Composition for synergistically improving cognitive impairment

Abstract

The invention provides a composition for synergistically improving cognitive impairment. The invention provides a composition. The composition is composed of 2 '-FL and 3'-SL in a mass ratio of (1: 2.5)-(1: 15). The invention further provides food and medicine containing the composition, application of the composition in preparation of food or medicine for improving cognitive impairment, application of the food or medicine for improving cognitive impairment and a method for improving cognitive impairment.

Description

Technical Field

[0001] The present invention belongs to the field of improving cognitive dysfunction, and more specifically, the present invention relates to a composition for synergistically improving cognitive dysfunction. Background Art

[0002] With the accelerating pace of modern social life, increasing work pressure, and the intensifying trend of global population aging, the problem of cognitive dysfunction has become increasingly prominent. Phenomena such as memory decline, attention deficit, and cognitive function decline not only affect the quality of personal life but may also develop into neurodegenerative diseases such as mild cognitive impairment (MCI) and Alzheimer's disease (AD).

[0003] Studies have shown that Ginkgo biloba extract has certain effects and potential in improving cognitive function. Ginkgo biloba extract can reduce the deposition of β-amyloid (Aβ), improve the memory of AD model mice; may delay age-related cognitive decline by enhancing hippocampal neurogenesis; increase NO release, inhibit PAF, dilate cerebral blood vessels, and enhance oxygen and glucose supply; up-regulate BDNF, promote synaptic plasticity, inhibit Aβ aggregation and tau protein phosphorylation, and thus protect neurons. However, the significant differences in the contents of its active ingredients, flavonoid glycosides and terpene lactones, lead to unstable efficacy; the impurity component ginkgolic acid may cause allergies and requires strict purification; the oral bioavailability of flavonoid glycosides is <10%, and new technologies such as nanocarriers are needed to improve it; the combination with anticoagulants (warfarin) or antiplatelet drugs may increase the risk of bleeding, etc., which limits its application.

[0004] Therefore, there is an urgent need in this field to explore a composition that can significantly improve cognitive dysfunction. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition for synergistically improving cognitive dysfunction.

[0006] In the first aspect of the present invention, a composition is provided, which is composed of 2′-FL and 3′-SL with a mass ratio of (1:2.5) to (1:15).

[0007] In one or more embodiments, relative to 1 part by mass of 2′-FL, the amount of 3′-SL is 2.5 to 14 parts by mass, preferably 3 to 13 parts by mass, 4 to 12 parts by mass, 5 to 11 parts by mass, 6 to 10 parts by mass, and more preferably 7 to 10 parts by mass.

[0008] In the second aspect of the present invention, a food is provided, which contains the composition according to any embodiment of the present invention.

[0009] In one or more embodiments, the food includes finished food products, semi-finished food products, food additives, and food supplements.

[0010] In one or more embodiments, the food further includes a pharmaceutically acceptable material.

[0011] In one or more embodiments, the pharmaceutically acceptable materials include: nutritional additives, ingredients homologous in medicine and food, excipients, and / or auxiliary materials.

[0012] In one or more embodiments, the nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins.

[0013] In one or more embodiments, the ingredients homologous in medicine and food include one or more of red dates, hawthorn, wolfberries, longans, lilies, poria cocos, and dried tangerine peels.

[0014] In one or more embodiments, the excipients or auxiliary materials include one or more of calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0015] In one or more embodiments, the food is infant food, children's food, juvenile food, adolescent food, young adult food, adult food, middle-aged food, or elderly food.

[0016] In one or more embodiments, the infants include babies, older babies, and toddlers.

[0017] In one or more embodiments, the food is infant formula milk powder, baby complementary food, children's formula milk powder, children's snacks, pregnant women's formulated milk powder, middle-aged and elderly milk powder, or nutritional or dietary supplements.

[0018] In a third aspect of the present invention, there is provided a drug comprising an effective amount of the composition according to any one of the embodiments of the present invention and a pharmaceutically acceptable carrier.

[0019] In one or more embodiments, in the composition: the effective amount of 2′-FL is 45~1000 μg / mL, and / or the effective amount of 3′-SL is 500~2000 μg / mL.

[0020] In a fourth aspect of the present invention, there is provided the use of the composition according to any one of the embodiments of the present invention in the preparation of a drug for improving cognitive dysfunction.

[0021] In a fifth aspect of the present invention, there is provided the use of the drug according to any one of the embodiments of the present invention in improving cognitive dysfunction, or the use of the food according to any one of the embodiments of the present invention in improving cognitive dysfunction for non-therapeutic purposes.

[0022] In one or more embodiments, the cognitive dysfunction is a neural and / or cognitive dysfunction.

[0023] In one or more embodiments, the cognitive dysfunction includes: executive dysfunction, learning and memory dysfunction, sensorimotor dysfunction, language disorder, complex attention disorder, social cognitive deficit, aphasia, apraxia, agnosia, and dyspraxia.

[0024] In one or more embodiments, the cognitive dysfunction is a learning and memory dysfunction.

[0025] In one or more embodiments, the learning and memory dysfunction includes: absence or reduction of the ability of zebrafish to prefer colors, increased acetylcholinesterase activity, decreased area of dopamine neurons, and decreased expression levels of genes related to cognitive function.

[0026] In one or more embodiments, the genes related to cognitive function include bdnf and gdnfa.

[0027] In a sixth aspect of the present invention, there is provided a method for improving cognitive dysfunction for non-therapeutic purposes, the method comprising: administering the composition according to any one of the embodiments of the present invention to an individual in need, or administering the food according to any one of the embodiments of the present invention or the drug according to any one of the embodiments of the present invention to an individual in need.

[0028] In one or more embodiments, the improvement of cognitive dysfunction is as defined in any one of the embodiments of the present invention.

[0029] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A , 2 Typical graph of the movement trajectory of zebrafish with cognitive dysfunction model after treatment with 3′-FL and 3′-SL, where the blue box is the blue area of the plus maze, which is the area for quantification.

[0031] Figure 1B , 2 Percentage of movement in the blue area of zebrafish with cognitive dysfunction model after treatment with 3′-FL and 3′-SL. Compared with the model control group, *p < 0.05, **p < 0.01.

[0032] Figure 2 , 2After treatment with ′-FL and 3′-SL, the fluorescence value of acetylcholinesterase (AchE) in zebrafish with cognitive impairment model, compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 3A , 2 Typical graph of the movement trajectory of zebrafish with cognitive impairment model after treatment with the composition of ′-FL and 3′-SL, where the blue square is the blue area of the plus maze, which is the area for quantification.

[0034] Figure 3B , 2 After treatment with the composition of ′-FL and 3′-SL, the proportion of movement in the blue area of zebrafish with cognitive impairment model, compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with formula 1, # p <0.05; compared with formula 2, & p < 0.05; compared with formula 3, @ p < 0.05, @@ p < 0.01; compared with formula 4, $ p <0.05, $$ p < 0.01.

[0035] Figure 4 , 2 After treatment with the composition of ′-FL and 3′-SL, the fluorescence value of acetylcholinesterase (AchE) in zebrafish with cognitive impairment model, compared with the model control group, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with formula 3, @ p < 0.05, @@ p < 0.01.

