Traditional Chinese medicine composition for treating or improving cognitive impairment and preparation method thereof

Abstract

The present invention discloses a traditional Chinese medicine composition for treating or improving cognitive impairment and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw medicinal materials: rhubarb, peach kernel, semen vaccariae, rhizoma sparganii, and dried ginger. The present invention further discloses a method for preparing the traditional Chinese medicine composition. The traditional Chinese medicine composition of the present invention has clear source and smooth flow, sufficient nourishment for the brain mansion, and the efficacy of the marrow sea and spiritual mechanism, and can effectively improve the symptoms of dementia and amnesia, achieving the therapeutic effects of promoting blood circulation and dredging collaterals, anti-inflammatory and removing stasis, and protecting neurons. Animal behavior experiments show that the traditional Chinese medicine composition of the present invention has a significant improvement effect on cognitive impairment and anxiety in mice. The results of immunofluorescence staining show that the traditional Chinese medicine composition of the present invention protects axons, myelin sheaths, and synapses and reduces neuronal apoptosis by inhibiting the proliferation of astrocytes and microglia. The results of toxicological experiments show that the traditional Chinese medicine composition of the present invention is safe and has no toxic and side effects.

Description

Technical Field

[0001] The present invention relates to a traditional Chinese medicine composition for treating or improving cognitive impairment, and further relates to a preparation method of the traditional Chinese medicine composition, belonging to the field of traditional Chinese medicine treatment of cognitive impairment. Background Art

[0002] Learning and memory impairment is a common symptom of Alzheimer's disease (AD), vascular dementia (VaD), and mild cognitive impairment (MCI), usually manifested as cognitive impairment in one or more fields. Its symptoms are caused by the deposition of amyloid plaques (Aβ) in the brain, neurofibrillary tangles (NFT), and the proliferation of astrocytes and microglia, resulting in the loss of synapses and neurons. Patients usually show manifestations such as time and space amnesia, difficulty in conceptual memory learning, and language recognition disorders, which seriously affect their quality of life and bring great troubles to families and society.

[0003] At present, foreign drug research on improving learning and memory impairment mainly targets the targets that occur in AD. However, the cause of AD is unknown. At present, several acetylcholinesterase (hAchE) inhibitors have been approved by the US Food and Drug Administration (FDA) for marketing. However, this type of drug needs to be taken in increasing doses over time, and strong side effects gradually appear. Most patients will experience strong gastrointestinal reactions such as nausea and vomiting, some patients will experience neurological disorders such as anxiety and headache, and a small number of patients will even experience syncope symptoms. If the drug is stopped or the dose is reduced, serious rebound phenomena will occur, including mental confusion, aggravation of the disease, and autonomic nerve disorders. Although these drugs can improve the learning and memory impairment of patients, they greatly reduce their quality of life.

[0004] Compared with Western medicine, traditional Chinese medicine has shown unexpected improvement effects on some intractable diseases in traditional treatments. Therefore, domestic experts have gradually turned to the research on improving learning and memory impairment with traditional Chinese medicine prescriptions. In traditional Chinese medicine theory, the fundamental pathogenesis of learning and memory impairment is the reduction of marrow and the consumption of the brain, and the failure of the spiritual mechanism to function. It is caused by various reasons such as insufficient marrow sea, deficiency of the spleen and kidney, phlegm stasis obstruction, and hyperactivity of the heart and liver fire. One of the main manifestations is forgetfulness. Based on traditional Chinese medicine theory, domestic experts have constructed many prescriptions, but there are problems such as few research teams, low sample size, short treatment time, low research quality, and heterogeneity of results. At present, no proprietary Chinese medicine has been widely recognized in improving learning and memory impairment.

[0005] AD is a Western medicine disease name. Traditional Chinese medicine believes that it is caused by the reduction of marrow and the consumption of the brain, and the failure of the spiritual mechanism to function. For those with similar syndromes, including Alzheimer's dementia, vascular dementia, normal pressure hydrocephalus, brain tumors, paralytic dementia, toxic encephalopathy, etc., syndrome differentiation and treatment can be referred to traditional Chinese medicine.

[0006] Previous treatments believed that it was mainly due to deficiency syndromes. Whether it was due to old age and physical decline, or due to loss of nourishment and prolonged accumulation of damage caused by illness, it led to deficiency of kidney essence. The kidney stores congenital essence and is responsible for bone growth and marrow production. When the essence is full, the marrow is full. This marrow not only refers to bone marrow but also to brain marrow, which is a broad concept. Therefore, the brain is also called the sea of marrow. As early as in "Classified Classic of Medicine", there was a related discussion that "among all the marrows in bones, the brain marrow is the largest, so all marrows belong to the brain". Therefore, in traditional Chinese medicine theory, the balance of kidney essence is closely related to the normal function of the brain. Once the kidney essence is deficient, for those with a short onset time and relatively mild condition, headache and dizziness may occur. For those with a long onset time and relatively severe condition, replenishing essence and marrow and tonifying the kidney and nourishing the brain are one of the main treatment methods. Summary of the Invention

[0007] One of the objectives of the present invention is a traditional Chinese medicine composition for treating or improving cognitive impairment;

[0008] Another objective of the present invention is to provide a method for preparing the above-mentioned traditional Chinese medicine composition for treating or improving cognitive impairment;

[0009] The above objectives of the present invention are achieved through the following technical solutions:

[0010] One aspect of the present invention is to provide a traditional Chinese medicine composition for treating or improving cognitive impairment, comprising the following raw materials: rhubarb, peach kernel, semen vaccariae, sparganium, dried ginger.

