0042] In one or more embodiments, the types of food include: plant-based foods, animal-based foods, microbially fermented foods, and processed foods.

[0043] In one or more embodiments, the food also includes food additives and / or nutritional fortifiers.

[0044] In one or more embodiments, the dosage form of the health product includes, but is not limited to, powder, tablet, granule, capsule, solution, emulsion, and suspension.

[0045] In one or more embodiments, the health product further includes additives and / or nutritional fortifiers.

[0046] In one or more embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0047] In one or more embodiments, the subjects of the enhancement of cognitive ability and memory and / or improvement of cognitive function decline are animals.

[0048] In one or more embodiments, the animal includes livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, rats, rabbits, and humans.

[0049] In one or more embodiments, the animal is a cypriniformes species belonging to the Cyprinidae family, including carp, crucian carp, grass carp, snakehead, and minnow, preferably zebrafish.

[0050] In one or more embodiments, the subject may or may not have experienced cognitive decline. In one or more embodiments, the subject is an infant, child, or adolescent. In one or more embodiments, the subject is a person aged 0-18 years.

[0051] The present invention also provides non-therapeutic applications of the compositions, formulations and / or products described in any embodiment herein, including: enhancing cognitive abilities and memory and / or improving cognitive decline.

[0052] In one or more embodiments, the enhanced cognitive ability and memory include: enhancing memory, promoting brain development, and promoting the expression of cognitive function-related genes.

[0053] In one or more embodiments, the cognitive decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes.

[0054] In one or more embodiments, the cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0055] In one or more embodiments, the subjects of the enhancement of cognitive ability and memory and / or improvement of cognitive function decline are animals.

[0056] In one or more embodiments, the animal includes livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, rats, rabbits, and humans.

[0057] In one or more embodiments, the animal is a cypriniformes species belonging to the Cyprinidae family, including carp, crucian carp, grass carp, snakehead, and minnow, preferably zebrafish.

[0058] In one or more embodiments, the subject may or may not have experienced cognitive decline. In one or more embodiments, the subject is an infant, child, or adolescent. In one or more embodiments, the subject is a person aged 0-18 years.

[0059] The present invention also provides a method for enhancing a subject’s cognitive abilities and memory and / or improving cognitive function decline, comprising applying the composition, food, health product, or pharmaceutical composition described in any embodiment herein to the subject.

[0060] In one or more embodiments, the object is an animal.

[0061] In one or more embodiments, the animal includes livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, rats, rabbits, and humans.

[0062] In one or more embodiments, the animal is a cypriniformes species belonging to the Cyprinidae family, including carp, crucian carp, grass carp, snakehead, and minnow, preferably zebrafish.

[0063] The present invention also provides the use of the compositions or formulations described herein in the preparation of health products that help improve memory.

[0064] The beneficial effects of this invention are: The composition proposed in this invention specifically includes phosphatidylserine and 3'-sialic acid lactose. This composition has health benefits of (a) enhancing cognitive ability and memory and / or (b) improving cognitive decline. Moreover, this composition has a synergistic effect compared with the individual components used alone. Attached Figure Description

[0065] Figure 1 This represents the total time zebrafish spent in the T-maze enrichment zone after sample treatment. Compared with the normal control group, p <0.05, p < 0.01.

[0066] Figure 2 This is a typical diagram of the zebrafish movement trajectory after sample treatment (dose-effect relationship), where the blue box represents the blue area of ​​the cross maze, which is the quantitative area.

[0067] Figure 3 The percentage of movement in the blue region of the zebrafish after sample treatment (dose-effect relationship) is compared with the model control group. p < 0.05, p < 0.01, p < 0.001.

[0068] Figure 4 This is a typical image of the zebrafish's movement trajectory after sample treatment. The blue box represents the blue area of ​​the cross maze, which is the quantitative area.

[0069] Figure 5 The percentage of blue color in zebrafish after sample treatment is compared with the model control group. p < 0.05, p < 0.01, p < 0.001.

[0070] Figure 6 This represents the total time zebrafish spent in the T-maze enrichment zone after sample treatment. Compared with the normal control group, p <0.05, p < 0.01, p < 0.001; compared with 3'-SL 60.0 μg / mL, # p < 0.05; compared with 3'-SL 300 μg / mL, && p < 0.01; compared with PS 31.2 μg / mL p < 0.05; compared with PS 125 μg / mL, ! p <0.05, !! p < 0.01; compared with PS 500 μg / mL, %% p < 0.01.

[0071] Figure 7 This is a typical image of the zebrafish's movement trajectory after sample treatment. Note: The blue box represents the blue area of ​​the cross maze, which is the quantitative area.

[0072] Figure 8 This represents the percentage of movement in the blue area of ​​the zebrafish after sample treatment. Compared with the normal control group, p < 0.05, p < 0.01, p < 0.001; compared with 3'-SL 60.0 μg / mL, # p < 0.05, ### p < 0.001; compared with PS 31.2 μg / mL, p < 0.05.

[0073] Figure 9 This is a typical image of the blue proportion of zebrafish after sample processing, where the blue boxes represent the blue areas of the cross maze, which are the quantitative regions.

[0074] Figure 10 The percentage of blue color in zebrafish after sample treatment is compared with the model control group. p < 0.05, p < 0.01, p < 0.001; with P Compared to S 31.2 μg / mL, p < 0.05, p < 0.001; with P Compared to S at 125 μg / mL, ! p < 0.05, !! p < 0.01; with P Compared to S 500 μg / mL, % p < 0.05, %% p < 0.01; compared with 3'-SL 60.0 μg / mL, # p < 0.05, ## p < 0.01; compared with 3'-SL 300 μg / mL, & p < 0.05.

[0075] Figure 11 The fluorescence value of acetylcholinesterase (AChE) in zebrafish after sample treatment was compared with that of the model control group. p <0.05, p < 0.01, p < 0.001; with P Compared to S 31.2 μg / mL, p < 0.05, p < 0.001; with P Compared to S 125 μg / mL, !! p < 0.01, !!! p < 0.001; with P Compared to S 500 μg / mL, %% p < 0.01, %%% p<0.001; compared with 3'-SL 60.0 μg / mL, # p < 0.05, ## p < 0.01; compared with 3'-SL 300 μg / mL, & p <0.05, && p < 0.01; compared with 3'-SL 1500 μg / mL, @ p < 0.05.

[0076] Figure 12 Typical area of ​​dopamine neurons in zebrafish after sample processing, with white arrows pointing to dopamine neurons in the zebrafish brain.

[0077] Figure 13 The area of ​​zebrafish dopamine neurons after sample treatment was compared with that of the model control group. p < 0.01, p < 0.001; with P Compared to S 31.2 μg / mL, p < 0.05; with P Compared to S at 125 μg / mL, ! p < 0.05, !! p <0.01; compared with 3'-SL 60.0 μg / mL, ## p < 0.01, ### p < 0.001; compared with 3'-SL 300 μg / mL, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with 3'-SL 1500 μg / mL, @@ p < 0.01.

[0078] Figure 14 for gdnfa Relative gene expression levels. Compared with the normal control group, p < 0.01, p < 0.001; compared with 3'-SL 60.0 μg / mL, ## p < 0.01, ### p < 0.001; compared with 3'-SL 300 μg / mL, & p <0.05, && p < 0.01; withP Compared to S 31.2 μg / mL, p < 0.05, p < 0.01; with P Compared to S at 125 μg / mL, ! p < 0.05, !! p < 0.01; with P Compared to S 500 μg / mL, %%% p < 0.001.

[0079] Figure 15 for bdnf Relative gene expression levels. Compared with the normal control group, p < 0.05, p < 0.01, p < 0.001; compared with 3'-SL 60.0 μg / mL, # p < 0.05, ## p < 0.01; compared with 3'-SL 300 μg / mL, & p < 0.05, && p < 0.01; compared with PS 31.2 μg / mL, p < 0.01, p < 0.001; compared with PS 500 μg / mL, %% p < 0.01.

[0080] Figure 16 Zebrafish after sample processing bdnf Relative gene expression levels, compared with the model control group, p < 0.05, p < 0.01, p < 0.001; with P Compared to S 31.2 μg / mL, p < 0.05, p < 0.01, p <0.001; and P Compared to S at 125 μg / mL, ! p< 0.05, !! p < 0.01; with P Compared to S 500 μg / mL, %%% p <0.001; compared with 3'-SL 60.0 μg / mL, # p < 0.05, ## p < 0.01; compared with 3'-SL 300 μg / mL, & p <0.05; compared with 3'-SL 1500 μg / mL, @@ p < 0.01, @@@ p < 0.001.

[0081] Figure 17 Zebrafish after sample processing gdnfa Relative gene expression levels, compared with the model control group, p < 0.05, p < 0.01, p < 0.001; with P Compared to S 31.2 μg / mL, p < 0.05, p < 0.001; with P Compared to S 125μg / mL, !! p < 0.01, !!! p < 0.001; with P Compared to S 500 μg / mL, % p < 0.05, %%% p < 0.001; compared with 3'-SL 60.0 μg / mL, ## p < 0.01, ### p < 0.001; compared with 3'-SL 300 μg / mL, && p <0.01; compared with 3'-SL 1500 μg / mL, @@@ p < 0.001. Detailed Implementation

[0082] The combination protected by this patent application consists of two functional substances, characterized in that the composition includes phosphatidylserine (PS) and 3'-sialyl lactose (3'-SL) or a salt thereof. PS, as a major component of brain cell membranes, participates in neural signal transduction and neuroprotection, providing a neural structural basis and signal transduction support; while 3'-SL plays a role in neural development, neuronal connectivity, and immune regulation, potentially optimizing the cognitive environment by promoting neuronal growth and immune regulation. Furthermore, developing scientifically validated nutritional compositions targeting the specific and critical developmental stages of infancy, childhood, and adolescents not only meets market demand for products that enhance cognitive function and improve cognitive decline but also provides this group with new and effective cognitive enhancement solutions.

