Composition for preventing and / or improving brain dysfunction

Abstract

The invention provides a composition for preventing and / or improving brain dysfunction, especially Alzheimer's disease, the composition comprises dihydromyricetin or a salt thereof, L-theanine, taurine and cerebroside, the components act together to enhance the intestinal stability and bioavailability of dihydromyricetin, and the bioavailability of dihydromyricetin is improved. The brain dysfunction is prevented and improved through multiple mechanisms of resisting inflammation, inhibiting DNA methylation, eliminating hematoma, clearing A beta and the like. In addition, the composition can be used in combination with a monoclonal antibody drug, so that adverse reactions such as encephaledema and cerebral hemorrhage possibly occurring in the treatment process of the Alzheimer's disease are reduced.

Description

Technical Field

[0001] The present invention relates to a composition for preventing and / or improving brain dysfunction, in particular to a composition for preventing and / or improving Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by neuronal loss, neurofibrillary tangles, and senile plaque formation in the cerebral cortex. The primary mechanisms of AD involve the deposition of β-amyloid protein (Aβ), abnormal phosphorylation of tau protein, neuroinflammation, and oxidative stress. These factors interact to lead to neuronal dysfunction and death, which in turn affects memory, cognition, and behavior.

[0003] Currently, AD treatment strategies primarily include pharmacological and non-drug therapies. In terms of pharmacological treatment, monoclonal antibodies such as aducanumab are used to target Aβ deposits. However, these drugs may cause adverse reactions, such as cerebral edema and cerebral hemorrhage, limiting their clinical application. Therefore, in addition to traditional drug therapies, novel adjunctive therapies are needed for the treatment of AD.

[0004] Looking ahead, the ideal therapeutic product should be able to ameliorate AD pathology, alleviate symptoms, or provide brain protection, while avoiding adverse reactions such as cerebral edema. As an adjunctive therapy, when used in conjunction with therapeutic drugs, it can mitigate or eliminate adverse drug reactions. Natural products show great potential in this area due to their safety and multi-target mechanisms of action. However, many natural products suffer from drawbacks such as poor solubility, unstable physicochemical properties, and low bioavailability, which limit their therapeutic efficacy.

[0005] In response to the above-mentioned shortcomings of existing AD treatment products and natural products with the potential to improve the progression of AD, the present invention aims to enhance their potential in the treatment of Alzheimer's disease through a new natural product composition, and to provide a safe and effective product with both therapeutic and auxiliary therapeutic effects, in order to play an important role in the treatment and management of AD. Summary of the Invention

[0006] In order to solve the problems of obvious adverse reactions of AD treatment products and low bioavailability of natural product compositions, the present invention provides a drug combination of natural products, which has preventive, therapeutic and ameliorative effects, can enhance the treatment effect and increase safety and stability.

[0007] Specifically, the present invention aims to provide a composition for preventing and / or improving brain dysfunction, wherein the composition comprises: dihydromyricetin or a salt thereof, L-theanine, taurine and cerebrosides.

[0008] Preferably, the composition comprises, by weight: 10-40 parts of dihydromyricetin or its salt, 1-5 parts of L-theanine, 1-5 parts of taurine and 1-10 parts of cerebroside.

[0009] Preferably, in the composition, the mass ratio of DHM or its salt to taurine is (2-40):1; and / or the mass ratio of DHM or its salt to L-theanine is (2-40):1; and / or the mass ratio of DHM or its salt to cerebroside is (1-40):1.

[0010] Preferably, the composition further comprises one or more of ascorbic acid, spinosum, linoleic acid or phosphatidylserine.

[0011] Preferably, the cerebroside is selected from one or more of sea cucumber cerebroside, cordycerebroside, pistachio cerebroside or soybean cerebroside; preferably 1-O-β-D-glucosyl-(2S, 3R, 4E, 8Z)-2-[(2-hydroxyoctadecanoyl)amino]-4,8-octadecadiene-1,3-diol and / or 1-O-β-D-glucosyl-(2S, 3R, 4E, 8Z)-2-[(2-hydroxyhexadecanoyl)amino]-4,8-octadecadiene-1,3-diol; more preferably 1-O-β-D-glucosyl-(2S, 3R, 4E, 8Z)-2-[(2-hydroxyoctadecanoyl)amino]-4,8-octadecadiene-1,3-diol.

[0012] Another object of the present invention is to provide a preparation for preventing and / or improving brain dysfunction, comprising dihydromyricetin or its salt, L-theanine, taurine and cerebrosides, as well as a carrier and / or excipients.

