A composition for synergistically promoting brain development

Abstract

The present invention provides a composition for synergistically promoting brain development. The present invention provides a composition comprising 2′-FL and 3′-SL in a mass ratio of (2.5:1) to (15:1). The present invention also provides food and medicine comprising the composition, use of the composition in preparing food or medicine for promoting brain development, use of the food or medicine for promoting brain development, and methods for promoting brain development.

Description

Technical Field

[0001] The present invention belongs to the field of brain development, and more specifically, the present invention relates to a composition for synergistically promoting brain development. Background Art

[0002] Brain development, especially the development of the nervous system, is crucial for the growth of infants, children, and adolescents. Incomplete brain development may lead to a variety of symptoms such as attention deficit hyperactivity disorder, anxiety disorders, and learning and memory difficulties.

[0003] Early studies have shown that Ginkgo biloba extract (GBE), containing 24% flavonoids and 6% terpene lactones, can enhance long-term potentiation by regulating the BDNF-TrkB signaling pathway in the hippocampus. Cistanche deserticola polysaccharides promote oligodendrocyte differentiation by activating the mTOR pathway. Ginkgo biloba and Cistanche deserticola tablets, developed based on a combination of Ginkgo biloba extract and Cistanche deserticola polysaccharides, have been shown to enhance memory, replenish brain nutrients, and boost brain cell vitality, potentially promoting brain development. However, studies have found that the effects of Ginkgo biloba and Cistanche deserticola tablets on brain development remain limited, with no significant effect in healthy individuals. The extraction process for Ginkgo biloba leaves may contain residual ginkgolic acid (an allergen), posing a risk of allergy. Furthermore, the long growth cycle of the ginkgo tree (leaves over 10 years old are useful) limits the availability of high-quality raw materials, resulting in a high price. These issues have limited the application of Ginkgo biloba extract.

[0004] Therefore, there is an urgent need in the art to explore a composition that can significantly promote brain development. Summary of the Invention

[0005] The object of the present invention is to provide a composition for synergistically promoting brain development.

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

[0007] In one or more embodiments, the amount of 2′-FL 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, 7-9 parts by mass, and more preferably 7-8 parts by mass, relative to 1 part by mass of 3′-SL.

[0008] The second aspect of the present invention provides a food comprising 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 product further comprises a food-sustainable material.

[0011] In one or more embodiments, the food-acceptable materials include nutritional additives, food-drug ingredients, 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 medicinal and edible ingredients include one or more of red dates, hawthorn, wolfberry, longan, lily, poria cocos, and tangerine peel.

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

[0015] In one or more embodiments, the food is an infant food, a children's food, a juvenile food, a teenager's food, a young adult's food, an adult's food, a middle-aged food, or a senior citizen's food.

[0016] In one or more embodiments, the infant includes infants, older infants, and toddlers.

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

[0018] The third aspect of the present invention provides a medicine comprising an effective amount of the composition described in any embodiment 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 500-1000 μg / mL, and / or the effective amount of 3′-SL is 45-2000 μg / mL.

[0020] The fourth aspect of the present invention provides use of the composition according to any embodiment of the present invention in the preparation of a medicine for promoting brain development.

[0021] The fifth aspect of the present invention provides the use of the drug described in any embodiment of the present invention in promoting brain development, or the use of the food described in any embodiment of the present invention in promoting brain development for non-therapeutic purposes.

[0022] In one or more embodiments, the promoting brain development is improving brain, neural and / or cognitive development.

[0023] In one or more embodiments, the promoting brain development includes preventing or improving brain development delay, nervous system development delay and / or cognitive development delay.

[0024] In one or more embodiments, the promoting brain development includes improving cognitive maturity and promoting the expression of genes related to brain development.

[0025] In one or more embodiments, the brain development-related genes include bdnf and gdnfa.

[0026] In a sixth aspect, the present invention provides a method for promoting brain development for non-therapeutic purposes, the method comprising: administering the composition described in any embodiment of the present invention to an individual in need, or administering the food described in any embodiment of the present invention or the medicine described in any embodiment of the present invention to an individual in need.

[0027] In one or more embodiments, the promoting brain development is as defined in any embodiment of the present invention.

[0028] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A Typical diagram of the movement trajectory of zebrafish after treatment with 2′-FL and 3′-SL, where the blue box is the blue area of ​​the plus maze and the quantitative area.

[0030] Figure 1B The movement ratio of the blue area of ​​zebrafish after treatment with , 2′-FL, and 3′-SL was compared with that of the normal control group, *p < 0.05, **p < 0.01.

[0031] Figure 2A A typical diagram of the movement trajectory of zebrafish after treatment with a combination of 2′-FL and 3′-SL, where the blue box is the blue area of ​​the plus maze and the quantitative area.

[0032] Figure 2B After treatment with the combination of 2′-FL and 3′-SL, the movement ratio of the blue area of ​​zebrafish was significantly decreased compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ## p < 0.01; compared with formula 2, && p < 0.01; compared with formula 3, @ p < 0.05, @@ p < 0.01; compared with formula 4,$ p < 0.05.

