Use of a traditional Chinese medicine composition in the preparation of a drug for improving cognitive impairment

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a traditional Chinese medicine composition in the preparation of drugs for improving cognitive impairment. By weight, the traditional Chinese medicine composition comprises: 225-300 parts rhubarb, 50-75 parts pig bile powder, 90-120 parts aster, 360-480 parts scrophularia, 180-240 parts immature bitter orange peel, and 180-240 parts areca nut. This invention is the first to demonstrate that a composition of six traditional Chinese medicines, including scrophularia, rhubarb, and areca nut, significantly improves memory acquisition (induced by scopolamine), consolidation (induced by sodium nitrite), and retrieval impairments, covering the entire process of memory formation, encoding, storage, and retrieval, thus overcoming the limitations of existing drugs that only target a single memory stage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a traditional Chinese medicine composition in the preparation of drugs to improve cognitive impairment. Background Technology

[0002] Cognitive impairment is a collective term for a group of diseases characterized by impairment in cognitive domains such as memory, attention, thinking, and executive function. It encompasses various diseases including degenerative neurodegenerative diseases (such as Alzheimer's disease, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease, etc.), vascular cognitive impairment (such as vascular dementia, mixed dementia, etc.), secondary cognitive impairment (such as metabolic / endocrine abnormalities, infectious diseases, poisoning or drug effects, trauma or tumors, etc.), mild cognitive impairment (MCI), and cognitive impairment related to mental illness. Its pathogenesis is closely related to cholinergic system disorders, cerebral hypoxia, neuroinflammation, and oxidative stress. With the increasing aging of the population, the incidence rate is rising year by year, placing a heavy burden on families and society.

[0003] Currently, clinically used drugs to improve cognitive impairment mainly include cholinesterase inhibitors (such as donepezil) and N-methyl-D-aspartate receptor antagonists (such as memantine). However, these chemical drugs have side effects such as gastrointestinal discomfort, dizziness, and insomnia, and can only relieve symptoms, not reverse cognitive decline. Their effectiveness in improving the entire process of memory acquisition, consolidation, and retrieval is limited. Given the limitations of current Western medicine in treating memory impairment, its adverse reactions, and the complexity of the causes of memory dysfunction, it is imperative and urgent to fully leverage the characteristics and advantages of traditional Chinese medicine (TCM) for holistic and comprehensive treatment, addressing multiple aspects and targets.

[0004] Scrophularia, rhubarb, and areca nut have the effects of "clearing heat and nourishing yin, regulating qi and relieving constipation, resolving phlegm and opening the orifices." Modern pharmacological studies have confirmed that their active ingredients can regulate the level of neurotransmitters in the central nervous system, improve the brain microenvironment, and inhibit neuroinflammation. However, existing Chinese medicine preparations mostly target single-stage memory impairments and lack a systematic improvement effect on the entire process of memory acquisition, consolidation, and retrieval.

[0005] There are many traditional Chinese medicine (TCM) compositions for improving and treating cognitive impairment. For example, Chinese invention patent application CN102716459A discloses a TCM composition for treating mild cognitive impairment in the elderly, made from 48 herbs including ginseng, astragalus, angelica, deer antler, chrysanthemum, and mint. Chinese invention patent application CN105943888A discloses a TCM composition for improving memory and mild cognitive impairment, including polygala, acorus, ginseng, gastrodia, paper mulberry, ginkgo leaves, and borneol. Chinese invention patent application CN102526624A discloses a TCM preparation for treating mild cognitive impairment, made from acorus, turmeric, echinops, sargentodoxa cuneata, poria cocos, angelica, polygonatum, and earthworm. The mechanisms of action of these TCM compositions are mostly focused on tonifying the kidneys and promoting sperm production, clearing phlegm and heat, and resolving blood stasis and unblocking the meridians, and they often contain a large number of herbs.

[0006] In summary, existing drugs for treating cognitive impairment suffer from significant side effects, limited improvement stages, and incomplete effects of traditional Chinese medicine preparations. There is a need to develop a safe and effective traditional Chinese medicine composition that can improve impairments in all stages of memory acquisition, consolidation, and retrieval. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an application of a traditional Chinese medicine composition in the preparation of drugs for improving cognitive impairment.

[0008] To achieve the above-mentioned objectives of this invention, the specific technical solution adopted by this invention is as follows: The application of a traditional Chinese medicine composition in the preparation of a drug for improving cognitive impairment, wherein the traditional Chinese medicine composition comprises, by weight, 225-300 parts of rhubarb, 50-75 parts of pig bile powder, 90-120 parts of aster, 360-480 parts of scrophularia, 180-240 parts of immature bitter orange peel, and 180-240 parts of areca nut.

[0009] Preferably, the traditional Chinese medicine composition comprises, by weight, 250-300 parts of rhubarb, 50-60 parts of pig bile powder, 100-120 parts of aster, 400-480 parts of scrophularia, 200-240 parts of immature bitter orange peel, and 200-240 parts of areca nut.