[0013] When the above-mentioned active ingredients are prepared as a single preparation, there is no particular limitation. For every 1 part by weight of dihydromyricetin or its salt, 0.01 to 10 parts by weight of L-theanine, taurine, and cerebroside can be prepared. Based on a comprehensive consideration of effect and cost, for every 1 part by weight of dihydromyricetin or its salt, 0.05 to 5 parts by weight of L-theanine, taurine, and cerebroside are each preferably used, and more preferably 0.05 to 0.3 parts by weight.

[0014] Another object of the present invention is to provide a composition for use in preparing a product for preventing and / or improving brain dysfunction, wherein the composition comprises: dihydromyricetin or a salt thereof, L-theanine, taurine and cerebrosides.

[0015] The products of the present invention include: food, beverages, medicines, health products or dietary supplements.

[0016] Another object of the present invention is to provide a method for preventing and / or improving brain dysfunction, comprising administering to a patient a composition comprising dihydromyricetin or a salt thereof, L-theanine, taurine and cerebroside.

[0017] The brain dysfunction described in the present invention includes: schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, Alzheimer's disease and Parkinson's disease, and related clinical symptoms include learning difficulties, memory loss, inability to concentrate, emotional apathy, emotional despair, irritability, loss of interest, lack of pleasure, sleep disorders, fatigue, decreased or increased appetite, weight loss or weight gain, personality changes, and lack of motivation.

[0018] In the present invention, "amelioration" refers to the improvement of disease symptoms and conditions, the prevention or delay of worsening of disease symptoms and conditions, the reversal, prevention or delay of disease symptom progression, or the treatment of a disease. "Prevention" refers to preventing the onset of a disease in a subject, delaying its onset, or reducing the risk of developing a disease in a subject.

[0019] In the present invention, "treatment" refers to any treatment of a disease (eg, improvement, alleviation, inhibition of progression, etc.), and also includes preventing the onset and / or progression of a disease.

[0020] In the present invention, "patient" refers to humans and other animals, such as dogs, cats, horses, etc. The patient is preferably a mammal, more preferably a human.

[0021] The present invention also aims to provide a use of a composition comprising dihydromyricetin or a salt thereof, L-theanine, taurine, and cerebrosides in preparing a product for eliminating cerebral hematomas, particularly in preparing a product for eliminating cerebral hematomas caused by administration of monoclonal antibodies for Alzheimer's disease.

[0022] The core components of the present invention can be extracted, separated, and purified from plants and animals, or directly from dried powders or extract mixtures of plants and animals. For example, dihydromyricetin is primarily derived from plants with high dihydromyricetin content, such as Ampelopsis pilosula and Hovenia dulcis; L-theanine is primarily derived from high-theanine teas, such as Camellia sinensis and Camellia lutea; taurine can be derived from marine plants and animals, and animal offal; and cerebrosides are primarily derived from plants and animals, such as brain tissue, Cordyceps sinensis, soybeans, sea cucumbers, starfish, flax roots, and Sterculia lychnophora. For example, one or more of sea cucumber cerebroside, cordycerebroside, pistachio cerebroside or soybean cerebroside; preferably 1-O-β-D-glucosyl-(2S, 3R, 4E, 8Z)-2-[(2-hydroxyoctadecanoyl)amino]-4,8-octadecadiene-1,3-diol and / or 1-O-β-D-glucosyl-(2S, 3R, 4E, 8Z)-2-[(2-hydroxyhexadecanoyl)amino]-4,8-octadecadiene-1,3-diol.

[0023] In addition, the core components of the present invention can also be derived from total synthesis or semi-synthesis by biological or chemical methods.

[0024] The composition of the present invention may be appropriately formulated with other active ingredients (eg, substances with auxiliary effects) and / or additives by methods known per se, as long as the effect of preventing and / or improving brain dysfunction is not impaired.

