Application of Allium macrostemon extract in the preparation of drugs for the prevention and / or treatment of Parkinson's disease and memory impairment

Abstract

This invention utilizes *Allium macrostemon* to obtain its aqueous extract, petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract. The effects of these aqueous extracts and extracts of different polarities on the learning and memory functions of D-galactose-induced aging model mice were investigated using Y-maze, darkness avoidance, Morris water maze, and laser speckle cerebral blood flow assays. Reactive oxygen species (ROS), SOD, and MDA were also measured. Multiple experiments demonstrated that *Allium macrostemon* can improve memory function in mice. Furthermore, the effects of *Allium macrostemon* extract on arecoline- or oxidative tremor-induced tremor behavior in mice were studied. The results showed that *Allium macrostemon* extract can effectively shorten the duration of tremors, proposing a new application for *Allium macrostemon* extract in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases, providing broad application prospects for the treatment or adjuvant therapy of neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the technical field of novel applications of Allium macrostemon, specifically to the application of Allium macrostemon extract in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases. Background Technology

[0002] With the world's population surpassing 8 billion and projected to increase further in the coming decades, the proportion of the elderly population will continue to rise, exacerbating global population aging. Neurodegenerative diseases will also intensify alongside this global population aging trend. Characterized by the death and loss of a large number of nerve cells, these are progressive and complex diseases that can lead to disability or death. They primarily include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). For many years, due to the complexity of brain function, the treatment of neurodegenerative diseases has been a challenge. There are no cures in clinical practice, and most treatments focus on symptom relief. Therefore, research into new drugs targeting these diseases has become a hot topic for scientific research and pharmaceutical companies worldwide.

[0003] Allium macrostemon, the dried bulb of *Allium tuberosum* or *Allium chinense*, is a traditional Chinese medicine used to warm the heart and promote the flow of qi. Modern pharmacological studies have shown that it has vasodilatory and anti-myocardial ischemia effects. However, no research has been reported on the effects of Allium macrostemon and its extracts on neurodegenerative diseases. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by providing the application of Allium macrostemon extract in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases, discovering a new use for Allium macrostemon extract, and showing broad application prospects in the treatment or adjuvant treatment of neurodegenerative diseases.

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

[0006] Application of Allium macrostemon extract in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases.

[0007] Furthermore, neurodegenerative diseases include at least one of memory impairment and Parkinson's disease.

[0008] Application of Allium macrostemon extract in the preparation of drugs that improve cognitive impairment.

[0009] Application of Allium macrostemon extract in the preparation of drugs that increase cerebral blood flow.

[0010] Application of Allium macrostemon extract in the preparation of drugs that reduce ROS, and / or drugs that enhance SOD activity, and / or drugs that specifically reduce MDA content.

[0011] Application of Allium macrostemon extract in the preparation of drugs that shorten the duration of tremor.

[0012] This invention utilizes *Allium macrostemon* to obtain its aqueous extract, petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract. The effects of these extracts with different polarities on the learning and memory function of a D-galactose-induced aging model mouse were investigated using Y-maze, darkness avoidance, Morris water maze, and laser speckle cerebral blood flow assays. Reactive oxygen species (ROS), SOD, and MDA were also measured. Multiple experiments demonstrated that *Allium macrostemon* can improve memory function in mice. Furthermore, the effects of *Allium macrostemon* extract on arecoline- or oxidative tremor-induced tremor behavior in mice were studied. The results showed that *Allium macrostemon* extract can effectively shorten the duration of tremors, proposing a new application for *Allium macrostemon* extract in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases, providing broad application prospects for the treatment or adjuvant therapy of neurodegenerative diseases.

[0013] Furthermore, the Allium macrostemon extract is at least one selected from Allium macrostemon aqueous extract, Allium macrostemon petroleum ether extract, Allium macrostemon dichloromethane extract, Allium macrostemon ethyl acetate extract, and Allium macrostemon n-butanol extract.

[0014] Furthermore, the Allium macrostemon extract is prepared by the following method:

[0015] Take *Allium macrostemon*, soak it in pure water, reflux and heat to boiling, filter through gauze to obtain the first filtrate and the first residue; add 8-10 times the amount of pure water to the first residue, reflux and heat to boiling again, filter through gauze to obtain the second filtrate and the second residue; heat 8-10 times the amount of pure water to the second residue, reflux and heat to boiling, filter through gauze to obtain the third filtrate and the third residue; combine the first, second, and third filtrates, centrifuge, filter the supernatant, and concentrate the filtrate using a rotary evaporator to obtain the water extract of *Allium macrostemon*.

[0016] Take the aqueous extract of Allium macrostemon, add petroleum ether, shake well, let stand, the upper layer is the petroleum ether fraction, the lower layer is extracted 2-4 times, combine the 2-4 extracts and recover the organic solvent by rotary evaporation under reduced pressure, pour the concentrated liquid into an EP tube, put it into a freeze dryer and freeze dry to obtain Allium macrostemon petroleum ether extract;

[0017] Take the lower layer of the remaining liquid after petroleum ether extraction, add dichloromethane for extraction, shake well, let stand, and wait for the liquid surface to separate into layers. The lower layer is the dichloromethane fraction, and the upper layer is extracted 2-4 times. Combine the 2-4 extracts and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the dichloromethane fraction extract of Allium macrostemon.

[0018] Take the remaining liquid in the upper layer after dichloromethane extraction, add ethyl acetate for extraction, shake well, let stand, and wait for the liquid surface to separate into layers, with the upper layer being the ethyl acetate fraction; repeat the extraction 2-4 times, combine the 2-4 extracts, and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the ethyl acetate extract of Allium macrostemon.

[0019] Take the lower layer of liquid remaining after ethyl acetate extraction, add n-butanol for extraction, shake well, let stand, and wait for the liquid surface to separate into layers. The upper layer is the n-butanol portion. Repeat the extraction 2-4 times, combine the 2-4 extracts, and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the n-butanol extract of Allium macrostemon.

[0020] Furthermore, the medicament comprises a therapeutically effective amount of the above-mentioned Allium macrostemon extract and pharmaceutically acceptable excipients; wherein the excipients are at least one of fillers, binders and lubricants.

[0021] Furthermore, the drug is in the form of capsules, tablets, powders, or granules.