[0033] Figure 3 After treatment with the combination of 2′-FL and 3′-SL, the relative expression of bdnf gene in zebrafish was significantly decreased compared with the normal control group, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ### 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 < 0.001.

[0034] Figure 4 After treatment with the combination of 2′-FL and 3′-SL, the relative expression of gdnfa gene in zebrafish was significantly decreased compared with the normal control group, **p < 0.01, ***p < 0.001; compared with formula 1, # p < 0.05, ## p < 0.01; 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.

[0035] Figure 5A Typical diagrams of zebrafish movement trajectories after treatment with a combination of 2′-FL and 3′-SL and other combinations, where the blue box represents the blue area of ​​the plus maze and the quantitative area.

[0036] Figure 5B After treatment with the combination of 2′-FL, 3′-SL and other combinations, the movement ratio of the blue area of ​​zebrafish was compared with the normal control group, ***p < 0.001; compared with the three raw material ratio groups, # p < 0.05. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the meanings of the terms in this application are the same as those generally 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, and can also refer to the specific examples given in the Examples section of this application.

[0038] In the present invention, unless otherwise specified, all ratios are weight ratios. It should be understood that the specific numerical values ​​(e.g., ratios) given herein are not only to be understood as individual numerical values, but should also be considered to provide endpoints of a range, and can be combined with each other to provide other ranges. For example, when the weight ratio of 2′-FL to 3′-SL is disclosed as (2.5:1) to (15:1), it is equivalent to disclosing that the weight ratio of the two can be 2.5:1, 15:1, or any ratio within this range, such as 7:1.

[0039] Some exemplary embodiments of the present application are described in detail below. It should be understood that these detailed descriptions are intended only to enable those skilled in the art to more clearly understand the contents of the present application and are not intended to limit the present application in any respect. Those skilled in the art will be able to make various modifications and variations to the embodiments described.

[0040] After in-depth research, the inventors discovered that only a combination of 2′-FL and 3′-SL within a specific ratio range of (2.5:1) to (15:1) exhibits a synergistic effect on promoting brain development. Compositions containing 2′-FL and 3′-SL supplemented with 3-FL, or compositions with ratios outside this range, fail to effectively promote brain development. Therefore, the present invention provides a composition comprising 2′-FL and 3′-SL, and also provides its use in promoting brain development.

[0041] Composition

[0042] The invention provides a composition, which consists of 2'-FL and 3'-SL in a mass ratio of (2.5:1) to (15:1).

[0043] As used in the present invention, "2'-FL" refers to 2'-fucosyllactose, which is a trisaccharide compound formed by linking β-D-galactose to D-glucose via a 1-4 glycosidic bond to form a lactose core structure, and fucose via an α1-2 glycosidic bond at the C2' hydroxyl position. Its chemical formula is C 18 H 32 O 15 , molecular weight is 488.44 g / mol, CAS number is 41263-94-9.

[0044] As used herein, "3'-SL" refers to 3'-sialyllactose, which is a tetrasaccharide compound formed by linking β-D-galactose to D-glucose via a 1-4 glycosidic bond to form a lactose core structure, and linking N-acetylneuraminic acid (Neu5Ac) via an α2-3 glycosidic bond at the C3' hydroxyl position. Its chemical formula is C 23 H 39 NO 19 , molecular weight is 633.56 g / mol, CAS number is 35890-38-1.

[0045] The present invention discovered that only a combination of 2′-FL and 3′-SL within a specific weight ratio range of (2.5:1) to (15:1) has a synergistic effect on promoting brain development. Therefore, the mass ratio of 2′-FL to 3′-SL can be (2.5:1) to (15:1), for example, (2.5:1) to (13:1), (2.5:1) to (12:1), (2.5:1) to (10:1), (2.5:1) to (9:1), (2.5:1) to (8:1), (2.5:1) to (7:1), (5:1) to (15:1), (5:1) to (10:1), or (5:1) to (7:1). In other words, when 2′-FL and 3′-SL are used in combination, the amount of 2′-FL relative to 1 part by mass of 3′-SL can be 2.5 to 15 parts by mass, for example, 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, 7 to 9 parts by mass, 7 to 8 parts by mass, or a range defined by any two values. When the weight ratio of 2′-FL to 3′-SL is within the above range, the effect on promoting brain development is more significant, and a synergistic effect is achieved between the two.

[0046] In some embodiments, the concentration of 2′-FL in the composition may be 500-1000 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. In other embodiments, the concentration of 3′-SL in the composition may be 45-2000 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.

[0047] The present invention also provides a method for preparing the composition, which comprises the step of mixing 2′-FL and 3′-SL.

[0048] food

[0049] The present invention also provides a food product comprising the composition.

[0050] The term "food" means an item or substance that can be taken into the body of an individual, including finished food products, semi-finished food products, food additives, food supplements and health products.

[0051] As used herein, the term "subject" refers to any animal of interest. In some embodiments, the subject is a mammal, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, or rats.

