A pharmaceutical composition for preventing and treating cognitive impairment in the elderly with bipolar disorder, and a preparation method and application thereof

Abstract

The application provides a pharmaceutical composition for preventing and treating senile cognitive impairment with bipolar disorder, a preparation method and application thereof, and belongs to the technical field of medicines.The pharmaceutical composition prepared by mixing volatile oil separated from Acorus calamus and Ligusticum chuanxiong and water extract separated from Acorus calamus, Ligusticum chuanxiong, Astragalus and Polygala is rich in volatile oil and various water-soluble active ingredients.Based on the characteristics of promoting nerve cell growth and memory impairment repair of the pharmaceutical composition, the application also provides application of the pharmaceutical composition in preparation of medicines for preventing and / or treating senile cognitive impairment and / or bipolar disorder.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for the prevention and treatment of cognitive impairment in the elderly with bipolar disorder, its preparation method, and its application. Background Technology

[0002] Cognitive impairment in the elderly is a broad term encompassing multiple cognitive domains, such as orientation, memory, calculation, attention, language, executive function, reasoning, and visuospatial function. It includes cognitive impairments of varying severity, ranging from mild cognitive impairment (MCI) to dementia. Current treatments for cognitive impairment in the elderly primarily focus on relieving symptoms and slowing disease progression. For example, some medications (such as piracetam, citicoline, ginkgo biloba preparations, and nimodipine) are used to improve cognitive function, but their efficacy is limited and they can lead to dependence.

[0003] Bipolar disorder, also known as manic-depressive illness, is a mental illness characterized by alternating mood states between mania (or hypomania) and depression. Common treatments include mood stabilizers (such as lithium, valproate, and carbamazepine), antipsychotics (such as olanzapine and risperidone), and antidepressants; however, these treatments often involve drug dependence and have limited effectiveness.

[0004] my country is the birthplace of traditional Chinese medicine and its largest producer and user. Although there are reports of traditional Chinese medicine compositions being used to treat Alzheimer's disease and early cognitive impairment, these compositions are all prepared by simple decoction, resulting in poor therapeutic effects. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a pharmaceutical composition in which the volatile oils of Acorus tatarinowii and Ligusticum chuanxiong are extracted by steam distillation, and the active ingredients are retained by decoction of Astragalus membranaceus and Polygala tenuifolia. The prepared traditional Chinese medicine composition has a good therapeutic effect on elderly cognitive impairment with bipolar disorder.

[0006] This invention provides a pharmaceutical composition comprising the following compounds in mass concentrations: astragaloside A 0.27~0.39 mg / g, polygala saponin 3.78~8.03 mg / g, verbascoside 0.04~0.17 mg / g, polygala succinate III 0.51~0.77 mg / g, 3,6-disinozylosyl sucrose 1.30~5.44 mg / g, ferulic acid 0.38~2.02 mg / g, asarone 0.51~1.83 mg / g, and ligustilide 0.01~0.15 mg / g.

[0007] Preferably, the compound comprises the following mass concentrations: astragaloside A 0.32 mg / g, polygala saponin 5.20 mg / g, verbascoside 0.10 mg / g, polygala succinate III 0.60 mg / g, 3,6-disinozylosyl sucrose 2.34 mg / g, ferulic acid 1.01 mg / g, asarone 0.85 mg / g, and ligustilide 0.10 mg / g.

[0008] Preferably, it is a mixture of volatile oils separated from Acorus tatarinowii and Ligusticum chuanxiong and water extracts separated from Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus and Polygala tenuifolia;

[0009] The mass ratio of Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus and Polygala tenuifolia is (1~10):(1~10):(1~10):(1~10).

[0010] Preferably, the mass ratio of Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus and Polygala tenuifolia is (3~4):(2~1):(3~4):(3~4).

[0011] This invention provides a method for preparing the pharmaceutical composition, comprising the following steps:

[0012] The volatile oils of Acorus tatarinowii and Ligusticum chuanxiong were extracted by steam distillation, and the first aqueous extract was collected. The volatile oils were then encapsulated with excipients to obtain volatile oil inclusion complexes.

[0013] Astragalus and Polygala were decocted in water, and the second water extract was collected. The second water extract and the first water extract were combined and the water was removed to obtain the third water extract.

[0014] The volatile oil inclusion complex and the third aqueous extract are mixed to obtain a pharmaceutical composition.

[0015] Preferably, the mass ratio of the total mass of Acorus tatarinowii and Ligusticum chuanxiong to water is 1:(6~10); the extraction time by steam distillation is not less than 4.5 hours;

[0016] During the inclusion process, the amount of excipients used is 2 to 6 times the mass of the volatile oil; the excipients include β-cyclodextrin;

[0017] Preferably, the mass ratio of the total mass of Acorus tatarinowii and Ligusticum chuanxiong to water is 1:(7~8); the extraction time by steam distillation is 5~8 hours.

[0018] During the encapsulation process, the amount of excipients added is 4 to 6 times the mass of the volatile oil.

[0019] Preferably, the mass ratio of the total mass of Astragalus membranaceus and Polygala tenuifolia to water is 1:(6~8);

[0020] The boiling time is 1-2 hours;

[0021] The number of times the water is boiled includes 1 to 3 times.

[0022] Preferably, the method for removing moisture includes alcohol precipitation concentration and / or drying;

[0023] The alcohol precipitation and concentration resulted in an extract with a relative density of 1.10~1.14 g / ml; the working volume concentration of ethanol used for alcohol precipitation and concentration was 70%.

[0024] The alcohol precipitation concentration time is less than 6 hours; the alcohol precipitation concentration temperature is less than 70°C.

[0025] The drying temperature is below 80°C.

[0026] This invention provides the use of the pharmaceutical composition or the pharmaceutical composition prepared by the preparation method in the preparation of a medicament for the prevention and / or age-related cognitive impairment and / or bipolar disorder.

[0027] Preferably, the cognitive impairment in the elderly includes cognitive impairment caused by Parkinson's disease and / or cognitive impairment caused by Alzheimer's disease.

[0028] This invention provides a pharmaceutical composition comprising astragaloside A, polygala saponin, isoflavone glucoside, polygala succinate III, and 3,6-disinoyl sucrose extracted from Astragalus membranaceus and Polygala tenuifolia, as well as ferulic acid extracted from Acorus tatarinowii and Ligusticum chuanxiong, and volatile oils asarone and ligustilide extracted from Acorus tatarinowii and Ligusticum chuanxiong. In embodiments of this invention, the effects of the pharmaceutical composition on a scopolamine-induced mouse memory impairment model were investigated. The results showed that the pharmaceutical composition significantly prolonged the latency period of errors and reduced the number of errors, showing a significant difference compared to the model group and control group. Simultaneously, it inhibited mouse memory impairment by reducing AChE and MDA levels, and its efficacy was superior to pharmaceutical compositions obtained through other extraction processes. This indicates that the volatile oil components extracted from Acorus tatarinowii and Ligusticum chuanxiong have a synergistic effect, as do the aqueous extracts of Astragalus membranaceus and Polygala tenuifolia. Meanwhile, experiments of this invention show that the pharmaceutical composition not only has good efficacy against cognitive impairment caused by Parkinson's disease and Alzheimer's disease, but also has a good therapeutic effect on bipolar disorder. Therefore, the pharmaceutical composition provided by this invention can exert a good therapeutic effect on cognitive impairment and bipolar disorder in the elderly, providing a new approach for the treatment of clinical diseases.

[0029] This invention provides a method for preparing the pharmaceutical composition. The method involves extracting volatile oils from *Acorus tatarinowii* and *Ligusticum chuanxiong* using steam distillation, while simultaneously collecting a first aqueous extract. The volatile oils are then encapsulated with excipients to obtain a volatile oil inclusion complex. A second aqueous extract is extracted from *Astragalus membranaceus* and *Polygala tenuifolia* using decoction. The second and first aqueous extracts are combined and the water is removed to obtain a third aqueous extract. The volatile oil inclusion complex and the third aqueous extract are mixed to obtain the pharmaceutical composition. This preparation method, which involves the combined extraction of volatile oils from *Acorus tatarinowii* and *Ligusticum chuanxiong*, demonstrates a more significant therapeutic effect on a scopolamine-induced mouse memory impairment model compared to preparations prepared by process 2 (extracting volatile oils from *Acorus tatarinowii* alone, and decocting the other three herbs) and process 3 (extracting volatile oils from *Polygala tenuifolia* alone with alcohol and *Astragalus membranaceus* alone with water, and extracting volatile oils from both *Acorus tatarinowii* and *Ligusticum chuanxiong*), effectively improving mouse memory. Attached Figure Description

[0030] Figure 1 This is the extraction process route 1 for the Astragalus and Ligusticum tablets of the present invention;

[0031] Figure 2 This is the extraction process route 2 for the Astragalus and Ligusticum tablets of the present invention;

[0032] Figure 3 This is the extraction process route 3 for the Astragalus and Ligusticum tablets of the present invention;

[0033] Figure 4 A bar chart showing the results of cytokine content detection in each group of mice;

[0034] Figure 5 A bar chart showing the results of target quadrant dwell time and latency after drug administration in the water maze test;

[0035] Figure 6 The trajectory diagrams of mice in each group during the water maze test;

[0036] Figure 7 A bar chart showing the latency test results of the water maze experiment.

[0037] Figure 8 A bar chart showing the results of the number of times the platform was traversed in the water maze experiment;

[0038] Figure 9 A histogram of new object preference detection results in the new object recognition experiment;

[0039] Figure 10 This is a graph showing the results of the mouse tail suspension test.

[0040] Figure 11 A bar chart showing the open arm dwell time results of the elevated cross maze test;

[0041] Figure 12 Pathological morphology of brain tissue in mice of each group (200×, n = 6);

[0042] Figure 13 The mean escape latency of the water maze for each group of mice (n = 6);

[0043] Figure 14 The number of times mice in each group passed through the water maze ( - (x±s, n = 6)

[0044] Figure 15 The activity time of each group of mice in the quadrant of the water maze platform ( - (x±s, n = 6)

[0045] Figure 16 The swimming path of each group of mice in the quadrant where the platform is located (n = 6);

[0046] Figure 17 The distance the mouse moved and the number of times it stood up in the open field test. - (x±s, n = 6)

[0047] Figure 18 The immobility time of mice in each group during the tail suspension test ( - (x±s, n = 6)

[0048] Figure 19 The content of inflammatory factors in the brain tissue of mice in each group ( - (x±s, n = 6)

[0049] Figure 20 The image shows the actual climbing pole (left) and the schematic diagram (right).

