Tea leaf theanine composition capable of improving memory function as well as preparation method and application of tea leaf theanine composition

Abstract

The invention relates to the field of health-care food, and particularly discloses a tea leaf theanine composition capable of improving a memory function, a preparation method and application of the tea leaf theanine composition. The composition is a pellet with a three-layer core-shell structure, the outer layer is a quick-release layer containing tea theanine, the middle layer is an enteric-coated layer containing phosphatidylserine and walnut oligopeptide, and the inner core is a slow-release layer containing a lion mushroom extract; according to the structure, through a sequential release mechanism of'quick release-enteric coating-slow release ', a multi-target synergistic effect of'quick attention starting-midway neurological function enhancing-long-term memory consolidation' is realized; the invention further provides a specific preparation method of the composition, hot melt extrusion rounding and fluidized bed coating technologies are combined, the process is controllable, and the preparation method is suitable for industrial production; experiments show that the composition can significantly improve memory defects, has better effects than a single component and a physical mixture, and is safe and efficient.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of health food, in particular to a tea leaf theanine composition for improving memory function, a preparation method and application thereof. BACKGROUND

[0002] At present, the intervention means for memory and cognitive impairment mainly includes two categories: chemical synthetic drugs and natural dietary supplements. In the field of drugs, donepezil, rivastigmine and other cholinesterase inhibitors are the first-line drugs for the clinical treatment of Alzheimer's disease. This kind of drug can improve the symptoms by increasing the concentration of acetylcholine in the brain, but its target is relatively single, and it often has side effects such as gastrointestinal discomfort, dizziness, bradycardia, etc. It is mainly suitable for patients who have been diagnosed, and is not suitable for healthy or sub-healthy people for daily brain health and cognitive improvement.

[0003] On the other hand, in the field of dietary supplements, the market is flooded with various products based on single natural ingredients, such as lecithin, docosahexaenoic acid, and extracts derived from traditional Chinese medicinal materials. Although these ingredients are generally safe, their mechanisms of action are often limited to providing raw materials for the synthesis of nerve cells or possessing preliminary antioxidant activity, and their improvement effect on the complex learning and memory process is limited. Moreover, due to the lack of multi-target synergistic effect, the effect is often not significant and unstable, which is difficult to meet the urgent needs of users for "effectiveness".

[0004] Theanine, as a unique non-protein free amino acid in tea leaves, has attracted attention in recent years due to its unique neuroregulatory function. Studies have shown that theanine can easily cross the blood-brain barrier and exert multiple beneficial effects in the central nervous system: it can promote the production of alpha brain waves in the brain, induce a relaxed and alert mental state, thereby helping to relieve anxiety and improve attention; at the same time, it can also regulate the levels of key neurotransmitters such as dopamine and 5-hydroxytryptamine, and may indirectly protect nerve cells by antagonizing the neurotoxicity caused by excessive excitation of glutamic acid. However, although theanine shows potential in improving mood and attention, its effect is still insufficient and limited in improving the complex memory encoding, consolidation and extraction process if used as a single component. The formation of memory is a complex system engineering involving neurotransmitter regulation, synaptic plasticity change, brain energy metabolism, and even brain blood circulation. Single-component tea theanine is difficult to fully and efficiently exert its effect in the whole system.

[0005] Based on the above statements, the present application proposes a tea leaf theanine composition for improving memory function, a preparation method and application thereof. SUMMARY

[0006] In order to solve the problems of single target of chemical synthetic drugs, large side effects, not suitable for health care in the prior art, and the limited efficacy of single natural ingredient for improving memory, and the inability to meet the high efficiency requirements, the application provides a tea theanine composition for improving memory function, a preparation method and application thereof.

[0007] In a first aspect, the application provides a tea theanine composition for improving memory function, which is a micro-pellet with a three-layer core-shell structure, the outer layer comprising tea theanine, the middle layer comprising phosphatidylserine and walnut oligopeptide, and the inner core comprising lion's mane mushroom extract.

[0008] Preferably, the outer layer is a quick-release layer composed of a water-soluble film-forming material, the middle layer is an enteric layer composed of a pH-dependent polymer, and the inner core is a sustained-release layer composed of a hydrophobic matrix.

[0009] Preferably, the water-soluble film-forming material is hydroxypropyl methyl cellulose, the pH-dependent polymer is shellac, and the hydrophobic matrix is a mixture of hydrogenated palm oil and glycerol monostearate.

[0010] Preferably, the tea theanine composition is composed of the following raw materials in mass fraction: tea theanine 10-15 parts, phosphatidylserine 2-5 parts, walnut oligopeptide 1-3 parts, and lion's mane mushroom extract 1-3 parts.

[0011] In a second aspect, the application provides a preparation method of a tea theanine composition for improving memory function, which adopts the following technical solution: The preparation method of the tea theanine composition for improving memory function comprises the following steps: S1, preparing the inner core: melt-blending the hydrophobic matrix, then mixing with the lion's mane mushroom extract, and preparing the inner core by hot melt extrusion and spheronization; S2, coating the middle layer: homogeneously dispersing the phosphatidylserine and the walnut oligopeptide in the pH-dependent polymer to obtain coating liquid A, and spraying and coating the coating liquid A on the surface of the inner core by fluidized bed coating technology to form an enteric middle layer, thereby obtaining a double-layer micro-pellet; S3, coating the outer layer: dissolving the tea theanine in the water-soluble film-forming material to obtain coating liquid B, and spraying and coating the coating liquid B on the surface of the enteric middle layer by fluidized bed coating technology to form a quick-release outer layer, thereby obtaining a three-layer micro-pellet; S4, post-treatment: drying the three-layer structure micro-pellet at low temperature, thereby obtaining the tea theanine composition for improving memory function.