[0052] The composition of the present invention can be formulated into a food product using standard techniques well known to those skilled in the art. For example, the composition can be added directly to a food-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-acceptable material.

[0053] The term "food-acceptable materials" refers to nutritional additives (such as dietary fiber, prebiotics, proteins, lipids, minerals, vitamins), ingredients with medicinal and edible properties (such as red dates, hawthorn, wolfberry), excipients or auxiliary materials (such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol), etc. that can be used in food.

[0054] The food of the present invention may be in any form suitable for oral administration, such as powder, tablet, capsule, granule, solution, suspension, etc.

[0055] The food of the present invention can generally be used by people of any age, such as infants (including infants, older infants, and toddlers), children, adolescents, teenagers, young adults, adults, middle-aged people, and the elderly. The term "infant" refers to a person aged 0 to 6 months. The term "older infant" refers to a person aged 6 to 12 months. The term "toddler" refers to a person aged 12 to 36 months. The term "infant" refers to a person aged 0 to 36 months. The term "child" refers to a person aged 3 to 6 years. The term "teenager" refers to a person aged 7 to 17 years. The term "adult" refers to a person aged 18 years and older. The term "young adult" refers to a person aged 18 to 40 years. The term "teenager" refers to a person aged 7 to 40 years. The term "middle-aged person" refers to a person aged 41 to 65 years. The term "elderly" or "elderly person" refers to a person aged 65 years and older.

[0056] In some embodiments, the food can be infant food (e.g., baby food, follow-on food, toddler food), children's food, adolescent food, or adult food, such as infant formula (e.g., infant formula, toddler formula), baby food, nutritional or dietary supplement, children's formula, children's snacks, milk powder for pregnant women, or milk powder for the middle-aged and elderly.

[0057] The composition can be added to the food in an amount sufficient to promote brain development. In one embodiment, the composition can comprise 0.001-80% by mass, preferably 0.01-50% by mass, for example 0.1-30% or 1-20% by mass, 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-scientifically acceptable materials.

[0058] drug

[0059] The present invention also provides a medicine comprising the composition, which may further comprise a pharmaceutically acceptable carrier.

[0060] As used herein, "pharmaceutically acceptable carriers" refer to carriers that have no significant irritating effect on an organism and do not impair the biological activity and properties of the agent in the administered pharmaceutical composition, such as, but not limited to, solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, or liposomes. In some embodiments, a pharmaceutically acceptable carrier can be an inert substance added to the pharmaceutical composition to further facilitate the administration of the agent, such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and the like.

[0061] The medicament of the present invention can be formulated into any suitable dosage form for oral administration or the like. 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 medicament of the present invention can also be stored in a sterile container suitable for injection or instillation.

[0062] The 2′-FL and 3′-SL combination described in the medicament is typically present in an effective amount (e.g., a therapeutically effective amount, a prophylactically effective amount). An effective amount is sufficient to ameliorate or alleviate in some way the symptoms associated with the disease, for example, to effectively ameliorate or eliminate one or more symptoms. This amount can be determined based on the subject's age, gender, and physical condition. While an administered amount may cure the disease, administration is typically intended to ameliorate disease symptoms. Repeated administration is generally required to achieve the desired symptom improvement.

[0063] In a specific embodiment of the present invention, zebrafish were used as experimental animals, and a dosage regimen for a composition of 2′-FL and 3′-SL to promote brain development was proposed. When zebrafish were used as experimental animals, the concentration of 2′-FL could be 500-1000 μg / mL, and the concentration of 3′-SL could be 45-2000 μg / mL. It should be understood that it is easy for a person skilled in the art to convert the zebrafish dosage into a dosage suitable for humans. For example, the theoretical human dosage can be calculated according to the formula: zebrafish (mg / L) = [human (g / day) × 1000] / 6, and the actual human dosage can be further calculated based on the multiple relationship between the theoretical human dosage and the actual human dosage of 0.1-4 times.

[0064] When necessary, the composition of 2′-FL and 3′-SL can also be administered in combination with other active ingredients or drugs. Exemplary active ingredients or drugs that promote brain development include, but are not limited to, cephalin, choline, PS, DHA, sialic acid, neuraminic acid, etc.

[0065] Application / Method

[0066] The present invention also provides use of the composition in preparing medicines for promoting brain development.

[0067] The present invention also provides the use of the drug in promoting brain development, and the use of the food in promoting brain development for non-therapeutic purposes.

[0068] In the present invention, "promoting brain development" can mean improving brain, nerve and / or cognitive development, such as preventing or improving brain retardation, nervous system retardation and / or cognitive retardation. In some embodiments, "promoting brain development" includes improving cognitive maturity and promoting the expression of genes related to brain development.

[0069] In the present invention, the brain development-related genes include: bdnf, gdnfa, SOX2, DCX, GRIN2B, etc., preferably bdnf and gdnfa.

[0070] In some embodiments, the "promoting brain development" is non-therapeutic and non-diagnostic.