[0010] More preferably, the traditional Chinese medicine composition comprises, by weight, 278 parts rhubarb, 56 parts pig bile powder, 111 parts aster, 444 parts scrophularia, 222 parts immature bitter orange peel, and 222 parts areca nut.

[0011] Rhubarb is the dried root and rhizome of *Rheum palmatum* L., *Rheum tanguticum* Maxim. ex Balf., or *Rheum officinale* Baill., all belonging to the Polygonaceae family. It is harvested in late autumn when the stems and leaves wither, or in the following spring before sprouting. Fine roots are removed, the outer skin is scraped off, and it is cut into segments or sections, strung together, and dried, or dried directly. Properties and Channels Entered: Bitter, cold. Enters the Spleen, Stomach, Large Intestine, Liver, and Pericardium channels. Functions and Indications: Purgative, clears heat and fire, cools blood and detoxifies, removes blood stasis and promotes menstruation, promotes diuresis and relieves jaundice. Used for constipation due to heat accumulation, hematemesis due to blood heat, red eyes and swollen throat, carbuncles and boils, intestinal abscesses and abdominal pain, amenorrhea due to blood stasis, postpartum blood stasis, traumatic injuries, damp-heat dysentery, jaundice with dark urine, urinary tract infections, and edema; externally used for burns and scalds.

[0012] Pig bile powder is the dried bile of the pig (Sus scrofa domestica Brisson.). Its properties and channels are: bitter and cold. It enters the liver, gallbladder, lung, and large intestine channels. Its functions and indications are: clearing heat and moistening dryness, relieving cough and asthma, and detoxifying. It is used for whooping cough, asthma, thirst due to fever, red eyes, sore throat, jaundice, diarrhea, dysentery, constipation, and carbuncles and boils.

[0013] Aster tataricus Lf, a plant in the Asteraceae family, is a dried root and rhizome. It is harvested in spring and autumn, with the noded rhizomes (commonly known as "mother roots") and dirt removed. The roots are braided and sun-dried, or simply sun-dried directly. Properties and Channels Entered: Pungent, bitter, warm. Enters the Lung channel. Functions and Indications: Moistens the lungs, lowers qi, resolves phlegm, and relieves cough. Used for cough with excessive phlegm, both acute and chronic, and cough with hemoptysis due to overwork.

[0014] Scrophularia, or dried root, is the dried root of *Scrophularia ningpoensis* Hemsl., a plant in the Scrophulariaceae family. It is harvested in winter when the stems and leaves wither. The rhizomes, young buds, fibrous roots, and dirt are removed. It is then sun-dried or oven-dried until semi-dry, piled up for 3-6 days, and this process is repeated several times until completely dry. Properties and Channels Entered: Sweet, bitter, salty, slightly cold. Enters the Lung, Stomach, and Kidney channels. Functions and Indications: Clears heat and cools the blood, nourishes Yin and reduces fire, detoxifies and dissipates nodules. Used for heat entering the blood level, febrile diseases with rashes, febrile diseases damaging Yin, red tongue with thirst, constipation due to fluid depletion, steaming cough, red eyes, sore throat, diphtheria, scrofula, carbuncles, and boils.

[0015] Citrus aurantium L., a plant of the Rutaceae family, is the dried, immature fruit of *Citrus aurantium* L. and its cultivated varieties. It is harvested in July when the peel is still green, cut in half horizontally from the middle, and then sun-dried or dried at low temperature. Its properties and channels are: bitter, pungent, sour, and slightly cold. It enters the spleen and stomach channels. Its functions and indications are: regulating qi and relieving chest tightness, promoting digestion and relieving bloating. It is used for qi stagnation in the chest and hypochondrium, abdominal distension and pain, indigestion, phlegm retention, and organ prolapse.

[0016] Areca catechu L., a plant in the palm family (Arecaceae), is the dried, mature seed. Mature fruits are harvested from late spring to early autumn, boiled in water, dried, the pericarp removed, and the seeds extracted and dried. Properties and Channels Entered: Bitter, pungent, warm. Enters the Stomach and Large Intestine channels. Functions and Indications: Kills parasites, eliminates food stagnation, promotes qi circulation, promotes diuresis, and treats malaria. Used for tapeworm infection, ascariasis, fascioliasis, abdominal pain due to intestinal parasites, diarrhea due to food stagnation, tenesmus, edema, beriberi, and malaria.

[0017] Preferably, the preparation method of the traditional Chinese medicine composition includes the following steps: (1) Half of the rhubarb was crushed and mixed with pig bile powder, ethanol was added, the mixture was stirred evenly, sealed, dried, and crushed to obtain fine powder 1; (2) Mix the other half of the rhubarb with the remaining other drugs, add water and decoct, filter, combine the filtrates, concentrate, add ethanol, let stand, and obtain mixture A; (3) Filter mixture A, concentrate the filtrate, dry it, and pulverize it to obtain fine powder 2; (4) Mix fine powder 1 and fine powder 2 to obtain the Chinese medicine composition.

[0018] More preferably, the ethanol in step (1) has a mass concentration of 70%-80%, is sealed for 1.5-2.5 hours, dried, and then pulverized through an 80-100 mesh sieve.