[0022] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] This invention utilizes *Allium macrostemon* to obtain its aqueous extract, petroleum ether extract, dichloromethane extract, ethyl acetate extract, and n-butanol extract. The effects of these extracts with different polarities on the learning and memory function of a D-galactose-induced aging model mouse were investigated using Y-maze, darkness avoidance, Morris water maze, and laser speckle cerebral blood flow assays. Reactive oxygen species (ROS), SOD, and MDA were also measured. Multiple experiments demonstrated that *Allium macrostemon* can improve memory function in mice. Furthermore, the effects of *Allium macrostemon* extract on arecoline- or oxidative tremor-induced tremor behavior in mice were studied. The results showed that *Allium macrostemon* extract can effectively shorten the duration of tremors, proposing a new application for *Allium macrostemon* extract in the preparation of drugs for the prevention and / or treatment of neurodegenerative diseases, providing broad application prospects for the treatment or adjuvant therapy of neurodegenerative diseases. Attached Figure Description

[0024] Figure 1 The effect of Allium macrostemon aqueous extract on the spontaneous rotation rate of the Y-maze in Example 1.

[0025] Figure 2 The effect of Allium macrostemon aqueous extract in Example 1 on the latency period (2a) and number of errors (2b) of dark avoidance escape in mice.

[0026] Figure 3 The effect of Allium macrostemon water extract on the latency period of directional cruise escape in Example 1 is shown as the changing trend.

[0027] Figure 4 The effects of the water extract of Allium macrostemon in Example 1 on the space exploration experiment; (4a) number of times the platform area was traversed; (4b) time spent in the platform area.

[0028] Figure 5 The effect of the aqueous extract of Allium macrostemon on cerebral blood flow in Example 1 is shown in Figure 5a and a representative figure (5b).

[0029] Figure 6 The effects of Allium macrostemon aqueous extract on ROS (6a), SOD (6b), and MDA (6c) in Example 1.

[0030] Figure 7 This is a diagram showing the influence of different polarity regions of Allium macrostemon on the spontaneous rotation rate of the Y-maze in Example 2.

[0031] Figure 8 The graph shows the effect of different polar parts of Allium macrostemon on the latency (8a) and number of errors (8b) of mice in Example 2.

[0032] Figure 9 This is a graph showing the changing trend of the influence of different polarity parts of Allium macrostemon on the latency period of directional cruise escape in Example 2.

[0033] Figure 10 The diagram shows the influence of different polar parts of Allium macrostemon on the space exploration experiment in Example 2. (10a) Number of times the platform area was traversed, and (10b) Time spent in the platform area.

[0034] Figure 11 The effects of different polarity regions of Allium macrostemon on cerebral blood flow in Example 2 are shown in Figure 11a and a representative figure (11b).

[0035] Figure 12 The diagram shows the effect of different polarity components of Allium macrostemon on ROS (12a), SOD (12b), and MDA (12c) in Example 2.

[0036] Figure 13 The figure shows the effect of different polar parts of Allium macrostemon on the latency (13a) and duration (13b) of tremor induced by arecoline hydrobromide in mice in Example 3.

[0037] Figure 14It is a graph showing the effects of different polar parts of Allium macrostemon Bunge on the tremor latency (14a) and tremor duration (14b) induced by oxotremorine in Example 3. Detailed implementation manners

[0038] The present invention will be described in detail below with reference to the accompanying drawings.

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Example 1

[0041] Using a D-galactose-induced cognitive aging mouse model, the effect of Allium macrostemon aqueous extract on improving cognitive aging was investigated.

[0042] 1. Experimental materials

[0043] 1.1 Experimental animals

[0044] SPF-grade KM mice, half male and half female, with a body weight of (20±2 g), 36 in number, were purchased from Beijing Specif Biological Technology Co., Ltd. The animal quality certificate number is: N0.110324220107338154, and the license number is: SCXK (Beijing) 2019-0010. The animal experiment was approved by the Animal Ethics Committee of the Institute of Integrative Transformation of Traditional Chinese Medicine for Encephalopathy, Chengdu University of Traditional Chinese Medicine (approval number No. IBD2022012). All animals were housed in the Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine under the State Administration of Traditional Chinese Medicine (Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine, number: TCM2032043). The temperature was controlled at 23±1°C, the humidity was controlled at 50±5%, and the 12-hour light-dark cycle was (light cycle from 8:00 to 20:00, dark cycle from 20:00 to 8:00).

[0045] 1.2 Drugs and reagents

[0046] Allium macrostemon, batch number: 2206149, Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd.; Memantine Hydrochloride Tablets, batch number: 918564, Lundbeck Pharmaceuticals, Denmark; D-galactose, batch number: HR859W8, Baoji Chenguang Biotechnology Co., Ltd.; Petroleum ether, batch number: 2021030301, Chengdu Kelong Chemical Co., Ltd.; Dichloromethane, batch number: 2021101401, Chengdu Kelong Chemical Co., Ltd.; Ethyl acetate, batch number: 2021102001, Chengdu Kelong Chemical Co., Ltd.; n-Butyl... Alcohol, batch number: 2021121701, Chengdu Kelong Chemical Co., Ltd.; Sodium carboxymethyl cellulose, batch number: 2022052301, Chengdu Kelong Chemical Co., Ltd.; Reactive oxygen species detection kit, batch number: 061821211102, Beyotime Biotechnology Co., Ltd.; SOD kit (WST-1 method), batch number: 20230131, Nanjing Jiancheng Biotechnology Co., Ltd.; Lipid oxidation (MDA) detection kit, batch number: 081722230316, Beyotime Biotechnology Co., Ltd.

[0047] 1.3 Instruments and Equipment

[0048] Analytical balance, Shenyang Longteng Electronics Co., Ltd., model JD210-4; Pure water system, Sichuan Chaochun Technology Co., Ltd., model: UPR-II-5TNZ; Heating mantle ZNHW-type, Gongyi Yuhua Instrument Co., Ltd., serial number 221190; Rotary evaporator, Gongyi Ruide Instrument Equipment Co., Ltd., model: RE-2000A; Benchtop low-speed large-capacity centrifuge, Lu Xiangyi, model: TD5N; Freeze dryer, Shanghai Tuo Technology Co., Ltd., model: YTLG-12-12A-80; EthoVision XTMorris water maze system, NOLDUS (Beijing) Information Technology Co., Ltd.; PAT-8 dark avoidance experiment video analysis system, Chengdu Taimeng Software Co., Ltd.; Laser speckle blood flow imaging system, model RFLSIⅢ, Shenzhen Ruiwode Life Technology Co., Ltd.

[0049] 2. Experimental Methods

[0050] 2.1 Drug Preparation

[0051] 2.1.1 Extraction and preparation of aqueous extract of Allium macrostemon

[0052] 40g of Allium macrostemon was soaked in 600ml of pure water for 60min, refluxed and heated to boiling, and then timed for 60min. The mixture was filtered through four layers of gauze to obtain filtrate 1. 10 times the amount of pure water (400ml) was added to the residue, refluxed and heated for 60min, and then filtered through four layers of gauze to obtain filtrate 2. 8 times the amount of pure water (320ml) was added to the residue, refluxed and heated for 60min, and then filtered through four layers of gauze to obtain filtrate 3. The three filtrates were combined, centrifuged at 3000rpm for 5min, and the supernatant was filtered through a Buchner funnel. The filtrate was concentrated to 80ml using a rotary evaporator to obtain Allium macrostemon aqueous extract concentrate, which was diluted to various concentrations using 0.5% CMC-Na.

