A kind of silver linden through pulse brain particles, preparation method and application

Abstract

The application provides a silver fungus vessel-activating and brain-strengthening granule, a preparation method and application, and comprises the following components in parts by weight: wine jujube 10-15 parts, ginkgo leaves 10-15 parts, female privet fruit 8-12 parts, salvia miltiorrhiza 8-12 parts, alocasia 5-7 parts, eucommia ulmoides 8-12 parts, poria cocos 8-12 parts, Uncaria 8-10 parts, Gastrodia 8-12 parts, fried hawthorn 5-7 parts, turmeric 8-10 parts, and chuanxiong 5-7 parts. The silver fungus vessel-activating and brain-strengthening granule has the effect of improving memory impairment of vascular dementia.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to Yinling Tongmai Jiannao Granules, their preparation method, and their application. Background Technology

[0002] Vascular dementia refers to dementia caused by brain damage due to cerebrovascular lesions, and it is the second most common type of dementia after Alzheimer's disease. Its main causes include cerebral infarction, cerebral hemorrhage, and cerebral venous lesions. In addition, white matter lesions, incomplete ischemic injuries, and local and distant ischemic functional changes are also associated with vascular dementia. Risk factors include those associated with cerebrovascular disease, such as hypertension, dyslipidemia, heart disease, diabetes, generalized arteriosclerosis, and smoking. Lesions may involve the frontal lobe, temporal lobe, and limbic system, or the lesions may damage a sufficient volume of brain tissue, leading to severe impairment of higher cognitive functions such as memory, attention, executive function, and language. Clinical manifestations include both cognitive impairment and neurological dysfunction related to cerebrovascular disease. Clinical characteristics include sudden onset, stepwise progression, fluctuating or chronic course, and a history of stroke. Treatment includes stroke prevention and treatment, improvement of cognitive function, and control of behavioral and psychiatric symptoms.

[0003] Traditional Chinese medicine (TCM) categorizes Alzheimer's disease under the "dementia" category and has a long history and rich experience in treating it. Clinically, the use of compound Chinese herbal formulas and single Chinese herbal extracts obtained through modern technology to treat vascular dementia patients has shown significant efficacy. Furthermore, TCM formulas and their combinations are guided by the TCM principle of syndrome differentiation and treatment, taking a holistic approach, and have advantages such as fewer toxic side effects and fewer withdrawal symptoms. Chinese herbal granules are produced by purifying drugs to achieve the same therapeutic effect as decocted Chinese medicine. The preparation method directly affects the type, content, and activity of the purified active ingredients. Therefore, improvements and innovations can be made to the preparation methods of traditional formulas for promoting blood circulation and strengthening the brain to obtain a Chinese herbal granule that can significantly improve memory impairment in vascular dementia. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide Yinling Tongmai Jiannao Granules, their preparation method, and their application. The Yinling Tongmai Jiannao Granules have the effect of improving memory impairment in vascular dementia.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0006] This invention provides Yinling Tongmai Jiannao Granules, comprising the following components in parts by weight: Cornus officinalis 10-15 parts, Ginkgo biloba leaf 10-15 parts, Ligustrum lucidum 8-12 parts, Salvia miltiorrhiza 8-12 parts, Acorus tatarinowii 5-7 parts, Eucommia ulmoides 8-12 parts, Poria cocos 8-12 parts, Uncaria rhynchophylla 8-10 parts, Gastrodia elata 8-12 parts, Crataegus pinnatifida 5-7 parts, Curcuma longa 8-10 parts, and Ligusticum chuanxiong 5-7 parts.

[0007] Preferably, the components include the following parts by weight: 12 parts Cornus officinalis, 12 parts Ginkgo biloba leaves, 10 parts Ligustrum lucidum, 10 parts Salvia miltiorrhiza, 6 parts Acorus tatarinowii, 10 parts Eucommia ulmoides, 10 parts Poria cocos, 9 parts Uncaria rhynchophylla, 10 parts Gastrodia elata, 6 parts Crataegus pinnatifida (fried), 9 parts Curcuma longa, and 6 parts Ligusticum chuanxiong.

[0008] This invention provides a method for preparing Yinling Tongmai Jiannao Granules, comprising the following steps:

[0009] 1) Take the prescribed amounts of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong, add water, and distill to extract the volatile oil. Then, perform β-CD inclusion on the volatile oil and grind the resulting β-CD inclusion complex into a fine powder for later use.

[0010] 2) Take the prescribed amounts of Cornus officinalis, Ginkgo biloba, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and stir-fried Crataegus pinnatifida. Add water and decoct twice. Combine the filtrates and concentrate under reduced pressure to a thick paste with a relative density of 1.20 or higher. Vacuum dry to obtain a dry paste, pulverize it into a fine powder, and set aside for later use.

[0011] 3) Add the β-CD inclusion complex obtained in step 1) to the dry extract powder in step 2), add dextrin, mix well, add appropriate amounts of steviol glycosides and povidone, moisten with ethanol, extrude and sieve to granulate, dry, granulate, and package to obtain the final product.

[0012] In one embodiment, in step 1), the acorus tatarinowii, turmeric, and chuanxiong are coarse powders that are passed through a sieve of ... mesh.

[0013] In one embodiment, the amount of water added in step 1) is 6 to 8 times, and the distillation extraction time for the volatile oil is 5 to 8 hours.

[0014] In one embodiment, in step 2), the first decoction is made with 8 to 10 times the amount of water and decocted for 2.0 to 2.5 hours, and the second decoction is made with 6 to 8 times the amount of water and decocted for 0.5 to 1.5 hours.

[0015] In one embodiment, the conditions for vacuum concentration in step 2) are 60–70°C and -0.06–-0.08 MPa.

[0016] In one embodiment, the vacuum drying conditions in step 2) are 50–60°C and -0.06–-0.08 MPa.

[0017] In one embodiment, the concentration of ethanol in step 3) is 92%.

[0018] In one embodiment, the preparation method of Yinling Tongmai Jiannao Granules includes the following steps:

[0019] 1.1) Take the prescribed amounts of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong, add 6 times the amount of water, and distill to extract the volatile oil for 5 hours. Then, encapsulate the volatile oil with β-CD. Grind the obtained β-CD inclusion complex into a fine powder and set aside.

[0020] 1.2) Take the prescribed amounts of Cornus officinalis, Ginkgo biloba, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and Crataegus pinnatifida, add water and decoct twice. For the first decoction, add 8 times the amount of water and decoct for 2 hours. For the second decoction, add 6 times the amount of water and decoct for 1.5 hours. Combine the filtrates and concentrate under reduced pressure at 60-70℃ and -0.06--0.08 MPa to a thick paste with a relative density of 1.20 or higher. Dry under vacuum at 50-60℃ and -0.06--0.08 MPa to obtain a dry paste. Grind the dry paste into a fine powder and set aside for later use.

[0021] 1.3) Add the β-CD inclusion complex obtained in step 1.1) to the dry extract powder in step 1.2), add dextrin, mix well, add appropriate amounts of steviol glycosides and povidone, moisten with 92% ethanol, extrude and sieve to granulate, dry, granulate, and package to obtain the final product.

[0022] This invention provides an application of the aforementioned Yinling Tongmai Jiannao Granules, which are used to prepare a drug for improving memory impairment in vascular dementia.

[0023] The beneficial effects of this invention are as follows: This invention uses a combination of Cornus officinalis, Ginkgo biloba leaves, Ligustrum lucidum, Salvia miltiorrhiza, Acorus tatarinowii, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, Crataegus pinnatifida (fried), Curcuma longa, and Ligusticum chuanxiong. First, Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong are coarsely crushed, water is added, and volatile oil is extracted by distillation. β-CD inclusion complex is obtained. Then, the β-CD inclusion complex is added to a dry extract powder made by decocting Cornus officinalis, Ginkgo biloba leaves, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and Crataegus pinnatifida (fried) with water, combining the filtrates, concentrating under reduced pressure, and vacuum drying. This effectively improves the efficacy of Yinling Tongmai Jiannao Granules in improving memory impairment in vascular dementia, significantly improves memory impairment in vascular dementia and improves blood rheology, and has a significant anti-vascular dementia effect.

[0024] Other advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The diagram shows the Morris water maze trajectory in the experiment; where A is the normal control group; B is the sham surgery group; C is the model control group; D is the donepezil group; F is the Yinling Tongmai Jiannao Granules Process No. 1 sample group; E is the Yinling Tongmai Jiannao Granules Process No. 2 sample group; and G is the Yinling Tongmai Jiannao Granules Process No. 3 sample group.