[0083] As used in this invention, "PS" refers to phosphatidylserine, a phospholipid compound formed by esterification of two fatty acid chains at the sn-1 and sn-2 positions with a glycerol backbone, and L-serine linked to the sn-3 position via a phosphodiester bond. A typical example is 1,2-distearatel-sn-glycerol-3-phosphate-L-serine (DSPS), with the chemical formula C. 42 H 82 NO 10 P has a molecular weight of approximately 792.07 g / mol and a corresponding CAS number of 51446-62-9.

[0084] Phosphatidylserine may be synthetic or commercially available, and its synthesis methods are known to those skilled in the art. Commercially available phosphatidylserine, such as Sharp-PS... ® International Flavors and Fragrances Inc. (IFF) is an American company.

[0085] As used in this invention, "3'-SL" refers to 3'-sialyllactose, a tetrasaccharide compound formed by a lactose core structure of β-D-galactose linked to D-glucose via a 1-4 glycosidic bond, and an N-acetylneuraminic acid (Neu5Ac) linked to the C3' hydroxyl site via an α2-3 glycosidic bond. Its chemical formula is C 23 H 39 NO 19 It has a molecular weight of 633.56 g / mol and a CAS number of 35890-38-1.

[0086] 3' Sialyllactose or its salt may be a sodium salt, and may be synthetic or commercially available. Its synthesis methods are known to those skilled in the art. Commercially available sodium sialyllactose salts include SYNAURA®, Hongmo Biotechnology (Shanghai) Co., Ltd.

[0087] This composition possesses health benefits such as enhancing cognitive abilities and memory and / or improving cognitive decline. Its functional properties were primarily validated through zebrafish experiments. Validation indicators included zebrafish color recognition (percentage of movement in the blue area), acetylcholinesterase activity, dopamine neuron area, memory testing (maze test), and related genes. bdnf and gdnfa ) Expression level.

[0088] The composition exhibits a synergistic effect compared to the individual components when used alone. It is characterized in that, by mass, the content ratio of PS and 3′-SL to exert a synergistic effect ranges from (1:0.12) to (1:48), and the above range includes the endpoint values.

[0089] Unless otherwise specified, the technical terms in this specification have the same meaning as those generally understood by those skilled in the art; however, in case of any conflict, the definitions in this specification shall prevail.

[0090] The term "infant" refers to people aged 0-36 months, including infants, follow-up infants, and toddlers. The term "infant" refers to people aged 0-6 months. The term "follower infant" refers to people aged 6-12 months. The term "toddler" refers to people aged 12-36 months.

[0091] The term "infant formula" encompasses infant formula, follow-up formula, and toddler formula. Generally, infant formula is used as a breast milk substitute from birth, follow-up formula is used from 6-12 months after birth, and toddler formula is used from 12-36 months after birth.

[0092] The term "Foods for Special Medical Purposes (FSMP)" refers to foods specially processed and formulated to meet the specific nutritional or dietary needs of individuals with restricted food intake, digestive and absorptive disorders, metabolic disorders, or specific disease states. These include: complete nutritional formula foods (such as liquid diets for individuals with swallowing difficulties), specific complete nutritional formula foods (such as formulas for diabetes or liver disease), and incomplete nutritional formula foods (such as electrolyte formulas or amino acid component formulas).

[0093] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0094] In this document, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0095] Unless otherwise specified, percentages refer to mass percentages, and proportions refer to mass ratios. In this document, the sum of the percentage contents of all components in the composition is 100%.

[0096] This invention is applicable to normal humans, including infants and / or older infants, and / or toddlers, and / or children, and / or young adults, and / or middle-aged adults, and / or the elderly. The food products of this invention are generally suitable for people of any age, such as infants (including infants, older infants, and toddlers), children, teenagers, adolescents, young adults, adults, middle-aged adults, or the elderly. The term "infant" refers to a person aged 0-6 months. The term "older infant" refers to a person aged 6-12 months. The term "toddler" refers to a person aged 12-36 months. The term "infant-toddler" refers to a person aged 0-36 months. The term "child" refers to a person aged 3-6 years. The term "teenager" refers to a person aged 7-17 years. The term "adult" refers to a person aged 18 years and older. The term "young adult" refers to a person aged 18-40 years. The term "adolescent" refers to a person aged 7-40 years. The term "middle-aged adult" refers to a person aged 41-65 years. The term “elderly” or “senior citizen” refers to people who are 65 years of age or older.

[0097] Composition or reagent

[0098] This invention provides a composition for (a) enhancing cognitive ability and memory and / or (b) improving cognitive decline, comprising phosphatidylserine (PS) and 3'-sialyllactose or a salt thereof (3'-SL). In one or more embodiments, the content of PS and 3'-SL in the composition, by mass, ranges from 1:0.05 to 1:60, preferably from 1:0.1 to 1:50, for example 1:0.1 to 1:10, 1:0.12 to 1:9.6, 0.12 to 1:0.6, 1:0.12 to 1:2.4, 1:0.12 to 1:12, 1:0.12 to 1:48, 1:0.48 to 1:10. :0.6, 1:0.48-1:2.4, 1:0.48-1:12, 1:0.48-1:48, 1:0.6-1:2.4, 1:0.6-1:12, 1:0.6-1:48, 1:2.4-1:12, 1:2.4-1:48, 1:12-1:48; and for example, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0 .2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.8, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, 1:12, 1: 14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:42, 1:44, 1:46, 1:48, 1:50, 1:52, 1:54, 1:56, 1:58, 1:60, or any range defined by both of these, as well as any values ​​and subranges encompassed within these ranges. Within the aforementioned ranges, phosphatidylserine (PS) and 3'-sialyllactose (3'-SL) exhibit significant synergistic effects, particularly in enhancing cognitive abilities and memory and / or improving cognitive decline.

[0099] In one or more embodiments, the mass ratio of phosphatidylserine and 3'-sialyl lactose or their salts in the composition is selected from any of the following ranges: (1:0.12)~(1:0.48), (1:0.12)~(1:0.6), (1:0.12)~(1:2.4), (1:0.12)~(1:12), (1:0.12)~(1:48), (1:0.48)~(1: 0.6), (1:0.48)~(1:2.4), (1:0.48)~(1:12), (1:0.48)~(1:48), (1:0.6)~(1:2.4), (1:0.6)~(1:12), (1:0.6)~(1:48), (1:2.4)~(1:12), (1:2.4)~(1:48), (1:12)~(1:48).

[0100] In some embodiments, the concentration of PS in the composition may be 125-2000 μg / mL, for example 125-1000 μg / mL, 250-800 μg / mL, 300-600 μg / mL, 350-500 μg / mL, or 450-480 μg / mL. In some embodiments, the concentration of 3'-SL in the composition may be 125-2000 μg / mL, for example 125-1000 μg / mL, 250-800 μg / mL, 300-600 μg / mL, 350-500 μg / mL, or 450-480 μg / mL.

[0101] In this paper, the enhancement of cognitive ability and memory includes: enhancing memory, promoting brain development, and promoting the expression of cognitive function-related genes. In this paper, the cognitive function decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes. The cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0102] In this document, the objects of the enhancement of cognitive ability and memory and / or improvement of cognitive function decline are animals, including livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, mice, rabbits, and humans. In one or more embodiments, the animal is a cypriniformes species of carp, including carp, crucian carp, grass carp, snakehead, and topmouth gudgeon, preferably zebrafish. In some embodiments, the mammal is a human. In one or more embodiments, the object may or may not have experienced cognitive function decline. In one or more embodiments, the object is an infant, child, or adolescent. In one or more embodiments, the object is a person aged 0-18 years.

[0103] The present invention also provides a formulation comprising the composition described in any embodiment herein. In one or more embodiments, the formulation is a powder, pill, capsule, granule, tablet, liquid formulation, or gel.

[0104] product

[0105] The present invention also provides products comprising the compositions and / or formulations described in any embodiment herein. In one or more embodiments, the products are food, health product, or pharmaceutical compositions.

[0106] In some implementations, the food type includes: plant-based foods, animal-based foods, microbially fermented foods, and processed foods. Further, the food also includes food additives and / or nutritional fortifiers. Specifically, the food additives may be selected from one or more of the following: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifiers may include one or more of the following: vitamins, minerals, amino acids, fatty acids, and dietary fiber.

[0107] The compositions described herein can be formulated into a food product using standard techniques well known to those skilled in the art. For example, the composition can be added directly to a food-grade acceptable material, or it can be used to prepare an intermediate composition (e.g., a food additive or premix) suitable for subsequent addition to a food-grade acceptable material.

[0108] The term "food-acceptable material" refers to nutritional additives (such as dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins), food-medicine homologous ingredients (such as jujubes, hawthorn berries, longan, lily bulbs, poria cocos, and dried tangerine peel), excipients or auxiliary materials (such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol) that can be used in food.

[0109] In one or more embodiments, the food includes one or more selected from: dairy products, soy products, probiotic powder, probiotic oil drops, dietary fiber supplements, nutrition bars, rice cereal, fruit puree, fruit and vegetable juice, solid beverages, fruit juice, ice cream, candy, biscuits, infant formula, infant food, and foods for special medical purposes. In one or more embodiments, the product is a solid beverage, probiotic oil drops, compressed candy, dairy products, modified milk powder, or infant formula. Among them, solid beverages refer to solid products with a moisture content of no more than 5 grams per 100 grams of finished product, such as instant coffee, fruit juice powder, milk tea powder, etc., which are easy to store and carry; dairy products refer to various foods made from fresh cow (sheep) milk and its products as the main raw materials; modified milk powder is a product based on milk powder with the addition of various nutritional fortifiers (such as vitamins, minerals, probiotics, etc.), such as milk powder for pregnant women, milk powder for middle-aged and elderly people, and milk powder for children's growth; infant formula is a milk powder specially designed for infants to meet the nutritional needs of infant growth and development, which contains a variety of nutrients such as protein, fat, carbohydrates, vitamins and minerals.

[0110] Foods applicable to this invention include ordinary foods and special foods (including health foods not intended to treat diseases, foods for special medical purposes, infant formula, etc.), such as fermented dairy products, non-dairy fermented foods (including fermented vegetable products, fermented soy products, fermented tea, fermented herbs, fermented fruits, probiotic beverages), formula milk or formula milk powder or food additives, such as yogurt, kefir, probiotic cheese (such as Swiss cheese, Gouda cheese, Cheddar cheese, mozzarella cheese), pickles, pickled vegetables, sauerkraut, fermented black beans, natto, fermented bean curd, apple cider vinegar, fermented soy milk, fermented fruit juice, probiotic bread, probiotic steamed buns, live bacteria lactic acid bacteria beverages, probiotic cereals, probiotic chocolate, etc.