[0053] 2.2 Grouping and Dosing

[0054] After one week of acclimatization, mice were randomly divided into four groups according to body weight: a control group (Cont.), a D-galactose model group (D-gal), a memantine (MEM) group, a low-dose (AM-L) group of Allium macrostemon aqueous extract, a medium-dose (AM-M) group of Allium macrostemon aqueous extract, and a high-dose (AM-H) group of Allium macrostemon aqueous extract, with six mice in each group. Animals were administered the corresponding drug via gavage at a dose of 10 mL / kg daily. One hour after gavage, all groups except the Cont. group were subcutaneously injected with D-galactose (500 mg / kg, dissolved in physiological saline). The Cont. group received an equal volume of physiological saline subcutaneously. This treatment continued for 42 consecutive days.

[0055] Table 1 Animal grouping and administration

[0056]

[0057] 2.3 Evaluation of Learning and Memory Ability

[0058] 2.3.1 Y Maze Experiment

[0059] The Y-maze experiment is primarily used to test animal discrimination learning, working memory, and reference memory. Spontaneous correct alternation rate is a method for measuring spatial working memory. The Y-maze consists of three identical arms, with an angle of 120° between each arm. Each arm measures 30cm x 15cm x 7cm (length x width x height), with a triangular area connecting the three arms in the center. A camera is installed directly above the maze to record the mouse's movement trajectory. The mouse is placed sequentially into the central triangular area of ​​the maze, and the order in which it enters each arm is recorded for 5 minutes. Each instance of entering an arm with a different number than the previous two is considered a correct spontaneous alternation. The spontaneous correct alternation rate is calculated using the following formula: Spontaneous correct alternation rate % = [Number of correct alternations / (Total number of arm entries - 2)] × 100%.

[0060] 2.3.2 Darkness Avoidance Experiment

[0061] The darkness avoidance test apparatus mainly consists of a bright box and a dark box separated by a partition. The dark box is electrified, while the bright box is not. Utilizing the mice's tendency to be attracted to darkness, mice entering the apparatus will move towards the dark box. However, once they touch the bottom of the dark box, they will receive an electric shock, inducing fear memory. When placed in the same environment again, their memory is evaluated based on their reaction. The specific experimental operation is divided into two stages: the first is an adaptation test, and the second is a detection test. On day 42 of drug administration, 2 hours after drug administration, the adaptation test begins. The mice are placed in the apparatus, the partition is opened, and the power is not connected, allowing the mice to move freely between the bright and dark boxes. After 5 minutes of adaptation, the mice are placed in the bright box, the power is connected, and the partition is opened. The time of the mouse's first electric shock is recorded, which is the escape latency. The number of times the mouse enters the dark box and is shocked within 5 minutes is the number of errors. If the mouse is not shocked within 5 minutes, the escape latency is recorded as 300 seconds. Twenty-four hours later, the experiment was conducted. Mice were placed in a bright box, while the dark box was powered on. The partition was removed immediately at the start of the experiment. The escape latency and the number of errors for each mouse were recorded.

[0062] 2.3.3 Morris Water Maze Experiment

[0063] Twenty-four hours after the passive avoidance experiment, the Morris water maze experiment was conducted. The water maze apparatus was divided into four sections, with a hidden, movable cylindrical platform (6.5 cm in diameter and 15 cm in height) in the third quadrant. Before the experiment, water was poured into the pool, just covering the cylindrical platform by 1 cm, and harmless black ink was added to help the system identify white mice. The Morris water maze experiment consisted of an orienteering navigation experiment (finding the platform) and a spatial exploration experiment.

[0064] Orientational navigation experiment: Mice were placed in a pool of water facing the tank wall from the first quadrant. The time from entry into the water to finding the hidden platform was recorded as the escape latency. Starting from day 44 of drug administration, the experiment was conducted once daily for 5 days. Each session was set to 2 minutes. The first four days were the training period; if the mouse could not find the platform within 2 minutes, it was guided to the platform and held there for 5 seconds. The fifth day was the testing period; if the mouse found the platform within 2 minutes, the system automatically recorded its escape latency. If the mouse did not find the platform within 2 minutes, the escape latency was automatically recorded as 120 seconds.

[0065] Space exploration experiment: 24 hours after the platform search experiment ended, the platform was removed, and the mice were slowly placed into the pool from the first quadrant with their faces facing the bucket wall. The system automatically recorded the cumulative swimming time in the area where the platform was located within 120 seconds (Time in the platform zoom) and the number of times the mice passed the platform area (Number of passing the platform zoom).

[0066] 2.4 Laser speckle cerebral blood flow detection

[0067] After the behavioral experiments, cerebral blood flow in mice was detected using a laser speckle imaging system, and pseudo-color images were used to simulate and visualize the cerebral blood flow. After anesthetizing the mice, the entire skull was exposed, the periosteum was carefully removed using ophthalmic forceps, and the skull was moistened with physiological saline, maintaining this moisture throughout the experiment. The mice were fixed to a foam board, and the head position and camera focus were adjusted until the image was clear. The magnification was adjusted according to the target imaging field of view, and recording was performed continuously for 20 seconds. A 10-second period of stable blood flow (10-20 seconds) was selected to extract the median blood flow value, and the corresponding speckle and pseudo-color images were saved.

[0068] 2.5 Indicator Testing

[0069] 2.5.1 Detection of reactive oxygen species

[0070] Fresh mouse brain tissue was weighed and a single-cell suspension was prepared. The tissue was placed in a culture dish (pre-filled with a small amount of PBS) and minced. 2 ml of trypsin was added. After mixing thoroughly, the mixture was transferred to a 10 ml centrifuge tube and digested at 37°C for 10 min on a shaker. During digestion, the centrifuge tube was removed and inverted twice to mix. After digestion, 6 ml of culture medium was added to stop the digestion. The tube was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 ml of PBS and passed through a cell sieve. The supernatant was discarded after centrifugation at 1500 rpm for 5 min. The pellet was then lysed with 3-5 times its volume of erythrocyte lysis buffer for 2 min. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of PBS. Cells were collected and DCFH-DA was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. The cells were resuspended and incubated at 37°C for 20 min, inverting to mix every 3-5 minutes. Cells were washed with serum-free medium to remove excess DCFH-DA, repeated three times, sieved, and analyzed using FlowJo analysis software.