[0050] Figure 21 The climbing time results for each group;

[0051] Figure 22 Total ion chromatogram of blank blood in positive ion mode;

[0052] Figure 23 This is the total ion chromatogram of drug-containing plasma in positive ion mode;

[0053] Figure 24 The total ion chromatogram of blank blood plasma under negative ion mode;

[0054] Figure 25 The total ion chromatogram of drug-containing plasma in negative ion mode;

[0055] Figure 26 Total ion flow map of brain tissue in the blank group under positive and negative ion modes;

[0056] Figure 27 The total ion flux diagram of the brain tissue of the drug administration group in positive ion mode;

[0057] Figure 28 Total ion flow map of brain tissue in the blank group under negative ion mode;

[0058] Figure 29 The total ion flow map of the brain tissue of the drug administration group under negative ion mode;

[0059] Figure 30 Total ion chromatogram for compound preparations analyzed in positive ion mode;

[0060] Figure 31 Total ion chromatogram analysis for compound preparations in negative ion mode. Detailed Implementation

[0061] This invention provides a pharmaceutical composition comprising the following compounds in mass concentrations: astragaloside A 0.27~0.39 mg / g, polygala saponin 3.78~8.03 mg / g, verbascoside 0.04~0.17 mg / g, polygala succinate III 0.51~0.77 mg / g, 3,6-disinozylosyl sucrose 1.30~5.44 mg / g, ferulic acid 0.38~2.02 mg / g, asarone 0.51~1.83 mg / g, and ligustilide 0.01~0.15 mg / g.

[0062] In this invention, the pharmaceutical composition preferably comprises the following compounds in mass concentrations: astragaloside A 0.32 mg / g, polygala saponin 5.20 mg / g, verbascoside 0.10 mg / g, polygala succinate III 0.60 mg / g, 3,6-disinozyl sucrose 2.34 mg / g, ferulic acid 1.01 mg / g, asarone 0.85 mg / g, and ligustilide 0.10 mg / g. The asarone preferably includes α-asarone and β-asarone.

[0063] In this invention, the pharmaceutical composition is prepared using Astragalus and Polygala tablets as raw materials. The Astragalus and Polygala tablets preferably include Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus, and Polygala tenuifolia. The mass ratio of Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus, and Polygala tenuifolia is (1~10):(1~10):(1~10), or it can be (3~4):(2~1):(3~4):(3~4). This invention does not have any special restrictions on the source of Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus, and Polygala tenuifolia; any source known in the art can be used. Explanation: This formula is mainly used to treat symptoms such as qi and blood deficiency, restlessness, forgetfulness, and insomnia. Composed of four herbs—Astragalus membranaceus, Polygala tenuifolia, Acorus tatarinowii, and Ligusticum chuanxiong—it has the effects of invigorating qi and nourishing the heart, calming the mind and stabilizing the will, and enhancing memory. Astragalus membranaceus: sweet in taste, slightly warm in nature, and enters the spleen and lung meridians. It has the functions of tonifying qi and raising yang, strengthening the defensive qi and consolidating the exterior, and promoting diuresis and reducing swelling. It is especially good at tonifying the spleen and stomach qi. The spleen and stomach are the foundation of acquired constitution and the source of qi and blood production. With a source of acquired qi and blood production, the innate kidney essence will be continuously nourished. Sufficient kidney essence will continuously fill the marrow sea, eliminating confusion and forgetfulness. Therefore, it is the chief herb in the formula. Acorus tatarinowii: "It is pungent and warm in nature, and enters the heart and stomach meridians. It opens the heart orifices, nourishes the five internal organs, opens the nine orifices, brightens the eyes and ears, and produces sound. Long-term use lightens the body, prevents forgetfulness and confusion, and prolongs life." It has the effects of opening the orifices and awakening the mind, resolving dampness and opening the stomach, and calming the mind and stabilizing the will. Polygala tenuifolia: It is warm in nature, pungent and bitter in taste, and enters the heart, kidney, and lung meridians. Shizhen said: Polygala tenuifolia enters the foot Shaoyin kidney meridian, not the heart meridian. Its function is to strengthen the will and benefit the essence, and treat forgetfulness. Since essence and will are both stored in the kidney meridian. If the kidney essence is insufficient, the will qi will weaken and cannot reach the heart, hence confusion and forgetfulness. The *Ling Shu Jing* states that the kidneys store essence, and essence unites with the will. Excessive anger in the kidneys, if uncontrolled, injures the will. Injured will leads to forgetfulness, inability to bend or straighten the waist and spine, and withered hair and a pale complexion. *Acorus tatarinowii* and *Polygala tenuifolia*, one opening the orifices and the other calming the mind, harmonize the heart and kidneys, benefiting intelligence and calming the mind; they are a classic herbal pair for improving intelligence and opening the orifices, serving as assistant herbs. *Ligusticum chuanxiong*: pungent and warm in nature. It enters the liver, gallbladder, and pericardium meridians. This herb is pungent and warm, able to descend to the blood sea, acting as a qi-regulating herb in the blood, with the effects of activating blood circulation, removing blood stasis, promoting qi circulation, and relieving pain. When the heart and kidneys are not in harmony, qi and blood stagnate, leading to blood stasis over time, affecting the circulation of qi and blood and the distribution of essence. Therefore, the sensory orifices are malnourished, resulting in confusion and forgetfulness. Therefore, *Ligusticum chuanxiong* is used to remove blood stasis, serving as the guiding herb. The entire formula, when used together, works to replenish qi and blood, harmonize the heart and kidneys, open the orifices, and awaken the mind, thus immediately eliminating forgetfulness and confusion.

[0064] In this embodiment of the invention, the Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus, and Polygala tenuifolia were all purchased from Beijing Tongrentang Pharmacy as processed medicinal slices. The preferred pharmaceutical composition is a mixture of volatile oils isolated from Acorus tatarinowii and Ligusticum chuanxiong, and water extracts isolated from Acorus tatarinowii, Ligusticum chuanxiong, Astragalus membranaceus, and Polygala tenuifolia. The volatile oils are extracted from Acorus tatarinowii and Ligusticum chuanxiong together, and the water-soluble components are extracted by decoction of Astragalus membranaceus and Polygala tenuifolia together. The combination of volatile oils and water-soluble components effectively prolongs the latency period of errors and reduces the number of errors, showing a significant difference compared to the model group and control group. Simultaneously, it inhibits memory impairment in mice by reducing AChE and MDA levels.

[0065] In this embodiment of the invention, three extraction process routes were designed and carried out using Astragalus and Polygala tablets as raw materials. First, based on the rich volatile oil components in Acorus tatarinowii and Ligusticum chuanxiong, preparation process one was designed (separating volatile oil from Acorus tatarinowii and Ligusticum chuanxiong, and extracting water from Astragalus membranaceus and Polygala tenuifolia). Based on the main medicinal component asarone in the volatile oil of Acorus tatarinowii, preparation process two was designed (separating volatile oil from Acorus tatarinowii alone, and extracting water from the other three Chinese herbs). Based on the main medicinal components saponins and polysaccharides in Astragalus membranaceus, and the main medicinal component saponins in Polygala tenuifolia, preparation process three was designed (ethanol extraction of Polygala tenuifolia, water extraction of Astragalus membranaceus, and separation of volatile oil from Acorus tatarinowii and Ligusticum chuanxiong). The efficacy of drug compositions prepared using three different processes was evaluated using a scopolamine-induced mouse memory impairment model. Results showed that, compared to the model group, all three drug compositions shortened the latency period and reduced the number of errors. However, significant differences were observed in certain dosages of the drug compositions prepared using processes 1 and 2, while process 3 showed no statistically significant difference compared to the model group. The medium and low dose groups of the drug composition prepared using process 1 showed significant differences compared to the model group, while only the low dose group of the drug composition prepared using process 2 showed a difference. This indicates that the drug composition prepared using process 1 has better efficacy in repairing memory impairment in mice.

[0066] This invention provides a method for preparing the pharmaceutical composition, comprising the following steps:

[0067] The volatile oils of Acorus tatarinowii and Ligusticum chuanxiong were extracted by steam distillation, and the first aqueous extract was collected. The volatile oils were then encapsulated with excipients to obtain volatile oil inclusion complexes.

[0068] Astragalus and Polygala were decocted in water, and the second water extract was collected. The second water extract and the first water extract were combined and the water was removed to obtain the third water extract.

[0069] The volatile oil inclusion complex and the third aqueous extract are mixed to obtain a pharmaceutical composition.

[0070] This invention extracts volatile oils from Acorus tatarinowii and Ligusticum chuanxiong using steam distillation, while simultaneously collecting the first aqueous extract. The volatile oils are then encapsulated with excipients to obtain volatile oil inclusion complexes.

[0071] In this invention, the preferred mass ratio of the total mass of Acorus tatarinowii and Ligusticum chuanxiong to water is 1:(6~10), but it can be 7~9 or 8. Experiments show that the amount of water added is a crucial factor affecting the extraction rate of volatile oils by steam distillation. Adding water at 6, 8, and 10 times the total mass of Acorus tatarinowii and Ligusticum chuanxiong affects the content and transfer rate of asarone and ligustilide in the volatile oil, and also affects the content and transfer rate of ferulic acid in the first aqueous extract. The volatile oil extracted with 8 times the amount of water has the highest asarone content, while the volatile oil extracted with 10 times the amount has the lowest asarone content. The ligustilide content obtained from the 10-fold water extraction is significantly higher than that from the 6-fold and 8-fold water extractions. The ferulic acid content obtained from the 10-fold water extraction is also higher than that from the 6-fold and 8-fold water extractions.

[0072] In this invention, the extraction time by steam distillation is no less than 4.5 hours, but can be 5 hours, 6 hours, 7 hours, or 8 hours. The extraction temperature by steam distillation is preferably 70-100℃, but can be 80-95℃ or 85-92℃. Experiments show that 5 hours of extraction can basically completely extract the volatile oil. During steam distillation, after extracting with 10 times the amount of water for 5 hours and collecting the first aqueous extract, the residue is extracted again with 8 times the amount of water for 1 hour, and the aqueous extract is collected again and combined with the first aqueous extract.

[0073] In this invention, during the inclusion process, the amount of excipient is preferably 2 to 6 times the mass of the volatile oil, and can be 4 to 5 times. The excipient includes β-cyclodextrin. The amount of excipient added affects the inclusion effect (component content and transfer rate). The inclusion effect was evaluated using a colloid mill method. The results showed that the volatile oil recovery rate and the content of asarone and ligustilide in the recovered volatile oil were the highest when 4 times the amount of β-cyclodextrin was used for inclusion. Therefore, colloid milling with 4 times the amount of β-cyclodextrin is the optimal process.

