A composition for synergistically improving cognitive dysfunction

Abstract

The present application provides a composition for synergistically improving cognitive dysfunction. The present application provides a composition consisting of 2'-FL and 3'-SL in a mass ratio of (1:2.5) to (1:15). The present application also provides a food or a drug comprising the composition, an application of the composition in the preparation of a food or a drug for improving cognitive dysfunction, an application of the food or the drug for improving cognitive dysfunction, and a method for improving cognitive dysfunction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of improving cognitive impairment, and more particularly, the present application relates to a composition for synergistically improving cognitive impairment. BACKGROUND

[0002] With the acceleration of modern social life pace, the increase of work pressure, and the intensification of global population aging trend, the problem of cognitive impairment is increasingly prominent. Memory decline, decreased attention, cognitive decline and other phenomena not only affect the quality of life of individuals, but also may develop into mild cognitive impairment (MCI) and even neurodegenerative diseases such as 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 β-amyloid protein (Aβ) deposition, improve memory in AD model mice; by enhancing hippocampal neurogenesis, it may delay age-related cognitive decline; increase NO release, inhibit PAF, dilate cerebral blood vessels, and improve oxygen and glucose supply; up-regulate BDNF, promote synaptic plasticity, inhibit Aβ aggregation and tau protein phosphorylation, and thus protect neurons. However, the content difference of its active ingredients flavonoid glycosides and terpene lactones leads to unstable efficacy; impurity components ginkgo acid may cause sensitization and need to be strictly purified; the oral bioavailability of flavonoid glycosides is less than 10%, which needs new technologies such as nanocarriers to improve; and the combination with anticoagulants (warfarin) or antiplatelet drugs may increase the risk of bleeding, which limits its application.

[0004] Therefore, there is an urgent need in the art to explore a composition capable of significantly improving cognitive impairment. SUMMARY

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

[0006] In a first aspect, the present application provides a composition consisting of 2'-FL and 3'-SL in a mass ratio of (1:2.5)~(1:15).

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

[0008] In a second aspect, the present application provides a food product comprising the composition according to any one of the embodiments of the present application.

[0009] In one or more embodiments, the food product comprises a food product, a food product intermediate, a food additive, a food supplement.

[0010] In one or more embodiments, the food product further comprises a foodstuff-acceptable material.

[0011] In one or more embodiments, the foodstuff-acceptable material comprises a nutritional additive, a medicinal food ingredient, an excipient, and / or a vehicle.

[0012] In one or more embodiments, the nutritional additive comprises one or more of dietary fiber, prebiotic, protein, lipid substance, mineral, and vitamin.

[0013] In one or more embodiments, the medicinal food ingredient comprises one or more of red dates, hawthorn, medlar, longan, lily, poria cocos, and dried tangerine or orange peel.

[0014] In one or more embodiments, the excipient or vehicle comprises one or more of calcium carbonate, calcium phosphate, sugar, starch, cellulose derivative, gelatin, vegetable oil, and polyethylene glycol.

[0015] In one or more embodiments, the food product is an infant food product, a child food product, an adolescent food product, a young adult food product, an adult food product, a middle-aged adult food product, or an elderly adult food product.

[0016] In one or more embodiments, the infant comprises an infant, a larger infant, and a young child.

[0017] In one or more embodiments, the food product is an infant formula, an infant complementary food, a child formula, a child snack, a pregnant woman formula, a middle-aged adult formula, or a nutritional or dietary supplement.

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

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

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

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

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

[0023] In one or more embodiments, the cognitive dysfunction comprises: executive dysfunction, learning and memory dysfunction, perceptual-motor dysfunction, language impairment, complex attention impairment, social cognitive deficits, aphasia, apraxia, agnosia, apraxia.

[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 comprises: loss or reduction of zebrafish color preference ability, increase of acetylcholinesterase activity, reduction of dopamine neuron area, reduction of expression level of cognitive function related genes.

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

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

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

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

[0030] Figure 1A 、 2 Typical diagram of the movement trajectory of the cognitive dysfunction model zebrafish after treatment with 3'-FL and 3'-SL, wherein the blue square is the blue area of the cross maze, which is the quantified area.

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

[0032] Figure 2 、 2The fluorescence value of acetylcholinesterase (AchE) of the cognitive dysfunction model zebrafish after treatment of 3'-SL and 3'-SL combination, compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 3A , 2 The typical diagram of the motion track of the cognitive dysfunction model zebrafish after treatment of 3'-SL and 3'-SL combination, wherein the blue square is the blue area of the cross maze, which is the quantitative area.

[0034] Figure 3B , 2 The proportion of motion in the blue area of the cognitive dysfunction model zebrafish after treatment of 3'-SL and 3'-SL combination, 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 The fluorescence value of acetylcholinesterase (AchE) of the cognitive dysfunction model zebrafish after treatment of 3'-SL and 3'-SL combination, 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 The typical diagram of the area of dopamine neurons of the cognitive dysfunction model zebrafish after treatment of 3'-SL and 3'-SL combination, wherein the white arrow points to the dopamine neurons in the brain of zebrafish.

[0037] Figure 5B , 2After treatment of the combination of 3'-FL and 3'-SL, the area of dopamine neurons of the cognitive dysfunction model zebrafish was 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 of the combination of 3'-FL and 3'-SL, the relative expression of bdnf gene of the cognitive dysfunction model zebrafish was 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 of the combination of 3'-FL and 3'-SL, the relative expression of gdnfa gene of the cognitive dysfunction model zebrafish was 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.

[0040] Figure 8A , 2 Typical zebrafish movement trajectories of a cognitive impairment model after treatment with the combination of 3′-FL and 3′-SL and other compositions, where the blue boxes represent the blue areas of the cross maze, which are quantitative regions.

[0041] Figure 8B , 2 After treatment with the combination of ′-FL and 3′-SL and other compositions, the percentage of blue color in zebrafish in the cognitive impairment model was significantly lower than that in the control group (*p < 0.05, **p < 0.01, ***p < 0.001) and compared with the three-ingredient formulation group (#p < 0.05). Detailed Implementation

[0042] Unless otherwise stated, the terms used in this application have the same meaning as commonly understood by those skilled in the art. For the various experimental techniques mentioned herein, those skilled in the art may refer to various textbooks, literature, and commercial product specifications, as well as the specific examples given in the Embodiments section of this application.