[0011] In a preferred specific implementation manner of the present invention, the weight parts of each raw material are: 15 - 20 parts of rhubarb, 8 - 12 parts of peach kernel, 18 - 25 parts of semen vaccariae, 18 - 25 parts of sparganium, 6 - 10 parts of dried ginger; most preferably, the weight parts of each raw material are: 18 parts of rhubarb, 10 parts of peach kernel, 20 parts of semen vaccariae, 20 parts of sparganium, 8 parts of dried ginger.

[0012] In order to achieve a better treatment effect, the rhubarb is preferably wine-processed rhubarb, which is prepared according to the conventional processing method in the art. For reference, the rhubarb slices are stir-fried dry by the wine roasting method to obtain it.

[0013] All the raw materials used in the present invention can be purchased from ordinary pharmacies, and their specifications meet the national pharmaceutical standards.

[0014] The occurrence and development of diseases are closely related to specific historical backgrounds. The present inventor preliminarily infers that kidney essence deficiency should not be the only pathogenesis of AD, so the effect of the method of tonifying the kidney and replenishing essence will also be reduced. "Plain Questions·On the Harmony between Man and Nature": "Excessive consumption of rich and fatty foods can lead to the occurrence of large boils." Zhang Jiebin believed that "gaoliang refers to rich and fatty foods", that is, various high-sugar and high-fat foods nowadays. Although these foods are rich in nutrition, if consumed for a long time beyond the body's needs, with consumption less than intake, they will accumulate in the body, resulting in problems of energy surplus, and then triggering a series of diseases such as obesity, hypertension, hyperlipidemia, and diabetes. "Plain Questions·On the General Assessment of Deficiency and Excess": "For the treatment of consumptive thirst (diabetes), stroke fainting, hemiplegia, flaccidity and syncope (limbs are weak and unable to move freely), qi fullness and reverse flow, those who are rich and noble and consume rich and fatty foods are suffering from diseases caused by excessive consumption of rich and fatty foods" are often recorded and elaborated. Slightly different is that there were fewer "rich and noble people" in the past, while now there are more and more "rich and noble people". Based on this, the present inventor speculates that among the main pathogenic mechanisms causing AD at present, excess syndrome - energy metabolism surplus plays an increasingly important role. Regarding the syndrome differentiation and treatment of AD caused by energy surplus, the present inventor believes that the spleen and stomach are the foundation of acquired constitution and the source of qi and blood generation. It has the function of transforming daily diet into nutrient essence, dispersing the essence to the lungs, transporting it throughout the body, and supplying various zang-fu organs and tissues. Once the energy intake is too high and the nutrient essence transformed cannot be consumed completely, it will accumulate in the Yangming meridian to form stagnant heat. The stagnant heat is scorched and transformed into phlegm and dampness (increased blood viscosity), which flows to the brain. Over time, it causes stasis. The two interact with each other and gradually lead to stasis and entanglement, which are difficult to heal, and finally lead to the failure of the mental mechanism. The treatment method should mainly focus on purging pathogenic factors, clearing heat and removing stasis, and slightly assisting in strengthening the healthy qi and warming and protecting the spleen and stomach. The stomach and intestine being congested with heat is the root cause, so rhubarb is used to purge the congested heat, phlegm and dampness in the qi aspect of the Yangming meridian. When processed with wine, it can also enter the blood aspect, and at the same time remove stasis downward, so that the stasis can come out through the stool; blood stasis obstruction is the flow of the meridian, so semen vaccariae and peach kernels are used to promote blood circulation and remove stasis, dredge the meridians. Peach kernels can also moisten the intestines and relieve constipation, and serve as an assistant to guide the stasis downward to the intestinal tract and come out with rhubarb through the stool; the stasis and entanglement in the brain is the end of the pathogenic factor, which is difficult to open for a long time, and ordinary drugs for promoting blood circulation and removing stasis are not powerful enough. It is necessary to use the powerful blood-breaking effect of sparganium to cut through the entanglement and remove stasis obstruction. Therefore, from the congestion and heat in the Yangming meridian, to blood stasis obstruction in the meridians, to stasis and entanglement in the brain, this is how the pathogenic factor comes into being; Sparganium opens the stasis and entanglement in the brain and disperses the pathogenic factor into the meridian, to semen vaccariae and peach kernels promoting blood circulation and dredging the meridian to guide the pathogenic factor to the intestine, to wine-processed rhubarb purging heat and promoting defecation to expel the pathogenic factor downward, this is how the pathogenic factor is removed. Finally, considering that AD is a chronic pathological process involving complex zang-fu organs, meridians and pathogenic factors, its treatment also requires a process and cannot be achieved overnight. And the rhubarb, semen vaccariae, sparganium and peach kernels used are all of a relatively cold nature. Especially rhubarb, although processed with wine and its nature is slightly alleviated, its strong and drastic nature still remains. Taking it for a long time may damage the spleen and stomach. If the stasis and entanglement in the brain have not been completely cleared and the spleen and stomach become weak, then it cannot continue to bear the purging effect of the medicine. Therefore, dried ginger is added as an assistant to warm and protect the spleen and stomach, so that the purging of pathogenic factors will not damage the healthy qi.