[0111] In the food, the amount of the composition added can enhance cognitive function. In some embodiments, the mass percentage of the composition relative to the total mass of the food can be 0.001-80%, preferably 0.01-50%, for example 0.1-30%, 1-20%. Besides PS and 3'-SL, the food may also contain other ingredients, such as other proteins / amino acids, carbohydrates, fats, vitamins, minerals, and other food-grade acceptable materials. For example, when the food is milk powder, in addition to the nutritional composition described in the first aspect of the invention, the milk powder may also include proteins such as α-lactalbumin and milk fat globule membrane protein; carbohydrates such as lactose; lipids; minerals such as calcium, iron, and phosphorus; vitamins; and other additives such as whey powder, choline tartrate, docosahexaenoic acid, arachidonic acid, and walnut oil.

[0112] In this article, the food is not intended for treatment, is not used for the treatment of diseases, and is intended to help (a) enhance cognitive abilities and memory and / or (b) improve cognitive decline.

[0113] In this document, health supplements are those that help enhance cognitive abilities and memory and / or improve cognitive decline. In one or more embodiments, the health supplement further includes additives and / or nutritional fortifiers. In one or more embodiments, the additives include one or more selected from: flavorings, stabilizers, thickeners, preservatives, antioxidants, emulsifiers, and / or the nutritional fortifiers include one or more selected from: vitamins, minerals, amino acids, fatty acids, dietary fiber. In one or more embodiments, the health supplement is a powder, pill, capsule, granule, tablet, oil drop, liquid formulation, or gel.

[0114] The present invention also provides a pharmaceutical composition comprising the composition described in any embodiment of the present invention and a pharmaceutically acceptable excipient. The term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0115] Examples of pharmaceutically acceptable excipients include binders (syrups, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), fillers (lactose, sucrose, starch, calcium phosphate, sorbitol, glycine, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, etc.), disintegrants (starch, microcrystalline cellulose, etc.), humectants (sodium lauryl sulfate, etc.), and suspending agents (sorbitol, syrups, methylcellulose, glucose syrup). Hydrates, gelatin, hydrogenated edible fats, emulsifiers (lecithin, sorbitan monooleate, gum arabic, etc.), non-aqueous carriers (almond oil, fractionated coconut oil or hydrophobic esters such as glycerin, propylene glycol, and ethanol), preservatives (methylparaben or propylparaben, sorbic acid, etc.), flavorings (synthetic fragrances, natural fragrances, etc.), sweeteners (sucrose, stevia, xylitol, etc.), pH adjusters (sodium bicarbonate, potassium carbonate, etc.), powders (pigments, dyes, resins, etc.), thickeners (gum arabic, methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), etc.

[0116] The product may also contain minerals and micronutrients, such as trace elements and vitamins recommended by government agencies like the USRDA. For example, the product may contain one or more of the following micronutrients in daily doses: calcium, magnesium, phosphorus, iron, zinc, copper, iodine, selenium, vitamin C, vitamin B1, vitamin B6, vitamin B2, niacin, vitamin B12, folic acid, biotin, vitamin D, and vitamin E.

[0117] Uses and methods

[0118] The present invention also provides the use of the compositions and formulations described in any embodiment herein in the preparation of products that enhance cognitive abilities and memory and / or improve cognitive decline.

[0119] The present invention also provides the use of the compositions, formulations and / or products described in any embodiment herein, the applications including: enhancing cognitive abilities and memory and / or improving cognitive decline.

[0120] Accordingly, this document also provides a method for enhancing a subject's cognitive abilities and memory and / or improving cognitive decline, comprising applying the composition, food, health product, or pharmaceutical composition described in any embodiment of this document to the subject.

[0121] In this paper, the enhancement of cognitive ability and memory includes: enhancing memory, promoting brain development, and promoting the expression of cognitive function-related genes. In this paper, the cognitive function decline includes: loss or weakening of color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes. The cognitive function-related genes include: gdnfa Gene, bdnf Gene.

[0122] In this document, the objects of the enhancement of cognitive ability and memory and / or improvement of cognitive function decline are animals, including livestock, poultry, or aquatic animals, or rodents or mammals such as pigs, cattle, sheep, chickens, ducks, geese, fish, shrimp, crabs, mice, rabbits, and humans. In one or more embodiments, the animal is a cypriniformes species of carp, including carp, crucian carp, grass carp, snakehead, and topmouth gudgeon, preferably zebrafish. In some embodiments, the mammal is a human. In one or more embodiments, the object may or may not have experienced cognitive function decline. In some embodiments, the object is a person aged 0-18 years, such as an infant, child, or adolescent.

[0123] In the methods described herein, the application frequency can be daily, every two days, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, every four weeks, every month, every three months, every six months, every year, or every two years. In some embodiments, the composition is applied daily for a predetermined number of days. In some embodiments, the treatment period is between about 1 day and about 30 days. In some embodiments, the treatment period is between about 1 month and about 6 months. In some embodiments, the subject applies the composition for a single treatment period. In some embodiments, the subject applies the composition for more than one treatment period. In one or more embodiments, in the composition: the effective amount of PS is 0.3-48 g / day, and / or, the effective amount of 3'SL is 0.18-48 g / day.

[0124] In some embodiments, the effective dosage of PS can be 0.3g / day, 0.4g / day, 0.5g / day, 0.6g / day, 0.7g / day, 0.8g / day, 0.9g / day, 1g / day, 2g / day, 3g / day, 4g / day, 5g / day, 6g / day, 7g / day, 8g / day, 9g / day, 10g / day, 11g / day, 12g / day, 13g / day, 14g / day, 15g / day, 16g / day, 17g / day, 18g / day, or 19g / day. 20g / day, 21g / day, 22g / day, 23g / day, 24g / day, 25g / day, 26g / day, 27g / day, 28g / day, 29g / day, 30g / day, 31g / day, 32g / day, 33g / day, 34g / day, 35g / day, 36g / day, 37g / day, 38g / day, 39g / day, 40g / day, 41g / day, 42g / day, 43g / day, 44g / day, 45g / day, 46g / day, 47g / day, 48g / day.

[0125] In some embodiments, the effective dosage of the 3'SL can be 0.18 g / day, 0.2 g / day, 0.22 g / day, 0.24 g / day, 0.26 g / day, 0.28 g / day, 0.3 g / day, 0.32 g / day, 0.34 g / day, 0.36 g / day, 0.38 g / day, 0.4 g / day, 0.42 g / day, 0.44 g / day, 0.46 g / day, 0.48 g / day, 0.5 g / day, 0.6 g / day, 0.7 g / day, 0.8 g / day, 0.9 g / day, 1 g / day, 2 g / day, 3 g / day, 4 g / day, 5 g / day, 6 g / day, 7 g / day, 8 g / day, 9 g / day, 10 g / day, etc. 1g / day, 11g / day, 12g / day, 13g / day, 14g / day, 15g / day, 16g / day, 17g / day, 18g / day, 19g / day, 20g / day, 21g / day, 22g / day, 23g / day, 24g / day, 25g / day, 26g / day, 27g / day, 28g / day, 29g / day, 30g / day, 31g / day, 32g / day, 33g / day, 34g / day, 35g / day, 36g / day, 37g / day, 38g / day, 39g / day, 40g / day, 41g / day, 42g / day, 43g / day, 44g / day, 45g / day, 46g / day, 47g / day, 48g / day.

[0126] In the method described herein, the route of administration can be oral, medicated bath (immersion) or rectal, and can be adjusted according to the dosage form of the composition or the target of administration. For example, it can be administered in water for fish, or directly orally for mice or other animals such as livestock and poultry.

[0127] In one or more embodiments, the present invention provides a composition comprising phosphatidylserine and 3'-sialyl lactose or a salt thereof in a mass ratio of (1:0.12) to (1:9.6); preferably, the mass ratio of phosphatidylserine and 3'-sialyl lactose or a salt thereof in the composition is selected from any of the following ranges: (1:0.12) to (1:0.48), (1:0.12) to (1:0.6), (1:0.12) to (1:1.92), (1:0.12) to (1:2.4), (1:0.12) to (1:9.6), (1:0.4... 8)~(1:0.6), (1:0.48)~(1:1.92), (1:0.48)~(1:2.4), (1:0.48)~(1:9.6), (1:0.6)~(1:1.92), (1:0.6)~(1:2.4), (1:0.6)~(1:9.6), (1:1.92)~(1:2.4), (1:1.92)~(1:9.6), (1:2.4)~(1:9.6). In one or more embodiments, the present invention provides a product characterized in that the product comprises the composition. For example, the product is a food for non-therapeutic purposes to enhance cognitive ability and memory; more preferably, the food comprises finished food products, semi-finished food products, food additives, and food supplements; even more preferably, the food further comprises food science-acceptable materials, such as nutritional additives, food-medicine homologous ingredients, excipients, and / or excipients. For example, the product is a health supplement used to assist in improving memory. Or, the product is a pharmaceutical composition used to enhance cognitive ability and memory; more preferably, the pharmaceutical composition contains an effective amount of phosphatidylserine and 3'-sialic acid lactose or their salts, and further contains a pharmaceutically acceptable carrier. The nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins, and / or the medicinal and edible ingredients include one or more of jujube, hawthorn, goji berries, longan, lily bulb, poria cocos, and dried tangerine peel, and / or the excipients or adjuvants include one or more of calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.In one or more embodiments, the present invention provides a composition comprising phosphatidylserine and 3'-sialic acid lactose or a salt thereof in a mass ratio of (1:0.12) to (1:48); preferably, the mass ratio of phosphatidylserine and 3'-sialic acid lactose or a salt thereof in the composition is selected from any of the following ranges: (1:0.12) to (1:0.48), (1:0.12) to (1:0.6), (1:0.12) to (1:2.4), (1:0.12) to (1:12), (1:0.12) to (1:48), (1:0.12) to (1:48), (1:0.12) to (1:48) to (1:0.12) to (1:48) to (1:0.12) to (1:48) to (1:0.12) to (1:48) to (1:0.12) to (1:0. ... (1:0.48)~(1:0.6), (1:0.48)~(1:2.4), (1:0.48)~(1:12), (1:0.48)~(1:48), (1:0.6)~(1:2.4), (1:0.6)~(1:12), (1:0.6)~(1:48), (1:2.4)~(1:12), (1:2.4)~(1:48), (1:12)~(1:48). In one or more embodiments, the present invention provides a product characterized in that the product comprises the composition. For example, the product is a food for non-therapeutic purposes to improve cognitive decline; more preferably, the food comprises finished food products, semi-finished food products, food additives, and food supplements; further preferably, the food also comprises food science-acceptable materials, such as nutritional additives, food-medicine homologous ingredients, excipients, and / or excipients. As another example, the product is a health supplement for assisting in improving memory. For example, the product is a pharmaceutical composition for improving cognitive decline; more preferably, the pharmaceutical composition contains an effective amount of phosphatidylserine and 3'-sialyl lactose or their salts, and further contains a pharmaceutically acceptable carrier. The nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins, and / or the medicinal and edible ingredients include one or more of jujube, hawthorn, goji berries, longan, lily bulb, poria cocos, and dried tangerine peel, and / or the excipients or adjuvants include one or more of calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.