[0071] The present invention also provides a method for promoting brain development for non-therapeutic purposes, comprising: administering the composition of the present invention to an individual in need, or administering the food or medicine of the present invention to an individual in need.

[0072] In the present invention, the term "administering" may refer to introducing, providing, or delivering a substance to a subject through any appropriate route to achieve its intended function.

[0073] The beneficial effects of the present invention include at least:

[0074] The present invention provides a composition that can promote brain development. The composition is composed of 2′-FL and 3′-SL in a mass ratio of (2.5:1) to (15:1). In this composition, 2′-FL and 3′-SL work synergistically to significantly improve cognitive maturity in zebrafish and promote the expression of brain development-related genes bdnf and gdnfa. This composition can be used to prevent or improve brain developmental delay and has promising application prospects.

[0075] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0076] Example 1: Determination of the maximum detectable concentration of 2′-FL and 3′-SL

[0077] In this example, the maximum detectable concentration (MTC) of 2′-FL and 3′-SL for promoting brain development was determined. The experimental method is as follows:

[0078] Zebrafish used in this experiment were maintained in aquaculture water at 28°C (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450-550 μS / cm, pH 6.5-8.5, and hardness 50-100 mg / L CaCO₃). This water was provided by the Hangzhou Huante Biological Company's fish farming center. Wild-type AB zebrafish, 5 days post-fertilization (dpf), were randomly selected and placed in beakers. Thirty zebrafish were treated in each beaker (experimental group). 2′-FL or 3′-SL (concentrations shown in Table 1) were administered in aquaculture water. A normal control group (no substance added, 20 mL of aquaculture water) was also established. Each beaker held 20 mL of aquaculture water. After 24 hours of treatment at 28°C, the maximum detectable concentration (MTC) of 2′-FL or 3′-SL in zebrafish was determined.

[0079] Table 1. Exploration of the concentrations of 2′-FL and 3′-SL in promoting brain development (n=30)

[0080]

[0081] At the end of the experiment, zebrafish in the normal control group showed no obvious abnormalities. Zebrafish treated with 2′-FL at concentrations of 125, 250, 500, and 1000 μg / mL showed no obvious abnormalities. The 2000 μg / mL treatment group showed more severe symptoms than the normal control group, manifested by sluggish movements. Zebrafish treated with 3′-SL at concentrations of 125, 250, 500, 1000, and 2000 μg / mL showed no obvious abnormalities.

[0082] The results showed that under the experimental conditions, the maximum detectable concentration (MTC) of 2′-FL for promoting brain development was 1000 μg / mL, and the MTC of 3′-SL for promoting brain development was 2000 μg / mL (Table 1).

[0083] Example 2: Dose-effect relationship study of 2′-FL and 3′-SL in promoting brain development

[0084] In this example, the dose-effect relationship of 2′-FL and 3′-SL in promoting brain development was investigated.

[0085] Color preference tests are often used to assess zebrafish brain development and evaluate their color perception. Zebrafish prefer short-wavelength colors and show a strong preference for blue compared to other colors such as red, yellow, and green. As the brain develops, the preference for blue becomes more pronounced. More movement in the blue area indicates greater cognitive maturity and a more mature brain.

[0086] The zebrafish used in the experiment and the culture conditions were the same as in Example 1. 5 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish per beaker. 2′-FL or 3′-SL (concentrations shown in Table 2) were administered in an aqueous solution. A positive control, Ginkgo Biloba and Cistanche Deserticola tablets, were administered at a concentration of 125 μg / mL (aqueous solution, Amway (China) Daily Products Co., Ltd., batch number 3313N903), and a normal control group was also established. Each beaker had a capacity of 20 mL. After one day of treatment at 28°C, five zebrafish were randomly selected from each experimental group and placed in a cross-shaped module. The module was divided into four zones: yellow, blue, red, and green. Each group had six modules. Data were collected using a behavioral analyzer. The percentage of the total distance traveled within the blue zone relative to the total distance traveled within the entire zone within 10 minutes was analyzed. This metric was used to statistically evaluate the efficacy of the samples in promoting brain development. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0087] Table 2. Dose-effect relationship study of 2′-FL and 3′-SL in promoting brain development (n = 6)

[0088]

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

[0090] The experimental results are shown in Table 2. When the 2′-FL treatment concentrations were 250 μg / mL, 500 μg / mL, 750 μg / mL, and 1000 μg / mL, the proportion of movement in the blue area of ​​zebrafish (p value compared with the normal control group) was 49.7 ± 2.20% (p > 0.05), 55.2 ± 4.33% (p > 0.05), 59.4 ± 0.765% (p < 0.05), and 59.8 ± 2.88% (p < 0.05), respectively. Therefore, when the 2′-FL treatment concentration was ≥ 750 μg / mL, there was a statistically significant effect on promoting the brain development of zebrafish. When the 3′-SL treatment concentrations were 500 μg / mL, 1000 μg / mL, 1500 μg / mL, and 2000 μg / mL, the proportion of movement in the blue area of ​​zebrafish (p value compared with the normal control group) was 52.5 ± 4.10% (p > 0.05), 54.9 ± 4.52% (p > 0.05), 61.9 ± 3.71% (p < 0.05) and 63.6 ± 3.00% (p < 0.01), so the promotion of zebrafish brain development by 3′-SL treatment at a concentration ≥ 1500 μg / mL was statistically significant.