[0015] Different from the traditional approach that focuses on tonifying deficiency and replenishing marrow, the treatment of this formula emphasizes eliminating pathogenic factors, promoting blood circulation to remove stasis, restoring the smooth flow of blood vessels, thereby nourishing the brain and enabling the revival of mental functions. Additionally, different from other prescriptions for promoting blood circulation to remove stasis in treating this disease, this formula not only focuses on promoting blood circulation and dredging collaterals to clear the flow, but also has the effect of purging the bowels. It is to purge heat and diarrhea to promptly remove the accumulated food stagnation with heat, and also to provide an outlet for the stagnated blood to discharge through the feces, fundamentally rectifying the source. With the source clear and the flow smooth, the brain is sufficiently nourished, the marrow and mental functions are effective, thereby effectively improving the symptom of amnesia, achieving the therapeutic effects of promoting blood circulation and dredging collaterals, anti-inflammatory and removing stasis, and protecting neurons.

[0016] The cognitive impairment described in the present invention includes Alzheimer's disease or vascular dementia.

[0017] After extracting the active ingredients from each component in the traditional Chinese medicine composition of the present invention according to the conventional extraction method, various excipients or carriers (such as disintegrants, lubricants or binders, etc.) required for preparing different dosage forms are added, and any clinically applicable oral preparation or injection preparation can be prepared. Among them, the oral preparation can be pills, capsules, tablets, powders, fine powders, mixtures, granules or oral liquids; the injection preparation can be freeze-dried powder injections or injections, etc.

[0018] For reference, the present invention provides a preparation method of a traditional Chinese medicine composition, and the method includes:

[0019] (1) Weigh each raw material drug;

[0020] (2) Soak each raw material drug in water and decoct it 1 - 3 times with water to obtain decoction liquid, and combine the decoction liquid to obtain.

[0021] For reference, the present invention provides another preparation method of a traditional Chinese medicine composition, and the method includes: decocting each raw material drug with water, filtering the decoction liquid, concentrating the filtrate into clear paste, drying to obtain dry powder; adding excipients for preparing the dosage form to the dry powder, mixing evenly to prepare the corresponding solid preparation; or decocting each raw material drug with water, filtering the decoction liquid, concentrating the filtrate to obtain concentrated liquid; adding excipients to the concentrated liquid, mixing evenly to prepare the corresponding liquid preparation.

[0022] Among them, the decocting is to decoct with water 1 - 3 times, preferably 2 times; among them, for the first time, add 5 - 12 times the weight of water and decoct for 1 - 3 hours; for the second time, add 4 - 10 times the weight of water and decoct for 1 - 2 hours.

[0023] For reference, the present invention also provides a preparation method of a traditional Chinese medicine composition, and the method includes:

[0024] (1) Mix each raw material drug evenly, pulverize and sieve to obtain fine powder for standby;

[0025] (2) Add excipients or carriers for preparation forming to the fine powder, mix evenly, and prepare the corresponding preparation.

[0026] The preparation of the traditional Chinese medicine composition can be administered to the desired subject by conventional means. Representative administration methods include (but are not limited to): oral administration, injection, aerosol inhalation, etc.

[0027] Usage and dosage of the traditional Chinese medicine composition of the present invention: The dosage of the medicine of the present invention depends on specific dosage forms and factors such as the age, weight, and health status of the patient. As a guide: for tablets, adults take 2.2 g orally each time, 3 times a day; one month is a treatment course, and 1 - 4 treatment courses are carried out.

[0028] Animal behavior experiments (open field, novel object recognition, Barnes maze) show that the traditional Chinese medicine composition of the present invention has a significant improvement effect on cognitive impairment and anxiety in mice. The results of immunofluorescence staining show that the traditional Chinese medicine composition of the present invention protects axons, myelin sheaths, and synapses and reduces neuronal apoptosis by inhibiting the proliferation of astrocytes and microglial cells; the results of toxicology experiments show that the traditional Chinese medicine composition of the present invention has no effect on the ratios of main organs in mice, blood biochemistry (ALT, AST, CK, Cr, Bun), and the morphological HE staining of organs, and has no toxic and side effects. Description of the Drawings

[0029] Figure 1 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving the abnormal behavior of samp8 mice.

[0030] Figure 2 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving synaptic damage in samp8 mice.

[0031] Figure 3 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving neuronal apoptosis in samp8 mice.

[0032] Figure 4 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving axonal and myelin sheath damage in samp8 mice.

[0033] Figure 5 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving axonal damage in samp8 mice.

[0034] Figure 6 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving astrocyte proliferation in samp8 mice.

[0035] Figure 7 These are the experimental results of the traditional Chinese medicine composition of the present invention in improving microglial cell proliferation in samp8 mice.