[0128] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. The methods and reagents used in the embodiments, unless otherwise stated, are conventional methods and reagents in the art. The invention will now be described by way of specific embodiments.

[0129] Example

[0130] Example 1, Determination of the maximum detectable concentration (MTC) of PS and 3′-SL

[0131] (1)Group 1

[0132] All zebrafish used in the experiment were raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3), and were provided by the fish-raising center of Hangzhou Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK (Zhe) 2022 - 0004, and the feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was: IACUC - 2025 - 10439 - 01.

[0133] Wild-type AB strain zebrafish at 7 days post-fertilization (7 dpf) were randomly selected into beakers, and 30 zebrafish were treated in each beaker (experimental group). The samples were administered in water (concentrations are shown in Table 1), and a normal control group was set up. The capacity of each beaker was 20 mL. After treatment at 28 °C for 24 h, the maximum test concentration (MTC) of the samples on the model zebrafish was measured.

[0134] Under the conditions of this experiment, when the treatment concentrations of PS were 62.5, 125, 250, 500, and 1000 μg / mL respectively, no obvious abnormalities were observed in the zebrafish; when the PS treatment concentration was 2000 μg / mL, the mortality rate of the zebrafish reached 50%. When the treatment concentrations of 3′-SL were 125, 250, 500, 1000, and 2000 μg / mL respectively, no obvious abnormalities were observed in the zebrafish.

[0135] To sum up, under the conditions of this experiment, the MTCs of the cognitive function disorder repair effects of PS and 3′-SL were 1000 and 2000 μg / mL respectively. See Table 1 for details.

[0136] Table 1. Results of the experiment on exploring the concentrations of the sample's cognitive function disorder repair effect (n = 30)

[0137] (2)Group 2

[0138] All zebrafish used in the experiment were raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3), and were provided by the fish-raising center of Hangzhou Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK (Zhe) 2022 - 0004, and the feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was: IACUC - 2025 - 10439 - 01.

[0139] Randomly selected 5 days after fertilization (5 dpf Wild-type AB strain zebrafish were placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples were administered (concentrations shown in Table 2). A normal control group and a model control group were also included, with each beaker containing 20 mL. Except for the normal control group, all other experimental groups were given water-soluble bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) to establish a zebrafish cognitive impairment model. After treatment at 28℃ for 24 h, the MTC of the samples in the model zebrafish was measured.

[0140] At the experimental endpoint, no obvious abnormalities were observed in the zebrafish of the model control group. No obvious abnormalities were observed in the zebrafish when the treatment concentrations of 3′-SL and PS were 125, 250, 500, 1000, and 2000 μg / mL, respectively.

[0141] In summary, under the conditions of this experiment, the MTC for the cognitive impairment repair efficacy of 3′-SL and PS was 2000 μg / mL. See Table 2 for details.

[0142] Table 2. Results of the concentration exploration experiment for the repair efficacy of samples for cognitive dysfunction (n = 30)

[0143] Example 2: Experimental study on the enhancement of memory in zebrafish by PS and 3′-SL monomers (dose-effect relationship)

[0144] Mazes are crucial for assessing various phenomena (such as learning and memory, anxiety, and preferences) in zebrafish neurological disease models. Various cognitive and learning tasks frequently utilize color or food reward cues within mazes to study zebrafish learning and memory. The T-maze, a type of maze induced by foraging motivation, guides animals to complete tasks. Zebrafish select rich areas based on remembering previously explored rich areas, i.e., spatial working memory. The correct alternation of target arms in zebrafish reflects their complete working memory capacity. As brain development matures, zebrafish exhibit a more pronounced preference for the distance and time spent in rich areas. In other words, the more movement and the longer the stay in a rich area, the better the zebrafish's memory.

[0145] The zebrafish used in the experiment and the breeding conditions were the same as in Group 1 of Example 1. Seven zebrafish were randomly selected. dpfWild-type AB strain zebrafish were placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples were administered (concentrations shown in Table 2), with a positive control of edaravone at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group was also included. Each beaker had a volume of 20 mL. After 24 h of treatment, the samples were placed in a T-maze for 1 h of enrichment zone (food) training. After training, treatment continued at 28 ℃ for another 24 h. Zebrafish from each experimental group were randomly selected and placed in the T-maze, with the enrichment zone consistent with the training zone. Video recordings were used to analyze the total time zebrafish spent in the T-maze enrichment zone over a period of time. The statistical analysis results of this index were used to evaluate the sample's memory-enhancing effect. Figure 1 Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0146] Table 3. Evaluation results of the samples' efficacy in promoting brain development (enhancing memory) (n = 3)

[0147] Example 3: Experimental study on the effects of PS and 3′-SL monomers on color perception in zebrafish.

[0148] Cognitive impairment is a mental health disorder affecting cognitive abilities, primarily manifested in six cognitive deficits: executive function, learning and memory, sensorimotor function, language, complex attention, and social cognition. It typically causes learning and memory impairments, accompanied by aphasia, apraxia, agnosia, and apraxia. Bisphenol A (BFA) exhibits neurodevelopmental toxicity, inhibiting motor behavior in zebrafish and damaging glial cells, affecting synapse formation and neurotransmitter release, ultimately leading to a decline in learning, memory, and cognitive abilities. Color preference tests can serve as an effective tool for memory assessment, cognitive impairment assessment, neurodegenerative disease assessment, and toxic behavior assessment. Furthermore, mazes are crucial for evaluating various phenomena (such as learning and memory, anxiety, and preferences) in zebrafish neurological disease models; color cues are frequently used in mazes to study learning and memory in zebrafish for various cognitive tasks. Color preference tests are frequently used in assessing cognitive impairment in zebrafish. Zebrafish prefer shorter wavelengths of color and show a strong preference for blue compared to other colors (such as red, yellow, and green). However, in zebrafish with cognitive impairment, changes in hormones and neurotransmitters in the brain cause them to lose their preference for blue. When a sample is effective in improving cognitive impairment in zebrafish, the proportion of movement in the blue region increases. A ratio of blue region movement proportion between the model control group and the normal control group, ranging from 0.42 to 0.82, is considered a successful model establishment.

[0149] The zebrafish culture conditions used in the experiment were the same as those in Group 2 of Example 1. Five dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples 3′-SL and PS (concentrations shown in Tables 4-5) were administered in water, with edaravone at a concentration of 5.00 μg / mL as a positive control (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group and a model control group were also set up, with each beaker containing 20 mL. Except for the normal control group, all other experimental groups were given bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive impairment model. After treatment at 28℃ for 24 h, five zebrafish were randomly selected from each experimental group and placed into a cross-shaped module. The module was divided into four areas: yellow, blue, red, and green. Six modules were placed in each group. Data were collected using a behavior analyzer, and the percentage (%) of the total movement distance of the zebrafish within the blue area to the total movement distance of the entire area within 10 min was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of cognitive function repair in the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0150] Under the conditions of this experiment, 3′-SL showed cognitive impairment repair efficacy within the range of the maximum tolerated concentration (2000 μg / mL), specifically manifested as an increase in the motor proportion of the blue area; the lowest effective concentration was 500 μg / mL. See Table 4 for details. Figure 2 and Figure 3 .

[0151] Table 4. Experimental Results of the Effect of Samples on Cognitive Impairment Repair (Motor Percentage in Blue Area) (n = 6)

[0152] Compared with the model control group, p < 0.05, p < 0.01

[0153] Similarly, under the conditions of this experiment, PS showed cognitive impairment repair efficacy within the range of the maximum tolerated concentration (2000 μg / mL), specifically manifested as an increase in the motor proportion of the blue area; the lowest effective concentration was 500 μg / mL. See Table 5 for details. Figure 4 and Figure 5 .

[0154] Table 5. Experimental Results of the Efficacy of Samples in Improving Cognitive Impairment (Blue Percentage) (n = 6)

[0155] Compared with the model control group, p < 0.05, p < 0.01, p < 0.001

[0156] Example 4: Effect of the combination of PS and 3′-SL on the memory of zebrafish

[0157] The zebrafish used in the experiment and the breeding conditions were the same as in Group 1 of Example 1. Seven zebrafish were randomly selected. dpf Wild-type AB strain zebrafish were treated in beakers, with 30 zebrafish per beaker (experimental group). Water-soluble samples (concentrations shown in Table 6) were administered, with a positive control of edaravone at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group was also included. Each beaker had a volume of 20 mL. Twenty-four hours after administration, the zebrafish were placed in a T-maze for one hour to train in the enrichment zone (food). After training, the zebrafish were treated at 28℃ for another 24 hours. Zebrafish from each experimental group were randomly selected and placed in the T-maze, with the enrichment zone consistent with the training zone. Video recordings were used to analyze the total time the zebrafish spent in the T-maze enrichment zone over a given period. The statistical analysis results of this indicator were used to evaluate the sample's efficacy in promoting brain development (enhancing memory). Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0158] As shown in Table 6, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the total time spent in the enriched area of ​​the T-maze in zebrafish in the corresponding groups were 213 ± 40.2 s, 239 ± 60.0 s, 274 ± 53.6 s, 287 ± 35.5 s, 376 ± 55.3 s, and 444 ± 33.9 s, respectively. Compared with the normal control group (106 ± 12.3 s), the p values ​​were < 0.05, < 0.05, < 0.05, < 0.01, < 0.01, and < 0.001, respectively. Therefore, formulas 1 to 6 have statistical significance in promoting the improvement of zebrafish memory.