[0019] More preferably, in step (2), the solid-liquid ratio of the decoction is 1g:8-12mL, the number of decoctions is 2-4 times, each time for 1-2 hours, the concentration temperature is 45-55℃, the concentration is to a relative density of 1.10-1.15, the mass concentration of the ethanol is 60%-80%, and the standing time is 10-14 hours.

[0020] More preferably, the concentration temperature in step (3) is 45-55℃, the concentration is carried out to a relative density of 1.30-1.35, and the material is pulverized through a 100-120 mesh sieve.

[0021] Preferably, the dosage form of the drug for improving cognitive impairment is tablets, pills, capsules, granules, powders, mixtures, emulsions, decoctions, or suspensions.

[0022] Preferably, the cognitive impairment is a memory acquisition impairment, a memory consolidation impairment, and / or a memory retrieval impairment.

[0023] More preferably, the memory acquisition impairment is induced by damage to the cholinergic system, and the memory consolidation impairment is induced by brain hypoxia damage.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention first confirms that the composition of six traditional Chinese medicines, such as Scrophulariae Radix, Rhei Radix et Rhizoma, and Arecae Semen, has a significant improvement effect on memory acquisition (induced by scopolamine), consolidation (induced by sodium nitrite), and retrieval impairment (induced by ethanol), covering the entire process of memory formation, encoding, storage, and retrieval, and making up for the limitation of existing drugs that only target a single memory stage. The whole formula takes "Rhei Radix et Rhizoma as the monarch drug, purging the fu-organs and removing blood stasis, clearing heat and detoxifying" as the core, and plays a role in targeting the pathogenesis characteristics of qi obstruction in the fu-organs, phlegm-heat accumulation in the interior, stasis-toxin blocking the orifices, and yin-fluid deficiency in cognitive impairment. Scrophulariae Radix and Arecae Semen are the minister drugs. Scrophulariae Radix nourishes yin and clears heat, detoxifies and dissipates nodules, assisting Rhei Radix et Rhizoma in clearing heat and detoxifying and preventing the bitter-cold nature from injuring yin; Arecae Semen promotes qi movement and induces qi stagnation, reduces accumulation and purges the fu-organs, assisting Rhei Radix et Rhizoma in purging the fu-organs and clearing heat. Asteris Radix and Fellis Suis are the assistant drugs. Asteris Radix moistens the lungs and lowers qi, resolves phlegm and promotes its dispersal and descent, and Fellis Suis clears heat and moistens dryness, detoxifies and promotes defecation. The two work together to assist in clearing heat and detoxifying, moistening dryness and protecting yin, and resolving phlegm and opening the orifices. Aurantii Fructus Immaturus is the assistant drug and also serves as the guiding drug, regulating qi and widening the middle, promoting qi stagnation and relieving distension, guiding the medicinal power into the gastrointestinal tract, and enabling the qi mechanism to be smooth and the qi in the fu-organs to be unobstructed. The compatibility of all the drugs achieves the effects of purging the fu-organs and removing blood stasis, clearing heat and detoxifying, nourishing yin and moistening dryness, promoting qi movement and resolving phlegm, so that the heat-toxin is cleared, phlegm and blood stasis are transformed, and the clear orifices are nourished, thus realizing the systematic improvement of memory acquisition, memory consolidation, and memory retrieval impairment.

[0025] (2) Through a specific preparation method, the present invention has controllable quality, does not require the development of a completely new production system, and is easy to achieve industrial transformation. Description of the Drawings

[0026] Figure 1 shows the effects of the composition of the present invention and the compositions of Comparative Examples 1-4 on the fear memory ability of mice. Among them, compared with the control group, P <0.05.

[0027] Figure 2 is a schematic diagram of the quadrant division of the Morris water maze.

[0028] Figure 3 shows the results of the effects of the composition of the present invention on the passive avoidance response ability of mice in the memory acquisition impairment model induced by scopolamine. Among them, Figure A is: the number of errors during the learning period; Figure B is: the latency of jumping off the platform during the test period. Compared with the control group, P <0.05; compared with the model group, # P <0.05.

[0029] Figure 4 shows the results of the effects of the composition of the present invention on the fear memory ability of mice in the memory acquisition impairment model induced by scopolamine. Among them, compared with the control group, P <0.05; compared with the model group, # P <0.05.

[0030] Figure 5 The results show the effect of the composition of this invention on the spatial learning and memory ability of mice with scopolamine-induced memory acquisition impairment, wherein A: line graph of escape latency during the learning period; B: AUC of escape latency during the learning period; C: escape latency during the testing period; D: time spent in the target quadrant; E: number of ring crossings; compared with the control group, P <0.01; compared with the model group, # P <0.05, ## P <0.01.

[0031] Figure 6 The results show the effect of the composition of this invention on the passive avoidance response ability of mice with a sodium nitrite-induced memory consolidation impairment model, wherein A: number of errors during the learning period; B: latency to jump off the platform during the testing period; compared with the control group, P <0.01; compared with the model group, # P <0.05.