[0036] Figure 8This is the result of the drug safety experiment of the traditional Chinese medicine composition of the present invention. Detailed implementation manners

[0037] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present invention without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0038] Example 1 Preparation of mixture

[0039] (1) Weigh each raw material medicine according to the following weights: 1.8 kg of Rheum officinale Baill. var. tanguticum Maxim. ex Balf. (processed with wine), 1.0 kg of Prunus persica (L.) Batsch, 2.0 kg of Vaccaria segetalis (Neck.) Garcke, 2.0 kg of Sparganium stoloniferum Buch.-Ham., 0.8 kg of Zingiber officinale Rosc.

[0040] (2) Soak each raw material medicine in water for 1 hour and boil it over high heat until it boils.

[0041] (3) After boiling over low heat for 40 minutes, filter and take out the first decoction.

[0042] (4) Add water to cover the residue of traditional Chinese medicine and boil it over high heat until it boils.

[0043] (5) Take out the second decoction after boiling over low heat for 25 minutes.

[0044] (6) Combine the two decoctions and boil them over low heat for concentration to obtain the mixture.

[0045] Example 2 Preparation of tablets

[0046] (1) Weigh each raw material medicine according to the following weights: 1.5 kg of Rheum officinale Baill. var. tanguticum Maxim. ex Balf. (processed with wine), 0.8 kg of Prunus persica (L.) Batsch, 1.8 kg of Vaccaria segetalis (Neck.) Garcke, 1.8 kg of Sparganium stoloniferum Buch.-Ham., 0.6 kg of Zingiber officinale Rosc.

[0047] (2) Soak each raw material medicine in water for 1 hour and boil it over high heat until it boils.

[0048] (3) After boiling over low heat for 40 minutes, filter and take out the first decoction.

[0049] (4) Add water to cover the residue of traditional Chinese medicine and boil it over high heat until it boils.

[0050] (5) Take out the second decoction after boiling over low heat for 25 minutes.

[0051] (6) Combine the two decoctions and boil them over low heat for concentration to an extract with a relative density of 1.12 - 1.18 (75 °C), and then spray-dry to obtain dry extract powder.

[0052] (7) Add pregelatinized starch to the dry extract powder, mix, dry-press into granules, add magnesium stearate and silicon dioxide to size the granules, tableting, film coating, sub-packaging, packaging to obtain the finished product.

[0053] Preparation of Capsules in Example 3

[0054] (1) Weigh each raw material medicine according to the following weights: Weigh each raw material medicine according to the following weights: 2.0 kg of Rheum palmatum, 1.2 kg of Prunus persica, 2.5 kg of Vaccaria segetalis, 2.5 kg of Sparganium stoloniferum, 1.0 kg of Zingiber officinale.

[0055] (2) Soak each raw material medicine in water for 1 hour and boil it over high heat until it boils.

[0056] (3) After boiling over low heat for 40 minutes, filter and take out the first decoction.

[0057] (4) Add water to cover the residue of traditional Chinese medicine and boil it over high heat until it boils.

[0058] (5) Take out the second decoction after boiling over low heat for 25 minutes.

[0059] (6) Combine the two decoctions and boil it over low heat to concentrate it into an extract with a relative density of 1.12 - 1.18 (75 °C), then spray dry to obtain the dry extract powder;

[0060] (7) Add starch to the dry extract powder, mix, dry-press into granules, mix thoroughly, fill into capsules, sub-packaging, packaging to obtain the finished product.

[0061] Experimental Example 1 Improvement of Cognitive Impairment and Toxicity Experiment of the Traditional Chinese Medicine Composition of the Invention in Samp8 Mice

[0062] 1 Materials and Methods

[0063] 1.1 Medicines and Grouping

[0064] 1.1.1 Decoction of Traditional Chinese Medicine

[0065] The traditional Chinese medicine for testing was provided by Beijing Bencaofangyuan Pharmaceutical Group Co., Ltd.

[0066] Soak the traditional Chinese medicine composition of the invention in distilled water for 1 h and boil it with 500 w until it boils. Then boil it with 200 w for 40 min and take out the first decoction. Add distilled water and boil it with 500 w until it boils. Then boil it with 200 w for 25 min and take out the second decoction. Combine the two decoctions and boil it with 200 w until it reaches 200 ml.

[0067] 1.1.2 Dose Setting

[0068] The dose settings for each sample are shown in Table 1. For the traditional Chinese medicine group, the dose was calculated based on 200 ml of the medicinal liquid per 60 kg of body weight per person, and the dose for mice was converted according to the body surface area calculation method for humans and animals. The control group was given an equal volume of distilled water. Donepezil was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. and the dose was converted according to the adult dose. The doses in each group were concentrated and fixed to an appropriate gavage volume with traditional Chinese medicine, and gavaged three times a week (Monday, Wednesday, and Friday).