[0091] The results show that under the experimental conditions, both 2′-FL and 3′-SL have the effect of promoting brain development, with the effective concentrations being 750 and 1500 μg / mL, respectively. Specifically, compared with the normal control group, the movement ratio of the blue area of ​​zebrafish increased, as shown in Table 2. Figure 1A and Figure 1B .

[0092] Example 3: Study on the efficacy of a combination of 2′-FL and 3′-SL in promoting brain development

[0093] In this example, the efficacy of the combination of 2′-FL and 3′-SL in promoting brain development was investigated, including its effects on color cognition in zebrafish and on the expression of brain development genes (bdnf gene, gdnfa gene).

[0094] Effects of the combination of 2′-FL and 3′-SL on color perception in zebrafish

[0095] The zebrafish used in the experiment and the culture conditions were the same as in Example 1. 5 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish per beaker. A water-soluble mixture of 2′-FL and 3′-SL at varying mass percentages (concentrations shown in Table 3) was administered. A positive control, Ginkgo Biloba and Cistanche Deserticola tablets at a concentration of 125 μg / mL (water-soluble, Amway (China) Daily Products Co., Ltd., batch number 3313N903), was also used. A normal control group was also established. Each beaker had a capacity of 20 mL. After one day of treatment at 28°C, five zebrafish were randomly selected from each experimental group and placed in a cross-shaped module. The module was divided into four zones: yellow, blue, red, and green. Each group was assigned to six modules. Data were collected using a behavioral analyzer. The percentage of the total distance traveled within the blue zone relative to the total distance traveled within the entire zone within 10 minutes was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of the 2′-FL and 3′-SL combination in promoting brain development. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0096] Table 3. Effects of 2′-FL and 3′-SL Combinations on Zebrafish Color Perception (n = 6)

[0097]

[0098] Note: The concentrations of formulas 1-7 in Table 3 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL;

[0099] Compared with the normal control group, *p < 0.05, **p < 0.01, ***p < 0.001;

[0100] Compared with formula 1, # p < 0.05, ## p < 0.01;

[0101] Compared with formula 2, && p < 0.01;

[0102] Compared with formula 3, @ p < 0.05, @@ p < 0.01;

[0103] Compared with formula 4, $ p < 0.05.

[0104] As shown in Table 3, the proportion of exercise in the blue area of ​​Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, and Formula 7 (p value compared with the normal control group) were 62.2 ± 2.66% (p < 0.01), 67.2 ± 3.27% (p < 0.001), 62.0 ± 2.23% (p < 0.01), 61.3 ± 4.17% (p < 0.05), 50.1 ± 2.52% (p > 0.05), 54.6 ± 1.76% (p < 0.05), and 54.3 ± 2.26% (p < 0.05), respectively.

[0105] Further inter-group comparisons showed that Formula 1 had statistically significant effects on promoting brain development. When compared with Formula 5, Formula 6, and Formula 7, the p values ​​were p < 0.01, p < 0.05, and p < 0.05, respectively, indicating that Formula 1 had a significant difference in promoting brain development compared with Formula 5, Formula 6, and Formula 7.

[0106] Formula 2 has statistical significance in promoting brain development. When compared with Formula 5, Formula 6, and Formula 7, the p values ​​are all p < 0.01, indicating that Formula 2 has a significant difference in promoting brain development compared with Formula 5, Formula 6, and Formula 7.

[0107] Formula 3 has statistical significance in promoting brain development. Compared with Formula 5, Formula 6 and Formula 7, the p values ​​are p < 0.01, p < 0.05 and p < 0.05 respectively, indicating that Formula 3 has a significant difference in promoting brain development compared with Formula 5, Formula 6 and Formula 7.

[0108] Formula 4 has statistical significance in promoting brain development, and the p value when compared with Formula 5 is p < 0.05, indicating that Formula 4 has a significant difference in promoting brain development compared with Formula 5.

[0109] The above results show that under the experimental conditions, the increase in the proportion of exercise in the blue area of ​​Formulas 1-4 compared to the normal control group was 16.3%, 21.3%, 16.1% and 15.4%, respectively, all showing the effect of promoting brain development, which is better than the positive control Ginkgo Biloba and Cistanche Tablets. In addition, Formulas 1-3 were more effective than Formulas 5-7, and Formula 4 was more effective than Formula 5, indicating that when the 2′-FL and 3′-SL in Formulas 1-4 were within the range of (2.5:1) to (15:1), they had a synergistic effect on promoting brain development, as shown in Table 3. Figure 2A and Figure 2B .