[0036] Figure 5A , 2 Typical graph of the area of dopamine neurons in zebrafish with cognitive impairment model after treatment with the composition of ′-FL and 3′-SL, and the white arrow points to the dopamine neurons in the zebrafish brain.

[0037] Figure 5B , 2After treatment with the composition of ′-FL and 3′-SL, the area of dopamine neurons in zebrafish with cognitive dysfunction model, compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with Formula 1, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with Formula 2, && p < 0.01, &&& p < 0.001; compared with Formula 3, @ p <0.05, @@ p < 0.01, @@@ p < 0.001; compared with Formula 4, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

[0038] Figure 6 、 2 After treatment with the composition of ′-FL and 3′-SL, the relative expression level of bdnf gene in zebrafish with cognitive dysfunction model, compared with the model control group, **p < 0.01, ***p < 0.001; compared with Formula 1, # p < 0.05, ## p < 0.01; compared with Formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with Formula 3, @ p < 0.05, @@ p < 0.01; compared with Formula 4, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

[0039] Figure 7 、 2 After treatment with the composition of ′-FL and 3′-SL, the relative expression level of gdnfa gene in zebrafish with cognitive dysfunction model, compared with the model control group, **p < 0.01, ***p < 0.001; compared with Formula 1, # p < 0.05, ## p <0.01, ### p < 0.001; compared with Formula 2, && p < 0.01, &&& p < 0.001; compared with Formula 3,@@ p < 0.01, @@@ p < 0.001; Compared with formulation 4, $$ p < 0.01, $$$ p < 0.001.

[0040] Figure 8A 、 2 Typical diagram of the movement trajectory of zebrafish with cognitive dysfunction model after treatment with the composition of 2′-FL and 3′-SL and other compositions, where the blue box is the blue area of the cross maze, which is the quantitative area.

[0041] Figure 8B 、 2 Percentage of blue in zebrafish with cognitive dysfunction model after treatment with the composition of 2′-FL and 3′-SL and other compositions. Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; Compared with the group with 3 raw material ratios, #p < 0.05. Detailed implementation manners

[0042] Unless otherwise specified, the meanings of the terms in this application are the same as those commonly understood by those skilled in the art. For the various experimental techniques mentioned herein, those skilled in the art can refer to various textbooks, literature, and commercial product descriptions, or they can also refer to the specific examples given in the embodiment part of this application.

[0043] In the present invention, unless otherwise specified, all ratios are weight ratios. It should be understood that the specific numerical values (such as ratios) given herein should not only be understood as individual numerical values, but should also be considered as providing the end point values of a certain range, and can be combined with each other to provide other ranges. For example, when the weight ratio of 2′-FL and 3′-SL is disclosed as (1:2.5)~(1:15), it is equivalent to disclosing that the weight ratio of the two can be 1:2.5, or 1:15, or any ratio within this range, such as 1:7.

[0044] The following details some exemplary embodiments of this application. It should be understood that these detailed descriptions are only for making those skilled in the art more clearly understand the content of this application, and are not intended to limit in any way. Those skilled in the art can make various changes and variations to the described embodiments.

[0045] After in-depth research, the present inventor found that only the composition of 2′-FL and 3′-SL has a synergistic effect on improving cognitive dysfunction when within a specific ratio range of (1:2.5) to (1:15), while the composition adding 3-FL on the basis of 2′-FL and 3′-SL and the composition not within this ratio range cannot play a good role in improving cognitive dysfunction. Therefore, the present invention provides a composition composed of 2′-FL and 3′-SL, and also provides its application in improving cognitive dysfunction.

[0046] Composition

[0047] The present invention provides a composition, which is composed of 2′-FL and 3′-SL with a mass ratio of (1:2.5) to (1:15).

[0048] As used in the present invention, "2′-FL" refers to 2'-Fucosyllactose, which is a trisaccharide compound formed by a lactose core structure in which β-D-galactose is linked to D-glucose through a 1-4 glycosidic bond and fucose is linked to the C2' hydroxyl site through an α1-2 glycosidic bond. Its chemical formula is C 18 H 32 O 15 , with a molecular weight of 488.44 g / mol and a CAS number of 41263-94-9.

[0049] As used in the present invention, "3′-SL" refers to 3'-Sialyllactose, which is a tetrasaccharide compound formed by a lactose core structure in which β-D-galactose is linked to D-glucose through a 1-4 glycosidic bond and N-acetylneuraminic acid (Neu5Ac) is linked to the C3' hydroxyl site through an α2-3 glycosidic bond. Its chemical formula is C 23 H 39 NO 19 , with a molecular weight of 633.56 g / mol and a CAS number of 35890-38-1.

[0050] The present invention has found that a composition containing only 2′-FL and 3′-SL has a synergistic effect on improving cognitive dysfunction when within a specific weight ratio range of (1:2.5) to (1:15). Therefore, the mass ratio of 2′-FL to 3′-SL can be (1:2.5) to (1:15), such as (1:2.5) to (1:12), (1:2.5) to (1:10), (1:2.5) to (1:8), (1:2.5) to (1:7), (1:2.5) to (1:6), (1:2.5) to (1:5), (1:3) to (1:10), (1:5) to (1:10), or (1:7) to (1:10). It can also be said that when 2′-FL and 3′-SL are used in combination, the amount of 3′-SL can be 2.5 to 15 parts by mass relative to 1 part by mass of 2′-FL, such as 2.5 to 14 parts by mass, 3 to 13 parts by mass, 4 to 12 parts by mass, 5 to 11 parts by mass, 6 to 10 parts by mass, 8 to 10 parts by mass, or a range defined by any two point values. When the weight ratio of 2′-FL to 3′-SL is within the above range, the effect on improving cognitive dysfunction is more significant, and there is a synergistic effect between the two.

[0051] In some embodiments, in the composition, the concentration of 2′-FL can be 45 to 1000 μg / mL, such as 45 to 1000 μg / mL, 45 to 800 μg / mL, 45 to 600 μg / mL, 45 to 500 μg / mL, 45 to 300 μg / mL, 45 to 250 μg / mL, or 45 to 220 μg / mL. In other embodiments, in the composition, the concentration of 3′-SL can be 500 to 2000 μg / mL, such as 500 to 900 μg / mL, 500 to 800 μg / mL, 500 to 750 μg / mL, 500 to 720 μg / mL, 500 to 710 μg / mL, 510 to 710 μg / mL, 520 to 710 μg / mL, or 530 to 710 μg / mL.

[0052] The present invention also provides a method for preparing the composition, the method comprising the step of mixing 2′-FL and 3′-SL.

[0053] Food

[0054] The present invention also provides a food comprising the composition.

[0055] The term "food" means an article or substance that can be ingested by an individual into their body, which includes finished food products, semi-finished food products, food additives, food supplements, and health products.

[0056] In the present invention, the term "individual" refers to any animal of interest. In some embodiments, the individual is a mammal, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, rats.

[0057] The compositions described in the present invention can be formulated into a food using standard techniques well known to those of ordinary skill in the art. For example, the composition can be directly added to a food pharmaceutically 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 pharmaceutically acceptable material.