[0069] Table 1 Dose settings of each sample for Samp8 mice

[0070] Group Mouse dose Multiple of equivalent adult dose Number of mice per group Low-dose group <![CDATA[0.224*(W) 2 / 3 g / ml]]> 1-fold 10 mice, 5 females and 5 males Medium-dose group <![CDATA[0.448*(W) 2 / 3 g / ml]]> 2-fold 10 mice, 5 females and 5 males Control group <![CDATA[0.224*(W) 2 / 3 g / ml]]> / 10 mice, 5 females and 5 males Donepezil group 0.003*W mg / g 1-fold 10 mice, 5 females and 5 males

[0071] (W - mouse body weight)

[0072] 1.2 Experimental animals

[0073] Forty 3 - month - old clean - grade Samp8 mice (purchased from Beijing Specific Pathogen Free Biotechnology Co., Ltd.), 20 males and 20 females, with a body weight of 20 - 25 g. They were housed in the Experimental Animal Department of Capital Medical University, with a breeding temperature of 18℃ - 22℃, a light duration of 12 h, and a relative humidity of 50% - 60%. They had free access to food and water. All operations in this research process complied with the standards and requirements of the animal ethics of our hospital [AEEI - 2017 - 125], and humane care was given in accordance with the 3R principle.

[0074] Samp8 is a kind of rapidly aging model mouse. After 6 months of age, the mice of this strain will age rapidly and show symptoms such as cognitive impairment. It is a good animal model for studying the occurrence mechanism of aging and learning and memory dysfunction and evaluating intelligence - improving drugs.

[0075] 1.3 Main reagents and instruments

[0076] Goat anti-GFAP antibody (abcam, ab53554), goat anti-iba-1 (abcam, ab48904), rabbit anti-Neun antibody (abcam, ab177487), rabbit anti-PSD95 antibody (abcam, ab18258), mouse anti-MBP antibody (abcam, ab62631), rabbit anti-NF-H antibody (abcam, ab207176), Citrate antigen retrieval solution pH6.0 (Wuhan Sevier Biotechnology Co., Ltd., G1202), EDTA antigen retrieval solution pH9.0 (Wuhan Sevier Biotechnology Co., Ltd., G1203), EDTA antigen retrieval solution pH8.0 (Wuhan Sevier Biotechnology Co., Ltd., G1206), PBS buffer (Wuhan Sevier Biotechnology Co., Ltd., G0002), Tissue autofluorescence quencher (Wuhan Sevier Biotechnology Co., Ltd., G1221), Bovine serum albumin BSA (Wuhan Sevier Biotechnology Co., Ltd., GC305010), DAPI staining reagent (Wuhan Sevier Biotechnology Co., Ltd., G1012), Anti-fluorescence quenching mounting medium (Wuhan Sevier Biotechnology Co., Ltd., G1401), Absolute ethanol (Sinopharm Chemical Reagent Co., Ltd., 100092683), Xylene (Sinopharm Chemical Reagent Co., Ltd., 10023418), HE staining solution set (Servicebio, G1003), Neutral balsam (Sinopharm Chemical Reagent Co., Ltd., 10004160), Golgi staining solution (Servicebio, G1069-1), Acidic hardening fixing solution (Servicebio, G1069-2).

[0077] Panoramic scanner (3D HISTECH), ultra-low temperature freezer (Sanyo Electric Group, Japan), tabletop centrifuge (Shanghai Anting Scientific Instrument Factory), electronic analytical balance (Shanghai Jingke Tianping), full-automatic gel image analysis system (Beijing Saizhi Chuangye Technology Co., Ltd.), laminar flow hood (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), optical microscope (Shanghai Leica Instruments Co., Ltd.), open field platform (Shanghai Xinruan Co., Ltd.), novel object recognition platform (Shanghai Xinruan Co., Ltd.), Barnes maze platform (Shanghai Xinruan Co., Ltd.), optical microscope (Shanghai Leica Instruments Co., Ltd.), dehydrator (DIAPATH, Donatello), embedding machine (Wuhan Junjie Electronics Co., Ltd., JB-P5), microtome (Shanghai Leica Instruments Co., Ltd., RM2016), freezing table (Wuhan Junjie Electronics Co., Ltd., JB-L5), tissue spreading machine (Jinhua Cody Instrument and Equipment Co., Ltd., Zhejiang Province, KD-P), staining machine (DIAPATH, Giotto), oven (Tianjin Laiborui Instrument and Equipment Co., Ltd., GFL-230), glass slide (Servicebio, G6004), upright optical microscope (Nikon, Japan, E100), imaging system (Nikon, Japan, DS-U3).

[0078] 1.4 Experimental methods

[0079] 1.4.1 Novel object recognition

[0080] Novel object recognition is an ethological method that uses the innate instinct of rodents to approach and explore novel objects to detect animal memory recognition.

[0081] This experiment was conducted for five days. On the first to the third days, the mice were placed in the center of the experimental box and allowed to freely adapt for 15 minutes. After each session, the box was wiped with 75% alcohol to eliminate the odor effect. On the fourth day, two cubes (3 cm × 3 cm × 3 cm) were placed in a semi-corner, and each mouse was placed in the center of the experimental box in turn to learn for 5 min. On the fifth day, one of the cubes was replaced with a sphere (diameter = 3 cm), and each mouse was placed in the center of the experimental box in turn to explore for 5 min. The time for the mouse to explore the cube was recorded as F (familiar), and the time to explore the sphere was recorded as N (new). The discrimination index (DI) was calculated as DI = (N - F) / (N + F).