[0012] Preferably, the specific operation method of step S1 is as follows: glycerol monostearate and hydrogenated palm oil are melt-blended at a mass ratio of 3-5:1 at 75-85℃ to obtain a hydrophobic matrix, the hydrophobic matrix is uniformly mixed with lion's mane extract at a mass ratio of 2-4:1, and then transferred to a hot melt extruder for melt extrusion at 30-50rpm and 65-75℃; the extrudate is placed in a spheronizer and spheronized at 1000-1500rpm for 10-15min, and then cooled and solidified at 4-10℃, and the pellets with a particle size of 20-24 meshes are screened to obtain the inner core.

[0013] Preferably, the specific operation method of step S2 is as follows: phosphatidylserine is mixed with walnut oligopeptide, and then mixed with a shellac solution with a mass-volume concentration of 3-5% at a mass ratio of 1:8-10, and homogenized at a pressure of 40-60MPa for 10-20min to obtain coating liquid A; the inner core is placed in a fluidized bed, the inlet air temperature is controlled at 35-40℃, the material temperature is controlled at 30-35℃, the atomization pressure is 0.8-1.2bar, and the coating liquid A is sprayed at a rate of 5-10mL / min to form an enteric intermediate layer, thereby obtaining double-layer pellets.

[0014] Preferably, the specific operation method of step S3 is as follows: theanine is mixed with a hydroxypropyl methyl cellulose solution with a mass-volume concentration of 2-4% at a mass ratio of 1:3-5 to obtain coating liquid B; the double-layer pellets are placed in a fluidized bed, the inlet air temperature is controlled at 30-35℃, the material temperature is controlled at 25-30℃, the atomization pressure is 0.6-1.0bar, and the coating liquid B is sprayed at a rate of 8-12mL / min to form a fast-release outer layer, thereby obtaining triple-layer pellets.

[0015] Preferably, the temperature for low-temperature drying in step S4 is 35-45℃, and the time is 1-2h.

[0016] In a third aspect, the application provides a theanine composition for improving memory function, and application of the theanine composition in the preparation of a functional food, a health food or a medicine for improving attention, improving learning and memory ability or delaying cognitive decline.

[0017] In summary, the application has the following beneficial effects: (1) Synergistic effect brought by the composition structure design: By designing the "fast release - enteric - sustained release" three-layer core-shell structure, the present application realizes the time sequence release and precise delivery of multiple functional ingredients for improving memory. The water-soluble fast-release layer in the outer layer ensures that theanine in tea can be quickly absorbed by the gastrointestinal tract, cross the blood-brain barrier in a short time, and play its role of "starting" to relieve tension and improve attention, creating the best brain state for subsequent cognitive activities. The enteric layer in the middle layer is like an "intelligent key" that can protect phosphatidylserine and walnut oligopeptide from the gastric acid environment and release them precisely in the intestinal neutral environment. The two ingredients work together to repair and build nerve cell membranes, greatly optimizing the transmission efficiency of nerve signals, thereby playing a key role in enhancing the "encoding and consolidation" stage of memory. The innermost sustained-release core ensures that the lion's mane mushroom extract can be released continuously for a long time at a low dose, like a "long-acting nutrient" that continuously nourishes neurons, and promotes nerve regeneration and long-term memory stability by inducing neurotrophic factors. This "start - enhance - consolidate" time sequence synergy mechanism solves the industry problems of limited target sites of single ingredients, mutual interference of ingredients, and inability to act at the most suitable time point, achieving "1 + 1 + 1 > 3" synergistic effect.

[0018] (2) Significant improvement in product safety and stability: The present application uses only natural functional ingredients or endogenous substances in the human body, fundamentally avoiding the side effects of chemical synthetic drugs, which is safe and suitable for long-term use by all groups of people to maintain brain health. At the same time, the structure of the preparation itself also improves the stability of the product. On the one hand, the active ingredients with unstable properties (such as lion's mane mushroom extract and phosphatidylserine) are encapsulated in the hydrophobic inner core, which can effectively isolate water, oxygen and light to prevent degradation. On the other hand, this physical isolation also avoids the interaction of ingredients during storage, ensuring the consistency of product efficacy throughout the shelf life.

[0019] (3) Advancement and controllability of the preparation process: The preparation method of "hot melt extrusion spheronization" combined with "fluidized bed coating" used in the present application is a process route very suitable for industrial production. The hot melt extrusion technology can form a dense and uniform sustained-release core without solvent residue, and the fluidized bed coating technology can accurately control the coating thickness and uniformity, thereby ensuring that each batch of products has highly consistent release behavior. The whole process is clear, the parameters are clear, and the reproducibility is good, providing reliable protection for large-scale production of high-quality and high-standard products.