[0025] Other active ingredients that can be incorporated into the composition of the present invention include, for example, vitamin A; carotenoids (excluding lutein); vitamin B; vitamin C; vitamin D; vitamin E; tocotrienols; glutathione and its derivatives or salts thereof; polyphenols such as spinosin, catechins, anthocyanins, tannins, rutin, isoflavones, chlorogenic acid, ellagic acid, curcumin, and coumarin; linoleic acid, α- or γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and their derivatives or salts thereof; proteins selected from collagen, elastin, fibronectin, and keratin and their derivatives and hydrolyzates; α-hydroxy acids such as glycolic acid, lactic acid, malic acid, citric acid, and salicylic acid and their corresponding derivatives or salts thereof; yeast, lactic acid bacteria, bifidobacteria, jujube seeds, ganoderma lucidum, ginseng, rosemary, phellodendron, Natural substances or extracts thereof such as garlic, cyperus chinensis, stephanine, aloe vera, sage, arnica, chamomile, birch, forsythia suspensa, eucalyptus, millettia reticulata, hyssopus, mulberry bark, angelica, sophora flavescens, hawthorn, coix seed, houttuynia cordata, seaweed, natto, lemongrass, ginkgo biloba extract, and phosphatidylserine; adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and other adenylate derivatives; minerals such as iron, vanadium, molybdenum, manganese, copper, potassium, magnesium, calcium, zinc, selenium, and iodine; monosaccharides such as mannitol, xylitol, and glucosamine; polysaccharides such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, glycogen, chitin, and chitosan; nucleic acids such as deoxyribonucleic acid and ribonucleic acid; other substances such as glycyrrhizic acid, guanine, mucin, ubiquinone, α-lipoic acid, octacosanol, aldehyde lysine, and alliin, and their mixtures.

[0026] The other active ingredients may be incorporated in an amount of, for example, 0.1 to 99% by weight, preferably 1 to 90% by weight, more preferably 5 to 80% by weight, further preferably 10 to 70% by weight, and particularly preferably 20 to 60% by weight relative to the total weight of the composition. Two or more other active ingredients may also be used in combination.

[0027] The compositions comprising the present invention are formulated for administration by a route selected from the group consisting of oral, parenteral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural.

[0028] The composition is preferably taken orally. The oral dosage form is not particularly limited and may be any oral dosage form known in the art, preferably tablets, capsules, suspensions, oral solutions, and the like. When used as an oral dosage form, the dosage standard used is, for example, 500-1500 mg / day, preferably 700-1200 mg / day, preferably 800-1000 mg / day, and most preferably 1000 mg / day.

[0029] The duration of administration of the composition of the present invention may depend on the severity of the disease, and is preferably at least 1 month, for example, 1, 2, 3, 4, 5 or 6 months, and may be lifelong if required by the disease.

[0030] According to an embodiment of the present invention, the composition may further comprise a pharmaceutically acceptable excipient; preferably, the excipient is selected from at least one of the following excipients, including but not limited to: a filler, a disintegrant, a binder, a lubricant, a surfactant, a flavoring agent, a wetting agent, a pH adjuster, a solubilizer or cosolvent, and an osmotic pressure regulator. Those skilled in the art can easily determine how to select the appropriate excipient and its corresponding dosage according to the requirements of the specific dosage form.

[0031] Beneficial effects

[0032] The composition of the present invention can enhance the stability of dihydromyricetin in the intestinal environment and improve the bioavailability of dihydromyricetin.

[0033] The composition of the present invention can prevent and improve brain dysfunction through multiple mechanisms of action, such as anti-inflammation, inhibition of NDA methylation, elimination of hematoma, and gentle clearance of Aβ.

[0034] The composition of the present invention is expected to be used in combination with monoclonal antibody drugs for treating AD, while helping to clear Aβ and reducing adverse reactions caused by antibody drugs, such as cerebral edema (ARIA-E), cerebral hemorrhage (ARIA-H) and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Stability test of DHM and its combination in SIF

[0036] Figure 2 : DHM and its composition in SIF+Fe 3+ Stability experiment in

[0037] Figure 3 : Hematoma volume on the first day of modeling

[0038] Figure 4 : Hematoma volume on the third day of modeling

[0039] Figure 5 :AGEs content detection

[0040] Figure 6 :Aβ1-42 content detection

[0041] Figure 7 :IL-1β content detection

[0042] Figure 8 : TNF-α content detection

[0043] Figure 9 :IL-6 content detection

[0044] Figure 10 DNMT1 activity assay

[0045] Definitions and Explanations of Terms

[0046] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The group definitions and compound structures resulting from such combinations and couplings should be understood to be within the scope of this specification and / or claims.

[0047] In this application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0048] References in the specification to "one embodiment," "an embodiment," etc. indicate that the described embodiment may include a particular aspect, feature, structure, part, or characteristic, but not every embodiment must include that aspect, feature, structure, part, or characteristic. Furthermore, such phrases may, but do not necessarily, refer to the same embodiment referenced in other parts of the specification. Furthermore, when a particular aspect, feature, structure, unit, or characteristic is described in conjunction with an embodiment, whether or not explicitly described, it is within the knowledge of those skilled in the art to influence or relate that aspect, feature, structure, unit, or characteristic to other embodiments.