[0159] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the total increase in the total residence time in the T-maze enrichment zone of zebrafish with Formula 1 was 107 s, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 60 μg / mL (57 s).

[0160] Formula 2 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the total residence time of zebrafish in the T-maze enrichment zone of Formula 2 increased by 133 s, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 300 μg / mL (105 s).

[0161] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the total increase in the residence time of zebrafish in the T-maze enrichment zone of Formula 3 was 168 s, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (61 s).

[0162] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the total increase in the residence time of zebrafish in the T-maze enrichment zone of Formula 4 was 181 s, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 300 μg / mL (109 s).

[0163] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the total increase in the residence time of zebrafish in the T-maze enrichment zone of Formula 5 was 270 s, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 60 μg / mL (181 s).

[0164] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the total increase in the residence time of zebrafish in the T-maze enrichment zone of Formula 6 was 338 s, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 300 μg / mL (229 s).

[0165] In summary, under the conditions of this experiment, formulations 1-6 and PS 500 μg / mL all effectively promoted memory enhancement in zebrafish brains, specifically by increasing the total time spent in the T-maze enrichment region. The effects of 3′-SL 60 μg / mL, 3′-SL 300 μg / mL, PS 31.5 μg / mL, and PS 125 μg / mL on improving zebrafish brain memory were not significant. Formulations 1-6 (PS + 3′-SL combination) showed a synergistic effect in enhancing zebrafish brain memory compared to the independent use of PS and 3′-SL. See Table 6 for details. Figure 6 .

[0166] Table 6. Total time spent in the T-maze enrichment zone of zebrafish after sample treatment (n = 3)

[0167] Compared with the normal control group, p < 0.05, p < 0.01, p < 0.001

[0168] Compared with 3'-SL 60.0 μg / mL, # p < 0.05

[0169] Compared with 3'-SL 300 μg / mL, && p < 0.01

[0170] Compared with PS 31.2 μg / mL p < 0.05

[0171] Compared with PS 125 μg / mL, ! p < 0.05, !! p < 0.01

[0172] Compared with PS 500 μg / mL, %% p < 0.01

[0173] Example 5: Effect of the composition of PS and 3′-SL on zebrafish color perception.

[0174] Color preference tests are frequently used in cognitive assessments of zebrafish. Zebrafish prefer shorter wavelengths of color, and compared to other colors (such as red, yellow, and green), they exhibit a strong preference for blue. As brain development progresses, this preference for blue becomes more pronounced; the more movement a zebrafish makes in the blue area, the more cognitively mature its understanding and the higher its brain development.

[0175] (1) Group 1: The zebrafish used in the experiment and the breeding conditions were the same as in Group 1 of Example 1. Five zebrafish were randomly selected. dpfWild-type AB strain zebrafish were placed in beakers, with 30 zebrafish treated in each beaker. PS and 3′-SL were administered to the samples in water (concentrations shown in Table 7). Edaravone was administered as a positive control at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group was also included. Each beaker had a volume of 20 mL. After treatment at 28℃ for 24 h, 5 zebrafish from each experimental group were randomly selected and placed in a cross-shaped module. The module was divided into four regions: yellow, blue, red, and green. Six modules were placed in each group. Data were collected using a behavioral analyzer, and the percentage (%) of the total movement distance of the zebrafish within the blue region relative to the total movement distance of the entire region within 10 min was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the samples in promoting brain development. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 was considered statistically significant.

[0176] As shown in Table 7, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the percentage of movement in the blue area of ​​zebrafish was 50.0 ± 1.45%, 54.0 ± 3.02%, 55.9 ± 3.51%, 57.7 ± 2.52%, 64.8 ± 1.35%, and 65.3 ± 2.16%, respectively. Compared with the normal control group (44.3 ± 1.99%), p < 0.05 & p < 0.05 & p < 0.05 & p < 0.01 & p < 0.001 & p < 0.001. Therefore, formulas 1-6 have statistical significance in promoting cognitive development in zebrafish brains.

[0177] Formula 1 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 1 is 5.7%, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 60 μg / mL (5.6%).

[0178] Formula 2 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 2 was 9.7%, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 300 μg / mL (6.2%).

[0179] Formula 3 is equivalent to a combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 3 was 11.6%, which is equal to the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (11.6%).

[0180] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 4 was 13.4%, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 300 μg / mL (12.2%).

[0181] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 5 was 20.5%, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 60 μg / mL (18.4%).

[0182] Formula 6 is equivalent to a combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 6 was 21%, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 300 μg / mL (19%).

[0183] In summary, under the conditions of this experiment, formulations 1-6, PS 125 μg / mL, and PS 500 μg / mL all promoted cognitive development in zebrafish brains, specifically manifested as an increase in the proportion of movement in the blue area. 3′-SL 60 μg / mL, 3′-SL 300 μg / mL, and PS 31.5 μg / mL did not show significant effects on promoting cognitive development in zebrafish brains. Formulations 1-6 (PS + 3′-SL combination) showed a synergistic effect on promoting cognitive development in zebrafish brains compared to the independent use of PS and 3′-SL. See Table 7 for details. Figure 7 and Figure 8 .

[0184] Table 7. Experimental results on the brain development promotion efficacy of the samples (blue percentage) (n = 6)

[0185] Compared with the normal control group, p < 0.05, p < 0.01, p < 0.001

[0186] Compared with 3'-SL 60.0 μg / mL, #p < 0.05, ###p < 0.001

[0187] Compared with 3'-SL 300 μg / mL, ∂p / ∂x < 0.05, ∂p / ∂x < 0.01, ∂p / ∂x < 0.001

[0188] Compared with 3'-SL 1500 μg / mL, @p < 0.05

[0189] Compared with PS 31.2 μg / mL, p < 0.05

[0190] (2) Group Two: The zebrafish were cultured under the same conditions as in Example 1, Group 2. Five zebrafish were randomly selected. dpf Wild-type AB strain zebrafish were placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples (concentrations shown in Table 8) were administered. The positive control, edaravone, was administered at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group and a model control group were also set up. Each beaker had a volume of 20 mL. Except for the normal control group, all other experimental groups were given water-soluble bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) to establish a zebrafish cognitive impairment model. After treatment at 28℃ for 24 h, 5 zebrafish were randomly selected from each experimental group and placed in a cross-shaped module. The module was divided into four areas: yellow, blue, red, and green. Six modules were placed in each group. Data were collected using a behavior analyzer, and the percentage (%) of the total movement distance of the zebrafish within the blue area relative to the total movement distance of the entire area within 10 min was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of the samples in repairing cognitive impairment. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0191] As shown in Table 8, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the percentage of movement in the blue area of ​​zebrafish was 59.8 ± 1.90%, 55.9 ± 2.93%, 56.6 ± 2.93%, 60.6 ± 2.00%, 60.8 ± 3.17%, and 65.3 ± 2.91%, respectively. Compared with the model control group (42.1 ± 2.24%), p < 0.001 & p < 0.01 & p < 0.001 & p < 0.001 & p < 0.001. Therefore, formulas 1-6 have statistical significance in improving the cognitive function decline in zebrafish.

[0192] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the increase in the proportion of motion in the blue area of ​​Formula 1 is 17.7%, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 1500 μg / mL (17.3%).

[0193] Formula 2 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the increase in the proportion of motion in the blue area of ​​Formula 2 was 13.8%, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (6.3%).

[0194] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the increase in the proportion of motion in the blue area of ​​Formula 3 was 14.5%, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 300 μg / mL (13.6%).

[0195] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the increase in the proportion of motion in the blue area of ​​Formula 4 is 18.5%, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 1500 μg / mL (18.3%).

[0196] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the increase in the proportion of motion in the blue area of ​​Formula 5 was 18.7%, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 60 μg / mL (15.5%).

[0197] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the model control group, the increase in the proportion of motion in the blue area of ​​Formula 6 was 23.2%, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 300 μg / mL (22.8%).

[0198] In summary, under the experimental conditions, formulations 1-6, PS 500 μg / mL, 3′-SL 300 μg / mL, and 3′-SL 1500 μg / mL all showed efficacy in improving cognitive decline in zebrafish, specifically manifested as an increase in the proportion of movement in the blue area compared to the model group. PS 31.2 μg / mL, PS 125 μg / mL, and 3′-SL 60 μg / mL did not show significant efficacy in improving cognitive decline in zebrafish. Formulations 1-6 (PS + 3′-SL combination) exhibited a synergistic effect in improving cognitive decline in zebrafish compared to the independent use of PS and 3′-SL. See Table 8 for details. Figure 9 and Figure 10 .

[0199] Table 8. Experimental results on the efficacy of samples in improving cognitive impairment (blue percentage) (n = 6)

[0200] Compared with the model control group, p < 0.05, p < 0.01, p < 0.001

[0201] Compared with PS 31.2 μg / mL, p < 0.05, p < 0.001

[0202] Compared with PS 125 μg / mL, !p < 0.05, !!p < 0.01

[0203] Compared with PS 500 μg / mL, %p < 0.05, %%p < 0.01

[0204] Compared with 3'-SL 60.0 μg / mL, #p < 0.05, ##p < 0.01

[0205] Compared with 3'-SL 300 μg / mL, p < 0.05

[0206] Example 6: Effect of the combination of PS and 3′-SL on the activity of acetylcholinesterase in zebrafish.