[0027] Figure 2 The results show the effects of different processing methods of Yinling Tongmai Jiannao Granules on the pathological examination of rat cortical tissue (HE, ×100); where: A is the normal control group, B is the sham-operated group, C is the model control group, D is the donepezil group, F is the Yinling Tongmai Jiannao Granules process ① sample group, E is the Yinling Tongmai Jiannao Granules process ② sample group, and G is the Yinling Tongmai Jiannao Granules process ③ sample group.

[0028] Figure 3 The following are the results of histopathological examination of rat hippocampal tissue (HE, ×100) under different processing methods of Yinling Tongmai Jiannao Granules in the experiment; where: A is normal control group; B is sham-operated group; C is model control group; D is donepezil group; F is sample group No. 1 of Yinling Tongmai Jiannao Granules processing method; E is sample group No. 2 of Yinling Tongmai Jiannao Granules processing method; G is sample group No. 3 of Yinling Tongmai Jiannao Granules processing method.

[0029] Figure 4 The following are the results of the different processing methods of Yinling Tongmai Jiannao Granules for histopathological examination of rat hippocampal tissue (Nissl, ×100); where: A is the normal control group; B is the sham-operated group; C is the model control group; D is the donepezil group; F is the Yinling Tongmai Jiannao Granules process ① sample group; E is the Yinling Tongmai Jiannao Granules process ② sample group; G is the Yinling Tongmai Jiannao Granules process ③ sample group.

[0030] Figure 5 The spatial exploration movement trajectory diagrams of dementia rats in Experiment 1 are shown below; A is the sham-operated group; B is the model control group; C is the donepezil hydrochloride tablet group; D is the compound Cistanche capsule group; F is the low-dose Yinling Tongmai Jiannao granules group; E is the medium-dose Yinling Tongmai Jiannao granules group; and G is the high-dose Yinling Tongmai Jiannao granules group.

[0031] Figure 6 The results of Experiment 1 showed the effect of Yinling Tongmai Jiannao Granules on the cortical tissue pathology of dementia rats (×400); among them, A was the sham-operated group; B was the model control group; C was the donepezil acid tablet group; D was the Cistanche deserticola Yizhi capsule group; E was the low-dose group of Yinling Tongmai Jiannao Granules; F was the medium-dose group of Yinling Tongmai Jiannao Granules; and G was the high-dose group of Yinling Tongmai Jiannao Granules.

[0032] Figure 7 The results of Experiment 1 showed the effects of Yinling Tongmai Jiannao Granules on the hippocampal tissue of dementia rats (×400); among them, A was the sham-operated group; B was the model control group; C was the donepezil acid tablet group; D was the Cistanche deserticola Yizhi capsule group; E was the low-dose Yinling Tongmai Jiannao Granules group; F was the medium-dose Yinling Tongmai Jiannao Granules group; and G was the high-dose Yinling Tongmai Jiannao Granules group.

[0033] Figure 8Nissl staining (×400) of hippocampal tissue in dementia rats was performed using Yinling Tongmai Jiannao Granules in Experiment 1. The groups included: A. sham-operated group; B. model control group; C. donepezil acid tablet group; D. Cistanche deserticola Yizhi capsule group; E. low-dose Yinling Tongmai Jiannao Granules group; F. medium-dose Yinling Tongmai Jiannao Granules group; and G. high-dose Yinling Tongmai Jiannao Granules group.

[0034] Figure 9 Nissl staining (×400) of Yinling Tongmai Jiannao Granules on the cortical tissue of demented rats in Experiment 1; A: sham-operated group; B: model control group; C: donepezil acid tablet group; D: Cistanche deserticola Yizhi capsule group; E: low-dose Yinling Tongmai Jiannao Granules group; F: medium-dose Yinling Tongmai Jiannao Granules group; G: high-dose Yinling Tongmai Jiannao Granules group;

[0035] Figure 10 The image shows the water maze trajectory of the scopolamine-induced cognitive impairment mouse model using Yinling Tongmai Jiannao Granules in Experiment 2. The groups are: A) sham-operated group; B) model control group; C) donepezil acid tablet group; D) Cistanche deserticola Yizhi capsule group; E) low-dose Yinling Tongmai Jiannao Granules group; F) medium-dose Yinling Tongmai Jiannao Granules group; and G) high-dose Yinling Tongmai Jiannao Granules group. Detailed Implementation

[0036] To further explain the technical solution of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1

[0038] Weigh the following raw materials according to the following proportions: Cornus officinalis 12 parts, Ginkgo biloba 12 parts, Ligustrum lucidum 10 parts, Salvia miltiorrhiza 10 parts, Acorus tatarinowii 6 parts, Eucommia ulmoides 10 parts, Poria cocos 10 parts, Uncaria rhynchophylla 9 parts, Gastrodia elata 10 parts, Crataegus pinnatifida (fried) 6 parts, Curcuma longa 9 parts, and Ligusticum chuanxiong 6 parts. Prepare the extract powder of Yinling Tongmai Jiannao Granules according to the following steps:

[0039] 1) Take the prescribed amount of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong, coarsely crush them, add 6 times the amount of water, and distill to extract the volatile oil for 5 hours. Then, encapsulate the volatile oil with β-CD. Grind the obtained β-CD inclusion complex into a fine powder and set aside.

[0040] 2) Take the prescribed amounts of Cornus officinalis, Ginkgo biloba, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and Crataegus pinnatifida, add water and decoct twice. For the first decoction, add 8 times the amount of water and decoct for 2 hours. For the second decoction, add 6 times the amount of water and decoct for 0.5 hours. Combine the filtrates and concentrate under reduced pressure at 60-70℃ and -0.06--0.08 MPa to a thick paste with a relative density of 1.20 or higher. Dry under vacuum at 50-60℃ and -0.06--0.08 MPa to obtain a dry paste, and pulverize it into a fine powder.

[0041] 3) Add the β-CD inclusion complex obtained in step 1) to the dry extract powder in step 2) for later use.

[0042] Comparative Example 1

[0043] Weigh the following raw materials according to the following proportions: Cornus officinalis 12 parts, Ginkgo biloba 12 parts, Ligustrum lucidum 10 parts, Salvia miltiorrhiza 10 parts, Acorus tatarinowii 6 parts, Eucommia ulmoides 10 parts, Poria cocos 10 parts, Uncaria rhynchophylla 9 parts, Gastrodia elata 10 parts, Crataegus pinnatifida (fried) 6 parts, Curcuma longa 9 parts, and Ligusticum chuanxiong 6 parts. Prepare the extract powder of Yinling Tongmai Jiannao Granules according to the following steps:

[0044] Take all the ingredients in the formula and boil them twice. For the first boil, add 8 times the amount of water and boil for 2 hours. For the second boil, add 6 times the amount of water and boil for 1.5 hours. Combine the decoctions and concentrate them to a thick paste with a relative density of 1.20 or higher. Dry the paste under vacuum at 50-60℃ and -0.06--0.08 MPa to obtain a dry paste. Grind the dry paste into a fine powder and set aside for later use.

[0045] Comparative Example 2

[0046] Weigh the following raw materials according to the following proportions: Cornus officinalis 12 parts, Ginkgo biloba 12 parts, Ligustrum lucidum 10 parts, Salvia miltiorrhiza 10 parts, Acorus tatarinowii 6 parts, Eucommia ulmoides 10 parts, Poria cocos 10 parts, Uncaria rhynchophylla 9 parts, Gastrodia elata 10 parts, Crataegus pinnatifida (fried) 6 parts, Curcuma longa 9 parts, and Ligusticum chuanxiong 6 parts. Prepare the extract powder of Yinling Tongmai Jiannao Granules according to the following steps:

[0047] Take the prescribed amounts of ginkgo leaves, privet fruit, salvia miltiorrhiza, and turmeric, coarsely crush them, and extract them twice with 70% ethanol. For the first extraction, add 7 times the amount of 70% ethanol and reflux for 2 hours. For the second extraction, add 6 times the amount of 70% ethanol and reflux for 1.5 hours. Combine the extracts, recover the ethanol, and decoct the residue once with 8 times the amount of water. Concentrate the extract and combine it with the ethanol extract. Concentrate the extract to a thick paste with a density of approximately 1.2 or higher. Vacuum dry the paste at 50–60℃ and -0.06–-0.08 MPa to obtain a dry paste. Grind the dry paste into a fine powder for later use.

[0048] I. Experiments were conducted based on Example 1, Comparative Example 1, and Comparative Example 2:

[0049] 1. Experimental Objective: This experiment established a vascular dementia model in rats by bilateral cervical vascular ligation and investigated the therapeutic effects of Yinling Tongmai Jiannao Granules processed in different ways on the rat vascular dementia model in Example 1, Comparative Example 1, and Comparative Example 2.