[0071] 2.5.2 Determination of SOD and MDA using reagent kits

[0072] The activity of SOD and the content of MDA in the brain tissue of mice in each group were determined strictly according to the instructions of the kit.

[0073] 3. Experimental Results

[0074] 3.1Y Maze

[0075] Table 2. Effects of Allium macrostemon aqueous extract on the spontaneous rotation rate of the Y-maze

[0076] Group N (animals) Spontaneous rotation rate (%) Cont. 6 72.34±3.77 D-gal 6 <![CDATA[60.13±3.77 ## ]]> MEM 6 <![CDATA[67.52±4.97 * ]]> AM-PE 6 <![CDATA[69.88±1.24 ** ]]> AM-EA 6 <![CDATA[71.87±8.82 * ]]> AM-NA 6 <![CDATA[72.44±3.11 ** ]]>

[0077] Note: Compared with the blank group, ## P<0.01; compared with the model group, ** P<0.01.

[0078] From Table 2, Figure 1 It was found that, compared with the blank group, the spontaneous rotation rate of mice in the model group was significantly reduced, and the difference was statistically significant (P<0.01). Compared with the model group, the spontaneous rotation rate of the treatment group was increased, and the difference was statistically significant (P<0.05 or P<0.05). This suggests that administration of Allium macrostemon aqueous extract has a positive effect on spatial working memory in D-galactose mice.

[0079] 3.2 Darkness Avoidance Experiment

[0080] Table 3. Effects of Allium macrostemon aqueous extract on escape latency and error count in mice.

[0081] Group N (animals) Avoiding the incubation period (S) Number of errors (times) Cont. 6 173.17±105.44 1.17±0.75 D-gal 6 <![CDATA[17.33±7.97 ## ]]> <![CDATA[3.33±1.21 ## ]]> MEM 6 85.00±109.23 <![CDATA[1.50±1.05 * ]]> AM-L 6 28.50±7.09 <![CDATA[1.83±0.98 * ]]> AM-M 6 <![CDATA[94.17±64.96 * ]]> <![CDATA[1.33±0.52 ** ]]> AM-H 6 <![CDATA[115.83±141.54 * ]]> <![CDATA[0.83±0.41 ** ]]>

[0082] Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0083] From Table 3, Figure 2 The results showed that, compared with the control group, the escape latency of mice in the model group was significantly shortened (P<0.05), and the number of errors was significantly increased (P<0.05). Compared with the model group, the number of errors in all treatment groups was significantly reduced (P<0.05, P<0.01). For escape latency, the medium and high doses significantly prolonged it (P<0.01), while the escape latency in the memantine positive control group was prolonged but not statistically significant (P>0.05). These results suggest that administration of Allium macrostemon aqueous extract can improve fear memory in D-galactosamine mice.

[0084] 3.3 Water Maze Experiment

[0085] 3.3.1 Oriented Cruise Experiment

[0086] Table 4. Effects of Allium macrostemon aqueous extract on the evasion latency of directional cruise.

[0087]

[0088] From Table 4, Figure 3 It can be seen that after five days of directional cruise experiment, the escape latency of each group shortened with the increase of days. Compared with the model group, the escape latency of the blank group was significantly shortened. Two-way ANOVA of group and training days showed F(1,16)=13.3, P=0.0021. The escape latency of the other groups decreased compared with the model group. Among them, the high-dose group showed F(1,18)=5.85, P=0.0263, which was statistically significant.

[0089] 3.3.2 Space Exploration Experiments:

[0090] Table 5. Effects of Allium macrostemon aqueous extract on space exploration experiments

[0091]

[0092]

[0093] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0094] From Table 5, Figure 4 It can be seen that in the spatial exploration experiment, compared with the blank group, the number of times the model group crossed the platform area and the time spent in the platform area were significantly reduced (P<0.05), while the number of times the model group crossed the platform area and the time spent in the platform area were significantly increased in the positive drug group and the high-dose group (P<0.05), suggesting that administration of Allium macrostemon aqueous extract can improve the spatial memory of D-galactosamine mice.

[0095] 3.4 Cerebral blood flow detection

[0096] Table 6. Effects of Allium macrostemon aqueous extract on cerebral blood flow

[0097] Group N (animals) Median blood flow Cont. 6 740±83.06 D-gal 6 <![CDATA[617.8±46.31 # ]]> MEM 6 <![CDATA[691.0±39.21 * ]]> AM-L 6 668.3±34.61 AM-M 6 <![CDATA[701.9±19.55 ** ]]> AM-H 6 <![CDATA[712.1±41.82 ** ]]>

[0098] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0099] From Table 6, Figure 5It was found that D-galactose administration significantly decreased cerebral blood flow in mice (P<0.01), while administration of Allium macrostemon aqueous extract significantly increased cerebral blood flow (P<0.05, P<0.01). This suggests that administration of Allium macrostemon aqueous extract can improve the decrease in cerebral blood flow induced by D-galactose in mice, and the improvement of D-galactose-induced cognitive aging by Allium macrostemon may be related to the improvement in cerebral blood flow.

[0100] 3.5 Indicator Testing

[0101] Table 7. Effects of Allium macrostemon aqueous extract on ROS, SOD, and MDA

[0102]

[0103]

[0104] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0105] From Table 7, Figure 6 It was found that, compared with the control group, the percentage of ROS-positive cells in the brain tissue of the model group was significantly increased, SOD activity was decreased, and MDA content was increased, all with statistical significance (P<0.01). After drug intervention, the percentage of ROS-positive cells decreased, SOD activity increased, and MDA content decreased in all groups. Except for the low-dose group where the percentage of ROS-positive cells and the memantine group showed no statistically significant change in SOD activity (P>0.05), all other groups showed statistical significance (P<0.05 or P<0.01). This suggests that the aqueous extract of Allium macrostemon can improve D-galactose-induced oxidative damage.

[0106] This experiment shows that the aqueous extract of Allium macrostemon can improve D-galactose-induced cognitive aging in mice, and its effect may be related to improving decreased cerebral blood flow and anti-oxidation.

[0107] Example 2

[0108] The preparation of different Allium macrostemon extracts is as follows:

[0109] Extraction and preparation of water extract of Allium macrostemon

[0110] 80g of Allium macrostemon was soaked in 1200ml of pure water for 60min, refluxed and heated to boiling for 60min, and filtered through four layers of gauze to obtain filtrate 1; 10 times the amount of pure water (800ml) was added to the residue, refluxed and heated for 60min, and filtered through four layers of gauze to obtain filtrate 2; 8 times the amount of pure water (640ml) was added to the residue, refluxed and heated for 60min, and filtered through four layers of gauze to obtain filtrate 3; the three filtrates were combined, centrifuged at 3000rpm for 5min, the supernatant was filtered through a Buchner funnel, and the filtrate was concentrated to 300ml using a rotary evaporator.