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

[0044] The following describes some exemplary embodiments of this application in detail. It should be understood that these detailed descriptions are only intended to provide a clearer understanding of the content of this application to those skilled in the art, and are not intended to limit it in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0045] The inventors have found that only the composition of 2'-FL and 3'-SL in a specific ratio range of (1:2.5)~(1:15) has a synergistic effect on improving cognitive dysfunction, and the composition of adding 3-FL on the basis of 2'-FL and 3'-SL, and the composition not in the ratio range, cannot play a good role in improving cognitive dysfunction. Therefore, the present application provides a composition consisting of 2'-FL and 3'-SL, and also provides its application in improving cognitive dysfunction.

[0046] Composition

[0047] The present application provides a composition consisting of 2'-FL and 3'-SL in a mass ratio of (1:2.5)~(1:15).

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

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

[0050] The present application found that only the composition of 2'-FL and 3'-SL has a synergistic effect on improving cognitive dysfunction when the weight ratio is in the range of (1:2.5)~(1:15). Therefore, the mass ratio of 2'-FL and 3'-SL can be (1:2.5)~(1:15), such as (1:2.5)~(1:12), (1:2.5)~(1:10), (1:2.5)~(1:8), (1:2.5)~(1:7), (1:2.5)~(1:6), (1:2.5)~(1:5), (1:3)~(1:10), (1:5)~(1:10) or (1:7)~(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 2.5~15 mass parts, such as 2.5~14 mass parts, 3~13 mass parts, 4~12 mass parts, 5~11 mass parts, 6~10 mass parts, 8~10 mass parts, or a range defined by any two point values, relative to 1 mass part of 2'-FL. When the weight ratio of 2'-FL and 3'-SL is in 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, the concentration of 2'-FL in the composition can be 45~1000 μg / mL, such as 45~1000 μg / mL, 45~800 μg / mL, 45~600 μg / mL, 45~500 μg / mL, 45~300 μg / mL, 45~250 μg / mL or 45~220 μg / mL. In other embodiments, the concentration of 3'-SL in the composition can be 500~2000 μg / mL, such as 500~900 μg / mL, 500~800 μg / mL, 500~750 μg / mL, 500~720 μg / mL, 500~710 μg / mL, 510~710 μg / mL, 520~710 μg / mL or 530~710 μg / mL.

[0052] The present application also provides a method for preparing the composition, which comprises the step of mixing 2'-FL and 3'-SL.

[0053] Food

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

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

[0056] In the present application, the term "individual" refers to any animal of interest. In some embodiments, the individual is a mammal, such as a human, monkey, cow, sheep, horse, pig, goat, dog, cat, mouse, rat.

[0057] The composition of the present application can be formulated into a food product using standard techniques well known to those of ordinary skill in the art. For example, the composition can be added directly to a foodstuff, or it can be used to prepare an intermediate composition (e.g., a food additive or premix) suitable for subsequent addition to a foodstuff.

[0058] The term "foodstuff" refers to a nutrition additive (e.g., dietary fiber, prebiotic, protein, lipid substance, mineral, vitamin), a medicinal ingredient (e.g., red date, hawthorn, Chinese wolfberry), an excipient or adjuvant (e.g., calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols), and the like, which can be applied to a food.

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

[0060] The food product of the present application can be suitable for any age group, such as an infant (including an infant, an older infant, a young child), a child, an adolescent, a teenager, a young adult, an adult, a middle-aged adult, or an elderly adult. The term "infant" refers to a human of 0-6 months of age. The term "older infant" refers to a human of 6-12 months of age. The term "young child" refers to a human of 12-36 months of age. The term "infant" refers to a human of 0-36 months of age. The term "child" refers to a human of 3-6 years of age. The term "adolescent" refers to a human of 7-17 years of age. The term "adult" refers to a human of 18 years of age or older. The term "young adult" refers to a human of 18-40 years of age. The term "teenager" refers to a human of 7-40 years of age. The term "middle-aged adult" refers to a human of 41-65 years of age. The term "elderly adult" or "old adult" refers to a human of 65 years of age or older.

[0061] In some embodiments, the food product can be an infant food product (e.g., an infant formula, an older infant formula, a young child formula), a child food product, a teenager food product, or an adult food product, such as an infant formula (e.g., an infant formula, a young child formula), an infant complementary food, a nutritional or dietary supplement, a child formula, a child snack, a pregnant woman formula, or an elderly adult formula.

[0062] In the food, the composition can be added in an amount that enables the food to improve cognitive dysfunction. In one embodiment, the mass percentage of the composition can be 0.001-80%, preferably 0.01-50%, for example, 0.1-30%, 1-20%, relative to the total mass of the food. 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 food-acceptable materials.

[0063] pharmaceutical

[0064] The present application also provides a medicament comprising the composition. The medicament can also comprise a pharmaceutically acceptable carrier.

[0065] Herein, "pharmaceutically acceptable carrier" refers to those carriers that have no apparent stimulating effect on organisms and do not impair the biological activity and performance of the agents in the administered pharmaceutical composition, such as but not limited to: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, or liposomes. In some embodiments, the pharmaceutically acceptable carrier can be an inert substance added to the pharmaceutical composition to further facilitate the administration of the agents, such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and the like.

[0066] The pharmaceuticals of the present application can be formulated in any suitable dosage form for administration in any manner, including orally. Suitable dosage forms for oral administration include, but are not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, drops, emulsions, syrups, elixirs, or slurries. The pharmaceuticals of the present application can also be stored in sterile containers suitable for injection or infusion.