[0207] Acetylcholinesterase (AChE) is an enzyme responsible for degrading acetylcholine (ACh), the main neurotransmitter of the cholinergic system. A decrease in ACh leads to memory decline and cognitive impairment. Measuring AChE activity reflects the degradation of ACh in the body; lower AChE activity indicates less ACh degradation and better memory and cognitive function. Bisphenol A (BFA) has neurodevelopmental toxicity, damaging zebrafish glial cells, affecting synapse formation and neurotransmitter release, and enhancing AChE activity, ultimately leading to decreased learning and memory abilities and cognitive impairment. When a sample demonstrates efficacy in improving cognitive impairment, the fluorescence value of acetylcholinesterase (AChE) decreases. An acetylcholinesterase index between 0.27 and 0.62 in the normal control / model control group is considered a successful model preparation.

[0208] The zebrafish culture conditions used in the experiment were the same as those in Group 2 of Example 1. Four dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered in water (concentrations shown in Table 9). The positive control, edaravone, was administered at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group and a model control group were also set up, with each beaker containing 20 mL. Except for the normal control group, all other experimental groups were given bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive impairment model. After treatment at 28℃ for 48 h, data were collected using a multi-functional microplate reader with acetylcholinesterase assay kits (batch numbers 3320281 and 3321701, AAT Bioquest, USA) to analyze the fluorescence value of acetylcholinesterase (AChE) in zebrafish. The statistical analysis results of this index were used to evaluate the efficacy of the samples in repairing cognitive impairment. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0209] As shown in Table 9, the fluorescence values ​​of acetylcholinesterase in zebrafish treated with formulations 1, 2, 3, 4, 5, and 6 were 3803 ± 248, 3722 ± 221, 3729 ± 212, 3418 ± 206, 3308 ± 227, and 3327 ± 212, respectively, which were significantly lower than those in the model control group (5559 ± 388), with p < 0.01, p < 0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively. Therefore, formulations 1-6 have statistical significance in improving the cognitive decline in zebrafish.

[0210] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the fluorescence value of acetylcholinesterase in Formula 1 decreased by 1756, which is greater than the sum of the decrease values ​​of PS 31.2 μg / mL and 3'-SL 1500 μg / mL (1635).

[0211] Formula 2 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the fluorescence value of acetylcholinesterase in Formula 2 decreased by 1837, which is the sum of the decrease values ​​of PS 125 μg / mL and 3'-SL 60 μg / mL (1323).

[0212] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the fluorescence value of acetylcholinesterase in Formula 3 decreased by 1830, which is greater than the sum of the decrease values ​​of PS 125 μg / mL and 3'-SL 300 μg / mL (1524).

[0213] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the fluorescence value of acetylcholinesterase in Formula 4 decreased by 2141, which is greater than the sum of the decrease values ​​of PS 125 μg / mL and 3'-SL 1500 μg / mL (2078).

[0214] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the fluorescence value of acetylcholinesterase in Formula 5 decreased by 2251, which is greater than the sum of the decrease values ​​of PS 500 μg / mL and 3'-SL 60 μg / mL (1854).

[0215] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the model control group, the fluorescence value of acetylcholinesterase in Formula 6 decreased by 2232, which is greater than the sum of the decrease values ​​of PS 500 μg / mL and 3'-SL 300 μg / mL (2055).

[0216] In summary, under the experimental conditions, formulations 1-6, PS 500 μg / mL, 3′-SL 300 μg / mL, and 3′-SL 1500 μg / mL all showed efficacy in improving cognitive decline in zebrafish, specifically by inhibiting acetylcholinesterase activity compared to the model group. PS 31.2 μg / mL, PS 125 μg / mL, and 3′-SL 60 μg / mL did not show significant efficacy in improving cognitive decline in zebrafish. Formulations 1-6 (PS + 3′-SL combination) exhibited a synergistic effect in improving cognitive decline in zebrafish compared to the independent use of PS and 3′-SL. See Table 9 for details. Figure 11 .

[0217] Table 9. Experimental results on the efficacy of the samples in improving cognitive dysfunction (acetylcholinesterase) (n = 10)

[0218] Compared with the model control group, p < 0.05, p < 0.01, p < 0.001

[0219] Compared with PS 31.2 μg / mL, p < 0.05, p < 0.001

[0220] Compared with PS 125 μg / mL, !!p < 0.01, !!!p < 0.001

[0221] Compared with PS 500 μg / mL, %%p < 0.01, %%%p < 0.001

[0222] Compared with 3'-SL 60.0 μg / mL, #p < 0.05, ##p < 0.01

[0223] Compared with 3'-SL 300 μg / mL, ·p < 0.05, ·&p < 0.01

[0224] Compared with 3'-SL 1500 μg / mL, @p < 0.05

[0225] Example 7, Effect of the combination of PS and 3′-SL on the area of ​​dopamine neurons in zebrafish

[0226] Dopamine plays a crucial regulatory role in the central nervous system and is widely distributed throughout it. Several dopamine pathways are closely related to motor control, cognitive function, and memory. Exposure to bisphenol A (BPA) affects neuronal differentiation and connection formation, especially dopamine neurons. This effect may cause cognitive impairment in early developmental stages. The area of ​​dopamine neurons can reflect their functional activity in vivo, as well as their activity in information processing and neurotransmission. Larger dopamine neuron areas are associated with stronger memory and cognitive function. When zebrafish exhibit cognitive impairment, the area of ​​dopamine neurons decreases. When samples show efficacy in improving cognitive impairment, the area of ​​dopamine neurons increases. A dopamine neuron area ratio between the model control group and the normal control group, ranging from 0.38 to 0.68, is considered a successful model establishment.

[0227] The zebrafish culture conditions used in the experiment were the same as those in Group 2 of Example 1. Four-day-fiber (dpf) transgenic monoaminergic neuron green fluorescent zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Samples were administered in water (concentrations shown in Table 10). The positive control, edaravone, was administered at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group and a model control group were also set up, with each beaker containing 20 mL. Except for the normal control group, all other experimental groups were given bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive impairment model. After treatment at 28℃ for 48 h, 10 zebrafish from each experimental group were randomly selected and photographed under a Zeiss fluorescence microscope. ImageJ software was used to analyze and collect data, and the area of ​​zebrafish dopamine neurons was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the samples in improving dopamine neuron damage. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0228] As shown in Table 10, the fluorescence values ​​of zebrafish dopamine neurons under the treatments of formulations 1, 2, 3, 4, 5, and 6 were 4312 ± 224, 4426 ± 204, 4527 ± 149, 4646 ± 183, 4598 ± 182, and 4649 ± 156, respectively, which were significantly lower than those of the model control group (3300 ± 207), with p < 0.01, p < 0.01, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively. Therefore, formulations 1-6 have statistical significance in improving the cognitive function decline in zebrafish.

[0229] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the fluorescence value of dopamine neurons in Formula 1 increased by 1012, which is greater than the sum of the increases of PS 31.2 μg / mL and 3'-SL 1500 μg / mL (860).

[0230] Formula 2 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the fluorescence value of dopamine neurons in Formula 2 increased by 1126, which is the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (787).

[0231] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the fluorescence value of dopamine neurons in Formula 3 increased by 1227, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 300 μg / mL (843).

[0232] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the fluorescence value of dopamine neurons in Formula 4 increased by 1346, which is greater than the sum of the increases of PS 125 μg / mL and 3'-SL 1500 μg / mL (954).

[0233] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the fluorescence value of dopamine neurons in Formula 5 increased by 1298, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 60 μg / mL (1254).

[0234] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the model control group, the fluorescence value of dopamine neurons in Formula 6 increased by 1349, which is greater than the sum of the increases of PS 500 μg / mL and 3'-SL 300 μg / mL (1310).

[0235] In summary, under the experimental conditions, formulations 1-6 and PS 500 μg / mL all showed efficacy in improving cognitive decline in zebrafish, specifically manifested as an increase in dopamine neuron area compared to the model group. PS 31.2 μg / mL, PS 125 μg / mL, 3′-SL 60 μg / mL, 3′-SL 300 μg / mL, and 3′-SL 1500 μg / mL did not show significant efficacy in improving cognitive decline in zebrafish. Formulations 1-6 (PS + 3′-SL combination) showed a synergistic effect in improving cognitive decline in zebrafish compared to the independent use of PS and 3′-SL. See Table 10 for details. Figure 12 and Figure 13 .

[0236] Table 10. Experimental results on the efficacy of improving cognitive dysfunction (area of ​​dopamine neurons) (n = 10)

[0237] Compared with the model control group, p < 0.01, p < 0.001

[0238] Compared with PS 31.2 μg / mL, p < 0.05

[0239] Compared with PS 125 μg / mL, !p < 0.05, !!p < 0.01

[0240] Compared with 3'-SL 60.0 μg / mL, ##p < 0.01, ###p < 0.001

[0241] Compared with 3'-SL 300 μg / mL, ∂p / ∂x < 0.05, ∂p / ∂x < 0.01, ∂p / ∂x < 0.001

[0242] Compared with 3'-SL 1500 μg / mL, p < 0.01

[0243] Example 8: Effect of the combination of PS and 3′-SL on the expression levels of genes related to cognitive function in zebrafish.

[0244] bdnfIts receptors are widely expressed in the nervous system, with the highest content in the hippocampus and cortex. Its specific mechanisms of action in the central nervous system are as follows: (1) increasing synaptic plasticity, thereby affecting long-term potentiation (nLTP), which is the basis of learning and memory formation (secondary memory); (2) promoting neurogenesis, especially in the hippocampus; (3) promoting cell survival, mainly manifested in maintaining and promoting the development, differentiation, growth, and regeneration of various neurons, especially 5-hydroxytryptamine (5-HT) and dopaminergic (DA) neurons. gdnfa Both genes promote the survival of different neuronal subsets at different stages of development in the central and peripheral nervous systems, supporting the generation of type I astrocytes, meningeal cells, neurons, and pineal gland cells, with a particularly significant effect on promoting the survival of spinal motor neurons. Therefore, upregulation of the expression levels of these two genes can promote central nervous system and neural development, thereby promoting the formation of learning and memory.