[0032] Figure 7 The results show the effect of the composition of this invention on fear memory ability in a mouse model of sodium nitrite-induced memory consolidation impairment, wherein, compared with the control group, P <0.01; compared with the model group, # P <0.05.

[0033] Figure 8 The results show the effect of the composition of this invention on the spatial learning and memory ability of mice with a sodium nitrite-induced memory consolidation impairment model. In the figures, A: Line graph of escape latency during the learning period; B: AUC of escape latency during the learning period; C: Escape latency during the testing period; D: Time spent in the target quadrant; E: Number of times the ring was crossed; compared with the control group, P <0.05, P <0.01; compared with the model group, # P <0.05.

[0034] Figure 9 The results show the effect of the composition of this invention on the passive avoidance response ability of mice with ethanol-induced memory retrieval disorder, where A: number of errors during the learning period; B: latency to jump off the platform during the testing period; compared with the control group, P <0.01; compared with the model group, # P <0.05. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0036] Example 1 A traditional Chinese medicine composition for improving cognitive impairment, comprising, by weight, the following ingredients: 278 parts rhubarb, 56 parts pig bile powder, 111 parts aster, 444 parts scrophularia, 222 parts immature bitter orange peel, and 222 parts areca nut.

[0037] The preparation method of the above-mentioned traditional Chinese medicine composition is as follows: (1) Half of the rhubarb was crushed and mixed with pig bile powder. Then, 75% ethanol was added, stirred evenly, sealed for 2 hours, dried, crushed, and passed through a 90-mesh sieve to obtain fine powder 1.

[0038] (2) Mix the other half of the rhubarb with the remaining other drugs (aster, scrophularia, immature bitter orange, areca nut), add water and decoct. The solid-liquid ratio is 1g:10mL. Decoction is performed 3 times, 1.5h each time. Filter and combine the filtrates. Concentrate at 50℃ to a relative density of 1.12 (measured at 50℃). Add ethanol to make the alcohol content reach 70%. Let stand for 12h and take the supernatant to obtain mixture A.

[0039] (3) Filter the mixture A, concentrate the filtrate at 50°C to a relative density of 1.32 (measured at 50°C), dry, pulverize, and pass through a 110-mesh sieve to obtain fine powder 2.

[0040] (4) Mix fine powder 1 and fine powder 2 to obtain the final product.

[0041] Example 2 A traditional Chinese medicine composition for improving cognitive impairment, comprising, by weight, the following ingredients: 225 parts rhubarb, 50 parts pig bile powder, 90 parts aster, 360 parts scrophularia, 180 parts immature bitter orange peel, and 180 parts areca nut.

[0042] The preparation method of the above-mentioned traditional Chinese medicine composition is as follows: (1) Half of the rhubarb was crushed and mixed with pig bile powder. Then, 70% ethanol was added, stirred evenly, sealed for 1.5 hours, dried, crushed, and passed through an 80-mesh sieve to obtain fine powder 1.

[0043] (2) Mix the other half of the rhubarb with the remaining other drugs, add water and decoct. The solid-liquid ratio is 1g:8mL. Decoction is carried out twice, 1h each time. Filter and combine the filtrates. Concentrate at 45℃ to a relative density of 1.10 (measured at 50℃). Add ethanol to make the alcohol content reach 60%. Let stand for 10h and take the supernatant to obtain mixture A.

[0044] (3) Filter the mixture A, concentrate the filtrate at 45°C to a relative density of 1.30 (measured at 50°C), dry, pulverize, and pass through a 100-mesh sieve to obtain fine powder 2.

[0045] (4) Mix fine powder 1 and fine powder 2 to obtain the final product.

[0046] Example 3 A traditional Chinese medicine composition for improving cognitive impairment, comprising, by weight, the following ingredients: 300 parts rhubarb, 75 parts pig bile powder, 120 parts aster, 480 parts scrophularia, 240 parts immature bitter orange peel, and 240 parts areca nut.

[0047] The preparation method of the above-mentioned traditional Chinese medicine composition is as follows: (1) Half of the rhubarb was crushed and mixed with pig bile powder. Ethanol with a mass concentration of 80% was added, stirred evenly, sealed for 2.5 hours, dried, crushed, and passed through a 100-mesh sieve to obtain fine powder 1.

[0048] (2) Mix the other half of the rhubarb with the remaining other drugs, add water and decoct. The solid-liquid ratio is 1g:12mL. Decoction is carried out 4 times, 2h each time. Filter and combine the filtrates. Concentrate at 55℃ to a relative density of 1.15 (measured at 50℃). Add ethanol to make the alcohol content reach 80%. Let stand for 14h and take the supernatant to obtain mixture A.

[0049] (3) Filter the mixture A, concentrate the filtrate at 55°C to a relative density of 1.35 (measured at 50°C), dry, pulverize, and pass through a 120-mesh sieve to obtain fine powder 2.

[0050] (4) Mix fine powder 1 and fine powder 2 to obtain the final product.