[0067] The combination of 2'-FL and 3'-SL in the pharmaceuticals is generally present in an effective amount (e.g., a therapeutically effective amount, a prophylactically effective amount). An effective amount is an amount sufficient to ameliorate or in some way reduce the symptoms associated with the disease, e.g., to effectively ameliorate or eliminate one or more symptoms of the disease, and can be determined based on the age, sex, physical condition, etc. of the subject. The amount administered can cure the disease, but administration is generally to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired amelioration of symptoms.

[0068] In specific embodiments of the present application, zebrafish are used as experimental animals, and a dosing regimen for the combination of 2'-FL and 3'-SL to improve cognitive dysfunction is proposed. When zebrafish are used 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 conversion of the dosing amount for zebrafish to a dosing amount suitable for humans is easily made by one skilled in the art, e.g., by calculating the theoretical human dose based on the formula: zebrafish (mg / L) = [human (g / day) x 1000] / 6, and further calculating the actual human dose based on the relationship between the theoretical human dose and the actual human dose being 0.1-4 times.

[0069] If necessary, the combination of 2'-FL and 3'-SL can also be administered in combination with other active ingredients or drugs. Exemplary active ingredients or drugs for improving cognitive dysfunction include, but are not limited to, DHA, theanine, PS, nervonic acid, imidazole dipeptide, etc.

[0070] Applications / Methods

[0071] The present application also provides the use of the combination in the preparation of a pharmaceutical product for improving cognitive dysfunction.

[0072] The present application also provides the use of the pharmaceuticals in improving cognitive dysfunction, or the use of the foodstuffs in improving cognitive dysfunction for non-therapeutic purposes.

[0073] In the present application, the "cognitive dysfunction" can be a dysfunction of neural and / or cognitive functions, such as a deficiency in cognitive abilities such as executive function, learning and memory, perceptual-motor function, language, complex attention, and social cognition, learning and memory impairment, aphasia, apraxia, agnosia, and apraxia. In some embodiments, the cognitive dysfunction is a learning and memory dysfunction. In some specific embodiments, the learning and memory dysfunction includes a deficiency or weakening in the color preference ability of zebrafish, an increase in acetylcholinesterase activity, a decrease in the area of dopamine neurons, and a decrease in the expression level of genes related to cognitive function (e.g., bdnf, gdnfa, etc.). It should be understood that the present application uses bisphenol AF to construct a model of cognitive dysfunction in zebrafish to evaluate the improvement of cognitive dysfunction by the composition of the present application, but the improvement of cognitive dysfunction by the composition is not limited to this model.

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

[0075] The present application also provides a method for improving cognitive dysfunction for non-therapeutic purposes, which comprises administering the composition of the present application to an individual in need thereof, or administering the food or drug of the present application to an individual in need thereof.

[0076] In the present application, the "administration" can be introducing, providing, or delivering a substance to an individual through any suitable route to achieve its intended function.

[0077] The present application has at least the following beneficial effects:

[0078] The present application provides a composition for improving cognitive dysfunction, which consists of 2'-FL and 3'-SL in a mass ratio of (1:2.5) to (1:15). In the composition, 2'-FL and 3'-SL synergistically improve the color preference ability of zebrafish in the cognitive dysfunction model constructed by bisphenol AF, reduce acetylcholinesterase activity, increase the area of dopamine neurons, and promote the expression of genes related to cognitive function such as bdnf and gdnfa. The composition can be used to prevent or improve cognitive dysfunction, especially learning and memory dysfunction, and has good application prospects.

[0079] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions or under the conditions recommended by the manufacturer.

[0080] Example 1, 2'-FL, 3'-SL maximum test concentration (MTC) determination

[0081] In this example, the maximum test concentration (MTC) of 2'-FL and 3'-SL for cognitive dysfunction repair was determined, respectively. The experimental method is as follows:

[0082] The zebrafish used in the experiment were bred in fish water at 28°C (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; the pH was 6.5-8.5; the hardness was 50-100 mg / L CaCO3), and were provided by the fish breeding center of Hangzhou Huan Te Biological Company. Randomly selected 5-day post-fertilization (5 dpf) wild-type AB strain zebrafish in beakers, 30 zebrafish were treated in each beaker (experimental group). 2'-FL or 3'-SL was dissolved in water (concentration see Table 1), and a normal control group (no additional substances were added in the normal control group, only 20 mL of fish water) and a model control group were set up, and the volume of each beaker was 20 mL. Except for the normal control group, the rest of the experimental groups were given bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Biochem Technology Co., Ltd., China) to establish a zebrafish cognitive dysfunction model. After 24 h of treatment at 28°C, the MTC of 2'-FL and 3'-SL for the model zebrafish was determined.

[0083] Table 1. Maximum test concentration (MTC) of 2'-FL and 3'-SL for cognitive dysfunction repair (n=30)

[0084]

[0085] At the end of the experiment, no obvious abnormalities were observed in the model control group zebrafish. When the treatment concentration of 2'-FL was 125, 250, 500, and 1000 μg / mL, no obvious abnormalities were observed in the zebrafish, and the 2000 μg / mL concentration treatment group was more severe than the model control group, with specific manifestations of slow movement. When the treatment concentration of 3'-SL was 125, 250, 500, 1000, and 2000 μg / mL, no obvious abnormalities were observed in the zebrafish.

[0086] The results showed that under the conditions of this experiment, the maximum test concentration (MTC) of 2'-FL for cognitive dysfunction repair was 1000 μg / mL, and the MTC of 3'-SL for cognitive dysfunction repair was 2000 μg / mL (Table 1).

[0087] Example 2, color cognition experiment of 2'-FL and 3'-SL

[0088] Cognitive impairment is a mental health disorder that affects cognitive ability, mainly with 6 kinds of cognitive ability defects of executive function, learning and memory, perceptual motor function, language, complex attention and social cognition, which usually causes learning and memory impairment, and is accompanied by aphasia, apraxia, agnosia, and other performances. Bisphenol AF has neurodevelopmental toxicity, which can inhibit the motor behavior of zebrafish, damage glial cells, affect the formation of synapses and the release of neurotransmitters, and ultimately lead to decreased learning and memory and cognitive ability. Color preference test can be used as an experimental scheme for memory evaluation, cognitive impairment evaluation, neurodegenerative disease evaluation, and toxicity behavior evaluation. At the same time, the maze is one of the important devices for evaluating different phenomena (such as learning and memory, anxiety, preference, etc.) in the zebrafish neurological disease model, and various cognitive tasks often use color cues in the maze to study the learning and memory of zebrafish. Color preference test is often used in the evaluation of cognitive impairment of zebrafish. Zebrafish prefer short-wavelength colors, and compared with other colors (such as red, yellow and green), zebrafish show a strong preference for blue. However, for zebrafish with cognitive impairment, the hormones and neurotransmitters in their brains change, causing 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 the cognitive function of zebrafish.