[0110] 2. Effects of the combination of 2′-FL and 3′-SL on gene expression during brain development

[0111] In this example, the bdnf gene and the gdnfa gene were used as representative genes for brain development to explore the effect of the combination of 2′-FL and 3′-SL on the expression of brain development genes.

[0112] BDNF and its receptors are widely expressed in the nervous system, with the highest levels in the hippocampus and cortex. Their specific modes of action in the central nervous system are as follows: (1) increasing synaptic plasticity, thereby affecting long-term potentiation (nLTP), which is the basis of the learning process and memory formation (second-order memory) process; (2) promoting neurogenesis, especially neurogenesis in the hippocampus; (3) promoting cell survival, which is mainly reflected in the maintenance and promotion of the development, differentiation, growth, and regeneration of various neurons, especially 5-hydroxytryptamine (5-HT) and dopamine (DA) neurons. GDNF can promote the survival of different neuronal subpopulations at different stages of development of the central and peripheral nervous systems, supporting the production of type 1 astrocytes, neural membrane cells, neurons, pineal cells, etc., and is particularly effective in promoting the survival of spinal motor neurons. Therefore, the upregulation of the expression of the two genes can promote the development of the central nervous system and nerves, and thus promote the formation of learning and memory. They are representative genes of brain development.

[0113] The zebrafish used in the experiment and the culture conditions were the same as in Example 1. Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). Water-soluble administration samples (concentrations shown in Tables 4 and 5) and a positive control, Ginkgo biloba and Cistanche deserticola tablets, were used at a concentration of 125 μg / mL. A normal control group was also established. Each beaker had a capacity of 20 mL. Three parallel experiments were performed. After one day of treatment at 28°C, total RNA was extracted from each group using a pre-loaded magnetic bead-based universal RNA extraction kit (Cat. No. TL2402001643C, ONREW, China). Total RNA concentration and purity were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from zebrafish samples was synthesized into 20.0 μL of cDNA using the first-strand cDNA synthesis kit (lot number: H9305270, Yisheng Biotechnology (Shanghai) Co., Ltd., China). q-PCR was used to analyze the expression of β-actin, bdnf, and gdnfa genes. β-actin was used as an internal control for gene expression, and the relative expression levels of bdnf and gdnfa genes were calculated. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0114] (1) Effect of the combination of 2′-FL and 3′-SL on bdnf gene expression

[0115] Table 4. Effects of the combination of 2′-FL and 3′-SL on bdnf gene expression (n=3)

[0116]

[0117] Note: The concentrations of formulas 1-7 in Table 4 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL;

[0118] Compared with the normal control group, **p < 0.01, ***p < 0.001;

[0119] Compared with formula 1, # p < 0.05, ### p < 0.001;

[0120] Compared with formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001;

[0121] Compared with formula 3, @ p < 0.05, @@ p < 0.01, @@@ p < 0.001;

[0122] Compared with formula 4, $ p < 0.05, $$$ p < 0.001.

[0123] As shown in Table 4, the relative expression levels of the bdnf gene in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, and Formula 7 (p values ​​compared with the normal control group) were 5.51 ± 0.117 (p < 0.001), 2.65 ± 0.194 (p < 0.01), 4.26 ± 0.265 (p < 0.001), 3.73 ± 0.161 (p < 0.001), 1.01 ± 0.065 (p > 0.05), 1.56 ± 0.046 (p < 0.001), and 1.48 ± 0.056 (p < 0.01), respectively. Therefore, compared with the blank control group, Formula 1, Formula 2, Formula 3, Formula 4, Formula 6, and Formula 7 had statistically significant effects on upregulating the relative expression level of the bdnf gene; Formula 5 had no statistically significant effect on upregulating the relative expression level of the bdnf gene.

[0124] The p-values ​​of formula 1 for upregulating the relative expression of the bdnf gene compared with formula 5, formula 6, and formula 7 were all p < 0.001, indicating that the efficacy of formula 1 in upregulating the relative expression of the bdnf gene was statistically significant compared with formula 5, formula 6, and formula 7.

[0125] The p values ​​of formula 2 for upregulating the relative expression of the bdnf gene compared with formula 5, formula 6, and formula 7 were p < 0.01, p < 0.01, and p < 0.01, respectively, indicating that formula 2 was statistically significant in upregulating the relative expression of the bdnf gene compared with formula 5, formula 6, and formula 7.

[0126] The p-values ​​of formula 3 for upregulating the relative expression of bdnf gene compared with formula 5, formula 6 and formula 7 were all p < 0.001, indicating that the efficacy of formula 3 in upregulating the relative expression of bdnf gene was statistically significant compared with formula 5, formula 6 and formula 7.

[0127] Formula 4 had a statistically significant effect on upregulating the relative expression of the bdnf gene, and the p-values ​​compared with Formula 5, Formula 6, and Formula 7 were all p < 0.001, indicating that Formula 4 had a synergistic effect on upregulating the relative expression of the bdnf gene compared with Formula 5, Formula 6, and Formula 7.