[0020] (4) Broad application prospects and market value: Based on the above-mentioned multi-target, high safety, and strong synergy characteristics, the composition can be flexibly applied in multiple fields such as functional food, health food, and medicine. It is not only suitable for healthy people such as students and mental workers to improve mental performance and relieve mental fatigue, but also has great application potential in preventing and assisting in delaying age-related cognitive decline and mild cognitive impairment, meeting the diversified and efficient needs of different consumer groups for brain health products, and has high market conversion value and broad social application prospects. DETAILED DESCRIPTION

[0021] The application will be further described in detail below in conjunction with the examples. It should be understood that the specific examples described herein are only intended to explain the application and not to limit the scope of the application.

[0022] Unless otherwise specified in the examples, the techniques or conditions are in accordance with those described in the literature in the art or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0023] In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

[0024] Among them, tea theanine is provided by Shanghai Nuo De Biological Industry Co., Ltd., batch number: 19111503; Lion's mane mushroom extract is purchased from Shaanxi Jiuyuan Biological Technology Co., Ltd.; Phosphatidylserine is purchased from Shenyang Tianfeng Biological Pharmaceutical Co., Ltd., CAS number: 51446-62-9; Walnut oligopeptide is purchased from Shanxi Yuan Sheng Peptide Technology Co., Ltd.; Shellac is purchased from Shanghai Linchen Pharmaceutical Technology Co., Ltd., CAS number: 9000-59-3; Aluminum chloride hexahydrate (AlCl3·6H2O) is purchased from Shengong Biological Engineering (Shanghai) Co., Ltd., CAS number: 7784-13-6; Donepezil hydrochloride tablets are purchased from Shengong Biological Engineering (Shanghai) Co., Ltd.

[0025] Example 1 The tea theanine composition is composed of the following mass fractions of raw materials: tea theanine 10 parts, phosphatidylserine 2 parts, walnut oligopeptide 1 part, and lion's mane mushroom extract 1 part.

[0026] A preparation method of a tea theanine composition for improving memory function, comprising the following steps: S1, preparing the inner layer core: melt blend glycerin monostearate and hydrogenated palm oil at 75°C at a mass ratio of 3:1 to obtain a hydrophobic matrix, mix the hydrophobic matrix and lion's mane extract at a mass ratio of 2:1, and transfer to a hot melt extruder for melt extrusion at 30 rpm and 65°C; place the extrudate in a rounding machine, round at 1000 rpm for 10 min, cool and solidify at 4°C, sieve the 20-mesh particle size pellets, and the inner layer core is obtained; S2, coating the intermediate layer: first mix phosphatidylserine and walnut oligopeptide, then mix with a shellac solution with a mass volume concentration of 3% at a mass ratio of 1:8, homogenize at a pressure of 40 MPa for 10 min to obtain coating liquid A; place the inner layer core in a fluidized bed, control the inlet air temperature at 35°C, the material temperature at 30°C, the atomization pressure at 0.8 bar, spray coating liquid A at a rate of 5 mL / min to form an enteric intermediate layer, and obtain double-layer pellets; S3, coating the outer layer: mix tea theanine and a hydroxypropyl methyl cellulose solution with a mass volume concentration of 2% at a mass ratio of 1:3 to obtain coating liquid B; place the double-layer pellets in a fluidized bed, control the inlet air temperature at 30°C, the material temperature at 25°C, the atomization pressure at 0.6 bar, and spray coating liquid B at a rate of 8 mL / min to form a fast-release outer layer, and obtain triple-layer pellets; S4, post-treatment: dry the triple-layer structure pellets at 35°C for 1 h, and the tea theanine composition for improving memory function is obtained.

[0027] Example 2 The tea theanine composition for improving memory function is composed of the following raw materials in mass fraction: tea theanine 12.5 parts, phosphatidylserine 3.5 parts, walnut oligopeptide 2 parts, and lion's mane extract 2 parts.

[0028] A preparation method of a tea theanine composition for improving memory function includes the following steps: S1, preparing the inner layer core: melt blend glycerin monostearate and hydrogenated palm oil at 80°C at a mass ratio of 4:1 to obtain a hydrophobic matrix, mix the hydrophobic matrix and lion's mane extract at a mass ratio of 3:1, and transfer to a hot melt extruder for melt extrusion at 40 rpm and 70°C; place the extrudate in a rounding machine, round at 1200 rpm for 12 min, cool and solidify at 8°C, sieve the 22-mesh particle size pellets, and the inner layer core is obtained; S2, coating the intermediate layer: first mix phosphatidylserine and walnut oligopeptide, then mix with a shellac solution with a mass volume concentration of 4% at a mass ratio of 1:9, homogenize at a pressure of 50 MPa for 15 min to obtain coating liquid A; place the inner layer core in a fluidized bed, control the inlet air temperature at 37°C, the material temperature at 32°C, the atomization pressure at 1.0 bar, and spray coating liquid A at a rate of 7.5 mL / min to form an enteric intermediate layer, and obtain double-layer pellets; S3, coating outer layer: tea theanine was mixed with a 3% hydroxypropyl methylcellulose solution with a mass ratio of 1:4 to obtain a coating solution B; the double-layer pellets were placed in a fluidized bed, the inlet air temperature was controlled at 32℃, the material temperature was 27℃, the atomization pressure was 0.8 bar, and the coating solution B was sprayed at a rate of 10 mL / min to form a rapid-release outer layer, thereby obtaining three-layer pellets; S4, post-processing: the three-layer structure pellets were dried at 40℃ for 1.5h to obtain a tea theanine composition for improving memory function.