[0082] 1.4.2 Barnes maze

[0083] The Barnes maze is established based on the characteristics of rodents to avoid light and prefer darkness and their love of exploration, and mainly evaluates the spatial memory ability of animals.

[0084] This experiment was conducted for five days. Training was carried out in the first four days. A layer of bedding was placed in the target hole to attract the mice to enter. First, the animals were placed in a plastic bucket in the center of the maze and restricted from moving for 10 s. Subsequently, the plastic bucket was removed, and the mice were allowed to freely explore for 4 min, and the number of times the mice explored the wrong holes before finding the target hole was recorded. If all four limbs of the mouse entered the target box, the plastic bucket was placed over the target hole, and the animal was allowed to stay in the box for 30 s. If the mouse did not enter the target hole all the time, after the mouse explored for 4 min, it was placed in the target box and the plastic bucket was covered and it stayed for 30 s. The mice were trained twice a day for 4 consecutive days. After each session, the bedding was replaced and the box was wiped with 75% alcohol to eliminate the odor effect. On the fifth day, a test was conducted. The target box was removed, and the other steps were the same as above. The mice were allowed to freely explore for 4 min, and the number of times the mice explored the target hole was recorded.

[0085] 1.4.3 Open field test

[0086] The open field test is used to detect the spontaneous activity and exploratory behavior of rodents, and it is a method to evaluate the autonomous behavior, exploratory behavior and anxiety of experimental animals in a new environment.

[0087] Each group of experimental mice was taken out in advance and allowed to adapt to the behavior room for half an hour. The box was wiped with 75% alcohol to eliminate the odor effect. Each group of mice was placed in the center of the box in turn, and they were allowed to freely explore for 15 min. The total movement distance, average movement speed, and time spent in the central area of the mice were counted and recorded. Subsequently, the mice were taken out, the number of feces was counted and recorded. After each experiment, the box was wiped with 75% alcohol.

[0088] 1.4.4 Sample collection

[0089] The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (0.1%, 2 ml / animal). Blood (1 - 2 ml) was collected from the orbital venous plexus. After coagulation at room temperature, it was centrifuged at 3000 rmp for 5 min at 4℃, and the supernatant was taken to obtain serum, which was delivered to Beijing Chengweiben Biotechnology Co., Ltd. for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), blood creatinine (Cr), and blood urea nitrogen (BUN). The mice were perfused intracardially with phosphate buffer (PBS, 0.1 M; pH 7.4), and then the brain tissue samples were obtained by dissection. The hippocampus of the left half of the brain was stored at -80℃ for later use. The right half of the brain was placed in 4% PFA at 4℃ overnight. The fixed brain tissue samples were embedded in paraffin, and coronal sections of 4 μm were prepared and stored for later use. The hearts, livers, spleens, and kidneys of the mice were weighed and recorded, placed in 4% PFA at 4℃ overnight, and embedded in paraffin for later use.

[0090] 1.4.5 Immunofluorescence staining

[0091] The mouse brain tissues were sent to Wuhan Saiweier Biotechnology Co., Ltd. for dehydration, embedding, sectioning, and immunofluorescence staining.

[0092] The brain sections were dewaxed, subjected to microwave antigen retrieval, and then treated with 3% formaldehyde solution for 10 minutes to inactivate endogenous peroxidase activity. After washing three times with PBS, the sections were incubated in 5% bovine serum albumin at room temperature for 20 minutes, followed by overnight incubation with the primary antibody at 4°C. Subsequently, the corresponding secondary antibody was incubated, and the sections were placed in acetone for 90 seconds and washed three times with PBS. Immunofluorescence-stained sections needed to be placed in a DAPI dish to maintain fluorescence. All images were scanned panoramically on a whole-slide scanner, the Panoramic MIDI digital slide scanner.

[0093] 1.4.6 HE staining

[0094] The sections were successively placed in xylene I for 20 minutes, xylene II for 20 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, 75% alcohol for 5 minutes, and washed with tap water. Subsequently, the sections were stained with hematoxylin solution for 3 - 5 minutes, washed with tap water, differentiated with the differentiating solution, washed with tap water, blued with the bluing solution, and rinsed with running water. Then the sections were dehydrated successively in 85% and 95% gradient alcohols for 5 minutes each, and stained with eosin solution for 5 minutes. Finally, the sections were successively placed in absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, absolute ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. The sections were sealed with neutral balsam, examined under a microscope, and the images were collected and analyzed.

[0095] 1.4.7 Golgi staining

[0096] The mice were sacrificed by cervical dislocation, and the brain tissues were immediately taken in a fume hood and placed in the Golgi staining fixative. The mouse brains were gently rinsed several times with normal saline, placed in a 50 ml round-bottom centrifuge tube, and 20 ml of Golgi staining solution was added to completely immerse the tissue blocks. They were placed in a cool, ventilated, and dark place for a total of 14 days (after soaking for 48 hours, a new staining solution was replaced, and then a new staining solution was replaced every 3 days). The mouse brains were gently taken out with a weighing spoon and washed several times with distilled water. The brain tissues were glued to the tray of the vibratome with 502 super glue and immersed in pure water. Sections were cut at 100 μm, and after cutting, they were assisted to stick onto the glass slides with a brush, then blotted dry with filter paper and left to dry overnight at 4°C. The sections were washed with pure water for 5 minutes, 10 minutes of concentrated ammonia water was added, and the glass slides were washed twice with pure water. The glass slides were immersed in the acidic hardening fixing solution and treated in the dark for 45 minutes, and the glass slides were washed twice with pure water. After sealing with glycerin jelly, they were stored at room temperature in a dark slide box.