[0089] The zebrafish breeding conditions used in the experiment are the same as 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 (concentration see Table 2) was administered in water, and the positive control ginkgo leaf podophyllum tablet 125 μg / mL concentration (water administration, Unilever (China) Daily Goods Co., Ltd., batch number 3313N903) was also administered, and a normal control group (no additional substances were added in the normal control group, only 20 mL of water for fish breeding) and a model control group were set up, and each beaker had a capacity of 20 mL. Except for the normal control group, bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Bio-Chem Technology Co., Ltd., China) was administered in water to establish a zebrafish cognitive impairment model in the remaining experimental groups. After 24 h of treatment at 28°C, 5 zebrafish were randomly selected from each experimental group and placed in a 'cross' module, the module was divided into four regions of yellow, blue, red and green, and six modules were placed in each group. The behavior analyzer was used to collect data, and the percentage of the total movement distance of zebrafish in the blue region to the total movement distance of the entire region was calculated within 10 min. The statistical analysis results of this index were used to evaluate the cognitive impairment repair efficacy of 2'-FL and 3'-SL. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicates that the difference is statistically significant.

[0090] Table 2. Effects of 2'-FL, 3'-SL on color recognition of cognitive dysfunction model zebrafish (n = 6)

[0091]

[0092] Note: *p < 0.05, **p < 0.01 compared with the model control group.

[0093] The results are shown in Table 2, Figure 1A and Figure 1B Under the conditions of this experiment, 3'-SL has cognitive dysfunction repair efficacy in the range of the maximum tolerated concentration (2000 μg / mL), and the effective concentration is 500 μg / mL, which is specifically manifested as an increase in the movement proportion of the blue area; 2'-FL has no significant cognitive dysfunction repair efficacy in the range of the maximum tolerated concentration (1000 μg / mL).

[0094] Example 3, 2'-FL, 3'-SL on zebrafish acetylcholinesterase activity

[0095] Acetylcholinesterase (AChE) is an enzyme responsible for degrading acetylcholine (ACh), which is the main neurotransmitter of the cholinergic system, and ACh reduction can cause memory decline and cognitive dysfunction. By detecting the activity of AChE, the degradation of ACh in the body can be reflected, and the lower the AChE activity, the less ACh is degraded, 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, ultimately lead to decreased learning and memory ability, and cause cognitive dysfunction. Therefore, this example uses bisphenol AF to construct a zebrafish cognitive dysfunction model to analyze the effects of 2'-FL, 3'-SL on zebrafish acetylcholinesterase activity, in order to evaluate the effects of 2'-FL, 3'-SL on zebrafish cognitive function.

[0096] The zebrafish used in the experiment were bred under the same conditions as in Example 1. Four dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). 2'-FL and 3'-SL were administered in water (concentrations are shown in Table 3), and the positive control Ginkgo Biloba Leaf Picrohila 125 μg / mL concentration (water-soluble administration, Unilever (China) Daily Goods Co., Ltd., batch number 3313N903) was also set up. A normal control group (no additional substances were added to the normal control group, only 20 mL of water for fish breeding) and a model control group were also set up, and each beaker had a capacity of 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 in water to establish a zebrafish cognitive dysfunction model in the remaining experimental groups. After 48 h of treatment at 28°C, an acetylcholinesterase assay kit (batch numbers 3320281 and 3321701, AAT Bioquest, USA) was used to collect data using a multifunctional enzyme label meter with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The fluorescence value of acetylcholinesterase (AchE) in zebrafish was analyzed, and the statistical analysis results of this indicator were used to evaluate the cognitive dysfunction repair efficacy of 2'-FL and 3'-SL. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicates that the difference is statistically significant.

[0097] Table 3. Effect of 2'-FL and 3'-SL on acetylcholinesterase activity in cognitive dysfunction model zebrafish (n = 10)

[0098]

[0099] Note: *p < 0.05, **p < 0.01, ***p < 0.001 compared with the model control group

[0100] As shown in Table 3 and Figure 2 Under the conditions of this experiment, 2'-FL and 3'-SL both have cognitive dysfunction repair efficacy, specifically by inhibiting acetylcholinesterase activity, with optimal dose concentrations of 750 μg / mL and 1500 μg / mL, respectively.

[0101] Example 4. Effect of a combination of 2'-FL and 3'-SL on color recognition in zebrafish

[0102] In this example, bisphenol AF was used to construct a zebrafish cognitive dysfunction model, and a color recognition experiment was used to evaluate the effect of a combination of 2'-FL and 3'-SL on the cognitive function of zebrafish.

[0103] The zebrafish used in the experiment were bred under the same conditions as 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 (concentration see Table 4) was administered in water, the positive control Ginkgo Biloba Leaf Picroside Tablets 125 μg / mL concentration (water administration, Unilever (China) Daily Necessities Co., Ltd., batch number 3313N903), and a normal control group (no additional substances were added in the normal control group, only 20 mL of water for fish) and a model control group were set up, and each beaker had a capacity of 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 in water to establish a zebrafish cognitive dysfunction model in the remaining experimental groups. After 24 h of treatment at 28°C, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" module, which was divided into four areas of yellow, blue, red and green. Six modules were placed in each group, and data were collected using a behavior analyzer to analyze the percentage (%) of the total movement distance of zebrafish in the blue area to the total movement distance of the entire area within 10 min. The statistical analysis results of this index were used to evaluate the cognitive dysfunction repair efficacy of the composition of 2'-FL and 3'-SL. 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.