[0029] Example 3 The tea theanine composition was composed of the following raw materials in mass fraction: tea theanine 15 parts, phosphatidylserine 5 parts, walnut oligopeptide 3 parts, and lion's mane mushroom extract 3 parts.

[0030] A preparation method of a tea theanine composition for improving memory function, comprising the following steps: S1, preparing an inner layer core: glycerol monostearate and hydrogenated palm oil were melt blended at a mass ratio of 5:1 at 85℃ to obtain a hydrophobic matrix, the hydrophobic matrix was uniformly mixed with lion's mane mushroom extract at a mass ratio of 4:1, and then transferred to a hot melt extruder for melt extrusion at 50 rpm and 75℃; the extrudate was placed in a roller compactor and rolled for 15 min at 1500 rpm, and then cooled and solidified at 10℃; pellets with a particle size of 24 mesh were sieved to obtain the inner layer core; S2, coating the middle layer: phosphatidylserine and walnut oligopeptide were mixed, and then mixed with a 5% shellac solution with a mass ratio of 1:10, and homogenized at a pressure of 60 MPa for 20 min to obtain a coating solution A; the inner layer core was placed in a fluidized bed, the inlet air temperature was controlled at 40℃, the material temperature was 35℃, the atomization pressure was 1.2 bar, and the coating solution A was sprayed at a rate of 10 mL / min to form an enteric middle layer, thereby obtaining double-layer pellets; S3, coating the outer layer: tea theanine was mixed with a 4% hydroxypropyl methylcellulose solution with a mass ratio of 1:5 to obtain a coating solution B; the double-layer pellets were placed in a fluidized bed, the inlet air temperature was controlled at 35℃, the material temperature was 30℃, the atomization pressure was 1.0 bar, and the coating solution B was sprayed at a rate of 12 mL / min to form a rapid-release outer layer, thereby obtaining three-layer pellets; S4, post-processing: the three-layer structure pellets were dried at 45℃ for 2h to obtain a tea theanine composition for improving memory function.

[0031] Comparative Example 1 The tea theanine composition was composed of the following raw materials in mass fraction: tea theanine 12.5 parts, phosphatidylserine 3.5 parts, walnut oligopeptide 2 parts, and lion's mane mushroom extract 2 parts.

[0032] A preparation method of a tea theanine composition for improving memory function, comprising the following steps: S1, preparing an inner core: melt blend glycerol monostearate and hydrogenated palm oil at 80°C at a mass ratio of 4:1 to obtain a hydrophobic matrix, mix the hydrophobic matrix and lion's mane mushroom extract uniformly at a mass ratio of 3:1, and transfer to a hot melt extruder for melt extrusion at 40 rpm and 70°C; place the extrudate in a spheronizer, spheronize at 1200 rpm for 12 min, cool and solidify at 8°C, sieve out the pellets with a particle size of 22 meshes, and the inner core is obtained; S2, coating an intermediate layer: first mix phosphatidylserine and walnut oligopeptide, then mix with a shellac solution with a mass and volume concentration of 4% at a mass ratio of 1:9, homogenize at a pressure of 50 MPa for 15 min to obtain coating liquid A; place the inner core in a fluidized bed, control the inlet air temperature at 37°C, the material temperature at 32°C, and the atomization pressure at 1.0 bar, spray coating liquid A at a rate of 7.5 mL / min to form an enteric intermediate layer, and obtain double-layer pellets; S3, coating an outer layer: mix the tea theanine and double-layer pellets directly and uniformly to obtain a mixture; S4, post-treatment: fill the mixture into a capsule shell to obtain the tea theanine composition for improving memory function.

[0033] Comparative Example 2 The tea theanine composition is composed of the following raw materials in mass parts: 12.5 parts of gamma-aminobutyric acid, 3.5 parts of phosphatidylserine, 2 parts of walnut oligopeptide, and 2 parts of lion's mane mushroom extract.

[0034] A preparation method of a tea theanine composition for improving memory function, comprising the following steps: S1, preparing an inner core: melt blend glycerol monostearate and hydrogenated palm oil at 80°C at a mass ratio of 4:1 to obtain a hydrophobic matrix, mix the hydrophobic matrix and lion's mane mushroom extract uniformly at a mass ratio of 3:1, and transfer to a hot melt extruder for melt extrusion at 40 rpm and 70°C; place the extrudate in a spheronizer, spheronize at 1200 rpm for 12 min, cool and solidify at 8°C, sieve out the pellets with a particle size of 22 meshes, and the inner core is obtained; S2, coating an intermediate layer: first mix phosphatidylserine and walnut oligopeptide, then mix with a shellac solution with a mass and volume concentration of 4% at a mass ratio of 1:9, homogenize at a pressure of 50 MPa for 15 min to obtain coating liquid A; place the inner core in a fluidized bed, control the inlet air temperature at 37°C, the material temperature at 32°C, and the atomization pressure at 1.0 bar, spray coating liquid A at a rate of 7.5 mL / min to form an enteric intermediate layer, and obtain double-layer pellets; S3, coating outer layer: dissolve theophylline and a 3% hydroxypropyl methylcellulose solution with a mass ratio of 1:4 to obtain coating liquid B; place the double-layer pellets in the fluidized bed, control the inlet air temperature at 32°C, the material temperature at 27°C, the atomization pressure at 0.8 bar, and spray the coating liquid B at a rate of 10 mL / min to form a rapid-release outer layer, thereby obtaining three-layer pellets; S4, post-processing: dry the three-layer structure pellets at 40°C for 1.5 h to obtain the theophylline composition for improving memory function.