[0097] 1.4.8 Data statistics

[0098] For immunofluorescence imaging, ImageJ software was used for fluorescence intensity, average length, and positive area analysis. GraphPad Prism software 8.0 was used to exclude extreme values from the original data, and the valid data were retained for statistical chart drawing and variance analysis. A p value < 0.05 was considered statistically significant.

[0099] 2 Experimental Results

[0100] 2.1 The Traditional Chinese Medicine Composition of the Invention Improves the Behavioral Abnormalities of Samp8 Mice

[0101] 2.1.1 The Traditional Chinese Medicine Composition of the Invention Improves the Memory Recognition Ability of Samp8 Mice

[0102] According to Figure 1 (A), the discrimination indices of the two groups of samp8 mice treated with traditional Chinese medicine were higher than those of the control group, and the medium-dose group increased significantly (p < 0.05), indicating that the memory recognition ability of samp8 mice was significantly improved after administration (n = 10).

[0103] 2.1.2 The Traditional Chinese Medicine Composition of the Invention Improves the Spatial Learning and Memory Ability of Samp8 Mice

[0104] According to Figure 1 (B), as the number of training days increased, the number of exploratory error holes of the samp8 mice in the medium-dose group was significantly decreased compared with the control group on the third, fourth, and fifth days (p < 0.05), indicating that the spatial learning ability of samp8 mice was significantly increased after administration. According to Figure 1 (C)(D), the number of times the mice in the medium-dose group explored the target hole on the last day was higher than that of the control group, and the activity range of the mice in the medium-dose group was concentrated near the target hole while the activity range of the control group covered the entire Barnes maze, indicating that the spatial memory ability of samp8 mice was improved after administration (n = 10).

[0105] 2.1.1 The Traditional Chinese Medicine Composition of the Invention Improves the Anxiety and Tension of Samp8 Mice

[0106] According to Figure 1 (E)(F)(G), the residence time of the samp8 mice in the medium-dose group in the central grid was significantly increased compared with the control group (p < 0.05). The total horizontal movement distance of the two groups of samp8 mice treated with traditional Chinese medicine in the behavior box was significantly lower than that of the control group (p < 0.01), and the defecation frequency of the two groups of traditional Chinese medicine and the donepezil group was significantly lower than that of the control group (p < 0.01), indicating that the anti-anxiety ability and tension of samp8 mice were significantly improved after administration (n = 10).

[0107] 2.2 The Traditional Chinese Medicine Composition of the Invention Improves the Neuropathological Abnormalities of Samp8 Mice

[0108] 2.2.1 The traditional Chinese medicine composition of the present invention improves synaptic and neuronal apoptosis in samp8 mice. Synaptic loss and neuronal apoptosis are common symptoms of cognitive impairment. Postsynaptic density protein 95 (PSD95) is the core scaffolding protein of synapses and plays a key role in maintaining normal synaptic function and plasticity, mediating and integrating the signals and signal transduction of N-methyl-D-aspartic acid receptors and glutamate receptors. According to Figure 2 (A), the fluorescence intensity of positive PSD95 in the whole brain of the traditional Chinese medicine group was higher than that of the control group after the samp8 mice were administered the medicine. According to Figure 2 (B), in the CA1 region of the mouse hippocampus related to long-term memory and spatial tasks (p < 0.01) and the CA3 region with neuronal reception tasks (p < 0.05), the fluorescence intensity of PSD95 in the traditional Chinese medicine group was significantly higher than that of the control group. According to Figure 2 (C), in the entorhinal cortex (Ect) of the mouse temporal lobe, which plays an important role in biological memory and navigation (p < 0.05), and the piriform cortex (Pir) related to innate genetic emotional motivation activities (p < 0.01), the fluorescence intensity of PSD95 in the traditional Chinese medicine group was significantly higher than that of the control group (n = 6).

[0109] Neuronal nuclei (NeuN) is a precursor mRNA alternative splicing regulatory molecule and is commonly used to specifically label the nuclei in most neurons of vertebrates. According to Figure 3 (A), the fluorescence intensity of NeuN in the traditional Chinese medicine group was significantly higher than that of the control group after the samp8 mice were administered the medicine. According to Figure 3 (B)(C), the number of neurons in the traditional Chinese medicine group was significantly higher than that of the control group in the CA1 region of the hippocampus and the Ect region of the temporal lobe (p < 0.05) (n = 6).

[0110] In summary, the traditional Chinese medicine composition of the present invention significantly improves synaptic damage and neuronal apoptosis in the hippocampus and temporal lobe of samp8 mice, thereby improving the abnormal behavior of mice.