[0245] When zebrafish exhibited cognitive impairment, the expression levels of both bdnf and gdnfa were significantly downregulated. When the samples demonstrated efficacy in improving cognitive impairment, the expression levels of both bdnf and gdnfa were upregulated. A relative expression level of bdnf between the model control group and the normal control group was considered a successful model establishment if the ratio was between 0.12 and 0.65. Similarly, a relative expression level of gdnfa between the model control group and the normal control group was considered a successful model establishment if the ratio was between 0.18 and 0.68.

[0246] (1) Group 1: The zebrafish and culture conditions used in the experiment were the same as in Group 1 of Example 1. Wild-type AB strain zebrafish with a 5 dpf growth rate were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples were administered (concentrations shown in Tables 11 and 12). Edaravone was used as a positive control at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group was also set up. Each beaker had a volume of 20 mL. The experiment was conducted in triplicate. After treatment at 28℃ for 24 h, total RNA was extracted from each group of zebrafish using a pre-loaded magnetic bead-based universal RNA extraction kit (catalog number TL2402001643C, ONREW, China). The concentration and purity of the total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from zebrafish samples was collected, and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit (batch number: H9305270, Yisheng Biotechnology (Shanghai) Co., Ltd., China). β-actin was detected by q-PCR. bdnf and gdnfaGene expression. Using β-actin as an internal reference for gene expression, calculations were performed. bdnf and gdnfa Relative gene expression levels. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0247] As shown in Table 11, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the corresponding groups of zebrafish... gdnfa The relative gene expression levels were 1.75 ± 0.058, 2.42 ± 0.038, 1.47 ± 0.029, 2.33 ± 0.109, 5.91 ± 0.464, and 5.44 ± 0.273, respectively. Compared with the normal control group (1.00 ± 0.061), p < 0.001 & p < 0.001 & p < 0.001 & p < 0.001, respectively. Therefore, formulations 1-6 have statistically significant effects on promoting cognitive development in zebrafish brains.

[0248] Formula 1 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 1 zebrafish... gdnfa The increase in relative gene expression was 0.75, which was greater than the sum of the increases in PS 31.2 μg / mL and 3'-SL 60 μg / mL (-0.172).

[0249] Formula 2 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 300 μg / mL. Compared to the normal control group, Formula 2 zebrafish... gdnfa The increase in relative gene expression was 1.42, which was greater than the sum of the increases in PS 31.2 μg / mL and 3'-SL 300 μg / mL (0.12).

[0250] Formula 3 is equivalent to a combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 3 zebrafish... gdnfa The increase in relative gene expression was 0.47, which was greater than the sum of the increases in PS 125 μg / mL and 3'-SL 60 μg / mL (0.268).

[0251] Formula 4 is equivalent to a combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared to the normal control group, Formula 4 zebrafish... gdnfaThe increase in relative gene expression was 1.33, which was greater than the sum of the increases of PS 125 μg / mL and 3'-SL 300 μg / mL (0.56).

[0252] Formula 5 is equivalent to a combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 5 zebrafish... gdnfa The increase in relative gene expression was 4.91, which was greater than the sum of the increases in PS 500 μg / mL and 3'-SL 60 μg / mL (0.42).

[0253] Formula 6 is equivalent to a combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared to the normal control group, Formula 6 zebrafish... gdnfa The increase in relative gene expression was 4.44, which was greater than the sum of the increases in PS 500 μg / mL and 3'-SL 300 μg / mL (0.71).

[0254] In conclusion, under the conditions of this experiment, formulations 1-6, PS 125 μg / mL, and PS 500 μg / mL all had the effect of promoting cognitive development in zebrafish brains, specifically by promoting the development of zebrafish brains. gdnfa The relative expression levels of genes were upregulated; however, the effects of 3′-SL 60 μg / mL, 3′-SL 300 μg / mL, and PS 31.5 μg / mL on promoting cognitive development in zebrafish brains were not significant; formulations 1-6 (PS+3′-SL combination) showed a synergistic effect on promoting cognitive development in zebrafish brains compared to the independent use of PS and 3′-SL. See Table 11 for details. Figure 14 .

[0255] Table 11. The brain development-promoting effects of the samples ( gdnfa Relative gene expression levels (n = 3) Experimental results

[0256] Compared with the normal control group, p < 0.01, p < 0.001

[0257] Compared with 3'-SL 60.0 μg / mL, ##p < 0.01, ###p < 0.001

[0258] Compared with 3'-SL 300 μg / mL, p < 0.01, p < 0.001

[0259] Compared with PS 31.2 μg / mL, p < 0.05, p < 0.01

[0260] Compared with PS 125 μg / mL, !p < 0.05, !!p < 0.01

[0261] Compared with PS 500 μg / mL, %%%p < 0.001

[0262] As shown in Table 12, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the corresponding groups of zebrafish... bdnf The relative gene expression levels were 1.97 ± 0.104, 2.66 ± 0.059, 2.61 ± 0.231, 1.54 ± 0.089, 3.17 ± 0.160, and 4.83 ± 0.308, respectively. Compared with the normal control group (1.00 ± 0.084), the p values ​​were all statistically significant. Therefore, formulations 1-6 have a statistically significant effect on promoting cognitive development in zebrafish brains.

[0263] Formula 1 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 1 zebrafish... bdnf The increase in relative gene expression was 0.97, which was greater than the sum of the increases in PS 31.2 μg / mL and 3'-SL 60 μg / mL (0.12).

[0264] Formula 2 is equivalent to a combination of PS 31.2 μg / mL and 3'-SL 300 μg / mL. Compared to the normal control group, Formula 2 zebrafish... bdnf The increase in relative gene expression was 1.66, which was greater than the sum of the increases in PS 31.2 μg / mL and 3'-SL 300 μg / mL (0.52).

[0265] Formula 3 is equivalent to a combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 3 zebrafish... bdnf The increase in relative gene expression was 1.61, which was greater than the sum of the increases in PS 125 μg / mL and 3'-SL 60 μg / mL (1.12).

[0266] Formula 5 is equivalent to a combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared to the normal control group, Formula 5 zebrafish... bdnfThe increase in relative gene expression was 2.71, which was greater than the sum of the increases in PS 500 μg / mL and 3'-SL 60 μg / mL (1.12).

[0267] Formula 6 is equivalent to a combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared to the normal control group, Formula 6 zebrafish... bdnf The increase in relative gene expression was 3.83, which was greater than the sum of the increases in PS 500 μg / mL and 3'-SL 300 μg / mL (1.52).

[0268] In conclusion, under the conditions of this experiment, formulations 1-6, PS 125 μg / mL, and PS 500 μg / mL all had the effect of promoting cognitive development in zebrafish brains, specifically by promoting the development of zebrafish brains. bdnf The relative expression levels of genes were upregulated; however, the effects of 3′-SL 60 μg / mL, 3′-SL 300 μg / mL, and PS 31.5 μg / mL on promoting cognitive development in zebrafish brains were not significant; formulations 1–6 (PS + 3′-SL combination) showed a synergistic effect on promoting cognitive development in zebrafish brains compared to the independent use of PS and 3′-SL. See Table 12 for details. Figure 15 .

[0269] Table 12. The brain development-promoting effects of the samples ( bdnf Relative gene expression levels (n = 3) Experimental results

[0270] Compared with the normal control group, p < 0.05, p < 0.01, p < 0.001

[0271] Compared with 3'-SL 60.0 μg / mL, #p < 0.05, ##p < 0.01

[0272] Compared with 3'-SL 300 μg / mL, ·p < 0.05, ·&p < 0.01

[0273] Compared with PS 31.2 μg / mL, p < 0.01, p < 0.001

[0274] Compared with PS 500 μg / mL, %%p < 0.01

[0275] (2) Group Two

[0276] The zebrafish culture conditions used in the experiment were the same as those in Group 2 of Example 1. Five dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). Water-soluble samples were administered (concentrations shown in Tables 13 and 14). The positive control, edaravone, was administered at a concentration of 5.00 μg / mL (water-soluble administration, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number B2217151). A normal control group and a model control group were also set up. Each beaker had a volume of 20 mL. Except for the normal control group, all other experimental groups were given water-soluble bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) to establish a zebrafish cognitive impairment model. Three parallel experiments were conducted. After treatment at 28℃ for 24 h, total RNA was extracted from zebrafish in each group using a pre-loaded magnetic bead-based universal RNA extraction kit (catalog number TL2402001643C, ONREW, China). The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from zebrafish samples was used to synthesize 20.0 μL of cDNA according to the instructions of a cDNA first-strand synthesis kit (batch number: H9305270, Yisheng Biotechnology (Shanghai) Co., Ltd., China). The expression of β-actin, bdnf, and gdnfa genes was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative expression levels of bdnf and gdnfa genes were calculated. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0277] As shown in Table 13, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the relative expression levels of the bdnf gene in zebrafish were 3.43 ± 0.242, 1.83 ± 0.122, 2.50 ± 0.148, 3.10 ± 0.216, 3.52 ± 0.042, and 3.77 ± 0.204, respectively. These levels were significantly higher than those in the model control group (1.00 ± 0.038), with p < 0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively. Therefore, formulas 1-6 have statistical significance in improving the cognitive function decline in zebrafish.

[0278] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the relative expression level of bdnf gene in Formula 1 increased by 2.43, which is greater than the sum of the individual increases of PS 31.2 μg / mL and 3'-SL 1500 μg / mL (1.168).

[0279] Formula 2 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the relative expression level of bdnf gene in Formula 2 increased by 0.83, which is the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (0.48).

[0280] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the relative expression level of bdnf gene in Formula 3 increased by 1.5, which is greater than the sum of the individual increases of PS 125 μg / mL and 3'-SL 300 μg / mL (1.17).

[0281] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the relative expression level of bdnf gene in Formula 4 increased by 2.1, which is greater than the sum of the individual increases of PS 125 μg / mL and 3'-SL 1500 μg / mL (1.48).

[0282] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the relative expression level of bdnf gene in Formula 5 increased by 2.52, which is greater than the sum of the individual increases of PS 500 μg / mL and 3'-SL 60 μg / mL (0.65).

[0283] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the model control group, the relative expression level of bdnf gene in Formula 6 increased by 2.77, which is greater than the sum of the individual increases of PS 500 μg / mL and 3'-SL 300 μg / mL (1.25).