[0035] Comparative Example 3 The theophylline composition is composed of the following raw materials in mass fraction: theophylline 12.5 parts, phosphatidylcholine 3.5 parts, walnut oligopeptide 2 parts, and lion's mane mushroom extract 2 parts.

[0036] A preparation method of a theophylline composition for improving memory function, comprising the following steps: S1, preparing the inner layer core: melt and blend glycerol monostearate and hydrogenated palm oil at a mass ratio of 4:1 at 80°C to obtain a hydrophobic matrix, mix the hydrophobic matrix and lion's mane mushroom extract at a mass ratio of 3:1, and transfer to a hot melt extruder for melt extrusion at 40 rpm and 70°C; place the extrudate in a roller compactor, roll for 12 min at 1200 rpm, and then cool and solidify at 8°C; screen the pellets with a particle size of 22 mesh to obtain the inner layer core; S2, coating the intermediate layer: first mix phosphatidylcholine and walnut oligopeptide, then mix with a 4% shellac solution with a mass ratio of 1:9, and homogenize at 50 MPa for 15 min to obtain coating liquid A; place the inner layer core in the fluidized bed, control the inlet air temperature at 37°C, the material temperature at 32°C, the atomization pressure at 1.0 bar, and spray the coating liquid A at a rate of 7.5 mL / min to form an enteric intermediate layer, thereby obtaining double-layer pellets; S3, coating the outer layer: dissolve theophylline and a 3% hydroxypropyl methylcellulose solution with a mass ratio of 1:4 to obtain coating liquid B; place the double-layer pellets in the fluidized bed, control the inlet air temperature at 32°C, the material temperature at 27°C, the atomization pressure at 0.8 bar, and spray the coating liquid B at a rate of 10 mL / min to form a rapid-release outer layer, thereby obtaining three-layer pellets; S4, post-processing: dry the three-layer structure pellets at 40°C for 1.5 h to obtain the theophylline composition for improving memory function.

[0037] Comparative Example 4 The theophylline composition is composed of the following raw materials in mass fraction: theophylline 12.5 parts, phosphatidylcholine 3.5 parts, walnut oligopeptide 2 parts, and lion's mane mushroom extract 2 parts.

[0038] A preparation method of a tea theanine composition for improving memory function, comprising the following steps: S1, preparing an inner core: melting and blending glycerol monostearate and hydrogenated palm oil at 80°C at a mass ratio of 4:1 to obtain a hydrophobic matrix, uniformly mixing the hydrophobic matrix and tea theanine at a mass ratio of 3:1, and transferring to a hot melt extruder for melt extrusion at 40 rpm and 70°C; placing the extrudate into a rounding machine, rounding for 12 min at 1200 rpm, and then cooling and solidifying at 8°C, and sieving the pellets with a particle size of 22 meshes to obtain the inner core; S2, coating an intermediate layer: mixing lion's mane extract and a shellac solution with a mass volume concentration of 4% at a mass ratio of 1:9, and homogenizing at a pressure of 50 MPa for 15 min to obtain a coating liquid A; placing the inner core in a fluidized bed, controlling the inlet air temperature at 37°C, the material temperature at 32°C, the atomization pressure at 1.0 bar, and spraying the coating liquid A at a rate of 7.5 mL / min to form an enteric intermediate layer, thereby obtaining double-layer pellets; S3, coating an outer layer: first mixing phosphatidylserine and walnut oligopeptide, and then mixing and dissolving with a hydroxypropyl methyl cellulose solution with a mass volume concentration of 3% at a mass ratio of 1:4 to obtain a coating liquid B; placing the double-layer pellets in a fluidized bed, controlling the inlet air temperature at 32°C, the material temperature at 27°C, the atomization pressure at 0.8 bar, and spraying the coating liquid B at a rate of 10 mL / min to form a rapid-release outer layer, thereby obtaining triple-layer pellets; S4, post-treatment: drying the triple-layer structure pellets at 40°C for 1.5 h to obtain the tea theanine composition for improving memory function.

[0039] Zebrafish experiment verification of tea theanine for improving memory function 1. Experimental purpose To evaluate the memory improvement effect and safety of core ingredient tea theanine on memory deficiency model zebrafish, and to provide direct and strong experimental basis for taking tea theanine as the core functional ingredient in the composition.