[0058] The term "food pharmaceutically acceptable material" refers to nutritional additives that can be applied to foods (e.g., dietary fiber, prebiotics, proteins, lipids, minerals, vitamins), ingredients that are both food and medicine (e.g., red dates, hawthorn, wolfberries), excipients or adjuvants (e.g., calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol), etc.

[0059] The food of the present invention can be in any form suitable for oral administration, such as powder, tablet, capsule, granule, solution, suspension, and so on.

[0060] The food of the present invention is generally applicable to people of any age group, such as infants and young children (including infants, older infants, toddlers), children, juveniles, adolescents, young people, adults, middle-aged people, 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 "infants and young children" refers to a person aged 0 - 36 months. The term "child" refers to a person aged 3 - 6 years. The term "juvenile" refers to a person aged 7 - 17 years. The term "adult" refers to a person aged 18 years or older. The term "young people" refers to a person aged 18 - 40 years. The term "adolescents" refers to a person aged 7 - 40 years. The term "middle-aged people" refers to a person aged 41 - 65 years. The term "elderly" or "the elderly" refers to a person aged 65 years or older.

[0061] In some embodiments, the food can be infant food (e.g., infant formula, older infant formula, toddler formula), children's food, adolescent food, or adult food, such as infant formula (e.g., infant formula, toddler formula), baby food supplements, nutritional or dietary supplements, children's formula, children's snacks, pregnancy-modified formula, or middle-aged and elderly formula.

[0062] In the food, the addition amount of the composition can enable the food to improve cognitive dysfunction. In one embodiment, relative to the total mass of the food, the mass percentage of the composition can be 0.001-80%, preferably 0.01-50%, and can be, for example, 0.1-30%, 1-20%. In addition to 2′-FL and 3′-SL, the food can also contain other ingredients, such as other proteins / amino acids, carbohydrates, fats, vitamins, minerals and other pharmaceutically acceptable materials.

[0063] Drug

[0064] The present invention also provides a drug comprising the composition. The drug can also include a pharmaceutically acceptable carrier.

[0065] As used herein, "pharmaceutically acceptable carrier" refers to those carriers that have no obvious stimulating effect on the organism and do not damage the biological activity and performance of the reagents in the administered pharmaceutical composition, such as but not limited to: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent or liposome. In some embodiments, the pharmaceutically acceptable carrier can be an inert substance added to the pharmaceutical composition to further facilitate the administration of the reagent, such as calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oil, polyethylene glycol, etc.

[0066] The medicament of the present invention can be formulated into any suitable dosage form for administration by oral or other means. Dosage forms suitable for oral administration include but are not limited to: sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or fine granules, solutions, suspensions, drops, emulsions, syrups, elixirs or slurries. The medicament of the present invention can also be stored in a sterilized device suitable for injection or infusion.

[0067] The composition of 2′-FL and 3′-SL in the medicament is usually present in an effective amount (such as a therapeutically effective amount, a prophylactically effective amount). The effective amount is a dosage sufficient to improve or in some way alleviate the symptoms related to the disease, for example, a dosage that effectively improves or eliminates one or more symptoms, and can be determined according to the age, gender, physical condition, etc. of the subject. The dosage may cure the disease, but the administration is usually for improving the symptoms of the disease. Generally, repeated administration is required to achieve the desired symptom improvement.

[0068] In a specific embodiment of the present invention, zebrafish are used as experimental animals, and a dosing regimen for the composition of 2′-FL and 3′-SL to improve cognitive dysfunction is proposed. When using zebrafish as experimental animals, the concentration of 2′-FL can be 45 - 1000 μg / mL, and the concentration of 3′-SL can be 500 - 2000 μg / mL. It should be understood that it is easy for those skilled in the art to convert the dosing dose of zebrafish to the dosing dose applicable to humans. For example, the theoretical human dose can be calculated according to the formula: zebrafish (mg / L) = [human (g / day) × 1000] / 6, and further, the actual human dose can be deduced based on the multiple relationship between the theoretical human dose and the actual human dose, which is 0.1 - 4 times.

[0069] When necessary, the composition of 2′-FL and 3′-SL can also be co-administered with other active ingredients or medicaments. Exemplary active ingredients or medicaments for improving cognitive dysfunction include but are not limited to: DHA, theanine, PS, nervonic acid, imidodipeptides, etc.

[0070] Application / Method

[0071] The present invention also provides the application of the composition in the preparation of a medicament for improving cognitive dysfunction.

[0072] The present invention also provides the application of the medicament in improving cognitive dysfunction, or the application of the food in improving cognitive dysfunction for non-therapeutic purposes.

[0073] In the present invention, the "cognitive dysfunction" may be a neurological and / or cognitive dysfunction, such as deficits in cognitive abilities such as executive function, learning and memory, sensorimotor function, language, complex attention, and social cognition, learning and memory disorders, aphasia, apraxia, agnosia, and dyspraxia. In some embodiments, the cognitive dysfunction is a learning and memory dysfunction. In some specific embodiments, the learning and memory dysfunction includes: loss or reduction of the color preference ability of zebrafish, increased acetylcholinesterase activity, decreased dopamine neuron area, and decreased expression levels of genes related to cognitive function (such as bdnf, gdnfa, etc.). It should be understood that the present invention constructs a zebrafish model of cognitive dysfunction using bisphenol AF to evaluate the improvement effect of the composition described in the present invention on cognitive dysfunction, but the improvement of the composition on cognitive dysfunction is not limited to this model.

[0074] In some embodiments, the "improvement of cognitive dysfunction" is non-therapeutic and non-diagnostic.

[0075] The present invention also provides a method for improving cognitive dysfunction for non-therapeutic purposes, the method comprising: administering the composition described in the present invention to an individual in need, or administering the food or drug described in the present invention to an individual in need.

[0076] In the present invention, the "administering" may be introducing, providing, or delivering a certain substance to an individual through any suitable route to achieve its intended function.

[0077] The beneficial effects of the present invention at least include: The present invention provides a composition that can improve cognitive dysfunction, which is composed of 2′-FL and 3′-SL with a mass ratio of (1:2.5) to (1:15). In this composition, 2′-FL and 3′-SL act synergistically to significantly improve the color preference ability of zebrafish in the cognitive dysfunction model constructed using bisphenol AF, reduce acetylcholinesterase activity, increase the dopamine neuron area, and promote the expression of genes related to cognitive function, bdnf and gdnfa. This composition can be used for preventing or improving cognitive dysfunction, especially learning and memory dysfunction, and has good application prospects.

[0078] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer.

[0079] Example 1. Determination of the maximum test concentration (MTC) of 2′-FL and 3′-SL

[0080] In this example, the maximum test concentration (MTC) for the repair of cognitive impairment by 2′-FL and 3′-SL was detected respectively. The experimental method is as follows:

[0081] All zebrafish used in the experiment were raised in fish culture 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 provided by the fish culture center of Hangzhou Huante Biotechnology Co., Ltd. Wild-type AB strain zebrafish at 5 days post-fertilization (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). 2′-FL or 3′-SL was administered by water solution (the concentrations are shown in Table 1). At the same time, a normal control group (no substances were added in the normal control group, only 20 mL of fish culture water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) was administered by water solution to the remaining experimental groups to establish a zebrafish cognitive impairment model. After treatment at 28°C for 24 h, the MTC of 2′-FL and 3′-SL on the model zebrafish was measured.