[0111] Separation of different polarity parts of Allium macrostemon

[0112] Petroleum ether fraction: After checking the separatory funnel for leaks, pour in 300 ml of total Allium macrostemon extract, add 300 ml of petroleum ether for extraction, shake well, and let stand for 15 minutes until the liquid surface separates into layers. The upper layer is the petroleum ether fraction. Repeat the extraction three times for the lower layer, combine the three extracts, and recover the organic solvent by rotary evaporation under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the petroleum ether fraction of the Allium macrostemon aqueous extract.

[0113] Dichloromethane fraction: Add the lower layer of the remaining liquid after petroleum ether extraction to a separatory funnel, add the same amount of dichloromethane for extraction, shake well, let stand for 15 minutes, and wait for the liquid to separate into layers; the lower layer is the dichloromethane fraction. Repeat the extraction 3 times, combine the 3 extracts, and recover the organic solvent by rotary evaporation under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the dichloromethane fraction of the Allium macrostemon aqueous extract.

[0114] Ethyl acetate fraction: Add the remaining liquid from the upper layer after dichloromethane extraction to a separatory funnel, add the same amount of ethyl acetate for extraction, shake well, and let stand for 15 minutes until the liquid layers separate; the upper layer is the ethyl acetate fraction. Repeat the extraction three times, combine the three extracts, and recover the organic solvent under reduced pressure using a rotary evaporator. Pour the concentrated liquid into an EP tube and freeze-dry it in a freeze dryer to obtain the ethyl acetate fraction of the Allium macrostemon aqueous extract.

[0115] n-Butanol fraction: Add the lower layer of the remaining liquid after ethyl acetate extraction to a separatory funnel, add the same amount of n-butanol for extraction, shake well, let stand for 15 minutes, and wait for the liquid to separate into layers. The upper layer is the n-butanol fraction. Repeat the extraction 3 times, combine the 3 extracts, and recover the organic solvent by rotary evaporation under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the n-butanol fraction of the Allium macrostemon aqueous extract.

[0116] Effects of Allium macrostemon on learning and memory function in D-galactose-induced aging model mice

[0117] 4. Experimental Materials

[0118] 4.1 Laboratory Animals

[0119] SPF-grade KM mice, with an equal number of males and females, weighing (20±2 g), 42 in total, were purchased from Spf (Beijing) Biotechnology Co., Ltd. The animal quality certificate number is: N0.110324220107338154, and the license number is: SCXK (Beijing) 2019-0010. The animal experiment was approved by the Animal Ethics Committee of the Institute of Integrative Transformation of Traditional Chinese Medicine for Encephalopathy, Chengdu University of Traditional Chinese Medicine (approval number No. IBD2022012). All animals were housed in the Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine (Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine, number: TCM2032043). The temperature was controlled at 23±1 °C, the humidity was controlled at 50±5%, and the 12-hour light-dark cycle was (light cycle from 8:00 to 20:00, dark cycle from 20:00 to 8:00).

[0120] 4.2 Drugs and Reagents

[0121] The same as in Example 1.

[0122] 4.3 Instruments and Equipment

[0123] The same as in Example 1.

[0124] 5. Experimental Methods

[0125] 5.1 Grouping and Drug Administration

[0126] After 1 week of adaptive feeding, the mice were randomly divided into blank (Cont.) group, D-galactose model (D-gal) group, memantine (MEM) group, petroleum ether fraction of Allium macrostemon Bunge (AM-PE) group, dichloromethane fraction of Allium macrostemon Bunge (AM-DC) group, ethyl acetate fraction of Allium macrostemon Bunge (AM-EA) group, and n-butanol fraction of Allium macrostemon Bunge (AM-NA) group, with 6 mice in each group. The animals were intragastrically administered 10 mL / kg of the corresponding drug every day. After 1 hour of intragastric administration, except for the Cont. group, the other groups were subcutaneously injected with D-galactose (500 mg / kg, dissolved in normal saline), and the Cont. group was subcutaneously injected with an equal volume of normal saline for 42 consecutive days.

[0127] Table 8 Animal Grouping and Drug Administration

[0128]

[0129] 5.2 Evaluation of Learning and Memory Ability

[0130] 5.2.1 Y-Maze Experiment

[0131] The Y-maze experiment is primarily used to test animal discrimination learning, working memory, and reference memory. Spontaneous correct alternation rate is a method for measuring spatial working memory. The Y-maze consists of three identical arms, with an angle of 120° between each arm. Each arm measures 30cm x 15cm x 7cm (length x width x height), with a triangular area connecting the three arms in the center. A camera is installed directly above the maze to record the mouse's movement trajectory. The mouse is placed sequentially into the central triangular area of ​​the maze, and the order in which it enters each arm is recorded for 5 minutes. Each instance of entering an arm with a different number than the previous two is considered a correct spontaneous alternation. The spontaneous correct alternation rate is calculated using the following formula: Spontaneous correct alternation rate % = [Number of correct alternations / (Total number of arm entries - 2)] × 100%.

[0132] 5.2.2 Darkness Avoidance Experiment

[0133] The darkness avoidance test apparatus mainly consists of a bright box and a dark box separated by a partition. The dark box is electrified, while the bright box is not. Utilizing the mice's tendency to be attracted to darkness, mice entering the apparatus will move towards the dark box. However, once they touch the bottom of the dark box, they will receive an electric shock, inducing fear memory. When placed in the same environment again, their memory is evaluated based on their reaction. The specific experimental operation is divided into two stages: the first is an adaptation test, and the second is a detection test. On day 42 of drug administration, 2 hours after drug administration, the adaptation test begins. The mice are placed in the apparatus, the partition is opened, and the power is not connected, allowing the mice to move freely between the bright and dark boxes. After 5 minutes of adaptation, the mice are placed in the bright box, the power is connected, and the partition is opened. The time of the mouse's first electric shock is recorded, which is the escape latency. The number of times the mouse enters the dark box and is shocked within 5 minutes is the number of errors. If the mouse is not shocked within 5 minutes, the escape latency is recorded as 300 seconds. Twenty-four hours later, the experiment was conducted. Mice were placed in a bright box, while the dark box was powered on. The partition was removed immediately at the start of the experiment. The escape latency and the number of errors for each mouse were recorded.

[0134] 5.2.3 Morris Water Maze Experiment

[0135] Twenty-four hours after the passive avoidance experiment, the Morris water maze experiment was conducted. The water maze apparatus was divided into four sections, with a hidden, movable cylindrical platform (6.5 cm in diameter and 15 cm in height) in the third quadrant. Before the experiment, water was poured into the pool, just covering the cylindrical platform by 1 cm, and harmless black ink was added to help the system identify white mice. The Morris water maze experiment consisted of an orienteering navigation experiment (finding the platform) and a spatial exploration experiment.