[0040] 2. Experimental materials (1) Test sample: tea theanine; (2) Experimental animals, feeding method and environment: wild type AB strain zebrafish, bred by natural pair mating, and the age is 4 days after fertilization (4dpf); The zebrafish were bred in fish water at 28°C, 200 mg of instant sea salt was added to each 1 L of reverse osmosis water, the conductivity was 480-510 μS / cm, the pH was 6.9-7.2, and the hardness was 53.7-71.6 mg / L CaCO3; The experimental animal use license number is: SYXK (Zhejiang) 2012-0171. The feeding management meets the requirements of international AAALAC certification.

[0041] (3) Modeling reagent: aluminum chloride hexahydrate (AlCl3·6H2O), prepared into a stock solution with a concentration of 33.8 mg / mL using ultrapure water before use, and the working solution has a concentration of 33.8 μg / mL; (4) Positive control: donepezil hydrochloride tablets, prepared into a stock solution with a concentration of 3.33 mg / mL using DMSO before use, and the working solution has a concentration of 3.33 μg / mL.

[0042] 3. Experimental method and results of maximum tolerance concentration (MTC) determination (1) Establishment of memory deficiency model: Modeling group: 4 dpf zebrafish were randomly selected and treated with 33.8 μg / mL aluminum chloride hexahydrate aqueous solution for 48 hours by water exposure to establish a memory deficiency model. This group was then divided into a model control group and various test sample groups; Normal control group: treated with the same volume of fish water at the same time; After the treatment, the zebrafish in the modeling group showed phenotypes such as reaction delay and lateral turning, and the acetylcholinesterase (AchE) fluorescence signal intensity was significantly higher than that of the normal control group, indicating that the memory deficiency model was successfully established.

[0043] (2) The grouping and treatment are as follows: Model control group: 30 zebrafish from the modeling group were given 3 mL of fish water; Tea theanine concentration gradient group: 180 zebrafish from the modeling group were randomly divided into 6 groups (n=30) and exposed to 3 mL of 62.5, 125, 250, 500, 1000, and 2000 μg / mL tea theanine aqueous solution, respectively; Normal control group: composed of 30 zebrafish that did not undergo modeling, and given 3 mL of fish water at the same time.

[0044] After 48 hours of treatment at 28°C, the number of dead zebrafish and abnormal phenotypes in each group were counted to determine the maximum tolerance concentration of tea theanine.

[0045] (3) Experimental results: As shown in Table 1, tea theanine at concentrations of 1000 μg / mL and 2000 μg / mL caused the death of all zebrafish (100% mortality rate). Within the concentration range of 62.5-500 μg / mL, no zebrafish died, and their state was similar to that of the model control group (showing reaction delay and lateral turning). Therefore, the maximum tolerance concentration (MTC) of tea theanine was determined to be 500 μg / mL under the experimental model.

[0046] Table 1. Results of concentration exploration experiment of tea theanine on memory deficiency model zebrafish (n=30)

[0047] 4. Memory improvement effect evaluation experiment method and results (1) Memory deficiency model establishment: Modeling group: 4 dpf zebrafish were randomly selected and treated with 33.8 μg / mL aluminum chloride hexahydrate aqueous solution for 48 hours by water exposure to establish a memory deficiency model. This group was then divided into a model control group, a positive control group, and various test sample groups; Normal control group: The same volume of fish water was used for treatment at the same time; After treatment, the zebrafish in the modeling group showed phenotypes such as reaction delay and lateral turning, and the acetylcholinesterase (AchE) fluorescence signal intensity was significantly higher than that of the normal control group, indicating that the memory deficiency model was successfully established.

[0048] (2) Grouping and administration: Model control group: 30 zebrafish from the modeling group were given 3 mL of fish water; Positive control group: 30 zebrafish from the modeling group were given 3 mL of 3.33 μg / mL donepezil solution; Low, medium, and high dose groups of tea theanine (test product group): 90 zebrafish from the modeling group were randomly divided into 3 groups (n=30) and given 3 mL of 125 μg / mL, 250 μg / mL, and 500 μg / mL tea theanine solution, respectively; Normal control group: 30 zebrafish that had not undergone modeling were used, and 3 mL of fish water was given at the same time.

[0049] After incubation in a 28°C incubator for 48 hours, the acetylcholinesterase (AchE) fluorescence signal intensity (S2) was detected using an AchE assay kit and a multifunctional enzyme marker. The memory improvement effect of tea theanine on the memory deficiency model zebrafish was evaluated based on the statistical analysis results of the AchE fluorescence signal intensity.

[0050] (3) The calculation formula for memory improvement effect is: Memory improvement effect (%) = [S2 (model control group) - S2 (test product group)] / S2 (model control group) x 100%; where S2 is the AchE fluorescence signal intensity. Experimental data was analyzed using GraphPad Prism software for single-factor analysis of variance, followed by Dunnett's T-test for comparison between groups, with p<0.01 indicating a highly significant difference.

[0051] (4) Experimental results: As shown in Table 2, the AchE fluorescence signal intensity of the model control group (22979±2368) was significantly higher than that of the normal control group, indicating that the memory deficiency model was successfully established. The signal intensity of the positive drug donepezil group decreased to 13302±1657, and the memory improvement effect was 42%, proving that the experimental system was reliable. The tea theanine groups of each concentration showed a very significant memory improvement effect: 125 μg / mL group: signal intensity decreased to 12824±2768, memory improvement effect reached 44%; 250 μg / mL group: signal intensity decreased to 12569±1999, memory improvement effect reached 45%; 500 μg / mL group: signal intensity decreased to 11853±1613, memory improvement effect reached 48%.