[0082] Table 1. Exploration of the maximum test concentration for the repair of cognitive impairment by 2′-FL and 3′-SL (n = 30)

[0083] At the end of the experiment, there were no obvious abnormalities in the zebrafish of the model control group. When the treatment concentrations of 2′-FL were 125, 250, 500, and 1000 μg / mL respectively, there were no obvious abnormalities in the zebrafish. The state of the treatment group at a concentration of 2000 μg / mL was more severe than that of the model control group, specifically manifested as slow movement. When the treatment concentrations of 3′-SL were 125, 250, 500, 1000, and 2000 μg / mL respectively, there were no obvious abnormalities in the zebrafish.

[0084] The results showed that under the experimental conditions, the maximum test concentration (MTC) for the repair of cognitive impairment by 2′-FL was 1000 μg / mL, and the MTC for the repair of cognitive impairment by 3′-SL was 2000 μg / mL (Table 1).

[0085] Example 2. Experimental study on color cognition of 2′-FL and 3′-SL

[0086] Cognitive dysfunction is a mental health disorder that affects cognitive ability. It mainly includes six cognitive deficits, including executive function, learning and memory, sensorimotor function, language, complex attention, and social cognition. It usually causes learning and memory disorders, accompanied by aphasia, apraxia, agnosia, and apraxia. Bisphenol AF has neurodevelopmental toxicity and has an inhibitory effect on the motor behavior of zebrafish. It also damages glial cells, affects the formation of synapses and the release of neurotransmitters, and ultimately leads to a decline in learning, memory, and cognitive ability. The color preference test can be used as an experimental protocol for memory assessment, cognitive dysfunction, neurodegenerative disease assessment, toxic behavior assessment, etc. At the same time, the maze is one of the important equipment for evaluating different phenomena (such as learning and memory, anxiety, preference, etc.) in zebrafish neurological disease models. Various cognitive tasks often use color clues in the maze to study zebrafish learning and memory. Color preference tests are often used in the evaluation of cognitive impairment in zebrafish. Zebrafish prefer short-wavelength colors. Compared with other colors (such as red, yellow, and green), zebrafish show a strong preference for blue. For zebrafish with cognitive impairment, hormones and neurotransmitters in their brains change, causing the zebrafish to lose their preference for blue. Therefore, this example uses bisphenol AF to construct a zebrafish cognitive impairment model, and uses color cognition experiments to evaluate the effects of 2′-FL and 3′-SL on zebrafish cognitive function.

[0087] The zebrafish culture conditions used in the experiment were the same as those in Example 1. 5 dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). 2′-FL or 3′-SL (concentrations are shown in Table 2) was administered in water, and the positive control Ginkgo biloba cistanche tablets were 125 μg / mL (water-soluble administration, Amway (China) Daily Products Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substance was added to the normal control group, only 20 mL of fish water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, the remaining experimental groups were all administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive dysfunction model. After 24 h of treatment at 28℃, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" 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 to analyze the percentage (%) of the total movement distance of zebrafish in the blue area within 10 min to the total movement distance in the entire area. The statistical analysis results of this indicator were used to evaluate the efficacy of 2′-FL and 3′-SL in cognitive dysfunction repair. Statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0088] Table 2. Effects of 2′-FL and 3′-SL on color recognition of zebrafish with cognitive dysfunction model (n = 6)

[0089] Note: Compared with the model control group, *p < 0.05, **p < 0.01.

[0090] The results are shown in Table 2 Figure 1A and Figure 1B As shown, under the experimental conditions, 3′-SL has the efficacy of repairing cognitive dysfunction within the maximum tolerated concentration range (2000 μg / mL), and the effective concentration is 500 μg / mL, specifically manifested as an increase in the movement proportion in the blue area; within the maximum tolerated concentration range (1000 μg / mL), the efficacy of 2′-FL in repairing cognitive dysfunction is not significant.

[0091] Example 3. Study on the effects of 2′-FL and 3′-SL on acetylcholinesterase activity in zebrafish

[0092] Acetylcholinesterase (AChE) is an enzyme responsible for degrading acetylcholine (ACh). ACh is the main neurotransmitter of the cholinergic system. A decrease in ACh will lead to memory decline and cognitive dysfunction. By detecting the activity of AChE, the degradation situation of ACh in the body can be reflected. The lower the activity of AChE, the less the degradation of Ach, and the better the memory and cognitive function. Bisphenol AF has neurodevelopmental toxicity, which can damage zebrafish glial cells, affect the formation of synapses and the release of neurotransmitters, enhance the activity of AChE, and ultimately lead to a decline in learning and memory ability and cause cognitive dysfunction. Therefore, in this example, a zebrafish cognitive dysfunction model was constructed using bisphenol AF to analyze the effects of 2′-FL and 3′-SL on the acetylcholinesterase activity of zebrafish, so as to evaluate the effects of 2′-FL and 3′-SL on the cognitive function of zebrafish.

[0093] The breeding conditions of zebrafish used in the experiment were the same as those in Example 1. Wild-type AB strain zebrafish at 4 dpf were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). 2′-FL and 3′-SL were administered by water solubilization (concentrations are shown in Table 3), and the positive control was Ginkgo biloba and Cistanche deserticola tablets at a concentration of 125 μg / mL (administered by water solubilization, Amway (China) Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substances were added in the normal control group, only 20 mL of fish-raising water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, all other experimental groups were administered bisphenol AF by water solubilization (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) to establish a zebrafish cognitive dysfunction model. After treatment at 28°C for 48 h, an acetylcholinesterase assay kit (batch numbers 3320281 and 3321701, AAT Bioquest, USA) was used, and data were collected using a multifunctional microplate reader. The detection conditions were: excitation wavelength 490 nm and emission wavelength 520 nm. The fluorescence values of acetylcholinesterase (AchE) in zebrafish were analyzed, and the repair efficacy of 2′-FL and 3′-SL on cognitive dysfunction was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0094] Table 3. Effects of 2′-FL and 3′-SL on the activity of acetylcholinesterase in zebrafish with cognitive dysfunction model (n = 10)

[0095] Note: Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001

[0096] The results are shown in Table 3 and Figure 2 As shown, under the experimental conditions in this experiment, both 2′-FL and 3′-SL have the efficacy of repairing cognitive dysfunction, specifically manifested as inhibiting the activity of acetylcholinesterase, and the optimal dose concentrations are 750 μg / mL and 1500 μg / mL, respectively.

[0097] Example 4. Effects of the composition of 2′-FL and 3′-SL on color recognition in zebrafish

[0098] In this example, a zebrafish cognitive dysfunction model was constructed using bisphenol AF, and a color recognition experiment was used to evaluate the effects of the composition of 2′-FL and 3′-SL on zebrafish cognitive function.