[0074] In this invention, Astragalus membranaceus and Polygala tenuifolia are decocted in water, and a second water extract is collected. The second water extract and the first water extract are combined and the water is removed to obtain a third water extract.

[0075] In this invention, the preferred mass ratio of the total mass of Astragalus membranaceus and Polygala tenuifolia to water is 1:(6-8), and can be 1:7. The preferred decoction time is 1-2 hours. The preferred number of decoctions is 1-3 times, and can be twice the original volume. The preferred decoction temperature is 90-100℃, and can be 95℃.

[0076] In one embodiment of the present invention, the optimal process for the number of extractions (1, 2, 3), extraction time (1h, 2h, 3h), and solvent ratio (1x, 2x, and 3x) was analyzed through orthogonal experiments. The results showed that the influencing factors were ranked as follows: number of extractions > extraction time > solvent ratio. The final determined process was: three extractions, each lasting 1 hour, with 8 times the volume of water added for the first extraction, and 6 times the volume of water added for the second and third extractions respectively.

[0077] In this invention, the method for removing moisture preferably includes alcohol precipitation concentration and / or drying. The alcohol precipitation concentration is preferably to a relative density of 1.10~1.14 g / ml; the working volume concentration of ethanol used for alcohol precipitation concentration is preferably 70%. The alcohol precipitation concentration time is preferably less than 6 hours, but can be 4 hours or 5 hours; the alcohol precipitation concentration temperature is preferably less than 70°C, but can be 68°C, 66°C, 65°C, 62°C, 60°C, 55°C, or 50°C. The drying temperature is preferably less than 80°C, but can be 78°C, 75°C, 73°C, 70°C, 68°C, 66°C, 65°C, 62°C, 60°C, 55°C, or 50°C.

[0078] In one embodiment of the present invention, the solvent concentration of alcohol precipitation affects the content of active ingredients and the yield of dry extract in the water extract. The alcohol precipitation process conditions are consistent, all involving concentration to a relative density of 1.10~1.14 g / ml, with the best effect achieved at an alcohol precipitation concentration of 70%. The effects of concentration time and concentration temperature on the content of active ingredients were also analyzed. The results showed that within 4 hours and 6 hours of concentration, the content of each indicator component decreased significantly when the temperature was above 70℃; therefore, the concentration temperature should be controlled below 70℃, and the concentration time should be within 6 hours. Furthermore, the present invention optimizes the drying temperature. The results showed that vacuum drying of the extract at 70, 80, and 90℃ did not significantly change the content of each compound; therefore, ≤80℃ is acceptable as a vacuum drying temperature.

[0079] This invention provides the use of the pharmaceutical composition or the pharmaceutical composition prepared by the preparation method in the preparation of a medicament for the prevention and / or age-related cognitive impairment and / or bipolar disorder.

[0080] In this invention, the dosage form of the drug preferably includes tablets, powders, oral liquids, capsules, etc. The drug preferably also includes excipients. This invention does not impose any special restrictions on the type of excipients; depending on the dosage form, commonly known excipients in the art can be selected to prepare the drug. The preferred dosage of the drug composition is not less than 11g crude drug / 60kg / day and 1.0g drug composition powder / kg / day, and can be 2.0 or 3.0g drug composition powder / kg / day.

[0081] In this invention, the age-related cognitive impairment preferably includes age-related cognitive impairment caused by Alzheimer's disease and / or age-related cognitive impairment caused by Parkinson's disease. In embodiments of this invention, experiments were conducted using a mouse model of Alzheimer's disease. Compared to the model group, the drug composition increased the time spent in the target quadrant and shortened the latency in the water maze test. In embodiments of this invention, experiments were conducted using a mouse model of Parkinson's disease. Compared to the model group, the drug composition significantly improved the limb coordination ability of mice in the pole climbing test.

[0082] In this embodiment of the invention, a bipolar disorder model in ouabain-induced rapid aging mice (SAMP8 mice) was used as the research subject. Behavioral tests revealed that the drug composition, after administration, shortened latency and reduced the number of errors in the water maze test, showing a significant difference compared to the model group. In the new object recognition test, administration of the drug composition in all three dosage groups increased the percentage of new object preference, showing a significant difference compared to the model group. The mouse tail suspension test showed that the immobility time was reduced in the low-to-medium dose groups of the drug composition, showing a significant difference compared to the model group. The elevated cross maze test showed that on day 60 of administration, compared to the model control group, the open-arm dwell time was increased in both the high- and medium-dose groups of the drug composition.

[0083] The following detailed description, in conjunction with embodiments, illustrates a pharmaceutical composition for preventing and treating cognitive impairment with bipolar disorder in the elderly, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0084] Example 1

[0085] Optimization of extraction process

[0086] 1. Design and selection of extraction process route

[0087] The formula of Qizhi tablets includes the following parts by weight: 10 parts of Acorus tatarinowii, 8 parts of Ligusticum chuanxiong, 10 parts of Astragalus membranaceus, and 10 parts of Polygala tenuifolia. Qizhi tablets are a clinically experienced formula. The volatile oil components in Acorus tatarinowii have aromatic and orifice-opening effects and are the effective components of the herb. In traditional processing, the four herbs in the formula are decocted together, resulting in a significant loss of volatile oil. Therefore, a volatile oil extraction process is used for Acorus tatarinowii. Ligusticum chuanxiong also contains aromatic and orifice-opening volatile oil components, and its combination with Acorus tatarinowii is a classic herbal pair for treating Alzheimer's disease. Therefore, a steam distillation method is designed to extract the oil from Acorus tatarinowii and Ligusticum chuanxiong, while the remaining herbs, Astragalus membranaceus and Polygala tenuifolia, are decocted (process route one, see...). Figure 1 The water extraction temperature is 100℃; when β-cyclodextrin is added, the amount added is 4 times the mass of the volatile oil.

[0088] Studies have reported that asarone, a key active ingredient in the volatile oil of Acorus tatarinowii, is widely used in the treatment of nervous system diseases. The pharmacopoeia specifies the content of this volatile oil. To verify the efficacy of the combination of Acorus tatarinowii and Ligusticum chuanxiong in this formula, a method was designed to extract the oil from Acorus tatarinowii using steam distillation alone, with the remaining herbs decocted in water (process route two, see...). Figure 2 The water extraction temperature is 100℃; when β-cyclodextrin is added, the amount added is 4 times the mass of the volatile oil.

[0089] The main active ingredients of Astragalus membranaceus are saponins and polysaccharides, while the main active ingredients of Polygala tenuifolia for calming the mind and improving intelligence are saponins. Given that polysaccharides are readily soluble in water, and saponins have higher solubility in alcohol, Polygala tenuifolia is extracted with alcohol. Therefore, the design involves extracting Polygala tenuifolia alone with 70% alcohol, extracting Astragalus membranaceus alone with water, and extracting oil together with Acorus tatarinowii and Ligusticum chuanxiong (process route three, see...). Figure 3 The water extraction temperature is 100℃; when β-cyclodextrin is added, the amount added is 4 times the mass of the volatile oil.

[0090] 2. Effects of drug samples obtained from different extraction processes on a scopolamine-induced mouse model of memory impairment.

[0091] 2.1 Test Materials

[0092] 2.2 Test Samples: Screening Sample (Extract Powder): Provided by the Preparation Center of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Characteristics: Sample 1 # It is a light brown powder, sample 2 # It is a light brown powder, sample 3 # It is a brownish-yellow powder. Content: 2.1g crude drug / g powder. Storage conditions: Store in a cool, dry place.

[0093] 2.3 Dosage design and drug preparation

[0094] 2.1 Test Drug

[0095] Table 1. Preparation methods of the test drugs

[0096]

[0097] 2.2 Positive control drug

[0098] Donepezil hydrochloride tablets are intended for human clinical use at a dose of 5 mg / 60 g / kg / day, or 0.083 mg / kg / day. In the experiments, the dosage in mice was 0.92 mg / kg / day, which is equivalent to a multiple of the human clinical dose.

[0099] One donepezil hydrochloride tablet (5mg) was thoroughly ground and then diluted with high-purity water to 109ml, which yielded a concentration of 0.046mg / ml. Mice were administered the solution via gavage at a dose of 0.2ml / 10g. The solution was prepared once every two weeks.

[0100] 3. Test Methods

[0101] 3.1 Experimental grouping and administration

[0102] One hundred and twenty ICR mice were randomly divided into 12 groups according to their weight class: a normal control group, a model control group, a donepezil hydrochloride control group, drug sample 1# prepared by process 1, drug sample 2# prepared by process 2, and drug sample 3# prepared by process 3. Each group had three dosage groups: 3 times, 2 times, and equal dosage, with 10 mice in each group. After grouping, except for the normal control group and the model control group, the other drug treatment groups began to be administered the drug once a day at a dose of 0.2 ml / 10 g by gavage for 28 consecutive days. The normal control group and the model control group were administered distilled water by gavage under the same conditions.

[0103] 3.3 Modeling and Testing

[0104] On day 28 of drug administration, scopolamine was administered intraperitoneally at a dose of 3 mg / kg (0.2 ml / 10 g) to establish an animal model of memory impairment. Normal control mice were intraperitoneally injected with an equal volume of physiological saline. Behavioral training was initiated 2 hours after model establishment, and formal behavioral testing was performed 24 hours later. Testing indicators included: behavioral assessment on day 28 of drug administration; and cytokine detection in brain tissue after dissection.

[0105] 3.4 Detection Methods

[0106] 3.4.1 Diving Platform Experiment

[0107] Adaptation period: The mice were placed on the platform inside the jumping reaction box to familiarize themselves with the environment and allowed to adapt freely in the box for 3 minutes.

[0108] Training period: Place the mouse on a metal panel, turn on the electric shock for 10 seconds, then turn it off. Repeat this training 5-10 times. When the electric shock is applied, the mouse will jump up and down its limbs. It will accidentally jump onto the platform to avoid the shock, and then jump off the platform again. Repeat this many times to form a memory and prevent it from jumping off the platform. If any mouse fails to jump onto the platform during training, it should be discarded.

[0109] Experimental period: Place the mouse inside the instrument and allow it to acclimatize for 3 minutes. Then, lift the mouse by its tail and place it on the jumping platform, keeping the movements as synchronized as possible. Apply electricity for 5 minutes at a current setting of 0.6 AM. Record the time when the animal first jumps off the platform (i.e., latency) and the number of times it jumps off the platform (number of errors).