[0052] Table 2 Evaluation of the memory improvement effect of tea theanine on memory deficiency model zebrafish

[0053] Note: compared with the model control group, *p<0.05, **p<0.01.

[0054] 5. Experimental conclusion The experimental results show that tea theanine at a concentration of 500 μg / mL and below shows good safety for memory deficiency zebrafish, and within the safe concentration range of 125-500 μg / mL, it can significantly reduce the activity of acetylcholinesterase (AchE) in the brain of zebrafish, and the memory improvement effect reaches 44-48%, which is comparable to the clinical first-line drug donepezil (42%), and even better in numerical value. From the aspects of safety and efficacy, this experiment fully verifies that tea theanine as the core component of the memory improvement functional composition of the present application has a solid scientific basis and significant effectiveness.

[0055] Verification of the effect of tea theanine composition on improving memory function 1. Experimental purpose Through standardized animal behavior experiments, the comprehensive effect of the tea theanine composition (Examples 1-3) provided in the present application on improving memory function is systematically evaluated, and through comparison with Comparative Examples 1-4, the unexpected technical advantages brought by "tea theanine fast-release outer layer", "the irreplaceability of tea theanine", "the specificity of phosphatidylserine", and "the scientificity of time-release structure" are verified.

[0056] 2. Experimental materials (1) Test sample: sample prepared in Examples 1-3; (2) Control sample: sample prepared in Comparative Examples 1-4; (3) Experimental animals: healthy adult ICR mice, male, body weight 18-22 g; (4) Main reagents and instruments: anisodamine, Morris water maze system, new object recognition experiment box.

[0057] 3. Test method (1) Animal grouping and administration: The mice were randomly divided into 9 groups, 12 in each group, and the grouping and treatment were as follows: Blank control group: intragastrically administered with the same volume of normal saline; Model control group: intragastrically administered with the same volume of normal saline; Example 1-3 groups: intragastrically administered with the sample suspensions of Examples 1-3 (calculated based on theanine, the dose was 50 mg / kg); Comparative Example 1-4 groups: intragastrically administered with the sample suspensions of Comparative Examples 1-4 (the total dose of functional components was equivalent to that of the example groups); All groups were continuously administered for 28 days, and the behavior test was performed after the last administration.

[0058] (2) Preparation of memory acquisition disorder model: 30 minutes before the behavior test, the mice in each group except the blank control group were intraperitoneally injected with anisodamine (2 mg / kg) to induce memory acquisition disorder. The blank control group was injected with the same volume of normal saline.

[0059] (3) Behavior test - Morris water maze experiment: Positioning navigation experiment: for 5 consecutive days, the mice were placed into the water from four different entry points with the wall facing them, and the time required to find the hidden platform under the water (escape latency) was recorded.

[0060] Spatial exploration experiment: on the 6th day, the platform was removed, and the mice were placed into the water from the opposite quadrant of the original platform, and the number of times they crossed the original platform location within 60 seconds and the time they spent in the target quadrant were recorded.

[0061] (4) Behavior test - new object recognition experiment: Adaptation period: the mice were placed individually into the experimental box with an empty field and allowed to explore freely for 5 minutes; Training period: 24 hours later, two identical objects (A and A') were placed, and the mice were allowed to explore for 10 minutes; Test period: 2 hours after training, one familiar object (A) was replaced with a new object (B), and the mice were allowed to explore again for 5 minutes; the time they spent exploring the new object and the familiar object was recorded.

[0062] Evaluation index: recognition index (RI) = exploration time of new object / (exploration time of new object + exploration time of familiar object) x 100%.

[0063] 4. Experimental results (1) Morris water maze experiment results and analysis: As shown in Table 3, the example groups showed significant advantages in escape latency, platform crossing times and target quadrant residence time.

[0064] Table 3. Morris water maze test results of mice in each group (n = 12, Mean ± SD)

[0065] Note: Compared with the model control group, *p <0.05, **p <0.01.

[0066] Result analysis: The escape latency of example groups 1-3 was significantly shorter than that of the model control group and all comparative example groups, and their spatial memory retention ability was better than that of all comparative example groups. Comparative example 1 and comparative example 4 had the worst effect, and there was no significant difference from the model control group, proving that the time sequence structure is the key to the effect. Comparative example 2 performed worse than the model group, proving the irreplaceability of theanine. Comparative example 3 performed better than comparative example 1, but significantly worse than the example groups, proving the structural specificity of phosphatidylserine.

[0067] (2) New object recognition experiment results and analysis: As shown in Table 4, the example groups also showed the best effect in new object recognition ability.

[0068] Table 4. New object recognition test results of mice in each group (n = 12, Mean ± SD)

[0069] Note: Compared with the model control group, *p <0.05, **p <0.01.

[0070] Result analysis: The recognition index of example groups 1-3 was the highest, and was significantly higher than that of the model control group and all comparative example groups. The effect of each comparative example group was significantly lower than that of the example group, and the effect ranking was: example > comparative example 3 > comparative example 1 ≈ comparative example 4 > comparative example 2. This result is highly consistent with the conclusion of the water maze experiment, and together confirms the core advantage of the present application.