[0050] 2. Experimental Materials

[0051] 2.1 Test sample

[0052] The extract powder prepared according to the preparation method of Example 1 is used as the first sample of the Yinling Tongmai Jiannao Granules process, batch number: 20211020-2, brown extract, content: 2.0206g crude drug / g extract, expiration date: 2022.10.30;

[0053] The extract powder prepared according to the preparation method of Comparative Example 1 was used as Sample No. 2 of the Yinling Tongmai Jiannao Granules, batch number: 20211019-1, appearance: brown extract, content: 1.9738 g of crude drug / g of extract, expiration date: October 30, 2022;

[0054] The extract powder prepared according to the preparation method of Comparative Example 2 was used as Sample No. 3 of the Yinling Tongmai Jiannao Granules, batch number: 20211020-3, brown extract, content: 1.9073 g of crude drug / g of extract, expiration date: October 30, 2022.

[0055] 2.2 Reference Substances

[0056] Donepezil Hydrochloride Tablets, batch number: 20210204, product of Zhejiang Huahai Pharmaceutical Co., Ltd.

[0057] 2.3 Experimental Animals

[0058] 80 SPF-grade SD rats, male, weighing 220-250 g, purchased from Changsha Tianqin Biotechnology Co., Ltd., experimental animal production license number: SCXK(Xiang)2019-0014; quality certificate number: 430726221100014685, raised in Area D of the barrier environment of Hunan Prima Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2020-0015.

[0059] 2.4 Main Reagents

[0060] Isoflurane, batch number: 20201201, product of Shandong Ant牧业科技有限公司 (There seems to be a typo here, it might be "Shandong Ant Animal Husbandry Technology Co., Ltd.").

[0061] Penicillin Sodium for Injection, batch number: F0042309, specification: 1.6 million units / bottle, appearance: white powder, product of North China Pharmaceutical Co., Ltd.

[0062] 2.5 Main Instruments

[0063] Morris water maze, product of Chengdu Taimeng Technology Co., Ltd.; EG1150H+C type tissue embedding machine, RM2235 type paraffin slicer, DM2000 type biological microscope, all are products of Leica company.

[0064] 3. Experimental Methods

[0065] 3.1 Model Establishment and Grouping

[0066] Eighty qualified male SD rats, weighing 220-250g, were selected. Eight rats were randomly selected as the normal control group, and eight as the sham-operated group. The remaining rats were used to establish a rat model of vascular dementia using a modified double-vessel occlusion (2-VO) method. The specific method was as follows: After anesthetizing the rats with isoflurane inhalation, the right common carotid artery was isolated, and both ends of the vessel were ligated at the proximal and distal ends. The common carotid artery was then cut at the ligation center, and the layers were sutured. Penicillin 400,000 U / rat was injected intramuscularly to prevent infection. Six days later, the left common carotid artery was treated in the same way. The common carotid artery in the sham-operated group was not ligated or cut, and the remaining procedures were the same as those for surgical modeling. Six weeks after model establishment, 40 surviving rats were randomly divided into three groups: a model control group, a donepezil group (0.9 mg / kg), a Yinling Tongmai Jiannao Granules sample group ① (9.9 g crude drug / kg), a Yinling Tongmai Jiannao Granules sample group ② (9.9 g crude drug / kg), and a Yinling Tongmai Jiannao Granules sample group ③ (9.9 g crude drug / kg), with eight rats in each group. The normal control group and the sham-operated group were administered pure water by gavage, while the other groups were administered the corresponding drug solution by gavage. The drug administration volume was 10 mL / kg, once a day, for 6 consecutive weeks. After the last administration, the Morris water maze test was performed. Subsequently, cortical and hippocampal tissues were collected for HE staining and Nissl staining to observe the pathological changes in the rat hippocampus and cortical tissues.

[0067] 3.2 Dosage Design

[0068] The clinical dosage of Yinling Tongmai Jiannao Granules (processes ①, ②, and ③) is 110g crude drug / day. Based on the conversion of human and rat body surface area, the equivalent rat dose is 110g crude drug * 0.018 / 0.2kg = 9.9g crude drug / kg. The clinical dosage of donepezil hydrochloride is 10mg / day. Based on the conversion of human and rat body surface area, the equivalent rat dose is 10mg * 0.018 / 0.2kg = 0.9mg / kg. The group and dosage settings are shown in Table 1.

[0069] Table 1 Animal grouping and dosage design

[0070]

[0071]

[0072] 3.3 Detection Indicators

[0073] 3.3.1 Morris Water Maze Test: After the last administration, rats in each group underwent a water maze test. Initially, they underwent navigation training for 4 days. A platform was fixed in the center of the second quadrant. Tap water was filled into the pool, and white dye was added to make the platform invisible. The water level was 1.0 cm above the platform. The water temperature was maintained at 25℃±1℃. Each animal was tested once daily. Before the test, the animal was placed on the platform for 10 seconds to acclimatize. Three entry points were randomly selected, and the test lasted 60 seconds. If the animal successfully found the platform, it was allowed to stay on the platform for 10 seconds before being returned to its cage. If it failed to find the platform, it was guided to the platform and allowed to stay for 10 seconds. The time it took to find the platform (escape latency / total time), total swimming distance, and average speed were recorded to evaluate the rats' learning and memory abilities. On day 5, spatial exploration was conducted, and the number of times the animal crossed the platform was measured.

[0074] 3.3.3 Cortical and hippocampal histopathological examination: After the behavioral test, rats in each group were anesthetized with isoflurane, and brain tissue was dissected and fixed in 10% formalin. HE staining and Nissl staining were performed to observe the morphological characteristics of nerve cells, such as their arrangement, apoptosis, and microvessels in the CA1 region of the cortex and hippocampus.

[0075] 3.4 Statistical Methods

[0076] Data in this experiment were rounded to the nearest whole number, and SPSS was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (Mean ± SEM), and Leven's test was used to test for normality and homogeneity of variance. If there was no statistical significance (P > 0.05), one-way ANOVA was used for statistical analysis; two-way ANOVA was used for water maze navigation data. If the ANOVA was statistically significant (P ≤ 0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P ≤ 0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P ≤ 0.05), Dunnett's test (non-parametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P ≤ 0.05 indicated statistical significance, and P ≤ 0.01 indicated a highly significant difference.

[0077] 4. Experimental Results

[0078] 4.1 Effects of different processing methods of Yinling Tongmai Jiannao Granules on local navigation in a rat model of vascular dementia

[0079] As shown in Table 2, compared with the normal control group, there was no significant difference in the latency of the model control group rats on days 1 and 2 of the navigation, but the latency of the model control group rats on days 3 and 4 of the navigation was significantly increased (P≤0.01); compared with the model control group, the latency of the model control group rats in the Yinling Tongmai Jiannao Granules process No. 1 and No. 3 groups was significantly reduced on days 3 and 4 of the navigation (P≤0.05), and the latency of the model control group rats in the donepezil hydrochloride group was reduced on days 3 and 4 of the navigation (P≤0.01).

[0080] Table 2. Effects of different processing methods of Yinling Tongmai Jiannao Granules on local navigation in a rat model of vascular dementia (Mean±SEM, n=8)

[0081]

[0082] Note: Compared with the normal control group and the sham surgery group. ++ P≤0.01; compared with the model control group, * P≤0.05.

[0083] 4.2 Effects of different processing methods of Yinling Tongmai Jiannao Granules on spatial exploration in a rat model of vascular dementia

[0084] As shown in Table 3, compared with the normal control group, the number of times rats in the model control group crossed the platform was significantly reduced (P≤0.01). Compared with the model control group, the number of times rats in the Yinling Tongmai Jiannao Granules No. 1 sample group crossed the platform was significantly increased (P≤0.01), and the number of times rats in the donepezil hydrochloride group crossed the platform was significantly increased (P≤0.01). The Morris water maze trajectory diagram is shown below. Figure 1 As shown.

[0085] Table 3. Effects of different processing methods of Yinling Tongmai Jiannao Granules on spatial exploration in a rat model of vascular dementia (Mean±SEM, n=8)

[0086]

[0087] Note: Compared with the normal control group and the sham surgery group, ++P≤0.01; compared with the model control group, **P≤0.01.

[0088] The data in Tables 2 and 3 show that the Yinling Tongmai Jiannao Granules process ① and ③ significantly reduced the latency of rats in positioning and navigation on days D3 and D4, while the Yinling Tongmai Jiannao Granules process ① significantly increased the number of times rats crossed the platform. The results suggest that the Yinling Tongmai Jiannao Granules process ① can simultaneously improve the acquisition and retention of spatial memory in dementia model rats.