[0284] In summary, under the experimental conditions, formulations 1-6, PS 500 μg / mL, 3′-SL 300 μg / mL, and 3′-SL 1500 μg / mL all showed efficacy in improving cognitive decline in zebrafish, specifically manifested as an increase in the relative expression level of the bdnf gene compared to the model group. PS 31.2 μg / mL, PS 125 μg / mL, and 3′-SL 60 μg / mL did not show significant efficacy in improving cognitive decline in zebrafish. Formulations 1-6 (PS + 3′-SL combination) exhibited a synergistic effect in improving cognitive decline in zebrafish compared to the independent use of PS and 3′-SL. See Table 13 for details. Figure 16 .

[0285] Table 13. Efficacy of the samples in improving cognitive impairment ( bdnf Relative gene expression levels (n = 3) Experimental results

[0286] Compared with the model control group, p < 0.05, p < 0.01, p < 0.001

[0287] Compared with PS 31.2 μg / mL, p < 0.05, p < 0.01, p < 0.001

[0288] Compared with PS 125 μg / mL, !p < 0.05, !!p < 0.01

[0289] Compared with PS 500 μg / mL, %%%p < 0.001

[0290] Compared with 3'-SL 60.0 μg / mL, #p < 0.05, ##p < 0.01

[0291] Compared with 3'-SL 300 μg / mL, p < 0.05

[0292] Compared with 3'-SL 1500 μg / mL, @@p < 0.01, @@@p < 0.001

[0293] As shown in Table 14, under the treatments of formulas 1, 2, 3, 4, 5, and 6, the relative expression levels of the gdnfa gene in zebrafish were 2.94 ± 0.152, 2.72 ± 0.186, 2.52 ± 0.093, 1.88 ± 0.160, 2.19 ± 0.079, and 2.23 ± 0.096, respectively. These levels were significantly higher than those in the model control group (1.00 ± 0.047), with p < 0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001, respectively. Therefore, formulas 1-6 have statistical significance in improving the cognitive function decline in zebrafish.

[0294] Formula 1 is equivalent to the combination of PS 31.2 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the relative expression level of gdnfa gene in Formula 1 increased by 1.94, which is greater than the sum of the individual increases of PS 31.2 μg / mL and 3'-SL 1500 μg / mL (0.373).

[0295] Formula 2 is equivalent to the combination of PS 125 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the relative expression level of gdnfa gene in Formula 2 increased by 1.72, which is the sum of the increases of PS 125 μg / mL and 3'-SL 60 μg / mL (0.014).

[0296] Formula 3 is equivalent to the combination of PS 125 μg / mL and 3'-SL 300 μg / mL. Compared with the normal control group, the relative expression level of gdnfa gene in Formula 3 increased by 1.52, which is greater than the sum of the individual increases of PS 125 μg / mL and 3'-SL 300 μg / mL (0.074).

[0297] Formula 4 is equivalent to the combination of PS 125 μg / mL and 3'-SL 1500 μg / mL. Compared with the model control group, the relative expression level of gdnfa gene in Formula 4 increased by 0.88, which is greater than the sum of the individual increases of PS 125 μg / mL and 3'-SL 1500 μg / mL (0.364).

[0298] Formula 5 is equivalent to the combination of PS 500 μg / mL and 3'-SL 60 μg / mL. Compared with the model control group, the relative expression level of gdnfa gene in Formula 5 increased by 1.19, which is greater than the sum of the individual increases of PS 500 μg / mL and 3'-SL 60 μg / mL (0.71).

[0299] Formula 6 is equivalent to the combination of PS 500 μg / mL and 3'-SL 300 μg / mL. Compared with the model control group, the relative expression level of gdnfa gene in Formula 6 increased by 1.23, which is greater than the sum of the individual increases of PS 500 μg / mL and 3'-SL 300 μg / mL (0.77).

[0300] In summary, under the experimental conditions, formulations 1-6, PS 500 μg / mL, and 3′-SL 1500 μg / mL all showed efficacy in improving cognitive decline in zebrafish, specifically manifested as an increase in the relative expression level of the gdnfa gene compared to the model group. PS 31.2 μg / mL, PS 125 μg / mL, 3′-SL 60 μg / mL, and 3′-SL 300 μg / mL did not show significant efficacy in improving cognitive decline in zebrafish. Formulations 1-6 (PS + 3′-SL combination) showed a synergistic effect in improving cognitive decline in zebrafish compared to the independent use of PS and 3′-SL. See Table 14 for details. Figure 17 .

[0301] Table 14. Efficacy of the samples in improving cognitive impairment ( gdnfa Relative gene expression levels (n = 3) Experimental results

[0302] Compared with the model control group, p < 0.05, p < 0.01, p < 0.001

[0303] Compared with PS 31.2 μg / mL, p < 0.05, p < 0.001

[0304] Compared with PS 125 μg / mL, !!p < 0.01, !!!p < 0.001

[0305] Compared with PS 500 μg / mL, %p < 0.05, %%%p < 0.001

[0306] Compared with 3'-SL 60.0 μg / mL, ##p < 0.01, ###p < 0.001

[0307] Compared with 3'-SL 300 μg / mL, p < 0.01

[0308] Compared with 3'-SL 1500 μg / mL, p < 0.001

[0309] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this invention, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A composition for (a) enhancing cognitive ability and memory and / or (b) improving cognitive function deterioration, which consists of phosphatidylserine and 3'-sialyllactose or a salt thereof.

2. The composition of claim 1, wherein The mass ratio of phosphatidylserine to 3'-sialyllactose or a salt thereof in the composition is 1:0.05 to 1:

60.

3. The composition of claim 1, wherein The mass ratio of phosphatidylserine to 3'-sialyllactose or a salt thereof in the composition is 1:0.1 to 1:

50.

4. The composition according to claim 1, wherein the composition is used for enhancing cognitive ability and memory, and the mass ratio of phosphatidylserine to 3'-sialyllactose or a salt thereof in the composition is (1:0.12) to (1:9.6); or the composition is used for improving cognitive function deterioration, and the mass ratio of phosphatidylserine to 3'-sialyllactose or a salt thereof in the composition is (1:0.12) to (1:48). The salt of 3'-sialyllactose is a sodium salt.

6. A preparation comprising the composition according to any one of claims 1 to 5.

5. The composition according to any one of claims 1 to 4, wherein The preparation is a powder, a pill, a capsule, a granule, a tablet, a liquid preparation, or a gel.

8. A product comprising the composition according to any one of claims 1 to 5 or the preparation according to claim 6 or 7.

7. The formulation of claim 6, wherein, The product is a food, a health food, or a pharmaceutical composition. The types of the food include a plant food, an animal food, a microorganism fermented food, and a processed food.

9. The product of claim 8, wherein, The food further includes a food additive and / or a nutritional fortifier.

10. The product of claim 9, wherein, The food additive includes one or more selected from the group consisting of a flavoring, a stabilizer, a thickening agent, a preservative, an antioxidant, and an emulsifier, and / or the nutritional fortifier includes one or more selected from the group consisting of a vitamin, a mineral, an amino acid, a fatty acid, and a dietary fiber.

11. The product of claim 10, wherein, The food includes one or more selected from the group consisting of a dairy product, a soy product, a probiotic powder, a probiotic oil droplet, a dietary fiber supplement, a nutrition bar, a rice powder, a fruit puree, a fruit-vegetable juice, a food solid beverage, a fruit juice, an ice cream, a candy, a cookie, an infant formula, a baby formula, and a food for special medical purposes.

12. The product of claim 11, wherein, The types of the health food include a powder, a pill, a capsule, a granule, a tablet, a solution, an emulsion, and a suspension.

13. The product of claim 9, wherein, The health food is a powder, a pill, a capsule, a granule, a tablet, an oil droplet, a liquid preparation, or a gel.

14. The product of claim 9, wherein, The health food further includes an additive and / or a nutritional fortifier.

15. The product of claim 9, wherein, The additive includes one or more selected from the group consisting of a flavoring, a stabilizer, a thickening agent, a preservative, an antioxidant, and an emulsifier, and / or the nutritional fortifier includes one or more selected from the group consisting of a vitamin, a mineral, an amino acid, a fatty acid, and a dietary fiber.

16. The product of claim 9, wherein, The pharmaceutical composition further includes a pharmaceutically acceptable excipient.

17. The product of claim 16, wherein 19. Use of the composition according to any one of claims 1 to 5 or the preparation according to claim 6 or 7 for the manufacture of a product for enhancing cognitive ability and memory and / or improving cognitive function deterioration in a subject.

18. The product of claim 9, wherein, The enhancement of cognitive ability and memory includes enhancement of memory, promotion of brain development, and promotion of expression of a gene related to cognitive function. ​ 20. The use of claim 19, wherein, ​ 21. The use of claim 19, wherein, The cognitive function deterioration includes loss or weakening of color preference ability, increase of acetylcholinesterase activity, decrease of dopamine neuron area, and decrease of expression level of genes related to cognitive function.

22. The use according to claim 20 or 21, characterized in that The cognitive function-related genes include: gdnfa gene, bdnf gene.

23. The use of claim 19, wherein, The animal includes livestock, poultry, or aquatic animals, or includes rodents or mammals.

24. The use of claim 23, wherein, The animal includes livestock, poultry, or aquatic animals, or includes rodents or mammals.

25. Non-therapeutic use of a composition as claimed in any of claims 1 to 5, a preparation as claimed in claim 6 or 7 and / or a product as claimed in any of claims 8 to 18, which use comprises: The cognitive function deterioration includes loss or weakening of color preference ability, increase of acetylcholinesterase activity, decrease of dopamine neuron area, and decrease of expression level of genes related to cognitive function.

26. The use of claim 25, wherein, gdnfa 27. The use of claim 25, wherein, bdnf 28. Use according to claim 26 or 27, wherein The cognitive function-related genes include The cognitive function deterioration includes loss or weakening of color preference ability, increase of acetylcholinesterase activity, decrease of dopamine neuron area, and decrease of expression level of genes related to cognitive function. Genes and / or gdnfa bdnf Genes.

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