[0104] Table 4. Effect of the composition of 2'-FL and 3'-SL on color cognition of cognitive dysfunction model zebrafish (n = 6)

[0105]

[0106] Note: The concentrations of Formulas 1-9 in Table 4 are expressed in the form of 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; 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.

[0107] The results are shown in Table 4, Figure 3A and Figure 3BAs shown, under the experimental conditions, the motion proportion of the blue area of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 8 and formula 9 (p value compared with the model control group) 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, so formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 8 and formula 9 all have cognitive dysfunction repair effect, and the motion proportion of the blue area is significantly increased compared with the model group; and the cognitive dysfunction repair effect of formula 7 is not obvious.

[0108] Further comparison of the differences between different formulas, the cognitive dysfunction repair effect of formula 1 is better than that of formula 5, formula 6, formula 7 and formula 9, and there is no obvious difference with formula 2, formula 3 and formula 4; the cognitive dysfunction repair effect of formula 2 is better than that of formula 5, formula 6, formula 7 and formula 9, and there is no obvious difference with formula 1, formula 3 and formula 4; the cognitive dysfunction repair effect of formula 3 is better than that of formula 5, formula 6, formula 7, formula 8 and formula 9, and there is no obvious difference with formula 1, formula 2 and formula 4; the cognitive dysfunction repair effect of formula 4 is better than that of formula 5, formula 6, formula 7 and formula 9, and there is no obvious difference with formula 1, formula 2 and formula 3.

[0109] Compared with the model control group, the motion proportion of the blue area of formula 1-4 (the total amount of 2'-FL and 3'-SL is 750 μg / mL) increased by 19.4%, 19.5%, 25% and 20.1% respectively. In contrast, the motion proportion of the blue area of formula 7 using 750 μg / mL 2'-FL alone increased by only 9.3% compared with the model control group, the motion proportion of the blue area of formula 9 using 750 μg / mL 3'-SL alone increased by only 10.5% compared with the model control group, and the motion proportion of the blue area of formula 8 using 1500 μg / mL 3'-SL alone increased by 12.8% compared with the model control group, but the motion proportion of the blue area was still significantly lower than that of formula 1-4. It can be seen that 2'-FL and 3'-SL in formula 1-4 have a synergistic effect in improving cognitive dysfunction, and the synergistic ratio range is (1:2.5)~(1:15).

[0110] Example 5, Effect of the combination of 2’-FL and 3’-SL on acetylcholinesterase activity of zebrafish

[0111] The zebrafish breeding conditions used in the experiment were the same as in Example 1. 30 wild-type AB strain zebrafish at 4 dpf were randomly selected in a beaker, and each beaker (experimental group) was treated with 30 zebrafish. The combination of 2’-FL and 3’-SL was administered in water (concentration see Table 5), and the positive control Ginkgo Biloba Leaf Picroside Tablets 125 μg / mL concentration (water administration, Unilever (China) Daily Necessities Co., Ltd., batch number 3313N903) was also set up. A normal control group (no additional substances were added in the normal control group, only 20 mL of water for fish) and a model control group were also set up, and each beaker had a capacity of 20 mL. Except for the normal control group, the rest of the 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 48 h of treatment at 28°C, an acetylcholinesterase assay kit (batch numbers 3320281, 3321701, AAT Bioquest, USA) was used to collect data using a multifunctional enzyme marker, and the fluorescence value of acetylcholinesterase (AchE) in zebrafish was analyzed. 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. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0112] Table 5. Effect of the combination of 2’-FL and 3’-SL on acetylcholinesterase activity of cognitive dysfunction model zebrafish (n = 10)

[0113]

[0114] Note: The concentrations of Formulas 1-8 in Table 5 are expressed in the form of 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 Formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001; compared with Formula 3, @ p < 0.05, @@ p < 0.01.

[0115] The results are shown in Table 5 and Figure 4As shown, under the experimental conditions, the acetylcholinesterase fluorescence values (p value compared with the model control group) of Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 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, so Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, and Formula 8 all had cognitive dysfunction repair efficacy, specifically manifested as a significant decrease in acetylcholinesterase fluorescence values relative to the model group.

[0116] Further comparison of the differences between different formulas showed that Formula 1 had better acetylcholinesterase inhibition efficacy than Formula 5, Formula 6, Formula 7, and Formula 8, and had no significant difference with Formula 2 and Formula 3; Formula 2 had better acetylcholinesterase inhibition efficacy than Formula 5, Formula 6, Formula 7, and Formula 8, and had no significant difference with Formula 1, Formula 3, and Formula 4; Formula 3 had better acetylcholinesterase inhibition efficacy than Formula 5, Formula 6, Formula 7, and Formula 8, and had no significant difference with Formula 1, Formula 2, and Formula 4.

[0117] Compared with the model control group, the decrease in acetylcholinesterase fluorescence values of Formula 1-4 (2'-FL + 3'-SL total dosage was 750 μg / mL) was 3124, 2988, 2744, and 2230, respectively. In comparison, Formula 7 using 750 μg / mL 2'-FL alone had a decrease in acetylcholinesterase fluorescence value of only 1738 compared with the model control group, and Formula 8 using 1500 μg / mL 3'-SL alone had a decrease in acetylcholinesterase fluorescence value of only 1866 compared with the model control group, which was significantly lower than Formula 1-4. Therefore, 2'-FL and 3'-SL in Formula 1-4 had a synergistic effect in improving cognitive dysfunction, and the synergistic ratio range was (1:2.5)~(1:15).

[0118] Example 6, Effect of the combination of 2'-FL and 3'-SL on the area of dopamine neurons in zebrafish

[0119] Dopamine plays a key regulatory role in the central nervous system and is widely distributed throughout the central nervous system. Several pathways involving dopamine 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 can cause cognitive dysfunction at an early developmental stage. By detecting the area of dopamine neurons, the functional activity of dopamine neurons in the body, as well as the level 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 is constructed using bisphenol AF, and the effect of the combination of 2'-FL and 3'-SL on the cognitive function of zebrafish is evaluated by detecting the area of dopamine neurons in zebrafish.