[0136] Orientational navigation experiment: Mice were placed in a pool of water facing the tank wall from the first quadrant. The time from entry into the water to finding the hidden platform was recorded as the escape latency. Starting from day 44 of drug administration, the experiment was conducted once daily for 5 days. Each session was set to 2 minutes. The first four days were the training period; if the mouse could not find the platform within 2 minutes, it was guided to the platform and held there for 5 seconds. The fifth day was the testing period. If the mouse found the platform within 2 minutes, the system automatically recorded its escape latency. If the mouse did not find the platform within 2 minutes, the escape latency was automatically recorded as 120 seconds.

[0137] Space exploration experiment: 24 hours after the platform search experiment ended, the platform was removed, and the mice were slowly placed into the pool from the first quadrant with their faces facing the bucket wall. The system automatically recorded the cumulative swimming time in the area where the platform was located within 120 seconds (Time in the platform zoom) and the number of times the mice passed the platform area (Number of passing the platform zoom).

[0138] 5.3 Laser speckle cerebral blood flow detection

[0139] After the behavioral experiments, cerebral blood flow in mice was detected using a laser speckle imaging system, and pseudo-color images were used to simulate and visualize the cerebral blood flow. After anesthetizing the mice, the entire skull was exposed, the periosteum was carefully removed using ophthalmic forceps, and the skull was moistened with physiological saline, maintaining this moisture throughout the experiment. The mice were fixed to a foam board, and the head position and camera focus were adjusted until the image was clear. The magnification was adjusted according to the target imaging field of view, and recording was performed continuously for 20 seconds. A 10-second period of stable blood flow (10-20 seconds) was selected to extract the median blood flow value, and the corresponding speckle and pseudo-color images were saved.

[0140] 5.4 Indicator Testing

[0141] 5.4.1 Detection of reactive oxygen species

[0142] Fresh mouse brain tissue was weighed and a single-cell suspension was prepared. The tissue was placed in a culture dish (pre-filled with a small amount of PBS) and minced. 2 ml of trypsin was added. After mixing thoroughly, the mixture was transferred to a 10 ml centrifuge tube and digested at 37°C for 10 min on a shaker. During digestion, the centrifuge tube was removed and inverted twice to mix. After digestion, 6 ml of culture medium was added to stop the digestion. The tube was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 ml of PBS and passed through a cell sieve. The supernatant was discarded after centrifugation at 1500 rpm for 5 min. The pellet was then lysed with 3-5 times its volume of erythrocyte lysis buffer for 2 min. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of PBS. Cells were collected and DCFH-DA was diluted 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. The cells were resuspended and incubated at 37°C for 20 min, inverting to mix every 3-5 minutes. Cells were washed with serum-free medium to remove excess DCFH-DA, repeated three times, sieved, and analyzed using FlowJo analysis software.

[0143] 5.4.2 Determination of SOD and MDA using reagent kits

[0144] Mouse brain tissue was homogenized by weight: physiological saline = 1:9, and centrifuged at 12000 r / min at 4℃. The activity of SOD and the content of MDA in the brain tissue of each group of mice were determined strictly according to the operation steps of the kit instructions.

[0145] 6. Experimental Results

[0146] 6.1Y Maze

[0147] Table 9. Influence of different polarity regions of Allium macrostemon on the spontaneous rotation rate of the Y-maze.

[0148] Group N (animals) Spontaneous rotation rate (%) Cont. 6 76.42±4.17 D-gal 6 <![CDATA[63.59±7.57 ## ]]> MEM 6 68.93±6.02 AM-PE 6 70.85±6.01 AM-DC 6 <![CDATA[76.29±4.18 ** ]]> AM-EA 6 72.64±7.30 AM-NA 6 66.08±5.00

[0149] Note: Compared with the blank group, ##P<0.01; compared with the model group, **P<0.01.

[0150] From Table 9, Figure 7 The results showed that, compared with the control group, the spontaneous rotation rate of mice in the model group was significantly reduced (P<0.01). Compared with the model group, the spontaneous rotation rate of the drug-treated groups was increased, with the DC group showing a significant difference (P<0.01). There were no significant differences between the other drug-treated groups and the model group (P>0.05). This suggests that administration of the dichloromethane fraction of Allium macrostemon has a positive effect on spatial working memory in D-galactosamine mice.

[0151] 6.2 Darkness Avoidance Experiment

[0152] Table 10. Effects of different polar parts of Allium macrostemon on escape latency and error rate in mice (X±S)

[0153] Group N (animals) Avoiding the incubation period (S) Number of errors (times) Cont. 6 137.67±129.63 1.00±1.10 D-gal 6 <![CDATA[9.00±5.76 # ]]> <![CDATA[3.33±1.21 ## ]]> MEM 6 121.33±129.65 <![CDATA[1.50±0.84 * ]]> AM-PE 6 41.00±44.01 <![CDATA[1.67±0.82 * ]]> AM-DC 6 <![CDATA[152.83±147.61 * ]]> <![CDATA[1.17±1.17 * ]]> AM-EA 6 <![CDATA[143.83±127.81 * ]]> <![CDATA[1.33±0.82 ** ]]> AM-NA 6 59.00±118.09 <![CDATA[1.00±0.63 ** ]]>

[0154] Note: Compared with the blank group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0155] From Table 10, Figure 8 The results showed that, compared with the control group, the escape latency of mice in the model group was significantly shortened (P<0.05), and the number of errors was significantly increased (P<0.05). Compared with the model group, the number of errors in all drug-treated groups was significantly reduced (P<0.05, P<0.01). Regarding escape latency, the DC and EA groups showed significant prolongation (P<0.01), while the escape latency in the memantine positive control group was prolonged but not statistically significant (P>0.05). The PE and NA groups showed no significant change in escape latency. These results suggest that the dichloromethane and ethyl acetate fractions of Allium macrostemon have a better effect on fear memory in D-galactosamine mice.

[0156] 6.3 Water Maze Experiment

[0157] 6.3.1 Oriented Cruise Experiment

[0158] Table 11. Influence of different polarity parts of Allium macrostemon on the latency period of directional cruising evasion.

[0159]

[0160]

[0161] From Table 11, Figure 9 It can be seen that after five days of directional cruise experiment, the escape latency of the blank group was significantly shortened compared with the model group. Two-way ANOVA of group and training days showed F(1,14)=6.064, P=0.0274. The escape latency of DC group and EA group decreased to a certain extent. Specifically, DC group F(1,15)=4.299, P=0.0558, and EA group F(1,14)=4.253, P=0.0583. The escape latency of other groups did not change significantly.