[0049] As will be understood by those skilled in the art, all numerical values, including those expressing the amounts of ingredients, properties such as molecular weight, reaction conditions, etc., are approximate and are understood to be optionally modified in all instances by the term "about". These values ​​may vary depending on the desired properties sought to be obtained by those skilled in the art utilizing the teachings described herein. It will also be understood that these values ​​inherently contain variability necessarily resulting from the standard deviation found in their respective testing measurements.

[0050] Whenever the term "comprising" is used herein, the option of using the term "consisting of," "comprising or consisting essentially of," is contemplated. As used herein, "comprising" is synonymous with "including" or "characterized by," and is inclusive or open-ended and does not exclude additional, unlisted elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel characteristics of the aspect. In each instance herein, any one of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms. The disclosure illustratively described herein may be appropriately implemented without any element or elements not specifically disclosed herein. DETAILED DESCRIPTION

[0051] The following examples are intended to illustrate the above invention and should not be construed as narrowing its scope. Those skilled in the art will readily appreciate that the embodiments have proposed many other ways of implementing the present invention. It should be understood that many changes and modifications may be made while remaining within the scope of the present invention.

[0052] Example 1 Stability test

[0053] Dihydromyricetin (DHM) is relatively stable in acidic environments with a pH of 3 to 6, but is easily decomposed in alkaline environments. In simulated intestinal fluid (SIF), DHM undergoes a degradation process similar to first-order kinetics. Metal ions such as Fe 3+ The presence of can accelerate the oxidation process of DHM, thereby reducing its stability in intestinal fluid. The purpose of this example is to study the effect of the interaction of the core components of the composition of the present invention on the stability of DHM.

[0054] Experimental plan:

[0055] Solutions AD and A1-D1 were prepared with the following components and concentrations, each with a volume of 10 mL. The mixed solutions were placed in a 37°C water bath and the residual DHM content was determined by HPLC at different time intervals.

[0056] Solution A: SIF + DHM (1 mg / mL);

[0057] Solution B: SIF + DHM (1 mg / mL) + taurine (0.3 mg / mL)

[0058] Solution C: SIF + DHM (1 mg / mL) + L-theanine (0.4 mg / mL);

[0059] Solution D: SIF + DHM (1 mg / mL) + taurine (0.1 mg / mL) + L-theanine (0.1 mg / mL)

[0060] Solution A1: SIF + DHM (1 mg / mL) + Fe 3+ (50 μmol / L);

[0061] Solution B1: SIF + DHM (1 mg / mL) + taurine (0.3 mg / mL) + Fe 3+ (50 μmol / L);

[0062] Solution C1: SIF + DHM (1 mg / mL) + L-theanine (0.4 mg / mL) + Fe 3+ (50 μmol / L);

[0063] Solution D1: SIF + DHM (1 mg / mL) + taurine (0.1 mg / mL) + L-theanine (0.1 mg / mL) + Fe 3+ (50μmol / L).

[0064] according to Figure 1 Data from the Institute of Pharmaceutical Sciences (IMPRESS) show that dihydromyricetin (DHM) is readily degraded in simulated intestinal fluid (SIF), with the remaining DHM amount dropping below 50% after a four-hour incubation period. Experiments have shown that adding only taurine or L-theanine to a formula can slow DHM degradation, while adding both taurine and L-theanine significantly slows DHM degradation.

[0065] Figure 2 It further demonstrated the presence of Fe in SIF solution. 3+ When the cells were incubated for 4 hours, the degradation rate of DHM would be accelerated. In this case, the delay effect of adding taurine or L-theanine alone was not obvious, but if taurine and L-theanine were added at the same time, the degradation of DHM could still be significantly delayed, so that the DHM retention after 4 hours of incubation exceeded 90%.

[0066] In addition, the experiment also studied the 3+The effects of different amounts of taurine and L-theanine on delaying DHM degradation in the presence of taurine and L-theanine were investigated. The results showed that when the mass ratio of DHM, taurine, and L-theanine was controlled within the range of 10-20:1-5:1-5, the degradation of DHM in SIF could be significantly delayed, ensuring that the retention of DHM after 4 hours of incubation was maintained at more than 90%.