[0099] The breeding conditions of zebrafish used in the experiment were the same as those in Example 1. Wild-type AB strain zebrafish at 5 dpf were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). A composition of 2′-FL and 3′-SL was administered in water (concentrations are shown in Table 4), and the positive control was Ginkgo biloba and Cistanche deserticola tablets at a concentration of 125 μg / mL (administered in water, Amway (China) Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substances were added in the normal control group, only 20 mL of fish-raising water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, all other experimental groups were administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive dysfunction model. After treatment at 28 °C for 24 h, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" module. The module was divided into four regions: yellow, blue, red, and green. Six modules were placed in each group, and a behavior analyzer was used to collect data. The percentage (%) of the total movement distance of zebrafish in the blue region within 10 min in the total movement distance of the entire region was analyzed, and the cognitive dysfunction repair effect of the composition of 2′-FL and 3′-SL was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0100] Table 4. Effects of the composition of 2′-FL and 3′-SL on color recognition of zebrafish with cognitive dysfunction model (n = 6)

[0101] Note: The concentrations of Formulas 1 - 9 in Table 4 are expressed as the concentration of 2′-FL + the concentration of 3′-SL, and the unit is μg / mL; compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with Formula 1, # p < 0.05; compared with Formula 2, & p < 0.05; compared with Formula 3, @ p < 0.05, @@ p < 0.01; compared with Formula 4, $ p < 0.05, $$ p <0.01.

[0102] The results are shown in Table 4, Figure 3A and Figure 3BAs shown, under the conditions of this experiment, the movement ratios (p-values compared with the model control group) of the blue regions in Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 8, and Formulation 9 were 62.3 ± 3.01% (p < 0.01), 62.4 ± 3.09% (p < 0.01), 67.9 ± 3.81% (p < 0.001), 63.0 ± 1.74% (p < 0.001), 52.1 ± 2.01% (p < 0.5), 52.9 ± 2.79% (p < 0.05), 52.2 ± 2.35% (p > 0.05), 55.7 ± 3.17% (p < 0.05), and 53.4 ± 2.59% (p < 0.05), respectively. Therefore, Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 8, and Formulation 9 all have the efficacy of repairing cognitive impairment, specifically manifested as a significant increase in the movement ratio of the blue region compared to the model group; while the efficacy of Formulation 7 in repairing cognitive impairment is not obvious.

[0103] Further comparing the differences between different formulations, the efficacy of Formulation 1 in repairing cognitive impairment is superior to that of Formulation 5, Formulation 6, Formulation 7, and Formulation 9, and there is no obvious difference from Formulation 2, Formulation 3, and Formulation 4; the efficacy of Formulation 2 in repairing cognitive impairment is superior to that of Formulation 5, Formulation 6, Formulation 7, and Formulation 9, and there is no obvious difference from Formulation 1, Formulation 3, and Formulation 4; the efficacy of Formulation 3 in repairing cognitive impairment is superior to that of Formulation 5, Formulation 6, Formulation 7, Formulation 8, and Formulation 9, and there is no obvious difference from Formulation 1, Formulation 2, and Formulation 4; the efficacy of Formulation 4 in repairing cognitive impairment is superior to that of Formulation 5, Formulation 6, Formulation 7, and Formulation 9, and there is no obvious difference from Formulation 1, Formulation 2, and Formulation 3.

[0104] Compared with the model control group, the increased values of the movement ratios of the blue regions in Formulation 1 - 4 (the total dosage of 2′-FL + 3′-SL is 750 μg / mL) were 19.4%, 19.5%, 25%, and 20.1%, respectively. In contrast, compared with the model control group, the increased value of the movement ratio of the blue region in Formulation 7 with a single use of 750 μg / mL 2′-FL was only 9.3%, and the increased value of the movement ratio of the blue region in Formulation 9 with a single use of 750 μg / mL 3′-SL was only 10.5%. The increased value of the movement ratio of the blue region in Formulation 8 with a single use of 1500 μg / mL 3′-SL was 12.8%, but this increased value of the movement ratio of the blue region was still significantly lower than that of Formulation 1 - 4. It can be seen that 2′-FL and 3′-SL in Formulation 1 - 4 have a synergistic effect in the direction of improving cognitive impairment function, and the synergistic ratio range is (1:2.5) - (1:15).

[0105] Example 5: Effect of the Combination of 2′-FL and 3′-SL on the Activity of Acetylcholinesterase in Zebrafish

[0106] The zebrafish culture conditions used in the experiment were the same as those in Example 1. 4 dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The composition of 2′-FL and 3′-SL was administered in water (concentrations are shown in Table 5), and the positive control Ginkgo biloba and Cistanche tablets were 125 μg / mL in concentration (water-soluble administration, Amway (China) Daily Products Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substance was added to the normal control group, only 20 mL of fish water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, the remaining experimental groups were all administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive dysfunction model. After treatment at 28°C for 48 h, the acetylcholinesterase assay kit (lot number 3320281, 3321701, AAT Bioquest, USA) was used to collect data using a multifunctional microplate reader to analyze the fluorescence value of acetylcholinesterase (AchE) in zebrafish. The statistical analysis results of this indicator were used to evaluate the cognitive dysfunction repair efficacy of the combination of 2′-FL and 3′-SL. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0107] Table 5. Effects of the combination of 2′-FL and 3′-SL on acetylcholinesterase activity in zebrafish with cognitive dysfunction model (n=10)

[0108] Note: The concentrations of formulas 1-8 in Table 5 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL; compared with the model control group, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ## p < 0.01, ### p <0.001; compared with formulation 2, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with formulation 3, @ p < 0.05, @@ p < 0.01.

[0109] The results are shown in Table 5 and Figure 4As shown, under the conditions of this experiment, the fluorescence values of acetylcholinesterase (p-values compared with the model control group) of Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 7, and Formulation 8 were 2104 ± 153 (p < 0.001), 2240 ± 229 (p < 0.001), 2484 ± 250 (p < 0.001), 2998 ± 336 (p < 0.001), 3291 ± 239 (p < 0.001), 3867 ± 398 (p < 0.01), 3490 ± 401 (p < 0.01), and 3362 ± 166 (p < 0.001), respectively. Therefore, Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 7, and Formulation 8 all have the efficacy of repairing cognitive dysfunction, specifically manifested as a significant decrease in the fluorescence value of acetylcholinesterase compared to the model group.

[0110] Further comparing the differences between different formulations, the efficacy of Formulation 1 in inhibiting acetylcholinesterase is better than that of Formulation 5, Formulation 6, Formulation 7, and Formulation 8, and there is no significant difference from Formulation 2 and Formulation 3; the efficacy of Formulation 2 in inhibiting acetylcholinesterase is better than that of Formulation 5, Formulation 6, Formulation 7, and Formulation 8, and there is no significant difference from Formulation 1, Formulation 3, and Formulation 4; the efficacy of Formulation 3 in inhibiting acetylcholinesterase is better than that of Formulation 5, Formulation 6, Formulation 7, and Formulation 8, and there is no significant difference from Formulation 1, Formulation 2, and Formulation 4.

[0111] Compared with the model control group, the decreased values of the fluorescence value of acetylcholinesterase for Formulation 1 - 4 (the total dosage of 2′-FL + 3′-SL is 750 μg / mL) were 3124, 2988, 2744, and 2230, respectively. In contrast, for Formulation 7 with 750 μg / mL of 2′-FL used alone, the decreased value of the fluorescence value of acetylcholinesterase compared with the model control group was only 1738, and for Formulation 8 with 1500 μg / mL of 3′-SL used alone, the decreased value of the fluorescence value of acetylcholinesterase compared with the model control group was only 1866, which was significantly lower than that of Formulation 1 - 4. Therefore, 2′-FL and 3′-SL in Formulation 1 - 4 have a synergistic effect in improving cognitive impairment, and the synergistic ratio range is (1:2.5) - (1:15).