[0071] 5. Experimental conclusion The experimental results show that the structure of theanine composition of tea leaves is crucial, the components are irreplaceable, and the release timing of "rapid start (theanine) → mid-way enhancement (phosphatidylserine + walnut oligopeptide) → long-term consolidation (lion's mane mushroom extract)" is the only optimal biological solution.

[0072] The present application delivers specific and irreplaceable functional components at precise time points through a unique time-release structure, resulting in unexpected synergistic effects in the whole memory formation cycle that cannot be achieved by simple mixing, random replacement or time reversal of the components, fully demonstrating the creativity and technical progressiveness of the present application.

[0073] The above specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the present embodiments without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A tea L-theanine composition for improving memory function, characterized by, The tea theanine composition is a micro-pellet with a three-layer core-shell structure, the outer layer contains tea theanine; the middle layer contains phosphatidylserine and walnut oligopeptide; and the inner layer core contains lion's mane mushroom extract.

2. The tea L-theanine composition for improving memory function according to claim 1, characterized by, The outer layer is a quick-release layer composed of water-soluble film-forming material; the middle layer is an enteric layer composed of pH-dependent polymer; and the inner layer core is a sustained-release layer composed of hydrophobic matrix.

3. The tea L-theanine composition for improving memory function according to claim 2, characterized by, The water-soluble film-forming material is hydroxypropyl methyl cellulose; the pH-dependent polymer is shellac; and the hydrophobic matrix is a mixture of hydrogenated palm oil and glycerol monostearate.

4. The tea L-theanine composition for improving memory function according to claim 1, wherein The tea theanine composition is composed of the following raw materials in mass fraction: tea theanine 10-15 parts, phosphatidylserine 2-5 parts, walnut oligopeptide 1-3 parts, and lion's mane mushroom extract 1-3 parts.

5. A method of preparing the tea L-theanine composition for improving memory function according to any one of claims 1 to 4, characterized by, The method comprises the following steps: S1, preparing the inner layer core: melt-blending the hydrophobic matrix, mixing with the lion's mane mushroom extract, and preparing the inner layer core by hot melt extrusion and spheronization; S2, coating the middle layer: homogeneously dispersing the phosphatidylserine and walnut oligopeptide in the pH-dependent polymer to obtain coating liquid A, and spraying the coating liquid A on the surface of the inner layer core by fluidized bed coating technology to form the enteric middle layer, thereby obtaining the double-layer micro-pellet; S3, coating the outer layer: dissolving the tea theanine in the water-soluble film-forming material to obtain coating liquid B, and spraying the coating liquid B on the surface of the enteric middle layer by fluidized bed coating technology to form the quick-release outer layer, thereby obtaining the three-layer micro-pellet; S4, post-treatment: drying the three-layer structure micro-pellet at low temperature, thereby obtaining the tea theanine composition for improving memory function.

6. The method of preparing the tea L-theanine composition for improving the memory function according to claim 5, characterized in that, The specific operation method of step S1 is as follows: melt-blending glycerol monostearate and hydrogenated palm oil at a mass ratio of 3-5:1 at 75-85°C to obtain the hydrophobic matrix, uniformly mixing the hydrophobic matrix and lion's mane mushroom extract at a mass ratio of 2-4:1, transferring to a hot melt extruder, melt-extruding at 30-50 rpm and 65-75°C, placing the extrudate in a spheronizer, spheronizing at 1000-1500 rpm for 10-15 min, cooling and solidifying at 4-10°C, and sieving the pellets with a particle size of 20-24 mesh, thereby obtaining the inner layer core.

7. The method for preparing the tea theanine composition for improving memory function according to claim 5, characterized in that, The specific operation method of step S2 is as follows: mixing the phosphatidylserine and walnut oligopeptide, mixing with a shellac solution with a mass and volume concentration of 3-5% at a mass ratio of 1:8-10, homogenizing at a pressure of 40-60 MPa for 10-20 min to obtain the coating liquid A; placing the inner layer core in a fluidized bed, controlling the inlet air temperature at 35-40°C, the material temperature at 30-35°C, the atomization pressure at 0.8-1.2 bar, spraying the coating liquid A at a rate of 5-10 mL / min, forming the enteric middle layer, and obtaining the double-layer micro-pellet.

8. The method for preparing the tea theanine composition for improving memory function according to claim 5, characterized in that, The specific operation method of step S3 is: mixing and dissolving tea theanine with a hydroxypropyl methylcellulose solution with a mass concentration of 2-4% at a mass ratio of 1:3-5 to obtain a coating solution B; placing the double-layer pellets in a fluidized bed, controlling the inlet air temperature at 30-35°C, the material temperature at 25-30°C, the atomization pressure at 0.6-1.0 bar, and spraying the coating solution B at a rate of 8-12 mL / min to form a rapid-release outer layer, thereby obtaining three-layer pellets.

9. The method for preparing the tea theanine composition for improving memory function according to claim 5, characterized in that, The temperature for low-temperature drying in step S4 is 35-45°C, and the time is 1-2 h.

10. Use of the tea theanine composition for improving memory function according to any one of claims 1-4 in the preparation of a functional food, a health food or a medicine for improving attention, improving learning and memory ability or delaying cognitive decline.

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