[0162] 6.3.2 Space Exploration Experiments:

[0163] Table 12. Influence of different polarity parts of Allium macrostemon on space exploration experiments

[0164] Group N (animals) Number of times the platform area was traversed (times) Platform area dwell time (s) Cont. 6 2.67±0.82 0.53±0.16 D-gal 5 <![CDATA[0.60±0.55 ## ]]> <![CDATA[0.12±0.11 ## ]]> MEM 5 <![CDATA[1.40±0.55 * ]]> <![CDATA[0.52±0.23 ** ]]> AM-PE 6 1.17±0.98 0.27±0.24 AM-DC 6 <![CDATA[2.17±0.98 * ]]> <![CDATA[0.53±0.24 ** ]]> AM-EA 6 <![CDATA[1.83±0.75 * ]]> <![CDATA[0.47±0.21 ** ]]> AM-NA 6 0.67±1.21 0.13±0.24

[0165] Note: Compared with the blank group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0166] From Table 12, Figure 10 It can be seen that in the spatial exploration experiment, compared with the blank group, the number of times the model group crossed the platform area and the time spent in the platform area were significantly reduced (P<0.01), while the number of times the model group crossed the platform area and the time spent in the platform area were significantly increased in the methimazole positive drug group, DC group and EA group (P<0.05, P<0.01), suggesting that administration of dichloromethane and ethyl acetate fractions of Allium macrostemon can improve the spatial memory of D-galactosamine mice.

[0167] 6.4 Cerebral blood flow detection

[0168] Table 13. Effects of different polarity sites of Allium macrostemon on cerebral blood flow

[0169]

[0170]

[0171] Note: Compared with the blank group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0172] From Table 13, Figure 11 It was found that D-galactose administration significantly decreased cerebral blood flow in mice (P<0.01), while administration of DC, EA, and NA from Allium macrostemon significantly increased cerebral blood flow (P<0.05, P<0.01). This suggests that administration of dichloromethane, ethyl acetate, and n-butanol fractions of Allium macrostemon can improve the D-galactose-induced decrease in cerebral blood flow in mice, and the improvement in D-galactose-induced cognitive aging by different polarity fractions of Allium macrostemon may be related to the improvement in cerebral blood flow.

[0173] 6.5 Indicator Testing

[0174] Table 14. Effects of different polarity fractions of Allium macrostemon on ROS, SOD, and MDA

[0175] Group N (animals) Percentage of ROS-positive cells (%) SOD activity (U / mgprot) MDA content (μM) Cont. 5 13.86±6.71 51.84±6.29 9.51±1.61 D-gal 5 <![CDATA[24.69±4.56 ## ]]> <![CDATA[27.10±4.71 ## ]]> <![CDATA[16.91±2.63 ## ]]> MEM 5 <![CDATA[9.20±2.35 ** ]]> <![CDATA[47.33±11.21 ** ]]> 12.86±3.91 AM-PE 5 <![CDATA[13.77±4.02 ** ]]> <![CDATA[45.41±10.43 ** ]]> <![CDATA[9.43±2.10 ** ]]> AM-DC 5 <![CDATA[11.80±1.91 ** ]]> <![CDATA[46.33±14.20 * ]]> <![CDATA[11.80±2.69 * ]]> AM-EA 5 <![CDATA[13.33±5.83 ** ]]> <![CDATA[39.36±10.87 * ]]> <![CDATA[9.96±1.87 ** ]]> AM-NA 5 18.33±13.19 <![CDATA[41.17±1.59 ** ]]> <![CDATA[10.78±1.51 ** ]]>

[0176] Note: Compared with the blank group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0177] From Table 14, Figure 12It can be seen that compared with the blank group, the percentage of ROS-positive cells in the brain tissue of the model group was significantly increased, the SOD activity was decreased, and the MDA content was increased, all of which were statistically significant (P<0.01). After drug intervention, the percentage of ROS-positive cells in each group decreased, the SOD activity increased, and the MDA content decreased. Except for the MDA content in the memantine group and the percentage of ROS-positive cells in the NA group, which were not statistically significant (P>0.05), the rest were statistically significant (P<0.05 or P<0.01).

[0178] Example 3

[0179] Effects of Allium macrostemon Bunge on the tremor behavior of mice induced by arecoline or oxotremorine

[0180] 7. Experimental materials

[0181] 7.1 Experimental animals

[0182] SPF-grade KM mice, all male, weighing (20±2 g), 72 in number, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The animal quality certificate numbers are: NO.430727230100582120, NO.430727230101382416, and the license number is: SCXK(Xiang)2019-0004. The animal experiment was approved by the Animal Ethics Committee of the Institute of Integrative Transformation of Traditional Chinese Medicine Encephalopathy Drugs, Chengdu University of Traditional Chinese Medicine (approval number No.IBD2023005). All animals were housed in the Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine (Pharmacology Third-level Laboratory of Chengdu University of Traditional Chinese Medicine, number: TCM2032043). The temperature was controlled at 23±1°C, the humidity was controlled at 50±5%, and the 12h light-dark cycle was (8:00-20:00 light cycle, 20:00-8:00 dark cycle).

[0183] 7.2 Drugs and reagents

[0184] Allium macrostemon Bunge, batch number: 2206149, Sichuan New Lotus Chinese Herbal Pieces Co., Ltd.; Madopar, batch number: SH5238, Roche Pharmaceuticals Shanghai Co., Ltd.; Arecoline hydrobromide, batch number: 170502S, Nanjing Dausif Bio-Tech Co., Ltd.; Oxotremorine, batch number: 033M4724V, Sigma, USA; Petroleum ether, batch number: 2021030301, Chengdu Kelong Chemical Co., Ltd.; Dichloromethane, batch number: 2021101401, Chengdu Kelong Chemical Co., Ltd.; Ethyl acetate, batch number: 2021102001, Chengdu Kelong Chemical Co., Ltd.; n-Butanol, batch number: 2021121701, Chengdu Kelong Chemical Co., Ltd.; Sodium carboxymethyl cellulose, batch number: 2022052301, Chengdu Kelong Chemical Co., Ltd.

[0185] 7.3 Instruments and Equipment

[0186] Analytical balance, Shenyang Longteng Electronics Co., Ltd., model JD210-4; Pure water system, Sichuan Chaochun Technology Co., Ltd., model: UPR-II-5TNZ; Heating mantle ZNHW-type, Gongyi Yuhua Instrument Co., Ltd., serial number 221190; Rotary evaporator, Gongyi Ruide Instrument Equipment Co., Ltd., model: RE-2000A; Benchtop low-speed large-capacity centrifuge, Lu Xiangyi, model: TD5N; Freeze dryer, Shanghai Tuo Technology Co., Ltd., model: YTLG-12-12A-80.