[0112] Example 6. Effect of the composition of 2′-FL and 3′-SL on the area of dopamine neurons in zebrafish

[0113] Dopamine plays a key regulatory role in the central nervous system and is widely distributed throughout the central nervous system. Several pathways in which dopamine is involved are closely related to motor control, cognitive function, and memory. Exposure to bisphenol AF can affect the differentiation and connection formation of neurons, especially dopamine neurons. This effect may cause cognitive dysfunction in the early developmental stage. By detecting the area of dopamine neurons, the functional activity of dopamine neurons in the body, as well as the degree of activity in information processing and neurotransmission can be reflected. The larger the area of dopamine neurons, the stronger the memory and cognitive function. In this embodiment, a zebrafish cognitive dysfunction model was constructed using bisphenol AF, and the effect of the composition of 2′-FL and 3′-SL on the cognitive function of zebrafish was evaluated by detecting the area of zebrafish dopamine neurons.

[0114] The zebrafish culture conditions used in the experiment were the same as those in Example 1. 4 dpf transgenic monoamine neuron green fluorescent strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The composition of 2′-FL and 3′-SL was administered in water (concentrations are shown in Table 6), and the positive control Ginkgo biloba cistanche tablets were 125 μg / mL in concentration (water-soluble administration, Amway (China) Daily Products Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substance was added to the normal control group, only 20 mL of fish water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, the remaining experimental groups were all administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive dysfunction model. After 48 h of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a Zeiss fluorescence microscope. The data were analyzed and collected using ImageJ software. The area of zebrafish dopamine neurons was analyzed, and the statistical analysis results of this index were used to evaluate the efficacy of the combination of 2′-FL and 3′-SL in improving dopamine neuron damage. The statistical processing results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0115] Table 6. Effects of the combination of 2′-FL and 3′-SL on the dopamine neuron area in zebrafish models of cognitive dysfunction (n = 10)

[0116] Note: The concentrations of formulas 1-8 in Table 6 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL; compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ##p < 0.01, ### p < 0.001; Compared with Formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001; Compared with Formula 3, @ p < 0.05, @@ p < 0.01, @@@ p < 0.001; Compared with Formula 4, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

[0117] The results are shown in Tables 6, Figure 5A and Figure 5B as follows. Under the experimental conditions, the dopamine neuron areas (p-values compared with the model control group) of Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8 are 4902 ± 244 pixels (p < 0.001), 4976 ± 117 pixels (p < 0.001), 5183 ± 296 pixels (p < 0.001), 5039 ± 262 pixels (p < 0.001), 4017 ± 219 pixels (p < 0.05), 4198 ± 225 pixels (p < 0.01), 3672 ± 167 pixels (p > 0.05), and 3766 ± 250 pixels (p > 0.05), respectively. Therefore, Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, and Formula 6 all have the efficacy of repairing cognitive dysfunction, specifically manifested as a significant increase in the pixel value of the dopamine neuron area relative to the model group; the efficacy of Formula 7 and Formula 8 in improving dopamine neuron damage is not obvious.

[0118] Further comparing the differences between different formulas, the efficacy of Formula 1 in improving dopamine neuron damage is better than that of Formula 5, Formula 6, Formula 7, and Formula 8, and there is no obvious difference from Formula 2, Formula 3, and Formula 4; the efficacy of Formula 2 in improving dopamine neuron damage is better than that of Formula 5, Formula 6, Formula 7, and Formula 8, and there is no obvious difference from Formula 1, Formula 3, and Formula 4; the efficacy of Formula 3 in improving dopamine neuron damage is better than that of Formula 5, Formula 6, Formula 7, and Formula 8, and there is no obvious difference from Formula 1, Formula 2, and Formula 4; the efficacy of Formula 4 in improving dopamine neuron damage is better than that of Formula 5, Formula 6, Formula 7, and Formula 8, and there is no obvious difference from Formula 1, Formula 2, and Formula 3.

[0119] For Formulas 1-4 (the total dosage of 2′-FL + 3′-SL is 750 μg / mL in all cases), the increased values of the pixel area of dopamine neurons compared with the model control group were 1753, 1827, 2034, and 1890 respectively. In contrast, for Formula 7 with only 750 μg / mL of 2′-FL, the increased value of the pixel area of dopamine neurons compared with the model control group was only 523; for Formula 8 with only 1500 μg / mL of 3′-SL, the increased value of the pixel area of dopamine neurons compared with the model control group was only 617. Therefore, 2′-FL and 3′-SL in Formulas 1-4 have a synergistic effect on improving cognitive impairment function, and the synergistic ratio range is (1:2.5) - (1:15).

[0120] Example 7. Effects of the composition of 2′-FL and 3′-SL on the expression levels of genes related to zebrafish cognitive function

[0121] Bdnf and its receptor are widely expressed in the nervous system, with the highest content in the hippocampus and cortex. The specific modes of action in the central nervous system are as follows: (1) increasing synaptic plasticity, and thus affecting long-term potentiation (LTP), which is the basis for the processes 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 dopamine (DA) neurons. Gdnfa can promote the survival of different neuron subsets at different stages of the development of the central and peripheral nervous systems, support the generation of type 1 astrocytes, Schwann cells, neurons, pinealocytes, etc., and has a particularly significant effect on promoting the survival of spinal motor neurons. Therefore, the up-regulation of the expression levels of these two genes can promote the development of the central nervous system and nerves, and thus promote the formation of learning and memory and improve cognitive function, belonging to representative genes of cognitive function. In this example, the bdnf gene and the gdnfa gene were used as representative genes of cognitive function, and a zebrafish cognitive impairment model was constructed using bisphenol AF to explore the effects of the composition of 2′-FL and 3′-SL on the gene expression of zebrafish cognitive function.

[0122] The zebrafish culture conditions used in the experiment were the same as those in Example 1. 5 dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). The composition of 2′-FL and 3′-SL was administered in water (concentrations are shown in Tables 7 and 8), and the positive control Ginkgo biloba and Cistanche tablets were 125 μg / mL in concentration (water-soluble administration, Amway (China) Daily Products Co., Ltd., batch number 3313N903). At the same time, a normal control group (no substance was added to the normal control group, only 20 mL of fish water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, the remaining experimental groups were all administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China) in water to establish a zebrafish cognitive dysfunction model. Three experiments were set up in parallel. After treatment at 28℃ for 24 h, the total RNA of zebrafish in each group was extracted using a universal RNA extraction kit with pre-loaded magnetic beads (Cat. No. TL2402001643C, ONREW, China), and the concentration and purity of total RNA were determined using a UV-visible spectrophotometer. 2.00 μg of zebrafish total RNA was taken and 20.0 μL of cDNA was synthesized according to the instructions of the cDNA first-strand synthesis kit (Batch No.: 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 reference for gene expression to calculate the relative expression levels of bdnf and gdnfa genes. The statistical results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0123] (1) Effect of the combination of 2′-FL and 3′-SL on the expression of the zebrafish bdnf gene