[0089] 4.3 Effects of different processing methods of Yinling Tongmai Jiannao Granules on the pathological characteristics of rat cortex and hippocampus tissues

[0090] like Figure 2 and Figure 3 As shown, in the normal control group and sham-operated group, no abnormalities were observed in the morphology of neurons, glial cells, and hippocampal pyramidal cells in the hippocampal tissue of rats. In the model control group, some pyramidal cells showed sparse and disordered arrangement, some pyramidal cells were necrotic, and interstitial edema was observed in the hippocampal tissue of rats processed with Yinling Tongmai Jiannao Granules ② and ③. Some pyramidal cells showed disordered arrangement and interstitial edema in the hippocampal tissue of rats processed with Yinling Tongmai Jiannao Granules ①. Some pyramidal cells in the hippocampal tissue of rats processed with Yinling Tongmai Jiannao Granules ① were tightly arranged, and a small amount of interstitial edema was observed. In the donepezil hydrochloride group, the hippocampal tissue of rats showed tightly arranged pyramidal cells and a small amount of interstitial edema. Process ① of Yinling Tongmai Jiannao Granules can significantly improve the pathological changes of sparse and disordered arrangement of pyramidal cells, pyramidal cell necrosis, and interstitial edema in dementia rats.

[0091] 4.3 Effects of different processing methods of Yinling Tongmai Jiannao Granules on Nissl staining of rat cortex and hippocampus tissues

[0092] like Figure 4 As shown, in the normal control group and sham-operated group, the neurons in the CA1 region of the hippocampus of rats were densely arranged, with clear and numerous Nissl bodies staining within the neurons. In the model group, the neurons in the CA1 region of the hippocampus of mice were loosely arranged, and some neurons showed a reduction or disappearance of Nissl bodies. Compared with the model control group, the number of Nissl bodies in the CA1 region of the hippocampus of rats in the Yinling Tongmai Jiannao Granules Process No. 1 group was significantly increased. Yinling Tongmai Jiannao Granules Process No. 1 can increase the number of Nissl bodies in the hippocampus of rats.

[0093] 5. Experiment Summary

[0094] The results showed that the Yinling Tongmai Jiannao Granules Process No. 1 significantly increased the number of times rats crossed the platform and significantly improved the pathological changes in the cortex and hippocampus, suggesting that the Yinling Tongmai Jiannao Granules Process No. 1 can significantly improve memory impairment in vascular dementia.

[0095] 6. Discussion and Summary

[0096] Vascular dementia (VD) refers to a heterogeneous group of diseases encompassing a range of clinical and neuropathological manifestations of cognitive impairment caused by cerebrovascular diseases. Currently, the construction of rat models of vascular dementia primarily employs methods such as cerebral vascular occlusion to reduce cerebral blood flow. Among these, permanent bilateral common carotid artery ligation is a reliable animal model of vascular dementia induced by chronic hypoperfusion in rats. It is currently the most commonly used classic method for inducing chronic global cerebral ischemia in rats and is widely applied in vascular dementia research. Its behavioral characteristics mainly manifest as learning disabilities and memory impairment.

[0097] The Morris water maze is a behavioral test used to evaluate spatial learning and memory abilities in animals. It examines both reference and working memory abilities through different modes. In this experiment, the escape latency of a rat model with vascular dementia was significantly prolonged during navigation tests D3 and D4. However, continuous administration of Yinling Tongmai Jiannao Granules (processes ① and ③) for 6 weeks significantly shortened this escape latency, indicating that Yinling Tongmai Jiannao Granules (processes ① and ③) significantly improved the spatial platform-finding ability of dementia rats. Simultaneously, Yinling Tongmai Jiannao Granules (process ①) significantly increased the number of platform crossings in the spatial exploration test, improving spatial exploration memory in dementia animals. Both experimental modes are based on reference memory; the former examines memory acquisition ability, while the latter examines memory retention ability. The results suggest that Yinling Tongmai Jiannao Granules (process ①) can simultaneously improve both the acquisition and retention abilities of spatial memory in dementia model rats.

[0098] In the central nervous system (CNS), the hippocampus is an important brain region within the limbic system closely related to higher cognitive functions such as emotion, learning, and memory. The hippocampus is a crucial component of the limbic system, participating in emotion and memory. Studies have shown that vascular dementia causes neuronal damage in the hippocampus of rats. This experiment found that in a rat model of vascular dementia, some pyramidal cells in the hippocampus were sparsely and disordered, some pyramidal cells were necrotic, interstitial edema was present, and the number of Nissl bodies was significantly reduced. The Yinling Tongmai Jiannao Granules process① significantly increased the number of Nissl bodies in the rat hippocampus.

[0099] In conclusion, the No. 1 process of Yinling Tongmai Jiannao Granules can significantly improve memory impairment in vascular dementia.

[0100] II. A vascular dementia model was established by vascular ligation, a scopolamine-induced cognitive impairment model was established in mice, and an adrenaline-induced blood stasis model was established in rats by ice water bath. The effects of Yinling Tongmai Jiannao Granules prepared in Example 1 on dementia were studied.

[0101] Experimental methods:

[0102] (1) Effects of Yinling Tongmai Jiannao Granules on a rat model of vascular dementia

[0103] One hundred SPF-grade, quarantine-qualified male SD rats, weighing 180.0–220.0 g, were selected. Ten rats were randomly selected based on weight as the sham-operated group, while the remaining rats underwent bilateral carotid artery ligation to establish a vascular dementia model. The methods were as follows: Rats were fasted and deprived of water for 12 hours before surgery and weighed. They were anesthetized with isoflurane inhalation, their skin was prepared, and the skin of the rat neck was disinfected with iodine and cut open. The bilateral common carotid arteries were separated and ligated. In the sham-operated group, only the bilateral common carotid arteries were separated without ligation. Six weeks after surgical modeling, rats were randomly divided into six groups based on their body weight: a model control group, donepezil hydrochloride tablet group, compound Cistanche deserticola capsule group, and low, medium, and high dose Yinling Tongmai Jiannao granules groups, with 10 rats in each group. Rats in the sham-operated group and the model control group were administered pure water orally at a dose of 10 mL / kg, while rats in the other groups were administered the corresponding drug solution by gavage at the same volume once a day for a total of 8 weeks. After the drug administration was completed, the Morris water maze behavioral test was performed. After the behavioral test, rats in each group were anesthetized with isoflurane inhalation, and blood was collected from the abdominal aorta for coagulation and hemorheological index testing. Hippocampal tissue was taken, and the contents of TNF-α, IL-6, IL-1β, ChAT, A-chE, and BDNF in the hippocampal tissue were detected using an ELISA kit. The histopathological changes of the hippocampal cortex were also observed.

[0104] (2) Effects of Yinling Tongmai Jiannao Granules on Scopolamine-Induced Cognitive Impairment in Mice

[0105] Seventy qualified male ICR mice, weighing 180.0–220.0 g, were randomly divided into seven groups based on their weight: a normal control group, a model control group, donepezil hydrochloride tablets, compound Cistanche deserticola capsules, and low, medium, and high dose Yinling Tongmai Jiannao granules, with ten mice in each group. Mice in the normal and model control groups were administered pure water by gavage at a volume of 20 mL / kg. The other groups were administered the corresponding drug solutions by gavage once daily for seven days. After the last administration, the normal control group received an intraperitoneal injection of 0.9% sodium chloride injection, while the other groups received an intraperitoneal injection of scopolamine 2 mg / kg at a volume of 20 mL / kg. Morris water maze behavioral tests were performed 20 minutes after injection.

[0106] (3) Effects of Yinling Tongmai Jiannao Granules on Hemorheology in Rats

[0107] Sixty SD rats qualified for quarantine were selected, with 30 males and 30 females, weighing 180.0 - 220.0 g. According to their body weights, the animals were randomly divided into 6 groups, namely the normal control group, the model control group, the enteric-coated aspirin tablet group, and the low, medium, and high-dose groups of Yinling Tongmai Jiannao Granules, with 10 rats in each group (5 males and 5 females). Rats in the normal control group and the model control group were orally gavaged with pure water at a dose of 10 mL / kg; rats in the other groups were gavaged with an equal volume of the corresponding medicinal solution once a day for 7 consecutive days. After the last administration, except for the normal control group, rats in the other groups were subcutaneously injected with 0.1% epinephrine at a dose of 0.08 ml / 100 g. Two hours later, rats in each group swam in ice water (4°C) for 5 minutes, and the same dose of epinephrine was administered again 4 hours after the first injection. After fasting for 18 hours without water deprivation, blood was collected from the abdominal aorta, and plasma viscosity was measured using a hemorheology testing instrument.

[0108] Ⅰ. Experiment 1: Effect of Yinling Tongmai Jiannao Granules on Rat Vascular Dementia Model

[0109] 1. Experimental Purpose

[0110] In this experiment, a vascular dementia model was established by double-vessel ligation to study the effect of Yinling Tongmai Jiannao Granules prepared according to the preparation method of Example 1 in treating dementia, providing an experimental basis for clinical research.