[0187] 8. Experimental Methods

[0188] 8.1 Drug Preparation

[0189] The drug preparation method is the same as in Example 1.

[0190] 8.2 Grouping and Dosing

[0191] Arecoline hydrobromide model mice were acclimatized for three days, weighed, numbered, and randomly divided into the arecoline hydrobromide model group, the levodopa group, the allium macrostemon dichloromethane extract (AM-DC) group, the allium macrostemon ethyl acetate extract (AM-EA) group, and a blank control group (n=6). The remaining groups each had 7 mice. The arecoline hydrobromide group was administered 0.5% CMCCNa by gavage, the levodopa group was administered 50 mg / kg levodopa by gavage, and the other groups received the corresponding drugs. All medications were administered once daily for 14 consecutive days. On the last day, arecoline (25 mg / kg) was administered intraperitoneally one hour after the initial administration.

[0192] The grouping and administration methods of the oxidative shock factor model were the same as those of the arecoline hydrobromide model. There were 6 blank group and 8 in each of the other groups. One hour after the last day of administration, oxidative shock factor (0.15 mg / kg) was administered via a single intraperitoneal injection. The specific results are shown in Tables 15 and 16.

[0193] Table 15 Grouping and Administration of Arecoline Hydrobromide Model Animals

[0194]

[0195] Table 16 Grouping and Administration of Oxidative Tremor-Induced Animal Models

[0196]

[0197] 8.3 Behavioral Observation

[0198] Record the time from intraperitoneal injection of arecoline hydrobromide or oxytetracycline to the onset of muscle tremors (tremor latency) and the time from the onset to the end of muscle tremors (tremor duration) in mice.

[0199] 9. Experimental Results

[0200] 9.1 Arecoline hydrobromide model

[0201] Table 17. Effects of different polar fractions of Allium macrostemon on arecoline hydrobromide-induced tremor behavior in mice.

[0202]

[0203]

[0204] Note: *P<0.05 compared to the model group.

[0205] From Table 17, Figure 13 It was found that, compared with the model group mice, the levodopa positive control group significantly prolonged the latency of tremor induced by arecoline bromate (P<0.05) and shortened the duration of tremor (P<0.05). Administration of the DC part of Allium macrostemon can shorten the duration of tremor induced by arecoline bromate (P<0.05), but has no significant effect on the latency of tremor (P>0.05). Administration of the EA part of Allium macrostemon has no significant effect on the latency and duration of tremor induced by arecoline hydrobromate in mice (P>0.05).

[0206] 9.2 Oxidative Tremor Model

[0207] Table 18. Effects of different polar parts of Allium macrostemon on tremor behavior induced by oxy-tremorin in mice.

[0208]

[0209] Note: Compared with the model group, *P<0.05, **P<0.01

[0210] From Table 18, Figure 14 It was found that, compared with the model group mice, the positive control group of levodopa significantly shortened the duration of tremor induced by oxidative tremor in mice (P<0.01), and administration of the DC and EA parts of Allium macrostemon could shorten the duration of tremor (P<0.05). None of the administration groups had a significant effect on the tremor latency (P>0.05).

[0211] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of Allium macrostemon extract as the sole active ingredient in the preparation of drugs for the prevention and / or treatment of Parkinson's disease; The Allium macrostemon extract is at least one of the following: dichloromethane extract of Allium macrostemon aqueous extract and ethyl acetate extract of Allium macrostemon aqueous extract.

2. The application of Allium macrostemon extract as the sole active substance in the preparation of drugs for the prevention and / or treatment of memory impairment, characterized in that, The Allium macrostemon extract is at least one of the following: dichloromethane extract of Allium macrostemon aqueous extract and ethyl acetate extract of Allium macrostemon aqueous extract.

3. The application according to claim 1 or 2, characterized in that, The Allium macrostemon extract was prepared by the following method: Allium macrostemon was soaked in pure water, refluxed and heated to boiling, and filtered through gauze to obtain a first filtrate and a first residue; 8-10 times the amount of pure water was added to the first residue, refluxed and heated to boiling again, and filtered through gauze to obtain a second filtrate and a second residue; 8-10 times the amount of pure water was added to the second residue, refluxed and heated to boiling, and filtered through gauze to obtain a third filtrate and a third residue; the first, second, and third filtrates were combined, centrifuged, the supernatant was filtered, and the filtrate was concentrated using a rotary evaporator to obtain the Allium macrostemon aqueous extract; Take the aqueous extract of Allium macrostemon, add petroleum ether, shake well, let stand, the upper layer is the petroleum ether fraction, the lower layer is extracted 2-4 times, combine the 2-4 extracts and recover the organic solvent by rotary evaporation under reduced pressure, pour the concentrated liquid into an EP tube, put it into a freeze dryer and freeze dry to obtain Allium macrostemon petroleum ether extract; Take the lower layer of the remaining liquid after petroleum ether extraction, add dichloromethane for extraction, shake well, let stand, and wait for the liquid surface to separate into layers. The lower layer is the dichloromethane fraction, and the upper layer is extracted 2-4 times. Combine the 2-4 extracts and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the dichloromethane fraction extract of Allium macrostemon. Take the remaining liquid in the upper layer after dichloromethane extraction, add ethyl acetate for extraction, shake well, let stand, and wait for the liquid surface to separate into layers, with the upper layer being the ethyl acetate fraction; repeat the extraction 2-4 times, combine the 2-4 extracts, and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the ethyl acetate extract of Allium macrostemon. Take the lower layer of liquid remaining after ethyl acetate extraction, add n-butanol for extraction, shake well, let stand, and wait for the liquid surface to separate into layers. The upper layer is the n-butanol portion. Repeat the extraction 2-4 times, combine the 2-4 extracts, and use a rotary evaporator to recover the organic solvent under reduced pressure. Pour the concentrated liquid into an EP tube and freeze dry it in a freeze dryer to obtain the n-butanol extract of Allium macrostemon.

4. The application according to any one of claims 1-2, characterized in that, The drug is prepared from a therapeutically effective amount of Allium macrostemon extract and pharmaceutically acceptable excipients; wherein the excipients are at least one of fillers, binders and lubricants.

5. The application according to claim 4, characterized in that, The drug is in the form of capsules, tablets, powders, or granules.

6. The application according to claim 3, characterized in that, The drug is prepared from a therapeutically effective amount of Allium macrostemon extract and pharmaceutically acceptable excipients; wherein the excipients are at least one of fillers, binders and lubricants.

7. The application according to claim 6, characterized in that, The drug is in the form of capsules, tablets, powders, or granules.

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