[0124] Table 7. Effects of the combination of 2′-FL and 3′-SL on the expression of bdnf gene in zebrafish with cognitive dysfunction model (n=3)

[0125] Note: The concentrations of formulas 1-8 in Table 7 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL; compared with the model control group, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ## p < 0.01; compared with formulation 2, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with formulation 3, @p < 0.05, @@ p < 0.01; Compared with Formula 4, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

[0126] The results are shown in Table 7 and Figure 6 As shown, under the experimental conditions, the relative expression levels of the bdnf gene of Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8 (p-values compared with the model control group) were 4.89 ± 0.379 (p < 0.001), 4.94 ± 0.325 (p < 0.001), 3.08 ± 0.137 (p < 0.001), 3.57 ± 0.063 (p < 0.001), 2.28 ± 0.152 (p < 0.01), 2.65 ± 0.236 (p < 0.01), 2.18 ± 0.249 (p < 0.01), and 1.93 ± 0.118 (p < 0.01), respectively. Therefore, Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8 all have the efficacy of repairing cognitive dysfunction, specifically manifested as a significant up-regulation of the relative expression level of the bdnf gene compared with the model control group.

[0127] Further comparing the differences between different formulas, the up-regulation of the relative expression level of the bdnf gene by Formula 1 is better than that of Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8, and there is no significant difference from Formula 2; the up-regulation of the relative expression level of the bdnf gene by Formula 2 is better than that of Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8, and there is no significant difference from Formula 1; the up-regulation of the relative expression level of the bdnf gene by Formula 3 is better than that of Formula 5, Formula 7, and Formula 8, and there is no significant difference from Formula 6. The up-regulation of the relative expression level of the bdnf gene by Formula 1, Formula 2, and Formula 4 is better than that of Formula 3; the up-regulation of the relative expression level of the bdnf gene by Formula 4 is better than that of Formula 3, Formula 5, Formula 6, Formula 7, and Formula 8, and the up-regulation of the relative expression level of the bdnf gene by Formula 1 and Formula 2 is better than that of Formula 4.

[0128] For Formulas 1-4 (with the total dosage of 2′-FL + 3′-SL being 750 μg / mL in all cases), the increased values of the relative expression level of the bdnf gene compared with the model control group were 3.89, 3.94, 2.08, and 2.57, respectively. In contrast, for Formula 7 with only 750 μg / mL of 2′-FL used alone, the increased value of the relative expression level of the bdnf gene compared with the model control group was only 1.18; for Formula 8 with only 1500 μg / mL of 3′-SL used alone, the increased value of the relative expression level of the bdnf gene compared with the model control group was only 0.93. Therefore, 2′-FL and 3′-SL in Formulas 1-4 have a synergistic effect in improving cognitive impairment function, and the synergistic ratio range is (1:2.5) - (1:15).

[0129] (2)Effect of the combination of 2′-FL and 3′-SL on the expression level of the gdnfa gene in zebrafish

[0130] Table 8. Effect of the combination of 2′-FL and 3′-SL on the expression level of the gdnfa gene in zebrafish with cognitive dysfunction model (n = 3)

[0131] Note: In Table 8, the concentrations of Formulas 1-8 are expressed as the concentration of 2′-FL + the concentration of 3′-SL, and the unit is μg / mL; compared with the model control group, **p < 0.01, ***p < 0.001; compared with Formula 1, # p < 0.05, ## p < 0.01, ### p <0.001; compared with Formula 2, && p < 0.01, &&& p < 0.001; compared with Formula 3, @@ p < 0.01, @@@ p < 0.001; compared with Formula 4, $$ p < 0.01, $$$ p < 0.001.

[0132] The results are shown in Table 8 and Figure 7As shown, under the conditions of this experiment, the relative expression levels of the gdnfa gene (p-values compared with the model control group) of Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 7, and Formulation 8 were 1.97 ± 0.050 (p < 0.001), 3.11 ± 0.125 (p < 0.001), 2.11 ± 0.059 (p < 0.001), 2.51 ± 0.022 (p < 0.001), 1.31 ± 0.045 (p < 0.01), 1.55 ± 0.093 (p < 0.01), 1.00 ± 0.094 (p > 0.05), and 1.49 ± 0.080 (p < 0.01), respectively. Therefore, Formulation 1, Formulation 2, Formulation 3, Formulation 4, Formulation 5, Formulation 6, and Formulation 8 all have the efficacy of repairing cognitive impairment, specifically manifested as a significant up-regulation of the relative expression level of the gdnfa gene compared with the model group. The efficacy of Formulation 7 in up-regulating the relative expression level of the gdnfa gene was not significant.

[0133] Further comparing the differences between different formulations, the up-regulation of the relative expression level of the gdnfa gene by Formulation 1 was better than that of Formulation 5, Formulation 6, Formulation 7, and Formulation 8, and there was no significant difference from Formulation 3; the up-regulation of the relative expression level of the gdnfa gene by Formulation 2 was better than that of Formulation 1, Formulation 3, Formulation 4, Formulation 5, Formulation 6, Formulation 7, and Formulation 8; the up-regulation of the relative expression level of the gdnfa gene by Formulation 3 was better than that of Formulation 5, Formulation 6, Formulation 7, and Formulation 8, and there was no significant difference from Formulation 1. The up-regulation of the relative expression level of the gdnfa gene by Formulation 2 and Formulation 4 was better than that of Formulation 3; the up-regulation of the relative expression level of the gdnfa gene by Formulation 4 was better than that of Formulation 1, Formulation 3, Formulation 5, Formulation 6, Formulation 7, and Formulation 8.

[0134] Compared with the model control group, the increments of the relative expression level of the gdnfa gene in Formulation 1 - 4 (the total amount of 2′-FL + 3′-SL was 750 μg / mL in each) were 0.97, 2.11, 1.11, and 1.51, respectively. In contrast, compared with the model control group, the increment of the relative expression level of the gdnfa gene in Formulation 7 with 750 μg / mL of 2′-FL alone was 0, and the increment of the relative expression level of the gdnfa gene in Formulation 8 with 1500 μg / mL of 3′-SL alone was only 0.49. Therefore, 2′-FL and 3′-SL in Formulation 1 - 4 have a synergistic effect in improving cognitive impairment, and the synergistic ratio range is (1:2.5) - (1:15).

[0135] Example 8. Comparison of the composition of 2′-FL and 3′-SL with other compositions

[0136] In the research process, through repeated experiments, the inventors found that only the combination of 2′-FL and 3′-SL has a synergistic effect on improving cognitive dysfunction when within a specific ratio range of (1:2.5) to (1:15). However, the combination of 3-FL added to the basis of 2′-FL and 3′-SL, and the combinations outside this ratio range cannot play a good role in improving cognitive dysfunction.

[0137] In this example, a combination with a ratio of 2′-FL and 3′-SL of 9:0:5.89 (i.e., a 2-ingredient ratio group), and a combination with a ratio of 2′-FL, 3-FL and 3′-SL of 9:1:5.89 (i.e., a 3-ingredient ratio group) were used as controls, and a combination with a ratio of 2′-FL and 3′-SL of 1:10 (i.e., the optimal ratio group) was used as an example of the composition of the present invention to compare the differences in improving cognitive dysfunction among these three compositions.