[0111] 2. Experimental Materials

[0112] 2.1 Tested Products

[0113] The extract powder of Yinling Tongmai Jiannao Granules prepared according to Example 1, batch number: 20221204, character: yellowish-brown powder, slightly fragrant smell, slightly sour taste, specification: 4.699 g crude drug / g dry extract powder, provided by Hunan Butian Pharmaceutical Co., Ltd.

[0114] 2.2 Positive Controls

[0115] Donepezil Hydrochloride, batch number: 0000047417, product of Zhejiang Huahai Pharmaceutical Co., Ltd.; Compound Cistanche and Alpinia Oxyphylla Capsules, batch number: 221113, product of Anhui Leyunsheng Pharmaceutical Co., Ltd.

[0116] 2.3 Experimental Animals

[0117] One hundred SPF-grade male SD rats, weighing 180.0 - 220.0 g, were purchased from Changsha Tianqin Biotechnology Co., Ltd. The production license number of experimental animals is SCXK(Xiang)2022 - 0014, and the quality certificate number of experimental animals is 430726231100078136. They were housed in Area D of the barrier environment of Hunan Prima Pharmaceutical Research Center Co., Ltd., and the license number for using experimental animals is SYXK(Xiang)2020 - 0015.

[0118] 2.4 Main Reagents

[0119] Rat Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1β (IL-1β), Choline Acetyltransferase (ChAT), Acetylcholinesterase (A-chE), and Brain-Derived Neurotrophic Factor (BDNF) ELISA Kit, Batch No.: 0612023; Manufacturer: Huangshi Ains Biotechnology Co., Ltd.

[0120] 2.5 Main Instruments

[0121] ME2002E electronic balance (Mettler AG); Morris water maze video analysis system (Chengdu Taimeng Co., Ltd.); SpectraMaxi3x multi-functional microplate reader (MD Inc., USA); AUY220 and AUW-220D analytical balances (Mettler AG); TD4 low-speed centrifuge (Nanjing Pulang Co., Ltd.); RM2235 paraffin sectioner, TP1020 fully automatic dehydrator, HI1220 slide roaster, HI1210 slide spreader, EG1150H+C tissue embedding machine, DM2000 biological microscope, and DFC420C pathological imaging system (all Leica AG, Germany).

[0122] 3. Research Content

[0123] 3.1 Grouping and Dosing

[0124] One hundred SPF-grade, quarantine-qualified male SD rats, weighing 180.0–220.0 g, were selected. Ten rats were randomly selected based on weight as the sham-operated group, while the remaining rats underwent bilateral carotid artery ligation to establish a vascular dementia model. The methods were as follows: Rats were fasted and deprived of water for 12 hours before surgery and weighed. They were anesthetized with isoflurane inhalation, their skin was prepared, and the skin of the rat neck was disinfected with iodine and cut open. The bilateral common carotid arteries were separated and ligated. In the sham-operated group, only the bilateral common carotid arteries were separated without ligation. Six weeks after surgical modeling, rats were randomly divided into six groups based on their body weight: a model control group, donepezil hydrochloride tablet group, compound Cistanche deserticola capsule group, and low, medium, and high dose Yinling Tongmai Jiannao granules groups, with 10 rats in each group. Rats in the sham-operated group and the model control group were administered pure water orally at a dose of 10 mL / kg, while rats in the other groups were administered the corresponding drug solution by gavage at the same volume once a day for a total of 8 weeks. After the drug administration was completed, the Morris water maze behavioral test was performed. After the behavioral test, rats in each group were anesthetized with isoflurane inhalation, and blood was collected from the abdominal aorta for coagulation and hemorheological parameters. Hippocampal tissue was taken, and the contents of TNF-α, IL-6, IL-1β, ChAT, A-chE, and BDNF in the hippocampal tissue were detected using an ELISA kit. The histopathological changes of the hippocampal cortex were also observed.

[0125] 3.2 Dosage setting

[0126] According to the data, the proposed clinical dosage of Yinling Tongmai Jiannao Granules is 55g of raw drug per day. Based on the conversion of animal and human surface area, the clinical equivalent dose is calculated as: 55g raw drug / day * 0.018 / 0.2kg = 4.95g raw drug / kg. The low, medium, and high doses of Yinling Tongmai Jiannao Granules are set at 1, 2, and 4 times the clinical equivalent dose, respectively: 4.95g raw drug / kg, 9.9g raw drug / kg, and 19.8g raw drug / kg. The clinical dosage of donepezil hydrochloride is 10mg / day. Based on the conversion of animal and human surface area, the clinical equivalent dose is: 10mg / day * 0.018 / 0.2kg = 0.9mg / kg. The clinical dosage of Compound Cistanche deserticola Yizhi Capsules is 3.6g / day. Based on the conversion of animal and human surface area, the clinical equivalent dose is: 3.6g / day * 0.018 / 0.2kg = 0.32g / kg. The group and dosage settings are shown in Table 1-1.

[0127] Table 1-1 Trial grouping and dosage design

[0128]

[0129] 3.3 Detection Indicators

[0130] 3.3.1 Morris Water Maze Test: After the last administration, rats in each group underwent navigation training for 5 days. A platform was fixed in the center of the second quadrant. Tap water was poured into a pool, and white fuel was added to obscure the platform, with the water level 1.5 cm above the platform. The water temperature was maintained at 25℃±1℃. Each animal was tested once daily. Before the test, the animal was placed on the platform for 10 seconds to acclimatize. Three entry points were randomly selected, and the test lasted 60 seconds. If the animal successfully found the platform, it was allowed to stay on the platform for 10 seconds before being returned to its cage. If it failed to find the platform, it was guided to the platform and allowed to stay for 10 seconds. The time it took to find the platform (escape latency), total swimming distance, and average speed were recorded to evaluate the mice's learning and memory abilities. On day 6, spatial exploration was conducted, and the total distance traveled and the number of times the animals crossed the platform were measured.

[0131] 3.3.2 Blood rheology test: After the behavioral test, rats in each group were anesthetized with isoflurane, and blood was collected from the abdominal aorta. The coagulation parameters such as fibrinogen (FIB), plasma prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were measured using a blood coagulation analyzer.

[0132] 3.3.3 Histopathological examination: Brain tissue was fixed in 10% neutral formalin and subjected to HE and Nissl staining to observe the morphological characteristics of nerve cells, such as their arrangement, apoptosis, and microvessels in the cortex and CA1 region of the hippocampus.

[0133] 3.4. Data Processing and Statistical Analysis

[0134] The data in this experiment were rounded to the nearest whole number and statistically analyzed according to the "Statistical Processing of Experimental Data" (SOP-Rd-003). SPSS software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (Mean ± SEM), and normality and homogeneity of variance were tested using Leven's test. If there was no statistical significance (P>0.05), one-way ANOVA was used. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), Dunnett's test (non-parametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P≤0.05 indicated statistical significance, and P≤0.01 indicated a highly significant difference.

[0135] 4. Experimental Results

[0136] 4.1 Effects of Yinling Tongmai Jiannao Granules on Navigation and Orientation in a Rats with Dementia

[0137] As shown in Table 1-2, compared with the normal control group, the latency of local navigation on days 4 and 5 in the model control group was significantly increased (P≤0.05 or P≤0.01). Compared with the model control group, the latency of local navigation on day 2 in the medium and high dose groups of Yinling Tongmai Jiannao Granules was significantly decreased (P≤0.05 or P≤0.01), the latency of local navigation on day 3 in the low and high dose groups of Yinling Tongmai Jiannao Granules was significantly decreased (P≤0.05 or P≤0.01), and the latency of local navigation on days 4 and 5 in the low, medium, and high dose groups of Yinling Tongmai Jiannao Granules was significantly decreased (P≤0.05 or P≤0.01). The latency of local navigation on days 3 to 5 in the positive control group (donepizil hydrochloride tablets) was significantly decreased (P≤0.05 or P≤0.01), and the latency of local navigation on days 4 to 5 in the compound Cistanche deserticola capsules group was significantly decreased (P≤0.05 or P≤0.01).

[0138] Table 1-2 Effects of Yinling Tongmai Jiannao Granules on the latency of local navigation in a rat model of dementia ( n=10)

[0139]

[0140]

[0141] Note: Compared with the sham surgery group, +P≤0.05, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0142] 4.2 Effects of Yinling Tongmai Jiannao Granules on Spatial Exploration in a Rats with Dementia

[0143] like Figure 5 As shown in Tables 1-3, compared with the normal control group, the total spatial exploration distance and the number of platform crossings in the model control group were significantly reduced (P≤0.05 or P≤0.01). Compared with the model control group, the total spatial exploration distance and the number of platform crossings in the low, medium, and high dose groups of Yinling Tongmai Jiannao Granules were significantly reduced and increased (P≤0.05 or P≤0.01). The total spatial exploration distance and the number of platform crossings in the positive control groups (Donepezil Hydrochloride Tablets and Compound Cistanche Yizhi Capsules) were significantly reduced and increased (P≤0.05 or P≤0.01).