[0110] 3.4.2 Factor Detection

[0111] After all animal behavior studies were completed, the animals were euthanized, and the hippocampus and cortex of the mice were collected. The levels of acetylcholine (ACH), acetylcholinesterase (AChE), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and malondialdehyde (MDA) were detected by ELISA.

[0112] 4. Experimental Results

[0113] 4.1 The results of the platform jump test are shown in Table 2 and Figure 4 .

[0114] Table 2. Effects of samples processed using different techniques on a scopolamine-induced mouse model of memory impairment.

[0115]

[0116] 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.

[0117] Table 2 shows that, after modeling on day 28 of drug administration, the latency period and the number of errors in the model control group were shortened, and there were significant differences compared with the normal control group. P<0.01 After 28 days of administration, the latency period was prolonged and the number of errors decreased in all dose groups of the three samples, with 1... # The latency period was significantly prolonged in the 2x and equal-dose groups, and the number of errors was significantly reduced in the 3x and equal-dose groups; 2 # The latency period and the number of errors were significantly prolonged in the equal-dose group of the sample, and both were significantly different from those in the model control group. P<0.05 );

[0118] Compared to 1 # The sample's efficacy was better than 2 # Sample 3 # There were no statistically significant differences between the samples and the model control group.

[0119] 4.2 The results of cytokine detection are shown in Table 3 and Figure 5 .

[0120] Table 3. Effects of Sample #1 on Scopolamine-Induced Memory Impairment Model in Mice

[0121]

[0122] 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.

[0123] Table 3 shows that the levels of ACH and SOD and the levels of MDA in the brain tissue of mice in the model control group were decreased and increased, which were significantly different from those in the normal control group. P<0.05, P<0.01 ); 1 # The 2-fold and equal-fold dose groups of the samples reduced AChE levels, and the equal-dose group reduced MDA levels, showing significant differences compared with the model control group. P<0.05, P<0.01 ).

[0124] Using a scopolamine-induced mouse model of memory impairment as the subject, 1 # 2 # 3 # After 28 consecutive days of administration, all three sample groups, regardless of dosage, showed varying degrees of increased error latency and decreased error frequency; among them, 1 # Sample, 2 # The sample showed a significant increase in latency and a reduction in the number of errors, with a statistically significant difference compared to the model control group. P<0.05, P<0.01 ); and compared to 1 # The sample's efficacy was better than 2 # Sample. In factor detection, 1 # The sample can inhibit memory impairment in mice by reducing AChE and MDA levels. Therefore, the experimental results show that: after the four herbs are combined, the volatile oil components of Acorus tatarinowii and Ligusticum chuanxiong have a synergistic effect; the water extracts of Astragalus membranaceus and Polygala tenuifolia have a synergistic effect; the four parts work together to exert their significant pharmacodynamic effects, which are related to reducing the content of AChE and MDA in brain tissue.

[0125] Example 2

[0126] Optimization of the extraction process of Astragalus and Zhizhi tablets

[0127] 1. Optimization of volatile oil extraction and inclusion process

[0128] 1.1 Determination of the contents of β-asarone, α-asarone, and ligustilide

[0129] (1) Chromatographic conditions for asarone: Welch Xtimate UPLC C18 (2.1×100mm, 1.8μm); flow rate: 0.3mL / min; injection volume: 1~2μL; column temperature: 35℃; detection wavelength: 260nm; gradient elution with acetonitrile (A)-0.1% phosphoric acid water (B) is shown in Table 4.

[0130] Table 4. Chromatographic gradient elution program for asarone

[0131]

[0132] (2) Chromatographic conditions for ligustilide: Welch Xtimate UPLC C18 (2.1×100mm, 1.8μm); flow rate: 0.3mL / min; injection volume: 1~2μL; column temperature: 35℃; detection wavelength: 325nm; gradient elution with acetonitrile (A)-0.1% phosphoric acid water (B) is shown in Table 5.

[0133] Table 5 Chromatographic gradient elution program for ligustilide

[0134]

[0135] (3) Preparation of reference solution: Take 2.31 mg of β-asarone reference standard and 2.02 mg of α-asarone reference standard respectively, put them in a 10 ml volumetric flask, add methanol to prepare a solution containing 0.231 mg of β-asarone and 0.202 mg of α-asarone per ml. Take 5.05 mg of ligustilide reference standard in a 10 ml volumetric flask, add methanol to prepare a solution containing 0.505 mg of ligustilide per ml, filter, and the solution is obtained.

[0136] (4) Preparation of the test solution: Place the oil obtained by steam distillation in a separatory funnel, add an appropriate amount of anhydrous diethyl ether and shake to extract. After the layers are clearly separated, discard the water layer and pour the ether layer from the bottle mouth into a pre-weighed constant-weight evaporating dish. Place the dish in a 37°C water bath to evaporate the ether until it is odorless, cool, weigh, and use a pipette to draw an appropriate amount of oil into a 10ml volumetric flask. Weigh the oil and dilute it to the mark with methanol. Mix well by sonication and filter to obtain the test solution.

[0137] 1.2 Determination of ferulic acid content

[0138] (1) Chromatographic conditions: Welch Xtimate UHPLC C18 (2.1×100mm, 1.8μm); flow rate: 0.3mL / min; injection volume: 1-2μL; column temperature: 35℃; detection wavelength: 325nm; gradient elution with acetonitrile (A)-0.1% phosphoric acid water (B) is shown in Table 6.

[0139] Table 6 Chromatographic gradient elution program

[0140]

[0141] (2) Preparation of reference solution: Weigh 2.25 mg of ferulic acid reference standard accurately, place it in a 20 ml brown volumetric flask, and add methanol to prepare a solution containing 0.1125 mg per ml.

[0142] (3) Preparation of test solution: Accurately pipette an appropriate amount of volatile oil water extract into two 3ml EP tubes, balance them, and centrifuge them in a centrifuge for 2min at 25℃ and 6000r. Take the supernatant, filter it, and the solution is obtained.

[0143] 1.3 Optimization of volatile oil extraction process

[0144] Take 30g of Acorus tatarinowii and 20g of Ligusticum chuanxiong, a total of 50g, and place them in a round-bottom flask. Add 8 times the amount of water and extract the volatile oil using steam distillation for different times, recording the oil yield per hour. The results are shown in Table 7. Table 8 shows that the volatile oil can be basically completely extracted after 5 hours of extraction.

[0145] Table 7. Investigation of oil extraction volume at different times

[0146]

[0147] Table 8 Content of Indicator Components in Medicinal Materials

[0148]

[0149] Under the optimized conditions described above, the oil extraction results were examined by adding solvent at multiples of 6, 8, and 10, and reducing the number of extractions to two. The results are shown in Table 9.

[0150] Table 9. Results of oil extraction with different water addition amounts and extraction times.

[0151]

[0152] The results showed that the volatile oil extracted with 8 times the amount of water had the highest content of asarone, while the volatile oil extracted with 10 times the amount of water had the lowest content of asarone. The content of ligustilide extracted with 10 times the amount of water was much higher than that extracted with 6 times or 8 times the amount of water. The content of ferulic acid extracted with 10 times the amount of water was higher than that extracted with 6 times or 8 times the amount of water.

[0153] Considering all factors, a 10-fold increase in water volume was selected for extracting the volatile oils from Acorus tatarinowii and Ligusticum chuanxiong. Furthermore, the ferulic acid content of the combined water extracts from the two extractions was significantly higher than that from a single extraction. Therefore, the final extraction process for the volatile oils was determined to be: two water extractions, the first with a 10-fold increase in water volume for 5 hours, and the second with an 8-fold increase in water volume for 1 hour.

[0154] 1.4 Investigation of the volatile oil encapsulation process

[0155] 900g of Acorus tatarinowii and 600g of Ligusticum chuanxiong, totaling 1500g of medicinal slices, were used to extract 30ml of oil using the optimized process (oil yield 2%~2.4%). The oil density was measured to be 0.97g / ml using a density flask, yielding a total of 29.15g of oil. The amount of oil used per sample was 8g. Considering the low efficiency and poor inclusion effect of the water-saturated solution method, the colloid mill method was directly used to investigate the amount of excipients used in the inclusion process. The content of asarone and ligustilide in the inclusion mixture and the transfer rate of the index components were used as evaluation indicators. β-CD was tested at 2, 4, and 6 times the amount of volatile oil, as detailed in Table 10.

[0156] Table 10 Investigation of Encapsulation Process

[0157]

[0158] Table 11 Total content of asarone and ligustilide in different amounts of β-CD inclusion complexes / mg

[0159]

[0160] Note: The sum of asarone and ligustilide.

[0161] The results are shown in Table 11. The results indicate that the recovery rate of volatile oil encapsulated with 4 times the amount of β-cyclodextrin in the colloid milling method was the highest, and the contents of asarone and ligustilide in the recovered volatile oil were the highest, confirming the effectiveness of using 4 times the amount of β-cyclodextrin for colloid milling.

[0162] 1.2 Process optimization of water extraction

[0163] 1.2.1 Orthogonal Design of Water Extraction Experiment

[0164] L9 (3) 4 Orthogonal experiments were conducted at three levels to investigate three factors: extraction times, extraction time, and solvent dosage, in order to determine the optimal process parameters. Astragaloside A, Polygala tenuifolia saponin, Verticillium isoflavone glucoside, Polygala tenuifolia sucrose, 3,6'-disinyl sucrose, and the yield of the dried extract were selected as evaluation indicators. A comprehensive scoring standard was then established for orthogonal analysis to determine the optimal levels of each influencing factor, as shown in Table 12.

[0165] Table 12 Orthogonal Factor Design Table for Water Extraction Process

[0166]

[0167] 1.2.2 Content determination method

[0168] 1.2.2.1 Determination of Astragaloside A Content

[0169] Chromatographic conditions:

[0170] Welch Ximate XBC18 (4.6×250mm, 5μm) was used with acetonitrile (A)-water (D) (32:68) as the mobile phase for isocratic elution (0–26 min, 32% A; 26.1–35 min, 80% A; 35.1–45 min, 32% A%); column temperature 35℃; flow rate 1.0 ml / min; injection volume: 5 μL, 10 μL; detection was performed using an evaporative light scattering detector under the following conditions: drift tube temperature 60℃; pressure: 313 kPa. The elution program is shown in Table 13.

[0171] Table 13 Chromatographic Elution Procedures

[0172]

[0173] Preparation of reference solution: Take 4.55g of astragaloside A reference standard, place it in a 10ml volumetric flask, add 80% methanol to dissolve and dilute to prepare a 0.44mg / ml (purity 96.9%) reference solution, filter through a membrane to obtain the solution.