[0128] The above results show that under the experimental conditions, the relative expression of bdnf gene in formulas 1-4 increased by 4.51, 1.65, 3.26 and 2.73 respectively compared with the normal control group, all of which promoted brain development, which was better than the positive control Ginkgo biloba and Cistanche tablets, and was better than formulas 5-7. This shows that when the 2′-FL and 3′-SL in formulas 1-4 are in the range of (2.5:1) to (15:1), they have a synergistic effect on promoting brain development, as shown in Tables 4 and Figure 3 .

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

[0130] Table 5. Effects of the combination of 2′-FL and 3′-SL on gdnfa gene expression (n=3)

[0131]

[0132] Note: The concentrations of formulas 1-7 in Table 5 are expressed as 2′-FL concentration + 3′-SL concentration, and the unit is μg / mL;

[0133] Compared with the normal control group, **p < 0.01, ***p < 0.001;

[0134] Compared with formula 1, #p < 0.05, ## p < 0.01;

[0135] Compared with formula 2, & p < 0.05, && p < 0.01, &&& p < 0.001;

[0136] Compared with formula 3, @ p < 0.05, @@ p < 0.01, @@@ p < 0.001;

[0137] Compared with formula 4, $ p < 0.05, $$ p < 0.01.

[0138] As shown in Table 5, the relative expression levels of the gdnfa gene in Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula 6, and Formula 7 (p values ​​compared with the normal control group) were 1.83 ± 0.062 (p < 0.001), 2.42 ± 0.098 (p < 0.001), 2.49 ± 0.143 (p < 0.001), 2.28 ± 0.136 (p < 0.001), 1.14 ± 0.060 (p > 0.05), 1.29 ± 0.027 (p < 0.01), and 1.58 ± 0.083 (p < 0.01), respectively. Therefore, Formula 1, Formula 2, Formula 3, Formula 4, Formula 6, and Formula 7 had statistically significant effects on upregulating the relative expression level of the gdnfa gene; Formula 5 had no statistically significant effect on upregulating the relative expression level of the gdnfa gene.

[0139] Formula 1 had a statistically significant effect on upregulating the relative expression of the gdnfa gene, and the p values ​​compared with Formula 5 and Formula 6 were p < 0.01 and p < 0.01, respectively, indicating that Formula 1 had a statistically significant effect on upregulating the relative expression of the gdnfa gene compared with Formula 5 and Formula 6.

[0140] Formula 2 had a statistically significant effect on upregulating the relative expression of the gdnfa gene. The p-values ​​compared with Formula 5, Formula 6, and Formula 7 were p < 0.001, p < 0.001, and p < 0.01, respectively, indicating that Formula 2 had a statistically significant effect on upregulating the relative expression of the gdnfa gene compared with Formula 5, Formula 6, and Formula 7.

[0141] Formula 3 had a statistically significant effect on upregulating the relative expression of the gdnfa gene. The p values ​​compared with Formula 5, Formula 6, and Formula 7 were p < 0.001, p < 0.01, and p < 0.01, respectively, indicating that Formula 3 had a statistically significant effect on upregulating the relative expression of the gdnfa gene compared with Formula 5, Formula 6, and Formula 7.

[0142] Formula 4 had a statistically significant effect on upregulating the relative expression of the gdnfa gene. The p values ​​compared with Formula 5, Formula 6, and Formula 7 were p < 0.01, p < 0.01, and p < 0.05, respectively, indicating that Formula 4 had a statistically significant effect on upregulating the relative expression of the gdnfa gene compared with Formula 5, Formula 6, and Formula 7.

[0143] The above results show that under the experimental conditions, the relative expression of gdnfa gene in formulas 1-4 increased by 0.83, 1.42, 1.49 and 1.28 respectively compared with the normal control group, all of which promoted brain development and were better than the positive control Ginkgo biloba and Cistanche tablets. They were also better than formulas 5-7, indicating that when the ratio of 2′-FL and 3′-SL in formulas 1-4 was within the range of (2.5:1) to (15:1), they had a synergistic effect on promoting brain development, as shown in Tables 5 and 6 for details. Figure 4 .

[0144] Example 4: Comparison of the composition of 2′-FL and 3′-SL with other compositions

[0145] During the research process, the inventors conducted repeated experiments and found that only the combination of 2′-FL and 3′-SL in a specific ratio range of (2.5:1) to (15:1) has a synergistic effect on promoting brain development. Compositions that add 3-FL to 2′-FL and 3′-SL, and compositions that are not within this ratio range, cannot effectively promote brain development.

[0146] In this example, a composition in which the ratio of 2′-FL to 3′-SL was 9:5.89 (i.e., a two-ingredient ratio group) and a composition in which the ratio of 2′-FL, 3-FL, and 3′-SL was 9:1:5.89 (i.e., a three-ingredient ratio group) were used as comparisons. A composition in which the ratio of 2′-FL to 3′-SL was 7:1 (i.e., an optimal ratio group) was used as an example of the composition of the present invention. The differences between these three compositions in promoting brain development were compared.