[0144] Table 1-3 Effects of Yinling Tongmai Jiannao Granules on Spatial Exploration in a Rat Model of Dementia n=10)

[0145]

[0146] Note: Compared with the sham surgery group, +P≤0.05, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0147] 4.3 Effects of Yinling Tongmai Jiannao Granules on Hemorheology in a Rat Model of Dementia

[0148] As shown in Tables 1-4, compared with the normal control group, there were no significant differences in PT, APTT, Fbg, and TT levels in the model control group rats. Compared with the model control group, the high-dose Yinling Tongmai Jiannao Granules group showed significantly increased blood PT and Fbg levels (P≤0.05), the low- and medium-dose groups showed significantly increased blood APTT levels (P≤0.01), and the medium- and high-dose groups showed significantly increased blood TT levels (P≤0.01 or P≤0.05). The Compound Cistanche Yizhi Capsule group showed significantly increased blood TT levels (P≤0.05).

[0149] Table 1-4 Effects of Yinling Tongmai Jiannao Granules on Coagulation Indicators in a Rat Model of Dementia n=10)

[0150]

[0151] Note: Compared with the model control group, *P≤0.05, **P≤0.01.

[0152] 4.4 Effects of Yinling Tongmai Jiannao Granules on Pathological Changes in Hippocampus and Cortical Tissues of a Rat Model of Dementia

[0153] like Figure 6 and Figure 7 As shown, in the normal control group rats, the pyramidal cells in the CA1 region of the hippocampus were tightly and uniformly arranged. Under high magnification, the pyramidal cells had normal morphology, large and round nuclei, clear nucleoli, and uniform cytoplasmic staining. The cortical tissue showed a large number of neurons with uniform staining, no obvious cell degeneration, and no inflammatory cell infiltration. In the model control group mice, the pyramidal cells in the CA1 region of the hippocampus were loosely arranged and disordered; the number of neurons was reduced, and some neurons showed deep nuclear staining and pyknosis. The number of normally morphological neurons in the cortical tissue was reduced, and a large number of irregularly shaped degenerated neurons were visible. In the low-dose group of Yinling Tongmai Jiannao Granules, the number of pyramidal cells in the CA1 region of the hippocampus increased, the cells were more neatly arranged, and some areas still showed loose arrangement. The cell morphology was relatively normal, and a small number of cells showed deep nuclear staining and pyknosis; small blood vessel dilation was observed. In rats in the low, medium, and high dose groups of Yinling Tongmai Jiannao Granules, the pyramidal cells in the CA1 region of the hippocampus were relatively well-arranged with regular cell morphology. Occasionally, deep staining and pyknosis of the nuclei were observed in neurons, but no obvious cell necrosis was seen. The number of normally morphological neurons in the cortical tissue was reduced, and some irregularly shaped degenerated neurons were visible. In rats in the donepezil and Cistanche Yizhi Capsule groups, the pathological damage in the CA1 region of the hippocampus and cortex was significantly improved. The pyramidal cells were tightly arranged with regular cell morphology and uniform cytoplasmic staining, and no obvious cell necrosis was seen. The number of normally morphological neurons in the cortical tissue was reduced, and some irregularly shaped degenerated neurons were visible.

[0154] 4.5 Effects of Yinling Tongmai Jiannao Granules on Nissl bodies in the hippocampus and cortical tissue of a rat model of dementia

[0155] As shown in Tables 1-5, compared with the normal control group, the number of Nissl bodies in the hippocampus and cortex of rats in the model control group was significantly reduced (P≤0.01). Compared with the model control group, the number of Nissl bodies in the hippocampus and cortex of rats in the low, medium, and high dose groups of Yinling Tongmai Jiannao Granules was significantly increased (P≤0.01). The number of Nissl bodies in the hippocampus and cortex of rats in the positive control groups (P≤0.05 or P≤0.01) was significantly increased.

[0156] Table 1-5 Effects of Yinling Tongmai Jiannao Granules on the number of Nissl bodies in brain tissue of a rat model of dementia ( n=10)

[0157]

[0158] Note: Compared with the sham surgery group, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0159] 5. Experiment Summary

[0160] The results showed that the medium and high doses of Yinling Tongmai Jiannao Granules could significantly reduce the latency on the 2nd day of the spatial navigation test in rats. The low and high doses of Yinling Tongmai Jiannao Granules could significantly reduce the latency on the 3rd day of the spatial navigation test in rats. The low, medium, and high doses of Yinling Tongmai Jiannao Granules could significantly reduce the latency on the 4th and 5th days of the spatial navigation test in rats, and could significantly increase the number of times the rats crossed the platform. The high dose of Yinling Tongmai Jiannao Granules could significantly increase the PT and Fbg in the rats' blood, and the low and medium doses could significantly increase the content of APTT in the rats' blood. The medium and high doses could significantly increase the TT content in the rats' blood. The low, medium, and high doses of Yinling Tongmai Jiannao Granules could significantly improve the pathological changes in the hippocampus and cortex tissues of the dementia rats, and increase the number of Nissl bodies in the hippocampus and cortex tissues of the rats. Yinling Tongmai Jiannao Granules could significantly improve the cognitive behavior of the vascular dementia model and had a significant effect on improving cognitive function.

[0161] II. Experiment 2: Effects of Yinling Tongmai Jiannao Granules on the Cognitive Impairment Model Induced by Scopolamine in Mice

[0162] 1. Experimental Purpose

[0163] Using the cognitive impairment model induced by scopolamine in mice, to study the effects of Yinling Tongmai Jiannao Granules prepared by the preparation method based on Example 1 on improving memory, and to provide experimental basis for clinical research.

[0164] 2. Experimental Materials

[0165] 2.1 Test Samples

[0166] The extract powder of Yinling Tongmai Jiannao Granules prepared according to Example 1, batch number: 20221204, character: yellowish brown powder, slightly fragrant smell, slightly sour taste, specification: 4.699 g of crude drug / g of dry extract powder, provided by Hunan Butian Pharmaceutical Co., Ltd.

[0167] 2.2 Positive Controls

[0168] Donepezil Hydrochloride, batch number: 0000047417, product of Zhejiang Huahai Pharmaceutical Co., Ltd.; Compound苁蓉益智胶囊, batch number: 221113, Anhui Leyunsheng Pharmaceutical Co., Ltd.

[0169] 2.3 Experimental Animals

[0170] 70 SPF-grade ICR mice, male, body weight 18.0 - 22.0 g, purchased from Changsha Tianqin Biotechnology Co., Ltd., experimental animal production license number: SCXK(Xiang)2022-0011, experimental animal quality certificate number: 430726231100119463, raised in Area D of the shielding environment of Hunan Prima Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2020-0015.

[0171] 2.4 Main Reagents

[0172] Scopolamine, batch number: D2017146, product of Aladdin Company.

[0173] 2.5 Main Instruments

[0174] ME2002E electronic balance, a product of Mettler; Morris water maze video analysis system, a product of Chengdu Taimeng Company.

[0175] 3. Experimental Methods

[0176] 3.1 Grouping and Dosing

[0177] Seventy qualified male ICR mice, weighing 18.0–22.0 g, were randomly divided into seven groups based on their weight: a normal control group, a model control group, donepezil hydrochloride tablets, compound Cistanche deserticola capsules, and low, medium, and high dose Yinling Tongmai Jiannao granules, with ten mice in each group. Mice in the normal and model control groups were administered pure water by gavage at a volume of 20 mL / kg. The other groups were administered the corresponding drug solutions by gavage once daily for seven days. After the last administration, the normal control group received an intraperitoneal injection of 0.9% sodium chloride injection, while the other groups received an intraperitoneal injection of scopolamine 2 mg / kg at a volume of 20 mL / kg. Morris water maze behavioral tests were performed 20 minutes after injection.

[0178] 3.2 Dosage setting

[0179] According to the data, the proposed clinical dosage of Yinling Tongmai Jiannao Granules is 55g of raw drug per day. Based on the conversion of animal and human surface area, the equivalent clinical dose is: 55g of raw drug per day × 0.0026 / 0.02kg = 7.15g of raw drug per kg. The low, medium, and high doses of Yinling Tongmai Jiannao Granules are set at 1, 2, and 4 times the equivalent clinical dose as follows: 7.15g of raw drug per kg, 14.3g of raw drug per kg, and 28.6g of raw drug per kg. The clinical dosage of donepezil hydrochloride is 10 mg / day. Based on the animal and human surface area conversion, the clinical equivalent dose is: 10 mg × 0.0026 / 0.02 kg = 1.3 mg / kg. The clinical dosage of compound Cistanche deserticola capsules is 3.6 g / day. Based on the animal and human surface area conversion, the clinical equivalent dose is: 3.6 g / day × 0.0026 / 0.02 kg = 0.47 g / kg. The group and dosage settings are shown in Table 2-1.