[0067] Example 2 Bioavailability Experiment

[0068] Dihydromyricetin (DHM) has low solubility in water and is unstable in alkaline environments, which limits its oral bioavailability. This experiment aims to evaluate the potential effect of the composition of the present invention on improving the bioavailability of DHM.

[0069] Experimental design:

[0070] Experimental subject groups: 24 male SD rats, weighing between 200-220 g, were randomly divided into four groups.

[0071] Dosage regimen:

[0072] Group A (control group): DHM was administered alone at a dose of 100 mg / kg.

[0073] Group B: DHM (100 mg / kg) and L-theanine (10 mg / kg) were administered in combination.

[0074] Group C: DHM (100 mg / kg) and taurine (10 mg / kg) were administered together.

[0075] Group D: DHM (100 mg / kg), L-theanine (10 mg / kg) and taurine (10 mg / kg) were administered in combination.

[0076] Dosage:

[0077] The drug was mixed with 3 mL of 0.5% CMC-Na solution and administered by oral gavage.

[0078] Sample collection and processing:

[0079] At 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours after administration, 200 μL of blood samples were collected through the tail vein and placed in heparinized EP tubes.

[0080] Blood samples were centrifuged at 5000 rpm for 15 min at 4°C, and plasma was collected and stored at -20°C.

[0081] Before analysis, 30 μL of plasma sample was taken, 30 μL of internal standard (rutin) solution and 140 μL of 1% formic acid-acetonitrile solution were added, vortexed for 1 minute, and centrifuged at 12000 rpm for 5 minutes. 150 μL of the supernatant was taken for LC-MS / MS analysis.

[0082] Purpose of analysis:

[0083] The concentration of DHM in plasma was measured at different time points to evaluate the effect of the composition on the bioavailability of DHM.

[0084] Table 1 Bioavailability experiment

[0085]

[0086] According to the data in Table 1:

[0087] Improved bioavailability: Compared with group A (DHM alone), the bioavailability of C in groups B, C, and D was significantly improved. max (maximum blood concentration), t 1 / 2 (half-life), AUC 0→t (area under the curve from administration to a specific time), AUC 0→∞ Key pharmacokinetic parameters such as area under the curve (area under the curve from dosing to infinity) and CL (clearance) were significantly improved, indicating that the addition of L-theanine and / or taurine can significantly enhance the bioavailability of DHM.

[0088] Combined effect: Group D (DHM combined with L-theanine and taurine) showed the best effect in all pharmacokinetic parameters. max It is more than 3 times that of group A. 1 / 2 It is more than 2.5 times that of group A, and AUC 0→∞ The CL of group D was significantly higher than that of group A, while the CL was significantly lower than that of group A. These results showed that the absorption, distribution, metabolism and excretion of DHM in group D were better than those in other groups.

[0089] Statistical significance: Group D compared with Group B and Group C, max , t 1 / 2 , AUC 0→∞ Statistically significant differences were also shown in parameters such as DHM and CL (P < 0.01), which further confirmed the superiority of the combined administration of DHM with L-theanine and taurine in improving bioavailability.

[0090] Example 3 Brain hematoma experiment

[0091] In the treatment of Alzheimer's disease (AD), the use of monoclonal antibody drugs may induce amyloid-associated imaging abnormalities (ARIA), which is an issue that needs to be strictly monitored during the treatment process. Studies have shown that these drugs quickly clear amyloid (Aβ) in the short term, which may cause Aβ deposition on the blood vessel walls, thereby affecting the integrity of the blood vessels and ultimately leading to the occurrence of ARIA. The main symptoms of ARIA include cerebral edema and cerebral hemorrhage, and the occurrence of these symptoms is directly related to the dosage and frequency of administration of the drug. The purpose of this experiment is to simulate and evaluate the effect of the composition of the present invention on cerebral hematoma, which is of great significance for improving AD treatment plans and reducing potential risks during treatment.

[0092] This study aimed to evaluate the effects of different compositions on hematoma volume in a C57BL / 6N mouse intracerebral hemorrhage (ICH) model. A total of 72 male C57BL / 6N mice, aged 8 to 10 weeks, were randomly divided into six groups and treated with oral gavage for 7 days. Subsequently, an ICH model was induced using the collagenase method in all groups except the sham-operated group (CON). The experimental groups and treatments were as follows:

[0093] CON group: CMC-Na solution was gavaged orally for 7 days, followed by injection of PBS into the basal ganglia.