[0120] The zebrafish used in the experiment were bred under the same conditions as in Example 1. 30 zebrafish of the 4 dpf transgenic monoaminergic neuron green fluorescent strain were randomly selected in a beaker. The combination of 2'-FL and 3'-SL was administered in water (concentration see Table 6), and the positive control ginkgo leaf icariin tablets 125 μg / mL concentration (water administration, Anhui (China) Daily Necessities Co., Ltd., batch number 3313N903) was also administered. A normal control group (no additional substances were added in the normal control group, only 20 mL of water for fish breeding) and a model control group were also set up, and each beaker had a capacity of 20 mL. Except for the normal control group, the rest of the experimental groups were administered bisphenol AF (1 μg / mL; batch number J1925142, Shanghai Aladdin Bio-Chem 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 placed under a Zeiss fluorescence microscope for photography. ImageJ software was used to analyze and collect data, and the area of dopamine neurons in zebrafish was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of the combination of 2'-FL and 3'-SL in improving dopamine neuron damage. The statistical processing results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicates that the difference is statistically significant.

[0121] Table 6. Effect of the combination of 2'-FL and 3'-SL on the area of dopamine neurons in cognitive dysfunction model zebrafish (n = 10)

[0122]

[0123] Note: The concentrations of Formulas 1-8 in Table 6 are expressed in the form of 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.

[0124] As shown in Table 6, Figure 5A and Figure 5B Under the experimental conditions, the dopamine neuron area (p value compared with the model control group) of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7 and formula 8 was 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, so formula 1, formula 2, formula 3 and formula 4 all had cognitive dysfunction repair efficacy, which was specifically manifested in that the pixel value of the dopamine neuron area was significantly increased relative to the model group; formula 7 and formula 8 had no obvious effect on improving dopamine neuron damage.

[0125] Further comparison of the differences between different formulas showed that formula 1 was more superior to formula 5, formula 6, formula 7 and formula 8 in improving dopamine neuron damage, and had no obvious difference with formula 2, formula 3 and formula 4; formula 2 was more superior to formula 5, formula 6, formula 7 and formula 8 in improving dopamine neuron damage, and had no obvious difference with formula 1, formula 3 and formula 4; formula 3 was more superior to formula 5, formula 6, formula 7 and formula 8 in improving dopamine neuron damage, and had no obvious difference with formula 1, formula 2 and formula 4; formula 4 was more superior to formula 5, formula 6, formula 7 and formula 8 in improving dopamine neuron damage, and had no obvious difference with formula 1, formula 2 and formula 3.

[0126] Compared to the model control group, formulations 1-4 (each with a total dosage of 750 μg / mL of 2′-FL and 3′-SL) showed increases in dopamine neuron area pixels of 1753, 1827, 2034, and 1890, respectively. In contrast, formulation 7, using 750 μg / mL of 2′-FL alone, showed an increase in dopamine neuron area pixels of only 523 compared to the model control group; and formulation 8, using 1500 μg / mL of 3′-SL alone, showed an increase in dopamine neuron area pixels of only 617 compared to the model control group. Therefore, 2′-FL and 3′-SL in formulations 1-4 have a synergistic effect in improving cognitive impairment, with a synergistic ratio ranging from (1:2.5) to (1:15).

[0127] Example 7: Effects of the combination of 2′-FL and 3′-SL on the expression levels of genes related to cognitive function in zebrafish.

[0128] bdnf and its receptors are widely expressed in the nervous system, with the highest content in the hippocampus and cortex. Their 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 for learning and memory formation (secondary memory); (2) promoting neurogenesis, especially in the hippocampus; and (3) promoting cell survival, mainly manifested in maintaining and promoting the development, differentiation, growth, and regeneration of various neurons, especially serotonergic (5-HT) and dopaminergic (DA) neurons. gdnfa, on the other hand, promotes the survival of different neuronal subsets at different stages of development in both the central and peripheral nervous systems, supporting the generation of type I astrocytes, neuronal membrane cells, neurons, pineal gland cells, etc., with a particularly significant effect on the survival of spinal motor neurons. Therefore, upregulation of the expression levels of these two genes can promote the development of the central nervous system and nerves, thereby promoting learning and memory formation, and enhancing cognitive function; they are representative genes of cognitive function. In this embodiment, the bdnf and gdnfa genes were used as representative genes of cognitive function. A zebrafish cognitive dysfunction model was constructed using bisphenol AF to investigate the effect of the combination of 2′-FL and 3′-SL on the expression of cognitive function genes in zebrafish.

[0129] The zebrafish used in the experiment were bred under the same conditions as 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 (the concentration is shown in Table 7 and Table 8), the positive control Ginkgo Biloba Leaf Picrohila 125 μg / mL concentration (water administration, Unilever (China) Daily Necessities Co., Ltd., batch number 3313N903), and a normal control group (no additional substances were added in the normal control group, only 20 mL of water for fish) and a model control group were set up, and each beaker had a capacity of 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 in water to establish a zebrafish cognitive dysfunction model in the remaining experimental groups. Three parallel experiments were set up. After 24 h of treatment at 28°C, total RNA was extracted from each group of zebrafish using a pre-loaded magnetic bead method universal RNA extraction kit (item number TL2402001643C, ONREW, China), and the concentration and purity of the total RNA were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish was used to synthesize 20.0 μL of cDNA according to the instructions of the cDNA first strand synthesis kit (batch number: H9305270, Yixing Biotechnology Co., Ltd. (Shanghai) Co., Ltd., China), and the expression of bdnf and gdnfa genes was detected by q-PCR. β-actin was used as an internal reference for gene expression, and the relative expression of bdnf and gdnfa genes was calculated. The results were statistically processed and expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0130] (1) Effect of the composition of 2'-FL and 3'-SL on the expression of bdnf gene in zebrafish

[0131] Table 7. Effect of the composition of 2'-FL and 3'-SL on the expression of bdnf gene in cognitive dysfunction model zebrafish (n = 3)

[0132]

[0133] Note: The concentrations of Formulas 1-8 in Table 7 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; 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.