[0180] Table 2-1 Trial grouping and dosage design

[0181]

[0182] 3.3 Detection Indicators

[0183] 3.3.1 Morris Water Maze Test: After the last administration, mice in each group underwent navigation training for 5 days. A platform was fixed in the center of the second quadrant. Tap water was filled into a pool, and white fuel was poured in to make the platform invisible, with the water level 1.5 cm above the platform. The water temperature was maintained at 25℃±1℃. Each animal was tested once daily. Before the test, the animal was placed on the platform for 10 seconds to acclimatize. Three entry points were randomly selected, and the test lasted 60 seconds. If the animal successfully found the platform, it was allowed to stay on the platform for 10 seconds before being returned to its cage. If it failed to find the platform, it was guided to the platform and allowed to stay for 10 seconds. The time it took to find the platform (escape latency), total swimming distance, and average speed were recorded to evaluate the mice's learning and memory abilities. On day 6, spatial exploration was conducted, and the total distance traveled, platform quadrant time and distance, and number of times the animal crossed the platform were measured.

[0184] 3.4 Statistical Methods

[0185] The experimental data were rounded to the nearest whole number and statistically analyzed according to the "Statistical Processing of Experimental Data" (SOP-Rd-003). SPSS software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation (Mean ± SEM), and normality and homogeneity of variance were tested using Leven's test. If there was no statistical significance (P>0.05), one-way ANOVA was used for statistical analysis; two-way ANOVA was used for water maze navigation data. If the ANOVA was statistically significant (P≤0.05), the LSD test (parametric method) was used for comparative analysis. If the variances were unequal (P≤0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P≤0.05), Dunnett's test (nonparametric method) was used for comparative analysis. The statistical results were compared with α = 0.05, where P ≤ 0.05 indicated statistical significance and P ≤ 0.01 indicated that the difference was highly significant.

[0186] 4. Experimental Results

[0187] 4.1 Effects of Yinling Tongmai Jiannao Granules on Water Maze Navigation in a Cognitive Impairment Model Mice

[0188] like Figure 10As shown in Table 2-2, compared with the normal control group, the latency period for local navigation on days 4 and 5 in the model control group was significantly increased (P≤0.05). Compared with the model control group, the latency period for local navigation on day 4 in the high-dose Yinling Tongmai Jiannao Granules group was significantly reduced (P≤0.05), the latency period for local navigation on day 5 in the medium- and high-dose Yinling Tongmai Jiannao Granules groups was significantly reduced (P≤0.05), and the latency period for local navigation on day 5 in the positive control groups (donepezil hydrochloride tablets and compound Cistanche deserticola capsules) was significantly reduced (P≤0.05).

[0189] Table 2-2 Effects of Yinling Tongmai Jiannao Granules on the Localization and Navigation Latency in a Cognitive Impairment Model Mice (Mean±SEM, n=10)

[0190]

[0191] Note: Compared with the normal control group, +P≤0.05, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0192] 4.3 Effects of Yinling Tongmai Jiannao Granules on Spatial Exploration in a Water Maze in a Mouse Model of Cognitive Impairment

[0193] As shown in Table 2-3, compared with the normal control group, the number of times mice in the model control group crossed the platform was significantly reduced (P≤0.01). Compared with the model control group, the number of times mice in the medium and high dose groups of Yinling Tongmai Jiannao Granules crossed the platform was significantly increased (P≤0.01). The number of times mice in the positive control groups of donepezil hydrochloride tablets and compound Cistanche deserticola capsules crossed the platform was significantly increased (P≤0.05 or P≤0.01).

[0194] Table 2-3 Effects of Yinling Tongmai Jiannao Granules on the number of times mice crossed the platform ( n=10)

[0195] Group Dosage (g crude drug / kg) Number of times the platform was traversed (times) normal control group -- 1.6±0.3 Model control group -- 0.2±0.1++ Donepezil hydrochloride tablets 1.3 mg / kg 1.3±0.4** Compound Cistanche and Alcohol-Enhancing Capsule Group 0.47g / kg 1.0±0.3* Low-dose group of Yinling Tongmai Jiannao Granules 7.15 0.6±0.2 Yinling Tongmai Jiannao Granules (medium dose group) 14.3 1.3±0.4* High-dose group of Yinling Tongmai Jiannao Granules 1.3 mg / kg 1.5±0.3**

[0196] Note: Compared with the normal control group, ++P≤0.01; compared with the model control group, *P≤0.05, **P≤0.01.

[0197] 5. Experiment Summary

[0198] The results showed that high doses of Yinling Tongmai Jiannao Granules significantly reduced the latency period of mice during orientation navigation on day 4. Medium and high doses significantly reduced the latency period of mice during orientation navigation on day 5 and significantly increased the number of times mice crossed the platform. Yinling Tongmai Jiannao Granules significantly improved the scopolamine-induced cognitive impairment model in mice and had a significant effect on improving learning and memory.

[0199] III. Experiment 3: Effects of Yinling Tongmai Jiannao Granules on Hemorheology in Rats

[0200] 1. Experimental purpose

[0201] A rat blood stasis model was established by subcutaneous injection of adrenaline in rats followed by ice water bath, and the effect of Yinling Tongmai Jiannao Granules prepared by the preparation method based on Example 1 on hemorheology was studied to provide an experimental basis for clinical research.

[0202] 2. Test materials

[0203] 2.1 Tested products

[0204] The extract powder of Yinling Tongmai Jiannao Granules prepared according to Example 1, batch number: 20221204, properties: yellowish brown powder, slightly fragrant smell, slightly sour taste, specification: 4.699 g crude drug / g dry extract powder, provided by Hunan Butian Pharmaceutical Co., Ltd.

[0205] 2.2 Positive control

[0206] Aspirin enteric-coated tablets, batch number: BJ70356, products of Bayer Healthcare Co., Ltd.

[0207] 2.3 Experimental animals

[0208] 60 SPF-grade SD rats, half male and half female, weighing 180.0 - 22 g, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., production license number of experimental animals: SCXK(Xiang)2019 - 0014, quality certificate number of experimental animals: 430727231100589435, raised in Area D of the barrier environment of Hunan Prima Pharmaceutical Research Center Co., Ltd., use license number of experimental animals: SYXK(Xiang)2020 - 0015.

[0209] 2.4 Main reagents

[0210] Adrenaline hydrochloride injection, batch number: 221107, products of Grand Pharmaceutical (China) Co., Ltd.; ZLC(Z1) cleaning solution, batch number: 202211 - 201B10, ZLC(Z2) cleaning solution, batch number: 202210 - 201L0, products of Beijing Zhongchi Weiye Technology Development Co., Ltd.

[0211] 2.5 Main instruments

[0212] ZL6000 full-automatic hemorheology tester, products of Beijing Zhongchi Weiye Co., Ltd.

[0213] 3. Research content

[0214] 3.1 Grouping and administration

[0215] Sixty qualified SD rats, half male and half female, weighing 180.0–220.0 g, were selected and randomly divided into 6 groups according to their weight: normal control group, model control group, enteric-coated aspirin tablet group, and low, medium, and high dose Yinling Tongmai Jiannao granules group, with 10 rats in each group. Rats in the normal control and model control groups were administered pure water orally at a dose of 10 mL / kg. Rats in the other groups were administered the corresponding drug solution by gavage once daily for 7 consecutive days. After the last administration, except for the normal control group, all other groups were subcutaneously injected with 0.1% adrenaline at a dose of 0.08 ml / 100 g. Two hours later, the rats swam in ice water (4℃) for 5 minutes. Four hours after the first injection, the same dose of adrenaline was administered again. After 18 hours of fasting but with unlimited water, blood was collected from the abdominal aorta, and plasma viscosity was measured using a hemorheology analyzer.

[0216] 3.2 Dosage setting

[0217] According to the data, the proposed clinical dosage of Yinling Tongmai Jiannao Granules is 55g of raw drug per day. Based on the conversion of animal and human surface area, the clinically equivalent dose is calculated as: 55g raw drug / day × 0.018 / 0.2kg = 4.95g raw drug / kg. The low, medium, and high doses of Yinling Tongmai Jiannao Granules are set at 1, 2, and 4 times the clinically equivalent dose: 4.95g raw drug / kg, 9.9g raw drug / kg, and 19.8g raw drug / kg, respectively. The clinical dosage of aspirin is 100mg / day. Based on the conversion of animal and human surface area, the clinically equivalent dose is: 100mg × 0.018 / 0.2kg = 9mg / kg. The group and dosage settings are shown in Table 3-1.