[0094] MOD group: mice were gavaged with CMC-Na solution for 7 days, and then collagenase was injected into the basal ganglia region, serving as the model control group.

[0095] Group G1: DHM (100 mg / kg·d) was administered orally for 7 days, followed by injection of collagenase into the basal ganglia.

[0096] Group G2: DHM (50 mg / kg·d), L-theanine (5 mg / kg·d), and taurine (5 mg / kg·d) were gavaged for 7 days, followed by injection of collagenase into the basal ganglia.

[0097] G3 group: DHM (30 mg / kg·d), L-theanine (3 mg / kg·d), taurine (3 mg / kg·d) and cerebroside (3 mg / kg·d) were gavage for 7 days, followed by injection of collagenase into the basal ganglia region.

[0098] G4 group: L-theanine (5 mg / kg·d), taurine (5 mg / kg·d) and cerebroside (10 mg / kg·d) were gavage for 7 days, followed by injection of collagenase into the basal ganglia.

[0099] On the first and third day after modeling, 6 mice were randomly selected from each group for decapitation and brain removal. The brain tissue was cut into slices on ice, and the hematoma volume was calculated using ImageJ software.

[0100] Based on the hematoma volume measurements on days 1 and 3, the following conclusions can be drawn (e.g. Figure 3 , as shown in 4):

[0101] Day 1: Compared with the MOD group, the hematoma volume of the G1 and G4 groups was reduced, while the hematoma volume of the G2 and G3 groups decreased more significantly, indicating that these compositions can reduce the hematoma caused by ICH to some extent.

[0102] Day 3: The hematoma volume of all treatment groups was significantly reduced compared with the MOD group, among which the effect of G3 group was the most significant, which may mean that the composition of G3 group has potential therapeutic effect in reducing hematoma caused by ICH.

[0103] Example 4 Brain Function Improvement Experiment

[0104] Alzheimer's disease (AD) is a complex neurodegenerative disease whose pathogenesis involves multiple factors that jointly promote the pathological process of AD. This example will evaluate the effects of different compositions on the pathological process of AD and their potential effects on improving brain function. By comparing the activity of biomarkers such as AGEs, Aβ1-42, inflammatory factors (IL-1β, TNF-α, IL-6) and DNA methylase (DNMT1) in the brain tissue of each group of rats, the improvement effect of the composition on the pathological characteristics of AD can be evaluated. In addition, the experiment will also explore the effects of different compositions on the cognitive function of rats to provide a scientific basis for the development of new AD treatment strategies.

[0105] Experimental plan

[0106] A total of 36 healthy male SD rats aged 6-8 weeks, weighing between 200-220 g, were randomly divided into 6 groups for an 8-week experiment.

[0107] CON group (normal control group): rats were gavaged with normal saline according to body weight and injected subcutaneously at the back of the neck with normal saline as normal control.

[0108] MOD group (model group): rats were gavaged with normal saline according to body weight and injected subcutaneously with 200 mg / kg D-galactose in normal saline at the back of the neck to induce AD ​​model.

[0109] Group G1 (treatment group 1): Rats were gavaged with a saline suspension containing DHM (50 mg / kg·d), L-theanine (5 mg / kg·d) and taurine (5 mg / kg·d) according to their body weight, and 200 mg / kg of D-galactose in saline solution was injected subcutaneously at the back of the neck.

[0110] Group G2 (treatment group 2): Rats were gavaged with a saline suspension containing DHM (20 mg / kg·d), L-theanine (3 mg / kg·d), taurine (3 mg / kg·d) and cerebrosides (5 mg / kg·d) according to their body weight, and 200 mg / kg of D-galactose in saline was injected subcutaneously at the back of the neck.

[0111] Group G3 (treatment group 3): The rats' feed was mixed with Hovenia dulcis powder (containing 50 mg of DHM), L-theanine (4 mg), taurine (4 mg) and Sterculia lychnophora powder (containing 1 mg of cerebrosides), and they were gavage-fed with normal saline according to their body weight. 200 mg / kg of D-galactose in normal saline was injected subcutaneously at the back of the neck.

[0112] Group G4 (treatment group 4): Rats were gavaged with a saline suspension containing L-theanine (10 mg / kg·d), taurine (10 mg / kg·d) and cerebrosides (10 mg / kg·d) according to their body weight, and 200 mg / kg of D-galactose in saline solution was injected subcutaneously at the back of the neck.