[0051] Comparative Example 1 The only difference between this comparative example and Example 1 is that the raw materials do not include Scrophularia ningpoensis; the amounts of the other raw materials and the preparation methods are the same as in Example 1. Specifically, the raw materials are: 278 parts rhubarb, 56 parts pig bile powder, 111 parts aster, 222 parts immature bitter orange peel, and 222 parts areca nut.

[0052] Comparative Example 2 The only difference between this comparative example and Example 1 is the preparation method, specifically: (1) Mix all the rhubarb (278 parts) with aster, scrophularia, immature bitter orange and areca nut, add water and decoct, the solid-liquid ratio is 1g:10mL, decoct 3 times, 1.5h each time, filter, combine the filtrates; concentrate, add ethanol to make the alcohol content reach 70%, let stand for 12h, and obtain the supernatant.

[0053] (2) Add pig bile powder directly to the supernatant above, mix evenly, concentrate the filtrate at 50°C to a relative density of 1.32 (measured at 50°C), dry, pulverize, and pass through a 110-mesh sieve to obtain the product.

[0054] Comparative Example 3 The only difference between this comparative example and Example 1 is that the amount of rhubarb was adjusted to 600 parts, while the amount of other raw materials and the preparation method are the same as in Example 1.

[0055] Comparative Example 4 The only difference between this comparative example and Example 1 is that the raw materials do not contain pig bile powder, and only rhubarb is treated with alcohol in step (1).

[0056] The specific ingredients are: 278 parts rhubarb, 111 parts aster, 444 parts scrophularia, 222 parts immature bitter orange peel, and 222 parts areca nut.

[0057] The preparation method has been adjusted as follows: (1) After crushing half of the rhubarb, add 75% ethanol, stir evenly, seal for 2 hours, dry, crush, and pass through a 90-mesh sieve to obtain fine powder 1.

[0058] (2) Mix the other half of rhubarb with aster, scrophularia, immature bitter orange and areca nut, add water and decoct, the solid-liquid ratio is 1g:10mL, decoct 3 times, 1.5h each time, filter, and combine the filtrates; concentrate at 50℃ to a relative density of 1.12 (measured at 50℃), add ethanol to make the alcohol content reach 70%, let stand for 12h, take the supernatant to obtain mixture A.

[0059] (3) Filter the mixture A, concentrate the filtrate at 50°C to a relative density of 1.32 (measured at 50°C), dry, pulverize, and pass through a 110-mesh sieve to obtain fine powder 2.

[0060] (4) Mix fine powder 1 and fine powder 2 to obtain the final product.

[0061] Effect test Test Example 1: Conditioned Fear Experiment This study used 2-month-old male Kunming mice as experimental subjects. The test drugs described in Example 1 and Comparative Examples 1-4 were administered via gavage. A conditioned fear test was used to evaluate the effect of each test drug on improving the memory function of the mice. Details are as follows: 1. Laboratory animals A total of 60 2-month-old male Kunming mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The production license number is SCXK (Beijing) 2021-0011, and they were raised in the barrier environment of the Laboratory Animal Center of Shandong University of Traditional Chinese Medicine. The use license number is SYXK (Shandong) 20220009. The raising environment: temperature 20-25°C, 12h light / 12h dark cycle, relative humidity 50%-65%. All animal feeds were provided by the Laboratory Animal Center of Shandong University of Traditional Chinese Medicine. All treatments of animals in this study complied with the regulations of Shandong University of Traditional Chinese Medicine regarding animal use and ethics. All animal experiments were approved by the Welfare Ethics Review Committee of Shandong University of Traditional Chinese Medicine, approval number: SDUTCM20240506002.

[0062] 2. Experimental Instruments The instruments used are shown in Table 1 below.

[0063] Table 1 Experimental Instruments

[0064] 3. Experimental Grouping The mice were randomly divided into 6 groups: control group (C), example 1 group (T), comparative example 1 group (D1), comparative example 2 group (D2), comparative example 3 group (D3), and comparative example 4 group (D4), with 10 mice in each group. During the preventive administration stage, animals in each group were administered by gavage, as shown in Table 2 specifically.

[0065] Table 2 Experimental Grouping

[0066] 4. Experimental Procedures After 3 days of adaptive feeding for each group of mice, gavage administration was started, and conditioned fear was performed on the 7th day of administration to detect the learning and memory functions of each group of mice.

[0067] 5. Experimental Steps Learning period (5 min / rat / day, for 1 day in total): Each mouse was placed in the conditioned fear test chamber to adapt for 2 min, and the mouse was allowed to move freely. Then, with a cycle of 30 s (sound stimulation (4 kHz, 80 dB, 15 s), and finally an electric shock (0.5 mA, 2 s) was given after the sound stimulation, and wait for 13 s after the sound and electric shock), after 6 cycles, the mouse was taken out of the test chamber, and the immobility time of the mouse in the last 3 min was recorded. Each time a new animal was changed, the test chamber was wiped with 75% alcohol.

[0068] Detection period (5 min / rat / day, for 1 day in total): The mouse was placed in the conditioned fear test chamber to adapt for 2 min, and the mouse was allowed to move freely. Then, with a cycle of 30 s (sound stimulation (4 kHz, 80 dB, 15 s), and wait for 15 s after the sound stimulation), after 6 cycles, the mouse was taken out of the test chamber. Each time a new animal was changed, the test chamber was wiped with 75% alcohol.