[0174] Preparation of the test solution: Accurately weigh about 2g of orthogonal aqueous extract powder and place it in a stoppered conical flask. Accurately add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 80% methanol to 100ml, and shake well). Stopper tightly, weigh, heat under reflux for 1 hour, cool, weigh again, and replenish the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution. Shake well, filter, accurately measure 25ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in 80% methanol, transfer to a 10ml volumetric flask, add 80% methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the test solution.

[0175] 1.2.2.2 Determination of saponin content in Polygala tenuifolia

[0176] Chromatographic conditions: ACQUITY UPLC BEH C18 column (Waters) (2.1×100mm, 1.7μm); flow rate: 0.3mL / min; injection volume: 1-2μL; column temperature: 35℃; detection wavelength: 210nm; isocratic elution with acetonitrile (A)-0.1% phosphoric acid water (B), the isocratic elution results are shown in Table 14.

[0177] Table 14 Chromatographic Elution Procedures

[0178]

[0179] Preparation of reference solution: Take 2.43 mg of Polygala tenuifolia saponin reference standard, place it in a 2 ml volumetric flask, add methanol to prepare a solution containing 1.215 mg per ml, filter, and the solution is obtained.

[0180] Preparation of the test solution: Accurately weigh about 1g of orthogonal aqueous extract powder and place it in a stoppered conical flask. Accurately add 25ml of 70% methanol, weigh the solution, and sonicate (400W power, 40kHz frequency) for 1 hour. Cool the solution and weigh it again. Make up the lost weight with 70% methanol, shake well, and filter. Accurately measure 10ml of the filtrate and place it in a round-bottom flask. Evaporate to dryness. Add 25ml of 2% sodium hydroxide solution to the residue and heat under reflux for 2 hours. Cool the solution and adjust the pH to 4-5 with hydrochloric acid. Extract the solution three times with 25ml of water-saturated n-butanol each time. Combine the n-butanol solutions and recover the solvent to dryness. Dissolve the residue in an appropriate amount of methanol, transfer it to a 5ml volumetric flask, add methanol to the mark, shake well, and filter to obtain the test solution.

[0181] 1.2.2.3 Determination of 3,6'-disinic sucrose content

[0182] Chromatographic conditions: ACQUITY UPLC BEH C18 column (Waters) (2.1×100mm, 1.7μm); flow rate: 0.3mL / min; injection volume: 1μL; column temperature: 35℃; detection wavelength: 320nm; isocratic elution with methanol (A)-0.1% phosphoric acid water (B), gradient elution as shown in Table 15.

[0183] Table 15 Chromatographic Elution Procedures

[0184]

[0185] Preparation of reference solution: Take 2.61 mg of 3,6'-disinyl sucrose reference standard, place it in a 25 ml volumetric flask, add methanol to prepare a solution containing 0.1044 mg per ml, filter, and the solution is obtained.

[0186] Preparation of the test solution: Take about 1g of orthogonal powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, sonicate for 0.5 hours, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0187] 1.2.2.4 Determination of the content of verbascoside isoflavone glucoside and polygalactosyl quinone III

[0188] Chromatographic conditions: ACQUITY UPLC BEH C18 column (Waters) (2.1×100mm, 1.7μm); flow rate: 0.3mL / min; injection volume: 1-2μL; column temperature: 35℃; detection wavelength: 260nm; gradient elution with acetonitrile (A)-0.1% phosphoric acid water (B), as shown in Table 16.

[0189] Table 16 Chromatographic gradient elution procedures

[0190]

[0191] Preparation of reference solution: Take 3.23 mg of verbenafil isoflavone glucoside and 3.14 mg of polygalactosorbide III reference standard and place them in a 25 ml volumetric flask. Add methanol to prepare a solution containing 0.1292 mg of verbenafil isoflavone glucoside and 0.1256 mg of polygalactosorbide III per ml. Filter to obtain the solution.

[0192] Preparation of the test solution: Take about 1g of orthogonal powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, sonicate for 0.5 hours, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and the test solution is obtained.

[0193] 1.2.3 Orthogonal Experiment Analysis

[0194] Weigh out 60g of Astragalus membranaceus and 60g of Polygala tenuifolia according to the prescription ratio, for a total of 120g of medicinal slices. Take 9 parallel samples and conduct orthogonal experiments according to Table 17.

[0195] Table 17 Results of orthogonal content determination in water extraction

[0196]

[0197] Table 18. Intuitive Analysis Table of Scoring Results for Water Lifting Orthogonal Experiment

[0198]

[0199] Table 19 Analysis of Variance of Water Lifting Orthogonal Experiment Results

[0200]

[0201] Note: F 0.05 (2,2)=19.

[0202] Comprehensive score = Amount of astragaloside A / Maximum amount of astragaloside A in the orthogonal table × 45 + Amount of Polygala tenuifolia saponin / Maximum amount of Polygala tenuifolia saponin in the orthogonal table × 20 + Amount of verbenacisin glucoside / Maximum amount of verbenacisin glucoside in the orthogonal table × 15 + Amount of polygala tenuifolia sucrose / Maximum amount of polygala tenuifolia sucrose in the orthogonal table × 5 + Amount of 3,6-disinoyl sucrose / Maximum amount of 3,6-disinoyl sucrose in the orthogonal table × 5 + Extract yield / Maximum extract yield in the orthogonal table × 10 Formula I.

[0203] Results Analysis: The orthogonal results table shows that the optimal process conditions for comprehensive scoring are A3B3C2. The range indicates that the influence of each factor is A > B > C. Analysis of variance shows that A and B both significantly affect the extraction effect. Since there are significant differences between the number of extractions, particularly between the first, second, and third extractions, three extractions were chosen. Simultaneously, there are significant differences in extraction time, with the highest K value observed at 1 hour; therefore, an extraction time of 1 hour was selected. There are no significant differences in water addition, and the differences are small, resulting in approximate K values. Considering the water absorption capacity of the medicinal materials and production costs, 8 times the amount of water was added for the first extraction, and 6 times the amount of water was added for the second and third extractions, respectively. The final determined process is: three extractions, each lasting 1 hour, with 8 times the amount of water added for the first extraction, and 6 times the amount of water added for the second and third extractions, respectively.

[0204] 2. Optimization of alcohol precipitation process

[0205] Water extraction and alcohol precipitation is a commonly used purification method for water extracts of traditional Chinese medicine in industry. Before alcohol precipitation, the medicinal solution needs to be concentrated to reduce the amount of ethanol used. It is necessary to examine the concentration density of the medicinal solution and the alcohol precipitation concentration.

[0206] 2.1 Optimization of the relative density of the aqueous extract of volatile oil and the alcohol precipitation process

[0207] Take 540g of Acorus tatarinowii slices and 360g of Ligusticum chuanxiong slices, totaling 900g. Prepare aqueous extracts according to the optimized process, concentrate to the required relative density according to Table 20, add the corresponding ethanol to a precipitation concentration of 65%, stir thoroughly, and filter to collect the supernatant. The ferulic acid content and extract yield in the supernatant were used as indicators to investigate the ethanol precipitation concentration, and the results are shown in Table 20.

[0208] Table 20 Results of the relative density study of the aqueous extract of volatile oils before alcohol precipitation.

[0209]

[0210] Three portions of raw medicinal slices, each weighing 133g, were taken and concentrated to a relative density of 1.12g / ml using the optimized process. A single-factor experiment was conducted, where the aqueous extract was extracted using the optimized process and then concentrated. Ethanol was added according to Table 21 to the corresponding alcohol precipitation concentration, and the mixture was thoroughly stirred and filtered to collect the supernatant. The alcohol precipitation concentration was investigated using the content of each component as an indicator.

[0211] Table 21 Results of alcohol precipitation in aqueous extracts of volatile oils

[0212]

[0213] The above results indicate that the ferulic acid content in the solution is highest and the dry extract yield is lowest when the relative density is 1.12 g / ml. Therefore, the concentration range is controlled at 1.12 ± 0.02 g / ml (50℃). Three portions of the solution with a relative density of 1.12 g / ml (50℃) were ethanol-precipitated to concentrations of 60%, 65%, and 70%, respectively. The results showed that the solid-liquid separation effect was good when the ethanol precipitation reached 70%, the ferulic acid content in the solution was highest, and the dry extract yield was lowest. Therefore, the ethanol precipitation concentration was determined to be 70%. Finally, the ethanol precipitation process conditions for the volatile oil water extract were determined to be concentrated to 1.10~1.14 g / ml and the ethanol precipitation concentration was 70%.

[0214] 2.2 Optimization of the relative density and alcohol precipitation process of the aqueous extract of Astragalus membranaceus and Polygala tenuifolia

[0215] The aqueous extracts of Astragalus membranaceus and Polygala tenuifolia were prepared by separating 150g of Astragalus membranaceus and 150g of Polygala tenuifolia, totaling 300g. The aqueous extract was prepared using an optimized process, filtered, and concentrated to a relative density of 1.08 (60℃). The concentrate was divided into five equal portions, each concentrated to the desired relative density according to Table 22. Ethanol was added to the corresponding alcohol precipitation concentration, and the mixture was thoroughly stirred and filtered to obtain the supernatant. The relative density and alcohol precipitation concentration of the extract were investigated using the content of each component as an indicator.

[0216] Table 22 Results of the alcohol precipitation process of Astragalus membranaceus and Polygala tenuifolia water extract

[0217]

[0218] Conclusion: The purification results of the aqueous extract of Astragalus membranaceus and Polygala tenuifolia were consistent with those of the aqueous extract of volatile oil. The purification processes for both fractions could be combined. The final determination of the alcohol precipitation conditions for both fractions was consistent: concentration to a relative density of 1.10–1.14 g / ml and an alcohol concentration of 70%. The yield of supernatant after alcohol precipitation was 23.2% (32% before precipitation).

[0219] 2.3 Concentration Condition Screening

[0220] The experiment included screening of concentration conditions. The concentration temperature was 60℃, 70℃, and 80℃, and the concentration time was 2h, 4h, and 6h, respectively, using the various index components in the aqueous extract as evaluation indicators. The results are shown in Tables 23 and 24.

[0221] Table 23 Results of Concentration Time and Concentration Temperature Study 1

[0222]

[0223] Table 24 Results of Concentration Time and Concentration Temperature Investigation

[0224]

[0225] The results showed that within 2 hours of concentration, the index components were relatively stable at different temperatures; within 4 and 6 hours of concentration, when the temperature was higher than 70°C, the contents of each index component decreased significantly; therefore, the concentration temperature should be controlled below 70°C and the concentration time within 6 hours would be sufficient.