[0113] After the experiment, all rats were fasted for 24 hours, but not water. They were then anesthetized, weighed, and perfused with saline to fix the brain tissue. The isolated brain tissue was placed in a container and frozen at -80°C for subsequent analysis.

[0114] Aβ1-42 and AGEs

[0115] Aβ1-42 is a key pathological hallmark of Alzheimer's disease (AD). It originates from the cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. These protein fragments accumulate in the brain, forming amyloid plaques, one of the most characteristic pathological changes in AD. The deposition of amyloid plaques is closely associated with neuronal dysfunction and death, which in turn affects memory, cognition, and behavior.

[0116] On the other hand, advanced glycation end products (AGEs) accumulate in the brains of AD patients, potentially contributing to the pathogenesis of AD by impairing neuronal function and promoting oxidative stress. The accumulation of AGEs is associated with neurodegeneration and may affect neuronal health through multiple pathways, including altering cell signaling, promoting inflammatory responses, and interfering with normal cellular metabolic processes.

[0117] In summary, the deposition of Aβ1-42 and the accumulation of AGEs play an important role in the pathological process of AD. They not only reflect the core pathological characteristics of the disease but may also be important factors driving disease progression. Therefore, the study of these pathological features and the development of therapeutic strategies are crucial for the management and treatment of AD.

[0118] Approximately 0.02 g of tissue sample was collected from the same part of the brain tissue of each rat, and the levels of AGEs and Aβ1-42 in the brain tissue were detected according to the instructions of the commercial kits (Abcam AGE (Advanced Glycation End) Assay Kit; Thermo Fisher Scientific Human Amyloid beta 42 ELISA Kit).

[0119] according to Figure 5 and Figure 6 Data from the study showed that compared with the normal control group (CON), the levels of AGEs and Aβ1-42 in the brain tissue of rats in the AD model group (MOD) were significantly increased, which is consistent with the pathological characteristics of AD. Compared with the model group, the levels of AGEs and Aβ1-42 in all treatment groups were significantly decreased, indicating a positive effect of the treatment.

[0120] In particular, the addition of cerebrosides to treatment group 2 (G2) showed the most significant reduction in AGEs and Aβ1-42, indicating that the combination has potential therapeutic effects in reducing AD-related biomarkers. This may be related to the neuroprotective effects of cerebrosides, which help improve the pathological progression of AD.

[0121] IL-1β, TNF-α, IL-6

[0122] In Alzheimer's disease (AD), IL-1β, TNF-α, and IL-6 play important roles. They are cytokines closely related to the inflammatory response in the pathological process of AD.

[0123] IL-1β is a multifunctional immunomodulatory cytokine. Its levels are significantly elevated in the cerebrospinal fluid and serum of AD patients, and it is overexpressed early in plaque formation. IL-1β can promote the transformation of diffuse amyloid plaques into neuritic plaques and facilitate the spread of these plaques in the cortex. It can also promote the degradation of APP to generate more Aβ and activate microglia, leading to neuronal damage.

[0124] TNF-α is produced by stimulated microglia. It not only significantly activates cultured astrocytes but also promotes the release of TNF-α, nitric oxide, glutamate, and other substances, which can have toxic effects on neurons. TNF-α can stimulate monocytes to produce IL-6 and IL-1β, as well as IL-8, and induce microglia to synthesize colony-stimulating factor, further amplifying inflammation.

[0125] IL-6 is primarily produced by monocytes and macrophages, Th2 cells, vascular endothelial cells, fibroblasts, and glial cells in response to inflammation or other stimuli. IL-6 is associated with the synthesis of APP and is abundant in senile plaques in AD patients, where its expression precedes their formation. Excessive IL-6 expression can lead to neuronal degeneration and accelerate the formation of neurofibrillary tangles.

[0126] These inflammatory factors not only participate in the inflammatory response of AD but also promote the progression of AD by affecting the production and deposition of Aβ, the phosphorylation of Tau protein, and the damage and death of nerve cells. Therefore, they play a key role in the pathogenesis of AD and may become potential targets for the treatment of AD.

[0127] like Figures 7-9 As shown in the figure, in this experiment, we used enzyme-linked immunosorbent assay (ELISA) to quantitatively analyze the inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus. The experimental results revealed that the levels of these inflammatory factors in D-galactose-induced AD model rats (MOD group) were significantly increased compared with the normal control group (CON group), which is consistent with the inflammatory response characteristics in AD pathology.