[0138] The zebrafish used in the experiment and the breeding conditions were the same as in Example 1. Wild-type AB strain zebrafish at 5 dpf were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). The combination of 2′-FL and 3′-SL or other combinations were administered by water solution (concentrations are shown in Table 9), and the positive control was Ginkgo biloba and Cistanche deserticola tablets at a concentration of 125 μg / mL. At the same time, a normal control group (no substances were added in the normal control group, only 20 mL of fish-raising water) and a model control group were set up, and the capacity of each beaker was 20 mL. Except for the normal control group, all other experimental groups were administered bisphenol AF by water solution to establish a zebrafish cognitive dysfunction model. After treatment at 28°C for 24 h, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" module. The module was divided into four regions: yellow, blue, red, and green. Six modules were placed in each group, and a behavior analyzer was used to collect data and analyze the percentage of the total movement distance of the zebrafish in the blue region within the total movement distance of the entire region within 10 min (%). The statistical analysis results of this index were used to evaluate the efficacy of the sample in repairing cognitive dysfunction. The statistical processing results were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0139] Table 9. Effects of the combination of 2′-FL and 3′-SL and other combinations on color recognition of zebrafish with cognitive dysfunction model (n = 6)

[0140] Note: The concentration of the best formulation group in Table 9 is expressed as the concentration of 2′-FL + the concentration of 3′-SL, and the concentrations of the 2-ingredient formulation group and the 3-ingredient formulation group are expressed as the concentration of 2′-FL + the concentration of 3-FL + the concentration of 3′-SL, with the unit of μg / mL; compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the 3-ingredient formulation group, #p < 0.05.

[0141] As can be seen from Table 9, the movement percentages of the blue area in the normal control group and the model control group were 56.8 ± 4.19% and 44.0 ± 2.08% respectively. The movement percentage of the blue area in the model control group was significantly decreased, indicating that the model was successfully established. The movement percentages of the blue area (p values compared with the model control group) in the best formulation group (2′-FL:3′-SL ratio of 1:10), the 2-ingredient formulation group, and the 3-ingredient formulation group were 62.7 ± 1.78% (p < 0.001), 53.3 ± 2.88% (p < 0.05), and 52.8 ± 2.70% (p < 0.05) respectively, indicating that the best formulation group, the 2-ingredient formulation group, and the 3-ingredient formulation group all had the effect of repairing cognitive dysfunction.

[0142] Further statistics found that the p value when the best formulation group was compared with the 3-ingredient formulation group was p < 0.05, suggesting that the best formulation group had a better effect on repairing cognitive dysfunction than the 3-ingredient formulation group. When the best formulation group, the 2-ingredient formulation group, and the 3-ingredient formulation group were respectively compared with the positive control Ginkgo biloba and Cistanche deserticola tablets, only the best formulation group was superior to the positive control Ginkgo biloba and Cistanche deserticola tablets, indicating that the composition of the present invention was more excellent in improving the color preference of zebrafish with cognitive dysfunction models and had a very significant function of improving cognitive dysfunction.

[0143] When the 2-ingredient formulation group and the 3-ingredient formulation group were compared, the result showed that the p value of the comparison between the 2-ingredient formulation group and the 3-ingredient formulation group was p > 0.05, suggesting that there was no significant difference in the effect of repairing cognitive dysfunction between the 2-ingredient formulation group and the 3-ingredient formulation group. It indicated that adding 3-FL on the basis of 2′-FL and 3′-SL could not further improve the function of the composition in improving cognitive dysfunction.

[0144] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application for reference, just as if each document is cited separately for reference.

Claims

1. A composition, which is composed of 2′-FL and 3′-SL with a mass ratio of (1:2.5) to (1:15).

2. The composition according to claim 1, wherein Relative to 1 part by mass of 2′-FL, the amount of 3′-SL is 2.5 to 10 parts by mass.

3. A food, characterized in that, The food contains the composition according to claim 1 or 2.

4. The food according to claim 3, characterized in that, The food includes finished food products, semi-finished food products, food additives, and food supplements.

5. The food according to claim 3, characterized in that, The food further includes food-grade acceptable materials.

6. The food according to claim 5, characterized in that, The food-grade acceptable materials include: nutritional additives, ingredients with both food and medicinal uses, excipients, and / or auxiliary materials.

7. The food according to claim 6, characterized in that, The nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipids, minerals, and vitamins, and / or the ingredients with both food and medicinal uses include one or more of red dates, hawthorn, wolfberries, longans, lilies, poria cocos, and dried tangerine peels, and / or the excipients or auxiliary materials include one or more of calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

8. The food according to any one of claims 3-7, characterized in that, The food is infant food, children's food, juvenile food, adolescent food, young adult food, adult food, middle-aged food, or elderly food.

9. The food according to claim 8, characterized in that, The infants include babies, older infants, and toddlers.

10. The food according to claim 8, characterized in that, The food is infant formula milk powder, baby complementary food, children's formula milk powder, children's snacks, pregnant women's formulated milk powder, middle-aged and elderly milk powder, or nutritional or dietary supplements.

11. A drug, characterized in that, The drug contains an effective amount of a composition composed of 2′-FL and 3′-SL with a mass ratio of (1:2.5) to (1:15), and a pharmaceutically acceptable carrier.

12. The drug according to claim 11, characterized in that, Relative to 1 part by mass of 2′-FL, the amount of 3′-SL is 2.5 to 10 parts by mass.

13. The drug according to claim 11, characterized in that, The effective amount of 2′-FL is 45 to 1000 μg / mL, and / or the effective amount of 3′-SL is 500 to 2000 μg / mL.

14. Use of the composition according to claim 1 or 2 in the preparation of a drug for improving cognitive dysfunction.

15. Use of the drug according to any one of claims 11 - 13 in improving cognitive dysfunction, or use of the food according to any one of claims 3 - 10 in improving cognitive dysfunction for non-therapeutic purposes.

16. The application according to claim 14 or 15, characterized in that, The cognitive dysfunction is neurological and / or cognitive dysfunction.

17. The application according to claim 16, characterized in that, The cognitive dysfunction includes: executive dysfunction, learning and memory dysfunction, sensorimotor dysfunction, language disorder, complex attention disorder, social cognitive ability deficiency, aphasia, apraxia, agnosia, and dyspraxia.

18. The application according to claim 16, characterized in that, The cognitive dysfunction is learning and memory dysfunction.

19. The application according to claim 18, wherein The learning and memory dysfunction includes: absence or weakening of zebrafish color preference ability, increase in acetylcholinesterase activity, decrease in dopamine neuron area, and decrease in the expression level of genes related to cognitive function.

20. The application according to claim 19, wherein The genes related to cognitive function include bdnf, gdnfa.

21. A method for improving cognitive dysfunction for non-therapeutic purposes, characterized in that, The method includes: administering the composition according to claim 1 or 2 to an individual in need, or administering the food according to any one of claims 3 - 10 or the drug according to any one of claims 11 - 13 to an individual in need.

22. The method according to claim 21, wherein The improvement of cognitive dysfunction is as defined in any one of claims 16 - 20.

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