[0134] As shown in Table 7 and Figure 6 Under the conditions of the present experiment, the relative expression amounts of bdnf gene (p value compared with the model control group) of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7, and formula 8 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, so formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7, and formula 8 all had cognitive dysfunction repair efficacy, specifically manifested in that the relative expression amount of bdnf gene was significantly up-regulated relative to the model control group.

[0135] Further comparison of the differences between different formulas showed that formula 1 was more superior than formula 3, formula 4, formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of bdnf gene, and had no obvious difference with formula 2; formula 2 was more superior than formula 3, formula 4, formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of bdnf gene, and had no obvious difference with formula 1; formula 3 was more superior than formula 5, formula 7, and formula 8 in up-regulating the relative expression amount of bdnf gene, and had no obvious difference with formula 6, and formula 1, formula 2, and formula 4 were superior to formula 3 in up-regulating the relative expression amount of bdnf gene; formula 4 was more superior than formula 3, formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of bdnf gene, and formula 1 and formula 2 were superior to formula 4 in up-regulating the relative expression amount of bdnf gene.

[0136] The increase in the relative expression of bdnf gene of Formulas 1-4 (total amount of 2'-FL + 3'-SL is 750 μg / mL) compared with the model control group was 3.89, 3.94, 2.08 and 2.57, respectively. In comparison, the increase in the relative expression of bdnf gene of Formula 7 using 750 μg / mL 2'-FL alone compared with the model control group was only 1.18; the increase in the relative expression of bdnf gene of Formula 8 using 1500 μg / mL 3'-SL alone 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 function, and the synergistic ratio range is (1:2.5)~(1:15).

[0137] (2) Effect of the combination of 2'-FL and 3'-SL on the expression of gdnfa gene of zebrafish

[0138] Table 8. Effect of the combination of 2'-FL and 3'-SL on the expression of gdnfa gene of cognitive dysfunction model zebrafish (n=3)

[0139]

[0140] Note: The concentration of Formulas 1-8 in Table 8 is expressed in the form of 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 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.

[0141] The results are shown in Table 8 and Figure 7As shown, under the experimental conditions, the relative expression amounts of gdnfa gene (p value compared with the model control group) of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7, and formula 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, so formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, and formula 8 all had cognitive dysfunction repair efficacy, specifically manifested as significant up-regulation of the relative expression amount of gdnfa gene relative to the model group. Formula 7 did not have significant up-regulation of the relative expression amount of gdnfa gene.

[0142] Further comparison of the differences between different formulas showed that formula 1 was more superior than formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of gdnfa gene, and had no significant difference with formula 3; formula 2 was more superior than formula 1, formula 3, formula 4, formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of gdnfa gene; formula 3 was more superior than formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of gdnfa gene, and had no significant difference with formula 1, and formula 2 and formula 4 were superior to formula 3 in up-regulating the relative expression amount of gdnfa gene; formula 4 was more superior than formula 1, formula 3, formula 5, formula 6, formula 7, and formula 8 in up-regulating the relative expression amount of gdnfa gene.

[0143] Compared with the model control group, the increase in the relative expression amount of gdnfa gene of formula 1-4 (total amount of 2'-FL+3'-SL was 750 μg / mL) was 0.97, 2.11, 1.11, and 1.51, respectively. In comparison, the increase in the relative expression amount of gdnfa gene of formula 7 using 750 μg / mL of 2'-FL alone was 0, and the increase in the relative expression amount of gdnfa gene of formula 8 using 1500 μg / mL of 3'-SL alone was only 0.49. Therefore, 2'-FL and 3'-SL in formula 1-4 have a synergistic effect in improving cognitive dysfunction, and the synergistic ratio range is (1:2.5)~(1:15).

[0144] Example 8, Comparison of the combination of 2'-FL and 3'-SL with other compositions

[0145] During the research, the inventors found through repeated experiments that only the composition of 2'-FL and 3'-SL has a synergistic effect on improving cognitive dysfunction in a specific ratio range of (1:2.5)~(1:15), while the composition of adding 3-FL on the basis of 2'-FL and 3'-SL and the composition not in the ratio range cannot well improve cognitive dysfunction.

[0146] In this embodiment, the composition with the ratio of 2'-FL and 3'-SL being 9:0:5.89 (i.e., 2-raw material ratio groups) and the composition with the ratio of 2'-FL, 3-FL and 3'-SL being 9:1:5.89 (i.e., 3-raw material ratio groups) are taken as the contrast, and the composition with the ratio of 2'-FL and 3'-SL being 1:10 (i.e., the best ratio group) is taken as the example of the composition of the application, to compare the differences of the three compositions in improving cognitive dysfunction.

[0147] The zebrafish used in the experiment and the breeding conditions are the same as 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 or other compositions (concentration see Table 9) was water-solubilized, and the positive control ginkgo leaf and cistanche tablet was 125 μg / mL in concentration, and a normal control group (no any substance was added in the normal control group, only 20 mL of water for fish) and a model control group were set up, and each beaker had a capacity of 20 mL. Except for the normal control group, the cognitive dysfunction model of zebrafish was established by water-solubilizing bisphenol AF in the rest of the experimental groups. After 24 h of treatment at 28℃, 5 zebrafish were randomly selected from each experimental group and placed in a "cross" module, and the module was divided into yellow, blue, red and green four areas, and 6 modules were placed in each group. The behavior analyzer was used to collect data, and the percentage (%) of the total movement distance of zebrafish in the blue area to the total movement distance of the whole area within 10 min was analyzed, and the statistical analysis results of the index were used to evaluate the cognitive dysfunction repair efficiency of the sample. The statistical processing results are expressed by mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicates that the difference is statistically significant.

[0148] Table 9. Effect of the composition of 2'-FL and 3'-SL and other compositions on color cognition of cognitive dysfunction model zebrafish (n=6)

[0149]

[0150] Note: The concentration of the best ratio group in Table 9 is expressed in the form of 2'-FL concentration + 3'-SL concentration, and the concentration of the 2-ingredient ratio group and the 3-ingredient ratio group is expressed in the form of 2'-FL concentration + 3-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 the 3-ingredient ratio group, #p < 0.05.