[0069] Observation index: Record the immobile time of mice for 15 s after acoustic stimulation in 6 cycles.

[0070] 6. Experimental results The results of the conditioned fear experiment showed that, as Figure 1 , compared with the control group, the immobile time of mice in the Example 1 group and the Comparative Example 1-4 groups showed a trend of varying degrees of prolongation during the test period, but only the Example 1 group had a statistical difference ( P <0.05), indicating that the composition of Example 1 of the present invention can improve the formation of contextual fear memory, and its effect is better than that of Comparative Examples 1-4.

[0071] Test Example 2 The present invention was confirmed by animal experiments that the traditional Chinese medicine composition can significantly improve memory acquisition disorders, memory consolidation disorders, and memory retrieval disorders by regulating the cholinergic system, improving brain hypoxia tolerance, and inhibiting neuroinflammation. Memory acquisition (induced by scopolamine), consolidation (induced by sodium nitrite), and retrieval (induced by ethanol) disorders. Specifically as follows: 1. Experimental animals A total of 99 2-month-old male KM mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. for this experiment. The production license number: SCXK (Beijing) 2021-0011. They were raised in the barrier environment of A209 in the Experimental Animal Center of Shandong University of Traditional Chinese Medicine. The use license number: SYXK (Shandong) 20220009. The feeding environment: temperature 20-25 °C, 12 h light / 12 h dark cycle, relative humidity 50%-65%. The animal feed was provided by the Experimental Animal Center of Shandong University of Traditional Chinese Medicine. All treatments of animals in this study complied with the regulations of Shandong University of Traditional Chinese Medicine regarding animal use and ethics. All animal experiments were approved by the Welfare Ethics Review Committee of Shandong University of Traditional Chinese Medicine, approval number: SDUTCM20240506002.

[0072] 2. Experimental instruments The instruments used are shown in Table 3 below.

[0073] Table 3 Experimental instruments

[0074] 3. Experimental reagents The reagents used are shown in Table 4 below.

[0075] Table 4 Experimental reagents

[0076] 4. Experimental grouping This study used three batches of experimental animals to evaluate the effects of a traditional Chinese medicine composition on memory acquisition, memory consolidation, and memory retrieval impairments. Each batch of animals was randomly divided into three groups: a control group (C), a model group (M), and a traditional Chinese medicine composition group (T), with 11 animals in each group. During the preventative administration phase, all groups were administered the medicine via gavage. The control and model groups were given an equal volume of purified water by gavage, while the traditional Chinese medicine composition group was given the composition described in Example 1 by gavage. For different types of memory impairment models, corresponding modeling treatments were used during the behavioral experiment phase: 5 mg / kg scopolamine was injected intraperitoneally 20 min before training to establish a memory acquisition impairment model; 80 mg / kg sodium nitrite was injected intraperitoneally immediately after training to establish a memory consolidation impairment model; and 25% ethanol was administered by gavage 30 min before testing to establish a memory retrieval impairment model. The corresponding control groups were treated with an equal volume of physiological saline or purified water at the same time points. All experimental procedures were performed synchronously on all groups of mice, using parallel treatment to reduce experimental error. Specific experimental groupings are shown in Table 5.

[0077] Table 5 Mouse experimental grouping

[0078] Note: Dosage was administered once daily. The models were established by intraperitoneal injection of scopolamine 20 minutes before training to create a memory acquisition impairment model, intraperitoneal injection of sodium nitrite immediately after training to create a memory consolidation impairment model, and gavage administration of 25% ethanol 30 minutes before testing to create a memory retrieval impairment model. The corresponding control group received physiological saline or purified water at the same time points. Each mouse underwent the procedures in parallel.

[0079] 5. Experimental Procedure After 3 days of acclimatization feeding, mice in each group were given oral medication. On days 12, 16, and 21 after administration, mice underwent jumping, conditioned fear, and Morris water maze tests to assess their learning and memory functions.

[0080] 6. Experimental Procedure 6.1 Diving Platform Adaptation: Place the animal on the platform and allow it to move freely up and down within the enclosure for 2 minutes to adapt. Then, return the animal to its original feeding box.

[0081] Training period (5 min / animal / day): After the animal has adapted, place it back on the platform and turn on the electric stimulator. The power supply is located at the bottom of the enclosure, with a voltage of 80V and a current of 0.4mA.

[0082] Record indicator: The number of times the animal repeatedly jumps off the platform within 5 minutes.

[0083] Testing period (5 min / animal / day): Test 24 hours after training, put the animal back on the platform (no power required) and observe the animal's behavior after getting off the platform.

[0084] Recording indicator: the incubation period from when the animal is placed on the platform until it jumps off the platform.