[0128] Specifically, the levels of IL-1β, TNF-α, and IL-6 were significantly increased in the MOD group compared to the CON group, indicating that D-galactose successfully induced an inflammatory response. However, the levels of these inflammatory factors were significantly decreased in all treatment groups (G1 to G4) compared to the MOD group, indicating a positive effect of the treatment.

[0129] Of particular note, treatment group 2 (G2) showed the most significant effect in reducing IL-1β, TNF-α, and IL-6 levels, with levels close to or even lower than those in the normal control group (CON). This suggests that the G2 treatment regimen has potential therapeutic effects in inhibiting AD-related inflammatory responses.

[0130] DNMT1 activity assay

[0131] DNMT1 is a key DNA methyltransferase responsible for maintaining DNA methylation patterns during the cell cycle. Alterations in DNMT1 expression may play a significant role in the pathogenesis of AD. In particular, accumulation of Aβ1-42 has been shown to reduce DNMT1 protein expression in brain cells, potentially disrupting normal DNA methylation processes and affecting gene expression and neuronal function.

[0132] To assess DNMT1 expression levels, this experiment used an EpiQuik extraction kit to extract protein from brain tissue, and the EpiQuik DNMT1 Assay Kit was used to measure DNMT1 activity. The percentage of DNMT1 activity inhibition was calculated by comparing the experimental results with the control DNMT1 activity provided by the kit.

[0133] Experimental results (such as Figure 10 Figure 3 (shown in Figure 3) showed that DNMT1 protein expression in brain tissue of the AD model group (MOD group) was significantly reduced compared with the normal control group (CON group). This is consistent with the expectation that Aβ1-42 induces a decrease in DNMT1 expression. However, compared with the model group, the DNMT1 protein expression levels of all treatment groups (G1 to G4 groups) were significantly increased, indicating that treatment can promote the recovery of DNMT1. In particular, treatment group 2 (G2 group) showed the most significant increase in DNMT1 expression, which may be related to its significant effect in reducing inflammatory factors.

[0134] Although specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are merely illustrative and do not limit the scope of the invention. Changes and modifications may be made according to those skilled in the art without departing from the broader aspects of the invention as defined in the appended claims. All publications, patents, and patent documents are incorporated herein by reference as if individually incorporated herein by reference. No limitations inconsistent with the present disclosure should be understood thereby. The present invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

1. A composition for preventing and / or improving brain dysfunction, characterized in that: The composition comprises: dihydromyricetin or its salt, L-theanine, taurine and cerebroside.

2. The composition for preventing and / or improving brain dysfunction according to claim 1, wherein The composition comprises, by weight, 10-40 parts of dihydromyricetin or its salt, 1-5 parts of L-theanine, 1-5 parts of taurine and 1-10 parts of cerebroside.

3. The composition for preventing and / or improving brain dysfunction according to claim 1, wherein The mass ratio of DHM or its salt to taurine is (2-40):1; and / or the mass ratio of DHM or its salt to L-theanine is (2-40):1; and / or the mass ratio of DHM or its salt to cerebroside is (1-40):

1.

4. The composition for preventing and / or improving brain dysfunction according to any one of claims 1 to 3, characterized in that: The composition further comprises one or more of ascorbic acid, spinosin, linoleic acid or phosphatidylserine.

5. The composition for preventing and / or improving brain dysfunction according to any one of claims 1 to 3, characterized in that: The cerebroside is selected from one or more of sea cucumber cerebroside, cordycerebroside, pistachio cerebroside or soybean cerebroside; preferably 1-O-β-D-glucosyl-(2S,3R,4E,8Z)-2-[(2-hydroxyoctadecanoyl)amino]-4,8-octadecadiene-1,3-diol and / or 1-O-β-D-glucosyl -(2S,3R,4E,8Z)-2-[(2-hydroxyhexadecanoyl)amino]-4,8-octadecadiene-1,3-diol.

6. A preparation for preventing and / or improving brain dysfunction, characterized in that: The invention comprises the composition as claimed in claim 1.

7. The preparation for preventing and / or improving brain dysfunction according to claim 6, characterized in that: The preparation further comprises a carrier and / or an auxiliary material.

8. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for preventing and / or improving brain dysfunction.

9. The use according to claim 8, characterized in that The brain dysfunction includes: schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, brain hematoma, Alzheimer's disease and Parkinson's disease.

10. A method for preventing and / or improving brain dysfunction, characterized in that: The method comprises administering to a patient the composition according to any one of claims 1 to 3 or the formulation according to claim 6 or 7.

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