[0147] The zebrafish used in the experiment and the culture conditions were the same as in Example 1. 5 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker (experimental group). A water-soluble combination of 2′-FL and 3′-SL or other combinations (concentrations shown in Table 6) was administered, along with a positive control of 125 μg / mL of Ginkgo Biloba and Cistanche tablets. A normal control group was also established. Each beaker held 20 mL of water. After 24 hours of treatment at 28°C, five zebrafish were randomly selected from each experimental group and placed in a cross-shaped module. The module was divided into four zones: yellow, blue, red, and green. Each group had six modules. Data were collected using a behavioral analyzer. The percentage of the total distance traveled within the blue zone relative to the total distance traveled within the entire zone within 10 minutes was analyzed. This metric was used to evaluate the efficacy of the samples in promoting brain development. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated statistical significance.

[0148] Table 6. Comparison of the effects of a combination of 2′-FL and 3′-SL on promoting brain development with other combinations (n=6)

[0149]

[0150] Note: Compared with the normal control group, ***p < 0.001, compared with the three raw material ratio groups, #p < 0.05

[0151] From Table 6, Figure 5A and Figure 5B It can be seen that the proportion of exercise in the blue area of ​​the optimal ratio group, the two-ingredient ratio group and the three-ingredient ratio group were 64.8±1.59%, 58.8±1.83% and 58.5±1.63%, respectively. When compared with the normal control group (45.4±2.10%), the p values ​​were all p < 0.001. Therefore, the optimal ratio group, the two-ingredient ratio group and the three-ingredient ratio group were statistically significant in promoting brain development.

[0152] Further statistical analysis revealed that the p-value for the optimal ratio group compared with the three raw material ratio groups was p < 0.05, indicating that the optimal ratio group was more effective in promoting brain development than the three raw material ratio groups. When the optimal ratio group, the two raw material ratio groups, and the three raw material ratio groups were compared with the positive control Ginkgo Biloba and Cistanche tablets, only the optimal ratio group outperformed the positive control Ginkgo Biloba and Cistanche tablets, demonstrating that the composition of the present invention is superior in enhancing color cognition in zebrafish and has a highly significant function in promoting brain development.

[0153] The two-raw material ratio group and the three-raw material ratio group were compared. The results showed that the p-value of the comparison between the two-raw material ratio group and the three-raw material ratio group was p > 0.05, indicating that there was no statistical difference in the brain development promoting effect between the two-raw material ratio group and the three-raw material ratio group, indicating that adding 3-FL on the basis of 2′-FL and 3′-SL cannot further enhance the function of the composition in promoting brain development.

[0154] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. Use of a composition in the preparation of a medicine for promoting brain development, the composition consisting of 2′-FL and 3′-SL in a mass ratio of (2.5:1) to (15:1).

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

3. Use of a food in the preparation of a substance for promoting brain development for non-therapeutic purposes, characterized in that: The food comprises the composition according to claim 1 or 2.

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

5. The use according to claim 3, characterized in that The food product also includes food-scientifically acceptable materials.

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

7. The use according to claim 6, 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 red dates, hawthorn, wolfberry, longan, lily, poria and tangerine peel, and / or the excipients or auxiliary materials include one or more of calcium carbonate, calcium phosphate, sugar, starch, cellulose derivatives, gelatin, vegetable oil and polyethylene glycol.

8. The use according to claim 3, characterized in that The food is infant food, children's food, juvenile food, young people's food, middle-aged people's food or elderly people's food.

9. The use according to claim 3, characterized in that The food is a juvenile food or an adult food.

10. The use according to claim 8, characterized in that The infants and young children include infants aged 0 to 6 months, older infants aged 6 to 12 months, and young children aged 12 to 36 months.

11. The use according to claim 8, wherein The food is infant formula milk powder, baby food supplement, children's formula milk powder, children's snacks, pregnant women's milk powder, middle-aged and elderly people's milk powder, or nutritional or dietary supplements.

12. Use of a drug in the preparation of a medicine for promoting brain development, characterized in that: The drug comprises an effective amount of a composition consisting of 2′-FL and 3′-SL in a mass ratio of (2.5:1) to (15:1), and a pharmaceutically acceptable carrier.

13. The use according to claim 12, characterized in that The amount of 2′-FL is 2.5 to 10 parts by mass relative to 1 part by mass of 3′-SL.

14. The use according to claim 12, characterized in that The effective amount of 2′-FL is 500 to 1000 μg / mL, and / or the effective amount of 3′-SL is 45 to 2000 μg / mL.

15. The use according to any one of claims 1 to 14, characterized in that The promoting of brain development is to improve brain, nerve and / or cognitive development.

16. The use according to claim 15, characterized in that The promoting of brain development includes preventing or improving brain development delay, nervous system development delay and / or cognitive development delay.

17. The use according to claim 15, characterized in that The promoting of brain development includes improving cognitive maturity and promoting the expression of genes related to brain development; wherein the genes related to brain development are bdnf or gdnfa.

Images