[0085] 6.2 Conditioned Fear Learning period (5 min / mouse / day, 1 day total): Each mouse was placed in the conditioned fear test chamber for 2 minutes to acclimatize, during which time the mice were allowed free movement. Then, in 30-second cycles (sound stimulation (4 kHz, 80 dB, 15 s), followed by electrical stimulation (0.5 mA, 2 s), with a 13-second wait after the sound and electrical stimulation), the mice were removed from the test chamber, and the immobility time of the mice in the last 3 minutes was recorded. The test chamber was wiped with 75% alcohol each time an animal was replaced.

[0086] Testing period (5 min / mouse / day, 1 day in total): Place the mice in the conditioned fear test chamber for 2 min to adapt, and let the mice move freely. Then, in 30-second cycles (sound stimulation (4 kHz, 80 dB, 15 s), wait 15 s after the sound stimulation), remove the mice from the test chamber after 6 cycles. Wipe the test chamber with 75% alcohol each time you change animals.

[0087] Observation indicators: Record the immobility time of mice 15 seconds after sound stimulation in 6 cycles.

[0088] 6.3 Morris Water Maze (1) Hidden platform training period (60 s / bird, 4 training sessions / bird, 5 days in total) The circular pool is divided into four quadrants, each with specific markers (★○△□) drawn on its wall. The target platform is fixed in the third quadrant (SW), as shown below. Figure 2As shown in the diagram. Water was then added to the water maze until the water level was 1 cm above the target platform, and food coloring was added to conceal the platform. Four training sessions were then conducted, with the animals placed in the pools from different entry points, heads facing the pool wall. Each quadrant was used only once per day, and the quadrant order was adjusted accordingly each day (see Table 6 below). The time required for the animal to climb onto the platform from entry into the water was recorded as the escape latency period; the animal's swimming trajectory in the water was also recorded as a basis for analyzing the strategies used by the animal when searching for the target. After climbing onto the platform, the animal was allowed to stand on it for 10 seconds. If the animal did not find the platform or fail to climb onto it within 60 seconds, it was led to stand on the platform for 10 seconds to form a memory, after which the animal was removed from the platform. The average escape latency period for each animal at the four entry points per day was calculated (the latency period for those who did not climb onto the platform was calculated as 60 seconds). Inter-group statistical comparisons were performed daily, and fluctuation curves were plotted to observe trends. During the training period, it is necessary to monitor the passing time of each mouse's escape latency. This passing time is used to determine whether to abandon training and conduct further testing on the mouse. The observation indicator during the hidden platform training period is the escape latency.

[0089] Table 6. Starting position of mice during each training session.

[0090] Note: NE represents the first quadrant, NW the second quadrant, SW the third quadrant, and SE the fourth quadrant. During the learning period, the target platform is always fixed in the third quadrant (SW), and during the testing period, the mouse is placed in the position opposite to the original platform in the quadrant (NE).

[0091] (2) Remove the platform testing period (60 seconds / animal, 1 test / animal, 1 day in total) On the last day of training, after 24 hours, the test began. The platform was removed from its original location, and the animal was placed in the pool from the NE. The time it took for the animal to first explore and swim past the original platform location, the number of times it explored and swam past the original platform location area within 60 seconds, and the swimming time of the animal in the target quadrant where the platform was located were recorded.

[0092] Remove the following observation indicators from the platform training period: escape latency; number of times the device is crossed; swimming time in the target quadrant.

[0093] 7. Experimental Results 7.1 The effect of the traditional Chinese medicine composition of the present invention on improving scopolamine-induced memory acquisition impairment (1) The effect of the traditional Chinese medicine composition (TLC) of this invention on the passive avoidance response ability of mice with scopolamine-induced memory acquisition impairment was evaluated using the platform drop test. The results showed that ( Figure 3 Compared with the control group, the number of errors made by mice in the model group during the learning period was significantly increased. P <0.05, the latency period during the testing period was significantly shortened (P <0.05); After intervention with the composition of the present invention, the number of errors in the TLC group mice was significantly reduced ( P <0.05, and the latency of jumping off the platform was longer than that of the model group ( P <0.05), indicating that the composition can effectively enhance the passive avoidance response ability of mice.

[0094] (2) Results of the conditioned fear experiment (see Figure 4 The results showed that, compared with the control group, the model group mice had a significantly shorter rigidity time during the test period. P <0.05, indicating that it failed to effectively establish the association between the environment and the fear stimulus; while the rigidity time of mice in the TLC group was significantly increased compared with that in the model group ( P <0.05), indicating that the composition of the present invention can improve the formation process of situational fear memories.

[0095] (3) Results of the Morris water maze experiment (see Figure 5 The display shows that during the positioning and navigation training phase ( Figure 5 In group A), the escape latency of mice in all groups gradually shortened with increasing training days, but the learning curve of the model group showed a slower downward trend. Compared with the control group, the escape latency of the model group was prolonged on days 2-5 of the learning period. P <0.01, the area under the curve of the avoidance latency period during the learning period increases ( P <0.01, the evasion latency period during the testing period is prolonged ( P <0.05, the time spent in the target quadrant is shortened ( P <0.05, the number of circumcisions decreased ( P <0.05); Compared with the model group, the area under the curve of escape latency was reduced in the TLC group mice ( P <0.01, shortened escape latency during the testing period ( P <0.05, the number of piercings increased ( P <0.05). The above results indicate that the traditional Chinese medicine composition of the present invention can significantly improve the spatial learning and memory abilities of mice with memory acquisition disorder.