[0226] 3 Optimization of drying process

[0227] Taking the contents of each component as indexes, through single-factor experiments, the main process parameters such as different drying methods (vacuum drying and spray drying), relative density of the extract before drying (the extract was concentrated to 1.25 g / mL for vacuum drying), temperature (70°C, 80°C, 90°C for vacuum drying), dosage of excipients (no excipients were added during spray drying, 3% and 10% of maltodextrin were added), water content, dry extract yield, etc. were investigated.

[0228] During the experiment, it was found that the wall sticking phenomenon was serious during spray drying, and adding excipients could not improve the wall sticking situation. Therefore, vacuum drying was selected for the investigation of the drying process. 240 g of Astragalus membranaceus (120 g) and Polygala tenuifolia (120 g) in total, and 200 g of Acorus tatarinowii (120 g) and Ligusticum chuanxiong (80 g) were taken respectively. After preparing the samples according to the above optimized process, the investigation of vacuum drying temperature was carried out at 70, 80, and 90°C respectively, and the content of each index component was used as the evaluation index for the selection of the best temperature. The results are shown in Table 25.

[0229] Table 25 Results of vacuum drying investigation at different temperatures

[0230]

[0231] The results showed that there was no significant change in the content of the index components of the vacuum drying samples at different temperatures. Considering from the actual production cost of the factory, ≤80°C was selected as the vacuum drying temperature.

[0232] Example 3

[0233] Effect of pharmaceutical composition 1# on cognitive function of AD model in rapidly aging mice (SAMP-8 mice)

[0234] 1 Test drugs

[0235] 1.1 Tested drug, composition extract 1# prepared by process 1, provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Storage conditions: at room temperature in a dry place.

[0236] 1.2 Test animals

[0237] SAMP-8 mice, SPF grade, 8 months old, 32 mice; SAMR-1 mice, SPF grade, 8 months old, 8 mice; all male, purchased from Peking University Health Science Center, animal license number: SCXK (Beijing) 2021-0013.

[0238] 2 Dosage design and drug preparation

[0239] 2.1 Test drug:

[0240] Table 26 Preparation methods of test drugs

[0241]

[0242] 2.2 Positive control drug

[0243] Donepezil hydrochloride tablets are intended for human clinical use at a dose of 5 mg / 60 g / kg / day, or 0.083 mg / kg / day. In the experiments, the dosage in mice was 0.92 mg / kg / day, which is equivalent to a multiple of the human clinical dose.

[0244] One donepezil hydrochloride tablet (5mg) was thoroughly ground and then diluted with high-purity water to 109ml, which yielded a concentration of 0.046mg / ml. Mice were administered the solution via gavage at a dose of 0.2ml / 10g. The solution was prepared once every two weeks.

[0245] 3. Test Methods

[0246] 3.1 Experimental grouping and administration

[0247] Thirty-two SAMP-8 mice were randomly divided into five groups according to their weight class: a model control group, a donepezil hydrochloride control group, and groups receiving equal, 1 / 2, and 1 / 4 times the dosage of drug composition #1, with six mice in each group. Eight SAMR-1 mice were used as a normal control group. Except for the normal control group and the model control group, the other drug groups were given the drug once a day at a dose of 0.2 ml / 10 g by gavage for 28 consecutive days. The normal control group and the model control group were given distilled water by gavage under the same conditions.

[0248] 3.2 Detection

[0249] Detection indicators: Behavioral studies were conducted on day 28 after drug administration.

[0250] Morris water maze experiment: Conducted in week 4, with 4 days of training followed by formal testing on day 5. Medication continued as usual during the experiment. Orientation and navigation experiment: Before the experiment, mice were placed in the water maze and swam for 30 seconds to acclimatize to the water stimulation, then placed on a platform for 10-20 seconds to acclimatize. Subsequently, mice were placed into the water sequentially from each of the four quadrants facing the pool wall, and the time required for the mouse to find the platform was recorded as the latency period for orientation and navigation. If a mouse could not find the platform within 60 seconds, the latency period was recorded as 60 seconds, and the mouse was induced to stay on the platform for 10 seconds. Spatial exploration experiment: After the orientation and navigation experiment, the platform was removed, and the mouse was placed into the water from the opposite side of the target quadrant (the quadrant where the platform was located). The time the mouse spent in the quadrant where the platform was located within 60 seconds was recorded.

[0251] 4 Test Results

[0252] 4.1 The results of the water maze experiment are shown in Table 27 and Figure 6 .

[0253] Table 27 Effects on the AD model of SAM mice

[0254]

[0255] 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.

[0256] The results in Table 27 show that: The residence time of the mice in the model control group in the target quadrant was shortened and the latency was prolonged, showing a significant difference compared with the normal control group ( P <0.05); The high and medium dose groups of Composition 1# could prolong the residence time of the mice in the target quadrant, and the high, medium and low dose groups shortened the latency, showing a significant difference compared with the model control group ( P <0.05, P <0.01).

[0257] Example 4

[0258] Effects of the pharmaceutical composition on cognitive impairment with bipolar disorder in senescence-accelerated mice (SAMP8 mice)

[0259] 1 Test drugs

[0260] 1.1 The extract of Composition 1# prepared by Process 1 was provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

[0261] 1.2 Test animals: SAMP8 / R1 mice, SPF grade, 5 months old, 120 in total, all female, purchased from the Health Science Center of Peking University, animal license number: SCXK (Beijing) 2022-0009, certificate number: No.110332241100031824.

[0262] 2 Dosage design and drug preparation

[0263] Table 28 Preparation methods of the test drugs

[0264]

[0265] 2.2 Positive control drug

[0266] Donepezil hydrochloride tablets are prescribed for human clinical use at doses of 5 mg / 60 g / kg / day and 0.083 mg / kg / day. In the experimental study, the dosage in mice was 0.92 mg / kg / day, equivalent to a multiple of the human clinical dose.

[0267] One donepezil hydrochloride tablet (5mg) was thoroughly ground and then diluted with high-purity water to 109ml, which yielded a concentration of 0.046mg / ml. Mice were administered the solution via gavage at a dose of 0.2ml / 10g. The solution was prepared once a week.

[0268] 3. Test methods

[0269] 3.1 Experimental grouping and administration

[0270] Twelve SAMR1 mice were used as the normal control group. Sixty SAMP8 mice were evenly divided into a model control group, a donepezil hydrochloride control group, and three dose groups (high, medium, and low) of the test substance, based on the latency of the water maze test, ensuring that the mean latency of each group was similar, with 12 mice in each group. Except for the normal control group and the model control group, the other treatment groups were given the drug once daily at a dose of 0.2 ml / 10 g via gavage for 90 consecutive days. The normal control group and the model control group were given distilled water via gavage under the same conditions. The following indicators were measured:

[0271] 3.2 Behavioral Testing

[0272] Before the experiment began, mice were grouped according to the latency period of the water maze. After the drug was administered, the water maze and new object recognition experiments were conducted on days 30, 60, and 90. The mouse tail suspension and elevated cross maze experiments were conducted on days 60 and 90.

[0273] 3.2.1 Water Maze Experiment

[0274] (1) Days 1-4 were for the orientation navigation experiment. The platform locations remained fixed in quadrant III. Except for the quadrant where the platform was located, the other three platforms served as the landing points for the mice. Each mouse was gently placed into the water facing the pool wall. The time from when the mouse landed in the water until it found the platform was recorded; this period was called the escape latency period. Each training session consisted of 3 repetitions, each lasting 1 minute.

[0275] (2) Day 5 is the space exploration experiment. After removing the platform, mice are placed back in the quadrant opposite the platform, and the number of times the mice cross the original platform position in quadrant III is recorded.

[0276] 3.2.2 New Object Recognition Experiment

[0277] Based on the rodents' innate tendency to explore new objects, a new object recognition experiment was conducted on mice to reflect their learning and memory abilities. The experiment was divided into an adaptation period, a training period, and a testing period.

[0278] (1) Adaptation period: On day 1, mice were placed in a 60 cm × 60 cm × 40 cm test box and allowed to explore freely for 5 minutes. After exploration, the mice were removed and returned to their cages. Before each animal replacement, the mice's feces and urine in the test box should be cleaned, and the box should be sprayed and wiped with 75% ethanol to eliminate odor.

[0279] (2) Training period: On the second day, place two identical cylinders A and B in the lower left and lower right corners of the test box, and put the mouse into the test box with its back to the two objects. Let the mouse explore freely in the test box for 5 minutes.

[0280] (3) Testing period: On the third day, remove object B from the lower right corner and replace it with a new cube C. Turn on the recording device and place the mouse into the test box. The object recognition test time is 5 minutes. The video is recorded by a camera installed above the test area. When the animal's nose is about 2 cm away from or in contact with the object, the time for the mouse to explore object A (TA) and the time for the mouse to explore object C (TC) are recorded. The new object recognition index is calculated according to Formula II.

[0281] New object recognition index = TC / (TA+TC)×100% Formula II.

[0282] 3.2.3 Mouse tail suspension test

[0283] Attach tape to the tail of the mouse 2 cm from the end, and suspend the mouse on a horizontal bar 30 cm high for 6 minutes. Record the mouse’s immobility time for the last 4 minutes.

[0284] 3.2.4 Elevated Cross Maze Experiment

[0285] Set up the cross maze test chamber and wipe it with 70% ethanol. Place the mice in a quiet environment for 30 minutes and then put them into the chamber. Ensure that the mice can move freely in the closed arms of the cross maze. After 5 minutes of the experiment, record the time the mice spend in the open arms to assess their anxiety level.

[0286] 4. Experimental Results

[0287] 4.1 The results of the water maze test are shown in Table 29. Figure 7 and Figure 8 .

[0288] Table 29 Results of the Water Maze Experiment

[0289]

[0290] Note: Compared with the normal control group # P <0.05、 ## P<0.01; compared with the model control group , .

[0291] Table 29 shows that on days 30, 60, and 90 after drug administration, the latency period and the number of platform crossings were prolonged in the model control group, with significant differences compared to the normal control group. P <0.05, P <0.01); After 60 days of administration, the latency period of the low-dose group and the medium- and low-dose groups of Qizhi tablets was shortened, and the difference was significant compared with the model control group. P <0.05); the number of platform crossings increased in the high-dose group of Qizhi tablets after 30 days of administration and in the high- and medium-dose groups of Qizhi tablets after 60 days, showing a significant difference compared with the model control group. P <0.05、 P <0.01).