[0111] 2.2.2 The traditional Chinese medicine composition of the present invention improves nerve axon, dendrite, and myelin sheath damage in samp8 mice

[0112] Nerve injury is one of the pathological symptoms of cognitive impairment. Neurofilament heavy chain (Nfh) is an important subunit of neuronal neurofilament protein (NF), and myelin basic protein (MBP) is an alkaline membrane protein in the central nervous system of vertebrates and is a component of myelin sheaths. According to Figure 4 (A)(C), the fluorescence intensities of Nfh and MBP in the whole brain of the traditional Chinese medicine group were higher than those of the control group after the samp8 mice were administered the medicine. According to Figure 4 (B)(D), the fluorescence intensities of Nfh and MBP in the traditional Chinese medicine group were significantly higher than those of the control group in the Ect region of the temporal lobe (p < 0.05) (n = 6).

[0113] Golgi staining is commonly used for staining neuron axons and dendrites. According to Figure 5 (B)(C), the average lengths of the processes in the CA1 region of the hippocampus and the Ect in the temporal lobe of the traditional Chinese medicine group of samp8 mice were significantly higher than those of the control group (p < 0.05) (n = 4).

[0114] In summary, the traditional Chinese medicine composition of the present invention significantly improves the synaptic and axonal injuries in the hippocampus and temporal lobe of samp8 mice, thereby improving the conduction of electrical signals and the transmission of neurotransmitters.

[0115] 2.2.3 Improvement of astrocyte and microglia hyperplasia in samp8 mice by the traditional Chinese medicine composition of the present invention

[0116] Glial fibrillary acidic protein (GFAP) is a marker of astrocyte activation. According to Figure 6 (A), the fluorescence intensity of positive astrocytes in the whole brain of the traditional Chinese medicine group was lower than that of the control group after administration to samp8 mice. According to Figure 6 (B)(C), the number of astrocytes in the traditional Chinese medicine group was significantly lower than that of the control group in the CA1 region, CA3 region of the hippocampus, and the Ect region, Pir region of the temporal lobe (p < 0.05) (n = 6).

[0117] Ionized calcium-binding adapter molecule 1 (Iba1) is a calcium-binding protein specific to microglia. According to Figure 7 (A), the fluorescence intensity of positive microglia in the whole brain of the traditional Chinese medicine group was lower than that of the control group after administration to samp8 mice. According to Figure 7 (B)(C), the number of microglia in the traditional Chinese medicine group was significantly lower than that of the control group in the CA3 region of the hippocampus and the Ect region of the temporal lobe (p < 0.05) (n = 6).

[0118] In summary, the traditional Chinese medicine composition of the present invention significantly improves the hyperplasia of astrocytes and microglia in the hippocampus and temporal lobe of samp8 mice, thereby reducing neuronal autophagy and protecting neurons.

[0119] 2.3 Results of the drug safety test of the traditional Chinese medicine composition of the present invention

[0120] According to Figure 8 (A)(B), by statistically comparing the ratios of the main organs and the blood biochemical indexes (ALT, AST, CK, Cr, BUN) of the two groups, no differences were found, indicating that the traditional Chinese medicine composition of the present invention does not have hepatotoxicity, nephrotoxicity, or cardiotoxicity. By observing the HE staining of the main organs of the traditional Chinese medicine group and the control group after administration, no significant differences or lesions were found in the morphology of the organs of the two groups. The above results prove that the traditional Chinese medicine composition of the present invention has good drug safety.

Claims

1. A traditional Chinese medicine composition for treating or improving cognitive impairment, characterized in that, it is made from the following raw materials by weight: 18 parts of Rheum palmatum L. var. tanguticum Maxim. ex Regel processed with wine, 10 parts of Prunus persica (L.) Batsch, 20 parts of Vaccaria segetalis (Neck.) Garcke, 20 parts of Sparganium stoloniferum Buch.-Ham., and 8 parts of Zingiber officinale Rosc.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, after extracting the active ingredients from each raw material by the conventional extraction methods in the art, various excipients or carriers required for preparing different dosage forms are added to prepare any clinically applicable preparation.

3. The traditional Chinese medicine composition according to claim 2, characterized in that, the preparation is selected from pills, capsules, tablets, powders, fine powders, mixtures, granules, oral liquids, freeze-dried powder injections or injections.

4. A method for preparing the traditional Chinese medicine composition according to claim 1, characterized in that, it includes: Decocting each raw material with water, filtering the decoction, concentrating the filtrate into a clear paste, drying to obtain a dry powder; adding excipients for preparing the dosage form to the dry powder, mixing evenly to prepare the corresponding solid preparation; or decocting each raw material with water, filtering the decoction, concentrating the filtrate to obtain a concentrated solution; adding excipients to the concentrated solution, mixing evenly to prepare the corresponding liquid preparation.

5. A method for preparing the traditional Chinese medicine composition according to claim 1, characterized in that, it includes: (1) Mixing each raw material evenly, pulverizing and sieving to obtain a fine powder for standby; (2) Adding excipients or carriers for preparing the dosage form to the fine powder, mixing evenly to prepare the corresponding preparation.

6. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a pharmaceutical preparation for treating or improving cognitive impairment.

7. The use according to claim 6, characterized in that, the cognitive impairment is the cognitive impairment caused by Alzheimer's disease.

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