[0218] Table 3-1 Trial grouping and dosage design

[0219]

[0220] 3.2 Detection Indicators

[0221] 3.2.1 Blood rheology test: After the last administration, rats in each group were anesthetized, and blood was collected from the abdominal aorta. The plasma viscosity was measured using a fully automated blood rheology tester.

[0222] 4. Experimental Results

[0223] 4.1 Effects of Yinling Tongmai Jiannao Granules on Hemorheology in Rats

[0224] As shown in Table 3-2, compared with the normal control group, the plasma viscosity shear rate of rats in the model control group was significantly increased (P≤0.01). Compared with the model control group, the plasma viscosity shear rate of rats in the medium and high dose groups of Yinling Tongmai Jiannao Granules was significantly decreased (P≤0.01). The plasma viscosity shear rate of rats in the positive control group of enteric-coated aspirin tablets was significantly decreased (P≤0.01).

[0225] Table 3-2 Effects of Yinling Tongmai Jiannao Granules on Hemorheology in Rats n=10)

[0226] Group Dosage (g crude drug / kg) plasma viscosity shear rate normal control group -- 1.34±0.05 Model control group -- 1.80±0.72++ Enteric-coated aspirin tablets 9mg / kg 1.47±0.04** Low-dose group of Yinling Tongmai Jiannao Granules 4.95 1.89±0.81 Yinling Tongmai Jiannao Granules (medium dose group) 9.9 1.4±0.02** High-dose group of Yinling Tongmai Jiannao Granules 19.8 1.4±0.07**

[0227] Note: Compared with the normal control group, ++P≤0.01; compared with the model control group, **P≤0.01.

[0228] 5. Experiment Summary

[0229] The results showed that medium and high doses of Yinling Tongmai Jiannao Granules could significantly reduce the plasma viscosity shear rate in rats, suggesting that Yinling Tongmai Jiannao Granules could significantly improve the hemorheology of rats with cold coagulation and blood stasis model.

[0230] IV. Summary

[0231] Vascular dementia (VD) refers to a heterogeneous group of diseases encompassing a range of clinical and neuropathological manifestations of cognitive impairment caused by cerebrovascular diseases. Currently, the construction of rat models of vascular dementia primarily employs methods such as cerebral vascular occlusion to reduce cerebral blood flow. Among these, permanent bilateral common carotid artery ligation is a reliable animal model of vascular dementia induced by chronic hypoperfusion in rats. It is currently the most commonly used classic method for inducing chronic global cerebral ischemia in rats and is widely applied in vascular dementia research. Its behavioral characteristics mainly manifest as learning disabilities and memory impairment.

[0232] The Morris water maze is a behavioral test used to evaluate spatial learning and memory abilities in animals. It examines reference memory and working memory abilities in different modes. In Experiment 1, the escape latency in the vascular dementia rat model was significantly prolonged during navigation on days 4 and 5. Medium and high doses of Yinling Tongmai Jiannao Granules significantly reduced the escape latency on day 2, low and high doses significantly reduced the escape latency on day 3, and low, medium, and high doses significantly reduced the escape latency on days 4 and 5, while significantly increasing the number of times rats crossed platforms. Similarly, in a scopolamine-induced learning and memory impairment mouse model, high doses of Yinling Tongmai Jiannao Granules significantly reduced the escape latency on day 4, and medium and high doses significantly reduced the escape latency on day 5, while significantly increasing the number of times mice crossed platforms. These findings suggest that Yinling Tongmai Jiannao Granules can improve spatial exploration memory abilities in dementia animals. Both navigation and spatial exploration experimental modes are based on reference memory; the former examines memory acquisition ability, while the latter examines memory retention ability. The results suggest that Yinling Tongmai Jiannao Granules can simultaneously improve the acquisition and retention of spatial memory in dementia model rats, and have a significant ameliorative effect on cognitive impairment in vascular dementia. A rat blood stasis model was established by subcutaneous injection of adrenaline followed by an ice-water bath. The results showed that Yinling Tongmai Jiannao Granules can significantly improve the hemorheology of rats with blood stasis.

[0233] In summary, the Yinling Tongmai Jiannao Granules prepared by the method in Example 1 can significantly improve memory impairment in vascular dementia and improve blood rheology, and have obvious anti-vascular dementia effects.

[0234] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in the details for the sake of brevity.

[0235] The embodiments of this invention are intended to cover all such substitutions, modifications, and variations falling within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia, characterized in that, It is made from the following Chinese herbal medicine components in parts by weight: Cornus officinalis 10-15 parts, Ginkgo biloba leaf 10-15 parts, Ligustrum lucidum 8-12 parts, Salvia miltiorrhiza 8-12 parts, Acorus tatarinowii 5-7 parts, Eucommia ulmoides 8-12 parts, Poria cocos 8-12 parts, Uncaria rhynchophylla 8-10 parts, Gastrodia elata 8-12 parts, Crataegus pinnatifida 5-7 parts, Curcuma longa 8-10 parts, and Ligusticum chuanxiong 5-7 parts.

2. The traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 1, characterized in that, It is made from the following Chinese herbal medicine components in the indicated weight proportions: 12 parts Cornus officinalis, 12 parts Ginkgo biloba leaf, 10 parts Ligustrum lucidum, 10 parts Salvia miltiorrhiza, 6 parts Acorus tatarinowii, 10 parts Eucommia ulmoides, 10 parts Poria cocos, 9 parts Uncaria rhynchophylla, 10 parts Gastrodia elata, 6 parts Crataegus pinnatifida (fried), 9 parts Curcuma longa, and 6 parts Ligusticum chuanxiong.

3. A method for preparing a traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 1 or 2, characterized in that, Includes the following steps: 1) Take the prescribed amounts of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong, add water, and distill to extract the volatile oil. Then, perform β-CD inclusion on the volatile oil and grind the resulting β-CD inclusion complex into a fine powder for later use. 2) Take the prescribed amounts of Cornus officinalis, Ginkgo biloba, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and Crataegus pinnatifida, decoct them twice with water, combine the filtrates, concentrate them under reduced pressure to a thick paste with a relative density of 1.20 or higher, dry them under vacuum to obtain a dry paste, pulverize it into a fine powder, and set it aside for later use. 3) Add the β-CD inclusion complex obtained in step 1) to the dry extract powder in step 2), add dextrin, mix well, add appropriate amounts of steviol glycosides and povidone, moisten with ethanol, extrude and sieve to granulate, dry, granulate, and package to obtain the final product.

4. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, In step 1), the coarse powder of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong is passed through a 60-mesh sieve.

5. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, In step 1), the amount of water added is 6 to 8 times, and the distillation extraction time for volatile oil is 5 to 8 hours.

6. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, In step 2), add 8 to 10 times the amount of water for the first decoction and decoct for 2.0 to 2.5 hours. For the second decoction, add 6 to 8 times the amount of water and decoct for 0.5 to 1.5 hours.

7. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, In step 2), the conditions for vacuum concentration are 60–70°C and -0.06–-0.08 MPa; the conditions for vacuum drying are 50–60°C and -0.06–-0.08 MPa.

8. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, In step 3), the volume concentration of ethanol is 92%.

9. The method for preparing the traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 3, characterized in that, Includes the following steps: 1.1) Take the prescribed amounts of Acorus tatarinowii, Curcuma longa, and Ligusticum chuanxiong, add 6 times the amount of water, and distill to extract the volatile oil for 5 hours. Then, encapsulate the volatile oil with β-CD. Grind the obtained β-CD inclusion complex into a fine powder and set aside. 1.2) Take the prescribed amounts of Cornus officinalis, Ginkgo biloba, Ligustrum lucidum, Salvia miltiorrhiza, Eucommia ulmoides, Poria cocos, Uncaria rhynchophylla, Gastrodia elata, and Crataegus pinnatifida, add water and decoct twice. For the first decoction, add 8 times the amount of water and decoct for 2 hours. For the second decoction, add 6 times the amount of water and decoct for 1.5 hours. Combine the filtrates and concentrate under reduced pressure at 60-70℃ and -0.06--0.08 MPa to a thick paste with a relative density of 1.20 or higher. Dry under vacuum at 50-60℃ and -0.06--0.08 MPa to obtain a dry paste. Grind the dry paste into a fine powder and set aside for later use. 1.3) Add the β-CD inclusion complex obtained in step 1.1) to the dry extract powder in step 1.2), add dextrin, mix well, add appropriate amounts of steviol glycosides and povidone, moisten with 92% ethanol, extrude and sieve to granulate, dry, granulate, and package to obtain the final product.

10. The use of a traditional Chinese medicine granule preparation for improving memory impairment in vascular dementia as described in claim 1 or 2 in the preparation of a medicament for improving memory impairment in vascular dementia.

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