[0292] 4.2 The results of the new object recognition experiment are shown in Table 30. Figure 9 .

[0293] Table 30 Experimental Results of Novel Object Recognition

[0294]

[0295] 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.

[0296] Table 30 shows that on days 60 and 90 after drug administration, the percentage of new object preference in the model control group decreased significantly compared with the normal control group. P <0.01); On days 60 and 90 of administration, the percentage of new object preference increased in all three dosage groups of Qizhi tablets, showing a significant difference compared with the model control group. P <0.05, P <0.01).

[0297] 4.3 The results of the mouse tail suspension test are shown in Table 31 and Figure 10 .

[0298] Table 31 Results of mouse tail suspension test

[0299]

[0300] 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.

[0301] Table 31 shows that on days 60 and 90 after drug administration, the immobility time of the suspended tail in the model control group was prolonged, which was significantly different from that in the normal control group. P <0.01); On day 60 of administration, the immobility time was shortened in the medium and low dose groups of Qizhi tablets and on day 90 of administration in the three dose groups of Qizhi tablets, with significant differences compared with the model control group. P <0.05、 P <0.01).

[0302] 4.4 The results of the elevated cross maze experiment are shown in Table 32. Figure 11 .

[0303] Table 32 Results of the elevated cross maze experiment

[0304]

[0305] 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.

[0306] Table 32 shows that on days 60 and 90 after administration, the open-arm residence time was prolonged in both the high- and low-dose groups of the composition, with significant differences compared to the model control group. p<0.01 ).

[0307] Example 5:

[0308] Effects of the drug composition on a bipolar disorder model in ouabain-induced rapid aging mice (SAMP8 mice)

[0309] 1. Experimental Materials

[0310] 1.1 Test Sample

[0311] 1.1.1 Complete Formula (Extract Powder): Provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The clinical dosage for humans is 11g crude drug / 60kg / day, or 0.18g crude drug / kg / day.

[0312] 1.1.2 Single crude drug

[0313] Astragalus membranaceus: The human dosage is 3 g of crude drug / 60 kg / d, 0.05 g of crude drug / kg / d;

[0314] Acorus tatarinowii: The human dosage is 3 g of crude drug / 60 kg / d, 0.05 g of crude drug / kg / d;

[0315] Polygala tenuifolia: The human dosage is 3 g of crude drug / 60 kg / d, 0.05 g of crude drug / kg / d;

[0316] Ligusticum chuanxiong; The human dosage is 2 g of crude drug / 60 kg / d, 0.03 g of crude drug / kg / d.

[0317] 1.2 Positive control drug: Lithium carbonate sustained-release tablets, Jiangsu Nhwa Pharmaceutical Co., Ltd. The dosage during the treatment period is 0.9 - 1.5 g (3 - 5 tablets) per day, taken in 1 - 2 divided doses, and the maintenance dosage is 0.6 - 0.9 g (2 - 3 tablets) per day

[0318] 1.3 Modeling drug: Ouabain, Shanghai TargetMol Bio-Tech Co., Ltd.

[0319] 1.4 Experimental animals: SAM mice, SPF grade, 25 g, 63 mice, all male, purchased from Beijing Zhong'an Funeng (Hebei Xiongan) Biotechnology Co., Ltd., animal license number: SCXK (Beijing) 2022 - 0009, certificate number: 110332241100052417.

[0320] 2 Dosage design and drug preparation

[0321] 2.1 Test drugs

[0322] 2.1.1 Whole formula (extract powder) is shown in Table 33.

[0323] Table 33 Preparation of the whole formula (extract powder)

[0324]

[0325] 2.1.2 Single crude drug

[0326] (1) The preparation and administration methods of Astragalus membranaceus, Acorus tatarinowii, and Polygala tenuifolia are shown in Table 34.

[0327] Table 34 Preparation and administration methods of Astragalus membranaceus, Acorus tatarinowii, and Polygala tenuifolia

[0328]

[0329] (2) The preparation and administration methods of Ligusticum chuanxiong are shown in Table 35.

[0330] Table 35 Preparation and administration methods of Ligusticum chuanxiong

[0331]

[0332] 2.2 Positive control drug

[0333] The proposed clinical dosage of lithium carbonate sustained-release tablets for humans is 1.2g / 60kg / day, while the dosage for mice in the experiment was 0.22g / kg / day, which is equivalent to a multiple of the clinical human dosage.

[0334] Two lithium carbonate sustained-release tablets (0.6 mg) were thoroughly ground and then diluted with pure water to 54.55 mL, which yielded a concentration of 0.011 g / mL. Mice were administered the solution via gavage at a dose of 0.2 ml / 10 g. The solution was prepared once every 7 days.

[0335] 3. Test Methods

[0336] 3.1 Animal selection: All animals were weighed and then subjected to acclimatization feeding in the animal room to adapt to the animal environment. Mice that were too small in weight were removed before modeling to allow for subsequent experiments.

[0337] 3.2 Experimental grouping and drug administration

[0338] SAMP8 mice were randomly divided into 10 groups according to body weight: normal control group, sham-operated group, model control group, lithium carbonate group, isoploid group of the whole formula, low-ploid group of the whole formula, Astragalus group, Ligusticum chuanxiong group, Acorus tatarinowii group, and Polygala tenuifolia group, with 6 mice in each group. After grouping, except for the normal control group, sham-operated group, and model group, the other groups were given the drug once a day after modeling, 0.2 mL / 10 g / time, for 15 consecutive days. The normal control group, sham-operated group, and model control group were given distilled water by gavage under the same conditions.

[0339] 3.3 Modeling Method

[0340] After acclimatization, mice were used to induce a bipolar disorder model by intraventricular injection of ouabain. Mice were fasted and deprived of water before surgery. After weighing, the mice were anesthetized via intraperitoneal injection. The mice's heads were fixed in a stereotaxic apparatus, the hair on the top of the head was removed, the skin was disinfected, and a midline incision was made to expose the fontanelle. Following a mouse brain atlas, 0.5 mm posterior to the anterior fontanelle and 1 mm to the left, a microsyringe was inserted vertically 2 mm deep. 0.5 μL of ouabain (50 μM) was injected into one ventricle at a rate of 0.5 µL / 2 min. The needle was left in place for 2 min and then slowly and evenly withdrawn. The wound was sutured, and lidocaine gel was applied to the suture site to relieve pain. Postoperatively, the mice were placed on a heated blanket to keep them warm. After the mice regained consciousness, they were returned to their cages and their condition was monitored. Penicillin was administered intraperitoneally for 3 consecutive days postoperatively to prevent infection. In the sham surgery group, only the fontanelle was exposed and sutured; no other surgical procedures were performed.

[0341] 3.4 Detection Indicators

[0342] 3.4.1 Water maze test: The method is the same as before.

[0343] 3.4.2 Open field test: The test was conducted on day 7 and day 15 after drug administration. The mice were placed in the square at the bottom center of the open field test chamber. The exploration behavior and movement of the mice were recorded for 5 minutes. The distance the mice moved in the open field, the number of squares they crossed, the number of times they stood up, and the number of times they visited the central area were recorded.

[0344] 3.4.3 Tail suspension test: The test was conducted on day 7 and day 15 after drug administration. The test duration was 6 minutes. The immobility time of the mice within 4 minutes after administration was recorded.

[0345] 3.4.4 Sugar Water Preference Test: After the test, the sugar water preference index of the mice was calculated, and the differences in the sugar water preference index among the groups were compared and analyzed. The sugar water preference index was calculated according to Formula III:

[0346] Sugar water preference index = (sugar water consumption / total liquid consumption) × 100% Formula III.

[0347] 3.4.5 Brain tissue pathological examination: A panoramic tissue scanner was used to scan the pathological sections of the brain tissue to observe the lesions in the mouse brain tissue.

[0348] 3.4.6 Cytokine detection:

[0349] After all animal behavior studies were completed, the animals were euthanized, and mouse brain tissue was collected. The levels of IL-6, IL-10, TNF-α, and CRP, as well as the activity of Na+-K+-ATPase in the brain tissue, were detected by ELISA.

[0350] 4. Experimental Results

[0351] 4.1 Effects of the drug on brain tissue pathology in ouabain-induced bipolar disorder model mice

[0352] See results Figure 12The normal control group and the sham-operated group showed normal brain tissue morphology, clear structure, and neat arrangement. Neuronal cell bodies were round or spindle-shaped, with large, lightly stained nuclei, clear nucleoli, intact nuclear membranes, and consistent nerve fiber course with uniform interstitial staining. Compared with the normal control group or the sham-operated group, the model control group showed pathological changes in brain tissue, with disordered tissue structure, reduced number of neurons, irregular arrangement, some cell bodies showing eosinophilic degeneration and wrinkling, and cell morphology becoming triangular or elongated. Nerve fiber course was disordered, but some basically normal neurons were still visible. Compared with the model control group, the positive drug lithium carbonate group showed improved brain tissue pathological morphology, with more neat cell arrangement and most surviving cells showing relatively normal morphology. The isoploid group, low-power group, Astragalus group, Ligusticum chuanxiong group, Acorus tatarinowii group, and Polygala tenuifolia group showed varying degrees of uniform improvement in brain tissue pathological morphology, with more neat cell arrangement, more normal neuronal morphology, and reduced cell body shrinkage and degeneration.

[0353] 4.2 Effects of drugs on learning and memory abilities in ouabain-induced bipolar disorder model mice

[0354] Figure 13 The results showed that the escape latency was prolonged on days 2-4 in the model control group, which was significantly different from that in the normal control group and the sham surgery group. P <0.05; The escape latency on days 2-4 was shortened in the whole formula low-power group, Astragalus group, and Acorus tatarinowii group, with significant differences compared with the model control group. P <0.01). Moreover, the combined effect of the whole formula was better than that of the Astragalus group and the Acorus tatarinowii group, indicating that the combination of drugs exerted a synergistic effect and increased the efficacy.

[0355] Figure 14 The results showed that, compared with the normal control group and the sham-operated group, the number of times mice in the model control group crossed the platform was reduced, and the difference was statistically significant. P <0.05); Compared with the model control group, the number of times mice crossed the platform increased in the low-dose group of the whole formula, the Chuanxiong group, and the Shichangpu group, and the differences were statistically significant. P <0.05).

[0356] Figure 15 The results showed that, compared with the normal control group and the sham-operated group, the activity time in the quadrant where the platform was located in the model control group mice was significantly shortened, and the difference was statistically significant. P <0.05); Compared with the model control group, the activity time in the quadrant where the plateau was located was prolonged in the low-dose group of mice, and the difference was statistically significant. P <0.05).