[0151] As can be seen from Table 9, the motion proportion of the blue area of the normal control group and the model control group was 56.8 ± 4.19% and 44.0 ± 2.08%, respectively, and the motion proportion of the blue area of the model control group was significantly reduced, indicating that the modeling was successful. The motion proportion of the blue area of the best ratio group (2'-FL:3'-SL ratio was 1:10), the 2-ingredient ratio group and the 3-ingredient ratio group (p value compared with the model control group) was 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 ratio group, the 2-ingredient ratio group and the 3-ingredient ratio group all had the effect of repairing cognitive dysfunction.

[0152] Further statistics found that the p value of the best ratio group compared with the 3-ingredient ratio group was p < 0.05, indicating that the effect of repairing cognitive dysfunction of the best ratio group was better than that of the 3-ingredient ratio group. The best ratio group, the 2-ingredient ratio group and the 3-ingredient ratio group were compared with the positive control ginkgo leaf and cistanche tablet, and only the best ratio group was better than the positive control ginkgo leaf and cistanche tablet, indicating that the composition of the application was more excellent in improving the color preference of the cognitive dysfunction model zebrafish and had a very significant function of improving cognitive dysfunction.

[0153] The 2-ingredient ratio group and the 3-ingredient ratio group were compared, and the results showed that the p value of the 2-ingredient ratio group compared with the 3-ingredient ratio group was p > 0.05, indicating that there was no statistical difference in the effect of repairing cognitive dysfunction between the 2-ingredient ratio group and the 3-ingredient ratio group. It was found that the addition of 3-FL based on 2'-FL and 3'-SL could not further improve the function of the composition in improving cognitive dysfunction.

[0154] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a separate reference.

Claims

1. A composition comprising 2′-FL and 3′-SL in a mass ratio of (1:2.5) to (1:15), wherein the composition is used to prepare a medicament for improving cognitive impairment.

2. The composition according to claim 1, characterized in that, The amount of 3′-SL is 2.5 to 10 parts by mass relative to 1 part by mass of 2′-FL.

3. A composition comprising 2′-FL and 3′-SL in a mass ratio of (1:2.5) to (1:15), wherein the composition is used for non-therapeutic purposes to improve learning and memory function.

4. The composition according to claim 3, characterized in that, The amount of 3′-SL is 2.5 to 10 parts by mass relative to 1 part by mass of 2′-FL.

5. A food product, characterized in that, The food product comprises the composition according to claim 3 or 4.

6. The food product as described in claim 5, characterized in that, The food products include finished food products, semi-finished food products, food additives, and food supplements.

7. The food product as described in claim 5, characterized in that, The food also includes food science acceptable materials.

8. The food product as described in claim 7, characterized in that, Food science acceptable materials include: nutritional additives, food and medicine homologous ingredients, excipients and / or auxiliary materials.

9. The food product as described in claim 8, characterized in that, The nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals and vitamins, and / or the medicinal and edible ingredients include one or more of jujube, hawthorn, wolfberry, longan, lily, poria and dried tangerine peel, and / or the excipients or excipients include one or more of calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives, gelatin, vegetable oil and polyethylene glycol.

10. The food product according to any one of claims 5-9, characterized in that, The food in question is infant food, children's food, adolescent food, or adult food.

11. The food product as described in claim 10, characterized in that, The adult food products include food for young people, food for middle-aged people, and food for the elderly.

12. The food product as described in claim 10, characterized in that, The infants and young children include infants aged 0-6 months, older infants aged 6-12 months, and toddlers aged 12-36 months.

13. The food product according to any one of claims 5-9, characterized in that, The food in question is for teenagers.

14. The food product as described in claim 10, characterized in that, The food products mentioned are infant formula, baby food, children's formula, children's snacks, formula milk powder for pregnant women, milk powder for middle-aged and elderly people, or nutritional or dietary supplements.

15. A drug, characterized in that, The drug comprises an effective amount of a composition consisting of 2′-FL and 3′-SL in a mass ratio of (1:2.5) to (1:15), and a pharmaceutically acceptable carrier, wherein the composition is used to prepare a medicament for improving cognitive impairment.

16. The medicament as claimed in claim 15, characterized in that, The amount of 3′-SL is 2.5 to 10 parts by mass relative to 1 part by mass of 2′-FL.

17. The medicament as claimed in claim 15, characterized in that, The effective amount of 2′-FL is 45~1000 μg / mL, and / or the effective amount of 3′-SL is 500~2000 μg / mL.

18. Use of the composition of claim 1 or 2 in the preparation of a medicament for improving cognitive impairment.

19. The use of the medicament of any one of claims 15-17 in the preparation of a medicament for improving cognitive impairment, or the use of the food of any one of claims 5-14 in the non-therapeutic purpose of improving learning and memory function.

20. The application as described in claim 18 or 19, characterized in that, The cognitive impairment mentioned above is a neurological and / or cognitive dysfunction.

21. The application as described in claim 20, characterized in that, The cognitive impairments mentioned include learning and memory impairments.

22. The application as described in claim 21, characterized in that, The learning and memory impairments include: loss or weakening of zebrafish color preference ability, increased acetylcholinesterase activity, reduced dopamine neuron area, and decreased expression levels of cognitive function-related genes.

23. The application as described in claim 19, characterized in that, The improvements in learning and memory function include: improving zebrafish color preference ability, inhibiting acetylcholinesterase activity, increasing the area of ​​dopamine neurons, and increasing the expression level of cognitive function-related genes.

24. The application as described in claim 22 or 23, characterized in that, The cognitive function-related genes include bdnf、 gdnfa.

25. A method for improving learning and memory function for non-therapeutic purposes, characterized in that, The method includes: giving the composition of claim 3 or 4 to an individual in need, or giving the food of any one of claims 5-14 to an individual in need.

26. The method as described in claim 25, characterized in that, The improvement in learning and memory function is as defined in claim 23 or 24.

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