[0096] 7.2 The effect of the traditional Chinese medicine composition of the present invention on improving sodium nitrite-induced memory consolidation impairment (1) Results of the platform jumping experiment and the conditioned fear experiment (see Figure 6 and Figure 7 This indicates that, in the memory consolidation disorder model, compared with the control group, the model group mice had a shorter latency in the step-down test. P <0.01, and the time of rigidity in the conditioned fear experiment was also significantly shortened ( P <0.01); while the latency of mice in the TLC group was significantly prolonged in the jumping test (P <0.05, significantly prolonging the time of rigidity in the conditioned fear experiment ( P The value is <0.05, indicating that the traditional Chinese medicine composition can promote the conversion of short-term memory into long-term memory and its consolidation process.

[0097] (2) Results of the Morris water maze experiment (see Figure 8 The results showed that, compared with the control group, the escape latency of mice in the model group was prolonged on days 4-5 of the learning period. P <0.01, the area under the curve increases during the learning period ( P <0.01, the evasion latency period during the testing period is prolonged ( P <0.01, while the time spent in the target quadrant is shortened ( P <0.05), number of circumcisions reduced ( P <0.01); Compared with the model group, the area under the curve of escape latency was reduced in the TLC group mice ( P <0.05, shortened escape latency during the testing period ( P <0.05). The above results further demonstrate that the traditional Chinese medicine composition of the present invention has a significant protective effect on the memory consolidation process.

[0098] 7.3 The effect of the traditional Chinese medicine composition of the present invention on ethanol-induced memory retrieval impairment Results of the diving platform experiment (see) Figure 9 The results showed that, in the memory retrieval disorder model, compared with the control group, the latency of mice in the model group was significantly shortened during the test period. P <0.01); After intervention with the composition of the present invention, the latency period of mice in the TLC group was significantly prolonged ( P The concentration of ethanol in the sample is <0.05%, indicating that the composition can effectively reverse the inhibitory effect of ethanol on the memory extraction process.

[0099] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for improving cognitive impairment, characterized in that, The traditional Chinese medicine composition comprises, by weight, 225-300 parts of rhubarb, 50-75 parts of pig bile powder, 90-120 parts of aster, 360-480 parts of scrophularia, 180-240 parts of immature bitter orange peel, and 180-240 parts of areca nut.

2. The application according to claim 1, characterized in that, The traditional Chinese medicine composition comprises, by weight, 250-300 parts of rhubarb, 50-60 parts of pig bile powder, 100-120 parts of aster, 400-480 parts of scrophularia, 200-240 parts of immature bitter orange peel, and 200-240 parts of areca nut.

3. The application according to claim 2, characterized in that, The traditional Chinese medicine composition comprises, by weight, 278 parts rhubarb, 56 parts pig bile powder, 111 parts aster, 444 parts scrophularia, 222 parts immature bitter orange peel, and 222 parts areca nut.

4. The application according to any one of claims 1, characterized in that, The preparation method of the traditional Chinese medicine composition includes the following steps: (1) Crush 1 / 3-1 / 2 of the rhubarb and mix it with pig bile powder, add ethanol, stir evenly, seal, dry, and crush to obtain fine powder 1; (2) Mix the remaining rhubarb with aster, scrophularia, immature bitter orange and areca nut, add water and decoct, filter, combine the filtrates, concentrate, add ethanol, let stand, and obtain mixture A; (3) Filter mixture A, concentrate the filtrate, dry it, and pulverize it to obtain fine powder 2; (4) Mix fine powder 1 and fine powder 2 to obtain the Chinese medicine composition.

5. The application according to claim 4, characterized in that, The ethanol in step (1) has a mass concentration of 70%-80%, is sealed for 1.5-2.5 hours, dried, and then pulverized through an 80-100 mesh sieve.

6. The application according to claim 4, characterized in that, In step (2), the solid-liquid ratio of the decoction is 1g:8-12mL, the number of decoctions is 2-4 times, each time for 1-2 hours, the concentration temperature is 45-55℃, the concentration is made to a relative density of 1.10-1.15, the mass concentration of the ethanol is 60%-80%, and the standing time is 10-14 hours.

7. The application according to claim 4, characterized in that, The concentration temperature in step (3) is 45-55℃, and the concentration is carried out to a relative density of 1.30-1.

35. The powder is then pulverized through a 100-120 mesh sieve.

8. The application according to any one of claims 1-7, characterized in that, The dosage form of the drug for improving cognitive impairment is tablets, pills, capsules, granules, powders, mixtures, emulsions, decoctions, or suspensions.

9. The application according to any one of claims 1-7, characterized in that, The cognitive impairments are memory acquisition impairment, memory consolidation impairment, and / or memory retrieval impairment.

10. The application according to claim 9, characterized in that, The memory acquisition disorder is induced by damage to the cholinergic system, and the memory consolidation disorder is induced by brain hypoxia damage.

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