[0357] Figure 16 The results showed that, compared with the normal control group and the sham-operated group, the swimming path of the model control group was chaotic and longer; the swimming path of the isoploid group, the low-power group and the Astragalus group was shorter.

[0358] The above results indicate that the learning and memory abilities of mice with ouabain-induced bipolar disorder were significantly reduced; the Astragalus and Acorus groups shortened the escape latency, while the Ligusticum and Acorus groups increased the number of times mice crossed platforms; the combined formula significantly affected the escape latency, the number of times mice crossed platforms, and the activity time within the quadrant, and its effect was superior to that of Astragalus, Ligusticum, and Acorus alone, indicating that the combined formula can significantly improve the learning and memory abilities of model mice and exert a comprehensive therapeutic effect.

[0359] 4.4 Effects of drugs on manic-like behavior in ouabain-induced bipolar disorder model mice

[0360] like Figure 17 The results showed that, in the open field test after drug administration, compared with the normal control group and the sham-operated group, the model group mice had significantly increased movement distance and number of standing times. P <0.05, exhibiting manic-like behavior; compared with the model group, the distance traveled by mice in the all-fold and Astragalus groups was significantly shortened ( P <0.05); The number of standing times was reduced in mice in the isopylogous and hypoploid groups. P <0.05).

[0361] 4.5.1 Results of the tail suspension test

[0362] Figure 18 The results showed that, compared with the normal control group and the sham-operated group, the immobility time of mice in the model control group increased after drug administration ( P <0.05, indicating that the mice exhibited depressive-like behavior; compared with the model group, the immobility time of mice in the full-dose group and the Astragalus-treated group was shortened ( P <0.05), suggesting that drug treatment can improve depressive-like behavior in model mice to some extent.

[0363] 4.6 Effects of drugs on inflammatory factors in brain tissue of mice with ouabain-induced bipolar disorder

[0364] Figure 19 The results showed that, compared with the normal group and the sham surgery group, the levels of CRP, IL-6 and TNF-α in the brain tissue of the model group mice were significantly increased. P <0.01) and IL-10 levels were significantly reduced ( P <0.01); After drug treatment, compared with the model group, the isoploid group, the low-power group, the Astragalus group, the Ligusticum chuanxiong group, the Acorus tatarinowii group, and the Polygala tenuifolia group were able to reduce the levels of CRP and IL-6 in brain tissue; the isoploid and low-power groups of the whole formula were able to significantly reduce the levels of TNF-α in brain tissue ( P <0.05); each treatment group was able to increase the IL-10 content in mouse brain tissue (P <0.01) indicates that each drug can inhibit the inflammatory response to a certain extent, and the combined effect of the drugs is enhanced, indicating a synergistic effect.

[0365] Example 6

[0366] Effects of the drug composition on an MPTP-induced mouse model of Parkinson's disease

[0367] 1. Experimental Materials

[0368] 1.1 Extract of drug composition No. 1, provided by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. Storage conditions: Store in a cool, dry place. The human clinical dosage is 11g crude drug / 60kg.

[0369] 1.2 Positive control drug: Rasagiline Mesylate Tablets, trade name: Anzilai; manufacturer: Teva Pharmaceutical Industries Ltd; production date: 2021-05; expiry date: 2024-04; batch number: 112530.

[0370] 1.3 The experimental animals are shown in Table 36.

[0371] Table 36. Description of the source of laboratory animals

[0372]

[0373] 2. Methods and Results

[0374] 2.1 Dosage Design

[0375] 2.1.1 The proposed human clinical dosage for the composition is 11g crude drug 60 / kg / day, which is equivalent to 0.18g crude drug / kg / day. In the experiment, the dosage for mice was 2.0g crude drug / kg / day and 4.0g crude drug / kg / day, which are equivalent to the same and twice the human clinical dosage, respectively.

[0376] 2.1.2 Positive control drug: The dosage was converted to the equivalent dose for humans and animals during the experiment.

[0377] 2.2 Animal modeling and drug administration

[0378] Fifty C57 / BL6JNifd mice were randomly divided into five groups: a normal control group, a model control group, a positive drug control group, and two dosage groups of the combined drug, with ten mice in each group. Except for the normal control group, the other animals were intraperitoneally injected with MPTP solution at a dose of 20 mg / kg, alternating between left and right sides every 2 hours for four consecutive times to establish an acute Parkinson's disease model. The normal control group received intraperitoneal injections of physiological saline under the same conditions. Each drug-treated group began receiving the drug 1 hour after the last modeling treatment, once daily for 7 consecutive days. The normal control group and the model control group received the same volume of physiological saline. The following indicators were measured: behavioral tests were performed before modeling and 7 days after drug administration.

[0379] 2.3 Results of the mouse pole climbing experiment

[0380] Preliminary experiments showed that the homemade pole-climbing tool, indicated by the arrow in the diagram, was used. Dimensions are as shown. Figure 20 As shown, all areas are wrapped with medical tape.

[0381] Figure 21 The results showed that the climbing time of mice in the model group was significantly prolonged, with a significant difference compared with the normal control group. P <0.01); Seven days after administration, the climbing time of composition #1 was significantly shortened, showing a significant difference compared with the model control group ( P <0.01);

[0382] Example 7

[0383] Analysis report of effective components of the composition

[0384] Sample preparation: Six rats were administered the extract 1# prepared by process 1 by gavage at a dose of 4g crude drug / kg / day, once daily for 7 consecutive days. On the 8th day, blood was collected to separate serum, and brain tissue was obtained by dissection. Three rats were used as blank controls and administered distilled water by gavage under the same conditions.

[0385] I. Sample Preparation

[0386] Serum sample processing: Before testing, reconstitute the plasma sample at 4℃ for 1 hour, take 90 μL, place it in a centrifuge tube, add 360 μL of acetonitrile to precipitate the protein, vortex for 2 min, centrifuge at 4000 r / min for 15 min, and dry the supernatant under nitrogen. Before injection, reconstitute the sample with 100 μL of methanol, vortex for 2 min, centrifuge at 4000 r / min for 10 min, and filter the supernatant through a 0.22 μm microporous membrane.

[0387] Brain tissue sample processing: Take 0.2 mL of brain homogenate, add 0.6 mL of ice-cold acetonitrile to precipitate proteins, mix well, incubate at 4 ℃ for 30 min, centrifuge at 4 ℃ for 5 min, and dry the supernatant under nitrogen. Before injection, reconstitute with 100 μL of methanol, vortex for 2 min, centrifuge at 4000 r / min for 10 min, and filter the supernatant through a 0.22 μm microporous membrane.

[0388] II. Liquid chromatography-mass spectrometry analysis of compound preparations and their blood-entering and brain-entering components

[0389] Chromatographic conditions

[0390] A Waters ACQUITY UPLC™ system was used. The column was an ACQUITY UPLC-HSS T3 column (2.1 mm × 100 mm, 1.7 μm). Mobile phase A was an aqueous solution containing 0.2% formic acid, and mobile phase B was acetonitrile. The flow rate was 0.30 mL / min, the column temperature was 35℃, the autosampler temperature was 10℃, and the injection volume was 5.0 μL. Gradient elution was performed: 0–28 min, 30%–95% B; 28–32 min, 95%–5% B.

[0391] Mass spectrometry conditions

[0392] Electrospray ionization (ESI) was used, with the main component analysis performed in MSe scanning mode, detecting in both positive and negative ion modes. Ion source operating parameters were: capillary voltage 3.0 kV, orifice voltage 30 V, and orifice backflush gas flow rate (N2) 50 L·h. -1 The desolvation gas flow rate is 800 L·h -1 The ion source temperature was 120 ℃, the desolvation gas temperature was 450 ℃, the spectrum acquisition interval was 0.2 s, and the scanning range was m / z 50 ~ 1500.

[0393] III. Result of the Examination

[0394] The results of the liquid chromatography-mass spectrometry test are shown below. Figures 22-31 .

[0395] 1. The results of the compound identification are shown in Table 37.

[0396] Table 37 Identification results of active compounds in compound preparations

[0397]

[0398]

[0399]

[0400]

[0401] 2. The results of blood component identification are shown in Table 38.

[0402] Table 38 Results of Blood Component Identification

[0403]

[0404]

[0405] 3. The results of the brain-entering component identification are shown in Table 39.

[0406] Table 39 Results of Brain-Entry Component Identification

[0407]

[0408]

[0409] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of age-related cognitive impairment with bipolar disorder, wherein the preparation method of the pharmaceutical composition comprises the following steps: The volatile oils of Acorus tatarinowii and Ligusticum chuanxiong were extracted by steam distillation, and the first aqueous extract was collected. The volatile oils were then encapsulated with excipients to obtain volatile oil inclusion complexes. Astragalus and Polygala were decocted in water, and the second water extract was collected. The second water extract and the first water extract were combined and the water was removed to obtain the third water extract. The volatile oil inclusion complex and the third aqueous extract are mixed to obtain a pharmaceutical composition.

2. The application according to claim 1, characterized in that, The total mass ratio of Acorus tatarinowii and Ligusticum chuanxiong to water is 1:(6~10); the extraction time by steam distillation is not less than 4.5 hours; During the inclusion process, the amount of excipients used is 2 to 6 times the mass of the volatile oil; the excipients include β-cyclodextrin.

3. The application according to claim 1, characterized in that, The mass ratio of the total mass of Acorus tatarinowii and Ligusticum chuanxiong to water is 1:(7~8); the extraction time by steam distillation is 5~8 hours; During the encapsulation process, the amount of excipients added is 4 to 6 times the mass of the volatile oil.

4. The application according to claim 1, characterized in that, The total mass ratio of Astragalus membranaceus and Polygala tenuifolia to water is 1:(6~8); The boiling time is 1-2 hours; The number of times the water is boiled includes 1 to 3 times.

5. The application according to claim 1, characterized in that, The methods for removing moisture include alcohol precipitation and / or drying; The alcohol precipitation and concentration resulted in an extract with a relative density of 1.10~1.14 g / ml; the working volume concentration of ethanol used for alcohol precipitation and concentration was 70%. The alcohol precipitation concentration time is less than 6 hours; the alcohol precipitation concentration temperature is less than 70°C. The drying process includes vacuum drying; the temperature of the vacuum drying is below 80°C.

6. The application according to claim 1, characterized in that, The cognitive impairment in the elderly includes cognitive impairment caused by Alzheimer's disease and / or cognitive impairment caused by Parkinson's disease, as well as associated depressive-anxious bipolar disorder.

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